The relative success from targeting inhibitory signals in vitro has led to much anticipation for the promotion of regeneration and functional recovery after CNS injury in vivo. Both genetic deletion and pharmacological interventions have been used towards this aim to block the inhibitory pathways, including ligands, receptor components and signalling intermediates (). These studies have primarily focused on the regeneration of long axonal fibres, such as those in the CST, dorsal columns or optic nerve in various nerve injury models.
Summary of in vivo studies targeting inhibitory signals in the adult CNS
At the ligand level, initial attempts to block Nogo-A activity met with some success. Treatment with IN-1 antibody induced some sprouting after a CST lesion13,94
. However, when CST regeneration was examined in Nogo-knockout mutants that were generated independently by three groups, the results were ambiguous. Whereas one group reported extensive sprouting of lesioned axons in young adult mice lacking Nogo-A/B37
, the other two observed little (in mice lacking only Nogo-A38
) to no (in two mouse lines lacking either NogoA/B or Nogo-A/B/C39
) improvement in regeneration. Studies using MAG-mutant mice also showed little to no detectable regeneration of optic nerve and CST fibres95
. Interestingly, although OMgp-knockout animals have not been examined in any models of injury, the animals were found to have abnormally wide nodes of Ranvier and signs of collateral sprouting35
, supporting the importance of this molecule in limiting aberrant sprouting under physiological conditions.
Despite the limited success achieved by genetic deletions of individual myelin components, targeting the NgR complex should neutralize all three major inhibitors. Indeed, early experiments involving the delivery of a Nogo-66 antagonist peptide (NEP1–40)96,97
or the function-blocking NgR ectodomain (NgR(310)ecto)98,99
after spinal cord hemisection promoted some axon regeneration and functional recovery. In addition, transgenic expression of a truncated NgR lacking its co-receptor binding site can enhance optic nerve regeneration if the retinal ganglion cells are in an active growth state70
. However, the recent characterization of two strains of mutant mice lacking NgR once again produced mixed outcomes. Although in one of the studies some regeneration was observed in the raphespinal tract and rubrospinal tract, no CST regeneration could be detected in either mutant strain71,72
. Similarly, initial studies showed that sympathetic neurons from p75-mutant mice overexpressing NGF can grow in extensively myelinated portions of the cerebellum100
or optic nerve101
. However, no CST or dorsal column repair could be detected in these p75 knockouts after spinal cord injury102
. Nevertheless, as neurons from both p75- and NgR-deficient mice still retain at least partial responses to myelin inhibitors in vitro67,72
, these in vivo
results might not faithfully reflect the contribution of myelin inhibition to regeneration failure. In fact, published results from genetic deletion studies have largely been less consistent than function-blocking or dominant-negative approaches in enhancing axon regeneration. The reason for such diver gent regenerative responses remains unclear, although it is probably attributable to compensatory upregulation of other inhibitory pathways. Neutralizing agents might also affect other mediators of inhibition that have not yet been identified. Nevertheless, these studies are beginning to help us to understand the relative contribution of myelin-associated inhibitors and their receptor components to regeneration failure.
Studies that target the glial scar have also shown some promise for the promotion of regeneration and recovery after CNS injury. Mutant mice that are deficient in both glial fibrillary acidic protein (GFAP) and vimentin (important cytoskeletal proteins that are induced in reactive astrocytes) show reduced astroglial reactivity, increased supraspinal sprouting and improved functional recovery after spinal cord hemisection103
. Intrathecal administration of ChABC following spinal cord injury promoted the regeneration of various axon tracts as well as some recovery of function104,105
. Interestingly, mutants with genetic deletion of EphA4, the cognate neuronal receptor for ephrin B3, also showed regrowth of corticospinal and rubrospinal tract fibres after spinal cord hemisection, although the phenotype was attributed to reduced astrocytic gliosis rather than a loss of outgrowth inhibition by ephrin B3 from oligodendrocytes106
Additional studies have been used to target intra cellular pathways common to both myelin and CSPGs. Application of C3 transferase, which ADP-ribosylates and inhibits RhoA, increases axon sprouting in the optic nerve after crush injury and in CST fibres after spinal cord hemisection84,86
. Pharmacological inhibition of Rho’s downstream effector ROCK using Y-27632 also promoted significant regeneration of CST fibres and an improvement in function84,85
. However, not all CNS fibre tracts may respond in the same way. Intrathecal infusion of the PKC inhibitor Gö6976 after dorsal hemisection promoted increased regeneration of only dorsal column, but not CST, fibres88
. Nevertheless, pharmacological treatments might ultimately be more useful than genetic approaches in the clinical context. For example, local application of EGFR kinase inhibitors such as PD168393 can promote dramatic retinal ganglion axon regeneration after optic nerve crush90
. Incidentally, the EGFR antagonist erlotinib (Tarceva) has already been approved for the treatment of lung cancer, and might therefore be readily tested in clinical trials for its efficacy in nerve repair.
It is difficult to reconcile the varying outcomes from these in vivo
studies. The effects of genetic deletions might be obscured by variations in the genetic back ground and differential compensatory mechanisms from the different mouse strains. Pharmacological treatments using small molecules, enzymes or function-blocking antibodies or peptides might depend on the pharma-cokinetic properties of the compound or method of delivery. In addition, evaluating these results could also be complicated by the heterogeneous nature of the complex surgical techniques used in these injury para digms. Even the criteria used to distinguish bona fide
regeneration from collateral sprouting or fibre sparing lack standardization107
Standardized criteria to assess true axon regeneration
studies on the promotion of nervous system repair often fail to distinguish regeneration of transected axons from compensatory sprouting that arises from preserved fibres (see figure). Although both types of axon growth might potentially enhance functional recuperation, local sprouting might be more important in physiological plasticity or conditions such as stroke, whereas long-distance regeneration of axons is required for recovery from spinal cord injury Quantifying the number of regenerated fibres is further plagued by the presence of spared fibres, which are easily mistaken for regenerated fibres107
. A potentially useful approach could be to monitor the sprouting and/or regenerative behaviours of lesioned axons by in vivo
Nevertheless, the mixed success from these studies has taught us much more about the complex nature of the mechanisms that prevent CNS axon regeneration. In particular, it is becoming clear that nerve fibre tracts have different intrinsic regenerative potentials, and might respond to various methods of intervention in different ways. For example, PKC inhibitor treatment failed to enhance CST regeneration, but might promote the repair of dorsal column fibres71,88
. In addition, treatments that show little effect in one injury paradigm could still have potential benefits under other conditions. This can be seen in a model of middle cerebral artery occlusion, in which both IN-1 antibody treatment and genetic deletion of NgR or Nogo-A/B have been shown to improve local axonal sprouting and stroke recovery108–111
. In fact, ischaemic or contusion injuries occur much more frequently in the clinical setting than complete nerve transection, highlighting the importance of testing different injury paradigms when evaluating the efficacy of a particular approach.
It is clear from these studies that no single comp onent is solely responsible for regeneration failure in the adult CNS. Although we cannot exclude the possibility of other key inhibitory molecules that have not yet been identified, a reasonable next step would be to use com binatorial approaches to simultaneously target multiple inhibitory pathways. In addition, treatments to enhance the intrinsic regenerative machinery of the damaged neurons, such as neurotrophin treatment112,113
, preconditioning injury114,115
or macrophage activation115
, might also be required. Studies combining ChABC treatment with a preconditioning lesion have already revealed markedly improved regeneration across the DREZ after dorsal root crush injury compared with either treatment alone116
. Further investigation along these lines will provide valu able information regarding the relative importance of different factors, as well as their therapeutic potential in the clinical setting.