Thrombopoietin Increases Peri-infarct Angiogenesis in Restoring Post-Stroke Sensory-Motor and Cognitive Functioning

Abstract

Ischemic stroke is a major cause of long-term sensory, motor, and cognitive deficits. Although recent clinical demonstrations of improved outcome following early intravascular clot removal, still only about 5% of stroke patients are able to meet the 4.5-hour time window for acute stroke intervention using tissue plasminogen activator (tPA), the only available FDA approved treatment for stroke. Clearly there is a need for new interventions that can improve post-stroke outcome when administered early or late post-stroke. Previously it has been shown that thrombopoietin (TPO), a hematopoietic growth factor, provides significant brain protection in rat experimental stroke using an optimum i.v. dose of 0.1 mg/kg. TPO administered to rats up to 4 hours after middle cerebral artery occlusion (MCAO) significantly decreased post-stroke brain infarction, swelling, inflammatory cytokines, blood brain barrier disruption and neurological deficits.

Here, TPO administration was delayed until after brain injury had fully evolved in order to determine if TPO can restore lost brain function and increase brain angiogenesis post-stroke. Male Sprague-Dawley rats underwent transient (2 hour) MCAO (stroke) and were studied for 1, 2, or 4 weeks. Vehicle or TPO (the optimum brain protective dose of 0.1mg/kg, i.v.) was administered at 1 and 4 days post-stroke (i.e., after brain injury had occurred). Sensory-motor and active place avoidance (APA; a cognitive task very sensitive to brain injury) performance was measured. Peri-infarct immunohistochemistry for CD31 (endothelial cell marker), vascular endothelial growth factor (VEGF), and double labeling of angiopoietin-1 with platelet derived growth factor receptor b (PDGFRb; pericyte specific marker) measured changes in microvessels and angiogenesis. In addition, peri-infarct MRI arterial spin labeling (ASL) measured blood flow at 4 weeks.

Results indicated that sensory-motor deficits improved significantly (p<0.001) over 4 weeks in TPO- compared to vehicle-treated rats. Although APA baseline performance prior to stroke was similar, post-stroke TPO treated rats performed the APA task at 4 weeks with significantly fewer errors (p<0.05) compared to vehicle treated rats that failed to learn the task. Following TPO, CD31-staining of microvascular endothelium verified increased cortical peri-infarct microvessels throughout the forebrain over 4 weeks (p<0.01). TPO also significantly increased peri-infarct cellular labeling of VEGF at 1 and 2 week post-stroke (p<0.05) and angiopoietin-1 in PDGFRb-labeled pericytes at 2 weeks (p<0.01), thus demonstrating the progression of angiogenesis that resulted in TPO-induced microvessel proliferation. ASL differences indicated increased peri-infarct blood flow 4 weeks post-stroke (p<0.05), thus demonstrated functioning of the TPO-induced microvessels.

In summary, delayed TPO administration significantly restores long-term sensory-motor and cognitive function and increases angiogenesis. Elevated blood flow in peri-infarct regions is due to the angiogenic effects of TPO, defined by increased VEGF in pericytes and astrocytes, and angiopoietin-1 in pericytes. Thus, TPO could be a promising drug for post-stroke recovery.