Background
Microvascular basal lamina damage is a regular result of experimental focal cerebral ischemia. Recombinant tissue plasminogen activator (rt-PA) is successfully used in human ischemic stroke. The main drawback is the development of hemorrhage. Hypothermia was proven to be able to reduce the basal lamina damage by almost 50%. The combination of hypothermia and rt-PA completely preserves the microvascular basal lamina following experimental focal cerebral ischemia. Matrix metalloproteinases (MMP) are critically involved in the degradation of the basal lamina after ischemia. Animals treated with rt-PA but not with hypothermia developed a dramatic increase in MMP-2, MMP-9. Hypothermia reduced the levels of both proteases, they did not differ between the groups of animals treated with or without rt-PA in combination with hypothermia. EMMPRIN, as a MMP inducer protein, was shown to be relevant in cerebral ischemia. The aim of this study was to test the influence of hypothermia and the combination of hypothermia and rt-PA to the MMP activator protein EMMPRIN.
Methods
Using the suture model, we subjected 30 rats to 3 hours of focal ischemia and 24 hours of reperfusion. Each six rats received post-ischemic normothermia (37oC) with and without rt-PA (i.v. administered 18 mg/kg bw at the end of ischemia), hypothermia (32–34oC) for the reperfusion period with and without rt-PA; a group of six sham operated animals with hypothermia was used as control. EMMPRIN expression was measured by Western blot of the ischemic and non-ischemic basal ganglia and cortex separately. Gelatine-zymography was used to detect MMP-2.
Results
With immunohistochemistry it was shown that EMMPRIN was present on cerebral microvessels in the rat. A significant increase of the MMP-inducer protein was demonstrated by Western blot analysis with anti-EMMPRIN antibody in the ischemic basal ganglia area after I3R24. Compared with the contralateral area, EMMPRIN expression was significantly increased in the ischemic hemisphere. Hypothermia reduces significantly the level of EMMPRIN: in basal ganglia 94±13% (hypothermia) vs. 169±29% (normothermia), and in cortex 83±7% vs. 109±26% (p<0.05). EMMPRIN expression parallels to MMP-2 expression: in basal ganglia 71±28% (hypothermia) vs. 107±4% (normothermia), and in cortex 53±11% vs. 121±5% (p<0.05) (all data are presented as ratio ischemic vs. contralateral nonischemic side).
Discussion
It was recently shown that additionally given exogenous rt-PA after the period of ischemia caused an significant increase of MMP-2 under hypothermic and normothermic conditions. However the amount of EMMPRIN was not significantly increased by administration of rt-PA. These results confirm that the expression of EMMPRIN may be responsible for the known increase of MMPs following cerebral ischemia and reperfusion. Following hypothermia there was less EMMPRIN in the ischemic brain tissue. Calculating the ratio protease (MMP-2) to its possible activator (EMMPRIN) showed a lower ratio under hypothermic conditions. This also points towards a lower influence of EMMPRIN in hypothermia.
Conclusions
Hypothermia provides a microvascular protection, this can be seen in the significant reduction of MMP-2. One explanation can be seen in the reduction of the MMP inducer protein EMMPRIN under hypothermic conditions.