DIFFERENT APPROACHES FOR NEURONAL REGENERATION

 

a. Electric Field Assisted Neuronal regeneration Approach:

 Effect of DC field (McCaig et.al.)

Ø     Application of electric fields to damaged neurons represents a new frontier in the treatment of nervous system injury.
Ø     Several attempts to enhance mammalian neuronal regeneration with electromagnetic fields induced by Helmoltz coils as well as with AC or DC fields.
Ø  The first indication that peripheral nerves responded to exogenous electric fields was discovered during attempts to stimulate regeneration of amputated frog limbs. When the cathode was positioned at the plane of amputation, as much as 20% of the distal area of the stump was composed of nerve.
Ø     The response was dependent on current polarity; in anode-distal stumps and those to which no current was applied, only 1% of the volume of the limb terminus consisted of nerve trunks. This result is consistent with the polarity of the field responses of frog neurones in vitro.

How do electric fields exert their effects in-vivo?

 The mechanism by which electric fields enhance neuronal regeneration remains unclear but several possibilities exist.
 (1) A distally negative- imposed field reduces inward flow of Ca 2+ at the cut end of neurons à reduces the level of retrograde degeneration following transection.
 Ø     There is evidence that an applied field (cathode distal) virtually eliminates Ca2+ influx in lamprey spinal axons and that applied fields augment regeneration of these same axons (Borgens et al.). Whether these effects are reproduced within mammalian systems is not known.
 
(2) The field may induce branch formation, as has been shown for intact rat peripheral neurons and cultured frog spinal neurons.
 Ø     Increased branch formation in damaged cells would allow numerous paths to be sampled, thereby increasing the chances of finding a favorable route for regeneration.
Ø     Alternatively, uninjured neighboring cells may be stimulated to branch, causing formation of deviant, yet potentially functional synapses.

 (3) Non-neuronal elements, such as glial cells, fibroblasts or Schwann cells may respond to the fields, thus affecting the physical composition of the scar at the lesion site.
Ø     For example, fibroblasts, which migrate towards the cathode of an applied field, may be arranged more diffusely. The resulting scar would be less dense, possibly permitting penetration by advancing growth cones.
Ø     Alternatively, exogenous fields, which increase capillary permeability, may affect the inflammatory response following injury.

 (4) More neuroblasts differentiate in the presence of an applied field in vitro so the field may act as a trophic factor, increasing cell survival and differentiation.

 

Example of in-vivo effects:

 

 

Drawing of the intraperitoneal battery implantation and subcutaneous electrode routing. The inset detail shows the approximation of the end of the wick electrode to the exposed spinal cord at the lamineetomy site. The electrode is sutured to the axial musculature.

 
Ø     Using an implanted battery and electrodes, a weak, steady electrical field across partially severed guinea pig spinal cords was imposed. Regeneration of dorsal column axons was observed in experimental animals and sham-treated controls at 50-60 days post-injury by anterograde filling of these axons with the intracellular marker horseradish peroxidase and by employing a marking device to identify precisely the original plane of transaction.
 
Ø     In response to electric field applications, axons grew into the glial scar, as far as the plane of transaction in most experimental animals. In a few animals, axons could be traced around the margins of the lesion (but never through it). Moreover, these fibers returned to their approximate positions within the rostral spinal cord before turning toward the brain.
 
Ø     In sham-treated controls, ascending axons were found to terminate caudal to the glial scar, and rarely were any fibers found within the scar itself. Axons were never observed to cross into the rostral cord segment. Thus an imposed electrical field promotes growth of axons within the partially severed mammalian spinal cord, that a steady voltage gradient may be an environmental component necessary for axonal development and regeneration, and that some component(s) of the scar impede or deflect axonal growth and projection.
 
Ø     Peripheral nerves within the limb stumps of adult frogs showed an exaggerated growth in the presence of an imposed field (using implanted DC stimulators). The regenerative response of lamprey reticu-lospinal neurons was enhanced in-vivo by placing a distally negative field across the transected spinal cord.
 

How do electric fields exert their effects in-vitro?

Ø     Applied extracellular steady electric fields of 0.1-10 V/cm have been found (Patel et. al) to have marked effects on the neurite growth of single dissociated neurons (Xenopus) in culture.
Ø      Neurites facing the cathode showed accelerated growth, while the growth of those facing the anode was reduced.
Ø      Neurites growing perpendicular to field axis were prompted to curve towards cathode.
Ø      More neurites appeared to be initiated from the cathodal side of the cell.
Ø      Number of neurite-bearing neurons per culture & average neurite length increased.
Ø      These effects are absent in cultures treated with electric fields of similar strength but alternating polarity and cannot be attributed either to a gradient of extracellular diffusible substances or to the flow of culture medium produced by the field.
Ø      Field effects are reversible: removal of electric field result in loss of neurite orientation in a few hours; reversal of polarity of the field led to rapid reversal in neurite orientation.
Ø      Incubation with concanavalin A (Con A) was found to abolish field effects completely.
Ø      Since binding of Con A to neuronal surface has been shown to prevent field-induced accumulation of Con A receptors toward cathodal side of these neurons, it is believed that cathodal accumulation of growth-controlling surface glycoproteins by field is the underlying mechanism of field-induced orientation of neurite growth toward the cathode.

Asymmetric neurite growth in vitro in an electric field (5V/cm)

 
Ø     A: bipolar neuron (1, 2) at onset of experiment. B: after 2 hr exposure to electric field.
Ø     Neurite 1 (facing the cathode) grew substantially, while neurite 2 retracted.
Ø   C: after 4 hr exposure to field. Neurite 1 grew further & branched, and neurite 2 disappeared. Polarity of  field was reversed immediately after photograph shown in C.
Ø  D: 2 hr after the field was reversed, the branched neurite 1 bent toward new cathode and neurite 2 reappeared on the new cathodal side of the cell.
Ø  Note: substantial migration of cell bodies was observed. Direction of cell migration was towards cathode & cell bodies appeared to be “dragged” along by growing neurites.
 

Neural injury regeneration:

During development, neurons extend long axonal processes to synapse on specific target neurons. This growth capacity is lost in the mature central nervous system of mammals so that when an axon such as an optic axon is severed, it can no longer regrow to restore functional connections.
 
However, culture conditions that allow adult mouse optic axons to regenerate in vitro have been found, indicating that they retain the inherent capacity to grow. Molecular comparison with embryonic optic fibers reveal that adult fibers differ in intracellular axonal proteins and cell surface receptors that regulate growth and mediate interactions with the glial cells of the mature nervous system. A specific axon-glia / interactions may be the cause of regenerative failure in adult mammals.
 
So what is the bottom line?

Ø     It is hard to dispute the evidence: nervous system tissues, both in vivo and in vitro, strikingly respond to an applied electric field.
Ø     Although it is not generally appreciated, field effects on cultured nervous tissue can equal or exceed effects of nerve growth factor (NGF). Cultured dorsal root ganglia (DRG) demonstrate an enhanced proliferation of processes toward the negative pole of an applied electric field.
Ø     Moreover, the very presence of the field has been seen to stimulate neuroblast development in culture.
Ø     Other responses of neural tissue to fields include increases in the rate of growth, increases in the amount of branching of axons, and a decrease in axonal dieback.


Light assisted neuronal regeneration Approach:


Ø     Ehrlicher et al. & Mohanty et al. used weak optical force produced in optical tweezers by focused laser beam, to guide direction taken by leading edge of growth cone of cell.
 
Ø     In actively extending growth cones, a laser spot placed in front of a specific area of nerve’s leading edge, enhances growth into beam focus and resulting in guided neuronal turns as well as enhanced growth.
 
Ø     Power of laser is so that the resulting gradient forces are sufficiently powerful to bias actin polymerization driven lamellipodia extension, but too weak to hold, move growth cone.
 
Ø     Laser Scissors and Laser Tweezers can proved very useful in studies on neural regeneration. As shown below in the Figure, a similar analogy can be applied to a neuron where, laser scissor perform the cutting of the axon, dendrites, etc and laser tweezers can be used to enhance the regeneration process of the axons and dendrites.

                                                                                                                          (cellular biophotonics lab, UCI, UCSD)


                                                                                                  (Yanik et.al Nature 2004)

hybrid approaches

 1. Opto-chemotaxis

  Light induces chemical modification or releases or activates caged compounds which act as cues for neuronal growth.
 
Laser Activated guiding in agarose hydrogel:






2.. Electro-chemotaxis




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3. Chemotaxis on patterned substrates:

Nerves have been directed by topographically structured artificial surfaces (silicon  wafers or nanotubes) and or by selectively patterning the substrate with materials that act as adhesives/ attractants for nerves.

  
   
Comparison of all approaches:

   Chemotaxis approach has high throughput, but addition and removal of chemical in localized spatial locations with high temporal resolution is very difficult to achieve.
            - It requires fabrication of special microfluidic channels, valves and pumps.
 
Guiding neurons with electrodes in a controlled way also requires pre-fabrication of microelectrodes on which neurons have to be grown.
              - Specific impact of induced electrophoresis effects is not well understood.
 
Though phototaxis approach has advantage of being non-contact in nature, it has very low throughput.
            - Using spatial light modulators (SLM), the laser beam can be split into  several hundreds of moveable trapping beams                     and thus throughput of neuronal growth cone manipulation can be enhanced.
 
Stem cell approach is very promising as it will rule out surgery and will be minimally invasive but time could be a constraint. Also it is still in the early research stage.