American Society for Peripheral Nerve

Spring 2015   •   Volume 5, Issue 1
Expert Opinion

A virtual interview with Dr. Tessa Gordon, PhD, University of Toronto on stimulation axons

Editor: Dr. Gordon, despite decades of bench research, there has been no significant change in clinical outcomes for major peripheral nerve repairs. Some of the things you've been working on seem to hold promise for breaking this stalemate... Would you agree and which of the concepts that you are currently working on do you think have the most potential for translation to the clinical arena?

Dr. Gordon: I do agree. Together with Ming Chan, we recently wrote a review in which we considered several agents that have potential to promote nerve regeneration. These include FK506 that has positive effects on nerve regeneration but studies remain to be carried out to provide good evidence of effect. Studies in Susan Mackinnon's laboratory have shown positive effects when the agent is administered prior to the injury but of course, the question is whether FK506 can promote nerve regeneration when applied after surgical repair of an injured nerve. Studies that I carried out with Dr. Wale Sulaiman some time ago demonstrated the efficacy of systemic FK506 in promoting the regeneration after delayed nerve repair but currently in our laboratory in Toronto more localized administration of FK506 is having a dramatic effect of regeneration after immediate nerve repair.

In our review, we also considered our findings with my PhD graduate student at the time Abdul Al-Majed, and with Tom Brushart that brief low frequency electrical stimulation of a transected nerve proximal to the site of immediate coaptation, has a dramatic accelerating effect on nerve regeneration. We published these findings in 2000 and 2002 in Journal of Neuroscience. What was very revealing to us in the study published in 2000 was that axon regeneration across the suture site is much slower than previously thought. The latent period of a few days actually pans out to be much longer with axons growing in a haphazard fashion across the suture site through disorganized extracellular matrix and Schwann cells that migrate into the site rather slowly.

Editor: Can you briefly review the theory behind the neurostimulatory effects of electrical stimulation?

Dr. Gordon: Our original rationale for determining the effect of brief electrical stimulation on nerve regeneration was based on findings of Nix and Hoft (1983) and Pockett and Gavin (1985). These authors published short papers that demonstrated that the electrical stimulation accelerated the recovery of muscle contractile force reflex and the return of a withdrawal, respectively. Whilst these studies were enticing and informing, the latter study indicating that electrical stimulation was most effective when administered within a half-hour of the injury and the former that a continuous low frequency stimulation regime was effective, neither study elucidated whether the stimulation affected nerve regeneration itself as opposed to the process of target reinnervation. It was for these reasons that we began our valued collaboration with Tom Brushart whose expertise in backlabeling of regenerated axons allowed the quantative assessment of the numbers of neurons that regenerated their axons to the point of application of the fluorescent dye. This expertise allowed us to count the numbers of motor and sensory neurons that regenerated their axons across the suture site as well as some distance into the distal nerve stump. Further collaboration with Melitta Schachnerís group demonstrated the accelerated recovery of function after electrical stimulation of the injured and repair nerve proximal to the site of the nerve repair (Eberhardt et al., 2006).

I must add that the rationale for the continuous 20Hz electrical stimulation for a 2 week period that we adopted was in light of the timing of preferential nerve regeneration that Tom Brushart had described for regeneration of femoral axons down the motor and sensory branches of the nerve. Brushart (1993) had demonstrated that the regenerating motor nerves that grow a distance of 25 mm into the quadriceps and the saphenous nerves within the first two weeks do so in a random fashion with equal numbers of motoneurons sending their axons into both branches. He reported that by 8 weeks, there was preferential motor nerve regeneration into the quadriceps branch to the denervated quadriceps muscle. We therefore began our studies with a continuous two week 20Hz stimulation paradigm. This paradigm being effective, we progressively reduced the period of stimulation with a 1 hour period being equally effective (Al-Majed et al, 2000). The 1 hour paradigm has been adopted in many studies that followed with the efficacy of the electrical stimulation being confirmed in several nerves. Kathryn Jones in her several papers on the efficacy of electrical stimulation accelerating regeneration and reinnervation of facial muscles has adopted a daily stimulation period of 20Hz electrical stimulation for a period of 30 minutes each daily after a crush injury to the facial nerve (Sharma et al, 2010; Hetzler et al, 2008).

When you ask about "The Theory Behind the Neurostimulatory Effects of Electrical Stimulation" I understand your question to include the basis for the neurostimulatory effects of electrical stimulation. As a continuation of Dr. Al-Majed's PhD thesis, he demonstrated the accelerated expression of brain derived neurotrophic factor and its receptor followed by accelerated expression of the regeneration associated genes of tubulin and actin that are cytoskeletal proteins essential for the elongation of the growing axons (Al-Majed et al, 2000b; 2004). In continuing studies we demonstrated a central role of cAMP and now, Drs Jones (Foecking et al, 2012; Sharma et al, 2008) and Art English (Liu et al, 2014; Thompson et al, 2014) are also demonstrating a role of androgens in the cascade to accelerated expression of regeneration associated genes and, in turn, accelerated axon outgrowth.

Editor: What do you see as the major obstacles to moving your electrical stimulation work from the lab to clinical settings? It seems to me that for any idea like this to achieve widespread adoption, a company needs to make a packaged "kit" or device... Many surgeons just want a tool

Dr. Gordon: In answer to your question, I would say that I am very hopeful that electrical stimulation of repaired peripheral nerves will move to clinical setting. Our work with Ming Chan on the electrical stimulation of median nerve after carpal tunnel release surgery required simply that one of the two stainless steel wires that were bared of their insulation at the end, were inserted close to the nerve proximal to the site of release. These wires were simply pulled out after the 1 hour 20Hz electrical stimulation was carried out within 15 minutes of the release surgery. These proof of principle studies provided amazing acceleration of reinnervation of the musculature of the median eminence with all the median nerves reinnervating the muscle within 6-8 months in contrast to no significant increase in the numbers of motor nerves innervating the muscle even after 12 months. The major outcome of the release surgery of relief of pain and the movement of the hand muscles being adequately compensated by the unaffected muscles in the lower arm apparently did not impress all surgeons. A paper in Annals of Neurology by Wong et al (2015) is currently in press that reports accelerated recovery of sensation after surgical repair of transected and surgically repaired digital nerves. Such recovery is more obvious but it is clear that the efficacy of the electrical stimulation in human nerve repair is certain and we are definitely ready for more widespread adoption. As you suggest, a packaged kit or device is the way to go and we are certainly going that way.

Editor: Are there settings where electrical stimulation of repaired nerves might not be appropriate?

Dr. Gordon: Electrical stimulation of repaired nerves, being feasible, can be applied within the operating room. We had in our experimental series in rats, demonstrated that it is essential for action potentials to be conducted toward the neuronal cell bodies, the effect of the electrical stimulation being eliminated when action potential conduction was prevented by application of a sodium channel blocker, tetrodotoxin (Al-Majed et al, 2000). We had used suprathreshold stimulation to stimulate the injured rat nerves but the electrical stimulation was adjusted for comfort of the awake patient in the study in which we performed the electrical stimulation after carpal tunnel release. Whilst it remains to be determined whether all the nerves need to be activated for effect, in surgery, the electrical stimulus should be of minimum duration (~100Ķs) and, under conditions where a branch of the nerve is intact the stimulus current can be adjusted to evoke muscle contractions. Because it is the electrical stimulation must be given to the nerve proximal to the injury, it is feasible to electrically stimulate the nerve some distance from the site of nerve repair. The questions of secondary incision sites come up and of course all conditions of safety are paramount. We are currently evaluating these issues.

Editor: The other area that you have been working on lately that is very exciting is supercharging or side-to-side grafting (or I call it reverse end-to-side nerve transfer). Can you briefly describe the theory behind why you think this will help improve nerve regeneration?

Dr. Gordon: The technique that you refer to as reverse end-to-side nerve transfer is exciting, especially because I believe that it can readily be translated to the clinical arena. I frequently give the intact ulnar and injured median nerves as examples of possible translation because, especially for brachial nerve injuries the period of time for regenerating nerves to "reach" the lower arm are extremely long and certainly well beyond the short window of time when the regenerative power of the injured neuron and the regenerative support of the denervated Schwann cells are prime. The expression of growth associated genes in the neurons and the Schwann cells is short lived with the highest expression declining within 3 months, many declining in even shorter periods of time. The basis for examining whether donor nerves that regenerate into a nerve stump can sustain a growth permissive state for longer periods of time was based on the release of many mitogens (agents that promote cell division) that promote cell division of Schwann cells. These mitogens are normally released from the growing nerve and promote a second phase of Schwann cell division when they encounter the cells in the distal nerve stump. This was an early finding of Pellegrino and Spencer, 1985).

We first examined the efficacy of the end-to-side nerve transfer in Edmonton where Dr. Ladak, an MSc student at the time compared the effect of one and three cross-bridges as we called them. Essentially, we used common peroneal autografts to insert 6 mm cross-bridges between the intact donor tibial nerve and the recipient denervated common peroneal nerve stump at right angles to the nerves. Small perineurial windows were opened to insert these cross-bridges. We found that 3 bridges were better than a single bridge and that common peroneal nerve regeneration through the 4 month chronically denervated common peroneal nerve stump was better when the bridges were inserted as compared to when they were not (Ladak et al, 2011). Further experimentation in Toronto explored the question of the whether or not a perineurial window was necessary and how many cross-bridges are optimal. We are just submitting the revised manuscript (Gordon et al, 2015). We are finding that, indeed a perineurial window is necessary with the number of tibial axons regenerating through the bridges increasing as a function of the diameter of the perineurial window. The issue is that an optimum number of axons is required. Because the capacity of nerves to sprout and reinnervate muscle is considerable with 20% of remaining nerves in partially denervated muscles being able to sprout and reinnervate denervated muscle fibers, many donor axons can enter into the recipient denervated nerve stump.

Editor: How close are we to clinical trials of some of these concepts?

Dr. Gordon: I believe that the first sentences in answer to your previous question, answer the currant question.

Editor: One last question... which area of the complex regeneration process do you think the next major breakthrough in nerve surgery will be and why?

Dr. Gordon: This is an interesting and important question. Dr. Susan Mackinnon frequently talks of major steps (-novel leaps forward but I canít quite remember her terminology), in which she includes nerve transfers. At this point I would like to comment that surgeons repeatedly refer to the irreversible replacement of chronically denervated muscles by fat. I have personally fought this idea for some time. My reason for doubting the conclusion of the irreversible fate of the denervated muscles comes from the studies of my graduate students Drs. Susan Fu and Mukaila Raji in 1995 and 2010 (Fu and Gordon, 1995a,b; Gordon et al, 2011). Their studies demonstrated that the short duration of the regenerative capacity of injured neurons and of the regenerative support of chronically denervated Schwann cells can together fully account for the poor nerve regeneration and/or the failure of functional recovery after delayed nerve repair and/or after repair requiring very long period for nerves to regenerate to reach denervated targets. Clearly denervated muscles atrophy but I always remember a wonderful chapter on muscle denervation by Sunderland in his 1978 book. He considered the ischemia in denervated limbs after nerve injury as an important and neglected issue when considering the atrophy of denervated muscles. This is a key question concerning the fate of denervated muscles and must be addressed. Recently Dr. Michael Willand has electrically stimulated muscles to find that the daily stimulation accelerates nerve regeneration (Willand et al, 2014). I personally was amazed by this finding. I know from the work of Kerns and others that electrical stimulation of denervated muscle can sustain the muscle fibers and the question I would address at this point in time is the consequence of the electrical stimulation in sustaining the blood flow to the denervated muscles in addition to the direct effect in sustaining the dimensions of the muscle fibers.

With respect to the direct question of nerve surgery, I anticipate that the work on delivery of agents to the nerves via microspheres is an interesting and important current endeavor. Whilst I am in favor of such approaches, my bent is always to "use" the biology of the system in order to attempt to replicate the optimal conditions that the body so often presents. My respect for the biology of living things is boundless.

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