293
Understanding Spinal Cord Regeneration
FIGURE 20.1 (Continued)
proliferation and restricted macrophage infltration; NR-stage shows limited macrophage infltration. At 2dpt, the R-stage animals show
abundant NSPC proliferation and the peak of macrophage invasion, and NR-stage shows disorganized and necrotic cells lining the central
canal and abundant infltration by red blood cells (RBCs). At 6 dpt, the R-stage animals have NSPC rosettes, NSPC differentiation into
neurons, and neurons elongating axons through the ablation gap, and NR-stage have abundant RBC and macrophage infltration into the
ablation gap. At 10 dpt, the R-stage animals still have NSPC rosettes and now also axons crossing the ablation gap, and the NR-stage showed
glial processes surrounding the stumps and macrophages at the ablation gap. Finally, at 20 dpt, the R-stage exhibits an almost complete
regenerated spinal cord, and, on the contrary, the NR-stage shows the presence of a glial scar and accumulation of ECM and glial processes.
TABLE 20.1
Cellular Response to Spinal Cord Injury in R- and
NR-Stages.
Cellular
R-Stages
NR-Stages
Response to SCI
Morphology of Almost healthy and
Damaged cells. Rupture of
cells lining the normal
cell membrane and
central canal
organelle loss (2–6 dpt)
Cell death
Reduced and controlled
Massive. Necrotic cells
(1dpt)
(2–6 dpt)
Stump closure
Early (2 dpt)
Late (20 dpt)
By cells lining the central By cells lining the central
canal
canal
Immune
Rapid inf ltration, debris
Slow inf ltration, prolonged
response
clearance by macrophages red blood cell clearance by
(2 dpt)
macrophages (6–10 dpt)
Proliferation
Early (2 dpt) by NSPCs
Late (6–10 dpt), mainly
other cells
Cells in the
NSPCs forming neural
Red blood/immune cells (6
ablation gap
tube-like structures
dpt)
(rosettes) (6–10 dpt)
Fibroblast-like cells (20 dpt)
ECM deposition (10–20 dpt)
Neurogenesis
Differentiation of NSPCs Not clearly def ned
into neurons and astrocytes
Axon
Axon tips in ablation gap Not achieved
regeneration
(6 dpt)
Bundle of axons crossing
the ablation gap (10 dpt)
Continuity of
Achieved (20 dpt)
Not achieved
ependymal canal
Border of stumps
surrounded by glial
processes (10–20 dpt)
such as Fibronectin and Collagen fll the injury site
(Table 20.1) (Edwards-Faret et al. 2021).
4. Late cellular response: In the R-stage at 20 dpt,
cells lining the central canal complete the reconstruction of the pseudo-stratif ed epithelium,
the central canal is continuous across the injury
site, and axons cross it (Figure 20.1; Table 20.1)
(Edwards-Faret et al. 2021), correlating with the
recovery of swimming capabilities (Muñoz et al.
Source : Muñoz et al. 2015 ; Edwards-Faret et al. 2018 , 2021
dpt: days post transection, NSPCs: neural stem and progenitor cells; ECM:
extracellular matrix.
2015). On the other hand, in the NR-stage, although
the cells lining the central canal have recovered a
pseudo-stratifed epithelial structure and the central canal of both stumps is fnally sealed, a glialscar like structure remains in the ablation gap, and
no reconnection between the stumps is observed.
This glial-scar like structure is characterized by
both stumps being surrounded by glial processes
and the injury gap being flled by f broblast-like
cells and a dense ECM containing Collagen and
Chondroitin Sulfate Proteoglycans (EdwardsFaret et al. 2021). The lack of axonal connections
between both stumps persists after 40 days, leading to the complete lack of swimming capacities in
NR-stage (Edwards-Faret et al. 2021; Muñoz et al.
2015 ).
20.3.3. THE ROLE OF NEURAL STEM AND
PROGENITOR CELLS AND NEUROGENESIS
The cells lining the central canal of the spinal cord have
a fundamental role during spinal cord regeneration. sox2,
which is a marker of stem cells and also neural stem cells
(Ellis et al. 2004; Pevny and Nicolis 2010), is expressed in
cells lining the central canal during R-stages, and a progressive decrease in the number of Sox2-expressing cells takes
place during metamorphosis (Gaete et al. 2012; Muñoz et
al. 2015). Sox2 protein and mRNA expression are upregulated after tail amputation, concomitant with a proliferative
response of Sox2-expressing cells (Gaete et al. 2012), which
is also observed in R-stages after spinal cord transection
(Muñoz et al. 2015). In vivo time-lapse imaging has shown,
for the frst time, their capacity to generate new neurons
in response to injury (Figure 20.1; Table 20.1), which correlates with an increase in neurogenic markers (Muñoz et
al. 2015). Consistent with this, Sox2 functional knockdown
using morpholino oligonucleotides or overexpressing a
dominant negative form of Sox2 results in spinal cord regeneration impairment (Muñoz et al. 2015) and in defective tail
regeneration (Gaete et al. 2012). The neural stem progenitor
cell identity of some sox2-expressing cells lining the central
canal was recently corroborated (Edwards-Faret et al. 2021).
Taking advantage of a reporter transgenic line (Xla.Tg(Dre.
gfap::EGFP) Larra ), which expresses EGFP in spinal cord cells
with radial glial cell morphology (Edwards-Faret et al. 2018,
2021), it was possible to perform a transcriptomic (RNAseq) analysis of these cells in isolation and show the enrichment of transcripts mainly related to neural precursor cell
Understanding Spinal Cord Regeneration
FIGURE 20.1 (Continued)
proliferation and restricted macrophage infltration; NR-stage shows limited macrophage infltration. At 2dpt, the R-stage animals show
abundant NSPC proliferation and the peak of macrophage invasion, and NR-stage shows disorganized and necrotic cells lining the central
canal and abundant infltration by red blood cells (RBCs). At 6 dpt, the R-stage animals have NSPC rosettes, NSPC differentiation into
neurons, and neurons elongating axons through the ablation gap, and NR-stage have abundant RBC and macrophage infltration into the
ablation gap. At 10 dpt, the R-stage animals still have NSPC rosettes and now also axons crossing the ablation gap, and the NR-stage showed
glial processes surrounding the stumps and macrophages at the ablation gap. Finally, at 20 dpt, the R-stage exhibits an almost complete
regenerated spinal cord, and, on the contrary, the NR-stage shows the presence of a glial scar and accumulation of ECM and glial processes.
TABLE 20.1
Cellular Response to Spinal Cord Injury in R- and
NR-Stages.
Cellular
R-Stages
NR-Stages
Response to SCI
Morphology of Almost healthy and
Damaged cells. Rupture of
cells lining the normal
cell membrane and
central canal
organelle loss (2–6 dpt)
Cell death
Reduced and controlled
Massive. Necrotic cells
(1dpt)
(2–6 dpt)
Stump closure
Early (2 dpt)
Late (20 dpt)
By cells lining the central By cells lining the central
canal
canal
Immune
Rapid inf ltration, debris
Slow inf ltration, prolonged
response
clearance by macrophages red blood cell clearance by
(2 dpt)
macrophages (6–10 dpt)
Proliferation
Early (2 dpt) by NSPCs
Late (6–10 dpt), mainly
other cells
Cells in the
NSPCs forming neural
Red blood/immune cells (6
ablation gap
tube-like structures
dpt)
(rosettes) (6–10 dpt)
Fibroblast-like cells (20 dpt)
ECM deposition (10–20 dpt)
Neurogenesis
Differentiation of NSPCs Not clearly def ned
into neurons and astrocytes
Axon
Axon tips in ablation gap Not achieved
regeneration
(6 dpt)
Bundle of axons crossing
the ablation gap (10 dpt)
Continuity of
Achieved (20 dpt)
Not achieved
ependymal canal
Border of stumps
surrounded by glial
processes (10–20 dpt)
such as Fibronectin and Collagen fll the injury site
(Table 20.1) (Edwards-Faret et al. 2021).
4. Late cellular response: In the R-stage at 20 dpt,
cells lining the central canal complete the reconstruction of the pseudo-stratif ed epithelium,
the central canal is continuous across the injury
site, and axons cross it (Figure 20.1; Table 20.1)
(Edwards-Faret et al. 2021), correlating with the
recovery of swimming capabilities (Muñoz et al.
Source : Muñoz et al. 2015 ; Edwards-Faret et al. 2018 , 2021
dpt: days post transection, NSPCs: neural stem and progenitor cells; ECM:
extracellular matrix.
2015). On the other hand, in the NR-stage, although
the cells lining the central canal have recovered a
pseudo-stratifed epithelial structure and the central canal of both stumps is fnally sealed, a glialscar like structure remains in the ablation gap, and
no reconnection between the stumps is observed.
This glial-scar like structure is characterized by
both stumps being surrounded by glial processes
and the injury gap being flled by f broblast-like
cells and a dense ECM containing Collagen and
Chondroitin Sulfate Proteoglycans (EdwardsFaret et al. 2021). The lack of axonal connections
between both stumps persists after 40 days, leading to the complete lack of swimming capacities in
NR-stage (Edwards-Faret et al. 2021; Muñoz et al.
2015 ).
20.3.3. THE ROLE OF NEURAL STEM AND
PROGENITOR CELLS AND NEUROGENESIS
The cells lining the central canal of the spinal cord have
a fundamental role during spinal cord regeneration. sox2,
which is a marker of stem cells and also neural stem cells
(Ellis et al. 2004; Pevny and Nicolis 2010), is expressed in
cells lining the central canal during R-stages, and a progressive decrease in the number of Sox2-expressing cells takes
place during metamorphosis (Gaete et al. 2012; Muñoz et
al. 2015). Sox2 protein and mRNA expression are upregulated after tail amputation, concomitant with a proliferative
response of Sox2-expressing cells (Gaete et al. 2012), which
is also observed in R-stages after spinal cord transection
(Muñoz et al. 2015). In vivo time-lapse imaging has shown,
for the frst time, their capacity to generate new neurons
in response to injury (Figure 20.1; Table 20.1), which correlates with an increase in neurogenic markers (Muñoz et
al. 2015). Consistent with this, Sox2 functional knockdown
using morpholino oligonucleotides or overexpressing a
dominant negative form of Sox2 results in spinal cord regeneration impairment (Muñoz et al. 2015) and in defective tail
regeneration (Gaete et al. 2012). The neural stem progenitor
cell identity of some sox2-expressing cells lining the central
canal was recently corroborated (Edwards-Faret et al. 2021).
Taking advantage of a reporter transgenic line (Xla.Tg(Dre.
gfap::EGFP) Larra ), which expresses EGFP in spinal cord cells
with radial glial cell morphology (Edwards-Faret et al. 2018,
2021), it was possible to perform a transcriptomic (RNAseq) analysis of these cells in isolation and show the enrichment of transcripts mainly related to neural precursor cell