Min Of Middle Cerebral Artery Occlusion Research Articles

Hyperglycemia exacerbates cerebral ischemia/reperfusion injury by up-regulating autophagy through p53-Sesn2-AMPK pathway

Hyperglycemia exacerbates ischemic brain injury by up-regulating autophagy. However, the underlying mechanisms are unknown. This study aims to determine whether hyperglycemia activates autophagy through the p53-Sesn2-AMPK signaling pathway. Rats were subjected to 30-min middle cerebral artery occlusion (MCAO) with reperfusion for 1- and 3-day under normo- and hyperglycemic conditions; and HT22 cells were exposed to oxygen deprivation (OG) or oxygen–glucose deprivation and re-oxygenation (OGD/R) with high glucose. Autophagy inhibitors, 3-MA and ARI, were used both in vivo and in vitro. The results showed that, compared with the normoglycemia group (NG), hyperglycemia (HG) increased infarct volume and apoptosis in penumbra area, worsened neurological deficit, and augmented autophagy. after MCAO followed by 1-day reperfusion. Further, HG promoted the conversion of LC-3I to LC-3II, decreased p62, increased protein levels of aldose reductase, p53, P-p53ser15, Sesn2, AMPK and numbers of autophagosomes and autolysosomes, detected by transmission electron microscopy and mRFP-GFP-LC3 molecular probe, in the cerebral cortex after ischemia and reperfusion injury in animals or in cultured HT22 cells exposed to hypoxia with high glucose content. Finally, experiments with autophagy inhibitors 3-MA and aldose reductase inhibitor (ARI) revealed that while both inhibitors reduced the number of TUNEL positive neurons and reversed the effects of hyperglycemic ischemia on LC3 and p62, only ARI decreased the levels of p53, P-p53ser15. These results suggested that hyperglycemia might induce excessive autophagy to aggravate the brain injury resulted from I/R and that hyperglycemia might activate the p53-Sesn2-AMPK signaling pathway, in addition to the classical PI3K/AKT/mTOR autophagy pathway.

Intranasal insulin helps overcome brain insulin deficiency and improves survival and post-stroke cognitive impairment in male mice.

Obesity increases the risk for stroke and is associated with worse post-stroke outcomes; however, the mechanisms are poorly understood. Diet-induced obesity leads to insulin resistance and subsequently, brain insulin deficiency. The purpose of this study was to investigate the potential impact of brain insulin deficiency on post-stroke outcomes. To accomplish this, brain insulin levels were assessed in male C57BL/6J (B6) mice placed on either a standard diet or 54% kcal high-fat diet, a known model of insulin resistance. Mice were subjected to either a sham surgery (control) or 30-min middle cerebral artery occlusion to induce an ischemic stroke and administered either intranasal saline (0.9%) or intranasal insulin (1.75 U) twice daily for 5 days beginning on day 1 post-stroke. High-fat diet-induced brain insulin deficiency was associated with increased mortality, neurological and cognitive deficits. On the other hand, increasing brain insulin levels via intranasal insulin improved survival, neurological and cognitive function in high-fat diet mice. Our data suggests that brain insulin deficiency correlates with worse post-stroke outcomes in a diet-induced mouse model of insulin resistance and increasing brain insulin levels may be a therapeutic target to improve stroke recovery.

Short-term beneficial effects of human dental pulp stem cells and their secretome in a rat model of mild ischemic stroke

Although cell therapy has been applied in regenerative medicine for decades, recent years have seen greatly increased attention being given to the use of stem cell-based derivatives such as cell-free secretome. Dental pulp stem cells (DPSCs) are widely available, easily accessible, and have high neuroprotective and angiogenic properties. In addition, DPSC-derived secretome contains a rich mixture of trophic factors. The current investigation evaluated the short-term therapeutic effects of human DPSCs and their secretome in a rat model of mild ischemic stroke.Mild ischemic stroke was induced by 30 min middle cerebral artery occlusion, and hDPSCs or their secretome was administered intra-arterially and intranasally. Neurological function, infarct size, spatial working memory, and relative expression of seven target genes in two categories of neurotrophic and angiogenic factors were assessed three days after stroke. In the short-term, all treatments reduced the severity of neurological and histological deficits caused by ischemic stroke. Moreover, transient middle cerebral artery occlusion led to the striatal and cortical over-expression of BDNF, NT-3, and angiogenin, while NGF and VEGF expression was reduced. Almost all interventions were able to modulate the expression of target genes after stroke. The obtained data revealed that single intra-arterial administration of hDPSCs or their secretome, single intranasal transplantation of hDPSCs, or repeated intranasal administration of hDPSC-derived secretome was able to ameliorate the devastating effects of a mild stroke, at least in the short-term.

The impact of anesthetic drugs on hemodynamic parameters and neurological outcomes following temporal middle cerebral artery occlusion in rats.

The induction of ischemic stroke in the experimental model requires general anesthesia. One of the factors that can be effective in the size of ischemic brain lesions and neurological outcomes is the type of anesthesia. So, the current study was designed to compare the impacts of the most important and widely used anesthetics including halothane, isoflurane, and chloral hydrate on the transient middle cerebral artery occlusion (MCAO) outcomes. Adult Male Sprague-Dawley rats were randomly divided into three groups as follows: (1) MCAO + halothane group, (2) MCAO + isoflurane group, and (3) MCAO + chloral hydrate group. After 24 h, the mortality rate, infarct size, tissue swelling, neurological function, hemodynamic, and arterial blood gas parameters were assessed. Our finding showed that 60 min MCAO rats anesthetized with chloral hydrate significantly increased mortality rate, infarct size, tissue swelling, and neurological deficits compared with halothane and isoflurane anesthetics after 24 h of MCAO. Also, chloral hydrate caused a significant decrease in mean arterial pressure and arterial pO2 compared to halothane and isoflurane anesthetics. On the basis of the current data, we concluded that chloral hydrate increased cerebral infarct volume and neurological outcomes and reduced hemodynamic and metabolic parameters compared with halothane and isoflurane-anesthetized rats temporal MCAO.

Abstract WP219: Repurposing The K Ca 3.1 Inhibitor Senicapoc For Treatment Of Acute Ischemic Stroke

Background: Acute ischemic stroke (AIS) is a leading cause of death and long-term disability. Both microglia (MG) and infiltrating macrophages (MP) are critical effector cell types in ischemic brain injury and recovery. K Ca 3.1 is a calcium-activated potassium channel that is upregulated in reactive MG and MP. Studies using either genetic deletion or pharmacological inhibition of K Ca 3.1 demonstrated that this channel is critical for pro-inflammatory activation of MG/MP as well as exacerbation of stroke pathophysiology. Senicapoc (ICA-17043) is a K Ca 3.1-specific inhibitor that has been used in human clinical trials for non-neurological indications (sickle cell anemia, asthma) and was proven safe. Here we evaluate the potential for repurposing senicapoc for AIS. Methods: Young adult male and female mice underwent 60 min middle cerebral artery occlusion (MCAO)/reperfusion. MCAO was monitored by laser doppler. Senicapoc’s pharmacokinetic (PK) profile was determined using high performance liquid chromatography - mass spectroscopy. Drug levels in plasma and brain were quantified at multiple time points post administration. Effects of senicapoc on post-stroke release of cytokines/chemokines was determined by multiplex ELISA. Inflammatory infiltrates were quantified with flow cytometry. Efficacy studies included: (i) infarct volume (MRI T2), white matter integrity (DTI) and longitudinal neurobehavioral outcomes (NBO). In-vitro chromogenic assay was used to assess senicapoc’s effect on proteolytic activity of tissue plasminogen activator (tPA). Results: Administration of senicapoc (40 mg/kg, i.p.) twice daily for seven days starting 12 h after MCAO resulted in ~55% reduction in infarct volume with corresponding improvements in NBO. Free senicapoc levels in brain ranged from 20 - 200 nM in stroked mice at 1, 4 & 12 hours following administration. All values exceeded senicapoc’s IC 50 (11 nM) for K Ca 3.1. Senicapoc, at up to 5 μM, had no effect on tPA’s proteolytic activity. Conclusions: We provide proof-of-concept data that senicapoc, administered in an extended temporal window, can markedly reduce infarct volume and improve NBO in a mouse model of AIS. Senicapoc demonstrated favorable PK and CNS penetration. It did not interfere with tPA.

Abstract TMP117: Housing Light Cycle Impacts Metabolic Profile, Stroke Survival, And Cognitive Outcomes In Diabetic Rats

Diabetes worsens stroke outcomes and cognitive function, but the underlying reasons are not understood. Long-term experimental studies had been limited by greater mortality in diabetic cohorts. Recent studies suggested circadian rhythms may modulate brain ischemia and hence mortality/recovery. The goal of this study was to determine the impact of stroke timing and housing light cycle conditions on survival and cognitive outcomes in control and diabetic rats. Methods: Control (C) and diabetic (D) male rats (n=7/group) were randomly grouped into normal light cycle (NLC 6 am/6 pm) or reverse light cycle (RLC 12 am/12 pm) starting at 4 w of age. At 12 w of age, rats were subjected to 60 min middle cerebral artery occlusion (MCAO, night stroke in NLC and morning stroke in RLC). A battery of behavioral tests was performed up to W8 post-stroke to assess motor, cognitive, and psychosocial status. Results: (Table). RLC conditions improved stroke surgery mortality and long-term survival in diabetic animals. Pre- and post-stroke body weight gain was affected only in the NLC/D group. High mortality in the NLC/D prevented 2x2 comparisons at W8 but suggested the worst performance the in NLC/D cohort. Comparisons of C and D rats in RLC showed a decline in recognition memory in both groups over time with trends for worse performance in diabetic animals. There was no difference in aversive learning or anxiety status. Conclusions: These results suggest that the light cycle and timing of MCAO affect the circadian rhythm, overall health state, and cognitive outcomes, especially in diabetes. RLC experiments may refine and improve the long-term studies in comorbid disease models.

Abstract 41: Synthesis And Pharmacological Validation Of Novel Neuroprotective Purinergic Receptor P2X4 Antagonists For The Treatment Of Ischemic Stroke

Background and Purpose: Identification of a novel treatment for ischemic stroke is urgently needed. We previously showed that genetic deletion as well as short term pharmacological inhibition of P2X4R, a purinergic receptor for adenosine triphosphate ATP, provides acute neuroprotection and thus can be potential drug targets to treat ischemic stroke. Here we synthesized and pharmacologically validated those experimental drugs for the treatment of ischemic stroke. Methods: We first performed the P2X4 receptor structure activity (SAR) relationship based on known heterocyclic antagonist P2X4 antagonists including NP-1815-PX Following extensive modification of the reported synthetic route, we synthesized several analogues that maintained or enhanced antagonist affinity. We used [Ca 2+ ] i influx assay in P2X4R overexpressing human HEK cells to identify potent and specific P2X4R antagonist. Two most potent leads (MRS4719 and 4596 given subcutaneously for 3 days post stroke) were then tested for their neuroprotective activity using middle cerebral artery occlusion (MCAo) model of ischemic stroke (60 min MCAo followed by 3 days of reperfusion) using both sexes of young and aged mice. MRS4719 was also tested in human primary monocyte derived macrophage obtained from the blood of de-identified aged healthy subjects (55±10 years). Results: Analogues MRS 4719 and MRS4596 were most potent at human (h) P2X4R (IC 50 0.503 and 1.38 μM, respectively), and were highly selective versus hP2X1R, hP2X2/3R, hP2X3R). Both MRS 4719 and MRS 4596 showed neuroprotective effects after 3 days of ischemic stroke in a dose dependent manner in young mice (8-12 weeks old). Optimal dose of MRS4719 (1.5 mg/kg/day) also significantly reduced infarct volume (59.6±5.3 vs. 27.47±10.23 mm 3 ; p<0.05) in middle-aged mice (11-12 months). MRS 4719 showed learning and memory enhancing activity when measured after 4 weeks of stroke. MRS 4719 inhibited ATP-induced [Ca 2+ ] i influx in primary human monocyte-derived macrophages, consistent with its blocking effect at endogenously expressed P2X4R. Conclusions: This study indicates MRS4719 is a potent P2X4R antagonist and has translational potential for the treating of ischemic stroke across the age.

Sphingosine-1-phosphate receptor modulation improves neurogenesis and functional recovery after stroke.

Cerebral ischemia activates neural progenitors that participate in brain remodeling following acute injury. Sphingosine-1-phosphate receptor (S1PR) signaling governs cell proliferation and mobilization, yet its potential impact on neural progenitors and stroke recovery remains poorly understood. The goal of this study was to investigate the impact of S1PR modulation on post-stroke neurogenesis and functional recovery, using a S1PR modulator BAF312. Mice were subjected to 60 min middle cerebral artery occlusion (MCAO) and received BAF312 starting from day 3 after MCAO until the end of experiment. BAF312 facilitated motor function recovery in MCAO mice until day 14 after surgery. Flow cytometry analysis revealed that BAF312 treatment led to an increase of type A cells in subventricular zone (SVZ), but not other progenitor cell subsets in MCAO mice. We found an increase of BrdU incorporation in SVZ DCX+ cells from MCAO mice receiving BAF312 and augmented proliferation of DCX+ cells in cultured neurospheres isolated from SVZ tissues. Notably, a S1PR1 antagonist W146 abolished BAF312-induced increase of SVZ type A cells from MCAO mice and proliferation of DCX+ cells in cultured neurospheres. Additionally, the benefit of BAF312 to improve neurogenesis and stroke recovery remains in Rag2-/- mice lacking of T and B cells. Our results demonstrate that S1PR modulation improves neurogenesis and functional recovery following brain ischemia.

Xiao-Xu-Ming decoction extracts promotes mitochondrial biogenesis and improves neurobehavioral deficits in cerebral ischemia/reperfusion rats

Introduction: Stroke is one of the leading causes of death and disability in the worldwide. Traditional Chinese medicine plays an important role in preventing and treatment of stroke. Xiao-Xu-Ming decoction (XXM) is an effective traditional Chinese prescription for the treatment of stroke and its sequelae. Previous studies have reported the extracts of XXM has a good therapeutic effect on experimental animal stroke. However, the mechanism is unclear. Our present study focused on metabolic remodeling in cerebral ischemia-reperfusion to investigate the mechanism of XXM on cerebral ischemia/reperfusion (I/R). Methods: The cerebral I/R rat model was established by 90 min middle cerebral artery occlusion following reperfusion. XXM at doses of 37.5, 75 and 150 mg/kg was administered for the continuous 7 day after cerebral I/R. Behavioral testing, cerebral infarction detected by TTC staining, Nissl staining, NeuN immunohistochemical staining, and MAP2 immunofluorescence staining were conducted to evaluate the effect of XXM treatment on cerebral I/R of rats. TUNEL staining was used to detect DNA damage. The expression of related proteins was detected by qPCR and western blotting. Results: 7 day XXM treatment improved neurological and behavioral deficits, regulated the biogenesis and dynamic process of mitochondria, promoted the recovery of mitochondrial function, and improved the energy metabolism disorder after cerebral I/R. Furthermore, XXM also had an improving effect on mitochondrial-dependent apoptosis after cerebral I/R. Conclusion: we found that XXM regulated metabolic remodeling in cerebral ischemia-reperfusion and improve neurological deficits.

Application of an atmospheric pressure plasma jet in a rat model of ischaemic stroke: Design, optimisation, and characteristics

AbstractRecent studies have shown the in vitro neuro‐protective functions of atmospheric pressure plasma (APP) against multiple pathological injuries during ischaemic stroke (IS). However, whether APP treatment exerts a therapeutic effect on a rat IS model remains unclear. Here, on the basis of needle‐to‐ring dielectric barrier discharge, an atmospheric pressure plasma jet (APPJ) was designed, with the Helium as the working gas which was driven by a sinusoidal voltage. Then, the treatment conditions were optimised for IS rat model treatment and the characteristics of this APPJ were further diagnosed. Subsequently, the rat IS model was established through 90 min middle cerebral artery occlusion (MCAO), and plasma was intermittently inhaled by rats via the nasal cavity for a 2 min period at 60 min of MCAO process. The therapeutic effects of this plasma jet device were then evaluated using biomedical analyses. According to our results, intermittent APP inhalation in the MCAO rats increased the serum NO content, improved the neurological function, enhanced regional cerebral blood flow, lowered brain infarction, and reduced the cell apoptosis in brain tissues of MCAO rats. Collectively, our data provides a novel potential strategy for IS treatment by using atmospheric‐pressure plasma inhalation.

Dl-3-n-Butylphthalide Reduced Neuroinflammation by Inhibiting Inflammasome in Microglia in Mice after Middle Cerebral Artery Occlusion.

The inflammatory response is one of the key events in cerebral ischemia, causing secondary brain injury and neuronal death. Studies have shown that the NLRP3 inflammasome is a key factor in initiating the inflammatory response and that Dl-3-n-butylphthalide (NBP) can attenuate the inflammatory response and improve neuronal repair during ischemic stroke. However, whether NBP attenuates the inflammatory response via inhibition of NLRP3 remains unclear. A 90 min middle cerebral artery occlusion was induced in 62 2-month-old adult male ICR mice, and NBP was administered by gavage zero, one, or two days after ischemia. Brain infarct volume, neurological deficits, NLRP3, microglia, and neuronal death were examined in sacrificed mice to explore the correction between NBP effects and NLRP3 expression. NBP significantly reduced infarct volume and attenuated neurological deficits after ischemic stroke compared to controls (p < 0.05). Moreover, it inhibited ASC+ microglia activation and NLRP3 and CASP1 expression in ischemic mice. In addition, neuronal apoptosis was reduced in NBP-treated microglia cultures (p < 0.05). Our results indicate that NBP attenuates the inflammatory response in ischemic mouse brains, suggesting that NBP protects against microglia activation via the NLRP3 inflammasome.

The impacts of anesthetic regimens on the middle cerebral artery occlusion outcomes in male rats.

The middle cerebral artery occlusion (MCAO) model was introduced more than 3 decades ago to simulate human stroke. Till now, it is the most common platform to investigate stroke-induced pathological changes as well as to discover new drugs and treatments. Induction of general anesthesia is mandatory to induce this model, and different laboratories are using various anesthetic drugs, which might affect MCAO results. Therefore, the present study was designed to compare the impacts of several widely used anesthetic regimens on the MCAO outcomes. Here, adult male rats were anesthetized by isoflurane inhalation, intraperitoneal injection of chloral hydrate (CH), intraperitoneal injection of ketamine-xylazine, or subcutaneous administration of ketamine-xylazine, then subjected to 30 min MCAO. Survival rate, body weight change, infarct size, as well as cognitive and neurological performance were evaluated up to 3 days after the surgery. Our findings revealed CH caused the highest, whereas subcutaneous ketamine-xylazine led to the lowest mortality. Meanwhile, there were no significant differences in the body weight loss, infarct size, cognitive impairments, and neurological deficits among the experimental groups. Based on the current results, we proposed that subcutaneous injection of ketamine-xylazine could be an effective anesthetic regimen in the rat model of MCAO with several advantages such as low mortality, cost-effectiveness, safety, ease of administration, and not requiring specialized equipment.

Liver vitronectin release into the bloodstream increases due to reduced vagal muscarinic signaling after cerebral stroke in female mice.

Vitronectin (VTN) is a glycoprotein enriched in the blood and activates integrin receptors. VTN blood levels increase only in female mice 24 h after an ischemic stroke and exacerbate brain injury through IL‐6‐driven inflammation, but the VTN induction mechanism is unknown. Here, a 30 min middle cerebral artery occlusion (MCAO) in female mice induced VTN protein in the liver (normally the main source) in concert with plasma VTN. Male mice were excluded as VTN is not induced after stroke. MCAO also increased plasma VTN levels after de novo expression of VTN in the liver of VTN−/− female mice, using a hepatocyte‐specific (SERPINA1) promoter. MCAO did not affect SERPINA1 or VTN mRNA in the liver, brain, or several peripheral organs, or platelet VTN, compared to sham mice. Thus, hepatocytes are the source of stroke‐induced increases in plasma VTN, which is independent of transcription. The cholinergic innervation by the parasympathetic vagus nerve is a potential source of brain‐liver signaling after stroke. Right‐sided vagotomy at the cervical level led to increased plasma VTN levels, suggesting that VTN release is inhibited by vagal tone. Co‐culture of hepatocytes with cholinergic neurons or treatment with acetylcholine, but not noradrenaline (sympathetic transmitter), suppressed VTN expression. Hepatocytes have muscarinic receptors and the M1/M3 agonist bethanechol decreased VTN mRNA and protein release in vitro via M1 receptors. Finally, systemic bethanechol treatment blocked stroke‐induced plasma VTN. Thus, VTN translation and release are inhibited by muscarinic signaling from the vagus nerve and presents a novel target for lessening detrimental VTN expression.

Inhibition of Ferroptosis Using UAMC‐3203 in the Post‐stroke Period Does Not Impact Cognitive Outcomes in Diabetic Rats

IntroductionPost‐stroke cognitive impairment (PSCI) contributes to significant long‐term disability in stroke victims. 30% of ischemic stroke victims in the United States also have diabetes, which increases the risk of hemorrhagic transformation as well as PSCI. Ferroptosis, an iron‐induced cell death can instigate increased oxidative stress and contribute to impaired neurovascular repair leading to PSCI in diabetes. In our previous studies, we were able to identify that treating diabetic animals with an iron chelator, deferoxamine (DFX), prevents post‐stroke vasoregression and improves functional outcomes. Furthermore, DFX prevented the activation of ferroptosis in brain microvascular endothelial cells in vitro. Untreated diabetic animals experienced progressive cognitive decline while being monitored for 8 weeks. These findings led us to speculate that endothelial ferroptosis also plays a role in vasoregression and impacts cognitive outcomes post‐stroke. Therefore, this study was designed to test the hypothesis that inhibiting ferroptosis in the post‐stroke period will improve cognitive recovery in diabetic animals.MethodsAnimals were housed in reverse light cycle. 8 weeks after diabetes onset, male rats underwent 60 min middle cerebral artery occlusion (MCAO). On Day 3, after stroke injury was confirmed by MRI, animals were randomized to UAMC‐3 (2mg/kg) or vehicle treatment for 2 weeks. Sensorimotor and cognitive behavioral tests were performed during the animals’ active hours up to 8 weeks post MCAO.Results(Table 1): 60 min occlusion caused significant acute neurological deficits. There were no differences between the groups in indices measured by novel object recognition (NOR), Y‐maze and sucrose preference tests. Interestingly, step through latency in passive avoidance test (PAT) was lower in the UAMC‐3203 group.ConclusionTreatment with a ferroptosis inhibitor for 2 weeks after stroke did not impact recognition and working memory but worsened aversive learning in diabetic male rats. Unlike our previous study in which behavior tests were performed during rats’ passive hours, there was no progressive cognitive decline in untreated animals. Further evaluation of behavior testing times as well as tissue markers of neurovascular degeneration, inflammation and ferroptosis are required to determine whether molecular and cellular markers are affected by the treatment before overt changes in behavioral outcomes.

Inhalation of Atmospheric-Pressure Gas Plasma Attenuates Brain Infarction in Rats With Experimental Ischemic Stroke

Previous studies suggest the potential efficacy of neuroprotective effects of gaseous atmospheric-pressure plasma (APP) treatment on neuronal cells. However, it remains unclear if the neuroprotective properties of the gas plasmas benefit the ischemic stroke treatment, and how to use the plasmas in the in vivo ischemic stroke models. Rats were subjected to 90 min middle cerebral artery occlusion (MCAO) to establish the ischemic stroke model and then intermittently inhaled the plasma for 2 min at 60 min MCAO. The regional cerebral blood flow (CBF) was monitored. Animal behavior scoring, magnetic resonance imaging (MRI), 2,3,5-triphenyltetrazolium chloride (TTC) staining, and hematoxylin and eosin (HE) staining were performed to evaluate the therapeutic efficacy of the gas plasma inhalation on MCAO rats. Intermittent gas plasma inhalation by rats with experimental ischemic stroke could improve neurological function, increase regional CBF, and decrease brain infarction. Further MRI tests showed that the gas plasma inhalation could limit the ischemic lesion progression, which was beneficial to improve the outcomes of the MCAO rats. Post-stroke treatment with intermittent gas plasma inhalation could reduce the ischemic lesion progression and decrease cerebral infarction volume, which might provide a new promising strategy for ischemic stroke treatment.

Xenograft of Human Umbilical Mesenchymal Stem Cells Promotes Recovery from Chronic Ischemic Stroke in Rats.

Stroke is a leading cause of adult disability. In our previous study, transplantation of human umbilical mesenchymal stem cells (HUMSCs) in Wharton’s jelly in the acute phase of ischemic stroke promotes recovery in rats. Unfortunately, there is no cure for chronic stroke. Patients with chronic stroke can only be treated with rehabilitation or supportive interventions. This study aimed to investigate the potential of xenograft of HUMSCs for treating chronic stroke in rats. Rats were subjected to 90 min middle cerebral artery occlusion and then reperfusion to mimic ischemic cerebral stroke. On day 14 following stroke, HUMSCs were transplanted into the damaged cerebral cortex. The motor function in rats of the Stroke + HUMSCs group exhibited significant improvement compared to that of the Stroke + Saline group, and the trend persisted until day 56 post stroke. The cerebral cortex changes were tracked using magnetic resonance imaging, showing that cerebral atrophy was found starting on day 7 and was reduced significantly in rats receiving HUMSCs compared to that in the Stroke + Saline group from day 21 to day 56. HUMSCs were found to be existed in the rats’ cerebral cortex on day 56, with signs of migration. The grafted HUMSCs did not differentiate into neurons or astrocytes and may release cytokines to improve neuroprotection, decrease inflammation and increase angiogenesis. Our results demonstrate that xeno-transplantation of HUMSCs has therapeutic benefits for chronic ischemic stroke. Most importantly, patients do not need to use their own HUMSCs, which is a gospel thing for clinical patients.

Ischemic Preconditioning Modulates the Peripheral Innate Immune System to Promote Anti-Inflammatory and Protective Responses in Mice Subjected to Focal Cerebral Ischemia.

The development of tolerance triggered by a sublethal ischemic episode (preconditioning, PC) involves a complex crosstalk between neurons, astrocytes and microglia, although the role of the peripheral immune system in this context is largely unexplored. Here, we report that severe cerebral ischemia caused by transient middle cerebral artery occlusion (MCAo) in adult male mice elevates blood counts of inflammatory neutrophils and monocytes, and plasma levels of miRNA-329-5p. These inflammatory responses are prevented by ischemic PC induced by 15 min MCAo, 72h before the severe insult (1h MCAo). As compared with sham-operated animals, mice subjected to either ischemic PC, MCAo or a combination of both (PC+MCAo) display spleen contraction. However, protein levels of Ym1 (a marker of polarization of myeloid cells towards M2/N2 protective phenotypes) are elevated only in spleen from the experimental groups PC and PC+MCAo, but not MCAo. Conversely, Ym1 protein levels only increase in circulating leukocytes from mice subjected to 1h MCAo, but not in preconditioned animals, which is coincident with a dramatic elevation of Ym1 expression in the ipsilateral cortex. By immunofluorescence analysis, we observe that expression of Ym1 occurs in amoeboid-shaped myeloid cells, mainly representing inflammatory monocytes/macrophages and neutrophils. As a result of its immune-regulatory functions, ischemic PC prevents elevation of mRNA levels of the pro-inflammatory cytokine interleukin (IL)-1β in the ipsilateral cortex, while not affecting IL-10 mRNA increase induced by MCAo. Overall, the elevated anti-inflammatory/pro-inflammatory ratio observed in the brain of mice pre-exposed to PC is associated with reduced brain infarct volume and ischemic edema, and with amelioration of functional outcome. These findings reaffirm the crucial and dualistic role of the innate immune system in ischemic stroke pathobiology, extending these concepts to the context of ischemic tolerance and underscoring their relevance for the identification of novel therapeutic targets for effective stroke treatment.

Zinc Accumulation Aggravates Cerebral Ischemia/Reperfusion Injury Through Inducing Endoplasmic Reticulum Stress.

Zinc is highly enriched in the central nervous system. Numerous evidences suggest that high concentration of zinc acts as a critical mediator of neuronal death in the ischemic brain, however, the possible mechanisms of neurotoxicity of zinc during cerebral ischemia/reperfusion (I/R) remain elusive. Endoplasmic reticulum (ER) is a storage location of intracellular zinc. ER stress related genes were up-regulated during zinc-induced neuronal death in vascular-type senile dementia. In the present study, we investigated whether intracellular accumulated zinc aggravates I/R injury through ER stress and ER stress-associated apoptosis. Male Sprague-Dawley rats were subjected to 90min middle cerebral artery occlusion (MCAO) and received either vehicle or zinc chelator TPEN 15mg/kg. The expression of ER stress related factors glucose-regulated protein 78 (GRP78) and phosphorylated eukaryotic initiation factor 2α (p-eIF2α), ER stress related apoptotic proteins CCAAT-enhancer-binding protein homologous protein (CHOP) and caspase-12, as well as anti-apoptotic factor B-cell lymphoma-2 (Bcl-2) were assessed 24h after reperfusion. Our results showed that the levels of GRP78 and p-eIF2α, as well as CHOP and caspase-12, were increased in ischemic brain, indicating that cerebral I/R triggers ER stress. Furthermore, GRP78, CHOP and caspase-12 were all colocalized with the zinc-specific dyes NG, suggesting that there is certain relationship between cytosolic labile zinc and ER stress following cerebral ischemia. Chelating zinc with TPEN reversed the expression of GRP78, p-eIF2α in ischemic rats. Moreover, CHOP and NeuN double staining positive cells, as well as caspase-12 and TUNEL double staining positive cells were also decreased after TPEN treatment, indicating that chelating zinc might inhibit ER stress and decreased ER stress associated neuronal apoptosis. In addition, TPEN treatment reversed the downregulated level of Bcl-2, which localized in the ER membrane and involved in the dysfunction of ER, confirming that the anti-apoptosis effects of chelating zinc following I/R are exerted via inhibition of the ER stress. Taken together, this study demonstrated that excessive zinc activates ER stress and zinc induced neuronal cell death is at least partially due to ER stress specific neuronal apoptosis in ischemic penumbra, which may provide an important mechanism of cerebral I/R injury.

Abstract 86: Dicer Ablation In Oligodendrocytes Impairs Oligodendrogenesis And Functional Recovery After Stroke

Introduction: Stroke-induced oligodendrogenesis contributes to ischemic brain remodeling and spontaneous functional recovery. Genes that regulate microRNA biogenesis in stroke-induced oligodendrogenesis remain little known. Methods: We employed a transgenic mouse line with conditional and inducible ablation of Dicer, a key gene of miRNA biogenesis, in myelin proteolipid protein (PLP) lineage oligodendrocytes (Dicer-KO). A PLP reporter mouse line was used as a control. Mice were subjected to transient (60min) middle cerebral artery occlusion (MCAO, n=8/group). An array of behavioral and cognitive tests were performed. All mice were sacrificed at 28 days after MCAO. Fluorescence-activated cell sorting (FACS) in combination with miRNA sequencing were used to profile the miRNA alteration. Results: Compared to ischemic reporter mice, ischemic Dicer-KO mice exhibited significantly increased infarct volumes and significantly impaired neurological outcomes and cognitive deficits post stroke as assayed by social recognition memory, novel object recognition, and Morris water-maze tests. Immunochemistry analysis revealed that ischemic Dicer-KO mice showed a significant reduction of the number of PLP-lineage NG2 + oligodendrocyte precursor cells (OPCs) and APC + myelinating oligodendrocytes compared to ischemic reporter mice. Moreover, Dicer-KO mice exhibited a robust reduction of myelinated axons measured by myelin binding protein (MBP) and Bielschowsky/Luxol Fast Blue assay. FACS analysis of PLP-lineage cells in peri-infarct regions showed that ablation of Dicer significantly decreased many miRNAs including the miR-200 family that are known to regulate oligodendrogenesis. Conclusion: Our data indicate that Dicer in PLP lineage oligodendrocytes is required for stroke-induced oligodendrogenesis and myelination, and that Dicer is involved in spontaneous functional recovery after stroke.

Protective effect of S-allyl cysteine against cerebral ischemia/reperfusion injury in experimental rats

Purpose: To investigate the protective effect of S-allyl cysteine (SAC) against cerebral ischemiareperfusion injury (CRI) in rats.Methods: The protective effect of SAC was determined in a male Wistar rat model of middle cerebral artery occlusion (MCAO)-stimulated transient focal ischemia, followed by reperfusion. Cerebral ischemia reperfusion injury was induced via 90 min of MCAO, followed by 24-h reperfusion. The cerebral infarct size was determined by staining with 2,3,5- triphenyl tetrazolium chloride. The onset of cellular derangement, neurological deficit score and neuronal oedema were determined. In addition, the expressions of CRI markers and inflammatory cytokines were measured by enzyme-linked immunosorbent assay (ELISA).Results: Rats subjected to CRI showed marked increases in cellular oxidative stress, as evidenced by significant increase in the levels of inflammatory markers, including MDA (p &lt; 0.05), MPO (p &lt; 0.05) and nitric oxide (p &lt; 0.01). In addition, CRI increased the mRNA expression levels of the marker genes TLR4, syndecan-1, CSF, aquaporin-1, OCT3, and RFX1. In contrast, rats pre-treated with SAC prior to CRI displayed reduced levels of inflammatory cytokines, with a near-normal cellular arrangement. SAC treatment significantly reduced the mRNA expression levels of the marker genes in CRI rats.Conclusion: These findings suggest that SAC may protect the brain of rats from cerebral ischemiareperfusion injury caused by amplification of oxidative stress and inflammatory signaling. Thus, S-allyl cysteine is a potential therapy for the management of CRI.