In seeking reasons for the poor regeneration of central axons, one critical question is whether the poor recovery reflects an inability of neurons themselves to grow or an inability of the environment to support axonal growth. This issue was addressed by Albert Aguayo and his colleagues in the early 1980s. They inserted segments of a central nerve trunk into a peripheral nerve graft, and segments of a peripheral nerve into the brain or spinal cord, to find out how the translocated axons would respond.
Aguayo found that the axons in the translocated segments promptly degenerated, leaving "distal stumps" containing glia, support cells, and extracellular matrix. The results were striking. Spinal axons that regenerated poorly following spinal cord injury grew several centi-meters when inserted into a peripheral nerve (Figure 57–4). Conversely, peripheral axons regenerated well through their own distal nerve trunk, but fared poorly when paired with a severed optic nerve (Figure 57–5).
A transplanted peripheral nerve provides a favorable environment for the regeneration of central axons.
Left: After sectioning of the spinal cord, ascending and descending axons fail to cross the lesion site. Right: Insertion of a bridging peripheral nerve graft that bypasses the lesion site promotes regeneration of both ascending and descending axons. (Adapted, with permission, from Aguayo 1981.)
Peripheral and central nerves differ in their ability to support axonal regeneration.
A. In the peripheral nervous system severed axons regrow past the site of injury. Insertion of a segment of optic nerve into a peripheral nerve suppresses the ability of the peripheral nerve to regenerate.
B. In the central nervous system severed axons typically fail to regrow past the site of injury. Insertion of a section of peripheral nerve into a central nerve tract promotes regeneration.
Aguayo extended these studies to show that axons from multiple regions, including the retinal ganglion, olfactory bulb, brain stem, and mesencephalon, could each regenerate long distances if provided with a suitable environment. As we will see later, it turns out that regrowth of central axons is intrinsically limited. Nevertheless, these pioneering experiments focused attention on components of the central environment that inhibit regenerative ability and motivated an intensive search for the molecular culprits.
Environmental Factors Support the Regeneration of Injured Axons
In probing the differences between peripheral and central growth environments, initial searches were influenced by the results of experiments performed by Ramón y Cajal's student Jorge Tello-Muñoz nearly a century before Aguayo's studies. Tello transplanted segments of peripheral nerves into the brains of experimental animals and found that injured central axons grew toward the implants, whereas they barely grew when implants were not available.
This result implied that peripheral cells provide growth-promoting factors to the injured areas; factors normally absent from the brain. Ramón y Cajal reasoned that central nerve pathways lacked "substances able to sustain and invigorate the indolent and scanty growth" similar to those provided by peripheral pathways. Numerous studies over the succeeding century identified constituents of peripheral nerves that are potent promoters of neurite outgrowth. These include components of Schwann cell basal laminae such as laminin, and cell adhesion molecules of the immunoglobulin superfamily. In addition, cells in denervated distal nerve stumps begin to produce neurotrophins and other trophic molecules. Together these molecules nourish neurons and guide growing axons in the embryonic nervous system, so it makes sense that they also promote the regrowth of axons. By contrast, central neuronal tissue is a poor source of these molecules, containing little laminin and low levels of trophic molecules. Thus in the embryo both central and peripheral nervous systems provide environments that promote axon outgrowth. But only the peripheral environment retains this capacity in adulthood, or is able to regain it effectively following injury.
The practical implications of this view are that supplementing the central environment with growth-promoting molecules might improve regeneration. To this end investigators have infused neurotrophins into areas of injury or inserted conduits rich in extracellular matrix molecules such as laminin. In some attempts Schwann cells themselves, or cells engineered to secrete trophic factors, have been grafted into sites of injury. In many of these cases injured axons grow slightly more extensively than they do under control conditions. Yet regeneration remains limited, with axons generally failing to extend long distances. More important, functional recovery is minimal.
What accounts for such disappointingly limited regeneration? One limitation appears to be the existence of inhibitory signaling pathways that block the growth-promoting activity of cytokine factors. In the optic nerve, for example, the poor regeneration of the axons of retinal ganglion neurons is accounted for in part by the state of activation of a cytokine signaling pathway. The axonal growth-promoting effects of cytokines such as ciliary neurotrophic factors (CNTFs) involve activation of a receptor GP130, and GP130 signaling is counteracted by the activity of a suppressor of cytokine signaling called SOCS3. Thus deletion of the SOCS3 gene in mice augments the ability of CNTF to promote regeneration of retinal ganglion cell axons in the optic nerve (Figure 57–6).
Signaling pathways that regulate axon regeneration in the optic nerve.
The regeneration of retinal ganglion cell axons in the optic nerve is normally constrained by neuronal expression of the gene for SOCS3, which blocks the ability of ciliary neurotrophic factor (CNTF) to bind its receptor GP130 and thus blocks CNTF from promoting regeneration. In SOCS3 mutant mice, ambient levels of CNTF are sufficient to improve optic nerve regeneration. Elimination of GP130 as well as SOCS3 blocks the capacity for regeneration. Addition of extra CNTF enhances the capacity for regeneration in SOCS3 mutant mice. (Adapted, with permission, from Smith et al. 2009.)
Components of Myelin Inhibit Neurite Outgrowth
Fragments of central myelin are potent inhibitors of neurite outgrowth. Sprouting of spinal axon collaterals following injury is enhanced in rats treated to prevent myelin formation in the spinal cord (Figure 57–7). These findings implied that although both central and peripheral environments might contain a supply of growth-promoting elements, central nerves also contain inhibitory components. That myelin inhibits neurite growth may seem peculiar, but in fact myelination normally occurs postnatally, after axon extension is largely complete.
Myelin inhibits regeneration of central axons.
(Adapted, with permission, from Schwegler, Schwab, and Kapfhammer 1995.)
A. Sensory fibers normally extend rostrally in a myelin-rich spinal cord.
B. Right dorsal root fibers were sectioned in 2-week-old normal rats. Regeneration of the fibers was assessed histochemically 20 days later. The central branches of the sectioned axons degenerated, leaving a portion of the spinal cord denervated. Little regeneration occurred in the myelin-rich cord.
C. Some littermates received local x-irradiation to block myelination. In these animals sensory fibers that entered the cord through neighboring uninjured roots sprouted new collaterals following denervation.
Searches for the inhibitory components of central myelin turned up an embarrassment of riches. Several classes of molecules found at higher levels in central myelin compared to peripheral myelin are able to inhibit neurite outgrowth when presented to cultured neurons. The first to be discovered was identified when an antibody generated against myelin proteins proved to be capable of partially neutralizing myelin's ability to inhibit neurite outgrowth. Use of this antibody to isolate the corresponding antigen yielded the protein now called Nogo. Two other proteins, myelin-associated glycoprotein (MAG) and oligodendrocyte-myelin glycoprotein (OMgp), initially isolated as major components of myelin, have been found to inhibit the growth of some neuronal types.
Intriguingly, Nogo, MAG, and OMgp each bind to common membrane receptors, NogoR and PirB (Figure 57–8). In mutant mice lacking PirB, regeneration of severed corticospinal axons is enhanced; the extent of axonal regeneration in mice lacking NogoR or its three ligands is uncertain. Identifying physiologically relevant constraints on regeneration may be complicated by the presence of still undiscovered inhibitors. But if many inhibitory components trigger the same intracellular signaling pathway, then interference with that pathway might neutralize the impact of many inhibitors in one fell swoop.
Myelin and glial scar components that inhibit regeneration of central axons.
Left: Myelin contains the proteins Nogo-A, oligodendrocyte-myelin glycoprotein (OMgp), and myelin-associated glycoprotein (MAG). All three proteins are exposed when myelin breaks down. They can bind a receptor protein Nogo R, which can associate with the neurotrophin receptor p75, as well as an immunoglobulin-like receptor protein PirB. Inactivation of PirB results in a modest enhancement of corticospinal axon regeneration. Right: Chondroitin sulphate proteoglycans (CSPG) are major components of the glial scar and are thought to suppress axon regeneration through interaction with the receptor tyrosine phosphatase PTP-sigma, which activates intracellular mediators such as Rho and ROCK. (Adapted, with permission, from Yiu et al. 2006.)
Injury-Induced Scarring Hinders Axonal Regeneration
Myelin debris is not the only source of growth-inhibiting material in the injured brain or spinal cord. As noted earlier, astrocytes become activated and proliferate following injury, acquiring features of reactive astrocytes that generate scar tissue at sites of injury. Scarring is an adaptive response that helps to limit the size of the injury, to reestablish the blood-brain barrier, and to reduce inflammation.
But the scar itself hinders regeneration in two ways: through mechanical interference with axon growth and through growth-inhibiting effects of proteins produced by cells within the scar. Chief among these inhibitors are a class of chondroitin sulfate proteoglycans (CSPG) that are produced in abundance by reactive astrocytes and directly inhibit axon extension by interaction with tyrosine phosphatase receptors on axons (Figure 57–8). Attention has therefore focused on ways of dissolving the glial scar by infusion of an enzyme called chondroitinase, which breaks down the sugar chains on CSPG. This treatment promotes axon regeneration and functional recovery in animals. Drugs that reduce inflammation and decrease scarring, notably prednisolone, are also beneficial if administered shortly after injury, before the scar forms.
An Intrinsic Growth Program Promotes Regeneration
So far we have emphasized differences between the local environments of peripheral and central axons. However, environmental differences cannot completely account for the poor regeneration of central axons. Even though they can regenerate in peripheral nerves, central axons grow much less well than peripheral axons when navigating the same path. Thus adult central axons may be less capable than peripheral axons of regeneration.
In support of this idea, experiments in tissue culture have shown that the growth potential of central neurons decreases with age, whereas mature peripheral neurons extend axons robustly in a favorable environment. One potential explanation for this difference is variation in the expression of proteins thought to be critical for optimal axon elongation, such as the 43 kDa growth-associated protein or GAP-43. This protein is expressed at high levels in embryonic central and peripheral neurons. In peripheral neurons the level remains high in maturity and increases even more following axotomy, whereas in central neurons its expression decreases as development proceeds.
Is this reduced ability of central axons to regenerate irreversible? Hope for reversibility is provided by studies involving "conditioning lesions." Recall that primary sensory neurons in dorsal root ganglia have a bifurcated axon, with a peripheral branch that extends to skin, muscle, or other targets, and a central branch that enters the spinal cord. The peripheral branch regenerates well following injury, whereas the central branch regenerates poorly. However, the central branch will regenerate successfully if the peripheral branch is damaged several days before the central branch is damaged (Figure 57–9). Somehow prior injury or conditioning lesion activates an axonal growth program.
A conditioning lesion promotes regeneration of the central branch of a primary sensory neuron axon.
After lesions of the spinal cord there is little regeneration of the central branch beyond the injury site. However, if the peripheral branch of the axon is sectioned before the central branch is damaged, the latter will grow beyond the lesion site. The impact of such a "conditioning lesion" can be mimicked by elevating levels of cyclic adenosine monophosphate (cAMP) or of the growth-associated protein GAP-43 in the peripheral branch.
One component of the growth program responsible for regeneration of the central branch appears to be cyclic adenosine monophosphate (cAMP). This second-messenger molecule activates enzymes that in turn promote neurite outgrowth. Levels of cAMP are high when neurons initially form circuits; they decline postnatally in central but not peripheral neurons. In some instances increased supplies of cAMP or proteins normally activated by cAMP can promote regeneration of central axons following injury. Accordingly, drugs that increase cAMP levels, or that activate targets of cAMP, are being actively considered as therapeutic agents to be administered following spinal cord injury. In addition, expression of GAP-43 can promote the regeneration of sensory axons past the site of a spinal cord lesion.
Formation of New Connections by Intact Axons Can Lead to Functional Recovery
So far we have discussed interventions designed to enhance the limited regenerative capacity of injured central axons. An alternative strategy focuses on the significant, although incomplete, functional recovery that can occur following injury even without appreciable regeneration of cut axons. If the basis for this limited recovery of function can be understood, it may be possible to enhance regeneration and function.
A rearrangement of existing connections in response to injury may contribute to recovery of function. We have learned that axotomy leads to changes in both the inputs to and the targets of the injured neuron. Although many of these changes are detrimental to function, some are beneficial. In particular, the central nervous system can, following injury, spontaneously undergo adaptive reorganization that helps it regain function. For example, after transection of the descending corticospinal pathway, which occurs with many traumatic injuries of the spinal cord, the cortex can no longer transmit commands to motor neurons below the site of the lesion. Over several weeks, however, intact corticospinal axons rostral to the lesion begin to sprout new terminal branches and form synapses on spinal interneurons whose axons extend around the lesion, thereby forming an intraspinal detour that contributes to limited recovery of function (Figure 57–10).
Function can be recovered after spinal cord injury through reorganization of spinal circuits.
Severed corticospinal axons can reestablish connections with motor neurons by sprouting axon collaterals that innervate propriospinal interneurons whose axons bypass the lesion and contact motor neurons located caudal to the lesion site. (Adapted, with permission, from Bareyre et al. 2004.)
Similar instances of functional reorganization have been demonstrated in the motor cortex and brain stem. These compensatory responses attest to the latent plasticity of the nervous system. The ability of the nervous system to rewire itself is most vigorous during the critical periods of early postnatal life (see Chapter 56) but can also be reawakened by traumatic events in adulthood.
How can the nervous system's rewiring ability be improved? It is possible that some of the beneficial effects of grafts in experimental animals reflect reorganization of intact axons rather than regeneration of transected axons. As the nervous system's plasticity becomes better understood, therapeutic strategies that promote specific changes in circuitry may become possible. Perhaps most promising is an approach in which cellular or molecular interventions that promote growth are combined with behavioral therapies that result in circuit rewiring.