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Mark Murphy

Publications and source records attributed to Mark Murphy.

17 recordsLinked to original sources
PubMed2006

Eph tyrosine kinase receptor EphA4 is required for the topographic mapping of the corticospinal tract.

Fine movement in the body is controlled by the motor cortex, which signals in a topographically specific manner to neurons in the spinal cord by means of the corticospinal tract (CST). How the correct topography of the CST is established is unknown. To investigate the possibility that the Eph tyrosine kinase receptor EphA4 is involved in this process, we have traced CST axons in mice in which the EphA4 gene has been deleted. The forelimb subpopulation of CST axons is unaffected in the EphA4-/- mice, but the hindlimb subpopulation branches too early within the cord, both temporally and spatially. EphA4 shows a dynamic expression pattern in the environment of the developing CST in the spinal cord: high at the time of forelimb branching and down-regulated before hindlimb branching. To examine whether the fore- and hindlimb subpopulations of CST axons respond differently to EphA4 in their environment, neurons from fore- and hindlimb motor cortex were cultured on a substrate containing EphA4. Neurons from the hindlimb cortex showed reduced branching on the EphA4 substrate compared with their forelimb counterparts. Neurons from the hindlimb cortex express ephrinA5, a high-affinity ligand for EphA4, at higher levels compared with forelimb cortex neurons, and this expression is down-regulated before hindlimb branching. Together, these findings suggest that EphA4 regulates topographic mapping of the CST by controlling the branching of CST axons in the spinal cord.

Animals↗
PubMed2006

High-molecular-weight hyaluronan inhibits macrophage proliferation and cytokine release in the early wound of a preclinical postlaminectomy rat model.

BACKGROUND CONTEXT: Failed back syndrome, a condition that affects 3-14% of postoperative spine patients, is characterized by the recurrence of radicular pain after spinal decompression. The source of this pain in some patients is thought by many investigators to be the result of epidural scarring and nerve root tethering, but this is controversial. We have previously demonstrated that in a disc-injury model the untreated postlaminectomy rats develop a significant proliferative fibrous response at 8 weeks with spinal nerve scarring to the disc and adjacent pedicle, and increased sensitivity to tactile allodynia testing in the related sensory dermatome. Topical high-molecular-weight hyaluronan (HMW HA) moderates both the proliferative fibrosis and the behavioral pain response. PURPOSE: Our purpose is to study the time-related changes in the proinflammatory cytokine and monocyte/macrophage profiles in the epidural space in the early postlaminectomy untreated and HMW HA gel treated groups. STUDY DESIGN/SETTING: A modified rat laminectomy with disc injury model was employed to assess epidural fibrosis between and around the spinal nerves using a quantitative immunohistochemistry assessment approach along with correlative enzyme-linked immunosorbent assay analysis. METHODS: Lumbar laminectomies at L5 and L6 with a L5-L6 disc injury were performed on 120 adult male Sprague-Dawley rats. The rats were then randomized into one of two groups: untreated and treated. The treatment group received a one-time topical application of 0.1 cc of HMW HA gel directly to the laminectomy site just before wound closure. The rats were then randomly subdivided into survival periods of 24 hours, 72 hours, and 7 days. Immunohistochemistry was performed on fresh frozen sections and stained for interleukin-1 beta (IL-1beta) and monocytes/macrophages (ED-1) using monoclonal antibodies and 3, 3' diaminobenzidine (DAB) chromogen. The amount of stain in each specimen was then quantified using the National Institutes of Health computer imaging analysis system. RESULTS: The semiquantified data from the histological specimens demonstrated significant decreases in the IL-1beta and IL-6 infiltration observed at 24 hours in the epidural space and around the right nerve root (p=.0296 and 0.0195, respectively) in the HA gel treated group. Additionally, significant decreases in the monocyte/macrophage infiltration were observed at 72 hours in the epidural space around the left nerve root (p=.0039) and right nerve root (p=.0072) in the HA gel treated group. At 7 days, IL-1beta, IL-6, and macrophage infiltration of the wound had declined in both the HA gel and the untreated groups. The enzyme-linked immunosorbent assay data support the same pattern as seen in the histological results. CONCLUSION: These results demonstrate that treatment of postlaminectomy wounds with HMW HA gel decreases the number of monocytes and macrophages and the concentration of certain cytokines in the early inflammatory phase of healing. There are several plausible explanations for this effect. First, the HMW HA may block the interaction of short-chain low-molecular-weight HA with proinflammatory cell surface receptors. The interaction of these short-chain oligo-HA fragments, upon cell-surface receptor binding, induces changes in inflammatory cells that lead to increased cell motility and migration into the wound area. Second, the addition of exogenous HMW HA may cause a dilution effect in the wound, thereby decreasing the concentration of inflammatory cells in the extracellular matrix of the region of injury. Finally, the migration of inflammatory cells may be decreased in the viscous environment of the HMW HA. The first explanation is believed by the authors of this paper to be the more likely mechanism. HMW HA probably mutes the proinflammatory effects of the low-molecular weight fragments, leading to decreased inflammation, and thus decreased fibrosis and scar formation noted in the chronic model.

Adjuvants, Immunologic↗
PubMed2005

A brightness-area-product-based protocol for the quantitative assessment of antigen abundance in fluorescent immunohistochemistry.

A problem frequently facing researchers examining abundance of expression of a given antigen is measurement. When the antigen is confined to the nucleus, absolute numbers of nuclei or a percentage of nuclei expressing the antigen in a given region can be estimated. When the antigen is localized to cytoplasm, cytoplasmic organelles or processes or membranes, the assessment becomes more difficult. In these settings, an observer/experimenter may assign a density score but intra- and inter-observer agreement using a three-tiered system, and finer resolution than this, is unlikely to be reproducible. Digital image analysis provides an opportunity to minimize observer bias in quantification of immunohistochemical staining. Previously, reported digital methods have mostly employed chromogen-staining methods and often report mean image brightness. We report a method for quantitatively assessing and expressing abundance of expression of an antigen in neural tissue stained with immunofluorescent methods by determining the brightness-area-product (BAP). The described protocol utilizes simple to use commercially available software and calculates BAP rather than mean brightness as a measure more representative of antigen abundance and visual interpretation. Accordingly, we propose this protocol as a useful adjunct to observer interpretation of fluorescent immunohistochemistry and its application to assessment of antigen abundance for varying patterns of antigen localization.

Animals↗
PubMed2004

Neural crest cell lineage segregation in the mouse neural tube.

Neural crest (NC) cells arise in the dorsal neural tube (NT) and migrate into the embryo to develop into many different cell types. A major unresolved question is when and how the fate of NC cells is decided. There is widespread evidence for multipotential NC cells, whose fates are decided during or after migration. There is also some evidence that the NC is already divided into subpopulations of discrete precursors within the NT. We have investigated this question in the mouse embryo. We find that a subpopulation of cells on the most dorsomedial aspect of the NT express the receptor tyrosine kinase Kit (previously known as c-kit), emigrate exclusively into the developing dermis, and then express definitive markers of the melanocyte lineage. These are thus melanocyte progenitor cells. They are generated predominantly at the midbrain-hindbrain junction and cervical trunk, with significant numbers also in lower trunk. Other cells within the dorsal NT are Kit-, migrate ventrally, and, from embryonic day 9.5, express the neurotrophin receptor p75. These cells most likely only give rise to ventral NC derivatives such as neurons and glia. The p75+ cells are located ventrolateral to the Kit+ cells in areas of the NT where these two cell types are found. These data provide direct in vivo evidence for NC lineage segregation within the mouse neural tube.

Animals↗
PubMed2004

Tracing functional circuits using c-Fos regulated expression of marker genes targeted to neuronal projections.

We have developed novel techniques to trace functionally activated circuits and synaptic plasticity within the brain. We have generated transgenic mice, FTL, which contain a tau-lacZ fusion gene regulated by the promoter for c-fos. Following a particular nervous system stimulation in these mice, only neurons, which are functionally activated, will express LacZ, which is targeted to neuronal processes by the tau protein. In the FTL mice, we found highly inducible expression of lacZ by a range of different stimuli, and successful targeting of expression to neuronal cell bodies, axons and dendrites. To test if a functionally activated circuit could be visualized, the mice were deprived of water, which activates nuclei involved in body fluid homeostasis. LacZ was induced in these nuclei and their projections, allowing the mapping of a neuroendocrine circuit. Further studies have employed these mice in the analysis of neurons and circuits activated in vision, and learning and memory. We have also developed methods to measure markers of synaptic plasticity in the brain, and found significant experience dependent changes in the levels of these markers in different parts of the brain. We believe these techniques will aid in the identification of circuits for many different brain functions, and within those circuits, the locations of synaptic plasticity.

Animals↗
PubMed2004

Fos-tau-LacZ mice expose light-activated pathways in the visual system.

We have employed fos-tau-LacZ (FTL) transgenic mice to examine functional activation in the visual areas of the nervous system. The FTL mice express the marker gene lacZ in neurons and their processes following many different stimuli, and allow the imaging of activation from the level of the entire brain surface through individual neurons and their projections. Analysis of FTL expression in the retinas of mice following diurnal exposure to light shows that bipolar cells, specific classes of amacrine cells, ganglion cells, and a dense network of processes in the inner plexiform layer are functionally activated. In animals deprived of light, there is almost no activity in the retina. In the lateral geniculate nucleus (LGN), light exposure appears responsible for FTL expression in dorsal nuclei, but not for expression in the ventral nuclei or the intergeniculate leaflet. In the superficial layers of the superior colliculus, FTL expression is highly dependent on light exposure. Similarly, light exposure is required for FTL expression in primary visual cortex (area 17), but some expression remains in area 18 of dark-adapted animals. Finally, using mice with one or both eyes missing, we have determined which parts of the visual system are dependent on the presence of a functional connectivity from the eye. These data demonstrate the usefulness of the FTL mice to map functional activation within the entire visual system. Furthermore, we can capture visual activation in a conscious animal. Our findings give an insight into the architecture of activity within the retina and throughout the visual system.

Animals↗
PubMed2004

Environmental enrichment results in cortical and subcortical changes in levels of synaptophysin and PSD-95 proteins.

Experience-dependent plasticity is thought to involve selective change in pre-existing brain circuits, involving synaptic plasticity. One model for looking at experience-dependent plasticity is environmental enrichment (EE), where animals are exposed to a complex novel environment. Previous studies using electron microscopy showed that EE resulted in synaptic plasticity in the visual cortex and hippocampus. However, the areas in the brain that have been examined following EE have been limited. The present study quantified potential synaptic plasticity throughout the brains of C57BL/6 mice using an enzyme-linked immunosorbent assay (ELISA) for two synaptic proteins, synaptophysin and PSD-95. EE resulted in increased synaptophysin and PSD-95 levels through major brain regions, including anterior and posterior areas of the forebrain, hippocampus, thalamus, and hypothalamus. However, no changes in synaptophysin were detected in the cerebellum. These results demonstrate that EE results in an increase in levels of both pre- and post-synaptic proteins in multiple regions of the brain, and it is possible that such changes represent the underlying synaptic plasticity occurring in EE.

Adaptation, Physiological↗
PubMed2004

Hypopharyngeal perforation near-miss during transesophageal echocardiography.

OBJECTIVES/HYPOTHESIS: The traditional blind passage of a transesophageal echocardiography probe transorally through the hypopharynx is considered safe. Yet, severe hypopharyngeal complications during transesophageal echocardiography at several institutions led the authors to investigate whether traditional probe passage results in a greater incidence of hypopharyngeal injuries when compared with probe passage under direct visualization. STUDY DESIGN: Randomized, prospective clinical study. METHODS: In 159 consciously sedated adults referred for transesophageal echocardiography, the authors performed transesophageal echocardiography with concomitant transnasal videoendoscopic monitoring of the hypopharynx. Subjects were randomly assigned to receive traditional (blind) or experimental (optical) transesophageal echocardiography. The primary outcome measure was frequency of hypopharyngeal injuries (hypopharyngeal lacerations or hematomas), and the secondary outcome measure was number of hypopharyngeal contacts. RESULTS: No perforation occurred with either technique. However, hypopharyngeal lacerations or hematomas occurred in 19 of 80 (23.8%) patients with the traditional technique (11 superficial lacerations of pyriform sinus, 1 laceration of pharynx, 12 arytenoid hematomas, 2 vocal fold hematomas, and 1 pyriform hematoma) and in 1 of 79 patients (1.3%) with the optical technique (superficial pyriform laceration) (P =.001). All traumatized patients underwent flexible laryngoscopy, but none required additional intervention. Respectively, hypopharyngeal contacts were more frequent with the traditional than with the optical technique at the pyriform sinus (70.0% vs. 10.1% [P =.001]), arytenoid (55.0% vs. 3.8% [P =.001]), and vocal fold (15.0% vs. 3.86% [P =.016]). CONCLUSION: Optically guided trans-esophageal echocardiography results in significantly fewer hypopharyngeal injuries and fewer contacts than traditional, blind transesophageal echocardiography. The optically guided technique may result in decreased frequency of potentially significant complications and therefore in improved patient safety.

Adult↗
PubMed2003

Localisation of specific heparan sulfate proteoglycans during the proliferative phase of brain development.

Early brain development is characterised by the proliferation of neural precursor cells. Several families of signalling molecules such as the fibroblast growth factors (FGFs) and Wnts are known to play important roles in this early phase of brain development. Accumulating evidence demonstrates that signalling of these molecules requires the presence of heparan sulfate chains attached to a proteoglycan core protein (HSPG). However, the specific identity of the HSPG components in the developing brain is unknown. To determine which HSPGs might be involved at this early phase, we analysed the expression of the major cell surface HSPG families in the developing brain at a time of most active proliferation. Syndecan-1 and glypican-4 were the most highly expressed in the developing brain during the time of peak proliferation and localise to ventricular regions of the brain, where the precursor cells are proliferating. Syndecan-4, although less abundant, also localises to cells in the ventricular zone. We have also examined HSPG involvement in brain development using cultures of embryonic neural precursor cells. We find that FGF2 stimulation of proliferation is inhibited in the presence of sodium chlorate, an inhibitor of heparan sulfate synthesis, and is rescued by addition of exogenous heparan sulfate. These data support a requirement for heparan sulfate in FGF signalling for proliferation of brain precursor cells. The expression of these specific HSPGs within the proliferative zone of the brain suggests that they may be involved in regulation of early brain development, such as FGF-stimulated proliferation.

Animals↗
PubMed2003

Human melanoblasts in culture: expression of BRN2 and synergistic regulation by fibroblast growth factor-2, stem cell factor, and endothelin-3.

The BRN2 transcription factor (POU3F2, N-Oct-3) has been implicated in development of the melanocytic lineage and in melanoma. Using a low calcium medium supplemented with stem cell factor, fibroblast growth factor-2, endothelin-3 and cholera toxin, we have established and partially characterised human melanocyte precursor cells, which are unpigmented, contain immature melanosomes and lack L-dihydroxyphenylalanine reactivity. Melanoblast cultures expressed high levels of BRN2 compared to melanocytes, which decreased to a level similar to that of melanocytes when cultured in medium that contained phorbol ester but lacked endothelin-3, stem cell factor and fibroblast growth factor-2. This decrease in BRN2 accompanied a positive L-dihydroxyphenylalanine reaction and induction of melanosome maturation consistent with melanoblast differentiation seen during development. Culture of primary melanocytes in low calcium medium supplemented with stem cell factor, fibroblast growth factor-2 and endothelin-3 caused an increase in BRN2 protein levels with a concomitant change to a melanoblast-like morphology. Synergism between any two of these growth factors was required for BRN2 protein induction, whereas all three factors were required to alter melanocyte morphology and for maximal BRN2 protein expression. These finding implicate BRN2 as an early marker of melanoblasts that may contribute to the hierarchy of melanocytic gene control.

Antigens, Neoplasm↗
PubMed2003

"Balanced" orbital decompression for severe Graves' orbitopathy: technique with treatment algorithm.

BACKGROUND: Graves' orbitopathy can produce proptosis, compressive optic neuropathy, and extraocular motility abnormalities; symptoms result from an increase in orbital volume due to expansion of intraorbital fat, with or without extraocular muscle involvement. STUDY DESIGN: We conducted a chart review of patients who underwent orbital decompression to treat Graves' orbitopathy. Twenty-two orbits (13 patients) underwent orbital bone decompression, of which 17 orbits (9 patients) underwent the combined medial and lateral "balanced" decompression. RESULTS: All patients had significant improvement with an average decrease in proptosis of 5.9 mm in the balanced decompression group. Restoration of normal optic nerve function was achieved in all patients with compressive optic neuropathy. Diplopia was noted in 4 patients (30.7%) preoperatively. Two patients had new postoperative diplopia (15.35%). CONCLUSION: In our experience, "balanced" decompression results in a reduction of proptosis and improved optic nerve function and has a low incidence of complications.

Adult↗
PubMed2002

Visualization of functionally activated circuitry in the brain.

We have used a transgenic approach to visualize functionally activated neurons and their projections. The transgenic mice contain a tau-lacZ fusion gene regulated by the promoter for c-fos, an immediate early gene that is rapidly induced in neurons after functional stimulation. Constitutive expression of beta-galactosidase (beta-gal), the lacZ product, was low and in accord with previous reports of c-fos expression. However, expression of beta-gal in positive neurons was clearly in cell bodies, axons, and dendrites. Treatment of the mice with kainic acid, a strong inducer of c-fos expression, resulted in high induction of beta-gal. beta-gal was induced in the same defined populations of neurons in the brain as those that express c-fos after kainic acid induction. Furthermore, the pattern of beta-gal expression within the neurons changed over time after kainic acid treatment. Early after kainate treatment, beta-gal was found mainly in cell bodies; at later times, expression extended further along the neuronal processes. This expression pattern is consistent with induction and anterograde transport of the Fos-Tau-beta-gal protein in the neurons. To test whether a functionally activated pathway could be visualized, transgenic mice were deprived of water, which activates nuclei involved in body fluid homeostasis. beta-gal induction was traced in neurons and their processes in the lamina terminalis, in magnocellular neurons of the supraoptic and paraventricular nuclei, and in their projections to the posterior pituitary gland. This strategy allowed the mapping of an activated osmoregulatory pathway. This transgenic approach may have general application in the mapping of functionally activated circuitry in the brain.

Animals↗
PubMed2002

An international study of hospital readmissions and related utilization in Europe and the USA.

This study concerns a comparative analysis of hospital readmission rates and related utilization in six areas, including three European countries (Finland, Scotland and the Netherlands) and three states in the USA (New York, California, Washington State). It includes a data analysis on six major causes of hospitalization across these areas. Its main focus is on two questions. (1) Do hospital readmission rates vary among the causes of hospitalization and the study populations? (2) Are hospital inpatient lengths of stay inversely related to readmissions rates? The study demonstrated that diagnoses such as chronic obstructive pulmonary disease (COPD) and congestive heart failure (CHF) were the major causes of hospital readmission rates. The data showed that (initial) hospital stays were generally longer for patients who were readmitted than for those who were not. As a result, short stays were not associated with a higher risk of readmission, meaning that hospital readmissions were not produced by premature hospital discharges in the study population. Furthermore, the spatial variation in readmission rates within 7 versus 8-30 days showed to be identical. Finally, it was found that countries or states with relatively shorter stays showed higher readmission rates and vice versa. Since patients with readmissions in all of the areas had on average longer initial stays, this finding at country level does illustrate that there seems to be a country specific trade off between length of stay and rate of readmission. An explanation should be sought in differences in health care arrangements per area, including factors that determine length of stay levels and readmission rates in individual countries (e.g. managed care penetration, after care by GP's or home care).

Adult↗
PubMed2002

Developmental expression of EphA4-tyrosine kinase receptor in the mouse brain and spinal cord.

Eph receptor tyrosine kinases and their ephrin ligands are involved in some of the most important steps during the development of the central nervous system, including cell migration, axon guidance, topographic mapping and synapse formation. Moreover, in the adult, they have been implicated in plasticity and regulation of neural stem cell function. One member of the Eph family, EphA4, can bind to both classes of ephrins and may have multiple functions in nervous system development. In order to look for potential sites of EphA4 action during central nervous system development, we conducted a spatio-temporal analysis of EphA4 protein expression. We used immunohistochemistry in preference to in situ hybridization because of the high likelihood that EphA4 protein is expressed on axon tracts, long distances from EphA4 mRNA. In the telencephalon, EphA4 was expressed in the developing cortex from embryonic day 11 (E11) and, later, on major cortical tracts including the corpus callosum and cortico-spinal tract. Robust EphA4 expression was also found in the hippocampus and fornix, and cells and tracts in the striatum. In the diencephalon, the thalamus, the hypothalamus and thalamo-cortical projection were strongly positive. In the mesencephalon, a number of different nuclei were weakly positive, most prominently the red nucleus. In the rhombencephalon, many nuclei were strongly positive including the cerebellum and one of its afferent paths, the inferior cerebellar peduncle, as well as the olivary region. In the spinal cord, there was a dynamic pattern of expression through development, with persistent expression in the dorsal funiculus and ventral grey matter.

Animals↗
PubMed2002

Developmental expression of EphA4-tyrosine kinase receptor in the mouse brain and spinal cord.

Eph receptor tyrosine kinases and their ephrin ligands are involved in some of the most important steps during the development of the central nervous system, including cell migration, axon guidance, topographic mapping and synapse formation. Moreover, in the adult, they have been implicated in plasticity and regulation of neural stem cell function. One member of the Eph family, EphA4, can bind to both classes of ephrins and may have multiple functions in nervous system development. In order to look for potential sites of EphA4 action during central nervous system development, we conducted a spatio-temporal analysis of EphA4 protein expression. We used immunohistochemistry in preference to in situ hybridization because of the high likelihood that EphA4 protein is expressed on axon tracts, long distances from EphA4 mRNA. In the telencephalon, EphA4 was expressed in the developing cortex from embryonic day 11 (E11) and, later, on major cortical tracts including the corpus callosum and cortico-spinal tract. Robust EphA4 expression was also found in the hippocampus and fornix, and cells and tracts in the striatum. In the diencephalon, the thalamus, the hypothalamus and thalamo-cortical projection were strongly positive. In the mesencephalon, a number of different nuclei were weakly positive, most prominently the red nucleus. In the rhombencephalon, many nuclei were strongly positive including the cerebellum and one of its afferent paths, the inferior cerebellar peduncle, as well as the olivary region. In the spinal cord, there was a dynamic pattern of expression through development, with persistent expression in the dorsal funiculus and ventral grey matter.

Animals↗
PubMed2002

Variant heparan sulfates synthesized in developing mouse brain differentially regulate FGF signaling.

Heparan sulfates (HSs) exert critical regulatory actions on many proteins, including growth factors, and are essential for normal development. Variations in their specific sulfation patterns are known to regulate binding and signaling of fibroblast growth factors (FGFs) via tyrosine kinase receptors (FGFRs). We previously reported differences in sulfation patterns between HS species expressed by embryonic day 10 (E10) and E12 mouse neural precursor cells. We have examined the abilities of the different HS species to support signaling of the relevant FGF-FGFR combinations expressed early during brain development. For FGF8, which only functions early (E8-E11), E10 HS showed preferential activation. The most potent signaling for FGF8 was via FGFR3c, for which E10 HS was strongly active and E12 HS had no activity. For FGF2, which functions from E10 to E13, HS from both stages showed similar activity and were more potent at activating FGFR1c than the other receptors. Thus, we find a stage-specific correlation with activation. To explore the potential mechanisms for the generation of these stage-specific HS species, we investigated the expression of the HS sulfotransferase (HSST) isozymes responsible for creating diverse sulfation motifs in HS chains. We find that there are stage-specific combinations of HSST isozymes that could underlie the synthesis of different HS species at E10 and E12. Collectively, these data lead us to propose a model in which differential expression of HSSTs results in the synthesis of variant HS species that form functional signaling complexes with FGFs and FGFRs and orchestrate proliferation and differentiation in the developing brain.

Alternative Splicing↗