Rat Model Of Intracerebral Hemorrhage Research Articles

Examining Transcriptomic Alterations in Rat Models of Intracerebral Hemorrhage and Severe Intracerebral Hemorrhage.

Intracerebral hemorrhage (ICH) is a life-threatening condition associated with significant morbidity and mortality. This study investigates transcriptomic alterations in rodent models of ICH and severe ICH to shed light on the genetic pathways involved in hemorrhagic brain injury. We performed principal component analysis, revealing distinct principal component segments of normal rats compared to ICH and severe ICH rats. We employed heatmaps and volcano plots to identify differentially expressed genes and utilized bar plots and KEGG pathway analysis to elucidate the molecular pathways involved. We identified a multitude of differentially expressed genes in both the ICH and severe ICH models. Our results revealed 5679 common genes among the normal, ICH, and severe ICH groups in the upregulated genes group, and 1196 common genes in the downregulated genes, respectively. A volcano plot comparing these groups further highlighted common genes, including PDPN, TIMP1, SERPINE1, TUBB6, and CD44. These findings underscore the complex interplay of genes involved in inflammation, oxidative stress, and neuronal damage. Furthermore, pathway enrichment analysis uncovered key signaling pathways, including the TNF signaling pathway, protein processing in the endoplasmic reticulum, MAPK signaling pathway, and Fc gamma R-mediated phagocytosis, implicated in the pathogenesis of ICH.

Cystatin C Attenuates Perihematomal Secondary Brain Injury by Inhibiting the Cathepsin B/NLRP3 Signaling Pathway in a Rat Model of Intracerebral Hemorrhage.

Secondary brain injury (SBI) is a noticeable contributor to the high mortality and morbidity rates associated with intracerebral hemorrhage (ICH), and effective treatment options remain limited. Cystatin C (CysC) emerges as a novel candidate for SBI intervention. The therapeutic effects and underlying mechanisms of CysC in mitigating SBI following ICH were explored in the current research. An in vivo ICH rat model was established by injecting autologous blood into the right caudate nucleus. Western blotting (WB) was utilized to assess the levels of CysC, cathepsin B (CTSB), and the NLRP3 inflammasome. Subsequently, the ICH rat model was treated with exogenous CysC supplementation or CysC knockdown plasmids. Various parameters, including Evans blue (EB) extravasation, brain water content, and neurological function in rats, were examined. RT-qPCR and WB were employed to determine the expression levels of CTSB and the NLRP3 inflammasome. The co-expression of CTSB, CysC, and NLRP3 inflammasome with GFAP, NeuN, and Iba1 was assessed through double-labeled immunofluorescence. The interaction between CysC and CTSB was investigated using double-labeled immunofluorescence and co-immunoprecipitation. The findings revealed an elevation of CysC expression level, particularly at 24h after ICH. Exogenous CysC supplementation alleviated severe brain edema, neurological deficit scores, and EB extravasation induced by ICH. Conversely, CysC knockdown produced opposite effects. The expression levels of CTSB and the NLRP3 inflammasome were significantly risen following ICH, and exogenous CysC supplement attenuated their expression levels. Double-labeled immunofluorescence illustrated that CysC, CTSB, and the NLRP3 inflammasome were predominantly expressed in microglial cells, and the interaction between CysC and CTSB was evidenced. CysC exhibited potential in ameliorating SBI following ICH via effectively suppressing the activation of the NLRP3 inflammasome mediated by CTSB specifically in microglial cells. These findings underscore the prospective therapeutic efficacy of CysC in the treatment of ICH-induced complications.

Activating transcription factor 6 alleviates secondary brain injury by increasing cystathionine γ-lyase expression in a rat model of intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) comprises primary and secondary injuries, the latter of which induces increased inflammation and apoptosis and is more severe. Activating transcription factor 6 (ATF6) is a type-II transmembrane protein in the endoplasmic reticulum (ER). ATF6 target genes could improve ER homeostasis, which contributes to cryoprotection. Hence, we predict that ATF6 will have a protective effect on brain tissue after ICH. The ICH rat model was generated through autologous blood injection into the right basal ganglia, the expression of ATF6 after ICH was determined by WB and IF. The expression of ATF6 was effectively controlled by means of intervention, and a series of measures was used to detect cell death, neuroinflammation, brain edema, blood-brain barrier and other indicators after ICH. Finally, the effects on long-term neural function of rats were measured by behavioral means. ATF6 was significantly increased in the ICH-induced brain tissues. Further, ATF6 was found to modulate the expression of cystathionine γ-lyase (CTH) after ICH. Upregulation of ATF6 attenuated neuronal apoptosis and inflammation in ICH rats, along with mitigation of ICH-induced brain edema, blood-brain barrier deterioration, and cognitive behavior defects. Conversely, ATF6 genetic knockdown induced effects counter to those aforementioned. This study thereby emphasizes the crucial role of ATF6 in secondary brain injury in response to ICH, indicating that ATF6 upregulation may potentially ameliorate ICH-induced secondary brain injury. Consequently, ATF6 could serve as a promising therapeutic target to alleviate clinical ICH-induced secondary brain injuries.

Electroacupuncture reduces oxidative stress response and improves secondary injury of intracerebral hemorrhage in rats by activating the peroxisome proliferator-activated receptor-γ/nuclear factor erythroid2-related factor 2/γ-glutamylcysteine synthetase pathway.

Intracerebral hemorrhage (ICH) is a severe stroke subtype. Secondary injury is a key factor leading to neurological deficits after ICH. Electroacupuncture (EA) can improve the neurological function after ICH, however, its internal mechanism is still unclear. The aim of this study is to investigate whether EA could ameliorate secondary injury after ICH through antioxidative stress and its potential regulatory mechanism. A rat model of ICH was established by injecting autologous blood into striatum. After the intervention of EA and EA combined with peroxisome proliferator-activated receptor-γ (PPARγ) blocker, Zea-longa scores, modified neurological severity scores and open field tests were used to evaluate the neurological function of the rats. Flow cytometry detected tissue reactive oxygen species (ROS) levels. Tissue tumor necrosis factor-α (TNF-α) levels were analyzed by enzyme-linked immunosorbent assays. The protein expressions of PPAR γ, nuclear factor erythroid2-related factor 2 (Nrf2) and γ-glutamylcysteine synthetase (γ-GCS) were detected by Western blot. Immunohistochemistry was used to observe the activation of microglia. The demyelination degree of axon myelin was observed by transmission electron microscope. Compared with the model group, EA intervention improved neurological function, decreased ROS and TNF-α levels, increased the protein expression of PPARγ, Nrf2 and γ-GCS, and reduced the activation of microglia, it also alleviated axonal myelin sheath damage. In addition, the neuroprotective effect of EA was partially attenuated by PPARγ blocker. EA ameliorated the neurological function of secondary injury after ICH in rats, possibly by activating the PPARγ/Nrf2/γ-GCS signaling pathway, reducing microglia activation, and inhibiting oxidative stress, thus alleviating the extent of axonal demyelination plays a role.

Unraveling the complex pathophysiology of white matter hemorrhage in intracerebral stroke: A single-cell RNA sequencing approach.

This study aims to elucidate the cellular dynamics and pathophysiology of white matter hemorrhage (WMH) in intracerebral hemorrhage (ICH). Using varying doses of collagenase IV, a consistent rat ICH model characterized by pronounced WMH was established. Verification was achieved through behavioral assays, hematoma volume, and histological evaluations. Single-cell suspensions from the hemorrhaged region of the ipsilateral striatum on day three post-ICH were profiled using single-cell RNA sequencing (scRNA-seq). Gene Ontology (GO) and gene set variation analysis (GSVA) further interpreted the differentially expressed genes (DEGs). Following WMH induction, there was a notable increase in the percentage of myeloid cells and oligodendrocyte precursor cells (OPCs), alongside a reduction in the percentage of neurons, microglia, and oligodendrocytes (OLGs). Post-ICH WMH showed homeostatic microglia transitioning into pro-, anti-inflammatory, and proliferative states, influencing lipid metabolic pathways. Myeloid cells amplified chemokine expression, linked with ferroptosis pathways. Macrophages exhibited M1 and M2 phenotypes, and post-WMH, macrophages displayed a predominance of M2 phenotypes, characterized by their anti-inflammatory properties. A surge in OPC proliferation aligned with enhanced ribosomal signaling, suggesting potential reparative responses post-WMH. The study offers valuable insights into WMH's complex pathophysiology following ICH, highlighting the significance and utility of scRNA-seq in understanding the cellular dynamics and contributing to future cerebrovascular research.

"Baihui" (DU20)-penetrating "Qubin" (GB7) acupuncture on blood-brain barrier integrity in rat intracerebral hemorrhage models via the RhoA/ROCK II/MLC 2 signaling pathway.

Blocking the RhoA/ROCK II/MLC 2 (Ras homolog gene family member A/Rho kinase II/myosin light chain 2) signaling pathway can initiate neuroprotective mechanisms against neurological diseases such as stroke, cerebral ischemia, and subarachnoid hemorrhage. Nevertheless, it is not clear whether and how disrupting the RhoA/ROCK II/MLC 2 signaling pathway changes the pathogenic processes of the blood-brain barrier (BBB) after intracerebral hemorrhage (ICH). The present investigation included the injection of rat caudal vein blood into the basal ganglia area to replicate the pathophysiological conditions caused by ICH. Scalp acupuncture (SA) therapy was performed on rats with ICH at the acupuncture point "Baihui"-penetrating "Qubin," and the ROCK selective inhibitor fasudil was used as a positive control to evaluate the inhibitory effect of acupuncture on the RhoA/ROCK II/MLC 2 signaling pathway. Post-assessments included neurological deficits, brain edema, Evans blue extravasation, Western blot, quantitative polymerase chain reaction, and transmission electron microscope imaging. We found that ROCK II acts as a promoter of the RhoA/ROCK II/MLC 2 signaling pathway, and its expression increased at 6 h after ICH, peaked at 3 days, and then decreased at 7 days after ICH, but was still higher than the pre-intervention level. According to some experimental results, although 3 days is the peak, 7 days is the best time point for acupuncture treatment. Starting from 6 h after ICH, the neurovascular structure and endothelial cell morphology around the hematoma began to change. Based on the changes in the promoter ROCK II, a 7-day time point was selected as the breakthrough point for treating ICH model rats in the main experiment. The results of this experiment showed that both SA at "Baihui"-penetrating "Qubin" and treatment with fasudil could improve the expression of endothelial-related proteins by inhibiting the RhoA/ROCK II/MLC 2 signaling pathway and reduce neurological dysfunction, brain edema, and BBB permeability in rats. This study found that these experimental data indicated that SA at "Baihui"-penetrating "Qubin" could preserve BBB integrity and neurological function recovery after ICH by inhibiting RhoA/ROCK II/MLC 2 signaling pathway activation and by regulating endothelial cell-related proteins.

Use of Network Pharmacology and Experiment Validation to Uncover the Mechanism of Jianshen Lishui Prescription in the Treatment of Intracerebral Hemorrhage.

As a Chinese medicinal formula, the Jianshen Lishui prescription has been clinically proven to be effective in treating intracerebral hemorrhage (ICH). Yet, the mechanisms involved are unknown. (1) Network pharmacology analysis: It involved the screening of active components in the Jianshen Lishui prescription, identification of potential targets for these components, and the screening of ICH-related targets. Common targets for both disease and drug were identified. Protein- protein interaction networks were constructed, followed by further screening of core targets. Gene Ontology(GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes( KEGG) pathway enrichment analysis were performed on these core targets. Finally, molecular docking verification was carried out using the active components and core targets. (2) Experimental verification: It was conducted using a rat model of intracerebral hemorrhage. This involved observing neurological deficit scores in the rats and measuring cerebral water content. The effects of Jianshen Lishui prescription on the neurological function, cerebral water content, and brain tissue core targets were observed through HE staining, Western blot and qPCR. (1) In this study, 29 common targets were obtained by intersecting 256 potential drug targets and 642 genes associated with ICH. 9 core targets were obtained by employing the protein- protein interaction (PPI) construction system to screen more specific targets. In addition, the findings revealed that the molecular mechanism of Jianshen Lishui prescription in treating ICH was mainly related to cancer signaling pathways and signal transduction pathways, based on the results of GO and KEGG enrichment analysis. Molecular docking results showed that the active constituent of Jianshen Lishui prescription mannitol has the highest binding activity with KRAS, luteolin, and Poria sterol with AR, INS1 and KRAS, cerebrosterol with GNB1, INS and ESR1, and sitosterol with AR, INS1 and KRAS. (2) Animal experiments verified that Jianshen Lishui prescription significantly alleviated encephaledema and improved nerve functions of the rat model of ICH. And INS1 expression levels were upregulated and the expression levels of AR, KRAS, PTGS2, and ESR1 were down-regulated by the prescription. Jianshen Lishui prescription protects the nerve function of ICH patients by inhibiting inflammation and reducing cerebral edema. This study provides more supportive evidences for the clinical use of traditional Chinese prescriptions in 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

Glibenclamide pretreatment attenuates early hematoma expansion of warfarin-associated intracerebral hemorrhage in rats by alleviating perihematomal blood–brain barrier dysfunction

BackgroundHematoma expansion is a determinant of poor outcome of intracerebral hemorrhage but occurs frequently, especially in warfarin-associated intracerebral hemorrhage (W-ICH). In the present study, we employ the warfarin-associated intracerebral hemorrhage (W-ICH) rat model, to explore the efficacy and potential mechanism of glibenclamide pretreatment on hematoma expansion after intracerebral hemorrhage, hoping to provide proof of concept that glibenclamide in stroke primary and secondary prevention is also potentially beneficial for intracerebral hemorrhage patients at early stage.MethodsIn the present study, we tested whether glibenclamide, a common hypoglycemic drug, could attenuate hematoma expansion in a rat model of W-ICH. Hematoma expansion was evaluated using magnetic resonance imaging; brain injury was evaluated by brain edema and neuronal death; and functional outcome was evaluated by neurological scores. Then blood–brain barrier integrity was assessed using Evans blue extravasation and tight junction-related protein.ResultsThe data indicated that glibenclamide pretreatment significantly attenuated hematoma expansion at 24 h after W-ICH, thus mitigating brain edema and neuronal death and promoting neurological function recovery, which may benefit from alleviating blood–brain barrier disruption by suppressing matrix metallopeptidase-9.ConclusionsThe results indicate that glibenclamide pretreatment in stroke primary and secondary prevention might be a promising therapy for hematoma expansion at the early stage of W-ICH.

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 Neuroprotective Effects and Probable Mechanisms of Everolimus in a Rat Model of Intracerebral Hemorrhage.

Mammalian target of rapamycin (mTOR) is a central regulator of cellular growth and homeostasis. Changes in mTOR activity are often observed in many neurological diseases, such as stroke. Intracerebral hemorrhage (ICH) is associated with high mortality and morbidity. However, there are currently no treatments that have been shown to enhance outcomes following ICH, so new treatments are urgently required. In this study, a selective mTOR inhibitor, everolimus, was applied to investigate the outcome after ICH and the possible underlying mechanism. The ICH model was established by autologous blood injection. Everolimus (50 and 100µg/kg) was administered intraperitoneally for 14 consecutive days' post-operation. The neurological functions were examined at 3, 7, and 14days' post-ICH. Samples of brain tissue were collected to perform histopathological and immunohistochemical (NF-k-positive cell) examinations. Besides, the striatum was used to evaluate parameters related to oxidative stress (superoxide dismutase (SOD) activity, malondialdehyde (MDA), and total thiol levels) and inflammation markers (TNF-α and NO). Everolimus ameliorated ICH-induced neurological deficits. In addition, treatment with everolimus reduced infarct volume and NF-k-β positive cells as compared to the ICH group. Furthermore, everolimus significantly increased total thiol content and SOD activity while significantly reducing MDA, NO, and TNF- levels as compared to the ICH group. Collectively, our investigation showed that everolimus improves ICH outcome and modulates oxidative stress and inflammation after ICH. Treatment with rapamycin reduced neurological deficient, oxidative stress, and inflammation in a rat model of intracerebral hemorrhage.

Modulation of autophagy in the rats with acute intracerebral hemorrhage by acupuncture based on JNK pathway

To investigate the effects of acupuncture on JNK pathway and autophagy level in rats with intracerebral hemorrhage （ICH） and explore the partial mechanism of acupuncture against ICH. SD rats were randomly divided into blank group, model group and acupuncture group. Each group was divided into Day 1, Day 3 and Day 7 subgroups respectively, with 5 rats in each group. The autologous blood injection was adopted to duplicate rat model of ICH. In the acupuncture group, the needle was inserted from "Baihui" （GV20） towards "Qubin" （GB7） on the affected side, stimulating for 30 min each time, once daily； the same acupuncture technique was opera-ted in each subgroup for 1, 3 and 7 days, separately. Using Bederson scale, the neurological deficit was evaluated in each group. Western blot was adopted to detect the protein expression levels of Beclin1, LC3Ⅰ/Ⅱ, phosphorylated c-Jun amino-terminal kinase （p-JNK） and the phosphorylated （p）-c-Jun around hematoma lesion of the brain tissue of rats in each group. After treatment, the neurological deficit score of rats in the model group was higher than that of the blank group at each time point （P<0.05）, and the score of the acupuncture group started declining since the 3rd day of treatment when compared with the model group （P<0.05）. At each time point, compared with the blank group, the protein expression levels of LC3Ⅰ/Ⅱ, Beclin1, p-c-Jun and p-JNK was increased （P<0.01）. Compared with the model group, the protein expression level of LC3Ⅰ/Ⅱ was reduced （P<0.05）； the protein expression levels of Beclin1, p-c-Jun and p-JNK was increased （P<0.05, P<0.01） on day 3 and 7 in the acupuncture group. Acupuncture can activate the JNK pathway in the brain tissue of rats with ICH and increase the level of autophagy, thereby improving the neurological function of the rats with ICH.

MIC19 Exerts Neuroprotective Role via Maintaining the Mitochondrial Structure in a Rat Model of Intracerebral Hemorrhage.

As an essential constituent of the mitochondrial contact site and cristae organization system (MICOS), MIC19 plays a crucial role in maintaining the stability of mitochondrial function and microstructure. However, the mechanisms and functions of MIC19 in intracerebral hemorrhage (ICH) remain unknown and need to be investigated. Sprague Dawley (SD) rats injected with autologous blood obtained from the caudal artery, and cultured neurons exposed to oxygen hemoglobin (OxyHb) were used to establish and emulate the ICH model in vivo and in vitro. Lentiviral vector encoding MIC19 or MIC19 short hairpin ribonucleic acid (shRNA) was constructed and administered to rats by intracerebroventricular injection to overexpress or knock down MIC19, respectively. First, MIC19 protein levels were increased after ICH modeling. After virus transfection and subsequent ICH modeling, we observed that overexpression of MIC19 could mitigate cell apoptosis and neuronal death, as well as abnormalities in mitochondrial structure and function, oxidative stress within mitochondria, and neurobehavioral deficits in rats following ICH. Conversely, knockdown of MIC19 had the opposite effect. Moreover, we found that the connection between MIC19 and SAM50 was disrupted after ICH, which may be a reason for the impairment of the mitochondrial structure after ICH. In conclusion, MIC19 exerts a protective role in the subsequent injury induced by ICH. The investigation of MIC19 may offer clinicians novel therapeutic insights for patients afflicted with ICH.

Induced neural stem cells suppressed neuroinflammation by inhibiting the microglial pyroptotic pathway in intracerebral hemorrhage rats

Intracerebral hemorrhage usually manifests as strong neuroinflammation and neurological deficits. There is an urgent need to explore effective methods for the treatment of intracerebral hemorrhage. The therapeutic effect and the possible mechanism of induced neural stem cell transplantation in an intracerebral hemorrhage rat model are still unclear. Our results showed that transplantation of induced neural stem cells could improve neurological deficits by inhibiting inflammation in an intracerebral hemorrhage rat model. Additionally, induced neural stem cell treatment could effectively suppress microglial pyroptosis, which might occur through inhibiting the NF-κB signaling pathway. Induced neural stem cells could also regulate the polarization of microglia and promote the transition of microglia from pro-inflammatory phenotypes to anti-inflammatory phenotypes to exert their anti-inflammatory effects. Overall, induced neural stem cells may be a promising tool for the treatment of intracerebral hemorrhage and other neuroinflammatory diseases.

Hyperbaric oxygen ameliorates neuronal injury and neurological function recovery in rats with intracerebral hemorrhage by silencing microRNA-204-5p-targeted chloride channel protein 3.

Hyperbaric oxygen (HBO) therapy is of clinical utility in patients with transient cerebral ischemia. The investigatory study was to identify the potential regulatory mechanism of HBO treatment on neuronal injury and neurological function recovery in rats with intracerebral hemorrhage (ICH). Firstly, the rat model of ICH was established by collagenase, and the experimental rats were treated with HBO at 2.5 absolute atmospheres for 60 min each time. Next, lentivirus interfering with microRNA (miR)-204-5p or chloride channel protein 3 (CLCN3) expression was injected via the tail vein. Afterward, neurological function assessment was conducted, serum S100β and NSE contents were detected by enzymer-linked immunosorbent assay, and pathological conditions of brain tissue were observed by hematoxylin-eosin staining. Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling staining was used to detect neuronal apoptosis. The results showed that HBO alleviated neuronal injury and neurological function recovery in ICH rats and reduced serum S100β and NSE content (all P<0.05). At the same time, overexpressing miR-204-5p or depleting CLCN3 further promoted the therapeutic effect of HBO on ICH rats (all P<0.05), while silencing miR-204-5p or elevating CLCN3 did oppositely (all P<0.05). In conclusion, HBO alleviates neuronal injury and neurological function recovery in ICH rats by silencing miR-204-5p-targeted CLCN3.

Brain-derived neurotrophic factor contributes to neurogenesis after intracerebral hemorrhage: a rodent model and human study.

Intracerebral hemorrhage (ICH) enhances neurogenesis in the subventricular zone (SVZ); however, the mechanism is not fully understood. We investigated the role of brain-derived neurotrophic factor (BDNF) in post-ICH neurogenesis in a rodent model and in patients with ICH using cerebrospinal fluid (CSF). A rat model of ICH was constructed via stereotaxic injection of collagenase into the left striatum. Patients with ICH receiving an external ventricular drain were prospectively enrolled. CSF was collected from rats and patients at different post-ICH times. Primary cultured rat neural stem cells (NSCs) were treated with CSF with or without BDNF-neutralized antibody. Immunohistochemistry and immunocytochemistry were used to detect NSC proliferation and differentiation. The BDNF concentration in CSF was quantified using enzyme-linked immunosorbent assays (ELISA). In the rat model of ICH, the percentage of proliferating NSCs and neuroblasts in SVZ was elevated in bilateral hemispheres. The cultured rat NSCs treated with CSF from both rats and patients showed an increased capacity for proliferation and differentiation toward neuroblasts. BDNF concentration was higher in CSF collected from rats and patients with ICH than in controls. Blocking BDNF decreased the above-noted promotion of proliferation and differentiation of cultured NSCs by CSF treatment. In patients with ICH, the BDNF concentration in CSF and the neurogenesis-promoting capacity of post-ICH CSF correlated positively with ICH volume. BDNF in CSF contributes to post-ICH neurogenesis, including NSC proliferation and differentiation toward neuroblasts in a rat model and patients with ICH.

Selective HDAC6 inhibitor TubA offers neuroprotection after intracerebral hemorrhage via inhibiting neuronal apoptosis.

A large body of evidence has demonstrated that neuronal apoptosis is involved in the pathological process of secondary brain injury following intracerebral hemorrhage (ICH). Additionally, our previous studies determined that the inhibition of HDAC6 activity by tubacin or specific shRNA can attenuate neuronal apoptosis in an oxygen-glucose deprivation reperfusion model. However, whether the pharmacological inhibition of HDAC6-attenuated neuronal apoptosis in ICH remains unclear. In this study, we used hemin-induced SH-SY5Y cells to simulate a hemorrhage state in vitro and adopted a collagenase-induced ICH rat model in vivo to assess the effect of the HDAC6 inhibition. We found a significant increase in HDAC6 during the early stages of ICH. As expected, the acetylated α-tubulin significantly decreased in correlation with the expression of HDAC6. Medium and high doses (25, 40 mg/kg) of TubA, a selective inhibitor of HDAC6, both reduced neurological impairments, histological impairments, and ipsilateral brain edema in vivo. TubA or HDAC6 siRNA both alleviated neuronal apoptosis in vivo and in vitro. Finally, HDAC6 inhibition increased the level of acetylated α-tubulin and Bcl-2 and lowered the expression of Bax and cleaved caspase-3 post-ICH. In general, these results suggested that the pharmacological inhibition of HDAC6 may act as a novel and promising therapeutic target for ICH therapy by up-regulating acetylated α-tubulin and reducing neuronal apoptosis.

Nox4-and Tf/TfR-mediated peroxidation and iron overload exacerbate neuronal ferroptosis after intracerebral hemorrhage: Involvement of EAAT3 dysfunction.

Intracerebral hemorrhage (ICH) induces high mortality and disability. Neuronal death is the principal factor to unfavourable prognosis in ICH. However, the mechanisms underlying this association remain unclear. In this study, we investigated the molecular mechanisms by which neuronal ferroptosis occurs after ICH and whether the use of corresponding modulators can inhibit neuronal death and improve early outcomes in a rat ICH model. Our findings indicated that Nox4 and TF/TfR were upregulated in the perihematomal tissues of ICH rats. Oxidative stress and iron overload induced by Nox4 and TF/TfR promoted neuronal ferroptosis post-ICH. In contrast, application of Nox4-siRNA and the deferoxamine (DFO) attenuated peroxidation and iron deposition in the hemorrhagic brain, alleviated neuronal ferroptosis, and improved sensorimotor function in ICH rats. Additionally, our findings indicated that the post-ICH neuronal reduced glutathione (GSH) depletion were not related to dysfunctional glutamine delivery in astrocytes but rather to downregulation of EAAT3 due to lipid peroxidation-induced dysfunction in the neuronal membrane. These findings indicate that ferroptosis is involved in neuronal death in model rats with collagenase-induced ICH. Oxidative stress and iron overload induced by Nox4 and TF/TfR exacerbate ferroptosis after ICH, while Nox4 downregulation and iron chelation exert neuroprotective effects. The present results highlight the cysteine importer EAAT3 as a potential biomarker of ferroptosis and provide insight into the neuronal death process that occurs following ICH, which may aid in the development of translational treatment strategies for ICH.

Identification of CCL20 as a Key Biomarker of Inflammatory Responses in the Pathogenesis of Intracerebral Hemorrhage.

Inflammatory responses after intracerebral hemorrhage (ICH) contribute to severe secondary brain injury, leading to poor clinical outcomes. However, the responsible genes for effective anti-inflammation treatment in ICH remain poorly elucidated. The differentially expressed genes (DEGs) of human ICH were explored by online GEO2R. Go and KEGG were used to explore the biological function of DEGs. Protein-protein interactions (PPI) were built in the String database. Critical modules of PPI were identified by a molecular complex detection algorithm (MCODE). Cytohubba was used to determine the hub genes. The mRNA-miRNA interaction network was built in the miRWalk database. The rat ICH model was applied to validate the key genes. A total of 776 DEGs were identified in ICH. Go and KEGG analyses indicated that DEGs were mainly involved in neutrophil activation and the TNF signaling pathway. GSEA analysis presented that DEGs were significantly enriched in TNF signaling and inflammatory response. PPI network was constructed in the 48 differentially expressed inflammatory response-related genes. The critical module of the PPI network was constructed by 7 MCODE genes and functioned as the inflammatory response. The top 10 hub genes with the highest degrees were identified in the inflammatory response after ICH. CCL20 was confirmed as a key gene and mainly expressed in neurons in the rat ICH model. The regulatory network between CCL20 and miR-766 was built, and the miR-766 decrease was confirmed in a human ICH dataset. CCL20 is a key biomarker of inflammatory response after intracerebral hemorrhage, providing a potential target for inflammatory intervention in ICH.