ABSTRACT
We evaluated effects of thrombosis of the superior sagittal sinus, its bridging and cortical veins (SVT) on the cerebral microvasculature in rats. Cryosections of brains ( n=7) were examined for venous infarction and microvascular basal lamina damage 3 h after SVT by immunohistochemical staining of microtubule-associated protein 2 and collagen type IV. Microvessels in the infarctions showed a decrease in the number (23.5+/-6.1%, P<0.002) and the total area (24.9+/-6.5%, P<0.011) of collagen type IV-positive vessels in contrast to control areas (21.7+/-12.4%, P<0.007; and 26.3+/-15.1%, P<0.026 in contrast to control areas of unoperated animals). This study showed a significant alteration of the cerebral microvasculature in SVT, which might contribute to edema and hemorrhagic transformation.
Subject(s)
Cerebrovascular Disorders/etiology , Sinus Thrombosis, Intracranial/complications , Venous Thrombosis/complications , Animals , Cell Count , Cerebrovascular Disorders/metabolism , Collagen Type IV/metabolism , Disease Models, Animal , Immunohistochemistry , Male , Microcirculation/metabolism , Microcirculation/pathology , Microtubule-Associated Proteins/metabolism , Rats , Rats, Wistar , Sinus Thrombosis, Intracranial/metabolism , Statistics, Nonparametric , Venous Thrombosis/metabolismABSTRACT
To determine the activity of matrix metalloproteinases (MMP), especially MMP-2 and MMP-9, which play an important role in ischemic stroke and intracerebral hemorrhage, we adapted a simple and rapid method for localizing gelatinase activity to a gelatin film in situ-overlay technique previously used in cancer research. Ten micrometer cryosections of rat brain from controls and animals subjected to 3 h of ischemia and 48 h of reperfusion (suture model for transient cerebral ischemia) were used. After thawing, a gelatin film with a polyester base was put on the slide, incubated for 24 h at 37 degrees C, stained with Ponceau S, and then discolored in bi-distilled water. Non-staining areas on the film corresponded to lysis zones, caused by activated MMPs. This was proven by MMP incubation at various concentrations on the plain gelatin film and pretreatment with EDTA (an MMP inhibitor), which prevents lysis zones in normal and ischemic brains. As confirmatory tests, SDS-PAGE zymography was used to define MMP activity, and also MMP-2 immunohistochemistry to detect the possibly cellular origin of MMPs. Normal rat brain exhibited a low background activity, which was visible as a light halo-like lysis zone over and around the brain. Areas in normal brain with medium MMP activity were within the white matter (corpus callosum, anterior commissure, and cerebellum). Ischemic brain exhibited high activity lysis zones within the infarcted area (detected by microtubuli associated protein-2 staining). These zones consisted of microscopically small lysis holes with a diameter of about 10-20 microm. Immunohistochemistry showed that especially microvessels expressed MMP antigen. SDS-PAGE zymography differentiated between a high level of activated MMPs in the ischemic area and a low level in the non-ischemic basal ganglia. The gelatin film in situ-overlay technique is able to localize MMP activity in ischemic rat brain tissue on a microscopic level.
Subject(s)
Brain Ischemia/enzymology , Brain/enzymology , Gelatin , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Animals , Brain/pathology , Brain Ischemia/pathology , Enzyme Activation , Immunohistochemistry , Male , Matrix Metalloproteinase 2/analysis , Matrix Metalloproteinase 9/analysis , Rats , Rats, WistarABSTRACT
In focal cerebral ischemia the plasminogen-plasmin system plays a role in the fibrinolysis of vessel-occluding clots and also in the proteolysis of extracellular matrix components, which potentially contributes to brain edema and bleeding complications. The authors investigated the plasminogen activation after middle cerebral artery occlusion with and without reperfusion (reperfusion intervals 9 and 24 hours) in rats by histologic zymography and compared areas of increased plasminogen activation to areas of structural injury, which were detected immunohistochemically. After 3 hours of ischemia, increased plasminogen activation was observed in the ischemic hemisphere. The affected area measured 5.2%+/-8.5% and 19.4%+/-30.1% of the total basal ganglia and cortex area, respectively. Reperfusion for 9 hours after 3 hours of ischemia led to a significant expansion of plasminogen activation in the basal ganglia (68.8%+/-42.2%, P < 0.05) but not in the cortex (43.0%+/-34.6%, P = 0.394). In the basal ganglia, areas of increased plasminogen activation were related to areas of structural injury (r = 0.873, P < 0.001). No such correlation was found in the cortex (r = 0.299, P = 0.228). In this study, increased plasminogen activation was demonstrated early in focal cerebral ischemia. This activation may promote early secondary edema formation and also secondary hemorrhage after ischemic stroke.
