Hemorrhage In Rats Research Articles

Endothelial pyroptosis-driven microglial activation in choroid plexus mediates neuronal apoptosis in hemorrhagic stroke rats

BackgroundSpontaneous intracerebral hemorrhage (ICH) is associated with alarmingly high rates of disability and mortality, and current therapeutic options are suboptimal. A critical component of ICH pathology is the initiation of a robust inflammatory response, often termed “cytokine storm,” which amplifies the secondary brain injury following the initial hemorrhagic insult. The precise sources and consequences of this cytokine-driven inflammation are not fully elucidated, necessitating further investigation. MethodsTo address this knowledge gap, our study conducted a comprehensive cytokine profiling using Luminex® assays, assessing 23 key cytokines. We then employed single-cell RNA sequencing and spatial transcriptomics at three critical time points post-ICH: the hyperacute, acute, and subacute phases. Integrating these multimodal analyses allowed us to identify the cellular origins of cytokines and elucidate their mechanisms of action. ResultsLuminex® cytokine assays revealed a significant upregulation of IL-6 and IL-1β levels at the 24-h post-ICH time point. Through the integration of scRNA-seq and spatial transcriptomics in the hemorrhagic hemisphere of rats, we observed a pronounced activation of cytokine-related signaling pathways within the choroid plexus. Initially, immune cell presence was sparse, but it surged 24 h post-ICH, particularly in the choroid plexus, indicating a substantial shift in the immune microenvironment. We traced the source of IL-1β and IL-6 to endothelial cells, establishing a link to pyroptosis. Endothelial pyroptosis post-ICH induced the production of IL-1β and IL-6, which activated microglial polarization characterized by elevated expression of Msr1, Lcn2, and Spp1 via the NF-κB pathway in the choroid plexus. Furthermore, we identified neuronal populations undergoing apoptosis, mediated by the Lcn2-SLC22A17 pathway in response to IL-1β and IL-6 signaling. Notably, the inhibition of pyroptosis using VX-765 significantly mitigated neurological impairments. ConclusionsOur study provides evidence that endothelial pyroptosis, characterized by the release of IL-1β and IL-6, triggers microglial polarization through NF-κB pathway activation, ultimately leading to microglia-mediated neuronal apoptosis in the choroid plexus post-ICH. These findings suggest that targeted therapeutic strategies aimed at mitigating endothelial cell pyroptosis and neutralizing inflammatory cytokines may offer neuroprotection for both microglia and neurons, presenting a promising avenue for ICH treatment.

An experimental study to estimate the early postmortem interval based on the degradation of lncRNAs in rat brain tissue

To study the degradation of lncRNAs in EPMI in rat brain tissue, this study provides a new direction for the estimation of EPMI. LncRNA high-throughput sequencing was performed on the brain tissues of hemorrhagic shock model rats at 0 h and 24 h, and the target lncRNAs were screened. Samples at 0, 1, 3, 6, 12, 18 and 24 h after death were collected, and miRNA-9 and miRNA-125b were used as reference genes. The relative expression levels of lncRNAs at each PMI were detected by RT–qPCR, and a functional model involving lncRNAs and EPMI was established. Samples were collected at 6, 9, 15, and 21 h after death for functional model verification. The expression of several lncRNAs decreased with the prolongation of EPMI, and the mathematical model established by several lncRNA indices exhibited good fit. The verification results of the multi-index joint function model are significantly better than those of the single-index function model, and the established model is more practical. There is a linear relationship between lncRNAs and EPMI, and the multi-index function model is significantly better than the single-index function model, which is important for EPMI inference in forensic pathology practice.

Trefoil factor 1 (TFF1) reduces cerebral edema and gastric mucosal injury by regulating the EGFR/Src/FAK pathway in an intracerebral hemorrhage rat model

The destruction of the blood-brain barrier and damage to the gastrointestinal mucosa after intracerebral hemorrhage (ICH) are important reasons for its high disability and mortality rates. However, the exact etiology is not yet clear. In addition, there are currently no effective treatments for improving cerebral edema and gastric mucosal damage after ICH. Trefoil factor 1 (TFF1) is a secretory protein that plays a crucial role in maintaining the integrity and barrier function of the gastric mucosa, and it has been reported to have a protective effect on brain damage induced by various causes. This study utilized a rat model of ICH induced by type IV collagenase was utilized, and intervened with recombinant TFF1 protein from an external institute to investigate the protective mechanisms of TFF1 against brain edema and gastric mucosal damage after ICH. The results demonstrated that TFF1 alleviated the neurological function and gastric mucosal damage in the rat model of ICH induced by type IV collagenase. TFF1 may ensure the integrity of the blood-brain and gastric mucosal barriers by regulating the EGFR (epidermal growth factor receptor)/Src (non-receptor tyrosine kinase)/FAK (focal adhesion kinase) pathway. Clearly, the disruption of the blood-brain barrier and the destruction of the gastric mucosal barrier are key pathological features of ICH, and TFF1 can improve the progression of blood-brain barrier and gastric mucosal barrier disruption in ICH by regulating the EGFR/Src/FAK pathway. Therefore, TFF1 may be a potential target for the treatment of ICH.

Modifications and Optimization of an Autologous Intracerebral Hemorrhage Rat Model

Objective: To date, no novel drug therapies have proven effective in improving outcomes after intracerebral hemorrhage (ICH). This has been slowed, in part, by a lack of reliable preclinical models that recapitulate the pathophysiological consequences of ICH. Limited descriptions of the methodology of preclinical models may play a role in this failed translation. The current study aimed to improve the autologous blood injection model of ICH and provide a comprehensive methodology to facilitate a reliable preclinical model that bridges the translational research gap. Methods: A modified single-injection protocol for an autologous-blood ICH model was developed and tested. This rodent ICH model produces injury in the basal ganglia of adult rats via a stereotaxically-assisted right-sided single-injection of fresh autologous whole blood. A comprehensive step-wise description of the methodology is presented and documented in a written and visual format with supporting pictorialmaterial, to prevent common mistakes that result in failed hematoma formation.Rates of successful hematoma formation using the single-injection protocol, outlined here, were compared to those using the traditional double-injection protocol.

Liangxue Tongyu prescription attenuates neuroinflammation by increasing cholecystokinin octapeptide in acute intracerebral hemorrhage rats

Inflammatory reactions after acute intracerebral hemorrhage (AICH) contribute significantly to a poor prognosis. Liangxue Tongyu Prescription (LTP) has been proven to be clinically effective in treating AICH. Numerous studies have shown that LTP suppresses brain inflammatory damage in AICH, while the internal mechanisms underlying its action remain unclear. The aim of this study was to verify the anti-inflammatory effects of LTP on an AICH rat model and investigate the potential mechanisms. The AICH rat models were created by injecting autologous blood into the right caudate nucleus. LTP markedly decreased cerebral hematoma and brain water content and recovered from neurological deficits. Meanwhile, LTP prevented microglial activation and reduced the inflammatory reaction caused by pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6). Notably, the expression of cholecystokinin octapeptide (CCK-8) in the brain and intestine was increased by LTP or CCK-8 treatment. LTP further suppressed nuclear factor kappa B (NF-κB) in the brains of rats with AICH. Moreover, LTP increased the protein and mRNA expression of Occludin and Claudin-1 in the intestine and decreased the levels of lipopolysaccharide (LPS) and diamine oxidase (DAO) in serum. Furthermore, the results showed that LTP increased the protein and mRNA expression of Claudin-5 and zonula occludens-1 (ZO-1) in the brain. CCK-8 receptor antagonists increased the expression of NF-κB and the concentration of pro-inflammatory cytokines. These findings suggested that LTP attenuated neuroinflammation by increasing CCK-8 in the brain and intestine, and its mechanism might be related to alterations in the gut-brain axis (GBA).

Nrf-2 modulates excitability of hippocampal neurons by regulating ferroptosis and neuroinflammation after subarachnoid hemorrhage in rats

Excitability of hippocampal neurons in subarachnoid hemorrhage (SAH) rats has not been well studied. The rat SAH model was applied in this study to explore the role of nuclear factor E2-related factor (Nrf-2) in the early brain injury of SAH. The neural excitability of CA1 pyramidal cells (PCs) in SAH rats was evaluated by using electrophysiology experiments. Ferroptosis and neuroinflammation were measured by ELISA, transmission electron microscopy and western blotting. Our results indicated that SAH induced neurological deficits, brain edema, ferroptosis, neuroinflammation and neural excitability in rats. Ferrostatin-1 treatment significantly decreased the expression and distribution of IL-1β, IL-6, IL-10, TGF-β and TNF-α. Inhibiting ferroptosis by ferrostatin-1 can attenuate neural excitability, neurological deficits, brain edema and neuroinflammation in SAH rats. Inhibiting the expression of Nrf-2 significantly increased the neural excitability and the levels of IL-1β, IL-6, IL-10, TGF-β and TNF-α in Fer-1-treated SAH rats. Taken together, inhibiting the Nrf-2 induces early brain injury, brain edema and the inflammatory response with increasing of neural excitability in Fer-1-treated SAH rats. These results have indicated that inhibiting ferroptosis, neuroinflammation and neural excitability attenuates early brain injury after SAH by regulating the Nrf-2.

The New Nano-Resuscitation Solution (TPP-MR) Attenuated Myocardial Injury in Hemorrhagic Shock Rats by Inhibiting Ferroptosis.

Hemorrhagic shock was a leading cause of death worldwide, with myocardial injury being a primary affected organ. As commonly used solutions in fluid resuscitation, acetated Ringer's (AR) and Lactate Ringer's solution (LR) were far from perfect for their adverse reactions such as lactic acidosis and electrolyte imbalances. In previous studies, TPP@PAMAM-MR (TPP-MR), a novel nanocrystal resuscitation fluid has been found to protect against myocardial injury in septic rats. However, its role in myocardial injury in rats with hemorrhagic shock and underlying mechanism is unclear. The hemorrhagic shock rats and hypoxia-treated cardiomyocytes (H9C2) were utilized to investigate the impact of TPP-MR on cardiac function, mitochondrial function, and lipid peroxidation. The expressions of ferritin-related proteins glutathione peroxidase 4 (GPX4), Acyl CoA Synthase Long Chain Family Member 4 (ACSL4), and Cyclooxygenase-2(COX2) were analyzed through Western blotting to explore the mechanism of TPP-MR on hemorrhagic myocardial injury. TPP-MR, a novel nanocrystalline resuscitation fluid, was synthesized using TPP@PAMAM@MA as a substitute for L-malic acid. We found that TPP-MR resuscitation significantly reduced myocardial injury reflected by enhancing cardiac output, elevating mean arterial pressure (MAP), and improving perfusion. Moreover, TPP-MR substantially prolonged hemorrhagic shock rats' survival time and survival rate. Further investigations indicated that TPP-MR improved the mitochondrial function of myocardial cells, mitigated the production of oxidative stress agents (ROS) and increased the glutathione (GSH) content. Additionally, TPP-MR inhibited the expression of the ferroptosis-associated GPX4 protein, ACSL4 and COX2, thereby enhancing the antioxidant capacity. The results showed that TPP-MR had a protective effect on myocardial injury in rats with hemorrhagic shock, and its mechanism might be related to improving the mitochondrial function of myocardial cells and inhibiting the process of ferroptosis.

Integrated Network Pharmacology and in vivo Experimental Validation Approach to Explore the Potential Antioxidant Effects of Annao Pingchong Decoction in Intracerebral Hemorrhage Rats.

Annao Pingchong decoction (ANPCD) is a traditional Chinese decoction which has definite effects on treating intracerebral hemorrhage (ICH) validated through clinical and experimental studies. However, the impact of ANPCD on oxidative stress (OS) after ICH remains unclear and is worth further investigating. To investigate whether the therapeutic effects of ANPCD on ICH are related to alleviating OS damage and seek potential targets for its antioxidant effects. The therapeutic candidate genes of ANPCD on ICH were identified through a comparison of the target genes of ANPCD, target genes of ICH and differentially expressed genes (DEGs). Protein-protein interaction (PPI) network analysis and functional enrichment analysis were combined with targets-related literature to select suitable antioxidant targets. The affinity between ANPCD and the selected target was verified using macromolecular docking. Subsequently, the effects of ANPCD on OS and the selected target were further investigated through in vivo experiments. Forty-eight candidate genes were screened, in which silent information regulator sirtuin 1 (SIRT1) is one of the core genes that has antioxidant effects and ICH significantly affected its expression. The good affinity between 6 compounds of ANPCD and SIRT1 was also demonstrated by macromolecular docking. The results of in vivo experiments demonstrated that ANPCD significantly decreased modified neurological severity scoring (mNSS) scores and serum MDA and 8-OHdG content in ICH rats, while significantly increasing serum SOD and CAT activity, complicated with the up-regulation of ANPCD on SIRT1, FOXO1, PGC-1α and Nrf2. Furthermore, ANPCD significantly decreased the apoptosis rate and the expression of apoptosis-related proteins (P53, cytochrome c and caspase-3). ANPCD alleviates OS damage and apoptosis after ICH in rats. As a potential therapeutic target, SIRT1 can be effectively regulated by ANPCD, as are its downstream proteins.

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.

Involvement of CD44 and MAPK14-mediated ferroptosis in hemorrhagic shock.

To elucidate the induction of ferroptotic pathways and the transcriptional modulation of pivotal genes in the context of hemorrhagic shock. The R software was used to analyze the GSE64711 dataset, isolating genes relevant to ferroptosis. Enrichment analyses and protein interaction networks were assembled. Using WGCNA hub genes were identified and intersected with ferroptosis-related genes, highlighting hub genes CD44 and MAPK14. In a rat hemorrhagic shock model, cardiac ROS, Fe2+, MDA, and GSH levels were assessed. Key ferroptotic proteins (SLC7A11/GPX4) in myocardial tissues were examined via western blot. Hub genes, CD44 and MAPK14, expressions were confirmed through immunohistochemistry. Analyzing the GSE64711 dataset revealed 337 differentially expressed genes, including 12 linked to ferroptosis. Enrichment analysis highlighted pathways closely related to ferroptosis. Using Genemania, we found these genes mainly affect ROS metabolism and oxidative stress response. WGCNA identified CD44 and MAPK14 as hub genes. Rat myocardial tissue validation showed significant cardiac damage and elevated ROS and MDA levels, and decreased GSH levels in the hemorrhagic shock model. The ferroptotic pathway SLC7A11/GPX4 was activated, and immunohistochemistry showed a significant increase in the expression levels of CD44 and MAPK14 in the hemorrhagic shock rat model. We demonstrated the presence of tissue ferroptosis in hemorrhagic shock by combining bioinformatics analysis with in vivo experimentation. Specifically, we observed the activation of the SLC7A11/GPX4 ferroptotic pathway. Further, CD44 and MAPK14 were identified as hub genes in hemorrhagic shock.

Hemorrhagic transformation in patients with large-artery atherosclerotic stroke is associated with the gut microbiota and lipopolysaccharide.

Abstract JOURNAL/nrgr/04.03/01300535-202407000-00032/figure1/v/2023-11-20T171125Z/r/image-tiff Hemorrhagic transformation is a major complication of large-artery atherosclerotic stroke (a major ischemic stroke subtype) that worsens outcomes and increases mortality. Disruption of the gut microbiota is an important feature of stroke, and some specific bacteria and bacterial metabolites may contribute to hemorrhagic transformation pathogenesis. We aimed to investigate the relationship between the gut microbiota and hemorrhagic transformation in large-artery atherosclerotic stroke. An observational retrospective study was conducted. From May 2020 to September 2021, blood and fecal samples were obtained upon admission from 32 patients with first-ever acute ischemic stroke and not undergoing intravenous thrombolysis or endovascular thrombectomy, as well as 16 healthy controls. Patients with stroke who developed hemorrhagic transformation (n = 15) were compared to those who did not develop hemorrhagic transformation (n = 17) and with healthy controls. The gut microbiota was assessed through 16S ribosomal ribonucleic acid sequencing. We also examined key components of the lipopolysaccharide pathway: lipopolysaccharide, lipopolysaccharide-binding protein, and soluble CD14. We observed that bacterial diversity was decreased in both the hemorrhagic transformation and non-hemorrhagic transformation group compared with the healthy controls. The patients with ischemic stroke who developed hemorrhagic transformation exhibited altered gut microbiota composition, in particular an increase in the relative abundance and diversity of members belonging to the Enterobacteriaceae family. Plasma lipopolysaccharide and lipopolysaccharide-binding protein levels were higher in the hemorrhagic transformation group compared with the non-hemorrhagic transformation group. lipopolysaccharide, lipopolysaccharide-binding protein, and soluble CD14 concentrations were associated with increased abundance of Enterobacteriaceae. Next, the role of the gut microbiota in hemorrhagic transformation was evaluated using an experimental stroke rat model. In this model, transplantation of the gut microbiota from hemorrhagic transformation rats into the recipient rats triggered higher plasma levels of lipopolysaccharide, lipopolysaccharide-binding protein, and soluble CD14. Taken together, our findings demonstrate a noticeable change in the gut microbiota and lipopolysaccharide-related inflammatory response in stroke patients with hemorrhagic transformation. This suggests that maintaining a balanced gut microbiota may be an important factor in preventing hemorrhagic transformation after stroke.

Electro-nape-acupuncture regulates the differentiation of microglia through PD-1/PD-L1 reducing secondary brain injury in acute phase intracerebral hemorrhage rats.

This study aimed to investigate the effect of electro-nape-acupuncture (ENA) on the differentiation of microglia and the secondary brain injury in rats with acute-phase intracerebral hemorrhage (ICH) through the programmed cell death protein-1/ligand-1 (PD-1/PD-L1) pathway. A total of 27 male Sprague-Dawley rats were randomly divided into three groups: sham group, ICH group, and ENA group. The autologous blood infusion intracerebral hemorrhage model was used to study the effects of ENA by administering electroacupuncture at GB20 (Fengchi) and Jiaji (EX-B2) acupoints on 24h after the modeling, once per day for 3 days. The neurological function damage, hematoma lesion, and inflammatory cell infiltration were measured by the beam walking test and hematoxylin-eosin staining. The expression of PD-1, PD-L1, CD86, CD206, and related cytokines around the hematoma was measured by western blot, quantitative reverse transcription polymerase chain reaction, and immunofluorescence. The ICH group had significant neurological deficits (p < .001), hematoma lesions, and inflammatory cell infiltration. The levels of CD86 protein, inflammatory factors tumor necrosis factors (TNF)-α, interleukin (IL)-1β, and IL-6 were increased (p < .001), while CD206 protein was reduced (p < .01), and the number of CD86+ /CD11b+ cells was also increased (p < .001) compared to the sham group. However, after ENA intervention, there was a significant reduction in neurological function damage (p < .05), infiltration of inflammatory cells, and the expression levels of CD86+ /CD11b+ cells (p < .05), resulting in the increased expression of PD-1 protein and differentiation of M2 phenotype significantly (p < .001). The study concludes that ENA could reduce neurological function damage, inhibit the expression of pro-inflammatory cytokines, and improve the infiltration of inflammatory cells to improve secondary brain injury in acute-phase intracerebral hemorrhage rats. These effects could be related to the increased expression of PD-1 around the lesion, promoting the differentiation of microglia from M1 to M2 phenotype.

Protective effects of Scutellariae Radix Carbonisata-derived carbon dots on blood-heat and hemorrhage rats.

As the charcoal processing product of Scutellariae Radix (SR), SR Carbonisata (SRC) has been clinically used as a cooling blood and hemostatic agent for thousands of years. However, the underlying active ingredients and mechanism of SRC still remained unspecified. In this study, SRC derived carbon dots (SRC-CDs) were extracted and purified from the aqueous solution of SRC, followed by physicochemical property assessment by series of technologies. The cooling blood and hemostatic effects of SRC-CDs were further evaluated via a blood-heat and hemorrhage (BHH) rat model. Results showed that the diameters of obtained fluorescent SRC-CDs ranged from 5.0nm to 10.0nm and possessed functional group-rich surfaces. Additionally, the as-prepared SRC-CDs showed remarkable cooling blood and hemostasis effects in BHH model, mainly manifested by significant improvement of elevated rectal temperature, inflammatory cytokines (TNF-α, IL-6, and IL-1β) levels, as well as protein expressions of myD88 and NF-κB p65, abnormal coagulation parameters (elevated APTT and FIB), hemogram parameters (RBC, HGB, and HCT), and histopathological changes in lung and gastric tissues. This study, for the first time, demonstrated that SRC-CDs were the cooling blood and hemostatic active components of SRC, which could inhibit the release of inflammatory cytokines by regulating myD88/NF-κB signaling pathway, and activating the fibrin system and endogenous coagulation pathway. These results not only provide a new perspective for the study of active ingredients of carbonized herbs represented by SRC, but also lay an experimental foundation for the development of next-generation nanomedicines.

Articles on hemorrhagic shock published between 2000 and 2021: A CiteSpace-Based bibliometric analysis

ObjectiveTo conduct a bibliometric analysis of literature on hemorrhagic shock published between 2000 and 2021 with the help of Citespace to explore the current status, hotspots and research trends in this regard, with the results presented in a visualized manner. MethodsThe data over the past 22 years were retrieved from the Web of Science (WOS) Core Collection database and downloaded as the “Full Record and Cited References”. Cooperative analysis, cluster analysis, co-citation analysis, and burst analysis were performed based on the data on countries/regions, institutions, journals, authors, and keywords through Citespace. ResultsA total of 2027 articles were retrieved. The number of annual publications fluctuated but was generally on an upward trend. The United States stands out as the most productive country (989 articles), the University of Pittsburgh the most productive publishing institution (109 articles), SHOCK the most cited journal (1486 articles), TAO LI the most productive author (40 articles), DEITCH EA the most cited author (261 times of citation), hemorrhagic shock the most frequent keyword (725 times of occurrence), and “traumatic brain injury” the most covered article in keyword clustering (29 articles). The burst analysis revealed Harvard University as the institution with the highest strength value and the Journal of Trauma and Acute Care Surgery the most important journal. It was also concluded that HASAN B ALAM, AARON M WILLIAMS, and LIMIN ZHANG may continue to publish high-quality articles in the future. In the meanwhile, both “protect” and “transfusion” were considered the hotspots and trends in current research. ConclusionsThe United States has been a major contributor to the publication of the articles over the past 22 years, with the most productive publishing institution, the most cited journal, and the most cited author all coming from the US. Hemorrhagic shock, injury, resuscitation, trauma, models, activation, expression, fluid resuscitation, rats, and nitric oxide are hot topics in relevant research. According to the keyword burst analysis, the areas related to “protect” and “transfusion” may rise as the research directions in the future. However, since the hotspots in the research of hemorrhagic shock are short-lived and fast-changing, the researchers should pay more attention to the development trend in this field.

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.

Annao Pingchong decoction alleviate the neurological impairment by attenuating neuroinflammation and apoptosis in intracerebral hemorrhage rats.

Intracerebral hemorrhage (ICH) is a central nervous system disease that causes severe disability or death. Even though Annao Pingchong decoction (ANPCD), a traditional Chinese decoction, has been used clinically to treat ICH in China, its molecular mechanism remains unclear. To study whether the neuroprotective effect of ANPCD on ICH rats is achieved by alleviating neuroinflammation. This paper mainly explored whether inflammation-related signaling pathways (HMGB1/TLR4/NF-κB P65) plays a role in ANPCD treatment of ICH rats. Liquid chromatography-tandem mass spectrometry was used to analyze the chemical composition of ANPCD. ICH models were established by injecting autologous whole blood into the left caudate nucleus of Sprague-Dawley (SD) rats. Modified neurological severity scoring (mNSS) was used to assess the neurological deficits. The levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 were analyzed using enzyme-linked immunosorbent assay (ELISA). Pathological changes in the rat brains were observed using hematoxylin-eosin, Nissl, and TUNEL staining. The protein levels of HMGB1, TLR4, NF-κB p65, B-cell lymphoma 2 (Bcl-2), and Bcl-2-associated X protein (Bax) were measured by western blotting and immunofluorescence analysis. Ninety-three ANPCD compounds were identified, including 48 active plasma components. Treatment with ANPCD effectively improved the outcome, as observed by the neurological function scores analysis and brain histopathology. Our results showed that ANPCD exerts its anti-inflammatory effects by significantly downregulating the expression of HMGB1, TLR4, NF-κB p65, TNF-α, IL-1β, and IL-6. ANPCD also exerted anti-apoptotic effects by significantly decreasing the apoptosis rate and Bax/Bcl-2 ratio. We found that ANPCD had neuroprotective effect in clinical work. Here, we also found that the action mechanism of ANPCD might be related to attenuate neuroinflammation and apoptosis. These effects were achieved by inhibiting the expression of HMGB1, TLR4 and NF-κB p65.

Zhilong Huoxue Tongyu Capsule attenuates hemorrhagic transformation through the let-7f/TLR4 signaling pathway

Ethnopharmacological relevanceHemorrhagic transformation after acute ischemic stroke is a life-threatening disease that currently has no effective chemotherapy. Zhilong Huoxue Tongyu Capsule (ZL) is an empirical prescription of traditional Chinese medicine that is used to prevent and treat cardiovascular and cerebrovascular diseases in China. However, only a few studies have addressed the mechanisms of ZL in treating hemorrhagic transformation. Aim of the studyTo evaluate the anti-inflammatory effects of ZL on hemorrhagic transformation model rats and lipopolysaccharide (LPS)-induced RAW264.7 macrophages and to explore the underlying molecular mechanisms. Materials and methodsMurine RAW264.7 cells were treated with ZL and LPS (1 μg/mL), and cell viability was detected by cell counting kit-8 assay. RT-qPCR was used to detect the expression of inflammatory chemokines, microRNA let-7a/e/i/f, toll like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and nuclear factor kappa-B (NF-κB) p65. The protein expression levels of TLR4, MyD88, NF-κB p65, and apoptosis related molecules were determined by Western blotting. The apoptosis rate of RAW264.7 macrophages was detected by Annexin V-FITC/PI double staining. A hemorrhagic transformation model in rats was established by intraperitoneal injection of high glucose solution combined with thread embolization. Then, the model rats were observed behaviourally, pathologically, and molecularly. The gene expression of TLR4, MyD88, and NF-κB p65 was measured by RT-qPCR and used to evaluate the protective effect of ZL against hemorrhagic transformation in rats. ResultsZL (5, 20, 40 μg/mL) was beneficial in cell proliferation. LPS (1 μg/mL) stimulated the production of inflammatory chemokines and inhibited the production of let-7a/e/i/f, with let-7f being influenced most strongly. Moreover, overexpression of let-7f decreased the gene and protein levels of TLR4, MyD88, and NF-κB p65, downregulated TLR4, and inhibited its transcriptional activity. ZL (5, 20, and 40 μg·mL-1) inhibited the production of TLR4, MyD88, and NF-κB p65 and promoted the production of let-7f in a concentration-dependent manner. Furthermore, the blockade of TLR4 antagonized the promoting effects of TLR4 pathway activation in cell inflammation and apoptosis by downregulating let-7f. Critically, it was confirmed in vivo and in vitro that ZL upregulated the expression of let-7f and inhibited the gene expression of TLR4, MyD88, and NF-κB p65 to reduce inflammatory cell infiltration, which determined the occurrence of hemorrhagic transformation. ConclusionsZL can reduce inflammatory response by upregulating let-7f and subsequently inhibiting the TLR4 signaling pathway, thereby decreasing the occurrence of hemorrhagic transformation.

Comparison of motor function recovery and brain changes in intracerebral hemorrhagic and ischemic rats with similar brain damage.

In this study, we compared the mechanisms of brain recovery in intracerebral hemorrhage and ischemia, focusing on synapses, glial cells, and dopamine expression, which are considered fundamental for neural recovery after stroke. Male Wistar rats were divided into intracerebral hemorrhage, ischemia, and sham surgery (SHAM) groups. The intracerebral hemorrhage group was injected with a collagenase solution, the ischemia group was injected with an endothelin-1 solution, and the SHAM group was injected with physiological saline. The motor function of these rats was evaluated using a rotarod test on days 7, 14, 21, and 28 post-surgery. On postoperative day 29, lesion volume was analyzed using Nissl staining. In addition, the protein expression levels of NeuN, GFAP, tyrosine hydroxylase, and PSD95 were analyzed in the striatum and motor cortex. There was no significant difference between the ischemia and intracerebral hemorrhage groups in terms of lesion volume in the striatum; however, the motor recovery of the intracerebral hemorrhage group occurred more rapidly than that of the ischemia group, and the intracerebral hemorrhage group exhibited higher GFAP protein expression in the motor cortex. The rapid motor recovery in intracerebral hemorrhage rats relative to that in ischemia rats may be associated with changes in astrocytes in brain regions remote from the injury site.

Anti-DEspR antibody treatment improves survival and reduces neurologic deficits in a hypertensive, spontaneous intracerebral hemorrhage (hsICH) rat model

Progressive secondary brain injury—induced by dysregulated neuroinflammation in spontaneous intracerebral hemorrhage (sICH)—underlies high sICH-mortality and remains without FDA-approved pharmacotherapy. Clinical insight that hematoma-directed interventions do not improve mortality prioritizes resolving acute secondary brain injury in sICH. As neutrophils are implicated in sICH secondary brain injury, we tested whether inhibition of a rogue neutrophil-subset expressing the dual endothelin-1/signal peptide receptor (DEspR) and associated with secondary tissue injury, DEspR+ CD11b+ immunotype, will attenuate mortality in a hypertensive-sICH (hsICH) rat model. We confirmed sICH-related deaths in hsICH-rats by T2*-weighted 9.4 T MRI and DEspR+ neutrophils in hsICH-rat brain perihematomal areas by immunostaining. At acute sICH, anti-DEspR muIgG1-antibody, mu10a3, treatment increased median survival in hsICH rats vs controls (p < 0.0001). In pre-stroke sICH, weekly 10a3-treatment did not predispose to infection and delayed sICH-onset vs controls (p < 0.0001). As potential sICH-therapeutic, we tested humanized anti-DEspR IgG4S228P-mAb, hu6g8. In vitro, hu6g8 reversed delayed-apoptosis in DEspR+ CD11b+ neutrophils. In vivo, hu6g8 increased median survival and reduced neurologic symptoms in male/female hsICH-rats vs controls (p < 0.0001). Altogether, preclinical efficacy of inhibition of DEspR+ CD11b+ neutrophils in acute sICH—without infection complications, supports the potential of anti-DEspR therapy in sICH. Data provide basis for clinical study of DEspR+ CD11b+ neutrophil-subset in sICH patients.

Alpha-Asarone Ameliorates Neurological Dysfunction of Subarachnoid Hemorrhagic Rats in Both Acute and Recovery Phases via Regulating the CaMKII-Dependent Pathways.

Early brain injury (EBI) is the leading cause of poor prognosis for patients suffering from subarachnoid hemorrhage (SAH), particularly learning and memory deficits in the repair phase. A recent report has involved calcium/calmodulin-dependent protein kinase II (CaMKII) in the pathophysiological process underlying SAH-induced EBI. Alpha-asarone (ASA), a major compound isolated from the Chinese medicinal herb Acorus tatarinowii Schott, was proven to reduce secondary brain injury by decreasing CaMKII over-phosphorylation in rats' model of intracerebral hemorrhage in our previous report. However, the effect of ASA on SAH remains unclear, and the role of CaMKII in both acute and recovery stages of SAH needs further investigation. In this work, we first established a classic SAH rat model by endovascular perforation and intraperitoneally administrated different ASA doses (10, 20, and 40mg/kg) 2h after successful modeling. Then, the short- and long-term neurobehavioral performances were blindly evaluated to confirm ASA's efficacy against SAH. Subsequently, we explored ASA's therapeutic mechanism in both acute and recovery stages using histopathological examination, TUNEL staining, flow cytometry, Western-blot, double-immunofluorescence staining, and transmission electron microscopy (TEM) observation. Finally, KN93, a selective CaMKII inhibitor, was applied in oxyhemoglobin-damaged HT22 cells to explore the role of CaMKII in ASA's neuroprotective effect. The results demonstrated that ASA alleviated short- and long-term neurological dysfunction, reduced mortality and seizure rate within 24h, and prolonged 14-day survival in SAH rats. Histopathological examination showed a reduction of neuronal damage and a restoration of the hippocampal structure after ASA treatment in both acute and recovery phases of SAH. In the acute stage, the Western-blot and flow cytometer analyses showed that ASA restored E/I balance, reduced calcium overload and CaMKII phosphorylation, and inhibited mitochondrion-involved apoptosis, thus preventing neuronal damage and apoptosis underlying EBI post-SAH. In the recovery stage, the TEM observation, double-immunofluorescence staining, and Western-blot analyses indicated that ASA increased the numbers of synapses and enhanced synaptic plasticity in the ipsilateral hippocampi, probably by promoting NR2B/CaMKII interaction and activating subsequent CREB/BDNF/TrkB signaling pathways. Furthermore, KN93 notably reversed ASA's neuroprotective effect on oxyhemoglobin-damaged HT22 cells, confirming CaMKII a potential target for ASA's efficacy against SAH. Our study confirmed for the first time that ASA ameliorated the SAH rats' neurobehavioral deterioration, possibly via modulating CaMKII-involved pathways. These findings provided a promising candidate for the clinical treatment of SAH and shed light on future drug discovery against SAH.