Haiying Li

Evaluation of the Protective Potential of Brain Microvascular Endothelial Cell Autophagy on Blood–Brain Barrier Integrity During Experimental Cerebral Ischemia–Reperfusion Injury

Brain microvascular endothelial cell (BMVEC) injury induced by ischemia–reperfusion (I/R) is the initial phase of blood–brain barrier (BBB) disruption, which results in a poor prognosis for ischemic stroke patients. Autophagy occurs in ischemic brain and has been shown to exhibit protective effects on endothelial cell against stress. However, the potential effects of BMVEC autophagy on BBB permeability during I/R and the mechanisms underlying these effects have yet to be elucidated. In the current study, we answered these questions by using chemical modulators of autophagy, including rapamycin and lithium carbonate acting, respectively, as mammalian target of rapamycin (mTOR)-dependent and mTOR-independent autophagy inducers and 3-methyladenine (3-MA) as an autophagy inhibitor. To mimic I/R injury, BMVECs were exposed to oxygen–glucose deprivation/reoxygenation (OGD/R), and a rat transient middle cerebral artery occlusion/reperfusion (MCAO/R) model was performed. All the drugs were given at 0.5xa0h before OGD/R or MCAO/R. First, enhancement of autophagy by rapamycin and lithium carbonate attenuated, whereas suppression of autophagy by 3-MA intensified BMVEC apoptosis and the high level of ROS induced by OGD/R. In addition, rapamycin and lithium carbonate pretreatments significantly reversed the decreased level of tight junction protein zonula occludens-1 (ZO-1) induced by OGD/R and promoted the distribution of ZO-1 on cell membranes. Finally, pretreatments with rapamycin and lithium carbonate reduced evans blue extravasation and brain water content in the ischemic hemisphere of the rat. In contrast, 3-MA pretreatment exerted opposite effects both in vitro and in vivo. These results may indicate a beneficial effect of BMVEC autophagy on BBB integrity during I/R injury.

Alterations in the time course of expression of the Nox family in the brain in a rat experimental cerebral ischemia and reperfusion model: effects of melatonin

Ischemia–reperfusion (I/R) injury induces the generation of reactive oxygen species (ROS), which results in a poor prognosis for ischemic stroke patients. This study was designed to evaluate the time course of expression of the Nox family, a major source of ROS, and whether melatonin, a potent scavenger of ROS, influences these parameters in a rat model of cerebral I/R caused by middle cerebral artery occlusion (MCAO). After 2‐hr occlusion, the filament was withdrawn to allow reperfusion. At 0, 3, 6, 12, 24, and 48 hr after reperfusion, brain tissue samples were obtained for assays. Among the Nox family, the mRNA and protein levels of Nox2 and Nox4 were increased both in the ischemic hemisphere and contralateral counterpart in the experimental I/R rats at 0 hr after reperfusion, peaked at 3 hr, and then returned to the basal level at 24 hr. Double‐immunofluorescence staining further confirmed the expressions of Nox2 and Nox4 in three major types of brain cells, including neurons, astrocytes, and endothelial cells. In addition, melatonin (5 mg/kg) or its vehicle was injected intraperitoneally at 0.5 hr before MCAO. Compared with I/R + vehicle group, melatonin pretreatment diminished the increased expression of Nox2 and Nox4, reduced ROS levels, and inhibited cell apoptosis. Our findings suggested that the inhibition of Nox2 and Nox4 expressions by melatonin may essentially contribute to its antioxidant and anti‐apoptotic effects during brain I/R.

Hydrogen Sulfide Ameliorates Early Brain Injury Following Subarachnoid Hemorrhage in Rats.

Increasing studies have demonstrated the neuroprotective effect of hydrogen sulfide (H2S) in central nervous system (CNS) diseases. However, the potential application value of H2S in the therapy of subarachnoid hemorrhage (SAH) is still not well known. This study was to investigate the potential effect of H2S on early brain injury (EBI) induced by SAH and explore the underlying mechanisms. The role of sodium hydrosulfide (NaHS), a donor of H2S, in SAH-induced EBI, was investigated in both in vivo and in vitro. A prechiasmatic cistern single injection model was used to produce experimental SAH in vivo. In vitro, cultured primary rat cortical neurons and human umbilical vein endothelial cells (HUVECs) were exposed to OxyHb at concentration of 10xa0μM to mimic SAH. Endogenous production of H2S in the brain was significantly inhibited by SAH. The protein levels of the predominant H2S-generating enzymes in the brain, including cystathionineb-synthase (CBS) and 3-mercaptopyruvate sulfur transferase (3MST), were also correspondingly reduced by SAH, while treatment with NaHS restored H2S production and the expressions of CBS and 3MST. More importantly, NaHS treatment could significantly attenuate EBI (including brain edema, blood–brain barrier disruption, brain cell apoptosis, inflammatory response, and cerebral vasospasm) after SAH. In vitro, H2S protects neurons and endothelial function by functioning as an antioxidant and antiapoptotic mediator. Our results suggest that NaSH as an exogenous H2S donor could significantly reduce EBI induced by SAH.

Cyclophilin A/Cluster of Differentiation 147 Interactions Participate in Early Brain Injury After Subarachnoid Hemorrhage in Rats.

Objectives:Cyclophilin A has been found to be involved in many inflammatory diseases via its receptor, cluster of differentiation 147 (CD147). This study was designed to estimate the potential role of cyclophilin A/CD147 in subarachnoid hemorrhage–induced early brain injury. Design:Controlled in vivo laboratory study. Setting:Animal research laboratory. Subjects:Two hundred ninety adult male Sprague-Dawley rats weighing 300–350u2009g. Interventions:A prechiasmatic cistern single-injection model was used to produce experimental subarachnoid hemorrhage in Sprague-Dawley rats. The expressions of cyclophilin A and CD147, the interaction between cyclophilin A and CD147, and the secretion of cyclophilin A were assessed using immunofluorescence staining, Western blot analysis, and coimmunoprecipitation analysis. Down-regulation of cyclophilin A expression by small interfering RNA was performed, and recombinant human cyclophilin A and monoclonal antibody of CD147 were exploited to study the role of cyclophilin A/CD147 in subarachnoid hemorrhage–induced early brain injury. Measurements and Main Results:The expressions of cyclophilin A and CD147 in neurons were higher than that of the sham group and peaked at 24 hours after subarachnoid hemorrhage. Compared with sham group, subarachnoid hemorrhage was found to increase the secretion of cyclophilin A and the interaction between cyclophilin A and CD147. Cyclophilin A small interfering RNA and anti-CD147 treatments were found to ameliorate subarachnoid hemorrhage–induced early brain injury, including cortical apoptosis and necrosis, brain edema, blood-brain barrier damage, and neurobehavioral deficits. Cyclophilin A small interfering RNA and anti-CD147 treatments also decreased the phosphorylation of extracellular signal-regulated protein kinase 1/2, the protein levels of p53 and caspase-3, and the level of active nuclear factor-&kgr;B. Finally, recombinant human cyclophilin A treatment resulted in an opposite effect, which was inhibited by anti-CD147 treatment. Conclusions:Cyclophilin A/CD147 interactions may participate in subarachnoid hemorrhage–induced early brain injury via increasing neuronal apoptosis pathway, at least partly through the ERK1/2-nuclear factor-&kgr;B pathway. Cyclophilin A/CD147 may be a suitable therapeutic target for subarachnoid hemorrhage.

Role of Neurexin-1β and Neuroligin-1 in Cognitive Dysfunction After Subarachnoid Hemorrhage in Rats

Background and Purpose— Neurexin-1&bgr; and neuroligin-1 play an important role in the formation, maintenance, and regulation of synaptic structures. This study is to estimate the potential role of neurexin-1&bgr; and neuroligin-1 in subarachnoid hemorrhage (SAH)-induced cognitive dysfunction. Methods— In vivo, 228 Sprague–Dawley rats were used. An experimental SAH model was induced by single blood injection to prechiasmatic cistern. Primary cultured hippocampal neurons were exposed to oxyhemoglobin to mimic SAH in vitro. Specific small interfering RNAs and expression plasmids for neurexin-1&bgr; and neuroligin-1 were exploited both in vivo and in vitro. Western blot, immunofluorescence, immunoprecipitation, neurological scoring, and Morris water maze were performed to evaluate the mechanism of neurexin-1&bgr; and neuroligin-1, as well as neurological outcome. Results— Both in vivo and in vitro experiments showed SAH-induced decrease in the expressions of neurexin-1&bgr; and neuroligin-1 and the interaction between neurexin-1&bgr; and neuroligin-1 in neurons. In addition, the interaction between neurexin-1&bgr; and neuroligin-1 was reduced by their knockdown and increased by their overexpression. The formation of excitatory synapses was inhibited by oxyhemoglobin treatment, which was significantly ameliorated by overexpression of neurexin-1&bgr; and neuroligin-1 and aggravated by the knockdown of neurexin-1&bgr; and neuroligin-1. More importantly, neurexin-1&bgr; and neuroligin-1 overexpression ameliorated SAH-induced cognitive dysfunction, whereas neurexin-1&bgr; and neuroligin-1 knockdown induced an opposite effect. Conclusions— Enhancing the expressions of neurexin-1&bgr; and neuroligin-1 could promote the interaction between them and the formation of excitatory synapses, which is helpful to improve cognitive dysfunction after SAH. Neurexin-1&bgr; and neuroligin-1 might be good targets for improving cognitive function after SAH.

The Neuroprotection of Lysosomotropic Agents in Experimental Subarachnoid Hemorrhage Probably Involving the Apoptosis Pathway Triggering by Cathepsins via Chelating Intralysosomal Iron.

Abstractα-Lipoic acid-plus (LAP), an amine derivative of α-lipoic acid (LA), could protect cells against oxidant challenges via chelating intralysosomal iron. However, the application of LAP in experimental subarachnoid hemorrhage (SAH) is still not well known. This study was designed to evaluate the potential neuroprotection of LAP on the early brain injury (EBI) and the underlying mechanisms in a rat model of SAH. The SAH models were induced in Sprague–Dawley rats. LA and LAP were oral administration and lasted for 72xa0h once a day. The brain tissue samples were obtained for assay at 72xa0h after SAH. In experiment 1, we found that lysosome amounts in neurons decreased significantly in SAH group, and LAP (100xa0mg/kg) could stabilize lysosomal membrane markedly based on lysosomal-associated membrane protein-1 (LAMP-1) expression in neurons by immunofluorescence. Hence, the LAP dosages of 100 and 150xa0mg/kg were applied in experiment 2. Firstly, Western blot analysis showed that the protein levels of cathepsin B/D, caspase-3, Bax, ferritin, and heme-oxygenase-1 (HO-1) markedly increased after SAH, which were further confirmed by double immunofluorescence staining and reversed by LA and LAP treatments. In addition, LA and LAP also reduced oxidative stress and iron deposition in brain tissue. Furthermore, LA and LAP significantly ameliorated brain edema, blood–brain barrier injury, cortical apoptosis, and neurological behavior impairment induced by SAH. Finally, it is noteworthy that LAP exerted more significant effects than LA on these parameters as described above. LAP probably exerted neuroprotective effects via targeting lysosomes and chelating intralysosomal iron in EBI post-SAH in rats.

Transient receptor potential channel 1/4 reduces subarachnoid hemorrhage-induced early brain injury in rats via calcineurin-mediated NMDAR and NFAT dephosphorylation

Transient receptor potential channel 1/4 (TRPC1/4) are considered to be related to subarachnoid hemorrhage (SAH)-induced cerebral vasospasm. In this study, a SAH rat model was employed to study the roles of TRPC1/4 in the early brain injury (EBI) after SAH. Primary cultured hippocampal neurons were exposed to oxyhemoglobin to mimic SAH in vitro. The protein levels of TRPC1/4 increased and peaked at 5 days after SAH in rats. Inhibition of TRPC1/4 by SKF96365 aggravated SAH-induced EBI, such as cortical cell death (by TUNEL staining) and degenerating (by FJB staining). In addition, TRPC1/4 overexpression could increase calcineurin activity, while increased calcineurin activity could promote the dephosphorylation of N-methyl-D-aspartate receptor (NMDAR). Calcineurin antagonist FK506 could weaken the neuroprotection and the dephosphorylation of NMDAR induced by TRPC1/4 overexpression. Contrarily, calcineurin agonist chlorogenic acid inhibited SAH-induced EBI, even when siRNA intervention of TRPC1/4 was performed. Moreover, calcineurin also could lead to the nuclear transfer of nuclear factor of activated T cells (NFAT), which is a transcription factor promoting the expressions of TRPC1/4. TRPC1/4 could inhibit SAH-induced EBI by supressing the phosphorylation of NMDAR via calcineurin. TRPC1/4-induced calcineurin activation also could promote the nuclear transfer of NFAT, suggesting a positive feedback regulation of TRPC1/4 expressions.

Expression and clinical significance of non-phagocytic cell oxidase 2 and 4 after human traumatic brain injury

The goal of this study was to examine NOX2 and NOX4 expression in clinical samples of patients with traumatic brain injury (TBI), and to explore the correlation of NOX2 and NOX4 expression with the severity of injury, duration of injury, and prognosis. Brain samples of 20 TBI patients within 1xa0cm of the contusion site were collected and grouped based on duration of injury, Glasgow Coma Scale (GCS) and Glasgow Outcome Scale (GOS), and immunofluorescence staining were performed to examine the expression levels of NOX2 and NOX4 in the neurons and astrocytes. We found that the expression level of NOX2 in neurons and positive rate of NOX2 expression in astrocytes peaked at 12–24 and 6–12xa0h after injury, respectively. The expression level of NOX4 in neurons peaked at 24–48xa0h, and the positive rate of NOX4 expression gradually increased with prolonged injury. We also found that the higher the GCS score, the lower the expression levels of NOX2 and NOX4 in neurons was, while higher the GCS score, the lower the positive rate of NOX4 expression in astrocytes was and the higher the GOS grade, the lower the positive rate of expression in astrocytes was. In conclusion, NOX2 and NOX4 expressions significantly increase at an early stage after TBI, and abnormal expressions of NOX2 and NOX4 are correlated to patient prognosis to certain extent.

Possible Role of Raf-1 Kinase in the Development of Cerebral Vasospasm and Early Brain Injury After Experimental Subarachnoid Hemorrhage in Rats.

This study aims to clarify the potential role of Raf-1 kinase in cerebral vasospasm (CVS) and early brain injury (EBI) after subarachnoid hemorrhage (SAH). Two experimental SAH models in rats, including cisterna magna double injection model for CVS study and prechiasmatic cistern single injection model for EBI study, were performed in this research. As a specific inhibitor of Raf-1, BAY 43-9006 was used in this study. In CVS study, time course study showed that the basilar artery exhibited vasospasm after SAH and became most severe at day 5, and the phosphorylation of Raf-1 had the same trends, while both vasospasm and the phosphorylation of Raf-1 induced by SAH were inhibited by BAY 43-9006 treatment. In addition, BAY 43-9006 treatment significantly reversed the phosphorylation of ERK1/2 and the activation of NF-κB induced by SAH and decreased the messenger RNA (mRNA) levels of IL-6 and IL-1β. In EBI study, BAY 43-9006 treatment significantly suppressed the brain injury induced by SAH. Besides, BAY 43-9006 inhibited the phosphorylation of Raf-1 and ERK1/2; decreased the protein levels of COX-2, VEGF, and MMP-9; and reversed the activation of NF-κB induced by SAH. These results demonstrate that Raf-1 kinase contributes to CVS and EBI after SAH by enhancing the activation of the Raf-1/ERK1/2 and Raf-1/NF-κB signaling pathways, and that the inhibition of these pathways might offer new treatment strategies for CVS and EBI.

Pramipexole-Induced Hypothermia Reduces Early Brain Injury via PI3K/AKT/GSK3β pathway in Subarachnoid Hemorrhage rats

Previous studies have shown neuroprotective effects of hypothermia. However, its effects on subarachnoid hemorrhage (SAH)-induced early brain injury (EBI) remain unclear. In this study, a SAH rat model was employed to study the effects and mechanisms of pramipexole-induced hypothermia on EBI after SAH. Dose-response experiments were performed to select the appropriate pramipexole concentration and frequency of administration for induction of mild hypothermia (33–36u2009°C). Western blot, neurobehavioral evaluation, Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and Fluoro-Jade B (FJB) staining were used to detect the effects of pramipexole-induced hypothermia on SAH-induced EBI, as well as to study whether controlled rewarming could attenuate these effects. Inhibitors targeting the PI3K/AKT/GSK3β pathway were administered to determine whether the neuroprotective effect of pramipexole-induced hypothermia was mediated by PI3K/AKT/GSK3β signaling pathway. The results showed that intraperitoneal injection of pramipexole at 0.25u2009mg/kg body weight once per 8u2009hours was found to successfully and safely maintain rats at mild hypothermia. Pramipexole-induced hypothermia ameliorated SAH-induced brain cell death, blood-brain barrier damage and neurobehavioral deficits in a PI3K/AKT/GSK3β signaling-dependent manner. Therefore, we may conclude that pramipexole-induced hypothermia could effectively inhibit EBI after SAH in rats via PI3K/AKT/GSK3β signaling pathway.