Current State of Affairs – Amping up Axonal Regeneration

Title

Current state of affairs – amping up axonal regeneration

Abstract

Introduction

• Significance
  • Why bother?
  • Is complete recovery of function in an injured spinal cord possible?
• Pathophysiology of spinal cord injury?
• Differentiating between axonal regeneration and cellular regeneration
• Current problem and paradigm?
  • Spinal cord is part of the CNS
  • CNS neurones are refractory to regeneration
    • Why?

Scope and significance

Spinal cord injuries (SCI) are a significant issue around in the world. SCI is estimated to affect 282,000 people in the U.S., with 17,000 newly diagnosed cases annually (1). Trauma is the main cause of SCI, most commonly following a motor vehicle accident, whilst other less common causes include spinal metastases or primary malignancies such as myeloma (see Figure 1). There is a substantial financial cost to SCI, with indirect costs averaging approximately 72,000 USD per year (1). SCI also have long lasting and poor clinical outcomes, ranging from motor deficits to sensory deficits or autonomic function impairment.

Current treatment for SCI involve neurosurgery (if indicated), continued neurological observations and high dose steroids to reduce inflammatory response in early stages (2). Late stage management relies on physiotherapy and outpatient rehabilitation. The use of high dose steroids is controversial due to side effects, which may include sepsis and gastrointestinal bleeding (3). This highlights the importance of developing safer and more effective treatment for SCI.

Figure 1: Causes of spinal cord injuries. Data taken from BMJ – Best Practice, last updated on April 2017 (4).

Background – current paradigm and problems

Neuronal output comes is facilitated by axons. Information, in the form of a membrane potential, travels down axons to reach the synaptic terminal where there is a release of neurotransmitters. Neurotransmitters then mediate the actions of effector tissues. Lesions in the spinal cord impair both ascending and descending axons.  The spinal cord itself houses a multiple nerve fibre tracts formed from axons.

The long-term impairment of SCI is dependent on the extent and level of axonal damage. The degree of axonal regeneration differs depending on the part of the nervous system in which damage resides. A neurone is considered to reside in the peripheral nervous system (PNS) if the cell body is found outside of the brain and spinal cord; whereas a neurone of the central nervous system (CNS) is defined by having their cell body is located within the brain and spinal cord. Peripheral neurones have a large capacity to regenerate from injury. This is due to their ability to retrogradely transport injury signals to their soma, thus activating transcription of repair and regeneration genes (5). They also have Schwann cells to support recovery through growth factors (6).

A combination of factors inhibit central neurones’ regeneration ability. Myelin proteins (such as Nogo, MAG and Omgp) can bind to a receptor on neurones (Nogo-66 Receptor), activating the Rho GTPase cascade. This prevents polymerisation of fibrous actin filaments thus preventing axon outgrowth (7). Reactive astrocytes form a physical barrier (a glial scar), and chondroitin sulphate proteoglycans (CSPG), act as a chemical barrier against axon regeneration. Moreover, mature CNS neurones have undergone intrinsic changes to their gene expressions to better fulfil their role of stable neurotransmission rather than growth. The extracellular environment has reduced cues to encourage axon growth. Additionally, the axon in the mature CNS has longer distances to regenerate compared to early development. These factors present a challenge for new regenerative strategies for axon repair and recovery.

Researchers are tackling this issue from various angles – pharmacological therapy, brain machine interfaces, stem cell therapy and electrical stimulation (ES). In the current molecular and genetic era, one must not neglect other older perspectives. ES is not an alien concept to neurological disorders. Electroconvulsive therapy, despite a significantly declined usage, was first used in 1938 (8). Currently, functional electrical stimulation is being used as a form of short-term management in aiding rehabilitation, and deep brain stimulation has been shown to be beneficial in patients with Parkinson’s disease in terms of symptomatic control (9). Recent studies have shown that ES is capable of stimulating axonal regeneration, thereby offering a potential new therapeutic option to CNS injuries, including SCI.

Body

• Mechanism
• Experimental evidence
  • What do they show?

Underlying concepts – endogenous electric currents already exist

In chick embryo studies carried out in 1990, it was shown that endogenous electrical currents formed voltage gradients. The manipulation of these currents led to changes in morphogenic fields of the embryo (10). Further studies also showed that these voltage gradients are a normal phenomena in early stages of development within the nervous system (11,12). Therefore suggesting that electrical currents have some form of contribution to axon growth.

Moreover, endogenous electrical currents have also been shown to facilitate recovery. In mice models, these endogenous electrical currents are rapidly established at the site of injury (13). Other studies have also shown that endogenous electrical fields not only guide axonal growth and increase the rate of wound healing (14). Given that endogenous electrical currents exist in both early development and during wound healing, this forms a basis upon utilising exogenous electrical currents to manipulate axonal regeneration.

External/exogenous electric currents

• Evidence
• What actually happens?
• Problems?
  • Can we get around it?

By creating an exogenous electric field, researchers have attempted to mimic these conditions to promote axonal growth and regeneration after SCI. Initial studies utilising guinea pig models showed that a direct current (DC) electric field stimulation showed enhanced indirect axonal growth compared to those in the control group (15). Some of these guinea pigs also showed faster functional recovery time (15,16). Similarly, in mice studies, ES through a DC field also showed functional improvement despite fewer studies showing significant histological differences (17,18). In other mammal models, synapses were also successfully formed at the distal end of the lesion (19).

The direction of current in ES is critical for appropriate axonal regeneration. Nerve fibres were shown to grow towards the cathode faster than towards the anode (20). Furthermore, the cathode was shown to be able to re-orientate axon growth depending on the strength of the ES. Nerve fibres that were growing parallel to the electric field would simply grow towards the cathode whilst fibres growing perpendicular to the electric field would turn to align themselves towards the cathode (21). This is further supported by the fact the removal of ES resulted in disorganised nerve fibre outgrowth, and reversal of direction led to subsequent change in orientation. The ability to use ES to direct axonal growth is crucial if ES is to be used as a form of SCI treatment. Abnormal or haphazard synapses can be as catastrophic as the lack of synapses.

However, whilst axonal growth can be directed using the cathode, a DC electric field will suppress axon growth and even induce regression at the anode if given sufficient time (22). This means axonal regeneration will be successful for either descending motor pathways or ascending sensory pathways in the spinal cord but not both. A strategy to allow simultaneous regeneration of both afferent and efferent pathways of the spinal cord is to use an alternating current (AC) electric field. This has been shown to be successful in dog models through the use of oscillating field stimulation (OFS). The study switched polarities of the ES every 15 minutes to provide opportunity for sufficient axonal growth without suppressing axonal regeneration or even regression (23,24). OFS treated dogs showed greater improvement even at six months compared to the control group. In fact, these dog studies were so successful they prompted Phase I human trials. Ten patients with acute traumatic SCI affecting both motor and sensory function had OFS implanted within three weeks post-insult, and underwent OFS therapy for 15 weeks. Despite a later erratum regarding inconsistencies in functional scoring, the trial still maintained there was improvement in both sensory and motor function (25,26).

The underlying mechanism of ES mediated axon regeneration is still not fully understood. However, there are a few theorised mechanisms by which ES encourages axon outgrowth after a SCI. Firstly, ES can upregulate growth associated proteins, neuronal growth factors (neurotrophin) and their receptors (27). In vitro studies showed DC ES and neurotrophins interact synergistically, with faster and more directed growth towards the cathode. Interestingly, stimulating neurones with specific neurotrophins can even cause directional changes despite an established DC electric field (28). Coupled with the fact that endogenous electrical currents are proven to exist during early development, a time where neurotrophins and other growth factors are in abundance, it should come as no surprise that ES can interact with neurotrophins or alter neuronal response to neurotrophins.

ES via OFS on mice models also showed a reduction in reactive astrocytes (29). After insult, astrocytes will become reactive, taking on a more fibrous morphology. This fibrous morphology is courtesy of GFAP, an intermediate filament found in astrocytes (30). Besides a change in morphology, they also aggregate around the site of injury to proliferate and form a glial scar that hinders axonal regeneration (31). Not only did OFS significantly reduce the number of astrocytes at the site of the lesion, it also reduced the number of astrocytes with cell processes orientated towards the lesion (29). As mentioned previously, reactive astrocytes also form CSPG, which bind to CSPG receptors to activate the Rho GTPase cascade, inhibiting axonal growth (32). This inhibition of glial scar formation is another way in which ES can aid axonal regeneration.

Another method by which ES can stimulate axon regeneration is through the activation of secondary messengers such as cAMP (33). Through the upregulation of cAMP, there is an increase in protein kinase A (PKA), which in turn increases transcription and translation of regeneration associated genes, neurotrophins and neurotrophin receptors (34). ES mediated cAMP pathway has only been shown in peripheral neurones, but there is little to suggest this mechanism is impossible in the CNS (35).

Discussion

• Advantages
• Disadvantages
• What else is there to offer?
• Other forms of electrical stimulation?

With the clinical implications in mind, the mechanisms of ES warrant further investigation. Uncovering the fundamental principles by which ES can stimulate CNS axonal growth will not only allow more applications to be revealed but ultimately a better clinical outcomes for patients. Another aspect that should be considered for future research is the relationship between delay of providing ES and axonal regeneration. Although acute SCI can usually be treated relatively quickly due to trauma being one of the highest causes, other forms of CNS axonal degeneration may not be this convenient. It is crucial to know if there is a decline in the number of nerve fibres capable of regenerating in the CNS post-injury or ability of CNS neurones to respond to ES; and if so, at what point is ES of no additional benefit. This resonates with the basic principle of maleficence. If there is no benefit from offering a form of treatment, perhaps withholding the treatment is in the patient’s best interest.

It must also be noted that that despite the overall better outcome in the aforementioned dog models, recovery was not perfect. The discrepancies in results of human trials do little do encourage the use of ES. OFS as a form of treatment is not without risks. It is invasive, involving surgical implantation stimulators across the lesion in the spinal cord. This carries the typical risks involved with any surgery (haemorrhage, infection), as well as more specific risks such as stimulator malfunction or battery life. Despite these risks, one must remember that overall, OFS is still a straightforward procedure, and most importantly, a safe procedure. General surgical risks are encountered on a daily basis and implantation of electrodes is not an utterly novel concept (through pre-existing forms of ES mentioned earlier and cardiac pacing). Healthcare professionals are capable of managing the risks that are associated with ES and OFS.

Does this mean ES therapy is the way forward? Highly unlikely. Combinations of therapies may help minimise risks of ES. Studies exploring the interplay between neurotrophins and ES on axonal regeneration have already shown that weaker ES are required to promote growth (28). In practical terms, this indicates a lower risk of battery failure. It also suggests that a lower dose of pharmacological therapy is required, hence potential side effects from such therapies may also be reduced.

ES research can also collaborate with neural stem cell (NSC) and nanotechnological research more regularly. Carbon nanotubes have shown to provide stable scaffolding for NSC differentiation compared to traditional culture mediums (36). Other studies have also shown that there is around 10% more neuronal growth from NSC grown on conductive polymers under ES compared to the lack of ES (37). The cumulative approach is most likely the future of SCI treatment and will inspire new main stead therapies for CNS degeneration.

For those who favour a more conservative approach, combining ES with the current guidelines for SCI treatment is also viable. If used in conjunction with steroid therapy, which reduces oedema in the spinal cord, ES is more efficient and shows earlier recovery (38).

Latest research in the role of reactive scar formation has also challenged the current dogma. A study carried out last year has suggested that glial scar formation actually encourages axon regeneration. This goes against the common belief that glial scar formation is a principal inhibitory factor of CNS regeneration. Through activation of axonal growth programs in the astrocyte genome, only CNS lesions with glial scars exhibited axonal regeneration. The physical isolation formed by the astrocytes may prove to serve more of a protective function than previously considered (39,40). This is not to say if ES reduces the number of astrocytes, it must hinder axonal regeneration. The tradition of classifying cell states as discrete morphologies is also dated. Reactive astrocytes more realistically exist on a spectrum ranging from neuro-protective to neurotoxic (41). Therefore more research should be focused on expanding our knowledge of the astrocytic response spectrum, and how their properties can be manipulated through external stimuli such as ES.

In conclusion,

As mentioned above, there are risks associated with ES. The sheer complexity of the nervous system itself contributes to the difficulty in developing an efficient and safe treatment for SCI.

ES on neural stem cells?

Conclusion

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