Carbonic anhydrase activity of human peripheral nerves: a possible histochemical aid to nerve repair

The return of usable function after injury of peripheral nerves depends upon the appropriate regeneration of axons to their end organs. Debridement trimmings of severed nerves harvested during surgery were stained to demonstrate carbonic anhydrase activity. This histochemical method can be accomplished within 3 to 4 hours of receiving the tissue. Nerve fascicles were readily discriminated from one another by the individual staining patterns of their constituent axons. Axoplasmic staining was predominantly a feature of sensory fibers, and myelin staining was characteristic of skeletal motor axons. Carbonic anhydrase histochemistry may provide a means of accurately matching fascicles in cut nerve ends.     

Techniques of peripheral nerve repair.

At present, the principles of microsurgical reconstruction of the peripheral nerve incorporate a clear understanding of the pathophysiology of the peripheral nerve, accurate preoperative assessment of the lesion, aggressive early treatment to avoid irreversible atrophy of the end organ, use of nontraumatic microtechniques for optimal alignment of individual fascicular bundles, introduction of a minimum amount of foreign material at the suture line, resection of the scar-producing epineurium, total avoidance of tension at the suture line, and placement of the nerve repair in a well-vascularized soft tissue bed. If tension is eliminated, a minimal amount of suture material is required to repair the nerve ends, because the bundles are maintained in anatomical alignment by a fibrin clot. We have reviewed the various nerve repair methods, stressing that with strict attention to microsurgical technique, the surgeon can hope to maximize reinnervation. Although the importance of all aspects of careful surgical technique cannot be overemphasized, we believe that it is unlikely that improved clinical results will come from further refinements in microsurgical techniques. We are not limited by a working knowledge and understanding of the details of the neurobiology and the neurochemistry of nerve regeneration.     

Engineering strategies for peripheral nerve repair

Tissue engineering in the peripheral nervous system unites efforts by physicians, engineers, and biologists to create either natural or synthetic tubular nerve guidance channels as alternatives to nerve autografts for the repair of peripheral nerve defects. Guidance channels help direct axons sprouting from the regenerating nerve end, provide a conduit for diffusion of neurotropic and neurotrophic factors secreted by the damaged nerve stumps, and minimize infiltration of fibrous tissue. In addition to efforts to control these physical characteristics of nerve guidance channels, researchers are optimizing the incorporation of biologic factors and engineering interactive biomaterial that can specifically stimulate the regeneration process. Current and future research will ultimately result in biologically active and interactive nerve guidance channels that can support and enhance peripheral nerve regeneration over longer, more clinically relevant defect lengths.     

Selective fascicular nerve repair: a rapid method for intraoperative motorsensory differentiation by acetylcholinesterase histochemistry

Summary The selective reunion of motor and sensory fascicles of severed mixed nerves appears indispensible for optimal recovery of the impaired motor function. Procedures available for rapid identification of motor and sensory fascicles rendered ambiguous results. The only highly reliable and simple method marking motor fascicles, namely acetylcholinesterase histochemistry, neccessitated two operations due to its long duration (28 h). In the present study rapid demonstration of acetylcholinesterase activity was accomplished by elevating the temperature and the concentration of 2 constituents of the incubation medium. Now incubation takes 2 h instead of 20 h. Thus, the time necessary for the entire diagnostic procedure could be reduced from 28 h to 4 h. Indeed, intraoperative motorsensory differentiation using acetylcholinesterase activity as a marker appears feasible. The new technique has already been applied to 6 clinical cases of acute nerve injuries of the forearm. The histochemical results obtained are comparable to those of the standard procedure indicating equally high reliability of both methods. Finally, opportunities for further reducing the duration of the modified histochemical procedure and its applicability to fascicular nerve grafting are discussed.     

Luminal fillers in nerve conduits for peripheral nerve repair

The use of nerve conduits as an alternative for nerve grafting has a long experimental and clinical history. Luminal fillers, factors introduced into these nerve conduits, were later developed to enhance the nerve regeneration through conduits. Though many luminal fillers have been reported to improve nerve regeneration, their use has not been subjected to systematic review. This review categorizes the types of fillers used, the conduits associated with fillers, and the reported performance of luminal fillers in conduits to present a preference list for the most effective fillers to use over specific distances of nerve defect.     

End-to-side nerve suture--a technique to repair peripheral nerve injury.

End-to-side nerve suture (ETSNS) has until recently been extensively researched in the laboratory animal (rat and baboon). Lateral sprouting from an intact nerve into an attached nerve does occur, and functional recovery (sensory and motor) has been demonstrated. We have demonstrated conclusively that ETSNS in the human is a viable option in treating peripheral nerve injuries, including injuries to the brachial plexus. Among the many advantages of this new technique are: (i) simple and short operation; (ii) shorter recovery time--suture is done closer to the target organs; (iii) nerve grafts to bridge injured gaps are eliminated, reducing the morbidity of nerve surgery to a minimum; (iv) innervation of paralysed muscles, for which there was previously thought to be no hope of recovery, opens up many new treatment options; and (v) certain aspects of nerve function and regeneration, unknown until recently, open new horizons and understanding. ETSNS has given us new dimensions in the management of peripheral nerve injuries.     

New fibrin conduit for peripheral nerve repair

An ideal substitute to treat a nerve gap has not been found. Initially, silicone conduits were employed. Later, conduits were fabricated from collagen or polyesters carbonates. More recently, it has been shown that a bioresorbable material, poly-3-hydroxybutyrate (PHB), can enhance nerve repair. The present investigation shows the use of fibrin as a conduit to guide nerve regeneration and bridge nerve defects. In this study we prepared and investigated a novel nerve conduit made from fibrin glue. Using a rodent sciatic nerve injury model (10-mm gap), we compared the extent of nerve regeneration through the new fibrin conduits versus established PHB conduits. After 2 and 4 weeks, conduits containing proximal and distal stumps were harvested. We evaluated the initial axon and Schwann cell stimulation using immunohistochemistry. The conduits presented full tissue integration and were completely intact. Axons crossed the gap after 1 month. Immunohistochemistry using the axonal marker PGP 9.5 showed a superior nerve regeneration distance in the fibrin conduit compared with PHB (4.1 mm versus 1.9 mm). Schwann cell intrusion (S100 staining) was similarly enhanced in the fibrin conduits, both from the proximal (4.2 mm versus 2.1 mm) and distal ends (3.2 mm versus 1.7 mm). These findings suggest an advantage of the new fibrin conduit for the important initial phase of peripheral nerve regeneration. The use of fibrin glue as a conduit is a step toward a usable graft to bridge peripheral nerve lesions. This might be clinically interesting, given the widespread acceptance of fibrin glue among the surgical community.     

Schwann cell strip for peripheral nerve repair

Many strategies have been investigated to provide an ideal substitute to treat a nerve gap injury. Initially, silicone conduits were used and more recently conduits fabricated from natural materials such as poly-3-hydroxybutyrate (PHB) showed good results but still have their limitations. Surgically, a new concept optimising harvested autologous nerve graft has been introduced as the single fascicle method. It has been shown that a single fascicle repair of nerve grafting is successful. We investigated a new approach using a PHB strip seeded with Schwann cells to mimic a small nerve fascicle. Schwann cells were attached to the PHB strip using diluted fibrin glue and used to bridge a 10-mm sciatic nerve gap in rats. Comparison was made with a group using conventional PHB conduit tubes filled with Schwann cells and fibrin glue. After 2 weeks, the nerve samples were harvested and investigated for axonal and Schwann cell markers. PGP9.5 immunohistochemistry showed a superior nerve regeneration distance in the PHB strip group versus the PHB tube group (> 10 mm, crossed versus 3.17+/- 0.32 mm respectively, P<0.05) as well as superior Schwann cell intrusion (S100 staining) from proximal (> 10 mm, crossed versus 3.40+/- 0.36 mm, P<0.01) and distal (> 10 mm, crossed versus 2.91+/- 0.31 mm, P<0.001) ends. These findings suggest a significant advantage of a strip in rapidly connecting a nerve gap lesion and imply that single fascicle nerve grafting is advantageous for nerve repair in rats.     

End-to-side nerve repair in peripheral nerve injury

In peripheral nerve injury, end-to-side neurorrhaphy has been reported as an alternative in cases that the proximal nerve stump is not accessible. Several hypotheses have been proposed to explain peripheral nerve regeneration after end-to-side neurorrhaphy. Recent evidence suggests that nerve regeneration occurs by collateral sprouting. Although a great number of humoral factors have been identified, molecular mechanism of nerve regeneration after end-to-side neurorrhaphy has not been completely clarified yet. The goal of this technique is to provide satisfactory functional recovery for the recipient nerve, without any deterioration of the donor nerve function. End-to-side technique has been investigated in detail in both experimental and clinical studies. Only a limited number of reported cases in clinical practice, until today, can reveal that end-to-side technique may become a viable means of repairing peripheral nerves in certain clinical situations.     

Progress of peripheral nerve repair

Study on repair of peripheral nerve injury has been proceeding over a long period of time.With the use of microsurgery technique since 1960s, the quality of nerve repair has been greatly improved.In the past 40 years,with the continuous increase of surgical repair methods,more progress has been made on the basic research of peripheral nerve repair.     

Octyl 2-cyanoacrylate for repair of peripheral nerve

The repair of peripheral nerves with sutures is time consuming. The aim of this study was to evaluate the benefits and functional outcome of repairing nerves with octyl 2-cyanoacrylate adhesive. The right peroneal nerve of 64 male, Lewis rats was sectioned and repaired. The rats were randomized into 3 experimental groups: A (n = 27), using only octyl 2-cyanoacrylate; B (n = 27), using 4, 10-0 nylon sutures; and C (n = 10), a sham operation. The recovery of nerve function was quantified through walking-track analyses; group A showed faster return of nerve function than B, especially at 15 days (P < 0.017). Histologic analysis showed a greater axonal regeneration in group A versus group B and no indication of tissue toxicity in group A. No dehiscence occurred during the 6-month study. Use of adhesive shortened the anastomosis time from 12 minutes to 4 minutes. These results indicate that the use of octyl 2-cyanoacrylate adhesive for nerve anastomoses is safe and effective and may have benefits compared with the use of sutures.     

Vein graft for repair of peripheral nerve gap

Ten millimeter gaps in sciatic nerves of rats were repaired by vein grafting (n = 30) and by nerve grafting (n = 30). Electrophysiologic evaluation demonstrated that the nerve-graft group had a significantly higher percentage of re-innervation and shorter delay of terminal latency in myoelectrical response evoked by electrical stimulation of the sciatic nerve, compared with the vein-graft group at two and three months. But there were no differences between the two grafted groups after six months. In both grafted groups, the numbers of myelinated nerve fibers increased substantially over time, although the vein-grafted segments had smaller numbers of fibers than the nerve-grafted segments at both three and 12 months. Microangiograms also demonstrated different patterns of revascularization between the two groups. Results suggest that, although autologous vein grafting requires a longer period of time for nerve regeneration, compared with autologous nerve grafting, its ability to repair nerve is considerable.     

Use of nerve conduits in peripheral nerve repair

Studies on nerve conduits for peripheral nerve regeneration have concentrated on the manipulation of various conduit materials to avoid sacrificing native nerve in the clinical situation. With the proliferation of available nerve growth-stimulating factors, the focus is shifting experimentally toward molecular biologic manipulation, with the addition of these materials as substrates within the conduit. The clinical use of conduits has concentrated on the use of autogenous tissue, with a few examples of polyglactin (PGA) mesh and silicone. Ultimately, as yet, conduit material does not seem to have a profound effect on outcome. Substrate manipulation has not yet had clinical application. An important problem that remains, both experimentally and clinically, is overriding the size of the maximal gap that can be bridged successfully, as well as obtaining good functional sensory and motor recovery, compared with the use of nerve grafts. Advances in molecular biology may reveal further details about the nerve growth phenomenon, the precise sequencing of the substrate materials that are effective in promoting nerve growth, and when they should be applied. Advances in chemical engineering may provide additional biologically stable materials that have the ability to integrate growth-enhancing agents or factors into the lumen of the conduit.     

Immunogenicity and regenerative potential of acellular nerve allografts to repair peripheral nerve in rats and rabbits

Summary This study describes the ability of acellular nerve allografts (genetically different) to repair injured peripheral nerve in rats and rabbits. We recently reported the regeneration supporting potential and immunogenicity of acellular nerve allografts in rats⁷. The present study extends our previous work and quantitates the extent of axonal regeneration through various nerve grafts in rats. In addition, the use of longer nerve grafts to repair rabbit peripheral nerve is described.Inbred strains of Fischer and Buffalo rats and New Zealand white and Dutch rabbits were used. Acellular grafts were prepared by repeated freezing and thawing of in situ degenerated nerves. Nonfrozen predegenerated nerves were used as cellular grafts for comparison. Nerve isografts (genetically identical) were also performed.The graft length was 2.0 cm in rats and 4.0 cm in rabbits. In both rats and rabbits the cellular isografts showed the most rapid regeneration and target muscle innervation. The cellular allografts were invariably rejected and only showed limited regeneration. In contrast, acellular allografts, in spite of their mild immunogenicity, allowed significant regeneration through them.It is concluded that acellular nerve allografts are capable of supporting axonal regeneration because of their reduced immunogenicity, and thus can be used to bridge nerve gaps after nerve injury.     

The repair of peripheral nerve lesions

The importance of exact and detailed methods of evaluating nerve deficit, and the careful recording of all factors which might influence nerve recovery is emphasized. In our hands the plasma clot repair of nerves has seemed to offer definite advantages over other methods of repair. We believe that the unavoidable obstacles to successful employment of grafts limits their use to small nerves and peripheral branches. Gentle and prolonged stretching of the central stump may be the answer to bridging the gap of major trunks.