Functional Assessment of Stroke-Induced Regulation of miR-20a-3p and Its Role as a Neuroprotectant

AbstractMicroRNAs have gained popularity as a potential treatment for many diseases, including stroke. This study identifies and characterizes a specific member of the miR-17–92 cluster, miR-20a-3p, as a possible stroke therapeutic. A comprehensive microRNA screening showed that miR-20a-3p was significantly upregulated in astrocytes of adult female rats, which typically have better stroke outcomes, while it was profoundly downregulated in astrocytes of middle-aged females and adult and middle-aged males, groups that typically have more severe stroke outcomes. Assays using primary human astrocytes and neurons show that miR-20a-3p treatment alters mitochondrial dynamics in both cell types. To assess whether stroke outcomes could be improved by elevating astrocytic miR-20a-3p, we created a tetracycline (Tet)-induced recombinant adeno-associated virus (rAAV) construct where miR-20a-3p was located downstream a glial fibrillary acidic protein promoter. Treatment with doxycycline induced miR-20-3p expression in astrocytes, reducing mortality and modestly improving sensory motor behavior. A second Tet-induced rAAV construct was created in which miR-20a-3p was located downstream of a neuron-specific enolase (NSE) promoter. These experiments demonstrate that neuronal expression of miR-20a-3p is vastly more neuroprotective than astrocytic expression, with animals receiving the miR-20a-3p vector showing reduced infarction and sensory motor improvement. Intravenous injections, which are a therapeutically tractable treatment route, with miR-20a-3p mimic 4 h after middle cerebral artery occlusion (MCAo) significantly improved stroke outcomes including infarct volume and sensory motor performance. Improvement was not observed when miR-20a-3p was given immediately or 24 h after MCAo, identifying a unique delayed therapeutic window. Overall, this study identifies a novel neuroprotective microRNA and characterizes several key pathways by which it can improve stroke outcomes.

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Abstract 59: Post-stroke Delivery of Microrna-363 to Middle-aged Female Rats is Internalized by Neurons and Suppresses the Cell Death Effector Pathway

Stroke ◽

10.1161/str.48.suppl_1.59 ◽

2017 ◽

Vol 48 (suppl_1) ◽

Author(s):

Amutha Selvamani ◽

Farida Sohrabji

Keyword(s):

Cell Death ◽

Caspase 3 ◽

Infarct Volume ◽

Cell Types ◽

Female Rats ◽

Middle Aged ◽

Stroke Outcomes ◽

Post Stroke ◽

Double Label ◽

Death Effector

Background: MicroRNAs serve as translational inhibitors and offer a unique therapeutic target for acute diseases such as stroke. Profiling of circulating miRNA after stroke identified mir363, whose expression was inversely correlated with infarct volume. Middle aged female rats show worse stroke outcomes than younger females and have much lower levels of mir363. Our recent studies showed that iv injections of miR363 mimic to middle aged females, significantly improved stroke outcome. The present study is designed to determine the mechanisms by which miR-363 acts as a therapeutic miR. Methods: Middle aged (12 mo) females were subject to MCAo. At 4h post-stroke, animals received a tail-vein injection of miR-363-3p FAM or scrambled control. Animals were terminated at 48h or 5d post-MCAo and perfused transcardially or processed for protein, respectively. To determine which neural cell types localized exogenous mir363-3p, combined immunofluorescence was performed for cell specific markers (neuronal (NeuN), astrocytic (GFAP), microglial (CD11b) and endothelial (PECAM)) and mir363-3p-FAM mimic on coronal brain sections (25 mm thickness). Protein lysates from the ischemic tissue was analyzed for caspase-3 expression by Western blot analysis. Results: FAM-labeled mir363-3p was widely detected in the forebrain. The majority of NeuN+ cells in the cortex and striatum were also labeled with FAM-363-3p, indicating a robust internalization of the mimic in neurons. FAM-mir363-3p was also localized to a few microglia (CD11b +), virtually no double-label was seen in astrocytes and endothelial cells. Mir363 decreased the expression and functional activity of caspase3 in the ischemic hemisphere. Conclusion: Collectively, the data suggests that exogenous miR-363-3p is shuttled to the brain and is preferentially internalized by neurons. Together with the caspase-3 regulation, our data suggests that mir363 may improve stroke outcomes by suppressing a cell death effector.

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Abstract WP96: The Histone Deacetylase Inhibitor, Sodium Butyrate, Exhibits Biphasic Neuroprotective Mechanisms in the Acute Phase of Ischemic Stroke in Middle-Aged Female Rats

Stroke ◽

10.1161/str.48.suppl_1.wp96 ◽

2017 ◽

Vol 48 (suppl_1) ◽

Author(s):

Min Jung Park ◽

Farida Sohrabji

Keyword(s):

Histone Deacetylase ◽

Acute Phase ◽

Sodium Butyrate ◽

Hdac Inhibitor ◽

Female Rats ◽

Middle Aged ◽

Peripheral Tissues ◽

Post Stroke ◽

Sensory Motor ◽

Anti Inflammatory

Introduction: Sodium butyrate (NaB) is a histone deacetylase (HDAC) inhibitor exhibiting anti-inflammatory and neuroprotective effects in a rat ischemic model of a myocardial ischemia as well as stroke. Although clinical evidence shows that older women are at higher risk for stroke occurrence and greater stroke severity, no studies have evaluated the effectiveness of NaB either in females or in older animals. Methods: To determine the effects of NaB on stroke in older females, acyclic middle-aged Sprague-Dawley female rats (10-12 months old, constant diestrus) were subject to middle cerebral artery occlusion (MCAo) by intracerebral injection of recombinant endothelin-1. Rats were treated with NaB (300 mg/kg, i.p.) at 6h and 30h following ET-1 injection. Animals were tested for sensory motor performance pre and post stroke. Subsequently, rats were sacrificed at the early (2d) or late (5d) acute phase after MCAo. Serum and tissue samples were collected for biochemical analyses. Results: NaB treatment reduced infarct volume and ameliorated stroke-induced sensory motor impairment in middle-aged female rats post MCAo. At the early acute phase, NaB treatment decreased brain lipid peroxides, and reduced serum levels of GFAP, a marker of blood brain barrier permeability. NaB also reduced expression of the inflammatory cytokine IL-1beta in circulation and IL-18 in the ischemic hemisphere. At the late acute phase, NaB treatment further suppressed MCAo-induced increase of IL-1beta, IL-17A, and IL-18 in brain lysates (cortex and striatum) from the ischemic hemisphere, and decreased ischemia-induced upregulation of IL-1beta and IL-18 in circulation, indicating a potent anti-inflammatory effect of the HDAC inhibitor. Moreover, NaB treatment also increased expression of IGF-1, a known neuroprotectant, in peripheral tissues including serum, liver, and spleen. Conclusions: These data provide the first evidence that delayed (> 6h) NaB treatment post-stroke is neuroprotective in older female rats. Importantly, these data also show that in addition to its well-known anti-inflammatory actions, NaB may exert a biphasic effect after stroke, operating initially to reduce oxidative stress in the brain, and later, elevating IGF-1 expression in peripheral tissues.

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Abstract TMP33: Repair of Ischemic Intestinal Epithelial Stem Cells: Potential Therapy to Improve Stroke Outcomes

Stroke ◽

10.1161/str.51.suppl_1.tmp33 ◽

2020 ◽

Vol 51 (Suppl_1) ◽

Author(s):

Kathiresh Kumar Mani ◽

Farida Sohrabji

Keyword(s):

Stem Cells ◽

Motor Function ◽

Stroke Recovery ◽

Female Rats ◽

Stem Cell Marker ◽

Left Middle Cerebral Artery ◽

Intestinal Epithelial ◽

Epithelial Stem Cells ◽

Stroke Outcomes ◽

Sensory Motor

Background: Nearly 50% of all stroke patients experience “leaky” gut, gut hemorrhage and gut epithelium damage. Gut leakiness may increase circulating inflammatory cytokines and other gut products such as endotoxins, which can impair stroke recovery. Here we tested the hypothesis that normalizing gut function via transplantation of intestinal epithelial stem cells (IESC) after stroke may stimulate repair of gut structures and improve stroke outcomes. Methods: Reproductive senescent female Sprague-Dawley rats used for this study and assigned to the following groups: Control (no stroke); stroke with sham transplant (vehicle); stroke with IESC transplantation. Rats were subjected to stereotaxic surgery to occlude the left middle cerebral artery by using Endothelin-1. Primary IECs were isolated from young female rats to prepare organoids cultures. Dissociated organoids were labeled with PKH26 and injected iv either once (48h after stroke) or 3 times (4h/24h/48h after stroke). Behavioral assays and saphenous blood draws were performed pre-stroke, 2d and 5d after stroke. Trunk blood, brain tissue and a segment of small intestine was collected at termination and processed for the expression of the stem cell marker Lgr5+, Na/K ATPase-α, and tight junction proteins. Results: Significant deterioration of the gut architecture was observed after stroke, including blunted or absent villi and irregular crypts. In animals that received PKH26-labeled organoid tranplants at 48h post stroke, labeled cells were seen in the center of the villus and a few organoid cells were immunositve for Lgr5+. Animals that received 3 organoid injections showed PKH26-labeled cells incorporated in both the villus and crypts. In these animals, villus were well formed and appeared no different from non-stroke controls. Sensory motor function assessed by adhesive removal test on the side contralateral to the infarction was severely impaired in the stroke/no transplant animals (120+secs/timed out), while this impairment was attenuated in the group that received 3 organoid injections (31±11 secs). Conclusion: These data suggest that transplantation of IESC after stroke may promote repair of gut villus and crypts, with a concomitant improvement in sensory motor function.

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Neuronal expression of the mitochondrial protein prohibitin confers profound neuroprotection in a mouse model of focal cerebral ischemia

Journal of Cerebral Blood Flow & Metabolism ◽

10.1177/0271678x17720371 ◽

2017 ◽

Vol 38 (6) ◽

pp. 1010-1020 ◽

Cited By ~ 3

Author(s):

Anja Kahl ◽

Corey J Anderson ◽

Liping Qian ◽

Henning Voss ◽

Giovanni Manfredi ◽

...

Keyword(s):

Brain Injury ◽

Cerebral Ischemia ◽

Focal Cerebral Ischemia ◽

Infarct Volume ◽

Mitochondrial Protein ◽

Motor Impairment ◽

Cell Types ◽

Viral Gene ◽

Sensory Motor ◽

The Brain

The mitochondrial protein prohibitin (PHB) has emerged as an important modulator of neuronal survival in different injury modalities . We previously showed that viral gene transfer of PHB protects CA1 neurons from delayed neurodegeneration following transient forebrain ischemia through mitochondrial mechanisms. However, since PHB is present in all cell types, it is not known if its selective expression in neurons is protective, and if the protection occurs also in acute focal ischemic brain injury, the most common stroke type in humans. Therefore, we generated transgenic mice overexpressing human PHB1 specifically in neurons (PHB1 Tg). PHB1 Tg mice and littermate controls were subjected to transient middle cerebral artery occlusion (MCAo). Infarct volume and sensory-motor impairment were assessed three days later. Under the control of a neuronal promoter (CaMKIIα), PHB1 expression was increased by 50% in the forebrain and hippocampus in PHB1 Tg mice. The brain injury produced by MCAo was reduced by 63 ± 11% in PHB1 Tg mice compared to littermate controls. This reduction was associated with improved sensory-motor performance, suggesting that the salvaged brain remains functional. Approaches to enhance PHB expression may be useful to ameliorate the devastating impact of cerebral ischemia on the brain.

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Sex differences in stroke outcome correspond to rapid and severe changes in gut permeability in adult Sprague-Dawley rats

Biology of Sex Differences ◽

10.1186/s13293-020-00352-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Yumna El-Hakim ◽

Kathiresh Kumar Mani ◽

Amir Eldouh ◽

Sivani Pandey ◽

Maria T. Grimaldo ◽

...

Keyword(s):

Sex Differences ◽

Tight Junction ◽

Infarct Volume ◽

Motor Impairment ◽

Gut Permeability ◽

Sprague Dawley ◽

Stroke Outcomes ◽

Sprague Dawley Rats ◽

Junction Protein ◽

Sensory Motor

Abstract Background Sex differences in experimental stroke outcomes are well documented, such that adult males have a greater infarct volume, increased stroke-induced mortality, and more severe sensory-motor impairment. Based on recent evidence that the gut is an early responder to stroke, the present study tested the hypothesis that sex differences in stroke severity will be accompanied by rapid and greater permeability of the gut-blood barrier and gut dysbiosis in males as compared to females. Method Male and female Sprague-Dawley rats (5–7 months of age) were subject to endothelin (ET)-1-induced middle cerebral artery occlusion (MCAo). Sensory-motor tests were conducted pre- and 2 days after MCAo. Gut permeability was assessed in serum samples using biomarkers of gut permeability as well as functional assays using size-graded dextrans. Histological analysis of the gut was performed with H&E staining, periodic acid-Schiff for mucus, and immunohistochemistry for the tight junction protein, ZO-1. Fecal samples obtained pre- and post-stroke were analyzed for bacterial taxa and short-chain fatty acids (SCFAs). Results After stroke, males displayed greater mortality, worse sensory-motor deficit, and higher serum levels of proinflammatory cytokines IL-17A, MCP-1, and IL-5 as compared to females. MCAo-induced gut permeability was rapid and severe in males as indicated by dextran extravasation from the gut to the blood in the hyperacute (< 2 h) and early acute (2 days) phase of stroke. This was accompanied by dysmorphology of the gut villi and dysregulation of the tight junction protein ZO-1 in the acute phase. Fecal 16s sequencing showed no differences in bacterial diversity in the acute phase of stroke. Predictive modeling indicated that markers of gut permeability were associated with acute sensory-motor impairment and infarct volume. Conclusions These data show that extensive leakiness of the gut barrier is associated with severe post-stroke disability and suggest that reinforcing this barrier may improve stroke outcomes.

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Abstract WMP42: Astrocyte Specific rAAV-mediated Insulin-Like Growth Factor-1 Expression in Aging Female Rats Increases Post-Stroke Survival and Sensory Motor Performance

Stroke ◽

10.1161/str.47.suppl_1.wmp42 ◽

2016 ◽

Vol 47 (suppl_1) ◽

Author(s):

Andre K Okoreeh ◽

Shameena Bake ◽

Farida Sohrabji

Keyword(s):

Motor Performance ◽

Low Dose ◽

Female Rats ◽

Specific Gene ◽

High Dose ◽

Middle Aged ◽

Post Stroke ◽

Post Surgery ◽

Virus Serotype ◽

Sensory Motor

Background and Purpose: Our previous work shows that middle aged female rats sustain larger strokes as compared to younger female rats. With age, circulating and brain parenchymal levels of IGF-1 are reduced. Exogenous IGF-1 treatment improves infarct volume in aging females. Our recent studies show that astrocytes from aging females synthesize less IGF-1. Here we tested the hypothesis that elevation of astrocyte derived IGF-1 would improve stroke impairment in older female rats. Methods: Middle-aged (10-12 month old; acyclic) female rats were injected with adeno-associated virus serotype 5 (rAAV5) into the cortex and striatum. rAAV5 was packaged with the coding sequence of the IGF-1 gene downstream of an astrocyte-specific gene (GFAP). The construct contained the mCherry reporter gene. Control rAAV consisted of an identical shuttle vector construct without the IGF-1 gene. In separate experiments, two titers of virus were injected: high dose (5 X 10 12 VP/mL) or low dose (5 X 10 11 VP/mL). Three to four weeks after injection, middle-cerebral artery occlusion via an intraluminal suture for ninety minutes was performed followed by reperfusion. Post-surgery survival was monitored as well as sensory motor function using the vibrissae evoked forelimb placement task. Results: Specificity of IGF-1 expression was confirmed by visualization of the mCherry reporter under fluorescent illumination and immunohistochemistry. Post stroke survival was improved in animals that received the high dose rAAV-IGF-1 animals a 5-day period (p<0.001). Low dose rAAV-IGF-1 did not affect post stroke survival, however sensory motor performance was preserved in this group. In low dose control animals, ischemic stroke impaired performance on the vibrissae evoked forelimb placement task. Impairment was seen in the same-side and cross-midline task performance on the limb contralateral to the infarct and cross midline task on the limb ipsilateral to the infarcted side (p<0.05). No significant deficits were seen in the rAAV-IGF-1 low dose treated animals. Conclusion: These data support the hypothesis that increasing astrocytic IGF-1 in aging females improves post stroke survival and behavior outcomes.

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Abstract T P240: Intravenous Injections of Microrna 363 Mimic Promotes Neuroprotection in Middle Aged Females in an Ischemic Stroke Model

Stroke ◽

10.1161/str.46.suppl_1.tp240 ◽

2015 ◽

Vol 46 (suppl_1) ◽

Author(s):

Amutha Selvamani ◽

Farida Sohrabji

Keyword(s):

Infarct Volume ◽

Expression Patterns ◽

Elevated Serum ◽

Treated Group ◽

Female Rats ◽

Pcr Analysis ◽

Circulating Microrna ◽

Middle Aged ◽

Post Stroke ◽

Male And Female Rats

Background: Analysis of circulating microRNA in young and middle aged male and female rats revealed distinct expression patterns of post-stroke microRNAs (Selvamani et al., 2014). Mir-363 expression was inversely related to infarct volume, such that adult females, who display the smallest infarct volumes, had the highest expression of miR363. Based on this association, we hypothesized that mir-363 may promote survival of ischemic neurons. As proof of concept, the present study utilized middle aged females to investigate the role of miR-363 in neuroprotection. Methods: Middle aged (12 mo) female rats were subject to middle cerebral artery occlusion (MCAo). At 4h post-stroke, animals received a tail-vein injection of miR-363 or scrambled control. Vibrissae-elicited forelimb placement (VIB) test was performed pre and post MCAo to assess motor deficits. Blood samples were drawn at 2d and 5d post stroke. All animals were terminated at 5d post MCAo and the brains processed for infarct analysis by standard histological procedures. Total RNA isolated from serum and brain was subject to QPCR amplification for miR-363 and U6 (normalization control) Results: IV injections of mir363 significantly elevated serum expression of this microRNA as compared to animals injected with scrambles control oligos, when measured 2d post stroke. Infarct volumes (cortex and striatum), at 5d post stroke, were significantly reduced in the miR-363 treated group as compared to controls (p ≤ 0.001). VIB-test indicated significant motor recovery post-stroke in the contralateral limb in miR-363 mimic treated group as compared to controls. RT-PCR analysis of brain tissue showed higher expression of miR-363 in the left (ischemic) hemisphere in the miR-363 mimic group while, no difference was observed in the non-ischemic, indicating that the mimetic is recruited to the ischemic site. Conclusion: The present study underscores the value of miRNA profiling in populations with different stroke outcomes as a strategy to identify new therapeutic targets for stroke.

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Abstract WP108: Astrocyte-specific rAAV-mediated Insulin-like Growth Factor-1 Expression in Aging Female Rats Alters Stroke-induced Brain Immune Profile and Reduces Motor Impairment

Stroke ◽

10.1161/str.48.suppl_1.wp108 ◽

2017 ◽

Vol 48 (suppl_1) ◽

Author(s):

Andre K Okoreeh ◽

Shameena Bake ◽

Farida Sohrabji

Keyword(s):

Flow Cytometry ◽

Growth Factor ◽

Immune Cells ◽

Motor Impairment ◽

Female Rats ◽

Insulin Like Growth Factor ◽

Middle Aged ◽

Post Stroke ◽

Sensory Motor ◽

The Brain

Introduction: Middle-aged female rats sustain larger strokes than younger female rats. This may be due to age-related loss of circulating and parenchymal brain levels of insulin like growth factor-1 (IGF-1). With age, IGF-1 synthesis is decreased in astrocytes, a critical cell type for post-stroke recovery. Here we tested the hypothesis that replenishing IGF-1 in aging astrocytes will improve stroke outcomes by influencing the type and extent of immune cells infiltration into the brain post-stroke. Methods: Middle-aged (10-12 mo old, acyclic) female rats were injected into the striatum and cortex with adeno-associated virus serotype 5 (rAAV5) packaged with the coding sequence of the hIGF-1 gene downstream of an astrocyte-specific promoter (GFAP). The rAAV-control consisted of an identical shuttle vector construct without the hIGF-1 gene. Three to four weeks later, animals underwent 90 minute transient middle cerebral artery occlusion via intraluminal suture. At 2d post stroke, flow cytometry was used to determine the type and extent of peripheral immune cells trafficked into the brain. In parallel studies, animals were tested for performance on sensory motor tasks at 2d and 5 days after MCAo. Results: rAAV-mediated IGF-1 expression was confirmed in astrocytes with RT-PCR. Flow cytometry analysis of immune cells in the brain at 24h post-stroke found that proportion of Treg cells was greater in animals with rAAV-IGF-1 as compared to rAAV-controls. Additionally, while there was no difference in the proportion of M2 microglia, rAAV-IGF-1 enhanced M2 infiltrating macrophages. At 2d and 5d post stroke, stroke-induced sensory motor impairment was reduced in animals with rAAV-IGF-1 as compared to rAAV-controls. Conclusion: Targeted enhancement of IGF-1 in astrocytes of middle-aged females improved stroke-induced behavioral impairment concomitant with recruitment of anti-inflammatory cell types to the ischemic brain.

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Abstract TP96: Neuroprotective Effects of IGF-1 in Middle-Aged Females Can Be Replicated by Antagomirs to microRNA (miR)-92b and miR-33a

Stroke ◽

10.1161/str.51.suppl_1.tp96 ◽

2020 ◽

Vol 51 (Suppl_1) ◽

Author(s):

Shameena Bake ◽

Homa Khosravian ◽

Farida Sohrabji

Keyword(s):

Infarct Volume ◽

Cell Monolayer ◽

Glucose Deprivation ◽

Stroke Recovery ◽

Female Rats ◽

Middle Aged ◽

Neuroprotective Effects ◽

Lectin Staining

Background: Our previous studies show that intracerebroventricular (ICV) delivery of Insulin-like growth factor (IGF)-1 reduces MCAo-induced infarction in middle-aged female rats. Analysis of ischemic tissue at 4h showed that IGF1 treatment significantly reduced the expression of a group of miRNAs. To determine if inhibiting these miRNAs could replicate IGF-1-mediated neuroprotection, we examined the effects of antagomirs to miR-33a (A-mir33a) and miR-92b (A-mir92b) in in vivo and in vitro models of ischemia. Methods: Middle-aged (12 mo) acyclic female rats were subject to intraluminal MCAo for 90 min followed by reperfusion. Animals received a single tail vein injection of A-miR-33a, A-miR-92b, A-miR-33a+A-miR-92b or scrambled oligos (7ug/kg bwt) after 4h of reperfusion. We evaluated infarct volume (TTC staining) at 2d post stroke and behavioral recovery using neurological scores and adhesive removal test (ART). In parallel, human brain endothelial cell cultures were exposed to oxygen-glucose deprivation (OGD) in the presence of A-mir-33a, A-mir-92b or scrambled oligos. The integrity of the cell monolayer was assessed by tomato-lectin staining and cell death was assayed by LDH. Results: Single injection of A-mir92b during the acute stroke phase reduced infarct volume (p<0.04) and improved sensorimotor behavior (p<0.04) while A-mir33a treated animals were not different from scrambled control. However, the combination of A-mir-33a+A-mir-92b resulted in significantly smaller infarct volumes (p<0.04), less motor impairment (neurological score; p<0.02) and sensorimotor deficits (ART; p<0.05). In vitro studies showed that neither of the antagomirs reduced media LDH under OGD. As reported previously, OGD caused cell retraction and A-mir33a notably preserved the cell geometry (continuous lectin staining) to maintain the monolayer integrity and was similar to the normoxic control. Conclusions: IGF-1 regulated microRNAs appear to modulate specific aspects of stroke recovery. IV delivery of a combination of miRNA inhibitors may replicate the neuroprotective actions of IGF-1, thus providing a safer alternate to ICV delivery of IGF-1. Supported by R56 NS074895 and RF1 AG04218906

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Abstract WP107: Igf-1-mediated Neuroprotection for Ischemic Stroke is Preceded by Its Actions at the Endothelial Cell Barrier

Stroke ◽

10.1161/str.48.suppl_1.wp107 ◽

2017 ◽

Vol 48 (suppl_1) ◽

Author(s):

Shameena Bake ◽

Andre Okoreeh ◽

Farida Sohrabji

Keyword(s):

Endothelial Cells ◽

Ischemic Stroke ◽

Acute Phase ◽

Ex Vivo ◽

Infarct Volume ◽

Female Rats ◽

Histological Evaluation ◽

Blue Dye ◽

Cell Contact ◽

Middle Aged

Background: Our studies show that Insulin like growth factor (IGF)-1 reduces MCAo-induced infarction in middle-aged female rats. We tested the hypothesis that IGF-1s neuroprotective effect is due to its action on the microvascular /endothelial barrier in the early acute phase of stroke. Methods: Middle-aged (12 mo) acyclic female rats were subject to intraluminal middle cerebral artery occlusion (MCAo) for 90 min followed by reperfusion and terminated 4h or 24h later. Animals received ICV infusion of artificial CSF, IGF-1 or IGF-1+JB-1. In separate experiments, we evaluated infarct volume, barrier assessment by uptake of Evan’s blue dye or histological evaluation of lectin stained microvessels. In parallel, cortical endothelial cells from middle-aged females were harvested and cultured ex vivo. Barrier properties were tested in cultures in transfer wells exposed to oxygen glucose deprivation (OGD) in the presence or absence of IGF-1 with FITC-labeled BSA in the media. Results: At 4h post MCAo, IGF-1 treatment significantly reduced brain uptake of Evan’s blue dye, while infarct volume was no different from controls. At 24h post MCAo, IGF-1 reduced infarct volume by 40% and decreased motor impairment (neurological score; p<0.05). Lectin stained microvessels in the ischemic hemisphere displayed wider lumens (normal uncompressed lumen, p<0.003) and more intense label (p<0.01) in IGF-1 treated animals as compared to either vehicle or IGF-1+JB-1 groups. Ex vivo, IGF-1 reduced the loss of cell-cell contact due to OGD and reduced the dye transfer across the monolayer. Conclusions: Our findings indicate that IGF-1 acts in the early acute phase of ischemic stroke to reduce barrier permeability, which precedes its beneficial effect on infarct volume and behavior. Further, brain endothelial cells play a crucial role in IGF-1 mediated neuroprotection in middle-aged females.

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