Sciatic nerve axotomy in aged rats: response of motoneurons and the effect of GM1 ganglioside treatment

The number, size, and staining intensity of choline acetyltransferase (ChAT)-immunopositive cells in the retrodorsal lateral nucleus (RDLN) of the spinal cord were studied in young (3-5 months old) and aged (22-24 months old) rats following left sciatic nerve distal transection (axotomy) and treatment with GM1 ganglioside. The cell size and the ChAT immunostaining density were decreased in the RDLN of non-manipulated as well as in the contralateral intact side of axotomized aged rats. Axotomy had no effect on the number of RDLN motoneurons in both aged and young rats. In the young rats, there was a decrease in the size of motoneurons 7 days post-axotomy and a partial spontaneous recovery occurred by 21 days. Axotomy did not reduce further the size of aged motoneurons, however. The ChAT staining intensity of the axotomized RDLN declined in both age groups after 7 days, and there was spontaneous near normal recovery by 21 days. In the aged rats, GM1 administration for 7 days corrected the cell size and ChAT immunoreactivity of the contralateral intact RDLN. With regard to axotomized RDLN neurons, 7 days of GM1 restored the cell size but not the ChAT immunostaining in young animals. The same treatment schedule, however, corrected both cell size and staining in aged rats. Administration of GM1 for 21 days had no further effect on the morphometric parameters of the axotomized motoneurons in aged rats, but slightly enhanced the recovery of ChAT immunostaining in young rats. Thus, it appears that GM1 facilitates the phenotypic recovery of RDLN motoneurons during aging and after axotomy.     

ULTRASTRUCTURE OF AXOTOMIZED ALPHA AND GAMMA MOTONEURONS IN THE CAT THORACIC SPINAL CORD

Using horseradish peroxidase as a retrograde marker, the ultrastructural response of alpha and gamma motoneuronal cell bodies in the cat thoracic spinal cord has been compared 1-8 days following intercostal nerve transection and ligation. By light microscopy, reduction of Nissl body size, together with nuclear and nucleolar alterations were seen in alpha motoneurons 4-8 days following axotomy, but not at any stage in axotomized gamma motoneurons. In the electron microscope, disorganization of Nissl body ultrastructure was seen in both alpha and gamma motoneurons 2 days following axotomy. Only in alpha motoneurons, however, did these disorganized Nissl bodies subsequently fragment into smaller pieces. Both alpha and gamma motoneurons lost synapses following axotomy, but the proportional loss from gamma motoneurons was two-fold greater than that from alpha motoneurons. Loss of synaptic terminals with flattened synaptic vesicles was two-fold higher than that of synaptic terminals with round synaptic vesicles from axotomized gamma motoneurons, whereas axotomized alpha motoneurons lost both types of synaptic terminal equally.     

Neurotrophin modulation of the monosynaptic reflex after peripheral nerve transection.

The effects of neurotrophin-3 (NT-3) and NT-4/5 on the function of axotomized group Ia afferents and motoneurons comprising the monosynaptic reflex pathway were investigated. The axotomized medial gastrocnemius (MG) nerve was provided with NT-3 or NT-4/5 for 8-35 d via an osmotic minipump attached to its central end at the time of axotomy. After this treatment, monosynaptic EPSPs were recorded intracellularly from MG or lateral gastrocnemius soleus (LGS) motoneurons in response to stimulation of the heteronymous nerve under pentobarbital anesthesia. Controls were preparations with axotomized nerves treated directly with vehicle; other axotomized controls were administered subcutaneous NT-3. Direct NT-3 administration (60 microgram/d) not only prevented the decline in EPSP amplitude from axotomized afferents (stimulate MG, record LGS) observed in axotomy controls but, after 5 weeks, led to EPSPs larger than those from intact afferents. These central changes were paralleled by recovery of group I afferent conduction velocity. Removal of NT-3 4-5 weeks after beginning treatment resulted in a decline of conduction velocity and EPSP amplitude within 1 week to values characteristic of axotomy. The increased synaptic efficacy after NT-3 treatment was associated with enhanced connectivity of single afferents to motoneurons. NT-4/5 induced modest recovery in group I afferent conduction velocity but not of the EPSPs they elicited. NT-3 or NT-4/5 had no effect on the properties of treated motoneurons or their monosynaptic EPSPs. We conclude that NT-3, and to a limited extent NT-4/5, promotes recovery of axotomized group Ia afferents but not axotomized motoneurons or the synapses on them.     

BDNF regulation of androgen receptor expression in axotomized SNB motoneurons of adult male rats

Brain-derived neurotrophic factor (BDNF) prevents the axotomy-induced loss of androgen receptor-like immunoreactivity (AR-LI) in the spinal nucleus of the bulbocavernosus (SNB) motoneurons of adult male rats. In this report, we investigated the dose-response effect of BDNF on androgen receptor expression in axotomized SNB motoneurons, and examined whether delayed application of BDNF to the cut SNB axons can completely reverse the axotomy-induced loss of androgen receptor expression. We also used autoradiography to test whether axotomy decreases the ability of SNB motoneurons to accumulate androgens. SNB motoneurons were axotomized bilaterally and BDNF or PBS was applied to the proximal ends of the axons. The percentage of SNB motoneurons expressing medium or high AR-LI was the major measure of androgen receptor expression. AR-LI was significantly higher on the BDNF-treated side than on the contralateral side treated with phosphate-buffered saline (PBS) for all three doses of BDNF (1.45, 2.9, and 5.8 mg/ml) and was higher than in rats treated bilaterally with PBS. Moreover, AR-LI at the highest dose of BDNF was not different from that in intact SNB motoneurons. Delayed application of BDNF to the axotomized SNB motoneurons restored the AR-LI to the intact level. The AR-LI decreased by axotomy started to increase significantly 4 days after BDNF application and returned to the intact level by 10 days. Furthermore, axotomy significantly decreased the percentage of SNB motoneurons to accumulate tritiated testosterone or its metabolites. In conclusion, our data demonstrate that BDNF completely prevents and reverses the axotomy-induced loss of AR-LI. Moreover, decrease of AR-LI by axotomy reflects the decrease in the ability of SNB motoneurons to accumulate androgens.     

A light and electron microscopic study of intracellularly HRP-labeled lumbar motoneurons after intramedullary axotomy in the adult cat.

In contrast to many other neurons in the central nervous system, spinal motoneurons in adult cats have been shown to regenerate their axons after an axotomy accomplished within the CNS compartment. This regenerative capacity may be the result of extrinsic influences, or intrinsic properties of the motoneurons themselves, or interactions between extrinsic and intrinsic factors. As part of the effort to establish circumstances of importance for this central regeneration, a detailed analysis of the morphology of lumbar motoneurons was performed 3-11 weeks following a ventral funiculus axotomy. Fourteen large neurons considered to be intramedullarly axotomized alpha motoneurons were labeled intracellularly with horseradish peroxidase. Twelve out of the fourteen analyzed neurons had an axonlike regenerating process. These twelve neurons could, in turn, be separated into two groups, based on the proximity of the axonal lesion and the proximal morphology of the regenerating process. Thus, after a comparatively proximal axotomy, new axons were produced, originating either from the cell soma or from a distal dendritic branch. After a more distal axotomy, but still intramedullarly, it seemed as if the proximal part of the original axon always persisted and subsequently regenerated. Analysis of the relation between the cell soma diameter and the diameter and number of its stem dendrites revealed that dendrites become thinner and also decrease in number after an intramedullary axotomy. In this way, it may be calculated that the total dendritic surface area of lesioned motoneurons will decrease by approximately half. In four neurons, most dendrites had an abnormal appearance in the light microscope with increasing diameter of distal branches. Ultrastructural analysis revealed that such dendrites were surrounded by myelin sheaths. Small filopodia in close relation to axon terminals were found to emerge from the cell membrane of the lesioned motoneurons. Their function may be to establish contact with presynaptic elements and then retract them to the cell membrane. We interpret the morphological changes of the motoneurons as signs of a large capacity for axonal regeneration, even after axotomy in the central nervous system.     

Reinnervation of developing rat muscle by non-axotomized motoneurons.

To study the ability of developing motoneurons to reinnervate their denervated muscle, axotomized motoneurons in rat neonates and pups were retrogradely labeled with two fluorescent tracers. Fluorogold (FG), a long-lasting fluorescent dye, was injected into intercostal muscle T8 to retrogradely label the motoneurons that innervated it. Two days later intercostal nerves T7-T9 were cut. The intercostal muscle denervated at birth was reinnervated within 10-20 days, as evidenced by nerve-evoked muscle contraction. Three weeks following axotomy, tetramethylrhodamine isothiocyanate (TRITC) was injected into the same muscle to label the motoneurons that reinnervated it. The motoneurons double-labeled with FG and TRITC were, therefore, axotomized motoneurons that regenerated to reinnervate T8. In neonates, axotomy resulted in a significant reduction in the number of FG-labeled motoneurons, which suggests that axon transection at early postnatal days causes a massive motoneuron death. The percentage of double-labeled motoneurons was significantly smaller than that in non-axotomized rats. TRITC-labeled motoneurons constituted the majority of stained motoneurons; these were located in different nuclei than the intercostal motoneurons. These findings suggest that muscle reinnervation is, at least in part, by motoneurons which originally did not innervate intercostal muscle T8. Unlike axotomy at birth, axotomy performed 2-3 weeks after birth did not result in a significant motoneuron loss. The number of stained motoneurons labeled with both FG and TRITC was significantly smaller, however, than in non-axotomized spinal cords. Our data indicate that in pups only a small percentage of axotomized motoneurons reinnervated the denervated muscle.     

Factors regulating AMPA-type glutamate receptor subunit changes induced by sciatic nerve injury in rats

Excitatory glutamatergic neurotransmission at Ia afferent-motoneuron synapses is enhanced shortly after physically severing or blocking impulse propagation of the afferent and/or motoneuron axons. We considered the possibility that these synaptic changes occur because of alterations in the number or properties of motoneuron alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors. Therefore, we quantitatively analyzed glutamate receptor (GluR)1, GluR2/3, and GluR4 AMPA subunit immunoreactivity (ir) in motoneurons 3, 7, or 14 days after axotomy or continuous tetrodotoxin (TTX) block of the sciatic nerve. GluR1-ir remained low in experimental and control motoneurons with either treatment and at any date. However, there was a large reduction of GluR2/3-ir (peak at 7 days >60% reduced) and a smaller, but statistically significant, reduction of GluR4-ir (around 10% reduction at days 3, 7, and 14) in axotomized motoneurons. TTX sciatic blockade did not affect AMPA subunit immunostainings. Axonal injury or interruption of the trophic interaction between muscle and spinal cord, but not activity disruption, appears therefore more likely responsible for altering AMPA subunit immunoreactivity in motoneurons. These findings also suggest that synaptic plasticity induced by axotomy or TTX block, although similar in the first week, could be related to different mechanisms. The effects of axotomy or TTX block on motoneuron expression of the metabotropic glutamate receptor mGluR1a were also studied. mGluR1a-ir was also strongly decreased after axotomy but not after TTX treatment. The time course of the known stripping of synapses from the cell somas of axotomized motoneurons was studied by using synaptophysin antibodies and compared with AMPA and mGluR1a receptor changes. Coverage by synaptophysin-ir boutons was only clearly decreased 14 days post axotomy and not at shorter intervals or after TTX block.     

A morphometric study on the early stages of dendrite changes in the axotomized motoneuron of the spinal cord in newborn rats

OBJECTIVES: To study the early effect of axotomy on spinal motoneurons and dendritic trees in the newborn rat.METHODS: The left sciatic nerve of 50 neonatal Sprague--Dawley rats aged 5 days old was transected and the right area kept as a control. The operated animals were killed 2, 4, 8, 12 and 24 hours after axotomy. The L4--L6 segments of the spinal cord were sampled, and stained either with Golgi-Cox or Cresyl fast violet methods. The number of motoneurons, the largest soma diameter and the different parameters of the dendritic trees such as: number, length and thickness of the primary, secondary and tertiary branches in the axotomized sides were estimated and compared statistically with that of the intact sides.RESULTS: The results indicated that in the axotomized sides, the number of motoneurons and the largest soma diameter were decreased, but these were significant only in 12- and 24-hour groups. The number of branches of the dendritic trees including primary, secondary and tertiary branches was not significantly decreased in the groups. The thickness of the dendrites showed a reduction; however, this was significant only for the tertiary branches in the 24-hour groups. The length of the primary, secondary and tertiary branches of the dendrites--especially the latter--were also decreased significantly in most of the groups.CONCLUSION: Axotomy at the early stages in newborn rats resulted in noticeable morphometrical changes in motoneurons and their dendrites.     

Influence of the axotomy to cell body distance in rat rubrospinal and spinal motoneurons: differential regulation of GAP-43, tubulins, and neurofilament-M.

Axotomized motoneurons regenerate their axons regardless of whether axotomy occurs proximally or distally from their cell bodies. In contrast, regeneration of rubrospinal axons into peripheral nerve grafts has been detected after cervical but not after thoracic injury of the rubrospinal tract. By using in situ hybridization (ISH) combined with reliable retrograde tracing methods, we compared regeneration-associated gene expression after proximal and distal axotomy in spinal motoneurons versus rubrospinal neurons. Regardless of whether they were axotomized at the iliac crest (proximal) or popliteal fossa (distal), sciatic motoneurons underwent highly pronounced changes in ISH signals for Growth Associated Protein 43 (GAP-43) (10-20x increase) and neurofilament M (60-85% decrease). In contrast, tubulin ISH signals substantially increased only after proximal axotomy (3-5x increase). To compare these changes in gene expression with those of axotomized rubrospinal neurons, the rubrospinal tract was transected at the cervical (proximal) or thoracic (distal) levels of the spinal cord. Cervically axotomized rubrospinal neurons showed three- to fivefold increases in ISH signals for GAP-43 and tubulins (only transient) and a 75% decrease for neurofilament-M. In sharp contrast, thoracic axotomy had only marginal effects. After implantation of peripheral nerve transplants into the spinal cord injury sites, retrograde labeling with the sensitive retrograde tracer Fluoro-Gold identified regenerating rubrospinal neurons only after cervical axotomy. Furthermore, rubrospinal neurons specifically regenerating into the transplants were hypertrophied and expressed high levels of GAP-43 and tubulins. Taken together, these data support the concept that, even if central nervous system (CNS) axons are presented with a permissive/supportive environment, appropriate cell body responses to injury are a prerequisite for CNS axonal regeneration.     

Semaphorin and neuropilin expression in motoneurons after intraspinal motoneuron axotomy

We have examined mRNA and protein distribution for the axon guidance molecules semaphorin3A, 3F, 4F and semaphorin receptors neuropilin-1 and 2, 1-21 days after intramedullary axotomy of rat lumbar spinal cord motoneurons. We show that semaphorin3A mRNA and protein are up-regulated in the scar and in motoneurons from 3 days and upto 3 weeks after injury. Neuropilin-1 mRNA showed no changed expression in axotomized motoneurons. Semaphorin3F mRNA expression was found in ventral roots after ventral funiculus lesion (VFL) and neuropilin-2 mRNA was found in affected motoneurons from 1 day after injury throughout the examined period. Semaphorin4F mRNA was first found in motoneurons 3 weeks after lesion. These results suggest semaphorin/neuropilin involvement in the injury response of intramedullary axotomized motoneurons.     

APOPTOSIS ONSET AND BAX PROTEIN DISTRIBUTION IN SPINAL MOTONEURONS OF NEWBORN RATS FOLLOWING SCIATIC NERVE AXOTOMY

Extensive apoptosis in spinal cord motoneuron was reported to occur in the newborn following sciatic nerve axotomy. The purpose of this study was to evaluate the onset of cell death at early stages of axotomy, and the changes in Bax protein distribution pattern in the apoptotic cell. Newborn rats were divided into seven groups, axotomized at day 5 postnatal and sacrificed at the following time points: 1, 3, 6, 12, 24, 48, and 72 h after surgery. The left sciatic nerve was transected while the right side was kept as a control. Three experiments were made for morphometric, immunohistochemical, and ultrastructural studies. Morphometric study showed sustained reduction in the number of neurons in the ventral horn. Neuronal losses onset occurred at the first hour after axotomy and the highest loss occurred in the 72-h group (33.7%). The percentage of survived motoneuron (PSM) was calculated. The test of linearity showed that neuron reduction pattern was nonlinear. A nonlinear curve fitting of PSM against time showed an exponential decline in the number of neurons. Immunohistochemistry results showed three Bax protein patterns; early stage increase in Bax gene expression; Bax protein punctation; and dense Bax protein immunoreactivity (DBI). The last two can either be early or late patterns. Bax gene expression increased as early as the first hour post-axotomy and the number of Bax-positive motoneurons continued to increase throughout the time course. Ultrastructural study of the cytoplasm showed an early vacuolation in Golgi apparatus and endoplasmic reticulum that was associated by mitochondrial and nuclear changes.­­     

Cell size and geometry of spinal cord motoneurons in the adult cat following the intramuscular injection of adriamycin: comparison with data from aged cats

Adriamycin (ADM), an antineoplastic antibiotic, when injected intramuscularly, is taken up by motoneuron axonal terminals and retrogradely transported to the motoneuron soma where it exerts its neurotoxic effect. In the present study, ADM was injected into the hindlimb muscles of five adult cats. Measurements of the electrophysiological properties of the lumbar motoneurons innervating these muscles were obtained using intracellular recording techniques. Based upon these data the equivalent cylinder model of motoneurons was employed to evaluate ADM-induced changes in cell size and cell geometry. The size of cell somas in the ventral horn was also measured using light microscopy and computer imaging software. There were significant increases in the membrane time constant (25%) and input resistance (50%) in motoneurons whose muscles were treated with ADM (ADM-MNs) compared with data from control motoneurons (control-MNs). The increase in membrane time constant is attributed to an increase in membrane resistance; the increase in input resistance appears to depend upon both an increase in membrane resistance and a decrease in total cell surface area. Cell capacitance, which is proportional to the total cell surface area, was significantly reduced (15%) in ADM-MNs. Calculations based on cable theory indicate that while there was no significant change in the length of the equivalent cylinder for ADM-MNs, there was a significant decrease (17%) in the diameter of the equivalent cylinder. These data indicate that there is a decrease in total cell surface area which can be attributed to the shrinkage of branches throughout the dendritic tree. There was also a small (7%) but statistically significant decrease in the electrotonic length of ADM-MNs. Morphological analysis also revealed that the mean cross-sectional area of the somas of those ventral horn neurons which are likely to correspond to the motoneuron population was significantly reduced on the ADM-treated side compared to that of neurons on the control side. We conclude that significant geometrical changes were induced in lumbar motoneurons of adult cats after ADM was injected into their target muscles. In old cats, spinal cord motoneurons exhibit similar patterns of changes in their electrophysiological characteristics which have also been suggested to be correlated with changes in cell geometry. The question then arises as to whether the response of motoneurons to ADM and the aging process reflects a stereotypic reaction of motoneurons to a variety of insults or whether the response to ADM mirrors specific aspects of the aging process.     

Regeneration‐associated genes decline in chronically injured rat sciatic motoneurons

Chronic nerve injuries are notorious for their poor regenerative outcomes. Here, we addressed the question of whether the established reduced ability of injured motoneurons to regenerate their axons with time of disconnection with targets (chronic axotomy) is associated with a failure of injured motoneurons to express and sustain their expression of regeneration‐associated genes. Sciatic motoneurons were prevented from regenerating by ligation of the transected nerves (chronic axotomy), and then subjected to a second nerve transection (acute axotomy) to mimic the clinical surgical procedure of refreshing the proximal nerve stump prior to delayed nerve repair. The expression of α₁‐tubulin, actin and GAP‐43 mRNA was analysed in axotomized sciatic motoneurons by the use of in situ hybridization followed by autoradiography and silver grain quantification. The expression of these regeneration‐associated genes by naive (acutely) axotomized motoneurons declined exponentially, to reach baseline levels within 6 months. These chronically injured motoneurons responded to a refreshment axotomy by elevating the expression of the genes to the same levels as in acutely (i.e. for the first time) axotomized sciatic motoneurons. However, the expression of these declined more rapidly than after acute axotomy. We conclude that a progressive decline in the expression of the regeneration‐associated genes in chronically axotomized motoneurons and the even more rapid decline in their expression in response to a refreshment axotomy may explain why the regenerative capacity of chronically axotomized neurons declines with time.     

A morphometric study on the early stages of dendrite changes in the axotomized motoneuron of the spinal cord in newborn rats

Abstract

Objectives: To study the early effect of axotomy on spinal motoneurons and dendritic trees in the newborn rat.

Methods: The left sciatic nerve of 50 neonatal Sprague–Dawley rats aged 5 days old was transected and the right area kept as a control. The operated animals were killed 2, 4, 8, 12 and 24 hours after axotomy. The L4–L6 segments of the spinal cord were sampled, and stained either with Golgi-Cox or Cresyl fast violet methods. The number of motoneurons, the largest soma diameter and the different parameters of the dendritic trees such as: number, length and thickness of the primary, secondary and tertiary branches in the axotomized sides were estimated and compared statistically with that of the intact sides.

Results: The results indicated that in the axotomized sides, the number of motoneurons and the largest soma diameter were decreased, but these were significant only in 12- and 24-hour groups. The number of branches of the dendritic trees including primary, secondary and tertiary branches was not significantly decreased in the groups. The thickness of the dendrites showed a reduction; however, this was significant only for the tertiary branches in the 24-hour groups. The length of the primary, secondary and tertiary branches of the dendrites—especially the latter—were also decreased significantly in most of the groups.

Conclusion: Axotomy at the early stages in newborn rats resulted in noticeable morphometrical changes in motoneurons and their dendrites.     

Implantation of PNS Graft Inhibits the Induction of Neuronal Nitric Oxide Synthase and Enhances the Survival of Spinal Motoneurons Following Root Avulsion

In a spinal root injury model, our previous studies have shown that induction of nitric oxide synthase (NOS) appears only in spinal motoneurons of the root-avulsed segment in which significant motoneuron loss occurs but not in those of the distal root-axotomized segment (root axotomy 5-10 mm from the spinal cord) in which most motoneurons survive the injury. One hypothesis for the different response of motoneurons to root avulsion and distal root axotomy is that neurotrophic factors produced by the remaining peripheral nervous system (PNS) component are available for the distally axotomized motoneurons but are not available following avulsion. This hypothesis is tested in the present study by implantation of a PNS graft following the root avulsion. Results of the present study show that implantation of a PNS graft significantly enhances the survival of motoneurons following avulsion. Expression of NOS due to avulsion injury is completely inhibited in all motoneurons that regrow into the PNS graft. These results indicate that induction of NOS in avulsed motoneurons may result from the deprivation of neurotrophic factors produced by the PNS component, and the survival promoting effects of neurotrophic factors may be achieved by modifying certain cellular molecules such as NOS.     

Response of facial and rubrospinal neurons to axotomy: changes in mRNA expression for cytoskeletal proteins and GAP-43.

Neurons confined within the mammalian CNS usually do not regenerate after axonal injury, while axonal regeneration is the rule in the PNS. It has been hypothesized that this may be related to differences in the microenvironment of the PNS versus CNS and to differences in the neuronal response to injury. In order to test the latter hypothesis, we compared changes in gene expression after axotomy in two populations of neurons: rat facial motoneurons and rat rubrospinal neurons. In situ hybridization with cDNA probes for the medium and light neurofilament protein revealed a reduced mRNA content in both facial and rubrospinal neurons at all times investigated (i.e., 1, 2, and 3 weeks after axotomy). On the other hand, mRNAs for actin and tubulin were increased in both neuronal populations during the first week after axotomy. While this increase was sustained in facial motoneurons for several weeks, total tubulin mRNA and actin mRNA were decreased in rubrospinal neurons at 2 and 3 weeks after axotomy, coincident with their atrophy. The developmentally regulated T alpha 1 tubulin mRNA, which was previously shown to be reexpressed in facial motoneurons after axotomy, was elevated severalfold in axotomized rubrospinal neurons, and increased levels persisted in some rubrospinal neurons as late as 7 weeks after axotomy. Similarly, the developmentally regulated GAP-43 mRNA increased in both axotomized facial and rubrospinal neurons, and increased levels were sustained in some axotomized rubrospinal neurons for at least 7 weeks. The response of rubrospinal neurons to axotomy in the cervical spinal cord is, in the first week, qualitatively similar to the response of facial motoneurons. However, by 2 weeks after axotomy there is a generalized reduction in mRNA levels for all three cytoskeletal proteins that is associated with neuronal atrophy. During this period, mRNA levels for the two specific markers of the growth state, T alpha 1 tubulin and GAP-43, remain elevated. Thus, axotomy of rubrospinal neurons appears to set in motion two independent events. First, an axotomy signal initiates a cell-body reaction similar to that of PNS neurons, including increased mRNA levels for T alpha 1 tubulin and GAP-43. Later, a generalized cellular atrophy and decrease in mRNA levels occur without reversing the specific responses of T alpha 1 and GAP-43 to axotomy. We conclude that the failure of rubrospinal neurons to regenerate is not due to a failure to initiate gene-expression changes characteristic of regenerating peripheral neurons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mechanism of delayed intracranial hypertension after cerebroventricular infusions in conscious rats

Prior studies showed that cerebroventricular infusions of artificial cerebrospinal fluid, 8 μl/min for 10 min, followed by a 10 min rest and a 24 h infusion of 0.5 μl/min, raised cerebrospinal fluid pressure (CSFp) of conscious, unrestrained rats after about 2 h. Here, we report that the 10 min infusion alone evoked a delayed, prolonged rise in CSFp. Pressure during the infusion itself rose and recovered quickly, as is usually reported. Pressure/volume tests, used to calculate resistance to outflow (R_o) and compliance (C), revealed that infusions increasedR_o and decreasedC, after a delay (P < 0.05). The rise in CSFp after infusion was blocked by pretreatment with acetazolamide + ouabain (P < 0.05), but the delayed changes in R_o andC were unaffected. We suggest that the 10 min infusion of a sterile, balanced salt solution has a primary effect that increasesR_o; as CSF synthesis continues, C is exhausted and the delayed rise in CSFp ensues. This non-traumatic method of raising CSFp may be a useful method to study intracranial fluid dynamics.     

Restorative effects of reinnervation on the size and dendritic arborization patterns of axotomized cat spinal alpha-motoneurons.

In a preceding paper [Br?nnstr?m, et al. (1992) J. Comp. Neurol. 318:439-451] a marked reduction in dendritic size was observed in cat spinal motoneurons following permanent axotomy. The aim of the present study was to analyse the possible restorative effects of peripheral reinnervation on the size and dendritic branching patterns of cat spinal motoneurons which had been deprived of neuromuscular contact for an extended period of time. In adult cats the medial gastrocnemius (MG) nerve was transected and ligated. After 6 weeks the nerve was allowed to reinnervate its muscle through a nerve graft. With approximately 6 weeks needed for muscle reinnervation [Foehring, et al. (1986) J. Neurophysiol. 55:947-965], the MG motoneurons were devoid of neuromuscular contact for altogether about 12 weeks. Two years later reinnervated MG alpha-motoneurons were intracellularly labelled with horseradish peroxidase to allow quantitative analyses of the cell bodies and dendritic trees. Comparisons were made with previous data from normal and permanently axotomized MG motoneurons. The reinnervated motoneurons exhibited positive correlations between dendritic stem diameter, on one hand, and combined length, volume, membrane area, and number of end branches of the whole dendrite, on the other. By using the regression equations for these correlations, the total dendritic size of whole reinnervated motoneurons could be estimated. Such calculations showed that in comparison with the reduction in dendritic size found at 12 weeks after permanent axotomy (Br?nnstr?m et al., see above), peripheral reinnervation caused the dendritic volume and membrane area to return to normal values. However, the values for combined dendritic length and number of dendritic end branches were still reduced by more than 25% as compared to the normal situation. The results indicate that following reinnervation of the target muscle, the axotomized motoneurons did not recover their original number of dendritic branches. The normalization of dendritic membrane area and volume was instead accomplished by two other mechanisms, namely an increase in dendritic diameters and an increased number of dendrites per neuron.     

A dose-dependent facilitation and inhibition of peripheral nerve regeneration by brain-derived neurotrophic factor

The time-dependent decline in the ability of motoneurons to regenerate their axons after axotomy is one of the principle contributing factors to poor functional recovery after peripheral nerve injury. A decline in neurotrophic support may be partially responsible for this effect. The up-regulation of BDNF after injury, both in denervated Schwann cells and in axotomized motoneurons, suggests its importance in motor axonal regeneration. In adult female Sprague-Dawley rats, we counted the number of freshly injured or chronically axotomized tibial motoneurons that had regenerated their axons 1 month after surgical suture to a freshly denervated common peroneal distal nerve stump. Motor axonal regeneration was evaluated by applying fluorescent retrograde neurotracers to the common peroneal nerve 20 mm distal to the injury site and counting the number of fluorescently labelled motoneurons in the T11-L1 region of the spinal cord. We report that low doses of BDNF (0.5-2 microg/day for 28 days) had no detectable effect on axonal regeneration after immediate nerve repair, but promoted axonal regeneration of motoneurons whose regenerative capacity was reduced by chronic axotomy 2 months prior to nerve resuture, completely reversing the negative effects of delayed nerve repair. In contrast, high doses of BDNF (12-20 microg/day for 28 days) significantly inhibited motor axonal regeneration, after both immediate nerve repair and nerve repair after chronic axotomy. The inhibitory actions of high dose BDNF could be reversed by functional blockade of p75 receptors, thus implicating these receptors as mediators of the inhibitory effects of high dose exogenous BDNF.