Michael J. Brenner

Controlled release of nerve growth factor enhances sciatic nerve regeneration

Based on previous studies demonstrating the potential of growth factors to enhance peripheral nerve regeneration, we developed a novel growth factor delivery system to provide sustained delivery of nerve growth factor (NGF). This delivery system uses heparin to immobilize NGF and slow its diffusion from a fibrin matrix. This system has been previously shown to enhance neurite outgrowth in vitro, and in this study, we evaluated the ability of this delivery system to enhance nerve regeneration through conduits. We tested the effect of controlled NGF delivery on peripheral nerve regeneration in a 13-mm rat sciatic nerve defect. The heparin-containing delivery system was studied in combination with three doses of NGF (5, 20, or 50 ng/mL) and the results were compared with positive controls (isografts) and negative controls (fibrin alone, NGF alone, and empty conduits). Nerves were harvested at 6 weeks postoperatively for histomorphometric analysis. Axonal regeneration in the delivery system groups revealed a marked dose-dependent effect. The total number of nerve fibers at both the mid-conduit level and in the distal nerve showed no statistical difference for NGF doses at 20 and 50 ng/mL from the isograft (positive control). The results of this study demonstrate that the incorporation of a novel delivery system providing controlled release of growth factors enhances peripheral nerve regeneration and represents a significant contribution toward enhancing nerve regeneration across short nerve gaps.

Effects of motor versus sensory nerve grafts on peripheral nerve regeneration.

Autologous nerve grafting is the current standard of care for nerve injuries resulting in a nerve gap. This treatment requires the use of sensory grafts to reconstruct motor defects, but the consequences of mismatches between graft and native nerve are unknown. Motor pathways have been shown to preferentially support motoneuron regeneration. Functional outcome of motor nerve reconstruction depends on the magnitude, rate, and precision of end organ reinnervation. This study examined the role of pathway type on regeneration across a mixed nerve defect. Thirty-six Lewis rats underwent tibial nerve transection and received isogeneic motor, sensory or mixed nerve grafts. Histomorphometry of the regenerating nerves at 3 weeks demonstrated robust nerve regeneration through both motor and mixed nerve grafts. In contrast, poor nerve regeneration was seen through sensory nerve grafts, with significantly decreased nerve fiber count, percent nerve, and nerve density when compared with mixed and motor groups (P < 0.05). These data suggest that use of motor or mixed nerve grafts, rather than sensory nerve grafts, will optimize regeneration across mixed nerve defects.

Binary imaging analysis for comprehensive quantitative histomorphometry of peripheral nerve.

Quantitative histomorphometry is the current gold standard for objective measurement of nerve architecture and its components. Many methods still in use rely heavily upon manual techniques that are prohibitively time consuming, predisposing to operator fatigue, sampling error, and overall limited reproducibility. More recently, investigators have attempted to combine the speed of automated morphometry with the accuracy of manual and semi-automated methods. Systematic refinements in binary imaging analysis techniques combined with an algorithmic approach allow for more exhaustive characterization of nerve parameters in the surgically relevant injury paradigms of regeneration following crush, transection, and nerve gap injuries. The binary imaging method introduced here uses multiple bitplanes to achieve reproducible, high throughput quantitative assessment of peripheral nerve. Number of myelinated axons, myelinated fiber diameter, myelin thickness, fiber distributions, myelinated fiber density, and neural debris can be quantitatively evaluated with stratification of raw data by nerve component. Results of this semi-automated method are validated by comparing values against those obtained with manual techniques. The use of this approach results in more rapid, accurate, and complete assessment of myelinated axons than manual techniques.

The spectrum of complications of immunosuppression: Is the time right for hand transplantation?

• Life-threatening complications of long-term immunosuppression include malignancy, infection, and metabolic disorders such as renal failure and diabetes. • Up to three-fourths or more of patients on chronic immunosuppressive medications experience an infectious complication. • The hand transplants to date have had multiple episodes of acute rejection. • The frequency and timing of episodes of acute rejection, even if the episodes are easily treated, are predictive of chronic allograft dysfunction and failure. • Chronic allograft rejection is not effectively treated with current immunosuppressive medications, and it has become a primary cause of long-term allograft failure. The introduction of cyclosporine A in the early 1980s revolutionized solid organ transplantation. There were marked improvements not only in preserving graft function, but also in prolonging patient survival following solid organ transplantation 1. As the viability of transplanted organs improved, the indications for clinical transplantation expanded to include not only acutely life-threatening conditions, but also more chronic conditions that adversely affected longevity and quality of life. Composite tissue allotransplantation involves the transplantation of nonvital tissues for reconstruction of deficits following trauma or tumor resection. If successful, composite tissue allotransplantation would facilitate the recovery of lost function through grafting of complex, disparate tissue types. However, composite tissue allotransplantation also poses unique challenges. Solid organs, such as the kidneys, may be more tolerant of rejection episodes than are composite tissues, for which the risk of rejection of one or more components is high. Parenchymal organs may have a tremendous functional reserve so that a substantial amount of tissue can be lost before organ function is compromised, but composite tissue allografts tend to have complex architecture with limited built-in redundancy. Thus, while the majority of kidney function may be lost before blood urea nitrogen or creatinine levels are elevated, comparable loss of the function of composite …

Repair of Motor Nerve Gaps With Sensory Nerve Inhibits Regeneration in Rats

Objective: Sensory nerve grafts are often used to reconstruct injured motor nerves, but the consequences of such motor/sensory mismatches are not well studied. Sensory nerves have more diverse fiber distributions than motor nerves and may possess phenotypically distinct Schwann cells. Putative differences in Schwann cell characteristics and pathway architecture may negatively affect the regeneration of motor neurons down sensory pathways. We hypothesized that sensory grafts impair motor target reinnervation, thereby contributing to suboptimal outcomes. This study investigated the effect of motor versus sensory grafts on nerve regeneration and functional recovery.

Use of cold-preserved allografts seeded with autologous Schwann cells in the treatment of a long-gap peripheral nerve injury.

Background: Limitations in autogenous tissue have inspired the study of alternative materials for repair of complex peripheral nerve injuries. Cadaveric allografts are one potential reconstructive material, but their use requires systemic immunosuppression. Cold preservation (≥7 weeks) renders allografts devoid of antigens, but these acellular substrates generally fail in supporting regeneration beyond 3 cm. In this study, the authors evaluated the reconstruction of extensive nonhuman primate peripheral nerve defects using 7-week cold-preserved allografts repopulated with cultured autologous Schwann cells. Methods: Ten outbred Macaca fascicularis primates were paired based on maximal genetic disparity as measured by similarity index assay. A total of 14 ulnar nerve defects measuring 6 cm were successfully reconstructed using autografts (n = 5), fresh allografts (n = 2), cold-preserved allografts (n = 3), or cold-preserved allografts seeded with autogenous Schwann cells (n = 4). Recipient immunoreactivity was evaluated by means of enzyme-linked immunosorbent spot assay, and nerves were harvested at 6 months for histologic and histomorphometric analysis. Results: Cytokine production in response to cold-preserved allografts and cold-preserved allografts seeded with autologous Schwann cells was similar to that observed for autografts. Schwann cell–repopulated cold-preserved grafts demonstrated significantly enhanced fiber counts, nerve density, and percentage nerve (p < 0.05) compared with unseeded cold-preserved grafts at 6 months after reconstruction. Conclusions: Cold-preserved allografts seeded with autologous Schwann cells were well-tolerated in unrelated recipients and supported significant regeneration across 6-cm peripheral nerve defects. Use of cold-preserved allogeneic nerve tissue supplemented with autogenous Schwann cells poses a potentially safe and effective alternative to the use of autologous tissue in the reconstruction of extensive nerve injuries.

Effect of tension on nerve regeneration in rat sciatic nerve transection model.

Excessive tension across a nerve repair is known to impair nerve regeneration. However, it is uncertain whether nerve grafting is necessary when end-to-end repair would result in only mild to moderate tension. This study investigated the effect of tension on nerve regeneration. Sciatic nerves of 48 Lewis rats were transected and then repaired primarily after resection of 0-, 3-, 6-, or 9-mm lengths of nerve. Postoperative tension levels were quantified using a tensometer. Robust nerve regeneration was observed at 4 weeks in all except the 9-mm repair group, which showed lower nerve fiber counts, percent neural tissue, and nerve density (P < 0.05) and decreased functional recovery. These data indicate that modest levels of tension are well tolerated, but nerve regeneration drops precipitously once a critical tension threshold is exceeded. This threshold was between 0.39 and 0.56 N in the model studied, corresponding to a nerve defect between 6 mm and 9 mm.

Motor neuron regeneration through end-to-side repairs is a function of donor nerve axotomy.

Background: Over the past decade, a growing body of literature has emerged supporting the use of end-to-side (terminolateral) neurorrhaphy for the treatment of selected peripheral nerve injuries. It remains unclear, however, whether injury to the donor nerve is necessary to achieve significant regeneration through such repairs. Methods: End-to-side repair was studied in a rodent model in which the terminal limb of a transected peroneal nerve was sutured to the lateral aspect of the tibial nerve. Twenty-eight Lewis rats were randomized to four groups of seven animals each corresponding to incrementally greater donor nerve injuries as follows: group 1, conventional end-to-side neurorrhaphy; group 2, end-to-side neurorrhaphy with proximal crush injury; group 3, end-to-side neurorrhaphy with neurotomy; and group 4, end-to-end repair of transected peroneal nerve (positive control). Results: At 12 weeks, retrograde labeling of cell bodies of the ventral horn demonstrated significant differences between experimental groups, with mean counts in group 4 (1237 ± 171) > group 3 (522 ± 204) > group 2 (210 ± 132) ≥ group 1 (126 ± 146). This association between nerve injury and motor neuron counts was closely mirrored in quantitative assessments of peripheral nerve regeneration and normalized wet muscle masses. Conclusions: These data support the hypothesis that donor nerve injury is a prerequisite for significant motor neuronal regeneration across end-to-side repairs. Motor neuron regeneration through end-to-side repairs is optimized by deliberate transection of donor nerve axons.

Ropivacaine-induced peripheral nerve injection injury in the rodent model.

BACKGROUND: Intraneural administration of local anesthetics has been associated with nerve damage. We undertook the present study to investigate histological changes induced by ropivacaine injection into rat sciatic nerve. METHODS: Fifty-four adult male Lewis rats were randomly distributed into 9 groups, 6 animals per group. Fifty microliters of normal saline, 10% phenol, or 0.75% ropivacaine were administered by intrafascicular injection, extrafascicular injection, or extraneural (topical) placement. At 2 weeks, animals were killed and the sciatic nerve at the injection site was evaluated with light microscopy, quantitative histomorphometry, and electron microscopy. RESULTS: On cross-sectional evaluation, extrafascicular ropivacaine injection and extraneural placement of ropivacaine were both associated with damage to the perineurium, with focal demyelination surrounded by edematous endoneurium. Intrafascicular injection of ropivacaine resulted in a wedge-shaped region of demyelination and focal axonal loss with some regeneration, bordered by a region of normally myelinated axons in a background of edematous endoneurium. Extrafascicular injection resulted in more significant damage than extraneural placement of ropivacaine, but less than intrafascicular injection as shown with quantitative histomorphometry. Quantitatively, ropivacaine-injured specimens had significantly lower nerve density than saline-injured specimens. Wallerian degeneration and perineural edema were also demonstrated qualitatively with electron microscopy. CONCLUSIONS: This study demonstrates that, in the rat model, ropivacaine is associated with marked histological abnormality, including edema of the perineurium and axonal destruction with wallerian degeneration, when injected into or extraneurally placed onto a nerve. Extrafascicular injection and extraneural placement were associated with similar, although milder, histological damage than intrafascicular injection. Further work is needed to investigate the functional implications, if any, of the histological abnormalities observed in this study.

Effect of FK506 on peripheral nerve regeneration through long grafts in inbred swine

Numerous small-animal studies have demonstrated that FK506 enhances nerve regeneration and accelerates functional recovery after nerve injury. However, no experimental study has corroborated these neuroregenerative effects in larger animals. This study investigated the effects of FK506 on nerve regeneration in inbred miniature swine. Eight animals received 8-cm ulnar nerve autografts and allografts. Treated animals received 0.1 to 0.4 mg/kg FK506 injections twice weekly to maintain immunosuppressive serum FK506 levels. At 24 weeks posttransplant, nerve grafts were harvested for histomorphometric analysis. Mixed lymphocyte cultures demonstrated alloreactivity in 1 treated animal and all untreated animals. In autografts, mean fiber count, nerve density, and percent neural tissue were doubled with FK506 therapy. In allografts, significant neuroregeneration was observed in animals treated with FK506, whereas untreated animals had no regeneration. Treatment with FK506 resulted in a trend toward enhanced axonal regeneration through nerve autografts and allografts in a large-animal model with defined histocompatibility barriers.