*3.7. Schwann Cell Senescence*

Axon regeneration across long nerve gaps requires Schwann cells of the proximal nerve stump to proliferate extensively so they can both promote and accompany the axons as they regenerate. The limited regeneration of axons across long nerve gaps, and when nerves are repaired at long times post-trauma, is in part, attributed to Schwann cell senescence [69]. Senescence is associated with increased expression of markers for β-galactosidase, p<sup>16</sup>INK4A, and interleukin (IL)6 [82]. Thus, over time, without contact with an axon, Schwann cells lose their ability to proliferate, synthesize, and release neurotrophic factors, which are required to promote axon regeneration [82,83].

#### *3.8. Neuron Loss of Ability to Regenerate*

Another explanation for the significant decrease in axon regeneration with increasing time of motor neuron axotomy is that many motor neurons lose the ability to extend axons [83]. The decrease in the ability of motor neurons to regenerate with increasing time of axotomy appears to be associated with the downregulation of neuregulin 1, which is required for axon regeneration [38,84,85]. However, it is important to note that while some motor neurons lose this capacity, others can regenerate even after many years of axotomy. In a clinical case, motor neurons that were axotomized for 22 years were able to extend axons to reinnervate newly denervated muscles [86].

It is not known what determines which motor neurons retain or lose the capacity to regenerate. However, understanding this regulation might provide insights into how neurons' gene expression can be modulated to promote enhanced axon outgrowth. Thus, it would be interesting to compare the gene expression between motor neurons with and without the capacity to regenerate after prolonged axotomy to determine the gene expression that underlies the capacity to regenerate normally. As is discussed later, techniques have been developed that induce motor neurons, which appear to have lost the ability to regenerate, to extend axons.

#### *3.9. Decreasing Recovery with Patient Age*

The recovery of function following nerve graft repair is best for patients < 20–25 years of age [87], and the extent of recovery decreases significantly with increasing patient age [87,88]. This is, in part, attributed to the decreasing capacity of sensory nerve grafts to promote axon regeneration (i.e., their Schwann cells becoming senescent) [69]. This change is also associated with the downregulation over time post-axotomy of the ability of neurons to synthesize and release neuregulin 1, which is required for axon regeneration [38,84,85].

Endoneurial vasculature is required for the outgrowth of axons from the proximal stump, and angiogenesis is essential for nerve regeneration [89]. However, clinically, with increasing age, there is a reduction in, or lack of, angiogenesis in response to injury [90]. This change is due to the decrease in nerve injury-induced upregulation of the expression and release of vascular endothelial growth factor (VEGF) [91,92]. VEGF is required for inducing vascularization, which, in turn, is required for axon regeneration. This suggests that vascular abnormalities might play a role in the decreasing ability of axons to regenerate with increasing age. This further suggests that promoting vascularization may increase the extent of neurological recovery in older patients.

#### *3.10. Enhancing the <sup>E</sup>*ffi*cacy of Autografts to Promote Axon Regeneration*

The extent and distance axons regenerate decreases with increasing graft length, increasing delays between nerve injury and repair, and increasing age. As already stated, these limitations are associated with (1) the inability of neurons to extend axons, (2) development of Schwann cell senescence, and (3) the failure, or slow process of, graft re-vascularization. Despite these challenges, various techniques overcome these limitations and promote axon regeneration.

Axon regeneration can be triggered from neurons that no longer extend axons, and the rate and extent of axon regeneration increases by refreshing central nerve stumps and then stimulating them electrically, or by applying neurotrophic factors [93–95]. These techniques restore the capacity of neurons to regenerate [96–98] while also inducing the senescent Schwann cells of the distal portion of the nerve to proliferate and release neurotrophic factors that promote the extension of axons from long-term axotomized neurons [99,100].
