Effects of various lengths of hypoglossal nerve resection on motoneuron survival.

We employed stereological analyses for whole quantification of hypoglossal (XII) motoneurons in adult rats that received varying degrees of resection of the XII nerve. Various lengths of nerve gaps (0.0-13.3 mm) were made at the main trunk of the unilateral XII nerve, and the total number of XII neurons on the injured and uninjured sides was counted 12 weeks after nerve resection. The stereologically estimated total number of XII neurons decreased after various lengths of nerve resection, and survival rates ranged from 34.4% to 87.1%. Statistically significant negative correlations were observed between increasing length of the resected nerve and decreasing XII neuron survival. It was concluded that the total number of XII neurons decreased after nerve resection and that survival rates of XII neurons were related to distances between resected nerve stamps.

Effects of repeated nerve injuries at different time intervals on functional recovery and nerve innervation.

Effects of repeated nerve injuries on functional recovery and nerve innervation were examined in rodents. Crush injuries of the sciatic nerve were inflicted on adult rats and repeated twice or thrice at different time intervals of 1, 2, 3, and 4 weeks. Motor function was assessed by the static sciatic index at 1, 7, 14, 21, 28, 35, 42, 49, and 56 days after the final crush. The rates of nerve innervation of the tibialis anterior muscle, a main muscle innervated by the common peroneal nerve, were evaluated by the quantification of ßIII-tubulin-positive nerve terminals and α-bungarotoxin-positive acetylcholine receptors 21 and 56 days after the final crush of triple nerve injuries at 1-, 2-, 3-, and 4-week intervals. Compared with single nerve crush injury, delayed recovery of motor function was observed in repeated crush injuries. In addition, recoveries in the triple crush groups were slower than those in the double crush groups. The rates of reinnervation were lower in the triple crush groups than in the single crush groups, both at 21 days (single: 59.7%; triple: 54.1%-56.1%) and 56 days (single: 88.8%; triple: 72.5%-83.0%) after the final crush, except in the groups with 1-week (triple: 73.8%) and 2-week (triple: 70.5%) intervals at 21 days after the final crush. We concluded that the recovery of motor function was delayed according to the number of repetitions of crush injuries, and that the rates of nerve innervation were still low in the triple crush groups 8 weeks after the final crush.

Acetyl-l-carnitine enhances myelination of regenerated fibers of the lateral olfactory tract.

It is well known that acetyl-l-carnitine (ALC) has various neuroprotective effects against neurodegenerative diseases. In addition, it has been reported that ALC facilitates myelination of regenerated axons after peripheral nerve injuries. We previously reported that spontaneous regeneration of the lateral olfactory tract (LOT), the main fiber tract of the central olfactory system, consistently occurred in newborn rats and a majority of these regenerated fibers were unmyelinated in neonatally LOT-transected young adult rats. To investigate the effects of ALC treatment on myelination in LOT, neonatal rats were treated with ALC after LOT transection. Immunohistochemistry for myelin basic protein showed more positive areas in ALC-treated rats than in control rats. Moreover, the number of myelinated axons of regenerated fibers was assessed using electron microscopy and was found to be statistically higher in ALC-treated rats compared to control rats. The study revealed that ALC accelerates myelination of regenerated fibers in neonatally LOT-injured young adult rats.

Stereological assessment of the total number of hypoglossal neurons after repeated crush injuries to the hypoglossal nerve in adult rats.

OBJECTIVE: Retrograde neuronal cell death does not occur in mature motoneurons following the axonal injury of peripheral nerves. However, a previous study suggested that retrograde neuronal cell death does occur in adult rats after the creation of double lesions on the hypoglossal (XII) nerve based on a substantial decrease in the number of XII neurons. Using stereological methods, we examined neuronal apoptosis in XII neurons and the total number of XII neurons following repeated crush injuries to the XII nerve. METHODS: The right XII nerve of adult rats was crushed three times at one-week intervals with a brain aneurysm clip. At 4 weeks after the final crush, the total numbers of XII neurons on the injured right and uninjured left sides were estimated stereologically. RESULTS: After repeated crush injuries, no apoptosis was evident in XII neurons as indicated by immunostaining for cleaved caspase-3. Moreover, immunohistochemistry for the vesicular acetylcholine transporter revealed axonal elongation in the tongue 4 weeks after repeated crush injuries. At 4 weeks, the total numbers of XII neurons were 7800 ± 290 on the injured right side and 8000 ± 230 on the uninjured left side, and no significant difference was evident between the injured and uninjured sides. CONCLUSION: Neuronal cell death does not occur in XII neurons and the total number of XII neurons does not decrease after repeated crush injuries of the XII nerve in adult rats.

Morphological analysis of regenerated bulbar fibers in relation to neonatal olfaction.

It was revealed that regeneration of the lateral olfactory tract (LOT) occurred in developing rats and the regenerated olfactory system was functional 4 weeks after transection. The aim of this study was to determine the earliest onset of functional recovery in LOT-injured rats and to quantify regenerated nerve components with functional correlation. Neonatal rats on postnatal day (P) 2 were subjected to unilateral transection of the left LOT and underwent unilateral removal of the right olfactory bulb on P11. Functional recovery of the tract injury was assessed by the suckling capability, which can be achieved by olfaction. Suckling capability was observed on P12 in most neonatally LOT-transected pups. Rat pups were subjected to unilateral transection of the left LOT on P2, and received injections of biotinylated dextran amine (BDA) into the bilateral olfactory bulb on P5 to quantify normal and regenerated nerve components in the olfactory cortices at the level of the olfactory tubercle. BDA(+) areas and density indices of the olfactory cortices in the neonatally LOT-transected P12 pups were 11.05×105µm2 and 0.35 on the normal right side and 4.34×105µm2 and 0.21 on the transected left side. We concluded that functional recovery of the LOT-transected neonatal rats occurred as early as 10days after tract transection and that areas and densities of regenerated nerve components essential for functional recovery were approximately 40% and 60% of the age-matched normal values in the olfactory cortices at the level of the olfactory tubercle.

Effect of graded nerve pressure injuries on motor function.

OBJECT: The purpose of this study was to determine the minimum amount of nerve fibers required to maintain normal motor function after nerve injury in rats. METHODS: The authors first confirmed that a common peroneal nerve injury caused more aggravating effects on lower limb motor function than tibial nerve injury, as assessed by the static sciatic index (SSI). Thereafter, rats were subjected to varying degrees of crush injury to the common peroneal nerve. At 48 hours after the injury, motor function was assessed using the SSI and slope-walking ability (with slope angles of 30° and 45°). The tibialis anterior muscle, a main muscle innervated by the common peroneal nerve, was removed. Muscle sections were co-labeled with neuronal class III ß-tubulin polyclonal antibody to identify the presence of axons and Alexa Fluor 488-conjugated α-bungarotoxin to identify the presence of motor endplates. RESULTS: The evaluation of neuromuscular innervation showed a correlation between SSI scores and ratios of residual axons (rs = 0.68, p < 0.01), and there was a statistically significant difference between slope-walking ability and ratios of residual axons (p < 0.01). Moreover, the ratios of residual axons in the nerve-crushed rats with normal motor function (SSI above -20) ranged from 36.5% to 88.7%, and those ratios in the success group with slope-walking angles of 30° and 45° ranged from 14.7% to 88.7% and from 39.8% to 88.7%, respectively. CONCLUSIONS: In this study of rodents, less than half of the motor axons were sufficient to maintain normal motor function of the lower limb.

Stereological estimation of olfactory receptor neurons in rats.

The total number of olfactory receptor neurons (ORNs) in the rat main olfactory epithelium (MOE) was estimated using stereological sampling. Skulls and noses of newborn (postnatal day 0), young adult (8 weeks), and adult (6 months) rats were decalcified, embedded in paraffin, cut into 10-µm thick sections serially at 100-µm intervals, and processed for immunohistochemistry for olfactory marker protein (OMP), a specific marker of mature ORNs with fiber connections to the olfactory bulb. The number of OMP (+) receptor neurons was measured using an optical fractionator with stereological software (Stereoinvestigator). The total number of OMP (+) receptor neurons in the unilateral MOE was 0.47 × 10(6) in newborns and 21 × 10(6) in young adults and adults. Our previous study showed that the corresponding unilateral total number of neurons was 5.2 × 10(6) in young adult mice. Accordingly, we concluded that rats had 4 times more OMP (+) receptor neurons than mice at the adult stage and that the number of these neurons increased approximately 45 times between birth and maturity.

Two specific tongue regions receive bilateral hypoglossal innervation: a study in neonatal rat pups.

OBJECTIVE: The purpose of this study was to investigate the functional role of bilateral hypoglossal (XII) nerve innervation of the tongue. MATERIALS AND METHODS: The possibility of bilateral innervation of XII nerve in the tongue was examined using postmortem fibre tracing in normal neonatal rats. In addition, immunohistochemical testing for synaptophysin or vesicular acetylcholine transporter (VAChT) was carried out in unilaterally XII nerve-resected neonatal rats. RESULTS: Postmortem fibre tracing revealed constant distribution of the nerve fibres extending across the midline and existence of bilateral innervated area in the rostroventral and caudodorsal regions of the tongue. Synaptophysin-positive and VAChT-positive nerve terminals were also distributed continuously from the XII nerve-intact to the nerve-resected side across the midline of the tongue. The contralaterally projecting VAChT-positive nerve terminals were more numerous in suckling P2 rats (6.6 ± 0.5/section) than those in non-suckling P2 rats (4.9 ± 0.3/section) 24 h after nerve resection. Furthermore, the contralaterally projecting VAChT-positive nerve terminals were more numerous in P7 rats with nerve resection on P1 (6.3 ± 0.2/section) than those in P7 rats with nerve resection on P6 (3.1 ± 0.8/section). CONCLUSION: We concluded that neonatal rats have two specific tongue regions receiving bilateral XII innervation, which allowed suckling in unilaterally XII nerve-resected neonatal rats.

Correlation between motor function and axonal morphology in neonatally sciatic nerve-injured rats.

The present study was conducted to investigate the correlation between motor function and axonal morphology in neonatally sciatic nerve-injured rats. The left sciatic nerve of newborn rats was transected or crushed, and functionality of the sciatic nerve was assessed by the static sciatic index after 8 weeks. After functional assessment, the common peroneal nerves in the control, nerve-transected, and nerve-crushed rats were removed and prepared for morphometric examinations. The cross-sectional area of the nerve, total number of myelinated axons, and size of each myelinated axon were analyzed for each group. The control rats showed normal motor function, whereas the nerve-transected rats showed severe motor dysfunction. The cross-sectional area of the nerve and total number of myelinated axons were reduced after nerve transection. Moreover, the percentage per size class of myelinated axons was almost uniform in the control rats, while the distribution was shifted to the left in the nerve-transected rats. Furthermore, no large myelinated axons were observed in the nerve-transected rats. The nerve-crushed rats showed various gait functions with various distribution patterns of axonal size, and the rats were divided into two groups with and without uninjured residual large axons. The results showed that the importance of regenerated medium-sized axons in cases without large axons and of residual large axons in cases with large axons in motor function. It was revealed that motor function was related closely to axonal size in neonatally nerve-injured rats.

Quantitative analysis of survival of hypoglossal neurons in neonatally nerve-injured rats: Correlation with milk intake.

INTRODUCTION: Tongue movement innervated by the hypoglossal (XII) nerve is essential for the survival of neonatal rats. The pups with bilateral XII nerve resection failed to suckle milk and did not survive, and the pups with unilateral XII nerve resection showed disturbed suckling capability and lower survival rates. The present study was performed to investigate the relation between neuronal population and milk intake of developing rats that had received various degrees of crush injuries to the unilateral XII nerve during the neonatal period. METHODS: The right XII nerve of postnatal day 1 (P1) pups was crushed and milk intake was estimated at 3 days and 6 days after the nerve injury. As nerve injury at the neonatal stage results in death of axotomized neurons, varying degrees of crushing was estimated by the number of survived motor neurons. RESULTS: In nerve-crushed rats, the populations of XII motor neurons and amounts of milk intake were reduced in a varied manner. Statistically significant positive correlations were observed between increasing XII neuron survival and increasing milk intake at 3 (r=0.62) and 6 (r=0.71) days after the nerve injury. CONCLUSION: The results indicate that there is a strong relationship between the number of XII motor neurons and the amount of milk intake in neonatally XII nerve-injured rats.

Regenerative capacity of bulbar projection neurons during development: a quantitative neuronal analysis with functional correlation.

Critical periods and degrees of regeneration in injured olfactory bulbar projection neurons (mitral cells) were examined in adult rats whose lateral olfactory tracts (LOTs) were transected at different postnatal (P) days. After the LOTs were transected at P7, P10, and P14, a retrograde fluorescent tracer, Fluoro-Gold (FG), was injected into the posterior olfactory cortex (the olfactory tubercle and the piriform cortex), a target brain region of mitral cells, 5 weeks after the transection. FG (+) mitral cells were observed in P7 LOT-transected bulbs and some of P10 LOT-transected bulbs but not in P14 LOT-transected bulbs. Neuron numbers of regenerated FG (+) mitral cells in P2 LOT-transected adult rats decreased to approximately 70% of the normal values (actually counted number: 804±46; stereologically estimated number: 49 700±4300), and 100% of these rats were demonstrated to exhibit olfactory discriminative ability in our previous study. Meanwhile, the numbers in P7 LOT-transected adult rats further decreased to approximately 40% of the normal values, and 78% of these rats showed olfactory discriminative ability. We conclude that the critical periods of spontaneous regeneration of the LOT are between P0 and P10 and that the proportions of regenerated mitral cells decreased as rats became older.

Analysis of spontaneous regeneration of olfactory structures with emphasis on myelination and re-innervation of cortical areas.

Regeneration of the lateral olfactory tract (LOT) occurs spontaneously after transection in developing rats. In neonatally LOT-transected rats, we observed a newly formed myelinated tract near the rhinal sulcus. The aim of this study was to analyze the precise re-innervated cortical areas and to demonstrate ectopic LOT myelination in neonatally LOT-transected rats. Neonatal rats were subjected to unilateral LOT transection and simultaneous injection of a retrograde fluorescent tracer into the posterior olfactory cortex to evaluate the degree of transection. After 8 weeks, bilateral olfactory bulbs of the rats were subjected to multiple injections of an anterograde neuronal tracer to determine the extent of the regenerated fibers. In the completely LOT-transected rats, the regenerated fibers were distributed in the anterior olfactory cortices; the anterior olfactory nucleus, the olfactory tubercle, and the rostral part of the piriform cortex. Ectopic myelination of LOT was evident immediately below the rhinal sulcus in the completely and incompletely LOT-transected rats. We concluded that the regenerated bulbar fibers were confined to the regions of the anterior olfactory cortices and that ectopic myelination of the regenerated LOT occurred only at a specific site near the rhinal sulcus.

Effects of repeated crush injuries on motor functional recovery of the sciatic nerve.

OBJECTIVES: The present study was conducted to examine whether repeated crush injuries have significant effects on motor functional recovery of peripheral nerves. METHODS: Repeated crush injuries of the sciatic nerve were inflicted on adult rats at 1-week intervals, and functionality of the sciatic nerve was assessed by the static sciatic index each week for 8 weeks after the final injury. To determine the effects of repeated crush injuries on motor functional recovery of the sciatic nerve, tibialis anterior muscle fibers from single and triple crush injuries were examined, and fiber size and fiber reinnervation during the 2- to 4-week period after the final injury were measured. RESULTS: Compared to single crush injuries, which completely recovered by post-injury week 4, double crush injuries resulted in retarded, but complete recovery by post-injury week 6, whereas triple crush injuries resulted in marked retardation in the regenerative process with incomplete recovery during week 8 of the experimental period. Muscle fiber size for rats with triple crush did not recover to normal range at post-injury week 4, despite its normal size for rats with single crush. The rate of reinnervation increased prominently between post-injury weeks 2 and 3 in both injuries, but the rate with triple crush was lower than that with single crush at post-injury week 3. DISCUSSION: These results, which contradict those of a previous study that reported early functional recovery, indicate that repeated crush injuries inhibit motor functional recovery of the damaged sciatic nerve, as evidenced by delayed and incomplete regeneration, atrophied muscle fibers, and delayed reinnervation.

Functional correlation between olfaction and various sectioning of the lateral olfactory tract.

The lateral olfactory tract (LOT) is a central olfactory pathway, and efferent projections from the olfactory bulb are conveyed to the olfactory-related cortical structures via the LOT. The purpose of the present study is to determine the exact site of the LOT causing functional impairment in animals. After ablation of the right olfactory bulb, rats received rostrocaudal transection injuries on the left LOT at different levels between the olfactory bulb and the middle cerebral artery. Olfactory function of LOT-transected rats was studied by examining their olfactory ability to discriminate between the smell of water and cycloheximide solution, a strong repellent for rodents. Rats were divided into two groups based on their olfactory discriminative abilities. The olfaction positive (+) group achieved 83%± 1% correct responses and the distances of the LOT-transected sites from the middle cerebral artery of this group ranged between 0.8 and 2.4mm (n=8). The olfaction negative (-) group achieved 48%± 1% correct responses and the distances of the LOT-transected sites from the middle cerebral artery ranged between 2.5 and 4.2mm (n=10). From these data, we concluded that the site of the LOT critical for olfactory function is located approximately 2.5mm from the middle cerebral artery.

Re-evaluation of spontaneous regeneration of the lateral olfactory tract.

Spontaneous regeneration of the lateral olfactory tract (LOT) was re-evaluated in newborn rats using a fluorescent retrograde neuronal tracer as objective indicators of complete LOT transection. Complete LOT transection was evidenced by the loss of the white myelinated band characteristic for adult LOT and the total lack of retrograde neuronal labeling of mitral cells by Fast Blue that was injected during LOT transection. In completely LOT-transected young adult rats, mitral cells were retrogradely labeled consistently only by Fluoro-Gold that was injected into the olfactory cortex at the adult stage. Moreover, an anterograde neuronal tracer, biotinylated dextran amine (BDA), was demonstrated to pass from the neonatally LOT-transected bulb, through the transected retrobulbar site, towards the olfactory cortex, far caudally at a level near the optic chiasm. The regenerated structures lacked immunoreactivity for myelin basic protein and electron-dense myelinated axon bundles, and were also characterized by the thinness of the BDA+ terminal zone within the olfactory cortex and the lack of its caudal extension. Young adult rats subjected to unilateral bulbectomy contralateral to the neonatally LOT-transected side showed perfect ability to discriminate cycloheximide solution by olfaction. From these findings, we conclude that the spontaneously regenerated olfactory system is functional despite structural incompleteness.

Determination of functionally essential neuronal population of the olfactory epithelium for nipple search and subsequent suckling behavior in newborn rats.

Neuronal population of the olfactory epithelium required for nipple search and subsequent suckling behavior was examined in newborn rats. After unilateral ablation of the olfactory bulb, different concentrations of ZnSO(4) were contralaterally injected into the nasal cavity to produce varying degrees of neuronal degeneration in the olfactory epithelium. The ZnSO(4)-treatment resulted in two populations of pups. One exhibited suckling while the other did not, and intact olfactory receptor neurons were quantified immunohistochemically using an antibody for olfactory marker protein (OMP), a marker protein for olfactory receptor neurons. The total numbers of the OMP (+) cells in the ZnSO(4)-treated pups with suckling capability ranged between 2457 and 4615, whereas those in the ZnSO(4)-treated pups without suckling capability ranged between 112 and 2398. With the mean value (4969) of the total numbers of the OMP (+) cells of the normal/control pups assumed to represent 100%, the total numbers of the OMP (+) cells accounted for 49-93% in the suckling (+) group and 2-48% in the suckling (-) group. From these findings, we conclude that approximately 50% of neuronal population of the olfactory epithelium is a critical value to distinguish between the two groups with and without suckling capability in the unilateral olfactory system of newborn rats.

Tyrosine hydroxylase-immunoreactive fibers in the human vagus nerve.

Sympathetic catecholaminergic fibers in the vagus nerve were immunohistochemically examined in formalin-fixed human cadavers using an antibody against the noradrenalin-synthetic enzyme tyrosine hydroxylase (TH). TH-positive fibers were extensively distributed in the vagal nerve components, including the superior and inferior ganglia, the main trunk and the branches (superior and recurrent laryngeal, superior and inferior cardiac, and pulmonary branches). The inferior ganglion and its continuous cervical main trunk contained numerous TH-positive fibers with focal or diffuse distribution patterns in each nerve bundle. From these findings, we conclude that sympathetic fibers are consistently included in the human vagus nerve, a main source of parasympathetic preganglionic fibers to the cervical, thoracic and abdominal visceral organs.

Neural plasticity of neonatal hypoglossal nerve for effective suckling.

The adaptive movement of the tongue after unilateral lesion of the hypoglossal (XII) nerve during the early postnatal days is essential for recovery of milk intake. The present study investigated the basic mechanisms underlying such adaptation, focusing on the neural plasticity that allows effective suckling. After resection of the ipsilateral XII nerve on P1, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlolate (DiI), a postmortem neuronal tracer, was applied to the contralateral uninjured XII nerve on P4 and P7. DiI-labeled fibers were traced successfully within the tongue and showed gradually increased extension over the XII nerve-injured side in the central core portion of the denervated tongue between P4 and P7. Systematic neuroanatomic experiments showed that contralateral axonal sprouting occurred as early as 1 day after nerve injury (P2), and that such axonal sprouting occurred exclusively from the medial branch of the XII nerve responsible for tongue protrusion, an essential movement for suckling. These findings provide direct evidence of functional neural plasticity that allows effective suckling in XII nerve-injured newborns with suckling disturbance.

Functional role of lingual nerve in breastfeeding.

Functional role of lingual nerve in breastfeeding was investigated in rat pups during the suckling period. DiI, a postmortem neuronal tracer, was used to confirm the immature lingual nerve (LN) responsible for tongue sensation and resulted in successful fiber labeling anterogradely to the tongue, which showed different distribution patterns from fiber labeling derived from the hypoglossal nerve. Unilaterally LN-injured pups did not show suckling disturbance with absence of any shortening (P11 pups: 559+/-16s; 105% of the control value) in nipple attachment time and the survival rate remained high (P11: 100%). Bilaterally LN-injured pups showed suckling disturbance with marked shortening (P11 pups: 220+/-54 s; 42% of the control value) in nipple attachment time and a low survival rate (P1: 33%; P11: 41%). Bilaterally infraorbital nerve-injured or bilaterally bulbectomized pups did not show any nipple attachment at all and there were no survivors, confirming the crucial roles of upper lip sensation and olfaction in suckling. Based on these findings, we conclude that tongue sensation is very important, but not essential for suckling.

Motor neurons essential for normal sciatic function in neonatally nerve-injured rats.

The present study was aimed to determine neuronal population essential for normal motor function in young adult rats receiving various degrees of crushing to the sciatic nerve at the neonatal stage. Motor function was estimated by the static sciatic index, and a neuronal tracer was applied to the common peroneal nerve. The total numbers of the tracer-labeled neurons of the nerve-crushed rats were 74-383 in the normal function group, 14-61 in the disordered function group, and 0-32 in the severely disordered function group. We conclude that normal motor function can be well preserved by a very small population of motor neurons (approximately 15% of the control value) in the neonatally sciatic nerve-injured rats.