Microthrombosis after experimental subarachnoid hemorrhage: Time course and effect of red blood cell-bound thrombin-activated pro-urokinase and clazosentan

Abstract

Delayed cerebral ischemia (DCI) is a significant cause of morbidity and mortality for patients surviving the rupture of an intracranial aneurysm. Despite an association between vasospasm and DCI, thrombosis and thromboembolism may also contribute to DCI. In this study we investigate the time course of intravascular microclot formation after experimental subarachnoid hemorrhage (SAH) and assess the effects of the following two drugs on microclot burden: mutant thrombin-activated urokinase-type plasminogen activator (scFv/uPA-T), which is bound to red blood cells for use as a thromboprophylactic agent, and clazosentan, an endothelin antagonist. In the first study, adult male C57BL/6 mice were sacrificed at 24 (n=5), 48 (n=6), 72 (n=8), and 96 (n=3) hours after SAH induced by filament perforation of the anterior cerebral artery. Sham animals (n=5) underwent filament insertion without puncture. In the second study, animals received scFv/uPA-T (n=5) 3 hours after hemorrhage, clazosentan (n=5) by bolus and subcutaneous pump after SAH just prior to skin closure, or a combination of scFv/uPA-T and clazosentan (n=4). Control (n=6) and sham (n=5) animals received saline alone. All animals were sacrificed at 48 hours and underwent intra-cardiac perfusion with 4% paraformaldehyde. The brains were then extracted and sliced coronally on a cryostat and processed for immunohistochemistry. An antibody recognizing thrombin–anti-thrombin complexes was used to detect microclots on coronal slices. Microclot burden was calculated for each animal and compared among groups. Following SAH, positive anti-thrombin staining was detected bilaterally in the following brain regions, in order of decreasing frequency: cortex; hippocampus; hypothalamus; basal ganglia. Few microclots were found in the shams. Microclot burden peaked at 48 hours and then decreased gradually. Animals receiving scFv/uPA-T and scFv/uPA-T+clazosentan had a lower microclot burden than controls, whereas animals receiving clazosentan alone had a higher microclot burden (p<0.005). The overall mortality rate in the time course study was 40%; mortality was highest among control animals in the second study. Intravascular microclots form in a delayed fashion after experimental SAH. Microclots may be safely reduced using a novel form of thromboprophylaxis provided by RBC-targeted scFv/uPA-T and represent a potential target for therapeutic intervention in the treatment of DCI.

The Role of Platelet Activation and Inflammation in Early Brain Injury Following Subarachnoid Hemorrhage

“…In a rat endovascular perforation model of SAH 23 , platelet aggregates have been observed to occur in microvessels within 10 minutes of SAH and continue to increase over the first 48 hours from vessel rupture. 5, 24 Platelet aggregates may cause microthrombosis, ischemia and resultant tissue death. Indeed, endothelial and parenchymal apoptosis and neuronal necrosis occur in animals within 10 minutes of SAH and continue to increase over 24 hours from ictus, concomitant with platelet aggregation within microvessels.…”

Delayed neurological deterioration after subarachnoid haemorrhage

“…mortality. 47 The discrepancy between these experimen tal findings and clinical trial results might be due to the fact that the specific mechanism targeted in the animal experiment was not targeted in the human studies, or because of other limitations of human trials in SAH ( discussed below).…”

Acute Microvascular Changes after Subarachnoid Hemorrhage and Transient Global Cerebral Ischemia

“…The importance of the microthrombi to brain injury and outcome in experimental SAH was suggested in an endovascular perforation model of SAH in mice [53]. The number of microthrombi decreased upon administration of a mutant thrombin-activated urokinase-type plasminogen activator, and this correlated with decreased mortality.…”