Wang Gai Qing, Yang Qi Dong, Tang Qing Ping, Li Guang Lai, Li Dong Fang, Hu Wei Min, Lian Xia and Pei Yu Heng
Brain edema formation following intracerebral hemorrhage (ICH) appears to be partly related to erythrocyte lysis and hemoglobin release. An increase of brain water content was associated with an increase of brain iron, which is an erythrocyte degradation product. Expression of AQP4 is highly modified in several brain disorders, and it can play a key role in cerebral edema formation. However, the question whether AQP4 is regulated by drugs lacks reliable evidence, and the interacting roles of iron overload and AQP4 in brain edema after ICH are unknown. The goal of this study was to clarify the relationship between iron overload and AQP4 expression and to characterize the effects of the iron chelator deferoxamine (DFO) on delayed brain edema after experimental ICH.
A total of 144 Sprague-Dawley rats weighing between 250 and 300 g were used in this work. The animals were randomly divided into 4 groups. The ICH models (Group C) were generated by injecting 100 μl autologous blood stereotactically into the right caudate nucleus; surgical control rats (Group B) were generated in a similar fashion, by injecting 100 μl saline into the right caudate nucleus. Intervention models (Group D) were established by intraperitoneal injection of DFO into rats in the ICH group. Healthy rats (Group A) were used for normal control models. Brain water content, iron deposition, and AQP4 in perihematomal brain tissue were evaluated over the time course of the study (1, 3, 7, and 14 days) in each group.
Iron deposition was found in the perihematomal zone as early as the 1st day after ICH, reaching a peak after 7 days and remaining at a high level thereafter for at least 14 days following ICH. Rat brain water content around the hematoma increased progressively over the time course, reached its peak at Day 3, and still was evident at Day 7 post-ICH. Immunohistochemical analysis showed that AQP4 was richly expressed over glial cell processes surrounding microvessels in the rat brain; there was upregulation of the AQP4 expression in perihematomal brain during the observation period, and it reached maximum at 3 to 7 days after ICH. The changes of brain water content were accompanied by an alteration of AQP4. The application of the iron chelator DFO significantly reduced iron overload, brain water content, and AQP4 level in the perihematomal area compared with the control group.
Iron overload and AQP4 may play a critical role in the formation of brain edema after ICH. In addition, AQP4 expression was affected by iron concentration. Importantly, treatment with DFO significantly reduced brain edema in rats and inhibited the AQP4 upregulation after ICH. Deferoxamine may be a potential therapeutic agent for treating ICH.
Hua-Jun Zhou, Tao Tang, Han-Jin Cui, A-Li Yang, Jie-Kun Luo, Yuan Lin, Qi-Dong Yang and Xing-Qun Li
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.
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.
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.
Thrombin can induce angiogenesis in rat brains and may be an important trigger for ICH-related angiogenesis.
Hua-Jun Zhou, Hai-Nan Zhang, Tao Tang, Jian-Hua Zhong, Yong Qi, Jie-Kun Luo, Yuan Lin, Qi-Dong Yang and Xing-Qun Li
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.
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.
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.
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.
Guang Yang, Zhendong Liu, Lu Wang, Xin Chen, Xiaoxiong Wang, Qi Dong, Daming Zhang, Zhao Yang, Qi Zhou, Jingxian Sun, Linmeng Xue, Xinzhuang Wang, Ming Gao, Lili Li, Ran Yi, Gareev Ilgiz, Jing Ai and Shiguang Zhao
It has been reported that microRNA-195 (miR-195) protects against chronic brain injury induced by chronic brain hypoperfusion. However, neither the expression profile of miR-195 nor its potential role during acute ischemic stroke has been investigated. In this study, the authors’ aim was to verify the mechanism of miR-195 in acute ischemic stroke.
The plasma levels of miR-195 expression were assessed using real-time PCR in 96 patients with acute ischemic stroke, and the correlation with the National Institutes of Health Stroke Scale score was evaluated. In addition, cerebral infarct volume, neurological score, and levels of miR-195 and CX3CL1/CX3CR1 mRNA and protein expression were assessed in mice subjected to middle cerebral artery occlusion (MCAO) with or without intra-cerebroventricular infusion of lentiviral vector. The inflammatory cytokines tumor necrosis factor–α (TNFα), interleukin (IL)–1β, and IL-6 of mouse brains after MCAO and BV2 cells treated with oxygen-glucose deprivation were measured using enzyme-linked immunosorbent assay, and apoptotic proteins were examined by Western blotting. Direct targeting of CX3CL1/CX3CR1 by miR-195 was determined by immunoblotting and dual luciferase assay.
In ischemic stroke patients, miR-195 was significantly downregulated and expression levels of miR-195 in these patients negatively correlated with the National Institutes of Health Stroke Scale score. In mice after MCAO, miR-195 overexpression decreased infarct volume, alleviated neurological deficits, and most importantly, suppressed an inflammatory response. Meanwhile, miR-195 suppressed the expression of the inflammatory cytokines TNFα, IL-1β, and IL-6 in vitro and in vivo. The authors further discovered that both CX3CL1 and CX3CR1 are direct targets of miR-195, but miR-195 exerts neuroprotective roles mainly through inhibiting CX3CR1-mediated neuroinflammation and subsequent neuronal cell apoptosis.
Taken together, these findings suggest that miR-195 promotes neuronal cell survival against chronic cerebral ischemic damage by inhibiting CX3CR1-mediated neuroinflammation. This indicates that miR-195 may represent a novel target that regulates neuroinflammation and brain injury, thus offering a new treatment strategy for cerebral ischemic disorders.