Anti-vascular endothelial growth factor drugs combined with laser photocoagulation maintain retinal ganglion cell integrity in patients with diabetic macular edema: study protocol for a prospective, non-randomized, controlled clinical trial

The integrity of retinal ganglion cells is tightly associated with diabetic macular degeneration that leads to damage and death of retinal ganglion cells,affecting vision.The major clinical treatments for diabetic macular edema are anti-vascular endothelial growth factor drugs and laser photocoagulation.However,although the macular thickness can be normalized with each of these two therapies used alone,the vision does not improve in many patients.This might result from the incomplete recovery of retinal ganglion cell injury.Therefore,a prospective,non-randomized,controlled clinical trial was designed to investigate the effect of anti-vascular endothelial growth factor drugs combined with laser photocoagulation on the integrity of retinal ganglion cells in patients with diabetic macular edema and its relationship with vision recovery.In this trial,150 patients with diabetic macular edema will be equally divided into three groups according to therapeutic methods,followed by treatment with anti-vascular endothelial growth factor drugs,laser photocoagulation therapy,and their combination.All patients will be followed up for 12 months.The primary outcome measure is retinal ganglion cell-inner plexiform layer thickness at 12 months after treatment.The secondary outcome measures include retinal ganglion cell-inner plexiform layer thickness before and 1,3,6,and 9 months after treatment,retinal nerve fiber layer thickness,best-corrected visual acuity,macular area thickness,and choroidal thickness before and 1,3,6,9,and 12 months after treatment.Safety measure is the incidence of adverse events at 1,3,6,9,and 12 months after treatment.The study protocol hopes to validate the better efficacy and safety of the combined treatment in patients with diabetic macula compared with the other two monotherapies alone during the 12-month follow-up period.The trial is designed to focus on clarifying the time-effect relationship between imaging measures related to the integrity of retinal ganglion cells and best-corrected visual acuity.The trial protocol was approved by the Medical Ethics Committee of the Affiliated Hospital of Beihua University with approval No.(2023)(26)on April 25,2023,and was registered with the Chinese Clinical Trial Registry(registration number:ChiCTR2300072478,June 14,2023,protocol version:2.0).

Cell replacement with stem cell-derived retinal ganglion cells from different protocols

Glaucoma,characterized by a degenerative loss of retinal ganglion cells,is the second leading cause of blindness worldwide.There is currently no cure for vision loss in glaucoma because retinal ganglion cells do not regenerate and are not replaced after injury.Human stem cell-derived retinal ganglion cell transplant is a potential therapeutic strategy for retinal ganglion cell degenerative diseases.In this review,we first discuss a 2D protocol for retinal ganglion cell differentiation from human stem cell culture,including a rapid protocol that can generate retinal ganglion cells in less than two weeks and focus on their transplantation outcomes.Next,we discuss using 3D retinal organoids for retinal ganglion cell transplantation,comparing cell suspensions and clusters.This review provides insight into current knowledge on human stem cell-derived retinal ganglion cell differentiation and transplantation,with an impact on the field of regenerative medicine and especially retinal ganglion cell degenerative diseases such as glaucoma and other optic neuropathies.

Small extracellular vesicles derived from human induced pluripotent stem cell-differentiated neural progenitor cells mitigate retinal ganglion cell degeneration in a mouse model of optic nerve injury

Several studies have found that transplantation of neural progenitor cells(NPCs)promotes the survival of injured neurons.However,a poor integration rate and high risk of tumorigenicity after cell transplantation limits their clinical application.Small extracellular vesicles(sEVs)contain bioactive molecules for neuronal protection and regeneration.Previous studies have shown that stem/progenitor cell-derived sEVs can promote neuronal survival and recovery of neurological function in neurodegenerative eye diseases and other eye diseases.In this study,we intravitreally transplanted sEVs derived from human induced pluripotent stem cells(hiPSCs)and hiPSCs-differentiated NPCs(hiPSC-NPC)in a mouse model of optic nerve crush.Our results show that these intravitreally injected sEVs were ingested by retinal cells,especially those localized in the ganglion cell layer.Treatment with hiPSC-NPC-derived sEVs mitigated optic nerve crush-induced retinal ganglion cell degeneration,and regulated the retinal microenvironment by inhibiting excessive activation of microglia.Component analysis further revealed that hiPSC-NPC derived sEVs transported neuroprotective and anti-inflammatory miRNA cargos to target cells,which had protective effects on RGCs after optic nerve injury.These findings suggest that sEVs derived from hiPSC-NPC are a promising cell-free therapeutic strategy for optic neuropathy.

Casein kinase-2 inhibition promotes retinal ganglion cell survival after acute intraocular pressure elevation

Intraocular pressure elevation can induce retinal ganglion cell death and is a clinically reversible risk factor for glaucoma,the leading cause of irreversible blindness.We previously demonstrated that casein kinase-2 inhibition can promote retinal ganglion cell survival and axonal regeneration in rats after optic nerve injury.To investigate the underlying mechanism,in the current study we increased the intraocular pressure of adult rats to 75 mmHg for 2 hours and then administered a casein kinase-2 inhibitor(4,5,6,7-tetrabromo-2-azabenzimidazole or 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole)by intravitreal injection.We found that intravitreal injection of 4,5,6,7-tetrabromo-2-azabenzimidazole or 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole promoted retinal ganglion cell survival and reduced the number of infiltrating macrophages.Transcriptomic analysis showed that the mitogen activated protein kinase signaling pathway was involved in the response to intraocular pressure elevation but was not modulated by the casein kinase-2 inhibitors.Furthermore,casein kinase-2 inhibition downregulated the expression of genes(Cck,Htrsa,Nef1,Htrlb,Prph,Chat,Slc18a3,Slc5a7,Scn1b,Crybb2,Tsga10ip,and Vstm21)involved in intraocular pressure elevation.Our data indicate that inhibition of casein kinase-2 can enhance retinal ganglion cell survival in rats after acute intraocular pressure elevation via macrophage inactivation.

Effect of Sonic hedgehog gene-modified bone marrow mesenchymal stem cells on graft-induced retinal gliosis and retinal ganglion cells survival in diabetic mice

AIM:To investigate the effects of Sonic hedgehog(Shh)gene-modified bone marrow mesenchymal stem cells(MSCs)on graft-induced retinal gliosis and retinal ganglion cells(RGCs)survival in diabetic mice.METHODS:Bone marrow-derived MSCs were genetically modified with the Shh gene to generate a stably transfected cell line of Shh-modified MSCs(MSC-Shh).Intravitreal injections of MSC-Shh and green fluorescent protein-modified MSCs(MSC-Gfp;control)were administered in diabetic mice.After 4wk,the effects of MSC-Shh on retinal gliosis were evaluated using fundus photography,and markers of gliosis were examined by immunofluorescence and Western blotting.The neurotrophic factors expression and RGCs survival in the host retina were evaluated using Western blotting and immunofluorescence.The mechanisms underlying the effects of MSC-Shh was investigated.RESULTS:A significant reduction of proliferative vitreoretinopathy(PVR)was observed after intravitreal injection of MSC-Shh compared to MSC-Gfp.Significant downregulation of glial fibrillary acidic protein(GFAP)was demonstrated in the host retina after MSC-Shh administration compared to MSC-Gfp.The extracellular signal-regulated kinase 1/2(ERK1/2),protein kinase B(AKT)and phosphatidylin-ositol-3-kinase(PI3K)pathways were significantly downregulated after MSC-Shh administration compared to MSC-Gfp.Brain-derived neurotrophic factor(BDNF)and ciliary neurotrophic factor(CNTF)levels were significantly increased in the host retina,and RGCs loss was significantly prevented after MSC-Shh administration.CONCLUSION:MSC-Shh administration reduces graft-induced reactive gliosis following intravitreal injection in diabetic mice.The ERK1/2,AKT and PI3K pathways are involved in this process.MSC-Shh also increases the levels of neurotrophic factors in the host retina and promoted RGCs survival in diabetic mice.

Regulatory mechanisms of retinal ganglion cell death in normal tension glaucoma and potential therapies

Normal tension glaucoma(NTG)is a multifactorial optic neuropathy characterized by normal intraocular pressure,progressive retinal ganglion cell(RGC)death,and glaucomatous visual field loss.Recent studies have described the mechanisms underlying the pathogenesis of NTG.In addition to controlling intraocular pressure,neuroprotection and reduction of RGC degeneration may be beneficial therapies for NTG.In this review,we summarized the main regulatory mechanisms of RGC death in NTG,including autophagy,glutamate neurotoxicity,oxidative stress,neuroinflammation,immunity,and vasoconstriction.Autophagy can be induced by retinal hypoxia and axonal damage.In this process,ischemia can cause mutations of optineurin and activate the nuclear factor-kappa B pathway.Glutamate neurotoxicity is induced by the over-stimulation of N-methyl-D-aspartate membrane receptors by glutamate,which occurs in RGCs and induces progressive glaucomatous optic neuropathy.Oxidative stress also participates in NTG-related glaucomatous optic neuropathy.It impairs the mitochondrial and DNA function of RGCs through the apoptosis signal-regulating kinase-JUN N-terminal kinase pathway.Moreover,it increases inflammation and the immune response of RGCs.Endothelin 1 causes endothelial dysfunction and impairment of ocular blood flow,promoting vasospasm and glaucomatous optic neuropathy,as a result of NTG.In conclusion,we discussed research progress on potential options for the protection of RGCs,including TANK binding kinase 1 inhibitors regulating autophagy,N-methyl-D-aspartate receptor antagonists inhibiting glutamate toxicity,ASK1 inhibitors regulating mitochondrial function,and antioxidants inhibiting oxidative stress.In NTG,RGC death is regulated by a network of mechanisms,while various potential targets protect RGCs.Collectively,these findings provide insight into the pathogenesis of NTG and potential therapeutic strategies.

Valproate reduces retinal ganglion cell apoptosis in rats after optic nerve crush

The retinal ganglion cells of the optic nerve have a limited capacity for self-repair after injury.Valproate is a histone deacetylase inhibitor and multitarget drug,which has been demonstrated to protect retinal neurons.In this study,we established rat models of optic nerve-crush injury and injected valproate into the vitreous cavity immediately after modeling.We evaluated changes in the ultrastructure morphology of the endoplasmic reticulum of retinal ganglion cells over time via transmission electron microscope.Immunohistochemistry and western blot assay revealed that valproate upregulated the expression of the endoplasmic reticulum stress marker glucose-regulated protein 78 and downregulated the expression of transcription factor C/EBP homologous protein,phosphorylated eukaryotic translation initiation factor 2α,and caspase-12 in the endoplasmic reticulum of retinal ganglion cells.These findings suggest that valproate reduces apoptosis of retinal ganglion cells in the rat after optic nerve-crush injury by attenuating phosphorylated eukaryotic translation initiation factor 2α-C/EBP homologous protein signaling and caspase-12 activation during endoplasmic reticulum stress.These findings represent a newly discovered mechanism that regulates how valproate protects neurons.

Inhibition of retinal ganglion cell apoptosis: regulation of mitochondrial function by PACAP

Pituitary adenylate cyclase-activating polypeptide（PACAP） is an endogenous peptide with neuroprotective effects on retinal neurons, but the precise mechanism underlying these effects remains unknown. Considering the abundance of mitochondria in retinal ganglion cells（RGCs）, we postulate that the protective effect of PACAP is associated with the regulation of mitochondrial function. RGC-5 cells were subjected to serum deprivation for 48 hours to induce apoptosis in the presence or absence of 100 nM PACAP. As revealed with the Cell Counting Kit-8 assay, PACAP at different concentrations significantly increased the viability of RGC-5 cells. PACAP also inhibited the excessive generation of reactive oxygen species in RGC-5 cells subjected to serum deprivation. We also showed by flow cytometry that PACAP inhibited serum deprivation-induced apoptosis in RGC-5 cells. The proportions of apoptotic cells and cells with mitochondria depolarization were significantly decreased with PACAP treatment. Western blot assays demonstrated that PACAP increased the levels of Bcl-2 and inhibited the compensatory increase of PAC1. Together, these data indicate protective effects of PACAP against serum deprivation-induced apoptosis in RGCs, and that the mechanism of this action is associated with maintaining mitochondrial function.

Lycium barbarum polysaccharides protects retinal ganglion cells against oxidative stress injury

The accumulation of excessive reactive oxygen species can exacerbate any injury of retinal tissue because free radicals can trigger lipid peroxidation,protein damage and DNA fragmentation.Increased oxidative stress is associated with the common pathological process of many eye diseases,such as glaucoma,diabetic retinopathy and ischemic optic neuropathy.Many studies have demonstrated that Lycium barbarum polysaccharides(LBP)protects against oxidative injury in numerous cells and tissues.For the model of hypoxia we used cultured retinal ganglion cells and induced hypoxia by incubating with 200μM cobalt chloride(CoCl2)for 24 hours.To investigate the protective effect of LBP and its mechanism of action against oxidative stress injury,the retinal tissue was pretreated with 0.5 mg/mL LBP for 24 hours.The results of flow cytometric analysis showed LBP could effectively reduce the CoCl2-induced retinal ganglion cell apoptosis,inhibited the generation of reactive oxygen species and the reduction of mitochondrial membrane potential.These findings suggested that LBP could protect retinal ganglion cells from CoCl2-induced apoptosis by reducing mitochondrial membrane potential and reactive oxygen species.

Role of endoplasmic reticulum stress in the loss of retinal ganglion cells in diabetic retinopathy

Endoplasmic reticulum stress is closely involved in the early stage of diabetic retinopathy. In the present study, a streptozotocin-induced diabetic animal model was given an intraperitoneal injection of tauroursodeoxycholic acid. Results from immunofluorescent co-localization experiments showed that both caspase-12 protein and c-Jun N-terminal kinase 1 phosphorylation levels significantly in- creased, which was associated with retinal ganglion cell death in diabetic retinas. The C/ERB ho- mologous protein pathway directly contributed to glial reactivity, and was subsequently responsible for neuronal loss and vascular abnormalities in diabetic retinopathy. Our experimental findings in- dicate that endoplasmic reticulum stress plays an important role in diabetes-induced retinal neu- ronal loss and vascular abnormalities, and that inhibiting the activation of the endoplasmic reticulum stress pathway provides effective protection against diabetic retinopathy.

Gender difference in the neuroprotective effect of rat bone marrow mesenchymal cells against hypoxiainduced apoptosis of retinal ganglion cells

Bone marrow mesenchymal stem cells can reduce retinal ganglion cell death and effectively prevent vision loss. Previously, we found that during differentiation, female rhesus monkey bone marrow mesenchymal stem cells acquire a higher neurogenic potential compared with male rhesus monkey bone marrow mesenchymal stem cells. This suggests that female bone marrow mesenchymal stem cells have a stronger neuroprotective effect than male bone marrow mesenchymal stem cells. Here, we first isolated and cultured bone marrow mesenchymal stem cells from female and male rats by density gradient centrifugation. Retinal tissue from newborn rats was prepared by enzymatic digestion to obtain primary retinal ganglion cells. Using the transwell system, retinal ganglion cells were co-cultured with bone marrow mesenchymal stem cells under hypoxia. Cell apoptosis was detected by flow cytometry and caspase-3 activity assay. We found a marked increase in apoptotic rate and caspase-3 activity of retinal ganglion cells after 24 hours of hypoxia compared with normoxia. Moreover, apoptotic rate and caspase-3 activity of retinal ganglion cells significantly decreased with both female and male bone marrow mesenchymal stem cell co-culture under hypoxia compared with culture alone, with more significant effects from female bone marrow mesenchymal stem cells. Our results indicate that bone marrow mesenchymal stem cells exert a neuroprotective effect against hypoxia-induced apoptosis of retinal ganglion cells, and also that female cells have greater neuroprotective ability compared with male cells.

β-Ⅲ-Tubulin: a reliable marker for retinal ganglion cell labeling in experimental models of glaucoma

·AIM: To evaluate the reliability of β-III-Tubulin protein as a retinal ganglion cell(RGC) marker in the experimental glaucoma model.·METHODS: Glaucoma mouse models were established by injecting polystyrene microbeads into the anterior chamber of C57BL/6J mice, then their retinas were obtained 14 d and 28 d after the intraocular pressure(IOP)was elevated. Retinal flat mounts and sections were double-labeled by fluorogold(FG) and β-III-Tubulin antibody or single-labeled by β-III-Tubulin antibody,then RGCs were counted and compared respectively.· RESULTS: IOP of the injected eyes were elevated significantly and reached the peak at 22.8 ±0.7 mm Hg by day 14 after injection, then dropped to 11.3 ±0.7 mm Hg by day 28. RGC numbers counted by FG labeling and β-III- Tubulin antibody labeling were 64 807 ± 4930 and64 614 ±5054 respectively in the control group, with no significant difference. By day 14, RGCs in the experimental group decreased significantly compared to the control group, but there was no significant difference between the FG labeling counting and the β-III-Tubulin antibody labeling counting either in the experimental group or in the control group. The result was similar by day 28, with further RGC loss.·CONCLUSION: Our result suggested that the β-III-Tubulin protein was not affected by IOP elevation and can be used as a reliable marker for RGC in experimental models of glaucoma.

Protective effect of Lycium barbarum polysaccharide on retinal ganglion cells in vitro

AIM: To observe the effect of Lycium barbarum polysaccharide (LBP) on rat retinal ganglion cells (RGCs) In vitro METHODS: Retinal cells of neonatal Sprague-Dawley rats were collected 1 to 3 days after birth, and co-cultured with different concentrations of LBP for 24 hours. Absorbance values (OD) were recorded using MTT assay for calculating survival rates. RESULTS: All the test groups had protective effects on RGCs. The group with 10mg/mL concentration of LOP had the most significantly difference of OD value compared with that in control group ( P<0.01). CONCLUSION: LBP can increase the survival rate and promote the growth of mixed cultured rat RGCs.

Alpha B-crystallin improved survival of retinal ganglion cells in a rat model of acute ocular hypertension

Increased endogenous αB-crystallin protein levels have been shown to reduce cell apoptosis, although the effects of exogenous aB-crystallin protein remain poorly understood. The present study established an acute ocular hypertension model in the right eye of Sprague-Dawley rats. Fluorogold retrograde tracing and immunofluorescence methods showed that the number of retinal ganglion cells decreased in the right eyes and caspase-3 expression increased following acute ocular hypertension. Intravitreal injection of aB-crystallin in the right eye increased the number of retinal ganglion cells and reduced caspase-3 expression. Results demonstrated that exogenous αB-crystallin protein inhibited caspase-3 expression and improved retinal ganglion cell survival following acute ocular hypertension.

Extraction(DSX) from Erigeron breviscapus modulates outward potassium currents in rat retinal ganglion cells

AIMTo investigate the effect of DSX, an active component extracted from Erigeron breviscapus, on the voltage-gated outward K+ channel currents in rat retinal ganglion cells (RGCs) by using electrophysiological method, and to explore the possible mechanisms of DSX on optic nerve protection.METHODSOutward K+ currents were recorded by using whole-cell patch-clamp techniques on acutely isolated rat RGCs. Outward K+ currents were induced by a series of depolarizing voltage pulses from a holding potential of -70 mV to +20 mV in an increment of 10 mV.RESULTSExtracellular application of DSX voltage-dependently suppressed both the steady-state and peak current amplitudes of outward K+ currents in rat RGCs. Furthermore, DSX reversibly and dose-dependently inhibited the amplitudes of outward K+ currents of the cells. At +20 mV membrane potential DSX at the concentrations of 0.02 g/L and 0.05 g/L showed no significant effects on the currents. In contrast, DSX at higher concentrations (0.1 g/L, 0.2 g/L and 0.5 g/L) significantly suppressed the current amplitudes.CONCLUSIONThese results suggest that DSX reversibly and dose-dependently suppress outward K+ channel currents in rat RGCs, which may be one of the possible mechanisms underlying Erigeron breviscapus prevents vision loss and RGC damage caused by glaucoma.

Structural impairment patterns in peripapillary retinal fiber layer and retinal ganglion cell layer in mitochondrial optic neuropathies

AIM：To evaluate the structural injure patterns in peripapillary retinal fiber layer （pRNFL）, retinal ganglion cell layer （RGCL） and their correlations to visual function in various mitochondrial optic neuropathies （MON） to offer help to their differential diagnosis.METHODS：Totally 32 MON patients （60 eyes） were recruited within 6mo after clinical onsets, including 20 Leber hereditary optic neuropathy （LHON） patients （37eyes）, 12 ethambutol-induced optic neuropathy （EON）patients （23 eyes）, and 41 age-gender matched healthy controls （HC, 82 eyes）. All subjects had pRNFL and RGCL examinations with optic coherence tomography （OCT） and visual function tests.RESULTS：In the early stages of MON, the temporal pRNFL thickness decreased （66.09±22.57μm）, but increased in other quadrants, compared to HC （76.95±14.81μm）. The other quadrants remaining stable for LHON and EON patients besides the second hour sector of pRNFL thickness reduced and the temporal pRNFL decreased （56.78±15.87μm） for EON. Total macular thickness in MON reduced remarkably（279.25±18.90μm; P=0.015）, which mainly occurring in the inner circle （3 mm diameter of circle） and the nasal temporal sectors in the outer circle （5.5 mm diameter of circle）, in contrast to those in HC. RGCL thickness reduced in each sector of the macula （61.90±8.73μm; P≤0.001）. It strongly showed the correlationship of best corrected visual acuity （R=0.50, P=0.0003） and visual field injury （R=0.54,P=0.0002） in MON patients.CONCLUSION：OCT is a potential tool for detecting structural alterations in the optic nerves of various MON. Different types of MON may have different damage patterns.

In vivo transfection of enhanced green fluorescent protein in rat retinal ganglion cells mediated by ultrasound-induced microbubbles

BACKGROUND： Studies have demonstrated that ultrasound-mediated microbubble destruction significantly improves transfection efficiency of enhanced green fluorescent protein （EGFP） in in vitro cultured retinal ganglial cells （RGCs）. OBJECTIVE： To investigate the feasibility of ultrasound-mediated microbubble destruction for EGFP transfection in rat RGCs, and to compare efficiency and cell damage with traditional transfection methods. DESIGN, TIME AND SETTING： In vivo, gene engineering experiment. The study was performed at the Central Laboratory, Institute of Ultrasonic Imaging, Chongqing Medical University from March to July 2008. MATERIALS： Eukaryotic expression vector plasmid EGFP and microbubbles were prepared by the Institute of Ultrasonic Imaging, Chongqing Medical University. The microbubbles were produced at a concentration of 8.7 × 10^11/L, with a 2-4 μm diameter, and 10-hour half-life in vitro. METHODS： A total of 50 Sprague Dawley rats were randomly assigned to four groups. Normal controls （n = 5） were infused with 5 μL normal saline to the vitreous cavity; the naked plasmid group （n = 15） was infused with 5 pL EGFP plasmid to the vitreous cavity; in the plasmid with ultrasound group （n = 15）, the eyes were irradiated with low-energy ultrasound wave （0.5 W/cm^2） for a total of 60 seconds （irradiated for 5 seconds, at 10-second intervals） immediately following infusion of EGFP plasmids to the vitreous cavities. In the microbubble-ultrasound group （n = 15）, the eyes were irradiated with the same power of ultrasonic wave immediately following infusion of microbubbles containing EGFP plasmids to the vitreous cavities. MAIN OUTCOME MEASURES： After 7 days, retinal preparations and EGFP expression in RGCs were observed by fluorescence microscopy. RGC quantification in the retinal ganglion cell layer was performed. In addition, EGFP mRNA expression was semi-quantitatively determined by RT-PCR. RESULTS： The transfection efficiency of EGFP to RGCs by microbubbles with ultrasound was significantly greater than the other groups, and no obvious damage was detected in the RGCs. CONCLUSION： Under irradiation of low-frequency ultrasound waves, ultrasound-mediated microbubble destruction was effective and resulted in safe transfection of the EGFP gene to the RGCs.

Characterization of intraocular pressure pattern and changes of retinal ganglion cells in DBA2J glaucoma mice

AIM： To characterize the pattern of intraocular pressure （lOP） change and the deficit of retinal ganglion cells （RGCs） in DBA2J, which is most well-characterized chronic glaucoma mouse model and wild type （WT） C57bl/6 mice, and to study the relationship between lOP change and RGCs deficit. METHODS： lOP was monitored with a rebound tonometer in C57bl/6 and DBA2J mice from 3 to 15-month-old. Retinal function was evaluated by dark -adapted electroretinogram （ERG） in DBA2J and WT mice of 15-month-old. A dye （Neurobiotin） was applied to optic nerve stump to retrograde label RGCs. TO-PRO-3 visualized all nuclei of cells in the RGC layer. RESULTS： The lOP in WT mice was 9.03-0.6 mm Hg on average and did not increase significantly as aging. The lOP in DBA2J mice, arranging from 7.2 to 28 mm Hg, was increasing significantly as aging, and it was normal at 3-month--old compared with WT mice, slightly increased from 7-month-old and increased in 50% animals at 11-month-old and in 38% animals at 15-month-old. The RGCs density in DBA2J mice started reducing by 7-month-old, continuously decreased until reached about 20% of RGC in WT retina by 15-month-old. RGC density was not linearly correlated with lOP in 15-month- old DBA2J mice. The amplitude of positive scotopic threshold response, and negative scotopic threshold response of ERG were significantly reduced in DBA2J mice of 15-month-old than that in age-paired WT mice. CONCLUSION： The present study found that DBA2J mice display pathological and functional deficits of the retina that was not linearly correlated with lOP.

Protective effects of ciliary neurotrophic factor on the retinal ganglion cells by injure of hydrogen peroxide

AIM： To explore the effect of ciliary neurotrophic factor （CNTF） on retinal ganglion cell （RGC）-5 induced by hydrogen peroxide （H2O2）. METHODS： After cell adherence, RGC-5 culture medium was changed to contain different concentrations of H2O2 from 50 to 150 μmol/L at four time points （0.5, 1, 1.5 and 2h） to select the concentration and time point for H2O2 induced model. Two different ways of interventions for injured RGC-5 cells respectively were CNTF as an addition in the culture medium or recombinant lentiviral plasmid carrying CNTF gene transfecting bone mesenchymal stem cells （BMSCs） for co-culture with RGC-5. RESULTS： Compared to the control group, H2O2 led to RGC-5 death closely associated with concentrations and action time of H2O2 and we chose 125 μmol/L and 2h to establish the H2O2-induced model. While CNTF inhibited the loss of RGC-5 cells obviously with a dose-dependent survival rate. Nevertheless two administration routes had different survival rate yet higher rate in recombinant lentiviral plasmid group but there were no statistically significant differences. CONCLUSION： Both the two administration routes of CNTF have effects on RGC-5 cells induced by H2O2. If their own advantages were combined, there may be a better administration route.

Retinal ganglion cell neuroprotection by growth factors and exosomes:lessons from mesenchymal stem cells

Retinal ganglions cells （RGCs） are responsible for propagating electrochemical information from the eye to the brain along their axons which make up the optic nerve. The loss of RGCs is characteristic in several conditions such as glaucoma and trau- matic optic neuropathy and leads to visual loss and blindness. While no therapy exists to directly treat RGCs, the use of bone marrow-derived mesenchymal stem cells （BMSCs） has shown promise in eliciting significant RGC neuroprotection.