2.1. Animals and experimental protocol
All experimental procedures were carried out in the Animal Experiments Laboratory of Bülent Ecevit University Faculty of Medicine. Approval was obtained from the local ethics committee of the university (decree no: 2020/06). A total of 32 Wistar albino female rats, weighing 350-450 g, were randomly seperated into four groups of equal numbers (n = 8, total: 32): C (Control + Vehicle), AME (Annona Muricata leaf extracts), RAD (Radiation + Vehicle), AME+RAD (Radiation + Annona Muricata leaf extracts). Sample size was determined by using G Power. The animals were kept below a constant temperature (18 °C-21 °C), and adequate nutrition and photoperiod (12 hours light/dark cycle) were provided for the duration of the experiment.
Rats in group C were injected intraperitoneally (IP) with 2ml/kg of normal saline per day for 1 week. In the AME group, 300 mg/kg AME was administered IP on the first day and 50 mg/kg AME every day for 7 days. Rats in the RAD and AME+RAD group exposed only one single IR dose (20 Gy) to their right legs without any treatment. The rats in AME+RAD group were given 300 mg/kg AME ip 30 minutes before IR application and 50 mg/kg AME IP every day for 7 days after the application. After 1 month, rats in all groups were sedated with xylazine (10 mg/kg, IP; Bioveta, Ankara, Turkey) and ketamine hydrochloride (80 mg/kg, IP; Ketalar, Pfizer, Istanbul, Turkey) anesthesia for biochemical examination from the tail arteries. Blood samples were taken, after which all rats were sacrificed with high-dose anesthetic drug (pentobarbital 200 mg/kg, IP; Bioveta, Ankara, Turkey). Right after this procedure, the right sciatic nerves of all rats were carefully separated from the surrounding tissues by blunt dissection and removed for histomorphological examination (Figure 1).
2.2. Irradiation
The rats in the AME+RAD and RAD groups were anesthetized using xylazine (10 mg/kg, IP; Bioveta, Ankara, Turkey) and ketamine hydrochloride (80 mg/kg, IP; Ketalar, Pfizer, Istanbul, Turkey) and placed in the prone position. Before the procedure, rat simulation was performed with a 1 mm section computed tomography scan; dose was calculated with Eclipse treatment planning system, version 8.9 (Varian Medical Systems, Palo Alto-CA, USA). After the simulation, other parts of their bodies were protected using lead and beam collimation. 6 mV linear accelerator (Clinac, Varian Medical Systems, Palo Alto CA, USA) was used to expose IR to the right legs of the rats in both groups with a 1.0 cm bolus source-skin (SSD) distance technique on the surface [26]. No rat was excluded due to death.
2.3. Chemical
Air-dried AM (Graviola) leaves were obtained from a local supplier in the Mediterranean region in Turkey. They cut into small pieces after washing with distilled water. 25 g samples were extracted in 70:70 ethanol for seven days with occasional shaking. The extract concentration procedure was done by removing ethanol. A rotary evaporator was used (Heidolph, Germany). After, the extract was lyophilized (Telstar- LyoQuest, Spain) overnight to prepare a dry extract, which was kept at -20 °C until use.
2.4. Biochemical analysis
Superoxide Dismutase (SOD, U/ml)
Superoxide dismutase accelerates the conversion to hydrogen peroxide and molecular oxygen in the elimination steps of reactive oxygen radicals produced in oxidative reactions. This method takes place by means of xanthine and xanthine oxidase. Here it is intended to generate superoxide radicals that react with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride to form a red formazan dye. The degree of inhibition of this reaction gives information about the Superoxide dismutase activity. (Relassay, Turkey)
Catalase (CAT, U/ml)
This colorimetric assay involves two steps. The sample is incubated with a certain hydrogen peroxide. hydrogen peroxide is converted to water and oxygen by this sample. catalase concentration is proportional to this ratio. Enzyme plant and a fixed incubation period are completed. The peroxide remaining after this is determined as a chromogen appearance. Absorbances at 405 nm are expressed in U/ml. (Relassay, Turkey)
Glutathione Peroxidase (GPx, U/ml)
This method is based on the Paglia and Valentine method. GPx catalyzes the oxidation of glutathione by cumene hydroperoxide. In the presence of glutathione (GSSG), it is immediately converted to the reduced form by oxidation of NADPH to NADP. The decrease in absorbance at 340 nm is the measure (Relassay, Turkey)
2.5. Histomorphologic examination
Automated tissue processing equipment (Leica ASP300S, Wetzlar, Germany) was used for routine processing of 32 sciatic nerve tissue samples fixed with 10% formalin. Afterwards, the tissues embedded in paraffin were cut using a Leica RM2255 rotary microtome (Wetzlar, Germany) to obtain 4 µm thick sections. Tissue sections stained with Hematoxylin-Eosin (HE) were evaluated under the microscope. The same procedure was performed for all samples. The camera used for the observation of all study materials was Nikon Digital DS-Ri2 and the microscope was Nikon Eclipse Ni-U equipped with the corresponding software. (Nikon, Tokyo, Japan).
In four separate groups, four parameters were examined for vasculature change, degenerative change, necrotic change, swelling in myelin sheath in sciatic nerve tissue. Histomorphological findings in tissues and cells were scored. Scoring is scored between 0 and 3 for each criterion. (0=normal, 1=Light, 2=medium, 3=heavy).
2.6. Statistical analysis
SPSS version 22.0 was used to analyze the data. Descriptive statistics for quantitative variables were expressed as mean and standard deviation, and categorical variables were expressed as numbers and percentages. Normal distribution assumptions for continuous variables were checked with the help of Skewness and Kurtosis coefficients, Shapiro-Wilk test, and distributions in q-q graphs. As the data show a normal distribution, One-way analysis of variance with LSD post hoc was used to compare the differences of the groups. For categorical variables, the differences between the groups were evaluated using Chi-Square analysis. Based on the p value <0.05, the results were considered statistically significant.