Intracerebral Hemorrhage Model Research Articles

P2Y6 Receptor Activation Aggravates NLRP3-dependent Microglial Pyroptosis via Downregulation of the PI3K/AKT Pathway in a Mouse Model of Intracerebral Hemorrhage.

Pro-inflammatory signals generated after intracerebral hemorrhage (ICH) trigger a form of regulated cell death known as pyroptosis in microglia. White matter injury (WMI) refers to the condition where the white matter area of the brain suffers from mechanical, ischemic, metabolic, or inflammatory damage. Although the p2Y purinoceptor 6 (P2Y6R) plays a significant role in the control of inflammatory reactions in central nervous system diseases, its roles in the development of microglial pyroptosis and WMI following ICH remain unclear. In this study, we sought to clarify the role of P2Y6R in microglial pyroptosis and WMI by using an experimental mouse model of ICH. Type IV collagenase was injected into male C57BL/6 mice to induce ICH. Mice were then treated with MRS2578 and LY294002 to inhibit P2Y6R and phosphatidylinositol 3-kinase (PI3K), respectively. Bio-conductivity analysis was performed to examine PI3K/AKT pathway involvement in microglial pyroptosis. Quantitative Real-Time PCR, immunofluorescence staining, and western blot were conducted to examine microglial pyroptosis and WMI following ICH. A modified Garcia test, corner turning test, and forelimb placement test were used to assess neurobehavior. Hematoxylin-eosin staining (HE) was performed to detect cells damage around hematoma. Increases in the expression of P2Y6R, NLRP3, ASC, Caspase-1, and GSDMD were observed after ICH. P2Y6R was only expressed on microglia. MRS2578, a specific inhibitor of P2Y6R, attenuated short-term neurobehavioral deficits, brain edema and hematoma volume while improving both microglial pyroptosis and WMI. These changes were accompanied by decreases in pyroptosis-related proteins and pro-inflammatory cytokines both in vivo and vitro. Bioinformatic analysis revealed an association between the PI3K/AKT pathway and P2Y6R-mediated microglial pyroptosis. The effects of MRS2578 were partially reversed by treatment with LY294002, a specific PI3K inhibitor. P2Y6R inhibition alleviates microglial pyroptosis and WMI and ameliorates neurological deficits through the PI3K/AKT pathway after ICH. Consequently, targeting P2Y6R might be a promising approach for ICH treatment.

Correction to: The Neuroprotective Effects and Probable Mechanisms of Everolimus in a Rat Model of Intracerebral Hemorrhage

Correction to: The Neuroprotective Effects and Probable Mechanisms of Everolimus in a Rat Model of Intracerebral Hemorrhage

Clearance of Neutrophils From ICH-Affected Brain by Macrophages Is Beneficial and Is Assisted by Lactoferrin and CD91.

Within hours after intracerebral hemorrhage (ICH) onset, masses of polymorphonuclear neutrophils (PMNs) infiltrate the ICH-affected brain. After degranulation involving controlled release of many toxic antimicrobial molecules, the PMNs undergo rapid apoptosis and then are removed by phagocytic microglia/macrophages (MΦ) through a process called efferocytosis. Effective removal of PMNs may limit secondary brain damage and inflammation; however, the molecular mechanisms governing these cleanup activities are not well understood. We propose that scavenger receptor CD91 on myeloid phagocytes especially in presence of CD91 ligand, LTF (lactoferrin, protein abundant in PMNs), plays an important role in clearance of dead apoptotic PMNs (ANs). Mice/rats were subjected to an autologous blood injection model of ICH. Primary cultured microglia were used to assess phagocytosis of ANs. Immunohistochemistry was employed to assess CD91 expression and PMN infiltration. CD91 knockout mice selectively in myeloid phagocytes (Mac-CD91-KO) were used to establish the CD91/LTF function in phagocytosis and in reducing ICH-induced injury, as assessed using behavioral tests, hematoma resolution, and oxidative stress. Masses of PMNs are found in ICH-affected brain, and they contain LTF. MΦ at the outer border of hematoma are densely packed, expressing CD91 and phagocytosing ANs. Microglia deficient in CD91 demonstrate defective phagocytosis of ANs, and mice deficient in CD91 (Mac-CD91-KO) subjected to ICH injury have increased neurological dysfunction that is associated with impaired hematoma resolution (hemoglobin and iron clearance) and elevated oxidative stress. LTF that normally ameliorates ICH injury in CD91-proficient control mice shows reduced therapeutic effects in Mac-CD91-KO mice. Our study suggests that CD91 plays a beneficial role in improving ANs phagocytosis and ultimately post-ICH outcome and that the beneficial effect of LTF in ICH is in part dependent on presence of CD91 on MΦ.

Kv1.3 Blockade Alleviates White Matter Injury through Reshaping M1/M2 Phenotypes via the NF-κB Signaling Pathway after Intracerebral Hemorrhage.

White matter injury (WMI) in basal ganglia usually induces long-term disability post intracerebral hemorrhage (ICH). Kv1.3 is an ion channel expressed in microglia and induces neuroinflammation after ICH. Here, we investigated the functions and roles of Kv1.3 activation-induced inflammatory response in WMI and the Kv1.3 blockade effect on microglia polarization after ICH. Mice ICH model was constructed by autologous blood injection. The expression of Kv1.3 was measured using immunoblot, real-time quantitative polymerase chain reaction (RT-qPCR), and immunostaining assays. Then, the effect of administration of 5-(4-Phenoxybutoxy) psoralen (PAP-1), a selectively pharmacological Kv1.3 blocker, was investigated using open field test (OFT) and basso mouse score (BMS). RT-qPCR, immunoblot, and enzyme-linked immunosorbent assay (ELISA) were taken to elucidate the expression of pro-inflammatory or anti-inflammatory factors around hematoma. PAP-1's function in regulating microglia polarization was investigated using immunoblot, RT-qPCR, and immunostaining assays. The downstream PAP-1 signaling pathway was determined by RT-qPCR and immunoblot. Kv1.3 expression was increased in microglia around the hematoma significantly after ICH. PAP-1 markedly improved neurological outcomes and the WMI by reducing pro-inflammatory cytokine accumulation and upregulating anti-inflammatory factors. Mechanistically, PAP-1 reduces NF-κB p65 and p50 activation, thus facilitating microglia polarization into M2-like microglia, which exerts this beneficial effect. PAP-1 reduced pro-inflammatory cytokines accumulation and increased anti-inflammatory factors by facilitating M2-like microglia polarization via the NF-κB signaling pathway. Thus, the current study shows that the Kv1.3 blockade is capable of ameliorating WMI by facilitating M2-like phenotype microglia polarization after ICH.

Astrocyte-Derived Extracellular Vesicular miR-143-3p Dampens Autophagic Degradation of Endothelial Adhesion Molecules and Promotes Neutrophil Transendothelial Migration after Acute Brain Injury.

Pivotal roles of extracellular vesicles (EVs) in the pathogenesis of central nervous system (CNS) disorders including acute brain injury are increasingly acknowledged. Through the analysis of EVs packaged miRNAs in plasma samples from patients with intracerebral hemorrhage (ICH), it is discovered that the level of EVs packaged miR-143-3p (EVs-miR-143-3p) correlates closely with perihematomal edema and neurological outcomes. Further study reveals that, upon ICH, EVs-miR-143-3p is robustly secreted by astrocytes and can shuttle into brain microvascular endothelial cells (BMECs). Heightened levels of miR-143-3p in BMECs induce the up-regulated expression of cell adhesion molecules (CAMs) that bind to circulating neutrophils and facilitate their transendothelial cell migration (TEM) into brain. Mechanism-wise, miR-143-3p directly targets ATP6V1A, resulting in impaired lysosomal hydrolysis ability and reduced autophagic degradation of CAMs. Importantly, a VCAM-1-targeting EVs system to selectively deliver miR-143-3p inhibitor to pathological BMECs is created, which shows satisfactory therapeutic effects in both ICH and traumatic brain injury (TBI) mouse models. In conclusion, the study highlights the causal role of EVs-miR-143-3p in BMECs' dysfunction in acute brain injury and demonstrates a proof of concept that engineered EVs can be devised as a potentially applicable nucleotide drug delivery system for the treatment of CNS disorders.

Multifunctional Carbon Quantum Dots: Iron Clearance and Antioxidation for Neuroprotection in Intracerebral Hemorrhage Mice.

Iron overload and oxidative stress are pivotal in the pathogenesis of brain injury secondary to intracerebral hemorrhage (ICH). There is a compelling need for agents that can chelate iron and scavenge free radicals, particularly those that demonstrate substantial brain penetration, to mitigate ICH-related damage. In this study, we have engineered an amine-functionalized aspirin-derived carbon quantum dot (NACQD) with a nominal diameter of 6-13 nm. The NACQD possesses robust iron-binding and antioxidative capacities. Through intrathecal administration, NACQD therapy substantially reduced iron deposition and oxidative stress in brain tissue, alleviated meningeal inflammatory responses, and improved the recovery of neurological function in a murine ICH model. As a proof of concept, the intrathecal injection of NACQD is a promising therapeutic strategy to ameliorate the ICH injury.

Establishment of a rat model of severe spontaneous intracerebral hemorrhage

BackgroundSevere intracerebral hemorrhage (ICH) is the most devastating subtype of stroke resulting in high mortality and disability. At present, the development of targeted treatments to minimize the high morbidity and mortality is limited partly due to the lack of a severe ICH animal model. In this study, we aimed to establish an accurate severe ICH model in rats and examine the pathological and physiological changes associated with ICH. MethodsA rat model of severe ICH model was established by intrastriatal injection of autologous blood using different blood volumes (ICH 100 µL group, ICH 130 µL group, ICH 160 µL group, ICH 170 µL group, and ICH 180 µL group). The mortality was assessed during the 28-day post-ICH period. Short- and long-term neurological deficits were evaluated using the Longa method, foot fault, falling latency, and Morris water maze tests. Brain water content, hematoma volume, hemoglobin content, and magnetic resonance imaging were assessed to determine the extent of brain injury. Immunofluorescence staining was conducted to examine microglial activation and neuronal apoptosis. Hematoxylin and eosin (H&E) staining, lung water content, and western blotting were used to assess lung injury following ICH. ResultsThe mortality of ICH rats increased significantly with an increase in autologous blood injection. The 28-day mortality in the 100 µL, 130 µL, 160 µL, 170 µL, and 180 µL ICH groups were 5%, 20%, 40%, 75%, and 100%, respectively. A significantly higher 28-day mortality was observed in the ICH 160 µL group compared to the ICH 100 µL group. The ICH 160 µL group exhibited significantly increased neurological deficits, brain edema, hematoma volume, and hemoglobin content compared to the sham group. Compared with the sham operation group, the activation of microglia and neuronal death in ICH 160 µL rats increased. The use of H&E staining and western blotting demonstrated that disruption of the intra-alveolar structure, alveolar edema, and infiltration of inflammatory cells and cytokines into the lung tissue were more severe in the ICH 160 µL group than the sham group. ConclusionsA severe ICH model in rats was successfully established using an injection of autologous blood at a volume of 160 µL. This model may provide a valuable tool to examine the pathological mechanisms and potential therapeutic interventions of severe ICH.

Knockdown of BCL-3 Attenuates Inflammatory Response in Intracerebral Hemorrhage Through an rBMECs/MGs Microenvironment

Intracerebral hemorrhage (ICH) is a severe disease with high mortality. Recently, the role of BCL-3 in ICH has started to gain attention, but its mechanism remains unclear. A collagenase injection method was used to establish an ICH model in rats, and the expression of BCL-3 were detected. Rat brain microvascular endothelial cells (rBMECs) were isolated and induced with Hemin to establish an in vitro ICH model. The expression of BCL-3 was assessed, followed by detection of cell apoptosis. In the cell model, the recruitment, polarization, and pro-inflammatory features of the microglia (MGs) were assessed after co-cultured with rBMECs. Finally, in the ICH animal model, after knockdown of BCL-3, comprehensive evaluations of inflammatory responses in brain tissue, polarization and recruitment of microglia, and apoptosis were conducted. Results revealed an upregulated expression of BCL-3 in brain tissue of the ICH animal model. In Hemin-treated rBMECs, an upward trend in BCL-3 expression was observed, accompanied by an increase of cell apoptosis. After co-culturing with the in vitro model, microglia exhibited enhanced M1 polarization and intensified inflammatory responses. However, when BCL-3 expression was inhibited in the in vitro model, a reversal occurred in the polarization tendency and inflammatory responses of microglia. Additionally, after knockdown of BCL-3 in the animal model, notable improvements occurred in M1 polarization, infiltration of macrophages, and inflammatory reactions in the brain tissue. Therefore, BCL-3 modulates the inflammatory response after ICH occurrence through the BMECs/MGs microenvironment. Additionally, BCL-3 might be a potential therapeutic target for ICH management.

The phosphokinase activity of IRE1ɑ prevents the oxidative stress injury through miR-25/Nox4 pathway after ICH.

Endoplasmic reticulum (ER) stress and oxidative stress are the major pathologies encountered after intracerebral hemorrhage (ICH). Inositol-requiring enzyme-1 alpha (IRE1α) is the most evolutionarily conserved ER stress sensor, which plays a role in monitoring and responding to the accumulation of unfolded/misfolded proteins in the ER lumen. Recent studies have shown that ER stress is profoundly related to oxidative stress in physiological or pathological conditions. The purpose of this study was to investigate the role of IRE1α in oxidative stress and the potential mechanism. A mouse model of ICH was established by autologous blood injection. The IRE1α phosphokinase inhibitor KIRA6 was administrated intranasally at 1 h after ICH, antagomiR-25 and agomiR-25 were injected intraventricularly at 24 h before ICH. Western blot analysis, RT-qPCR, immunofluorescence staining, hematoma volume, neurobehavioral tests, dihydroethidium (DHE) staining, H2O2 content, brain water content, body weight, Hematoxylin and Eosin (HE) staining, Nissl staining, Morris Water Maze (MWM) and Elevated Plus Maze (EPM) were performed. Endogenous phosphorylated IRE1α (p-IRE1α), miR-25-3p, and Nox4 were increased in the ICH model. Administration of KIRA6 downregulated miR-25-3p expression, upregulated Nox4 expression, promoted the level of oxidative stress, increased hematoma volume, exacerbated brain edema and neurological deficits, reduced body weight, aggravated spatial learning and memory deficits, and increased anxiety levels. Then antagomiR-25 further upregulated the expression of Nox4, promoted the level of oxidative stress, increased hematoma volume, exacerbated brain edema and neurological deficits, whereas agomiR-25 reversed the effects promoted by KIRA6. The IRE1α phosphokinase activity is involved in the oxidative stress response through miR-25/Nox4 pathway in the mouse ICH brain.

Screening and identification of differential-expressed RNAs in thrombin-induced in vitro model of intracerebral hemorrhage.

Survival of olfactory mucosal mesenchymal stem cells (OM-MSCs) remains the low level in the cerebral microenvironment during intracerebral hemorrhage (ICH). This article aims to reveal the differential expression profile of ICH-stimulated OM-MSCs based on whole transcriptome sequence analysis. OM-MSCs were isolated from 6-week C57BL/6 mice. Morphology and surface markers of OM-MSCs were investigated by light microscope and flow cytometry, respectively. OM-MSCs were incubated with 20 U/mL thrombin for 24h to mimic ICH-induced injury in vitro. Total RNA was extracted for whole transcriptome sequencing and qPCR. OM-MSCs were characterized by negative for CD45 and CD34, and positive for CD44, CD90 and CD29. Thrombin led to decrease in cell viability and increase in senescence and apoptosis in OM-MSCs. In total, 736 lncRNAs (upregulated: 393; downregulated: 343), 21 miRNAs (upregulated: 7; downregulated: 14) and 807 mRNAs (upregulated: 422; downregulated: 385) were identified. GO and KEGG pathways were enriched in protein heterodimerization activity, trans-synaptic signaling, membrane pathway, alcohol metabolic process, organic hydroxy compound biosynthesis process, secondary alcohol metabolic process, alcoholism, neutrophil extracellular trap formation, systemic lupus erythematosus, metabolic process, steroid biosynthesis and drug metabolism-cytochrome P450. 200 lncRNA-miRNA-mRNA were predicted in thrombin-induced OM-MSCs. Based on qPCR, we validated COMMD1B, MOAP1, lncRNA CAPN15, lncRNA ALDH1L2, miR-3473b and miR-1964-3p were upregulated in thrombin-stimulated OM-MSCs, and GM20431, lncRNA GAPDH and miR-122b-3p were downregulated. Our findings provide novel understanding for thrombin-induced injury in OM-MSCs. Differently-expressed RNAs can be the targets of improving therapeutic application of OM-MSCs.

Group 2 innate lymphoid cells suppress neuroinflammation and brain injury following intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) mobilizes circulating leukocytes that contribute to neuroinflammation and neural injury. However, little is known about the endogenous regulatory immune mechanisms to restrict neuroinflammation following ICH. We examined the role of group 2 innate lymphoid cells (ILC2) that are a specialized subset of innate immune modulators in a mouse model of ICH. We found accumulation of ILC2 in the brain following acute ICH and a concomitant increase of ILC2 within the peripheral lymph nodes. Depletion of ILC2 exacerbated neurodeficits and brain edema after ICH in male and female mice. This aggravated ICH injury was accompanied by augmented microglia activity and leukocyte infiltration. In contrast, expansion of ILC2 using IL-33 led to reduced ICH injury, microglia activity and leukocyte infiltration. Notably, elimination of microglia using a colony stimulating factor 1 receptor inhibitor diminished the exacerbation of ICH injury induced by depletion of ILC2. Brain-infiltrating ILC2 had upregulation of IL-13 after ICH. Results from in vitro assays revealed that ILC2 suppressed thrombin-induced inflammatory activity in microglia-like BV2 cells. Thus, our findings demonstrate that ILC2 suppress neuroinflammation and acute ICH injury.

Wnt5a/Ror2 promotes Nrf2-mediated tissue protective function of astrocytes after brain injury.

Astrocytes, a type of glial cells, play critical roles in promoting the protection and repair of damaged tissues after brain injury. Inflammatory cytokines and growth factors can affect gene expression in astrocytes in injured brains, but signaling pathways and transcriptional mechanisms that regulate tissue protective functions of astrocytes are still poorly understood. In this study, we investigated the molecular mechanisms regulating the function of reactive astrocytes induced in mouse models of stab wound (SW) brain injury and collagenase-induced intracerebral hemorrhage (ICH). We show that basic fibroblast growth factor (bFGF), whose expression is up-regulated in mouse brains after SW injury and ICH, acts synergistically with inflammatory cytokines to activate E2F1-mediated transcription of a gene encoding the Ror-family protein Ror2, a receptor for Wnt5a, in cultured astrocytes. We also found that subsequent activation of Wnt5a/Ror2 signaling in astrocytes results in nuclear accumulation of antioxidative transcription factor Nrf2 at least partly by increased expression of p62/Sqstm1, leading to promoted expression of several Nrf2 target genes, including heme oxygenase 1. Finally, we provide evidence demonstrating that enhanced activation of Wnt5a/Ror2 signaling in astrocytes reduces cellular damage caused by hemin, a degradation product of hemoglobin, and promotes repair of the damaged blood brain barrier after brain hemorrhage.

Microglial FoxO3a deficiency ameliorates ferroptosis-induced brain injury of intracerebral haemorrhage via regulating autophagy and heme oxygenase-1.

Microglial HO-1 regulates iron metabolism in the brain. Intracerebral haemorrhage (ICH) shares features of ferroptosis and necroptosis; hemin is an oxidized product of haemoglobin from lysed red blood cells, leading to secondary injury. However, little is known about the underlying molecular mechanisms attributable to secondary injury by hemin or ICH. In this study, we first show that FoxO3a was highly co-located with neurons and microglia but not astrocytes area of ICH model mice. Hemin activated FoxO3a/ATG-mediated autophagy and HO-1 signalling resulting in ferroptosis invitro and in a mice model of brain haemorrhage. Accordingly, autophagy inhibitor Baf-A1 or HO-1 inhibitor ZnPP protected against hemin-induced ferroptosis. Hemin promoted ferroptosis of neuronal cells via FoxO3a/ATG-mediated autophagy and HO-1 signalling pathway. Knock-down of FoxO3a inhibited autophagy and prevented hemin-induced ferroptosis dependent of HO-1 signalling. We first showed that hemin stimulated microglial FoxO3a/HO-1 expression and enhanced the microglial polarisation towards the M1 phenotype, while knockdown of microglial FoxO3a inhibited pro-inflammatory cytokine production in microglia. Furthermore, the microglia activation in the striatum showed significant along with a high expression level of FoxO3a in the ICH mice. We found that conditional knockout of FoxO3a in microglia in mice alleviated neurological deficits and microglia activation as well as ferroptosis-induced striatum injury in the autologous blood-induced ICH model. We demonstrate, for the first time, that FoxO3a/ATG-mediated autophagy and HO-1 play an important role in microglial activation and ferroptosis-induced striatum injury of ICH, identifying a new therapeutic avenue for the treatment of ICH.

Intracerebral Administration of a Novel Self-Assembling Peptide Hydrogel Is Safe and Supports Cell Proliferation in Experimental Intracerebral Haemorrhage

Intracerebral haemorrhage (ICH) is the deadliest form of stroke, but current treatment options are limited, meaning ICH survivors are often left with life-changing disabilities. The significant unmet clinical need and socioeconomic burden of ICH mean novel regenerative medicine approaches are gaining interest. To facilitate the regeneration of the ICH lesion, injectable biomimetic hydrogels are proposed as both scaffolds for endogenous repair and delivery platforms for pro-regenerative therapies. In this paper, the objective was to explore whether injection of a novel self-assembling peptide hydrogel (SAPH) Alpha2 was feasible, safe and could stimulate brain tissue regeneration, in a collagenase-induced ICH model in rats. Alpha2 was administered intracerebrally at 7 days post ICH and functional outcome measures, histological markers of damage and repair and RNA-sequencing were investigated for up to 8 weeks. The hydrogel Alpha2 was safe, well-tolerated and was retained in the lesion for several weeks, where it allowed infiltration of host cells. The hydrogel had a largely neutral effect on functional outcomes and expression of angiogenic and neurogenic markers but led to increased numbers of proliferating cells. RNAseq and pathway analysis showed that ICH altered genes related to inflammatory and phagocytic pathways, and these changes were also observed after administration of hydrogel. Overall, the results show that the novel hydrogel was safe when injected intracerebrally and had no negative effects on functional outcomes but increased cell proliferation. To elicit a regenerative effect, future studies could use a functionalised hydrogel or combine it with an adjunct therapy.

Long non-coding RNA MIR17HG impedes FOSL2-mediated transcription activation of HIC1 to maintain a pro-inflammatory phenotype of microglia during intracerebral haemorrhage.

Activation and polarization of microglia play decisive roles in the progression of intracerebral haemorrhage (ICH), and lactate exposure correlates with microglia polarization. This study explores molecules influencing lactate production and microglia phenotype alteration following ICH. A murine model of ICH was induced by intracerebral injection of collagenase. The mice experienced autonomous neurological function recovery, haematoma resolution and rapid lactate production, along with a gradual increase in angiogenesis activity, neuronal recovery and an M1-to-M2 phenotype change of microglia. Galloflavin, a lactate dehydrogenase antagonist, suppressed this phenotype change and the functional recovery in mice. FOS like 2 (FOSL2) was significantly upregulated in the brain tissues from day 7 post-ICH. Overexpression of FOSL2 induced an M1-to-M2 phenotype shift in microglia and accelerated lactate production in vivo and in haemoglobin-treated microglia in vitro. Long non-coding RNA MIR17HG impeded FOSL2-mediated transcription activation of hypermethylated in cancer 1 (HIC1). MIR17HG overexpression induced pro-inflammatory activation of microglia in mice, which was blocked by further HIC1 overexpression. Overall, this study demonstrates that MIR17HG maintains a pro-inflammatory phenotype of microglia during ICH progression by negating FOSL2-mediated transcription activation of HIC1. Specific inhibition of MIR17HG or upregulation of FOSL2 or HIC1 may favour inflammation inhibition and haematoma resolution in ICH.

EAAT3 impedes oligodendrocyte remyelination in chronic cerebral hypoperfusion-induced white matter injury.

Chronic cerebral hypoperfusion-induced demyelination causes progressive white matter injury, although the pathogenic pathways are unknown. The Single Cell Portal and PanglaoDB databases were used to analyze single-cell RNA sequencing experiments to determine the pattern of EAAT3 expression in CNS cells. Immunofluorescence (IF) was used to detect EAAT3 expression in oligodendrocytes and oligodendrocyte progenitor cells (OPCs). EAAT3 levels in mouse brains were measured using a western blot at various phases of development, as well as in traumatic brain injury (TBI) and intracerebral hemorrhage (ICH) mouse models. The mouse bilateral carotid artery stenosis (BCAS) model was used to create white matter injury. IF, Luxol Fast Blue staining, and electron microscopy were used to investigate the effect of remyelination. 5-Ethynyl-2-Deoxy Uridine staining, transwell chamber assays, and IF were used to examine the effects of OPCs' proliferation, migration, and differentiation in vivo and in vitro. The novel object recognition test, the Y-maze test, the rotarod test, and the grid walking test were used to examine the impact of behavioral modifications. A considerable amount of EAAT3 was expressed in OPCs and mature oligodendrocytes, according to single-cell RNA sequencing data. During multiple critical phases of mouse brain development, there were no substantial changes in EAAT3 levels in the hippocampus, cerebral cortex, or white matter. Furthermore, neither the TBI nor ICH models significantly affected the levels of EAAT3 in the aforementioned brain areas. The chronic white matter injury caused by BCAS, on the other hand, resulted in a strikingly high level of EAAT3 expression in the oligodendroglia and white matter. Correspondingly, blocking EAAT3 assisted in the recovery of cognitive and motor impairment as well as the restoration of cerebral blood flow following BCAS. Furthermore, EAAT3 suppression was connected to improved OPCs' survival and proliferation in vivo as well as faster OPCs' proliferation, migration, and differentiation in vitro. Furthermore, this study revealed that the mTOR pathway is implicated in EAAT3-mediated remyelination. Our findings provide the first evidence that abnormally high levels of oligodendroglial EAAT3 in chronic cerebral hypoperfusion impair OPCs' pro-remyelination actions, hence impeding white matter repair and functional recovery. EAAT3 inhibitors could be useful in the treatment of ischemia demyelination.

The neuroprotective effect of dl-3-n-butylphthalide on the brain with experimental intracerebral hemorrhage

Intracerebral hemorrhage (ICH) is the most devastating subtype of stroke, nevertheless specific treatments with conclusive clinical benefit in improving outcomes of ICH remain lacking. The present study applied dl-3-n-butylphthalide (NBP), a compound approved for the treatment of ischemic stroke and rarely studied in ICH, to an experimental animal model of ICH, aiming to evaluate the therapeutic effects of NBP on ICH and the potential mechanisms. The results showed that rats receiving NBP administration exhibited a structural and functional restoration of brain after ICH mainly manifested as alleviation of neuronal apoptosis, suppression of neuroinflammation and oxidative stress, neurovascular remodeling, and eventually improvement of neurological deficits. In addition, several protein targets of NBP were revealed, which mainly play molecular functions of ribonucleoside triphosphate phosphatase activity, pyrophosphatase activity, hydrolase activity and GTPase activity, and participate in the biological process of brain development by regulating the formation of cellular components such as spindles, polymeric cytoskeletal fibers, microtubules and synapses, through mediating pathways such as VEGF signaling pathway, Fc epsilon RI signaling pathway, ECM-receptor interaction, Fc gamma R-mediated phagocytosis, peroxisome and so on, guiding the mechanism exploration of NBP therapy to some extent. Taken together, the study added some new evidence to the application of NBP in ICH treatment.

Therapeutic effect of allicin in a mouse model of intracerebral hemorrhage

Natural compounds with sulfur moiety produce various biological actions that may be beneficial for the therapies of several devastative disorders of the central nervous system. Here we investigated potential therapeutic effect of allicin, an organosulfur compound derived from garlic, in a mouse model of intracerebral hemorrhage (ICH) based on intrastriatal collagenase injection. Daily intraperitoneal administration of allicin (50 mg/kg) from 3 h after induction of ICH afforded neuroprotective effects, as evidenced by the increase of surviving neurons in the hematoma, reduction of axonal transport impairment, and prevention of axon tract injury. In addition, allicin inhibited accumulation of activated microglia/macrophages around the hematoma and infiltration of neutrophils within the hematoma. Allicin also suppressed ICH-induced mRNA upregulation of pro-inflammatory factors such as interleukin 6 and C-X-C motif ligand 2 in the brain, suggesting its anti-inflammatory effect. Moreover, ICH-induced increase of malondialdehyde as well as decrease of total glutathione in the brain was attenuated by allicin. Finally, allicin-treated mice showed better recovery of sensorimotor functions after ICH than vehicle-treated mice. These results indicate that allicin produces a therapeutic effect on ICH pathology via alleviation of neuronal damage, inflammatory responses and oxidative stress in the brain.

Deubiquitylating Enzyme OTUB1 Facilitates Neuronal Survival After Intracerebral Hemorrhage Via Inhibiting NF-κB-triggered Apoptotic Cascades.

The deubiquitylase OTU domain-containing ubiquitin aldehyde-binding protein 1 (OTUB1) has been implicated in the pathogenesis of various human diseases. However, the molecular mechanism by which OTUB1 participates in the pathogenesis of intracerebral hemorrhage (ICH) remains elusive. In the present study, we established an autologous whole blood fusion-induced ICH model in C57BL/6J mice. We showed that the upregulation of OTUB1 contributes to the attenuation of Nuclear factor kappa B (NF-κB) and its downstream apoptotic signaling after ICH. OTUB1 directly associates with NF-κB precursors p105 and p100 after ICH, leading to attenuated polyubiquitylation of p105 and p100. Moreover, we revealed that NF-κB signaling was modestly activated both in ICH tissues and hemin-exposed HT-22 neuronal cells, accompanied with the activation of NF-κB downstream pro-apoptotic signaling. Notably, overexpression of OTUB1 strongly inhibited hemin-induced NF-κB activation, whereas interference of OTUB1 led to the opposite effect. Finally, we revealed that lentiviral transduction of OTUB1 markedly ameliorated hemin-induced apoptotic signaling and HT-22 neuronal death. Collectively, these findings suggest that the upregulation of OTUB1 serves as a neuroprotective mechanism in antagonizing neuroinflammation-induced NF-κB signaling and neuronal death, shed new light on manipulating intracellular deubiquitylating pathways as novel interventive approaches against ICH-induced secondary neuronal damage and death.

A systematic review and meta-analysis on the efficacy of glibenclamide in animal models of intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) is a devastating stroke with many mechanisms of injury. Edema worsens outcome and can lead to mortality after ICH. Glibenclamide (GLC), a sulfonylurea 1- transient receptor potential melastatin 4 (Sur1-Trpm4) channel blocker, has been shown to attenuate edema in ischemic stroke models, raising the possibility of benefit in ICH. This meta-analysis synthesizes current pre-clinical (rodent) literature regarding the efficacy of post-ICH GLC administration (vs. vehicle controls) on behaviour (i.e., neurological deficit, motor, and memory outcomes), edema, hematoma volume, and injury volume. Six studies (5 in rats and 1 in mice) were included in our meta-analysis (PROSPERO registration = CRD42021283614). GLC significantly improved behaviour (standardized mean difference (SMD) = -0.63, [-1.16, -0.09], n = 70-74) and reduced edema (SMD = -0.91, [-1.64, -0.18], n = 70), but did not affect hematoma volume (SMD = 0.0788, [-0.5631, 0.7207], n = 18-20), or injury volume (SMD = 0.2892, [-0.4950, 1.0734], n = 24). However, these results should be interpreted cautiously. Findings were conflicted with 2 negative and 4 positive reports, and Egger regressions indicated missing negative edema data (p = 0.0001), and possible missing negative behavioural data (p = 0.0766). Experimental quality assessed via the SYRCLE and CAMARADES checklists was concerning, as most studies demonstrated high risks of bias. Studies were generally low-powered (e.g., average n = 14.4 for behaviour), and future studies should employ sample sizes of 41 to detect our observed effect size in behaviour and 33 to detect our observed effect in edema. Overall, missing negative studies, low study quality, high risk of bias, and incomplete attention to key recommendations (e.g., investigating female, aged, and co-morbid animals) suggest that further high-powered confirmatory studies are needed before conclusive statements about GLC's efficacy in ICH can be made, and before further clinical trials are performed.