Emmanouil Skouras

Superior muscle reinnervation after autologous nerve graft or poly-L-lactide-?-caprolactone (PLC) tube implantation in comparison to silicone tube repair

Recovery after peripheral nerve injury depends not only on the amount of reinnervation, but also on its accuracy. The rat sciatic nerve was subjected to an 8 mm long gap lesion repaired either by autograft (AG, n = 6) or tubulization with impermeable silicone tube (SIL, n = 6) or permeable tube of poly‐L‐lactide‐ϵ‐caprolactone (PLC, n = 8). Recordings of the compound muscle action potential (CMAP) from gastrocnemius (mGC), tibialis anterior (mTA) and plantar (mPL) muscles were performed 90 days after injury to assess the amount of muscle reinnervation. The CMAP amplitude achieved in mGC, mTA and mPL was similar in after nerve autograft (39%, 42%, 22% of control values) and PLC tube implantation (37%, 36%, 24%) but lower with SIL tube (29%, 30%, 14%). The nerve fascicles projecting into each of these muscles were then transected and retrograde tracers (Fluoro Gold, Fast Blue, DiI) were applied to quantify the percentage of motoneurons with single or multiple branches to different targets. The total number of labeled motoneurons for the three muscles did not differ in autografted rats (1186 ± 56; mean ± SEM) with respect to controls (1238 ± 82), but was reduced with PLC tube (802 ± 101) and SIL tube (935 ± 213). The percentage of neurons with multiple projections was lower after autograft and PLC tube (6%) than with SIL tube (10%). Considering the higher CMAP amplitude and lower number of neurons with multiple projections, PLC nerve conduits seem superior to SIL tubes and a suitable alternative to autografts for the repair of long gaps. J. Neurosci. Res. 63:214–223, 2001.

Peripheral and spinal motor reorganization after nerve injury and repair.

Functional recovery after peripheral nerve injury depends on the amount as well as on the accuracy of reinnervation by regenerative axons. In this study, the rat sciatic nerve was subjected to crush injury or complete transection repaired by either (1) straight nerve suture, (2) crossed nerve suture of tibial and peroneal fascicles, or (3) silicone tubulization leaving a gap of 4 mm. The compound muscle action potentials (CMAP) of gastrocnemius, tibialis anterior and plantar muscles were recorded 90 days post operation to assess functional reinnervation and Fast Blue, Fluoro Gold and DiI were applied to the nerve branches projecting into these muscles to quantify morphological reinnervation. The CMAP amplitude achieved in gastrocnemius, tibialis anterior and plantar muscles was higher after nerve crush (86%, 82%, 65% of control) than after any surgical nerve repair (straight suture: 49%, 53%, 32%; crossed suture: 56%, 50%, 31%; silicone tube: 42%, 44%, 25%). The total number of labeled motoneurons, however, did not significantly differ between groups (control: 1238 +/- 82, crush: 1048 +/- 49, straight suture: 1175 +/- 106, crossed suture: 1085 +/- 84, silicone tube: 1250 +/- 182). The volume occupied by labeled motoneurons within the spinal cord was larger after surgical nerve repair than in crush or normal control animals, and fewer neurons showed abnormal multiple projections after crush (2.5%) or straight suture (2.2%) than following crossed suture (5%) or silicone tube (6%). In conclusion, nerve repair with a silicone tube leaving a short gap does not increase accuracy of reinnervation.

Contralateral trigeminal nerve lesion reduces polyneuronal muscle innervation after facial nerve repair in rats

Functional recovery after facial nerve surgery is poor. Axotomized motoneurons (hyperexcitable upon intracellular current injections, but unable to discharge upon afferent stimulation) outgrow supernumerary branches which are misrouted towards improper muscles. We hypothesized that alterations in the trigeminal input to axotomized electrophysiologically silent facial motoneurons might improve specificity of reinnervation. To test this we compared, in the rat, behavioural, electrophysiological, and morphological parameters after transection and suture of the buccal facial nerve (buccal–buccal anastomosis, BBA) with those after BBA plus excision of the ipsi‐ or contralateral infraorbital nerve (ION). After BBA, the mystacial vibrissae dropped and remained motionless until 18–21 days post operation (days PO). After BBA plus ipsilateral ION excision, there was no recovery of vibrissae whisking at all. Following BBA plus contralateral ION excision, full restoration of whisking occurred at 7–10 days PO. Electromyography of whiskerpad muscles showed normal waveform and amplitude was also most rapidly restored after BBA plus contralateral ION excision. Neuron counts after retrograde tracing showed that the intact buccal nerve contained axons of the superior (91%) and inferior (9%) buccolabial nerves. After BBA, the superior nerve comprised 56%, the inferior 21%, and 23% of the motoneurons projected within both nerves. After BBA plus ipsilateral ION excision, misdirection worsened and values changed to 48, 39 and 13%, respectively. After BBA plus contralateral ION excision, portions improved to 69, 23 and 8%. We conclude that, by reducing the redundant axon branching, lesion of contralateral ION provides the best conditions for recovery of vibrissae rhythmical whisking after reconstructive surgery on the facial nerve.

Opposite impacts of tenascin-C and tenascin-R deficiency in mice on the functional outcome of facial nerve repair

The glycoproteins tenascin‐C (TNC) and tenascin‐R (TNR) are extracellular matrix proteins involved in the development, plasticity and repair of the nervous system. Altered expression patterns after nerve lesions in adult animals have suggested that these molecules influence axonal regeneration. To test this hypothesis, we investigated adult mice constitutively deficient in the expression of TNC, TNR or both, using the facial nerve injury paradigm. Quantitative analysis of vibrissal movements prior to nerve transection and repair (facial–facial anastomosis) did not reveal genotype‐specific differences, and thus impacts of the mutations on motor function in intact animals. Two months after nerve repair, recovery of vibrissal whisking was poor in wild‐type mice, a typical finding after facial–facial anastomosis in rodents. Differential effects of the mutations on whisking were found: recovery of function was worse in TNC‐deficient and better in TNR null mice compared with wild‐type littermates. In double‐knockout animals, vibrissal performance was insufficient, but to a lesser extent compared with TNC null mutant mice. Retrograde labelling of motoneurons in the same animals showed that similar numbers of motoneurons had reinnervated the whisker pads in all experimental groups precluding varying extents of motoneuron death and/or axon regeneration failures as causes for the different outcomes of nerve repair. Our results provide strong evidence that TNC promotes and TNR impedes recovery after nerve lesion. These findings are of particular interest with regard to the scanty knowledge about factors determining success of regeneration in the peripheral nervous system of mammals.

Electrical stimulation of paralyzed vibrissal muscles reduces endplate reinnervation and does not promote motor recovery after facial nerve repair in rats.

The outcome of peripheral nerve injuries requiring surgical repair is poor. Recent work has suggested that electrical stimulation (ES) of denervated muscles could be beneficial. Here we tested whether ES has a positive influence on functional recovery after injury and surgical repair of the facial nerve. Outcomes at 2 months were compared to animals receiving sham stimulation (SS). Starting on the first day after end-to-end suture (facial-facial anastomosis), electrical stimulation (square 0.1 ms pulses at 5 Hz at an ex tempore established threshold amplitude of between 3.0 and 5.0V) was delivered to the vibrissal muscles for 5 min a day, 3 times a week. Restoration of vibrissal motor performance following ES or SS was evaluated using the video-based motion analysis and correlated with the degree of collateral axonal branching at the lesion site, the number of motor endplates in the target musculature and the quality of their reinnervation, i.e. the degree of mono- versus poly-innervation. Neither protocol reduced collateral branching. ES did not improve functional outcome, but rather reduced the number of innervated motor endplates to approximately one-fifth of normal values and failed to reduce the proportion of poly-innervated motor endplates. We conclude that ES is not beneficial for recovery of whisker function after facial nerve repair in rats.

Manually-stimulated recovery of motor function after facial nerve injury requires intact sensory input

We have recently shown in rat that daily manual stimulation (MS) of vibrissal muscles promotes recovery of whisking and reduces polyinnervation of muscle fibers following repair of the facial nerve (facial-facial anastomosis, FFA). Here, we examined whether these positive effects were: (1) correlated with alterations of the afferent connections of regenerated facial motoneurons, and (2) whether they were achieved by enhanced sensory input through the intact trigeminal nerve. First, we quantified the extent of total synaptic input to motoneurons in the facial nucleus using synaptophysin immunocytochemistry following FFA with and without subsequent MS. We found that, without MS, this input was reduced compared to intact animals. The number of synaptophysin-positive terminals returned to normal values following MS. Thus, MS appears to counteract the deafferentation of regenerated facial motoneurons. Second, we performed FFA and, in addition, eliminated the trigeminal sensory input to facial motoneurons by extirpation of the ipsilateral infraorbital nerve (IONex). In this paradigm, without MS, vibrissal motor performance and pattern of end-plate reinnervation were as aberrant as after FFA without MS. MS did not influence the reinnervation pattern after IONex and functional recovery was even worse than after IONex without MS. Thus, when the sensory system is intact, MS restores normal vibrissal function and reduces the degree of polyinnervation. When afferent inputs are abolished, these effects are eliminated or even reversed. We conclude that rehabilitation strategies must be carefully designed to take into account the extent of motor and/or sensory damage.

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.

Manual stimulation, but not acute electrical stimulation prior to reconstructive surgery, improves functional recovery after facial nerve injury in rats.

UNLABELLED The outcome of peripheral nerve injuries requiring surgical repair is poor. Recent work suggested that electrical stimulation (ES) of the proximal nerve stump to produce repeated discharges of the parent motoneurons for one hour could be a beneficial therapy if delivered immediately prior to reconstructive surgery of mixed peripheral nerves. PURPOSE We tested whether ES has a positive influence on functional recovery after repair of a purely motor nerve, the facial nerve. METHODS Electrical stimulation (20 Hz) was delivered to the proximal nerve stump of the transected facial nerve for 1 hour prior to nerve reconstruction by end-to-end suture (facial-facial anastomosis, FFA). For manual stimulation (MS), animals received daily rhythmic stroking of the whisker pads. Restoration of vibrissal motor performance following ES or MS was evaluated using video-based motion analysis. We also assessed the degree of collateral axonal branching at the lesion site, by counting motoneuronal perikarya after triple retrograde labeling, and estimated the quality of motor end-plate reinnervation in the target musculature. Outcomes at 4 months were compared to animals receiving sham stimulation (SS) or MS. RESULTS Neither protocol reduced the degree of collateral sprouting. ES did not improve functional outcome and failed to reduce the proportion of polyinnervated motor end-plates. By contrast, MS restored normal whisking function and reduced polyinnervation. CONCLUSION Whereas acute ES is not beneficial for facial nerve repair, MS provides long-term benefits.

Manual stimulation of the orbicularis oculi muscle improves eyelid closure after facial nerve injury in adult rats

We have shown that manual stimulation of rat whisker‐pad muscles following facial–facial‐anastomosis (FFA) restores normal whisking by lowering the proportion of polyinnervated motor endplates. Here we examined whether manual stimulation of the orbicularis oculi muscle (OOM) after FFA would also improve outcome. Blink responses to standardized air puffs were analyzed using video‐based motion analysis. Two months after FFA, blink capacity was impaired, as indicated by a largely increased minimum distance between the eyelids after air‐puff stimulation compared with intact rats (2.7 ± 0.4 vs. 0.2 ± 0.01 mm). Manual stimulation reduced this deficit by a factor of two (1.3 ± 0.5 mm). The functional improvement after manual stimulation was associated with a 2‐fold decrease in the proportion of polyinnervated OOM endplates (21 ± 10% vs. 42 ± 10% without manual stimulation, 0% in intact rats). We conclude that manual stimulation is a noninvasive and simple procedure with immediate potential for clinical rehabilitation of eyelid closure following facial nerve injury. Muscle Nerve, 2008

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.