ABSTRACT
In the central nervous system (CNS), three types of myelin-associated inhibitors (MAIs) have major inhibitory effects on nerve regeneration. They include Nogo-A, myelin-associated glycoprotein, and oligodendrocyte-myelin glycoprotein. MAIs possess two co-receptors, Nogo receptor (NgR) and paired immunoglobulin-like receptor B (PirB). Previous studies have confirmed that the inhibition of NgR only results in a modest increase in regeneration in the CNS; however, the inhibitory effects of PirB with regard to nerve regeneration after binding to MAIs remain controversial. In this study, we demonstrated that PirB is expressed in primary cultures of retinal ganglion cells (RGCs), and the inhibitory effects of the three MAIs on the growth of RGC neurites are not significantly decreased after direct PirB knockdown using adenovirus PirB shRNA. Interestingly, we found that retinal Müller cells expressed PirB and that its knockdown enhanced the regeneration of co-cultured RGC neurites. PirB knockdown also activated the JAK/Stat3 signaling pathway in Müller cells and upregulated ciliary neurotrophic factor levels. These findings indicate that PirB plays a novel role in retinal Müller cells and that its action in these cells may indirectly affect the growth of RGC neurites. The results also reveal that PirB in Müller cells affects RGC neurite regeneration. Our findings provide a novel basis for the use of PirB as a target molecule to promote nerve regeneration.
ABSTRACT
In the central nervous system (CNS), three types of myelin-associated inhibitors (MAIs) have major inhibitory effects on nerve regeneration. They include Nogo-A, myelin-associated glycoprotein, and oligodendrocyte-myelin glycoprotein. MAIs possess two co-receptors, Nogo receptor (NgR) and paired immunoglobulin-like receptor B (PirB). Previous studies have confirmed that the inhibition of NgR only results in a modest increase in regeneration in the CNS; however, the inhibitory effects of PirB with regard to nerve regeneration after binding to MAIs remain controversial. In this study, we demonstrated that PirB is expressed in primary cultures of retinal ganglion cells (RGCs), and the inhibitory effects of the three MAIs on the growth of RGC neurites are not significantly decreased after direct PirB knockdown using adenovirus PirB shRNA. Interestingly, we found that retinal Müller cells expressed PirB and that its knockdown enhanced the regeneration of co-cultured RGC neurites. PirB knockdown also activated the JAK/Stat3 signaling pathway in Müller cells and upregulated ciliary neurotrophic factor levels. These findings indicate that PirB plays a novel role in retinal Müller cells and that its action in these cells may indirectly affect the growth of RGC neurites. The results also reveal that PirB in Müller cells affects RGC neurite regeneration. Our findings provide a novel basis for the use of PirB as a target molecule to promote nerve regeneration.
ABSTRACT
Mitochondria play an important role in energy ( ATP ) production through oxidative phosphorylation pathway and the regulation of cell death by apoptosis. Mitochondrial dysfunction has been implicated in the pathophysiology of a number of neurodegenerative diseases. Glaucoma as a neurodegenerative disorder, mitochondrial oxidative stress in the pathogenesis of glaucoma and the damage of RGCs has received close attention in recent years. ln this article, we reviewed the current evidences and recent advances in the relationship between mitochondrial oxidative stress and the RGCs apoptosis.
ABSTRACT
Background Toll-like receptor 4 (TLR4) is an immune related receptor.It plays an important role in inducing inflammation response.The inflammatory response secondary to optic nerve crush will results in serious retinal damage.It is worthy of studying the expression and effect of TLR4 in retina after optic nerve crush.Objective This experimental study was to explore the role of TLR4 in the loss of retinal ganglion cells(RGCs) after optic nerve crush.Methods Twenty-four SPF adult health Sprague-Dawley (SD) rats were used in the study and radomized into two groups based to the experimental time.The optical nerve crush models were established by crushing the optical nerve in the right eyes of the rats,and the left normal eyes served as controls.The rats were sacrificed by over anesthesia and retinas were isolated 3 days and 7 days after operation.Expression of TLR4 in the retinas was detected using immunofluorescence method.Reverse trancription PCR (RT-PCR) and Western blot were applied respectively for the detection of TLR4 mRNA and protein in the retinas.The apoptosis of RGCs was evaluated using TUNEL staining.The use and care of experimental animals followed theGuide for the Care and Use of Laboratory Animals of NIH.This study was approved by the Institutional Animal Care and Use Committee at the Zhongshan Ophthalmic Center.Results The expression of TLR4 in rat retinas presented with green fluorescence mainly in the inner layer of retinas.The fluorescence was enhanced in the model 3 days group and the model 7 days group compared with the corresponding control groups.The relative expressing values of TLR4 mRNA in the retinas were 2.92±0.06and 3.92±0.12 in the model 3 days and 7 days groups,respectively,which were significantly higher than 2.87±0.12and 3.44±0.17 in the control 3 days and 7 days group (t3d =-12.888,P＜0.001 ;t7d =-4.669,P=0.010).In the model 3 days group and model 7 days group,the grey values of TLR4 protein were 1.14±0.05 and 1.49±0.03,and those in the control 3 days and 7 days groups were 0.99 ± 0.09 and 1.38 ± 0.07,showing significant differences between them(t3d =-11.324,P＜0.001 ;t7d =-5.638,P=0.005).Apoptotic RGCs were obviously increased in the optic nerve damage group in comparison with the control group.Conclusions The TLR4 is over-expressed in the inner layer of retina after optic nerve crush,which suggestes that TLR4 is probably involved in the loss of RGCs after optic nerve crush.
ABSTRACT
Background Neuroglobin (Ngb) is a newly discovered member of globin superfamily.It is thought to regulate cell survival under hypoxia or oxidative stress condition.Ngb is expressed at a high level in retinal neuron,suggesting that retina may be one of important functional sites of Ngb.Objective The aim of this study was to investigate the protective role of endogenous Ngb on retina ganglion cells (RGCs) following chronic high intraocular pressure(IOP)in mice and the underlying mechanisms.Methods This study included the in vitro and in vivo experiment.RGCs derived from adult C57BL/6J wild type(WT) mice and Ngb-transgenic(Ngb-Tg) mice which cultivated by our laboratory were incubated with 5.0,7.5,10.0 mmol/L glutamic acid for 3 days.RGCs survival rate was calculated for the ration of dead and survival cells using a double labeling kit to evaluate the influence of Ngb on RGCs survival rate in the addition of glutamic acid.Chronic ocular hypertension models were established by injection of fluorescent microballon(MB) (10 μm)into the anterior chamber of WT mice and Ngb-Tg mice,The mice were divided into WT control group(n=18),Ngb-Tg control group(n=30),WT+MB single injection group(n=38),Ngb-Tg+MB single injection group (n =38),WT+MB twice injection group (n =6) and Ngb-Tg + twice injection group (n=6).In addition,WT+PBS injection group (n =6) and Ngb-Tg+ PBS injection group (n =6) were designed as negative controls to identify if it can affect IOP or not.The mice were sacrificed on 0 day(control group),3 days and 1,4,8 weeks followed the MB anterior chamber injection.Real-time PCR,Western blot and immunoytochemistry were used respectively for the analysis of the expressions of Ngb mRNA and protein in mouse retina,and the survival rates of RGCs were compared between the two types mice.Dihydroethidium (DHE) in retina was detected after cardiac perfusion and ATP level in mouse retina homogenate was analyzed.Results The RGCs survival rate was significantly higher in Ngb mice compared with WT mice in 5.0,7.5 and 10.0 mmol/L glutamic acid treatcd groups (t =2.810,3.020,3.110,P＜ 0.01).IOP of WT+ MB single injection group and Ngb-Tg+ MB single injection group were elevated in comparison with the WT control group and WT+PBS injection group and the high IOP remained for 4 weeks.Ngb level was raised in the WT+MB single injection group on the third day following injection,but the Ngb concentration remained a high level in the Ngb-Tg mice during the period of the experiment duration.The RGCs apoptosis rate was elevated both in the WT mice and Ngb-Tg mice 1,4,8 weeks after injection of MB,however,the cell apoptosis rate was higher in WT mice than that of Ngb-Tg mice(P＜0.05).DHE content in retina in the Ngb-Tg+MB single injection group was significantly lower than that in the WT+MB single injection group (t =3.212,P=0.008),and ATP content in retina was elevated in the Ngb-Tg+MB single injection group compared with WT+MB single injection group(t =2.864,P＜0.01).Conclusions It is suggested that Ngb might be a neuroprotective molecules against RGCs death by decreasing oxidative stress and improving mitochondria function.
ABSTRACT
Background Glaucoma is common blinding eye diseases characterized by chronic loss of retinal ganglion cells(RGCs).Currently glaucoma pathogenesis is not completely understood,heat shock protein 27 ( HSP27 )may be associated with the pathogenesis of glaucomatous optic neuropathy. Objective Through the establishment of a rat model of high intraocular pressure,detection of the expression of HSP27 antibody in serum and RGCs apoptosis to investigate the role of HSP27 in RGCs apoptosis. Methods Fifty-one clean Wistar rats were divided into high intraocular pressure group (34 rats)and sham operation group( 17 rats)using a random number table.An animal model of high intraocular pressure was established in the right eye by the application of bipolar underwater electrocoagulation on vein of sclera surface in the experimental group,and rats with conjunctiva incision only without electric coagulation were served as sham operation (control).The intraocular pressure of rats of the both groups including experimental and control rats was measured 1,2,4,6,8 weeks after operation and then the rats were sacrificed.1 ml serum was collected from these rats to determine the concentration of HSP27 antibody.The retinas of the rats were isolated and homogenated for the extraction and analysis of the retinal protein by Western blot.Apoptosis of RGCs were assayed by TUNEL.The use of the experimental animals followed the Regulations for the Administration of Affair Concerning Experimental Animals by Kunming Medical Collegc. Results Intraocular pressure was elevated significantly after modeling and remained a high value during the expcrimental duration,showing a significant difference among the different groups ( F =318.502,P＜0.01 ).However,no significant change in intraocular pressure before and after operation in the sham-operation group ( F =2.076,P ＞ 0.05 ).The serum concentration of HSP27 antibody was increased significantly overtime from 1,2,4,6 to 8 weeks after the surgery applied,with a significant difference among various time points( F =154.221,P＜0.01 ),but no obvious difference was seen in the shamoperation group( F =0.422,P＞0.05 ).Importantly,Apoptosis rate of RGCs in the experimental eyes was significant higher as the time prolong after intraocular elevated(x2=856.12,P＜0.05 ).The positive rate of RGCs apoptosis in the rats with high intraocular pressure group was elevated significantly compared to the rats in the sham operation group(P＜0.05). Conclusions High intraocular pressure induces the expressions of HSP27 in retina and the accumulation of HSP27 antibody in rats serum;the elevated HSP27 is associated with the increased cell death in RGCs.
ABSTRACT
In order to evaluate the efficacy of traditional paeonia extract paeoniflorin against optic nerve crush, 16 Brown Norway rats were divided into two groups at random, with 8 rats in each group. In paeoniaflorin-treated group, 2 mg paeoniaflorin (total volum: 1 mL) was injected into rat's peritoneum one time a day for a period of 8 days. Rats in untreated group were given a single dose of vehicle. The optic nerve was crushed by a special forceps for 30 s in the left eye and a sham proce-dure was performed in the right eye on the 2nd day after the first injection. The retrograde fluorogold labeling of ganglion cells was conducted 5 days after optic nerve crush. The whole retina was flat-mounted thereafter. The ganglion cells that survived the crush were counted under fluorescent microscope by using an automatic counting software. As compared with the contralateral eye, the survival rate of ganglion cells in the left eye increased from 40.22% to 64.53% with a significant dif- ference found between them (t=2.55, P=0.023). The results suggested that the paeonia extract paeoniflorin possessed a protective effect against optic nerve crush.