Background and Purpose
Our recent evidences suggest that neurogenesis following ischemic brain injury might not only lead to the replacement of damaged cells but can also contribute to functional recovery, thereby supporting the notion that strategies which augment endogenous neurogenesis may hold clues for the development of stroke therapy. It is known that activated microglia localized in close proximity to the newly formed neurons compromise the survival of new hippocampal neurons, and blocking inflammation has been shown to restore hippocampal neurogenesis after cranial irradiation and seizure. The aim of our current study is to determine the effect of minocycline on neurogenesis and functional recovery after focal cerebral ischemia.
Methods
Adult male rats underwent 60 minutes of middle cerebral artery occlusion (MCAO) via the intraluminal suture method. Minocycline was administered intraperitoneally (i.p.) starting at 6 h (early treatment) or 5d (delayed treatment) after reperfusion for a continuous period of 4 weeks. During the first week of minocycline injection, animals received a daily dose of 50 mg/kg, followed by daily 25 mg/kg for the remaining period. BrdU (50 mg/kg) was administered twice daily on days 5–8 after MCAO. The number of surviving BrdU-labeled cells, Ki67 and ED1 positive cells were quantified in the dentate gyrus (DG) or hilus following immunohistochemistry, and the phenotype of newborn cells was determined by confocal microscopy. Infarct volume was evaluated by unbiased stereology using the Cavalieri's principal based on NeuN staining. Motor and cognitive functions were evaluated by open field, cylinder, horizontal ladder, rotor rod and Morris water maze tests on a separate group of animals receiving the delayed treatment paradigm.
Results
Among the early treatment groups (6 h after reperfusion), infarct volume was significantly reduced in minocyline treated MCAO rats (McI) (72.6±8.4 mm3) with a concurrent decrease in progenitor cell proliferation and neurogenesis (BrdU: 128.8±20.2/DG), compared to rats with MCAO alone (I) (infarct volume: 34.5±12.3 mm3; BrdU: 217.0±43.0/DG). On the other hand, delayed treatment (5d after reperfusion) of minocycline did not result a difference in infarct size, but caused a significant decrease in the number of activated microglia in DG (McI: 14.4±2.1/DG) compared to vehicle treatment (I: 53.1±4.7/DG). Although there was no statistical difference in the number of total surviving new cells in the DG between minocycline and vehicle treated MCAO rats, minocycline significantly increased the number of surviving newborn neurons that co-expressing BrdU and NeuN (McI: 165.0±27.5/DG; I: 90.2±24.4/DG). Furthermore, minocycline did not affect progenitor cell proliferation at the SGZ, consistent with previous reports. Behavioral testing showed that there was no difference in spontaneous activity in an open field, however, minocycline significantly improved motor coordination on the rotor rod, reduced the preferential use of the unaffected limb, reduced the frequency of footfalls in the affected limb when traveling on a horizontal ladder, and improved spatial learning and memory.
Conclusion
Our data suggest that minocylin reduces functional impairment after cerebral focal ischemia, and the improved function is associated with enhanced neurogenesis in the DG.