The expression of a motoneuron-specific serine protease, motopsin (PRSS12), after facial nerve axotomy in mice.

Motopsin (PRSS12) is a mosaic serine protease that is preferentially expressed in motor neurons. To study the relationship between motopsin and motoneuron function, we investigated the expression of motopsin mRNA in facial nerve nuclei after facial nerve axotomy at the anterior margin of the parotid gland in mice. Neuronal function was monitored by assessing vibrissal motion in 3 months. Vibrissal behaviour on the injured side disappeared until the day 14 post-operation, and then recovered between the day 21 and 35. Motopsin expression decreased at the day 14, but markedly recovered by the day 21. In contrast, expression of growth-associated protein-43 (GAP-43) was induced at the day 3. These results suggest that the recovery of motopsin expression is correlated with the recovery of the facial motor neuronal function.

Effects of axotomy on telomere length, telomerase activity, and protein in activated microglia.

The adult central nervous system (CNS) is generally thought of as a postmitotic organ. However, DNA labeling studies have shown that one major population of nonneuronal cells, called microglia, retain significant mitotic potential. Microglial cell division is prominent during acute CNS injury involving neuronal damage or death. Prior work from this laboratory has shown that purified microglia maintained in vitro with continual mitogenic stimulation exhibit telomere shortening before entering senescence. In the current study, we sought to investigate whether telomere shortening occurs in dividing microglia in vivo. For this purpose, we used a nerve injury model that is known to trigger localized microglial proliferation in a well-defined CNS region, the facial motor nucleus. Adult Sprague-Dawley rats underwent facial nerve axotomy, and facial motor nuclei were microdissected after 1, 4, 7, and 10 days. Whole tissue samples were subjected to measurements of telomere length, telomerase activity, and telomerase protein. Results revealed a tendency for all of these parameters to be increased in lesioned samples. In addition, microglial cells isolated directly from axotomized facial nuclei with fluorescence-activated cell sorting (FACS) showed increased telomerase activity relative to unoperated controls, suggesting that microglia are the primary cell type responsible for the increases observed in whole tissue samples. Overall, the results show that microglia activated by injury are capable of maintaining telomere length via telomerase during periods of high proliferation in vivo. We conclude that molecular mechanisms pertaining to telomere maintenance are active in the injured CNS.

[Effects of aminoguanidine on the expression of NOS in facial nerve and surrounding tissues of traumatic facial paralysis rats].

OBJECTIVE: To study the effects of inducible NOS inhibitor aminoguanidine on the expression of NOS in facial nerve and surrounding tissue of traumatic facial paralysis rats. METHODS: A small dose of aminoguanidine were intraperitoneally injected into rats before and after facial paralysis. The facial nerve and surrounding tissues were cut at different time point. Immunohistochemical ABC method was used to study the changes of NOS expression in facial nerve and surrounding tissues. RESULTS: The inducible NOS immunoreactivity was obvious inhibited in the facial nerve and surrounding tissues in aminoguanidine group. CONCLUSION: Aminoguanidine chronic treatment can obvious inhibit the inducible NOS expression in the facial nerve and surrounding tissues. Aminoguanidine can improve the regeneration of facial nerve and the recovery of traumatic tissues.

[Changes of trkB and trkC mRNA in facial nucleus after axotomy and end-to-end anastomosis in the rat].

OBJECTIVE: To explore the changes of receptor tyrosine kinase B and C (trkB and trkC) mRNA in facial motoneurons following facial nerve injury and repair. METHODS: The time course of trkB and trkC mRNA in facial motoneurons following facial nerve transection and anastomosis was analyzed using in situ hybridization (ISH) and reverse transcriptase-polymerase chain reaction(RT-PCR) in adult rats. RESULTS: In the facial nerve simple transection group, the intensity of trkB mRNA signal began to increase to 3d post-operation and to its maximum at 7d. Then, the intensity started to decrease at 14d, but it was still higher than that in the contralateral side at 35 d. After facial nerve injury, the changing trend of trkC mRNA was in the contrary to that of trkB. In the group of anastomosis, the changing trend of the intensity of trkB and trkC mRNA signal in the lesioned side was almost the same as that in the transection group. However, the intensity of trkB and trkC mRNA signal in the lesioned side returned to normal level at 35d. The finding obtained by RT-PCR was consistent with that of ISH. The statistic t-test showed significant differences between the two groups for the relative density level of positive band for trkB and trkC at 21d, 35d post-operation, respectively, (ttrkB,21 = 13.795, ttrkC,21 = 7.445, ttrkB,35 = 8.560, ttrkC,35 = 10.132, P < 0.01). CONCLUSIONS: Transection of the facial nerve results in a upregulation of trkB mRNA, but a downregulation of trkC mRNA in axotomized facial motoneurons. The immediate anastomosis can't counteract and attenuate the changes of trkB and trkC induced by the nerve injury, but whether the changes of trkB and trkC return to normal level depends on the regeneration facial nerve.

Involvement of nitric oxide synthase in the physiology and pathophysiology of facial nerve function and dysfunction.

To date few reports have discussed the presence and function of nitric oxide (NO) in structures of the facial nerve. We performed nicotinamide adenine dinucleotide phosphate (NADPH-d)-diaphorase-histochemistry and immunohistochemistry on the intratemporal portion of the facial nerve, including the geniculate ganglion, of guinea pigs using specific antibodies to the three known isoforms of NO synthase and soluble guanylyl-cyclase (sGC). Normal facial nerves were compared to those treated intratympanically with bacterial lipopolysaccharides (LPS) and tumor necrosis factor-alpha (TNF-alpha). Both constitutive NOS isoforms and sGC could be detected in the bipolar ganglion cells of normal animals, while the inducible isoform (iNOS or NOS II) was not found. Endothelial NOS (NOS III) and sGC were present in blood vessels and were predominantly found in the perineurial sheath and less in the endoneurium. sGC could be detected in all fibers in a cross section of the facial nerve. LPS and TNF treatment led to the detection of iNOS in the perikaryia of the geniculate ganglion and the perineural sheath. These findings imply that NO may be involved in neurotransmission at least in the visceroafferent system. NO regulates vascular tone of nutrient blood vessels in the perineural sheath and endoneurium. The presence of sGC indicates that NO acts via its second messenger cGMP. NOS II expression may be a contributing factor to facial nerve palsy via two different mechanisms: NOS II-generated NO may lead to an overstimulation of the visceroefferent nerve fibers and motor fibers of the facial nerve. Dysregulation in facial nerve blood vessels could lead to edema and elevated pressure on the nerve within its osseous canal.

Activation and response to axotomy of microglia in the facial motor nuclei of G93A superoxide dismutase transgenic mice.

Mice over-expressing a human mutation of Cu(2+)/Zn(2+) superoxide dismutase (SOD1) provide a model of amyotrophic lateral sclerosis. Using tomato lectin histochemistry, we analyzed microglia in the facial nuclei of SOD1(G93A) transgenic mice in the late stage of disease. In these animals, microglia was markedly activated, and ensheathed facial motoneurons as observed in wild-type mice 1 week after nerve transection. In the axotomized facial nucleus of transgenic mice at the same time point, microglia activation was enhanced and exhibited phagocytic features. The findings show that in the facial nucleus microglial cells react to motoneuron disease caused by the SOD1 mutation and to axotomy-induced damage of facial motoneurons.

The effect of age and injury on the expression of inducible nitric oxide synthase in facial motor neurons in F344 rats.

Nitric oxide has been implicated in both normal neuronal aging as well as nerve repair events because of its known roles in synaptic plasticity, synaptogenesis and neuroplathologic processes. In this study, we have determined the effect of aging, by comparing brainstem facial motor neurons (FMNs) as well as blood vessels, from adult F344 rats to those in old animals. Inducible nitric oxide synthase (iNOS) expression was determined both by immunohistochemistry using an antibody to iNOS on tissue sections and slot blots. In adult rats, iNOS expression was detectable only in FMNs and not in blood vessels. In old rats, there were robust levels of iNOS protein in blood vessels, while iNOS protein was not detectable in FMNs from old rats. There was also a 12-fold increase in iNOS expression in isolated blood vessels from old rats compared to vessels from adult animals. To determine the effect of injury on iNOS expression, the facial nerve was transected and immunocytochemistry performed as above. After nerve transection in adult rats, iNOS was demonstrable only in blood vessels after 1 day, but by 7 days iNOS protein immunoreactivity was robust in FMNs. In old animals, iNOS protein expression was observed only in FMNs at 1 day, but by 7 days after injury, protein immunoreactivity was localized to the blood vessels. These data suggest that aging and injury differentially affect the expression of iNOS and that the up-regulation of iNOS may be important for the availability of nitric oxide in the aged or injured nervous system.

Organization of choline acetyltransferase-containing structures in the cranial nerve motor nuclei and spinal cord of the monkey.

Cholinergic structures in the cranial nerve motor nuclei and ventral and lateral horns of the spinal cord of the monkey, Macaca fuscata, were investigated immunohistochemically with a monoclonal antibody against monkey choline acetyltransferase (ChAT). ChAT-immunoreactive perikarya and dendrites were present in the oculomotor, trochlear, abducent, trigeminal motor, facial and hypoglossal nuclei, nucleus of Edinger-Westphal, nucleus ambiguus, dorsal nucleus of the vagus, lamina IX of the cervical, thoracic and lumbar spinal cords, and intermediolateral nucleus of the thoracic spinal cord. The neuropil of the trigeminal motor, facial and hypoglossal nuclei, nucleus ambiguus and lamina IX of the cervical, thoracic and lumbar spinal cords contained many ChAT-positive bouton-like structures and they were seemingly in contact with perikarya and dendrites of motoneurons, suggesting that motoneurons in these nuclei are cholinoceptive as well as cholinergic. The oculomotor, trochlear and abducent nuclei, nucleus of Edinger-Westphal, dorsal nucleus of the vagus and intermediolateral nucleus of the thoracic spinal cord contained a small number of ChAT-immunoreactive bouton-like structures, but they did not contact with perikarya and dendrites of ChAT-positive neurons. These observations suggest that the organization of the motor nuclei is complex, at least regarding the cholinoceptivity.

Cytochemical localization of ouabain-sensitive, K(+)-dependent p-nitrophenylphosphatase activity in the facial nerve of reserpinized guinea pigs.

Ion-transporting Na,K-ATPase plays an essential role in nerve conduction. To clarify the cytochemical effects of reserpine on transport Na,K-ATPase activity, the localization of ouabain-sensitive, K(+)-dependent p-nitrophenylphosphatase (K-NPPase) activity was investigated in the facial nerves of normal and reserpinized guinea pigs using a cerium-based method. In the normal facial nerve, the reaction product of K-NPPase activity was observed on the internodal axolemma and Schmidt-Lanterman incisures. In the Ranvier nodes, enzyme activity was localized to the paranodal and nodal axolemma. In the reserpinized nerves, reaction product was detectable on the nodal axolemma but was undetectable on the other parts of the axolemma. Nodal K-NPPase was not affected by reserpine treatment. Therefore, the transport Na,K-ATPase on the nodal axolemma might differ from that on the other parts of the axolemma. Allowing reserpinized animals to survive. Two different ouabain-sensitive K-NPPase reactivities, "reserpine-sensitive" and "reserpine-resistant," might be present in the facial nerve of guinea pigs.

Prevention of motoneuron death by adenovirus-mediated neurotrophic factors.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of motoneurons, and has no effective treatment. Experimental studies in rodents have shown that motoneurons respond to a variety of molecules including brain-derived neurotrophic factor (BDNF). and the glial-cell line-derived neurotrophic factor (GDNF). Here we investigated the neuroprotective effect of these growth factors, encoded by an adenovirus, on the death of axotomized facial motoneurons in newborn rats. We used a new gene therapy strategy that involves gene transfer to motoneurons by intramuscular injection of an adenoviral vector, which is retrogradely transported from injected target muscle (Finiels et al.,: NeuroReport 7:373-378, 1995). A significant increased survival of motoneurons was observed in animals pretreated with adenovirus encoding BDNF (34.5%, P < 0.05) ou GDNF (41.9%, P < 0.05) 1 week after axotomy. These results indicate that pretreatment with BDNF or GDNF, using this therapeutic strategy, is able to prevent the massive death of motoneurons that normally follows axotomy in the neonatal period, opening new perspectives to limit neuronal death in degenerative disorders.

Induction of urokinase-type plasminogen activator in rat facial nucleus by axotomy of the facial nerve.

The response of plasminogen activator activity in the CNS to peripheral nerve axotomy was examined in vivo. After transection of the rat facial nerve, a transient increase in plasminogen activator activity was observed in the facial nucleus on the operated side with maximal activity 3-5 days after lesion. This activity was inhibited by the urokinase-specific inhibitor amiloride but not by antibodies against tissue plasminogen activator. The molecular mass of the induced form of plasminogen activator was estimated to be approximately 48 kDa. An in vitro assay of plasminogen hydrolysis also demonstrated an increase in amiloride-sensitive plasminogen activator activity in facial nerve extracts following facial nerve axotomy. These data indicate that the plasminogen activator activity induced in the facial nucleus following axotomy of facial motoneurons is of the urokinase type. It is suggested that the urokinase-type plasminogen activator might play a role in the events accompanying injury and regeneration in the facial nucleus following motoneuron lesion.

Neurotransmitters and peptides in the developing human facial nucleus.

The human facial nucleus can be sub-divided into five structurally discrete regions. Immunohistochemistry was used to locate various neurotransmitters and neuropeptides in the neurons and nerve fibres of the human facial nucleus at 14 and 27 weeks of gestation and in the neonate. Whilst choline acetyltransferase-positive neurons were observed in the facial nucleus at all stages of development, dopamine beta-hydroxylase-positive neurons were only found in the neonate. In addition, afferent nerve fibres positive for choline acetyl-transferase, enkephalin and substance P were observed at all stages of development. In the younger specimens these fibres were evenly distributed; however, in the neonates the fibres were asymmetrically distributed as the different types became concentrated in the various structurally distinct regions of the facial nucleus.

Expression of estrogen receptor in the facial nucleus is suppressed by estradiol, but not by testosterone, indicating a lack of requirement for aromatization.

The transient expression of estrogen receptor (ER) in the ventromedial subnucleus of the facial nucleus was previously detected in the newborn rat, and the expression of ER molecules was down-regulated by daily injections of estradiol. Here we examined possible involvement of aromatization in this process. ER molecules were measured by immunohistochemistry and in situ hybridization histochemistry after daily injections of testosterone propionate (TP; 100 micrograms/0.02 ml) and estradiol benzoate (EB; 10 micrograms/0.02 ml) in the male pups castrated within 24 h of birth. Daily injections of TP for 5 consecutive days did not suppress ER and ER mRNA in the facial nucleus, while they were both suppressed by daily injections of EB. Moreover, aromatase immunoreactivity was not detected in the facial nucleus of both castrated, TP injected and intact control males at 6 days of age. The present findings therefore suggest that ER molecules expressed transiently in the facial nucleus are not directly involved in masculine sexual differentiation of the brain in newborn rat.

BDNF-mediated rescue of axotomized motor neurones decreases with increasing dose.

Direct application of brain-derived neurotrophic factor (BDNF) to the cut end of axotomized immature motor neurones had only transient survival-promoting effects. Therefore, we have examined whether additional delivery of BDNF with repeated subcutaneous injections (1 mg/ml) could potentiate this short-term rescue of the lesioned sciatic and facial motor neurones in neonatal rats. Direct application of BDNF combined with intermittent (3-day intervals) injections slightly improved motor neurone survival. However, when BDNF was injected daily in addition to the direct application, the number of surviving lesioned motor neurones was markedly reduced. These findings, corroborated by results in embryonic spinal cord cultures, show that a dose-dependent reversal of BDNF-mediated positive effects on motor neurones occurs in vivo.

Astrocytes but not microglia express NADPH-diaphorase activity after motor neuron injury in the rat.

The purpose of this study was to identify cellular sources of nitric oxide (NO) after injury to rat facial motor neurons using NADPH-diaphorase histochemistry. We employed intraneural injections of either saline or toxic ricin, followed by nerve crush, in order to produce regeneration or degeneration of facial motor neurons (FMNs), respectively. Reactive astrocytes responding to ricin-induced degeneration of FMNs showed increased NADPH-diaphorase activity while reactive astrocytes responding to axotomy (saline injection) did not. Reactive microglial cells were found not to express NADPH-diaphorase in either one of these two paradigms. We conclude that irreversible neuron injury resulting in neurodegeneration causes increased production of NO by reactive astrocytes.

Cytochemical study of ouabain-sensitive, K(+)-dependent p-nitrophenylphosphatase activity in guinea pig facial nerve.

The localization of ouabain-sensitive, K(+)-dependent p-nitrophenylphosphatase (K-NPPase) activity, the second dephosphorylative property of the Na-K adenosine triphosphatase complex, was cytochemically studied in the intra-temporal portion of the facial nerve in normal guinea pigs using a cerium-based method. A fine-granular reaction product of the K-NPPase activity was observed on the cytoplasmic side of the axolemma of the axon cylinder, of the incisures of Schmidt-Lanterman, and of the nodes of Ranvier. No reaction product was detected on the periaxonal and outermost plasma membrane of Schwann cells and in the myelin sheath. In control tissue samples, the enzyme activity was almost completely inhibited by 10 mM ouabain, and no reaction was noted in medium without K+.

Axotomy induces intranuclear immunolocalization of neuron-specific enolase in facial and hypoglossal neurons of the rat.

Neuron-specific enolase as an enzyme of the glycolytic pathway is localized in the cytoplasm of nerve cells, but not in the cell nucleus. We have applied immunocytochemistry with 1:64,000 polyclonal anti-rat neuron-specific enolase to the brainstem of male and female adult Wistar rats following: (a) transection of the facial nerve with immediate microsurgical nerve suture (facial-facial anastomosis), (b) transection of the hypoglossal nerve with immediate suture (hypoglossal-hypoglossal anastomosis) and (c) transection of the facial and hypoglossal nerve with immediate suture of the proximal hypoglossal to the distal facial nerve stump (hypoglossal-facial anastomosis). Studying the intracellular immunolocalization of neuron-specific enolase in neurons of the facial and hypoglossal nucleus we detected that (1) in normal rats about 20% of all facial and hypoglossal neurons display not only cytoplasmic, but also intranuclear neuron-specific enolase-like immunoreactivity and (2) following any axotomy of the facial or hypoglossal peripheral nerve, the perikarya of all injured motoneurons react by an outstanding increase of neuron-specific enolase-like immunoreactivity in the karyoplasm. Similar findings were obtained in experiments on non-fixed cultured Neuro-2a cells that had been lesioned with hydrogen peroxide. Counting the absolute numbers of normal and reactive neurons at 1-365 days post axotomy revealed that the increase of neuron-specific enolase in neuronal cell nuclei is temporary and reversible. It is first detected at 2 days post axotomy, reaches its maximum at 10-18 days post axotomy and is no longer evident 56 days following surgery.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultracytochemical demonstration of ouabain-sensitive, K(+)-dependent, p-nitrophenylphosphatase (Na-K ATPase) activity in cat facial nerve.

Ouabain-sensitive, K(+)-dependent, p-nitrophenylphosphatase (K-NPPase) is the second dephosphorylative property of the Na-K ATPase complex. Localization of its activity in the horizontal portion of the facial nerve in 11 normal cats was studied ultracytochemically using a cerium-based method. The fine granular reaction product of the K-NPPase activity was observed on the cytoplasmic side of the axolemma of the axon cylinder. Enzyme activity was also detected on the cytoplasmic side of the plasma membrane of Schmidt-Lanterman incisures and nodes of Ranvier. No reaction product was detected on the periaxonal and outermost plasma membrane of Schwann cells and in the myelin sheath. In control tissue samples, enzyme activity was almost completely inhibited by 10 mM ouabain, and no reaction was noted in medium without K+. The present findings indicate that localization of Na-K ATPase in the cat facial nerve simulates that of other peripheral and cranial nerves.

Tyrosinase protein is expressed also in some neural crest derived cells which are not melanocytes.

Some neural crest cells give rise to pigment cells in early ontogenesis. We tested here whether tyrosinase--a key enzyme in melanogenesis--was present in some pigment neural crest derivatives in adult hamsters. Interestingly enough, inactive tyrosinase protein was detected, using indirect immunofluorescence, in the satellite cells of spinal ganglia and Schwann cells of sciatic and facial nerves in normal adult animals. The results of cell blotting from spinal ganglia were similar to the fluorescence findings. Thus, our results seem to support the hypothesis that Schwann cells, satellite cells of spinal ganglia, and melanocytes may be more intimately related developmentally than other neural-crest-derived cells. Moreover, since we detected tyrosinase protein in cells which normally do not produce melanosomes, it could be deduced that, during the melanocyte's differentiation from its cell precursor, the expression of tyrosinase protein might precede the point when melanosomes begin to differentiate from known cytoplasmic structures.