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PubMed2000

Induction of postnatal schwann cell death by the low-affinity neurotrophin receptor in vitro and after axotomy.

Schwann cells express the low-affinity neurotrophin receptor (p75), but no role for either the neurotrophins or their cognate receptors in Schwann cell development has been established. We have found that Schwann cells isolated from postnatal day 1 (P1) or P2 mice that were p75-deficient exhibited potentiated survival compared to wild-type cells after growth factor and serum withdrawal. There was, however, no disparity in the survival of p75-deficient and wild-type Schwann cells isolated at embryonic day 15, suggesting that the death-inducing effects of p75 are developmentally regulated. A comparable degree of cell death was also observed in the sciatic nerves of both wild-type and p75-deficient mice at P1. However, 24 hr after axotomy, there was a 13-fold increase in the percentage of apoptotic nuclei in the distal nerve stumps of the transected sciatic nerves of neonatal wild-type but not p75-deficient mice. The expression of both the p75 and nerve growth factor (NGF) genes was upregulated after axotomy in neonatal wild-type nerves. Collectively, these results suggest that NGF-mediated activation of p75 is likely to be an important mediator of Schwann cell apoptosis in the context of peripheral nerve injury.

Age Factors↗
PubMedDate not supplied

[Neuronal survival following axotomy: a model for the experimental section of the optic nerve].

INTRODUCTION AND OBJECTIVES: Retina is a privileged tissue to study the central nervous system (CNS) due to the disposition of different cell types and synaptic plexus. Moreover, the retina and the optic nerve are accessible for manipulation and surgery. Neuronal consequences of axotomy have been widely studied in this portion of the CNS. This paper offers a brief review of the morphological changes experienced by axotomized cells. We show these consequences in retinal ganglion cells (RGCs) injured by optic nerve section. DEVELOPMENT: Neurons transmit information to other cells through their axons; by cutting the axon not only the synaptic transmission is interrupted, but cell death is induced in axotomized neurons. In many cases degeneration of pre and postsynaptic neurons is also triggered. However, in the retinal model, some RGCs are able to survive to axotomy; they experience profound changes trying to adapt to a new situation. Such changes have been observed in labeled axotomized cells. CONCLUSION: Knowledge of the qualitative and quantitative changes after injury, as well as the determination of no return point in the evolution of the lesion are aspects that must be known before to start a repairing therapy of the function.

Animals↗
PubMed1998

Axotomy upregulates the anterograde transport and expression of brain-derived neurotrophic factor by sensory neurons.

In addition to the known retrograde transport of neurotrophins, it is now evident that endogenous brain-derived neurotrophic factor (BDNF) is transported in the anterograde direction in peripheral and central neurons. We used a double-ligation procedure that distinguishes between anterograde and retrograde flow to quantify the anterograde transport of endogenous neurotrophins and neuropeptides in the peripheral nervous system before and after axotomy. BDNF accumulation proximal to the ligation (anterograde transport) was twice that distal to the ligation (retrograde direction). Anterograde transport of nerve growth factor and neurotrophin-3 was not evident. Furthermore, BDNF anterograde transport increased 3.5-fold within 24 hr after sciatic nerve injury or dorsal rhizotomy. Anterograde transport of substance P and calcitonin gene-related peptide decreased after peripheral nerve lesion, demonstrating that there was no generalized increase in anterograde transport. To determine the source of the anterogradely transported BDNF, we performed in situ hybridization in a variety of tissues before and after axotomy. Expression of BDNF mRNA in proximal nerve segments did not change with treatment, showing that the increased accumulation of BDNF was not a result of increased local synthesis. BDNF mRNA and protein were expressed by dorsal root ganglion sensory neurons but not by motor neurons. BDNF mRNA expression was increased 1 d after nerve injury, and BDNF protein was also increased twofold to threefold, suggesting that sensory neurons are the major contributing source of the increased BDNF traffic in the sciatic nerve. Our results suggest that increased anterogradely transported BDNF plays a role in the early neuronal response to peripheral nerve injury at sites distal to the cell body.

Animals↗
PubMed1998

Axotomy reduces the effect of analgesic opioids yet increases the effect of nociceptin on dorsal root ganglion neurons.

There is some doubt as to the effectiveness of opioids in the management of neuropathic pain. We therefore examined the actions of morphine and the opioid-like peptide nociceptin (both 1 mu) on dorsal root ganglion (DRG) neurons that were isolated from control or from nerve-injured rats. Both substances reduced omega-conotoxin (CTX) GVIA-sensitive, N-type Ca2+ channel current and small persistent nifedipine/ CTX-insensitive (non-N, non-L type) current. Nifedipine-sensitive L-type current was unaffected. The effect of nociceptin was antagonized by naloxone benzoylhydrazone (nalbzoh) but not by naloxone. Sciatic nerve section (axotomy) profoundly reduced the effects of morphine and the mu-receptor agonist D-ala2, N-Me-Phe4,Gly-ol5 enkephalin (DAMGO). The effect of the kappa-agonist [(+)-(5alpha,7alpha, 8beta)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxaspiro(4, 5)dec-8-yl)-benzeneacetamide] (U69593) was unchanged, whereas the effect of nociceptin was increased. All agonists produced their strongest effects on the small, putative nociceptive cells and their weakest effects on the largest cells. The delta-receptor agonist, enkephalin D-pen2,5 (DPDPE), was without effect on control or on axotomized cells. These and other data suggest that the functional downregulation of mu-opioid receptors on sensory nerves contributes to the poor efficacy of opioids in neuropathic pain. Also, the increased effectiveness of nociceptin after axotomy supports the hypothesis that its actions are mediated via a "non-opioid" receptor. Pronounced suppression of Ca2+ channel current in axotomized DRG neurons by nociceptin led to a reduction in Ca2+-dependent K+ conductance and a marked increase in excitability. Despite this, the spinal administration of nociceptin or agonists that activate ORL1 (opioid-like orphan receptor) may prove to be of clinical interest in the management of neuropathic pain.

Analgesics↗
PubMed1990

Effects of advancing age on the central response of rat facial neurons to axotomy: light microscope morphometry.

Following axotomy, the regrowth of peripheral axons takes longer in older individuals than in young ones. The present study compares central responses of facial motor neurons to a crush injury of the facial nerve in 3-month-old and 15-month-old male rats sampled through 28 days post-crush (dpc). Neuronal somata, nuclei, and nucleoli were measured in 30 microns brain stem sections within subdivisions of the facial nucleus that contain the cell bodies responsible for the movement of the vibrissae. The temporal patterns of change in the size of the three structures were interpreted with reference to the re-establishment of functional connections, i.e., the return of voluntary vibrissae activity, which is delayed by 4 days in the older animals relative to the younger ones. There was no age-related difference in the pattern of somal swelling and recovery, nor was there an age-related difference in the response of nuclei and nucleoli to axotomy through 4 dpc. Both nuclei and nucleoli increased in size in animals of both age groups, but after 4 dpc in the older animals nuclear enlargement was prolonged and the nucleolar increases were less robust compared to the younger animals. The greatest age difference appeared with the re-establishment of functional connections. In the 3-month-old animals, the resumption of whisker activity coincided with vigorous transient increases in the sizes of nuclei and nucleoli; in the 15-month-old animals, there was little nuclear response to functional recovery and a comparatively small increase in nuclear sizes.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed2001

Motor neurons rapidly accumulate DNA single-strand breaks after in vitro exposure to nitric oxide and peroxynitrite and in vivo axotomy.

The mechanisms of neuronal degeneration in motor neuron disease are not fully understood. We tested the hypothesis that oxidative stress in vitro and axotomy in vivo induce single-strand breaks (SSB) in DNA, a form of early DNA damage, in adult motor neurons early during their degeneration. We developed and characterized a novel cell suspension system enriched in motor neurons from adult rat spinal cord ventral horn. This cell system is approximately 84% neurons, with approximately 86% of these neurons being motor neurons; approximately 72% of these motor neurons are alpha-motor neurons. Motor neuron viability in suspension is approximately 100% immediately after isolation and approximately 61% after 12 hours of incubation. During incubation, isolated motor neurons generate high levels of superoxide. We used single-cell gel electrophoresis (comet assay) to detect DNA-SSB in motor neurons. Exposure of motor neurons to nitric oxide (NO) donors (sodium nitroprusside or NONOate), H2O2, or NO donor plus H2O2 rapidly induces DNA-SSB and causes motor neuron degeneration, the occurrence of which is dose and time related, as represented by comet formation and cell loss. Motor neuron toxicity is potentiated by cotreatment with NO donor and H2O2 (at nontoxic concentrations alone). Peroxynitrite causes DNA-SSB in motor neurons. The DNA damage profiles (shown by the comet morphology and moment) of NO donors, NO donor plus H2O2, and peroxynitrite are similar. In an in vivo model of motor neuron apoptosis, DNA-SSB accumulate slowly in avulsed motor neurons before apoptotic nuclear features emerge, and the comet fingerprint is similar to NO toxicity. We conclude that motor neurons challenged by oxidative stress and axotomy accumulate DNA-SSB early in their degeneration and that the formation of peroxynitrite is involved in the mechanisms.

Animals↗
PubMed1987

Response of septal cholinergic neurons to axotomy.

In the present study we employed quantitative morphometric techniques to assay the response of septal cholinergic neurons following unilateral transection of the fimbria/fornix and supracallosal stria. Analysis of 50-micron-thick tissue sections with a Quantimet 920 image analysis system demonstrated a reduction in ChAT immunoreactivity as early as 1 day following denervation. This decrease was associated with a drop in the number of labeled cells ipsilateral to the lesion and a decrease in the area of cholinergic perikarya on the lesioned and nonlesioned side of the septum. The response at 1 day, however, was transient, and at 4 days the number of labeled neurons was not significantly different from controls. By 8 days we observed a dramatic reduction in the number and size of ChAT-positive cells ipsilateral to the lesion and a reduction in the size of cholinergic perikarya on the contralateral (i.e., nonlesioned) side. These values persisted throughout the remainder of the study. To assess more completely the morphologic response of neurons to axotomy than can be determined in 50-micron-thick tissue sections, we embedded the adjacent immunolabeled tissue section in Epon and then serially sectioned it to a thickness of 0.75-1.0 micron. By using this method, we were able to measure the area, length, and width of the cell, the area of the nucleus and nucleolus, and the position of the nucleus (i.e., eccentricity). Measurements were performed on ChAT-labeled and nonlabeled cells. The results of our studies demonstrate that cholinergic and noncholinergic cells responded to axotomy in a characteristic yet different fashion from each other and that this response could be quantitatively assayed. In general, labeled and nonlabeled cells on the lesioned side of the septum shrink in response to denervation. This shrunken state was reflected in measurements of cellular area, length, width, and nuclear area. Moreover, other measurements of cellular morphology (i.e., area of the nucleolus, position of the nucleus) indicate that none of the neuronal populations examined in the present study displayed morphologic evidence of regeneration. Our results indicate a dramatic loss of cholinergic perikarya ipsilateral to the lesion. Moreover, although a few neurons do persist they do so in a shrunken state. These data provide an essential baseline for the second study in this series, which will evaluate the effect of nerve growth factor on the survival of denervated septal neurons.

Animals↗
PubMed1993

Nitric oxide synthase-like immunoreactivity in lumbar dorsal root ganglia and spinal cord of rat and monkey and effect of peripheral axotomy.

With the immunofluorescence technique, nitric oxide synthase (NOS)-like immunoreactivity (LI) was found in a few medium-sized and small sensory neurons in lumbar (L) 4 and L5 dorsal root ganglia (DRG) of normal rat, and in most of these neurons, NOS-LI coexisted with calcitonin gene-related peptide and sometimes with substance P and galanin. NOS-immunoreactive nerve fibers, terminals and small neurons were also located in the dorsal horn of the segments 4 and 5 of the rat lumbar spinal cord with the highest density in inner lamina II. Many NOS-positive neurons and fibers were seen in the area around the central canal. A sparse network of NOS-immunoreactive nerve fibers was found in the ventral horn. After unilateral sciatic nerve cut in the rat, the number of NOS-positive neurons increased in the ipsilateral L4 and L5 DRGs, mainly in medium and small neurons, but also in some large neurons and very small neurons. NOS-LI could now also be seen in the ipsilateral dorsal roots, and in an increased number of fibers and terminals in both outer and inner lamina II of the ipsilateral dorsal horn. The number of NOS-immunoreactive neurons in lamina II of the ipsilateral dorsal horn was reduced. In the monkey L4 and L5 DRGs, many small neurons were NOS-immunoreactive, but only a few weakly stained nerve fibers and terminals were found in laminae I-IV of the dorsal horn at L4 and L5 lumbar levels. A few NOS-positive neurons were present in lamina X. The number of NOS-immunoreactive neurons was somewhat reduced in DRGs 14 days after peripheral axotomy, but no certain effect was seen in the dorsal horn. These results, together with earlier in situ hybridization studies, demonstrate that axotomy in rat induces a marked upregulation of NOS synthesis in primary sensory neurons, thus suggesting a role for NO in lesioned sensory neurons. In contrast, no such effect was recorded in monkey, perhaps indicating distinct species differences.

Amino Acid Oxidoreductases↗
PubMed1994

Lack of Purkinje cell loss in adult rat cerebellum following protracted axotomy: degenerative changes and regenerative attempts of the severed axons.

The cerebellar Purkinje cells, due to their geometrical disposition and their high calbindin content, offer an optimal system in which to test the adequacy of current opinions on axotomy effects. We have, therefore, analyzed with calbindin immunostaining the morphological changes of Purkinje cells from 1 day to 6 months after axonal section in the cerebellar white matter. This method allows us to study the morphological changes in their dendrites, cell bodies, and axons. We have also searched for simultaneous changes in glial cells and vascularization by using cell type-specific markers. In addition, an ultrastructural study of Purkinje cells, 7 days after large electrolytic lesions affecting the white matter and the overlying granular layer, was carried out to determine whether amputation of the recurrent collateral system provokes a fast neuronal death. Neither the Purkinje cells axotomized close to their cell bodies (electrolytic lesions) nor those axotomized in the white matter (cerebellar transection) degenerated. Thus, this study demonstrates that Purkinje cells are extremely resistant to axotomy; those severed in the white matter at distances varying from 100 microns to 3 mm remain alive for as long as 6 months. At all survival times studied, axotomized Purkinje cells exhibited few changes in their somata and dendrites, as well as in their glial microenvironment. The major changes occurred in the axonal compartment. Axonal alterations, namely the presence of torpedoes and hypertrophy of the recurrent collateral system, were early events already noticeable 24 hours after the lesion, although they later differed in their time course and spatial distribution. It is remarkable that the distal segments of the central stumps of the cut axons survived in large numbers without any apparent retraction, with their terminal varicosities apposed to the wall of the wound cavity even 6 months after the lesion. Nevertheless, these segments were thinner than normal Purkinje cell axons (axonal atrophy). Despite this apparent immutability, some regenerative attempts did occur in the severed axons, such as axonal sprouts penetrating the deeper region of the granular layer in zones close to the lesion, presence of arciform axons, and hypertrophy of the recurrent collateral system. However, the Purkinje cell axons did not regenerate, and these neurons remained separated from their targets by a cavity in virtually all cases.

Animals↗
PubMed1995

Neurofilament sidearm proteolysis is a prominent early effect of axotomy in lamprey giant central neurons.

In the accompanying paper, it was shown that axotomy of lamprey spinal axons induces the rapid formation of condensed neurofilamentous masses in the proximal axon stump near the lesion. In this study, we used immunocytochemical and Western blot analysis to characterize these masses further and to determine the time course of their formation and dispersal. We show that monoclonal antibodies specific to the "rod" domain of lamprey neurofilament protein strongly stain such masses in tissue sections without staining other axonal neurofilaments. Antibodies specific for the neurofilament "sidearm" domain fail to recognize neurofilamentous masses but stain other axonal neurofilaments. Western blots of spinal cord segments from the lesion site were compared to unlesioned cord and to samples of cord distant from the lesion. We found that a neurofilament rod-specific antibody identified breakdown products of the same size as the rod domain in samples from the lesion site, but not elsewhere. Other lesion-specific neurofilament breakdown products were recognized by a sidearm-specific antibody. This lesion-specific pattern of neurofilament proteolysis was visible at 1 day postlesion and was still present 3 weeks later. Immunocytochemistry showed masses of rod-staining neurofilaments in axon stumps by 12 hours postlesion that remained for 1-2 weeks postaxotomy; these dispersed with the onset of regeneration. Such neurofilament rod staining was also prominent in distal axon stumps undergoing Wallerian degeneration. We conclude that axotomy induces neurofilament sidearm proteolysis near the lesion, permitting antibody access to the rod domain. We suggest that sidearm loss causes the high packing density of neurofilaments within neurofilamentous masses near the lesion site and that neurofilament sidearm proteolysis can be used to distinguish degenerative from regenerative changes in lesioned lamprey axons.

Animals↗
PubMed1990

Astrocytic reactions in spinal gray matter following sciatic axotomy.

Astrocytic responses following unilateral sciatic nerve axotomy were examined in the spinal gray matter. Using an antiserum to glial fibrillary acidic protein (GFAP), immunoreactive astrocytes were studied in both dorsal and ventral gray matter at intervals from 2 days through 34 days post-axotomy. In all axotomized animals, increased numbers of strongly immunoreactive astrocytes were present in the gray matter ipsilateral to the surgery. Such astrocytes were absent from the contralateral intact side and from gray matter bilaterally in adjacent spinal segments not involved in formation of the sciatic nerve. These GFAP-positive astrocytes occurred not only in association with large motor neurons in the ventral gray matter but also in association with central processes of dorsal root ganglion neurons in the dorsal gray matter. The response was quite rapid, being discernible both dorsally and ventrally as early as the second post-operative day. This increased GFAP immunoreactivity persisted throughout the entire observation period, with the perikarya of large ventral motor neurons appearing to become surrounded or encapsulated by the immunoreactive processes. A further alteration noted at the longest post-operative intervals was the presence in the ventral gray matter of astrocytes appearing to be binucleate. The data obtained indicate that the astrocytic response is not related solely to reactions in motor neurons and, furthermore, the rapidity with which it develops in the dorsal gray matter suggests that its induction is not dependent upon transganglionic degeneration, which others have reported to occur weeks after peripheral nerve injury.

Animals↗
PubMed1994

Motoneuron survival is not affected by the proximo-distal level of axotomy but by the possibility of regenerating axons to gain access to the distal nerve stump.

The aim of this study was to examine whether axotomy-induced motoneuron death in adult mammals differ: (1) with the distance between the site of injury and the nerve cell body, and (2) if contact between the transected nerve stumps is established after the injury, compared with cases where contact is prevented. The hypoglossal nerve of adult rats was transected either proximally in the neck (proximal injury) or close to the tongue (distal injury). The nerve stumps were then either deflected from each other in order to prevent axon regeneration into the distal nerve stump, or sutured. Three months later, the extent of nerve cell loss was examined bilaterally in cresyl violet-stained sections of the hypoglossal nucleus. In addition, we examined hypoglossal neuron survival twelve months after a proximal nerve transection with prevented regeneration. Our results show that there was no significant difference in neuronal survival after a proximal nerve transection compared with a distal one, neither if contact between the nerve stumps was established nor if it was prevented. However, contact between the transected nerve stumps increased the likelihood of neuronal survival significantly after both proximally and distally located injury compared to nerve injury with prevented regeneration. There was no significant decrease in nerve cell survival after twelve months with prevented reinnervation compared with survival after three months. These observations indicate that the extent of axotomy-induced motoneuron death in adult mammals does correlate with the proximo-distal level of peripheral injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1996

Attenuation and recovery of nerve growth factor receptor mRNA in dorsal root ganglion neurons following axotomy.

The actions of nerve growth factor (NGF) are mediated by two receptor proteins, trk and p75. Recent evidence indicates that NGF upregulates the expression of both trk and p75 in responsive neurons including rat dorsal root ganglion (DRG) neurons. Axotomy by disconnecting the neuron from its source of target-derived NGF is predicted to lead to the downregulation of trk and p75 expression. However, previous studies of the effects of axotomy on trk and p75 mRNA expression in rat DRG have yielded discrepant results. We report that following sciatic nerve crush, trk and p75 mRNA levels in L4-L6 DRG decrease to approximately 50% of control levels at 4-14 days, return to control levels by 30 days, and are increased by approximately 30% at 60 days. Similar changes are observed following nerve transection although mRNA levels are slower in returning to normal and do not exceed control levels at later timepoints. Thus, trk and p75 expression decline early following target disconnection and later recover irrespective of target reinnervation. These observations indicate that target derived NGF is required for the maintenance of NGF receptor expression in adult rat DRG neurons and that non-target derived factors can appropriate this function following peripheral nerve injury.

Animals↗
PubMed1989

Laser microbeam axotomy and conduction block show that electrical transmission at a central synapse is distributed at multiple contacts.

Touch (T) sensory neurons in the leech innervate defined regions of skin and synapse on other neurons, including other T cells, within the ganglionic neuropil. The cells' receptive fields in the periphery are comprised of a central region, innervated by thick axons, and adjoining regions (minor fields) innervated by thinner axons. Secondary branches, known to be sites of synapses, emerge from the thinner and thicker axons. Pairs of T cells appear to make up to 200 separate contacts distributed within the neuropil. When the T cell is hyperpolarized, as occurs during natural stimulation of the cell, action potentials generated in the minor field and travelling into the ganglion along the thin axons may fail to conduct at central branch points. Evidence is presented, using axon conduction block and laser axotomy of cells filled with 6-carboxy-fluorescein, that synapses between separate groups of branches can function independently. Thus, selective activation of branches of the thin anterior axon produced a synaptic potential 36 +/- 6% of control amplitude, which was consistent with counts of 39 +/- 6% of contacts made by these branches. Laser axotomy of postsynaptic neurons showed that the anterior contacts indeed made the principal or only contacts activated during anterior conduction block. The results show that conduction block can modulate transmission within the ganglion, and it operates by silencing particular contacts between cells.

Animals↗
PubMed1992

Modulation of sprouting in organ culture after axotomy of an identified molluscan neuron.

We examined a variety of factors that might modulate the initiation of neurite outgrowth in an attempt to identify means by which its initiation might be accelerated. We examined this initiation from an identified molluscan neuron, Helisoma trivolvis buccal neuron B5 after axotomy, and determined whether the site of injury, temperature, ion channel blockers, pH, the second messenger cAMP, and protein synthesis affect the initiation of neurite outgrowth. Neurite outgrowth was assayed from axotomized neurons by filling the neurons intracellularly with Lucifer Yellow and examining the percentage of axons that extended (sprouted) new process after 9 or 24 h in organ culture. About one-third (31%) of axotomized neurons sprouted from the site of injury after 9 h (n = 22), and 88% (n = 20) sprouted after 24 h in saline at 22 degrees-24 degrees C when the injury was located 800 microns from the soma. Elevating the temperature to 32 degrees C or moving the lesion site to 400 or 1500 microns from the soma did not significantly alter the incidence of sprouting. Blocking sodium channels with tetrodotoxin [TTX (2 x 10(-5) M)] did not significantly reduce the incidence of sprouting, whereas the sodium channel agonist, veratridine (10(-5) M) did. The calcium channel blocker lanthanum (10(-6)-10(-4) M), stimulated neurite outgrowth; however, the organic calcium channel blocker verapamil (10(-3)-10(-5) M), and the calcium ionophore A23187 (10(-5) M), had no effect on sprouting. Exposure of neurons to the potassium channel blocker tetraethylammonium [TEA (20 mM)], elevation of intracellular pH with NH4Cl (5 mM), or treatment with the adenylate cyclase activator forskolin (10(-5) M) reduced the incidence of sprouting, whereas dideoxy-forskolin (10(-5) M) had no effect. Inhibition of protein synthesis with anisomycin (2 x 10(-4) to 2 x 10(-6) M) did not significantly suppress sprouting 24 h after axotomy. Both D and L isomers of glutamate (300 microM) stimulated sprouting. The present results suggest that the initiation of sprouting is regulated locally at or near the site of injury, and that blocking specific ion channels may either inhibit or enhance the initiation of neurite outgrowth.

Animals↗
PubMed1994

Regulation of vasoactive intestinal peptide expression in sympathetic neurons in culture and after axotomy: the role of cholinergic differentiation factor/leukemia inhibitory factor.

Vasoactive intestinal peptide (VIP) expression increases in sympathetic neurons when they are grown in dissociated cell or explant cultures and when they are axotomized in vivo. In dissociated cell culture, the magnitude of the VIP increase was reduced when nonneuronal cells were removed and medium conditioned by ganglionic nonneuronal cells increased VIP in neuron-enriched cultures. Antiserum against cholinergic differentiation factor (also leukemia inhibitory factor; CDF/LIF), but not against ciliary neurotrophic factor, immunoprecipitated this activity. Medium conditioned by sympathetic ganglion explants also contained a VIP-stimulatory molecule that was immunoprecipitated by CDF/LIF antiserum, and CDF/LIF antiserum partially blocked VIP induction in explants. CDF/LIF mRNA was increased in dissociated cell cultures, in ganglion explants and in vivo after axotomy. Our results suggest that CDF/LIF released from ganglionic nonneuronal cells plays an important role in regulating VIP after axotomy.

Animals↗
PubMed1994

Neurotrophic regulation of mouse muscle beta-amyloid protein precursor and alpha 1-antichymotrypsin as revealed by axotomy.

Kunitz-inhibitor containing forms of the beta-amyloid precursor protein (beta APP), known also as protease nexin II (PNII), and alpha 1-antichymotrypsin (alpha 1-ACT), a serpin, are important components of the serine protease and inhibitor balance in many tissues. In the nervous system, this balance may have trophic or growth factor activity at different stages of development, after injury and in disease states. In the current study, using immunocytochemistry and Western blotting with antibodies against the human homologues, we analyzed whether denervation affected the localization of beta APP and alpha 1-ACT in adult mouse muscle following axotomy. In mouse muscle, anti-human alpha 1-ACT antibody detected a 60 kD immunoreactive band and anti-human beta APP antibody a band at 92 kD in both normal and denervated extracts. beta APP was present in normal mouse muscle at both neuromuscular junctions and within intramuscular nerves. alpha 1-ACT was also detected at neuromuscular junctions, on the perineurium and endothelial cell surfaces. Following axotomy, both beta APP and alpha 1-ACT disappeared from intramuscular nerves simultaneously. However, at the neuromuscular junction, alpha 1-ACT decreased more rapidly with beta APP lingering before disappearing. Since both alpha 1-ACT as well as beta APP are present within senile plaques in Alzheimer's disease brains such experiments with the nicotinic, cholinergic neuromuscular synapse in denervated muscle may help to focus experiments on the mechanism of synapse loss as well as plaque deposition in this disease.

Amyloid beta-Protein Precursor↗
PubMed1995

Inhibition of formation of filopodia after axotomy by inhibitors of protein tyrosine kinases.

The activity of motile protrusions of the growth cone--filopodia, veils, and lamellipodia--is essential for directed growth of a neuronal process. The regulation of the formation of these protrusions is not well understood. Numerous filopodia and veils or lamellipodia form within minutes of transection of an Aplysia axon in culture, as the initial components of growth cones of regenerating neurites. Axotomy, therefore, provides a robust and reliable protocol for analyzing the formation of these protrusions. We evaluated the involvement of protein phosphorylation in the regulation of protrusive activity. Of the inhibitors of protein kinases assayed, only the inhibitors of protein tyrosine kinases--genistein, lavendustin A, herbimycin A, and erbstatin analogue--suppressed the formation of protrusions, as assessed by high magnification video microscopy. These drugs did not work by preventing resealing of the axon, as evident from visual inspection and by the unimpaired effectiveness of genistein or lavendustin in preventing formation of filopodia when applied after resealing. Inhibition of protein tyrosine kinases not only prevented the formation of actin-based protrusions, but also caused deterioration of the actin network underlying the protrusive area of preexisting growth cones. Consistent with an involvement of protein tyrosine phosphorylation in the generation of protrusive structures, immunocytochemistry revealed that aggregates of phosphotyrosine appeared at the margins of the axon, from which protrusions emerge shortly after axotomy. These results suggest a role for protein tyrosine phosphorylation in the formation and maintenance of actin-based protrusive structures.

Actins↗