High glucose suppresses the corneal limbal stem cell migration and changes the cell phenotype / 眼科新进展

Objeetive To explore the influence of extracellular high glucose on the proliferation,migration and biomarkers of corneal limbal stem cells.Methods Establishment of a model of high glucose in cultured human limbal stem cells to observe and investigate the effects of extracellular high glucose on the proliferation and migration of corneal limbal stem cells by immunoflurescence,CCK-8 and Transwell assay,respectively.Totally 16 SPF rats were collected and induced diabetic model by streptozotocin as the high-glucose group,and the normal rats of the same age served as the control group.Corneal epidermises of rats in both groups were scraped to observe the repair of corneal epithelium.And the corneas were treated with HE staining and immunohistochemical staining to detect the expression of biomarkers of corneal limbal stem cells and the modality changes of cells.Results The proliferation rate of human limbal epithelial cells was significantly decreased when exposed to high glucose,and the rate at 24 h,48 h and 72 h was 0.728,0.345 and 0.395,respectively,which was markedly lower than that in the control group,with a significant difference (P ＜ 0.05);meanwhile the cell migration rate of the high-glucose group was 17.6％ at 48 h,which was significantly slower than that of the control group (100％).And the inhibition was accompanied by the decreased expression of β-catenin and vimentin.Furthermore,the expression levels of β-catenin and vimentin mRNA and protein were down-regulated,with abnormal location,in the high-glucose group.And diabetic rats had poor corneal epithelial healing.The epithelial layer became thinner and the structures were disorganized in diabetic rats through HE staining.The immunohistochemical assay revealed the expression of β-catenin and vimentin of cornea limbal stem cells was down-regulated in high-glocose group when compared with the control group.Conclusion High glucose can significantly inhibit the proliferation and migration of cornea limbal stem cells,and its main damage mechanism is correlated with the abnormalities of β-catenin and vimentin.

A study of polyvinyl alcohol hydrogel simulating human vitreous body / 眼科新进展

Objective To screen a kind of polyvinyl alcohol (PVA) hydrogel with similar physical properties to human vitreous body as to provide basis for exploring new vitreous substitutes.Methods Different concentrations (1％,3％,7％) PVA solution were crosslinked by γ-irradiation (Co60),and 1％,3％ and 7％ PVA hydrogels were harvested.Then PVA hydrogels with similar properties to natural vitreous were screened by detecting the physical and optical properties (density,water content,light transmittance,refractive index,pH value,swelling property) and rheological properties and cytotoxicity of PVA hydrogels.Results As for the physical and optical properties of the hydrogels,the water content,refractive index,light transmittance,pH value and density of 1％,3％ and 7％ PVA hydrogel was 98.90％ vs.98.10％ vs.93.50％,1.335 5 vs.1.336 1 vs.1.342 5,94.80％ vs.93.20％ vs.88.20％,7.22 vs.7.25 vs.7.41 and 1003.9 g · L-1d vs.1014.4 g · L-1 vs.1114.7 g · L-1,respectively.Finally,1％ and 3％PVA hydrogels had the similar physical and optical properties to the human vitreous.As for rheological properties,the storage modulus (G') of the three PVA hydrogels was larger than the loss modulus (G"),indicating that these three hydrogels were viscoelastic gel,and the elastic properties was greater than the viscosity.1％ PVA:G'was (3.2 ±1.1)Pa,G" was (0.8 ±0.5)Pa;3％ PVA:G'was (6.1 ± 1.3)Pa,G" was (1.3 ±0.9)Pa;7％ PVA:G'was (106.5 ± 18.6) Pa and G" was (18.3 ± 12.8) Pa.According to resilience,the hydrogels can be ranked as follows:7％ PVA ＞ 3％ PVA ＞ 1％ PVA;in terms of creep,3％ PVA ＞ 7％ PVA ＞ 1％ PVA.Additionally,MTT colorimetric assay showed that there was no significant difference in A values among the three groups by cells culture in 1％,3％ and 7％ PVA for 72 hours (all P ＞0.05),suggesting that all the three hydrogels showed no obvious cytotoxicity and had good biocompatibility.Conelusion 3％ PVA hydrogel has similar optical,physical and rheological properties to the human vitreous,and shows good biocompatibility,which can be one of vitreous substitutes.

Effect of FTY720 inhibiting corneal neovascularition induced by sphingosine 1-phosphate / 国际眼科杂志(Guoji Yanke Zazhi)

AlM: To explore the inhibiting effect of FTY720 on corneal neovascularization ( CNV) of rat.METHODS: MTT assay and cells scratch were adopted to observe hyperplasia of human umbilical vein endothelial cells ( HUVECs ) and cell migration induced by sphingosine-1-phosphate(S1P) after using FTY720 of different concentration. The effect of FTY720 on CNV induced by S1P in a rat corneal micropocket model was detected. 30SD rats were randomly divided into group A, group B and group C with 10 rats per group. S1P and 0μg, 5μg, and 20μg FTY720 controlled-released particles were implanted into the corneal stroma. The growth of CNV and having pathological examination on 12d after the operation was observed. Findings was analyzed by one-way ANOVA.RESULTS: 10, 102 , 103 , and 104 nmol/L FTY720 and HUVECs co-incubate 72h could inhibit cell proliferation (P < 0. 01 ), 24h after the function of 10, 100nmol/L FTY720, it could inhibit S1P-induced cell migration and the ability of restricting cell proliferation and cell migration was enhanced with increasing concentration of FTY720. On 12d, after rat corneal micropocket controlled-release particles was implanted into groups A, B, C, the CNV area were respectively 10. 05±1. 19, 6. 59±0. 95, 2. 70± 0.68mm2(F=145. 155, P<0. 01), group A and group B was statistically different and this was the same case between group B and group C (P<0. 01).CONCLUSlON:FTY720 can inhibit S1P-induced corneal neovascularization.

Concentration change of TGF -β 1 in aqueous humor of rabbits

OBJECTIVE@#To observe the influence of the the transforming growth factor β1 (TGF-β1) eye drops on rabbit aqueous humor TGF-β1 concentration, and to analyze the best drug concentration.@*METHODS@#A total of 30 New Zealand white rabbits were randomly divided into 5 groups with 6 in each. Rabits in control group had PBS eye drops, group A, B, C, D adopted TGF-β1 eye drops at 0.5, 1.0, 2.0, 4.0 mg/L, respectively, 4 times a day. Aqueous humor of right eye was extracted 1 week after administration to detect concentration changes of TGF-β1 by ELISA; rabbits in fpur hroups adopted 2.0 mg/L eye drops to left eyes 4 times a day, 0.2 mL aqueous humor was extracted left eye at the scheduled time point 0, 30 min, 2 h, 4 h, 24 h for testing, the slit lamp was used to observe the cornea, chamber and lens.@*RESULTS@#No obvious pathological changes in conjunctiva, cornea, rabbit conjunctival, anterior chamber, and the lens was found. Concentration of TGF-β1 in rabbit aqueous humor in C, D group was significantly higher than the control group (P<0.05).@*CONCLUSIONS@#TGF-β1 eye drops at 2.0 mg/L, 4.0 mg/L can significantly increase concentration of TGF-β1 in rabbit aqueous humor, withe good ocular surfac permeability.

Pathological changes of the cornea in rabbits with hyphema and concurrent ocular hypertension / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the impact of hyphema secondary to high intraocular pressure on corneal pathology in rabbits.</p><p><b>METHODS</b>Thirty adult New Zealand rabbit were randomized into 3 equal groups, and in each rabbit, one eye served as the experimental eye with the other as the control eye. In the experimental eye, autoblood was injected into the anterior chamber to induce high intraocular pressure maintained for 3, 5, or 8 days. Only saline was injected into the control eye. After the injections, the cornea was observed with slit-lamp microscopy, and at 3, 5, or 8 days, the experimental and control eyes were taken from the 3 groups for microscopic examination of the corneas to detect the occurrence of cornea bloodstain with prolonged high intraocular pressure. Corneal edema, elastic fibers changes, growth of new blood vessels, changes of eosinophils, fibroblasts, lymphocytes and plasma cells, as well as the pathological changes of the corneal layers were observed and compared between the experimental and control eyes.</p><p><b>RESULTS</b>Maintenance of high intraocular pressure for 8 days resulted in the most severe corneal edema and thickening, and histopathologically, the corneal stroma showed widened space between the elastic fibers and obvious fiber distortion. Neovascularization was seen in the marginal cornea where eosinophil infiltration occurred with a small number of lymphocytes, plasma cells and fiber cells. All the three groups showed more obvious edema in the posterior than in the anterior cornea.</p><p><b>CONCLUSION</b>Prolonged hyphema with ocular hypertension results in aggravation of corneal edema, and corneal blood staining does not occur until 8 days of high intraocular pressure but corneal elastic fiber disruption can be seen, suggesting the impending irreversible pathological changes of cornea.</p>

Role of Toll-like receptor 2 in corneal graft rejection following penetrating keratoplasty / 南方医科大学学报

<p><b>OBJECTIVE</b>To gain insight into the role of Toll-like receptor 2 (TLR2) in graft rejection following penetrating keratoplasty, and investigate the expression of TLR2 mRNA in the corneal graft.</p><p><b>METHODS</b>Penetrating keratoplasty was performed in 3 groups of rats for orthotopic autologous corneal transplantation (group A), allograft corneal transplantation (group B), or allograft corneal transplantation with hormone treatment (C). The transparency and neovascularization of the cornea were observed using a slit-lamp microscope and scored according to the rejection index, with normal cornea serving as the control. The corneal tissues were sampled at 5, 7, and 9 days after the transplantation for histopathological examination and detection of TLR2 mRNA expression using RT-PCR.</p><p><b>RESULTS</b>With the passage of time, edema, opacities and neovascularization of the corneal graft occurred after the operation in all the groups. Seven days after the operation, the rejection index of group B, but not that of groups A and C, met the diagnostic criteria for graft rejection with also support by histopathological evidence. The expression of TLR2 mRNA was detected in normal corneas and augmented in the corneal grafts in the 3 transplantation groups. TLR2 mRNA expression in group B was significantly higher than that of group A, and the expression in group C decreased significantly in comparison with that in group B (P<0.05).</p><p><b>CONCLUSION</b>As the recognition receptors of native immune system, TLR2 in the rejected corneal grafts may recognize the allograft antigen and play a role in acute graft rejection after penetrating keratoplasty.</p>

Neovascularization profile in rat allogenic penetrating keratoplasty / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To observe the neovascularization process with no intervention in rat allogenic penetrating keratoplasty. METHODS: Allogenic penetrating keratoplasties were successfully performed in 34 female SPF SD rats with no intervention after operations. Corneal neovascularization(CNV) process was noted on day 4, 7, 15 and 30 with operating microscope. The vascular area surface was calculated using the formula C/12×3.14×[r2-(r-I)2].RESULTS: CNV was noted in 29 out of 34 rats (85%). Firstly, the new vessels distributed around the cornea like a brush then gradually extended towards the center. The vessels were distorted and massive with branched tails, they continued growing to reticulated veins in peak time then gradually atrophied. The average neovascularization area (SE) on day 4, 7, 15 and 30 was 11.8±3.5mm2, 18.5±4.0mm2,14.4±4.3mm2 and 6.0±1.8mm2 respectively and 12.7±1.9mm2 in total. The average percentage that new vessels accounting the whole cornea area(SE) was 30.8%±8.7%, 65.3%±12.8%, 59.4%±14.5%,36.2%±10.9% and 48.7%±6.4% in total.CONCLUSION: In rat allogenic penetrating keratoplasties without intervention, CNV presented on day 4 and reached the maximum area on day 7. Then the vessels gradually atrophied, about 50% of the maximum area still remained on day 30.

Recombinant type 1 adeno-associated virus-mediated transfection of ECV304 cells with enhanced green fluorescent protein gene in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To assess the feasibility of recombinant type 1 adeno-associated virus (rAAV1) as a vector for gene therapy of corneal neovascularization.</p><p><b>METHODS</b>The rAAV1 vector carrying enhanced green fluorescence protein (EGFP) gene (rAAV1-EGFP) was transfected into ECV304 cells at different multiplicities of infection (MOI=5 x 10(3), 5 x 10(4), 5 x 10(5)). EGFP expression in the cells was observed under inverted fluorescence microscope, and the EGFP-positive cell percentage determined by flow cytometry. MTT assay was used to assess the proliferation of the transfected cells.</p><p><b>RESULTS</b>The cells with rAAV1-EGFP transfection at MOI of 5 x 10(5) began to exhibit GFP expression 2 days after transfection, and the fluorescence intensity increased with the MOI used for transfection. GFP expression reached the maximum on day 7, at the point of which the transduction efficiency of rAAV1-EGFP in ECV304 cells was 45.90%, 58.56% and 68.31% corresponding to MOIs of 5 x 10(3), 5 x 10(4), and 5 x 10(5), respectively. MTT assay did not reveal significant difference in the absorbance between the transfected cells and the control cells at 72 and 96 h after transfection.</p><p><b>CONCLUSION</b>arAAV1-EGFP gene can be stably and efficiently expressed in ECV304 cells without causing cell growth inhibition, suggesting the potential of rAAV1 as a safe and efficient vector for gene therapy of corneal neovascularization.</p>

Effects of anti-CD25 monoclonal antibody on the level of cytokines in aqueous humour after rat penetrating keratoplasty / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the toxicity of anti-CD25 monoclonal antibody (mAb) to rat cornea and its effects on the cytokines in the aqueous humour after penetrating keratoplasty (PKP), thereby evaluating the effect of anti-CD25 mAb in preventing corneal allograft rejection.</p><p><b>METHODS</b>The corneal toxicity of anti-CD25 mAb at 50, 100 and 200 microg administered via subconjunctival injection was evaluated in 12 SD rats by histological examination and transmission electron microscopy. Another 93 SD rats were randomized into 5 groups, and transplantation of corneal allograft from Wistar rats was performed in 4 groups with the other group as the normal control. The 4 allograft groups were treated with saline, 100 microg anti-CD25 mAb, 100 microg anti-CD25 mAb with 50 microg dexamethasone, and 50 microg dexamethasone, respectively. The graft rejection was observed, the aqueous humour levels of IFN-gamma and IL-4 were measured with ELISA, and IFN-gamma mRNA expressions in the grafts detected with RT-PCR.</p><p><b>RESULTS</b>anti-CD25 mAb at 50 or 100 microg did not show significant toxicity on the cornea, but at 200 microg, the mAb caused swelling of the corneal stromal cells and endothelial cells. After corneal allograft transplantation, a significant delay in allograft rejection was observed in the 3 groups with mAb or dexamethasone treatment as compared with that in saline group (P<0.05). IFN-gamma mRNA expression in the allograft on days 11 after PKP and in the aqueous humour on days 6 and 11 was markedly increased in saline group compared with that in the 3 treatment groups (P<0.05). The mean IL-4 level in the aqueous humour was significantly higher in the mAb group than in saline group (P<0.05), but markedly lower in anti-CD25 mAb+dexamethasone and dexamethasone groups than in anti-CD25 mAb group (P<0.05).</p><p><b>CONCLUSIONS</b>Anti-CD25 mAb at 20 and 100 microg does not obviously affect the rat corneas. Anti-CD25 mAb inhibits IFN-gamma expression and promotes IL-4 the expression to reduce corneal allograft rejection, whereas anti-CD25 mAb with low-dose dexamethasone inhibits both IFN-gamma and IL-4 expressions to more effectively promote the graft survival.</p>

Role of dendritic cells in graft rejection after penetrating keratoplasty / 南方医科大学学报

<p><b>OBJECTIVE</b>To gain insight into the role of dendritic cells in graft rejection following penetrating keratoplasty by investigating their distribution in rat cornea.</p><p><b>METHODS</b>Orthotopical corneal transplantation was performed and immunohistochemical staining of the whole-mount cornea and the spleen tissue specimen employed to determine the distribution of the dendritic cells in the cornea.</p><p><b>RESULTS</b>Graft rejection occurred in all rats following the transplantation. No OX-62(+) dendritic cells were found in normal cornea but they were present in the epithelium of the cornea graft with allograft rejection.</p><p><b>CONCLUSION</b>OX-62(+) dendritic cells presenting in the rejected cornea may be related to acute graft rejection after penetrating keratoplasty.</p>

Clinical observation of the treatment of male sexual dysfunction by yangjingjiaonang / 中华男科学杂志

<p><b>OBJECTIVE</b>To observe the therapeutic effect of Yangjingjiaonang on the treatment of male sexual dysfunction.</p><p><b>METHODS</b>Two hundred and ten patients with male sexual dysfunction were divided into 2 groups, the treatment group orally delivered with Yangjingjiaonang, and the control group administrated with Wuziyanzongwan. The treatment lasted for 60 days, and the changes of sexual function were compared and measured by the total scores of IIEF.</p><p><b>RESULTS</b>After taking orally drugs for 60 days, the total scores of IIEF-5 and the total scores of the questions 11 and 12 of IIEF in treatment group elevated from (11.26 +/- 2. 72) score and (5.12 +/- 1.16) score to (16.84 +/- 3.12) score and (8.50 +/- 1.02) score, respectively, showing a significant improvement in the treatment group compared with the control (P <0.01). There was no significant difference for the treatment of premature ejaculation between the 2 groups.</p><p><b>CONCLUSION</b>Yangjingjiaonang has been demonstrated to have apparent therapeutic effect on improving male sexual function.</p>

Application of digital subtraction angiography and type B ultrasonography in the evaluation of vascular injury in patients with high voltage electrical injury / 中华烧伤杂志

<p><b>OBJECTIVE</b>To compare the difference between digital subtraction angiography (DSA) and type B ultrasonography in the evaluation of vascular injury in patients inflicted with high voltage electrical injury.</p><p><b>METHODS</b>Nineteen patients with high voltage electrical injury of upper limbs were enrolled in the study as burn group, and another 12 healthy volunteers as controls. The endovascular membrane, vascular wall thickness, intra-vascular blood flow and endovascular thrombosis formation of ulnar and radial arteries at wound site and in regions 5, 10 and 15 cm proximal to the wounds were examined by DSA and type B ultrasonography and compared with imagings of healthy volunteers as control. The injury degree of the ulnar and radial arteries was examined during operation for evaluation to corroborate with DSA and ultrasonography findings. Necrotic and/or thrombotic vessels were excised and sent for pathomorphological examination.</p><p><b>RESULTS</b>By DSA images abnormal signs as thrombosis, vascular lumen stenosis and blood flow deceleration were found in 14 ulnar and 11 radial arteries, and the signs were more pronounced in ulnar arteries. By type B ultrasonography, abnormal signs as roughing of tunica intima, swelling or exfoliation, thickening of vascular wall, lumen stenosis, decreased blood flow, even necrosis of vascular wall and thrombosis were identified in 19 ulnar and 16 radial arteries in burn group (P < 0.05 approximately 0.01). The blood flow in ulnar artery 5 cm to the approximal part of the wound edge was obvious lower than that of the control (31.60 +/- 13.90 ml/min vs 47.70 +/- 9.60 ml/min, P < 0.05).</p><p><b>CONCLUSION</b>Type B ultrasonography and DSA could be helpful in the evaluation of vascular injury in patients inflicted with high voltage electrical injury.</p>