Comparison of the therapeutic effects of bone marrow mononuclear cells and microglia for permanent cerebral ischemia

Abstract

In this study we transplanted bone marrow mononuclear cells (BM-MNCs) or microglia into rats that had undergone permanent cerebral ischemia and observed the distribution or morphology of transplanted cells in vivo. In addition, we compared the effects of BM-MNCs and microglia on infarct volume, brain water content, and functional outcome after permanent cerebral ischemia. BM-MNCs and microglia were obtained from femur and brain, respectively, of newborn rats. Adult rats were injected with vehicle or 3 million BM-MNCs or microglia via the tail vein 24h after permanent middle cerebral artery occlusion (pMCAO). The distribution or morphologic characteristics of transplanted BM-MNCs (double stained with BrdU/Cd34 or BrdU/CD45) and microglia (double stained with BrdU/Iba-1) were detected with immunofluorescent staining at 3 or 7 and 14 days after pMCAO. Functional deficits were assessed by the modified neurologic severity score at 1, 3, 7 and 14 days after pMCAO. Brain water content was assessed at 3 days, and infarct volume was determined at 14 days. We observed more BrdU/CD45 and BrdU/Iba-1 double-stained cells than BrdU/CD34 double-stained cells around the infarcted area. Some infused microglia showed the morphology of innate microglia at 7 days after pMCAO, and the number increased at 14 days. BM-MNC-treated rats showed significantly reduced infarct volume and brain water content compared to vehicle- and microglia-treated rats. In addition, BM-MNC treatment reduced neurologic deficit scores compared to those in the other groups. The results provide evidence that infusion of BM-MNCs, but not microglia, is neuroprotective after permanent cerebral ischemia.

Fig. 1

Migration of microglia and BM-MNCs in vivo. Immunofluorescence staining for BrdU-labeled cells revealed that both microglia (A) and BM-MNCs (B) migrated to the boundary zone of the injured cortex in rats after infusion via the tail vein on day 3 after pMCAO. There are more CD45-positive cells (D) around the infarcted area than CD34 cells (C) (n = 4 rats per group). Scale bar = 50 μm.

Fig. 2

The number of transplanted microglia and BM-MNCs in vivo. There are more BrdU/double-stained CD45 and Iba-1 cells than BrdU/double-stained CD34 around the infarcted area at 3 days after pMCAO (A–I). Scale bar = 50 μm.

Fig. 3

The morphologic characteristics of transplanted primary microglia in vivo. The transplanted primary microglia did not show a ramified or an activated/amoeboid morphology at 3 days after pMCAO and only a few of them show a ramified or an activated/amoeboid morphology from 7 days after pMCAO. More infused primary microglia can be detected to show a ramified or an activated/amoeboid morphology (A–I). Scale bar = 50 μm.

Fig. 4

Effect of BM-MNC and microglia treatment on brain water content. Statistical analysis showed significant difference among the five groups (F = 7.37, n = 6 rats per group, P < 0.001). Post hoc analysis revealed that only BM-MNC treatment produced a significant reduction in brain water content. *P < 0.05 vs. untreated, vehicle-treated, and microglia-treated groups.

Fig. 5

Treatment with BM-MNCs reduces infarct volume after pMCAO. (A) Representative images of TTC-stained brain slices from vehicle-treated (top), microglia-treated rats (middle) and BM-MNC-treated rats (bottom) at 14 days post-pMCAO. (B) Quantification shows that the infarct volume of BM-MNC-treated rats was significantly smaller than that of untreated, vehicle-treated, and microglia-treated rats (F = 30.80, n = 6 rats per group, P < 0.001). *P < 0.05 vs. any other group.

Fig. 6

Treatment with BM-MNCs reduces neurologic deficit after pMCAO. ANOVA revealed a significant difference in neurologic function among untreated, vehicle-treated, microglia-treated, and BM-MNC-treated groups on days 1, 3, 7 and 14 after pMCAO (F value for tests of between-subject effect = 9.33, P < 0.001). Treatment with BM-MNCs significantly improved neurologic function on day 14 compared to that in untreated, vehicle-treated, and microglia-treated groups (n = 10 rats per group). *P < 0.05 vs. all other groups.