Facial Nerve Repair by Muscle-Vein Conduit in Rats: Functional Recovery and Muscle Reinnervation

Abstract

The facial nerve is the most frequently damaged nerve in head and neck traumata. Repair of interrupted nerves is generally reinforced by fine microsurgical techniques; nevertheless, regaining all functions is the exception rather than the rule. The so-called “postparalytic syndrome,” which includes synkinesia and altered blink reflexes, follows nerve injury. The purpose of this study was to examine if nerve-gap repair using an autologous vein filled with skeletal muscle would improve axonal regeneration, reduce neuromuscular junction polyinnervation, and improve the recovery of whisking in rats with transected and sutured right buccal branches of the facial nerve. Vibrissal motor performance was studied with the use of a video motion analysis. Immunofluorescence was used to visualize and analyze target muscle reinnervation. The results taken together indicate a positive effect of muscle-vein-combined conduit (MVCC) on the improvement of the whisking function after reparation of the facial nerve in rats. The findings support the recent suggestion that a venal graft with implantation of a trophic source, such as autologous denervated skeletal muscle, may promote the monoinnervation degree and ameliorate coordinated function of the corresponding muscles.

Impact statement

This is the first attempt for facial nerve repair with muscle-vein-combined conduit. The findings indicate that a venal graft with implantation of a trophic source, such as autologous denervated skeletal muscle, may promote the monoinnervation degree and improve coordinated activity of the corresponding muscles.

Introduction

Facial nerve is the nerve damaged more often after injuries of the head or neck. Facial nerve lesions may occur during car accidents causing fractures of the temporal bone or face traumata. Most facial nerve injuries, however, occur after surgical operations and constitute a complication of parotidectomy, vestibular schwannoma surgery, or petrous bone surgery. After a peripheral nerve is damaged, there is consistently a local effort of the traumatized axons to regenerate,¹ a process that involves a wide spectrum of reactions.²

Repair of interrupted nerves is usually reinforced by fine microsurgical techniques. Nevertheless, functional recovery is rarely fully succeeded and regaining of facial expressions and of function of all 42 facial muscles remains poor.^3–5 The so-called “postparalytic syndrome,” which includes synkinesia and modified blink reflexes,^6–8 follows nerve injury. The so-called “misdirected” reinnervation,^9,10 the transmission of abnormally intensive stimuli from one axon to another,¹¹ and the alterations in synaptic input of the facial motoneurons^12–15 are the main parameters responsible for the syndrome.

After the nerve is cut, both ends are pulled back and create a gap that is often surgically bridged by nerve grafts.¹⁶ Blood vessels and striated muscles are the conduits that received the highest interest from study groups. A milestone for their use was the demonstration in the 1980s that vein grafts carrying injured nerves resulted in similar effects to autologous nerve grafting.^17–21

Striated muscle autografts used to restore nerve damage were documented a long time ago,²² but were investigated more thoroughly in the beginning of 1980s.^23–25 The thought to employ muscular tissue for axonal regrowth was based on the resemblance between the basal lamina (BL) around muscles cells and the BL outside the endoneurial tubes.²³ Although the effectiveness of both autologous vein and muscle conduits has been separately demonstrated both in animal models and human patients,^26–33 their positive outcome is usually related only to restoration of small nerve gaps.³⁴ To enable neural entubulation over longer distances, a conduit made out of venous pieces filled with striated muscular tissue has been previously described.^35–37 Another rationale for this approach was that vein collapse can be impeded by the muscle content, while axon dispersion can be hindered by the vein wall.³⁷ Interestingly, the use of fresh muscle is especially effective when combined with vein conduits, whereas its effectiveness is limited when used with synthetic conduits.^38,39

In our previous studies, we attempted to ameliorate axonal navigation using surgical reconstruction with an isogeneic aortic Y-tube and with the use of an aortic three-way guide. Both techniques reduced collateral branching of the axons at the lesion site; nevertheless, polyinnervation at the neuromuscular junction (NMJ) remained high and promoted poor vibrissal motor function (whisking).^15,40,41

The purpose of this study was to investigate whether a nerve-gap repair using an autologous vein filled with skeletal muscle would improve axonal regeneration, reduce neuromuscular junction (NMJ-) polyinnervation, and improve the recovery of whisking. Although similar facial nerve reconstructions have been tested before by methods such as fascicular tubulization,⁴² synthetic conduits,⁴³ or venous ensheathment,⁴⁴ this is the first attempt for facial nerve repair with muscle-vein-combined conduit (MVCC).

Materials and Methods

Animals and experimental design

A total of 40 adult, female Wistar rats (175–200 g; strain HsdCpb:WU; Harlan Winkelmann, Borchen, Germany) were used. We used only female rats because testosterone has been shown to beneficially affect peripheral nerve regeneration.⁴⁵ In addition to that, doing this since 1990 provides us the possibility to compare data among different experimental sets. All rats were provided with tap water and were fed with common food (Sniff, Soest, Germany) ad libitum. A 12-h light cycle with lights on, followed a 12-h dark cycle, with lights off. The procedures were all approved by the animal care committee (Approval number 2133/2.27.201) and were performed according to the German Law for Animal Protection. The recommendations and regulations of the National Institute of Health about animal care were thoroughly followed.

The rats were separated into 4 groups, each of 10 rats. Group 1 consisted of intact (not surgically treated) rats. In animals from group 2, the right buccal branch of the facial nerve (BBFN) was transected and sutured (buccal-buccal anastomosis, BBA). In group 3, both ends of the transected BBFN were inserted into a fragment of an empty autologous external jugular vein, measuring 1 cm. In group 4, the vein conduit contained additionally a small piece of muscle, more specifically, the autologous pectoralis major, thus creating an MVCC.

All surgical processes were conducted with the help of a microscope, after the animals were intraperitoneally injected with Ketamin/Xylazin (100 mg Ketanest and 5 mg Rompun per kg body weight) as described in previous work.^46,47

A postoperative interval of 4 weeks was chosen to evaluate vibrissal function of all animals. This was the time after surgery, where the behavioral recordings began to demonstrate a limited restitution of the rhythmical whisking in some animals. Thereafter, we documented the type of motor end plate reinnervation in the levator labii superioris (LLS) muscle, which is the biggest vibrissal muscle.

Surgery for BBA in the rats of group 2

All operations were performed by a trained microsurgeon (Prof. Dr. Nektarios Sinis), under an operating microscope.

In group 2, BBFN was uncovered, cut, and directly stitched with two sutures, type 11-0, epineurally and atraumatically (Ethicon, Braunschweig, Germany). In groups 2–4, the study emphasized on the precision reinnervation after transection by the BBFN; therefore, the aim was to remove possible extra innervation by the marginal mandibular branch.⁴⁸ This is the reason why the latter was transected, followed by proximal ligature, to prevent its regeneration when performing BBA (Fig. 1).

FIG. 1.

Schematic drawing illustrating the sites of transection and suture in the buccal branch (BBA) and of the transection and ligature of the marginal mandibular branch of the facial nerve. The cervical branch of the facial nerve is indicated by a dotted line. BBA, buccal-buccal anastomosis. Adapted from Dörfl⁵⁴ and Semba and Egger.⁴⁸

Surgery: entubulation of the transected BBFN

The right BBFN was transected and the two stumps, proximal and distal, were placed into an empty conduit that had been obtained from an autologous external jugular vein (Fig. 2). To avoid tension, the two nerve ends were attached to the vein wall with two epineural sutures, keeping a distance of about 5–8 mm. Previous studies demonstrate that cut axons placed in artificial nerve conduits in rats are capable to bridge gaps that measure up to 10 mm.⁴⁹ Phosphate-buffered saline 0.1 M at pH 7.4 filled the area between the two nerve stumps in group 3, while a piece of the ipsilateral m. pectoralis major was added in group 4 (Fig. 3).

FIG. 2.

Schematic drawing of the infratemporal portion of the rat facial nerve indicating the entubulation site of BBFN. BBFN, buccal branch of the facial nerve. Adopted from Angelov et al.¹¹²

FIG. 3.

Schematic drawing illustrating the external jugular vein of a rat (A) and its preparation (B). Following dissection, the vein was rinsed and a matching piece of the ipsilateral pectoralis major muscle was prepared (C). The BBFN was exposed (D) and transected, and its proximal stump entubulated in the empty vein conduit (E). The pectoralis-major specimen was inserted through the distal end of the conduit and the distal stump of the BBFN sutured to the vein (F). Color images are available online.

Analysis of motor performance

Analysis of the movement of vibrissae was performed with a Video, a procedure that has been conducted many times in previous experimental studies and is very familiar to our research group.^41,50–53

Under light anesthesia, on both sides of the face, all vibrissal hairs were cut off, apart from two big ones in row C. We employed a digital camera (Panasonic NV DX-110 EG) and animals were recorded for 3–5 min during active exploration.

We reviewed captured video sequences and 1.5 s sequence fragments from every animal were chosen for examination of whisking biometrics. Selection criteria included the stable position of the animal's head, where whisking frequency and vibrissae protraction degree were clearly documented. The tip of the rat's nose and the inner angles of both eyes were defined as reference points. We evaluated the following: (1) protraction, (2) the whisking frequency, (3) the amplitude, (4) the angular velocity during protraction, and (5) the angular acceleration during protraction. The angle between the midsagittal plane and the hair shaft is a rostrally opening angle, showing the forward movement of the vibrissae, thus the protraction. It is measured in degrees. The cycles of protraction and the passive backward movement, which is acknowledged as retraction, correspond to the whisking frequency. The difference between maximal retraction and maximal protraction in degrees constitutes the amplitude. During protraction, the angular velocity and the angular acceleration are measured in degrees per second (Fig. 4).

FIG. 4.

The developed spatial model allows precise measurement of angles, angular velocity, and angular acceleration on the intact (left) and operated side (right) during protraction (A) and retraction (B) of the vibrissae. Note the significant change in angle between the sagittal line Fr-Occ during protraction and retraction on the intact side. The vibrissae on the operated side remain spastic.

Analysis of target muscle reinnervation

Four weeks after all surgical treatments, the rats were deeply anesthetized and transcardially perfused with 0.9% NaCl in distilled water for 60 s. They were then fixated with 4% formaldehyde in 0.1 M phosphate buffer at pH 7.4 for 20 min. The LLS muscle (Fig. 5a) of the face of both operated and unoperated side was dissected free, using a surgical microscope. Specimens were cryoprotected in 30% sucrose. Longitudinal sections (30 μm thick) were cut on a cryostat and were mounted on SUPERFROST/Plus slides (Carl Roth, Karlsruhe, Germany) to visualize intramuscular axons and motor end plates. For this purpose, every third longitudinal section through the muscle was stained with anti-neuronal class III β-tubulin (No. PRB-435P; Covance, Richmond, CA), diluted at 1:1000, and Alexa Fluor 488-conjugated α-bungarotoxin, diluted at 1:1000 (B-13422; Molecular Probes) (Guntinas-Lichius et al. 2005). Specificity controls were performed.

FIG. 5.

(a) Schematic drawing of the extrinsic vibrissae muscles. α–δ: the four caudal hair follicles; the muscle slings that “straddle” the five vibrissae rows (A–E); T: m. transversus nasi; L: m. LLS; N: m. nasalis; M: m. maxilolabialis; O: orbit; S: septum intermusculare. (b, c) Superimposed stacks of confocal images of end plates in LLS muscles of intact and surgically treated rats visualized by staining of the motor end plates with Alexa Fluor 488-conjugated α-bungarotoxin (green fluorescence) and immunostaining of the intramuscular axons for neuronal class III β-tubulin (Cy3 red fluorescence). (b, c) Show examples of a polyinnervated and a monoinnervated end plate, respectively. Three axonal branches (arrows in b) reach the boundaries of the polyinnervated end plate delineated by the α-bungarotoxin staining. In contrast, the monoinnervated end plate is reached by a single axon (empty arrow in c) with several preterminal rami. In both examples, the whole end plates are within the stack of confocal images. The scale bar shown in (c) indicates 125 μm. LLS, levator labii superioris. Adopted from Guntinas-Lichius et al.¹¹³ Color images are available online.

Sections were examined with a Zeiss Axioskop 50 epifluorescence microscope through the “rhodamine” filter (No. 15 of Carl Zeiss, excitation BP 546/12, beamsplitter FT 580, and emission LP 590) and through the “fluorescein” filter (No. 9 of Carl Zeiss, excitation BP 450–490, beamsplitter FT510, and emission LP 520). We documented the axonal branches with beta-tubulin staining, entering, or in some cases leaving the boundaries of individual end plates, which were demonstrated with acetylcholine receptor staining, alpha-bungarotoxin. Entries of branches from one axon were accounted as single events. The quality of end plate reinnervation was determined by counting the number of these axonal branches. The end plates were characterized as “monoinnervated” (one axon), “polyinnervated” (two or more axons), or denervated (no visible axonal associated with the receptor staining). All end plate countings were realized directly under the microscope (objective × 400) in a blind manner (Fig. 5b, c). We excluded from the count all end plates that appeared to be cut. The frequencies of mono innervated, poly innervated, and noninnervated end plates were expressed in percentage of the total population and group mean values were compared with the two-sided t test for independent groups. Photographic documentation was made on an LSM 510 confocal microscope (Zeiss).

All the counts were made on sections marked with codes, hiding the information about the surgical procedure used on each animal. All values are presented in the tables as mean ± standard deviation (SD) or as percentages of the total number of labeled motoneurons.

Statistical evaluation

A one-way analysis of variance was used, followed by a post hoc Bonferroni-Holm correction, to investigate the labeled neurons between the control and the experimental subgroups. A p-value of <0.05 was considered to indicate statistically significant differences. If significant effects were detected (p < 0.05), comparisons with the control group 2 were performed using the post hoc test of Dunnett at a significance level of 0.05. For analysis, Statistica 6.0 software (StatSoft, Tulsa, OK) was used.

Results

Application of MVCC improved whisking function

The movement of the large caudal whiskers is regulated by two kinds of muscles. One type is responsible for the movement of the whole mystacial pad, while the other for every individual hair follicle. The second type of musculature is absent in the rostrally situated follicles.⁵⁴ The musculature responsible for protraction creates a sling surrounding the rostral part of each hair follicle and its function is controlled by branches of the facial nerve. During contraction, each follicle at its root is pulled downwards, thus shifting the hair upwards/forwards. Retraction of the hair on the other hand mainly depends on the elasticity of the deep connective tissue and occurs passively.^54,55

In the control group, the whiskers were upright, oriented anteriorly, and during exploration, they moved back and forth with a frequency around 6 Hz. The highest protraction, which corresponds to the rostrally open angle between the vibrissal shaft and the median sagittal plane, was 70°. The amplitude of whisking, which corresponds to the difference between maximal retraction and maximal protraction, measured 50°. These movements were performed at a sagittal angular velocity of about 500°/s and a sagittal angular acceleration of ∼20.000°/s².

The biometric analysis of vibrissal movements on operated rats demonstrated that MVCC ameliorated the amplitude of whisking to 22.67° ± 4.93°, which was higher than 15.67° ± 2.25° after BBA and higher than 15.21° ± 4.82° after bridging with an empty vein (mean ± SD, n = 10 rats per group). In all operated rats, all the corresponding values remained lower than the values measured in the intact controls (60–70°) (Fig. 6).

FIG. 6.

Graphic representation of the results from our biometric analysis of vibrissae movements. Application of MVCC promoted a clear trend for increase in the amplitude (22.67° ± 4.93°), which was larger than that after BBA (15.67° ± 2.25°) and that after bridging with an empty vein (15.21° ± 4.82°; mean ± SD, n = 10 rats per group). Anyway all these values remained significantly lower than those measured in the intact controls (*). MVCC, muscle-vein-combined conduit; SD, standard deviation. Color images are available online.

MVCC improved the quality of target reinnervation

The quality of target muscle reinnervation was evaluated in m. LLS, which is an extrinsic vibrissal muscle. It is innervated the same way as the intrinsic vibrissae muscles, by the “six longitudinal branches” of the BBFN (the short common trunk of the fused ramus buccolabialis superior and ramus buccolabialis inferior).⁵⁴

Examinations of the longitudinal sections revealed that MVCC promoted a monoinnervation degree of 74.05% ± 3.91%, whereas after BBA and bridging with an empty vein, the corresponding values were 64.51% ± 15.71% and 67.74 % ± 5.92 %, respectively (Fig. 7).

FIG. 7.

Graphic representation of the results from the analysis of quality of target reinnervation. Application of MVCC promoted an increased monoinnervation degree (74.05% ± 3.91%), whereas after BBA and bridging with an empty vein, these values were 64.51/% ± 15.71% and 67.74 ± 5.92, respectively. All these values remained significantly lower than those determined in the intact controls (*). Color images are available online.

Discussion

The results taken together indicate a positive effect of muscle-vein-combined conduit (MVCC) on the improvement of whisking, following reconstruction of the facial nerve in rats. To our knowledge, this is the first study of such a conduit on the facial nerve. Our findings support the recent suggestion that a venal graft with an implanted trophic source, such as autologous denervated skeletal muscle, may promote the monoinnervation degree and ameliorate the coordinated function of the corresponding muscle fibers.

Misdirected target reinnervation: intramuscular sprouting

It has been established that there are three ways for the post-transectional “misdirected” or “aberrant” reinnervation of muscles to take place.¹⁰ First, the regenerated axons may simply be misrouted along “false” endoneural tubes toward incorrect muscles because of false axon guidance.^56–59 The second way is caused by the plethora of branches arising from the cut and misguided axons.^60–62 In the course of embryonic development, there is an accurate target-directed pathfinding of single motor axons.⁶³ During regeneration, however, many branches, rather than one only, arise from the cut axon,^8,64–66 and finally, a muscle fiber may be reinnervated by a number of axonal branches coming from separate, individual motoneurons.⁶⁷ This is a condition known as “polyneuronal innervation.”^68,69 Finally, misdirection can occur within the target, with intramuscular or terminal sprouting of axons.⁷⁰ Although thought to be temporary,⁷¹ this abnormal innervation may continue for long time^61,72 with harmful consequences on muscle performance.

Sprouting of terminal axons is an impressive occurrence that takes place after neuronal injury and aims to regeneration. Motor units of remaining neurons increase their size aiming to reinnervate as many muscles as possible, thus compensating for reduced functional capacity.^73–75 Both collateral branching and terminal axonal sprouting are naturally and automatically realized following segmentation of a peripheral nerve, so it is crucial to understand their influence on the recovery of function. A muscle fiber is regulated by at least two motoneurons, which are often functionally different and may operate asynchronously.^73–79 In our previous studies, results show that reduced collateral branching does not promote a decrease of terminal sprouting or an amelioration of the coordinated muscular function, so we suggest that sprouting within the muscle is critical.^15,40,41 In this way, presumptions of reports are supported, which demonstrate that enhanced neuromuscular activity ameliorates the quality of target reinnervation by restraining terminal axonal sprouting and by bridge formation of perisynaptic Schwann cell processes.^75,80–83

The use of tubes to improve reinnervation/MVCCs

Following nerve transection at the injury site, the majority of neurons react with branching, meaning that each parental axon divides into as many as 25 daughter axons or branches.^64,84 Axonal branching is initiated within 3 h after injury^85,86 and is considered a pursuit for local navigating cues required for axonal piloting. It is established that if the sectioned nerve ends are placed very close to each other, a large part of the traumatized axons will be entrapped at the lesion area. Overall, guided axonal growth is compromised, mainly because the proceeding Wallerian degeneration is performed slower than axonal elongation. Therefore, the newly developed branches will start growing backwards or will alternatively create a disorganized, disarranged, confused tangled terminal aggregation, called neuroma.^87,88 This is the reason why we attached the cut nerve stumps to each other using a tube, enabling an undisturbed axonal regrowth and avoiding a facial-facial anastomosis.

The preferred method used to bridge big peripheral nerve defects is still the autograft, which, however, has serious restrictions, such as limited nerve availability and donor morbidity, presenting mainly with neuroma formation, scarring, and persistent impairment of function.⁸⁹ This is why alternatives are investigated and tested. Arterial grafts⁹⁰ and especially venal grafts^91–95 have been selected based on specific characteristics/abilities: they are able to assist neuronal regrowth at the transected nerve end, they offer a duct where neurotropic and neurotrophic agents that are produced by the traumatized nerve ends can diffuse, and they restrict penetration of fibrous tissue.⁹⁴

Every axon along with its myelinating Schwann cell is surrounded by a BL canal, consisting of collagen, laminin, heparan sulfate proteoglycans, and fibronectin.⁹⁶ These BL tubes are disrupted by nerve injuries, they then degenerate, and finally they are phagocyted. The notion for utilizing muscle tissue for axonal restoration was based on the resemblance of the muscular BL to the endoneurial tubes.²³ Confocal imaging studies of such conduits demonstrated that the muscle-vein grafts, even from the early days after surgery, were extensively colonized by Schwann cells migrating from cut nerve ends, actively increasing rapidly in number.^97–99 In addition to that, 14 days after surgery within the graft, axon regeneration was easily detected. It seems that for sufficient regeneration, it is absolutely necessary for Schwann cells to be very closely situated to the BL.¹⁰⁰ Another rationale for the incorporation of muscle in the vein was that it averts breakdown of the vessel, while the vein itself impedes neuronal spreading.³⁷ In vitro and in vivo studies have shown that fresh skeletal muscle fibers can dismiss important gliotrophic factors aiming to promote axonal reconstruction and Schwann cell activity.⁸⁹

Such combined ducts have been applied in the clinical practice to repair gaps measuring up to 6 cm (mean 2.5), both for sensory and mixed nerves.^35,101 In 21 studied cases, both sensory and mixed nerve gaps were bridged, and in 85%, favorable results were reported with a follow-up of at least 14 months. These outcomes appear to surpass those described when different conduits are used, either artificial or biological.³⁵ Moreover, the muscle-vein conduits are free of cost and can be produced after examining and documenting the gap extent, according to the specific demands, after taking into account the type of nerve and the extend of the damage.

In a following study, Battiston et al.¹⁰² studied 47 treated patients with very restricted indications. A total of 13 patients appeared with sensory nerve damage, 9 with motor nerve, and 25 with mixed injuries. All patients received treatment in emergency, and in all cases, the gaps were too extended and the nerve graft was not sufficient for surgical treatment. There was a satisfying or very satisfying recovery in 52% of the patients with mixed nerve lesions, while 13% exhibited an unsatisfactory recovery. A limited improvement was observed in 35% of the participants in the study with combined nerves injuries; either just the motor or only the sensory nerve fibers exhibited a satisfying improvement. In the cases where nerve lesions were solely motor or sensory, 90% of patients had good recovery results.¹⁰² Overall, favorable results were achieved in a group of 13 patients with crush traumata of sensory and mixed nerves of the upper limb in emergency. In all these patients, a muscle-vein-combined graft was applied for nerve reconstruction.¹⁰³

Taking everything into account, Battiston et al.¹⁰⁴ suggest that reconstruction of a traumatized nerve in primary care using a muscle-vein-combined graft is reasonable and promising. Moreover, if the nerve repair fails in this stage, it is still feasible to attempt a secondary reconstruction with a positive outcome.¹⁰⁵

The role of trophic factors in axonal regrowth, future targets

Under normal conditions, the target tissue generates the majority of neurotrophic agents, which are transported retrogradely along the axons to access the motoneurons. Following axotomy, the traumatized axons pick up the trophic factors to maintain the neurons trophically satisfied for as long as the target-derived source is not fully functional.^106–110

Neural regeneration constitutes a complicated procedure, which requires the participation of many agents, elements, and signaling cues for it to be effective. The exact extracellular proteins and/or neurotrophic factors that play a key role on the restoration of axons are not fully specified; however, in neurological diseases, the most frequently used and suggested therapies include the administration of neurotrophic factors, since it is known that they enhance motoneuron cell survival during embryonic and early postnatal development. For each type of nerve (motor, sensory, or both) there is a subset of neurotrophic factors recommended. For axonal regeneration, the neurotrophic factors commonly used are NGF, GDNF, brain-derived neurotrophic factor (BDNF), neurotrophin-3,4/5 (NT-3,4/5), and ciliary neurotrophic factor (CNTF). At the time, there are various methods applied for neurotrophic factor transportation, such as matrices, microspheres, hydrogels, or combined muscle conduits.¹¹¹

Conclusion

Peripheral nerve regeneration along the distal nerve stump is a fruitless process, unless the surviving axons succeed to reinnervate the original targets. Increasing evidence suggests that investigation should mostly be aiming to find ways to minimize polyinnervation at the NMJ, since recent studies that succeeded to do so, also achieved improvement in function.⁴¹ Our findings support the recent suggestion that a venal graft with implantation of a trophic source, such as autologous denervated skeletal muscle, may promote the monoinnervation degree and ameliorate coordinated function of the corresponding muscles.

Footnotes

Disclosure Statement

The authors declare that no competing financial interests exist.

Funding Information

No funding was received for this project.

References

Wilson

D.L.

, and Perry

G.W.

Some hypotheses concerning axon regeneration. Restor Neurol Neurosci, 1, 197, 1990.

Moran

L.B.

, and Graeber

M.B.

The facial nerve axotomy model. Brain Res Brain Res Rev, 44, 154, 2004.

Vaughan

E.D.

, and Richardson

Facial nerve reconstruction following ablative parotid surgery. Br J Oral Maxillofac Surg, 31, 274, 1993.

Ferreira

M.C.

, Besteiro

J.M.

, and Tuma

Jr . Results of reconstruction of the facial nerve. Microsurgery, 15, 5, 1994.

Goodmurphy

C.W.

, and Ovalle

W.K.

Morphological study of two human facial muscles: orbicularis oculi and corrugator supercilii. Clin Anat, 12, 1, 1999.

Bento

R.F.

, and Miniti

Anastomosis of the intratemporal facial nerve using fibrin tissue adhesive. Ear Nose Throat J, 72, 663, 1993.

Kimura

, Rodnitzky

R.L.

, and Okawara

S.H.

Electrophysiologic analysis of aberrant regeneration after facial nerve paralysis. Neurology, 25, 989, 1975.

Baker

R.S.

, Stava

M.W.

, Nelson

K.R.

, May

P.J.

, Huffman

M.D.

, and Porter

J.D.

Aberrant reinnervation of facial musculature in a subhuman primate: a correlative analysis of eyelid kinematics, muscle synkinesis, and motoneuron localization. Neurology, 44, 2165, 1994.

Montserrat

, and Benito

Facial synkinesis and aberrant regeneration of facial nerve. Adv Neurol, 49, 211, 1988.

10.

Sumner

A.J.

Aberrant reinnervation. Muscle Nerve, 13, 801, 1990.

11.

Sadjadpour

Postfacial palsy phenomena: faulty nerve regeneration or ephaptic transmission? Brain Res, 95, 403, 1975.

12.

Bratzlavsky

, and vander Eecken

Altered synaptic organization in facial nucleus following facial nerve regeneration: an electrophysiological study in man. Ann Neurol, 2, 71, 1977.

13.

Graeber

M.B.

, Bise

, and Mehraein

Synaptic stripping in the human facial nucleus. Acta Neuropathol, 86, 179, 1993.

14.

Moran

C.J.

, and Neely

J.G.

Patterns of facial nerve synkinesis. Laryngoscope, 106(Pt 1), 1491, 1996.

15.

Ozsoy

, Hizay

, Demirel

B.M.

, et al. The hypoglossal-facial nerve repair as a method to improve recovery of motor function after facial nerve injury. Ann Anat, 193, 304, 2011.

16.

Lundborg

, Dahlin

, Danielsen

, and Zhao

Trophism, tropism, and specificity in nerve regeneration. J Reconstr Microsurg, 10, 345, 1994.

17.

Chiu

D.T.

, and Strauch

A prospective clinical evaluation of autogenous vein grafts used as a nerve conduit for distal sensory nerve defects of 3 cm or less. Plast Reconstr Surg, 86, 928, 1990.

18.

Chiu

D.T.

, Janecka

, Krizek

T.J.

, Wolff

, and Lovelace

R.E.

Autogenous vein graft as a conduit for nerve regeneration. Surgery, 91, 226, 1982.

19.

Risitano

, Cavallaro

, and Lentini

Autogenous vein and nerve grafts: a comparative study of nerve regeneration in the rat. J Hand Surg Br, 14, 102, 1989.

20.

Suematsu

Tubulation for peripheral nerve gap: its history and possibility. Microsurgery, 10, 71, 1989.

21.

Walton

R.L.

, Brown

R.E.

, Matory

W.E.

Jr ., Borah

G.L.

, and Dolph

J.L.

Autogenous vein graft repair of digital nerve defects in the finger: a retrospective clinical study. Plast Reconstr Surg, 84, 944; discussion 950–952, 1989.

22.

Kraus

H.R.H.

Results after treatment of injured peripheral nerves with special consideration to gunshot wounds [in German]. Arch Klin Chir, 199, 318, 1940.

23.

Fawcett

J.W.

, and Keynes

R.J.

Muscle basal lamina: a new graft material for peripheral nerve repair. J Neurosurg, 65, 354, 1986.

24.

Keynes

R.J.

, Hopkins

W.G.

, and Huang

L.H.

Regeneration of mouse peripheral nerves in degenerating skeletal muscle: guidance by residual muscle fibre basement membrane. Brain Res, 295, 275, 1984.

25.

Kong

J.M.

, Zhong

S.Z.

, Bo

, and Zhu

S.X.

Experimental study of bridging the peripheral nerve gap with skeletal muscle. Microsurgery, 7, 183, 1986.

26.

Lundborg

, and Richard

Bunge memorial lecture. Nerve injury and repair—a challenge to the plastic brain. J Peripher Nerv Syst, 8, 209, 2003.

27.

Meek

M.F.

, and Coert

J.H.

Clinical use of nerve conduits in peripheral-nerve repair: review of the literature. J Reconstr Microsurg, 18, 97, 2002.

28.

Pereira

J.H.

, Bowden

R.E.

, Gattuso

J.M.

, and Norris

R.W.

Comparison of results of repair of digital nerves by denatured muscle grafts and end-to-end sutures. J Hand Surg Br, 16, 519, 1991.

29.

Pereira

J.H.

, Palande

D.D.

, Subramanian

, Narayanakumar

T.S.

, Curtis

, and Turk

J.L.

Denatured autologous muscle graft in leprosy. Lancet, 338, 1239, 1991.

30.

Pereira

J.H.

, Bowden

R.E.

, Narayanakumar

T.S.

, and Gschmeissner

S.E.

Peripheral nerve reconstruction using denatured muscle autografts for restoring protective sensation in hands and feet of leprosy patients. Indian J Lepr, 68, 83, 1996.

31.

Pogrel

M.A.

, and Maghen

The use of autogenous vein grafts for inferior alveolar and lingual nerve reconstruction. J Oral Maxillofac Surg, 59, 985; discussion 988–993, 2001.

32.

Stahl

, and Goldberg

J.A.

The use of vein grafts in upper extremity nerve surgery. Eur J Plast Surg, 22, 255, 1999.

33.

Terzis

J.K.

, and Kostas

Vein grafts used as nerve conduits for obstetrical brachial plexus palsy reconstruction. Plast Reconstr Surg, 120, 1930, 2007.

34.

Chiu

D.T.

Autogenous venous nerve conduits. A review. Hand Clin, 15, 667, ix, 1999.

35.

Battiston

, Tos

, Cushway

T.R.

, and Geuna

Nerve repair by means of vein filled with muscle grafts I. Clinical results. Microsurgery, 20, 32, 2000.

36.

Battiston

, Tos

, Geuna

, Giacobini-Robecchi

M.G.

, and Guglielmone

Nerve repair by means of vein filled with muscle grafts. II. Morphological analysis of regeneration. Microsurgery, 20, 37, 2000.

37.

Brunelli

G.A.

, Battiston

, Vigasio

, Brunelli

, and Marocolo

Bridging nerve defects with combined skeletal muscle and vein conduits. Microsurgery, 14, 247, 1993.

38.

Varejao

A.S.

, Cabrita

A.M.

, Geuna

, et al. Functional assessment of sciatic nerve recovery: biodegradable poly (DLLA-epsilon-CL) nerve guide filled with fresh skeletal muscle. Microsurgery, 23, 346, 2003.

39.

Varejao

A.S.

, Cabrita

A.M.

, Meek

M.F.

, Fornaro

, and Geuna

Nerve regeneration inside fresh skeletal muscle-enriched synthetic tubes: a laser confocal microscope study in the rat sciatic nerve model. Ital J Anat Embryol, 108, 77, 2003.

40.

Hizay

, Ozsoy

, Demirel

B.M.

, et al. 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. Neurosurgery, 70, 1544; discussion 1556, 2012.

41.

Bendella

, Rink

, Manthou

, et al. Effect of surgically guided axonal regrowth into a 3-way-conduit (isogeneic trifurcated aorta) on functional recovery after facial-nerve reconstruction: experimental study in rats. Restor Neurol Neurosci, 37, 181, 2019.

42.

Rosen

J.M.

, Hentz

V.R.

, and Kaplan

E.N.

Fascicular tubulization: a cellular approach to peripheral nerve repair. Ann Plast Surg, 11, 397, 1983.

43.

Ljungberg

, Johansson-Ruden

, Bostrom

K.J.

, Novikov

, and Wiberg

Neuronal survival using a resorbable synthetic conduit as an alternative to primary nerve repair. Microsurgery, 19, 259, 1999.

44.

Chen

, Knox

C.J.

, Yao

, Li

, and Hadlock

T.A.

The effects of venous ensheathment on facial nerve repair in the rat. Laryngoscope, 127, 1558, 2017.

45.

W.H.

, and Yu

M.C.

Acceleration of the regeneration of the crushed hypoglossal nerve by testosterone. Exp Neurol, 80, 349, 1983.

46.

Dohm

, Streppel

, Guntinas-Lichius

, et al. Local application of extracellular matrix proteins fails to reduce the number of axonal branches after varying reconstructive surgery on rat facial nerve. Restor Neurol Neurosci, 16, 117, 2000.

47.

Guntinas-Lichius

, Angelov

D.N.

, Tomov

T.L.

, Dramiga

, Neiss

W.F.

, and Wewetzer

Transplantation of olfactory ensheathing cells stimulates the collateral sprouting from axotomized adult rat facial motoneurons. Exp Neurol, 172, 70, 2001.

48.

Semba

, and Egger

M.D.

The facial “motor” nerve of the rat: control of vibrissal movement and examination of motor and sensory components. J Comp Neurol, 247, 144, 1986.

49.

Lundborg

, Dahlin

L.B.

, Danielsen

, et al. Nerve regeneration in silicone chambers: influence of gap length and of distal stump components. Exp Neurol, 76, 361, 1982.

50.

Angelov

D.N.

, Guntinas-Lichius

, Wewetzer

, Neiss

W.F.

, and Streppel

Axonal branching and recovery of coordinated muscle activity after transection of the facial nerve in adult rats. Adv Anat Embryol Cell Biol, 180, 1, 2005.

51.

Bendella

, Brackmann

D.E.

, Goldbrunner

, and Angelov

D.N.

Nerve crush but not displacement-induced stretch of the intra-arachnoidal facial nerve promotes facial palsy after cerebellopontine angle surgery. Exp Brain Res, 234, 2905, 2016.

52.

Bischoff

, Grosheva

, Irintchev

, et al. Manual stimulation of the orbicularis oculi muscle improves eyelid closure after facial nerve injury in adult rats. Muscle Nerve, 39, 197, 2009.

53.

Grosheva

, Rink

, Jansen

, et al. Early and continued manual stimulation is required for long-term recovery after facial nerve injury. Muscle Nerve, 57, 100, 2018.

54.

Dörfl

The innervation of the mystacial region of the white mouse: a topographical study. J Anat, 142, 173, 1985.

55.

Wineski

L.E.

Facial morphology and vibrissal movement in the golden hamster. J Morphol, 183, 199, 1985.

56.

Thomander

Reorganization of the facial motor nucleus after peripheral nerve regeneration. An HRP study in the rat. Acta Otolaryngol, 97, 619, 1984.

57.

Aldskogius

, and Thomander

Selective reinnervation of somatotopically appropriate muscles after facial nerve transection and regeneration in the neonatal rat. Brain Res, 375, 126, 1986.

58.

Brushart

T.M.

, and Seiler

W.A.

, 4th. Selective reinnervation of distal motor stumps by peripheral motor axons. Exp Neurol, 97, 289, 1987.

59.

Zhao

, Dahlin

L.B.

, Kanje

, Lundborg

, and Lu

S.B.

Axonal projections and functional recovery following fascicular repair of the rat sciatic nerve with Y-tunnelled silicone chambers. Restor Neurol Neurosci, 4, 13, 1992.

60.

Al-Majed

A.A.

, Neumann

C.M.

, Brushart

T.M.

, and Gordon

Brief electrical stimulation promotes the speed and accuracy of motor axonal regeneration. J Neurosci, 20, 2602, 2000.

61.

Mackinnon

S.E.

, Dellon

A.L.

, and O'Brien

J.P.

Changes in nerve fiber numbers distal to a nerve repair in the rat sciatic nerve model. Muscle Nerve, 14, 1116, 1991.

62.

Morris

J.H.

, Hudson

A.R.

, and Weddell

A study of degeneration and regeneration in the divided rat sciatic nerve based on electron microscopy. II. The development of the “regenerating unit.” Z Zellforsch Mikrosk Anat, 124, 103, 1972.

63.

Liu

D.W.

, and Westerfield

The formation of terminal fields in the absence of competitive interactions among primary motoneurons in the zebrafish. J Neurosci, 10, 3947, 1990.

64.

Shawe

G.D.

On the number of branches formed by regenerating nerve-fibres. Br J Surg, 42, 474, 1955.

65.

Esslen

Electromyographic findings on two types of misdirection of regenerating axons. Electroencephalogr Clin Neurophysiol, 12, 738, 1960.

66.

Brushart

T.M.

, and Mesulam

M.M.

Alteration in connections between muscle and anterior horn motoneurons after peripheral nerve repair. Science, 208, 603, 1980.

67.

Ito

, and Kudo

Reinnervation by axon collaterals from single facial motoneurons to multiple muscle targets following axotomy in the adult guinea pig. Acta Anat (Basel), 151, 124, 1994.

68.

Brown

M.C.

, and Hopkins

W.G.

Role of degenerating axon pathways in regeneration of mouse soleus motor axons. J Physiol, 318, 365, 1981.

69.

Rich

M.M.

, and Lichtman

J.W.

In vivo visualization of pre- and postsynaptic changes during synapse elimination in reinnervated mouse muscle. J Neurosci, 9, 1781, 1989.

70.

Son

Y.J.

, Trachtenberg

J.T.

, and Thompson

W.J.

Schwann cells induce and guide sprouting and reinnervation of neuromuscular junctions. Trends Neurosci, 19, 280, 1996.

71.

Hennig

, and Dietrichs

Transient reinnervation of antagonistic muscles by the same motoneuron. Exp Neurol, 130, 331, 1994.

72.

Madison

R.D.

, Archibald

S.J.

, Lacin

, and Krarup

Factors contributing to preferential motor reinnervation in the primate peripheral nervous system. J Neurosci, 19, 11007, 1999.

73.

Grimby

, Einarsson

, Hedberg

, and Aniansson

Muscle adaptive changes in post-polio subjects. Scand J Rehabil Med, 21, 19, 1989.

74.

Trojan

D.A.

, Gendron

, and Cashman

N.R.

Electrophysiology and electrodiagnosis of the post-polio motor unit. Orthopedics, 14, 1353, 1991.

75.

Tam

S.L.

, and Gordon

Mechanisms controlling axonal sprouting at the neuromuscular junction. J Neurocytol, 32, 961, 2003.

76.

Barry

J.A.

, and Ribchester

R.R.

Persistent polyneuronal innervation in partially denervated rat muscle after reinnervation and recovery from prolonged nerve conduction block. J Neurosci, 15, 6327, 1995.

77.

Friede

R.L.

, and Bischhausen

The fine structure of stumps of transected nerve fibers in subserial sections. J Neurol Sci, 44, 181, 1980.

78.

Gorio

, Carmignoto

, Finesso

, Polato

, and Nunzi

M.G.

Muscle reinnervation—II. Sprouting, synapse formation and repression. Neuroscience, 8, 403, 1983.

79.

Schröder

J.M.

Hyperneurotization of Bungnerscher ligaments during experimental isoniazid neuropathy: phase contrast-electron microscope studies [in German]. Virchows Archiv Abteilung, 1, 131, 1968.

80.

Brown

M.C.

, Goodwin

G.M.

, and Ironton

Prevention of motor nerve sprouting in botulinum toxin poisoned mouse soleus muscles by direct stimulation of the muscle [proceedings]. J Physiol, 267, 42P, 1977.

81.

Brown

M.C.

, and Holland

R.L.

A central role for denervated tissues in causing nerve sprouting. Nature, 282, 724, 1979.

82.

Tam

S.L.

, Archibald

, Jassar

, Tyreman

, and Gordon

Increased neuromuscular activity reduces sprouting in partially denervated muscles. J Neurosci, 21, 654, 2001.

83.

Love

F.M.

, Son

Y.J.

, and Thompson

W.J.

Activity alters muscle reinnervation and terminal sprouting by reducing the number of Schwann cell pathways that grow to link synaptic sites. J Neurobiol, 54, 566, 2003.

84.

Jenq

C.B.

, Jenq

L.L.

, Bear

H.M.

, and Coggeshall

R.E.

Conditioning lesions of peripheral nerves change regenerated axon numbers. Brain Res, 457, 63, 1988.

85.

Bisby

M.A.

, and Pollock

Increased regeneration rate in peripheral nerve axons following double lesions: enhancement of the conditioning lesion phenomenon. J Neurobiol, 14, 467, 1983.

86.

Sjoberg

, and Kanje

The initial period of peripheral nerve regeneration and the importance of the local environment for the conditioning lesion effect. Brain Res, 529, 79, 1990.

87.

Horch

K.W.

, and Lisney

S.J.

On the number and nature of regenerating myelinated axons after lesions of cutaneous nerves in the cat. J Physiol, 313, 275, 1981.

88.

Ashur

, Vilner

, Finsterbush

, Rousso

, Weinberg

, and Devor

Extent of fiber regeneration after peripheral nerve repair: silicone splint vs. suture, gap repair vs. graft. Exp Neurol, 97, 365, 1987.

89.

Crosio

, Fornasari

B.E.

, Gambarotta

, et al. Chitosan tubes enriched with fresh skeletal muscle fibers for delayed repair of peripheral nerve defects. Neural Regen Res, 14, 1079, 2019.

90.

Büngner

Nerve degeneration and regeneration process after injuries [in German]. Beitr pathol Anat allg Pathol, 10, 21, 1891.

91.

Fansa

, Keilhoff

, Plogmeier

, Frerichs

, Wolf

, and Schneider

Successful implantation of Schwann cells in acellular muscles. J Reconstr Microsurg, 15, 61, 1999.

92.

Fansa

, Keilhoff

, Wolf

, Schneider

, and Gold

B.G.

Tissue engineering of peripheral nerves: a comparison of venous and acellular muscle grafts with cultured Schwann cells. Plast Reconstr Surg, 107, 495, 2001.

93.

Fansa

, Schneider

, Wolf

, and Keilhoff

Influence of insulin-like growth factor-I (IGF-I) on nerve autografts and tissue-engineered nerve grafts. Muscle Nerve, 26, 87, 2002.

94.

Hudson

T.W.

, Evans

G.R.

, and Schmidt

C.E.

Engineering strategies for peripheral nerve repair. Orthop Clin North Am, 31, 485, 2000.

95.

Johnson

E.O.

, and Soucacos

P.N.

Nerve repair: experimental and clinical evaluation of biodegradable artificial nerve guides. Injury, 39(Suppl 3), S30, 2008.

96.

Tohyama

, and Ide

The localization of laminin and fibronectin on the Schwann cell basal lamina. Arch Histol Jpn, 47, 519, 1984.

97.

Fornaro

, Tos

, Geuna

, Giacobini-Robecchi

M.G.

, and Battiston

Confocal imaging of Schwann-cell migration along muscle-vein combined grafts used to bridge nerve defects in the rat. Microsurgery, 21, 153, 2001.

98.

Geuna

, Raimondo

, Nicolino

, et al. Schwann-cell proliferation in muscle-vein combined conduits for bridging rat sciatic nerve defects. J Reconstr Microsurg, 19, 119; discussion 124, 2003.

99.

Raimondo

, Nicolino

, Tos

, et al. Schwann cell behavior after nerve repair by means of tissue-engineered muscle-vein combined guides. J Comp Neurol, 489, 249, 2005.

100.

Hall

S.M.

Regeneration in the peripheral nervous system. Neuropathol Appl Neurobiol, 15, 513, 1989.

101.

Battiston

, Geuna

, Ferrero

, and Tos

Nerve repair by means of tubulization: literature review and personal clinical experience comparing biological and synthetic conduits for sensory nerve repair. Microsurgery, 25, 258, 2005.

102.

Battiston

B.T.P.

, Conforti

L.G.

, Ciclamini

, Artiaco

, and Geuna

End-to-side nerve suture. Basic principles and clinical applications [in Italian]. GIOT Giornale Italiano di Ortopedia e Traumatologia, 34, 111, 2008.

103.

Battiston

, Raimondo

, Tos

, et al. Chapter 11: tissue engineering of peripheral nerves. Int Rev Neurobiol, 87, 227, 2009.

104.

Battiston

, Papalia

, Tos

, and Geuna

Chapter 1: peripheral nerve repair and regeneration research: a historical note. Int Rev Neurobiol, 87, 1, 2009.

105.

Tos

, Battiston

, Ciclamini

, Geuna

, and Artiaco

Primary repair of crush nerve injuries by means of biological tubulization with muscle-vein-combined grafts. Microsurgery, 32, 358, 2012.

106.

DiStefano

P.S.

, Friedman

, Radziejewski

, et al. The neurotrophins BDNF, NT-3, and NGF display distinct patterns of retrograde axonal transport in peripheral and central neurons. Neuron, 8, 983, 1992.

107.

Unsicker

, Grothe

, Westermann

, and Wewetzer

Cytokines in neural regeneration. Curr Opin Neurobiol, 2, 671, 1992.

108.

Yan

, Elliott

, and Snider

W.D.

Brain-derived neurotrophic factor rescues spinal motor neurons from axotomy-induced cell death. Nature, 360, 753, 1992.

109.

Friedman

, Kleinfeld

, Ip

N.Y.

, et al. BDNF and NT-4/5 exert neurotrophic influences on injured adult spinal motor neurons. J Neurosci, 15, 1044, 1995.

110.

Sendtner

, Gotz

, Holtmann

, and Thoenen

Endogenous ciliary neurotrophic factor is a lesion factor for axotomized motoneurons in adult mice. J Neurosci, 17, 6999, 1997.

111.

Nectow

A.R.

, Marra

K.G.

, and Kaplan

D.L.

Biomaterials for the development of peripheral nerve guidance conduits. Tissue Eng Part B Rev, 18, 40, 2012.

112.

Angelov

D.N.

, Skouras

, Guntinas-Lichius

, Streppel

, Popratiloff

, Klein

, Walther

, Stennert

, Neiss

W.F.

Contralateral trigeminal stimulation improves axonal pathfinding after reconstructive surgery on the facial nerve in rats. Eur J Neurosci, 11, 1369, 1999.

113.

Guntinas-Lichius

, Irintchev. A., Streppel, M., Lenzen, M., Grosheva, M., Wewetzer, K., Neiss, W.F., and Angelov, D.N. Factors limiting motor recovery after facial nerve transection in the rat: combined structural and functional analyses. Eur J Neurosci, 21, 391, 2005.