Jonathan Isaacs

Overcoming short gaps in peripheral nerve repair: conduits and human acellular nerve allograft

Nerve conduits and acellular nerve allograft offer efficient and convenient tools for overcoming unexpected gaps during nerve repair. Both techniques offer guidance for migrating Schwann cells and axonal regeneration though utilizing very different scaffolds. The substantially greater amount of animal and clinical data published on nerve conduits is marked by wide discrepancies in results that may be partly explained by a still poorly defined critical repair gap and diameter size. The available information on acellular allografts appears more consistently positive though this tool is also hampered by a longer but also limited critical length. This article reviews the current relative literature and examines pertinent parameters for application of both acellular allograft and nerve conduits in overcoming short nerve gaps.

Modification of commercially available image analysis software for semi-automated qualitative analysis of axon regeneration and myelination in the rat sciatic nerve.

BACKGROUND Manual measurement of three routine parameters of axon regeneration and myelination, such as axon counts, axon caliber and G-Ratio, is tedious and time consuming. An automated or semi-automated computer program could improve the efficiency of this process. The concern, however, is that the automated method will lack the accuracy of manual counting and measuring. METHOD We introduce semi-automated axon analysis software utilizing customized Image Pro Plus software designed to identify, count, and measure axons and their surrounding myelin sheaths. Histologic specimens were subsequently analyzed using either conventional manual techniques (manual counting and computer assisted measurements) or this semi-automated software (software performed counting and measurements with human supervision and adjustment of axon identification). RESULTS G-Ratios, axon numbers, and axon diameter values did not differ between the two methods though the semi-automated method took approximately 50% less time. COMPARISON WITH EXISTING METHODS Other described semi-automated nerve analysis tools require complex, original software development. CONCLUSIONS Commercially available software can be modified to improve nerve histomorphology analysis time while maintaining accuracy.

Consequences of oversizing: nerve-to-nerve tube diameter mismatch.

BACKGROUND Although commercially available nerve conduits are an accepted tool for overcoming short gaps in peripheral nerve repair, unexplained inconsistencies in clinical outcomes are not uncommon. Although exceeding the critical gap size and nerve caliber are most frequently cited as the source of these failures, oversizing of the nerve conduit in relation to the nerve diameter may be a previously unrecognized factor as well. METHODS Sixty female Sprague-Dawley rats underwent excision of a 10-mm section midway between the sciatic notch and the sciatic nerve division of one hindlimb. The defect was immediately repaired by reversing the resected nerve section and suturing it back in place (reverse autograft) (group A, n = 13) or it was repaired with a 14-mm nerve tube of 3 mm in diameter (group B, n = 12), 2 mm in diameter (group C, n = 15), or 1.5 mm in diameter (group D, n = 15). At twelve weeks, the rodents underwent muscle strength testing before harvest of muscle and nerve (including the conduit) for histomorphologic assessment. RESULTS Most conduits from group B were collapsed at final inspection and demonstrated diminished nerve regenerate. Muscle atrophy was most pronounced in groups B and C (p < 0.05), although normalized muscle contraction force was weakest in group B (p < 0.05), indicating inferior reinnervation. The axon counts, axon diameter, and G-ratios at the midpart of the conduit or graft demonstrated more axons and lower G-ratios in the autologous graft group. Among the conduit groups, the axon counts were lowest in group B (p < 0.05) followed by group C (p < 0.05). The G-ratio was highest in group B (p < 0.05), although the axon diameter was highest in group B (p < 0.05) as well. CONCLUSIONS Repair of a 10-mm gap in a rodent nerve with an oversized, poorly fitted nerve conduit resulted in tube collapse, poor nerve regenerate, and decreased muscle reinnervation compared with the findings in the animals treated with more accurately fitted nerve conduits. CLINICAL RELEVANCE Accurate sizing of nerve conduits to the nerve-stump diameter improves nerve recovery.

A rodent model of partial muscle re-innervation.

BACKGROUND There is limited appreciation of the risks and benefits of the few salvage treatment options available for inadequate motor function following incomplete spontaneous recovery or surgical repair of major peripheral nerve injuries. The lack of a reliable and economical animal model has hindered laboratory investigation into this difficult clinical problem. We propose a straightforward and reproducible rodent model of partial re-innervation of a hind limb muscle. NEW METHOD Twelve Sprague-Dawley rats underwent identical surgical manipulations: the left tibial nerve was isolated, partially transected (2/3rds), and the remaining intact portion crushed. Eight weeks later, bilateral (1) gastrocnemius (2) soleus and (3) flexor digitorum longus muscles underwent maximal isometric contraction force testing before being excised and weighed. RESULTS Only the gastrocnemius muscles were statistically weaker (p<0.05) in the experimental limb compared with the contralateral (control) limb. There was no difference in muscle weights between experimental and control sides. COMPARISON WITH EXISTING METHODS Our model differs from other published models by: allowing time for compensatory axonal sprouting as would be seen in clinical scenarios, precisely identifying the portion of the tibial nerve to be transected to ensure reproducibility, and achieving temporary but complete denervation by crushing the intact portion of the nerve. CONCLUSIONS Controlled partial transection and crushing of the rodent tibial nerve results in a consistently partially re-innervated and clinically weakened gastrocnemius muscle that can serve as a model in studying incomplete recovery following nerve injury.

Athymic rat model for studying acellular human allograft.

BACKGROUND Although human acellular nerve allograft is a promising nerve repair tool, optimizing graft application and understanding effective graft dimensions has been hampered by lack of an appropriate animal model. Rodent nerve acellular allograft can be tested in the utilitarian rodent nerve repair model, but testing different size options is limited by the size of the rodent donor animal. Human acellular nerve allograft offers the variety of sizes desired for more complete study but poses a high risk of rejection as xenograft tissue in the rodent model. Athymic nude rats are less prone to reject xenograft tissue due to their immunocompromised state and may offer an animal model for testing human acellular allograft. METHODS Fifteen athymic nude and 15 Sprague-Dawley rats underwent unilateral excision and repair of a 10mm tibial nerve segment using 10mm of human acellular nerve graft. Testing at 3 months consisted of muscle force measurements, wet muscle weight, and histological assessment from the middle of the nerve grafts. RESULTS Athymic rats repaired with human acellular xenograft demonstrated higher reinnervated muscle weight Gross inspection of the xenograft in euthymic rats revealed a brown and scarred center and histological inspection demonstrated larger axon diameters, and higher midgraft axon counts in the grafts of athymic rats. COMPARISON WITH EXISTING METHODS The athymic rat has been used in many studies that require an immunocompromised host, including implantation of foreign nervous tissue. Previous attempts at implanting acellular nerve xenograft into immunocompetent rats have yielded suboptimal results when compared to allograft. This study is the first to test acellular human nerve allograft in an athymic rat. CONCLUSION The nerve regeneration was better in human acellular nerve allograft implanted into immunocompromised athymic rats when compared to euthymic rats supporting a potential role of this model in studying acellular human nerve tissue.

Revision of Failed Distal Interphalangeal Arthrodesis Complicated by Retained Headless Screw

Arthrodesis of the distal interphalangeal joint is a well-accepted treatment for painful arthritis. Although headless screw fixation across the distal interphalangeal joint is an overall effective method for achieving bony union, failures and nonunions still occur. We present a salvage technique to address both the retained headless screw within the medullary canal as well as the persistent nonunion.

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.

Soft Tissue Atrophy Related to Corticosteroid Injection: Review of the Literature and Implications for Hand Surgeons

Corticosteroid injections (CIs) are frequently used by hand surgeons to treat a wide range of pathology including de Quervain tenosynovitis and lateral epicondylitis. Although generally viewed as a benign modality, and a way to potentially avoid or postpone surgical intervention, common complications from CI should be considered and discussed with patients before the procedure. One such complication is local soft tissue atrophy and hypopigmentation after injection. We discuss the incidence of soft tissue-related adverse effects from CI, the pathophysiology and influence of different steroid preparations on soft tissues, and potential treatment options once atrophy has occurred.

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

Abstract 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.

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.