Manual stimulation of the whisker pad after hypoglossal-facial anastomosis (HFA) using a Y-tube conduit does not improve recovery of whisking function.

Facial nerve injury is a common clinical trauma involving long-term functional deficits with facial asymmetry leading to associated psychological issues and social hardship. We have recently shown that repair by hypoglossal-facial or facial-facial nerve surgical end-to-end anastomosis and suture [hypoglossal-facial anastomosis (HFA) or facial-facial anastomosis (FFA)] results in collateral axonal branching, polyinnervation of neuromuscular junctions (NMJs) and poor function. We have also shown that another HFA repair procedure using an isogenic Y-tube (HFA + Y-tube) and involving a 10-mm gap reduces collateral axonal branching, but fails to reduce polyinnervation. Furthermore, we have previously demonstrated that manual stimulation (MS) of facial muscles after FFA or HFA reduces polyinnervation of NMJs and improves functional recovery. Here, we examined whether HFA + Y-tube and MS of the vibrissal muscles reduce polyinnervation and restore function. Isogenic Y-tubes were created using abdominal aortas. The proximal hypoglossal nerve was inserted into the long arm and sutured to its wall. The distal zygomatic and buccal facial nerve branches were inserted into the two short arms and likewise sutured to their walls. Manual stimulation involved gentle stroking of the vibrissal muscles by hand mimicking normal whisker movement. We evaluated vibrissal motor performance using video-based motion analysis, degree of collateral axonal branching using double retrograde labeling and the quality of NMJ reinnervation in target musculature using immunohistochemistry. MS after HFA + Y-tube reduced neither collateral branching, nor NMJ polyinnervation. Accordingly, it did not improve recovery of function. We conclude that application of MS after hypoglossal-facial nerve repair using an isogenic Y-tube is contraindicated: it does not lead to functional recovery but, rather, worsens it.

Whole-body vibration improves functional recovery in spinal cord injured rats.

Whole-body vibration (WBV) is a relatively novel form of exercise used to improve neuromuscular performance in healthy individuals. Its usefulness as a therapy for patients with neurological disorders, in particular spinal cord injury (SCI), has received little attention in clinical settings and, surprisingly, even less in animal SCI models. We performed severe compression SCI at a low-thoracic level in Wistar rats followed by daily WBV starting 7 (10 rats) or 14 (10 rats) days after injury (WBV7 and WBV14, respectively) and continued over a 12-week post-injury period. Rats with SCI but no WBV training (sham, 10 rats) and intact animals (10 rats) served as controls. Compared to sham-treated rats, WBV did not improve BBB score, plantar stepping, or ladder stepping during the 12-week period. Accordingly, WBV did not significantly alter plantar H-reflex, lesion volume, serotonergic input to the lumbar spinal cord, nor cholinergic or glutamatergic inputs to lumbar motoneurons at 12 weeks after SCI. However, compared to sham, WBV14, but not WBV7, significantly improved body weight support (rump-height index) during overground locomotion and overall recovery between 6-12 weeks and also restored the density of synaptic terminals in the lumbar spinal cord at 12 weeks. Most remarkably, WBV14 led to a significant improvement of bladder function at 6-12 weeks after injury. These findings provide the first evidence for functional benefits of WBV in an animal SCI model and warrant further preclinical investigations to determine mechanisms underpinning this noninvasive, inexpensive, and easily delivered potential rehabilitation therapy for SCI.

Use of a Y-tube conduit after facial nerve injury reduces collateral axonal branching at the lesion site but neither reduces polyinnervation of motor endplates nor improves functional recovery.

BACKGROUND: Despite increased understanding of peripheral nerve regeneration, functional recovery after surgical repair remains disappointing. A major contributing factor is the extensive collateral branching at the lesion site, which leads to inaccurate axonal navigation and aberrant reinnervation of targets. OBJECTIVE: To determine whether the Y tube reconstruction improved axonal regrowth and whether this was associated with improved function. METHODS: We used a Y-tube conduit with the aim of improving navigation of regenerating axons after facial nerve transection in rats. RESULTS: Retrograde labeling from the zygomatic and buccal branches showed a halving in the number of double-labeled facial motor neurons (15% vs 8%; P < .05) after Y tube reconstruction compared with facial-facial anastomosis coaptation. However, in both surgical groups, the proportion of polyinnervated motor endplates was similar (≈ 30%; P > .05), and video-based motion analysis of whisking revealed similarly poor function. CONCLUSION: Although Y-tube reconstruction decreases axonal branching at the lesion site and improves axonal navigation compared with facial-facial anastomosis coaptation, it fails to promote monoinnervation of motor endplates and confers no functional benefit.

Hypoglossal-facial anastomosis (HFA) over a 10 mm gap bridged by a Y-tube-conduit enhances neurite regrowth and reduces collateral axonal branching at the lesion site.

PURPOSE: The outcome of severe peripheral nerve injuries requiring surgical repair (transection and suture) is usually poor. Recent work suggests that direct suture of nerves increases collagen production and provides unfavourable conditions for a proper axonal regrowth. We tested whether entubulation of the hypoglossal nerve into a Y-tube conduit connecting it with the zygomatic and buccal facial nerve branches would improve axonal pathfinding at the lesion site, quality of muscle reinnervation and recovery of vibrissal whisking. METHODS: For hypoglossal-facial anastomosis (HFA) over a Y-tube (HFA-Y-tube) the proximal stump of the hypoglossal nerve was entubulated and sutured into the long arm of a Y-tube (isogeneic abdominal aorta with its bifurcation). The zygomatic and buccal facial branches were entubulated and sutured to the short arms of the Y-tube. Restoration of vibrissal motor performance, degree of collateral axonal branching at the lesion site and quality of neuro-muscular junction (NMJ) reinnervation were compared to animals receiving HFA-Coaptation (no entubulation) after 4 months. RESULTS: HFA-Y-tube reduced collateral axonal branching. However it failed to reduce the proportion of polyinnervated NMJ and did not improve functional outcome when compared to HFA-Coaptation. CONCLUSION: Elimination of compression by tightly opposed nerve fragments improved axonal pathfinding. However, biometric analysis of vibrissae movements did not show positive effects suggesting that polyneuronal reinnervation - rather than collateral branching - may be the critical limiting factor. Since polyinnervation of muscle fibers is activity-dependent and can be manipulated, the present findings raise hopes that clinically feasible and effective therapies after HFA could be soon designed and tested.

Objective measures of motor dysfunction after compression spinal cord injury in adult rats: correlations with locomotor rating scores.

Precise assessment of motor deficits after traumatic spinal cord injury (SCI) in rodents is crucial for understanding the mechanisms of functional recovery and testing therapeutic approaches. Here we analyzed the applicability to a rat SCI model of an objective approach, the single-frame motion analysis, created and used for functional analysis in mice. Adult female Wistar rats were subjected to graded compression of the spinal cord. Recovery of locomotion was analyzed using video recordings of beam walking and inclined ladder climbing. Three out of four parameters used in mice appeared suitable: the foot-stepping angle (FSA) and the rump-height index (RHI), measured during beam walking, and for estimating paw placement and body weight support, respectively, and the number of correct ladder steps (CLS), assessing skilled limb movements. These parameters, similar to the Basso, Beattie, and Bresnahan (BBB) locomotor rating scores, correlated with lesion volume and showed significant differences between moderately and severely injured rats at 1-9 weeks after SCI. The beam parameters, but not CLS, correlated well with the BBB scores within ranges of poor and good locomotor abilities. FSA co-varied with RHI only in the severely impaired rats, while RHI and CLS were barely correlated. Our findings suggest that the numerical parameters estimate, as intended by design, predominantly different aspects of locomotion. The use of these objective measures combined with BBB rating provides a time- and cost-efficient opportunity for versatile and reliable functional evaluations in both severely and moderately impaired rats, combining clinical assessment with precise numerical measures.

Schwann cells overexpressing FGF-2 alone or combined with manual stimulation do not promote functional recovery after facial nerve injury.

PURPOSE: To determine whether transplantation of Schwann cells (SCs) overexpressing different isoforms of fibroblast growth factor 2 (FGF-2) combined with manual stimulation (MS) of vibrissal muscles improves recovery after facial nerve transection in adult rat. PROCEDURES: Transected facial nerves were entubulated with collagen alone or collagen plus naïve SCs or transfected SCs. Half of the rats received daily MS. Collateral branching was quantified from motoneuron counts after retrograde labeling from 3 facial nerve branches. Quality assessment of endplate reinnervation was combined with video-based vibrissal function analysis. RESULTS: There was no difference in the extent of collateral axonal branching. The proportion of polyinnervated motor endplates for either naïve SCs or FGF-2 over-expressing SCs was identical. Postoperative MS also failed to improve recovery. CONCLUSIONS: Neither FGF-2 isoform changed the extent of collateral branching or polyinnervation of motor endplates; furthermore, this motoneuron response could not be overridden by MS.

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.

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.

Manual stimulation of the suprahyoid-sublingual region diminishes polynnervation of the motor endplates and improves recovery of function after hypoglossal nerve injury in rats.

BACKGROUND: Using the rat facial nerve axotomy model, the authors recently showed that manual stimulation of denervated whiskerpad muscles reduced the posttransectional polyinnervation at the neuromuscular junctions and promoted full recovery of vibrissal whisking. OBJECTIVE: Prompted by implications for rehabilitation therapy, the authors examined whether manual stimulation of denervated supra- and infrahyoid muscles would also improve recovery after unilateral lesion on the hypoglossal nerve. METHODS: Adult rats underwent transection of the right hypoglossal nerve. Half of the animals received no postoperative treatment, and the other half were subjected to daily manual stimulation of the suprahyoid/sublingual region for 2 months. Recovery was assessed by measuring the angle of tongue-tip deviation from the midline, degree of collateral axonal branching at the lesion site (counts after retrograde labeling with 2 fluorescent dyes), synaptic input to the hypoglossal motoneurons using synaptophysin immunocytochemistry, tongue-muscles motor representation in the cerebral cortex after c-Fos immunocytochemistry, and portion of polyinnervated neuromuscular junctions. RESULTS: In animals receiving manual stimulation, the tongue-tip deviation was 37.0 +/- 49.37 degrees , whereas values in control nonstimulated rats were significantly higher (50.1 +/- 9.01 degrees ; P < .05; mean +/- SD). Improved recovery was not associated with reduced collateral axonal branching; there were also no differences in tongue-muscles representation in the motor cortex. However, manual stimulation restored the total synaptic input to levels in intact animals and reduced the proportion of polyinnervated neuromuscular junctions compared with nonstimulated animals. CONCLUSION: The data show that manual stimulation of denervated muscles improves functional outcome following peripheral nerve injury. This suggests immediate potential for enhancing clinical rehabilitation strategies.

Bone marrow-derived mesenchymal stem cell transplantation does not improve quality of muscle reinnervation or recovery of motor function after facial nerve transection in rats.

Recently, we devised and validated a novel strategy in rats to improve the outcome of facial nerve reconstruction by daily manual stimulation of the target muscles. The treatment resulted in full recovery of facial movements (whisking), which was achieved by reducing the proportion of pathologically polyinnervated motor endplates. Here, we posed whether manual stimulation could also be beneficial after a surgical procedure potentially useful for treatment of large peripheral nerve defects, i.e., entubulation of the transected facial nerve in a conduit filled with suspension of isogeneic bone marrow-derived mesenchymal stem cells (BM-MSCs) in collagen. Compared to control treatment with collagen only, entubulation with BM-MSCs failed to decrease the extent of collateral axonal branching at the lesion site and did not improve functional recovery. Post-operative manual stimulation of vibrissal muscles also failed to promote a better recovery following entubulation with BM-MSCs. We suggest that BM-MSCs promote excessive trophic support for regenerating axons which, in turn, results in excessive collateral branching at the lesion site and extensive polyinnervation of the motor endplates. Furthermore, such deleterious effects cannot be overridden by manual stimulation. We conclude that entubulation with BM-MSCs is not beneficial for facial nerve repair.

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.

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.

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.

Improved outcome of facial nerve repair in rats is associated with enhanced regenerative response of motoneurons and augmented neocortical plasticity.

Within a recent study on the vibrissae motor performance after facial nerve repair in strains of blind (SD/RCS) and sighted (SD) rats we found that, despite persisting myotopic disorganization in the facial nucleus, the blind animals fully restored vibrissal whisking. Here we searched for morphological substrates of better recovery in the regenerating motoneurons and in the cerebral motor cortex. Expression analyses of the neurite growth-related proteins f-actin, neuronal class III beta-tubulin and plasticity-related gene-1, and stereological estimates of growth cone densities revealed a more vigorous regenerative response in the proximal nerve stump of blind SD/RCS rats compared with SD animals at 5-7 days after buccal nerve transection. Using c-Fos immunoreactivity as a marker for neuronal activation, we found that the volume of the cortex acutely responding to nerve transection (facial muscles reactive volume, FMRV) in both hemispheres of intact sighted rats was twofold smaller than that measured in blind animals. One month after transection and suture of the right facial nerve (FFA) we found a twofold increase in the FMRV in both rat strains compared with intact animals. The FMRV in SD/RCS animals, but not in SD rats, returned to the values in intact rats 2 months after FFA. Our findings suggest that enhanced plasticity in the CNS and an augmented regenerative response of the injured motoneurons contribute to better functional recovery in blind rats.