Expression of Cannabinoid Receptors in the Trigeminal Ganglion of the Horse.

Cannabinoid receptors are expressed in human and animal trigeminal sensory neurons; however, the expression in the equine trigeminal ganglion is unknown. Ten trigeminal ganglia from five horses were collected post-mortem from an abattoir. The expression of cannabinoid receptors type 1 (CB1R) and type 2 (CB2R), and the cannabinoid-related receptors like transient receptor potential vanilloid type 1 (TRPV1), peroxisome proliferator-activated receptor gamma (PPARÉ£), and G protein-related receptor 55 (GPR55) in the trigeminal ganglia (TG) of the horse were studied, using immunofluorescence on cryosections and formalin-fixed paraffin-embedded (FFPE) sections. Neurons and glial cells were identified using fluorescent Nissl staining NeuroTrace® and an antibody directed against the glial marker glial fibrillary acidic protein (GFAP), respectively. Macrophages were identified by means of an antibody directed against the macrophages/microglia marker ionized calcium-binding adapter molecule 1 (IBA1). The protein expression of CB1R, CB2R, TRPV1, and PPARÉ£ was found in the majority of TG neurons in both cryosections and FFPE sections. The expression of GPR55 immunoreactivity was mainly detectable in FFPE sections, with expression in the majority of sensory neurons. Some receptors were also observed in glial cells (CB2R, TRPV1, PPARγ, and GPR55) and inflammatory cells (PPARγ and GPR55). These results support further investigation of such receptors in disorders of equine trigeminal neuronal excitability.

Endocannabinoid System Receptors at the Hip and Stifle Joints of Middle-Aged Dogs: A Novel Target for the Therapeutic Use of Cannabis sativa Extract in Canine Arthropathies.

The endocannabinoid system (ECS) has emerged as a potential therapeutic target in veterinary medicine due to its involvement in a wide range of physiological processes including pain, inflammation, immune function, and neurological function. Modulation of the ECS receptors has been shown to have anti-inflammatory, analgesic, and immunomodulatory effects in various animal models of disease, including dogs with osteoarthritis. The goal of this study was to identify and compare the cellular expression and distribution of cannabinoid receptor type 1 (CB1R) and type 2 (CB2R) and the cannabinoid-related G protein-coupled receptor 55 (GPR55) on the synovial cells of hip and stifle joints of seven dogs of different breeds without overt signs of osteoarthritis (OA). The synovial membranes of seven hips and seven stifle joints were harvested post mortem. The expression of the CB1R, CB2R, and GPR55 present in the synovial tissues was investigated using qualitative and quantitative immunofluorescence and Western blot (Wb) analysis. Synoviocytes of the stifle and hip joints expressed CB1R, CB2R, and GPR55 immunoreactivity (IR); no significant differences were observed for each different joint. Cannabinoid receptor 2- and GPR55-IR were also expressed by macrophages, neutrophils, and vascular cells. The ECS receptors were widely expressed by the synovial elements of dogs without overt signs of OA. It suggests that the ECS could be a target for the therapeutic use of Cannabis sativa extract in canine arthropathies.

Expression of cannabinoid (CB1 and CB2) and cannabinoid-related receptors (TRPV1, GPR55, and PPARα) in the synovial membrane of the horse metacarpophalangeal joint.

Background: The metacarpophalangeal joint undergoes enormous loading during locomotion and can therefore often become inflamed, potentially resulting in osteoarthritis (OA). There are studies indicating that the endocannabinoid system (ECS) modulates synovium homeostasis, and could be a promising target for OA therapy. Some cannabinoid receptors, which modulate proliferative and secretory responses in joint inflammation, have been functionally identified in human and animal synovial cells. Objective: To characterize the cellular distribution of the cannabinoid receptors 1 (CB1R) and 2 (CB2R), and the cannabinoid-related receptors transient receptor potential vanilloid type 1 (TRPV1), G protein-related receptor 55 (GPR55) and peroxisome proliferator-activated receptor alpha (PPARα) in the synovial membrane of the metacarpophalangeal joint of the horse. Animals: The dorsal synovial membranes of 14 equine metacarpophalangeal joints were collected post-mortem from an abattoir. Materials and methods: The dorsal synovial membranes of 14 equine metacarpophalangeal joints were collected post-mortem from an abattoir. The expression of the CB1R, CB2R, TRPV1, GPR55, and PPARα in synovial tissues was studied using qualitative and quantitative immunofluorescence, and quantitative real-time reverse transcriptase PCR (qRT-PCR). Macrophage-like (MLS) and fibroblast-like (FLS) synoviocytes were identified by means of antibodies directed against IBA1 and vimentin, respectively. Results: Both the mRNA and protein expression of the CB2R, TRPV1, GPR55, and PPARα were found in the synoviocytes and blood vessels of the metacarpophalangeal joints. The synoviocytes expressed the mRNA and protein of the CB1R in some of the horses investigated, but not in all. Conclusions and clinical importance: Given the expression of the CB1R, CB2R, TRPV1, GPR55, and PPARα in the synovial elements of the metacarpophalangeal joint, these findings encouraged the development of new studies supporting the use of molecules acting on these receptors to reduce the inflammation during joint inflammation in the horse.

The use of cannabidiol as a novel treatment for oral stereotypic behaviour (crib-biting) in a horse.

Behaviour is the response of living things to their environment and external stimulation, and is one of the parameters to be observed when assessing animal welfare. Any alteration from the conditions found in nature can lead to the occurrence of some specific behaviours, called stereotypies which are characterised as repetitive, consistent patterns of behaviour usually defined as having no apparent ultimate or proximal functions. It has been reported that once stabled or subjected to stressful activities, horses have more susceptibility of developing behavioural disturbances; therefore, behavioural disorders in horses are a strong indicator of poor welfare. Cannabis spp.-derived molecules have been studied under different medical conditions; the therapeutic potentials of phytocannabinoids are related to the effects of delta-9-tetrahydrocannabinol, cannabidiol (CBD), and other compounds. Cannabidiol has many activities within the central nervous system, such as anxiolytic, antidepressant, antipsychotic, anticonvulsant, and anti-inflammatory activities. Some studies have recently shown the potential and successful therapeutic use of phytocannabinoids in veterinary medicine. This clinical case report described a 22-year-old mare suffering from chronic crib-biting and wind-sucking, and the successful outcome of four weeks-therapy with CBD. This is the first report of the successful therapeutic use of phytocannabinoids in equine behavioural disorders.

Cannabinoid receptors in the inflammatory cells of canine atopic dermatitis.

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.

Distribution of Cannabinoid Receptors in Keratinocytes of Healthy Dogs and Dogs With Atopic Dermatitis.

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.

Ex vivo ultrasonographic and histological morphometry of small intestinal wall layers in horses.

Ultrasonographic morphometry of wall layers is commonly used in veterinary patients with suspected small intestinal disease, however published studies comparing this method with histopathology in horses are limited. This prospective, methods comparison study compared the qualitative and quantitative characteristics of small intestinal wall layers using ex vivo high-frequency ultrasound versus histopathology in a sample of 16 horses. Transverse section images of duodenum, distal jejunum, and ileum were acquired with a high-frequency linear transducer (7-15 MHz). Transverse histological cryosections were obtained at the same level. Appearance and measurements of the intestinal wall layers were assessed on the ultrasonographic and histological images. High-frequency scanning with the probe in close contact with the serosal surface of the equine intestinal wall allowed a clear and detailed definition of wall layers. A hyperechoic line was consistently detected within the tunica muscularis in all the intestinal tracts, corresponding histologically to the interface between its longitudinal and circular muscle layers. The overall trend of the values for wall layers thickness was comparable between ex vivo ultrasonography and histology. However, a poor agreement was found between the two methods for all layers. The ultrasonographic measurements were thicker compared to histological measurements, with the exception of the total wall and the muscular layer thicknesses. These layers were thinner on ultrasonography in the duodenum and in all the intestinal segments, respectively. Findings from the current study can be used as background for future ultrasonographic investigations of small intestinal diseases in horses.

Morphological variability of the atrioventricular valve cusps in the equine heart.

BACKGROUND: The morphometric studies of the atrioventricular valves are still limited in the horse. OBJECTIVES: To investigate the anatomy of the atrioventricular valves in the horse, focusing on the morphometric features of the valvular leaflets and the tendinous cords. We hypothesised that accessory leaflets occur commonly and exist as independent structures in the atrioventricular valves of the horse. STUDY DESIGN: Descriptive anatomical study. METHODS: Twenty normal hearts from slaughtered half-bred horses were used. The cardiac weight and circumference were recorded. The atrioventricular valves were exposed by excision of the atria, and the tricuspid and mitral annular diameters and circumferences were measured; the number of leaflets and tendinous cords for each atrioventricular ostium were then counted. The atrioventricular valves were isolated and the width, height and thickness of each leaflet were measured. RESULTS: In addition to the principal leaflets, accessory leaflets were identified in 39 of 40 cardiac valves, 2 to 6 accessory leaflets for the mitral valve and 1 to 4 for the tricuspid valve. All the accessory leaflets were separated from the adjacent leaflets at their insertion. They were narrower and thinner than the principal leaflets, and were attached to a single papillary muscle; 95% of the accessory leaflets had two tendinous cords shared with the adjacent leaflets while a minority (34%) had their own specific tendinous cord. MAIN LIMITATIONS: Lack of signalment data from the study population. CONCLUSIONS: Supernumerary leaflets occurred commonly in the atrioventricular valves of the horse and appeared as independent structures. The clinical relevance of increased numbers of commissures that result from accessory leaflets and their relationship with valvular regurgitation are currently unknown.

A Double Histochemical/Immunohistochemical Staining for the Identification of Canine Mast Cells in Light Microscopy.

Immunohistochemistry (IHC) is a widely used technique in diagnostic pathology, but the simultaneous analysis of more than one antibody at a time with different chromogens is rather complex, time-consuming, and quite expensive. In order to facilitate the identification of mast cells (MCs) during immunohistochemical analysis of membrane and/or nuclear markers, we propose a new staining method that includes the association of IHC and toluidine blue as a counterstain. To achieve this goal, we tested c-kit, Ki67, and cannabinoid receptor 2 on several cases of cutaneous canine mast cell tumors (MCTs), cutaneous mastocytosis, and atopic dermatitis. The results obtained show how this double staining technique, although limited to non-cytoplasmic markers and of little use in poorly differentiated MCTs in which MC metachromasia is hard to see, can be used during the evaluation of nuclear and/or membranous immunohistochemical markers in all canine cutaneous disorders, especially if characterized by the presence of a low number of MCs. It can help to evaluate those MCTs in which neoplastic MCs must be clearly distinguished from inflammatory cells that can infiltrate the tumor itself, in facilitating the calculation of the Ki67 index. Moreover, it can be used to study the expression of new markers in both animal and human tissues containing MCs and in MC disorders.

Cellular distribution of cannabinoid-related receptors TRPV1, PPAR-gamma, GPR55 and GPR3 in the equine cervical dorsal root ganglia.

BACKGROUND: The activation of cannabinoid and cannabinoid-related receptors by endogenous, plant-derived or synthetic cannabinoids may exert beneficial effects on pain perception. Of the cannabinoids contained in Cannabis sativa, cannabidiol (CBD) does not produce psychotropic effects and seems to represent a molecule having great therapeutic potential. Cannabidiol acts on a great number of cannabinoid and cannabinoid-related G-protein-coupled receptors and ionotropic receptors which have, to date, been understudied in veterinary medicine particularly in equine medicine. OBJECTIVES: To localise the cellular distribution of four putative cannabinoid-related receptors in the equine cervical dorsal root ganglia (DRG). STUDY DESIGN: A qualitative and quantitative immunohistochemical study. METHODS: The cervical (C6-C8) DRG of six slaughtered horses were obtained from a local slaughterhouse. The tissues were fixed and processed for immunohistochemistry, and the resulting cryosections were used to investigate immunoreactivity for the following putative CBD receptors: Transient receptor potential vanilloid type 1 (TRPV1), nuclear peroxisome proliferator-activated receptor gamma (PPARγ), and G protein-coupled receptors 55 (GPR55) and 3 (GPR3). RESULTS: Large percentages of neuronal cell bodies showed immunoreactivity for TRPV1 (80 ± 20%), PPARγ (100%), GPR55 (64 ± 15%) and GPR3 (63 ± 11%). The satellite glial cells (SGCs) were immunoreactive for TRPV1, PPARγ and GPR55. In addition, GPR55 immunoreactivity was expressed by DRG interneuronal macrophages. In addition, microglia cells were observed surrounding the neuron-SGC complex. MAIN LIMITATIONS: The limited number of horses included in the study. CONCLUSIONS: Cannabinoid-related receptors were distributed in the sensory neurons (TRPV1, PPARγ, GPR55 and GPR3), SGCs (TRPV1, PPARγ and GPR55), macrophages (GPR55) and other interneuronal cells (PPARγ and GPR55) of the equine DRG. Given the key role of DRG cellular elements and cannabinoid receptors in the pathophysiology of pain, the present findings provided an anatomical basis for additional studies aimed at exploring the therapeutic uses of non-psychotropic cannabinoid agonists for the management of pain in horses.

Localisation of Cannabinoid and Cannabinoid-Related Receptors in the Horse Ileum.

Colic is a common digestive disorder in horses and one of the most urgent problems in equine medicine. A growing body of literature has indicated that the activation of cannabinoid receptors could exert beneficial effects on gastrointestinal inflammation and visceral hypersensitivity. The localisation of cannabinoid and cannabinoid-related receptors in the intestine of the horse has not yet been investigated. The purpose of this study was to immunohistochemically localise the cellular distribution of canonical and putative cannabinoid receptors in the ileum of healthy horses. Distal ileum specimens were collected from six horses at the slaughterhouse. The tissues were fixed and processed to obtain cryosections which were used to investigate the immunoreactivity of canonical cannabinoid receptors 1 (CB1R) and 2 (CB2R), and three putative cannabinoid-related receptors: nuclear peroxisome proliferator-activated receptor-alpha (PPARα), transient receptor potential ankyrin 1 and serotonin 5-HT1a receptor (5-HT1aR). Cannabinoid and cannabinoid-related receptors showed a wide distribution in the ileum of the horse. The epithelial cells showed immunoreactivity for CB1R, CB2R and 5-HT1aR. Lamina propria inflammatory cells showed immunoreactivity for CB2R and 5-HT1aR. The enteric neurons showed immunoreactivity for CB1R, transient receptor potential ankyrin 1 and PPARα. The enteric glial cells showed immunoreactivity for CB1R and PPARα. The smooth muscle cells of the tunica muscularis and the blood vessels showed immunoreactivity for PPARα. The present study represents a histological basis which could support additional studies regarding the distribution of cannabinoid receptors during gastrointestinal inflammatory diseases as well as studies assessing the effects of non-psychotic cannabis-derived molecules in horses for the management of intestinal diseases.

Nitrergic and Substance P Immunoreactive Neurons in the Enteric Nervous System of the Bottlenose Dolphin (Tursiops truncatus) Intestine.

Compared with other mammals, the digestive system of cetaceans presents some remarkable anatomical and physiological differences. However, the neurochemical features of the enteric nervous system (ENS) in these animals have only been described in part. The present study gives a description of the nitrergic and selected peptidergic systems in the myenteric plexus (MP) and submucosal plexus (SMP) of the intestine of the bottlenose dolphin (Tursiops truncatus). The distribution and morphology of neurons immunoreactive (IR) for the neuronal nitric oxide synthase (nNOS) and Substance P (SP) were immunohistochemically studied in formalin-fixed specimens from the healthy intestine of three animals, and the data were compared with those described in the literature on other mammals (human and non-human). In bottlenose dolphins, the percentages of nitrergic neurons (expressed as median and interquartile range-IQR) were 28% (IQR = 19-29) in the MP and 1% (IQR = 0-2) in the SMP, while the percentages of SP-IR neurons were 31% (IQR = 22-37) in the MP and 41% (IQR = 24-63) in the SMP. Although morphological features of nNOS- and SP-IR neurons were similar to those reported in other mammals, we found some noticeable differences in the percentages of enteric neurons. In fact, we detected a lower proportion of nNOS-IR neurons in the SMP and a higher proportion of SP-IR neurons in the MP compared to other mammals. To the best of the authors' knowledge, this study represents the first description and quantification of nNOS-IR neurons and the first quantification of SP-IR neurons in the intestine of a cetacean species. As nNOS and SP are important mediators of intestinal functions and the nitrergic population is an important target for many neuroenteropathies, data obtained from a healthy intestine provide a necessary basis to further investigate and understand possible functional differences and motor intestinal dysfunctions/alterations in these special mammals.

Design-based stereological study of the guinea-pig (Cavia porcellus) cerebellum.

Guinea pigs have proved useful as experimental animal models in studying cerebellar anatomical and structural alterations in human neurological disease; however, they are also currently acquiring increasing veterinary interest as companion animals. The morphometric features of the normal cerebellum in guinea pigs have not been previously investigated using stereology. The objective of the present work was to establish normal volumetric and quantitative stereological parameters for cerebellar tissues in guinea pigs, by means of unbiased design-based stereology. Cerebellar total volume, gray and white matter volume fractions, molecular and granular layers volume fractions, cerebellar surface area, Purkinje cellular and nuclear volumes, and the Purkinje cell total count were stereologically estimated. For this purpose, cerebellar hemispheres from six adult male guinea pigs were employed. Isotropic, uniform random sections were obtained by applying the orientator method, and subsequently processed for light microscopy. The cerebellar total volume, the white and grey matter volume fractions, and the molecular and granular layer volumes were estimated using the Cavalieri's principle and the point counting system. The cerebellar surface area was estimated through the use of test lines; Purkinje cellular and nuclear volumes were analysed using the nucleator technique, whereas the Purkinje cell total count was obtained by means of the optical disector technique. The mean ± standard deviation total volume of a guinea-pig cerebellar hemisphere was 0.11 ± 0.01 cm3 . The mean volumetric proportions occupied by the gray and white matters were, respectively, 78.0 ± 2.6% and 22.0 ± 2.6%, whereas their mean absolute volumes were found to be 0.21 ± 0.02 cm3 and 0.059 ± 0.006 cm3 . The volumes of the molecular and granular layers were estimated at 112.4 ± 20.6 mm3 and 104.4 ± 7.3 mm3 , whereas their mean thicknesses were calculated to be 0.184 ± 0.020 mm and 0.17 ± 0.02 mm. The molecular and granular layers accounted for 40.7 ± 3.9% and 37.4 ± 1.8% of total cerebellar volume respectively. The surface area of the cerebellum measured 611.4 ± 96.8 mm2 . Purkinje cells with a cellular volume of 3210.1 µm3 and with a nuclear volume of 470.9 µm3 had a higher incidence of occurrence. The mean total number of Purkinje cells for a cerebellar hemisphere was calculated to be 253,090 ± 34,754. The morphometric data emerging from the present study provide a set of reference data which might prove valuable as basic anatomical contribution for practical applications in veterinary neurology.

The Influence of Skin Thickness on Flash Glucose Monitoring System Accuracy in Dogs with Diabetes Mellitus.

A flash glucose monitoring system (FGMS) has been validated for use in diabetic dogs. However, it is unknown whether skin thickness affects FGMS measurements. The aim of this study was to evaluate whether FGMS accuracy is affected by skin thickness. Fourteen client-owned diabetic dogs on insulin treatment were prospectively enrolled in the study. The dogs were divided into two groups according to their ultrasound-measured skin thickness: dogs with skin thickness < 5 mm (Group 1) and dogs with skin thickness > 5 mm (Group 2). On days 1, 7 and 14, glucose curves were obtained simultaneously using the FGMS and a validated portable blood glucose meter. Paired measurements were used to calculate the mean bias and to determine accuracy according to ISO 15197:2013 criteria. The mean bias was significantly inversely correlated (p = 0.02; r = -0.6) with the mean skin thickness. Clinical accuracy was observed only in Group 2, with 99% of the results in zone A + B of the Parkes consensus error grid analysis. In conclusion, skin thickness seems to affect FGMS measurements, and the device is accurate in dogs with thicker skin (>5 mm); in dogs with thin skin (<5 mm), the clinical accuracy is low, and the results should be interpreted with caution.

Cannabinoid and Cannabinoid-Related Receptors in the Myenteric Plexus of the Porcine Ileum.

An important piece of evidence has shown that molecules acting on cannabinoid receptors influence gastrointestinal motility and induce beneficial effects on gastrointestinal inflammation and visceral pain. The aim of this investigation was to immunohistochemically localize the distribution of canonical cannabinoid receptor type 1 (CB1R) and type 2 (CB2R) and the cannabinoid-related receptors transient potential vanilloid receptor 1 (TRPV1), transient potential ankyrin receptor 1 (TRPA1), and serotonin receptor 5-HT1a (5-HT1aR) in the myenteric plexus (MP) of pig ileum. CB1R, TRPV1, TRPA1, and 5-HT1aR were expressed, with different intensities in the cytoplasm of MP neurons. For each receptor, the proportions of the immunoreactive neurons were evaluated using the anti-HuC/HuD antibody. These receptors were also localized on nerve fibers (CB1R, TRPA1), smooth muscle cells of tunica muscularis (CB1R, 5-HT1aR), and endothelial cells of blood vessels (TRPV1, TRPA1, 5-HT1aR). The nerve varicosities were also found to be immunoreactive for both TRPV1 and 5-HT1aR. No immunoreactivity was documented for CB2R. Cannabinoid and cannabinoid-related receptors herein investigated showed a wide distribution in the enteric neurons and nerve fibers of the pig MP. These results could provide an anatomical basis for additional research, supporting the therapeutic use of cannabinoid receptor agonists in relieving motility disorders in porcine enteropathies.

Degenerative Myelopathy in Hovawart Dogs: Molecular Characterization, Pathological Features and Accumulation of Mutant Superoxide Dismutase 1 Protein.

Degenerative myelopathy (DM) is an adult-onset, progressive neurological disease affecting several breeds of dog. Homozygosity or compound heterozygosity for the canine superoxide dismutase 1 (SOD1) gene mutations, possibly modulated by the modifier SP110 locus, are associated with a high risk for DM. Although the pathophysiological mechanisms are largely unknown, a role for mutant SOD1 in causing neuronal degeneration has been postulated. Three Hovawart dogs, 9-12 years of age, developed slowly progressive incoordination and weakness of the pelvic limbs leading to non-ambulatory flaccid paraparesis and muscle atrophy. Neuropathological lesions comprised axonal degeneration and loss of ascending and descending spinal pathways, which were most severe in the mid- to caudal thoracic segments. Accumulation of mutant SOD1 protein in neurons and reactive astrocytes was demonstrated by immunolabelling with the 16G9 antibody against the mutant SOD1 protein (p.E40K amino acid substitution). All three dogs were homozygous for the c.118A allele, but none had the SP110 'risk' haplotype, suggesting a weak association of SP110 with the onset of DM in this breed. Our data suggest that the Hovawart breed is predisposed to the SOD1:c.118G>A mutation, which is associated with the development of DM. Prevention of DM could be achieved with the help of strategies based on epidemiological and genetic testing.

Localisation of cannabinoid and cannabinoid-related receptors in the equine dorsal root ganglia.

BACKGROUND: Growing evidence recognises cannabinoid receptors as potential therapeutic targets for pain. Consequently, there is increasing interest in developing cannabinoid receptor agonists for treating pain. As a general rule, to better understand the actions of a drug, it would be of extreme importance to know the cellular distribution of its specific receptors. The localisation of cannabinoid receptors in the dorsal root ganglia of the horse has not yet been investigated. OBJECTIVES: To localise the cellular distribution of canonical and putative cannabinoid receptors in the equine cervical dorsal root ganglia. STUDY DESIGN: Qualitative and quantitative immunohistochemical study. METHODS: Cervical (C6-C8) dorsal root ganglia were collected from six horses (1.5 years of age) at the slaughterhouse. The tissues were fixed and processed to obtain cryosections which were used to investigate the immunoreactivity of canonical cannabinoid receptors 1 (CB1R) and 2 (CB2R), and for three putative cannabinoid-related receptors: nuclear peroxisome proliferator-activated receptor alpha (PPARα), transient receptor potential ankyrin 1 (TRPA1) and serotonin 5-HT1a receptor (5-HT1aR). RESULTS: The neurons showed immunoreactivity for CB1R (100%), CB2R (80% ± 13%), PPARα (100%), TRPA1 (74% ± 10%) and 5-HT1aR (84% ± 6%). The neuronal satellite glial cells showed immunoreactivity for CB2R, PPARα, TRPA1 and 5-HT1aR. MAIN LIMITATIONS: The low number of horses included in the study. CONCLUSIONS: This study highlighted the expression of cannabinoid receptors in the sensory neurons and glial cells of the dorsal root ganglia. These findings could be of particular relevance for future functional studies assessing the effects of cannabinoids in horses to manage pain.

Localization of the Serotonin Transporter in the Dog Intestine and Comparison to the Rat and Human Intestines.

Serotonin is crucial in gastrointestinal functions, including motility, sensitivity, secretion, and the inflammatory response. The serotonin transporter (SERT), responsible for serotonin reuptake and signaling termination, plays a prominent role in gastrointestinal physiology, representing a promising therapeutic target in digestive disorders. Serotonin transporter expression has been poorly investigated in veterinary medicine, under both healthy and pathological conditions, including canine chronic enteropathy, in which the serotonin metabolism seems to be altered. The aim of the present study was to determine the distribution of SERT immunoreactivity (SERT-IR) in the dog intestine and to compare the findings with those obtained in the rat and human intestines. Serotonin transporter-IR was observed in canine enterocytes, enteric neurons, lamina propria cells and the tunica muscularis. Data obtained in dogs were consistent with those obtained in rats and humans. Since the majority of the serotonin produced by the body is synthesized in the gastrointestinal tract, SERT-expressing cells may exert a role in the mechanism of serotonin reuptake.

Expression of cannabinoid and cannabinoid-related receptors in the oral mucosa of healthy cats and cats with chronic gingivostomatitis.

OBJECTIVES: Feline chronic gingivostomatitis (FCGS) is an oral disease. Cats with FCGS experience intense oral pain. Some cats remain refractory to current therapies based on dental extraction and adjuvant medical treatment; it is therefore necessary to investigate alternative therapeutic targets involved in inflammatory mechanisms and pain, namely the endocannabinoid system (ECS). The present study investigated the expression of cannabinoid receptors type 1 (CB1R) and 2 (CB2R), and cannabinoid-related receptors G protein-coupled receptor 55 (GPR55), transient receptor potential ankyrin 1 (TRPA1) and serotonin 1a receptor (5-HT1aR), in the oral mucosa of healthy cats to determine whether there was altered expression and distribution in cats with FCGS. METHODS: Samples of caudal oral mucosa were collected from eight control cats (CTRL cats) and from eight cats with FCGS (FCGS cats). Tissue samples were processed using an immunofluorescence assay with cat-specific antibodies, and the immunolabelling of the receptors studied was semiquantitatively evaluated. RESULTS: The mucosal epithelium of the CTRL cats showed CB1R, TRPA1 and 5-HT1aR immunoreactivity (IR), while CB2R and GPR55 IR were generally not expressed. In the CTRL cats, the subepithelial inflammatory cells expressed CB2R, GPR55 and 5-HT1aR IR. In the FCGS cats, all the receptors studied were markedly upregulated in the epithelium and inflammatory infiltrate. CONCLUSIONS AND RELEVANCE: Cannabinoid and cannabinoid-related receptors are widely expressed in the oral mucosa of healthy cats and are upregulated during the course of FCGS. The presence of cannabinoid and cannabinoid-related receptors in healthy tissues suggests the possible role of the ECS in the homeostasis of the feline oral mucosa, while their overexpression in the inflamed tissues of FCGS cats suggests the involvement of the ECS in the pathogenesis of this disease, with a possible role in the related inflammation and pain. Based on the present findings, ECS could be considered a potential therapeutic target for patients with FCGS.

ACE2 Expression in the Cat and the Tiger Gastrointestinal Tracts.

Angiotensin-converting enzyme 2 (ACE2) has been identified as the functional receptor for Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2). It has been identified in the human gastrointestinal tract (GIT), and SARS-CoV-2 has been isolated in human and animal fecal samples. The aim of the present study was to investigate the expression of ACE2 in the gastrointestinal tract of domestic (cat) and wild (tiger) felines. Samples of the pylorus, duodenum, and distal colon were collected from six cats and one tiger. The tissues were processed for immunofluorescence assay with an anti-human ACE2 antibody. Angiotensin-converting enzyme 2 was widely expressed in the gastrointestinal mucosa of the cats and the tiger. In both the species, ACE2-immunoreactivity (ACE2-IR) was expressed by the mucosal epithelial cells of the GIT and by the enteric neurons. In the cats, ACE2-IR was also expressed by the smooth muscle cells of the blood vessels and the tunica muscularis. The expression of the ACE2 receptor in enteric neurons may support the potential neurotropic properties of SARS-CoV-2. Although the evidence of ACE2-IR in the feline GIT does not necessarily indicate the possibility of viral replication and SARS-CoV-2 spread with stool, the findings in the present study could serve as an anatomical basis for additional studies considering the risk of the SARS-CoV-2 fecal-oral transmission between cats/felids, and between cats/felids and humans.