B lymphocytes infiltrate ischemic brain forming follicle-like structures and contributing to autoreactive responses after experimental stroke
K. Winek1, T. Zhang1, C. Dames2, E. Andrzejak1, C. Meisel2 and A. Meisel1
1Charité - Universitätsmedizin Berlin, Department of Experimental Neurology, Berlin, Germany
2Charité - Universitätsmedizin Berlin, Institute for Medical Immunology, Berlin, Germany
Abstract
Central nervous system (CNS) injury including stroke leads to local immune reaction (microglia) and infiltration of peripheral immune cells (neutrophils, monocytes/macrophages and lymphocytes) to the brain. Accumulating evidence suggests a pivotal role of CNS-infiltrating B cells in the late phase of local inflammatory reaction after cerebral ischemia and production of autoantibodies, possibly contributing to post-stroke cognitive impairment.
In our experiments, we investigated CNS infiltration of B lymphocytes, presence of autoantibodies and cognitive phenotype in C57BL/6J mice 14 and 49 days after experimental stroke (middle cerebral artery occlusion, MCAo).
We found accumulation of B cells in the ischemic brain on both time points after MCAo with higher B cell-counts on day 49 compared to day 14. Within CD19+ cells ca. 10% were plasmablasts and plasma cells. Interestingly, CNS-infiltrating B lymphocytes formed aggregates similar to follicle-like structures. Whereas T cells were scattered in the ischemic hemisphere, B cells clustered together in the core of injured tissue. Importantly, infiltration of B cells was dependent on the presence of CD4+ T cells, since we did not find B cells in brains of animals after CD4+ T cells depletion. Additionally, we found serum antibodies reactive to CNS antigens in ca. 18% of MCAo mice. We observed a trend towards decreased cognitive performance in behavioral tests (novel object recognition and Y maze) in mice after cerebral ischemia.
In summary, our data demonstrate a T cell dependent delayed B cell infiltration into the ischemic brain tissue. Moreover, our data support findings of other groups suggesting that CNS-infiltrating B cells are involved in post-stroke cognitive impairment. Autoreactive T and B cell responses may provide targets for immunomodulatory approaches for such delayed post-stroke complications.
PS05-002
Poster Viewing Session V
Supplementary motor area, but not primary motor cortex - graded real-time fMRI neurofeedback training and its translation to stroke patients
D. Mehler1, A. Williams1, F. Krause2, M. Luehrs2, H. Shetty3, D. Turner4, D. Linden1 and J. Whittaker1
1Cardiff University, CUBRIC, Cardiff, United Kingdom
2Maastricht University, Department of Cognitive Neuroscience, Maastricht, Netherlands
3University Hospital of Wales, Cardiff, United Kingdom
4University of East London, School of Health, Sport and Bioscience, London, United Kingdom
Abstract
Objectives: Real-time fMRI neurofeedback has been suggested as a potential tool to improve motor recovery in stroke [1]. We first aimed to identify the best training protocol comparing the up-regulation success of healthy participants in two motor areas involved in stroke recovery: the supplementary motor area (SMA) and the primary motor cortex (M1). Based on the best protocol, we will then present the first data on the feasibility of this approach in stroke patients with middle cerebral artery infarct (MCA).
Methods: Healthy participants (n = 20) were tested in a within-subject design containing 5 training runs of bilateral SMA and M1 neurofeedback, respectively. Further, we employed a graded neurofeedback training which involves discrete target levels of activation and to test the ability of fine self-regulation [2]. Participants were instructed to use kinesthetic motor imagery. Data from 3 subjects was excluded due to technical reasons.
Results: The data suggest that healthy participants can up-regulate the SMA, a sustained plateau BOLD response was found. In contrast, M1 was on average down-regulated despite positive reinforcement. The data further suggest a trend for a parametric effect of the target levels for SMA training. These results inform the protocol for a study with MCA stroke patients, of which preliminary results will be compared and discussed in light of the pathology caused by the MCA stroke.
Conclusions: This is the first study that compared SMA and M1 real-time fMRI neurofeedback training in healthy participants. The SMA was identified as a promising target for patients with MCA stroke.
References
[1] Linden, D., Turner, D., Real-time functional magnetic resonance imaging neurofeedback in motor neurorehabilitation, Current Opinion in Neurology.
[2] Sorger, B., Kamp, T., Weiskopf, N., Peters, J.C., Goebel, R., When the brain takes ‘BOLD' steps: Real-time fMRI neurofeedback can further enhance the ability to gradually self-regulate regional brain activation, Neuroscience.
PS05-003
Poster Viewing Session V
Rivaroxaban limits early hematoma expansion after experimental intracerebral hemorrhage compared with warfarin
S. Sawada1,2, Y. Egashira1,2, Y. Ono2, M. Shimazawa2, T. Iwama1 and H. Hara2
1Gifu University Graduate School of Medicine, Neurosurgery, Gifu City, Japan
2Gifu Pharmaceutical University, Biofunctional Evaluation, Molecular Pharmacology, Gifu, Japan
Abstract
Objectives: Intracerebral hemorrhage (ICH) occurring during oral anticoagulation with vitamin K antagonist is life-threatening complication. However, it remains controversial whether direct oral anticoagulants (DOACs) are associated with larger hematoma volume and higher mortality rates. The aim of this study was to evaluate hemorrhagic volume and pathophysiology of ICH during anticoagulation with rivaroxaban compared with warfarin.
Methods: Mice were orally pretreated with rivaroxaban (10 or 30 mg/kg), warfarin (4 mg/kg), or vehicle. ICH was induced by intrastriatal collagenase-injection. Hemorrhagic volume, neurological outcome, and survival rate were examined. We evaluated blood brain barrier (BBB) permeability 6 hours after ICH-induction using Evans blue spectrophotometry. To estimate the mechanism of hematoma expansion and BBB permeability between warfarin and rivaroxaban, we focused thrombin, which was clot-derived factors and one of major contributor to ICH-induced brain injury. To investigate effects of anticoagulant agents to thrombin-induced injuries, we evaluated human brain endothelial cells of vessel components in vitro, using membrane permeability assays with TEER and FITC-dextran leakage.
Results: Hematoma volume and neurological deficit 24 hours after ICH-induction were significantly decreased in rivaroxaban-pretreated group compared with warfarin-pretreated group (p < 0.05 and p < 0.01, respectively). Rivaroxaban did not increase hemorrhagic volume compared with vehicle. Survival rate 7 days after ICH-induction was improved in rivaroxaban group (p < 0.05 vs. warfarin). Evans blue extravasation at perihematomal lesion was significantly reduced in rivaroxaban compared with warfarin (p < 0.01). In vitro studies, TEER and FITC-dextran leakage were detectably altered by thrombin exposure. Rivaroxaban significantly mitigated thrombin-induced paracellular permeability of brain endothelial cells, but warfarin did not affect.
Conclusions: ICH occurring during rivaroxaban anticoagulation was smaller than warfarin anticoagulation in the experimental models. Rivaroxaban decreases the BBB disruption after ICH, and limits early hematoma expansion compared with warfarin. Our study suggests that rivaroxaban has advantage over warfarin in consideration of ICH, the important complication of long-term anticoagulation.
PS05-004
Poster Viewing Session V
Longitudinal monitoring of mesoscopic cortical activity using a fluorescent bead mouse model of small vessel disease and GCaMP6 imaging
M. Balbi1, G. Silasi1, M.P. Vanni1, Y. Sekino1, J. LeDue1 and T.H. Murphy1
1University of British Columbia, Psychiatry, Vancouver, Canada
Abstract
Introduction: Small vessel diseases (SVDs) comprise the majority of cases of vascular dementia. Microinfarcts usually escape detection by conventional magnetic resonance imaging. Reliable animal models that allow high-resolution functional imaging are needed to study brain function in SVD.
Objectives: Our work explores a SVD model where occlusions of small penetrating arterioles are reproduced in mice by endovascular injection of fluorescent microspheres. Occlusions in this model cause gross motor impairments, cell loss and axonal disruption throughout the brain. While the cortex itself is relatively spared from structural damage —less than 1% of occlusions result in microinfarcts1— subcortical, white matter damage may disrupt cortical function.
Methods: Hence, we evaluated functional connectivity in the mouse cortex using genetically encoded calcium indicators. Mesoscopic functional connectivity was mapped longitudinally in awake GCaMP6 mice using transcranial wide-field calcium imaging through a bilateral chronic window. Spontaneous activity was recorded over 4 weeks before and 6 weeks after injection of red fluorescent microspheres (2000, 20 µm microspheres in 100µl PBS) into the left common carotid artery.Changes in cerebral blood flow were assessed with laser speckle imaging. To assess motor function we used the clasping and neurodeficit scores (NDS).
Results: Stroke mice showed a significantly higher NDS (p = 0.03) than sham mice and a trend to a higher clasping score. Seed pixel correlation and standard deviation maps within somatosensory, visual, and motor cortices showed no significant differences between sham and stroke groups.
Conclusions: A possible interpretation of our findings is that impairments to functional circuits in subcortical structures such as the basal ganglia or thalamus affect motor function without alterations to functional connectivity in the cortex detectable at the mesoscopic scale. Analysis at the microscopic scale may bring to light more subtle relationships and provide a unifying theory spanning microscopic, mesoscopic and behavioral phenomena.
References
Silasi et al., JCBFM (2015)35, 734–738
PS05-005
Poster Viewing Session V
Usefulness of bright arterial appearance in ASL MR perfusion image
Y. Kanazawa1,2,3, S. Arakawa2, T. Morioka2, T. Ago3, T. Kitazono3 and H. Ooboshi1
1Fukuoka Dental College Medical and Dental Hospital, Department of Internal Medicine, Fukuoka, Japan
2Japan Labor Health and Welfare Organization Kyushu Rosai Hospital, Department of Cerebrovascular Disease, Fukuoka, Japan
3Kyushu University, Department of Medicine and Clinical Science, Graduate School of Medical Sciences, Fukuoka, Japan
Abstract
Objectives: Arterial spin-labeling magnetic resonance perfusion imaging (ASL MRI) allows noninvasive measurement of cerebral blood flow but depends on arterial transit time. On the other hand, if labeled blood stagnates at the site of responsible artery, it may be identified as a dotted or linear high signal. We defined this signal as bright arterial appearance (BAA) and examined clinical features of BAA positive cases.
Methods: Patients with acute stroke-like symptoms, who underwent ASL on admission with high intensity lesions on diffusion-weighted image (DWI), were investigated during 2012–2013. Presence of BAA was diagnosed with ASL. ASL findings in the DWI high intensity area were also classified as increased, invariant, and decreased. Stenosis or occlusion of the responsible artery was evaluated by MR angiography. The clinical diagnosis was classified as atherothrombotic brain infarction, cardioemboric stroke, lacunar infarction, infarction of other etiology, or epileptic disorder.
Results: One hundred and thirty patients (average age 72 ± 13; 71 males) were enrolled in this study. Thirty cases were diagnosed as BAA positive. There were no lacunar infarction or epileptic disorders in the BAA positive group. In the BAA positive group, more cases had the stenosis or occlusion of the responsible artery compared to the negative group (88% vs. 33%, p < 0.001), and more cortical lesions (73% vs. 51%, p = 0.03). In addition, decreased ASL signal was more common in the perfusion area (77% vs. 26%, p < 0.001). Most of BAA was found at the proximal site of vascular lesion.
Conclusions: Imaging of ASL and detection of BAA may be useful for the exclusion of the epileptic disorder and the diagnosis of hypoperfusion with stagnating of proximal arterial flow.
References
Detre JA, et al: Magn Reson Med 23:375,1992
Yoo RE et al. Stroke 46:564,2015.
Akiyama T. et al: J Stroke Cerebrovasc Dis 25:2099,2016
PS05-006
Poster Viewing Session V
Improving stem cell delivery to the stroke brain using self-assembling silk hydrogels
O. Ibrahim1, C. McKirrtick1, J. Totten1, N. Gorenkova1, T. Wongpinyochit1, P. Seib1 and H. Carswell1
1Strathclyde University, SIPBS, Glasgow, United Kingdom
Abstract
Objectives: The biomaterial silk is self-assembling, biocompatible and biodegradable with potential tissue engineering applications. The present study assessed the capacity of self-assembling silk hydrogels to improve stem cell delivery in vitro and in an in vivo stroke model.
Methods: We exploited the self-assembly properties of silk by fine tuning processing parameters to generate a hydrogel system that shows predictable and controllable solution-gel kinetics ranging as assessed by light scattering and circular dichroism. Viability and 3D distribution of mesenchymal stem cells (MSCs) encapsulated in hydrogels were assessed using cell viability assays and haematoxylin and eosin staining. Space conformity in the stroke cavity was assessed histologically after injecting hydrogels post-middle cerebral artery occlusion (MCAo) in male C57BL/6 mice.
Results: For 1–5% w/v silk hydrogels, light scattering increased during sol-gel transition, accompanied by increased β-sheets content without any swelling following solution-gel transition. MSCs embedded in silk hydrogels showed good cytocompatibility and proliferated by 3–4 fold in 3% (w/v) and 4% (w/v) silk hydrogels over 10 days (p < 0.0001 and p < 0.001, respectively, one way ANOVA then Dunnett's test). MSCs exhibited a 3D distribution in the hydrogels when cells were added up to 10 minutes prior to the completion of the solution-gel transition. MSC viability significantly improved ∼1.4 fold when injected through a 30G needle prior to the onset of gelation compared to preformed silk hydrogels (p < 0.005, one way ANOVA then Dunnett's test). Finally, silk hydrogels were able to fill the stroke cavity indicating good conformity after MCAo.
Conclusions: It was possible to fine-tune self-assembling silk hydrogels to achieve uniform cell distribution and viability as well as space conformity in the stroke cavity in the absence of any silk hydrogel swelling. Overall, this study has demonstrated that silk hydrogels are emerging as promising cell delivery platform.
This work was sponsored by Taif University, Saudi Arabia.
PS05-007
Poster Viewing Session V
Identifying targets of neuronal auto-reactive responses to improve stroke recovery
U.M. Selvaraj1, P. Pandiyan2, X. Kong3, F. Miró4, X. Urra4,5, S. Ortega3, E. Plautz3, A. Planas4,6,7 and A. Stowe3
1UTSW Medical Center, Immunology, Dallas, United States
2UTSW Medical Center, Pediatrics, Dallas, United States
3UTSW Medical Center, Neurology and Neurotherapeutics, Dallas, United States
4August Pi i Sunyer Biomedical Research Institute (IDIBAPS), Barcelona, Spain
5Functional Unit of Cerebrovascular Diseases, Hospital Clínic, Barcelona, Spain
6Instituto de Investigaciones Biomédicas de Barcelona (IIBB), Department of Brain Ischemia and Neurodegeneration, Barcelona, Spain
7Consejo Superior de Investigaciones Científicas (CSIC), Barcelona, Spain
Abstract
Introduction: Stroke patients exhibiting increased colocalization of antigen-presenting cells to the neuronal antigens microtubule-associated protein 2 (MAP2) and N-methyl-D-aspartate (NMDA) receptor subunit 2A (GluN2A) in lymph nodes had smaller infarctions at day 7, and better long-term improvement at 3 months. However, patients with poor outcome exhibited more myelin-reactive colocalization. We hypothesized that post-stroke T-cell responses to MAP2 and GluN2A have a neuroprotective profile after focal stroke in mice.
Methods: Male C57BL/6 mice (B6, Jackson Labs, 8–10 wks old) were subjected to 60-min transient middle cerebral artery occlusion. Spleen and cervical lymph nodes were harvested 4, 8 and 10 days post-stroke, stained with CFSE, and cultured with GluN2A and MAP2 peptides for 6 days. Cell cultures were stained with fluorescently-tagged antibodies (TCRβ, CD4, CD19, CD8, CD25, TNF-α, IFN-γ, IL-10, IL-6, IL-4, IL-17) and quantified by flow cytometry. CD25+/CFSE-low responses were considered positive when ΔPF (test condition-non-stimulated condition) exceeded 1% and stimulation index (SI, test condition/non-stimulated condition) was greater than 2.
Results: Stroke mice had higher CNS-autoreactive T-cells in spleen compared to sham mice (10/12 mice vs 4/7 mice). Further, in stroke mice, CD8+ T-cells (10/12 mice) had higher GluN2A-specific responses in spleen compared to CD4+T-cells (4/12 mice). GluN2A-15 peptide-specific CD4+ (17% and 4%) and CD8+ (35% and 22%) splenic T-cells primarily produced pro-inflammatory cytokines TNF-α and IFN-γ, respectively. Animals with lower infarct volumes had higher number of IL-10- and TNF-α-producing neuronal antigen-specific CD4 and CD8 T-cells in spleen.
Conclusion: CNS-derived antigens induce autoimmune responses as early as four days after stroke in outbred mice. We now identified GluN2A and MAP2 peptides that elicit strong positive responses from the CD4+ and CD8+ T-cells at 8 and 10 days post-stroke in B6 mice. Future experiments will determine cytokine profile of neuronal antigen-specific cells in brain and establish if they are protective after stroke using in-vitro and in-vivo models.
PS05-008
Poster Viewing Session V
A role for brain pericytes in revascularization after stroke revealed by a novel reporter mouse
L.-P. Bernier1, J. Hefendehl1, C.-A. Lewis1, W. Scott1, L. Dissing-Olesen1, F. Rossi1, M. Underhill1 and B. Macvicar1
1University of British Columbia, Vancouver, Canada
Abstract
Brain pericytes are vascular mural cells that are a critical component of the neurovascular unit, essential for blood brain barrier integrity, blood flow regulation and vessel maturation during development. However, the role of CNS pericytes in pathologies such as stroke remains unclear, partly because their identification has traditionally relied on protein markers that are transiently expressed by other cell types, an issue particularly problematic under pathological conditions. We therefore generated a novel transgenic mouse to achieve inducible, stable and heritable expression of a tdTomato reporter driven by a pericyte-specific promoter. A remarkable coverage of pericytes was observed, allowing clear visualization of thin-strand ramified capillary pericytes and mesh-like pericytes on larger vessels. The mice were subjected to a photothrombotic focal stroke and the involvement of pericytes in post-stroke recovery was followed for up to 40 days. Cell death occurred within 24 hours however, significant revascularization occurred in the tissue surrounding the ischemic area, as visualized with optical coherence tomography and in vivo imaging of IV-injected dextran-conjugated fluorescein. Prior to local tissue revascularization, activated pericytes detached from the vasculature, proliferated and migrated to accumulate within the ischemic area, on the lesion side of the astroglial border. Accumulation of tdTomato+ pericytes in that region coincided with a localized overproduction of basal lamina components laminin and collagen type IV. Immature pericytes showed early signs of ramification along vessels and associated with endothelial cells before functional blood flow returned, suggesting the presence of active angiogenesis driven by pericytes well inside the astroglial scar border. Pericytes that had proliferated post-stroke showed normal coverage of the capillaries in the revascularized area, and supported the formation of a mature blood-brain barrier. In conclusion, we provide a novel transgenic model for pericyte investigation and demonstrate a surprising role for pericytes in promoting angiogenesis and tissue revascularization following ischemic injury.
PS05-009
Poster Viewing Session V
Circulating miR-126-3p and miR-126-5p are increased after acute ischemic stroke and expressed in platelets and T cells
N. Schieferdecker1, S. Tiedt1,2, V. Kautzky1, M. Prestel1, M. Duering1, M. Klein3 and M. Dichgans1,4
1Institute for Stroke and Dementia Research, Munich, Germany
2Graduate School of Systemic Neurosciences, Ludwig-Maximilians-University LMU, Munich, Germany
3University Hospital of the Ludwig-Maximilians-University LMU, Department of Neurology, Munich, Germany
4Munich Cluster for Systems Neurology (SyNergy), Munich, Germany
Abstract
Objectives: Recent studies suggest important roles of miR-126-3p and miR-126-5p in platelet aggregation and atherosclerosis - two processes strongly linked to ischemic stroke (IS). We investigated whether expression levels of circulating miR-126-3p and miR-126-5p are altered after acute ischemic stroke (IS). We further aimed at identifying the cellular source of these miRNAs in human plasma.
Methods: The study population consisted of 60 patients with acute IS recruited within 24 hours of symptom onset through the emergency department of a single stroke center. 60 healthy individuals matched for both demographic and vascular risk factors as well as previous use of antiplatelet medication served as controls. Non-fasting blood samples from IS patients were collected before the receipt of any medication. Small RNAs were isolated from platelet-poor plasma and analysed by qRT-PCR. Magnetic-activated cell sorting and platelet spike-in experiments were used to identify cell types contributing to circulating miRNA levels.
Results: The mean time from symptom onset to hospital arrival in IS patients was 5,4 hours and the mean infarct volume was 18,6 ml (ranging from 0.04 ml to 161 ml). Expression levels of miR-126-3p and miR-126-5p in plasma were significantly higher in IS patients compared to healthy matched controls (miR-126-3p: p = 0,0025, miR-126-5p: p = 0,0005), but did not correlate with infarct volume. We found platelets and circulating T-cells to be a major source of both circulating miR-126-3p and miR-126-5p.
Conclusions: Circulating miR-126-3p and -5p are significantly elevated after acute IS and with platelets and T cells being the major cellular source in blood. We are currently performing expression analyses in an independent validation sample as well as longitudinal measurements up to 90 days after IS to explore the clinical utility of miR-126-3p and -5p as diagnostic and predictive marker.
PS05-010
Poster Viewing Session V
The role of transforming growth factor-β in post-stroke glymphatic impairment
M. Howe1, L. McCullough1 and A. Urayama1
1UT Health, Neurology, Houston, United States
Abstract
The recently discovered glymphatic system may protect the brain from dementia by clearing toxic amyloid-β (Aβ) along the basement membrane (BM) via bulk flow of cerebrospinal fluid (CSF). Previous work has shown that stroke enhances astrocytic transforming growth factor-β (TGF-β) signaling, which could increase the expression of pro-fibrotic BM proteins such as fibronectin. BM fibrosis may be a novel mechanism of glymphatic impairment, driving Aβ accumulation and neurodegenerative disease after stroke. I hypothesize that stroke induces BM fibrosis via enhanced TGFβ-Smad2 signaling in perivascular astrocytes, impairing glymphatic flow. To test this hypothesis, the glymphatic influx of CSF tracer in mice following stroke or TGF-β treatment (ICV, 500 ng) was assessed. TGF-β signaling was measured by Smad2 phosphorylation. Brain expression of fibronectin and GFAP (astrocytes) were assessed by western blot and immunohistochemistry. Stroke impaired the glymphatic distribution of FITC-dextran bilaterally. TGF-β treatment also inhibited glymphatic distribution, measured by 14C-Inulin uptake (38 fold-reduction, p < .05). Increased fibronectin (5.5 ± 0.7 fold-increase) and GFAP (2.3 ± .2 fold-increase) expression was found after stroke (p < .05), localized to the glial scar and around vessels bilaterally. Finally, Smad2 phosphorylation was increased (2.5 ± 0.2 fold) in cortex contralateral to injury (p < .01). The present study shows that stroke impairs glymphatic flow, which may be mediated through increased TGF-β signaling and global BM fibrosis. TGF-β inhibition could rescue post-stroke fibrosis, glymphatic flow and Aβ clearance, and may present a novel therapeutic target for post-stroke dementia.
PS05-011
Poster Viewing Session V
In vivo silk hydrogel distribution, biocompatibility and biodegradation in the brain after experimental stroke
N. Gorenkova1, C. McKittrick1, S. White1, O. Ibrahim1, F.P. Seib1 and H. Carswell1
1University of Strathclyde, Strathclyde Institute of Pharmacy and Biomedical Sciences, Glasgow, United Kingdom
Abstract
Background: Silk has been used in humans for centuries and is a FDA approved biomaterial for load bearing applications. Silk has a strong clinical track record and is widely regarded as biocompatible and biodegradable; reverse engineered silk can be processed into many material formats, including self-assembling silk hydrogels. These silk hydrogels are emerging as a promising delivery platform for both drugs and cells, but have never been tested in the brain.
Aim: To investigate in vivo distribution, biocompatibility and biodegradation of the self-assembling silk hydrogel in the brain after experimental stroke.
Methods: 22 male adult Sprague Dawley rats underwent transient middle cerebral artery occlusion and randomly selected to receive no treatment, vehicle or silk hydrogel (4 % w/v) implant 2 weeks later. Animals were randomly selected for termination at 1 week (subacute) or 7 weeks (chronic) time point. Tissue loss and silk graft were detected using haematoxylin and eosin (H&E) staining and quantified by image analysis. Glial scar formation and graft distribution were measured using GFAP and DAPI nuclear staining.
Results: Self-assembling silk hydrogels were able to fill the stroke cavity, were present at the site of application 7 weeks post application and were well integrated into the host tissues. GFAP and H&E detected no inflammation in the brain in response to the silk hydrogel (1 or 7 weeks postgrafting). There was partial host cell infiltration into the silk hydrogel as revealed by nuclei staining.
Conclusions: Silk hydrogels are emerging as a biocompatible biomaterials in the stroke setting. Furthermore, silk hydrogels showed excellent space conformity; all necessary requirements to successfully deliver a therapeutic playload. Further studies are ongoing to establish if host cell infiltration is due to neurogenesis or inflammatory cells. Post?graft survival within the cavity at 7 weeks indicates the potential of silk hydrogels to promote new neurovascular niche formation.
This work was sponsored by Daphne Jackson and Medical Research Scotland
PS05-012
Poster Viewing Session V
Predictors of ischemic stroke on 18F-FDG PET-CT in patients with cancer
K. Choi1 and J. Kim2
1Chonnam National University Hwasun Hospital, Hwasun, Korea, Republic of
2Chonnam National University Hospital, Gwangju, Korea, Republic of
Abstract
Background:18F-FDG PET/CT can acquire both anatomical and functional images in a single scanning session. The purpose of the present study was to investigate which factors are associated with the occurrence of future ischemic stroke in patients with cancer using 18F-FDG PET/CT.
Methods: A total of 134 patients with cancer were enrolled. Future ischemic stroke was defined as any new infarct lesion in brain MRI within 1 year after FDG PET/CT. Arterial inflammation on PET imaging was defined as a target-to-background (TBR) for each arterial segment and abdominal obesity was defined as area and proportion of visceral adipose tissue (VAT), subcutaneous adipose tissue (SAT), and total adipose tissue (TAT) on a single CT slice at the umbilical level.
Results: Thirty patients experienced the occurrence of future ischemic stroke confirmed by brain MRI, and 104 patients did not have a stroke during follow up period. As for PET, patients with stroke had higher TBRs of both carotid arteries and abdominal aorta (P < 0.001) than patients without stroke. As for CT, patients with stroke had higher VAT (P = 0.021) and TAT (P = 0.041) proportions, compared with patients without stroke. TBRs of right and left carotid arteries and abdominal aorta, VAT and TAT proportions, and metabolically active tumor were significantly associated with the future ischemic stroke in multiple logistic regression analysis. The combination of both PET and CT variables had a stronger predictive power for future ischemic stroke.
Conclusion: Our findings suggest that arterial FDG uptake and hypermetabolic malignancy on PET and visceral adipose tissue proportion on CT could be independent predictors of future ischemic stroke in patients with cancer. Oncologic PET/CT might be used to identify patients at risk of future ischemic stroke and who would benefit from the medical treatment.
PS05-013
Poster Viewing Session V
Perineuronal nets structures and modulators in the rat somatosensory cortex are molecular substrates for experience-dependent plasticity during stroke recovery
M.J. Quattromani1, M. Pruvost2, C. Guerreiro1, F. Backlund1, E. Englund3, A. Aspberg4, T. Jaworski5, J. Hakon1, K. Ruscher1, L. Kaczmarek5, D. Vivien2 and T. Wieloch1
1Lund University, Laboratory for Experimental Brain Research, Lund, Sweden
2INSERM UMR-S U919, Serine Proteases and Pathophysiology of the Neurovascular Unit, Université Caen Basse Normandie, GIP Cyceron, Caen, France
3Lund University Hospital, Division of Oncology and Pathology, Lund, Sweden
4Lund University, Rheumatology and Molecular Skeletal Biology, Lund, Sweden
5Nencki Institute of Experimental Biology, Laboratory of Neurobiology, Warsaw, Poland
Abstract
Objectives: Following stroke, complete cellular death in the ischemic brain may ensue, with remaining brain areas undergoing tissue remodelling. Experience-dependent plasticity exerted through an enriched environment (EE) promotes remodelling and neuroplasticity after CNS injury, such as stroke. Post-stroke tissue reorganization is modulated by growth-inhibitory molecules differentially expressed within the ischemic hemisphere, such as chondroitin sulfate proteoglycans (CSPGs) found in perineuronal nets (PNNs). PNNs enwrap parvalbumin-containing GABAergic (PV/GABA) cells, important in sensori-information processing. A recent study demonstrates that during stroke recovery EE induces a reduction in the number of PNNs in the peri-infarct and regions remote from the infarct. Here we investigate how extracellular matrix (ECM) proteases and their inhibitors may participate in the regulation of PNN integrity during stroke recovery.
Methods: Rats were subjected to photothrombotic stroke (PT) in the motor cortex and functional deficits assessed at 7 days of recovery. Shams and stroked rats were housed in either standard (STD) or EE conditions for 5 days and infarct volumes calculated. PNNs were visualized by immunohistochemistry and counted by bright-field and confocal microscopy in the somatosensory cortex of both hemispheres. ECM proteases and protease inhibitors mRNA expression levels were assessed by qRT-PCR and their activity analyzed by gel zymography. PNNs and protease activity were also studied in brains from stroke patients.
Results: EE starting 2 days after PT did not influence the size of the infarct, but promoted behavioral recovery of limb-placement ability after stroke. The multisensory stimulation exerted through an EE promoted a decrease of PNNs around PV/GABA neurons, an increased proteolytic activity of the ECM proteases MMP-9 and tPA and a modulation of the mRNA expression of ECM-proteases and protease inhibitors in the somatosensory cortex after PT.
Conclusions: Elucidating the mechanisms of ECM remodelling after experimental stroke may provide new therapies supporting rehabilitation of stroke patients.
PS05-014
Poster Viewing Session V
Alarmin HMGB1 induces systemic and brain inflammatory exacerbation in post-stroke infection rat model
I.-D. Kim1,2, S.-W. Kim1,2, H.-K. Lee1,2, H. Lee1,2, L. Luo1,2, P.-L. Han3 and J.-K. Lee1,2
1Inha University School of Medicine, Anatomy, Incheon, Korea, Republic of
2Inha University School of Medicine, Medical Research Center, Incheon, Korea, Republic of
3Ewha Womans University, Brain and Cognitive, Seoul, Korea, Republic of
Abstract
Post-Stroke Infection (PSI) is known to worsen functional outcomes of stroke patients and accounts one third of stroke-related deaths in hospital. In our previous reports, we demonstrated that massive release of High mobility group box protein 1 (HMGB1), an endogenous danger signal molecule, is promoted by NMDA-induced acute damage in the postischemic brain, exacerbating neuronal damage by triggering delayed inflammatory processes. Moreover, augmentation of proinflammatory function of LPS by HMGB1 via direct interaction has been reported. The aim of this study was to investigate the role of HMGB1 to aggravating inflammation in the post-stroke infection by exacerbating the function of LPS. Post-stroke infection animal model was produced by administrating a low dose LPS at 24 hrs post-MCAO (middle cerebral artery occlusion). Profound aggravations of brain and systemic inflammation, deterioration of behavioral outcomes, and infarct expansion were observed in LPS-injected MCAO animals, in which serum HMGB1 surge occurred immediately after LPS administration and aggravated brain and systemic inflammations probably by acting in synergy with LPS. In contrast, Rhodobacter sphaeroides LPS (LPS-RS), a toll like receptor 4 (TLR4) selective antagonist, failed to exert these effects, indicating its TLR4-dependence. Furthermore, blockage of HMGB1 function by delayed administrations of therapeutic peptides known to inhibit HMGB1 (HMGB1 A box, HPep1) at 21 hrs post-MCAO, which was 3 hrs prior to LPS injection, or treatment with LPS after pre-incubation with HMGB1 A box significantly ameliorated damages observed in the post-stroke infection, demonstrating that HMGB1 plays a crucial role. Together these results indicated that HMGB1 might play an important role in TLR-4-dependent exacerbation of systemic and brain inflammations in a rat model of post-stroke infection, and modulation of HMGB1 might provide a valuable therapeutic strategy.
PS05-015
Poster Viewing Session V
Integrative analysis of transcriptomics and proteomics data for the molecular characterization of human brain after ischemic stroke
T. García-Berrocoso1, F. Briansó2, A. Simats1, V. Llombart1, A. Hainard3, J.-C. Sanchez3, A. Sánchez-Pla2 and J. Montaner1
1VHIR, Universitat Autònoma de Barcelona, Neurovascular Research Laboratory, Barcelona, Spain
2VHIR, Universitat Autònoma de Barcelona, Statistics and Bioinformatics Unit, Barcelona, Spain
3University Medical Center of Geneva, Translational Biomarker Group, Geneva, Switzerland
Abstract
Objectives: The use of neuroprotective drugs to avoid infarct growth after stroke has not succeeded yet. To better understand the pathophysiology of stroke and how to reduce its dramatic outcomes by new therapeutic strategies, our aim was to identify new molecular changes that happen in brain after stroke integrating both proteomics and transcriptomics data and interpreting these results in a biological context.
Methods: Flash-frozen post-mortem brain samples from infarct-core (IC) and healthy contralateral (CL) areas of 6 stroke patients were processed to obtain protein and total RNA. Proteomes were analyzed by mass-spectrometry and a label-free comparative quantification of ICvsCL using Progenesis LC-MS software was done. Affymetrix® Human Transcriptome Arrays were employed to explore gene expression changes between ICvsCL samples. Relevant features (p < 0.05, fold-change > 1) in each dataset were integrated through multiple co-inertia analysis and regularized canonical correlation analysis (made4, mogsa & miXomics R packages). Finally, they were combined with biological annotations from the Gene Ontology consortium.
Results: Datasets of 1900 proteins and 6800 genes were included to compare ICvsCL samples. From these, 120 proteins showed up as relevant and were involved in pathways of cell assembly and synaptogenesis. In regard to genes, 720 showed statistically significant differences and were involved in the immune response, metabolic processes and signaling pathways. Once these datasets were integrated, best gene-protein correlations were highlighted and several enriched biological categories were found to be related either to gene, protein, or both datasets, from which TNF-alpha signaling and hypoxia signatures were particularly associated with IC samples.
Conclusions: We have integrated changes due to ischemia at protein and transcript level in the same human brain samples. This data will help us to find flagship molecules for the understanding of this pathology and showing candidates to be further explored as biomarkers or neuroprotective therapeutic targets in stroke.
PS05-016
Poster Viewing Session V
LncRNA FosDT mediates ischemic brain damage in both sexes
S. Mehta1, T. Kim1 and R. Vemuganti1
1University of Wisconsin, Neurological Surgery, Madison, United States
Abstract
Transient focal ischemia induces extensive spatio-temporal changes in expression levels of protein-coding and many classes of noncoding RNAs including long non-coding RNAs (lncRNAs >200 nt). However, the significance of lncRNAs expression to the neurologic outcome after stroke is essentially unknown. Recently, we have reported that one of the lncRNAs called Fos downstream transcript (FosDT) is significantly altered after transient focal ischemia and promotes ischemic brain injury by interacting with REST-associated chromatin modifying proteins. Presently using a focal ischemia model in SHR rats, we investigated whether 1) FosDT expression is independent of age and sex, 2) whether post-ischemic targeting of FosDT is a viable strategy to curtail stroke brain damage and 3) whether rodent knockouts of FosDT are viable, fertile and developmentally normal to study the role of FosDT in stroke. We found that transient focal ischemia induced the expression levels of FosDT independent of age and sex. We also found that post-ischemic targeting of FosDT using siRNA injected intracerebrally resulted in smaller infarcts compared to control siRNA group in both young adult male and female rats. Interestingly, deletion of FosDT using CRISPR/Cas9 had no effect on normal development, body weight and fertility in knockout compared to wildtype rats. Thus, FosDT induction could modulate ischemic brain damage and targeting lncRNAs such as FosDT is a viable strategy to minimize post-stroke brain damage.
PS05-017
Poster Viewing Session V
The neuroprotective peptide poly-arginine-12 (R12) reduces cell surface levels of NMDA NR2B receptor subunit in cortical neurons
G. MacDougall1,2, R.S. Anderton1,2, A.B. Edwards1,2, N.W. Knuckey1,3 and B.P. Meloni1,4
1WANRI, Perth, Australia
2The University of Notre Dame, Perth, Australia
3Sir Charles Gairdner Hospital, QEII Medical Centre, Neurosurgery, Perth, Australia
4Centre for Neuromuscular and Neurological Disorders, The University of Western Australia, Perth, Australia
Abstract
At present, there are no clinically available neuroprotective agents that can be administered early after acute neurological events, such as stroke, global ischemia, and brain trauma, to minimize injury and improve outcomes. However, we have previously reported that cationic poly-arginine and arginine-rich cell-penetrating peptides display high-level neuroprotection and reduce calcium influx following in vitro excitotoxicity, as well as reduce brain injury in transient and permanent animal stroke models. Using the neuroprotective peptides poly-arginine R12 (R12) and the NR2B9c peptide fused to the arginine-rich carrier peptide TAT (TAT-NR2B9c; also known as NA-1), we investigated the mechanisms whereby poly-arginine and arginine-rich peptides reduce glutamate-induced excitotoxic calcium influx. Using cell surface biotin protein labeling and western blot analysis, we demonstrated that R12 and TAT-NR2B9c significantly reduced cortical neuronal cell surface expression of the NMDA receptor subunit NR2B. Chemical endocytic inhibitors used individually or in combination prior to glutamate excitotoxicity did not significantly affect R12 peptide neuroprotective efficacy. Similarly, pretreatment of neurons with enzymes to degrade anionic cell surface proteoglycans, heparan sulfate proteoglycan (HSPG), and chondroitin sulfate proteoglycan (CSPG), as well as sialic acid residues, did not significantly affect peptide neuroprotective efficacy. While the exact mechanisms responsible for R12 peptide-mediated NMDA receptor NR2B subunit cell surface downregulation were not identified, an endocytic process could not be ruled out. The study supports our hypothesis that arginine-rich peptides reduce excitotoxic calcium influx by reducing the levels of cell surface ion channels.
PS05-018
Poster Viewing Session V
Hypoxic preconditioning augments the therapeutic efficacy of bone marrow stromal cells in a rat ischemic stroke model
L. Shen1, J. Chen1, W. Ding1, X. Chen1,2, L. Ding2 and G. Wang2,3
1Affiliated Hospital of Nantong University, Neurology, Nantong, China
2Institute of Nautical Medicine, Nantong University, Neuropharmacology, Nantong, China
3University of Pittsburgh School of Medicine, Neurology, Pittsburgh, United States
Abstract
Objectives: Transplantation of bone marrow stromal cells (BMSCs) is a promising therapy for ischemic stroke, but the poor oxygen environment in brain lesions limits the efficacy of cell-based therapies. Here, we tested whether hypoxic preconditioning (HP) could augment the efficacy of BMSC transplantation in a rat ischemic stroke model and investigated the underlying mechanism of the effect of HP.
Methods:In vitro, BMSCs were divided into 5 passage (P0, P1, P2, P3 and P4) groups, and HP was applied to the groups by incubating the cells with 1% oxygen for 0 h, 4 h, 8 h, 12 h and 24 h, respectively. We demonstrated that the expression of hypoxia-inducible factor-1α (HIF-1α) was increased in the HP-treated BMSCs, while their viability was unchanged. We also found that HP decreased the apoptosis of BMSCs during subsequent simulated ischemia-reperfusion (I/R) injury, especially in the 8-h HP group. In vivo, a rat transient focal cerebral ischemia model was established. These rats were administered normal cultured BMSCs (N-BMSCs), HP-treated BMSCs (H-BMSCs) or DMEM cell culture medium (Control) at 24 h after the ischemic insult.
Results: Compared with the DMEM control group, the two BMSC-transplanted groups exhibited significantly improved functional recovery and reduced infarct volume, especially the H-BMSC group. Moreover, HP decreased neuronal apoptosis and enhanced the expression of BDNF and VEGF in the ischemic brain. Survival and differentiation of transplanted BMSCs were also increased by HP, and the quantity of engrafted BMSCs was significantly correlated with neurological function improvement.
Conclusions: These results suggest that HP may enhance the therapeutic efficacy of BMSCs in an ischemic stroke model. The underlying mechanism likely involves the inhibition of caspase-3 activation and an increasing expression of HIF-1α, which promotes angiogenesis and neurogenesis and thereby reduces neuronal death and improves neurological function.
PS05-020
Poster Viewing Session V
Sesn3 and post-ischemia seizures in diabetes
Z. Shi1, Z. Lei1, F. Wu1 and Z. Xu1
1Indiana University School of Medicine, Indianapolis, United States
Abstract
Seizures are the most common neurological sequelaee of stroke. Diabetes mellitus has been identified as an independent predictor of acute seizures in stroke patients. The mechanisms of post-stroke seizures under diabetic condition remain unclear. Recent studies indicated that epilepsy could be mediated by energy metabolism related proteins such as Sestrin3 (Sesn3). The present study attempted to reveal the contribution of Sesn3 to seizure generation in diabetic condition after ischemia.
Transient global ischemia was produced in adult Wistar rats and mice. Diabetes was induced by i.p. injection of 50 mg/kg streptozotocin (STZ). The seizure activity was defined by the Racine scale III-V. The neuronal death in the brain was determined by hematoxylin-eosin staining. The expression levels of Sesn3 were analyzed by Western blotting and immunohistochemistry. The neuronal excitability was recorded using electrophysiological approaches.
The blood glucose levels was >300 mg/dL in animals one week after STZ injection. The seizure rate significantly increased from zero of naive animals to 100% in diabetic rats 24 hr after 15 min ischemia. No obvious neuronal damage was observed in hippocampus and cerebral cortex at the time of seizure. Sesn3 expression in hippocampus was significantly increased in diabetic animals with post-ischemia seizures. The level of phosphorylated p70S6 significantly increased accordingly. The potassium channel expression and currents in hippocampal neurons were decreased and neuronal excitability increased in these animals. The seizure rate was significantly decreased from 60% of wild type mice to 15% of Sesn3 knockout mice after 15 min ischemia.
These results indicate that increase of Sesn3 expression might be responsible to post-ischemia seizure in diabetic animals through inflammatory mTOR pathways.
PS05-021
Poster Viewing Session V
Danegaptide enhances astrocyte gap junctional coupling and reduces ischemic reperfusion brain injury in mice
J. Bechberger1, M. Freitas-Andrade1, J. Wang2, K. Yeung2, S. Whitehead2, R. Schultz Hansen3 and C. Naus1
1University of British Columbia, Cellular and Physiological Sciences, Vancouver, Canada
2Western University, Anatomy and Cell Biology, London, Canada
3Zealand Pharma A/S, Copenhagen, Denmark
Abstract
Objectives: To explore the therapeutic potential of the antiarrhythmic dipeptide ZP1609 (danegaptide), to increase astrocyte gap junctional coupling (GJC) and provide neuroprotection in ischemic stroke.
Methods: To assess the effect of ZP1609 on astrocyte GJC, primary cultures of mouse astrocytes were treated with ZP1609 (247 nM - 2.47 mM) and GJC measured. For in vivo stroke studies, we used a 60 min transient unilateral middle cerebral artery occlusion (MCAO) followed by reperfusion in 3–4 month old C57BL6 male mice. At 50 min, saline (pH 7.4) or ZP1609 (75ug/kg) was injected into the tail vein. After 60 min, the clamp on the carotid artery was released to allow reperfusion. Mice were treated with subsequent ZP1609 injections at 1, 2 and 3 hr after the initial injection. The mice were euthanized at 48 hrs post-stroke and the volume of the stroke infarct measured. To assess ZP1609 levels in the brain, 10 µm thick coronal brain sections were thaw mounted onto ITO glass slides and processed for imaging mass spectrometry in positive reflectron mode on the 5800 TOF/TOF ABSciex instrument.
Results: ZP1609 increased astrocyte GJC by 22.2% at 247nM concentration. There was a significant reduction (P < 0.002) in infarct size following ZP1609 treatment. In saline treated mice, infarct volume was 26.2 + 2.9 mm3 (n = 10), while ZP1609 treatment reduced infarct volume to 14.3 + 1.3 mm3 (n = 11). Using mass spectrometry imaging induced ion mages, the spatial distribution of ZP1609 was observed throughout the brain tissue sectioned from mice injected with the peptide, consistent with passage through the blood brain barrier.
Conclusions: Our results show that ZP1609 enhances GJC in mouse astrocytes, and provides a protective effect in transient brain ischemia with reperfusion.
PS05-022
Poster Viewing Session V
Loss of EphB2 decreases edema and infarct size during ischemic stroke
A.-S. Ernst1, L.-I. Böhler2, R. Kunze2, H.H. Marti2 and T. Korff1
1Heidelberg University/Institute of Physiology and Pathophysiology, Cardiovascular Research, Heidelberg, Germany
2Heidelberg University/Institute of Physiology and Pathophysiology, Neurovascular Research, Heidelberg, Germany
Abstract
Introduction: Stroke is one of the most relevant causes of death worldwide. However, molecular determinants controlling the mechanisms triggered during brain ischemia are only partially characterized. In this context, the role of the receptor tyrosine kinase EphB2 has not been investigated so far, although it is crucial for neuronal synapse formation/function, angiogenesis and immune responses. Given the relevance of these functional implications for stroke, this study was intended to unravel the role of EphB2 during experimental brain ischemia in mice.
Results: Transient middle cerebral artery occlusion (tMCAo) followed by 12, 24 and 48 h reperfusion, resulted in reduced infarct and edema sizes as well as attenuated neurological deficits in EphB2−/− mice as compared to EphB2+/+ mice. Experimental approaches investigating possible vascular alterations revealed no differences between EphB2−/− and EphB2+/+ mice regarding cerebrovascular anatomy (whole-mount staining, CT scans), pericyte-coverage of brain vessels, reduction of regional cerebral blood flow (Laser-Doppler), or capillary permeability (Evans Blue extravasation, capillary gap formation). While analyses of brain transcriptomes (Affymetrix microarray) also indicated no major differences under baseline conditions, expression of stroke-related gene products was significantly attenuated in EphB2−/− mice (48 h reperfusion). In line with this, brain lysates from mice subjected to tMCAo and 12 h reperfusion showed a decrease in the abundance of proteins such as VEGF, MCP-1, MMP-9 and CXCL1 in EphB2−/− mice indicating reduced inflammation and angiogenesis. However, differences in the expression of synapse-related gene products and pathways involving NMDA receptor point towards a neuronal contribution of the attenuated stroke outcome in EphB2−/− mice.
Conclusion: Collectively, the attenuation of stroke in EphB2−/− mice is most likely not mediated by alterations of the vascular system. Considering the relevance of EphB2 for synapse-formation and NMDA receptor function, future experiments will investigate as to whether EphB2 contributes to ischemia-induced neuronal cell death via NMDA receptor signaling.
PS05-023
Poster Viewing Session V
Recovery of grey and white matter requires the DNA base excision repair enzyme APE1 following transient cerebral ischemia
S. Hassan1, Y. Gao1,2, Z. Wang1, L. Zhang1, H. Pu1, Y. Shi1, G. Cao1,2, M.V.L. Bennett2,3, J. Chen1,2 and R.A. Stetler1,2
1University of Pittsburgh, Pittsburgh Institute for Brain Disorders and Recovery, Pittsburgh, United States
2Fudan University, State Key Laboratory of Medical Neurobiology and Institutes of Brain Science, Shangai, China
3Albert Einstein College of Medicine, Dominick P. Purpura Department of Neuroscience, Bronx, United States
Abstract
Cerebral ischemia has long been associated with increased oxidative DNA damage, which necessitates the activation of the base excision repair pathway (BER) for DNA repair. The contribution of BER to ischemic recovery has not been well defined due to the limitation of available tools, including the specific targeting of critical repair enzymes in animal models. We created a conditionally targeted knockout of the BER enzyme, apurinic/apyrimidinic endonuclease 1 (APE1-cKO) to determine whether APE1 is involved in ischemic recovery. Conditional deletion of APE1 was accomplished by tamoxifen injection. Using a model of mild transient focal ischemia (30 minutes, tFCI), we assessed infarct volume and sensorimotor and cognitive behavioral outcomes in APE-cKO and wildtype mice. Oxidized DNA damage and functional cellular recovery in both grey and white matter was assessed using a variety of histological and electrophysiological techniques. APE1-cKO led to increased early mortality rates following 60 min tFCI. Therefore, we used a 30 min tFCI model with no significant differences in mortality between genotypes. APE1-cKO increased infarct volume (p ≤ 0.01), and behavioral assessments indicated that APE1-cKO animals had impaired sensorimotor recovery and worsened cognitive performance following tFCI. Impairment in conduction along the CC/EC was observed in ischemic APE1-cKO brain (p ≤ 0.001). Neurons and oligodendrocytes displayed heightened sensitivity to ischemic injury in the APE1-cKO, as well as pronounced accumulation of DNA damage, whereas no change in astrocytic and microglial sensitivity was observed between genotypes. Exacerbated loss of myelin occured in corpus callosum, and extended to striatum and cortex, in APE-cKO brain. Cell death in oligodendrocytes was associated with increased PARP1 activity, whereas PUMA activation occurred in neuronal populations. Our study highlights the finding that the DNA repair enzyme APE1 is critical to the protection of both neurons and oligodendrocytes following ischemic injury, and contributes to functional recovery following mild stroke.
PS05-025
Poster Viewing Session V
In vivo cerebral blood flow imaging in small animals with three-dimensional, high-density speckle contrast optical tomography
T. Dragojević1, J.L. Hollmann1, H.M. Varma1, C.P. Valdes1, J.P. Culver2,3, C. Justicia4,5 and T. Durduran1,6
1ICFO - The Institute of Photonic Sciences, Castelldefels, Spain
2Washington University in St. Louis School of Medicine, Department of Radiology, St. Louis, United States
3Washington University in St. Louis School of Medicine, Department of Physics, St. Louis, United States
4Institute of Biomedical Research (IIBB), Department of Brain Ischemia and Neurodegeneration, Barcelona, Spain
5Institute d'Investigacions Biomedical Research August Pi i Sunyer (IDIBAPS), Spanish Research Council (CSIC), Barcelona, Spain
6Institucio Catalana de Reserca i Estudis Avancats (ICREA), Barcelona, Spain
Abstract
Objectives: Novel high-density speckle contrast optical tomography (SCOT) [1] is introduced for non-invasive, three-dimensional cerebral blood flow (CBF) imaging in small animals.
Methods: SCOT [1] provides high-density optical tomography by scanning a laser into 25 positions over the skull surface and uses a CCD camera (Basler,Germany) to collect light from thousands of detectors in parallel.
Nine, male mice (C57/BL6, 4 months, 30g) were anesthetized with isoflurane and placed on a stereotaxic frame. Scalp was removed and a baseline measurement acquired. Photosensitive dye (Rose Bengal) was injected through the intraperitoneal cavity and localized activation of the dye was done on the right hemisphere (Fig.1(A)). Measurement of the perturbed CBF was acquired one hour after stroke induction. For comparison, 7T MRI (Bruker,Germany) T2 multi-slice multi-echo (MSME) maps were acquired.
Results: Fig.1(B) shows the reconstructed and thresholded SCOT data superimposed over the MRI image. For all animals the CBF change is shown in Fig.1(C).A significant CBF decrease was seen on all the animals affected by the stroke (stroke(MRI)) and confirmed with the MRI (Fig.1(D)). Animals with a small change in CBF (no stroke (MRI)) did not have an infarct. For the animals without the MRI (stroke (no MRI)), stroke was visually confirmed. Of these, two animals (*) that show the highest CBF change did not survive.
Conclusion: SCOT is a new, in vivo, non-invasive method for high-density tomographic reconstruction of deep tissue CBF in small animals with a potential to be translated to the adult human brain.
(A) Illustration of mouse brain. (B) A slice compared to the MRI measured zone of the infarct after twenty-four hours - shown in light gray; rCBF change for nine animals (C) and MRI volume (D) of stroke and ipsilateral side.
References
[1] Varma H.,BOE (2014).
PS05-026
Poster Viewing Session V
Restitution of hippocampal long-term potentiation following global cerebral ischemia: ghrelin function as a mitochondrial anti-apoptotic
and B. Sadeghi1,2
1Shahid Beheshti University of Medical Sciences, Physiology, Tehran, Iran, Islamic Republic of
2University of Tehran, International Institute of Biochemistry and Biophysics (IBB), Tehran, Iran, Islamic Republic of
Abstract
Ghrelin receptors (growth hormone secretagogue receptors) are expressed in the brain, including the hippocampus. The activation of ghrelin receptors facilitates high frequency stimulation (HFS)-induced long-term potentiation (LTP) and also improves learning and memory. Here, it is reported that chronic intraperitoneal administration of ghrelin (10 µg/kg for 2 weeks) before and after cardiac arrest prevented ischemia-induced impairment of LTP and led to restitution of long-lasting potentiation of excitatory postsynaptic potentials (EPSPs) and population spikes (PSs) in the dentate gyrus (DG) area of anesthetized rats. Animals were subjected to 4-VO ischemia following a single dose of ghrelin and received daily ghrelin injections for a week after surgery. To assess cognitive performance, the Morris water maze task was performed for 5 consecutive days. Intrahippocampal field potential recordings were done, brains were removed and immunohistochemistry to Bcl-2 and Bax was examined to observe the expression of these proteins in DG neurons. The results showed that ghrelin enhanced spatial memory by significantly reducing escape latencies in ischemic rats and also increased the EPSP slope and PS amplitude, suggesting the involvement of ghrelin in postsynaptic mechanisms of hippocampal LTP. Additionally, it seems that the Bcl-2/Bax ratio is enhanced and the expression of Bcl-2 was sufficient to prevent apoptosis of hippocampal neurons. It is revealed that neuroprotective effects of chronic ghrelin not only can enhance but also can restore LTP in DG area of global cerebral ischemic rats through inhibition of the mitochondrial pathway of apoptosis. Therefore, it is suggested that the ghrelin signaling pathway could have therapeutic value in cognitive deficits.
References
1. Miao, Yanying, et al. “Ghrelin protects cortical neuron against focal ischemia/reperfusion in rats.” Biochemical and biophysical research communications 359.3 (2007): 795–800.
PS05-027
Poster Viewing Session V
Prolyl-4-hydroxylase 2 activity is crucial for cognitive function and stroke outcome
H.H. Marti1, L. Li1, D. Gruneberg1, P. Barteczek1, S. Reischl1 and R. Kunze1
1University of Heidelberg, Institute of Physiology and Pathophysiology, Heidelberg, Germany
Abstract
Objectives: Tissue hypoxia activates an adaptive protective response which is initiated by inhibition of prolyl-4-hydroxylases (PHDs). PHD inhibition results in stabilization and activation of hypoxia-inducible transcription factors, HIFs, which in turn activate cytoprotective factors. As ischemic stroke is associated with tissue hypoxia, we sought to characterize the role of the PHD-HIF-system for stroke outcome.
Methods: Various mouse lines with neuron-specific deletion of PHD2 and/or HIF were generated and subjected to transient or permanent occlusion of the middle cerebral artery, systemic hypoxia or oligemia. Additionally, pharmacologic inhibitors of PHDs were applied. Outcome was characterized on a molecular, histological and functional level.
Results: Neuronal deletion of HIFα improved survival early, but impaired brain function at late stage upon stroke. Neuron-specific PHD2 deletion reduced infarct and edema size, improved sensorimotor and cognitive functions, and reduced expression of pro-inflammatory cytokines. On a cellular level, PHD2 inhibition increased microvascular density, and the number of resting microglia and reactive astrocytes. These beneficial reactions were completely absent when mice were additionally deficient for both HIF1α and HIF2α. On a molecular level, PHD2 inhibition was associated with increased expression of erythropoietin and VEGF. Inhibition of PHD function by the drug FG-4497 reduced brain tissue injury and edema formation upon ischemic stroke, also when applied 1 h after onset of vessel occlusion. Blood-brain barrier function was preserved, as FG-4497 prevented the ischemia-induced rearrangement and gap formation of tight junctions.
Conclusion: Our findings suggest that lack of PHD in neurons or pharmacological inhibition of PHD activity improves histological and functional outcome from stroke, and that these effects are - at least partly - dependent on the presence of HIF transcription factors. Our results may provide the basis for a new therapeutic approach to treat ischemic pathologies such as stroke.
PS05-028
Poster Viewing Session V
Effects of Exendin-4 on nitric oxide production, hydroxyl radical metabolism and ischemic change of hippocampal CA1 neuron during cerebral ischemia and reperfusion in mice
H. Kawasaki1, C. Kitabayashi2, Y. Ito2, M. Yamazato2, R. Nishioka2, M. Hirayama2, T. Yamamoto2, K. Takahashi2 and N. Araki2
1Saitama Medical University Hospital, Moroyama, Japan
2Saitama Medical University Hospital, Neurology, Moroyama, Japan
Abstract
Introduction: It is suggested that Exendin-4 protects brain tissue during cerebral ischemia. The purpose of this study is to investigate the effects of Exendin-4 on NO production, hydroxyl radical metabolism and ischemic change of hippocampal CA1 neuron during cerebral ischemia and reperfusion in mice.
Methods: (1) C57BL/6 mice [n = 16] were used. Exendin-4 (50 µg/kg) was injected to intraperitoneal in 6 mice 30 minutes before ischemia, and others were used as contol group. Both NO production and hydroxyl radical metabolism in the bilateral striatum were continuously monitored by in vivo microdialysis. The in vivo salicylate trapping method was applied for monitoring hydroxyl radical formation via 2,3 dihydroxybenzoic acid (DHBA), and 2,5-DHBA. A Laser doppler probe was placed on the skull surface. Forebrain cerebral ischemia was produced by occlusion of both common carotid arteries for 10 minutes. Levels of NO metabolites, nitrite (NO2−) and nitrate (NO3−) in the dialysate were determined using the Griess reaction.
(2) 8-OHdG immunopositive cell: To evaluate the oxidative stress in Hippocampal CA1 neurons, the ratio of the number of 8-OHdG immunopositive cell was calculated in 72 hours after the start of reperfusion.
Results: (1) NO2−, NO3−; There were no significant differences between the two groups.
(2) Total DHBA (2,3-DHBA+2,5-DHBA); Exendin-4 group showed consistently lower than that of the control group after repurfusion 60 minutes, although there was no significant difference. (3) 8-OHdG immunopositive cell counts: Exendin-4 group (36.0 ± 19.9%) showed lower than that of the control group (71.9 ± 36.9), although there was no significant difference.
Conclusions: Total DHBA and 8-OHdG immunopositive cell counts of Exendin-4 groups showed lower than those of the control groups, however there were no significant difference. The number of animals in this study may not be enough to get significant differences, thus we would like to continue the experiment.
[8-OHdG immunopositive cell]
PS05-029
Poster Viewing Session V
Combining robotic training and inactivation of the healthy hemisphere restores pre-stroke motor patterns in mice
C. Spalletti1, C. Alia1, S. Lai2, A. Panarese2, S. Micera2,3 and M. Caleo1
1National Research Council, Neuroscience Institute, Pisa, Italy
2Scuola Superiore Sant'Anna, The Biorobotic Institute, Pontedera, Italy
3Ecole Polytechnique Federale de Lausanne, Bertarelli Foundation Chair in Translational NeuroEngineering Laboratory, Center for Neuroprosthetics and Institute of Bioengineering, Lausanne, Switzerland
Abstract
Introduction: Focal cortical stroke often leads to persistent motor deficits affecting limbs and there is a great interest in developing new rehabilitative treatments. Robotic devices are now common in neurorehabilitation therapies with promising clinical results. It is also increasingly recognized that physical therapy should be combined with “plasticizing” interventions and transcallosal pathways is a promising target, but the variable results obtained in clinical trials (Di Pino et al. 2014; Lefaucheur et al. 2014) indicate the need for more investigations with appropriate animal models.
Methods: We used mice expressing channelrhodopsin-2 in pyramidal neurons and optogenetic stimulation to assess transcallosal influence from the intact to the lesioned hemisphere after stroke. We combined training on a robotic platform (Spalletti et al. 2014) with inactivation of the contralesional motor cortex via delivery of the synaptic blocker botulinum neurotoxin E (BoNT/E) and we evaluated the effect on general motor tests and kinematic analyses of skilled reaching (Lai et al. 2015). We also evaluated the expression of plasticity "brakes" in perilesional tissue.
Results: We found an increased transcallosal inhibition exerted over the perilesional tissue. After the combined treatment we found a significant recovery of forelimb function in general motor tasks and in the skilled test with re-establishment of pre-lesion kinematic patterns and a rehabilitation-induced decrease of plasticity "brakes" in peri-infarct areas.
Conclusions: For the first time, we demonstrated that a transient inactivation of the healthy hemisphere, in combination with the robotic training, restores the general forelimb functionality and allows true recovery of pre-lesion movement pattern instead of the development of compensatory, possibly maladaptive, motor strategies. This approach is able to overcome major limitations of current rehabilitative protocols as the lack of generalization to untrained motor tasks. Our data pave the way for innovative combined therapies to be implemented in clinical practice using robotics and neuromodulatory approaches.
PS05-030
Poster Viewing Session V
Manganese-enhanced MRI for the study of post-stroke cognitive impairment in a rodent stroke model
M. El Amki1, P. Baumgartner1, O. Bracko1, A. Luft1 and S. Wegener1
1University Hospital Zurich, Department of Neurology, Zurich, Switzerland
Abstract
Introduction: Cognitive impairment is a serious consequence of stroke in the middle cerebral artery (MCA) territory. However, cognitive symptoms are not easily explained by the stroke lesion itself, which usually does not affect hippocampal or parahippocampal areas. Our goal was to reveal an anatomical and/ or functional link between the sensorimotor and the hippocampal network in a rodent stroke model using manganese enhanced MRI (MEMRI). Furthermore, the effect of a motor rehabilitation algorithm on cognitive symptoms after stroke was investigated.
Methods: Rats were subjected to intraluminal MCAO or sham surgery. Sensorimotor and cognitive deficits were assessed during a 4 week period. After 28 days, we performed structural MRI, including MEMRI, where MnCl2 was injected into the entorhinal cortex and MEMRI was performed directly and 1 day after the injection. In a subset of rats, motor rehabilitation was performed between days 5 (d5) and d12 after MCAO.
Results: Stroke induced sensorimotor and cognitive impairment after MCAO from d7. MEMRI revealed a targeted distribution of the tracer within the hippocampal network. The Manganese signal pattern was altered after MCAO, with signal enhancement in the ipsilateral thalamus. Motor rehabilitation improved motor but not cognitive deficits.
Conclusions: MEMRI indicates alterations of the hippocampal network after stroke and involvement of thalamic structures in post-stroke cognitive impairment.
Further histological studies are under way to evaluate whether this effect is due to reorganization of networks and the potential effects of training on this phenomenon.
PS05-031
Poster Viewing Session V
Electrophysiological evidence of a peri-ischemic transition zone in the rat photothrombosis model
K. Schoknecht1,2,3, A. Friedman4,5 and J.P. Dreier3,6
1Charité - University Medicine Berlin, Neuroscience Research Center, Berlin, Germany
2Charité - University Medicine Berlin, Institute for Neurophysiology, Berlin, Germany
3Charité - University Medicine Berlin, Neurology & Experimental Neurology, Berlin, Germany
4Ben-Gurion University of the Negev, Physiology & Cell Biology, Cognitive & Brain Sciences, The Zlotowski Center for Neuroscience, Beer-Sheva, Israel
5Dalhousie University, Department of Medical Neuroscience, Halifay, Canada
6Charité - University Medicine Berlin, Center for Stroke Research Berlin (CSB), Berlin, Germany
Abstract
Stroke is one of the leading causes of death and disability. In hospital, the ischemic penumbra may either recover or undergo delayed injury and cell death. The penumbra is hence potentially salvageable. Unfortunately, the mechanisms of delayed injury are still largely unknown. Here we investigated the influence of epileptic seizures and spreading depolarizations (SDs) on the progression of injury in the penumbra using the photothrombosis model (PT).
Cerebral ischemia was induced by the Rose bengal PT model in anaesthetized male Wistar rats. Blood-brain barrier (BBB) permeability, cerebral perfusion and cell damage were assessed through a cranial window following intravenous injection of fluorescein sodium salt (BBB permeability and cerebral perfusion) and propidium iodide (cell damage) until 4 hrs post PT. Intracortical field potentials and extracellular potassium concentrations were measured using ion-sensitive microelectrodes. Seizures and SDs were induced by topical perfusion of 4-aminopyridine (4-AP).
Topical 4-AP administration led to rapid stroke progression as hypoperfusion, BBB dysfunction and cellular damage spread beyond the primary ischemic core. Following PT, 4-AP induced repetitive seizures and superimposed SDs and SD clusters, while 4-AP never induced SDs in control, lesion-free animals. As an indicator of metabolic disturbance, peri-ischemic interictal extracellular potassium levels rose from 3 to 6.1 ± 2.0 mM (p < 0.05, n = 13), whereas interictal potassium levels remained normal in lesion-free animals. Interestingly, increased extracellular potassium levels correlated with increased BBB permeability (R = 0.57, p = 0.042, n = 13).
Together, our data suggest a metabolically impaired peri-ischemic transition zone in the PT model and propose a link between BBB dysfunction and extracellular potassium accumulation.
PS05-032
Poster Viewing Session V
Ovarian inflammasome activation: Possible cause of exacerbated ischemic brain damage in reproductive senescence female rats
A. Raval1 and J.P. de Rivero Vaccari2
1University of Miami, Miller School of Medicine, Neurology, Miami, United States
2University of Miami, Miller School of Medicine, Neurological Surgery, Miami, United States
Abstract
Objectives: Woman's risk of cerebral ischemia increases after menopause. The decline in ovarian function at menopause is associated with an increase in the pro-inflammatory cytokine, interleukin (IL)-1β. The processing of IL-1β is carried out by activated caspase-1 that is a part of the inflammasome, a key component of the innate immune response - the major contributor to the pathomechanism of cerebral ischemia. Recently, we observed increased inflammasome proteins (IP) in the brains of reproductively senescent female rats (PMID: 26490364). In the current study, we hypothesized that reproductive senescence triggers inflammation in the ovaries that spreads to the brain, making it more susceptible to ischemic damage.
Methods: Young adult (Y; 6–7 months) or retired breeder (9–12 months) Sprague-Dawley male and female rats were used. Retired breeder rats in constant diestrus (14–20 days) were considered reproductively senescent (RS). Rats (n = 4–7) were sacrificed; hippocampus, gonads, serum and cerebrospinal fluid (CSF) were collected. Extracellular vesicles (EV) were isolated from serum and CSF using an Invitrogen kit.
Results: We observed significant increases in IP (caspase-1, caspase recruitment domain (ASC) and IL-1β) in the hippocampi, serum and ovaries of RSF as compared to YF (p < 0.05). Increases in IP were not observed in the hippocampi or gonads of age-matched males. Importantly, EV from RSF contain higher levels of IP as compared to YF (p < 0.05).
Conclusions: EV containing IP originate in the ovaries of RSF and are hematogenously carried to the brain. We believe that this increase in IP in the brain contributes to the inflammation present in the brain of RSF, and may be responsible for the exacerbated ischemic damage. Future studies exploring the role of ovarian EV in post-ischemic inflammation are under way to understand how modulating EV trafficking can reduce the incidence and impact of cerebral ischemia in post-menopausal women.
PS05-033
Poster Viewing Session V
Reduction in microglial activation, selective neuronal loss and sensorimotor deficits by the KCa3.1-blocker TRAM-34 following temporary middle-cerebral artery occlusion (MCAo) in the spontaneously hypertensive rat
and J.-C. Baron1
1Inserm U874, Paris, France
Abstract
Introduction: Fifteen-minute distal MCAo in the spontaneously hypertensive rat (SHR) consistently induces slowly recovering sensori-motor deficits (SMD) together with isolated selective neuronal loss (SNL) (i.e., without infarction) and topographically congruent microglial activation (MA) (Ejaz et al, Stroke, 2015). We hypothesized that preventing MA by means of the KCa3.1-blocker TRAM-34 would curtail SNL and in turn SMD in this model.
Methods: 12 adult male SHRs underwent 15min distal MCAo under isoflurane anesthesia and were randomized into two groups of n = 6 each, a control group and a treated group that received TRAM-34 (40 mg/kg i.p. bd for 1 week starting 12 hrs post-MCAo, then od for 3 weeks). The sticky label test was administered 3 times a week for 28 days. Immunoflorescence (IF) using both NeuN and Iba4 was then performed. Areas displaying SNL and MA were manually delineated by two independent assessors (blinded to subject's group) on randomly presented IF sections, and volumes of SNL and MA were determined.
Results: There was significant SMD in both groups, but this was less significant in the treated than control group (p < .001 and .01, respectively), with a trend for faster recovery from day 7 onward and significant Group x Time interaction (p = 0.004; repeated-measures ANOVA). The volume of MA was markedly reduced in the TRAM-34 treated group relative to the control group (1.33 ± 1.7 mm3 vs 0.27 ± 0.46 mm3, respectively; p = 0.018 by two-sample t-test), with a similar reduction in SNL (1.41 ± 1.79 mm3 vs 0.28 ± 0.41 mm3; p = 0.015).
Conclusion: As expected, TRAM34 markedly prevented MA, which was associated with concomitant reductions in SNL and a trend for faster SMD recovery. Thus, early administration of an MA blocker following reperfusion therapy in the clinical setting might reduce cell damage in the salvaged penumbra and in turn foster neurological recovery.
PS05-034
Poster Viewing Session V
Stroke syndromics: Multivariate analyses for outcome measures after middle cerebral artery occlusion in mice
R. Bernard1, N. Wenger1, A. Rex1, L. Mosch1, M. Dopatka1, C. Harms1, M. Endres1 and U. Dirnagl1
1Charite University Medicine, Experimental Neurology, Berlin, Germany
Abstract
Preclinical models of stroke are important tools for studying the course of disease and development of treatment strategies. We hypothesized that an unbiased multifactorial statistical analysis of multiple behavioral and general health outcome measures will improve identification of parameters that reliably capture the disease state. Here, we used multifactorial syndromic analysis to identify functional clusters of outcome measures that predict disease severity after stroke.
Methods: Adult, male C57Bl6 mice were subjected to a 60 min MCAO (N = 17) and sham (N = 20) surgery. Three days later various outcome measures were assessed including general health parameters, behavioral parameters (RotaRod, Rotating Pol, CatWalk) as well as histological measures, which resulted in a total of 69 outcome parameters per animal. CatWalk customized analysis initially provided a total of 225 gait parameters. To avoid redundancy, experimental data from Catwalk were analysed for inherent autocorrelations. Parameters with high autocorrelation were then excluded from analysis, which resulted in a comprehensive number of 50 parameters. Multifactorial syndromic analysis was then applied to identify distinct functional clusters of outcome parameters after stroke. These functional clusters were captured in different principle components.
Results: Principle component 1 (PC1) explained 21%, PC2 14% and PC3 10% of the total variance in the original dataset. PC1 factor loading contained mainly gait measures related to speed, such as stand duration for all individual paws. Additional factors were weight loss and DeSimoni health sum score. PC2 factor loadings were composed of two subparameters of the DeSimoni Score and speed variation of all four paws. PC3 factor loadings exhibited correlations with right hindlimb and forelimb area and the two paw support parameters.
Conclusion: Multifactorial syndromic analysis accurately captured the severity of stroke deficits after 60 min MCAO. This analysis may serve as a reliable strategy to identify therapeutic effects of stroke treatments in preclinical and clinical research.
PS05-035
Poster Viewing Session V
Mass cytometry profiling of inflammatory cells in ischemic stroke mice
Y. Li1, T. Tao1, B. Xu1 and H. Zhao1
1Stanford University, Stanford, United States
Abstract
Background: How stroke-induced inflammatory leukocytes modulate brain injury is not fully understood. In this study, we established a novel technique, CyTOF, to identify the functions and roles of inflammatory cells in ischemic stroke mice by a 16 metal-labeled cell surface markers panel, with no fluorescence cross contaminations when using flow cytometry.
Methods: 25 male C57BL/6 mice underwent 90 min of middle cerebral artery occlusion. Leukocytes were isolated from the sham, 1, 3, 7, 14 days of ischemic brain hemisphere, then simultaneously stained with 16 mental-labeled immune cell phenotype markers. CyTOF was used to detect the cell surface marker expressions. Manual gating analysis and unsupervised spanning-tree progression analysis of density-normalized events (SAPDE) algorithm were used for quantification.
Results: The total number of isolated-leukocytes from the ischemic brain was significantly increased at 1 day and peaked at 3 days post ischemia and decreased thereafter, of which MiMΦs, neutrophils, pDCs, and B cells peaked at 1 day post ischemia. Meanwhile MoMΦs, cDCs, γδ T, CD4 T, CD8 T, Eff/mem CD4 T, Eff/mem CD8 T and NK cells peaked at 3 days post ischemia. The expression densities of CD45 and CD11b in MiMΦs, Gr-1 in MoMΦs, B220 in B cells, CD3 in γδ T cells, and CD44 in Eff/mem CD8 T cells were all robustly upregulated in 3 days of the ischemic brain. Furthermore, the expression densities of Gr-1 in MoMΦs, CD4 in CD4 T, CD8 in CD8 T and CD44 in Eff/mem CD4 T cells were significantly activated at 1 day of ischemic brain.
Conclusions: CyTOF is a powerful tool to quantify leukocyte cell types and protein expressions in individual cells. Our results suggest that effect memory T cells are involved in brain injury, and the upregulation of cell surface receptors in individual leukocytes may be important for their effects on brain injury.
PS05-036
Poster Viewing Session V
Maternal obesity exacerbates brain injury responses following neonatal hypoxia-ischaemia in rat offspring
N.M. Jones1, J.D. Teo1 and M.J. Morris1
1University of New South Wales, Pharmacology, School of Medical Sciences, Sydney, Australia
Abstract
In humans, maternal obesity is associated with an increase in the incidence of birth related difficulties, but the impact on brain injury severity in offspring is not known. Recent studies have shown enhanced inflammation in brains of obese rodents. Here, we have studied the effects of maternal obesity on various brain injury responses following neonatal hypoxia-ischaemia (HI) in offspring. Adult female Sprague Dawley rats were allocated to high fat (HFD, n = 7) or chow (n = 4) diets and once a 10% difference in weight was observed, mated with lean males. On postnatal day 7 (P7), pups were randomly assigned to HI injury (right carotid artery occlusion followed by 8% oxygen (3 h)) or control (C) groups. Control pups were kept under ambient conditions for 3h. Brain injury was quantified in brain sections from P14 pups using cresyl violet staining and immunohistochemistry for neuronal, glial and microglial markers. The Chow-HI pups (n = 31) showed an 8.9 ± 3.3% loss in ipsilateral brain tissue while the HFD-HI group had a significantly greater reduction in ipsilateral brain size (20.5 ± 3.2% n = 44, p < 0.05, 2-way ANOVA). There was no significant injury detected in pups from either of the control groups (HFD-C (n = 16) and Chow-C (n = 25)). When analysed on a per litter basis, the size of the injury was significantly correlated with maternal weight (r = 0.89; P < 0.05). We observed a similar pattern of exacerbated injury responses in the glial markers. Astrocytes were more hypertrophic and a greater number of microglia were present in the injured hemisphere of HFD-HI compared to Chow-HI pups. Our data clearly demonstrate that maternal obesity can exacerbate brain injury severity caused by HI in neonatal offspring. Given that previous studies have shown enhanced inflammatory responses in offspring of obese mothers, these factors including gliosis and microglial infiltration may contribute to the enhanced brain injury.
PS05-037
Poster Viewing Session V
Microglia control central neurotrophic virus infection via P2Y12-mediated signaling
R. Fekete1, B. Orsolits1, B. Sperlágh2, M. Baranyi2, Z. Boldogkői3, Á. Kittel2, S. Ferenczi4, K. Kovács4, E. Méhes5, Z. Környei1 and Á. Dénes1
1Institute of Experimental Medicine, Hungarian Academy of Sciences, Laboratory of Neuroimmunology, Budapest, Hungary
2Institute of Experimental Medicine, Hungarian Academy of Sciences, Laboratory of Molecular Pharmacology, Budapest, Hungary
3Faculty of Medicine, University of Szeged, Department of Medical Biology, Szeged, Hungary
4Institute of Experimental Medicine, Hungarian Academy of Sciences, Laboratoy of Molecular Neuroendocrinology, Budapest, Hungary
5ELTE, Budapest, Department of Biological Physics, Budapest, Hungary
Abstract
Objectives: Understanding immune mechanisms that control viral infection in the CNS is essential to develop appropriate therapies for diseases such as herpes simplex encephalitis or viral meningitis. We have reported earlier that microglia form barriers around viral infected neurons in the brain. However, the functional role of microglia in defense against neurotrophic viral infection and the mechanisms controlling microglia recruitment to infected neurons remained unclear.
Materials and methods: The role of microglia in controlling neurotrophic virus infection was studied by selective microglia depletion prior to peripheral injection of pseudorabies virus (PRV), which results in retrograde transsynaptic spread of the virus into the brain. Central propagation of PRV infection was also assessed in wild type, P2X7 and P2Y12 knock out (KO) mice. We have established in vitro cultures of microglia and infected neurons or astrocytes to further explore the mechanisms involved.
Results: Selective elimination of microglia resulted in markedly increased numbers of infected neurons in the brain, which was associated with high levels of extracellular virus particles. Microglia recruitment to infected cells was associated with release of ATP and increased Ecto-ATPase activity both in vitro and in vivo. In contrast, the expression of inflammatory chemokines that facilitate the recruitment of monocytes and macrophages was negligible. Microglia were rapidly recruited to infected neurons in the brain and phagocytosed cells showing signs of late infection, whereas deficient recruitment of microglia and increased number of infected neurons were found in P2Y12 KO, but not in P2X7 KO mice.
Conclusion: Our results suggest that ATP released from infected neurons and/or nearby cells could mediate rapid recruitment of microglia to the sites of neurotrophic viral infecion in the brain. Therapeutic interventions facilitating the phagocytosis of virally infected neurons by microglia could be used to support the control of viral infection in the CNS.
PS05-038
Poster Viewing Session V
Mafb prevents excess inflammation after ischemic stroke by accelerating clearance of danger signals through MSR1
T. Shichita1,2,3, H. Ooboshi4, S. Takahashi5, T. Kodama6 and A. Yoshimura2
1Tokyo Metropolitan Institute of Medical Science, Stroke Renaissance Project, Tokyo, Japan
2Keio University, School of Medicine, Department of Microbiology and Immunology, Tokyo, Japan
3Kyushu University, Graduate School of Medical Sciences, Department of Medicine and Clinical Science, Fukuoka, Japan
4Fukuoka Dental College Medical and Dental Hospital, Department of Internal Medicine, Fukuoka, Japan
5University of Tsukuba, Faculty of Medicine, Department of Anatomy and Embryology, Tsukuba, Japan
6The University of Tokyo, Research Center for Advanced Science and Technology, Laboratory for Systems Biology and Medicine, Tokyo, Japan
Abstract
Inflammation is an essential step for the pathology of ischemic stroke. Since no pathogen exists within brain, post-ischemic inflammation will be triggered by some endogenous molecules (DAMPs: danger associated molecular patterns), which were released from dying brain cells. High mobility group box 1 (HMGB1) and peroxiredoxin (PRX) have been recently identified as DAMPs in the ischemic brain. HMGB1 exaggerates the disruption of blood brain barrier; on the other hand, PRX directly activates macrophage/neutrophil and induces the inflammatory cytokine production through the stimulation of Toll-like receptor (TLR2/4). Both the extracellular release of PRX and the infiltration of immune cells reach the peak within 1 to 3 days after the onset of ischemic stroke, and thereafter they decrease. This will lead to the resolution of post-ischemic inflammation.
We clarified the molecular mechanisms for the clearance of DAMPs from ischemic brain. Using random mutagenesis method, MSR1 (scavenger receptor) and Mafb (transcription factor) were identified as key factors for DAMP clearance. MSR1 expression levels in infiltrating mononuclear phagocytes increased from day 1 to day 3 after stroke onset, which was dependent on Mafb. These MSR1-high cells efficiently removed DAMPs and produced IGF-1, a neurotrophic factor, indicating that Mafb-MSR1 pathway played pro-resolving roles in delayed phase of ischemic stroke. The deficiency of Msr1 or Mafb in inflitrating immune cells resulted in exacerbation of inflammation and neuronal damages. Thus, Mafb-MSR1 pathway is important for the resolution of cerebral post-ischemic inflammation. We also found that Am80, a retinoic acid receptor agonist, enhanced MSR1 expression in infiltrating immune cells through Mafb and revealed neuroprotective effects against ischemic brain injury.
Our results indicate that DAMPs regulate not only the induction but also the resolution of post-ischemic inflammation. The novel neuroprotective strategy for ischemic stroke can be developed by accelerating the endogenous pro-resolving mechanisms to prevent the excess inflammatory responses.
PS05-039
Poster Viewing Session V
Cytoprotective role of microglia-derived nitric oxide induced by TLR4 stimulation in concert with astroglial pentose-phosphate pathway activation through the Keap1/Nrf2 system
S. Takahashi1, T. Iizumi1, K. Mashima1, K. Minami1, Y. Izawa1, T. Abe2, T. Hishiki3,4,5, M. Suematsu3,5, M. Kajimura3,5 and N. Suzuki1
1Keio University School of Medicine, Neurology, Tokyo, Japan
2Osaka City University Graduate School of Medicine, Neurology, Osaka, Japan
3Keio University School of Medicine, Biochemistry, Tokyo, Japan
4Keio University School of Medicine, Clinical and Translational Research Center, Tokyo, Japan
5Keio University School of Medicine, ERATO Suematsu Gas Biology Project, Tokyo, Japan
Abstract
Objectives: Both astroglia and microglia express toll-like receptor 4 (TLR4) that plays a pivotal role in the stroke-induced inflammation. Endogenous ligands for TLR4 produced in the ischemic brain induce inflammatory responses. Both reactive oxygen species (ROS) and nitric oxide (NO) produced by TLR4 activation play harmful roles in neurovascular unit damage. Although astroglia exhibit pro-inflammatory responses upon TLR4 stimulation by lipopolysaccharide (LPS), they may also play cytoprotective roles via the activation of the pentose-phosphate pathway (PPP), reducing oxidative stress with glutathione. We investigated the mechanisms by which astroglia reduce oxidative stress via the activation of PPP in concert with microglia.
Methods: In vitro experiments were performed using cells prepared from Sprague-Dawley rats. Coexisting microglia in the astroglial culture were chemically eliminated using L-leucine methyl ester (LME). Cells were exposed to LPS (0.01 µg/mL) for 12–15 h. PPP activity was measured using [1-14C]glucose and [6-14C]glucose. ROS and NO production were measured using fluorescent indicators. The involvement of nuclear factor-erythroid-2-related factor 2 (Nrf2) that regulates glucose 6-phosphate dehydrogenase, the rate-limiting enzyme of PPP and glutathione synthesis was evaluated using immunohistochemistry.
Results: Cultured astroglia exposed to LPS elicited 20% increases in PPP flux, and these actions of astroglia appeared to involve Nrf2. However, the chemical depletion of coexisting microglia eliminated both increases in PPP and astroglial nuclear translocation of Nrf2. LPS induced ROS and NO production in the astroglial culture containing microglia but not in the microglia-depleted astroglial culture. U0126, an upstream inhibitor of mitogen-activated protein kinase, eliminated LPS-induced NO production, whereas ROS production was unaffected. U0126 also eliminated LPS-induced PPP activation in astroglial-microglial culture, indicating that microglia-derived NO mediated astroglial PPP activation. SNAP, an NO donor, did indeed induce PPP activation in astroglia.
Conclusions: Astroglia in concert with microglia may play a cytoprotective role for countering oxidative stress in stroke.
PS05-040
Poster Viewing Session V
Playing with omega-3 Alpha-linolenic acid in the diet to prevent the toxic CCL2 post-stroke inflammatory response
M. Bourourou1, E. Gouix1, C. Nguemeni1, A. Petit-Paitel1, J. Cazareth1, J.-L. Nahon1, C. Heurteaux1, C. Rovère1 and N. Blondeau1
1Université Côte d’Azur, CNRS, IPMC, Institut de Pharmacologie Moléculaire et Cellulaire, Valbonne, France
Abstract
Objectives: Omega-3 polyunsaturated fatty acids (ω-3) are increasingly foreseen as nutraceuticals or disease-modifying therapy in stroke. Stroke is a leading cause of death and disability, an outcome strongly influenced by neuroinflammation. Pro-inflammatory chemokines may be the primary determinant in injury evolution by promoting BBB disruption, MMP-9 release, leukocyte transmigration and microglial activation. We investigated how modification of the alpha-linolenic acid (ALA, the w-3 contained in plant-derived edible products), in diet reduced neuronal damage and proinflammatory response post-stroke, and which proinflammatory chemokine was the target.
Methods: C57/BL6 mice were fed either an ALA-diet (0.75% ALA, w-6/ w-3 of 2.3) or a regular rodent diet (0.25% ALA, w-6/ w-3 of 6.4) during 6 weeks. Mice were then subjected to 60-minute middle cerebral artery occlusion (MCAo). Brain coronal sections were analyzed by Cresyl violet staining and immunostaining to assess infarct volume, IgG leakage, MMP9 and Iba1 expression. Leucocyte infiltration and microglia activation were studied by FACs. Chemokine CCL2 mRNA and protein were analyzed by quantitative RT-PCR and CBA techniques. An in vitro approach on primary cultured neurons was used to dissect the correlation of ALA protective effect and CCL2 neurotoxicity during oxygen-glucose deprivation (OGD).
Results:
1) Restoring the dietary w-6/ w-3 ratio to below 5 by increasing the ALA concentration protected the brain against stroke and dampened post-stroke proinflammatory response, especially preventing CCL2 overexpression.
2) Prevention of CCL2 overexpression after stroke alters its contribution to neuronal damage synergistically exerted directly by cytotoxicity on neurons and indirectly by stimulation of MMP-9 up-regulation, BBB disruption, leucocyte infiltration and microglial activation.
Conclusion: Here we document the relevance of increasing the ALA consumption to promote brain resistance to stroke preventing neurotoxic CCL2 proinflammatory response. That results may be transferable to the public promoting the need of increasing absolute intakes of ALA for correcting the omega-6/omega-3 ratio.
PS05-041
Poster Viewing Session V
Vitamin D supplementation reduces brain injury and inflammation following ischemic stroke
M.A. Evans1, H.A. Kim1, Y.H. Ling1, S. Uong1, G.R. Drummond1,2, B.R.S. Broughton1 and C.G. Sobey1,2
1Monash University, Cardiovascular Disease Program, Biomedicine Discovery Institute, Department of Pharmacology, Melbourne, Australia
2Monash University, Department of Surgery, Southern Clinical School, Melbourne, Australia
Abstract
Objectives: Inflammation is a major contributor to secondary brain injury after ischemic stroke. Beyond its well-characterized role in calcium metabolism, the active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25-VitD3), has been shown to elicit anti-inflammatory effects. Therefore we aimed to determine whether 1,25-VitD3 supplementation can reduce brain injury and associated inflammation following stroke.
Methods: Male C57Bl6 mice (7–10 weeks) were randomly assigned to be administered either 1,25-VitD3 (n = 32; 100 ng/kg) or vehicle (n = 30; mixture of dH2O, propylene glycol and ethanol) i.p. daily for 5 d prior to stroke. Stroke was induced via middle cerebral artery occlusion for 1 h followed by 23 h reperfusion. Hanging grip and parallel rod tests were then used to assess grip strength and locomotor activity, respectively. In addition, infarct volume was assessed by thionin staining and cerebral inflammation was evaluated using quantitative PCR and immunohistochemistry.
Results: Supplementation with 1,25-VitD3 reduced infarct volume by 50% compared to vehicle (18 ± 3 mm3 versus 36 ± 6 mm3 respectively, p < 0.05). However, at this early timepoint there were no differences in functional outcomes, with hanging grip time and total time mobile being similar in 1,25-VitD3- and vehicle-supplemented groups. Expression of key pro-inflammatory cytokines, IL-6, IL-1β and IL-23a, were reduced in brains of mice that received 1,25-VitD3 versus vehicle. Brain expression of the T regulatory cell marker, Foxp3, was higher in mice supplemented with 1,25-VitD3 versus vehicle, while expression the transcription factor, ROR-γ, was decreased, suggestive of a reduced Th17 response. Immunohistochemistry revealed that numbers of neutrophils and T cells infiltrating the ischemic hemisphere were similar in 1,25-VitD3- and vehicle-supplemented animals. Ongoing experiments are assessing other immune cell types.
Conclusions: These data indicate that administration of exogenous vitamin D to vitamin D-replete mice can attenuate infarct development and exert anti-inflammatory actions, and thus may represent a direction for acute stroke therapy.
1Nanjing Drum Tower Hosptial of Nanjing University, Neurology, Nanjing, China
2Nanjing Drum Tower Hosptial of Nanjing Medical University, Nanjing, China
Abstract
Histone deacetylases (HDACs) regulate acetylation states of histone and non-histone proteins and could be a potential regulator of inflammatory process. However, individual subtypes of HDACs remain largely unknown. RGFP966 is a newly discovered selective HDAC3 inhibitor. In this study, we aimed to profile molecular alternations in LPS-treated primary microglia with the application of RGFP966. We used protein mass spectrometry to analyze protein discrepancies in LPS group and LPS+RGFP966 group. RT-PCR and western blot were involved to detect mRNA and protein level of Toll like receptor 2 (TLR2), TLR3, TLR6, CD36, interferon regulatory factor (IRF)-3, spleen tyrosine kinase (SYK). Supernatant tumor necrosis factor (TNF)-α, Interleukin 6 (IL-6), interferon (IFN)-β of various time points were collected and detected by CBA kit. We also stained microglia with CD16 in different groups by immunofluorescence to observe microglia activation. Phosphorylation and total level of STAT3 and STAT5 were measured by western blotting. Generally, about 2000 proteins were studied. 168 of 444 (37.8%) LPS-induced proteins were significantly reduced with the treatment of RGFP966, which mainly concentrated on Toll-like receptor signaling pathway. In this regard, we selected TLR2, TLR3, TLR6, CD36, IRF-3 and SYK for further validation, and found that they were all significantly up-regulated after LPS stimulation and down-regulated in the presence of RGFP966. Additionally, RGFP966 inhibited supernatant TNF-α, IL-6 and IFN-β concentrations. Activation of STAT3, STAT5 was partially blocked by RGFP966. The fluorescence intensity of CD16/32 was significantly decreased in LPS-treated group. In conclusion, our data provided a hint that RGFP966 may be a potential therapeutic medication combating microglia activation and inflammatory response in neurological diseases, which was probably related to its repressive impacts on TLR signaling pathways and STAT3/STAT5 pathways.
PS05-043
Poster Viewing Session V
White matter inflammation as a biomarker of cognitive impairment induced by mild diabetes in a mouse model of cerebrovascular disease
M. Lacalle-Aurioles1, J. Royea1, L.J. Trigiani1, X.K. Tong1 and E. Hamel1
1Laboratory of Cerebrovascular Research, Montreal Neurological Institute, McGill University, Montréal, QC, Canada
Abstract
Epidemiological studies suggest a 2 fold risk of suffering from vascular dementia and late onset Alzheimer's disease in patients with type 2 diabetes (DB). Although it is well known that DB contributes to cerebrovascular dysfunction and neuroinflammation, the specific mechanisms by which DB can trigger cognitive impairments (CI) are still unknown. We hypothesize that cerebrovascular pathology, commonly observed in aging and patients with cardiovascular diseases, may reduce the threshold required for high glucose levels to trigger CI, and that there might be a gender susceptibility for developing CI. We induced mild DB by combined administration of streptozotocin-nicotinamide in adult (6 months at term) littermate male and female transgenic transforming growth factor-β1 (TGF) mice with a compromised cerebrovasculature without overt CI, and wild-type (WT) control mice. In female TGF mice, mild DB induced learning deficits in the Morris water maze (MWM) and impaired memory on the novel object recognition (NOR) test, without worsening the already present cerebrovascular deficits: impaired endothelium-dependent dilation and whisker-evoked neurovascular coupling. CI was accompanied by an exacerbated brain inflammatory response, most prominent in the white matter (WM), as detected by increased Mac-2 immunopositive microglial cells, which have been involved in myelin phagocytosis. DB impaired dilatory function and neurovascular coupling in WT mice but not cognitive function. In male WT and TGF mice, mild DB did not induce cognitive deficits, and there was no worsening of the cerebrovascular pathology in TGF mice. Our results indicate that a compromised cerebrovasculature facilitates the appearance of CI associated with mild DB, that females may be more susceptible than males to cognitive failure induced by DB, and that WM inflammation may play an important role in this manifestation.
Sponsored by the CIHR-CVN and ASRP grants (EH) and by a CIHR-CVN scholar award and Jeanne Timmins Costello fellowship (ML).
PS05-044
Poster Viewing Session V
Neuronal interleukin-4 promotes M2-like microglia activation and neuroprotection following focal cerebral ischemia
S.-H. Choi1 and A.C. Silva1
1NINDS/NIH, Bethesda, United States
Abstract
Microglia-mediated inflammation is a near universal response to injuries of the central nervous system. In the presence of neuronal stress, microglia can react rapidly and interact with the local environment according to either one of two phenotypes: a pro-inflammatory, harmful (M1); or a pro-restaurative, healing (M2) phenotype. However, the specific sequence of events, the signaling pathways and the role of M1 and M2 microglia phenotypes associated with the inflammatory response following brain injury are not fully understood. Using in vivo and in vitro models of ischemic injury, we demonstrate here that anti-inflammatory interleukin-4 (IL-4), released from injured neurons, induces M2-like microglia activation. We found robust induction of IL-4 expression both in neuronal cells surrounding the ischemic core as well as in the primary neuronal culture media after exposing them to transient oxygen-glucose deprivation. To investigate whether expression of IL-4 at an early stage after focal cerebral ischemia could affect microglia polarization and the corresponding functional outcome of brain tissue in vivo, we designed a plasmid driven by the mouse phosphoglycerate kinase promoter containing IL-4 and green fluorescence protein and injected it into the mouse brain using recombinant lentiviruses. Immunostaining of brain sections from lentivirus-injected mice with several cellular markers revealed that neurons were the primary cell type transduced by the viral vector. Real-time PCR measurements revealed the up-regulation of multiple M2-like activation markers. Finally, using an endothelin-1 model of focal cerebral ischemia, we determined that lentivirus-derived IL-4 reduces ischemic lesion, improves functional outcome, and is associated with elevated M2-like microglia activation. Our results indicate that IL-4 promotes M2-like microglia activation and neuroprotection of injured brain tissue, and suggest that strategies to enhance local IL-4 expression have a strong potential to attenuate neuronal damage and accelerate tissue repair in ischemic brain injury.
PS05-045
Poster Viewing Session V
Time-course analysis of infiltrating dendritic cell subpopulations in a murine model of ischemic stroke
M. Gallizioli1, M. Gelderblom2, E. Orthey2, A.M. Planas1 and T. Magnus2
1Instituto de Investigaciones Biomedicas de Barcelona IIBB-CSIC, Cerebral Ischemia and Neurodegeneration, Barcelona, Spain
2Universitätsklinikum Hamburg-Eppendorf, Experimental Research in Stroke and Inflammation Unit, Hamburg, Germany
Abstract
Objective: Dendritic cells (DCs) are professional antigen presenting cells that are normally present in low numbers in the brain parenchyma under physiological conditions. DC numbers increase in the brain following ischemia, but their phenotype and role are still largely unknown. Recent advances allowed to better discriminate various subpopulations of DCs with renewed specificity and reliability (1). We applied these new definitions to identify different DC subtypes in the mouse brain after ischemia.
Methods: C57Bl/6J mice underwent transient cerebral ischemia by filament-mediated occlusion of the middle cerebral artery as described (2).
We analyzed by flow cytometry single cells suspensions derived from the ipsilesional hemisphere at 12h, 1, 3 and 7 days after reperfusion. We localized DCs by immunofluorescence in reporter CD11c-eYFP transgenic mice at the same time points after ischemia. Gene expression of subpopulation-defining transcription factors was analyzed by RT-PCR on DCs isolated by fluorescence-activated cell sorting.
Results: We defined conventional dendritic cells (cDC) as CD45hiCD11c+MHCII+Ly6G-CD64-Ly6C-F4/80- and subclassified this population into cDC1 cells as XCR1+ and cDC2 cells as Sirpα+. We also identified monocyte-derived DCs (moDC) as CD45hiCD11c+MHCII+CD64+Ly6Cint and plasmacytoid DCs (pDCs) as CD45hiCD11c+MHCII+Ly6G-B220+.
DCs infiltrated already at 12h after reperfusion and located initially at the border of the lesion to later invade the ischemic core. cDCs were the prevalent population at all time points analyzed. Already at 12h, cDC2 Sirpα+ cells were more abundant than cDC1 XCR1+ cells. Sorted Sirpα+ cells expressed typical cDC2 population-defining genes, such as ZBTB46, BATF3 and IRF4.
Conclusions: With this data we show for the first time an infiltration pattern of DC subpopulations in the brain after ischemia. In particular the cDC2 subpopulation enters the brain shortly after ischemia and could possibly regulate the neuroinflammatory response.
References
1. Guilliams et al., Frontiers in Immunology. 2015. 6:1–12
2. Gelderblom et al., Stroke. 2009. 40:1849–57
PS05-046
Poster Viewing Session V
Microglial cells phagocyte neutrophils after brain ischemia
A. Otxoa-de-Amezaga1,2, F. Miró-Mur1,2, N. Gaja-Capdevila1, J. Pedragosa1,2, M. Calvo3 and A.M. Planas1,2
1Institut d’Investigacions Biomèdiques de Barcelona, Consejo Superior de Investigaciones Científicas, Barcelona, Spain
2Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain
3Unitat de Microscòpia Òptica Avançada. Universitat de Barcelona, Barcelona, Spain
Abstract
Introduction: Ischemic stroke causes an acute inflammatory response involving microglial activation and leukocyte infiltration. An important function of microglia is phagocytosis of death cells and cell debris. Recent findings show that microglia phagocyte neutrophils after brain ischemia (Neumann et al., 2015). The aim of this study is to further characterize phagocytosis of neutrophils by microglia and investigate the underlying mechanisms.
Methods: To differentiate microglia from infiltrating leukocytes we generated chimeric mice by transferring bone marrow of DsRed reporter mice to WT mice (C57BL/6) and permanent middle cerebral artery occlusion (MCAo) was induced 8 weeks later. Flow cytometry and confocal microscopy (n = 5 mice each) was carried out 4 days after MCAo. For in vitro studies, microglial cells isolated from the adult mouse brain and cultured for 7 days were exposed to neutrophils extracted from the bone marrow of DsRed reporter mice or obtained from wild type mice and stained with carboxyfluorescein succinimidyl ester. Cultures were treated with purinergic receptor antagonists and were studied by time-lapse confocal microscopy.
Results: Neutrophils (CD11b+Ly6G+) recruited to the ischemic brain were DsRed+ (98.3 ± 1.3%) and accounted for 16.5 ± 4.8% of the infiltrating DsRed cells, whereas microglia (CD45lowCD11blow) remained non-fluorescent. Reactive microglial cells bordering the infarcted core showed prolongations surrounding DsRed+Ly6G+ cells, suggesting phagocytosis of neutrophils. In the in vitro culture preparation, microglia engulfed 20% of the neutrophils. The P2Y12 antagonist PSB0937 reduced by 30% (n = 3) the numbers of phagocyted neutrophils, potentially through affecting microglia movement. Some neutrophils formed extracellular traps involving nuclear restructuration, swelling, and expulsion of the intracellular content, but this process seems to be independent of microglial activity.
Conclusion: The results show that microglia phagocyte neutrophils after ischemia and in culture suggesting the involvement of purinergic receptors in this process.
Reference
Neumann et al. Acta Neuropathol, 2015:129:259–7.
Supported by MINECO (SAF2014-56279R). AO has a FPI fellowship.
PS05-047
Poster Viewing Session V
Angiotensin receptor type 1 (ATR1) deficiency or inhibition by candesartan improves outcome after experimental traumatic brain injury and inhibits inflammation
J.Y. Kim1,2, N. Kim1,2, Z. Zheng1,2, M. Johnson3,4 and M. Yenari1,2
1University of California, San Francisco, Neurology, San Francisco, United States
2San Francisco Veterans Affairs Medical Center, Neurology, San Francisco, United States
3University of California, San Francisco, Medicine, San Francisco, United States
4San Francisco Veterans Affairs Medical Center, Medicine, San Francisco, United States
Abstract
Angiotensin II receptor type 1 (ATR1) mediates vasoconstriction, and its inhibition has been widely used to treat hypertension; however, recent work has suggested that it may also modulate apoptosis, and neuroinflammation. Thus, treatment with already available ATR1 blockers may have additional neuroprotective value. We explored the contribution of ATR1 to neuroprotection and brain hemorrhage in a model of TBI. Male, wildtype (Wt) and ATR1 knockout (Ko) mice were subjected to TBI using controlled cortical impact (CCI). Sensorimotor function (adhesive removal & elevated body swing tests), brain hemorrhage and lesion size were assessed out to 14d post insult. Wt mice were also treated with an ATR1 inhibitor (candesartan, 0.1 mg/kg IP). ATR1 deficient mice were protected from CCI as evidenced by decreased lesion volumes (decreases of ∼40% in lesion size amongst Ko mice, n = 6/group, p < 0.05), improved neurobehavioral outcomes (n = 6/group, p < 0.05) and fewer activated microglia in Ko mice (p < 0.05). This was also associated with decreased TNF-alpha and IL-6 cytokine expression relative to Wt. Further, the ATR1Ko mice suffered less brain hemorrhage (p < 0.05) and this was correlated to reduced MMP-9 expression (p < 0.01). Candesartan similarly protected against brain injury, brain hemorrhage and improved neurological outcome out to 14 days post CCI (n = 6/group, p < 0.05). Candesartan, while often used to treat hypertension in humans, did not reduce blood pressure in mice at the dose studied. These data are consistent with the notion that ATR1 contributes negatively to traumatic brain injury, and its inhibition or deficiency leads to improved outcomes and decreased immune responses. Considering the clinical availability of ATR1 inhibitors, this approach may be a promising novel therapeutic target against TBI and related conditions.
PS05-048
Poster Viewing Session V
An in vitro microfluidic model of microglia migration after stroke
S. White1,2, R. Plevin1, M. Zagnoni2 and H. Carswell1
1University of Strathclyde, Strathclyde Institute of Pharmacy and Biomedical Sciences, Glasgow, United Kingdom
2University of Strathclyde, Electronic and Electrical Engineering, Glasgow, United Kingdom
Abstract
Objectives: Microglia migrate to the site of ischemic insult in response to mediators such as glutamate and ADP being released from damaged or stressed cells and can exert both protective and detrimental effects1. Our present objective is to characterise microglia migration in vitro using a microfluidic model which allows precise chemical concentration gradients to be established over time, mimicking the release of mediators after stroke in vivo.
Methods: Microglial cell line, SIM-A9, were seeded in microfluidic culture chambers at 2.5 × 106 cells/ml for 24 hrs prior to exposure to concentration gradients of glutamate (100 µM) or vehicle (DMEM, control), with and without direct LPS (1µg/ml). Real time time-lapse imaging and cell tracking software were used to quantify cell migration velocity, and accumulated and Euclidean distance. Preliminary experiments analysed an average of 24 cell tracks per group (mean ± SD).
Results: Microglia were observed to migrate towards increasing chemical concentration gradients compared to control. This directionality effect was supported by an increased average number of cells entering the microchannels and an increased Euclidean distance towards the glutamate gradient versus control (170.36 ± 108.19 µM vs 35.5 ± 36.9 µm, respectively). Interestingly, the addition of direct LPS dampened down the increased Euclidean distance to 75.26 ± 53.5 µm. Compared to vehicle, a concentration gradient of glutamate induced a substantial increase in velocity which was further increased by the additional direct application of LPS (0.33 ± 0.18 µm/min vs 0.58 ± 0.15 µm/min vs 0.65 ± 0.18 µm/min, respectively). A similar pattern was observed for accumulated distance (372.8 ± 203.12 µm vs 651.02 ± 169.4 µm vs 730.4 ± 205.47 µm, respectively).
Conclusions: These results suggest that a pro-inflammatory environment limits glutamate-induced directionality and provide novel insight into dynamics of microglia responses after stroke.
References
1Patel et al., Int J Physiol Pathophysiol Pharmacol, 2013; 5, 73–90
This project is funded by BBSRC DTP.
PS05-049
Poster Viewing Session V
Stroke and traumatic acute brain injury line indicator system for emergent recognition (STABILISER-!) phase i feasibility study: preliminary results of the tbi cohort
J. Fraser1,2,3, M. Bradley-Whitman4, A. Bernard5, N. Timoney1, B. Eckerle2, J. Lee2, R. Humphries6, G. Bix1,2,3 and M. Lovell4
1University of Kentucky, Neurological Surgery, Lexington, United States
2University of Kentucky, Neurology, Lexington, United States
3University of Kentucky, Anatomy and Neurobiology, Lexington, United States
4University of Kentucky, Sanders Brown Center for Aging, Lexington, United States
5University of Kentucky, Surgery, Lexington, United States
6University of Kentucky, Emergency Medicine, Lexington, United States
Abstract
Objectives: Early diagnosis of Traumatic Brain Injury (TBI) is a major health issue. We developed a point-of-care lateral flow device to rapidly detect a neuron-specific biomarker, visinin-like-protein-1 (VILIP-1), that is released into the blood after TBI. The STABILISER study is a first-in-human evaluation of feasibility and efficacy in rapid diagnosis of patients with acute brain injury. While the results of the study linking device detection to clinical and radiographic diagnosis remain blinded, we present the preliminary results of feasibility and biomarker detection after TBI.
Methods: Subjects diagnosed with TBI presenting to the emergency department within 8 hours of injury were enrolled. 10 mL of serum was applied to the device, which was processed to the laboratory for quantitative evaluation of the biomarker using an automated reader. Similarly, a group of volunteer control subjects with no recent history of brain injury underwent testing.
Results: There were no device application failures. There were no differences in gender or age among the groups (control N = 13; mild N = 10, moderate N = 3). Overall, there was a difference between control subjects (9.5 pg/mL, range 0.0–39.3) and those with any TBI (121.4 pg/mL, range 3.6–232.5; p < 0.005). In addition, the median VILIP-1 concentration remained significantly different when TBI subjects were separated by severity: controls (9.5 pg/mL, range 0.0–39.3) versus mild TBI (83.0 pg/mL, range 3.7–189.2) versus moderate TBI (173.5 pg/mL; range 158.5–232.5, difference p < 0.005). Finally, there was a significant inverse correlation between Glasgow Coma Score (GCS) and VILIP-1 concentration (R = -0.625, p = 0.02).
Conclusions: The preliminary results of the TBI cohort in STABILISER demonstrate a significant association between elevated VILIP-1 levels within 8 hours of injury and TBI diagnosis. Further, there is an inverse correlation between biomarker concentration and GCS. These data support the VILIP-1 Lateral Flow Device as a diagnostic tool with high clinical potential.
PS05-050
Poster Viewing Session V
Repetitive anodal transcranial direct current stimulation (tDCS) improves neurologic outcome in mice after traumatic brain injury (TBI) by increasing cerebral blood flow and tissue oxygenation
O.A. Bragina1, Y. Yang1, C.W. Shuttleworth1, E.M. Nemoto1, O.V. Semyachkina-Glushkovskaya2 and D.E. Bragin1
1University of New Mexico, Albuquerque, United States
2Saratov State University, Saratov, Russian Federation
Abstract
Objectives: tDCS electrotherapy is being tested clinically for TBI but mechanisms and optimal stimulation parameters have not yet been determined. We examined the repetitive tDCS in a mouse model of TBI.
Methods: TBI was induced by controlled cortical impact; for sham-control craniotomy only. Repetitive tDCS (0.1 mA/15min) or Sham stimulation was done under anesthesia over 4 weeks for 4 consecutive days with 3-day intervals beginning 1 or 3 weeks after TBI. The anode was placed around the craniotomy and the counter electrode on the thorax. CBF was obtained pre and post stimulation by MRI. Cortical microvascular tone, cerebral blood flow (mCBF) and tissue oxygenation (NADH) were measured by two-photon microscopy (2PLSM). Rotarod, passive avoidance, Y-maze and novel object recognition were used to evaluate neurological recovery.
Results: TBI induced cortical and hippocampal lesions, CBF was 35 ± 8% lower in the ipsilateral hemisphere. tDCS increased CBF bilaterally in the cortex and hippocampi in both sham-control and TBI. In the peri-lesion cortex 2PLSM showed decreased arteriolar diameters, number of functioning capillaries, mCBF and tissue oxygenation. tDCS dilated arterioles, increased mCBF and tissue oxygenation in both sham-control and TBI. Motor function assessed by Rotarod was better in tDCS-stimulated mice compared to Sham-stimulated in both sham-control and TBI (P < 0.05). Step-through retention latency of tDCS-stimulated mice in Passive Avoidance was longer than that of Sham-stimulated mice (P < 0.5). In the Y-maze, tDCS-stimulated mice entered in to the new-opened arm more frequently compared to Sham-stimulated (P < 0.05).The difference between tDCS-stimulated mice and sham-stimulated in the novel object recognition was not significant (P = 0.39). The group in which stimulation started at three weeks after TBI recovered better than when stimulation started one week after TBI.
Conclusions: Repetitive tDCS improved motor and cognitive deficits in mice after TBI possibly involving vasodilation dependent increases in CBF and tissue oxygenation.
Support: NIH-NIGMS:P20GM109089-01A1
PS05-051
Poster Viewing Session V
Induced intracranial pressure (iPRx) and cerebrovascular reactivity (iCVRx) in the assessment of cerebral autoregulation after traumatic brain injury (TBI) with intracranial hypertension
D. Bragin1, G. Statom1 and E. Nemoto1
1University of New Mexico, Albuquerque, United States
Abstract
Objectives: High ICP in a healthy brain shifts capillary flow (CAP) to microvascular shunts (MVS) resulting in hypoxia, edema and blood brain barrier (BBB) damage (1). MVS flow was associated with loss of cerebrovascular autoregulation detected by induced ICP (iPRx) and cerebrovascular (iCVRx) reactivity (2). We hypothesized that iPRx and iCVRx correlate with MVS/CAP ratio after TBI with high ICP.
Materials: TBI was induced by fluid percussion in rats resulting in: I) No ICP increase; and II) 35 mmHg ICP increase. Using in vivo 2PLSM over the parietal cortex, microvascular cerebral blood flow (mCBF), hypoxia (NADH autofluoresence), and blood brain barrier (BBB) permeability (fluorescein extravasation) were measured before and during 4 hours after TBI. Doppler flux, temperatures, ICP and mean arterial pressure (MAP), blood gases and electrolytes were monitored. Every 30 minutes, a transient 10 mmHg MAP rise was induced by i.v. bolus of dopamine. iPRx = ΔICP/ΔMAP; iCVRx = ΔCBF/ΔMAP.
Results: TBI without high ICP showed reduced mCBF from 0.74 ± 0.09 at baseline to 0.48 ± 0.08 mm/s, increased hypoxia (154.5 ± 8.3%) and damaged BBB (ΔF/Fo = 9.15 ± 0.98, n = 10, p < 0.05). iPRx and iCVRx increased to 0.26 ± 0.02 and 0.37 ± 0.02, respectively (p < 0.05). TBI with increased ICP to 35.9 ± 4.4 mmHg (p < 0.05) showed a progressive rise in the MVS/CAP ratio from 0.41 ± 0.07 to 1.41 ± 0.32 (p < 0.01). Tissue hypoxia and BBB permeability increased to 172.8 ± 9.6% (NADH) and ΔF/Fo = 12.43 ± 1.02 (p < 0.01). iPRx and iCVRx increased to 0.35 ± 0.02 and 0.61 ± 0.03 from −0.02 ± 0.07 and −0.02 ± 0.05, respectively, reflecting impaired CBF autoregulation (p < 0.01).
Conclusions: iPRx and iCVRx revealed greater impaired CBF autoregulation after TBI with raised ICP.
References
1. Bragin et al, J Neurotrauma, 2011.
2. Bragin et al, Crit. Care Med., 2014.
PS05-052
Poster Viewing Session V
Interleukin 4 promotes white matter integrity and functional recovery after traumatic brain injury via enhanced microglia/macrophage M2 polarization
M. Bennett1,2, H. Pu2, Y. Shi2, Y. Xia2, X. Jiang2, Y. Gao2, X. Hu2,3 and J. Chen2,3
1Albert Einstein College of Medicine, Dominick P. Purpura Department of Neuroscience, Bronx, United States
2University of Pittsburgh, Pittsburgh Institute of Brain Disorders & Recovery, Pittsburgh, United States
3Veterans Affairs Pittsburgh Health Care System, Geriatric Research, Educational and Clinical Center, Pittsburgh, United States
Abstract
Objectives: White matter (WM) impairment contributes to long-term sensorimotor and cognitive deficits after traumatic brain injury (TBI). A persistent pro-inflammatory microenvironment hinders the differentiation of surviving precursor cells (OPCs) into mature oligodendrocytes essential for WM restoration. This study tested the hypothesis that interleukin 4 (IL-4) promotes microglia/macrophage polarization into the inflammation-resolving M2 phenotype and thereby enhances WM restoration and long-term neurological recovery after TBI.
Methods: Adult male C57/BL6 mice were randomly assigned to sham surgery or TBI induced by controlled cortical impact (CCI). Nanoparticle-encapsulated IL-4 was administered intranasally (50µg/kg) beginning 2h after CCI, and then daily for 14d. Sensorimotor and cognitive functions were assessed up to 35d after TBI. WM structural and functional integrity was evaluated by immunohistochemistry of myelin basic protein (MBP) in the cortex, striatum and corpus callosum (CC) and electrophysiological measurement of compound action potentials (CAPs) in the CC. Mechanistic studies were performed using both in vitro (primary OPC and microglia co-cultures) and in vivo (OPC-specific PPARγ conditional knockout mice) models.
Results: IL-4 facilitated the recovery of sensorimotor functions (rotarod and cylinder tests) and spatial learning and memory (Morris water maze) after TBI (n = 6/group, p < 0.05 vs. vehicle by ANOVA). IL-4 also promoted WM structural (increased MBP) and functional (increased CAP amplitude) integrity at 35d after TBI (n = 4–6/group, p < 0.001 vs. vehicle). In cultures, conditioned medium from IL-4-treated microglia facilitated OPC differentiation via PPARγ activation (n = 4, p < 0.001). In CCI mice, IL-4 enhanced microglia/macrophage M2 polarization and oligodendrocyte maturation. Moreover, IL-4-afforded WM protection, and beneficial effects on neurological recovery after TBI were greatlly diminished in OPC-specific PPARγ knockout mice (n = 8/group, p < 0.01 vs. wild-type).
Conclusions: IL-4 enhances microglia/macrophage M2 polarization, which promotes OPC differentiation and WM integrity via PPARγ signaling in OPC, thereby improving long-term TBI outcomes.
[IL-4 TBI mechanism]
PS05-053
Poster Viewing Session V
A rapid blood test for the early detection of mild traumatic brain injury
M.A. Bradley-Whitman1, K.N. Roberts1, S.W. Scheff1, J.F. Fraser2, B.C. Lynn3 and M.A. Lovell1
1University of Kentucky, Sanders Brown Center on Aging, Lexington, United States
2University of Kentucky, Kentucky Neuroscience Institute, Lexington, United States
3University of Kentucky, Department of Chemistry, Lexington, United States
Abstract
Objectives: Rapid diagnosis and triage of traumatic brain injury (TBI) can be vital in determining treatment pathway. We evaluated serum levels of visinin-like protein 1 (VILIP-1), a neuron-specific calcium sensor protein released into blood following injury, in a controlled cortical impact (CCI) model of TBI by Western Blot and by novel lateral flow device (LFD).
Methods: Serum was prepared from blood collected via tail nicks prior to and 4 h post-injury from young, male Sprague-Dawley rats in three groups: naïve, sham, or mild CCI (n = 6 per group). Samples were analyzed by Western Blot with antibodies raised against full-length VILIP-1, a C-terminal VILIP-1 peptide, and ubiquitin, and also by a novel LFD designed with antibodies raised against full-length VILIP-1 and a C-terminal VILIP-1 peptide.
Results: Western blot identified three distinct molecular weight (MW) bands immuno-positive for both VILIP-1 antibodies, as well as the ubiquitin antibody. Although there was no significant differences for any of the 3 distinct MW bands using the full-length VILIP-1 antibody 4 h post-injury, one distinct MW band was identified using the C-terminal VILIP-1 antibody that was significantly (p ≤ 0.05) elevated post-injury in mild CCI compared to sham and naïve animals. LFDs prepared with a combination of full-length and C-terminal VILIP antibodies demonstrated a pg/mL limit of detection with minimal sample input volume (10 µL) and a short analysis time (≤20 m). The measured LFD response was significantly elevated (p ≤ 0.05) post-injury in TBI compared to sham and naïve animals.
Conclusion: The current study demonstrates that our novel LFD rapidly detects a specific ubiquitin positive sub-species of VILIP-1 associated with TBI in a CCI model within a clinically relevant time frame. This combination of biomarker and rapid device detection could provide a vital tool in the diagnosis and management of mild TBI.
PS05-054
Poster Viewing Session V
Extended therapeutic window of a novel peptide inhibitor of TRPM2 channels on memory following traumatic brain injury
J. Orfila1, R. Dietz2, O. Patsos1, R. Schmidt3, R. Traystman1 and P. Herson1
1University of Colorado, Denver School of Medicine, Anesthesiology, Aurora, United States
2University of Colorado, Denver School of Medicine, Department of Pediatrics, Aurora, United States
3University of Colorado, Denver School of Medicine, Modern Human Anatomy, Aurora, United States
Abstract
Traumatic Brain Injury (TBI) is the leading cause of mortality and morbidity in adults, with significant sequelae including memory deficits. Despite intense research, no pharmacological interventions are currently available to improve outcome following TBI. Studies suggest that the non-selective transient receptor potential M2 (TRPM2) channels may contribute to brain injury, specifically in males. This study used stereology, electrophysiology and neurobehavior to assess the role of TRPM2 on memory impairments following TBI. Stereologic analysis of mice treated with a novel TRPM2 inhibitor peptide, tat-M2NX, did not reduce TBI-induced injury volume from a 2 mm controlled cortical impact when compared to tat-scrambled (tat-SCR) peptide. To assess the functional role of TRPM2 channels, tat-M2NX was administered intravenously 2 hours after TBI and contextual fear conditioning was performed to measure memory function in mice 7 days after TBI. Sham operated mice exhibited a 60.3 ± 5.1% (n = 8) freezing whereas TBI reduced freezing behavior (32.7 ± 6.2%, n = 6; p < 0.05). Administration of tatM2NX 2 hours after TBI resulted in improved memory function (62.3 ± 5.6%, n = 7) compared to tatSCR-treated mice (30.8 ± 10.9%, n = 5, p < 0.05). To assess the effect of TBI on hippocampal long-term potentiation (LTP), a well-accepted model of learning and memory, extracellular field recordings of CA1 neurons were performed in acute hippocampal slices prepared 7 days after TBI. Under control conditions, a physiological theta burst stimulation (40 pulses, 100Hz) resulted in LTP that increased the slope of fEPSP to 167.5 ± 12.4% (n = 5) of baseline. TBI injured mice had impaired LTP (123.1 ± 8.6%, n = 6, p < 0.05). However, mice treated with tat-M2NX preserved LTP (166.1 ± 1.9%, n = 2), consistent with improved memory function above. This study suggests that TRPM2 channels contribute to the functional memory impairment seen after TBI. Therefore, targeting TRPM2 channel activity may be a potential therapeutic approach to improve long-term functional outcome following TBI.
PS05-055
Poster Viewing Session V
The salutary effects of omega-3 fatty acids on cognition and tissue repair after traumatic brain injury in rats
L. Belayev1, L. Khoutorova1, L.M. Pizarro Cabral1, S. Marcell1, L. Cong1, R. Semikov1, A. Obenaus2 and N.G. Bazan1
1Louisiana State University Health Sciences Center, Neuroscience Center of Excellence, New Orleans, United States
2Loma Linda University, Loma Linda, United States
Abstract
Objectives: Traumatic brain injury (TBI) is characterized by cerebral damage leading to impairment of neurobehavioral function. Docosahexaenoic acid (DHA; 22:6n-3) is an omega-3 essential fatty acid family member and the precursor of neuroprotectin D1 (NPD1), a lipid mediator that downregulates apoptosis and promotes cell survival. DHA shows therapeutic potential in neurotrauma, however the effect of NPD1 was never studied in a TBI model. The purpose of this study was to determine whether treatment with DHA and NPD1 would be beneficial in a rat model of TBI.
Methods: Male SD rats were subjected to fluid-percussion injury. DHA and NPD1 (5 mg/kg) or saline treatment was administered i.v. at 1 h after TBI (n = 6 per group). Behavior was evaluated on days 1, 2, 3 and 7 after TBI. Ex vivo imaging of the brains was conducted on 11.7T MRI on day 7. T2WI and apparent diffusion coefficient (ADC) maps were generated. After completion of the MRI study on day 7, histopathology was conducted and the contusion areas, number of NeuN-, GFAP- and ED1-positive cells were quantitated in anterior and posterior areas next to the lesion.
Results: Treatment with DHA and NPD1 significantly improved the neurological score compared to saline on day 3 (by 33 or 42%) and day 7 (by 35 or 33%, respectively). Total lesion volumes computed from T2WI were dramatically reduced by DHA treatment compared to saline (by 73%). Histopathology revealed that lesion areas were significantly smaller in DHA- and NPD1-treated rats compared to the saline group, and lesion volume was reduced by 78%. In addition, DHA administration decreased ED1-positive microglia/microphages and increased GFAP-positive astrocytes and NeuN-positive neurons.
Conclusion: These results suggest that DHA and NPD1 therapy is neuroprotective in rats following TBI and might provide the basis for future therapeutics in patients suffering from TBI.
PS05-056
Poster Viewing Session V
Sex impacts outcome after traumatic brain injury in mice
A. Clevenger1, H. Kim2, E. Salcedo3, K. Rodgers4, J. Yonchek4, J. Orfila4, P. Herson5 and R. Traystman5
1University of Colorado, Denver, Pediatrics, Aurora, United States
2Chungbuk National University Hospital, Emergency Medicine, Cheongju, Korea, Republic of
3University of Colorado, Denver, Cell and Developmental Biology, Aurora, United States
4University of Colorado, Denver, Anesthesiology, Aurora, United States
5University of Colorado, Denver, Anesthesiology & Pharmacology, Aurora, United States
Abstract
The role of sex in short-term and long-term changes after traumatic brain injury (TBI) remains controversial, but sex differences seen in some clinical studies suggest that sex steroids may play a role in outcome. We used the controlled cortical impact (CCI) model of TBI in mice to help evaluate sex differences. Adult male and female C57Bl/6 mice (8–12 weeks) were exposed to CCI under isoflurane anesthesia. A subset of female mice underwent ovariectomy (OVX) prior to CCI and were recovered for seven days. Seven days after injury, animals underwent repeat behavioral testing and were then perfused for tissue collection. H&E-stained tissue sections were analyzed stereologically and injury volume calculated. Immunohistochemistry was additionally performed using Iba-1 and glial fibrillary acidic protein (GFAP) to analyze changes in microglia and reactive astrocytes, respectively. Differences between groups were analyzed using 2-way ANOVAs and Tukey multiple comparisons. Adult females exhibit histological protection (3.7 ± 0.5 mm3) compared to male mice (6.8 ± 0.6 mm3) and OVX females showed increased injury compared to intact females. Consistent with histology, sensorimotor deficits measured as reduced contralateral limb use was most pronounced in male mice (31.9 ± 6.9% reduced limb use) compared to 12.7 ± 3.8% in female mice. Ovariectomized mice demonstrated behavioral deficits similar to males (3.15 ± 3.9%). Upon immunohistochemical analysis, ovariectomized females demonstrated increased Iba-1 i compared to females in both the peri-injury cortex and the reticular thalamic nucleus. Ovariectomized females also demonstrated increased GFAP in comparison to both females and males in the peri-injury cortex. These data indicate that female sex steroids reduce brain sensitivity to TBI and that reduced acute neuroinflammation may contribute to the relative protection observed in females.
PS05-057
Poster Viewing Session V
Resuscitation fluid with drag reducing polymer enhances cerebral microcirculation after traumatic brain injury complicated by hemorrhagic shock
D. Bragin1, D. Lara1, O. Bragina1, Y. Yang1, M. Kameneva2 and E. Nemoto1
1University of New Mexico, Albuquerque, United States
2University of Pittsburgh, Pittsburgh, United States
Abstract
Objectives: Traumatic brain injury (TBI) is frequently accompanied by hemorrhagic shock (HS) and arterial hypotension significantly worsening morbidity and mortality. Existing resuscitation fluids (RF) for volume expansion do not adequately mitigate impaired microvascular cerebral blood flow (mCBF). We hypothesized that nanomolar quantities of drag reducing polymers in resuscitation fluid (DRP-RF), would improve mCBF by rheological modulation of hemodynamics. Due to viscoelasticity, DRP reduce blood flow microvortices and separations at vessel bifurcations resulting in a reduction in the pressure gradient across arterioles thereby increasing precapillary pressure and the number of erythrocytes entering capillaries thus reducing capillary stasis.
Methods: TBI was induced in rats by fluid percussion (1.5 ATA, 50 ms) followed by a controlled hemorrhage to a mean arterial pressure (MAP) = 40 mmHg. DRP-RF or RF was infused to MAP = 60 mmHg for one hour (pre-hospital), followed by blood re-infusion to MAP = 70 mmHg (hospital). Temperatures, MAP, blood gases and electrolytes were monitored. In vivo 2-photon microscopy over the parietal cortex was used to monitor microvascular blood flow, hypoxia (NADH) and necrosis (i.v. propidium iodide) for 5 hours after TBI/HS followed by MRI for CBF and lesion volume.
Results: TBI/HS compromised brain microvascular flow leading to capillary microthrombosis, tissue hypoxia and neuronal necrosis. DRP-RF reduced microthrombosis, restored collapsed capillary flow and improved mCBF (82 ± 9.7% vs. 62 ± 9.7%, DRP-RF vs. RF, respectively from pre-TBI baseline, p < 0.05, n = 10). DRP-RF better decreased tissue hypoxia (77 ± 8.2% vs. 60 ± 10.5%, DRP-RF vs. RF, respectively from baseline, p < 0.05), and neuronal necrosis (21 ± 7.2% vs. 36 ± 7.3%, respectively, p < 0.05). MRI revealed better CBF improvement and reduced lesion volumes in the DRP-RF group.
Conclusions: Rheological modulation of blood flow using DRP-RF effectively restores mCBF, reduces hypoxia and protects neurons compared to conventional volume expansion with RF after TBI/HS.
PS05-058
Poster Viewing Session V
Glutamate and D-serine dynamics during trauma-induced cortical spreading depolarizations
B. Balanca1, T. Lieutaud1, A. Meiller1 and S. Marinesco1
1Universite de Lyon, CRNL Team TIGER, Lyon, France
Abstract
Cortical spreading depolarizations (SDs) are waves of massive depolarization travelling through the cortex at a speed of 2–3 mm/min. They are observed after severe brain injury in humans and animals and associated with poor outcome. N-methyl-D-aspartate receptors (NMDAR) are involved in SD induction and/or propagation, and excitotoxic NMDAR overactivation could play an important role in neuronal injury following severe traumatic brain injury (TBI). However monitoring glutamate extracellular concentration remains a difficult technical challenge and the range of glutamate elevation during SDs remains a matter of debate. In addition to glutamate, D-serine is another endogenous NMDAR agonist and its concentrations during SDs and after TBI remain unknown. Here, we monitored glutamate and D-serine dynamics in the cortex of male Wistar rats subjected to severe (3.8 atmosphere) lateral fluid percussion. We used enzyme microelectrode biosensors with an external diameter of 40 µm that are known to preserve blood-brain barrier integrity. In addition, cortical perfusion was assessed by laser Doppler flowmetry (LDF) and local field potentials were monitored in direct current (DC) mode to detect SDs. SDs occurred spontaneously after severe TBI at a rate of about 2 SDs in the first 5h following TBI, and were associated with a hyperemic response. The extracellular glutamate and D-serine concentrations increased in response to SDs. However, their basal extracellular level between SDs remained at near-physiological levels: below 1 µM for glutamate and around 2–3 µM for D-serine. These data indicate that NMDAR overactivation after severe TBI does not appear to result from a tonic elevation of basal glutamate and/or D-serine. Instead, elevations in glutamate and D-serine extracellular concentrations appear limited to times and areas affected by the passage of an SD. These findings suggest that excitotoxic damage following severe TBI is mainly inflicted by SDs.
PS05-059
Poster Viewing Session V
Measuring exercise-induced cerebrovascular changes in Huntington's disease using arterial spin labelling (ASL) fMRI
J. Steventon1, H. Furby1, J. Ralph1, P. O'Callaghan2, A. Rosser3, M. Busse4 and K. Murphy5
1Cardiff University, CUBRIC, School of Psychology, Cardiff, United Kingdom
2Cardiac Services, University Hospital of Wales (UHW), Cardiff, United Kingdom
3Cardiff University, Psychological Medicine, Cardiff, United Kingdom
4Cardiff University, Centre for Trials Research, Cardiff, United Kingdom
5Cardiff University, School of Physics and Astronomy, Cardiff, United Kingdom
Abstract
Objectives: Exercise is potentially therapeutic via vascular adaptations (angiogenesis, improved cerebral perfusion and metabolism) however the underlying dynamics are not fully understood. In Huntington's disease (HD), cerebral vasculature alterations have been reported. Here we use arterial spin labelling (ASL) to examine the acute effect of aerobic exercise on the cerebrovasculature in HD patients.
Methods: 18 HD patients (11 males, 45.0 ± 8.5 years old) and 18 healthy age-matched controls underwent a baseline MRI scan (3T GE HDx system) which included a multi-inversion time ASL sequence (TIs: 400, 500, 600, 700, 1100, 1400, 1700, 2000 ms, TE = 0.27ms; slice delay = 52 ms, QUIPSS II cutoff TI > 700 ms) to measure cerebral blood flow (CBF, ml / 100 g/min) and a structural FSPGR (1 mm3 resolution). Participants then completed 20-minutes of moderate intensity aerobic exercise on a cycle ergometer. Immediately after, participants underwent a repeat MRI session with the ASL sequence occurring 15 minutes after exercise cessation. Perfusion quantification was performed on a voxel-by-voxel basis using a two-compartment model1 with partial volume correction. Median grey matter CBF values were compared pre- and post-exercise using a repeated-measure ANOVA.
Results: No difference in CBF between HD and control participants pre- or post-exercise was found (Fig1a). However at both time points CBF was significantly correlated with CAG repeat length (∼genetic mutation load) in HD (Fig1b,d, n = 13). The absolute exercise-induced change in CBF was negatively related to CAG repeat length (Fig 1f). The partial volume fraction of grey matter was significantly lower in HD participants (Fig1c), however this fraction was not related to CAG repeat length (p > 0.05).
[Cerebrovascular response to exercise in HD]
Conclusions: We demonstrate that genetic load in HD affects baseline CBF and exercise-induced CBF change. Importantly, the relationship between genetic load and CBF does not appear to be driven by atrophy-derived partial volume effects.
PS05-060
Poster Viewing Session V
Investigating temporal characteristics of atrophy, amyloid deposition and quantitative magnetization transfer parameters in mild cognitive impairment and Alzheimers disease
B.A. Thomas1, M.C. Stephenson1, C.K. Wong1, T.Y. Wong2, C. Cheung3, N. Venketasubramanian2, J.J. Totman1, D.W. Townsend1 and C. Chen4
1Agency for Science Technology and Research - National University of Singapore, Clinical Imaging Research Centre, Singapore, Singapore
2National University of Singapore, Singapore Eye Research Institute, Singapore, Singapore
3The Chinese University of Hong Kong, Opthalmology and Visual Science, Hong Kong, Hong Kong
4National University of Singapore, Memory, Ageing and Cognition Centre, Singapore, Singapore
Abstract
Objectives: The mechanisms leading to neurodegeneration in Alzheimer's Disease (AD) remain unclear. In this study we investigate markers of amyloid deposition, atrophy, and metabolism in patients with mild cognitive impairment (MCI) and AD, to aid understanding of the pathway to neurodegeneration.
Methods: 8 patients with probable AD, 29 with MCI and four age-matched controls underwent cognitive testing (MMSE) and were scanned using a Siemens 3T Biograph mMR system following infusion of 370 MBq of 11C-Pittburgh Compound-B (PIB). PET data was acquired 40–70 minutes post-injection. MR data was acquired simultaneously and included a T1-weighted magnetization prepared (MPRAGE) image and Quantitative Magnetization Transfer (qMT) images, for measurement of kinetic parameters of proton exchange between macromolecules and water.
Anatomical images were analysed using Freesurfer. Mean qMT and PET SUVR (relative to cerebellar grey matter) values were calculated from Freesurfer parcellations of hippocampus, temporal lobes, posterior cingulate (PCC), frontal, and occipital cortex.
Values were correlated with MMSE to assess disease effects. T-tests were then used to identify early changes between groups (stratified by MMSE score).
Results: Brain volumetrics were significantly correlated with MMSE scores in hippocampus, temporal lobes and PCC, but not in parietal and occipital cortex. Amyloid content was inversely correlated with MMSE score in temporal lobes, PCC, parietal and occipital cortex, but not hippocampus. qMT parameters were correlated with MMSE in PCC (T1a and T2a, the relaxation parameters of the free pools).
Group differences indicate changes occur at an early stage for temporal volumes (p = 0.02) and T2a in PCC (p = 0.05) with significant amyloid deposition occurring later.
Conclusion: This study indicates that early qMT changes and reduced temporal volumes can predict decreases in cognition in early MCI. These appear to precede amloid deposition.
References
[1] Sinclair, C.D.J. et al; Quantitative Magnetization Transfer in In vivo Healthy Human Skeletal Muscle, MRM (2010).
PS05-061
Poster Viewing Session V
Efficient clearance of Amyloid-β protofibrils in APP-transgenic mice treated with a brain penetrating bifunctional antibody
D. Sehlin1, G. Hultqvist1, A. Gumucio1, H. Laudon2, L. Söderberg2, L. Lannfelt1 and S. Syvänen1
1Uppsala University, Public Health and Caring Sciences, Uppsala, Sweden
2BioArctic AB, Biochemistry, Stockholm, Sweden
Abstract
Objectives: Amyloid-β (Aβ) immunotherapy is one of the most promising strategies to battle Alzheimer's disease (AD). Despite recent progress targeting aggregated Aβ [1], low antibody brain penentrance remains a challenge. Here we used transferrin receptor (TfR) mediated transcytosis to facilitate brain uptake of mAb158 [2–3], recombinantly fused to a fragment of TfR antibody 8D3 [4], with the aim to increase the efficacy of immunotherpy targeting soluble Aβ protofibrils.
[Transcytosis]
Methods: Four groups of 14 month old APP-transgenic mice (tg-ArcSwe [3]) were intravenously injected with a single dose of either:
· low dose of mAb158-8D3 fusion protein (equivalent to 5 mg/kg of mAb158)
· low dose of mAb158 (5 mg/kg)
· high dose of mAb158 (50 mg/kg)
· placebo (PBS)
Three days after injection, mice were saline perfused and brains were isolated for Aβ ELISA and immunohistochemistry analyses.
Results: ELISA analysis in TBS brain extracts demonstrated a 40% reduction of Aβ protofibril levels in tg-ArcSwe mice treated with the bispecific mAb158-8D3 in comparison with placebo. The same dose (5 mg/kg) of unmodified mAb158 did not have any effect whereas the high dose (50 mg/kg) displayed a similar reduction as mAb158-8D3. Total Aβ burden was unaffected by this short term treatment.
Conclusions: By engineering mAb158 to be actively transported into the brain, we demonstrate a rapid and specific clearance of soluble Aβ aggregates with only 10% of the dose required for unmodified mAb158. This strategy could dramatically increase the efficiency of Aβ immunotherapy, thereby also reducing side effects and treatment cost.
2. Englund H, et al. J Neurochem 2007. 103(1):334–45
3. Sehlin D, et al. Nat Commun 2016 Feb 19;7:10759
4. Kissel K, et al. Histochem Cell Biol. 1998 Jul;110(1):63–72
PS05-062
Poster Viewing Session V
Gut microbiome associated with cognitive and brain structural outcomes in apolipoprotein E4 variant
I. Parikh1,2, V. Bakshi1, J. Hoffman1,2, S. Green3, A. Hartz1,4 and A.-L. Lin1,2
1University of Kentucky, Sanders Brown Center on Aging, Lexington, United States
2University of Kentucky, Pharmacology and Nutritional Sciences Center, Lexington, United States
3University of Illinois at Chicago, Chicago, United States
4University of Kentucky, Molecular and Biomedical Pharmacology, Lexington, United States
Abstract
Alzheimer's Disease (AD) is the most common cause of dementia. The cause of sporadic AD has genetic, as well as environmental, components. A primary genetic risk factor for AD is the alleles of apolipoprotein E (APOE). The three most common APOE variants are E2, E3, and E4; one copy of E4 increases AD risk by 4 fold and two copies by 12-fold. Gut microbiome can be an integral accessory to the human genome. Pronounced differences in microbial diversity can shape the brain through the gut-brain axis. We studied the gut microbiome of mice expressing E3 and E4 variants. Our results show that expression of E4 allele compared to E3 allele, resulted in significantly different microbiotic diversity (P < 1 × 10−5 R = 0.3632, ANOSIM) and composition (p < 4 × 10−4, Mann-Whitney test) in the gut, with E4 mice characterized by significant changes in Actinobacteria, Bacteroidetes, Cyanobacteria and Firmicutes. Furthermore, previous studies have shown increased microbiota from Firmicutes phyla alters blood brain barrier (BBB) permeability. In our study, presence of E4 allele led to increased errors in radial arm water maze test (p < 0.01) and decreased expression of P-gp, a marker for BBB permeability. In conclusion, our study suggests there are microbiome composition dissimilarities between APOE genotypes and that presence of E4 allele is associated with cognitive deficits and decreased BBB integrity. These microbiota differences provide novel insights into differential functions of APOE genetics' influence on microbiome that may modulate AD.
PS05-063
Poster Viewing Session V
Glucose and glycogen metabolism in the brain of insulin resistant Goto-Kakizaki rats
A.F. Soares1, S.S. Nussbaum1, R. Gruetter1,2,3 and J.M.N. Duarte1
1École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
2University of Lausanne, Lausanne, Switzerland
3University of Geneva, Geneva, Switzerland
Abstract
Objectives: Diabetes impacts the central nervous system predisposing to cognitive decline. While glucose is the main source of energy fuelling the adult brain, brain glycogen is necessary for adequate neuronal function, for synaptic plasticity, and for learning and memory. We investigated diabetes-induced alterations of glycogen metabolism in the living brain by means of magnetic resonance spectroscopy (MRS).
Methods: MRS experiments in vivo were performed on a 14.1 T spectrometer using a home-built surface coil. [1-13C]glucose was infused into adult Wistar and insulin-resistant Goto-Kakizaki (GK) rats under isoflurane anaesthesia. Localised 13C MRS was performed with a modified SIRENE pulse sequence to measure brain glucose and glycogen signals over 8 hours. Then, rats were sacrificed with a focused microwave fixation device, and fractional enrichment (FE) and content of glucose and glycogen were determined in brain extracts. Time courses of glucose and glycogen 13C labelling measured in vivo and FE and concentration determined in brain extracts served to determine kinetic parameters of glucose homeostasis and glycogen turnover.
Results: Brain glucose uptake and the cerebral metabolic rate of glucose were similar in both experimental groups. Glycogen was 5.5 ± 0.9 µmol/g in controls and 5.0 ± 0.4 µmol/g in diabetic rats. However, FE of brain glycogen was lower in GK rats than in Wistar rats, suggesting that insulin resistance reduces 13C incorporation from glucose. Indeed, glycogen labelling from [1-13C]glucose occurred at 0.48 ± 0.09 µmol/g/h in controls and 0.24 ± 0.05 µmol/g/h in GK rats, representing turnovers of 15 ± 2 h and 26 ± 5 h (P = 0.039), respectively.
Conclusions: These study demonstrates impaired brain glycogen metabolism in insulin resistance despite normal brain glycogen content, suggesting that mobilisation of available glycogen stores in astrocytes is impaired in diabetes. This may have implications for the adequate support of neuronal function, especially during increased brain activity or during reduced energy availability such as hypoglycaemia.
PS05-064
Poster Viewing Session V
Evaluation of higher cortical functions of Alzheimer's disease
and M. Salohiddinov1
1Tashkent Medical Academy, Neurology, Tashkent, Uzbekistan
Abstract
Purpose: Evaluate the effectiveness of diagnostic disorders of higher cortical functions in patients with Alzheimer's disease using scales GDR (Global Deterioration Rating) and MMSE (MiniMentalScaleExamination).
Material and Methods: Study involved 52 patients (32 men and 20 women) aged 65 to 77 years (mean age 70,8 ± 3,3 years) divided into 2 groups: group 1 (basic group) - Alzheimer's disease 45 patients, group 2 (comparison group) - 27 patients with chronic cerebral ischemia (II- IIIst.) with mild cognitive impairment. GDR and MMSE were used for assessing the severityof cognitive impairment.
Results: These neuropsychological studies indicate the results of cognitive functions: in group I - GDR 5,0 ± 0,5 scores, MMSE 21,8 ± 4,05 scores. In group II - GDR 2,0 ± 0,5 scores, MMSE 27,0 ± 0,5 scores. Senile dementia develops in commonly characterized by a relatively sparse confabulation product. Confabulation shifted to a more or less distant past ideas about the environmental situation and the self (amnestic confabulation). At the stage of mild dementia clearly identified the most features of amnestic aphasia, amnestic disorder component of praxis, and in some cases, signs of constructive dyspraxia. There is a long preservation of motor component of praxis.
Conclusions: The total scores on the MMSE and the GDR is a sensitive indicator of cognitive deficits and higher cortical functions of mild to moderate Alzheimer's diseasebefore, is effective in determining therapeutic approaches and tactics of early prevention in patients with Alzheimer's disease.
2. Todd S, Barr S, Roberts M. “Survival in dementia and predictors of mortality”
3. Forbes D, Thiessen EJ, Blake CM, Forbes SC, “Exercise programs for people with dementia”
PS05-065
Poster Viewing Session V
PET CT applications in Parkinson and radiotracers
and J.M. Isusi1
1Hospital Angeles del Pedregal, Neuroradiologia y PET CT, Mexico, Mexico
Abstract
Between 15 and 25% of diagnoses indicative of Parkinson's disease have proven over time to be wrong. Multiple system atrophy, entity in which the atrophy or degeneration striatonigral, olivopontocerebellar atrophy and Shy Drager syndrome included are an example of this.
To characterize the degree of presynaptic degeneration has been used primarily 18F-dopa and 11C-DOPA.
By using these tracers it is possible to measure enzyme activity dopa decarboxylase, which converts dopa to dopamine level axons synapse with the neostriatum system.
Another method for determining the degree of presynaptic degeneration is the use of:
•11C-β-CIT76,
•S-11C-nomifensina77 and
•11C-dihydrotetrabenazin78.
These substances demonstrate affinity for monoamine reuptake (among which there are dopamine) located in the synaptic terminals of neurons of the substantia nigra.
In combination with 11C-DOPA is for the moment, FDG, the best clinical differential diagnosis method. In cases of dystonia it is possible to observe a change in the balance of production of dopamine which can be very high in the ventral parts of the putamen and caudate in
PS05-067
Poster Viewing Session V
Influence of diabetes mellitus on CSF and brain characteristics
E. Schmidt1, Z. Czosnyka2 and L. Balardy3
1University Hospital, Neurosurgery, Toulouse, France
2Brain Physic Lab, Neurosurgery, Cambridge, United Kingdom
3University Hospital, Geriatry, Toulouse, France
Abstract
Introduction: Diabetes mellitus (DM) represents a major cause of morbidity and mortality worldwide. DM is an independent risk factor for cerebrovascular disease. DM is associated with an increase in the risk of brain atrophy and dementia. The WHO diagnostic criteria for DM is fasting plasma glucose ≥7.0 mmol/L. As well, glycated haemoglobin (HbA1c) reflects average plasma glucose over the previous 2–3 months. An HbA1c of 6.5% is recommended as the cut off point for diagnosing diabetes. DM is considered as a co morbidity of normal pressure hydrocephalus (NPH). We hypothesize that DM has an influence on ICP, CSF circulation and brain biophysical characteristics.
Methods: In a prospective cohort of 91 patients suspected of NPH, we performed lumbar infusion tests and measured at the same time plasma glucose and HbA1C. To understand the influence of DM on ICP and CSF/brain characteristics we dichotomized our cohort into 2 sub-groups: one diabetic and one non-diabetic. According the WHO, we characterized our 2 sub-groups with glycemia and HbA1C: patients with normal glycemia (i.e.<7.0 mmol/L) or high glycemia (i.e. ≥7.0 mmol/L) and patients with normal HbA1c (i.e.<6.5%) or high HbA1c (i.e. ≥6.5%). Then we compared the data in the diabetic and non-diabetic subgroups with Student test.
Results: The table 1 gives mean ± sd values of various parameters in each diabetic and non-diabetic subgroup.
Conclusions: Our data suggest that patients with DM have stiffer brain with less CSF production rate and more resistive CSF outflow.
PS05-068
Poster Viewing Session V
Arterial pressure, heart rate, and cerebral hemodynamics across the adult life span
C. Xing1,2,3, T. Tarumi1,4, R. Meijers1, L. Yuan3 and R. Zhang1,2,4
1Institute for Exercise and Environmental Medicine, Dallas, United States
2University of Texas Southwestern Medical Center, Department of Internal Medicine, Dallas, United States
3Tangdu Hospital, Fourth Military Medical University, Department of Ultrasound Diagnostics, Xi’an, China
4University of Texas Southwestern Medical Center, Department of Neurology and Neurotherapeutics, Dallas, United States
Abstract
Objectives: Aging is the strongest risk factor for cardio- and cerebrovascular disease; however, the underlying pathophysiological mechanism(s) remains poorly understood. The purpose of this study is to characterize age-related alterations in systemic and cerebral hemodynamics and the underlying regulatory mechanisms, particularly cardiac baroreflex function and dynamic cerebral autoregulation (dCA).
Methods: We studied 136 healthy adults aged 21–80 years (60% women). Beat-to-beat blood pressure (BP), heart rate (HR), and cerebral blood flow (CBF) velocity were measured at rest and during sit-stand maneuvers to mimic postural changes in daily life. Transfer function analysis was used to assess baroreflex sensitivity (BRS) and dCA. Central arterial stiffness was assessed with carotid-femoral pulse wave velocity (cfPWV).
Results: Aging was associated with significant elevations in cfPWV, systolic BP, pulse pressure, cerebrovascular resistance and CBF pulsatility, but reductions in mean CBF velocity. Compared to young and middle-aged subjects, older adults had lower beat-to-beat BP, HR and CBF velocity variability under resting conditions, but higher BP and CBF variability during sit-stand maneuvers. BRS was reduced while dCA transfer function gain was elevated in older adults. Multiple linear regression analysis indicated that systolic BP variability is positively correlated with cfPWV independent of HR variability.
Conclusions: Aging is associated with significant alterations in both systemic and cerebral hemodynamics which are closely related to each other. The augmented BP and CBF variability in older adults during sit-stand maneuvers reflect diminished cardio- and cerebrovascular regulatory capability with aging.
PS05-069
Poster Viewing Session V
Rejuvenation of microglia in the aged central nervous system
E. Koellhoffer1,2, J. d'Aigle1, M. Howe1,2, D. Morales-Scheihing1 and L. McCullough1,2,3
1McGovern Medical School, Neurology, Houston, United States
2The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, United States
3Mischer Neuroscience Institute, Houston, United States
Abstract
Microglia, the resident innate immune cells of the central nervous system (CNS), play a crucial role in maintaining homeostasis. However, microglia of the aged CNS demonstrate decreased mobility, reduced phagocytosis of debris, and a primed phenotype. Understanding the molecular mechanisms that underlie the aging process of microglia will allow us to identify the factors responsible for this transition in phenotype with age and offer novel targets for interventional therapies. Our lab has previously shown that two histone modifiers, Jmjd3 and Ezh2, antagonize each other in microglia polarization. Ezh2 is associated with H3K27me3 and pro-inflammatory polarization, while Jmjd3 demethylates this mark to H3K27me1 and is essential for anti-inflammatory polarization. We hypothesized that the transition of microglia to an aged phenotype is influenced by the periphery through epigenetic mechanisms and is reversible. We show that the aged brain has a higher H3K27me3:H3K27me1 ratio compared to young mice. Primary microglia stimulated with LPS+IFNγ resulted in increased global levels of H3K27me3. Inhibition of Ezh2 abrogated H3K27me3 deposition and up-regulation of pro-inflammatory genes. To determine if peripheral factors are responsible for the epigenetic dysregulation in the aged CNS and if these changes are reversible, we utilized a model of heterochronic parabiosis. In this model a young and aged mouse are surgically attached and come to share a common blood supply. Western blot analysis indicates that these epigenetic perturbations observed with age are reversible, with a rejuvenation effect of the H3K27me3:H3K27me1 ratio in aged heterochronic mice (an aged mouse attached to a young mouse) compared to aged isochronic surgical controls. Together, our results suggest that Ezh2 and the H3K27me3 mark are central to the pro-inflammatory polarization of microglia. This epigenetic imbalance in favor of higher H3K27me3:H3K27me1 with aging is reversible through heterochronic parabiosis, suggesting that peripheral factors are responsible for these changes observed with age.
PS05-070
Poster Viewing Session V
Heart-brain coupling: Resting heart rate variability is associated with network architecture in the resting brain
D. Kumral1,2, F. Beyer1, D. Husser3, M.L. Schroeter1,3, M. Loeffler3, V. Witte1,4, A. Villringer1,2,3 and M. Gaebler1,2,3
1Max Planck Institute for Human Cognitive and Brain Sciences, Neurology, Leipzig, Germany
2Humboldt University Berlin, Berlin School of Mind & Brain, Berlin, Germany
3Leipzig University, Leipzig Research Center for Civilization Diseases, Leipzig, Germany
4Leipzig University, Faculty of Medicine, Collaborative Research Centre, “Obesity Mechanisms”, Leipzig, Germany
Abstract
Heart rate variability (HRV) is an index for parasympathetic cardioregulation. Resting HRV is an individual trait marker and has been associated with physiological and psychological well-being (Thayer et al., 2012). The aim of this study was to investigate the relationship between HRV as a proxy for parasympathetic cardioregulation and brain connectivity over the adult lifespan. While temporal fluctuations in HRV and RSFC have been associated in previous studies (Chang et al., NeuroImage 2013), we treated average values of these measures as trait markers. We used two data sets: Sample 1: 273 healthy subjects (young: 28 ± 4 y, middle: 47 ± 6 y, old: 67 ± 5 y) from a large cohort study in Leipzig (Löffler et al., BMC Pub Health 2015) and for confirmatory analysis (Sample 2) 53 young subjects (24 ± 3 y) from an independent dataset in whom ECG and resting state fMRI were available. We calculated the root mean square of successive differences (RMSSD) of inter beat intervals as an index of trait HRV. RSFC was analyzed using eigenvector centrality mapping that captures neural connectivity at a voxel-level (Lohmann et al., PLOS One 2010). In Sample 1, HRV strongly decreased with age. Higher HRV was associated with increased centrality in right posterior cingulate cortex (PCC) in all age groups and in bilateral ventromedial prefrontal cortex (vmPFC) in young subjects only. In sample 2, we confirmed the vmPFC finding in young subjects (r = 0.24, p < 0.05). These regions have previously been associated with fluctuations in HRV and largely overlap with the default-mode network (Beissner et al., J Neurosci 2013); specifically vmPFC has been shown to modulate the vagal efferent outflow to the heart (Wong et al., NeuroImage 2007). Our finding that vmPFC only is related to HRV in young subjects supports the view that the well-known decrease of HRV with aging is related to attenuated central cardiovagal control.
[centrality in young subjects related with HRV]
PS05-071
Poster Viewing Session V
IGF-1 deficiency exacerbates hypertension-induced cerebral microhemorrhages in mice, mimicking the aging phenotype
S. Tarantini1, M.N. Valcarcel-Ares1, A. Yabluchanskiy1, Z. Springo1, G.A. Fulop1, N. Ashpole2, T. Gautam1, C. Giles1, J.D. Wren1, W.E. Sonntag1, A. Csiszar1 and Z. Ungvari1
1University of Oklahoma Health Sciences Center, Reynolds Oklahoma Center on Aging Research, Oklahoma City, United States
2University of Mississippi, Oxford, United States
Abstract
Clinical and experimental studies show that aging exacerbates hypertension-induced cerebral microhemorrhages (CMHs), which progressively impair neuronal function. There is growing evidence that aging promotes IGF-1 deficiency, which compromises multiple aspects of cerebromicrovascular and brain health. To determine the role of IGF-1 deficiency in the pathogenesis of CMHs, we induced hypertension in mice with liver-specific knockdown of IGF-1 (Igf1f/f + TBG-Cre-AAV8) and control mice by angiotensin-II plus L-NAME treatment. In IGF-1 deficient mice the same level of hypertension led to significantly earlier onset and increased incidence and neurological consequences of CMHs, as compared to control mice, as shown by neurological examination, gait analysis and histological assessment of CMHs in serial brain sections. Previous studies showed that in aging increased oxidative stress-mediated MMP activation importantly contributes to the pathogenesis of CMHs. Thus, it is significant that hypertension-induced cerebrovascular oxidative stress and MMP activation were increased in IGF-1 deficient mice. We found that IGF-1 deficiency, by impairing hypertension-induced adaptive media hypertrophy and extracellular matrix remodeling, also perturbs arteriolar remodeling processes, which act to restore circumferential stress, preventing mechanical damage to the vascular wall. Collectively, IGF-1 deficiency promotes the pathogenesis CMHs (6.2 ± 3 bleeds/mouse in control vs. 22.3 ± 7 bleeds/mouse in IGF-1 deficient mice), mimicking the aging phenotype, which likely contribute to its deleterious effect on cognitive function. Therapeutic strategies that up-regulate IGF-1 signaling in the cerebral vessels and/or reduce microvascular oxidative stress and MMP activation may be useful for the prevention of CMHs, protecting neurocognitive function in high-risk elderly patients.
PS05-072
Poster Viewing Session V
Blood flow through the cerebral microcirculation exhibits wide variability of hemodynamic states
I. Gould1 and A. Linninger1
1University of Illinois at Chicago, Bioengineering, Chicago, United States
Abstract
Objectives: Biochemical signaling between the cerebral capillary bed and the cerebral tissue regarding oxygen supply is critical for normal brain function as well as disease states including ischemia, micro-infarcts, and functional hyperemia. The cortical angioarchitecture is a key factor in controlling cerebral blood flow and oxygen metabolism. We create a new methodology combining advanced microscopy with large scale simulations to quantify the effect of differing angioarchitecture. We propose a 2-dimensional simplification of the microcirculatory system to increase its utility in large-scale hemodynamic simulations.
Methods: High-resolution images of the mouse primary somatosensory cortex were combined with a computational model of cerebral perfusion and oxygen supply ranging from the pial vessels to individual brain cells. A biphasic non-Newtonian blood flow and oxygen distribution model was used. The mathematical model for hemodynamics, oxygen transport across the blood brain barrier and metabolism in the extravascular space was derived.
Results: Computational analysis of microcirculatory blood flow and pressure indicates arteriole-side capillaries (d < 10µm) are the largest contributor to hydraulic resistance. These simulations also suggest that a series of resistors is an inadequate model for cortical blood supply. Rather, flow through a conductive plate network best describes the microcirculatory blood flow patterns in the cerebral cortex. This conductive plate network gave reliable hydraulic resistance estimates and accurately predicted blood flow and pressure drops in the microcirculation.
Conclusions: Simulations of blood flow, hematocrit and oxygen tension show that the wide variation of hemodynamic states in the tortuous, randomly organized capillary bed is responsible for uniform cortical tissue perfusion and oxygenation. This supports that the commonly suggested serial resistance circuit is not apt to describe microcirculatory resistance and flow patterns and the conductive plate network can serve as an effective surrogate for predicting hemodynamics in the cortical microcirculation.
[Simulation Results of Mouse Microvasculature]
PS05-073
Poster Viewing Session V
Linking macro-scale voxel behaviour to the microvasculature using a multi-scale model
W.K. El-Bouri1 and S.J. Payne1
1University of Oxford, Institute of Biomedical Engineering, Oxford, United Kingdom
Abstract
The aim of this work is to quantify the link between the topology of the microvasculature and the macro-scale perfusion seen in perfusion-based imaging. Perfusion-based imaging does not have a small enough resolution to pick out individual vessels of the microvasculature and hence the link between the micro-scale architecture and the macro-scale perfusion is not well understood. As a result, a microvascular model of an MRI-sized voxel is developed here in order to quantify this link.
We have previously developed statistically accurate models of the capillary bed and penetrating arteriolesvenules using data from imaged segments of the temporal lobe in the prefrontal cortex of humans. The capillary bed was mathematically homogenized and so can be treated as a porous medium, significantly reducing computation time. The penetrating vessels are treated as discrete vessels with 1-dimensional flow.
This work couples the capillary network with the model of the penetrating vessels in an MRI voxel-sized model (1 × 1 × 2.5 mm) - see figure. This is roughly the depth of the grey matter in the temporal lobe. A PDE/ODE solver is developed which treats the arterioles/venules as sources/sinks embedded in a porous medium that is the capillary bed. This solver then allows us to solve for blood and oxygen perfusion in a given voxel, and as a result allows us to see how the microvasculature affects macro-scale perfusion and metabolism.
The novel multi-scale model of the microvasculature developed here is very easily adaptable to different regions of the brain - in any region where the relevant statistical data are available. As more data become available, more of the microvasculature in the brain can be modelled accurately and we can therefore better quantify how the microvasculature affects brain perfusion and metabolism, and hence better understand the neurovascular coupling.
[Penetrating vessels embedded in a capillary bed]
PS05-074
Poster Viewing Session V
Decreasing cerebral tissue oxygen saturation during deep hypothermic circulatory arrest
J.M. Lynch1, C.D. Mavroudis2, T. Ko2, K. Mensah-Brown2, D.R. Busch2, S.C. Nicolson2, L.M. Montenegro2, S. Fuller2, T.L. Spray2, J.W. Gaynor2, A.G. Yodh3 and D.J. Licht2
1New York University School of Medicine, New York, United States
2The Children's Hospital of Philadelphia, Philadelphia, United States
3University of Pennsylvania, Philadelphia, United States
Abstract
Introduction: Approximately half of school-age survivors of complex congenital heart disease exhibit neurobehavioral symptoms, such as inattention, hyperactivity, and impaired executive function. Underlying these neurodevelopmental outcomes is the high prevalence of hypoxic-ischemic white matter brain injury seen in this patient population. Although the exact timing and mechanism of this injury are unknown, previous studies have shown duration of deep hypothermic circulation arrest (DHCA) to be a risk factor. In this study, we employed non-invasive optical spectroscopy to quantify cerebral tissue oxygen saturation (ScO2) during DHCA in a cohort of infants with complex CHD.
Methods: Term neonates (N = 7) with the following cardiac diagnoses were recruited: hypoplastic left heart syndrome (n = 4), interrupted aortic arch (n = 1), coarctation of the aorta (n = 1), and double inlet left ventricle (n = 1). A non-invasive optical probe was placed on the forehead for the duration of the surgery, and frequency-domain near-infrared spectroscopy (FD-NIRS) was employed to quantify ScO2 during DHCA.
Results: The average duration of DHCA in this cohort was 38.3 ± 12.2. The average initial ScO2 at the start of DHCA was 60.7 ± 12.3%. We observed a decrease in ScO2 during DHCA in all patients, with an average decrease of 39.0 ± 15.3%. A linear fit of the data yielded an average slope of −1.5 ± 0.8%/min.
Conclusions: Cerebral tissue oxygen saturation decreases during circulatory arrest, even under deep hypothermia. This trend is in agreement with previous studies that link the duration of DHCA with risk of hypoxic-ischemic injury.
PS05-075
Poster Viewing Session V
Protective effect of Rutin in experimental paradigms of STZ induced diabetic neuropathy
and R. Mittal1
1Panjab University, University Institute of Pharmaceutical Sciences, Chandigarh, India
Abstract
Introduction: Diabetes mellitus is a serious global health problem and diabetic neuropathy affects more than 50% of diabetic patients and is a major cause of disability. Rutin has been demonstrated in number of pharmacological activities including anti-diabetic, anti-oxidant and anti-inflammatory activities.
Material and methods: Streptozotocin (STZ, 55 mg/kg) dissolved in 0.1 M citrate buffer (pH 4.5) was administered i.p. Animals with blood glucose level more than 250g/dl are considered diabetic and are used for further studies.Rutin (100 and 200 mg/kg, i.p.) (Sigma-Aldrich, USA) and Ramipril (0.2 and 2.3 mg/kg, p.o.) (IPCA, Mumbai) was suspended in 0.25% sodium carboxy methyl cellulose. All the behavioural parameters (Measurement of body weight, Mechanical allodynia, Cold allodynia, Mechanical hyperalgesia, Thermal hyperalgesia) were performed on day 0, 2nd, 4th, 6th and 8th week. On last day (of 8th week), blood was collected retro-orbitally and mean nerve conduction velocity was assessed. The animals were then sacrificed sciatic nerves were isolated for further biochemical estimations (Lipid peroxidation, Nitrite estimation, Superoxide dismutase activity, reduced glutathione (GSH) estimation, and Catalase estimation).
Results: Rutin (100 and 200 mg/kg) for 8 weeks significantly protected all the behavioral alterations (loss in body weight, mechanical allodynia, cold allodynia, mechanical hyperalgesia, thermal hyperalgesia), oxidative damage (lipid peroxidation, nitrite estimation, superoxide dismutase activity, reduced glutathione (GSH) estimation, and Catalase estimation) and change in mean nerve conduction velocity induced by STZ. Further, combination of Rutin (100 and 200 mg/kg) with ramipril (0.2 mg/kg) significantly reversed all the behavioural, biochemical and changes in nerve conduction velocity as compared to their effect per se in STZ-induced diabetic neuropathy.
Conclusion: The present study suggests the protective effect of Rutin against STZ-induces diabetic neuropathy. Study further provides a an evidence that rutin produces better effect in combination with rampril against STZ-induces diabetic neuropathy.
PS05-076
Poster Viewing Session V
The effect of changes in brain lactate uptake and BDNF production on executive function after high-intensity interval exercise in humans
S. Takenaka1, H. Tsukamoto1, N. Olesen2, L. Petersen2, H. Sørensen2, H. Nielsen2, N. Secher2, S. Ogoh3 and T. Hashimoto1
1Ritsumeikan University, Shiga, Japan
2The Copenhagen Muscle Research Centre, Copenhagen, Denmark
3Toyo University, Saitama, Japan
Abstract
Objective: We postulate that lactate, which is an important energy substrate for the brain particularly during exercise, may have a pivotal role for brain function. Indeed, we previously found that repeated bouts of high-intensity interval exercise (HIIE), which decreases systemic lactate concentration in a later HIIE session, dampened the positive effect of exercise on executive function (EF) during the post-exercise recovery (1). However, we did not examine whether brain lactate metabolism is also diminished with decreased systemic lactate accumulation. In addition, we could not exclude the effect of other physiological factors on EF in relation to repeated bouts of HIIE. Brain-derived neurotrophic factor (BDNF) may be such a factor to affect EF (2). Interestingly, lactate infusion causes an increase in blood BDNF concentration (3), suggesting the possibility that the brain BDNF production would be associated with changes in brain lactate uptake and would mediate post-exercise EF. Here, we aimed to investigate this possibility.
Methods: According to our previous study (1), 14 healthy male subjects performed the two HIIE protocols (four 4-min bouts of cycling exercise at 80–90% peak power output with 3-min active recovery period) with a 60 min resting period between 1st and 2nd HIIEs. Arterial and internal jugular venous catheterization was used to determine concentration differences for lactate and BDNF across the brain.
Results: Brain lactate uptake was diminished in the later HIIE session with EF impairment during post-exercise recovery. However, brain BDNF production was not diminished in parallel with decreased brain lactate uptake in response to later HIIE session.
Conclusion: These findings suggest that brain lactate metabolism is associated with positive effect of exercise on EF, while BDNF may not be a crucial mediator for EF.
References
(1) Tsukamoto, Suga, Takenaka et al., 2016.
(2) Ferris et al., 2007.
(3) Schiffer et al., 2010.
PS05-077
Poster Viewing Session V
Relationship of brain lactate uptake, cerebral hemodynamics, and executive function after high intensity interval exercise in humans
H. Tsukamoto1,2, N. Olesen3, L. Petersen3, H. Sørensen3, H. Nielsen3, N. Secher3, S. Ogoh4 and T. Hashimoto1
1Ritsumeikan University, Kusatsu, Shiga, Japan
2Japan Society for the Promotion of Science, Tokyo, Japan
3The Copenhagen Muscle Research Centre, Copenhagen, Denmark
4Toyo University, Kawagoe, Saitama, Japan
Abstract
Objectives: Executive function (EF) is improved during exhaustive dynamic exercise despite a decrease in cerebral perfusion (1). Recently, we demonstrated that a repeated high-intensity interval exercise (HIIE) inhibited exercise-induced EF improvement and this response seems to be related to decreases in blood lactate concentration (2). The purpose of the present investigation was to examine whether decreases in brain lactate uptake affects human brain function such as post-exercise EF.
Methods: 14 healthy male subjects performed two HIIE protocols (four 4-min bouts of high-intensity exercise at 80–90% peak power output with 3-min bouts of moderate-intensity cycling exercise period) separated by a 60 min resting period (2). Blood samples were obtained from the bulb of the right internal jugular vein and a brachial artery to determine concentration differences for O2 (a-v diffoxygen), glucose (a-v diffglucose) and lactate (a-v difflactate) across the brain. Transcranial Doppler determined middle cerebral artery blood flow velocity (MCA Vmean) as an index of regional cerebral blood flow. To evaluate EF, color-word Stroop tasks were performed.
Results: The EF improvement after the 1st HIIE was sustained while the improvement was diminished after the 2nd HIIE. Although a-v diffoxygen and a-v diffglucose were similar between 1st and 2nd HIIEs, a-v difflactate was significantly lower for 2nd than for 1st HIIE, associated with arterial lactate concentration and cerebral metabolic rate of oxygen (CMRO2) calculated as MCA Vmean × a-v diffoxygen, respectively. Interestingly, chronological reduction in EF after either HIIE was associated with decreases in CMRO2.
Conclusion: These results suggest that the decreases in brain lactate uptake may lead to reduction in EF improvement after HIIE, and might be related to decreases in CMRO2.
Reference
(1) Ogoh S, Tsukamoto H et al. Physiol Rep. 2014;2(9):e12163.
(2) Tsukamoto H et al. Physiol Behav. 2016;160:26–34.
PS05-078
Poster Viewing Session V
Lactate does not fully substitute glucose in powering gamma oscillations
J.-O. Hollnagel1, T. Cesetti1, J. Schneider1, A. Lewen1 and O. Kann1
1University of Heidelberg, Institute of Physiology and Pathophysiology, Heidelberg, Germany
Abstract
Neuronal information processing not only relies on a well-balanced interplay between excitation and inhibition but also on the availability of energy substrates to empower neuronal activity. Although glucose is the preferred exogenous energy substrate under normal conditions it has been argued that lactate might be sufficient to supply the energy underlying higher brain functions.
We investigated the capability of lactate to fuel two types of naturally occurring fast network oscillations. We recorded local field potentials and oxygen concentrations in the hippocampal subfield CA3 from rat organotypic slice cultures and acute slices.
Our main findings are: (I) Gamma oscillations have less power in the presence of lactate (2 - 20 mM) compared to glucose (10 mM) in slice cultures. (II) Equicaloric lactate (20 mM) leads to epileptiform discharges during gamma oscillations in acute slices. (III) Sharp wave-ripples remain stable in presence of either lactate (20 mM) or glucose (10 mM) in acute slices. (IV) Switches from sharp wave-ripples to gamma oscillations are accompanied by an increased oxygen consumption rate (OCR) in acute slices. (V) OCR during sharp wave-ripples is higher in the presence of lactate (20 mM) compared to glucose (10 mM). (VI) Gamma oscillations have a higher OCR while displaying lower power in presence of lactate (20 mM) in slice cultures.
We conclude that lactate does not meet the energy demand required to fully power gamma oscillations whereas sharp wave-ripples can be maintained during episodes of reduced energy supply.
PS05-079
Poster Viewing Session V
Characterizing the impacts of early interventional ketogenic diet on brain vasculature, energy metabolism, and cognition
D. Ma1, A. Wang1, J. Hoffman1, V. Bakshi1, I. Parikh1, J. Guo1, R. Armstrong1, A. Hartz1, B. Bauer1 and A.-L. Lin1
1University of Kentucky, Lexington, United States
Abstract
The ketogenic diet (KD), or the low-carbohydrate and high-fat diet, has served as a therapeutic for medically intractable epilepsy for the past ninety years. Recent studies have shown the neuroprotective qualities of ketone bodies. However, KD's impact on overall in vivo brain function remains largely unexplored. The aim of this study was to characterize the interaction between specialized nutrition and in vivo brain function. Subjects were age-matched and gender-matched young wild type mice models, divided into two groups based on diet: Western (control) diet or KD. We employed multimodal, non-invasive neuroimaging (MRI/MRS) to determine in vivo brain cerebral blood flow and energy metabolites. We also assessed the animal's memory and learning ability with the Radial Arm Water Maze and the Novel Object Recognition Test. In addition, we performed western blots and BBB function analysis. Blood glucose, blood ketone bodies, and body weight were also measured. We found distinct patterns between Western and Ketogenic diet - the KD group exhibited significantly higher cerebral blood flow in the dorsal thalamus and the hypothalamus compared to the control. We observed significant modulation of the metabolites alanine and lactate, which suggest a more diverse metabolic profile in KD mice. When examining the behavioral test results, there is indication that KD fortifies various memory and sensory functions, consist with our CBF data. Furthermore, KD mice exhibited significantly higher brain endothelial NOS and brain capillary P-glycoprotein, as well as a significantly lower expression of the mechanistic target of Rapamycin. In accordance with our CBF results, there is significant evidence that KD improves vascular brain function. Our novel findings demonstrate KD produces noticeable shifts in brain vascular and metabolic function, while maintaining cognition in a young mice model. These results provide rationale for KD as a viable early interventional dietary measure.
PS05-080
Poster Viewing Session V
Circulating microRNAs as biomarkers of delayed cerebral infarction after aneurysmal subarachnoid hemorrhage
and G. Wong1
1The Chinese University of Hong Kong, Surgery, Hong Kong, Hong Kong
Abstract
Background and Purpose: Delayed cerebral infarction (DCI) is a major cause of morbidities after aneurysmal subarachnoid hemorrhage (SAH) and typically starts at day 4 to 7 after initial hemorrhage. Multiple mechanisms have been proposed for the development of DCI, and these had held back the development of diagnostic biomarkers and therapeutics strategy. MicroRNAs (miRNA) play important role in posttranscriptional gene expression control, and distinctive patterns of circulating miRNA changes have been identified for some diseases. We aimed to establish miRNA indicators to characterize SAH patients with DCI as compared to those without DCI.
Methods: Circulating miRNAs were collected on day 7 post-SAH in a 60-subject cohorts namely the healthy controls (N = 20), SAH patients with DCI (N = 20) and SAH patients without DCI (N = 20). We developed a bioinformatics pipeline to integrate multi-miRNAs classifier approach with the LASSO regression model to classify SAH patients with and without DCI.
Results: In the 28 dysregulated miRNAs associated with DCI and SAH, we found that a four-miRNAs indicator (miR-4532, miR-4463, miR-1290, and miR-4793) could differentiate SAH patients with DCI from without DCI with an AUC of 100% (95% CI: 1.000 – 1.000, p < 0.001). The four miRNAs can also distinguish SAH patients with or without DCI from healthy controls with an AUC of 99.3% (95% CI: 0.977 - 1.000, p < 0.001) and of 82.0% (95% CI: 0.685 ̶ 0.955, p < 0.001) respectively.
Conclusion: We developed a four-miRNA-based classifier system with the LASSO Cox regression model to differentiate SAH patients with or without DCI.
PS05-081
Poster Viewing Session V
Study of early coagulation activation, inhibition, thrombin generation and fibrinolysis in isolated severe traumatic brain injury (iSTBI) induced coagulopathy and its influence on immediate outcome
V. Albert1, A. Subramanian1, D. Agrawal2, P. Hara Prasad3, A. Mukhopadhayay1 and S.D. Gupta4
1All India Institute of Medical Science, Laboratory Medicine, New Delhi, India
2All India Institute of Medical Science, Neurosurgery, New Delhi, India
3All India Institute of Medical Science, Hematology, New Delhi, India
4All India Institute of Medical Science, Pathology, New Delhi, India
Abstract
Objectives & Background: Early coagulopathy in isolated severe traumatic brain injury occurs despite lack of severe bleeding, shock and fluid administration. We aimed to correlate coagulation activation/inhibition, thrombin generation & fibrinolysis with the development of acute trauma induced coagulopathy (ATIC) and its effects on early mortality in iSTBI patients
Methods: A prospective screening of iSTBI patients was done for two years. H/O anticoagulants, liver disease, hypotension, extracranial injuries, transfusion, brain death were excluded. ATIC was defined as INR ≥ 1.27 &/or PT ≥ 16.7 sec &/or aPTT ≥ 28.8 sec on admission. Analysis of TF, TFPI, PC, PS, TAT, sFM, TPA and PAI-1 was done. Cases were categorised as presence or absence of ATIC and 20 healthy controls included.
Results: 120 cases met the inclusion criteria, aged 35.7 ± 12.12 years, 96% males. ATIC was identified in 50 (41.6%). ATIC occurred independently of age, gender, GCS but was associated acidosis (60%; p 0.01). Following iSTBI significant decline was seen in coagulation activation. Thrombin generation and fibrinolysis were markedly increased. TF, TFPI, PC & PS were low in ATIC compared to control. Significant depletion of PS was seen in ATIC vs. No-ATIC. iSTBI patients with depleted PS had an OR of 7.10 (1.61–31.2) for ATIC. ROC analysis depicted AUC of 0.73 [95% Conf. Interval 0.63–0.84] with a cut off of ≥74 of PS (specificity 63.9%, sensitivity 72.7%). Mortality was higher in ATIC group (44%) compared to No ATIC (20%) with OR of 4.73 (1.68–13.3)].
Conclusion: Incidence of ATIC in iSTBI is 41.6%, with 4.7 times odds for mortality. Traumatic Brain injury causes enhanced coagulation activation, inadequate inhibition, exacerbation of thrombin generation and subsequent increased fibrinolysis ROC curve analysis revealed a cut-off of Protein S ≤ 74, with odds of 7.10 (1.61–31.2 95% CI) for development of ATIC.
PS05-082
Poster Viewing Session V
Assessment of cerebral autoregulation using near infrared spectroscopy during squat-stand maneuvers in healthy subjects with experiencing frequent symptoms of orthostatic hypotension
J.-M. Kim1, H.-W. Jung1, S. Yu1, M. Kim1, J.-K. Choi1, M.-G. Choi1, G. Hwang1, M. Ji1, K.-S. Kim2, S.-B. Ko3 and H.-M. Bae1
1KAIST, Electrical Engineering, Daejeon, Korea, Republic of
2Seoul National University Bundang Hospital, Department of Emergency Medicine, Seoul, Korea, Republic of
3Seoul National University Hospital, Department of Neurology, Seoul, Korea, Republic of
Abstract
Objectives: Orthostatic hypotension (OH) is caused by autonomic dysfunction compromising cerebral autoregulation (CA). The variations of blood pressure (BP) during postural changes are known to denote OH [1]. However, since the BP is an indirect indicator of CA, only patients with severe OH demonstrate meaningful changes in the BP [2]. In this study, we used near-infrared spectroscopy (NIRS) to evaluate CA during squat-stand maneuvers.
Methods: Twenty young healthy volunteers (age, 22.1 ± 3.4, 12 Males) were studied. Subjects are categorized into control and symptom groups (experiencing symptoms of OH at least once a month, n = 13). All subjects signed written informed consent for the study, which was approved by the IRB. A continuous wavelength NIRS system (NIRSIT, OBELAB Inc.) was used to measure the changes of oxy- and deoxy- hemoglobin concentration in the forehead, which are derived into total hemoglobin (HbT = HbO2+HHb) and the time derivative of HbT (dHbT/dt). Beat-to-beat BP was also measured using Finometer®. For the maneuvers, 1min squatting followed by 3min standing, were repeated 5 times.
Results: The mean change of the systolic/diastolic BP under the maneuvers showed no significant difference in the control and symptom groups (8.76 ± 2.67, 9.96 ± 1.04 mmHg; p = 0.621 and 1.86 ± 0.7, 1.11 ± 0.53 mmHg; p = 0.412 respectively). However, significant reduction in the total variation of dHbT/dt was observed in the symptom group (-3.166 ± 0.203, −2.438 ± 0.1021 uM/sec; p = 0.0019 and 2.032 ± 0.07804, 1.679 ± 0.08522 uM/sec; p = 0.0026 respectively). Moreover, such variation appeared with increased time delay in the symptom group as shown in Figure (3.787 ± 0.1604, 4.650 ± 0.1438 sec; p = 0.0001 and 13.44 ± 0.2621, 15.29 ± 0.2705 sec; p < 0.0001 respectively).
Conclusions: Significant difference in dHbT/dt between two groups suggests that NIRS could be used to evaluate CA for the prognosis of OH.
References
[1] Freeman, R., et al. Clin Auton Res 21:69–78 (2011)
[2] Sun, Zhanfang, et al. Scientific reports 6 (2016).
PS05-083
Poster Viewing Session V
Activation of the lectin complement pathway is associated with vulnerability of atherosclerotic plaques
S. Fumagalli1, C. Perego1, R. Zangari1, D. De Blasio1, M. Oggioni1, F. De Nigris2, F. Snider2, P. Garred3, A. Ferrante2 and M.-G. De Simoni1
1IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Neuroscience, Milan, Italy
2Catholic University of Sacred Heart, Rome, Italy
3University of Copenhagen, Copenhagen, Denmark
Abstract
The molecular mechanisms resulting in atherosclerotic plaque rupture are incompletely understood, but inflammatory mechanisms could be important. Thus we hypothesized that activation of the lectin complement pathway, a major arm of the inflammatory response, could represent an index of plaque instability.
Plaques from 42 consecutive patients undergoing carotid endarterectomy were stained with hematoxylin-eosin and the lipid core, cholesterol clefts, hemorrhagic content, thickness of tunica, including or not infiltration of cellular debris and cholesterol, were determined. The presence of ficolin-1, -2 and -3 and mannose-binding lectin (MBL), initiator molecules of the lectin pathway, was assessed in the plaques by immunofluorescence and in plasma by ELISA. The activation of the lectin pathway was assessed in plasma through functional in vitro assays.
Patients presenting moderate stenosis (≤75%) had higher hemorrhagic content than those with severe stenosis (>75%), indicating increased erosion. Increased tunica media thickness, decreased lipid core and infiltrated content were associated with vulnerable plaques and therefore used for stratification. Ficolins and MBL were found both in plaques' necrotic core and tunica media. Plasma levels of ficolin-1 decreased in symptomatic patients, and those of ficolin-2 decreased in patients with vulnerable plaques. In contrast, the activation of the lectin pathway by MBL increased in plasma of patients with vulnerable plaques.
The lectin pathway initiators are present within the plaques and their plasma levels change in patients with rupture-prone plaques, indicating their potential use as markers for cardiovascular risk assessment in atherosclerotic patients.
PS05-084
Poster Viewing Session V
Multi-modal MRI of vascularity, cellularity, and acidosis in brain tumors
M. Parent1, D. Coman1, Y. Huang1, J.U. Rao1, S. Maritim2, J.J. Walsh2 and F. Hyder1,2
1Yale University, Radiology and Biomedical Imaging, New Haven, United States
2Yale University, Biomedical Engineering, New Haven, United States
Abstract
Objectives: Glioblastoma multiforme (GBM) is the most common primary brain tumor, and its prognostic remains dismal. The WHO grading system of GBMs is based on histological criteria such as nuclear pleomorphism, but also increased cellularity, vascular proliferation, and/or necrosis. Rapid proliferation of cancer cells is supported by glycolysis even in presence of oxygen (Warburg effect), generating acids that are extruded to the extracellular space. Since it is unknown how morphological characteristics are related to metabolic features of tumors, we used MRI to measure vascular permeability, tissue cellularity, and extracellular acidosis (pHe) for GBM models in rat brain.
Methods: Rats with either RG2 or U251 tumors were imaged on a 9.4T, 3 to 5 weeks following intraparenchymal tumor cell injections (n = 9). Vascular permeability (Ktrans) was measured by dynamic contrast enhanced (DCE) MRI with a bolus i.v. injection of 0.25mmol/kg Gadobutrol. Cellularity was measured by apparent diffusion coefficient (ADC) with diffusion-weighed MRI. pHe was assessed using BIRDS with the thulium paramagnetic ion agent TmDOTP5−.
Results: For small tumors (∼3mm diameter), mean values of Ktrans, ADC, and pHe were 0.03 min−1, 1.34, and 6.77, respectively (Figure A), whereas for larger tumors (6–7mm), mean values were 0.07 min−1, 0.93, and 6.56, respectively (Figure B). The spatial gradient for Ktrans (0.02 min−1/mm) was not significantly different during tumor growth. However the spatial gradient for pHe doubled with tumor growth (0.2/mm vs. 0.4/mm).
Conclusion: As tumors grew, they exhibited higher capillary permeability (shown by increased Ktrans), higher cellularity (decreased ADC) and higher acidosis (increased pHe). Spatial distribution of vascularity and acidosis (but not cellularity) showed specific gradients; in the case of vascularity, the gradient remained comparable in small and large tumors, while the pHe spatial gradient doubled in the larger tumors. Such multi-modal measurements of GBMs are a promising tool for assessing various cancer treatments.
PS05-086
Poster Viewing Session V
Minimal microbiota differences between commercial breeders impacts post-stroke immunity
R. Sadler1,2, V. Singh1,2, C. Benakis1,2,3, D. Brea-Lopez3, T. Hünig4, J. Anrather3, B. Stecher5,6 and A. Liesz1,2
1Klinikum der Universität München, Institute for Stroke and Dementia Research (ISD), Munich, Germany
2Munich Cluster for Systems Neurology (SyNergy), Munich, Germany
3Feil Family Brain and Mind Research Institute, Weill Cornell Medical College at Burke Medical Research Institute, New York, United States
4Institute for Virology and Immunobiology, Würzburg, Germany
5Max-von-Pettenkofer Institute, Munich, Germany
6German Center for Neurodegenerative Disease (DZNE) Center for Infection Research (DZIF), Munich, Germany
Abstract
Experimental reproducibility between laboratories is a major translational obstacle, particularly in studies investigating immunomodulatory therapies in relation to brain disease. In recent years increasing attention has been drawn towards the gut microbiota as a highly influential area of immune cell polarisation. Manipulation of the gut microbiota has been found to mediate beneficial or detrimental immune effects in various models of stroke, autoimmune brain disorders and psychiatric diseases. In this study we have investigated the impact of minimal microbiota differences and the immune response of mice from three commercial breeders with the same genetic background (C57Bl6). While overall microbiota composition and species diversity was comparable, we observed distinct differences at species level. Most prominently, segmented filamentous bacteria (SFB)-which are known to promote Th17 cell polarization-were absent in mice from one breeder but abundant in others. Merely SFB presence was directly correlated to the ratio of Treg to Th17 cells in the periphery. Moreover, recolonization of SFB-free mice with SFB resulted in a T-cell shift which mimicked the ratios found in SFB-positive mice. We next tested the response to immunotherapeutic approaches by using a CD28 superagonist (CD28SA) which has previously been shown to expand Treg. CD28SA treatment had differing effects between breeders and was found to be ineffective at inducing Treg expansion in SFB-free mice. These changes directly corresponded to stroke outcome as SFB-free mice had significantly larger infarcts. This study unequivocally demonstrates that minimal microbiota differences have a major impact on naïve immune homeostasis and therefore immunotherapeutics responses.
PS05-087
Poster Viewing Session V
Circulating CX3CR1low classical monocytes are an immunologic feature of cerebral ischaemia
S. Krishnan1,2, C. O'Boyle1,2, M. Haley1, J. Konkel1,2, S. Allan1, C. Smith3, J. Grainger1,2 and C. Lawrence1
1University of Manchester, Faculty of Biology, Medicine and Health, Manchester, United Kingdom
2University of Manchester, Manchester Collaborative Centre for Inflammation Research (MCCIR), Manchester, United Kingdom
3University of Manchester, Manchester Academic Health Science Centre, Stroke and Vascular Research Centre, Institute of Cardiovascular Sciences, Salford, United Kingdom
Abstract
Objectives: Infections in the post-acute phase of stroke are a significant source of mortality and have been ascribed to the onset of an immunosuppressive syndrome driven by the ischaemic damage. Monocytes have been implicated in neuroinflammation but their role in modulating infection susceptibility remains elusive. Our study aimed to characterise the temporal changes in monocyte surface phenotype and functional responsiveness in a cohort of stroke patients and employed a mouse model of stroke to explore underlying mechanisms critical to these alterations.
Methods: Peripheral blood drawn from stroke patients was immune-phenotyped on admission and 3 subsequent days using flow cytometry. The middle cerebral artery occlusion (MCAO) method was used to model focal cerebral ischaemia in mice. The surface phenotype and functional responsiveness of monocytes was assessed 24 h and 72 h post-stroke using flow cytometry.
Results: On admission following stroke, a dramatic reduction was evident in the expression of the chemokine receptor, CX3CR1 on circulating classical (CD14++CD16−) monocytes in patients, correlating with stroke severity in males. Modelling stroke in-vivo revealed that murine equivalent of classical monocytes (Ly6Chi) in circulation also mirrored this CX3CR1low surface phenotype which was transcriptionally regulated. This altered surface phenotype was prolonged in bone marrow monocytes and their direct progenitors up to 72 h following MCAO. CX3CR1 was also acutely downregulated on bone marrow and circulating monocytes in response to endotoxin challenge, sterile peritonitis, certain infections and surgical wounding in-vivo.
Conclusions: Collectively, our data suggest that pathways tuning expression of CX3CR1 on monocytes may be initiated in a diverse array of contexts but are prolonged or subverted post-stroke. We hypothesise that disrupted neural signalling post-stroke shapes systemic immunity by altering monocyte surface phenotype which could modulate their trafficking, function and anti-bacterial defence. Future work will employ single cell RNASeq to broadly investigate the functional characteristics of CX3CR1low-classical monocytes in stroke-induced immunosuppression.
PS05-088
Poster Viewing Session V
mTOR regulates brain vascular PICALM levels in a model of Alzheimer's disease
A. Olson1, J. Jahrling1, S. Hussong1 and V. Galvan1
1University of Texas Health at San Antonio, Physiology, San Antonio, United States
Abstract
Advanced age is the greatest known risk factor for the development of Alzheimer's disease (AD). A hallmark of AD is the accumulation of amyloid beta (Aβ) in brain, the majority of which is cleared from brain into the bloodstream across the blood-brain barrier (BBB) via transcytosis mediated by the low-density lipoprotein receptor-related protein 1 (LRP1). Several recent genome-wide association studies (GWAS) have implicated the allele rs3851179A near the phosphatidylinositol-binding clathrin assembly protein (PICALM) gene as a protective against the development of AD. This allele is associated with an increase in mRNA expression of PICALM, which is abundant in brain endothelial cells (EC). PICALM binds to LRP1 to initiate transcytosis by recruiting clathrin to form clathrin-coated vesicles, ultimately removing toxic Aβ from the brain.
In the present study, we tested the hypothesis that mTOR may contribute to increased Aβ brain levels by reducing LRP1-mediated transport across BBB via a reduction in PICALM. To test this hypothesis, we measured protein levels and mRNA expression of PICALM and LRP1 in cultured endothelial cells and in brain microvasculature of transgenic AD mice that were treated with control or with the mTOR inhibitor rapamycin. While mTOR attenuation did not affect PICALM nor LRP1 mRNA expression levels, our studies demonstrated significant increases in PICALM protein levels in rapamycin-treated cultured EC and in microvasculature of rapamycin-treated AD mice, suggesting that mTOR attenuation may increase PICALM in vitro and in vivo. The observed increases in PICALM levels as a result of rapamycin treatment were recapitulated by knock-down of Raptor, the obligatory companion of mTOR complex 1. Our data suggest that mTOR-mediated reduction of PICALM may contribute to increased brain Aβ levels in mice modeling AD, thus revealing a novel mechanism by which mTOR attenuation may reduce brain Aβ levels and significantly delay or stop the progression of AD.
PS05-089
Poster Viewing Session V
Brain angiogenesis in chronic pain
V. Del Grosso1,2, A.G. Zippo2, A. Patera3, A. Bravin1 and G.E.M. Biella2
1European Synchrotron Radiation Facility, Grenoble, France
2Istituto di Bioimmagini e Fisiologia Molecolare, Consiglio Nazionale delle Ricerche, Segrate (MI), Italy
Objectives: Chronic pain (CP) is a complex sensory disorder, lasting from three to six months, characterized by structural changes, i.e. severe anatomical rearrangements of somatosensory cortex, and functional changes, i.e. anomalies in network functional connectivity and in information transmission (1). The aim of this work is to investigate the microstructural anomalies in the brain cortical glio-vascular compartments in the genesis and progression of CP (2,3).
Methods: We applied the experimental Seltzer Model (partial constriction of the sciatic nerve) on rats to induce a neuropathic degenerative syndrome. At three different time stages from the neuropathy onset the animals are sacrificed and perfused with special techniques and samples of the involved somatosensory cortex have been excised. These samples have been then analyzed at Synchrotron resources by X-ray Nano Computer Tomography (NanoCT) Imaging (TOMCAT Villigen and ESRF, Grenoble). Volume renderings allowed for a detailed visualization of cortical vasculature at nano-scale. Quantitative and morphological analyses have then been performed of micro-vascular structures at the different stages from the neuropathy onset with comparative studies between CR (control) and CP rats. Histological and immuno-histochemical evaluations have been done to validate the results obtained through NanoCT.
Results: an increased number of vessels and a reduced mean value of vessel diameter have been found in CP samples, suggesting a neo-genesis at the capillary level in CP condition. These angiogenic events have been confirmed by the positivity to vessel neogenesis antibodies on same samples analysed through NanoCT.
Conclusion: Evidences of vascular neo-genesis at capillary level have been found in CP conditions. These findings could shed light on new pathogenetic mechanisms and potential novel therapeutic approaches.
Baicalin reduces hemorrhagic transformation in ischemic stroke rats with delayed t-PA treatment: involvement of ONOO–MMP-9 pathway
H. Chen1 and J. Shen1
1The University of Hong Kong, School of Chinese Medicine, Hong Kong, China
Abstract
Objective: Hemorrhagic transformation (HT) is the severe complication of delayed tissue plasminogen activator (t-PA) treatment in ischemic stroke. Previously we found that peroxynitrite (ONOO-) plays an important role in HT after t-PA treatment. Here we test the hypothesis that baicalin, a highly-safe natural compound capable of scavenging peroxynitrite, could attenuate HT in a rat cerebral ischemia-reperfusion model with delayed t-PA treatment.
Methods: Male Sprague-Dawley (SD) rats were subjected to middle cerebral artery occlusion (MCAO) 5 hours plus reperfusion 19 hours. T-PA (10 mg/kg) or t-PA plus baicalin (50, 100, 150 mg/kg) was administrated via femoral vein at 4.5 hours after MCAO.
Results: Delayed t-PA treatment significantly increased the mortality rate, induced hemorrhagic transformation, and exacerbated blood-brain barrier (BBB) permeability, brain edema, and neurological deficit at 24 hours after ischemia. Such deleterious effects of t-PA were dose-dependently and significantly attenuated by baicalin co-treatment. Delayed t-PA treatment substantially increased 3-NT (a biomarker of peroxynitrite) formation in ischemic hemispheres, leading to active MMP-9 overproduction, which were also greatly prevented by baicalin co-treatment.
Conclusion: Baicalin is a potential candidate for combination therapy in ischemic stroke to reduce HT and other complications induced by delayed t-PA treatment, possibly via inhibiting ONOO- mediated active MMP-9 production.
[2] Xu, M.; Chen, X.; Gu, Y.; Peng, T.; Yang, D.; Chang, R.C.-C.; So, K.-F.; Liu, K.; Shen, J. Baicalin can scavenge peroxynitrite and ameliorate endogenous peroxynitrite-mediated neurotoxicity in cerebral ischemia-reperfusion injury. J Ethnopharmacol, 2013, 150(1), 116–124.
PS05-091
Poster Viewing Session V
Blood brain barrier (BBB) protection by let-7 microRNAs during cerebral stroke
V. Zuluaga-Ramirez1, S. Gaghate1, N. Reichenbach1, Y. Persidsky1,2 and S. Rom1,2
1Temple University School of Medicine, Pathology, Philadelphia, United States
2Temple University School of Medicine, Center for Substance Abuse Research, Philadelphia, United States
Abstract
Most neurological diseases, including stroke, multiple sclerosis, Alzheimer's and Parkinson's diseases, brain infections and epilepsy lead to blood brain barrier (BBB) dysfunction. A significant role in BBB injury belongs to inflammation, due to pro-inflammatory factors produced in the brain or leukocyte engagement of brain endothelium. Brain microvascular endothelial cells (BMVEC) are active contributors and regulators of inflammatory activities at a site of inflammation. Inflammatory responses in brain endothelium comprise hundreds of genes whose expression entails fine-tuned regulation. microRNAs (miRNAs) have lately appeared as major regulators of gene expression. Very little information exists about their contribution in inflammatory responses triggered by ischemia/reperfusion (I/R) events in brain endothelial cells. Based on our studies of BBB dysfunction during neuroinflammation, we suggest that barrier protection is best achieved when the intervening agents possess anti-inflammatory properties and can stabilize tight junctions. We have identified, recently, highly modified let-7 miRNAs, which are important for endothelial maintenance. let-7 miRNAs were predicted to target the inflammatory molecules, CCL2, CCL5, IL8 and IP10. Overexpression of let-7 in vitro and in vivo resulted in reduced leukocyte adhesion to and migration across brain endothelium and diminished expression of pro-inflammatory cytokines. In an in vitro I/R model, oxygen glucose deprivation (OGD), followed by reperfusion (OGD/R), overexpression of these miRNAs led to increased BBB tightness, thus attenuating barrier 'leakiness'. Overexpression of these miRNAs resulted in decreased infarct volume and neutrophil infiltration in to the brain in transient Middle Cerebral Artery Occlusion (tMCAO), an in vivo I/R stroke animal model. Our study provides identification and functional assessment of miRNAs in brain endothelium and leads to future therapeutic developments for prevention of deleterious effects of ischemia/reperfusion on the brain.
PS05-092
Poster Viewing Session V
Establishment of a human blood-brain barrier co-culture model based on human multipotent and pluripotent stem cells mimicking the neurovascular unit
A. Appelt-Menzel1,2, A. Cubukova2, W. Neuhaus3, J. Piontek4, H. Walles1,2 and M. Metzger1,2
1University Hospital Würzburg, Department Tissue Engineering and Regenerative Medicine (TERM), Würzburg, Germany
2Fraunhofer-Institute Interfacial Engineering and Biotechnology IGB, Translational Center Würzburg ‘Regenerative therapies in oncology and musculoskelettal diseases’ Würzburg, Würzburg, Germany
3AIT Austrian Institute of Technology GmbH, Competence Center Health and Bioresources, Competence Unit Molecular Diagnostics, Vienna, Austria
4Charité Berlin, Institute of Clinical Physiology, Berlin, Germany
Abstract
In vitro models of the blood-brain barrier (BBB) are very strong tools in drug development and to study physiological as well as pathophysiological mechanisms. In order to closely mimic the BBB in vivo and to optimize model characteristics, we analyzed a set of different BBB co-culture models based on primary cells, human induced pluripotent stem cells (hiPSCs) and multipotent fetal neural stem cells (NSCs). The use of hiPSCs allows a very standardized, robust and reproducible model establishment. Further, they pose an effective cell source to generate functional brain cells and have the advantage to be independent of postnatal brain tissue biopsies, including their variations and limitations during in vitro culture.
The aim of this study was to systematically investigate the individual impact of different cell types (astrocytes, pericytes, NSCs) on hiPSC-derived BBB endothelial cell (EC) function and gene expression. Differentiation protocols to generate brain endothelial cells as well as human astrocytes from iPSCs and NSCs mimicking the in vivo embryogenesis are performed as described recently (Lippmann et al. 2014, Wilson et al. 2015, Yan et al. 2013, Reinhardt 2013).
With these cells in hand, ten different BBB-(co)-culture setups were performed and characterized by proving the cell morphology and functionality, analyzing the gene and protein expression, ultrastructural analyses, measurement of the trans-endothelial electrical resistance (TEER) and drug permeability using reference substances.
Finally, we were able to present a quadruple BBB culture model with improved BBB characteristics compared to the monoculture and most of the co-cultures. TEER up to 2500 Ω*cm2 was achieved and at least 1.5-fold up regulation of characteristic BBB genes. Further, the important BBB related TJ proteins, CLDN1, CLDN3, CLDN4, CLDN5, could be analyzed at protein and gene level as well as by freeze fracturing. Moreover, the functionality of these proteins was investigated.
PS05-093
Poster Viewing Session V
Evidence for a complex connectivity gradient in the prefrontal cortex
S. Bedwell1 and C. Tinsley1
1Nottingham Trent University, Nottingham, United Kingdom
Abstract
Prefrontal cortex (PFC) is associated with high-order functions and has been implicated in a number of neurological and psychological deficits e.g. schizophrenia, depression and autism. Despite its clear importance in neuropathology, PFC remains relatively little understood.
In a traditional model of hierarchical organisation, PFC is positioned at the top of a processing hierarchy. In a hierarchical model, connections travel from primary sensory cortex, followed by secondary sensory cortex and association areas, then reaching the top of the processing hierarchy e.g. PFC. This is followed by return connections travelling to secondary motor cortex followed by primary motor cortex, it is understood that reciprocal connections exist between source and target regions at each level of the hierarchy. Based on this understanding, it is thought that all cortical networks must contain a significant level of reciprocity in order to function, making it a fundamental structural component.
Anterograde and retrograde tracers were injected into sub-regions of PFC in the rat. At anterior and central coronal levels, injections produced a similar pattern of connectivity in temporal and sensory-motor cortices. However, at the posterior level the connectivity pattern was reversed. This provides evidence for changes in ordering of connections in the anterior-posterior axis of PFC, indicating a possible connectivity gradient in terms of functional complexity. Such a connectivity gradient is supported by imaging studies and suggestions of a gradient of abstraction in humans.
These findings imply that the functions associated with PFC, and the circuitry underlying these processes, become more complex towards anterior regions. For example, decision-making networks may become more complex and involve more abstract processing as we move towards the anterior pole. When considered alongside observations of functional gradients, our observed gradient suggests that more abstract processes require connections to be non-reciprocal and involve more complex circuitry.
PS05-094
Poster Viewing Session V
Intracerebral hemorrhage and ischemic stroke have different transcriptome architecture in human blood
B. Stamova1, G. Jickling1,2, B. Ander1, X. Zhan1, D. Liu1, H. Hull1, C. Dykstra-Aiello1, M. Durocher1, N. Shroff1, M. Orantia1, F. Hamade1 and F. Sharp1
1University of California at Davis, Neurology, Sacramento, United States
2University of Alberta, Edmonton, Canada
Abstract
Objectives: It is essential to differentiate ischemic stroke (IS) from intracerebral hemorrhage (ICH) in the acute setting. Moreover, future treatment and prevention strategies would be improved by understanding how the blood transcriptome architecture differs between IS, ICH and matched controls (CTRL).
Methods: RNA from 99 peripheral whole-blood samples (33 IS, 33 ICH, 33 CTRL) was hybridized on Affymetrix Human Transcriptome Arrays (HTA 2.0). Probesets were mapped to human genome hg19 and quantified to Ensembl75 transcriptome in Partek Flow for transcript-level analyses. We used a Mixed Regression Model including Diagnosis, Batch, Sex, Age and Time. Transcripts with FDR-corrected p(Diagnosis) < 0.05, and with p < 0.005 and |FC| > 1.2 for the individual comparisons (IS vs. CTRL, ICH vs. CTRL) were considered significant. For temporal analyses (in a cross-sectional design), subjects were binned for time since event into 3 time-points - less than 24h (TP1), 24–48h (TP2) and over 48h (TP3).
Results: 396 transcripts were differentially expressed between IS and CTRL, and 489 between ICH and CTRL. The transcript architecture was different in IS and ICH. More non-coding RNA were differentially expressed in IS than in ICH, and more T-cell receptor transcripts were differentially expressed in ICH than in IS. Additionally, there were differences in genes from cytokine signaling, cellular and humoral immune response pathways. Temporal profiles also differed. 209 transcripts changed expression levels over time in IS, while 1129 changed in ICH. The predominant pattern in IS was decreasing expression of transcripts (65%) from TP1 to TP2 and increasing levels to TP3. In ICH, temporally regulated transcripts (38%) often increased over time.
Conclusions: The blood transcriptome architecture differs between IS and ICH. This important difference will be useful in development of diagnostic and prognostic biomarkers, and in understanding differences of clotting, immune and other pathways for treatment and/or prevention of IS and ICH.
PS05-095
Poster Viewing Session V
Subarachnoid hemorrhage severely impairs brain parenchymal cerebrospinal fluid circulation in the non-human primate
R. Goulay1, J. Flament2, M. Gauberti1, M. Naveau3, N. Pasquet1, C. Gakuba1, E. Emeri1, P. Hantraye2, R. Aron-Badin2, D. Vivien1 and T. Gaberel1
1INSERM U919 / University of Caen Normandy, Caen, France
2MIRCen, Fontenay Aux Roses, France
3UMS Cyceron, Caen, France
Abstract
Background and Purpose: Subarachnoid hemorrhage (SAH) is a devastating form of stroke with neurological outcomes dependent of the occurrence of delayed cerebral ischemia (DCI). It has been shown in rodents that some of the mechanisms leading to DCI are related to a decreased circulation of the cerebrospinal fluid (CSF) within the brain parenchyma. Here, we evaluated the cerebral circulation of the CSF in a non-human primate (NHP) in physiological condition and after SAH.
Methods: We first evaluated the circulation of the brain CSF in Macaca Facicularis, in physiologic condition, using magnetic resonance imaging (MRI) of the temporal DOTA-Gd distribution after its injection into the CSF (N = 3). Then, animals were subjected to a minimally invasive SAH prior to an MRI evaluation of the impact of SAH on the parenchymal CSF circulation as described above.
Results: We first demonstrate that the CSF actively penetrates the brain parenchyma, beginning by the most superficial part of the brain. Two hours after injection, almost the entire brain is labeled by gadolinium. We also show that our proposed model of SAH in NHP displays the typical characteristics of SAH in humans, and leads to a dramatic impairment of the brain parenchymal circulation of the CSF.
Conclusions: The CSF actively penetrates within the brain parenchyma in the gyrencephalic brain, as described for the glymphatic system in rodent. This parenchymal CSF circulation is severely impaired by SAH. In vivo characterization of the glymphatic system in healthy and injured primate brains
PS05-096
Poster Viewing Session V
Magnesium pre-exposure induces mitoprotection in the immature brain
G. Koning1, A.-L. Leverin1, S. Nair1, C. Mallard1, X. Wang1, C. Thornton2, P. Gressens3 and H. Hagberg1,2,4
1University of Gothenburg, Neuroscience and Physiology, Gothenburg, Sweden
2King’s College London, Division of Imaging Sciences and Biomedical Engineering, London, United Kingdom
3Inserm, U1141, Paris, France
4University of Gothenburg, Clinical Sciences, Gothenburg, Sweden
Abstract
Objectives: Antenatal MgSO4 given at preterm labor reduces cerebral palsy occurrence in the offspring1. Cerebral preconditioning (PC) is a phenomenon whereby pharmacologic agents or sub-lethal exposures induce tolerance against subsequent severe insults. The aim was to explore whether MgSO4 induces PC to hypoxia-ischemia (HI) and investigate its underlying mechanisms.
Methods: A model of HI-induced neonatal brain injury in PND7 rat was combined with single intraperitoneal bolus MgSO4 (1.1mg/g) administration at different time-points prior to/post HI. At PND14 brains were collected for evaluation of macroscopical and immunohistochemical injury. Mitochondrial respiration was measured and mRNA/miRNA expression as well as metabolomic changes were analysed.
Results: MgSO4 administered between 6 days-12h before HI, induces powerful PC in the immature brain (p < 0.0001) by as much as 50–80%. However, MgSO4 given between 3h before-1h post-HI had no effect on brain injury. Gene analysis showed that mRNA/miR related to mitochondria were affected and the mitochondrial respiratory control ratio during HI was significantly higher after MgSO4 compared to control (p < 0.05). Significant downregulation of metabolic pathways was detected and both succinate-and malate dehydrogenase were significantly regulated (p < 0.05). H+-NMR revealed significant increase of succinate (p < 0.01) and γ-aminobutyric acid (GABA) (p < 0.0001) in vehicle-treated samples during HI, MgSO4 pre-treatment attenuated these increases. MgSO4 also sustained the levels of ATP/ADP (p < 0.02) and phosphocreatine (p < 0.0002) at 60min of HI.
Conclusions: MgSO4 administered as a bolus 6 days-12h before the insult induces PC of the immature brain conferring resistance to HI. Global mRNA/miRNA analysis and experiments in isolated mitochondria indicate that MgSO4 suppresses mitochondrial metabolism rendering mitochondria more resistant to respiratory deterioration during HI. Subsequent metabolism alteration and downregulation reduces succinate toxicity2, increases mitochondrial protection and sustains high-energy phosphate reserves in the brain tissue following HI.
References
1. Doyle LW et al., CochraneSystRev.2009(1)
2. Chouchani et al. Nature.014,Nov 20;515:431–5.
PS05-097
Poster Viewing Session V
Neuronal prothrombin mediated astrocyte activation in ischemia
P.S. Rajput1, J. Lamb1, S. Kothari1 and P.D. Lyden1
1Cedars-Sinai Medical Center, Neurology, Los Angeles, United States
Abstract
Astrocytes protect neurons during cerebral injury through several postulated mechanisms. The signaling mechanism that activates astrocytes could be the injury if the insult is sub-lethal, or adjacent neurons could signal astrocytes to initiate protective functions. Our previous studies have shown that neurons contain prothrombin (FII) gene and transcribed protein. We hypothesized a signaling role of FII from injured neurons in ischemia. We used invivo and invitro techniques to determine the role of neuronal FII. Oxygen glucose deprivation experiments were performed in primary neuronal cultures isolated from rats and neuronal FII knockout mice. The effect of released thrombin in media was studied on primary astrocytes cultures from rats, wild type mice and PAR-1 knockout mice. Conditional knockout and wild type mice were subjected to middle cerebral occlusion to study the effect on astrocytes activation and infarct volume. Our studies demonstrated that stressed neurons secrete thrombin into the culture media; and causes astrocyte activation; such astrocyte activation can be reproduced with low doses of thrombin; and is blocked by either thrombin inhibitor or by knocking down or knocking out PAR-1 from the astrocytes. In animal studies neuronal FII knockout, the region of MCAo induced infarction significantly enlarged and reduction of astrocyte activation in the ischemic bed and was partially restored by Lentivirus mediated prothrombin gene restoration. Taken together, these results suggest thrombin constitutes a necessary and sufficient signal from neurons to activate adjacent astrocytes in a PAR-1 dependent manner.
PS05-098
Poster Viewing Session V
The role of T-cells in endotoxin/hypoxic-ischemic neonatal brain injury
E. Rocha Ferreira1, A. Nazmi1, A. Zelco1, P. Svedin1, A.-L. Leverin1, C. Mallard1, X. Wang1 and H. Hagberg1
1University of Gothenburg, Gothenburg, Sweden
Abstract
Objectives: Materno-fetal infection and hypoxia-ischemia (HI) around birth are contributing factors to neonatal encephalopathy, affecting up to 20/1000 babies annually in low-income countries1. This synergy involves peripheral immune response, with T-cells playing an important role as a mediator of injury2.
In this study, we investigated potential infiltration of T-cell subtypes into the immature brain at multiple time points following LPS/HI injury. We also used T-cell deficient mice to investigate its role in this condition.
Methods: P9 pups underwent unilateral carotid ligation followed by 30min/10% oxygen exposure. 14h prior, animals received intraperitoneal LPS (0.3µg/g) or saline. At 6h, 24h, 48h, 72h, 7d, 30d and 90d brains were collected for FACS analysis of CD3, CD4, CD8 and gammadelta (gd) T-cells infiltration. Additionally nude (no T-cells), CD4-/-, CD8-/-, alphabeta (ab)-/- and gd-/- mice were assessed 7d post LPS/HI injury for grey and white matter injury and statistically analysed using one-way ANOVA followed by post-hoc TUKEY.
Results: LPS/HI injury resulted in significant up-regulation of CD4+ in the ipsilateral brain hemisphere (p < 5%). This effect was lost by 48h, where gd T-cells were now significantly present in the ipsilateral brain hemisphere (p < 5%). By 7d post-injury, there was a significant bilateral infiltration of CD3 (p < 5%), CD8 (p < 1%) and gd T-cells (p < 5%), which persisted over time.
Histological assessment using MAP2 and MBP 7d post-HI demonstrated no effect in CD4-/- and CD8-/- LPS-treated groups when compared to WT controls. However, ab deletion was detrimental (p < 5%), whereas gd-/- mice were no longer LPS-sensitized. Nude mice showed an abolishment of LPS-sensitization (p < 5%).
Conclusions: Different T-cell subtypes enter the brain at different time points following LPS/HI injury. This supports our histological results showing that LPS sensitization requires T-cell function, particularly gd T-cells.
References
1. Pauliah SS et al., PONE 2013;8(3):E588834.
2. Lai JC et al., BBI 2016;doi: 10.1016/j.bbi.2016.10.022.
PS05-099
Poster Viewing Session V
Optogenetic modulation of sleep slow wave after focal ischemic stroke
L. Facchin1,2, C. Schöne1, M. Bandarabadi1,3, A. Mensen1, K. Schindler3, C. Bassetti1,3 and A. Adamantidis1,2
1Inselspital University Hospital, Experimental Neurology, Bern, Switzerland
2Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland
3Inselspital University Hospital, Neurology, Bern, Switzerland
Abstract
Objectives: Disturbances of sleep-wake cycle and brain state oscillations are frequent after stroke and are associated to negative outcomes. Experimental studies demonstrated that sleep supports the reorganization of neuronal connections and neuroplasticity during stroke recovery. We hypothesize that stroke causes an increase in sleep-like 'up' and 'down' states in electroencephalogram and local field potential recordings and that this so-called bistability is critical for stroke recovery. To investigate the role of sleep oscillations on brain plasticity following stroke we directly target the neuronal populations in layer V of forelimb somatosensory cortex by combining cell-type specific optogenetic techniques with in vivo electrophysiology.
Methods: We expressed ChR2 (activation), ArchT (inhibition) or mCherry (control) in inhibitory (VGAT) or excitatory (CamKII) deep layer cortical cells of the peri-infarct area to render them light sensitive. Animals were chronically implanted with optical fibers and multiple tetrodes in ipsi and controlateral cortical layer V. Experimental stroke was induced by Middle Cerebral Artery Occlusion (MCAO).
Results: Indeed stroke caused sleep disturbances. 24h after stroke down state rate was reduced during slow-wave-sleep in the peri-infarct area, while rapid-eye-movement sleep duration was increased. To optogenetically investigate the contribution of excitatory versus inhibitory cortical neurons to altered sleep oscillations we confirmed the presence of transfected cells within the layer V, forelimb somatosensory cortex through immunohistochemistry. Amongst all the stimulation protocols tested, optical silencing of pyramidal cells in layer V of the cortex robustly induced both LFP and single unit spike activity similar to a down-state of the neuronal network.
Conclusions: We successfully targeted layer V neuronal cells of the somatosensory cortex in both transgenic and wild type mice and showed that optogenetical induction of down-state is possible and represents the first step in the modulation of sleep-like oscillations. Comparing stimulation before and after stroke will reveal possible altered susceptibility to down state-induction.
PS05-101
Poster Viewing Session V
Effect of mechanical reperfusion outcome on the expression of matrix metalloproteinase-9 (MMP-9) in rat brain following an acute occlusive stroke
J. Shearer1, A. Douglas1, A. Pandit2 and K. Doyle1
1National University of Ireland, Physiology and CURAM, Galway, Ireland
2National University of Ireland, CURAM, Galway, Ireland
Abstract
Reperfusion of ischaemic tissue following occlusive stroke can result in serious complications such as haemorrhagic transformation, due to abnormal blood brain barrier (BBB) permeability. We investigated the effect of mechanical reperfusion outcomes on the expression of MMP-9, a marker of BBB dysfunction.
Middle cerebral artery occlusion (2h) was induced in male Sprague Dawley rats (n = 5–8 per group). Cerebral blood flow, pressure, gases, glucose and body temperature were continuously monitored. Rats underwent varying reperfusion for a further 2 hours (full, partial or no reperfusion), simulating restoration of flow through a clot that may occur during thrombectomy. In sham-operated animals, surgery was performed as described but insertion of the occluder was omitted. After reperfusion, rats were killed and the brain removed for histological analysis and measurement of MMP-9 expression by immunohistochemistry and ELISA.
Reperfusion approach had a significant effect on cerebral blood flow (F(2,11) = 78.6, p < 0.001; CBF of 95%, 70%, 47% of sham levels for full, partial and no reperfusion respectively). All other physiological parameters remained stable. The no reperfusion group had significantly higher hemispheric lesion volume than those fully or partially reperfused (F(2,13) = 20.98, p < 0.001). MMP-9 expression was significantly increased in the lesioned hemisphere in comparison to unlesioned hemisphere and sham operated animals. Expression was significantly higher in animals with no reperfusion than those fully or partially reperfused, particularly in the frontal (F(3,13) = 5.2, p < 0.05) and temporal cortex (F(3,13) = 7.2, p < 0.01).
In those animals not reperfused there was greater infarct volume and higher MMP-9 expression compared to full or partially reperfused animals, but no significant difference was observed between the partial and full reperfusion groups. This supports the clinical benefits of attempting reperfusion as soon as possible and suggests that even partial success may be beneficial.
Funding from Enterprise Ireland through the European Regional Development fund and our industrial partners, Neuravi.
PS05-102
Poster Viewing Session V
Protection of glyoxalase anti-glycation system in porcine brain post-cardiac arrest by pyruvate
G. Scott1 and R. Mallet1
1University of North Texas Health Science Center, Institute for Cardiovascular and Metabolic Diseases, Fort Worth, United States
Abstract
Background: Glyoxalase-1 (GLO1) degrades the toxic glycolytic byproduct methylglyoxal, thereby preventing irreversible glycation of proteins and nucleic acids. However, the glyoxalase system may be inactivated by oxidative stress. Cardiac arrest (CA) and cardiocerebral resuscitation (CCR) provokes intense oxidative stress in the brain. However, the impact of CA/CCR on the brain's anti-glycation machinery is unknown.
Purpose: To analyze the impact of ischemia-reperfusion on the cerebrocortical glyoxalase system in a porcine model of CA/CCR, and to determine the ability of the natural antioxidant pyruvate to protect this crucial anti-glycation system.
Methods: Domestic pigs (25–35 kg) were subjected to 10 min CA, followed by 4 min closed-chest CCR, defibrillation and 4 h recovery. Sodium pyruvate (Figures: Pyr) or NaCl control was infused iv to 3.5–4 mM during CA/CCR and the first 60 min recovery. GLO1 content, activities of GLO1 and glutathione reductase (GR), and protein glycation (Arg-pyrimidine immunoblot) were analyzed in frontal cortical homogenates collected at 4 h recovery.
Results: Compared with non-arrested sham pigs (n = 4), GLO1 content and GLO1 and GR activities (Figures 1–3) fell sharply after CA/CCR (n = 4), but were preserved by pyruvate (n = 4). Also, CA/CCR caused protein glycation, particularly at 65 kDa, the MW of the blood-brain-barrier component occludin, while pyruvate reversed this glycation.
Conclusions: Cardiac arrest-resuscitation impaired the brain's GLO1/GR anti-glycation defenses in a manner prevented by pyruvate treatment. Thus, the brain's GLO1 system is disabled by acute ischemia-reperfusion, yet pyruvate intervention protected this pivotal, cerebroprotective system.
PS05-102a
Poster Viewing Session VI
Time-dependent targeting and accumulation of liposomes into the lesioned brain intravenously administered post ischemic stroke
Z. Al-Ahmady1, D. Jasim1, P. Bonijol1, S. Ahmad1, R. Wong2, M. Haley2, G. Coutts2, S.M. Allan2 and K. Kostarelos1
1University of Manchester, Division of Pharmacy, Manchester, United Kingdom
2University of Manchester, Division of Neuroscience & Experimental Psychology, Manchester, United Kingdom
Abstract
Despite decades of active experimental and clinical research, therapeutic options for stroke are extremely limited and the development of new therapies is facing repeated translational failures. Poor delivery of drugs to the brain has long been a critical obstacle limiting treatment options, therefore development of targeted drug delivery agents in post-stroke lesioned brain and other selected tissues is highly warranted.
In this study, we have shown that clinically-used nanoscale (around 100nm in mean diameter) liposomes can be targeted to ischemic brain areas by careful understanding of their post-stroke pharmacokinetic profile. A combination of both in vivo real-time imaging (SPECT/CT and IVIS) and histological analysis has been used to track the early (0.5h & 4h) and late (24h & 48h) liposomal brain accumulation after intravenous injection into mice exposed to 20 minutes MCAO model followed by reperfusion (n=3-4). Our data have shown time-dependent selective recruitment of liposomes into the ischemic brain that is linked to blood brain barrier (BBB) hyperpermeability. We have also obtained initial evidence that selective liposomal accumulation into the ischemic area is co-localised with areas of endothelial cells hyperpermeability mediated by increased transcellular vesicle (termed caveolae) formation. Furthermore, we have observed that cerebral accumulation of liposomes, administered in the early phase after reperfusion (up to 4h post stroke), can be detected as early as 2h and even before clear histological evidence of neuronal damage. Whole-body SPECT/CT imaging and quantification of liposomal accumulation into peripheral organs using 111In-DTPA-liposomes indicated significant selective liposomal recruitment and accumulation into the spleen, particularly when administered in the late phase post stroke (>24h).
In summary, our study highlights the potential of nanoscale liposomal delivery systems for time-dependent selective drug delivery to ischemic brain and other peripheral orangs (e.g. spleen) to maximise therapeutic agent transport and consequently enhance possible therapeutic options.
PS05-103
Poster Viewing Session V
Benefit of optimal cerebral perfusion pressure targeted treatment for traumatic brain injury patients
A. Preiksaitis1,2,3, V. Petkus4, E. Zubaviciute2, S. Krakauskaite4, S. Rocka2,3, D. Rastenyte1, S. Vosylius2,3 and A. Ragauskas4
1Academy of Medicine, Lithuanian University of Health Sciences, Department of Neurology, Kaunas, Lithuania
2Faculty of Medicine, Vilnius University, Clinic of Neurology and Neurosurgery, Vilnius, Lithuania
3Republic Vilnius University Hospital, Department of Neurosurgery, Vilnius, Lithuania
4Kaunas University of Technology, Health Telematics Science Institute, Kaunas, Lithuania
Abstract
Introduction: The maintenance of patient-specific optimal cerebral perfusion pressure (CPPopt) is crucial for patients with traumatic brain injury (TBI). The goal of the prospective study was to explore the influence of CPP declination from CPPopt value on the TBI patients' outcome.
Methods: The CPP and cerebrovascular autoregulation (CA) monitoring of 52 TBI patients was performed. Patient-specific CPPopt has been identified and the associations between the patients' outcome and complex influence of time of CPP declination from CPPopt value, age, and the duration of CA impairment events has been analyzed.
Results: The multiple correlation coefficient between the Glasgow Outcome Scale (GOS), duration of CA impairment events and percentage time, when 0 < ΔCPPopt < 10 mmHg was r = -0.643 (P < 0.001). The multiple correlation coefficients between GOS, age, and percentage time of ΔCPPopt when 0 < ΔCPPopt < 10 mmHg was r = -0.587 (P < 0.001).
Conclusion: The CPPopt-targeted patient-specific management might be useful for stabilizing CA in TBI patients as well as for improving their outcome. The CPP management depends on the patients' CA status and the CPPopt range. Better outcomes were obtained for younger patients (age < 47 years) by avoiding longer CA impairment events and by maintaining CPP in the range of CPPopt to CPPopt + 10 mmHg when CPPopt is in the range of 60 - 80 mmHg and within the range of CPPopt ± 5 mmHg when CPPopt is above 80 mmHg.
Acknowledgement: This research has been funded by the grant MIP-087/2015 from the Research Council of Lithuania.
PS05-104
Poster Viewing Session V
Transcriptomic profiling of long non-coding RNAs after traumatic brain injury
K. Morris-Blanco1, V. Nakka1, R. Chandran1, G. Pandi1, S. Mehta1 and R. Vemuganti1
1University of Wisconsin-Madison, Neurological Surgery, Madison, United States
Abstract
Objectives: The molecular mechanisms underlying secondary damage after traumatic brain injury (TBI) are poorly understood. Previous research indicates that TBI rapidly alters the expression of hundreds of genes in the brain that are associated with functional outcome after TBI. Long non-coding RNAs (lncRNAs) have been implicated in the pre- and post-transcriptional regulation of gene expression. We currently profiled the expression of lncRNAs and mRNAs following TBI.
Methods: Adult Sprague-Dawley rats were subjected to moderate controlled cortical impact injury (CCI). At 3h, 6h, and 24h post-CCI, the cortex surrounding the injury site was collected and total RNA was extracted. Microarray analyses were used to profile the expression of both lncRNAs and mRNAs followed by in silico pathways analysis.
Results: A total of 666 lncRNAs were upregulated (>2 fold) and 850 lncRNAs were downregulated (<0.5 fold) between 3 hours and 24 hours post-CCI. There were 84 lncRNAs that were altered at all three time points after CCI, which were transcribed from both intergenic and intragenic regions of the rat genome. We further identified several protein-coding genes in the TBI-responsive lncRNA loci involved in various molecular and biological functions including transcription factor activity, neurodegeneration, and inflammation. CCI also altered the expression of 2,972 mRNAs between 3 hours and 24 hours with defined roles in several cellular process involved in neurotransmitter signaling, neurodegenerative disorders and inflammation.
Conclusions: TBI induces extensive changes in the transcription of lncRNAs and mRNAs. Post-TBI-transcriptomic changes indicate that inflammation and neurodegeneration pathways are induced which may lead to neuronal death. Studying the functional significance of lncRNAs after TBI may identifying novel therapeutic approaches to minimize secondary brain damage and to promote neurological recovery.
PS05-105
Poster Viewing Session V
Alterations of the hippocampal microvascular network in Alzheimer's Disease
M. Montero-Crespo1,2, L. Blázquez-Llorca2,3 and J. De Felipe1,2
1Instituto Cajal - Consejor Superior de Investigaciones Cientificas (CSIC), Madrid, Spain
2Laboratorio Cajal de Circuitos Corticales (Centro de Tecnología Biomédica), Universidad Politécnica de Madrid (UPM), Madrid, Spain
3Facultad de Psicología, Universidad Nacional de Educación a Distancia (UNED), Madrid, Spain
Abstract
Alzheimer's disease (AD) is a neurodegenerative disease which constitutes the main cause of dementia. It is characterized by a progressive and persistent impairment in the upper cerebral functions, such as memory, language or orientation, producing a loss of autonomy and a decline in the social and working life of the patient. Several hypothesis have been proposed trying to explain the etiology of this disorder. In the recent time, the vascular hypothesis have gained importance due to the vast evidence of data suggesting that vascular pathology plays a key role in the onset of AD. Taking that into account, we tried to analyze the condition of the microvascular network in the hippocampus of human AD autopsies by evaluating the state of endothelial cells in the brain capillaries and its association with astrocytes. Specifically, light and confocal microscopy were used to determine the distribution of collagen Type IV and alkaline phosphatase activity and to study the allocation of collagen Type IV, Factor VIII and GFAP. Preliminary results show a loss of microvessels in the hippocampus of AD patients. These data suggest that blood vessel alterations are a pathological correlate of AD and therefore might be related to the pathogenesis of this condition.
PS05-106
Poster Viewing Session V
In vivo detection of hypothalamic glucose metabolism in HFD and regular fed mice
B. Lizarbe1, H. Lei2,3, A. Cherix1 and R. Gruetter1,3,4
1Laboratory for Functional and Metabolic Imaging, EPFL, Lausanne, Switzerland
2Animal Imaging and Technology Core (AIT), Center for Biomedical Imaging (CIBM), EPFL, Lausanne, Switzerland
3Department of Radiology, University of Geneva, Geneva, Switzerland
4Department of Radiology, University of Lausanne, Lausanne, Switzerland
Abstract
Objective: Recent investigations have associated consumption of high fat diets (HFD) with inflammation in the hypothalamus1. In the present study, we aimed to follow the incorporation of 13C-labeled glucose into its metabolites in the hypothalamus of regular or HFD-fed mice using in vivo1H-[13C] magnetic resonance spectroscopy (MRS), and to investigate the HFD-induced effects on glucose metabolism and metabolic fluxes.
Methods: C57BL/6 mice were fed either with a 60% kcal fat diet (n = 6) or with regular food chow (n = 5) during two months. We performed MRS in a 14.1T/26cm magnet, using a homemade 1H-[13C] surface coil. The hypothalamus voxel (9µL) was selected based on anatomical MR images and field homogeneity was improved using FASTMAP2. 1H-[13C] was achieved using BISEP-SPECIAL3 combined with OVS and VAPOR water supression4, with a 11min time resolution during the infusion of [1,6-13C2] glucose (20% w/v, bolus = 9mL/kg, infusion = 15mL/kg/h). Metabolites were quantified using LCModel, and the fractional enrichment (FE) in glucose, lactate, glutamate and glutamine were estimated. Results were fitted to a 1-compartment model of glucose metabolism5 using MATLAB.
Results: Body weight and fasting glucose levels increased significantly (45.8 ± 2.6g/13 ± 1.2 mmol in HFD Vs 25.6 ± 1g/5.25 ± 1.2 mmol in control, p < 0.005, t-test). Figure 1 summarizes the results obtained after fitting the data to the 1-compartment model.
Conclusion: HDF animals, which show prediabetic fasting blood glucose levels, display increased VTCA and Vdil fluxes that are in agreement with up-regulated neuronal metabolism and increased fatty acid uptake in the brain, in agreement with previous findings in diabetic animals and metabolic syndrome patients6
