Adapted Smart-seq3xpress Facilitates Selective Microglial Transcriptomic Profiling From Frozen Brain Tissue.

After a stroke, the neurogenic response from the subventricular zone (SVZ) to repair the brain is limited. Microglia, as an integral part of the distinctive SVZ microenvironment, control neural stem / precursor cell (NSPC) behavior. Here, we show that discrete stroke-associated SVZ microglial clusters negatively impact the innate neurogenic response, and we propose a repository of relevant microglia–NSPC ligand–receptor pairs. After photothrombosis, a mouse model of ischemic stroke, the altered SVZ niche environment leads to immediate activation of microglia in the niche and an abnormal neurogenic response, with cell-cycle arrest of neural stem cells and neuroblast cell death. Pharmacological restoration of the niche environment increases the SVZ-derived neurogenic repair and microglial depletion increases the formation and survival of newborn neuroblasts in the SVZ. Therefore, we propose that altered cross-communication between microglial subclusters and NSPCs regulates the extent of the innate neurogenic repair response in the SVZ after stroke.

BackgroundIschemic stroke is a devastating disease without a cure. The available treatments for ischemic stroke, thrombolysis by tissue plasminogen activator, and thrombectomy are suitable only to a fraction of patients and thus novel therapeutic approaches are urgently needed. The neuroinflammatory responses elicited secondary to the ischemic attack further aggravate the stroke-induced neuronal damage. It has been demonstrated that these responses are regulated at the level of non-coding RNAs, especially miRNAs.MethodsWe utilized lentiviral vectors to overexpress miR-669c in BV2 microglial cells in order to modulate their polarization. To detect whether the modulation of microglial activation by miR-669c provides protection in a mouse model of transient focal ischemic stroke, miR-669c overexpression was driven by a lentiviral vector injected into the striatum prior to induction of ischemic stroke.ResultsHere, we demonstrate that miR-669c-3p, a member of chromosome 2 miRNA cluster (C2MC), is induced upon hypoxic and excitotoxic conditions in vitro and in two different in vivo models of stroke. Rather than directly regulating the neuronal survival in vitro, miR-669c is capable of attenuating the microglial proinflammatory activation in vitro and inducing the expression of microglial alternative activation markers arginase 1 (Arg1), chitinase-like 3 (Ym1), and peroxisome proliferator-activated receptor gamma (PPAR-γ). Intracerebral overexpression of miR-669c significantly decreased the ischemia-induced cell death and ameliorated the stroke-induced neurological deficits both at 1 and 3 days post injury (dpi). Albeit miR-669c overexpression failed to alter the overall Iba1 protein immunoreactivity, it significantly elevated Arg1 levels in the ischemic brain and increased colocalization of Arg1 and Iba1. Moreover, miR-669c overexpression under cerebral ischemia influenced several morphological characteristics of Iba1 positive cells. We further demonstrate the myeloid differentiation primary response gene 88 (MyD88) transcript as a direct target for miR-669c-3p in vitro and show reduced levels of MyD88 in miR-669c overexpressing ischemic brains in vivo.ConclusionsCollectively, our data provide the evidence that miR-669c-3p is protective in a mouse model of ischemic stroke through enhancement of the alternative microglial/macrophage activation and inhibition of MyD88 signaling. Our results accentuate the importance of controlling miRNA-regulated responses for the therapeutic benefit in conditions of stroke and neuroinflammation.

Background. Inflammation plays an important role in the pathophysiology of ischemic stroke and may contri-bute to secondary damage following ischemic stroke. Recently, it has been shown that synthetic oligopeptides related to human chorionic gonadotropin (hCG) and various other synthetic oligopeptides may have immuno-modulatory effects. We aimed to investigate the effects of two promising synthetic anti-inflammatory tetrapeptides, namely the hCG-related peptide AQGV and the p53-related peptide EPPE on infarct volume and on inflammatory gene expres-sion in a mouse model of focal cerebral ischemia. Methods. In a randomized single-blinded fashion, mice received two intravenous injections of either AQGV (30 mg/kg bodyweight) or EPPE (30mg/kg bodyweight) or phosphate buffered saline. The first dose was adminis-tered directly before 90-minutes middle cerebral artery occlusion, and the second dose was injected directly af-ter reperfusion. Infarct volume and gene expression levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), E-selectin and intercellular adhesion molecule-I (ICAM-1) were measured 24 hours after reperfusion. Results. We found no significant effect of either AQGV (108.7 +/- 12.1 mm3 versus PBS 114.9 +/- 23.9 mm3; P=0.7) or EPPE (131.8 +/-12.8 mm3 versus PBS 114.9 +/- 23.9 mm3 Conclusion. AQGV and EPPE did not show any beneficial effects in this mouse model of acute ischemic stroke. Further studies are needed to investigate the nature and dynamics of the immunomodulatory effects of synthetic oligopeptides in acute ischemic stroke. ; P=0.1) on direct lesion volume. All three experimental groups displayed similar mRNA expression levels of TNF-α, IL-6, E-selectin and ICAM-1 at 24 hours after the ischemic insult.

Sesamin attenuates neurotoxicity in mouse model of ischemic brain stroke

Background. Inflammation plays an important role in the pathophysiology of ischemic stroke and may contri-bute to secondary damage following ischemic stroke. Recently, it has been shown that synthetic oligopeptides related to human chorionic gonadotropin (hCG) and various other synthetic oligopeptides may have immuno-modulatory effects. We aimed to investigate the effects of two promising synthetic anti-inflammatory tetrapeptides, namely the hCG-related peptide AQGV and the p53-related peptide EPPE on infarct volume and on inflammatory gene expres-sion in a mouse model of focal cerebral ischemia. Methods. In a randomized single-blinded fashion, mice received two intravenous injections of either AQGV (30 mg/kg bodyweight) or EPPE (30mg/kg bodyweight) or phosphate buffered saline. The first dose was adminis-tered directly before 90-minutes middle cerebral artery occlusion, and the second dose was injected directly af-ter reperfusion. Infarct volume and gene expression levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), E-selectin and intercellular adhesion molecule-I (ICAM-1) were measured 24 hours after reperfusion. Results. We found no significant effect of either AQGV (108.7 +/- 12.1 mm3 versus PBS 114.9 +/- 23.9 mm3; P=0.7) or EPPE (131.8 +/-12.8 mm3 versus PBS 114.9 +/- 23.9 mm3 Conclusion. AQGV and EPPE did not show any beneficial effects in this mouse model of acute ischemic stroke. Further studies are needed to investigate the nature and dynamics of the immunomodulatory effects of synthetic oligopeptides in acute ischemic stroke. ; P=0.1) on direct lesion volume. All three experimental groups displayed similar mRNA expression levels of TNF-α, IL-6, E-selectin and ICAM-1 at 24 hours after the ischemic insult.

Previous studies have used sodium magnetic resonance imaging (MRI) to investigate the increase in tissue sodium concentration that occurs during a stroke by using various animal models of brain ischemia. However, most of these studies have involved rats, cats, or nonhuman primates. Although studies involving mice are relatively scant, mice have become the principal animal model for studying many human diseases, particularly in the field of genetics. Accordingly, this study employed sodium MRI to monitor changes in the intensity of sodium signals in a mouse model of ischemic stroke. The experiments were conducted using a 7-T MRI system, and a commercial double-tuned sodium/proton transmit-receive surface coil was used to capture the sodium and proton signal images. Sodium MRI was performed using a fast low-angle shot pulse sequence. The mice underwent middle cerebral artery occlusion to induce focal brain ischemia 48 h before the MRI scans were performed. The signal intensity of the sodium image was determined for a region of interest (ROI) in the ischemic area, and an ROI contralateral to this area. The average signal intensity in the sodium images of the mouse brains exhibited a 2.51-fold increase and a standard deviation was 0.93. The results of this study demonstrate the feasibility of using a 7-T MRI system to perform sodium MRI of a mouse model of ischemic stroke. The sodium signal intensity of the mouse brain revealed a substantial increase in sodium levels in the ischemic area compared with that in the contralateral brain hemisphere.

JOURNAL/nrgr/04.03/01300535-202410000-00025/figure1/v/2024-02-06T055622Z/r/image-tiff In situ direct reprogramming technology can directly convert endogenous glial cells into functional neurons in vivo for central nervous system repair. Polypyrimidine tract-binding protein 1 (PTB) knockdown has been shown to reprogram astrocytes to functional neurons in situ. In this study, we used AAV-PHP.eB-GFAP-shPTB to knockdown PTB in a mouse model of ischemic stroke induced by endothelin-1, and investigated the effects of GFAP-shPTB-mediated direct reprogramming to neurons. Our results showed that in the mouse model of ischemic stroke, PTB knockdown effectively reprogrammed GFAP-positive cells to neurons in ischemic foci, restored neural tissue structure, reduced inflammatory response, and improved behavioral function. These findings validate the effectiveness of in situ transdifferentiation of astrocytes, and suggest that the approach may be a promising strategy for stroke treatment.

Identifying novel neuroprotectants that can halt or reverse the neurological effects of stroke is of interest to both clinicians and scientists. We and others previously showed the pre-clinical neuroprotective efficacy of neuregulin-1 (NRG-1) in rats following focal brain ischemia. In this study, we examined neuroprotection by exogenous and endogenous NRG-1 using a mouse model of ischemic stroke. C57BL6 mice were subjected to middle cerebral artery occlusion (MCAO) followed by reperfusion. NRG-1 or vehicle was infused intra-arterially (i.a.) or intravenously (i.v.) after MCAO and before the onset of reperfusion. NRG-1 treatment (16μg/kg; i.a.) reduced cerebral cortical infarct volume by 72% in mice when delivered post-ischemia. NRG-1 also inhibited neuronal injury as measured by Fluoro Jade B labeling and rescued NeuN immunoreactivity in neurons. Neuroprotection by NRG-1 was also observed in mice when administered i.v. (100μg/kg) in both male and female mice. We investigated whether endogenous NRG-1 was neuroprotective using male and female heterozygous NRG-1 knockout mice (NRG-1+/-) compared with wild-type mice (WT) littermates. NRG-1+/- and WT mice were subjected to MCAO for 45min, and infarct size was measured 24h following MCAO. NRG-1+/- mice displayed a sixfold increase in cortical infarct size compared with WT mice. These results demonstrate that NRG-1 treatment mitigates neuronal damage following cerebral ischemia. We further showed that reduced endogenous NRG-1 results in exacerbated neuronal injury in vivo. These findings suggest that NRG-1 represents a promising therapy to treat stroke in human patients.

Tissue factor (TF), the primary initiator of the extrinsic coagulation cascade, is highly expressed by astrocytes surrounding blood vessels in the central nervous system. Using mouse models of hemorrhagic and ischemic stroke, we previously demonstrated that astrocyte TF plays a critical role in brain hemostasis while also contributing to neuronal damage. Importantly, using mice with different levels of astrocyte TF expression, we determined that an astrocyte TF activity around 5% of the wild type level was sufficient to limit bleeding in hemorrhagic stroke and at the same time was low enough to reduce infarct size and behavioral deficits in ischemic stroke, without increased hemorrhagic transformation. Therefore, we hypothesized that pharmacologic inhibition of TF may improve outcomes after ischemic stroke and have a wide safety margin. To test this hypothesis, C56Bl/6J mice were subjected to middle cerebral artery occlusion (MCAO) for 1 hour followed by 24 hours of reperfusion. Inhibitory rat anti- mouse TF antibody (1H1) and control IgG were administered as a bolus intraperitoneal injection immediately after ischemia. Inhibition of TF dose-dependently reduced brain infarct size in female mice (36.6±1.4% of total brain volume in IgG group [n=19] vs 28.3 ± 1.7% in 25mg/kg 1H1 group [n=9, p<0.01] and 18.5 ±2.3% in 75mg/kg 1H1 group [n=8, p<0.001]; mean±SEM). Importantly, 1H1 did not exacerbate hemorrhagic transformation during the reperfusion phase. Furthermore, TF inhibition significantly reduced microvascular thrombosis (analyzed by fibrin staining), expression of inflammatory markers in the injured brain (IL-6, MCP-1 and KC levels- analyzed by ELISA) and attenuated behavioral deficit after ischemic stroke. Subjecting male mice to the same MCAO condition resulted in partial lethality in IgG treated group (4 out of 6 mice died with 24 hours) which was prevented by 1H1 (25mg/kg) treatment (0 out of 5, Log-rank test p=0.031). Altogether, our data suggest that the overabundance of astrocyte TF expression resulted from selective pressure to limit intracerebral hemorrhage after traumatic brain injury, but in the modern era poses the additional risk of increased injury during ischemic stroke.

Ischemic stroke causes severe neurological disability, with some patients still presenting with residual neurological dysfunction. Aerobic exercise shows potential for post-stroke recovery. However, the mechanism behind its beneficial effects on the brain remains to be further explored. This study aimed to explore aerobic exercise's role in the neurological recovery of ischemic stroke mice and to clarify the specific protective mechanisms involved. A mouse model of ischemic stroke was employed in this study. Mice were subjected to aerobic exercise intervention, with additional exogenous lactate administration and RNA-sequencing (RNA-seq) analysis conducted to explore the molecular mechanisms. Results demonstrated that aerobic exercise significantly elevated protein lactylation levels in ischemic stroke mice and facilitated neurological recovery. In addition, aerobic exercise attenuated neuroinflammation in the ischemic penumbra and promoted neural cell proliferation in the subventricular zone. Mechanistically, aerobic exercise-induced lactylation of α-tubulin may enhance the dynamic properties of microtubules, thereby facilitating neural repair. Exogenous lactate intervention was found to augment α-tubulin lactylation, promote hippocampal neurogenesis, and increase dendritic spine density as well as synaptic plasticity in both the ischemic penumbra and hippocampus. Consistent with these findings, exogenous lactate administration improved neurological function in ischemic stroke mice. RNA-seq analysis further revealed that elevated lactate levels exerted a promotive effect on neural repair. Aerobic exercise promotes post-ischemic stroke neurological recovery via elevating protein lactylation (α-tubulin lactylation), attenuating neuroinflammation, and enhancing neural repair. These findings highlight the potential of targeting lactate metabolism and protein lactylation as therapeutic strategies for facilitating neural repair and improving neurological outcomes following ischemic stroke.

Mouse models have become increasingly popular in the field of behavioral neuroscience, and specifically in studies of experimental stroke. As models advance, it is important to develop sensitive behavioral measures specific to the mouse. The present protocol describes a skilled motor task for use in mouse models of stroke. The Pasta Matrix Reaching Task functions as a versatile and sensitive behavioral assay that permits experimenters to collect accurate outcome data and manipulate limb use to mimic human clinical phenomena including compensatory strategies (i.e., learned non-use) and focused rehabilitative training. When combined with neuroanatomical tools, this task also permits researchers to explore the mechanisms that support behavioral recovery of function (or lack thereof) following stroke. The task is both simple and affordable to set up and conduct, offering a variety of training and testing options for numerous research questions concerning functional outcome following injury. Though the task has been applied to mouse models of stroke, it may also be beneficial in studies of functional outcome in other upper extremity injury models.

Introduction: Astrocytes are non-neuronal cells in the CNS that play crucial roles in synaptic formation, maturation, and maintenance through the synthesis and secretion of various proteins. We identified Chordin-like 1 (Chrdl1) as an astrocyte-secreted protein that limits synaptic plasticity by stabilizing GluA2-containing AMPA receptors (GluA2-AMPARs) at synaptic sites in the cortex. In a mouse model of ischemic stroke, we found that Chrdl1 is excessively upregulated in astrocytes in the peri-infarct area during the post-stroke plasticity window (2–30 days post-stroke in the mouse). We found that Chrdl1 KO mice displayed enhanced synaptic plasticity, and in response to stroke, deficits such as dendritic spine degeneration, impaired GluA2-AMPAR distribution, and motor dysfunction were significantly alleviated. Seeking a pharmacological approach to mitigate stroke-induced Chrdl1 upregulation, we explored the use of sub-anesthetic ketamine, known to enhance plasticity and alter astrocytic function, potentially affecting protein secretion. However, its impact on post-stroke astrocyte-regulated plasticity and recovery has not yet been explored. We hypothesize that sub-anesthetic ketamine may restrict the upregulation of astrocytic Chrdl1 during the post-stroke plasticity window and reduce stroke-induced deficits. Methods: To generate ischemia in vivo , we used photothrombosis to induce distal middle cerebral artery occlusion (PT-dMCAO). Male and female adult mice (2 months old) received a daily dose of sub-anesthetic ketamine (10mg/kg) via intranasal injection for one week. We used single molecule fluorescence in situ hybridization (smFISH) and immunohistochemistry to assess Chrdl1 mRNA and protein levels in peri-infarct astrocytes, synaptic staining and high-resolution confocal microscopy to analyze GluA2-AMPARs distribution, Golgi stainings to study dendritic branching and spine density, and several behavioral tests to assess motor performance, sensorimotor function, and learning and memory. Results: We found that sub-anesthetic ketamine treatment reverts stroke-induced Chrdl1 upregulation in peri-infarct astrocytes and mitigates molecular, structural and behavioral deficits. Conclusions: Our study suggests that sub-anesthetic ketamine treatment is a promising approach to limit astrocyte-driven molecular deficits during the critical post-stroke plasticity window, which may otherwise hinder recovery, and holds great promise for clinical translation.

Background Stroke is a leading cause of disability worldwide. Focused training of the impaired limb has been shown to improve its functional outcome in animal models. However, most human stroke survivors exhibit persistent motor deficits, likely due to differences in rehabilitation intensity between experimental (animal) and clinical (human) settings. Objective The current study investigated the effect of training intensity on behavioral outcome in a mouse model of stroke. Methods Mice were trained preoperatively on a skilled reaching task. After training, mice received a unilateral photothrombotic stroke. Postoperatively, animals received either daily rehabilitative training (traditional intensity), intermittent rehabilitative training (every other day), or no rehabilitative training (control). Assessment of the impaired limb occurred after 14 training sessions (14 days for the Traditional group; 28 days for the Intermittent group). Results Assessment of the impaired limb illustrated that traditional, daily training resulted in significantly better performance than no training, while intermittent training offered moderate performance gains. Mice receiving intermittent training performed significantly better than control mice but did not exhibit reaching performance as strong as that of animals trained daily. Conclusions The intensity of rehabilitation is important for optimal recovery. Although intermediate intensity offers some benefit, it is not intensive enough to mimic the performance gains traditionally observed in animal models. These results suggest that intensive training, which is often unavailable for human stroke survivors, is necessary to achieve an optimal functional outcome. The lower bounds of training intensity for functional benefit still need to be determined.

Acute ischaemic stroke can induce secondary brain injury by activating an inflammatory response that contributes to clinical impairment. As a specificinhibitor of the immunoproteasome subunit low molecular weightpolypeptide 7 (LMP7), PR-957 may participate in regulating pathophysiological and inflammatory responses in multiple diseases of the central nervous system (CNS). We investigated the neuroprotective properties of PR-957 in a mouse model of stroke, induced by middle cerebral artery occlusion (MCAO). After MCAO and injections of PR-957 or vehicle, we evaluated mice behaviourally using modified Neurological Severity Scores (mNSS) and sensorimotor tests, including the adhesive-removal test, a foot-fault test and an inclined plane test. Infarct volume was measured 24 and 72h after MCAO. Infiltration by different lymphocyte subpopulations was evaluated by flow cytometry and immunofluorescent staining of brain tissue from the penumbral area. Quantitative real-time polymerase chain reaction analysis and enzyme-linked immunosorbent assay were used to measure the expression of proinflammatory cytokines: interkeukin (IL)-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IL-17A, interferon (IFN)-γ, tumour necrosis factor (TNF)-α, granulocyte colony-stimulating factor (GCSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Expression of phosphorylated signal transducer and activator of transcription 3 (pSTAT-3) protein levels in brain was measured by immunoblot. MCAO mice treated with PR-957 showed a significant decrease in infarct volume and had mild neurological deficits compared to vehicle-treated mice. PR-957 administration also significantly decreased IL-1β, IL-6, IL-12, IL-17A and TNF-α. PR-957 provides neuroprotection via inhibiting T lymphocyte infiltration and decreasing T helper type 17 (Th17) cell differentiation in MCAO mice, which may result from the reduced expression of pSTAT-3. The neuroprotective effect of PR-957 indicates its potential utility as anti-inflammatory therapy for ischaemic stroke.

GPR37 modulates progenitor cell dynamics in a mouse model of ischemic stroke