Stroke leads to atherosclerotic plaque rupture and recurrent events via exacerbating inflammation and inflammasome activation
Stefan Roth1, Jiayu Cao1, Christina Fürle1, Gerrit Große2 and Arthur Liesz1,3
1Institute for Stroke and Dementia Research, University Medical Center Munich, Munich, Germany
2Department of Neurology, Medical University Hannover, Hannover, Germany
3Munich Cluster for Systems Neurology (SyNergy), Munich, Germany
Abstract
Background and aims: Patients with large artery atherosclerosis have the highest risk of recurrent event after stroke onset. However, the mechanism of how stroke leads to atherosclerotic plaque rupture and secondary ischemia is largely unknown.
Methods: Here we induced rupture-prone plaques by a tandem stenosis (TS) ligation of the right common carotid artery (RCCA) in high fat diet-fed ApoE−/− mice. 4 weeks after TS surgery, transient left middle cerebral artery occlusion (MCAO) surgery of the ApoE−/− mice are conducted. Brain secondary lesions were first detected by MRI scan 7 days after stroke, further confirmed by histology and immunofluorescence stainings. To analyze the morphological features of the plaques, RCCA were harvested 7 days after stroke and processed for histology analysis. Vascular inflammatory response was studied by western blot, flow cytometry and immunofluorescence stainings. In order to investigate the inflammasome impact, we used systemic in vivo administration of the caspase-1 inhibitor — VX765.
Result: Stroke induced recurrent ischemia in 30.5% ApoE−/− mice. As markers of plaque vulnerability, we observed by CCA histology analysis that stroke increased macrophage amounts and necrotic area, as well as decreased smooth muscle cells and collagen content, which make the plaque more vulnerable. Further detailed experiments showed by immunofluorescence staining that increased macrophage counts were due to increased proliferation rate; but in addition using a combination of adoptive cell transfer and flow cytometry we also observed higher number of monocytes recruitment. Caspase-1 as the central effector enzyme of the inflammasome, was observed elevated expression of both pro and active cleavage isoforms in RCCA tissue lysate after stroke, indicating post-stroke inflammasome activation within atherosclerotic plaques.
Stroke leads to plaque inflammasome activation which could be associated with increased macrophage counts and plaque vulnerability. Therefore, we aimed to target this pathway as a therapeutic approach. After systemic inhibition of caspase-1, stroke caused secondary event in 10.52% ApoE−/− mice. Examining the markers of plaque vulnerability by histology, we observed that VX765 treatment was able to reduce plaque inflammation while enhance stability. Further experiments showed that caspase-1 inhibition reduced macrophage proliferation rate as well as mitigated myeloid cell infiltration to plaques. Besides, plaque samples from symptomatic patients showed increased numbers of myeloid cells as well as elevated expression of pro-caspase-1 and its active isoforms when compared to asymptomatic patients. These results further confirm our pre-clinical findings.
Conclusion: Here we provide for the first time a mechanistic explanation for high recurrence rate of atherosclerotic stroke. Our study reveals that stroke leads to exacerbated plaque inflammation and ultimately plaque rupture by activation of the inflammasome. Additionally, inhibition of the inflammasome could be a novel and potent therapy to prevent recurrent ischemic events beyond currently established secondary prevention treatments.
582
BK channel activity in the cerebrovasculature is compromised in a mouse model of Alzheimer’s disease
Jade Taylor1,2, Harry Pritchard1,2, Katy Walsh1,2, Grant Hennig3, Mark Nelson3, Stuart Allan1,2 and Adam Greenstein1,2
1University of Manchester, Manchester, UK
2Geoffrey Jefferson Brain Research Centre, Manchester, UK
3University of Vermont, Burlington, USA
Abstract
Background: Mounting evidence suggests that cerebral microcirculatory dysfunction is key to Alzheimer’s disease (AD). Despite reduced cerebral blood flow (CBF) being an early pathogenic marker of AD, the underlying mechanisms behind this defect is currently unknown.
Aim: Here we aimed to determine if changes to vascular ion channels contribute to altered vascular function in a mouse model of AD.
Method: The APP23 mouse model has a seven-fold increase in amyloid precursor protein, leading to substantial amyloid-β (Aβ) plaque accumulation within the brain, as is described in AD patients. During this study we used a range of physiological techniques; pressure myography, electrophysiology and confocal microscopy, to investigate in depth, vascular ion channel function within the cerebral microvasculature of these mice.
Results/Conclusions: We show that attenuation of large conductance calcium–activated potassium (BK) channel function in this model, due to a reduced frequency of calcium sparks, is responsible for the reduction in cerebral blood flow described. Further investigation showed that wild-type vessels exposed to the Aβ1–40 peptide for 30 minutes exhibited significantly reduced BK channel function at both the cellular and vascular level. Acute exposure to Aβ1–40 resulted in an increase in calcium waves. Both a decrease in calcium sparks (as seen in the APP23 model) and an increase in calcium waves (as seen with the Aβ1–40 peptide) results in contraction of cerebral arteries and a subsequent reduction of CBF. The data directs future research into preventative strategies that may restore microvasculature function and promote a healthy brain environment to limit disease progression in AD patients.
583
The Alzheimer’s risk factor CD2AP drives defects across the cerebrovascular network
Milene Vandal1, Colin Gunn1, Adam Institoris1, Philippe Bourassa2, Ben Korin3, Suzie Lee1, Ramesh Mishra1, Govind Peringod1, Sotaro Hirai1, Yulan Jiang1, Camille Belzil1, Louise Reveret2, Cyntia Tremblay2, Mada Hashem1, Wilten Nicola1, Jakob Körbelin4, Jeff Dunn1, Andrew Braun1, David Bennett5, Andrey Shaw3, Grant Gordon1, Frédéric Calon2 and inh Dang Nguyen1
1Hotchkiss Brain Institute, University of Calgary, Calgary, Canada
2Axe Neurosciences, Centre de recherche du centre Hospitalier de l’Université Laval (CHUL), Quebec, Canada
3Departments of Research Biology, Genentech, Inc., South San Francisco, USA
4Department of Oncology, Hematology and Bone Marrow Transplantation, University Medical Center Hamburg-Eppendorf, Germany
5Rush Alzheimer’s disease Center, Rush University Medical Center, USA
Abstract
Background: Cerebrovascular impairment is amongst the earliest defects observed in Alzheimer’s disease (AD). Molecular mapping of the human brain vasculature suggests that brain endothelial cell (BEC) gene expression is differentially modulated across the vascular network in AD. CD2-associated protein (CD2AP), an important predisposing factor for the disease, is enriched in BEC but its function in the brain vasculature remains undefined.
Aim: To investigate the roles of CD2AP in cerebrovascular dysfunction in AD.
Method: We used diverse approaches including: human brain samples from AD volunteers, genetically modified mouse models, awake mouse two-photon microscopy and cell culture to study the vascular function of CD2AP.
Results/Conclusions: We show that AD individuals with lower levels of brain vascular CD2AP displayed the poorest cognitive performance. Genetic deletion of CD2AP in BEC of two distinct mouse models impairs memory function. Additionally, endothelial CD2AP deletion reduces resting cerebral blood flow (CBF) and neurovascular coupling (NCV) in pial vessels, arterioles, and capillaries. In BEC, CD2AP controls the levels and signaling of ApoER2. Activation of this pathway with Reelin glycoprotein mitigates the toxic effects of Aβ on capillary red blood cells speed and on vasomotion of arterioles deleted of CD2AP, but not during NVC. In sum, our data unveil that CD2AP drives cerebrovascular function through a novel pathway elicited by Reelin and counteracted by Aβ in specific brain vessel types.
585
Sleep efficiency is associated with white matter hyperintensity burden in non-demented, amyloid-beta negative individuals
Kristine Wilckens, Minjie Wu, Howard Aizenstein, Beth Snitz, Dana Tudorascu, Davneet Minhas, Brian Lopresti, Oscar Lopez, Charles Laymon, Victor Villemagne, Chester Mathis, William Klunk and Ann Cohen
University of Pittsburgh School of Medicine, Pittsburgh, USA
Abstract
Background: Preliminary evidence suggests that poor sleep is associated with both cerebrovascular disease and Alzheimer’s disease pathology. However, it is unclear whether associations between sleep and cerebrovascular disease depend on extent of Alzheimer’s disease pathology. Addressing this question is important to identify the individuals in which sleep interventions may be most effective for mitigating cerebrovascular disease.
Aim: Test cross-sectional associations between actigraphically assessed sleep time and sleep efficiency and white matter hyperintensities among individuals characterized on amyloid-beta (Aβ).
Method: 47 participants with normal cognition or mild cognitive impairment (MMSE > 24) completed a 11C-PiB- positron emission tomography to assess Aβ status (29 Aβ negative and 18 Aβ positive) and a T2-weighted magnetic resonance imaging scan for white matter hyperintensities. Partial correlation analyses were controlled for age and sex.
Results/Conclusions: Across all participants, there were no significant associations between sleep efficiency and white matter hyperintensities. Analyses stratified by Aβ status demonstrated significant associations between higher sleep efficiency and lower white matter hyperintensities in Aβ negative participants, across the whole brain, r = 0.48, p = 0.014, and in periventricular, r = 0.50, p = 0.010, and bilateral temporal regions, r’s > 0.4, p < 0.05. In Aβ positive participants, there were no significant associations between sleep and white matter hyperintensity burden. We conclude that sleep may be more related to cerebrovascular disease in individuals at low risk for Alzheimer’s disease, or before Aβ pathology develops. Future studies should examine these associations in Aβ positive individuals with a broader range of cognitive impairments and dementia.
588
Can sleep architecture affect brain clearance?
Alexandra Vallet1, Laura Bojarskaite2, Daniel Bjørnstad2, Miroslav Kuchta3, Rune Enger2 and Kent Mardal1,3
1Department of mathematics, University of Oslo, Oslo, Norway
2Institute of Basic Medical Sciences – University of Oslo, Oslo, Norway
3Department of Numerical Analysis and Scientific Computing – Simula Research Laboratory, Oslo, Norway
Abstract
Background: Recent studies showed that waste clearance from the brain is more efficient during sleep than wakefulness.1,2 Sleep stages are associated with specific oscillatory vasomotion patterns that subsequently affect the flow of cerebrospinal fluid (CSF).3 These oscillations are believed to affect the so-called ‘glymphatic system’ where brain metabolic wastes are transported by CSF in perivascular spaces (PVSs).
Aim: To quantify how different stages in sleep affect brain clearance through the mechanical coupling between the cerebral vascular system and the CSF.
Method: We developed a mathematical model of CSF flow and tracer transport around an oscillating vessel wall. The simulations based on the mathematical model (Figure 1(a)) were used to interpret in vivo measurements, with two-photon laser imaging, of vessel and astrocyte end foot displacements in unanesthetized mice across the sleep-wake states (Figure 1(b)).
Results/Conclusions: During NREM sleep in mice, we observed low-frequency oscillations that enhanced solute transport in PVS in the associated simulations (Figure 1(c)). During REM sleep, we observed large dilation of the vessels which leads to an increased brain clearance. Sleep architecture may therefore play an important role in brain clearance during sleep.Figure 1. Simulation of fluid flow and tracer transport around a vessel during sleep in mice. a – Geometry and typical velocity field. b – Two-photon imaging of PVS deformation. c – Diffusion transport enhancement (in pourcentage).
Neuronal Serpina3n is an endogenous protector against blood brain barrier damage following cerebral ischemic stroke
Feng Shi Li, Pei ying Li and Jie qing Wan
Shanghai Jiaotong University, Shanghai, China
Abstract
Backgrounds and aims: Cerebral ischemic stroke results in blood-brain barrier (BBB) disruption, in which the reciprocal interaction between ischemic neurons and components of the BBB appears to play a critical role. However, the underlying mechanisms for BBB protection remain largely unknown.
Methods and results: In this study, using RNA-seq, immunofluorescence, and flow cytometry, we found that Serpina3n, a serine protease inhibitor, was significantly upregulated in the ischemic brain, predominantly in ischemic neurons 6 hours to 3 days after stroke. Using neuron-specific adeno-associated virus (AAV), intranasal delivery of recombinant protein, and immune-deficient Rag1 mice, we demonstrated that Serpina3n attenuated BBB disruption and immune cell infiltration following stroke by inhibiting the activity of granzyme B (GZMB) and neutrophil elastase (NE) secreted by T cells and neutrophils. Furthermore, we found that intranasal delivery of rSerpina3n significantly attenuated the neurologic deficits after stroke. In conclusion, Serpina3n is a novel ischemic neuron-derived proteinase that counterbalances the BBB disruption induced by peripheral T cell and neutrophil infiltration after stroke.
Conclusions: The above finding reveals a novel endogenous protective mechanism of BBB damage after cerebral ischemia and Serpina3n is a potential therapeutic target for ischemic stroke.
592
Stroke induces long-term innate immune memory in hematopoietic stem cells via IL1b-driven mechanisms
Alba Simats1, Sijia Zhang1, Eduardo Beltrán2,3,4 and Arthur Liesz1,3
1Institute for Stroke and Dementia Research (ISD), University Hospital, LMU Munich, Munich, Germany
2Biomedical Center Munich, Faculty of Medicine, LMU Munich, Munich, Germany
3Munich Cluster of Systems Neurology (SyNergy), Munich, Germany
4Institute of Clinical Neuroimmunology, University Hospital, LMU Munich, Munich, Germany
Abstract
Background: Stroke has a profound impact on systemic immunity. Peripheral immune changes after stroke might have a potential impact on long-term stroke recovery and secondary comorbidities.
Aim: Understand the long-term consequences of stroke on systemic immunity.
Method: Stroke was induced in C57BL6/J adult mice via transient middle cerebral occlusion (tMCAo). Quantitative and phenotypic analyses of myeloid immune cells in blood and bone marrow (BM) were performed using multiparametric flow cytometry and single-cell RNA sequencing (scRNA-Seq). To unravel potential gene regulatory interactions, BM myeloid lineage cells were analyzed via the single-nuclei sequencing assay for transposase-accessible chromatin (scATAC-Seq) and scRNA-Seq, simultaneously. Furthermore, BM cells isolated from stroke mice and enriched in myeloid progenitors were transplanted into BM-depleted naïve recipients and evaluated by scRNA-Seq 1 month later to seek for cell-intrinsic immune memory traits. Finally, we assessed the role of IL1β on post-stroke long-term changes in BM cellularity via the administration of a neutralizing antibody against IL1β within the hyper-acute phase after stroke.
Results/Conclusions: Our findings suggest that stroke increases absolute myeloid progenitors and mature immune cell counts not only within one day but for at least one month after stroke. Stroke also results in long-term phenotypic changes of myeloid lineage cell populations in the BM, which chronically retain a pro-inflammatory phenotype. These changes are cell-intrinsic adaptations that persist over time and after being transplanted into naïve mice. This myeloid immune memory might be potentially mediated by IL1β-driven mechanisms, and could have translational relevance as a driver of secondary inflammatory comorbidities after stroke.
606
Evidence that CD206+LYVE1+ border-associated macrophages accumulate beyond the period of infarct development after ischaemic stroke
Gabriela Gerganova, Arun Flynn, Emily Gallen, Ashton Bernard and Alyson Miller
University of Glasgow, Glasgow, UK
Abstract
Background: Our understanding of the roles of CNS border-associated macrophages (BAMs) in the diseased brain are only beginning to emerge. Recent studies indicate that BAMs accumulate and possibly migrate from perivascular spaces to the parenchyma in the early phases of infarct development following ischaemic stroke and may make a deleterious contribution. However, it is unknown if BAMs accumulate beyond the period of infarct development and whether they also have reparative roles like their distant cousins (microglia).
Aim: Determine if ischaemic stroke is accompanied by an increase in BAM number and/or altered localisation during and following the period of infarct development.
Method: Ischaemia was induced by middle cerebral artery occlusion (MCAo) followed by reperfusion for 1, 3, or 7 days. BAM (CD206+LYVE1+) numbers and their localisation were assessed by flow cytometry and double-label immunofluorescence.
Results/Conclusions: We found that the number of CD206+LYVE1+ cells increased ∼4-fold in the ischaemic hemisphere during the period of infarct development (day 1–3) relative to the contralateral hemisphere or sham-controls (sham: 270 ± 79; contralateral: 188 ± 57; ischaemic: 835 ± 146; cells per hemisphere, n = 9–10, P < 0.05). Furthermore, this increase was sustained (∼20-fold) beyond the period of infarct development (day 7; sham: 54 ± 37; contralateral: 92 ± 32; ischaemic: 1814 ± 681, n = 3, P < 0.05). Immunofluorescent analyses thus far suggest that CD206+LYVE1+ cells primarily accumulate around vessels located in the peri-infarct zone (day 3). The findings of this study so far suggest that BAMs might accumulate in the ischaemic brain beyond the period of infarct development, raising the possibility that they may also have roles in neurovascular repair.
607
Network hubs revealed by “metabolic connectivity” mapping from [18F]FDG kinetic parameters
Tommaso Volpi1,2, Giulia Vallini3, John Lee4, Manu Goyal4, Andrei Vlassenko4, Maurizio Corbetta1,2 and Alessandra Bertoldo1,3
1Padova Neuroscience Center, University of Padova, Padova, Italy
2Department of Neuroscience, University of Padova, Padova, Italy
3Department of Information Engineering, University of Padova, Padova, Italy
4Department of Radiology, Washington University School of Medicine, Saint Louis, USA
Abstract
Background: Research on ‘metabolic connectivity’ (MC), conventionally assessed as across-subject covariation of [18F]FDG PET measurements, is expanding, but so far only semi-quantitative indices, i.e., standard uptake value ratio (SUVR), have been employed.1
Aim: Here, we estimate across-subject MC from SUVR and [18F]FDG kinetic model parameters, i.e., tracer uptake (Ki), inflow (K1), and phosphorylation (k3), and assess their network structure by identifying their “hub” nodes.2
Method: Dynamic [18F]FDG data (60 min) were acquired from 54 healthy subjects (57.4 ± 14.8 yo). Image-derived input functions were extracted and corrected for spillover.3 Voxel-wise quantification was performed using Sokoloff’s model.4SUVR (40–60 min) relative to whole-brain mean [18F]FDG uptake was calculated. Ki, K1, k3, and SUVR voxel-wise maps were parcelled using the Hammersmith atlas4 and within-subject z-scored. Across-subject Pearson’s correlation matrices of each parameter were thresholded (20% density). For each MC matrix, node degree (DEG) and eigenvector centrality (EC) were calculated to assess “hub” nodes2 (Figure 1(a)). Combination hubs were identified as the nodes in the top 20% of both DEG and EC distributions (Figure 1(b)).
Results/Conclusions: The identified combination hubs are consistent with those previously reported for SUVR MC.1 Notably, SUVR, Ki, and K1 MC display similar distribution of hub nodes (temporal, insular and cingulate cortex), while the k3 network structure is remarkably different, highlighting frontoparietal and subcortical areas. How these results relate to single-subject MC estimates5 remains to be explored.Figure 1. ▪.
Predictability of the molecular MRI, and the role of perivascular macrophages in intracerebral aneurysm outcome
Martina Glavan1, Damien Levard1, Bart Franx2, Maxime Gauberti1,3, Denis Vivien1,4 and Marina Rubio1
1INSERM, Physiopathology and Imaging of Neurological Disorders, UMR-S 1237, GIP Cyceron, Institut Blood and Brain @ Caen-Normandie (BB@C), Normandie Université, Caen, France
2Biomedical MR Imaging and Spectroscopy Group, Center for Image Sciences, University Medical Center Utrecht and Utrecht University, Utrecht, Netherland
3CHU Caen, Department of Radiology, Caen University Hospital, Caen, France
4Caen-Normandie University Hospital (CHU), Department for Clinical Research, Caen, France
Abstract
Background: Intracranial aneurysms (IAs) are brain vascular malformations which rupture leads to subarachnoid haemorrhage, a significant cause of disability and death. Consequently, a considerable effort has been placed in determining the mechanisms of aneurysm formation, growth, and rupture. A chronic vascular inflammation is the common key element of these processes.1
Aim: The aim of our research is to assess the inflammatory mechanisms concerning aneurysms, focusing on vascular inflammatory markers (VCAM-1, P-selectin), and the perivascular macrophages (PVMs). For this reason, we first developed a new mouse model of IAs at the Middle Cerebral Artery.
Method: As an original tool to non-invasively assess the vascular inflammation, our group developed molecular MRI approach to target adhesion molecules expressed at the surface of activated brain endothelial cells.2 Role of PVMs is studied by their global depletion (injecting the clodronate-liposomes), MRI and the immunohistochemistry analysis.
Results/Conclusions: We obtained 3D reconstructions of the brains at different time points, with aneurysms at different stages and the vessel inflammatory markers (Figure 1(a)). Quantifying the expression of the vascular adhesion molecules, we were able to predict the outcome of the aneurysm rupture in our mouse model. Moreover, a global PVM depletion showed to reduce the incidence of ruptured aneurysms. In addition, PVM depleted group showed significantly smaller haemorrhages in compared to the control group. This data shed a light on the interesting unexplored role of PVMs in the aneurysm pathophysiology, as well as the molecular MRI potential to become a future prognostic and diagnostic tool in clinic.
Figure 1. Molecular MRI imaging of vascular inflammation in MCA aneurysm mouse model. (a) Representative MRI images before and after intravenous injection of MPIO-αP-selectin and MPIO-αVCAM-1 at Day3 post-aneurysm induction. (b) Strong correlation between the P-selectin signal on the MRI at Day3, and the haemorrhage volume after the rupture (Day5) (c) Strong correlation between the VCAM-1 signal on the MRI at Day3, and the haemorrhage volume after the rupture (Day5)
References
Chalouhi, et al. Biology of intracranial aneurysms: role of inflammation. J Cereb Blood Flow Metab 2012 doi: 10.1038/jcbfm.2012.84.a-11Gauberti, et al. Ultra-sensitive molecular MRI of vascular cell adhesion molecule-1 reveals a dynamic inflammatory penumbra after strokes. Stroke 2013 doi: 10.1161/STROKEAHA.111.000544.a-12
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Three-photon imaging of neurovascular coupling across neocortical layers to determine the neural basis of fMRI
Prakash Kara1, Arani Roy1, Chao Liu1, Shinho Cho1, Philip O’Herron2, Wei Chen1, Xiao-Hong Zhu1, Austin Leikvoll1, Deano Farinella1, Harishankar Jayakumar1, Erik Tilseth1 and Kamil Ugurbil1
1University of Minnesota, Minneapolis, USA
2Augusta University, Augusta, USA
Abstract
Background: The visual cortex of the non-rodent brain is ideally suited to study the spatial organization of neurovascular coupling at the level of synapses, neurons, individual blood vessels and laminar-resolution fMRI because it is organized in functional neural columns and the array of visual stimuli which are needed to optimally drive neurons are established. Specifically, feature selectivity such as orientation tuning emerges in layer 4 neurons from untuned thalamic inputs. Tuning is further specialized in layer 2/3 neurons. Thus, in a specific cortical layer, neuro-vascular coupling can be precisely quantified in terms of tuning curves of neural and vascular selectivity.
Aim: To determine the extent to which different types of neural (spiking, synaptic) and vascular signals (blood flow from individual vessels and fMRI voxels) are coupled across cortical layers.
Method: Multi-photon optical imaging and cerebral blood volume weighted fMRI in cat visual cortex.
Results/Conclusions: In layer 2/3 single blood vessel responses were partially decoupled from spiking and synaptic activity. Specifically, individual blood vessel responses were tuned for stimulus orientation but neural responses were far more selective. These differences are likely due to the propagation of dilation from one orientation column into adjacent ones. In pial and layer 4 vessels, the responses were completely untuned for stimulus orientation. For hemodynamic responses, both fMRI and optical imaging showed a consistent laminar response pattern in which orientation selectivity in cortical layer 4 was lower compared to layer 2/3. This systematic change in selectivity across cortical layers has a clear underpinning in neural circuitry.
639
Cerebral ischemia following Middle cerebral artery occlusion in rats measured by near infrared spectroscopy
Kelly Drew1, Ardy Wong2, Mohammad Iqbal3, Jeffrey Rothman1, Kambiz Pourrezaei4, Dandan Sun3 and Zeinab Bahareh Barati3
1University of Alaska Fairbanks, Fairbanks, USA
2Barati Medical, Fairbanks, USA
3University of Pittsburgh, Pittsburgh, USA
4Drexel University, Philadelphia, USA
Abstract
Background: Timely and sensitive in vivo estimation of ischemic stroke-induced brain infarction is necessary to guide diagnosis and evaluate treatment efficacy. The gold standard for estimation of the cerebral infarction volume is magnetic resonance imaging, which is not readily accessible. Measuring rCBF with Laser Doppler flowmetry is the status quo for confirming reduced blood flow in experimental ischemic stroke models. However, rCBF often does not correlate with subsequent infarct volume_ENREF_7.
Aim: In the present study, we ask if continuous-wave near infrared spectroscopy (NIRS) measurement of cerebral oxygenation during intraluminal middle cerebral artery occlusion (MCAO) in Sprague-Dawley rats (n = 8-male) will predict infarct volume.
Methods: The NIRS device consisted of a controller module and an optical sensor with two LED light sources and two photodiodes making up two parallel channels for monitoring left and right cerebral hemispheres. Optical intensity measurements were converted to deoxyhemoglobin (Hb) and oxyhemoglobin (HbO2) changes relative to a 2-min window prior to MCAO. Area under the curve for Hb and HbO2 were calculated for the 90-min occlusion period for each hemisphere as a measure of total ischemia. Infarct volume (IV) was calculated by triphenyl tetrazolium chloride staining at 24 h reperfusion.
Results/Conclusion: Results showed a negative correlation (r = −0.77, p = 0.03) between total deoxygenation measured as normalized Hb (auc(Hbips) – auc(Hbcon)) and IV. Our results show feasibility of using NIRS to monitor cerebral ischemia in a rodent stroke model. This cost-effective, non-invasive technique may improve experimental models of ischemic stroke by enabling in vivo and longitudinal assessment of ischemic injury. P20GM130443, 1R43NS100174
Poster – Late breaking
579
Sub-dose anesthesia combined with chloride regulator protects the brain resist chronic ischemia-hypoxia injury
Ye Wang, Chenyi Yang and Haiyun Wang
Tianjin Third Central Hospital, Tianjin, China
Abstract
Background: Cerebral ischemia-hypoxia leads to excitotoxicity-mediated neuronal damage and cognitive dysfunction, especially in the elderly. Excessive intracellular [Cl−]i accumulation weakens γ-aminobutyric acid (GABA) compensatory effects. Sub-anesthetic dose of propofol protected the brain against ischemia-hypoxia, which was abolished by blocking Cl− efflux transporter K+/Cl− cotransporter 2 (KCC2). We aimed to determine whether low dose anesthetic combined with [Cl−]i regulators could restore the compensatory GABAergic system and improve cognitive function.
Methods: Chronic cerebral hypoxia (CCH) model was established by bilateral carotid artery ligation in aged rats. Sub dose of anesthetics (propofol and sevoflurane) with or without KCC2 agonist N-ethylmaleimide (NEM) or Na+/K+/Cl− cotransporter 1 (NKCC1) antagonist bumetanide (BTN) were administered systemically 30 days post-surgery. Primary rat hippocampal neuronal cultures were subjected to hypoxic injury with or without drug treatment. Memory function, hippocampal neuronal survival, GABAergic system functioning and brain-derived neurotrophic factor (BDNF) expressions were evaluated.
Results/Conclusions: Sub-anesthetic dose of combined propofol (1.2 μg/ml) and sevoflurane [0.7 MAC (minimum alveolar concentration)] did not aggravate the hypoxic brain injury in rats or cell damage in neuronal cultures. Adding either BTN or NEM protected against hypoxic injury, associated with improved cognitive function in vivo, less intracellular accumulation of [Cl−]i, reduced cell death, restored GABAergic compensation, and increased BDNF expression both in vivo and in vito.
Sub-anesthetic dose of propofol and sevoflurane is a recommended anesthesia regimen in at-risk patients. Restoration of [Cl−]i homeostasis and GABAergic could further reduce the brain damage caused by ischemia-hypoxia.
580
Cellular mechanisms after transient ischemic attack
Gemma Llovera1, Nicolett Lenart2, Steffanie Heindl1, Daniel Varga1, Ádam Dénes2 and Arthur Liesz1,3
1Institute for Stroke and Dementia Research, München, Germany
2Momentum Laboratory of Neuroimmunology, Institute of Experimental Medicine, Budapest, Hungary
3Munich Cluster for Systems Neurology (SyNergy), Munich, Germany
Abstract
Background: Although transient ischemic attack (TIA) symptoms last 24 h and do not cause permanent brain damage, 20% of total ischemic stroke cases are preceded by transient ischemic symptoms and nearly half of the strokes will occur within the first days after a TIA.
Aim: Identify the cellular mechanisms underlying TIA.
Methods: Adult male C57BL/6J mice were subjected to 5 min transient middle cerebral artery occlusion (tMCAo). Cortical connectivity was studied by wide-field Ca2+ imaging in male Thy1-GCaMP6s mice and Golgi-Cox and synapse staining (VGLUT/Homer) further assessed neuronal function. In addition, microglia morphology and motility analysis were performed, as well as microglia transcriptomics to investigate microglial activation. To study a possible reverse of TIA, two microglia therapeutic approaches were tested: complete microglia ablation via PLX5622 and inhibition of microglial activation using P2Y12R antagonist (PSB0739).
Results/Conclusions: Although memory deficits and focal deficits were detected up to 3 days after TIA, a significant reduction in cortical connectivity, spine density and cortical synapses were undetectable after 24 hours after TIA. In-depth analysis of microglia revealed “transient” changes in morphology and motility only up to 24 hours after TIA, but “permanent” transcriptomic changes up to 7 days after TIA. Our study demonstrates for the first time that microglial cells not only play a key role in regulating neuronal function early in TIA, but also undergo permanent transcriptomic changes. Furthermore, only P2Y12R inhibition was able to improve the TIA phenotype. These findings should be taken into account in future studies examining neuroinflammatory mechanisms and immunotherapies for TIA.
584
Development of a flow-phantom for transcranial doppler ultrasound quality assurance
Fatmah Alablani1,2,*, Justyna Janus1,3,* and Emma M.L. Chung1,3,4,5
1Radiology and Medical Imaging Department, College of Applied Medical Sciences, Prince Sattam bin Abdulaziz University, Kharj, Saudi Arabia
2Cerebral Haemodynamics in Aging and Stroke Medicine (CHIASM) Group, Department of Cardiovascular Sciences, University of Leicester, Leicester, UK
3Medical Physics Department, University Hospitals of Leicester NHS Trust, Leicester, UK
4National Institute for Health Research Leicester Biomedical Research Centre, Leicester, UK
5Faculty of Life Sciences and Medicine, Guy’s Campus, King’s College London, London, UK
*Joint authors.
Abstract
Introduction: Transcranial Doppler (TCD) ultrasound blood flow velocity estimates are highly variable due to variations in probe placement with respect to anatomy and the absence of B-mode angle correction. Additional potential sources of variation occur due to differences in hardware and software between manufacturers. The aim of this study was to use a Middle Cerebral Artery (MCA) flow phantom to facilitate comparison of TCD velocity estimates between manufacturers under controlled conditions.
Method: An MCA flow phantom was fabricated to include 3 mm internal diameter c-flex tubing, embedded within a validated tissue-mimicking material at an angle of 30°. A programmable pump was used to circulate blood mimicking fluid (BMF), reproducing a clinically relevant range of steady and pulsatile flow values. Time-averaged velocity estimates were obtained under identical conditions using two TCD devices: an ST3 Doppler system (Spencer Technologies, MA) and a TCD DopplerBox (DWL, Germany). Timed collection of fluid, and angle-corrected velocity estimates obtained using Zonare duplex scanner (Zonare Medical Systems Inc., Mountain View, CA, USA), were used to provide reference (gold standard) velocity estimates for comparison with TCD.
Results: Velocity estimates from DWL TCD were ≈3% higher than Spencer device in Bland-Altman analysis, with a significant mean difference (P = 0.009). This difference in TAMMV estimates increasing significantly with high velocities reaching 5.7% in ≈200 cm/s values.
Conclusion: This MCA phantom provides a valuable tool for quantifying differences in TCD velocity estimates between manufacturers. Extra care may be needed to adjust for bias between manufacturers when using high velocity measurements to inform clinical care.
Xinyue Zhang, Beibei Dong, Yi Jiang, Yuanyuan Bai and Yonghao Yu
Tianjin Medical University General Hospital, Tianjin, China
Abstract
Background: Exosome secretion is an important paracrine way of endothelial progenitor cells (EPCs) to modulate resident endothelial cells, which have been demonstrated curative potential in multiple diseases, including ischemic diseases. Ischemic preconditioning (IPC) can improve the prognosis of cerebral ischemia-reperfusion (I/R) injury via establishing ischemia tolerance.
Aim: We focused on the effects of EPCs-derived exosomes in cerebral I/R injury and further studied whether ischemic pretreatment of EPCs could obtain more effective exosomes.
Method: We established oxygen glucose deprivation (OGD) model in vitro and isolated exosomes from different EPCs-conditioned medium (EPCs cultured under normal condition or OGD treatment for 3 h). Neuronal viability, apoptosis and inflammatory cytokines were tested. In vivo, mouse models of middle cerebral artery occlusion (MCAO) were established. Exosomes were engineered with RVG peptide on the surface for brain targeting and were injected to mice intravenously. Neurological deficits, infarct volumes, neural plasticity and angiogenesis were examined.
Results: Our findings demonstrated that EPCs-derived exosomes could effectively alleviated OGD-induced neuron death and apoptosis in vitro. In addition, intravenous administration of these exosomes in mice could decrease the infarct volume, enhance neurogenesis and angiogenesis, as well as alleviate BBB impairment. Exosomes derived from ischemic preconditioned EPCs exerted more effective neuroprotection than that derived from normal EPCs.
Conclusions: Our results indicated that exosomes derived from EPCs can promote neurovascular repair and alleviate cerebral I/R injury. Besides, ischemic preconditioning of EPCs could enhance the efficacy of derived exosomes, which might contribute to a better therapeutic strategy for cerebral I/R treatment.
587
Role of autotaxin in central nervous system autoimmunity
Background: Multiple sclerosis (MS) is an immune-mediated inflammatory disease of the central nervous system (CNS). A defining characteristic of MS is the ability of autoreactive T lymphocytes to cross the blood brain barrier (BBB) and mediate inflammation within the CNS. Previous study from our lab has found the gene ENPP2 to be highly upregulated in encephalitogenic T cells in the mouse model of MS. ENPP2 codes for the secreted protein autotaxin which promotes transendothelial migration of T cells from the blood stream into the lymphatic system. Our hypothesis is that inhibiting autotaxin signaling may prevent autoreactive T cells from crossing the BBB and causing neuroinflammation.
Aim: Aim 1: Do MS patient CD4+ T cells show differential expression of autotaxin compared to healthy controls?
Aim 2: Does pharmacological inhibition of autotaxin ameliorate the mouse model of MS, experimental autoimmune encephalomyelitis (EAE)?
Method: Peripheral blood mononuclear cells from MS patients and healthy controls were activated and analyzed for changes in autotaxin expression via flow cytometry. For EAE studies, mice were immunized to induce EAE and then treated with autotaxin inhibitor HA-130. Changes to disease severity were measured by tracking clinical score.
Results/Conclusions: Our data show that MS patients’ CD4+ T cells differentially express autotaxin compared to healthy controls. In the context of EAE, we have found that treatment with autotaxin inhibitor HA-130 decreases EAE severity but does not affect the ability of CD4+ T cells to clear viral infection, suggesting autotaxin inhibition may be a viable therapeutic target.
593
The profile of extracellular vesicles in intracerebral hemorrhage patients
Ji Bihl1, Harshal Sawant1, Doan Nguyen2, Trevor Bihl3 and Ifeanyi Iwuchukwu2
1Marshall University, Huntington, USA
2Ochsner Medical Center, Jefferson, USA
3Wright State University, Dayton, USA
Abstract
Background: Intracerebral hemorrhage (ICH) is one of the leading life-threatening types of strokes with high mortality. A prominent feature of ICH is neuroinflammation involving neutrophils and macrophages. Circulating microvesicles (MV) and exosomes (EX) are used as biomarkers for different diseases.
Aim: We aimed to determine the concentration of MVs/EXs from neutrophils and microglia in ICH patients and analyze the correlation of these MVs/EXs with clinical parameters.
Method: MVs/EXs were isolated from the plasma of ICH patients (n = 39) by using the serial centrifuge methods. Nanoparticle tracking analysis (NTA, NS300) was used to determine the type and concentration of neutrophil and macrophage-released MVs/EXs. Specific antibodies, CD66b, and P2RY12 were used for neutrophil and microglia, respectively.
Results/Conclusions: A predictive relationship between both hospital length of stay (R2 = 0.83) and Intensive care units (ICU) length of stay (R2 = 0.88) was found with MVs and EXs and patient data [including low-density lipoprotein (LDL), ICH volume, etc,]. Further predictive multiple linear regression relationship was seen between MV and EX concentrations and MSRV3 (outcome at 90 days) (R2 = 0.46) and MSRV5 (outcome at 180 days) (R2 = 0.51). Further, slight multiple linear regression relationships were seen between MV and EX concentrations and both hospital length of stay (R2 = 0.18) and ICU length of stay (R2 = 0.26). LDL and systolic blood pressure at admission were found to have a slight predictive relationship with neutrophils-MVs (R2 = 0.31) and age, race, hypertension, and the ICH location were found to be predictive of MV concentration (R2 = 0.46). This study found predictive relationships between patient outcomes and MVs and EXs.
594
3-D optogenetic control of arteriole diameter in vivo
Philip OHerron1,2, David Hartmann2,3, Kun Xie1, Prakash Kara2,4 and Andy Shih2,5,6
1Augusta University, Augusta, USA
2Medical University of South Carolina, Charleston, USA
3Stanford University, Stanford, USA
4University of Minnesota, Minneapolis, USA
5Seattle Children’s Research Institute, Seattle, USA
6University of Washington, Seattle, USA
Abstract
Arteries in the brain rapidly dilate in a spatially localized fashion when there is an increase in local neural activity – a process called functional hyperemia – which is the basis of hemodynamic imaging techniques like fMRI. Altered vascular function, including impaired functional hyperemia and reduced blood flow, is now seen as a contributing factor in many neurological disorders. Thus, there is a critical need for an accurate understanding of vascular dynamics and of how neural activity interacts with and shapes hemodynamic responses.
Here we present an experimental paradigm to optically probe the contractile function of arterioles in vivo with high spatiotemporal precision, independently of activating neurons and without the use of pharmacological agents. This was done by expressing the excitatory opsin ReaChR in vascular smooth muscle cells and pericytes. Using a 594 nm light-emitting diode we were able to evoke widespread vasoconstriction across the cranial window. With a 1040 nm focused, pulsed laser for two-photon stimulation, we were able to evoke highly localized constrictions targeted to individual pial artery branches or penetrating arterioles. Our dual light-path imaging system allowed the optogenetic stimulation to be performed with simultaneous two-photon imaging to monitor vessel activity. Using a spatial light modulator, we were also able to constrict vessels both above and below the imaging plane. This is a powerful tool to assay vasoconstrictive function of single arterioles across 3-dimensional vascular networks in vivo. We demonstrate its usefulness for studying the conductance of vascular signals and manipulating blood flow during functional hyperemia.
595
Computational fluid dynamics identified the effect of blood transfusion on cerebral hemodynamics and vascular topology
Russell Sawyer1, Sirjana Pun2, Michael DeBaun3, Riccardo Barille2 and Hyacinth I Hyacinth1
1Department of Neurology and Rehabilitation Medicine, University of Cincinnati, Cincinnati, USA
2Department of Biomedical Engineering, University of Cincinnati, Cincinnati, USA
3Vanderbilt-Meharry Center of Excellence in Sickle Cell Disease, Vanderbilt University Children’s Hospital, Nashville, USA
Abstract
Background: Stroke and Silent cerebral infarct (SCI) are frequent complications of Sickle Cell Disease (SCD), though chronic blood transfusion (cRBC) therapy constitutes the most effective therapy for primary and secondary prevention of stroke/SCIs. High (≥200 cm/s) transcranial Doppler ultrasound (TCD) velocity of blood flow identifies patients SCD who are at high risk for stroke/SCIs, even patients with normal TCD velocity also develop stroke/SCIs.
Aim: To use computational fluid dynamics (CFD) modeling to examining the impact of cRBC transfusion on vascular topology and hemodynamics.
Methods: Baseline, pre-randomization and study exit magnetic resonance angiogram (MRA) images from two participants in the SIT trial, were analyzed using CFD modeling. Patient 1 received standard care and Patient 2 received cRBC transfusion. We reconstructed the intracranial portion of the carotid artery and branches and extracted the geometry using Mimics Research 18. We cut specific potions of the large intracranial artery to include the ICA, MCA, and ACA, such that the vessel segment analyzed, extends from the intracranial beginning of the ICA up to immediately after the emergence of the MCA and ACA (Figure 1). Cut models were imported into Ansys and laminar and time-dependent simulation was performed for CFD assessment.
Results and Conclusion: Figure 1(a) shows the velocity streamline results of both patients. Patient 2 showed a steady decrease in velocity in the MCA from baseline to study exit while there were no velocity changes for Patient 1. Similarly, Patient 2 showed less areas of low wall shear stress (Figure 1(b)) and improvement of vessel tortuosity (Table 1) from baseline to study exit. The results of this study identify potential mechanism(s) of the benefit of cRBC transfusion therapy in decreasing the TCD velocity that is associated with an increased risk of SCIs and strokes.
596
Intravenous hematopoietic stem-cell transplantation in aged mice improves neurogenesis at dentate gyrus and short-term memory
Sheraz Gul1, Carsten Claussen1, Rie Akamatsu2, Yuka Okinaka2, Johannes Boltze3 and Akihiko Taguchi2
1Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, Germany
2Department of Regenerative Medicine Research, Foundation for Biomedical Research and Innovation, Kobe, Japan
3School of Life Sciences, University of Warwick, Coventry, UK
Abstract
The development of therapies for Alzheimer’s disease has largely been based on the concept of (a) inhibiting and removing toxic substances such as amyloid-β, (b) suppressing existing neuronal cell death, and (c) preventing the onset of Alzheimer’s disease. However, clinical trials using various therapies to target these have shown unsatisfactory results. A hallmark of Alzheimer’s disease is short-term memory loss and the absence of newborn neurons in the hippocampus. Therefore, we considered that a novel therapeutic target for Alzheimer’s disease are the newborn neurons in the hippocampus.
We previously reported that the mechanism of action of hematopoietic and mesenchymal stem-cell therapies, which are widely used in regenerative medicine, involves cell-cell interaction via gap junction. Therefore, stem-cell approaches may be useful in activating hippocampal neurogenesis as an experimental treatment Alzheimer’s disease. Our experimental data supporting this is:
The number of newborn neurons in the hippocampus of aged mice that exhibit short-term memory deficit was drastically reduced.
Intravenous administration of stem-cells into aged mice improved short-term memory and significantly increased the number of newborn neurons in the hippocampus.
Metabolism-related gene expression in peripheral blood leukocyte cells is altered in aged mice.
Intravenous administration of stem-cells into aging mice induced metabolic status of peripheral blood leukocytes similar to that of young mice through cell-cell interactions mediated by gap junction.
RNA expression of gap junction is decreased in hippocampal tissues & peripheral blood leukocytes in aged mice and cultured vascular endothelial cells even following passage.
RNA expression of gap junction in cultured vascular endothelial cells was increased when co-cultured with hematopoietic stem-cells.
In conclusion, we showed that (a) a rational target for Alzheimer’s disease is the activation of newborn neurons in the hippocampus, (b) the stem-cell based therapy offers the potential to cure dementia rather than prevent it, and (c) the central mechanism of its efficacy is cell-cell interaction via gap junction. This is a new perspective to treat Alzheimer’s disease. Cell-cell interactions via gap junction play an important and direct role in multicellular organisms and this function declines with age. Stem-cell based regeneration enhances these interactions, suggesting that cell-cell interactions via gap junction have a key role during the aging of multicellular organisms. We believe that cell-cell interactions via gap junction will shed a new light on the process of aging science and the development of therapies.
597
Effects of PCSK9 inhibition or deletion on intracerebral hemorrhage lesion volume in mice
Paul Fischer1, Frieder Schlunk1, Hans MG Princen2, André Rex1, Vincent Prinz3, Marco Foddis1, Dieter Lütjohann4, Ulrich Laufs5 and Matthias Endres1,6,7,8
1Department of Neurology with experimental Neurology, Center for Stroke Research Berlin and Neurocure Charité University Medicine Berlin, Berlin, Germany
2Metabolic Health Research, The Netherlands Organization of Applied Scientific Research (TNO), Gaubius Laboratory, Leiden, The Netherlands
3Department of Neurosurgery, Charité University Medicine Berlin, Berlin, Germany
4Department of Clinical Pharmacology, University of Bonn, Bonn, Germany
5Department of Cardiology, University Medicine Leipzig, Leipzig, Germany
6DZHK (German Center for Cardiovascular Research), Berlin, Germany
7Berlin Institute of Health (BIH), Berlin, Germany
8German Center for Neurodegenerative Diseases (DZNE), Berlin, Germany
Abstract
Background: Anti-PCSK9-antibodies prevent cardiovascular events by effectively lowering plasma cholesterol levels. An inverse correlation between plasma cholesterol levels and risk of intracerebral haemorrhage (ICH) has been suggested.
Aim: In a mouse model of ICH, we test whether PCSK9 inhibition using an anti-PCSK9-antibody or PCSK9 knock out affect hemorrhagic lesion volume and investigate the mechanistic role of plasma cholesterol levels.
Method: APOE*3Leiden.CETP or B6129SF1/J mice were treated with anti-PCSK9-antibody or vehicle for 6 weeks and fed western type diet. A separate cohort of PCSK9−/− mice or B6129SF1/J controls received normal chow. Plasma cholesterol levels were computed using a colorimetric assay, ICH induced by stereotactic intrastriatal injection of collagenase VIIs and hemorrhage volumes measured spectrophotometrically 6 or 24 h after haemorrhage induction.
Results/Conclusions: Compared to severely hypercholesterolemic vehicle-treated controls plasma cholesterol levels were markedly decreased in APOE*3Leiden.CETP mice treated with anti-PCSK9-antibody while hemorrhagic lesion volume was increased by 50%. In contrast no difference in hemorrhagic lesion volume between groups was observed in mildly hypercholesteremic B6129SF1/J mice receiving vehicle or anti-PCSK9-antibody. Likewise, PCSK9 knock out did not affect lesion volumes, while yielding very low cholesterol levels compared to normocholesteremic B6129SF1/J controls.1
Using a well-established mouse model we shed new light on the role of PCSK9 in ICH. We show inhibition or deletion of PCSK9 to not affect ICH volumes under hypo- to mildly hypercholesteremic conditions. Instead our data suggest a protective effect of very high cholesterol levels in the setting of ICH independent of PCSK9 inhibition.Figure 1. Experimental timeline, plasma cholesterol levels and intracerebral hemorrhage (ICH) volumes. a. Left panel shows timeline as described in methods section for markedly hypercholesteremic APOE*3Leiden.CETP mice. After 6 weeks of western type diet (WTD) and treatment with anti-PCSK9-antibody (Ab) or vehicle (Veh.), blood samples were taken to analyze blood cholesterol levels. ICH was then induced using bacterial collagenase VIIs and ICH volumes computed 6 or 24 h later. Anti-PCSK9-antibodies reduced plasma cholesterol levels by ∼30% (left bar graph), vice versa increased ICH volumes 1.5-fold (right bar graph) b. Same experimental protocol was used in B6129SF1/J mice (left panel) in which mildly increased plasma cholesterol levels could be observed in both anti-PCSK9-antibody and vehicle treated animals after 6 weeks of WTD (left bar graph). Here PCSK9 Inhibition did not affect ICH volume (right bar graph). C. PCSK9−/− mice (KO) and B6129SF1/J controls (WT) were fed normal chow to investigate effects of PCSK9 knockout in normocholesteremic mice (left panel). While PCSK9 knockout resulted in hypocholesteremic plasma levels (left bar graph), ICH volumes remained unchanged when compared to controls (right bar graph).
Reference
SchlunkFFischerPPrincenHMG, et al.
Effects of inhibition or deletion of PCSK9 (proprotein convertase subtilisin/kexin type 9) on intracerebral hemorrhage volumes in mice. Stroke2020;
51: e297–e298.
599
Application of AI in metabolomics analyses for novel diagnostic approach for acute ischemic stroke
Kazuya Matsuo1,2, Kohkichi Hosoda3, Yusuke Yamamoto4 and Takashi Sasayama1
1Department of Neurosurgery, Kobe University Graduate School of Medicine, Kobe, Japan
2Department of Neurosurgery, Hyogo Emergency Medical Center & Japanese Red Cross Kobe Hospital, Kobe, Japan
3Department of Neurosurgery, Kobe City Nishi-Kobe Medical Center, Kobe, Japan
4Department of Neurosurgery, Toyooka Hospital, Toyooka, Japan
Abstract
Background: Early diagnosis and treatment are essential to improve the outcome of patients with acute ischemic stroke. However, MRI, the only established method of diagnosing acute ischemic stroke, has a drawback that some time lag exists between stroke onset and abnormal MRI signal appearance.
Aim: To develop a new diagnostic method for acute ischemic stroke, we applied AI/machine learning techniques to metabolomics analyses in the cerebral cortex of a rat model.
Method: The male rats were subjected to the MCAO (n = 40) with suture-occluded method or sham-MCAO (n = 10). Gas chromatograph-mass spectrometry (GC-MS) was performed on the affected cerebral cortex. The data were divided into a training set (n = 40) and a test set (n = 10) by stratified randomization. After data pre-processing, XGBoost models were developed by optimizing hyperparameters on the training set. Metabolites contributing to classification were determined by sequential reduction with reference to the SHAP values of the XGBoost model while maintaining classification performance.
Results/Conclusions: GC-MS identified 74 relevant metabolites, of which three were finally selected as necessary for classification. The machine learning model trained on these three metabolites could classify subjects into ischemic and normal groups with 96.7% sensitivity, 80.0% specificity, 94.3% positive predictive value, 92.5% accuracy, and ROC-AUC of 0.892 in the training set. On the test set, the model could correctly classify all samples.
Further investigation of the metabolic pathways associated with these three metabolites may provide a basis for the development of new diagnostic methods for acute ischemic stroke.
601
Development of an ischemic stroke model using self-assembling spheroids from human induced pluripotent stem cells
Stefano Fumagalli, Francesca Pischiutta, Davide Comolli, Alessandro Mariani, Joe Kelk, Ilaria Lisi, Gianluigi Forloni, Elisa Roncati Zanier and Massimilano De Paola
Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Milan, Italy
Abstract
Background: The use of human induced pluripotent stem cells (iPSC)-derived cultures for disease modelling offers new opportunities to obtain translational insights into pathobiology of ischemic stroke.
Aim: To develop a human 3D self-assembling iPSC-derived human brain spheroids (hBS) to characterize the effects of an in vitro model of ischemia by oxygen-glucose deprivation (OGD).
Method: Neural progenitor cells were derived from iPSCs by inhibition of the TGF-β/BMP-dependant SMAD signalling and grown to hBS over two months. hBS were exposed to 2 or 8 hour OGD. We assessed: cell death by lactate dehydrogenase (LDH) release and propidium iodide (PI) assay; neuronal and astrocytic damage as neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP) release in culture media by simoa immunoassay; hBS microenvironmental and morphological changes by qPCR and immunohistochemistry.
Results/Conclusions: We observed persistent cell death induced by OGD, indicated by increased PI incorporation in hBS and LDH release in culture media. OGD impaired the neuronal network complexity as measured in whole-mounting MAP-2-immunostained hBS. Neuronal damage corresponded to Map-2 mRNA reduction and an increased release of NfL in culture media. GFAP gene or protein levels did not change in hBS, but its release in medium increased after OGD. All effects were proportional to OGD duration.
We provide a human 3D iPSC-based in vitro model of brain ischemic injury, characterized by overt neuronal damage that is proportional to the duration of OGD. We show that the translational biomarker NfL closely reflects neuronal injury with implication for studies aimed at testing neuroprotective strategies.
602
Ripk2: A novel target for post-stroke pharmacotherapy
Jonathan Larochelle1, Ryland Tishko2, Bianca Lavayen2, Changjun Yang2, Lei Liu2 and Eduardo Candelario-Jalil2
1Department of Pharmacology and Therapeutics, University of Florida, Gainesville, USA
2Department of Neuroscience, University of Florida, Gainesville, USA
Abstract
Background: Receptor interacting serine/threonine protein kinase 2 (RIPK2) is a propagator of inflammatory and cell death pathways via its interaction with members of the NOD-like family of pattern recognition receptors (PRRs), NOD1 and NOD2, and caspase-1 respectively. Ischemic stroke is characterized by initial hypoxia/ischemia resulting in immediate cell death of brain cells directly downstream of the occluded cerebral artery and subsequent inflammation which contributes to the growth of the stroke injury. Dampening neuroinflammatory processes may serve to preserve functional brain tissue after stroke.
Aim: We hypothesize that abrogation of RIPK2 activity either by selective degradation via a proteolysis targeting chimera (PROTAC) or kinase inhibitor will be beneficial in treating post-stroke injury by dampening neuroinflammation and delaying hypoxia/ischemia-induced cell death.
Methods: Young (3-month-old) male mice were subjected to 45-min of middle cerebral artery occlusion (MCAO). Mice were randomly assigned to receive either the RIPK2 PROTAC, RIPK2 Inhibitor, or Vehicle upon reperfusion. Mice were subjected to behavioral testing and assessed for neurological deficits 24 h after MCAO. Mice were euthanized 24 h post-stroke for infarct size calculation by TTC staining.
Results: PROTAC and Inhibitor treatment reduced infarct size relative to the Vehicle-treated group at 24 h post-stroke. PROTAC- and Inhibitor-treated animals traveled further in an open field behavioral test, displayed less neurological deficits, showed greater strength preservation by the weight test, and greater ability to descend a vertical grid compared to Vehicle-treated animals at 24 h post-stroke.
Conclusion: Our preliminary data indicates that RIPK2 may be a suitable target for treating post-stroke injury.
603
Preliminary evidence of two dimensional image translation to estimate beta-amyloid PET from MRI
Fernando Vega1,2, Abdoljalil Addeh1,2 and Ethan MacDonald1,2
1Departments of Electrical & Software Engineering and Radiology, University of Calgary, Calgary, Canada
2Hotchkiss Brain Institute, Calgary, Canada
Abstract
Background: It is widely believed that beta-amyloid is one of the root causes of Alzheimer’s Disease (AD) pathophysiology. Over the last 15-years, beta-amyloid targets have gained significance with the use of PET tracers. PET have some disadvantages such as: cost, invasiveness, radiation and advanced tracers are not available in all jurisdictions. Structural MRI is about 10-times cheaper, and can aid to assess AD as well by targeting structural changes and atrophy driven by AD. Currently, MRI does not provide the valuable molecular information as PET does, however, this may be possible by building image translation models that yield synthetic PET images from MRI.
Aim: Implement an image-to-image translation model, which is an advanced form of machine learning, to generate beta-amyloid synthetic PET images from structural MRI.
Method: We implemented a Convolutional Neural Network and Generative Adversarial Network model that uses pairs of MRI and Pittsburgh-Blue-PET images from the Open Access of Imaging Studies (OASIS-3) database. A custom loss function was implemented that masks the images during training and to penalize information outside of the brain.
Results/Conclusions: Figure 1 shows two examples of the structural MRI, PET, synthetic PET images and the difference between the synthetic PET and PET. Preliminary results are encouraging and show that the model can generate synthetic PET images that have a high degree of similarity with the real PET images, however, the estimation in pixel intensity still requires improvement, therefore, future research will address this problem by implementing a more complex model architectures and tuning metrics.Figure 1. ▪.
604
Temporal brain transcriptome analysis reveals key pathological events after germinal matrix hemorrhage in neonatal rats
Xiaoyang Wang1, Gisela Nilsson2, Juan Song2,3, Yiran Xu3, Aura Zelco2, Eridan Rocha-Ferreira1, Joakim Ek2, Henrik Hagberg1 and Changlian Zhu4
1Centre for Perinatal Medicine and Health, Institute of Clinical Sciences, University of Gothenburg, Gothenburg, Sweden
2Centre for Perinatal Medicine and Health, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
3Henan Key Laboratory of Child Brain Injury and Henan Pediatric Clinical Research Center, Third Affiliated Hospital, and Institute of Neuroscience, of Zhengzhou University, Zhengzhou, China
4Center for Brain Repair and Rehabilitation, Institute of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
Abstract
Background: Germinal matrix hemorrhage (GMH) is a common complication in preterm infants and is associated with high risk of adverse neurodevelopmental outcomes. Thus, studies focusing on potential mechanisms of GMH in preterm infants are important in order to devise new therapies.
Aim: To provide an overall picture and a deeper understanding of the mechanisms of brain injury caused by GMH in order to support the development of effective therapeutic targets for this debilitating condition.
Method: A model of preterm GMH in postnatal day 5 rats was used, and RNA sequencing (RNA-seq) was performed at different time points after insult to identify changes in the transcription profile in the brain after GMH.
Results/Conclusions: At 6 hours after GMH, gene expression indicated an increase in mitochondrial activity such as ATP metabolism and oxidative phosphorylation along with upregulation of heme metabolism. At 24 hours after GMH, the expression pattern suggested an increase in cell cycle progression and downregulation of neurodevelopmental-related pathways. At 72 hours after GMH, there was an increase in genes related to inflammation and an upregulation of ferroptosis. Hemoglobin components and genes related to heme metabolism and ferroptosis were among the most upregulated genes. We observed dysregulation of processes involved in development, mitochondrial function, cholesterol biosynthesis, and inflammation, all of which contribute to neurodevelopmental deterioration following GMH. This study is the first temporal transcriptome profile providing a comprehensive overview of the molecular mechanisms underlying brain injury following GMH, and it provides useful guidance in the search for therapeutic interventions.
605
Evaluation of novel, data-driven metrics of amyloid β deposition
Ariane Bollack1, Pawel Markiewicz1, Alle Meije Wink2, Hugh G Pemberton3, Lloyd Prosser4, William Coath4, Johan Lilja5, Pierrick Bourgeat6, Gill Farrar3, Frederik Barkhof1,2,7 and David M Cash7,8
1Centre for Medical Image Computing, Department of Medical Physics and Biomedical Engineering, UCL, London, UK
2Amsterdam UMC, Vrije Universiteit Amsterdam, Department of Radiology and Nuclear Medicine, Amsterdam, the Netherlands
3GE Healthcare, Amersham, UK
4Dementia Research Centre, UCL Queen Square Institute of Neurology, London, UK
5Hermes Medical Solutions, Stockholm, Sweden
6CSIRO The Australian e-Health Research Centre, Australia
7UCL Institute of Neurology, London, UK
8UK Dementia Research Institute at University College London, London, UK
Abstract
Background: Positron emission tomography (PET) provides in vivo quantification of amyloid-β (Aβ) pathology. The most common method for assessing PET is standardized uptake value ratio (SUVr), which can be affected by physiological and technical factors. Novel, data-driven metrics have been developed to address some of these sources of variability.
Aim: To evaluate the performance of four data-driven metrics of Aβ deposition against established metrics against a common set of criteria.
Method: Three cohorts were used for evaluation: Insight 46, a sub-study of the 1946 British birth cohort (florbetapir), AIBL (flutemetamol), and a test-retest flutemetamol dataset. Four data-driven metrics were extracted: the Centiloid derived from non-negative matrix factorisation (CLNMF), the Aβ PET pathology accumulation index (Aβ-index), the amyloid load (Aβ-load), and the amyloid pattern similarity score (AMPSS). These data-driven metrics were compared to a global composite SUVr, a Centiloid (CL) score, and a non-displaceable binding potential (BPND). They were evaluated by measures of reliability in test-retest data, associations with BPND, and sample size estimates to detect a 20% slowing in Aβ accumulation.
Results/Conclusions: All metrics show good reliability and strong correlation to the BPND (R2: 0.8 to 0.94), with SUVr and CL explaining more variance in BPND than the data-driven metrics. Sample size estimates were lowest in CL and CLNMF compared to the SUVr (see Table 1). Novel data-driven metrics provide comparable performance in terms of accuracy and performance to more established quantification methods of Aβ PET tracer uptake, but with additional benefits, such as being MRI-free or reference region independent.
608
Altered brain structural similarity networks in migraine without aura
XiaoWang, yutong Zhang, tao Xu, xiu Ni, xu Ouyang, shan Gao and ling Zhao
Chengdu University of Traditional Chinese Medicine, Chengdu, China
Abstract
Background: Chronic migraine is a serious neurological disorder characterized by abnormal connections between brain regions. Recently, many studies have suggested that migraine is associated with cortical organization abnormalities in the brain. But the altered trajectory of brain development and maturation in migraine remains unknown.
Method: To address this issue, 30 individuals with migraine without aura (MWoA) (drug-naive) and 20 healthy controls (HC) were scanned with high-resolution T1-weighted anatomical images. Brain similarity network (BSN) was adopted to assess the microscale cortical organization in MWoA and HC groups. Network-based statistic was utilized to assess the alteration of developmental trajectories in migraine. Spearman rank correlation analysis was conducted between the edge in the altered BSN and clinical scores (i.e., frequency of migraine attacks, duration, headache impact test) in patients with MWoA.
Results: A remarkable different developmental trajectories network was identified including the edges with inferior frontal gyrus opercular to the middle cingulate gyrus, insula to the middle cingulate gyrus, superior frontal gyrus medial to the posterior cingulate gyrus, superior frontal gyrus medial to paraHippocampal gyrus, inferior frontal gyrus opercular to vermis9, middle frontal gyrus, orbital to vermis9, and insula to vermis9. In addition, increased covariance connectivity between the insula and middle cingulate gyrus, superior frontal gyrus medial to vermis9, superior frontal gyrus medial to parahippocampal gyrus were positively correlated with clinical symptoms in patients with MWoA.
Conclusion: The current findings point to the migraine-related alteration in trajectories of brain maturation and highlight the significance of the coordinated development between the cingulate, medial frontal gyrus, insula, and cerebellum in migraine.
609
Ischemic preconditioning enhanced repair processes and iron uptake during the recovery phase after ischemic stroke
Hye Won Kim, Jin A Shin, Hyun-Jung Kim and Eun-Mi Park
Department of Pharmacology, College of Medicine, Ewha Womans University, Seoul, Republic of Korea
Abstract
Background: It is well known that ischemic preconditioning (IP) reduces ischemic brain damage and neurological deficits in rodents after subsequent ischemic strokes, by activating endogenous protective mechanisms. Since transient ischemic attack (TIA) induces ischemic tolerance in the human brain after ischemic stroke, defining mechanisms of IP effects may provide therapeutic targets to improve recovery of patients with ischemic strokes.
Aim: This study aimed to determine whether IP effects were sustained and increased repair processes by modulating iron metabolism during the chronic phase after a stroke.
Method: C57BL5 male mice were divided into sham and IP groups, and IP was induced 24 h before a transient focal ischemic stroke. Sensorimotor recovery was observed over 8 weeks after the stroke, and brain volumes and levels of proteins related to repair processes and iron metabolism in the ischemic brains were examined 8 weeks after the stroke.
Results/Conclusions: There was significantly less ischemic brain atrophy in the IP group than in the sham group, with no differences in sensorimotor recovery between the groups. Levels of blood-brain barrier tight junction proteins, neurites outgrowth markers, and myelin sheath proteins and markers for mature oligodendrocytes were significantly increased in the IP group. Iron import proteins transferrin receptor 1 and DMT1 were also increased in the IP group. These results indicate that IP increases brain repair processes and iron uptake during the chronic phase after an ischemic stroke, and provide new insights to understand the molecular mechanisms of TIA effects on post-stroke recovery.
610
Functional segmentation of occupancy images improves precision of EC50 images
Alaaddin Ibrahimy1, Jocelyn Hoye2, Bart de Laat2, HaoWu3, David Wilson3 and Evan D Morris1,2
1Yale University, Department of Biomedical Engineering, New Haven, USA
2Yale University, Department of Radiology and Biomedical Imaging, New Haven, USA
3Case Western University, Department of Biomedical Engineering, Cleveland, USA
Abstract
Background: PET is used productively by pharmaceutical companies to measure drug occupancy, in vivo. The outcome measure is usually a whole-brain EC50. We recently introduced voxel-level occupancy images (Occ) from PET which can be used to generated EC50images by applying an Emax model at each voxel (de Laat 2020; 2021)
Aim: Our goal is to evaluate image segmentation via the SLICR algorithm to denoise the Occ and produce more precise EC50images.
Method: We created a 4D digital phantom containing 10 Occ (corresponding to 10 different plasma concentrations of drug). The Occ were constructed to include two bilateral local hot-spots of high EC50 (region-1: 25; region-2: 50; Background: [6-10] ng/ml) . An established noise model was applied to the simulated images to make noisy Occ (de Laat 2021). SLICR, a k-means clustering algorithm, was modified to segment Occ into “k” clusters. EC50, images were created by nonlinear estimation at each voxel. Coefficient of variation images, CV(EC50), were estimated from the covariance matrix of the parameters at each voxel. EC50, and CV(EC50) images were created from the noisy and the segmented versions of the Occ.
Results/Conclusions: Variability in EC50 values was lower using segmented Occ with SLICR while the increase in bias was minimal (see Figure 1). Using SLICR, the overall computation time decreases by orders of magnitude. Our results suggest that segmentation of Occ with SLICR could produce more precise EC50images and improve our ability to identify ‘hot-spots’ of high effective affinity of a drug for target(s).Figure 1. ▪.
611
Roles of dimethylarginine dimethylaminohydrolase 1 (DDAH1) in cerebral endothelial function and angiogenesis
Arun Flynn1,2, Alexandra Riddell1,2, Laura Dowsett1,2, James Leiper1,2 and Alyson Miller1,2
1University of Glasgow, Glasgow, UK
2British Heart Foundation, London, UK
Abstract
Background: Asymmetric dimethyl arginine (ADMA) is an endogenously produced inhibitor of nitric oxide (NO) synthesis and is primarily metabolised by the DDAH enzymes (DDAH1/2). Elevations in ADMA levels or loss-of-function DDAH1 polymorphisms are associated with cerebrovascular disease, however causal evidence is lacking. Furthermore, although there is evidence that DDAH1 is important in the systemic circulation, its functional importance in the cerebral circulation is largely unknown.
Aim: Determine the importance of DDAH1 for key NO-regulated processes: cerebrovascular reactivity and angiogenesis.
Methods: Common carotid arteries and middle cerebral arteries (MCA) were isolated from male C57BL6/J mice and treated with saline, the selective DDAH1 inhibitor L-257 (100 µM), or exogenous ADMA (0.5, 3, and 100 µM). Cerebrovascular reactivity and angiogenesis were evaluated by myography and a vessel sprouting assay, respectively.
Results/Conclusions: L-257 impaired endothelium-dependent relaxation responses (acetylcholine) of carotid arteries (Rmax: saline: −98 ± 5% vs L-257: −56 ± 17%; P < 0.05, n = 3) without affecting responses to sodium nitroprusside. Carotid rings treated with L-257 or ADMA (100 µM) had significantly fewer vessel sprouts than saline-treated rings (saline 176 ± 23 vs L-257 129 ± 22 per ring/mm2; saline 158 ± 15 vs ADMA [100 µM] 90 ± 17, *P < 0.05, n = 6–7), and developed fewer branches per sprout. Preliminary data shows that inhibition of DDAH1 also shortens sprout length (saline 0.97 ± 0.2 mm/mm2 vs L-257 0.52 ± 0.2 mm/mm2; *P < 0.05, n = 5) but not number of sprouts of MCA. These findings provide evidence that DDAH1 may play an important role in cerebrovascular regulation and may ultimately provide a mechanism for the association between ADMA, DDAH1 polymorphisms, and cerebrovascular disease.
612
Protein synthesis rate measured by [11C]leucine PET imaging in TgF344-AD rats
Daniela Bochicchio1, Karl Herholz1, Christine A Parker2, Rainer Hinz1 and Hervé Boutin1
1University of Manchester, Manchester, UK
2GlaxoSmithKline, Stevenage, UK
Abstract
Background: Brain protein synthesis (PS) is essential for long term memory formation. Memory alteration is one of the main hallmarks of Alzheimer’s disease. Changes in PS pathways have been shown in AD animal models and patients ex vivo, however such changes have not been assessed in vivo in AD.
Aim: We aimed to investigate the quantification of the PSR in transgenic AD rat (TgF344-AD (TG) compared to wild-types (WT).
Method: All procedures were approved by the Home Office and carried out in accordance with the Animals (Scientific Procedures) Act 1986. Under isoflurane anaesthesia, rats were scanned with [11C]leucine for 60 min. TG and WT were scanned at 6, 12 and 18 months of age. Arterial blood activity was measured online and by γ-counting of discrete blood samples in some rats. PS inhibition was measured in Wistar rats by injection of anisomycin (60 mg/kg) 10 min prior PET acquisition. For the longitudinal study, averaged population-based whole-blood and plasma input functions (IF) and unlabelled leucine concentrations were used to calculate PS.
Results/Conclusions: Anisomycin administration significantly reduced PS (-89%) proving the sensitivity of the method. In the longitudinal study, we found a modest but significant genotype effect at ANOVA level in the globus pallidus (TG < WT: −15% at 18 months). However, data modelling in rodents remains challenging; population-based IF was feasible but the data suggested using cross-sectional studies with individual IF instead. Our findings provide valuable methodological information for future preclinical and clinical studies where these methods could be utilised to investigate further the effect of PS in AD.
613
Early alterations in synaptic density in TgF344-AD rats measured by [18F]UCB-H PET imaging
Daniela Bochicchio1, James Minshull1, Christine A Parker2, Federico Roncaroli1, Rainer Hinz1 and Hervé Boutin1
1University of Manchester, Manchester, UK
2GlaxoSmithKline, Stevenage, UK
Abstract
Background: Alterations in synapses are associated with a variety of neurodegenerative disorders including Alzheimer’s disease (AD). 18F-UCB-H is a tracer able to bind to synaptic vesicles 2 A (SV2A), involved in vesicle trafficking and exocytosis.
Aim: We here investigate synaptic density with 18F-UCB-H in TgF344-AD (AD transgenic (TG)) rats and wild-type (WT) littermates at 7 and 15 months of age.
Method: All procedures were approved by the Home Office and carried out in accordance with the Animals (Scientific Procedures) Act 1986. Under isoflurane anaesthesia, WT and TG rats were scanned with 18F-UCB-H at 7 and 15 months of age. To measure the non-specific binding, some WT and TG rats were injected with cold UCB-J (1 mg/kg) 10 min before 18F-UCB-H injection. Data were then expressed as normalised-uptake values (NUVND) of the ratio between the 9–22 min averaged baseline SUV over the blocked (non-displaceable) SUV for each ROI.
Results/conclusion: Without pre-saturation, similar ROIs heterogeneities were observed in TG and WT. UCB-J pre-saturation blocked ∼70% of the specific binding and regional differences were abolished but non-specific binding tended to be higher in TG. Individual and group averages NUVND were significantly correlated, we therefore used the population-based SUVUCB-J to calculate the NUVND for all rats. NUVND were significantly lower in TG than WT at 7 m of age (−12% to −17%). There was an age effect so that WT and TG were no longer significantly different at 15 months. We here demonstrate an early alteration in synaptic density in TG vs WT rats, followed by an age-effect in WT.
614
The effect of a novel AQP4-facilitator, TGN-073, on glymphatic transport captured by diffusion-MRI and DCE-MRI
Alaa Alghanimy, William Holmes, Conor Martin and Lindsay Gallagher
University of Glasgow, Glasgow, UK
Abstract
Background: The glymphatic system is a low resistance pathway, by which cerebrospinal fluid enters the brain parenchyma along perivascular spaces via AQP4 channels. It is hypothesised that the resulting convective flow of the interstitial fluid provides an efficient mechanism for the removal of waste toxins from the brain. Therefore, enhancing AQP4 function might protect against neurodegenerative diseases such as Alzheimer’s disease (AD), in which the accumulation of harmful proteins and solutes is a hallmark feature.
Aim: We test the effect of an AQP4 facilitator, TGN-073, on glymphatic transport in a normal rat brain by employing different MRI techniques.
Method: Male Wistar rats were randomly assigned to either TGN-073, vehicle, or aCSF groups. First, surgical procedures were undertaken to catheterise the cisterna magna, thereby enabling infusion of the MRI tracer. Then, an intraperitoneal injection of either TGN-073, or the vehicle (200 mg/kg in 20 ml/kg body weight), was administered. Using paramagnetic contrast agent (Gd-DTPA) as the MRI tracer, dynamic 3D T1 weighted imaging of the glymphatic system was undertaken over two hours. Further, the apparent diffusion coefficient was measured in different brain regions using diffusion-weighted imaging (DWI).
Results: The study results indicate that rats treated with TGN-073 showed the distribution of Gd-DTPA was more extensive and parenchymal uptake was higher compared with the vehicle group. Water diffusivity was increased in the brain of TGN-073 treated group, which may indicate greater water flux. Also, MRI showed the glymphatic transport and distribution in the brain is naturally heterogeneous, which is consistent with previous studies.
Conclusions: Our results indicate that compounds such as TGN-073 can improve glymphatic water flux in the brain. Since glymphatic impairment due to AQP4 dysfunction is potentially associated with several neurological disorders such as AD, dementia and traumatic brain injury, enhancing AQP4 functionality might be a promising therapeutic target.
615
Automatic segmentation with deep neural networks for the choroid plexus in multiple sclerosis patients
Valentina Visani1, Valerio Natale2, Agnese Tamanti3, Alessandra Bertoldo1, ssa Francesca Benedetta Pizzini2, Massimiliano Calabrese3 and Marco Castellaro1
1Department of Information Engineering, University of Padova, Padova, Italy
2University Hospital of Verona, Verona, Italy
3Department of Neuroscience, Biomedicine and Movement, University of Verona, Verona, Italy
Abstract
Background: The Choroid Plexus (ChP) is a vascular tissue involved in inflammatory processes. ChP volume (ChPV) measured by T1-w MRI has been observed altered in several neurological disorders. Therefore, ChPV can become a promising biomarker to improve the understanding of neurological diseases. However, the manual segmentation of ChP is time-consuming and affected by inter-operator variability.
Aim: The aim of this work is to propose a method for the completely automatic, accurate and reliable segmentation of the ChP based on novel deep learning neural networks (DNNs).
Method: The dataset is composed by 60 Relapsing Remitting-Multiple Sclerosis patients (Training/Validation: 45/15). Ground truth was depicted on the T1-w sequence. Several DNNs were tested: 3D U-Net, nnU-Net and UNETR. Several DNNs parameters (i.e., input number (T1, FLAIR), data augmentation, patch size (64×64×64−128×128×128 voxels), loss function (Dice, CrossEntropy, weightedCE, Dice + CE)) were tested for a total of 144 parameter combinations. Goodness of segmentation was assessed with: Dice Coefficient, 95% Hausdorff Distance, Percentage Volume Difference. We tested also FreeSurfer and the GMM method.
Results/Conclusions: Overall, nnU-Net provided the best results, its segmentation was not particularly affected by changing the DNN parameters (Dice (min-max): 0.749–0.769). The UNETR showed slightly lower performance than nnU-Net (Dice: 0.723–0.757). U-Net performance (Dice: 0.672–0.738) were lower than nnU-Net and UNETR. Both FreeSurfer (Dice: 0.319 ± 0.066) and GMM (Dice: 0.513 ± 0.054) provided poor results compared to DNNs.
The nnU-Net is a reliable tool for the segmentation of the ChP and shows its promising usefulness to establish a new neuroimaging biomarker.
616
Vascular infectivity of SARS CoV-2 is increased by APOE genotype and existing inflammation
Nikolaos Zervogiannis, Luca Biasetti, Louise Serpell, Edward Wright and Catherine Hall
University of Sussex, Brighton, UK
Abstract
Background: Severity of COVID-19 varies widely, with existing inflammation and expression of the ε4 allele of the apolipoprotein E (APOE) gene both increasing severity. Vascular permeability is also increased by these factors, and vascular dysfunction is increasingly understood to be a key factor in COVID19 disease. However, the site of vascular infection is unclear: endothelial cells become dysfunctional but pericytes express much higher levels of ACE2, which mediates SARS CoV-2 entry. Understanding SARS CoV-2 infectivity of vascular cells in different conditions is however likely to be vital for understanding and combatting COVID-19 disease.
Aim: Ascertain levels of SARS-CoV-2 infectivity of endothelial cells and pericytes, and test if risk factors that increase COVID-19 severity also modulate vascular infectivity.
Method: Pseudotyped SARS-CoV-2 variants expressing luciferase were used to infect human and mouse endothelial and pericyte cultures, as well as control HEK cells, allowing us to measure infectivity levels. We used lipopolysaccharide (LPS) to induce inflammation prior to SARS-CoV-2 infection and cultures from transgenic mice expressing human APOE3 or APOE4 to test the effect of APOE genotype.
Results/Conclusions: In mice, brain pericytes were more readily infected than brain endothelial cells, and APOE4 genotype and pre-existing LPS-induced inflammation both increased infection levels. In humans, LPS also increased infectivity of vascular cells, but the opposite effects were seen in HEK cells, suggesting this is a cell-specific effect. Our results show that factors that increase severity of COVID-19 disease also increase vascular infectivity of SARS CoV-2, suggesting that vascular infectivity could, in part, mediate COVID-19 severity.
617
Alpha-synuclein modulates vascular responses following cerebral ischemia-reperfusion injury
1Division of Pharmacology, Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy
2Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Department of Neuroscience, Milan, Italy
3Biotechnology Division, Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy
4Laboratory for Preventive and Personalized Medicine, Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy
Abstract
Background: The vascular damage after ischemic stroke correlates with worse outcomes in patients. Alpha-synuclein (α-synuclein) has been demonstrated to mediate secondary brain injury in experimental models of ischemic stroke. This is notwithstanding whether and how α-synuclein mediates vascular injury.
Aim: We investigated the hypothesis that α-synuclein contributes to vascular damage or limits vascular recovery in acute and subacute phases of ischemic stroke.
Method: Ischemia/ reperfusion injury was induced in wt (C57BL/6J) and α-synuclein null (C57BL/6J Ola) mice by transient occlusion of middle cerebral artery (tMCAo). Sensorimotor deficits at 2 days, anxiety test by elevated plus maze at 7 days and survival analysis were assessed as primary outcomes. Histology-based neuronal count and infarct volume were measured to determine the lesion size. Real-time PCR and western blot were used to determine the expression of vessel-associated genes and proteins in the mice cortex 2 and 7 days after the tMCAo.
Results/Conclusions: The α-synuclein null mice showed improved survival and less anxiety compared to wt mice at 7 days, though this was not supported by differences in neuronal loss. No differences were observed in the sensorimotor deficits between genotypes at 2 days. In the acute phase, i.e., 2 days after tMCAo, ischemic null mice had decreased expression of ICAM1, HIF1α, VEGFs, VEGFRs, MMP9 compared to wt. In the subacute phase (7 days), null mice had increased expression of VEGFR2 and MMP9. These results suggests that α-synuclein may influence functional recovery in later phases of stroke by modulating vascular injury and repair.
618
Hypoxia versus asphyxia induced brain interstitial pH changes in normothermic or hypothermic newborn pigs
Gabor Remzso, Viktória Kovács, Valéria TTóth-Szűki and Ferenc Domoki
University of Szeged, Albert Szent-Györgyi Medical School, Department of Physiology, Szeged, Hungary
Abstract
Background: Brain interstitial pH (pHbrain) alterations contribute to neuronal injury in neonatal hypoxic-ischemic encephalopathy (HIE), the severe neurodevelopmental consequence of birth asphyxia affecting millions of neonates each year. In various HIE animal models, normocapnic hypoxia or asphyxia are both employed as insults to elicit HIE. However, hypoxia or asphyxia may trigger neuronal injury by distinct pathomechanisms.
Aim: We wanted to determine quantitatively the magnitude and temporal dynamics of the pHbrain changes during both asphyxia/hypoxia and investigating the potential confounding effect of body temperature alteration.
Method: Anesthetized, mechanically ventilated P1 piglets (n = 21) were equipped with an open cranial window to determine pHbrain using pH-selective microelectrodes inserted into the parietal cortex. Asphyxia or hypoxia was induced by swithching the ventilation to a 6%O2–20%CO2 gas mixture or to 6%O2 for 20 min, respectively in either normothermic (38.5 °C) or hypothermic (33.5 °C) pigs followed by 60 min reoxygenation/reventilation.
Results/Conclusions: Both arterial blood pH (pHa) and pHbrain showed an acidotic shift during the insult in all four experimental conditions. Cerebral acidosis was similar in normothermic animals exposed to either asphyxia or hypoxia. In hypothermic conditions, however, the acidotic pHbrain shift was greatly attenuated only in the hypoxia but not in the asphyxia group. Our findings suggest that using hypoxia or asphyxia insults result in similar cerebral but not systemic (blood) acidosis in normothermic piglets. However, the degree of developing cerebral acidosis to hypoxia is greatly attenuated in hypothermic animals, although an alkalotic shift reported in rats could not be observed.
619
Impact of comorbidities, prediabetes and chronic hyperuricemia, on inflammation and motor recovery after ischemic stroke
Andrew Clarkson
University of Otago, Dunedin, New Zealand
Abstract
Background: Recovery from stroke is difficult to predict, with stroke comorbidities contributing to poor neurological outcome. Diabetes increases the risk of ischemic stroke and is associated with poor recovery. A hallmark of both diabetes and stroke is inflammation. Further, hyperuricaemia is associated with metabolic dysfunctions including obesity, diabetes and poor vascular health.
Aim: We aimed to assess whether obese prediabetic (POUND (Lepr(db/lb)) and chronic hyperuricemic, (PLT2) mice would have aberrant inflammation that would contribute to poor vascular health and impaired stroke recovery.
Method: Focal stroke to the motor cortex was induced using the photothrombosis method in POUND, PLT2 or wild-type (WT) mice. Circulating cytokines levels were assessed from tail tip blood samples as were blood lipids, HbA1c levels and basal fasting glucose. Behavioural assessments were performed weekly on both cylinder and grid-walking tasks.
Results/Conclusions: PLT2 mice (lean, hyperuricemic) had significantly lower HbA1c and basal glucose levels compared to WT controls indicating they were hypoglycaemic. Assessment of PLT2 mice revealed increased circulating cytokine levels, expansion of infarct volume and impaired motor recovery. Further PLT2 mice had increased total cholesterol and LDL levels. Assessment of POUND mice (obese, hyperuricemic) revealed normal basal fasting glucose and elevated HbA1c levels confirming they are prediabetic. A significant increase in total cholesterol and LDL level were observed compared to WT controls. POUND mice also revealed increase in circulating cytokine levels and impaired motor recovery.
These studies highlight that both POUND and PLT2 mice have aberrant inflammation and elevated lipids contributing to coronary risk and poor recovery.
620
Multimodal imaging of microstructural cerebral changes and loss of synaptic density in Alzheimer’s disease
Soodeh Moallemian, Eric Salmon, Mohamed Ali Bahri, Nikita Beliy, Emma Delhaye, Evelyne Balteau, Christian Degueldre, Christophe Phillips and Christine Bastin
GIGA-Cyclotron Research Centre-in Vivo imaging, University of Liège, Liège, Belgium
Abstract
Background: Multiple neuropathological changes are involved in Alzheimer’s disease (AD) progression. The hallmark biomarkers are amyloid-beta, tau pathology, neuronal and synaptic loss. Other potential biomarkers, such as the level of iron and myelin content in the brain, have not been thoroughly studied. Nevertheless, these can be estimated in vivo thanks to tissue magnetic resonance (MR) properties measured through quantitative MR imaging (qMRI) techniques.
Aim: We aimed to assess the co-occurrence of neurodegeneration (as measured with synaptic density), increased iron content, and decreased myelin content in Alzheimer’s disease.
Method: Data include 24 amyloid-positive Alzheimer’s patients (AD-11/13 males/females) and 19 healthy controls (HC-9/11 males/females). They underwent a multiparameter qMRI protocol used to generate quantitative maps sensitive to microstructural changes in myelin, iron deposits, and water content in grey matter (GM). Synaptic density was indexed by [18F]UCB-H-PET imaging using the distribution volume density (VT) maps. First, we applied univariate statistical analyses to investigate variation between AD and HC groups for each modality individually. Then, a multivariate GLM approach was used to compare the two groups pooling all modalities.
Results/Conclusions: In GM univariate analyses, there was no significant difference between the AD and HC groups in any map at corrected statistical threshold. Conversely, the multivariate analysis on GM, combining MT, R2s, and synaptic density, provided significant group differences (FWEcorr P-value <0.05) see Figure 1. These variations are observed in the right amygdala (at voxel level) and in 5 distinct clusters covering the bilateral anterior hippocampal structures. These show that patients with AD present convergence of neuropathological changes in the hippocampal area, suggesting that different pathological mechanisms co-exist in areas known to harbor early-stage neuronal death.
621
Impact of cerebral blood vessels on volume analysis of brain tissues at 7T MRI
Shin-ichi Yamazaki1, Hideto Kuribayashi2 and Ikuhiro Kida1,3
1Center for Information and Neural Networks, National Institute of Information and Communications Technology, Suita, Japan
2Siemens Healthcare K.K., Shinagawa, Japan
3Graduate School of Frontier Biosciences, Osaka University, Suita, Japan
Abstract
Background: Voxel-based morphometry (VBM) for T1 weighted (T1w) images in MRI is a powerful tool to estimate volume and thickness of gray matter to assess structural changes such as brain pathology. Although ultra-high magnetic field strength provides higher spatial resolution to estimate a precise gray matter volume, the signals of blood vessels might be compatible with those in the gray and white matter due to the T1 extension of blood.
Aim: We investigated an impact of cerebral blood vessels on the volume analysis of T1w image by MP2RAGE at 7 T.
Method: Vendor-supplied MP2RAGE sequence as works in progress (WIP) packages was used.
Cerebral blood vessels were segmented by MP2rase-CA [1] and enhanced the signal onto the T1w image. Two T1w images were analyzed by VBM12 toolbox in SPM12. The AAL2ROI was used for volume analysis.
Results/Conclusions: The total volume of gray matter was reduced in the T1w corrected with cerebral blood vessel, 1.2 ± 0.4%, compared to the T1w images without correction, but the volume of white matter was slightly increased by 0.3 ± 0.4%. ROI analysis indicated that most regions were reduced in gray matter, especially medial and posterior orbitofrontal cortex, by 2.6 ± 1.3% and 2.4 ± 1.5%, respectively. The results suggest that the signal of blood vessels in T1w images at ultra-high magnetic fields should be considered for the analysis of gray and white matter volume and thickness.
622
Minimum functional unit of the cerebral capillaries for neural activation in the somatosensory cortex
1Graduate School of Informatics and Engineering, University of Electro-Communications, Chofugaoka, Chofu, Japan
2Department of Functional Brain Imaging, National Institutes for Quantum and Radiological Science and Technology, Anagawa, Inage, Japan
3Molecular Imaging Center, National Institute of Radiological Sciences, Anagawa, Inage, Japan
4Brain Science Inspired Life Support Research Center, University of Electro-Communications, Chofugaoka, Chofu, Japan
Abstract
Background: The cerebral capillary responds to neural activation, which plays a key role in the adjustment of regional blood flow coupled with neural demand. However, the mechanism for regulating the capillary flow, specifically a capillary reaction length is largely unknown.
Aim: To determine coherent responses of the spatiotemporal changes in capillary diameters at variable frequencies of sensory stimulation, two-photon microscopy was used to capture volumetric changes in capillary morphology in the somatosensory cortex of mice expressing the calcium indicator in cortical neurons.
Method: The experimental protocol was approved by the Animal Ethics Committee of the University of Electro-Communications. Air-puff stimulation was applied with varying frequencies on the contralateral side of the whiskers, while activated neurons and microvessels were imaged simultaneously with two-photon microscopy. All acquired images were analyzed using custom code written in Matlab software.
Results/Conclusions: The images were reconstructed in three dimensions and capillary diameters and number of active neurons were quantified. For the volume image of capillaries ( <8 μm in diameter), a total of 3,000–7,000 measuring points were extracted. A mean diameter of 4.9 ± 0.3 µm at rest and a coefficient of variation of 0.172 ± 0.025 were observed. Neural activation caused dilation (14% ± 3% relative to the baseline) and constriction (14% ± 2%). The capillary response to neural activity examined based on the consistent direction of response and the temporal coefficient of variation demonstrated a minimum functional unit of capillary flow control with a length of 6 µm.
623
Identification of brain-autonomous neural repair triggered through lipid metabolism
Akari Nakamura, Seiichiro Sakai and Takashi Shichita
Stroke Renaissance Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
Abstract
Background: Stroke is a major cause of disability worldwide. Since rehabilitation promotes functional recovery after brain injury, the brain has endogenous self-recovery mechanisms triggering a broad range of neural repair that have not been identified.
Aim: We tried to identify the cerebral endogenous molecules that trigger neural repair after ischemic stroke by the comprehensive study of brain endogenous lipid and single-cell RNA-seq analysis of neurons in the ischemic brain.
Method: By the comprehensive study of phospholipid metabolites and their generating enzyme, phospholipase A2 (PLA2), we have identified the pivotal enzyme and phospholipid metabolites for functional recovery after ischemic stroke. Through this lipid metabolism, the endogenous molecular mechanisms triggering a broad range of neural repair were investigated by single-cell RNA-seq analysis of peri-infarct neurons collected from the murine ischemic brain after transient middle cerebral artery occlusion (MCAO).
Results/Conclusions: We have identified dihomo-g-linolenic acid (DGLA) and its metabolites generated by PLA2G2E, a secreted PLA2 from peri-infarct neurons, as essential for functional recovery. Poor functional prognosis in Pla2g2e-deficient mice was explained by the loss of peptidyl arginine deiminase 4 (Padi4) in peri-infarct neurons. Single-cell RNA-sequencing analysis demonstrated that Padi4 was pivotal for the transcriptional regulation in peri-infarct neurons to promote survival and neural repair. The administration of DGLA and its metabolites induced Padi4 in the peri-infarct neurons and accelerated functional recovery after ischemic stroke. Thus, our research clarifies the promising potential of brain-autonomous neural repair triggered by the specialized lipids that initiate self-recovery processes after brain injury.
625
Post-Stroke learning impairment is augmented in hypertensive mice
Michael De Silva, David Wong Zhang, Grant Drummond and Christopher Sobey
La Trobe University, Bundoora, Australia
Abstract
Background: Cognitive impairment is disorder that can arise as a result of cardiovascular pathology or cerebrovascular injury. Due to the aging population, the incidence of cognitive impairment is expected to rise. Hypertension is a major risk factor for stroke and cognitive impairment, but it is unclear whether it may affect post-stroke cognitive outcomes.
Aim: This study aims to assess the effects of hypertension and/or stroke on the brain and its effect on cognitive impairment.
Method: C57BL/6J mice (n = 117) aged 3–5 months received infusion of either saline or angiotensin II (0.7 mg/kg/day for 14d or 0.28 mg/kg/day for 28d). 7 d after minipump implantation mice received either sham or photothrombotic stroke surgery targeting the prefrontal cortex. The Barnes maze was utilised to assess cognitive function. RNA sequencing was used to quantify transcriptomic changes in the brain.
Results/Conclusions: Angiotensin II increased blood pressure (normotensive: 118 ± 1 mmHg vs. hypertensive: 149 ± 2 mmHg; P < 0.05). In the Barnes maze, hypertensive mice that received stroke surgery had increased latency to enter the escape hole when compared to other groups (day 3 escape latency: hypertensive + stroke = 166.6 ± 6.0 s vs. hypertensive + sham = 122.8 ± 13.8 s vs. normotensive + stroke = 139.9 ± 10.1 s vs. normotensive + sham = 101.9 ± 16.7 s), consistent with a greater learning impairment. RNA sequencing revealed >1500 differentially expressed genes in hypertensive + stroke vs. hypertensive + sham, including genes related to inflammation and synaptic function. These findings indicate that the combination of hypertension and stroke resulted in greater learning impairment and brain injury.
626
Nimodipine limits neurovascular dysfunction after spreading depolarization in mice
Ferenc Bari1, Peter Kozak1,2, Akos Menyhart2,3 and Eszter Farkas2,3
1Department of Medical Physics and Informatics, Albert Szent-Györgyi Medical School, Faculty of Science and Informatics, University of Szeged, Szeged, Hungary
2Department of Cell Biology and Molecular Medicine, Albert Szent-Györgyi Medical School, Faculty of Science and Informatics, University of Szeged, Szeged, Hungary
3Hungarian Centre of Excellence for Molecular Medicine – University of Szeged, Cerebral Blood Flow and Metabolism Research Group, Szeged, Hungary, Szeged, Hungary
Abstract
Background: Depression of neuronal activity and long-lasting vasoconstriction, called spreading oligemia (SO) are the pathological correlates of spreading depolarization (SD) in migraine aura. SD disrupts cellular signaling within the neurovascular unit, leaving neurovascular coupling (NVC) diminished during the 1–2 hours of oligemia.
Aim: We aimed to improve NVC after SD by attenuating SO.
Methods: Male, adult C57BL/6 mice (n = 9) were used. Anesthesia was induced with isoflurane (1–1.5%) and then switched to isoflurane (0.1%) – medetomidine (0.1 mg/kg i.p.). To monitor NVC function, whisker stimulation (2 Hz) – evoked potentials (EVPs) and functional hyperemia (FH) were recorded (silver ball electrodes, laser Doppler flowmetry). After 3 baseline stimulations, SD was induced by 1 M KCl. Then stimulations were repeated 5-times at 15 min intervals. The L-type voltage-gated Ca2+ channel blocker nimodipine was administered i.p. (10 mg/kg).
Results/Conclusions: EVP and FH amplitudes were markedly reduced after SD (EVP: 132.24 ± 35.48 vs. 247.68 ± 39.8 µV, after vs. before SD; FH: 11.98 ± 6.78 vs. 28.48 ± 11.06%, after vs. before SD). The amplitude of FH completely recovered 75 min after SD (28.48 ± 11.06 vs. 30.92 ± 6.93%, after vs. before SD). Nimodipine treatment tempered the duration of neuronal depression (8.6 ± 1.48 vs. 13.2 ± 0.6 min, Nimo vs. Control) and oligemia (26.63 ± 3.78 vs. 49.17 ± 9.79 min, Nimo vs. Control) after SD. Finally, nimodipine increased the amplitude of FH after SD (22.06 ± 7.19 vs. 11.98 ± 6.78%, Nimo vs. Control). We propose nimodipine treatment to curtail the SD-caused sensory dysfunctions in migraine patients.
Acknowledgements
NKFIH (K134334, K134377), Horizon 2020 (739593).
628
Relation between changes in cardiorespiratory, cerebrovascular and cognitive health across ten years in older adults
Ralf Weijs1, Janneke Vloet1, Daria Shkredova1,2, Marit Sanders3, Madelijn Oudegeest-Sander1, Rianne de Heus3, Daan de Jong3, Maria Hopman1, Jurgen Claassen3 and Dick Thijssen1,4
1Department of Physiology, Radboud Institute for Health Sciences, Radboud University Medical Center, Nijmegen, The Netherlands
2Centre for Heart, Lung, and Vascular Health, School of Health and Exercise Sciences, University of British Columbia Okanagan, Kelowna, Canada
3Department of Geriatrics, Radboudumc Alzheimer Center, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands
4Research Institute for Sport and Exercise Sciences, Liverpool John Moores University, Liverpool, UK
Abstract
Background: Several studies explored the relation between cardiorespiratory health and cerebrovascular perfusion in cognitive aging. This evidence is mostly based on cross-sectional findings and scarce longitudinal studies with relatively short follow-up.
Aim: To examine changes in cerebral hemodynamics and cardiorespiratory fitness, and their relation with the development of subjective memory complaints, across 10 years in cognitively healthy older adults.
Method: At baseline and after 10 years of observational follow-up, twenty-nine cognitively healthy older adults (median baseline age 69 years) underwent transcranial Doppler evaluation of cerebral blood flow (CBF) and cerebrovascular resistance index (CVRi), and a cycle ergometer test to estimate maximal oxygen consumption (VO2max). At follow-up, subjects were evaluated for the presence of subjective memory complaints. Linear mixed model analyses were performed to examine (changes in) CBF, CVRi and VO2max and their relation with subjective memory complaints. Moreover, Pearson’s correlation analyses were performed to examine potential linear relations between changes in these measures.
Results/Conclusions: Overall, while VO2max decreased significantly across 10 years of follow-up, CBF and CVRi remained stable. Changes in CVRi from baseline differed significantly however between subjects with (n = 15) versus subjects without (n = 14) subjective memory complaints (+0.72% versus –0.09%; P = 0.023), whereas no differences between groups were found for changes in CBF and VO2max. No significant correlations were found between changes in VO2max, CBF and CVRi. In conclusion, these findings suggest a potential relation between an increase in cerebrovascular resistance across time and development of cognitive impairment. Further analyses regarding cerebrovascular stiffness, autoregulation and carbon dioxide reactivity are ongoing.
630
ERα mediated recovery of synaptic plasticity in juvenile female mice after global cerebral ischemia
Jose Vigil1, Erika Tiemeier1, Nicholas Chalmers1, Rob Dietz1, James Orfila2, Paco Herson2 and Nidia Quillinan1
1University of Colorado Anschutz Medical Campus, Denver, USA
2Ohio State University, Columbus, USA
Abstract
Introduction: We previously showed that global cerebral ischemia (GCI) in juvenile male mice results in cognitive impairments and corresponding hippocampal long-term potentiation (LTP) impairments which endogenously recover within 30 days. It is also important to determine if the juvenile female brain undergoes endogenous recovery and the mechanisms by which this recovery occurs.
Hypothesis: GCI in juvenile female mice results LTP impairments that endogenously recover as a result of estrogen signaling that increases during puberty.
Methods: Female mice were subjected to cardiac arrest (CA) or sham procedure at p21-25 and ovariectomy (OVX) performed 5–7-days later. At time of OVX, hormone replacement (E2, DPN, PPT) was performed via subcutaneous osmotic pumps. 30-days after CA, hippocampal slices were prepared and LTP recordings were performed.
Results/Conclusions: GCI in juvenile female mice produces acute deficits in LTP that endogenously recover on a timescale that coincides with the onset of sexual maturity. LTP in sham animals reaches 145.3 ± 6.9% of baseline while GCI animals only reach 118.3 ± 7.25% (p < 0.05 vs. shams) 7-days after GCI. Interestingly, 30-days after GCI, LTP returns to 143.2 ± 7.13% of baseline (p > 0.05 vs. shams). Endogenous recovery was ablated in OVX animals (130.6 ± 6.9%), however, OVX and prolonged hormone replacement therapy increased LTP (E2 = 45.8 ± 7.48; DPN = 160.0 ± 12.6%) with the ERα agonist PPT restoring LTP to the greatest extent (170.4 ± 12.1%; p < 0.05 vs sham). This suggests that endogenous recovery of LTP in the juvenile female brain after GCI is mediated by the surge of sex hormones that occurs during the onset of puberty by acting upon ERα receptors.
Amelia Burch1, Joshua Garcia2, Heather Caballes1, James Orfila3, Erika Tiemeier1, Nidia Quillinan1, Katharine Smith2 and Paco Herson3
1Department of Anesthesiology, University of Colorado Anschutz Medical Campus, Aurora, USA
2Department of Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, USA
3Department of Neurological Surgery, The Ohio State University, Columbus, USA
Abstract
Background: Excitatory-inhibitory (E/I) imbalance underlies perturbations in circuit function following numerous neurologic diseases, leading to cognitive and motor sequelae. In the context of cerebral ischemia, recent studies have demonstrated increased phasic GABA signaling in the peri-infarct region of the cortex during the chronic repair phase following stroke. Here, we assessed GABAergic signaling in the hippocampus following global cerebral ischemia (GCI) and tested the hypothesis that the oxidative stress-activated calcium channel TRPM2 regulates post-ischemic GABA signaling, resulting in changes is plasticity and repair.
Aim: To assess TRPM2-mediated phasic GABA changes in the hippocampus during the chronic phase following ischemia.
Methods: Dissociated rat hippocampal neurons were exposed to 20-minute oxygen-glucose deprivation (OGD), fixed at 96 hours, and stained for GABAergic synaptic proteins, GABAA receptor γ2 subunit, gephyrin, and vGAT. For in vivo experiments, hippocampal slices were prepared 7 days following cardiac arrest/cardiopulmonary resuscitation in young adult mice (CA/CPR; GCI model) for whole-cell patch-clamp electrophysiology. Spontaneous inhibitory post-synaptic currents (sIPSC) were recorded in CA1 pyramidal neurons.
Results/Conclusions: CA/CPR and OGD increased density and clustering of GABAergic synaptic markers (22%-increase gephyrin, 25% γ2, 30% vGAT; p < 0.05). CA/CPR mice also exhibit increased sIPSC amplitude in CA1 neurons (sham: 58.5 ± 4.5 pA; CA/CPR: 91.2 ± 8.2 pA, p < 0.05). Inhibition of TRPM2 rapidly reduced clustering of GABAergic synapses and restored sIPSC amplitude to sham levels (66.25 ± 6.0 pA). These data suggest enhanced phasic GABA signaling may be a significant contributor to long-term GCI-induced E/I imbalance and hippocampal dysfunction resulting from persistent TRPM2 activity.
632
Diffuse traumatic brain injury induces progressive and long-term pericyte loss in the white matter
Ilknur Ozen and Niklas Marklund
Lund University, Lund, Sweden
Abstract
Traumatic brain injury (TBI) is a complex disease causing long-term neurological complications that can be observed in patients for several months and years after the initial injury. TBI leads to vascular dysfunction that can initiate a cascade of events leading to neurodegeneration and cognitive dysfunction. Although the mechanisms linking TBI to neurodegeneration are far from established, vasculo-centric pattern of axonal damage and neuroinflammation may be key pathological contributors to the accumulation of factors associated with neurodegeneration. Pericytes are versatile mural cells around the capillaries responsible for numerous microvascular functions in the CNS Acute effects of TBI on the vasculature and pericytes have been reported, yet long-term impacts of diffuse TBI on degeneration of pericytes and capillaries in ageing white matter (WM) remain unknown. We therefore investigated whether pericyte alterations in the WM are a time-dependent processes that starts with the initial injury or whether this becomes prominent with aging. We examined long-term effects of a single diffuse TBI on pericytes and capillaries of the WM, in particular, corpus callosum (CC) and external cortex (EC). We induced in C57/BL6 mice (age 10 to 12 weeks, 20–25 g) a central fluid percussion injury (cFPI) model or sham injury. Mice were sacrificed for analysis at 2, 7, 14, and 30 days as well as at 6 and 18 months post-injury. Immunohistochemical analyses were used to assess the distribution and quantification of pericytes in the WM capillaries. Pericytes were identified by platelet-derived growth factor receptor-β (PDGFR-β) antibody. When compared to sham-injured controls, there was a progressive decrease in the number of pericytes following cFPI, starting at 2 days post injury that continued up to 18 months in the white matter (p < 0.05 for all time-points).
These findings suggest that TBI causes chronic pathological white matter vascular changes, evident by progressive pericytes loss up to 18 months post-injury, which may contribute to the for development of neurodegenerative disease at long-term post-injury.
633
Possible vascular contributions to O-(2-[18F]-fluoroethyl)-L-tyrosine activity in patients with treated high grade gliomas
Otto Mølby Henriksen, Aida Muhic, Michael J Lundemann, Henrik BW Larsson, Adam E Hansen, Hans S Poulsen, Vibeke A Larsen and Ian Law
Rigshospitalet, Copenhagen, Denmark
Abstract
Background: Although uptake of amino acid positron emission tomography (PET) tracers in gliomas is considered to be mediated by specific transporters on tumor cells, tracer accumulation may also depend on tissue perfusion and permeability.
Aim: To investigate the association of dynamic contrast enhanced (DCE) perfusion MRI parameters to O-(2-[18F]-fluoroethyl)-L-tyrosine ([18F]FET) uptake in patients with treated gliomas.
Method: A total of 161 contrast enhancing lesions in 108 hybrid [18F]FET PET/MRI scans with DCE MRI from 98 adult patients with treated high-grade glioma were analyzed. Associations of median [18F]FET tumor-to-background ratio (TBRmed) with median BV (BVmed), blood flow (Fmed), permeability (Kimed) within contrast enhancing volumes (VOLCE) were investigated. Influence on prediction of progressive disease was assessed by logistic regression and ROC analysis.
Results/Conclusions: BVmed, Fmed and Kimed were all positively correlated with TBRmed. In mixed model analysis the combined effects of Fmed, BVmed, Kimed , VOLCE and IDH status explained 52% of the total variance in TBRmed, and the DCE parameters alone accounted for 32% of the residual variance when accounting for lesion volume. In multivariate logistic regression models progression was predicted by TBRmed, but by DCE parameters only in models without TBRmed. Adjusting TBRmed for activity accounted for by DCE parameters tended to lower ROC AUC for separation of progressive from non-progressive lesions.
In conclusion, tumor perfusion and permeability may contribute to a considerable fraction of the [18F]FET activity in contrast enhancing lesions in patient with treated gliomas, but may also possibly contribute to the diagnostic value of [18F]FET PET imaging.
635
Utility of PET/SPECT imaging as non-invasive method to quantify brain uptake of antibody transport vehicles
Paul McQuade1, Robert Thorne2, Michelle Pizzo2, Sarah DeVos2, Kirk Henne2, Darren Chan2, Mark Dennis2, Joy Zuchero2 and Christopher Winkelmann1
1Takeda Pharmaceuticals, Cambridge, USA
2Denali Therapeutics, San Francisco, USA
Abstract
Background: The blood–brain barrier limits the passage of biologics into the brain, with mAbs typically present in the brain at < 0.01–0.1% of blood concentration. One way to increase brain accumulation is to actively transport molecules through Receptor-Mediated Transcytosis (RMT). Antibody Transport Vehicles (ATVs) have been engineered to bind specifically to human transferrin receptor (hTfR) and increase brain accumulation via RMT.
Aim: To investigate whether imaging could be a suitable translatable biomarker to quantify regional brain distribution of ATVs, a tool ATV (ATV:Ctrl) was radiolabeled and imaging experiments conducted in both Wild Type (WT) and human/mouse transferrin receptor knock-in (TfRmu/hu KI) mice.
Method: ATV:Ctrl was radiolabled via N-succinimidyl 3-[125I]iodobenzoate ([125I]SIB). Then, imaging studies at tracer-only concentrations were conducted in both WT and TfRmu/hu KI mice, including a dose escalation study in TfRmu/hu KI mice. Following [125I]SIB-ATV:Ctrl administration, mice were imaged at 2, 24, and 48 h, with animals euthanized following 48 h timepoint. Ex-vivo biodistribution, as well as autoradiography, on perfused brain sections were then conducted.
Results/Conclusions: Under tracer-only conditions, the TfRmu/hu KI mice had significantly higher brain accumulation of [125I]SIB-ATV:Ctrl as compared to WT. A dose-dependent reduction in [125I]SIB-ATV:Ctrl brain accumulation was also observed in the TfRmu/hu KI. The imaging results were confirmed by both ex vivo autoradiography and biodistribution analysis. Together, this data confirms that increased brain accumulation of ATVs in TfRmu/hu KI mice can be directly assessed via imaging and supports continued investigation into imaging as a suitable translatable biomarker to quantify regional brain accumulation of ATV constructs.
636
Brain serotonin transporter is associated with cognitive affective biases in healthy individuals
Patrick Fisher1, Sophia Armand1,2, Brice Ozenne1,2, Nanna Svart1,2, Vibe Frokjaer1,2, Gitte Knudsen1,2 and Dea Stenbæk1,2
1Neurobiology Research Unit, Copenhagen, Denmark
2University of Copenhagen, Copenhagen, Denmark
Abstract
Background: Cognitive affective biases articulate tendencies to attend more to negative or positive environmental information. These biases are sensitive to alterations in extracellular brain serotonin (5-HT) levels following, e.g., pharmacological blocking and/or down-regulating the 5-HT transporter (5-HTT), which ameliorates negative affective biases. This suggests that 5-HTT level is positively associated with a cognitive priority of negative information over positive, but this link has not been evaluated in-vivo in healthy individuals.
Aim: Evaluate the association between brain 5-HTT level and cognitive affective bias in healthy humans. We hypothesised a negative association between brain 5-HTT level and cognitive bias.
Method: Ninety-eight healthy humans completed a 90-min [11C]DASB positron emission tomography scan to estimate regional binding potential (BPND). Cognitive affective bias was measured using the Emotional Faces Identification Task (EFIT) by subtracting the percent hit rate for happy from that of sad faces (i.e., negative values reflect negative bias and vice versa). We evaluated the association between [11C]DASB BPND and EFIT in a linear latent variable model where a 5-HTT-related latent variable (5-HTT-LV) was estimated from [11C]DASB BPND in fronto-striatal and fronto-limbic regions implicated in affective cognition.
Results/Conclusions: The estimated 5-HTT-LV was inversely associated with EFIT (estimate = −8% EFIT per unit 5-HTTLV, CI = −14% to −3%, p = 0.002). That is, in healthy individuals, higher 5-HTT level was associated with more negative cognitive bias. 5-HTT putatively contributes to higher 5-HT clearance; as such, a negative bias could stem from lower extracellular 5-HT. This observation more concretely implicates an association between brain 5-HTT and cognitive bias.
637
Cerebral and peripheral tissue oxygenation monitoring during arousal from torpor in arctic ground squirrels
Kelly Drew1, Hoshi Sugiura1, ArdyWong2, Caroline Vitovitsky2, Oivind Toien1 and Kambiz Pourrezaei3
1University of Alaska Fairbanks, Fairbanks, USA
2Barati Medical, Fairbanks, USA
3Drexel University, Fairbanks, USA
Abstract
Background: Cerebral blood flow in hibernating ground squirrels falls to 7 ± 4 ml 100 g−1 min−1, but sO2 of 57% during arousal suggests that arousal from hibernation poses the greatest risk of cerebral ischemia/reperfusion. Arousal from hibernation in arctic ground squirrels (Urocitellus parryii, AGS) is well known for profound increases in metabolic rate, core body temperature (from −3 to 37 °C) and heart rate (from 5 to 300bpm), but data related to cerebral and hind-limb tissue oxygenation during arousal is unequivocal.
Aim: We ask if AGS experience cerebral ischemia during arousal.
Method: We employed a noninvasive optical imaging technique, (near infrared spectroscopy; NIRS), to monitor oxygenation of the hind leg and brain during arousal in 8 AGS.
The NIRS device consisted of a controller module and two optical sensors: one for the head and one for the hind leg, and each contained an array of LED light source and photodiodes. Optical intensity measurements were converted to deoxyhemoglobin (Hb) and oxyhemoglobin (HbO2) changes relative to the first 30 s of recording at the beginning of arousal. O2 consumption was recorded by indirect calorimetry.
Results/Conclusion: Data shows an increase in deoxyhemoglobin and decrease in oxyhemoglobin concentration at both hind leg and brain during arousal. These results complement other findings suggesting that arousal from hibernation carries risk of ischemia/reperfusion. Results also demonstrate the feasibility of monitoring changes in cerebral oxygenation using NIRS. This will open new possibilities for studying brain and muscle metabolism and oxygenation in wild and laboratory animal species.
Supported P20GM130443 and 1R43NS100174.Figure 1. Changes in oxyhemoglobin (HbO2), deoxyhemoglobin (Hb), and (VO2) during arousal from torpor in the Arctic ground squirrel. Smooth black traces are the 9th order polynomial curve fits.
640
The importance of M1 muscarinic receptor phosphorylation in learning and memory
Aisling McFall1, Colin Molloy1, Craig Lindsley2, P. Jeffrey Conn2 and Andrew Tobin1
1Centre for Translational Pharmacology University of Glasgow, Glasgow, UK
2Vanderbilt Center for Neuroscience Drug Discovery, Nashville, USA
Abstract
Background: Recently published data reveal that agonism of the G-protein coupled receptor, muscarinic acetylcholine receptor 1 (mAChR1), restores learning and memory (LM) deficits and improves survival in a murine prion model of terminal neurodegeneration which, importantly, displays many of the hallmarks of Alzheimer’s disease. In addition, it has been demonstrated that phosphorylation of mAChR1 is involved in this neuroprotective response. Here we further test the hypothesis that the phosphorylation of mAChR1 is required for the neuroprotective effect of mAChR1 agonism.
Aim: Assess if the phosphorylation of mAChR1 is required for the rescue effect of VU846 (mAChR1 positive allosteric modulator) in LM deficits in contextual fear conditioning.
Method: A memory deficit was induced in male mice expressing HA-tagged mAChR1 (M1-WT) (n = 8–11) or HA-tagged phosphorylation deficient mAChR1 (M1-PD) (n = 5–9) with 1.5 mg/kg of scopolamine (i.p) +/− VU846 (10 mg/kg), or vehicle (10% Tween 80) as control, 30 mins prior to training in a fear conditioning (FC) paradigm. After 24 hrs, contextual memory retrieval was assessed by measuring the level of freezing when returned to the FC context.
Results/Conclusions: Preliminary results indicate that VU846 restores LM deficits in M1-WT mice but not in M1-PD mice. In addition, while scopolamine induced LM deficits in both groups, M1-PD mice (freezing: Veh 30%, Scop 15%) display a deficit in contextual fear memory compared to M1-WT mice (Veh 58%, Scop 38%). This suggests that phosphorylation of mAChR1 is required for the LM benefits of mAChR1 agonism and further work will assess if the same is true for mAChR1 agonism in prion neurodegeneration.
641
Prmt4 methylation of NOTCH1 causes BBB compromise and neurovascular uncoupling in Alzheimer’s disease
Garrett Clemons1, Christina Acosta1, Mariana Sayuri Berto Udo2, Cristiane T Citadin1, Vesna Tesic2, Reggie HC Lee2, Celeste CY Wu2 and Kevin (Hung Wen) Lin2
1Department of Cell Biology and Anatomy, Shreveport, USA
2Department of Neurology, Shreveport, USA
Abstract
Background: Alzheimer’s disease (AD) is a leading cause of mortality, disability, and long-term care burden in the United States; and women are more likely to develop a rapid progression of dementia than men. While AD-related dementia is associated with tau and beta-amyloid accumulation, concurrent derangements in cerebral blood flow (CBF) have been observed alongside these proteinopathies in humans/mice. Cerebral vascular dysfunction can exacerbate blood brain barrier (BBB) dysfunction, loss of junctional proteins, and ionic imbalance altogether contributes to neurovascular uncoupling and cognitive decline. Altered NOTCH1 expression has been associated with AD, as NOTCH1 is lower in AD patients. NOTCH1 knockdown can enhance BBB permeability resulting in further damage. Therefore, neuronal/vascular NOTCH1 is a promising target against AD-related cerebrovascular pathologies. Protein arginine methyltransferase 4 (PRMT4) is a specific methylator of the Proline, Glycine, Methionine motif in the intracellular domain of NOTCH1, leading to degradation.
Aim: The aims of this study are to 1) elucidate the link between PRMT4 and NOTCH1, 2) restore CBF in AD and preserve functional learning/memory.
Method: Real-time qPCR and capillary-phoresis (protein detection) were used to quantify mRNA and proteins levels, respectively. Neurovascular coupling was measured using two-photon laser scanning microscopy with whisker stimulation. Novel texture discrimination task, novel object recognition, and T-maze were used to assess cognitive and behavioral outcomes.
Results/Conclusion: Our data suggest that 3×Tg-AD female mice have 1) increased hippocampal levels of PRMT4 and decreased NOTCH1, 2) increased type-1 PRMT methylation of NOTCH1, 3) reduced endothelial junctional proteins, 4) neurovascular coupling, and 5) functional outcomes.
642
Quantitative relationship between the cerebrovascular network and neuronal cell types in mice
Yuan-Ting Wu1, Hannah Bennett1, URee Chon6, Daniel Vanselow2, Qingguang Zhang3, Rodrigo Muñoz-Castañeda4, Keith Cheng2, Pavel Osten4, Patrick Drew3,5 and Yongsoo Kim1
1Department of Neural and Behavioral Sciences, The Pennsylvania State University, Hershey, USA
2Department of Pathology, The Pennsylvania State University, Hershey, USA
3Center for Neural Engineering, Department of Engineering Science and Mechanics, The Pennsylvania State University, University Park, USA
4Cold Spring Harbor Laboratory, Cold Spring Harbor, USA
5Biomedical Engineering, and Neurosurgery, The Pennsylvania State University, University Park, USA
6Stanford University, Stanford, USA
Abstract
Background: The importance of cerebrovasculature and its mural cells in maintaining brain regional energy demands is well understood, but how their spatial relationship with different neuronal cell types varies across the brain remains largely unknown.
Aim: We apply brain-wide mapping methods to define quantitative relationships between cerebrovasculature, capillary pericytes, and glutamatergic and GABAergic neurons, including neuronal nitric oxide synthase-positive (nNOS+) neurons and their subtypes.
Method: Our brain-wide mapping is achieved through cellular resolution imaging using serial two photon tomography (STPT) in adult mice. For vessel tracing, we filled microvessels from C57BL/6 mice with cardiac perfusion of fluorescein isothiocyanate (FITC)-conjugated albumin gel. For mural and neuronal cell type mapping, a genetic labelling method using cell type specific Cre drivers was crossed with conditional reporter mice. For nNOS subtype populations, we also utilized genetic intersection methods, using Cre and Flp dependent reporter mice.
Results/Conclusions: Our results show strikingly high densities of vasculature with high fluid conductance and capillary pericytes in primary motor-sensory cortices (compared to association cortices), that also show significantly positive and negative correlations with energy demanding parvalbumin+ and vasomotor nNOS+ neurons, respectively. Moreover, sensory-motor associated thalamo-striatal areas also show high densities of vasculature and pericytes, suggesting dense energy support for sensory-motor processing. Our cellular-resolution resource offers opportunities to examine these spatial relationships in largely under-studied subcortical areas. To further understand the anatomical organization of the brain vasculature and mural cells, we are now incorporating whole brain clearing, labelling, and light sheet fluorescent microscopy.
643
Repurposing the cough expectorant ambroxol to a neuroprotective drug for ischemic stroke
Kristin Patzwaldt1, Georgy Berezhnoy1, Tudor Ionescu1, Linda Schramm1, Yi Wang2, Miriam Owczorz1, Eduardo Calderon3, Irene Gonzalez-Menendez4, Sven Poli2, Kristina Herfert1, Leticia Quintanilla-Martinez4, Bernd Pichler1, Christoph Trautwein1 and Salvador Castaneda-Vega1,3
1Werner Siemens Imaging Center, University of Tuebingen, Tübingen, Germany
2Department of neurology, University Clinic Tuebingen, Tübingen, Germany
3Department of Nuclear Medicine and Clinical Molecular Imaging, University Clinic Tuebingen, Tübingen, Germany
4Department of pathology and neuropathology, University Clinic Tuebingen, Tübingen, Germany
Abstract
Background: Rehabilitation and functional recovery following ischemic stroke remains a difficult clinical challenge. Ambroxol is an over-the-counter drug with a well-known safety profile and brain penetration. It is a powerful inhibitor of sodium and calcium channels, increases oxygen radical scavengers and is therefore an optimal candidate for drug-repurposing as a neuroprotectant.
Aim: To evaluate the effects of ambroxol administration on brain function recovery after ischemic stroke from the sub-acute to chronic phase combining advanced in-vivo neuroimaging techniques with ex-vivo tissue metabolomics.
Method: Longitudinal evaluation of 53 rats using a comprehensive framework of neuroimaging tools measuring T2-weighted images, diffusion tensor images and resting state fMRI for up to one month after ischemic stroke induction using the temporary middle cerebral artery occlusion model. Neuroimaging data was acquired in parallel to longitudinal behavioral evaluations. Finally, we explored metabolomic changes in the stroke brain following Ambroxol administration using NMR in a subgroup of animals.
Results/Conclusions: Stroke rats receiving one week-course of Ambroxol after stroke onset presented significantly reduced stroke volumes, reduced cytotoxic edema, reduced white matter deterioration in the corpus callosum, improved behavioral outcomes and complex changes in functional brain connectivity as measured by resting state fMRI. In the chronic stroke phase, we found reductions in necrosis in Ambroxol administered animals in comparison to Controls. NMR tissue metabolomics at 24 h post stroke identified several altered metabolites that are capable of minimizing post-ischemic damage, which were increased in the stroke regions of Ambroxol-treated animals, such as pantothenate and ascorbate, ketone bodies and glutathione.
645
Does the combination of principal component analysis/logistic regression improve the prediction of Parkinson’s disease?
Lyna El Haffaf1,2, Lucas Ronas1,2 and Alexandru Hanganu1,2
1Université De Montréal, Laval, Canada
2Centre de recherche de l’institut universitaire de gériatrie de montréal, Montral, Canada
Abstract
Background: Machine learning is a potential tool in identifying relevant features that are not traditionally used in the clinical diagnosis of Parkinson’s disease (PD).
Aim: We aim to compare two machine learning models based on MRI and neuropsychiatric variables.
Method: The Parkinson’s Progressive Markers Initiative database was used to analyze data from 166 healthy controls and 372 patients with PD. Neuropsychiatric (apathy, anxiety, depression and hallucination) and 3 T MRI data were extracted. Statistical MRI data (cortical thickness, areas, curvatures, folding index, and volumes) were extracted with FreeSurfer 7.1.1. We performed principal component analysis and binary logistic regression models with IBM Statistics SPSS 27.0 software with the clinical group as a dependent variable and demographic, neuropsychiatric, and neuroimaging data as covariates.
Results/Conclusions: A Principal Component Analysis (PCA) approach applied on 301 variables (9 neuropsychiatric variables and 288 brain structures), extracted 54 components that explained 83% of the total group variance. These 54 components were correlated with 146/301 variables (R = 0.4–0.98). Based on the PCA results, logistic regression models were used to classify PD participants from the controls with 72.1% accuracy. Based on the full set of variables, the regressions showed 74.2% accuracy of classifications. Reducing the dimensionality using the PCA algorithm reported an almost similar accuracy level compared to the method without the PCA. Nevertheless, the analysis on the whole group of features seems to provide a slightly increased accuracy. These results outline that both methods – with and without dimensionality reduction, can be included in predictive models.
646
Cerebral regional pharmacokinetics and distribution at the steady-state of haloperidol and clozapine in healthy human
Hye Won Kim1,2, Su Bin Kim1,2, Byung Chul Lee1,3, Byung Seok Moon4, Hyun Soo Park1,5 and Sang Eun Kim1,5,6
1Department of Nuclear Medicine, Seoul National University Bundang Hospital, Seoul National University College of Medicine, Seongnam, Korea
2Department of Applied Bioengineering, Graduate School of Convergence Science and Technology, Seoul National University, Suwon, Korea
3Center for Nanomolecular Imaging and Innovative Drug Development, Advanced Institutes of Convergence Technology, Suwon, Korea
4Department of Nuclear Medicine, Ewha Womans University Seoul Hospital, Ewha Womans University College of Medicine, Seoul, Korea
5Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul National University, Suwon, Korea
6Advanced Institutes of Convergence Technology, Suwon, Korea
Abstract
Background: Haloperidol (HAL) and clozapine (CLZ) are widely used antipsychotic drugs fundamentally sharing the mechanism of action for dopaminergic hyperactivity; however, their cerebral regional pharmacokinetics and distribution at the steady-state have not yet been fully elucidated. Positron emission tomography (PET) allows analysis of the pharmacokinetic characteristics of radiolabeled molecules in vivo.
Aim: The aim of the present study was to examine and compare the cerebral regional in vivo pharmacokinetics and distribution at the steady-state of HAL and CLZ using PET and 18F-HAL and 11C-CLZ.
Method: The cerebral regional time-courses of 18F-HAL and 11C-CLZ distribution were determined using brain PET/CT images dynamically acquired following the single intravenous administration in human volunteers. Compartmental analysis with image-derived input function for reversible and irreversible binding models was performed to quantitatively describe pharmacokinetics in the non-specific and specific binding compartment and subsequently estimate the distribution volume (DV). To illustrate the differences between 18F-HAL and 11C-CLZ distribution at the steady-state, voxel-wise analysis for the DV parametric images was performed with statistical parametric mapping.
Results/Conclusions: The cerebral regional time-courses of 18F-HAL and 11C-CLZ distribution was considerably different. 11C-CLZ showed varied pharmacokinetics across regions, while the variation across regions was minor for 18F-HAL. The reversible two-tissue four-parameter model best described the pharmacokinetics in the striatal non-specific and specific binding compartment for both 18F-HAL and 11C-CLZ. DVs across regions were ranged 5.1–5.7 and 2.9–4.2 for 18F-HAL and 11C-CLZ, respectively, and the greatest differences were found in the voxels constructed the cerebellar gray matter.
647
In vivo characterization of [11C](+)3-MPB binding to mAChR in the brain of living mice
Su Bin Kim1,2, Hye Won Kim1,2, Byung Seok Moon3, Byung Chul Lee2, Hyun Soo Park2,4 and Sang Eun Kim2,4,5
1Department of Applied Bioengineering, Graduate School of Convergence Science and Technology, Seoul National University, Seoul, South Korea
2Department of Nuclear Medicine, Seoul National University College of Medicine, Seoul National University Bundang Hospital, Seongnam, South Korea
3Department of Nuclear Medicine, Ewha Womans University Seoul Hospital, Ewha Womans University College of Medicine, Seoul, South Korea
4Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul National University, Seoul, South Korea
5Advanced Institutes of Convergence Technology, Suwon, South Korea
Abstract
Background: Positron emission tomography (PET) radiotracers targeting muscarinic acetylcholine receptors (mAChR) could have an enormous impact on enhancing therapeutic strategies for various neurological diseases including dementia. (+)N-[11C]methyl-3-piperidyl benzilate ([11C](+)3-MPB) was developed as a PET radiotracer for mAChR.
Aim: The aim of the present study is to characterize binding of [11C](+)3-MPB to mAChR in the brain of living mice.
Method: A total of 24 ICR mice underwent dynamic [11C](+)3-MPB PET/CT studies after treatment with vehicle and different doses of mAChR antagonists, solifenacin and oxybutynin. Regional brain non-displaceable binding potential (BPND) of [11C](+)3-MPB to mAChR was estimated using time-course of [11C](+)3-MPB distribution in cortical and subcortical structures and a simplified reference tissue model with cerebellar reference tissue input function. The differences in regional brain BPND of [11C](+)3-MPB were examined to establish the dose-response relationship in terms of the effective dose (ED50) using non-linear regression analysis for the Emax model.
Results/Conclusions: [11C](+)3-MPB PET quantitatively visualized cortical and subcortical mAChR function and its dose-dependent changes after solifenacin and oxybutynin treatment in the brain of living mice. Solifenacin and oxybutynin showed a maximal 61% and 60% difference compared to BPND of vehicle treatment for the highest doses of those drugs (10 mg/kg and 1 mg/kg, respectively) in the striatum. ED50 across regions ranged 3.67–12.54 mg/kg and 0.42–1.18 mg/kg for solifenacin and oxybutynin, respectively. In vivo quantitative PET analysis of brain mAChR occupancy may provide a fundamental basis for managing therapeutic strategies of potential drugs that exert mAChR antagonism implicated in neurological diseases.
648
Cognitive impairment in aging population is associated with decreased MTL synaptic density
Alexandra DiFilippo1, Yangchun Xin1, Andrew McVea1, Max McLachlan1, Mary-Elizabeth Pasquesi2, Nancy Davenport-Sis2, Tobey Betthauser2, Todd Barnhart2, Jonathan Engle2, Sterling Johnson2, Barbara Bendlin2 and Bradley Christian1
1University of Wisconsin-Madison Waisman Center, Madison, USA
2University of Wisconsin-Madison School of Medicine and Public Health, Madison, USA
Abstract
Background: Cognitive impairment in the ageing population is believed to associate with synaptic loss, but data are limited due to few in vivo studies. The PET radiotracer [11C]UCB-J is used in this work to quantify synaptic density in the mesial temporal lobe and globally. Group differences were assessed between cognitively unimpaired and impaired participants.
Methods: Participants (N = 45) were recruited from the Wisconsin ADRC and the Wisconsin Registry for Alzheimer’s Prevention study. Cognitive impairment (dementia or mild cognitive impairment) was determined using the NINDS/ADRDA criteria (McKahnn et al, 2011). Participants underwent PET imaging of beta-amyloid plaques, tau tangles, and synapses. Synaptic density was quantified from UCB-J PET data using LGA (DVRCb(T) =DVRCS(T)/DVRCS(cerebellum)). Unpaired one-sided t-tests with ⍺ = 0.05 used for group comparisons.
Results: Participants were separated into cognitively unimpaired (CU, N = 31, 23 F/8M, 65.5 ± 6.9 yo) and cognitively impaired (CI, N = 12, 3 F/9M, 74.3 ± 6.9 yo). Group differences were significant in the mesial temporal lobe (P = 0.03), but not in the global ROI (P = 0.13). Exploratory analyses between the sub-regions of the global ROI revealed significant group differences in the hippocampus (P = 0.01) and the medial occipital lobe (P = 0.01).
Conclusion: Comparing regional synaptic density in the mesial temporal lobe in cognitively unimpaired and impaired participants revealed significant differences, but density did not differ globally. Future work will incorporate PET data on beta-amyloid plaques and tau tangles when evaluating cognitive impairment and synaptic density.
649
Resting cerebral blood flow and respiratory parameters immediately post concussion
Stephanie Iring-Sanchez1, Paul Breen2, Ganesh Naik2, Michael Falvo1,3 and Jorge Serrador1,2
1Department of Pharmacology, Physiology and Neuroscience, Rutgers Biomedical Health Sciences, Newark, USA
2Western Sydney University, Australia
3War Related Illness and Injury Study Center, Department of Veterans Affairs, East Orange, USA
Abstract
Objectives: Sports-related concussions have become a major public health issue due to their common occurrence in recreational sports and the increased awareness of injuries in high-profile athletes. The clinical symptoms of concussions are reported to resolve within two weeks, but increasing evidence indicates that many athletes continue to experience symptoms and cognitive problems beyond this time frame. The development of an objective concussion assessment protocol that can be administered immediately after a suspected concussion has occurred is critical. In developing this assessment, it would be useful to understand the physiological changes that occur following a concussion. The objective of our research study was to investigate changes in cerebral blood flow and respiratory parameters in recreational rugby players.
Methods: Data was obtained in a mobile tent-based laboratory during a recreational rugby tournament. Athletes were assessed acutely after a head injury, within a median of 60 minutes, or following participation in a rugby match. Of the 50 rugby players (39 male and 11 female) that participated in our study 22 were concussed and 28 were control players. Participants ranged in age between 18 and 66 years (33 years ± 13 years). Participants who experienced an impact to the head were assessed by the medical team, and if they did not need further treatment, they were offered the opportunity to participate in this study. Next, the Sports Concussion Assessment Tool 3 (SCAT3) was administered by trained personnel. To assess the autonomic effects of concussion we measured beat-by-beat blood pressure (Finometer), heart rate, common and internal carotid blood flow, and end-tidal CO2 using a nasal cannula. To assess respiratory parameters, we used a T-shirt embedded with four non-invasive, non-contact, and unobtrusive electro-resistive bands (Gargiulo, O’Loughlin, & Breen, 2015). Measures were taken during rest, both in the seated and supine positions.
Results: There were no significant differences in the SCAT3 symptom severity scores among players who experienced a head impact following a match and the control players who just played a match (18 ± 19 vs 18 ± 18, p=.95). There were significant decreases in the internal carotid artery (ICA) blood flow, in the supine position in players with a head impact (507 ± 107ml/min) compared to the control players (663 ± 250ml/min), p = .03. However, there were no differences in ICA blood flow in the seated position (689 ± 222 vs 634 ± 196, p = 0.41). Players with a head impact had significantly higher Respiratory Rates (RR) (19.26 ± 2.63 vs 16.75 ± 2.94, p < .001) than control players as well as significantly lower Respiratory Rate Variability (RRV) (2.54 ± 0.34 vs. 2.09 ± 0.82, p < 0.001) than control players. Similarly, Respiratory Effort (RE) was significantly lower (0.69 ± 0.08 vs 0.54 ± 0.1; p < 0.001) in players with a head impact compared to control players.
Conclusions: This data suggests that players with head injuries demonstrate reduced cerebral blood flow, indicated by lower ICA flow, when supine. In addition, they had significant changes in respiratory patterns with higher rates, lower variability and reduced effort. These physiological changes were not associated with significantly different symptom reporting. Thus, these data suggest that physiological indicators of head injury may provide a better diagnostic indicator than self-reported symptoms. Future work is needed to examine how acute post injury measures of respiratory parameters and brain blood flow in both men and women are predictive of concussion outcomes.
650
Effect of carbonic anhydrase inhibitors in the neurovascular disturbances of the Tg-SwDI murine AD model
Boris Wied, Signe Mikkelsen, Dmitry Postnov, Alberto Gonzalez Olmos, Thomas Beck Lindhart, Vladimir Matchkov, Sylvia Forsati, Leif Østergaard and Eugenio Gutierrez
Center for Functionally Integrative Neuroscience CFIN, Aarhus University, Aarhus N, Denmark
Abstract
Background: Alzheimer’s Disease (AD) is the most prevalent type of dementia, characterized by an accumulation of amyloid-beta (Aβ) and hyperphosphorylated tau protein. Unfortunately, current treatments of AD are inadequate and show limited efficiency. Much evidence suggests that AD has a cerebrovascular component. Patients with AD show decreased cerebral blood flow, accompanied by impaired neurovascular coupling (NVC) and changes in pulsatility index (PI). These changes hinder the delivery of oxygen and nutrients to the brain tissue. Therefore, drugs targeting the regulation of brain hemodynamics might be beneficial for treating AD. Carbonic anhydrase inhibitors (CAIs) are vasodilator drugs that reduce amyloid-beta neurovascular toxicity and might prevent cerebrovascular disturbances in AD.
Methods: In this study, we performed in-vivo LSCI imaging in awake mice to test the effect of 10-months treatment with CAIs on vascular changes of the Tg-SwDI mouse model of AD. Both males and females were included. Treatment started at 6-months of age, and a chronic cranial window was implanted at 12-months. All mice were imaged three times in the following four months. The scans performed evaluated 1. Neurovascular coupling during whisker stimulation, 2. Pulsatility index, and 3. Vasoreactivity to CAI bolus with Acetazolamide.
Results: We hope to show how disturbances in NVC and PI are characteristic of Tg-SwDI mice, and may be prevented by CAIs. We also expect vasoreactivity to CAIs to be intact in all mice, showing flow impairment to be functional rather than mechanically limited. Our results highlight the prospect of utilizing CAIs for improving hemodynamics disturbances in AD.
651
Automated segmentation of neck arteries on phase contrast images using deep learning
Britney Campbell1, Dhruv Yadav2, Moss Zhao3, Ramy Hussein3, Michael Moseley3 and Greg Zaharchuk3
1University of South Florida, Tampa, USA
2University of Washington, Seattle, USA
3Stanford University, Stanford, USA
Abstract
Background: As cerebral blood flow (CBF) is a component of brain health, its accuracy is of great importance.1 Patients’ CBF can be determined using phase contrast (PC) MRI imaging which computes blood vessels’ flow.2 Moyamoya patients experience low CBF which leads to strokes due to narrowing of their arteries.3 Analysis of patients like those with Moyamoya disease require segmentation of PC MRI images, traditionally done manually.
Aim: Deep learning (DL) allows for automated segmentation with improved accuracy.4 In this study, DL models auto-segment neck arteries in PC MRI images using 107 healthy subjects (HC) and 46 Moyamoya (MM) patients.
Method: PC MRI images were averaged across 10 cardiac phases to form the mean magnitude and phase images, the two inputs for each model.5 Three U-Net segmentation models, standard, Attention, and Nested, were trained on the dataset for 1000 epochs, optimized by Adam with an initial learning rate of 0.001. The learning rate was divided by 2 per 30 epochs when validation stagnated, and extensive data augmentation was applied as affine image transforms. The Wilcoxon rank-sum test quantified significant differences between the volume flows calculated by the models and Arterys, an AI web-based platform.
Results/Conclusions: The standard, Nested, and Attention U-Net achieved average dice scores of 0.814, 0.846, and 0.845, respectively. Of the three models, the Nested U-Net showed no significant difference (p > 0.05) in volume flows compared to Arterys for the HC and MM subsets. Therefore, attaining accuracy in both segmenting neck arteries and calculating blood flow.Figure 1. Here we see the predicted segmentation by the Nested U-Net on a sample in the validation dataset. The input to the model consists of the two channels shown in the first column, and the ground truth and prediction are shown in the second and third column, respectively.
References
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Phase-contrast magnetic resonance imaging measurements of cerebral autoregulation with a breath-hold challenge: a feasibility study. Stroke2004;
35: 1350–1354.KhanMALiuJTarumiT, et al.
Measurement of cerebral blood flow using phase contrast magnetic resonance imaging and duplex ultrasonography. J Cereb Blood Flow Metab2017;
37: 541–549.a-15SteinbergGKGooderhamPA. Intracranial-extracranial bypass surgery for moyamoya disease. Neurovasc Surg2015; in 2nd edition., Y. Kalani, P. Nakaji, and R. F. Spetzler, Eds. New York: Thieme.a-16ZaharchukGGongEWintermarkD, et al. Deep learning in neuroradiology. Am J Neuroradiol 2018, doi: 10.3174/ajnr.A5543.a-17ZhaoMYFanAPChenDY-T, et al.
Cerebrovascular reactivity measurements using simultaneous 15O-water PET and ASL MRI: impacts of arterial transit time, labeling efficiency, and hematocrit. NeuroImage2021;
233: 117955.a-18
652
Covid status is related to clot burden during thrombectomy in acute stroke patients
Justin Fraser1, Alan Dabney2, James Vicari3, Dennis Rivet4, B Keith Woodward5, Ashish Nanda6, David Fiorella7, Selva Baltan8, Farida Sohrabji2, Keith Pennypacker1 and Christopher Kellner3
1University of Kentucky, Lexington, USA
2Texas A&M University, College Station, USA
3Mount Sinai School of Medicine, New York, USA
4Virginia Commonwealth University School of Medicine, Richmond, USA
5Vista Radiology, Knoxville, USA
6SSM Health, Fenton, USA
7SUNY Stony Brook, Stony Brook, USA
8Oregon Health & Science University, Portland, USA
Abstract
Background: The INSIGHT Registry, a multicentered ‘multi-omic’ analysis of thrombi associated with acute hemorrhagic and ischemic stroke is currently enrolling, with over 300 subjects already enrolled. Through this effort, we can evaluate ischemic stroke thrombi in the setting of concurrent medical conditions. While it has been reported that COVID positivity can affect clotting mechanisms, there is a paucity of data on histopathology of stroke clot in this setting.
Aim: Our aim in this analysis was to study the relationship between COVID positivity and clot histopathology in large vessel occlusive stroke.
Methods: Patients, aged ≥18 years, treated frontline with the Penumbra System® for thrombectomy are included in this analysis. Patient demographics, medical history, radiographic, and procedural information are collected in conjunction with extracted clot and concurrent extracranial arterial blood. While the protocol includes analysis for proteomics and transcriptomics, for this analysis we used histopathological evaluation of the thrombi.
Results/Conclusion: Of 230 patients included in this interim analysis across 24 centers in the United States, between 02/2021 and 01/2022, 16 patients had a history of COVID. Comparing COVID-positive to non-positive, patients with COVID were younger (median 66.5 years vs 71.5 years, p 0.042), and had more intracranial clot by weight (median 101.0 mg vs 42.0 mg, p 0.047). There was a trend toward COVID positive patients to have more clot volume (median 180 cc vs 77.5 cc p 0.08), suggesting the difference was a higher clot burden instead of more density to the thrombus. These results suggest different histopathological characteristics in clot formation in COVID patients with large vessel occlusive stroke.
aPvalues based on the Wilcoxon Signed Rank Test for continuous outcomes, Fisher’s Exact Test for categorical outcomes.
653
Ephrin-Receptor-Beta-1 single nucleotide polymorphism in association with age in acute stroke patients undergoing thrombectomy
Justin Fraser1, Alan Dabney2, James Vicari3, Dennis Rivet4, B. Keith Woodward5, Ashish Nanda6, David Fiorella7, Selva Baltan8, Farida Sohrabji2, Keith Pennypacker1 and Christopher Kellner3
1University of Kentucky, Lexington, USA
2Texas A&M University, College Station, USA
3Mount Sinai School of Medicine, New York, USA
4Virginia Commonwealth University, Richmond, USA
5Vista Radiology, Knoxville, USA
6SSM Health, Fenton, USA
7SUNY Stony Brook, Stony Brook, USA
8Oregon Health and Science University, Portland, USA
Abstract
Background: Ephrin receptors and their ligands, the ephrins, mediate numerous developmental processes, particularly in the nervous system. The Ephrin Receptor Beta 1 (EPHB1) is one of several developmentally associated growth inhibitors induced during ischemic models. Using the INSIGHT Registry, a multicentered ‘multi-omic’ analysis of thrombi removed during human thrombectomy, we can analyze variances in single nucleotide polymorphisms for EPHB1.
Aim: Our aim in this analysis was to evaluate SNPs previously correlated with ischemic stroke. In this abstract, we present data on EPHB1 single nucleotide polymorphisms (SNP) as it relates to age during acute infarction.
Methods: Patients, aged ≥18 years, treated frontline with the Penumbra System® for thrombectomy are included in this analysis. Patient demographics, medical history, radiographic, and procedural information are collected in conjunction with extracted clot and concurrent extracranial arterial blood. For this analysis, we evaluated SNPs previously correlated with ischemic stroke.
Results/Conclusion: 193 patients were included in this interim analysis across 23 centers in the United States, between 02/2021 and 11/2021. For the EPHB1 SNP (rs13073658), Table 1 shows the numbers and proportions of patients with varying expressions of the SNP. While no patients 50 or older were homozygous for the SNP, 16% of those under 50 years old were. The potential relationship between EPHB1 SNP and stroke in younger patients requires further evaluation.
Table 1. Numbers and proportions of patients with varying expressions of the SNP.
638
The influence of PRMT7 in the pathological progression of repetitive and mild traumatic brain injury
Mrs Christina Acosta1, Dr. Cristiane Citadin1, Mr. Garrett Clemons1, Mr. William Carr2, Dr. Mariana Udo2, Dr. Celeste Yin-Chieh Wu2, Dr. Reggie Hui-Chao Lee2 and Dr. Hung Wen (Kevin) Lin2
1LSU Health Shreveport – Cell Biology & Anatomy, Shreveport, United States,
2LSU Health Shreveport – Neurology, Shreveport, United States
Abstract
Background: Traumatic brain injury (TBI) occurs as an impact to the head that causes brain tissue alterations. Mild and repetitive TBI (rmTBI) accounts for the largest proportion of TBI-cases, leading to long-term cognitive and behavioral impairment.
Aim: We sought to identify novel molecules that could contribute to the pathological progression of rmTBI.
Method: rmTBI Hits: A clinically relevant closed-head impact model of engineered rotational acceleration (CHIMERA).
Results/Conclusions: We assessed diffuse axonal injury and demonstrated enhanced silver deposition (dark stained regions) throughout the brain. Next, PRMT7 protein expression was significantly decreased in the cortex at 3 days (0.5815 ± 0.04701) and 7 days (0.5696 ± 0.03154) relative to sham (0.7780 ± 0.03064). Glutamate levels were enhanced at 3 days (6714 ± 107.8) relative to SHAM (5522 ± 235.3) within the hippocampus. In addition, there was a significant increase in DRP1 protein expression at 3 (5.248 ± 0.2749) and 7 days (5.720 ± 0.2031) relative to sham (2.571 ± 0.1839) in the cortex and a significant increase of DRP1 at 1 day (4.120 ± 0.1551), 3 day (5.802 ± 0.3690), and 7 days (4.861 ± 0.4092) in the hippocampus compared to sham (2.790 ± 0.1325).
Seahorse XF24 analyzer demonstrated a significant decrease 1 day (70.28 ± 3.476) in ATP-linked respiration followed by an increase 3 days (102.1 ± 4.991) post-rmTBI. In addition, there was a significant increase of maximal respiration at 3 days (176.7 ± 8.430) relative to 1 day (130.7 ± 6.614) post-rmTBI mice. Finally, learning, working memory, and locomotor skills were significantly impaired post-rmTBI. Overall, our data suggests PRMT7 may be influencing mitochondrial health and contributing to the pathological progression of repetitive and mild TBI in mice.
