TRPML1 Inhibited Photoreceptor Apoptosis and Protected the Retina by Activation of Autophagy in Experimental Retinal Detachment.

OBJECTIVE: In this study, we used a rat model of retinal detachment (RD) to investigate the effects of transient receptor potential mucolipin 1 (TRPML1) on photoreceptor cells and the underlying mechanism. METHODS: An RD model was established by subretinal injection of sodium hyaluronate, and mucolipin synthetic agonist 1 (ML-SA1) and dimethyl sulphoxide were subretinally injected after RD induction. Retinal morphology was observed using haematoxylin-eosin staining, and the apoptosis of photoreceptor cells was detected by transmission electron microscopy. Reactive oxygen species (ROS) were examined with an ROS detection kit. The retinal expression levels of TRPML1, the autophagy-related protein microtubule-associated protein 1 light chain 3 (LC3), Beclin 1, and cleaved caspase 3 were detected by Western blotting. The Morris water maze was used to test vision-dependent behaviour. RESULTS: We found that retinal structure and the outer nuclear layer were improved and that the apoptosis of photoreceptor cells was reduced after ML-SA1 injection. The expression of ROS was reduced, and the loss of TRPML1 was inhibited after ML-SA1 treatment. The LC3-II to LC3-I ratio and Beclin 1 expression were enhanced, and cleaved caspase 3 expression was decreased after ML-SA1 treatment. Treatment with ML-SA1 also improved vision-dependent behaviour. CONCLUSIONS: Our findings suggest that ML-SA1 attenuates photoreceptor apoptosis and improves vision-dependent behaviour by activation of autophagy.

TRPV4 regulates migration and tube formation of human retinal capillary endothelial cells.

BACKGROUND: Ca2+ entry plays an important role in modulating endothelial cell migration and tube formation. Transient receptor potential cation channel subfamily V member 4 (TRPV4) is a Ca2+-permeable channel that is widely expressed in endothelial cells. It has been reported that TRPV4 is expressed in HRCECs and regulates Ca2+ entry. However, the function of TRPV4 in human retinal capillary endothelial cells (HRCECs) remains unknown. METHODS: In this study we used western blot and immunostaining assay to verify TRPV4 expression in HRCECs. And then we pretreated HRCECs with HC067047 and transfected with specific shRNA of TRPV4. The functional presence of TrpV4 was determined by using fluorescence, migration and tube formation assay in TrpV4 knockdown cells or control cells. RESULTS: Using western blot and immunostaining, we confirmed TRPV4 expression in HRCECs. Moreover, inhibition of TRPV4 using the specific inhibitor HC067047 and the knockdown of TRPV4 with shRNA significantly suppressed tube formation and migration by HRCECs. CONCLUSIONS: TRPV4 is essential for HRCEC migration and tube formation, and maybe a potential therapeutic target for retinal vascular diseases.

[Effects of blocking activation of IGF-1-Stat3 signaling pathway in guinea pig sclera fibroblast by AG490 on expression of MMP-2 and Integrinß(1)].

OBJECTIVE: To explore the effect of Blocking activation of IGF-1-Stat3 signaling pathway in guinea pig sclera fibroblast (GSFs) by AG490 on expressions of MMP-2, Integrinß(1). METHODS: Cultured GSFs were divided into four groups: group A(control group: only DMEM without IGF-1), group B (only IGF-1 group), group C(IGF-1 + PBS group), group D (IGF-1 + 25 µmol/L AG490 group). The expressions of Stat3, p-Stat3, MMP-2, Integrinß(1) protein induced by IGF-1 and inhibited by AG490 in GSFs were detected by Western blot. The levels of Stat3, MMP-2 and Integrinß(1)mRNA were detected by RT-PCR. RESULTS: Compared with Groups A and D, Stat3, p-Stat3, MMP-2 protein expression in groups B and C were expressed at higher level (t(pr) = -32.324, -26.284, -32.876, -26.345, -68.668, -58.724, -187.481, -58.842, -110.264, -120.256, -121.345, -120.286; t(mRNA) = -31.554, -31.178, -31.286, -31.198, -12.076, -14.969, -11.896, -14.546, P < 0.05), but the expression levels were not obviously different between groups B and C (t(p) = -32.720, -32.816, -68.668, -187.481, -110.264, -121.345; t(mRNA) = -0.692, -0.579, P > 0.05), which were similar to mRNA level. The Integrinß(1) protein and mRNA were expressed in groups A, B, C and D but no significant difference among them respectively (F(pr) = 0.214;F(mRNA) = 0.045, P > 0.05). CONCLUSIONS: Activation of Stat3 signaling pathway may be involved in up-modulating the expression of MMP-2 in GSFs, and not affect the Integrinß(1) protein and mRNA changes. The results reveal that Stat3 signaling transduction pathway may play a critical role in sclera remodeling by means of modulating MMP-2 expression.

[Supracapsular implantation with optic capture of posterior chamber intraocular lens in aphakic school-age children after traumatic cataract].

OBJECTIVE: To assess the clinical effects of supracapsular implantation with optic capture of the posterior chamber intraocular lens in school-age children with traumatic cataract. METHOD: It was a retrospective case series study. Thirteen cases (13 eyes) received posterior curvilinear capsulorhexis with optic capture of the posterior chamber intraocular lens. Pre- and post-operative visual acuities were recorded. Intra-o and post-operative complications were observed. The follow-up period ranged from 6 to 42 months. RESULTS: Implantation of optic capture of the posterior chamber intraocular lens was successfully performed in 13 eyes. The best-corrected-visual acuity ranged from 0.2 to 1.0. No optic axis opaque was found in 10 eyes with optic capture. The major complications of optic capture were lenticular precipitates and posterior synechia of the iris. Intraocular dislocation was found in one case two weeks after the operation. CONCLUSIONS: Supracapsular implantation with optic capture of the posterior chamber intraocular lens is safe and effective for the treatment of traumatic cataract in school-age children.

Expression and role of specificity protein 1 in the sclera remodeling of experimental myopia in guinea pigs.

AIM: To study the expression of collagen I and transcription factor specificity protein 1 (Sp1), a transforming growth factor-ß1 (TGF-ß1) downstream target, and reveal the impact of the TGF-ß1-Sp1 signaling pathway on collagen remodeling in myopic sclera. METHODS: Seventy-five 1-week-old guinea pigs were randomly divided into normal control, form deprivation myopia (FDM), and self-control groups. FDM was induced for different times using coverage with translucent latex balloons and FDM recovery was performed for 1wk after 4wk treatment; then, changes in refractive power and axial length were measured. Immunohistochemistry and reverse transcription-polymerase chain reaction were used to evaluate dynamic changes in collagen I and Sp1 expression in the sclera of guinea pigs with emmetropia and experimental myopia, and the relationship between collagen I and Sp1 levels was analyzed. RESULTS: In the FDM group, the refractive power was gradually changed (from 2.09±0.30 D at week 0 to -1.23±0.69 D, -4.17±0.59 D, -7.07±0.56 D, and -4.30±0.58 D at weeks 2, 4, 6, and 1wk after 4wk, respectively; P<0.05), indicating deepening of myopia. The axial length was increased (from 5.92±0.39 mm at week 0 to 6.62±0.36 mm, 7.30±0.34 mm, 7.99±0.32 mm, and 7.41±0.36 mm at weeks 2, 4, 6, and 1wk after 4wk; P<0.05). The mRNA and protein expression of Sp1 and collagen I in the sclera of the FDM group was lower than that of the control groups (P<0.05), and the reduction was eye-coverage time-dependent. Furthermore, correlation between Sp1 and collagen I down-regulation in the myopic sclera was observed. CONCLUSION: Our data indicate that transcription factor Sp1 may be involved in the regulation of type I collagen synthesis/degradation during myopic sclera remodeling, suggesting that TGF-ß1 signaling plays a role in the development and progression of myopia.

Effects of scleral encircling surgery on vitreous cavity length and diopter.

AIM: To observe the changes of vitreous cavity length and diopter after scleral encircling (SE) produce. METHODS: This prospective study included 68 eyes of 68 non-consecutive patients with macula-off retinal detachment who were operated by SE surgery. The corneal refractive power, ocular axial length and diopter were measured by keratometer, A-mode ultrasonic meter and computed dioptometer. RESULTS: There was no significant difference in corneal refractive power among preoperative and postoperative 1, 3 and 6 mo (0.57±0.54 D at pre-surgery;0.72±0.26 D at 1mo; 0.71±0.34 D at 3mo; 0.69±0.31 D at 6mo; all P>0.05 ). Axial lengths were obviously lengthened, especially in vitreous cavity length (17.87±3.09 mm, 19.69±3.12 mm, 18.97±3.56 mm, 18.76±3.47 mm, 18.68±3.42 mm at pre-surgery, 1wk, 1, 3 and 6mo postoperatively, P <0.05) and diopter also increased at beginning and then recovered gradually. After 1 and 3 mo, axial length (vitreous cavity length) and myopia were more and in higher degree than before surgery. CONCLUSION: The change of postoperative vitreous cavity length is the main factor that results in the changes of axial length and then makes the change of diopter.

Risk factors of rhegmatogenous retinal detachment associated with choroidal detachment in Chinese patients.

AIM: To comprehensively analyze the risk factors of rhegmatogenous retinal detachment (RRD) associated with choroidal detachment (CD). METHODS: A total of 265 eyes of 265 consecutive cases of RRD were retrospectively analyzed. All patients had systemic and ophthalmologic examination. CD was diagnosed by indirect ophthalmoscopy, B-scan ultrasonography, and ultrasound biomicroscope (UBM). Each parameter was compared between patients of RRD and rhegmatogenous retinal detachment associated with choroidal detachment (RRDCD). Logistic regression analysis was used to determine the independent risk factors of CD. RESULTS: There were 52 eyes (19.62%) with CD. Pseudophakia was more commonly seen in RRDCD (21.15% vs 6.10%, P=0.002). Intraocular pressure (IOP) was lower (8.60±3.62 vs 12.96±3.55, P<0.001), best-corrected visual acuity was worse [3.00 (2.00 to 3.00) vs 1.92 (1.22 to 3.00), P=0.001], and refractive error was more myopic [-4 (-9 to -2) vs -2 (-6 to 0), P=0.007] in RRDCD. Eyes with RRDCD had larger extent of retinal detachment (P=0.007). In RRDCD, 34.62% of eyes presented with multiple holes (P=0.044) and 25.00% with macular holes (P=0.012), compared with 20.66% and 14.08% in RRD. High myopia (P=0.039), low IOP (P=0.017), and larger extent of retinal detachment (P<0.001) were significant and independent risk factors for developing CD. CONCLUSION: For CD in RRD, related factors include BCVA, IOP, lens status, refractive error, extent of retinal detachment, number of holes, and macular hole. Larger extent of retinal detachment, high myopia, and low IOP are significant and independent risk factors.

Activation of autophagy in photoreceptor necroptosis after experimental retinal detachment.

AIM: To investigate whether photoreceptor necroptosis induced by z-VAD-FMK (pan caspase inhibitor) was involved the activation of autophagy and whether Necrostatin-1, a specific necroptosis inhibitor, could inhibit this induction of autophagy after experimental retinal detachment. METHODS: Experimental retinal detachment models were created in Sprague-Dawley rats by subretinal injection of sodium hyaluronate and subretinal injections of z-VAD-FMK, vehicle or z-VAD-FMK plus Necrostatin-1. Three days after retinal detachment, morphologic changes were observed by transmission electron microscopy. In other animals, retinas were subjected to immunoprecipitation and Western Blotting, then probed with anti-RIP1, phosphoserine, LC-3II or caspase 8 antibody. RESULTS: It was proved by immunoprecipitation and western blotting, that photoreceptor necroptosis was mediated by caspase-8 inhibition and receptor interacting protein kinase (RIP1) phosphorylation activation. Transmission electron microscope and western blotting results indicated that photoreceptor necroptosis was involved the LC-3II and autophagosomes induction. We also discovered Necrostatin-1 could inhibit RIP1 phosphorylation and LC-3II induction. CONCLUSION: These data firstly indicate photoreceptor necroptosis is associated with the activation of autophagy. Necrostatin-1 protects photoreceptors from necroptosis and autophagy by down-regulation of RIP1 phosphorylation and LC-3II.