Animals and ethics of study:
The rats are provided by Fırat University Experimental Research Center. In this study, 8-10 weeks old Wistar Albino male rats weighing 225-300 g were used. The rats were kept at room temperature at 22-25 °C for 12 hours in light (7.00-19.00) and in the dark for 12 hours (19.00-7.00). Add-libitum water and food were provided to all groups.
Group 1 (Control Group); No procedure was applied during the experiment.
Group 2 (Negative Control Group); On the first day of the experiment (FDE), 2 µl of 0.1M PBS was administered intravitreally with a Hamilton injector. No treatment was applied.
Group 3 (Positive Control Group); FDE, 2 µl of 160 nmol/µl NMDA solution was administered intravitreally with a Hamilton injector. No treatment was applied.
Group 4 (Oral TG Group); FDE, 2 µl of 160 nmol/µl NMDA solution was administered intravitreally with a Hamilton injector and 100mg/kg daily dose of TG was administered with oral gavage method for 21 days.
Group 5 ( Drop TG Group); FDE, 2 µl of 160 nmol/µl NMDA solution was administered intravitreally with a Hamilton injector. 20 mg/ml (1mg in each drop) one drop of eye drops was applied in the mornings and evenings for 21 days.
Group 6 (Drop BT Group); FDE, 2 µl of 160 nmol/µl NMDA solution was administered intravitreally with a Hamilton injector. BT (Allergan ALPHAGAN-P %0.15 5 ml) one drop of eye drops was applied in the mornings and evenings for 21 days.
A combination of 50 mg/kg ketamine hydrochloride (Ketalar, Eczacıbaşı, Turkey) and 5 mg/kg xylazine hydrochloride (Rompun, Bayer, Turkey) was used for anesthesia and analgesia. This procedure was administered to all groups except the control group (Group 1). After intramuscular injection, topical 1% proparacaine hydrochloride was instilled into the right and left eyes of all rats. After the application, 0.1 M 2 µl of PBS was injected at 1 mm behind the limbus with a 30-gauge Hamilton injector into both eyes of the rats in the group 2. 2 µl of 160 nmol/µl NMDA solution was administered intravitreally at 1 mm behind the limbus to both eyes of the rats in groups 3, 4, 5, and 6 with a 30-gauge Hamilton injector. By this means, retinal excitotoxicity was induced in Groups 3, 4, 5 and 6. After the operation, antibiotic drops were applied to all the eyes. Rats were decapitated on the 21st day of the experiment. Enucleation was performed on both eyes of the rats. Before the enucleation procedure, the orientation suture was passed at the 12 o'clock position in the right eyes of the rats. The right eyes of the rats were evaluated in the medical pathology department, and the left eyes were evaluated in the medical biochemistry department.
The right eyes of the rats were marked with black ink on the temporal side 2mm ahead of the optic disc. Tissue samples were taken from the marked area in vertical sections to include all enucleation tissue. Hematoxylin-eosin staining was performed on 3-4 μm thick sections.
Sections of 3-4 μm thickness were obtained from formalin-fixed paraffin-embedded tissues. The sections were subjected to dehydration and rehydration. The tissues were incubated with 0.05% proteinase K for 10 minutes. It was incubated with 3% hydrogen peroxide for 5 minutes to inhibit endogenous peroxidase activity. The tissues were washed again with PBS. It was incubated with equilibration buffer for 6 minutes and incubated for 60 minutes with working solution (70 µl ReactionBuffer + 30% TdTEnzyme) in a humid environment at 37ºC. ABP Biosciences TUNEL Chromogenic Apoptosis Detection kit was used.
Tissues kept in Stop/Wash Buffer for 10 minutes were treated with Anti-Digoxigenin-Perosidase for 30 minutes. It was coated with diaminobenzidine (DAB) substrate. Sections that were counterstained with Mayer's hematoxylin were closed with the appropriate closure solution. The preparations were evaluated under an Olympus BX50 light microscope. In the evaluation of TUNEL staining, nuclei stained blue with Mayer hematoxylin were considered normal, cells with brown nuclear staining were considered apoptotic. 100 normal and apoptotic cells were counted in randomly selected areas at 40x magnification. Statistical analyzes were performed by calculating the apoptotic index (AI) by the ratio of apoptotic cells to total cells (Apoptotic Index = Total number of apoptotic cells / 100).
Caspase 3 and Brn3a Immunohistochemical Evaluation:
Sections of 3-4 μm thickness obtained from formalin-fixed paraffin-embedded tissues were taken on polylysine slides. Caspase 3(Fine test) and Brn3a(Bioss) antibodies were applied to these slides with Ventana brand Ultraview Universal DAB Detection Kit and Ultra DAB chromogen protocol in Ventana Bench Mark Ultra model automatic immunohistochemistry device. The preparations were evaluated with an Olympus BX50 light microscope.
The prevalence of staining with Caspase 3 and Brn3a in 10 randomly selected areas at 40x magnification was scored as follows :
Grade 0: There are no immune positive cells.
Grade 1: The immune positive cell is between 1% and 10%.
Grade 2: The immune positive cell is between 11% and 30%.
Grade 3: The immune positive cell is between 31% and 50%.
Grade 4: More than 51% immune positive cells.
Evaluation of Biochemical Samples:
Eye tissue samples were washed with 0.9% cold (+4ºC) sodium chloride (NaCl) and dried with blotting paper. It was homogenized in a 0.01M PBS (1/10) solution with a homogenizer at 16000 rpm for 4 minutes under appropriate conditions. The homogenate was centrifuged at 5000 xg for 1 hour (+4ºC) and the supernatants were separated and stored at –80°C until analysis. The amount of protein in the supernatants was determined in the Qubit Fluorometer device (Invitrogen, USA) using the Qubit protein measurement kit. SOD level from supernatant samples was determined by Enzyme-Linked Immuno Sorbent Assay (ELISA) method. Protein levels in the supernatants were determined according to the Lowry method. The principle of this method; It is based on the formation of a blue color by the Folin-Phenol reagent of proteins in alkaline environment .
Determination of Supernatant SOD Levels by ELISA Method:
Supernatant SOD levels were studied in accordance with the kit procedure using the rat SOD ELISA kit (Andy Gene Biotechnology Co., Ltd.). Absorbances were read spectrophotometrically at 450 nm on EPOCH 2 (BioTek Instrument, Inc, USA) microplate reader. Results were expressed as ng/L. The measuring range of the kit was 10-200 ng/L and the sensitivity was 1.0 ng/L. Intra-Assay CV was <8%; Inter Assay CV was <10%.
Tissue MDA Measurement:
MDA was measured using the method determined by Ohkawa et al. The principle of this method; It is based on the spectrophotometric measurement of the pink complex formed with thiobarbituric acid, at a wavelength of 532 nm, of the MDA released as a result of lipid peroxidation under the effect of temperature of the supernatants obtained from the tissue in an acidic environment (pH: 3.5) and under aerobic conditions. The results are given in nmol/mg protein by dividing the total protein concentrations .
Evaluation of Tissue iNOs Protein Levels by Western Blot Method:
Western blot; The transfer of the proteins migrated in the polyacrylamide gel by electrophoresis to the support membrane and the demonstration of the proteins in the membrane by immunological methods. Western blot technique is performed in four steps following electrophoresis. These steps; transferring the proteins in the gel to the nitrocellulose membrane (blotting), covering the nonprotein-bound regions in the nitrocellulose membrane with irrelevant proteins (blocking), reaction with specific antibodies, and imaging of the proteins in the last step.
Obtained data were determined as mean ± standard deviation. SPSS version 22 program was used for statistical analysis. Inter group evaluation was done with One-way ANOVA and Posthoc Tukey test. P ˂0.05 values were considered statistically significant.