Investigation of the therapeutic effects of mesenchymal stem cells i̇solated from wharton's jelly in cows with subclinical mastitis by pathological and molecular methods

In this study, the therapeutic efficacy of intramammary administration of mesenchymal stem cells (WJ-MSCs) isolated from Wharton's jelly in dairy cows with subclinical mastitis (SCM) was investigated at the molecular level using somatic cell count in milk, haematological parameters, microbiological analyses, histopathological and immunohistochemical examinations, and quantitative PCR (qPCR). In the study, 20 Holstein cows kept under equal care and feeding conditions during the same lactation period were used. The California Mastitis Test (CMT) was applied to detect SM animals. The animals were divided into 4 groups: healthy control (n=4, 3 ml DMEM solution, intramammary, twice at 7-day intervals), WJ-MKH control (n=4, 2,5 x 107 WJ-MKH, intramammary, twice at 7-day intervals), SM+Antibiotic (n=6, 3 ml intramammary DMEM solution and Ceftiofur, 1mg/kg/IM twice at 7-day intervals), and SM+WJMKH (n=6, 2,5 x 107 WJ-MKH, intramammary twice at 7-day intervals). On days 1, 8, 15, 20, and 30 of the study, venous blood samples were collected directly from the jugular vein under aseptic conditions into EDTA tubes containing anticoagulant for hemogram determination, and milk samples were collected into karyotypes for molecular analysis. After fixation of mammary tissue biopsy samples in 10% formaldehyde solution on the 30th day of the study for histopathological and immunohistochemical (PCNA) examinations, routine tissue processing was performed. In the hemogram analysis, it was determined that the reference ranges of the RBC, HGB, HCT, MCV, MCH, MCHC, Lym%, Neu%, and PLT parameters varied. It was determined that the WJMKH application balanced the immune response, bringing the WBC value, which was above the reference range on the 15th day of the study, closer to normal limits by the end of the study. It was found that the severity of histopathological findings caused by SM in breast tissue was significantly reduced with WJ-MKH treatment (p<0.05). It was found that the PCNA expression level in IHC staining was expressed at a higher level in the WJ-MKH treatment group compared to the antibiotic treatment group (p<0.05). According to the qPCR analysis results from milk samples, no significant difference was observed in the expression levels of ACTA2 and EGF. However, it was determined that the expression levels of DEFB1, TLR2, which were high at the beginning of the study, and VEGFA, which were low at the beginning of the study, showed values close to those of the control group at the end of the study in both treatment groups. Additionally, it was found that WJ-MKH administration in SM animals successfully and gradually reduced GM-CSF expression levels. In the treatment of SM, it was determined that WJ-MKH treatment showed at least a similar level of efficacy to antibiotic treatment. It was also concluded that stem cells can regulate the inflammatory response through their immunomodulatory effects, contribute to the healing process of damaged breast tissue by increasing epithelial cell proliferation, and support tissue regeneration.