The therapeutic potential of Pluronic F127 (PF127) hydrogel loaded with adipose-derived stromal vascular fraction (AdSVF), mesenchymal stem cells (AdMSC), and conditioned media (AdMSC-CM) for repairing full-thickness skin wounds was evaluated using a rabbit model. The rabbits were randomly divided into eight groups with six animals each and treatment was given as per the predetermined protocol (3 doses at one-week interval): Group A (Control), Group B (AdSVF), Group C (AdMSC), Group D (AdMSC-CM), Group E (PF127), Group F (AdSVF + PF127), Group G (AdMSC + PF127), and Group H (AdMSC-CM + PF127). Skin tissue samples were collected from the healing wounds on day 28 for staining and collagen quantification. Collagen density (Area %) was quantified using tissue sections stained with Masson's Trichrome (MT) and Picrosirius Red (PSR) stain using the Colour Deconvolution plugin of ImageJ and RGB stack method, respectively. These techniques function based on separating different colour channels in the stained tissue sections to isolate the collagen fibers and then quantifying them through thresholding and image analysis. Across the treatment groups, both staining methods generally showed a trend of increased collagen density compared to the control group. For most groups, PSR staining consistently indicated slightly lower collagen densities than MT staining. However, the overall trends were similar in both staining. The comparison between PSR and MT staining methods revealed that both techniques effectively assess collagen density in healing wounds. However, there were subtle differences in the absolute values obtained, with PSR staining tending to yield slightly lower collagen density measurements than MT. These differences can be attributed to the distinct mechanisms of these staining methods. Therefore, both staining methods can digitally quantify collagen density in wound healing research.
The neuropeptide kisspeptin (Kp) through its receptor Kiss1r regulates the HPG axis by controlling GnRH release. Since buffalo is a seasonal breeder with problems of delayed puberty and postpartum anestrus, we characterized the Kiss1 and Kiss1r and investigated the immunolocalization in the hypothalamus and corpus luteum (CL). Kiss1 and Kiss1r genes were amplified from gDNA covering the coding region, cloned and sequenced with accession numbers MF168937 and MG820539, respectively. The Kiss1 DNA sequence had two exonic segment contained coding sequence (cds); 408 bp encoding a predicted protein of 136 aa with conservation of Kp-10 and shared 94.5–98.3% identity with ruminants. Kiss1r DNA sequence consisted of five exons with a cds of 1134 bp encoding a protein of 378 aa. Phylogenetic analysis of Kiss1 and Kiss1r revealed that it formed a monophyletic clade with cattle, which branched from sheep and goat. Immunofluorescence study revealed the presence of Kiss1 and Kiss1r in the neuronal soma and perinuclear area of preoptic and arcuate regions of the hypothalamus and luteal cells of the CL. This is the first report on molecular characterization of bubaline Kiss1 and Kiss1r genes that confirmed the presence of conserved Kp-10 like other ruminants and kisspeptinergic system is present in the hypothalamus and CL.