Cannabinoid receptor-2 expression in canine multicentric diffuse large B-cell lymphoma: An immunohistochemical, digital pathology and clinical analysis.

The cannabinoid 2 receptor (CB2R) is a crucial element of the endocannabinoid system (ECS), which is predominantly expressed on cells of the reticuloendothelial system. Alterations in CB2R expression have shown a prognostic role in various human neoplastic diseases and its expression has been studied in canine mast cell tumours (MCT). Canine diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma in dogs and has a variable clinical behaviour. Expression of CB2R was assessed by means of immunohistochemistry in fifteen dogs with proven histological diagnosis of DLBCL. A semiquantitative and quantitative assessment of immunoreactivity (IR) by digital analysis was performed in all cases. Our results indicate that CB2R expression is conserved in canine DLBCL but does not correlate with clinical outcome.

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.

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.

The KIT gene is associated with the english spotting coat color locus and congenital megacolon in Checkered Giant rabbits (Oryctolagus cuniculus).

The English spotting coat color locus in rabbits, also known as Dominant white spotting locus, is determined by an incompletely dominant allele (En). Rabbits homozygous for the recessive wild-type allele (en/en) are self-colored, heterozygous En/en rabbits are normally spotted, and homozygous En/En animals are almost completely white. Compared to vital en/en and En/en rabbits, En/En animals are subvital because of a dilated ("mega") cecum and ascending colon. In this study, we investigated the role of the KIT gene as a candidate for the English spotting locus in Checkered Giant rabbits and characterized the abnormalities affecting enteric neurons and c-kit positive interstitial cells of Cajal (ICC) in the megacolon of En/En rabbits. Twenty-one litters were obtained by crossing three Checkered Giant bucks (En/en) with nine Checkered Giant (En/en) and two en/en does, producing a total of 138 F1 and backcrossed rabbits. Resequencing all coding exons and portions of non-coding regions of the KIT gene in 28 rabbits of different breeds identified 98 polymorphisms. A single nucleotide polymorphism genotyped in all F1 families showed complete cosegregation with the English spotting coat color phenotype (θ=0.00 LOD  =75.56). KIT gene expression in cecum and colon specimens of En/En (pathological) rabbits was 5-10% of that of en/en (control) rabbits. En/En rabbits showed reduced and altered c-kit immunolabelled ICC compared to en/en controls. Morphometric data on whole mounts of the ascending colon showed a significant decrease of HuC/D (P<0.05) and substance P (P<0.01) immunoreactive neurons in En/En vs. en/en. Electron microscopy analysis showed neuronal and ICC abnormalities in En/En tissues. The En/En rabbit model shows neuro-ICC changes reminiscent of the human non-aganglionic megacolon. This rabbit model may provide a better understanding of the molecular abnormalities underlying conditions associated with non-aganglionic megacolon.

Ocular glioneuroma with medulloepitheliomatous differentiation in a goldfish (Carassius auratus).

An intraocular mass in the left eye causing chronic severe exophthalmia in an adult female goldfish (Carassius auratus) is described. The fish shared an aquarium with another goldfish found dead with gross and microscopic lesions consistent with mycobacteriosis. Histological examination of the left eye, histochemical (periodic acid-Schiff [PAS], Alcian blue, Ziehl-Neelsen) and immunohistochemical tests (glial fibrillary acidic protein, human neuronal protein, vimentin, and cytokeratin AE1/AE3) were carried out on the intraocular mass. Neoplastic cells forming an unencapsulated highly cellular proliferation partially covered by an intact corneal epithelium were stained with Alcian blue, which demonstrated an abundant hyaluronic acid-rich extracellular matrix. Multifocally, there were cyst-like dilatations bordered by neuroepithelial cells, which were PAS-positive. The complex neoplastic proliferation was composed of glial-like cells, neuronal-like cells (immunoreactive to glial fibrillary acidic protein and human neuronal protein, respectively) and neuroepithelium, which suggested a retinal origin.

Identification of neuron types in the submucosal ganglia of the mouse ileum.

The continuing and even expanding use of genetically modified mice to investigate the normal physiology and development of the enteric nervous system and for the study of pathophysiology in mouse models emphasises the need to identify all the neuron types and their functional roles in mice. An investigation that chemically and morphologically defined all the major neuron types with cell bodies in myenteric ganglia of the mouse small intestine was recently completed. The present study was aimed at the submucosal ganglia, with the purpose of similarly identifying the major neuron types with cell bodies in these ganglia. We found that the submucosal neurons could be divided into three major groups: neurons with vasoactive intestinal peptide (VIP) immunoreactivity (51% of neurons), neurons with choline acetyltransferase (ChAT) immunoreactivity (41% of neurons) and neurons that expressed neither of these markers. Most VIP neurons contained neuropeptide Y (NPY) and about 40% were immunoreactive for tyrosine hydroxylase (TH); 22% of all submucosal neurons were TH/VIP. VIP-immunoreactive nerve terminals in the mucosa were weakly immunoreactive for TH but separate populations of TH- and VIP-immunoreactive axons innervated the arterioles in the submucosa. Of the ChAT neurons, about half were immunoreactive for both somatostatin and calcitonin gene-related peptide (CGRP). Calretinin immunoreactivity occurred in over 90% of neurons, including the VIP neurons. The submucosal ganglia and submucosal arterioles were innervated by sympathetic noradrenergic neurons that were immunoreactive for TH and NPY; no VIP and few calretinin fibres innervated submucosal neurons. We conclude that the submucosal ganglia contain cell bodies of VIP/NPY/TH/calretinin non-cholinergic secretomotor neurons, VIP/NPY/calretinin vasodilator neurons, ChAT/CGRP/somatostatin/calretinin cholinergic secretomotor neurons and small populations of cholinergic and non-cholinergic neurons whose targets have yet to be identified. No evidence for the presence of type-II putative intrinsic primary afferent neurons was found.

Adenosine A1 and A3 receptor agonists inhibit nonadrenergic, noncholinergic relaxations in the guinea pig isolated trachea.

BACKGROUND: Adenosine affects the tone and reactivity of airways by activating specific membrane receptors, named A(1), A(2a), A(2b) and A(3). It affects cellular activities either directly by regulating membrane ion exchanges and polarization, or indirectly by modifying neurotransmitter release. OBJECTIVES: We assessed the effect of A(1) and A(3) receptor activation on electrically induced nonadrenergic, noncholinergic (NANC) relaxations in the guinea pig isolated trachea and the localization of A(1) and A(3) receptors in tracheal inhibitory neurons. METHODS: NANC responses at 3 Hz were evaluated in the presence of 2-chloro-N(6)-cyclopentyladenosine (CCPA), a selective A(1) agonist, and 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA), a selective A(3) agonist, before and after the administration of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), a selective A(1) antagonist, or 9-chloro-2-(2-furanyl)-5-((phenylacetyl)amino[1,2,4]triazolo[1,5-c])quinazoline (MRS 1220), a selective A(3) antagonist, respectively. For immunohistochemistry, tissues were exposed to antibodies to HuC/D, a general neuronal marker, neuronal nitric oxide synthase (nNOS), and A(1) or A(3) adenosine receptors and processed by indirect immunofluorescence. RESULTS: CCPA (10 nM-3 microM) inhibited NANC relaxations. DPCPX (10 nM) failed to antagonize the effect of CCPA, but inhibited per se NANC relaxations (range 0.1-100 nM). CCPA (10 nM-10 microM) contracted unstimulated tracheal preparations, an effect antagonized by 10 nM DPCPX, with a pK(B) value of 8.43. Cl-IB-MECA (10 nM-3 microM) inhibited NANC relaxations through a mechanism antagonized by MRS 1220 (100 nM). A(1)- and A(3)-positive neurons containing nNOS were detected in tracheal sections. CONCLUSIONS: Enogenous adenosine may induce airway hyperresponsiveness by inhibiting NANC relaxations via A(1) and A(3) receptors.

The distribution of P2X3 purine receptor subunits in the guinea pig enteric nervous system.

Adenosine 5'-triphosphate (ATP) excites 70-90% of enteric neurons through P2X type purine receptors, and is likely to be an enteric neurotransmitter. Recent studies indicate that the P2X2 subunit is expressed by specific subgroups of enteric neurons, and that there are enteric neurons that are responsive to ATP but lack this subunit. In the present work, we have investigated whether the P2X3 subunit is similarly localised to specific subgroups of neurons, and whether these are different from the P2X2 subunit-expressing neurons. The P2X3 subunit was localised by immunohistochemistry to nerve cells of the myenteric ganglia of the stomach, small and large intestines, and nerve cells of the submucosal ganglia in the small and large intestines of the guinea pig. All immunoreactivity was absorbed with the P2X3 receptor peptide against which the antiserum was raised. In myenteric ganglia of the ileum, P2X3 receptor immunoreactivity was in calretinin, enkephalin and nitric oxide synthase (NOS)-immunoreactive neurons. In submucosal ganglia, all calretinin-immunoreactive nerve cells were P2X3 receptor immunoreactive. In the submucosal ganglia of the ileum, 13 +/- 3% of neuropeptide Y (NPY)-immunoreactive neurons were also P2X3 receptor immunoreactive, whereas in the distal colon, almost all NPY-expressing nerve cells were P2X3 receptor immunoreactive. The localisation of the P2X3 subunit was largely distinct from that of the P2X2 subunit, although both subunits occur in some NOS neurons, where P2X2 and P2X3 subunits may form heteromeric receptors. Unlike the P2X2 subunit, the P2X3 subunit is not expressed in intrinsic sensory neurons in the ileum. It is concluded that the P2X3 receptor subunit is expressed in specific functional groups of neurons; the major types are excitatory and inhibitory muscle motor neurons, ascending interneurons and cholinergic secretomotor neurons.