Human retinal secretome:A cross-link between mesenchymal and retinal cells

In recent years,mesenchymal stem cells(MSC)have been considered the most effective source for regenerative medicine,especially due to released soluble paracrine bioactive components and extracellular vesicles.These factors,collectively called the secretome,play crucial roles in immunomodulation and in improving survival and regeneration capabilities of injured tissue.Recently,there has been a growing interest in the secretome released by retinal cytotypes,especially retinal pigment epithelium and Müller glia cells.The latter trophic factors represent the key to preserving morphofunctional integrity of the retina,regulating biological pathways involved in survival,function and responding to injury.Furthermore,these factors can play a pivotal role in onset and progression of retinal diseases after damage of cell secretory function.In this review,we delineated the importance of cross-talk between MSCs and retinal cells,focusing on common/induced secreted factors,during experimental therapy for retinal diseases.The cross-link between the MSC and retinal cell secretomes suggests that the MSC secretome can modulate the retinal cell secretome and vice versa.For example,the MSC secretome can protect retinal cells from degeneration by reducing oxidative stress,autophagy and programmed cell death.Conversely,the retinal cell secretome can influence the MSC secretome by inducing changes in MSC gene expression and phenotype.

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.

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.

Neuroprotective effects of resveratrol on retinal ganglion cells in glaucoma in rodents:A narrative review

Glaucoma,an irreversible optic neuropathy,primarily affects retinal ganglion cells(RGC)and causes vision loss and blindness.The damage to RGCs in glaucoma occurs by various mechanisms,including elevated intraocular pressure,oxidative stress,inflammation,and other neurodegenerative processes.As the disease progresses,the loss of RGCs leads to vision loss.Therefore,protecting RGCs from damage and promoting their survival are important goals in managing glaucoma.In this regard,resveratrol(RES),a polyphenolic phytoalexin,exerts antioxidant effects and slows down the evolution and progression of glaucoma.The present review shows that RES plays a protective role in RGCs in cases of ischemic injury and hypoxia as well as in ErbB2 protein expression in the retina.Additionally,RES plays protective roles in RGCs by promoting cell growth,reducing apoptosis,and decreasing oxidative stress in H_(2)O_(2)-exposed RGCs.RES was also found to inhibit oxidative stress damage in RGCs and suppress the activation of mitogen-activated protein kinase signaling pathways.RES could alleviate retinal function impairment by suppressing the hypoxia-i nducible factor-1 alpha/vascular endothelial growth factor and p38/p53 axes while stimulating the PI3K/Akt pathway.Therefore,RES might exert potential therapeutic effects for managing glaucoma by protecting RGCs from damage and promoting their survival.

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.

Etomidate protects retinal ganglion cells from hydrogen peroxide-induced injury via Nrf2/HO-1 pathway

AIM:To determine whether etomidate(ET)has a protective effect on retinal ganglion cells(RGCs)injured with hydrogen peroxide(H_(2)O_(2))and to explore the potential mechanism underlying the antioxidative stress effect of ET.METHODS:Cultured RGCs were identified by double immunofluorescent labeling of microtubule-associated protein 2 and Thy1.1.An injury model of H_(2)O_(2)-induced RGCs oxidative stress was established in vitro.Cells were pretreated with different concentrations of ET(1,5,and 10μmol/L)for 4h,followed by further exposure to H_(2)O_(2)at 1000μmol/L.Cell counting kit 8 and Annexin V/propidium iodide assays were applied to detect the viabilities and apoptosis rates of the RGCs at 12,24,and 48h after H_(2)O_(2)stimulation.The levels of nitric oxide,malondialdehyde,and glutathione in culture media were measured at these time points.Quantitative reverse transcription polymerase chain reaction(qRT-PCR)and Western blot were performed to observe the effects of ET on the messenger RNA and protein expression of inducible nitric oxide synthase(iNOS),nuclear factor erythroid 2-related factor 2(Nrf2),heme oxygenase 1(HO-1),glutathione peroxidase 1 and the level of conjugated acrolein in RGCs at 12,24,and 48h after H_(2)O_(2)stimulation and in the retina at 12h after optic nerve transection(ONT).RESULTS:The applications of 5 and 10μmol/L of ET significantly increased the viability of RGCs.Results from qRT-PCR indicated a decrease in the expression of iNOS and an increase in the expressions of Nrf2 and HO-1 in ETpretreated RGCs at 12,24 and 48h after H_(2)O_(2)stimulation,as well as in ET-treated retinas at 12h after ONT.Western blot analysis revealed a decrease in the expression of iNOS and levels of conjugated acrolein,along with an increase in the expressions of Nrf2 and HO-1 in ET-pretreated RGCs in vitro and ET-treated retinas in vivo.CONCLUSION:ET is a neuroprotective agent in primary cultured RGCs injured by H_(2)O_(2).The effect of ET is dosedependent with the greatest effect being at 10μmol/L.ET plays an antioxidant role by inhibiting iNOS,up-regulating Nrf2/HO-1,decreasing the production of acrolein,and increasing the scavenge of acrolein.

Inhibition of viability of human retinal microvascular endothelial cells by vialinin A under high glucose condition

AIM:To investigate the effects of vialinin A on viability of human retinal endothelial cells(HRECs)under high glucose condition and its potential mechanism.METHODS:The HRECs were divided into four groups:normal glucose control group(NG,5 mmol/L D-glucose),high glucose group(HG,30 mmol/L D-glucose),HG+1μmol/L vialinin A group,and HG+5μmol/L vialinin A group.The cell viabilities were measured with cell counting kit-8(CCK-8)assay for proliferation,with scratch assay for migration,and tube formation,for evaluation of the impact of vialinin A on cellular behaviour.Real-time PCR and Western blotting were used to determine the expression level of vascular endothelial growth factor(VEGF).RESULTS:The proliferative capacity and migration of HRECs was reduced by 5μmol/L vialinin A in high glucose environment(both P<0.05).Vialinin A also inhibited highglucose-induced tube formation of HRECs.The expression level of VEGF and PI3K in HRECs was also significantly decreased by vialinin A(P<0.05).CONCLUSION:Vialinin A inhibits the cell viability of HRECs.It may serve as a potential target for anti-angiogenic therapy.

Cone-rod homeobox transcriptionally activates TCF7 to promote the proliferation of retinal pigment epithelial and retinoblastoma cells in vitro

AIM:To investigate the proliferation regulatory effect of cone-rod homeobox(CRX)in retinal pigment epithelium(RPE)and retinoblastoma(RB)cells to explore the potential application and side effect(oncogenic potential)of CRXbased gene therapy in RPE-based retinopathies.METHODS:Adult human retinal pigment epithelial(ARPE)-19 and human retinal pigment epithelial(RPE)-1 cells and Y79 RB cell were used in the study.Genetic manipulation was performed by lentivirus-based technology.The cell proliferation was determined by a CellTiter-Glo Reagent.The mRNA and protein levels were determined by quantitative real-time polymerase chain reaction(qPCR)and Western blot assay.The transcriptional activity of the promoter was determined by luciferase reporter gene assay.The bindings between CRX and transcription factor 7(TCF7)promoter as well as TCF7 and the promoters of TCF7 target genes were examined by chromatin immunoprecipitation(ChIP)assay.The transcription of the TCF7 was determined by a modified nuclear run-on assay.RESULTS:CRX overexpression and knockdown significantly increased(n=3,P<0.05 in all the cells)and decreased(n=3,P<0.01 in all the cells)the proliferation of RPE and RB cells.CRX overexpression and knockdown significantly increased and deceased the mRNA levels of Wnt signaling target genes[including MYC proto-oncogene(MYC),JUN,FOS like 1(FOSL1),CCND1,cyclin D2(CCND2),cyclin D3(CCND3),cellular communication network factor 4(CCN4),peroxisome proliferator activated receptor delta(PPARD),and matrix metallopeptidase 7(MMP7)]and the luciferase activity driven by the Wnt signaling transcription factor(TCF7).TCF7 overexpression and knockdown significantly increased and decreased the proliferation of RPE and RB cells and depletion of TCF7 significantly abolished the stimulatory effect of CRX on the proliferation of RPE and RB cells.CRX overexpression and knockdown significantly increased and decreased the mRNA level of TCF7 and the promoter of TCF7 was significantly immunoprecipitated by CRX antibody.CONCLUSION:CRX transcriptionally activates TCF7 to promote the proliferation of RPE and RB cells in vitro.CRX is a potential target for RPE-based regenerative medicine.The potential risk of this strategy,tumorigenic potential,should be considered.

Postnatal development of rat retina:a continuous observation and comparison between the organotypic retinal explant model and in vivo development

The organotypic retinal explant culture has been established for more than a decade and offers a range of unique advantages compared with in vivo experiments and cell cultures.However,the lack of systematic and continuous comparison between in vivo retinal development and the organotypic retinal explant culture makes this model controversial in postnatal retinal development studies.Thus,we aimed to verify the feasibility of using this model for postnatal retinal development studies by comparing it with the in vivo retina.In this study,we showed that postnatal retinal explants undergo normal development,and exhibit a consistent structure and timeline with retinas in vivo.Initially,we used SOX2 and PAX6 immunostaining to identify retinal progenitor cells.We then examined cell proliferation and migration by immunostaining with Ki-67 and doublecortin,respectively.Ki-67-and doublecortin-positive cells decreased in both in vivo and explants during postnatal retinogenesis,and exhibited a high degree of similarity in abundance and distribution between groups.Additionally,we used Ceh-10 homeodomain-containing homolog,glutamate-ammonia ligase(glutamine synthetase),neuronal nuclei,and ionized calcium-binding adapter molecule 1 immunostaining to examine the emergence of bipolar cells,Müller glia,mature neurons,and microglia,respectively.The timing and spatial patterns of the emergence of these cell types were remarkably consistent between in vivo and explant retinas.Our study showed that the organotypic retinal explant culture model had a high degree of consistency with the progression of in vivo early postnatal retina development.The findings confirm the accuracy and credibility of this model and support its use for long-term,systematic,and continuous observation.

LEF1 influences diabetic retinopathy and retinal pigment epithelial cell ferroptosis via the miR-495-3p/GRP78 axis through lnc-MGC

BACKGROUND Diabetic retinopathy(DR)is one of the major eye diseases contributing to blindness worldwide.Endoplasmic reticulum(ER)stress in retinal cells is a key factor leading to retinal inflammation and vascular leakage in DR,but its mechanism is still unclear.AIM To investigate the potential mechanism of LEF1 and related RNAs in DR.METHODS ARPE-19 cells were exposed to high levels of glucose for 24 hours to simulate a diabetic environment.Intraperitoneally injected streptozotocin was used to induce the rat model of DR.The expression levels of genes and related proteins were measured by RT-qPCR and Western blotting;lnc-MGC and miR-495-3p were detected by fluorescent in situ hybridization;CCK-8 and TUNEL assays were used to detect cell viability and apoptosis;enzyme-linked immunosorbent assay was used to detect inflammatory factors;dual-luciferase gene assays were used to verify the targeting relationship;and the retina was observed by HE staining.RESULTS LEF1 and lnc-MGC have binding sites,and lnc-MGC can regulate the miR-495-3p/GRP78 molecular axis.In high glucose-treated cells,inflammation was aggravated,the intracellular reactive oxygen species concentration was increased,cell viability was reduced,apoptosis was increased,the ER response was intensified,and ferroptosis was increased.As an ER molecular chaperone,GRP78 regulates the ER and ferroptosis under the targeting of miR-495-3p,whereas inhibiting LEF1 can further downregulate the expression of lnc-MGC,increase the level of miR-495-3p,and sequentially regulate the level of GRP78 to alleviate the occurrence and development of DR.Animal experiments indicated that the knockdown of LEF1 can affect the lnc-MGC/miR-495-3p/GRP78 signaling axis to restrain the progression of DR.CONCLUSION LEF1 knockdown can regulate the miR-495-3p/GRP78 molecular axis through lnc-MGC,which affects ER stress and restrains the progression of DR and ferroptosis in retinal pigment epithelial cells.

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.

Wharton's jelly mesenchymal stem cells differentiate into retinal progenitor cells

Human Wharton＇s jelly mesenchymal stem cells were isolated from fetal umbilical cord. Cells were cultured in serumfree neural stem cellconditioned medium or neural stem cellconditioned medium supplemented with Dkk1, a Wnt/13 catenin pathway antagonist, and LeftyA, a Nodal signaling pathway antagonist to induce differentiation into retinal progenitor cells. Inverted microscopy showed that after induction, the spindleshaped or fibroblastlike Wharton＇s jelly mesenchymal stem cells changed into bulbous cells with numerous processes. Immunofluorescent cytochemical stain ing and reversetranscription PCR showed positive expression of retinal progenitor cell markers, Pax6 and Rx, as well as weakly downregulated nestin expression. These results demonstrate that Wharton＇s jelly mesenchymal stem cells are capable of differentiating into retinal progenitor cells in vitro.

In vitro Culture of Bone Marrow Mesenchymal Stem Cells in Rats and Differentiation into Retinal Neural-like Cells

In order to study the in vitro culture and expansion of bone marrow mesenchymal stem cells in rats （rMSCs） and the possibility of rMSCs differentiation into retinal neural cells, the bone marrow-derived cells in SD rats were isolated and cultured in vitro. The retinal neural cells in SD rats were cultured and the supernatants were collected to prepare conditioned medium. The cultured rMSCs were induced to differentiate by two steps. Immunofluorescence method and anti-nestin, anti-NeuN, anti-GFAP and anti-Thyl. 1 antibodies were used to identify the cells derived from the rMSCs. The results showed that the in vitro cultured rMSCs grew well and expanded quickly. After induction with two conditioned media, rMSCs was induced to differentiate into neural progenitor cells, then into retinal neural-like cells which were positive for nestin, NeuN, GFAP and Thyl. 1 detected by fluorescence method. The findings suggested that rMSCs could be culture and expanded in vitro, and induced to differentiate into retinal neural-like cells.

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.

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.

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.

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.

PI3K/AKT/mTOR signaling pathway inhibitors in proliferation of retinal pigment epithelial cells

AIM: To determine whether the PI3K/AKT/mTOR pathway is activated in proliferative vitreoretinopathy (PVR) in homo-sapiens. METHODS: The retina of controls and patients with PVR were collected and their levels of PI3K, phospho-AKT, phospho-mTOR, phospho-p70S6k and phospho-4EBP-1 were determined by Western blot. The cultured human retinal pigment epithelial cell line D407 was treated with a specific mTOR inhibitor, rapamycin (RAPA) or a PI3K inhibitor, LY294002, of various concentrations and durations. Cell morphology was observed by phase contrast microscopy and the proliferation and apoptosis of treated cells were determined by MTT assay and flow cytometry. RESULTS: Levels of PI3K, phospho-AKT, phospho-mTOR, phospho-P70S6K and phospho-4EBP1 was increased in the retina in PVR (P <0.05). In D407 cells, both RAPA and LY294002 significantly inhibited cell proliferation and cell cycle progression, and promoted apoptosis (P <0.05); morphologically, the cells became smaller. Both RAPA and LY294002 reduced levels of phospho-AKT, phospho-mTOR, phospho-p70S6k and phospho-4EBP1 expression (P <0.05). RAPA, but not LY294002, had no significant effect on PI3K expression. CONCLUSION: PI3K/AKT/mTOR signaling pathway is highly activated in the retinal pigment epithelial cells of PVR. The inhibitors of PI3K/AKT/mTOR signaling pathway, RAPA and LY294002, could inhibited the PI3K/AKT/mTOR signaling pathway by reducing the levels of phosphorylation of mTOR pathway components.

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.