Univocal identification of retinal ganglion cells(RGCs) is an essential prerequisite for studying their degeneration and neuroprotection. Before the advent of phenotypic markers, RGCs were normally identified using re...Univocal identification of retinal ganglion cells(RGCs) is an essential prerequisite for studying their degeneration and neuroprotection. Before the advent of phenotypic markers, RGCs were normally identified using retrograde tracing of retinorecipient areas. This is an invasive technique, and its use is precluded in higher mammals such as monkeys. In the past decade, several RGC markers have been described. Here, we reviewed and analyzed the specificity of nine markers used to identify all or most RGCs, i.e., pan-RGC markers, in rats, mice, and macaques. The best markers in the three species in terms of specificity, proportion of RGCs labeled, and indicators of viability were BRN3A, expressed by vision-forming RGCs, and RBPMS, expressed by vision-and non-vision-forming RGCs. NEUN, often used to identify RGCs, was expressed by non-RGCs in the ganglion cell layer, and therefore was not RGC-specific. γ-SYN, TUJ1, and NF-L labeled the RGC axons, which impaired the detection of their somas in the central retina but would be good for studying RGC morphology. In rats, TUJ1 and NF-L were also expressed by non-RGCs. BM88, ERRβ,and PGP9.5 are rarely used as markers, but they identified most RGCs in the rats and macaques and ERRβ in mice. However, PGP9.5 was also expressed by non-RGCs in rats and macaques and BM88 and ERRβ were not suitable markers of viability.展开更多
AIM:To find a new concept to show whether or not apoptosis of retinal ganglion cells(RGCs)canbe determined in the histology of acute hyperglycemia in the role of expressed Brn3b gene related to nitric oxide(NO),caspas...AIM:To find a new concept to show whether or not apoptosis of retinal ganglion cells(RGCs)canbe determined in the histology of acute hyperglycemia in the role of expressed Brn3b gene related to nitric oxide(NO),caspase-3,nuclear factor kappa-B(NF-κB),and tumor necrosis factor-α(TNF-α)as an early predictor of primary open angle glaucoma(POAG)eyes and their associations.METHODS:Experimental in vivo study was carried out using adult male,white Sprague-Dawley rats aged≥2 mo,weighing 150-200 g.The animals were divided into two groups,one group receiving intraperitoneal injection of streptozotociz 50 mg/kg in 0.01 mol/L citricbuffer and p H 4.5 and a comparison made with the control group.Retinal tissue was divided into two parts(both experimental and control groups respectively):a)right retina for immunohistochemistry(IHC;caspase-3 and TNF-α);b)left retina was divided into two parts for the purpose of real-time polymerase chain reaction(PCR)test(RNA extraction for Brn3b gene expression analysis)and ELISA test(NO and NF-κB).RESULTS:The experimental group showed a decrease in Brn3b gene expression compared to the control group(1.3-fold lower in 2nd month;1.1-fold lower in 4th month and 2.5-fold lower in 6th month).However,there was a decrease of NO,caspase-3,and an increase of NF-κB and TNF-αquantity.CONCLUSION:The expression of m RNA Brn3b gene is inversely proportional to apoptosis in RGCs.The quantity of NO,caspase-3,NF-κB and TNF-αis influential in expression of Brn3b in RGCs caused by hyperglycemia in diabetic rats.展开更多
We explored the appropriate inducing conditions needed to facilitate the differentiation of bone marrow mesenchymal stem cells（BMSCs） into retinal ganglion cells（RGCs）.Math5,a pro-neural basic helixloop-helix（bHL...We explored the appropriate inducing conditions needed to facilitate the differentiation of bone marrow mesenchymal stem cells（BMSCs） into retinal ganglion cells（RGCs）.Math5,a pro-neural basic helixloop-helix（bHLH） gene,was constructed in an adenoviral vector and then infected into the 3rd passage BMSCs.An inverted fluorescence microscope was used to observe the morphological changes of the infected cells.The expressions of Math5,the neuromarkers neuron-specific enolase（NSE）,neurofilament（NF）,Thy1.1,and the RGC-related genes GAP-43 and Brn3b were examined by Western blot and reverse transcription-polymerase chain reaction（RT-PCR）.The results show that cells infected with Math5 adenoviral vector were able to stably express Math5 and presented with a typical morphology of RGCs.Moreover,these cells expressed NSE,NF,Thy1.1,and GAP-43.Under the synergistic induction conditions of retinal conditioned differentiation medium in combination with epidermal growth factor（EGF） and basic fibroblast growth factor（BFGF）,BMSCs infected with Math5 adenoviral vector had a more typical morphology of RGCs,with a greater number of longer axons that connected with each other and formed a net.In addition,the number of NF positive cells was higher,the expression of Brn3b was detected,and the expressions of NSE,NF,and GAP-43 were significantly up-regulated compared to those of them in the control.These results indicate that BMSCs infected with Math5 are able to differentiate into retinal ganglion-like cells.Moreover,Math5 is a stronger activator of the downstream gene Brn3b than the cytokine,which suggests that it is possible to regulate the survival and axon path determination of these differentiated cells.展开更多
基金supported by the Spanish Ministry of Economy and Competitiveness(PID2019-106498GB-I0)Instituto de Salud Carlos III,Fondo Europeo de Desarrollo Regional“Una manera de hacer Europa”(PI19/00071)+2 种基金Fundación Séneca,Agencia de Ciencia y Tecnología Región de Murcia(19881/GERM/15)Spanish Ministry of Science and Innovation(PID 2019-106498 GB-I00)Intramural Research Program of the National Eye Institute,National Institutes of Health(NIH/NEI RO1 EY029087)。
文摘Univocal identification of retinal ganglion cells(RGCs) is an essential prerequisite for studying their degeneration and neuroprotection. Before the advent of phenotypic markers, RGCs were normally identified using retrograde tracing of retinorecipient areas. This is an invasive technique, and its use is precluded in higher mammals such as monkeys. In the past decade, several RGC markers have been described. Here, we reviewed and analyzed the specificity of nine markers used to identify all or most RGCs, i.e., pan-RGC markers, in rats, mice, and macaques. The best markers in the three species in terms of specificity, proportion of RGCs labeled, and indicators of viability were BRN3A, expressed by vision-forming RGCs, and RBPMS, expressed by vision-and non-vision-forming RGCs. NEUN, often used to identify RGCs, was expressed by non-RGCs in the ganglion cell layer, and therefore was not RGC-specific. γ-SYN, TUJ1, and NF-L labeled the RGC axons, which impaired the detection of their somas in the central retina but would be good for studying RGC morphology. In rats, TUJ1 and NF-L were also expressed by non-RGCs. BM88, ERRβ,and PGP9.5 are rarely used as markers, but they identified most RGCs in the rats and macaques and ERRβ in mice. However, PGP9.5 was also expressed by non-RGCs in rats and macaques and BM88 and ERRβ were not suitable markers of viability.
文摘AIM:To find a new concept to show whether or not apoptosis of retinal ganglion cells(RGCs)canbe determined in the histology of acute hyperglycemia in the role of expressed Brn3b gene related to nitric oxide(NO),caspase-3,nuclear factor kappa-B(NF-κB),and tumor necrosis factor-α(TNF-α)as an early predictor of primary open angle glaucoma(POAG)eyes and their associations.METHODS:Experimental in vivo study was carried out using adult male,white Sprague-Dawley rats aged≥2 mo,weighing 150-200 g.The animals were divided into two groups,one group receiving intraperitoneal injection of streptozotociz 50 mg/kg in 0.01 mol/L citricbuffer and p H 4.5 and a comparison made with the control group.Retinal tissue was divided into two parts(both experimental and control groups respectively):a)right retina for immunohistochemistry(IHC;caspase-3 and TNF-α);b)left retina was divided into two parts for the purpose of real-time polymerase chain reaction(PCR)test(RNA extraction for Brn3b gene expression analysis)and ELISA test(NO and NF-κB).RESULTS:The experimental group showed a decrease in Brn3b gene expression compared to the control group(1.3-fold lower in 2nd month;1.1-fold lower in 4th month and 2.5-fold lower in 6th month).However,there was a decrease of NO,caspase-3,and an increase of NF-κB and TNF-αquantity.CONCLUSION:The expression of m RNA Brn3b gene is inversely proportional to apoptosis in RGCs.The quantity of NO,caspase-3,NF-κB and TNF-αis influential in expression of Brn3b in RGCs caused by hyperglycemia in diabetic rats.
基金Supported by the Postdoctoral Science Foundation of China(No.20110490731)
文摘We explored the appropriate inducing conditions needed to facilitate the differentiation of bone marrow mesenchymal stem cells（BMSCs） into retinal ganglion cells（RGCs）.Math5,a pro-neural basic helixloop-helix（bHLH） gene,was constructed in an adenoviral vector and then infected into the 3rd passage BMSCs.An inverted fluorescence microscope was used to observe the morphological changes of the infected cells.The expressions of Math5,the neuromarkers neuron-specific enolase（NSE）,neurofilament（NF）,Thy1.1,and the RGC-related genes GAP-43 and Brn3b were examined by Western blot and reverse transcription-polymerase chain reaction（RT-PCR）.The results show that cells infected with Math5 adenoviral vector were able to stably express Math5 and presented with a typical morphology of RGCs.Moreover,these cells expressed NSE,NF,Thy1.1,and GAP-43.Under the synergistic induction conditions of retinal conditioned differentiation medium in combination with epidermal growth factor（EGF） and basic fibroblast growth factor（BFGF）,BMSCs infected with Math5 adenoviral vector had a more typical morphology of RGCs,with a greater number of longer axons that connected with each other and formed a net.In addition,the number of NF positive cells was higher,the expression of Brn3b was detected,and the expressions of NSE,NF,and GAP-43 were significantly up-regulated compared to those of them in the control.These results indicate that BMSCs infected with Math5 are able to differentiate into retinal ganglion-like cells.Moreover,Math5 is a stronger activator of the downstream gene Brn3b than the cytokine,which suggests that it is possible to regulate the survival and axon path determination of these differentiated cells.
