Bioinformatics analysis of potential essential genes that response to the high intraocular pressure on astrocyte due to glaucoma.

AIM: To study the gene expression response and predict the network in cell due to pressure effects on optic nerve injury of glaucoma. METHODS: We used glaucoma related microarray data in public database [Gene Expression Omnibus (GEO)] to explore the potential gene expression changes as well as correspondent biological process alterations due to increased pressure in astrocytes during glaucoma development. RESULTS: A total of six genes were identified to be related with pressure increasing. Through the annotation and network analysis, we found these genes might be involved in cell morphological remodeling, angiogenesis, mismatch repair. CONCLUSION: Increasing pressure in glaucoma on astrocytes might cause gene expression alterations, which might induce some cellular responses changes.

HIF­1 signaling pathway involving iNOS, COX­2 and caspase­9 mediates the neuroprotection provided by erythropoietin in the retina of chronic ocular hypertension rats.

This study aimed to investigate the impacts of erythropoietin (EPO) on the electroretinogram b­wave (ERG­b), and on the mRNA and protein expression levels of hypoxia­inducible factor­1α (HIF­1α), inducible nitric oxide synthase (iNOS), cyclooxygenase­2 (COX­2) and caspase­9 in chronic ocular hypertension rats. Episcleral vein cauterization (EVC) was used to establish the chronic ocular hypertension rat model based on the intraocular pressure (IOP) value. ERG­b and mRNA and protein expression levels of HIF­1α, iNOS, COX­2 and caspase­9 in normal, EVC­treated and EVC combined with EPO (EVC+EPO)­treated rats were measured by electroretinography, RT­PCR and western blotting, respectively. Moreover, the correlations of HIF­1α with IOP, ERG­b, iNOS, COX­2 and caspase­9 were evaluated. The mRNA and protein expression levels of HIF­1α, iNOS, COX­2 and caspase­9 in EVC­treated rats were increased significantly compared with normal rats. The peak expression levels of HIF­1α, iNOS, COX­2 and caspase­9 were respectively obtained 7, 7, 7 and 14 days postoperatively. Compared with EVC­treated rats, EPO administration weakened the mRNA and protein expression levels of HIF­1α, iNOS, COX­2 and caspase­9. The mRNA expression level of HIF­1α demonstrated a significant positive correlation with IOP and ERG­b. HIF­1α was positively correlated with iNOS, COX­2 and caspase­9 at the mRNA and protein levels. The protective effect of EPO on the retina of chronic ocular hypertension rats may be mediated by the HIF­1 signaling pathway involving iNOS, COX­2 and caspase­9.

Inhibition of choroidal neovascularization by anti-EphB4 monoclonal antibody.

The aim of this study was to determine the effect of the EphB4 monoclonal antibody on experimental choroidal neovascularization (CNV) progression. Experimental CNV was established by argon laser photocoagulation. In the experimental group, the EphB4 monoclonal antibody was injected into the vitreous space in the eye specimens on days 0, 3, 6 and 9 after CNV model establishment. In the control group, an equal amount of balanced salt solution was injected at the same time points. On day 10 after CNV model establishment, fluorescein isothiocyanate-dextran endocardial perfusion and choroidal stretched preparation were conducted, respectively, for the two groups. The CNV area in each light spot and the mean values were determined. Histopathological examination was conducted and the ratio of the maximum thickness of the CNV in each light spot to the surrounding normal choroidal thickness, as well as the mean ratio, were calculated. Choroidal stretched preparation confirmed that the CNV of the experimental group was smaller, whereas the CNV of the control group was wider and larger. Quantitative analysis revealed that CNV in the experimental group was significantly inhibited (t=11.84, P<0.01) and that CNV progression in the experimental group was significantly suppressed (t=7.45, P<0.01). Histopathological examination revealed that CNV in the experimental group was thinner and smaller. Vitreous injection of the EphB4 monoclonal antibody inhibits experimental CNV progression. However, its specific mechanism remains unclear. Endogenous EphrinB2/EphB4 regulates ocular neovascularization and may become a new target in treating CNV diseases.

Overexpression of cytoglobin gene inhibits hypoxic injury to SH-SY5Y neuroblastoma cells.

A plasmid for cytoglobin expression, pAcGFP1-C1-cytoglobin, was transfected into SH-SY5Y cells. Cobalt chloride was used to establish a model of hypoxia. Western blotting indicated that cytoglobin was overexpressed and there was low expression of hypoxia-inducible factor-1α in SH-SY5Y cells after transfection. Following cobalt chloride-induced hypoxia, cytoglobin and hypoxia-inducible factor-1α expression gradually increased in SH-SY5Y cells. Flow cytometry showed that with increasing duration of hypoxia, the proportion of normal cells significantly diminished in the transfected and non-transfected groups. The proportion of cells in the early stages of apoptosis increased. However, the proportion of apoptotic cells was significantly lower in the transfected group compared with the non-transfected group. These results demonstrate that cytoglobin and hypoxia-inducible factor-1α are strongly up-regulated by hypoxia, and that there is a strong relationship between hypoxia-inducible factor-1α and cytoglobin during hypoxic injury.

An AD-related neuroprotector rescues transformed rat retinal ganglion cells from CoCl2-induced apoptosis.

Some ocular diseases characterized by apoptotic death of retinal ganglion cells (RGCs) and Alzheimer's disease (AD) are chronic neurodegenerative disorders and have similarities in neuropathology. Humanin (HN) is known for its ability to suppress neuronal death induced by AD-related insults. In present study, we investigated the neuroprotective effects of HN on hypoxia-induced toxicity in RGC-5 cells. Hypoxia mimetic compound cobalt chloride (CoCl2) could increase the cell viability loss and apoptosis, whereas HN can significantly attenuate these effects. This finding may provide new therapeutics for the retinal neurodegenerative diseases targeting neuroprotection.

Fasudil, a Rho-associated protein kinase inhibitor, attenuates retinal ischemia and reperfusion injury in rats.

Abnormal activation of Rho kinase (ROCK) plays a vital role in the pathogenesis of ischemia/reperfusion (I/R)-induced retinal injury. The aim of this study was to investigate whether fasudil, a potent inhibitor of ROCK, has a protective effect on retinal I/R injury in rats and to explore the possible underlying mechanisms. Forty adult Sprague-Dawley rats were randomly assigned into sham, I/R injury model (I/R), model plus normal saline (control), and model plus fasudil (fasudil) groups. Rats in the control and fasudil groups were intravitreously injected with normal saline and fasudil, respectively, 5 min prior to the induction of ischemia. Retinal ischemia was induced by increasing the intraocular pressure to 100 mmHg for 60 min. Overall retinal thickness and retinal cell apoptosis was evaluated by histological analysis and the TUNEL assay, respectively. The protein expression of caspase-3 and the Bax/Bcl-2 mRNA ratio were also examined. Moreover, the retinal expression of inducible nitric oxide synthase (iNOS) was determined by immunohistochemical staining, quantitative real-time RT-PCR and Western blot analysis. Fasudil attenuated the I/R-induced apoptosis of retinal cells in the inner nuclear and ganglion cells of the rat retina. Fasudil significantly decreased the Bax/Bcl-2 mRNA ratio and the expression of caspase-3 and iNOS compared to the control group (P<0.05). Seven days after I/R, the overall retinal thickness in the fasudil group was significantly greater compared to that in the control group (P<0.05). In conclusion, fasudil can protect the rat retina from I/R injury by inhibiting apoptosis and iNOS expression, suggesting that fasudil may have a therapeutic potential for the prevention of retinal diseases associated with I/R.

Effect of erythropoietin on the expression of HIF-1 and iNOS in retina in chronic ocular hypertension rats.

AIM: To research the effect of erythropoietin (EPO) to the HIF-1\iNOS signal transduction path in retina in chronic ocular hypertension rat. METHODS: One hundred and twenty Wistar rats were divided into 12 groups randomly. Two episcleral veins were coagulated unilaterally in rats with electric coagulator to establish the glaucoma model. PT-PCR and Western Blot analysis were used to examine the expression of Caspase-9 genes in retina. And the changes of ERG-b wave before and after were detected using EPO. RESULTS: In EPO drug treatment group, the amplitude of ERG-b wave of retina restored remarkably. There was significant difference between two groups (P<0.05). The expressions of HIF-1\iNOS mRNA and protein in EPO drug treatment group were weakened remarkably. It was statistically different compared with the non-drug treatment group. CONCLUSION: One of protect mechanisms of EPO to injured retina caused by chronic intraocular hypertension is through HIF-1\iNOS signal conduct path.

IOP-lowering effects for the application of human umbilical vein in non-penetrating deep sclerostomy in rabbits.

AIM: To estimate the effects of human umbilical vein (HUV) implanted under the sclera of glaucoma model on intraocular pressure (IOP) lowering and to investigate its related mechanisms METHODS: A total of 20 human umbilical veins (HUV) were collected from healthy fetus umbilical core. After the establishment of glaucoma model in rabbits, human freeze-dried umbilical vein was implanted under the sclera during NPDS, while for control group, sclerostomy was performed without implant. The formation of the filtration bleb and IOP were detected every 24 hours before surgery and on day 3, 7, 10 and 14 after surgery. Handheld pen-type Tono-pen II tonometer was used to measure IOP after topical anesthesia treatment. Each measurement has three duplicates. The incision recovery, filtration, conjunctiva congestion and anterior chamber inflammation were observed everyday after surgery. RESULTS: IOP was decreased dramatically with less inflammation than traditional sclerostomies with the application of HUV. The significant differences of IOP between the NPDS with and without HUV implant groups were shown up from 10 days after surgery. The average IOP in NPDS without HUV implant was 14.25mmHg, while for NPDS with HUV implant group, it was 12.30mmHg. This structure of filtration bleb, which allowed the aqueous humor to leave the eye, was formed for any type of surgery. However, 1-2 weeks later, filtration bleb was still existed in the group of sclerostomy with HUV implant and more stable than that of the surgery without HUV implant. Histological observations were performed on day 3, 7 and 14 after surgery. For the eyes under sclerostomy with HUV implant, HUV lumina was shown up on 3 days after surgery with few fibroblast cells near the sclera. On 7 days after surgery, HUV lumina was stably maintained but with obvious fibroblast cells and inflammatory cell. On 14 days after surgery, HUV lumina was still clearly observed but with scarring formation, which suggests that the IOP lowering effects might result from an effective drainage structure formation. CONCLUSION: HUV might be an alternative material to make the drainage pathway for non-penetrating deep sclerostomy.

Effect of aminoguanidine on caspase-3 expression in rat retina after ischemia-reperfusion injury.

AIM: To investigate the effect of aminoguanidine (AG) on the expression of caspase-3 in rat retina after ischemia- reperfusion injury. METHODS: The rats were anesthetized with 30mg/kg sodium pentobarbital introperitoneal(ip) injections. After topical application of 10g/L dicaine, the anterior chamber was punctured with a 5-gauge needle connected to a bottle containing normal saline. Intraocular pressure was raised to 100 mmHg by elevating the saline container. The infusion needle was removed from the anterior chamber 60 minutes later. Reperfusion of the retinal vasculature was confirmed by fundus examination. AG 100mg/kg was ip injected in drug group. The rats were then euthanatized at 6, 24, and 72 hours after reperfusion, and their eyes were enucleated for immunohistochemistry. RESULTS: No specific staining was detected by using the caspase-3 antibody in the retina of control group. In ischemia group, the protein of caspase-3 was over-expressed at 6 hours and relieved at 24 hours and 72 hours, while with drug treatment, the expression of protein of caspase-3 was decreased at each time point. CONCLUSION: AG provides retinal protection against ischemia-reperfusion injury in rat retina, probably through an inducible NOS-dependent mechanism.

TGF-ß(1) in retinal ganglion cells in rats with chronic ocular hypertension: its expression and anti-apoptotic effect.

AIM: To investigate the anti-apoptotic effect of transforming growth factor beta-1 (TGF-ß(1)) on chronic ocular hypertension. METHODS: The expression of TGF-ß(1) in retinal ganglion cells (RCGs) was measured using the immunohistochemiscal S-P method and real-time PCR in the normally control group, the ocular hypertension group (experimental group A), the ocular hypertension plus antibody intervention group (experimental group B) and the ocular hypertension plus antigen intervention group (experimental group C) at 1, 2, 3 and 4 weeks postoperatively. The count of apoptotic RCGs was measured using the TUNEL method. RESULTS: The expression of TGF-ß(1 )was significantly higher in experimental group C than that in other three groups (P<0.05). The expression was the lowest in experimental group B (4.17%). A statistically significant difference was noted between the four groups (P<0.01). The count of apoptotic RCGs was statistically significantly lower in experimental group C than that in the experimental groups A and B (P<0.01). A statistically significant difference was noted in the count of apoptotic RCGs between these three experimental groups (P<0.01). CONCLUSION: TGF-ß(1) can inhibit the apoptosis of RCGs in rats with chronic ocular hypertension.

Expression of Nogo-A on the retina in rat model with chronic ocular hypertension.

AIM: To study the expressive variation of Nogo-A on rat retina in the process of chronic ocular hypertension. METHODS: Thirty-six healthy adult male Wistars were randomly divided into control group (6 rats) and chronic hypertension group (30 rats). Chronic hypertension was created by cauterizing the superficial scleral veins. Immunohistochemistry technique was used to evaluate the expressive varieties of Nogo-A at different time points during the course of chronic ocular hypertension. RESULTS: The success of the model was indicated by over 40% of increase in the IOP as compared with normal rats. Compared with control group, as time passed chronic hypertension group gradually had detectable morphology changes in the retina. At the 21st day of chronic ocular hypertension, retinas became thinner and the quantity of retinal ganglion cells (RGC) decreased (P<0.05). Assoicated with the morphological changes, the expression of Nogo-A was strongly increased (P<0.05). CONCLUSION: Myelin associated protein Nogo-A plays a part in the process of chronic ocular hypertension.

[The expression of extracellular matrix in epithelial ingrowth after LASIK].

PURPOSE: To observe the expression of extracellular matrix in cornea and its relationship with corneal opacity after epithelial ingrowth in LASIK. METHODS: Corneal flap was made on rabbit eyes, and epithelial cells mechanically scraped from surroundings the flap were implanted underneath. The corneas were harvested 1, 3, 7 and 30 days following surgery, histological and immunohistochemical examination was performed. RESULTS: On slit-lamp examination, corneal opacity was observed in the area where epithelial cells were implanted. Histological study revealed clusters of epithelial cells at the flap and stroma bed interface. One week and one month post-operation intense immunoreactivity for collagen type IV was detected at the perimeters of the intrastromal epithelial island, but not in the ordinary interface area, strong staining for collage type III was also observed around the epithelial islands. CONCLUSION: The marked deposition of collagen type IV surrounding the ingrowth epithelial cells indicated that it might be the essential component of interface haze in epithelial ingrowth after LASIK.

[The results and analysis of making corneal flaps with Moria M(2) microkeratome in LASIK].

OBJECTIVE: To investigate the results and technology of making corneal flaps with Moria M(2) microkeratome. METHODS: Eight hundred and six corneal flaps (in 409 cases) were made using Moria M(2) microkeratome in LASIK. The group consisted of 205 males (405 eyes), whose mean age was (23.61 +/- 5.44) years, mean refractive spherical equivalent was (-6.32 +/- 3.61) D, and mean corneal thickness was (542.72 +/- 29.54) micro m. The 130-micron head option was chosen, the pressure was less than 250 mm Hg (1 mm Hg = 0.133 kPa), the stop-ring was adjusted to the 8.0 mm position and the suction ring was chosen according to corneal curvatures. RESULTS: All 806 corneal flaps were made successfully at one time with suitable hinge size, and without ruptured or thin or incomplete flaps. Flaps could be turned and reattached easily. There was no breaking down during the surgical procedure. The flaps showed smooth edges with smooth keratectomy beds. COMPLICATIONS: (1) Free flaps occurred in 3 eyes (0.37%). (2) Island phenomenon occurred in 2 eyes (0.25%). (3) Failure to make the flaps occurred in 3 eyes (0.37%). CONCLUSION: All 806 corneal flaps were made successfully at one time with no serious complications. Choosing the suction ring according to corneal curvature, checking blades preoperatively and inserting blades correctly may help to avoid complications when making corneal flaps with Moria M(2) microkeratome.

[Suppression effects of interleukin-10 on mouse herpetic stromal keratitis].

OBJECTIVE: To investigate the role of interleukin-10 (IL-10) in herpetic stromal keratitis (HSK). METHODS: Eighty BALB/c mice were divided into two groups and infected with HSV-1 Mckrae strain by corneal scarification. Murine rIL-10 (20 ng/ml) was inoculated intracorneally 6 h before and again on days 0, 2 and 4 after topical HSV-1 corneal infection. rIL-10 (500 ng) was given intraperitoneally in treated mice simultaneously. The same amount of saline was injected into the control mice. The effects of IL-10 on HSK were evaluated. RESULTS: In the IL-10-treated animals, the onset of HSK was delayed, corneal stromal opacification and neovascularization were reduced, extensive cellular infiltrates in the cornea were prevented and delayed type hypersensitivity (DTH) was weakened. Examination of pro-inflammatory cytokine levels in the cornea 10 days after the infection revealed that the amounts of IL-2 and IL-6 were lower than those found in the controls. IL-10 did not suppress the viral replication nor did it eliminate the virus from IL-10-treated eyes, as compared with the controls. CONCLUSIONS: IL-10 treatment can suppress DTH responsiveness and the production of certain cytokines by corneal cells. It delays the onset and decreases the severity of HSK.