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Background: Atopic dermatitis (AD) is one of the most common cutaneous inflammatory and pruritic diseases in dogs. Considering its multifactorial nature, AD can be a challenging disease to manage, and the therapeutic strategy must often be multimodal. In recent years, research has been moving toward the use of natural products which have beneficial effects on inflammation and itching, and no side effects. Cannabinoid receptors have been demonstrated to be expressed in healthy and diseased skin; therefore, one of the potential alternative therapeutic targets for investigating AD is the endocannabinoid system (ECS)., Objective: To immunohistochemically investigate the expression of the cannabinoid receptor type 2 (CB2R), and the cannabinoid-related receptors G protein-coupled receptor 55 (GPR55), transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1) in mast cells (MCs), macrophages, dendritic cells (DCs), T cells, and neutrophils of the skin of dogs with AD., Animals: Samples of skin tissues were collected from eight dogs with AD (AD-dogs)., Materials and Methods: The immunofluorescent stained cryosections of the skins of 8 dogs with AD having antibodies against CB2R, GPR55, TRPV1, TRPA1 were semiquantitatively evaluated. The inflammatory cells were identified using antibodies against tryptase (mast cells), ionized calcium binding adaptor molecule 1 (IBA1) (macrophages/DCs), CD3 (T cells), and calprotectin (neutrophils). The proportions of MCs, macrophages/DCs, T cells, and neutrophils expressing CB2R, GPR55, TRPV1 and TRPA1 were evaluated., Results: The cells of the inflammatory infiltrate showed immunoreactivity (IR) for all or for some of the cannabinoid and cannabinoid-related receptors studied. In particular, MCs and macrophages/DCs showed CB2R-, GPR55-, TRPA1-, and TRPV1-IR; T cells showed CB2R-, GPR55- and TRPA1-IR, and neutrophils expressed GPR55-IR. Co-localization studies indicated that CB2R-IR was co-expressed with TRPV1-, TRPA1-, and GPR55-IR in different cellular elements of the dermis of the AD-dogs., Conclusions and Clinical Importance: Cannabinoid receptor 2, and cannabinoid-related receptors GPR55, TRPV1 and TRPA1 were widely expressed in the inflammatory infiltrate of the AD-dogs. Based on the present findings, the ECS could be considered to be a potential therapeutic target for dogs with AD, and may mitigate itch and inflammation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Chiocchetti, Salamanca, De Silva, Gobbo, Aspidi, Cunha, Galiazzo, Tagliavia, Sarli and Morini.)

It is commonly accepted that some form of skin barrier dysfunction is present in canine atopic dermatitis (AD), one of the most common cutaneous pruritic inflammatory diseases of dogs. The impaired skin barrier function facilitates the penetration of allergens and subsequently stronger sensitization responses. The role of the endocannabinoid system (ECS) in the physiology and pathology of the skin is becoming increasingly established. It has been demonstrated that cannabinoid receptors are expressed in healthy and diseased skin and, based on current knowledge, it could be stated that cannabinoids are important mediators in the skin. The present study has been designed to immunohistochemically investigate the expression of the cannabinoid receptors type 1 (CB1R) and 2 (CB2R) and the cannabinoid-related receptors G protein-coupled receptor 55 (GPR55), transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1), peroxisome proliferator-activated receptors alpha (PPARα), and serotoninergic receptor 1a (5-HT1aR) in keratinocytes of healthy dogs and of dogs with AD. Samples of skin tissues were collected from 7 healthy controls (CTRL-dogs) and from 8 dogs with AD (AD-dogs). The tissue samples were processed using an immunofluorescence assay with commercially available antibodies, and the immunolabelling of the receptors studied was quantitatively evaluated. The keratinocytes of the CTRL- and the AD-dogs showed immunoreactivity for all the receptors investigated with a significant upregulation of CB2R, TRPA1, and 5-HT1aR in the epidermis of the AD-dogs. The presence of cannabinoid and cannabinoid-related receptors in healthy keratinocytes suggested the possible role of the ECS in canine epidermal homeostasis while their overexpression in the inflamed tissues of the AD-dogs suggested the involvement of the ECS in the pathogenesis of this disease, having a possible role in the related skin inflammation and itching. Based on the present findings, the ECS could be considered a potential therapeutic target for dogs with AD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Chiocchetti, De Silva, Aspidi, Cunha, Gobbo, Tagliavia, Sarli and Morini.)

Large colon volvulus in horses is associated with a poor prognosis, especially when ischemic-reperfusion injury of the affected intestinal tract develops. Proteinase-activated receptor 2 (PAR 2 ) plays an important role in the pathogenesis of inflammation in the gastrointestinal tract. The aim of this study was to evaluate the distribution and expression of PAR 2 in colonic pelvic flexure of horses spontaneously affected by large colon volvulus (CVH group). Eight horses admitted for severe abdominal colon volvolus and which underwent surgery were included. Colon samples were collected after enterotomy. Data previously obtained from healthy horses were used as a control group. Histologic evaluation was carried out to grade the severity of the colon lesions. Immunofluorescence, western blot and quantitative polymerase chain reaction (RT-qPCR) were carried out on colon samples to evaluate PAR 2 expression. In addition, the transcriptional profile of cytokines and chemokines was evaluated using RT 2 Profiler™ PCR Array Horse Cytokines & Chemokines. Three out of the eight patients were euthanised due to clinical deterioration. Immunostaining for PAR 2 was observed in the enterocytes, intestinal glands and neurons of the submucosal and myenteric plexi. In the CVH horses, the expression of PAR 2 mesenger RNA (mRNA) did not differ significantly from that of the healthy animals; western blots of the mucosa of the colon tracts showed a clear band of the expected molecular weight for PAR 2 (~44 kDa) and a band smaller than the expected molecular weight for PAR 2 (25kDa), suggesting its activation. The gene expressions for C-X-C motif ligand 1 (CXCL1); interleukin 8 (IL8), macrophage inflammatory protein 2 beta (MIP-2BETA) were upregulated in the colic horses as compared with the colons of the healthy horses. Therefore, in the present study, the expression and activation of PAR 2 in the colons of horses in the presence of an inflammatory reaction like that occurring in those with spontaneous colon volvulus was confirmed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Lambertini, Zannoni, Romagnoli, Bombardi, Morini, Dondi, Bernardini, Forni, Rinnovati and Spadari.)

Proteinase activated receptor 4 (PAR 4 ) in the gastrointestinal tract is involved in the regulation of inflammation and pain pathways. The aim of the present study was to evaluate the distribution and expression of PAR 4 in the jejunum of healthy horses and in the pathologic tracts from horses undergoing surgery for herniation of the small intestine through the epiploic foramen. Eight healthy horses (Group H) and eight horses with epiploic hernia (Group EH) were included; the jejunum samples were collected at the slaughter or intraoperatively after enterectomy, respectively. To evaluate PAR 4 expression in sections of the jejunum, immunofluorescence, western blot and quantitative polymerase chain reaction (qRT-PCR) were performed. Immunohistochemistry of PAR 4 in the jejunum of the healthy horses showed that receptors are predominantly expressed in the immune cell population scattered throughout the lamina propria of the mucosa and in the submucosa. Quantitative PCR data demonstrated that PAR 4 mRNA was detectable in all of the samples analyzed without any difference between the H and the EH groups, however the PAR 4 protein level was significantly lower in the jejunums of the EH horses. In the Group EH horses, PAR 4 immunoreactivity was mainly expressed in the mast cells and was extensively distributed in the sierosa. In the lamina propria of mucosa of Group EH, leukocytes were less abundant than in Group H. In this study, the distribution and expression of PAR 4 in the jejunums of the healthy horses and in those with spontaneous occurring epiploic hernia was demonstrated., (Copyright © 2020 Lambertini, Bombardi, Zannoni, Bernardini, Dondi, Morini, Rinnovati, Spadari and Romagnoli.)