Male Sprague Dawley (SD) rats (weighting 200–250 g, purchased from Vital River, Beijing, China) were randomly assigned to 6 groups (n=24 per group): i) Control group, ii) STZ group, iii) STZ+control siRNA group, iv) STZ+NgR siRNA group, v) STZ+CNTF group and vi) STZ+NgR siRNA+CNTF group. The rats in the STZ group were treated intraperitoneally with a single injection of 65 mg/kg STZ (Solarbio, Beijing, China) dissolved in 0.01 M cold sodium citrate buffer and treated with saline. The control siRNA or NgR siRNA (5 µl and 3 µM) (GenePharma, Shanghai, China) were injected into the vitreous of diabetic rats in the STZ+control siRNA group or STZ+NgR siRNA group, respectively (This injection was repeated after 6 weeks). CNTF (1 µg) (Sino Biological, Beijing, China) were administrated into the vitreous of diabetic rats in the STZ+CNTF group once a week for 12 weeks. The rats in the STZ+NgR siRNA+CNTF group received NgR siRNA and CNTF after induction of diabetes. The rats in the control group received sodium citrate buffer and saline. Blood glucose level (mM) was detected 3 days post-STZ administration for diabetes induction confirmation (>16.7 mmol/l). All the rats were sacrificed after 12 weeks. The retinal tissues were excised and fixed in 4% paraformaldehyde for further analyses. The experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by Animal Care and Use Committee of Guangxi Medical University (Nanning, China).

The paraffin-embedded retinal tissues were sectioned into 5-µm slices. The sections were deparaffinized with xylene and graded ethanol (100, 95, 85 and 75% ethanol). The sections were stained with hematoxylin for 5 min and incubated with 1% hydrochloric acid alcohol for 3 sec, followed by staining with eosin for 3 min. Subsequently, the sections were dehydrated with graded ethanol (75, 85, 95, and 100% ethanol) and xylene. The sections were mounted with neutral gum and captured by OLYMPUS DP73 microscope (Olympus Corp., Tokyo, Japan).

The paraffin-embedded sections (5-µm thick) of retinal tissues were deparaffinized, treated with 0.1% Triton X-100 and blocked with 3% H₂O₂. After washing with PBS, the sections were incubated with the mixture of terminal deoxynucleotidyl transferase (TdT) and fluorescein-labeled dUTP and then treated with converter-POD according to the manufacturer's protocol (Roche, Basel, Switzerland). DAB was added and the sections were counterstained with hematoxylin. Finally, the sections were dehydrated and photographed under OLYMPUS DP73 microscope.

The retinal tissues were lysed on the ice, followed by protein extraction. The protein concentration was determined by BCA assay kit (Wanleibio, Shenyang, China). The proteins were subjected to SDS-PAGE (8–13%) and transferred to PVDF membranes. Then, membranes were blocked with non-fat milk dissolved in Tween-20/TBS buffer. Subsequently, the membranes were incubated with primary antibodies against NgR (1:10,000; ab184556; Abcam, Cambridge, UK), RhoA (1:200; sc-197; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), Rock1 (1:200; sc-374388; Santa Cruz Biotechnology, Inc.), Bcl-2 (1:400; BA0412; Boster, Wuhan, China), Bax (1:400; BA0315; Boster), Caspase-3 (1:1000; ab2302; Abcam), F-actin (1:500; ab205; Abcam), growth-associated protein-43 (GAP-43) (1:200; sc-33705; Santa Cruz Biotechnology, Inc.) at 4°C overnight and then with secondary antibody (1:5,000; horseradish peroxidase-labeled IgG; WLA023 and WLA024; Wanleibio) for 45 min at 37°C. The protein bands were visualized via ECL reagent and analyzed by Gel-Pro Analyzer (Media Cybernetics, Rockville, MD, USA).

Retinal tissue sections (5-µm thick) were deparaffinized with xylene and gradient ethanol (100, 95, 85 and 75% ethanol). After antigen retrieval, the sections were treated with H₂O₂ and blocked with goat serum. Then, sections were incubated with primary antibodies against F-actin (1:200; bs-1571R; Bioss, Beijing, China) and GAP-43 (1:200; D163002; Sangon Biotech, Shanghai, China) at 4°C overnight and biotin-labeled secondary antibody (1:200; A0277; Beyotime Institute of Biotechnology, Haimen, China) at 37°C for 30 min, followed by incubation with HRP-labeled avidin (A0303; Beyotime) at 37°C for 30 min. The sections were visualized with DAB, counterstained with hematoxylin and imaged under a microscope (Olympus Corp.).

Total RNAs were extracted from the retinal tissues using RNApure Total RNA Extraction kit (BioTeke, Beijing, China) and reverse-transcribed to cDNAs using M-MLV Reverse Transcriptase (BioTeke). The volume of obtained cDNAs was 20 µl. qPCR analysis was carried out following the reaction conditions on Real-Time PCR system (BIONEER, Daejeon, Korea): 95°C for 10 min, followed by 40 cycles of 95°C for 10 sec, 60°C for 20 sec and 72°C for 30 sec. The primers used were as follows: NgR forward, 5′-GTCCCTTCCAGACCAATCAGC-3′ and reverse, 5′-GCCATTGCCTGGTGGAGTGT-3′; RhoA forward, 5′-TCGGAATGATGAGCACACAA-3′ and reverse, 5′-GCTTCACAAGATGAGGCAC-3′; Rock1 forward, 5′-GTGATGGCTATTATGGACG-3′ and reverse, 5′-AGGAAGGCACAAATGAGAT-3′; F-actin forward, 5′-GAAGAGAAAGCAGCAGTGTTA-3′ and reverse, 5′-GGAGCCAGAGGGTGGTTAT-3′; GAP-43 forward, 5′-AGGGAGATGGCTCTGCTAC-3′ and reverse, 5′-CACATCGGCTTGTTTAGGC-3′; GAPDH forward, 5′-CGGCAAGTTCAACGGCACAG-3′ and reverse, 5′-CGCCAGTAGACTCCACGACAT-3′. The primers were synthesized by Sangon Biotech. Gene expression was normalized to GAPDH and calculated using the 2^−ΔΔCq formula.

Data are expressed as the mean ± SD and analyzed by Student's t-test for the comparison of every two groups. P<0.05 was considered to indicate a statistically significant difference.

Histopathological examination showed that the cell numbers in GCL (Fig. 1) were decreased in diabetic rats compared with those in non-diabetic control rats. We found that NgR siRNA or CNTF injection alone increased the number of cells in GCL. Similarly, combination treatment further enhanced the ability of single treatment, although the difference was not statistically significant.

Death of RGCs is one of the earliest events in DR occurrence and progression (18). Therefore, we evaluated cell apoptosis using TUNEL assay. As expected, diabetic rats presented higher apoptosis rate of the cells in the retina than control rats (Fig. 2A). However, NgR knockdown or CNTF incubation significantly protected RGCs against apoptosis in diabetic rats. Combined injection of NgR siRNA and CNTF decreased the apoptotic rate of RGCs in diabetic rats in comparison with the STZ+NgR siRNA group or STZ+CNTF group, however, no significant difference was observed between the single treatment group and the combination group. Next, we measured the levels of apoptosis-related proteins (Bax, Bcl-2 and Caspase-3) in the retinal tissues using western blotting. We demonstrated that Bax and Caspase-3 levels were significantly upregulated in the retinal tissues of diabetic rats compared with control rats, whereas anti-apoptotic protein Bcl-2 was downregulated (Fig. 2B). NgR siRNA alone and CNTF injection alone notably reversed diabetes-induced cell apoptosis by decreasing Bax and Caspase-3 levels and increasing Bcl-2 level. Compared with the STZ+NgR siRNA group or STZ+CNTF group, NgR siRNA injection combined with CNTF further enhanced the anti-apoptotic effect of NgR knockdown and CNTF.

To evaluate the effect of NgR knockdown and CNTF treatment, either alone or in combination, on growth cone cytoskeleton and axonal regeneration, we measured the expression levels of F-actin and GAP-43 in the retinal tissues. We observed the downregulated expression of F-actin and GAP-43 at both protein (Fig. 3A) and mRNA levels (Fig. 3B) in the retinal tissues of diabetic rats. However, NgR siRNA or CNTF incubation significantly elevated the levels of these two proteins. Moreover, the combination of NgR siRNA and CNTF further increased F-actin and GAP-43 levels. We then performed IHC analysis (Fig. 3C) and verified the results obtained from western blotting and qPCR.

We then examined the effect of NgR knockdown and CNTF treatment, either alone or in combination, on the activation of NgR/RhoA/Rock1 signaling pathway in diabetic rats. The results showed that NgR, RhoA and Rock1 levels were elevated in diabetic rats in comparison with control rats, as demonstrated by western blotting (Fig. 4A) and qPCR (Fig. 4B). After injection with NgR siRNA or CNTF, the diabetic rats expressed lower levels of NgR, RhoA and Rock1 in the retinal tissues than the corresponding model rats. As expected, the expression of NgR, RhoA and Rock1 were significantly inhibited by the combined administration of NgR siRNA and CNTF compared with the single treatment group.