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PubMed1990

The role of ornithine decarboxylase and polyamines in regeneration of the frog sciatic nerve.

The current study examined both in vivo and in vitro the effects of alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC), on regeneration of sensory axons from a local crush of the adult frog sciatic nerve. If daily injections of DFMO started at the same time as crushing and continued throughout the regeneration period (7 days) the outgrowth in vivo of new sensory axons was reduced by about 30%. If DFMO injections started 2 days after crushing, the outgrowth distance did not differ from control values. The sensory axons of a cultured frog sciatic nerve with the attached spinal ganglia start to regenerate from a local crush applied 7 days after the start of the incubation. Five days after crushing the outgrowth distance was 4.5 mm. At the end of the culturing period (7 + 5 days) both the putrescine and spermidine concentrations in the ganglia had increased about 2.5 times, whereas the spermine concentration remained constant. The presence of 10 mM DFMO throughout the culturing period, 7 + 5 days, almost depleted putrescine and prevented the spermidine increase in the ganglia without affecting the regeneration distance. In the nerve putrescine was only reduced by 55% and the other polyamines were unaffected by DFMO. The results show that DFMO influences the early onset of regeneration in vivo. The in vitro results indicate that this is not due to a close mechanistic relationship between the perikaryonal ODC/polyamine system and nerve regeneration. The question of whether polyamines are of local importance for regeneration of the frog sciatic nerve cannot be answered by the present results.

Animals↗

PubMed1983

The axon reaction in motor and sensory neurones of mice studied by a monoclonal antibody marker of neurofilament protein.

Following unilateral sciatic nerve crush in mice, changes in the neurofilament content of neuronal perikarya were studied, using a monoclonal antibody to neurofilament protein (RT97). In the spinal cord, anterior horn motor neurones, normally unstained, showed a positive staining reaction with immunoperoxidase on the operated side. This reaction was short lived and maximal on the 11th post-operative day. In spinal ganglia, the proportion of positively staining sensory neurones showed an earlier but otherwise similar increase. In both cases, the response was well defined and contrasted with the changes on Nissl staining, which were markedly different in the two populations of neurones. In the nerve crush region, although regenerating axons were visible with silver staining only 5 days post-operatively, neurofilament protein was not demonstrated in these axons until several days later, after the peak perikaryal increase. These results suggest that an increase in perikaryal neurofilament protein is a consistent and quantifiable event following distal axon trauma, possibly indicating either synthesis of protein subunits or repolymerization of neurofilaments prior to their transport distally down the regenerating axons. The findings may be useful in identifying neurones with distal axon lesions in experimental and other neuropathological material.

Animals↗

PubMed1983

P2 protein-induced experimental allergic neuritis. An ultrastructural study.

Experimental allergic neuritis (EAN) was induced in 2 groups of inbred Lewis rats by sensitization with P2 protein and peripheral nervous system (PNS) myelin, both purified from bovine intradural roots. Light- and electronmicroscopic study of P2-induced EAN revealed demyelinative lesions in spinal ganglia and root nerves and less frequently in peripheral nerves and root entry zones. Both small and large myelinated fibers were demyelinated, contradictory to the reported selective binding of anti-P2 antibodies to myelin of large fibers. The early lesions were characterised by perivenular lymphocytic infiltration, and subsequent "dissolution" of myelin sheath was associated with invasive of phagocytic cells. The distribution of demyelinative lesions and patterns of demyelination resembled those of PNS myelin-induced EAN except that the disease was milder and dissolution of myelin and intramyelinic edema were more frequently found in P2-induced EAN. The abundance of demyelination in P2-induced EAN strikes contrast to the scarcity of myelin change in experimental allergic encephalomyelitis (EAE) induced by myelin basic protein immunization.

Animals↗

PubMed1990

Axonal transport of substance P-like immunoreactivity in ganglioside-treated diabetic rats.

This study examined the effect of treatment of control and streptozotocin-diabetic rats with a mixture of gangliosides, derived from bovine brain, on parameters of axonal transport of substance P-like immunoreactivity (SPLI) and its levels in sciatic nerve and lumbar spinal ganglia. Rats were treated daily (10 mg/kg i.p.) for 28 days and compared with untreated control and diabetic groups. The duration of diabetes was 28 days in both cases. Untreated diabetic rats showed deficits in accumulation of axonally transported SPLI proximal (59% of controls) and distal (34% of controls) to sciatic nerve ligations (left in place for 12 h). Rates of accumulation were unaltered by diabetes. There were small numerical reductions in the SPLI content of unconstricted sciatic nerve and of L4 and L5 dorsal root ganglia in diabetic rats. None of these diabetes-associated changes was altered by ganglioside treatment, nor was there any indication of an effect of gangliosides on substance P in non-diabetic rats. The implications are discussed in relation to the possible pathogenesis of diabetic neuropathy.

Animals↗

PubMed1979

Differential response of embryonic cells to culture on tissue matrices.

Cell responses to different natural substrates have been followed by scanning microscopy in order to evaluate the role of these substrates in morphogenesis. Matrix has been isolated then repopulated with suspensions of embryonic cells from chick skin, spinal ganglia, duodenal epithelium and heart. In some cases outgrowth from amphibian embryonic tissue was used. Basal lamina of the Xenopus tail may be exposed by freezing and thawing the tissue, or by EDTA treatment. The underlying lamella of orthogonally oriented collagen fibers may be exposed by use of trypsin or hyaluronidase. Trypsin causes more clumping of collagen fibers and a coarser texture of the matrix. On trypsin isolated basement lamella, nerve cell processes grow out on the surface and show no strong tendency to penetrate the lamella while skin mesenchymal cells commonly burrow among the collagen plies. Epithelial cells remain on the surface. On the basal lamina mesenchymal cells ruffle in early stages of culture, then flatten. Epithelial cells flatten rapidly on the lamina. These differences in cell response are in some cases closely related to cell behavior in vivo and suggest that cells show a selective response to the chemical composition of the substrate as well as to its physical conformation.

Animals↗

PubMed1986

A herpes simplex virus mutant is temperature sensitive for reactivation from the latent state: evidence for selective restriction in neuronal cells.

When the replicative defect in the HSV-1 temperature sensitive mutant tsI was repaired, the agent derived (RI-1) was found to possess an additional temperature sensitive lesion limiting its reactivation from the latent state. Thus, when spinal ganglia from latently infected mice were scored for reactivation by cocultivating them with indicator cells in vitro, significantly more were found to be positive at 31 degrees than at 38.5 degrees. To assess a possible relationship between reactivation and replication in neurons, the replication of RI-1 in murine C1300 neuroblastoma cells was studied. In these cells, RI-1 was severely restricted, and viral replication was delayed at 38.5 degrees. Serial passage of RI-1 in neuroblastoma cells at the restrictive temperature resulted in selection of an agent which gained both the capacity to replicate efficiently in neuroblastoma cells and reactivate from the latent state at 38.5 degrees. However, the replication pattern of this neuron adapted virus in mouse embryo fibroblasts remained unchanged from the parental RI-1. Taken together, these results indicate that RI-1 possesses a neuron specific temperature sensitive replicative lesion which is also manifest during reactivation from the latent state.

Animals↗

PubMed1989

Peripheral competition in the control of sensory neuron numbers in Xenopus frogs reared with a single bilaterally innervated hindlimb.

Sensory neurons were counted in the hind-limb innervating spinal ganglia on both sides of juvenile Xenopus frogs which, as tadpoles, had had one hind limb bud amputated prior to innervation, and a channel made to allow innervation of the remaining limb bud from both sides. The total number of sensory neurons surviving on the two sides approximated the number on one side of normal frogs, the ipsilateral and contralateral numbers being negatively correlated. These effects differ markedly from the effects on motoneuron numbers, suggesting different control mechanisms of cell death in the two neuronal classes.

Animals↗

PubMed1985

Localization of chromatographically characterized oxytocin and arginine-vasopressin to sensory neurones in the rat.

Following treatment with colchicine 50-60% of all neurones in rat trigeminal and L5 spinal ganglia showed oxytocin (OXT)- and arginine-vasopressin (AVP)-like immunoreactivity. Further, OXT and AVP, together with their associated neurophysins, could be isolated from trigeminal ganglia by reversed-phase high-performance liquid chromatography followed by radioimmunoassay.

Animals↗

PubMed1988

Cationic channels activated by extracellular ATP in rat sensory neurons.

Single channels activated by externally applied ATP were investigated in cultured sensory neurons from nodosal and spinal ganglia of rat using patch clamp and concentration clamp methods. Mean conductance of single ATP-activated channels was 17 pS when measured at a holding potential of -75 mV in saline containing 3 mM Ca2+ and 1 mM Mg2+. Sublevels of conductance were detected in some cases. The current-voltage relationship for a single channel is highly non-linear and demonstrates inwardly directed rectification. The I-V curve obtained for single channels was identical to that for macroscopic current. ATP activated the channels in the absence of divalent cations (in ethylenediaminetetra-acetate-containing medium) as well as in their presence. This indicates that ATP as a free anion can activate the receptor. Ca2+ ions decreased both macro- and microscopic ATP-activated currents. The concentration dependence of this Ca2+ effect does not fit a single site binding isotherm. The single channel current demonstrated prominent fluctuations. When measured in the 0-4 kHz frequency band the amplitude of fluctuations evaluated as a double r.m.s. was about 30% of the mean amplitude of current. The autocorrelation function for the current fluctuations in an open channel could be approximated by a single exponential with the time constant of 0.4 ms. These fluctuations did not depend on the presence of divalent cations in the external medium. The open time distribution for the investigated channels could be described by a sum of two exponentials. Presumably this reflects the existence of two subtypes of ATP-activated channels.

Adenosine Triphosphate↗

PubMed1990

Responses of type A cat spinal ganglion neurons to repetitive stimulation of their central and peripheral processes.

Intracellular recordings were made from L7-S1 type A spinal ganglion neurons of anesthetized cats while electrical stimulation was delivered repetitively to their associated dorsal root and the sciatic nerve. The general response pattern of these neurons changed during stimulation at progressively higher rates. The changes were observable as jitter in onset latency of the evoked spikes, inability of evoked responses to follow electrical stimuli in a 1:1 manner (spike failure), reduction in action potential amplitude, and decomposition of the full spike into its non-myelinated and myelinated components. The frequency following ability of these spike components was in the order of full spike less than non-myelinated less than myelinated. In jitter in onset latency and inability to follow high frequency stimulation was determined only for the full spike, as is typical for antidromicity criteria, a wide frequency following spectrum was obtained for our sample of spinal ganglion neurons. Less than a third of the cells were able to follow stimulation rates in excess of 200 Hz, and about a fifth of the neurons failed to follow any rates greater than 20 Hz. Most of the neurons activated from both the dorsal root and sciatic nerve responded with the same pattern of stimulus-evoked responses. However, some of these cells exhibited strikingly different patterns to dorsal root and sciatic stimulation, including the presence of prepotentials following stimulation of one, but not the other, process. These prepotentials occurred in the depolarizing direction, at threshold stimulation were all-or-none in nature, generated spikes that varied in onset latency, and failed to occur at even low-to-moderate rates of stimulation. The results indicate that the frequency following spectrum of cat type A ganglion neurons is wide, and that it is their somata that are most vulnerable to high frequency stimulation. It is possible that some of the observed prepotentials are functional manifestations of synaptic contacts in spinal ganglia.

Action Potentials↗

PubMedDate not supplied

Spinal cord grafts: an intraocular approach to enigmas of nerve growth regulation.

The possible usefulness of intraocular transplantation in studies of spinal cord growth and regeneration has been evaluated. Defined segments of fetal rat spinal cord were grafted to the anterior chamber of the eye of adult rats. Such grafts become vascularized from the host iris, grow and develop neuron types, myelinated fiber bundles, astroglial populations (as shown by GFA-immunoreactivity), and electrical activity reminiscent of such features in normal spinal cord tissue. The intraocular technique permits studies of intrinsic circuitries as well as conditions for formation of afferent and efferent connections with the host iris and with other central or peripheral tissues which can be grafted into contact with the spinal cord grafts. One example of an intrinsic system preserved in the grafts is a rich network of nerve fibers with enkephalin-like immunoreactivity. When combined with cerebral cortex, the enkephalin-positive neurons of the spinal cord graft are able to form only very limited projections to the cortex graft. Special emphasis was given the possible formation of adrenergic afferents to spinal cord grafts. No appreciable ingrowth of peripheral sympathetic nerves occurred. Locus coeruleus grafts have many organotypical electrophysiological characteristics and were able to innervate adjacent spinal cord grafts provided that the sensory innervation of the host iris was removed. Experiments such as these suggest that "negative neurotropic" factors may be present in spinal cord and possibly relate to the unique relationship between spinal ganglia and spinal cord.

Adrenergic Fibers↗

PubMed1986

The biphasic effect of phospholipase A2 inhibitors on axon elongation.

We have analyzed changes in axon elongation rate in vitro induced by three different phospholipase A2 inhibitors, namely, bromophenacyl bromide (BPB), quinacrine and Sr2+. Spinal ganglia were obtained from 19.5-day-old rat fetuses and explanted onto polyornithine substrata. Axon length was measured at 1, 2 and 3 days in vitro (d.i.v.). We have previously shown that BPB added at 5 hr in vitro (h.i.v.) modifies the structure of growth cones and inhibits or promotes axon elongation according to its concentration. Now, we have observed that 0.5 x 10(-6) M BPB also stimulates axon elongation when added at 1 d.i.v. Culture media with different Ca2+ concentrations were used to test Sr2+ added at 1 d.i.v. In 2.3 mM Ca2+ only an inhibitory effect was observed with 8 mM Sr2+. On the other hand, in 0.3 mM Ca2+, axon growth was stimulated by 0.6-1.2 mM Sr2+ but inhibited by 6 mM Sr2+. Quinacrine, added at 5 h.i.v. was inhibitory at 10(-5) M and showed no effects at 10(-6) M. However, after washing quinacrine, normal elongation rate was recovered by those previously treated with 10(-5) M and axon growth was enhanced in those treated with 10(-6) M. Since three different phospholipase A2 inhibitors, tested in different situations, produce a similar biphasic effect on axon elongation rate, we postulate that this enzymatic activity is involved in the motility of axon growth cones.

Acetophenones↗

PubMed1993

Transition of localization of the N-Myc protein from nucleus to cytoplasm in differentiating neurons.

N-myc is a developmentally regulated proto-oncogene encoding a putative sequence-specific DNA-binding protein. Previous studies on tissue distribution of N-myc transcripts indicated that one of the major sites of N-myc expression is the CNS and neural crest derivatives in developing embryos. We investigated N-Myc protein expression in embryonic neural tissues and found that the protein was usually localized in the nucleus, but accumulated in the cytoplasm upon differentiation of specific classes of neurons, e.g., retinal ganglion cells, neurons of spinal ganglia, and Purkinje cells of the cerebellum. The change of localization of N-Myc from the nucleus to the cytoplasm indicates a novel feature of regulation of myc family proteins and suggests functions of N-myc in the cytoplasm of maturing neurons.

Amino Acid Sequence↗

PubMed2004

Expression of WW domain-containing oxidoreductase WOX1 in the developing murine nervous system.

WW domain-containing oxidoreductase WOX1, also known as WWOX or FOR, is a proapoptotic protein and a putative tumor suppressor. Hyaluronidases such as PH-20, Hyal-1 and Hyal-2 induce the expression of WOX1, and hyaluronidases and hyaluronan are involved in the embryonic development. In the present study, we document the expression of WOX1 in the developing murine nervous system. Immunohistochemical analysis revealed that WOX1 was differentially expressed in early dividing cells from all three germ layers from embryonic to perinatal stages. In murine fetuses, WOX1 was present prevalently in the brainstem, spinal cord and peripheral nerve bundles, but its expression decreased after birth. In parallel, the expression of WOX1, as determined by Western blotting, was significantly reduced in the brain stem and spinal cord of adult mice. Notably, high levels of WOX1 immunoreactivity was observed in the neural crest-derived structures such as cranial and spinal ganglia and cranial mesenchyme during the late fetal stage. In the adult brain, WOX1 is abundant in the epithelial cells of the choroids plexus and ependymal cells, while a low to moderate level of WOX1 is observed within white matter tracts, such as axonal profiles of the corpus callosum, striatum, optic tract, and cerebral peduncle. WOX1 is shown to mediate apoptosis synergistically with p53 in vitro. Nonetheless, the expression profiles of WOX1 were found to be similar in both p53 wild type and knockout mice, suggesting that WOX1 expression is not controlled by p53-mediated gene transcription. Taken together, in this study we have shown the expression and distribution of WOX1 in developing and adult murine nervous system. The potential role of WOX1 in the neuronal differentiation is discussed.

Aging↗

PubMed2006

Transient receptor potential vanilloid 1-immunopositive neurons in the mouse are more prevalent within colon afferents compared to skin and muscle afferents.

Previous studies in our laboratories found that isolectin B(4)(IB(4))-positive polymodal nociceptors in the mouse do not express transient receptor potential vanilloid 1 (TRPV1), nor does deletion of TRPV1 compromise the ability of these afferents to detect thermal stimuli. Considering that IB(4)-positive afferents account for over 70% of cutaneous nociceptors and that 30-50% of all mouse primary afferents express TRPV1, it is highly likely that many TRPV1-positive fibers project to non-cutaneous structures. To investigate this issue, Alexa Fluor-conjugated wheat germ agglutinin (WGA) or IB(4) was injected into the nerves innervating quadriceps muscle (femoral) or hindlimb skin (saphenous) of male C57Bl/6 mice. Similarly, Alexa Fluor-conjugated cholera toxin-beta was injected subserosally into the distal colon. Spinal ganglia at the appropriate level (L2-3 for saphenous and femoral nerves; L6 for colon) were processed for TRPV1, calcitonin gene-related peptide (CGRP), neurofilament heavy chain (NHF) and IB(4) visualization and examined on a confocal microscope. Colon afferents contained the highest percentage of both TRPV1- and CGRP-positive neurons, followed by femoral (WGA) and saphenous afferents (WGA and IB(4)). In contrast, NHF staining was more prevalent among femoral afferents, followed by saphenous (WGA) and colon afferents. IB(4) binding was observed in very few colon or saphenous (WGA) afferents, with no femoral afferents binding or transporting IB(4). Considering that the largest percentages of TRPV1-positive neurons observed in this study were within visceral and muscle afferent populations (neurons that typically are not subject to noxious temperatures), these results suggest that TRPV1 may not function primarily as a temperature sensor but rather as a detector of protons, vanilloid compounds or through interactions with other membrane proteins.

Animals↗

PubMed1997

Hoxb-8 gain-of-function transgenic mice exhibit alterations in the peripheral nervous system.

To understand the developmental role of Hoxb-8, this relatively 5' Hoxb gene was ectopically expressed in embryonic regions where only more 3' Hox genes are normally expressed. Hoxb-8 coding sequences driven by a retinoic acid receptor beta2 promoter fragment were introduced in the mouse germ line by pronuclear injection. The promoter was chosen with the aim to extend rostrally the expression domain of the gene in neurectoderm and mesoderm at the time of development when Hox gene expression domains are being established. Embryos developing from DNA-injected zygotes, and from transgenic mouse lines were analyzed. Pattern alterations were observed in transgenic embryos, some of which involved the peripheral nervous system. Spinal ganglia in the mouse are first detectable around embryonic day 9.5. By day 11.5, the first of these ganglia (C1, Froriep's ganglion) has degenerated in the mouse and other amniotes. In contrast, this first ganglion did persist in the Hoxb-8 gain-of-function transgenic mice. We have started to take advantage of the phenotype of transgenic versus wild-type embryos to understand the mechanisms underlying the ontogeny and degeneration of Froriep's ganglion in wild-type mice, and the role of Hoxb-8 in C1 maintenance in transgenic embryos. The present work describes a morphological, histological and immunocytological analysis of both the degenerating and the permanent C1, and a preliminary characterization of the axonal extensions from the transgenic C1. We discuss the methodology of generating gain-of-function transgenic mice to study the genetics of pattern formation along the antero-posterior axis, and the usefulness of analyzing these particular Hoxb-8 transgenic embryos to understand some aspects of the ontogenesis and development of the upper cervical dorsal root ganglia.

Animals↗

PubMed1999

In vivo predegeneration of peripheral nerves: an effective technique to obtain activated Schwann cells for nerve conduits.

In vivo predegeneration of peripheral nerves is presented as a convenient and effective method to obtain activated Schwann cells and an enhanced cell yield following in vitro cultivation. The experiments conducted in rats were aimed at clinical use in gaining Schwann cell suspensions for filling artificial conduits in order to bridge peripheral nerve gaps. The rat sciatic nerve used as a model was transected distally to the spinal ganglia. Predegeneration in vivo was allowed to take place for 1, 2, 3 and 4 days and up to 1, 2 and 3 weeks. The nerve was then resected and prepared for cell cultivation. Schwann cells cultivated from the contralateral untreated nerve served as control. Immunostaining for S100, nerve growth factor receptor and the adhesion molecules N-cadherin and L1 was used to characterize the general state of the cultures. Viability was assessed by fluorescein fluorescence staining, and the proliferation index was determined by bromodeoxyuridine-DNA incorporation. The Schwann cells from predegenerated nerves revealed an increased proliferation rate compared to the control, whereas fibroblast contamination was decreased. Best results were obtained 1 week after predegeneration.

Animals↗

PubMed1998

Age-related decrease in the overall extent of perikaryal projections in rabbit spinal ganglion neurons.

The overall extent of the perikaryal projections of sensory neurons from spinal ganglia of young adult and aged rabbits was estimated by a stereological method using the electron microscope. The extent of perikaryal projections was significantly smaller in the aged animals. This age-related decrease did not seem to depend on factors intrinsic to the neuron, but on the absence of a satellite cell covering over extensive portions of the nerve cell body surface. This decrease may influence the organization of the subplasmalemmal cytoskeleton, metabolic exchange between the nerve cell body and its environment and perhaps also neuronal metabolism.

Aging↗