Spontaneous thrombosis and decreased tPA activity following tHI insults.

<p>(<b>A–L</b>) Immunostaining performed at 1 h after tCCAo, tHI or permanent carotid ligation (pCCAo) plus hypoxia showed deposits of fibrin(ogen) and platelets (detected by anti-Integrin α2β/CD41) and P-Selectin-positive blood vessels (a marker for endothelial activation) and in the ipsilateral hemisphere following tHI insults (C, G, K). Permanent carotid ligation plus hypoxia produced the same staining pattern, but with stronger signals of all three markers (D, H, L). This staining pattern was absent in the brain after tCCAo (A, E, I) or on the contralateral hemisphere following tHI (B, F, J) (n>4 for each). (<b>M</b>) Plasminogen-zymogram showed definitive induction of the brain uPA and ∼50% increase of tPA activity in the ipsilateral hemisphere (R) at 4 h post-HI in neonates. No uPA-induction was found in adult brains at 4 h following tCCAo or tHI insults. The tPA activity in the ipsilateral hemisphere (R) following tHI was reduced to ∼67% of the level in unchallenged (UN) brains (p<0.05). The right panel is the quantification of zymogram band intensity (n = 4 for each). (<b>N</b>) The plasminogen-zymogram from plasma showed the lack of tPA activity and a similar degree of uPA activity at 4 h after tCCAo or tHI insults in adult mice. Immunoblot detection of transferrin (Tf) served as the internal control. (<b>O</b>) Direct measurement of tPA activity using a fluorescent substrate kit also showed significant reduction in the ipsilateral hemisphere at 4 h following tHI insult in adult brains (n = 3-4 for each). (<b>P–Q</b>) Immunoblot analysis showed minimal change of the tPA protein level and no sign of plasminogen activator inhibitor-1 (PAI-1) up-regulation after tCCAo or tHI insult in adult brains (n = 4 for each). Scale bar: 200 μm in A–L.