Relationship between ribosomal RNA gene transcription activity and motoneuron death: observations of avulsion and axotomy of the facial nerve in rats.

Motoneuron number and expression of cytoplasmic RNA and ribosomal RNA (rRNA) gene transcription activity in the facial nucleus were examined quantitatively and chronologically for up to 4 weeks in rats after facial nerve axotomy and avulsion in order to elucidate interrelationships in axonal changes. The right facial nerves of adult Fischer rats were avulsed at a portion of the outlet or axotomized at a portion of the foramen stylomastoideus. The number of large motoneurons in the facial nucleus was reduced by 40% 2 weeks after avulsion and by 70% 4 weeks after avulsion but displayed a 19% loss even 4 weeks after axotomy. The amount of cytoplasmic RNA decreased significantly and progressively from 1 day after avulsion. rRNA gene transcription activity in the large motoneurons of the facial nucleus decreased significantly beginning 30 min after both axotomy and avulsion, but the severity of the decrease was far more marked in the avulsion group, showing a 59% loss from the control value 4 weeks after avulsion. These findings indicate that rRNA gene transcription activity, expression of cytoplasmic RNA, and the number of motoneurons that survive are interrelated and that the decrease in rRNA gene transcription activity is a very early event in the phenomena observed in the axonal reactions of motoneurons.

Adenoviral gene transfer of hepatocyte growth factor prevents death of injured adult motoneurons after peripheral nerve avulsion.

Hepatocyte growth factor (HGF) exhibits strong neurotrophic activities on motoneurons both in vitro and in vivo. We examined survival-promoting effects of an adenoviral vector encoding human HGF (AxCAhHGF) on injured adult rat motoneurons after peripheral nerve avulsion. The production of HGF in COS1 cells infected with AxCAhHGF and its bioactivity were confirmed by ELISA, Western blot and Madin-Darby canine kidney (MDCK) cell scatter assay. The facial nerve or the seventh cervical segment (C7) ventral and dorsal roots of 3-month-old Fischer 344 male rats were then avulsed and removed from the stylomastoid or vertebral foramen, respectively, and AxCAhHGF, AxCALacZ (adenovirus encoding beta-galactosidase gene) or phosphate-buffered saline (PBS) was inoculated in the lesioned foramen. Treatment with AxCAhHGF after avulsion significantly prevented the loss of injured facial and C7 ventral motoneurons as compared to AxCALacZ or PBS treatment and ameliorated choline acetyltransferase immunoreactivity in these neurons. These results indicate that HGF may prevent the degeneration of motoneurons in adult humans with motoneuron injury and motor neuron diseases.

Peripheral nerve avulsion injuries as experimental models for adult motoneuron degeneration.

We have used adult rat peripheral nerve avulsion models to evaluate the effects of neuroprotective molecules on motoneuron degeneration. The right facial nerves of adult Fischer 344 male rats were avulsed and adenoviral vectors encoding glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), transforming growth factor-beta2 (TGFbeta2), and growth inhibitory factor (GIF) were injected into the facial canal. The treatment with the vectors significantly prevented the loss of lesioned facial motoneurons, improved choline acetyltransferase (ChAT) immunoreactivity and suppressed the induction of nitric oxide synthase activity in these neurons. In separate experiments, animals were orally administered a solution of a neuroprotective compound T-588 after avulsion. Both free oral administration and oral tube administration of T-588 improved the survival of injured motoneurons and ameliorated their ChAT immunoreactivity. These results indicate that the gene transfer of GDNF, BDNF, TGFbeta2, and GIF and oral administration of T-588 may prevent the degeneration of motoneurons in adult humans with motoneuron injury and motor neuron diseases.

Motoneuron degeneration after facial nerve avulsion is exacerbated in presymptomatic transgenic rats expressing human mutant Cu/Zn superoxide dismutase.

We investigated motoneuron degeneration after proximal nerve injury in presymptomatic transgenic (tg) rats expressing human mutant Cu/Zn superoxide dismutase (SOD1). The right facial nerves of presymptomatic tg rats expressing human H46R or G93A SOD1 and their non-tg littermates were avulsed, and facial nuclei were examined at 2 weeks postoperation. Nissl-stained cell counts revealed that facial motoneuron loss after avulsion was exacerbated in H46R- and G93A-tg rats compared with their non-tg littermates. The loss of motoneurons in G93A-tg rats after avulsion was significantly greater than that in H46R-tg rats. Intense cytoplasmic immunolabeling for SOD1 in injured motoneurons after avulsion was demonstrated in H46R- and G93A-tg rats but not in their littermates. Facial axotomy did not induce significant motoneuron loss nor enhance SOD1 immunoreactivity in these tg rats and non-tg littermates at 2 weeks postoperation, although both axotomy and avulsion elicited intense immunolabeling for activating transcription factor-3, phosphorylated c-Jun, and phosphorylated heat shock protein 27 in injured motoneurons of all these animals. The present data indicate the increased vulnerability of injured motoneurons after avulsion in the presymptomatic mutant SOD1-tg rats.

Growth inhibitory factor prevents degeneration of injured adult rat motoneurons.

We examined neuroprotective effects of growth inhibitory factor (GIF) on injured adult rat facial motoneurons. The right facial nerves of adult rats were avulsed and removed from the stylomastoid foramen, and an adenoviral vector encoding rat GIF and Myc epitope (AxCArGIFM) were injected into the facial canal. Animals treated with AxCArGIFM showed intense immunolabeling for GIF/Myc in injured facial motoneurons. Treatment with AxCArGIFM after avulsion significantly prevented the loss of injured facial motoneurons, improved choline acetyltransferase immunoreactivity and prevented the induction of nitric oxide synthase activity in these neurons. These results indicate that GIF may have therapeutic potential against degeneration of motoneurons in adult humans with motoneuron injury and motor neuron diseases.

Oral administration of a neuroprotective compound T-588 prevents motoneuron degeneration after facial nerve avulsion in adult rats.

OBJECTIVE: R(-)-1-(benzo[b]thiophen-5-yl)-2-[2-(N,N-diethylamino) ethoxylethanol hydrochloride (T-588), a synthetic compound, has been shown to have neuroprotective potentials for neuronal cells. We investigated whether orally administered T-588 can rescue injured motoneurons after facial nerve avulsion in adult rats. METHODS: The right facial nerves of adult Fischer 344 male rats were avulsed and the animals were freely administered solution of 0.05% (w/v) T-588 or received T-588 (3-30 mg/kg/day) through an oral tube for 1-4 weeks. Facial motoneurons on both sides of the facial nuclei were counted in Nissl-stained sections, and choline acetyltransferase (ChAT) immunoreactivity in injured motoneurons and ChAT enzyme activities in the ventral brain stem tissue containing the facial nuclei were examined. RESULTS: Both free oral administration of 0.05% T-588 solution and oral tube administration of T-588 (30mg/kg/day) improved the survival of facial motoneurons at 3 or 4 weeks after avulsion. These treatments ameliorated ChAT immunoreactivity in injured motoneurons and the tissue ChAT enzyme activities at 1-week postoperation examined. CONCLUSION: These results indicate that oral administration of T-588 ameliorates the survival of injured motoneurons and supports their neuronal function after facial nerve avulsion in adult rats. T-588 may have therapeutic potential in motoneuron injury or motor neuron diseases in humans.

Tissue culture methods to study neurological disorders: establishment of immortalized Schwann cells from murine disease models.

Previously, the authors have established spontaneously immortalized cell lines from long-term cultures of normal adult mouse Schwann cells. Establishment of such Schwann cell lines derived from murine disease models may greatly facilitate studies of the cellular mechanisms of their peripheral nervous system lesions in the relevant diseases. Recently, the authors have established immortalized Schwann cell lines derived from Niemann-Pick disease type C mice (NPC; spm/spm) and globoid cell leukodystrophy mice (twitcher). In the present study, long-term cultures were maintained of Schwann cells derived from dorsal root ganglia and consecutive peripheral nerves of another NPC mouse (npc(nih)/npc(nih), npc(nih)/+), myelin P0 protein-deficient mice (P0-/-, P0+/-) with their wild-type littermates (P0+/+), and neurofibromatosis type 1 gene (NF1)-deficient mice (Nf1(FCr)/+) for 8-10 months, and immortalized cell lines from all these animals established spontaneously. These cell lines had spindle-shaped Schwann cell morphology and distinct Schwann cell phenotypes and retained genomic and biochemical abnormalities, sufficiently representing the in vivo pathological features of the mutant mice. These immortalized Schwann cell lines can be useful in studies of nervous system lesions in these mutant mice and relevant human disorders.

Adenoviral gene transfer of GDNF, BDNF and TGF beta 2, but not CNTF, cardiotrophin-1 or IGF1, protects injured adult motoneurons after facial nerve avulsion.

We examined neuroprotective effects of recombinant adenoviral vectors encoding glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT1), insulin-like growth factor-1 (IGF1), and transforming growth factor-beta2 (TGFbeta2) on lesioned adult rat facial motoneurons. The right facial nerves of adult Fischer 344 male rats were avulsed and removed from the stylomastoid foramen, and adenoviral vectors were injected into the facial canal. Animals avulsed and treated with adenovirus encoding GDNF, BDNF, CNTF, CT1, IGF1 and TGFbeta2 showed intense immunolabeling for these factors in lesioned facial motoneurons, respectively, indicating adenoviral induction of the neurotrophic factors in these neurons. The treatment with adenovirus encoding GDNF, BDNF, or TGFbeta2 after avulsion significantly prevented the loss of lesioned facial motoneurons, improved choline acetyltransferase immunoreactivity and prevented the induction of nitric oxide synthase activity in these neurons. The treatment with adenovirus encoding CNTF, CT1 or IGF1, however, failed to protect these neurons after avulsion. These results indicate that the gene transfer of GDNF and BDNF and TGFbeta2 but not CNTF, CT1 or IGF1 may prevent the degeneration of motoneurons in adult humans with motoneuron injury and motor neuron diseases.