Experimental Stroke Models Research Articles

Berberine Facilitates Angiogenesis Against Ischemic Stroke Through Modulating Microglial Polarization via AMPK Signaling.

Evidence suggests that microglia/macrophages can change their phenotype to M1 or M2 and participate in tissue damage or repair. Berberine (BBR) has shown promise in experimental stroke models, but its effects on microglial polarization and long-term recovery after stroke are elusive. Here, we investigated the effects of BBR on angiogenesis and microglial polarization through AMPK signaling after stroke. In the present study, C57BL/6 mice were subjected to transient middle cerebral artery occlusion (tMCAO), intragastrically administrated with BBR at 50mg/kg/day. Neo-angiogenesis was observed by 68Ga-NODAGA-RGD micro-PET/CT and immunohistochemistry. Immunofluorescent staining further exhibited an increase of M2 microglia and a reduction of M1 microglia at 14days after stroke. In vitro studies, the lipopolysaccharide (LPS)-induced BV2 microglial cells were used to confirm the AMPK activation effect of BBR. RT-PCR, Flow cytometry, and ELISA all demonstrated that BBR could inhibit M1 polarization and promote M2 polarization. Furthermore, treatment of human umbilical vein endothelial cells (HUVEC) with conditioned media collected from BBR-treated BV2 cells promoted angiogenesis. All effects stated above were reversed by AMPK inhibitor (Compound C) and AMPK siRNA. In conclusion, BBR treatment improves functional recovery and promotes angiogenesis following tMCAO via AMPK-dependent microglial M2 polarization.

Cell-Based Therapies for Stroke: Promising Solution or Dead End? Mesenchymal Stem Cells and Comorbidities in Preclinical Stroke Research

Stroke is a major health problem worldwide. It has been estimated that 90% of the population attributable risk of stroke is due to risk factors such as aging, hypertension, hyperglycemia, diabetes mellitus and obesity, among others. However, most animal models of stroke use predominantly healthy and young animals. These models ignore the main comorbidities associated with cerebrovascular disease, which could be one explanation for the unsuccessful bench-to-bedside translation of protective and regenerative strategies by not taking the patient's situation into account. This lack of success makes it important to incorporate comorbidities into animal models of stroke in order to study the effects of the various therapeutic strategies tested. Regarding cell therapy, the administration of stem cells in the acute and chronic phases has been shown to be safe and effective in experimental animal models of stroke. This review aims to show the results of studies with promising new therapeutic strategies such as mesenchymal stem cells, which are being tested in preclinical models of stroke associated with comorbidities and in elderly animals.

Analysis of the Effect of Phototherapy on the Inflammatory Mediators in an Experimental Model of Ischemic Vascular Accident

Cerebral Vascular Accident (CVA) is a sudden neurological condition, which mostly results from cerebral ischemia. Responsible for a large number of deaths and functional disability worldwide, it is extremely important to implement preventive strategies and identify new therapies. Phototherapy has been an excellent therapeutic modality for several diseases, few studies have explored this therapeutic modality for the treatment of stroke. The aim of this study was to analyze whether the treatment with LLLT is able to modulate the inflammatory mediators in the experimental model of ischemic stroke (AVCI). The study was approved by the Ethics Committee on Animal Use of the Federal University of São Paulo (CEUA / UNIFESP) under the number 3316110516. The investigation was conducted in conformance with the FASEB Statement of Principles for the use of Animals in Research and Education. The AVC was induced by the photothrombosis method in 8 male Wistar rats (230 ± 20g) randomly assigned to two experimental groups: Group AVC Control (AVCC) and Group AVC Laserterapia (AVCL). The application of transcranial LLLT was done using a laser diode (MM Optics), with wavelength of 780 ± 5nm, power density of 10 mW / cm2, for 2 minutes, 3× / week for a period of 2 months . The animals were euthanized with anesthetic overdoses of Barbituric / Thiopental Sodium (Cristália) at a dose of 80 mg / kg intraperitoneally; transcardiac perfusion was performed and then the biological tissue (brain) was extracted. The tissues were frozen in liquid nitrogen. The levels of TNF‐α, IL‐1β, IL‐6, IL‐10, VEGFA, BDNF, IGF1, TGFB1 and ANGIOTENSIN II in the samples were determined by the enzyme‐linked immunosorbent assay (ELISA), following commercial kit instructions (R & D System, USA). ). Data were analyzed by the Shapiro‐Wilk normality test in order to verify that the samples come from populations with normal distribution; then the variance test was performed using the Snedecor F test, and then the unpaired T‐test was used; for all tests was considered α = 5%. Our results indicate that there was modulation of the IL1‐b mediators (AVCC 0.4075 ± 0.156659 × AVCL 0.232091 ± 0.083417 p <0.01); IL‐6 (AVCC 180.5753 ± 49.70076 × AVCL 25.47956 ± 15.58209; p <0.001); TNF (40.06277 ± 17.23243 × AVCL 18.2546 ± 5.8065; p <0.01), and in relation to IL‐10 and TGF‐β there were no significant changes. We can conclude that phototherapy modulates the inflammatory response , This result may indicate that the area of shadow of the lesion could be smaller favoring recovery after AVC.Support or Funding InformationFinancial Support: Foundation for Research Support of the State of São Paulo (FAPESP) 2016 / 10286‐7This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Melatonin Protects MCAO-Induced Neuronal Loss via NR2A Mediated Prosurvival Pathways.

Stroke is the significant cause of human mortality and sufferings depending upon race and demographic location. Melatonin is a potent antioxidant that exerts protective effects in differential experimental stroke models. Several mechanisms have been previously suggested for the neuroprotective effects of melatonin in ischemic brain injury. The aim of this study is to investigate whether melatonin treatment affects the glutamate N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor signaling in cerebral cortex and striatum 24 h after permanent middle cerebral artery occlusion (MCAO). Melatonin (5 mg/kg) attenuated ischemia-induced down regulation of NMDA receptor 2 (NR2a), postsynaptic density-95 (PSD95) and increases NR2a/PSD95 complex association, which further activates the pro-survival PI3K/Akt/GSK3β pathway with mitigated collapsin response mediator protein 2 (CRMP2) phosphorylation. Furthermore, melatonin increases the expression of γ-enolase, a neurotrophic factor in ischemic cortex and striatum, and preserve the expression of presynaptic (synaptophysin and SNAP25) and postsynaptic (p-GluR1845) protein. Our study demonstrated a novel neuroprotective mechanism for melatonin in ischemic brain injury which could be a promising neuroprotective agent for the treatment of ischemic stroke.

Combined Adipose Tissue-Derived Mesenchymal Stem Cell Therapy and Rehabilitation in Experimental Stroke.

Background/Objective: Stroke is a leading global cause of adult disability. As the population ages as well as suffers co-morbidities, it is expected that the stroke burden will increase further. There are no established safe and effective restorative treatments to facilitate a good functional outcome in stroke patients. Cell-based therapies, which have a wide therapeutic window, might benefit a large percentage of patients, especially if combined with different restorative strategies. In this study, we tested whether the therapeutic effect of human adipose tissue-derived mesenchymal stem cells (ADMSCs) could be further enhanced by rehabilitation in an experimental model of stroke.Methods: Focal cerebral ischemia was induced in adult male Sprague Dawley rats by permanently occluding the distal middle cerebral artery (MCAO). After the intravenous infusion of vehicle (n = 46) or ADMSCs (2 × 106) either at 2 (n = 37) or 7 (n = 7) days after the operation, half of the animals were housed in an enriched environment mimicking rehabilitation. Subsequently, their behavioral recovery was assessed by a neurological score, and performance in the cylinder and sticky label tests during a 42-day behavioral follow-up. At the end of the follow-up, rats were perfused for histology to assess the extent of angiogenesis (RECA-1), gliosis (GFAP), and glial scar formation.Results: No adverse effects were observed during the follow-up. Combined ADMSC therapy and rehabilitation improved forelimb use in the cylinder test in comparison to MCAO controls on post-operative days 21 and 42 (P < 0.01). In the sticky label test, ADMSCs and rehabilitation alone or together, significantly decreased the removal time as compared to MCAO controls on post-operative days 21 and 42. An early initiation of combined therapy seemed to be more effective. Infarct size, measured by MRI on post-operative days 1 and 43, did not differ between the experimental groups. Stereological counting revealed an ischemia-induced increase both in the density of blood vessels and the numbers of glial cells in the perilesional cortex, but there were no differences among MCAO groups. Glial scar volume was also similar in MCAO groups.Conclusion: Early delivery of ADMSCs and combined rehabilitation enhanced behavioral recovery in an experimental stroke model. The mechanisms underlying these treatment effects remain unknown.

Tissue regulatory T cells and neural repair.

Inflammation and immune responses after tissue injury play pivotal roles in the pathology, resolution of inflammation, tissue recovery, fibrosis and remodeling. Regulatory T cells (Tregs) are the cells responsible for suppressing immune responses and can be activated in secondary lymphatic tissues, where they subsequently regulate effector T cell and dendritic cell activation. Recently, Tregs that reside in non-lymphoid tissues, called tissue Tregs, have been shown to exhibit tissue-specific functions that contribute to the maintenance of tissue homeostasis and repair. Unlike other tissue Tregs, the role of Tregs in the brain has not been well elucidated because the number of brain Tregs is very small under normal conditions. However, we found that Tregs accumulate in the brain at the chronic phase of ischemic brain injury and control astrogliosis through secretion of a cytokine, amphiregulin (Areg). Brain Tregs resemble other tissue Tregs in many ways but, unlike the other tissue Tregs, brain Tregs express neural-cell-specific genes such as the serotonin receptor (Htr7) and respond to serotonin. Administering serotonin or selective serotonin reuptake inhibitors (SSRIs) in an experimental mouse model of stroke increases the number of brain Tregs and ameliorates neurological symptoms. Knowledge of brain Tregs will contribute to the understanding of various types of neuroinflammation.

Pharmacological Manipulation of Trk, p75NTR, and NGF Balance Restores Memory Deficit in Global Ischemia/Reperfusion Model in Rats.

Long-term memory impairment is reported in more than 50% of cardiac arrest survivors. Monosialoganglioside (GM1) provided neuroprotection in experimental models of stroke but failed to replicate its promise clinically for unknown reasons. GM1 stimulates the release of nerve growth factor (NGF), which is synthesized as a precursor protein (pro-NGF) that either mediates apoptosis through the p75 neurotrophin receptor (p75NTR) or is cleaved by the protease furin (FUR) to yield mature NGF, the latter supporting survival through tropomyosin kinase receptor (Trk). The flavanol epicatechin (EPI) inhibits p75NTR-mediated signaling and apoptosis by pro-NGF. The aim of the current work is to test whether these two drugs affect, or communicate with, each other in the setting of CNS injuries. Using the two-vessel occlusion model of global ischemia/reperfusion (I/R), we tested if pharmacological modulation of Trk, p75NTR, and NGF balance with GM1, EPI, and their combination, can correct the memory deficit that follows this insult. Finally, we tested if FUR insufficiency and/or p75NTR-mediated apoptosis negatively affect the neurotherapeutic effect of GM1. Key proteins for Trk and p75NTR, FUR, and both forms of NGF were assessed. All treatment regiments successfully improved spatial memory retention and acquisition. A week after the insult, most Trk and p75NTR proteins were normal, but pro/mature NGF ratio remained sharply elevated and was associated with the poorest memory performance. Pharmacological correction of this balance was achieved by reinforcing Trk and p75NTR signaling. GM1 increased FUR levels, while concomitant administration of EPI weakened GM1 effect on pro-survival Trk and p75NTR mediators. GM1 neuroprotection is therefore not limited by FUR but could be dependent on p75NTR. Graphical Abstract "."

Neuroprotective Effects of BHDPC, a Novel Neuroprotectant, on Experimental Stroke by Modulating Microglia Polarization

This study mainly investigated the therapeutic effects of BHDPC on ischemic stroke and its underlying mechanisms. In vivo, the transient middle cerebral artery occlusion (MCAO) was used to induce ischemic model. In vitro, oxygen and glucose deprivation/reperfusion (OGD/R)-induced ischemic stroke in BV-2 microglia and primary neurons, and bEnd.3 mouse cerebral microvascular endothelial cells (ECs) were also used. First, we found that BHDPC exerts considerable neuroprotection against MCAO-induced ischemic injury to mice via alleviating neurological deficits and brain infarcts, inhibiting neuronal cell loss and apoptosis, and attenuating blood-brain barrier disruption and tight junction protein changes. Next, we observed that BHDPC significantly reduced microglial M1 activation but enhanced M2 polarization in MCAO-induced ischemic brain. Further experiments in vitro indicated that BHDPC suppressed microglial activation but promoted M2 microglial polarization in OGD/R-induced BV-2 microglia. In addition, conditioned medium (CM) experiments showed that CM from BHDPC-treated BV-2 microglia provided protections against OGD/R-induced ischemic damage in primary neurons and bEnd.3 ECs. Moreover, we found that BHDPC actions on microglial inflammation were associated with the inactivation of NF-κB signaling. Interestingly, we also found that BHDPC enhanced phosphorylation of protein kinase A (PKA) and cAMP-response element-binding protein (CREB). The pharmacological inhibition or gene knockdown of PKA/CREB signaling diminished BHDPC-promoted microglial M2 polarization. In summary, BHDPC conferred neuroprotection against ischemic injury in experimental stroke models. Modulating microglial activation and polarization contributes to BHDPC-mediated neuroprotective actions, which in part were mediated by nuclear factor kappa B and PKA/CREB signaling pathway.

Нарушение распределения лимфоцитов селезенки по фазам клеточного цикла при экспериментальном остром нарушении мозгового кровообращения различной степени тяжести

Актуальность. Нарушение работы иммунной системы при инсульте играет важную патогенетическую роль в течении данного заболевания и складывается в целом на системном уровне из депрессии неспецифического и клеточного звена иммунитета, активации гуморального иммунитета, развития аутоиммунных реакций и дисбаланса в системе цитокинов. Однако в литературе не представлены данные, посвященные изучению статуса активности иммунокомпетентных клеток в зависимости от степени тяжести острой цереброваскулярной патологии. Цель исследования: изучение пролиферативной активности и распределения лимфоцитов по фазам клеточного цикла при остром нарушении мозгового кровообращения (ОНМК) различной степени тяжести. Материалы и методы: в экспериментальном исследовании на животных (крысы линии Вистар, массой 200-220 г, n = 55) изучали пролиферативную активность и распределение Т- и В-лимфоцитов селезенки по фазам митотического цикла при экспериментальном ОНМК в левом полушарии (область внутренней капсулы) легкой, средней и тяжелой степени. У животных также оценивали неврологический статус с помощью шкалы Stroke-index McGraw в модификации И.В. Ганнушкиной. Результаты: при оценке неврологического статуса на 3-и сутки от момента воспроизведения ОНМК выявлено, что тяжесть экспериментальной модели соответствует тяжести неврологического статуса животных. При моделировании ОНМК обнаружено изменение в распределении В-лимфоцитов по фазам клеточного цикла в виде снижения пролиферативной активности, на что указывает снижение пула клеток в группах ОНМК в фазе S, в которой происходит репликация ДНК (ОНМК легкой степени: 55,3% [52,7; 58,1]; ОНМК средней степени: 53,2% [50,9; 54,7]; ОНМК тяжелой степени: 46,2% [44,2; 50,0]), в фазе G2/M, в которой происходит деление клетки (ОНМК легкой степени: 1,1% [0,9; 1,3]; ОНМК средней степени: 0,8% [0,7; 1,1]; ОНМК тяжелой степени: 0,5% [0,5; 0,7]), и увеличение количества клеток в фазе G0/G1, в которой происходит синтез мРНК и белка (ОНМК легкой степени: 43,4% [40,8; 46,2]; ОНМК средней степени: 46,1% [44,4; 48,3]; ОНМК тяжелой степени: 53,3% [49,6; 55,4]), с достоверными отличиями от контроля. Указанные изменения нарастают с утяжелением модели ОНМК. Подобная тенденция отмечена в отношении Т-лимфоцитов в группах ОНМК в виде снижения процента клеток в фазе S - в повышении пула клеток в фазах G0/G1, но при этом отмечается повышение количества лимфоцитов в фазах G2/M (ОНМК легкой степени: 2,9% [2,6; 3,2]; ОНМК средней степени: 3,8% [3,5; 4,1]; ОНМК тяжелой степени: 4,4% [4,1; 4,8] с достоверными отличиями от контроля, что указывает на активное деление клеток. Данные изменения нарастают, при повышении степени тяжести ОНМК. Заключение: острая цереброваскулярная патология приводит к нарушению распределения Т- и В-лимфоцитов селезенки по фазам клеточного цикла и снижению их пролиферативной активности с нарастанием данных изменений с утяжелением степени тяжести инсульта. Background. Impairment of the immune system in stroke plays an important pathogenic role in development of this disease and generally consists at the systemic level of depressed non-specific and cellular immunity, activated humoral immunity, autoimmune reactions, and imbalance of the cytokine system. However, information about the relationship between activity of immunocompetent cells and severity of acute cerebrovascular disease is unavailable. Aim: To study proliferative activity and distribution of lymphocytes over phases of the cell cycle in stroke of different severity. Materials and methods: This experimental study was performed on Wistar rats weighing 200-220 g (n=55). Proliferative activity and distribution of spleen T and B lymphocytes in the mitotic cycle were studied in the left hemisphere (capsula interna) on experimental models of mild, moderate and severe stroke. Neurological status of animals was evaluated using the Stroke-index McGraw scale modified by I.V. Gannushkina. Results: Assessment of the neurological status on the 3rd day after the onset of experimental stroke showed that the severity of experimental model corresponded to the severity of neurological status. Stroke was associated with changes in the distribution of B-lymphocytes over the cell cycle phases. This was evident as a decrease in proliferative activity shown by decreased pools of cells in the S-phase, in which DNA replication occurs (mild stroke, 55.3% [52.7; 58.1]; moderate stroke, 53.2% [50.9; 54.7]; severe stroke, 46.2% [44.2; 50.0]) and in the G2/M-phase, in which mitosis occurs (mild stroke, 1.1% [0.9; 1.3]; moderate stroke, 0.8% [0.7; 1.1]; severe stroke, 0.5% [0.5; 0.7]); and by increased pool of cells in the G0/G1 phase, in which mRNA and protein are synthesized (mild stroke, 43.4% [40.8; 46.2]; moderate stroke, 46.1% [44.4; 48.3]; severe stroke, 53.3% [49.6; 55.4]). These differences were significant compared with the control group. These changes increased with severity of the stroke model. A similar tendency was noted for T-lymphocytes in stroke groups - a decreased proportion of cells in the S-phase and an increased cell pool in the G0/G1-phases. However, in the G2/M-phases, the number of lymphocytes was increased (mild stroke, 2.9% [2.6; 3.2]; moderate stroke, 3.8% [3.5; 4.1]; severe stroke, 4.4% [4.1; 4.8]) and significantly different from the control, which indicated active mitosis. These changes increased with the severity of stroke. Conclusion: acute cerebrovascular pathology results in disordered distribution of splenic T and B lymphocytes over cell cycle phases and decreased lymphocyte proliferative activity; these changes increased with the severity of experimental stroke.

Study of transcranial therapy 904nm in experimental model of stroke.

Cerebrovascular accidents (CVAs), commonly known as strokes, can damage the brain through vascular injuries caused by either blood vessel blockages (ischemic stroke) or ruptures (hemorrhagic stroke) which disrupt regular brain blood supply and can cause severe damage to the individual. The objective of the present study was to evaluate the effects of photobiomodulation with a light-emitting diode (LED) device (904nm, 110mW, 7J/cm2) on neurogenesis, muscle resistance, and motor behavior in animals submitted to an experimental model of hemiplegia. The sample consisted of 30 Wistar rats, divided into two groups: control group (GC) and 904-nm LED-treated group (TG). All animals underwent stereotactic surgery for electrode implant and subsequent electrolytic injury to induce an ischemic stroke. TG was subjected to daily LED irradiation (904nm, 110mW, 7J/cm2) for 63s. Suspension test results indicate an improvement of TG muscle resistance when compared with baseline evaluation (BLT); a reduction in open-field freezing time and the number of fecal bolus pellets suggest diminished anxiety induced by 904-nm LED treatment on treatment days 7 and 21 (TG7 and TG21) compared with the baseline results; and lastly, histological analysis showed important signs of neurogenesis in TG in comparison to CG, especially on treatment days 7 and 21 (TG7 and TG21). In conclusion, the present study suggests that 904-nm LED irradiation may beneficially affect neurogenesis, muscle resistance, and animal motor behavior following ischemic CVA.

Experimental pediatric stroke shows age-specific recovery of cognition and role of hippocampal Nogo-A receptor signaling

Ischemic stroke is a leading cause of death worldwide and clinical data suggest that children may recover from stroke better than adults; however, supporting experimental data are lacking. We used our novel mouse model of experimental juvenile ischemic stroke (MCAO) to characterize age-specific cognitive dysfunction following ischemia. Juvenile and adult mice subjected to 45-min MCAO, and extracellular field recordings of CA1 neurons were performed to assess hippocampal synaptic plasticity changes after MCAO, and contextual fear conditioning was performed to evaluate memory and biochemistry used to analyze Nogo-A expression. Juvenile mice showed impaired synaptic plasticity seven days after MCAO, followed by full recovery by 30 days. Memory behavior was consistent with synaptic impairments and recovery after juvenile MCAO. Nogo-A expression increased in ipsilateral hippocampus seven days after MCAO compared to contralateral and sham hippocampus. Further, inhibition of Nogo-A receptors reversed MCAO-induced synaptic impairment in slices obtained seven days after juvenile MCAO. Adult MCAO-induced impairment of LTP was not associated with increased Nogo-A. This study demonstrates that stroke causes functional impairment in the hippocampus and recovery of behavioral and synaptic function is more robust in the young brain. Nogo-A receptor activity may account for the impairments seen following juvenile ischemic injury.

Mapping tissue pH in an experimental model of acute stroke – Determination of graded regional tissue pH changes with non-invasive quantitative amide proton transfer MRI

pH-weighted amide proton transfer (APT) MRI is sensitive to tissue pH change during acute ischemia, complementing conventional perfusion and diffusion stroke imaging. However, the currently used pH-weighted magnetization transfer (MT) ratio asymmetry (MTRasym) analysis is of limited pH specificity. To overcome this, MT and relaxation normalized APT (MRAPT) analysis has been developed that to homogenize the background signal, thus providing highly pH conspicuous measurement. Our study aimed to calibrate MRAPT MRI toward absolute tissue pH mapping and determine regional pH changes during acute stroke. Using middle cerebral artery occlusion (MCAO) rats, we performed lactate MR spectroscopy and multi-parametric MRI. MRAPT MRI was calibrated against a region of interest (ROI)-based pH spectroscopy measurement (R2 = 0.70, P < 0.001), showing noticeably higher correlation coefficient than the simplistic MTRasym index. Capitalizing on this, we mapped brain tissue pH and semi-automatically segmented pH lesion, in addition to routine perfusion and diffusion lesions. Tissue pH from regions of the contralateral normal, perfusion/diffusion lesion mismatch and diffusion lesion was found to be 7.03 ± 0.04, 6.84 ± 0.10, 6.52 ± 0.19, respectively. Most importantly, we delineated the heterogeneous perfusion/diffusion lesion mismatch into perfusion/pH and pH/diffusion lesion mismatches, with their pH being 7.01 ± 0.04 and 6.71 ± 0.12, respectively (P < 0.05). To summarize, our study calibrated pH-sensitive MRAPT MRI toward absolute tissue pH mapping, semi-automatically segmented and determined graded tissue pH changes in ischemic tissue and demonstrated its feasibility for refined demarcation of heterogeneous metabolic disruption following acute stroke.

Mechanisms Underlying Neuroprotection by the NSAID Mefenamic Acid in an Experimental Model of Stroke

Stroke is a devastating neurological event with limited treatment opportunities. Recent advances in understanding the underlying pathogenesis of cerebral ischemia support the involvement of multiple biochemical pathways in the development of the ischemic damage. Fenamates are classical non-steroidal anti-inflammatory drugs but they are also highly subunit-selective modulators of GABAA receptors, activators of IKS potassium channels and antagonists of non-selective cation channels and the NLRP3 inflammosome. In the present study we investigated the effect of mefenamic acid (MFA) in a rodent model of ischemic stroke and then addressed the underlying pharmacological mechanisms in vitro for its actions in vivo. The efficacy of MFA in reducing ischemic damage was evaluated in adult male Wistar rats subjected to a 2-h middle cerebral artery occlusion. Intracerebroventricular (ICV) infusion of MFA (0.5 or 1 mg/kg) for 24 h, significantly reduced the infarct volume and the total ischemic brain damage. In vitro, the fenamates, MFA, meclofenamic acid, niflumic acid, and flufenamic acid each reduced glutamate-evoked excitotoxicity in cultured embryonic rat hippocampal neurons supporting the idea that this is a drug class action. In contrast the non-fenamate NSAIDs, ibuprofen and indomethacin did not reduce excitotoxicity in vitro indicating that neuroprotection by MFA was not dependent upon anti-inflammatory actions. Co-application of MFA (100 μM) with either of the GABAA antagonists picrotoxin (100 μM) or bicuculline (10 μM) or the potassium channel blocker tetraethylammonium (30 mM) did not prevent neuroprotection with MFA, suggesting that the actions of MFA also do not depend on GABAA receptor modulation or potassium channel activation. These new findings indicate that fenamates may be valuable in the adjunctive treatment of ischemic stroke.

Abstract WMP78: Hypertensive Animals Develop Increased Post-Stroke Cognitive Impairment Associated With Bilateral Enlargement of Brain Ventricles

Introduction: Cognitive deficits are a common occurrence in both patients and experimental models of stroke. However, there is still a gap in our understanding of the time course and mechanisms involved in the progression of cognitive dysfunction after stroke in part due to the insufficient integration of risk factors for vascular cognitive impairment such as hypertension. Methods: 50 male rats (26 Wistar and 24 SHR), 14 weeks of age, were subjected to 1 h middle cerebral artery occlusion (MCAO) using a silicone-coated monofilament or sham surgery. Behavioral testing (Bederson, Open Field, Novel Object Recognition, Y-maze, Passive Avoidance and Morris Water Maze) was done (blinded) to examine the progression and resolution of motor and cognitive deficits, weekly for the first 6 weeks post stroke and again at 12 weeks. A brain MRI was done at week 12, animals were sacrificed and brains collected for Western blotting and immunologic staining. Results: Wistar animals showed a high rate of spontaneous recovery 24h after stroke. Both Wistar and SHR animals demonstrated total motor recovery by 2 weeks post stroke. Both groups showed evidence of cognitive dysfunction starting from the 4 th week after stroke. Stroke, especially in the SHR animals, caused a bilateral enlargement of brain ventricles (by MRI) at week 12 suggesting ongoing tissue loss. Conclusions: Normotensive animals show more resilience to the initial insult and a higher rate of 24hr recovery. However, both Wistar and SHR animals show similar patterns in the development and resolution of motor and cognitive impairment. Hypertensive animals showed evidence of an ongoing extensive brain tissue loss up to 3 months after stroke.

Abstract WP134: Calcium Release-activated Calcium Channel Inhibition Improves Neurological Outcome and Suppresses Inflammation in Female Mice Following Cerebral Ischemic Injury

Purpose: Calcium release-activated calcium (CRAC) channels activate microglia via store-operated Ca 2+ entry. We previously reported that the specific CRAC channel inhibitor (CM-EX-137) had neuroprotective effects in a model of experimental stroke due to suppression of microglial activation in male mice. However, it is well known that sex differences can influence stroke outcome as well as immune responses. Whether CRAC channel inhibition in female mice is similarly neuroprotective is unknown. The purpose of this study is to determine whether female mice respond similarly CRAC channel inhibition in a stroke model Subjects and Methods: C57/BL6 female mice, aged 2 months were subjected to distal middle cerebral artery occlusion (dMCAO) were treated with CM-EX-137 (5mg/kg IP; dMCAO-CM) or vehicle control (dMCAO-vehicle) daily for 3d. Stroke surgeries were carried out during the same phase of the estrus cycle (diestrus) to avoid potential confounds due to varying estrogen levels. These groups were also compared to sham groups. CM-EX-137 was injected immediately after occlusion and at 24 &amp; 48h. Brains were extracted 3d post dMCAO. Neurological functions were evaluated before surgery, and 1 &amp; 3d later. Lesion size was estimated from cresyl violet stains. Microglia and astrocyte activation were assessed using immunohistochemistry. Results: Neurological function at both 24 and 72 h post ischemia were significantly improved in dMCAO-CM group, compared with dMCAO-vehicle group (n=7/group; p&lt;0.001). CM-EX-137 also significantly reduced infarct volume at 3dafter dMCAO (p&lt;0.001). CM-EX-137 inhibited microglial/monocyte/macrophage activation (p&lt;0.001; isolectin B4, CD68) and led to decreased iNOS positive cells (p&lt;0.01). CM-EX-137 also decreased expression of CRAC channel components STIM1 and Orai1 on CD68+ macrophages (p&lt;0.001). However, GFAP staining indicated that CM-EX-137 had no effect on astrocyte activation. Conclusion: CRAC channel inhibition reduce inflammatory responses and improve neurological outcome following experimental stroke in female animals. These results are in line with those that we observed in male mice. Thus, CRAC channel inhibition seems to be similarly effective and anti-inflammatory in both genders in a stroke model.

Protection against stroke with glucagon-like peptide 1 receptor agonists: a systematic review and meta-analysis.

In experimental stroke models pretreatment with the newly introduced antidiabetic agents, glucagon-like peptide 1 receptor (GLP-1R) agonists, has been shown to exert neuroprotective effects. Published evidence with regard to the effect of treatment with GLP-1R agonists on the risk of stroke was evaluated. Data from prospective randomized placebo-controlled trials up to October 2018 involving GLP-1R agonists which reported cardiovascular outcomes as primary end-points of efficacy and/or safety were meta-analysed. Five eligible multicentre randomized placebo-controlled trials (ELIXA, LEADER, SUSTAIN, EXSCEL and HARMONY) were included. The pooled analysis (n=42358) showed a significant reduction by 13% in the risk of total stroke from treatment with GLP-1R agonists versus placebo (risk ratio 0.87, 95% confidence interval 0.78-0.98, P=0.021) with no significant heterogeneity between trials (Q = 4.094, P=0.393, I2 =2.307%). When only fatal stroke was included (this applied for the ELIXA, LEADER, EXSCEL and HARMONY trials), active treatment was associated with a non-significant reduction by 16% compared with placebo (risk ratio 0.84, 95% confidence interval 0.60-1.17, P=0.29). The findings of this meta-analysis support the evidence from earlier experimental studies calling attention to potential 'stroke protective' effects from treatment with GLP-1R.

Spatio-temporal overview of neuroinflammation in an experimental mouse stroke model

After ischemic stroke, in the lesion core as well as in the ischemic penumbra, evolution of tissue damage and repair is strongly affected by neuroinflammatory events that involve activation of local specialized glial cells, release of inflammatory mediators, recruiting of systemic cells and vascular remodelling. To take advantage of this intricate response in the quest to devise new protective therapeutic strategies we need a better understanding of the territorial and temporal interplay between stroke-triggered inflammatory and cell death-inducing processes in both parenchymal and vascular brain cells. Our goal is to describe structural rearrangements and functional modifications occurring in glial and vascular cells early after an acute ischemic stroke. Low and high scale mapping of the glial activation on brain sections of mice subjected to 30 minutes middle cerebral artery occlusion (MCAO) was correlated with that of the neuronal cell death, with markers for microvascular changes and with markers for pro-inflammatory (IL-1β) and reparative (TGFβ1) cytokines. Our results illustrate a time-course of the neuroinflammatory response starting at early time-points (1 h) and up to one week after MCAO injury in mice, with an accurate spatial distribution of the observed phenomena.

Middle Cerebral Artery Occlusion Allowing Reperfusion via Common Carotid Artery Repair in Mice.

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue plasminogen activator (tPA) as an approved drug treatment to target ischemic strokes. Current research in the field of ischemic stroke focuses on better understanding the pathophysiology of stroke, to develop and investigate novel pharmaceutical targets. Reliable experimental stroke models are crucial for the progression of potential treatments. The middle cerebral artery occlusion (MCAO) model is clinically relevant and the most frequently used surgical model of ischemic stroke in rodents. However, the outcomes of this model, such as lesion volume, are associated with high levels of variability, particularly in mice. The alternative MCAO model described here allows the reperfusion of the common carotid artery (CCA) and the increased perfusion of the middle cerebral artery (MCA) territory, using a tissue pad with fibrinogen-based sealant to repair the vessel, and the improved welfare of the mice by avoiding external carotid artery (ECA) ligation. This reduces the reliance on the Circle of Willis, which is known to be highly anatomically variable in mice. Representative data show that using this alternative surgical approach decreases the variability in lesion volumes between the traditional MCAO approach and the alternative approach described here.

Rapid loss of perihematomal cell viability in the collagenase intracerebral hemorrhage model

The effective time window of any therapeutic in an experimental stroke model is limited by the rate of injury progression. Intracerebral hemorrhage in rodents is commonly induced by striatal injection of either autologous blood or bacterial collagenase, which digests local blood vessels. During time window studies of the heme oxygenase-1 inducer hemin, which is protective when administered within 1–3 h in both models, the rate of perihematomal injury was directly compared after striatal blood or collagenase injection. Surprisingly, about 80% of the loss of perihematomal cell viability as measured by MTT reduction assay occurred within 6 h of collagenase injection. In contrast, significant viability loss was not observed at this time point after autologous blood injection, but rather it progressed over the subsequent four days to a level similar to that produced by collagenase. Consistent with these observations, systemic hemin therapy reduced blood-brain barrier disruption and perihematomal cell injury when initiated at 6 h after striatal injection of blood but not collagenase. These results indicate that the rate of early cell injury differs markedly in the collagenase and blood injection ICH models, which may contribute to inconsistent results in time window studies. The blood injection model may be more appropriate for prolonged time window studies of a neuroprotective agent.

Identification of Proteins Differentially Expressed in the Striatum by Melatonin in a Middle Cerebral Artery Occlusion Rat Model-a Proteomic and in silico Approach.

Ischemic stroke is characterized by permanent or transient obstruction of blood flow, which initiates a cascading pathological process, starting from acute ATP loss to subsequent membrane depolarization, glutamate excitotoxicity, and calcium overload. Melatonin is a potent antioxidant that exerts protective effects in different experimental stroke models. In this study, melatonin effects were demonstrated by a proteomic and in silico approach. The proteomic study identified differentially expressed proteins by 2D gel electrophoresis in the striatum 24 h after middle cerebral artery occlusion. Proteomic analysis revealed several proteins with aberrant expression and was validated by western blot and immunofluorescence analysis. Homology modeling was performed to build 3D structures for γ-enolase, thioredoxin (TRX), and heat shock 60 (HSP60) by the template crystal structures using a protein data bank as a sequence database. The structure refinement of each model was achieved by energy minimization via molecular dynamic simulation, and the generated models were further assessed for stability by Procheck and ProSA. The models were processed for docking analysis using AutoDock Vina, and post-docking analysis was determined by discovery studio. The proteomic study showed decreased expression of γ-enolase, TRX, and protein phosphatase 2A subunit B and increased expression of collapsin response mediator protein 2 and HSP60 in the striatum after ischemic injury. Treatment with melatonin modulated the expression profiles of these proteins. This study demonstrated the neuroprotective role of melatonin in the ischemic striatum using a proteomic and in silico approach. Collectively, melatonin may act in a multimechanistic way by modulating the expression of several proteins in the ischemic striatum.