Axonal regeneration and physiological activity following transection and immunological disruption of myelin within the hatchling chick spinal cord

Abstract

Transections of the chicken spinal cord after the developmental onset of myelination at embryonic day (E) 13 results in little or no functional regeneration. However, intraspinal injection of serum complement proteins with complement-binding GalC or 04 antibodies between E9-E12 results in a delay of the onset of myelination until E17. A subsequent transection of the spinal cord as late as E15 (i.e., during the normal restrictive period for repair) results in neuroanatomical regeneration and functional recovery. Utilizing a similar immunological protocol, we evoked a transient alteration of myelin structure in the posthatching (P) chicken spinal cord, characterized by widespread "unravelling" of myelin sheaths and a loss of MBP immunoreactivity (myelin disruption). Myelin repair began within 7 d of cessation of the myelin disruption protocol. Long term disruption of thoracic spinal cord myelin was initiated after a P2-P10 thoracic transection and maintained for > 14 d by intra-spinal infusion of serum complement proteins plus complement-binding GalC or 04 antibodies. Fourteen to 28 d later, retrograde tract tracing experiments, including double-labeling protocols, indicated that approximately 6-19% of the brainstem-spinal projections had regenerated across the transection site to lumbar levels. Even though voluntary locomotion was not observed after recovery, focal electrical stimulation of identified brainstem locomotor regions evoked peripheral nerve activity in paralyzed preparations, as well as leg muscle activity patterns typical of stepping in unparalyzed animals. This indicated that a transient alteration of myelin structure in the injured adult avian spinal cord facilitated brainstem-spinal axonal regrowth resulting in functional synaptogenesis with target neurons.

Setting the stage for functional repair of spinal cord injuries: a cast of thousands

“…We have used a complement-fixing antibody to galactocerebroside (GalC), coadministered intraspinally with serum complement, to achieve transient focal demyelination at the site of SCI. [67][68][69] Within the CNS, GalC is restricted to the cell membrane of oligodendrocytes and myelin; specifically, it is present in the outer layer of the myelin membrane. Binding of the GalC antibody triggers activation of the complement cascade, which results in disruption of myelin membranes and recruitment of endogenous microglia or invading macrophages to phagocytically clear CNS myelin within the injured region.…”

The role of complement in immunological demyelination of the mammalian spinal cord

“…Recent studies have shown that either the selective disruption of myelin or the targeted functional inhibition of the aforementioned myelin proteins within either the developing and adult avian or mammalian spinal cord can facilitate functional axonal sprouting and/or regeneration. [10][11][12][13][14][15][16] To reduce transiently the axonal growth inhibition activity associated with CNS myelin, we have developed an immunological intraspinal infusion protocol. 17 The immunological process is activated by infusing a myelinspecific IgG antibody to an outer cell surface myelinspecific epitope (eg anti-galactocerebroside or GalC) that has a high affinity for activating the accompanying infusion of serum complement.…”

“…Only when combined will these two reagents selectively target CNS myelin to focally demyelinate an injured spinal cord region in a concentration-dependent, diffusion-limited manner. 11,12,14,17,18 When applied to the embryonic chick spinal cord, prior to transection of the cord, 12,17 this treatment suppressed CNS myelin development and extended the normal permissive period for axonal regeneration and functional recovery to later stages of development. 12,17 In the mature spinal cord of posthatchling birds, the same immunological protocol transiently demyelinates *Correspondence: JK Dyer, International Collaboration on Repair Discoveries (ICORD), University of British Columbia, 6270 University Boulevard, Vancouver, BC, Canada V6T 1Z4 and disrupts compact myelin, thereby facilitating some functional axonal regeneration of axotomized avian brainstem-spinal neurons, albeit at a reduced level of repair when compared to the injured developing spinal cord.…”

“…12,17 In the mature spinal cord of posthatchling birds, the same immunological protocol transiently demyelinates *Correspondence: JK Dyer, International Collaboration on Repair Discoveries (ICORD), University of British Columbia, 6270 University Boulevard, Vancouver, BC, Canada V6T 1Z4 and disrupts compact myelin, thereby facilitating some functional axonal regeneration of axotomized avian brainstem-spinal neurons, albeit at a reduced level of repair when compared to the injured developing spinal cord. 14 The compacted form of myelin subsequently reappears after the immunological protocol is discontinued. 14,19 More recent work in the adult rat spinal cord indicates that a similar immunological treatment after a thoracic cord lesion also facilitates anatomical regrowth of axonal projections from specific brainstem-spinal nuclei, 11 as well as ascending dorsal column neurons.…”

“…14 The compacted form of myelin subsequently reappears after the immunological protocol is discontinued. 14,19 More recent work in the adult rat spinal cord indicates that a similar immunological treatment after a thoracic cord lesion also facilitates anatomical regrowth of axonal projections from specific brainstem-spinal nuclei, 11 as well as ascending dorsal column neurons. 18 These studies indicated that this immunological protocol is effective in transiently removing the inhibitory effects of CNS myelin against axonal repair in both the developing and adult spinal cord of different species and will facilitate functional axonal regeneration.…”