Neuron-generated thrombin induces a protective astrocyte response via protease activated receptors.

Astrocytes protect neurons during cerebral injury through several postulated mechanisms. Recent therapeutic attention has focused on enhancing or augmenting the neuroprotective actions of astrocytes but in some instances astrocytes can assume a neurotoxic phenotype. The signaling mechanisms that drive astrocytes toward a protective versus toxic phenotype are not fully known but cell-cell signaling via proteases acting on cell-specific receptors underlies critical mechanistic steps in neurodevelopment and disease. The protease activated receptor (PAR), resides in multiple brain cell types, and most PARs are found on astrocytes. We asked whether neuron-generated thrombin constituted an important astrocyte activation signal because our previous studies have shown that neurons contain prothrombin gene and transcribed protein. We used neuron and astrocyte mono-cell cultures exposed to oxygen-glucose deprivation and a model of middle cerebral artery occlusion. We found that ischemic neurons secrete thrombin into culture media, which leads to astrocyte activation; such astrocyte activation can be reproduced with low doses of thrombin. Media from prothrombin-deficient neurons failed to activate astrocytes and adding thrombin to such media restored activation. Astrocytes lacking PAR1 did not respond to neuron-generated thrombin. Induced astrocyte activation was antagonized dose-dependently with thrombin inhibitors or PAR1 antagonists. Ischemia-induced astrocyte activation in vivo was inhibited after neuronal prothrombin knockout, resulting in larger strokes. Restoring prothrombin to neurons with a lentiviral gene vector restored astrocyte activation and reduced stroke damage. We conclude that neuron-generated thrombin, released during ischemia, acts via PAR1 and may cause astrocyte activation and paracrine neuroprotection.

Neuroprotection and vasculoprotection using genetically targeted protease-ligands.

Thrombin and activated protein C (APC) are known coagulation factors that exhibit profound effects in brain by acting on the protease activated receptor (PAR). The wild type (WT) proteases appear to impact cell survival powerfully, and therapeutic forms of APC are under development. Engineered recombinant thrombin or APC were designed to separate their procoagulant or anticoagulant effects from their cytoprotective properties. We measured vascular disruption and neuronal degeneration after a standard rodent filament stroke model. For comparison to a robust anticoagulant, we used a GpIIb/IIIa inhibitor, GR144053. During 2 h MCAo both WT murine APC and its mutant, 5A-APC, significantly decreased neuronal death 30 min after reperfusion. During 4 h MCAo, only 5A-APC significantly protected neurons but both WT-APC and 5A-APC exacerbated vascular disruption during 4 h MCAo. Human APC mutants appeared to reduce 24 h neuronal injury significantly when given after 2 h delay after MCAo. In contrast, 24 h vascular damage was worsened by high doses of WT and mutant APCs, although only statistically significantly for high dose 3K3A-APC. Mutated thrombin worsened vascular damage significantly without affecting neuron damage. GR144053 failed to ameliorate vascular disruption or neuronal injury despite significant anticoagulation. Differential effects on neurons and the vasculature were demonstrated using wild-type and mutated proteases. The mutants murine 3K3A-APC and 5A-APC protected neurons in this rodent model but in high doses worsened vascular leakage. Cytoactive effects of plasma proteases may be separated from their coagulation effects. Further studies should explore impact of dose and timing on cytoactive and vasculoactive properties of these drugs.

Direct thrombin inhibitor argatroban reduces stroke damage in 2 different models.

BACKGROUND AND PURPOSE: We showed previously robust neuroprotection with the thrombin inhibitor argatroban and now sought additional support for its neuroprotective potential. METHODS: We used behavioral and histological end points; rigorously blinded the study groups; extended the treatment window to 3 hours after ischemia onset; and used 2 separate models. First, 2-hour filament middle cerebral artery occlusion in 64 male Sprague-Dawley rats was followed by learning and memory testing and quantitative histomorphometry. Randomly assigned treatment was 0.45 mg argatroban, saline, or 0.4 U thrombin. Second, we used the quantal bioassay (n=272) after 2-hour middle cerebral artery occlusion to detect the longest time delay after which therapy failed. RESULTS: Argatroban powerfully and significantly reversed learning and memory deficits because of focal ischemia compared with saline or thrombin (P<0.03; ANOVA). Argatroban was significantly (P<0.05; t test with Bonferroni) protective when given immediately or after 1, 2, 3, but not 4 hours delay. CONCLUSIONS: We obtained supportive evidence for argatroban protection of the neurovascular unit using behavioral and histological measurements at realistic therapeutic time windows.

Clinical use of computed tomographic perfusion for the diagnosis and prediction of lesion growth in acute ischemic stroke.

BACKGROUND: Computed tomography perfusion (CTP) mapping in research centers correlates well with diffusion-weighted imaging (DWI) lesions and may accurately differentiate the infarct core from ischemic penumbra. The value of CTP in real-world clinical practice has not been fully established. We investigated the yield of CTP-derived cerebral blood volume (CBV) and mean transient time (MTT) for the detection of cerebral ischemia and ischemic penumbra in a sample of acute ischemic stroke (AIS) patients. METHODS: We studied 165 patients with initial clinical symptoms suggestive of AIS. All patients had an initial noncontrast head CT, CTP, CT angiogram (CTA), and follow-up magnetic resonance imaging (MRI) of the brain. The obtained perfusion images were used for image processing. CBV, MTT, and DWI lesion volumes were visually estimated and manually traced. Statistical analysis was conducted using R and SAS software. RESULTS: All normal DWI sequences had normal CBV and MTT studies (N = 89). Seventy-three patients had acute DWI lesions. CBV was abnormal in 23.3% and MTT was abnormal in 42.5% of these patients. There was a high specificity (91.8%) but poor sensitivity (40.0%) for MTT maps predicting positive DWI. The Spearman correlation was significant between MTT and DWI lesions (ρ = 0.66; P > .0001) only for abnormal MTT and DWI lesions >0 cc. CBV lesions did not correlate with final DWI. CONCLUSIONS: In real-world use, acute imaging with CTP did not predict stroke or DWI lesions with sufficient accuracy. Our findings argue against the use of CTP for screening AIS patients until real-world implementations match the accuracy reported from specialized research centers.

Concurrent middle cerebral artery occlusion and intra-arterial drug infusion via ipsilateral common carotid artery catheter in the rat.

Pre-clinical development of therapy for acute ischemic stroke requires robust animal models; the rodent middle cerebral artery occlusion (MCAo) model using a nylon filament inserted into the internal carotid artery is the most popular. Drug screening requires targeted delivery of test substance in a controlled manner. To address these needs, we developed a novel method for delivering substances directly into the ischemic brain during MCAo in the awake rat. An indwelling catheter is placed in the common carotid artery ipsilateral to the occlusion at the time of the surgical placement of the occluding filament. The internal and common carotid arteries are left patent to allow superfusion anterograde. The surgeries can be completed quickly to allow rapid recovery from anesthesia; tests substances can be infused at any given time for any given duration. To simulate clinical scenarios, the occluding filament can be removed minutes or hours later (reperfusion) followed by therapeutic infusions. By delivering drug intra-arterially to the target tissue, "first pass" loss in the liver is reduced and drug effects are concentrated in the ischemic zone. To validate our method, rats were infused with Evans blue dye either intra-arterially or intravenously during a 4 h MCAo. After a 30 min reperfusion period, the dye was extracted from each hemisphere and quantitated with a spectrophotometer. Significantly more dye was measured in the ischemic hemispheres that received the dye intra-arterially.

Thrombin activity associated with neuronal damage during acute focal ischemia.

Mechanisms of ischemic neuronal and vascular injury remain obscure. Here we test the hypothesis that thrombin, a blood-borne coagulation factor, contributes to neurovascular injury during acute focal ischemia. Stroke was induced in adult Sprague Dawley rats by occluding the middle cerebral artery. Intra-arterial thrombin infusion during ischemia significantly increased vascular disruption and cellular injury. Intravenous infusion of argatroban, a direct thrombin inhibitor, alleviated neurovascular injury. Immunostaining showed thrombin on neurons in the ischemic core. Using an activatable cell-penetrating peptide engineered to detect thrombin activity, we discovered that thrombin proteolytic activity was specifically associated with neuronal damage during ischemia. Protease activated receptor-1, the presumptive thrombin receptor, appeared to mediate ischemic neurovascular injury. Furthermore, rats receiving thrombin during ischemia showed cognitive deficit, whereas rats receiving argatroban retained intact learning and memory. These results suggest a potential role for thrombin contributing to neurovascular injury and several potential avenues for neuroprotection.