The Manipal Academy of Higher Education, Kasturba Medical College animal ethics committee approved the experimental protocol (IAEC/02/2017). Animal handling and investigational procedures were carried out as per IAEC and CPCSEA (No:94/PO/Re Bi/5/99/ CPCSEA) guidelines. The healthy animals (n=24) are divided into three experimental groups (n=6 in each group), one serving as an (unexposed) control group (n=6). We have included healthy rats for the study and the control rat’s retinas were selected from the same pool of rats. However, it’s safe to assume that the experimental control rats will also not have any degeneration. Both eyes of rats were exposed to blue, white, and yellow LED light with the maintenance of homogeneousness and intensity of light. The light standard was measured and standardized using Asensetek Lighting Passport Pro, New Taipei City, Taiwan. The spectral sensitivity of different wavelengths of light visible spectrum 400-780nm) used for this experiment (450-500 lux) light. The spectral properties of light transmittance of the front surface of light, scotopic sensitivity, flicker index and melatonin suppression of each light were measured. The total light exposure per day of (450-500 lux). The height from the source to the animals (cage) was 50 cm and the exposure time (12:12 dark & light cycle routine for 90 days) was matched with the nocturnal time of the rodents. The light exposure group animals were subjected to the blue LED (400 - 490nm), white LED (380-780nm) and yellow LED (400-780nm) light exposure (Ack LED Panels, 3W, Epistar, ES-EMBCF22L-A, InGan -series Blue LED chip, ES-SMYLPX42C, UHB-PX, AlGaInP PN-series LED Chip). The light source was fitted on the top of the cage (L=100cm, W=70cm and H=50cm) the mounting of the light kept it at 50 cms (100 % system level light output at standard operating voltage range, the total light output of the cage at the testing area (510 lumens). Post-exposure, all animals were sacrificed with a lethal dose of pentobarbital intraperitoneal injection (i.p. 100 mg/kg) (Euthasol®) and xylazine (10 mg/kg) (Proxylaz®). Then eyes were enucleated by using watchmaker forceps (number 5) and Sklar's blunt enucleation scissors. Immediately after enucleation, the vitreous was extracted by aspiration (21-gauge hollow needle) technique and stored in -20o C for LC-MS and the retinal tissue stored in 4% paraformaldehyde (7 days) for immunofluorescence (IF) and Hematoxylin and eosin (H&E) stain.
Rat whole vitreous:
For the analysis of the chronic light exposure model, the whole vitreous sample was collected by aspiration technique. The samples were sonicated and stored at −20 °C until LC-MS analysis.
Vitreous Metabolomic Analysis
Untargeted quantitative metabolomics was used to analyze the vitreous fluid samples using an LC-MS Agilent LC-QTOF system (Agilent Technologies, Santa Clara, CA, USA). It consisted of an Agilent 1200 LC system coupled online with an Agilent 6520 time-of-flight mass spectrometer. The vitreous samples were defrosted on ice, vortexed, and centrifuged at 12,000 rpm for 15 min. An 8 µl aliquot was injected into an Agilent 1290 LC system coupled to an ESI-Q-TOF instrument (Agilent 6520, Agilent Technologies, Santa Clara, CA, USA). HPLC column (Phenomenex, Torrance, CA, USA (P/No:00G-4601-E0; Desc: Kinetex 5μm C18 100A; Size: LC Column 250X 4.6 mm; S/No H18-343854; B/No 5701-0060) was maintained was at 25°C. The injected vitreous data were collected in the positive mode of electrospray ionization (ESI) technique. Basic and neutral metabolites were eluted in positive mode using subsequent gradient at 400 µl/min using mobile phase A: 0.1% formic acid in water and mobile phase B: 0.1% formic acid in 90% acetonitrile (2% to 98% B in 25 minutes, 98% B for 10 minutes and equilibrated to 2% B for 10min). ESI spray voltage was maintained at 3.5kV with MS interface capillary at 350°C. Fragmentor was set at 140V. Drying gas and nebulizer were maintained at 8 litres/minute and pressure of 40 psig respectively. Data was acquired at an acquisition rate of 2Hz in a range of 50-1700 m/z. Based on precise mass value and retention time, an untargeted mass spectrometer approach was employed for confirmation of the most abundant and significant metabolites.
Data processing and analysis:
The raw data obtained from each run was processed using a molecular feature extraction tool in Qualitative Mass Hunter Analysis Software B.04.00 (Agilent Technologies). The data files containing monoisotopic mass, respective abundance and retention time were used for data alignment and filtering in Mass Profiler Plus software (MPP) (Agilent Technologies, version B.12.5). Raw data files were segregated aligned, transformed to log10, and baselined to the median of all samples in MPP. Features that were present in at least 75% of individuals in each group were considered for further analysis. Partial least square discriminant analysis (PLS-DA) was performed on metabolome data to discriminate the study groups.Compounds were identified in METLIN and HMDB databases based on isotopic pattern distribution and accurate mass within a specified tolerance (15 ppm error). Peaks obtained from raw MS data were aligned and subjected to bioinformatics and statistical analysis. The metabolite peaks were attained in NC, BLE, WLE and YLE groups separately (Figure 1:).
The twenty-four retinal tissues were embedded in 4% paraformaldehyde immediately after the following enucleation. The retinal tissues were sectioned after calibrating the cryostat for 40µ thickness and the sections were mounted on the gelatin-coated slides serially. Frozen sections (Leica cm3050 s) of 40 microns thickness were made in the sagittal plane and kept at -80°C before further processing. Sections were fixed with 2% paraformaldehyde in 0.01M phosphate buffer solution at 24°C for 1 hour, followed by incubation with 1% H2O2, 2% sodium azide, 0.1% saponin, 10 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) in EBSS -saponin for 36 hours at 24°C in the dark. Sections were then rinsed and incubated in a moist chamber overnight at 24°C with primary rabbit polyclonal to active + pro Caspase 3 antibody directed against cleaved caspase-3 (1:200 aliquoted 4µl of antibody is added to 1.6ml of PBS.TX) with working buffer and approximately 200µl of diluted (Abcam, Cambridge, UK). The retinal tissue sections were washed and incubated with a secondary antibody (1:100, Anti-Rabbit IgG (whole molecule) F(ab)2 fragment-Cy3 antibody, Abcam) for 16 hours at 2°C.
Immunofluorescence imaging and quantification: Caspase-3 (active + pro Caspase 3) stained retinae were imaged using a Dmi8- SP8 Confocal Microscope (Leica Microsystems) using a 63 X objective. The laser power offset and gain, and other acquisition parameters were determined by using isotopic control samples. The values of these parameters were then fixed across all retinal samples. Retinal mosaics were created in Adobe Photoshop 7.0 (Mountain View, CA, USA). The area of each retinal layer was segmented by a masked observer, and the average intensity was calculated after subtracting the background using ImageJ software (To overcome the background intensity staining we have accounted brightness in each layer, and it was expressed relative intensity to the entire retina, and the total brightness was averaged). A total of 12 images, 2 from each eye were analyzed in each group and the intensity of IF was quantified in a masked method.
Measurement of hematoxylin and eosin-
Measurement of hematoxylin and eosin-stained retinal layers was conducted as follows. Images of a sagittal plane containing the optic nerve on paraffin-embedded blocks of H&E images were taken using a Motic Images Plus 3.0 for Windows 32 (2021-10-29) Microscope (Leica Microsystems, Germany) at 40× magnification then Retinal mosaics were created in Adobe Photoshop 7.0 (Mountain View, CA, USA). Images were analyzed with the built-in measuring tool in Image-J software. The thicknesses of the whole retina (excluding the outer segments) and the ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL) and outer nuclear layer (ONL) were measured for each layer in each picture in three different areas. Data were collected in Excel and then imported to R software (version 3.6.3) for statistical analysis
Data preparation and statistical application
The LC-MS/MS spectral region 5.00 ppm was segmented into bins of 0.05 ppm width Agilent pro software. The region anything above 5.00 ppm error was excluded from the analysis as it might have a remnant noise signal. A total column (bins) of metabolites were obtained and the integrated area within each bin was normalized to a constant sum of 500 for each range to minimize the effects of variable concentration among different samples.