Three-Dimensional Cultivation a Valuable Tool for Modelling Canine Mammary Gland Tumour Behaviour In Vitro.

Cell cultivation has been one of the most popular methods in research for decades. Currently, scientists routinely use two-dimensional (2D) and three-dimensional (3D) cell cultures of commercially available cell lines and primary cultures to study cellular behaviour, responses to stimuli, and interactions with their environment in a controlled laboratory setting. In recent years, 3D cultivation has gained more attention in modern biomedical research, mainly due to its numerous advantages compared to 2D cultures. One of the main goals where 3D culture models are used is the investigation of tumour diseases, in both animals and humans. The ability to simulate the tumour microenvironment and design 3D masses allows us to monitor all the processes that take place in tumour tissue created not only from cell lines but directly from the patient's tumour cells. One of the tumour types for which 3D culture methods are often used in research is the canine mammary gland tumour (CMT). The clinically similar profile of the CMT and breast tumours in humans makes the CMT a suitable model for studying the issue not only in animals but also in women.

Canine Mammary Tumors: Classification, Biomarkers, Traditional and Personalized Therapies.

In recent years, many studies have focused their attention on the dog as a proper animal model for human cancer. In dogs, mammary tumors develop spontaneously, involving a complex interplay between tumor cells and the immune system and revealing several molecular and clinical similarities to human breast cancer. In this review, we summarized the major features of canine mammary tumor, risk factors, and the most important biomarkers used for diagnosis and treatment. Traditional therapy of mammary tumors in dogs includes surgery, which is the first choice, followed by chemotherapy, radiotherapy, or hormonal therapy. However, these therapeutic strategies may not always be sufficient on their own; advancements in understanding cancer mechanisms and the development of innovative treatments offer hope for improved outcomes for oncologic patients. There is still a growing interest in the use of personalized medicine, which should play an irreplaceable role in the research not only in human cancer therapy, but also in veterinary oncology. Moreover, immunotherapy may represent a novel and promising therapeutic option in canine mammary cancers. The study of novel therapeutic approaches is essential for future research in both human and veterinary oncology.

Two Amnion-Derived Mesenchymal Stem-Cells Injections to Osteoarthritic Elbows in Dogs-Pilot Study.

The aim of the study was to investigate the potential of cell-based regenerative therapy for elbow joints affected by osteoarthritis. Interest was focused on two intra-articular applications of amnion-derived mesenchymal stem cells (A-MSCs) to a group of different breeds of dogs with elbow osteoarthritis (13 joints). Two injections were performed 14 days apart. We evaluated synovial fluid biomarkers, such as IFN-γ, IL-6, IL-15, IL-10, MCP-1, TNF-α, and GM-CSF, by multiplex fluorescent micro-bead immunoassay in the treated group of elbows (n = 13) (day 0, day 14, and day 28) and in the control group of elbows (n = 9). Kinematic gait analysis determined the joint range of motion (ROM) before and after each A-MSCs application. Kinematic gait analysis was performed on day 0, day 14, and day 28. Kinematic gait analysis pointed out improvement in the average range of motion of elbow joints from day 0 (38.45 ± 5.74°), day 14 (41.7 ± 6.04°), and day 28 (44.78 ± 4.69°) with statistical significance (p < 0.05) in nine elbows. Correlation analyses proved statistical significance (p < 0.05) in associations between ROM (day 0, day 14, and day 28) and IFN-γ, IL-6, IL-15, MCP-1, TNF-α, and GM-CSF concentrations (day 0, day 14, and day 28). IFN-γ, IL-6, IL-15, MCP-1, GM-CSF, and TNF- α showed negative correlation with ROM at day 0, day 14, and day 28, while IL-10 demonstrated positive correlation with ROM. As a consequence of A-MSC application to the elbow joint, we detected a statistically significant (p < 0.05) decrease in concentration levels between day 0 and day 28 for IFN-γ, IL-6, and TNF-α and statistically significant increase for IL-10. Statistical significance (p < 0.05) was detected in TNF-α, IFN-γ, and GM-CSF concentrations between day 14 and the control group as well as at day 28 and the control group. IL-6 concentrations showed statistical significance (p < 0.05) between day 14 and the control group.

Impact of Canine Amniotic Mesenchymal Stem Cell Conditioned Media on the Wound Healing Process: In Vitro and In Vivo Study.

The aim of this study was to provide a beneficial treatment effect of mesenchymal stem cell products derived from the canine amniotic membrane (AM-MSC) on the complicated wound healing process in dogs. AM-MSCs were characterized in terms of morphology, phenotypic profile, and multilineage differentiation potential. The in vitro study of the effect of canine amniotic mesenchymal stem cell conditioned media (AMMSC-CM) on a primary skin fibroblast cell culture scratch assay showed a decrease in the measured scratch area of about 66.39% against the negative control (Dulbecco's Modified Eagle's Medium-32.55%) and the positive control (Dulbecco's Modified Eagle's Medium supplemented with FGF2, N2, B27, and EGF-82.077%) after 72 h treatment. In the experimental study, seven dogs with complicated nonhealing wounds were treated with a combination of antibiotics, NSAIDs, and local AMMSC-CM application. After 15 days of therapy, we observed a 98.47% reduction in the wound surface area as opposed to 57.135% in the control group treated by conventional therapy based on debridement of necrotic tissue, antibiotic therapy, pain management, and change of wound dressing.

Retrospective analysis of tracheal hypoplasia in brachycephalic and non-brachycephalic dogs: Inter- and intra-observer agreement of measurements.

This retrospective study was undertaken on the records of intraluminal diameter of the trachea in 185 dogs, in which hypoplasia of the trachea had been suspected. The relative size of the trachea was measured using the tracheal diameter (TD), thoracic inlet distance (TI), thoracic tracheal diameter (TT) and the width of the third rib (3R), expressed as ratios TD:TI and TT:3R. Thirty-five dogs were diagnosed as having tracheal hypoplasia. Bulldogs and non-bulldog brachycephalic dogs had significantly smaller measured trachea diameters compared to the predicted values calculated on the basis of their body weight. Radiographs of each dog were investigated by four observers. Inter- and intra-observer reliability (ICCinter, ICCintra) was based on the measurements taken by four observers to evaluate the reproducibility of the protocol. There was a good ICCinter (0.8) and ICCintra (0.89) agreement. Craniocaudal tangential radiographs, centred on the cranial thoracic aperture, did not show a significant difference in tracheal diameter measurements compared to the right lateral radiographs. In conclusion, our findings indicate that bulldogs and non-bulldog brachycephalic dogs have smaller tracheal diameters than non-brachycephalic dogs.

A Comparative Study of Canine Mesenchymal Stem Cells Isolated from Different Sources.

In this study, we provide comprehensive analyses of mesenchymal stem cells (MSCs) isolated from three types of canine tissues: bone marrow (BM-MSCs), adipose tissue (AT-MSCs) and amniotic tissue (AM-MSCs). We compare their morphology, phenotype, multilineage potential and proliferation activity. The BM-MSCs and AM-MSCs showed fibroblast-like shapes against the spindle shape of the AT-MSCs. All populations showed strong osteogenic and chondrogenic potential. However, we observed phenotypic differences. The BM-MSCs and AT-MSCs revealed high expression of CD29, CD44, CD90 and CD105 positivity compared to the AM-MSCs, which showed reduced expression of all the analysed CD markers. Similarly, the isolation yield and proliferation varied depending on the source. The highest isolation yield and proliferation were detected in the population of AT-MSCs, while the AM-MSCs showed a high yield of cells, but the lowest proliferation activity, in contrast to the BM-MSCs which had the lowest isolation yield. Thus, the present data provide assumptions for obtaining a homogeneous MSC derived from all three canine tissues for possible applications in veterinary regenerative medicine, while the origin of isolated MSCs must always be taken into account.

Impact of mesenchymal stem cells derived conditioned media on neural progenitor cells.

Neurodegenerative diseases are common problem for companion animals. Due to the limited ability of injured axons to regenerate, innovative therapies combined with rehabilitation have been applied and evaluated. Among them, stem cells and their conditioned media implantation, which can ameliorate damaged tissue has been suggested as a promising treatment strategy. The main goal of our study was to characterize mesenchymal stem cells (MSC) derived from canine adipose tissue (AT-MSC) and umbilical cord (UC-MSC) and analyse effect of their conditioned media (CM) on neurite outgrowth of neural progenitor cells isolated from the brain cortex of neonatal rats. MSC from both sources showed high osteogenic and chondrogenic potential and expression of CD90 and CD29. Furthermore, both UC-MSCCM and AT-MSCCM stimulated neurite growth. Interestingly, this effect was more pronounced with UC-MSCCM when compared to AT-MSCCM in vitro, which may be related to the different content of neurotrophic factors included in the CM.

Novel Diagnostic Tools for Identifying Cognitive Impairment in Dogs: Behavior, Biomarkers, and Pathology.

Canine cognitive dysfunction syndrome (CCDS) is a progressive neurodegenerative disorder in senior dogs that is mainly associated with decreased ability to learn and respond to stimuli. It is commonly under-diagnosed because behavioral changes are often attributed to the natural process of aging. In the present study, we used for the first time a comprehensive approach enabling early diagnosis of canine patients with mild cognitive disorders (MiCI). We included CAnine DEmentia Scale (CADES) questionnaires, biochemical parameters, and biomarkers in blood serum, and correlated them with post-mortem histopathological changes. The CADES questionnaires enabled us to identify MiCI dogs developing changes mainly in domains corresponding to social interaction and spatial orientation, which seems to be crucial for delineating early cognitive disorders. Biochemical analyses in these dogs showed slightly elevated liver enzyme parameters (AST and ALT) and significantly decreased sodium and chloride levels in blood serum. Furthermore, we describe for the first time a significant increase of neurofilament light chain (NFL) in blood serum of MiCI dogs, compared to normal aging seniors and young controls, but no changes in TAU protein and amyloid-ß (Aß42) peptide levels. In canine brains with cognitive impairment, amyloid plaques of mainly diffuse and dense types were detected. Furthermore, activated microglia with amoeboid body and dystrophic processes occurred, in some cases with spheroidal and bulbous swellings. On the other hand, no TAU pathology or neurofibrillary tangles were detected. These results suggest that a combination of CADES questionnaire mainly with CNS injury biomarker (NFL) and with biochemical parameters (ALT, AST, Na, and Cl) in blood serum may predict CCDS in senior dogs.

A comprehensive study of disorder of sex development in Staffordshire bull terrier dog.

An 8-month-old female Staffordshire bull terrier was clinically examined because of external sexual organs abnormality-clitoral hypertrophy. As stated by the owner, the female dog had not been in heat yet. Serum profile of testosterone (3.39 ng/ml), as well as an anti-Mullerian hormone (24.0 ng/ml), suggested the presence of testicular tissue. On the contrary, the estimated level of 17ß-oestradiol (24.6 pg/ml) was approximately two times higher when compared with the normal anoestrus values (5-10 pg/ml). A midline laparotomy was performed to detect the cranial parts of the genital system. Gonads resembling testicle or ovotestis (left) and hypoplastic testicle (right) was visible. Cranial portion of gonads was attached to structures indicative of bilateral epididymidis. The next tubular structures-oviducts were resected along with adherent parts of a hypoplastic uterus. Histological evaluation confirmed that the examined gonad samples were testicles with modified interstitial testicular tissue. Hypertrophy of interstitial space was predominantly formed by Leydig cells. Examination of a cross-section through the head of suspected epididymidis confirmed their characteristic structures. In addition, the characteristic configuration of the oviducts was presented. The uterus consisted of three walls, in which the endometrium was hypoplastic with the presence of endometrial glands. No Y chromosome was detected by chromosomal analysis using CFA Y probe and the amplification of SRY-gene coding region (813 bp) indicated genotype 78, XX; SRY-negative. Sequencing of SOX9 gene exons 1-3 did not reveal any differences in exon 1 and 3. On the contrary, a few changes were determined in the SOX9 exon 2 sequences: G instead of A at position 103; C instead of reference T at position 115; GCG instead of reference CGC at position 138-140; T instead of reference C at positions 161, 164 and 167.