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PubMed1997

Deprenyl induces GFAP immunoreactivity in the intact and injured dopaminergic nigrostriatal system but fails to counteract axotomy-induced degenerative changes.

There is increasing evidence of a trophic-like mechanism for some effects ascribed to deprenyl therapy in the central nervous system. For that, we studied the effect of chronic treatment with deprenyl in an animal model of Parkinson's disease induced by unilateral knife transection of the medial forebrain bundle (MFB) in adult rats. The experimental conditions included a 3-week pretreatment with deprenyl before stereotaxic transection of the MFB. Following surgery, deprenyl treatment was maintained for 3 weeks. Neurochemical and immunohistochemical procedures were used to study the dopaminergic system and reactive astrocytes in the nigrostriatal system. Deprenyl treatment failed to counteract the axotomy-induced degenerative changes of the nigrostriatal dopaminergic system. However, it was effective in increasing the density of reactive astrocytes in terms of glial fibrillary acidic protein (GFAP) immunoreactivity in the intact contralateral substantia nigra and also in further enhancing the axotomy-induced increase of GFAP immunolabeled astrocytes in the lesioned substantia nigra. This deprenyl-induced effect on GFAP immunoreactivity was confined to substantia nigra without effect in striatum. In addition, we found a medial to lateral gradient decrease in the distribution pattern of GFAP immunolabeled astrocytes. Axotomy increased the number of reactive astrocytes in either striatal area examined, but yet the preferential distribution pattern of reactive astrocytes in striatum was still evident.

Animals↗
PubMed1997

Targeted overexpression of the neurite growth-associated protein B-50/GAP-43 in cerebellar Purkinje cells induces sprouting after axotomy but not axon regeneration into growth-permissive transplants.

B-50/GAP-43 is a nervous tissue-specific protein, the expression of which is associated with axon growth and regeneration. Its overexpression in transgenic mice produces spontaneous axonal sprouting and enhances induced remodeling in several neuron populations (; ). We examined the capacity of this protein to increase the regenerative potential of injured adult central axons, by inducing targeted B-50/GAP-43 overexpression in Purkinje cells, which normally show poor regenerative capabilities. Thus, transgenic mice were produced in which B-50/GAP-43 overexpression was driven by the Purkinje cell-specific L7 promoter. Uninjured transgenic Purkinje cells displayed normal morphology, indicating that transgene expression does not modify the normal phenotype of these neurons. By contrast, after axotomy numerous transgenic Purkinje cells exhibited profuse sprouting along the axon and at its severed end. Nevertheless, despite these growth phenomena, which never occurred in wild-type mice, the severed transgenic axons were not able to regenerate, either spontaneously or into embryonic neural or Schwann cell grafts placed into the lesion site. Finally, although only a moderate Purkinje cell loss occurred in wild-type cerebella after axotomy, a considerable number of injured transgenic neurons degenerated, but they could be partially rescued by the different transplants placed into the lesion site. Thus, B-50/GAP-43 overexpression substantially modifies Purkinje cell response to axotomy, by inducing growth processes and decreasing their resistance to injury. However, the presence of this protein is not sufficient to enable these neurons to accomplish a full program of axon regeneration.

Animals↗
PubMed1997

BDNF and NT-4/5 prevent atrophy of rat rubrospinal neurons after cervical axotomy, stimulate GAP-43 and Talpha1-tubulin mRNA expression, and promote axonal regeneration.

Rubrospinal neurons (RSNs) undergo a marked atrophy in the second week after cervical axotomy. This delayed atrophy is accompanied by a decline in the expression of regeneration-associated genes such as GAP-43 and Talpha1-tubulin, which are initially elevated after injury. These responses may reflect a deficiency in the trophic support of axotomized RSNs. To test this hypothesis, we first analyzed the expression of mRNAs encoding the trk family of neurotrophin receptors. In situ hybridization revealed expression of full-length trkB receptors in virtually all RSNs, which declined 7 d after axotomy. Full-length trkC mRNA was expressed at low levels. Using RT-PCR, we found that mRNAs encoding trkC isoforms with kinase domain inserts were present at levels comparable to that for the unmodified receptor. TrkA mRNA expression was not detected in RSNs, and the expression of p75 was restricted to a small subpopulation of axotomized cells. In agreement with the pattern of trk receptor expression, infusion of recombinant human BDNF or NT-4/5 into the vicinity of the axotomized RSNs, between days 7 and 14 after axotomy, fully prevented their atrophy. This effect was still evident 2 weeks after the termination of BDNF treatment. Moreover, BDNF or NT-4/5 treatment stimulated the expression of GAP-43 and Talpha1-tubulin mRNA and maintained the level of trkB expression. Vehicle, NGF, or NT-3 treatment had no significant effect on cell size or GAP-43 and Talpha1-tubulin expression. In a separate experiment, infusion of BDNF also was found to increase the number of axotomized RSNs that regenerated into a peripheral nerve graft. Thus, in BDNF-treated animals, the prevention of neuronal atrophy and the stimulation GAP-43 and Talpha1-tubulin expression is correlated with an increased regenerative capacity of axotomized RSNs.

Animals↗
PubMed1998

Ribosomal RNA transcriptional activation and processing in hamster facial motoneurons: effects of axotomy with or without exposure to testosterone.

A key step in the ability of neurons to survive injury and successfully regenerate involves ribosomal RNA production. Testosterone propionate (TP), augments facial nerve regeneration in the adult hamster. TP modulates the nucleolar reaction in injured facial motoneurons, such that mature ribosome levels increase more rapidly and in greater magnitude than with injury only. In this study, molecular and electron microscopic stereologic approaches were used to determine the effects of axotomy and steroid treatment on ribosomal transcription and processing in facial motoneurons. Castrated adult male hamsters were subjected to right facial nerve transection at the stylomastoid foramen. Half the animals were subcutaneously implanted with one Silastic TP capsule, with the remainder sham implanted. For the in situ hybridization experiments, postoperative survival times were 0.5, 2, or 6 hours. In situ hybridization with a ribosomal DNA probe specific to the external transcribed spacer region located at the 5' end of the ribosomal gene was accomplished. Transcriptional activation of the rRNA gene occurred rapidly, within 2 hours, after injury only. Unexpectedly, TP treatment did not alter the time course or magnitude of rRNA transcriptional activity. For the electron microscope experiments, the postoperative time of 12 hours was selected. Stereologic analysis of 3 nucleolar subcomponents, fibrillar centers (site of rRNA transcription), nucleolonema (site of rRNA processing), and granular material (site of preribosome storage), was accomplished. TP decreased the nucleolonemal strands and the granular material, relative to injury only. These results suggest that, although rRNA transcription is rapidly activated by axotomy, rRNA processing is temporarily stalled. TP does not affect the early, axotomy-induced transcriptional activation of the ribosomal gene, but may, instead, prevent the subsequent disruption in rRNA processing. An hypothesis for the molecular mechanism by which steroids augment the regenerative capabilities of injured facial motoneurons is presented.

Animals↗
PubMed1999

Increased uptake and transport of cholera toxin B-subunit in dorsal root ganglion neurons after peripheral axotomy: possible implications for sensory sprouting.

In the present study we show that, in contrast to the rat, injection of cholera toxin B-subunit (CTB) into the intact sciatic nerve of Macaca mulatta monkey gives rise to labelling of a sparse network of fibers in laminae I-II of spinal cord and of some mainly small dorsal root ganglion (DRG) neurons. Twenty days after sciatic nerve cut, the percentage of CTB-positive lumbar 5 (L5) DRG neuron profiles increased from 11% to 73% of all profiles. In the spinal cord, a marked increase in CTB labelling was seen in laminae I, II, and the dorsal part of lamina III. In the rat L5 DRGs, 18 days after sciatic nerve cut, the percentage of CTB- and CTB conjugated to horseradish peroxidase (HRP)-labelled neuron profiles increased from 45% to 81%, and from 54% to 87% of all neuron profiles, respectively. Cell size measurements in the rat showed that most of the CTB-positive neuron profiles were small in size after axotomy, whereas most were large in intact DRGs. In the rat spinal dorsal horn, a dense network of CTB-positive fibers covered the whole dorsal horn on the axotomized side, whereas CTB-labelled fibers were mainly seen in laminae III and deeper laminae on the contralateral side. A marked increase in CTB-positive fibers was also seen in the gracile nucleus. The present study shows that in both monkey and rat DRGs, a subpopulation of mainly small neurons acquires the capacity to take up CTB/CTB-HRP after axotomy, a capacity normally not associated with these DRG neurons. These neurons may transganglionically transport CTB and CTB-HRP. Thus, after peripheral axotomy, CTB and CTB-HRP are markers not only for large but also for small DRG neurons and, thus, possibly also for both myelinated and unmyelinated primary afferents in the spinal dorsal horn. These findings may lead to a reevaluation of the concept of sprouting, considered to take place in the dorsal horn after peripheral nerve injury.

Animals↗
PubMed1996

Assembly of microfilaments and microtubules from axonally transported actin and tubulin after axotomy.

The slow component (SC) of axonal transport conveys structural proteins, regulatory proteins, and glycolytic enzymes toward the axon tip at 1-6 mm/day. Following axon interruption (axotomy), the rate of outgrowth corresponds to the rate of SCb-the fastest subcomponent of SC. Both axonal outgrowth and SCb accelerate 20-25% after axotomy. Tubulin and actin are the major proteins being carried by SCb. To further characterize the acceleration of SCb, we measured the equilibrium between subunits and polymers for both actin and tubulin. We radiolabeled newly synthesized proteins in rat motor neurons by microinjecting [35S]methionine into the spinal cord 7 days after crushing the sciatic nerve (85 mm from the spinal cord). Nerves were removed 7 days later for homogenization in polymer-stabilizing buffer (PSB) and centrifugation, followed by SDS-PAGE of supernatants (S) and pellets (P). We removed beta-tubulin, actin, and the medium-weight neurofilament protein (NF-M) from each gel by using the fluorogram as a template. After solubilizing gel segments for liquid scintillation spectrometry, we expressed counts as a polymerization ratio: P/[S+P]. In the nerve segments that contained radiolabeled Scb proteins, located 24-36 mm from the spinal cord, axotomy increased the polymerization ratio of SCb actin from 0.23 to 0.36 (P < 0.05) but had no effect on SCb beta-tubulin. In a separate experiment, we added 12 microM taxol to PSB to stabilize newly assembled microtubules. Adding taxol did not alter the polymerization ratio for SCb beta-tubulin in sham-axotomized nerves but aid increase the ratio in axotomized nerves, from 0.44 to 0.63 (P < 0.05); polymerization ratios for SCb actin were unaffected. We conclude that the assembly of microfilaments and microtubules increases to provide cytoskeletal elements for axon sprouts. The resulting loss of actin and tubulin subunits may play a role in the acceleration of SCb.

Actin Cytoskeleton↗
PubMed1994

Effects of peripheral axotomy on presynaptic axon terminals with GABA-like immunoreactivity.

The facial nerve was unilaterally crushed at its exit from the stylomastoid foramen in three 3-month old male rats. After 10 days survival, before the regenerating axons had reinnervated their target muscles, the facial nucleus was examined to determine central patterns of response in material prepared to demonstrate the presence of GABA-like immunoreactivity with postembedding procedures using gold-labeled secondary antibody. The uninjured nucleus served as a control. In both control and injured nuclei, the GABAergic terminals synapse with all parts of the motor neurons, except the axon, and exhibit diverse morphologies. GABAergic axon terminals vary in their size and in the electron density of their axoplasm and the majority of the terminals contain pleomorphic vesicle profiles that display a range in their packing density and size. In both control and injured facial nuclei, only approximately 40% of the axon terminal profiles with pleomorphic vesicles exhibit GABA immunoreactivity. A morphometric analysis of the synaptic vesicle profiles in the GABA-positive terminals reveals that following axotomy there is no change in the mean number of synaptic vesicle profiles per GABAergic terminal profile. However, the mean size of the synaptic vesicle profiles in these terminals shows an axotomy-induced 50% increase, without change in the shapes of the enlarged vesicle profiles. Also, the numerical density of gold particles associated with the GABA-positive terminals is consistently greater in the injured than the control axon terminals. In the control animals quantitative analysis of the relative distribution of all axon terminal profiles in the neuropil categorized by the shape of their vesicle profiles as round, pleomorphic, or flat is 57:37:6. Ten days after axotomy the ratio of these categories in the injured nucleus has shifted to 35:60:5. This study demonstrates that the functional state of a postsynaptic target can influence the morphology of vesicle profiles in presynaptic elements as well as patterns of its afferent input.

Animals↗
PubMed1984

Replication and differentiation of olfactory receptor neurons following axotomy in the adult hamster: a morphometric analysis of postnatal neurogenesis.

Regeneration of olfactory receptor neurons following unilateral olfactory nerve section was studied in Syrian golden hamsters by morphometric procedures. Characteristic structural and histochemical features of olfactory receptor neurons were compared on the sectioned and intact sides of the nasal septum at 6, 12, 18, 33, and 130 days following axotomy. The parameters measured were epithelial thickness and the numbers of nuclei, hematoxylin-staining olfactory vesicles, olfactory marker protein (OMP)-containing neuronal perikarya, and OMP-containing olfactory vesicles. The olfactory receptor neuron population was severely depleted 6 days after axotomy. In the succeeding 12-day period there was an initially rapid, then slower return of receptor neuron numbers. Though the regenerating olfactory epithelium appeared normal by gross inspection 33 days after the lesion, morphometric analysis revealed a substantial increase in the number of olfactory receptor neurons between 33 and 130 days postlesion. At our longest survival interval, all quantitative parameters had returned to 91-99% of control values. The numbers of OMP-containing perikarya and olfactory vesicles on the sectioned side were unchanged between the sixth and 12th postlesion days, thus suggesting that neural turnover was depressed for several days following surgery. Marker protein developed in newly formed receptor neurons between 6 and 12 days after the formation of the olfactory vesicle. Consequently, the ratio between OMP-containing olfactory vesicles and hematoxylin-staining vesicles (O/H) was used as an index of neural differentiation. The O/H ratio on the sectioned side was minimal 12 days following axotomy when stem-cell division was well under way but OMP antigenicity had not yet been expressed in newly formed neurons. At 33 days postlesion, O/H ratios were slightly but significantly greater than the control value, reflecting a degree of synchrony in the regenerating receptor neuron population. The average O/H ratio on the unlesioned side was 0.667. This value, together with our other observations, was used to derive an estimate of the life span of hamster olfactory receptor neurons, approximately 25-35 days.

Animals↗
PubMed1994

Plasticity of postganglionic sympathetic neurons in the rat superior cervical ganglion after axotomy.

The neuropeptides galanin (GAL) and vasoactive intestinal polypeptide (VIP) are upregulated in spinal and vagal sensory as well as in cranial motor neurons after axonal transection. In this study an increase of both peptides is demonstrated in axotomized principal ganglionic neurons (PGN) of the rat sympathetic superior cervical ganglion by use of double-labeling immunofluorescence. Compared to control ganglia that do not contain more than 1% GAL- or VIP-positive cells, about 26% of all PGN exhibit GAL immunoreactivity by day 1 after transection of the major postganglionic branches. The proportion of immunoreactive neurons reaches its maximum after 30 days (40%) and decreases to about 27% within the second month after axotomy. The percentage of VIP-positive neurons is much lower than for GAL: 2% of the PGN exhibit VIP immunoreactivity at day 1 and about 7% are observed 30 and 60 days after axotomy. In order to further characterize newly GAL- and VIP-positive PGN, their cell diameters were determined 12 days after axotomy. Compared to the mean overall neuron diameter of 24.8 microns, GAL-immunoreactive neurons are predominantly of small and intermediate size (22.2 microns), whereas VIP occurs mainly in larger neurons (26.1 microns). Besides cell bodies, many intraganglionic nerve fibers stain positive for GAL or VIP, particularly at day 6. Most likely, these fibers represent axons, as indicated by the absence of MAP2, a cytoskeletal protein found in neuronal somata and dendrites. They establish direct membrane contacts with postganglionic perikarya, as revealed by pre-embedding immuno-electron microscopy. Some cell bodies and fibers contain both peptides. Colocalization of GAL or VIP with tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine synthesis, reveals a reduced immunoreactivity for TH in intensely GAL- or VIP-positive cells, and vice versa at day 6. However, no difference in staining intensity for VIP or GAL, and TH, is observed after 30 and 60 days. Possible implications of GAL and VIP for peripheral nerve regeneration and their regulation by target-derived factors are discussed.

Animals↗
PubMed1991

Changes in glial fibrillary acidic protein mRNA expression after corticospinal axotomy in the adult hamster.

We examined changes in the expression of glial fibrillary acidic protein (GFAP) mRNA during Wallerian degeneration in the corticospinal system of the adult Golden hamster following axotomy. GFAP is the product of a type III intermediate filament (IF) gene that is expressed specifically in mature astrocytes. A well-studied component of a complex response termed reactive astrogliosis that occurs after various types of CNS injury is the increased production of astrocytic processes filled with GFAP-containing IFs. While increased expression of GFAP during reactive astrogliosis has been well established at the protein level, little is known about whether or not changes in GFAP mRNA levels occur after CNS injury. In the present study we used in situ hybridization methods to examine this issue. A 35S-labeled mouse GFAP cDNA probe was used for in situ hybridizations of sections of the brain stem obtained 2, 7, and 14 days after unilateral transections of the corticospinal tract in the caudal medulla. Film as well as emulsion autoradiography showed a dramatic increase in GFAP mRNA labeling associated with the degenerating corticospinal tract. GFAP mRNA levels were already dramatically increased in the injured corticospinal tract by 2 days post axotomy and remained elevated at 14 days. Interestingly, in addition to the robust increase in GFAP mRNA levels specifically associated with the degenerating tract, a diffuse increase in GFAP mRNA labeling was observed throughout the grey matter of the brain stem at 2 days post-axotomy, but not after this time. Immunoblotting and immunocytochemical experiments verified that the increased GFAP mRNA levels in the degenerating corticospinal system were accompanied by an increased expression of the protein. These results demonstrate that an increase in GFAP mRNA levels occurs during Wallerian degeneration in the CNS and suggest that increased expression of the GFAP gene is a major contributor to CNS scarring that results after direct traumatic injury.

Animals↗
PubMed1994

(-)-Deprenyl alters the survival of adult murine facial motoneurons after axotomy: increases in vulnerable C57BL strain but decreases in motor neuron degeneration mutants.

The effect of (-)-deprenyl on the survival of axotomized adult murine facial motoneurons was investigated. Previously, (-)-deprenyl was shown to increase the number of rat facial motoneurons (FMns) surviving after axotomy at postnatal day 14, apparently by compensating for the loss of muscle-derived trophic factor. In the present study, three different strains of adult mice--A/J, C57BL/6J, and a congenic substrain of the C57BL/6J mice, the C57BL/Mnd mutants--underwent unilateral facial nerve transection. FMns were counted from serial sections taken through the entire length of the facial nuclei ipsilateral and contralateral to the facial nerve transections in animals sacrificed 21 days after axotomy. Subgroups of C57BL/6J and Mnd mutants were treated with either saline or 1.0 mg/kg (-)-deprenyl for 21 days. Another subgroup of Mnd mutants were treated with the metabolites of (-)-deprenyl, a mixture of (-)-amphetamine and (-)-methamphetamine, at a dosage equimolar to 1.0 mg/kg (-)-deprenyl. The number of surviving facial motoneurons in the A/J strain was 90% of unlesioned, control values which supports previous findings that adult FMns receive adequate trophic support and thus can survive loss of muscle-derived trophic support. In the C57BL/6J strain, the facial motoneuron survival was 35% and (-)-deprenyl increased the survival to 50.5%. Mnd mutants showed 62.4% survival; however, (-)-deprenyl decreased the number of motoneurons to 54.9% and amphetamine and methamphetamine treatment further decreased the motoneuron survival to 41.1%. These findings show that FMns in the Mnd mutants and their parental strain, C57BL/6J mice, show greater vulnerability to axotomy as compared to other adult strains of mice. The vulnerability is similar to that found in early postnatal life. (-)-Deprenyl increases the survival of the axotomized C57BL/6J FMns but its major metabolites, (-)-methamphetamine and (-)-amphetamine, further decrease FMn survival in the C57BL/Mnd mutants, possibly due to the induction of neurotoxic proteins causing programmed neuronal death. The efficacy of (-)-deprenyl in increasing the survival of damaged neurons would be expected to decrease as dosage increased above the dosage sufficient to induce maximum neuronal rescue (approximately 0.01 mg/kg) but would decrease as the dosage exceeded that necessary to produce toxic concentrations of the metabolites of (-)-deprenyl (1.0 mg/kg in this study).

Amphetamine↗
PubMed1978

Changes in synthesis of specific proteins following axotomy: detection with two-dimensional gel electrophoresis.

Changes in protein synthesis during development and following axotomy were analyzed by two-dimensional gel electrophoresis. The two major postganglionic nerves emerging from the superior cervical sympathetic ganglia (SCSG) of adult rats were either cut or crushed unilaterally. At intervals ranging from 1 to 112 days after surgery both SCSG were removed and incubated for 1 hr in the presence of 14C-leucine. Proteins were extracted and subjected to two-dimensional electrophoretic separation and autoradiography. With this technique, proteins are separated on the basis of isoelectric point and molecular weight. Also, intact SCSG from 1, 2, 7, and 14 day old rats were labeled and analyzed. It was found that a minority of the separated proteins exhibited some detectable change in relative rate of synthesis following axotomy. Actin exhibited a slight (less than 20%) increase in relative synthesis rate while tubulin did not change significantly. There were small but significant differences in the protein patterns following nerve crush, as opposed to nerve cut. Comparison of protein synthesis patterns from developing rat SCSG with those from intact and from axotomized adult SCSG failed to demonstrate any marked similarity between the developmental and the axotomy patterns.

Actins↗
PubMed1993

Rapid and protracted phases of retinal ganglion cell loss follow axotomy in the optic nerve of adult rats.

To investigate the short- and long-term effects of axotomy on the survival of central nervous system (CNS) neurons in adult rats, retinal ganglion cells (RGCs) were labelled retrogradely with the persistent marker diI and their axons interrupted in the optic nerve (ON) by intracranial crush 8 or 10 mm from the eye or intraorbital cut 0.5 or 3 mm from the eye. Labelled RGCs were counted in flat-mounted retinas at intervals from 2 weeks to 20 months after axotomy. Two major patterns of RGC loss were observed: (1) an initial abrupt loss that was confined to the first 2 weeks after injury and was more severe when the ON was cut close to the eye; (2) a slower, persistent decline in RGC densities with one-half survival times that ranged from approximately 1 month after intraorbital ON cut to 6 months after intracranial ON crush. A small population of RGCs (approximately 5%) survived for as long as 20 months after intraorbital axotomy. The initial loss of axotomized RGCs presumably results from time-limited perturbations related to the position of the ON injury. A persistent lack of terminal connectivity between RGCs and their targets in the brain may contribute to the subsequent, more protracted RGC loss, but the differences between intraorbital cut and intracranial crush suggest that additional mechanisms are involved. It is unclear whether the various injury-related processes set in motion in both the ON and the retina exert random effects on all RGCs or act preferentially on subpopulations of these neurons.

Animals↗
PubMed1994

Galanin and vasoactive intestinal peptide messenger RNAs increase following axotomy of adult sympathetic neurons.

The adult rat superior cervical ganglion (SCG) contains low levels of galanin- and vasoactive intestinal peptide-(VIP) like immunoreactivity, with very few immunostained principal neurons. Immunoreactivity for both neuropeptides increases in these neurons after explantation or postganglionic axotomy in vivo. Northern blot analysis has demonstrated concomitant increases in mRNAs encoding these peptides. To localize cells in axotomized ganglia which increase their expression of these mRNAs, we performed in situ hybridization studies. In control SCG, only a few principal neurons contained mRNA for either galanin or VIP. After 48 h in organ culture, galanin mRNA was expressed in the majority of principal neurons. At 48 h after in vivo axotomy of both postganglionic trunks of the SCG, the internal and external carotid nerves, the distribution and number of neurons, expressing galanin mRNA increased similarly to that seen in culture. Lesioning either trunk alone produced increases in galanin mRNA localized to those regions of the ganglion containing neurons that project into the lesioned trunk. Transection of the predominantly preganglionic cervical sympathetic trunk increased galanin mRNA expression in a small population of neurons near that nerve trunk. The distributions of these labeled neurons, together with previous neuroanatomical studies, suggests that they had been axotomized by the lesions. Similar studies examining VIP mRNA expression demonstrated that although considerably fewer VIP mRNA expressing neurons than galanin mRNA expressing neurons were present after axotomy, the distribution of neuropeptide mRNA-positive cells were similar in both cases. These observations suggest that increases in the peptides galanin and VIP after nerve transection result from changes in the levels of their mRNAs in those neurons that have been axotomized.

Animals↗
PubMed1994

Gene expression in central cholinergic neurons in response to axotomy and deafferentation.

Although the molecular and cellular responses to injury in the central nervous system (CNS) have been widely investigated, few studies have examined the potential variations between direct and indirect neuronal injury. To differentiate between the response to axotomy and deafferentation, two central cholinergic populations were analyzed: the horizontal limb of the diagonal band of Broca (HLDB) and the interneurons in the corpus striatum (CS). At time points from one hour to eight weeks postinjury the levels of choline acetyltransferase (ChAT) mRNA and protein were assessed by in situ hybridization and immunohistochemistry. Also examined was the expression of the immediate early gene product, c-fos. One week post axotomy, neurons in the HLDB exhibited an increase in the levels of ChAT mRNA without a concomitant increase in ChAT protein, followed by a steady decrease reaching a nadir in both parameters at eight weeks. In contrast, a transient increase occurred at one week postdeafferentation in the levels of both ChAT mRNA and protein in the interneurons of the CS. Axotomized neurons in the HLDB did not exhibit either c-fos mRNA or protein expression, while robust fos induction occurred after one hour in deafferented neurons in the CS. These data demonstrate that the molecular and cellular responses differ following direct and indirect neuronal injury. Furthermore, they suggest that in these central cholinergic populations deafferentation may result in cellular hyperactivity and cell survival while axotomy results in decreased cellular activity and subsequent cellular regression.

Animals↗
PubMed1994

Axotomy-induced changes in ribosomal RNA levels in female hamster facial motoneurons: differential effects of gender and androgen exposure.

Following facial nerve injury, female hamster facial motoneurons (HFMN) regenerate faster than their male counterparts. Testosterone propionate (TP) markedly accelerates the rate of facial nerve regeneration in males, but has a relatively reduced effect in females. In the present study, we utilized in situ hybridization in conjunction with ribosomal DNA probes to test the hypothesis that in females HFMN axotomy produces a less dramatic cell body response than in males and to examine gender differences in the effects of steroids on peripheral nerve regeneration at the molecular level. The results indicate that in females axotomy alone induced moderate increases in rRNA levels, beginning 2 days postoperatively. These changes were both slower in onset and lower in magnitude than those in the males. TP augmented the effects of axotomy on rRNA levels at the later postoperative times. There were no early, rapid effects of TP, like those observed in males. These molecular data substantiate our previous findings of inherent sex differences in neuronal regeneration and the ability of gonadal steroids to augment the reparative response of peripheral neurons to injury.

Animals↗
PubMed1996

Two novel pyrrolopyrimidine lipid peroxidation inhibitors U-101033E and U-104067F protect facial motor neurons following neonatal axotomy.

Recent reports suggest that oxygen radical-induced lipid peroxidation plays a role in the retrograde degeneration of motor neurons following facial nerve axotomy in the neonatal rat. The purpose of the present study was to explore this notion further by testing the neuroprotective properties of two novel brain-penetrating, lipid peroxidation inhibitors, U-101033E and U-104067F, in this model of neuronal degeneration. In Experiment 1, 14-day-old rats were pretreated with 3, 10, or 30 mg/kg U-101033E (po) 10 min before right facial nerve axotomy (Day 0) and then posttreated once a day from Day 1 to Day 6, and once every other day from Day 8 to Day 21. Rats were sacrificed 21 days postaxotomy and surviving cholinergic cell bodies were identified using choline acetyltransferase immunocytochemistry. Both 10 and 30 mg/kg U-101033E significantly enhanced motor neuron survival, with survival rates of 65.9-88.9% being noted in comparison to 51.7-62% survival in vehicle controls (P < or = 0.05). Experiment 2 demonstrated a significant neuroprotective effect of 10 and 30 mg/kg U-104067F using the same dosing schedule. Experiment 3 was designed to test whether shorter periods of drug exposure (e.g., 5 or 7 days) would be sufficient to preserve motor neurons in rats treated with 10 mg/kg U-101033E. The results suggested that as little as 5 days of drug treatment is sufficient to enhance motor neuron survival. Finally, Experiment 4 demonstrated an 18-19% increase in motor neuron survival in rats treated with 10 and 30 mg/kg U-104067F for 5 consecutive days postaxotomy. Taken together, the attenuation of motor neuron degeneration by the two pyrrolopyrimidine lipid peroxidation inhibitors, U-101033E and U-104067F, lends support to the notion that lipid peroxidation contributes to the pathogenesis of axotomy-induced neurodegeneration.

Animals↗
PubMed1997

Age-dependent induction of nitric oxide synthase activity in facial motoneurons after axotomy.

The facial nerve was transected in rats at different postnatal ages, from birth to early adulthood. NADPH-diaphorase histochemistry was performed to analyze the induction of nitric oxide synthase, the synthetic enzyme of the free radical nitric oxide, in injured facial motoneurons. In addition, in situ nick-end labeling of DNA fragmentation (TUNEL technique) was performed after axotomy at birth, to verify the occurrence of apoptosis in the damaged facial motoneurons. A striking age-dependency was found in the induction of nitric oxide synthase activity in axotomized facial motoneurons. NADPH-diaphorase positivity was not detectable in these neurons 1 and 2 days after axotomy at birth, when apoptotic changes were evident and marked. In addition, NADPH-diaphorase staining was hardly detectable in the facial nucleus 4 days after axotomies at birth, when extensive motoneuron loss was evident. NADPH-diaphorase positivity was instead induced in the facial motoneurons axotomized from the end of the first postnatal week to adulthood, when the nerve cell loss was less severe than in newborns. However, the time course of the enzyme activity induction varied considerably in relation to the animals' age. These findings are discussed in relation to the role of nitric oxide in motoneuron death or protective response to injury and of oxidative stress in neurodegeneration.

Age Factors↗