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Objective:To dynamically observe the effect of N-acetylserotonin (NAS) on the expression of tumor necrosis factor-α (TNF-α) protein in retina of retinal ischemia reperfusion injury (RIRI) rats, and to explore the mechanism.Methods:By using random number table method, 90 healthy male Sprague-Dawley rats were divided into sham operation group ( n=10), RIRI group ( n=40), and NAS group ( n=40). The right eye was as the experimental eye. In the RIRI group and NAS group, the anterior chamber high intraocular pressure method was used to establish the RIRI model. In the NAS group, 10 mg/kg NAS was injected intraperitoneally before modeling and 30 minutes after modeling. At 6, 12, 24, 72 h after modeling, hematoxylin-eosin staining was used to observe the pathological changes of the retina, and the retinal ganglion cells (RGC) were counted. Each group was detected by immunohistochemical staining and Western blot about the relative expression of TNF-α, nuclear factor E2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) protein in the rat retina. Oneway analysis of variance was used for differences between groups. The general linear regression method was used to analyze the correlation between the relative expression changes of TNF-α protein and the changes of Nrf2 and HO-1 protein expression after NAS intervention. Results:Optical microscope observation revealed that the retinal edema of rats in the RIRI group was observed at 6, 12, and 24 h after modeling; the thickness of the retina in the NAS group was significantly thinner than that in the RIRI group, and the difference was statistically significant ( F=9.645, 477.150, 2.432; P<0.01). At 6, 12, 24, and 72 h after modeling, the retinal RGC counts in the NAS group were significantly higher than those in the RIRI group, and the difference was statistically significant ( F=12.225, 12.848, 117.655, 306.394; P<0.05). The results of immunohistochemical staining and Western blot showed that 6 h after modeling, the relative expression of TNF-α protein in the retina of the RIRI group increased significantly compared with that in the sham operation group, reaching a higher level at 12 h, and decreased at 24 and 72 h. But all were significantly higher than the sham operation group, the difference was statistically significant (immunohistochemical staining: F=105.893, 1 356.076, 434.026, 337.351; P<0.01; Western blot: F=92.906, 534.948, 327.600, 385.324; P<0.01). At different time points after modeling, the relative expression of TNF-α protein in the retina of the NAS group was significantly lower than that of the RIRI group (immunohistochemical staining: F=15.408, 570.482, 21.070, 13.767; P<0.05; Western blot: F=12.618, 115.735, 13.176, 111.108; P<0.05), but still higher than the sham operation group (immunohistochemical staining: F=40.709, 151.032, 156.321, 216.035; P<0.01; Western blot: F=33.943, 79.729, 74.057, 64.488; P<0.01), the difference was statistically significant; 12 h after modeling, Nrf2 in the retina of the NAS group (immunohistochemical staining: F=51.122, P<0.05; Western blot: F=33.972, P<0.05), HO-1 (immunohistochemical staining: F=30.750, P<0.05; Western blot: F=18.283, P<0.05) protein relative expression was significantly higher than that of RIRI group, and the differences were statistically significant. The results of linear regression analysis showed that the difference in the number of TNF-α + cells in the RIRI group and the NAS group was negatively correlated with the difference in the number of Nrf2 + and HO-1 + cells ( r 2=0.923, 0.936; P<0.01). Conclusions:NAS can inhibit the expression of TNF-α protein in the retina of RIRI rats and reduce RIRI. The mechanism may be related to the Nrf2/HO-1 pathway.
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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.
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Objective To investigate the effects of N-acetylserotonin (N-AS) on the expression of active caspase-3,Bcl-2 and Bax in rat retinas induced by retinal ischemia-reperfusion injury (RIRI).Methods Adult male Sprague-Dawley rats were randomly divided into the normal control group (6 cases),RIRI group (30 cases) and NAS group (30 cases),RIRI models in NAS group were established after giving NAS,the groups were sub-divided into 6 hours,12 hours,24 hours,48 hours and 72 hours group based on the time of RIRI.Morphologic changes were evaluated by HE staining.The expression of active caspase-3,Bcl-2 and Bax protein in the retina of rats was detected by immunohistochemistry.Results HE staining showed that the retinal structure in the normal control group was clear,and the cells in each layer were tightly packed;Each layer of retina was edema in the RIRI group after 6 hours and 12 hours,the edema gradually alleviated after 24 hours,the ganglion cells decreased gradually,the distribution was in disorder,with the prolongation of time,the retinal ganglion cells were defected;drug group of as Compared with RIRI group,the cell edema in the NAS group at 6 hours and 12 hours were obvious reduced,the cells in 24 hours,48 hours,72 hours group arranged regularly,the loss number of ganglion cells were reduced.The number of active caspase-3 positive cells in RIRI group increased at 6 hours after peffusion,the number was (561.15 ±37.19) cell ·mm-2,and reached the high level at 24 hours,the number was (1522.61 ±84.36) cell · mm-2,and then decreased gradually.The number of active caspase-3 positive cells in NAS group was significantly lower than that in RIRI group,the difference was statistically significant (all P ＜ 0.05).The expression of Bcl-2 positive cells in RIRI group began to decrease after 6 hours,and decreased to a low level at 24 hours,and the number of Bcl-2 positive cells in NAS group was significantly higher than that in RIRI group at each time point,the differences were statistically significant (all P ＜ 0.05).There were almost no Bax positive cells in the retina of the control normal group,and the Bax positive cells were found to be higher of the RIRI group at the 6 hours after RIRI,and reached the higher level at 24 hours,and decreased at 48 hours.The Bax positive cells of NAS group were significantly less than those in the RIRI group at different time points,and the differences were statistically significant (all P ＜0.05).Conclusion NAS can promote the expression of Bcl-2 protein in rat retina after RIRI,inhibit the expression of Bax protein,decrease the expression of active caspase-3 protein,alleviate cell apoptosis,and have neuroprotective effects.
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Objective To analyze the application value of enhanced depth imaging optical coherence tomography (EDI-OCT) in measuring the lamina cribrosa thickness(LCT) in patients with branch retinal vein occlusion (BRVO).Methods The clinical data of 65 patients with unilateral BRVO treated in our hospital from September 2014 to March 2016 were selected as the observation group.The single healthy eyes of 40 healthy individuals who received physical examination in the hospital during the same period were selected as the control group.The changes of retinal nerve fiber layer (RNFL) thickness,LCT,central corneal thickness,axial length,transverse diameter of optic disc,vertical diameter of optic disc,diopter and extent of visual field defect in the two groups were determined by EDI-OCT.Results There was no significant difference in the central corneal thickness,axial length,transverse diameter of optic disc,vertical diameter of optic disc and diopter between the two groups (all P ＞ 0.05).LCT of different regions of optic disc in the observation group were lower than those in the control group (all P ＜ 0.05).The range of visual field defects in the observation group was larger than that in the control group,and the RNFL thickness was lower than that in the control group (P ＜ 0.05).LCT was positively correlated with the thickness of whole RNFL in patients with BRVO,and was negatively correlated with the visual field defects (P ＜ 0.05).Conclusion EDI-OCT is an effective means for measuring LCT.LCT of patients with glaucoma BRVO is thinner than that of normal healthy people,and LCT is positively correlated with RNFL.thickness,and negatively correlated with visual field defects.
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Objective Based on middle cerebral artery occlusion (MCAO) model,to investigate the effects of melatonin (MT) on DWI and expression of Fas,FasL and cleaved Caspase-3 proteins in rat model with focal cerebral ischemia.Methods Eighty SD rats were randomly divided into Sham group (n=16),MCAO group (n=32) and MT group (n=32).The rats in sham group were treated with sham-operation.And the rats in MCAO and MT groups were peritoneally injected with saline and MT respectively.The behavioral scores were assessed in the three groups.The rats in MCAO and MT group with the behavioral scores of 1 3 points were selected in the study.The DWI relative signal intensity (rDWI-SI),Fas,FasL and cleaved Caspase-3 proteins were respectively examined by MR scaning and immunohistochemical staining in all rats of each group at 6 h,24 h,72 h and 7 days after ischemia reperfusion (IR) or sham-operation.And the DWI and immunohistochemical results for each group were compared.Results At last,there were 16 rats in sham group,29 rats in MCAO group and 30 rats in MT group,respectively.There was significant difference of the behavioral scores among the three groups (x2 =50.125,P＜0.01).The behavioral scores of MT and MCAO groups were higher than those of sham group (all P ＜0.05).And the behavior scores of the MT group were lower compared with MCAO group after IR.Compared with the rDWI-SI values measured at 6 h,24 h and 72 h,7 days in sham group,the rDWLSI values of MT and MCAO groups were significantly higher (all P＜0.01).And the rDWI-SI was higher in MCAO group than those in MT group at 6 h,24 h and 72 h after IR (all P＜0.01).And there was no significant difference of rDWI-SI at 7 days after IR between MT and MCAO groups (P＞0.05).The immunohistochemical staining results showed that the number of Fas,FasL and cleaved Caspase-3 positive cells in MCAO and MT groups were significantly higher than those in sham group (all P＜0.01).And there were less Fas,FasL and cleaved Caspase-3 positive cells in MT groups compared with MCAO group (all P＜0.05) at 6 h,24 h and 72 h after IR.There was no significant difference of Fas,FasL and cleaved Caspase-3 positive cells among the three groups at 7 days after IR (P＞0.05).Conclusion MT can effectively alleviate the rDWI-SI value and inhibit the expression of Fas,FasL and cleaved Caspase-3 proteins in rats of focal cerebral ischemia.
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Background Retinal ichemic-reperfusion injury (RIRI) affects vision and life quality and has no ideal treatment outcome up to now.The mechanism of RIRI is associated with apoptosis.Researches showed that subretinal transplantation of bone mesenchymal stem cells (BMSCs) can relieve RIRI,but its mechanism is unclear.Objective This study was to observe the influence of BMSCs subretinal transplantation on the expressions of apoptosis protein Fas/FasL,caspases-3 after RIRI,and to explore the neuroprotecitve mechanism of BMSCs transplantation to treat RIRI.Methods Bone marrow was isolated and extract form femur and tibia of SD rat by the density gradient centrifugation method and then the BMSCs were collected.BMSCs suspension was prepared using DMEM containing low-glucose with the cell density 1 x 106-2× 106/ml.RIRI models were established by ligation of optical nerve (Daniel M method).Sixty-four SD rats were randomly divided into the normal control group,model control group,BMSCs transplantation group and PBS control group.Twenty-four hours after RIRI,BMSCs suspension with the cells density 5× 104 cells was subretinally injected in the rats of the BMSCs transplantation group,and equal volume of PBS was injected in the same way in the rats of the PBS control group.The expressions of Fas/FasL and caspase-3 were dynamically detected using immunohistochemistry at 6,24,48 and 72 hours after BMSCs transplantation.Results The positive response cells for Hoechst33324 were seen in subretina 72 hours after BMSCs transplantation.A few of positive cells for Fas,FasL,caspase-3 proteins were expressed in the rats of the normal control group at 6,24,48 and 72 hours after BMSCs transplantation.However,the positive cells for Fas,FasL,caspase-3 protein were significantly increased in the model control group and the BMSCs transplantation group compared with the normal control group at various time points after injection (all at P＜0.01),and the number of positive cells was significantly less in the BMSCs group than those of the RIRI group and the BMSCs transplantation group (P＜0.05,P＜0.01).No significant differences were found in the numbers of positive cells of Fas,FasL and caspase-3 proteins at various time points between the model control group and the PBS control group (all at P＞0.05).Conclusions The subretinal transplantation of BMSCs down-regulate the expression of Fas/FasL and caspases-3 proteins in RIR1 rats,and thus alleviate the apoptosis of retinal neural cells.
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Objective To investigate the effect of umbilical cord blood monocytes(UCBMC) transplantation in neonatal rats with hypoxic-ischemic brain damage (HIBD) on rat astrocyte proliferation and its correlation with bone morphogenetic protein(BMP) 4.Methods Forty 7-day-old SD rats with the random number table,were divided into normal control(CON) group,hypoxic-ischemic(HI) group,normal(N) + UCBMC group,HI + UCBMC group,10 rats in each group.HIBD model was prepared according to the Rice method.Twenty-four hours after hypoxia,the UCBMC group and HI + UCBMC group were injected with 3 × 106 UCBMC via the lateral ventricle.Seven days after transplantation,changes in the number of neurons were observed by Nissl staining;the expression of glial fibrillary acidic protein (GFAP) was observed by Western blot;the astrocyte proliferation was observed by proliferating cell nuclear antigen (PCNA)/GFAP,BMP4/GFAP immunofluorescence double staining,and their correlation was analyzed.Results Nissl staining showed that the neurons at cerebral cortex and hippocamp were irregularly arranged and decreased in the HI group,and the number of Nissl-stained cells were significantly less than that in the CON group(tcortex =26.54,thippocamp =32.26,all P ＜0.05) ;but the Nissl-stained cells were well arranged in the HI + UCBMC group and more Nissl-stained cells were observed as compared with the HI group (tcortex =10.18,thippocamp =12.56,all P ＜ 0.05) ; Western blot showed that the expression of GFAP in the HI group was significantly higher than that in CON group(t =5.50,P ＜ 0.05) ;but the expression of GFAP in the HI + UCBMC group was significantly lower than that in the HI group (t =3.04,P ＜ 0.05) ; immunofluorescence double staining showed that there were more PCNA + GFAP + cells in the HI group compared with the CON group(t =10.39,P ＜ 0.05),but fewer PCNA + GFAP + cells were observed in the HI + UCBMC group than those in the HI group(t =3.72,P ＜ 0.05).And there were more BMP4 + cells in the HI group compared with the CON group (t =5.52,P ＜ 0.05),but fewer BMP4 + cells were observed in the HI + UCBMC group than those in the HI group(t =2.33,P ＜0.05).The fluorescence intensity of GFAP were correlated with that of BMP4 in HI + UCBMC group (r =0.84,P ＜ 0.05).Conclusions UCBMC transplantation can decrease the proliferation of the astrocytes,thus promote brain damage repair and its mechanism might be collected with the decreased expression of BMP4.
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Objective To investigate the effects of bone mesenchymal stem cell (BMSC) transplantation on neuronal nuclei (NeuN) and neurogenin 1 (Ngnl) in focal cerebral ischemic rats.Methods A total of 64 healthy adult male Sprague-Dawley rats were randomly divided into normal (N) + phosphate-buffered solution (PBS),middle cerebral occlusion (MCAO)+ PBS,N + BMSC and MCAO + BMSC groups (n =16 in each group).A rat model was induced by the intraluminal suture method.BMSC was cultured in vitro.At 24 h after modeling,brain transplantation was conducted.Magnetic resonance imaging (MRI) was used to detect infarct volume in vivo.NeuN/DAP,Ngnl/DAPIimmunofluorescence double-labeling and Western blot were used to detect the expression of NeuN and Ngnl around ischemic brain tissue.Results On day 14 after transplantation,the T1-and T2-weighted imaging revealed that the cerebral cortex and striatum had abnormal signal areas in the rats of the MCAO group.The infarct volume of the MCAO + BMSC group was significantly less than that of the MCAO + PBS group (32.5％ ± 4.2％ vs.47.9％ ± 7.9％ ; P ＜ 0.01).Immunofluorescence doublelabeling assay showed that the numbers of cells of NeuN+/DAPI+ (976.2 ± 87.5/mm2 vs.1 908.3 ±127.8/mm2; P ＜ 0.01) and Ngn1 +/DAPI + (251.6 ± 23.1/rmm2 vs.285.1 ± 25.2/mm2 ; P ＜ 0.01) of the MCAO + PBS group were significantly less than those of the N + PBS group,but those of NeuN+/DAPI +(1 439.9 ± 101.7/mm2; P ＜ 0.01) and Ngn1 +/DAPI + (356.3 ± 35.6/mm2; P ＜ 0.01) of the MCAO + BMSC group were significantly more than the MCAO + PBS group.Western blot analysis showed that the protein expression levels of NeuN (0.69 ±0.06 vs.0.91 ±0.09; P ＜0.01) and Ngn1 (0.53 ±0.05 vs.0.62 ±0.07;P ＜0.01) of the MCAO +PBS group were significantly lower than those of the N +PBS group,but those of NeuN (0.82 ± 0.07; P ＜ 0.01) and Ngn1 (0.77 ± 0.09; P ＜ 0.01) of the MCAO + BMSC group were significantly higher than the MCAO + PBS group.Conclusions BMSC transplantation may promote the expression of NeuN and Ngn1,and alleviate MCAO caused brain injury.
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Objective To explore the relationship between the proliferation of neural stem cells (NSCs)and the expression of β-catenin protein in neonate rats with hypoxic ischemic brain damage (HIBD) after hyperbaric oxygen (HBO) therapy. Methods One hundred and eighty Sprague-Dawley rats aged 7 days were randomly divided into a normal control group (CON) , a HIBD model group and a HBO treatment group. The HIBD model was induced using Rice's method. Beginning 3h after the HIBD, HBO was administered to the HBO treatment group at 2 atmospheres for 60 min, once daily for 7 days. The HIBD model group was not given any treatment. The expression of nestin/β-catenin protein in the subventricular zone of the ischemic brain was double-stained for immunofluorescence and analyzed by confocal scanning microscopy dynamically at 3 hours, 21 hours, and then on the 3rd, 5th, 7th and 14th day of HBO therapy. The expression of whole cell β-catenin and nuclear β-catenin protein in the left brain were also examined by Western blotting at these 6 time points. Linear correlation was used to analyze the correlation between β-catenin and nestin protein. Results The expression of β-catenin protein in NSCs increased initially at the 21st hour after HBO therapy in the model group and the HBO group as compared with the normal control group.β-catenin protein in the model group reached a higher level, though there was no significant difference between model group and the HBO group. At the 5th day of HBO therapy β-catenin protein in the HBO group had reached a significantly higher level than in the model group. At the 14th day the average expression of β-catenin in the HBO group began to decrease. The expression of nestin protein began to increase 21 hours after HBO therapy began, and it peaked at the 7th day of HBO therapy and then decreased. In the HBO group the increase in nestin protein was linearly correlated with that of β-catenin protein. The whole cell β-catenin protein and β-catenin nucleic protein readings increased initially by the 21st hour of HBO therapy and by the 5th day were significantly higher than the levels in the model group. Conclusion HBO treatment is capable of stimulating the proliferation of NSCs in HIBD neonate rats.The proliferation of NSCs is correlated with the activation of β-catenin protein.