Tessa Gordon

The cellular and molecular basis of peripheral nerve regeneration

Functional recovery from peripheral nerve injury and repair depends on a multitude of factors, both intrinsic and extrinsic to neurons. Neuronal survival after axotomy is a prerequisite for regeneration and is facilitated by an array of trophic factors from multiple sources, including neurotrophins, neuropoietic cytokines, insulin-like growth factors (IGFs), and glial-cell-line-derived neurotrophic factors (GDNFs). Axotomized neurons must switch from a transmitting mode to a growth mode and express growth-associated proteins, such as GAP-43, tubulin, and actin, as well as an array of novel neuropeptides and cytokines, all of which have the potential to promote axonal regeneration. Axonal sprouts must reach the distal nerve stump at a time when its growth support is optimal. Schwann cells in the distal stump undergo proliferation and phenotypical changes to prepare the local environment to be favorable for axonal regeneration. Schwann cells play an indispensable role in promoting regeneration by increasing their synthesis of surface cell adhesion molecules (CAMs), such asN-CAM, Ng-CAM/L1, N-cadherin, and L2/HNK-1, by elaborating basement membrane that contains many extracellular matrix proteins, such as laminin, fibronectin, and tenascin, and by producing many neurotrophic factors and their receptors. However, the growth support provided by the distal nerve stump and the capacity of the axotomized neurons to regenerate axons may not be sustained indefinitely. Axonal regeneration may be facilitated by new strategies that enhance the growth potential of neurons and optimize the growth support of the distal nerve stump in combination with prompt nerve repair.

Neurotrophic factors and their receptors in axonal regeneration and functional recovery after peripheral nerve injury.

Over a half a century of research has confirmed that neurotrophic factors promote the survival and process outgrowth of isolated neurons in vitro. The mechanisms by which neurotrophic factors mediate these survival-promoting effects have also been well characterized. In vivo, peripheral neurons are critically dependent on limited amounts of neurotrophic factors during development. After peripheral nerve injury, the adult mammalian peripheral nervous system responds by making neurotrophic factors once again available, either by autocrine or paracrine sources. Three families of neurotrophic factors were compared, the neurotrophins, the GDNF family of neurotrophic factors, and the neuropoetic cytokines. Following a general overview of the mechanisms by which these neurotrophic factors mediate their effects, we reviewed the temporal pattern of expression of the neurotrophic factors and their receptors by axotomized motoneurons as well as in the distal nerve stump after peripheral nerve injury. We discussed recent experiments from our lab and others which have examined the role of neurotrophic factors in peripheral nerve injury. Although our understanding of the mechanisms by which neurotrophic factors mediate their effects in vivo are poorly understood, evidence is beginning to emerge that similar phenomena observed in vitro also apply to nerve regeneration in vivo.

Contributing factors to poor functional recovery after delayed nerve repair: prolonged axotomy

The contribution of prolonged motoneuron axotomy to the poor functional recovery after delayed nerve repair was determined by means of a nerve cross-anastomosis paradigm in the rat. The tibial nerve was axotomized up to 12 months before it was cross-sutured to the distal stump of the freshly cut common peroneal nerve to innervate the freshly denervated tibialis anterior muscle. Three to 17 months later, muscle and motor unit (MU) forces were measured to quantify the number of axons that had successfully regenerated and reinnervated the muscle. The extent of axonal branching was estimated by the innervation ratio (IR) (i.e., the number of muscle fibers innervated by each axon), which was obtained directly by counting muscle fibers in a single glycogen-depleted MU in each muscle and indirectly by calculation. The total number of MUs in each muscle significantly decreased with progression of axotomy and was only 35% of the control when axotomy was prolonged more than 3 months. Concurrently, MU force and IR increased exponentially, with a mean increase of threefold when axotomy was more than 3 months, which largely compensated for the reduction in the number of axons that reinnervated the muscle. Consequently, muscles reinnervated by tibial motor axons that had been axotomized up to 12 months produced as much force as those reinnervated by freshly axotomized tibial motor axons. Muscle weight, size, and muscle fiber size were similar to those after immediate nerve suture. Although prolonged axotomy does not compromise the number of muscle fibers innervated by each axon, it does reduce the capacity of motor axons to regenerate and thus is an important contributing factor to the poor functional recovery in delayed nerve repair.

Experimental strategies to promote functional recovery after peripheral nerve injuries

Abstract  The capacity of Schwann cells (SCs) in the peripheral nervous system to support axonal regeneration, in contrast to the oligodendrocytes in the central nervous system, has led to the misconception that peripheral nerve regeneration always restores function. Here, we consider how prolonged periods of time that injured neurons remain without targets during axonal regeneration (chronic axotomy) and that SCs in the distal nerve stumps remain chronically denervated (chronic denervation) progressively reduce the number of motoneurons that regenerate their axons. We demonstrate the effectiveness of low‐dose, brain‐derived neurotrophic and glial‐derived neurotrophic factors to counteract the effects of chronic axotomy in promoting axonal regeneration. High‐dose brain‐derived neurotrophic factor (BDNF) on the other hand, acting through the p75 receptor, inhibits axonal regeneration and may be a factor in stopping regenerating axons from forming neuromuscular connections in skeletal muscle. The immunophilin, FK506, is also effective in promoting axonal regeneration after chronic axotomy. Chronic denervation of SCs (>1 month) severely deters axonal regeneration, although the few motor axons that do regenerate to reinnervate muscles become myelinated and form enlarged motor units in the reinnervated muscles. We found that in vitro incubation of chronically denervated SCs with transforming growth factor‐β re‐established their growth‐supportive phenotype in vivo, consistent with the idea that the interaction between invading macrophages and denervated SCs during Wallerian degeneration is essential to sustain axonal regeneration by promoting the growth‐supportive SC phenotype. Finally, we consider the effectiveness of a brief period of 20 Hz electrical stimulation in promoting the regeneration of axons across the surgical gap after nerve repair.

Electrical stimulation promotes sensory neuron regeneration and growth-associated gene expression

Brief electrical stimulation enhances the regenerative ability of axotomized motor [Nix, W.A., Hopf, H.C., 1983. Electrical stimulation of regenerating nerve and its effect on motor recovery. Brain Res. 272, 21-25; Al-Majed, A.A., Neumann, C.M., Brushart, T.M., Gordon, T., 2000. Brief electrical stimulation promotes the speed and accuracy of motor axonal regeneration. J. Neurosci. 20, 2602-2608] and sensory [Brushart, T.M., Jari, R., Verge, V., Rohde, C., Gordon, T., 2005. Electrical stimulation restores the specificity of sensory axon regeneration. Exp. Neurol. 194, 221-229] neurons. Here we examined the parameter of duration of stimulation on regenerative capacity, including the intrinsic growth programs, of sensory neurons. The effect of 20 Hz continuous electrical stimulation on the number of DRG sensory neurons that regenerate their axons was evaluated following transection and surgical repair of the femoral nerve trunk. Stimulation was applied proximal to the repair site for 1 h, 3 h, 1 day, 7 days or 14 days at the time of nerve repair. Following a 21-day regeneration period, DRG neurons that regenerated axons into the muscle and cutaneous sensory nerve branches were retrogradely identified. Stimulation of 1 h led to a significant increase in DRG neurons regenerating into cutaneous and muscle branches when compared to 0 h (sham) stimulation or longer periods of stimulation. Stimulation for 1 h also significantly increased the numbers of neurons that regenerated axons beyond the repair site 4 days after lesion and was correlated with a significant increase in expression of growth-associated protein 43 (GAP-43) mRNA in the regenerating neurons at 2 days post-repair. An additional indicator of heightened plasticity following 1 h stimulation was elevated expression of brain-derived neurotrophic factor (BDNF). The effect of brief stimulation on enhancing sensory and motoneuron regeneration holds promise for inducing improved peripheral nerve repair in the clinical setting.

A dose-dependent facilitation and inhibition of peripheral nerve regeneration by brain-derived neurotrophic factor.

The time‐dependent decline in the ability of motoneurons to regenerate their axons after axotomy is one of the principle contributing factors to poor functional recovery after peripheral nerve injury. A decline in neurotrophic support may be partially responsible for this effect. The up‐regulation of BDNF after injury, both in denervated Schwann cells and in axotomized motoneurons, suggests its importance in motor axonal regeneration. In adult female Sprague–Dawley rats, we counted the number of freshly injured or chronically axotomized tibial motoneurons that had regenerated their axons 1 month after surgical suture to a freshly denervated common peroneal distal nerve stump. Motor axonal regeneration was evaluated by applying fluorescent retrograde neurotracers to the common peroneal nerve 20 mm distal to the injury site and counting the number of fluorescently labelled motoneurons in the T11–L1 region of the spinal cord. We report that low doses of BDNF (0.5–2 µg/day for 28 days) had no detectable effect on axonal regeneration after immediate nerve repair, but promoted axonal regeneration of motoneurons whose regenerative capacity was reduced by chronic axotomy 2 months prior to nerve resuture, completely reversing the negative effects of delayed nerve repair. In contrast, high doses of BDNF (12–20 µg/day for 28 days) significantly inhibited motor axonal regeneration, after both immediate nerve repair and nerve repair after chronic axotomy. The inhibitory actions of high dose BDNF could be reversed by functional blockade of p75 receptors, thus implicating these receptors as mediators of the inhibitory effects of high dose exogenous BDNF.

Effects of short- and long-term Schwann cell denervation on peripheral nerve regeneration, myelination, and size

Poor functional recovery after peripheral nerve injury has been generally attributed to inability of denervated muscles to accept reinnervation and recover from denervation atrophy. However, deterioration of the Schwann cell environment may play a more vital role. This study was undertaken to evaluate the effects of chronic denervation on the capacity of Schwann cells in the distal nerve stump to support axonal regeneration and to remyelinate regenerated axons. We used a delayed cross‐suture anastomosis technique in which the common peroneal (CP) nerve in the rat was denervated for 0–24 weeks before cross‐suture of the freshly axotomized tibial (TIB) and chronically denervated CP nerve stumps. Motor neurons were backlabeled with either fluoro‐ruby or fluorogold 12 months later, to identify and count TIB motor neurons that regenerated axons into chronically denervated CP nerve stumps. Number, size, and myelination of regenerated sensory and motor axons were determined using light and electron microscopy. We found that short‐term denervation of ≤4weeks did not affect axonal regeneration but more prolonged denervation profoundly reduced the numbers of backlabeled motor neurons and axons in the distal nerve stump. Yet, atrophic Schwann cells retained their capacity to remyelinate regenerated axons. In fact, the axons were larger and well myelinated by long‐term chronically denervated Schwann cells. These findings demonstrate a progressive inability of chronically denervated Schwann cells to support axonal regeneration and yet a sustained capacity to remyelinate the axons which do regenerate. Thus, axonal interaction can effectively switch the nonmyelinating phenotype of atrophic Schwann cells back into the myelinating phenotype. GLIA 32:234–246, 2000.

Glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor sustain the axonal regeneration of chronically axotomized motoneurons in vivo

In contrast to injuries in the central nervous system, injured peripheral neurons will regenerate their axons. However, axotomized motoneurons progressively lose their ability to regenerate their axons, following peripheral nerve injury often resulting in very poor recovery of motor function. A decline in neurotrophic support may be partially responsible for this effect. The initial upregulation of glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) by Schwann cells of the distal nerve stump after nerve injury has led to the speculation that they are important for motor axonal regeneration. However, few experiments directly measure the effects of exogenous BDNF or GDNF on motor axonal regeneration. This study provided the first direct and quantitative evidence that long-term continuous treatment with exogenous GDNF significantly increased the number of motoneurons which regenerate their axons, completely reversing the negative effects of chronic axotomy. The beneficial effect of GDNF was not dose-dependent. A combination of exogenous GDNF and BDNF on motor axonal regeneration was significantly greater than either factor alone, and this effect was most pronounced following long-term continuous treatment. The ability of GDNF, either alone or in combination with BDNF, to increase the number of motoneurons that regenerated their axons correlated well with an increase in axon sprouting within the distal nerve stump. Thus long-term continuous treatment with neurotrophic factors, such as GDNF and BDNF, can be used as a viable treatment to sustain motor axon regeneration.

A decline in glial cell-line-derived neurotrophic factor expression is associated with impaired regeneration after long-term Schwann cell denervation.

In the peripheral nervous system, regeneration of motor and sensory axons into chronically denervated distal nerve segments is impaired compared to regeneration into acutely denervated nerves. In order to find possible causes for this phenomenon we examined the changes in the expression pattern of the glial cell-line-derived neurotrophic factor (GDNF) family of growth factors and their receptors in chronically denervated rat sciatic nerves as a function of time with or without regeneration. Among the GDNF family of growth factors, only GDNF mRNA expression was rapidly upregulated in Schwann cells as early as 48 h after denervation. This upregulation peaked at 1 week and then declined to minimal levels by 6 months of denervation. The changes in the protein expression paralleled the changes in the expression of the GDNF mRNA. The mRNAs for receptors GFRalpha-1 and GFRalpha-2 were upregulated only after maximal GDNF upregulation and remained elevated as late as 6 months. There were no significant changes in the expression of GFRalpha-3 or the tyrosine kinase coreceptor, RET. When we examined the expression of GDNF in a delayed regeneration paradigm, there was no upregulation in the distal chronically denervated tibial nerve even when the freshly axotomized peroneal branch of the sciatic nerve was sutured to the distal tibial nerve. This study suggests that one of the reasons for impaired regeneration into chronically denervated peripheral nerves may be the inability of Schwann cells to maintain important trophic support for both motor and sensory neurons.

Electrical stimulation accelerates and enhances expression of regeneration-associated genes in regenerating rat femoral motoneurons.

Abstract1. In this study we investigated whether electrical stimulation accelerates the upregulation of Tα1-tubulin and GAP-43 (regeneration-associated genes; RAGs) and the downregulation of the medium-molecular-weight neurofilament (NFM), in concert with stimulation-induced acceleration of BDNF and trkB gene expression and axonal regener- ation.2. Two weeks prior to unilateral femoral nerve transection and suture, fluorogold (Fluorochrome Inc., Denver) or fluororuby (Dextran tetramethylrhodamine, Mol. Probes, D-1817, Eugene, OR) was injected into quadriceps muscles of the left and right hindlimbs to label the femoral motoneuron pools as previously described. Over a period of 7 days, fresh spinal cords were processed for semiquantitation of mRNA by using in situ hybridi- zation.3. There was an increase in Tα1-tubulin and GAP-43 mRNA and a decline in the NFM mRNA at 7 days after nerve suture and sham stimulation but not in intact nerves. In contrast, 1-h stimulation of sutured but not intact nerves dramatically accelerated the changes in gene expression: mRNA levels of Tα1-tubulin and GAP-43 were significantly elevated above control levels by 2 days while NFM mRNA was significantly reduced by 2 days in the sutured nerves. Thereby, the neurofilament/tubulin expression ratio was reduced at 2 days after suture and stimulation, possibly allowing more tubulin to be transported faster into the growing axons to accelerate the elongation rate following stimulation. Importantly, the changes in RAGs and NFM gene expression were delayed relative to the accelerated upregulation of BDNF and trkB mRNA by electrical stimulation.4. The temporal sequence of upregulation of BDNF and trkB, altered gene expression of RAGs and NFM, and accelerated axonal outgrowth from the proximal nerve stump are consistent with a key role of BDNF and trkB in mediating the altered expression of RAGs and, in turn, the promotion of axonal outgrowth after electrical stimulation.