Vascular integrin immunoreactivity is selectively lost on capillaries during rat focal cerebral ischemia and reperfusion.

The alpha1-integrin cell adhesion molecules, the principal endothelial receptors for basal lamina (BL) components disappear during transient ischemia. The current study investigated the localization of integrins, the time dependency and vessel size selectivity in the normal rat brain before and after 3 h of cerebral ischemia (I3) and reperfusion (R). Additionally we looked for a correlation to the amount of extravasation and hemorrhage. In the normal brain, there was a clear immunoreactivity for the alpha1, alpha6, and beta1 integrins on the endothelial perivascular cells. After I3 followed by variable reperfusion intervals of 0, 9, and 24 h (R0, R9 and R24; respectively), the number of vessels and staining intensity indicating immunoreactivity in the ischemic area were compared with the contralateral side. The number of the beta1-immunoreactive capillaries was steadily decreasing with the reperfusion time: -12+/-5%, -15+/-7% and -43+/-8% at I3R0, I3R9 and I3R24 (all p<0.05). The beta1-staining intensity decreased homogeneously to -21% at I3R24 (p<0.05). Vascular staining for alpha1 was affected similarly. Interestingly, the alpha6-positive arterioles/venules were also reduced by -21% at I3R24 (p<0.05) in a diameter-selective way on vessels with diameters larger than 15 mum. The correlated break-down of the blood-brain-barrier was demonstrated by the significant rise of the extravasation of BSA from the perfusion solution as well as the increased hemorrhage after MCAO/R (hemoglobin: 103+/-4% versus 330+/-17%; BSA 101+/-3% versus 132+/-9% in I0R0 and I3R24, respectively). The prominent capillary vulnerability contributes significantly to the impairment of the microvascular integrity and after ischemia and reperfusion.

Characterization of microvascular basal lamina damage and blood-brain barrier dysfunction following subarachnoid hemorrhage in rats.

Vasogenic brain edema is one of the major determinants for mortality following subarachnoid hemorrhage (SAH). Although the formation of vasogenic brain edema occurs on the microvascular level by opening of endothelial tight junctions and disruption of the basal lamina, microvascular changes following experimental SAH are poorly characterized. The aim of the present study was therefore to investigate the time course of blood-brain barrier (BBB) dysfunction and basal lamina damage following SAH as a basis for the better understanding of the pathophysiology of SAH. SAH was induced in Sprague-Dawley rats by an endovascular filament. Animals were sacrificed 6, 24, 48, and 72 h thereafter (n=9 per group). Microvascular basal lamina damage was quantified by collagen type IV immunostaining. Western blotting was used to quantify collagen IV protein content and bovine serum albumin (BSA) extravasation as a measure for basal lamina damage and blood-brain barrier disruption, respectively. BSA Western blot revealed significant (p<0.05) BBB opening in the cerebral cortex ipsilateral to the hemorrhage beginning 6 h and peaking 48 h after SAH. Significant (p<0.05) basal lamina damage occurred with gradual increase from 24 to 72 h. Basal lamina damage correlated significantly with BBB dysfunction (r=-0.63; p=0.0001). Microvascular damage as documented by collagen IV degradation and albumin extravasation is a long lasting and ongoing process following SAH. Due to its delayed manner microvascular damage may be prone for therapeutic interventions. However, further investigations are needed to determine the molecular mechanisms responsible for basal lamina degradation and hence damage of the microvasculature following SAH.

Doxycycline inhibits MMPs via modulation of plasminogen activators in focal cerebral ischemia.

Tetracyclines inhibit matrix metalloproteinases (MMPs) and reduce infarction volume following cerebral ischemia. In this thesis an involvement of urokinase could be proven. Cerebral ischemia in rats was induced for 3 h followed by 24 h reperfusion (suture model). Each 6 animals received orally either doxycycline or water. Doxycycline treatment began 10 days before ischemia. MMP-2 and MMP-9 were substantially decreased. The possibility of involvement of the endogenous MMP inhibitors in the MMP inhibiting mechanisms was excluded. The plasminogen activator uPA was significantly decreased by doxycycline indicating an MMP inhibiting mechanism including the plasminogen/plasmin system. In the doxycycline group, this resulted in a decreased damage to the cerebral microvessels and less loss of the basal lamina antigen collagen type IV. Hemoglobin extravasation was also significantly reduced. Our results suggest that doxycycline may have a potential use as an anti-ischemic compound since it provides microvascular protection by inhibiting the plasminogen system.

Pravastatin reduces microvascular basal lamina damage following focal cerebral ischemia and reperfusion.

Transient ischemia has been shown to damage the basal lamina of the cerebral microvasculature. Other studies proved statins to be beneficial to non-cerebral microvessels. The aim of this study was to determine whether pravastatin pretreatment ameliorates microvascular basal lamina damage following transient ischemia. Using the suture model, we subjected 15 rats to focal ischemia (3 h) and reperfusion (24 h). Rats received pravastatin (20 mg/kg/day) or saline for 4 weeks prior to the experiment. The outcome was determined by a behavior test and the infarct size. Collagen type IV, a marker for an intact basal lamina, and hemoglobin extravasation were measured by Western blot analysis. A ratio (in percentage) between ischemic and contralateral hemispheres was calculated. Pravastatin pretreatment resulted in a significantly better neurological outcome and reduced infarct size (15 +/- 0.5 and 59 +/- 10 mm(3), respectively) compared with controls (12.25 +/- 0.4 and 167 +/- 13 mm(3), respectively, P < 0.01 for both). In controls, loss of collagen type IV was seen in the basal ganglia and in the cortex (43 +/- 4 and 64 +/- 5%, respectively). Pravastatin prevented significant collagen loss (basal ganglia: 106 +/- 17%; cortex: 112 +/- 14%, P < 0.01 for both) and significantly reduced the hemoglobin extravasation compared with controls in the basal ganglia (198 +/- 49 vs. 553 +/- 47%, P < 0.01). Pravastatin pretreatment resulted in a reduction of microvascular basal lamina damage and hemoglobin extravasation following transient ischemia. Pravastatin seems to protect the cerebral microvascular system.