ABSTRACT
Objective To explore the effects of two routes of melatonin (MT) administration including intraperitoneal and caudal vein injection on the behavior,histopathology and the expression of myelin basic protein (MBP) and active caspase-3 protein in focal cerebral ischemic rats.Methods 84 male Sprangue-Dawley rats were randomly divided into normal control group (CON,n=12),middle cerebral artery occlusion group(MCAO,n=24),MT-intraperitoneal group (n=24) and MT-intravenous injection group (n=24) by random number table.Twenty-four hours after ischemia reperfusion (IR),Morris water maze was used to observe the effects of two routes of MT administration on behavior in focal cerebral ischemic rats.7 d after IR,MBP immunohistochemical and hematoxylin eosin (HE) staining were used to examine the expression of MBP in striatum and histopathological changes in hippocampal CA1 region.24 h,72 h and 7 d after IR,the expression of active caspase-3 in hippocampal CA1 region was observed by immunohistochemical staining.Results The average escape latencies in Morris water maze in MT-intravenous injection group at different time points were all lower than those of the MT-intraperitoneal,and they were all lower than those of the MCAO group.Swimming time percentage of target quadrant in MT-intravenous injection group were higher than those of the MT-intraperitoneal,and they were all higher than those of the MCAO group (all P＜0.01);7 d after IR,the results of HE staining showed that the hippocampus cells in MCAO group were disarranged with hyperchromatic nucleus and cytoplasm.More hippocampal cells were observed in MT-intraperitoheal and MT-intravenous injection groups,and they were relatively well arranged.The optical density (OD)of MBP in MT-intravenous injection group (105.60±4.04) was significantly higher than those in MCAO group (95.60±2.07) and MT-intraperitoneal injection group (98.00±4.18) (both P＜0.01).Immunohistochemical results showed that the number of active caspase-3 positive cells in MT-intravenous injection group ((116.93± 12.58)/mm2,(130.16±21.22)/mm2,(88.25±7.80)/mm2) at each time point were significantly lower than those in MT-intraperitoneal injection group ((156.64± 32.54)/mm2,(176.49± 17.44)/mm2,(127.96±16.73)/mm2) (all P＜0.05).At the time points of 24 h and 72 h after IR,there were less active caspase-3 positive cells in MT-intraperitoneal and MT-intravenous injection group compared with those in MCAO group((273.56±32.54)/mm2,(288.63±35.17)/mm2)(all P＜0.01).Conclusion MT administration by both intraperitoneal and intravenous injection can significantly improve the behavior and attenuate the histopathology and white matter damage,and reduce the cell apoptosis in hippocampal CA1 region in focal cerebral ischemic rats,and the therapeutic effects of MT-intravenous injection are better than MT-intraperitoneal injection.
ABSTRACT
Objective To observe the effect of melatonin (MT) on retinal apoptosis in rats with ischemia-reperfusion injury (RIRI).Methods A total of 54 male healthy Sprague-Dawley adult rats were randomly divided into the normal control (CON) group (6 rats), RIRI group (24 rats) and MT group (24 rats). The rats of RIRI and MT group were induced using suture-occluded methods to establish RIRI model. The rats of MT group were injected with MT in the left carotid artery 30 minutes after RIRI, and RIRI group was injected with the same amount of saline. On 6, 24 hours and 3, 7 days after RIRI, the morphological changes of retina were evaluated by hematoxylin and eosin (HE) staining; the effects of MT on retinal cell apoptosis and Nrf2,HO-1 proteins were examined by immunohistochemistry staining. The correlation between active Caspase-3 and Nrf2 protein, active Caspase-3 and HO-1 protein in MT group were analyzed by linear regression analysis. Results HE staining results showed that the morphology of retinal cells was regular and retinal cells were well arranged in the CON and MT group. In the RIRI group, both the thickness of inner retinal layer and the number of retinal ganglion cells (RGC) were decreased. On 6, 24 hours and 3, 7 days after RIRI, the thickness of inner retinal layer (F=16.710, 62.303, 68.389, 57.132;P<0.01) and RGC number (F=24.250, 11.624, 14.155, 32.442;P<0.05) in MT group were more than those in RIRI group. Immunohistochemistry staining results showed that less active Caspase-3+ cells were observed in MT group as compared with those in RIRI group at each time points (F=49.118, 134.173, 76.225, 18.385;P<0.01). There were more Nrf2+ (F=11.041, 31.480, 59.246, 6.740;P<0.05) and HO-1+ cells (F=128.993, 21.606, 51.349, 8.244;P<0.05) in MT group as compared with those in RIRI group at each time points. Linear regression analysis results showed that the difference of active Caspase-3+cells were all linearly correlated with the Nrf2+ cells and HO-1+cells in the MT group (r2=0.810, 0.730;P<0.01). Conclusion MT could reduce retinal cell apoptosis in RIRI rats, and its mechanism may be associated with increased Nrf2 and HO-1 expression, reduced active Caspase-3 expression.
ABSTRACT
Objective To investigate the effects of melatonin on the proliferation of neural stem cells (NSCs) in cerebral ischemia reperfusion (IR) rats, and to explore the possible mechanisms. Methods Seventy-two rats were randomly divided into the normal control group (n=12), model group (n=30) and melatonin group (n=30) according to the random number table. The rats in the model group and melatonin group were divided into four subgroups: 6 h, 24 h, 72 h and 7 d subgroups according to the time after IR. The morphological changes of the subventricular zone (SVZ) were examined by HE staining;the effects of melatonin on NSCs proliferation were examined by immunofluorescence staining;the effects of melatonin on toll-like receptor 4 (TLR4) and nuclear factor (NF)-κB p65 protein were examined by immunohistochemistry staining and Western blotting analysis. The correlation between the proliferating NSCs and TLR4 protein or the NF-κB p65 protein was analyzed by linear regression analysis. Results HE staining showed that the cells in the SVZ of rats in the model group were in disorder and irregular in shape. In the melatonin group, the cells in the SVZ of the injured side were relatively well arranged. Immunofluorescence staining showed that the number of proliferating cell nuclear antigen (PCNA)+Nestin+4',6-diamidino-2-phenylindole (DAPI)+cells in the SVZ of the model (498.47 ± 26.44/mm2) and melatonin groups (623.10 ± 39.70/mm2) increased gradually, and reached a higher level after IR for 7 days, which were significantly higher than the normal control group (203.91 ± 32.23/mm2) (F=35.193, 170.344, 277.536, 285.947, all P＜0.01). The number of PCNA+Nestin+DAPI+cells in the melatonin group rats at each time points was significantly higher than that in the model group (F=102.561, 91.244, 168.502, 38.013, all P＜0.01). Immunohistochemistry staining showed that the numbers of TLR4+and NF-κB p65+cells in the SVZ of the model (740.02±31.63/mm2;710.01± 26.59/mm2) and melatonin groups (555.57 ± 25.28/mm2;528.85 ± 30.60/mm2) increased gradually, and reached a higher level 7 d after IR, which were significantly higher than the normal control group (107.97±12.84/mm2;109.80±13.89/mm2) (F=21.413, 263.059, 873.691, 1 037.098, all P＜0.01;F=26.374, 372.940, 854.826, 929.018, all P＜0.01). There were less TLR4+(F=7.641, 25.135, 66.094, 103.753, all P＜0.05) and NF-κB p65+cells (F=18.612, 69.597, 113.113, 119.814, all P＜0.01) in the melatonin group as compared with those in the model group at each time points. Western blotting analysis showed that the expression of TLR4 (0.87±0.08;0.68±0.06) and NF-κB p65 (0.72±0.05;0.58±0.05) protein was higher in the model and melatonin groups as compared with the normal control group (0.35±0.04, 0.31±0.03;F=107.43, F=132.51, both P＜0.01). The expression of the TLR4 and NF-κB p65 protein was lower in the melatonin group as compared with that in the model group (P＜0.01). Linear regression analysis showed that the differences of PCNA+Nestin+DAPI+cells were all negatively correlated with that of the TLR4+cells and NF-κB p65+cells in the melatonin group (r2=0.838, r2=0.813, both P＜0.01). Conclusion Melatonin can inhibit the expression of TLR4 and NF-κB p65 protein, thus promote the proliferation of endogenous NSCs in cerebral ischemia reperfusion rats.