Hemorrhage In Rats Research Articles

Hepatic Cytochrome P450 Profiles in Hemorrhagic Shock Model Rats After Transfusion With Stored Red Blood Cells

Red cell transfusions, which deteriorate in quality during storage, triggers several negative biological responses. However, little is known regarding the effects of stored red cell transfusion on cytochrome P450 (P450) profiles. To clarify this issue, we investigated hepatic P450 profiles in hemorrhagic shock model rats after resuscitation with stored packed red cells (PRC). The pharmacokinetics data for P450-metabolizing substrates showed that the clearance of substrates for Cyp1A2 and Cyp3A2 in the stored PRC resuscitation group were decreased compared to sham group. The protein expression, metabolic activity and mRNA expression of the P450 isoforms in the stored PRC resuscitation group were lower than the corresponding values for the sham group. However, these changes would be expected to have weak effects on the in vivo pharmacokinetics of the concomitant drugs based on the criteria stated in the guideline on drug interactions. In contrast, the results of these P450 profiles in the stored PRC and fresh PRC resuscitation group exhibited a similar trend. These results suggest that the stored PRC transfusion has an influence on the hepatic P450 profiles, but is of little clinical significance, not by the deterioration of the quality of red cells but pathophysiological alterations following the hemorrhage and transfusion.

Semi-Interpenetrating Polymer Network of Hyaluronan and Chitosan Self-Healing Hydrogels for Central Nervous System Repair.

The repair of the central nervous system (CNS) is a major challenge because of the difficulty for neurons or axons to regenerate after damages. Injectable hydrogels have been developed to deliver drugs or cells for neural repair, but these hydrogels usually require conditional stimuli or additional catalysts to control the gelling process. Self-healing hydrogels, which can be injected locally to fill tissue defects after stable gelation, are attractive candidates for CNS treatment. In the current study, the self-healing hydrogel with a semi-interpenetrating polymer network (SIPN) was prepared by incorporation of hyaluronan (HA) into the chitosan-based self-healing hydrogel. The addition of HA allowed the hydrogel to pass through a narrow needle much more easily. As the HA content increased, the hydrogel showed a more packed nanostructure and a more porous microstructure verified by coherent small-angle X-ray scattering and scanning electron microscopy. The unique structure of SIPN hydrogel enhanced the spreading, migration, proliferation, and differentiation of encapsulated neural stem cells in vitro. Compared to the pristine chitosan-based self-healing hydrogel, the SIPN hydrogel showed better biocompatibility, CNS injury repair, and functional recovery evaluated by the traumatic brain injury zebrafish model and intracerebral hemorrhage rat model. We proposed that the SIPN of HA and chitosan self-healing hydrogel allowed an adaptable environment for cell spreading and migration and had the potential as an injectable defect support for CNS repair.

Neuroprotective Effects of Early Brain Injury after Subarachnoid Hemorrhage in Rats by Calcium Channel Mediating Hydrogen Sulfide.

The present study explored the modulating apoptosis effect of hydrogen sulfide (H2S) in subarachnoid hemorrhage (SAH) rats and its exact mechanism. A rat SAH model established by intravascular puncturing was used for the present study. After giving NaHS (donor of H2S), an L-type calcium channel opener (Bay K8644), or a calcium channel agonist (nifedipine), the neurological function of the rats, associated pathological changes, and expression of apoptosis-related proteins (Bcl-2, Bax, and caspase-3) and microtubule-associated protein (MAP-2) were examined. The concentration of H2S and expression of cystathionine beta synthase in the hippocampus changed upon early brain injury (EBI) after SAH. Compared with the SAH group, the neurological function of the rats and microstructure observed by electron microscopy were better in the SAH + NaHS group and SAH + Bay K8644 group. It was observed that apoptosis was more obvious in the SAH group than in the control group and was alleviated in the SAH + NaHS group. Furthermore, the alleviating effect of NaHS was partially weakened by nifedipine, indicating that the effect of anti-apoptosis in H2S might be correlated with the calcium channel. The expression of Bax and caspase-3 was elevated, while the expression of Bcl-2 decreased in the SAH group but improved in the SAH + NaHS and SAH + Bay K8644 group. Compared with the SAH + NaHS group, the expression of pro-apoptotic proteins was higher in the SAH + NaHS + nifedipine group. Therefore, upon EBI following SAH, the H2S system plays an important neurological protective effect by modulating the function of the L-type calcium channel and inhibiting apoptosis.

Integration of organ metabolomics and proteomics in exploring the blood enriching mechanism of Danggui Buxue Decoction in hemorrhagic anemia rats

Ethnopharmacological relevanceDanggui Buxue Decoction (DBD), as a classical Chinese medicine prescription, is composed of Danggui (DG) and Huangqi (HQ) at a ratio of 1:5, and it has been used clinically in treating anemia for hundreds of years. Aim of the studyThe aim of this study was to explore the treatment mechanisms of DBD in anemia rats from the perspective of thymus and spleen. Materials and methodsIn this study, a successful hemorrhagic anemia model was established, and metabolomics (UPLC-QTOF-MS/MS) and proteomics (label-free approach) together with bioinformatics (Gene Ontology analysis and Reactome pathway enrichment), correlation analysis (pearson correlation matrix) and joint pathway analysis (MetaboAnalyst) were employed to discover the underlying mechanisms of DBD. ResultsDBD had a significant blood enrichment effect on hemorrhagic anemia rats. Metabolomics and proteomics results showed that DBD regulated a total of 10 metabolites (lysophosphatidylcholines, etc.) and 41 proteins (myeloperoxidase, etc.) in thymus, and 9 metabolites (L-methionine, etc.) and 24 proteins (transferrin, etc.) in spleen. With GO analysis and Reactome pathway enrichment, DBD mainly improved anti-oxidative stress ability of thymocyte and accelerated oxidative phosphorylation to provide ATP for splenocyte. Phenotype key indexes were strongly and positively associated with most of the differential proteins and metabolites, especially nucleosides, amino acids, Fabp4, Decr1 and Ndufs3. 14 pathways in thymus and 9 pathways in spleen were obtained through joint pathway analysis, in addition, the most influential pathway in thymus was arachidonic acid metabolism, while in spleen was the biosynthesis of phenylalanine, tyrosine and tryptophan. Furthermore, DBD was validated to up-regulate Mpo, Hbb and Cp levels and down-regulate Ca2+ level in thymus, as well as up-regulate Fabp4, Ndufs3, Tf, Decr1 and ATP levels in spleen. ConclusionDBD might enhance thymus function mainly by reducing excessive lipid metabolism and intracellular Ca2+ level, and promote ATP production in spleen to provide energy.

Evaluation of cytochrome P450-based drug metabolism in hemorrhagic shock rats that were transfused with native and an artificial red blood cell preparation, Hemoglobin-vesicles

Hemoglobin-vesicles (Hb-V) are being developed as red blood cell (RBC) substitutes. In this study, we report on quantitative and qualitative alterations of hepatic cytochrome P450 (CYPs) and the pharmacokinetics of CYP-metabolizing drugs, with a focus on four CYP isoforms (CYP1A2, CYP2C11, CYP2E1 and CYP3A2), after Hb-V resuscitation from a massive hemorrhage. The results of proteome analysis and western blot data indicate that resuscitation with both Hb-V and packed RBC (PRBC) resulted in a decrease in the protein levels of CYPs. Along with a decrease in the protein expression of CYPs, pharmacokinetic studies showed that the elimination of CYP-metabolizing drugs was prolonged in the Hb-V and PRBC resuscitation groups. It is also noteworthy that the CYP-metabolizing drugs in the Hb-V resuscitation group was retained for a longer period compared to the PRBC resuscitation group, and this is attributed to the CYP isoforms having a lower metabolic activity in the Hb-V resuscitation group than that for the PRBC resuscitation group. These findings suggest that resuscitation with Hb-V after a massive hemorrhage has a slight but not clinically significant effect on drug metabolism via CYPs in the liver due to decreased protein levels and the metabolic activity with respect to the CYPs.

Mechanism of Fisetin Inhibiting Oxidative Stress to Alleviate the Damage of Blood Brain Barrier, Around the Intracerebral Hemorrhag Lesions

Background: The paper determined the brain water content, blood-brain barrier permeability, oxidative stress-related indicators, expressions of tight junction-associated proteins ZO-1 and Occludin, as well as expressions of ERK, p38, and JNK proteins in cerebral hemorrhage rats. The paper explored whether Fisetin reduced the damage of blood-brain barrier caused by cerebral hemorrhage through inhibiting oxidative stress, and investigated whether MAPK pathway is the main pathway for BBB damage caused by oxidative stress. Material and Methods: Rat ICH model was established. After 72 hours, mNSS score was used to measure the behavior of rats. The water content of brain tissue was determined by dry-wet weight method. Evans blue method was applied to measure the blood-brain barrier permeability. Oxidative stress index was detected by spectrophotometer. The expressions of ZO-1, Occludin, ERK, p38 and JNK proteins in rat brain tissue were determined by Western blot. Results: Fisetin significantly reduced the mNSS score, brain water content, bloodbrain barrier permeability, oxidative stress index and the expressions of ERK, p38 and JNK in rat ICH model, and also significantly increased the antioxidant index of rat ICH model, as well as the expressions of ZO-1 and Occludin. The effect is becoming more significant as the therapeutic dose is increased. Conclusion: Fisetin can inhibit the oxidative stress and alleviate the damage of BBB around the lesions of rat ICH model, thus alleviating secondary brain injury. In the rat ICH model, the expressions of ERK, p38 and JNK in the brain tissue around the lesion are increased and oxidized. The stress response is enhanced, and Fisetin inhibits oxidative stress and the expressions of ERK, p38, and JNK. MAPK is imperative in the process of oxidative stress-induced BBB damage in rat ICH model.

Thrombin-induced miRNA-24-1-5p upregulation promotes angiogenesis by targeting prolyl hydroxylase domain 1 in intracerebral hemorrhagic rats.

Thrombin is a unique factor that triggers post-intracerebral hemorrhage (ICH) angiogenesis by increasing hypoxia-inducible factor-1α (HIF-1α) at the protein level. However, HIF-1α mRNA remains unchanged. MicroRNAs (miRNAs) mediate posttranscriptional regulation by suppressing protein translation from mRNAs. This study aimed to determine if miRNAs might be involved in thrombin-induced angiogenesis after ICH by targeting HIF-1α or its upstream prolyl hydroxylase domains (PHDs). The study was divided into two parts. In part 1, rats received an injection of thrombin into the right globus pallidus. An miRNA array combined with miRNA target prediction, luciferase activity assay, and miRNA mimic/inhibitor transfection were used to identify candidate miRNAs and target genes. Part 2 included experiments 1 and 2. In experiment 1, rats were randomly divided into the sham group, ICH group, and ICH+hirudin-treated (thrombin inhibitor) group. In experiment 2, the rats were randomly divided into the sham group, ICH group, ICH+antagomir group, ICH+antagomir-control group, and ICH+vehicle group. Western blotting and quantitative real-time polymerase chain reaction were used to determine the expression of protein and miRNA, respectively. The coexpression of miR-24-1-5p (abbreviated to miR-24) and von Willebrand factor was detected by in situ hybridization and immunohistochemical analysis. The angiogenesis was evaluated by double-labeling immunofluorescence. Neurological function was evaluated by body weight, modified Neurological Severity Scores, and corner turn and foot-fault tests. In part 1, it was shown that miR-24, which is predicted to target PHD1, was upregulated (fold-change of 1.83) after thrombin infusion, and that the miR-24 mimic transfection decreased luciferase activity and downregulated PHD1 expression (p < 0.05). miR-24 inhibitor transfection increased PHD1 expression (p < 0.05). In part 2, it was shown that miR-24 was expressed in endothelial cells. The HIF-1α protein level and proliferating cell nuclear antigen-positive (PCNA+) nuclei in vessels were increased, while the PHD1 protein level was decreased after ICH, and these effects were reversed by hirudin (p < 0.05). The antagomiR-24-treated rats exhibited a markedly lower body weight and significantly poorer recovery from neurological deficit compared with those in ICH groups (p < 0.05). AntagomiR-24 intervention also led to lower miR-24 expression, a higher PHD1 protein level, and fewer PCNA+ nuclei in vessels compared with those in ICH groups (p < 0.05). The present study suggests that thrombin reduces HIF-1α degradation and initiates angiogenesis by increasing miR-24, which targets PHD1 after ICH.

Effect of Polydatin on Neurological Function and the Nrf2 Pathway during Intracerebral Hemorrhage.

This study investigates the effect of polydatin on the neurological function of cerebral hemorrhage rats and on the Nrf2 pathway of the endogenous antioxidant system in tissues around cerebral hematoma. Further, the study also aims to provide solid insights for clinical diagnosis and treatment. A total of 54 SPF grade male Wistar rats were divided into three groups: the sham group, model group, and polydatin group. Various parameters such as neurological deficit score, brain water content, pathological morphology, oxidative stress index content, Nrf2, NQO1, HO-1 mRNA expression, and the expression of HO-1, Nrf2, and kelch-like epichlorohydrin-1 (Keap1) protein were observed. Compared with the sham group, the mNSS score and brain water content of rats in the model group increased significantly after dosing (P < 0.05). When compared with the model group, the mNSS score and brain water content of rats in the polydatin group decreased significantly after dosing (P < 0.05). Compared with the other group, the serum NSE content of rats in the polydatin group decreased (P < 0.05). An increase was observed in the contents of NO, SOD, MDA, GSSG, and GSH in the brain tissue of rats in the model group when compared with the sham group. Compared with the model group, the contents of NO and MDA in the brain tissue of rats in the polydatin group decreased, while the contents of SOD, GSSG, and GSH increased (P < 0.05). The relative expressions of Nrf2, NQO1, and HO-1mRNA in the brain tissue of rats in the polydatin group was relatively high compared with both groups (P < 0.05). Polydatin can improve the neurological function of ICH rats and reduce the oxidative stress response by regulating the Nrf2-ARE pathway and downstream gene expression. This study preliminarily discussed the relevant mechanism of polydatin in the treatment of ICH rats, thus providing a theoretical reference to ICH treatment.

The Beneficial Effect of HES on Vascular Permeability and Its Relationship With Endothelial Glycocalyx and Intercellular Junction After Hemorrhagic Shock.

BackgroundVascular leakage is a common complication of hemorrhagic shock. Endothelial glycocalyx plays a crucial role in the protection of vascular endothelial barrier function. Hydroxyethyl starch (HES) is a commonly used resuscitation fluid for hemorrhagic shock. However, whether the protective effect of HES on vascular permeability after hemorrhagic shock is associated with the endothelial glycocalyx is unclear.MethodsUsing hemorrhagic shock rat model and hypoxia treated vascular endothelial cells (VECs), effects of HES (130/0.4) on pulmonary vascular permeability and the relationship to endothelial glycocalyx were observed.ResultsPulmonary vascular permeability was significantly increased after hemorrhagic shock, as evidenced by the increased permeability of pulmonary vessels to albumin-fluorescein isothiocyanate conjugate (FITC-BSA) and Evans blue, the decreased transendothelial electrical resistance of VECs and the increased transmittance of FITC-BSA. The structure of the endothelial glycocalyx was destroyed, showing a decrease in thickness. The expression of heparan sulfate, hyaluronic acid, and chondroitin sulfate, the components of the endothelial glycocalyx, was significantly decreased. HES (130/0.4) significantly improved the vascular barrier function, recovered the thickness and the expression of components of the endothelial glycocalyx by down-regulating the expression of heparinase, hyaluronidase, and neuraminidase, and meanwhile increased the expression of intercellular junction proteins ZO-1, occludin, and VE-cadherin. Degradation of endothelial glycocalyx with degrading enzyme (heparinase, hyaluronidase, and neuraminidase) abolished the beneficial effect of HES on vascular permeability, but had no significant effect on the recovery of the expression of endothelial intercellular junction proteins induced by HES (130/0.4). HES (130/0.4) decreased the expression of cleaved-caspase-3 induced by hemorrhagic shock.ConclusionsHES (130/0.4) has protective effect on vascular barrier function after hemorrgic shock.The mechanism is mainly related to the protective effect of HES on endothelial glycocalyx and intercellular junction proteins. The protective effect of HES on endothelial glycocalyx was associated with the down-regulated expression of heparinase, hyaluronidase, and neuraminidase. HES (130/0.4) had an anti-apoptotic effect in hemorrhagic shock.

The role of serum Dickkopf-1 in predicting 30-day death in severe traumatic brain injury.

ObjectiveDickkopf‐1 (DKK‐1), an inhibitor of the canonical/‐catenin cascade of the Wnt pathway, was upregulated in brain tissues of hemorrhagic stroke rats, and its rising circulating levels were associated with poor prognosis of acute ischemic stroke patients. We attempted to ascertain the relationship between serum DKK‐1 levels and 30‐day death after severe traumatic brain injury (sTBI).Materials and methodsSerum DKK‐1 levels were gauged in a total of 94 sTBI patients and 94 healthy controls. Trauma severity was assessed using Glasgow Coma Scale (GCS) and Rotterdam classification based on head computerized tomography scan. Prognostic variable was 30‐day death.ResultsCompared with controls, serum DKK‐1 levels were substantially elevated in patients (median value, 3.7 versus 1.0 ng/ml). Area under receiver operating characteristic curve was 0.802 (95% confidence interval (CI), 0.708–0.877) for predicting 30‐day death. Adjusted logistic regression showed that serum DKK‐1 levels above 3.7 ng/ml remained as an independent marker of 30‐day death (odds ratio, 8.573; 95% CI, 1.386–53.020) and overall survival (hazard ratio, 7.322; 95% CI, 1.320–40.622). An intimate correlation existed between DKK‐1 levels and GCS scores (r = −.649) in addition to Rotterdam classification (r = .664).ConclusionsHigh serum levels of DKK‐1 are closely associated with increasing severity and rising short‐term mortality of sTBI.

Mitochondrial-Derived Vesicles Protect Cardiomyocytes Against Hypoxic Damage.

Myocardial ischemia is a condition with insufficient oxygen supporting the heart tissues, which may result from myocardial infarction or trauma-induced hemorrhagic shock. In order to develop better preventive and therapeutic strategies for myocardial ischemic damage, it is important that we understand the mechanisms underlying this type of injury. Mitochondrial-derived vesicles (MDVs) have been proposed as a novel player in maintaining mitochondrial quality control. This study aimed to investigate the role and possible mechanisms of MDVs in ischemia/hypoxia-induced myocardial apoptosis. H9C2 cardiomyocytes were used for the cellular experiments. A 40% fixed blood volume hemorrhagic shock rat model was used to construct the acute general ischemic models. MDVs were detected using immunofluorescence staining with PDH and TOM20. Exogenous MDVs were reconstituted in vitro from isolated mitochondria under different hypoxic conditions. The results demonstrate that MDV production was negatively correlated with cardiomyocyte apoptosis under hypoxic conditions; exogenous MDVs inhibited hypoxia-induced cardiomyocyte apoptosis; and MDV-mediated protection against hypoxia-induced cardiomyocyte apoptosis was accomplished via Bcl-2 interactions in the mitochondrial pathway. This study provides evidence that MDVs protect cardiomyocytes against hypoxic damage by inhibiting mitochondrial apoptosis. Our study used a novel approach that expands our understanding of MDVs and highlights that MDVs may be part of the endogenous response to hypoxia designed to mitigate damage. Strategies that stimulate cardiomyocytes to produce cargo-specific MDVs, including Bcl-2 containing MDVs, could theoretically be helpful in treating ischemic/hypoxic myocardial injury.

Intracerebral Hemorrhage Induced Brain Injury Is Mediated by the Interleukin-12 Receptor in Rats.

BackgroundIL-12 inhibition of the endothelial cell functions and angiogenesis is mediated by the cross-talk between the lymphocyte and the endothelial cells, which plays a key role in inhibiting the process of angiogenesis in the eyeballs and in malignant tumors.MethodsWe established the intracerebral hemorrhage (ICH) rat model, and IL-12 receptor beta monoclonal antibody was injected into the ICH rats. Western blot, immunofluorescence and RT-qPCR were used to detect the gene expression. Brain water content, EB staining, Garcia test, Beam walking test and wire hanging test were used to assess the injury of brain in ICH rats.ResultsIL-12 gene was significantly increase in hematoma border tissue of ICH rats, and IL-12 protein mainly localized in monocytes. Anti-IL-12 treatment with IL-12 monoclonal antibodies could not only significantly decrease the brain water content and EB content in brain tissues of ICH rats, but also significantly increase the score of the Garcia, Beam balance and the Wire hanging test in ICH rats. Moreover, anti-IL-12 treatment significantly decrease the expression of pro-inflammatory gene, inflammatory gene, p-JAK2/JAK2 and p-STAT4/STAT4 protein, but significantly increase the expression anti-inflammatory gene and CD31 protein, and M2 macrophage ratio in hematoma border tissues of ICH rats. In vitro, rmIL-12 inhibited the tube formation of brain microvascular endothelial cells (BMVES) in BMVES and bone marrow-derived monocytes (BMDM) co-culture systems, but not work in a separately cultured BMVES system. In addition, Fedratinib not only reduced p-JAK2/JAK2 and p-STAT4/STAT4 protein expression in BMDM after treating with b-FGF and rmIL-12, but also significantly increased the tube formation of BMVES in BMVES and BMDM co-culture systems after treating with b-FGF and rmIL-12.ConclusionBlockade of IL-12 receptor attenuated brain injury after ICH in rat by promoting angiogenesis, and the mechanism might be related to blocking IL-12 could inhibit M2 cell activation via the JAK2/STAT4 pathway.

Penetrative needling of scalp-points improves neurological function by promoting stem cell proli-feration and activating Shh/Gil1 signaling in intracerebral hemorrhage rats

To observe the effect of penetrative needling from "Baihui" (GV20) to "Qubin" (GB7) on neural stem cell proliferation and sonic hedgehog (Shh) signaling in subventricular zone (SVZ) in intracerebral hemorrhage (ICH) rats so as to explore its mechanisms underlying improvement of ischemic injury of brain. Male SD rats were randomly divided into blank control, model, acupuncture and agonist (Purmorphamine, an activator of Shh signaling pathway) groups (n＝18 in each group, 6 for H．E. stain, 6 for examining neuronal cell proliferation, and 6 for immunohistochemistry). The ICH model was established by injecting autogenous blood (50 µL) into the right caudate nucleus. The neurological defect was scored with refe-rence to Bederson's method. Penetrative needling from GV20 to GB7 was performed by manipulating the needle for 6 min (repeated 3 times in 30 min), once daily for 7 days. Intraperitoneal injection of Purmorphamine (1 mg/mL, 1 mg/kg) was performed, once daily for 7 days. Histopathological changes of the hemorrhagic penumbra region were observed under microscope after H．E. stain, the newborn neural stem cell proliferation (BrdU＋/Nestin＋ double labeled cells) in the SVZ was observed by immunofluorescence after intraperitoneal injection of BrdU (50 mg/kg), and the expression of Shh and glioma-associated hemolog-1 (Gli1) detected by immunohistochemistry. After modeling, the neurological score and expression levels of Shh and Gil1 proteins were significantly increased in the model group relevant to the blank control group (P<0.001). Following the interventions, the neurological score was evidently decreased (P<0.05), while the number of BrdU＋/Nestin＋ double labeled cells and the expression levels of Shh and Gil1 proteins were significantly increased in both acupuncture and agonist groups in comparison with the model group (P<0.001). No significant differences were found between the acupuncture and agonist groups in down-regulating the neurological score and in up-regulating the number of BrdU＋/Nestin＋ double labeled cells and the expression of Shh and Gil1 proteins (P>0.05). Outcomes of H．E. stain showed obvious edema, disordered arrangement of cells, infiltration of inflammatory cells and red blood cells with glial cell hyperplasia around the hematoma area in the model group, which was relatively milder in both acupuncture and agonist groups such as in basic disappearance of edema and inflammatory reaction. Penetrative needling from GV20 to GB7 can obviously improve neurological function in ICH rats, which is related to its effects in activating Shh/Gil1 signaling and in further promoting neural stem cell proliferation in the SVZ region.

Molecular mechanism whereby paraoxonase-2 regulates coagulation activation through endothelial tissue factor in rat haemorrhagic shock model.

We investigated the molecular mechanism of paraoxonase-2 (PON-2) in regulating blood coagulation activation in rats with haemorrhagic shock through endothelial tissue factor (TF). Thirty adult Sprague Dawley rats were randomly divided into three groups: healthy control group (group A), the haemorrhagic shock PON-2 treatment group (group B), and the haemorrhagic shock group (group C). After the model was established, blood was withdrawn from the inferior vena cava of all rats. The difference in plasma thrombomodulin (TM) levels of the three groups was determined by Western blotting. The expression of transcription factors Egr-1 and Sp1 was detected by Western blotting assays. reverse transcription-polymerase chain Reaction (RT-PCR) was used to determine the mRNA expression of t-PA, PAI-1, TM, and PON-2 in the serum of three groups of rats. Endothelial TF was measured by enzyme linked immunosorbent assay (ELISA), and coagulation assay was used to detect the activity of coagulation factor VIII. Histopathological examination of the arteries of the rats was performed. The molecular mechanism of PON-2 in regulating blood coagulation activation in haemorrhagic shock model rats by endothelial tissue factor was analysed. The expression of thrombin was determined by electrophoresis. Compared with the healthy control group, the expression of TM in groups B and C decreased, both 188.64 ± 12.47 and 137.48 ± 9.72, respectively, with a significant difference. The mRNA expression of TM and PON was determined by RT-PCR. The mRNA expression of TM and PON in group B was 0.97 ± 0.07 and 1.14 ± 0.09, compared with the control group, and the mRNA expression of TM and PON in group C was 0.86 ± 0.38 and 1.12 ± 0.41, both of which increased, and there were significant differences. By measuring the expression of endothelial TF, the expression of TF in groups B and C was elevated to 12.69 ± 1.07 and 11.59 ± 0.87, with significant differences. The enzyme activities of PON-2 in groups B and C, which were 110.34 ± 14.37 and 52.37 ± 8.06, respectively, were increased compared with the healthy control group and there were significant differences. PON-2 regulates the activation of coagulation in rats with haemorrhagic shock by regulating the expression of endothelial tissue-related genes such as plasma TM and endothelial TF under hypoxic and ischaemic conditions.

Effects of voluntary and forced exercises on motor function recovery in intracerebral hemorrhage rats.

Motor paralysis is a severe consequence of intracerebral hemorrhage (ICH) that reduces patient quality of life. Rehabilitation is beneficial for stroke patients. However, functional recovery depends on the exercise type, and which factors are effective during rehabilitation are unknown. We aimed to clarify the effect of voluntary and forced exercises for functional recovery in ICH rats. Male Sprague-Dawley rats were divided into three groups: forced treadmill running (F-Ex.), voluntary wheel cage running (V-Ex.) and no exercise (Non-Ex.). The effects of the two exercises on motor recovery were analyzed by determining the motor deficit score and using the beam walking test. Stress and motivation status after rehabilitation were determined by corticosterone concentrations (ELISA) and immunoreactivity of ΔFosB (immunohistochemistry) in the nucleus accumbens, respectively. Significantly enhanced motor functional recovery was observed in the two trained groups compared with that in the Non-Ex. group. Of note, recovery in the V-Ex. group was greater than that in the F-Ex. group. To investigate the motivation and stress related to the exercises, the expression of ΔFosB in the nucleus accumbens and corticosterone concentration were compared after rehabilitation. In the V-Ex. group, there was a significant increase of ΔFosB, and in the F-Ex. Group, there was a high concentration of corticosterone. These data suggest that the effect of training for motor recovery was enhanced by motivation and reduced by stress.

Neuroprotective mechanism of L-cysteine after subarachnoid hemorrhage.

Hydrogen sulfide, which can be generated in the central nervous system from the sulfhydryl-containing amino acid, L-cysteine, by cystathionine-β-synthase, may exert protective effects in experimental subarachnoid hemorrhage; however, the mechanism underlying this effect is unknown. This study explored the mechanism using a subarachnoid hemorrhage rat model induced by an endovascular perforation technique. Rats were treated with an intraperitoneal injection of 100 mM L-cysteine (30 μL) 30 minutes after subarachnoid hemorrhage. At 48 hours after subarachnoid hemorrhage, hematoxylin-eosin staining was used to detect changes in prefrontal cortex cells. L-cysteine significantly reduced cell edema. Neurological function was assessed using a modified Garcia score. Brain water content was measured by the wet-dry method. L-cysteine significantly reduced neurological deficits and cerebral edema after subarachnoid hemorrhage. Immunofluorescence was used to detect the number of activated microglia. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the levels of interleukin 1β and CD86 mRNA in the prefrontal cortex. L-cysteine inhibited microglial activation in the prefrontal cortex and reduced the mRNA levels of interleukin 1β and CD86. RT-PCR and western blot analysis of the complement system showed that L-cysteine reduced expression of the complement factors, C1q, C3α and its receptor C3aR1, and the deposition of C1q in the prefrontal cortex. Dihydroethidium staining was applied to detect changes in reactive oxygen species, and immunohistochemistry was used to detect the number of NRF2- and HO-1-positive cells. L-cysteine reduced the level of reactive oxygen species in the prefrontal cortex and the number of NRF2- and HO-1-positive cells. Western blot assays and immunohistochemistry were used to detect the protein levels of CHOP and GRP78 in the prefrontal cortex and the number of CHOP- and GRP78-positive cells. L-cysteine reduced CHOP and GRP78 levels and the number of CHOP- and GRP78-positive cells. The cystathionine-β-synthase inhibitor, aminooxyacetic acid, significantly reversed the above neuroprotective effects of L-cysteine. Taken together, L-cysteine can play a neuroprotective role by regulating neuroinflammation, complement deposition, oxidative stress and endoplasmic reticulum stress. The study was approved by the Animals Ethics Committee of Shandong University, China on February 22, 2016 (approval No. LL-201602022).

Recombinant Human Milk Fat Globule-Epidermal Growth Factor 8 Attenuates Microthrombosis after Subarachnoid Hemorrhage in Rats

BackgroundMicrothrombosis after subarachnoid hemorrhage has an adverse effect on prognosis. Milk fat globule-epidermal growth factor 8 promotes phagocytosis of phagocytic cells and may reduce microthrombosis. This study investigated the effects of recombinant human milk fat globule-epidermal growth factor 8 on microthrombosis and neurological function after subarachnoid hemorrhage. MethodsRats subarachnoid hemorrhage model was induced by intravascular puncture method. Western blot was performed to measure the expression of endogenous milk fat globule-epidermal growth factor 8 after subarachnoid hemorrhage. Microthrombosis was quantified by microthrombi count using immunohistochemistry and immunofluorescence. The neuroprotective effect of recombinant human milk fat globule-epidermal growth factor 8 administration was evaluated by modified Garcia score, beam balance, Rotarod test, and Morris water maze. ResultsEndogenous milk fat globule-epidermal growth factor 8 protein level increased after subarachnoid hemorrhage. Microthrombosis was significantly increased in subarachnoid hemorrhage rats brain, while recombinant human milk fat globule-epidermal growth factor 8 dramatically reduced microthrombosis as well as improve short- and long- term neurobehavior after subarachnoid hemorrhage. ConclusionsRecombinant human milk fat globule-epidermal growth factor 8 reduces microthrombosis and improves neurological function after subarachnoid hemorrhage, which may be an effective strategy for treating subarachnoid hemorrhage.

Edaravone combined with cinepazide maleate on neurocyte autophagy and neurological function in rats with subarachnoid hemorrhage.

Effects of edaravone combined with cinepazide maleate on neurocyte autophagy and neurological function in rats with subarachnoid hemorrhage were investigated. Eighty SD rats were selected to establish subarachnoid hemorrhage (SAH) rat models, which were divided into sham operation group, SAH group, MCI group and combined group. Hippocampal tissue of each group was taken to observe the number of neurocytes. The expression levels of Beclin-1 and (light chain LC3)-II of rats in each group were detected by ELISA. Pearson's correlation factors were used to analyze the correlation between Beclin-1 and LC3-11, and neurological function tests were carried out on rats of each group 14 and 28 days after administration. The morphological and structural damage of nerve cells in the combined group was further alleviated, and the survival rate of neurons significantly increased at all time points (P<0.05). The expression levels of Beclin-1 and LC3-11 in combined group was significantly higher than those in SAH group and CMI group (P<0.05), and Beclin-1 was positively correlated with LC3-11 (r=0.9454). Longa score of the combined group was significantly lower than that of the other two groups, and muscle strength score was significantly higher than that of the other two groups (P<0.05). Edaravone combined with cinepazide maleate can effectively increase the survival rate of brain cells and promote the volatilization of neurological function in the treatment of hemorrhage in the subretinal space of the omentum, which is worthy of popularization and application.

LncRNA Snhg3 contributes to dysfunction of cerebral microvascular cells in intracerebral hemorrhage rats by activating the TWEAK/Fn14/STAT3 pathway

LncRNA small nucleolar RNA host gene 3 (Snhg3) has been involved in cell proliferation and migration in malignant cells. However, its role in regulating functions of non-malignant cells has been hardly reported. Here, we found Snhg3 expression was sharply induced in primary brain microvascular endothelial cells (BMVECs) treated with oxygen-and-glucose-deprivation (OGD) plus hemin, an in vitro model of intracerebral hemorrhage (ICH). Downregulation of Snhg3 by siRNA transfection improved cell proliferation and migration abilities and reduced cell apoptosis and monolayer permeability in BMVECs under treatment with OGD plus hemin. Snhg3 overexpression suppressed cell proliferation and migration and increased cell apoptosis and monolayer permeability under normal condition. In ICH rats, downregulation of Snhg3 by siRNA injection improved behavioral and histological manifestations, including number of right turns, limb placement score, integrity of blood-brain barrier (BBB), brain water content and cell apoptosis in vivo. In the mechanism exploration, we found that, TWEAK and Snhg3 displayed a positive correlation with each other. Snhg3 overexpression increased expression of TWEAK protein and its receptor Fn14, that were also induced by OGD plus hemin, activating the downstream neuroinflammatory pathway STAT3 and enhancing the secretion of MMP-2/9. Finally, the TWEAK-siRNA, the Fn14 inhibitor ATA and the STAT3 blocker AG490 were respectively used to treat BMVECs under treatment with OGD plus hemin. Our results showed either TWEAK downregulation, Fn14 inhibition, or STAT3 blockade, could rescue Snhg3-induced impairment of BMVEC functions. In conclusion, the lncRNA Snhg3 contributes to dysfunction of cerebral microvascular cells in ICH rats by activating the TWEAK/Fn14/STAT3 pathway.

Altered long noncoding RNA profile after intracerebral hemorrhage.

ObjectiveWe investigated the expression pattern of long noncoding RNAs (lncRNA) and messenger RNAs (mRNA) from two different intracerebral hemorrhage (ICH) rat models, and performed gene ontology and gene/protein interaction analyses.MethodsWe harvested hemorrhagic brain 1, 3, and 7 days after ICH induction by stereotactic collagenase injection. We performed microarray analyses with Agilent array platform to compare the expression of lncRNA and mRNAs from hemorrhagic and normal brains. The RNA expression patterns were also examined from the autologous blood injection ICH model at days 1 and 3, and significantly altered lncRNAs from two ICH models were validated by quantitative reverse transcriptase‐polymerase chain reaction. Gene ontology analysis and pathway analysis were performed with differentially expressed mRNAs after ICH. Gene and protein interaction analysis was performed to elucidate the functional role of upregulated lncRNA in neuronal damage.ResultsAmong the 13,661 lncRNAs studied, 83, 289, and 401 lncRNAs were significantly elevated after 1, 3, and 7 days after collagenase‐induced ICH, respectively. NR_027324, or H19, was the most upregulated lncRNA after 1 day from the two ICH models and its elevation persisted until the 7th day. Gene ontology analysis revealed that immune‐related biological processes such as immune response, immune system process, and defense response were upregulated from both ICH models. Gene and protein interaction study demonstrated that NR_027324 was closely related to the type I interferon signaling pathway.InterpretationThis study illustrates the dynamic expression pattern of the lncRNA profile following ICH, and that H19 is the most consistently upregulated lncRNA after ICH.