Abstract
M. M. Acharya, V. Martirosian, V. K. Parihar, L.-A. Christie, and C. L. Limoli
Department of Radiation Oncology, University of California, Irvine, CA, USA
Advancements in cancer therapies have increased the number of cancer survivors significantly. As a more chronic disease, nearly 60% of the patients routinely survive over 20 years. With increased survival, factors impacting quality of life become increasingly important. For those afflicted with primary and secondary malignancies of the CNS, radiotherapy remains the frontline treatment for forestalling disease progression. Approximately one third of the 1.5 million cancers diagnosed will develop brain metastases, and along with primary tumors of the CNS, nearly 200,000 patients/year receive partial or whole brain irradiation. The majority of these patients suffer from cognitive dysfunction, which is a particular problem in pediatric cases. The deteriorating cognitive health experienced by those subjected to cranial irradiation becomes a greater concern in the absence of any satisfactory long-term solutions. Mechanisms responsible for radiation-induced cognitive decline are certain to be complex, but one of the causal factors is likely to involve disruptions to hippocampal neurogenesis. Given this potential therapeutic target, we explored the use of cell replacement strategies for ameliorating radiation-induced dementia. For these studies we utilized induced pluripotent stem cell (iPSC)-derived human neural stem cells (iPS-hNSCs). Previously, we have shown that transplantation of human embryonic and embryonic-derived neural stem cells at 2-days postirradiation (IRR) restored radiation-induced cognitive dysfunction (Acharya et al., 2009, 2011, 2012) in a hippocampal-dependent novel place recognition task (NPR). We have now extended this study by transplanting iPS-hNSCs at 2 days, 2 weeks, or 4 weeks after 10-Gy head-only λ-IRR, followed by cognitive testing using hippocampus-dependent NPR and fear conditioning (FC) tasks at 1 month after transplantation surgery. Transplanted iPS-NSCs significantly improved cognition at all three post-IRR time points on the NPR task. iPS-hNSC transplanted animals explored the novel place more than expected by chance as compared to the IRR sham surgery group. Importantly, the 4-week group showed significant improvements in a hippocampus-dependent contextual FC test. On the other hand, neither IRR nor iPS-hNSC transplantation affected freezing behavior during the cue test phase of FC, indicating intact amygdala function. Immunohistochemical analyses using human-specific nuclear antigen marker (Ku80) revealed that transplanted cells migrated extensively throughout the host hippocampus. Ongoing dual immunofluorescence staining and confocal microscopy analyses thus far indicated that transplant-derived cells differentiated along glial and neuronal lineages. These findings provide evidence that cell replacement strategy using iPS-hNSCs can either stop, reverse, or prevent radiation-induced cognitive dysfunction when transplanted at protracted post-IRR time points. iPS-hNSCs can survive and differentiate along neural lineages in the irradiated tissue bed. These data suggest that similar strategies can be tailored using good laboratory practice (GLP)-derived human neural stem cells to revert the devastating side effects of clinical radiation exposure on cognitive function.
Research supported by NIH-NINDS, RO1 NS074388581, C.L.L.
S. A. Acosta,* N. Tajiri,* M. Shahaduzamman,* K. Shinozuka,* H. Ishikawa,* C. Metcalf,* T. Dailey,* G. Franyuti,* M. Pabon,* D. Kim,* D. G. Hernandez-Ontiveros,* J. Vasconcellos,* M. Staples,* L. Gould,†‡ N. Patel,†§ D. R. Cooper,†§ Y. Kaneko,* C. V. Borlongan,* and P. C. Bickford*†‡
*Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
†James A. Haley Veterans Affairs Medical Center, Tampa, FL, USA
‡Department of Molecular Pharmacology and Pharmacology, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
§Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
Traumatic brain injury (TBI) survivors exhibit neurological, motor, and neurocognitive symptoms from the primary injury, which can aggravate over time due to secondary cell death. In the present in vivo study, we examined the beneficial effects of human adipose stem cells (hADSCs) in a controlled cortical impact (CCI) model of mild TBI using young (3 months) and aged (20 months) F344 rats. Young and aged male F344 rats were treated with 4×106 ADSCs (Tx), conditioned media (CM), or control media (M) at 3 h post CCI mild TBI. A separate cohort of animals with the same treatments received 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DiR)-labeled hADSCs. hADSCs labeled with DiR were imaged using the In Vivo Imaging System (IVIS) imager at 1, 4, 12, 24, 48, and 72 h posttransplant, with organs separately imaged at the end of the study. At day 7, post TBI, all groups underwent motor and cognitive assessment tests, then at day 11, all groups were euthanized and brain tissues were harvested. Significant amelioration of motor and cognitive functions was revealed in young Tx and CM groups, but there was less improvement in aged Tx rats relative to TBI rats that received the control media treatment (M). Fluorescence (FL) imaging revealed that hADSCs had moved to organs and brain within 1–12 h following TBI. In aged rats, decreased FL was seen in spleen; however, higher FL was observed in brain at 12–72 h post TBI. The impact volume in cortex was found to be significantly reduced in young Tx and CM relative to control treatment. However, in aged rats, this effect was decreased, and only Tx reduced impact volume. Furthermore, the percentage of intact peri-impact area in the cortex revealed a significant amelioration in both young Tx and CM treated and old Tx and CM compared with M rats. In addition, there was a decrease of hippocampal CA3 pyramidal neuron loss in both Tx young and Tx old rats compared to M rats. Results show that hADSCs is a promising therapeutic intervention to rescue against TBI-induced behavioral and histological impairments with better functional recovery in young animals, likely due to robust migration of the transplanted cells to peripheral organs quickly in young animals despite increased stem cell recruitment to the aged ischemic brain.
J. An,* S. S. Choi,* H. J. Lee,*† and S. U. Kim*†
*Medical Research Institute, Chung-Ang University College of Medicine, Seoul, Korea
†Department of Medicine, University of British Columbia, Vancouver, Canada
Human neural stem cells (hNSCs) hold great promise for treating a variety of human neurological diseases including spinal cord injury. In spite of the great benefits of cell therapy, there are some obstacles such as genetic background of cell source. To overcome this limitation, immortalized NSCs overexpressing human telomerase reverse transcriptase (hTERT) gene (H1 hNSCs) were established and characterized in vitro and in vivo. SD rats were severely injured at the T9 region of the spinal cord by an MIO impactor and transplanted with 1×105 cells into the two T8 and T10 regions. H1 hNSCs showed the high telomerase activity and expressed sodium channels from NaV 1.1 to 1.9 as well as several neural stem cell and neuronal cell markers but not sex-determining region Y box 1 (Sox1) and myelin basic protein (MBP). H1 hNSCs improved scoring in rota-rod experiment and Basso, Beattie, Bresnahan locomotor test (BBB) from 4 and 6 weeks in recipient rats, respectively, and promoted the survival and neuronal differentiation in vivo. Results suggested that H1 hNSCs may be effective tools of cell therapy for a broad spectrum of neurological disorders as well as for human clinical trials.
A. Andrzejewska, A. Jablonska, and B. Lukomska
Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland
Mesenchymal stem cell (MSC) transplantation has been explored as a new clinical approach to repair injured tissue. Numerous in vivo studies have shown that MSCs could migrate to damaged, inflamed tissues from blood and exert therapeutic effects. The detailed mechanism of MSC migration might involve the different receptors expressed on these cells. It seems that integrin α4β1 can mediate initial capture, rolling, and firm attachment of transplanted MSCs to the endothelial cells of microvessels; however, the recruitment of MSCs into injured tissue is mediated by the stromal derived factor-1/chemokine (C-X-C motif) receptor 4 (SDF-1/CXCR4) axis. Reliable testing of transplantation efficacy and maintenance of cell graft requires development of methods for cell visualisation in vivo. The technique cannot disrupt the multipotency and biological activity of MSCs. The possible approaches are to use the vital stains as 5-chloromethylfluorescein diacetate (CMFDA) or super paramagnetic iron oxide (SPIO) nanoparticles for cell labeling. The goal of the study was to investigate the effect of CMFDA and SPIO labeling on human bone marrow mesenchymal cell (hBM-MSC) proliferation, expression of different markers, adhesion molecules, and growth factors in vitro during cell culture. hBM-MSCs were cultured in mesenchymal stem cell basal medium (MSCBM) supplemented with 10% mesenchymal cell growth supplement (MCGS), l-glutamine, and gentamicin. For experiments, hBM-MSCs were transferred to 24-well plates at a density of 5,000 cells/well and then labeled with CMFDA stain or tagged with SPIO nanoparticles. Cell analysis was performed daily for 7 days. The proliferation potential of hBM-MSCs was measured in a cell counting kit-8 (CCK-8) assay. Expression of different markers and adhesion molecules was defined by immunocytochemistry. The activity of certain growth factors has been evaluated using real-time PCR. CMFDA and SPIO labeling procedure diminished proliferation of hBM-MSCs in vitro in comparison to untreated cells. The results obtained from the proliferation assay CCK-8 was confirmed by immunostaining, using anti-Ki67 antibodies. The vital observation of hBM-MSCs under the microscope revealed morphological changes in cell population labeled with CMFDA but not with SPIO and low cell survival during in vitro culture. Regardless of different labeling, hBM-MSCs expressed stage-specific embryonic antigen4 (SSEA4). The expression of pluripotency markers was seen during the entirety of the culture. Immunohistochemical analysis of CXCR4 proteins revealed their presence in all experimental groups of cells. However, the high activity of β1 integrin found on hBM-MSCs labeled with SPIO was much lower on their counterparts labeled with CMFDA. Our results have shown that SPIO nanoparticles used for tagging do not exert a toxic effect on hBM-MSCs. The vital labeling with SPIO does not alter the expression of pluripotent markers and proteins involved in cell migration. It suggests that SPIO is a better substance used for the visualisation of transplanted hBM-MSCs in vivo than CMFDA. Further analysis of different trophic factors expressed by examining the mRNA levels of these cells is in progress.
Supported by a National Center for Research and Development Grant No. 101 in ERA-NET NEURON project: “MEMS-IRBI.”
I. Antonucci
Department of Oral Sciences, Nano and Biotechnologies, Gabriele d'Annunzio University of Chieti, Chiety, Italy
Great attention has been recently devoted to stem cells obtained from amniotic fluid (amniotic fluid stem cells, AFSCs), which represent an interesting model due to their unique features and the possible advantages of their use in basic research and regenerative medicine. AFSCs are known to express specific markers of pluripotency even at high passage numbers, to proliferate rapidly, and to show a remarkable ability to differentiate into cells of all three embryonic germ layers. On the other hand, these cells do not form tumors when transplanted in nude mice. Due to these features, AFS cells are considered as a new class of stem cells with properties intermediate between embryonic stem cells and lineage-restricted adult progenitor cells and could represent the gold standard for cell therapy, bringing together the differentiation capabilities of embryonic stem cells and the therapeutic safety of adult stem cells. Our studies and those of other groups have evidenced that AFS cells have characteristics similar to pluripotent cells of the epiblast, since it has been hypothesized that during embryogenesis some cells have migrated from the epiblast into the amniotic fluid. To verify this hypothesis, we investigated the ability of human AFS cells to form in vitro three-dimensional aggregates, known as embryoid bodies, widely considered as an optimal starting point for the differentiation of pluripotent stem cells into three germ layers. Moreover, we have studied transcriptional profiles of genes abundantly and uniquely expressed in human embryonic stem cells and primordial germ cells as well as the expression of alternatively spliced genes implicated in development and differentiation and in particular in epigenetic mechanisms. Finally, the results of some preclinical studies carried out by using AFSCs as a cellular therapy for stroke in animal models will be discussed.
N. L. Arias,* L. M. Mercier,* E. J. Gonzalez-Rothi,* L. M. Guimond,* M. S. Sandhu,† P. J. Reier,* D. D. Fuller,† and M. A. Lane*
*University of Florida, Department of Neuroscience, McKnight Brain Institute, Gainesville, FL, USA
†University of Florida, Department of Physical Therapy, McKnight Brain Institute, Gainesville, FL, USA
The majority of traumatic spinal cord injuries (SCIs) occur at cervical levels, resulting in a wide range of motor and sensory deficits. Of these, impaired respiratory function is one of the most life threatening. While experimental and clinical studies have revealed spontaneous functional recovery within weeks, the extent of this plasticity remains limited for months. Furthermore, the long-term spinal and supraspinal changes mediating respiratory plasticity post-SCI are poorly understood. It is likely that the most effective treatments for SCI will need to work synergistically with underlying neuroplasticity. Accordingly, a detailed understanding of spontaneous functional and anatomical plasticity within the central nervous system is essential. While a number of ongoing studies have begun to examine spinal plasticity post-SCI, few have investigated changes at supraspinal levels. The objective of this study was to investigate supraspinal plasticity-associated respiratory function following an experimental cervical SCI. Adult female Sprague–Dawley rats were surgically anesthetized and received a lateral cervical (C2) hemisection (Hx). Two (n = 6) or 12 weeks (n = 6) later, animals were reanesthetized and electrodes were stereotaxically placed into the caudal medulla to map the distribution of inspiratory and expiratory phase activity. In a separate group of animals, supraspinal connectivity with spinal phrenic circuitry was examined using retrograde, transynaptic tracing with pseudorabies virus. Immunohistochemistry for c-fos expression in brainstem tissue was used to gain insight into changes in neuronal activity. Preliminary data reveal that regions of inspiratory and expiratory phase activity are altered post-SCI. Brainstem neurons normally active during inspiration are instead active during expiration in injured animals. These ongoing studies document a functional reorganization within respiratory-related neurons that has not been previously described. Ongoing anatomical experiments seek to better elucidate this reorganization and determine whether these changes may be impacting respiratory function following cervical spinal cord injury.
E. R. Aurand,* J. Wagner,† and K. B. Bjugstad*‡
*Neuroscience Program, University of Colorado-Denver, Anschutz Medical Campus, Aurora, CO, USA
†Department of Bioengineering, University of Colorado-Denver, Anschutz Medical Campus, Aurora, CO, USA
‡Department of Pediatrics, University of Colorado-Denver, Anschutz Medical Campus, Aurora, CO, USA
Neural tissue engineering could benefit from the use of hydrogels; however, the hydrogels must be customizable to mimic the various properties of brain tissue in addition to being therapeutically functional. Currently, hydrogels used for neural tissue engineering are not thoroughly characterized for their chemical, physical, and mechanical properties. The goal of this research was to create a well-characterized hydrogel using materials suitable for the implantation of neural precursor cells (NPCs) derived from fetal and adult sources. Twenty-five hydrogels were created with a range of ratios of hyaluronic acid (HA) and poly(ethylene glycol) (PEG). Hydrogels were assessed for their physical properties, the mechanical property of compressive modulus, and the cytocompatibility with encapsulated NPCs. Physical properties were a function of the amount of HA incorporated into the hydrogel. With increasing amounts of HA, the rate of polymerization increased (>120 to <2 min) and the rate of degradation decreased (<12 h to >10 days). The compressive modulus was also a function of the HA content when the PEG levels were low but had less of an affect at higher PEG concentrations. Compressive moduli ranged from 0.1 to 32 kPa. Comparably, rodent brain tissue compressive moduli ranged from 2 to 10 kPa. NPCs derived from fetal and adult rats (fNPCs and aNPCs, respectively) were encapsulated within a three-dimensional (3D) HA:PEG hydrogel environment. Cell survival was measured at 24-h postencapsulation and compared to NPCs plated on standard tissue culture plates (TCP; controls). Results demonstrated that 3D cultures of fNPCs had good survival/proliferation, with an average survival rate of 122.51 ± 2.69% compared to only 34.62 ± 3.47% in TCP controls. fNPC survival was dependent on the relative proportion of HA or PEG. At low concentrations of PEG, fNPC survival was high, regardless of HA content. At high concentrations of PEG, fNPC survival was dependent on the amount of HA, with greater survival at higher HA concentrations. aNPC survival was decreased compared with controls. The three-week fNPC and aNPC differentiation was influenced by mechanical properties. With fNPCs, greater numbers of astrocytes were observed on stiffer hydrogels, while increased numbers of neurons were seen on softer hydrogels. Greater numbers of aNPCs differentiated compared to fNPCs, with more cells becoming neuronal. These results indicate that there can be a wide range of HA:PEG-based hydrogels, which are compatible with NPCs. The results also suggest that NPCs respond differently to hydrogel encapsulation depending on the age of their tissue source (fetal vs. adult). Lastly, hydrogel properties can be readily altered by changing the proportion of HA without substantially altering NPC survival. This bodes well for neural tissue engineering strategies, allowing for the possibility to create customized hydrogels for specific applications.
K. S. Bankiewicz, A. P. Kells, L. Samaranch, W. P. San Sebastian, J. Forsayeth, M. Aminoff, P. Starr, P. Larson, and C. W. Christine
Interventional Neurology Center, Department of Neurological Surgery, University of California-San Francisco, San Francisco, CA, USA
We have been developing aromatic l-amino acid decarboxylase (AADC) gene transfer technology for Parkinson's disease (PD) and AADC deficiency, the latter a rare debilitating recessive genetic disorder in which mutations in the AADC gene lead to an inability to synthesize catecholamines [norepinephrine (NE), epinephrine (EP), dopamine (DA)] and serotonin. Putaminal administration of adeno-associated virus-AADC (AAV2-AADC) has been used to treat 16 PD patients and 8 AADC-deficient children. Results so far demonstrate encouraging long-term safety and clinical efficacy that justify initiation of phase 2 clinical trials. Magnetic resonance (MR) will be used to guide delivery of AAV2-AADC by convection-enhanced delivery into the putamen in PD patients and substantia nigra (SN) and ventral tegmental area (VTA) in AADC-deficient children. In the latter indication for the first time, we will exploit the anterograde axonal transport properties of AAV2 vector to disseminate AADC in the terminal regions of the striatum. Our most recent results from ongoing and upcoming clinical trials will be presented.
K. B. Bjugstad,* D. Miyagishima,† and K. Estes‡
*Department of Pediatrics, University of Colorado-Denver, Aurora CO, USA
†BRAiN Program, New Mexico State University, Las Cruces, NM, USA
‡UPPs Program, University of Colorado-Denver, Denver, CO, USA
Transplantation of neural cells into the brain to repair neurodegenerative disorders itself creates traumatic brain injury (TBI). TBI, along with Down syndrome (DS), is one of the biggest risk factors for developing Alzheimer's disease (AD). By attempting to repair one neurological disorder by transplantation, it is possible that we are putting the brain at risk for developing AD. The aggregation of tau and amyloid into the classic plaques and tangles of AD occurs only in the presence of sulfated proteoglycans, the primary components of the extracellular matrix (ECM). By understanding the changes that occur to the ECM as a function of TBI or DS, we might be able to develop better transplantation approaches that minimize the risk for AD. Hyaluronic acid (HA), a nonsulfated proteoglycan, is the foundation of the brain's ECM. Neurocan (NCN) is expressed in juvenile brain tissue and during times of neurogenesis. Using DS modeling and control mice, we measured changes in the brain HA and NCN 4 and 12 months after a neonatal penetration-type TBI. In the hippocampus and the septum, NCN changed primarily as a function of injury and time postinjury. In the septum, NCN greatly increased in mice 12 months postinjury compared to mice 4 months postinjury. CA1 and dentate gyrus (DG) had an age-associated increase in NCN from 4 to 12 months, which was amplified by TBI. CA3 was the only area with a karyotype effect in NCN expression. While age (12 months) and injury increased neurocan, the increase was greater in DS-modeling mice especially in the pyramidal cell layer. In the septum, HA expression decreased as a function of age with no effects of injury or DS modeling. In contrast, age significantly increased HA expression in CA1, especially in the oriens and lacunosum moleculare, and in the DG. These age effects were primarily expressed in the DS-modeling mice. CA3 again revealed both aging, injury, and DS effects. Injury increased HA expression more so in control mice than in DS-modeling mice. This effect was mostly found in mice 12 months postinjury. Thus, enhanced NCN expression is mostly associated with TBI and time postinjury, whereas HA expression is more consistently altered by DS modeling. By understanding the differences in ECM component in two models at risk for developing AD, we might be able to approach neural transplantation in a way that limits subsequent brain damage. For example, by adding certain growth factors to the transplants, which regulate ECM activities or by developing biomaterials based on ECM components, which do not promote tau/amyloid aggregation.
L. J. Blair,* B. Nordhues,* S. E. Hill,† K. M. Scaglione,‡ J. C. O'Leary III,* Z. Davey,* L. Breydo,* B. Zhang,* P. Li,* L. Wang,* C. Cotman,§ H. L. Paulson,‡ M. Muschol,† T. E. Golde,¶ V. N. Uversky,*# E. Binder,** N. Berchtold,§ and C. A. Dickey*
*Department of Molecular Medicine, University of South Florida, Tampa, FL, USA
†Department of Physics, University of South Florida, Tampa, FL, USA
‡Department of Pathology, University of Michigan, Ann Arbor, MI, USA
§Institute for Memory Impairments and Neurological Disorders (MIND), Univeristy of California, Irvine, CA, USA
¶Department of Neuroscience, University of Florida, Gainesville, FL, USA
#Institute for Biological Instrumentation, Russian Academy of Sciences, Moscow Region, Russia
**Max Planck Institute of Psychiatry, Munich, München, Germany
FK506 binding protein 51 kDa (FKBP51/FKBP5) is part of a mature heat shock protein 90 kDa (Hsp90) complex that preserves tau. In fact, mice deficient in FKBP51 have reduced tau levels in the brain. We have defined the mechanism for how tau is stabilized by this complex and the consequence of this stabilization for human tauopathy diseases. FKBP51 and Hsp90 synergistically prevent tau clearance through the proteasome. This profolding chaperone complex not only prevents tau clearance but also produces a tau conformation that alters fibril formation kinetics and reduces amyloidogenic properties of these aggregates. These biophysical observations were confirmed in vivo. Overexpression of FKBP51 in a tau mouse model revealed that FKBP51 selectively preserved phosphorylated and oligomeric tau species but decreased the amyloidogenic tangle load. These tau alterations culminated in enhanced neurotoxicity. In human brains, FKBP51 levels were found to increase with age due to methylation changes, while other Hsp90 cochaperones were unchanged or reduced. FKBP51 levels were also elevated in Alzheimer's disease brain, which correlated with Braak pathological staging. Moreover, increased FKBP51 levels outcompeted the association of other prodegradation Hsp90 cochaperones, resulting in tau accumulation. We conclude that age-associated increases in FKBP5 promote tau neurotoxicity through altering the composition of the Hsp90 complex. Strategies aimed at disrupting the FKBP51/Hsp90 association or attenuating FKBP51 levels could be therapeutically relevant for Alzheimer's disease and other tauopathies.
C. V. Borlongan,* N. Tajiri,* R. Pilla,† K. Shinozuka,* H. Ishikawa,* M. R. Steele,‡ P. R. Sanberg,* W. S. Quillen,§ and J. Dean†
*Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
†Hyperbaric Biomedical Research Laboratory, Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
‡Veterans Reintegration Steering Committee, Veterans Research, University of South Florida, Tampa, FL, USA
§School of Physical Therapy and Rehabilitation Sciences, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
The primary injury induced by traumatic brain injury (TBI) is largely unavoidable but triggers secondary brain cell death that may be readily treatable. This proposal examined an urgent health-related issue of high military significance, which will have direct impact on treating soldiers at risk of presenting with TBI or have succumbed to TBI. Previously, we demonstrated the potential of hyperbaric oxygen therapy (HBOT; Borlongan and colleagues, Exp. Neurol., 1998) and rehabilitation therapy (RT; Borlongan and colleagues, Neuroscience 2007) individually in animal models of stroke. Here, we evaluated their therapeutic effects in TBI either as stand-alone treatments or combination therapy. Adult, male Sprague–Dawley rats (8 weeks of age) were exposed to the TBI model of controlled cortical impact (CCI) as previously described (Borlongan and colleagues, PLoS One, 2013). Rats were then randomly assigned to HBOT of 1.5 atmospheres absolute (ATA) for 90 min either as a single treatment (day 3 post-TBI) or multiple treatments (days 3, 4, and 5 post-TBI) (Harch et al., 1997; Borlongan and colleagues, 2000). In addition, randomly selected animals were subsequently exposed to RT either as single (90-min running wheel exercise) or multiple exposure (days 3, 4, and 5 post-TBI) as described previously (Borlongan and colleagues, 2007). Results revealed that TBI-induced histological deficits in the frontal cortex were significantly reduced in TBI animals exposed to a single HBOT and/or RT. Quantitative analyses revealed that the single exposure to HBOT with or without RT resulted in reduction of the impacted cortical damage. RT alone did not significantly reduce cortical damage. There was also a trend in protection of the peri-impact cortical area with HBOT with or without RT, but did not reach statistical significance. These histological benefits corresponded with significant amelioration of TBI-induced motor and neurological deficits in animals exposed to HBOT with or without RT. These results demonstrated the efficacy of combination therapy of HBOT and RT in TBI. Additional experiments are warranted to show the optimal regimen and long-term effects of this combination therapy.
This work is supported by a U.S. Department of Defense Research Grant.
K. M. Braun,* N. Schultz-Darken,* M. Schneider,*† and M. E. Emborg*‡
*Wisconsin National Primate Research Center, University of Wisconsin-Madison, WI, USA
†Department of Kinesiology, University of Wisconsin-Madison, WI, USA
‡Department of Medical Physics, University of Wisconsin-Madison, WI, USA
Nonhuman primate (NHP) models of Parkinson's disease (PD) are essential to understand disease pathology and to test novel and invasive therapies. As spontaneous PD is not observed in NHPs, the most widely used models rely on neurotoxin administration to induce parkinsonism. Although useful, neurotoxic models have limitations including acute onset and a focus on dopaminergic nigrostriatal neurodegeneration. Transgenic PD animals present the opportunity to assess the impact of genetic mutations in PD and to evaluate the early onset of both motor and nonmotor PD signs and pathology. Common marmoset (Callitrix jacchus) monkeys present several advantages for transgenic studies as compared to rhesus monkeys (Macaca mullatta). They frequently give birth to twins, increasing the number of subjects per litter, and they additionally have shorter life spans than rhesus monkeys, thus facilitating studies where age may impact disease onset. Although rhesus neurodevelopment milestones are well known, marmoset neurodevelopment is not yet characterized with appropriate evaluation scales, which makes it difficult to assess changes. In this experiment, 14 newborn common marmoset monkeys were evaluated using a novel marmoset Postnatal Neurodevelopmental Assessment scale (PNNA). The scale was based on current available scales for human and rhesus. The neonatal assessments involved 10-min testing administered at two time points: one during total dependency at 15 days and a second during emerging independence at 30 days. The PNNA scale was designed to assess orienting, affective, and neuromotor responses that mature during the first month of life. We found clear differences in the responses at the two time points, including a more pronounced response in parachute and rotation reflexes at the 3-day mark. Additionally we observed increases in coordination and aggression and decreases in activity, consolability, and fearfulness from age 15 to 30 days. Compared to rhesus, marmoset babies did not show much distress during orienting tests, reflective of marmoset habit of carrying the babies on the back and transiently passing them between family members. Our results demonstrate the sensitivity and cross-species translational validity of this scale. Additional experiments to characterize developmental stages in the first year of life and its application to transgenic animals are currently ongoing.
Acknowledgments: Welton Summer Sophomore Research Apprenticeship Research Grant (K.M.B.), P51 RR000167 (NIH-NCRR), to the Wisconsin National Primate Research Center, University of Wisconsin-Madison. This research was conducted in part at a facility constructed with support grants RR15459-01 and RR020141-01.
C. R. Bye, J. A. Kauhausen, L. H. Thompson, and C. L. Parish
Florey Neurosciences Institutes, Melbourne Brain Centre, The University of Melbourne, Parkville, Australia
Clinical trials have provided proof of principle that new dopamine (DA) neurons isolated from the developing ventral midbrain (VM) and transplanted into the denervated striatum can functionally integrate and alleviate symptoms in Parkinson's disease (PD) patients. However, extensive variability across patients has been observed, ranging from long-term motor improvement to the absence of symptomatic relief and development of dyskinesias. Heterogeneity of the donor tissue, including the number and types of cells present, is likely to be a significant underlying factor. There is currently little knowledge on how variables such as donor age, which has been poorly controlled for in clinical trials, will impact on the final neuronal composition of fetal VM grafts. Here we performed a birth dating study to identify the time course of DA neurogenesis within the various VM DA subpopulations in an effort to enrich donor tissue for transplantation. The results show that A9 DA neurons, important for motor function, precede the birth of the A10 subpopulation. Subsequent ectopic or homotopic grafting of younger VM donor tissue revealed significantly larger grafts, enriched with A9 neurons capable of greater innervation of the target dorsolateral striatum and DA release. Additionally, exposure of these grafts to the neurotrophin glial-derived neurotrophic factor (GDNF) enhanced graft size and integration. These findings demonstrate the importance of standardized methods to improve cell therapy for PD and have significant implications for the generation and selectivity of DA neurons from stem cell-based sources.
A. Cerreta,* G. B. Grossl,* B. Creary,* M. A. Lane,† and D. J. Hoh*†
*Department of Neurosurgery, College of Medicine and McKnight Brain Institute, University of Florida, Gainesville, FL, USA
†Department of Neuroscience, College of Medicine and McKnight Brain Institute, University of Florida, Gainesville, FL, USA
Urological complications remain a significant source of morbidity and mortality for individuals with chronic spinal cord injury (SCI), with recovery of urinary function rated as a high priority among the spinal cord injured population for future neural repair research. While urinary dysfunction after chronic human SCI has been well documented, preclinical studies have revealed conflicting results, complicating further translational investigation. Prior studies of complete thoracic transection injury disrupt spinal cord pathways regulating urinary function with resulting pathological changes of increased bladder muscle compliance, weight, and elastin content post-SCI. Animal studies of more clinically relevant incomplete injuries, however, have paradoxically shown normal or spontaneous recovery of urinary function post-SCI. Investigating potential neural repair strategies for chronic SCI relies on identifying translational models that parallel clinical observations. Therefore, the purpose of this study was to demonstrate evidence of urinary dysfunction after experimental SCI by assessing for pathological changes in the bladder muscle wall in a clinically relevant model of incomplete cervical SCI. We used a rodent midcervical (C4–C5) contusion injury model, assessing three groups: uninjured (n = 22), moderate SCI (n = 51), and severe SCI (n = 34). Injuries were performed using the Infinite Horizon pneumatic impactor with a unilateral 200 kDyne impact (moderate SCI) or a bilateral 100 kDyne impact (severe SCI). Bladder tissue was harvested at either 7, 14, 28, or 56 days postinjury and assessed for bladder weight and elastin concentration. Uninjured animals had a mean bladder weight of 150 mg. After mild SCI, mean bladder weight was increased at 168, 158, 154, and 189 mg at 7, 14, 28, and 56 days post-SCI, respectively. Severe SCI resulted in a greater increase in mean bladder weight at 212, 192, 203, and 206 mg, at the same time points. Similarly, elastin concentration also correlated with severity of SCI. Uninjured animals had a mean elastin concentration of 0.277 mg/g tissue. After mild SCI, mean elastin concentration increased from 0.262, 0.276, 0.289, to 0.330 mg/g tissue at 7, 14, 28, and 56 days post-SCI. Severe SCI demonstrated a greater increase in mean elastin concentration from 0.318, 0.302, 0.297, and 0.344 mg/g tissue, at the same time points. Significant increases in bladder weight and elastin concentration were observed after experimental chronic cervical SCI, representing pathological changes in bladder muscle tissue as a result of disruption of normal neural pathways. These changes appeared to correlate with injury severity, extending into the chronic phase. This model may serve as valuable model for assessing neural repair strategies for recovering urinary function after chronic SCI.
D.-P. Chao,* W.-T. Chiu,*† Y.-H. Chiang,‡ S.-F. Chu,§ T.-N. Lui,¶ J.-W. Lin,# H.-W. Lin,** C.-C. Wu,‡†† and Y.-S. Yang‡
*Graduate Institute of Injury Prevention and Control, Taipei Medical University, Taipei, Taiwan, ROC
†Department of Health, Executive Yuan, Taipei, Taiwan, ROC
‡Department of Neurosurgery, Taipei Medical University Hospital, Taipei, Taiwan, ROC
§School of Public Health, Taipei Medical University, Taipei, Taiwan, ROC
¶Department of Neurosurgery, Taipei Medical University-Wan Fang Hospital, Taipei, Taiwan, ROC
#Department of Neurosurgery, Taipei Medical University-Shuang-Ho Hospital, Taipei, Taiwan, ROC
**Biostatistics and Research Consulting Center, Taipei Medical University, Taipei, Taiwan, ROC
††Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan, ROC
Serum albumin levels have been considered an independent predictor of neurological outcome in head trauma with various degrees of severities. Stabilization of serum albumin of patients with various diseases is always one of the treatment goals recommended to avoid secondary injury. The aim of this study was to examine the effect of albumin treatment on survival rate and outcome of severe traumatic brain injury (sTBI) patients at acute and long-term stages. Medical records of s TBI patients with Glasgow coma scale (GCS) 3–8 were recruited from 12 hospitals in Taiwan, excluding patients with multiple trauma. During 2006–2010, data from 32 albumin-treated patients receiving appropriate albumin within the first 7 days in the intensive care unit (ICU) were collected and compared with 127 nonalbumin controls matched for gender, age, body weight, initial GCS, and operative status. There was a significant correlation between albumin level and GCS score in sTBI patients immediately after injury. The albumin-treated patients had a significantly greater survival rate over the first 7 days and significantly more survival and favorable outcome at 3 and 6 months postinjury compared with the nonalbumin patients. After subgrouping of patients into initial GCS 3–5 and 6–8, the survival rate during the first 7 ICU days and the rate of favorable outcome from 1 to 6 months postinjury were still markedly improved in albumin-treated patients with initial GCS 3–5. In patients with initial GCS 6–8, these benefits were not significant between the two groups. This finding demonstrates that albumin treatment in acute and long-term stage seem to be associated with better survival and outcome among sTBI patients, particularly in those with an initial GCS 3–5 who are likely to have insufficient initial albumin levels.
Y.-H. Chiang,*†‡§ K.-Y. Chen,*†‡ C.-C. Wu,‡¶# Y.-W. Yu,*†§ J.-W. Lin,† and W.-T. Chiu†
*Translational Research Laboratory, Cancer Center, Taipei Medical University Hospital, Taipei, Taiwan
†Department of Surgery, College of Medicine, Taipei Medical University, Taipei, Taiwan
‡Department of Neurosurgery, Taipei Medical University Hospital, Taipei, Taiwan
§Neural Regenerative Program, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan
¶Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan
#Taitung Christian Hospital, Taitung, Taiwan
Emodin has previously been reported to exhibit neuroprotective effects in cerebral ischemia-reperfused injury and glutamate-induced neuronal damage. It is extracted from Polygonum cuspidatum Sieb. et Zucc, a traditional Chinese medicinal herb widely used to treat acute hepatitis. This study investigates the neuroprotective effects of Emodin in a traumatic brain injury (TBI) model both in vitro and in vivo. Primary cortical neurons were exposed to hypoxic conditions to cause neural injury and involve signaling pathways. Emodin treatment was evaluated by Western blot and MTT assay. In addition, cortical impact was induced in Male Sprague–Dawley (SD) rats to establish an TBI model. Results of the Emodin treatment 30 min after cortical impact were evaluated with behavioral examinations with water maze and modified neurological severity score (mNSS) tests. The mechanisms of Emodin treatment in animal models were also analyzed with Western blot. Results show neuroprotective effects in an in vitro study as decreased cell death was observed in cultures treated with Emodin. Furthermore, neuroprotective effects were observed as animals showed a more rapid recovery of memory in the group treated with Emodin. Finally, Western blot analysis shows that the p38-mitogen-activated protein kinase (MAPK) pathway is involved in the neuroprotective effects of Emodin. In conclusion, Emodin demonstrates neuroprotective effects in TBI model both in vitro and in vivo via p38-MAPK pathway.
S. S. Choi,* J. An,* H. J. Lee,*† and S. U. Kim*†
*Medical Research Institute, Chung-Ang University College of Medicine, Seoul, Korea
†Department of Medicine, University of British Columbia, Vancouver, Canada
Spinal cord injuries caused by trauma are still difficult to treat due to the low efficiency of neuronal cell recovery. Transplantation of human neural stem cells (NSCs) has shown promise for improving functional recovery after spinal cord injury (SCI). Glial cell line-derived growth factor (GDNF) is a neurotrophic factor that promotes neuron survival and neuronal differentiation and has a neuroprotective effect in the recovery of degenerating motorneurons. To examine the therapeutic effect of GDNF for spinal cord injury, GDNF-overexpressing H1.GDNF neural stem cells were generated and transplanted into T8 and T10 regions of rat spinal cord and monitored for 12 weeks. H1.GDNF cells expressed nestin and neural stem cell markers and induced the expression of some markers for neuronal cells and oligodendrocytes; neurofilament (NF)-L, NF-M, and myelin basic protein (MBP) but not NF-H or glial fibrillary acidic protein (GFAP). These cells also expressed several sodium ion channels—NaV1.1, 1.2, 1.3, 1.7, and 1.9 based on electrophysiological analysis. The progress of hind leg movement was significantly improved in functional tests of H1.GDNF rats. Transplanted cells survived and migrated into injured regions for 12 weeks and differentiated into neuronal cells expressing neurofilament but not GFAP. GDNF induced cell survival and promoted neuronal differentiation. These results suggested that H1.GDNF may be effective and helpful cells for spinal cord regeneration and a useful tool for cell therapy for SCI model as well as various degenerative disorders in CNS.
C.-H. Chou*† and M. Modo†
*Department of Neuroscience, Institute of Psychiatry, King's College London, London, UK
†McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, USA
The concept of the neurovascular unit (fundamentally comprising neurons, astrocytes, and endothelium) extends the framework of a purely neurocentric focus that is not sufficient to interpret the mechanism of some neurovascular diseases, such as stroke, as well as the therapeutic efficacy of cell-based therapies. Surrounding the cellular elements, extracellular matrix (ECM) also contributes to the development and maintenance of the neurovascular unit. Not only the cellular components but also acellular matrix, a bioscaffold, is essential for promoting neurovascular restoration and reconstructing brain tissues. To further the application of neural stem cells (NSCs) in cell transplantation and regenerative medicine, the clinical grade human NSC lines (STROC05 and CTXOE03; ReNeuron) and human cerebral microvascular endothelial cells (hCMECs/D3) were cocultured with direct physical contact to form a neurovascular network. The process of developing a highly organized capillary-like cytoarchitecture, in which the neuronal and oligodendrocyte differentiation was increased, was very dependent on the ratio of endothelial cells (ECs) and NSCs. Importantly, these ratios differed between both NSC lines. Additionally, the expression of ECM proteins (neurocan, nidogen, laminin, collagen, fibronectin) was measured to determine how these change during the development of a neurovascular network. Decellularized ECM bioscaffold was then used to culture ECs with NSCs in a three-dimensional environment to illustrate that a specific composition of ECM is required in endothelial morphogenesis. A neurovascular microenvironment generated with a well-organized cytoarchitecture, in a fashion beneficial for both neural and endothelial differentiation may be favourable to replace diseased tissues in future animal studies. Further studies are required to elucidate the specific cellular and molecular mechanisms involved in the development of these neurovascular units. Vascular endothelial growth factor and platelet-derived growth factor signalling pathways may be involved in this process.
A. T. Crane,* K. D. Fink,*† J. Rossignol,*‡§ R. Wyse,* D. J. Dues,* P. A. Starski,* and G. L. Dunbar*‡§
*Central Michigan University Program in Neuroscience, Mount Pleasant, MI, USA
†INSERM 1043, University of Nantes, Nantes, France
‡College of Medicine, Central Michigan University, Mount Pleasant, MI, USA
§Field Neuroscience Institute, Saginaw, MI, USA
Mesenchymal stem cells (MSCs) are stromal cells that are characterized by in vitro expansion through plastic adherence and the ability to differentiate into multiple lineages. MSCs have been shown to be present in multiple sources, including the bone marrow (BM) and umbilical cord blood (UCB). However, it has become apparent that the characteristics of MSCs can change from source to source and through increased in vitro passaging. Due to beneficial properties, such as immune suppression and trophic support, the use of MSCs as a potential therapy in neurodegenerative diseases shows promise. The goal of the present study was to compare the behavioral-sparing and life span-altering effects of MSCs isolated from two sources over a range of passage numbers. MSCs were isolated from C57/B16 background mice from the BM and UCB, expanded in vitro, and characterized through flow cytometry and RT-PCR for over 100 passages. Flow cytometry results indicate that both sources of MSCs go through dramatic changes in protein expression within the first 10 passages, while subsequent passaging produces more gradual fluctuations. The cell types were then bilaterally transplanted, intrastriatally, into 5-week-old R6/2 mice, which have exon 1 of the Huntington's disease gene and display a rapid onset of motor and cognitive deficits, weight loss, and a significantly reduced life span. For behavioral analysis, R6/2 mice transplanted with either low (4–10) or high (40–60) passage BM-MSCs or UCB-MSCs underwent weekly testing on a fixed-speed rotorod and a Morris water maze task. At 11 weeks of age, transplanted and sham-operated R6/2 and wild-type mice were sacrificed, and their brains were preserved for immunohistochemical, gene, and protein analyses. Results from these analyses show R6/2 mice transplanted with high-passaged BM-MSCs displayed reduced motor and cognitive deficits, while protecting against striatal shrinkage observed in untreated R6/2 mice. Further, transplants of high-passaged UCB-MSCs displayed a transient improvement in the behavioral phenotype, early following transplantation. Interestingly, transplants of low-passaged UCB-MSCs showed a greater glial response and fewer surviving cells 6 weeks following transplant. For life span analysis, a second group of R6/2 mice was transplanted with either low, high, or very high (100–140) passaged BM-MSCs or UCB-MSCs and then monitored daily for changes in weight and survivability (with righting ability used as a humane endpoint). The R6/2 mice that were transplanted with the very high-passage BM-MSCs showed a modest trend in increased survivability and high-passage BM-MSCs had less weight loss by the end stage. These results suggest that BM-MSCs at higher passages tend to provide more protection against neuropathological and behavioral deficits in the R6/2 model of HD. Further research on the optimal source and number of passages is warranted to optimize the therapeutic potential of MSC transplants.
Funding was provided by the John G. Kulhavi Professorship in Neuroscience and the Field Neuroscience Institute (to G.L.D.).
A. C. Cuello,*†‡ M. F. Iulita,* and J. Busciglio§
*Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada
†Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada
‡Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec, Canada
§Department of Neurobiology and Behavior, University of California, Irvine, CA, USA
Cholinergic dysfunction is a central feature of the cognitive decline in Alzheimer's disease (AD). Another hallmark aspect of AD is the preferential vulnerability of nerve growth factor (NGF)-dependent cholinergic neurons, which become atrophic (and eventually die as the disease progresses). Paradoxically, the synthesis of NGF mRNA is not affected by AD pathology and the levels of its precursor molecule (proNGF) are increased in the AD brain. To gain further insight into this apparent contradiction, we revisited the modality of NGF release in the CNS applying classical pharmacology superfusion to ex vivo cortical tissue. Upon tissue stimulation, we observed, unexpectedly, the consistent activity-dependent release of proNGF, as opposed to its mature form (mNGF). We further described a metabolic pathway responsible for the extracellular conversion and subsequent degradation of NGF. The activity-dependent release of NGF occurs with the concomitant release of enzymes, zymogens, and regulators, which process it to mNGF and eventually degrade it (Bruno and Cuello, PNAS 2006). This pathway is relevant in the context of AD. Using human postmortem brains from the Netherlands Brain Bank, we have found that the extracellular maturation of NGF is impaired in AD (hence proNGF accumulates) and that its degradation is enhanced (Bruno et al., JNEN 2009a). Thus, alterations in this metabolic pathway explain the paradoxical atrophy of cholinergic neurons in a context of proNGF abundance and normal NGF synthesis. We have further investigated this pathway in postmortem brains from mild cognitive impairment (MCI) cases from the Religious Orders Study and found that the activity of the NGF-degrading enzyme (matrix metalloproteinase 9; MMP-9) is enhanced already at this prodromal stage (Bruno et al., JNEN 2009b). The study of AD-related changes in Down syndrome (DS) brains may offer important clues related to the progression of the disease. With this in mind, we investigated the status of the NGF metabolic pathway in postmortem brain samples from the Brain Tissue Repository at the University of California, Irvine, from DS patients and age-matched controls. Our studies reveal that alterations in the NGF pathway, such as elevated proNGF levels and increased MMP-9 activity are also present in this condition, before the onset of overt dementia.
The authors would like to acknowledge support from the Canadian Institutes of Health and Research (MOP-97776) to A.C.C. and from the National Institutes of Health grants (HD38466 and AG16573) to J.B. M.F.I is the recipient of a Biomedical Doctoral Award from the Alzheimer Society of Canada.
L. Cui* and L.-R. Zhao*†
*Department of Neurology, Louisiana State University Health Sciences Center, Shreveport, LA, USA
†Department of Cellular Biology and Anatomy, Louisiana State University Health Sciences Center, Shreveport, LA, USA
Stroke, a leading cause of adult permanent disability, is a critical medical condition with limited treatment. Chronic stroke is the phase beyond 3 months after stroke onset. Targeting brain repair in chronic stroke is highly important but is poorly investigated in the field of stroke research. In an earlier study, we demonstrated that the combination of two hematopoietic growth factors, stem cell factor (SCF), and granulocyte-colony stimulating factor (G-CSF) (SCF + G-CSF), induced stable and long-term functional improvement with the enhancement of neuronal activity in the lesion side brain in an animal model of chronic stroke. It remains unclear, however, how SCF + G-CSF repairs the brain during chronic stroke. Recently, we have revealed that SCF + G-CSF treatment in chronic stroke enhances neuronal network remodeling in the cortex adjacent to the infarct cavity and that SCF + G-CSF promotes neurite outgrowth through the regulation of nuclear factor k-light-chain-enhancer of activated B cells (NF-κB) pathway. The aim of this study, therefore, was to determine whether SCF + G-CSF rebuilds synaptic networks in the peri-infarct cortex through NF-κB pathway. Seven-day injections of SCF + G-CSF were initiated 7 months after induction of cortical brain ischemia. NF-κB inhibitor (Bay 11-7082) or saline was infused into the contralateral ventricle for 7 days by a microosmotic pump starting at 1 h before SCF + G-CSF treatment. The apical dendrites of layer V pyramidal neurons in the peri-infarct cortex were scanned before treatment, 2 and 6 weeks after treatment in the same animals with a multiphoton microscope. The postsynaptic element was detected using immunohistochemistry at the end of brain imaging. Before treatment, the mushroom dendritic spines were significantly reduced in both stroke groups (vehicle control and SCF + G-CSF) as compared to the intact controls but there was no difference between the two stroke groups. However, 2 and 6 weeks after treatment, mushroom spines were significantly increased by SCF + G-CSF when compared to the vehicle controls, and the SCF + G-CSF-induced increase in mushroom spines was significantly blocked by the NF-κB inhibitor. In addition, the size of spine heads was also increased by SCF + G-CSF and the effects of SCF + G-CSF in enlarging the size of spine heads were prevented by intracerebral infusion of the NF-κB inhibitor. Furthermore, the NF-κB inhibitor significantly diminished the effects of SCF + G-CSF-induced postsynaptic density protein 95 (PSD-95) puncta formation in the peri-infarct cortex. These data suggest that systemic administration of SCF + G-CSF during chronic stroke can stimulate surviving neurons in the peri-infarct cortex to generate functional synaptic connections with other neurons and that NF-κB signaling is required for SCF + G-CSF-induced neuronal network remodeling in the preinfarct cortex. This study provides supportive evidence for the therapeutic role of SCF + G-CSF in chronic stroke.
This study was supported by the National Institutes of Health, National Institute of Neurological Disorders and Stroke (NINDS), R01 NS060911 to L.R.Z.
T. Dailey, Y. Kaneko, N. Tajiri, H. Ishikawa, K. Shinozuka, T. Malapira, L. Gemma, F. Vale, and C. V. Borlongan
The Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
Epilepsy is the third most common neurological disorder in the United States, imposing an economic burden of $15.5 billion with its associated costs. Our team seeks to explore both the therapeutic efficacy and mechanism of action in stem cell media promoting cell survival during excitotoxic kainic acid exposure, an established epilepsy model both in vivo and in vitro. Cell culture was conducted using a standard rat primary hippocampal neuron in vitro cell model. The cells were incubated in 100-μl cell media with 1 μM kainic acid, using a dose-responsive concentration. Cells were then treated with a 100-μl aliquot of stem cell media for a duration ranging from 1 to 4 days. Following treatment, an MTT assay was performed to establish cell viability. To further reveal the therapeutic benefits of stem cells, Sprague–Dawley rats with kainic acid-induced epilepsy received intra-hippocampal transplants of stem cells derived from cortical tissues of epileptic patients. Graft fate and its effects on the epileptic brain were examined. Over the 4-day protocol, all groups exposed to the stem cell media had a significantly greater survival than the control group. Cells in the 3-day treatment group demonstrated the greatest viability; double that of the kainic acid-only control. Transplantation of stem cells derived from the neocortex of epileptic patients not only survived in the amygdala but reduced the kainic acid-induced hippocampal cell loss in epileptic rats. Transplanted human neocortical cells migrated from the amygdala to the lesioned hippocampus, specifically to the CA1 and CA3 regions. However, the rates of graft survival and migration were low, despite the robust rescue of hippocampal cells, suggesting a trophic factor mechanism as seen in the cell culture experiments. The administration of stem cell media provided a significant reduction in kainic acid-induced cell death and promoted cell survival beyond the control group of cells. Transplantation of stem cells derived from epileptic patients afforded rescue of the epileptic hippocampus, likely via a trophic factor pathway. Ongoing projects are now exploring specific neurotrophic factors, such as BDNF and NGF, involved in these observed therapeutic benefits of stem cell transplantation for treatment of epilepsy.
D. Darlington,*1 J. Deng,*†1 B. Giunta,‡§ H. Hou,* C. D. Sanberg,¶ N. Kuzmin-Nichols,¶ H. D. Zhou,† T. Mori,# J. Ehrhart,¶ P. R. Sanberg,§ and J. Tan*†§
*Rashid Laboratory for Developmental Neurobiology, Silver Child Development Center, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
†Department of Neurology, Daping Hospital, Third Military Medical University, Chongqing, China
‡Neuroimmunology Laboratory, Department of Psychiatry and Behavioral Neurosciences, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
§Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
¶Saneron CCEL Therapeutics, Inc., Tampa, FL, USA
#Departments of Biomedical Sciences and Pathology, Saitama Medical Center and Saitama Medical University, Kawagoe, Saitama, Japan
1These authors contributed equally.
Alzheimer's disease (AD) is the most common progressive age-related dementia in the elderly and the fourth major cause of disability and mortality in that population. The disease is pathologically characterized by deposition of β-amyloid plaques and neurofibrillary tangles (NFT) in the brain. Current strategies for the treatment of AD are symptomatic only. As such, they are less than efficacious in terms of significantly slowing or halting the underlying pathophysiological progression of the disease. Modulation by cell therapy may be new promising disease-modifying therapy. Recently, we showed reduction in amyloid-β (Aβ) levels/β-amyloid plaques and associated astrocytosis following low-dose infusions of mononuclear human umbilical cord blood cells (HUCBCs). Our current study extended our previous findings by examining cognition via the: (1) rotarod test, (2) a 2-day version of the radialarm water maze test, and (3) a subsequent observation in an open pool platform test to characterize the effects of monthly peripheral HUCBC infusion (1×106 cell/μl) into the transgenic PSAPP mouse model of cerebral amyloidosis [bearing mutant human amyloid precursor protein (APP) and presenilin-1 transgenes] from 6 to 12 months of age. We show that HUCBC therapy correlates with decreased: (1) cognitive impairment, (2) Aβ levels/β-amyloid plaques, (3) amyloidogenic APP processing, and (4) reactive microgliosis after a treatment of 6 or 10 months. As such, this report lays the groundwork for HUCBC therapy as a potentially novel alternative to oppose AD at the disease-modifying level.
This work was supported by the NIH/NIA [R01AG032432 and R42AG031586 (J.T.)]. P.R.S. is a cofounder and J.T. is a consultant for Saneron CCEL Therapeutics, Inc., and are inventors on a patent application submitted by USF. P.R.S. was not involved in any data acquisition and analysis.
M. Das,*† C. Wang,*‡ R. Bedi,† S. Mohapatra,*†§ and S. Mohapatra*‡§
*Nanomedicine Research Center, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
†Department of Internal Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
‡Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
§James A. Haley Veteran's Hospital and Medical Research Center, Tampa, FL, USA
Traumatic brain injury (TBI) causes significant mortality, long-term disability and psychological symptoms. However, the mechanism underlying TBI pathogenesis is poorly understood. Although TBI compromises the blood–brain barrier (BBB), drug delivery to the brain remains a challenge with mild TBI (mTBI). Gene therapy is a promising approach for treatment of different pathological conditions. Here we tested super paramagnetic iron oxide containing dual-purpose chitosan and polyethyleneimine (PEI)-coated magnetic micelles (CPMMs), a potential MRI contrast agent, to deliver a reporter DNA to the brain after mTBI. Adult male Sprague–Dawley rats were subjected to mTBI using a lateral fluid percussion injury device. Control animals were sham-operated. CPMMs were conjugated with a plasmid (tomato plasmid, tdt) expressing a red fluorescent protein (RFP). CPMM-tdt conjugate was administered intranasally immediately after mTBI or sham surgery, and rats were subjected to magnetic field around the head region for 1 h postadministration of CPMM. Twenty-four or 48 h later, rats were transcardially perfused with 4% paraformaldehyde. Brains, lungs, livers, kidneys, and spleens were removed, processed, and sectioned for different staining experiments. Fluoro-Jade staining was performed on brain sections to observe neuronal injury. Prussian Blue staining was performed to identify the presence of iron oxide particles in different tissues. Immunostaining was performed using anti-DsRed antibody to see reporter gene expression, anti-interleukin-1β (IL-1β), anti-IL-6, or anti-tumor necrosis factor-α (TNF-α) antibodies to observe presence of inflammatory cytokine expressions in different tissues. Evans blue extravasation combined with intranasal CPMM administration suggested a possible route of CPMM entry into the brain parenchyma via the compromised BBB. Intranasally administered CPMM-tdt nanoparticles with chitosan/PEI at a weight ratio of 1:1 showed RFP expression in the cortex and hippocampus 48 h after mTBI. Magnetofection increased the concentration of CPMM nanoparticles in the brain. The expression was more pronounced in the brain parenchyma of the ipsilateral side compared to the contralateral side. RFP expression was insignificant in sham animals. CPMM particles did not evoke any inflammatory response by themselves and were excreted from the body. Although CPMM nanoparticles were observed in lung, liver, kidney, or spleen, no RFP expression was observed in these organs. These results show that intranasally administered CPMM can target the brain following mild TBI and successfully deliver and express the reporter gene in the brain tissue. Therefore, these particles may be useful as a theranostic agent for mTBI.
This work is supported by the Office of Naval Research grant (N000140810914) to Shyam Mohapatra and VA reintegration grant to Subhra Mohapatra.
M. Denham,*†1 C. L. Parish,*†1 B. Leaw,*† J. Wright,*† C. A. Reid,*† S. Petrou,*† M. Dottori,* and L. H. Thompson*†
*Centre for Neuroscience, University of Melbourne, Parkville, Australia
†Florey Neuroscience Institute, University of Melbourne, Parkville, Australia
1These authors contributed equally.
Human pluripotent stem cells have the capacity for directed differentiation into a wide variety of neuronal subtypes that may be useful for brain repair. While a substantial body of research has lead to a detailed understanding of the ability of neurons in fetal tissue grafts to structurally and functionally integrate after intracerebral transplantation, we are only just beginning to understand the in vivo properties of neurons derived from human pluripotent stem cells. Here we have utilized the human embryonic stem (ES) cell line Envy, which constitutively expresses green fluorescent protein (GFP), in order to study the in vivo properties of neurons derived from human ES cells. Rapid and efficient neural induction, followed by differentiation as neurospheres resulted in a GFP+ neural precursor population with traits of neuroepithelial and dorsal forebrain identity. Ten weeks after transplantation into neonatal rats, GFP+ fiber patterns revealed extensive axonal growth in the host brain, particularly along host white matter tracts, although innervation of adjacent nuclei was limited. The grafts were composed of a mix of neural cell types including differentiated neurons and glia, but also dividing neural progenitors and migrating neuroblasts, indicating an incomplete state of maturation at 10 weeks. This was reflected in patch-clamp recordings showing stereotypical properties appropriate for mature functional neurons, including the ability to generate action potentials, as well as profiles consistent for more immature neurons. These findings illustrate the intrinsic capacity for neurons derived from human ES cells to integrate at a structural and functional level following transplantation.
N. S. Duchamp,*† L. Cui,* D. J. Boston,† X. Ren,* H. Hu,* and L.-R. Zhao*‡
*Department of Neurology, Louisiana State University Health Sciences Center, Shreveport, LA, USA
†School of Medicine, Louisiana State University Health Sciences Center, Shreveport, LA, USA
‡Department of Cellular Biology and Anatomy, Louisiana State University Health Sciences Center, Shreveport, LA, USA
Stroke is a leading cause of adult permanent disability worldwide. The phase of chronic stroke usually starts 3–6 months after stroke onset. Currently no pharmaceutical therapy is available for the treatment of chronic stroke. Stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF) are the essential members of hematopoietic growth factor family and play a key role in the regulation of blood cell production. Recent findings suggest that SCF and G-CSF may be also involved in neuronal plasticity in the central nervous system. Our early study has revealed that SCF in combination with G-CSF (SCF + G-CSF) improves functional outcome when administered in the chronic phase of stroke in animal models. However, the mechanism by which SCF + G-CSF repairs the brain in chronic stroke remains poorly understood. Recently, we have revealed that the nuclear factor k-light-chain-enhancer of activated B-cells (NF-kB) pathway is required for SCF + G-CSF-induced enhancement of neurite outgrowth in primary cortical neurons. In this study, we have determined the contribution of NF-κB pathway in the SCF + G-CSF-induced brain repair in chronic stroke. We observed that SCF + G-CSF significantly improved functional outcome in chronic stroke mice. The axonal sprouting in the peri-infarct area, which projected from both the contralateral and ipsilateral lesioned cortex, was significantly increased by SCF + G-CSF treatment. Moreover, the functional postsynaptic element and the blood vessel density in the cortex adjacent to the infarct cavities were also increased by SCF + G-CSF treatment. Remarkably, the effects of SCF + G-CSF on functional restoration, neuronal network remodeling, and cerebrovascular regeneration in the brain of chronic stroke were prevented by the NF-kB inhibitor. These results suggested that SCF + G-CSF treatment improves functional restoration in chronic stroke though rewiring neural networks and regenerating new blood vessels and that NF-κB pathway is critically involved in SCF + G-CSF-induced brain repair in chronic stroke. This study provides new insights into the contribution of hematopoietic growth factors in brain repair and helps in developing a new pharmaceutical therapy for treatment of chronic stroke.
This study was supported by the National Institutes of Health, National Institute of Neurological Disorders and Stroke (NINDS), R01 NS060911 to L.R.Z.
K. D. Fink,*† J. Rossignol,*‡§ A. T. Crane,* T. D. Hulse,* X. Lévêque,*§ R. Wyse,*‡ D. Dues,* L. D. Huffman,* A. C. Moore,* D. T. Story,* R. Dejonge,* M. Lu,* L. Lescaudron,† and G. L. Dunbar*‡§
*Brain Research and Integrative Neuroscience Center, Department of Psychology, Central Michigan University, Mount Pleasant, MI, USA
†Faculté des Sci. et des Techniques, Université de Nantes, Nantes, France
‡College of Medicine, Central Michigan University, Mount Pleasant, MI, USA
§Field Neuroscience Institute, Saginaw, MI, USA
Huntington's disease (HD) is an autosomal dominant disorder caused by an expanded CAG trinucleotide repeat that causes a progressive degeneration of neurons in the putamen and caudate nucleus. Long-term clinical studies have demonstrated that embryonic stem cell (ESC) transplantation can slow the progression of the disease. The use of induced pluripotent stem cells (iPSCs) also show considerable therapeutic promise, while circumventing the ethical issues surrounding ESCs. The iPSCs are more readily available, but some methods of producing these cells involve pro-oncogenes, which become permanently integrated into the genome during reprogramming. The use of a nonintegrating adenovirus avoids this problem. It was shown in our lab that tail-tip fibroblasts reprogrammed with two adenoviruses to become iPSCs, could survive and differentiate into neuronal lineages, in the absence of tumor formation, following transplantation into healthy adult rats. However, the ability of these cells to survive, differentiate, and provide functional recovery in a disease-state brain is unknown and was the focus of this study. Adult rats received a regimen of 3-nitropropionic acid (3-NP) to induce HD behavior and neuropathology and then received bilateral transplantation of iPSCs into the striatum at either 7, 21, or 42 days after initiation of 3-NP injections representing the early, middle, and late stages of HD. Animals were tested on the accelerating rotarod and all rats receiving 3-NP displayed significant motor impairment [F(4, 31) = 6.021, p < 0.001]. However, animals that received iPSC transplantation had preserved motor function. Following behavioral analysis, the brains were analyzed for graft survival, inflammatory response, and neuronal differentiation of the iPSCs. Significant striatal atrophy [F(4, 64) = 14.770, p < 0.001], lateral ventricle enlargement [F(4, 32) = 13.377, p < 0.001], and increased striosome size [F(4, 119) = 3.783, p < 0.001] was observed in all rats receiving 3-NP when compared to sham rats. Rats with transplantations of iPSCs at 7 or 21 days following 3-NP administration did not have neuropathological deficits. Transplantation of iPSCs at 42 days following 3-NP treatment did not protect against the 3-NP-induced neuropathological deficits, despite preserving motor function. Transplanted iPSCs were found to survive and differentiate into region-specific neurons in the striatum of 3-NP rats, irrespective of the timing of the transplant. Results from this study indicate that rats given transplantations of iPSCs had preserved anatomical and motor function, at 7 and 21 days after injections of 3-NP. Further, rats receiving iPSC transplants at 42 days after initiation of 3-NP injections had a restoration of motor function despite significant neuropathological deficits. Taken together, these results suggest that iPSC transplantation may be a viable therapeutic treatment for HD.
Support for this project was provided by a PUF grant (to K.D.F.) and funding from the John G. Kulhavi Professorship and Field Neurosciences Institute (to G.L.D.).
S. Garbuzova-Davis*†‡ and P. R. Sanberg*‡§
*Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
†Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
‡Department of Pathology and Cell Biology, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
§Department of Psychiatry, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
Vascular pathology, including blood–brain/spinal cord barrier (BBB/ BSCB) alterations, has recently been recognized as a key factor possibly aggravating motor neuron damage and identifying amyotrophic lateral sclerosis (ALS) as a neurovascular disease. The majority of findings on microvascular pathology in ALS have been determined in mutant superoxide dismutase 1 (SOD1) rodent models, identifying barrier damage during disease development, which might similarly occur in familial ALS (FALS) patients carrying the SOD1 mutation. However, little is known about BBB/BSCB competence in sporadic ALS (SALS). We recently showed BBB/BSCB structural and functional impairment in postmortem gray and white matter microvessels of medulla and spinal cord tissue from SALS patients, suggesting pervasiveness of barrier damage. Although numerous signs of BBB/BSCB damage (endothelial cell degeneration, capillary leakage, perivascular edema, and tight junction protein downregulations) are indicated in both mutant SOD1 rodent models of ALS and SALS patients, other pathogenic features possibly linked to barrier alterations have so far only been identified in SALS patients. The pericyte degeneration, perivascular collagen IV expansion, and white matter capillary abnormalities and other barrier related pathologies observed in SALS patients have not yet been seen in ALS SOD1 animal models. Similarities and differences of CNS barrier alterations between human ALS and an animal model of the disease relate to whether barrier damage is an initial factor and/or component in ALS pathogenesis. Understanding these differences may also be crucial for development of new therapeutic strategies for ALS.
This study was supported by the Department of Neurosurgery and Brain Repair and the Muscular Dystrophy Association (Grant #92452).
J. Garcia,* N. C. Kuhn,* J. J. Galligan,†‡ and F. P. Manfredsson*‡
*Department of Translational Science and Molecular Medicine, Michigan State University, Grand Rapids, MI, USA
†Department of Pharmacology and Toxicology, Michigan State University, Grand Rapids, MI, USA
‡The Neuroscience Program, Michigan State University, Grand Rapids, MI, USA
The enteric nervous system (ENS) is a neuronal population that has become increasingly relevant to the field of neurodegenerative disorders. For instance, the classical Braak staging of Parkinson's disease (PD) describes a peripheral origin, potentially the ENS, of the disease. Moreover, PD-related pathology in the ENS may be the source of gastrointestinal (GI) dysfunction, a very common comorbidity in PD. Finally, the PD-associated protein α-synuclein undergoes potentially pathological changes in enteric neurons. However, tools in place to study the molecular mechanisms underlying ENS dysfunction are limited. PD transgenic animals do indeed display GI abnormalities; however, data collected from these models may be confounded by the fact that the genetic manipulations are systemic and are present from development. Here we sought to evaluate the use of virally mediated gene therapy delivered directly to the enteric neurons of the colon as an alternative method. Recombinant adeno-associated virus (rAAV) serotypes AAV 1, 2, 5, and 6 with either a single stranded or double stranded (self-complementary) genome expressing green fluorescent protein (GFP) was injected directly into the myenteric plexus of the descending colon of adult male rats (5 × 5 ml). One month following the injections the animals were sacrificed and the colon was removed for histological analysis. The mucosal layer was removed and whole mounts of the longitudinal muscle-myenteric plexus and submucosal plexus were analyzed for GFP expression. Our results show robust transduction of neurons in myenteric and submucosal ganglia. Moreover, GFP-positive nerve fibers can be observed distally to the injection area and transduced neurons. Efficacy differed greatly between the various serotypes with rAAV2 and rAAV6 exhibiting the greatest level of transduction, both in terms of vector spread, as well as transgene expression. Ongoing studies include a quantitative analysis of transduction. Our results show that neurons of the colon are amenable to viral vector transduction and will be useful for studying neuropathology caused by α-synuclein transduction.
E. J. Gonzalez-Rothi,*† L. Fernandez,† A. M. Rombola,‡ M. S. Sandhu,† N. J. Doperalski,† M. A. Lane,‡ P. J. Reier,‡ and D. D. Fuller*†
*College of Public Health and Health Professions Graduate Program in Rehabilitation Science, University of Florida, Gainesville, FL, USA
†Department of Physical Therapy, University of Florida, Gainesville, FL, USA
‡Department of Neuroscience, McKnight Brain Institute, University of Florida, Gainesville, FL, USA
Phrenic motoneurons (PhrMNs) control phrenic nerve output to the diaphragm. The respiratory-related control of phrenic motoneurons (PhrMNs) is regulated primarily by monosynaptic inputs from the medulla, but there is accumulating evidence that suggests that cervical interneurons (INs) are synaptically coupled to PhrMNs and may modulate phrenic output following cervical spinal cord injury. Therefore, we hypothesize that cervical INs are part of the anatomical substrate regulating plasticity in respiratory-related phrenic motor output. Spinal serotonin (5-HT) receptor activation is necessary to trigger changes in phrenic motor output, hence our interest on the influence of 5-HT on cervical INs. The results of initial experiments show that prephrenic cervical INs receive serotonergic innervation and express serotonin (5-hydroxytryptamine; 5-HT) receptors. A retrograde transynaptic tracer, Pseudorabies virus (PRV), was applied to the diaphragm of adult female Sprague–Dawley rats to label PhrMNs and prephrenic cervical INs. Using fluorescent, immunohistochemical dual-labeling techniques we confirmed the presence of extensive 5-HT innervation of PhrMNs. In addition, we demonstrated robust 5-HT immunoreactivity on or near pre-phrenic INs in laminae VII and X. Furthermore, prominent 5-HT2a receptor immunoreactivity was observed on PhrMNs, and modest 5-HT2a receptor expression was observed in cervical laminae VII and X. Ongoing experiments are investigating expression of additional 5-HT receptor subtypes on pre-phrenic INs. Based on these findings, we conclude that prephrenic cervical INs are innervated by serotonergic neurons and that these cells likely play a role in initiating or maintaining 5-HT dependent phrenic motor plasticity.
C. Gordon, S. Jergova, S. Gajavelli, and J. Sagen
Miami Project, University of Miami, Miller School of Medicine, Miami, FL, USA
Chronic neuropathic pain induced by spinal cord injury or peripheral nerve injury insufficiently responds to current pharmacotherapy; therefore, there is a need for novel therapeutic targets and approaches. One of the hypothesized mechanisms underlying the development of chronic pain is increased excitatory signaling through glutamate N-methyl-d-aspartate (NMDA) receptors. Unfortunately, directly antagonizing NMDA receptors has classically been limited by adverse effects such as hallucinations and motor dysfunction. Opiates have also been used in the treatment of chronic pain but with limited success due to unwanted side effects. Previous studies in our lab have shown that the naturally occurring peptide histogranin and its stable analog serine-histogranin (SHG) have NMDA receptor antagonist activity and do not interfere with locomotion. Additionally, this peptide can enhance opioid antinociceptive potency, thus reducing opioid doses and side effects. Endomorphins (EMs), endogenous opioid peptides highly selective for μ-opioid receptors, are suitable for gene therapy in combination with SHG. The aim of this study was to evaluate the antinociceptive potency of combined SHG-EM therapy in models of chronic neuropathic pain. Peripheral nerve injury and spinal cord clip compression was used to induce neuropathic pain in rats. Animals were monitored weekly for pain related behavior. One week post peripheral nerve injury or 5 weeks post spinal cord injury, lenti-EM1 constructs were intraspinally injected, and reduction of tactile and cold allodynia was observed. To evaluate the potential synergistic effect of combined therapy, SHG peptide was intrathecally delivered to lenti-EM1 injected animals 10 days post peripheral nerve injury or 7 weeks post-spinal cord injury. This further reduced pain-related behavior compared to EM1-only treated animals and stabilized hypoalgesic response. In another set of spinal cord injury animals, intraspinal injection of both EM1 and SHG lenti-plasmids together nearly completely abolished cold allodynia and modestly attenuated tactile allodynia. Due to these findings, we are engineering compound constructs encoding both SHG and EM1 peptides in hopes of expanding on the previous success of dual treatment. The two constructs will contain SHG-EM1 as well as hexSHG-EM1 (a 6 copy multi-SHG construct). The compound will be transduced into a viral vector and intraspinally injected into peripheral nerve injury and spinal cord injury rats in anticipation of achieving better long-term analgesic outcomes to manage chronic neuropathic pain.
This work was supported by NS51667 and CNF 190926.
A.-Ch. Granholm, M. Buhusi, J. Lockrow, and A. M. Fortress
Center on Aging, Department of Neurosciences, Medical University of South Carolina, Charleston, SC, USA
Individuals with Down syndrome (DS) develop Alzheimer's-like (AD) neuropathology by the fourth decade of life, followed by a significantly increased risk of dementia later in life. The neuropathological manifestations of DS-AD include neurofibrillary tangles, amyloid plaques, altered growth factor signaling, and degeneration of basal forebrain cholinergic and hippocampal neurons. In addition, it has been shown that brain stem neurons including the locus coeruleus noradrenergic (LC-NE) and the raphe serotonergic neurons degenerate early in the disease process. Recent translational studies have suggested that the loss of LC-NE neurons may exacerbate AD pathology and also might be responsible for early cognitive deficits noted in DS-AD patients, including working memory and executive function deficits. Our studies suggest that LC-NE degeneration may exacerbate working memory deficits observed in a mouse model for DS, the Ts65Dn mice. In addition, novel studies including designer receptors (DREADDs) also suggest that stimulation of LC-NE signaling may counteract these working memory deficits, perhaps suggesting a novel avenue for pharmaceutical treatment in patients with DS-AD. These findings are important based on recent clinical findings demonstrating that DS-AD patients respond as well to pharmacological interventions commonly used for AD, including the cholinesterase inhibitor Aricept and the N-methyl-d-aspartate (NMDA) antagonist Memantine, making drug discovery important for successful intervention in DS-AD.
This study was supported by NIA grant AG12122 and a grant from the Sie Foundation.
A. Granic and H. Potter
Alzheimer's Disease Programs, Department of Neurology and Linda Crnic Institute for Down Syndrome, University of Colorado-Denver, Denver, CO, USA
Individuals with Down Syndrome (trisomy 21) develop Alzheimer's disease (AD) pathology by age 30. We hypothesized that both sporadic and familial AD might exhibit cell cycle defects that generate trisomy 21 and other aneuploidy during life and, more generally, that chromosome missegregation/instability may underlie many disease of aging. Experimentally, we found that all forms of AD exhibit elevated trisomy 21 and borderline elevated trisomy 18 in skin fibroblasts and that mutant amyloid precursor protein and presenilin genes that cause familial AD induce chromosome missegregation and aneuploidy of multiple chromosomes in neurons and other cells in transgenic mice and in cultures. Confirmatory results from other labs showed that 30% of neurons in early stage sporadic AD are aneuploid, including 10% trisomy 21. Furthermore, the specific loss of aneuploid neurons in late AD accounts for 90% of the neuronal loss observed at autopsy, indicating that the birth and death of aneuploidy neurons underlies AD neurodegeneration. We also found that adding amyloid β (Aβ to cells or Xenopus egg extracts impairs the formation and stability of mitotic spindles by inhibiting kinesins Kinesin family member 11 (Eg5), kinesin-family member 4A (KIF4A), and mitotic centromere-associated kinesin (MCAK), which are essential for mitotic spindle structure and function. Like Aβ, the Eg5 inhibitor monastrol disrupts the mitotic spindle and leads to chromosome missegregation. Aneuploid neurons also accumulate in brains of frontotemporal dementia (FTD) patients, in mouse models of FTD, and in cells expressing FTD-causing mutant Tau genes. Finally, elevated low-density lipoproteins (LDL)/cholesterol is a risk factor for both AD and cardiovascular disease, in which each atherosclerotic plaque harbors a monoclonal growth of aneuploid smooth muscle cells. LDL or solubilized cholesterol [but not high-density lipoproteins (HDL)] cause chromosome missegregation/aneuploidy in cultured cells, in mice fed a high cholesterol diet and in brain neurons of Niemann pick C patients. Ethanol or calcium chelation prevents cholesterol from inducing mitotic spindle abnormalities and chromosome missegregation. These data suggest that genome instability may underlie the pathogenesis of both neurodegenerative and cardiovascular disease and that rectifying normal mitotic spindle function is a promising approach to prevention and/or therapy.
J. D. Heiss
National Institutes of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA
The pathological hallmark of Parkinson's disease (PD) is progressive loss of nigral dopaminergic (DA) neurons, which results in progressive neurological dysfunction and death. Glial cell derived neurotrophic factor (GDNF) infusion in animal models of PD has reversed this degenerative process but clinical trials of GDNF infusions have not been therapeutic. Recent animal studies suggest that gene transfer via direct delivery of viral vectors would be better tolerated and more efficacious than GDNF infusion for the treatment of PD. To test this hypothesis, we have begun enrolling Parkinson's disease patients for a Phase 1 single-center, open-label, dose escalation, safety and tolerability study of adeno-associated virus, serotype 2 vector (AAV2) containing human GDNF complementary DNA, bilaterally delivered by convection-enhanced delivery (CED) to the putamen. Clinical, anatomical [by magnetic resonance imaging (MRI)], and neurochemical effects [by positron emission tomography (PET)] will be evaluated at regular intervals after vector delivery during a 5-year observation period.
D. G. Hernandez-Ontiveros, N. Tajiri, S. A. Acosta, M. Pabon, K. Shinozuka, H. Ishikawa, Y. Kaneko, and C. V. Borlongan
Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
Traumatic brain injury (TBI) has escalated over the years due to intense military combat worldwide. The brain suffers massive cell loss, neighboring cells undergo progressive neurodegeneration, inflammatory, and cell death events. If subsequent incidents are not controlled on time, they may extend to normal areas beyond the core of injury; exacerbating the central nervous system's immune response. Cluster of differentiation 36 (CD36)/fatty acid translocase (FAT), a class B scavenger receptor of modified low-density lipoproteins (mLDLs) in macrophages, has been associated in lipid metabolism, atherosclerosis, oxidative stress, inflammatory response, tissue injury in cerebral ischemia, TBI, and in certain neurodegenerative diseases. CD36 may play a key pathological role in mediating the neuroinflammatory response in the animal model of TBI. Here, we show that the soluble receptor of advanced glycation end products RAGE (sRAGE) effectively blocked the cell association of hypochlorite-modified low-density lipoprotein HOCl-LDL to monocytes/macrophages (RAW cells, derived from mouse tumor). sRAGE exhibited the same inhibitory activity on cell association of HOCl-LDL to stably transfected Chinese hamster ovary (CHO) cells expressing murine scavenger receptor class B, type I (SR-BI, termed CHO-SR-BI), or human CD36 (termed CHO-CD36). sRAGE also displayed robust antagonistic effects on cell association of HOCl-LDL to human spleen-derived monocytes/macrophages (MD cells). Thus, sRAGE was shown here to interfere with Class B scavenger receptor CD36-mediated uptake of HOCl-LDL. All treatment conditions performed in quadruplicates. Next, we asked the question whether spleen immune function, similar to stroke, could also alter TBI outcome, but more specifically we sought to reveal the interaction of CD36 and spleen in TBI with emphasis on age as a factor. Adult (2 months old) or neonatal (7 days old) Sprague–Dawley rats subjected to TBI, respectively, displayed upregulated and downregulated expression of CD36 in the spleen and brain at the supra-acute phase of injury (15 min after TBI). Sample size was n = 20 per treatment group. These data revealed a close interaction of CD36 in spleen and brain, as well as the influence of age in CD36 expression, which likely plays a key role in TBI pathological outcome. That the neonatal brain mounts anti-CD36 response in both spleen and brain at supra-acute phase of TBI prompted us to further examine this unique neonatal CD36 temporal profile. Neonatal (7 days old) Sprague–Dawley rats subjected to TBI displayed downregulated expression of CD36 in the spleen and brain at 15 min and 3 h after TBI, then reached normal levels at 12 and 24 h, and finally upregulated by 48 h post-TBI. Different cohorts of TBI rats were randomly euthanized at each time point to reveal CD36 expression. Sample size is n = 6 treatment group. Controls were age-matched rats that received sham surgery. The critical timing (i.e., 24–48 h) of CD36 expression shift (from downregulation to upregulation) may signal the transition of functional effects of this immune response from prosurvival to cell death. This dynamic CD36 expression also suggests the therapeutic window for TBI. Indeed, this immune response parallels our observed downstream prosurvival (neurogenesis) and cell death (neurovascular alterations revealed by matrix metalloproteinaises breakdown and inflammatory response) over the same 24- to 48-h period. Based on our in vitro data demonstrating the capability of sRAGE to block CD36-mediated cell association of HOCl-LDL to human spleen-derived monocytes/ macrophages, we next embarked on testing whether sRAGE could modulate CD36 in an in vivo model of neonatal TBI. sRAGE treatment (25 mg/ 0.25 ml IP at 15 min, 3 h, 12 h, and 24 h) or its equivalent volume of diluent (PBS) was performed in neonatal Sprague–Dawley rats (7 days old). Animals were euthanized at 48 h post-TBI for spleen and brain analysis of CD36 expression. Data revealed that levels of CD36 expression were significantly downregulated in sRAGE-treated TBI animals compared to vehicle-treated TBI animals. Controls were age-matched rats that received sham surgery. Sample size was n = 20 per group. Altogether, our results demonstrate the intimate involvement of CD-36 in TBI neuroinflammation. Targeting CD-36 may prove effective in attenuating neuroinflammation and therefore stands as a potent therapeutic pathway for treatment of TBI.
Y.-C. Hsu,* C.-Y. Kao,*† J.-W. Liu,*‡ Y.-F. Chung,* D.-C. Lee,* and I.-M. Chiu*†‡§
*Division of Regenerative Medicine, Institute of Cellular and System Medicine, National Health Research Institutes, Miaoli, Taiwan
†Graduate Program of Biotechnology in Medicine, Institute of Biotechnology and Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan
‡Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan
§Department of Internal Medicine, The Ohio State University, Columbus, OH, USA
Aurora-A kinase (AurA) is a centrosome and mitotic spindle-associated, cell cycle-regulated serine/threonine kinase and is a key regulator of asymmetric cell division and cell fate determination. Fibroblast growth factor 1 (FGF1) has been suggested as an important growth factor for many cell types. FGF1 and FGF-1B promoter (-540 to +31)-driven green fluorescent protein (F1BGFP) have been used to isolate neural stem/progenitor cells (NSPCs) and glioblastoma stem cells from developing mouse brains, human glioblastoma cell lines, and tissues, respectively. In this study, we provide several lines of evidence to demonstrate that AurA activation is required for self-renewal and multipotency in F1BGFP(+) stem cells: (i) Inhibition of AurA activation disrupted the maintenance of mouse embryonic stem cells (ESCs), mouse ESC-derived NSPCs, primary mouse brain NSPCs, and human glioblastoma stem cells; (ii) F1BGFP reporter could be used to isolate GFP(+) NSPCs with significantly higher levels of AurA activation, cell proliferation, and neurosphere formation; (iii) Autocrine/paracrine activation of AurA in F1BGFP(+) cells could be blocked by FGF1-neutralizing antibody, FGF receptor inhibitor SU5402, and AKT inhibitor; (iv) Inhibition of AurA activation reduced neurosphere formation of F1BGFP(+) NSPCs; (v) Markers of NSPC-multipotency also decreased upon AurA inhibition. These results suggest that AurA kinase regulates the growth and self-renewal of stem cells, such as ESCs and NSPCs. Using the F1BGFP reporter, we have provided a novel method to identify and enrich a subset of ESCs and NSPCs with AurA activation from different cellular and tissue sources.
M. F. Iulita,* A. Ower,* L. Breuillaud,* M. Hanna,† A. M. Fortress,‡ A. C. Granholm,‡ J. Busciglio,† and A. C. Cuello*§¶
*Department of Pharmacology and Therapeutics, McGill University, Montreal, Canada
†Department of Neurobiology and Behavior, University of California, Irvine, CA, USA
‡Department of Neurosciences, Medical University of South Carolina, Charleston, South Carolina, USA
§Department of Anatomy and Cell Biology, McGill University, Montreal, Canada
¶Department of Neurology and Neurosurgery, McGill University, Montreal, Canada
Our studies focus on a novel pathway we have described, responsible for the CNS metabolism of nerve growth factor (NGF) (Bruno and Cuello, PNAS 2006). We have recently demonstrated that the Alzheimer's disease (AD) amyloid pathology interferes with the extracellular metabolism of NGF, such that NGF fails to mature and its degradation is enhanced. This is evidenced by increased levels of its precursor molecule (proNGF) and increased activity of matrix metalloprotease 9 (MMP-9), the NGF degrading enzyme (Bruno et al., JNEN 2009a). Similar alterations occur at the mild cognitive impairment stage (Bruno et al., JNEN 2009b). These changes are relevant in the context of AD, as NGF is essential for cholinergic neurons and cholinergic dysfunction contributes to a great extent to the marked cognitive impairments seen in AD patients. To further explore whether NGF deregulation occurs in other conditions of pathological amyloid-β (Aβ) accumulation before the onset of AD, we embarked on an extensive study with Down's syndrome (DS) brain material involving the use of human postmortem brains (47–64 years) from the frontal, parietal, and temporal regions, the use of human DS fetal cortical neurons (17–21 gestational age weeks) and frozen frontal cortex tissue from the Ts65Dn mouse model (8–12 months), including age-matched controls in all cases. We investigated the expression and gelatinolytic activity of MMP-9 by qPCR and zymography, respectively. The levels of proNGF and of Aβ species were analyzed by Western blotting and ELISA. DS brains exhibited significantly increased levels of APP, Aβ 42, Aβ 40, and Aβ 40/42 ratios in frontal cortex compared to control cases. In this region, proNGF was found significantly elevated together with upregulated MMP-9 activity. Conditioned media from DS neurons exhibited significantly higher APP and proNGF levels as well as upregulated MMP-9 zymographic activity. We are currently investigating whether other NGF pathway markers are altered in an AD-like manner, specifically, analyzing the expression and activity of the enzymes responsible for proNGF maturation (plasmin and tissue plasminogen activator). We are also complementing these studies by looking at NGF pathway markers in the Ts65Dn mouse model of DS. Investigating NGF dysfunction before dementia onset is relevant, as these changes could be early signals of a silent, preclinical, ongoing AD pathology. Our investigations in DS provide further evidence to support that AD and DS share common pathogenic mechanisms, in particular regarding the role of Aβ in the deregulation of NGF's extracellular metabolism. The study of DS brains provides a compelling platform to investigate early molecular changes related to AD progression.
The authors would like to acknowledge support from the Canadian Institutes of Health and Research (MOP-97776) to A.C.C., the National Institutes of Health (HD38466 and AG16573 grants) to J.B., the National Institute on Aging (AG012122) and the Sie Foundation to A.C.G. M.F.I. is the recipient of a Biomedical Doctoral Award from the Alzheimer Society of Canada. L.B. is the recipient of a Postdoctoral Fellowship from the Centers of Excellence in Neurodegeneration.
P. Jendelová,*† N. Romanyuk,*† T. Amemori,* B. Onteniente,‡§ J. Price,¶ and E. Syková*†
*Institute of Experimental Medicine, ASCR, Prague, Czech Republic
†Department of Neuroscience, 2nd Faculty of Medicine, Charles University, Prague, Czech Republic
‡INSERM UMR861, Université d'Evry-Val d'Essonne, Evry, France
§INSERM UMR894, Université Paris Descartes, Paris, France
¶Institute of Psychiatry, Kings College London, UK
Despite advances in medical and surgical care, current clinical therapies for spinal cord injury (SCI) are limited. Here, we compared the effect of neural precursors obtained from conditionally immortalized neural stem cell lines from human fetal spinal cord tissue (SPC-NPs) and human induced pluripotent stem cell-derived neural precursors (iPS-NPs) in the treatment of a balloon-induced spinal cord lesion. Prior to in vivo experiments, we tested the ability of the cells to terminally differentiate in vitro. Three weeks after the induction of differentiation, the neuron/astrocyte ratio was higher in iPS-NP cultures than in SPC-NP cultures. However, the percentage of NK homeobox 1 (Nkx 6.1)-positive cells was higher in SPC-NP cultures—8.09 + 1.73%—than in iPS-NP cultures—4.15 + 0.65%. In the in vivo experiments, suspensions of stem cells (5 × 105 cells in 5 μl of culture medium) were implanted into the lesion 1 week after SCI (n = 22 for SPC-NPs and n = 18 for iPS-NPs), while the control groups (n = 16 and n = 14, respectively) were injected with saline. Locomotor (Basso, Beattie, and Bresnahan; BBB) and sensitivity (plantar) tests were performed weekly for 2 months. Animals transplanted with either cell type displayed significant motor improvement compared to the controls and a stable physical recovery. Animals transplanted with SPC-NPs also demonstrated significant improvement in the plantar test. Two months postimplantation (PI), both types of cells robustly survived in the lesion, but with a low Ki67 index. Compared to SPC-NPs, which partially filled the lesion cavity, iPS-NPs interacted more with the host tissue. Besides differentiating into 5-hydroxytryptamine (5-HT)-positive neurons, iPS-NPs also differentiated into doublecortin+ dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDa (DARPP32)+, calbindin+, parvalbumin+ and choline acetyltransferase (ChAT+) neurons. Some cells were positive for 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) and chondroitin sulfate proteoglycan 4 (NG2)+ protein. SPC-NPs cells expressed mainly glial fibrillary acidic protein (GFAP); however, at 2 months PI we found 25% of the cells to be positive for Nkx 6.1, and at 4 months PI the cells were positive for ChAT and Islet2, motor neuron-specific markers. Both cell types upregulated host neurotrophin genes [nerve growth factor (NGF) in the case of SPC-NPs, neurotrophin-3 (NT3) in the case of iPS-NPs] and spared the white matter in the lesion; the gray matter was spared only in iPS-NP transplanted animals. Our results demonstrate that the transplantation of SPC-NPs or iPS-NPs into the lesioned rat spinal cord improves functional outcome by bridging the spinal cord lesion and providing trophic support to the spared axons in the injured tissue. In addition, iPS-NPs undergo tissue-specific differentiation and can serve as a tool for autologous or allogenic cell transplantation therapy.
This study was supported by GA CR 13-00939S, LH12024.
C. M. Kelley,* B. E. Powers,† R. Velazquez,† J. A. Ash,† S. D. Ginsberg,‡§¶ B. J. Strupp,† and E. J. Mufson*
*Department of Neurological Sciences, Rush University Medical Center, Chicago, IL, USA
†Division of Nutritional Sciences and Department of Psychology, Cornell University, Ithaca, NY, USA
‡Center for Dementia Research, Nathan Kline Institute, Orangeburg, NY, USA
§Department of Psychiatry, New York University Langone Medical Center, New York, NY, USA
¶Department of Physiology and Neuroscience, New York University Langone Medical Center, New York, NY, USA
Down syndrome (DS) is a condition marked by mental retardation and high incidence of early Alzheimer's disease (AD)-type pathology including degeneration of the basal forebrain cholinergic neuron (BFCN) system. Using a mouse model for DS and AD, the Ts65Dn mouse, we explored the effects of perinatal choline supplementation on the BFCN system. Ts65Dn and disomic male mice were perinatally exposed to a choline supplemented (5.1 g/kg choline chloride in AIN-76A, Dyets, Inc.) or unsupplemented, choline-controlled (1 g/kg) diet from conception to weaning via maternal diet. Postweaning, mice were maintained on the unsupplemented diet until sacrifice at 14–18 months (n = 16–23 per group). Mice were transcardially perfused with paraformaldehdye, and brains were removed from the skull, sectioned, and immunolabeled for choline acetyltransferase (ChAT), p75-neurotrophin receptor (p75NTR), and tyrosine receptor kinase A (TrkA). Stereology was used to determine number, size, regional area, and neuron density in the four BFCN subregions. Optical density was used to evaluate hippocampal cholinergic fiber innervation. An additional series was immunolabeled for ChAT and BFCNs were extracted using laser capture microdissection (LCM). The BFCN transcriptome was determined using 33P-labeled samples hybridized to membrane arrays embedded with >550 probes. Ts65Dn unsupplemented mice displayed decreased ChAT-positive BFCN numbers in the medial septum (MS) and horizontal diagonal band (HDB) compared with unsupplemented disomic mice; choline supplementation normalized these decreases. In contrast, genotype and treatment did not affect mean BFCN size; although, when divided into bins, choline supplementation increased the percentage of large BFCNs. Region areas were consistently larger in Ts65Dn mice compared with treatment-matched disomic mice, whereas BFCN density was decreased in Ts65Dn mice only in the MS and nucleus basalis of Meynert (NBM). Perinatal choline supplementation increased cholinergic innervation in Ts65Dn mice across all strata of the dentate gyrus and the rostral hippocampus proper; however, choline supplementation did not have any effect on altered patterns of innervation observed between the genotypes. Transcriptome differences were found between Ts65Dn and disomic unsupplemented mice with normalization of many genes observed in mice that received perinatal choline supplementation. We did not find a clear pattern of up- or downregulation of genes between genotypes or a strong effect for a single class of genes. Interestingly, the genotype difference and treatment-mediated normalization was most marked in the MS/vertical diagonal band (VDB). Overall, our findings suggest an influence of genotype on BFCNs and the cholinoceptive hippocampal formation. Perinatal choline supplementation effectively normalizes some of the genotype-mediated differences and may prove an effective costrategy in the treatment or prevention of BFCN pathology.
D. W. Kim, N. Tajiri, H. Ishikawa, K. Shinozuka, S. A. Acosta, M. Pabon, Y. Kaneko, and C. V. Borlongan
Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
Wharton's jelly is a gelatinous tissue within the umbilical cord that contains myofibroblast-like stromal cells. A unique cell population of Wharton's jelly that has been suggested as displaying the stemness phenotype is the mesenchymal stromal cells or MSCs. The stemness and immune properties of MSCs appear to be more robustly expressed and functional from fetal compared with adult-derived MSCs and so MSCs harvested from the Wharton's jelly are considered much more proliferative, immunosuppressive, and even therapeutically active stem cells than those isolated from older, adult tissue sources such as the bone marrow or adipose. The present review discusses the phenotypic character, therapeutic applications, and optimization of experimental protocols for Wharton's jelly-derived stem cells. MSCs derived from Wharton's jelly display promising transplantable characteristics including ease of sourcing, in vitro expandability, differentiation abilities, immune evasion, and immune regulation features. Thus, Wharton's jelly-derived stem cells possess many potential advantages as transplantable cells for treatment of various diseases (e.g., cancer, chronic liver disease, cardiovascular diseases, nerve, cartilage and tendon injury). We are witnesses to many different tissue sources of stem cells. Here, we provide scientific evidence of the many appealing features of a unique set of stem cells those from the Wharton's jelly.
S. W. Kim,*† D. Yu,†‡ W. P. Kuo,* H. Haragopal,†‡ X. Zheng,†‡ and Y. D. Teng†‡
*Department of Oral Medicine, Infection, and Immunity, Harvard School of Dental Medicine, Boston, MA, USA
†Division of SCI Research, Veterans Affairs Boston Healthcare System, Boston, MA, USA
‡Department of Neurosurgery, Harvard Medical School, Brigham and Women's Hospital and Children's Hospital Boston, Boston, MA, USA
There are several published reports that the localized or systematic delivery of autogenous mesenchymal stromal cells to neural injury patients could achieve better and more prompt functional outcomes of treatment. This has been repeatedly reported in animal models. However, the cellular and molecular level of mechanisms for this phenomenon is not well elucidated. We hypothesized that the developmental role of human mesenchymal stromal cells on human neural stem cells is mediated by direct cell to cell interaction. We took the whole transcriptome analysis approach to see the differential expression of genes in coculture of human mesenchymal stromal cells and human neural stem cells in vitro. We demonstrated that coculture of human mesenchymal stromal cells and human neural stem cells resulted in an increase of the proliferation of human neural stem cells. The whole transcriptome analysis with gene set enrichment analysis revealed a significant increase of antioxidative stress-related genes, indicating the neuroprotective role of human mesenchymal stromal cells. In addition, we report that significant increases in decorin (DCN), lumican (LUM), microfibrillar-associated protein 2 (MFAP2), and fibulin 5 (FBLN5) in direct coculture further substantiate that the interaction of two stem cells are mediated by a formation of glycoprotein complex. Understanding of mesenchymal stromal stem cells mediated molecular mechanism and signals involved in neural repair will allow for engineering of mesenchymal stromal cells that will potentially enhance the host's capacities to promote the neural repair.
J. H. Kordower
Rush University Medical Center, Chicago, IL, USA
Neurturin is a trophic factor in the glial family of ligands, a family of trophic factors that includes GDNF. Ceregene, Inc., both alone and in collaboration with my lab, has performed a large series (>17) of preclinical studies that lead to a filing of an IND and the performance of multiple Phase I and Phase II clinical trials for AAV2-neurturin (CERE-120) in patients with Parkinson's disease (PD). Along the way, our study of the PD brain have revealed significant challenges to trophic factor delivery protocols. This presentation will include a summary of all relevant preclinical studies demonstrating the power of neurturin gene delivery in experimental models of PD, present all available clinical trials data in which AAV2-neurturin was tested in PD patients, and discuss the status of the PD brain at the time of gene therapy/trophic factor delivery and the challenges the PD brain provides in moving this therapy forward.
S. M. Kousik,* T. C. Napier,* and P. M. Carvey*†
*Department of Pharmacology and the Center for Compulsive Behavior and Addiction, Rush University Medical Center, Chicago, IL, USA
†Department of Neurology, Rush University Medical Center, Chicago, IL, USA
Methamphetamine (meth) dysregulates the neurovascular unit (NVU). Persistent reductions in regional cerebral blood flow (rCBF) occur in the striatum of meth-treated rats and detoxified human meth abusers. Similar changes are documented in patients with Parkinson's disease (PD) and may underlie the increased risk for PD in meth abusers. The current study had three objectives: (1) determine which vessels in the striatum and substantia nigra are most affected by meth using micro-computed tomography (μCT); (2) examine the duration of vascular dysregulation in meth-withdrawn rats; and (3) ascertain which dopamine (DA) receptor subtype is responsible for these effects. Rats were trained to self-administer meth (3 h/day) for 14 days. Saline-yoked rats served as controls. Following withdrawal for 1, 14, 28, and 56 days, rats were anesthetized and perfused for μCT. Meth decreased the mean vessel diameter in the striatum after 1 day (0.021 ± 0.002; p < 0.001) and 14 days (0.020 ± 0.002; p < 0.001) compared with controls (0.042 ± 0.003). The distribution of vessel diameter changes in the striatum revealed that microvessels (10–30 μm), primarily regulated by the NVU, were most sensitive to meth-induced constriction while larger vessels remained largely unchanged. Meth also decreased the overall vascular volume fraction in the striatum after 1 (0.061 ± 0.003; p < 0.01), 14 (0.062 ± 0.003; p < 0.01) and 28 days (0.077 ± 0.004; p < 0.05). No vascular alterations were observed in the substantia nigra or parietal cortex (negative control). We previously demonstrated loss of tyrosine hydroxylase (TH) in the striatum of rats 10 days following meth self-administration and are currently assessing both TH and Fluoro-Gold (retrograde tracer) to determine if the decrease in TH is a result of nigral cell loss or phenotypic suppression. As meth markedly increases DA, a second set of animals were treated acutely with either a dopamine 1 receptor (D1R; SCH23390; 0.5 mg/kg) or D2R (L741,626; 1 mg/kg) antagonist 30 min prior to meth (2.5 mg/kg) and perfused for μCT 2 h following meth. Meth alone reduced vascular vessel diameter (0.026 ± 0.001; p < 0.05) and volume fraction (0.044 ± 0.007; p < 0.05). D2R antagonist pretreatment attenuated the meth-induced decreases in both diameter (0.047 ± 0.004; p < 0.05) and volume (0.115 ± 0.006; p < 0.05); D1R antagonist pretreatment slightly attenuated the volume reductions (0.089 ± 0.004; p < 0.05). These data suggest that meth abuse produces long-lived reductions in striatal rCBF that are likely a consequence of a D2R > D1R mediated interaction with the NVU responsible for tissue perfusion. Prolonged hypoperfusion may render the striatum more susceptible to degenerative changes explaining the risk of meth abuse for subsequently developing PD. Alterations in NVU function may also underlie the degenerative changes in PD itself.
This study was funded by Kenneth Douglas Fdn, Daniel and Ada Rice Fdn, and Center for Compulsive Behavior and Addiction at Rush University, Chicago, IL.
X. Leveque,*† R. Welchko,*† G. Shall,*† K. Fink,*† M. Lu,*† J. Rossignol,*†‡ and G. Dunbar*†
*Field Neurosciences Institute Laboratory for Restorative Neuroscience, Department of Psychology, Central Michigan University, Mount Pleasant, MI, USA
†Field Neurosciences Institute, Saginaw, MI, USA
‡College of Medicine, Central Michigan University, Mount Pleasant, MI, USA
Parkinson's disease (PD) is a late-stage neurodegenerative disorder characterized by a loss of dopaminergic neurons within the nigrostriatal pathway. Loss of this pathway leads to significant motor impairments. Although an effective long-term therapy has not been developed, there have been promising results using transplantation of human fetal neuroblasts in the neostriatum of PD patients. However, issues of tissue availability and ethical concerns limit the utility of this approach. As an alternative to transplanting human fetal neuroblasts, the use of induced pluripotent stem cells (iPSCs) has gained recent attention. In this study, we tested the efficacy of transplanted iPSCs after differentiation in vitro into the striatum of rats given the dopaminergic toxin, 6-hydroxydopamine (6-OHDA), into the nigrostriatal pathway. The iPSCs were generated from somatic stem cells derived from rat tail-tip fibroblasts (TTFs), using a combination of adenoviruses [AD; octamer-binding transcription factor 4 (Oct4), sex-determining region Y Box 2 (Sox2), krüppel-like factor 4 (Klf4), and c-Myc]. The pluripotency of these cells were confirmed using flow cytometry and immunocytochemistry for the pluripotent markers stage-specific embryonic antigen 3 (SSEA3), SSEA4, Tra-1-60, Nanog, and Oct4. Differentiation of TTF-AD iPSCs into mature dopaminergic neurons was induced in vitro and different stages of differentiation were determined using immunocytochemistry and molecular analysis to identify various phenotypically expressed dopaminergic markers, such as tyrosine hydroxylase (TH), forkhead box A2 (FoxA2), LIM homeobox transcription factor 1α (Lmx1a), and nuclear receptor related 1 (Nurr1). To determine the most efficacious stage of iPSC differentiation for graft integration and recovery of motor improvement after transplantation, undifferentiated iPSCs and immature neurons from iPSCs were unilaterally transplanted into the striatum of 6-OHDA-treated rats. All the rats were tested using cylinder test and apomorphine-induced rotations every 4 weeks and sacrificed 4 months posttransplantation. The initial findings from this study suggest that iPSCs provide behavioral sparing in the 6-OHDA rat model of PD and that the stage of differentiation is a critical factor in the therapeutic efficacy of the transplants.
This work was supported by the John G. Kulhavi, Endowed Professorship in Neuroscience, Central Michigan University Neuroscience Program, and the Field Neurosciences Institute.
M. de C. Lima,* M. Cordeiro,† M. Richardson,† M. V. Gomez,‡ M. Moraes,* and A. Massensini*
*Department of Physiology and Biophysics, Universidade Federal de Minas Gerais, Minas Gerais, Brazil
†Fundação Ezequiel Dias, Belo Horizonte, Minas Gerais, Brazil
‡School of Medicine, Universidade Federal de Minas Gerais, Minas Gerais, Brazil
Stroke is an important cause of disability in the world. Despite this fact, there is no effective drug therapy to protect neurons against death induced by ischemic and excitotoxic insults. The peptide Tx3–4 is a voltage-dependent calcium channel blocker purified from the Phoneutria nigriventer spider venom. This peptide demonstrated an in vitro neuroprotective effect against hypoxia-induced neuronal death. The aim of this work was to investigate the effects of Tx3–4 on neurological injury following focal cerebral ischemia in rats. Heart rate, blood pressure, and electroencephalographic activity were recorded after intracerebroventricular injection of Tx3–4 in the rats. Doses of 0.16, 0.32, and 0.75 μg were used to investigate its neuroprotective effect. Middle cerebral artery occlusion was induced for 60 min and the functional status of the animals was evaluated using cylinder test, bilateral asymmetry test, foot fault, and parallel bars test (before, 7 and 14 days after surgery). Magnetic resonance images T2-weighted and histological analyses (H&E, neutral red) were performed to evaluate infarct volume. Electroencephalographic recordings confirmed arterial occlusion and evaluated cerebral activity during experiments. Our results showed that Tx3–4 did not induce significant changes on heart rate and blood pressure within this dose range. Sham-operated animals centrally injected with Tx3–4 did not show functional disabilities. Tx3–4 also protected rats against brain lesions and loss of weight after transient cerebral ischemia. These animals also showed a decrease in functional impairment. Thus, the peptide Tx3–4 was able to provide functional and structural neuroprotection in rats after transient cerebral ischemia without relevant toxic effects.
This work was supported by grants from Instituto do Milênio, FAPEMIG, CNPq, and CAPES.
L. N. Little,* S. Hussey,* D. E. Sanchez,* B. E. Osteen,* D. D. Fuller,† M. A. Lane,* and P. J. Reier*
*Department of Neuroscience, University of Florida College of Medicine, Gainesville, FL, USA
†Departments of Physical Therapy, College of Public Health and Health Professions, University of Florida, Gainesville, FL, USA
Cervical spinal cord injuries (SCIs) at or above the C4 level are often accompanied by severe respiratory complications due to secondary alterations in the complex neural, neuromuscular, pulmonary, and dynamic processes that control respiration. To date, the most extensive studies of post-SCI respiratory dysfunction and neuroplasticity have utilized a popular C2 hemisection model. However, SCIs are more common at or near the level of the phrenic nucleus (C3–C5/6) where a neuroprotective strategy may be of benefit. The goals of this study were to examine temporal changes in diaphragm electromygraphy (diaEMG) activity and overall ventilatory function following lateralized C3/4 contusion injuries and to characterize how the extent and time-course of immediate and secondary phrenic motoneuron (PhMN) loss relates to functional outcomes postinjury. Bilateral diaphragm activity was assessed in adult female Sprague–Dawley rats using implanted telemetric electrodes. To assess the relationship between diaphragm activity and ventilatory function, diaphragm EMG recordings were paired with whole-body plethysmography in awake, unanesthetized animals prior to and at weekly intervals postinjury. Measurements were made under baseline (normoxic, normocapnic) or hypercapnic (7% CO2) breathing conditions. In separate, ongoing studies, we are characterizing the extent and time course of PhMN loss following injury by retrograde labeling PhMNs with cholera toxin. Initial studies have demonstrated that under baseline conditions, diaphragm activity was markedly reduced immediately after injury but showed modest recovery over the first 2 days postinjury. Conversely, ventilation appeared unaffected by the injury. During exposure to hypercapnic challenge, ventilatory patterns were transiently affected by C3/4 contusion. In contrast, diaphragm activity in response to hypercapnia is dramatically and persistently attenuated. Preliminary results indicate loss of a subset of phrenic neurons at 0 h, which does not increase significantly at 4 weeks postinjury. These initial results suggest the persistence of normal ventilatory patterns most likely reflects compensatory spinal respiratory plasticity. Secondary PhMN loss, however, does not appear to contribute to diaphragm dysfunction when increased respiratory drive is required. Whether impaired diaphragm activity is due to primary PhMN loss, reduced innervation of PhMNs caudal to the injury, or loss of other phrenic circuit constituents remains to be determined.
This study was supported by NIH RO1 NS054025 (P.J.R.), NIH 1R01 NS080180-01A1 (D.D.F.), and the Anne and Oscar Lackner Chair in Medicine.
F. P. Manfredsson,* S. E. Gombash,*† C. J. Kemp,* D. L. Fischer,* N. C. Kuhn,* A. Cole-Strauss,* R. J. Mandel,‡ S. M. Fleming,§ T. J. Collier,* J. W. Lipton,* and C. E. Sortwell*
*Department of Translational Science and Molecular Medicine, Michigan State University, Grand Rapids, MI, USA
†Graduate Program in Neuroscience, University of Cincinnati, Cincinnati, OH, USA
‡Department of Neuroscience, University of Florida, Gainesville, FL, USA
§Department of Psychology, University of Cincinnati, Cincinnati, OH, USA
Mutations in the gene encoding α-synuclein (α-syn) have been linked to familial Parkinson's disease (PD). Viral vector-mediated α-syn overexpression in the nigrostriatal dopamine (DA) system recapitulates key pathological features of PD. Aging is the single most important risk factor for PD yet no study to date has examined nigrostriatal pathology associated with viral vector-mediated α-syn overexpression in the aged brain to determine whether the aged brain has enhanced susceptibility. Elevated levels of oxidative stress and neuroinflammation have been linked to both α-syn mechanisms of neurotoxicity as well as the aging process. Therefore, we hypothesized that α-syn-mediated neurotoxicity will be amplified in the aged nigrostriatal system. We examined recombinant adeno-associated virus serotype 2/5 (rAAV2/5)-mediated human wildtype (WT) α-syn or green fluorescent protein (GFP) expression and resulting nigral degeneration in young (3 month) and aged (20 month) rats. The impact of intranigral injections of three separate titers of rAAV2/5 α-syn (1012 = Low, 1013 = Medium, 1014 = High) and one titer (1013 = Medium) of rAAV2/5 GFP was examined. Overexpression of α-syn or GFP occurred in both young and aged rats with the greatest magnitude of nigrostriatal system toxicity observed with increasing rAAV2/5 α-syn vector titer. However, aged rats with matched titer rAAV2/5 α-syn or rAAV2/5 GFP resulted in significantly less human WT α-syn or GFP protein expression in the aged brain in both the striatum and substantia nigra (SN). Despite this approximate 50% decrease in human WT α-syn protein expression, the aged nigrostriatal system was equivalently degenerated compared to young rats injected with the identical rAAV2/5 α-syn titer. Initial quantitation of transduced neuron numbers indicates equivalent infectivity of SN neurons in young and aged rats. Ongoing studies are examining whether differences exists between mRNA expression in young versus aged rats resulting from rAAV2/5 α-syn or rAAV2/5 GFP injections. Collectively, our results demonstrate that, under conditions of equivalent α-syn protein expression, the aged nigrostriatal system is significantly more susceptible to α-syn-mediated toxicity. Further, gene therapy strategies for the aged brain may require higher vector titers to achieve therapeutic expression levels.
P. K. Menon,* D. F. Muresanu,† A. Sharma,* Z. P. Aguilar,‡ Y. A. Wang, ‡ J. V. Lafuente,§ H. Mössler,¶ R. Patnaik,# and H. S. Sharma*
*Laboratory of Cerebrovascular Research, Department of Surgical Sciences, Anesthesiology and Intensive Care Medicine, Uppsala University Hospital, Uppsala, Sweden
†Department of Neurosciences, University Hospital, University of Medicine and Pharmacy, Cluj-Napoca, Romania
‡Ocean NanoTech, Springdale, AR, USA §Department of Neurosciences, University of Basque Countries, Bilbao, Spain
¶Ever NeuroPharma, Oberburgau, Austria
#Department of Biomaterials, Institute of Technology, Banaras Hindu University, Varanasi, India
Iron oxide magnetic nanoparticles (IOMNPs) are used for diagnostic purposes in cancer studies. In animal models, IOMNPs showed lower uptake in many organs. However, the effect of these NPs per se on the central nervous system (CNS) is not well known. In this study, we examined whether IOMNPs (10 nm in diameter) given in rats (0.25 or 0.50 mg/ml in100 μl, IV) could influence the blood–brain barrier (BBB) permeability and neuronal or glial changes within 4–24 h after administration. Furthermore, whether the IOMNPs given in identical situations after a focal spinal cord injury (SCI) would affect SCI induced pathophysiology. Finally, whether cerebrolysin, a combination of several neurotrophic factors and active peptide fragments in a dose of 2.5 ml/ kg, IV, will affect the IOMNPs induced changes in CNS pathology. The SCI was inflicted in rats by making a longitudinal incision into the right dorsal horn of the T10–11 segments, and the animals were allowed to survive 4 or 24 h after trauma. Cerebrolysin (2.5 ml/kg, IV) was given either 30 min before IOMNP injection in the 4 h survival group or 4 h after injury in the 24 h survival groups. In the control group cerebrolysin was administered in an identical situation following IOMNP administration. In all groups, leakage of serum albumin in the CNS as a marker of BBB breakdown and activation of astrocytes using glial fibrillary acidic protein (GFAP) was evaluated by immunohistochemistry. The neuronal injury was examined by Nissl staining. The results indicated that the IOMNPs in low or high doses did not induce albumin or GFAP immunoreactivity or neuronal changes in the CNS following 4 or 24 h after administration. This suggests that IOMNPs were innocuous within the CNS. A focal SCI significantly induced increased albumin and GFAP immunoreactivity in the spinal cord after 4 h and also in some areas of the brain following 24 h after trauma. Abnormal neuronal reactions were observed in SCI in both spinal cord and in the brain at this time. Administration of IOMNPs in SCI group slightly enhanced the pathological changes in the CNS after 24 h but not 4 h after trauma. Cerebrolysin treatment alone did not alter neuronal, glial or BBB function but markedly attenuated NP and/or SCI induced or neuronal and glial cell changes and thwarted albumin leakage indicating protection of the BBB. These observations are the first to show that IOMNPs are safe for the CNS and cerebrolysin treatment prevented CNS pathology following a combination of trauma and IOMNP injection. This indicated that cerebrolysin might be used as adjunct therapy during IOMNP administration in disease conditions.
L. M. Mercier,* N. L. Arias,* E. J. Gonzalez-Rothi,† L. N. Little,* D. D. Fuller,† E. M. Muir,‡ J. H. Rogers,‡ M. B. Bunge,§ M. A. Lane,* and P. J. Reier*
*Department of Neuroscience, College of Medicine, University of Florida, Gainesville, FL, USA
†Department of Physical Therapy, College of Public Health and Health Professions, University of Florida, Gainesville, FL, USA
‡Department of Physiology, Development and Neuroscience, School of the Biological Sciences, University of Cambridge, Cambridge, UK
§The Miami Project to Cure Paralysis, Miller School of Medicine, University of Miami, Miami, FL, USA
Chondroitin sulfate proteoglycans (CSPGs), which are associated with glial scar formation, represent a major impediment to axonal growth and restoration of functional connectivity after spinal cord injury (SCI). Recent evidence suggests that the combination of peripheral nerve [i.e., Schwann cell (SC)] grafts and delivery of exogenous chondroitinase (Ch'ase) can promote improved respiratory outcomes in a high cervical hemisection model (Alilain et al., Nature 2011). However, limited availability of the enzyme and its progressive denaturation at normal body temperature complicate its therapeutic use. To eliminate the need for infusion of the enzyme, SCs were engineered to generate Ch'ase (Kanno et al., SfN, 2012). The present study compared the efficacy of transplanting SCs with SCs generating Ch'ase (Ch'ase-SCs) in a midcervical contusion injury model. Adult female rats received lateralized C3/C4 contusion injuries, which lead to compromised diaphragm function and a rapid shallow breathing pattern (RSB). At 1 week postinjury, animals were separated into three groups and received injections directly into the lesion site of either (1) SCs (~2.5 million) expressing either mCherry or green fluorescent protein (gfp) or (2) mCherry- or gfp- expressing Ch'ase-SCs or (3) were injured but not injected. Three days prior to transplantation and throughout the length of the study animals were immunosuppressed with daily injections of cyclosporine (10 mg/kg s.q.). Respiratory function under eupneic and hypercapnic breathing conditions was assessed by routine plethysmography and terminal diaphragmatic electromy-graphy (EMG) recordings. Histological analyses confirmed survival of SCs throughout the injury site. Our initial findings show that in contrast to injury-only animals, RSB was absent in recipients of SCs. Functional improvement in diaphragmatic activity during respiratory challenge was likewise observed in SC recipients. Ch'ase-SCs did not produce any additional effect on either ventilation or diaphragm activity despite immunostaining that revealed overt CSPG degradation. These preliminary results suggest that SC transplantation alone can promote recovery of diaphragm function following experimental cervical contusion injuries.
This study was supported by University of Florida Opportunity Fund, the Anne and Oscar Lackner Chair in Medicine (P.J.R.), The Brain & Spinal Cord Injury Research Trust (BSCIRT) Development Funds, NIH NS09923 (M.B.B.), NIH NS081112 (M.A.L.).
K. Minor,* K. Jasper,* S. Fulgham,* J. E. Davies,* and S. J. A. Davies*†‡
*Department of Neurosurgery, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, CO, USA
†Department of Neurology, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, CO, USA
‡Gates Center for Regenerative Medicine and Stem Cell Biology, University of Colorado School of Medicine, Anschutz Medical Campus, Aurora, CO, USA
Decorin is a small leucine-rich repeat protein that has been shown to have both anti-inflammatory and antifibrotic properties in many types of tissue and represents a promising agent for promoting recovery of the traumatically injured adult central nervous system (CNS). Previous studies have shown that treatment of brain injuries with decorin can suppress fibrotic scarring (Logan et al., 1999) and the expression of semaphorin 3A, a major inhibitor of axon growth (Minor et al., 2010). Infusion of decorin directly into acute spinal cord injuries has been shown to promote a major suppression of multiple inhibitory chondroitin sulfate proteoglycans (CSPGs) and the growth of adult sensory axons across acute spinal cord injuries (Davies et al., 2004). In addition to suppressing the formation of axon growth inhibitory CNS scar tissue, decorin has been shown to also effectively “desensitize” neurons to the inhibitory effects of both multiple axon growth inhibitory scar-associated CSPGs and myelin associated molecules (Minor et al., 2008). To further explore the therapeutic potential of decorin in treating CNS injuries, we have tested the ability of decorin to promote recovery in a clinically relevant cervical spinal cord contusion model. Unilateral contusion spinal cord injuries were carried out on adult Sprague–Dawley rats at the C4/C5 level of the spinal cord. In two separate experiments, decorin treatment was initiated at either 12 days or 6 months after spinal cord injury. Intrathecal decorin infusion beginning at 12 days after injury promoted robust recovery of locomotor function (Catwalk and Horizontal ladder), accompanied by plasticity of corticospinal tract axons within adjacent spinal gray matter and synaptic plasticity within lamina 8/9 motor neuron pools caudal to sites of injury. Intrathecal decorin infusion at 6 months postinjury promoted significant improvements in multiple measures of fore and hind limb performance, recovery of normal gait patterns and synaptic plasticity within motor neuron pools. Our results demonstrate that intrathecal infusion of decorin can promote functional recovery at both subacute and long-term chronic time points after injury and provide further support for the development of decorin as a therapy for the injured human spinal cord.
Funding support: Hong Kong SCI Fund, Lone Star Paralysis Foundation, Lloyd and Floyd Holman Fund, David Van Wagener SCI Research Fund, members of the SCI community.
D. F Muresanu,* A. Sharma,† R. Patnaik,‡ H. Mössler,§ and H. S. Sharma‡
*Department of Clinical Neurosciences, University Hospital, University of Medicine and Pharmacy, Cluj-Napoca, Romania
†Laboratory of Cerebrovascular Research, Department of Surgical Sciences, Anesthesiology and Intensive Care Medicine, University Hospital, Uppsala University, Uppsala, Sweden
‡School of Biomedical Engineering, Indian Institute of Technology, Banaras Hindu University, Varanasi, India
§Ever NeuroPharma, Oberburgau, Austria
Previous reports from our laboratory showed that diabetes or hypertension aggravates heat stroke-induced brain pathology. However, this is not clear whether a combination of diabetes and hypertension will further enhance brain damage following hyperthermia. In this investigation, possible exacerbation of brain pathology and edema formation in diabetic-hypertensive (DBHY) animals after heat stroke (HS) was examined in a rat model. Since hyperthermia upregulates nitric oxide synthase (NOS) upregulation, the role of nitric oxide (NO) in exacerbation of HS-induced brain pathology was also evaluated using immunoreactivity of neuronal (n) or inducible (i) NOS expression. Hypertensive rats (produced by two-kidney one clip (2K1C) method) were made diabetic with streptozotocine (50 mg/kg IP/day for 3 days) treatment. After 6 weeks, DBHY rats show 20–30 mM/L blood glucose and hypertension (180–200 mmHg). Subjection of these rats to 4 h HS resulted in six- to eightfold higher blood–brain barrier (BBB) breakdown, brain edema formation, and brain pathology. At this time, neuronal or inducible NOS expression was four- to sixfold higher in DBHY rats compared to controls. Interestingly, iNOS expression was higher than nNOS in DBHY rats. In these animals, cerebrolysin in high doses (10 ml/kg IV instead of 5 or 2.5 ml/kg) induced significant neuroprotection and downregulation of nNOS and iNOS in DBHY animals whereas normal animals need only 5 ml/kg doses for this purpose. However, when TiO2 nanowired cerebrolysin was administered (in a dose of 5 ml/kg IV) pronounced neuroprotection and downregulation of iNOS and nNOS was observed in DBHY rats after heat stroke. On the other hand TiO2 nanowired alone did not induce any neuroprotection against heat stroke. This suggests that nanowires themselves do not induce any beneficial or adverse effects on brain pathology in heat stroke. Taken together, our observations demonstrate that comorbidity factors exacerbate brain damage in HS through NOS expression and alter treatment modalities of neuroprotective drugs. Thus, modulation of drug dose or enhanced drug delivery using nanotechnologies is needed to induce sufficient neuroprotection in such situations. The potential use of nanowired cerebrolysin seems to be quite appropriate in heat stroke associated with comorbidity factors, for example, hypertension and diabetes together in clinical situations.
M. Pabón, N. Tajiri, K. Shinozuka, S. Acosta, D. W. Kim, H. Ishikawa, D. G. Hernandez-Ontiveros, J. Vasconcellos, T. Dailey, C. Metcalf, M. Staples, C. Tamboli, Y. Kaneko, and C. V. Borlongan
Center for Excellence in Aging and Brain Repair, Department of Neurosurgery and Brain Repair, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
Traumatic brain injury (TBI) occurs when the head is impacted by an external force causing either a closed or penetrating head injury through a direct or accelerating impact. Approximately 1.7 million people sustain a type of TBI annually, which is a major cause of disability and even death in the US. TBI has become the signature of wars for our soldiers returning from the Middle East. Technology has provided many methods of protection, such as helmets, but morbidity and mortality resulting from TBI remains high. TBI, depending on the severity or type of object, can be focal or widespread. In laboratory research, most of the TBI animal models focus on a specific trajectory affecting specific areas of the brain; but traumatic injuries in patients do not always impact the same brain regions. Indeed, TBI can result from different angles/trajectories of impact, which may alter specific areas of the brain and may lead to varying levels of disabilities. For this reason, we opted to examine the histopathological effects of different angles of mechanical injury by manipulating the trajectory of the controlled cortical impact injury (CCI) model in adult Sprague–Dawley rats. We hypothesized that different types of impact trajectories would produce different levels and extent of brain damage. The following manipulations of the CCI model were performed: conventional rat model of TBI targeting the frontal cortex (AP = 2.0 mm, ML = 2.00, DV = 1.0 mm), farthest right angle targeting the frontal cortex (FRA) (AP = 2.0 mm, ML = 2.0 mm, DV = 1.0 mm), closest right angle targeting the frontal cortex (CRA) (AP = 2.0 mm, ML = 2.0 mm, DV = 1.0 mm), olfactory bulb (OB) injury (AP = 6.2 mm, ML = 1.0 mm, DV = 1.0 mm), and cerebellar injury (CB) (AP = −11.6, ML = 0.2 mm, DV = 1 mm). Animals were euthanized at 3 days after CCI and perfused, and then brains were harvested and processed for immunohistochemical analyses. Our preliminary data confirmed typical cell loss in the M1 cortical region in the conventional TBI group as determined by H&E-positive histological staining and Cavalieri method of unbiased stereology. Interestingly, although we did not detect significant cell death in M1 region of the animals that received OB or CB injury, the FRA and CRA types of injury displayed comparable cortical damage to the conventional TBI group. Further examination of the H&E positive expression in the hippocampus, although not significant, revealed cell loss in the CA3 region for all types of impact trajectories in the ipsilateral side, without any apparent differences across TBI groups. Coincidentally, an increased expression of the inflammatory marker OX-6 in the M1 region of the cortex accompanied the decrease in H&E-positive cells in this cortical region after conventional, FRA and CRA TBI groups, suggesting a critical role for neuroinflammation. Altogether, these results suggest that varying the trajectory target of the CCI may result in different levels and extent of brain damage, but manipulating the angle of impact to the cortical region appears to consistently produce similar core and peri-impact damage to M1 and CA3, respectively, at least for the short 3-day period following our experimental TBI. Additional studies are warranted to assess long-term secondary cell death effects of varying impact trajectories which will guide the management and treatment of TBI patients.
Funding: Department of Defense, USF Neuroscience Collaborative Funds, USF-VA Reintegrations Funds.
S. Palfi,* J.-M. Gurruchaga,* G. S. Ralph,† C. Watts,‡ P. Buttery,‡ S. Scorer,† H. Lepetit,* J. Miskin,† H. Iwamuro,* S. Lavisse,§ A. Kas,§ A. L. Ramelli,* N. Tani,* P. Dolphin,* G. Fenelon,* P. Brugiere,* S. Kingsman,† S. Naylor,† P. Hantraye,§ P. Remy,*§ R. Barker,‡ and K. Mitrophanous†
*Henri Mondor Hospital, Paris University, Paris, France
†Oxford BioMedica (UK) Ltd, Oxford Science Park, Oxford, UK
‡Addenbrooke hospital (UK) Ltd., Cambridge, UK §CEA-CNRS MirCen Fontenay aux Roses, France
Parkinson's disease (PD) is a neurodegenerative disease that results in a progressive degeneration of dopaminergic neurons. The dopamine precursor l-Dopa and dopamine agonists provide the primary standard of care and demonstrate good therapeutic benefit in the early stages of disease. However, their long-term use is associated with severe motor side effects that are at least partially caused by the fluctuating nature of dopaminergic stimulation that arises from oral drug administration. As such, a therapy that provides a more continuous and local supply of dopamine to the site of pathology provides a potential approach for the development of new therapeutic strategies. ProSavin® is a gene therapy product that utilizes a lentiviral vector to transfer three genes that are critical for dopamine biosynthesis to the the striatum, that is depleted of dopamine in PD. Clinical evaluation of the safety and efficacy of ProSavin in mid to late stage PD patients is currently ongoing. In the study 15 patients have received ProSavin® in three dose cohorts. ProSavin® has been demonstrated to be safe and well tolerated at all doses evaluated to date. There have been no serious adverse events related to Prosavin® or the administration procedure and no inflammatory responses. In terms of efficacy, an improvement in the primary endpoint, UPDRS Part III, has been observed in all cohorts relative to their baseline scores. Furthermore, an improvement has been maintained out to the latest timepoints evaluated to date (up to 4 years for the earliest cohort). Secondary endpoints including the patients concommitant dopaminergic medications have also demonstrated improvements.
K. Parsha, B. Yang, O. Mir, K. Schaar, P. J. Hanley, X. Xi, and S. I. Savitz
University of Texas at Houston, Houston, TX, USA
Human bone marrow mesenchymal stem cells (hMSCs) are being widely investigated as a therapeutic approach for ischemic stroke. However, most studies have used MSCs expanded in media containing animal serum, which hinders their use in the clinical setting. Recently, studies have shown that hMSCs grown in human platelet lysate (HPL) media maintain their phenotypic and in vitro properties. With a view to translate this therapy into clinical trials, we used clinically viable hMSCs and their efficacy and mechanisms of action were tested in an aged rodent stroke model. Human bone marrow from a healthy donor was harvested and expanded in HPL media up to P4 in a current good manufacturing practice (cGMP) facility. Retired breeder male Long-Evan rats (612 ± 56 g) were subjected to transient middle cerebral artery occlusion (MCAo) for 75 min. On day 7 after stroke, the animals were randomly divided into two groups and treated with hMSCs or vehicle (1×106 per animal, n = 12/ group). All animals were serially evaluated on circling and corner tests up to 28 days. Brain lesion sizes were measured 28 days after stroke. In a separate experiment, the spleen and brains were removed on day 7 from stroked or sham animals (n = 3/group). Splenocytes and microglia from these animals were isolated and cocultured, respectively, with vehicle or hMSCs for 48 h, following which, assessment of their phenotype and cytokine release profile was performed. hMSCs significantly improved functional recovery compared with saline on day 28 on both neurological tests. There was no significant difference in infarct size. Coculture with hMSCs significantly reduced microglial induced nitric oxide synthase (iNOS) expression and reduced release of interleukin-6 (IL-6) from microglia. hMSCs also significantly increased transforming growth factor (TGF)-β expression in splenocytes but had no effect on their interferon (IFN)-γ expression. MSCs expanded with HPL improved long-term neurological deficits in aged animals when administered 7 days after stroke. The possible mechanisms may be related to a reduction in proinflammatory microglia and improvement in the Th2 type response in the spleen. These mechanisms are being investigated further in vivo.
K. L Paumier,* S. J. Sukoff Rizzo,* Z. Berger,* Y. Chen,* C. Gonzales,* E. Kaftan,*† L. Li,* S. Lotarski,* W. Shen,* P. Stolyar,* D. Vasilyev,*† M. Zaleska,* J. Dunlop,*‡ and W. D. Hirst*
*Neuroscience Research Unit, Pfizer Global Research and Development, Cambridge, MA, USA
†Current address: Department of Pharmacology, Yale University, New Haven, CT, USA
‡Current address: AstraZeneca, Cambridge, MA, USA
Parkinson's disease (PD) pathology is characterized by the formation of intraneuronal inclusions called Lewy bodies, which are comprised of α-synuclein (α-syn). Duplication, triplication or genetic mutations in α-syn (A53T, A30P, and E46K) are linked to autosomal dominant PD, thus implicating its role in the pathogenesis of PD. Furthermore, preclinical studies indicate that overexpression of wild-type α-syn impairs macroautophagy and the A53T mutation impairs chaperone-mediated autophagy. Therefore, we hypothesize that promoting autophagic clearance of α-syn will reduce PD-related pathology and improve PD-related motor and cognitive impairments. We utilized the A53T transgenic mouse that expresses the human A53T α-syn variant (full-length, 140 amino acid isoform) directed by the mouse prion promoter. This model has been extensively studied in the context of α-syn aggregation and toxicity; however, the temporal course of phenotypic deficits has not been thoroughly characterized. Here we performed a systematic evaluation, from behavior to synaptic indices, of the temporal progression of neuronal dysfunction in the A53T mouse. We examined spontaneous locomotor activity and thigmotaxis in an open field, stress induced thermoregulation (SIH), gait abnormalities, rotarod performance, nesting behavior, acoustic startle response and Y-maze in homozygous and wild-type littermates at 2, 6, and 12 months. Additionally, we evaluated the impact of A53T α-syn on hippocampal synaptic physiology in young (1–2 months) mice, which has not been previously characterized in this model. Our findings demonstrate that the A53T mice develop early- and late-onset behavioral and synaptic impairments similar to PD patients; making the A53T model a useful tool with tangible biochemical and behavioral endpoints for testing therapeutics relevant to human disease. To this end, we utilized the autophagy enhancer, trehalose, to examine whether chronic autophagy induction can alter disease progression in this model. Contrary to our hypothesis, trehalose did not attenuate motor or sensorimotor deficits in A53T mice, nor did it delay the onset of disease compared to water or sucrose treatment. Furthermore, biochemical analyses indicate both trehalose and sucrose treated animals show increased levels of microtubule-associated protein 1 light chain 3α-phosphatidylethanolamine conjugate (LC3-II), sequestosome 1 (p62), and insoluble α-syn in brain, suggesting autophagy blockade. However, mass spectrometry and radiolabeled studies indicate trehalose does not get into the brain, which suggests chronic trehalose treatment cannot directly impact α-syn pathology within the CNS; yet it appears to block autophagy and increase insoluble α-syn in the A53T mouse brain. Further studies are required to elucidate the indirect or peripheral effects of chronic trehalose in the A53T mouse model of PD.
C. Perez,* S. Jergova,* S. Gajavelli,* L. Imperial,† B. Olivera,† and J. Sagen*
*Miami Project, University of Miami, Miami, FL, USA
†University of Utah, Salt Lake City, UT, USA
With the limitations of current long-term pharmacotherapy for chronic pain, the identification of alternative approaches and new therapeutic targets is essential. Cannabinoid (CB) receptor agonists have emerged as potential therapeutic targets, as robust antinociceptive effects have been reported in various pain models. However, most of the available drugs that interact with CB receptors are derived from cannabis and considered clinically unacceptable for long-term therapy due to psychoactive side effects. Therefore, specific compounds interacting with CB receptors without aversive side effects are of clinical interest. The venoms of marine snail genus Conus are a natural source of various peptides (conopeptides) with potent analgesic effects, some of them already FDA approved. In this study, we evaluated the ability of several Conus venom extracts to interact with CB1 receptor. The venom extracts of six Conus species were analyzed in vitro. HEK293 cells expressing CB1 receptors were treated with venom extracts for 30 min, and the rate of CB1 receptor internalization was analyzed by immunofluorescence. Results showed the highest rate of CB1 internalization in HEK293 cells after treatment with venoms of C. textile and C. miles. HPLC fractions (seven per species) of these venoms were subsequently analyzed using a similar approach. Based on the analysis, C. tex fraction 5 and C. mil fraction 4 with the highest CB1 agonist activity was evaluated in a formalin test. IT injection of the C. tex and C. mil fractions reduced flinching/licking behavior during the second phase of formalin test. The results indicate the presence of CB1 agonists within the Conus venom extract and their potential analgesic effects. To identify the nature of CB1 agonists, the C. tex and C. mil fractions was treated with proteolytic enzymes. The effect of treatment on CB1 internalization was subsequently evaluated. Results showed that proteotolytic treatment attenuated the ability of venom fraction to induce CB1 receptor internalization. Currently, subfractions of C. tex and C. mil are being analyzed to find the most CB1 effective fraction of the venom. The final peptide compounds of screened venoms with CB1 receptor affinity could subsequently be used as a new analgesic agent in recombinant cell or gene therapy approaches.
This study was supported by NS51667.
S. M. Portis, J. Tian, J. Ehrhart, H. Hou, A. R. Bailey, D. Obregon, and J. Tan
Rashid Laboratory for Developmental Neurobiology, Silver Child Development Center, Department of Psychiatry and Behavioral Neurosciences, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
Fragile X Syndrome (FXS) is the most common form of genetic intellectual disability and is an autism spectrum disorder (ASD). Glycogen synthase kinase-3 (GSK-3) is a ubiquitous kinase that has been found to be overactive in murine models of FXS [fragile X mental retardation 1 (fmr1) knockout mice]. Recent studies have utilized lithium as a GSK-3 inhibitor in these animal models. The studies revealed that lithium administration induced the inhibitory phosphorylation of GSK-3α and -β isoforms at serine residues Ser-21 and Ser-9, respectively. This inhibitory phosphorylation led to improved performance on tests of cognition and social interaction as well as amelioration of brain pathology associated with FXS. Lithium is currently being tested as a therapeutic drug for FXS in a clinical trial. Though lithium is an effective GSK3 inhibitor, the drug is known to have a deleterious effect on hepatic function. Thus, long-term lithium administration could be a highly toxic and risky therapeutic option, particularly for small children. The current study found that the bioflavonoid luteolin also decreases GSK3 activity by directly binding the active sites on GSK3-α and GSK3-β at Tyr-216 and Tyr-279 in mouse embryonic fibroblast (MEF) cells derived from fmr1 KO mice. In addition, luteolin treatment increased inhibitory serine phosphorylation of both isoforms in MEF cells. Use of a bioflavonoid as a GSK3 inhibitor may prove a safer, more effective therapy for FXS.
N. Porz, S. Seiler, H. R. Widmer, and S. Di Santo
Department of Neurosurgery, University of Berne, Inselspital, Berne, Switzerland
Current strategies for neuronal replacement therapy in neurodegenerative diseases include cell transplantation and interventions aimed to enhance endogenous neurogenesis. In line with these notions, there is increasing evidence for a regenerative potential of soluble factors released from stem and progenitor cells. We have previously shown that endothelial progenitor cell-derived factors (EPC-CM) promoted angiogenesis and enhanced the activity of cultured endothelial cells. In the present study, we investigated the effects of EPC-CM on the neuronal stem cell niche in the subventricular zone. EPCs were isolated from the peripheral blood of healthy human donors by gradient centrifugation. Cells were cultured under hypoxic conditions (1.5% O2) for 2 days to enhance the secretion of growth factors. EPC-CM was infused by means of mini osmotic pumps implanted into the right lateral ventricle of anesthetized adult Sprague–Dawley rats. Basal cell culture medium was used as control. The infusion rate was 0.5 ml/h, and the cannula was left in place for 7 days. Animals were injected daily with the proliferation marker bromodeoxyuridine (BrdU). At the end of the experimental period, the rats were perfusion fixed using 4% paraformaldehyde (PFA) and the brains were sectioned on a cryostat. Brain slices were immunostained for BrdU and markers of neuronal progenitor cells. Intraventricular infusion of EPC-CM was observed to significantly increase the number of BrdU-positive cells in the subventricular zone compared to controls. Furthermore, we could demonstrate that the number of doublecortin, KI-67, and vimentin-positive cells were significantly higher in the EPC-CM-treated group as compared to controls. Taken together, our findings demonstrate that EPC-CM administration resulted in enhanced cell proliferation and promoted endogenous neurogenesis. These observations indicate that EPC-CM may be offering a new therapeutic strategy to induce neuroregeneration.
B. Priddy, S. Jergova, S. Gajavelli, and J. Sagen
Miami Project to Cure Paralysis, University of Miami, Miami, FL, USA
Current treatments for chronic neuropathic pain are limited and usually not very efficacious. Most treatments are limited to opioids and nonsteroidal anti-inflammatory drugs. Unfortunately, their regular usage is limited by both central nervous system and autonomic-mediated adverse side-effects; these problems combined with the low efficacy of pharmacotherapies implies the need for alternatives and new therapeutic targets. Researchers have implicated voltage gated ion channels and voltage gated calcium channels as contributing mediators of neuropathic pain. This study focused on a novel sodium channel (Nav) antagonist, μ-conotoxin SIIIA peptide from the marine snail Conus striatus. SIIIA complimentary DNA was cloned and amplified, a secretion signal sequence was added (ssPAM), and then SIIIA was ligated to adeno-associated viral vector 2/8 hybrid serotype (AVV-2/8). ssPAM-SIIIA in AAV-2/8 was then sequenced and the sequence matched that of the expected theoretical sequence. Ten Sprague–Dawley male rats were prepared by performing intrathecal catheter surgery to place a catheter into the intrathecal space of the spinal cord for direct drug delivery. Additionally, 15 Sprague–Dawley male rats were prepared by performing direct intradorsal root ganglion (DRG) injections of either saline (control) or AAV-2/8 ssPAM-SIIIA viral vector (treatment). Human embryonic kidney cells were also transduced with this viral vector, and the supernatant was collected several times in phosphate buffer solution. The group of 10 rats with catheters was injected with the collected SIIIA supernatant, and all rats were challenged with 5% formalin in their right hindpaw. SIIIA supernatant and AAV injection treatment groups significantly reduced flinching responses compared against their respective control groups. The rats with the intra-DRG injections showed a higher number of time intervals with reduced pain behaviors compared with the supernatant groups. Direct DRG injection of the viral vector led to reduced flinch and pain-like responses at multiple intervals implying an analgesic effect. If SIIIA is a specific antagonist of Nav1.8 channels, then pure SIIIA could serve as an efficacious analgesic with minimal side effects.
This study was supported by Buoniconti Fund and NIH grant NS72769 and Craig H. Neilsen Foundation 190848.
A. Roholt,* G. Kidd,*† E. Benson,* S. Medicetty,* B. Bai,* and B. Trapp*†
*Renovo Neural, Inc., Cleveland, OH, USA
†Cleveland Clinic, Cleveland, OH, USA
Remyelination is a process of generating new myelin, which is an actively pursued therapeutic objective of treating demyelinating diseases such as multiple sclerosis (MS). Existing methods for assessing the extent of myelin loss and recovery offer limited information. Techniques such as transmission electron microscopy (TEM) have the spatial resolution necessary to measure axon diameter, myelin fiber thickness, and the ratio of these (g-ratio), but rarely provide myelin internodal length measurements. Although confocal microscopy is capable of measuring internodal length, it lacks the resolution to discriminate between densely packed myelinated fibers in CNS tissues. Serial block face scanning electron microscopy (SBF-SEM) has the spatial resolution necessary to measure myelin and axons but additionally provides a wide field of view (0.5 mm or more) and serial images that allow tracing of individual axons in 3D and evaluation of their internodal length metrics. In this study, we evaluated the extent of remylination in mouse corpus callosum at different time points after demyelination and compared the results to traditional single section g-ratio measurements. Mice were subjected to a 12-week course of cuprizone chow and daily rapamycin injections in order to induce and maintain demyelination. They were subsequently allowed to recover for a period of either 0, 3, or 6 weeks. Traditional g-ratios were similar between TEM and SBF-SEM groups and did not discriminate well between recovery time points. Myelin thickness and internodal lengths obtained by SBF-SEM showed significant differences between groups and provided complementary data. In addition, internodal length measurement identified the few myelin internodes that had resisted demyelination. We conclude that SBF-SEM approach provides sensitive new metrics to assess therapeutics that model demyelination and remyelination.
A. Rombola,* E. J. Gonzalez-Rothi,†‡ L. Fernandez,‡ L. M. Mercier,* C. A. Rousseau,* B. E. O'Steen,* P. J. Reier,* D. D. Fuller,†‡ and M. A. Lane*
*Department of Neuroscience, McKnight Brain Institute, University of Florida, Gainesville, FL, USA
†College of Public Health and Health Professions Graduate Program in Rehabilitation Science, University of Florida, Gainesville, FL, USA
‡Department of Physical Therapy, University of Florida, Gainesville, FL, USA
Injury to the cervical spinal cord (cSCI) can lead to devastating consequences, including significant impairments in upper extremity function. Although some capacity for spontaneous functional improvement has been demonstrated, the extent of recovery is limited and the underlying mechanisms of this plasticity are ill-defined. Using a well-established rodent model of cSCI, lateral spinal cord hemisection at C2 (C2Hx), the present works examines the neuroanatomical substrates underlying upper extremity dysfunction and recovery that is observed following incomplete cSCI. Gross forelimb motor function was assessed prior to and at 2, 4, 6, 8, 12, and 16 weeks postinjury using the limb use asymmetry (cylinder) test. Forelimb locomotor function was also assessed at similar time points using the Forelimb Locomotor Assessment Scale (FLS). The neuroanatomical circuitry of the extensor carpi radialis muscle, a distal forelimb muscle, was assessed using retrograde transneuronal tracing techniques. Significant deficits in forelimb motor function were observed at early time points postinjury. Moderate functional recovery occurred over the first 6–8 weeks postinjury, reaching an apparent plateau by 10 weeks postinjury. Initial tracing studies revealed the location and distribution of forelimb motoneurons and interneurons in the cervical spinal cord as well as labeling in the brainstem and motor cortex in uninjured animals. Ongoing studies are exploring changes in the forelimb neuroanatomical circuitry that occur following C2Hx injury. These experiments are the first to examine the neuroanatomical substrates underlying upper limb neuroplasticity and functional recovery following cSCI. We predict that a better definition of these substrates will enable identification of potential therapeutic targets.
T. Rossetti,* F. Nicholls,*† and M. Modo*
*University of Pittsburgh, Departments of Radiology and Bioengineering, McGowan Institute for Regenerative Medicine, Pittsburgh, PA, USA
†King's College London, Institute of Psychiatry, London, UK
Cell transplantation as a treatment for neurodegenerative diseases is an engaging field, which is potentially widely applicable. However, the number of cells surviving after implantation is rather low compared to the number of cells injected. Although a significant effort has investigated the apoptosis of cells after implantation, very little optimization of cell preparation has so far been undertaken. Moreover, there is a general neglect of the biophysical aspects of cell injection. Cell transplantation can only be an efficient therapeutic approach if we can ensure an optimal transfer of cells from the dish to brain. We therefore here focused on the in vitro aspects of cell preparation for intracerebral cell implantation. To ensure the clinical relevance of our study, we evaluated the clinical grade human foetal STR0C05 cell line (derived from a 10-week-old ganglionic eminence) suspended in five different vehicles [PBS, Dulbecco's modified Eagle's medium: N-acetylcysteine (DMEM:NAC), artificial cerebrospinal fluid (CSF), Hypothermosol and Pluronic F68] at a concentration of 50,000 cells/μl. Viability of cells was measured over 8 h at different temperatures (0–2°, 20–22°, and 37°). Room temperature (20–22°) consistently produced good viability for all vehicles. Hypothermosol always yielded good survival at all temperatures, whereas the other vehicles saw a gradual and dramatic decrease in viability on ice (0–2°) as well as physiological temperature (37°). Resuspension of the cells by means of pipetting (1, 3, 5, or 10 times) once per hour indicated that three times pipetting preserved a good viability, whereas 5 and 10 times decreased viability and pipetting only once increased the number of large cell clumps (>5 cells aggregates). Preparation of cell suspension volumes <50 ml did not yield a constant suspension of cells for injection, whereas 100 ml provided a consistent cell number. The volume fraction of different cell concentrations was further determined and we established the effect of cell concentration on viability over time, as well as the interaction between cell concentration and vehicle on sedimentation while cells are prepared for injection. The optimal cell preparation and injection will be a combination of outcomes from all these measures and will allow us to investigate how these factors influence cell survival after intracerebral implantation.
R. Rumpel,* M. Alam,† A. Klein,* M. Oezer,*‡ M. Wesemann,* J. K. Krauss,†‡ K. Schwabe,†‡ A. Ratzka,* and C. Grothe*‡
*Institute of Neuroanatomy, Hannover Medical School, Hannover, Germany
†Department of Neurosurgery, Hannover Medical School, Hannover, Germany
‡Centre for Systems Neuroscience (ZSN), Hannover Medical School, Hannover, Germany
In Parkinson's disease (PD), as well as in 6-hydroxydopamine (6-OHDA)-lesioned rats, dopamine (DA) depletion in the nigrostriatal system leads to basal ganglia dysfunction with neuronal hyperactivity in the subthalamic nucleus (STN), that is, enhanced firing rate and burst activity, together with enhanced β oscillatory activity. Intrastriatal transplantation of DA neurons has been shown to functionally reinnervate the host striatum and restore DA input. To better understand the effect of intrastriatal transplantation of DA cells on the STN, we combined behavioral and histological findings with electrophysiological extracellular recordings, as well as qRT-PCR analyses of λ-aminobutyric acid (GABA)ergic and glutamatergic transporter and receptor genes. In animals that were transplanted with cells derived from the ventral mesencephalon of E12 rat embryos, the rotational behavior after amphetamine injection was improved or even overcompensated by 116% in rats with large grafts (2,000–6,000 cells). While burst activity was not affected, STN neuronal firing rate, as well as β oscillatory activity was fully normalized in rats with these grafts, while small grafts were less effective. Although grafted rats displayed restored expression of the GABA synthesizing enzymes glutamate decarboxylase 65 and 67 (Gad65 and Gad67) in the striatum compared to naive rats, the grafts induced a decrease in N-methyl-d-aspartate (NMDA) receptor subunit expression. Interestingly, the NMDA receptor subunit 2B was also less expressed in the STN, both compared to 6-OHDA-lesioned and naive rats. In summary, DA grafts restore functional deficits and partially neuronal activity of the STN in PD rats. Incomplete recovery, however, may be due to changes in receptor gene expression induced by DA grafts.
K. L. Schaar, B. Yang, and S. I. Savitz
Department of Neurology, University of Texas Medical School, Houston, TX, USA
Environmental experience during stroke recovery can markedly affect brain function. To better simulate the clinical situation of testing new therapies to promote stroke recovery, we aimed to generate a more clinically relevant paradigm in a rodent stroke model by applying a physical therapy (PT) program that is well-established in the literature for rodents. Thirty Long-Evans male rats were subjected to middle cerebral artery (MCA) suture occlusion for 90 min. Day 5 poststroke, animals remained in standard cages (single housed, n = 15) or were moved to enrichment cages (five per cage, n = 15). Enrichment rats also underwent 3-h voluntary wheel running sessions twice a week, beginning 5 days after stroke. Behavioral evaluations were performed preoperatively and on weeks 1, 3, and 5 after stroke by measuring food handling behaviors on the vermicelli pasta test and reaching ability on the staircase test. At week 3, standard housing rats took significantly less time to consume pasta when compared to enrichment rats (p = 0.029). There was a trend suggesting improvement on the lowest step reached with the impaired limb in the staircase test, in the enrichment group when compared to the standard group. There were no significant differences between the groups in the number of pellets retrieved in the staircase or number of adjustments made when eating pasta. Standard PT programs in rodent models, consisting of social housing and wheel running, were actually not effective at promoting recovery and might have even adversely affected fine digit motor and grasping recovery following stroke. Wheel running might have had a positive effect on limb extension, as observed in the staircase test. These results have important implications for the design of future rehabilitation paradigms for rodent stroke. In designing future PT programs in rodent stroke, it may be more important to investigate the effects of modality-specific components of therapy such as daily skilled reach training and increasing the frequency and intensity of rehabilitation. Tasks that require complex digit movements such as grasping and food manipulation may need other modality-specific PT. Testing novel restorative therapies should take into account rehabilitation paradigms that are appropriate for specific deficits.
M. Shahaduzzaman,* C. Hudson,† X. Lin,‡ G. Bai,‡ H. Liu,‡ C. Cao,‡§ and P. C. Bickford*†
*Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
†James A. Haley Veterans Administration Hospital, Tampa, FL, USA
‡USFHealth Byrd Alzheimer's Institute, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
§Department of Pharmaceutical Sciences, College of Pharmacy, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
Abnormal accumulation of α-synuclein (α-syn) plays a central role in the pathogenesis of Parkinson's disease (PD) and thus has become a valuable target for developing novel therapeutics for PD. Several approaches have been investigated to inhibit aggregation and enhance clearance of α-syn. Although immunotherapy shows promise, very few have been developed for preclinical testing. In this study, we have investigated whether injecting with novel anti-α-syn antibodies (A1, A2) halts PD progression and improves behavioral outcomes. We injected adeno-associated virus 9 (AAV9)-α-syn into the substantia nigra of Fisher344 rats as a PD model. Rats were randomly assigned into four groups: AAV-α-Syn-IgG (antibody control), AAV-α-Syn-Ab1 (treatment 1), AAV-α-Syn-Ab2 (treatment 2), and AAV-green fluorescent protein (GFP) only. Beginning 1 week after AAV9-α-syn injection rats were treated IP with either IgG, A1, or A2 at 1 mg/rat in 200 μl 1 × PBS and then boosted every 3 weeks for 3 months. Blood sam-ples were drawn to check the injected antibody level. The cylinder test was performed monthly to assess behavioral deficits. Beginning at 2 months following AAV9-α-syn-IgG rats demonstrated paw bias in the cylinder test when compared with AAV-GFP controls. Although there was no significant difference between A1-or A2-treated rats compared with AAV-GFP, there was also no difference from AAV9-α-syn-IgG; thus, there was intermediate improvement in paw bias with the cylinder test. Furthermore, we have observed that anti-α-syn antibodies (A1, A2) confer neuroprotection from α-syn as unbiased stereological estimation revealed a significantly higher number of tyrosine hydroxylase positive (TH+) cells in the substantia nigra compacta (SNc) (p = 0.004) in the brains from rats treated with anti-α-syn A1 and A2 antibody compared tononimmune IgG. Our results suggested that anti-α-syn antibodies (A1, A2) could be potential immunotherapeutic against α-synucleinopathies.
This work was supported by the Veterans Administration Medical Research Service, Merit award to P.C.B.
A. Sharma,* D. F. Muresanu,† R. Patnaik,‡ and H. S. Sharma*
*Laboratory of Cerebrovascular Research, Department of Surgical Sciences, Anesthesiology and Intensive Care Medicine, University Hospital, Uppsala University, Uppsala, Sweden
†Department of Clinical Neurosciences, University Hospital, University of Medicine and Pharmacy, Cluj-Napoca, Romania
‡School of Biomedical Engineering, Indian Institute of Technology, Banaras Hindu University, Varanasi, India
Military personnel are often subject to sleep deprivation (SD) during combat operations or peacekeeping missions abroad. Frequent SD in these military personals may affect their brain function and reduce their ability to perform at optimal levels. Whether disturbances in brain function following SD may further be affected by additional exposure to nanoparticles (NPs) from the environment or work related exposure is not known. In the present investigation we examined whether SD could affect blood–brain barrier (BBB) breakdown and alter cognitive and sensory motor function in a rat model following chronic intoxication of engineered metal NPs, for example, Cu and Ag of two different sizes (20–30 or 50–60 nm). SD was induced in rats (age 12–14 weeks) using an inverted flowerpot model (ca. 7 cm diameter) placed in a water pool where the water levels are just 3 cm below the surface. Animals can go to sleep for brief periods but could not achieve deep sleep, as they would fall into the water disrupting their sleep. Leakage of Evans blue is seen in the cerebellum, hippocampus, caudate nucleus, parietal, temporal, occipital and cingulate cerebral cortices, and brain stem following 12–48 h after SD in normal rats. The ventricular walls of the lateral and fourth ventricles were also stained blue. Abnormal behavior on Rota Rod, inclined plane angle test as well as walking on a wired mesh was also seen in these animals. The breakdown of the BBB and blood–cerebrospinal fluid (CSF) barrier (BCSFB) and behavioral disturbances were progressive in nature from 12 to 48 h. However, when Cu or Ag NP-treated rats (50 mg/kg IP daily for 7 days) were subjected to identical SD, profound exacerbation of BBB disruption to Evans blue was seen in identical brain regions. The behavioral dysfunction in the NP-intoxicated animals after SD was also exacerbated compared to normal animals. Interestingly, small sized NPs induced most pronounced effects on BBB disruption and behavioral dysfunction compared to their larger counterpart. The Ag NPs irrespective of their sizes exerted the most pronounced effects on BBB and BCSFB disruption compared to Cu NPs of similar sizes. This indicates that small sized NPs could induce greater devastating effects on brain and behavioral dysfunctions in SD.
H. S. Sharma,* D. F. Muresanu,† R. Patnaik,‡ and A. Sharma*
*Laboratory of Cerebrovascular Research, Department of Surgical Sciences, Anesthesiology and Intensive Care Medicine, University Hospital, Uppsala University, Uppsala, Sweden
†Department of Clinical Neurosciences, University Hospital, University of Medicine and Pharmacy, Cluj-Napoca, Romania
‡School of Biomedical Engineering, Indian Institute of Technology, Banaras Hindu University, Varanasi, India
Concussive brain injury (CBI) as a result of roadside blast or missile explosion is quite common in military personnel engaged in combat operations across the World. CBI is often associated with mild to moderate brain dysfunction and alterations in cognitive and sensory motor functions. However, these CBI symptoms could be further altered by acute or chronic exposure to various nanoparticles present in the environment or due to gunpowder explosion. In the present investigation effects of SiO2 and carbon nanoparticles (CNPs) on CBI induced pathophysiology was examined in a rat model. The CBI was produced in Equithesin anesthetized (3 ml/kg IP) rats by inflicting a blunt head injury on right parietal skull bone using a 114.6 g of cylindrical iron rod tapering towards one end (2 mm2) not pointed enough to pierce the skull from a height of 20 cm. This weight and distance will result in an impact injury of 0.224 N on the right parietal skull surface without breaking it. In a separate group of rats SiO2 (50–60 nm) or CNPs (45–50 nm) were administered daily in a suspension of Tween 80 at a dose of 50 mg/ kg (IP) for 7 days. In these nanoparticle-treated animals, CBI was inflicted on the 8th day. CBI in normal animals resulted in a marked increase in blood–brain barrier (BBB) disruption and brain edema formation that was most marked in the contralateral left half as compared to the injured ipsilateral side. Interestingly SiO2 treatment resulted in the most marked aggravation of BBB leakage and brain edema compared to CNPs intoxication. However, in nanoparticle-treated injured rats, the left hemisphere was also more adversely affected than the right injured hemisphere. These pathological changes increased over time. Thus, pronounced changes were seen after 8 h CBI and increased further at 24 h after CBI. CBI in normal animals did not result in any marked changes in cognitive and sensory motor disturbances up to 5 h. However, behavioral dysfunctions were prominent after 8 and 24 h CBI in a progressive manner. On the other hand nanoparticle-treated rats showed profound behavioral changes at 5 h after CBI that worsen further with the advancement of time. Taken together, our observations show that nanoparticle intoxication exacerbates CBI-induced brain pathology and behavioral disturbances. These pathophysiological changes following CBI largely depend on the nature of nanoparticles intoxication and duration of trauma.
K. Shinozuka, N. Tajiri, H. Ishikawa, Y. Kaneko, and C. V. Borlongan
Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, Morsani College of Medicine, University of South Florida, Tampa FL, USA
Stress is a major exacerbating factor for stroke, including hyperglycemia in response to the secretion of stress hormones (O'Neill et al., 1991). The present study investigated social modulatory interactions between stroke, stress, and expression of advanced glycation end products (AGE)-related proteins in the thymus, an important organ of the immune system (i.e., T-lymphocyte maturation). Social conditions in the housing colony of paired rats were manipulated as follows: Stroke with Stroke (SS), Stroke with Naïve (SN), Naïve with Stroke (NS), or Naïve with Naïve (NN). Stroke rats received middle cerebral artery occlusion (MCAo). For behavioral analysis, the subjects were videotaped for 15 min at 1 day before and 0, 1, and 3 days after MCAo and then sacrificed. General activity and pain perception in accordance with the Rat Grimace Scale (Sotocinal et al., 2011) were analyzed using digital video. Concentrations of plasma corticosterone and methylglyoxal (MG) were analyzed with ELISA kits. Expressions of AGE-related proteins in the thymus were analyzed by Western blotting. Following MCAo, SS, SN, and NS subjects displayed significantly lower activity. Higher pain scores were observed in SS and SN. Concentration of plasma corticosterone was significantly upregulated only in SS. MG concentration was significantly elevated in SS and SN. Interestingly, changes in expressions of AGE-related proteins were shown to be modulated by social conditions. We demonstrated that some of the MCAo-induced pathophysiological changes were affected by social conditions, in that stroke was accompanied by social inhibition of general activity and social facilitation of plasma corticosterone. We also demonstrated that MCAo altered expression of AGE-related proteins in the thymus. These results suggest that stroke affects immune function via activation of AGE-related cascades in the thymus, and such molecular and cellular alterations could be modulated by appropriate settings of social environments.
A. J. Smith,*† P. Kavuru,‡ K. K. Arora,‡ S. Kesani,‡ J. Tan,† M. J. Zaworotko,‡ and R. D. Shytle*†
*Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
†Neuroimmunology Laboratory, Silver Child Development Center, Department of Psychiatry and Neurosciences, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
‡Department of Chemistry, College of Arts and Sciences, University of South Florida, Tampa, FL, USA
The most abundant polyphenol in green tea, epigallocatechin-3-gallate (EGCg), has recently received considerable attention due to the discovery of numerous health-promoting bioactivities. Despite reports of its poor oral bioavailability, EGCg has been included in many dietary supplement formulations. Conventional preformulation methods have been employed to improve the bioavailability of EGCg. However, these methods have limitations that hinder the development of EGCg as an effective therapeutic agent. In this study, we evaluated the efficacy of crystal engineering for modulating the pharmacokinetics of EGCg. We synthesized and characterized seven previously undescribed crystal forms of EGCg. The aqueous solubility profiles of four new EGCg cocrystals were determined. These cocrystals were subsequently dosed at 100 mg EGCg per kg body weight in rats and the plasma levels were monitored over the course of 8 h following the single oral dose. Our findings suggest that modulation of the pharmacokinetic profile of EGCg is possible using cocrystallization and that it offers certain opportunities that could be useful during its development as a therapeutic agent.
G. A. Smith, E. N. Mangano, M. Hayes, J. Beagan, T. Brand, S. C. Izen, T. B. Brown, O. Isacson, and P. J. Hallett
Neuroregeneration Research Institute, Harvard Medical School/McLean Hospital, Belmont, MA, USA
Regulation of synaptic function, vesicular transport, and organelle dynamics is fundamental to the normal function of the neuron. These processes are dysregulated in many neurodegenerative diseases prior to cell death, therefore providing a target for therapeutic intervention at early stages. Synaptic dysfunction and disturbances in protein transport and energy homeostasis have been reported in Huntington's disease (HD) patients and in experimental models with a CAG expansion in the huntingtin gene. These changes are modulated by specific direct and indirect protein interactions with huntingtin and the cytoskeleton, synapses, and organelles, leading to both gain and loss of function. We have previously shown that delivery of Rab3b (a member of the RAS oncogene family) by gene therapy can improve neurotransmitter handling and storage capacity at presynaptic terminals and prevent synaptic neurodegenerative changes in the dopaminergic system. We hypothesize that modulation of early pathophysiological changes by Rab proteins can enhance neuronal function in experimental models of HD. We first examined motor and cognitive functional deficits, biochemical changes, inflammation, inclusion formation, and the synaptic and axonal transport dyregulation of 22 proteins in a novel Q175 knock in (Q175KI) mouse model of HD at 1, 6, 12, and 16 months of age. Q175KI mice exhibited motor and cognitive deficits at 12 months, which were paralleled to striatal atrophy and microglial activation. Intracellular inclusions of mutant huntingtin were present from 6 months of age. Striatal λ-aminobutyric acid (GABA) levels were increased at 1 and 6 months, and a reduction in striatal dopamine was observed from 6 to 16 months. Proteins involved in synaptic and transport functions were differently dysregulated in the striatum, motor cortex, prefrontal cortex, and hippocampus regions at 12 months. Rab3b and Rab1a protein levels are decreased in cortical regions. We hypothesize that the overexpression of Rab1a and Rab3b by adeno-associated virus 2/5 (AAV2/5) vectors will improve secretory transport and synaptic functions of the corticostriatal pathway, thereby preventing key predegenerative changes associated with HD.
Funding: CHDI Foundation.
V. M. Spruance, D. E. Sanchez, E. J. Gonzalez-Rothi, G. B. Grossl, B. E. O'Steen, D. D. Fuller, P. J. Reier, and M. A. Lane
Department of Neuroscience, College of Medicine, University of Florida McKnight Brain Institute, Gainesville, FL, USA
Midcervical spinal cord injuries (SCI) result in devastating and life-threatening respiratory deficits. This is due in part to the disruption of the phrenic motor system, which controls the primary inspiratory muscle—the diaphragm. It is known that spontaneous functional recovery of the diaphragm can occur following injury, though the extent of recovery is limited. Given that impaired breathing ranks as one of the leading causes of death among SCI patients, there is an urgent need for the development of approaches for improving respiratory function. Midcervical (C3–4) contusion injury results in combined white and gray matter damage (including a partial loss of phrenic interneurons and motoneurons) and impaired diaphragm function. Previous studies of the injured spinal cord have revealed that interneurons are critical for the formation of new pathways that facilitate functional plasticity postinjury, albeit limited. Building upon this principle, the present study tests whether transplantation of neural progenitor tissue—rich in interneuronal precursors—can also provide an anatomical substrate for repair of phrenic pathways and optimize functional plasticity and diaphragm recovery. Adult, female Sprague–Dawley rats (n = 20) received C3/C4 lateralized contusions using the Infinite Horizons Device (preset force = 200 kdyne) and were allowed to recover for 1 week. At this time, a suspension of rat fetal spinal cord (FSC) tissue was injected intraspinally at the site of contusion and cavitation. One month posttransplantation, either a intramuscular (diaphragm) or intratransplant injection of pseudorabies virus (PRV; a retrograde transynaptic tracer) was made 72 h prior to perfusion. Transynaptic tracing from the diaphragm revealed that donor tissue synaptically integrated with the phrenic circuit ipsilateral to injury. In addition, PRV injection into transplanted tissue confirmed synaptic integration between the host and donor neurons. This is further supported by the presence of serotonergic axons within transplanted tissue. Immunohistochemistry also showed an increased c-fos expression within transplanted cells. Ongoing analyses are using dual-labeling methods to identify the neurotransmitter phenotype of those donor neurons that innervate the host phrenic circuitry. Terminal diaphragm electromyography (EMG) revealed that transplantation of FSC tissue increased activity during eupneic breathing and during respiratory challenge (hypoxia or hypercapnia). These results suggest that transplanted FSC tissue may provide a neuronal relay to enhance lasting recovery of phrenic function following midcervical contusion injury.
M. Staples,* N. Tajiri,* T. Dailey,* Y. I. Mosley,* T. Lau,* H. van Loveren,* S. U. Kim,† T. Yamashima,‡ T. Yasuhara,§ I. Date,§ Y. Kaneko,* and C. V. Borlongan*
*Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
†Department of Neurology, University of British Columbia, Vancouver, Canada
‡Department of Restorative Neurosurgery, Kanazawa University Graduate School of Medical Science, Kanazawa, Japan
§Department of Neurological Surgery, Okayama University Graduate School of Medicine, Okayama, Japan
Stroke is the fourth leading cause of mortality in the US, responsible for about 160,000 deaths per year. More than 750,000 Americans have a new or recurrent stroke each year. There have been multiple animal models, mainly rodent models, of stroke demonstrating that novel therapeutics can help improve the clinical outcome. However, these results have failed to show the same outcomes when translated into human clinical trials. While rodent models are very widely utilized due to their low cost and large sample sizes, they have substantial limitations with regards to translational research. Nonhuman primate models are not currently as widely utilized due to the lack of rigorous data in support of a well-validated set of stroke histopathological deficits and the expense associated with caring for a larger animal. However, nonhuman primate models offer a more appropriate representation of human brains due to their comparable primate anatomy. To date, further guidance and research is needed to advance nonhuman primate models in terms of stroke and expand their role in translational stroke research.
I. Strömberg, A. Rehnmark, G. Orädd, and A. Virel
Department of Integrative Medical Biology, Umeå University, Umeå, Sweden
Neuroinflammation is a chronic event in neurodegenerative disorders. To control the inflammatory process, an antioxidant treatment has been tried in an animal model of Parkinson's disease. The neuroinflammatory response was surprisingly stronger in the short term after inducing the dopamine lesion in animals fed with antioxidant-enriched diet compared to the inflammation found in animals given control diet. The antioxidant-enriched diet was 2% bilberries. However, at longer time points, the strong neuroinflammatory response became weaker in the antioxidant-treated animals while the microglia activation became stronger in the control diet-fed animals. The attenuation of microglia activation in antioxidant-fed animals was followed by regeneration of the dopamine nerve fibers in the striatum. It is still elusive why heavy accumulation of microglia was found early after the lesion in antioxidant-treated animals. Therefore, magnetic resonance imaging (MRI) was used to study if the inflammatory response was generated from the blood or activated brain microglia. Activated microglia were visualized using ED1 [antibody for cluster of differentiation (CD) 68] immunoreactivity. Superparamagnetic iron oxide (SPIO) particles were IV injected prior to a striatal 6-OHDA injection in Sprague–Dawley rats to tag phagocytes in the blood. T2-and T2*-weighted scans were performed at 1 week after the lesion. The results revealed that the strong accumulation of monocytes in the bilberry-treated animals was derived from the blood, while less blood-derived monocytes were found in the control-fed animals. Thus, antioxidant-enriched diet promoted the strong accumulation of bone marrow-derived ED1-positive cells at 1 week after a dopamine lesion. The study emphasizes the importance of following both the acute and long-term events of neuroinflammation.
N. Tajiri, H. Ishikawa, Y. Kaneko, T. Malapira, C. Gemma, F. Vale, and C. V. Borlongan
Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
Over three million Americans suffer from some form of epilepsy, with mesial temporal lobe epilepsy (MTLE) being the most common, arising from the temporal lobe structures of amygdala, hippocampus and parahippocampal gyrus. While many antiepileptic drugs are available, temporal lobectomy is the definitive treatment for intractable TLE. The present study sought to further understand the pathophysiology of epilepsy in an effort to develop more effective treatments for this disorder. Under USF IRB approval, tissues from multiple brain regions were obtained from consenting TLE patients undergoing hippocampal resection. Specimens were processed for microRNA and RT-qPCR analysis, while alternate tissues were grown in culture for immunohistochemical assays. Approximately 800 microRNAs were examined. Results revealed that miR-34b and miR-34c were significantly upregulated in hippocampus and amygdala compared to the neocortex, with miR-34-b/c expression highest in the amygdala. In contrast, levels of miR-592 were significantly downregulated in the hippocampus and amygdala compared to the neocortex. Paired brain tissue from profiled microRNA samples were then processed for RT-qPCR focusing on the identified novel microRNAs. RT-qPCR data generally support microRNA data. Notably levels of miR-34b/c were significantly upregulated, while levels of miR-592 were downregulated in the hippocampus and amygdala compared to neocortex. In tandem, immunocytochemical staining against stem cell markers and cell survival/death provided insights into the function of these miRs, in that distinct cell growth patterns were recognized from stem cells harvested from brain-specific regions with cell proliferation and differentiation reduced in the hippocampus and amygdala compared to the neocortex of TLE patients. Intra-amygdalar transplants of stem cells derived from the neocortex (i.e., reduced miR-34b/c but elevated miR-592 expression) of epileptic patients not only survived in the amygdala and migrated to the hippocampal lesions but also reduced hippocampal cell loss in rats subjected to kainic acid-induced epilepsy. This study elucidates the key role of microRNAs in the disease pathology of epilepsy and their utility as a stem cell optimization tool for identifying efficacious stem cells for transplantation therapy. A better understanding of the role of microRNAs in the disease pathology of TLE may lead to novel (i.e., stem cell-based) biomarkers and treatments for epilepsy and relevant disorders.
C. Tamboli,* T. Dailey,* N. Tajiri,* H. Ishikawa,* K. Shinozuka,* J. Vasconcellos,* S. A. Acosta,* D. G. Hernandez-Ontiveros,* M. Pabon,* J. G. Allickson,† N. Kuzmin-Nichols,‡ P. R. Sanberg,* D. J. Eve,* Y. Kaneko,* and C. V. Borlongan*
*Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
†Cryo-Cell International, Inc., Tampa, FL, USA
‡Saneron-CCEL Therapeutics, Inc., Tampa, FL, USA
We reviewed the experimental and clinical application of menstrual blood stem cells and Sertoli cells to treat stroke from cell isolation to administration. This meta-analysis discusses phenotypic characterization, mechanisms of repair in neurological disorders with emphasis in ischemic stroke, and presents transplantable features of menstrual blood stem cells and Sertoli cells as autologous cell donors for personalized medicine. The assessment of menstrual blood cells and Sertoli cells as viable therapy for stroke was conducted by analyzing previous studies pertaining to menstrual blood stem cells and Sertoli cells to understand the underlying mechanisms of the different cells and how they may aid in the process of recovery from ischemic stroke. Menstrual blood stem cells and Sertoli cells are two gender-specific sources of viable transplantable cells for stroke therapy. The use of autologous cells for the subacute phase of stroke offers practical clinical application. Menstrual blood stem cells are readily available, display proliferative capacity, pluripotency and angiogenic features, and, following transplantation in stroke models, have the ability to migrate to the infarct site, regulate the inflammatory response, secrete neurotrophic factors, and have the possibility to differentiate into neural lineage. Similarly, the testis-derived Sertoli cells secrete many growth and trophic factors, are highly immunosuppressive, and exert neuroprotective effects in animal models of neurological disorders. Menstrual blood stem cells and Sertoli cells display novel transplantable features for stroke therapy. Stem cells may be linked to repairing stroke brain by a modulated immune system and trophic factors. Cryopreservation of autologous cells may be sensible for stroke patients.
This study was supported by USF Department of Neurosurgery and Brain Repair Funds.
J. Vasconcellos, H. Ishikawa, N. Tajiri, K. Shinozuka, S. A. Acosta, Y. Kaneko, and C. V. Borlongan.
Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
Ischemic stroke is a leading cause of mortality and morbidity in the world. Cardiac myocyte vulnerability may be associated with ischemic stroke. However, it remains uncertain how an ischemic brain condition links to cardiac alterations. Here, we employed experimental stroke models, using the in vitro oxygen-glucose deprivation (OGD) condition and the in vivo middle cerebral artery occlusion (MCAO) rat paradigm in order to reveal the pathological effects of the ischemic brain on the heart. For the in vitro study, primary rat neuronal cells (PRNCs) and rat cardiac myocytes (RCMs) were grown in culture for 5 days. PRNCs were subjected to 90 min OGD condition, and 2 h after, the supernatant was collected and cryopreserved. Primary RCMs were cultured with the PRNC-derived supernatant for 2, 6, 24, and 48 h. The RCM was processed for MTT, cAMP, and calcein assays to reveal mitochondrial activity and cell viability. For the in vivo study, focal cerebral ischemia was induced by MCAO in rats. Animals were euthanized 3 months poststroke and processed immunohistochemically for cell death markers including necrosis, apoptosis, and autophagy. In vitro results demonstrated that exposure for at least 24 h to the supernatant from OGD-exposed PRNCs caused significant reduction of both mitochondrial activity and cell viability in RCM. In vivo results showed cardiac cells immunopositive for all cell death markers in chronic stroke animals. In tandem, the brains of chronic stroke animals displayed immunopositive neuronal cells against all cell death markers involving necrosis in the ipsilateral side and autophagy-positive neuronal cells in the contralateral side. Both in vitro and in vivo models of chronic stroke were accompanied by cardiac cell death, indicating a close pathological link between brain and heart. These results suggest a vigilant assessment of the heart condition in stroke patients, likely requiring the need to treat systemic cardiac symptoms following an ischemic brain episode.
Research supported by the USF Department of Neurosurgery and Brain Repair Funds.
T. Vorobyeva, E. Veselovskaya, and A. Shlyakhova
Department of Neurophysiology and Immunology, Institute of Neurology, Psychiatry, and Narcology, National Academy of Medical Science, Kharkiv, Ukraine
Our aim was to study cannabis addiction in rats and the effect of distant embryonic tissue implants from leeches upon the addiction. Tissue was gathered from embryos of Hirudo medicinalis, the European medical leech. Selected tissue contained saliva glands and rostral ganglion, areas known to contain anandamide, a ligand of the CB1 cannabinoid receptor. Four to five tissue segments were implanted under the skin of cannabis-addicted rats at the level of the third neck vertebra and the procedure was repeated 7 days later. Effect of implantations was determined upon performance of electrophysiological, hemodynamic, and behavioral tests. Results after the two tissue implantations showed that electrogenesis resumed in emotiogenic brain structures, the threshold for negative emotional reactions rose, and systolic pressure decreased. This normalization of test results indicated a reduction in cannabis addiction and in the severity of abstinence syndrome.
B. Yang, K. Schaar, X. Xi, and S. I. Savitz
Department of Neurology, University of Texas HSC, Medical School, Houston, TX, USA
Autologous bone marrow-derived mononuclear cells (MNCs) had been reported by us and other investigators to have benefits of recovery after stroke, and clinical trials have commenced testing the safety of MNCs in stroke patients. Mice have many genes in common with humans and are considered as “model organisms” to study human diseases. We aimed to determine if we could develop a stroke model to test the effects of autologous MNCs on recovery after stroke in mice. C57 or SV-129 mice were subjected to tandem middle cerebral artery occlusion/common cerebral artery occlusion (MCAo/CCAo) or suture occlusion of the MCA. Animals (n = 10 per group) at 22 h after stroke were randomized to receive intravenous injection (IV) of saline or undergo a bone marrow harvest of the tibia followed by intravenous reinfusion of autologous MNCs (1 million cells each animal). Mice participated in a series of neurological behavior tests and were evaluated up to 28 days after stroke. In another experiment, C57 mice with MCAo/CCAo were divided into three groups: (1) treated with saline IV; (2) treated with MNCs from wild-type (WT) mice IV; (3) treated with MNCs from interleukin-10 (IL-10) knockout (KO) mice I V. Serum was collected at day 3 after stroke for serum cytokines measurement (n = 3 per group). Brains were harvested at day 5 for infarction sizes assay (n = 5 per group). Bone marrow harvest was successfully completed in all mice without hind limb impairment. Compared to saline group, autologous MNC treatment significantly improved functional recovery in the cylinder and corner tests at 28 days after stroke (p < 0.05). At day 5 after stroke, MNCs from WT mice treatment significantly reduced infarct maturation (7.2 ± 2.3 mm3) compared with saline controls (11.9 ± 2.0 mm3) (p < 0.05). However, there was no difference in infarct size in animals treated with MNCs from IL-10 KO mice versus saline control. MNCs significantly reduced serum levels of IL-1β and IL-6 and increased IL-10 at day 3 after stroke. Compared to animals treated with wild-type MNCs, animals treated with MNCs from IL-10 KO mice had increased levels of IL-1β and IL-6 and reduced levels of IL-10 and Granulocyte-macrophage colony-stimulating factor (GM-CSF) in serum. We successfully established a stroke model to test poststroke autologous bone marrow-derived MNCs in mice. MNCs reduced infarct maturation and improved functional recovery after stroke in the mice stroke model. Our data suggest that IL-10 is released from MNCs and is a pivotal cytokine involved in the underlying neuroprotective effects of MNCs. MNCs may promote recovery through its effects on the poststroke immune response.
D. Yu,*† D. K. Thakor,*† I. B. Han,*† A. E. Ropper,*† H. Haragopal,*† R. L. Sidman,‡ R. Zafonte,‡ S. C. Schachter,‡§ and Y. D. Teng*†¶
*Department of Neurosurgery, Harvard Medical School/Brigham and Women's Hospital/Boston Children's Hospital, Boston, MA, USA
†Division of SCI Research, Veteran Affairs Boston Healthcare System, Boston, MA, USA
‡Department of Neurology, Harvard Medical School/Beth Israel Deaconess Medical Center, Boston, MA, USA
§Center for Integration of Medicine and Innovative Technology (CIMIT), Boston, MA, USA
¶Department of Physical Medicine and Rehabilitation, Harvard Medical School/Spaulding Rehabilitation Hospital, Boston, MA, USA
Diverse mechanisms including activation of N-methyl-d-aspartate (NMDA) receptors as well as microglial activation, reactive astrogliosis, loss of descending inhibition, and spasticity are responsible for intractable neuropathic pain post-spinal cord injury (SCI). Since conventional treatments blocking individual mechanisms elicit only short-term effectiveness, a simultaneous multimodal approach against major pathophysiologic processes may offer a fresh approach with translational value. We hypothesize that [–]-Huperzine A (HUP-A), an alkaloid isolated from the club moss Huperzia serrata that is a potent reversible inhibitor of the enzyme acetylcholinesterase (AChE) and NMDA receptors, could mitigate pain without invoking drug tolerance or dependence by stimulating cholinergic interneurons to impede pain signaling, inhibiting inflammation via microglial cholinergic activation, and blocking NMDA-mediated central hypersensitization. We tested our hypothesis by administering repeated HUP-A injections intraperitoneally (IP) or intrathecally (IT) to female Sprague–Dawley rats (200–235 g) after moderate static compression (35g x 5 min) of T10 spinal cord. Compared to controls, HUP-A treatment demonstrates significant analgesic effects in both regimens. SCI rats manifested no drug tolerance following repeated bolus IP or chronic IT HUP-A dosing. The pain-ameliorating effect of HUP-A is cholinergic-dependent. HUP-A administration, relative to vehicle treatment, also reduced neural inflammation, retained higher numbers of calcium-impermeable GluR2-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, and prevented Homer1a upregulation in dorsal horn sensory neurons. Therefore, HUP-A provides safe and effective management for chronic pain following rat compression SCI by reestablishing homeostasis of sensory neural circuits. We conclude that multimodel actions provide a fresh translational approach to reduce chronic pain.
Y. Zhang,*† G. E. Meredith,* N. Mendoza-Elias,*† D. J. Rademacher,‡ K-Y. Tseng,† and K. Steece-Collier‡
*Department of Pharmaceutical Sciences, College of Pharmacy, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA
†Department of Cellular and Molecular Pharmacology, Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA
‡Department of Translational Science and Molecular Medicine, Michigan State University, Grand Rapids, MI, USA
We examined the structural plasticity of excitatory synapses from corticostriatal and thalamostriatal pathways and their postsynaptic targets in order to understand how these striatal circuits change in subjects experiencing levodopa-induced dyskinesia (LID). We have used detailed electron and light microscopic analyses and show for the first time how striatal medium spiny neurons (MSNs) adapt in subjects with or without LIDs. Numerous previous studies have implicated the striatum, enhanced glutamate signaling and persistent long-term potentiation (LTP) as central to this behavioral side effect of antiparkinsonian medication. Further, behavioral alterations are thought to involve structural modifications, particularly alterations in patterns of synaptic connectivity. Thus, we hypothesized that in the striatum of rats with LIDs, one of two major glutamatergic afferent pathways would form new or altered contacts, especially onto the spines of MSNs, a primary target of these excitatory afferents. Our data provide structural evidence of dramatic rewiring of the striatum of dyskinetic rats and demonstrate that this rewiring involves specifically corticostriatal, but not thalamostriatal contacts, onto MSNs. Specifically, our data corroborate that LID behaviors do not necessarily correlate with the degree of nigral dopamine neuron loss when the loss is >90%, but instead correlate with specific pre- and postsynaptic changes in the striatum. In particular, the initial synaptic loss that occurs with the lesion is followed by a restoration of corticostriatal synapses to control levels in dyskinetic rats; however, this involves reestablishment of atypical input patterns onto spines and excessive inputs onto dendrites. There is also evidence that the postsynaptic targets (i.e., MSNs) adapt structurally to the reestablishment of synaptic inputs by increasing distal, but not proximal spines, and increasing the number of mushroom spines that accommodate more than one excitatory synapse. Finally, these data show that there is a loss of multisynaptic boutons (MSBs) in all parkinsonian conditions, regardless of dyskinesia status. It is relevant that the increased surface area of mushroom spines has previously been shown to result in changes in plasticity (enhanced long-term potentiation; LTP). A dopaminergic lesion is known to lead to impairments in motor memory and the loss of both LTP and long-term depression (LTD) plasticity. Interestingly, levodopa treatment has been previously shown to result in reinstatement of LTP but not LTD, which our data suggest could be due to the multiple new spine synapses, particularly from the cortex. Thus, the enduring motor dysfunctions and aberrant LTP seen in rat LIDs are possibly associated with newly sprouted long-range connections that form multiple asymmetric connections with spines (presumably mushroom spines), rather than remodeling of existing contacts as MSBs.
This work was supported by R01 NS04513 and P50 NS058830 (K.S.-C.).