Quantitative magnetic resonance imaging and histological hoof wall assessment of 3-year-old Quarter Horses.

BACKGROUND: Few noninvasive methods are available for equine hoof wall evaluation. The highly organised wall structures and composition of proteoglycans and collagens may make this region amenable to quantitative MRI (qMRI) techniques of T1ρ and T2 mapping to identify pathology related to proteoglycan content and collagen organisation respectively. OBJECTIVE: To establish normative T1ρ and T2 values of the equine hoof wall of 3-year-old Quarter Horses with histological comparison. STUDY DESIGN: Cadaveric anatomical study. METHODS: Six cadaveric left thoracic feet from 3-year-old racing Quarter Horses with no reported lameness were evaluated using T1ρ and T2 mapping. Mapping was performed at six regions of interest at the toe of each hoof including proximal and distal regions of the inner epidermis, stratum lamellatum and corium. Histology was evaluated for standard hoof morphology and proteoglycan staining. RESULTS: T2 values of the stratum lamellatum and corium were similar (42.9 [95% CI: 41.6-44.2] ms and 44 [95% CI: 42.7-45.3] ms respectively), but both were significantly different to the inner epidermis (35.8 [95% CI: 34.5-37.1] ms, P<0.001). T1ρ values for the inner epidermis, stratum lamellatum and corium were significantly different (25.1 [95% CI: 23.1-27.1] ms, 44.4 [95% CI: 42.4-46.4] ms and 50.1 [95% CI: 48.1-52.1] ms, respectively, P<0.001). Histology demonstrated normal organised morphology. Proteoglycan staining was only visible in the stratum lamellatum and corium. MAIN LIMITATIONS: Cadaveric study with frozen samples used. CONCLUSIONS: Variation of qMRI metrics through the depth of the equine hoof wall was found. Although the highly ordered environment of collagen may contribute to T2 values, there was lack of evidence to support proteoglycan content as a major contributor of T1ρ values. It is possible T1ρ values had a greater dependence on total water content as the lowest values were seen in the epidermis. Additional research using qMRI is needed to determine mapping values in different disease states.

The microvasculature in the equine distal phalanx: implications for fracture healing.

OBJECTIVES: To describe the intra-osseous microvasculature of the distal phalanx of the equine forelimb with regard to its potential clinical relevance. METHODS: Eleven clinically normal equine forelimbs were used from six adult horses (range: 4 to 18 years old) euthanatized for reasons unrelated to lameness. In each limb the median artery was catheterized at the level of the carpus and India ink was injected under constant manual pressure. The limbs were frozen and 5 mm thick sections of the foot were cut in the sagittal, coronal, or transverse planes on a band saw. The sections were fixed in 10% formalin and cleared using a modified Spalteholz technique. Once cleared, the sections were photographed and the microvascular anatomy identified. RESULTS: The vascular injections revealed a rich intra-osseous microvascular supply of the distal phalanx originating from the medial and lateral palmar digital arteries. In addition, numerous smaller vessels from the terminal arch, formed by anastomosis of the medial and lateral palmar digital arteries, could be seen branching into the distal aspects of the distal phalanx. This distal portion of the distal phalanx appeared more densely vascularized than the proximal part in all specimens examined. CLINICAL SIGNIFICANCE: The increased vascularity demonstrated in the distal portion of the distal phalanx appears to correlate with improved fracture healing reported in this area. This may also explain why healing fractures which involve both the distal and proximal portions of the distal phalanx have been described as progressing from distal-to-proximal.

Effects of barefoot trimming on hoof morphology.

OBJECTIVE: To monitor changes in hoof morphology in response to barefoot trimming. METHODS: Seven horses were trimmed every 6 weeks according to barefoot trimming principles, which involved levelling the hoof to live sole, lowering the heels, bevelling the toe and rounding the peripheral wall, while leaving the sole, frog and bars intact. A 4-month period was allowed to lower the heels sufficiently to achieve a hoof shape representative of the barefoot trim. This was regarded as the starting point for morphological adaptations in response to maintenance of the trim. Hoof morphology was measured from lateral, dorsal and solar view photographs and lateromedial radiographs taken at 0, 4 and 16 months. Changes from 0 to 4 months represented differences between a natural hoof shape and the trim, while changes from 4 to 16 months represented adaptive effects during hoof growth. RESULTS: Establishment of the barefoot trim involved significant shortening of the toe, heel and medial and lateral walls, with increases in angulation at the toe, medial and lateral walls, but not at the heel. Maintenance of the trim resulted in a palmar/plantar migration of the heels, with increases in support length, heel angle and solar angle of the distal phalanx (P3). CONCLUSIONS: Bevelling the toe and engaging the frog and bars in the weight-bearing function of the foot resulted in elevation of the heel angle and solar angle of P3. These changes may be beneficial in treating under-run heels and negative solar plane angulation of P3.

Guidelines for the investigation and management of a reduced level of consciousness in children: implications for clinical biochemistry laboratories.

Whenever a child presents to hospital with a reduced level of consciousness, admitting clinicians have to decide the underlying cause rapidly so that the correct emergency treatment can be initiated. Unfortunately, the clinical presentations of many of the possible diagnoses are very similar. The diagnosis often results from investigations within the clinical biochemistry laboratory. In the past, clinicians have had limited guidance on which tests to request when presented with a child with a reduced level of consciousness. Guidelines have recently been developed relating to all aspects of management of the child in a coma. Due to a lack of evidence in the literature regarding the most appropriate first line tests for children with a reduced level of consciousness, a formal consensus process ('Delphi consensus') was performed using a large multidisciplinary panel of experts. The recommendations reached by this process include the list of initial ('core') tests to request for all children with a reduced level of consciousness (excluding those immediately after suffering a convulsion and those involved in obvious trauma). Depending upon the results of these 'core' tests and the clinical condition of the child, further tests may be requested later. The key point is that all the samples have been taken at the time of presentation to provide the best chance of reaching a diagnosis and correctly treating the child. The article reviews the recommended core investigations and further tests and discusses how individual laboratories can help to implement the guidelines jointly with their Emergency and Paediatric Departments.

Effect of contact stress in bones of the distal interphalangeal joint on microscopic changes in articular cartilage and ligaments.

OBJECTIVE: To examine articular cartilage of the distal interphalangeal (DIP) joint and distal sesamoidean impar ligament (DSIL) as well as the deep digital flexor tendon (DDFT) for adaptive responses to contact stress. SAMPLE POPULATION: Specimens from 21 horses. PROCEDURE: Pressure-sensitive film was inserted between articular surfaces of the DIP joint. The digit was subjected to a load. Finite element models (FEM) were developed from the data. The navicular bone, distal phalanx, and distal attachments of the DSIL and DDFT were examined histologically. RESULTS: Analysis of pressure-sensitive film revealed significant increases in contact area and contact load at dorsiflexion in the joints between the distal phalanx and navicular bone and between the middle phalanx and navicular bone. The FEM results revealed compressive and shear stresses. Histologic evaluation revealed loss of proteoglycans in articular cartilage from older horses (7 to 27 years old). Tidemark advancement (up to 14 tidemarks) was observed in articular cartilage between the distal phalanx and navicular bone in older clinically normal horses. In 2 horses with navicular syndrome, more tidemarks were evident. Clinically normal horses had a progressive increase in proteoglycans in the DSIL and DDFT. CONCLUSIONS AND CLINICAL RELEVANCE: Load on the navicular bone and associated joints was highest during dorsiflexion. This increased load may be responsible for microscopic changes of tidemark advancement and proteoglycan depletion in the articular cartilage and of proteoglycan production in the DSIL and DDFT Such microscopic changes may represent adaptive responses to stresses that may progress and contribute to lameness.

Digital angiography of the feet of horses.

OBJECTIVE: To describe the vascular anatomy of the palmar digital artery and its major branches in the equine foot and to quantify the diameter of these vessels by use of digital angiograms. Sample Population-6 thoracic limbs obtained from 6 horses. PROCEDURE: Distal portions of each limb were perfused with aerated Krebs-Henseleit solution. Digital angiograms were acquired in standing and lateral recumbent positions, following an intra-arterial injection of iopamidol. Select vessels were measured on radiographic views, and values were corrected for magnification. RESULTS: The palmar digital artery tapered from 2.28 mm at the coronary region to 1.61 mm at the entrance to the solar canal, and the major arterial branches ranged in diameter from 0.71 to 1.42 mm in the standing position. CONCLUSIONS AND CLINICAL RELEVANCE: Digital angiography is useful for imaging small vessels, but penumbra limits the image resolution of the macrovasculature of the foot. The palmarodorsal projection is more useful for evaluation of the terminal arch and solar branches, but 2 projections are necessary for a thorough examination of the foot. Image magnification, position of horse, and vascular response to contrast medium must be considered in the quantitative assessment of vessel diameter. Digital angiography may be performed in clinical cases and research models for examination of vascular perfusion of the distal portion of the limb.

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.

Functional anatomy of the cartilage of the distal phalanx and digital cushion in the equine foot and a hemodynamic flow hypothesis of energy dissipation.

OBJECTIVES: To examine macro- and microscopic characteristics of cartilage of the distal phalanx (ungual cartilage [UC]) and digital cushion in the equine foot and to relate them to the foot's function of energy dissipation. ANIMALS: 85 horses and 5 foals of various breeds and ages. PROCEDURE: Feet, obtained at necropsy, were perfused with India ink (n = 30), latex (5), or polymer plastic (10). Select feet were examined histologically for tissue architecture and to identify elastic fibers. Immunochemistry to identify substance P peptides in nerves (feet from foals) and gold chloride impregnation of axons (n = 10) were performed. Feet were sectioned transversely (n = 27) or coronally (62 feet in a matched-paired study). Ungual cartilage was measured at the navicular bone. Digital cushions were examined for relative tissue composition between forefeet and hind feet. RESULTS: Ungual cartilage formed an axial projection that extended towards the midline to overlie the bars, and dorsally along the semilunar line of the distal phalanx. Ungual cartilage of forefeet was significantly larger than that of hind feet. The digital cushion was composed of fat and elastic tissues in feet with thin UC, or fibrous and fibrocartilaginous tissue and elastic tissue in feet with thicker UC. Sensory nerves and an extensive network of venovenous anastomoses were found in the UC. CONCLUSIONS AND CLINICAL RELEVANCE: Ungual cartilage and the digital cushion provide the basis for a hemodynamic flow hypothesis of energy dissipation. Maximum energy dissipation depends on proper hoof preparation and shoeing.

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.

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).

Relationship between Legionella spp. and antibody titres at a therapeutic thermal spa in Portugal.

The presence of Legionella spp. in the water of a Portuguese spa was ascertained during the spa season, between May and November. Simultaneously the prevalence of anti-legionella antibodies in people attending the spa was also investigated. The antibody titres of 172 randomly selected patients and 42 therapists were determined, and compared with a control group of 503 blood donors. Legionellae were present in the spa water at low concentrations, generally lower than 10(3) c.f.u./l. A total of 92 strains representing eight different species or serogroups were isolated; the predominant isolates belonged to Legionella pneumophila serogroup 6 and to L. londiniensis. During the study, no clinical cases of Legionnaires' disease were observed, and the antibody titres were generally low in the groups studied. However, the antibody titres of the patients increased slightly during their stay at the spa, approaching the values for the therapists. Mean antibody titres in the groups related with the spa were significantly higher than those in the blood donors against five of the seven legionella antigens tested. The largest number of elevated antibody titres in the exposed groups were to the L. pneumophila sg 5 and sg 6 antigens.

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).