10 hpf wild type zebrafish (Danio rerio) specimens were used in this study. Adult zebrafish used for embryo generation were kept in aquaria under standard conditions of temperature (28 ºC), light-dark cycles (14 h of light and 10 h of darkness), and pH (7.0). Adult fish of both sexes were crossed to generate embryos, which were raised using standard conditions until being used for experimental procedures (at 10 hpf). The study was conducted according to the regulations and laws established by the European Union (2010/63/UE) and by the Spanish Royal Decree 1386/2018 for the care and handling of animals in research and was approved by the Bioethics Committee of the University of Santiago de Compostela and the Xunta de Galicia (project reference MR 110250).
Generation of embryonic nutritional hyperglycemia
10 hpf embryos were placed in groups of 5 into 24 well-plates containing 2 mL per well of reverse osmosis purified water supplemented (hyperglycemic fish) or not (control embryos) with 50 mM D-glucose (Sigma, St Louis, MO, USA). The water (with or without D-glucose) was replaced once a day for 5 days. At 5 dpf, larvae were anesthetized with 0.02% of ethyl 3-aminobenzoate methanesulfonate salt (MS-222, Tricaine; Sigma) and fixed by immersion in 4% paraformaldehyde in 0.1 M phosphate-buffered saline pH 7.4 (PBS) for 2 hours at 4 ºC. After rinsing in PBS, the larvae were cryoprotected with 30% sucrose in PBS, embedded in Neg-50™ (Thermo Scientific, Kalamazoo, MI, USA), and frozen with liquid nitrogen-cooled isopentane. Transverse sections of the head containing the eyes (18 µm thickness) were obtained on a cryostat and mounted on Superfrost Plus slides (Menzel-Glasser, Madison, WI, USA).
The TUNEL staining was performed according to the manufacturer’s protocol with minor modifications (In situ Cell Death Detection Kit, TMR red; catalogue number 12156792910; Roche, Mannheim, Germany). Briefly, the sections were incubated in methanol for 15 minutes at -20°C to permeabilize the lipid membranes, followed by brief washes in PBS and another incubation in 0.01 M citrate buffer pH 6.0 for 30 min at 90 ºC. After several washes in PBS, the sections were incubated in the TUNEL reaction mix, containing the Labelling Solution (TMR red labelled nucleotides) and the Enzyme Solution (terminal deoxynucleotidyl transferase), for 90 minutes at room temperature (RT). Slides were washed in PBS and distilled water, allowed to dry for 45 min at 37 ºC, and mounted with MOWIOL® 4–88 (Calbiochem, Darmstadt, Germany). Negative controls were obtained by incubating slides only with the Labelling solution (without terminal deoxynucleotidyl transferase).
Sections were first treated with 0.01 M citrate buffer pH 6.0 for 30 min at 90 ºC for heat-induced epitope retrieval, allowed to cool for 20 min at RT, and rinsed in 0.05 M Tris-buffered saline (TBS) pH 7.4 for 5 min. Then, the sections were incubated overnight at RT with the following combinations of primary antibodies: 1) a mouse monoclonal anti-PCNA antibody (1:500; Sigma; catalogue number P8825; RRID: AB_477413) and a rabbit polyclonal anti-pH3 antibody (1:300; Millipore; Billerica, MA, USA; catalogue number 06-570; RRID: AB_310177); 2) rabbit polyclonal anti-bovine rod rhodopsin antibody [CERN-922; 1:500; generated by W. J. Degrip (Radboudumc, The Netherlands)] and a mouse monoclonal anti-glutamine synthetase (GS) (1:500; Millipore; catalogue number MAB302; RRID: AB_2314617). Sections were then rinsed 3 times in TBS for 10 min each and incubated for 1 hour at RT with a combination of fluorescent dye-labelled secondary antibodies: a Cy3-conjugated goat anti-rabbit (1:200; Invitrogen, Waltham, MA, USA; catalogue number A10520; RRID: AB_2534029) and a FITC-conjugated goat anti-mouse (1:200; Invitrogen; catalogue number F2761; RRID: AB_1500661). All antibody dilutions were made in TBS containing 15% normal goat serum (Millipore), 0.2% Triton X-100 (Sigma) and 2% BSA (Sigma). Finally, sections were rinsed 3 times in TBS for 10 min each and in distilled water for 30 min, allowed to dry for 45 min at 37 ºC, and mounted in MOWIOL® 4–88 (Calbiochem).
Specificity of antibodies
Anti-PCNA antibodies (including the one used in this study) have been traditionally used to label proliferating cells in the retina of different fish species (including zebrafish): Oryzias latipes (Negishi et al. 1990); Haplochromis burtoni (Mack and Fernald 1995, 1997); Onchorynchus mykiss (Julian et al. 1998); Tinca tinca (Velasco et al. 2001; Cid et al. 2002; Jimeno et al. 2003); Carassius auratus (Negishi et al. 1990; Cid et al. 2002); Salmo trutta fario (Candal et al. 2005); Astyanax mexicanus (Alunni et al. 2007); D. rerio (Cid et al. 2002; Amini et al. 2019; Hernández-Núñez et al. 2021b); Scyliorhinus canicula (Ferreiro-Galve et al. 2010a, 2010b, 2012; Sánchez-Farías and Candal 2015, 2016; Hernández-Núñez et al. 2021a); and Petromyzon marinus (Villar-Cheda et al. 2008). Anti-pH3 antibodies (including the one used in this study) have been also commonly used to label mitotic cells in the retina of various fish species (including zebrafish): C. auratus (Otteson et al. 2001); S. canicula (Ferreiro-Galve et al. 2010a; Bejarano-Escobar et al. 2012; Hernández-Núñez et al. 2021a); and D. rerio (Jensen et al. 2001; Godinho et al. 2007; Weber et al. 2014; Hernández-Núñez et al. 2021b). The location of labeled cells with both antibodies (see results) in stereotypical locations containing proliferating cells in the highly organized retina also ensures the specificity of labeling. The anti-bovine rod rhodopsin antibody (CERN-922) has been previously shown to label specifically rod photoreceptors in the retina of lampreys (Meléndez-Ferro et al. 2002; Villar-Cheda et al. 2008) and teleosts (trout: Candal et al. 2005; tench: Bejarano-Escolar et al. 2009). The morphology and location of labelled rods in the zebrafish retina also ensures the specificity of labeling. Anti-GS antibodies have been widely used as specific markers of Müller glia cells in the retina of fish, including teleosts as H. burtoni (Mack et al. 1998) and D. rerio (Peterson et al. 2001; Thummel et al. 2008); and in the elasmobranch fish S. canicula (Sánchez-Farías and Candal 2016; Hernández-Núñez et al. 2021a). As with other antibodies, the location and morphology of labelled cells in the highly organized zebrafish retina ensures the specificity of labeling.
Images of fluorescent labelled sections were taken with Leica TCS-SP2 (CERN-922) or Leica Stellaris 8 (pH3, PCNA, TUNEL and GS) confocal microscopes with blue and green excitation lasers. 20x and 40x objectives were used for imaging. Confocal optical sections were taken at steps of 1 µm along the z-axis. Collapsed images of the whole retinal sections (18 µm) were obtained with LITE or LAS X software (Leica, Wetzlar, Germany). For figure preparation, and always after cell quantifications, contrast and brightness of the images were minimally adjusted using Adobe Photoshop CS4 (Adobe, San Jose, CA, USA).
Cell quantification and statistical analyses
We quantified the number of apoptotic (TUNEL+) and mitotic cells (pH3+) in the whole retina (CMZ and central retina), and the number of cells progressing through the cell cycle (PCNA+) in the central retina. The numbers of TUNEL+, pH3 + and PCNA + cells were manually counted under an Olympus fluorescence microscope in 1 out of each 2 consecutive retinal sections (thickness of 18 µm). The boundary between the CMZ and the central retina was established based on the expression pattern of PCNA, which shows intense CMZ labeling, and the different morphology of the CMZ and the central retina (which shows a characteristic layered structure). With the number of cells for each quantified section we calculated the mean number of cells per section for each retina (each dot in the graph represents 1 retina from 1 animal). We also quantified the differential distribution of PCNA + cells in the different cell layers of the central retina: the ganglion cell layer (GCL), the inner nuclear layer (INL), and the outer nuclear layer (ONL).
The number of GS-ir or CERN-922-ir positive pixels was quantified in confocal photomicrographs of entire retinal sections using the Feature J plugin in the Fiji software (Schindelin et al. 2012) and as previously described for quantification of immunoreactive (ir) profiles in lamprey spinal cord sections (Fernández-López et al. 2014; Romaus-Sanjurjo et al. 2018). Briefly, the retinal images were always taken with the same confocal microscope and acquisition software parameters for control and hyperglycemic retinal sections. The number of positive pixels was quantified in 1 out of each 2 consecutive retinal sections. Then, the mean number of positive pixels per section was obtained for each retina (each dot in the graphs represents 1 retina from 1 animal). A threshold was established to have the most representative images when converting them to binary B&W images for pixel quantification. The same threshold was used for all the photomicrographs with the same labeling (GS or CERN-922).
Statistical analyses were performed with Prism 9 (GraphPad software, La Jolla, CA, USA). Normality of the data was determined with the D'Agostino-Pearson test. To determine significant differences (p < 0.05) between control and hyperglycemic animals, a Mann-Whitney test was used for non-normally distributed data and a Student’s (unpaired) t-test for normally distributed data. Each dot in the graphs represents 1 retina from 1 animal and all analyses were carried out in at least 2 different batches of animals.