Hypoglossal Neurons Research Articles

Trigemino-Hypoglossal Silent Period – A New Pontomedullary Brainstem Reflex

Introduction: Inhibitory connections between neurons of the sensory trigeminal nucleus and the hypoglossal nucleus have been described in the cat (Tomioka et al., 1999). We investigated if these inhibitory pontomedullary projections are also present in man by applying a newly developed stimulation and recording technique. Methods: In a preliminary study we examined 14 healthy subjects (9 males, 5 females, age: 42±13 years) with a specially designed enoral stimulation and recording device. The device allowed unilateral electrical stimulation of the trigeminal (V2) innervated palatal mucosa and simultaneous recording of the electromyographic activity from both halves of the tongue using surface electrodes. The stimulation intensity was at least 5-fold the perception threshold and was increased up to a maximum of 20 mA. During stimulation the subject pressed his/her tongue against the recording device. Five recordings were registered and the electromyographic activity was averaged and rectified. Results: The examination was well tolerated by all subjects without adverse events. In all subjects we observed one bilateral suppression period of tongue muscle activity following unilateral electrical stimulation of the mucosal V2 afferents. The silent period started at 41.1±4.7 ms and ended at 82.4±12.5 ms. The mean duration of the silent period was 41.4±10.2 ms. Conclusion: Analogously to animal studies, we demonstrated that inhibitory pontomedullary projections between sensory neurons of the enoral endings of the 2nd trigeminal nerve and hypoglossal neurons are also present in man. The silent period was bilaterally organized and showed, in contrast to the exteroceptive suppression of masseter muscle activity, only one suppression period. The trigemino-hypoglossal silent period is most probably a nociceptive protection reflex and further investigations in patients with defined brainstem lesions have to clarify its diagnostic relevance.

Changes of high-affinity choline transporter CHT1 mRNA expression during degeneration and regeneration of hypoglossal nerves in mice

The high-affinity choline transporter CHT1 works for choline uptake in the presynaptic terminals of cholinergic neurons. We examined its expression in the hypoglossal nucleus after unilateral hypoglossal nerve transection in mice by fluorescent in situ hybridization. One week after axotomy, CHT1 mRNA expression was lost in all hypoglossal motoneurons in the lesioned side. Two weeks after axotomy, CHT1 mRNA started to be re-expressed in a few motoneurons that recovered connections to tongue muscles as revealed by retrograde labeling with Fast Blue. After 4 weeks, most of axotomized hypoglossal motoneurons were reconnected and re-expressed CHT1 mRNA as strongly as control neurons, and the regenerating cholinergic axons established mature neuromuscular junctions. These results suggest that the establishment of motor innervation is critical for CHT1 mRNA expression in hypoglossal neurons after axotomy.

Increased expression of insulin-like growth factor I augments the progressive phase of synaptogenesis without preventing synapse elimination in the hypoglossal nucleus.

The in vivo actions of insulin-like growth factor I (IGF-I) on synaptogenesis in the hypoglossal nucleus were investigated in transgenic mice that overexpress IGF-I in the brain postnatally and in normal nontransgenic littermate controls. In a previous study using these mice, we found that IGF-I increases the total volume of the hypoglossal nucleus by increasing the volume of neuropil rather than by increasing total neuron number; therefore, the progressive and regressive phases of synaptogenesis could be evaluated without the confounding effects of altered neuron number. The volume of the hypoglossal nucleus was significantly increased by 28% to 59% in transgenic mice after postnatal day (P) 7, whereas the total number of hypoglossal neurons did not differ significantly from controls. The numerical density of neurons was significantly decreased by 21% to 38% after P7, and the density of myelinated axons was significantly increased by 19%. Although the numerical density of synapses did not differ between groups at any age, the total number of synapses in transgenic mice was increased by 42% to 52% after P14. Total synapse number in controls increased from P7 (7.9 million) to peak values at P21 (36.0 million), followed by a significant decrease (33%) at P130 (24.2 million). In transgenic mice, total synapses increased from 8.2 million on P7 to 51.1 million on P21, followed by a significant decrease (28%) to 36.7 million at P130. Our results demonstrated that IGF-I can stimulate a persistent increase in the number of hypoglossal synapses, thereby augmenting the progressive phase of synaptogenesis without preventing synapse elimination during the regressive phase.

Connections between respiratory neurones in the neonatal rat transverse medullary slice studied with cross-correlation.

In the transverse medullary slice prepared from neonatal rats the hypoglossal nerve rootlets exhibit a bursting 'respiratory' rhythm as do neurones in the pre-Bötzinger complex (PBC). We used cross-correlation analysis of the rhythmic multiunit discharges recorded from hypoglossal nerve rootlets, hypoglossal nucleus neurones and PBC neurones to investigate the connections between these groups. All cross-correlograms computed between left and right hypoglossal nerves, and between hypoglossal neurones and contralateral hypoglossal nerves, displayed central peaks with broad half-amplitude widths (mean +/- S.D. of 29.6 +/- 10.4 and 37.3 +/- 6.0 ms, respectively), which we interpreted as evidence for activation from a common source. Five of the 18 cross-correlograms computed between left and right PBC neurones displayed peaks either side of time zero with narrower half-amplitude widths (mean +/- S.D. of 9.3 +/- 1.9 ms) superimposed on broader central peaks, which we interpreted as evidence for mutual excitation and common activation, respectively. Cross-correlograms computed between PBC neurones and contralateral hypoglossal neurones or nerves did not display consistent features, but some of those computed between PBC and ipsilateral hypoglossal neurones (two of eight) or nerves (two of five) displayed peaks with broad half-amplitude widths (mean +/- S.D. of 36.8 +/- 6.9 ms), offset from time zero by 6 ms (except for one at 18 ms), which we interpreted as evidence for excitation of hypoglossal neurones and motoneurones by PBC neurones. We concluded that rhythm is synchronised between left and right sides by mutual excitatory connections between left and right PBC neurones. The rhythm is transmitted to ipsilateral hypoglossal neurones by a paucisynaptic pathway. Both hypoglossal neurones and PBC neurones receive a common activation from as yet unidentified sources.

Immunohistochemical detection of wheat germ agglutinin-horseradish peroxidase (WGA-HRP)

Traditional histochemical detection of wheat germ agglutinin-horseradish peroxidase (WGA-HRP) can impose substantial technical limitations on studies requiring co-localization of neurotransmitters, receptors and other neural antigens. The goal of our experiments was to establish the ideal conditions and reagents for immunohistochemical detection of WGA-HRP. WGA-HRP was injected into the tongues and vibrissae pads of adult rats to characterize labeling of somas and synapses, respectively. Rats were perfused with either 4% paraformaldehyde (for light microscopy, LM) or 4% paraformaldehyde/0.15% glutaraldehyde (for electron microscopy, EM) after survival times of 2, 3, 4, 5 or 6 days. For LM, brainstem tissue was cut on a cryostat at 20 μm and collected onto glass slides. For EM, tissue was sectioned with a vibratome at 50 μm and processed free floating. For LM, WGA-HRP was detected with goat anti-HRP, goat anti-WGA, biotinylated goat anti-HRP or biotinylated goat anti-WGA antibodies. For EM, WGA-HRP was detected with biotinylated goat anti-WGA and anti-HRP antibodies. Survival intervals of 3 days were ideal for staining of hypoglossal neurons, whereas an interval of 4 days produced the strongest staining of synapses within the spinal trigeminal nucleus. For LM, the biotinylated antibodies resulted in better signal-to-noise ratios than the unconjugated antibodies. At both the LM and EM levels, the biotinylated antibody to WGA produced better quality staining than the biotinylated antibody to HRP.

Olfactory-hypoglossal connections.

Natural olfactory stimulation with amyl acetate significantly modulates the electrical activity of hypoglossal neurons and the electromyographic responses of the tongue musculature. The aim of the present study was to identify and characterize, using neuroanatomical and neurophysiological approaches, the pathways involved in the transmission of the olfactory information to the hypoglossal nucleus (XIIn). The neuroanatomical findings provided the initial demonstration that olfactory information is conveyed from the olfactory bulb to the hypoglossal nucleus via the interpeduncular nucleus (IPn) by both fast disynaptic and different polysynaptic pathways. The latter, in particular, involve many of the brain structures that process olfactory information. The electrophysiological studies demonstrated that the IPn neurons respond with a variety of patterns to natural stimulation of the olfactory receptors, thus supporting the hypothesis that the IPn is a crucial relay station for the elaboration and transmission of olfactory stimuli to XIIn.

Synaptic activation of hypoglossal respiratory motorneurons during inspiration in rats

Recent work has suggested glutamatergic and cholinergic synapses, and electric coupling may be involved in the activation of hypoglossal motorneurons during inspiration, however their relative importance is unknown. In this study we examined the excitatory inputs to hypoglossal motorneurons in a brainstem slice preparation. Focal application of d-2-amino-5-phosphonovalerate significantly inhibited a long lasting inward current evoked during inspiration. 6-Cyano-7-nitroquinoxaline-2,3-dione completely blocked the post-synaptic currents that increased in frequency and amplitude during inspiration and also reduced the long lasting inward current. Nicotinic receptors and gap junctional communication, blocked by d-tubocurare and carbenoxolone, respectively, contributed significant but smaller inputs to hypoglossal motorneurons during inspiration. In summary, non- N-methyl- d-aspartate (NMDA) receptors constitute the largest excitatory drive to hypoglossal neurons during inspiration, while NMDA, nicotinic receptors and gap junctions are also actively involved.

Morphometrical reappraisal of motor neuron system of Pick's disease and amyotrophic lateral sclerosis with dementia

The conventional concept of Pick's disease does not distinguish Pick's disease with Pick bodies (Pick body disease, PBD) from Pick's disease without Pick bodies [lobar atrophy without Pick bodies, LA-PB(-)]. Recently, intraneuronal ubiquitin-positive inclusions (ub-inclusions), which are thought to be a hallmark of amyotrophic lateral sclerosis with dementia (ALS-D), have been found also in LA-PB(-). We reconfirmed that ub-inclusions are consistently detected in LA-PB(-) as well as ALS-D. Subsequently, morphometric evaluation for involvement of the upper and lower motor neuron systems were performed in seven cases each of PBD, LA-PB(-), ALS-D and controls. As an indicator of upper motor neuron involvement, the total number of axons through the pyramis of the medulla oblongata was employed and for lower motor neuron involvement, the number of hypoglossal neurons per unit area was calculated. In LA-PB(-), axons of the pyramidal tract were significantly reduced in comparison to PBD and controls, while the lower motor neurons were preserved. Contrary to LA-PB(-), ALS-D revealed significant reduction of hypoglossal neurons but its pyramidal tract tends to be relatively preserved. These results seem to indicate that LA-PB(-) and ALS-D belong to the same spectrum and consist of subgroups with ub-inclusions and involvement of motor neuron system in common. The involvement of the upper motor neuron system is emphasized in LA-PB(-), while ALS-D accentuates the lower motor neuron system. However, the border between the two group is not always clear and there are patients who can not be definitively classified.

Macrophage-stimulating protein is a neurotrophic factor for embryonic chicken hypoglossal motoneurons.

Macrophage-stimulating protein (MSP) exerts a variety of biological actions on many cell types, but has no known functions in the brain. MSP is structurally related to hepatocyte growth factor (HGF), another pleiotropic factor whose many functions include promoting neuronal survival and growth. To investigate whether MSP is also capable of acting as a neurotrophic factor, we purified hypoglossal motoneurons from the embryonic chicken hindbrain because these neurons are known to express the MSP receptor tyrosine kinase RON. MSP promoted the in vitro survival of these neurons during the period of naturally occurring neuronal death and enhanced the growth of neurites from these neurons. MSP mRNA was detected in the developing tongue whose musculature is innervated by hypoglossal neurons. Our study demonstrates that MSP is a neurotrophic factor for a population of developing motoneurons.

GDNF gene delivery to injured adult CNS motor neurons promotes axonal growth, expression of the trophic neuropeptide CGRP, and cellular protection

Glial-cell-line--derived neurotrophic factor (GDNF) has been identified as a potent survival and differentiation factor for several neuronal populations in the central nervous system (CNS), but to date, distinct effects of GDNF on motor axon growth and regeneration in the adult have not been demonstrated. In the present study, ex vivo gene delivery was used to directly examine whether GDNF can influence axonal growth, expression of neuronal regeneration-related genes, and sustain the motor neuronal phenotype after adult CNS injury. Adult Fischer 344 rats underwent unilateral transections of the hypoglossal nerve, followed by intramedullary grafts of fibroblasts genetically modified to secrete GDNF. Control animals received lesions and grafts of cells expressing a reporter gene. Two weeks later, GDNF gene delivery (1) robustly promoted the growth of lesioned hypoglossal motor axons, (2) altered the expression and intracellular trafficking of the growth-related protein calcitonin gene-related peptide (CGRP), and (3) significantly sustained the cholinergic phenotype in 84 +/- 6% of hypoglossal neurons compared with 39 +/- 6% in control animals (P < 0.001). This is the first neurotrophic factor identified to increase the in vivo expression of the trophic peptide CGRP and the first report that GDNF promotes motor axonal growth in vivo in the adult CNS. Taken together with previous in vitro studies, these findings serve as the foundation for a model wherein GDNF and CGRP interact in a paracrine manner to regulate neuromuscular development and regeneration.

Oscillations and hypoxic changes of mitochondrial variables in neurons of the brainstem respiratory centre of mice.

We studied the functions of mitochondria and their hypoxic modulation in the brainstem slices of neonatal mice (postnatal day (P)6-11). The measurements were made in the preBotzinger complex (pBC), a part of the respiratory centre, and in the hypoglossal (XII) nucleus. Using a CCD camera, changes in the redox state were assessed from cell autofluorescence produced by NADH and FAD, while alterations in mitochondrial membrane potential ([Delta][psi]) and free Ca2+ concentration ([Ca2+]m) were obtained from fluorescence signals after loading the cells with Rh123 and Rhod-2, respectively. In the pBC, the cells were functionally identified by correlating the oscillations in [NADH], [FAD], [Delta][psi] and [Ca2+]m with the respiratory motor output recorded simultaneously from XII rootlets. In the inspiratory cells, NADH fluorescence showed a brief decrease followed by a slow and long-lasting increase during one oscillation period. The initial decrease in NADH fluorescence was accompanied by an increase in FAD fluorescence and coincided with [Delta][psi] depolarization. The slow secondary increase in NADH fluorescence had a time course similar to that of the Rhod-2 signal, indicating the role of Ca2+ uptake by mitochondria in NAD and FADH reduction. Brief (2-4 min) hypoxia reversibly abolished rhythmic changes in mitochondrial variables and brought them to new steady levels. In parallel, ATP-sensitive K+ (KATP) channels were activated and the respiratory output was depressed. The hypoglossal neurons showed much bigger increases in [Delta][psi] and [NADH] during hypoxia than the pBC neurons, which may explain their extreme vulnerability to hypoxia. We show here that mitochondrial function can be monitored in vitro in neurons constituting the respiratory neural network in slice preparations. Since mitochondrial variables demostrate specific, stereotypic fluctuations during a respiratory cycle, we suggest that mitochondrial function is modulated by spontaneous activity in the respiratory network. Therefore mitochondrial depolarization and Ca2+ uptake can contribute to the biphasic reaction of the respiratory network during hypoxia.

CEP-1347 (KT7515), a Semisynthetic Inhibitor of the Mixed Lineage Kinase Family

CEP-1347 (KT7515) promotes neuronal survival at dosages that inhibit activation of the c-Jun amino-terminal kinases (JNKs) in primary embryonic cultures and differentiated PC12 cells after trophic withdrawal and in mice treated with 1-methyl-4-phenyl tetrahydropyridine. In an effort to identify molecular target(s) of CEP-1347 in the JNK cascade, JNK1 and known upstream regulators of JNK1 were co-expressed in Cos-7 cells to determine whether CEP-1347 could modulate JNK1 activation. CEP-1347 blocked JNK1 activation induced by members of the mixed lineage kinase (MLK) family (MLK3, MLK2, MLK1, dual leucine zipper kinase, and leucine zipper kinase). The response was selective because CEP-1347 did not inhibit JNK1 activation in cells induced by kinases independent of the MLK cascade. CEP-1347 inhibition of recombinant MLK members in vitro was competitive with ATP, resulting in IC(50) values ranging from 23 to 51 nm, comparable to inhibitory potencies observed in intact cells. In addition, overexpression of MLK3 led to death in Chinese hamster ovary cells, and CEP-1347 blocked this death at doses comparable to those that inhibited MLK3 kinase activity. These results identify MLKs as targets of CEP-1347 in the JNK signaling cascade and demonstrate that CEP-1347 can block MLK-induced cell death.

Melatonin attenuates neuronal NADPH-d/NOS expression in the hypoglossal nucleus of adult rats following peripheral nerve injury

Oxidative stress and massive production of nitric oxide (NO) have been implicated in the neuropathogenesis following peripheral nerve injury. This study was aimed to ascertain whether melatonin would exert its neuroprotective effect on the lesioned hypoglossal neurons after peripheral axotomy, since it is known to reduce the oxidative damage in a variety of experimental neuropathologies in which NO is involved. Right-sided hypoglossal nerve transection was performed in adult rats following which the animals were given two different doses of melatonin administered intraperitoneally for 3, 7, 14, 21 and 30 successive days. Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and neuronal nitric oxide synthase (nNOS) immunohistochemistry were carried out to detect the neuronal NADPH-d/NOS expression in the hypoglossal nucleus (HN). At various time intervals following axotomy, the neurons in the affected HN were induced to express NADPH-d/NOS reactivity on the lesioned side peaking at 14 days. However, the enzyme expression was markedly depressed by melatonin treatment in a dose-dependent manner in terms of frequency of labelled neurons and staining intensity. It is suggested that the suppressive effect of melatonin on NADPH-d/NOS expression may be attributed to its antioxidant properties. Hence, in consideration of therapeutic strategies for reducing the oxidative stress following peripheral nerve injury, melatonin may prove to be beneficial.

Akt/Protein Kinase B Prevents Injury-Induced Motoneuron Death and Accelerates Axonal Regeneration

Motoneurons require neurotrophic factors for their survival and axonal projection during development, as well as nerve regeneration. By using the axotomy-induced neuronal death paradigm and adenovirus-mediated gene transfer, we attempted to gain insight into the functional significances of major growth factor receptor downstream cascades, Ras-extracellular signal-regulated kinase (Ras-ERK) pathway and phosphatidylinositol-3 kinase-Akt (PI3K-Akt) pathway. After neonatal hypoglossal nerve transection, the constitutively active Akt-overexpressing neurons could survive as well as those overexpressing Bcl-2, whereas the constitutively active ERK kinase (MEK)-overexpressing ones failed to survive. A dominant negative Akt experiment demonstrated that inhibition of Akt pathway hastened axotomy-induced neuronal death in the neonate. In addition, the dominant active Akt-overexpressing adult hypoglossal neurons showed accelerated axonal regeneration after axotomy. These results suggest that Akt plays dual roles in motoneuronal survival and nerve regeneration in vivo and that PI3K-Akt pathway is probably more vital in neuronal survival after injury than Ras-ERK pathway.

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.

Delayed Rat Facial Nerve Repair Leads to Accelerated and Enhanced Muscle Reinnervation with Reduced Collateral Axonal Sprouting during a Definite Denervation Period Using a Cross-Anastomosis Paradigm

To establish the influence of prolonged denervation on the recovery of a motor nerve, the rat facial nerve was transected and denervated for 0 to 224 days. Then, the freshly transected hypoglossal nerve was sutured to the predegenerated facial nerve (hypoglossal–facial nerve anastomosis, HFA). Using this nerve cross-anastomosis paradigm we analyzed the nerve regeneration and muscle reinnervation 7 to 112 days post-suture operation (DPSO). After HRP injection into the whiskerpad 931 ± 27 hypoglossal neurons were labeled at 112 DPSO after immediate HFA. Following 14 to 112 days denervation the number of labeled neurons increased to 138% (14 days delay), 154% (56 days), and 145% (112 days). In contrast, the reinnervation was poorer after 7 days denervation with the number of neurons increasing to 84%, and after long-term denervation of 224 days the number of neurons increased to 81%. The increase in amplitude of evoked electromyography wave after nerve suture correlated with the number of labeled neurons. After immediate HFA each regenerated motoneuron established on average 5.1 myelinated sprouts at 112 DPSO. The number of sprouts remained constant after delayed suture of 14 to 112 days, whereas the slower reinnervation after 7 or 224 days delay was accompanied by a massive sprouting of 9.1 or 8.1, respectively, sprouts per neuron. The muscles showed recovery after any denervation time. The muscle cross-sectional area continuously decreased with longer denervation time. This decrease was only significant after 224 days denervation (67% of the normal value). We conclude that motor nerve reconstruction achieves better functional results after a definite period of denervation when using a nerve cross-anastomosis paradigm.

Behavior of hypoglossal inspiratory premotor neurons during the carbachol-induced, REM sleep-like suppression of upper airway motoneurons

Behavior of hypoglossal inspiratory premotor neurons during the carbachol-induced, REM sleep-like suppression of upper airway motoneurons

Prevention of death of axotomized hypoglossal neurones and promotion of regeneration by chitin grafting.

1. Chitin is known to promote skin wound healing. In this study, chitin, prepared from Zuwai crab shell, was used as a bridge between the proximal and distal stumps of cut hypoglossal nerves in shrews. We compared the effects of chitin on the regeneration of transected right hypoglossal nerve axons, with those of porcine dermis, bovine dermal aterocollagen, and autologous nerve bundles. 2. To assess the survival of neurones, the size of neuronal cell body, and number of motoneurones were determined in the absence of any bridged material and in the presence of porcine dermis, bovine dermal aterocollagen, chitin, or autologous nerve bundles as a bridge. 3. Our results revealed a significantly better outcome in chitin and autologous nerve bridged groups; the size of neuronal cell body and number of hypoglossal neurones were higher than in the other groups. Chitin also enhanced the regeneration of neurones; the number of horseradish peroxide positive neurones indicative of repaired axonal processes was significantly higher in chitin and autologous nerve-bridged groups than in other groups. 4. Our results demonstrated that the use of chitin sheet or autograft successfully prevented the death of severed neurones and promoted the regeneration of the lesioned nerve. Although the mechanisms underlying the effects of chitin are still unknown, chitin seems to be a potentially useful biocompatible material for nerve repair and regeneration.

Analysis of neurotrophic effects of hepatocyte growth factor in the adult hypoglossal nerve axotomy model.

Recent studies have shown that hepatocyte growth factor (HGF) promotes the survival of embryonic motor neurons. However, it remains unclear whether HGF has trophic effects on mature motor neurons. In the present study, we examined the effects of HGF on adult motoneurons using the hypoglossal nerve transection model. In adult rats, neurons in the hypoglossal nucleus show a dramatic loss of choline acetyltransferase (ChAT) protein and mRNA after the axotomy. This reduction of ChAT was markedly prevented when HGF was administered continuously at the cut end of the nerve using an osmotic pump. The HGF receptor, c-met, protein and mRNA, which were faintly expressed in hypoglossal neurons under normal conditions, gradually increased and reached maximal levels 2 weeks after the axotomy. Administration of HGF reduced this c-met upregulation almost to normal levels. We also quantified HGF mRNA in the tongue and hypoglossal nucleus. The tongue contained abundant HGF mRNA, whereas the nucleus contained only low levels. Interestingly, the HGF mRNA level in the nucleus did not increase after the axotomy. These findings suggest that HGF is principally produced in the tongue and contributes to maintain ChAT expression in the nucleus. HGF produced in the hypoglossal nucleus alone after disconnection from the tongue may not be sufficient for the maintenance of the motor neuron function. Thus, exogenously applied HGF was effective to prevent the downregulation of ChAT activities. These findings provide a strong rationale for the potential clinical use of HGF for the treatment of motor neuron degenerative disease.

Prevention of the death of the rat axotomized hypoglossal nerve and promotion of its regeneration by bovine brain gangliosides.

We have examined the time course of the neuronal death and regeneration of rat axotomized hypoglossal nerve with various conditions of the nerve resection, and established a useful system to measure neurotrophic activities of bioactive substances. In this system, neuronal death can be evaluated by counting surviving neurons in the nucleus of hypoglossal neuron at the brain stem, and the degree of the regeneration can be measured by counting horseradish peroxidase-positive cells at the same region after injection of horseradish peroxidase into tongue. Using this system, the effects of brain gangliosides on rat hypoglossal nerve regeneration following 5 mm transection were examined. The addition of a ganglioside mixture from bovine brain as well as the autograft strongly prevented the death of neurons and promoted the regeneration of the lesioned nerve at 10 weeks after the operation. Further analyses on the dose effects and injection sites of gangliosides were performed. Although the mechanisms of the neurotrophic effects of the gangliosides are unknown, the therapeutic application of gangliosides for neuronal degeneration is a promising approach.