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Abstract: We describe a novel experimental set-up that allows biochemical, immunohistochemical and morphometric recording of multiple parameters from a single rat brain. The whole brain was cut (coronal sectioning) in a volumetric manner, and 100 cryo-sections (10μm) were collected from the region of infarction. By use of a scalpel to dissect the cryosection, crude brain material was obtained from the cortical and basal ganglia areas of ischemic and non-ischemic hemispheres. Material from four 10μm thick sections of the same animal was pooled. About 30μg protein lysate was extracted per four sections with various lysis buffers; this sufficed for one biochemical or enzymatic test called “micro-Western-blots” or “micro-zymographies”. Scraping brain material from cryosections allows the detection of up to 25 parameters from adjacent brain sections of one single rat brain. Different analysis are possible, we have chosen, e.g. to compare factors affecting the basal lamina of cerebral microvessels like the content of the metalloproteinases-2/-9, their tissue inhibitors, the plasminogen activators, collagen type IV, parameters to test the blood–brain barrier: hemoglobin and the protein of the perfusion solution BSA and the infarction volume. On the basis of these parameters it was possible to compare the interactions of the complex processes in the ischemic brain in the same animal in adjacent sections. Thus, this method increases the validity of data comparisons and reduces significantly the number of animals needed in various experimental settings. [Copyright &y& Elsevier]

Focal cerebral ischemia leads to the gradual disruption of the extracellular matrix. A key role in the turnover of the extracellular matrix is played by the system of matrix metalloproteinases (MMPs). In this study we describe changes of the MMP inducer protein (EMMPRIN) following experimental cerebral ischemia (induced for 3 h and followed by 24 h reperfusion, suture model) in rats. Extracellular EMMPRIN was measured by Western blot of the ischemic and nonischemic basal ganglia and cortex separately. Compared with the contralateral nonischemic area, the ischemic hemisphere showed a significant increase in EMMPRIN: basal ganglia, 158% ± 4% ( P < 0.05); cortex, 128% ± 25% ( P < 0.05). Immunohistochemistry was used to localize EMMPRIN on cerebral microvessels. EMMPRIN-positive microvascular structures were quantified by automatic morphometric video-imaging analysis and a significant increase in the number of cerebral microvessels staining positive for EMMPRIN in the ischemic basal ganglia was shown. The significant loss of microvascular basal lamina antigen collagen type IV in ischemic cortex and basal ganglia was calculated by Western blot. Measured by gelatin zymography, we demonstrated an MMP-2 and MMP-9 increase in the ischemic brain regions ( P < 0.05). For the first time the MMP activation system EMMPRIN was shown to be relevant in cerebral ischemia. These results raise the possibility that the increased expression of EMMPRIN, the increase in MMPs and the damage of the basal lamina following cerebral ischemia are connected and part of a network of related changes. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the effects of different doses of exogenous recombinant human tissue plasminogen activator (rt-PA) on the endogenous cerebral plasminogen–plasmin system in focal ischemia in rats. Ischemia was induced using the suture model. Each group of rats (n = 6) received either treatment (0.9, 9 or 18 mg rt-PA/kg body weight) or saline (control group) at the end of ischemia; a sham-operated group was added. The activity of the plasminogen activators was measured by casein-dependent plasminogen zymography. In the cortex urokinase (u-PA) rose from sham (no ischemia), 91 ± 7% to ischemia, 176 ± 10% (P < 0.005). Increasing rt-PA doses led to further significant (P < 0.001) cortical u-PA activation which was maximal at 18 mg: 249 ± 13%. An extreme increase in the u-PA activity was observed in the basal ganglia to 1019 ± 22% (P < 0.001). This increase was further aggravated by higher rt-PA doses (18 mg, 1236 ± 15%;P < 0.001). The t-PA level did not change I3R24 during (3 h ischemia followed by reperfusion for 24 h); however, during low and moderate doses of rt-PA, endogenous t-PA was reduced. In conclusion, while ischemia leads to a significant increase in u-PA, mainly in the basal ganglia, t-PA is not altered. Increasing doses of rt-PA lead to a further elevation of u-PA. Thus, u-PA seems to play a major role in the endogenous plasminogen activator system following focal cerebral ischemia. [ABSTRACT FROM AUTHOR]