Nerve compound action current (NCAC) measurements and morphometric analysis in the proximal segment after nerve transection and repair in a rabbit model

In the evaluation of nerve regeneration using magneto-neurography (MNG), the proximal segment showed a reproducible decrease in peak-peak amplitude of the nerve compound action current's (NCAC) of 60%. To explain these changes, morphometry of myelinated axons in the proximal segment is compared to the MNG signals. A standardised nerve transection and reconstruction was performed in rabbits. NCACs were measured approximately 5 cm proximal to the lesion from operated and control nerves after 12 weeks. Histological samples were taken from the same area of the nerve where the NCACs were obtained. Results showed a decrease of the peak-peak amplitude of the NCAC of 57% compared to the control. Conduction velocity decreased 15% (not significant). Morphometry elicited a decrease in larger (10-15 microm) axons (284 +/- 134 vs 82 +/- 55) and an increase in smaller (2-5 microm) axons (1445 +/- 360 vs 1921 +/- 393). A strong correlation existed between the decrease in amplitude and the decrease in larger axons (0.85). Peak-peak amplitude varies approximately with the square of the diameter axon. Therefore, because peak-peak amplitude is mainly dependent on the larger-diameter axons, the decrease in peak-peak amplitude of the NCACs may be explained by a decrease in numbers of 10-15-microm axons.     

Myelinated sensory and alpha motor axon regeneration in peripheral nerve neuromas

Histochemical staining for carbonic anhydrase and cholinesterase (CE) activities was used to analyze sensory and motor axon regeneration, respectively, during neuroma formation in transected and tube-encapsulated peripheral nerves. Median–ulnar and sciatic nerves in the rodent model permitted testing whether a 4 cm greater distance of the motor neuron soma from axotomy site or intrinsic differences between motor and sensory neurons influenced regeneration and neuroma formation 10, 30, and 90 days later. Ventral root radiculotomy confirmed that CE-stained axons were 97% alpha motor axons. Distance significantly delayed axon regeneration. When distance was negligible, sensory axons grew out sooner than motor axons, but motor axons regenerated to a greater quantity. These results indicate regeneration differences between axon subtypes and suggest more extensive branching of motor axons within the neuroma. Thus, both distance from injury site to soma and inherent motor and sensory differences should be considered in peripheral nerve repair strategies. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21: 1748–1758, 1998     

Rolipram-induced elevation of cAMP or chondroitinase ABC breakdown of inhibitory proteoglycans in the extracellular matrix promotes peripheral nerve regeneration

The inhibitory growth environment of myelin and extracellular matrix proteoglycans in the central nervous system may be overcome by elevating neuronal cAMP or degrading inhibitory proteoglycans with chondroitinase ABC (ChABC). In this study, we asked whether similar mechanisms operate in peripheral nerve regeneration where effective Wallerian degeneration removes myelin and extracellular proteoglycans slowly. We repaired transected common peroneal (CP) nerve in rats and either elevated cAMP in the axotomized neurons by subcutaneous rolipram, a specific inhibitor of phosphodiesterase IV, and/or promoted degradation of proteoglycans in the distal nerve stump by local ChABC administration. Rolipram treatment significantly increased the number of motoneurons that regenerated axons across the repair site at 1 and 2 weeks, and increased the number of sensory neurons that regenerated axons across the repair site at 2 weeks. Local application of ChABC had a similar effect to rolipram treatment in promoting motor axon regeneration, the effect being no greater when rolipram and ChABC were administered simultaneously. We conclude that blocking inhibitors of axon regeneration by elevating cAMP or degrading proteoglycans in the distal nerve stump promotes peripheral axon regeneration after surgical repair of a transected nerve. It is likely that elevated cAMP is sufficient to encourage axon outgrowth despite the inhibitory growth environment such that simultaneous enzymatic proteoglycan degradation does not promote more axon regeneration than either elevated cAMP or proteoglycan degradation alone.     

Regenerative axonal sprouting in the cat trochlear nerve

Following peripheral trochlear nerve axotomy in the cat, the normal number of myelinated axons is restored despite significant motor neuron death, suggesting regulation of the number of myelinated axons in the regenerated nerve. In this study we used light and electron microscopy to examine the production and maintenance of axonal sprouts at different locations in the nerve and at different postoperative intervals. Despite proliferative sprouting and an overproduction of nonmyelinated axons in the regenerating trochlear nerve, the number of myelinated axons was strictly regulated. Only ～1,000 regenerated axons were eventually remyelinated, but many nonmyelinated axons were still present 6–8 months postaxotomy. Regenerated axons were remyelinated in a proximal-to-distal direction between 3 and 4 weeks postaxotomy. We also examined the maturation of regenerated myelinated axons by measuring axon diameter and myelin index (an expression of myelin thickness). Mean myelinated axon diameter remained significantly below normal in long-term regenerated nerves. Mean myelin index was not different from normal at 4 weeks postaxotomy but was significantly decreased at long postoperative intervals, reflecting a slightly thicker myelin sheath relative to the axon diameter. This relative increase in mean myehn thickness could serve to restore normal conduction velocity despite the decrease in mean axon diameter. We suggest that the regulation of the number of myelinated axons at the normal number despite cell death and the increase in mean myelin thickness may both be compensatory mechanisms that function to restore preoperative conditions and maximize functional recovery. © 1995 Wiley-Liss, Inc.     

Glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor sustain the axonal regeneration of chronically axotomized motoneurons in vivo

In contrast to injuries in the central nervous system, injured peripheral neurons will regenerate their axons. However, axotomized motoneurons progressively lose their ability to regenerate their axons, following peripheral nerve injury often resulting in very poor recovery of motor function. A decline in neurotrophic support may be partially responsible for this effect. The initial upregulation of glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) by Schwann cells of the distal nerve stump after nerve injury has led to the speculation that they are important for motor axonal regeneration. However, few experiments directly measure the effects of exogenous BDNF or GDNF on motor axonal regeneration. This study provided the first direct and quantitative evidence that long-term continuous treatment with exogenous GDNF significantly increased the number of motoneurons which regenerate their axons, completely reversing the negative effects of chronic axotomy. The beneficial effect of GDNF was not dose-dependent. A combination of exogenous GDNF and BDNF on motor axonal regeneration was significantly greater than either factor alone, and this effect was most pronounced following long-term continuous treatment. The ability of GDNF, either alone or in combination with BDNF, to increase the number of motoneurons that regenerated their axons correlated well with an increase in axon sprouting within the distal nerve stump. Thus long-term continuous treatment with neurotrophic factors, such as GDNF and BDNF, can be used as a viable treatment to sustain motor axon regeneration.     

A magnetic evaluation of peripheral nerve regeneration: II. The signal amplitude in the distal segment in relation to functional recovery

Motor and sensory function in a healthy nerve is strongly related to the number of neuronal units connecting to the distal target organs. In the regenerating nerve the amplitudes of magnetically recorded nerve compound action currents (NCACs) seem to relate to the number of functional neuronal units with larger diameters regenerating across the lesion. The goal of this experiment was to compare the signal amplitudes recorded from the distal segment of a reconstructed nerve to functional recovery. To this end, the peroneal nerves of 30 rabbits were unilaterally transected and reconstructed. After 6, 8, 12, 20, and 36 weeks of regeneration time the functional recovery was studied based on the toe-spread test, and the nerve regeneration based on the magnetically recorded NCACs. The results demonstrate that the signal amplitudes recorded magnetically from the reconstructed nerves increase in the first 12 weeks from 0% to 21% of the amplitudes recorded from the control nerves and from 21% to 25% in the following 23 weeks. The functional recovery increases from absent to good between the 8th and the 20th week after the reconstruction. A statistically significant relation was demonstrated between the signal amplitude and the functional recovery (P < 0.001). It is concluded that the magnetic recording technique can be used to evaluate the quality of a peripheral nerve reconstruction and seems to be able to predict, shortly after the reconstruction, the eventual functional recovery. © 1998 John Wiley & Sons, Inc. Muscle Nerve 21:750–755, 1998.     

Collateral sprouting of sensory axons after end-to-side nerve coaptation--a longitudinal study in the rat

The end-to-side nerve coaptation is able to induce collateral sprouting of axons from the donor nerve and to provide functional reinnervation of the target tissue. Sensory axon sprouting and its effects on the donor nerve up to 9 months after the end-to-side nerve coaptation were studied in the rat. Peroneal, tibial and saphenous nerves were transected and ligated, and the distal stump of the transected peroneal nerve was sutured to the side of the uninjured sural nerve. The average skin area of the residual sensitivity to pinch due to the axons sprouting through the recipient peroneal nerve did not change statistically significantly between 4 and 9 months after surgery. Axon counting, measurements of compound action potentials and retrograde neuron labeling indicate that the sprouting of the myelinated sensory axons and unmyelinated axons through the recipient nerve was largely completed by 2 months and 4 months after the end-to-side nerve coaptation, respectively, and remained stable thereafter for at least 9 months. A decrease in the amplitude and area of the CAP of myelinated fibers, observed in the donor nerve up to 4 months after surgery, was probably due to mild degeneration of nerve fibers and a tendency of the diameter of myelinated axons to decline. However, no significant changes in functional, electrophysiological or morphological properties of the donor nerve could be observed at the end of the observational period, indicating that end-to-side nerve coaptation has no detrimental effect on the donor nerve on a long-term scale.     

Remodeling of the proximal segment of crayfish motor nerves following transection

Transected crustacean motor axons consist of a soma-endowed proximal segment that regenerates and a soma-less distal segment that survives for up to a year. We report on the anatomical remodeling of the proximal segment of phasic motor nerves innervating the deep flexor muscles in the abdomen of adult crayfish following transection. The intact nerve with 10 phasic axons and its two branches with subsets of 6 and 7 of these 10 axons undergo several remodeling changes. First, the transected nerve displays many more and smaller axon profiles than the 6 and 7 axons of the intact nerve, approximately 100 and 300 profiles in the two branches of a preparation transected 8 weeks previously. Serial images of the transected nerve denote that the proliferation of profiles is due to several orders of axon sprouting primary, secondary, and tertiary branches. The greater proliferation of axon sprouts, their smaller size, and the absence of intervening glia in the one nerve branch compared with the other branch denote that sprouting is more advanced in this branch. Second, the axon sprouts are regionally differentiated; thus, although in most regions the sprouts are basically axon-like, with a cytoskeleton of microtubules and peripheral mitochondria, in some regions they appear nerve terminal-like and are characterized by numerous clear synaptic vesicles, a few dense-core vesicles, and dispersed mitochondria. Both regions possess active zone dense bars with clustered synaptic vesicles found opposite other sprouts, glia, hemocytes, and connective tissue, but because the opposing membranes are not differentiated into a synaptic contact, the active zones are extrasynaptic. Third, some of the transected axons display a glial cell nucleus denoting assimilation of an adaxonal glial cell by the transected axons. Fourth, within the nerve trunk are a few myocytes and muscle fibers. These most likely originate from adjoining and intimately connected hemocytes, because such transformation occurs during muscle repair. In a crustacean nerve, however, where muscle is clearly misplaced, its presence implies an instructive role for motor nerves in muscle formation.     

Nerve conduction and electromyography studies

Nerve conduction studies (NCS) and electromyography (EMG), often shortened to 'EMGs', are a useful adjunct to clinical examination of the peripheral nervous system and striated skeletal muscle. NCS provide an efficient and rapid method of quantifying nerve conduction velocity (CV) and the amplitude of both sensory nerve action potentials (SNAPs) and compound motor action potentials (cMAPs). The CV reflects speed of propagation of action potentials, by saltatory conduction, along large myelinated axons in a peripheral nerve. The amplitude of SNAPs is in part determined by the number of axons in a sensory nerve, whilst amplitude of cMAPs reflects integrated function of the motor axons, neuromuscular junction and striated muscle. Repetitive nerve stimulation (RNS) can identify defects of neuromuscular junction (NMJ) transmission, pre- or post-synaptic. Needle EMG examination can detect myopathic changes in muscle and signs of denervation. Combinations of these procedures can establish if motor and/or sensory nerve cell bodies or peripheral nerves are damaged (e.g. motor neuronopathy, sensory ganglionopathy or neuropathy), and also indicate if the primary target is the axon or the myelin sheath (i.e. axonal or demyelinating neuropathies). The distribution of nerve damage can be determined as either generalised, multifocal (mononeuropathy multiplex) or focal. The latter often due to compression at the common entrapment sites (such as the carpal tunnel, Guyon's canal, cubital tunnel, radial groove, fibular head and tarsal tunnel, to name but a few of the reported hundred or so 'entrapment neuropathies').     

Misdirection of regenerating motor axons after nerve injury and repair in the rat sciatic nerve model

Misdirection of regenerating axons is one of the factors that can explain the poor results often found after nerve injury and repair. In this study, we quantified the degree of misdirection and the effect on recovery of function after different types of nerve injury and repair in the rat sciatic nerve model; crush injury, direct coaptation, and autograft repair. Sequential tracing with retrograde labeling of the peroneal nerve before and 8 weeks after nerve injury and repair was performed to quantify the accuracy of motor axon regeneration. Digital video analysis of ankle motion was used to investigate the recovery of function. In addition, serial compound action potential recordings and nerve and muscle morphometry were performed. In our study, accuracy of motor axon regeneration was found to be limited; only 71% (± 4.9%) of the peroneal motoneurons were correctly directed 2 months after sciatic crush injury, 42% (± 4.2%) after direct coaptation, and 25% (± 6.6%) after autograft repair. Recovery of ankle motion was incomplete after all types of nerve injury and repair and demonstrated a disturbed balance of ankle plantar and dorsiflexion. The number of motoneurons from which axons had regenerated was not significantly different from normal. The number of myelinated axons was significantly increased distal to the site of injury. Misdirection of regenerating motor axons is a major factor in the poor recovery of nerves that innervate different muscles. The results of this study can be used as basis for developing new nerve repair techniques that may improve the accuracy of regeneration.     

Sensory axon targeting is increased by NGF gene therapy within the lesioned adult femoral nerve

Even though peripheral nerves regenerate well, axons are often misrouted and reinnervate inappropriate distal pathways post-injury. Misrouting most likely occurs at branch points where regenerating axons make choices. Here, we show that the accuracy of sensory axon reinnervation is enhanced by overexpression of the guidance molecule nerve growth factor (NGF) distal to the bifurcation. We used the femoral nerve as a model, which contains both sensory and motor axons that intermingle in the parent trunk and distally segregate into the saphenous (SB) and motor branches (MB). Transection of the parent trunk resulted in misrouting of axon reinnervation to SB and MB. To enhance sensory axon targeting, recombinant adenovirus encoding NGF was injected along the SB close to the bifurcation 1 week post-injury. The accuracy of axon reinnervation was assessed by retrograde tracing at 3 or 8 weeks after nerve injury. NGF overexpression significantly increased the accuracy of SB axon reinnervation to the appropriate nerve branch, in a manner independent of enhancing axon regeneration. This novel finding provides in vivo evidence that gradient expression of neurotrophin can be used to enhance targeting of distal peripheral pathways to increase axon regeneration into the appropriate nerve branch.     

Prior Collateral Sprouting of Sensory Axons Delays Recovery of Pain Sensitivity after Subsequent Nerve Crush

Regeneration of motor axons is enhanced if they have sprouted prior to nerve injury. We examined whether sensory axon regeneration and recovery of pain response was affected by previous collateral sprouting. In the experimental group of rats, the right saphenous, tibial, and sural nerves were transected and ligated. The peroneal nerve was left to sprout into the adjacent denervated skin. Two months later, the axons of the peroneal nerve were crushed in the sciatic nerve. In the control group, the right sciatic nerve was crushed at the same time that the saphenous, tibial, and sural nerves were transected. Recovery of pain response in the foot was determined by the skin pinch test. Sensory axon elongation rate was measured by the nerve pinch test. The number of myelinated axons was determined in nerve cross sections stained by Azur blue. Recovery of pain sensitivity in the animals of the experimental group was delayed for 2–3 weeks in comparison to the control group. Moreover, the spatial pattern of pain response in the experimental group was irregular, displaying residual regions of insensitive skin which were not present in controls. The elongation rate of regenerating sensory axons in the experimental group was not decreased, and the number of myelinated axons in the peroneal nerves was even about 10% higher than in the control group. Therefore, we assume that the terminal arborization of the neurilemmal tubes pertaining to the former axon sprouts delayed regrowth of sensory axon terminals in the skin.     

Conduction slowing in diabetic distal polyneuropathy

The pathophysiologic significance of motor conduction slowing observed in diabetic distal symmetrical polyneuropathy (DSP) remains controversial. We have used multiple linear regression analysis of compound muscle action potential (CMAP) amplitude vs. motor conduction velocity (CV) and distal latency (DL) in 57 patients with diabetic DSP and 34 patients with amyotrophic lateral sclerosis (ALS) to determine whether motor conduction slowing in diabetic DSP is due mainly to loss of large axons as in ALS or whether there is an additional demyelinative component. We found amplitude-dependent slowing of CV and DL in both diabetic DSP and ALS in the upper and lower extremities, consistent with a loss of large myelinated fibers. However, in diabetic DSP, there was also significant amplitude-independent slowing in intermediate but not distal nerve segments, supportive of an additional demyelinative component. CMAP amplitude vs. CV and DL regression analyses using ALS as a control group for relatively pure axon loss may provide pathophysiologic information about motor nerves in other neuropathic disorders.     

End-to-end and end-to-side neurorrhaphy between thick donor nerves and thin recipient nerves:an axon regeneration study in a rat model

During nerve reconstruction,nerves of different thicknesses are often sutured together using end-to-side neurorrhaphy and end-to-end neurorrhaphy techniques.In this study,the effect of the type of neurorrhaphy on the number and diameter of regenerated axon fibers was studied in a rat facial nerve repair model.An inflow-type end-to-side and end-to-end neurorrhaphy model with nerve stumps of different thicknesses(2:1 diameter ratio) was created in the facial nerve of 14 adult male Sprague-Dawley rats.After 6 and 12 weeks,nerve regeneration was evaluated in the rats using the following outcomes:total number of myelinated axons,average minor axis diameter of the myelinated axons in the central and peripheral sections,and axon regeneration rate.End-to-end neurorrhaphy resulted in a significantly greater number of regenerated myelinated axons and rate of regeneration after 6 weeks than end-to-side neurorrhaphy;however,no such differences were observed at 12 weeks.While the regenerated axons were thicker at 12 weeks than at 6 weeks,no significant differences in axon fiber thickness were detected between end-to-end and end-toside neurorrhaphy.Thus,end-to-end neurorrhaphy resulted in greater numbers of regenerated axons and increased axon regeneration rate during the early postoperative period.As rapid reinnervation is one of the most important factors influencing the restoration of target muscle function,we conclude that end-to-end neurorrhaphy is desirable when suturing thick nerves to thin nerves.     

Functional aspects of the regeneration of unmyelinated axons in the rat saphenous nerve

Electrophysiological experiments have been carried out to investigate aspects of unmyelinated axon regeneration in a transected cutaneous nerve. Some comparisons with regeneration of myelinated axons in the same nerve have also been made.

By 3 months after injury approximately 80% of the unmyelinated axons that had survived in the proximal stump had regenerated into the distal stump. About the same proportion of myelinated axons had regrown into the distal stump by this time. With both groups of axons there was no marked increase in the amount of regeneration across the injury site with longer recovery times. Conduction velocities in the regenerated unmyelinated axons tended to be slower across the injury site than proximally; the proximal conduction velocities did not differ from those in control nerves. The unmyelinated axons seemed to take longer to resupply the skin than did the myelinated ones, but in both cases the extent of skin innervation had reached about 60% of control values by 6 months after the injury.     

Retinal ganglion cell axons regenerate in the presence of intact sensory fibres

A novel allograft paradigm was used to test whether adult mammalian central axons regenerate within a peripheral nerve environment containing intact sensory axons. Retinal ganglion cell axon regeneration was compared following anastomosis of dorsal root ganglia grafts or conventional peripheral nerve grafts to the adult rat optic nerve. Dorsal root ganglia grafts comprised intact sensory and degenerate motor axons, whereas conventional grafts comprised both degenerating sensory and motor axons. Retinal ganglion cell axons were traced after 2 months. Dorsal root ganglia survived with their axons persisting throughout the graft. Comparable numbers of retinal ganglion cells regenerated axons into both dorsal root ganglia (1053+/-223) and conventional grafts (1323+/-881; P>0.05). The results indicate that an intact sensory environment supports central axon regeneration.     

Characteristics of regenerating horizontal semicircular canal afferent and efferent fibers in the toadfish, Opsanus tau.

The horizontal semicircular canal nerve of the toadfish, Opsanus tau, was transected and allowed to regenerate. The time course, morphometrics, and projection patterns of regenerating afferent and efferent vestibular fibers were determined. Nerve transections were performed both pre- and postganglionically, and regeneration was assessed in afferent and efferent fibers by bulk labeling the peripheral axons of the horizontal semicircular canal nerve with biocytin after nerve regrowth. Afferent fibers regrew through the transection site within 14 days and projected to all vestibular nuclei within 3 weeks. Bouton and branch number, axon length, surface area, volume, fiber diameter, and internodal distance were quantified for afferent fibers from eight sites within the vestibular nuclei, and axon number and soma size was quantified for the efferent fibers. Extensive regeneration was seen within 5 weeks of transection in all nuclei, and most morphometric parameters approached or exceeded control levels within 10 weeks. Regeneration appeared to recapitulate morphogenesis with an initial overproduction of boutons and branch points followed by elimination of presumably superfluous structures. Internodal distance remained significantly shorter in regenerating afferent axons than in control fish throughout the 15-week observation period. Efferent fibers also were observed to regenerate. Efferent axon number, diameter, and soma size were indistinguishable from those in controls from 3 weeks posttransection through week 15. Electrophysiological recordings from the horizontal canal nerve during mechanical stimuli of the canal confirmed that the regenerated axons transmitted normal signals. The return of normal equilibrium and behavior coincided with the projection of afferent fibers into the central vestibular nuclei, indicating that functional connections had been reestablished.     

Maximum number of collaterals developed by one axon during peripheral nerve regeneration and the influence of that number on reinnervation effects

This study investigated the maximum number of collaterals that can be maintained by 1 axon during regeneration of rat peripheral nerve. The tibial nerve was transected, the proximal residual, with its variable number of axons, was fixed to the distal stump and served as the donor nerve. The number of myelinated axons was calculated after 12 weeks. An increasing ratio of distal stump axon numbers to proximal donor nerve axon numbers of 1.0, 1.83, 3.64 and 7.97 yielded ratios of regenerative myelinated axon numbers to proximal donor axon numbers of 0.98, 1.51, 2.39, 2.89, respectively, with an estimated maximum value of approximately 3.3 using the Hill function. The tibial function indexes and nerve conduction velocities of the regenerated tibial nerve were -44.1 +/- 5.1 and 43.2 +/- 5.3 m/s, -57.5 +/- 4.7 and 18.6 +/- 4.3 m/s, -80.2 +/- 7.1 and 12.7 +/- 3.7 m/s, and -85.4 +/- 5.7 and 10.5 +/- 3.9 m/s, respectively. It has been suggested that 1 axon can regenerate and maintain up to 3 or 4 collaterals in regenerated rat peripheral nerve.     

Effects of basic fibroblast growth factor (bFGF)-neutralizing antibody and platelet factor 4 on facial nerve regeneration

Exogenous basic fibroblast growth factor (bFGF) has been shown to prevent death of injured cholinergic neurons and stimulate neurite outgrowth from the proximal stump of the transected sciatic nerve. The present study was designed to examine the role of endogenous bFGF, rather than exogenous bFGF in the regenerative process of the transected facial nerve of guinea pig, by using the so-called silicone tubulization model which enabled us to bridge the transected facial nerve with a silicone tube and to inject into the tube bFGF-neutralizing antibody, normal IgG, saline, or platelet factor 4 (an antagonist for bFGF receptor). Under light microscopy, treatment with bFGF-neutralizing antibody caused significant decreases in vascular number, vascular area, and regenerating axons in the middle point of regeneration chambers at the third week after facial nerve transection, even though electron microscopy revealed that the bFGF-neutralizing antibody increased the number of thin axons with caliber smaller than 1 micrometer. Treatment with platelet factor 4 exhibited similar but more conspicuous effects on facial nerve regeneration. These findings suggest that endogenous bFGF not only facilitates angiogenesis within the transected facial nerve but also acts as a neurotrophic agent during facial nerve regeneration; it appears that endogenous bFGF contributes to the enlargement of axon caliber and increases the number of relatively large caliber axons.