Adult Rat Spinal Motoneurons Research Articles

Survival of Injured Spinal Motoneurons in Adult Rat upon Treatment with Glial Cell Line–Derived Neurotrophic Factor at 2 Weeks but Not at 4 Weeks after Root Avulsion

We conducted a study of whether treatment with glial cell line-derived neurotrophic factor (GDNF) initiated at 2 or 4 weeks after spinal-root avulsion could promote survival and regulate neuronal nitric oxide synthase (nNOS) expression in adult rat spinal motoneurons. By 6 weeks after root avulsion, the treatment given at 2 weeks not only increased motoneuron survival (86.1% vs. 27.9%), but also reversed the atrophy of injured motoneurons and increased their somatic size (101.3% vs. 52.9%) in comparison to the untreated control group of animals. All surviving motoneurons in the GDNF-treated group showed immunoreactivity for choline acetyltransferase. In contrast, GDNF treatment at 4 weeks post-injury failed to promote motoneuron survival (33.1% vs. 27.9%) at 6 weeks compared to the control group. Both the 2- and 4-week post-injury treatments downregulated nNOS expression. This finding suggests that injured adult motoneurons die shortly (a few weeks in the rat) after root avulsion injury, but can be saved from degeneration by treatment within the proper time frame after injury, which in the case of GDNF treatment in rats, appears to be within 2 weeks of the avulsion injury of the spinal root. These findings provide useful information for choosing the best time frame for the potential clinical treatment of brachial plexus avulsion.

Microarray analysis of gene expression patterns in adult spinal motoneurons after different types of axonal injuries

Three experimental models of axonal injuries in adult rat spinal motoneurons were established to investigate changes of gene expression in response to such injuries. We took advantage of cDNA microarray analysis to determine the differential expression of genes in injured motoneurons following distal axotomy or root avulsion in the absence or presence of BDNF. The major finding was that, in response to proximal axonal injury (avulsion), expression of genes that are known to facilitate neuronal survival and axonal regeneration (e.g., IGFRII, PI3K, IGFBP-6, GSTs, GalR2) were down-regulated; but following treatment with BDNF they were up-regulated. In addition, the expression of genes known to be involved in apoptosis and DNA damage (e.g., ANX5, TS, ALR) were down-regulated in BDNF-treated animals with avulsion. Furthermore, many functional families of genes previously shown to play roles in the pathophysiology of axonal injury, including SNAP-25A, SV2B, Ras-related ras3a/4b, ERK1/2, 14-3-3 proteins, proteasome proteins, oncogenes, GAP-43, and NMDAR1, were altered after either distal axotomy or avulsion injury. Some of the changes in gene expression, including Lim-2, FRAG1, GlaR2, GSTs, ALR, TS, ANX3/5, and nhe1/2, are first reported here in injured motoneurons. The differential expression of genes identified by the expression arrays was confirmed by gene-specific RT-PCR for eight genes (GAP-43, IGFR II, Lim-2, MIF, NDAP1, TS, PCC3, and FRAG1) and by in situ hybridization for Lim-2. These results suggest that abnormal regulation of particular biochemical pathways may induce motoneuron death after ventral root avulsion in adult animals. This study presents an approach for selecting specific genes and their products that may be involved in motoneuron degeneration following axonal injuries.

Expression of tenascin R and J1 mRNA in motoneurons after a traumatic lesion in the spinal cord.

We demonstrate, using in situ hybridization, that mRNA for the anti-adhesive molecules tenascin R and J1 in the adult rat spinal motoneurons are down-regulated rapidly as a reaction after a ventral funiculus lesion. Tenascin-R was significantly down-regulated at day 1 and normalized after 3 weeks. Tenascin-J1 declined to its lowest value at day 3 and returned to the initial level after 3 weeks. In adjacent sections, the distribution of macrophages was studied with immuno histochemistry. The density of macrophages reached a maximum 3 days after the injury. Thus, the density of macrophages appeared to be inversely related to the level of tenascin mRNA. These data are compatible with the notion that neuronal tenascins may modulate the adhesion of perincurial inflammatory cells.

Exogenous brain-derived neurotrophic factor regulates the synaptic composition of axonally lesioned and normal adult rat motoneurons

Brain-derived neurotrophic factor has previously been shown to promote survival and axonal regeneration in injured spinal motoneurons and, also, to modulate synaptic transmission and regulate the density of synaptic innervation in a variety of neurons. The present light and electron microscopic study demonstrates synaptotrophic effects of exogenously applied brain-derived neurotrophic factor on the synaptic composition of both normal and axonally lesioned adult rat spinal motoneurons. After L5–L6 ventral root avulsion, a massive loss of all types of boutons occurred on the somata of the lesioned motoneurons which persisted for at least 12 weeks postoperatively. We found that (i) intrathecal infusion of brain-derived neurotrophic factor during the first postoperative week did not prevent the synaptic detachment and activation of glial cells; (ii) prolonged treatment for four weeks restored synaptic covering and significantly reduced microglial reaction; (iii) the synaptotrophic effect remained significant for at least eight weeks after cessation of the treatment; (iv) brain-derived neurotrophic factor mainly supported F-type boutons with presumably inhibitory function, while it had little effect on S-type boutons associated with excitatory action; and (v) in normal unlesioned motoneurons, four weeks of treatment with brain-derived neurotrophic factor induced sprouting of F-type boutons, a loss of S-type boutons and motoneuron atrophy. The present data show that exogenous neurotrophins not only help to restore synaptic circuitry in axonally injured motoneurons, but also strongly influence the synaptic composition in normal motoneurons.

Neurotrophins and trk-receptors in adult rat spinal motoneurons: differences related to cell size but not to ‘slow/fast’ specialization

We have studied the mRNA expression of the neurotrophins brain-derived neurotrophic factor (BDNF), NT-3 and NT-4 and of their receptors trkB and trkC in individual retrogradely labeled lumbar spinal motoneurons of the adult rat, using quantitative non-radioactive in situ hybridization (ISH). We measured soma size and the optical density of the ISH reaction. We analyzed mRNA expression patterns in predominantly ‘slow’ or ‘fast’ motoneurons by comparing retrogradely labelled cells that innervated the slow m.soleus (Sol) with those innervating the fast m.extensor digitorum longus (EDL). Within individual motoneuron pools, there was a considerable variation in the intensity of mRNA expression encoding for BDNF, NT-3 and NT-4, as well as for the receptors trkB and trkC. The expression of trkC mRNA was significantly higher in small, presumed fusimotor neurons than in the larger α-motoneurons. Surprisingly, none of the variations in the level of motoneuronal mRNA expression for trkB, BDNF, NT-3 and NT-4 were related to soma size and no differences were found between EDL and Sol motoneurons. Apparently, the considerable variations in neurotrophin and neurotrophin receptor mRNA expression represent other parameters of motoneuronal specialization than those related to the well-established ‘fast’ vs. ‘slow’ categories of motoneurons.

Rescue of lesioned adult rat spinal motoneurons by adenoviral gene transfer of glial cell line-derived neurotrophic factor.

Glial cell line-derived neurotrophic factor (GDNF) has been shown to protect cranial and spinal motoneurons, that suggests potential uses of GDNF in the treatment of spinal cord injury and motor neuron diseases. We examined neuroprotective effect of human GDNF encoded by an adenovirus vector (AxCAhGDNF) on the death of lesioned adult rat spinal motoneurons. The seventh cervical segment (C7) ventral and dorsal roots and dorsal root ganglia of adult Fisher 344 rats were avulsed, and AxCAhGDNF, AxCALacZ (adenovirus encoding beta-galactosidase gene) or PBS was inoculated in C7 vertebral foramen. One week after the avulsion and treatment with AxCALacZ, the animals showed expression of beta-galactosidase activity in lesioned spinal motoneurons. Animals avulsed and treated with AxCAhGDNF showed intense immunolabeling for GDNF in lesioned spinal motoneurons and expression of virus-induced human GDNF mRNA transcripts in the lesioned spinal cord tissue. Nissl-stained cell counts revealed that the treatment with AxCAhGDNF significantly prevented the loss of lesioned ventral horn motoneurons 2 to 8 weeks after avulsion, as compared to AxCALacZ or PBS treatment. Furthermore, the AxCAhGDNF treatment ameliorated choline acetyltransferase immunoreactivity in the lesioned motoneurons after avulsion. These results indicate that the adenovirus-mediated gene transfer of GDNF may prevent the degeneration of motoneurons in adult humans with spinal cord injury and motor neuron diseases.

Rescue of lesioned adult rat spinal motoneurons by adenoviral gene transfer of glial cell line‐derived neurotrophic factor

Rescue of lesioned adult rat spinal motoneurons by adenoviral gene transfer of glial cell line‐derived neurotrophic factor

Brain-derived neurotrophic factor promotes axonal regeneration and long-term survival of adult rat spinal motoneurons in vivo

This study shows that in adult rat spinal motoneurons brain-derived neurotrophic factor exerts a neuroprotective effect which extends several weeks beyond the duration of treatment. In addition, brain-derived neurotrophic factor strongly enhances regeneration of avulsed motor axons across the border between the central and peripheral nervous systems. Treatment with brain-derived neurotrophic factor is known to rescue adult rat spinal motoneurons from retrograde cell death induced by ventral root avulsion. The present experiments were designed to test whether this survival effect remains over an extended period of time following cessation of treatment and, also, whether brain-derived neurotrophic factor promotes regeneration of avulsed motor axons. After avulsion of a spinal ventral root, four weeks of treatment with brain-derived neurotrophic factor (10 μg/day) or vehicle was initiated. By using different retrograde tracers to obtain pre- and postoperative labelling of avulsed and regenerating motoneurons, respectively, the number of surviving motoneurons as well as the extent of motor axonal regeneration could be analysed. The expression of nitric oxide synthase in the lesioned motoneurons was also studied. In the vehicle-treated rats, only 10% of the avulsed motoneurons remained at 12 weeks postoperatively, 20–40% of which displayed nitric oxide synthase activity. Treatment with brain-derived neurotrophic factor during the initial four postoperative weeks resulted in 45% motoneuron survival and a complete blockage of nitric oxide synthase expression at 12 weeks postoperatively. Brain-derived neurotrophic factor also induced abundant regeneration of the avulsed motor axons, which formed extensive fibre bundles along the surface of the spinal cord and adjacent ventral roots. The long-term effect by brain-derived neurotrophic factor seemed to be even stronger on motor axonal regeneration than on motoneuron survival. The present results indicate a therapeutic potential for brain-derived neurotrophic factor in the early treatment of traumatic injuries to spinal nerves and roots.

Potential Roles of Gene Expression Change in Adult Rat Spinal Motoneurons Following Axonal Injury: A Comparison among c-jun, Low-Affinity Nerve Growth Factor Receptor (LNGFR), and Nitric Oxide Synthase (NOS)

The present study investigates expression of nitric oxide synthase (NOS), immediate early genes (IEGs, c-jun, and c-fos), and low-affinity nerve growth factor receptor (LNGFR) in adult rat spinal motoneurons in response to three conditions of axonal injury: distal axotomy, root avulsion, and root avulsion followed by a peripheral nerve (PN) graft implantation. Expression of c-jun and LNGFR were predominately observed in motoneurons of the distal axotomized segment where most motoneurons survived. In contrast, expression of NOS was exclusively found in motoneurons of the root avulsed segment where most motoneurons died. c-fos was not expressed in motoneurons following either distal axotomy or root avulsion. In animals with PN graft implantation, a double fluorescent labeling technique was used to evaluate motoneuron regeneration. Expression of NOS was completely inhibited in all motoneurons that regenerated into the PN graft, but was not inhibited in those that did not regenerate. Moreover, regenerated motoneurons expressed LNGFR and c-jun while the nonregenerated motoneurons expressed NOS. Results of the present study have shown that motoneurons undergo changes in expression of cellular molecules in response to the axonal injury. The expression of c-jun and LNGFR may be related to the regenerative process while expression of NOS is more likely involved in the degenerative process. The results also show that PN graft implantation can alter the expression of cellular molecules and reduce motoneuron death due to root avulsion. The survival-promoting effects of PN graft implantation (presumably the effects of neurotrophic factors) may be achieved by modifying certain cellular molecules such as NOS.

Brain-derived neurotrophic factor promotes survival and blocks nitric oxide synthase expression in adult rat spinal motoneurons after ventral root avulsion

In adult spinal motoneurons, retrograde cell death is induced by ventral root avulsion. A lethal effect of nitric oxide has been implicated, since nitric oxide synthase (NOS) is expressed in the motoneurons destined to die. Our study investigates the effects of brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) on the retrograde cell death and NOS expression of adult rat spinal motoneurons. Following ventral root avulsion and 4 weeks of continuous treatment, BDNF, but not CNTF, was found to prevent cell death and NOS expression in the lesioned motoneurons. This suggests a therapeutic potential for BDNF in the adult nervous system, possibly through blockage of nitric oxide synthesis.