Functional electrical stimulation following nerve injury in a large animal model.

INTRODUCTION: Controversy exists over the effects of functional electrical stimulation (FES) on reinnervation. We hypothesized that intramuscular FES would not delay reinnervation after recurrent laryngeal nerve (RLn) axonotmesis. METHODS: RLn cryo-injury and electrode implantation in ipsilateral posterior cricoarytenoid muscle (PCA) were performed in horses. PCA was stimulated for 20 weeks in eight animals; seven served as controls. Reinnervation was monitored through muscle response to hypercapnia, electrical stimulation and exercise. Ultimately, muscle fiber type proportions and minimum fiber diameters, and RLn axon number and degree of myelination were determined. RESULTS: Laryngeal function returned to normal in both groups within 22 weeks. FES improved muscle strength and geometry, and induced increased type I:II fiber proportion (p = 0.038) in the stimulated PCA. FES showed no deleterious effects on reinnervation. DISCUSSION: Intramuscular electrical stimulation did not delay PCA reinnervation after axonotmesis. FES can represent a supportive treatment to promote laryngeal functional recovery after RLn injury. Muscle Nerve 59:717-725, 2019.

Delay modulates the immune response to nerve repair.

Effective regeneration after peripheral nerve injury requires macrophage recruitment. We investigated the activation of remodeling pathways within the macrophage population when repair is delayed and identified alteration of key upstream regulators of the inflammatory response. We then targeted one of these regulators, using exogenous IL10 to manipulate the response to injury at the repair site. We demonstrate that this approach alters macrophage polarization, promotes macrophage recruitment, axon extension, neuromuscular junction formation, and increases the number of regenerating motor units reaching their target. We also demonstrate that this approach can rescue the effects of delayed nerve graft.

Revitalizing the common peroneal function index for assessing functional recovery following nerve injury.

BACKGROUND AND AIMS: Peripheral nerve injury is common with poor functional recovery and consequent high personal and societal costs. Sciatic nerve transection and assessment of recovery using sciatic functional index (SFI) are widely used. SFI is biologically limited as axonal misdirection of axons supplying flexors and extensors in the hindlimb, after nerve injury can lead to synkinetic innervation and function which does not correspond to the degree of axonal regeneration. METHODS: We reevaluated the use of traditional metrics such as print length (PL), toe spread (TS), and intermediate toe spread (ITS) as well as hock angle at mid-swing as approaches for determining recovery. We used two alternative approaches in discrete cohorts of rats following common peroneal crush injury, transection with repair and critical gap, using transection with ligation as a negative control. We compared walking track analysis (print) with digital capture and kinematics. RESULTS: PL, TS, and ITS varied as expected after injury. The traditional functional index for common peroneal injury using inked prints failed to describe recovery and we derived new indices to describe recovery (all R2  > 0.88, p < .0001) although pre-injury PFI was never attained by any of the models. Kinematic analysis identified hock angle at mid-swing as a useful predictor of recovery (p < .0001). INTERPRETATION: Using complementary approaches.

Approach for semi-automated measurement of fiber diameter in murine and canine skeletal muscle.

Currently available software tools for automated segmentation and analysis of muscle cross-section images often perform poorly in cases of weak or non-uniform staining conditions. To address these issues, our group has developed the MyoSAT (Myofiber Segmentation and Analysis Tool) image-processing pipeline. MyoSAT combines several unconventional approaches including advanced background leveling, Perona-Malik anisotropic diffusion filtering, and Steger's line detection algorithm to aid in pre-processing and enhancement of the muscle image. Final segmentation is based upon marker-based watershed segmentation. Validation tests using collagen V labeled murine and canine muscle tissue demonstrate that MyoSAT can determine mean muscle fiber diameter with an average accuracy of ~92.4%. The software has been tested to work on full muscle cross-sections and works well even under non-optimal staining conditions. The MyoSAT software tool has been implemented as a macro for the freely available ImageJ software platform. This new segmentation tool allows scientists to efficiently analyze large muscle cross-sections for use in research studies and diagnostics.

RetroDISCO: Clearing technique to improve quantification of retrograde labeled motor neurons of intact mouse spinal cords.

BACKGROUND: Quantification of the number of axons reinnervating a target organ is often used to assess regeneration after peripheral nerve repair, but because of axonal branching, this method can overestimate the number of motor neurons regenerating across an injury. Current methods to count the number of regenerated motor neurons include retrograde labeling followed by cryosectioning and counting labeled motor neuron cell bodies, however, the process of sectioning introduces error from potential double counting of cells in adjacent sections. NEW METHOD: We describe a method, retroDISCO, that optically clears whole mouse spinal cord without loss of fluorescent signal to allow imaging of retrograde labeled motor neurons using confocal microscopy. RESULTS: Complete optical clearing of spinal cords takes four hours and confocal microscopy can obtain z-stacks of labeled motor neuron pools within 3-5min. The technique is able to detect anticipated differences in motor neuron number after cross-suture and conduit repair compared to intact mice and is highly repeatable. COMPARISON WITH EXISTING METHOD: RetroDISCO is inexpensive, simple, robust and uses commonly available microscopy techniques to determine the number of motor neurons extending axons across an injury site, avoiding the need for labor-intensive cryosectioning and potential double counting of motor neuron cell bodies in adjacent sections. CONCLUSIONS: RetroDISCO allows rapid quantification of the degree of reinnervation without the confounding produced by axonal sprouting.