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Early Microvascular Responses to Experimental Focal Cerebral Ischemia

G.J. del Zoppo, B.R. Copeland,* T. Abumiya, M. Tagaya, J.H. Heo, J. Hallenbeck,** T.E. Hugli, E. Berg***

* Scripps Clinic, La Jolla, CA
** National Institutes of Health, Bethesda, MD
*** Protein Design Laboratories, Mountain View, CA

Both clinical observations in patients with signs and symptoms of focal cerebral ischemia and experimental work in primate and rodent models of experimental stroke increasingly suggest that neuronal injury is tied to microvascular events. Recent work in our laboratory has focused on the hypothesis that early microvascular endothelial cell and astrocyte responses to ischemia contribute to neuronal injury. In particular, we continued our study of alterations in structural and signaling relationships within the microvascular complex, including the relationship between integrin expression and changes in the matrix (basal lamina) after occlusion and reperfusion of the middle cerebral artery. This work has centered on the responses of the basal ganglia to ischemic injury, because we propose that interventions that salvage tissue from injury in this region most likely will also have a beneficial effect on ischemic cortex.

Continuing experimental studies have shown that marked rapid reductions in expression of integrin ₁ß₁ in endothelium, integrin ₆ß₄ in astrocyte end-feet, and laminin in the matrix occur in a graded fashion relative to neuronal injury. That is, by 2 hours after occlusion of the middle cerebral artery, a successive decrease in integrin expression occurs that corresponds to the degree of neuronal injury in the ischemic basal ganglia. Furthermore, changes in expression of the integrin transcripts do not completely account for the losses in integrin protein. Moreover, the topographic distribution of expression of integrin ß₁ mRNA was novel. For instance, it was significantly reduced in the ischemic core (region of most severe neuronal injury) by 2 hours after occlusion. It was significantly upregulated on microvessels within the border of circular arrays of microvessels within the ischemic core. No expression of integrin ß₁ mRNA was detected in the center of the microvascular arrays, areas that had no expression of integrin ß₁ protein. One important implication of this observation is a modification in the long-held concept that tissue peripheral to the ischemic core (the "penumbra") may be recoverable with reperfusion. In fact, the recovery may actually involve subregions of the ischemic zone within the basal ganglia in a time-dependent manner. This finding suggests that tissue recovery may occur in other areas as well as in the periphery.

We investigated the relevance of early microvascular responses to ischemia and reperfusion by examining expression of integrin _Vß₃. Previously, we found a highly significant relationship between expression of this integrin and the formation of fibrin within the same microvessels by 2 hours after occlusion of the middle cerebral artery. This finding was the first observation of a very early microvascular response to ischemia in the CNS. The nature of the relationship between _Vß₃ and its ligand, however, remains uncertain.

We determined the expression of proliferating cell nuclear antigen, vascular endothelial growth factor (VEGF), and _Vß₃ at various times after occlusion of the middle cerebral artery. The results indicated a highly significant and orderly relationship among expression of these 3 molecules on microvessels within the ischemic core. Expression of VEGF and _Vß₃ could be detected as early as 1 hour after occlusion. This finding suggests that very early expression of VEGF and very early expression of _Vß₃ in response to ischemia are tied together, as they are in several other vascular responses in selected organ systems.

Furthermore, time as a variable was not relevant to the outcome. This result suggests that because of the variable responses to ischemic injury of the tissue at risk, no strict time sequence of vascular response could be detected. However, signals for increased vascular endothelial permeability (VEGF) and vascular endothelial cell and smooth muscle reactivity (_Vß₃) are generated early during the ischemic event. The implications for vascular recovery and resolution of injury must be explored.

Clinical work on the use of plasminogen activators to treat acute stroke is based on the hypothesis that recovery of neuronal injury may be augmented by early arterial reperfusion. In animal experiments in which the middle cerebral artery was occluded for 2--3 hours, no recovery of lost integrin expression occurred with reperfusion. The feasibility of establishing clinical reperfusion by intraarterial infusion of recombinant pro-urokinase and heparin within 6 hours of the onset of symptoms in patients with acute ischemic stroke was examined in a prospective multicenter placebo-controlled study. This study was the first placebo-controlled trial of direct intraarterial infusion of a plasminogen activator in patients with acute stroke.

The results indicated that the frequency of recanalization was significantly higher in patients treated with recombinant pro-urokinase than in patients treated with the placebo. Both the frequency of recanalization and the frequency of hemorrhagic transformation were significantly affected by the use of heparin anticoagulation. Whether recanalization within this time frame by direct methods will produce significant neuronal recovery remains to be seen. However, this clinical experiment reinforces the need to examine microvascular integrity and its effects on neuronal injury and recovery during reperfusion in very early focal cerebral ischemia.

PUBLICATIONS

del Zoppo, G.J. Antithrombotic therapy of acute stroke: Thrombolytic agents. Thromb. Haemost. 78:183, 1997.

del Zoppo, G.J. Clinical responses to thrombolysis in acute ischemic stroke, and the importance of the microvasculature. Jpn. J. Stroke 19:169, 1997.

del Zoppo, G.J. Thrombolytic therapy in the treatment of stroke. Drugs 54(Suppl. 3):90, 1997.

del Zoppo, G.J., Hacke, W. Thrombolytic treatment of thrombotic stroke. In: Cardiovascular Thrombosis: Thrombocardiology and Thromboneurology, 2nd ed. Verstraete, M., Fuster, V., Topol, E.J. (Eds.). Lippincott-Raven, Philadelphia, 1998, p. 597.

del Zoppo, G.J., Higashida, R.T., Furlan, A.J., Pessin, M., Rowley, H., Gent, M., the PROACT Investigators. A randomized trial of recombinant pro-urokinase by direct arterial delivery in acute middle cerebral artery stroke. Stroke 29:4, 1998.

del Zoppo, G.J., Wagner, S., Tagaya, M. Trends and future developments in the pharmacological treatment of acute ischemic stroke. Drugs 54:9, 1997.

Hosomi, N., Tsuda, Y., Ichihara, S.-I., Kitadai, M., Ohyama., H., Matsuo, H. Duration threshold of induced hypertension on cerebral blood flow, energy metabolism, and edema after transient forebrain ischemia in gerbils. J. Cereb. Blood Flow Metab. 16:1224, 1996.

Tagaya, M., Liu, K.-F., Copeland, B., Seiffert, D., Engler, R., Garcia, J.H., del Zoppo, G.J. DNA scission after focal brain ischemia: Temporal differences in two species. Stroke 28:1245, 1997.