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A Novel therapy for Damage to the Peripheral Nervous System (PNS)



A Novel therapy for Damage to the Peripheral Nervous System (PNS)
Introduction and Epidemiology

While more attention is often focused on damage to the Central Nervous System (CNS: Brain and Spinal Cord) because of the catastrophic loss of function and the inability of nerves to regenerate, severe damage to the nerves of our trunk and extremities can also be devastating and also without a useful program of clinical management.

In the 25th Anniversary Presentation of Peripheral Nerve Surgery (J. Hand Surg. 25: 2000), Professor Goran Lundborg laments “New experimental laboratory data based on evolving neuroscientific concepts however, unfortunately have not resulted in a corresponding development in the clinical field leading to improved results from nerve repair.” (page391).

He also states that “techniques for clinical approximation of severed nerve ends have reached an optimal technical refinement” (page 392).

If indeed the technical aspects of surgical manipulation of the injured PNS is highly refined, but clinical outcomes still very poor, then we have to return our attention to biology for the missing ingredients. This short essay suggests one avenue to exploit is the Biophysics and Electrophysiology of nerve repair.

Even though severe CNS injuries have been historically considered to be ‘hopeless’, the outlook for severe PNS injuries is no less troublesome. Such injuries leading to severe loss of nerve related functions in the face, extremities, etc.., necrosis of muscle mass, (a certain consequence of sustained denervation) and in some cases severe Peripheral Nerve Injury (PNI) can lead to even amputation of the limb. Factoring in the incidence of severe PNI numbers in the hundreds of thousands of cases per year (compare SCI at 10,000/ year), this area of Neurology/ Neurosurgery requires new approaches and innovation.

Estimates suggest there are ~ 800,000 PNIs per year, about half of this number are likely candidates for repair as they are due to laceration (severing or cutting of the nerve trunks). This latter group represents an ideal population to target a novel therapy for the reasons that:

a. The origin and character of the injury is evident
b. Laceration of the extremities, face or trunk, will require some level of surgical management
c. Transection is a severe form of PNI compared to focal compression injuries or stretch injuries
d. Transections require immediate intervention up to a delay of several days

This latter fact needs be evaluated in light of the approximately 3 months elapsed time routinely allowed for non- laceration PNIs prior to the physician implementing a plan of therapy. Below we briefly review the biological events occurring after PNI; conventional medical management; and the basis for a new approach via electrophysiology.

The Injury and Regeneration of Peripheral Nerves

First, let us briefly review the relevant anatomy of nerve trunks of the PNS, beginning with the Microscopic Anatomy. It is often confusing to lay persons that the terminology ‘nerves’ or ‘nerve fibers’ is often applied to the extensions of single nerve cells (properly called axons), as well as the bundles of thousands of nerve axons arranged in long ‘fibers (very poor usage) or trunks (better yet) coursing through our extremities. The nerve trunks servicing the extremities can be as large or larger in diameter than a finger, while single nerve processes, axons, are microscopic in size, ranging from large myelinated axons ( ~ 10 m in diameter ) to many types of pain fibers (unmyelinated and less than 1m in diameter). These comparatively large, and often long, nerve trunks are the easily visualized components of the PNS. It is also important to remind ourselves that the nerve cell bodies of Mammals (containing the nucleus of the nerve cell and other vital machinery and organelles) giving rise to the axons contained within these trunks are primarily located in two places: 1. within the spinal cord itself, or 2. next to – but outside of it.

For example: 1. Motor Neurons of the spinal cord gray matter give rise to axons that exit the cord via the venteral roots and project into the limbs as components of the PNS. 2. Sensory axons of the extremities arise from cell bodies of the Dorsal Root Ganglia residing within protected cavities in the boney spine next to the cord. Each of these cells has an axon that feeds the extremities and another that enters the spinal cord as part of CNS sensory tract. These nerve cells are therefore called ‘bi-polar’. Even nerve axons of the involuntary nervous system (the autonomic system) still arise from ganglia (clusters of nerve cell bodies) that lie next to the cord. All of this is to emphasize that in Mammals, nerve cell bodies are centrally placed in the torso.1. This anatomy belies ONE very important consequence:

A cut or a crush to a nerve trunk anywhere in the torso renders it into a Proximal Segment – in continuity with the nerve cell bodies residing near the center of the torso as explained – and a Distal Segment. The distal segment is thus separated from the cell bodies that provide it sustenance and, in mammals, this segment always dies and decays 2. This is called Wallerian Degeneration after the 19th Century Anatomist who first described the process in detail. Regeneration – new nerve growth – occurs at or near the tip of the Proximal Segment. Within a few days, there will not be any distal segments of nerve axons within the injured nerve trunk as they will have decayed and been removed by phagocytic cells.

1. There is one main exception to this rule, not particularly relevant to this discussion: Nerve Cells of the Enteric Nervous System of the Stomach and Gut lie within the linings of this viscera.

2. Interestingly the rapid Wallerian degeneration of distal segments is largely a feature of mammals. Distal segments of non- mammalian animals can persist for sometimes months to years.

Gross Anatomy


The picture above shows the proximal and distal segment after a clean cut to a nerve trunk. If this was instead a severe localized crush, there will still be the two ‘segments’. With time, i.e., days to months, changes at the crush site may actually lead to local decay, separation of the axons, and the formation of ‘true’ segments – the distal one to die and disappear as explained. The picture also provides a guide to relevant macroscopic anatomy. Axons – often projecting to shared parts of the arm or hand (alternately: upper /lower limb or foot) are bundled together in “fascicles”. They reside in bundles by a tough connective tissue sheath called the Perineureum . All of the fascicles comprising one large nerve trunk are ensheathed by the outermost covering of connective tissue, the Epineureum. Not Shown for sake of clarity is another layer of a much more delicate and loose connective tissue substance, the endoneureum. This material provides some support for blood vessels and tiny capalaries that coarse throughout the nerve trunk. It is wise to point out here that:

1. Even these connective tissue structures and fascicles begin to shrivel and shrink in the distal segment after injury, and

2. the myelin wrapping outlast the axons that they insulate, but damaged myelin degenerates with time. Myelin is produced by Schwann cells, and the many cell processes initiated by injury together with the products of cell decomposition actually stimulate Schwann cell multiplication in the distal segment. Older anatomist noticed the increased numbers and their arrangement referring to them as the ‘Bands of Bügner’. These bands and the cells forming them are attractive to regenerating nerve processes and are conduits helping to entrain growing nerves within the distal segment.

The Changing Anatomy of Injury

Pictures 2 – 4 reveal the stepwise degeneration of the distal segment isolated by a growing mass of scar tissue that becomes more dense and more of a barrier with time. Not shown for sake of clarity is the mass of blood - born and other cells, macrophages, leucocytes, fibroblasts, to name only a few that invade the local region of injury. These cells such as fibroblasts actually lay down part of the initial connective tissue scar along with clotting blood. In time, this morphs into a more mature scar of connective tissue, granular tissue, and fibrin – forming what is often described as a true ‘barrier’ to nerve regeneration from the Proximal Segment. Actually it is not a true ‘barrier’ but rather, a ‘confusing’ factor. Growing nerve fibers love to grow along these surfaces of connective tissue – even blood clots. Unfortunately, the chaotic organization of the scar carries them in various and useless directions, as shown, and discussed further below.





This set of Graphics also directs our attention to changes in the proximal segment. The nerve axons that will live and regenerate at their tips do not usually begin to do so from the plane where they were cut or where they eventually separated after a severe crush and localized decay. Rather, they “dieback” away from this plane – a distance that has to be made up. Dieback is also called ‘Retrograde Degeneration’ and is quite variable in extent – depending on many factors such as the caliber of the injured axon, whether it is myelinated or unmyelinated and other local issues. The Myelin at the plane of injury, though contained in the tip of the proximal segment, still decays and forms debris that acts as a signal or beacon for scar forming and phagocytic cells to move into the local area. None of this bodes well for functional nerve regeneration.



The Idealized Situation
1. …. would be that the tips of the proximal and distal segments of the nerve trunk lie close together naturally (or after being brought together by the surgeon) so that 2. ….growing axons from the proximal segment would enter the distal segment, and use the persisting fascicles as tubes or tunnels to guide them near to their target areas or tissues. 3…. the rate of growth would be sufficient to innervate targets before the atrophy and loss of target organs and tissues and 4. …. that sufficient directional guidance be provided so that fibers come close to original targets so functional responses would be more appropriate.

The Actual Situation ….
1. Fibers dieback away from the region of damage ALL-THE-WHILE scar formation builds progressively to become a significant obstacle to their entering the distal segment 2. The Gap separating the Proximal and Distal Segment may be small, but is often quite large owing to extensive mechanical damage, progressive decay and degeneration of the damaged trunk and the elastic properties of nerve trunks such that their segments pull away from each other after separation. This Gap problem requires a surgical remedy as the gap must be spanned by growing fibers. 3. The rate of growth of fibers limits how effective the functional outcome may be EVEN if they are ‘steered’ in the direction of target tissues. In humans ~ 1mm / day (i.e. approximately 1 inch/month) is the average speed fibers may regenerate. It becomes immediately obvious that the greater distance up the limb the damage occurs, the longer time it will take whatever reconnection may be induced to occur. Thus reasonable outcomes can be expected with damage to nerve trunks very close to the target muscles and sensory receptors in the hand or foot. The farther up the limb the damage occurs – the more perilous the situation, as target muscles deprived of their nerve supply begin to atrophy and die too. 4. Misdirected fibers at the level of the scar may not just be benign – they can form whorls of redundant growth that become extremely sensitive to stimuli. Such “neuromas” can cause excruciating pain to the patient and require surgical removal, often reforming once again.

Conventional Management of Severe PNI

Modern surgical techniques have attempted to remedy the issues described above. In the following picture, simple epineural sutures are used to draw the proximal and distal segments together. This may be sufficient if the nerve transection is clean …if the anatomy of the trunk is not complicated (little branching and few fascicles) … if the gap is small ….. and the target tissues are close to the injury. More complicated nerve trunk anatomy may require fascicular repair, where an attempt is made to close adjacent fascicles helping to insure more targeted growth of fibers.





Large gaps may require the insertion of a bridging material, often a segment of a nerve trunk removed from elsewhere on the patient’s body (such as the sural nerve of the leg ). Gaps may also spanned by suturing a polymeric tube – called tubulization – end to end with the proximal and distal nerve trunks. There is an enormous literature on “nerve conduits” believed to be useful in helping regenerating nerves span this distance.

As stated in the introductory paragraph however, none of these techniques are very useful given severe injuries at even modest distances from the target tissues.



Applied Voltages and Peripheral Nerve Regeneration

Background

The modern literature on neurotrophic (growth inducing) and neurotropic (guidance) cues often fails to mention the role of natural and artificially applied electric fields (voltage gradients) favoring instead tissue, cell, and molecular cues. Knowledgeable scholars such at Purves and Lichtman (Principles of Neural Development, Sinauer Associates, pages 90-104) recognize this bias, and have decried it – knowing that at its basis is a lack of training in biophysics/biomedical engineering by those interested in the neuroscience of nerve regeneration. Most investigations prior to 1979 revealed clear positive effects of voltages on nerve growth in culture dishes and animals, but the modern literature is, to our knowledge, is nearly free of negative reports. Moreover, in the last 2 decades, a strong basis in understanding the mechanisms of action of applied voltages has emerged making the issue of initiated nerve regeneration and guidance by applied electric fields incontrovertible (reviewed by Borgens 1982; 1988; 2003; and McCaig et al 2005. This methodology frames the basis for the treatment of acute human spinal cord injury by oscillating voltages (OFS; Shapiro et al 2005).

Vertebrate Peripheral Nerve Regeneration.

Studies of methods meant to enhance peripheral nerve regeneration in vertebrate animals have always been complicated. This is because the single nerve fibers (axons; both sensory and motor) regenerate with facility – in marked contrast to their counterparts in brain and spinal cord. Therefore an investigator must be able to discriminate an enhancement of regeneration which already is quite rapid, ca. 1 – 5 mm / day. This is not the case in spinal cord or brain where the assay is required to discriminate the occurrence of regeneration compared to the complete lack of it in Control animals. This situation mirrors that in long bone fracture repair where normally bones heal quite rapidly and it is sometimes difficult to determine an enhancement by certain therapies (such as drugs or applied voltages). Thus the most convincing human studies focus on enhancing poor fracture repair associated with chronic non-union, diabetes, or advanced age.

Frogs and Toads

Measurements of naturally produced injury currents and voltages, and means to enhance peripheral nerve regeneration, date to the early part of the 20th century, beginning with the famous student of Nobel Lauriat S. Ramon y Cahal – Raephael Lorrente de No (Lorrente do No, 1947).

The first unequivocal demonstration that an applied voltage could enhance Vertebrate PNS regeneration waited until 1979 as part of a series of reports on amphibian regeneration by Borgens and his colleagues. Borgens et al 1979a investigated a distally negative voltage applied within the forelimb stump of large adult African clawed Toads (Xenopis laeivis) with an implantable stimulator. This was done to initiate a significant amount of limb regeneration that ordinarily would not occur (Borgens et al. 1979b; reviewed by Borgens 1989a).





Unlike frogs of the genus Rana (common grassfrogs) that do not regenerate limbs at all as adults, the clawed toad does produce a unbroken “spike” of unbranched solid cartilage at the end of the stump after amputation. By 6 months to 1 year after the electrical application, it was discovered that the cartilage core of the regenerate was perforated throughout its entire length by peripheral nerves stemming from the stump tissues. Since this is: 1. not natural for such frog growths, and 2. Since cartilage is inhospitable to nerve regeneration (Snow et al. 1991); 3. ...... the parsimonious explanation for the presence of such a robust regeneration of nerves is that they were facilitated to grow into the regenerating limb prior to cartilage formation – which normally occurs first in these cases. This constituted an enhancement of the rate and branching of nerves remaining in the limb stump after amputation of the limb –those that were bathed by the applied voltage –and in only the current –treated animals. These results fit nicely with other facts about amphibian limb regeneration. The presence of nerves in the stump is fundamental and absolutely required for the remarkable natural ability of salamanders to regrow their limbs after amputation (Spallazani 1766). If nerves are removed from the limb prior to amputation – the regeneration of the limb will not occur until nerves grow back into the limb stump. Frog tadpoles regenerate their limbs but loose this ability as they transform into young frogs. Fully adult frogs do not regenerate their limbs. Abnormal limb regeneration in adult frogs can be induced however by deviating and transplanting living nerve trunks from the hind limb to the forelimbs (Singer 1954).



Thus, it was the opinion of Borgens et al 1979b that Clawed Toad limb regeneration was induced by enhancing the presence of nerve tissue in their stumps by electrical means.

The Laboratory Rat

In part, timed to the studies of enhanced fracture repair by Electrical Fields by Carl Brighton and C.A.L. Bassett in the 1980’s, a variety of reports showed positive responses on PNS regeneration in the adult rat by application of distally negative electrical fields (for example: Aebischer et al 1987; Kerns et al 1986, 1987, 1991; McDevitt et al 1987; Pomeranz 1986; Pomeranz et al 1984; Rowley et al 1985; Roman et al 1987. to name just a few.) Given the aforementioned difficulty of this chore, the best studies employed multiple means of determining regeneration other than by anatomy.
For example James Kerns and his colleagues tested a small indwelling DC stimulator system on PNS regeneration in the Rat Sciatic Nerve. They knew that injured nerves drive a sodium /calcium current of injury into their severed or degenerating tips (first described by Borgens using a non-invasive vibrating electrode for the detection of extracellular electric current (Borgens et al 1980)). They used this type of instrument, built by John Freeman at Vanderbilt, to scan crushed or cut sciatic nerves of the hindlimb in adult rats. They detected two peaks of naturally produced current – one at the site of the soft tissue injury, and another that was associated with the tips of degenerating or regenerating nerves which were invisible since they were inside the epineural sheath. In this way they focused on specific regions of the nerve to sample for anatomy. The location of the current peaks themselves were distinctly different in the control and electrically treated groups and by itself, evidence of enhanced regeneration by the applied electric voltage gradient. This was confirmed by careful electron microscopy of these “fronts” of nerve endings within the proximal segment (Kerns et al 1991).
Other groups used preformed guidance channels set into the gap produced by transection of a peripheral nerve, and a combination of electrophysiology and anatomy to assay PNS regeneration. In total these reports testify to the positive effects of electric fields on PNS regeneration. Of all of the reports (numbering approximately two dozen between 1987and 1992), only one claimed there was no effect by applied electric fields on PNS regeneration (McGinnis and Murphy, 1992). This paper arrogantly claimed that their results called “ into question” all of the other reports– even though they never replicated any one study or used an application that could be reasonably understood to mimic any other experiments. They admit this (Hanson and McGinnis 1994) reporting that their experiment “did not exactly duplicate any previous experiment”. Thus in summary: a distally negative weak applied electric field is known to enhance the regeneration of severed or crushed peripheral nerves.

These positive experiments gave way to new methods of application, including electrically charged guidance channels (championed by Peter Aebischer’s group), pulsed electromagnetic fields (championed by Betty Siskens Group at the University of Kentucky), and Spinal Cord Regeneration (Borgens group). To our knowledge, no one has repeated experiments placing simple DC Fields over PNS injuries with implantable stimulators since the early 1990’s as it’s ephicacy is unquestioned.

An implantable OFS system for the Enhancement of Peripheral Nerve Repair





The accompanying graphic shows a newly fabricated OFS stimulator capable of being implanted under the skin of the arm or leg, with the very small electrodes affixed to regions proximal and distal of a peripheral nerve injury. In certain circumstances this could find use in facial nerve injuries as well – given its small size. The unit comes with a newly developed external amplifier and wand that is able to scan frequencies of OFS “beacon” operation and detect if the unit is nominally working after implantation into the patient. This unit can be fabricated to output 5 to 50 µA total current to account for any differences in the geometry of the patient’s body where it will be used. Since the system is OFS (with reversing duty cycle of 15 minutes), the polarity of either electrode is a moot point.



This stimulator can also be integrated into a tubulization therapy. A loop of electrodes around the distal perimeter of the tube help “draw” axons in this direction. This has already been proven, as a active cathode within a silicone tube was able to coax robust regeneration of both spinal cord axons and motor axons of gray matter into end of the tube (Borgens 1999). This potential therapy also makes use of a new form of wrapped or lamellar conduit (see above) with the tube developed by CPR.

While OFS was developed to influence nerve regeneration in both directions in Spinal Cord - unnecessary in the PNS - it is both instructive and convenient to summarize the special properties of OFS that make it more ideal than simple DC field application as a therapy for PNS injury.

Significant Enhanced Window of Electrical Application over DC

Animal studies in nerve and bone employed stimulators ranging in output from 0.5 – 20 µA total current. While this current output could produce a therapeutically useful current density (µA /cm2) within the small diameter of a rat’s leg, it is highly unlikely this could produce therapeutic fields (ca 50 –500 mV /mm) within the limb of humans possessing a limb diameter orders of magnitude greater than a rat leg. OFS stimulators have already been tested to safely deliver 200-µA total current in humans (Shapiro et al 2005) through only one pair of electrodes.

Enhanced Safety of Higher Magnitude Electrical Currents

The highest 20µ A total current used in animal PNS studies has been safely increased to 50 in SCI (Borgens et al 1990), but this range in the tens of µAs, marks the upper limit of current that can be delivered through tissues without risking destruction of the tissue, either bone, cartilage, or nerve (see Science review by Borgens 1984). This is because there are electrode products generated by electrolysis at the metallic electrode/ tissue interface that can be very destructive: 1. at the Cathode (-pole) hydrogen and other gasses are produced in association with a precipitous rise in Ph to highly alkaline conditions. This is not good but less destructive to tissues than 2. at the annode (+pole), where a precipitous fall in Ph to highly acidic conditions is experienced. Moreover this is associated with the production of metal salts that are toxic, the generation of peroxides, and other damaging chemicals. When OFS is employed, the chemical reactions producing these unwanted tissue changes are reversed every 15 minutes. Since such electrolytic reactions are relatively slowly initiated within the soft tissues where electrodes are fastened, a 15 minute polarity reversal also reverses this chemistry and imposes a Ph right about neutral (~ 7.4) during the entire period of stimulation (Borgens, 1989b). Said another way, the only way to impose high DC current (i.e. hundreds of µAs) through tissues without damaging them is by OFS. This is vitally necessary because of the significantly increased cross sectional are of a human limb, and the requirement to increase current to support therapeutic electric fields. (This is given by Ohms law for extended media where the electrical Field (in V/cm) is equal to the Current Density (in µA /cm2) times the resistivity of the tissues (ohms cm)).

OFS can support higher current densities required to reduce / eliminate retrograde dieback of proximal nerve fibers after injury. It is fact that a DC electric field can reduce the retrograde degeneration of severed nerves (first demonstrated by Roederer et al 1983). This is due to a reversal of the naturally produced electric currents entering damaged portion of the nerve carried largely by Na+ and Ca++ ions (Borgens et al 1980). These ions are increased in the cytoplasm of the local nerve injury where they cause direct destruction of the architecture of the nerve at this site – i.e. “dieback”. A distally negative DC field can reverse this movement of cations into the nerve (Straughtman et al 1992), and is the mechanism by which DC fields can inhibit dieback. OFS has a profound effect on eliminating dieback, since the 10-fold increase in total current and density (see above points) can do what a simple DC field cannot. Recall that current densities entering nerves naturally are on the order of 100’s of µA /cm2 (Borgens et al 1980; Borgens 1988; Borgens 2003) and require significant applied current density to “buck” or neutralize this damaging flow of electricity.

OFS has been shown to significantly inhibit the production of scar tissue after nerve injury

This effect was reported after spinal cord injury where the character of spinal cord scar is both glial and collageneous. The former component is characteristic of CNS where glial cells increase in number forming a “glial scar”. These types of cells are not found in the PNS. In damaged spinal cord significant numbers of fibroblasts invade the cord from the outside producing a collagen based scar, typical of the scar tissue that forms in the PNS. In Spinal Cord the two part scar tissue is properly called a fibro-glial scar (Reier et al 1988). In cord, the numbers of cells that form this is cut in half by an OFS (Moriarty et al 2001). Thus it is reasonable to expect a reduction of scar tissue at the site of PNS repair in association with OFS stimulation.

OFS does not increase the accumulation of damaging cells at the site of soft tissue damage. Surgical intervention can induce more accumulation of damaging cells at the site of repair. Macrophages are phagocytes that play an important function in removing dead cells and cell debris after soft tissue injury. Large accumulations in local areas on- the- other- hand can produce even more injury. Macrophages are significantly increased by emigration into a tissue injury zone, as well as via cell division. Significant numbers of these phagocytes increase local tissue acidity, release catabolic enzymes damaging to healthy cells, and are overall not helpful to nervous tissue repair where abnormal accumulation of these cells cause bystander damage if their numbers are enhanced by surgical manipulation. While DC fields are know to guide many different types of cells (reviewed by Robinson 1985; Borgens 1989), OFS has no affect on the accumulation of macrophages thus not making a poor situation worse (Moriarty et al 2001).

In summary, OFS can safely provide a 10 – 50 fold increase in Current required to treat PNS injuries in human patients compared to DC fields alone; can reduce retrograde dieback – a serious complication facing conventional repair of PNS nerve damage; perhaps reduce scar formation; while not increasing phagocyte accumulations to damaging levels.
Finally, there are no known untoward effects of applying OFS to soft tissues. Thus OFS could feasibly be used as a “therapeutic insurance policy”, during the 2 – 3 months delay in therapy commonly employed in contusion, stretch, or compression injuries to the human PNS.

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