Significance of Clinical Treatments on Peripheral Nerve and its Effect on Nerve Regeneration

Some neurological disorders that cannot be managed with medication can alternatively be directly treated either at the sensory and/or motor nerve(s). In particular, chronic pain and/or muscle spasticity are a couple types of symptoms that can potentially be treated at the associated nerve. Various chemicals and medical devices are currently being employed in the clinic to treat peripheral nerves. Treatment of sensory nerves is typically used to attenuate chronic pain symptoms, while motor nerves are targeted for spasticity symptoms. A majority of treatment methods currently used in the field involve nerve injury and denervation of the target tissue through different mechanisms of action. Depending on the type and severity of the treatment, the nerve may or may not fully regenerate to a normal state. This article will review the various types of clinical methods used to treat neurological disorders and discuss the possible outcomes of nerve regeneration.


Introduction
Millions of people worldwide suffer from neurological diseases relating to chronic pain and/or spasticity [1][2][3]. Medication may not be a suitable option for some patients due to unacceptable systemic side effects and/or ineffectiveness in reducing the symptom(s). Some of the side effects of using pain medications include nausea, addiction, drowsiness, depression, and decreased effectiveness over time. While side effects of anti-spasticity drugs include muscle weakness, drowsiness, nausea, and psychological effects. Alternative methods may be employed in the direct treatment of the peripheral motor or sensory nerve implicated in the disease. The main objective of treating the peripheral nerves is to disrupt either the pain signaling of the sensory nerve or the involuntary signaling of muscle contraction by the motor nerves. Treatments directly targeting the nerve helps reduce the possibility of systemic side effects associated with either pain or anti-spasticity medications.
While the neurons in the central nervous system (CNS) are unable to regenerate after injury, axonal regeneration in peripheral nerves can be robust [4]. In the CNS, various inhibitory factors (ie Nogo, MAG, OMgp, etc) expressed by the surrounding cells (ie myelin) and present in the extracellular space, prevent nerve regeneration from occurring [4]. In contrast, the peripheral nerve system (PNS) responds to injury by upregulating various neurotrophic and axon guidance signaling factors to promote regeneration [5]. An injured nerve axon undergoes a complex process to restore normal function of conducting signal to/ from its target tissue. Upon injury, the axon begins a degeneration process at the injury site and progresses distally. A small distance of retrograde degeneration occurs, but the proximal portion of the nerve body and axon largely remains intact [6]. Concurrently, the myelin sheath also undergoes a degeneration phase. Macrophages and Schwann cells function to clear the cellular debris. Regeneration of the axon immediately follows with the Schwann cells undergoing a proliferation and differentiation phase to form the scaffolding for the axon. Various cell signaling factors occur between the neuron, Schwann cells, and the surrounding environment promotes the elongation and guidance of the regenerating axon. Greater detail of the molecular and cellular mechanisms of peripheral nerve regeneration can be found in literature [7][8][9]. Depending on the nature of the damage, the peripheral nerve may or may not be able to fully regenerate to a normal morphological and/or functional state [10]. The ability of the nerve to regenerate predominantly relies on how well the surrounding nerve structure (endo-, peri-, and epi-neurium) remains intact. This article will compare some common treatment methods employed in the clinic and its effect on the peripheral nerve's regenerative outcome.

Nerve Injury Classification and Expected Recovery
Characterization and classification of nerve injuries have been well established for many decades. Several research and review articles examine the molecular, pathological, and clinical characteristics of nerve injury in great detail and two commonly known nerve injury severity scales have been published. Sir Herbert Seddon first published a 3-point nerve injury scale in 1943 [11]. Later, in 1951 Sir Sydney Sunderland published a 5-point scale which gave more resolution to Seddon's scale [12] (Table 1).

Description
Seddon Scale

Sunderland Scale
Neuropraxia -Disruption of nerve conduction, but nerve structure and axon remain intact. I I Axonotmesis -Disruption of nerve conduction and axon degeneration, but remaining nerve structure (endoneurium, perineurium, and epineurium) remains intact.

II II
Axonotmesis with disruption of the endoneurium, but the perineurium and epineurium remains intact. II III Axonotmesis with disruption of the endoneurium and perineurium, but epineurium remains intact. II IV Neurotmesis -Total transection of the nerve fiber. III V First degree (neuropraxia) is the mildest form of nerve injury and is characterized by a disruption of the nerve's ability to transmit an action potential along the axon. Damaged myelin sheath localized at the injury site may occur, but the entirety of the nerve structure and the axon largely remains intact and unchanged. Full functional recovery from neuropraxia can range from minutes to a few weeks. Although this form nerve injury is not as widely studied as the more severe injuries, but pressure block and non-freezing low temperatures are typical methods used in basic research to study temporary nerve conduction blocks [12].
Second degree nerve injury is characterized by a degeneration process of the axon, also known as Wallerian degeneration. Nerve crush injury is typically the cause of Wallerian degeneration and is used as a model in basic research. Degeneration of the axon and myelin sheath occurs initially at the injury site, shortly followed by degeneration of the remaining distal portions of the axon and associated myelin sheath. The retrograde degeneration of the axon is limited to injury site while the nerve body remains unchanged. Wallerian degeneration results in a longer duration of functional loss as compared to neuropraxia. Functional recovery relies on the time it takes for the axon to regenerate and re-innervate the target organ. Depending on the distance of the axon injury site to the target site, the disruption of signaling can last from weeks to months. Numerous factors can affect the rate of axon regeneration which can range from 1 to 5 mm per day [13][14][15]. Reliability of axon regeneration also relies on the integrity of the surrounding endoneurial, perineurial, and epineurial structure. These structures acts as a scaffold to guide the regenerating axon for optimal recovery, while signaling factors promote axon regeneration. Depending on the extent of damage to these surrounding structures, the degree of nerve injury would be higher (see below). Furthermore, axonal regeneration relies on a complex interaction between the Schwann cells, macrophages, and the axon aid in regeneration and re-myelination of the nerve [16]. Some of the interactions among these cell types involve various signaling pathways, including Toll-like receptor activation, NF-κB activation, and up-regulation of various interleukin cytokines [17]. The proper regulation of the various signaling factors helps coordinate the clearance of the cellular debris and direct the axon to its target site. Through this complex molecular and cellular interaction, the nerve is able to reliably regenerate and re-innervate its target organ for normal function.
Third degree injury involves Wallerian degeneration of the axon but also includes damage to the endoneurium. This type of injury is common with nerve stretch injuries. The endoneurium primarily consist of various collagen types, reticular fibers, and an extracellular matrix (ECM). The primary role of the endoneurium is to provide a scaffold for the Schwann cells and axon. Injury to the endoneurium can induce fibrosis and scarring within the endoneurial tube [18]. Due to the disruption of this path, axonal regeneration rate is diminished. Additionally, studies have shown axon regeneration to deviate from the disrupted endoneurial tube and into the intrafasicular structure; which, could lead to a potential neuroma formation [18]. Functional, but not complete, innervation of the target site can occur over a period of months to year(s) [12,18,19].
Fourth degree injury involves Wallerian degeneration but also includes damage to the endoneurium, perineurium, and epineurium. This type of injury can be found in laceration or severe stretch injuries. The severity of this injury allows for the regenerating axon to grow into the extrafasicular structure, resulting in growth termination [20]. Increased scarring and fibrosis is more prevalent; therefore, promoting neuroma formation [12]. Without surgical intervention functional recovery is minimal, while surgical repair will allow for improved chances of useful functional recovery, but with incomplete reinnervation of the target tissue [21].
Fifth degree injury is the total transection of the nerve. This type of injury is commonly found in lacerations or amputations. Without surgical repair, total lack of functional recovery with scar and neuroma formation occurs. Due to the lack of the nerve scaffolding, axons will regenerate aberrantly at the injury site and will not be able to continue to the distal nerve stump [22]. Additionally, spontaneous and aberrant signaling from the injured sensory nerve result in neuropathic pain symptoms, commonly observed in amputation stump pain [23,24]. Some surgical methods used to repair nerve transection include suturing to join the proximal and distal stump, auto-or allo-grafting of nerve gaps, or use of biological or biosynthetic conduits [25]. These various methods allows for some percentage of the axons to regenerate along a scaffold and be able to reach the target organ. These methods allow for very limited success in functional and useful recovery in sensory and/or motor function. Continued research and development in the field is being carried out to determine the best materials and methods to promote a robust neuroregenerative response [26,27].
In general, the degree of nerve injury determines the ability for the nerve to regenerate over time. The milder forms of nerve injury (i.e. neuropraxia and axonotmesis) allow for normal nerve regeneration as the ability to minimize any structural changes to the scaffolding of the nerve allows for more complete regeneration of the axon. More severe injuries (i.e. axonotmesis with structural damage or neurotmesis) result in disruption of the nerve path which makes it more difficult or impossible for functional regeneration of the nerve.

Treatments Utilized in the Clinic
In cases where symptoms of a neurological disorder cannot be treated with medication, or an alternative treatment method is desired, a physician may consider directly treating the nerve. For example, chronic pain patients can be treated by disruption of the sensory nerve eliciting the pain. Similar methods can be used to treat the motor nerve for spasticity patients. Various methods are currently being employed to treat disorders related to the sensory and/or motor nerves. Typically the methods are used to disrupt signaling of these peripheral nerves to attenuate symptoms of the disorder. Each method produces vastly different mechanisms of action for disruption of nerve signaling, typically through some form of nerve injury. Below we will discuss the methods employed in the clinic and describe the mechanism involved and implications for the ability for nerve regeneration.

Phenol and Alcohol Nerve Blocks
Injection of either phenol or alcohol to the nerve has been used to treat pain and/or spasticity symptoms. The use of these neurolytic agents have been report since at least the early 1900's [28]. Examples of their use include, but are not limited to, the application for post-stroke related spasticity [29], neuromas [30], and pain [31]. These methods have demonstrated a loss of, or reduction in, nerve function which can last from 2 months to 3 years [32]. Both chemicals have different methods of action, but with similar clinical results.
At low concentrations (<2%) phenol can be used as a short term anesthetic with minimal nerve injury [29]. The primary mechanism of action at lower concentrations of phenol is believed to be an disruption of ability for the nerve to produce an action potential across the axon membrane [33]. Phenol at the higher concentrations is generally known to cause non-selective destruction to tissue. Depending on the concentration used, phenol can induce axonal degeneration with some damage to the acellular nerve structure. It is not fully understood as to whether the mechanism of action is due to the denaturation of proteins in the nerve or by an ischemic injury to the neurovasculature [34]. Morphological analysis of nerves treated with phenol has found Wallerian degeneration does occur, while the epineurium remains intact. However, loss of cellular fatty elements, separation of myelin sheath, and axonal edema also occurs [35]. Based on these histological findings, the data indicate a minimum of second degree nerve injury with minimal to no loss of the endoneurial nerve structure at concentrations of ~5-7%. Further examination demonstrated that even though function recovery appears normal, force measurements showed a long term decrease in maximum tension as compared to both untreated control and the crush injury model [34]. In particular, higher concentrations 7.5-10% has been found to produce permanent anaesthesia and/or muscle weakness, thus indicating a disruption of the nerve scaffold structure [36]. This data indicates that, depending on concentration used, phenol can produce a second and/or third degree injury, where there may be a mild deficit in nerve regeneration.
Similar to phenol, use of different concentrations of alcohol will also give differing results. Low concentrations (4% to 29%) of alcohol are able to temporarily interrupt nerve conduction with minimal damage to the nerve. Higher concentrations of alcohol (30% to 100%) inhibit signaling and degenerates the nerve. At particularly high percentages (90% to 100%), histological examination indicates fibrosis surrounding the treatment site with a slower regeneration rate [28]. Similarly to phenol, alcohol is generally known to cause non-selective destruction of proteins in tissue types [29]. Additional reports indicate that the function of the treated nerves will have normal functional return over time [37]. Similarly to phenol, clinical duration of effect has been reported to last from a few months to years. This variable range of duration is likely to be due to numerous factors ranging from targeting of the nerve, type of nerve, and concentration used. Furthermore, both phenol and alcohol have been known to cause vascular injury; thus, playing a possible role in nerve degeneration and/or regeneration process [32]. Alcohol produces a second to third degree nerve injury. While concentrations closer to 30% are likely to produce a second degree injury, there is increasing likelihood of third degree injury at concentrations near 100%.
The known evidence demonstrates that phenol and alcohol can create a temporary degeneration followed with normal regeneration both histologically and functionally. Higher concentrations may allow for a more effective treatment, but with increasing chance of third degree nerve injury and collateral damage to surrounding tissue structures.

Cryoneuromodulation
The use of freezing temperatures to treat chronic pain, also known as cryotherapy or cryoanalgesia, has been in use for many decades. Numerous reports have demonstrated the efficacy of freezing temperatures in attenuating various chronic pain symptoms ranging from trigeminal neuralgia to neuroma, and post-thoracotomy pain [38][39][40][41][42][43]. These reports have demonstrated pain relief ranging from a couple of months to a few years [40,43].
Though the concept of using cold to treat sensory nerves has been in use since the mid-1800's [44], application to motor nerves had not been employed until recently. Cryoneuromodulation, a term which encompasses treatment of either sensory of motor nerves, occurs over a wide range of temperatures with treatment often occurring at temperatures as cold as -196°C (liquid nitrogen coolant) or -88°C (nitrous oxide coolant). While clinical case reports have demonstrated the use of non-freezing low temperatures to demonstrate transient attenuation of muscle spasticity or pain associated with sports-related injuries [45][46][47]. A recent preclinical report described the feasibility of using freezing temperatures to treat motor nerves for potentially treating movement disorders [48]. These results could be expected given that the mechanism of denervation and regeneration is similar for sensory and motor nerves [49]. Clinical trials are currently underway to determine the potential use of freezing temperatures as a viable option for treatment of upper limb spasticity (http:// www.clinicaltrials.gov; NCT01863901). Due to the short duration of non-freezing temperature cryotherapy, we will focus on freezing temperature cryoneuromodulation as this is a more common method for treating chronic neurological disorders.
The mechanism of action for tissues treated with temperatures as cold as -196°C is based on both the final freezing temperature as well as the rate of cooling [50]. For this reason, there are multiple proposed modalities of injury including physical cell damage by intracellular ice formation, which occurs during cooling at a high rate. If the cooling occurs at a slow rate then an increase in extracellular solute concentration during the freezing process can cause the cells in the tissues to dehydrate thereby damaging cytoplasmic proteins [51]. In addition to either of the described mechanisms of action there may also be ischemic injury due to vascular damage [51,52].
Histological studies have shown that for treatment of nerves with temperatures from -20° to -60°C Wallerian degeneration occurs while leaving the endoneurium intact [53]. Additionally, a comparative study between crush lesion (a common model of Wallerian degeneration) and cryoneuromodulation treatment on a rat sciatic nerve showed comparable histological results of second degree injury [54]. Examination of tissue exposed to freezing temperature has found that cells are injured, but the surrounding acellular structures remain intact [55]. This feature aids in the preservation of the acellular structures of the endo-, peri-, and epi-neurium; allowing for normal axonal regeneration and return of function. Based on the morphological findings and reported normal return of function supports second degree degeneration at temperatures as low as -60°C.
At temperatures at -100°C to -180°C, histological analysis show extended damage to the basal membrane and collagen proliferations. These markers indicated endoneurial and perineurial damage, but the epineurium appeared to remain intact [53]. The lower temperatures have also demonstrated a longer duration of electrophysiological and functional disruption when compared to temperatures warmer than -60°C [53,55]. These findings demonstrate that lower temperatures (<-100°C) creates a third degree nerve injury. These colder temperatures can create a longer term efficacy of reduced muscle spasticity or pain attenuation as compared to the milder freezing temperatures. Due to the acellular structure damage of the endoneurium and perineurium, there is a risk of neuroma formation and reduced motor or sensory function of the target tissue.

Radiofrequency
Radiofrequency is commonly used to treat pain chronic pain syndromes with some use for motor nerves. Conventional (CRF) and pulsed (PRF) radiofrequency are 2 forms of RF technologies currently employed in the clinic. CRF is continuously applies radiofrequency in a continuous fashion and heats the tissue to approximately 60 -80°C. PRF applies the radiofrequency at a higher voltage in a pulsed fashion and heats the tissue to approximately 40-42°C. In between bursts of current, the inactive phase allows the heat to dissipate. Ongoing research has shown that these two different methods of energy application have a different mechanism of action [56,57].
The high temperatures employed by CRF are the primary cause of tissue injury. The exposure temperatures are known to cause a coagulative necrosis to both cellular and acellular structures. The temperatures reached in CRF are known to cause collagen hyalinization [58], and histological studies have shown both axonal degeneration and collagen fiber destruction of the endo-, peri-, and epi-neurium structure occurs [59,60]. Clinical reports for the treatment of chronic pain have also shown that CRF produces lasting effects for years, but with reported long term side effects. Some of the side effects include deafferentation pain syndrome, neuritis, and paresthesia; all of which could indicate abnormal regeneration of the treated nerve [61]. Based on the extensive damage on the nerve structure and prolonged length of functional loss and side effects, these characteristics indicate that CRF produces a third or fourth degree injury with a potential for neuroma formation. Although CRF may produce long term efficacy, this method produces a higher chance of neuroma pain or reduced motor function in the long term.
PRF is a more recent development of the radiofrequency technology that is commonly used and is presented as a less destructive alternative to CRF by reducing the temperature to approximately 40 to 42°C. Morphological analysis has shown transient endoneurial edema with a return to normal morphology by 7 days post-treatment, while clinical duration of effect has been found to last for months to years [60,62]. Since histological examination has determined that insignificant changes occur to the nerve and its surrounding structures, a distinctive mechanism of action is occurring [60]. At this point, the mechanism of action has not been identified. It appears that thermal energy does not create a neurodegenerative process to occur, it is believed that the electromagnetic field may be affecting the nerves, Schwann cells, and/or satellite cells [60,63]. Various studies have implied that PRF may either disrupt the neuronal membrane and function, gene expression change (c-Fos), and/or cytokine up-regulation [57,59]. At this point, the mechanism of action remains vague and more studies are required to elucidate the mechanism. Based on the known information, PRF does not fit into the five degrees of nerve injury and its mechanism of action vastly unique from the other treatment methods described in this article.

Surgical Transection
Nerve transection (neurectomy) is a permanent disruption of nerve function. Nerve transection is rarely used and considered a last resort treatment method for pain management or spasticity. As mentioned above, transection is a fifth degree nerve injury which results in a permanent loss of function with a likelihood of neuroma formation [64]. Clinical reports have indicated that this method has a reported pain recurrence rate ranging from 68% -78% [65,66]. The recurrence of pain is likely due to neuroma formation, which leads to neuropathic pain.
Morphological studies have shown that the regenerating axon forms a non-neoplastic neuroma at the end of the transected nerve stump. The neuroma consist of disorganized myelinated axons wrapped in perineurial tissue [67]. Additionally, an increase in smaller disorganized myelinated fibers was observed post-treatment indicating the formation of nerve sprouts occurring within the neuroma [68]. Currently, various techniques for the prevention of neuroma formation are being studied with varying outcomes [67,69].

Discussion
The methods described in this article detail several of the approaches that can be used in the treatment of neurological disorders relating to peripheral nerves. This article did not cover additional therapies, such as neurotoxins or high frequency nerve stimulation, as they fall outside the scope of nerve injury and regeneration. Table 2 provides an overview of each treatment method, as well as their associated degree of nerve injury and regeneration ability, based on the analysis of available literature. With increasing degree of injury severity, the ability and rate of axon regeneration diminishes; thus, producing a longer duration of clinical effect. The consequences of increased degree of injury would also increase the deficit in the nerves ability to regenerate; while, the likelihood of neuroma formation and aberrations increases. The mildest treatments (i.e. lower concentrations of phenol/alcohol and cryoneuromodulation above -60°C) produce a second degree nerve injury, effective in reduction of pain or muscle spasticity with normal recovery of function over time.
More aggressive treatments (i.e. higher concentrations of phenol/ alcohol, CRF, and cryoneuromodulation below -100°C) that produce third to fourth degree nerve injuries can also produce an effective loss of nerve function, but with a deficit in the nerves ability to regenerate and increased probability of neuroma formations. While nerve transection produces the most severe type of nerve injury which results in a permanent effect with high likelihood of neuroma formation.  In deciding on the appropriate treatment method, the goals of the clinical results must be considered. Successful treatment of the symptoms will depend on a variety of factors including patient selection, effects on surrounding tissue, proper 'dosage', nerve type, proper nerve targeting, and/or application method. Depending on the mechanism of action initiated by each method, short and long term nerve regeneration results can vary markedly and should be considered in deciding a treatment for any nerve treatment. For example, post-thoracotomy pain is expected to last about a year. A treatment with a shorter duration of effect would be desired and a physician may choose a method that will cause a second degree nerve injury (such as cryoneuromodulation (<-60°C) or lower concentrations of phenol/alcohol injection) which would last for months to a year, as opposed to CRF which tends to last for multiple years. Although it may seem desirable to use a more aggressive treatment for chronic long term pain symptoms, there is an increased risk of causing a neuroma; thus increasing the chance of overall pain symptoms. Therefore, repeat milder treatment may likely be a more desirable method as compared to a single aggressive treatment.
Another factor to consider is proximity of the targeted treatment site to other nearby tissues. In all the above mentioned methods, the treatments are non-selective and can disrupt any tissue type in the body. While cryoneuromodulation and radiofrequency treatments typically affect areas surrounding the probes of the device, injection of phenol and/or alcohol can spread to nearby structures. This is particularly important when unwanted injury to sensitive tissue that can cause unwanted side effects. At the same time, the localized nature of cryoneuromodulation and RF can create a challenging treatment when the precise location of a target nerve is not known. The small treatment zone surrounding the probes of these technologies requires that the tip be placed within a few millimeters from the target nerve. This can be mitigated through the use of imaging/guidance devices to help target the nerve location, such as transcutaneous electrical nerve stimulation, ultrasound, fluoroscopy, or other imaging technologies.
It is likely, in most treatment cases, that 2nd degree nerve injury would be the most desirable treatment outcome. Second degree injury appears to be best 'balanced' treatment when compared to 1st or 3rd degree nerve injury. Although 1st degree injury is associated with no risk of nerve regeneration, it is also has the shortest expected duration of effect. On the other end of the nerve injury spectrum, 3rd degree and higher nerve injury may produce longer term results, but with a risk of reduced re-innervation of the target organ and/or neuroma formation. Additionally, the risk is can be cumulative with repeat treatments over time. Due to the robust regenerative capacity of the axon in 2nd degree nerve injury, there is a more complete regeneration and re-innervation, along with a reasonable length of treatment efficacy.

Conclusion
This article details the various known mechanisms of action associated with a variety of treatments for peripheral nerve disorders and characterizes their degree of nerve injury as well as ability for nerve regeneration. Based on the known and expected results of the various mechanisms of action, a treatment that produces a 2nd degree nerve injury will provide acceptable duration of treatment efficacy while allowing the nerve to regenerate and re-innervate its target organ. A 'higher dosage' of most of these methods (i.e high percentage phenol/alcohol or extremely low temperature cryoneuromodulation) can lead to 3rd or higher degree of injury; therefore, care must be taken to apply a safe and effective treatment. Additionally, a higher degree nerve injury can accumulate over time with repeated treatments; thus, leading to reduced motor or sensory function. Determining the appropriate method for a specific patient should be based on the clinical use and desired result as well as the potential for long-term healthy nerve regeneration.