| Intracerebral hemorrhage (ICH) represents common and one of the most devastating types of stroke. It occurs when a blood vessel or vascular anomaly ruptures, releasing blood into the surrounding brain tissue. It accounts for 10% to 15% of all strokes, but is responsible for approximately one half of all stroke-related deaths. It is associated with a 30-day mortality rate of 43% and imparts some form of disability on 88% of its survivors. Despite much effort directed at clarifying the roles of surgical and medical therapy for ICH, the most appropriate management is still controversial. This may be partly due to poor understanding of the mechanisms of brain edema after ICH. Brain edema contributes directly and independently to the neurological deterioration after ICH. The peak of ICH-induced death often occurs during the first few days following the ictus in association with the progressive de-velopment of brain edema. Many patients with an ICH deteriorate progres-sively after the hemorrhage because of the accumulation of edema fluid around the mass. Better understanding of the pathophysiology of the edema formation due to ICH might lead to improved clinical management of the ICH patients. Because of the great importance of edema on the outcome of ICH pa-tients, it is critical to identify the potential factors that may associate with the edema formation, aiming at the improved strategies for therapeutic interven-tion. Although the mechanisms of brain injury after ICH are not fully under-stood, several mechanisms appear to contribute to edema development. In-flammatory processes have increasingly been shown to be involved in the process of cardiovascular and cerebrovascular disease (CVD). The weight of evidence supporting a link between the inflammatory response and vascular disease has grown considerably in recent years. Accumulating evidence has suggested that inflammatory response may be involved in promoting edema formation and secondary cell death in cerebral ischemia with reperfusion. But less effort has been directed toward a link between the inflammatory response and ICH. The purpose of the present study was to investigate whether inflamma-tory response contributed to the brain edema formation and progression after ICH. Besides the observation on the changes of neurobehavioral scores and brain water content, we chose to measure the dynamic changes of the proin-flammatory cytokines such as tumor necrosis factor-α(TNF-α), interleukin-6 (IL-6) and nuclear factor-κB (NF-κB), and complement receptor 2 in the ICH model. Part 1 Behavioral and brain edema after intracerebral hemorrhage in rats Objective: To make an intracerebral hemorrhage (ICH) model in rats by autologous tail artery blood, which can be used in the field of the behavioral and brain edema research. Methods: 60 male Sprague-Dawley rats weighing from 350 to 400 g were used in this experiment. All the rats were anesthetized with pentobarbital (40 mg/kg IP). They were positioned in a stereotaxic frame and a cranial burr hole (1 mm) was drilled on the right coronal suture 3.5 mm lateral to the mid-line. ICH were induced in the rats: 50 μl autologous tail artery blood was sam-pled and then infused stereotacticlly into the right caudate nucleus at the rate of 10 μl/min through a 26-gauge needle (coordinates: 0.2 mm anterior, 5.5 mm ventral, and 3.5 mm lateral to bregma). About sham control rats, they had only a needle insertion. After the infusion, the needle was removed, the burr hole was sealed with bone wax, the wound was sutured, and the animal was placed in a warm box with free access to food and water. Physiological parameters were maintained in the normal range in this experiment. Behavioral evaluation: all tests were done by a single observer without knowledge of neurological condition. Motor behavior was evaluated using 4 tests in each rat at 6 h, 24 h, 48 h, 72 h and 7 d after operation. The specific 4 tests included (1) Longa be-havioral test; (2) Berderson behavioral test; (3) Beam walking test; and (4) Footfault asymmetry test. The scores from all 4 tests were added to give a motor deficit score. Brain Water Content: All the rats were used for this ex-periment. At 6 h, 24 h, 48 h, 72 h and 7 d, after the motor-behavior tests were done, each rat was killed by pentobarbital overdose. The brain was quickly removed and placed on a cooled surface, and the frontal pole (approximately 3 mm thick), the cerebellum and brain stem were removed. The cerebrum was coronally cut into 3 parts: the first cut was through the needle entry site and the second through the midpoint of the posterior remnant. The first part (al-most 2 mm thick) was used for brain edema measurement and was cut into two sections: ipsilateral and contralateral of ICH. Each section was wrapped in preweighed aluminum foil and weighed to obtain the wet weight (WW), then dried for 72 h in an oven at 95～110℃and weighed again to obtain the dry weight (DW). Brain water content was calculated as the percentage change between wet weight and dry weight using the following formula: (WW-DW)/WW×100%. Results: The results showed that, the neurological deficit scores were the highest at 6 h after operation in the Longa, Berderson and Beam Walking test-ing methods, and at 24 h in the footfault Asymmetry test because of the anes-thesia and injuries during the operation. At 24 h~72 h, the scores of the ICH group leveled high until all the rats recovered and the scored fell to the lowest at 7d. At all time points, the scores of the Sham-operated rats were lower than that of the ICH rats and significant differences existed between the two groups. The line graph showed the dynamic tendency of brain water content in the ip-silateral, contralateral and sham groups at different time points after intracere-bral infusion. At 6 h and 24 h, brain water contents of the ICH and sham groups had no significant differences, P>0.05. But at 48 h and 72 h, the brain water content of ICH group was much higher than sham group, at 48 h 78.46%±0.47% verse 77.57%±0.39%,P<0.05;and at 72 h was 78.11%±0.41% verse 77.71%±0.18%, P<0.05. And at 48 h, it was the highest. Conclusion: ICH model in rats by infusing autologous caudate arteryblood was reproducible. Following the hemorrhage there were some changes on rat behavioral and brain edema. Part 2 Morphological observation of perihematoma in experimental in-tracerebral hemorrhage Objective: To observe dynamic pathological changes of perihematoma in experimental intracerebral hemorrhage(ICH) in rat. Methods: In this study, 24 Sprague-Dawley rats were used. Autologous blood were injected into the right caudate nucleus in experimental groups. Sham injections served as controls. Tissues from perihematoma were collected and observed under the light mi-croscope. Methods: The brain tissues were perfused with 4% paraformaldehyde in 0.1 mol/L PBS (pH 7.4) for three days and then were dehydrated and embed-ded in paraffin. Sections (5 μm) were cut, and stained with hematoxylin and eosin. The rest paraffin sections were stained by routine immunohistochemical methods using primary antibodies against TNF-α,IL-6, NF-κB, CR2. Anti-rat IgG was revealed with secondary antibodies, and secondary bioti-nylated conjugates were demonstrated using the Vector ABC kit and diamino-benzidine. The immunohistochemical labeling was performed on sections at the coronal level of the maximum hematoma diameter. Areas with labeled cells were compared between ipsilateral and contralateral side in the cortex and caudate nucleus. Results: In ICH group, there were obvious changes in brain tissue. Mi-croscopically, a single roughly spherical hematoma could be clearly seen in the caudate nucleus area at all time points after the blood injection. At 6 h, around the periphery of the hematoma were a few rounded scattered neutro-phils and the shape of neurons had no obvious changes. At 24 h, brain tissues around the hematoma were obviously swollen and necrotic, and the infiltrated inflammatory cells were mainly mononuclear, and several neutrophils could also be seen. At 48 h, the brain tissues around hematoma were highly swollen with pale cytoplasm, and they were diffluent, necrotic and were surrounded by a compact band of cells including viable neutrophils, some cell debris, a fewmacrophages, and rare clusters of intact erythrocytes. The neurons were de-generated with vague nuclear and disappearing nissl bodies, and neurono-phagia existed. At 72 h, surrounded the hematoma were scattered microglia containing hemosiderin, hyperplasia of glial cell and neovascularization. At 7 d, the hematoma was resolving and the glial cell hyperplasia and neovascu-larization were obvious. Neutrophils were no longer evident and there were many microglia. In sham group, there was no hematoma and significant changes in the brain tissue. Conclusions: The results showed that some neurons in perihematoma displayed necrosis. Several scattered inflammatory cells were observed in perihematoma at 6 h after ICH and the inflammatory infiltration characterized by neutrophils reached its maximum at 48 h. At 72 h after ICH gliosis and capillary hyperplasia were observed in and around hematoma. The area of hematoma reduced markedly, accompanied by distinct glial and capillary hy-perplasia at 7 days after ICH. All those showed that inflammatory infiltration, neuron necrosis, glial and capillary hyperplasia in perihematoma of experi-mental ICH rats may be one of the causes of brain edema and neurobehavioral changes. Part 3 The role of IL-6 expression in secondary neuronal injury and brain edema following experimental intracerebral hemorrhage in rat Objective: Intracerebral hemorrhage (ICH) is a severe disease to endan-ger the people's life because it can cause high mortality and serious disabil-ity.So there are a lot of researches on pathophysiology and therapy of ICH. But it depends on setting up the constant model of ICH first. The model of in-jection autologus blood into basal ganglia is well-used in the researches of ICH .It has more advantages than other models, especially for the study on the brain edema surronding the hemotoma. Inflammatory response occurs follow-ing ICH. It is more evident in hemorrhagic event than in non-hemorrhagic event. Inflammation contributes to the secondary brain injury and brain edema formation after ICH. Cytokine Interleukin-6 (IL-6) is an important preinflam-matory factor. It can enhance the toxicity of neutrophil, elevate the expression of intercellular adhesion molecule (ICAM) on endothelial cell, attract more inflammatory cells to the injury region by inducing chemokine production. So IL-6 can induce intense inflammatory response. We hypothesize that IL-6 invloves in acute secondary neuronal damage and brain edema formation of ICH. The present study will observe the expression of IL-6 in the rat brain of ICH at different time point. We intend to test the hypothesis that IL-6 can play a role in the brain injury and brain edema formation of ICH. Methods: A medium volume of hematoma was formed by stereotactic infusing 50 μl autologous caudate artery blood into the right basal ganglia of rat .We evaluated the rat's behavior at 6,24,48,72 h and 7 d after surgery and recorded the scores. Samples of tail arterial blood were collected. Then rats were decapitated for testing the brain water content. Sham rats underwent in-tracranial needle placement without blood infusion. IL-6 expression in rat brain at different time point was investigated by immunohistochemistory. We observed the pathological changes including hematoma, brain edema, the morphologic changes of neuron and glial cell by HE staining. We counted the intact neuronal numbers around hematoma and in hippocampus according to Nissl staining at different time point. Results: Immunohistochemistry results showed that IL-6 positive area ratio were 9.8±4.6(mean±standard error) versus 5.0±1.58 between the ICH group and the sham group at 6 h, P=0.05; 11.0±4.30 versus 5.6±1.95 at 24 h, P<0.05; 19.6±2.30 versus 6.2±1.30 at 48 h, P<0.01; 17.0±5.15 versus 5.8±1.92 at 72 h, P<0.01; In ICH group, the positive area ratio of 48 h and 72 h were different from 6 h and 24 h, whereas in sham group, no difference existed. So IL-6 expression in rat brain elevated at 24 h, peaked at 48 h, re-mained elevated at 72 h. It consisted with the brain water content. Conclusion: The high expression of IL-6 in the rat brain after ICH con-tributed to the brain edema formation and the secondary neuronal injury. Part 4 Expression of TNF-αin the brain tissue after experimental intracerebral hemorrhage in ratObjective: The present study will observe the expression of TNF-αin the rat brain of ICH at different time point. We intend to test the hypothesis that TNF-αcan play a role in the brain injury and brain edema formation of ICH. Methods: 56 male SD rats were randomly divided into two group: ICH group and sham group. The rats were anaesthetized and perfused at 3 h, 6 h, 12 h, 24 h, 48 h, 72 h, 7 d after operation, then the removed brain tissue was fixed, imbedded and incised. TNF-αin the brain tissue around the homatoma were measured by immunochemistry at different time point. Positive cells in the cerebral tissue around the hematoma were countered by light microscope at 400×Magnification.5 different scopes were randomly chosen at every time-point, then the data were processed by statistics. Results: Immunohistochemical dying result of TNF-αpositive cells in the ICH group and the sham group were:6.8±1.63 verse 1.2±0.84 at 3 h after operation, P<0.01,;9.2±1.79 verse 1.6±1.14 at 6 h, P<0.01; 13.4±2.07 verse 1.8±0.84 at 12 h, P<0.01; 44.8±4.55 verse 2.6±1.40 at 24 h, P<0.01; 58.4±6.19 verse 2.0±1.12 at 48 h, P<0.01; 49±2.91 verse 1.2±0.84 at 72 h, P<0.01; 6.4±1.14 verse 1.1±0.62 at 7 d, P<0.01. The results demonstrated expression of TNF-αjust at 3 h after operation, peak at 48 h and last 1 week. Conclusion: The experiment demonstrated there existed inflammation around the cerebral tissue after ICH. TNF-αexpression raised in the inflam-matory response. Part 5 The expression of NF-κB and the neural cells'aoptosis in brain tissue after ICH in rat Objective: Nuclear factor-κB (NF-κB) is a ubiquitous transcription fac-tor and a member of a family of proteins that are critical regulators of a variety of responses, This results in the transcriptional induction of genes for many proinflammatory substances, such as TNF-α, IL-6 and IL-8, transforming growth factor, ICAM-1, the iNOS, epoxide enzyme -2 and so on. Its activation has been played a key role in the inflammatory response in the cerebral dis-eases, In this study, we examined the rules of NF-κB expression in rat brainafter ICH in order to explore correlation between NF-κB and apoptosis after ICH. Method: 60 rats Adult male Sprague-Dawley were randomly divided into 2 groups, ICH group and sham group. According to the methods of Hua,Lee et al, the rats were subjected to stereotactic frame, and a cranial burr hole (1.0 mm) was drilled in the skull (1 mm anterior, 4.0 mm right to the bregma). 50 μl autologous caudate artery blood was infused into right caudate nucleus of the rats, and the shams served as controls. After ICH for 6 h,24 h,48 h,72 h and 7 d, the successful ICH models of each group were measured by behav-ioral tests and then part of the successful ICH models were anesthetized with pentobarbital and perfused through the left ventricle with 0.9% saline followed by 4% paraformaldehyde in 0.1 M phosphate-buffered (pH7.4). The removed brain were cut from the hole coronally, and the hematoma can be seen in the right caudate nucleus .We cut the brain into 4mm thick and kept them in 4% paraformaldehyde for 24~48 hours at 4℃, through dehydration, transparency, immersed in wax, embed, finally kept by wax mass. For another part, the suc-cessful ICH models were killed by decapitation without perfusing and their brains were removed. The fresh brains were frozen by the liquid nitro-gen-isopentane method in embedding compound and then cut into in 20μm segments with a cryostat section cutter. We can observe inflammatory cells immersing by HE staining and NF-κB expression. Result: Immunohistochemistry demonstrated that NF-κB expressed very little in sham group, but in the model group NF-κB expressed increasingly at 6 h, reached maximum at 48 h, continued to 7 d. NF-κB mostly expressed in glial, the positive cells'cytoplasm or nucleus are stained brown yellow. There are no statistic difference about NF-κB expressing between ipsi-hemotoma and contral-hemotoma, P>0.05;but there are statistic statistics about NF-κB expressing between the ICH group and the sham group, P<0. 05. Conclusion: The study suggests that there were a correlation between NF-κB expression and the brain edema formation. Part 6 The role of CR2 in secondary brain injury and brain edema after...
|
PDF Full Text Request