Ying Su

Axonal regeneration of optic nerve after crush in Nogo66 receptor knockout mice

Mature retinal ganglion cells (RGCs) cannot regenerate injured axons because some neurite growth inhibitors, including the C-terminal of Nogo-A (Nogo66), myelin-associated glycoprotein (MAG) and Omgp, exert their effects on neuron regeneration through the Nogo receptor (NgR). In this study, the axonal regeneration of retinal ganglion cells (RGCs) after optic nerve (ON) crush was investigated both in vivo and in vitro in NgR knockout mice. We used NgR knockout mice as the experimental group, and C57BL/6 mice as the control group. Partial ON injury was induced by using a specially designed ON clip to pinch the ON 1mm behind the mouse eyeball with 40g pressure for 9s. NgR mRNA was studied by in situ hybridization (ISH). NgR protein was studied by Western blot. Growth Associated Protein 43 (GAP-43), a plasticity protein expressed highly during axon regeneration, was studied by immunofluorescence staining on the frozen sections. RGCs were cultured and purified. The axonal growth of RGCs was calculated by a computerized image analyzer. We found that compared with the control group, the GAP-43 expression was significantly higher and the axonal growth was significantly more active at every observation time point in the experimental group. These results indicate that NgR genes play an important role in the axonal regeneration after ON injury, while knockout of NgR is effective for eliminating this inhibition and enhancing axonal regeneration.

Inhibition of cell proliferation of Tenon's capsule fibroblast by S-phase kinase-interacting protein 2 targeting siRNA through increasing p27 protein level.

PURPOSE Although antiproliferative drugs have been used to prevent scarring after filtration surgery in patients with glaucoma, there are complications associated with their use. In the present study, the authors investigated whether small interfering RNA (siRNA)-mediated gene silencing of Skp2 can be used to increase p27(kip1) level and inhibit cell proliferation in rabbit Tenons capsule fibroblast (rTF). METHODS A plasmid containing Skp2 siRNA was used to decrease the high constitutive level of Skp2 protein in rTF, which can lead to consequent degradation of p27(kip1). Cell proliferation was assayed by immunocytochemistry using antibodies against 59-bromodeoxyuridine (BrdU) and proliferating cell nuclear antigen (PCNA). Skp2 siRNA was delivered to a trabeculectomy animal model to study the effect on rTF proliferation in vivo. RESULTS Immunocytochemistry and Western blot analysis showed a decreased level of Skp2 and an increased level of p27(kip1) in cells transfected with pSkp2 siRNA but not in vehicle transfection and uninfected cells in vitro and in vivo. MTT assay showed that cell viability significantly declined in rTF transfected with Skp2 siRNA. Skp2 siRNA-transfected cells showed significantly less BrdU- and PCNA-positive staining than control cells in vitro and in vivo. Infiltration bleb was detected in the Skp2 siRNA group 14 days after trabeculectomy. CONCLUSIONS Skp2 siRNA inhibited cell proliferation and decreased cell viability of rTF in vivo and in vitro. These findings suggest that siRNA-mediated gene silencing of Skp2 can be a novel gene therapy to treat scarring after glaucoma surgery by the suppression of p27(kip1) downregulation.

Expression change of PirB in mice retina after optic nerve injury.

The aim of this study was to observe the location of paired immunoglobulin-like receptor B (PirB) in the retina and to evaluate the expressive varieties of PirB in the retina of mice after optic nerve injury. In situ hybridization was used to observe the location of PirB mRNA in the retina of mice. Western blotting was used to analyze the levels of PirB protein in retina 7 days after optic nerve crush. Expression of PirB was located in the retinal ganglion cells of mice. The level of PirB protein increased significantly in the retina after optic nerve crush compared to the control group. PirB plays an important role in the inhibition of axonal regeneration after optic nerve injury. We conclude that the inhibition of PirB expression may enhance axonal regeneration after optic nerve traumas.

Arsenic Trioxide Inhibits Proliferation of Rabbit Tenon's Capsule Fibroblasts After Trabeculectomy by Downregulating Expression of Extracellular Matrix Proteins.

PURPOSE The purpose of this study was to study the effectiveness of arsenic trioxide (ATO) in regulation of trabeculectomy in an animal model. METHODS This study was designed as random, comparative, and prospective study. Primary Tenons capsule fibroblasts (TFs) were cultured and their viability after arsenic trioxide (ATO) treatment was detected using 3-(4, 5-dimethylthiazolyl-2-)-2, 5-diphenyltetrazoliumbromide (MTT) and bromodeoxyuridine incorporation (BrdU) assays. The dose of ATO was determined by 50% growth inhibition (IC50 values) and cell-cycle analysis. The expression of extracellular matrix (ECM) components and proliferating cell nuclear antigen (PCNA) was determined by Western blotting analysis. The status of filtration bleb was detected by hematoxylin and eosins (HE) stain. RESULTS Based on the MTT and BrdU assays, IC50 values was observed in rabbit TFs (rTFs) cells after treatment with ATO at a concentration of 6 μM arsenic trioxide for 72 hours. In addition, the ATO treatment caused not only a significant reduction in the expression of ECM proteins, fibronectin, collagen IV, and laminin in rTFs, but also reduced the expression of PCNA expression in a time dependent manner. The histologic observation showed high-level proliferation of rTFs in both vehicle and control groups, and proliferative ability of rTFs of experimental group was less than in vehicle and control group. Moreover, no inflammation was observed in the experimental group, and the filtering bleb was detected in the experimental group after 14 days of trabeculectomy. CONCLUSIONS Arsenic trioxide inhibited the proliferation of rTFs after trabeculectomy and may improve the success ratio of trabeculectomy in an animal model.