Thrombin-triggered angiogenesis in rat brains following experimental intracerebral hemorrhage.

OBJECT: Angiogenesis occurs after intracerebral hemorrhage (ICH). Thrombin mediates mitogenesis and survival in endothelial cells and induces angiogenesis. The present study aimed to clarify whether thrombin is involved in triggering ICH-related angiogenesis. METHODS: In the first part of the experiment, autologous blood (with or without hirudin) was injected to induce ICH. In the second part, rats received either 1 U (50 µl) thrombin or 50 µl 0.9% sterile saline. In both parts, 5-bromo-2-deoxyuridine (BrdU) was administered intraperitoneally. Brains were perfused to identify BrdU-positive/von Willebrand factor (vWF)-positive nuclei. The expression of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1) and Ang-2 was evaluated by immunohistochemistry and quantitative real-time reverse transcription polymerase chain reaction. RESULTS: After ICH, the number of BrdU-/vWF-positive nuclei increased until Day 14, and vessels positive for HIF-1α, VEGF, Ang-1, and Ang-2 were observed around the clot. Quantitative analysis showed that ICH upregulated expression of HIF-1α, VEGF, Ang-1, and Ang-2 notably compared with that in sham controls (p < 0.05). However, hirudin significantly inhibited these effects. After thrombin treatment, many BrdU-positive/vWF-positive nuclei and HIF-1α-, VEGF-, Ang-1- and Ang-2-positive vessels could be detected around the affected region. CONCLUSIONS: Thrombin can induce angiogenesis in rat brains and may be an important trigger for ICH-related angiogenesis.

Salvianolic acid B protects SH-SY5Y neuroblastoma cells from 1-methyl-4-phenylpyridinium-induced apoptosis.

Parkinson's disease (PD) is associated with mitochondrial dysfunction, oxidative stress, and activation of the apoptotic cascade. In the study, we investigated the effects of salvianolic acid B (Sal B) on 1-methyl-4-phenylpyridinium (MPP(+))-treated SH-SY5Y cells, a classic in vitro model for PD. We found Sal B inhibited the loss of cell viability by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The underlying mechanisms of Sal B action were further studied. Treatment of SH-SY5Y cells with MPP(+) caused a loss of cell viability and mitochondrial membrane potential, condensation of nuclei, elevation in the level of reactive oxygen species (which was associated with cytochrome c release), an increase in the Bax/Bcl-2 mRNA ratio, and activation of caspase-3. Sal B ameliorated the MPP(+)-altered phenotypes. These results indicate that the Sal B protected SH-SY5Y cells against MPP(+)-induced apoptosis by relieving oxidative stress and modulating the apoptotic process. Our findings suggest that salvianolic acid B may be a promising agent to prevent PD.

Alteration of thrombospondin-1 and -2 in rat brains following experimental intracerebral hemorrhage. Laboratory investigation.

OBJECT: Spontaneous intracerebral hemorrhage (ICH) is among the most intractable forms of stroke. Angiogenesis, an orchestrated balance between proangiogenic and antiangiogenic factors, is a fundamental process to brain development and repair by new blood vessel formation from preexisting ones and can be induced by ICH. Thrombospondin (TSP)­1 and TSP-2 are naturally occurring antiangiogenic factors. The aim of this study was to observe their expression in rat brains with ICH. METHODS: Intracerebral hemorrhage was induced in adult male Sprague-Dawley rats by stereotactic injection of collagenase VII or autologous blood into the right globus pallidus. The expression of TSP-1 and -2 was evaluated by immunohistochemistry and quantitative real-time reverse transcription­polymerase chain reaction analysis. RESULTS: After the induction of ICH, some TSP1- or TSP2-immunoreactive microvessels resided around the hematoma for ~ 7 days and extended into a clot thereafter. Cerebral endothelial cells expressed the TSPs. The expression of TSP-1 and TSP-2 mRNA peaked at 4 and 14 days after collagenase-induced ICH, respectively. CONCLUSIONS: Findings in this study suggest that ICH can alter the expression of TSP-1 and TSP-2, which may be involved in modulating angiogenesis in brains following ICH.

[Effects of Xiehuo Bushen Decoction on survival and differentiation of transplanted neural stem cells in brains of rats with intracerebral hemorrhage].

OBJECTIVE: To investigate the effects of Xiehuo Bushen Decoction (XHBSD), a compound Chinese herbal medicine, on the survival and differentiation of transplanted neural stem cells (NSCs) in brains of rats with intracerebral hemorrhage, and to explore the mechanism of Xiehuo Bushen formula in promoting the survival of transplanted NSCs. METHODS: NSCs separated from hippocampuses of neonatal SD rats were cultured. Sixty-five panel reactive antibody (PRA) positive SD rats were selected by lymphocytotoxicity methods. The PRA positive rats were made into intracerebral hemorrhagic model and divided into three groups: cerebral hemorrhage group (n=15), NSCs transplanted group (n=25) and XHBSD group (n=25). XHBSD was orally administered after 5-bromodeoxyuridine (BrdU)-marked NSCs were transplanted in brains of rats with intracerebral hemorrhage in the XHBSD group. Rats in the other two groups were administered distilled water. The expressions of interferon gamma (IFN-gamma) and interleukin-4 (IL-4) mRNAs were measured by reverse transcription polymerase chain reaction (RT-PCR); the numbers of BrdU and 200 kD neurofilament (NF200) positive cells were detected by double-labeling immunofluorescence method. RESULTS: The expression of IFN-gamma mRNA was down-regulated significantly in the XHBSD group, but the expression of IL-4 mRNA was up-regulated significantly (P<0.05). The numbers of BrdU and NF200 positive cells were also increased remarkably in the XHBSD group. CONCLUSION: XHBSD can promote the survival and differentiation of transplanted NSCs, which may be related to inducing the expression of IL-4 mRNA and inhibiting the expression of IFN-gamma mRNA.

Cerebral angiogenesis after collagenase-induced intracerebral hemorrhage in rats.

Spontaneous intracerebral hemorrhage (ICH) is one of the most devastating subtypes of stroke. Since angiogenesis is a fundamental process to brain development and repair by new blood vessel formation from pre-existing ones, mediated by numerous angiogenic factors including vascular endothelial growth factor (VEGF), the goal of the present work is to establish whether there is cerebral angiogenesis in rat brains with collagenase-induced ICH. Investigations were also performed to evaluate whether ICH alters expression of VEGF and its receptors Flt-1 and Flk-1. ICH was induced on adult male Sprague-Dawley rats by stereotactic injection of collagenase type VII into right globus pallidus. Angiogenesis was identified by hematoxylin-eosin stain and double immunolabeling method, and expression of VEGF and the receptors was evaluated by immunohistochemistry and quantitative real time reverse transcription-polymerase chain reaction. New vessels appeared around the hematoma and extended into it from 7 days, and 5-Bromo-2-Deoxyuridine-labeled nuclei in cerebral endothelial cells resided around the hematoma and the labeling peaked from 7 to 14 days. Expression of VEGF, Flt-1 and Flk-1 was observed in cerebral endothelial cells at the hemorrhagic basal ganglion, and increases of their mRNA persisted to 28 days. These findings suggest that ICH can induce cerebral angiogenesis and upregulation of VEGF, Flt-1 and Flk-1 and that modulation of angiogenesis via altering expression of VEGF and its receptors may be a potential strategy for promoting ICH repair.

Effect of qi-tonifying and stasis-eliminating therapy on expression of vascular endothelial growth factor and its receptors Flt-1, Flk-1 in the brain of intracerebral hemorrhagic rats.

OBJECTIVE: To investigate the effects and mechanism of qi-tonifying and stasis-eliminating (QTSE) therapy on the expression of vascular endothelial growth factor (VEGF) and its receptors Flt-1 and Flk-1 in the brains of intracerebral hemorrhagic (model) rats. METHODS: One hundred and eighty Sprague-Dawley rats were randomly divided into six groups: the normal group (n=5), the sham-operative (SO) group (n=35), the model group (n=35), the QTSE group (n=35), the QT group (n=35) and the SE group (n=35). All the rats except those in the normal group and SO group were established into an intracerebral hemorrhage(ICH) model by intracerebral injection of collagenase type VII and the latter three were orally administered with Buyang Huanwu Decoction (a classical recipe for QTSE) or with some of its components for qi-tonification and for stasis-elimination, respectively. To the other three groups, normal saline solutions were given instead. Behavioral tests were carried out in the animals randomly chosen from each group on days 1, 2, 4, 7, 14, 21 and 28 after modeling. The expressions of VEGF, Flk-1 and Flt-1 were determined by immunohistochemistry and the number of vascular segments with positive expression in the injured brain area of the rats was calculated. RESULTS: From day 7 onwards, the asymmetric forelimb use rate in the QTSE group recovered more significantly than that in the other model groups. In the model group, the expressions of VEGF, Flk-1 and Flt-1 appeared on day 1 and reached a peak on day 21, then weakened gradually. In the QTSE group, as compared with the other model groups, a higher level of VEGF expression was shown from day 7 (P<0.01) and a higher level of Flt-1 expression was shown from the 7th day to the 21st day (P<0.01). CONCLUSION: QTSE therapy can up-regulate the expressions of VEGF and its receptors (Flk-1 and Flt-1) and improve the recovery of kinetic function in the ICH rats, which may be correlated with its action in modulating vascular regeneration to promote the reconstruction of microvascular networks in the damaged areas.

[Effects of naoyian serum on VEGF protein expression in cultured rat cerebral microvascular endothelial cell with hypoxia].

OBJECTIVE: To determine the effects of naoyian (NYA) serum on the expression of vascular endothelial growth factor (VEGF) protein in cultured rat cerebral microvascular endothelial cell (RCMEC) with hypoxia. METHODS: NYA serum was separated from rat heart which had been filled stomach with NYA successively for 3 days. The rat cerebral microvascular endothelial cells were taken from the Sprageu-Dawley rat brain at postborn 7 days. The rat cerebral microvascular endothelial cells were incubated at anaerobic incubator to establish the hypoxia models. The vigo of RCMEC was determined by MTT. The level of expression of VEGF protein was measured by cell immunohistochemistry and Western blot. RESULTS: The OD value of NYA serum group was higher than the control groups after hypoxia for 18 hours. VEGF protein was increased by hypoxia in cerebral microvascular endothelial cells (P < 0.05). The content of VEGF protein in NYA serum containing medium was more significantly elevated than those cultured in other control media (P < 0.01). CONCLUSION: VEGF protein was induced by hypoxia in rat cerebral microvascular endothelial cells, and NYA could upregulate the expression of VEGF protein, which may be one of the protection mechanisms for cerebral microvascular endothelial cells.

Activation of endogenous neural stem cells in experimental intracerebral hemorrhagic rat brains.

BACKGROUND: Many researchers suggest that adult mammalian central nervous system (CNS) is incapable of completing self-repair or regeneration. And there are accumulating lines of evidence which suggest that endogenous neural stem cells (NSCs) are activated in many pathological conditions, including stroke in the past decades, which might partly account for rehabilitation afterwards. In this study, we investigated whether there was endogenous neural stem cell activation in intracerebral hemorrhagic (ICH) rat brains. METHODS: After ICH induction by stereotactical injection of collagenase type VII into globus pallidus, 5-Bromo-2 Deoxyuridine (BrdU) was administered intraperitoneally to label newborn cells. Immunohistochemical method was used to detect Nestin, a marker for neural stem cells, and BrdU. RESULTS: Nestin-positive or BrdU-Labeled cells were predominantly located at 2 sites: basal ganglion around hemotoma, ependyma and nearby subventricular zone (SVZ). No positive cells for the 2 markers were found in the 2 sites of normal control group and sham group, as well as in non-leisioned parenchyma, both hippocampi and olfactory bulbs in the 4 groups. Nestin+ cells presented 4 types of morphology, and BrdU+ nucleus were polymorphologic. Positive cell counting around hemotoma showed that at day 2, Nestin+ cells were seen around hemotoma in model group, the number of which increased at day 4, day 7 (P <0.01), peaked at day 14 (P <0.05), and reduced significantly by day 28 (P <0.01). CONCLUSION: Endogenous neural stem cells were activated in experimental intracerebral hemorrhagic rat brains.

[Effect of naoyian on phosphorylated Akt in the neural stem cell injured with anoxia].

OBJECTIVE: To determine the effect of naoyian (NYA) on the expression of phosph-Akt in neural stem cells injured with anoxia. METHODS: The hippocampal neural stem cells (NSC) taken from the Sprageu-Dawley rat brain at 5 days after birth were cultured in DMEM/F12 solution containing EGF and bFGF. After being treated with anoxia for 6 hours, the neural stem cells were incubated in solution containing the NYA serum or normal serum. The neural stem cells were then detected for phosph-Akt and TUNEL stain. RESULTS: After the neural stem cells were injured with anoxia, the expression of the phosph-Akt in NYA serum group was higher than that in the normal serum group and the serum-free group. Treated with NYA serum, the amount of apoptotic NSC decreased. CONCLUSION: After the neural stem cell injured by anoxia was treated with NYA serum, the phosphorylation of Akt increased, the apoptosis of NSC was inhibited.

[Effects of naoyian granule on the expression of IL-6 mRNA and protein in the experimental intracerebral hemorrhagic brain in rats].

OBJECTIVE: To explore the protective mechanisms of the traditional Chinese medicine complex Naoyian granule (NYA) on intracerebral hemorrhagic (ICH). METHODS: ICH rat models were established by stereotaxically injecting 0.4 U type VII collagenase into the right globus pallidus (GP), the expression of IL-6mRNA and protein were investigated by in situ hybridization and Western blotting. RESULTS: In the NYA group the IL-6 mRNA level was higher than that of the model group on 4 - 7 d and the IL-6 protein level was remarkably higher than that of the model group on 6 h - 7 d after ICH injury (P <0.01). CONCLUSION: Up-regulating the expression of IL-6 protein, and stabilizing the increasing expression of IL-6 mRNA and protein may be the protective mechanisms of NYA.

[Characteristics of Bcl-2 and Bax expressions in rat brain after experimental intracerebral hemorrhage and intervention of naoyian granule].

OBJECTIVE: To investigate the characteristics of Bcl-2 and Bax expressions in rat brain after experimental intracerebral hemorrhagic (ICH), and to determine the protective mechanisms of the Traditional Chinese medicine complex, naoyian granule (NYA). METHODS: ICH models were induced according to Rosenbergs method. The rats were divided into the model group, the NYA-treatment group and the sham group. Bcl-2 and Bax expressions in the rat brain after ICH were detected by immunohistochemistry method. RESULTS: Bax expressed in the hemorrhagic core, and the immunoreactive neurons were seriously injuried and the shape varied. Bcl-2 expressed in the penumbra with the normal shape of the immunoreactive neurons. Both Bax and Bcl-2 expressed simultaneously in the marginal region of the cerebral hemorrhagic core. Bcl-2 expression in the rat brain reached the peak at the 2nd day and maintained at a relatively high level until the 7th day, and NYA could increase it. Bax expression peaked at the 1st day, and NYA could down-regulate it. CONCLUSION: The expressions of Bcl-2 and Bax after the cerebral hemorrhage are related to the degree of injury, the time, and the location of the brain tissues. NYA can up-regulate the Bcl-2 expression and down-regulate the Bax expression, which may be one of its neuroprotective mechanisms.

[Effect of naoyi'an on basic fibroblastic growth factor mRNA expression and tumor necrosis factor protein expression in brain of rats following intracerebral hemorrhage].

OBJECTIVE: To investigate the effect of Naoyi'an granule (NYAG) on basic fibroblastic growth factor (bFGF) mRNA expression and tumor necrosis factor (TNF) protein expression following intracerebral hemorrhage and provide the theoretical evidence of NYAG in treating intracerebral hemorrhage and promoting the rehabilitation of neural function. METHODS: Model rats of intracerebral hemorrhage induced by infusion of collagenase VII into the caudate-putamen were used to determine the related parameters of behavior scores (BS), Northern blot, Western blot assay and optical density (OD) scanning in the model and the model treated with NYAG. And the data got from the two groups were compared. RESULTS: BS in the model group began to lower 24 hrs after modeling and a significant decrease was shown 7 days later, while in the NYAG group, it decreased significantly three days after modeling, the difference between the two groups was significant (P < 0.05). Levels of bFGF mRNA expression and TNF protein expression increased after modeling, it reached the peak in three days and began to decrease gradually in seven days in both groups. However, the level in the NYAG group was higher than that in the model group in various times of the experimental process. CONCLUSION: NYAG could enhance the bFGF expression and suppress the TNF expression so as to improve the behavior deficit in treating intracerebral hemorrhage, which may be one of the main mechanisms of NYAG for promoting the rehabilitation of neural function.

[Effects of nao-yi-an granule on bcl-2 expression in the rat brain after experimental cerebral hemorrhage].

OBJECTIVE: To investigate the protective effects and mechanisms of the traditional Chinese medicine complex, nao-yi-an granule (NYA) on experimental intracerebral hemorrhage (ICH) in rats. METHODS: ICH models anesthesized were induced by stereotaxical injection of 2 microliters normal saline containing 0.4 U bacterial collagenase(type VII) into the right globas pallidus(GP), and the protein expression of bcl-2 in the rat brain after ICH was detected with the immunohistochemistry method, with which the effects of NYA were observed. RESULTS: Bcl-2 positive cells were found in the cerebral cortex and basal ganglia area after ICH. Immunoreactivity was localized in the cytoplasm as a fine granular precipitate. The levels of bcl-2 expression in the model group varied at different time-points, and reached the peak at 2 d. In the NYA group, the protein expression of bcl-2 was increased significantly both in the cortex and in the basal ganglia compared with those in the model group. CONCLUSION: NYA can significantly upregulate the protein expression of bcl-2.

[Effects of nao-yi-an granule on the expression of heme oxygenase-1 in brains of intracerebral hemorrhagic rats].

OBJECTIVE: To investigate the effects of the traditional Chinese medicine complex nao-yi-an granule(NYA) on the expression of heme oxygenase-1(HO-1) in the brains of intracerebral hemorrhagic (ICH) rats. METHODS: After inducing ICH rat models with collagenase VII, we used the immunohistochemical method and HO-1 immunoreactive cell count to observe the HO-1 expression. RESULTS: Following ICH, the expression of HO-1 in the rat brains was observed at 12 h, peaking at 2 d and persisting until 7 d; and NYA could increase the expression at 24 h obviously. CONCLUSION: Expression of HO-1 increases following ICH,; upregulation of HO-1 expression may be one of the neuroprotective mechanisms of NYA.

[Effects of nao-yi-an on tumor necrosis factor alpha and insulin resistance of acute intracerebral hemorrhagic patients].

OBJECTIVE: To investigate the effects of nao-yi-an on tumor necrosis factor alpha (TNF-alpha) and insulin resistance of acute intracerebral hemorrhagic patients. METHODS: The patients were randomly divided into two groups: the medicine control group and the nao-yi-an treatment group. TNF-alpha and insulin were checked by radioimmunoassay before and after the treatment. Fifteen healthy people were made as the negative control group. RESULTS: TNF-alpha of both treatment groups was higher while insulin sensitivity index(ISI) was remarkably lower compared with the health group (P < 0.01). After the treatment ISI increased and TNF-alpha was remarkably lower in the treatment group (P < 0.01). Differences of TNF-alpha and ISI between the treatment group and medicine control group were significant (P < 0.05 or P < 0.01). CONCLUSION: nao-yi-an can decrease TNF-alpha and increase ISI, which is a mechanism of reducing cerebral ischemic-reperfusion injury.

[Effects of nao-yi-an granule on the intercellular expression of IL-6 in the experimental intracerebral hemorrhagic brain of rats].

OBJECTIVE: To define the cellular localization of IL-6 and the effects of the traditional Chinese medicine complex nao-yi-an granule (NYA) on the intercellular expression of IL-6 in the experimental intracerebral hemorrhagic (ICH) brain of rats, so as to investigate the efficacy mechanisms of NYA on ICH. METHODS: ICH rat models were established by stereotaxically injecting 0.4 U type VII collagenase into the right globus pallidus (GP). The distribution and content of IL-6 protein expression together with the reaction of glial cells were investigated with the immunohistochemistry method. RESULTS: Microglial activation was detected from 6 h to 4 d after ICH in the hematoma region, and reactive astrocytes were found from 6 h to 7 d after ICH around the hematoma region; there was an up-regulation of IL-6 in the hematoma at 6 h after ICH; it reached its peak at 12 h, and disappeared on 7 d. CONCLUSION: The major sources of IL-6-positive protein were neurons and the activated microglia in the brain of ICH rats. Stabilizing the increasing expression of IL-6 may be one of the neuroprotective mechanisms of NYA.