The human G93A-superoxide dismutase-1 mutation, mitochondrial glutathione and apoptotic cell death.

Mutations in Cu/Zn superoxide dismutase are a cause of motor neuron death in about 20% of cases of familial amyotrophic lateral sclerosis (ALS). Although the molecular mechanism of which these mutations induce motor neuron cell death is to a large extent unknown, there is significant evidence that effects on mitochondrial function and development of oxidative stress make a major contribution to the selective death of motor neurons in this disease. In this overview article we review the current understanding of mutant SOD1-mediated motor neuron degeneration in ALS with focus on oxidative damage and mitochondrial dysfunction. We also present novel information on the role of mitochondrial glutathione for the survival of NSC-34 cells stably transfected with the human SOD1(G93A) mutation, putting forward the hypothesis that this antioxidant pool provides a potentially useful target for therapeutic intervention.

Targeted silencing of TrkA expression in rat forebrain neurons via the p75 receptor.

Basal forebrain neurons express the neurotrophin receptors, p75NTR and tyrosine kinase receptor A (TrkA). We tested the hypothesis that impairment of memory in rats could be achieved by RNA interference (RNAi) -induced silencing of TrkA specifically within these neurons. A novel fusogenic, karyophilic immunoporter (fkAb(p75)-ipr) was constructed from the antibody, MC192 (monoclonal antibody to the rat neurotrophin receptor p75NTR, Ab(p75)), poly-l-lysine together with the hemagglutinin 2 and VP1 nuclear localization peptides of influenza and SV40 virus, respectively. Plasmid DNA constructs containing short hairpin sequences inhibitory to tyrosine kinase receptor A expression (TrkAi) and the gene encoding cGFP (green fluorescent protein from coral fish) was produced. These TrkAi plasmids were mixed with the immunoporter, forming the immunogene, TrkAi-fkAb(p75). A control TrkAsc complexed with fkAb(p75) (TrkAsc-fkAb(p75)) immunogene was constructed from a scrambled sequence (TrkAsc) and fkAb(p75)-ipr. Rats were infused using an osmotic mini-pump into the third ventricle with either TrkAi-fkAb(p75) or TrkAsc-fkAb(p75). Naive rats were also included as additional controls. After 7 days, examination of gene expression on forebrain sections of some rats revealed cGFP expression in TrkA neurons. Fifteen to 19 days after infusion, rats were tested in a Morris water maze apparatus. Animals that received TrkAi-fkAb(p75) showed significantly impaired spatial memory learning ability compared with naive or TrkAsc-fkAb(p75)-treated rats. Western blot and immunofluorescence analysis showed that TrkA protein levels and numbers of TrkA positive neurons were reduced by 60% and 55% respectively in TrkAi-fkAb(p75)-infused rats compared with infused controls or naive animals. We conclude that p75-receptor-mediated RNAi-induced silencing of genes offers a novel and powerful way to study the function of specific endogenous genes within distinct neuronal subpopulations of the brain.

CD 271 (P75 neurotrophin receptor).

P75NTR (or CD271) is a member of the Tumor Necrosis Factor receptor (TNFR) super family of transmembrane proteins that share significant homology in their extracellular domains. Subsets of TNF receptors, including CD271, have a cytoplasmic death domain, although CD271 has unique intracellular structure and downstream signaling partners. CD271 is also differentiated from other members of the TNFR receptor family in that it binds pro and mature neurotrophins and affects the growth, differentiation and death of the nervous system. The ligands for CD271 are neurotrophins, which are Nerve Growth Factor (NGF), Brain-Derived Growth factor (BDNF), Neurotrophin 3 (NT3) and Neurotrophin 4/5 (NT4/5). Recent studies have provided evidence that CD271 also serves as a receptor for the pro-forms of these neurotrophins.

The regulation of nerve growth factor synthesis and delivery to peripheral neurons.

Recent experiments have provided new insights into the way in which the synthesis of nerve growth factor (NGF) is regulated. In particular, a variety of agents have been found to influence the cellular concentration of mRNANGF and NGF both in vitro and in vivo. However, no clear mechanism has been found to indicate the existence of a feedback regulation of NGF synthesis by effector tissue innervation. We have argued in this review that some form of feedback control is likely to exist between the innervation and the cells producing the factor. One such possibility, the regulation of the availability of NGF by control of its secretion, is discussed.

Functional roles of neurotrophin 3 in the developing and mature sympathetic nervous system.

Nerve growth factor (NGF) is a potent regulator of sympathetic neuronal function in both developing and adult animals. This article reviews the evidence published in recent years indicating that another member of the NGF family, neurotrophin 3 (NT3), plays both a complementary and overlapping role in the development and maturation of sympathetic neurons. In migratory neural crest cells, expression of the high-affinity receptor, trkC, and promotion of mitosis by NT3 suggest an involvement in gangliogenesis, since sympathetic neuroblasts express both NT3 and trkC and require NT3 for their proliferation, differentiation, and survival, it has been proposed that the factor acts at this developmental stage as an autocrine or paracrine factor. However, NT3 also acts in parallel with NGF to promote the survival of postmitotic neurons during late development. Both trkC and trkA are expressed in sympathetic neurons and function as high-affinity receptors for NT3. NT3 is synthesized in sympathetic effector tissues and the endogenous factor is retrogradely transported to accumulate within the cell soma. Thus, in addition to its role in the differentiation of sympathetic neurons, NT3, like NGF, is also an effector tissue-derived neurotrophic factor for these neurons in maturity.

Peripheral projections of rat primary sensory neurons immunoreactive for neurotrophin 3.

Sensory neurons can be classified into subpopulations based on a variety of characteristics, including their morphology and physiological modalities. Whether any of these classifications correlates with neurotrophic sensitivities has not been determined. We have recently reported that a subpopulation of large diameter sensory neurons of the rat contain neurotrophin 3-like immunoreactivity (NT3-ir). In this study, we have further characterised NT3-ir sensory neurons by their size, segmental localization, and peripheral projections by combined techniques of retrograde tracing and immunohistochemistry. The size distribution showed that NT3-ir was localised to a subpopulation of large-diameter neurons ranging from 560 to 3,120 microns2. Greater numbers of NT3-ir neurons reside in trigeminal (43% of total), cervical (36%), and lumbar (39%) than in thoracic spinal ganglia (13-17%). In combination with Fluoro-Gold retrograde tracing, it was found that about 30% of sensory neurons projecting to the tibial muscle were NT3-ir, compared with 39% for tendon, 50% for whisker hair follicles, 17% for subdermis or epidermis, and only 1% for kidney or adrenal gland. These studies indicate that NT3-ir sensory neurons mainly project to skin and muscles but not viscera. Thus, the characterization of NT3-ir spinal sensory neurons suggests that large sensory neurons subserving proprioception and mechanoception require NT3 for the maintenance of normal function.

Endogenous nerve growth factor is required for regulation of the low affinity neurotrophin receptor (p75) in sympathetic but not sensory ganglia.

During development, many sympathetic and sensory neurons are dependent on nerve growth factor (NGF) for survival. The low affinity neurotrophin receptor (p75), expressed in these neurons, is regulated by exogenous NGF in vitro and in vivo. However, whether p75 expression in vivo is under the control of endogenous NGF has not been determined. The role of NGF in regulating the expression of p75 in sympathetic and sensory nerves was investigated in Sprague-Dawley rats treated with an antiserum specific for NGF. P75 was differentially regulated. P75 immunoreactivity (-ir) within sympathetic neurons in the superior cervical ganglia (SCG) was reduced after 2 days, and disappeared after 5 days, of treatment with the NGF antiserum. In contrast, a significant increase in p75-ir was detected in nerve bundles within and close to the SCG from 3 to 14 days after treatment. A similar pattern of p75 expression was observed in the stellate and coeliac ganglia. In contrast, p75 expression in nerve terminals of the mesenteric arteries and irides was reduced. However, in the same animals the expression of p75 was not significantly affected by the treatment in dorsal root, trigeminal or nodose ganglia, salivary gland or small intestine. In contrast to p75, the NGF high affinity receptor trkA was little affected in sympathetic neurons by depletion of endogenous NGF for 2 weeks. These results indicate that endogenous NGF is required in sympathetic ganglia for the expression of p75 but not trkA in neurons, but for the down-regulation of p75 in glia. In contrast, endogenous NGF is not essential for the regulation of p75 in neurons or glia within sensory ganglia.

Neurotrophin 3 is increased in the spontaneously hypertensive rat.

OBJECTIVE: To determine whether the noradrenergic sympathetic hyperinnervation in the spontaneously hypertensive rat (SHR), a genetic model of essential hypertension, is associated with changes in neurotrophin 3 (NT3) concentrations. METHODS: NT3 levels were measured using a sensitive enzyme-linked immunosorbent assay (ELISA) in the superior cervical ganglia (SCG), heart, mesenteric artery (MA) and blood of postnatal and mature SHR and normotensive Wistar-Kyoto (WKY) rats. RESULTS AND CONCLUSIONS: NT3 levels in SHR are significantly higher in the SCG during the first 4 postnatal weeks, and in the heart and MA from 2 to 10 weeks of age, compared with levels in WKY rats. The elevated NT3 found in the sympathetic ganglia and hyperinnervated organs of SHR indicates that NT3 may play an important role in the development of hyperinnervation, possibly by enhancing the survival and/or nerve sprouting of sympathetic neurons.

Endogenous brain-derived neurotrophic factor is anterogradely transported in primary sensory neurons.

Neurotrophins are a family of proteins which act as survival and differentiative factors in the developing and mature nervous system. Extensive evidence has been provided for their retrograde action following incorporation into nerve terminals and transport to the cell body. In contrast, we now demonstrate that one neurotrophin, brain-derived neurotrophic factor, is transported anterogradely via both peripheral and central processes of spinal sensory neurons. Using newly generated antisera, we have examined the distribution of brain-derived neurotrophic factor immunoreactivity and found it to be present within a subpopulation of sensory somata, primarily those with a small-to-medium diameter. The immunoreactivity was accumulated on both the distal and proximal sides of a ligature on the sciatic nerve. The accumulation on the distal side, but not on the proximal side, was substantially reduced by pretreatment with brain-derived neurotrophic factor antibodies in vivo. In contrast to the periphery, the immunoreactivity only accumulated on the proximal side of a lesion of the dorsal root. In the spinal cord, most nerve terminals immunoreactive for brain-derived neurotrophic factor were identified in lamina II. Lesion of the dorsal root led to a reduction of these nerve terminals. These studies indicate that the factor is transported not only retrogradely to, but also anterogradely from, the spinal ganglia to terminals in the periphery and spinal cord. The findings add a new dimension to the role of neuronal growth factors, since anterograde transport has not been observed previously for any endogenous survival factor.

Endogenous neurotrophin-3 supports the survival of a subpopulation of sensory neurons in neonatal rat.

Neurotrophin-3 promotes the differentiation and supports the survival of neuroblasts derived from the neural crest in early development. Neurotrophin-3 also plays an important role in the differentiation and survival of a subpopulation of large sensory neurons after their axons arrive at their targets. Proprioception and mechanoception are lost after gene deletion of neurotrophin-3 or its high-affinity receptor, TrkC. However, the function of neurotrophin-3 during late development and in mature animals is not clear. We have used an antiserum, specific for neurotrophin-3, to neutralize endogenous neurotrophin-3 in postnatal rats to determine its role in late sensory neuron development. Administration of the antiserum for a period of two weeks, but not one week, resulted in a 20% reduction in the number of primary sensory neurons in the dorsal root ganglia and a 19% reduction in the number of myelinated axons in the saphenous nerve. The size distribution histogram also indicated that a subpopulation of large neurons was lost by the neurotrophin-3 antiserum treatment. This neuronal loss was accompanied by reduced cell soma sizes and weights of the ganglia. Immunoreactivities for calbindin and calretinin were reduced in the trigeminal and dorsal root ganglia and nerve fibres surrounding whisker hair follicles. The number of Merkel cells in touch domes labelled with quinacrine and the number of parvalbumin-immunoreactive neurons in the dorsal root ganglia were significantly reduced by the antibody treatment. In contrast, the number of muscle spindles in the gastrocnemius muscle is not reduced by the neurotrophin-3 antiserum. Together, these results indicate that a subpopulation of primary sensory neurons in the neonatal rat requires neurotrophin-3 for their survival and expression of calcium binding proteins. In addition, Merkel cells in touch domes also require neurotrophin-3 for their survival. Thus, endogenous neurotrophin-3 in neonatal rats is critical for the survival and function of a subpopulation of primary sensory neurons and Merkel cells.

Endogenous nerve growth factor and neurotrophin-3 act simultaneously to ensure the survival of postnatal sympathetic neurons in vivo.

For many years nerve growth factor was the only factor known to influence embryonic and postnatal development of sympathetic neurons. Its deprivation by antibody neutralization or gene mutation results in extensive neuron death. Recently it has been shown that these neurons also require neurotrophin-3 for survival in the late developmental period. Using neurotrophin-3 antiserum to neutralize endogenous factor in newborn rats. Our laboratory has shown that extensive numbers of neurons are lost from both pre- and paravertebral ganglia, indicating a continuing requirement for neurotrophin-3. In the present study we sought to determine whether neurons could survive in vivo in the presence of excess amounts of either nerve growth factor or neurotrophin-3 alone. Consistent with previous findings, administration of antiserum to nerve growth factor or neurotrophin-3 to newborn rats for eight days, resulted in an extensive loss of sympathetic neurons. Interestingly, administration of neurotrophin-3 together with nerve growth factor antiserum or nerve growth factor with neurotrophin-3 antiserum reversed this neuronal loss. However the latter combination was less effective than the former. Furthermore, the ability of exogenous nerve growth factor to increase both the number and size of sympathetic neurons was prevented by the simultaneous deprivation of endogenous neurotrophin-3. Unlike nerve growth factor, exogenous neurotrophin-3 failed to rescue the naturally occurring neuronal death in these newborn rats. Further evidence for a physiological role for both nerve growth factor and neurotrophin-3 was found by the detection of both trkA and trkC immunoreactivity in neurons of the superior cervical ganglion. Taken together, these results suggest that sympathetic neurons do not have an absolute requirement for either nerve growth factor or neurotrophin-3 and that the endogenous supply of either factor alone is insufficient to support neuronal survival postnatally. However, while each factor may play similar roles in the regulation of postmitotic neuronal function, some evidence for distinct functions has been identified.

Injured primary sensory neurons switch phenotype for brain-derived neurotrophic factor in the rat.

Peripheral nerve injury results in plastic changes in the dorsal root ganglia and spinal cord, and is often complicated with neuropathic pain. The mechanisms underlying these changes are not known. We have now investigated the expression of brain-derived neurotrophic factor in the dorsal root ganglia with histochemical and biochemical methods following sciatic nerve lesion in the rat. The percentage of neurons immunoreactive for brain-derived neurotrophic factor in the ipsilateral dorsal root ganglia was significantly increased as early as 24 h after the nerve lesion and the increase lasted for at least two weeks. The level of brain-derived neurotrophic factor messenger RNA was also significantly increased in the ipsibut not contralateral dorsal root ganglia. Both neurons and satellite cells in the lesioned dorsal root ganglia synthesized brain-derived neurotrophic factor messenger RNA after the nerve lesion. There was a dramatic shift in size distribution of positive neurons towards large sizes seven days after sciatic nerve lesion. Morphometric analysis and retrograde tracing studies showed that no injured neurons smaller than 600 microm2 were immunoreactive for brain-derived neurotrophic factor, whereas the majority of large injured neurons were immunoreactive in the ipsilateral dorsal root ganglia seven days postlesion. The brain-derived neurotrophic factor-immunoreactive nerve terminals in the ipsilateral spinal cord were reduced in the central region of lamina II, but increased in more medial regions or deeper into laminae III/IV. These studies indicate that sciatic nerve injury results in a differential regulation of brain-derived neurotrophic factor in different subpopulations of sensory neurons in the dorsal root ganglia. Small neurons switched off their normal synthesis of brain-derived neurotrophic factor, whereas larger ones switched to a brain-derived neurotrophic factor phenotype. The phenotypic switch may have functional implications in neuronal plasticity and generation of neuropathic pain after nerve injury.

Electron immunocytochemical localization of enkephalin-like material in catecholamine-containing cells of the carotid body, the adrenal medulla, and in pheochromocytomas of man and other mammals.

Enkephalin-like immunoreactivity has been localized to electron-dense secretory granules of cat and piglet carotid bodies and adrenal medullae, horse adrenal medulla, and also to human adrenal medulla and pheochromocytomas using a gold-labeled antibody technique performed at the electron microscopic level. The same granules were also demonstrated to exhibit dopamine-beta-hydroxylase-like immunoreactivity, which suggests a granular colocalization of amines and peptides in catecholamine-storing cells.

Localization of laminin to retinal vessels of the rat and mouse using whole mounts.

Using a whole mount procedure in adult and neonatal mice and adult rats, we have developed an immunohistochemical method for the localization of laminin-like immunoreactivity (LLIR) to the retinal vessels. LLIR was localized to the vascular basement membrane, permitting a clear three-dimensional view of the retinal vasculature. Positive stain was seen in the inner limiting membrane, in retracted capillaries, encasing pericytes, and in a banding pattern on retinal arterioles. The major findings with the whole mount preparations were confirmed using paraffin-embedded material, with the additional observation of LLIR in the lens capsule. In whole mounts of retinas from neonatal mice, LLIR was present from the earliest stages of capillary growth, indicating that laminin is likely to be secreted by endothelial cells during retinal angiogenesis. LLIR within the retinal nerve fiber layer does not precede capillary ingrowth, so no evidence was found that laminin acts as a tracker signal for retinal angiogenesis.

The thoracic sympathetic neurons of the chick: normal development and the effects of nerve growth factor.

The generation and degeneration of sympathetic neurons in the third thoracic ganglion (segment 19) of the chick were studied between embryonic days (E) 7-18 using 3H-Thymidine autoradiography and routine cell counts. Cumulative radiolabelling experiments indicated that few sympathetic neurons were generated on E6-7. 10% of the sympathetic neurons were generated on E8 and a further 20% on E9. The final 70% of neurons completed the mitotic cycle between E10-12. Cell counts demonstrated that the neuronal population increased from 10,166 +/- 423 (mean +/- SEM) to 22,291 +/- 767 between E8-10 and remained stable up to E14. The population subsequently declined by 37%, to 14,157 +/- 831, by E18. Pyknotic neurons were found at all stages of development, but were most apparent between E7-15. The effects of Nerve Growth Factor (NGF) on the number of both surviving and pyknotic neurons in the ganglion were also examined. E9 embryos treated with NGF from E5-8 showed a 57% increase in the number of sympathetic neurons. This increase therefore occurred prior to the decline in neuronal number and was not accompanied by a decrease in the number of visibly pyknotic neurons. It is therefore possible that early NGF treatment increases the number of sympathetic neurons through a mechanism other than the attenuation of cell death.

Increase in neuronal Jun immunoreactivity and epidermal NGF levels following UV exposure of rat skin.

Effects of cutaneous ultraviolet (UV) irradiation on superficial nerve fibres are controversial. In the present study we demonstrate by electron microscopy that single high-dose UV exposure of rat skin results in membrane damage in peripheral nerves. Furthermore, a UV-induced accumulation of nerve growth factor (NGF) within the irradiated epidermis and a delayed increase in nuclear Jun immunoreactivity in dorsal root ganglion neurones were detected by immunohistochemistry. Our results suggest that UV-mediated alterations of axonal membranes leads to an activation of Jun transcription factor within affected neurones despite an increase in neurotrophic NGF within their innervation area.

Small primary sensory neurons innervating epidermis and viscera display differential phenotype in the adult rat.

Target tissues contribute to the phenotype and function of sensory neurons. Due to lack of appropriate markers for trkA expressing sensory axons and terminals, the detailed peripheral projection of these neurons is unclear. In this study, the peripheral projections of trkA immunoreactive neurons are characterized using the combined techniques of immunohistochemistry and retrograde tracing. We found approximately 65% of all neurons projecting to the adrenal gland and kidney are trkA immunoreactive, whereas 6, 14 and 37% of neurons innervating whisker follicle, epidermis and footpad, respectively, are immunoreactive for trkA. A low proportion of trkA immunoreactive neurons innervating epidermis indicates that the majority of sensory neurons innervating epidermis are independent of trkA signalling for their normal function. We further investigated whether these epidermal projecting neurons can bind isolectin IB4. We found approximately 70% of all neurons innervating epidermis are IB4 binding neurons, but they did not express trkA. Thus, NGF sensitive neurons primarily project to viscera but not epidermis or other skin structures, whereas IB-4 positive neurons primarily project to epidermis in the adult rat.

Ultrastructural localization of brain-derived neurotrophic factor in rat primary sensory neurons.

In a previous study we have shown that brain-derived neurotrophic factor (BDNF) is present in a subpopulation of small- to medium-sized sensory neurons in the dorsal root ganglia (DRG) and is anterogradely transported in both the peripheral and central processes. Within the spinal cord, BDNF is localized to varicosities of sensory nerve terminals in laminae I and II of the dorsal horn. This study raised the question of whether BDNF is localized in synaptic vesicles of the afferent nerve terminals. Using immunohistochemical and immunocytochemical techniques we have now investigated the ultrastructural localization of BDNF in the spinal cord of the rat. In addition, its colocalization with the low affinity neurotrophin receptor, p75, and calcitonin gene related peptide (CGRP) was also investigated. In lamina II of the spinal cord, BDNF immunoreactivity was restricted to nerve terminals. The reaction product appeared associated with dense-cored and clear vesicles of terminals superficial laminae. Double labelling experiments at the light microscopic level showed that 55% of BDNF immunoreactive neurons in DRG are colocalized with CGRP and many nerve terminals in laminae I and II of the spinal cord contained both BDNF and CGRP immunoreactivities. The results of double labelling at the ultrastructural level showed that most BDNF-ir (immunoreactive) nerve terminals contained CGRP or the low affinity neurotrophin receptor, p75, but not vice versa. These results point to the conclusion that BDNF may be released in parallel with neurotransmitters from nerve terminals in the spinal cord from a subpopulation of nociceptive primary afferents.

An improved procedure for the immunohistochemical localization of nerve growth factor-like immunoreactivity.

Nerve growth factor (NGF) is a survival factor required by a number of neuronal populations including most post-ganglionic sympathetic neurones. NGF has been detected and quantified in many tissues but there is little information regarding its cellular localization. Although it has been argued that histological detection has proven difficult due to the low levels of NGF present, other factors may contribute to prevent its identification. In the present study, we report a method for the histological detection of NGF-like immunoreactivity in the rat superior cervical ganglia (SCG). Adult Wistar-Kyoto rats were perfused briefly with either a high or low pH buffer prior to fixation and routine immunohistochemistry. Polyclonal antibodies to native mouse NGF used in the present study recognized mouse NGF but not recombinant human neurotrophin 3 (rhNT3) or brain-derived neurotrophic factor (rhBDNF) by immunoblot analysis. NGF-like immunoreactivity was localized to most sympathetic neurones. Immunoreactivity was detected in the cytoplasm with dense labelling around nuclei. No stain was seen in sections incubated with normal sheep IgG or from animals perfused with phosphate buffer (pH 7.4) prior to fixation. In addition, axotomy resulted in the disappearance of NGF immunoreactivity which was confirmed by biochemical quantification. Finally, no NGF immunoreactivity was found in neurones of rats treated systemically with NGF antiserum 3 days earlier. Possible mechanisms underlying the improvement of NGF immunohistochemistry by pH manipulation before fixation are discussed.

Comparison of wheat germ agglutinin-horseradish peroxidase and biotinylated dextran for anterograde tracing of corticospinal tract following spinal cord injury.

Established methods for monitoring regeneration of the corticospinal tract involve anterograde labelling of the cortical motor neuron. While wheat germ agglutinin-horseradish peroxidase conjugate has been used to anterogradely label these neurons, we demonstrate that this technique may not completely label the whole axon and fine terminal processes when this tracer is administered in dried form. An alternative method is described for anterograde labelling of cortical motor neurons using biotinylated dextran. This tracer may be applied by either microinjection of 10% biotinylated dextran or implanting small globules of the dried tracer into the motor cortex. While more laborious, microinjection results in better anterograde labelling than implantation of dried biotinylated dextran. A procedure is also described for preparing serial coronal sections through the entire spinal cord and thaw-mounted on a minimum number of slides. The labelled nerve processes in these tissue sections can be visualised in the spinal cord under a fluorescent microscope following incubation with cy3-streptavidin complex. Permanent labelling of the biotinylated nerve processes is achieved by incubation of tissue sections with streptavidin-horseradish peroxidase conjugate followed by stringent washes and staining with tetramethylbenzidine. Use of tetramethylbenzidine allows resolution of a greater number of finer labelled processes than diaminobenzindine and allows clear visualisation of individual regenerating corticospinal tract processes. Using these procedures, we demonstrate that the corticospinal tract is completely lesioned by a standardised contusion spinal cord injury produced by the New York University weight-drop device.