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.

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.

Suppressor cell activity in melanoma-draining lymph nodes.

We previously demonstrated that the cells of lymph nodes near to a melanoma respond well to stimulation by mitogens, alloantigens, and interleukin 2 than do nodes further away. In this study we examined suppressor T-cell activity in nodes at different distances from primary melanoma, using a concanavalin A (Con A) suppressor cell assay. Tumor-free regional nodes were classified as proximal, intermediate, and distal relative to primary melanoma. Lymph node lymphocytes (LNL) were stimulated with 50 micrograms/ml Con A for 48-72 h, inactivated, and then mixed with autologous peripheral blood lymphocytes. The peripheral blood lymphocyte-LNL mixtures were stimulated with phytohemagglutinin for 3 days. Proliferation was measured by [3H]thymidine uptake during the final 18 h of culture. In 13 patients, Con A-treated LNL from nodes near to tumor were more suppressive of the peripheral blood lymphocyte response to phytohemagglutinin than those from nodes located further from tumor. T-lymphocyte subset assessment before and after Con A treatment of LNL showed no significant changes in T4:T8 ratios. Con A-induced suppressor cells could be maintained in culture in the presence of recombinant interleukin 2 and retained their suppressive activity. LNL not exposed to Con A and maintained in culture with interleukin 2 did not show suppressor cell activity. Suppressor cell activity thus contributes to the weak immune reactivity of lymph nodes near to melanoma.

Retrograde transport of horseradish peroxidase is specifically impaired in bulbospinal serotonin axons during experimental allergic encephalomyelitis.

Bulbospinal monoamine-containing axons appear to be severely damaged in rats with the inflammatory and demyelinating disease, experimental allergic encephalomyelitis (EAE). This paper reports that although bulbospinal serotonin axons are damaged in the disease, cell bodies of origin in the medulla oblongata retain normal morphology. However, these serotonin cells are not able to retrogradely transport the enzyme horseradish peroxidase (HRP) from terminals in the lumbar spinal cord. Most non-serotonin-containing cells in the medulla which project to the lumbar spinal cord retain the ability to retrogradely transport HRP from the lumbar cord during the disease. These findings suggest that there is some specificity to spinal cord axonal damage during EAE.

The relationship of the medullary catecholamine containing neurones to the vagal motor nuclei.

We have re-examined in the rat the nuclear localization of the medullary catecholamine-containing cell groups (A1 and A2) and their relation to the vagal motor nuclei using a double labeling method. The vagal nuclei were defined by the retrograde transport of horseradish peroxidase applied to the cervical vagus, and noradrenergic and adrenergic neurons were stained with the peroxidase-antiperoxidase immunocytochemical method using an antibody to dopamine beta-hydrolase. The method allows visualization of both labels within single neurons. The neurons of the A2 group are primarily distributed in both the nucleus of the solitary tract and the dorsal motor nucleus of the vagus in a complex interrelationship that depends on the rostrocaudal level. Caudal to the obex, cells of the dorsal motor nucleus of the vagus are scattered among cells immunoreactive for dopamine beta-hydroxylase in the area considered to be the commissural subnucleus of the nucleus of the solitary tract. At levels near and slightly rostral to the obex, the dopamine beta-hydroxylase-positive cells are largely confined to nucleus of the solitary tract. However, the rostral third of the A2 group lies predominantly within dorsal motor nucleus, as defined by horseradish peroxidase labeled cells, with only a few cells in the nucleus of the solitary tract. A subset of the dopamine beta-hydroxylase positive cells within the rostral dorsal motor nucleus of the vagus are also vagal efferents. Our results suggest that a second population of dopamine beta-hydroxylase positive vagal efferents may exist ventrolaterally where neurons of the AI cell group intermingle with those of nucleus ambiguus.

Transmitters of the raphe-spinal complex: immunocytochemical studies.

The localization of serotonergic and various peptidergic neurons in the medullary raphe nuclei that project to the lumbosacral spinal cord have been studied using a retrograde transport method combined with immunocytochemistry. Spinally projecting neurons stained for serotonin-like, substance P-like, enkephalin-like and thyrotropin-releasing hormone-like immunoreactivity were all observed in the raphe nuclei of the medulla, as well as in the adjacent ventrolateral reticular formation. The distribution of the descending serotonergic and peptidergic neurons in the raphe nuclei as well as quantitative data on their relative numbers suggest that a large fraction of raphe-spinal neurons contain serotonin co-existing with one or more peptides in the same cell.

A retrograde double label tracing technique using horseradish peroxidase and the fluorescent dye 4'-,6-diamidino-2-phenylindole 2HC1 (DAPI).

Retrograde transport of HRP and the fluorescent dye DAPI were used to double label spinothalamic tract cells with dual projections to thalamus and cerebellum. Cells containing both retrograde tracers were found in the intermediate gray zone and in the lateral spinal nucleus of the rat lumbosacral spinal cord. Double labeled cells were easily identified by the presence of granular HRP reaction, product and the blue fluorescence of DAPI. The use of HRP and DAPI provides an effective double labeling technique for the localization of neurons with nonoverlapping projections in the central and/or peripheral nervous system.

Intrinsic 5HT-immunoreactive neurons in the spinal cord of the fetal non-human primate.

Serotonin (5HT) immunoreactive neurons were identified in the late-term fetal spinal cord of normal non-human primates. These neurons were distributed throughout the spinal cord, being concentrated in lamina X and the subjacent ventral median fissure, while their immunoreactive fibers and terminals innervated the zone surrounding the central canal and the ventral spinal artery. Even at this late fetal stage, the dorsal and ventral spinal gray matter was virtually devoid of any positive 5HT immunoreactivity, in contrast to that seen in the adult primate. These findings suggest that the intrinsic 5HT neurons of the primate during development may modulate CSF composition or provide cues for spinal cord differentiation rather than regulate sensorimotor functions as they do in the adult.

Serotonergic and peptidergic inputs to the primate ventral spinal cord as visualized with multiple chromagens on the same tissue section.

Primate spinal cord tissue sections were sequentially incubated in serotonin (5-HT), substance P (SP) and methionine-enkephalin antisera and reacted immunocytochemically. The findings indicate that virtually all SP-like immunoreactivity coexists with 5-HT in the ventral horn, while an additional group of 5-HT terminals do not contain SP immunoreactivity.

Spinal origins of cardiac afferents from the region of the left anterior descending artery.

The origins of coronary artery afferents coursing in sympathetic nerves was determined using retrograde axonal transport methods. Cells labeled with horseradish peroxidase were found bilaterally in dorsal root ganglia (DRG) C8 to T6 were smaller in size than non-labeled DRG neurons. The findings indicate a more extensive segmental distribution of cardiac afferents than was previously believed to exist.

The origins and trajectories of somatostatin reticulospinal neurons: a potential neurotransmitter candidate of the dorsal reticulospinal pathway.

Somatostatin (SOMA)-immunoreactive neurons present in the nucleus gigantocellularis and nucleus raphe magnus formed the origins of a descending pathway from the rostral medulla to the spinal cord. This descending pathway was traced through the central medullary tegmentum and dorsolateral funiculus to innervate the dorsal horn. The location, trajectories and presumed inhibitory nature of SOMA in the spinal cord suggest that SOMA maybe a potential neurotransmitter candidate for the classic dorsal reticulospinal pathway.

Quantitative re-evaluation of descending serotonergic and non-serotonergic projections from the medulla of the rodent: evidence for extensive co-existence of serotonin and peptides in the same spinally projecting neurons, but not from the nucleus raphe magnus.

A quantitative analysis of serotonin (5-HT) and spinally-projecting neurons was re-evaluated in the rodent. The findings indicate that most (nearly 90%) of the medullary 5-HT neurons projected to the lumbar spinal cord, and most (up to 85%) of the spinally projecting neurons within the distribution of the serotonergic neurons contained 5-HT immunoreactivity. However, in nucleus raphe magnus (NRM) only about two-thirds of the projection cells were 5-HT immunoreactive. These data support two general conclusions: (1) the raphe-spinal system consists primarily of an extensive 5-HT pathway with neuronal subsets containing the co-localized peptides. Only the NRM contains a major non-5-HT projection. (2) As most of the medullary 5-HT neurons project to the caudal spinal segments of the rodent, collateralization of individual 5-HT neurons is extensive and widespread, existing to different spinal cord levels, as well as to other medullary nuclei, e.g., cranial nerve and inferior olivary nuclei. These findings argue that although differences are present within the 5-HT distributions, the raphe-spinal system as a whole should be considered to be a relatively homogeneous pathway containing 5-HT as the common element rather than as separate populations containing major projections of 5-HT alone, 5-HT co-localized with peptides and peptides without 5-HT.

Origins of serotonergic projections to the spinal cord in rat: an immunocytochemical-retrograde transport study.

The origins of the serotonergic projections to the spinal cord in the rat were determined by employing the retrograde cell marker HRP coupled with the unlabeled antibody, peroxidase-antiperoxidase immunocytochemical method of Sternberger. Large numbers of stained neurons (greater than 70%) in the medullary raphe nuclear complex were found to contain both HRP retrogradely transported from the spinal cord and positive 5-HT staining. These serotonergic cell groups, including the nucleus raphe obscurus, raphe pallidus, raphe magnus, and the ventral parts of the reticular formation, project to all spinal cord levels. In addition, some neurons contained HRP granules, but were unstained for 5-HT, suggesting that they may contain other non-serotonergic neurotransmitters. More rostrally in the midbrain reticular formation, many 5-HT neurons were found to have projections exclusively to the cervical spinal cord. These findings indicate that the descending serotonin inputs to the spinal cord originate not only from the serotonergic neurons located in the medullary raphe complex, but also from other new sources located in the central gray and reticular formation of the midbrain.

Serotonergic and peptidergic projections to the spinal cord demonstrated by a combined retrograde HRP histochemical and immunocytochemical staining method.

A simple technique is described for simultaneously visualizing the cells of origin of descending brain stem-spinal systems and immunocytochemically defined neurotransmitters. The combination of horseradish peroxidase (HRP) histochemistry, enhanced by the use of CoCl2, with the unlabeled antibody, peroxidase-antiperoxidase (PAP) immunocytochemical staining method, revealed double labeled cells containing black punctate HRP granules within a homogeneous brown cytoplasm stained positively for serotonin (5-HT), substance P or enkephalin-like immunoreactivity. The advantages of this method over other double labeling techniques are discussed.

Differential labeling of the vestibular complex following unilateral injections of horseradish peroxidase into the cat and rat locus coeruleus.

Following unilateral injections of horseradish peroxidase into the cat and rat locus coeruleus (LC), bilateral retrograde labeling was evident in all 4 vestibular nuclei. In both species, the major contributions of LC afferents arose from the ipsilateral rather than the contralateral vestibular nuclei. Quantitative analysis in the rat has indicated that approximately 90% of the total vestibular-LC projections originated from the ipsilateral vestibular nuclear complex, with 10% from the contralateral. The frequency distribution was in the order of lateral greater than medial greater than superior greater than inferior vestibular nuclei.

Origins and terminations of descending noradrenergic projections to the spinal cord of monkey.

This report describes the distribution of noradrenergic cells in the brainstem and the pattern of terminal varicosities in the spinal cord of monkey using the immunocytochemical localization of dopamine-beta-hydroxylase (DBH). Using two separate and equally reliable techniques, retrograde transport of the antibody to DBH and a double-labeling method, the cells of origin of noradrenergic fibers in the spinal cord have been identified. The results of these studies indicate that 79% of all noradrenergic cells with axons projecting to the spinal cord are located in the nucleus subcoeruleus and nucleus locus coeruleus. Other pontine noradrenergic cell groups contribute the remainder of the fibers to the cord. No medullary cells contribute to the noradrenergic innervation of the spinal cord.

Noradrenergic projections to the spinal cord of the rat.

Noradrenergic terminals were identified in the spinal cord of rats by immunocytochemical staining for dopamine-beta-hydroxylase. Although immunoreactive fibers and terminals were observed throughout the spinal grey matter, heavier accumulations of terminal labeling were observed in the marginal layer of the dorsal horn, in the ventral horn among motoneurons, and in the autonomic lateral cell columns of the thoracic and sacral spinal cord. Two specific retrograde transport techniques were employed to identify the origins of these noradrenergic terminations in the spinal cord. Cells of origin were observed in the locus coeruleus, the subcoeruleus, the medial and lateral parabrachial, and the Kölliker-Fuse nuclei, as well as adjacent to the superior olivary nucleus. These regions correspond to the A5-A7 cell groups of the pons. No spinally projecting noradrenergic cells were ever observed in the medulla. It was concluded that pontine noradrenergic cell groups are the sole source of noradrenergic terminals in the spinal cord.

Descending serotonergic, peptidergic and cholinergic pathways from the raphe nuclei: a multiple transmitter complex.

The localization of serotonergic, various peptidergic and possibly cholinergic neurons in the medullary raphe nuclei that project to the lumbosacral spinal cord have been studied using a retrograde transport method combined with immunocytochemical and histochemical techniques. Spinally projecting neurons stained for serotonin-like, substance P-like, enkephalin-like and thyrotropin-releasing hormone-like immunoreactivity and for the histochemical marker acetylcholinesterase were all observed in each of the raphe nuclei of the medulla, as well as in the adjacent ventrolateral reticular formation. The similar distributions of the descending serotonergic and peptidergic neurons in the raphe nuclei as well as quantitative data on their relative numbers suggest that a large fraction of raphe-spinal neurons contain serotonin co-existing with one or more peptides in the same cell.

Serotonergic projections to the caudal brain stem: a double label study using horseradish peroxidase and serotonin immunocytochemistry.

Cells of origin of serotonergic and non-serotonergic projections to the caudal brain stem in the primate were examined using a double label technique. Following HRP injections into medullary raphe nuclei and the adjacent reticular formation double labeled cells were found in the dorsal raphe nucleus, the central superior nucleus and the ventrolateral tegmentum. Retrogradely labeled cells that did not stain for serotonin-like immunoreactivity were found primarily in the periaqueductal gray (PAG) and the mesencephalic and pontine reticular formation. The results are discussed in relation to the descending pathway(s) mediating the effects of PAG stimulation.