Measured Infarct Volume Research Articles

Clinical application of the modified Pullicino formula in endovascular thrombectomy in acute anterior circulation large cerebral infarction-A two-center retrospective study.

This study aimed to assess whether the modified Pullicino formula could be used as a screening indicator for measuring infarct volume before endovascular thrombectomy (EVT) and observe baseline factors affecting neurological recovery in patients with acute anterior circulation large-core cerebral infarction. The Pullicino formula × (1-22%) was applied to anterior circulation large cerebral infarctions with ASPECTS <6 to screen patients with infarct volumes of 70-150 mL. The outcomes were assessed by mRS score at 90 days, modified Treatment in Cerebral Ischemia (mTICI) score, symptomatic intracranial hemorrhage within 48 h, and death rate within 90 days. Significantly more patients in the EVT group with infarct volumes 70-150 mL had mRS ≤3 at 90 days than did the standard drug therapy (OR, 2.93; 95% CI, 1.08-7.89, p=0.03) and a significantly decreased mortality rate (OR, 0.36; 95% CI, 0.13-0.98; p=0.04), with 88.2% in patients with mTICI ≥2b. There was no significant difference in the intracranial hemorrhage. EVT (adjusted OR [aOR], 2.84; 95% CI, 1.15-7.01, p=0.02), infarct volume <150 mL (aOR, 3.47; 95% CI, 1.20-10.14, p=0.02), and intracranial atherosclerosis (aOR, 4.20; 95% CI, 1.24-14.04, p=0.02) were significantly associated with patient prognosis. Without commercial artificial intelligence software, the modified Pullicino formula can be used to calculate infarct volume and, in combination with ASPECTS <6, to select patients who may benefit from EVT.

Abstract TP311: Preclinical Replication Study of Psd-95 Inhibitor NA-1

Background &amp; Objective: Nerinate (NA-1), a postsynaptic density protein-95 inhibitor, is a clinical-stage stroke neuroprotectant. Recently, in a phase-III clinical trial (Efficacy and safety of nerinetide for the treatment of acute ischaemic stroke [ESCAPE-NA1]), a single intravenous dose of NA-1 did not improve the proportion of patients achieving favorable 3-month functional outcomes after endovascular thrombectomy compared with patients receiving placebo. Thus, we conducted a preclinical replication study to test therapeutic efficacy of NA-1 in a mouse model of transient middle cerebral artery occlusion (tMCAO). Methods: After 30-minute or 60-minute MCAO with laser Doppler flowmetry (LDF) monitoring, mice were randomly assigned to a group treated with either vehicle control (n=10 and 21, respectively) or 10 nM/g NA-1 (n=10 and 21, respectively), which was intravenously administered once via penile vein in a volume of 200 μL at the beginning of recanalization. In a different set of mice (n=6 for vehicle and n=6 for NA-1), we performed MCAO and then allowed animals to wake up before reperfusion at 60 minutes to account for continuous anesthesia-related confounding. At 1-day poststroke, brain was harvested and sliced in 2-mm thickness for 2,3,5-triphenyl-tetrazolium chloride (TTC) staining and infarct volume measurement after neurological scoring. Results: LDF data indicated that stroke modeling was executed proficiently, without inter-group differences. The vehicle group and the NA-1 group showed similar infarct volumes at 1 day after either 30 minutes (32.5±6.4 mm 3 and 44.7±8.7 mm 3 ; t-test P =0.27) or 60 minutes of MCAO (113.4±7.6 mm 3 and 108.2±7.6 mm 3 ; P =0.63). Awakening between occlusion and reperfusion did not change the negative Results: 109.4±11.7 mm 3 and 115.8±10.6 mm 3 ( P =0.69). Neurological scores did not show significant inter-group differences, either (all P &gt;0.05). Conclusions: We were unable to reproduce neuroprotective effects of NA-1 to reduce infarct volume at 1 day after tMCAO for 30 or 60 minutes. We suggest that implementation of multicenter animal studies is warranted to enhance the reproducibility and generalizability of preclinical observations.

Abstract WP318: Increased Synapse Elimination by Inflammatory Cells Contribute to Lon-Lasting Post-Stroke Memory Dysfunction in Aged Mice

Background and Purpose: Memory impairments are common after stroke. Memory dysfunction lasted longer in aged than in young mice, but the mechanisms are still unclear. Microglia maintain normal memory via removing excessive synapses and increase turnover of synaptic connections after stroke. Long-term memory deficits in aged mice may be associated with increased microglia in the brain. Hypothesis: Increased synapse removal by inflammatory cells contribute to the long-lasting post-stroke memory dysfunction of aged mice. Methods: Permanent distal middle cerebral artery occlusion (pMCAO) was induced in young and aged mice. The memory functions were analyzed weekly for 8 weeks by Y-maze test and at 8 weeks post stroke by novel objective recognition (NOR) test. Brain samples were collected 8 weeks after pMCAO induction. RNAseq analysis was performed to study gene expression profiles in peri-infarct area and hippocampus. Nissl staining was used to measure infarct volume and immunostaining was done to detect CD68 + and GFAP + cells and synapse in peri-infarct region and hippocampus. Results: RNAseq data shown up-regulation of CD68, GFAP, pro-inflammatory cytokines, immune response, microglia chemotaxins and phagocytosis genes; and down-regulation of synaptic assembly and transmission, synaptic plasticity, and long-term potentiation genes in aged mice at peri-infarct region and ipsilateral hippocampi. Aged mice had larger infarct volumes, more CD68 + and GFAP + cells in the peri-infarct region and ipsilateral hippocampi than young mice and had more CD68 + cells in the ipsilateral than the contralateral hippocampi. In Y-maze test, aged stroke mice made fewer spontaneous alternations from 3 to 8 weeks after pMCAO than young stroke and sham operated aged mice. In the NOR test, aged stroke mice spent less time on the novel objects than young stroke and sham aged mice. Increased synaptophysin positive CD68 + cells were detected in the hippocampi of aged stroke mice. Conclusions: Increase of synaptic elements by inflammatory cells contribute to the long-lasting post-stroke memory deficit in the aged mice, and reduction of neuroinflammation may improve their memory function.

Isolation of a novel isoprenylated phenolic compound and neuroprotective evaluation of Dodonaea viscosa extract against cerebral ischaemia–reperfusion injury in rats

Dodonaea viscosa grows widely in Saudi Arabia, but studies evaluating its neuroprotective activity are lacking. Thus, this study aimed to isolate and identify the secondary metabolites and evaluate the neuroprotective effects of D. viscosa leaves. The isolation and identification of phytochemicals were performed using chromatographic and spectroscopic techniques. The neuroprotective potential of the extract was evaluated against focal cerebral ischaemia–reperfusion injury in rat model. Neurobehavioural deficits in the rats were evaluated, and their brains were harvested to measure infarct volume and oxidative biomarkers. Results revealed the presence of three compounds: a novel isoprenylated phenolic derivative that was elucidated as 4-hydroxy-3-(3ʹ-methyl-2ʹ-butenyl) phenyl 1-O-β-D-apiosyl-(1ʹʹʹ → 6ʹʹ)- β-D-glucopyranoside (named Viscomarfadol) and two known compounds (isorhamnetin-3-O-rutinoside and epicatechin (4–8) catechin). Pre-treatment of the rats with the extract improved neurological outcomes. It significantly reduced neurological deficits and infarct volume; significantly reduced lipid peroxidation, as evidenced by decreased malondialdehyde levels; and significantly elevated antioxidant (superoxide dismutase, catalase, and glutathione) activities. These results indicate that D. viscosa is a promising source of bioactive compounds that can improve neurological status, decrease infarct volume, and enhance antioxidant activities in rats with cerebral ischaemic injury. Thus, D. viscosa could be developed into an adjuvant therapy for ischaemic stroke and other oxidative stress-related neurodegenerative disorders. Further investigations are warranted to explore other bioactive compounds in D. viscosa and evaluate their potential neuroprotective activities.

Long-term effect of indomethacin on a rat model of neonatal hypoxia ischemic encephalopathy through behavioral tests.

Many medical experts prescribe indomethacin because of its anti-inflammatory, analgesic, tocolytic, and duct closure effects. This article presents an evaluation of the enduring impact of indomethacin on neonatal rats with hypoxic-ischemic (HI) insults, employing behavioral tests as a method of assessment. The experiment was conducted on male Wistar-Albino rats weighing 10 to 15 g, aged between seven and 10 days. The rats were divided into three groups using a random allocation method as follows: hypoxic ischemic encephalopathy (HIE) group, HIE treated with indomethacin group (INDO), and Sham group. A left common carotid artery ligation and hypoxia model was applied in both the HIE and INDO groups. The INDO group was treated with 4mg/kg intraperitoneal indomethacin every 24 h for 3days, while the Sham and HIE groups were given dimethylsulfoxide (DMSO). After 72 h, five rats from each group were sacrificed and brain tissue samples were stained with 2,3,5-Triphenyltetrazolium chloride (TCC) for infarct-volume measurement. Seven rats from each group were taken to the behavioral laboratory in the sixth postnatal week (PND42) and six from each group were sacrificed for the Evans blue (EB) experiment for blood-brain barrier (BBB) integrity evaluation. The open field (OF) test and Morris water maze (MWM) tests were performed. After behavioral tests, brain tissue were obtained and stained with TCC to assess the infarct volume. The significant increase in the time spent in the central area and the frequency of crossing to the center in the INDO group compared with the HIE group indicated that indomethacin decreased anxiety-like behavior (p < 0.001, p < 0.05). However, the MWM test revealed that indomethacin did not positively affect learning and memory performance (p > 0.05). Additionally, indomethacin significantly reduced infarct volume and neuropathological grading in adolescence (p < 0.05), although not statistically significant in the early period. Moreover, the EB experiment demonstrated that indomethacin effectively increased BBB integrity (p < 0.05). In this study, we have shown for the first time that indomethacin treatment can reduce levels of anxiety-like behavior and enhance levels of exploratory behavior in a neonatal rat model with HIE. It is necessary to determine whether nonsteroidal anti-inflammatory agents, such as indomethacin, should be used for adjuvant therapy in newborns with HIE.

A differential detailed diffusion-weighted imaging-ASPECTS for cerebral infarct volume measurement and outcome prediction.

Diffusion-weighted imaging-Alberta Stroke Program Early Computed Tomography Score (DWI-ASPECTS) has been used to estimate infarct core volume in acute stroke. However, the same and indiscriminate score deduction for punctate or confluent DWI high-intensity lesion might lead to variation in performance. To develop and evaluate a differential detailed DWI-ASPECTS method in comparison with the conventional DWI-ASPECTS in core infarct volume measurement and clinical outcome prediction. We retrospectively recruited patients with acute ischemic stroke (AIS) treated with endovascular treatment between April 2013 and October 2019. In differential detailed DWI-ASPECTS, restricted diffusion lesion that was punctate or less than half of a cortical region (M1-M6) would not lead to subtraction of point. A favorable outcome was modified Rankin Scale score ⩽2 at 90 days after stroke onset. Among 298 AIS patients, mean age was 75 years (interquartile range (IQR) 67-82), and 194 patients (65%) were males. Mean infarct core volume was 11 mL (IQR 3-37). Overall, the score by detailed DWI-ASPECTS was significantly higher than conventional DWI-ASPECTS (8 (7-9) vs. 7 (5-9); P < 0.01). The detailed DWI-ASPECTS resulted in a higher correlation coefficient (r) for core infarct volume estimation than the conventional DWI-ASPECTS (r = 0.832 vs. 0.773; P < 0.01). Upon re-classification of those scored ⩽6 in conventional DWI-ASPECTS (n = 134) by detailed DWI-ASPECTS, the rate of favorable outcome in patients with detailed DWI-ASPECTS >6 was significantly higher than those with ⩽6 (29 (48%) vs. 14 (19%); P < 0.01). Detailed DWI-ASPECTS appeared to provide a more accurate infarct core volume measurement and clinical outcome correlation than conventional DWI-ASPECTS among AIS patients treated with endovascular therapy.

Intracerebral hirudin injection alleviates cognitive impairment and oxidative stress and promotes hippocampal neurogenesis in rats subjected to cerebral ischemia.

Cerebral ischemia starts with cerebral blood flow interruption that causes severely limited oxygen and glucose supply, eliciting a cascade of pathological events, such as excitotoxicity, oxidative stress, calcium dysregulation, and inflammatory response, which could ultimately result in neuronal death. Hirudin has beneficial effects in ischemic stroke and possesses antioxidant and anti-inflammatory properties. Therefore, we investigated the biological functions of hirudin and its related mechanisms in cerebral ischemia. The ischemia-like conditions were induced by transient middle cerebral artery occlusion (MCAO). To investigate hirudin roles, intracerebroventricular injection of 10 U hirudin was given to the rats. Cognitive and motor functions were examined by beam walking and Morris water maze tests. 2,3,5-triphenyl tetrazolium chloride-stained brain sections were used to measure infarct volume. Oxidative stress was determined by assessment of oxidative stress markers. The proliferated cells were labeled by BrdU and Nestin double staining. Western blotting was performed to measure protein levels. Hirudin administration improved cognitive and motor deficits post-ischemia. Hirudin reduced brain infarction and neurological damage in MCAO-subjected rats. Hirudin alleviated oxidative stress and enhanced neurogenesis in ischemic rats. Hirudin facilitated the promotion of phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and serine-threonine kinase. In sum, hirudin alleviates cognitive deficits by attenuating oxidative stress and promoting hippocampal neurogenesis through the regulation of ERK1/2 and serine-threonine kinase in MCAO-subjected rats.

Role of Polymorphonuclear Myeloid-Derived Suppressor Cells and Neutrophils in Ischemic Stroke.

Background Immune cells play avital role in the pathology of ischemic stroke. Neutrophils and polymorphonuclear myeloid-derived suppressor cells share a similar phenotype and have attracted increasing attention in immune regulation research, yet their dynamics in ischemic stroke remain elusive. Methods and Results Mice were randomly divided into 2 groups and intraperitoneally treated with anti-Ly6G (lymphocyte antigen 6 complex locus G) monoclonal antibody or saline. Distal middle cerebral artery occlusion and transient middle cerebral artery occlusion were applied to induce experimental stroke, and mice mortality was recorded until 28 days after stroke. Green fluorescent nissl staining was used to measure infarct volume. Cylinder and foot fault tests were used to evaluate neurological deficits. Immunofluorescence staining was conducted to confirm Ly6G neutralization and detect activated neutrophils and CD11b+Ly6G+ cells. Fluorescence-activated cell sorting was performed to evaluate polymorphonuclear myeloid-derived suppressor cell accumulation in brains and spleens after stroke. Anti-Ly6G antibody successfully depleted Ly6G expression in mice cortex but did not alter cortical physiological vasculature. Prophylactic anti-Ly6G antibody treatment ameliorated ischemic stroke outcomes in the subacute phase. Moreover, using immunofluorescence staining, we found that anti-Ly6G antibody suppressed activated neutrophil infiltration into parenchyma and decreased neutrophil extracellular trap formation in penumbra after stroke. Additionally, prophylactic anti-Ly6G antibody treatment reduced polymorphonuclear myeloid-derived suppressor cell accumulation inthe ischemic hemisphere. Conclusions Our study suggested a protective effect of prophylactic anti-Ly6G antibody administration against ischemic stroke by reducing activated neutrophil infiltration and neutrophil extracellular trap formation in parenchyma and suppressing polymorphonuclear myeloid-derived suppressor cell accumulation in thebrain. This study may provide a novel therapeutic approach for ischemic stroke.

Neuroprotective Effects of Geopung-Chunghyuldan Based on Its Salvianolic Acid B Content Using an In Vivo Stroke Model.

Geopung-Chunghyuldan (GCD) has neuroprotective properties. Salviae miltiorrhizae Radix plays an essential role in GCD's effect. The Salviae miltiorrhizae Radix marker compound is salvianolic acid B; however, its content is not uniform among samples. This study aimed to evaluate the neuroprotective effects of GCD based on salvianolic acid B content. The neuroprotective effects of GCD based on the salvianolic acid B content were evaluated by measuring infarct volume 24 h after permanent middle cerebral artery occlusion in an in vivo stroke model. For the experimental group, each GCD was administered immediately before surgery. The control groups were administered distilled water and aspirin (30 mg/kg) in the same way. The salvianolic acid B content in five types of Salviae Miltiorrhizae Radix (two Chinese and three Korean regions) based on different cultivation regions was analyzed by high-performance liquid chromatography. Three samples met the Korean and Chinese Pharmacopeia standards for salvianolic acid B. However, two samples did not. GCDs with high salvianolic acid B showed marked neuroprotective effects compared to the control groups, whereas GCDs with low salvianolic acid B did not. The salvianolic acid B content of Salviae miltiorrhizae Radix affects the neuroprotection effect of GCD. Stable, raw Salviae miltiorrhizae Radix is essential for GCD homogenization.

Abstract WP234: Mitochondria Therapy Mitigates Neuroinflammation And Provides Neuroprotection Against Ischemic Stroke In Mice

Background: Ischemic stroke is the second prevalent life-threat problem associated with long-lasting neurological disabilities and a high mortality rate worldwide. Despite this, therapeutic options remain limited. Recently, mitochondria transplantation therapy has emerged as a potential approach for increasing brain protection. This study aimed to investigate the protective role of mitochondria transplantation against ischemic stroke-induced behavioral deficits and molecular alterations in a photothrombotic stroke model. Methods: Ischemic stroke was induced by a Rose Bengal photothrombosis method in young adult C57BL/6 mice. Repeated doses of isolated liver mitochondria (100μg/mouse) were administrated intranasally at 1 h, 3 h, 24 h, and 48 h post-stroke. After 72 h, mice were tested for behavioral outcomes and euthanized, and tissues were harvested to measure infarct volume, brain edema, and molecular analysis. Results: Mitochondria-treated mice showed a significant improvement in behavioral outcomes, reduction in infarct size, and brain edema compared to the vehicle-treated group. Additionally, mitochondria significantly inhibited the protein expression of NOD-like receptor protein-3, phospho-nuclear factor kappa-B, cleaved-interleukin-1β, apoptosis-associated speck-like protein, glial fibrillary acidic protein, and ionized calcium-binding adapter molecule-1 proteins. Furthermore, mitochondria treatment showed a lower expression of nitrotyrosine, and 4-hydroxynonenal and higher expression of endothelial nitric oxide synthase and sulfotransferase-4A1 proteins, oxidative stress markers ( Table 1 ). Conclusion: Taken together, these data indicate that mitochondria reveal neuroprotection through the attenuation of inflammation against stroke. Further investigations into the role of neuroprotection of mitochondria transplantation are needed to determine whether it can be an effective therapy for stroke patients.

Evaluating blood-brain barrier disruption and infarction volume concurrently in rats subjected to ischemic stroke using an optical imaging system

BackgroundBlood-brain barrier (BBB) disruption is pivotal in the pathophysiological process of ischemic stroke and is often measured in rodent stroke studies. Traditionally, rodent BBB permeability increase is determined by measuring cerebral leakage of certain dyes such as Evans Blue or sodium fluorescein (NaFL). However, due to the special processing of samples for BBB permeability measurement, they cannot be used afterward for determining other essential parameters such as cerebral infarction volume. Therefore, using different batches of animals for assessing BBB permeability and infarction volume is typical. However, this would limit the stroke study’s statistical power and scientific value while hindering the implementation of procedures for high standard animal welfare. New methodThe rats subjected to middle cerebral artery occlusion (MCAO) were intraperitoneally injected with NaFL during the reperfusion phase. The brains were sliced and measured for BBB permeability using the small animal optical imaging system (IVIS® Lumia series III). Afterward, the same brain samples were either sliced or homogenized for tests that assessed infarction volume or other molecular changes. ResultsThe sum fluorescence intensity of the ischemic brain slices under the IVIS® Lumia series Ⅲ showed a strong correlation with the infarction volume determined by 2,3,5-triphenyl tetrazolium chloride (TTC) staining (r = 0.7440, P = 0.0087). The fluorescence intensity of the whole ischemic brain was correlated with the NaFL concentration of brain tissue homogenates (r = 0.8653, P = 0.0026) and cerebral infarction volume (r = 0.7282, P = 0.0072). Comparison with existing methodsThe new method enables concurrent measurement of BBB permeability and infarction volume on the same batch of brain tissue samples without affecting most downstream biochemical assays. ConclusionsBy applying the new method, we could use the same batch of ischemic rodent brain tissue for multiple assays, including BBB permeability and infarction volume. Through this, we would reduce the animal numbers in each study and help to maximize the scientific and statistical potential of future rodent ischemic studies.

Automatic Segmentation in Acute Ischemic Stroke: Prognostic Significance of Topological Stroke Volumes on Stroke Outcome.

Stroke infarct volume predicts patient disability and has utility for clinical trial outcomes. Accurate infarct volume measurement requires manual segmentation of stroke boundaries in diffusion-weighted magnetic resonance imaging scans which is time-consuming and subject to variability. Automatic infarct segmentation should be robust to rotation and reflection; however, prior work has not encoded this property into deep learning architecture. Here, we use rotation-reflection equivariance and train a deep learning model to segment stroke volumes in a large cohort of well-characterized patients with acute ischemic stroke in different vascular territories. In this retrospective study, patients were selected from a stroke registry at Houston Methodist Hospital. Eight hundred seventy-five patients with acute ischemic stroke in any brain area who had magnetic resonance imaging with diffusion-weighted imaging were included for analysis and split 80/20 for training/testing. Infarct volumes were manually segmented by consensus of 3 independent clinical experts and cross-referenced against radiology reports. A rotation-reflection equivariant model was developed based on U-Net and grouped convolutions. Segmentation performance was evaluated using Dice score, precision, and recall. Ninety-day modified Rankin Scale outcome prediction was also evaluated using clinical variables and segmented stroke volumes in different brain regions. Segmentation model Dice scores are 0.88 (95% CI, 0.87-0.89; training) and 0.85 (0.82-0.88; testing). The modified Rankin Scale outcome prediction AUC using stroke volume in 30 refined brain regions based upon modified Rankin Scale-relevance areas adjusted for clinical variables was 0.80 (0.76-0.83) with an accuracy of 0.75 (0.72-0.78). We trained a deep learning model with encoded rotation-reflection equivariance to segment acute ischemic stroke lesions in diffusion- weighted imaging using a large data set from the Houston Methodist stroke center. The model achieved competitive performance in 175 well-balanced hold-out testing cases that include strokes from different vascular territories. Furthermore, the location specific stroke volume segmentations from the deep learning model combined with clinical factors demonstrated high AUC and accuracy for 90-day modified Rankin Scale in an outcome prediction model.

Fraxetin alleviates microglia-mediated neuroinflammation after ischemic stroke

BackgroundNeuroinflammation, which is mainly mediated by excessive microglia activation, plays a major role in ischemic stroke. Overactivated microglia secrete numerous inflammatory cytokines, causing excessive inflammatory responses and ultimately exacerbating ischemic brain injury. Hence, compounds that attenuate neuroinflammation could become promising drug candidates for ischemic stroke. Fraxetin has an anti-inflammatory effect in many inflammatory diseases. However, whether it possesses an anti-inflammatory capacity in microglia-mediated neuroinflammation after ischemic brain injury is unknown. Our study aimed to investigate the suppression effect of fraxetin on neuroinflammation in lipopolysaccharide (LPS)-activated microglia and establish whether fraxetin could alleviate ischemic brain injury in a rodent model of ischemic stroke.MethodsFor the in vitro experiment, primary microglia were obtained from 1-day-old C57/BL6J mice. The cells were activated with LPS and treated with fraxetin at a non-cytotoxic concentration. Real-time PCR, enzyme-linked immunosorbent assays, and immunofluorescence staining were used to evaluate the anti-inflammatory effects of fraxetin. The potential molecular mechanisms were explored and verified through RNA-sequencing analysis, western blotting and real-time PCR. For the in vivo experiment, focal ischemia was induced by middle cerebral artery occlusion (MCAO) in 8-week-old male C57/BL6J mice. Fraxetin (5 mg/kg) or an equal volume of saline was injected into mice intraperitoneally after MCAO, and 2% 2,3,5-triphenyltetrazolium chloride staining was applied to measure infarct volume. Behavioral tests were conducted to measure neurological deficits in the mice. Real-time PCR, western blotting, and immunofluorescence staining were used to examine the expression of inflammatory cytokines and microglia activation in the ischemic penumbra.ResultsFraxetin effectively inhibited the expression of proinflammatory cytokines including inducible nitric oxide synthase, tumor necrosis factor-α, interleukin-1 beta, and interleukin-6 in LPS-activated microglia. Fraxetin also suppressed the PI3K/Akt/NF-κB signaling pathway in activated microglia, which contributed to its anti-inflammatory effects. Furthermore, the administration of fraxetin attenuated ischemic brain injury and behavioral deficits after stroke. Finally, fraxetin was found to attenuate the activation of microglia both in vitro and in vivo.ConclusionsOur results suggest that fraxetin has a suppression effect on microglia-mediated neuroinflammation, and this effect is associated with the PI3K/Akt/NF-κB signaling pathway. Fraxetin may therefore have potential neuroprotective properties for ischemic stroke.

Abstract TMP118: Novel Regulation Of Peripheral Derived Immune Cells In The Remodeling Of Pial Collateral Vessels Following Ischemic Stroke

Introduction: Stroke is a leading cause of neurological morbidity and mortality in the United States. Remodeling of pial collateral (PC) vessels (arteriogenesis) is vital to restore blood perfusion into the ischemic penumbra to reduce damage. Previous reports showed perivascular monocyte /macrophage (MM) accumulation in hind limb ischemia and myocardial infarction, suggesting that MM are key regulators of inflammatory milieu following stroke. We reported EphA4 as a negative regulator of PC remodeling. However, the role of EphA4 during peripheral derived immune cell (PDI) mediated arteriogenesis after permanent middle cerebral artery occlusion (pMCAO) remains unknown. This study explores the role of EphA4 in regulating PDI-mediated arteriogenesis after pMCAO and interrogates if MM-specific EphA4 contributes to the neuroinflammatory milieu during remodeling process. Methods: GFP reporter EphA4 bone marrow chimeric knockout (KO BMCs ) and wild-type mice (WT BMCs ) were euthanized 1d post pMCAO. Vessel painted whole brains were used for immunostaining, infarct volume measurement, and confocal histological analysis including PC remodeling and GFP+ perivascular immune cell recruitment. Blood from sham and pMCAO mice was collected for flow cytometry to analyze EphA4 expression on circulating immune cell types. We performed rotarod test to assess behavioral recovery, and used clodronate liposome for MM depletion in both WT BMCs and KO BMCs to further explore the function of EphA4 MM after stroke. Results: This study developed a novel methodology to evaluate MM-specific EphA4 role after pMCAO. EphA4 was highly expressed on Ly6G+ neutrophils, Ly6Chi, and Ly6Clo MM in whole blood 24h after pMCAO. KO BMCs mice had enhanced growth of ipsilateral PC compared to WT BMCs mice after pMCAO. These findings correlate with KO BMCs reduced infarct volume and improved motor function compared to WT BMCs . Finally, WT BMCs displayed a significant increase in GFP+ PDI recruitment to the PC wall in KO BMCs compared to WT BMCs mice. Conclusions: PDI-specific EphA4 expression negatively regulates arteriogenesis and suppresses PDI accumulation on PC. The acute neurovascular inflammation may be dependent on the EphA4 expression of MM in the peripheral circulation after stroke.

Abstract WP141: Effect Of Non-invasive Vagus Nerve Stimulation In Hemorrhagic Brain Injury And Permanent Ischemic Stroke In Rats

Introduction: Non-invasive vagus nerve stimulation (nVNS) reduces infarct volume after transient ischemia in rats. Here, we tested the effect of nVNS in intracerebral hemorrhage (ICH) and permanent ischemic brain injury models. Methods: ICH was induced by whole blood (n=24) or collagenase (n=24) injection into the striatum of adult male and female Wistar rats. 1-hour sham stimulation (n=16) or nVNS (n=32) was initiated 30 minutes after injection. Spontaneous circling, hindlimb retraction, and grasp ability were tested at 24 hours to measure functional deficit. Rats were euthanized 24 hours or one week after injury, and brain tissue was processed for water content analysis (n=24) and histology (n=24), respectively. Permanent ischemia was induced by filament occlusion of the right middle cerebral artery in adult male Wistar rats (n=18). Sham stimulation and nVNS initiated 30 minutes after the induction of ischemia were applied for 1 hour and 3 hours (n=6 per group). Rats were euthanized 24 hours later to measure infarct volume. Results: 1-hour nVNS was associated with a non-significant reduction in hematoma volume in both models of ICH (Fig). Animals treated with nVNS demonstrated better functional outcome at 24 hours after ICH ( p =0.042). There was no difference in brain edema between control and active treatment arms in both models of ICH. 3-hour nVNS reduced infarct volume by 21% in permanent ischemia ( p =0.047, Fig). Conclusion: The results of this study support prior evidence that nVNS may have a therapeutic potential in ischemic stroke. nVNS is currently being evaluated in two clinical studies (NCT03733431 and NCT04050501) for the treatment of acute ischemic stroke. The lack of adverse events in two different models of ICH in this study suggests that nVNS could be safely administered as early as an ambulatory setting before the stroke etiology (ischemic vs. hemorrhagic) has been determined.

Antioxidant and anti-apoptotic effects of cannabidiol in model of ischemic stroke in rats

One of the main non-psychoactive phytocannabinoids of cannabis is cannabidiol (CBD), which has attracted much attention for its neuroprotective roles. The present study was designed to assess whether pretreatment of CBD can attenuate two of the destructive processes of cerebral ischemia, including oxidative stress and cell death. The male rats were randomly divided into 6 main groups (control, MCAO, vehicle, and CBD-treated groups). Using stereotaxic surgery, a cannula was inserted into the right lateral ventricle of the rat brain. CBD was injected at doses of 50, 100 and 200 ng/rat for five consecutive days. After pretreatment, middle cerebral artery (MCA) was blocked for 60 min using the intraluminal filament technique. 24 h after reperfusion, each main group was considered for measurement of infarct volume, superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), p53 gene expression, pathological alterations, and expression of Bax, Bcl-2, cytochrome C, and caspase-3 proteins. The results revealed that CBD at dose of 100 ng/rat reduced the infarction volume and MDA level in cortical and striatal areas of rat brain compared with vehicle group. In addition, the CBD at dose of 100 ng/rat elevated the activity of SOD enzyme in cortex and striatum. The increase in the activity of CAT was also seen at dose of 100 ng/rat in cortex. Furthermore, the Bcl-2/Bax ratio was significantly diminished by the dose of 100 ng/rat CBD in cortex. Moreover, a decrease in expression of cytosolic cytochrome C was observed by CBD at doses of 100 and 200 ng/rat in cortex. CBD at doses 100 and 200 ng/rat also reduced the expression of caspase-3 in cortical and striatal areas, respectively. P53 was downregulated following administration of CBD at dose of 100 ng/rat. Moreover, histological analysis showed the decrease in the percentage of pyknotic neurons in 100 and 200 ng/rat CBD-received groups. CBD played the anti-apoptosis and anti-oxidant roles in cerebral ischemia by affecting the pathways of intrinsic apoptosis, endogenous antioxidant enzymes, and lipid peroxidation.

Behavioral and histopathological consequences of transient ischemic stroke in the Flinders Sensitive Line rat, a genetic animal model of depression

Patients with depression have an increased risk for stroke, higher mortality rates following stroke and worse functional outcomes among survivors. Preclinical studies may help to better understand the underlying mechanisms linking these two diseases, but only a few animal studies have investigated the effects of prestroke depression. The present study investigates whether Flinders Sensitive Line (FSL) rats, a genetic depression model, respond differently to focal ischemic stroke compared to control strains (Flinders Resistant Line [FRL] and Sprague-Dawley [SD]). Male adult FSL, FRL and SD rats received a unilateral injection of either vehicle or Endothelin-1 (ET-1) adjacent to the middle cerebral artery (MCA). Motor function was assessed at 48 h followed by euthanasia and infarct volume measurement using 2,3,5-triphenyltetrazolium chloride (TTC) staining and image analysis. In a separate cohort behavior was assessed using standard tests for motor function, locomotor activity, cognition, anxiety- and depression-like behavior beginning at 10 days post-injection followed by infarct quantification. We found that ET-1-induced MCA occlusion produced significant infarcts in all three strains. Stroke animals had slightly impaired motor function, but there was no clear interaction effects between strain and stroke surgery on behavioral outcomes. We conclude that FSL rats show no increased susceptibility to brain damage or behavioral deficits following ET-1-induced focal ischemic stroke compared to controls.

Protective effects of Dimethyl malonate on neuroinflammation and blood-brain barrier after ischemic stroke.

After ischemic stroke, microglia will be activated and play a key role in neuroinflammation and the destruction of the blood-brain barrier (BBB), and activated microglia could polarize into pro-inflammation M1 phenotype and anti-inflammation M2 phenotype. Dimethyl malonate (DMM) could reduce reactive oxygen species and we speculate DMM could regulate microglia to protect ischemic brain. We used transient middle cerebral artery occlusion (tMCAO) mouse model to simulate ischemic stroke and adult male C57BL/6 mice were used in our study. 2,3,5-triphenyltetrazolium chloride staining was used to measure infarct volume. Evans Blue and Brain water content were used to evaluate the destruction of BBB. We used a five-point scale to assess the neurologic function of mice. Western blot and Immunofluorescence were used to measure microglia, pericytes and the expression of related proteins. DMM reduced cerebral infarct volume, Evans blue leakage, brain water content and improved neurologic deficits after tMCAO. The number of activated microglia and M1 microglia were decreased and the number of M2 microglia and pericytes were increased after DMM treatment. The expression of tumor necrosis factor-α was reduced while protein levels of IL-10 and ZO-1 were increased through DMM treatment. DMM could regulate activation and polarization of microglia to inhibit neuroinflammation and protect BBB.

PI3KC2β inactivation stabilizes VE-cadherin junctions and preserves vascular integrity.

Endothelium protection is critical, because of the impact of vascular leakage and edema on pathological conditions such as brain ischemia. Whereas deficiency of class II phosphoinositide 3-kinase alpha (PI3KC2α) results in an increase in vascular permeability, we uncover a crucial role of the beta isoform (PI3KC2β) in the loss of endothelial barrier integrity following injury. Here, we studied the role of PI3KC2β in endothelial permeability and endosomal trafficking in vitro and in vivo in ischemic stroke. Mice with inactive PI3KC2β showed protection against vascular permeability, edema, cerebral infarction, and deleterious inflammatory response. Loss of PI3KC2β in human cerebral microvascular endothelial cells stabilized homotypic cell-cell junctions by increasing Rab11-dependent VE-cadherin recycling. These results identify PI3KC2β as a potential new therapeutic target to prevent aggravating lesions following ischemic stroke.

MRI Diffusion-Weighted Imaging to Measure Infarct Volume: Assessment of Manual Segmentation Variability.

Manual segmentation of infarct volume on follow-up MRI diffusion-weighted imaging (MRI-DWI) is considered the gold standard but is prone torater variability. We assess the variability of manual segmentations of MRI-DWI infarct volume. Consecutive patients (May 2018 to May 2019) with the anterior circulation stroke and endovascularly treated were enrolled. All patients underwent 24- to 32-hour follow-up MRI. Three users manually segmented DWI infarct volumes slice by slice twice. The reference standard of DWI infarct volume was generated by the STAPLE algorithm. Intra- and interrater reliability was evaluated using the intraclass correlation coefficient (ICC) by comparing manual segmentations with the reference standard. Spatial measurements were evaluated using metrics of the Dice similarity coefficient (DSC). Volumetric measurements were compared using the lesion volume. The dataset consisted of 44 patients, mean (SD) age was 70.1 years (±10.3), 43% were women, and median baseline NIHSS score was 16. Among three users, the mean DSC for MRI-DWI infarct volume segmentations ranged from 80.6%±11.7% to 88.6%±7.5%, and the mean absolute volume difference was 2.8±6.8 to 13.0±14.0 ml. Interrater ICC among the users for DSC and infarct volume was .86 (95% confidence interval [95% CI]: .78-.91) and .997 (95% CI: .995-.998). Intrarater ICC for the three users was .83 (95% CI: .69-.93), .84 (95% CI: .72-.91), and .80 (95% CI: .64-.89) for DSC, and .99 (95% CI: .987-.996), .991 (95% CI: .983-.995), and .996 (95% CI: .993-.998) for infarct volume. Manual segmentation of infarct volume on follow-up MRI-DWI shows excellent agreement and good spatial overlap with the reference standard, suggesting its usefulness for measuring infarct volume on 24- to 32-hour MRI-DWI.