Permanent Middle Cerebral Artery Research Articles

Abstract TP235: Delayed FGF21 Administration Improves Cerebrovascular Remodeling And White Matter Repair After Focal Stroke In Diabetic Mice

Introduction: Type 2 diabetes mellitus (T2DM) is a major comorbidity exacerbating ischemic brain injury and impairing post-stroke recovery. Our previous study suggested that recombinant human fibroblast growth factor (rFGF) 21 might be a potent therapeutic targeting multiple aspects of pathophysiology in T2DM stroke. As a continuation of our previous work investigating the therapeutic effects of rFGF21 in T2DM stroke, this study aims to evaluate the potential effects of rFGF21 on cerebrovascular remodeling during a delayed phase after T2DM stroke. Methods: Permanent distal middle cerebral artery occlusion was performed in heterozygous non-diabetic db/+ and homozygous diabetic db/db mice. Daily rFGF21 administration was initiated one week after stroke induction and maintained for up to two weeks thereafter. Multiple markers associated with post-stroke recovery, including angiogenesis, oligodendrogenesis, white matter integrity and neurogenesis, were assessed up to three weeks after stroke. Results: Our results showed an impairment in post-stroke vascular remodeling under T2DM condition, reflected by the decreased expression of trophic factors in brain microvessels and impairments of angiogenesis. The defected cerebrovascular remodeling was accompanied by the decreased oligodendrogenesis and neurogenesis. However, delayed rFGF21 administration normalized post-stroke hyperglycemia and improved neurological outcomes, which may partially via the promotion of pro-angiogenic trophic factor expression in brain microvessels and cerebrovascular remodeling. The better cerebrovascular remodeling may also contribute to oligodendrogenesis, white matter integrity and neurogenesis after T2DM stroke. Conclusion: Delayed rFGF21 administration may improve neurological outcomes in T2DM stroke mice, at least in part by normalizing the metabolic abnormalities and promoting cerebrovascular remodeling and white matter repair.

Abstract WP247: Two Novel Interferon Signaling Proteins (Ifi27l2a And Ifitm3) Regulate Microglial Inflammation With Brain Aging And Following Stroke.

Interferon signaling drives neuroinflammation in AD and stroke. Microglia (MG) play a critical role in initiating and spreading inflammation and are involved in the chronic recovery phase following stroke. We compared the MG transcriptomic response to stroke in young and aged mice by single-cell RNA sequencing (scRNAseq) to identify novel key players in inflammation. Methods: Permanent distal middle cerebral artery occlusion (pdMCAO) or sham surgery was performed in young and aged mice (3 & 20 months). scRNAseq was performed to define the transcriptional signature in brains at 14-days after stroke versus sham surgery. Primary MG culture was used to compare the mRNA levels of Ifi27l2a and Ifitm3 after cytokine treatment. We used qRT-PCR to validate scRNAseq findings and compare specific MG genes. Results: Using comprehensive scRNAseq analysis, our unbiased approach identified Ifi27l2a and Ifitm3 as the most highly up-regulated genes in aged MG following stroke. Further analysis showed a positive correlation between Ifi27l2a and Ifitm3 in MG ( R =0.4, p<0.001) as well as in all cells. Our qRT-PCR results validated scRNAseq findings and further showed that Ifi27l2a was significantly up-regulated in the thalamus of aged brains (p=0.04; n=4 young/aged). Expression was increased in cortex, but below significance level (p=0.23). We also found upregulation of Ifitm3 in brain at PSD 3 ( p =0.01), and trending at PSD 14 ( p =0.08) (n=5-6 stroke vs. sham). In contrast, Ifitm1 and Ifitm2 were not changed. Interestingly, Ifitm3 was recently proposed as an activator to γ-secretase. In vitro exposure of MG to inflammatory cytokines (TNFα and IFNγ, 6 hrs) caused significant increase in Ifi27l2a ( p =0.03, n=5-6) and Ifitm3 mRNA (p=0.02, n=5-6) compared to untreated MG. The in vitro co-expression of these genes was also well correlated ( R =0.5). Conclusions: We identified Ifi27l2a and Ifitm3 as highly up-regulated and correlated genes in MG in aging and following stroke. Our findings suggest that co-expression of Ifi27l2a and Ifitm3 may contribute to neuroinflammation. Further studies will be required to identify the specific functions of these genes and to determine the therapeutic potential of targeting these proteins to regulate neuroinflammation in aging and stroke.

Abstract TP258: Assessing Post-stroke Motor Impairments Using Staircase Reaching Test

Background: Behavior tests that assess side-specific motor impairments after unilateral lesion such as focal ischemic stroke have translational applications, as 8 out of 10 stroke survivors suffer from hemiparesis. Accurate measurements of neurological functions are therefore important for assessing the effectiveness of various treatment strategies. Here, we demonstrate the feasibility of the staircase test to objectively evaluate lateralized deficits in coordinated paw reaching in adult mice after experimental stroke. Methods: A cohort of adult male C57BL/6J mice (12-14 weeks of age) were subjected to a 14-day training period, where they were kept on a 21-hour food deprivation regime and at 85-90% of their original body weight. They were then subjected to permanent distal middle cerebral artery occlusion (dMCAO, n=10). The number of steps reached and pellets grasped/eaten were evaluated at pre-stroke baseline and post-stroke days (PD) 7-8 and 14-15; behavior data from PD7-8 and PD14-15 were averaged between the two days. Brains were collected at PD16 and sections immunostained with antibodies targeting neurons (MAP2) and/or activated microglia/macrophage (CD68). Results: Mice attained a stable baseline for reaching steps and consuming pellets after a 14-day training period. At PD7-8, stroked mice showed a significant decrease in their ability to grasp/consume pellets on the affected side (right limb) compared to pre-stroke baseline (p=0.001). At PD14-15, stroked mice exhibited a significant decline in their ability to reach longer distances on the affected side (right limb), attaining only 65% of their baseline performance (p=0.006). No significant deficit was shown on the non-affected side (left limb). Conclusions: Our results show that the staircase test can detect side-specific motor deficits up to PD15 in a dMCAO model. Ongoing studies with a larger cohort are evaluating longer-term deficits up to one-month post-stroke and assessing the effects of optogenetic cortico-thalamic circuit stimulations on grasping behavior using the staircase test.

Abstract 19: Long-lasting Post-stroke Memory Dysfunction In Aged Mice Is Like Due To Exacerbated Hippocampal Inflammation And Synapses Removing

Background and Purpose: Stroke can cause memory dysfunction. Long-term memory dysfunction of mice with tibia fracture (BF)+stroke is associated with accumulation of CD68 + cells in the hippocampus, which can be alleviated by activation of alpha-7 nicotinic acetylcholine receptor (α-7 nAchR). Microglia maintain normal memory via removing excessive synapses. We hypothesize that aged mice would develop long-lasting memory dysfunction after stroke, which is associated with increased CD68 + cells and synapses removing in the hippocampi. Methods: Permanent distal middle cerebral artery occlusion (pMCAO) was performed in young (2-month-old) and aging (15-18-month-old) mice, or 6 hours before pMCAO in young mice. The memory functions were analyzed weekly for 8 weeks by Y-maze and at 8 weeks post pMCAO by NOR tests. Atrophic volumes, CD68 + cells and microglial phagocytosis of synapses were quantified at 8 weeks after pMCAO. Results: Aged mice had larger atrophic volumes, more CD68 + cells in the hippocampi ipsilateral to stroke side than young mice, and also had more CD68 + cells in the ipsilateral hippocampus than the contralateral. In Y-maze test, the aged stroke mice made fewer spontaneous alternations from 3 to 8 weeks after pMCAO than the young stroke mice and sham aged mice. In NOR test, aged stroke mice spent less time on the novel object than young stroke mice and sham aged mice. α-7 nAchR agonist treatment reduced the number of CD68 + cells in the hippocampi in the BF+stroke mice. Almost all CD68 + cells were synaptophysin positive. Therefore, increased CD68 + cells in the ipsilateral hippocampus will increase synaptic removal. Reduction of neuroinflammation could reduce synaptic loss and improve post-stroke memory function. Conclusions: Increased CD68 + cells in the hippocampus is associated with long-lasting post-stroke memory dysfunction in aged mice, and reduced neuroinflammation could improve post-stroke memory function.

Abstract WMP116: Age-related Dysfunction In Leptomeningeal Collateral Remodeling After Ischemic Stroke

Introduction: The leptomeningeal collaterals (LMCs) are specialized brain surface arterioles that interconnect the major cerebral artery territories. Good outcome following ischemic stroke is highly correlated with high collateral number and function. Unfortunately, with aging and disease, the LMCs demonstrate impaired dilatory capacity, reduced vessel diameter, and even complete vessel loss (rarefaction). The present study examined the chronic remodeling capacity of LMCs following permanent stroke in young and aged mice. Methods: Young and aged C57BL6 mice (5 and 20 months) were subjected to permanent distal middle cerebral artery occlusion (pdMCAO). At 2 weeks or 3 months, LMCs were evaluated for inner diameter and VWF expression (% LMC coverage). We compared the LMCs feeding the occluded MCA territory (LMC ipsi) to the contralateral equivalent vessels (LMC contra). Diameter change is reported as fold change relative to the distal-most arteriole (DMA) associated with each LMC. Results: Young male mice demonstrated increased diameter of LMC ipsi relative to DMA at both 2 weeks (1.36 ± 0.075; n=24 vessels/4 mice, P<0.01) and 3 months after stroke (1.27 ± 0.073, n=19 vessels/2 mice, P<0.01). LMC contra were 0.901 ± 0.064 and 1.01 ± 0.053 for the same time points (n=19 and 10 vessels, P=NS). VWF expression was significantly increased in LMC ipsi at 2 weeks (ipsi 10.9 ± 2.87 vs. contra 2.91 ± 0.82, P<0.05) and neared significance at 3 months (ipsi 6.45 ± 1.54 vs. contra 2.06 ± 0.264, P=0.051). Aged mice demonstrated increased diameter of LMC ipsi relative to DMA at 2 weeks (1.24 ± 0.052, n=25 vessels from 3 mice, P<0.01), but not at 3 months (1.08 ± 0.044, n=14 from 2 mice, P=NS). Notably, the 3 month LMC ipsi vessels demonstrated irregular diameter and characteristics of rarefaction. LMC contra were 0.897 ± 0.032 and 0.967 ± 0.092 for the same time points (n=17 and 8 vessels, P=NS). VWF expression was significantly elevated in LMC ipsi vs. LMC contra at 2 weeks (6.74 ± 1.33 vs. 1.87 ± 0.361, P<0.01), but not at 3 months (2.27 ± 0.387 vs. 1.81 ± 0.398, P=NS). Conclusions: Our data show lasting LMC remodeling in young stroke brain, consisting of increased LMC diameter and expression of VWF. With aging, LMC remodeling is transient and evidence of LMC rarefaction is apparent by 3 months.

Trillium tschonoskii rhizomes' saponins induces oligodendrogenesis and axonal reorganization for ischemic stroke recovery in rats

Ethnopharmacological relevanceTrillium tschonoskii Maxim. is one of traditional Chinese medical herbs that has been utilized to treat brain damages and cephalalgia. The neuroprotective effect of total saponins from Trillium tschonoskii rhizome (TSTT) has been demonstrated efficacy in rats following ischemia. However, the axonal remodeling effect of TSTT and the detailed mechanisms after ischemic stroke have not been investigated. Aim of the studyWe aimed to estimate therapeutic role of TSTT in axonal remodeling using magnetic resonance imaging (MRI) technique, and explored possible mechanisms underlying this process followed by histological assays in ischemic rats. MethodsMale Sprague-Dawley (SD) rats underwent permanently focal cerebral ischemia induced by occluding right permanent middle cerebral artery. TSTT was intragastrically administrated 6 h after surgery and once daily for consecutive 15 days. Neurological function was assessed by the motor deficit score and beam walking test. T2 relaxation mapping and diffusion tensor imaging (DTI) were applied for detecting cerebral tissues damages and microstructural integrity of axons. Luxol fast blue (LFB) and transmission electron microscope (TEM) were performed to evaluate histopathology in myelinated axons. Double immunofluorescent staining was conducted to assess oligodendrogenesis. Furthermore, the protein expressions regarding to axonal remodeling related signaling pathways were detected by Western blot assays. ResultsTSTT treatment (65, 33 mg/kg) markedly improved motor function after ischemic stroke. T2 mapping MRI demonstrated that TSTT decreased lesion volumes, and DTI further confirmed that TSTT preserved axonal microstructure of the sensorimotor cortex and internal capsule. Meanwhile, diffusion tensor tractography (DTT) showed that TSTT elevated correspondent density and length of fiber in the internal capsule. These MRI measurements were confirmed by histological examinations. Notably, TSTT significantly increased Ki67/NG2, Ki67/CNPase double-labeled cells along the boundary zone of ischemic cortex and striatum. Meanwhile, TSTT treatment up-regulated the phosphorylation level of Ser 9 in GSK-3β, and down-regulated phosphorylated β-catenin and CRMP-2 expression. ConclusionTaken together, our findings indicated that TSTT (65, 33 mg/kg) enhanced post-stroke functional recovery, amplified endogenous oligodendrogenesis and promoted axonal regeneration. The beneficial role of TSTT might be correlated with GSK-3/β-catenin/CRMP-2 modulating axonal reorganization after ischemic stroke.

Abstract P148: Role of Endothelin B Receptors in Neuronal Progenitor Cell Differentiation and Mitochondrial Function in Ischemic Stroke

Neuronal progenitor cells (NPCs) and mitochondria are well known to play roles in regeneration and functions of the brain, respectively. However, stimulation of endothelin-B receptors (ETBR) using an agonist, sovateltide (IRL-1620) on their fate in the brain after stroke remains elusive. We evaluated the effect of sovateltide mediated ETBR stimulation on differentiation of NPCs and fate of mitochondria after stroke. Ischemic stroke was induced by performing permanent right middle cerebral artery occlusion (MCAO) in rats. Sovateltide (5 μg/kg) or saline (equal volume) was injected intravenously; neurological and motor function evaluation was done at 24 hrs and day 7 post MCAO. Brain tissues were analyzed for NPCs differentiation and mitochondrial fate using techniques such as western blots, immunofluorescence, transmission electron microscopy and in situ PCR. In vitro hypoxia experiment was carried out to confirm sovateltide mediated neuronal differentiation. Neurological and motor functions were significantly improved in sovateltide treated rats at 24 hrs and day 7 post MCAO. Differentiation of NPCs was evident with upregulation of neuronal differentiation markers Neuro D1 (p=0.00002) as well as HuC/HuD (p=0.0037) along with neuronal marker Doublecortin (DCX) (p=0.00011) at 24 hrs post MCAO. However, significant upregulation only in HuC/HuD (p=0.043) was observed at day 7. Better preserved mitochondrial fate was observed in sovateltide rat brains with downregulation of mitochondrial fission marker, DRP1 (p<0.001), increase in fusion marker, MFN2 (p<0.0001) and increase in cross-sectional area x number (p<0.05) as well as mitochondrial/tissue area (p<0.05) at 24 hrs and day 7 post MCAO. In situ PCR analysis showed increased mitochondrial DNA (p=0.0418), indicating better mitochondrial biogenesis at day 7 post MCAO. In vitro exposure of sovateltide and hypoxia to cultured NPCs showed higher NeuroD1 and NeuN (a mature neuronal marker) expression confirming NPCs differentiation. Sovateltide mediated ETBR stimulation promotes differentiation of NPCs and mitochondrial fusion as well as biogenesis and helps in neuronal regeneration and function restoration in acute ischemic brains.

Abstract P727: Single-Cell RNA Sequencing Identifies Ifi27l2a as a Novel Gene Involved in Regulating Microglial Inflammation in the Context of Aging and Stroke

Microglial cells (MG) serve as resident immune cells in the brain and play a critical role in the acute response and chronic recovery to stroke. However, how the transcriptional signature of these cells is altered in aging and stroke remains poorly defined. Here, we compared the MG transcriptomic response to stroke in young and aged mice by single-cell RNA sequencing (scRNAseq). Our unbiased approach identified Ifi27l2a (a member of the interferon signaling family) as one of the most highly upregulated genes in MG with aging or ischemic stroke. Further analyses suggested that this gene may be a novel regulator of MG inflammation in aging and stroke. Methods: Permanent distal middle cerebral artery occlusion (pdMCAO) or sham surgery was performed in young and aged mice of both sexes (3 & 20 months). scRNAseq was performed to explore the transcriptional signature in brains at 14-days post-stroke and sham controls. We used qRT-PCR to validate our scRNAseq findings and compare regional transcriptional changes. Human microglial cells (HMC3; subjected to OGD) and human autopsy brain sections (stroke versus non-stroke) were used to examine IFI27l2 protein expression. Results: Using comprehensive scRNAseq analysis, we identified Ifi27l2a as the most highly up-regulated gene in aged MG following stroke. Further analysis showed a positive correlation between Ifi27l2a and multiple “MG activation” genes. In contrast, a negative correlation was shown with genes involved in promoting the phagocytic MG phenotype (e.g. Spp1, Cst7, Lpl, and Itgax). Our qRT-PCR results validated the central scRNAseq findings and further showed that Ifi27l2a was significantly up-regulated in thalamus of aged brains (p = 0.0412) and trending in cortex (p = 0.23) (n=4 young/aged). Following stroke, Ifi27l2a and other markers of MG activation were significantly increased in both brain regions (n= 4-6). In human tissues, we observed an increase in IFI27l2 protein in HMC3 cells with OGD and in brain of stroke patients. Conclusions: We identified Ifi272la as a highly up-regulated gene in MG in aging and following stroke. Our findings suggest that Ifi27l2a may be a novel regulator of neuroinflammation in MG, and thus provide the basis for a new therapeutic target to reduce inflammation in aging and stroke.

Abstract P795: Activating Transcription Factor 3 Improves Stroke Recovery Through Reduction of Neuronal Damage and Neuroinflammation

Background and Purpose: Activating transcription factor 3 (ATF3), a member of the ATF/CREB family, is upregulated in the early phase of various stresses including ischemic stroke. The role of ATF3 in ischemic stroke injury has not been fully elucidated. Hypothesis: ATF3 improves stroke outcomes through reduction of neuronal damage and neuroinflammation. Methods: Permanent distal middle cerebral artery occlusion (pMCAO) was performed in 8-week-old male and female C57BL/6J (WT) and ATF3 knockout (ATF3 -/- ) mice. The sensorimotor functions were analyzed 3 days after pMCAO through adhesive removal and corner tests. Infarct volumes and injured neurons were quantified on Nissl stained and Fluoro-Jade C (FJC) stained sections. The cell-specific expression of ATF3 was analyzed by co-staining ATF3 antibody with antibodies specific to NeuN (Neuron), CD68 (microglia/macrophage) and GFAP (astrocyte). The expression of ATF3 in injured neurons was analyzed by ATF3 and FJC double labeling. Results: ATF3 expression was detected exclusively in neurons in the infarct area 1 day after pMCAO. There were a few ATF3 + CD68 + microglia/macrophages at the peri-infarct regions 2 and 3 days after pMCAO. Almost all FJC + neurons were ATF3 positive. No ATF3 expression was detected in astrocytes. Compared to WT mice, ATF3 -/- mice took longer time to remove the adhesive from their right paws (p<0.001) in adhesive removal test and more left turns in corner test (p<0.001) 3 days after pMCAO. ATF3 -/- mice also had a larger infarct volume (p = 0.014), more FJC + neurons (p=0.002) and CD68 + microglia/macrophages (p=0.003) in the peri-infarct regions than WT mice. Conclusions: Deletion of ATF3 exacerbates neuronal injury, neuroinflammation, and sensorimotor dysfunction in stroke mice, suggesting that upregulation of ATF3 at early stage of stroke improves ischemic stroke recovery through reduction of neuronal damage and neuroinflammation.

Abstract P769: Remodeling of the Leptomeningeal Collaterals Following Stroke Involves Selective Upregulation of Von Willebrand Factor (VWF)

Introduction: The leptomeningeal collaterals (LMCs) are arterial anastomoses that interconnect the distal branches of the major cerebral arteries. They distribute blood flow from adequately to poorly perfused vascular territories when needed. In normal conditions, the LMCs experience little net flow. However, during ischemic stroke, the LMCs are recruited to provide rescue flow to the ischemic territory. During prolonged or permanent stroke, the LMCs can rapidly remodel to provide increased perfusion capacity. Von Willebrand factor (VWF) is a secreted protein expressed in cerebral endothelium and has recently been implicated in angiogenesis. We sought to determine if VWF expression was increased in remodeling LMCs in the post-stroke brain. Methods: C57BL6 mice were subjected to permanent distal middle cerebral artery occlusion (pdMCAO). At 3 days after pdMCAO, mice were perfused with fluorescent tomato lectin to label vascular endothelium. The cortical surface was “planed off” by vibratome to provide largely contiguous cortical surface vessel preparations containing distalmost arterioles (DMAs) and the connected LMCs. VWF immunofluorescence intensity was measured in LMCs and the connecting DMAs of hemispheres ipsilateral and contralateral to the stroke. Inner diameter of the LMCs and DMAs were measured with Image J. Results: At 3 days post-stroke, LMCs of the stroke hemisphere demonstrated significant outward remodeling. Mean inner diameter of the ipsilateral versus contralateral LMCs were 44.5 ± 5.9 um versus 28.2 ± 1.2 um, respectively (p<0.02, 7-8 vessels from 3 mice). In contrast, the DMAs were unaltered in diameter, 35.2 ± 4.0 and 35.7 ± 3.1 um, respectively. VWF expression was significantly increased in the ipsilateral LMCs versus contralateral LMCs (2.8 fold, p<0.05) and versus ipsilateral DMAs (3.7 fold, p<0.02). The DMAs showed significantly lower VWF expression, which was unchanged between ipsilateral and contralateral hemispheres. Conclusions: We demonstrate that VWF is highly expressed in the remodeling LMCs (but not connecting DMAs) of the post-stroke brain. These findings suggest a novel and selective role for endothelial VWF in the LMC response to prolonged recruitment during ischemic stroke.

Abstract TP98: Aging-Related Impairment in Post-Ischemic O-GlcNAcylation Activation Critically Contributes to Stroke Outcome

Introduction: Activity of the unfolded protein response (UPR) declines with age. The most conserved UPR branch is mediated by inositol- requiring enzyme-1 (IRE1). Once activated, IRE1 is converted to an endonuclease that splices X-box binding protein-1 (Xbp1) mRNA, leading to production of a new 54-kDa protein, XBP1s. XBP1s was recently shown to be capable of up-regulating expression of all major hexosamine biosynthetic pathway (HBP) enzymes. HBP generates UDP-GlcNAc, the sugar donor for O-GlcNAc modification (O-GlcNAcylation). These pathways together form the IRE1/XBP1s/HBP/O-GlcNAc axis. Our previous data demonstrated a pivotal role of this axis in stroke outcome, especially in the aged brain. Here, we expanded on our previous findings and further elucidated that aging-related impairment in post-ischemic O-GlcNAcylation activation critically contributes to stroke outcome. Methods: Permanent and transient middle cerebral artery occlusion (MCAO) surgeries were performed on young and aged animals. Conditional XBP1 transgenic (XBP1s-TG) and knockout (Xbp1-cKO) mice were used to determine the role of the XBP1 branch in stroke outcome. Thiamet-G was used to increase O-GlcNAcylation. Short- and long-term stroke studies were conducted. Functional outcome was evaluated by various behavioral tests. Results and Conclusions: In line with our previous data, XBP1s-TG mice exhibited improved stroke outcome, and boosting O-GlcNAcylation with thiamet-G rescued worse stroke outcome observed in Xbp1-cKO mice. Moreover, intervention with thiamet-G to enhance O-GlcNAcylation in the brain improved not only long-term outcome after transient MCAO in young mice, but also long-term functional recovery after permanent MCAO in aged rats, likely due to its rescue effect on impaired O-GlcNAcylation activation in the aged brain. Finally, we discovered that UDP-GlcNAc production under the physiologic state and after ischemia was compromised in the aged brain, which constitutes a potential mechanism responsible for impaired O-GlcNAcylation activation after stroke. Together, the findings strengthen our notion that targeting pro-survival pathways that are activated in the young, but not the aged, ischemic brain is a new promising therapeutic strategy in stroke.

Abstract TMP31: A Novel Shift of Notch Ligand, Dll4, Expression From Endothelial to Glial Cells in the Post-Stroke Brain May Contribute to Reparative Angiogenesis and Improved Functional Outcome

Delta-like 4 (Dll4) is a Notch receptor ligand classically thought to be expressed exclusively in endothelial cells. However, little is known about the cellular expression or functional role of Dll4 following ischemic stroke. Here, we present novel findings that Dll4 expression is shifted from endothelial cells to glial cells in the sub-acute stroke phase. Method: The permanent distal middle cerebral artery occlusion (dMCAO) model was performed to induce stroke. To investigate expression patterns of Dll4 protein and mRNA, immunostaining as well as single-cell RNA sequencing and single-cell fluorescent in situ hybridization were performed. To verify the role of endothelial Dll4 , tamoxifen-inducible, endothelial-specific Dll4 KO mice (Dll4iecKO) and Dll4 fl/fl (“floxed-only”) female mice (3 mos) were injected with tamoxifen (n=4) at 24 and 48 hrs post-stroke. To determine the effect of Notch inhibition at different time points, DAPT (100 mg/kg) or vehicle was administered at 24 or 72 hours post-stroke (n=4-5). Behavioral tests (foot fault) were conducted at Post Stroke Day (PSD) 1, 3, 7, and 14 and functional angiogenesis was assessed at PSD 14. Results: Elevated expression of Dll4 protein was observed in the microvasculature of the peri-infarct cortex (piCtx) as early as PSD 1. By PSD 3, however, we discovered Dll4 expression in microglia within the piCtx, with robust expression by PSD 14. Post-stroke deletion of endothelial Dll4 resulted in significant improvement in neurological function and increased capillary density in piCtx[l1] . DAPT given at 24 hours post-stroke improved outcome and capillary density; however, DAPT given at 72 hours (when microglia Dll4 expression is evident) appeared to worsen outcome. Conclusions: After stroke, expression of Dll4 shifts from exclusively endothelial (PSD 1) to mixed endothelial/microglial (PSD 3) to primarily microglial (PSD 14). Notch inhibition beginning at 24 hours post-stroke or endothelial-only deletion of Dll4 leads to increased angiogenesis and improved functional recovery. In contrast, Notch inhibition initiated at 72 hours post-stroke leads to worse outcome[l2] . These findings suggest a potential novel role of microglia in supporting reparative angiogenesis through Dll4/Notch signaling.

Abstract WP142: Targeting VCAM1 to Reduce Acute and Chronic Neuroinflammation After Stroke

Background: Ischemia-induced neuroinflammmation is associated with worse outcomes after stroke. The innate immune response can mediate infarct expansion and impair short-term recovery, while adaptive responses lead to chronic neuroinflammation, neurodegeneration remote to the ischemic lesion, and consequent vascular dementia. Vascular cell adhesion molecule 1 (VCAM1) is an endothelial protein that facilitates vascular-immune cell interactions by binding very late antigen-4 (VLA-4). Soluble VCAM1 plasma levels are elevated in stroke patients and in aging people and mice. In aging mice, blocking VCAM1 ameliorates age-induced neuroinflammation and cognitive impairment. Objective: To determine if acute anti-VCAM1 treatment in mice would reduce microgliosis and astrogliosis, while delayed treatment would reduce B and T lymphocyte infiltration. Methodology: Adult (3-month-old) C57BL/6 mice (N=10/group) underwent permanent distal middle cerebral artery occlusion and were dosed with anti-VCAM1 antibody 4h post-stroke, then sacrificed at 72h. Microgliosis and astrogliosis were quantified as percent area immunostained in the infarct border by Iba1 and GFAP, respectively. Results: Acute treatment reduced microgliosis by 30% (p=0.0476) and astrogliosis 39% (p<0.03). Additionally, adult mice (N=10/group) were treated with anti-VCAM1 immediately (4h) or later (4d) post-stroke and euthanized 3 weeks later. B cell infiltration was quantified as percent stroke core immunostained by B220, while T lymphocyte infiltration was quantified as CD3+ cells in the core. Delayed anti-VCAM1 significantly reduced B and T cell infiltration by approximately 25% (p=0.0015) and 50% (p=0.0192), respectively. In contrast, early anti-VCAM1 had no effect on B/T cell infiltration. Conclusions: Together, these findings establish VCAM1 as a possible therapeutic target to prevent stroke-induced neuroinflammation in both acute and chronic settings.

Abstract TP115: Nrf2 Plays an Essential Role in Long-Term Neuroprotection of Ginseng in Permanent Cerebral Ischemia

Introduction: Cerebral ischemia is a devastating disease with a high incidence of death and disability; however, effective therapeutics remain limited. The transcriptional factor Nrf2 has been shown to play a pivotal role in the endogenous defense against brain oxidative stress and inflammation; therefore, it represents a promising target for stroke intervention. The natural potential Nrf2-inducer ginseng, extracted from the root of Panax ginseng C.A.Meyer, has been widely used in East Asia for thousands of years, exhibiting potent anti-inflammatory and antioxidative properties. However, the long-term effects of Nrf2 and the standardized Korean red ginseng extract (ginseng) on permanent cerebral ischemic damage have not yet been reported. Methods: Wildtype (WT) and Nrf2 –/– adult mice were treated with either vehicle (water) or ginseng for 7 days and subjected to the permanent distal middle cerebral artery occlusion (pdMCAO). The infarct volume, the reactive astrogliosis and microgliosis, and water regulatory protein aquaporin 4 (AQP4) were examined 28 days after the initiation of stroke. Data acquisition and analyses were performed by an observer blinded to the treatment. Results: When compared with the WT matched controls, the Nrf2 disruption significantly enlarged the infarct volume by 40.4±10.1% (n=7 per group; p<0.05), increased levels of reactive gliosis and AQP4 protein after pdMCAO. Interestingly, ginseng significantly reduced the infarct volume and attenuated the reactive gliosis and AQP4 in the ischemic WT mice (47.3±6.9%, n=12; p<0.01), but not in the Nrf2 -/- mice (25.5±5.6%, n=11; p=0.241). Conclusion: Nrf2 plays an important role in the long-term recovery of permanent cerebral ischemic damage and neuroprotection of ginseng.

Abstract TP107: Midline Shift Predicts Functional Outcomes in Porcine Ischemic Stroke Model

In this study, we sought to identify acute MRI parameters which are predictive of long-term functional outcomes as well as assess the effects of a neural stem cell extracellular vesicle (NSC EV) therapeutic in a large animal model of ischemic stroke. In this randomized study, stroke was induced through a permanent right-sided middle cerebral artery occlusion (MCAO) on 16 male landrace pigs, which were divided into either treatment or control group. NSC EVs or PBS was administered at 2, 14, and 24 hours, and MRI was conducted at day 1 and 84 post-stroke. Data on 65 gait and 25 behavior parameters were collected pre-stroke and at multiple timepoints over 84 days following MCAO. Of all 15 measured MRI parameters, axial and coronal midline shift (MLS), at day 1 post stroke, had the highest total number of significant correlations (52 parameters at p<0.05) to acute and chronic functional measurements in control animals such as step time in the left front limb (p=0.0322) and cycle time in the right hind limb (p=0.0011) respectively. This suggests MLS is the best overall predictor of specific functional deficits at both acute and chronic timepoints, which to our knowledge has never been shown in an animal model. Additionally, the parameters found to be correlated to MLS in control animals were not correlated in NSC EV-treated animals, suggesting NSC EV treatment disrupts this natural correlation between degree of MLS and functional outcomes. NSC EVs and control pigs were binned into either high or low MLS groups and their survival and recovery was assessed by the modified Rankin Scale (mRS). While there was a significant difference in mRS scores of control animals with high and low MLS at day 6 post-MCAO (p=0.0008), there was not in NSC EV-treated animals (p=0.6754). Further, there was a significant difference in survival of control animals with high and low axial MLS (p=0.0401), but not in the NSC EV group (p=0.4142). Additionally, mRNA expression of GFAP was significantly correlated with increasing MLS in non-treated but not NSC EV-treated animals. These findings show although NSC EV treatment does not significantly alter the degree of MLS 1-day post-MCAO, it does alter gene expression, increase survival, and improve functional recovery following large MLS alterations.

Abstract TP321: Role of PGE 2 EP1 Receptor Antagonist on Stroke Outcomes in Alzheimer Disease Mouse Models

Background: AD is the most common memory disorder and its prevalence is expected to triple by the year 2050. Neuroinflammation is recognized as an important player in the pathogenesis of AD. One of the most recognized pathways in mediating the neuroinflammation is of prostaglandin E 2 (PGE 2 ) EP1 receptor pathway. Role of PGE 2 EP1 receptor in AD mouse models following stroke has not been studied. In this study, we examined the efficacy of a selective EP1 antagonist, ONO-8713, on lesion volumes and behavioral indexes in AD mouse models after stroke. Methods: Two cohorts of transgenic, APP/PS1 and 3xTg, and wildtype (WT) mice were subjected to permanent distal middle cerebral artery occlusion (pdMCAO) and sham surgeries. EP1 antagonist ONO-8713 or vehicle was then administered to analyze its effect on anatomical and functional outcomes. Results: The functional outcomes were significantly deteriorated in the APP/PS1 and the 3xTg mice after stroke. Interestingly, the ONO-8713-treated groups of WT mice performed significantly (p<0.001) better in open field test than the respective groups of 3xTg mice. For the passive avoidance test, APP/PS1+ONO-8713 mice exhibited significant (p<0.05) difference in retention as compared with the vehicle group; whereas, there was a significant difference in acquisition between WT+ONO-8713 and 3xTg+ONO-8713 (p<0.05). There was a significantly lower tissue injury in APP/PS1+Veh mice when compared to the APP/PS1+ONO-8713 mice (p<0.02). Percent tissue injury was significantly higher in APP/PS1+ONO-8713 mice when compared to WT+ONO-8713 mice (p<0.05). Similarly, in the 3xTg cohort, percent tissue injury and percent tissue loss were significantly higher in 3xTg+ONO-8713 mice than in WT+ONO-8713 mice (p<0.02). Conclusion: The EP1 receptor antagonist ONO-8713 shows some beneficial effects on functional and anatomical outcomes after stroke in APP/PS1 and 3xTg mice models of AD; though, the effects were not significant in all outcomes investigated. Further studies are needed to understand the role and optimal timing of EP1 receptor blockade in the context of AD etiopathology. Funding support: Grants from the São Paulo Research Foundation, FAPESP (FRM), NIH, and the Ed and Ethel Moore Alzheimer's Disease Research Program (SD).

Abstract 62: Disruption of DLL4/Notch Signaling in the Post Stroke Period Results in Enhanced Angiogenesis and Improved Outcome Following Stroke

Delta-like 4 (DLL4) is an endothelial transmembrane ligand for the Notch family receptors. Dll4 contributes to vascular development and angiogenesis, specifically as a negative regulator of capillary sprouting. We hypothesized endothelial-specific deletion of Dll4 or inhibition of Notch receptor activity in the post-stroke brain would result in enhanced reparative angiogenesis and improved functional outcome following permanent stroke in young and aged mice. Methods: We used the permanent distal middle cerebral artery occlusion (dMCAO) model to induce primary injury in the cortex with delayed secondary injury in the thalamus. DLL4 protein expression (IHC) and Notch receptor activity ( in vivo biosensor, CBF::H2B-Venus ) were determined through 2 weeks post-stroke. Tamoxifen-inducible, endothelial-specific Dll4 KO mice ( Dll4 iecKO ) and Dll4 fl/fl (“floxed-only” control) female mice (3 mos) were treated with tamoxifen (n=4 each) at 24 and 48 hrs post-stroke. In a separate cohort, young (3 mos, n=5 each) and aged (20 mos, n=3-4) male mice were treated with DAPT (Y-secretase inhibitor; 100 mg/kg) at 24 hours post-stroke to inhibit Notch activity. Functional angiogenesis was assessed histologically following in vivo perfusion of fluorescent lectin. Behavioral tests (foot fault) were conducted at post-stroke days 1, 3, 7, and 14. Results: Elevated Dll4 expression and Notch activation were observed in peri-infarct cortex (piCtx) and ipsilateral thalamus (iTh) by 24 hours after dMCAO. Post-stroke endothelial Dll4 deletion led to increased perfusion-competent microvessels (e.g. vessel area, length, etc.) at 2 weeks post-stroke in piCtx and iTh. Post-stroke DAPT treatment trended toward increased total microvascular length in piCtx (P=0.051 young/P=0.126 aged), but not iTh. Importantly, both treatment methods demonstrated significant improvement in neurological function at 7 and 14 days post-stroke. Conclusions: Deletion of endothelial Dll4 at 24 hours post-stroke leads to increased angiogenesis and improved functional recovery. Notch inhibition demonstrated improved outcome (young and aged), but increased angiogenesis was only suggestive in the cortex. DAPT treatment appears to have translational value as a delayed treatment option.

Dickkopf-3 Causes Neuroprotection by Inducing Vascular Endothelial Growth Factor.

Dickkopf-3 (Dkk3) is an atypical member of the Dkk family of Wnt inhibitors, which has been implicated in the pathophysiology of neurodegenerative disorders. However, the role of Dkk3 in mechanisms of cell degeneration and protection is unknown. We used Dkk3 knockout mice to examine how endogenous Dkk3 influences ischemic brain damage. In addition, we used primary cultures of astrocytes or mixed cultures of astrocytes and neurons to investigate the action of Dkk3 on cell damage and dissect the underlying molecular mechanisms. In a model of focal brain ischemia induced by permanent middle cerebral artery (MCA) occlusion (MCAO) Dkk3−/− mice showed a significantly greater infarct size with respect to their wild-type counterparts at all time points investigated (1, 3 and 7 days after MCAO). Immunohistochemical analysis showed that Dkk3 expression was enhanced at the borders of the ischemic focus, and was predominantly detected in astrocytes. This raised the possibility that Dkk3 produced by astrocytes acted as a protective molecule. We tested this hypothesis using either primary cultures of cortical astrocytes or mixed cortical cultures containing both neurons and astrocytes. Genetic deletion of Dkk3 was permissive to astrocyte damage induced by either oxidative stress or glucose deprivation. In addition, application of human recombinant Dkk3 (hrDkk3) was highly protective against oxidative stress in cultured astrocytes. We tested the hypothesis that the protective activity of Dkk3 was mediated byvascular endothelial growth factor (VEGF). Interestingly, glucose deprivation up-regulated both Dkk3 and VEGF in cultured astrocytes prepared from wild-type mice. VEGF induction was not observed in astrocytes lacking Dkk3 (i.e., in cultures prepared from Dkk3−/− mice). In mixed cultures of cortical cells, excitotoxic neuronal death induced by a brief pulse with N-methyl-D-aspartate (NMDA) was significantly enhanced when Dkk3 was lacking in astrocytes, whereas post-NMDA addition of hrDkk3 was neuroprotective. Neuroprotection by hrDkk3 was significantly reduced by pharmacological blockade of type-2 VEGF receptors and was mimicked by hrVEGF. These data offer the first evidence that Dkk3 protects both neurons and astrocytes against a variety of toxic insults, and at least in culture, protection involves VEGF induction.

Abstract 336: MiR-126 Modified Endothelial Progenitor Cells Transplantation Contributes to Angiogenesis after Brain Focal Ischemia in SHR Rats

Background and Purpose: Transplantation of endothelial progenitor cells (EPCs) leads to better outcomes in experimental stroke, while improve the EPCs survival rate in ischemia area is still a challenge. MiR-126 modulates vascular development and angiogenesis. Here we overexpressed miR-126 in transplanted EPCs, to investigate the function of gene modified EPCs in angiogenesis after brain ischemia. Methods: Adult male SHR rats underwent permanent suture middle cerebral artery occlusion (MCAO). One week after middle cerebral artery occlusion, the animals received tail vein injection of miR-126 modified EPCs as treatment or EPCs as control and were monitored for 5 weeks. Brain water content, infarct volume, neurological score, neurogenesis and angiogenesis were examined. Results: Neurological score was greatly improved and brain atrophy was greatly reduced in miR-126 modified EPCs-treated SHR rats compared with the control rats 5 weeks after MCAO (P<0.05). The number of bromodeoxyuridine+/CD31+ microvessels are significantly increased. EPCs migration and proliferation were promoted after miR-126 modified in vitro. Conclusions: Our results showed that miR-126 modified EPCs therapyreduced ischemic brain injury, along with increased angiogenesis and neurogenesis in SHR rats, suggesting miR-126 significantly improved EPCs function in angiogenesis after MCAO. Gene modified EPCs represents a promising avenue for ischemic stroke stem cell therapy.

Progressive Assessment of Ischemic Injury to White Matter Using Diffusion Tensor Imaging: A Preliminary Study of a Macaque Model of Stroke.

Background:Previous Diffusion Tensor Imaging (DTI) studies have demonstrated the temporal evolution of stroke injury in grey matter and white matter can be characterized by DTI indices. However, it still remains not fully understood how the DTI indices of white matter are altered progressively during the hyperacute (first 6 hours) and acute stage of stroke (≤ 1 week). In the present study, DTI was employed to characterize the temporal evolution of infarction and white matter injury after stroke insult using a macaque model with permanent ischemic occlusion.Methods and materials:Permanent middle cerebral artery (MCA) occlusion was induced in rhesus monkeys (n=4, 10-21 years old). The brain lesion was examined longitudinally with DTI during the hyperacute phase (2-6 hours, n=4), 48 hours (n=4) and 96 hours (n=3) post-occlusion.Results:Cortical infarction was seen in all animals. The Mean Diffusivity (MD) in lesion regions decreased substantially at the first time point (2 hours post stroke) (35%, p <0.05, compared to the contralateral side) and became pseudo-normalized at 96 hours. In contrast, evident FA reduction was seen at 48 hours (39%, p <0.10) post-stroke. MD reduction in white matter bundles of the lesion area was much less than that in the grey matter during the hyper-acute phase but significant change was observed 4 hours (4.2%, p < 0.05) post stroke . Also, MD pseudonormalisation was seen at 96 hours post stroke. There was a significant correlation between the temporal changes of MD in white matter bundles and those in whole lesion areas during the entire study period. Meanwhile, no obvious fractional anisotropy (FA) changes were seen during the hyper-acute phase in either the entire infarct region or white matter bundles. Significant FA alteration was observed in entire lesion areas and injured white matter bundles 48 and 96 hours post stroke. The stroke lesion in grey matter and white matter was validated by pathological findings.Conclusion: The temporal evolution of ischemic injury to the grey matter and white matter from 2 to 96 hours after stroke onset was characterized using a macaque model and DTI. Progressive MD changes in white matter bundles are seen from hyperacute phase to acute phase after permanent MCA occlusion and temporally correlated with the MD changes in entire infarction regions. MD reduction in white matter bundles is mild in comparison with that in the grey matter but significant and progressive, indicating it may be useful to detect early white matter degeneration after stroke.