Intraluminal Middle Cerebral Artery Research Articles

Selective Sphingosine 1-Phosphate Receptor 1 Agonist Is Protective Against Ischemia/Reperfusion in Mice.

Growing evidence supports that the immunomodulatory drug fingolimod is protective in stroke. Fingolimod binds to 4 of 5 sphingosine-1-phosphate (S1P) receptors and, among other actions, it induces lymphopenia. In this study, we investigated whether a selective S1P1 agonist is protective in experimental stroke. Drug selectivity was studied in vitro in cells overexpressing the human S1P receptors. Mice (n=54) received different doses of LASW1238 (3 or 10 mg/kg), fingolimod (1 mg/kg), or the vehicle intraperitoneal, and lymphopenia was studied at different time points. After intraluminal middle cerebral artery occlusion for 45 minutes and immediately after reperfusion, mice (n=56) received the drug treatment. At 24 hours, a neurological test was performed and infarct volume was measured. Treatment and all the analyses were performed in a blind fashion. In vitro functional assays showed that LASW1238 is a selective agonist of the S1P1 receptor. At 10 mg/kg, this compound induced sustained lymphopenia in mice comparable with fingolimod, whereas at 3 mg/kg it induced short-lasting lymphopenia. After ischemia, both LASW1238 (10 mg/kg) and fingolimod reduced infarct volume, but only LASW1238 (10 mg/kg) showed statistically significant differences versus the vehicle. The neurological function and plasma cytokine levels were not different between groups. The selective S1P1 agonist LASW1238 reduces infarct volume after ischemia/reperfusion in mice, but only when lymphopenia is sustained for at least 24 hours. S1P1 and lymphocytes are potential targets for drug treatment in stroke. Defining the best drug dosing regimens to control the extent and duration of lymphopenia is critical to achieve the desired effects.

A modification of intraluminal middle cerebral artery occlusion/reperfusion model for ischemic stroke with laser Doppler flowmetry guidance in mice.

Stroke is one of the common causes of death and disability in the world. The intraluminal middle cerebral artery occlusion/reperfusion (MCAO/R) model is a “gold standard” in surgical ischemic stroke models. Here, we optimized the procedure of this model by ligating on external carotid artery (ECA) stump and two ligatures prepared on internal carotid artery, which could improve the success and survival rate in mice. The results show that ECA approach was superior to common carotid artery approach. Meanwhile, we found that the exposure of pterygopalatine artery was not an essential step for MCAO/R model in mice.

Post-acute delivery of memantine promotes post-ischemic neurological recovery, peri-infarct tissue remodeling, and contralesional brain plasticity

The NMDA antagonist memantine preferentially inhibits extrasynaptic NMDA receptors, which are overactivated upon stroke and thought to disturb neuroplasticity. We hypothesized that memantine enhances post-ischemic neurological recovery, brain remodeling, and plasticity. C57BL6/j mice were exposed to intraluminal middle cerebral artery occlusion. Starting 72 hours post-stroke, vehicle or memantine (4 or 20 mg/kg/day) were subcutaneously delivered over 28 days. Neurological recovery, perilesional tissue remodeling and contralesional pyramidal tract plasticity were evaluated over 49 days. Memantine, delivered at 20 but not 4 mg/kg/day, persistently improved motor-coordination and spatial memory. Secondary striatal atrophy was reduced by memantine. This delayed neuroprotection was associated with reduced astrogliosis and increased capillary formation around the infarct rim. Concentrations of BDNF, GDNF, and VEGF were bilaterally elevated by memantine in striatum and cortex. Anterograde tract tracing studies revealed that memantine increased contralesional corticorubral sprouting across the midline in direction to the ipsilesional red nucleus. In the contralesional motor cortex, the NMDA receptor subunit GluN2B, which is predominantly expressed in extrasynaptic NMDA receptors, was transiently reduced by memantine after 14 days, whereas GluN2A and PSD-95, which preferentially co-localize with synaptic NMDA receptors, were increased after 28 days. Our data suggest the utility of memantine for enhancing post-acute stroke recovery.

The glial scar is more than just astrocytes

Cellular responses in glia play a key role in regulating brain remodeling post-stroke. However, excessive glial reactivity impedes post-ischemic neuroplasticity and hampers neurological recovery. While damage-associated molecular patterns and activated microglia were shown to induce astrogliosis, the molecules that restrain astrogliosis are largely unknown. We explored the role of tenascin-C (TnC), an extracellular matrix component involved in wound healing and remodeling of injured tissues, in mice exposed to ischemic stroke induced by transient intraluminal middle cerebral artery occlusion. In the healthy adult brain, TnC expression is restricted to neurogenic stem cell niches. We previously reported that TnC is upregulated in ischemic brain lesions. We herein show that the de novo expression of TnC post-stroke is closely associated with reactive astrocytes, and that astrocyte reactivity at 14 days post-ischemia is increased in TnC-deficient mice (TnC−/−). By analyzing the three-dimensional morphology of astrocytes in previously ischemic brain tissue, we revealed that TnC−/− reduces astrocytic territorial volume, branching point number, and branch length, which are presumably hallmarks of the homeostatic regulatory astrocyte state, in the post-acute stroke phase after 42 days. Interestingly, TnC−/− moderately increased aggrecan, a neuroplasticity-inhibiting proteoglycan, in the ischemic brain tissue at 42 days post-ischemia. In vitro in astrocyte-microglia cocultures, we showed that TnC−/− reduces the microglial migration speed on astrocytes and elevates intercellular adhesion molecule 1 (ICAM1) expression. Post-stroke, TnC−/− did not alter the ischemic lesion size or neurological recovery, however microglia-associated ICAM1 was upregulated in TnC−/− mice during the first week post stroke. Our data suggest that TnC plays a central role in restraining post-ischemic astrogliosis and regulating astrocyte-microglial interactions.

Abstract 69: Cerebellar Dentate Nucleus is an Effective Brain Stimulation Target for Post-stroke Recovery

Objective: Functional recovery after stroke has been observed in both human and animal studies. Post-stroke brain stimulations are promising neurorestorative techniques as they allow direct manipulation of the target area’s excitability. Previously we have demonstrated that optogenetic neuronal stimulation of the ipsilesional primary motor cortex (iM1) promotes functional recovery. To determine an optimal brain stimulation target, we test whether optogenetic neuronal stimulation of the contralesional cerebellar dentate nucleus (cLCN) can promote recovery. We hypothesize that stimulation of cLCN may be more effective, as it sends excitatory outputs to multiple motor and premotor areas. Methods: Thy-1-ChR2-YFP line-18 transgenic male mice were used. Mice underwent stereotaxic surgery to implant an optical fiber in cLCN or iM1, followed by an intraluminal middle cerebral artery suture occlusion. Three groups of mice were used: control non-stimulated stroke mice, short stimulated stroke mice (short-stim, day5-14 post-stroke) and long stimulated stroke mice (long-stim, day5-28 post-stroke). Sensorimotor behavior tests were used to assess their recovery. Results: Our data showed that cLCN-stimulated stroke mice recovered quickly, with significant improvement in distance traveled as early as day7 (p<0.05), and faster rotating beam spole speed at day14 post-stroke (p<0.001). The effect of cLCN stimulation was persistent, as the short stim group continued to recover after day14 without further stimulations. The long stim group did not further enhance recovery. Interestingly, cLCN-stimulated mice performed significantly faster than iM1-stimulated mice. Analysis of pCREB activation showed that cLCN stimulation activates the dentatothalamocortical pathway. Conclusion: Our data suggest that cLCN stimulations post-stroke can promote persistent functional recovery. Furthermore, cLCN-stimulated mice recovered faster than iM1-stimulated mice, indicate that cLCN may be a more effective brain stimulation target. Current studies examine the brain activation patterns of cLCN-stimulated mice, as well as the mechanisms of cLCN-induced recovery.

Abstract WP106: Optogenetic Neuronal Stimulation Reduces Neuronal Nitric Oxide Synthase After Stroke

Objective: Nitric oxide (NO) is a gaseous messenger that mediates many physiological functions, including neuronal excitability, vascular tone and angiogenesis. Nitric oxide synthases are a family of enzymes that catalyzes the production of NO. While activation of endothelial NOS (eNOS) has been shown to be neuroprotective, activation of neuronal NOS (nNOS) and inducible NOS (iNOS) worsens ischemic damage. Although the role of NOS in acute stroke is well-established, their contribution in stroke recovery is less clear. Our lab recently demonstrated that optogenetic neuronal stimulation can enhance post-stroke recovery. As NOS mediates many physiological processes important for stroke recovery, we investigate whether NOS is involved in stimulation-enhanced recovery. We hypothesize that neuronal stimulations promote recovery by altering NOS expression. Methods: Thy-1-ChR2-YFP line-18 transgenic male mice were used. Mice underwent experimental stroke induced by an intraluminal middle cerebral artery suture occlusion. Repeated optogenetic stimulations in the contralesional cerebellar dentate nucleus (cLCN) were administered from post-stroke day5-14. Functional recovery was evaluated by sensory-motor behavior tests. Mice were sacrificed at post-stroke day15, ipsilesional and contralesional motor and sensory cortices were dissected. qPCR was performed to examine expression of nNOS, iNOS and eNOS. Results: Our data showed that stimulated stroke mice recovered quickly, with significantly faster rotating beam speed at day14 post-stroke (p<0.001). Quantitative PCR revealed that stimulations significantly reduced nNOS expression, while no change was detected for eNOS and iNOS. Interestingly, nNOS was significantly reduced in the contralesional primary motor cortex in cLCN-stimulated stroke mice (p<0.001), suggesting the involvement of nNOS in the contralesional cortex in stroke recovery. Conclusion: Optogenetic stimulations promote post-stroke recovery and selectively reduced nNOS expression in the contralesional motor cortex. Current ongoing studies examine the nNOS signaling pathway after stimulation and the effect of manipulating nNOS expression in stroke recovery.

Intracranial Pressure and Collateral Blood Flow.

Leptomeningeal collateral vessels, linking the 3 major arterial territories over the surface of the brain, have been recognized for >140 years.1 More widespread use of advanced clinical imaging in the past decade has led to increasing recognition of the key importance of collaterals in ischemic stroke outcome.2 However, recent studies from several groups indicate that failure of initially good collateral supply is a key feature of patients with delayed infarct expansion.3,4 This clinically challenging problem typically occurs in the first 1 to 2 days after hospital admission in patients with initially mild stroke symptoms. Rethrombosis of reperfused vessels was previously thought to be the likely cause of delayed infarct expansion in most patients. However, this theory is not supported by more recent evidence from imaging studies. Despite the important recent observations, there is limited understanding of the dynamic control of the collateral circulation, in particular, the cause of collateral blood flow failure. In this article, we will discuss recent observations from our experimental stroke model, indicating a dramatic increase in intracranial pressure (ICP) occurring around 24 hours after onset of even small stroke.5,6 We have also shown a significant linear reduction of collateral blood flow in response to progressive ICP elevation.7 We believe that a similar transient ICP elevation occurring during the first 1 to 2 days post stroke is a likely mechanism to explain delayed infarct expansion in patients with minor stroke. Perhaps surprisingly, we can find no published data on ICP at 24 hours in patients with minor stroke. The preclinical findings suggest that gathering such data should be a priority. ### Human Stroke There is a strong association between the extent of leptomeningeal collaterals and clinical stroke outcome. Initial studies using digital subtraction angiography permitted direct visualization of collateral vessels and …

Systemic Proteasome Inhibition Induces Sustained Post-stroke Neurological Recovery and Neuroprotection via Mechanisms Involving Reversal of Peripheral Immunosuppression and Preservation of Blood-Brain-Barrier Integrity.

In view of its profound effect on cell survival and function, the modulation of the ubiquitin-proteasome-system has recently been shown to promote neurological recovery and brain remodeling after focal cerebral ischemia. Hitherto, local intracerebral delivery strategies were used, which can hardly be translated to human patients. We herein analyzed effects of systemic intraperitoneal delivery of the proteasome inhibitor BSc2118 on neurological recovery, brain injury, peripheral and cerebral immune responses, neurovascular integrity, as well as cerebral neurogenesis and angiogenesis in a mouse model of transient intraluminal middle cerebral artery occlusion. Systemic delivery of BSc2118 induced acute neuroprotection reflected by reduced infarct volume when delivered up to 9h post-stroke. The latter was associated with reduced brain edema and stabilization of blood-brain-barrier integrity, albeit cerebral proteasome activity was only mildly reduced. Neuronal survival persisted in the post-acute stroke phase up to 28days post-stroke and was associated with improved neurological recovery when the proteasome inhibitor was continuously delivered over 7days. Systemic proteasome inhibition prevented stroke-induced acute leukocytosis in peripheral blood and reversed the subsequent immunosuppression, namely, the reduction of blood lymphocyte and granulocyte counts. On the contrary, post-ischemic brain inflammation, cerebral HIF-1α abundance, cell proliferation, neurogenesis, and angiogenesis were not influenced by the proteasome inhibitor. The modulation of peripheral immune responses might thus represent an attractive target for the clinical translation of proteasome inhibitors.

Effects of normobaric oxygen and melatonin on reperfusion injury: role of cerebral microcirculation.

In order to protect the brain before an irreversible injury occurs, penumbral oxygenation is the primary goal of current acute ischemic stroke treatment. However, hyperoxia treatment remains controversial due to the risk of free radical generation and vasoconstriction. Melatonin is a highly potent free radical scavenger that protects against ischemic stroke. Considering its anti-oxidant activity, we hypothesized that melatonin may augment the survival-promoting action of normobaric oxygen (NBO) and prevent brain infarction. Herein, we exposed mice to 30 or 90 min of intraluminal middle cerebral artery occlusion (MCAo) and evaluated the effects of NBO (70% or 100% over 90 min), administered either alone or in combination with melatonin (4 mg/kg, i.p.), on disseminate neuronal injury, neurological deficits, infarct volume, blood-brain barrier (BBB) permeability, cerebral blood flow (CBF) and cell signaling. Both NBO and particularly melatonin alone reduced neuronal injury, neurological deficits, infarct volume and BBB permeability, and increased post-ischemic CBF, evaluated by laser speckle imaging (LSI). They also improved CBF significantly in the ischemic- core and penumbra, which was associated with reduced IgG extravasation, DNA fragmentation, infarct volume, brain swelling and neurological scores. Levels of phosphorylated Akt, anti-apoptotic Bcl-xL, pro-apoptotic Bax and endothelial nitric oxide synthase (NOS) were re-regulated after combined oxygen and melatonin delivery, whereas neuronal and inducible NOS, which were increased by oxygen treatment, were not influenced by melatonin. Our present data suggest that melatonin and NBO are promising approaches for the treatment of acute-ischemic stroke, which encourage proof-of-concept studies in human stroke patients.

Sustained neurological recovery induced by resveratrol is associated with angioneurogenesis rather than neuroprotection after focal cerebral ischemia

According to the French paradox, red wine consumption reduces the incidence of vascular diseases even in the presence of highly saturated fatty acid intake. This phenomenon is widely attributed to the phytoalexin resveratrol, a red wine ingredient. Experimental studies suggesting that resveratrol has neuroprotective properties mostly used prophylactic delivery strategies associated with short observation periods. These studies did not allow conclusions to be made about resveratrol's therapeutic efficacy post-stroke. Herein, we systematically analyzed effects of prophylactic, acute and post-acute delivery of resveratrol (50mg/kg) on neurological recovery, tissue survival, and angioneurogenesis after focal cerebral ischemia induced by intraluminal middle cerebral artery occlusion in mice. Over an observation period of four weeks, only prolonged post-acute resveratrol delivery induced sustained neurological recovery as assessed by rota rod, tight rope and corner turn tests. Although prophylactic and acute resveratrol delivery reduced infarct volume and enhanced blood–brain-barrier integrity at 2days post-ischemia by elevating resveratrol's downstream signal sirtuin-1, increasing cell survival signals (phosphorylated Akt, heme oxygenase-1, Bcl-2) and decreasing cell death signals (Bax, activated caspase-3), a sustained reduction of infarct size on day 28 was not observed in any of the three experimental conditions. Instead, enhanced angiogenesis and neurogenesis were noted in animals receiving post-acute resveratrol delivery, which were associated with elevated concentrations of GDNF and VEGF in the brain. Thus, sustained neurological recovery induced by resveratrol depends on successful brain remodeling rather than structural neuroprotection. The recovery promoting effect of delayed resveratrol delivery opens promising perspectives for stroke therapy.

Bone marrow stromal cell transplantation through tail vein injection promotes angiogenesis and vascular endothelial growth factor expression in cerebral infarct area in rats

Background aimsThis study sought to identify correlations between vascular endothelial growth factor (VEGF) expression after tail-vein injection of rat-derived bone marrow stromal cells (BMSCs) and neurogenesis and angiogenesis in cerebral infarct of rats. MethodsRats with intraluminal middle cerebral artery occlusion were injected in a tail vein with Hoechst-labeled BMSCs. Functional recovery from cerebral infarction was observed through the use of a locomotion score. The brains of injected rats were sliced, and Hoechst-labeled BMSCs in the infarct and peri-infarct areas and subventricular zone (SVZ) were detected with the use of fluorescence microscopy. Ki-67 (as a cell proliferation marker) and VEGF expression were determined by means of immunohistochemistry. Neurofibril formation and angiogenesis were examined by means of Bielschowsky staining. ResultsWithin 1 to 2 weeks after BMSC injection, rats showed significantly improved locomotion scores compared with rats without BMSC injection (P < 0.01). Viable BMSCs were found in the peri-infarct area. The numbers of Ki-67–positive and VEGF-positive cells in the peri-infarct area and SVZ of injected rats were significantly increased compared with the control group (P < 0.01). Numerous new vessels, neurofibrils and anastomosed vasculatures were present in the infarct area. These neurofibrils mainly surrounded the neovasculatures. ConclusionsThese results indicate that BMSC-transplantation in rats through tail vein injection can increase neurogenesis, perhaps as the result of VEGF-mediated and/or Ki-67–mediated angiogenesis.

Neuroprotective effect of interleukin-6 in a rat model of cerebral ischemia.

Interleukin (IL)-6 is known to be a key cytokine in immune regulation in addition to serving crucial functions in various autoimmune diseases; however, the neuroprotective potential of IL-6 has not been fully investigated. The aim of the present study was to investigate the neuroprotective effects of the inflammatory cytokine IL-6 in a rat model of cerebral ischemia. Rat cerebral ischemia was induced by intraluminal middle cerebral artery occlusion. Following treatment with 500 or 50 ng IL-6, the infarct volumes and symptoms of neurological deficit were ameliorated. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining suggested that the IL-6 treatment reduced neuronal apoptosis in vivo, which was consistent with a lower percentage of annexin V- and caspase-3-positive cortical neurons. In addition, IL-6 in vitro induced the phosphorylation of signal transducer and activator of transcription (STAT) 3 and the expression of induced myeloid leukemia cell differentiation protein Mcl-1, but not the expression of B-cell lymphoma 2, suggesting the activation of the Janus kinase/STAT pathway by IL-6. IL-6 also appeared to be involved in the regulation of cytokine secretion and blood-brain barrier (BBB) integrity in cerebral ischemia. IL-6 downregulated a number of inflammatory cytokines, including tumor necrosis factor-α and IL-1β, as well as myeloperoxidase activity, indicating the accumulation of granulocytes in the ischemic brain tissue. IL-6 was also observed to support the integrity of the BBB by reducing Evans blue leakage in vivo and suppressing the expression of matrix metalloproteinase-9 in ischemic brain tissue. In conclusion, the results of the present study indicate that the neuroprotective effects of IL-6 in cerebral ischemia are the result of a range of processes, including the modulation of cell apoptosis, cytokine secretion and the integrity of the BBB. IL-6 could therefore be used as a therapeutic agent in clinical practice.

Intracranial Pressure Elevation Reduces Flow through Collateral Vessels and the Penetrating Arterioles they Supply. a Possible Explanation for ‘Collateral Failure’ and Infarct Expansion after Ischemic Stroke

Recent human imaging studies indicate that reduced blood flow through pial collateral vessels ('collateral failure') is associated with late infarct expansion despite stable arterial occlusion. The cause for 'collateral failure' is unknown. We recently showed that intracranial pressure (ICP) rises dramatically but transiently 24 hours after even minor experimental stroke. We hypothesized that ICP elevation would reduce collateral blood flow. First, we investigated the regulation of flow through collateral vessels and the penetrating arterioles arising from them during stroke reperfusion. Wistar rats were subjected to intraluminal middle cerebral artery (MCA) occlusion (MCAo). Individual pial collateral and associated penetrating arteriole blood flow was quantified using fluorescent microspheres. Baseline bidirectional flow changed to MCA-directed flow and increased by >450% immediately after MCAo. Collateral diameter changed minimally. Second, we determined the effect of ICP elevation on collateral and watershed penetrating arteriole flow. Intracranial pressure was artificially raised in stepwise increments during MCAo. The ICP increase was strongly correlated with collateral and penetrating arteriole flow reductions. Changes in collateral flow post-stroke appear to be primarily driven by the pressure drop across the collateral vessel, not vessel diameter. The ICP elevation reduces cerebral perfusion pressure and collateral flow, and is the possible explanation for 'collateral failure' in stroke-in-progression.

Abstract T MP19: Optogenetic Stimulation of Cerebellar Dentate Nucleus Promotes Persistent Functional Recovery After Stroke

Objective: Functional recovery after stroke has been observed in both human and animal studies. Post-stroke brain stimulations are promising neurorestorative techniques as they allow direct manipulation of the target area’s excitability. Previously we have demonstrated that optogenetic neuronal stimulation of the ipsilesional primary motor cortex promotes functional recovery. To determine an optimal brain stimulation target, we test whether optogenetic neuronal stimulation of the contralesional cerebellar dentate nucleus (cLCN) can promote recovery. We hypothesize that stimulation of cLCN may be more effective, as it sends excitatory outputs to multiple motor and premotor areas. Methods: Thy-1-ChR2-YFP line-18 transgenic male mice were used. Mice underwent stereotaxic surgery to implant a fiber cannula in cLCN, followed by an intraluminal middle cerebral artery suture occlusion. Three groups of mice were used: control non-stimulated stroke mice, short stim stimulated stroke mice (day5-14 post-stroke) and long stim stimulated stroke mice (day5-28 post-stroke). Sensorimotor behavior tests (rotating beam tests) were used to assess their recovery at day 0, 4, 7, 10, 14, 21 and 28 post-stroke. Results: Our data showed that stimulated stroke mice recovered quickly, with significant improvement in distance traveled as early as day7 (p&lt;0.05), and faster speed at day14 post-stroke (p&lt;0.001). To evaluate whether the effect of cLCN stimulation was persistent, we tested the effects of short stim (day5-14) and long stim (day5-28) on recovery. Interestingly, the short stim group continued to recover after day14 without further stimulations and the long stim group did not further enhance recovery, indicating that functional outcome of cLCN stimulation is persistent, and prolonged stimulations may not be necessary to achieve permanent recovery. Analysis of pCREB activation showed that cLCN stimulation activates the dentatothalamocortical pathway. Conclusion: Our data suggest that cLCN stimulations post-stroke can promote functional recovery, and this pro-recovery effect is persistent. Current studies examine the mechanisms of cLCN-induced recovery, including cortical excitability and synaptic/plasticity markers.

A blinded randomized assessment of laser Doppler flowmetry efficacy in standardizing outcome from intraluminal filament MCAO in the rat.

Laser Doppler flowmetry (LDF) is widely used for estimating cerebral blood flow changes during intraluminal middle cerebral artery occlusion (MCAO). No investigation has systematically examined LDF efficacy in standardizing outcome. We examined MCAO histologic and behavioral outcome as a function of LDF measurement. Rats were subjected to 90min MCAO by 4 surgeons having different levels of MCAO surgical experience. LDF was measured in all rats during ischemia. By random assignment, LDF values were (Assisted) or were not (Blinded) made available to each surgeon during MCAO (n=12-17 per group). Neurologic and histologic outcomes were measured 7 days post-MCAO. A second study examined LDF effects on 1-day post-MCAO outcome. Pooled across surgeons, intra-ischemic %LDF change (P=0.12), neurologic scores (Assisted vs. Blinded=14±6 vs. 13±7, P=0.61, mean±standard deviation) and cerebral infarct volume (162±63mm(3)vs. 143±86mm(3), P=0.24) were not different between groups. Only for one surgeon (novice) did LDF use alter infarct volume (145±28mm(3)vs. 98±61mm(3), P=0.03). LDF use decreased infarct volume coefficient of variation (COV) by 35% (P=0.02), but had no effect on neurologic score COV. We compared intraluminal MCAO outcome as a function of LDF use. LDF measurement altered neither neurologic nor histologic MCAO outcome. LDF did not decrease neurologic deficit COV, but did decrease infarct volume COV. LDF may allow use of fewer animals if infarct volume is the primary dependent variable, but is unlikely to impact requisite sample sizes if neurologic function is of primary interest.

Recombinant T cell receptor ligand treatment improves neurological outcome in the presence of tissue plasminogen activator in experimental ischemic stroke.

RTL1000 is a partial human MHC molecule coupled to a human myelin peptide. We previously demonstrated that RTL1000 was protective against experimental ischemic stroke in HLA-DR2 transgenic (DR2-Tg) mice. Since thrombolysis with recombinant tissue plasminogen activator (t-PA) is a standard therapy for stroke, we determined if RTL1000 efficacy is altered when combined with t-PA in experimental stroke. Male DR2-Tg mice underwent 60min of intraluminal middle cerebral artery occlusion (MCAO). t-PA or vehicle was infused intravenously followed by either a single or four daily subcutaneous injections of RTL1000 or vehicle. Infarct size was measured by 2, 3, 5-triphenyltetrazolium chloride staining at 24 or 96h of reperfusion. Our data showed that t-PA alone reduced infarct size when measured at 24h but not at 96h after MCAO. RTL1000 alone reduced infarct size both at 24 and 96h after MCAO. Combining RTL1000 with t-PA did not alter its ability to reduce infarct size at either 24 or 96h after MCAO and provides additional protection in t-PA treated mice at 24h after ischemic stroke. Taken together, RTL1000 treatment alone improves outcome and provides additional protection in t-PA-treated mice in experimental ischemic stroke.

Abstract 312: Transcriptional and Posttranscriptional Circuitry in the Brain After Transient and Permanent Ischemia

Background: Stroke is a major neurovascular disease and a leading cause of mortality and long-term disability. Within cells of the brain, short non-encoding microRNAs (miRNAs) serve to modulate gene expression and likely contribute to most, neurological processes. However, miRNA changes in the brain tissue in response to stroke have not been reported. Aim: To investigate the functional roles of brain miRNAs and gene regulatory networks in stroke injury. Methods: Adult (8-12 weeks old) male C57Bl/6 mice underwent intraluminal filament-induced middle cerebral artery (MCA) occlusion. Permanent ischemia (ischemia no reperfusion, InoR; n=8) was achieved by occlusion for 24 h, and ischemia with reperfusion (IR; n=8) was completed after 30 min of MCA followed by 23.5 h of reperfusion. Sham-operated mice (n=8) were used as controls. Total RNA was isolated from mouse brains and gene arrays (Affymetrix) and miRNA arrays (TaqMan OpenArray microRNA) were performed. Validation studies were performed using RT-PCR and TaqMan Individual Assays. Results: Relative to the sham-operated mice, InoR significantly altered (p≤0.05; fold-change≥1.5) the levels of 471 genes (mRNA) in the brain. By contrast, IR resulted in only 114 significant changes in gene expression after 24 h. Brain miRNAs were also very sensitive to both ischemia and reperfusion. 28 miRNAs (11 down, 17 up) were significantly altered by InoR compared to the sham procedure. Likewise, 12 miRNAs (3 down, 9 up) were significantly altered with reperfusion compared to the sham procedure. Interestingly, we found 10 miRNAs to be significantly altered (5 up, 5 down) with ischemia (InoR/Sham), but were also significantly corrected towards normal Sham levels by 23.5 h reperfusion (IR/InoR). Validation studies confirmed that levels of multiple miRNAs were significantly altered with InoR. Reperfusion increased the levels of all these miRNAs. 48% (327/680) of the mRNAs that were altered were predicted targets of significantly altered miRNAs, and our results showed inverse directional changes. Conclusion: Results from our study show the role of miRNAs and post-transcriptional circuits in both adaptive and maladaptive responses to ischemic stroke and reperfusion.

Abstract W P88: Impact of Post Stroke Isolation on Sociability and Depressive Behavior

Background and Purpose: Social isolation (SI) has been linked epidemiologically with high rates of morbidity and mortality following stroke. Clinically, social support and coping strategies have been shown to be significant factors in caregiver’s health-related quality of life. In order to discover the biological mechanisms underlying the benefits of social interaction the development of pre-clinical animal models are needed. The goal of our study was to examine the effect of post stroke pair housing and SI in different behavioral paradigms, which can be used as a validated model to assess chronic functional recovery. Methods: C57BL/6 male mice (8-12 weeks) were paired for 14 days before 60 min of transient intraluminal middle cerebral artery occlusion (MCAO) or sham surgery (n=8-10/group) and randomly assigned to six different housing conditions: 1. Stroke isolated (STiso), 2. Stroke pair housed (PH) with sham, 3. Stroke PH with stroke and their respective sham controls (Gps 4, 5, and 6). Behavioral assessments (3 Chamber sociability task, tail suspension test and sucrose consumption test), were performed at 42 days (Cohort 1) or every 7Th day for 6 weeks (Cohort 2). Brain atrophy was calculated using cresyl violet staining and blood samples were collected for interleukin-6 (IL-6) measurements. Results: Post stroke PH mice, either with sham or stroke partner showed significantly higher (p&lt;0.05) sociability after MCAO. In the 2nd cohort, a two-factor interaction ‘group versus days’ showed a significant difference between post stroke SI and PH groups (F 3,24 = 5.41; p&lt; 0.01, RM ANOVA with Bonferroni correction). Post stroke SI mice showed reduced sucrose consumption (p&lt;0.05) and increased immobility (p&lt;0.05) indicating a depressive phenotype. Post stroke SI mice had significantly more tissue loss (40.8 ± 1.3% in SI vs. 17.5 ± 1.5% in PH; p&lt;0.001) and showed higher levels of IL-6, a biomarker of social stress and inflammation. Conclusions: Our findings suggest that post stroke isolation reduces sociability and exacerbates depressive behavior, whereas pair housing aids early functional recovery. Moreover, the sociability (“empathy”) task can be a useful model for chronic post stroke behavioral recovery.

Abstract W P96: Optogenetic Neuronal Stimulation Enhances Neurotrophin Expression in the Contralesional Motor Cortex After Stroke

Objective: Functional recovery after stroke has been observed and is currently attributed to both brain remodeling and plasticity. One form of cortical reorganization involves the balance of interhemispheric interactions between ipsilesional and contralesional cortex. Stimulation of ipsilesional primary motor cortex (iM1) has been shown to be beneficial, however, the role of the contralesional M1 (cM1) remains controversial. Recently we showed that optogenetic stimulations of iM1 post-stroke promote functional recovery. In this study, we investigate the role of contralesional cortex in recovery by optogenetically stimulating iM1 or cM1 and examine the involvement of activity-dependent neurotrophins. Methods: Thy-1-ChR2-YFP line-18 transgenic male mice were used. Mice underwent stereotaxic surgery to implant a fiber cannula in either iM1 or cM1, followed by an intraluminal middle cerebral artery suture occlusion. Optogenetic stimulation began at day5 post-stroke and continued until day14 post-stroke. Sensorimotor behavior tests were used to assess their recovery at day 0, 2, 7, 10 and 14 post-stroke. Mice were sacrificed at day15 post-stroke and neurotrophin expressions were examined using quantitative PCR. Results: Repeated iM1 stimulations promoted functional recovery at day14 post-stroke, with improved motor performance on the rotating beam test (p&lt;0.01). Real-time PCR revealed significant increases of neurotrophin expressions in contralesional M1 at day15 post-stroke, including brain-derived neurotrophic factor (BDNF) (p&lt;0.05), nerve growth factor (NGF) (p&lt;0.05) and neurotrophin 3 (NTF3) (p&lt;0.05). BDNF and NTF3 expression were also significantly increased in the contralesional S1 of stimulated mice (p&lt;0.05). Conclusion: Our data suggest that activity-dependent neurotrophins in the contralesional cortex may be an important mechanism mediating stroke recovery. Current studies include specific stimulation and inhibition of the iM1 or cM1 post-stroke to elucidate the neurocircuitry mediating stroke recovery. In addition, the expression of neurotrophins will be examined in these studies to elucidate their role in the recovery process.

Promotive effect of neovascularization on rats with cerebral infarction by intranasal administration of granulocyte colony-stimulating factor

Objective To study the promotive effect of neovascularization on rats with cerebral infarction by nasal administration of granulocyte colony-stimulating factor. Methods A blinded, vehicle-controlled study of ING-CSF and IHG-CSF administration was performed by intraluminal middle cerebral artery occlusion (MCAO) model.All Sprague-Dawley rats were randomly divided into sham-operation group, model group, INNS group, IHG-CSF group and ING-CSF group.The neurologic behavioral tests were assessed after reperfusion 72 h. After 72 h of MCAO, the brains of rats were stainned with TTC and the infarcted volume was calculated by computer image analysis. The expression of vascular endothelial growth factor (VEGF) in the brain was determined by immune-histochemistry.The density of angiogenesis in the brain was counted under fluorescence microscope. Results The score of neurological function of ING-CSF group(3.90±1.65)was improved significantly compared with the IHG-CSF group (10.55±2.19)at the point of 72 h after cerebral infarction (P 0.05). The expression of VEGF in the brains of ING-CSF group was significantly higher than other groups at 72 h. Conclusion Intranasal administration G-CSF can improve neurological function and vascular angiogenesis in rats following MCAO. Key words: Granulocyte colony-stimulating factor; Cerebral infarction; Angiogenesis; Intranasal administration