**4. Electrical Stimulation**

#### *4.1. Promoting Axotomized Neurons to Extend Axons*

As mentioned previously, within increasing the time of axotomy, neurons lose their ability to extend axons [83]. However, these neurons can be induced to extend axons by electrical stimulation of the proximal portion of the transected nerves for as little as one hour [94,101,102] This results in a 34–50% increase in the number of neurons that extend axons [98,103,104]. Electrical stimulation also induces a 2.3-fold increase in the extent of axon sprouting from transected axons [105] while increasing the speed of axon regeneration [101,103,106]. At the same time, in animal models, electrical stimulation increases the distance axons regenerate across nerve gaps, the accuracy of sensory vs. motor axon innervation of their appropriate targets, and extent of functional recovery [107–110]. Electrical stimulation of peripheral nerve clinically also induces enhanced axon regeneration [102].

Electrical stimulation acts by inducing neurons to upregulate their level of cyclic-AMP [89,103]. This, in turn, induces motor neurons to upregulate their expression and synthesis of the neurotrophic factor BDNF and its trkB receptor mRNA, as well as the mRNA for other factors that enhance axon regeneration [99,111] These actions make neurons more receptive to regeneration-promoting factors [112].

#### *4.2. Activating Senescent Schwann Cells*

The decrease in axon regeneration across long nerve grafts, and with increasing time between nerve injury and repair, is attributed in part to Schwann cells becoming senescent when they lose contact with axons [69,75]. Thus, they stop proliferating and releasing neurotrophic factors that are required to promote axon regeneration. Electrical stimulation enhances axon regeneration by inducing the senescent Schwann cells to proliferate, migrate, and upregulate their synthesis and release of neurotrophic factors, which act to promote axon regeneration [99,113].

Schwann cells can also be induced to exit their senescent state by the application of VEGF [76,112,114] and marrow-derived mesenchymal stem cells. Thus, the Schwann cells reinitiate their ability to proliferate and to express and release neurotrophic factors [80].

#### *4.3. Vascularized Nerve Grafts and Promoting Vascularization*

The standard sensory nerve graft is cut from a donor nerve without maintaining its vasculature. As stated earlier, non-vascularized grafts become necrotic, which creates a toxic environment that inhibits axons regeneration until re-vascularization occurs. However, re-vascularized takes days

to develop, and takes more time for longer nerve grafts. This is because vascularization normally progresses from one end of the graft to the other.

The limitation of using non-vascularized grafts is avoided by using vascularized nerve grafts. In the rat sciatic nerve model, vascularized grafts induce significantly greater neurological recovery than non-vascularized nerve grafts [115]. Clinically, vascularized nerve grafts are required for axons to regenerate across gaps of longer than 6 cm [116]. Although vascularized nerve grafts are more e ffective in restoring function than non-vascularized grafts, they are not commonly used because the surgery is more complicated and time-consuming.

An alternative technique to using vascularized autografts is to induce the rapid re-vascularization of non-vascularized grafts. This can be done by pre-treating nerve grafts with VEGF before using them [117]. This treatment stimulates neovascularization of the graft, and Schwann cell invasion into the graft [117] and can reduce the time of graft ischemia by three days [118].

Axon regeneration can also be enhanced by using autografts with cells overexpressing VEGF, which leads to hyper-vascularization [119]. This enhances axon regeneration by reducing endoneurial scarring, by maintaining the viability of Schwann and other cells within the graft, and by decreasing fibroblast infiltration. This results in a good nutritional environment for supporting axonal regeneration.

The decrease in axon regeneration through nerve grafts with increasing age is also attributed to reduced graft vascularization. This is because aging is associated with a decrease in the upregulation of the expression, and release of VEGF following nerve injury [91]. Following nerve injury to aged mice, there is a significant reduction in the upregulation of VEGF synthesis and release, and thus, a failure of axons to regenerate. These findings sugges<sup>t</sup> that, with increasing age, vascular abnormalities might play a role in the decreasing ability of axons to regenerate. They also sugges<sup>t</sup> that clinically, inducing enhanced vascularization might enhance axon regeneration and functional recovery.

#### *4.4. Conclusion about Sensory Nerve Grafts*

The use of sensory nerve grafts leads to a permanent neurological deficit of the donor nerve. While such grafts can induce good to excellent functional recovery, such recovery is only for young patients [120,121], short gaps [65,67] (Aszmann et al., 2008b), and 3 when the repairs are performed within a short time post-trauma [77,79,81]. As any one of these values increases, the extent of recovery decreases precipitously. When the values of two or all three of these parameters increase, there is minimal to no functional recovery. However, even when nerve repair surgery is o ffered to patients who are considered good candidates for recovery function, <50% of them recover function [122]. Thus, most individuals who su ffer peripheral nerve traumas su ffer permanent neurological deficits and commonly chronic neuropathic pain. Therefore, there is a need for novel techniques that induce recovery under conditions where sensory nerve grafts are not e ffective.

The following sections examine other techniques that have been tested for their e fficacy in enhancing the extent of axon regeneration and neurological recovery across nerve gaps. They also examine methods tested for increasing their e fficacy in enhancing the extent of axon regeneration. The final section briefly discusses a novel technique that holds promise for restoring function, even when simultaneously the values of all three nerve injury parameters far exceed those when sensory nerve grafts are e ffective in promoting axon regeneration.
