Biomedical subjects

R D Madison

Publications and source records attributed to R D Madison.

At least 19 recordsLinked to original sources

PubMed2004

Axotomized mouse retinal ganglion cells containing melanopsin show enhanced survival, but not enhanced axon regrowth into a peripheral nerve graft.

Melanopsin is found in only approximately 2% of mouse retinal ganglion cells (RGCs), making these RGCs uniquely and directly photosensitive. Given that the majority of RGCs die after axotomy and that grafting of a peripheral nerve to the eye provides a permissive environment for axon regrowth, the present study examined the survival and axonal regrowth of melanopsin-containing RGCs in mice. One month after optic nerve transection and grafting, RGCs with regrown axons were labeled from the grafts and retinae were processed to visualize melanopsin and TUJ1. Melanopsin-positive and negative RGCs were counted and compared to axotomized RGCs from ungrafted eyes and uninjured RGCs. Melanopsin-positive RGCs showed a 3-fold increase in survival rate compared to non-melanopsin RGCs. Despite this enhanced survival, melanopsin-containing RGCs did not show increased axon regrowth into nerve grafts.

Animals↗

PubMed2000

Netrin-1 and peripheral nerve regeneration in the adult rat.

Axonal guidance during development of the nervous system is thought to be highly regulated through interactions of axons with attractive, repulsive, and trophic cues. Similar mechanisms regulate axonal regeneration after injury. The netrins have been shown to influence the guidance of several classes of developing axons. Although netrins have been implicated as axonal guidance cues in the developing peripheral nervous system, there has been no direct evidence of netrin-1 expression in either developing or adult peripheral nerve. The present study utilized competitive PCR and immunohistochemistry to demonstrate the localization of netrin-1 within adult rat sciatic nerve. The expression of netrin-1 mRNA and protein was compared for normal or regenerated sciatic nerve 2 weeks following either a crush or a transection and repair injury. The PCR data show that netrin-1 mRNA is normally expressed at low levels in peripheral nerve, and similar low levels are found 2 weeks following a crush injury. However, 2 weeks following nerve transection and repair there is approximately a 40-fold increase in netrin-1 mRNA levels. Immunohistochemistry data show that Schwann cells are the major source of netrin-1 protein in peripheral nerve. Our results suggest that netrin-1 mRNA levels are profoundly affected during peripheral nerve injury and regeneration. The localization of netrin-1 to Schwann cells suggests that this protein is strategically situated to influence axon regeneration in adult peripheral nerve.

Animals↗

PubMed2000

Survival of adult rat retinal ganglion cells with regrown axons in peripheral nerve grafts: a comparison of graft attachment with suture of fibrin glue.

OBJECT: The goal of this study was to examine whether the method of attachment of a peripheral nerve graft would have an effect on retinal ganglion cell (RGC) regeneration. METHODS: The number of adult rat RGCs with regrown axons in a peripheral nerve graft was compared under two grafting conditions: 1) attachment of the graft to the optic nerve stump made using a suture; and 2) attachment made using fibrin glue. Counts of RGCs retrogradely labeled with FluoroGold from the grafts 1 month after attachment revealed approximately seven times the number of RGCs in the fibrin-glue group compared with the suture group. CONCLUSIONS: The use of fibrin glue may be a useful tool for enhancing the regrowth of central nervous system neuron axons into peripheral nervous system grafts.

Animals↗

PubMed1999

Factors contributing to preferential motor reinnervation in the primate peripheral nervous system.

Functional recovery after nerve lesions in the peripheral nervous system requires the accurate regeneration of axons to their original target end organs. This paper examines axonal regeneration of the primate median nerve lesioned at the wrist over nerve gap distances of up to 50 mm. Nerve gaps were bridged by either a sural nerve graft or a biodegradable collagen nerve guide tube, and recovery was followed for up to 1100 d. Nondestructive physiological methods were used to serially examine the number of regenerated motor units, and binomial statistics were used to compare the observed number of regenerated motor units with that expected if axonal regeneration of motor neurons were random. We found up to twice the number of motor units expected by random regeneration in direct suture and sural cable graft groups but not in nerve guide repairs of 20 or 50 mm. In all repaired nerves, aberrant motor axon collaterals were detected in digital sensory nerve territory. The results support the contention that the aberrant fibers represent collaterals of an alpha-motor axon, which also innervates muscle. Although the aberrant motor axon collaterals remained in digital sensory nerve territory for long periods, they remained relatively immature compared with their sibling collateral projecting to muscle, or sensory axons within the digital nerve. The number of such aberrant motor axon collaterals decreased over time in some repair groups, suggesting a selective pruning of the inappropriate collateral under certain conditions.

Action Potentials↗

PubMed1998

lambda RNA internal standards quantify sensitivity and amplification efficiency of mammalian gene expression profiling.

There is an increasing interest in being able to document simultaneous levels of multiple mRNAs from limited amounts of mammalian tissue. The combination of amplified antisense RNA (aRNA) and reverse Northern blot analysis is one technology that allows the measurement of relative levels of multiple mRNAs. However, potential problems exist with this approach, such as (i) unknown amplification efficiencies and sensitivity of detection, (ii) an inherent 3' bias of amplified products and (iii) cross-hybridization of homologous mRNAs with the gene targets. Each of these potential problems was addressed experimentally by the use of poly(A) RNA internal standards synthesized from lambda phage (lambda) DNA. The results showed detection levels of as few as 10 copies of the poly(A) RNA internal standards. The internal standards aid in the optimization of reaction conditions and also reduce dependence on traditional "housekeeping" genes whose mRNA levels might or might not change. The overall results of these experiments highlight and extend the general usefulness of amplified antisense aRNA and reverse Northern blot analysis to study mRNA expression profiles.

Animals↗

PubMed1997

Monoclonal antibody interaction with the third immunoglobulin-like domain of N-CAM is sufficient to cause cell migration.

Cellular adhesion molecules can influence a variety of biological mechanisms in the nervous system. These range from the processes of normal development and maintenance to neural plasticity and recovery following injury. The elucidation of the intricate contributions of these molecules will require the correlation of functional assays with specific molecules and the specific binding domains of such molecules with multiple signaling pathways. The data presented in this paper show that the monoclonal antibody anti-NCAM16, directed against the third immunoglobulin-like domain of the neural cell adhesion molecule N-CAM, is capable of stimulating the complex biological process of cell migration in primary embryonic motor neurons and human neuronal cell lines.

Amino Acid Sequence↗

PubMed1996

Reinnervation accuracy of the rat femoral nerve by motor and sensory neurons.

Previous studies in the rat femoral nerve have shown that regenerating motor neurons preferentially reinnervate a terminal nerve branch to muscle as opposed to skin, a process that has been called preferential motor reinnervation. However, the ability of sensory afferent neurons to accurately reinnervate terminal nerve pathways has been controversial. Within the dorsal root ganglia, sensory neurons projecting to muscle are interspersed with sensory neurons projecting to skin. Thus, anatomical studies assessing the accuracy of sensory neuron regeneration have been hampered by the inability to reliably determine their original innervation status. A sensory neuron that regenerated an axon into a terminal nerve branch to muscle might represent either an appropriate return of an original sensory afferent to muscle stretch receptors or the inappropriate recruitment of a cutaneous sensory afferent that originally innervated skin. The current experiments used a labeling strategy that effectively labels motor and sensory neurons projecting to a terminal nerve branch before experimental manipulation of the parent mixed nerve. Our results confirm previous observations concerning preferential motor reinnervation for motor neurons, and show for the first time anatomical evidence of specificity during regeneration of sensory afferent projections to muscle. In addition, the accuracy of sensory afferent regeneration was highly correlated with the accuracy of motor regeneration. This suggests that these two distinct neuronal populations that project to muscle respond in parallel to specific guidance factors during the regeneration process.

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PubMed1995

Selective laser-activated lesioning of prelabeled fetal hippocampal grafts by intracellular photolytic chromophore.

Selective removal of grafted tissue is critical to assess the functional role of that tissue in the host, yet is technically difficult for well-dispersed neural grafts. We labeled fetal hippocampal cells with both a nuclear marker (5'-bromodeoxyuridine) and a cytoplasmic marker (latex microspheres) before grafting into normal adult hippocampus. A nontoxic chromophore, chlorin e6, was conjugated on to the surface of latex microspheres of the treatment group. Chlorin e6 produces cytotoxic singlet oxygen only during photoactivation. Grafted animals received transcranial exposure to various intensities of near-infrared laser light. Following laser exposure, grafts were observed in all groups except in transplants prelabeled with chlorin e6 latex microspheres. An optimal laser exposure of 50-100 J/cm2 (4-8 min) was found to selectively remove only the chlorin e6-containing grafted cells. With increasing doses of laser illumination, non-specific lesions of the host tissue surrounding the graft were induced. Quantitative graft analysis, in the absence of laser exposure, indicated that the survival of grafted cells was similar between control transplants labeled with latex microspheres alone and grafts labeled with latex microspheres plus chlorin e6. This is further evidence that chlorin e6 by itself was not toxic without laser exposure. The results clearly demonstrate that singlet oxygen-induced cell photolysis can result in selective, non-invasive removal of dispersed grafted cells located in adult hippocampus. This technique may facilitate defining specific mechanisms of action of grafted cells which mediate functional recovery in different host conditions.

Animals↗

PubMed1995

Monkey median nerve repaired by nerve graft or collagen nerve guide tube.

Nerve regeneration was followed in 15 median and 1 ulnar nerve of eight Macaca fascicularis monkeys by serial electrophysiological assessments over a period of three and a half years. Nerve gaps of 5 mm at the wrist were bridged by collagen-based nerve guides, nerve autografts, or direct suture repairs. Thenar muscle reinnervation occurred between 50 and 70 d for all groups, indicating axonal elongation rates of approximately 1 mm/d. The recovery rates of the compound muscle action potential (CMAP) and the compound sensory action potential (CSAP) amplitudes were significantly slower after direct suture repair compared to the other two procedures, although the final levels of recovery were all comparable. Similar results were achieved in one median and one ulnar nerve following nerve guide repair of a 15 mm nerve gap. The functional reinnervation of Pacinian corpuscles was detected in all cases following either nerve graft or nerve guide repair, with similar amplitudes and latencies of the tactile evoked CSAP for both types of repair. Histological analysis demonstrated a significant increase in the number of myelinated axons in the median nerve distal to the nerve lesions following both nerve graft and nerve guide repairs compared to proximal and normal controls, with significant reductions of fiber diameter and corresponding increases in g-ratio. The return of a bimodal frequency distribution of myelinated axon fiber diameter was confirmed by three-dimensional surface plots which illustrate the frequency distribution of the relationship between fiber diameter and g-ratio. These combined results demonstrate that nerve regeneration after repair of a 5 mm nerve gap with a collagen nerve guide in the nonhuman primate is similar to that after graft repair, and the final level of physiological recovery for both repair procedures is comparable to direct suture repair of the median nerve.

Action Potentials↗

PubMed1994

Quantitative graft integration of fetal hippocampal transplants labeled with 5' bromodeoxyuridine into normal adult hippocampus.

Quantitative studies of neural graft development require: (1) a cell label which is both preferential for neurons and can be measured in terms of specific labeling; (2) a serial reconstruction method for identifying labeled cells in a three-dimensional pattern in the host; and (3) measurements of cell dispersion which indicate the specificity of cell movement in the host. We have used 5' bromodeoxyuridine (BrdU) as a nuclear marker of transplanted fetal hippocampal cells, performing daily injections into the donor to label the majority of cells in the hippocampus between Gestation Days 15 and 19. After harvesting, the BrdU-labeled hippocampal cell suspensions were either cultured or grafted into normal adult hippocampus. The labeling index of these fetal hippocampal cells was calculated to be 90% in both smears of cell suspensions and 48-h cultures and these were predominantly (77%) neurons by immunostains. Quantitative studies of potential toxicity of the BrdU in culture also revealed no differences in neurite development or survival, compared to unlabeled cells. The survival and migration of grafted cells were quantitatively evaluated by three-dimensional serial reconstruction of host brain sections. Absolute graft cell survival (cells recovered/cells injected) varied according to location: grafts in close proximity to the ventricle and white matter tracts (corpus callosum or fimbria) showed improved survival (20 and 25%, respectively) compared to grafts localized entirely within the (normal) hippocampus (only 9% survival). Although the grafts expanded considerably from the initial injection site, the graft cell dispersion appeared nonspecific. Thus, this format of quantitative graft assessment indicates that under normal (nonlesioned) host conditions the location of the grafts critically influenced graft development and there was minimal specific migration of grafted cells into cell body regions.

Animals↗

PubMed1994

Point sources of Schwann cells result in growth into a nerve entubulation repair site in the absence of axons: effects of freeze-thawing.

Axonal regeneration in the peripheral nervous system requires the comigration of Schwann cells along with or ahead of the growing neurites. The present studies were undertaken to elucidate some of the parameters that influence Schwann cell migration into a nerve wound site. We used a modification of an entubulation repair model with two isolated segments of denervated nerve to show that Schwann cells in vivo can bridge a 10-mm distance in the absence of axons. Two pieces of isolated nerve served as point sources of Schwann cells. A crucial finding was that in order for Schwann cells to bridge this distance both isolated/denervated nerve stumps had to be living. In cases where one nerve stump was frozen Schwann cells were not able to bridge the gap distance, although other cell types made up a tissue bridge between both stumps. We have further shown that as Schwann cells grow into this nerve wound site they stain intensely for the low-affinity NGF receptor p75NGFR. Related in vitro experiments showed that Schwann cells actively migrate on cryostat sections of either intact or previously lesioned sciatic nerve, suggesting that they can grow on either axon-containing or axonless areas of nerve tissue. Contrary to migration on sciatic nerve, although Schwann cells attached and survived on cryostat sections of optic nerve, they did not migrate on this substrate. The results of the present experiments suggest that Schwann cells can respond to specific diffusible and substrate bound signals which influence their migration into a nerve repair site.

Animals↗

PubMed1993

Noninvasively induced degeneration of neocortical pyramidal neurons in vivo: selective targeting by laser activation of retrogradely transported photolytic chromophore.

Interactions among neuronal subpopulations determine brain development and function. The present study illustrates the ability to noninvasively and selectively lesion targeted subpopulations of neurons in a highly specific, temporally defined, and geographically localized manner. This method provides a fundamental advance toward rigorous investigation of the importance of identified neuronal subtypes. Projecting pyramidal neurons of rats and mice were targeted by retrograde transport of latex nanospheres from contralateral motor cortex containing a nontoxic chromophore (chlorin e6) that produces cytotoxic singlet oxygen during photoactivation by deeply penetrating 670-nm light. Geographically defined regions of cortex were exposed to laser illumination at 670 nm to activate singlet oxygen production by the intracellular chromophore, and animals were sacrificed from 4 h to 7 days following laser exposure. Brains were processed to display degenerating neurons using sensitive silver-staining procedures. Selective damage occurred to pyramidal neurons with known callosal projections, solely within layers II/III and V of the illuminated region. Such subpopulation specificity provides models for neural transplantation and analysis of anatomically distributed neuronal networks.

Animals↗

PubMed1993

Induction of neuronal morphology in adrenal chromaffin cells cocultured with denervated Schwann cells.

We have studied the interactions of adrenal chromaffin and Schwann cells in a coculture system to observe whether denervated Schwann cells induce and support chromaffin cell differentiation in a manner analogous to nerve growth factor (NGF). Schwann cells induce both the accumulation of intense clumps of cocultured chromaffin cells on their surfaces and intense neurite outgrowth. This interaction is not blocked by antibodies to NGF or laminin. Conditioned medium from Schwann cell cultures fosters neurite outgrowth in chromaffin cells in a fashion qualitatively similar to NGF. Our data indicate that denervated Schwann cells exert a profound aggregating and differentiating effect upon chromaffin cells, inducing the expression of a neuronal phenotype via a predominantly NGF-independent mechanism.

Adrenal Glands↗

PubMed1992

Anatomical and physiological localization of prelabeled grafts in rat hippocampus.

Dissociated rat fetal hippocampal cells were grafted into normal adult rats. The fetal cells were incubated with one of a number of fluorescent compounds at the time of the dissociation to facilitate identification of the individual grafted cells. The fluorescent labels which were analyzed for this purpose included rhodamine latex microspheres, Cascade blue latex beads, rhodamine-dextran-amine, DiI, and carboxyfluorescein ester. The labeled cells were stereotaxically placed as a suspension into normal rat host hippocampi. The rats were sacrificed 2 to 6 weeks after the grafting for in vitro physiological recordings, and the prelabeled grafts were located using fluorescence optics. During intracellular recordings neurons within the prelabeled grafts were injected with Lucifer yellow to visualize the morphology and integration of neuronal processes within the host. Following the recordings the host slices with the grafts were fixed in 4% paraformaldehyde for anatomical analysis. The ability to prelabel cellular grafts allows subsequent anatomical and physiological analysis of the integration of grafted neurons at the resolution of a single neuron. Such an analysis will improve our understanding of the survival, differentiation, migration, and integration of the grafted neurons and their potential to replace lost function in the lesioned hippocampus.

Animals↗

PubMed1992

Fluorescent labeling of dissociated fetal cells for tissue culture.

The ability to pre-label cells used in transplantation experiments would have the potential benefits of identification of cell type and associated processes and the analysis of graft migration in the host. We have used an in vitro tissue culture system as a model to test several fluorescent dyes for this application. Fetal rat hippocampal tissue (E17-E18) was dissociated and incubated in the presence of carboxyfluorescein ester (CFSE), rhodamine-B dextran amine (RBD), DiI, or rhodamine-labeled latex microspheres. Cells were cultured in defined medium for up to 1 month. Cells labeled with CFSE were initially bright but faded over several days. RBD labeled the soma of cells, but fluorescence intensity was lost over a period of a few weeks. Cells labeled with DiI possessed brilliant staining of neuronal processes for weeks. Latex microspheres brightly labeled the soma but not the processes of neurons; fluorescent debris and sterility were problems with this label. We conclude that CFSE and DiI have significant potential usefulness in vitro as markers of cell viability and process formation with mammalian fetal CNS cells, whereas RBD is much less permanent. Latex microspheres may be suitable for pre-labeling of cells for transplantation if purification and sterility can be enhanced over present preparations.

Animals↗

PubMed1992

Peripheral nerve repair with collagen conduits.

This paper describes the repair of peripheral nerves with a tubular conduit fabricated from collagen. The tubular collagen matrix was made semipermeable to permit nutrient exchange and accessibility of neurotrophic factors to the axonal growth zone during regeneration. In-vitro studies showed that the semipermeable collagen conduit allowed rapid diffusion of molecules the size of bovine serum albumin and was adequately cross-linked for controlled resorption in vivo. Studies on primates suggest that collagen conduits worked as effectively as nerve autografts in terms of physiological recovery of motor and sensory responses. The results of in-vitro and in-vivo studies of the collagen conduit represent a significant step towards our specific aim of developing suitable off-the-shelf prostheses for clinical repair of damaged peripheral nerves.

Animals↗

PubMed1991

A collagen-based nerve guide conduit for peripheral nerve repair: an electrophysiological study of nerve regeneration in rodents and nonhuman primates.

When a peripheral nerve is severed and left untreated, the most likely result is the formation of an endbulb neuroma; this tangled mass of disorganized nerve fibers blocks functional recovery following nerve injury. Although there are several different approaches for promoting nerve repair, which have been greatly refined over recent years, the clinical results of peripheral nerve repair remain very disappointing. In this paper we compare the results of a collagen nerve guide conduit to the more standard clinical procedure of nerve autografting to promote repair of transected peripheral nerves in rats and nonhuman primates. In rats, we tested recovery from sciatic nerve transection and repair by 1) direct microsurgical suture, 2) 4 mm autograft, or 3) entubulation repair with collagen-based nerve guide conduits. Evoked muscle action potentials (MAP) were recorded from the gastrocnemius muscle at 4 and 12 weeks following sciatic nerve transection. At 4 weeks the repair group of direct suture demonstrated a significantly greater MAP, compared to the other surgical repair groups. However, at 12 weeks all four surgical repair groups displayed similar levels of recovery of the motor response. In six adult male Macaca fascicularis monkeys the median nerve was transected 2 cm above the wrist and repaired by either a 4 mm nerve autograft or a collagen-based nerve guide conduit leaving a 4 mm gap between nerve ends. Serial studies of motor and sensory fibers were performed by recording the evoked MAP from the abductor pollicis brevis muscle (APB) and the sensory action potential (SAP) evoked by stimulation of digital nerves (digit II), respectively, up to 760 days following surgery. Evoked muscle responses returned to normal baseline levels in all cases. Statistical analysis of the motor responses, as judged by the slope of the recovery curves, indicated a significantly more rapid rate of recovery for the nerve guide repair group. The final level of recovery of the MAP amplitudes was not significantly different between the groups. In contrast, the SAP amplitude only recovered to the low normal range and there were no statistically significant differences between the two groups in terms of sensory recovery rates. The rodent and primate studies suggest that in terms of recovery of physiological responses from target muscle and sensory nerves, entubulation repair of peripheral nerves with a collagen-based nerve guide conduit over a short nerve gap (4 mm) is as effective as a standard nerve autograft.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1991

Optic nerve tissue sections derived from myelin-deficient mutant mice as culture substrata for fibroblast adhesion and spreading.

The substrate properties were compared between normal and myelin-deficient central nervous system (CNS) tissues by an in vitro assay of cell attachment and spreading. Fibroblasts (3T3) were plated onto culture substrata consisting of optic nerve tissue sections cut from normal or two myelin-deficient mutant mice, Shiverer and Quaking. Optic nerve sections from either of the mutant animals supported more 3T3 fibroblast spreading and adhesion than sections derived from animals with normal myelin. These results demonstrate that CNS myelin influences the ability of cells to attach and spread and that it is the actual presence of myelin which is inhibitory rather than the presence of optic nerve axons or oligodendrocytes.

Animals↗