Animal care and use

Ten- to twelve-week-old male wild-type C57BL/6J mice were obtained from the Experimental Animal Center of Central South University. Mice were maintained in a specific pathogen-free (SPF) facility under a 12-h light/dark cycle with free access to food and water. All animal procedures were approved by the Institution of Animal Care and Use Committee of the Second Xiangya Hospital and adhered to the Guidelines for the Care and Use of Laboratory Animals. The retinal I/R model was established as previously reported (Li et al. 2014; Ge et al. 2020; Kim et al. 2013). Briefly, mice were randomly assigned to groups (n = 6 per group) and then sedated by intraperitoneal injection of sodium pentobarbital. Next, a 33-gauge infusion needle containing 0.9% NaCl was inserted into the anterior chamber to maintain the intraocular pressure at 120 mmHg (measured with a TonoLab tonometer). Transient retinal ischemia was achieved for 60 min. Then, the needle was removed, and reperfusion was initiated. The contralateral eye was cannulated to maintain a normal IOP and served as a nonischemic control (Li et al. 2014; Deng et al. 2020; Souza Monteiro de Araujo et al. 2020). lncRNA uc007nnj.1 siRNA or scramble or lncRNA uc007nnj.1 plasmid or vector was delivered directly into the vitreous chamber of mice (1 μL per injection, 1 μg/μL) 24 h before the retinal I/R model was constructed as previously described (Zhang et al. 2021; Kleinman et al. 2008; You et al. 2017; Hou et al. 2016).

Isolation and culture of mouse primary RGCs

Primary retinal ganglion cells were isolated from the retinas of 1- to 4-days-old neonatal mice according to the previously published protocols (Zhang et al. 2021; Ge et al. 2021; Huang et al. 2003; Chintalapudi et al. 2016). Routine asepsis was performed. Briefly, 24-well plates were precoated with poly-d-lysine (P6407; Sigma, St. Louis, MO, USA) and laminin (L-6274; Sigma) 1 day in advance. Then, 75 cm2 cell culture dishes were coated with purified donkey anti-rabbit IgG (H&L) (ab150075; Abcam, Cambridge, MA) for anti-macrophage elution, and 100 cm2 plates were coated with donkey anti-rat IgG (H&L) for immobilization of Thy1.2 antibody (BE0066; Bio X Cell, West Lebanon, NH, USA). All of the above plates were incubated overnight at 4 °C. After dissection of the retina and a wash with DPBS at 37 °C for 15 min in papain (G8430; Solarbio, Beijing, China) containing DNase I (Sigma, St. Louis, MO), the papain solution was carefully aspirated, and Lo Ovo solution containing anti-macrophage antibody (AIA31240; Accurate Chemical, Westbury, NY, USA) was added. The cells were incubated for 5 min at RT. The supernatant was centrifuged and discarded, the cells were resuspended with panning buffer, and the cell suspension was transferred into a preprepared purified donkey anti-rabbit IgG (H&L)-coated culture dish and incubated at room temperature for 40 min. Nitex mesh (352,350; BD Biosciences, Franklin Lakes, NJ) was used to filter the cell suspension, which was transferred into a culture dish precoated with an anti-Thy1.2 antibody (M7898; Sigma) and incubated for 1 h. Nonadherent cells were removed, and the RGCs in the culture dish were digested with 4 mL of trypsin/Earle's balanced salt solution (EBSS) for 5 min. After centrifugation, the cells were resuspended in a complete medium and grown in 24-well plates. RGCs were cultured in a humidified atmosphere at 37 °C with 5% CO2 and 95% O2; 50% of the medium was replaced with a prewarmed complete growth medium every 3 days.

In vitro ischemia simulation

We established an in vitro mimic of ischemic injury in RGCs according to our previously applied calcium ionophore/ATP depletion injury model (Zhang et al. 2021; Ge et al. 2020; Lee and Emala 2002). When RGCs were confluent over 90% of the plate, ischemia was simulated for 2 h by changing the medium to Hanks’ balanced salt solution (HBSS) with 10 mM antimycin A (a complex III inhibitor of mitochondrial electron transport; ab141904; Abcam, Cambridge, MA, US) and 2 mM calcium ionophore (A2318; Aladdin, Shanghai, China), which were dissolved in dimethylsulfoxide (DMSO). The complete growth medium was reapplied, and the cells were sustained for 0–4 h. For gene knockdown and overexpression experiments, 24 h before ischemic injury, when the density of the RGCs on the 24-well plate reached 80% to 90%, the cells were transfected lncRNA uc007nnj.1 siRNA/plasmid, miR-155-5p mimic or inhibitor, Tle4 siRNA, and scramble siRNA (Ribobio) using Lipofectamine 2000. After 6–8 h, the transfection solution was removed and replaced with a complete growth medium. Immunofluorescence staining of Tuj1 (1:500, GB11139; Servicebio, Wuhan, China) was used to identify the purity of RGCs.

Fluorescence In Situ Hybridization (FISH)

A Fluorescence In Situ Hybridization Kit and probes were purchased from RiboBio Corporation (C10910; Guangzhou, China). In short, paraformaldehyde-fixed RGCs were prepared and then hybridized with a Cy3-labeled lncRNA uc007nnj.1 probe and FAM-labeled miR-155-5p probe. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI), and fluorescence images were taken via laser-scanning confocal microscopy (TCS SP5; Leica, Wetzlar, Germany). 18 s and U6 served as positive cytoplasmic and positive cytosolic controls, respectively.

Luciferase reporter assay

The wild-type or mutant sequence of the Tle4 and lncRNA uc007nnj.1 3′-UTR (3′-untranslated region) was inserted into a pmirGLO vector and named Tle4 3′UTR WT, Tle4 3′UTR MUT, uc007nnj.1 WT or uc007nnj.1 MUT, as appropriate. RGCs were cotransfected with miR-155-5p mimic or miR-155-5p inhibitor or scramble and these reporter plasmids. After 48 h, the cells were collected with Passive Lysis Buffer, and a dual luciferase assay system was used to detect firefly and Renilla luciferase activities (Promega) according to the manufacturer’s instructions. Firefly luciferase activities were normalized according to Renilla luciferase levels. All plasmids were constructed by RuQi Biotechnology (Guangzhou, Guangdong Province, China).

Western blotting

According to our previous studies, total protein was extracted from retinal tissues or RGCs using lysis buffer (Zhang et al. 2017a, b). Protein concentrations were measured using a NanoPhotometer N50 Touch spectrophotometer (IMPLEN, Heidelberg, Germany). Equal amounts of protein per lane (30 µg) were run on a 10% SDS–PAGE gel and then transferred to polyvinylidene difluoride membranes. The blots were probed with primary antibodies against caspase-3 ([1:1000], 9662; CST, Danvers, MA, US), cleaved caspase-3 ([1:1000], 9661; CST, Danvers, MA, US), Tle4 (15140, [1:1500]; Novusbio, Minneapolis, MN, US), and β-actin (66009-I-Ig, [1:1000]; Proteintech, Rosemont, IL, US) followed by incubation with secondary antibodies (goat anti-rabbit IgG (H + L) HRP [1:5000] or goat anti-mouse IgG (H + L) HRP [1:5000], Affinity, Cincinnati, OH, US). The expression of target proteins was normalized to the corresponding β-actin expression level in the same sample and quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Each immunoblot was repeated three times to confirm the results.

Reverse transcription-quantitative real-time PCR (RT-qPCR)

According to the manufacturer’s protocol, total RNA was isolated from retinas or cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Complementary DNA (cDNA) synthesis was performed using a Prime Script RT Reagent Kit and gDNA Eraser Kit (RR047A; TaKaRa, Tokyo, Japan). RT-qPCR was performed using SYBR Green (K0221; Thermo Fisher Scientific, Waltham, MA, USA), and the results were quantitated with StepOne Software (Applied Biosystems, Carlsbad, CA, USA). β-Actin was used as a standard for each sample to determine relative expression levels. The primer sequences for lncRNA uc007nnj.1, miR-155-5p, and Tle4 are shown in Additional file 1: Table S2. Relative RNA levels were calculated using the 2−ΔΔCt method.

Flow cytometry (FCM)

RGCs were trypsinized, washed twice with cold PBS, and then resuspended in 1× Binding Buffer. According to the manufacturer’s protocol, 100 µL of each solution was transferred to a 1.5 mL Eppendorf tube and then incubated with YF 488-annexin V and PI (Everbright, Suzhou, China, USA) in the dark for 15 min at RT. Finally, 400 μL of 1× binding buffer was added to the tube. Flow cytometry was then performed within 1 h using a BD FACSCalibur flow cytometer (San Diego, CA, USA). The results were obtained using FlowJo V10 software (BD, San Diego, CA, USA).

Immunofluorescence and TUNEL assays

RGCs were identified and quantified utilizing Tuj1 immunofluorescence staining. Briefly, mice were killed 24 h after reperfusion. Eyes were enucleated within 10 min of death and fixed in 4% paraformaldehyde for 1 h. After carefully removing the anterior section, we placed the eyecup in FAS eye-fixation buffer (Servicebio; G1109) at 4 °C for 24 h and dehydrated it in a 30% source solution. Following fixation, some retinas were evaluated as flat mounts; the others were embedded in optimum cutting temperature compound (OCT) to obtain 10-µm-thick cryosections using a freezing microtome. For immunofluorescence analysis in retinal flat-mount and frozen sections, the retina was permeabilized and blocked with 10% Triton X-100 and 10% BSA for 1 h and immunolabeled with primary antibody (Tuj1 [1:500], GB11139; Servicebio, Wuhan, Hubei Province, China) overnight at 4 °C, followed by a 2-h incubation with a secondary antibody (goat anti-rabbit IgG (H + L) Alexa Fluor 488 [1:1000], ab150077; Abcam) in the dark at room temperature (RT). After being washed with PBS, the retina was stored in the dark at 4 °C until microscopic observation. For TUNEL staining, a terminal-deoxy-transferase-mediated 20-deoxyuridine, 50-triphosphate (dUTP) nick end-labeling (TUNEL) (#1684795; Roche, Basel, Switzerland) assay was performed in cryosections according to the manufacturer’s instructions. The sections were incubated with TUNEL reaction solution (including 50 µL Enzyme Solution [TdT] and 450 µL Label Solution [fluorescein-dUTP]) at 37 °C for 1 h and then mounted. DAPI staining was performed to visualize nuclei. Images were taken with a fluorescence microscope (Leica DMI3000B) in two different regions at distances of 1 and 2 mm from the disc of each quadrant in the retina to analyze the mean number of surviving RGCs. Four randomly selected fields were analyzed in each section.

Electroretinography (ERG)

The b-wave amplitudes in ERG have been considered a sensitive parameter for detecting the degree of retinal injury induced by ischemia (Dang and Zhang 2018; Grozdanic et al. 2003; Ji et al. 2021). Therefore, scotopic ERG was recorded in mice after 6 h of dark exposure as previously described. The animals were anesthetized by intraperitoneal injection of sodium pentobarbital. After topically applying 0.4% levofloxacin and 1% tropicamide ophthalmic solution, we placed five electrodes on the subcutaneous tissue by the tails, beneath each eye, and at the apex of the corneas in both eyes. Flash ERG was recorded at an intensity of 3.0 cd·s/m2 using a Ganzfeld Q450 (ROLAND ELECTRONIC, Keltern, Germany) system. The evaluated b-waves were analyzed with RETI-Port/Scan 21 software (ROLAND ELECTRONIC, Keltern, Germany).

Statistical analysis

All the results are presented as the mean ± SD of six independent experiments. Student’s t test was used to compare the means between two groups. One-way ANOVA followed by Tukey’s post-hoc test was used to compare multiple treatment groups. The data were considered to be statistically significant when the p value was less than 0.05. All statistical analyses were performed using SPSS software and GraphPad Prism software (GraphPad Software, La Jolla, CA, USA).