Intracerebral Hemorrhage Model Group Research Articles

Suppression of HMGB1 inhibits neuronal autophagy and apoptosis to improve neurological deficits in rats following intracerebral hemorrhage

To investigate the effect of suppressing high-mobility group box 1 (HMGB1) on neuronal autophagy and apoptosis in rats after intracerebral hemorrhage (ICH) in rats. Rat models of ICH induced by intracerebral striatum injection of 0.2 U/mL collagenase Ⅳ were treated with 1 mg/kg anti-HMGB1 mAb or a control anti-IgG mAb injected via the tail immediately and at 6 h after the operation (n=5). The rats in the sham-operated group (with intracranial injection of 2 μL normal saline) and ICH model group (n=5) were treated with PBS in the same manner after the operation. The neurological deficits of the rats were evaluated using modified neurological severity score (mNSS). TUNEL staining was used to detect apoptosis of the striatal neurons, and the expressions of HMGB1, autophagy-related proteins (Beclin-1, LC3-Ⅱ and LC3-Ⅰ) and apoptosis-related proteins (Bcl-2, Bax and cleaved caspase-3) in the brain tissues surrounding the hematoma were detected using Western blotting. The expression of HMGB1 in the striatum was detected by immunohistochemistry, and serum level of HMGB1 was detected with ELISA. The rat models of ICH showed significantly increased mNSS (P < 0.05), which was markedly lowered after treatment with anti- HMGB1 mAb (P < 0.05). ICH caused a significant increase of apoptosis of the striatal neurons (P < 0.05), enhanced the expressions of beclin-1, LC3-Ⅱ, Bax and cleaved caspase-3 (P < 0.05), lowered the expressions of LC3-Ⅰ and Bcl-2 (P < 0.05), and increased the content of HMGB1 (P < 0.05). Treatment with anti-HMGB1 mAb obviously lowered the apoptosis rate of the striatal neurons (P < 0.05), decreased the expressions of Beclin-1, LC3-Ⅱ, Bax and cleaved caspase-3 (P < 0.05), increased the expressions of LC3-Ⅰ and Bcl-2 (P < 0.05), and reduced the content of HMGB1 in ICH rats (P < 0.05). Down- regulation of HMGB1 by anti-HMGB1 improves neurological functions of rats after ICH possibly by inhibiting autophagy and apoptosis of the neurons.

Metabolic Insight Into the Neuroprotective Effect of Tao-He-Cheng-Qi (THCQ) Decoction on ICH Rats Using Untargeted Metabolomics

Tao-He-Cheng-Qi decoction (THCQ) is an effective traditional Chinese medicine used to treat intracerebral hemorrhage (ICH). This study was performed to investigate the possible neuroprotective effect of THCQ decoction on secondary brain damage in rats with intracerebral hemorrhage and to elucidate the potential mechanism based on a metabolomics approach. Sprague-Dawley (SD) rats were randomly divided into five groups: the sham group, collagenase-induced ICH model group, THCQ low-dose (THCQ-L)-treated group, THCQ moderate-dose (THCQ-M)-treated group and THCQ high-dose (THCQ-H)-treated group. Following 3 days of treatment, behavioral changes and histopathological lesions in the brain were estimated. Untargeted metabolomics analysis with multivariate statistics was performed by using ultrahigh-performance liquid chromatography–mass spectrometry (UPLC-Q-Exactive Orbitrap MS). THCQ treatment at two dosages (5.64 and 11.27 g/kg·d) remarkably improved behavior (p < 0.05), brain water content (BMC) and hemorheology (p < 0.05) and improved brain nerve tissue pathology and inflammatory infiltration in ICH rats. Moreover, a metabolomic analysis demonstrated that the serum metabolic profiles of ICH patients were significantly different between the sham group and the ICH-induced model group. Twenty-seven biomarkers were identified that potentially predict the clinical benefits of THCQ decoction. Of these, 4 biomarkers were found to be THCQ-H group-specific, while others were shared between two clusters. These metabolites are mainly involved in amino acid metabolism and glutamate-mediated cell excitotoxicity, lipid metabolism-mediated oxidative stress, and mitochondrial dysfunction caused by energy metabolism disorders. In addition, a correlation analysis showed that the behavioral scores, brain water content and hemorheology were correlated with levels of serum metabolites derived from amino acid and lipid metabolism. In conclusion, the results indicate that THCQ decoction significantly attenuates ICH-induced secondary brain injury, which could be mediated by improving metabolic disorders in cerebral hemorrhage rats.

Integrated Multiomics Analysis Identifies a Novel Biomarker Associated with Prognosis in Intracerebral Hemorrhage.

Existing treatments for intracerebral hemorrhage (ICH) are unable to satisfactorily prevent development of secondary brain injury after ICH and multiple pathological mechanisms are involved in the development of the injury. In this study, we aimed to identify novel genes and proteins and integrated their molecular alternations to reveal key network modules involved in ICH pathology. A total of 30 C57BL/6 male mice were used for this study. The collagenase model of ICH was employed, 3 days after ICH animals were tested neurological. After it, animals were euthanized and perihematomal brain tissues were collected for transcriptome and TMT labeling-based quantitative proteome analyses. Protein-protein interaction (PPI) network, Gene Set Enrichment Analysis (GSEA), and regularized Canonical Correlation Analysis (rCCA) were performed to integrated multiomics data. For validation of hub genes and proteins, qRT-PCR and Western blot were carried out. The candidate biomarkers were further measured by ELISA in the plasma of ICH patients and the controls. A total of 2218 differentially expressed genes (DEGs) and 353 differentially expressed proteins (DEPs) between the ICH model group and control group were identified. GSEA revealed that immune-related gene sets were prominently upregulated and significantly enriched in pathways of inflammasome complex, negative regulation of interleukin-12 production, and pyroptosis during the ICH process. The rCCA network presented two highly connective clusters which were involved in the sphingolipid catabolic process and inflammatory response. Among ten hub genes screened out by integrative analysis, significantly upregulated Itgb2, Serpina3n, and Ctss were validated in the ICH group by qRT-PCR and Western blot. Plasma levels of human SERPINA3 (homologue of murine Serpina3n) were elevated in ICH patients compared with the healthy controls (SERPINA3: 13.3 ng/mL vs. 11.2 ng/mL, p = 0.015). Within the ICH group, higher plasma SERPINA3 levels with a predictive threshold of 14.31 ng/mL (sensitivity = 64.3%; specificity = 80.8%; AUC = 0.742, 95% CI: 0.567-0.916) were highly associated with poor outcome (mRS scores 4-6). Taken together, the results of our study exhibited molecular changes related to ICH-induced brain injury by multidimensional analysis and effectively identified three biomarker candidates in a mouse ICH model, as well as pointed out that Serpina3n/SERPINA3 was a potential biomarker associated with poor functional outcome in ICH patients.

Proteomics and metabolomics of raw rhubarb and wine-processed rhubarb in the treatment of rats with intracerebral hemorrhage.

BackgroundTo explore the mechanisms of raw rhubarb and wine-processed rhubarb treatment in a rat model of intracerebral hemorrhage (ICH).MethodsAfter adapting to their environment, 30 male Wistar rats were divided into 5 treatment groups: blank control group (CK) (normal saline), sham operation group (SICH) (normal saline), ICH model group (ICH) (normal saline), ICH + raw rhubarb enema group (RO) (raw rhubarb 3.60 g/kg), and ICH + wine-processed rhubarb enema group (WRO) (wine-processed rhubarb 3.60 g/kg). The rhubarb enema (once a day) was given since 3 days before ICH treatment, and was consistently given until 12 hours, 24 hours, 3 days, and 7 days post operation. Serum oxidative stress and inflammatory markers were detected by ELISA, and differences between raw rhubarb and wine-processed rhubarb treatment in ICH rats were screened by proteomics and metabolomics.ResultsBoth rhubarb treatments effectively improved oxidative stress and inflammatory responses. After ICH, the proteins and metabolites in the brain tissue were significantly altered. Compared with raw rhubarb, wine-processed rhubarb had a better protective effect by dysregulating amino acid metabolism in the ICH model.ConclusionsOur study provides a basis for further research into the mechanisms of rhubarb treatment from different processing technologies, and promotes the application of rhubarb as a therapeutic approach to ICH.

Effect of miR-130a on neuronal injury in rats with intracranial hemorrhage through PTEN/PI3K/AKT signaling pathway.

The aim of this study was to investigate the effect of micro-ribonucleic acid (miR)-130a on neuronal injury in rats with intracerebral hemorrhage (ICH) through the phosphatase and tensin homolog deleted on chromosome ten/phosphatidylinositol 3-hydroxy kinase/protein kinase B (PTEN/PI3K/AKT) signaling pathway. A total of 30 healthy male rats were randomly divided into three groups, including the blank control group, ICH model group (ICH group) and ICH model + miR-130a treatment group (miR-130a treatment group). The differences in neurological injury, the number of apoptotic cells in brain tissues, the activity of Caspase-9 and protein expressions of PTEN/PI3K/AKT were analyzed among the three groups, respectively. Neurological function was normal without injury in the control group. However, the neurological injury was severe in the ICH group and mild in the miR-130a treatment group. There were statistically significant differences in neurological function in the control group relative to those of the ICH group and miR-130a treatment group (p<0.05). Meanwhile, the neurological injury was markedly milder in the miR-130a treatment group than that of the ICH group, showing a statistically significant difference (p<0.05). The number of apoptotic cells was remarkably smaller in the control group when compared with the ICH group and miR-130a treatment group. However, it was markedly larger in the ICH group than that of the miR-130a treatment group, showing significant differences (p<0.05). The activity of Caspase-9 was significantly lower in the control group than ICH group and miR-130a treatment group (p<0.05). However, it increased remarkably in the ICH group compared with that of the miR-130a treatment group (p<0.05). Moreover, the protein level of PTEN in the ICH group was significantly higher than control group and miR-130a treatment group, displaying statistically significant differences (p<0.05). However, no marked difference in the protein level of PTEN was observed between the control group and miR-130a treatment group (p>0.05). The protein levels of the phosphorylated 3-hydroxy kinase (p-PI3K) and phosphorylated protein kinase B (p-AKT) were remarkably lower in the ICH group than those of the control group and miR-130a treatment group, displaying statistically significant differences (p<0.05). However, they were remarkably higher in the miR-130a treatment group than that of the control group (p<0.05). MiR-130a promotes neuronal growth in brain tissues in ICH rats and alleviates neuronal injury after ICH through the PTEN/PI3K/AKT signaling pathway. Our findings suggest that miR-130a exerts important clinical significance in the treatment of ICH.

Buyang Huanwu Decoction () Attenuates Glial Scar by Downregulating the Expression of Leukemia Inhibitory Factor in Intracerebral Hemorrhagic Rats.

To evaluate the effect of Buyang Huanwu Decoction (, BYHWD) on glial scar after intracerebral hemorrhage (ICH) and investigate the underlying mechanism. Collagenase type VII (0.5 U) was injected stereotaxically into right globus pallidus to induce ICH model. One hundred and twenty Sprague-Dawley rats were randomly divided into 3 groups according to a random number table, including normal group (n=40), ICH model group (n=40) and BYHWD group (n=40), respectively. After ICH, the rats in the BYHWD group were intragastrically administered with BYHWD (4.36 g/kg) once a day for 21 days, while the rats in ICH group were administered with equal volume of distilled water for 21 days, respectively. Double immunolabeling was performed for proliferating cell nuclear antigen (PCNA)+/glial fibrillary acidic protein (GFAP)+ nuclei. The expression of GFAP and leukemia inhibitory factor (LIF) was evaluated by immunohistochemistry and quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). The astrocytes with hypertrophied morphology around the hematoma was observed on day 3 after ICH. The number of GFAP positive cells and GFAP mRNA levels increased notably on day 3 and reached the peak on day 14 post-ICH (P<0.01). PCNA+/GFAP+ nuclei were observed around the hematoma and reached the peak on day 14 post-ICH (P<0.01). In addition, LIF-positive astrocytes and LIF mRNA level in the hemorrhagic region increased significantly till day 14 post-ICH (P<0.01). However, BYHWD not only reduced the number of PCNA+/GFAP+ nuclei, but also decreased GFAP and LIF levels (P<0.05). BYHWD could attenuate ICH-induced glial scar by downregulating the expression of LIF in the rats.

Role of flunarizine hydrochloride in secondary brain injury following intracerebral hemorrhage in rats.

This study aimed to explore the role and mechanism(s) of flunarizine hydrochloride in the intracerebral hemorrhage (ICH) rats. The 32 adult male Sprague Dawley (SD) rats were randomly assigned into four groups: control group, sham group, ICH group, and FLU + ICH group. The effects of flunarizine hydrochloride were assessed on the basis of hematoma volume, blood–brain barrier (BBB) integrity, and brain water content in the ICH rat models. The role of flunarizine hydrochloride in cell recovery was assessed by behavioral scores, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot assay. Involvement of PI3K/AKT pathway in exerting the effect of flunarizine hydrochloride was also determined. Results showed that the hematoma volume, BBB integrity, and brain water content were significantly decreased in the FLU + ICH group. Cell apoptosis significantly increased in the ICH model group, while flunarizine hydrochloride decreased this increase. The expressions of glial cell line-derived neurotrophic factor (GDNF), neuroglobin (NGB), and p-AKT were increased after flunarizine hydrochloride treatment in ICH rats. In conclusion, flunarizine hydrochloride has protective effects against ICH by reducing brain injury, cell apoptosis, and the activation of P13K/AKT pathway. These findings provide a theoretical basis for the treatment of flunarizine hydrochloride in ICH.

Changes of glucose metabolism in mouse models of intracerebral hemorrhage: 18F-FDG microPET/CT study

Objective To investigate the brain glucose metabolism with 18F-FDG microPET/CT in mouse models of intracerebral hemorrhage (ICH). Methods A total of 12 healthy adult male mice were randomly divided into sham operation group (group A, n=6) and ICH model group (group B, n=6) by simple random sampling method. The animal models were established by injecting collagenase Ⅳ into the caudate nucleus of mice. Thereafter (5.5±0.3) MBq of 18F-FDG was injected into caudal vein at 6 h, 24 h, 48 h and 3 d, 5 d, 8 d, 14 d, respectively, following anesthesia. 18F-FDG microPET/CT scans were acquired 30 min after the trace injection. SUV in the perihematomal brain tissue of ICH was measured and analyzed. Two-sample t test was used to compare SUV between groups. Results (1) Some mice had mild neurologic deficit after the sham operation in group A, while all mice had a marked neurologic deficit in group B, especially at 24 h after 18F-FDG injection. (2)After 6 h, FDG uptake in perihematomal brain tissue decreased(SUV=0.80±0.04), which significantly lower than that in the opposite side(SUV=1.10±0.04; t=2.69, P<0.05) and decreased to the minimum at 24 h(SUV=0.50±0.05). 18F-FDG uptake in perihematomal brain tissue began to increase at 3 d(SUV=1.20±0.05) and kept increasing during the 14 d observation. Compared with the group A, glucose metabolism in group B was significantly lower at each time point(t=37.67-86.60, all P<0.05). Conclusions 18F-FDG microPET/CT may dynamically reflect the changes of brain glucose metabolism in ICH mouse models. The FDG uptake in the center of ICH may disappear and the volume of hematoma with decreased uptake may shrink during the observation period. Key words: Cerebral hemorrhage; Tomography, emission-computed; Tomography, X-ray computed; Deoxyglucose; Mice

Effects of melatonin on microglia activation in rats with acute intracerebral hemorrhag

Objective To investigate the effects of melatonin on activation of microglia and the changes of superoxide dismutase (SOD) and malondialdehyde (MDA) after intracerebral hemorrhage (ICH) in rats. Methods One hundred and thirty male SD rats were randomly divided into four groups: normal group,sham-operated group,intracerebral hemorrhage model group (Model group) and melatonin intervention group (MT group). Each group was further divided into 5 subgroups respectively at 12 h,1 d, 2 d,4 d and 7 d after modeling. The ICH models were made in SD rats by using Rosenberg methods. Melatonin in dose of 10 mg/ kg in solution of 1 mg/ mL was given intraperitoneally to rats of MT group. The morphology of microglia was observed under electron microscope. OX42-positive cells were detected by immunohistochemical (ABC) methods. The contents of MDA or the activity of SOD was measured respectively by thiobarbituric acid or xanthine oxidation method. Results Electron microscope showed that the activation of microglia cell displayed in ameboid shape and swelling of neuron in hemisphere cortex in model group at 2 d after ICH,the activation of microglia cell of the cortex was insignificant in MT group. OX42-positive microglia cell in large amount surrounded the hematoma at 12 h after ICH,peaked at 1 d and lasted for 7 d. OX42-positive microglia cells in MT group were significantly fewer than that in model group at different intervals after ICH (P <0.05). The content of MDA in model group increased significantly after ICH,and higher than that in normal group at 7 d,[(0.875 ±0.098) nmol/ mg prot vs. (0.725 ±0.061) nmol/ mg prot,P <0.05,whereas the activity of SOD changes in the opposite direction,(70.46 ±3.12) U/ mg prot vs. (85.86 ±4.95) /U/ mg prot,P <0.05. Compared with model group,the content of MDA were lower and the activity of SOD were higher in MT group after ICH (P <0.05). Conclusion Melatonin provides a protective effect on the damage of nerve cell after ICH. The mechanism might be associated with melatonin by reducing the level of oxidative stress in brain tissue and attenuating the activation of microglia after ICH. Key words: Melatonin; Intracerebral hemorrhage; Microglia; Oxidative stress; Superoxide dismutase; Rats

Long-time course of protease-activated receptor-1 expression after intracerebral hemorrhage in rats

Thrombin plays an important role in brain injuries associated with intracerebral hemorrhage (ICH). The protease-activated receptor (PAR)-1 is responsible for the vast majority of the thrombin's cellular activation functions. We tested the hypothesis that thrombin-induced brain damage after ICH, at least in part, is mediated by PAR-1. We report that there are significant differences between PAR-1 positive cell number and PAR-1 mRNA absorbance ratio between ICH model group (at 6 h, 24 h, 3 d, 7 d and 14 d) and normal group ( P < 0.05). These results suggest that the long-time course of PAR-1 expression may be partly involved in the mechanism of thrombin-induced brain damage after ICH.