The experimental protocol was carried out in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Academy of Sciences and ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The protocol was approved by the Ethics Committee of the Faculty of Medicine (S-1105-224-15), University of Sakarya, Turkey. The study was reported in accordance with ARRIVE guidelines.
Forty Sprague-Dawley male rats weighting 250–300 grams of 10–12 weeks of age were used. Rats were placed in cages under standard laboratory conditions at 22 ° C and 50–60% humidity for six weeks in light and dark cycles for 12/12 hours. All rats were given standard tap water and fed with pellet feed. The rats were randomly divided into four groups, with ten rats per group. The experimental groups were: Control Group (C): non- diabetic rats; Diabetes Mellitus Group (DM): rats induced to diabetes without treatment; Melatonin and Diabetes Mellitus Group (Mel + DM): rats induced to diabetes and after confirmation, treated with melatonin; Melatonin Group (Mel): rats were not induced to diabetes, treated with melatonin. At the end of the experiment, rats were sacrifced under general anesthesia with 65 mg/kg (i.p.) ketamine and 7 mg/kg xylazine (i.p.) injection.
Diabetes induction
It was induced by intraperitoneal administration of streptozotocin solution (Cayman Chemical product number, 13104, USA) after 12 h food fasting. Streptozotocin was diluted in 10 mM sodium citrate buffer and pH 4.5 at the single dosage of 60 mg/kg animal weight. Non-diabetic and diabetic animals without treatment received equivalent doses of saline. After 30 min all animals were fed normally. Diabetes was confirmed seven days after induction. Only animals that presented blood glucose above 250 mg/dL (Media Smart, Switzerland) were included in the study, except for the control group.
Melatonin administration
For eight weeks after the diabetes was induced, melatonin (Cayman Chemical East Ellsworth rd. Item No. 14427, USA) was prepared in isotonic NaCl solution containing 10% ethanol daily and subcutaneous injection at a dose of 10 mg / kg.
RPE isolation
Eyes were cut and excess tissues were removed in cold − 4 degrees phosphate buffered saline (PBS). Eyes were excised and then kept in a large petri dish at 37°C for 1 hour in 20 U/ml papain solution (Turklab). Place two eyes in the petri dish and add 1 ml of papain solution and cover the eyes. Obtained eyes were transferred to Dulbecco’s modified eagle media supplemented with 10% fetal bovine serum to inhibit papain absorption. A recess close to the lens and ora serrata was created with a fine needle and an incision was made through the ora serrata. The absorption induced by papain allowed the retina to disappear easily and led to the appearance of the choroid-sclera complex. retina-attached RPE cells were left to absorb in 1 ml of 20 U/ml papain for an average of 8 minutes at 37°C. RPE layers were separated from the retina. The plates were incubated in trypsin-PBS and then pulverized and ready to use to obtain smaller RPE cells.
Telomere Length Measurement
In epidemiological studies, TL analysis and chain reaction (Q-PCR) used to measure polymerase are the two most common methods for such analysis.
Q- PCR Application
The number of 36B4 copies from rat RPE was determined to use as a nuclear genome standard. DNA absorbance at 260 and 280 nm was examined using an ultraviolet spectrophotometer. and the DNA concentration was measured using a Nano-Drop 2000 spectrophotometer. The relative length of telomere was measured using quantitative polymerase chain reaction (qPCR), with the primers synthesized by Sango Biotech Co., Ltd. (Shanghai, China). DNA in RPE of rats was isolated from plates using a DNA extraction kit (Tiangen, Beijing, China). Relative telomere lengths were measured by qPCR method with modifications. Briefly, qPCR (36B4) was used for the master mix obtained by creating a total volume of 20 µL of Power SYBR Master mix, including DNA samples for single copy genes and telomeres. Table 1 demonstrates the detailed content of Power SYBR Master mix.
Table 1
Power SYBR Master Mix Preparation.
Reagents | Volumes for one sample (µl) | Final concentration |
Power SYBR Green master mix (2x) | 10 | 1x |
Primer 36B4 F (2 µM)) | 1 | 0.1 µM |
Primer 36B4 R (2 µM) | 1 | 0.1 µM |
H20 | 4 | |
DNA (5 ng/µl DNA) | 4 | 20 ng total |
µl: microliter; µM: micrometer; ng: nanogram |
Analysis of the results was performed using an integrated software to determine the threshold cycle (Ct) value with the negative control and baseline, and to determine the availability of the Ct value according to the dissolution curve. Triple reactions were performed simultaneously for each sample and the mean Ct value of the samples was calculated. The negative control reaction was also used for each round of PCR. The telomere / copy (T / S) ratio was calculated according to the formula:
△Ct1 = CtTL – Ct36B4, relative T/S ratio = 2–(△ Ct telomere – △ Ct 36B4) = 2–△△Ct .
△Ct1 stands for T/S ratio, and △Ct2 stands for the T/S ratio of the reference DNA.
Statistical analysis
The covariance analysis of repeated measurements by the mixed model approach was used for the telomere length analysis. The comparison of averaged data among groups was performed using least significant difference (LSD) and Kruskal – Wallis Test and One way ANOVA test.