Distribution of the neurokinin-1 receptor in equine intestinal smooth muscle.

REASON FOR PERFORMING STUDY: Tachykinins have profound effects on equine intestinal motility, but the distribution of the neurokinin receptors (NKRs) through which they act is unknown. This study reports the distribution of one of these receptors, the neurokinin-1 receptor (NK1R), in smooth muscle throughout the equine intestinal tract. OBJECTIVES: To quantify the distribution of the NK1R, based upon mRNA expression, in smooth muscle of different regions of the equine intestinal tract. METHODS: Nine regions of the intestinal tract were sampled in 5 mature horses. Total RNA was isolated from smooth muscle and reverse transcribed; NK1R mRNA was then quantified using real-time PCR. RESULTS: NK1R mRNA was found at all levels of the sampled intestinal tract. The smooth muscle of the proximal small intestine and the ventral colon exhibited the highest level of NK1R mRNA expression in the equine intestinal tract. CONCLUSIONS: Tachykinins probably affect intestinal contractility and propulsion in the proximal small intestine and in the ventral colon.

Mast cell density and substance P-like immunoreactivity during the initiation and progression of lung lesions in ovine Mannheimia (Pasteurella) haemolytica pneumonia.

To determine the density of mast cells (MCs) and the extent of substance P (SP) immunoreactivity during initiation and progression of pneumonic pasteurellosis (PP), 18 lambs were inoculated intrabronchially with Mannheimia (Pasteurella) haemolytica or saline, and lung tissue was collected at 1, 15 and 45 days post-inoculation (n=3, each group). Additionally, the left (non-inoculated) contralateral lungs in bacteria-inoculated animals were collected as controls. At 1 day after bacterial inoculation the lungs had typical M. haemolytica lesions. These pneumonic lesions had fewer numbers of MCs and reduced histamine content. Macrophages infiltrating some of the inflamed areas were strongly immunoreactive for SP. At 15 days, MCs remained scarce at sites where lung damage persisted, i.e. pyogranulomatous foci, but were increased in number in areas of interstitial damage. Pulmonary ganglion neurons were strongly immunoreactive for SP. By 45 days the fibrosing changes became more defined as pleural fibrosis, fibrosing alveolitis, alveolar epithelial hyperplasia and bronchiolitis obliterans. These lungs had increased numbers of MCs, but histamine content was not different from saline- and non-inoculated left lungs. Substance P immunoreactivity occurred only in nerves and was scarce and mild. This work demonstrates that MC density decreases initially with PP, but increases with progression of PP. SP fibres tend to be decreased during the initiation and at 45 days of PP, but other cells, such as macrophages and neuronal ganglion cells, produce substance P during progression of PP and thereby constitute an additional source of substance P.

Flow cytometric analysis of colonic and small intestinal mucosal lymphocytes obtained by endoscopic biopsy in the healthy dog.

Flow cytometric analysis of the lymphocyte population of the gut could provide useful information on the immune cells present in the gut that would not be easily obtained in tissue sections. However, little is known of the normal lymphocyte population in the canine gut as determined by flow cytometry, which allows for simultaneous staining of multiple cell surface antigens and identification of specific lymphocytic subsets. Therefore, intraepithelial lymphocytes were obtained from biopsies of the healthy canine proximal small intestine and colon taken with an endoscope, and flow cytometric analysis was used to characterize the lymphocyte subsets present. Endoscopic biopsy of the intestine is a minimally invasive technique commonly used for diagnostic purposes. Although CD3+ lymphocytes were the most abundant subset in both colon and small intestine, CD3+/CD8- lymphocytes predominated in the proximal small intestine, whereas CD3+/CD8+ lymphocytes did in the colon. Canine CD8+ intraepithelial lymphocytes were predominantly CD8alphabeta+ in both small intestine and colon. CD4+ intraepithelial lymphocytes were always much less numerous than CD8+ intraepithelial lymphocytes. As in man, a majority of intraepithelial lymphocytes expressed the T-cell receptor, TCRalphabeta, but TCRgammadelta was expressed by a third of intraepithelial T-cells in the proximal small intestine, and approximately 15% of those in the colon. Very few CD21+ lymphocytes were detected in samples of healthy canine colon and small intestinal intraepithelial cells. We have showed that canine intraepithelial lymphocytes are regionally specialized, and that those from the small intestine are unique in comparison to those of other species such as man and rodents due to the large numbers of CD3+/CD8- intraepithelial lymphocytes. This study provides a baseline for comparison with intraepithelial lymphocytes obtained from canine patients with intestinal disease.

Dihydrocapsaicin treatment depletes peptidergic nerve fibers of substance P and alters mast cell density in the respiratory tract of neonatal sheep.

In the present study we administered dihydrocapsaicin (DHC) to neonatal lambs to deplete C-fibers of neuropeptides. We measured the density of substance P (SP)-fibers in nasal septum to assess the effectiveness of the treatment at 3, 9, and 21 days. The numbers of mast cells in the upper and lower respiratory tract were determined at the same time points and histamine content was determined from lung tissue. DHC treatment depleted SP-fibers for up to the 21 day time point. This depletion was estimated as 85% in comparison with controls. In vehicle-treated lambs, the density of SP-fibers decreased progressively with age, but not to the degree of DHC-treated lambs whose SP-fibers were depleted from the initial 3-day measurement. In both, vehicle- and DHC-treated lambs, numbers of mast cells increased progressively with time; however, the density of mast cells was augmented in the entire respiratory tract of DHC-treated animals. Apparently, DHC treatment exerts a single and initial effect in increasing mast cells whereas time maintains a continuous influence; both factors exert their influence independently. Despite large numbers of mast cells in DHC-treated animals, histamine content in the lung had similar levels as controls. Our study provides fundamental data for a better understanding of conditions that may influence defense mechanisms dependent on the mast cell-nerve axis in the respiratory tract.

Distribution of substance P binding sites in equine airways.

Autoradiography with [125I]-Bolton Hunter substance P ([I]-BHSP) was used to detect substance P binding sites in the equine lung. Specific [I]-BHSP binding sites were very dense over small bronchial vessels, tracheobronchial glands and airway epithelium in large and small airways. The density of [I]-BHSP binding sites over airway smooth muscle was much lower than in the preceding tissues. Competition with an excess of either a specific neurokinin 1 receptor agonist, or a specific neurokinin 2 receptor agonist indicated that most specific [I]-BHSP binding sites in the equine lung represent neurokinin 1 receptors. The receptor-mediated effects of substance P in the equine lung are most likely to involve regulation of vascular tone and airway secretions based upon the density of specific [I]-BHSP binding sites in these tissues. Activation of intrapulmonary afferent nerves containing Substance P by noxious stimuli such as inhaled allergens or irritants may lead to increased mucus secretion and decreased airway diameter due to vascular congestion.

Flow cytometric analysis of canine colonic mucosal lymphocytes from endoscopically obtained biopsy specimens.

OBJECTIVE: To validate use of canine colonic biopsy specimens obtained via endoscopy as a source of mucosal lymphocytes (ML) for flow cytometric analysis. SAMPLE POPULATION: Mucosal biopsy specimens from 10 adult dogs. PROCEDURE: Mucosal lymphocyte subsets obtained from excised colon were compared with ML subsets obtained from biopsy specimens obtained by use of an endoscopic forceps (6 dogs). Endoscopic colonic biopsy specimens from 4 other dogs were used to define whether obtained ML were predominantly of intraepithelial or lamina propria origin. Mucosal lymphocytes were isolated and labeled, using commercially available monoclonal antibodies directed against canine cell surface antigens. Lymphocyte subsets (cytotoxic or helper T cells; B cells) were determined by use of flow cytometric analysis. RESULTS: A large number of viable ML was obtained after dissociation of the colonic epithelium from excised colon (45.5 + 21.5 X 10(6)) and endoscopic (7.2+/-3.4 X 10(6)) biopsy specimens. Lymphocyte subsets obtained with both methods were identical for each dog and consisted predominantly of intraepithelial lymphocytes, with some lymphocytes from the lamina propria. Collagenase digestion of excised colon also yielded a large number of viable lymphocytes from the lamina propria (56.7+/-20.4 X 10(6)), but collagenase digestion of endoscopic biopsy specimens was less rewarding. CONCLUSION AND CLINICAL RELEVANCE: A representative sample of viable intraepithelial ML is obtainable from endoscopic biopsy specimens. Flow cytometric analysis, a minimally invasive technique, can be used to study ML of client-owned animals.

Tachykinin receptors in the equine pelvic flexure.

Tachykinins, of which substance P (SP) is the prototype, are neuropeptides which are widely distributed in the nervous systems. In the equine gut, SP is present in enteric nerves and is a powerful constrictor of enteric muscle; in other species, SP is also known to have potent vasodilatory and pro-inflammatory effects. The specific effects of SP are determined by the subtype of receptor present in the target tissue. There are 3 known subtypes of tachykinin receptors, distinguished by their relative affinities for SP and other tachykinins. The distribution of SP binding sites in the equine pelvic flexure was determined using 125I-Bolton Hunter SP (I-BHSP) autoradiography. Most I-BHSP binding sites were determined to be saturable and specific, therefore presumably representing tachykinin receptors. The greatest degree of I-BHSP binding occurred over very small vessels, and over the muscularis mucosae; I-BHSP binding was also intense over the circular muscle of the muscularis externa and mucosa, and present, although less intense, over the longitudinal muscle of the muscularis externa. Competition of I-BHSP with specific receptor agonists for binding sites in the equine pelvic flexure were used to determine the subtypes of tachykinin receptors present. The neurokinin-1 receptor subtype predominated in the equine pelvic flexure, followed by the neurokinin-3 receptor subtype.

Ultrasonographic pregnancy detection and inhalation anesthesia in the gray short-tailed opossum (Monodelphis domestica).

Female gray short-tailed opossums (Monodelphis domestica) with known breeding dates were anesthetized with isoflurane for ultrasonographic pregnancy detection. Beginning on gestational day 9, gravid females could be identified using a 9-MHz mechanical sector-scanning transducer equipped with a standoff pad. Fluid-filled vesicles 3 to 4 mm in diameter were seen within the thick-walled uteri on gestational days 9 and 10. Visualization revealed loss of individual vesicles, with replacement by thick, irregular uterine walls and some free luminal fluid by gestational days 12 and 13. On the basis of subsequent birth of pups, sonographic diagnosis of pregnancy was accurate in 27 of 28 oppossums examined.

Ontogeny of androgen receptor-like immunoreactivity in the reproductive tract of male Monodelphis domestica.

The distribution of nuclear androgen receptor-like immunoreactivity was studied in the reproductive tract of the developing and adult gray short-tailed Brazilian opossum (Monodelphis domestica), using the well-characterized rabbit polyclonal androgen receptor antibody, PG21. Androgen receptor-like immunoreactivity was first detected on the fifth day of postnatal age, in the mesenchymal tissues of the ductus deferens, gubernaculum testis, inguinal, and scrotal areas; the urogenital sinus; and the genital tubercle. Androgen receptor-like immunoreactivity was first seen in the interstitial cells of the epididymis at 45 days of age; the testes developed androgen receptor-like immunoreactivity at 60 days of age. The epithelium of prostatic glands contained androgen receptor-like immunoreactivity only in the adult. The presence of androgen receptor-like immunoreactivity during development correlated well with the known androgen dependence of the differentiation of most reproductive organs.

Anatomy of the distal interphalangeal joint of the mature horse: relationships with navicular suspensory ligaments, sensory nerves and neurovascular bundle.

The anatomy of the distal interphalangeal (DIP) joint in the adult horse is described in relationship to the suspensory ligaments of the navicular bone, the neurovascular bundle and the sensory nerves to these periarticular regions. Using polymer plastic injections, the synovial cavity of the DIP joint was observed to have a complex relationship to the proximal suspensory or collateral sesamoidean ligament (CSL) of the navicular bone with the cavity forming cranial and caudal compartments around the CSL abaxially. Sensory nerves, as identified by peptide immunocytochemistry and silver/gold chloride axonal impregnation, were present superficially throughout the dorsal and palmar parts of the CSL, the distal sesamoidean impar ligament and in the periarticular connective tissues. These anatomical observations provide support for the idea that a DIP joint injection of local anaesthetic cannot be considered to be selective for only the joint surfaces, but must be considered also to desensitise much of the navicular suspensory apparatus, navicular bone and proximal intramedullary portions of the distal phalanx. However, while a DIP joint injection is not selective for only the joint surfaces, we believe that it is useful in terms of the diagnosis of painful sensations arising from parts of the navicular bone, the suspensory apparatus and proximal positions of the distal phalanx. These anatomical findings are discussed in terms of the potential effects of local anaesthesia injected into the DIP joint with known clinical and necropsy observations of Dyson and Kidd (1993).

Sensory innervation of the navicular bone and bursa in the foal.

The sensory innervation of the navicular bone (os sesamoideum distale) and its suspensory ligaments [ligamenta sesamoidea collateralia (CSL) and ligamentum sesamoideum distale impar or distal sesamoidean impar (DS-impar) ligament] and the navicular bursa (podotrochlearis) was examined in the neonatal foal using immunocytochemistry. With antisera raised to substance P (SP) and human calcitonin gene-related peptide (CGRP), immunoreactive nerves were demonstrated to innervate the CSL and navicular bursa. Within CSL, and SP- and CGRP-like nerves were present in the synovial lining of the navicular bursa, appearing to reach the surface lining. These nerves appeared to enter the CSL and navicular bursa via the abaxial regions of the foot. Both peptides were present in the deep digital flexor tendon (DDf) along the palmar border of the navicular bursa, as well as in the DS-impar ligament. More nerve fibres were present in the dorsal part of CSL bordering the distal interphalangeal joint than was observed palmarly in CSL along the navicular bursa. Both peptides were observed to innervate the cartilage canals within the navicular bone. In terms of relative densities of immunoreactive SP- and CGRP-like peptides, the CSL dorsally and the DS-impar ligament had the highest relative densities of nerve fibres followed by the navicular bone, the palmar aspect of CSL and the DDf tendon bordering the navicular bursa. These results are discussed in relationship to local anaesthetic injections into the navicular bursa.

Distribution of SP- and CGRP-like immunoreactive nerve fibers in the lower respiratory tract of neonatal foals: evidence for loss during development.

The lungs of neonatal foals contain many nerves immunoreactive for substance P and calcitonin gene-related peptide. These nerves are closely associated with the epithelium, bronchial and pulmonary vessels and the airway smooth muscle of all intrathoracic airways, including non-cartilaginous bronchioles. Activation of sensory nerves in the respiratory epithelium could thus potentially affect, via local axon reflexes, vascular and respiratory smooth muscle in neonatal equine airways. Nerves immunoreactive for these peptides are much more widely distributed within the lung than in adult horses; they may thus play a trophic role before birth, or contribute to the post-natal adaptation to breathing.

Substance P and calcitonin gene-related peptide-like immunoreactive nerve fibers in lungs from adult equids.

Distribution of pulmonary nerves immunoreactive for either substance P or calcitonin gene-related peptide was determined, using immunohistochemical methods on healthy lungs from adult equids. The overall patterns of distribution of substance P and calcitonin gene-related peptide-like immunoreactivity were similar. Distribution of immunoreactive nerves was not uniform throughout the lungs; nerve fibers immunoreactive for these peptides were more frequently observed near the hilus of the lung than in the caudal lobes or in the periphery of the lung. Nerve fibers immunoreactive for substance P or calcitonin gene-related peptide were most abundant in the lamina propria of the trachea and larger airways, particularly within and directly below the airway epithelium; they were also frequently associated with bronchial and pulmonary vessels. Presence of nerve fibers immunoreactive for substance P and calcitonin gene-related peptide in peribronchial neural ganglia indicated that these sensory nerves may modulate parasympathetic regulation of pulmonary function. Nerve fibers immunoreactive for substance P and calcitonin gene-related peptide were, therefore, well placed to detect inhaled agents and to contribute to the pulmonary response to irritants and pathogens.

A silver-impregnation and immunocytochemical study of innervation of the distal sesamoid bone and its suspensory ligaments in the horse.

The innervation of the navicular bone (os sesamoideum distale) and its suspensory ligaments (ligamenta sesamoidea collateralia) (CSL) or proximal suspensory ligament and the ligamentum sesamoideum distale impar or the distal sesamoidean impar ligament (DS-impar ligament) was examined using combined anatomical techniques of silver impregnation and immunocytochemistry. Silver impregnation studies revealed an abundance of nerve fibres present in both the CSL and DS-impar ligament with the latter having relatively more nerve fibres. These silver-impregnated nerves coursed parallel to and were associated with the vasculature rather than appearing to innervate the vessels. Immunocytochemistry identified several sensory-related neuropeptides (calcitonin gene-related peptide (CGRP), substance P (SP) and neurokinin A (NKA)) in the nerves of the navicular bone and suspensory ligaments. More peptidergic nerves were evident within the synovial membrane and loose connective tissue in the dorsal part than in the palmar aspect of the CSL. In the CSL along the synovial membrane bordering the distal interphalangeal joint, the CGRP, SP and NKA were present in the nerves of vessels as well as the intimal layer of the distal interphalangeal joint. In the DS-impar ligament, there were many more nerves innervating vessels and the synovial membrane between the navicular bone and the third phalanx than were present in these structures in the CSL. Nerves with all 3 peptides entered the navicular bone via the proximal border and the distal groove to innervate the perichondrium, trabeculae and osteons. SP-like nerves also innervated the cortical bone underlying the articular cartilage. We suggest that these sensory nerve peptides contribute to the pathology of the navicular syndrome. The distribution of the nerves in the CSL and the DS-impar ligament could explain the clinical effects of local anaesthetics injected into the distal interphalangeal joint.

Immunocytochemical and dye distribution studies of nerves potentially desensitized by injections into the distal interphalangeal joint or the navicular bursa of horses.

To determine whether the distal interphalangeal (DIP) joint directly or indirectly communicates with the navicular bursa (bursa podotrochlearis) and to identify sensory nerves in these synovial structures that might be desensitized by intra-articular injections of anesthetics, Evans blue dye in physiologic saline solution, Luxol fast blue dye with mepivicaine, or commercial latex was injected into the DIP joint (5 ml) or the navicular bursa (3 ml) of 152 digits obtained from horses or ponies at necropsy. The digits were frozen, cut with a band saw, and examined for distribution of dye or latex. Of 122 digits that had injections into the DIP joint, 120 did not have evidence of a communication between the DIP joint and either the navicular bursa or digital flexor tendon sheath. Of 16 digits that had injections into the navicular bursa, 14 did not have evidence of a direct communication with the DIP joint. Injection of dye into the DIP joint resulted in diffusion of dye and staining of other structures, including the synovial linings of the collateral sesamoidean ligaments and of the distal sesamoidean impar ligament and the medullary cavity of the navicular bone. In addition, a blue tinge was observed in the navicular bursa after dye was injected into the DIP joint, suggesting an indirect, and potentially functional, communication between the DIP joint and the navicular bursa. Injection of dye into the navicular bursa resulted in staining only of the bursa's synovial lining. Immunocytochemical analysis revealed nerves immunoreactive for the peptidergic neurotransmitters substance P, and calcitonin gene-related peptide located in structures that were stained after dye was injected into the DIP joint.(ABSTRACT TRUNCATED AT 250 WORDS)

Substance P innervation of equine synovial membranes: joint differences and neural and nonneural receptor localizations.

Substance P (SP) immunocytochemistry and receptor autoradiography were used to define the innervation of the equine synovial membrane of joints equivalent to the wrist and knuckle of man. SP-immunoreactive fibers were mainly concentrated around blood vessels in the subsynovial layer, although not exclusively, while in the more distal joint, SP fibers were more frequently seen in the synovial surface layer. Iodinated SP receptor autoradiography studies revealed silver grain concentrations in the advential layer of blood vessels associated with the vasa vasorum, on the vascular endothelium and in the synovial surface. These findings suggest that SP has various sites of action within the synovial membrane, each of which may contribute both a sensory function and a different component of the inflammatory process to the joint.

Sensory receptors in the equine foot.

Two types of sensory receptors were located in the equine foot, using anatomic techniques. Histologic examination of stained hoof sections revealed lamellated corpuscles in the hoof dermis, which had many of the morphologic characteristics of Pacinian corpuscles. These sensory receptors were restricted to the palmar (caudal) aspects of the solar dermis of the heel. A second type of receptor was detected by use of immunocytochemistry, indicating apparently naked nerve endings containing the neuropeptide calcitonin gene-related peptide-like immunoreactivity in skin, solar dermal tubules, and the digital cushion. This peptide is an example of a sensory neurotransmitter contained in dorsal root ganglion cells and is believed to exist only in unmyelinated sensory nerve fibers. These 2 morphologic structures may be used for detection of sensory stimuli, such as pressure (or vibratory senses) and pain, respectively, in horses during various locomotory gaits.

Neuropeptidergic innervation of equine synovial joints.

Immunocytochemical analysis of equine synovial membranes revealed presence of several neuropeptides, including substance P (SP), neurokinin A, and neuropeptide Y, in nerves of the radiocarpal, middle carpal, and metacarpophalangeal (fetlock) joints. Within the subsynovium, these neuropeptides were located perivascularly, whereas in the fronds, only neuropeptide Y was restricted to the vessels of the synovial membrane. Only SP and neurokinin A were found in the intimal layer. The intimal layer of the metacarpophalangeal joint contained more SP-immunoreactive fibers than were observed in the intimal layer of the radiocarpal joint. Substance P also was detected in the synovial fluid from all 3 joints, but mean +/- SD concentrations were significantly different only between the middle carpal joint (37.56 +/- 5.48 fmol/ml; n = 6) and the metacarpophalangeal joint (55.80 +/- 8.33 fmol/ml; n = 5) and between the middle carpal joint and the radiocarpal joint (52.43 +/- 14.60 fmol/ml; n = 7).

Adrenergic and peptidergic innervation of the trachealis muscle in the normal horse: a preliminary report.

The tone of respiratory smooth muscle is largely determined by the input from autonomic nerves. The distribution of adrenergic and selected nonadrenergic, non-cholinergic (NANC) nerves in the normal equine trachealis muscle was investigated using immunohistochemistry. The smooth muscle of the trachealis was found to contain numerous nerves immunoreactive for an enzymatic marker of adrenergic nerves, as well as many nerves immunoreactive for a putative NANC neurotransmitter, peptide histidine isoleucine, a potent bronchodilator. The tissue surrounding the respiratory smooth muscle contained numerous nerves immunoreactive for the neuropeptides substance P and calcitonin gene-related peptide, which can cause marked vasodilation and bronchoconstriction. The complex innervation of the equine trachea should be kept in mind when interpreting the results of physiological experiments.

Distribution of dopamine beta-hydroxylase and neuropeptide Y-immunoreactive nerves in healthy equine lungs.

Immunohistochemical methods were used to determine the distribution of pulmonary nerves containing either an enzymatic marker of adrenergic nerves, dopamine beta hydroxylase, or the putative neurotransmitter neuropeptide Y in 7 equids with healthy lungs. Nerves immunoreactive for these substances were found on airway smooth muscle in nearly all the samples of healthy equine lung examined. These nerves were generally more numerous in the larger airways but could be detected even in noncartilaginous bronchioles. Pulmonary and bronchial vessels also contained numerous immunoreactive nerves. On the basis of these findings, we suggest that dopamine beta-hydroxylase- and neuropeptide Y-immunoreactive nerves may be involved in the regulation of airway and vascular diameter in equids.