Yumei Tian

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.

Hypoxia stimulates proliferation of rat neural stem cells with influence on the expression of cyclin D1 and c-Jun N-terminal protein kinase signaling pathway in vitro

Ischemia/hypoxia is known to induce the neural stem cells proliferation and neural differentiation in rodent and human brain; however its mechanisms remain largely unknown. In this study we investigated the effect of hypoxia on neural stem cells (NSCs) proliferation with the expression of cyclin D1 and the phosphorylation of mitogen-activated protein kinases (MAPK) signaling molecules. NSCs were cultured from cortex of fetal Sprague-Dawley rats on embryonic day 5.5. The hypoxia was made using a microaerophilic incubation system. The NSCs proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, diameter measurement of neurospheres, bromodeoxyuridine (BrdU) incorporation assay and cell cycle analysis. The cell death of NSCs was evaluated by terminal dUTP nick-end labeling (TUNEL) assay. The expression of cyclin D1, phosphorylated extracellular signal regulated kinase (ERK), c-Jun N-terminal protein kinase (JNK) and p38 were analyzed by immunoblotting assay. The results showed that hypoxia increased NSCs proliferation in cell amount, diameter of neurospheres, BrdU incorporation and cell division, and the highest proliferation of the NSCs was observed with 12 h hypoxic treatment; hypoxia did not decrease cell death of NSCs; after hypoxic treatment, the expression of cyclin D1 increased, meanwhile P-JNK2 level increased, P-p38 decreased, and no significant change in P-ERK2 level compared to normoxic cultures. JNK inhibitor SP600125 attenuated the increase of cyclin D1 induced by hypoxia. These findings propose that hypoxia increases cyclin D1 expression through activation of JNK in NSCs of rat in vitro, suggesting a novel possible mechanism for hypoxia-induced proliferation of NSCs.

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.

The quantification of ADAMTS expression in an animal model of cerebral ischemia using real-time PCR.

Background:  ADAMTS1 and ADAMTS8 are proteases involved in extracellular matrix proteolysis and antiangiogenesis, but little is known about their expression and function in cerebral ischemia. We investigated the changes in ADAMTS1 and ADAMTS8 in a rat model of permanent middle cerebral artery occlusion (pMCAO). The expressions of glyseraldehyde‐3‐phosphate dehydrogenase (GAPDH), β‐actin, cyclophilin, and RPL13A were examined in order to validate the appropriate housekeeping genes for a long duration after inducing cerebral ischemia.

Effect of histamine on regional cerebral blood flow of the parietal lobe in rats.

Histamine is a powerful modulator that regulates blood vessels and blood flow. The effect of histamine on the extracortical vessels has been well described, while much less is known about the effect of histamine on intracortical vessels. In this study, we investigated the effect of histamine on regional cerebral blood flow in rat parietal lobe with laser Doppler flowmetry. The pharmacological characteristics of distinct ways (intracerebroventricular injection, intraperitoneal injection, and cranial window infusion) in applying histamine to the brain were also obtained and compared. Histamine applied in three ways all produced a decrease of rCBF in parietal lobe in a concentration-dependent manner. Cranial window infusion was the most effective way and intraperitoneal injection of L-histidine was the most ineffective, although it is a simple and applied way. To determine which type of receptor takes part in the vessel contraction induced by histamine, H1 receptor antagonist, diphenhydramine, and H2 receptor antagonist, cimetidine, were applied, respectively, before histamine administration. When the injection of cimetidine was conducted in advance, histamine still resulted in a decrease of infusion amount; while the injection of diphenhydramine was conducted in advance, the infusion of blood amount wasn’t changed. These findings indicated that histamine could result in a reduction of rCBF in the rat parietal lobe and this effect of histamine may attribute partly to its combination with H1 receptor.

Neonatal Ventral Hippocampus Lesion Changes Nuclear Restricted Protein/Brain (NRP/B) Expression in Hippocampus, Cortex and Striatum in Developmental Periods of Rats.

Schizophrenia is conceptualized as a neurodevelopmental disorder in which developmental alterations in immature brain systems are not clear. Rats with neonatal ventral hippocampal lesions (NVHL) can exhibit schizophrenia-like behaviors, and these rats have been widely used to study the developmental mechanisms of schizophrenia. The nuclear restricted protein/brain (NRP/B) is a nuclear matrix protein that is critical for the normal development of the neuronal system. This study assessed the effect of NVHL induced by the administration of ibotenic acid on the protein expression of NRP/B in the hippocampus, cortex and striatum in pre- and post-pubertal rats. The expressions of NeuN in various developmental periods were assessed accordingly. Sprague-Dawley rat pups were administered ibotenic acid at postnatal day (PD) 7. Western blotting and an immunofluorescence staining analysis showed that the expression of NRP/B was significantly decreased in the hippocampus, cortex and striatum of the NVHL rats at PD14, 28 and 42. The expressions of NeuN were decreased accordingly. In vitro experiment showed the NRP/B knockdown can decrease the Tuj1 expression in cultured cortical neurons. The data suggest that NVHL induces a change in NRP/B expression that affects neurons in the developmental period.