Collateral Axonal Branching Research Articles

Vascular smooth muscle cell-derived nerve growth factor regulates sympathetic collateral branching to innervate blood vessels in embryonic skin.

Blood vessels serve as intermediate conduits for the extension of sympathetic axons towards target tissues, while also acting as crucial targets for their homeostatic processes encompassing the regulation of temperature, blood pressure, and oxygen availability. How sympathetic axons innervate not only blood vessels but also a wide array of target tissues is not clear. Here we show that in embryonic skin, after the establishment of co-branching between sensory nerves and blood vessels, sympathetic axons invade the skin alongside these sensory nerves and extend their branches towards these blood vessels covered by vascular smooth muscle cells (VSMCs). Our mosaic labeling technique for sympathetic axons shows that collateral branching predominantly mediates the innervation of VSMC-covered blood vessels by sympathetic axons. The expression of nerve growth factor (NGF), previously known to induce collateral axon branching in culture, can be detected in the vascular smooth muscle cell (VSMC)-covered blood vessels, as well as sensory nerves. Indeed, VSMC-specific Ngf knockout leads to a significant decrease of collateral branching of sympathetic axons innervating VSMC-covered blood vessels. These data suggest that VSMC-derived NGF serves as an inductive signal for collateral branching of sympathetic axons innervating blood vessels in the embryonic skin.

Microtubule-binding protein MAP1B regulates interstitial axon branching of cortical neurons via the tubulin tyrosination cycle

Regulation of directed axon guidance and branching during development is essential for the generation of neuronal networks. However, the molecular mechanisms that underlie interstitial (or collateral) axon branching in the mammalian brain remain unresolved. Here, we investigate interstitial axon branching in vivo using an approach for precise labeling of layer 2/3 callosal projection neurons (CPNs). This method allows for quantitative analysis of axonal morphology at high acuity and also manipulation of gene expression in well-defined temporal windows. We find that the GSK3β serine/threonine kinase promotes interstitial axon branching in layer 2/3 CPNs by releasing MAP1B-mediated inhibition of axon branching. Further, we find that the tubulin tyrosination cycle is a key downstream component of GSK3β/MAP1B signaling. These data suggest a cell-autonomous molecular regulation of cortical neuron axon morphology, in which GSK3β can release a MAP1B-mediated brake on interstitial axon branching upstream of the posttranslational tubulin code.

Drebrin Regulates Collateral Axon Branching in Cortical Layer II/III Somatosensory Neurons.

Proper cortical lamination is essential for cognition, learning, and memory. Within the somatosensory cortex, pyramidal excitatory neurons elaborate axon collateral branches in a laminar-specific manner that dictates synaptic partners and overall circuit organization. Here, we leverage both male and female mouse models, single-cell labeling and imaging approaches to identify intrinsic regulators of laminar-specific collateral, also termed interstitial, axon branching. We developed new approaches for the robust, sparse, labeling of Layer II/III pyramidal neurons to obtain single-cell quantitative assessment of axon branch morphologies. We combined these approaches with cell-autonomous loss-of-function (LOF) and overexpression (OE) manipulations in an in vivo candidate screen to identify regulators of cortical neuron axon branch lamination. We identify a role for the cytoskeletal binding protein drebrin (Dbn1) in regulating Layer II/III cortical projection neuron (CPN) collateral axon branching in vitro LOF experiments show that Dbn1 is necessary to suppress the elongation of Layer II/III CPN collateral axon branches within Layer IV, where axon branching by Layer II/III CPNs is normally absent. Conversely, Dbn1 OE produces excess short axonal protrusions reminiscent of nascent axon collaterals that fail to elongate. Structure-function analyses implicate Dbn1S142 phosphorylation and Dbn1 protein domains known to mediate F-actin bundling and microtubule (MT) coupling as necessary for collateral branch initiation upon Dbn1 OE. Taken together, these results contribute to our understanding of the molecular mechanisms that regulate collateral axon branching in excitatory CPNs, a key process in the elaboration of neocortical circuit formation.SIGNIFICANCE STATEMENT Laminar-specific axon targeting is essential for cortical circuit formation. Here, we show that the cytoskeletal protein drebrin (Dbn1) regulates excitatory Layer II/III cortical projection neuron (CPN) collateral axon branching, lending insight into the molecular mechanisms that underlie neocortical laminar-specific innervation. To identify branching patterns of single cortical neurons in vivo, we have developed tools that allow us to obtain detailed images of individual CPN morphologies throughout postnatal development and to manipulate gene expression in these same neurons. Our results showing that Dbn1 regulates CPN interstitial axon branching both in vivo and in vitro may aid in our understanding of how aberrant cortical neuron morphology contributes to dysfunctions observed in autism spectrum disorder and epilepsy.

Effects of Electrical Stimulation on Facial Paralysis Recovery after Facial Nerve Injury: A Review on Preclinical and Clinical Studies.

Various methods have been used to improve function and manage facial nerve injury. Although electrical stimulation therapy is frequently used to treat facial paralysis, its effects have been found to vary and no clear standards have been developed. The current review describes the results of preclinical and clinical studies evaluating the effectiveness of electrical stimulation therapy in promoting the recovery of a peripheral facial nerve injury. Evidence is presented showing the efficacy of electrical stimulation in promoting nerve regeneration after peripheral nerve injuries in both animal models and human patients. The ability of electrical stimulation to promote the recovery of facial paralysis was found to depend on the type of injury (compression or transection), the species of animal tested, the type of disease, the frequency and method of electrical stimulation, and the duration of the follow-up. Electrical stimulation, however, can also have potential negative outcomes, such as reinforcing synkinesis, including mistargeted axonal regrowth via inappropriate routes; excessive collateral axonal branching at the lesion site; and multiple innervations at neuromuscular junctions. Because of the inconsistencies among studies and the low quality of evidence, electrical stimulation therapy is not currently regarded as a primary treatment of facial paralysis in patients. However, understanding the effects of electrical stimulation, as determined in preclinical and clinical studies, is important for the potential validity of future research on electrical stimulation.

Fitting Splines to Axonal Arbors Quantifies Relationship Between Branch Order and Geometry.

Neuromorphology is crucial to identifying neuronal subtypes and understanding learning. It is also implicated in neurological disease. However, standard morphological analysis focuses on macroscopic features such as branching frequency and connectivity between regions, and often neglects the internal geometry of neurons. In this work, we treat neuron trace points as a sampling of differentiable curves and fit them with a set of branching B-splines. We designed our representation with the Frenet-Serret formulas from differential geometry in mind. The Frenet-Serret formulas completely characterize smooth curves, and involve two parameters, curvature and torsion. Our representation makes it possible to compute these parameters from neuron traces in closed form. These parameters are defined continuously along the curve, in contrast to other parameters like tortuosity which depend on start and end points. We applied our method to a dataset of cortical projection neurons traced in two mouse brains, and found that the parameters are distributed differently between primary, collateral, and terminal axon branches, thus quantifying geometric differences between different components of an axonal arbor. The results agreed in both brains, further validating our representation. The code used in this work can be readily applied to neuron traces in SWC format and is available in our open-source Python package brainlit: http://brainlit.neurodata.io/.

Giant ankyrin-B suppresses stochastic collateral axon branching through direct interaction with microtubules.

Giant ankyrin-B (gAnkB) is a 440-kD neurospecific ankyrin-B isoform and a high-confidence target for autism mutations. gAnkB suppresses axon branching through coordination of cortical microtubules, and autism-related mutation of gAnkB results in ectopic neuronal connectivity. We identified a bipartite motif from gAnkB, which bundles and avidly binds to microtubules in vitro. This motif is composed of a module of 15 tandem repeats followed by a short, conserved fragment also found in giant ankyrin-G (BG-box). Combination of these two parts synergistically increases microtubule-binding avidity. Transfection of astrocytes (which lack gAnkB) with WT gAnkB resulted in prominent bundling of microtubules, which did not occur with mutant gAnkB with impaired microtubule-binding activity. Similarly, rescue of gAnkB-deficient neurons with WT gAnkB suppressed axonal branching and invasion of EB3-tagged microtubules into filopodia, which did not occur with the same mutant gAnkB. Together, these findings demonstrate that gAnkB suppresses axon collateral branching and prevents microtubule invasion of nascent axon branches through direct interaction with microtubules.

Assessment of axonal sprouting and motor performance after hypoglossal-facial end-to-side nerve repair: experimental study in rats.

Hypoglossal-facial nerve anastomosis (HFA) aims to reanimate denervated mimic muscles with hypoglossal axons when the transected facial nerve is not accessible. The aim of this study was to evaluate the recovery of HFA using a "Y" tube in two variants: (1) the proximal stump of the hypoglossal nerve was entubulated to the "Y" tube (classic "Y" tube HFA) and (2) the "Y" tube was sutured to an epineurial window of a slightly damaged hypoglossal nerve (end-to-side "Y" tube HFA). A total of 48 adult female rats were divided into four groups: intact controls (group 1), sham operated (group 2), classic "Y" tube HFA (group 3) and end-to-side "Y" tube HFA (group 4). The abdominal aorta with both common iliac arteries of isogeneic male rats served as the Y-tube conduit. Animals from group 4 recovered better than those from group 3: the degree of collateral axonal branching (3 ± 1%) was significantly lower than that determined in group 3 (13 ± 1%). The mean deviation of the tongue from the midline was significantly smaller in group 4 (6 ± 4°) than that measured in animals from group 3 (41 ± 6°). In the determination of vibrissal motor function in group 3 and group 4, a decrease in amplitude was found to be - 66% and - 92%, respectively. No differences in the reinnervation pattern of the target muscles were detected. As a result, these surgical models were not determined to be able to improve vibrissal movements. It was concluded that performance of end-to-side "Y" tube HFA diminishes collateral axonal branching at the lesion site, which in turn, promotes better recovery of tongue- and vibrissal-motor performance.

Effect of Pulsed and Continuous Ultrasound Therapy on the Degree of Collateral Axonal Branching at the Lesion Site, Polyinnervation of Motor End Plates, and Recovery of Motor Function after Facial Nerve Reconstruction.

The aim of the present study is to test whether ultrasound therapy of muscles denervated by nerve injury would improve the quality of their reinnervation by reduction of the collateral axonal branching at the lesion site and poly-innervation degree at the neuromuscular junctions. After transection and suture of the buccal branch of the facial nerve, pulsed or continuous type of ultrasound therapy was applied to the paralyzed whisker pad muscles of rats in the course of 2 months. Instead of reduction, we found a significant increase in the collateral axonal branching after continuous ultrasound therapy when compared to the branching determined after pulsed or sham ultrasound therapy. Both types of ultrasound therapy also failed to reduce the proportion of polyinnervated end plates in the reinnervated facial muscles. Accordingly, continuous ultrasound therapy failed to restore any parameter of the motor performance of the vibrissal hairs. Application of pulsed ultrasound therapy promoted slight improvements of the functional parameters angular velocity and acceleration. The inhomogeneous structural and functional results achieved after both types of ultrasound therapy let us conclude that further studies are required to evaluate its effects on peripheral nerve regeneration. Anat Rec, 302:1314-1324, 2019. © 2019 Wiley Periodicals, Inc.

Experimental Studies on Facial Nerve Regeneration

Insufficient recovery after injury of a peripheral motor nerve is due to (1) inappropriate pathfinding as a result of axonal regrowth to inappropriate targets, (2) excessive collateral axonal branching at the lesion site, and (3) polyinnervation of the neuromuscular junctions (NMJs). The rat facial nerve model is often used because of its simple and reliable readout to measure recovery of function (vibrissal whisking). Over the last decades scientists have concentrated their efforts to combat mostly NMJ polyinnervation, because it turned out to be very difficult to reduce collateral axonal branching and impossible to navigate thousands of axons toward the original fascicles. In the past, several groups of scientists concentrated their efforts to reduce the activity-dependent polyinnervation of NMJs by electrical stimulation of the muscles (square 0.1 msec pulses at 5 Hz). The results showed no recovery of functions and a severe reduction in the number of innervated NMJs to approximately one fifth of those observed in intact animals. More recent experiments, however, have shown that motor recovery improved significantly following mechanical stimulation of the denervated facial muscles (vibrissal and orbicularis oculi) and that restored functions could invariably be linked to reduced polyinnervation at the NMJ while the number of innervated NMJ remained the same. These results suggest that clinically feasible and effective therapies could be developed and tested in the near future. Anat Rec, 302:1287-1303, 2019. © 2019 Wiley Periodicals, Inc.

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.

The "post-paralytic syndrome" after facial nerve reconstruction has been attributed to (i) malfunctioning axonal guidance at the fascicular (branches) level, (ii) collateral branching of the transected axons at the lesion site, and (iii) intensive intramuscular terminal sprouting of regenerating axons which causes poly-innervation of the neuromuscular junctions (NMJ). The first two reasons were approached by an innovative technique which should provide the re-growing axons optimal conditions to elongate and selectively re-innervate their original muscle groups. The transected facial nerve trunk was inserted into a 3-way-conduit (from isogeneic rat abdominal aorta) which should "guide" the re-growing facial axons to the three main branches of the facial nerve (zygomatic, buccal and marginal mandibular). The effect of this method was tested also on hypoglossal axons after hypoglossal-facial anastomosis (HFA). Coaptational (classic) FFA (facial-facial anastomosis) and HFA served as controls. When compared to their coaptation (classic) alternatives, both types of 3-way-conduit operations (FFA and HFA) promoted a trend for reduction in the collateral axonal branching (the proportion of double- or triple-labelled perikarya after retrograde tracing was slightly reduced). In contrast, poly-innervation of NMJ in the levator labii superioris muscle was increased and vibrissal (whisking) function was worsened. The use of 3-way-conduit provides no advantages to classic coaptation. Should the latter be impossible (too large interstump defects requiring too long interpositional nerve grafts), this type of reconstruction may be applied. (230 words).

Breast cancer induced nociceptor aberrant growth and collateral sensory axonal branching.

The tumour and neuron interaction has a significant impact upon disease progression and the patients quality of life. In breast cancer patients, it is known that there is an interaction between the tumour microenvironment and the sensory neurons to influence the progression of cancer as well as pain, though these mechanisms still need to be clearly defined. Here it is demonstrated that in a rodent orthotopic model of breast cancer (MDA MB 231) there was an increase in nerve fibre innervation into the tumour microenvironment (protein gene product 9.5), which were calcitonin gene related peptide positive C fibre nociceptors. In contrast, there was a reduction in myelinated nerve fibres (NF200). A sensory neuronal cell line was cultured in response to conditioned media from MDA MB231 and MCF7 as well as vascular endothelial growth factor-A (VEGF-A). All these experimental conditions induced sensory neuronal growth, with increased formation of collateral axonal branches. Furthermore, it was demonstrated that MDA MB231 and VEGF-A induced sensory neuronal sensitisation in response to capsaicin a TRPV1 agonist. MDA MB231 induced neuronal growth was suppressed by VEGFR2 inhibition (ZM323881 and neutralising antibody DC101), in addition both MDA MB231 and VEGF-A induced neurite growth was attenuated by the inhibition of ARP2/3 complex through co-treatment with CK666. This demonstrates that breast cancer can interact with the sensory nervous system to drive neuritogenesis through a VEGF-A/VEGFR2/ARP2/3 mediated pathway.

Characterization of the central neural projections to brown, white, and beige adipose tissue.

The functional recruitment of classic brown adipose tissue (BAT) and inducible brown-like or beige fat is, to a large extent, dependent on intact sympathetic neural input. Whereas the central neural circuits directed specifically to BAT or white adipose tissue (WAT) are well established, there is only a developing insight into the nature of neural inputs common to both fat types. Moreover, there is no clear view of the specific central and peripheral innervation of the browned component of WAT: beige fat. The objective of the present study is to examine the neural input to both BAT and WAT in the same animal and, by exposing different cohorts of rats to either thermoneutral or cold conditions, define changes in central neural organization that will ensure that beige fat is appropriately recruited and modulated after browning of inguinal WAT (iWAT). At thermoneutrality, injection of the neurotropic (pseudorabies) viruses into BAT and WAT demonstrates that there are dedicated axonal projections, as well as collateral axonal branches of command neurons projecting to both types of fat. After cold exposure, central neural circuits directed to iWAT showed evidence of reorganization with a greater representation of command neurons projecting to both brown and beiged WAT in hypothalamic (paraventricular nucleus and lateral hypothalamus) and brainstem (raphe pallidus and locus coeruleus) sites. This shift was driven by a greater number of supraspinal neurons projecting to iWAT under cold conditions. These data provide evidence for a reorganization of the nervous system at the level of neural connectivity following browning of WAT.-Wiedmann, N. M., Stefanidis, A., Oldfield, B. J. Characterization of the central neural projections to brown, white, and beige adipose tissue.

Axon Dynamics during Neocortical Laminar Innervation.

SUMMARYThe cerebral cortex is a densely interconnected structure with neural circuits that form between cortical laminae and also between distinct cortical areas. However, the precise cell biological and developmental mechanisms that underlie the formation of these neural circuits remain unknown. Here, we visualize laminar innervation of the developing mouse cerebral cortex by layer II/III pyramidal neurons in real time, describing cytoskeletal dynamics during this process. We find that layer II/III pyramidal neurons achieve local laminar-specific innervation through the stabilization of collateral axon branches in target laminae. We also find that loss of neural activity does not abolish local laminar-specific innervation and that cells within the local environment are the likely source of cues that direct layer-specific cortical innervation.

Evolution of the hippocampus in reptiles and birds.

Although the hippocampus is structurally quite different among reptiles, birds, and mammals, its function in spatial memory is said to be highly conserved. This is surprising, given that structural differences generally reflect functional differences. Here I review this enigma in some detail, identifying several evolutionary changes in hippocampal cytoarchitecture and connectivity. I recognize a lepidosaurid pattern of hippocampal organization (in lizards, snakes, and the tuatara Sphenodon) that differs substantially from the pattern of organization observed in the turtle/archosaur lineage, which includes crocodilians and birds. Although individual subdivisions of the hippocampus are difficult to homologize between these two patterns, both lack a clear homolog of the mammalian dentate gyrus. The strictly trilaminar organization of the ancestral amniote hippocampus was gradually lost in the lineage leading to birds, and birds expanded the system of intrahippocampal axon collaterals, relative to turtles and lizards. These expanded collateral axon branches resemble the extensive collaterals in CA3 of the mammalian hippocampus but probably evolved independently of them. Additional examples of convergent evolution between birds and mammals are the loss of direct inputs to the hippocampus from the primary olfactory cortex and the general expansion of telencephalic regions that communicate reciprocally with the hippocampus. Given this structural convergence, it seems likely that some similarities in the function of the hippocampus between birds and mammals, notably its role in the ability to remember many different locations without extensive training, likewise evolved convergently. The currently available data do not allow for a strong test of this hypothesis, but the hypothesis itself suggests some promising new research directions.

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.

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

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. To determine whether the Y tube reconstruction improved axonal regrowth and whether this was associated with improved function. We used a Y-tube conduit with the aim of improving navigation of regenerating axons after facial nerve transection in rats. 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. 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.

Intrinsic and therapeutic factors determining the recovery of motor function after peripheral nerve transection

Insufficient recovery after peripheral nerve injury has been attributed to (i) poor pathfinding of regrowing axons, (ii) excessive collateral axonal branching at the lesion site and (iii) polyneuronal innervation of the neuromuscular junctions (NMJ). The facial nerve transection model has been used initially to measure restoration of function after varying therapies and to examine the mechanisms underlying their effects. Since it is very difficult to control the navigation of several thousand axons, efforts concentrated on collateral branching and NMJ-polyinnervation. Treatment with antibodies against trophic factors to combat branching improved the precision of reinnervation, but had no positive effects on functional recovery. This suggested that polyneuronal reinnervation--rather than collateral branching--may be the critical limiting factor. The former could be reduced by pharmacological agents known to perturb microtubule assembly and was followed by recovery of function. Because muscle polyinnervation is activity-dependent and can be manipulated, attempts to design a clinically feasible therapy were performed by electrical stimulation or by soft tissue massage. Electrical stimulation applied to the transected facial nerve or to paralysed facial muscles did not improve vibrissal motor performance and failed to diminish polyinnervation. In contrast, gentle stroking of the paralysed muscles (vibrissal, orbicularis oculi, tongue musculature) resulted in full recovery of function. This manual stimulation was also effective after hypoglossal-facial nerve suture and after interpositional nerve grafting, but not after surgical reconstruction of the median nerve. All these findings raise hopes that clinically feasible and effective therapies could be soon designed and tested.

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

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. 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. 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. 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.

Experimental studies on post-transectional facial nerve regrowth and functional recovery of paralyzed muscles of the face in rats and mice

Insufficient recovery after peripheral nerve injury has been attributed to (i) lack of axonal navigation and poor pathfinding of regrowing axons to improper targets, (ii) excessive collateral axonal branching at the lesion site and (iii) polyneuronal reinnervation of the neuromuscular junctions (NMJ). The facial nerve transection model in rat and mice has been used to measure the restoration of function after varying therapies and to examine the mechanisms underlying their effects. Since it is still very difficult to combat the first reason and control/correct the navigation of several thousand axons, several groups of scientists concentrated our efforts on the postlesional branching (extremely strong in adult rats) and NMJ-polyinnervation. Since polyneuronal innervation of muscle fibers is activity-dependent attempts to reduce it were performed applying electrical stimulation. Unfortunately, the highly recommended intraoperative electrical stimulation of the proximal nerve fragment (square 0.1 ms pulses at 20 Hz using suprathreshold amplitudes) prior to suture and the postoperative electrical stimulation (square 0.1 ms pulses at 5 Hz) not only did not improve functional outcome, but reduced the number of innervated NMJ to approximately one fifth of normal values. Finally, recent experiments demonstrated that it was the mechanical (but not electrical) stimulation of denervated facial muscles (vibrissal and orbicularis oculi) which improved motor performance. This beneficial effect of mechanical stimulation was also detected after hypoglossal-facial anastomosis and inter-positional nerve grafting. The beneficial effect of manual stimulation on target muscle reinnervation was not present in mice deficient in the expression of insulin-like growth factor 1 and also eliminated or even reversed when trigeminal afferent inputs were abolished. All these findings raise hopes that clinically feasible and effective therapies could be soon designed and tested.

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

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. We tested whether ES has a positive influence on functional recovery after repair of a purely motor nerve, the facial nerve. 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. 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. Whereas acute ES is not beneficial for facial nerve repair, MS provides long-term benefits.