Axonal degeneration in dorsal columns of spinal cord does not induce recruitment of hematogenous macrophages.

It is generally accepted that there are two populations of macrophages that respond to neural injuries and successful recruitment of hematogenous macrophages has been shown to help the process of nerve repair in the peripheral nervous system (PNS). Meanwhile, the recruitment of circulating macrophages after central nerve system (CNS) injuries is considered mild and delayed. We compared the recruitment of circulating macrophages in the peripheral nerves and spinal cord after dorsal root ganglionectomies, which induce selective and approximately similar extent of sensory fiber degeneration in PNS and CNS, in bone marrow chimeric mice. Our results showed that circulating macrophages were efficiently recruited in PNS but virtually no recruitment in CNS despite degeneration of peripheral and central sensory projections emanating from the same dorsal root ganglion (DRG) neurons. The mechanisms that prevent recruitment of circulating macrophages in CNS after injury remain poorly elucidated.

Reduced BACE1 activity enhances clearance of myelin debris and regeneration of axons in the injured peripheral nervous system.

ß-Site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) is an aspartyl protease best known for its role in generating the amyloid-ß peptides that are present in plaques of Alzheimer's disease. BACE1 has been an attractive target for drug development. In cultured embryonic neurons, BACE1-cleaved N-terminal APP is further processed to generate a fragment that can trigger axonal degeneration, suggesting a vital role for BACE1 in axonal health. In addition, BACE1 cleaves neuregulin 1 type III, a protein critical for myelination of peripheral axons by Schwann cells during development. Here, we asked whether axonal degeneration or axonal regeneration in adult nerves might be affected by inhibition or elimination of BACE1. We report that BACE1 knock-out and wild-type nerves degenerated at a similar rate after axotomy and to a similar extent in the experimental neuropathies produced by administration of paclitaxel and acrylamide. These data indicate N-APP is not the sole culprit in axonal degeneration in adult nerves. Unexpectedly, however, we observed that BACE1 knock-out mice had markedly enhanced clearance of axonal and myelin debris from degenerated fibers, accelerated axonal regeneration, and earlier reinnervation of neuromuscular junctions, compared with littermate controls. These observations were reproduced in part by pharmacological inhibition of BACE1. These data suggest BACE1 inhibition as a therapeutic approach to accelerate regeneration and recovery after peripheral nerve damage.

A conditioning lesion induces changes in gene expression and axonal transport that enhance regeneration by increasing the intrinsic growth state of axons.

Injury of axons in the peripheral nervous system (PNS) induces transcription-dependent changes in gene expression and axonal transport that promote effective regeneration by increasing the intrinsic growth state of axons. Regeneration is enhanced in axons re-injured 1-2 weeks after the intrinsic growth state has been increased by such a prior conditioning lesion (CL). The intrinsic growth state does not increase after axons are injured in the mammalian central nervous system (CNS), where they lack the capacity for effective regeneration. Sensory neurons in the dorsal root ganglion (DRG) have two axonal branches that respond differently to injury. Peripheral branches, which are located entirely in the PNS, are capable of effective regeneration. Central branches regenerate in the PNS (i.e., in the dorsal root, which extends from the DRG to the spinal cord), but not in the CNS (i.e., the spinal cord). A CL of peripheral branches increases the intrinsic growth state of central branches in the dorsal columns of the spinal cord, enabling these axons to undergo lengthy regeneration in a segment of peripheral nerve transplanted into the spinal cord (i.e., a peripheral nerve graft). This regeneration does not occur in the absence of a CL. We will examine how changes in gene expression and axonal transport induced by a CL may promote this regeneration.

Diffusion tensor magnetic resonance imaging of Wallerian degeneration in rat spinal cord after dorsal root axotomy.

Diffusion tensor imaging (DTI) and immunohistochemistry were used to examine axon injury in the rat spinal cord after unilateral L(2)-L(4) dorsal root axotomy at multiple time points (from 16 h to 30 d after surgery). Three days after axotomy, DTI revealed a lesion in the ipsilateral dorsal column extending from the lumbar to the cervical cord. The lesion showed significantly reduced parallel diffusivity and increased perpendicular diffusivity at day 3 compared with the contralateral unlesioned dorsal column. These findings coincided with loss of phosphorylated neurofilaments, accumulation of nonphosphorylated neurofilaments, swollen axons and formation of myelin ovoids, and no clear loss of myelin (stained by Luxol fast blue and 2'-3'-cyclic nucleotide 3'-phosphodiesterase). At day 30, DTI of the lesion continued to show significantly decreased parallel diffusivity. There was a slow but significant increase in perpendicular diffusivity between day 3 and day 30, which correlated with gradual clearance of myelin without further significant changes in neurofilament levels. These results show that parallel diffusivity can detect axon degeneration within 3 d after injury. The clearance of myelin at later stages may contribute to the late increase in perpendicular diffusivity, whereas the cause of its early increase at day 3 may be related to changes associated with primary axon injury. These data suggest that there is an early imaging signature associated with axon transections that could be used in a variety of neurological disease processes.

Cyclic AMP elevates tubulin expression without increasing intrinsic axon growth capacity.

Exposing rat dorsal root ganglion (DRG) neurons to dibutyryl cAMP (db-cAMP) enables central branches to regenerate in the spinal cord by nullifying the ability of CNS myelin to inhibit elongation. A conditioning lesion (CL) promotes similar regeneration of central branches in the spinal cord by increasing neuronal cAMP levels. It is a matter of speculation whether any of the other effects of a CL are triggered by elevated cAMP. We found that like a CL, intraganglionic injection of db-cAMP increases the expression of growth-associated tubulin isotypes. However, unlike a CL, db-cAMP does not increase the velocity at which tubulin is delivered to the tips of growing axons by slow component b (SCb). db-cAMP also fails to increase intrinsic axon growth capacity enough to raise the rate of regeneration of peripheral branches in the sciatic nerve or enable central branches to elongate long distances in an environment free of all CNS inhibitors of elongation (i.e., a peripheral nerve graft transplanted into the spinal cord at the site of dorsal column transection). Thus, the increase in cAMP induced by a CL induces some, but not all, of the changes that may be necessary to increase intrinsic axon growth capacity.

Intraocular injection of dibutyryl cyclic AMP promotes axon regeneration in rat optic nerve.

The optic nerve is a CNS pathway containing molecules capable of inhibiting axon elongation. The growth program in embryonic retinal ganglion cell (RGC) neurons enables axons to regenerate in the optic nerve through at least two mechanisms. Namely, high cyclic AMP (cAMP) levels abrogate the ability of CNS molecules to inhibit elongation, and the pattern of gene expression enables axons to undergo rapid, sustained, and lengthy elongation. In adult mammals, recovery of visual function after optic nerve injury is limited by both the death of most RGC neurons and the inability of surviving axons to regenerate. We now report that a single intraocular injection of the membrane-permeable cAMP analogue dibutyryl cAMP (db cAMP) promotes the regeneration of RGC axons in the optic nerves of adult rats, but does not prevent the death of RGC neurons. This regeneration in optic nerves crushed within the orbit (2 mm from the eye) was equally effective either 1 day before or 1 day after db cAMP injection. The number of regenerating axons, which was maximal 14 days after crush, declined with increasing time after injury (i.e., 28, 56, and 112 days) and distance beyond the crush site (i.e., 0.25, 0.5, and 1.0 mm). Thus, db cAMP promotes optic nerve regeneration without increasing the survival of axotomized RGC neurons. Furthermore, since db cAMP does not enable axons to undergo rapid, sustained, and lengthy elongation, strategies that increase survival and promote these changes in elongation may critically complement the ability of db cAMP to promote regeneration.

Spinal axon regeneration induced by elevation of cyclic AMP.

Myelin inhibitors, including MAG, are major impediments to CNS regeneration. However, CNS axons of DRGs regenerate if the peripheral branch of these neurons is lesioned first. We show that 1 day post-peripheral-lesion, DRG-cAMP levels triple and MAG/myelin no longer inhibit growth, an effect that is PKA dependent. By 1 week post-lesion, DRG-cAMP returns to control, but growth on MAG/myelin improves and is now PKA independent. Inhibiting PKA in vivo blocks the post-lesion growth on MAG/myelin at 1 day and attenuates it at 1 week. Alone, injection of db-cAMP into the DRG mimics completely a conditioning lesion as DRGs grow on MAG/myelin, initially, in a PKA-dependent manner that becomes PKA independent. Importantly, DRG injection of db-cAMP results in extensive regeneration of dorsal column axons lesioned 1 week later. These results may be relevant to developing therapies for spinal cord injury.