Temporal profile of angiogenesis and expression of extracellular matrix-related genes in rat brains following experimental intracerebral hemorrhage.

BACKGROUND: Angiogenesis is essential for the repair process after intracerebral hemorrhage (ICH). METHODS: Given the importance of the extracellular matrix (ECM) in angiogenesis, we analysed the temporal profile of angiogenesis in rat brains on days 4, 7, and 21 after ICH. To this end, we compared the expression of ECM-related genes between ICH-induced and sham-operated groups using a complementary DNA (cDNA) array. We further measured protein expression using western blot and immunohistochemistry assays. Fluorescein isothiocyanate (FITC)-dextran was injected into the tail vein to examine the angioarchitecture in the perihematomal region. RESULTS: Among the 88 ECM-related genes, we identified 42, 50, and 38 genes that were significantly upregulated on days 4, 7, and 21 after ICH, respectively (P < 0.05). Particularly, collagens, integrins, and matrix metalloproteinases (MMPs) were significantly increased on day 4 post-ICH and continued to increase at the other time points. Western blot and immunohistochemistry analyses showed a comparable trend in the upregulation of MMPs. Compared to the sham group, FITC-dextran labelling demonstrated decreased perfusion and increased vascular permeability in the perihematomal region in the ICH group. Doxycycline, an MMP inhibitor, significantly reduced angiogenesis (P < 0.05). CONCLUSIONS: The results of this study indicate that MMPs are involved in modulating angiogenesis following ICH.

Electroacupuncture ameliorates cerebral ischemia/reperfusion injury by suppressing autophagy via the SIRT1-FOXO1 signaling pathway.

Cerebral ischemia/reperfusion (CIR) injury occurs when blood flow is restored in the brain, causing secondary damage to the ischemic tissues. Previous studies have shown that electroacupuncture (EA) treatment contributes to brain protection against CIR injury through modulating autophagy. Studies indicated that SIRT1-FOXO1 plays a crucial role in regulating autophagy. Here we investigated the mechanisms underlying the neuroprotective effect of EA and its role in modulating autophagy via the SIRT1-FOXO1 signaling pathway in rats with CIR injury. EA pretreatment at "Baihui", "Quchi" and "Zusanli" acupoints (2/15Hz, 1mA, 30 min/day) was performed for 5 days before the rats were subjected to middle cerebral artery occlusion, and the results indicated that EA pretreatment substantially reduced the Longa score and infarct volume, increased the dendritic spine density and lessened autophagosomes in the peri-ischemic cortex of rats. Additionally, EA pretreatment also reduced the ratio of LC3-II/LC3-I, the levels of Ac-FOXO1 and Atg7, and the interaction of Ac-FOXO1 and Atg7, but increased the levels of p62, SIRT1, and FOXO1. The above effects were abrogated by the SIRT1 inhibitor EX527. Thus, we presume that EA pretreatment elicits a neuroprotective effect against CIR injury, potentially by suppressing autophagy via activating the SIRT1-FOXO1 signaling pathway.

Leukemia Inhibitory Factor Decreases Neurogenesis and Angiogenesis in a Rat Model of Intracerebral Hemorrhage.

Neurogenesis and angiogenesis can improve the neurologic function after intracerebral hemorrhage (ICH). Leukemia inhibitory factor (LIF) plays an important role in neurogenesis and angiogenesis. In this study, a rat model of autologous blood-induced ICH was used to evaluate the effect of LIF on the neurogenesis and angiogenesis following ICH. After ICH, LIF-positive neurons and dilated vessels were detected in the peri-hematomal region. It was found that LIF levels increased significantly and peaked 14 days after ICH induction. Double immunofluorescence confirmed that LIF was expressed in neurons and endothelial cells. ICH also led to increases of doublecortin (DCX)- and von Willebrand factor (vWF)-positive cells as well as proliferation of cell nuclear antigen (PCNA)+/DCX+ and PCNA+/vWF+ nuclei. All these ICH-induced increases were significantly attenuated by exogenous LIF infusion. These data suggested that LIF was a negative regulator of neurogenesis and angiogenesis after ICH.

Buyang Huanwu Decoction () Attenuates Glial Scar by Downregulating the Expression of Leukemia Inhibitory Factor in Intracerebral Hemorrhagic Rats.

OBJECTIVE: To evaluate the effect of Buyang Huanwu Decoction (, BYHWD) on glial scar after intracerebral hemorrhage (ICH) and investigate the underlying mechanism. METHODS: Collagenase type VII (0.5 U) was injected stereotaxically into right globus pallidus to induce ICH model. One hundred and twenty Sprague-Dawley rats were randomly divided into 3 groups according to a random number table, including normal group (n=40), ICH model group (n=40) and BYHWD group (n=40), respectively. After ICH, the rats in the BYHWD group were intragastrically administered with BYHWD (4.36 g/kg) once a day for 21 days, while the rats in ICH group were administered with equal volume of distilled water for 21 days, respectively. Double immunolabeling was performed for proliferating cell nuclear antigen (PCNA)+/glial fibrillary acidic protein (GFAP)+ nuclei. The expression of GFAP and leukemia inhibitory factor (LIF) was evaluated by immunohistochemistry and quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The astrocytes with hypertrophied morphology around the hematoma was observed on day 3 after ICH. The number of GFAP positive cells and GFAP mRNA levels increased notably on day 3 and reached the peak on day 14 post-ICH (P<0.01). PCNA+/GFAP+ nuclei were observed around the hematoma and reached the peak on day 14 post-ICH (P<0.01). In addition, LIF-positive astrocytes and LIF mRNA level in the hemorrhagic region increased significantly till day 14 post-ICH (P<0.01). However, BYHWD not only reduced the number of PCNA+/GFAP+ nuclei, but also decreased GFAP and LIF levels (P<0.05). CONCLUSIONS: BYHWD could attenuate ICH-induced glial scar by downregulating the expression of LIF in the rats.

Puerarin protects rat brain against ischemia/reperfusion injury by suppressing autophagy via the AMPK-mTOR-ULK1 signaling pathway.

Puerarin suppresses autophagy to alleviate cerebral ischemia/reperfusion injury, and accumulating evidence indicates that the AMPK-mTOR signaling pathway regulates the activation of the autophagy pathway through the coordinated phosphorylation of ULK1. In this study, we investigated the mechanisms underlying the neuroprotective effect of puerarin and its role in modulating autophagy via the AMPK-mTOR-ULK1 signaling pathway in the rat middle cerebral artery occlusion model of cerebral ischemia/reperfusion injury. Rats were intraperitoneally injected with puerarin, 50 or 100 mg/kg, daily for 7 days. Then, 30 minutes after the final administration, rats were subjected to transient middle cerebral artery occlusion for 90 minutes. Then, after 24 hours of reperfusion, the Longa score and infarct volume were evaluated in each group. Autophagosome formation was observed by transmission electron microscopy. LC3, Beclin-1 p62, AMPK, mTOR and ULK1 protein expression levels were examined by immunofluorescence and western blot assay. Puerarin substantially reduced the Longa score and infarct volume, and it lessened autophagosome formation in the hippocampal CA1 area following cerebral ischemia/reperfusion injury in a dose-dependent manner. Pretreatment with puerarin (50 or 100 mg/kg) reduced Beclin-1 expression and the LC3-II/LC3-I ratio, as well as p-AMPK and pS317-ULK1 levels. In comparison, it increased p62 expression. Furthermore, puerarin at 100 mg/kg dramatically increased the levels of p-mTOR and pS757-ULK1 in the hippocampus on the ischemic side. Our findings suggest that puerarin alleviates autophagy by activating the APMK-mTOR-ULK1 signaling pathway. Thus, puerarin might have therapeutic potential for treating cerebral ischemia/reperfusion injury.

2-Methoxyestradiol Inhibits Intracerebral Hemorrhage- Induced Angiogenesis in Rats.

AIM: Angiogenesis occurs after intracerebral hemorrhage (ICH). Hypoxia-inducible factor-1? (HIF-1?) is a critical regulator of angiogenesis. However, its role in the central nervous system remains controversial. 2-Methoxyestradiol (2ME2), a natural metabolite of estrogen, is known to inhibit HIF-1?. In the present study, we investigated the effect of 2ME2 in a rat model of ICH-induced angiogenesis. MATERIAL AND METHODS: Sprague-Dawley male rats (n=50) were randomly divided into 5 groups: Sham operated group; ICH; ICH+2ME2; and ICH+Vehicle groups. ICH model was induced by stereotactic injection of collagenase type VII into the right globus pallidus. 2ME2 or vehicle (10% dimethyl sulfoxide) was administered intraperitoneally 10 min after ICH. Angiogenesis and expression of HIF-1? was evaluated by immunohistochemistry, quantitative real time-reverse transcription polymerase chain reaction and western blot, respectively. RESULTS: Proliferating cell nuclear antigen (PCNA)-labeled nuclei were detected in cerebral endothelial cells (ECs) around the hematoma. The labeling peaked at 14 days post-ICH. HIF-1?-immunoreactive microvessels with dilated outline were detected in the perihematomal tissues. The vessels extended into the clot from the surrounding tissues from day 7 onwards. HIF-1? protein levels increased, while no change was observed in HIF-1? mRNA expression after ICH. 2ME2 decreased the PCNA-labeled nuclei in cerebral ECs and down-regulated the expression of HIF-1? protein as well, while it had little effect on the mRNA expression of HIF-1?. CONCLUSION: HIF-1? inhibitor, 2ME2, inhibited post-ICH angiogenesis by suppressing HIF-1? expression, thus exerting detrimental effects in ICH.

Activation of the Notch-1 signaling pathway may be involved in intracerebral hemorrhage-induced reactive astrogliosis in rats.

OBJECTIVE Reactive astrogliosis, a key feature that is characterized by glial proliferation, has been observed in rat brains after intracerebral hemorrhage (ICH). However, the mechanisms that control reactive astrogliosis formation remain unknown. Notch-1 signaling plays a critical role in modulating reactive astrogliosis. The purpose of this paper was to establish whether Notch-1 signaling is involved in reactive astrogliosis after ICH. METHODS ICH was induced in adult male Sprague-Dawley rats via stereotactic injection of autologous blood into the right globus pallidus. N-[ N-(3,5-difluorophenacetyl)-l-alanyl]- S-phenylglycine t-butyl ester (DAPT) was injected into the lateral ventricle to block Notch-1 signaling. The rats' brains were perfused to identify proliferating cell nuclear antigen (PCNA)-positive/GFAP-positive nuclei. The expression of GFAP, Notch-1, and the activated form of Notch-1 (Notch intracellular domain [NICD]) and its ligand Jagged-1 was assessed using immunohistochemical and Western blot analyses, respectively. RESULTS Notch-1 signaling was upregulated and activated after ICH as confirmed by an increase in the expression of Notch-1 and NICD and its ligand Jagged-1. Remarkably, blockade of Notch-1 signaling with the specific inhibitor DAPT suppressed astrocytic proliferation and GFAP levels caused by ICH. In addition, DAPT improved neurological outcome after ICH. CONCLUSIONS Notch-1 signaling is a critical regulator of ICH-induced reactive astrogliosis, and its blockage may be a potential therapeutic strategy for hemorrhagic injury.

Fermented Chinese formula Shuan-Tong-Ling attenuates ischemic stroke by inhibiting inflammation and apoptosis.

The fermented Chinese formula Shuan-Tong-Ling is composed of radix puerariae (Gegen), salvia miltiorrhiza (Danshen), radix curcuma (Jianghuang), hawthorn (Shanzha), salvia chinensis (Shijianchuan), sinapis alba (Baijiezi), astragalus (Huangqi), panax japonicas (Zhujieshen), atractylodes macrocephala koidz (Baizhu), radix paeoniae alba (Baishao), bupleurum (Chaihu), chrysanthemum (Juhua), rhizoma cyperi (Xiangfu) and gastrodin (Tianma), whose aqueous extract was fermented with lactobacillus, bacillus aceticus and saccharomycetes. Shuan-Tong-Ling is a formula used to treat brain diseases including ischemic stroke, migraine, and vascular dementia. Shuan-Tong-Ling attenuated H2O2-induced oxidative stress in rat microvascular endothelial cells. However, the potential mechanism involved in these effects is poorly understood. Rats were intragastrically treated with 5.7 or 17.2 mL/kg Shuan-Tong-Ling for 7 days before middle cerebral artery occlusion was induced. The results indicated Shuan-Tong-Ling had a cerebral protective effect by reducing infarct volume and increasing neurological scores. Shuan-Tong-Ling also decreased tumor necrosis factor-α and interleukin-1ß levels in the hippocampus on the ischemic side. In addition, Shuan-Tong-Ling upregulated the expression of SIRT1 and Bcl-2 and downregulated the expression of acetylated-protein 53 and Bax. Injection of 5 mg/kg silent information regulator 1 (SIRT1) inhibitor EX527 into the subarachnoid space once every 2 days, four times, reversed the above changes. These results demonstrate that Shuan-Tong-Ling might benefit cerebral ischemia/reperfusion injury by reducing inflammation and apoptosis through activation of the SIRT1 signaling pathway.

Hyperbaric oxygen for experimental intracerebral haemorrhage: Systematic review and stratified meta-analysis.

OBJECTIVE: Hyperbaric oxygen (HBO) is widely used in treating various neurological diseases. However, HBO for treatment of intracerebral haemorrhage (ICH) remains controversial, in either animal or clinical studies. Therefore, we conducted this systematic review and meta-analysis on studies describing the efficacy of HBO in animal models of ICH. METHODS: Studies were identified by searching mainstream databases through November 2015. The efficacy of HBO in animal models of ICH was assessed by changes in the brain water content (BWC), neurobehavioural outcome (NO) or both. Subgroup analyses were performed according to different design characteristics. RESULTS: In total 15 studies met our inclusion criteria. HBO can reduce the BWC (-0.982, 95% CI, -1.148 to -0.817; P < 0.01; 57 comparisons), and improve NO (-0.767, 95% CI, -1.376 to -0.159; P < 0.01; eight comparisons). HBO was most effective in reducing BWC when given 72 h after ICH for a 4- to 5-day consecutive treatment at the chamber pressure of 3.0 atmosphere absolute. Efficacy was higher with phenobarbital anaesthesia, the blood infusion model and in rabbits. CONCLUSION: Although HBO was found to be effective in experimental ICH, additional confirmation is needed due to possible publication bias, poor study quality and the limited number of studies conducting clinical trials.

Leukemia Inhibitory Factor Contributes to Reactive Astrogliosis via Activation of Signal Transducer and Activator of Transcription 3 Signaling after Intracerebral Hemorrhage in Rats.

Reactive astrogliosis has occurred after intracerebral hemorrhage (ICH). Leukemia inhibitory factor (LIF) can act as a modulator for glial gene expression. Signal transducer and activator of transcription 3 (STAT3) is a critical regulator of reactive astrogliosis. The present study tested whether endogenous LIF acted on ICH-induced reactive astrogliosis via the STAT3 signaling pathway. Rats were divided into three experimental groups: 1) Rats received either an ICH or a needle insertion (sham), 2) Rats received 100 ng LIF or an equal volume of phosphate-buffered saline (PBS) by direct infusion into the lateral ventricle (LV) after ICH, and 3) AG490 (0.25 mg/kg) was injected into the LV to block STAT3 signaling. Brains were perfused to identify proliferating cell nuclear antigen (PCNA)+/glial fibrillary acidic protein (GFAP)+nuclei. The expression of GFAP, LIF, LIF receptor (LIFR), glycoprotein 130 (gp130), and phospho-STAT3 (p-STAT3) was evaluated by immunohistochemistry and Western blot, respectively. After ICH, the number of the PCNA+/GFAP+ nuclei and the expression of GFAP, LIF, LIFR, gp130, and p-STAT3 were increased. Moreover, LIF increased the number of PCNA+/GFAP+ nuclei and the expression of GFAP, LIFR, gp130, and p-STAT3. The number of PCNA+/ GFAP+ nuclei and GFAP protein levels were attenuated markedly after inhibition of p-STAT3. Together, these data suggest that LIF contributes to ICH-related reactive astrogliosis via activation of STAT3 signaling.

Efficacy of deferoxamine in animal models of intracerebral hemorrhage: a systematic review and stratified meta-analysis.

Intracerebral hemorrhage (ICH) is a subtype of stroke associated with high morbidity and mortality rates. No proven treatments are available for this condition. Iron-mediated free radical injury is associated with secondary damage following ICH. Deferoxamine (DFX), a ferric-iron chelator, is a candidate drug for the treatment of ICH. We performed a systematic review of studies involving the administration of DFX following ICH. In total, 20 studies were identified that described the efficacy of DFX in animal models of ICH and assessed changes in the brain water content, neurobehavioral score, or both. DFX reduced the brain water content by 85.7% in animal models of ICH (-0.86, 95% CI: -.48- -0.23; P < 0.01; 23 comparisons), and improved the neurobehavioral score by -1.08 (95% CI: -1.23- -0.92; P < 0.01; 62 comparisons). DFX was most efficacious when administered 2-4 h after ICH at a dose of 10-50 mg/kg depending on species, and this beneficial effect remained for up to 24 h postinjury. The efficacy was higher with phenobarbital anesthesia, intramuscular injection, and lysed erythrocyte infusion, and in Fischer 344 rats or aged animals. Overall, although DFX was found to be effective in experimental ICH, additional confirmation is needed due to possible publication bias, poor study quality, and the limited number of studies conducting clinical trials.

Buyang huanwu decoction promotes angiogenesis via vascular endothelial growth factor receptor-2 activation through the PI3K/Akt pathway in a mouse model of intracerebral hemorrhage.

BACKGROUND: Intracerebral hemorrhage (ICH) is a fatal subtype of stroke that lacks effective treatments. Angiogenesis following ICH is an important response mediating brain recovery and repair. Phosphorylation of vascular endothelial growth factor receptor 2 (pVEGFR2) via PI3K/Akt signaling plays a key role in mediating cellular processes involved in repair, such as mitogenesis, angiogenesis, and vascular permeability. This study aimed to investigate the potential effects of Buyang Huanwu Decoction (BYHWD), a traditional Chinese medicine formula, on angiogenesis by VEGFR2 activation through the phosphatidylinositol 3 kinase (PI3K)/Akt signaling pathway in a mouse model of ICH. METHODS: Adult male Kunming mice (n = 50) were randomly assigned into sham and ICH-operated groups and treated with one of the followings SU5416 (VEGFR2 inhibitor), BYHWT and BYHWT + SU5416. ICH was induced in mice by injecting collagenase (type VII) into the right globus pallidus of the mouse brain. BYHWD (4.36 g/kg) was administrated in mice by intragastric infusion. Neurological function was evaluated in mice by a modified Neurological Severity Scores (mNSS) as well as corner turn and foot-fault tests. Angiogenesis was examined by intraperitoneal injection of 5-bromodeoxyuridine (BrdU) in mice to quantify new brain vessel growth. SU5416 treatment and assessment of VEGFR2 phosphorylation as well as alterations in PI3K/Akt signaling were performed to determine whether the effect of BYHWD on angiogenesis was partly mediated by phosphorylation of VEGFR2 via the PI3K/Akt signaling pathway. RESULTS: We show that BYHWD treated mice exhibited (i) significantly better recovery from neurological dysfunction, (ii) increased BrdU(+) nuclei in vWF(+) dilated brain vessels and (iii) higher VEGFR2 phosphorylation immunoreactivity in brain microvessels (P <0.05), (iv) higher expression of PI3K and pAkt at the protein level (P <0.05) when compared to untreated ICH mice. These beneficial effects were reversed by SU5416 (P <0.05). CONCLUSIONS: BYHWD promoted neurological recovery and angiogenesis after ICH in mice by enhancing VEGFR2 phosphorylation through the PI3K/Akt signaling pathway.

Electroacupuncture improves recovery after hemorrhagic brain injury by inducing the expression of angiopoietin-1 and -2 in rats.

BACKGROUND: Angiopoietin (Ang) is one of the major effectors of angiogenesis, playing a critical role in neurovascular remodeling after stroke. Acupuncture has been widely used for treating stroke in China for a long time. Recently, we have demonstrated that electroacupuncture (EA) can accelerate intracerebral hemorrhage (ICH)-induced angiogenesis in rats. In the present study, we investigated the effect of EA on the expression of Ang-1 and Ang-2 in the brain after ICH. METHODS: ICH was induced by stereotactic injection of collagenase type VII into the right globus pallidus. Adult male Sprague-Dawley rats were randomized into the following four groups: sham-operation (SHAM), stroke-no electroacupuncture (SNE), stroke-EA at the Zusanli acupoint (SEZ), and stroke-EA at a nonacupoint (SEN). EA was applied to the bilateral Zusanli (ST36) acupoint in the SEZ group and a nonacupoint in the SEN group. The expression of Ang-1 and Ang-2 was evaluated by immunohistochemistry and quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Some Ang-1 and Ang-2 immunoreactive microvessels with a dilated outline were detected in the perihematomal tissues after ICH, and the vessels extended into the clot from the surrounding area since day 7. The expression of Ang-1 increased notably as long as 2 weeks after ICH, while Ang-2 immunoreactivity declined at about 7 days following a striking upregulation at 3 days. EA at the Zusanli (ST36) acupoint upregulated the expression of Ang-1 and Ang-2 at both the protein and mRNA levels. However, EA at a nonacupoint had little effect on the expression of Ang-1 and Ang-2. CONCLUSIONS: Our data suggest that EA at the Zusanli (ST36) acupoint exerts neuroprotective effects on hemorrhagic stroke by upregulation of Ang-1 and Ang-2.

Angiogenesis opens a way for Chinese medicine to treat stroke.

Based on the pathophysiology of the brain, advance in angiogenesis induced by stroke, and evidences of Chinese-medicine-mediated angiogenesis, the possibility to study the stroke-treating mechanism of Chinese medicine in angiogenesis was discussed. And regarding our previous work on angiogenesis modulated by qi-tonifying and stasis-eliminating therapy following intracerebral hemorrhage, we proposed some questions, which should be taken into account in the further work.

Electroacupuncture at Zusanli (ST36) accelerates intracerebral hemorrhage-induced angiogenesis in rats.

OBJECTIVE: To investigate the effects of electro-acupuncture on intracerebral hemorrhage (ICH)-induced angiogenesis and hypoxia-inducible factor-1α (HIF-1α) expression in rats. METHODS: Adult male Sprague-Dawley rats were randomly divided into 4 groups of 24 rats each. ICH was induced in 3 groups by stereotactic injection of collagenase type VII into the right globus pallidus; of these, one group was not further treated, the second group underwent Zusanli (ST36)-acupuncture, and the third group underwent non-acupoint acupuncture. The fourth group underwent sham operations. Acupuncture was performed by stimulation with electrical needles at frequencies of 2-20 Hz for 30 min per day. Angiogenesis on days 3, 7 and 14 was assessed by double immunolabeling, and expression of HIF-1α was evaluated by immunohistochemistry, quantitative real time reverse transcription-polymerase chain reaction and Western blotting. RESULTS: 5-Bromo-2-deoxyuridine (BrdU)-labeled nuclei in cerebral endothelial cells (ECs) resided around the hematoma and the labeling peaked from 7 to 14 days (P<0.01). HIF-1α positive microvessels with a dilated outline were detected in perihematomal tissues after ICH, with the vessels extending into the clot from the surrounding area beginning on day 7. Following ICH, HIF-1α protein levels increased (P<0.05), but HIF-1α mRNA levels did not change. Electro-acupuncture at the Zusanli (ST36) acupoint increased BrdU-labeled nuclei in cerebral ECs (P<0.05) and up-regulated the expression of HIF-1α protein (P<0.05), but had little effect on the spatial distribution of HIF-1α or on HIF-1α mRNA levels. CONCLUSIONS: Electro-acupuncture treatment at the Zusanli (ST36) acupoint may accelerate ICH-induced angiogenesis by up-regulating HIF-1α protein, and may enhance recovery following hemorrhagic cerebral injury.

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.

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.

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.

[Effect of buyang huanwu decoction on expressions of angiopoietin-1 and its receptor mRNA in brain of rat after intracerebral hemorrhage].

OBJECTIVE: To investigate the mechanisms of Buyang Huanwu Decoction (BYHWD) by observing its effects on expressions of angiopoietin-1 (Ang-1) and the endothelial-specific receptor tyrosine kinase (Tie-2) mRNA in damaged region of rats' brain after intracerebral hemorrhage (ICH). METHODS: One hundred and sixty Sprague-Dawley rats were randomly divided into four groups, 10 in the normal control group, 60 in the sham-operative group, 60 in the ICH model group, and 30 in the BYHWD-treated group. The ICH model was established by injecting collagenase type VII 0.5 U stereotaxically into right globus pallidus. Animals in the BYHWD-treated group were administered orally with BYHWD, while animals in the sham-operative group and the ICH model group were administered orally with equal volume distilled water, and those in the normal control group drank water freely. The positional variations of the expression of Ang-1 and Tie-2 in the sham-operative group and the model group were assayed by immunohistochemistry on dayl, 4, 7, 14, 21 and 28 after modeling, in the meantime, the dynamic changes of Ang-1 and Tie-2 mRNA expressions in all groups were assayed by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: No significant expression of Ang-1 and Tie-2 in brain of rats in the normal or the sham-operative group was found during the experiment. In the model group, the Ang-1 and Tie-2 positive micrangio-segments appeared at the edge of clot on day 1 to day 4, they gradually penetrated to hematoma area from day 7; with Ang-1 and Tie-2 mRNA expressed from day 1, but very weak until day 4, showing no significant difference to that on day 1; thereafter, they increased gradually, and reached the peak on day 28 (P <0.05). While the two expressions in the BYHWD treated group reached the peak on day 21, and from day 7 to day 28, they were all significantly higher than those in ICH model group at the corresponding time points (P <0.01). CONCLUSION: BYHWD can promote the up-regulation of Ang-1 and Tie-2mRNA expressions in brain of intracerebral hemorrhagic rats, which might accelerate the angiogenesis in the reconstruction of microvascular network in the damaged zone, and thus facilitating the repairing of damaged tissue.