Qindong Shi

Adenovirus-mediated brain-derived neurotrophic factor expression regulated by hypoxia response element protects brain from injury of transient middle cerebral artery occlusion in mice

Some gene expression may be regulated by hypoxia-responsive element (HRE) that is bound by hypoxia-inducible factor-1 (HIF-1) which is up-regulated during cerebral ischemia. To explore ischemia/hypoxia-controlled expression and the neuroprotective effects of brain-derived neurotrophic factor (BDNF) after ischemic brain injury, an adenoviral vector using five copies of hypoxia response element (HRE) in the vascular endothelial growth factor gene to regulate the expression of BDNF gene (Ad5HRE:BDNF) was constructed, and its efficacy was verified for driving BDNF expression in cultured Hela cells under hypoxic condition by ELISA. We found that the concentration of BDNF in the Ad5HRE:BDNF-transfected culture media was 28-fold greater in a hypoxic condition than under normoxia. To examine the effect of Ad5HRE:BDNF on ischemic brain injury in vivo, Ad5HRE:BDNF was injected into right caudate putamen of adult mice 7 days prior to 60 min transient middle cerebral artery occlusion (MCAO). It was found that exogenous BDNF expression was increased in the Ad5HRE-BDNF-treated group and infarct volume of the Ad5HRE:BDNF-treated group at 3 days after MCAO was significantly smaller than that of vehicle- or AdNull-treated groups. Moreover, Ad5HRE:BDNF injection resulted in significantly improved sensorimotor scores 7 days after MCAO and induced a reduction in the number of Fluoro-Jade B-positive neurons and TUNEL-positive cells, compared with vehicle- or AdNull-injection. Our findings suggest that BDNF expression could be regulated in hypoxia/ischemia condition with five copies of HRE and ameliorates ischemic brain injury in a mouse focal cerebral ischemia model.

Tetramethylpyrazine promotes proliferation and differentiation of neural stem cells from rat brain in hypoxic condition via mitogen-activated protein kinases pathway in vitro

This study investigated the effects of tetramethylpyrazine (TMP), an active element of traditional Chinese medicine Ligusticum Chuanxiong, on proliferation and differentiation of neural stem cells (NSCs) from rat brain in hypoxia condition and the activation of mitogen-activated protein kinases (MAPKs) signaling pathway during the processes. The results showed that TMP promoted the proliferation and differentiation of the NSCs into neurons. TMP increased the phosphorylation of ERK1/2 and decreased the phosphorylation of p38 at different time points. ERK inhibitor (U0126) in part blocked the differentiation of the NSCs into neurons induced by TMP. Our findings demonstrated that TMP enhanced the proliferation and differentiation of NSCs of rat after hypoxia in vitro, in which the phosphorylation of ERK and p38 was involved.

AMN082 promotes the proliferation and differentiation of neural progenitor cells with influence on phosphorylation of MAPK signaling pathways

Metabotropic glutamate receptors (mGluRs) are expressed in neural progenitor cells (NPCs) and may play important roles in the neurogenesis during embryonic development and adult brain repair following injuries. In the present study, we investigated the expression of metabotropic glutamate receptor 7 (mGluR7) and the possible roles of this receptor in the proliferation and differentiation of NPCs isolated from embryonic Sprague-Dawley (SD) rats. The results showed that under the normal culture and the hypoxic condition, both mRNA and protein of mGluR 7 are expressed in NPCs. Administration of AMN082, a selective agonist ofmGluR7, promoted the proliferation and differentiation of NPCs. We also demonstrated that activation of JNK and ERK signaling pathways are involved in the differentiation of NPCs into neurons following AMN082 treatment. AMN082 stimulated p-ERK and p-JNK2 expression in both normal and hypoxic conditions at different time points. But p-p38 decreased in normoxia and increased in hypoxia condition at 6h following treated with AMN082 activation. In conclusion, mGluR7 possesses the potential in promoting rat NPCs proliferation and differentiation in vitro with changes in phosphorylation of mitogen-activated protein kinases (MAPK) signaling pathways, suggesting that mGluR7 may exert an important role in brain development and repair of the central nervous system after injury.

Selection of housekeeping genes for normalization of RT-PCR in hypoxic neural stem cells of rat in vitro

Gene expression analysis under various conditions using real-time reverse transcription polymerase chain reaction (RT-PCR) needs reliable control genes. Housekeeping genes are commonly used as the control. However, no validated housekeeping gene is available for study of hypoxic neural stem cell culture. To choose appropriate internal control genes, the expression of eight commonly used housekeeping genes was examined in rat neural stem cell model to find one or more stably expressed genes under hypoxic/ischemic conditions. Two genes, HPRT and RPL13A were identified as the most confidential housekeeping genes in this research by geNorm and NormFinder softwares. As a groundwork, the most stable housekeeping genes for neural stem cells under hypoxic/ischemic conditions are initially investigated and validated in this experiment, which might provide a better understanding for the gene expression study in ischemic and necrotic neural stem cell cultures or in ischemic diseases of the central nervous system (CNS).

Cell proliferation in ependymal/subventricular zone and nNOS expression following focal cerebral ischemia in adult rats

Abstract Neuronal nitric oxide synthase (nNOS) regulates neurogenesis in normal developing brain, but the role of nNOS in neurogenesis in the ischemic brain remains unclear. To investigate the temporal and spatial relationship between cell proliferation of the ependymal/subventricular zone (SVZ), a principal neuroproliferative region in the adult brain, and nNOS expression, the male Sprague–Dawley rats weighing 250–350 g were used. The focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO). 10 μl of 0.2% fluorescence dye DiI was injected into the right lateral ventricle to prelabel ependymal/subventricular zone cells before ischemia. The rats were killed immediately after ischemia and days 1, 3, 7, 11, 14, 21 and 28 after ischemia. DiI-labeled cell counting was employed to assess cell proliferation. Immunohistochemistry and grayscale analysis were performed to determine nNOS localization and its quantity in the specific regions. Compared with control, the density of DiI-labeled cells in the ipsilateral ependyma/SVZ was significantly higher at days 1, 3, 7 and 11 after ischemia, whereas the quantity of nNOS expression in the ependyma/SVZ adjacent regions was significantly lower at the above time points. Additionally, nNOS positive cells were largely excluded from SVZ, and their long processes did not enter the ependyma/SVZ. Our results indicate that after focal cerebral ischemia, decreased nNOS expression in the ipsilateral ependymal/SVZ adjacent regions might be related to cell proliferation in the ependymal/SVZ.

Neuroprotection of neurotrophin-3 against focal cerebral ischemia/reperfusion injury is regulated by hypoxia-responsive element in rats.

Exogenous delivery of the neurotrophin-3 (NT-3) gene may provide a potential therapeutic strategy for ischemic stroke. To investigate the neuroprotective effects of NT-3 expression controlled by 5HRE after focal cerebral ischemia, we constructed a recombinant retrovirus vector (RV) with five copies of hypoxia-responsive elements (5HRE or 5H) and NT-3 and delivered it to the rat brain. Three groups of rats received RV-5H-NT3, RV-5H-EGFP or saline injection. Three days after gene transfer, the rats underwent 90min of transient middle cerebral artery occlusion (tMCAO), followed by 1-28days of reperfusion. Three days after tMCAO, brain NT-3 expression was significantly increased in the RV-5H-NT3-transduced animals compared with the RV-5H-EGFP or saline group, and brain infarct volume was smaller in the RV-5H-NT3-transduced group than the RV-5H-EGFP or saline group. The percentage of TUNEL-positive cells was reduced in RV-5H-NT3-transduced brains compared with the RV-5H-EGFP or saline group 3 and 7days after tMCAO. Furthermore, the neurological status of RV-5H-NT3-transduced rats was better than that of RV-5H-EGFP- or saline-transduced animals from 1day to 4weeks after tMCAO. Our results demonstrated that 5HRE could modulate NT-3 expression in the ischemic brain environment and that the up-regulated NT-3 could effectively improve neurological status following tMCAO due to decreased initial damage. To avoid unexpected side effects, 5HRE-controlled gene expression might be a useful tool for gene therapy of ischemic disorders in the central nervous system.

Vasoactive intestinal peptide in rats with focal cerebral ischemia enhances angiogenesis

We studied the effect of vasoactive intestinal peptide (VIP) on angiogenesis in the ischemic boundary area after focal cerebral ischemia. Adult male Sprague-Dawley rats underwent middle cerebral artery occlusion for 2 h. A single dose of VIP was given via i.c.v. injection at the beginning of reperfusion. Immunohistochemistry and Western blotting were performed to assay angiogenesis and brain levels of vascular endothelial growth factor (VEGF) protein, respectively. In addition, the expression of VEGF and its receptors (flt-1 and flk-1), as well as endothelial proliferation, was measured using rat brain microvascular endothelial cells. Immunohistochemical analyses revealed significant (P<0.05) increases in the numbers of bromodeoxyuridine (BrdU) positive endothelial cells and microvessels at the boundary of the ischemic lesion in rats treated with VIP compared with rats treated with saline. Western blotting analysis showed that treatment with VIP significantly (P<0.05) raised VEGF levels in the ischemic hemisphere. In addition, treatment with VIP increased flt-1 and flk-1 immunoreactivity in endothelial cells. In vitro, incubation with VIP significantly (P<0.01) increased the proliferation of endothelial cells and induced the expression of VEGF, flt-1 and flk-1 in endothelial cells. The stimulatory effect of VIP on the proliferation of endothelial cells was significantly (P<0.01) inhibited by SU5416, a selective inhibitor of VEGF receptor tyrosine kinase. Our data suggest that treatment with VIP enhances angiogenesis in the ischemic brain, and this effect may be mediated by increases in levels of VEGF and its receptors.

Decreased neuronal nitric oxide synthase expression and cell migration in the peri-infarction after focal cerebral ischemia in rats.

Neuronal nitric oxide synthase (nNOS) regulates neurogenesis in the normal developing brain, but the role of nNOS in neurogenesis of the adult ischemic brain remains unclear. The aim of this study was to investigate the temporal and spatial relationship between cell migration from the ependymal/subventricular zone (SVZ) to peri‐infarction and nNOS expression in the rat. Ependymal/subventricular zone cells were prelabeled with fluorescence dye DiI. Focal cerebral ischemia was induced by occlusion of the left middle cerebral artery. At 1, 3, 7, 14 and 21 days after ischemia, the rats were killed in order to determine the number of migrating cells, the colocalization of DiI and nNOS as well as nNOS quantity in specific regions. Compared to non‐ischemic control and 1 day post‐ischemia, the number of DiI‐labeled cells in the selected regions increased at 3 days and peaked 14 days following ischemia. During 3–7 days post‐ischemia, none of the migrating cells expressed nNOS and decreased nNOS expression was observed in the regions where migrating cells passed through. These results suggest the possible association between ependymal/SVZ cell migration and decreased nNOS expression within the areas including the migrating routes towards the peri‐infarction.

Retroviral vector-mediated hypoxia-regulated neurotrophin-3 gene transfer reduces apoptosis induced by hypoxia in PC12 cells

Background Gene therapy for ischemic diseases is a prospective strategy. However, excessive expression of therapeutic genes may produce undesired side effects. Recently, multiple copies hypoxia response elements (HRE) were developed to conditionally regulate gene expression under hypoxia. As a nerve growth factor, Neurotrophin3 (NT-3) possesses neural protect effects either in vitro or in vivo. To explore hypoxia-controlled NT-3 expression, we constructed a recombinant retrovirus vector with 5HRE and NT-3, and generated a gene transferred cell line PC12-5HRE-NT3 to determine effects of conditionally expressed NT-3 on apoptosis induced by hypoxia in PC12 cells.

Retrovirus mediated hypoxia-responsive element-regulated neurotrophin-3 transduction attenuates brain injury following focal cerebral ischemia in rats

Background Exogenous delivery of Neurotrophin-3 (NT-3) gene may provide a potential therapeutic strategy for ischemic stroke. But uncontrolled expression of NT-3 may cause deleterious side effects. Recently, hypoxia-specific gene expression systems have been developed in various ischemic diseases. To explore ischemia/hypoxia-controlled expression of NT-3 in rats, we constructed a recombinant retrovirus vector with 5HRE and NT-3 and delivered it to rat brain to investigate the neuroprotective effects of hypoxia induced NT-3 overexpression on focal cerebral ischemia.