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 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 .

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.

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.

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.

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 investigate the possibility that transplanting the full-thickness neonatal piggy retinas that are completely differentiated but immature improves the retinal function after light-induced retinal degeneration in pigs.Methods Retinas from normal Guangxi Bama pigs aged 1-6 days were used as donor tissues.Neuroretina-RPE cografts were obtained from newborn pigs by using excimer laser for microablation of choroidal tissue and transplanted into the subretinal cavity of adult Bama pigs after light-induced retinal degeneration through vitrectomy and retinotomy.On days 5-7 and in 1st to 5th month after retinal transplantation,the survival of the cografts in the recipients and whether the host retinas have rejection were observed by ophthalmoscope,colour fundus photography and fundus fluorescein angiography,and the amplitude and lantency of N1,P1 waves between different periods were measured by mfERG.Results The retinal transplantation was performed in 15 eyes of 8 Bama pigs after light-induced retinal degeneration.The subretinal transplantation of the cografts was performed successfully in 11 eyes,with the operation success rate of 84.6%(11/15).In host retina,the gray-black graft inside transplantation bed could be seen clearly in 1st to 2nd month after transplantation and the leaked fluorescence in transplants was checked with FFA.The vertical comparison between different periods showed the amplitude of N1,P1 waves of retinal transplants rose with the extension of the survival time,and the areas where active response was observed were ring 2 and ring 3;but the latency of N1,P1 waves was shortened significantly in each ring as compared with that before operation,especially in late survival period.Conclusion The function measurement and the observation of living body together confirm the transplantation of completely differentiated retina from newborn pigs improves the retinal function of pigs after light-induced retinal degeneration.

Objective To study whether noggin alone can induce human bone marrow mesenchymal stem cells (MSCs) to differentiate into neural cells for the treatment of neural degenerative diseases.Methods Human bone marrow MSCs were primarily cultured and then divided into control,Ad group of empty adenovirus vector and Ad-noggin group of adenovirus vector with noggin gene.Their morphological changes were observed with fluorescence microscopy and laser confocal microscopy,and the results of 48 h,4,7,10 d were analyzed with statistical methods.Results Both the vector made human bone marrow MSCs carry green fluorescence.Ad group didn’t show any morphological changes.While for Ad-noggin group,a few neural-like cells appeared in 48 h after transfection.The number of such cells was increased,and there were some neurites around the cells after 4 d.The cells amplified greatly after 10 d.Compared to those of 7 d after transfection,the soma and dendritic diameters of Ad-noggin group were significantly increased after 10 d (P

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 investigate the relationship between electrophysiological and morphological properties of neurons in visual cortex of developing rat, speculate the coincided degree between electrophysiological and morphological change and realize the mechanism of normal visual development. Methods Whole cell patch-clamp recording and intracellular staining were used to acquire cellular microelectrode recording in visual cortex from Sprague-Dawley rats (4~28 days old). The histological process was made. Results The differences of electrical feature between pyramidal cells and non-pyramidal cells were significant. The morphological maturity degree is different in developing visual cortex. Conclusion The different function of pyramidal and non- pyramidal cells in local integrition is reflected by their electrical feature in the process of visual development. In critical period of visual development, the coincision degree of the electrophysiological and morphological change in visual cortex is larger than that in the subcortex constructure.