| The cerebrovascular diseases have badly threatened the health of the aged, In a long time, the incidence of cerebrovascular diseases in our country is higher than that of many other countries in the world. At present, the number of aging population in our country has been rapidly increasing. Therefore, we will face increasingly severe challenges of cerebrovascular diseases. The intracerebral hemorrhage (ICH) , which incidence, mortality and disability are high, is a common type of bleeding stroke. But up to now, there are still many questions which need be elucidated on cerebrovascular diseases, especially on the mechanism of pathophysiological procedure of hemorrhagic cerebrovascular diseases. And the key factors to induce brain injuries after ICH are not clear.The injuries of ICH included the primary and secondary injuries. The deterioration of neurological functions during early phase and later after ICH was regarded as the results of secondary brain injury around hematoma. There exist an area where brain tissue injury and edema have increasingly aggravated around hematoma after ICH. And the pathological changes in the area are reversible in definite time. If appropriate treatments may be administered in definite time window, the functions of injuried brain tissue may renew. Thus the area is called penumbra area around hematoma. How to retrieve thelatent, reversible, injuried brain tissue in penumbra area around hematoma in a length time after ICH hold the balance to the prognosis of ICH. Therefore, the studies of pathophysiology mechanism in penumbra area around hematoma have important significance.In recent years, the studies showed that the pathophysiology mechanisms in penumbra area around hematoma after ICH mainly comprise such several aspects as brain edema, the change of regional cerebral blood flow, inflammation and cell apoptosis. Although in the past the studies in these fields obtain some achievement and offer important basis and clue for the improvement of ICH treatment, there are still many questions that need be solved, especially on the study of the regional inflammation after ICH. Many studies on the inflammation after ICH barely begin and the markers that were observed in the fields are limited. And the studies in the original position on the regional inflammation after ICH are very few. There have still not reports on the role of astrocytic reaction in the pathophysiology procedure in penumbra area around hematoma after ICH.This thesis comprised two parts of contents. The first part comprised five experiments, which respectively studied the expressions of inflammatory factors and astrocytic reactions in the penumbra area around hematoma after ICH in rat ICH models that were produced by injecting collagenase into the striatum of the rats. We tried to study the key factors to induce the secondary injuries after ICH in order to offer new clues for the clinical ICH treatment. The second part cloned COX-2 gene and produced its polyclonal antibody, and observed COX-2's expression in neuron and glial cells in vitro and these studies may provide tools for further researches on the inflammation-immune reactions in the brain..The main results of the studies are the follows:1.There were more IL-1β , IL-6 and TNF α -like immunopositive cells in penumbra area around hematoma at the peak time of edema after ICH than that in controls. EL-1β and TNF α -like immunopositive cells were glia-like cells. IL-6-like immunopositive cells were neuron-like. There were clear IL-1β immunopositive cells around pathological changes in striatum at 4h after ICH. At 14h group, the number of IL-1β immunopositive cells were more densely than that of 4h's. At 24h, the number of IL-1β positive cells was maximal. And then after, the expression of EL-1β gradually decreased. After 72h, the expression of EL-1β almost disappeared.2. In the stratum hemorrhage model, the number of three kinds of adhesion molecules immunopositive cells in penumbra area around hematoma was nearly the same as that of the non-hemorrhagic side at 4h after ICH. At the 14 team there were more ICAM-1 and E-selectin positive cells than that of 4h team at hemorrhage sides. And at 24 team there were much more ICAM-1 and E-selectin positive cells than that of 14h team, and the distribution of positive cells was more widely than before. The number of VCAM-1 immunopositive cells also increased. The numbers of the three kinds of adhesion molecules immunopositive cells at the hemorrhage sides in the 72h team all decreased. The vascular endothelial cell in focus displayed immunofluorescence labeling for all three kinds of adhesion molecules. The results obtained with western blotting method were accorded with that of immunohistochemical method. And the thesis also observed that the effect of naloxone injected by intraperitoneal route on expressions of adhesion molecules in penumbra area around hematoma in ICH model. The results showed that there were many densely distributed adhesion molecules immunoreactive cells around the blood clot in saline treatment control group after ICH, and the number of adhesion molecules positive cells was decreased by naloxone injection, which had a significant difference with that of control.3.The expression of GFAP in penumbra area around hematoma was most strong during the early phases of hemorrhage (3~5h after ICH) . Positive cells were corpulent and had crassitude processes and were densely distributed. Then the expression of GFAP weakened slowly till 7d after ICH the expression increased appreciably.4.The results of the studies on the ultrastnictural changes and level of glycogen in penumbra area around hematoma in ICH model showed that there existed fine structural changes in different kinds of cells around pathological changes 24h after ICH. The most significant finding was that large numbers of glycogen accumulated in astrocytes cytoplasm and their processes around blood clot in brain. Especially, in the processes of astrocytes around the capillary vessel the number of glycogen granule was maximum.5.COX-2 gene was amplified by PCR from normal adult rat brain cDNA library. Expression vectors of whole COX-2 gene and its carboxyl terminal partial gene sequence were constructed respectively. The recombinant fusion protein was expressed in E.coli DH5 α . The rabbits were immunized with COX-2 fusion proteins coded by carboxyl terminal gene sequence to produce COX-2's polyclonal antibody. The COX-2 antiserum were characterized by ELISA, Western blotting, and immunocytochemistry methods. The results showed that we obtained COX-2 polyclonal antibody of high titer,... |
