Clarke's Nucleus Neurons Research Articles

Regulation of stearoyl-CoA desaturase-1 after central and peripheral nerve lesions

BackgroundInterruption of mature axons activates a cascade of events in neuronal cell bodies which leads to various outcomes from functional regeneration in the PNS to the failure of any significant regeneration in the CNS. One factor which seems to play an important role in the molecular programs after axotomy is the stearoyl Coenzyme A-desaturase-1 (SCD-1). This enzyme is needed for the conversion of stearate into oleate. Beside its role in membrane synthesis, oleate could act as a neurotrophic factor, involved in signal transduction pathways via activation of protein kinases C.ResultsIn situ hybridization and immunohistochemistry demonstrated a strong up-regulation of SCD at mRNA and protein level in regenerating neurons of the rat facial nucleus whereas non-regenerating Clarke's and Red nucleus neurons did not show an induction of this gene.ConclusionThis differential expression points to a functionally significant role for the SCD-1 in the process of regeneration.

Transplants of cells genetically modified to express neurotrophin-3 rescue axotomized Clarke's nucleus neurons after spinal cord hemisection in adult rats.

To test the idea that genetically engineered cells can rescue axotomized neurons, we transplanted fibroblasts and immortalized neural stem cells (NSCs) modified to express neurotrophic factors into the injured spinal cord. The neurotrophin-3 (NT-3) or nerve growth factor (NGF) transgene was introduced into these cells using recombinant retroviral vectors containing an internal ribosome entry site (IRES) sequence and the beta-galactosidase or alkaline phosphatase reporter gene. Bioassay confirmed biological activity of the secreted neurotrophic factors. Clarke's nucleus (CN) axons, which project to the rostral spinal cord and cerebellum, were cut unilaterally in adult rats by T8 hemisection. Rats received transplants of fibroblasts or NSCs genetically modified to express NT-3 or NGF and a reporter gene, only a reporter gene, or no transplant. Two months postoperatively, grafted cells survived at the hemisection site. Grafted fibroblasts and NSCs expressed a reporter gene and immunoreactivity for the NGF or NT-3 transgene. Rats receiving no transplant or a transplant expressing only a reporter gene showed a 30% loss of CN neurons in the L1 segment on the lesioned side. NGF-expressing transplants produced partial rescue compared with hemisection alone. There was no significant neuron loss in rats receiving grafts of either fibroblasts or NSCs engineered to express NT-3. We postulate that NT-3 mediates survival of CN neurons through interaction with trkC receptors, which are expressed on CN neurons. These results support the idea that NT-3 contributes to long-term survival of axotomized CN neurons and show that genetically modified cells rescue axotomized neurons as efficiently as fetal CNS transplants.

Site of injury‐directed induction of heme oxygenase‐1 and ‐2 in experimental spinal cord injury: differential functions in neuronal defense mechanisms?

The heme oxygenase (HO) isozymes catalyze oxidation of the heme molecule to biliverdin and carbon monoxide (CO) with the release of chelated iron. Presently, we have defined, for the first time, propensity for site of injury-directed induction of isozymes--the stress-inducible isozyme, HO-1, responds distal (below) and the glucocorticoid (GC)-inducible HO-2 responds proximal (above) to the site of injury. We have also shown that reactive iron (Fe3+) and cGMP staining spatially resemble that of HO-1; which, in turn, colocalizes in motor neurons with transcription factors: Fas-associated protein containing death domain (FADD), tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and p53. Spinal cord injury (SCI) was inflicted by clip compression for 30 min, and analyses were carried out after 4 h or 16 h. When compared with spinal cord segments proximal to the site of injury, northern blot analysis showed remarkably higher levels of HO-1 mRNA distal (below) to the site of injury at both time points. In contrast, HO-2 mRNA levels were elevated proximal (above) to the site of injury and more prominently at 16 h post SCI. Immunohistochemical analyses were carried out using 2 x 5 mm segments above and below the compression site. When compared with segments above the site of injury, the intensity of HO-1 immunostaining and the number of HO-1 positive neurons in the ventral horn motor neurons were prominently increased in segments below the injury. Western blot analysis confirmed the observations. HO-2 protein was mapped to the ventral horn motor neurons, oligodendrocytes, the Clarke's nucleus neurons and the ependymal cells. When compared with segments below the site of injury, neuronal HO-2 staining intensity was increased above the site of injury, and most notably at 16 h. These observations were also confirmed by western blotting and HO activity measurements. Tissue Fe3+ and cGMP staining were increased and prominently mapped below the site of injury, where cGMP colocalized with HO-1 in the nucleus of the motor neurons. Also, a site of injury-directed pattern of induction of FADD, TRAIL, and p53 immunoreactivity, and a widespread colocalization of the oncogenes with HO-1 protein, were found within motor neurons below the level of injury. We forward the hypothesis that HO-1 and HO-2 have different roles in the defense mechanisms of the injured nervous system. We hypothesize that HO-1 protects against further damage by contributing to controlled cell death through their intrinsic suicide program, while HO-2 is involved in suppression of inflammatory response by NO derived radicals.

Single Injections of a DNA Plasmid That Contains the Human Bcl-2 Gene Prevent Loss and Atrophy of Distinct Neuronal Populations after Spinal Cord Injury in Adult Rats

Spinal cord injury in adult mammals causes atrophy or loss of axotomized neurons. We have previously found that the product of the antiapoptotic gene Bcl-2, delivered by intraspinal injection of a DNA plasmid, reduces atrophy and loss of axotomized Clarke's nucleus neurons in adult rats. Here we studied whether the same treatment protects axotomized red nucleus (RN) neurons. Two months after the right dorsolateral funiculus was ablated in adult Sprague-Dawley rats by C3/C4 subtotal hemisection, there was approximately 48% loss of RN neurons in the magnocellular portion of the RN contralateral to the lesion and atrophy of many surviving neurons. When a DNA plasmid encoding the human Bcl-2 gene and the bacterial reporter gene LacZ, complexed with cationic lipids, was injected just rostral to the subtotal hemisection site, 87% of RN neurons survived, and there was partial, but robust, protection from atrophy. These and our previous results indicated that intraspinal administration of the Bcl-2 gene can prevent retrograde cell loss and reduce atrophy of axotomized RN and Clarke's nucleus neurons in adult rats and provide an effective means to rescue neurons whose survival depends on different growth factors.

Chondroitinase ABC promotes axonal regeneration of Clarke's neurons after spinal cord injury.

We examined whether enzymatic digestion of chondroitin sulfate (CS) promoted the axonal regeneration of neurons in Clarke's nucleus (CN) into a peripheral nerve (PN) graft following injury of the spinal cord. After hemisection at T11, a segment of PN graft was implanted at the lesion site. Either vehicle, brain-derived neurotrophic factor (BDNF) or chondroitinase ABC was applied at the implantation site. The postoperative survival period was 4 weeks. Treatment with vehicle or BDNF did not promote the axonal regeneration of CN neurons into the PN graft. Application of 2.5 unit/ml chondroitinase ABC resulted in a significant increase (12.8%) in the number of regenerated CN neurons. Double labeling with Fluoro-Gold and NADPH-diaphorase histochemistry showed that the regenerated CN neurons did not express nitric oxide synthase (NOS). Our results suggest that CS is inhibitory to the regeneration of CN neurons following injury of the spinal cord.

Peripheral but not central axotomy induces changes in Janus kinases (JAK) and signal transducers and activators of transcription (STAT).

Nerve injury leads to the release of a number of cytokines which have been shown to play an important role in cellular activation after peripheral nerve injury. The members of the signal transducer and activator of transcription (STAT) gene family are the main mediators in the signal transduction pathway of cytokines. After phosphorylation, STAT proteins are transported into the nucleus and exhibit transcriptional activity. Following axotomy in rat regenerating facial and hypoglossal neurons, a transient increase of mRNA for JAK2, JAK3, STAT1, STAT3 and STAT5 was detected using in situ hybridization and semi-quantitative polymerase chain reaction (PCR). Of the investigated STAT molecules, only STAT3 protein was significantly increased. In addition, activation of STAT3 by phosphorylation on position Tyr705 and enhanced nuclear translocation was found within 3 h in neurons and after 1 day in astrocytes. Unexpectedly, STAT3 tyrosine phosphorylation was obvious for more than 3 months. In contrast, none of these changes was found in response to axotomy of non-regenerating Clarke's nucleus neurons, although all the investigated models express c-Jun and growth-associated protein-43 (GAP-43) in response to axonal injury. Increased expression of Janus kinase (JAK) and STAT molecules after peripheral nerve transection suggests changes in the responsiveness of the neurons to signalling molecules. STAT3 as a transcription factor, which is expressed early and is activated persistently until the time of reinnervation, might be involved in the switch from the physiological gene expression to an 'alternative program' activated only after peripheral nerve injury.

BDNF abolishes the survival effect of NT-3 in axotomized Clarke neurons of adult rats.

Neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) have previously been shown to support survival and axonal regeneration in various types of neurons. Also, synergistic neuroprotective effects of these neurotrophins have been reported in descending rubrospinal neurons after cervical spinal cord injury (Novikova et al., [2000] Eur. J. Neurosci. 12:776-780). The present study investigates the effects of intrathecally delivered NT-3 and BDNF on the survival and atrophy of ascending spinocerebellar neurons of Clarke nucleus (CN) after cervical spinal cord injury in adult rats. At 8 weeks after cervical spinal cord hemisection, 40% of the axotomized CN neurons had been lost, and the remaining cells exhibited marked atrophy. Microglial activity was significantly increased in CN of the operated side. Intrathecal infusion of NT-3 for 8 weeks postoperatively resulted in 91% cell survival and a reduction in cell atrophy, but did not reduce microglial activity. In spite of the fact that the CN neurons expressed both TrkC and TrkB receptors, only NT-3 had a neuroprotective effect, whereas BDNF was ineffective. Furthermore, when a combination of BDNF and NT-3 was administered, the neuroprotective effect of NT-3 was lost. The present results indicate a therapeutic potential for NT-3 in the treatment of spinal cord injury, but also demonstrate that in certain neuronal populations the neuroprotection obtained by a combination of neurotrophic factors may be less than that of a single neurotrophin.

Peripheral Nerve Graft and Neurotrophic Factors Enhance Neuronal Survival and Expression of Nitric Oxide Synthase in Clarke's Nucleus after Hemisection of the Spinal Cord in Adult Rat

The present study examined the effects of peripheral nerve (PN) graft and neurotrophic factors on the expression of nitric oxide synthase (NOS) and the survival of Clarke's nucleus (CN) neurons at the first lumbar spinal segment (L1) 15 days after hemisection of the spinal cord at T11. Normal intact CN neurons did not express NOS. Forty-one percent of the ipsilateral CN neurons survived after hemisection at T11, and 48% of the surviving neurons expressed NOS. Transplantation of PN graft at the lesion site promoted the survival of CN neurons to 71% and increased the expression of NOS to 70%. Continuous infusion of brain-derived neurotrophic factor, ciliary neurotrophic factor, and neurotrophic-3, but not glial cell-derived neurotrophic factor, at the lesion site enhanced the survival of CN neurons to about 65%. Among the surviving neurons about 70% were NOS-positive. These results indicated that transplantation of autologous PN graft or continuous infusion of neurotrophic factors could enhance the survival of axotomized CN neurons. In addition, the survival-promoting function of the neurotrophic agents was coincided with the upregulation of the expression of NOS. However, whether the upregulation of NOS expression in injured CN neurons is related to the rescue function or is a side effect of the neurotrophic factors is not clear and needed further investigation.

DNA plasmid that codes for human Bcl-2 gene preserves axotomized Clarke's nucleus neurons and reduces atrophy after spinal cord hemisection in adult rats

Spinal cord injury in adult mammals causes atrophy or death of some axotomized neurons. The product of the antiapoptotic gene Bcl-2 prevents neuron death in vivo. We delivered Bcl-2 by intraspinal injection of a DNA plasmid encoding this gene to determine if axotomized neurons destined to undergo retrograde death could be rescued. Axons of the right side Clarke's Nucleus (CN) were cut unilaterally in adult Sprague-Dawley rats by T8 hemisection, leaving the contralateral (left) CN as an intact control. Two months postoperatively, there was approximately 35% loss of total CN neurons in the right L1 segment. Only 15% of large CN neurons (>400 microm2), whose axons project to the cerebellum, survived--indicating atrophy and/or death of 85% of these cells. We injected a DNA plasmid encoding the human Bcl-2 gene and the bacterial reporter gene LacZ, which was complexed with cationic lipids, into the right side of segment T8 of the normal spinal cord, or just caudal to the hemisection site. The reporter gene was expressed in the perikarya of right CN neurons at L1 for up to 7 days, but not 14 days. Two months following T8 hemisection and Bcl-2/LacZ DNA injection, there was no significant loss of CN neurons ipsilateral to the lesion. Surprisingly, 61% of large neurons survived, indicating partial protection from atrophy. In contrast, a DNA plasmid that codes for the LacZ reporter gene, but not Bcl-2, did not prevent CN neuron death or atrophy. Administration of the Bcl-2 gene in adult rats and its expression in these CNS neurons prevents retrograde cell death, and also minimizes atrophy. These results may serve as the basis for developing novel gene therapy strategies for patients with spinal cord injury.

Neurotrophin‐3 prevents death of axotomized Clarke's nucleus neurons in adult rat

In the present investigation, we studied whether neurotrophin-3 (NT-3) contributes to the rescue of axotomized Clarke's nucleus (CN) neurons in adult rats. A significant (24%) loss of CN neurons occurred at L-1 ipsilateral to T-8 hemisection by 14 days, which reached 31% at 2 months and then stabilized. Axotomized CN neurons had also atrophied by 14 days, but mean cell size did not decrease further. Animals that received gelfoam soaked in nerve growth factor, brain derived neurotrophic factor, or ciliary neurotrophic factor at the lesion site also showed a 30% neuron loss at 2 months, and a 40% reduction in average cell area. Rats receiving NT-3 showed a 15% neuron loss, which was not improved by additional neurotrophins in combination with NT-3. None of the treatments prevented neuron atrophy. Bioassay of the gelfoam showed that NT-3 bioactivity remained at 5 days after surgery but not at 14 days. Additional rats with hemisections that received NT-3 continuously via mini-pump for 2 months showed a 15% neuron loss, the same as with NT-3 given via gelfoam. These results indicate that even limited exposure of axotomized CN neurons to NT-3 produces permanent rescue of 50% of the neurons. The virtually complete rescue that we had previously observed with transplants of fetal central nervous system (CNS) tissues may, therefore, be due at least in part to NT-3, but the exogenous administration of a single neurotrophic factor or a combination of neurotrophic factors is less effective than transplants in producing long-term survival of axotomized CNS neurons. J. Comp. Neurol. 390:102–111, 1998. © 1998 Wiley-Liss, Inc.

Neurotrophin-3 prevents death of axotomized Clarke's nucleus neurons in adult rat

In the present investigation, we studied whether neurotrophin-3 (NT-3) contributes to the rescue of axotomized Clarke's nucleus (CN) neurons in adult rats. A significant (24%) loss of CN neurons occurred at L-1 ipsilateral to T-8 hemisection by 14 days, which reached 31% at 2 months and then stabilized. Axotomized CN neurons had also atrophied by 14 days, but mean cell size did not decrease further. Animals that received gelfoam soaked in nerve growth factor, brain derived neurotrophic factor, or ciliary neurotrophic factor at the lesion site also showed a 30% neuron loss at 2 months, and a 40% reduction in average cell area. Rats receiving NT-3 showed a 15% neuron loss, which was not improved by additional neurotrophins in combination with NT-3. None of the treatments prevented neuron atrophy. Bioassay of the gelfoam showed that NT-3 bioactivity remained at 5 days after surgery but not at 14 days. Additional rats with hemisections that received NT-3 continuously via mini-pump for 2 months showed a 15% neuron loss, the same as with NT-3 given via gelfoam. These results indicate that even limited exposure of axotomized CN neurons to NT-3 produces permanent rescue of 50% of the neurons. The virtually complete rescue that we had previously observed with transplants of fetal central nervous system (CNS) tissues may, therefore, be due at least in part to NT-3, but the exogenous administration of a single neurotrophic factor or a combination of neurotrophic factors is less effective than transplants in producing long-term survival of axotomized CNS neurons.

A novel early component of the cell body response in axotomized Clarke's nucleus neurons revealed by monoclonal antibody Py.

The monoclonal antibody Py was initially developed as a tool for the identification of subpopulations of hippocampal neurons. Recently it has also been demonstrated to be a useful marker for other populations of midbrain and spinal cord neurons in which the antigen showed a strong colocalization with cytoskeletal elements. To assess the possible usefulness of Py as a tool for studying lesion-induced cell body changes, densitometric analysis of altered Py-immunoreactivity (Py-IR) has been compared with that of microtubule-associated protein 2 (MAP2) in Clarke's nucleus following axotomy. One week after a unilateral transection of the dorsal spinocerebellar tract at Th9-10, Py-IR in the Clarke's nucleus ipsilateral and caudal to the lesion was reduced by approximately 40%. By 21 days, Py-IR was reduced by approximately 50% (a near maximal reduction) and remained constant up to 5 months after the lesion (the longest survival time studied). Alterations of MAP2-IR in Clarke's nucleus were later in onset, slower to develop, and less marked. The differential distribution of the Py antigen in the CNS and its rapid and long lasting loss indicate that the Py antibody is a sensitive tool for studying novel early alterations of the cytoskeleton which may be important molecular events in axotomy-induced pathological processes.

Critical interval for rescue of axotomized neurons by transplants.

To determine whether embryonic spinal cord transplants retained the ability to prevent retrograde death of Clarke's nucleus (CN) neurons if supplied after a delay, we hemisected adult rats at the T8 spinal cord segment and placed transplants of fetal tissue into the hemisection cavity immediately or up to 14 days later. Transplants provided in the first 7 days after injury prevented virtually all of the 30% loss of CN neurons at L1 ipsilateral to hemisection that occurs without a transplant. Transplants supplied at 14 days post-hemisection were ineffective. Because prevention of retrograde neuron death is one mechanism by which transplants may contribute to locomotor recovery after spinal cord injury, this window of effectiveness should be considered in the design of clinical trials.

Differential Distribution of Immunoreactivity in the Adult Rat Spinal Cord Revealed by the Monoclonal Antibody, Py: A Light and Electron Microscopic Study

The monoclonal antibody Py has previously been shown to be a useful marker for subpopulations of neurons in the rat brain. However, the distribution of Py immunoreactivity in other regions of the CNS and PNS is not known. Here, we present a light and electron microscopic investigation into the distribution of Py immunoreactivity in the adult rat spinal cord, dorsal root ganglia, and peripheral nerves. Py immunoreactivity was associated with cytoskeletal elements in the cell body and dendrites of large-diameter neurons (particularly motoneurons, Clarke's nucleus neurons, and some dorsal root ganglion cells). Small-diameter neurons of lamina II (substantia gelatinosa) were Py negative. Py immunoreactivity was also detected in some populations of nerve fibers, notably axons located in the corticospinal tract, axons in the region of the white matter bordering the gray matter (presumably propriospinal axons), and also motor axons of the ventral root, but not in peripheral nerve. Dorsal roots were largely unstained. The present observations suggest a possible involvement of the Py antigen in the function or maintenance of the cytoskeleton of some populations of neurons and that the antibody may be a potentially useful tool for studying lesion-induced cytoskeletal alterations, particularly in α-motoneurons and Clarke's nucleus neurons.

NMDA receptor blockade rescues Clarke's and red nucleus neurons after spinal hemisection

Hemisection of the adult rat spinal cord at T9 transects the ascending ipsilateral axons of Clarke's nucleus (CN) neurons and the descending contralateral axons of red nucleus (RN) neurons. Eight weeks following axotomy, 30% of CN neurons and 22% of RN neurons die. Since both nuclei receive glutamatergic input, we wished to examine the possibility that glutamatergic excitotoxicity contributes to axotomy-induced neuronal death in these nuclei. To test this we studied the effects of administration of the NMDA receptor antagonist MK-801 on cell survival after axotomy. When 1 mg/kg body weight MK-801 is administered subcutaneously every day for 1-8 weeks to hemisected rats, cell death is prevented. Treatment with 0.5 mg/kg body weight MK-801 over the same time periods results in only partial rescue of axotomized neurons. Paradoxically, when 1 mg/kg MK-801 administration is restricted to the first week of an 8 week survival period, cell death in both the RN and CN is greatly exaggerated over the cell loss found in saline-treated animals. Withdrawal of 1 mg/kg MK-801 after 1 week of administration induces the loss of 92% of CN neurons, which is 63% greater than that occurring after axotomy alone. If, however, 1 mg/kg MK-801 is withdrawn after 2 weeks post-axotomy in the RN and 3 weeks postaxotomy in CN, all axotomized neurons survive. This rescue is found at 6 months postsurgery, the longest survival period studied, and therefore appears to be permanent. These results suggest that glutamatergic afferent input contributes significantly to the death of axotomized red nucleus and Clarke's nucleus neurons via NMDA receptors located on these neurons.

Neurotrophic factors prevent the death of CNS neurons after spinal cord lesions in newborn rats.

The aim of this study was to determine if the exogenous administration of neurotrophic factors can rescue immature axotomized CNS neurons in vivo. After spinal cord hemisection in newborn rats, the exogenous administration of neurotrophic factors brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and nerve growth factor (NGF), prevents the retrograde cell death of axotomized red nucleus neurons (and other brain stem spinal neurons) in vivo. Rescue of red nucleus neurons was maintained in the presence of BDNF, but only transiently maintained by NT-3 and NGF. Neurons within the nucleus dorsalis (Clarke's nucleus) of the spinal cord are also axotomized by this lesion. The application of exogenous NT-3, but not NGF or BDNF, rescued Clarke's nucleus neurons. These observations indicate that neurotrophic factors play a crucial role in the survival of CNS neurons in vivo during development and after injury. Furthermore, these results indicate that particular populations of neurons are dependent upon specific neurotrophic support after injury.

Grafts of fetal central nervous system tissue rescue axotomized clarke's nucleus neurons in adult and neonatal operates

Many conditions are thought to contribute to neuron death after axotomy, including immaturity of the cell at the time of injury, inability to reestablish or maintain target contact, and dependence on trophic factors produced by targets. Exogenous application of neurotrophic factors and transplants of peripheral nerve and embryonic central nervous system (CNS) tissue temporarily rescue axotomized CNS neurons, but permanent rescue may require transplants that are normal targets of the injured neurons. We examined the requirements for survival of axotomized Clarke's nucleus (CN) neurons. Two months after hemisection of the spinal cord at the T8 segment, there was an ipsilateral 30% loss of neurons at the L1 segment in adult operates and a 40% loss in neonates. Transplants of embryonic spinal cord, cerebellum, and neocortex inserted into the T8 segment at the time of hemisection prevented virtually all of the cell death in both adults and neonates, but transplants of embryonic striatum were ineffective. None of the grafts prevented the somal atrophy of CN neurons caused by axotomy. Retrograde transport of fluoro-gold from the cerebellum demonstrated that 33% of all CN neurons at L1 project to the cerebellum, 50% of these died following a T8 hemisection, but all these projection neurons were rescued by a transplant of embryonic spinal cord. These results suggest that the rescue of axotomized CN neurons is relatively specific for the normal target areas of these neurons, but this specificity is not absolute and may depend on the distribution and synthesis of particular neurotrophic agents.

Removal of Dorsal Root Afferents Prevents Retrograde Death of Axotomized Clarke's Nucleus Neurons in the Cat

We investigated the effect of axotomy, deafferentation, and deafferentation plus axotomy on cell survival and cell size in Clarke's nucleus of the cat spinal cord. Hemisection of the adult spinal cord at T 9 leads to retrograde cell death of 40% of the neurons in Clarke's nucleus at L 3, as well as to a reduction in the mean soma size of the survivors. In contrast, deafferentation of Clarke's nucleus neurons by L 1-S 2 dorsal rhizotomy produces no cell loss and no shrinkage of the somata. These results indicate that dorsal root afferent input is not required for Clarke's nucleus cell survival. To test whether afferents may be required by the 60% of neurons that survive axotomy, we deafferented Clarke's nucleus prior to axotomy. Surprisingly, removal of primary afferents to Clarke's nucleus neurons prior to axotomy prevented the death of all neurons that would normally have died from axotomy. These results suggest that dorsal root afferent input is not required for Clarke's nucleus neuron survival after axotomy and may in fact be toxic to these axotomized neurons. This afferent toxicity is likely to be mediated through the dorsal root afferent neurotransmitter glutamate.