Rat Model Of Ischemic Stroke Research Articles

Ginkgo biloba extract (EGb-761) confers neuroprotection against ischemic stroke by augmenting autophagic/lysosomal signaling pathway

Ginkgo biloba extract (EGb-761) is well-recognized to have neuroprotective properties. Meanwhile, autophagy machinery is extensively involved in the pathophysiological processes of ischemic stroke. The EGb-761 is widely used in the clinical treatment of stroke patients. However, its neuroprotective mechanisms against ischemic stroke are still not fully understood. The present study was conducted to uncover whether the pharmacological effects of EGb-761 can be executed by modulation of the autophagic/lysosomal signaling axis. A Sprague-Dawley rat model of ischemic stroke was established by middle cerebral artery occlusion (MCAO) for 90 min, followed by reperfusion. The EGb-761 was then administered to the MCAO rats once daily for a total of 7 days. Thereafter, the penumbral tissues were acquired to detect proteins involved in the autophagic/lysosomal pathway including Beclin1, LC-3, SQSTM1/p62, ubiquitin, cathepsin B, and cathepsin D by western blot and immunofluorescence, respectively. Subsequently, the therapeutic outcomes were evaluated by measuring the infarct volume, neurological deficits, and neuron survival. The results showed that the autophagic activities of Beclin1 and LC3-II in neurons were markedly promoted by 7 days of EGb-761 therapy. Meanwhile, the autophagic cargoes of insoluble p62 and ubiquitinated proteins were effectively degraded by EGb-761-augmented lysosomal activity of cathepsin B and cathepsin D. Moreover, the infarction size, neurological deficiencies, and neuron death were also substantially attenuated by EGb-761 therapy. Taken together, our study suggests that EGb-761 exerts a neuroprotective effect against ischemic stroke by promoting autophagic/lysosomal signaling in neurons at the penumbra. Thus, it might be a new therapeutic target for treating ischemic stroke.

Downregulation of Histone H4 Lysine 16 Acetylation Ameliorates Autophagic Flux by Resuming Lysosomal Functions in Ischemic Neurons.

Autophagic/lysosomal dysfunction was a critical pathogenesis of neuronal death after an ischemic stroke, but what drove the impairment of autophagic flux remained elusive. Studies indicated that histone H4 lysine 16 acetylation (H4K16ac) drastically modulated the autophagic/lysosomal signaling pathway. Herein, we investigated whether the autophagic/lysosomal dysfunction in neurons could be restored by altering H4K16ac levels after cerebral ischemia. The rat model of ischemic stroke and the cell ischemia model in HT22 neurons were prepared by middle cerebral artery occlusion (MCAO) and oxygen-glucose deprivation (OGD), respectively. The result showed that H4K16ac could be effectively reduced by intracerebroventricular administration with MG149 (a H4K16ac inhibitor) after an ischemic stroke. Moreover, attenuated H4K16ac greatly alleviated the autophagic/lysosomal dysfunction in penumbral neurons, as indicated by decreased autophagic substrates of LC3-II, insoluble SQSTM1, and ubiquitinated proteins, accompanied by increased lysosomal cathepsin D. Conversely, treatment with trichostatin A (TSA, a H4K16ac facilitator) aggravated the impairment of autophagic flux. This regulative machinery of H4K16ac on the autophagic/lysosomal signaling pathway was also manifested in the OGD model of HT22 neurons. Furthermore, H4K16ac attenuation-ameliorated autophagic flux significantly alleviated stroke brain injury, as reflected by decreased infarct size, neuron loss, and neurological deficits. Similarly, the H4K16ac inhibition-mitigated autophagic/lysosomal dysfunction markedly promoted neuron survival and cell viability in OGD HT22 neurons. However, H4K16ac downregulation-ameliorated autophagic flux in neurons and thereby induced neuroprotection could be greatly counteracted by the lysosomal inhibitor bafilomycin A1 (Baf-A1). Our data indicate that cerebral ischemia-elevated H4K16ac creates the autophagic/lysosomal dysfunction due to lysosomal inefficiency, suggesting that H4K16ac attenuation benefits poststroke neuroprotection by resuming lysosomal functions in neurons.

Post-stroke Depression is Alleviated by Apelin-13 via Regulating BDNF Mediated Phosphorylation of Forkhead box O 3A Protein in Rat Model of Ischemic Stroke (P3-5.004)

<h3>Objective:</h3> To explore the role of Apelin-13 in alleviating post-stroke depression via regulation of BDNF mediated phosphorylation of forkhead box O 3A protein (FOXO3A) in rodent model of ischemic stroke. <h3>Background:</h3> Post-stroke depression (PSD) is a common complication in patients. Studies suggest that patients developing depression is associated with low-level serum brain derived neurotrophic factor (BDNF) that may increase the susceptibility to develop PSD. Treatment of PSD utilizes conventional drugs that are used to treat non stroke related mood disorders. However, limited success of these drugs have led researchers to investigate novel therapeutic strategies that can treat PSD. Apelin-13 is an adipocytokine that has been reported to exhibit beneficial effects in stroke as well as depression. Therefore, the present study aims to explore the role of Apelin-13 in alleviating PSD via regulation of BDNF mediated phosphorylation of forkhead box O 3A protein (FOXO3A) in rodent model of ischemic stroke. <h3>Design/Methods:</h3> Apelin-13 was administered to male SD rats post stroke and acute depression was induced. Rats were evaluated for functional and motor outcome at 24 h of reperfusion. Brains were harvested for infarct size estimation, biochemical assays, comparative histology and molecular studies. <h3>Results:</h3> Apelin-13 rendered neuroprotection in PSD rats and alleviated depressive like behavior. It reduced infarct size and improved functional, and behavioral outcome. Normalization of biochemical parameters were achieved in Apelin-13 treated group. Reduction of FOXO3A phosphorylation with relation to BDNF expression was evident by relevant protein expression studies. <h3>Conclusions:</h3> This study provides a preliminary evidence of the role of Apelin-13 in ameliorating PSD which can further provide a rationale to explore it as a treatment approach. <b>Disclosure:</b> Aishika Datta has nothing to disclose. Miss Sarmah has nothing to disclose. Mr. Sahu has received research support from National Institute Of Pharmaceutical Education and Research, Ahmedabad. Ms. Ghosh has received research support from National Institute of Pharmaceutical Education and Research, Ahmedabad. Ms. Rana has received research support from NIPER-Ahmedabad. Dr. Bhattacharya has nothing to disclose.

Treatment with rhFGF18 Analog Results in Cerebroprotection and Recovery of Motor and Memory Function in a Rat Model of Acute Ischemic Stroke (P3-5.005)

Treatment with rhFGF18 Analog Results in Cerebroprotection and Recovery of Motor and Memory Function in a Rat Model of Acute Ischemic Stroke (P3-5.005)

Cyclo (MQCNS) has the potential to treat ischemic stroke.

We previously found that monocyte locomotion inhibitory factor has a neuroprotective effect on ischemic brain injury during the acute phase of stroke. Therefore, we modified the structure of an anti-inflammatory monocyte locomotion inhibitory factor peptide to construct an active cyclic peptide-Cyclo (MQCNS) (LZ-3)-and investigated its effects on ischemic stroke. In this study, we established a rat model of ischemic stroke by occluding the middle cerebral artery and then administered LZ-3 (2 or 4 mg/kg) via the tail vein for 7 consecutive days. Our results showed that LZ-3 (2 or 4 mg/kg) substantially decreased infarct volume, reduced cortical nerve cell death, improved neurological function, reduced cortical and hippocampal injury, and decreased the levels of inflammatory factors in the blood and brain tissues. In a well-differentiated, oxygen-glucose deprivation/reoxygenation-induced BV2 cell model of post-stroke, LZ-3 (100 μM) inhibited the JAK1-STAT6 signaling pathway. LZ-3 regulated microglia/macrophage polarization from the M1 to the M2 type and inhibited microglia/macrophage phagocytosis and migration via the JAK1/STAT6 signaling pathway. To conclude, LZ-3 regulates microglial activation by inhibiting the JAK1/STAT6 signaling pathway and improves functional recovery post-stroke.

Evaluation of therapeutic effects of tetramethylpyrazine nitrone in Alzheimer's disease mouse model and proteomics analysis.

The pathophysiology of Alzheimer's disease (AD) is multifactorial with characteristic extracellular accumulation of amyloid-beta (Aβ) and intraneuronal aggregation of hyperphosphorylated tau in the brain. Development of disease-modifying treatment for AD has been challenging. Recent studies suggest that deleterious alterations in neurovascular cells happens in parallel with Aβ accumulation, inducing tau pathology and necroptosis. Therefore, therapies targeting cellular Aβ and tau pathologies may provide a more effective strategy of disease intervention. Tetramethylpyrazine nitrone (TBN) is a nitrone derivative of tetramethylpyrazine, an active ingredient from Ligusticum wallichii Franchat (Chuanxiong). We previously showed that TBN is a potent scavenger of free radicals with multi-targeted neuroprotective effects in rat and monkey models of ischemic stroke. The present study aimed to investigate the anti-AD properties of TBN. We employed AD-related cellular model (N2a/APPswe) and transgenic mouse model (3×Tg-AD mouse) for mechanistic and behavioral studies. Our results showed that TBN markedly improved cognitive functions and reduced Aβ and hyperphosphorylated tau levels in mouse model. Further investigation of the underlying mechanisms revealed that TBN promoted non-amyloidogenic processing pathway of amyloid precursor protein (APP) in N2a/APPswe in vitro. Moreover, TBN preserved synapses from dendritic spine loss and upregulated synaptic protein expressions in 3×Tg-AD mice. Proteomic analysis of 3×Tg-AD mouse hippocampal and cortical tissues showed that TBN induced neuroprotective effects through modulating mitophagy, MAPK and mTOR pathways. In particular, TBN significantly upregulated PINK1, a key protein for mitochondrial homeostasis, implicating PINK1 as a potential therapeutic target for AD. In summary, TBN improved cognitive functions in AD-related mouse model, inhibited Aβ production and tau hyperphosphorylation, and rescued synaptic loss and neuronal damage. Multiple mechanisms underlie the anti-AD effects of TBN including the modulation of APP processing, mTOR signaling and PINK1-related mitophagy.

Apolipoprotein E mimetic peptide COG1410 alleviates blood‑brain barrier injury in a rat model of ischemic stroke.

Blood‑brain barrier (BBB) damage is one of the main causes of poor outcomes and increased mortality rates following cerebral ischemia‑reperfusion injury. ApolipoproteinE (ApoE) and its mimetic peptide have been previously reported to exhibit potent neuroprotective properties in various central nervous system disease models. Therefore, the present study aimed to investigate the possible role of the ApoE mimetic peptide COG1410 in cerebral ischemia‑reperfusion injury and its potential underlying mechanism. Male SD rats were subjected to 2h middle cerebral artery occlusion followed by 22h reperfusion. Evans blue leakage and IgG extravasation assays results revealed that COG1410 treatment significantly reduced BBB permeability. In addition, insitu zymography and western blotting were used to prove that COG1410 was able to downregulate the activities of MMPs and upregulate the expression of occludin in the ischemic brain tissue samples. Subsequently, COG1410 was found to significantly reverse microglia activation while also suppressing inflammatory cytokine production, according to immunofluorescence signal of Iba‑1 and CD68 and protein expression of COX‑2. Consequently, this neuroprotective mechanism mediated by COG1410 was further tested using the BV2 cell line invitro, which was exposed to oxygen glucose deprivation followed by reoxygenation. The mechanism of COG1410 was found to be mediated, as least partly, through the activation of triggering receptor expressed on myeloid cells 2. In conclusion, the data suggest that COG1410 can alleviate BBB injury and neuroinflammation following ischemic stroke.

Enriched environment promotes post-stroke angiogenesis through astrocytic interleukin-17A.

Our previous studies have revealed that the protective effect of an enriched environment (EE) may be linked with astrocyte proliferation and angiogenesis. However, the relationship between astrocytes and angiogenesis under EE conditions still requires further study. The current research examined the neuroprotective effects of EE on angiogenesis in an astrocytic interleukin-17A (IL-17A)-dependent manner following cerebral ischemia/reperfusion (I/R) injury. A rat model of ischemic stroke based on middle cerebral artery occlusion (MCAO) for 120 min followed by reperfusion was established, after which rats were housed in either EE or standard conditions. A set of behavior tests were conducted, including the modified neurological severity scores (mNSS) and the rotarod test. The infarct volume was evaluated by means of 2,3,5-Triphenyl tetrazolium chloride (TTC) staining. To evaluate the levels of angiogenesis, the protein levels of CD34 were examined by means of immunofluorescence and western blotting, while the protein and mRNA levels of IL-17A, vascular endothelial growth factor (VEGF), and the angiogenesis-associated factors interleukin-6 (IL-6), JAK2, and STAT3 were detected by western blotting and real-time quantitative PCR (RT-qPCR). We found that EE promoted functional recovery, reduced infarct volume, and enhanced angiogenesis compared to rats in standard conditions. IL-17A expression in astrocytes was also increased in EE rats. EE treatment increased the levels of microvascular density (MVD) and promoted the expression of CD34, VEGF, IL-6, JAK2, and STAT3 in the penumbra, while the intracerebroventricular injection of the IL-17A-neutralizing antibody in EE rats attenuated EE-mediated functional recovery and angiogenesis. Our findings revealed a possible neuroprotective mechanism of astrocytic IL-17A in EE-mediated angiogenesis and functional recovery after I/R injury, which might provide the theoretical basis for EE in clinical practise for stroke patients and open up new ideas for the research on the neural repair mechanism mediated by IL-17A in the recovery phase of stroke.

Zhuang medicine Shuanglu Tongnao Compound Recipe treats stroke by affecting the intestinal flora regulated by the TLR4/NF-κB signaling pathway.

The standardized treatment of ischemic stroke (IS) with Shuanglu Tongnao Compound Recipe (SLTNCR) combined with Western medicine has improved the life quality and neurological function of patients and achieved a satisfactory clinical effect. However, the underlying mechanisms of SLTNCR in the treatment of IS remain unclear. A rat model of IS was prepared using Longa's wire bolus method. SLTNCR was administered by gavage with following doses: low dose, 7.16 g·kg-1; middle dose, 14.33 g·kg-1; high dose, 28.66 g·kg-1. The expressions of toll-like receptor 4 (TLR4), tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), IL-6, nuclear factor-κB (NF-κB), etc., brain neuron damage, small intestine structure, and the structure of intestinal flora of rats in the high, medium, and low dose SLTNCR groups as well as the Injury + Clostridium butyricum and Injury + Edaravone groups were detected by 16SrRNA gene sequencing, western blot, hematoxylin-eosin (HE) staining, enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR). SLTNCR significantly reduced the brain water content, decreased the cerebral infarct size, and improved the neurological deficits, neuronal damage, small bowel tissue damage, and expression of inflammatory factors [B-cell CLL/lymphoma 2 (Bcl-2), BCL2 associated agonist of cell death (Bad), cleaved-caspase-3] in brain tissue. SLTNCR administration significantly inhibited expressions of TLR4, NF-κB, and inhibitor of nuclear factor kappa B (IκB), and decreased phosphorylation levels of NF-κB and IκB in the small intestinal tissues of IS rats. Moreover, SLTNCR also significantly upregulated the expression of intestinal barrier function-related molecules [zona occludens 1 (ZO-1), occludin, claudin-5] and regulated the expression of colonic TLR4, TNF-α, IL-6, and IL-1β. SLTNCR can improve the symptoms of IS rats by improving brain and small intestinal function, particularly by regulating the TLR4/NF-κB signaling pathway, apoptotic proteins, and inflammatory factors in brain tissue. Gut microbiota analysis helped to identify the pharmacological mechanisms underlying the effects of SLTNCR on intestinal bacterial diversity and flora structure in IS rats. SLTNCR can alleviate symptoms of IS and the potential mechanism of its effect is to protect brain tissue by suppressing inflammation. SLTNCR can also alter the structure and diversity of the bacterial community in IS.

Effects of Bacterial Biofilm on Regulation of Neurovascular Unit Functions and Neuroinflammation of Patients with Ischemic Cerebral Stroke by Immunocyte.

In this experiment, the effects of biofilm on neurovascular unit functions and neuroinflammation of patients with ischemic cerebral stroke were investigated. For this purpose, 20 adult male rats were purchased from Taconic (8 to 10 weeks old, weighing between 20 and 24g) and selected as the research objects. Then, they were randomly divided into an experimental group (10 rats) and a control group (10 rats). Ischemic cerebral stroke rat models were established. Besides, pseudomonas aeruginosa (PAO1) was prepared manually and implanted into the bodies of rats in the experimental group. mNSS scores, cerebral infarction area, and the release of inflammatory cytokines of rats in the two groups were compared. Results showed that mNSS scores for rats in the experimental group at all periods were remarkably higher than those for rats in the control group (P<0.05), which demonstrated that the rats in the experimental group suffered much severer neurological impairment than those in the control group. In addition, the release of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β, inducible nitric oxide synthase (iNOS), and IL-10 were all higher than those of the control group (P<0.05). The cerebral infarction area of the experimental group at all periods was remarkably larger than that of the control group (P<0.05). In conclusion, the formation of biofilm led to the aggravation of neurological impairment and inflammatory reactions among patients with ischemic cerebral stroke.

From Hair to the Brain: The Short-Term Therapeutic Potential of Human Hair Follicle-Derived Stem Cells and Their Conditioned Medium in a Rat Model of Stroke.

The short-term therapeutic impacts of stem cells and their derivatives were frequently reported in preclinical investigations of ischemic stroke (IS); however, several drawbacks including accessibility, abundancy, and ethical concerns limited their clinical application. We describe here for the first time the therapeutic potential of human hair follicle-derived stem cells (hHFSCs) and their conditioned medium (CM) in a rat model of IS. Furthermore, we hypothesized that a combination of cell therapy with repeated CM administration might enhance the restorative efficiency of this approach compared to each treatment alone. Middle cerebral artery occlusion was performed for 30min to induce IS. Immediately after reperfusion, hHFSCs were transplanted through the intra-arterial route and/or hHFSC-CM administered intranasally. The neurological outcomes, short-term spatial working memory, and infarct size were evaluated. Furthermore, relative expression of seven target genes in three categories of neuronal markers, synaptic markers, and angiogenic markers was assessed. The hHFSCs and hHFSC-CM treatments improved neurological impairments and reduced infarct size in the IS rats. Moreover, molecular data elucidated that IS was accompanied by attenuation in the expression of neuronal and synaptic markers in the evaluated brain regions and the interventions rescued these expression changes. Although there was no considerable difference between hHFSCs and hHFSC-CM treatments in the improvement of neurological function and decrement of infarct size, combination therapy was more effective to reduce infarction and elevation of target gene expression especially in the hippocampus. These findings highlight the curative potential of hHFSCs and their CM in a rat model of IS.

Synergistic therapeutic effects of intracerebral transplantation of human modified bone marrow-derived stromal cells (SB623) and voluntary exercise with running wheel in a rat model of ischemic stroke

BackgroundMesenchymal stromal cell (MSC) transplantation therapy is a promising therapy for stroke patients. In parallel, rehabilitation with physical exercise could ameliorate stroke-induced neurological impairment. In this study, we aimed to clarify whether combination therapy of intracerebral transplantation of human modified bone marrow-derived MSCs, SB623 cells, and voluntary exercise with running wheel (RW) could exert synergistic therapeutic effects on a rat model of ischemic stroke.MethodsWistar rats received right transient middle cerebral artery occlusion (MCAO). Voluntary exercise (Ex) groups were trained in a cage with RW from day 7 before MCAO. SB623 cells (4.0 × 105 cells/5 μl) were stereotactically injected into the right striatum at day 1 after MCAO. Behavioral tests were performed at day 1, 7, and 14 after MCAO using the modified Neurological Severity Score (mNSS) and cylinder test. Rats were euthanized at day 15 after MCAO for mRNA level evaluation of ischemic infarct area, endogenous neurogenesis, angiogenesis, and expression of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF). The rats were randomly assigned to one of the four groups: vehicle, Ex, SB623, and SB623 + Ex groups.ResultsSB623 + Ex group achieved significant neurological recovery in mNSS compared to the vehicle group (p < 0.05). The cerebral infarct area of SB623 + Ex group was significantly decreased compared to those in all other groups (p < 0.05). The number of BrdU/Doublecortin (Dcx) double-positive cells in the subventricular zone (SVZ) and the dentate gyrus (DG), the laminin-positive area in the ischemic boundary zone (IBZ), and the mRNA level of BDNF and VEGF in SB623 + Ex group were significantly increased compared to those in all other groups (p < 0.05).ConclusionsThis study suggests that combination therapy of intracerebral transplantation SB623 cells and voluntary exercise with RW achieves robust neurological recovery and synergistically promotes endogenous neurogenesis and angiogenesis after cerebral ischemia, possibly through a mechanism involving the up-regulation of BDNF and VEGF.

Dynamic MRI of the Mesenchymal Stem Cells Distribution during Intravenous Transplantation in a Rat Model of Ischemic Stroke.

Systemic transplantation of mesenchymal stem cells (MSCs) is a promising approach for the treatment of ischemia-associated disorders, including stroke. However, exact mechanisms underlying its beneficial effects are still debated. In this respect, studies of the transplanted cells distribution and homing are indispensable. We proposed an MRI protocol which allowed us to estimate the dynamic distribution of single superparamagnetic iron oxide labeled MSCs in live ischemic rat brain during intravenous transplantation after the transient middle cerebral artery occlusion. Additionally, we evaluated therapeutic efficacy of cell therapy in this rat stroke model. According to the dynamic MRI data, limited numbers of MSCs accumulated diffusely in the brain vessels starting at the 7th minute from the onset of infusion, reached its maximum by 29 min, and gradually eliminated from cerebral circulation during 24 h. Despite low numbers of cells entering brain blood flow and their short-term engraftment, MSCs transplantation induced long lasting improvement of the neurological deficit, but without acceleration of the stroke volume reduction compared to the control animals during 14 post-transplantation days. Taken together, these findings indicate that MSCs convey their positive action by triggering certain paracrine mechanisms or cell-cell interactions or invoking direct long-lasting effects on brain vessels.

α7 nicotinic acetylcholine receptor agonist improved brain injury and impaired glucose metabolism in a rat model of ischemic stroke.

Vagus nerve stimulation through the action of acetylcholine can modulate inflammatory responses and metabolism. α7 Nicotinic Acetylcholine Receptor (α7nAChR) is a key component in the biological functions of acetylcholine. To further explore the health benefits of vagus nerve stimulation, this study aimed to investigate whether α7nAChR agonists offer beneficial effects against poststroke inflammatory and metabolic changes and to identify the underlying mechanisms in a rat model of stroke established by permanent cerebral ischemia. We found evidence showing that pretreatment with α7nAChR agonist, GTS-21, improved poststroke brain infarction size, impaired motor coordination, brain apoptotic caspase 3 activation, dysregulated glucose metabolism, and glutathione reduction. In ischemic cortical tissues and gastrocnemius muscles with GTS-21 pretreatment, macrophages/microglia M1 polarization-associated Tumor Necrosis Factor-α (TNF-α) mRNA, Cluster of Differentiation 68 (CD68) protein, and Inducible Nitric Oxide Synthase (iNOS) protein expression were reduced, while expression of anti-inflammatory cytokine IL-4 mRNA, and levels of M2 polarization-associated CD163 mRNA and protein were increased. In the gastrocnemius muscles, stroke rats showed a reduction in both glutathione content and Akt Serine 473 phosphorylation, as well as an elevation in Insulin Receptor Substrate-1 Serine 307 phosphorylation and Dynamin-Related Protein 1 Serine 616 phosphorylation. GTS-21 reversed poststroke changes in the gastrocnemius muscles. Overall, our findings, provide further evidence supporting the neuroprotective benefits of α7nAChR agonists, and indicate that they may potentially exert anti-inflammatory and metabolic effects peripherally in the skeletal muscle in an acute ischemic stroke animal model.

Knockdown of ATF3 suppresses the progression of ischemic stroke through inhibiting ferroptosis.

Current therapies towards ischemic stroke (IS) are still not satisfied, and alternative strategies targeting ferroptosis may be another choice. The purpose of this study is to screen potential ferroptosis-related genes involving in IS. A rat model of IS was established via middle cerebral artery occlusion. Differentially expressed genes (DEGs) were screened from the model rats through transcriptional sequencing. Among the isolated DEGs, the expression of several attractive DEGs relating with ischemic injury was confirmed by qRT-PCR. Then, ATF3 relating with both IS and ferroptosis was selected a candidate gene for functional assays. After knockdown of ATF3 in the model rats, the infarction, histopathology, apoptosis, and ferroptosis in brain tissues were evaluated. IS model was successfully established in rats, exhibiting the emergence of infarction area, histopathological injury, and enhanced cell apoptosis. Total 699 up-regulated DEGs and 461 down-regulated DEGs were screened from the model rats. qRT-PCR verified the up-regulation of Hspa1b, Tfpi2, Ptx3, and Atf3, and the down-regulation of Smyd1 and Tacr2 in the Model group compared with those in the Sham group. It is noteworthy that knockdown of ATF3 decreased the infarction area, relieved histopathological injury, weakened apoptosis, and inhibited ferroptosis in the model rats. Several candidate genes in relation with IS were revealed. More importantly, knockdown of ATF3 may relieve IS through inhibiting ferroptosis.

β2-Microglobulin exacerbates neuroinflammation, brain damage, and cognitive impairment after stroke in rats.

β2-Microglobulin (β2M), a component of the major histocompatibility complex class I molecule, is associated with aging-related cognitive impairment and Alzheimer's disease. Although upregulation of β2M is considered to be highly related to ischemic stroke, the specific role and underlying mechanistic action of β2M are poorly understood. In this study, we established a rat model of focal cerebral ischemia by occlusion of the middle cerebral artery. We found that β2M levels in the cerebral spinal fluid, serum, and brain tissue were significantly increased in the acute period but gradually decreased during the recovery period. RNA interference was used to inhibit β2M expression in the acute period of cerebral stroke. Tissue staining with 2,3,5-triphenyltetrazolium chloride and evaluation of cognitive function using the Morris water maze test demonstrated that decreased β2M expression in the ischemic penumbra reduced infarct volume and alleviated cognitive deficits, respectively. Notably, glial cell, caspase-1 (p20), and Nod-like receptor pyrin domain containing 3 (NLRP3) inflammasome activation as well as production of the inflammatory cytokines interleukin-1β, interleukin-6, and tumor necrosis factor-α were also effectively inhibited by β2M silencing. These findings suggest that β2M participates in brain injury and cognitive impairment in a rat model of ischemic stroke through activation of neuroinflammation associated with the NLRP3 inflammasome.

Vagus nerve stimulation is a potential treatment for ischemic stroke.

Microglia are the brain's primary innate immune cells, and they are activated and affect pro-inflammatory phenotype or regulatory phenotype after ischemic stroke. Vagus nerve stimulation was shown to activate microglial phenotypic changes and exhibit neuroprotective effects in ischemia/reperfusion injury. In this study, we established rat models of ischemic stroke by occlusion of the middle cerebral artery and performed vagus nerve stimulation 30 minutes after modeling. We found that vagus nerve stimulation caused a shift from a pro-inflammatory phenotype to a regulatory phenotype in microglia in the ischemic penumbra. Vagus nerve stimulation decreased the levels of pro-inflammatory phenotype markers inducible nitric oxide synthase and tumor necrosis factor α and increased the expression of regulatory phenotype markers arginase 1 and transforming growth factor β through activating α7 nicotinic acetylcholine receptor expression. Additionally, α7 nicotinic acetylcholine receptor blockade reduced the inhibition of Toll-like receptor 4/nuclear factor kappa-B pathway-associated proteins, including Toll-like receptor 4, myeloid differentiation factor 88, I kappa B alpha, and phosphorylated-I kappa B alpha, and also weakened the neuroprotective effects of vagus nerve stimulation in ischemic stroke. Vagus nerve stimulation inhibited Toll-like receptor 4/nuclear factor kappa-B expression through activating α7 nicotinic acetylcholine receptor and regulated microglial polarization after ischemic stroke, thereby playing a role in the treatment of ischemic stroke. Findings from this study confirm the mechanism underlying vagus nerve stimulation against ischemic stroke.

Infiltration of meningeal macrophages into the Virchow-Robin space after ischemic stroke in rats: Correlation with activated PDGFR-β-positive adventitial fibroblasts.

Macrophages play a crucial role in wound healing and fibrosis progression after brain injury. However, a detailed analysis of their initial infiltration and interaction with fibroblasts is yet to be conducted. This study aimed to investigate the possible route for migration of meningeal macrophages into the ischemic brain and whether these macrophages closely interact with neighboring platelet-derived growth factor beta receptor (PDGFR-β)-positive adventitial fibroblasts during this process. A rat model of ischemic stroke induced by middle cerebral artery occlusion (MCAO) was developed. In sham-operated rats, CD206-positive meningeal macrophages were confined to the leptomeninges and the perivascular spaces, and they were not found in the cortical parenchyma. In MCAO rats, the number of CD206-positive meningeal macrophages increased both at the leptomeninges and along the vessels penetrating the cortex 1 day after reperfusion and increased progressively in the extravascular area of the cortical parenchyma by 3 days. Immunoelectron microscopy and correlative light and electron microscopy showed that in the ischemic brain, macrophages were frequently located in the Virchow-Robin space around the penetrating arterioles and ascending venules at the pial surface. This was identified by cells expressing PDGFR-β, a novel biomarker of leptomeningeal cells. Macrophages within penetrating vessels were localized in the perivascular space between smooth muscle cells and PDGFR-β-positive adventitial fibroblasts. In addition, these PDGFR-β-positive fibroblasts showed morphological and molecular characteristics similar to those of leptomeningeal cells: they had large euchromatic nuclei with prominent nucleoli and well-developed rough endoplasmic reticulum; expressed nestin, vimentin, and type I collagen; and were frequently surrounded by collagen fibrils, indicating active collagen synthesis. In conclusion, the perivascular Virchow-Robin space surrounding the penetrating vessels could be an entry route of meningeal macrophages from the subarachnoid space into the ischemic cortical parenchyma, implying that activated PDGFR-β-positive adventitial fibroblasts could be involved in this process.

Therapeutic Effect of Rapamycin on TDP-43-Related Pathogenesis in Ischemic Stroke.

Stroke is a major cause of death and disability across the world, and its detrimental impact should not be underestimated. Therapies are available and effective for ischemic stroke (e.g., thrombolytic recanalization and mechanical thrombectomy); however, there are limitations to therapeutic interventions. Recanalization therapy has developed dramatically, while the use of adjunct neuroprotective agents as complementary therapies remains deficient. Pathological TAR DNA-binding protein (TDP-43) has been identified as a major component of insoluble aggregates in numerous neurodegenerative pathologies, including ALS, FTLD and Alzheimer's disease. Here, we show that increased pathological TDP-43 fractions accompanied by impaired mitochondrial function and increased gliosis were observed in an ischemic stroke rat model, suggesting a pathological role of TDP-43 in ischemic stroke. In ischemic rats administered rapamycin, the insoluble TDP-43 fraction was significantly decreased in the ischemic cortex region, accompanied by a recovery of mitochondrial function, the attenuation of cellular apoptosis, a reduction in infarct areas and improvements in motor defects. Accordingly, our results suggest that rapamycin provides neuroprotective benefits not only by ameliorating pathological TDP-43 levels, but also by reversing mitochondrial function and attenuating cell apoptosis in ischemic stroke.

Effects of the New Thrombolytic Compound LT3001 on Acute Brain Tissue Damage After Focal Embolic Stroke in Rats.

LT3001 is a novel synthetic small molecule with thrombolytic and free radical scavenging activities. In this study, we tested the effects of LT3001 as a potential alternative thrombolytic in focal embolic ischemic stroke rat model. Stroked rats received intravenous injection of 10mg/kg LT3001 or tPA at 1.5, 3, or 4.5h after stroke, respectively, and the outcomes were measured at different time points after stroke by performing multi-parametric MRI, 2,3,5-triphenyltetrazolium chloride (TTC) staining, and modified neurological severity score. Lastly, we assessed the effect of LT3001 on the tPA activity in vitro, the international normalized ratio (INR), and the serum levels of active tPA and plasminogen activator inhibitor-1 (PAI-1). LT3001 treated at 1.5h after stroke is neuroprotective by reducing the CBF lesion size and lowering diffusion and T2 lesion size measured by MRI, which is consistent with the reduction in TTC-stained infarction. When treated at 3h after stroke, LT3001 had significantly better therapeutic effects regarding reduction of infarct size, swelling rate, and hemorrhagic transformation compared to tPA. When treated at 4.5h after stroke, tPA, but not LT3001, significantly increased brain swelling and intracerebral hemorrhagic transformation. Lastly, LT3001 did not interfere with tPA activity in vitro, or significantly alter the INR and serum levels of active tPA and PAI-1 in vivo. Our data suggests that LT3001 is neuroprotective in focal embolic stroke rat model. It might have thrombolytic property, not interfere with tPA/PAI-1 activity, and cause less risk of hemorrhagic transformation compared to the conventional tPA. Taken together, LT3001 might be developed as a novel therapy for treating thrombotic ischemic stroke.