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Objective:To investigate the effect of pressure on the differentiation of rabbit retinal stem cells (RSCs) co-cultured with retinal ganglion cells (RGCs).Methods:SPF grade New Zealand rabbits on the day 22 of gestation were selected, and embryos were removed to obtain retinal ciliary margin pigment epithelial tissue and culture primary RSCs.Six SPF grade newborn New Zealand rabbits were selected, and retinal neuroepithelial layer tissues were isolated to culture primary RGCs.Rabbit RSCs cultured in vitro were identified by immunofluorescence staining of nestin antibody, bromodeoxyuridine (BrdU) cell proliferation assay kit, RSCs spontaneously differentiated cells immunofluorescence detection and flow cytometry.RGCs were identified through immunofluorescence staining of Brn3b antibody and Thy1.1 antibody.A co-culture system of RGCs and RSCs cultured in the upper and lower layers of a transwelll plate respectively was constructed.The mRNA and protein expression levels of nestin and Thy1.1 in RSCs and differentiated cells under pressures of 0, 20, 40, 60, 80 mmHg (1 mmHg=0.133 kPa) were detected by real-time fluorescence quantitative PCR and Western blot.The feeding and use of laboratory animals were in accordance with the Regulations on the Administration of Laboratory Animals promulgated by the State Science and Technology Commission.The study protocol was approved by the Ethics Committee of Yunnan University Affiliated Hospital (No.KPRC-IACUC17008). Results:RSCs cultured in vitro were nestin-positive.The percentage of BrdU-positive isolated RSCs was (92.26±3.28)%.Some cells differentiated from RSCs were Brn3b-positive, accounting for (13.00±3.06)%, and some were GS-positive, accounting for (31.60±3.67)%.RGCs cultured in vitro were Brn3b- and Thy1.1-positive.There were statistically significant differences in the relative mRNA and protein expressions of nestin and Thy1.1 between RSCs and differentiated cells under different pressures (mRNA: F=127.600, 137.400; both at P<0.01; protein: F=82.480, 158.700; both at P<0.001). The relative mRNA and protein expressions of nestin were significantly reduced in RSCs, and relative mRNA and protein expressions of Thy1.1 were significantly increased in differentiated cells at 20, 40, 60 and 80 mmHg in comparison with 0 mmHg (all at P<0.05). When the pressure was 40 mmHg, the relative mRNA and protein expressions of nestin were lowest in RSCs, and the relative mRNA and protein expressions of Thy1.1 in differentiated cells were highest. Conclusions:Within a certain range, pressure can promote the differentiation of RSCs co-cultured with RGCs into ganglion-like cells, and excessive pressure can inhibit the differentiation of RSCs.
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Objective:To observe the multimodal imaging characteristics of combined hamartoma of the retina and retinal pigment epithelium (CHRRPE).Methods:A cross-sectional study was conducted.Sixteen eyes of 16 patients with CHRRPE were enrolled in Second People's Hospital of Yunnan Province from March 2013 to July 2019.Fundus color photography, fundus autofluorescence (FAF), fluorescein fundus angiography (FFA), indocyanine green angiography (ICGA), optical coherence tomography (OCT), optical coherence tomography angiography (OCTA) and multicolor imaging were performed in all patients.The multimodal imaging characteristics were analyzed.This study protocol adhered to the Declaration of Helsinki and was approved by an Ethics Committee of Second People's Hospital of Yunnan Province (No.20130106). Written informed consent was obtained from patients or their guardians prior to any medical examination.Results:Tumors were located in the posterior optic disc, and translucent glial lesions with unclear borders and slight elevations were observed.The surface of the lesions was attached by different degrees of fibroproliferative membrane, and the adjacent vessels were twisted and dilated.The tumors presented flat bulging green reflexes on the retina at the posterior pole by multicolor imaging, and OCT image showed thickened optic disc and retina near the optic disc, structural disorder, high reflectance of the surface, and low reflectance of the deep retina below the periretinal membrane.OCTA showed irregular blood flow signals, and the signal of retinal blood vessels was twisted and dilated.FAF showed that the autofluorescence intensity of tumors was weakened to different degrees.Early lesions presented different degrees of blocked fluorescence in FFA.Deformed and tortuous blood vessels were found in the eyes, and telangiectasia showing needle-like punctate strong fluorescence leakage was observed in severe eyes by FFA.ICGA showed no abnormal choroidal vessels.Conclusions:The main imaging features of CHRRPE include abnormal retinal blood vessels in the tumor area and fibrous proliferative membranes on the surface in color image; OCT shows that the retina and the retinal pigment epithelium are involved, and the retina in the tumor is thickened with disordered structure; high reflection OCTA shows irregular internal blood flow signals inside the tumor; FFA examination shows fluorescence obscuration and obviously tortuous retinal blood vessels.Multimodal imaging examinations are helpful for the diagnosis of CHRRPE.
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Objective To investigate the stimulating effects of conjunctival sac implants following eye enucleation on orbital bone tissue in rabbits model.Methods Eighty 1-month-old New Zealand rabbits were randomly divided into normal control group,only eyeball enucleation group,2-week interval group and 4-week interval group.The right eyeballs were enucleated in the rabbits in the only eyeball enucleation group,2-week interval group and 4-week interval group,and conjunctival sac implants were placed intraoperatively.Then a larger implant was replaced in a 2-week interval or 4-week interval in different groups,respectively.Spiral CT scan was used to evaluate the orbital bone development with aging.The peripheral blood of 3 ml was collected in the rabbits for the detection of osteocalcin and alkaline phosphatase using osteocalcin test kit (N-MID Osteocalcin) and alkaline phosphatase assay kit (NPP substrate-AMP buffer method),respectively.The animals were sacrificed at 3 months after operation for the histopathological examination of orbital bone tissue.Results The operation was successful in all the rabbits and no infection occurred after operation.The variance of orbital bone CT value was obvious in all the rabbits.The intercalated osteocalcin concentration was (48.55 ± 7.99),(59.80 ± 2.96),(57.94 ± 5.20) and (55.96 ± 3.22) μg/L and the alkaline phosphatase concentration was (284.66± 132.69),(232.96±54.39),(232.40± 118.23),(284.20± 130.41) μg/L in the normal control group,only eyeball enucleation group,2-week interval group and 4-week interval group,respectively,showing insignificant differences among the four groups (F =2.710,0.281,both at P ＞ 0.05).Similar pathological findings were in the 2-week interval group and 4-week interval group,such as no obvious orbital bone malformations,weak bone absorption and few bone osteoclasts under the optical microscope.Varying degrees of orbital deformities and bone tissue absorption were found in the only eyeball enucleation group.No orbital bone developing abnormality was seen in the normal control group and the left eyes in various groups under the optical microscope.Conclusions Compared with the only eyeball enucleated eyes,the orbital bone tissue has a well developed process in conjunctiva sac implant eyes following eyeball enucleation.Conjunctiva sac implant is an effective method to stimulate orbital growth and orbital volume increasing.
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Objective To compare the treatment effect of intraocular lens ciliary sulcus implantation combined with posterior capsular incarceration of intraocular lens and simple intraocular lens ciliary sulcus implantation.Methods We retrospectively analyzed 60 eyes of 60 children with traumatic cataract from June 2012 to June 2015 in the Second People's Hospital of Yunnan Province,including 44 males and 16 females and the average age ranged from 1 to 14 years.The patients were divided into two groups according to the situation of posterior capsular,30 eyes of 30 children for each group.The patients in combined surgery group were performed intraocular lens ciliary sulcus implantation combined with posterior capsular incarceration of intraocular lens,and the patients in simple surgery group were performed simple intraocular lens ciliary sulcus implantation.The follow-up time ranged from 1 month to 6 months.The best corrected visual acuity(BCVA),grade of optic axis turbid,dislocation of intraocular lens,postoperative complications and surgical duration were compared between the two groups.Results The BCVA after surgery was significantly different from that before surgery in both groups (combined surgery group:x2 =7.548,P＜ 0.05;simple surgery group:x2 =5.579,P＜0.05).The postoperative turbidity of central axis area and BCVA were not significantly different between the 2 groups (x2 =0.362,P＞0.05).The dislocation degree of intraocular lens and proportion of postoperative complications were lower in the combined surgery group than those in the simple surgery group (x2 =9.858,P＜0.05;x2 =7.200,P＜0.05).The duration of surgery was not significantly different between the 2 groups (x2 =0.658,P ＞ 0.05).Conclusions Posterior capsular incarceration of intraocular lens has fewer complications,lower dislocation degree of intraocular lens and more effective for children with traumatic cataract than intraocular lens ciliary sulcus implantation.
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Objective To establish a tree shrew model of Fusarium solani keractitis by injecting Fusarium solani conidia into the corneal stroma.Methods Fusarium solani was inoculated into Sabouraud culture medium and incubated at 26℃ for 7 days.Fungal suspension was collected and the number of spores was adjusted to 1 × 1010 CFU /mL on the blood cell count plate.Forty healthy tree shrews were randomly divided into experimental group (n=30) and control group (n=10).In the experimental group, 50 μL of fungal spore suspension was injected into the cornea center with a 29G needle, and 50 μL saline was injected in the control group.The models were evaluated by anterior segment photography, in vivo confocal microscopy, histopathology, and corneal tissue culture.Results The fungal infiltration, the degree of edema of corneal epithelial and endothelial cells, and the number of mycelium were positively correlated with time.The number of infiltrating inflammatory cells, mainly, neutrophils, reached a peak on the 7th day after modeling.The mycelial growth was parallel to the stromal fibers.After the successful establishment of the model, the corneal tissue culture showed the growth of Fusarium solani.The successful rate of modeling was 86%.Conclusions The tree shrew model of Fusarium solani keratitis is established by injecting spores of Fusarium solani into the cornea.
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Objective To observe the changes of Th1 / Th2 inflammatory factors in the aqueous humor of tree shrews with fusarium solani keratitis,as well as to explore the relationship between Thl / Th2 inflammatory factors and inflammatory response in fusarium solani keratitis.Methods Forty healthy tree shrews were randomly divided into experimental group(n =30) and control group (n =10).Fusarium solani was inoculated into sabina culture medium and cultured at 26 ℃ for 7 days,and then the fungal suspension was collected and the density of spores was adjusted to 10 × 109 CFU · mL-1.In the experimental group,50 μL fungal spore suspension was injected into the center of the cornea stroma,while the control group received the same amount of saline.Next the levels of cytokines including interleukin (IL)-1 β,IL-6,IL-4 and IL-10 were analyzed by flow cytometry on day 3,day 7,day 14 after successful modeling,and the changes in types of infiltrating cells were observed by histopathological examination.Results The expression level of IL-1 β and IL-6 (Th1 type cytokines) was the highest on day 7,and the difference was statistically significant at each time point when compared with the control group (all P ＜ 0.05).The expression level of IL-10 (Th2 type cytokines) was the highest on day 14,and the difference was statistically significant at each time point when compared with the control group (all P ＜ 0.05).The difference in IL-4 expression was statistically significant on day 7 (P ＜ 0.05).In addition,histopathological examination showed that the number of infiltration cell reached its peak on day 7,mainly neutrophils,and fungal hyphae were observed to be parallel to the matrix fibers at each time point.Conclusion The proinflammatory cytokines IL-1β and IL-6 and the anti-inflammatory cytokines IL-10 may play an important role in the molecular mechanism of inflammatory response of fusarium solani keratitis in tree shrews.
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Objective To analyze and compare the characteristics and differences of corneal endothelial cells of rhesus monkey and tree shrew eyes.Methods Corneal endothelial cells of 6 healthy rhesus monkeys (12 eyes) and 20 healthy tree shrews (40 eyes) were measured using a non-contact SP3000P specular microscope.Eight parameters were de-termined and compared with relevant parameters of human eyes reported in the literature, including minimum cell area (Smin), maximum cell area (Smax), average cell area (Savg), standard deviation of cell area (SD), coefficient of variabili-ty ( CV) , cell density ( CD) , hexagonality percentage ( HG%) and central corneal thickness ( CCT) .Results The ima-ging and measurement of all parameters could be completed in a short time both in rhesus monkeys and tree shrews.The time spent in the two kinds of animals was not significantly different.The CCT was ( 449.2 ±12.8 ) μm and ( 262.4 ± 24.6) μm, Smin was (120.4 ±26.3) S/μm2 and (153.2 ±42.9) S/μm2 , Smax was (705.0 ±130.8) S/μm2 and (468.7 ±109.3) S/μm2 , Savg was (351.1 ±26.1) and (295.4 ±18.9) S/μm2 , SSD was (113.1 ±27.4) and (75.9 ±27.3) S/μm2, CV was (31.9 ±6.0) and (25.3 ±8.3), CD was (2874.2 ±203.8) p/cell· mm-2 and (3399.3 ±224.7) p/cell· mm-2 , and the HG% was (58.6 ±9.1) and (94.0 ±9.7) in the rhesus monkeys andt tree shrews, respectively. The differences of all the above parameters between rhesus monkeys and tree shrews were statistically significant ( P<0.05 for all) .The cornea of tree shrews was significantly thinner than that of rhesus monkeys.The area and coefficient of varia-bility of tree shrews were smaller to those of rhesus monkeys, while the cell density and hexagonality percentage were higher than those of rhesus monkeys.Compared with human eyes, the CCT, CV and HG%in rhesus monkeys were highly simi-lar, while the CD was lower than that of human eyes.The CCT in tree shrew was only 60%of the rhesus monkey eyes and 50%of human eyes, while the CD and Savg were similar to that of human eyes in the 10-20 years old group.Conclu-sions The morphology and parameters of corneal endothelial cells in rhesus monkeys and tree shrews are significantly dif-ferent.There are similarities and differences among the human, rhesus monkey and tree shrew corneal endothelial cells. Both rhesus monkeys and tree shrews are appropriate experimental animals feasible for researches on human corneal endo-thelial diseases.
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Objective To observe the clinical effect oflamellar keratectomy+conjunctival flap+implanted artificial eyein stimulating the orbital and conjunctival sac growth. Methods A retrospective case study: 12 cases (12 eyes) with congenital microphthalmos in the Fourth Affiliated Hospital of Kunming Medical University in 2009-2013 were selected. In these cases, there were 11 cases of microphthalmos, and 1 patient due to congenital absence of the eye without surgery, were given direct implant of the artificial eye;7 patients without significant stenosis in conjunctival sac,received thelamellar keratectomy+conjunctival Flap+implantation of artificial eye, 4 patients with conjunctival sac stenosis recieved thelamellar keratectomy+conjunctival flap+implanted artificial eye+eyelid suture. Results For stunted children who couldn't wear a prosthetic eye, after treated withlamellar keratectomy + conjunctival flap + artificial eye implantation, the conjunctival sac developed well, cornea was covered with conjunctiva well and no exposure,the appearance and volume of orbit was also improved. ConclusionLamellar keratectomy+conjunctival flap+artificial eye implantsurgery is an effective way to promote orbital and conjunctival sac development of the children with congenital microphthalmos.
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With the development of biological techniques,the study on the pathogenesis of disease-causing genes of congenital cataracts has substantial progress.Some positive results of screen of mutation gene in congenital cataract family has been reported,but the report of negative result is rate.ObjectiveThe present study attempts to screen the mutation of CRYAA,CRYAB,CRYA1/A3,CRYBB2,CRYGC and CRYGD gene in a Chinese family with autosomal dominant congenital cataract. MethodsThe periphery blood samples were exacted from 8 patients of 4 generations of with congenital cataract in this family.The complete coding region and intron spliced sites of CRYAA,CRYAB,CRYA1/A3,CRYBB2,CRYGC and CRYGD were amplified with polymerase chain reaction (PCR),and the products of PCR were directly sequenced.The control blood samples were from 10 normal subjects.This study followed the Declaration of Helsinki.Written informed consent was obtained from all of the patients.ResultsThe patients were found in each generation in this family and the mode of inheritance was in accordance with the characteristic of autosomal dominant inheritance.The sequence of amplified genetic fragments of CRYAA,CRYAB,CRYA1/A3,CRYBB2,CRYGC and CRYGD genes were inaccordance with those of normal subjects and GeneBank.No any mutation loci was found in all of the patients of this family.ConclusionCRYAA,CRYAB,CRYA1/A3,CRYBB2,CRYGC and CRYGD genes is not the causing-disease genes in this family.
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Background Elevated intraocular pressure leads to the loss of retinal ganglion cells and vigorous reaction of retinal glial cells.The expression of nestin in retinal glial cells secondary to hypertention and its significance are unclear.ObjectiveThis study aim to investigate the expression of nestin in retinal glial cells (RGCs) in ocular hypertention rats.Methods The ocular hypertention models were established by cauterizing the limbus-draining veins in the right eyes of 42 SD rats,and a conjunctival incision in the left eyes of the rats served as the sham group.The intraocular pressure (IOP) was measured with the Tono-Pen XL tonometer.The number of RGCs in the rats with ocular hypertention was counted.The expression of the nestin protein in RGCs was semi-quantitatively analyzed using Western by immunochemistry.Double immunofluorescence was carried out to evaluate the the confocal laser scaning microscope.Results Significant differences were found in the IOP between the model group and the sham group at various time points (P＜0.05).In 1 week to 3 weeks after operation,the number of RGCs significantly declined in the model group compared with the sham group (P＜0.05).Immunochemistry showed that from 2 hours through 1 week after operation,the expression of nestin was gradually enhanced in the model group in comparison with the sham group.Western blot revealed that the expression of the nestin protein reflected a similar tendency to that of immunofluorescence.The increased introcular pressure as manifested by the induced expression of nestin.Immunoelectron microscopy also confirmed the induced expression of nestin especially at their end-feet suggests a potential neuroprotective mechanism in neuronal degeneration.Nestin may be a useful biomarker for retinal injury study.
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Objective Amniotic membrane as the carrier to culture the vascular endothelial cells was investigated in this study in order to explore whether the corneal endothelial cells superseded by the vascular endothelial cells is feasible on the account of directing a kind of the original method to handle the bullous keratopathy.Methods The vascular endothelial cells adhering to the sphagitides lumen of the experimental rabbits were digested and gained by means of the perfusion method with 0.25%tripsin plus 0.02%EDTA.Fresh amniotic membrane without the amniotic epithelial cells was cut into the square tissue about 1.5 cm?1.5 cm and spread evenly in the 24-well culture dish.Primary cultured cells were subcultured on the amniotic membrane.We would not transplant the vascular endothelial cells feeding on the amniotic membrane until cells is full of the whole amniotic membrane surface.One to two months after the operation,the change of corneal diaphaneity was observed.Results About 12 days since the cell transplantation was performed,the corneal transparency alteration between the experimental groups and the control one is different.The corneal buttons in the experimental group show the severe edema and opacity,and the anterior chamber couldn't be seen unclearly.But,10 days after the operation,the corneal oedema which begins to extenuate was judged through the indicatrix that the corneal edema in the experimental group has been recovering slowly,among which the anterior chamber tissue of 7 animals was visible through the implant.The corneal edema in the control groups intensified evidently,even,the part of these appeared the ulcerous necrosis as result of the corneal severely edema.There is the existence of difference between two groups(P
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In order to investigate the effect of nerve growth factor (NGF) on the proliferation of rabbit corneal endothelial cells and epithelial cells, the in vitro cultured rabbit corneal endothelial cells and epithelial cells were treated with different concentrations of NGF.MTT assay was used to examine the clonal growth and proliferation of the cells by determining the absorbency values at 570nm. The results showed that NGF with three concentrations ranging from 5 U/mL to 500 U/mL enhanced the proliferation of rabbit corneal endothelial cells in a concentration-dependent manner.50 U/mL and 500 U/mL NGF got more increase of proliferation than that of 5 U/mL NGF did.Meanwhile, 50 U/mL and 500 U/mL NGF could promote the proliferation of the rabbit corneal epithelial cells significantly in a concentration-dependent manner. However, 5 U/mL NGF did not enhance the proliferation of epithelial cells. It was suggested that exogenous NGF can stimulate the proliferation of both rabbit corneal endothelial and epithelial cells, but the extent of modulation is different.
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In order to investigate the effect of nerve growth factor (NGF) on the proliferation of rabbit corneal endothelial cells and epithelial cells, the in vitro cultured rabbit corneal endothelial cells and epithelial cells were treated with different concentrations of NGF. MTT assay was used to examine the clonal growth and proliferation of the cells by determining the absorbency values at 570 nm. The results showed that NGF with three concentrations ranging from 5 U/mL to 500 U/mL enhanced the proliferation of rabbit corneal endothelial cells in a concentration-dependent manner. 50 U/mL and 500 U/mL NGF got more increase of proliferation than that of 5 U/mL NGF did. Meanwhile, 50 U/mL and 500 U/mL NGF could promote the proliferation of the rabbit corneal epithelial cells significantly in a concentration-dependent manner. However, 5 U/mL NGF did not enhance the proliferation of epithelial cells. It was suggested that exogenous NGF can stimulate the proliferation of both rabbit corneal endothelial and epithelial cells, but the extent of modulation is different.
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Cell Proliferation/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Endothelium, Corneal/cytology , Epithelium, Corneal/cytology , Nerve Growth Factor/pharmacologyABSTRACT
To study the secretion of epidermic growth factor (EGF) in tears from patients with herpes simplex virus keratitis (HSVK), radio-immunoprecipitation was used to detect the value of EGF in human reflex tears from 40 cases as control group (group A), 49 cases of acute HSVK (group B) and 31 cases of convalescence HSVK (group C). Results showed that the value of EGF in reflex tears from the patients in the group B was obviously increased as compared with that in the group A and group C (P<0.001). The value of EGF in tears in the group C was higher than in group A (P<0.05). It was concluded that the value of EGF in reflex tears from HSVK patients was higher than that from normal people.