Permanent Focal Cerebral Ischemia Research Articles

Dim light at night shifts microglia to a pro-inflammatory state after cerebral ischemia, altering stroke outcome in mice

Circadian rhythms are endogenous biological cycles that regulate physiology and behavior and are set to precisely 24-h by light exposure. Light at night (LAN) dysregulates physiology and function including immune response; a critical component that contributes to stroke pathophysiological progression of neuronal injury and may impair recovery from injury. The goal of this study is to explore the effects of dim LAN (dLAN) in a murine model of ischemic stroke to assess how nighttime lighting from hospital settings can affect stroke outcome. Further, this study sought to identify mechanisms underlying pathophysiological changes to immune response after circadian disruption. Male and female adult Swiss Webster (CFW) mice were subjected to transient or permanent focal cerebral ischemia, then were subsequently placed into either dark night conditions (LD) or one night of dLAN (5 lx). 24 h post-stroke, sensorimotor impairments and infarct sizes were quantified. A single night of dLAN following MCAO increased infarct size and sensorimotor deficits across both sexes and reduced survival in males after 24 h. Flow cytometry was performed to assess microglial phenotypes after MCAO, and revealed that dLAN altered the percentage of microglia that express pro-inflammatory markers (MHC II+ and IL-6) and microglia that express CD206 and IL-10 that likely contributed to poor ischemic outcomes. Following these results, microglia were reduced in the brain using Plexxikon 5622 (PLX 5622) a CSFR1 inhibitor, then the mice received an MCAO and were exposed to LD or dLAN conditions for 24 h. Microglial depletion by PLX5622 resulted in infarct sizes that were comparable between lighting conditions. This study provides supporting evidence that environmental lighting exacerbates ischemic injury and post-stroke mortality by a biological mechanism that exposure to dLAN causes a fundamental shift of activated microglial phenotypes from beneficial to detrimental at an early time point after stroke, resulting in irreversible neuronal death.

Palmatine, a natural alkaloid, attenuates memory deficits and neuroinflammation in mice submitted to permanent focal cerebral ischemia

Ischemic stroke is one of the major causes of human morbidity and mortality. The pathophysiology of ischemic stroke involves complex events, including oxidative stress and inflammation, that lead to neuronal loss and cognitive deficits. Palmatine (PAL) is a naturally occurring (Coptidis rhizome) isoquinoline alkaloid that belongs to the class of protoberberines and has a wide spectrum of pharmacological and biological effects. In the present study, we evaluated the impact of Palmatine on neuronal damage, memory deficits, and inflammatory response in mice submitted to permanent focal cerebral ischemia induced by middle cerebral artery (pMCAO) occlusion. The animals were treated with Palmatine (0.2, 2 and 20 mg/kg/day, orally) or vehicle (3% Tween + saline solution) 2 h after pMCAO once daily for 3 days. Cerebral ischemia was confirmed by evaluating the infarct area (TTC staining) and neurological deficit score 24 h after pMCAO. Treatment with palmatine (2 and 20 mg/kg) reduced infarct size and neurological deficits and prevented working and aversive memory deficits in ischemic mice. Palmatine, at a dose of 2 mg/kg, had a similar effect of reducing neuroinflammation 24 h after cerebral ischemia, decreasing TNF-, iNOS, COX-2, and NF- κB immunoreactivities and preventing the activation of microglia and astrocytes. Moreover, palmatine (2 mg/kg) reduced COX-2, iNOS, and IL-1β immunoreactivity 96 h after pMCAO. The neuroprotective properties of palmatine make it an excellent adjuvant treatment for strokes due to its inhibition of neuroinflammation.

Poststroke Intravenous Transplantation of Human Mesenchymal Stem Cells Improves Brain Repair Dynamics and Functional Outcomes in Aged Mice.

Stroke is the primary cause of chronic disability in the elderly, as there are no neurorestorative treatments for those who do not qualify for recanalization therapy. Experimental evidence in stroke animals suggests that transplantation of bone marrow-derived human mesenchymal stem cells (hMSCs) holds promise, but hMSC transplantation has not been systematically tested in aged animals. We tested the hypothesis that poststroke hMSC transplantation improves stroke recovery in aged mice by promoting brain repair. Permanent focal cerebral ischemia was induced in 20-month-old C57BL/6 male mice by distal middle cerebral artery occlusion. Bone marrow-derived hMSCs were expanded in vitro and then administrated intravenously into mice (1×106 cells in PBS) 24 hours after distal middle cerebral artery occlusion. Sensorimotor and cognitive functions, brain atrophy, and brain repair processes (neurogenesis, angiogenesis, oligodendrogenesis) were assessed for up to 56 days after stroke. Poststroke hMSC transplantation did not mitigate brain atrophy or improve neuronal survival at 56 days after distal middle cerebral artery occlusion. However, hMSC-treated mice displayed superior neurobehavioral performances in the open field, rotarod, adhesive removal, novel object, and Morris water maze tests compared with PBS-treated controls. hMSCs promoted white matter integrity and enhanced angiogenesis and oligodendrogenesis-but not neurogenesis-in the stroke brain. Positive correlations between neurobehavioral performance and brain repair profiles or white matter integrity were observed in stroke mice. Poststroke hMSC transplantation improves long-term stroke recovery in aged mice, likely via mechanisms involving enhanced microvascular regeneration and white matter restoration.

Research advances in the application of vagus nerve electrical stimulation in ischemic stroke.

Stroke seriously endangers human well-being and brings a severe burden to family and society. Different post-stroke dysfunctions result in an impaired ability to perform activities of daily living. Standard rehabilitative therapies may not meet the requirements for functional improvement after a stroke; thus, alternative approaches need to be proposed. Currently, vagus nerve stimulation (VNS) is clinically applied for the treatment of epilepsy, depression, cluster headache and migraine, while its treatment of various dysfunctions after an ischemic stroke is still in the clinical research stage. Recent studies have confirmed that VNS has neuroprotective effects in animal models of transient and permanent focal cerebral ischemia, and that its combination with rehabilitative training significantly improves upper limb motor dysfunction and dysphagia. In addition, vagus-related anatomical structures and neurotransmitters are closely implicated in memory-cognition enhancement processes, suggesting that VNS is promising as a potential treatment for cognitive dysfunction after an ischemic stroke. In this review, we outline the current status of the application of VNS (invasive and non-invasive) in diverse functional impairments after an ischemic stroke, followed by an in-depth discussion of the underlying mechanisms of its mediated neuroprotective effects. Finally, we summarize the current clinical implementation challenges and adverse events of VNS and put forward some suggestions for its future research direction. Research on VNS for ischemic stroke has reached a critical stage. Determining how to achieve the clinical transformation of this technology safely and effectively is important, and more animal and clinical studies are needed to clarify its therapeutic mechanism.

Fingolimod Does Not Reduce Infarction After Focal Cerebral Ischemia in Mice During Active or Inactive Circadian Phases.

It has been reported that the S1P (sphingosine 1-phosphate) receptor modulator fingolimod reduces infarction in rodent models of stroke. Recent studies have suggested that circadian rhythms affect stroke and neuroprotection. Therefore, this study revisited the use of fingolimod in mouse focal cerebral ischemia to test the hypothesis that efficacy might depend on whether experiments were performed during the inactive sleep or active wake phases of the circadian cycle. Two different stroke models were implemented in male C57Bl/6 mice-transient middle cerebral artery occlusion and permanent distal middle cerebral artery occlusion. Occlusion occurred either during inactive or active circadian phases. Mice were treated with 1 mg/kg fingolimod at 30- or 60-minute postocclusion and 1 day later for permanent and transient middle cerebral artery occlusion, respectively. Infarct volume, brain swelling, hemorrhagic transformation, and behavioral outcome were assessed at 2 or 3 days poststroke. Three independent experiments were performed in 2 different laboratories. Fingolimod decreased peripheral lymphocyte number in naive mice, as expected. However, it did not significantly affect infarct volume, brain swelling, hemorrhagic transformation, or behavioral outcome at 2 or 3 days after transient or permanent focal cerebral ischemia during inactive or active circadian phases of stroke onset. Outcomes were not improved by fingolimod in either transient or permanent focal cerebral ischemia during both active and inactive circadian phases. These negative findings suggest that further testing of fingolimod in clinical trials may not be warranted unless translational studies can identify factors associated with fingolimod's efficacy or lack thereof.

Effects of Long-Term Vagus Nerve Electrical Stimulation Therapy on Acute Cerebral Infarction and Neurological Function Recovery in Post MCAO Mice.

Background Vagus nerve stimulation therapy is proven to produce neuroprotective effects against central nervous system diseases and reduce neurological injury, having a positive effect on the recovery of neurological functions in mouse model of stroke. Objective This study was aimed at exploring a wider time window for VNS treatment, investigating the long-term behavioral improvement of long-term VNS in mice after pMCAO, and exploring the antiapoptotic properties of VNS and its role in autophagy, all of which may be a permanent deficiency potential mechanism of neuroprotection in hemorrhagic stroke. Methods Permanent focal cerebral ischemia and implantation of vagus nerve stimulator were performed through intracavitary occlusion of the right middle cerebral artery (MCA). The vagus nerve stimulation group received five times vagus nerve stimulation from 6 h after surgery for 5 days. Adhesive removal test and NSS neurological score were used to evaluate the neurological deficit of mice. TTC staining of mouse brain tissue was performed one week after surgery in order to assess the area of cerebral infarction. Additionally, frozen sections were stained with Fluoro-Jade B to observe the apoptotic cells in the ischemic penumbra of brain tissue. Finally, Western blot was used to detect the changes in the levels of apoptosis-related proteins such as cleaved-caspase3 and Bcl-2 and autophagy-related proteins such as mTOR, Beclin-1, and LC3-II in brain tissue. Results VNS can effectively reduce the behavioral score of pMCAO mice; TTC results showed that VNS could effectively reduce the infarct area after pMCAO (P < 0.05). After VNS intervention of the pMCAO group compared with the pMCAO+VNI group, the FJB-positive cells in the VNS group were significantly decreased (P < 0.05); Western Blot analysis showed that the expression of cleaved-caspase3 in the brain tissue of mice increased after pMCAO (P < 0.05), and the expression of Bcl-2 decreased (P < 0.05). This change could be effectively reversed after VNS intervention (P < 0.05). Conclusion VNS could effectively improve the behavioral performance of mice after permanent stroke in addition to significantly reducing the infarct size of the brain tissue. The mechanism may be related to the effective reduction of cell apoptosis and excessive autophagy after pMCAO by VNS.

MiR-330 regulates Drp-1 mediated mitophagy by targeting PGAM5 in a rat model of permanent focal cerebral ischemia

Growing evidence have suggested that mitophagy could exert a neuroprotective role in brain ischemia by removing the damaged mitochondria. However the upstream mechanisms of mitophagy are remain unclear. We previously observed a decrease of miR-330 in a miRNA profile of plasma from patients within 3 h after a stroke. Our study further focused on the role and mechanism of miR-330 in mitophagy induced by hypoxia-ischemia (H/I) in rats. Cerebral ischemia model in rats was made with permanent middle cerebral artery occlusion (pMCAO). In vitro, ischemic model in primary neurons was established with oxygen-glucose deprivation. Various methods, including TTC staining, immunofluorescence staining, Western blot, ELISA, flow cytometry, and transmission electron microscopy were used to clarify the role of miR-330 after H/I, and whether miR-330/phosphoglycerate mutase family member 5 (PGAM5) axis could regulate dynamin-related protein 1 (Drp-1) mediated mitophagy. MiR-330 levels decreased both in rat plasma and in ipsilateral brain tissues after H/I. Pretreating animals with miR-330 antagomir could decrease cerebral infarction, edema, mortality, and apoptosis after 6-h pMCAO. PGAM5 was validated as a target of miR-330. MiR-330 agomir and antagomir transfection respectively decreased and increased the PGAM5 protein expression. MiR-330 could down-regulate mitophagy by inhibiting PGAM5-induced Drp1 dephosphorylation, thus reducing the recruitment of Drp1 to mitochondrial outer membrane and Drp1-mediated mitophagy after H/I. Our results suggest a role of miR-330 in regulating mitophagy. Our study suggested a novel miR-based intervention strategy for stroke.

RNA sequencing reveals novel macrophage transcriptome favoring neurovascular plasticity after ischemic stroke

Blood monocytes/macrophages infiltrate the brain after ischemic stroke and critically influence brain injury and regeneration. We investigated stroke-induced transcriptomic changes of monocytes/macrophages by RNA sequencing profiling, using a mouse model of permanent focal cerebral ischemia. Compared to non-ischemic conditions, brain ischemia induced only moderate genomic changes in blood monocytes, but triggered robust genomic reprogramming in monocytes/macrophages invading the brain. Surprisingly, functional enrichment analysis of the transcriptome of brain macrophages revealed significant overrepresentation of biological processes linked to neurovascular remodeling, such as angiogenesis and axonal regeneration, as early as five days after stroke, suggesting a previously underappreciated role for macrophages in initiating post-stroke brain repair. Upstream Regulator analysis predicted peroxisome proliferator-activated receptor gamma (PPARγ) as a master regulator driving the transcriptional reprogramming in post-stroke brain macrophages. Importantly, myeloid cell-specific PPARγ knockout (mKO) mice demonstrated lower post-stroke angiogenesis and neurogenesis than wild-type mice, which correlated significantly with the exacerbation of post-stroke neurological deficits in mKO mice. Collectively, our findings reveal a novel repair-enhancing transcriptome in brain macrophages during post-stroke neurovascular remodeling. As a master switch controlling genomic reprogramming, PPARγ is a rational therapeutic target for promoting and maintaining beneficial macrophage functions, facilitating neurorestoration, and improving long-term functional recovery after ischemic stroke.

Macrophages reprogram after ischemic stroke and promote efferocytosis and inflammation resolution in the mouse brain.

AimsBlood‐borne monocytes/macrophages infiltrate the brain in massive numbers after ischemic stroke, but their impact on poststroke brain injury and recovery remains elusive. This study examined the transcriptomic changes in monocytes/macrophages after ischemic stroke and the functional implications of these changes, particularly with regards to the contribution of these cells to the phagocytic clearance of dead/dying cells (efferocytosis) in the poststroke brain.MethodsWe performed whole‐genome RNA sequencing on the monocyte/macrophage population sorted from mouse brain and peripheral blood 5 days after permanent focal cerebral ischemia. In addition, the spatial and temporal profiles of macrophage efferocytosis were examined in vivo by immunohistochemistry 3‐7 days after brain ischemia.ResultsRobust transcriptomic changes occurred in monocytes/macrophages upon infiltrating the poststroke brain. Functional enrichment analysis revealed a transcriptome of brain macrophages that strongly favored efferocytic activity. A large number of efferocytosis‐related genes were upregulated in brain macrophages, the products of which are essential components involved in various steps of efferocytosis, such as chemotaxis, recognition of dead cells, engulfment, and processing of phagosomes. The efferocytic activity of brain macrophages were verified by immunohistochemistry, wherein Iba1‐labeled microglia/macrophages effectively cleared apoptotic neurons in the infarct during the subacute stage after brain ischemia. We also identified PPARγ and STAT6 as potential upstream regulators that shaped this proefferocytic and inflammation‐resolving transcriptome of macrophages in the poststroke brain.ConclusionMacrophages play a crucial role in the phagocytic clearance of dead neurons after ischemic stroke and promote the resolution of inflammation in the brain. Molecular therapies that enhance macrophage efferocytic capability may be promising treatments for ischemic stroke by facilitating inflammation resolution, brain repair, and recovery of neurological functions.

JLX001 Ameliorates Ischemia/Reperfusion Injury by Reducing Neuronal Apoptosis via Down-Regulating JNK Signaling Pathway

JLX001, a novel compound with similar structure with cyclovirobuxine D (CVB-D), has been proved to exert therapeutical effects on permanent focal cerebral ischemia. However, the protective effects of JLX001 on cerebral ischemia/reperfusion (I/R) injury and its anti-apoptotic effects have not been reported. We investigated the efficacy of JLX001 in two pharmacodynamic tests (pre-treatment test and post-treatment) with rats subjected to middle cerebral artery occlusion/reperfusion (MCAO/R). The pharmacodynamic tests demonstrated that JLX001 ameliorated I/R injury by reducing infarct sizes and brain edema. The results of Morris water maze, neurological scores, cylinder test and posture reflex test implied that JLX001 improved the learning, memory and motor ability after MCAO/R in the long term. Anti-apoptotic effects of JLX001 and its regulation of cytosolic c-Jun N-terminal Kinases (JNKs) signal pathway were confirmed in vivo by co-immunofluorescence staining and western immunoblotting. Furthermore, primary cortical neuron cultures were prepared and exposed to oxygen glucose deprivation/reoxygenation (OGD/R) for in vitro studies. Cytotoxicity test and mitochondrial membrane potential (MMP) test showed that JLX001 enhanced cell survival rate and maintained MMP. Flow cytometry and TdT-mediated dUTP-X nick end labeling (TUNEL) staining demonstrated the anti-apoptotic effects of JLX001 in vitro. Likewise, JLX001 regulated JNK signal pathway in vivo, which was also confirmed by western immunoblotting. Collectively, this study presents the first evidence that JLX001 exerted protective effects against I/R injury by reducing neuronal apoptosis via down-regulating JNK signaling pathway.

Argon Inhalation for 24 Hours After Onset of Permanent Focal Cerebral Ischemia in Rats Provides Neuroprotection and Improves Neurologic Outcome.

We tested the hypothesis that prolonged inhalation of 70% argon for 24 hours after in vivo permanent or temporary stroke provides neuroprotection and improves neurologic outcome and overall recovery after 7 days. Controlled, randomized, double-blinded laboratory study. Animal research laboratories. Adult Wistar male rats (n = 110). Rats were subjected to permanent or temporary focal cerebral ischemia via middle cerebral artery occlusion, followed by inhalation of 70% argon or nitrogen in 30% oxygen for 24 hours. On postoperative day 7, a 48-point neuroscore and histologic lesion size were assessed. After argon inhalation for 24 hours immediately following "severe permanent ischemia" induction, neurologic outcome (neuroscore, p = 0.034), overall recovery (body weight, p = 0.02), and infarct volume (total infarct volume, p = 0.0001; cortical infarct volume, p = 0.0003; subcortical infarct volume, p = 0.0001) were significantly improved. When 24-hour argon treatment was delayed for 2 hours after permanent stroke induction or until after postischemic reperfusion treatment, neurologic outcomes remained significantly improved (neuroscore, p = 0.043 and p = 0.014, respectively), as was overall recovery (body weight, p = 0.015), compared with nitrogen treatment. However, infarct volume and 7-day mortality were not significantly reduced when argon treatment was delayed. Neurologic outcome (neuroscore), overall recovery (body weight), and infarct volumes were significantly improved after 24-hour inhalation of 70% argon administered immediately after severe permanent stroke induction. Neurologic outcome and overall recovery were also significantly improved even when argon treatment was delayed for 2 hours or until after reperfusion.

Neuroprotective effects of oleic acid in rodent models of cerebral ischaemia

Oleic acid (OA) is released from brain phospholipids after cerebral ischaemia; however, its role in ischaemic injury remains unknown. We hypothesised that OA has neuroprotective effects after cerebral ischaemia, which may be exerted through peroxisome proliferator-activated receptor gamma (PPAR-γ) activation, since OA is an endogenous ligand of PPAR-γ. The effects of OA administration were evaluated in rodent models of middle cerebral artery occlusion (MCAO), photothrombosis, and four-vessel occlusion (4-VO). We determined the time window of therapeutic opportunity and examined the ability of the PPAR-γ antagonist GW9662 to reverse OA’s protective effects after MCAO. We found that OA administration decreased the MCAO-induced infarct volume and functional deficits, photothrombosis-induced infarct volume, and 4-VO-induced hippocampal neuronal death. Additionally, OA was highly efficacious when administered up to 3 h after MCAO. Pre-treatment with GW9662 abolished the inhibitory effects of OA on the infarct volume and immunoreactivity of key inflammatory mediators in the ischaemic cortex. Our results indicate that OA has neuroprotective effects against transient and permanent focal cerebral ischaemia, as well as global cerebral ischaemia. It may have therapeutic value for the ischaemic stroke treatment with a clinically feasible therapeutic window. The OA-mediated neuroprotection might be attributable to its anti-inflammatory actions through PPAR-γ activation.

Protease-independent action of tissue plasminogen activator in brain plasticity and neurological recovery after ischemic stroke

Emerging evidence suggests that tissue plasminogen activator (tPA), currently the only FDA-approved medication for ischemic stroke, exerts important biological actions on the CNS besides its well-known thrombolytic effect. In this study, we investigated the role of tPA on primary neurons in culture and on brain recovery and plasticity after ischemic stroke in mice. Treatment with recombinant tPA stimulated axonal growth in culture, an effect independent of its protease activity and achieved through epidermal growth factor receptor (EGFR) signaling. After permanent focal cerebral ischemia, tPA knockout mice developed more severe sensorimotor and cognitive deficits and greater axonal and myelin injury than wild-type mice, suggesting that endogenously expressed tPA promotes long-term neurological recovery after stroke. In tPA knockout mice, intranasal administration of recombinant tPA protein 6 hours poststroke and 7 more times at 2 d intervals mitigated white matter injury, improved axonal conduction, and enhanced neurological recovery. Consistent with the proaxonal growth effects observed in vitro, exogenous tPA delivery increased poststroke axonal sprouting of corticobulbar and corticospinal tracts, which might have contributed to restoration of neurological functions. Notably, recombinant mutant tPA-S478A lacking protease activity (but retaining the EGF-like domain) was as effective as wild-type tPA in rescuing neurological functions in tPA knockout stroke mice. These findings demonstrate that tPA improves long-term functional outcomes in a clinically relevant stroke model, likely by promoting brain plasticity through EGFR signaling. Therefore, treatment with the protease-dead recombinant tPA-S478A holds particular promise as a neurorestorative therapy, as the risk for triggering intracranial hemorrhage is eliminated and tPA-S478A can be delivered intranasally hours after stroke.

Neuroprotective effects of targeting BET proteins for degradation with dBET1 in aged mice subjected to ischemic stroke

Neuroinflammation after stroke significantly contributes to neuronal cell death. Bromodomain and Extra Terminal Domain (BET) proteins are essential to inflammatory gene transcription. BET proteins (BRD2, BRD3, BRD4, and BRDT) have varied effects including chromatin remodeling, histone acetyltransferase activity, and as scaffolds to recruit transcription factors; they couple chromatin remodeling with transcription. BRD2/4 are of particularly interest to stroke-induced neuroinflammation that contributes to delayed cell death as they are required for NF-κB-dependent gene transcription. We hypothesized that targeting BET proteins for degradation with dBET1, a proteolysis targeting chimera (PROTAC) that combines the highly selective BET inhibitor JQ1 and a ligand for cereblon E3 ubiquitin ligase, will reduce brain injury in ischemic stroke. Male aged mice (18–20 months old) were subjected to permanent occlusion of the middle cerebral artery and received either vehicle or dBET1 (10 mg/kg; i.p.) at various times after stroke. Neurobehavioral tests were performed before (baseline) and at 24 and 48 h after stroke induction. Infarct volume was quantified at 48 h. Data showed that BET degradation significantly reduced infarct volume in permanent focal cerebral ischemia in aged mice, and this was associated with reduced brain levels of pro-inflammatory mediators including TNF-α, CXCL1, CXCL10, CCL2, and matrix metalloproteinase-9. Treatment with dBET1 significantly reduced blood-brain barrier damage and infiltration of neutrophils into the ischemic brain. Importantly, treatment with the BET degrader dBET1 resulted in a significant improvement in stroke-induced neurological deficits. Collectively, these data indicate that BET proteins are a novel target for neuroprotection in ischemic stroke.

Increased Peripheral CD137 Expression in a Mouse Model of Permanent Focal Cerebral Ischemia.

Various studies demonstrate that CD137 (TNFRSF9, 4-1BB) promotes atherosclerosis and vascular inflammation in experimental models via interactions with the CD137 ligand (CD137L). However, the exact role of CD137 in ischemic stroke remains unclear. In this study, we analyzed dynamic changes of peripheral CD137 expression on T cells in a mouse model of cerebral ischemia-middle cerebral artery occlusion (MCAO), as well as alternation of neurological function, infarct size and cerebral inflammatory status after inhibition of the CD137/CD137L pathway using an anti-CD137L monoclonal antibody. MCAO mice showed elevated surface expression of CD137 on T cells in both peripheral blood and lymphoid tissues during early cerebral ischemia. Remarkably, blockade of the CD137/CD137L pathway reduced the post-ischemic brain damage. Our findings indicate that enhanced CD137 costimulation occurs in early cerebral ischemia and promotes T cell activation, which in turn upregulates inflammatory immune response and possibly exerting deleterious effects on cerebral ischemia.

Role of rno-miR-124-3p in regulating MCT1 expression in rat brain after permanent focal cerebral ischemia

This study aimed to assess the role of microRNAs (miRNAs) in regulating monocarboxylate transporter-1 (MCT1) expression in rat brain after permanent focal cerebral ischemia to identify a new target for early treatment of cerebral ischemia. Focal cerebral ischemia was induced by permanent middle cerebral artery occlusion (pMCAO) in rats. Morphology and protein expression levels of MCT1 were assessed by immunofluorescence and Western blotting. Using bioinformatics and double luciferase reporter assays, rno-miR-124-3p was selected as a direct target for rat MCT1. Expression of rno-miR-124-3p after pMCAO was detected. Then, rats were treated with rno-miR-124-3p agomir via lateral ventricle injection, and after 6 h or 24 h ischemia, rno-miR-124-3p expression and gene and protein expression of MCT-1 were detected by qRT-PCR and Western blotting. Brain infarction was identified by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. Results showed that pMCAO induced brain infarction and increased the expression of MCT1. The levels of rno-miR-124-3p after pMCAO were in contrast to those of MCT1 protein in ischemic region, while declined after 3, 6 and 12 h of pMCAO in ischemic penumbra. After administration of rno-miR-124-3p agomir, MCT1 mRNA and protein levels were increased after 6 h of pMCAO, while decreased after 24 h of pMCAO. Meanwhile, rno-miR-124-3p levels increased after both times. TTC staining showed treatment with rno-miR-124-3p agomir reduced brain infarction. The role of rno-miR-124-3p in regulating MCT1 was as a positive regulator after 6 h of pMCAO, while a negative regulator after 24 h of pMCAO, however, both activities had protective effects against cerebral ischemia.

Post-stroke gastrodin treatment ameliorates ischemic injury and increases neurogenesis and restores the Wnt/β-Catenin signaling in focal cerebral ischemia in mice

Cerebral ischemic stroke is one of the leading causes of death and disability worldwide, and the only available drug treatment is limited to a short window following the ischemic event. Gastrodin is the major bioactive constituent extracted from thetuberGastrodia elata, and is currently used to treat dizziness in the clinic. “Early” application of gastrodin (before modeling or immediately after ischemic injury) has shown antioxidative and neuroprotective effects in a transient focal brain ischemia model in rodents; however, it is not known whether the delayed administration of gastrodin after permanent focal cerebral ischemia ameliorates neural injury and increases neurogenesis. In this study, we performed a permanent middle cerebral artery occlusion (MCAO) model for the study of cerebral ischemic stroke in adult male mice to examine the effects of gastrodin. Gastrodin treatment that was started “late” (one day after the ischemic injury) significantly improved neural function, reduced infarct volume and apoptosis, and increased the number of DCX/BrdU double-positive cells in permanent MCAO mice. Moreover, gastrodin treatment markedly preserved the Wnt/β-Catenin signaling pathway, which could promote neurogenesis and provide neuroprotection brain injury. Our findings suggest that gastrodin treatment following ischemic injury can induce neuroprotection, promote neurogenesis and restored the Wnt /β-Catenin signaling pathway.

Ferulic Acid Exerts Anti-apoptotic Effects against Ischemic Injury by Activating HSP70/Bcl-2- and HSP70/Autophagy-Mediated Signaling after Permanent Focal Cerebral Ischemia in Rats.

This study assessed the anti-apoptotic effects of the administration of ferulic acid (FrA) in rats 30 min before middle cerebral artery occlusion (MCAo) followed by 3 d of ischemia and the involvement of 70 kDa heat shock protein (HSP70)-mediated signaling in the penumbral cortex. Our results demonstrated that FrA pretreatment at doses of 80 mg/kg (FrA-80 mg) and 100 mg/kg (FrA-100 mg) effectively ameliorated neurological functions and reduced the numbers of cytochrome c-, cleaved caspase-3-, and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells in the penumbral cortex 3 d after ischemia. Moreover, FrA-80 mg and FrA-100 mg pretreatment markedly upregulated cytosolic HSP70, Beclin-1, microtubule-associated protein 1 light chain 3 (LC3) A/B-II and autophagy-related protein 5 (Atg5) expression; cytosolic and mitochondrial X-linked inhibitor of apoptosis (XIAP) expression and the Bcl-2/Bax ratio. FrA pretreatment downregulated cytosolic cytochrome c, apoptosis-inducing factor (AIF), procathepsin B, and cathepsin B expression and mitochondrial and cytosolic second mitochondria-derived activator of caspase/direct inhibitor of apoptosis protein-binding protein with a low isoelectric point (Smac/DIABLO) expression in the penumbral cortex. Pretreatment with VER155008, a HSP70 family inhibitor, significantly inhibited the effects of FrA-100 mg on the expression of the aforementioned proteins expression in the penumbral cortex. FrA-80 mg and FrA-100 mg pretreatment exerts neuroprotective effects against caspase-dependent and -independent apoptosis through activating HSP70/Bcl-2- and HSP70/autophagy-induced signaling pathways. Furthermore, the HSP70/Bcl-2- and HSP70/autophagy-induced anti-apoptotic effects of FrA pretreatment can be attributed to the regulation of Bax/cytochrome c/Smac/DIABLO/XIAP/ caspase-3- (or Bax/AIF-) and Beclin-1/LC3A/B-II/Atg5-mediated signaling, respectively, in the penumbral cortex 3 d after permanent MCAo.

Behavioral tests that reveal long-term deficits after permanent focal cerebral ischemia in mouse

Efforts are still needed regarding the research of therapeutics for ischemic stroke. While in experimental studies the protective effect of pharmacological agents is often highlighted by a reduction of the lesion size evaluated in the short term (days), in clinical studies a functional recovery of patients suffering from stroke is expected on the long-term (months and years). Long-term functional preclinical studies are highly recommended to evaluate potential neuroprotective agents for stroke, rather than an assessment of the infarction size at a short time point. The present study thus aimed to select among various behavioral tests those able to highlight long-term deficits (3 months) after cerebral ischemia in mice.Permanent focal cerebral ischemia was carried out in male Swiss mice by intraluminal occlusion of the left middle cerebral artery (MCA). Fourteen behavioral tests were assessed from 7 days to 90 days after ischemia (locomotor activity, neurological score, exit circle test, grip and string tests, chimney test, adhesive removal test, pole test, beam-walking tests, elevated plus maze, marble burying test, forced swimming test, novel object recognition test).The present study clearly identified a battery of behavioral tests able to highlight deficits up to 3 months in our mouse model of permanent MCA occlusion (locomotor activity, neurological score, adhesive removal test, pole test, beam-walking tests, elevated plus maze, marble burying test, forced swimming test and novel object recognition test).This battery of behavioral tests highlighting long-term deficits is useful to study future neuroprotective strategies for stroke treatment.

(-)-α-bisabolol prevents neuronal damage and memory deficits through reduction of proinflammatory markers induced by permanent focal cerebral ischemia in mice

The pathophysiology of ischemic stroke involves multiple events such as inflammation and oxidative stress which will lead to neuronal death and cognitive deficits. The (-)-α-bisabolol is a monocyclic sesquiterpene alcohol found in various plants and mainly in Matricaria chamomilla, which exerts antioxidant, anti-inflammatory, and anti-apoptotic activities. The aim of this work was to investigate the neuroprotective effects of (-)-α-bisabolol in mice underwent permanent occlusion of the middle cerebral artery (pMCAO). Animals were treated with (-)-α-bisabolol (50, 100 and 200 mg/kg/day, orally) or vehicle (3% tween 80) one day before and 1 h after pMCAO and the treatment continued once daily for the following five days. The treatment with (-)-α-bisabolol (100 and 200 mg/kg) significantly reduced the infarcted area and neurological deficits caused by pMCAO. (-)-α-bisabolol at the 200 mg/kg dose increased cell viability and decreased neuronal degeneration, as evaluated by cresyl violet and Fluoro-Jade C stainings, respectively. (-)-α-bisabolol also increased the locomotor activity which was reduced by cerebral ischemia and improved pMCAO-induced working, spatial, object recognition, and aversive memories deficits. (-)-α-bisabolol (200 mg/kg) significantly prevented the increase of myeloperoxidase (MPO) activity, TNF-α immunoreactivity in the temporal cortex, and the increase of iNOS both in the temporal cortex and in the striatum. (-)-α-bisabolol treatment also prevented astrogliosis in these areas. These data showed that (-)-α-bisabolol provides neuroprotective action probably due to its anti-inflammatory activity, although other mechanisms cannot be discarded.