Bryostatin improves survival and reduces ischemic brain injury in aged rats after acute ischemic stroke - PubMed (original) (raw)

Bryostatin improves survival and reduces ischemic brain injury in aged rats after acute ischemic stroke

Zhenjun Tan et al. Stroke. 2013 Dec.

Abstract

Background and purpose: Bryostatin, a potent protein kinase C (PKC) activator, has demonstrated therapeutic efficacy in preclinical models of associative memory, Alzheimer disease, global ischemia, and traumatic brain injury. In this study, we tested the hypothesis that administration of bryostatin provides a therapeutic benefit in reducing brain injury and improving stroke outcome using a clinically relevant model of cerebral ischemia with tissue plasminogen activator reperfusion in aged rats.

Methods: Acute cerebral ischemia was produced by reversible occlusion of the right middle cerebral artery (MCAO) in 18- to 20-month-old female Sprague-Dawley rats using an autologous blood clot with tissue plasminogen activator-mediated reperfusion. Bryostatin was administered at 6 hours post-MCAO, then at 3, 6, 9, 12, 15, and 18 days after MCAO. Functional assessment was conducted at 2, 7, 14, and 21 days after MCAO. Lesion volume and hemispheric swelling/atrophy were performed at 2, 7, and 21 days post-MCAO. Histological assessment of PKC isozymes was performed at 24 hours post-MCAO.

Results: Bryostatin-treated rats showed improved survival post-MCAO, especially during the first 4 days. Repeated administration of bryostatin post-MCAO resulted in reduced infarct volume, hemispheric swelling/atrophy, and improved neurological function at 21 days post-MCAO. Changes in αPKC expression and εPKC expression in neurons were noted in bryostatin-treated rats at 24 hours post-MCAO.

Conclusions: Repeated bryostatin administration post-MCAO protected the brain from severe neurological injury post-MCAO. Bryostatin treatment improved survival rate, reduced lesion volume, salvaged tissue in infarcted hemisphere by reducing necrosis and peri-infarct astrogliosis, and improved functional outcome after MCAO.

Keywords: aging; blood-brain barrier; infarction; protein kinase C; tissue plasminogen activator.

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Figures

Figure 1

Figure 1

A.) Administration of bryostatin increased the survival rate of aged rats from 2 to 17 d post-MCAO and tPA reperfusion. B.) Neurological function was improved in bryostatin-treated aged rats at 21 d post-MCAO. No difference in neurological score between saline and bryostatin treated rats was measured at 2, 7, and 14 d post-MCAO. (C.) Latency speed and (D.) distance traveled were measured at 21 d post-MCAO. Results showed a reduction in latency speed and distance traveled in bryostatin-treated rats as compared to saline-treated rats. Both treatment groups demonstrated a reduction in distance traveled when using the 300% platform. Data presented as mean ± SEM.

Figure 2

Figure 2

A.) Repeated administration of bryostatin decreased lesion volume in striatum and total hemisphere at 7 post-MCAO. At 21 d following MCAO, lesion volume was reduced in the cortex, striatum, and total hemisphere. No difference in lesion volume was measured at 2 d post-MCAO. B.) Bryostatin reduced hemispheric swelling at 2 and 7 d following MCAO. C.) Bryostatin reduced cerebral atrophy in the ischemic hemisphere as compared to saline-treated rats at 21 d post-MCAO. Data presented as mean ± SEM.

Figure 3

Figure 3

A.) Representative photomicrographs of GFAP-staining positive cells at 7 and 21 d following MCAO. Astrogliosis was observed in saline-treated brain at 7 and 21 d following MCAO, bryostatin mitigated the degree of astrogliosis. B.) At 21 d after MCAO, cavitation and necrosis was evident in saline-treated rats. Administration of bryostatin decreased lesion volume and reduced atrophy as compared to saline-treated rats. Cell nuclei were stained using DAPI.

Figure 4

Figure 4

Comparison of TUNEL staining in aged rats showed a marked reduction in TUNEL positive cells in bryostatin-treated rats at 7 and 21 d post-MCAO as compared to saline-treated rats. Cell nuclei were stained using DAPI.

Figure 5

Figure 5

Representative images of tri-color confocal immunofluorescent microscopy demonstrating colocalization of NeuN with αPKC (A) and εPKC (B) in the penumbra of the infarcted hemisphere of saline and bryostatin treated aged rats at 24 h after MCAO. (C) In neurons, a 68% decrease in αPKC expression and a 202% increase in εPKC expression were measured at 24 h following MCAO in bryostatin treated rats as compared to saline-treated rats. Data expressed as mean ± SEM, * p<0.05. Cell nuclei were stained using DAPI.

Figure 6

Figure 6

Representative images of tri-color confocal immunofluorescent microscopy demonstrating colocalization of CD31 (endothelial cells) with αPKC (A) and εPKC (B) in the penumbra of the infarcted hemisphere of saline and bryostatin treated aged rats at 24 h after MCAO. (C) In endothelial cells, no difference (p>0.05) in αPKC and εPKC expression were observed at 24 h following MCAO between bryostatin- and saline-treated rats. Data expressed as mean ± SEM, *p<0.05. Cell nuclei were stained using DAPI.

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