The inhibitory effect of small interference RNA protein kinase C-alpha on the experimental proliferative vitreoretinopathy induced by dispase in mice

Abstract

Aim: To evaluate the effects of small interference RNA protein kinase C-alpha (siRNA-PKCα) on experimental proliferative vitreoretinopathy (PVR) induced by dispase in mice.

Methods: C57BL/6 mice PVR models (4-6 weeks old) were induced by intravitreal injection of dispase and then equally divided into six groups. After 1 week, the five treatment groups received 2 μL, intravitreal injections of siRNA-PKCα at a concentration of 250 nM, 500 nM, 750 nM, 1000 nM, and 1500 nM, respectively, while the negative control group received 2 μL of 500 nM no-silencing siRNA. SiRNA-PKCα was transfected by a square wave electroporator. Postoperative ophthalmic observations of lens clarity and the fundus of the eyes were performed periodically. The eyeballs of the mice were enucleated and imbedded in optimal cutting temperature to perform histological and immunofluorescence analysis at the end of a 4-week observation period.

Results: Four weeks after the siRNA-PKCα injections, there are 100% lens dissolution and 100% PVR in the 250 nM group and 70%, 70%, 70%, and 50% PVR in the 500 nM, 750 nM, 1000 nM, and 1500 nM groups, respectively, which is significantly different from the negative group. Abnormalities in fundus appearance were related to the concentrations of siRNA-PKCα; a higher concentration of siRNA-PKCα resulted in a more normal fundus. Histological sections by hematoxylin-eosin staining of the eyes support the clinical observation. Immunofluorescence analysis showed that RPE65, glutamine synthase, glial acidic fibrillary protein, and α-smooth muscle actin were increasing in the retina with the decreasing concentration of siRNA-PKCα, indicating that intraocular siRNA-PKCα can partly inhibit changes of markers for glia cells, fibroblast cells, retinal pigment epithelium cells, and Müller cells in the process of PVR.

Conclusion: Gene therapy with siRNA-PKCα could effectively inhibit PVR in mice and provide us with a novel therapeutic target on PVR.

Keywords: dispase; proliferative vitreoretinopathy; protein kinase Cα; small interference RNA.

Figure 1

siRNA-PKCα intravitreal injection and transfection in mice. (A) Intravitreal injection with a Hamilton syringe, fitted with a 30 G needle. (B) Electricity transfection by square wave electroporator. Abbreviation: siRNA-PKCα, small interference RNA-protein kinase C-alpha.

Figure 2

PVR development at 4 weeks after siRNA-PKCα injection. (A) Clinical PVR fundus photographs in the 250 nM and 1500 nM siRNA-PKCα, and in the negative control at the end of the 4-week observation period. Obvious retinal folds, epiretinal membranes, and uneven irises are observed in the 250 nM siRNA-PKCα treatment group, similar to those in the negative group; however, the radial distribution of the retinal arteries and veins are shown in the 1500 nM siRNA-PKCα. (B) Percentage in the five treatment groups and negative control. Note: The percentages in the 250 nM and negative groups are significantly different from those in the other groups. *P < 0.05. Abbreviations: PVR, proliferative vitreoretinopathy; siRNA-PKCα, small interference RNA-protein kinase C-alpha.

Figure 3

RT-PCR analysis after injection of 1500 nM siRNA-PKCα. Notes: PKCα messenger RNA was significantly down regulated following siRNA-PKCα injection when compared with those that were dispase-injected or in the control groups (ANOVA, **P = 0.00018 < 0.01, *P = 0.00010 < 0.01). The GAPDH band is used for quantitation. Abbreviations: RT-PCR, reverse transcription polymerase chain reaction; siRNA-PKCα, small interference RNA-protein kinase C-alpha; PKCα, protein kinase C-alpha; RNA, ribonucleic acid; ANOVA, analysis of variance; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 4

PKCα protein changes after siRNA-PKCα injection. Notes: Western blot analysis shows that PKCα decreased compared to those from the dispase-injected and control groups (ANOVA, *P = 0.00220 < 0.01, **P = 0.00490 < 0.01). The β-actin band with 42 kDa is used for quantitation. Abbreviations: PKCα, protein kinase C-alpha; siRNA-PKCα, small interference RNA-protein kinase C-alpha; ANOVA, analysis of variance.

Figure 5

HE staining of eyes at 4 weeks after siRNA-PKCα injection. Notes: A proliferative membrane and retinal detachment in the vitreous cavity were observed in the negative control, 250 nM, 500 nM, and 750 nM groups; however, normal retinal structures were found in the 1000 nM and 1500 nM groups when compared with normal eyes. Scale bar: 100 μm. Abbreviations: HE, hematoxylin and eosin; siRNA-PKCα, small interference RNA-protein kinase C-alpha.

Figure 6

Immunofluorescence analysis of RPE65 and GS 4 weeks after siRNA-PKCα injection. Notes: There are faint expressions of RPE65 (red) and GS (green) in normal retinas; these expressions seemed most pronounced in the epiretinal membranes in the negative control, as well as in the 250 nM, 500 nM, and 750 nM groups when compared to the 1000 and 1500 nM groups. Scale bar: 100 μm. Abbreviations: RPE, retinal pigment epithelium; GS, glutamine synthetase.

Figure 7

Immunofluorescence analysis of GFAP and α-SMA at 4 weeks after siRNA-PKCα injection. Notes: There are faint expressions of GFAP (red) and (green) in normal retinas; these expressions seemed more pronounced in the epiretinal membranes in the negative control, as well as in the 250 nM, 500 nM, and 750 nM groups than in the 1000 nM and 1500 nM groups. Scale bar: 100 μm. Abbreviations: GFAP, glial fibrillary acidic protein; α-SMA, α-smooth muscle antibody; siRNA-PKCα, small interference RNA-protein kinase C-alpha.