Murine Stroke Research Articles

Abstract W P105: Lipopolysaccharide Exacerbates Stroke Severity and Compromises Mitochondrial Function in Cerebral Endothelium

Stroke is the fourth leading cause of death in the U.S, with many cases leading to severe disabilities. Statistics reports present that more than 40% of ischemic stroke patients have recent prior infections. Acute infections initiate rapid inflammation and can be a trigger for acute ischemic stroke in people. Ischemia-reperfusion injury following ischemic stroke induces blood-brain barrier (BBB) breakdown, hyperpermeability and brain edema. Cerebral vascular endothelial cells (CVEC) lining the BBB play a key role in fluid leakage into the brain tissue. The expression of toll-like receptor 4 (TLR4, specific for bacteria-sourced lipopolysaccharide, LPS) on CVEC implicates that acute bacterial infections may directly target CVEC and regulate BBB permeability and integrity. High mitochondrial contents in BBB capillaries suggest mitochondrial mechanisms are important to maintain BBB integrity. In order to test the hypothesis that bacterial infections regulate BBB permeability via mitochondrial mechanisms in stroke, we designed a series of studies both in vivo and in vitro. First, we used LPS challenged mice, then evaluated stroke outcomes including: infarct size, neurological deficits, BBB permeability and infiltration of neutrophils in the brain after transient middle cerebral artery occlusion (tMCAO). Second, we treated cultured CVEC with various concentrations of LPS and evaluated their mitochondrial function. Third, we impaired mitochondrial function in CVEC and investigated the BBB permeability in vitro. Our results demonstrated that LPS (100ug/kg) prior to tMCAO administration significantly exacerbates stroke infarct volume, BBB permeability and neutrophil infiltration in the brain, and worsens neurological deficits in murine experimental stroke in vivo. In vitro, LPS significantly impaired mitochondrial oxidative phosphorylation and decreased maximal respiration and spare capacity in CVEC. Finally, pharmacological inhibition of mitochondria respiration disrupted tight junctions in CVEC monolayers and increased CVEC monolayer permeability in vitro. These data are particularly important because they suggest a novel mitochondrial mechanism of the interactions between stroke and infections on BBB integrity.

Abstract T P79: Rapamycin Improves Outcome Post Stroke Injury by Enhancing Autophagy in Neuronal Cells

Background and Purpose: Previous literature and studies from our laboratory indicate that pharmacological inhibition and enhancement of autophagy are both effective in reducing infarct volume after stroke injury. Methods: Mice were treated with rapamycin to enhance, and chloroquine to suppress autophagy in mice immediately after stroke injury and again 24 hours post stroke injury in a murine stroke model. Survival data and neurological score were taken before sacrifice at 48 hours and lesion size was determined by TTC. HT22 neuronal mouse hippocampal cells were treated for one hour with H2O2 to mimic an oxidative stress injury and treated concurrently with rapamycin to enhance autophagy, chloroquine to inhibit autophagy or ZVAD to inhibit apoptosis. The treatment with pharmacological agents continued for 24 hours. Cells were counted with trypan blue on a hemocytometer. Western blot analysis was used to monitor protein changes. shRNA to Atg5 was used to genetically inhibit autophagy. Results: Both pharmacological agents showed decreased lesion size, though enhancement of autophagy with rapamycin exhibited a greater reduction in lesion size as well as increased survival and an improved neurological score. Rapamycin improved survival of neurons after oxidative injury and genetic inhibition of autophagy abrogated rapamycin's positive effect. Conclusions: Rapamycin improves stroke outcome by enhancing autophagy in neuronal cells.

A 2 × 2 factorial design for the combination therapy of minocycline and remote ischemic perconditioning: efficacy in a preclinical trial in murine thromboembolic stroke model.

BackgroundAfter the failure of so many drugs and therapies for acute ischemic stroke, innovative approaches are needed to develop new treatments. One promising strategy is to test combinations of agents in the pre-hospital setting prior to the administration of intravenous tissue plasminogen activator (IV-tPA) and/ or the use of mechanical reperfusion devices in the hospital.MethodsWe performed a 2 × 2 factorial design preclinical trial where we tested minocycline (MINO), remote ischemic perconditioning (RIPerC) and their combination treatment in a thromboembolic clot model of stroke in mice, without IV-tPA or later treated with IV-tPA at 4 hours post-stroke. Cerebral blood flow (CBF) was measured by laser speckle contrast imaging (LSCI), behavioral outcomes as neurological deficit score (NDS) and adhesive tape removal test, and infarct size measurement were performed at 48 hours post-stroke. Mice within the experimental sets were randomized for the different treatments, and all outcome measures were blinded.ResultsRIPerC significantly improved CBF as measured by LSCI in both with and without tPA treated mice (P < 0.001). MINO and RIPerC treatment were effective alone at reducing infarct size (p < 0.0001) and improving short-term functional outcomes (p < 0.001) in the tPA and non-tPA treated animals. The combination treatment of MINO and RIPerC significantly reduced the infarct size greater than either intervention alone (p < 0.05). There were trends in favor of improving functional outcomes after combination treatment of MINO and RIPerC; however combination treatment group was not significantly different than the individual treatments of MINO and RIPerC. There was no “statistical” interaction between minocycline and RIPerC treatments indicating that the effects of RIPerC and MINO were additive and not synergistic on the outcome measures.ConclusionIn the future, combining these two safe and low cost interventions in the ambulance has the potential to “freeze” the penumbra and improve outcomes in stroke patients. This pre-clinical 2 × 2 design can be easily translated into a pre-hospital clinical trial.

Transient receptor potential melastatin subfamily member 2 cation channel regulates detrimental immune cell invasion in ischemic stroke.

Brain injury during stroke results in oxidative stress and the release of factors that include extracellular Ca(2+), hydrogen peroxide, adenosine diphosphate ribose, and nicotinic acid adenine dinucleotide phosphate. These alterations of the extracellular milieu change the activity of transient receptor potential melastatin subfamily member 2 (TRPM2), a nonselective cation channel expressed in the central nervous system and the immune system. Our goal was to evaluate the contribution of TRPM2 to the tissue damage after stroke. In accordance with current quality guidelines, we independently characterized Trpm2 in a murine ischemic stroke model in 2 different laboratories. Gene deficiency of Trpm2 resulted in significantly improved neurological outcome and decreased infarct size. Besides an already known moderate neuroprotective effect of Trpm2 deficiency in vitro, ischemic brain invasion by neutrophils and macrophages was particularly reduced in Trpm2-deficient mice. Bone marrow chimeric mice revealed that Trpm2 deficiency in the peripheral immune system is responsible for the protective phenotype. Furthermore, experiments with mixed bone marrow chimeras demonstrated that Trpm2 is essential for the migration of neutrophils and, to a lesser extent, also of macrophages into ischemic hemispheres. Notably, the pharmacological TRPM2 inhibitor, N-(p-amylcinnamoyl)anthranilic acid, was equally protective in the stroke model. Although a neuroprotective effect of TRPM2 in vitro is well known, we can show for the first time that the detrimental role of TRPM2 in stroke primarily depends on its role in activating peripheral immune cells. Targeting TRPM2 systemically represents a promising therapeutic approach for ischemic stroke.

A dual-labeled Annexin A5 is not suited for SPECT imaging of brain cell death in experimental murine stroke.

Cell death is one of the pathophysiological hallmarks after stroke. Markers to image cell death pathways in vivo are highly desirable. We previously showed that fluorescently labeled Annexin A5 (AnxA5), which binds specifically to phosphatidylserine (PS) on dead/dying cells, can be used in experimental stroke for monitoring cell death with optical imaging. Here we investigated whether dual-labeled AnxA5 (technetium and fluorescence label) can be used for single-photon emission computed tomography (SPECT) of cell death in the same model. C57Bl6/N mice were subjected to 60-minute middle cerebral artery occlusion (MCAO) and underwent SPECT imaging at 24, 48, and 72 hours afterwards. They were injected intravenously with either PS-binding AnxA5 or the nonfunctional AnxA5 (negative control), labeled with 99mTc and Alexa Fluor 568, respectively. After SPECT imaging, brain sections were cut for autoradiography and fluorescence microscopy. Ethanol-induced cell death in the femur muscle was used as positive control. We detected dual-labeled AnxA5 in the model of ethanol-induced cell death in the femur muscle, but not after MCAO at any time point, either with SPECT or with ex vivo autoradiography or fluorescence microscopy. Dual-labeled AnxA5 appears to be unsuited for visualizing death of brain cells in this MCAO model.

Optical imaging of disrupted functional connectivity following ischemic stroke in mice

Recent human neuroimaging studies indicate that spontaneous fluctuations in neural activity, as measured by functional connectivity magnetic resonance imaging (fcMRI), are significantly affected following stroke. Disrupted functional connectivity is associated with behavioral deficits and has been linked to long-term recovery potential. FcMRI studies of stroke in rats have generally produced similar findings, although subacute cortical reorganization following focal ischemia appears to be more rapid than in humans. Similar studies in mice have not been published, most likely because fMRI in the small mouse brain is technically challenging. Extending functional connectivity methods to mouse models of stroke could provide a valuable tool for understanding the link between molecular mechanisms of stroke repair and human fcMRI findings at the system level. We applied functional connectivity optical intrinsic signal imaging (fcOIS) to mice before and 72h after transient middle cerebral artery occlusion (tMCAO) to examine how graded ischemic injury affects the relationship between functional connectivity and infarct volume, stimulus-induced response, and behavior. Regional changes in functional connectivity within the MCA territory were largely proportional to infarct volume. However, subcortical damage affected functional connectivity in the somatosensory cortex as much as larger infarcts of cortex and subcortex. The extent of injury correlated with cortical activations following electrical stimulation of the affected forelimb and with functional connectivity in the somatosensory cortex. Regional homotopic functional connectivity in motor cortex correlated with behavioral deficits measured using an adhesive patch removal test. Spontaneous hemodynamic activity within the infarct exhibited altered temporal and spectral features in comparison to intact tissue; failing to account for these regional differences significantly affected apparent post-stroke functional connectivity measures. Thus, several results were strongly dependent on how the resting-state data were processed. Specifically, global signal regression alone resulted in apparently distorted functional connectivity measures in the intact hemisphere. These distortions were corrected by regressing out multiple sources of variance, as performed in human fcMRI. We conclude that fcOIS provides a sensitive imaging modality in the murine stroke model; however, it is necessary to properly account for altered hemodynamics in injured brain to obtain accurate measures of functional connectivity.

Pre-existing hypertension dominates γδT cell reduction in human ischemic stroke.

T lymphocytes may play an important role in the evolution of ischemic stroke. Depletion of γδT cells has been found to abrogate ischemia reperfusion injury in murine stroke. However, the role of γδT cells in human ischemic stroke is unknown. We aimed to determine γδT cell counts and γδT cell interleukin 17A (IL-17A) production in the clinical setting of ischemic stroke. We also aimed to determine the associations of γδT cell counts with ischemic lesion volume, measures of clinical severity and with major stroke risk factors. Peripheral blood samples from 43 acute ischemic stroke patients and 26 control subjects matched on race and gender were used for flow cytometry and complete blood count analyses. Subsequently, cytokine levels and gene expression were measured in γδT cells. The number of circulating γδT cells was decreased by almost 50% (p = 0.005) in the stroke patients. γδT cell counts did not correlate with lesion volume on magnetic resonance diffusion-weighted imaging or with clinical severity in the stroke patients, but γδT cells showed elevated levels of IL-17A (p = 0.048). Decreased γδT cell counts were also associated with older age (p = 0.004), pre-existing hypertension (p = 0.0005) and prevalent coronary artery disease (p = 0.03), with pre-existing hypertension being the most significant predictor of γδT cell counts in a multivariable analysis. γδT cells in human ischemic stroke are reduced in number and show elevated levels of IL-17A. A major reduction in γδT lymphocytes also occurs in hypertension and may contribute to the development of hypertension-mediated stroke and vascular disease.

Abstract T P78: Remote Ischemic Conditioning (RIC), a Combined Regimen of Per- and Post- Conditionings (RIPerC and RIPostC), Provides Long-term Motor and Cognitive Benefits in Murine Embolic Stroke Model (eMCAO)

The majority of stroke sufferers need rehabilitation and institutional grade long-term care even after IV-tPA therapy. RIC therapy is easier to perform, safe, promising and ideal for post-stroke rehabilitation. Long-term benefits of RIC have never been reported after eMCAO. Our objective here was to determine if RIC provides long-term behavioral benefits with and without IV-tPA. Methods: eMCAO was induced in WT C57/Bl male mice (~6-mo old). Animals were randomized for the treatments (RIC, NO vs. YES; and IV-tPA, NO vs. YES, n=20/group) after eMCAO. Either IV-tPA (10 mg/kg b.wt.) or IV-saline was infused at 4 hours post-eMCAO. RIC was performed non-invasively on the left hind limb at 2, 8 and 24 hours post-eMCAO, followed by every alternate day till day 15. Neurological deficit score (NDS), and adhesive tape test were performed on day 2, 7 and 15 post-stroke to assess the motor function. Cognitive function was assessed by novel object recognition (NOR) test on day 8 and 16 post-stroke. Results: RIC improved NDS and sensorimotor functions significantly (p&lt;0.05) on day 2 and 7 with and without IV-tPA, but late IV-tPA alone was not effective. On day 15, there was a trend towards improved motor function in RIC treated groups, which was not significant. However, cognitive function was improved significantly (p&lt;0.05) on day 8 and 16 in RIC-treated groups with and without IV-tPA. IV-tPA alone did not provide any significant improvement in cognition. RIC also improved the survival with and without IV-tPA. Conclusion: RIC is a promising therapy for improved motor and cognitive functions during post-stroke rehabilitation and provides survival benefits. Further work is needed to determine the potential of RIC in aged animals and in animals with comorbidities.

Abstract 11: Ultrasonic Vocalizations as a Tool to Model Aphasia in Murine Experimental Stroke

BACKGROUND: Stroke is the leading cause of aphasia. One in four stroke survivors experience some form of speech impairment. Although rodents do not produce speech like humans, male mice vocalize at ultrasonic frequencies. By analyzing changes in ultrasonic vocalization (USV) patterns we assessed correlates of speech impairment in the rodent model. Further, to identify for any lateralization effect in vocalization we compared left and right sided strokes. Finally, changes in FOXP2 expression, a gene involved in the language production, were assessed. METHODS: Stroke was induced by 60-min middle cerebral artery occlusion (MCAO) in male C57BL/6 mice. All mice were pre-screened for baseline USVs one day prior to stroke, with an ultrasonic microphone and amplifier from Avisoft Bioacoustics for two 120-second trials, and analyzed using SAS-lab Pro. The first cohort determined if stroke led to changes in USVs and were tested on the day of stroke, post-stroke day (psd) 2, 7 and 14. The second cohort was used to assess differences between left and right MCAO (LMCAO/RMCAO) and shams, USVs were recorded psd 1 and 3. “High vocalizing” mice (&gt;30 USVs / 120sec) were used to avoid baseline bias (n=45). FOXP2 levels were assessed by western blot at 6 and 24 hrs after stroke. RESULTS: Repeated measures ANOVA revealed a significant effect of stroke on USVs across the days [F(1,8)=8.181; p&lt;0.05] in the first cohort. Stroke reduced USVs on psd 2 and psd 7, but mice recovered to sham levels by psd 14. Results from the second cohort identified that, on psd1 LMCAO mice had significantly reduced USVs compared to mice subjected to RMCAO (p&lt;0.10), and RMCAO mice vocalized less than sham mice (p&lt;0.05). Infarct analysis showed no difference between RMCAO and LMCAO. Shams showed full recovery to baseline USVs by psd 3. FOXP2 levels were elevated at 6 hrs after stroke, but fell to below sham levels at 24 hrs. CONCLUSIONS: The findings of this study suggest that screening for USVs may offer a novel tool for post-stroke behavioral recovery assessment. Stroke induced differential changes in USVs between RMCAO and LMCAO indicate lateralization of vocalization in rodents. In conclusion, decreased USVs parallels FOXP2 expression at 24 hrs suggests that rodent MCAO could be a practical model to study aphasia.

Abstract W P244: Effect of Remote Limb Ischemic Conditioning on CBF as Measured by Arterial Spin Labeling in Human Stroke

Background: Remote limb ischemic conditioning (RLIC) is a promising treatment for ischemic stroke. We have previously shown that RLIC increases cerebral blood flow, reduces infarct size, and improves outcome in a murine thromboembolic stroke model (Hoda N et al. Stroke. 2012; 43:2794). We hypothesized that RLIC will increase CBF in hypoperfused areas in acute stroke patients as detected by MRI Arterial Spin Labeling (ASL) Methods: Patients with an acute ischemic stroke within 14 days and with a persistent mismatch defined by a perfusion defect measured by ASL that was at least 20% larger than the area of infarction as measured by diffusion weighted imaging (DWI) were eligible. Patients underwent 4 cycles of blood pressure cuff inflation (x200 mm Hg) for 5 minutes and deflation for 5 minutes on the leg or arm, 1- 2 times per day. MRI ASL, DWI, and 3D T1-weighted Fast Spoiled Gradient-Echo (FSPGR) images were taken before and 60 minutes after the last cycle of RLIC to measure CBF. Two neuroradiologists analyzed the images. Results: After 3 of 4 RLIC sessions, the CBF improved and the mismatch decreased by at least 15%. In the one patient without a reduction in mismatch, the RLIC was performed on the arm and not the leg. Conclusions: 1.) RLIC increased CBF as measured by ASL in stroke patients with a persistent diffusion-perfusion mismatch. 2.) Since it can be repeated multiple times on the same patient, ASL may be a useful biomarker of the conditioning response in humans. 3.) Further work in a larger sample of patients is needed to define the optimal regimen of RLIC and to determine the utility of ASL as a biomarker.

Abstract W P220: Suppression of Interleukin 33 Limits Ischemic Brain Injury and Improves Functional Outcome in Murine Stroke Model

Background and purpose: Ischemic stroke is a leading cause of death and disability worldwide, and the treatment options are limited. Interleukin-33 (IL-33) is a newly recognized IL-1 family cytokine which signals via its ST2 receptor, and acts as a key regulator of inflammation. However, the expression of IL-33 in the brain was not well studied and its expression in ischemic stroke remains to be elucidated. In the present study, we measured IL-33 and ST2 levels and examine the correlation of IL-33 expression with brain damage and functional outcome following ischemic stroke. Methods: IL-33 expression was examined in ischemic brain hemisphere. Mice were subjected to middle cerebral artery occlusion (MCAO) for 1 hr using a filament model, followed by 23 hrs reperfusion. Briefly, mice were anesthetized with 1-1.5% isoflurane mixed with medical oxygen. Body temperature was maintained at 37°C ± 1.0 using a heating pad. At 23 hours after ischemia/reperfusion, mice were tested for neurological scores and were sacrificed for the estimation of IL-33 and ST2 expression. Expression of IL-33 and its receptor ST2 was monitored by ELISA, Western blot and immunohistochemistry. The neurobehavioral scores, infarction volumes, expression of NF-kB and proinflammatory cytokines were evaluated after ischemia/reperfusion. Results: We found significantly increased expression level of IL-33 and ST2 in the MCAO mice as compare to the saline treated control mice. Moreover, treating the MCAO mice with recombinant IL-33 increases the brain injury and worsens neurological deficits in MCAO mice as compare to control mice. Interestingly, increased ischemic brain damage and neurological deficits were largely abrogated in mice treated with IL-33 neutralizing antibody. Conclusion: These findings provide the first evidence that IL-33/ST2 signaling plays an important role in the pathogenesis of stroke. Moreover, IL-33 exacerbates inflammatory brain injury after ischemic stroke and treatment with specific IL-33 neutralizing antibody inhibited the ischemic brain injury. Therefore, blocking the IL-33 may represent an efficient therapy in stroke.

Adamts-18 Is a Novel Candidate Gene Of Vascular Development That Is Related To Aggravated Thrombosis: Evidence From a Adamts-18 Knock Out Mice

HIV-ITP patients have a unique antibody (Ab) against platelet GPIIIa49-66 which induces oxidative platelet fragmentation in the absence of complement (Cell 106: 551, 2001; JCI 113: 973, 2004). The search for a physiologic ligand that could induce this reaction was undertaken by panning the GPIIIa49-66 peptide with a phage surface display 7-mer peptide library. From 20 positive clones, 1 had 70% identity with a C-terminal region of ADAMTS-18 (a disintegrin and metalloproteinase with thrombospondin (TSR)-like motif 18), which is secreted by endothelial cell (EC). The recombinant C-terminal fragment of ADAMTS-18 can completely dissolve platelet aggregates formed in vitro. Moreover, this fragment lyses thrombi formed in the cerebral artery of mice and reduces infarction and neurologic impairment in murine ischemic stroke model (Blood 113: 6051, 2009). However, whether ADAMTS-18 represents the dominant physiologic mechanism controlling thrombus dissolution in vivo remains to be clarified.Here, we used ADAMTS-18-deficient (ADAMTS-18-/-) mice to study the contributions of ADAMTS-18 to thrombus formation in vivo. To investigate possible functional differences between WT and ADAMTS-18-/- platelets, we tested WT and ADAMTS-18-/- platelets in a model of pulmonary thromboembolism induced by infusion of a mixture of platelet agonist collagen (25 µg per mouse) and epinephrine (1 µg per mouse). In lung tissue Hematoxylin and eosin-stained (HE) slides, the mean number of thrombi per lung was same in the ADAMTS-18-/- group compared with WT group (163.7 ±14.38 vs 174.9 ±11.73, n=30/group, P=0.5480). In vitro, there is no difference between WT and ADAMTS-18-/- platelet aggregation trace and activation initiated by various platelet agonists ADP (10 µM) or collagen (2 µg/mL). No difference was noted on WT and ADAMTS-18-/- platelet adhesion on immobilized ligand (fibrinogen). These results indicated ADAMTS-18 had no effect on platelet function.We next evaluate the effect of ADAMTS-18 on thrombus formation in a second well-established carotid artery thrombosis model, which is induced by 10% FeCl3 patch. In the process of surgical operation, we unexpectedly observed that all ADAMTS-18-/- mice have premature common carotid artery bifurcation compared with WT mice. A Doppler flow monitor showed ADAMTS-18-/- mice exhibited significantly reduced carotid artery blood flow than WT mice (ADAMTS-18-/- vs WT, 0.5 ± 0.11 vs 0.75 ± 0.21 mL/min, n=7/group, P=0.0298), which results in shortened time of thrombus formation (ADAMTS-18-/- vs WT, 452.17 ± 68.88 vs 611.43 ± 92.02 sec, n=7/group, P=0.0005 ). Immunohistochemistry staining showed that the common carotid artery of ADAMTS-18-/- mice had increased adventitial collagen deposition compared with WT mice. In vivo matrigel plug assay demonstrated that ADAMTS-18-/- mice had significantly lower density of blood vessels compared to the WT mice. Since the middle cerebral artery arises from the internal carotid artery, we conjecture that ADAMTS-18-/- mice would have aggravated brain infarction for the less cerebral blood flow supplying. This proved to be true. In transient middle cerebral artery occlusion (tMCAO) model, the infarction size in ADAMTS-18-/- mice was significantly larger than in WT mice (mean infarction %, 25.68 ± 4.13 vs 17.41 ± 3.24, n=8, P=0.0012). Taken together, these observations suggest vasculature is the potential site of action of ADAMTS-18. To our knowledge, this is the first validation study of linkage and association of ADAMTS-18 as a pro-vasculature gene that is related to aggravated thrombosis. Disclosures:No relevant conflicts of interest to declare.

Highly efficient differentiation of neural precursors from human embryonic stem cells and benefits of transplantation after ischemic stroke in mice

IntroductionIschemic stroke is a leading cause of death and disability, but treatment options are severely limited. Cell therapy offers an attractive strategy for regenerating lost tissues and enhancing the endogenous healing process. In this study, we investigated the use of human embryonic stem cell-derived neural precursors as a cell therapy in a murine stroke model.MethodsNeural precursors were derived from human embryonic stem cells by using a fully adherent SMAD inhibition protocol employing small molecules. The efficiency of neural induction and the ability of these cells to further differentiate into neurons were assessed by using immunocytochemistry. Whole-cell patch-clamp recording was used to demonstrate the electrophysiological activity of human embryonic stem cell-derived neurons. Neural precursors were transplanted into the core and penumbra regions of a focal ischemic stroke in the barrel cortex of mice. Animals received injections of bromodeoxyuridine to track regeneration. Neural differentiation of the transplanted cells and regenerative markers were measured by using immunohistochemistry. The adhesive removal test was used to determine functional improvement after stroke and intervention.ResultsAfter 11 days of neural induction by using the small-molecule protocol, over 95% of human embryonic stem-derived cells expressed at least one neural marker. Further in vitro differentiation yielded cells that stained for mature neuronal markers and exhibited high-amplitude, repetitive action potentials in response to depolarization. Neuronal differentiation also occurred after transplantation into the ischemic cortex. A greater level of bromodeoxyuridine co-localization with neurons was observed in the penumbra region of animals receiving cell transplantation. Transplantation also improved sensory recovery in transplant animals over that in control animals.ConclusionsHuman embryonic stem cell-derived neural precursors derived by using a highly efficient small-molecule SMAD inhibition protocol can differentiate into electrophysiologically functional neurons in vitro. These cells also differentiate into neurons in vivo, enhance regenerative activities, and improve sensory recovery after ischemic stroke.

MR elastography in a murine stroke model reveals correlation of macroscopic viscoelastic properties of the brain with neuronal density

The aim of this study was to investigate the influence of neuronal density on viscoelastic parameters of living brain tissue after ischemic infarction in the mouse using MR elastography (MRE). Transient middle cerebral artery occlusion (MCAO) in the left hemisphere was induced in 20 mice. In vivo 7-T MRE at a vibration frequency of 900 Hz was performed on days 3, 7, 14 and 28 (n = 5 per group) after MCAO, followed by the analysis of histological markers, such as neuron counts (NeuN). MCAO led to a significant reduction in the storage modulus in the left hemisphere relative to contralateral values (p = 0.03) without changes over time. A correlation between storage modulus and NeuN in both hemispheres was observed, with correlation coefficients of R = 0.648 (p = 0.002, left) and R = 0.622 (p = 0.003, right). The loss modulus was less sensitive to MCAO, but correlated with NeuN in the left hemisphere (R = 0.764, p = 0.0001). In agreement with the literature, these results suggest that the shear modulus in the brain is reduced after transient ischemic insult. Furthermore, our study provides evidence that the in vivo shear modulus of brain tissue correlates with neuronal density. In diagnostic applications, MRE may thus have diagnostic potential as a tool for image-based quantification of neurodegenerative processes.

Protective effects of angiopoietin-like 4 on cerebrovascular and functional damages in ischaemic stroke

Given the impact of vascular injuries and oedema on brain damage caused during stroke, vascular protection represents a major medical need. We hypothesized that angiopoietin-like 4 (ANGPTL4), a regulator of endothelial barrier integrity, might exert a protective effect during ischaemic stroke. Using a murine transient ischaemic stroke model, treatment with recombinant ANGPTL4 led to significantly decreased infarct size and improved behaviour. Quantitative characteristics of the vascular network (density and branchpoints) were preserved in ANGPTL4-treated mice. Integrity of tight and adherens junctions was also quantified and ANGPTL4-treated mice displayed increased VE-cadherin and claudin-5-positive areas. Brain oedema was thus significantly decreased in ANGPTL4-treated mice. In accordance, vascular damage and infarct severity were increased in angptl4-deficient mice thus providing genetic evidence that ANGPTL4 preserves brain tissue from ischaemia-induced alterations. Altogether, these data show that ANGPTL4 protects not only the global vascular network, but also interendothelial junctions and controls both deleterious inflammatory response and oedema. Mechanistically, ANGPTL4 counteracted VEGF signalling and thereby diminished Src-signalling downstream from VEGFR2. This led to decreased VEGFR2-VE-cadherin complex disruption, increased stability of junctions and thus increased endothelial cell barrier integrity of the cerebral microcirculation. In addition, ANGPTL4 prevented neuronal loss in the ischaemic area. These results, therefore, show ANGPTL4 counteracts the loss of vascular integrity in ischaemic stroke, by restricting Src kinase signalling downstream from VEGFR2. ANGPTL4 treatment thus reduces oedema, infarct size, neuronal loss, and improves mice behaviour. These results suggest that ANGPTL4 constitutes a relevant target for vasculoprotection and cerebral protection during stroke.

Neurotoxicity of the anticoagulant-selective E149A-activated protein C variant after focal ischemic stroke in mice

Wild type (WT) activated protein C (APC) and cytoprotective-selective APC variants such as 3K3A-APC (<10% anticoagulant but normal cytoprotective activity) are neuroprotective in murine focal ischemic stroke models. Here we compared the neuroprotective effects of the anticoagulant-selective E149A-APC variant (>3-fold increased anticoagulant activity but defective cytoprotective activities) to those of the cytoprotective-selective 5A-APC variant (<10% anticoagulant activity). After transient distal middle cerebral artery occlusion, mice received a vehicle, E149A-APC or 5A-APC at 0.2mg/kg at 4h after stroke. Treatment with 5A-APC was neuroprotective, as it improved performance on forelimb use asymmetry test and foot fault test (P<0.05), reduced by 48% and 50% the infarct and edema volumes, respectively (P<0.05), and was not associated with an increased risk of bleeding as indicated by normal hemoglobin levels in the ischemic brain at day 7. In contrast, E149A-APC treatment worsened neurological outcome determined by foot fault tests and forelimb use asymmetry tests, and increased significantly by 44% and 60% infarct and edema volume, respectively (P<0.05). At 7days after treatment, E149A-APC compared to vehicle or 5A-APC notably increased by ~5-fold the hemoglobin level in the ischemic hemisphere suggesting it provoked significant intracerebral bleeding. Thus, the enhanced anticoagulant activity of E149A-APC increased post-ischemic accumulation of neurotoxic erythrocyte-derived hemoglobin which likely worsened the neurological and neuropathological outcomes after stroke. Our data emphasize that APC's cytoprotective activities, but not its anticoagulant activity, are key for APC neuroprotection after transient ischemic stroke.

Neuroprotection by inhaled nitric oxide in a murine stroke model is concentration and duration dependent

Inhaled nitric oxide (iNO) has been shown to reduce ischemia/reperfusion (I/R) injury in several different organ systems including the brain. We investigated whether iNO was neuroprotective in a mouse model of transient focal ischemia. Male Swiss Webster mice underwent middle cerebral artery occlusion for 1h followed by reperfusion for 47h. Mice were divided into 5 concentration groups and administered nitric oxide (NO) at either 10, 20, 40, 60 or 80ppm. Each of the 5 concentration groups was subdivided into 4 duration groups which were treated with iNO for 5, 8, 16 or 24h beginning immediately after artery occlusion. Results showed both concentration and duration determined efficacy. At 10ppm only the 24hr duration group exhibited reduced infarct volume while at 20, 40 and 60ppm only 8 and 16h of exposure led to smaller infarctions. At these concentrations the dose response curves were strongly U shaped indicating a loss of benefit at long durations. At 80ppm, reduction in infarct volume was not observed at any duration. Additional experiments showed that 60ppm iNO could be transported from lung to brain and that iNO administered for 8h improved recovery from subarachnoid hemorrhage and reduced the inflammatory response accompanying ischemic stroke. Enhanced blood flow during reperfusion may be an important mediator of these effects.

Spectratype analysis of clonal T cell expansion in murine experimental stroke

T lymphocytes are major contributors to post-ischemic neuroinflammation following ischemic stroke. However, the mode of postischemic T cell activation remains a controversial issue. Therefore, this study aimed to directly investigate antigen-dependent clonal T cell expansion after experimental brain ischemia using spectratype/immunoscope analysis. We detected clonal T cell expansion in ischemic brains and to a lesser extent in secondary lymphatic organs and characterized its time course. This is the first study presenting direct evidence of clonal T cell expansion after stroke — however, with delayed kinetics that make antigen-dependent mechanisms unlikely in acute post-ischemic neuroinflammation.

Abstract 136: Deleterious Effects of Synaptic Zn2+ in a Murine Distal Middle Cerebral Artery Occlusion Model

Background: Previous work has implicated accumulation of Zn2+ as a contributor to ischemic brain injury, however the sources of toxic Zn2+ accumulation are not fully understood. Previous data report that ICV delivery of a Zn2+ chelator limited mild focal stroke (Neuroscience 115 (2002) 871-8). We have recently demonstrated substantial synaptic Zn2+ release following spreading depolarization (JCBFM 31 (2011) 1073-84), and thus it is possible that repetitive spreading depolarization_ known to occur following stroke_ could be a major source of toxic Zn2+. In the present study, we examined whether synaptic Zn2+ release contributes to neuronal injury in a murine focal stroke model. Methods The effect of synaptic Zn2+ was assessed using mice lacking synaptic Zn2+, due to genetic deletion of the synaptic vesicle transporter ZnT3 (ZnT3 KO). Wild type C57Bl/6 mice were age and sex matched as controls. Mice were anesthetized with isofluorane and the cortical branch of the right middle cerebral artery was cauterized via a temporal burrhole. A single surgeon performed the occlusions. Body temperature was maintained at 37±0.5oC during the procedure and recovery. Animals were allowed to recover for 1 week before euthanasia. Stroke volume was assessed by Fluoro-Jade staining. Statistical analysis with 2-way ANOVA was used to determine the effects of sex and genotype on stroke volume. Results: There was no peri- or post-procedural mortality. Infarct was confirmed histologically in all animals. Stroke volume was reduced in the ZnT3 KO mice compared to wild type (mean volume 3.54mm3 versus 8.42mm3 respectively, n=7 for each arm). Only genotype (not sex) had a significant effect on stroke volume by 2-way ANOVA (p=0.0042). Conclusion: Synaptic Zn2+ release leads to increased stroke volume, likely due to toxic Zn2+accumulation. Therapies targeting synaptic release of Zn2+ or repetitive spreading depolarization events (which cause Zn2+ release) may decrease delayed ischemic injuries.

Protective effects of bifunctional platelet GPIIIa49-66 ligand on myocardial ischemia-reperfusion injury in rats

Current antiplatelet drugs mainly focus on prevention rather than the more clinically relevant issue of clearance of an existing thrombus. We recently described a novel and effective therapeutic strategy for dissolution of preexisting platelet thrombus in a murine ischemic stroke model with a bifunctional platelet GPIIIa49-66 ligand (Single-chain antibody Linked first Kringle 1 of plasminogen, named SLK), which homes to newly deposited fibrin strands tangled of platelet thrombus and induces aggregated platelet fragmentation. In this study, we perform in-depth analysis of the effect of SLK on myocardial ischemia-reperfusion (IR) injury in rats. We show that SLK dose-dependently reduces lactate dehydrogenase (LDH) release as well as mean infarction size of left ventricle. Histological observation demonstrates that the arterial thrombi in coronary arteries of rat almost disappear after SLK injection. Optimal dose of SLK (37.5 μg/ individual) provides the myocardial protection at 2 hours post-infusion. However, there are no significant protective effects if SLK was given at 4 or 8 hours post-infusion. The combined application of SLK and urokinase (UK) demonstrates greater myocardial protection than UK alone at 2 hours post-infusion. Thus, SLK could be used as a thrombolytic alternative in other arterial vascular beds associated with thrombosis to enhance fibrinolysis.