| Stroke is a devastating injury caused by interruption of the blood supply to the brain. Spatial cognitive deficits including spatial memory impairment are very common after ischemic stroke. About 30% of the stroke survivors suffer spatial cognitive deficits. However, there is no effective pharmacotherapy at present.Recent advances in the study of neural regeneration suggest that neurogenesis may be a critical element in brain repair. Spatial memory is largely dependent on hippocampal formation. Neurogenesis in the adult dentate gyrus (DG) is important in learning and memory processes. In adult rodents, experimental cerebral ischemia enhances neurogenesis in the brain's neuroproliferative zones, the subgranular zone (SGZ) of the hippocampal DG and the subventricular zone (SVZ) of the lateral ventricle. There is also evidence for stroke-induced neurogenesis in the human brain. Recently, these newborn neurons after ischemic injury were shown to migrate to injured brain regions, become actively integrated into the existing circuitry, and form appropriate synapses, which contributed to ameliorate neurological deficits and form hippocampal-dependent memory. These studies raise the possibility that spatial cognitive deficits after ischemic stroke may be improved through enhancing hippocampal neurogenesis. Fluoxetine is a widely used antidepressant compound, whose primary action is based on the inhibition of serotonin-reuptake in the central nervous system (CNS). Recent studies have shown that chronic fluoxetine treatment can promote the proliferation as well as the survival and differentiation of newborn cells in the hippocampus of the adult mammalian brain and in some neurological diseases, such as depression. It is important to determine whether chronic fluoxetine treatment can enhance neurogenesis and neuronal remodeling in the hippocampus and improve spatial cognitive functional recovery after ischemic stroke, as this question remains to be answered. Aiming to determine the contribution of fluoxetine on ischemic stroke, we examined the effects of a chronic fluoxetine regimen on hippocampal neurogenesis and functional recovery, especially spatial cognitive function after focal cerebral ischemia.To determine whether fluoxetine treatment improves sensorimotor functional recovery and attenuates spatial memory impairment, mice received i.p. injections of fluoxetine (dissolved in 0.9% NaCl and administered at a dose of 10 mg·kg-1) daily starting on day 8 after ischemia induction and continuing to 35 days after MCAO (28 days). Vehicle control mice and sham-operated mice were given an equivalent volume of vehicle (saline). Neurological severity scores and foot fault tests were performed 7, 14, 21, 28, 35, 42, 49 and 59 days after MCAO, and spatial cognitive performance was tested in the Morris water maze during day 22-28 and day 50-59 after MCAO. The animals subjected to MCAO exhibited significant and sustained neurological deficits compared with sham-operated mice, while the mean modified neurological severity scores showed no significant difference between fluoxetine- and vehicle-treated mice throughout the testing period. Unilateral foot faults were expressed by the number of contralateral foot faults as a percentage of the total errors made, and a value of 50% represents an equal number of errors made by both sides. In sham-operated animals, no functional deficit was observed as they made approximately the same number of errors on the contralateral side as they did on the ipsilateral side. In the mice subjected to MCAO, there was a significant increase in the number of contralateral errors. However, fluoxetine treatment did not significantly reduce this functional deficit compared with vehicle. These results suggest that chronic fluoxetine treatment does not significantly improve sensorimotor functional recovery after stroke. In the test of Morris water maze, we first exposed animals to water maze task after fluoxetine treatment for 14-20 days (day 22-28 after MCAO). In the hidden platform trials, with regard to escape latency, repeated-measures two-way ANOVA revealed a group difference. Post hoc analysis using LSD test revealed that sham-operated group had a significantly reduced escape latency compared with the two ischemic groups, with vehicle and with fluoxetine. However, there was no significant difference between fluoxetine- and vehicle-treated mice in escape latency. Ischemic groups had significantly prolonged swimming length also compared with sham-operated mice. Repeated-measures two-way ANOVA of the swimming speed showed that there were no differences between three groups, suggesting that the impaired spatial cognitive ability of ischemic mice on the water maze task is not caused by motor ability changes. During the spatial probe trials, in which the platform was removed, ischemic mice exhibited reduced time spent in the target quadrant and number of crossing platform position compared with sham-operated mice. Therefore, fluoxetine treatment for 14 days does not ameliorate the MCAO-induced spatial learning and memory impairment. When animals were exposed to the water maze task after 28 days of fluoxetine treatment and 14 days of drug withdrawal, however, the escape latency in fluoxetine-treated group was significantly shorter than that in vehicle-treated group, and was similar to that in sham-operated group. Similarly, fluoxetine-treated mice had significantly reduced swimming length compared with vehicle-treated mice. Swimming speed was also measured, and there were no significant differences between each group. In the spatial probe trials, Sham-operated and fluoxetine-treated mice exhibited markedly increased time spent in the target quadrant) and number of crossing the platform position compared with vehicle-treated mice. To exclude the possibility that the improved memory by chronic fluoxetine treatment was confounded by motivational or sensorimotor factors, we performed a water maze task with visible platform in which the platform was elevated 0.5 cm above the water level. Repeated-measures two-way ANOVA of escape latency demonstrated no significant differences between each group, suggesting that the improved memory by fluoxetine is spatial memory. At the end of behavioral tests, the infarct volume was determined in mice from the three groups by TTC staining. Fluoxetine treatment for 4 weeks starting day 8 after MCAO did not lessen infarct volume, suggesting that the improved memory by fluoxetine is not due to infarct volume reduction.To examine whether fluoxetine increases neurogenesis, mice received i.p. injections of 5-bromo-2'-deoxyuridine (BrdU; Sigma) 50 mg·kg-1 twice daily during day 19-21 after MCAO. For the cellular proliferation study, animals were sacrificed 24 hours after the last BrdU injection to examine the number of newly formed cells in the DG. To determine the survival of the newly born cells, animals were sacrificed 28 days after the last BrdU injection. The number of newborn cells in the DG of fluoxetine-treated mice did not differ from that of vehicle both in ipsilateral and contralalteral, suggesting that chronic fluoxetine treatment has no effect on progenitor cell proliferation in the DG of ischemic mice. Neither fluoxetine nor vehicle treatment changed the number of newborn cells in the DG of ischemic mice compared with sham-operated mice in both the ipsilateral and contralalteral region. Moreover, there was no significant difference in the numbers of BrdU-positive cells between the ipsilateral and contralateral sides in fluoxetine-, vehicle-treated and sham-operated groups. However, chronic fluoxetine treatment facilitated the survival of newborn cells in the DG, the number of BrdU-positive cells both in the ipsilateral and contralateral DG was significantly increased in the animals treated with fluoxetine compared with vehicle or sham-operated mice, suggesting that chronic fluoxetine treatment increased the survival of newly born cells in the hippocampus after stroke. There was no significant difference in the numbers of BrdU-positive cells between vehicle-treated mice and sham-operated mice both in the ipsilateral and contralateral DG.To determine whether hippocampal neurogenesis is necessary for the effect of fluoxetine on spatial cognitive performance after MCAO, we used a telomerase inhibitor, 3'-azido-deoxythymidine (AZT), to disrupt neurogenesis. The mice were treated with 100 mg/kg AZT per day i.p. during the period of fluoxetine treatment (from day 8 to day 35 after MCAO). Spatial cognitive performance and hippocampal neurogenesis were tested 49 d after MCAO (28 d after the last BrdU administration) by the same approach as was mentioned previously. In the mice treated with fluoxetine combined with AZT, the number of BrdU-positive cells in the DG was significantly fewer than that in the mice treated with fluoxetine alone, and similar to that in vehicle-treated animals, suggesting that the increased neurogenesis by fluoxetine treatment was neutralized by the negative action of AZT. In water maze task, AZT completely abolished the effects of fluoxetine on escape latency and swimming length, compared with fluoxetine alone group, even the speeds of fluoxetine and AZT treatment mice were rapid than fluoxetine-treated and vehicle-treated mice. Thus, hippocampal neurogenesis is required for the beneficial effect of fluoxetine on ischemia-induced spatial cognitive deficits.Thus, our data suggest that although chronic fluoxetine treatment does not significantly improve sensorimotor functional recovery after stroke and has no effect on progenitor cell proliferation in the DG of ischemic mice, it benefits spatial cognitive function recovery following ischemic insult, and the improved cognitive function is associated with enhanced newborn cell survival in the hippocampus. Moreover, hippocampal neurogenesis is essential for fluoxetine-improved spatial cognitive deficits. |
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