Follistatin Protein Enhances Satellite Cell Counts in Reinnervated Muscle.

Background Muscle recovery following peripheral nerve repair is sup-optimal. Follistatin (FST), a potent muscle stimulant, enhances muscle size and satellite cell counts following reinnervation when administered as recombinant FST DNA via viral vectors. Local administration of recombinant FST protein, if effective, would be more clinically translatable but has yet to be investigated following muscle reinnervation. Objective The aim of this study is to assess the effect of direct delivery of recombinant FST protein on muscle recovery following muscle reinnervation. Materials and Methods In total, 72 Sprague-Dawley rats underwent temporary (3 or 6 months) denervation or sham denervation. After reinnervation, rats received FST protein (isoform FS-288) or sham treatment via a subcutaneous osmotic pump delivery system. Outcome measures included muscle force, muscle histomorphology, and FST protein quantification. Results Follistatin treatment resulted in smaller muscles after 3 months denervation ( p = 0.019) and reduced force after 3 months sham denervation ( p < 0.001). Conversely, after 6 months of denervation, FST treatment trended toward increased force output ( p = 0.066). Follistatin increased satellite cell counts after denervation ( p < 0.001) but reduced satellite cell counts after sham denervation ( p = 0.037). Conclusion Follistatin had mixed effects on muscle weight and force. Direct FST protein delivery enhanced satellite cell counts following reinnervation. The positive effect on the satellite cell population is intriguing and warrants further investigation.

Implantation of Acellular Nerve Allograft Using Nerve Connectors.

Background: Nerve connectors are short nerve conduits used to approximate nerve ends. Acellular nerve allografts are viable alternatives when direct repair is not possible but do not produce exudate essential for fibrin clot formation. We hypothesize that acellular nerve allograft implanted using nerve connectors must have end-to-end contact with the in situ nerve stumps to support nerve regeneration. Methods: Sixty Sprague Dawley rats underwent a 14-mm unilateral tibial nerve injury and subsequent repair using various combinations of acellular nerve grafts and nerve connectors. Proximal repairs for all groups utilized direct contact with the nerve stump within connector. Variations in distal repair methods (allograft length, nerve gap, and connector length) defined our 4 groups-group A: 14 mm allograft, no distal gap, and distal connector; group B: 11.5 mm allograft, 2.5 mm distal gap, and distal connector; group C: 9 mm allograft, 5 mm distal gap, and distal connector; group D: 14 mm allograft, no distal gap, and no distal connector. At 3 months post-repair, function and histomorphology were assessed. Results: Developed muscle force was significantly lower in group C (0.073 ± 0.077 N) compared with the other 3 groups (group A = 0.529 ± 0.312 N, group B = 0.461 ± 0.462 N, and group D = 0.409 ± 0.327 N). Axon counts were significantly lower in group C (2121 ± 389) compared with group A (6401 ± 855), group B (4710 ± 755), and group D (4450 ± 126). There was no statistically significant difference in G-ratios (myelination) between groups (P > .05). Conclusion: Nerve regeneration was significantly impaired as the gap distance between the distal end of the allograft and the distal nerve stump increased to 5 mm.

Side-to-side supercharging nerve allograft enhances neurotrophic potential.

INTRODUCTION: Critical limitations of processed acellular nerve allograft (PNA) are linked to Schwann cell function. Side-to-side bridge grafting may enhance PNA neurotrophic potential. METHODS: Sprague-Dawley rats underwent tibial nerve transection and immediate repair with 20-mm PNA (n = 33) or isograft (ISO; n = 9) or 40-mm PNA (n = 33) or ISO (n = 9). Processed acellular nerve allograft groups received zero, one, or three side-to-side bridge grafts between the peroneal nerve and graft. Muscle weight, force generation, and nerve histomorphology were tested 20 weeks after repair. Selected animals underwent neuron back labeling with fluorescent dyes. RESULTS: Inner axon diameters, g-ratios, and axon counts were smaller in the distal vs proximal aspect of each graft (P < .05). Schwann cell counts were greater, with a lower proportion of senescent cells for groups with bridges (P < .05). Retrograde labeling demonstrated that 6.6% to 17.7% of reinnervating neurons were from the peroneal pool. DISCUSSION: Bridge grafting positively influenced muscle recovery and Schwann cell counts and senescence after long PNA nerve reconstruction.

Melatonin Effects on Non-Alcoholic Fatty Liver Disease Are Related to MicroRNA-34a-5p/Sirt1 Axis and Autophagy.

Melatonin, an indole produced by pineal and extrapineal tissues, but also taken with a vegetarian diet, has strong anti-oxidant, anti-inflammatory and anti-obesogenic potentials. Non-alcoholic fatty liver disease (NAFLD) is the hepatic side of the metabolic syndrome. NAFLD is a still reversible phase but may evolve into steatohepatitis (NASH), cirrhosis and carcinoma. Currently, an effective therapy for blocking NAFLD staging is lacking. Silent information regulator 1 (SIRT1), a NAD+ dependent histone deacetylase, modulates the energetic metabolism in the liver. Micro-RNA-34a-5p, a direct inhibitor of SIRT1, is an emerging indicator of NAFLD grading. Thus, here we analyzed the effects of oral melatonin against NAFLD and underlying molecular mechanisms, focusing on steatosis, ER stress, mitochondrial shape and autophagy. Male C57BL/6J (WT) and SIRT1 heterozygous (HET) mice were placed either on a high-fat diet (58.4% energy from lard) (HFD) or on a standard maintenance diet (8.4% energy from lipids) for 16 weeks, drinking melatonin (10 mg/kg) or not. Indirect calorimetry, glucose tolerance, steatosis, inflammation, ER stress, mitochondrial changes, autophagy and microRNA-34a-5p expression were estimated. Melatonin improved hepatic metabolism and steatosis, influenced ER stress and mitochondrial shape, and promoted autophagy in WT HFD mice. Conversely, melatonin was ineffective in HET HFD mice, maintaining NASH changes. Indeed, autophagy was inconsistent in HET HFD or starved mice, as indicated by LC3II/LC3I ratio, p62/SQSTM1 and autophagosomes estimation. The beneficial role of melatonin in dietary induced NAFLD/NASH in mice was related to reduced expression of microRNA-34a-5p and sterol regulatory element-binding protein (SREBP1) but only in the presence of full SIRT1 availability.

Viral vector delivery of follistatin enhances recovery of reinnervated muscle.

INTRODUCTION: Poor recovery following nerve repair is due to progressive temporal loss of muscle function. Follistatin (FS), a glycoprotein with anabolic properties, may enhance muscle recovery following reinnervation. METHODS: Seventy-two male Sprague-Dawley rats underwent temporary (3 or 6 month) denervation or sham denervation. After reinnervation, rats were administered adeno-associated viral vectors expressing FS deoxyribonucleic acid (isoform FS-317) injected into the target muscle or sham treatment. Final assessment included muscle function testing, muscle histomorphology, nerve histomorphology, and FS protein quantification. RESULTS: FS improved muscle mass and type IIB muscle fiber size, and increased G-ratios and mean axon diameter in the 6-month temporary denervation group (P < .05). Elevated FS protein levels were detected in treated muscle (P < .05). FS increased satellite cell counts following temporary denervation and repair (P < .05). DISCUSSION: FS treatment had anabolic, neurotrophic, and satellite cell stimulatory effects when administered following prolonged (6-month) temporary denervation and repair.

Effect of Reverse End-to-Side (Supercharging) Neurotization in Long Processed Acellular Nerve Allograft in a Rat Model.

PURPOSE: Processed acellular nerve allograft (PNA) has been suggested as a convenient tool for overcoming short and medium nerve defects. Although the clinical implications are unclear, animal data suggest that PNA becomes less effective at longer lengths. Although reverse or supercharging end-to-side nerve transfer may improve the neurotrophic potential in chronically denervated nerve tissue, the application of this strategy to long acellular nerve allograft has not been previously investigated. We hypothesized that supercharging acellular nerve allograft would increase its effective length. METHODS: Sprague-Dawley and Thy1-green fluorescent protein Sprague-Dawley rats underwent transection of the tibial nerve, followed by immediate repair with 20-, 40-, or 60-mm acellular nerve allografts processed identically to commercially available human acellular nerve allograft (AxoGen, Inc., Alachua, FL) or isograft. Half of the allograft group was supercharged with a reverse end-to-side transfer from the ipsilateral peroneal nerve. At 10 weeks, the reconstructed nerve in the Thy1-green fluorescent rat groups were exposed and examined under a fluorescence-enabled microscope. At 20 weeks, the remaining rats underwent motor testing and tissue harvest for morphological examination. RESULTS: In comparison with a nonenhanced allograft, supercharging had a statistically significant positive impact on the reinnervated muscle normalized force generation and distal axon counts for all graft sizes. Muscles in the supercharged group were heavier than those in the allograft group for the 40-mm-length grafts and G-ratio measurements were higher in the supercharged allograft group for 60-mm-length grafts only. CONCLUSIONS: This study supports that hypothesis that supercharging nerve transfer improves axon regeneration within PNA. CLINICAL RELEVANCE: When an appropriate donor nerve is available, supercharging nerve transfer may improve nerve regeneration in PNA across long nerve defects.

Tendon Adhesions: A Novel Method of Objectively Measuring Adhesions by Assessing Tendon Glide Through a Soft Tissue Envelope in a Rat Model.

PURPOSE: To develop a rat model of extra-synovial tendon adhesions that will enable accurate testing of scar barriers and adhesion inhibiting treatments to facilitate future research. METHODS: Thirty-six 6-month-old male Sprague-Dawley rats were randomized to one of the 3 groups of 12. In Group A, the middle one-third portion of the left Achilles tendon was excised. In Group B, the tendon and soft tissue bed was abraded with steel wool. In Group C, a silk suture was sewn along the tendon. The right hind limbs served as controls. At 4 weeks, biomechanical testing was performed on the bilateral hind limbs. The Achilles tendon was cut at the gastrocnemius-tendon junction proximal to the "adhesion zone" (or analogous level in the control limb). The calcaneal insertion of the Achilles was attached to a tensiometer. The force needed to pull the tendon out of its soft tissue envelope at a fixed rate was measured. RESULTS: Three rats were excluded because of complications during data collection. Pair-wise comparison testing was performed, comparing the mean peak force to pull the Achilles tendon from its soft tissue envelope in 33 control limbs and the contralateral limb from each group. The average peak force for the cut tendon group (A) was 20.1 N, 18.8 N in the steel wool group (B), and 21.1 N in the suture group (C). The average peak force in the control limbs was 15.6 N. There was a significant difference noted in peak forces between the control limbs and each experimental group. CONCLUSIONS: A consistent and statistically increased force was necessary to pull a rodent Achilles tendon from an adhesion-induced tissue bed compared with controls. No statistical difference was detected between experimental groups. CLINICAL RELEVANCE: Our study demonstrates an objective method of biomechanical tendon adhesion assessment in a rat model.

Micropuncture and pressure assisted Schwann cell seeding of nerve allograft.

BACKGROUND: Tissue processing to create immunotolerant nerve allograft removes neurosupportive cells. Few strategies have been described for implanting new cells into the graft to support axonal regeneration. NEW METHOD: Micropuncture of the nerve allograft surface combined with immersion into a pressurized cell-rich solution to potentiate the introduction of viable Schwann cells (SC) into processed nerve allograft. Allografts were used to repair rodent sciatic nerve defects. At 3, 7, and 21days, grafts were harvested, stained for SCs, and analyzed using total cross sectional area (CSA) occupied by SCs to quantify SC presence. RESULTS: At days 3 and 7, SC CSA was significantly greater for the injection group compared to all other groups. At day 21, SC CSA for the injection group (0.2252%±0.2730) was significantly greater compared to following groups: pressurized-punctured (0.0653%±0.0934), nonpressurized-nonpunctured (0.0607%±0.0709), punctured-control (0.0246%±0.0398), and nonpunctured-control (0.0126%±0.0151). A significant decrease in percent CSA occupied by SCs from day 3 to day 21 was noted in nonpressurized-punctured group (p=0.0106), pressurized-nonpunctured group (p=0.0477), and injection group (p=0.0010). COMPARISON WITH EXISTING METHOD(S): Most studies have used small caliber hypodermic needles to inject the cells into grafts. CONCLUSIONS: Despite a presumed decrease in cell viability over the three weeks of the study, the large initial inoculum achieved by injection technique results in higher levels of final SC seeding in acellular nerve allograft compared with bathing techniques with or without micropuncture or pressurization.

Does partial muscle reinnervation preserve future re-innervation potential?

INTRODUCTION: Late revision nerve surgery for incomplete motor recovery due to partial reinnervation would improve muscle function if all muscle fibers were protected from developing denervation atrophy. METHODS: Sixty immature Sprague-Dawley rats underwent the following tibial nerve manipulations (n = 15/group): group A, partial denervation (two thirds of nerve resected and the remaining one third crushed), revision repair at 8 months; group B, partial denervation; group C, complete denervation, immediate reconstruction; group D, complete denervation, reconstruction at 8 months; and group E, control. Final testing at 11 months included muscle force, weight, and histology. RESULTS: Muscle weight was significantly (P < 0.05) different among all groups (highest to lowest: E > B > C > A > D), and force was significantly lower in groups A and D compared with E. Muscle fiber cross-sectional area was statistically smaller in group A than in groups B, C, or E. DISCUSSION: Partial reinnervation still allowed substantial muscle recovery, but it did not preserve the non-innervated muscle fibers. Muscle Nerve 56: 1143-1148, 2017.

Introduction of neurosupportive cells into processed acellular nerve allografts results in greater number and more even distribution when injected compared to soaking techniques.

OBJECTIVES: Processing necessary to remove immunogenic components of nerve allograft renders it acellular. Seeding with supportive cells may improve axon regeneration. We aim to identify the method associated with implantation of the greatest volume and most even distribution of cells. METHODS: Hypodermic needle injection was compared to soaking in solution under both normal and pressurized conditions after micropuncture of the allograft. Distribution within the allograft was measured using an in vitro model of fluorescent beads, as well as cultured Schwann cells. RESULTS: Injection treatment resulted in larger volumes and a more uniform cross-sectional distribution of implanted cells. Beads and cells behaved similarly relative to the measured outcomes. CONCLUSIONS: Injection instills more cells in a more uniform distribution. In vivo testing may evaluate whether these techniques vary relative to cell survival, cell migration, and clinical outcomes. Size- and concentration-matched fluorescent beads may represent a viable model for analyzing cell implantation.

Guiding intracortical brain tumour cells to an extracortical cytotoxic hydrogel using aligned polymeric nanofibres.

Glioblastoma multiforme is an aggressive, invasive brain tumour with a poor survival rate. Available treatments are ineffective and some tumours remain inoperable because of their size or location. The tumours are known to invade and migrate along white matter tracts and blood vessels. Here, we exploit this characteristic of glioblastoma multiforme by engineering aligned polycaprolactone (PCL)-based nanofibres for tumour cells to invade and, hence, guide cells away from the primary tumour site to an extracortical location. This extracortial sink is a cyclopamine drug-conjugated, collagen-based hydrogel. When aligned PCL-nanofibre films in a PCL/polyurethane carrier conduit were inserted in the vicinity of an intracortical human U87MG glioblastoma xenograft, a significant number of human glioblastoma cells migrated along the aligned nanofibre films and underwent apoptosis in the extracortical hydrogel. Tumour volume in the brain was significantly lower following insertion of aligned nanofibre implants compared with the application of smooth fibres or no implants.

Molecular sequelae of topographically guided peripheral nerve repair.

Peripheral nerve injuries cause severe disability with decreased nerve function often followed by neuropathic pain that impacts the quality of life. Even though use of autografts is the current gold standard, nerve conduits fabricated from electrospun nanofibers have shown promise to successfully bridge critical length nerve gaps. However, in depth analysis of the role of topographical cues in the context of spatio-temporal progression of the regenerative sequence has not been elucidated. Here, we explored the influence of topographical cues (aligned, random, and smooth films) on the regenerative sequence and potential to successfully support nerve regeneration in critical size gaps. A number of key findings emerged at the cellular, cytokine and molecular levels from the study. Higher quantities of IL-1α and TNF-α were detected in aligned fiber based scaffolds. Differential gene expression of BDNF, NGFR, ErbB2, and ErbB3 were observed suggesting a role for these genes in influencing Schwann cell migration, myelination, etc. that impact the regeneration in various topographies. Fibrin matrix stabilization and arrest of nerve-innervated muscle atrophy was also evident. Taken together, our data shed light on the cascade of events that favor regeneration in aligned topography and should stimulate research to further refine the strategy of nerve regeneration using topographical cues.

Effect of modulating macrophage phenotype on peripheral nerve repair.

Peripheral nerve repair across long gaps remains clinically challenging despite progress made with autograft transplantation. While scaffolds that present trophic factors and extracellular matrix molecules have been designed, matching the performance of autograft-induced repair has been challenging. In this study, we explored the effect of cytokine mediated 'biasing' of macrophage phenotypes on Schwann cell (SC) migration and axonal regeneration in vitro and in vivo. Macrophage phenotype was successfully modulated by local delivery of either Interferon-gamma (IFN-γ) or Interleukin-4 (IL-4) within polymeric nerve guidance channels, polarizing them toward pro-inflammatory (M1) or pro-healing (M2a and M2c) phenotypes, respectively. The initial polarization of macrophages to M2a and M2c phenotype results in enhanced SC infiltration and substantially faster axonal growth in a critically-sized rat sciatic nerve gap model (15 mm). The ratio of pro-healing to pro-inflammatory population of macrophages (CD206+/CCR7+), defined as regenerative bias, demonstrates a linear relationship with the number of axons at the distal end of the nerve scaffolds. The present results clearly suggest that rather than the extent of macrophage presence, their specific phenotype at the site of injury regulates the regenerative outcomes.