Objective To observe the distribution and concentration of ~(125)I-nerve growth factor (NG-F) in rabbits' eyes after intravitreal injection and posterior juxtascleral injection.Methods Intravitreal injection(group A) and posterior juxtascleral injection (group B) were performed with the dosage of 30μg/100μl ~(125)I-NGF on left and right eyes in 45 white rabbits respectively.The γ-counts and the concentration of ~(125)I-NGF (%ID/g) of each ocular tissue was determined 15 and 30 minutes,and 1,3,6,12,24,and 48hours after injection.Results The ~(125)I-NGF diffusion in group A was faster in ocular content and ocular inner wall.The vitreous content of ~(125)I-NGF decreased gradually in group A,the curve changes in other eye tissues were normal.The concentration of ~(125)I-NGF reached the peak 3 hours after injection in aqueous humor,iris and ciliary body,retina,and choroids,but 6 hours after injection in sclera and 8 hours in cornea.The changes of concentration of ~(125)I-NGF in group B showed normal curve change.The peak time in group B were all 6 hours,in all the tissues except aqueous humor (3 hours).Except the high concentration in vitreous body caused by intravitreal injection,the concentration of ~(125)I-NGF in retina was the highest in group A.Conclusion Intravitreal injection of ~(125)I-NGF can gain higher concentration in each ocular tissue than posterior juxtaseleral injection,especially in retina.So intravitreal injection of NGF is a better ocular delivery method to treat the ocular fundus diseases.

Objective To observe the mutifocal electroretinogram (mfERG) characteristics of rod and cone cells in patients with retinitis pigmentosa (RP) and to evaluate the function of photosensory cell.Methods The mfERG recording technique for rod cell in eight normal subjects (eight eyes) were established and the influence of different brightness lightstimulus in P1 wave amplitude were analyzed.The cone and rod cells mfERG of 38 eyes in 19 patients were recorded and then calculated positive ratio from local signal-noise ratio.The average visual acuity and P1 wave amplitude density of cone mtERG in different types were compared and statistically analyzed.Meanwhile,the changes in P1 wave amplitude of cone and rod mfERG in four quadrants also compared and analyzed.Results Rod cell mfERG in normal subjects can be recorded stably by using blue flashes with low light intensity as 0.04 cd/m~2.In patients with RP,the cone and rod cells mfERG can he detectd 65.790./00 and 10.51% respectively.P1 wave amplitude density in type I of cone cell mfERG was significantly higher than that in type Ⅱ (t=5.21,P=0.000).There were no differences in average visual acuity (t=1.15,P=0.612).P1 wave amplitude density in type Ⅰ was negatively related to IogMAR visual acuity (r=-0.48,P=0.04 ).The comparison of rod and cone cells mfERG in local wave characteristics showed that P_1 wave amplitude densities had spatial relationship in each area.Conclusions The results suggested highly variable central responses in cone cell in RP patients,higher positive recorded ratio in cone cell than rod cell and spatial correspondence between the function of reserved cone and rod cells.

Objective To observe safety of intravitreal injection of mouse nerve growth factor and its distribution in retina in rabbits .Methods The behavioral observation ,slit lamp examination ,fundus examination ,eye B ultrasonic and histopathological ex‐amination were carried out on 1 ,7 and 30 d after intravitreal injection 30 μg/100 μL mNGF to determine the safety in eye .The dis‐tribution and peak time in retina were investigated at 15 ,30 min ,1 ,3 ,6 ,8 ,12 ,24 ,48 h after intravitreal injection 125 I‐NGF 30 μg/100 μL .Results No abnormal changes were found in their cornea ,lens ,vitreous body and retina after mNGF intravitreal injection . And the each layer of retinal cells layout were regular according to the result of morphological observation on 30 days after treat‐ment .The peak concentration of mNGF in retina and the highest in whole eye was (118 .32 ± 18 .74)% ID/g and the peak time was at 3 hour after injection .Conclusion It is safe for intravitreal injection of mNGF and mNGF could gather in retina quickly after in‐travitreal injection .

Background Researches showed that stem cells can rescue damaged cells through mitochondrial transfer.This mode has been used to regenerative cell-based therapy.Retinal pigment degeneration is an eye disease of retinal pigment epithelial (RPE) cell apoptosis as pathogenetic mechanism.Whether stem cells can repair the target cells by above mechanism has not been clarified.Objective This study was to investigate the influence of mitochondrial transfer on the function of RPE cells.Methods The RPE cells of Long-Evans rats were isolated and cultured and the third generation of cells were used in sequential experiment.The cells were identified by detecting the expressions of RPE65 and Bestrophin proteins with immunofluorescence stain.Mouse neural stem cells (NSCs) (C17.2 strain) with green fluorescence protein (GFP) and without GFP were cultured.Mitotracker-green and Mitotracker-red staining were separately used to labeled the mitochondria of RPE cells and NSCs.RPE cells were cocultured with NSCs,and wheat germ agglutinin (WGA) was used to mark the tunneling nanotubes (TNT) between the two kinds of cells,and then the mitochondrial migration in TNT was exhibited by the laser scanning confocal microscope.The proportion of RPE cells in different cycles was assayed after marked with propidium iodide (PI) by flow cytometry.The contents of ATP,ADP and AMP in RPE cells were detected by high performance liquid chromatography (HPLC).Results The third-generation of RPE cells grew well with the RPE65-and Bestrophinpositive rate ＞85％.The Mitotracker-red-labeled rates of NSCs and RPE cells were no less than 95％.TNT structure was seen to appear the blue fluorescence between RPE cells and NSCs 24 hours after co-cultured and the red dye mitochondria from NSCs migrated toward red dye mitochondria from RPEs with the lapse of time.The RPE cell proportion reduced in G1 phase and increased by 5％ and 2％ in the S phase and G2/M phase respectively after mitochondrial transfer than before (P=0.016,0.114,0.189).The contents of ATP,ADP and AMP in the RPE cells were (8.77 ±3.68),(2.76±0.92) and (1.07 ±0.65) μg/mg after cell co-culture,and those before co-culture were (11.29±2.29),(3.12±0.95) and (1.59± 1.22) μg/mg,without significant differences between them (P =0.370,0.668,0.553).Conclusions NSCs can transfer normal mitochondria to co-cultured RPEs via TNT structure.Mitochondrial exchange might be one of therapeutic mechanisms of NSCs recuing damaged RPE cells.

Stem cells are a group of undifferentiated cells with indefinite self-renewal and pluripotent differentiation ability.They are able to differentiate into precursor/progenitor cells and a variety of cell types and further regenerate new cells to be involved in the repair and rebuilding of injured tissue.Therefore,stem cells are becoming the major research objects in the study on tissue engineering therapy and regenerative medicine.As a sense organ composed of several kinds of neurons and other cells,eyes possesse the dominant superiority in stem cell transplantation therapy because of its good operation controllability and visuality,less demand for seed cells and low rejection after transplantation.These advantages have aroused growing interesting of the fundamental research and clinical trail in stem cell transplantation for irreversible eye diseases.Some exciting advances in the field of stem cell fundamental research,several phase Ⅰ/Ⅱ clinical trials are in progress.The patients with degenerative eye diseases for phase Ⅰ clinical trials are in recruiting in China to evaluate future curative effect and security of stem cell-based therapies.Currently,several issues in stem cell-based eye disease therapies are still pending.We discuss the updated development of stem cell-based transplantation in ophthalmology and future researching direction in order to help ophthalmological researchers to understand the concepts and research strategies.

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

Objective To explore the distribution and features of the optic cup stem cells in embryonic rat at tailbud stage. Methods The distribution of optic cup stem cells in optic cup tissue in 12.5-embryonic-day-old rats was observed by immunohistochemistry. The separated cells from optic cup were cultured with serum-free media, and immunofluorescence technique was used to detect the ability of hyperplasia of stem cells and expression of CHX10 antigen and specific antigens of mature retinal cells before and after differentiation. Results The optic cup stem cells in embryonic rat at tailbud stage were mainly located at inner, outer, and marginal layer of optic cup. No expression of specifically marked protein of mature retinal cells was detected. The cells separated from optic cup had the ability of single-cell clone, positive expression of CHX10 and expression of several specific antigens of mature retinal cells after the inducement, including Thy1.1, glial fibrillary acidic protein (GFAP), protein kinase C (PKC) ?, and rhodopsin. Conclusion Optic cup of 12.5-embryonic-day-old rats composes of undifferentiated cells, and the stem cells are mainly located in optic cup inner and marginal. High ability of hyperplasia of the optic cup stem cells cultured in vitro is found. The cells, which are retinal stem cells, can express several specifically marked proteins of mature retinal cells after inducement and differentiation.

Objective To describe cultured human retinal pigment epithelial (RPE) cells transdifferentiation and investigate the effects of human vitreous fluid on the morphologic and cytoskeleton changes of RPE cells in vitro. Methods Cytoskeleton characteristics in the 2 nd, 5 th, 8 th passage of RPE cells in normal culture, which included cytokeratin 18 (CK18) and ?-smooth muscle actin (?-SMA) were analyzed by Western blot. RPE cells were cultured in human vitreous-conditioned medium (VCM) at the concentration of 1∶4 for 6 days, morphologic changes were examined by light and electron microscopy, and cytoskeleton characteristics were analyzed by imunocytochemistry and Western blot. Results During culture in vitro, RPE cells lost epithelial characteristics and aquired fibroblast-like phenotype. The expression of CK18 was the highest at the 5 th passage, and it decreased in the following passage, but ?-SMA increased gradually. The morphologic transdifferentiation from epithelial to fibroblast-like cells of RPE was accelerated by VCM. Ultrastructural changes such as decreased microvilli and gradually increased rough endoplasmic reticulum and Golgi complex were found during the cultivation. CK18 produced by RPE cells decreased in VMC (P

Objective To observe the electrophysiological and morphological features of retinal ganglion cells (RGCs) in rats, and investigate its effect on the visual signal conduction. Methods Whole cell recordings were obtained from 112 RGCs of 30 rats at the age of 7-30 days. Resting membrane potential (RMP) was recorded, and input impedance was noted after given 2 mV hyperpolarizing current by voltage clamp. The action potential (AP) was induced by deplorizing current at different densities. The histological staining was actualized by injecting with biotin into the RGCs, and the diameter of the cells was measured. Results Three different discharge patterns of RGCs in response to maintained depolarizing currents were recorded: single spike (25 RGCs), transient firing (40 RGCs), and sustained firing (47 RGCs). The diameter was 14-16 ?m in 57.14% transient firing RGCs, and 10-12 ?m in 62.50% sustained firing RGCs. The maximum frequency of AP of sustained firing RGCs was significantly higher than that of transient firing RGCs (P

Objective To study the transplantation of embryonic full-thickness retina, including neural retina and retinal pigment epithelial (RPE), into the subretinal space of rats and to investigate the development of the grafts. Methods A total of 34 eyes from 3-week-old pigmented rabbit embryos were treated with the dispase to obtain pieces of the full-thickness retina, and then grafts were injected into the subretinal space of the right eyes of 20 Wistar rats and 14 RCS (Royal college of surgeons) rats. Host rats were sacrificed at 3, 7, 14, and 28 d after transplantation. Sections were stained with hematoxylin eosin and immunohistochemistry. Results The rabbit embryonic retina was capable of developing and differentiating in the subretinal space of the host rats. Conclusion The rabbit embryonic retina is capable of developing and differentiating in the subretinal space of host rats. Transplantation of xenografts of embryonic full-thickness retina is practicable, but the methods of transplantation need to be improved.