Abstract
The Rose Bengal (RB) dye-based photothrombotic stroke (PTS) model has many methodological advantages including consistent location and size of infarct, low mortality, and relatively simple surgical procedures. However, the standard PTS has the caveat of poor responses to tissue-type plasminogen activator (tPA)-mediated lytic treatment, likely as a result of the platelet-rich, fibrin-poor content of the blood clots. Here we tested whether the admixture of thrombin (80 U/kg) and RB dye (50 mg/kg) in the proximal middle cerebral artery (MCA)-targeted PTS will modify the clot composition and elevate the responsiveness to tPA-lytic treatment (Alteplase, 10 mg/kg). Indeed, intravital imaging, immunostaining, and immunoblot analyses showed less-compacted platelet aggregates with a higher fibrin content in the modified thrombin (T) plus RB photothrombotic stroke (T+RB-PTS) model compared with the standard RB-PTS-induced clots. Both RB-PTS and T+RB-PTS showed steady recovery of cerebral blood flow (CBF) in the ischemic border from 1 day after infarction, but without recanalization of the proximal MCA branch. Intravital imaging showed high potency of restoring the blood flow by tPA after single vessel-targeted T+RB-PTS. Further, although intravenous tPA failed to restore CBF or attenuate infarction in RB-PTS, it conferred 25% recovery of CBF and 55% reduction of the infarct size in T+RB-PTS (P < .05) if tPA was administered within 2 hours postphotoactivation. These results suggest that T+RB-PTS produces mixed platelet:fibrin clots closer to the clinical thrombus composition and enhanced the sensitivity to tPA-lytic treatment. As such, the modified photothrombosis may be a useful tool to develop more effective thrombolytic therapies of cerebral ischemia.
Highlights
The search for acute stroke therapies requires multiple preclinical models to address different aspects of the pathological mechanisms
Rose Bengal (RB)-based photothrombosis largely remains a model of permanent ischemia, as it is resistant to the tissue-type plasminogen activator–lytic treatment.[3,4,6]
Mice were randomized for treatments (10 mg/kg Alteplase, 50% injected as a bolus and 50% infused over the course of 30 minutes through tail vein, as previously described19), and the infarct size was analyzed using triphenyl tetrazolium chloride (TTC) stain at 24 hours recovery by investigators who were blinded to the treatment
Summary
The search for acute stroke therapies requires multiple preclinical models to address different aspects of the pathological mechanisms. The photodynamic dye (eg, Rose Bengal [RB])–based photothrombotic stroke (PTS) model has many strengths, including consistent location and size of infarct, relatively simple surgical procedures, and a low mortality rate of animals.[1,2,3,4] The introduction of middle cerebral artery (MCA)–targeted photoactivation (as opposed to aiming the laser beam at a large brain area) further widened the ischemic penumbra to study peri-infarct neovascularization.[5] Yet RB-based photothrombosis largely remains a model of permanent ischemia, as it is resistant to the tissue-type plasminogen activator (tPA)–lytic treatment.[3,4,6] The poor responses to tPA have prevented the application of RB photothrombosis for studying the pathology of transient ischemia and the search for better thrombolytic therapies.
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