Cunfang Qi

Neuroprotection and enhanced neurogenesis by tetramethylpyrazine in adult rat brain after focal ischemia

Abstract Objective: To investigate the effects of tetramethylpyrazine (TMP) on neural cell proliferation and differentiation in brain of rat after focal cerebral ischemia. Methods: The focal cerebral ischemia of rat was induced by middle cerebral artery occlusion (MCAO). Infarction volume was evaluated by TTC staining method. Immunohistochemistry was used to identify proliferating and differentiating cells. Results: TMP protected brain from damage by reducing volume of infarction, neuronal loss and water content. TMP not only increased the number of BrdU positive cell in SVZ, but also stimulated the cell differentiation after ischemia. The nNOS expression in cortex and dentate gyrus was reduced by treatment of TMP. Conclusion: These results indicate that TMP could protect ischemic brain damage, and promote cell proliferation and differentiation stimulated by ischemia, which might be related to the reduction of nNOS expression after treatment with TMP in rat cerebral ischemia model.

Post-stroke estradiol treatment enhances neurogenesis in the subventricular zone of rats after permanent focal cerebral ischemia.

Pretreatment with estrogen has been shown to increase subventricular zone (SVZ) neurogenesis and improve neurological outcome after cerebral ischemia reperfusion injury in mice. However, the potential of post-stroke estrogen administration to enhance neurogenesis is largely unknown. In this study, we explored whether post-stroke estradiol administration had any effect on SVZ neurogenesis in a rat model of permanent focal cerebral ischemia and elucidated the potential mechanism of its effects. Male Sprague-Dawley rats (250-280 g) were divided into sham-operated (n=10), control (n=40), and estradiol-treated (n=40) groups. 5-Bromo-2-deoxyuridine (BrdU) was used to label proliferating cells. Immunohistochemistry was used to detect neurogenesis in the ischemic ipsilateral SVZ at 1, 3, 7 and 14 days following ischemia. The protein levels of hypoxia-inducible factor 1α (HIF-1α), and vascular endothelial growth factor (VEGF) in ischemic ipsilateral SVZ were determined by Western blotting at 6, 12, 24 and 72h after ischemia. Improved behavioral deficits and reduced infarct size were seen in estradiol-treated rats (P<0.05). Post-stroke estradiol administration increased BrdU-labeled cells, nestin-positive cells, doublecortin (DCX)-positive cells and BrdU+/DCX+ cells in the ischemic ipsilateral SVZ at all time points (P<0.05). Treatment with estradiol also increased HIF-1α and VEGF protein levels in the ischemic ipsilateral SVZ at all time points examined (P<0.05). These findings indicate that post-stroke estradiol administration promotes SVZ neurogenesis in rats, probably by increasing HIF-1α and VEGF protein expression.

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.

Pre- and Posttreatment With Edaravone Protects CA1 Hippocampus and Enhances Neurogenesis in the Subgranular Zone of Dentate Gyrus After Transient Global Cerebral Ischemia in Rats:

Edaravone is clinically used for treatment of patients with acute cerebral infarction. However, the effect of double application of edaravone on neurogenesis in the hippocampus following ischemia remains unknown. In the present study, we explored whether pre- and posttreatment of edaravone had any effect on neural stem/progenitor cells (NSPCs) in the subgranular zone of hippocampus in a rat model of transient global cerebral ischemia and elucidated the potential mechanism of its effects. Male Sprague-Dawley rats were divided into three groups: sham-operated (n = 15), control (n = 15), and edaravone-treated (n = 15) groups. Newly generated cells were labeled by 5-bromo-2-deoxyuridine. Immunohistochemistry was used to detect neurogenesis. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling was used to detect cell apoptosis. Reactive oxygen species (ROS) were detected by 2,7-dichlorofluorescien diacetate assay in NSPCs in vitro. Hypoxia-inducible factor-1α (HIF-1α) and cleaved caspase-3 proteins were quantified by western blot analysis. Treatment with edaravone significantly increased the number of NSPCs and newly generated neurons in the subgranular zone (p < .05). Treatment with edaravone also decreased apoptosis of NSPCs (p < .01). Furthermore, treatment with edaravone significantly decreased ROS generation and inhibited HIF-1α and cleaved caspase-3 protein expressions. These findings indicate that pre- and posttreatment with edaravone enhances neurogenesis by protecting NSPCs from apoptosis in the hippocampus, which is probably mediated by decreasing ROS generation and inhibiting protein expressions of HIF-1α and cleaved caspase-3 after cerebral ischemia.

Hypoxia stimulates neural stem cell proliferation by increasing HIF‑1α expression and activating Wnt/β-catenin signaling.

Evidence indicates that after brain injury, neurogenesis is enhanced in regions such as hippocampus, striatum, and cortex. To study the role of hypoxia-inducible factor-1 (HIF‑1α) and Wnt signaling in cerebral ischemia/hypoxia-induced proliferation of neural stem cells (NSCs), we investigated the proliferation of NSCs, expression of HIF‑1α, and activation of Wnt signaling under conditions of pathologic hypoxia in vitro. NSCs were isolated from 30-day-old Sprague-Dawley rats and subjected to 0.3% oxygen in a microaerophilic incubation system. Cell proliferation was evaluated by measuring the diameter of neurospheres and by bromodeoxyuridine incorporation assays. Real-time quantitative PCR and Western blotting were used to detect mRNA and protein levels of HIF-1α, β-catenin, and cyclin D1 in the NSCs. The results showed that hypoxia increased NSC proliferation and the levels of HIF-1α, β‑catenin, and cyclin D1 (p < 0.05). Blockade of the Wnt signaling pathway decreased hypoxia-induced NSC proliferation, whereas activation of this pathway increased hypoxia-induced NSC proliferation (p < 0.05). Knockdown of HIF-1α with HIF-1α siRNA decreased β‑catenin nuclear translocation and cyclin D1 expression, and inhibited proliferation of NSCs (p < 0.05). These findings indicate that pathologic hypoxia stimulates NSC proliferation by increasing expression of HIF-1α and activating the Wnt/β-catenin signaling pathway. The data suggest that Wnt/β-catenin signaling may play a key role in NSC proliferation under conditions of pathologic hypoxia.

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.