Source of Early Regenerating Axons in Lamprey Spinal Cord Revealed by Wholemount Optical Clearing with BABB

Abstract

Many studies of axon regeneration in the lamprey focus on 18 pairs of large identified reticulospinal (RS) neurons, whose regenerative abilities have been individually quantified. Their axons retract during the first 2 weeks after transection (TX), and many grow back to the site of injury by 4 weeks. However, locomotor movements begin before 4 weeks and the lesion is invaded by axons as early as 2 weeks post-TX. The origins of these early regenerating axons are unknown. Their identification could be facilitated by studies in central nervous system (CNS) wholemounts, particularly if spatial resolution and examination by confocal microscopy were not limited by light scattering. We have used benzyl alcohol/benzyl benzoate (BABB) clearing to enhance the resolution of neuronal perikarya and regenerated axons by confocal microscopy in lamprey CNS wholemounts, and to assess axon regeneration by retrograde and anterograde labeling with fluorescent dye applied to a second TX caudal or rostral to the original lesion, respectively. We found that over 50% of the early regenerating axons belonged to small neurons in the brainstem. Some propriospinal neurons located close to the TX also contributed to early regeneration. The number of early regenerating propriospinal neurons decreased with distance from the original lesion. Descending axons from the brainstem were labeled anterogradely by application of tracer to a second TX close to the spinal–medullary junction. This limited contamination of the data by regenerating spinal axons whose cell bodies are located rostral or caudal to the TX and confirmed the regeneration of many small RS axons as early as 2 weeks post-TX. Compared with the behavior of axotomized giant axons, the early regenerating axons were of small caliber and showed little retraction, probably because they resealed rapidly after injury.

The Composition and Cellular Sources of CSPGs in the Glial Scar After Spinal Cord Injury in the Lamprey

“…Immediately post-TX, blood elements, and fibroblasts fill the lesion gap. By 10 days, glial cells in the rostral and caudal stumps send processes into the lesion across (Lurie et al, 1994 ), followed by axons of small RS neurons and local interneurons (Zhang et al, 2020 ). By 4 weeks post-TX, as the lesion scar matures, the cellular elements decline and are replaced by an accessible longitudinal-glial fibrotic scar.…”

“…These generally are worse regenerators than the smaller RS neurons, and they also usually undergo a very delayed retrograde cell death (Shifman et al, 2008 ; Zhang et al, 2018b ). Axon retraction is greater in large caliber than small caliber RS axons, many of which already have regenerated into or past the lesion site by 2 weeks post-TX (Zhang et al, 2020 ). Moreover, RS neurons with poor regenerating probability selectively express the RPTP receptors for CSPGs (Zhang et al, 2014a ).…”

“…Early after SCI, CSPGs might promote retraction and inhibit the growth of giant axons that express CSPG receptors and participate in glial process bridge formation. Upregulation of lectican D was particularly strong and persistent, at least to 10 weeks post-TX, when many smaller propriospinal and reticulospinal axons are already regenerating into the scar (Zhang et al, 2020 ). This suggests that post-SCI, some CSPGs, particularly lectican D, might actually promote axon growth or guidance.…”

Cyclic Stretch of Either PNS or CNS Located Nerves Can Stimulate Neurite Outgrowth

“…Swimming led to an increase of 5% strain on the spinal cord from non-stress situations [ 65 ]. Moreover, small caliber reticulospinal and propriospinal axons were the first to regrow before larger reticulospinal axons [ 66 ]. It can be postulated that smaller caliber axons may be more receptive to mechanical strain.…”

Transcriptomes of Injured Lamprey Axon Tips: Single-Cell RNA-Seq Suggests Differential Involvement of MAPK Signaling Pathways in Axon Retraction and Regeneration after Spinal Cord Injury

“…Of special interest, the identified reticulospinal neurons vary widely in their response to axotomy [ 21 , 22 ]. Those that are poor regenerators (i.e., the probability that their axons will regenerate beyond the lesion is low) show the greatest amount of early retraction and are most likely to undergo very delayed apoptosis [ 23 , 24 , 25 , 26 , 27 ]. By reimaging living axons in exposed spinal cords over a period of 2–4 h and locating the positions of the axons relative to fiduciary landmarks, we have been able to determine the growth status (growing, static or retracting) of individual axon tips [ 28 , 29 ] at the time of micro-aspiration.…”