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.

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.

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.

[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.

[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.