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Experimental Neurotisation of the Brachial Plexus by Means of Nerve Grafting from the Corticospinal Tract of the Proximal Spinal Cord at the T3–T4 LevelFondazione Midollo Spinale, Brescia, Italy Correspondence: Prof. Giorgio A. Brunelli, Fondazione Midollo Spinale, Via Galvani 26, 25123 Brescia, Italy. E-mail:giorgio.brunelli{at}midollospinale.com
This paper reports experimental work in rats in which the radial nerve was cut at its origin from the brachial plexus and connected by means of a nerve graft to the corticospinal tract of the spinal cord at the T3–T4 level. After surgery, the triceps and extensor muscles of the forearm were able to extend the elbow and the wrist and gave almost normal responses on EMG. This reinnervation was no longer cholinergic, as demonstrated by the Vecuronium test. Because of the drawbacks associated with implantation into the spinal cord, this technique could not be used in all cases of total avulsion of the brachial plexus but it might be useful in those rare cases in which avulsion of all the cervical nerves of the brachial plexus is associated with paraplegia: such cases are rare but are occasionally seen.
Key Words: spinal cord • brachial plexus avulsions • neurotisation • nerve grafting • corticospinal tract Traumatic brachial plexus lesions with avulsion of all the roots of the cervical nerves cannot be repaired by means of classical nerve suture or grafting. Extraplexal neurotisations can be done using various nerves, including the eleventh cranial nerve, the intercostal nerves, the anterior nerves of the cervical plexus, the seventh contralateral cervical nerve and others (Brunelli, 1980; Merle and Lim, 2001). However, the results of these procedures are, in general, fair or poor, even if they achieve some improvement relative to the pre-operative situation. They also leave functional defects of the donor nerves of varying degrees of significance. Over the last 27 years, we have carried out experimental and clinical neurotisations of the lower limbs after spinal cord lesions by means of grafting from the corticospinal tract of the cord (Brunelli, 1980; Brunelli and Brunelli, 1996; Brunelli et al., 1996) with good results. These results have been confirmed by other workers (Bertelli et al., 1994; Horvat et al., 1989) and by more recent studies by a multidisciplinary team in the University of Brescia (Brunelli et al., 2005; Pizzi et al., 2006). Based on this experience of grafting from upper motor neurones in paraplegics to reinnervate muscles of the lower limb, this paper reports the results of a study carried out in rats in which an attempt was made to achieve reinnervation of the brachial plexus by grafting from axons of upper motor neurones of the corticospinal tract of the spinal cord.
Fifteen adult male albino Sprague Dawley rats of weight 250–275 g underwent surgery between November 2006 and February 2007. Anaesthesia included an initial dose of Carprofen (8 mg/kg) as an analgesic, then administration of intramuscular Tiletamine (16 mg/kg) and Zolazepan (16 mg/kg). All experimental and surgical procedures conformed to the National Research Guide for the Care and Use of Laboratory Animals and were approved by the Animal Research Committees of the University of Brescia.
Surgical procedure
A laminectomy at the T3/T4 level was carried out and the other end of the nerve graft was inserted into the posterior bundle of the spinal cord, which, in rats, is the equivalent of the human corticospinal tract. The insertion was carried out by means of a 2 mm long longitudinal incision of the dura mater, through which the proximal end of the graft was introduced for 2 mm. The epineurium of the graft was then sutured to the dura mater. Following this surgery, the muscles originally innervated by the radial nerve could receive only axons from the upper presynaptic neurones. The surviving rats were examined, both from the clinical point of view and by EMG testing, 4 to 5 months after operation.
Clinical assessment The rats which had shown an extensor palsy of the radial nerve after operation and then regained extension of the wrist after 4 to 5 months were anaesthetised to allow electrical stimulation of the radial nerve at the axillary and arm levels in order to record the degree of recovery of the muscles innervated by the radial nerve.
Electrophysiological assessment The EMG response was then recorded after administration of IV Vecuronium Bromide (800 mg/kg), injected into the long saphenous vein in the groin. Vecuronium Bromide is a neuromuscular blocking agent which blocks Acetylcholine receptors at the neuromuscular junctions, so paralysing the cholinergic excitation of normal skeletal muscles. Because it will cause death from paralysis of the respiratory muscles, tracheotomy and artificial ventilation are necessary before it is administered. The Vecuronium test was used to identify whether the reinnervation of muscles was cholinergic or glutamatergic. Then GYKI 52466, a glutamate AMPA receptor antagonist, was administered intraperitoneally and the EMG responses recorded again to identify whether GYKI 52466 paralysed the muscles, supposedly now innervated by upper motor neurones. This was followed by more sophisticated analyses, including examination of compound action potentials, ChAT immunoreactivity, VGluT1 and VGlutT2 expression in control and grafted nerves, immunoblot analysis of ChAT and Glu receptors, AMPA receptors in reinnervated muscles and CTB retrograde tracing of upper neurones to confirm that the neuromuscular junctions of the reinnervated muscles had changed their receptors from cholinergic to glutamatergic (Brunelli et al., 2005; Pizzi et al., 2006).
Complications
Fifteen rats underwent surgery using this protocol. Eight (53%) survived. Three of the eight surviving rats showed no recovery of the flexion contracture of the foreleg digits and were not investigated further. The other five rats recovered from the flexion contracture of the foreleg digits (Fig 3). After 4 to 5 months, these five rats were re-anaesthetised and underwent electrical stimulation of the radial nerve at the axillary and arm levels. All five exhibited good responses of the reinnervated muscles with extension of the elbow and the wrist (Fig 4).
In two out of these five rats, the reinnervated muscles (along with all other muscles) were paralysed after administration of Vecuronium Bromide (Fig 5).
The other three rats showed persistence of function of the reinnervated muscles after administration of Vecuronium, indicating a glutamatergic reinnervation of the tested muscles innervated by the radial nerve (Fig 6). When GYKI 52466 was administered to the three rats which showed persistence of function of the reinnervated muscles after administration of Vecuronium, the reinnervated muscles were paralysed (Fig 7). The more sophisticated analyses confirmed that the neuromuscular junctions of the reinnervated muscles had changed their receptors from cholinergic to glutamatergic.
Two of the eight surviving rats, exhibited motor and sensory impairment to varying degrees of the ipselateral hindleg and one exhibited motor and sensory impairment of both hindlegs.
We have reported experimental and clinical neurotisations of the lower limbs after spinal cord lesions by means of grafting from the corticospinal tract of the cord (Brunelli, 1980; Brunelli and Brunelli, 1996; Brunelli et al., 1996) with good results. These results have been confirmed by other researchers (Bertelli et al., 1994; Horvat et al., 1989) and further by more recent studies by a multidisciplinary team in the University of Brescia (Brunelli et al., 2005; Pizzi et al., 2006). In this research, we grafted from the lateral bundle of the spinal cord to nerves innervating peripheral muscles of the lower limb using autologous nerve grafts. Clinical observation, as well as EMG studies and action potentials after magnetic stimulation of the brain (after craniotomy), demonstrated functional neurotisation of the reinnervated muscles by the upper motor neurones. This is a preliminary report describing the results of connecting the distal parts of the rat radial nerve to the corticospinal tract in the dorsal bundle of the spinal cord at the T3–T4 level. The operating shock sustained by opening the cord at the T3–T4 level is severe and maintaining life in rats after such a procedure is difficult. Nevertheless, eight rats survived to provide useful information. This microsurgical procedure led to extensor muscle reinnervation and recovery in the ipselateral upper limb in five of the eight rats which survived. This motor activity was insensitive to Vecuronium blockade of cholinergic transmission in three of these five rats and sensitive to Vecuronium blockade in two rats. The muscles of the three rats which were sensitive to Vecuronium were, on the contrary, paralysed by GYKI 52466. Upper motor neurones use the neurotransmitter Glutamate and peripheral muscles normally respond to the neurotransmitter Acetylcholine. Although more detailed histological and molecular analysis are required to demonstrate the neurotransmitter involved, the previous research detailed above would suggest that the muscle reinnervation in the three rats in which Vecuronium failed to block transmission was due to nerve fibres originating from glutamatergic upper neurones of the pyramidal tract or rubrospinal tract. The fact that two of the five rats developed palsy of the reinnervated forefeet after Vecuronium administration is interpreted as being due to persistent cholinergic innervation, probably depending on contact of the graft with the grey matter. In fact, proximal re-connection of the grafts cannot always be perfect because of difficulty of positioning the end of the graft within the spinal cord. As a consequence, the grafts may make contact with the grey, instead of the white, matter, resulting in cholinergic reinnervation. In animals, it is not possible to see if the voluntary movements obtained are selective. However, our experience in one paraplegic patient in whom connection of the proximal central nervous system to the peripheral nervous system allowed her to walk and to selectively activate one, or other, of the muscles connected to the corticospinal tract without disturbing co-contractions (Brunelli et al., 2005), would suggest that, in humans, an operation of this kind might give at least a moderate recovery of function of the upper limb. This is, of course, major surgery which might cause problems for the lower limb. As envisaged from the above experiment, without refinement, it could not be done in patients with isolated brachial plexus lesions. In rarer cases of total avulsion of the brachial plexus in paraplegics, concerns about interfering with the function of the lower limb would not, of course, exist. Also, in cases of bilateral brachial plexus lesions with total loss of function of both the upper limbs, this operation might be acceptable, even at risk of some disabling side-effects in the lower limbs. Needless to say, this is still an experimental procedure with a doubtful prognosis at this stage.
Manuscript received July 3, 2007. Accepted for publication August 24, 2007.
Bertelli JA, Orsal D, Mira JC (1994). Median nerve neurotization by peripheral nerve grafts directly implanted into the spinal cord: anatomical, behavioural and electrophysiological evidences of sensorimotor recovery. Brain Research, 644: 150–159.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]Brunelli GA (1980). Neurotisation of avulsed roots of the brachial plexus by means of anterior nerves of the cervical plexus. Journal of Microsurgery, 2: 55–58.Brunelli GA, Brunelli GR (1996). Experimental surgery in spinal cord lesions by connecting upper motoneurons directly to peripheral targets. Journal of the Peripheral Nervous System, 1: 111–118.[Medline] [Order article via Infotrieve]Brunelli GA, Brunelli GR, Mattiuzzo V et al. Spinal cord experimental repair: CNS PNS grafting gives functional connection, Brescia, Gruppo editoriale Delfo, 1996.Brunelli GA, Spano PF, Barlati S et al (2005). Glutamatergic reinnervation through peripheral nerve graft dictates assembly of glutamatergic synapses at rat skeletal muscle. Proceeding of the National Academy of Science, 102: 8752–8757.[CrossRef]Horvat JC, Pécot-Dechavassine M, Mira JC et al (1989). Formation of functional endplates by spinal axons regeneration through a peripheral nerve graft. A study in the adult rat. Brain Research Bulletin, 22: 103–114.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]Merle M, Lim A. Surgical techniques: neurolysis, sutures, grafts, neurotisations. In: Gilbert A (Ed) Brachial plexus injuries, London, Martin Dunitz, 2001: 15–110.Pizzi M, Brunelli G, Barlati S et al (2006). Glutamatergic innervation of rat skeletal muscle by supraspinal neurons: a new paradigm in spinal cord injury repair. Current Opinion in Neurobiology, 16: 323–328.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
Journal of Hand Surgery (European Volume), Vol. 32, No. 6,
620-625 (2007)
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Abstract
MATERIALS AND METHODS
