Post-stroke Day Research Articles

Long-Term Accumulation of T Cytotoxic 1, T Cytotoxic 17, and T Cytotoxic 17/1 Cells in the Brain Contributes to Microglia-Mediated Chronic Neuroinflammation After Ischemic Stroke.

Post-stroke neuroinflammation affects the damage and recovery of neurological functions. T cells including CD8+ T cells were present in the ipsilateral hemisphere in the subacute and late phases of ischemic stroke. However, the potential roles of CD8+ T cell subsets in the progression of neuroinflammation have not been characterized. In the current mouse transient middle cerebral artery occlusion model, we investigated the existence of CD8+ T cell subsets in the ipsilateral hemisphere in the subacute and late phases of stroke. We found that ipsilateral CD8+ T cells were present on post-stroke day 3 and increased on post-stroke day 30. The day-3 ipsilateral CD8+ T cells predominantly produced interferon-γ (IFN-γ), while the day-30 ipsilateral CD8+ T cells co-expressed IFN-γ and interleukin-17A (IL-17A). In addition, evaluation of cytokines and transcription factors of the day-30 ipsilateral CD8+ T cells revealed the presence of T cytotoxic 1 (Tc1), T cytotoxic 17 (Tc17), and T cytotoxic 17/1 (Tc17/1) cells. Furthermore, based on the expression of a series of chemokine/cytokine receptors, viable ipsilateral Tc1, Tc17, and Tc17.1 cells were identified and enriched from the day-30 ipsilateral CD8+ T cells, respectively. Co-culture of microglia with ipsilateral Tc1, Tc17, or Tc17.1 cells indicated that the three CD8+ T cell subsets up-regulated the expression of pro-inflammatory mediators by microglia, with Tc17.1 cells being the most potent cell in doing so. Collectively, this study sheds light on the contributions of Tc1, Tc17, and Tc17.1 cells to long-term neuroinflammation after ischemic stroke.

Temporal changes in regulatory T cell subsets defined by the transcription factor Helios in stroke and their potential role in stroke-associated infection: a prospective case–control study

BackgroundRegulatory T cells (Tregs) are involved in the systemic immune response after ischemic stroke. However, their role remains unclear, and the effect appears to be both neuroprotective and detrimental. Treg suppressor function may result in immunodepression and promote stroke-associated infection (SAI). Thus we assume that the bidirectional effects of Tregs may be in part attributed to the intracellular transcription factor Helios. Tregs with Helios expression (H+ Tregs) constitute 70–90% of all Treg cells and more frequently than Helios-negative Tregs (H− Tregs) express molecules recognized as markers of Tregs with suppressor abilities.Methods and resultsWe prospectively assessed the circulating Treg population with flow cytometry in 52 subjects on days 1, 3, 10 and 90 after ischemic stroke and we compared the results with those obtained in concurrent age-, sex- and vascular risk factor-matched controls. At all studied time points the percentage of H+ Tregs decreased in stroke subjects—D1: 69.1% p < 0.0001; D3: 62.5% (49.6–76.6), p < 0.0001; D10: 60.9% (56.5–72.9), p < 0.0001; D90: 79.2% (50.2–91.7), p = 0.014 vs. controls: 92.7% (81.9–97.0) and the percentage of H− Tregs increased accordingly. In patients with SAI the percentage of pro-suppressor H+ Tregs on post-stroke day 3 was higher than in those without infection (p = 0.03). After adjustment for confounders, the percentage of H+ Tregs on day 3 independently correlated with SAI [OR 1.29; CI 95%: 1.08–1.27); p = 0.02]. Although the percentage of H+ Tregs on day 3 correlated positively with NIHSS score on day 90 (rS = 0.62; p < 0.01) and the infarct volume at day 90 (rS = 0.58; p < 0.05), in regression analysis it was not an independent risk factor.ConclusionsOn the first day after stroke the proportion of H+ vs. H− Tregs changes in favor of pro-inflammatory H− Tregs, and this shift continues toward normalization when assessed on day 90. A higher percentage of pro-suppressive H+ Tregs on day 3 independently correlates with SAI and is associated positively with NIHSS score, but it does not independently affect the outcome and stroke area in the convalescent phase of stroke.

Expansion of plasma MicroRNAs over the first month following human stroke

Few have characterized miRNA expression during the transition from injury to neural repair and secondary neurodegeneration following stroke in humans. We compared expression of 754 miRNAs from plasma samples collected 5, 15, and 30 days post-ischemic stroke from a discovery cohort (n = 55) and 15-days post-ischemic stroke from a validation cohort (n = 48) to healthy control samples (n = 55 and 48 respectively) matched for age, sex, race and cardiovascular comorbidities using qRT-PCR. Eight miRNAs remained significantly altered across all time points in both cohorts including many described in acute stroke. The number of significantly dysregulated miRNAs more than doubled from post-stroke day 5 (19 miRNAs) to days 15 (50 miRNAs) and 30 (57 miRNAs). Twelve brain-enriched miRNAs were significantly altered at one or more time points (decreased expression, stroke versus controls: miR-107; increased expression: miR-99-5p, miR-127-3p, miR-128-3p, miR-181a-3p, miR-181a-5p, miR-382-5p, miR-433-3p, miR-491-5p, miR-495-3p, miR-874-3p, and miR-941). Many brain-enriched miRNAs were associated with apoptosis over the first month post-stroke whereas other miRNAs suggested a transition to synapse regulation and neuronal protection by day 30. These findings suggest that a program of decreased cellular proliferation may last at least 30 days post-stroke, and points to specific miRNAs that could contribute to neural repair in humans.

A novel stroke rehabilitation strategy and underlying stress granule regulations through inhibition of NLRP3 inflammasome activation.

Dynamic changes in ischemic pathology after stroke suggested a "critical window" of enhanced neuroplasticity immediately after stroke onset. Although physical exercise has long been considered a promising strategy of stroke rehabilitation, very early physical exercise may exacerbate brain injury. Since remote ischemic conditioning (RIC) promotes neuroprotection and neuroplasticity, the present study combined RIC with sequential exercise to establish a new rehabilitation strategy for a better rehabilitative outcome. A total of 120 adult male Sprague-Dawley rats were used and divided into five groups: (1) sham, (2) stroke, (3) stroke with exercise, (4) stroke with RIC, and (5) stroke with RIC followed by exercise. Brain damage was evaluated by infarct volume, neurological deficit, cell death, and lactate dehydrogenase (LDH) activity. Long-term functional outcomes were determined by grid walk tests, rotarod tests, beam balance tests, forelimb placing tests, and the Morris water maze. Neuroplasticity was evaluated through measurements of both mRNA and protein levels of synaptogenesis (synaptophysin [SYN], post-synaptic density protein-95 [PSD-95], and brain-derived neurotrophic factor [BDNF]) and angiogenesis (vascular endothelial growth factor [VEGF], angiopoietin-1 [Ang-1], and angiopoietin-2 [Ang-2]). Inflammasome activation was measured by concentrations of interleukin-18 (IL-18) and IL-1β detected by enzyme-linked immunosorbent assay (ELISA) kits, mRNA expressions of NLR pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC), IL-18 and IL-1β, and protein quantities of NLRP3, ASC, cleaved-caspase-1, gasdermin D-N (GSDMD-N), and IL-18 and IL-1β. Stress granules (SGs), including GTPase-activating protein-binding protein 1 (G3BP1), T cell-restricted intracellular antigen-1 (TIA1), and DEAD-box RNA helicase 3X (DDX3X) were evaluated at mRNA and protein levels. The interactions between DDX3X with NLRP3 or G3BP1 were determined by immunofluorescence and co-immunoprecipitation. Early RIC decreased infarct volumes, neurological deficits, cell death, and LDH activity at post-stroke Day 3 (p < 0.05). All treatment groups showed significant improvement in functional outcomes, including sensory, motor, and cognitive functions. RIC and exercise, as compared to RIC or physical exercise alone, had improved functional outcomes after stroke (p < 0.05), as well as synaptogenesis and angiogenesis (p < 0.05). RIC significantly reduced mRNA and protein expressions of NLRP3 (p < 0.05). SGs formation peaked at 0 h after ischemia, then progressively decreased until 24 h postreperfusion, which was reversed by RIC (p < 0.05). The assembly of SGs consumed DDX3X and then inhibited NLRP3 inflammasome activation. RIC followed by exercise induced a better rehabilitation in ischemic rats, while early RIC alleviated ischemia-reperfusion injury via stress-granule-mediated inhibition of NLRP3 inflammasome.

Exploratory Transcriptomic Profiling Reveals the Role of Gut Microbiota in Vascular Dementia.

Stroke is the second most common cause of cognitive impairment and dementia. Vascular dementia (VaD), a cognitive impairment following a stroke, is common and significantly impacts the quality of life. We recently demonstrated via gut microbe transplant studies that the gut microbe-dependent trimethylamine-N-oxide (TMAO) pathway impacts stroke severity, both infarct size and long-term cognitive outcomes. However, the molecular mechanisms that underly the role of the microbiome in VaD have not been explored in depth. To address this issue, we performed a comprehensive RNA-sequencing analysis to identify differentially expressed (DE) genes in the ischemic cerebral cortex of mouse brains at pre-stroke and post-stroke day 1 and day 3. A total of 4016, 3752 and 7861 DE genes were identified at pre-stroke and post-stroke day 1 and day 3, respectively. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated pathways of neurodegeneration in multiple diseases, chemokine signaling, calcium signaling, and IL-17 signaling as the key enriched pathways. Inflammatory response genes interleukin-1 beta (Il-1β), chemokines (C-X-C motif chemokine ligand 10 (Cxcl10), chemokine ligand 2 (Ccl2)), and immune system genes (S100 calcium binding protein 8 (S100a8), lipocalin-2 (Lcn2)) were among the most significantly upregulated genes. Hypocretin neuropeptide precursor (Hcrt), a neuropeptide, and transcription factors such as neuronal PAS domain protein 4 (Npas4), GATA binding protein 3 (Gata3), and paired box 7 (Pax7) were among the most significantly downregulated genes. In conclusion, our results indicate that higher plasma TMAO levels induce differential mRNA expression profiles in the ischemic brain tissue in our pre-clinical stroke model, and the predicted pathways provide the molecular basis for regulating the TMAO-enhanced neuroinflammatory response in the brain.

Life-threatening Neutropenia After a Short Course of Dual Antiplatelet Therapy for Minor Stroke (P7-5.017)

<h3>Objective:</h3> We present a case of unexpected severe neutropenia in a patient prescribed a 21-day course of double antiplatelet therapy (aspirin and clopidogrel) for ischemic stroke. <h3>Background:</h3> Antiplatelet agents are the cornerstone of atherosclerotic stroke prevention. In recent years, trials such as CHANCE and POINT have popularized the use of a short (3–4 week) course of dual antiplatelet therapy (aspirin plus a P2Y12 inhibitor) after minor stroke or high-risk transient ischemic attack. While some P2Y12 inhibitors such as ticlopidine have been associated with neutropenia, this side effect is exceedingly rare with clopidogrel and has primarily been reported with prolonged use after coronary stenting. <h3>Design/Methods:</h3> N/A <h3>Results:</h3> An 88-year-old woman presented with acute onset of left face, arm, and leg numbness. National Institutes of Health Stroke Scale was 1 for mild sensory loss. Non-contrast computed tomography (CT) of head showed no evidence of hemorrhage or acute ischemia. Brain magnetic resonance imaging revealed a small area of diffusion positivity in the right thalamus consistent with acute ischemic stroke. She was already taking aspirin 81mg daily; clopidogrel 75mg daily was added for a 21-day course. WBC count during admission had ranged from 4.11–6.27 K/uL. On post-stroke day 23, she presented with three days of malaise, abdominal pain, and fever to 102 °F. WBC and absolute neutrophil count were now markedly low (0.67, &lt;0.01 K/uL, respectively). CT scan of abdomen revealed enterocolitis and early appendicitis. She was diagnosed with febrile neutropenia and treated with broad-spectrum antibiotics and filgrastim. As there was no evidence of hematologic malignancy, it was concluded that her neutropenia was drug-induced secondary to clopidogrel. She gradually recovered and cell counts had normalized at follow-up. <h3>Conclusions:</h3> While rare, severe neutropenia can occur with even brief use of clopidogrel. With the widespread adoption of short course dual antiplatelet therapy after stroke, neurologists should be aware of this association. <b>Disclosure:</b> Dr. Culbertson has nothing to disclose. Dr. Blaszkowsky has nothing to disclose.

Abstract TP206: Scrnaseq Revealed Increased Microglial Expression Of Interferon-induced Transmembrane 3 (ifitm3) In Stroke And Other Inflammatory Conditions In The Brain.

Microglia (MG) play critical roles in modulating inflammation and interferon signaling is a driver of neuroinflammation in various neurodegenerative diseases. Recently, Ifitm3 (a member of the “interferon-inducible transmembrane” family) has been revealed as a molecular mediator between neuroinflammation and neurodegeneration. However, expression of Ifitm3 in MG under inflammatory conditions, is not well described. Methods: To define the expression of Ifitm3 following stroke, permanent distal middle cerebral artery occlusion was performed with aged mice (18-22 mos). Brains were isolated at post-stroke day (PSD) 14 for single-cell RNAseq (scRNAseq), and at PSD 3, 7, and 14 for immunostaining and qRT-PCR. Primary microglial culture was established to evaluate mRNA levels of Ifitm3 and other pro-inflammatory genes to cytokine treatment. Results: Our scRNAseq revealed that Ifitm3 is among the top 10 genes upregulated in MG following stroke in aged brains. To validate these findings, we utilized a stroke model in aged mice and primary cell culture. We found that ischemic injury causes an induction of Ifitm3 in the striatum and peri-infarcted area following stroke, with peak induction PSD 7 (n=3-5, p&lt;0.01). We also examined the induction of other Ifitm gene mRNA in brains following stroke. Interestingly, among Ifitm family members, only Ifitm3 was markedly induced in the damaged cortex of PSD 3 brains (n=4-6). We found that Ifitm3 expression is induced in primary MG following treatment with proinflammatory mediators (TNFα/IFNγ) for 6 hours (n=5-6, p&lt;0.05) or for 24 hours (n=7-8, p&lt;0.01). TNFα/IFNγ treatment also induced increased expression of inflammasome component gene, Il1b and Casp1 . There was a close correlation between Ifitm3 and Casp1 expression, suggesting a potential of Ifitm3 in inflammasome activation. Conclusions: We demonstrated the profound induction of Ifitm3 in activated MG within the stroke brain in aged mice and in response to inflammatory factors in primary microglia culture. These studies identify Ifitm3 as a novel player involved in microglial modulation in response to a variety of inflammatory conditions. Future studies are warranted to determine if Ifitm3 deletion or inhibition can reduce neuroinflammation in the stroke brain.

Abstract WMP111: MicroRNAs As A Therapeutic Target To Reduce Microglial Activation After Post-stroke Social Isolation

Introduction: Social isolation (SI) and loneliness are linked to all-cause mortality, as well as mortality from stroke and other vascular diseases. However, the mechanisms mediating the effects of social factors on stroke recovery are unknown. We hypothesized that differential expression of miRNAs contributes to the deleterious effects of post-stroke SI. Methods: Aged (18-20 months) C57BL/6 male mice were used to examine the detrimental effects of post-stroke SI on miRNA profiles in the brain. Mice were randomly assigned to either pair housing (PH), or single housing (SI) three days after a 60-minute transient right middle cerebral artery occlusion (MCAO). At this time point (post-stroke day 3), the infarct is complete, and was equivalent between groups, avoiding potential changes seen with differing infarct sizes. Temporal miRNA profiling of the ipsilateral hemisphere was assessed at two-time points (post-stroke SI D4 and D27). Brain cells were analyzed by flow cytometry. Results: Post-stroke SI resulted in significant alterations of distinct miRNA profiles within the brain across both acute and chronic time points (n=4/grp, FDR adjusted * p &lt;0.05). MiRNA-mRNA interactional analysis revealed miR-10a-5p and miR-10b-5p as pivotal nodes within the pool of miRNAs that interacted with the largest subset of miRNAs for post-stroke at SI D4 and D27, respectively. Downstream pathway analysis utilizing an independent repository, the KEGG pathway showed 4 days of isolation resulted in the enrichment of pathways related to microglial activation and 27 days of isolation lead to the activation of neuronal-specific pathways that regulate cognition and motivation (FDR adjusted * p &lt;0.05). Independent validation cohorts demonstrated significant activation of microglia at post-stroke SI D4 as assessed by the median fluorescence intensity (MFI) of purinergic receptor P2Y12 (P2RY12), in CD45 int CD11b + P2RY12 + cells in the brain. MFI of P2RY12 was significantly downregulated in post-stroke SI mice at D4 (n=7-8/grp, * p &lt;0.05) compared to PH mice. Conclusions: These results support our hypothesis that post-stroke SI exacerbates microglia activation, and results in the differential expression of microglial pathway-related miRNAs.

Abstract WMP30: Expansion Of Plasma Micrornas Over The First Month Following Human Stroke

Introduction: Stroke induces changes in plasma microRNA (miRNA) expression in the first 24hrs post-injury, but few have characterized miRNA expression during the early recovery phase. Hypothesis: We hypothesized that plasma miRNA expression would evolve over the first month post-stroke with fewer significantly altered miRNAs over time as inflammation and edema resolve. Methods: Longitudinal samples were collected 5, 15, and 30 days post-stroke from 2 studies (BIOREC and MORSE, n=55 combined at each time point) and compared to controls matched for age, sex, race and cardiovascular comorbidities. Stroke samples collected on average around day 15 from a second study (CPASS, n=48) were also compared to matched controls and used as a validation cohort. MiRNA expression was determined with OpenArray qRT-PCR quantifying 758 miRNAs. Significant miRNAs had a corrected p-value less than 0.05 and fold-change greater than |1.5|. Results: Eight miRNAs remained significantly altered across all 3 time points in the discovery cohort and in the day 15 validation cohort (decreased expression compared to controls: miR-101-3p, miR-107, miR-16-2-3p, miR-19a-3p, miR-19b-3p, and miR-26b-5p; increased expression: miRNA-199a-3p and miR-652-3p). The number of significant miRNAs more than doubled from post-stroke day 5 to days 15 and 30 (19, 53, and 45 miRNAs respectively in BIOREC/MORSE, 49 miRNAs at day 15 in CPASS). At day 5 most significant miRNAs (14/19) showed decreased expression compared to controls whereas by day 30 most demonstrated increased expression (36/45). Many of the significant miRNAs, including miR-107 and miR-652-3p, and many others at days 15 and 30 were enriched in brain or CSF in prior studies. Conclusions: While several miRNAs show significant change in expression consistently from 5 to 30 days post-stroke, there is also an expansion of significant miRNAs at days 15 and 30. This expansion includes many miRNAs enriched in brain or CSF and could represent a shift from inflammation to neural repair processes, but this requires further study.

Abstract TP238: Optogenetic Stimulation Of Cortico-thalamic Circuits Promotes Oligodendrogenesis In The Degenerative Thalamus After Stroke

Introduction: Loss of neuronal activity after stroke can impact many activity-dependent processes including oligodendrocyte maturation and myelination. The cortico-thalamic circuit is particularly disrupted after cortical stroke, resulting in secondary axonal degeneration in the thalamus. This secondary thalamic injury is associated with worsened functional outcomes. Increasing neuronal activity by optogenetic stimulation promotes oligodendrogenesis and myelination in normal conditions. In this study, we use optogenetic stimulation to selectively re-activate the cortico-thalamic circuit after stroke and examine its effects on oligodendrocytes in the degenerative thalamus. Methods: C57BL6 male mice (6-8 weeks old) were injected AAV1-CaMKIIa-hChR2 virus in the ipsilesional somatosensory cortex and implanted an optic fiber in the ipsilesional thalamus. After six weeks, the left middle cerebral artery was permanently occluded to generate infarct in iS1. Stimulated mice were given daily optogenetic stimulations between Post-stroke Day (PD) 4 to 14. Brain sections were immunostained to detect primary cortical injury and secondary thalamic injury (NeuN), and neuroinflammation (CD68) and oligodendrogenesis (Olig2 and CC1) in the thalamus. Results: ChR2 is specifically expressed in the somatosensory cortico-thalamic tract. Neuronal loss and robust neuroinflammation were observed in iTH after stroke, but there was no significant difference between non-stimulated and stimulated mice. However, stimulated mice exhibited 5.5 times increase in the numbers of mature oligodendrocytes (Olig2 + CC1 + , p=0.02, n=5/group), and 5.4 times increase in the numbers of oligodendrocyte progenitor cells (Olig2 + , p=0.03) in the degenerative thalamus. Conclusion: Our data demonstrate that selective re-activation of the cortical-thalamic circuits promoted oligodendrogenesis in the secondary degenerative thalamus after stroke. As the secondary thalamic injury allows an extended treatment window, targeting secondary injury is a promising approach to improve stroke recovery. Current studies are testing the effects of cortico-thalamic circuit stimulation in myelination in the degenerative thalamus and functional outcome after stroke.

Status Of Dysphagia After Ischemic Stroke: A Korean Nationwide Study

ObjectiveTo identify the incidence of dysphagia after ischemic stroke and determine factors affecting the presence of dysphagia. DesignRetrospective case-control study. This was an interim analysis of a prospective multicenter Korean stroke cohort. SettingAcute care university hospitals. ParticipantsPatients (N=6000) with first-ever acute ischemic stroke. Patients were divided into 2 groups according to the presence or absence of dysphagia confirmed at 7 days after onset using the American Speech-Language-Hearing Association National Outcomes Measurement System (ASHA-NOMS) scale, which was determined after conducting screening or standardized tests. InterventionsNot applicable. Main Outcome MeasuresAge at stroke onset, body mass index (BMI), premorbid modified Rankin Scale (mRS), brainstem lesions, National Institutes of Health Stroke Scale (NIHSS), poststroke mRS, and ASHA-NOMS swallowing level at poststroke day 7 were evaluated. ResultsAmong patients with ischemic stroke, 32.3% (n=1940) had dysphagia at 7 days after stroke onset. At discharge, 80.5% (n=1561) still had dysphagia. The prediction model for the presence of dysphagia identified age at onset, underweight (BMI <18.5 kg/m2), premorbid mRS, brainstem lesions, and NIHSS as independent predictors. The odds ratio (OR) for the presence of dysphagia significantly increased with underweight (OR, 1.6684; 95% confidence interval [CI], 1.27-2.20), increased age at onset (OR, 1.0318; 95% CI, 1.03-1.04), premorbid mRS (OR, 1.1832; 95% CI, 1.13-1.24), brainstem lesions (OR, 1.6494; 95% CI, 1.39-1.96), and NIHSS (OR, 1.2073; 95% CI, 1.19-1.23). ConclusionsThe incidence of dysphagia after ischemic stroke was 32.3%. The prediction model for the presence of dysphagia identified age, low BMI, premorbid disabilities, brainstem lesions, and NIHSS as predictive factors.

Multimodal Optical Imaging to Investigate Spatiotemporal Changes in Cerebrovascular Function in AUDA Treatment of Acute Ischemic Stroke.

Administration of 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA) has been demonstrated to alleviate infarction following ischemic stroke. Reportedly, the main effect of AUDA is exerting anti-inflammation and neovascularization via the inhibition of soluble epoxide hydrolase. However, the major contribution of this anti-inflammation and neovascularization effect in the acute phase of stroke is not completely elucidated. To investigate the neuroprotective effects of AUDA in acute ischemic stroke, we combined laser speckle contrast imaging and optical intrinsic signal imaging techniques with the implantation of a lab-designed cranial window. Forepaw stimulation was applied to assess the functional changes via measuring cerebral metabolic rate of oxygen (CMRO2) that accompany neural activity. The rats that received AUDA in the acute phase of photothrombotic ischemia stroke showed a 30.5 ± 8.1% reduction in the ischemic core, 42.3 ± 15.1% reduction in the ischemic penumbra (p < 0.05), and 42.1 ± 4.6% increase of CMRO2 in response to forepaw stimulation at post-stroke day 1 (p < 0.05) compared with the control group (N = 10 for each group). Moreover, at post-stroke day 3, increased functional vascular density was observed in AUDA-treated rats (35.9 ± 1.9% higher than that in the control group, p < 0.05). At post-stroke day 7, a 105.4% ± 16.4% increase of astrocytes (p < 0.01), 30.0 ± 10.9% increase of neurons (p < 0.01), and 65.5 ± 15.0% decrease of microglia (p < 0.01) were observed in the penumbra region in AUDA-treated rats (N = 5 for each group). These results suggested that AUDA affects the anti-inflammation at the beginning of ischemic injury and restores neuronal metabolic rate of O2 and tissue viability. The neovascularization triggered by AUDA restored CBF and may contribute to ischemic infarction reduction at post-stroke day 3. Moreover, for long-term neuroprotection, astrocytes in the penumbra region may play an important role in protecting neurons from apoptotic injury.

Enhanced Spontaneous Motor Recovery After Stroke in Mice Treated With Cerebrolysin

Background Motor recovery after stroke in humans and in rodent models is time sensitive. Recovery in patients is a result of biological spontaneous recovery via endogenous repair mechanisms and is likely improved by enhancing the synaptic plasticity required for endogenous repair. Cerebrolysin is a polypeptide preparation known to enhance neuroplasticity and may improve recovery in patients. In mice, we tested the hypothesis that Cerebrolysin can act poststroke to enhance both spontaneous and training-associated motor recovery. Methods Mice were trained to perform a skilled prehension task. We then induced a photothrombotic stroke in the caudal forelimb area, after which we retrained animals on the prehension task in the presence or absence of Cerebrolysin after a 2-day or 8-day delay. Mice received daily intraperitoneal Cerebrolysin or saline injections starting poststroke day 1 or poststroke day 7. Results Prior studies showed that poststroke recovery of prehension can occur if animals receive rehabilitative training during an early sensitive period but is incomplete if rehabilitative training is delayed. In contrast, we show complete recovery of prehension, despite a delay in rehabilitative training, when mice receive daily Cerebrolysin administration starting on poststroke day 1 or on poststroke day 8. When Cerebrolysin is given on poststroke day 1, recovery occurred even in the absence of training. Stroke volumes were similar across groups. Conclusions Poststroke Cerebrolysin administration leads to recovery of motor function independent of rehabilitative training without a protective effect on stroke volume. This is one of the first demonstrations of training-independent motor recovery in rodent stroke models.

Delayed Exercise-induced Upregulation of Angiogenic Proteins and Recovery of Motor Function after Photothrombotic Stroke in Mice

Treatments promoting post-stroke functional recovery continue to be an unmet therapeutic problem with physical rehabilitation being the most reproduced intervention in preclinical and clinical studies. Unfortunately, physiotherapy is typically effective at high intensity and early after stroke – requirements that are hardly attainable by stroke survivors. The aim of this study was to directly evaluate and compare the dose-dependent effect of delayed physical rehabilitation (daily 5 h or overnight voluntary wheel running; initiated on post-stroke day 7 and continuing through day 21) on recovery of motor function in the mouse photothrombotic model of ischemic stroke and correlate it with angiogenic potential of the brain. Our observations indicate that overnight but not 5 h access to running wheels facilitates recovery of motor function in mice in grid-walking test. Western blotting and immunofluorescence microscopy experiments evaluating the expression of angiogenesis-associated proteins VEGFR2, doppel and PDGFRβ in the peri-infarct and corresponding contralateral motor cortices indicate substantial upregulation of these proteins (≥2-fold) in the infarct core and surrounding cerebral cortex in the overnight running mice on post-stroke day 21. These findings indicate that there is a dose-dependent relationship between the extent of voluntary exercise, motor recovery and expression of angiogenesis-associated proteins in this expert-recommended mouse ischemic stroke model. Notably, our observations also point out to enhanced angiogenesis and presence of pericytes within the infarct core region during the chronic phase of stroke, suggesting a potential contribution of this tissue area in the mechanisms governing post-stroke functional recovery.

Abstract P758: Ischemic Stroke Induced Cardiac Dysfunction in Aged Mice

Introduction: Acute ischemic stroke patients with cardiac complications have increased in-hospital mortality. Most of these cardiac complications have been attributed to pre-existing cardiac comorbidities. Aging is a known risk factor for both stroke and myocardial injury. It is being increasingly recognized that ischemic injury to the brain in itself may lead to cardiac injury. However, the post stroke brain- heart interaction is not well defined in the aged. This study was designed to fill this gap in our knowledge. Methods: C57BL6/J young (3 months) and aged (18 months) male mice were subjected to 60 minutes of middle cerebral artery occlusion (MCAO). Mice had electrocardiogram (EKG, using rodent surgical monitor) and Echocardiogram (using vevo 3100 imaging system) pre-stroke, post stroke day 3, 7, and 14. All mice were sacrificed at day 14. EKG and ECHO results were analyzed using generalized estimated equation method with post hoc group comparison test at each time point, Bonferroni correction. Results: There was a significant decrease in cardiac output after stroke in aged but not young mice at three days post stroke, as compared to baseline (young, baseline:18.5±1.6ml/min vs day 3:16.6±5.16 ml/min, n=8; aged, baseline 25.4±6.8 6 ml/min vs. day 3, 16.8±6.8 n=6, p=0.0038). The cardiac output normalized to baseline in young but remained significantly low in aged mice even at day 14. Interestingly, the Left ventricular (LV) mass decreased significantly after stroke in aged mice at day 3, 118.4±23.2 mg as compared to baseline, 160.7±22.9 mg. This remained significantly lower than baseline at post stroke day seven, 124.6±32.9 mg and fourteen, 129.5±37.2 mg, p&lt;0.05. A similar trend was seen in left ventricular anterior and posterior wall thickness. There was no significant change in LV mass in young mice, p&gt;0.05. Conclusion: Our data indicates that cardiac output decreases after stroke in aged mice. Interestingly, there is a significant decrease in left ventricular mass and ventricular wall thickness in aged mice after MCAO, indicating cardiac atrophy. We speculate this may be secondary to cardiac ischemia caused by stroke. Ongoing work in our laboratory is aimed at examining the histological changes in cardiomyocytes after MCAO.

Abstract P736: Memantine, an NMDA Antagonist and Aging Alter the Thalamic Gliosis in Experimental Stroke in Mice

Secondary injury in the thalamus has been observed following cortical stroke in rodents and humans and is associated with worsened recovery. Interruption of this progressive injury reflects an important therapeutic goal. However, the mechanisms whereby primary cortical infarction leads to remote injury in distant regions of brain are not well defined. We used a mouse model of cortical stroke (which demonstrates delayed thalamic injury) to define the time course of thalamic gliosis and neuronal injury and then test the potential of delayed memantine treatment (an NMDA receptor antagonist) to attenuate this secondary injury. Methods: Cortical infarction was induced by permanent occlusion of the distal middle cerebral artery (pdMCAO) in male C57BL/6J mice (young and aged) and CCR2-RFP mice. Brain infarct, cell-specific injury, and gliosis were measured by cresyl violet, Fluoro-jade C (FJC), TTC, FACS, and immunofluorescence. In young mice, memantine was injected at 4 and 24 hours post-stroke (100 and 50 mg/kg, ip). Brains were evaluated at post-stroke day 3 and 14 (PSD3 and PSD14). Results: At PSD3, the primary infarct was restricted to the cortex of the MCA territory, with no infarct detected in the thalamus of young mice. However, by PSD 14, neurons in the ipsilateral thalamus exhibited significant injury (FJC positive, condensed pyknotic nuclei). Gliosis was first detectable in the ipsilateral thalamus at PSD3 and progressively increased to PSD14 (anti-GFAP and Iba1). Infiltration of peripheral-derived monocytes was determined to be one source of the activated microglia in the thalamus (CCR2-RFP reporter mice, n=3). Interestingly, pdMCAO mice allowed to recover for two years demonstrated persistent astrogliosis (cortex and thalamus), though microgliosis was no longer evident (n=2). Aged mice subjected to pdMCAO also demonstrated gliosis in thalamus at PSD14, albeit to a lesser extent than young mice (n=5 each age). Finally, delayed treatment with memantine resulted in significantly attenuated gliosis and neuronal loss in the thalamus at PSD14 (young mice, n=9 each). Conclusions: These results further define gliosis in the mechanism of secondary injury and importantly demonstrate attenuation of secondary injury by delayed NMDA receptor antagonism.

The Effect of Histone Deacetylase Inhibitors Panobinostat or Entinostat on Motor Recovery in Mice After Ischemic Stroke.

Using rigorous and clinically relevant experimental design and analysis standards, in this study, we investigated the potential of histone deacetylase (HDAC) inhibitors panobinostat and entinostat to enhance recovery of motor function after photothrombotic stroke in male mice. Panobinostat, a pan-HDAC inhibitor, is a FDA-approved drug for certain cancers, whereas entinostat is a class-I HDAC inhibitor in late stage of clinical investigation. The drugs were administered every other day (panobinostat-3 or 10mg/kg; entinostat-1.7 or 5mg/kg) starting from day 5 to 15 after stroke. To imitate the current standard of care in stroke survivors, i.e., physical rehabilitation, the animals run on wheels (2h daily) from post-stroke day 9 to 41. The predetermined primary end point was motor recovery measured in two tasks of spontaneous motor behaviors in grid-walking and cylinder tests. In addition, we evaluated the running distance and speed throughout the study, and the number of parvalbumin-positive neurons in medial agranular cortex (AGm) and infarct volumes at the end of the study. Both sensorimotor tests revealed that combination of physical exercise with either drug did not substantially affect motor recovery in mice after stroke. This was accompanied by negligible changes of parvalbumin-positive neurons recorded in AGm and comparable infarct volumes among experimental groups, while dose-dependent increase in acetylated histone 3 was observed in peri-infarct cortex of drug-treated animals. Our observations suggest that add-on panobinostat or entinostat therapy coupled with limited physical rehabilitation is unlikely to offer therapeutic modality for stroke survivors who have motor dysfunction.

Delayed atomoxetine or fluoxetine treatment coupled with limited voluntary running promotes motor recovery in mice after ischemic stroke

Currently, there is an unmet need for treatments promoting post-stroke functional recovery. The aim of this study was to evaluate and compare the dose-dependent effect of delayed atomoxetine or fluoxetine therapy (starting on post-stroke day 5), coupled with limited physical exercise (2 hours daily voluntary wheel running; post-stroke days 9 to 42), on motor recovery of adult male mice after photothrombotic stroke. These drugs are selective norepinephrine or serotonin reuptake inhibitors indicated for disorders unrelated to stroke. The predetermined primary end-point for this study was motor function measured in two tasks of spontaneous motor behaviors in grid-walking and cylinder tests. Additionally, we quantified the running distance and speed throughout the study, the number of parvalbumin-positive neurons in the medial agranular cortex and infarct volumes. Both sensorimotor tests revealed that neither limited physical exercise nor a drug treatment alone significantly facilitated motor recovery in mice after stroke. However, combination of physical exercise with either of the drugs promoted restoration of motor function by day 42 post-stroke, with atomoxetine being a more potent drug. This was accompanied by a significant decrease in parvalbumin-positive inhibitory interneurons in the ipsilateral medial agranular cortex of mice with recovering motor function, while infarct volumes were comparable among experimental groups. If further validated in larger studies, our observations suggest that add-on atomoxetine or fluoxetine therapy coupled with limited, structured physical rehabilitation could offer therapeutic modality for stroke survivors who have difficulty to engage in early, high-intensity physiotherapy. Furthermore, in light of the recently completed Assessment oF FluoxetINe In sTroke recoverY (AFFINITY) and Efficacy oF Fluoxetine-a randomisEd Controlled Trial in Stroke (EFFECTS) trials, our observations call for newly designed studies where fluoxetine or atomoxetine pharmacotherapy is evaluated in combination with structured physical rehabilitation rather than alone. This study was approved by the Texas Tech University Health Sciences Center Institutional Animal Care and Use Committee (protocol # 16019).

Optogenetic Stimulation Reduces Neuronal Nitric Oxide Synthase Expression After Stroke

Post-stroke optogenetic stimulation has been shown to enhance neurovascular coupling and functional recovery. Neuronal nitric oxide synthase (nNOS) has been implicated as a key regulator of the neurovascular response in acute stroke; however, its role in subacute recovery remains unclear. We investigated the expression of nNOS in stroke mice undergoing optogenetic stimulation of the contralesional lateral cerebellar nucleus (cLCN). We also examined the effects of nNOS inhibition on functional recovery using a pharmacological inhibitor targeting nNOS. Optogenetically stimulated stroke mice demonstrated significant improvement on the horizontal rotating beam task at post-stroke days 10 and 14. nNOS mRNA and protein expression was significantly and selectively decreased in the contralesional primary motor cortex (cM1) of cLCN-stimulated mice. The nNOS expression in cM1 was negatively correlated with improved recovery. nNOS inhibitor (ARL 17477)-treated stroke mice exhibited a significant functional improvement in speed at post-stroke day 10, when compared to stroke mice receiving vehicle (saline) only. Our results show that optogenetic stimulation of cLCN and systemic nNOS inhibition both produce functional benefits after stroke, and suggest that nNOS may play a maladaptive role in post-stroke recovery.

Molecular profile of the rat peri-infarct region four days after stroke: Study with MANF

The peri-infarct region after ischemic stroke is the anatomical location for many of the endogenous recovery processes; however, -the molecular events in the peri-infarct region remain poorly characterized. In this study, we examine the molecular profile of the peri-infarct region on post-stroke day four, a time when reparative processes are ongoing. We used a multiomics approach, involving RNA sequencing, and mass spectrometry-based proteomics and metabolomics to characterize molecular changes in the peri-infarct region. We also took advantage of our previously developed method to express transgenes in the peri-infarct region where self-complementary adeno-associated virus (AAV) vectors were injected into the brain parenchyma on post-stroke day 2. We have previously used this method to show that mesencephalic astrocyte-derived neurotrophic factor (MANF) enhances functional recovery from stroke and recruits phagocytic cells to the peri-infarct region. Here, we first analyzed the effects of stroke to the peri-infarct region on post-stroke day 4 in comparison to sham-operated animals, finding that strokeinduced changes in 3345 transcripts, 341 proteins, and 88 metabolites. We found that after stroke, genes related to inflammation, proliferation, apoptosis, and regeneration were upregulated, whereas genes encoding neuroactive ligand receptors and calcium-binding proteins were downregulated. In proteomics, we detected upregulation of proteins related to protein synthesis and downregulation of neuronal proteins. Metabolomic studies indicated that in after stroke tissue there is an increase in saccharides, sugar phosphates, ceramides and free fatty acids and a decrease of adenine, hypoxantine, adenosine and guanosine. We then compared the effects of post-stroke delivery of AAV1-MANF to AAV1-eGFP (enhanced green fluorescent protein). MANF administration increased the expression of 77 genes, most of which were related to immune response. In proteomics, MANF administration reduced S100A8 and S100A9 protein levels. In metabolomics, no significant differences between MANF and eGFP treatment were detected, but relative to sham surgery group, most of the changes in lipids were significant in the AAV-eGFP group only. This work describes the molecular profile of the peri-infarct region during recovery from ischemic stroke, and establishes a resource for further stroke studies. These results provide further support for parenchymal MANF as a modulator of phagocytic function.