Comparative dose of intracarotid autologous bone marrow mononuclear therapy in chronic ischemic stroke in rats

BACKGROUND: Research on chronic ischemic stroke is limited. One of the more promising approaches showing positive effects in the acute stage is mononuclear bone marrow cell therapy. This research may be the first which presents data about the optimum dose of bone marrow mononuclear cells (BM-MNCs) for chronic ischemic stroke in rats and discusses factors influencing recovery in the chronic stage. AIM: To elucidate the optimum dose of BM-MNCs for chronic ischemic stroke and to demonstrate factors influencing recovery in chronic stage of stroke ischemia. METHODS: Thirty-two male Sprague-Dawley rats sourced from the Kalbe Farma Institution (Bandung, Indonesia), aged 6–10 aged months, weighing 350–450 g were used in this study. We performed temporary middle cerebral artery occlusion (MCAO) procedures on the rats which were then randomly assigned to one of two experimental groups in which they were given either low or high doses of autologous BM-MNCs (5 million or 10 million cells per kg body weight intracarotid), after 4 week of MCAO. At 8^th or 12 week, rats were necropsied and rat brains were fixed for HE, cluster of differentiation (CD) 31, and doublecortin staining for analysis of the effects. Rat behavior was assessed weekly using the cylinder test and a modified neurological severity score (NSS) test. Cylinder test scores and NSS scores were analyzed by one-way ANOVA repeated measures and post hoc Bonferroni. The size of the infarct zone, the CD 31 vessels, and the DCX-neuroblast were analyzed using one-way ANOVA and a post hoc Bonferroni test. To investigate the degree of correlation between time and dose, two-way ANOVA and simple mass effect analyses were conducted. A linear regression test was used to evaluate the correlation between CD34 and other variables. RESULTS: In the 4 weeks before administration of BM-MNC, cylinder test scores improved to near normal, and NSS test scores improved moderately. The infarct zone decreased significantly (p < 0.01), there was an improvement in angiogenesis (p = 0.1590) and a significant improvement in neurogenesis (p < 0.01). Reduction of the infarct zone was associated with a higher dose whereas both higher and lower doses were found to have a similar effect on improving angiogenesis, and neurogenesis. Recovery was superior after 12 weeks compared with the recovery assessment at 8 weeks. CONCLUSION: A dose of 10 million cells was more effective than a dose of 5 million cells per kg body weight for reducing the infarct zone and ameliorating neurogenesis. There was an improvement of histopathological parameters associated with the longer infarct period.