Search

Peripheral nerve injuries (PNIs) that consist of simple nerve severance often result in severe motor impairment and permanent loss of function. Such patients face significant costs and pose major burdens to healthcare systems. Currently, the most promising surgical technique to achieve the best clinical outcome after such PNIs is immediate primary coaptation of severed nerve ends by microsutures (neurorrhaphy). However, recovery is often poor and delayed for many months due to Wallerian degeneration (WD) and slow (1–2 mm/day) axonal outgrowths from severed proximal axons that may not properly reinnervate denervated afferent/efferent targets that have atrophied. In contrast, recent pre-clinical studies using polyethylene glycol (PEG) to facilitate primary nerve repair have greatly improved the rate and extent of sensory and motor recovery and prevented much WD and muscle atrophy. That is, PEG-fused axons rapidly establish proximal–distal axoplasmic/axolemmal continuity, which do not undergo WD and maintain the structure and function of neuromuscular junction (NMJ). PEG-fused axons rapidly reinnervate denervated NMJs, thereby preventing muscle atrophy associated with monthslong denervation due to slowly regenerating axonal outgrowths. We now describe PEG-mediated fusion repair of a digital nerve in each of two patients presenting with a digital laceration resulting in total loss of sensation. The first patient’s tactile perception improved markedly at 3 days postoperatively (PO). Two-point discrimination improved from greater than 10 mm at initial presentation to 4 mm at 11-week PO, and the Semmes–Weinstein monofilament score improved from greater than 6.65 to 2.83 mm, a near-normal level. The second patient had severe PO edema and scar development requiring a hand compression glove and scar massage, which began improving at 11-week PO. The sensory function then improved for 4 months PO, with both two-point discrimination and Semmes–Weinstein scores approaching near-normal levels at the final follow-up. These case study data are consistent with data from animal models. All these data suggest that PEG-fusion technologies could produce a paradigm shift from the current clinical practice of waiting days to months to repair ablation PNIs with autografts, anucleated nerve allografts, or conduits in which the patient outcome is solely dependent upon axon regeneration over months or years.

Additional file 1: Fig. S1. (FigS1_Exp_Design.pdf). Outline of the experimental design of the study. PEG-fused sciatic nerve allografts (“PEG”, n=3 animals), negative control sciatic nerve allografts (“NC”, n=3 animals) from outbred Sprague Dawley rats were excised and sampled 14d post-operatively. Both treatment groups were compared to Unoperated Control sciatic nerves (“Unop”, n=2 animals). Total RNA was extracted from each sample via homogenization with TRIzol (Ambion) and a RNeasy Mini kit (Qiagen). Libraries were prepared using a TruSeq Stranded mRNA kit (Illumina), which were then sequenced on a HiSeq 3000 unit (Illumina). Analysis of read results was carried out sequentially by FastQC, Tophat2, HTSeq-count, and DESeq2 software. Subsequent analyses were performed via DAVID and the STRING application for Cytoscape software. Validation of candidate genes was performed via RT-qPCR, using the same treatment groups.

Additional file 7: Fig. S3. (FigS3_BiNGO_Diagram_Clusters.pdf). Enlarged versions of the BiNGO cluster diagrams displayed in Figs. 3A-D, in which each cluster can be zoomed-in on with clarity to view all associated GO annotations and their interrelations. (Page 1) Cluster 1 (enriched in PEG); (Page 2) Cluster 2 (enriched in PEG and Unop); (Page 3) Cluster 3 (enriched in Unop); (Page 4) Cluster 4 (enriched in NC).

Additional file 3: Fig. S2. (FigS2_Count_Analyses.pdf). Distribution and correlation of normalized read counts among samples. (A) Violin plots showing the distribution of all log2 normalized read counts for each gene transcript in each sample. The median is indicated by a solid horizontal line, and the upper quartile is indicated by a dashed horizontal line. The plots show that the majority of genes in each sample share similar expression profiles and are not differentially expressed. (B) Principle component analysis (PCA) allowing clustering of samples based on variation in expression profiles of all protein coding genes. Samples with similar expression profiles cluster together more closely. (C) Sample-to-sample correlation matrix comparing the gene expression profiles of all coding genes between all individual samples, using normalized read counts. White indicates a Pearson correlation coefficient of

Complete crush or cut severance of sciatic nerve axons in rats and other mammals produces immediate loss of axonal continuity. Loss of locomotor functions subserved by those axons is restored only after months, if ever, by outgrowths regenerating at ∼1 mm/day from the proximal stumps of severed axonal segments. The distal stump of a severed axon typically begins to degenerate in 1-3 days. We recently developed a polyethylene glycol (PEG) fusion technology, consisting of sequential exposure of severed axonal ends to hypotonic Ca(2+) -free saline, methylene blue, PEG in distilled water, and finally Ca(2+) -containing isotonic saline. This study examines factors that affect the PEG fusion restoration of axonal continuity within minutes, as measured by conduction of action potentials and diffusion of an intracellular fluorescent dye across the lesion site of rat sciatic nerves completely cut or crush severed in the midthigh. Also examined are factors that affect the longer-term PEG fusion restoration of lost behavioral functions within days to weeks, as measured by the sciatic functional index. We report that exposure of cut-severed axonal ends to Ca(2+) -containing saline prior to PEG fusion and stretch/tension of proximal or distal axonal segments of cut-severed axons decrease PEG fusion success. Conversely, trimming cut-severed ends in Ca(2+) -free saline just prior to PEG fusion increases PEG fusion success. PEG fusion prevents or retards the Wallerian degeneration of cut-severed axons, as assessed by measures of axon diameter and G ratio. PEG fusion may produce a paradigm shift in the treatment of peripheral nerve injuries. © 2016 Wiley Periodicals, Inc.