Retinitis pigmentosa (RP) is a large group of common hereditary eye diseases with highlyheterogeneous genetic background. Over forty genes with diverse functionalities are associated with RP and they include a set of ubiquitously expressed genes. These include five genes involved in the precursor messenger RNA( premRNA) splicing. Recent progress in disease gene identification for RP has established the involvement of pre-mRNA splicing as one important mechanism in the disease etiology and has shed light on the splicing process itself, a fundamental biological process. To this date, studies in this field have been focused on two major issues. First, how do the mutations of the adRP associated splicing factors (adRP-SF) affect the splicing function? Second, how do the mutations in these ubiquitously expressed genes lead to specific retinopathy? The two topics fit with the two continuous important steps of the disease pathogenesis. Recently, researchers have made a dramatic progress in the first topic. The identification of the SNRNP200 gene,the fifth adRP-SF and its relevant functional study has shown significance to the progress in the study of RP. Numerous investigations are also being carried out in addressing the second issue.Generation of a variety of models led to a better description of the pathological process of the disease. However, in respect to the key pathogenic mechanism,researchers are still puzzled with a number of confusing questions. In this commentary,the results from the latest investigations were summarized, and in particular,the difficulties in studying the molecular mechanism by which the pre-mRNA splicing deficiency causes RP were detailed.

Background Nomal binocular vision is achieved through accommodation and vergence.Anisometropia is associated with abnormal visual development,but its impact on accommodation and vergence is unknown.This study is helpful to make a reaonable correction plan for anisometropia.Objective This clinical study was to examine the binocular vision function in myopic anisometropia and evaluate the influence of myopic anisometropia on binocular vision function.Methods Seventy subjects with myopic anisometropia ≥ 1.0 D were recruited in Tianjin Eye Hospital and Tianjin Vocational Institute from 2006 to 2009.The subjects were assigned to the low anisometropia group with the 1.0 D ≤ spherical equivalent difference ＜ 2.5 D and the moderate and high anisometropia group with spherical equivalent difference ≥2.5D,and other 35 individuals with spherical equivalent difference ＜ 1.0 D were enrolled as the without anisometropia group.Binocular vision function parameters were examined in all the individuals,including comprehensive refractometer to check the diopter,Worth 4 dot to examine the binocular vision function,different distance of vertical fusional amplitudes,accommodative convergence/accommodation (AC/A) and stereoscopic vision.Written informed consent was obtained from each subject prior to the study.Results After anisometropia was completely corrected,the normal rate of Worth 4 dot examination was 94.3％,80.0％ and 60.0％ in the without anisometropia group,low anisometropia group,moderate and high anisometropia group,respectively,showing a statistical difference among groups (x2 =12.137,P＜0.05),and the normal rate of 4 dot was less,and that of 5 dot was higher in the moderate and high anisometropia group compared with the without anisometropia group and low anisometropia group (P＜0.05).No significant difference was found in the normal rate for phoria of distance among the three groups (x2 =4.489,P=0.344).The normal rate for near phoria of distance was 65.7％,42.9％ and 37.1％ in the without anisometropia group,low anisometropia group,moderate and high anisometropia group,respectively,with significant difference among them (x2 =6.045,P＜0.05).The normal rate of stereopsis was 91.4％,77.1％ and 54.3％ in the without anisometropia group,low anisometropia group,moderate and high anisometropia group,respectively,which was statistically different among groups (x2 =12.863,P =0.002),and that in the moderate and high anisometropia group was significant declined in comparison with the without anisometropia group (x2 =12.208,P ＜ 0.05).The difference of distance phoria,AC/A,negative relative accommodation (NRA),positive relative accommodation (PRA),accommodation amplitude and binocular accommodative lag were statistically different among groups (P＞0.05).Conclusions The normal rate of the stereopsis,Worth 4 dot and the phoria of near distance are decreased with the increase of anisometropia,but other ocular motor parameters are normal in anisometropic subject.

Background Rhodopsin (RHO) gene is the most common disease gene for autosomal dominant retinitis pigmentosa (adRP),one of the main pathogenesis is that misfolded mutant RHO proteins accumulate in the endoplasmic reticulum and cause endoplasmic reticulum stress (ERS).Objective This study aimed to determine the genetic basis for a consanguineous Chinese Han adRP family.Methods This study procedure complied with Helsinki Declaration.All participants in the family were investigated under the informed consent.Regular ocular examination was performed on the patients in this family.Next-generation sequencing (NGS) was carried out to screen the mutations in 189 genes associated with hereditary retinal diseases (HRDs).After being analyzed and filtered,variations detected by NGS were validated by Sanger sequencing and evaluating of pathogenicity.The wild-type RHOWT and mutant RHOP53Rwere cloned into the vector pEGFP-N1.Then the two plasmids were transfected into adult retinal pigmentosa epithelium cell line(ARPE19) and human embryo kidney 293 line (HEK293) to observe the location of rhodopsin-GFP fusion protein in cells,and the expression of ERS related protein XBP1 in the cells was detected by quantitative-PCR and Western blot.Results This family included 5 generations with the typical adRP characteristics.Genetic analysis identified a heterozygous variation,p.P53R in RHO gene,which was fully cosegregated in the family.Wild-type RHOWT-GFP fusion proteins showed the green fluorescence on the endoplasmic reticulum and cytomembrane,but the misfolded mutant RHO-GFP fusion protein gathered only in endoplasmic reticulum.Compared to wild-type RHOWT,the XBP1 was activated and increased by (1.28 ±0.09) fold.The introns of 26 bases in XBP1 mRNA were removed in the HEK293 cells with mutant RHO-GFP fusion protein,and the expression of XBP1 was stronger in the HEK293 cells with mutant RHO-GFP than that in HEK293 cells with wild type RHO-GFP and cells with blank pEGFP-N1 plasmid.Conclusions Heterozygous variant RHO p.P53R is very likely the pathogenical mutation in the adRP family.The RHOP53R mutant rhodopsin protein can not be delivery effectively from the endoplasmic reticulum to the cell membrane,and these proteins accumulate in the endoplasmic reticulum,which causes ERS.

BackgroundThe study of the molecular mechanism of visual plasticity is helpful for the explaining and prevention of strabismus and amblyopia.The effect and significance of NogoA in the strabismus and amblyopia formation are attracting more attention.ObjectiveThe present study was to investigate the expression of NogoA in 21a area of visual cortex in strabismic-induced amblyopia cats and explore the possible molecular mechanism of strabismic-induced amblyopia.MethodsSixteen 4-week old clean cats were randomizedly divided into normal group and strabismic-induced amblyopia group and eight for each group.The strabismic-induced amblyopia models were created by cutting off the external rectus in 8 cats.Pattern visual evoked potentials ( P-VEP ) were recorded 1 week after operation and compared with normal cats,and depression of amplitude and prolongation of implied time of P100 wave were as the successful criterion of model.The 200 ml paraformaldehyde was infused via heart to fax the brain under the deep anesthesia and then the cats were sacrificed and the brain cortex sections were prepared.The morphology of 21a zone of cat visual cortex was examined by haematoxylin and eosin staining,and the expressions of NogoA in 21a area of visual cortex were detected by immunohistochemistry with a monoclonal antiNogoA antibody.The use of the animals complied with the Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.ResultsThe implied time and amplitude ratio of VEP P100 wave were (98.10±7.07)ms and (0.83±0.14) in normal group,and those in strabismic-induced amblyopia group were (108.50±6.95 )ms and (0.35 ±0.09 ),showing significant differences between two groups (t=4.450,P=0.005 ; t =5.970,P =0.005 ).The numbers of neurons were similar in 21a area of visual cortex between the two groups,but the volume of the neurons was lessen in strabismic-induced amblyopia group.The positive cell densities for NogoA were ( 387.37±2.01 ) cells/mm2,( 354.58± 1.85 ) cells/mm2 and ( 289.68± 1.81 ) cells/mm2 in layer Ⅱ/Ⅲ,Ⅳ and V/V[ respectively in strabismic-induced amblyopia group,and those in normal group were ( 161.39± 1.98 ) cells/mm2,( 128.93 ± 1.26 ) cells/mm2 and ( 96.25 ± 1.49 ) cells/mm2,indicating a significant increase in model cats ( t =- 160.400,- 201.890,- 164.740,P =0.000 ).Conclusions NogoA may play an important role in the regulation of the sensitive developmental period of visual sense and its plasticity.

Background The development of retinopathy of prematurity(ROP) is associated with many regulatory cytokines related to neovascularization;however,the retinal expression and regulated mechanism of stromal cell-derived factor-1 (SDF-1) in mouse model of oxygen-induced retinopathy (OIR) remain uncertain.Objective This study was to investigate the expression of SDF-1 in retina of mouse model of OIR.Methods Forty 7-day-old C57BL/6J mice were divided into OIR group and control group.In OIR group,20 mice were exposed to 75% oxygen for 5 days and then to room air for 5 days.In control group,20 mice were raised in room air.The expression of SDF-1 in retina of mice was studied by immunochemistry and quantified by real time reverse transcriptase polymerase chain reaction (RT-PCR).Results The positive immunohistochemical staining for SDF-1 was found mainly locating at the ganglion cell layer in 12-day-old mice of OIR group;the stronger positive immunohistochemical staining for SDF-1 was noted mainly locating at the ganglion cell layer,vascular endothelial cells of inner retina,neovascular endothelial cells in 17-day-old mice of OIR group;the delicate positive immunohistochemical staining for SDF-1 was both found mainly locating at the inner retina and being around the retinal vascular in 12-day-old mice of control group and 17-day-old mice of control group.The expression of SDF-1 mRNA in 17-day-old mice of OIR group was higher than that of 12-day-old mice of OIR group (t=8.072,P＜0.05)and 17-day-old mice of control group(t=10.026,P＜0.05),respectively.The expression of SDF-1 mRNA in 12-day-old mice of OIR group was lower than that of 12-day-old mice of control group (t=4.336,P＜0.05).Conclusion SDF-1 might improve the onset of retinal neovascularization of OIR.

Background The extraocular muscles (EOMs) Pulley is considered as the functional origins of the recti EOMs,it is determinants of ocular motility.Objective The structure of the connective tissue around EOMs in cat was studied,and the role of EOMs Pulley to ocular movement was investigated.Methods Five adult cats were involved.The gross anatomy of an orbit in each cat was observed.The other orbit was processed with paraffin imbedding and coronal serial sections.A murine monocolonal antibody to α-smooth muscle actin (α-SMA) was used to show smooth muscle,while Masson trichrome stain was used to show muscle and collagen,and Weigert stain to show elastin.Results An encircling ring of collagen circled EOM was thin and connected to the orbital layer of muscle fiber loosely.Less elastin fibers and little smooth muscle cells were embedded in the collagen ring and connective band.Collagen ring around medial rectus and the connective band between medial rectus and inferior rectus was not more significantly developed than other bands.Conclusions The connective tissue around EOMs in cat may be related to its function of ocular movement,which is not developed.

Background The afferent signals of proprioceptor in extraocular muscles play an important role in controlling eye position and conjugate movement. Palisade ending in the extraocular muscles is the main source of proprioceptive information, and its abnormalities in structure and function may be associated with the occurrence of nystagmus. Objective This study was to observe the changes of palisade ending in the extraocular muscles of patients with congenital nystagmus ( CN) and discuss the probable mechanism. Methods Modified Kestenbaum procedure was performed on 10 patients with CN, and the extraocular muscle samples were collected during the operation. Normal extraocular muscle samples were obtained from the enucleated eyeballs after ocular wound. The ultrathin sections of extraocular muscles were prepared and double-staining by uranyl acetate and lead citrate. The morphological changes of the palisade ending of extraocular muscles were examined under the transmission electron microscopy. Written informed consent was obtained from each subject before surgery. Results The ultrastructure of palisade ending in the extraocular muscle of CN subjects showed the different degrees of alterations. The mild changes included the collapse and disconnection of external capsules and the nonhomogeneous electron-dense substracts. The degeneration and dissociation of myelin in nerve endings, swelling and vacuolation of mitochondria were also exhibited. Myeloid body was found in axon. In the severe patients,the necrosis of Schwann' s cells,dissolve of axon and disappear of capsules were seen. Conclusion The palisade ending of extraocular muscle in the patients with CN are obviously abnormal in comparison with normal one. These alterations are probably associated with the etiology and pathogenesis of CN.

BackgroundSeveral cornea collagen crosslinking methods have been used to treat keratoconus.However,the safety of these methods is dissatisfactory.Glyceraldehyde is a very potent and highly reactive crosslinking agent,with little toxicity,but its effect on corneal biomechanical property is poorly clear.ObjectiveThe aim of this study was to evaluate the biomechanical effects of glyceraldehyde collagen crosslinking on rats cornea.Methods Fifteen clean SD rats were randomly divided into 0.005 mol/L glyceraldhyde group,0.050 mol/L glyceraldhyde group and blank control group.Glyceraldhyde drops was topically administered in the right ryes 2 times per day for consecutive 7 days in the 0.005 mol/L and 0.050 mol/L glyceraldhyde groups,and no any eye drops was used in the blank control group.Seven days later,the rats were sacrificed.Transparency of corneal buttons in these different groups was evaluated.The central corneal strips of 2 mm×6 mm with 2 mm scleral tiasue were obtained for the biomeehanical stress-strain measurement,including ultimate stress ( MPa),ultimate strain (％) and 6％ elastic modulus (MPa).Corneal collagen fibril density was assessed by histological examination under a light microscopy.The use of the animals followed the Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.ResultsThe words could be clearly displayed transcorneally in all the three groups.When strain was 6％,the stress was (0.463±0.065 ) MPa in 0.005 mol/L glyceraldehyde group,(0.846±0.240) MPa in 0.050 mol/L glyceraldehyde group,both showing a significant increase in comparison with (0.195±0.103 ) MPa of the blank control group (P=0.029,0.000 ).Following the crosslinking treatment,the ultimate stress was significant elevated in 0.050 mol/L glyceraldehydes group compared with the blank control group ( ( 10.759 ± 3.337 ) MPa vs.(5.295± 1.313 ) MPa,P =0.007 ),but no significant change between the 0.005 mol/L glyceraldehydes group and the blank control group ( ( 6.043 ±2.084) M Pa vs.(5.295 ± 1.313 ) MPa,P =0.660 ).Corneal ultimate strain was lower in the 0.005 mol/L glyceraldehyde group and 0.050 mol/L glyceraldehyde group than the blank control group (36.57％ ±3.09％ vs.43.87％ ± 1.89％,P =0.009;28.53％ ±1.89％ vs.43.87％ ± 1.89％,P =0.000).However,significantly increased 6％ elastic modulus were seen in the 0.005 mol/L glyceraldehyde group and 0.050 moL/L glyceraldehyde group compared with the blank control group ( ( 7.718 ± 1.076 ) MPa,( 14.102 ± 4.011 ) MPa vs.( 3.252 ± 1.717 ) M Pa),with statistically significant differences ( P =0.029,0.000).Histological examination showed a increase of collagen fiber density in the 0.050 mol/L glyceraldehyde group.Conclusions Corneal collagen crosslinking induced by glyceraldehyde strengthens biomechanical intensity and increases the density of corneal collagen fiber.But the safety of glyceraldehyde crosslinking for keratoconus needs further study.

BackgroundRecent researches suggested that properties of neurons in the lateral geniculate neuron (LGN) may represent an important neural limitation on the development of basic spatial and temporal vision,and even binocular rivalry.However,previous studies on the properties of spatiotemporal frequency tuning of LGN were rather concentrated on a monkey or cat,whereas little is known about rat.ObjectiveThis study was to examine the development of spatiotemporal frequency tuning in rats LGN.MethodsTwenty Wistar rats were collected and divided into 14-16 day,20-22 day,27-30 day and 60 day groups according to the different ages after their eyes opened and 5 rats were assigned for each group.Extracellular single neuron recording was carried out in the rats to study the spatio-temporal receptive field properties of neurons in LGN by sinusoidal gratings visual stimuli.Dynamic changes of the spatio-temporal receptive field properties of neurons in LGN with the development of Wistar rats were evaluated.ResultsThe differences between band-pass and low-pass distribution of temporal frequency or spatial frequency of rat LGN were not statistically significant (x2 =0.68,0.47,P＞0.05 ).The optimal temporal frequency of receptive field in rat LGN went up to the maximum value until 60 day in Wistar rats.The mean optimal temporal frequencies of neurons in the four different age groups were ( 2.5 ± 1.3 ),( 2.6± 1.2),(2.6± 1.1 ) and ( 3.6± 1.1 ) Hz with significant differences among the 4 groups (F=4.53,P＜0.05 ),and those in the 14-16 day group,20-22 day group,27- 30 day group were significantly lower than in the 60 days group ( q =4.43,4.10,4.03,P ＜ 0.05 ).No significant differences were seen among the 14-16 day group,20-22 day group and 27-30 day group ( P＞0.05 ).The optimal spatial frequency values in the four groups were ( 0.04 ± 0.04 ),( 0.04 ± 0.03 ),( 0.05 ± 0.03 ),( 0.05 ± 0.04 ) cpd,respectively without statistical difference among them ( F =0.58,P ＞ 0.05 ).The temporal and spatial bandwidth values in the various age groups were not statistically significant among the four groups ( F =0.37,1.22,P＞0.05). Conclusions The development of temporal and spatial frequency characteristics of the rat LGN receptive field may be related to its functional visual pathway.

Background Arresting the overexpression of vascular endothelial growth factor (VEGF) will be a new approach to the inhibition of neovascularization.RNA interference (RNAi) can inhibit the expression of specific gene,and its application in eye has little interference to other gene expression.Objective This study was to investigate the effect of small interference RNA (siRNA) targeting VEGF on the expression of VEGF and retinal neovascularization in oxygen-induced retinopathy (OIR) model.Methods psi-HITM/enhanced green fluorescent protein (EGFP)/VEGF siRNA was designed and prepared in vitro.Mouse endothelioma (EOMA) were cultured in DMEM without antibiotic and divided into 5 groups.The cells were incubated in DMEM only in the blank control group;while 1 μl of LipofectamineTM 2000 + psi-HITM/EGFP,1 μl LipofectamineTM 2000 + 40,50 or 60 nmol/L of psi-HITM/EGFP/VEGF siRNA was added into DMEM in the negative control group and siRNA groups,respectively.The expression of VEGF mRNA and protein was detected by real time PCR (RT-PCR) and Western blot.The optimal effective concentration of VEGF siRNA was assessed.OIR models were established in 48 7-day-old C57BL/6J mice by raising them at an oxygen concentration of (75±2) ％ for 5 days and then to normal air.The mice were randomized into the model group,null vector group and VEGF siRNA group.1 μl of a mixture of psi-HITM/EGFP or VEGF siRNA (60 nmol/L) and LipofectamineTM 2000 was intravitreally injected,respectively,in the null vector group and VEGF siRNA group.The normal mice were used as the normal control group.Expression of VEGF mRNA and protein in the mouse retinas was detected by RT-PCR and Western blot,respectively,and FITC-dextran stretched retinal preparation was examined to evaluate the neovascularization,and retinal sections were examined to quantify the number of vascular endothelial cell nuclei extending beyond the internal limiting membrane (ILM).Results The in vitro transfection test showed that the expression of VEGF mRNA and protein in the EOMA cells was significantly different among the blank control group,negative control group and 40,50,60 nmol/L VEGF siRNA groups (F =148.890,P ＜ 0.001; F =306.960,P ＜ 0.001),and the expression of VEGF mRNA was lower in different concentrations of VEGF siRNA groups than that in the blank control group (t=73.950,119.890,156.480,all at P＜0.001).Also,the expression of VEGF protein was less in different concentrations of VEGF siRNA groups than that in the blank control group (t =15.452,23.344,42.119,all at P＜0.001).The optimal inhibitory concentration of VEGF siRNA was 60 nmol/L.In vivo study determined that compared to the model group and null vector group,the non-perfusion zones and neovascular net in the retina were decreased,and the number of vascular endothelial cell nuclei extending beyond the ILM was less in the VEGF siRNA group.The relative expression level of VEGF mRNA in the retinas was 1.23±0.18,4.02±0.16,3.98±0.19 and 1.98±0.12 in the normal control group,model group,null vector group and VEGF siRNA group,respectively,with a significant difference among them (F=369.780,P＜0.001),and the relative expression levels of VEGF mRNA in the model group and null vector group were higher than that in the normal control group (t=37.880,37.336,both P＜0.001),and the expression of VEGF mRNA in the VEGF siRNA group declined by 50.8％ (t=10.183,P＜0.001).The difference in the expression levels of VEGF protein also was assayed among the various groups (F=408.980,P＜0.001),and VEGF level in the retina was lowered by 68.0％ in the VEGF siRNA group compared to the model group (t =11.473,P＜0.001).However,VEGF level in the VEGF siRNA group remained at a high level in comparison with the normal control group (t =2.422,P＜0.001).Conclusions Intravitreal injection of VEGF siRNA can attenuate retinal neovascularization by effectively downregulate the expression VEGF mRNA and protein in the retina.