Learning curve of a trained vitreo-retinal surgeon in sub-retinal injections in a rat model: Implications for future clinical trials

Purpose: The sub-retinal injections are not very commonly performed procedures in vitreoretina, but form a crucial step in any cell replacement therapy for retinal diseases. The purpose of this study is to describe the learning curve of a trained vitreo-retinal surgeon in sub-retinal injections in a rat model and its implications in future clinical trials. Methods: This is an in-vivo retrospective animal study using Wistar rats. All ARVO guidelines regarding animal handling were followed. After anesthetization, aspectic preparation and dilating the pupils with 1% tropicamide eye drops, subretinal injection of 10 ?l saline was done via a limbal entry. Data recorded included time taken for the procedure, success of injection, associated complications, post-operative infections and complications. The rats were followed up for 1 month post procedure. A trend analysis was done for the above factors to look for improvement in ease of procedure, reduction in procedure time and reduction in complications for the clinician using a novel objective scale. Results: About 20 eyes were studied. Mean weight of the rats was 188 ± 12.82 gram. Mean time taken for the procedure was 14.1 ± 5.07 minutes. There was a significant inverse co-relation between the serial number of the eye and time taken for the procedure (r = ?0.89, P < 0.0001). Comparative complications noted between the first ten and the last ten eyes were: conjunctival tear 30% versus 10% (P = 0.27), lens touch 50% versus 10% (P = 0.05), subretinal hemorrhage 40% versus 0% (P = 0.13), vitreous loss 30% versus 0% (P = 0.06). The successful subretinal injection without intraocular complications was achieved in 40% versus 90% (P = 0.02). There was a significant co-relation between the serial number of the eye and ease of the procedure (r = 0.87, P < 0.0001). Post operatively none of the eyes had any infection. Six eyes (12%) developed cataract and 3 eyes (6%) had non-resolving retinal detachment at the last examination visit. Conclusion: Subretinal injections in rats have a definite learning curve even for a trained vitreoretinal surgeon. This should be accounted for and resources allocated accordingly to achieve good technical comfort and negate confounding by the surgeon factor in the results of future clinical trials

Retinal ganglion cell-conditioned medium alters the differentiation of rat retinal progenitor cells / 眼科新进展

Objective To investigate the effects of retinal ganglion cell-conditioned medium on the differentiation of retinal stem cells.Methods Rettial stem cells and retinal ganglion cells were isolated from rats,and immunofluorescence staining was applied to identify rat retinal stem cells and retinal ganglion cells with Nestin and Thy-1 antibody,respectively.Retinal stem cells were cultured in presence or absence of retinal ganglion cell-conditioned medium for 72 h,followed by detection of Nestin,PAX6,Thy-1 and Bin-3 gene expression in retinal stem cells by qPCR.Results isolated retinal stem cells were Nestin positive,and retinal ganglion cells were Thy-1 positive,indicating the success of isolation.Compared to retinal stem cells cultured without ganglion cellconditioned medium,ones cultured with ganglion-conditioned medium had significantly downregulated expression of Nestin and PAX6 (both P ＜ 0.000 1),and markedly upregulated expression of Thy-1 and Brn-3 (both P ＜ 0.05).Conclusion Retinal ganglion cell-conditioned medium can induce the differentiation of retinal progenitor cells into retinal ganglion-like cells.

Therapeutic effect of Ginkgobalide B and retinal stem cells transplantation on glaucoma in rats / 国际眼科杂志(Guoji Yanke Zazhi)

AIM:To investigate the combination therapeutic effect of Ginkgobalide B (GKB) and retinal stem cells (RSCs)transplantation on glaucoma in rats.METHODS:Rats were divided randomly into five groups:control group,glaucoma group,RSCs group,GKB group and RSCs combination therapy group.A chronic glaucoma model was established in rats,accordingly.The morphological changes in ocular tissues were analyzed by HE staining.Retinal ganglion cells apoptosis were analyzed by TUNEL staining.The protein expressions of Bcl-2,Bax,Cleaved caspase-3 and Cleaved caspase-9 were determined by Western blot.The mRNA levels of Bcl-2 and Bax were determined by qPCR.RESULTS:HE staining revealed that RSCs transplantation or GKB treatment decreased fiber interstitial edema and vacuole,as compared to glaucoma group.Furthermore,this improvement was more pronounced in combination therapy group than in single treatment alone.Combination therapy significantly inhibited retinal ganglion cells apoptosis,increased Bcl-2 mRNA and protein expression,but decreased Bax mRNA and protein expression.Moreover,the protein expression of Cleaved caspase-3 and Cleaved caspase-9 expression were decreased after combination therapy.CONCLUSION:Our data demonstrate that combination of Ginkgobalide B and retinal stem cells transplantation can inhibit retinal ganglion cells apoptosis and protect against glaucoma.These effects may be associated with the regulation of Bcl-2,Bax,Cleaved caspase-3 and Cleaved caspase-9 expression.

Effect of RSCs combined with COP-1 on optic nerve damage in glaucoma rat model

OBJECTIVE@#To explore effect of retinal stem cells (RSCs) combined with copolymer-1 (COP-1) immunotherapy on optic nerve damage in glaucoma rat model.@*METHODS@#A total of 40 SD rats were selected for glaucoma model and were randomly divided into 4 groups to observe protective effects of RSCs transplantation combined with COP-1.@*RESULTS@#Brain-derived neurotrophic factor (BDNF) and insulin like growth factor-1 (IGF-1) were either positive in retina of RSCs transplanted or COP-1 immunological treated rat. Positive rate of BDNF and IGF-1 and expression of mRNA and protein were significantly higher in RSCs transplantation combined with COP-1 immunotherapy treated rats compared with the other 3 groups, in which amount of apoptotic RGCs was lowest.@*CONCLUSIONS@#RSCs transplantation combined with COP-1 immunotherapy can promote the secretion of BDNF and IGF-1. They protect RGCs in glaucoma rats in coordination, significantly reduce the number of apoptosis RGCs so as to alleviate the optic nerve damage. It ponits a new research direction for treatment of glaucoma.

Terapia celular nas doenças oftalmológicas / Cell therapy in ophthalmology diseases

Avanços no campo das células-tronco proporcionaram muita perspectiva para uso destas células na regeneração dos tecidos oculares danificados por doenças as quais não possuem tratamento disponível até o momento. Terapia baseada com células-tronco para regeneração e reparo ocular constitui uma esperança para a restauração da função visual em indivíduos com tecido oculares irreversivelmente danificados por doença ou trauma. Até o presente momento, somente as células LESC para o reparo da córnea apresentam aplicação clínica reconhecida no campo da oftalmologia. Experiências adquiridas com esta abordagem poderão potencialmente ajudar com o desenho de terapias baseadas em células-tronco para regenerar outros tecidos oculares particularmente a retina. Utilização de células-tronco adultas para tratamento de doenças degenerativas da retina tem sido testada e também sua viabilidade para utilização em humanos. Apesar de muitos problemas práticos, existe um otimismo geral entre a comunidade médica e científica que a terapia baseada nas células-tronco para restaurar a função visual pode tornar-se realidade em um futuro não muito distante. Neste manuscrito, foi realizada uma revisão do estado atual e limitações na aplicação de células-tronco para terapia ocular e consideramos as perspectivas futuras de seu uso na restauração da visão.

Advances in the field of stem cell therapy have provided promising results in the regeneration of tissues damaged by eye diseases for which treatment is so far unavailable. Therapy based on stem cells to regenerate and repair is a hope for the restoration of visual function in individuals with ocular tissue irreversibly damaged by disease or trauma. Until now, only limbal epithelial stem cells have a recognized clinical application in ophthalmology for the repair of the cornea. Experience gained with this approach, may potentially help with the design of therapies based on stem cells to regenerate other eye tissues, in particular the retina. The use of adult stem cells to treat degenerative diseases of the retina has been tested in animals, as has their feasibility for use in humans. Although many practical problems exist, there is general optimism among the medical and scientific community that therapy based on stem cells to restore visual function can become a reality in the not too distant future. This article reviews the current status and limitations of the application of stem cell therapy in the eye and considers future prospects of its use in the restoration of vision.

Activation of endogenous retinal stem cells in RCS rats during onset and development of retinal degeneration / 第三军医大学学报

Objective To investigate whether endogenous retinal stem cells can be activated in the retina of Royal College of Surgeons (RCS) rats during the onset and development of retinitis pigmentosa. Methods The RCS-p+ rats with inherited retinal dystrophy were divided into 3 groups: the initial stage group (15-day RCS rats), the mid-stage group (30-day RCS rats) and the advanced stage group (90-day RCS rats) according to the severity of degeneration (n=4 in each group). RCS-rdy+p+ rats without retinal degeneration served as controls, and divide into three groups (15-day control, 30-day control, 90-day control) matched with RCS-p+ rats. A transcription factor (Chx10) expressed by embryonic retinal progenitors was detected using immunofluorescence and Western blotting. Results All of the retinal layers in the three control groups and in the 15-day RCS rats did not express Chx10, while the positive expression was observed in the photoreceptor layers of the 30-day and 90-day RCS rats. Chx10 protein could be detected by Western blotting in all RCS groups, but expressed higher in 30-day RCS rats than in 15-day and 90-day RCS rats (P