Lucas Vinícius de Oliveira Ferreira

Mesenchymal stem cells (MSCs) exhibit immunomodulatory and regenerative properties that have been extensively investigated in domestic species. However, knowledge of their biological characteristics in wildlife species remains limited and is critical for advancing translational research and conservation medicine. Thus, this study aimed to isolate, characterize, and evaluate the immunomodulatory and neuroregenerative potential of adipose tissue-derived MSCs (AT-MSCs) from crab-eating foxes ( Cerdocyon thous ). Subcutaneous adipose tissue samples were collected from five healthy adult animals during elective surgeries, following prior ethical approval. All procedures were conducted in accordance with institutional and national ethical guidelines. MSCs were isolated by enzymatic digestion, expanded in vitro, and characterized based on morphology, immunophenotype, and mesodermal multipotency. Cell viability was assessed following inflammatory stimulation with lipopolysaccharide (LPS), and the gene expression of immunomodulatory cytokines and neurotrophic factors was evaluated by RT-qPCR. The cells displayed typical fibroblast-like morphology, plastic adherence, expression of mesenchymal markers CD90, CD29, and CD44, absence of hematopoietic markers CD14 and CD45, and successful differentiation into adipogenic, chondrogenic, and osteogenic lineages. LPS stimulation significantly upregulated the expression of indoleamine-2,3-dioxygenase (IDO), as well as the neurotrophic and regenerative factors glial cell line-derived neurotrophic factor (GDNF), hepatocyte growth factor (HGF), and vascular endothelial growth factor A (VEGF-A). These findings indicate that AT-MSCs derived from Cerdocyon thous possess immunomodulatory and neuroregenerative potential, supporting their relevance for translational research and future applications in conservation veterinary medicine, particularly in the context of inflammatory and neurological disorders.

Background: Horses hold significant value in sports and the economy, requiring rapid recovery from musculoskeletal injuries. Cell-based therapies, particularly those using mesenchymal stem cells (MSCs), have emerged as promising tools due to their regenerative, anti-inflammatory, and immunomodulatory properties, as well as their capacity for self-renewal. However, their therapeutic efficacy may be influenced by host immune responses, particularly in allogeneic settings. Thus, this study aimed to morphologically evaluate the acute inflammatory response in equine muscle tissue following intramuscular transplantation of autologous and allogeneic bone marrow-derived MSCs (EqBM-MSCs). Materials, Method & Results: Fifteen clinically healthy adult horses (6-12 years; 300-500 kg) were randomly assigned to 3 groups (n = 5 each): Control (intramuscular injection of phosphate-buffered saline - PBS), Auto-MSCs (intramuscular transplantation of autologous EqBM-MSCs), and Allo-MSCs (intramuscular transplantation of allogeneic EqBM-MSCs). EqBM-MSCs were isolated from bone marrow, expanded under standard culture conditions, and characterized by plastic adherence, fibroblastoid morphology, expression of CD44 and CD90, absence of CD34, and their differentiation potential into adipogenic, osteogenic, and chondrogenic lineages. An average of 2×10⁶ cells in 100 µL of PBS were transplanted into the right gluteus medius muscle under ultrasound guidance. Two muscle biopsies were collected per horse before transplantation (D0) and 24 h post-transplantation (D24). Histological hematoxylin and eosin (H&E); Gomori’s trichrome (GT)) and histochemical (nicotinamide adenine dinucleotide tetrazolium reductase (NADH-tr)) analyses demonstrated preserved muscle architecture and normal fiber organization in both MSC-treated groups. No evidence of inflammation, such as fiber morphological alterations or inflammatory cell infiltration, was observed in any group. Discussion: MSCs possess self-renewal capacity and secrete bioactive factors that support tissue repair and modulate inflammation. In equines, bone marrow is a commonly used MSC source, and intramuscular delivery has been associated with efficient administration and prolonged local retention. The absence of acute muscle inflammatory changes observed in this study is consistent with previous reports in equine tendon following MSC transplantation. In contrast, inflammatory responses have been described in rat muscle after autologous bone marrow MSC administration, typically characterized by muscle fiber alterations and inflammatory cell infiltration. Such changes were not observed in the present study, and NADH-tr staining confirmed preserved metabolic activity. These findings indicate that intramuscular administration of both autologous and allogeneic EqBM-MSCs is well-tolerated and does not induce acute inflammation. The lack of acute response may be related to the immunomodulatory properties of MSCs, including secretion of factors such as transforming growth factor beta (TGF-β) and prostaglandin E2 (PGE2). The absence of inflammatory changes supports the immunomodulatory properties of MSCs and may reinforces their potential for clinical application in equine muscle injury. Nevertheless, the evaluation performed at 24 h reflects only the acute phase, and longer-term responses remain to be elucidated. Further studies are necessary to investigate the long-term safety and therapeutic potential of intramuscular MSC transplantation in equine musculoskeletal disorders. Keywords: autologous, cell therapy, gluteus medius, histology, histochemistry.

Heart rate variability (HRV) is an established biomarker of autonomic nervous system activity, yet its profile in miniature cattle remains poorly understood despite their growing importance in sustainable farming. This study compared HRV parameters between miniature and Holstein cows and assessed the influence of age on these profiles. Eighty clinically healthy female cattle (40 miniature, 40 Holstein), aged 2 to 8 years, were evaluated under field conditions using a Polar H10 heart rate monitor. RR intervals were analyzed using Kubios HRV software to obtain time- and frequency-domain indices. Miniature cows exhibited significantly lower heart rates and higher time-domain measures (RMSSD and SDNN) compared to Holsteins, while frequency-domain analysis revealed significant differences in LF, HF, and LF/HF ratio, suggesting group-associated differences in proportional autonomic balance. Age-stratified analysis revealed that these physiological distinctions were more pronounced in older cows (6–8 years). However, given the observational cross-sectional design of this study, confounding factors—specifically the different farm environments, management systems, and the active lactation status of the Holstein group—preclude attributing these differences solely to breed or body size. Therefore, these results suggest an associative physiological pattern rather than a definitive autonomic adaptation. Despite these limitations, portable HRV monitoring proved feasible under farm conditions, providing valuable preliminary baseline data that can inform future controlled studies on bovine cardiovascular welfare.

Summary: Peripheral nerve injuries (PNI) in horses represent a significant clinical challenge, frequently associated with functional deficits and reduced athletic performance. Conventional therapeutic approaches, primarily based on anti‐inflammatory treatments and rehabilitation, offer limited efficacy, driving the search for innovative alternatives. In this context, mesenchymal stem cells (MSCs) have gained attention for their robust paracrine activity, which underlies their anti‐inflammatory, immunomodulatory and neuroregenerative effects. In vitro studies demonstrate that equine MSCs can transdifferentiate into Schwann‐like cells and neural‐like cells, further expanding their therapeutic potential. However, in vivo applications remain poorly explored, with few studies available in horses and divergent outcomes that may have been influenced by differences in injury type, dosage and route of administration. Thus, this perspective critically examines the available knowledge, highlighting key advances, ongoing challenges and future directions needed to support the safe and effective implementation of MSC‐based therapies in equine PNI.

Peripheral nerve injuries (PNI) often lead to long-term functional impairment. Mesenchymal stem cells (MSCs) and cannabidiol (CBD) have shown anti-inflammatory and neuroprotective effects in vitro, which may be relevant for PNI research. The aim of this study was to evaluate CBD-rich cannabis extract’s potential to induce anti-inflammatory and neurotrophic gene expression in equine adipose tissue-derived MSCs (EqAT-MSCs) in an inflammatory in vitro environment. The morphology and metabolic activity of EqAT-MSCs ( n = 4) were assessed after CBD-rich extract priming at concentrations of 3, 5, 7, and 9 µM for 24 and 48 h. Cytokine and neurotrophic gene expression was evaluated under these conditions: DMEM (unprimed), DMEM + LPS (lipopolysaccharide) (10 ng/ml), and LPS (10 ng/ml) + DMEM + CBD at 3, 5, and 7 µM for 24 and 48 h. No morphological changes were observed in primed EqAT-MSCs versus unprimed cells. EqAT-MSCs showed a reduction in metabolic activity at 9 µM after 24 h. CBD priming following LPS stimulation led to statistically significant changes in EqAT-MSC gene expression. BDNF expression increased after 48 h (3 and 5 µM), while NGF expression decreased at both 24 and 48 h (3, 5, and 7 µM). IL-1β expression decreased after 24 h (3 and 7 µM), and IL-6 levels decreased at both 24 (5 and 7 µM) and 48 h (3, 5, and 7 µM). No significant changes were observed in GDNF, TNF-ɑ, IFN-ɣ, or IL-10. These results indicate that CBD-rich extract selectively modulates inflammatory and neurotrophic gene expression in EqAT-MSCs while maintaining metabolic integrity.

Neurological disorders in dogs pose a significant challenge in veterinary medicine, often leading to permanent deficits and reduced quality of life. Traditional therapeutic approaches frequently offer limited efficacy, especially in neuroimmune or degenerative conditions. In this context, mesenchymal stem cells (MSCs) have emerged as a promising therapeutic strategy due to their immunomodulatory, anti-inflammatory, neuroprotective, and neuroregenerative properties. This chapter provides a comprehensive review of MSC-based therapy for canine neurological disorders, with an emphasis on their application in meningoencephalomyelitis of unknown origin (MUO), peripheral nerve injuries, and spinal cord injury (SCI). Key aspects discussed include MSC sources, mechanisms of action, delivery routes, neurological improvement, functional recovery, and limitations. In addition, future directions and perspectives are explored to support the development of safe and effective MSC-based therapies in veterinary neurology.

Mesenchymal stem cells (MSCs) have emerged as a promising alternative for treating neurological disorders due to their neuroprotective, neuroregenerative, immunomodulatory, anti-inflammatory, and anti-apoptotic properties. The use of allogeneic MSCs offers advantages such as the selection of specific cells and their immediate availability. This study aimed to evaluate the safety and feasibility of intrathecal transplantation of allogeneic equine bone marrow-derived MSCs (EqBM-MSCs) in healthy horses. Ten healthy horses (five–12 years old) were randomly divided into two groups. In the control group (n = 5), phosphate-buffered saline was administered intrathecally, while the MSCs group (n = 5) received 3 × 10⁶ allogeneic EqBM-MSCs. Safety and feasibility were assessed through physical examinations monthly for one year, cerebrospinal fluid (CSF) analysis before and six days after transplantation, and magnetic resonance imaging (MRI) one-year post-transplantation. No significant differences were found between the groups in physical examinations or CSF analysis before and six days after transplantation. Furthermore, MRI showed no changes after one year. Matrix Metalloproteinase-2 (MMP-2) expression was detected in the CSF before and after transplantation but showed no significant differences. These findings suggest that intrathecal transplantation of allogeneic EqBM-MSCs is safe and viable, offering a potential therapeutic approach for neurological disorders in horses.

The endocannabinoid system regulates key biological functions such as neuroprotection, pain modulation, inflammation, and immunomodulation. Cannabis-based therapies have gained attention due to the therapeutic potential of their bioactive compounds, particularly phytocannabinoids like cannabidiol (CBD), which exhibit anti-inflammatory, neuroprotective, and immunomodulatory properties. Mesenchymal stem cells (MSCs) are widely studied for their regenerative and immunomodulatory potential. This study evaluated the effects of priming canine adipose tissue-derived MSCs (cAT-MSCs) with a CBD-rich cannabis extract on cell morphology, viability, neurotrophic factor gene expression, and cytokine gene and protein expression. cAT-MSCs (n = 5) were primed for 24 h and divided into three groups: Control (C, unprimed), D1 (2.25 µM CBD), and D2 (225 nM CBD). No morphological or viability changes were observed. Gene expression analysis showed that groups D1 and D2 exhibited increased HGF expression. D1 also showed increased IDO and decreased BDNF expression. In contrast, no significant changes were observed in GDNF, IL-10, TNF-α, IFN-γ, or PTGES2. Regarding the cytokine profile, GM-CSF, IL-2, and IL-10 were undetectable. Notably, IL-8 and MCP-1 levels were significantly reduced in D1 compared to the control. These findings suggest that CBD priming modulates key regenerative and inflammatory mediators in cAT-MSCs, supporting its potential application in enhancing the efficacy of cell-based therapies.

A Cannabis sativa L. e seus derivados têm se destacado em estudos científicos pelas suas propriedades terapêuticas, especialmente no campo da medicina regenerativa. Nosso projeto investiga os efeitos do extrato full spectrum de CBD em células tronco mesenquimais derivadas do tecido adiposo canino. Os receptores canabinoides são conhecidos por influenciar processos como apoptose, migração celular e a expressão de citocinas inflamatórias e fatores neurotróficos, demonstrando um potencial significativo para aplicações terapêuticas em doenças inflamatórias e degenerativas. Ao investigar esses efeitos, buscamos gerar conhecimento que possa contribuir para o desenvolvimento de novas terapias imunomoduladoras, com impacto direto na medicina veterinária e humana. Investigar o impacto do CBD em células tronco mesenquimais caninas, avaliando a expressão dos receptores CB1 e CB2, e analisar se o CBD influencia a viabilidade e secreção de fatores anti-inflamatórios e neurotróficos, visando otimizar terapias regenerativas. As células tronco mesenquimais foram isoladas de tecido adiposo canino, cultivadas e caracterizadas em laboratório. Em seguida, foram tratadas com extrato full spectrum de CBD nas doses de 2,25 μM e 225 nM. Os ensaios incluíram as análises de viabilidade celular (MTT) e expressão gênica de citocinas inflamatórias (como PTEG2, IDO, IL-10, TNF-α e INF-ɣ) e de fatores neurotróficos (HGF, BDNF e GDNF) por meio da técnica de qtPCR. Também foi avaliada morfologia celular por meio de fotomicrografia. A estimulação in vitro por 24 h das AT-MSCs caninas com o extrato de Cannabis rico em CBD nas doses de 2,25 μM e 225 nM não alterou a morfologia celular, não prejudicou sua viabilidade e não influenciou a expressão gênica da PTGES2, IDO, TNF-α, INF-γ, IL-10, BDNF, GDNF e HGF. Palavras-chave: Terapia celular; Imunomodulação; Fitocanabinoides.