Postischemic Brain Research Articles

Inhibition of LPA5 Activity Provides Long-Term Neuroprotection in Mice with Brain Ischemic Stroke.

Stroke is a leading cause of long-term disability in ischemic survivors who are suffering from motor, cognitive, and memory impairment. Previously, we have reported suppressing LPA5 activity with its specific antagonist can attenuate acute brain injuries after ischemic stroke. However, it is unclear whether suppressing LPA5 activity can also attenuate chronic brain injuries after ischemic stroke. Here, we explored whether effects of LPA5 antagonist, TCLPA5, could persist a longer time after brain ischemic stroke using a mouse model challenged with tMCAO. TCLPA5 was administered to mice every day for 3 days, starting from the time immediately after reperfusion. TCLPA5 administration improved neurological function up to 21 days after tMCAO challenge. It also reduced brain tissue loss and cell apoptosis in mice at 21 days after tMCAO challenge. Such long-term neuroprotection of TCLPA5 was associated with enhanced neurogenesis and angiogenesis in post-ischemic brain, along with upregulated expression levels of vascular endothelial growth factor. Collectively, results of the current study indicates that suppressing LPA5 activity can provide long-term neuroprotection to mice with brain ischemic stroke.

Caveolin-1 Derived from Brain Microvascular Endothelial Cells Inhibits Neuronal Differentiation of Neural Stem/Progenitor Cells In Vivo and In Vitro

Caveolin-1 (Cav-1) is an important modulator for adult neurogenesis in post stroke brain repair but its underlying mechanisms are largely unknown. In the present study, we report that endothelial Cav-1 inhibits neuronal differentiation of neural stem/progenitor cells (NSCs/NPCs) in post ischemic brain via regulating vascular endothelial growth factor (VEGF) and NeuroD1 signaling pathway. We first investigated the dynamic change of Cav-1 and its impact on neuronal differentiation in rat and mouse models of 2 h transient middle cerebral artery occlusion (MCAO) plus 1, 7, 14, 21 and 28 day of reperfusion. We then studied the roles of endothelial Cav-1 in modulating the neuronal differentiation of NPCs which were co-cultured with brain microvascular endothelial cells (BMVECs) under 2 h oxygen-glucose deprivation plus 5 days reoxygenation (OGD/R). The major discoveries include: (1) Cav-1 expression in the hippocampal dentate gyrus (DG) was down-regulated on day 1 after 2 h cerebral ischemia, and gradually recovered with reperfusion time, accompanied with transient increased but gradually reduced neuronal differentiation of NPCs marked by doublecortin (DCX). (2) Cav-1 knockout mice exhibited the increased DCX and VEGF at the granular cell layers of hippocampal DG in post-ischemic brains. (3) Co-cultured with BMVECs, NPCs had remarkably decreased neuronal differentiation under OGD/R. Knockdown of Cav-1 in the BMVECs increased VEGF secretion into the medium and NeuroD1+ cells, and rescued the neuronal differentiation of NPCs without affecting astroglial and oligodendroglial differentiation. (4) Cav-1 exosomes released from BMVECs inhibited neuronal differentiation of NPCs via decreasing the expression of VEGF, p44/42MAPK phosphorylation and NeuronD1 upon OGD/R insults. Taken together, endothelial Cav-1 serves as a niche regulator to inhibit neuronal differentiation via negatively modulating VEGF, p44/42MAPK phosphorylation and NeuronD1 signaling pathway.

Preventive Triple Gene Therapy Reduces the Negative Consequences of Ischemia-Induced Brain Injury after Modelling Stroke in a Rat.

Currently, the main fundamental and clinical interest for stroke therapy is focused on developing a neuroprotective treatment of a penumbra region within the therapeutic window. The development of treatments for ischemic stroke in at-risk patients is of particular interest. Preventive gene therapy may significantly reduce the negative consequences of ischemia-induced brain injury. In the present study, we suggest the approach of preventive gene therapy for stroke. Adenoviral vectors carrying genes encoding vascular endothelial growth factor (VEGF), glial cell-derived neurotrophic factor (GDNF) and neural cell adhesion molecule (NCAM) or gene engineered umbilical cord blood mononuclear cells (UCB-MC) overexpressing recombinant VEGF, GDNF, and NCAM were intrathecally injected before distal occlusion of the middle cerebral artery in rats. Post-ischemic brain recovery was investigated 21 days after stroke modelling. Morphometric and immunofluorescent analysis revealed a reduction of infarction volume accompanied with a lower number of apoptotic cells and decreased expression of Hsp70 in the peri-infarct region in gene-treated animals. The lower immunopositive areas for astrocytes and microglial cells markers, higher number of oligodendrocytes and increased expression of synaptic proteins suggest the inhibition of astrogliosis, supporting the corresponding myelination and functional recovery of neurons in animals receiving preventive gene therapy. In this study, for the first time, we provide evidence of the beneficial effects of preventive triple gene therapy by an adenoviral- or UCB-MC-mediated intrathecal simultaneous delivery combination of vegf165, gdnf, and ncam1 on the preservation and recovery of the brain in rats with subsequent modelling of stroke.

Inhibition of cyclooxygenase-1 does not reduce mortality in post-ischemic stroke rats

BackgroundIschemic stroke is one of the leading causes of mortality and morbidity. The currently available non-invasive therapeutic options are not sufficiently efficacious. Post-ischemic brain is characterized by a prominent inflammatory response. Little is known about the involvement of cyclooxygenase (COX)-1 in the pathophysiology of ischemic stroke. ObjectiveThis study was undertaken to examine the effects of a highly selective COX-1 inhibitor – mofezolac – on clinical outcomes and brain inflammatory markers in post-stroke rats. MethodsStroke was induced by subjecting rats to permanent middle cerebral artery occlusion (MCAO). Control rats underwent a sham surgery. Rats were treated with mofezolac (50 mg/kg, intraperitoneally [ip]) once daily for 14 days. Control animals were treated with vehicle. Body temperature (BT), neurological score (NS) and cumulative mortality were monitored at different time points. At the end of the experiment, rats were euthanized and three brain regions (hypothalamus, hippocampus and frontal cortex) were extracted. Levels of interleukin (IL)-6, prostaglandin (PG)E2 and tumor necrosis factor (TNF)-α in these brain regions were determined by ELISA kits. ResultsBT, NS and cumulative mortality were all significantly higher in post-MCAO rats than in sham-operated rats, irrespective of the treatment given. BT, NS and mortality rate did not differ significantly between mofezolac-treated and vehicle-treated sham-operated animals. BT was significantly lower in mofezolac-treated as compared to vehicle-treated post-MCAO rats. Mofezolac did not significantly alter NS in post-MCAO rats at any time-point. Cumulative 14-day mortality was non-significantly higher in mofezolac-treated as compared to vehicle-treated post-MCAO rats (48 % vs. 21 %, respectively; P = 0.184). Mostly, IL-6 and TNF-α levels did not differ between post-MCAO and sham-operated rats and were not affected by mofezolac treatment. In contrast, mofezolac significantly decreased PGE2 levels in post-MCAO rats’ brains. ConclusionOverall, these results suggest that chronic treatment with the selective COX-1 inhibitor mofezolac did not reduce morbidity or mortality in post-stroke rats.

Proteomics-Guided Study on Buyang Huanwu Decoction for Its Neuroprotective and Neurogenic Mechanisms for Transient Ischemic Stroke: Involvements of EGFR/PI3K/Akt/Bad/14-3-3 and Jak2/Stat3/Cyclin D1 Signaling Cascades.

Buyang Huanwu Decoction (BHD), a classic traditional Chinese medicine (TCM) formula, has been used for recovering neurological dysfunctions and treating post-stroke disability in China for 200years. In the present study, we investigated the effects of BHD on inhibiting neuronal apoptosis, promoting proliferation and differentiation of neural stem cells (NSCs) and neurite formation and enhancing learning and memory functional recovery in an experimental rat ischemic stroke model. BHD significantly reduced infarct volume and decreased cell apoptosis in the ischemic brain. BHD enhanced neuronal cell viability in vitro. BHD dose-dependently promoted the proliferation of NSCs in ischemic rat brains in vivo. Moreover, BHD promoted neuronal and astrocyte differentiation in primary cultured NSCs in vitro. Water maze test revealed that BHD promoted the recovery of learning function but not memory functions in the transient ischemic rats. We then investigated the changes of the cellular signaling molecules by using two-dimension (2D) gel electrophoresis and focused on the PI3K/Akt/Bad and Jak2/Stat3/cyclin D1signaling pathway to uncover its underlying mechanisms for its neuroprotective and neurogenetic effects. BHD significantly upregulated the expression of p-PI3K, p-Akt, and p-Bad as well as the expression of p-Jak, p-Stat3, and cyclin D1 in vitro and in vivo. In addition, BHD upregulated Hes1 and downregulated cav-1 in vitro and in vivo. Taken together, these results suggest that BHD has neuroprotective effects and neurogenesis-promoting effects via activating PI3K/Akt/Bad and Jak2/Stat3/Cyclin D1 signaling pathways. Graphical Abstract Buyang Huanwu Decoction (BHD) activates the PI3K-AKT-BAD pathway in the ischemic brain for neuroprotection. BHD also activates JAK2/STAT3/Cyclin D1 signaling cascades for promoting neurogenesis in the hippocampus of post-ischemic brains. Moreover, BHD inhibits the expression of caveolin-1 and increases the expression of HES1 for promoting neuronal differentiation. The neuroprotective and neurogenesis-promoting effects in the hippocampus of post-ischemic brains promote learning ability.

Sustained Increases in Immune Transcripts and Immune Cell Trafficking During the Recovery of Experimental Brain Ischemia.

Stroke is a major cause of chronic neurological disability. There is considerable interest in understanding how acute transcriptome changes evolve into subacute and chronic patterns that facilitate or limit spontaneous recovery. Here we mapped longitudinal changes in gene expression at multiple time points after stroke in mice out to 6 months. Adult C57BL/6 mice were subjected to transient middle cerebral artery occlusion. Longitudinal transcriptome levels were measured at 10 time points after stroke from acute to recovery phases of ischemic stroke. Localization and the number of mononuclear phagocytes were determined in the postischemic brain. Whole-mount brain imaging was performed in asplenic mice receiving GFP+ (green fluorescent protein)-tagged splenocytes. Sustained stroke-induced mRNA abundance changes were observed in both hemispheres with 2989 ipsilateral and 822 contralateral genes significantly perturbed. In the hemisphere ipsilateral to the infarct, genes associated with immune functions were strongly affected, including temporally overlapping innate and adaptive immunity and macrophage M1 and M2 phenotype-related genes. The strong immune gene activation was accompanied by the sustained infiltration of peripheral immune cells at acute, subacute, and recovery stages of stroke. The infiltrated immune cells were found in the infarcted area but also in remote regions at 2 months after stroke. The study identifies that immune components are the predominant molecular signatures and they may propagate or continuously respond to brain injury in the subacute to chronic phase after central nervous system injury. The study suggests a potential immune-based strategy to modify injury progression and tissue remodeling in ischemic stroke, even months after the initiating event.

Gonadal Hormones E2 and P Mitigate Cerebral Ischemia-Induced Upregulation of the AIM2 and NLRC4 Inflammasomes in Rats.

Acute ischemic stroke (AIS) is a devastating neurological condition with a lack of neuroprotective therapeutic options, despite the reperfusion modalities thrombolysis and thrombectomy. Post-ischemic brain damage is aggravated by an excessive inflammatory cascade involving the activation and regulation of the pro-inflammatory cytokines IL-1β and IL-18 by inflammasomes. However, the role of AIM2 and NLRC4 inflammasomes and the influence of the neuroprotective steroids 17β-estradiol (E2) and progesterone (P) on their regulation after ischemic stroke have not yet been conclusively elucidated. To address the latter, we subjected a total of 65 rats to 1 h of transient Middle Cerebral Artery occlusion (tMCAO) followed by a reperfusion period of 72 h. Moreover, we evaluated the expression and regulation of AIM2 and NLRC4 in glial single-cell cultures (astroglia and microglia) after oxygen–glucose deprivation (OGD). The administration of E2 and P decreased both infarct sizes and neurological impairments after cerebral ischemia in rats. We detected a time-dependent elevation of gene and protein levels (Western Blot/immunohistochemistry) of the AIM2 and NLRC4 inflammasomes in the post-ischemic brains. E2 or P selectively mitigated the stroke-induced increase of AIM2 and NLRC4. While both inflammasomes seemed to be exclusively abundant in neurons under physiological and ischemic conditions in vivo, single-cell cultures of cortical astrocytes and microglia equally expressed both inflammasomes. In line with the in vivo data, E and P selectively reduced AIM2 and NLRC4 in primary cortical astrocytes and microglial cells after OGD. In conclusion, the post-ischemic elevation of AIM2 and NLRC4 and their down-regulation by E2 and P may shed more light on the anti-inflammatory effects of both gonadal hormones after stroke.

Participation of Amyloid and Tau Protein in Neuronal Death and Neurodegeneration after Brain Ischemia.

Current evidence indicates that postischemic brain injury is associated with the accumulation of folding proteins, such as amyloid and tau protein, in the intra- and extracellular spaces of neuronal cells. In this review, we summarize protein changes associated with Alzheimer’s disease and their gene expression (amyloid protein precursor and tau protein) after brain ischemia, and their roles in the postischemic period. Recent advances in understanding the postischemic mechanisms in development of neurodegeneration have revealed dysregulation of amyloid protein precursor, α-, β- and γ-secretase and tau protein genes. Reduced expression of the α-secretase gene after brain ischemia with recirculation causes neuronal cells to be less resistant to injury. We present the latest data that Alzheimer’s disease-related proteins and their genes play a crucial role in postischemic neurodegeneration. Understanding the underlying processes of linking Alzheimer’s disease-related proteins and their genes in development of postischemic neurodegeneration will provide the most significant goals to date for therapeutic development.

Lysophosphatidic Acid Receptor 5 Plays a Pathogenic Role in Brain Damage after Focal Cerebral Ischemia by Modulating Neuroinflammatory Responses.

Receptor-mediated lysophosphatidic acid (LPA) signaling has come to be considered an important event for various diseases. In cerebral ischemia, LPA1 has recently been identified as a receptor subtype that mediates brain injury, but the roles of other LPA receptor subtypes remain unknown. Here, we investigated the potential role of LPA5 as a novel pathogenic factor for cerebral ischemia using a mouse model of transient middle cerebral artery occlusion (tMCAO). LPA5 was upregulated in the ischemic core region after tMCAO challenge, particularly in activated microglia. When TCLPA5, a selective LPA5 antagonist, was given to tMCAO-challenged mice immediately after reperfusion, brain damage, including brain infarction, functional neurological deficit, and neuronal and non-neuronal apoptosis, was reduced in mice. Similarly, delayed TCLPA5 administration (at three hours after reperfusion) reduced brain infarction and neurological deficit. The histological results demonstrated that TCLPA5 administration attenuated microglial activation, as evidenced by the decreased Iba1 immunoreactivities, the number of amoeboid cells, and proliferation in an injured brain. TCLPA5 administration also attenuated the upregulation of the expression of pro-inflammatory cytokines at mRNA levels in post-ischemic brain, which was also observed in lipopolysaccharide-stimulated BV2 microglia upon LPA5 knockdown. Overall, this study identifies LPA5 as a novel pathogenic factor for cerebral ischemia, further implicating it as a promising target for drug development to treat this disease.

Effects of β-Adrenergic Blockade on Metabolic and Inflammatory Responses in a Rat Model of Ischemic Stroke.

Ischemic stroke provokes an inflammatory response concurrent with both sympathetic nervous system activation and hyperglycemia. Currently, their crosstalk and consequences in stroke outcomes are of clinical attraction. We have provided experimental evidence showing the suppressive effects of the nonselective β-adrenoreceptor antagonist propranolol on hyperglycemia, inflammation, and brain injury in a rat model experiencing cerebral ischemia. Pretreatment with propranolol protected against postischemic brain infarction, edema, and apoptosis. The neuroprotection caused by propranolol was accompanied by a reduction in fasting glucose, fasting insulin, glucose tolerance impairment, plasma C-reactive protein, plasma free fatty acids, plasma corticosterone, brain oxidative stress, and brain inflammation. Pretreatment with insulin alleviated—while glucose augmented—postischemic brain injury and inflammation. Additionally, the impairment of insulin signaling in the gastrocnemius muscles was noted in rats with cerebral ischemia, with propranolol improving the impairment by reducing oxidative stress and tumor necrosis factor-α signaling. The anti-inflammatory effects of propranolol were further demonstrated in isoproterenol-stimulated BV2 and RAW264.7 cells through its ability to decrease cytokine production. Despite their potential benefits, stroke-associated hyperglycemia and inflammation are commonly linked with harmful consequences. Our findings provide new insight into the anti-inflammatory, neuroprotective, and hypoglycemic mechanisms of propranolol in combating neurodegenerative diseases, such as stroke.

Lentivirally administered glial cell line-derived neurotrophic factor promotes post-ischemic neurological recovery, brain remodeling and contralesional pyramidal tract plasticity by regulating axonal growth inhibitors and guidance proteins

Owing to its potent longterm neuroprotective and neurorestorative properties, glial cell line-derived neurotrophic factor (GDNF) is currently studied in neurodegenerative disease clinical trials. However, little is known about the longterm effect of GDNF on neurological recovery, brain remodeling and neuroplasticity in the post-acute phase of ischemic stroke. In a comprehensive set of experiments, we examined the effects of lentiviral GDNF administration after ischemic stroke. GDNF reduced neurological deficits, neuronal injury, blood-brain barrier permeability in the acute phase in mice. As compared with control, enhanced motor-coordination and spontaneous locomotor activity were noted in GDNF-treated mice, which were associated with increased microvascular remodeling, increased neurogenesis and reduced glial scar formation in the peri-infarct tissue. We observed reduced brain atrophy and increased plasticity of contralesional pyramidal tract axons that crossed the midline in order to innervate denervated neurons in the ipsilesional red and facial nuclei. Contralesional axonal plasticity by GDNF was associated with decreased abundance of the axonal growth inhibitors brevican and versican in contralesional and ipsilesional brain tissue, reduced abundance of the growth repulsive guidance molecule ephrin b1 in contralesional brain tissue, increased abundance of the midline growth repulsive protein Slit1 in contralesional brain tissue and reduced abundance of Slit1's receptor Robo2 in ipsilesional brain tissue. These data indicate that GDNF potently induces longterm neurological recovery, peri-infarct brain remodeling and contralesional neuroplasticity, which are associated with the fine-tuned regulation of axonal growth inhibitors and guidance molecules that facilitate the growth of contralesional corticofugal axons in the direction to the ipsilesional hemisphere.

Reduced Post-ischemic Brain Injury in Transient Receptor Potential Vanilloid 4 Knockout Mice.

Background and PurposeIn the acute phase of ischemia-reperfusion, hypoperfusion associated with ischemia and reperfusion in microvascular regions and disruption of the blood–brain barrier (BBB) contribute to post-ischemic brain injury. We aimed to clarify whether brain injury following transient middle cerebral artery occlusion (tMCAO) is ameliorated in Transient receptor potential vanilloid 4 knockout (Trpv4–/–) mice.MethodstMCAO was induced in wild-type (WT) and Trpv4–/– mice aged 8–10 weeks. Ischemia-induced lesion volume was evaluated by 2,3,5-triphenyltetrazolium chloride staining at 24 h post-tMCAO. Tissue water content and Evans blue leakage in the ipsilateral hemisphere and a neurological score were evaluated at 48 h post-tMCAO. Transmission electron microscopy (TEM) was performed to assess the morphological changes in microvasculature in the ischemic lesions at 6 h post-tMCAO.ResultsCompared with WT mice, Trpv4–/– mice showed reduced ischemia-induced lesion volume and reduced water content and Evans blue leakage in the ipsilateral hemisphere alongside milder neurological symptoms. The loss of zonula occludens-1 and occludin proteins in the ipsilateral hemisphere was attenuated in Trpv4–/– mice. TEM revealed that parenchymal microvessels in the ischemic lesion were compressed and narrowed by the swollen endfeet of astrocytes in WT mice, but these effects were markedly ameliorated in Trpv4–/– mice.ConclusionThe present results demonstrate that TRPV4 contributes to post-ischemic brain injury. The preserved microcirculation and BBB function shortly after reperfusion are the key neuroprotective roles of TRPV4 inhibition, which represents a promising target for the treatment of acute ischemic stroke.

Shared Genomic and Proteomic Contribution of Amyloid and Tau Protein Characteristic of Alzheimer's Disease to Brain Ischemia.

Post-ischemic brain damage is associated with the deposition of folding proteins such as the amyloid and tau protein in the intra- and extracellular spaces of brain tissue. In this review, we summarize the protein changes associated with Alzheimer’s disease and their gene expression (amyloid protein precursor and tau protein) after ischemia-reperfusion brain injury and their role in the post-ischemic injury. Recent advances in understanding the post-ischemic neuropathology have revealed dysregulation of amyloid protein precursor, α-secretase, β-secretase, presenilin 1 and 2, and tau protein genes after ischemic brain injury. However, reduced expression of the α-secretase in post-ischemic brain causes neurons to be less resistant to injury. In this review, we present the latest evidence that proteins associated with Alzheimer’s disease and their genes play a key role in progressive brain damage due to ischemia and reperfusion, and that an ischemic episode is an essential and leading supplier of proteins and genes associated with Alzheimer’s disease in post-ischemic brain. Understanding the underlying processes of linking Alzheimer’s disease-related proteins and their genes in post-ischemic brain injury with the risk of developing Alzheimer’s disease will provide the most significant goals for therapeutic development to date.

Abstract 361: The Significance of Higher Mean Arterial Pressure After Inpatient Cardiac Arrests in Improving Neurological Outcome and Mortality

Following cardiac arrest and the return of spontaneous circulation (ROSC), hemodynamic status can be critically unstable which may lead to the hypoperfusion of vital organs and poor clinical outcomes. In post-cardiac arrest survivors, studies have shown improved outcomes with a higher mean arterial pressure (MAP) compared with a lower MAP, however an ideal range of MAP post-ROSC is rarely explicitly defined in post-resuscitation care studies. The purpose of this study was to observe neurological and mortality outcomes in cardiac arrest patients with a lower range of post-ROSC MAP compared to a higher range of post-ROSC MAP.A retrospective single-center cohort study was used to design the project. Patients who met the inclusion criteria suffered a cardiac arrest while admitted to the hospital, achieved ROSC, and survived for at least 48 hours post-ROSC. Patients whose status was changed to DNR by 48 hours post-ROSC were excluded. The remaining patients were divided into two groups. The lower MAP group had an average MAP of 60 to 80 mmHg and the higher MAP group had an average MAP of 80 to 100 mmHg at 48 hours post-ROSC. The primary outcome analyzed was the presence of anoxic brain injury noted on EEG. Secondary outcomes were the length of intubation, ICU length of stay (LOS), and mortality rate. Of the total of 129 patients, 18 patients met our inclusion criteria. Of these, 10 patients met the lower MAP group and 8 patients met the higher MAP group. Anoxic brain injury was 20% in the lower MAP group compared to 12.5% in the higher MAP group (p>0.05). There was a 40% mortality in the lower MAP group, compared to 12.5% mortality in the higher MAP group (p>0.05) 48 hours post-ROSC. The mean length of intubation was 3.5 days in the higher MAP group compared to 4.9 days in the lower MAP group (p>0.05). There was no difference in the ICU LOS amongst the two groups. Our results showed a clinically significant difference between the two groups but could not reach statistical significance due to the small sample size. The optimal MAP for post-cardiac arrest patients has not been clearly defined by clinical trials. The simultaneous need to perfuse the post-ischemic brain adequately without putting unnecessary strain on the post-ischemic heart is unique to the post-cardiac arrest syndrome. The findings of this study show post-ROSC MAP maintained between 80 to 100 mmHg had a statistically insignificant tendency toward better neurological outcomes, decreased length of intubation and improved mortality compared to the group whose MAP was maintained between 60 to 80 mmHg at 48 hours. The small sample size is a limitation for this study, however, this preliminary study has shown promising results and it is predicted that a bigger population study with similar parameters will extrapolate similar results.

C3a receptor antagonist therapy is protective with or without thrombolysis in murine thromboembolic stroke.

Intravenous thrombolysis (IVT) after stroke enhances C3a generation, which may abrogate the benefits of reperfusion. The C3aR antagonist SB290157 is neuroprotective following transient but not permanent middle cerebral artery occlusion (MCAo). SB290157 remains untested in thromboembolic (TE) models, which better approximate human stroke and also facilitate testing in combination with IVT. We hypothesized SB290157 would confer neuroprotection in TE stroke with and without "late" IVT. We used two different models of TE stroke to examine the efficacy of SB290157 alone and in combination with late IVT. We evaluated the benefit of SB290157 in attenuating post-ischaemic behavioural deficits, infarction, brain oedema and haemorrhage. Plasma C3a was elevated 6 hr after TE stroke alongside increased cerebrovascular C3aR expression, which was sustained to 4 weeks. Increased C3aR expression also was visualized in human ischaemic brain. In a photothrombotic (PT) stroke model, which exhibits rapid spontaneous reperfusion, SB290157 given at 1 hr post-PT significantly improved neurofunction and reduced infarction at 48 hr. In an embolic (eMCAo) model, SB290157 administered at 2 hr improved histological and functional outcomes. Conversely, late IVT administered 4.5 hr post-eMCAo was ineffective likely due to increased haemorrhage and brain oedema. However, SB290157 administered prior to late IVT ameliorated haemorrhage and oedema and improved outcomes. We conclude that SB290157 is safe and effective with and without late IVT following TE stroke. Therefore, C3a receptor antagonist therapy represents a promising candidate for clinical translation in stroke, particularly as an adjuvant to IVT.

High fructose diet-induced obesity worsens post-ischemic brain injury in the hippocampus of female rats

ABSTRACT Objectives: Cerebral ischemia is caused by a reduction of the blood flow in a specific area in the brain, triggering cellular cascades in the tissue that result in neuronal death. This phenomenon leads to neurological decline in patients with stroke. The extent of the injury after stroke could be related to the condition of obesity. Thus, we aim to analyze the effect of obesity induced by a high fructose diet (HFD) on the brain after cerebral ischemia in rats. Methods: We induced the obesity model in female Wistar rats with 20% fructose in water for 11 weeks. We then performed cerebral ischemia surgery (2-vessel occlusion), carried out the neurological test 6, 24 and 48 h post-ischemia and analyzed the histological markers. Results: The HFD induced an obese phenotype without insulin resistance. The obese rats exhibited worse neurological performance at 6 h post-ischemia and showed neuronal loss and astroglial and microglial immunoreactivity changes in the caudate putamen, motor cortex, amygdala and hippocampus at 48 h post-ischemia. However, the most commonly affected area was the hippocampus, where we found an increase in interleukin 1β in the blood vessels of the dentate gyrus, a remarkable disruption of MAP-2+ dendrites, a loss of brain-derived neurotrophic factor and the presence of PHF-tau. In conclusion, a HFD induces an obese phenotype and worsens the neuronal loss, inflammation and plasticity impairment in the hippocampus after cerebral ischemia.

The Expression of GLAST and GLT1 in a Transient Cerebral Ischemia Mongolian Gerbil Model.

PurposeExcitatory amino acid transporters (EAATs) have an indispensable function in the reuptake of extracellular glutamate. To investigate the relationship and the expression of neuronal and astrocytic markers after brain ischemia, the temporal profile of glial EAATs in both peripheral and core regions of the cortex was examined.MethodsTransient common carotid artery occlusion was used to induce unilateral transient forebrain ischemia of Mongolian gerbils, and post-ischemic brains (6 h to 2 w) were collected and prepared for immunohistochemical and Western blotting analysis of glutamine synthetase (GS), GLT-1, GLAST, S100β, and NeuN, and for Alizarin red staining of calcium deposits.ResultsThe expression of GLAST and GLT-1 were significantly escalated at 6 h both in the core and periphery regions, while reduced from 12 h to 2 w in the core region post-ischemia. GS-positive cells increased at 6 h both in the core and periphery regions, while the density of Alizarin red-positive cells increased and peaked at 12 h in the ischemic cortex. The density of S100β-positive cells decreased in the ischemic core and increased in the periphery region. Immunofluorescence staining showed that S100β and TUNEL double-positive cells increased at 12 h in the core region.ConclusionThe results of GLT-1 and GLAST expression in the cortex indicate that their up-regulation was time-dependent and occurred in the acute post-ischemia period, whereas their down-regulation was region-dependent and it is involved in the pathological progress of nerve cell and glial cell death, and has a series of cascade reactions.

Abstract 111: Myeloid Specific Deletion of CD36 Increases Microglia-Like CD45 Low Subset and Exacerbates Stroke-Induced Brain Injury

Objective: CD36 regulates biological and pathological processes including inflammation, resolution, phagocytosis, and angiogenesis. Highly expressed in monocytes/macrophages (MM), MM CD36 has been implicated in both detrimental and beneficial effects in acute stroke outcome and recovery. Using myeloid specific deficiency of CD36 (CD36KO MM ) mice, the present study addresses the role of MM CD36 on subset composition of MM in the acute and recovery phases of stroke. Methods: WT (CD36 fl/fl ) and CD36KO MM (M/F, 12w old) mice were subjected to transient focal ischemia. Brain immune cells were collected at 3d, 7d and 2m (n=8-12/timepoint). Infarct volume and hemispheric swelling were measured at 3d (n=13/genotype). In the isolated brain immune cells, CD11b+ cells were further distinguished by CD45 Hi and CD45 Low subsets. The extent of MM trafficking was determined by an intravenous infusion of control GFP+ splenocytes into the stroked WT and CD36KO MM mice 1d prior to sacrifice and MM subset analyses. Results: Compared to WT mice, CD36KO MM mice showed no difference in CD45 Low subset at 3d, 7d and 2m after stroke. However, CD36KO MM mice displayed a significant reduction of CD45 Hi subset, presumably infiltrating cells, at these time points (n=4-8 /group, P<0.01). Adoptive transfer of control GFP+ splenocytes to stroked CD36KO MM mice resulted in an increase of GFP+ cells in CD45 Low but a profound reduction in CD45 Hi subset. Despite the near absence of CD45 Hi and increased CD45 Low population in the post-ischemic brain, CD36KO MM mice displayed exacerbation of brain injury (15.1±13.7 vs 25.8±17.5 mm 3 , p<0.05) and brain swelling (12.1±10.3 vs 18.4±12.7 %, p=0.1). Conclusion: The observed reduction of CD45 Hi subset and exacerbation of brain injury in CD36KO MM mice suggests a beneficial role of infiltrated CD45 Hi MMs in limiting stroke injury. The sustained trafficking at 2m indicates potential involvement of MM CD36 in the recovery process. Further studies to assess long-term outcomes of CD36KO MM mice will provide critical insights on the role of macrophage CD36 in stroke recovery.

Abstract TP120: Complement Activation Contributes to Malignant Transformation of Stroke and Limits Cognitive Recovery After Reperfusion Therapy

Introduction: Ischemic stroke involves an ischemic event followed by post-ischemic neuroinflammation, leading to acute mortality, neuronal loss, and impaired recovery. Standard of care reperfusion therapy (thrombolysis or thrombectomy) has a limited treatment window due to risk of hemorrhage, and leads to functional independence in only a subset of patients. We studied the role of complement and neuroinflammation in the pathophysiology behind the major clinical challenges of reperfusion therapy; the risk of hemorrhage and limited cognitive recovery. Methods: We used 3 stroke models, namely transient middle cerebral artery occlusion (MCAO), permanent MCAO and microembolic models, to investigate the effects of t-PA, mechanical reperfusion, rehabilitation therapy, and complement inhibition in mice. We used B4-Crry, a site-targeted inhibitor of C3 activation that specifically localizes to neoepitopes expressed in the post-ischemic brain following its intravenous administration. A retrospective analysis was also performed for cognitive and motor recovery in human patients following reperfusion therapy. Results: Complement inhibition with B4-Crry improved the safety, efficacy and treatment window of reperfusion therapy, decreased hemorrhagic transformation, and improved cognitive recovery. Despite reperfusion with t-PA, there was ongoing complement-dependent microglial activation and phagocytosis of hippocampal synapses for at least 30 days leading to pronounced long-term cognitive deficits. Administration of B4Crry, alone or in combination with thrombolytic therapy, limited complement deposition in the perilesional brain, and reduced microgliosis and synaptic uptake. Reperfusion therapy alone did not improve cognitive outcomes, even when combined with rehabilitation therapy. Reperfusion therapy did not improve cognitive outcomes in stroke patients. Conclusions: Although post-stroke reperfusion therapy limits the size of injury and improves motor deficits, post-reperfusion complement activation and neuroinflammation contribute to the malignant transformation of stroke acutely and seeds a chronic neurodegenerative inflammatory response. Complement modulation may be a promising adjuvant to reperfusion therapy.

Abstract WP314: Reduced Post-Ischemic Brain Tissue Injury Following Transient Focal Cerebral Ischemia in Transient Receptor Potential Vanilloid 4 Knockout Mice

Background: In the acute phase of ischemia-reperfusion, post-ischemic delayed hypoperfusion in microvascular regions contributes to post-ischemic brain tissue injury. Transient receptor potential vanilloid 4 (TRPV4) is a nonselective cation channel expressed on astrocyte endfeet regulating vascular tone, which may contribute to ischemia-induced brain tissue injury. Objective: To clarify whether brain tissue injury following transient middle cerebral artery occlusion (tMCAO) is ameliorated by Trpv4 knockout ( Trpv4 -/- ). Methods: tMCAO was induced by an insertion of silicon-coated filament from right common carotid artery to occlude right MCA for 30 minutes for wild type (WT) and Trpv4 -/- mice aged 8-10 weeks (median [interquartile] weight: 23.3 [21.7-24.9] g for WT mice and 24.1 [20.8-26.2] g for Trpv4 -/- mice). Ischemia-induced lesion volume was evaluated by 2,3,5-Triphenyltetrazolium chloride staining at 24 hours post-tMCAO, and tissue water content and Evans blue leakage in the ipsilateral hemisphere alongside 18-point neurological score were evaluated at 48 hours post-tMCAO. Transmission electron microscopy (TEM) was performed to assess the morphological changes in the microvasculature in the ischemic lesions at 6 hours post-tMCAO. Results: Compared with WT mice, Trpv4 -/- mice showed reduced ischemia-induced lesion volumes (median 40 mm 3 vs. 53 mm 3 , p < 0.05) at 24 hours and reduced tissue water content by subtraction of the contralateral from the ipsilateral hemisphere (median 4.9% vs. 2.8%, p < 0.05) and Evans blue leakage by ratio of ipsilateral to contralateral hemisphere (median 2.3 vs. 3.2, p < 0.05) alongside milder neurological score (median 12 vs. 10, p < 0.01) at 48 hours post-tMCAO. TEM revealed that parenchymal microvessels in the ischemic lesion were compressed and narrowed by the swollen endfeet of astrocytes in a WT mouse, whereas those were markedly reduced in a Trpv4 -/ - mouse. Conclusions: Trpv4 -/- mice showed reduced post-ischemic brain tissue injury. Suppressing the astrocyte endfeet swelling shortly after reperfusion is one of the key neuroprotective mechanisms of TRPV4 inhibition, which is a promising target for the treatment of acute ischemic stroke.