| Intracerebral hemorrhage (ICH) is a common disease in the nervous systemcharacterized by high mortality and unability. Since the pathogenesis of ICH isnot very clear and there are no fundamental therapeutic methods, it has becomeone of hot points on research. Animal experiments have confirmed that thederivates of blood clot, haematoma location effect and its physical injury were theimportant reasons of brain tissue damage. Around haematoma, there occurred aseries of physical and pathological changes, including edema, decreased localcerebral blood flow, neuronal injury and glial hypertrophy and proliferation. Someresearches indicated that blood clotting reaction (especially the release ofthrombin), products cleaved by hemoglobin and inflammatory reaction played animportant role in ICH and can be the new therapeutic target. Objective: In the present, by establishing the stable and reliable animalmodel of ICH and TB in Wistar rats, we observed the pathological changes oftissues around lesion and detected the water contents of brain tissues. After that,we approached the dynamic changes of MMP-9 and MMP-2 in brain from proteinlevel by the advanced techniques to provide theoretical basis and new clinicaltherapeutic direction. Methods: Wistar rats were used to create ICH and TB models by injectingblood and TB, respectively, to caudate putamen under sterological method. 3h, 6h,12h, 24h, 3d, 5d, 7d and 14d after operation we observed the pathological changesof brain tissue around lesion stained by HE method under light microscope anddetected water contents of brain tissues by dry-wet weight method. MMP-9 andMMP-2 expression were investigated by immunohistochemistry and Western blotat each time point and linear regression analysis was used to approach therelationship between MMP-9/MMP-2 expression and water contents of braintissues.Results: Water contents of brain tissues: In ICH group, water contentsincreased at 3h after operation, reached the peak at 3d, and then decreasedgradually to the level of the control group at 14d. In TB group, the change ofwater contents was not obvious at 3h, but occurred at 6h, peaked at 3d, quicklydecreased at 5d and 7d. Compared to TB group, water contents in ICH groupincreased significantly at 3h, 5d and 7d.The pathological changes under light microscope: In ICH group, 3h afterhaematoma, brain tissues around lesion became loose and matrix had the slightedema, which became obvious at 12h with the broken and necrosis of Nissl's body,and karyopyknosis. Neutrophil, swollen astrocytes, myelin and axondisaggregation could be found. At 24h, brain edema, necrotic neurons, a varioussizes of vacuoles between cells, neutrophil and macrophage infiltration wereobvious, which reached the peak at 3d. From 5d to 14d, the edema deceasedgradually and proliferated glia repaired. In TB group, the structure was stillcomplete at 3h after injection. There were edema, cells necrosis, and inflammatorycell infiltrate at 12-24d, which were most obvious at 3d, similar to ICH group. Theedema lessened at 5d, but more proliferated glia cells, foam cells and a fewleukocytes infiltration were still observed. At 7d, the edema disspeared.The results of immunohistochemistry and Western blot: In ICH group, weobserved a few sporadic MMP-9 positive neurons and gliocytes at 3h. Thepositive cells increased rapidly at 24h, peaked at 3d and concentrated oncircumvascular cells. At 14d a few positive cells could still be found. There wassignificant difference of the number of positive cells in ICH group compared tosham group at each time point (P<0.01). In addition, at 3d a few MMP-9 werefound to express in the endotheliocytes. A few MMP-2 positive cells was firstlyobserved at 3h, but a slightly decreased at 6h and increased again at 24h, with thepeak level at 5d after ICH. At 14d, obvious MMP-2 expression still was found.During the whole period, there were no MMP-2 positive endotheliocytes. Westernblot results indicated that the number of MMP-9 expression increased at 3h,peaked at 3d, then decreased gradually. Nevertheless, the number of MMP-2heightened obviously at 3h, declined at 6h, increased again at 24h, peaked at 5d.We can still observe higher gray scale of MMP-2 at 14d. The results fromWestern blot were consistent to the results from immunohistochemistry.Until 6h after TB injection into brain, the MMP-9 positive cells were found,increased quickly at 24h, peaked at 3d, and then rapidly decreased, only a few at14d. Compared with ICH group, the expression of MMP-9 positive cells in TBgroup delayed, the number of positive cells at 3h, 5d, 7d, and 14d was much more.However, MMP-2 postive cells (gliocytes and neurons) could be observed at 3h,declined at 6h, increased again at 24h, peaked at 5d. At 14d, we can still find afew MMP-2 positive cells. Compared with ICH group, the expression of MMP-2delayed, the number of cells was less. At 3h, 6h, 12h, 24h, 3d, and 5d, the numberof MMP-2 positive cells in TB group was obviously less than ICH group. Westernblot results showed that the expression of MMP-9 didn't increase at 3h,up-regulated at 24h, peaked at 3d, and then quickly declined, only a few at 14d.There were significant difference at 3h, 6h, 5d, 7d, and 14d when compared toICH group. Similar to the tendency of ICH group, in TB group MMP-2 expressionbegan at 3h, decreased at 6h, increase at 24h, peaked at 5d, and then inclined, until14d. There was remarkable difference on MMP-2 expression at the time point of3h, 6h, 12h, 24h, 3d, and 5d between TB group and ICH group.The relationship between gray scales of MMP-9/MMP-2 and water contentsof brain tissue was investgated by linear regression analysis method. Within 142. TB is a noxious substance in brain edema after ICH, especially in theperiod of 24h-3d, which provides the theoretical basis for clinical treatment.3. We clarified the relationship between the expression of MMP-9/MMP-2and brain edema, and their functions after ICH, which provides the possibility andprospect to apply MMPs antagonists into clinical treatment of ICH in future. |
PDF Full Text Request