A rodent model of partial muscle re-innervation.

BACKGROUND: There is limited appreciation of the risks and benefits of the few salvage treatment options available for inadequate motor function following incomplete spontaneous recovery or surgical repair of major peripheral nerve injuries. The lack of a reliable and economical animal model has hindered laboratory investigation into this difficult clinical problem. We propose a straightforward and reproducible rodent model of partial re-innervation of a hind limb muscle. NEW METHOD: Twelve Sprague-Dawley rats underwent identical surgical manipulations: the left tibial nerve was isolated, partially transected (2/3rds), and the remaining intact portion crushed. Eight weeks later, bilateral (1) gastrocnemius (2) soleus and (3) flexor digitorum longus muscles underwent maximal isometric contraction force testing before being excised and weighed. RESULTS: Only the gastrocnemius muscles were statistically weaker (p<0.05) in the experimental limb compared with the contralateral (control) limb. There was no difference in muscle weights between experimental and control sides. COMPARISON WITH EXISTING METHODS: Our model differs from other published models by: allowing time for compensatory axonal sprouting as would be seen in clinical scenarios, precisely identifying the portion of the tibial nerve to be transected to ensure reproducibility, and achieving temporary but complete denervation by crushing the intact portion of the nerve. CONCLUSIONS: Controlled partial transection and crushing of the rodent tibial nerve results in a consistently partially re-innervated and clinically weakened gastrocnemius muscle that can serve as a model in studying incomplete recovery following nerve injury.

Comparison of the fastest regenerating motor and sensory myelinated axons in the same peripheral nerve.

Functional outcome after peripheral nerve regeneration is often poor, particularly involving nerve injuries far from their targets. Comparison of sensory and motor axon regeneration before target reinnervation is not possible in the clinical setting, and previous experimental studies addressing the question of differences in growth rates of different nerve fibre populations led to conflicting results. We developed an animal model to compare growth and maturation of the fastest growing sensory and motor fibres within the same mixed nerve after Wallerian degeneration. Regeneration of cat tibial nerve after crush (n = 13) and section (n = 7) was monitored for up to 140 days, using implanted cuff electrodes placed around the sciatic and tibial nerves and wire electrodes at plantar muscles. To distinguish between sensory and motor fibres, recordings were carried out from L6-S2 spinal roots using cuff electrodes. The timing of laminectomy was based on the presence of regenerating fibres along the nerve within the tibial cuff. Stimulation of unlesioned tibial nerves (n = 6) evoked the largest motor response in S1 ventral root and the largest sensory response in L7 dorsal root. Growth rates were compared by mapping the regenerating nerve fibres within the tibial nerve cuff to all ventral or dorsal roots and, regardless of the lesion type, the fastest growth was similar in sensory and motor fibres. Maturation was assessed as recovery of the maximum motor and sensory conduction velocities (CVs) within the tibial nerve cuff. Throughout the observation period the CV was approximately 14% faster in regenerated sensory fibres than in motor fibres in accordance with the difference observed in control nerves. Recovery of amplitude was only partial after section, whereas the root distribution pattern was restored. Our data suggest that the fastest growth and maturation rates that can be achieved during regeneration are similar for motor and sensory myelinated fibres.

Evaluation of morphological and functional regeneration of rat nerve-muscle units after temporary and permanent tubulization.

We compared the ability of temporary and permanent tubing to achieve morphological and functional recovery of nerve-muscle units, following experimental nerve transection (8-mm gap) in rat tibial nerve. Electrical stimulation of the sciatic nerve was used to analyze tension output, evoked electromyogram and conduction-transmission time (CTT) of denervated nerve-muscle units. Morphological analysis of the nerve and muscle was also performed. Within 6 weeks, the nerve gap had been bridged by a thin nerve trunk, and a few myelinated fibers were observed, although there was still no functional recovery. The rats were divided into two groups: permanent tubing (PT) and temporary tubing (TT; tubing subsequently removed). At 10 weeks after the operation, the TT group showed apparently greater thickness of regenerated nerve trunks, significantly higher tension output of plantar flexors, shorter CTT, and heavier muscle mass. These results were consistent with the presence of myelinated fibers in the regenerated nerve trunks, as shown histologically. Thus, removal of the silicone chamber results in faster and better recovery than tubing left permanently in place.

[Growth and differentiation of epi- and perineural structures under graded stretching]. / Rost i differentsirovka struktur épi- i perinevriia v usloviiakh dozirovannogo rastiazheniia.

Light and electron microscopic and ultrastructural studies of the epi- and perineurium of sciatic and tibial nerves were performed in 63 adult mongrel dogs during lengthening the femur and leg at rates of 0.5 and 1 mm a day (no more than 0.25 mm per dose) by the Il-izarov procedure. Hypertrophy of the biosynthetic apparatus, complication of intercellular and cytostromal contacts were observed in the fibroblasts of the epineurium, in the cell elements of vascular walls and perineurium during distraction. There was growth and associated changes in the volume ratios of the wall components of epineural structures, as well as new formation of structures of the intrinsic innervation apparatus of epi- and perineural sheaths. At all levels (organ, tissue, cellular, and subcellular) the new formed structures incorporated into the preexisting ones without disintegrating them, which is a characteristic feature for intercalar growth.

Automatic image analysis of the postnatal growth of axons and myelin sheaths in the tibial and peroneal nerves of the rabbit.

The numerous morphometric studies on the myelinated fibers in the peripheral nerves have presented varying results. Only a few studies deal with the peripheral nerves from rabbits. In this work, a morphometric study was carried out on the tibial and peroneal nerves of new-born, 10-, 15-, 20-, 30-, 60-, 90- and 240-day-old rabbits. The bilateral proximal segments of both nerves were investigated. Negatives of semi-thin cross sections were used for myelinated fiber morphometric analysis, carried out by an OLYMPUS Video image analyser. Two morphometric parameters, the average axon diameter (AD) (the average length of Feret's diameters) and the specific width of the myelin sheaths (SWMS) (specifying the total width of the myelin sheath), were evaluated for every age group. In the tibial and peroneal nerves a bimodal distribution of the average AD appeared on the 20th day, and of the SWMS on the 10th day postnatum. A tight correlation was obtained when comparing mean AD (mAD) and mean SWMS (mSWMS) in new-born and 240-day-old rabbits. From birth to adulthood the mAD increased in both nerves by about 270% and the mSWMS by about 280%. The mAD/mSWMS ratio in both new-born and 8-month-old rabbits was found to be 4 in the tibial and 5 in the peroneal nerves. According to the available data, an approach to the measurement of AD as an average length of Feret's diameters and the measurement of the myelin thickness as a SWMS has not yet been employed. An extension of this methodological approach could help to understand the growth and myelinization of peripheral nerve fibers.

Natural course of diabetic peripheral neuropathy in spontaneous-onset diabetic Chinese hamsters.

We investigated metabolic and pathological changes in the peripheral nerve of the spontaneous-onset diabetic Chinese hamster. Electrophysiological examination revealed that the motor nerve conduction velocity was significantly decreased at 10 months and afterwards, however, the F-wave latency was significantly increased at 5 months and afterwards. Concerning sciatic nerve contents of sorbitol, myo- and scyllo-inositol, the content of sorbitol was not significantly increased at 5 months, but, myo- and scyllo-inositol were significantly decreased at 5 months and thereafter. At 10 and 15 months, however, sciatic nerve content of sorbitol was significantly increased. On morphological examination, loss of large myelinated fiber and reciprocal increase in degenerative fiber were also seen in sciatic nerve, but not in tibial nerve, at 5 months. At 15 months, these morphological changes were also found in the tibial as well as the sciatic nerve. Thus, we may hypothesize that F-wave latency is useful in the detection of initial diabetic neuropathy, and that the initial pathological changes in diabetic neuropathy of diabetic Chinese hamsters are predominantly found in the proximal site of peripheral nerves.

Acetyl-L-carnitine prevents age-dependent structural alterations in rat peripheral nerves and promotes regeneration following sciatic nerve injury in young and senescent rats.

Morphologic and morphometric alterations of the sciatic nerve from old Sprague-Dawley rats and of lesioned tibial nerve from young and senescent Sprague-Dawley rats were studied. The possible therapeutical effects of treatment with acetyl-L-carnitine (ALCAR) were also investigated, ALCAR being a compound shown to exert a beneficial pharmacological action on diabetic neuropathies. Nerve sections from animals sacrificed under anesthesia were stained with toluidine blue. In old rats, a 6-month treatment with ALCAR markedly reduced the percentage of myelinated fibers (MF) characterized by age-dependent morphologic alterations (4% in treated rats versus 11% in untreated ones), such as myelin balloons, infolded loops of myelin, myelin reduplication, and ovoids. In the lesioned animals, ALCAR-treatment (15-60 days for young rats and 6-9 months for senescent rats) produced a significant increase versus controls in the density of regenerating myelinated fibers (RMF) at 15 days (young rats) and at 30 days (senescent rats) after crush, as well as an increase in the axon diameter in both young and senescent rats at 60 days after nerve crush. The MF diameter (sheath + axons) was significantly larger in treated senescent rats than in controls at 100 days after nerve crush. In ALCAR-treated rats, both young and senescent, the density of degenerative elements was lower and the RMF ratio (RMF density/RMF density + density of degenerative elements) was higher than that in controls at all detection times.

Effects of predegeneration on nerve regeneration through silicone Y-chambers.

The effects of nerve predegeneration on the preferential growth of regenerating axons were studied using a silicone Y-chamber model. This system provided a choice for axons to grow towards two distal nerve options, either a 7-day predegenerated nerve segment (PNS) or a fresh nerve segment (FNS). The rat peroneal or tibial nerve was inserted into the proximal intlet and the PNS and FNS of the corresponding nerve were inserted into the distal outlets. At 28 days postoperative, the size of the distal regenerate was significantly greater (26%) towards the PNS for the tibial nerve group. The density and number of regenerated myelinated axons in the distal nerve segment was greater on the PNS for both the tibial (97 and 88%, respectively) and peroneal (221 and 221%, respectively) nerve groups. In contrast, the elevated density and number of nonvascular nuclei was relatively constant for both PNS and FNS. Immunocytochemical and ultrastructural evidence support the hypothesis that the early activation of Schwann cells is primarily responsible for the enhanced regeneration and maturation observed in PNS. It is suggested that PNS might improve the outcome after clinical repair of injured peripheral nerves.

Maturational changes in F waves in growing chickens.

The F waves evoked by supramaximal stimulation of distal tibial nerve were evaluated in chickens aged 2 to 15 weeks. Latency of these potentials increased from mean +/- SD 11.4 +/- 0.12 ms at week 2 to mean 12.88 +/- 0.65 ms at week 15. The F-wave latency increased linearly with age. When this latency was corrected for a standard distance to compensate for the increasing limb length with age, latency decreased with maturity.

Maturation of lower extremity EMG responses to postural perturbations: relationship of response-latencies to development of fastest central and peripheral efferents.

EMG responses to toe-up tilt perturbations on a movable platform system were analysed in 86 children between the age of 12 months and 13 years. To assess the relative contribution of peripheral and central nerve conduction properties, a concomitant recording of the fastest efferent pathways in the central and peripheral motor system was made using non-invasive transcranial magnetic stimulation of motor cortex and peripheral nerve roots. This allowed the determination of the fastest downstream efferent connection times from motor cortex to lumbar motor neuron pools and to measure the fastest efferent conduction from these motor neuron pools to effector muscles in the lower leg. The sequence observed for stance stabilizing EMG responses was similar to that obtained in earlier studies with short latency (SL) and middle latency (ML) companents occurring in the stretched triceps surae muscle and long latency (LL) responses occurring in the non-stretched tibialis anterior muscle. Homologous responses were also obtained in upper leg muscles, being recruited consistently later than those in lower leg muscles across all age groups. In the short latency range two different SL1- and SL2-responses were obtained in children of all age groups as well as in adult controls. Both the SL1- and the SL2-responses showed a flat developmental profile, reaching adult values between 20 and 30 months of age which correlated with that of the fastest efferents from lumbar motor neuron pools to leg muscles, i.e. the final motor path. ML-responses showed a steeper developmental profile.(ABSTRACT TRUNCATED AT 250 WORDS)

Success of regeneration of peripheral nerve axons in rats after injury at different postnatal ages.

Electrophysiological experiment have been carried out on rats to see if the age at which a peripheral nerve injury occurs influences the success of regeneration. The assessment was made on the basis of two measures of peripheral nerve regeneration; the extent to which axons manage to grow across the injury site and into the distal stump, and their ability to resupply cutaneous structures with functional endings. Regeneration after nerve transection of both myelinated and unmyelinated axons was studied. The results showed that, apart from rats injured when 2 weeks old, the age at which injury occurred, over the range 4-40 weeks, had little bearing on the overall success of skin reinnervation. The 2-week-old rats showed significantly poorer recovery.

Postnatal development of adrenergic innervation in the tibial and vagus nerves of Fischer-344 rats.

Adrenergic innervation of rat tibial and vagus nerves was studied in male Fischer-344 rats between 1 and 84 days of age, using sucrose-phosphate-glyoxylic acid (SPG) histochemistry and the formaldehyde-induced fluorescence (FIF) method. Adrenergic nerve fibers were found in epi-perineurial blood vessels of the vagus nerve at one day of age, whereas blood vessels in the tibial nerve received the first adrenergic nerve fibers at 3 days. A few adrenergic nerve fibers were seen in the endoneurium of both tibial and vagus nerves at 7 days. The densities of adrenergic innervation increased gradually during the first 4 postnatal weeks, and at 21 days the distributions of adrenergic innervation in both nerves resembled those in adult animals. The results suggest that development of adult adrenergic innervation in rat peripheral nerves occurs during the first postnatal month and that sympathetic innervation becomes available to regulate nerve blood flow within this period.

Adrenergic innervation of the tibial and vagus nerves in rats of different ages.

Adrenergic nerve fibers innervating blood vessels in the epi-perineurium and endoneurium of the tibial and vagus nerves of male Fischer-344 rats of different ages were examined using formaldehyde-induced fluorescence technique and fluorescence microscopy. Between 6 and 24 months of age, no significant difference in the mean density of perivascular innervation in the epi-perineurium of the nerves was found, whereas between 24 months and 30 months the density decreased in both nerve types. The intensity of noradrenaline fluorescence of nerve fibers also was decreased in the oldest age group. In the endoneurium, the density of adrenergic nerve fibers was reduced by 57% at 24 months, and by more than 98% at 30 months as compared with 18 months. Age-related differences were similar in the tibial and vagus nerves. The results suggest that adrenergic neuronal control of the microcirculation in the rat tibial and vagus nerves is lost during aging as it is in other organs of the cardiovascular system.

Relative growth and maturation of axon size and myelin thickness in the tibial nerve of the rat. 1. Normal animals.

Morphometric observations have been made on the medial plantar division of the tibial nerve (MPD) and on the motor branches of the tibial nerve to the calf muscles (MBC) in rats ranging in age from weaning (3 weeks) to 12 months. Axon size, assessed by measurements of circumference and cross-sectional area, increased rapidly until 3 months with further slight increases between 3 and 9 months and a slight fall between 9 and 12 months. Axon size distributions were unimodal throughout in the MPD but bimodal for the MBC except at 3 weeks. Distributions of myelin thickness were bimodal throughout for both nerves. Scatter plots of g ratios (axon diameter:total fibre diameter) confirmed the presence of two fibre populations: a group of small fibres with relatively thin myelin sheaths, and a group of larger fibres within which sheath thickness was relatively less on the larger than on the smaller axons. These two fibres populations were less easily separable in the MBC than in the MPD nerves. These results document morphometrically the normal growth changes in the rat tibial nerve and also provide control data for the analysis of the effects of experimental procedures on the growth and maturation of peripheral nerve fibres.

Relative growth and maturation of axon size and myelin thickness in the tibial nerve of the rat. 2. Effect of streptozotocin-induced diabetes.

The relative changes in the growth and maturation of axon size and myelin thickness were studied in the medial plantar division of the tibial nerve in the lower leg and in the motor branches of the tibial nerve to the calf muscles in rats in which diabetes mellitus had been induced with streptozotocin at the time of weaning. Observations were made at 6 weeks and 3, 6, 9 and 12 months of diabetes for comparison with age-matched controls. Similar changes were observed in both nerves. Growth in body weight and skeletal growth was severely retarded from the time of induction of diabetes but at the 6-week stage axon size was not reduced, suggesting that neural growth may initially be relatively protected. At later stages axon size was consistently reduced in the diabetic animals as compared with the controls and showed an absolute reduction at 12 months, as compared with 9 months, that was greater than in the controls. Myelin thickness became reduced earlier and was more severely affected than axon size so that the fibers were relatively hypomyelinated. The myelin changes were greater in larger than in smaller fibers. The index of circularity of axons was reduced in the diabetic nerves. These results show that induction of diabetes in prepubertal rats produces effects on peripheral nerve fibers which differ from those resulting from diabetes induced in adult animals. The effects also differ between large and small nerve fibres. These observations may explain some of the disparate findings obtained in previous studies on experimental diabetes in rats.

Maturational study of short-latency somatosensory evoked potentials after posterior tibial nerve stimulation in infants and children.

SSEPs produced in response to PTN stimulation were studied in 41 normal infants and children from 4 months to 16 years in age. SSEPs were recorded on the scalp with reference electrodes attached to the contralateral knee, shoulder and earlobe. Four positive SSEPs, PI, PII, PIII and PIV, named in order of appearance, and one negative SSEP, N0, were recorded as FFPs on the scalp with the cKn reference. Following these FFPs, the cortical component P1 which corresponded to P37 in adults was recorded. Preceding P1, another negative wave, N1, could be recognized solely at Cz' mainly at the onset of P1. P1 and N1 could be identified in all children with derivations with noncephalic references, although they could not be identified in 5 of 41 children with a Cz' - Fpz derivation. PI, bilobed in configuration, was considered to originate at the sacral plexus or entry to the spinal canal. PII was the least reproducible potential and was considered to originate at the dorsal root, dorsal horn or conus medullaris. PIII, PIV and N0 were considered to originate at the cervical cord, brain stem and thalamus, respectively. With the peak latencies of PI, PII, PIII, PIV, N0, N1 and P1, the RV was calculated in order to eliminate the influence of body height. The RV of the later appearing components leveled off in the older age categories. The RV of P1 reached a steady level at 3 years of age. RVs of PII and PIII appeared to level off by the age of 6 years. The RV of PIV leveled off by the age of 9 years. RVs of N0 and N1 leveled off by the age of 12 years, and that of P1 decreased until over 12 years of age. Furthermore, to eliminate the influence of naturation in the peripheral nerves, the RV was obtained from PI-PIV, PI-P1, PIV-P1 and N1-P1 interpeak latencies. The RVs of these 4 interpeak latencies all decreased until over 12 years of age. Accordingly, the maturation of afferent conduction in the central nervous system after PTN stimulation appeared to be complete after 12 years of age.

Influence of malnutrition on developing rat peripheral nerves.

Animals with experimentally induced neuropathies frequently fail to gain weight normally and appear poorly nourished. To determine whether or not malnutrition alone contributes to peripheral nerve dysfunction in these disorders, we subjected healthy 40-day-old rats to three grades of food restriction. After 4 weeks, food-restricted rats weighed 203 +/- 5 (mean +/- SE), 152 +/- 12, and 97 +/- 5 g, respectively, whereas control rats having free access to food weighed 379 +/- 22 g. Posterior tibial nerves of food-deprived rats had smaller endoneurial areas, fewer large-diameter fibers, and shorter internodes than did the nerves of control animals. However, the number of fibers was similar to controls, and there was no evidence of active degeneration or demyelination. Motor and mixed nerve conduction velocities along the same nerves increased with age and did not differ among control and malnourished groups. In growing rats, food deprivation sufficient to impair somatic growth interferes with the maturation of large-caliber myelinated fibers, but does not effect standard electrophysiologic measurements of nerve function.

Glycogen accumulation in tibial nerves of experimentally diabetic and aging control rats.

Tibial nerves of streptozotocin-diabetic, alloxan-diabetic, and age-matched control rats were examined at 2 weeks and 2, 4, 8, and 12 months following the induction of diabetes. Glycogen-like granules accumulated within perineurial and Schwann cells of only the diabetic animals. This accumulation may reflect a metabolic abnormality in these cells which could account for the reduced conduction velocities seen in the peripheral nerves of these same diabetic rats (Moore et al. 1980a). Glycogen-like granules were also present and increased with age in myelinated axons of both diabetic and control rats. Quantitative data suggest that axonal accumulation of glycogen-like granules is related to aging or injury related phenomena to which diabetic axons may be more susceptible.

Age changes in the tibial and plantar nerves of the rat.

Observations have been made on the changes in the myelinated fibres of the rat tibial and plantar nerves between 2 and 24 months of age. There is an initial rapid increase in fibre diameter followed by a later more gradual increase, which ceases after approximately 9 months of age in the tibial nerve but which continues for longer in the medial plantar nerve. The fibre size distribution remains substantially unimodal throughout. In both nerves maximal and average fibre diameter become reduced by 24 months. Total fibre number shows considerable variability between animals, but no definite systematic alteration with age is detectable. Teased fibre preparations demonstrate a low level of abnormality in the tibial nerve until after 18 months of age, but by 24 months approximately 30% of fibres display abnormalities. Although both paranodal and segmental demyelination and remyelination, and axonal degeneration and regeneration occur, the latter type of change predominates. By contrast, in the lateral plantar nerve paranodal and segmental demyelination become detectable to a significant extent from 6 months of age. Axonal degeneration and regeneration also become evident after 15 months, and by 24 months of age 55% of fibres show abnormalities. The possible explanation of these changes is discussed, as is their relevance to the frequent use of the tibial nerve for studies on experimental neuropathies.

[Contribution of Hoffmann's reflex, evoked on the soleus muscle, to the study of nerve conduction velocity from birth to age 5]. / Apport du réflexe d'Hoffmann, évoqué sur le muscle soléaire, à l'étude des vitesses de conduction nerveuse chez l'enfant de la naissance à 5 ans.

We tried to appreciate the maturation of the peripheric nervous system, in children from two days to five years old, by electrophysiological technics. Hoffmann's monosynaptic reflex is recorded on the soleus muscle and conduction velocity is calculated on the motor tibial nerve. So, we can follow the conduction velocities on the proximal and distal part of this nerve, from birth to five years of life. The conduction velocities develop in a parallel and approximately exponential way. The proximal conduction velocity remains always faster than the distal one but their difference decreases during the first 18 months of life and then remains constant. The evolution of the proximal conduction velocity explains that the conduction time slightly but significantly decreases during the first year of life in contrast to the increase of the nerve length. So, during the first year of life, nervous system maturation gets on very quickly and anticipates the skeletal growth.