Qi-Dong Yang

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.

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 transcription–polymerase 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.

The effect of hyperbaric oxygen on intracephalic angiogenesis in rats with intracerebral hemorrhage

Healthy SD rats were randomly divided into 3 groups as sham operation (group A), ICH (group B), and HBO2 (group C). The behavioral change and angiogenesis in brain tissue of rats in each group were observed. The protein expression of PCNA, vWF, HIF1-α, and VEGF in rat brain was measured by immunohistochemistry, while the mRNA expression level of HIF1-α and VEGF was determined using quantitative real-time PCR. This study has investigated the effect of HBO2 on intracephalic angiogenesis in rats with intracerebral hemorrhage (ICH). There were significant differences in behavior score between HBO2 and ICH groups at 14, 21, and 28 days. A large number of vessel-like structures and microvessels were observed in perihematomal brain tissues in HBO2 group. There were significant differences in HIF1-α and VEGF protein and HIF1-α mRNA level between HBO2 and ICH groups at 14, 21, and 28 days; at 7, 14, 21, and 28 days, the differences in PCNA and vWF protein expression between the 2 groups were statistically significant. At 21 and 28 days, the expression levels of VEGF mRNA in the 2 groups differed significantly from each other. Our results indicate that HBO2 can significantly promote the expression of HIF1-α and VEGF at both mRNA and protein levels in rats with ICH, increase the protein expression of both PCNA and vWF, promote the formation of new blood vessels, and promote the recovery of behavioral ability, hence resulting in a rapid rehabilitation.

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.

Thrombin promotes the expression of thrombospondin-1 and ‐2 in a rat model of intracerebral hemorrhage

Spontaneous intracerebral hemorrhage (ICH) is one of the most severe types of stroke. Thrombin has been reported to participate in brain repair following ICH and play an important role in angiogenesis. Our previous studies have shown that ICH induces angiogenesis in damaged rat brain, accompanied by upregulation of expression of thrombospondin (TSP)-1 and TSP-2. The aim of the present study was to investigate whether the expression of TSP-1 and TSP-2 was regulated by thrombin in rat brain following ICH. A rat model of ICH was induced by injection of autologous blood into the right globus pallidus (GP). Hirudin, a thrombin specific inhibitor, or thrombin was injected into the GP. Immunohistochemistry, quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) and western blot assays were applied. Results showed that ICH induced an increase in the expression of TSP-1 mRNA and TSP-2 mRNA after ICH, whereas hirudin significantly inhibited the expression of TSPs mRNA after ICH (P<0.05). In contrast, sole thrombin treatment in normal rats induced strong expression of TSP-1 or TSP-2 in the blood vessels around the damaged brain region when compared with those without thrombin treatment. Western blot analysis data confirmed that the protein levels of TSPs were significantly increased when compared with those in the sham control group (P<0.01). These findings support that thrombin positively regulates the expression of TSP-1 and TSP-2 after ICH, which may be involved in modulating angiogenesis in injured brains following ICH.

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.

ObjectiveTo 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.MethodsOne 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 (补阳还五汤, 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.ResultsFrom 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).ConclusionQTSE 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.

Vascular protective effects of KLF2 on Aβ-induced toxicity: Implications for Alzheimer’s disease

Alzheimers disease (AD) is characterized by excessive amounts of senile plaques and neurofibrillary tangles in the brain, and cerebrovascular pathologies in AD are attracting increasingly more attention. Krüppel-like factor (KLF) 2, a transcription regulator expressed in the mouse embryonic vasculature and involved in the regulation of vascular gene expression, serves as a protective factor in endothelial cells. However, whether KLF2 is involved in neurodegenerative disease, and especially in AD, remains unknown. In the present study, the effects of KLF2 in Aβ-induced neurotoxicity were investigated. Firstly, we found that KLF2 expression decreased at both the RNA and protein levels in AD cases. The following results show that KLF2 was found to be decreased in both a time- and dose-dependent manner in response to Aβ1-42 treatment in primary mouse brain microvascular endothelial cells. Overexpression of KLF2 attenuated Aβ-induced oxidative stress, improved mitochondrial function, and reduced the rate of apoptosis. Furthermore, inhibition of KLF2 promoted Aβ1-42-induced oxidative stress, exacerbated mitochondrial dysfunction, and increased the rate of apoptosis. Our data imply that the promotion of KLF2 expression in cerebral endothelial cells has the potential to be developed as a novel therapeutic strategy for the treatment of cerebral vascular dysfunction in AD.

Corrigendum to “Thrombin promotes the expression of thrombospondin-1 and -2 in a rat model of intracerebral hemorrhage” [J Neurol Sci 323 (2012) 141–146]

A-Li Yang , Hua-Jun Zhou, Yuan Lin , Jie-Kun Luo , Han-Jin Cui , Tao Tang ⁎, Qi-Dong Yang a Institute of Integrative Medicine, Xiangya Hospital, Central South University, Changsha 410008, Hunan, PR China b Institute of Neurology, Xiangya Hospital, Central South University, Changsha 410008, Hunan, PR China c Key Laboratory of Chinese Gan of State Administration of Traditional Chinese Medicine of China, Changsha 410008, Hunan, PR China