Animals and Experimental Design
Wistar rats, 6-8 months old albino male, were obtained from Animal Care and Research Unit of University and all experiments were performed according to the principles and guidelines of Animal Ethical Committee’s approval (HADYEK 64583101/2020/075). A total number of 44 rats were used in this study.
The study was conducted in two stages. In the first stage, the application dose of DTX, which was chosen in accordance with the purpose of the study, to cause hair follicle loss without causing lethal toxicity was determined. While establishing the experimental model, we aimed to mimic the clinical condition of DTX (Doxitax® sol, Kocak Farma, Istanbul, Turkey) treatment, well known for the side effect on hair growth. Dykes et al. stated that maximum tolerable dosage of DTX was 33mg/kg and lethal dosage was 50 mg/kg on mice (8). These investigators administered DTX once weekly to the rats for three times. Another research that was performed by Bissery et al. showed that non-lethal maximum dose of DTX was 32.2 mg/kg (9). In another study, DTX produced oxidative stress at 10 mg/kg dosages (10). Based on these previous studies, we have planned the first stage of experimental study groups as 10 mg/kg (low dose) and 30 mg/kg (high dose) weekly DTX, for three weeks, to assess the hair tissue response.
Taking this data as the basis, three groups were created for the first stage.
- Control Group (n=6): Serum physiologic (%09) was administered intra-peritoneal (IP) to the rats in this group once a week for three weeks, it was planned to sacrifice the animals at the 22nd
- DTX10 Group (n=8): DTX 10 mg/kg was administered IP to the rats in this group once a week (days 0, 8 and 15) of the study; it was planned to sacrifice the animals at the 22nd
- DTX30 Group (n=8): DTX 30 mg/kg was administered IP to the rats in this group once a week (days 0, 8 and 15) of the study; it was planned to sacrifice the animals at the 22nd
Using the findings obtained in the first stage of the study, the second stage was started. At this stage, three groups were created with the aim of determining the effectiveness and safety of the solution to be applied experimentally. Considering the results obtained in the first stage, the application dose for DTX was taken as 10 mg.
On the second stage, HDDPiW-jSB solution was applied on the one cm2 interscapular region of hairy area, starting one week before the administration of DTX in all groups. HDDPiW-jSB is a vitamin-based product (a mixture of multi-vitamin in specifically adjusted concentrations, developed by the authors S. & D. Barutca, depending on their clinical and patient experiences), developed to protect the patient from CIA. Each rat was individually caged to avoid licking of the solution. The second stage of experimental groups was as follows:
- HDDPiW-jSB Group (n=8): The solution was applied to the previously described dorsal area of the rats (no-DTX administration), twice a week (on Mondays and Thursdays), for four weeks, to watch the safety of the solution. It was planned to sacrifice the animals at the 28th
- DTX10 + HDDPiW-jSB I Group (n=7): The solution was applied once a week (on Mondays), for four weeks (days 0, 8,15 and 21); meanwhile IP DTX 10 mg/kg was administered on days 8, 15, 21 of the study; it was planned to sacrifice the animals at the 28th
- DTX10 + HDDPiW-jSB II Group (n=7): The solution was applied twice a week (on Mondays and Thursdays), for four weeks; meanwhile IP DTX 10 mg/kg was administered on days 8, 15, 21 of the study; it was planned to sacrifice the animals at the 28th
Ketamine and Xylasine (50 mg/kg and 5 mg/kg, respectively) were used for the scarification process.
Immuno-Histopathological Staining
The hair skin samples were fixed in 10% neutral-buffered formalin solution (Chempur, Poland), treated with graded alcohol, xylol series, and blocked-in paraffin (Histowax, Histo-Lab. Ltd, Goteborg, Sweden). Obtained 4 μm sections of skin from paraffin blocks were stained according to the hematoxylin-eosin (H&E) method. These sections were evaluated under the microscope and images were obtained (Olympus Stream Start BX 51, Olympus SP 350 digital camera, Japan).
On the first stage of study, the dose response of 10 mg/kg and 30 mg/kg DTX on hair follicles were evaluated. Then, the study was continued with 10 mg/kg DTX in order to reveal and interpret the possible protective effects of HDDPiW-jSB solution. Tissues were stained with H&E and follicles counted as anagen, catagen, telogen and terminal hair follicles in five areas (x40) of each sample. Next, immuno-histopathological staining of CD34 (Clone QBEnd10, Dako, Denmark), Bcl-2 oncoprotein (b cell leukemia protein-2) (Clone 124, Dako, Denmark), apoptosis-related cysteine protease (caspase 3) (mouse monoclonal antibody, Vision Biosystems Novocastra, Newcastle, United Kingdom) were performed in three areas of each sample (x400). Olympus Stream Start BX51 microscope and Image J software were used for the analyses.
Statistical Analyses
Statistical analyses were performed using the NCSS 2007 program (Number Cruncher Statistical System, Kaysville, Utah, USA). Data are expressed with the descriptive statistics (mean, standart deviation, minimum value, maximum value, median, quartiles, frequency, percent). Shapiro-Wilk test and graphical evaluation were performed to assess the normality of quantitative data. If the variances were heterogeneous, between the groups Mann-Whitney U test; amongst the group Kruskal-Wallis test and Dunn-Bonferroni test were used to evaluate for significance. Fisher-Freeman-Halton exact test was used for qualitative data. In all cases, p < 0.05 was considered as significant.