Manual stimulation of facial muscles improves functional recovery after hypoglossal-facial anastomosis and interpositional nerve grafting of the facial nerve in adult rats

The facial nerve in humans is often prone to injuries requiring surgical intervention. In the best case, nerve reconstruction is achieved by a facial-facial anastomosis (FFA), i.e. suture of the proximal and distal stumps of the severed facial nerve. Although a method of choice, FFA rarely leads to a satisfactory functional recovery. We have recently devised and validated, in an established experimental paradigm in rats, a novel strategy to improve the outcome of FFA by daily manual stimulation (MS) of facial muscles. This treatment results in full recovery of facial movements (whisking) and is achieved by reducing the proportion of functionally detrimental poly-innervated motor end-plates. Here we asked whether MS could also be beneficial after two other commonly used surgical methods of clinical facial nerve reconstruction namely hypoglossal-facial anastomosis (HFA) and interpositional nerve grafting (IPNG) which, however, seem to have a poorer outcome compared to FFA. Compared to FFA, daily MS for 2 months after HFA and IPGN did not completely restore function but, nevertheless, significantly improved the amplitude of whisker movements by 50% compared with untreated animals. Functional improvement was associated with a reduction in the proportion of polyinnervated end-plates. MS did not reduce the extent of axonal branching at the lesion site nor the subsequent misdirected axonal regrowth to inappropriate targets. Our data show that a simple approach leading to improved quality of muscle fiber reinnervation is functionally beneficial after different types of clinically relevant surgical interventions.     

Focal application of neutralizing antibodies to soluble neurotrophic factors reduces collateral axonal branching after peripheral nerve lesion

A major reason for the insufficient recovery of function after motor nerve injury are the numerous axonal branches which often re-innervate muscles with completely different functions. We hypothesized that a neutralization of diffusable neurotrophic factors at the lesion site in rats could reduce the branching of transected axons. Following analysis of local protein expression by immunocytochemistry and by in situ hybridization, we transected the facial nerve trunk of adult rats and inserted both ends into a silicon tube containing (i) collagen gel with neutralizing concentrations of antibodies to NGF, BDNF, bFGF, IGF-I, CNTF and GDNF; (ii) five-fold higher concentrations of the antibodies and (iii) combination of antibodies. Two months later, retrograde labelling was used to estimate the portion of motoneurons the axons of which had branched and projected into three major branches of the facial trunk. After control entubulation in collagen gel containing non-immune mouse IgG 85% of all motoneurons projecting along the zygomatic branch sprouted and sent at least one twin axon to the buccal and/or marginal-mandibular branches of the facial nerve. Neutralizing concentrations of anti-NGF, anti-BDNF and anti-IGF-I significantly reduced sprouting. The most pronounced effect was achieved after application of anti-BDNF, which reduced the portion of branched neurons to 18%. All effects after a single application of antibodies were concentration-dependent and superior to those observed after combined treatment. This first report on improved quality of reinnervation by antibody-therapy implies that, in rats, the post-transectional collateral axonal branching can be reduced without obvious harmful effects on neuronal survival and axonal elongation.     

Myo-GDNF increases non-functional polyinnervation of reinnervated mouse muscle

Glial cell line-derived neurotrophic factor (GDNF) promotes neuronal survival and influences the development and function of synaptic connections. To test the role of GDNF on the stabilisation of synapses, we examined reinnervated neuromuscular junctions in myo-GDNF mice that over-express GDNF under control of a myogenin promoter. The level of polyneuronal innervation was increased following reinnervation in these mice, although many converging inputs were extremely fine and contained few neurofilaments. Electrophysiological experiments confirmed that some inputs were weak or non-functional by showing that the increased morphological levels of polyneuronal innervation were not reflected in the functional responses of endplates to nerve stimulation. Thus, myo-GDNF over-expression enhances reinnervation, but at the expense of both neurofilament integrity, and functional reliability.     

Effects of aging on nerve sprouting and regeneration

We examined the effects of aging on nerve terminal sprouting and regeneration in the peripheral nervous system. Motor end-plates were demonstrated in rat soleus muscles by means of a cholinesterase-silver stain which permitted measurement of end-plate length and nerve terminal branching. Terminal sprouting was elicited by “pharmacological denervation” brough about by botulinum toxin treatment. In the youngest (2-month-old) animals, the end-plates were small, and the terminals had simple branch patterns. Botulinum toxin treatment elicited a striking increase in end-plate length and terminal arborization. In the older (10- and 18-month-old) animals, the end-plate length was greater, and nerve terminal branching patterns were more complex. Botulinum toxin elicited a sprouting response that was less marked than in the youngest animals. In the oldest (28-month-old) animals, the end-plates were shorter and the terminals less complex. Botulinum failed to elicit any significant sprouting response. Regeneration of motor and sensory axons after a crush lesion of the sciatic nerve was measured using radiolabeled proteins carried by axonal transport as a marker of axonal outgrowth. Our results showed that the average rate of regeneration slowed with increasing age, although a small population of axons continued to regenerate rapidly regardless of age. Evidence implicating inherent defects in the ability of nerves to sprout and regenerate with increasing age is presented. Our findings in the peripheral nervous system may shed light on similar functional changes occurring in the central nervous system.     

Functional recovery and collateral neuronal sprouting examined in young and aged rats following a partial neural lesion

The rat pineal gland was chosen as a model system to study how aging affects the capacity of surviving neurons to compensate for partial destruction of a neural pathway. The pineal gland receives bilateral overlapping sympathetic innervation from the two internal carotid nerves, whose activity regulates several aspects of pineal metabolism in a circadian fashion. The most dramatic of these is the marked nighttime increase in the activity of N-acetyltransferase, the rate-limiting enzyme in melatonin synthesis. These features allow for the pineal gland to be used as a model system for studies on neuronal plasticity, since it is possible to create specific partial neural lesions and to evaluate functional recovery subsequently at the cellular level. We examined the activity of N-acetyltransferase and the content of melatonin in the pineal gland as indices of pineal function at various time points after unilateral surgical denervation (lesion of one of the two internal carotid nerves) in 4-month-(young) and 25-month-old (aged) rats. At both ages, the nighttime levels of the two parameters were significantly lower 8 h after this lesion than in sham-operated animals of the same age, indicating impaired function. When examined at later time points (i.e., 1.5 and 10 days after this lesion), both young and aged animals exhibited full recovery in these two parameters. Measurement of specific neuronal uptake of [³H]norepinephrine was utilized as an index of the number of sympathetic varicosities innervating the pineal gland. One and a half days after unilateral denervation. [³H]norepinephrine uptake in 4-, 10-, 16-, and 25-month-old animals was approximately 50% of that obtained in sham-operated animals of the same age, as expected if half of the innervation of the gland was lesioned. Ten days after surgery, [³H]norepinephrine uptake in 4- and 10-month-old animals was 78% of the value found in sham-operated animals of the same age, suggesting collateral sprouting of the remaining sympathetic nerve fibers. However, in 16- and 25-month-old animals, uptake remained at 50%. These data indicate that recovery of pineal function after unilateral denervation occurs to a similar extent and with a similar time course in young and aged animals. In contrast, the data suggest that collateral sprouting is impaired in aged animals. The fact that functional recovery occurs in young animals sooner after the lesions than does collateral sprouting and that recovery occurs in the aged animals in the apparent absence of collateral sprouting suggests that the relationship between this anatomical form of plasticity and functional recovery may be complex. Among other possibilities, collateral sprouting may play a role in the maintenance of recovered function rather than in the initial recovery process or sprouting may be functionally important after nerve losses greater than those represented by a 50% lesion.     

Trophic dependence of fiber diameter in a crustacean muscle

Denervation or immobilization of crayfish opener muscles produces little or no atrophic change in fiber diameter within 250 days whereas tenotomy of the same muscle leads to significant fiber atrophy within 50 days. The observations that opener nerve terminals probably degenerated 40–90 days prior to sampling denervated muscles, and that opener muscle fibers are 10–30% shorter in tenotomized muscles than for muscles immobilized in the fully open position, lead to the conclusion that opener fiber diameter is much more dependent on muscle length (or passive tension) than upon neurotrophic factors or muscle activity. These results differ from those reported for most vertebrate twitch muscles which undergo significant atrophy after denervation, immobilization, or tenotomy. These data from a polyneuronal, multiterminal, tonic muscle of a crayfish closely resemble results obtained from avian muscles having similar innervation patterns and suggest that one or more of their common functional properties may determine their similar trophic dependencies.     

Recovery, innervation profile, and contractile properties of reinnervating fast muscles following postnatal nerve crush and administration of L-Dopa

Muscle and peripheral nerve development is clearly dependent on their interaction during early postnatal life. Furthermore, muscle or peripheral nerve activity plays a crucial role in the maturation of the neuromuscular system. In this study, the possible involvement of spinal catecholamines in fast muscle recovery after nerve crush is investigated. Sciatic nerve crush was performed on the fourth to fifth postnatal day. Following that, L-Dopa was administered daily [150 mg/kg body weight (BW)] i.p., until the 21st day after birth. L-Dopa-treated and control groups were then examined electrophysiologically for the contractile properties of extensor digitorum longus (EDL) muscles. Two experimental groups were included in this study: (i) rats whose sciatic nerve was crushed and were treated with L-Dopa and (ii) rats whose sciatic nerve was crushed and were not treated with L-Dopa. The number of motoneurones for both groups was estimated by HRP retrograde labelling. The results showed that the operated L-Dopa-treated EDL muscles of the rats exhibited limited atrophy, slighter impairment of maximal tetanic tension, lesser resistance to fatigue, and polyneuronal innervation than the controls. The number of motoneurones was the same for the operated muscles in both groups of animals and was within the normal ranges. Our findings suggest that catecholamines of locomotion during the early stages of development may have a beneficial effect on fast muscle recovery following nerve crush. The action of L-Dopa is attributed to noradrenaline, which acts through descending spinal noradrenergic pathways, possibly via a(2)-adrenergic receptors at the spinal level.     

Repair of peripheral nerve defects with chemically extracted acellular nerve allografts loaded with neurotrophic factors-transfected bone marrow mesenchymal stem cells

Chemically extracted acellular nerve allografts loaded with brain-derived neurotrophic factor-transfected or ciliary neurotrophic factor-transfected bone marrow mesenchymal stem cells have been shown to repair sciatic nerve injury better than chemically extracted acellular nerve allografts alone, or chemically extracted acellular nerve allografts loaded with bone marrow mesenchymal stem cells. We hypothesized that these allografts compounded with both brain-derived neurotrophic factor- and ciliary neurotrophic factor-transfected bone marrow mesenchymal stem cells may demonstrate even better effects in the repair of peripheral nerve injury. We cultured bone marrow mesenchymal stem cells expressing brain-derived neurotrophic factor and/or ciliary neurotrophic factor and used them to treat sciatic nerve injury in rats. We observed an increase in sciatic functional index, triceps wet weight recovery rate, myelin thickness, number of myelinated nerve fibers, amplitude of motor-evoked potentials and nerve conduction velocity, and a shortened latency of motor-evoked potentials when allografts loaded with both neurotrophic factors were used, compared with allografts loaded with just one factor. Thus, the combination of both brain-derived neurotrophic factor and ciliary neurotrophic factor-transfected bone marrow mesenchymal stem cells can greatly improve nerve injury.     

Mechanical stimulation of paralyzed vibrissal muscles following facial nerve injury in adult rat promotes full recovery of whisking

Many patients suffer lifelong disabilities after peripheral nerve injury. Insufficient recovery has been attributed to excessive axonal branching, axonal regrowth to improper targets and polyneuronal reinnervation of motor endplates. We used the rat facial nerve transection/suture model to quantify the effects of mechanical stimulation on the paralyzed whisker musculature. "Manual" stimulation involved briskly stroking the whiskers by hand in a manner that specifically mimicked normal whisker movement. "Environmental" stimulation involved enhanced whisker use as rats encountered objects in an enriched environment. Manual and environmental stimulation were also combined. Video-based motion analysis of vibrissal motor performance showed that daily manual, but not environmental, stimulation for 2 months resulted in full recovery of whisking. Polyneuronal reinnervation of motor endplates was reduced but not misdirected axonal regrowth. Our findings indicate the potential of use-specific training to enhance appropriate functional outcome after peripheral nerve injury and may be useful in a clinical rehabilitation setting.     

The axotomy-induced neuropeptides galanin and pituitary adenylate cyclase-activating peptide promote axonal sprouting of primary afferent and cranial motor neurones

The neuropeptides galanin and pituitary adenylate cyclase-activating peptide (PACAP) are markedly up-regulated in response to peripheral nerve lesion. Both peptides are involved in neuronal differentiation and neurite outgrowth during development. In this study, we investigated the effects of galanin and PACAP on axonal elongation and sprouting by adult rat sensory neurones in vitro and facial motor neurones in vivo. Dissociated rat dorsal root ganglion neurones were plated on laminin substrate and analysed morphometrically. Both the mean axonal length and the number of branch points significantly increased in the presence of galanin or PACAP (2-5 microm). Effects on axonal collateralization were investigated in the rat facial nerve lesion model by direct application of the peptides to collagen-filled conduits entubulating the transected facial nerve stumps. Triple retrograde labelling of brainstem neurones confirmed that the peptides potently induce axonal sprouting of cranial motor neurones. The number of neurones regenerating into identified rami of the facial nerve increased up to fivefold. Biometrical analysis of whisking behaviour revealed that galanin and PACAP impaired the functional outcome when compared with vehicle-treated animals 8 weeks after surgery. In conclusion, although galanin and PACAP have been established as neurotrophic molecules with respect to axonal development and regeneration, their potential as treatments for peripheral nerve lesions appears limited because of the extensive stimulation of collateral axon branching. These branches are misrouted towards incorrect muscles and cause impairment in their coordinated activity.     

Recovery of locomotor function in adult paraplegic frogs by inductive lability in the distal isolated spinal cord neural networks

We postulated (Krishnan, 1991) that in a spinal cord transected adult paraplegic mammal locomotor functions can be revived if polyneuronal innervation is reinduced in the paralyzed hind limb muscles. This procedure destabilizes the neural networks and induces new synaptic growth in the distal isolated cord. In this pilot project we tested the hypothesis in cord-transected adult paraplegic frogs. Polyneuronal innervation was reinduced by crushing the sciatic nerve in the right upper thigh. Left limb sciatic nerve was not crushed and served as control. Another group of adult frogs had only cord transection without nerve crush. Five to seven weeks postnerve crush, full powered flexion-extension movements in the hip and knee joints appeared in the right hind limb and were used for swimming and surface progression. Movements gradually declined over the next weeks, which in some animals was seen preserved even beyond 120 days. Paraplegic frogs without nerve crush did not show any recovery of locomotor function. Interestingly, the uncrushed contralateral limb also produced transient, weak locomotor-like movements. This lasted for 4 to 6 days and waned out completely thereafter. These results validate our hypothesis on methods to generate new synaptic sprouts and reconnections to redrive the locomotor system. We had recommended earlier that destabilization procedure should be included as an essential component in treatment strategies for spinal cord injury repair for effective relinking of the severed cord ends.     

Impacts of lesion severity and tyrosine kinase receptor B deficiency on functional outcome of femoral nerve injury assessed by a novel single-frame motion analysis in mice

Functional recovery after peripheral nerve injury is often poor. Comprehension of cellular and molecular mechanisms limiting or promoting restoration of function and design of efficient therapeutic approaches remain serious challenges for neuroscience and medicine. Progress has been restricted by the lack of reliable methods for evaluation of motor functions in laboratory animals. We describe a novel approach for assessment of muscle function in mice after femoral nerve damage, an injury causing impairment of knee extension. The functional deficit can be precisely estimated by angle and distance measurements on single video frames recorded during movements of the animals with or without body weight support. Using this method we describe here the precise time-course and degree of functional recovery after femoral nerve crush and transection. In addition, we show that restoration of function is considerably impaired in mice with a reduced expression level of the tyrosine kinase receptor B, a cognate receptor for the neurotrophin brain-derived neurotrophic factor. This finding is consistent with known functions of brain-derived neurotrophic factor and tyrosine kinase receptor B and demonstrates the potential of the method. The principles of the approach are highly relevant for the development of novel functional assays in other peripheral and, in particular, central nervous system injury paradigms.     

Transplantation of olfactory ensheathing cells stimulates the collateral sprouting from axotomized adult rat facial motoneurons.

Axon regrowth after CNS and PNS injury is only the first step toward complete functional recovery which depends largely on the specificity of the newly formed nerve-target projections. Since most of the studies involving the application of glial cells to the lesioned nervous system have focused primarily on the extent of neurite outgrowth, little is known regarding their effects on the accompanying processes of axonal sprouting and pathfinding. In this study, we analyzed the effects of transplanted olfactory ensheathing cells (OECs) on axonal sprouting of adult facial neurons by using triple fluorescent retrograde tracing and biometrical analysis of whisking behavior. We found that 2 months after facial nerve axotomy and immediate implantation of OECs in between both nerve stumps fixed in a silicon tube, the total number of labeled neurons was increased by about 100%, compared to animals with simple facial nerve suture or entubulation in an empty conduit. This change in the number of axon sprouts was not random. The highest increase in axon number was observed in the marginal mandibular branch, whereas no changes were detected in the zygomatic branch. This increased sprouting did not improve the whisking behavior as measured by biometric video analysis. Our results demonstrate that OECs are potent inducers of axonal sprouting in vivo. Hence OEC-filled nerve conduits may be a powerful tool to enforce regeneration of a peripheral nerve under adverse conditions, e.g., after long delay between injury and surgical repair. In mixed nerves, increased axonal sprouting will improve specificity since inappropriate nerve-target connections are pruned off during preferential motor innervation. In pure motor nerves, however, OEC-mediated axonal sprouting may result in polyneuronal innveration of target muscles.     

Neurotrophins from dorsal root ganglia trigger allodynia after spinal nerve injury in rats

Injury to peripheral nerves often results in chronic pain which is difficult to relieve. The mechanism underlying the pain syndrome remains largely unknown. In previous studies we showed that neurotrophins are up-regulated in satellite cells around sensory neurons following sciatic nerve lesion. In the present study, we have examined whether the neurotrophins in the dorsal root ganglia play any role in allodynia after nerve injury. Antibodies to different neurotrophins, directly delivered to injured dorsal root ganglia, significantly reduced (with different time sequences) the percentage of foot withdrawal responses evoked by von Frey hairs. The antibodies to nerve growth factor acted during the early phase but antibodies to neurotrophin-3 and brain-derived neurotrophic factor were effective during the later phase. Exogenous nerve growth factor or brain-derived neurotrophic factor, but not neurotrophin-3, directly delivered to intact dorsal root ganglia, trigger a persistent mechanical allodynia. Our results showed that neurotrophins within the dorsal root ganglia after peripheral nerve lesion are involved in the generation of allodynia at different stages. These studies provide the first evidence that ganglia-derived neurotrophins are a source of nociceptive stimuli for neuropathic pain after peripheral nerve injury.     

Leukemia inhibitory factor augments neurotrophin expression and corticospinal axon growth after adult CNS injury.

The cytokine leukemia inhibitory factor (LIF) modulates glial and neuronal function in development and after peripheral nerve injury, but little is known regarding its role in the injured adult CNS. To further understand the biological role of LIF and its potential mechanisms of action after CNS injury, effects of cellularly delivered LIF on axonal growth, glial activation, and expression of trophic factors were examined after adult mammalian spinal cord injury. Fibroblasts genetically modified to produce high amounts of LIF were grafted to the injured spinal cords of adult Fischer 344 rats. Two weeks after injury, animals with LIF-secreting cells showed a specific and significant increase in corticospinal axon growth compared with control animals. Furthermore, expression of neurotrophin-3, but not nerve growth factor, brain-derived neurotrophic factor, glia cell line-derived neurotrophic factor, or ciliary neurotrophic factor, was increased at the lesion site in LIF-grafted but not in control subjects. No differences in astroglial and microglial/macrophage activation were observed. Thus, LIF can directly or indirectly modulate molecular and cellular responses of the adult CNS to injury. These findings also demonstrate that neurotrophic molecules can augment expression of other trophic factors in vivo after traumatic injury in the adult CNS.     

Local stabilization of microtubule assembly improves recovery of facial nerve function after repair

Within a recent study on the recovery of vibrissae motor performance after facial nerve repair in blind (strain SD/RCS) and sighted (strain SD) rats, we found that, despite persisting myotopic disorganization in the facial nucleus, the blind animals fully restored vibrissal whisking. Searching for the morphological substrates of this improved recovery, we compared the amount of cytoskeletal proteins in the leading edge of elongating axons between both strains. Since our results showed an enhanced expression of neuronal class III beta-tubulin in the blind rats, we wondered whether this was due to an increased synthesis or to a delayed turnover of microtubules. In the present report, we approached this question applying established pharmacological agents to the transected buccal branch of the facial nerve in sighted Wistar rats perturbing either microtubule assembly towards stabilization (enhanced polymerization with 10 microg/ml taxol) or towards increased synthesis (challenged by destabilization with 100 microg/ml nocodazole and 20 microg/ml vinblastine). Evaluation of the effect(s) 2 months later included estimation of (i) vibrissae motor performance by video-based motion analysis, (ii) the degree of collateral axonal branching by double retrograde neuronal labeling with crystals of Fluoro-Gold and DiI and (iii) the pattern of motor end-plate reinnervation (proportions of mono- and poly-reinnervated) in the largest extrinsic vibrissal muscle, the m. levator labii superioris. We found that only stabilization of microtubules with 10 microg/ml taxol reduced intramuscular axonal sprouting and polyinnervation of the motor end-plates, which was accompanied by improved restoration of function.     

Time course of sprouting during muscle reinnervation in vitamin E-deficient rats

A typical aspect of motoneuron plasticity is the sprouting which occurs during muscle reinnervation, resulting in a transitory multiple innervation of the muscle cells. In order to verify the effect of a decreased protection from free radical attack on the sprouting, the multiple innervation in the extensor digitorum longus muscle, following sciatic nerve crush and regeneration, was studied in vitamin E-deficient rats. Thus, the innervated end-plates and the end-plates with multiple innervation were studied with histochemical and electrophysiological techniques. The percentage of innervated end-plates was similar in both groups at 30 as well as at 60 days after nerve crush. Nevertheless, multiple innervation was found in a larger part of the muscle and it lasted longer in the deficient rats. This finding is discussed in relation to some of the major hypotheses of sprouting; it may be relevant in the treatment of some lesions of peripheral nerve.     

Motoneurons innervating facial muscles after hypoglossal and hemihypoglossal-facial nerve anastomosis in rats

Hypoglossal-facial nerve anastomosis (HFA) is the most popular surgical procedure to reinnervate facial muscles after injury of the facial nerve. Section of the hypoglossus causes paralysis and atrophy of the hemi-tongue. In the attempt to overcome this consequence, the hemihypoglossal-facial nerve anastomosis (HHFA) has been proposed and only a half of the main trunk of the hypoglossus is connected to the distal stump of the facial nerve. In the rat, we have studied experimentally the anatomical nuclear changes after HFA and HHFA with the aim of establishing the quantitative motoneuron innervation of facial muscles obtained with each one of the two operative options. Horseradish peroxidase (HRP) injected in both types of anastomosis labeled not only hypoglossal motoneurons, but also facial motoneurons. HFA appeared to offer a significant quantitative motoneuron innervation higher than HHFA and then a higher probable better functional recovery. Both HFA and HHFA performed immediately after section of the facial nerve in rats did not result in a phenomenon of motor hyperinnervation. In our experimental model, the proximal facial nerve stump was coagulated at the stylomastoid foramen to avoid regeneration. Then, the labeled motoneurons into the facial nucleus could really be the expression of axonal projections from facial motoneurons to the hypoglossus nerve and facial muscles. No labeled motoneurons were seen contralaterally as we observed previously after section and repair of several nerves.     

Neuromuscular contacts in the developing rat soleus depend on muscle activity.

Neuromuscular transmission of the rat soleus muscle was interrupted by blocking the response of the postsynaptic membrane with alpha-bungarotoxin (alpha-BTX) at two different stages of postnatal development, i.e., at birth and at 10 days. The effect of this treatment on the maintenance of synaptic contacts was studied using histological and electrophysiological criteria. Following treatment at birth fewer muscle fibres were polyneuronally innervated 5-7 days later. After this initial loss of synaptic contacts, the subsequent rate of synapse elimination was slower than in control animals, so that even at 3 weeks muscles treated with alpha-BTX at birth had higher levels of polyneuronal innervation than their unoperated controls. Thus, interference with the response of the postsynaptic membrane at birth has prolonged effects on synaptic development. In muscles treated with alpha-BTX at 10 days the elimination of polyneuronal innervation was arrested and more neuromuscular contacts preserved.