G. J. Del Zoppo

Polymorphonuclear leukocytes occlude capillaries following middle cerebral artery occlusion and reperfusion in baboons.

Background and Purpose Microvascular perfusion defects may accompany sustained occlusion and subsequent reperfusion of the middle cerebral artery; however, the nature of such “no-reflow” defects remains unclear. Methods In the absence of antithrombotic pretreatment, we documented lenticulostriatal microvascular flow integrity following 3-hour middle cerebral artery occlusion and 1-hour reperfusion in a baboon occlusion/reperfusion model by two methods identifying 1) microvascular occlusion and 2) microvascular patency. Results Microvascular “no-reflow” involved capillaries (vessels of 4.0-7.5 μm diameter) of the lenticulostriatal territory. Capillary reflow included 27-39% of all capillaries in two subjects, indicating a significant reduction of perfusion from normal (2p=0.045). In identical experimental preparations, single polymorphonuclear leukocytes completely occluded 4.7% of microvessels of capillary diameter in randomly selected fields, partially occluded 33% of postcapillary venules, and occluded 40% (four of 10) of capillaries in linear reconstruction along a 110 μ length. Circumferential contact between polymorphonuclear leukocytes and the luminal endothelial cell membranes was documented, with an intercellular gap of, at most, 160 nm. Fibrin was found with degranulated platelets when the latter were associated with granulocytes, but not with polymorphonuclear leukocytes alone. Conclusions The rinding of capillary-obstructing polymorphonuclear leukocytes in the microvascular bed following middle cerebral artery reperfusion in focal ischemia in this model satisfies an essential requirement for postulating their role in early microvascular injury and the “no-reflow” phenomenon.

P-selectin and intercellular adhesion molecule-1 expression after focal brain ischemia and reperfusion.

Background and Purpose Polymorphonuclear leukocytes have been implicated in the development of the “no-reflow” phenomenon after focal cerebral ischemia and reperfusion. To further understand the role of granulocytes in microvascular occlusions, the responses of the granulocyte-endothelial cell adhesion molecules P-selectin and intercellular adhesion molecule-1 during middle cerebral artery ischemia and reperfusion were examined in a primate model. Methods Twelve adolescent male baboons were used for 2-hour middle cerebral artery occlusion (n=3) or for 3-hour occlusion with 1-hour (n=3), 4-hour (n=3), and 24-hour (n=3) reperfusion, and three separate unoperated primates served as controls. A quantitative immunohistochemical study of the microvascular distribution of P-selectin and intercellular adhesion molecule-1 was performed using 10-μm frozen sections from basal ganglia analyzed with computerized light microscopy video imaging. Results Significant (P <.05) persistent upregulation of P-selectin (beginning during ischemia) and transient upregulation of intercellular adhesion molecule-1 (at 1 and 4 hours of reperfusion) were observed on endothelium of selected post-capillary microvessels of the ischemic lenticulostriate artery territory. Platelet accumulation also occurred in this territory and was responsible for a significant proportion of the nonendothelial P-selectin signal at 24 hours after reperfusion. Conclusions Focal cerebral ischemia/reperfusion stimulates endothelial P-selectin and intercellular adhesion molecule-1 expression in brain microvessels in the ischemic zone, which may contribute to enhanced leukocyte adherence and persistent activation.

Inhibition of polymorphonuclear leukocyte adherence suppresses no-reflow after focal cerebral ischemia in baboons

Background and Purpose While polymorphonuclear leukocytes may contribute to the “no-reflow” phenomenon after focal cardiac and skeletal muscle ischemia/reperfusion, their contribution to acute focal cerebral ischemia is unresolved. We have examined the role of polymorphonuclear leukocytes in microvascular perfusion defects after focal cerebral ischemia/reperfusion in a baboon model of reversible middle cerebral artery occlusion with the anti-CD18 monoclonal antibody IB4, which inhibits neutrophil adherence to endothelium. Methods Microvascular patency in the basal ganglia after 3-hour middle cerebral artery occlusion and 1-hour reperfusion (by india ink tracer perfusion) was quantified by computerized video imaging. Animals were randomized to receive intravenous IB4 infusion 15 minutes before reperfusion (n=7) or to receive no treatment (n=6). Binding of IB4 to baboon leukocytes was maximal within 5 minutes of infusion. Results In the untreated group, a significant reduction in patency was observed in mlcrovessels <30 μn diameter mean percent reflow was 51% in the capillary diameter class (4.0–7.5 μm) and 39% in the precapillary arteriole and postcapillary venule diameter class (7.5–30 μm). Infusion of IB4 before middle cerebral artery reperfusion increased reflow in microvessels of all size classes, most significantly in those 7.5–30 μm (p=0.049) and 30–50 μm (p=0.034) in diameter. Conclusions These results suggest that CD18-mediated polymorphonuclear leukocyte-endothelium adherence contributes to no-reflow predominantly in noncapillary microvessels and at least partially to that in capillaries.

Treatment of Acute Ischemic Stroke Challenging the Concept of a Rigid and Universal Time Window

Acute stroke treatment has recently become a major area for clinical trials. Each year, thousands of patients with first-ever middle cerebral artery (MCA) territory stroke are enrolled in therapeutic trials, testing the hypothesis that the investigational treatment might improve functional outcome. In addition to trials of therapeutic thrombolysis, other classes of agents such as glutamate antagonists, free radical scavengers, anticytokines, and calcium channel blockers are presently being tested for therapeutic efficacy. Whereas most trials in the 1970s and 1980s enrolled patients within 24 or even 48 hours after stroke onset, at the present time it would be almost unthinkable to extend the time of inclusion beyond the “magic limit” of 6 hours. There are four reasons for this: First, experiments conducted in the nonhuman primate that led to the concept of the ischemic penumbra clearly documented that “time is brain,” with apparently only little penumbra remaining beyond 3 hours.1 2 Second, thus far many clinical trials have reported negative, only marginally significant, and/or positive but poorly reproducible findings; late inclusion of patients was considered to be responsible for treatment failure.3 4 Third, recent studies have demonstrated that it is possible to have stroke patients seen in the hospital within 3 to 6 hours after onset.5 6 Fourth, the success of intravenous therapeutic thrombolysis in less than 6 hours after myocardial infarction stimulated a similar design for acute stroke.7 For organizational reasons it has been operationally justified to apply a rigid time window for screened patients in multicenter, international trials. Furthermore, it is reasonable to consider that a cohort of stroke patients recruited within certain time limits may be more homogeneous. We would like to examine this view and call into question that therapy in individual patients within the 6-hour time limit would necessarily …

Ischaemic damage of brain microvessels: inherent risks for thrombolytic treatment in stroke

Recent trials of plasminogen activators in acute ischaemic stroke underscore the delicate balance between promise of benefit and risk. Attempts to manage clinical outcome by systemic infusion of recombinant tissue plasminogen activator (rt-PA) have so far had mixed success.1 2 The benefits seen in the National Institutes of Neurological Diseases and Stroke (NINDS) trial of rt-PA in acute ischaemic stroke were a significant absolute improvement in disability outcome.2 3 However, the risks in that study and in the European Cooperative Acute Stroke Study (ECASS)1 included further disability and mortality associated with haemorrhagic consequences of the plasminogen activator (PA).1 2 Three recent studies of streptokinase in acute ischaemic stroke were terminated because of safety concerns.4-9 In all five trials, the risk of symptomatic haemorrhage associated with the PA was significantly increased over placebo (table 1). In addition, approaches which effect recanalisation of documented cerebral arterial thrombotic occlusions by intravenous or intra-arterial PA infusion have yet to be rigorously shown to promote overall benefit, although anecdotal evidence suggests that individual patients may improve clinically.10-24 The reasons are practical. The perceived risks, those of angiography and the interventional procedures required for intra-arterial PA delivery, have not been fully evaluated,25 although the relative risks of diagnostic angiography are low.26-28 Clearly, this assessment hides many important differences in PA behaviour, study design and conduct, patient populations, diagnostic and therapeutic procedures, disease severity, comorbidity, and a host of other potential contributors. None the less, concerns which may erode the promise of benefit of thrombolytic agents in acute ischaemic stroke include the accentuation of innate risks associated with the evolution of ischaemic injury after the stroke event itself.29-34 These include early mortality caused by severe brain oedema and the development of haemorrhagic transformation which causes clinical deterioration or …

Fibrin contributes to microvascular obstructions and parenchymal changes during early focal cerebral ischemia and reperfusion.

Ischemic cerebral injury is associated with activation of the blood coagulation cascade. To elucidate the contribution of fibrin formation to microvascular injury during focal cerebral ischemia and reperfusion, we have studied the time course and the localization of fibrin deposition in cerebral microvessels and the surrounding tissues during ischemia/reperfusion in a well-described nonhuman primate model. Methods Cerebral tissues from adolescent male baboons were examined after 2-hour middle cerebral artery occlusion (n=3) and after 3 hours of middle cerebral artery occlusion and 1-hour (n=6), 4-hour (n=3), and 24-hour (n=4) reperfusion; tissues from control primates (n=3) also were examined. Fibrin deposition was detected by immunohistochemical techniques using the fibrin-specific monoclonal antibody MH-1. The number and size distribution of microvessels associated with fibrin were quantified by video-imaging microscopy. Results Fibrin was associated with microvessels only in the ischemic zone where severe neuronal injury was documented, its frequency increasing with the reperfusion period (F4.26=3.80, P<.05). Extravascular fibrin deposition was significantly increased by 24-hour reperfusion compared with the other subjects (P<.05). Preischemia infusion of the anti-tissue factor monoclonal antibody TF9-6B4 resulted in significant reduction of intramicrovascular fibrin (P<.038 versus no intervention) at 1-hour reperfusion but had no effect on extravascular fibrin deposition. Conclusions These results suggest that microvascular fibrin deposition accumulates in a time-dependent manner during focal cerebral ischemia/reperfusion and that exposure of plasma to perivascular tissue factor is partially responsible for occlusion formation. During ischemia the large plasma protein fibrinogen extravasates and interacts with parenchymal tissue factor, forming significant extravascular fibrin by 24 hours of reperfusion.

Experimental acute thrombotic stroke in baboons.

To study the effects of antithrombotic therapy in experimental stroke, we have characterized a baboon model of acute cerebrovascular thrombosis. In this model an inflatable silastic balloon cuff has been implanted by transorbital approach around the right middle cerebral artery (MCA), proximal to the take-off of the lenticulostriate arteries (LSA). Inflation of the balloon for 3 hours in six animals produced a stereotypic sustained stroke syndrome characterized by contralateral hemiparesis. An infarction volume of 3.2 +/- 1.5 cm3 in the ipsilateral corpus striatum was documented by computerized tomographic (CT) scanning at 10 days following stroke induction and 3.9 +/- 1.9 cm3 (n = 4) at 14 days by morphometric neuropathologic determinations of brain specimens fixed in situ by pressure-perfusion with 10% buffered formalin. Immediate pressure-perfusion fixation following deflation of the balloon was performed in 16 additional animals given Evans blue dye intravenously prior to the 3 hour MCA balloon occlusion. Light microscopy and transmission electron microscopy consistently confirmed the presence of thrombotic material occluding microcirculatory branches of the right LSA in the region of Evans blue stain, but not those of the contralateral corpus striatum. When autologous 111In-platelets were infused intravenously in four animals from the above group prior to the transient 3 hour occlusion of the right MCA, gamma scintillation camera imaging of each perfused-fixed whole brain demonstrated the presence of a single residual focus of 111In-platelet activity involving only the Evans blue-stained right corpus striatum. Focal right hemispheric activity was equivalent to 0.55 +/- 0.49 ml of whole blood, and the occlusion score derived from histologic examination of the microcirculation of the Evans blue-stained corpus striatum averaged 34.8 +/- 2.8. Similar 111In-platelet imaging and histologic scoring experiments carried out in four animals pretreated with the antithrombotic combination heparin and ticlopidine showed marked reduction of both 111In-platelet activity (0.01 +/- 0.03 ml vs. 0.55 +/- 0.49 ml; p less than 0.01) and thrombotic occlusion of the microcirculation (10.8 +/- 7.4 units vs. 34.8 +/- 2.8 units; p less than 0.01) in the right corpus striatum following 3 hours of MCA occlusion. In separate control experiments 111In-labeled autologous platelets were infused after the 3 hour period of right MCA occlusion and subsequent balloon deflation in two animals; no focus of 111In-platelet activity was demonstrated in fixed whole brain.(ABSTRACT TRUNCATED AT 400 WORDS)

The beneficial effect of intracarotid urokinase on acute stroke in a baboon model.

The capacity of intracarotid infusion of urokinase to salvage neurologic function in a baboon model of acute thrombotic stroke has been studied. The model consists of reversible eccentric balloon compression (3 hours) of the right middle cerebral artery (MCA) proximal to the take-off of the lenticulostriate arteries (LSA), resulting in in situ thrombosis of perforating branches supplying the right corpus striatum. Neurologic endpoints included quantitative assessment of neurologic function (NE), estimation of cerebral infarction volume by computerized tomographic (CT) scan, and carotid angiography. In untreated acute stroke control animals (n = 6), a persistent decrease in functional score (from 100 to 36 +/- 11) at 14 days and a defined region of cerebral infarction (volume = 3.2 +/- 1.5) were detected at 10 days. Intracarotid urokinase administered to five animals (1.2 X 10(6) IU over 60 min) following the 3 hour period of MCA occlusion improved neurologic function (NE = 50, 55, 85, 100, 100) and reduced infarction size (0.3, 0.5, 0.8, 0.7, 1.1 cm3, respectively) without evidence of intracranial hemorrhage. Systemic fibrinogenolysis was produced in all five treated animals. We conclude that thrombolytic therapy given within 3 hours of experimental thrombotic occlusion may salvage neurologic function and reduce cerebral infarction volume without CT scan detectable intracranial bleeding.

Tissue factor contributes to microvascular defects after focal cerebral ischemia.

Background and Purpose Microvascular perfusion defects occur after occlusion and reperfusion of the middle cerebral artery in examples of focal cerebral ischemia. In addition to cellular (eg, polymorphonuclear leukocyte) contributors to the focal “no-reflow” phenomenon, activation of coagulation may also play a role. We have tested a potential role of tissue factor-mediated coagulation in the microvascular perfusion defects seen after focal cerebral ischemia-reperfusion in a baboon model of reversible middle cerebral artery occlusion with the murine anti-tissue factor monoclonal antibody TF9-6B4. Tissue factor is the principal resident procoagulant substance in cerebral tissues and has a distinct perivascular distribution. Methods Microvascular patency in the basal ganglia after 3-hour middle cerebral artery occlusion and 1-hour reperfusion was quantified by computerized video imaging of carbon-tracer perfused tissues. Animals were randomized to receive intravenous TF9-6B4 (10 mg/kg) 10 minutes before middle cerebral artery occlusion (n=6) or no treatment (n=6) in an open study. Results In the control animals, a significant decrease in patency was confirmed in microvessels less than 30 μUm in diameter. Infusion of TF9-6B4 before middle cerebral artery occlusion produced a stable maximal level of circulating antibody within 10 minutes, which lasted the duration of ischemia and reperfusion. An increase in reflow in microvessels of all size classes occurred after TF9-6B4 infusion, which was significant in those 7.5 to 30 μUm (P=.038) and 30 to 50 μUm (P=.013) in diameter. Conclusions These results indicate that tissue factor-mediated events may also contribute to no-reflow in noncapillary microvessels after focal cerebral ischemia.

Vascular matrix adhesion and the blood-brain barrier

The integrity of the cerebral microvasculature depends on the interaction between its component cells and the extracellular matrix, as well as reorganized cell-cell interactions. In the central nervous system, matrix adhesion receptors are expressed in the microvasculature and by neurons and their supporting glial cells. Cells within cerebral microvessels express both the integrin and dystroglycan families of matrix adhesion receptors. However, the functional significance of these receptors is only now being explored. Endothelial cells and astrocytes within cerebral capillaries co-operate to generate and maintain the basal lamina and the unique barrier functions of the endothelium. Integrins and the dystroglycan complex are found on the matrix-proximate faces of both endothelial cells and astrocyte end-feet. Pericytes rest against the basal lamina. In the extravascular compartment, select integrins are expressed on neurons, microglial cells and oligodendroglia. Significant alterations in both cellular adhesion receptors and their matrix ligands occur during focal cerebral ischaemia, which support their functional significance in the normal state. We propose that matrix adhesion receptors are essential for the maintenance of the integrity of the blood-brain permeability barrier and that modulation of these receptors contributes to alterations in the barrier during brain injury.