Adipose mesenchymal stem cells(ADSCs)have protective effects against glutamate-induced excitotoxicity,but ADSCs are limited in use for treatment of optic nerve injury.Studies have shown that the extracellular vesicles...Adipose mesenchymal stem cells(ADSCs)have protective effects against glutamate-induced excitotoxicity,but ADSCs are limited in use for treatment of optic nerve injury.Studies have shown that the extracellular vesicles(EVs)secreted by ADSCs(ADSC-EVs)not only have the function of ADSCs,but also have unique advantages including non-immunogenicity,low probability of abnormal growth,and easy access to target cells.In the present study,we showed that intravitreal injection of ADSC-EVs substantially reduced glutamate-induced damage to retinal morphology and electroretinography.In addition,R28 cell pretreatment with ADSC-EVs before injury inhibited glutamate-induced overload of intracellular calcium,downregulation ofα-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor(AMPAR)subunit GluA2,and phosphorylation of GluA2 and protein kinase C alpha in vitro.A protein kinase C alpha agonist,12-O-tetradecanoylphorbol 13-acetate,inhibited the neuroprotective effects of ADSC-EVs on glutamate-induced R28 cells.These findings suggest that ADSCEVs ameliorate glutamate-induced excitotoxicity in the retina through inhibiting protein kinase C alpha activation.展开更多
·AIM: To explore the correlation between the retinal nerve fiber layer (RNFL) thickness by using optical coherence tomography (OCT) and by histological measurements in normal adult rats and optic nerve transected...·AIM: To explore the correlation between the retinal nerve fiber layer (RNFL) thickness by using optical coherence tomography (OCT) and by histological measurements in normal adult rats and optic nerve transected rats. · METHODS: The RNFL thickness of 36 rats was scanned in a circle 3.46mm far from the optic disc by OCT. The two experimental groups were the normal group ( =20 rats) and the optic nerve transected group ( =16 rats). The latter group included 4 groups ( =4 /group) surviving for 1 day, 3, 5 and 7 days. Then the RNFL thickness of the same retina area was also measured by NF -200 immunohistochemical staining method. Linear regression was used to analyze the correlation between the data obtained from these two methods. ·RESULTS: The RNFL thickness of normal right eyes around optic disc by OCT was 72.35 ±5.71μm and that of the left eyes was 72.65 ±5.88μm ( =0.074). The RNFL thickness of the corresponding histological section by immunohistochemistry was 37.54 ±4.05μm (right eyes) and 37.38 ±4.23μm (left eyes) ( =0.059). There was a good correlation between the RNFL thickness measured by OCT and that measured by histology (R 2 =0.8131). After optic nerve transection, the trend of the RNFL thickness was thinner with the prolonged survival time. The correlation of the thickness detected by the above two methods was approximately (R 2 =0.8265). Value of the RNFL thickness in rats around optic disc measured by OCT was obviously higher than that measured by common histological measurement in normal adult rats and optic nerve transected rats. ·CONCLUSION: The RNFL thickness measured by OCT has a strong correlation with that measured by histological method. Through OCT scanning, we found that the thickness of RNFL gradually becomes thinner in a time-dependent manner.展开更多
Objective Numerous studies have indicated that excitatory amino acid toxicity,such as glutamate toxicity,is involved in glaucoma.In addition,excessive glutamate can lead to an intracellular calcium overload,resulting ...Objective Numerous studies have indicated that excitatory amino acid toxicity,such as glutamate toxicity,is involved in glaucoma.In addition,excessive glutamate can lead to an intracellular calcium overload,resulting in regulated necrosis.Our previous studies have found that the calpastatin(CAST)-calpain pathway plays an important role in retinal neuron-regulated necrosis after glutamate injury.Although inhibition of the calpain pathway can decrease regulated necrosis,necrotic cells remain.It has been suggested that there are other molecules that participate in retinal neuron-regulated necrosis.CAST is an important regulator of dynamin-related protein 1(Drp1)-mediated mitochondrial defects.Thus,the aim of this study was to determine whether the CAST-Drp1 pathway may be an underlying signaling axis in neuron-regulated necrosis.Methods Using cultured retinal neurons and in an in-vivo glaucoma model induced by glutamate overload,members of the CAST-Drp1 pathway were assessed by immunofluorescence,Western blotting,Phos-tagTM SDS-PAGE,and co-immunoprecipitation assays.Moreover,the black and white box test was performed on the rats.Results We found that more retinal neuron-regulated necrosis and Drp1 activation as well as lower CAST levels were present in the glutamate-induced glaucoma model.Rats with glutamate-induced glaucoma exhibited impaired visual function.We also observed retinal neuron-regulated necrosis and Drp1 activity decreased,and impaired vision recovered after CAST active peptide application,indicating that the CAST-Drp1 pathway plays a critical role in retinal neuron-regulated necrosis and visual function.Conclusion The results of this study indicate that the CAST-Drp1 pathway protects against retinal neuron-regulated necrosis,which may expand the therapeutic targets for the treatment of neurodegenerative disorders involving dysfunction of glutamate metabolism,such as glaucoma.展开更多
AIM: To clarify the role of inducible nitric oxide synthase(i NOS) in blood-retinal barrier(BRB) injury after acute high intraocular pressure(IOP) in rats.METHODS: Forty-two Sprague-Dawley(SD) rats were randomized int...AIM: To clarify the role of inducible nitric oxide synthase(i NOS) in blood-retinal barrier(BRB) injury after acute high intraocular pressure(IOP) in rats.METHODS: Forty-two Sprague-Dawley(SD) rats were randomized into 7 groups [control(Cont), 3, 6, 12, 24, 48, and 72 h, n=6]. Except Cont group, other groups’ retina tissue was obtained at corresponding time points after a model of acute high IOP have been established in rats. The expression of i NOS and tight junction protein zonula occludens(ZO)-1 was detected by Western blotting. Evans blue(EB;3%) was injected into the great saphenous vein to detect the leakage of EB by spectrophotometer. Nine rats were divided into Cont, 6 h, 12 h groups, the expression of i NOS was localized by immunofluorescence. In order to verify the role of i NOS in the damage to BRB, thirty-six rats were randomly divided into 4 groups [Cont, Cont+inhibitor(Inh), 6 h and 6 h+Inh, n=9]. After treatment with the i NOSspecific inhibitor 1400 W, the expression of i NOS and ZO-1 and the leakage of BRB were detected again.RESULTS: The immunofluorescence results showed that the expression of i NOS was observed in the Cont group and 6 h group, but not in the 12 h group. i NOS was mainly expressed in the retinal nerve fiber layer, ganglion cell layer and inner nuclear layer and that it did not colocalize with the retinal ganglion cell marker Neu N but was co-expressed with the vascular endothelial cell marker CD31. Western blotting showed that in the early period(3 h, 6 h) after acute high IOP, the expression of i NOS was upregulated, then the down-regulation of i NOS were tested in the follow-up timing spots. ZO-1 expression showed a continuous downregulation after 6 h. The quantitative results for EB showed that the amount of EB leakage began to increase at 3 h after acute high IOP. At 6 h, the leakage of EB was lower, but at 12 h, the leakage of EB was highest, after which it gradually recovered but remained higher than that in the Cont group. The expression of i NOS was down-regulated after 1400 W treatment. ZO-1 expression was not significantly changed in the Cont+Inh group and the 6 h group, and significantly down-regulated in the 6 h+Inh group, and the leakage of EB was significantly increased after 1400 W treatment.CONCLUSION: These results suggest that the upregulation of i NOS expression in the early stage after acute high IOP may have a protective effect on BRB injury.展开更多
Because of a lack of sensitive biomarkers,the diagnosis of Alzheimer＇s disease（AD） cannot be made prior to symptom manifestation.Therefore,it is crucial to identify novel biomarkers for the presymptomatic diagnosis...Because of a lack of sensitive biomarkers,the diagnosis of Alzheimer＇s disease（AD） cannot be made prior to symptom manifestation.Therefore,it is crucial to identify novel biomarkers for the presymptomatic diagnosis of AD.While brain lesions are a major feature of AD,retinal pathological changes also occur in patients.In this study,we investigated the temporal changes in β-site APP-cleaving enzyme 1（BACE1） expression in the retina and brain to determine whether it could serve as a suitable biomarker for early monitoring of AD.APP/PS-1 transgenic mice,3,6 and 8 months of age,were used as an experimental group,and age-matched C57/BL6 wild-type mice served as the control group.In the Morris water maze test,there were no significant differences in escape latency or in the number of crossings in the target area among mice of different ages.Compared with wild-type mice,no changes in learning or memory abilities were detected in transgenic mice at 3 months of age.However,compared with wild-type mice,the escape latency was significantly increased in transgenic mice at 6 months,starting on day 3,and at 8 months,starting on day 2,during Morris water maze training.In addition,the number of crossings of the target area was significantly decreased in transgenic mice.The learning and memory abilities of transgenic mice were further worsened at 8 months of age.Immunohistochemical staining revealed no BACE1 plaques in wild-type mice at 3,6 or 8 months or in transgenic mice at 3 months,but they were clearly found in the entorhinal cortex,hippocampus and prefrontal cortex of transgenic mice at 6 and 8 months.BACE1 expression was not detected in the retina of wild-type mice at 3 months,but weak BACE1 expression was detected in the ganglion cell layer,inner plexiform layer and outer plexiform layer at 6 and 8 months.In transgenic mice,BACE1 expression in the ganglion cell layer was increased at 3 months,and BACE1 expression in the ganglion cell layer,inner plexiform layer and outer plexiform layer was significantly increased at 6 and 8 months,compared with age-matched wild-type mice.Taken together,these results indicate that changes in BACE1 expression appear earlier in the retina than in the brain and precede behavioral deficits.Our findings suggest that abnormal expression of BACE1 in the retina is an early pathological change in APP/PS-1 transgenic mice,and that BACE1 might have potential as a biomarker for the early diagnosis of AD in humans.展开更多
基金supported by the National Key R&D Program of China,No.2016YFC1201800(to JFH)the Key Research and Development Program of Hunan Province,Nos.2018SK2090(to JFH),2022SK2079(to JFH)+2 种基金the Natural Science Foundation of Hu nan Province,No.2021JJ30891(to DC)the Human Resource Bank Program of Hunan Province,No.2020TP3003(to JFH)the School-Enterprise Joint Program of Central South University,No.2021XQLH092(to TQD)。
文摘Adipose mesenchymal stem cells(ADSCs)have protective effects against glutamate-induced excitotoxicity,but ADSCs are limited in use for treatment of optic nerve injury.Studies have shown that the extracellular vesicles(EVs)secreted by ADSCs(ADSC-EVs)not only have the function of ADSCs,but also have unique advantages including non-immunogenicity,low probability of abnormal growth,and easy access to target cells.In the present study,we showed that intravitreal injection of ADSC-EVs substantially reduced glutamate-induced damage to retinal morphology and electroretinography.In addition,R28 cell pretreatment with ADSC-EVs before injury inhibited glutamate-induced overload of intracellular calcium,downregulation ofα-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptor(AMPAR)subunit GluA2,and phosphorylation of GluA2 and protein kinase C alpha in vitro.A protein kinase C alpha agonist,12-O-tetradecanoylphorbol 13-acetate,inhibited the neuroprotective effects of ADSC-EVs on glutamate-induced R28 cells.These findings suggest that ADSCEVs ameliorate glutamate-induced excitotoxicity in the retina through inhibiting protein kinase C alpha activation.
基金National Natural Science Foundation of China (No.81070729,No.81100663)Doctoral Foundation of Ministry of Education of China (No.20100162110067)+1 种基金Natural Science Foundation of Hunan Province (No.11JJ2020)Young Teachers Training Program of University of Hunan Province
文摘·AIM: To explore the correlation between the retinal nerve fiber layer (RNFL) thickness by using optical coherence tomography (OCT) and by histological measurements in normal adult rats and optic nerve transected rats. · METHODS: The RNFL thickness of 36 rats was scanned in a circle 3.46mm far from the optic disc by OCT. The two experimental groups were the normal group ( =20 rats) and the optic nerve transected group ( =16 rats). The latter group included 4 groups ( =4 /group) surviving for 1 day, 3, 5 and 7 days. Then the RNFL thickness of the same retina area was also measured by NF -200 immunohistochemical staining method. Linear regression was used to analyze the correlation between the data obtained from these two methods. ·RESULTS: The RNFL thickness of normal right eyes around optic disc by OCT was 72.35 ±5.71μm and that of the left eyes was 72.65 ±5.88μm ( =0.074). The RNFL thickness of the corresponding histological section by immunohistochemistry was 37.54 ±4.05μm (right eyes) and 37.38 ±4.23μm (left eyes) ( =0.059). There was a good correlation between the RNFL thickness measured by OCT and that measured by histology (R 2 =0.8131). After optic nerve transection, the trend of the RNFL thickness was thinner with the prolonged survival time. The correlation of the thickness detected by the above two methods was approximately (R 2 =0.8265). Value of the RNFL thickness in rats around optic disc measured by OCT was obviously higher than that measured by common histological measurement in normal adult rats and optic nerve transected rats. ·CONCLUSION: The RNFL thickness measured by OCT has a strong correlation with that measured by histological method. Through OCT scanning, we found that the thickness of RNFL gradually becomes thinner in a time-dependent manner.
基金supported by the National Natural Science Foundation of China(No.82101126)the Natural Science Foundation of Hunan Province(No.2021JJ40873).
文摘Objective Numerous studies have indicated that excitatory amino acid toxicity,such as glutamate toxicity,is involved in glaucoma.In addition,excessive glutamate can lead to an intracellular calcium overload,resulting in regulated necrosis.Our previous studies have found that the calpastatin(CAST)-calpain pathway plays an important role in retinal neuron-regulated necrosis after glutamate injury.Although inhibition of the calpain pathway can decrease regulated necrosis,necrotic cells remain.It has been suggested that there are other molecules that participate in retinal neuron-regulated necrosis.CAST is an important regulator of dynamin-related protein 1(Drp1)-mediated mitochondrial defects.Thus,the aim of this study was to determine whether the CAST-Drp1 pathway may be an underlying signaling axis in neuron-regulated necrosis.Methods Using cultured retinal neurons and in an in-vivo glaucoma model induced by glutamate overload,members of the CAST-Drp1 pathway were assessed by immunofluorescence,Western blotting,Phos-tagTM SDS-PAGE,and co-immunoprecipitation assays.Moreover,the black and white box test was performed on the rats.Results We found that more retinal neuron-regulated necrosis and Drp1 activation as well as lower CAST levels were present in the glutamate-induced glaucoma model.Rats with glutamate-induced glaucoma exhibited impaired visual function.We also observed retinal neuron-regulated necrosis and Drp1 activity decreased,and impaired vision recovered after CAST active peptide application,indicating that the CAST-Drp1 pathway plays a critical role in retinal neuron-regulated necrosis and visual function.Conclusion The results of this study indicate that the CAST-Drp1 pathway protects against retinal neuron-regulated necrosis,which may expand the therapeutic targets for the treatment of neurodegenerative disorders involving dysfunction of glutamate metabolism,such as glaucoma.
基金Supported by the National Natural Science(No.81660217)National College Students Innovation and Entrepreneurship Training Program Project (No.201911810004)。
文摘AIM: To clarify the role of inducible nitric oxide synthase(i NOS) in blood-retinal barrier(BRB) injury after acute high intraocular pressure(IOP) in rats.METHODS: Forty-two Sprague-Dawley(SD) rats were randomized into 7 groups [control(Cont), 3, 6, 12, 24, 48, and 72 h, n=6]. Except Cont group, other groups’ retina tissue was obtained at corresponding time points after a model of acute high IOP have been established in rats. The expression of i NOS and tight junction protein zonula occludens(ZO)-1 was detected by Western blotting. Evans blue(EB;3%) was injected into the great saphenous vein to detect the leakage of EB by spectrophotometer. Nine rats were divided into Cont, 6 h, 12 h groups, the expression of i NOS was localized by immunofluorescence. In order to verify the role of i NOS in the damage to BRB, thirty-six rats were randomly divided into 4 groups [Cont, Cont+inhibitor(Inh), 6 h and 6 h+Inh, n=9]. After treatment with the i NOSspecific inhibitor 1400 W, the expression of i NOS and ZO-1 and the leakage of BRB were detected again.RESULTS: The immunofluorescence results showed that the expression of i NOS was observed in the Cont group and 6 h group, but not in the 12 h group. i NOS was mainly expressed in the retinal nerve fiber layer, ganglion cell layer and inner nuclear layer and that it did not colocalize with the retinal ganglion cell marker Neu N but was co-expressed with the vascular endothelial cell marker CD31. Western blotting showed that in the early period(3 h, 6 h) after acute high IOP, the expression of i NOS was upregulated, then the down-regulation of i NOS were tested in the follow-up timing spots. ZO-1 expression showed a continuous downregulation after 6 h. The quantitative results for EB showed that the amount of EB leakage began to increase at 3 h after acute high IOP. At 6 h, the leakage of EB was lower, but at 12 h, the leakage of EB was highest, after which it gradually recovered but remained higher than that in the Cont group. The expression of i NOS was down-regulated after 1400 W treatment. ZO-1 expression was not significantly changed in the Cont+Inh group and the 6 h group, and significantly down-regulated in the 6 h+Inh group, and the leakage of EB was significantly increased after 1400 W treatment.CONCLUSION: These results suggest that the upregulation of i NOS expression in the early stage after acute high IOP may have a protective effect on BRB injury.
基金supported by the National Natural Science Foundation of China(to JFH,DC,JBT),No.81371011,81400399,81471107a grant from the Project of Innovation-driven Plan of Central South University(to DC),No.2015CXS022+2 种基金a grant from the National Key Technologies Research and Development Program of China(to JFH),No.2012BAK14B03Fundamental Research Funds of Central South University of China(to HW),No.2010QZZD022Graduate Thesis Innovation Foundation of Central South University of China(to LL),No.2011ssxt106
文摘Because of a lack of sensitive biomarkers,the diagnosis of Alzheimer＇s disease（AD） cannot be made prior to symptom manifestation.Therefore,it is crucial to identify novel biomarkers for the presymptomatic diagnosis of AD.While brain lesions are a major feature of AD,retinal pathological changes also occur in patients.In this study,we investigated the temporal changes in β-site APP-cleaving enzyme 1（BACE1） expression in the retina and brain to determine whether it could serve as a suitable biomarker for early monitoring of AD.APP/PS-1 transgenic mice,3,6 and 8 months of age,were used as an experimental group,and age-matched C57/BL6 wild-type mice served as the control group.In the Morris water maze test,there were no significant differences in escape latency or in the number of crossings in the target area among mice of different ages.Compared with wild-type mice,no changes in learning or memory abilities were detected in transgenic mice at 3 months of age.However,compared with wild-type mice,the escape latency was significantly increased in transgenic mice at 6 months,starting on day 3,and at 8 months,starting on day 2,during Morris water maze training.In addition,the number of crossings of the target area was significantly decreased in transgenic mice.The learning and memory abilities of transgenic mice were further worsened at 8 months of age.Immunohistochemical staining revealed no BACE1 plaques in wild-type mice at 3,6 or 8 months or in transgenic mice at 3 months,but they were clearly found in the entorhinal cortex,hippocampus and prefrontal cortex of transgenic mice at 6 and 8 months.BACE1 expression was not detected in the retina of wild-type mice at 3 months,but weak BACE1 expression was detected in the ganglion cell layer,inner plexiform layer and outer plexiform layer at 6 and 8 months.In transgenic mice,BACE1 expression in the ganglion cell layer was increased at 3 months,and BACE1 expression in the ganglion cell layer,inner plexiform layer and outer plexiform layer was significantly increased at 6 and 8 months,compared with age-matched wild-type mice.Taken together,these results indicate that changes in BACE1 expression appear earlier in the retina than in the brain and precede behavioral deficits.Our findings suggest that abnormal expression of BACE1 in the retina is an early pathological change in APP/PS-1 transgenic mice,and that BACE1 might have potential as a biomarker for the early diagnosis of AD in humans.
