Much attention and effort has been devoted to devising methods of promoting axonal regeneration over the past century and longer. It has certainly been appreciated that the regeneration of axons can and does occur over long distances in the adult mammalian peripheral nervous system and, to a much more limited extent, if at all, in the adult mammalian central nervous system (5
). Refinements in experimental techniques and methodology have also shown that even when regeneration of peripheral nerves occurs, the number of axons finally reaching their original target structures is only a fraction of what it was originally, and the accuracy of reinnervation is quite degraded (3
). Therefore, although axonal regeneration with recovery of function can and does occur in the peripheral nervous system of animals and patients, new methods of increasing both the quantity and specificity of regrowing axons are needed and are being actively pursued.
The earliest report of the idea of suture repairing a damaged peripheral nerve was written by Paul von Aegina in the 7th century (6
). Ironically, it was not until a millennium later that the surgical repair of a severely damaged peripheral nerve was shown to have clinical benefit (6
). Only recently have advances in surgical technique been made that significantly improve clinical outcome. One such important advance, made by Dr. David Kline, was the intraoperative application of electrophysiological techniques to determine whether or not a damaged nerve segment should be resected and repaired (4
). Dr. Kline and his colleagues showed that intraoperative nerve conduction studies could distinguish a neuroma in continuity with regenerating axons giving rise to a recordable nerve conduction response (axonotmetic grade of injury) from one without conducting axons and a recordable response (neurotometic grade of injury) several months after a traumatic nerve injury. This distinction is critically important, because the axonotmetic injury does not require a direct nerve repair, whereas the neurotometic injury requires surgical resection of the intraneural scar tissue impeding regeneration followed by anastomosis of the nerve with or without a graft, depending on the length of the gap. Such advances in peripheral nerve surgery have brought us to the point at which further progress that relies on recovery through axonal regeneration is likely to depend more on biological discoveries than technical innovations.
With the advent of micro- and nanotechnologies, an alternate and novel strategy has recently been proposed that does not rely on the regeneration of axons following damage sufficient to cause loss of axonal continuity (i.e., at least an axonotmetic grade of injury) (1
). Instead, this strategy is based on re-establishing axonal continuity soon after the injury and before distal Wallerian degeneration of axons occurs. It relies on acutely re-approximating the proximal and distal stumps of axons and splicing their membranes back together. This idea is analogous to splicing electrical wires back together instead of laying down an entirely new electrical cable. With the help of newly developed microscale surgical tools, the peripheral nerve surgeon would reach down to the subcellular level and manipulate the “wires” of the nerve. The obvious advantage of such an approach is that, if successful, the original pathways are restored and therefore, at least in theory, recovery of function could approach normal baseline values if all or most axons could be biologically spliced back together. The other advantage is the speed of recovery without having to rely on regeneration that occurs at a maximum of 1 mm/d.
Of course, this ambitious goal relies on a surgical “tool belt” that is not yet available. However, advances in micro- and nanotechnology are making it possible to both visualize and manipulate biological structures at the cellular and subcellular levels. In addition, experimental methodologies already exist for fusing together cellular membranes. Therefore, we think that the time is ripe to combine these old and new technologies to take surgery to the cellular and subcellular levels and consider splicing disrupted axons back together instead of and/or in combination with ongoing efforts to promote and enhance axonal regeneration.
We envision that axonal splicing would require at least 3 major steps: 1) trimming the cut ends of axons, 2) moving axons so that their proximal and distal cut ends can be placed in close approximation, and 3) fusing the axonal membranes so as to re-establish both anatomic and functional continuity of the axon (). All of these steps would need to be performed soon after an injury with disruption of axonal continuity to avoid having to deal with the consequences of distal Wallerian degeneration of axons. Combining the expertise of a multidisciplinary group, which includes neurobiologists, with the necessary anatomic and electrophysiological tools, engineers with the capacity to apply appropriate micro- and nanotechnologies, and peripheral nerve surgeons with clinical perspective and experience, we have succeeded in demonstrating proof of concept for each of these steps in vitro and, in some cases, in vivo. , demonstrate the use of a microknife to cut groups or individual axons both in vitro and in vivo. , demonstrate the capacity using changing electrical fields (dielectrophoresis) to move an axon in vitro without requiring physical contact. , demonstrate the capacity to disrupt and then fuse axonal membranes using electrofusion in vitro.
FIGURE 1 In the direct repair scheme recently proposed, novel microsurgical instruments directly manipulate individual axons in an injured nerve. A microcutting tool cuts each axon to remove damaged segments, leaving a discrete, resealing stump. Electrostatic (more ...)
In order to bring this technology in to the clinical arena, many additional steps are necessary. It will be necessary to demonstrate each of these steps on single axons and then demonstrate that the spliced axons survive and are capable of function. In order to make this novel approach of repairing axons practical, it will be necessary to repair axons quickly, which will likely require that many axons be repaired simultaneously in an automated fashion using computer robotic systems that the surgeon will control and oversee (7
). To do so, it will also be necessary to create a user-friendly interface that is compatible with the operating room environment. It will also be necessary to operate on these severe nerve injuries within a short amount of time, before the distal axons degenerate, which may not always be possible. New methods of keeping the distal axons viable for longer periods of time would make it possible to operate on a greater number of severe injuries Although many challenges remain, we think that the goals and their potential clinical benefits are sufficiently important to warrant our continuing with efforts to further develop and eventually make this approach and technology a clinical reality in the not too distant future (1
). If we succeed, our efforts will be a tribute to Dr. Kline, who has made so many important contributions to the field of peripheral nerve surgery, inspired so many peripheral nerve surgeons, and provided the essential foundation for these future advances.