291
Understanding Spinal Cord Regeneration
to identify the signals and genetic mechanisms involved
in tissue regeneration. BMP, FGF, Wnt, and Notch signaling play an important role in tail regeneration (Beck et al.
2003). Furthermore, the Amaya lab has reported a key role
for reactive oxygen species (ROSs) in the initiation of the
regenerative response that seems to be evolutionarily conserved among many species (Love et al. 2011; Love et al.
2013; Phipps et al. 2020).
Although fruitful to understand the basic and general
mechanism of tissue regeneration, the tail amputation
paradigm presents caveats for SCI studies. On one hand,
it involves regeneration of multiple tissues, not just of the
spinal cord, and on the other hand, it is different from the
situation that occurs in humans who suffer SCI. For these
reasons, the second model of injury, spinal cord transection and resection, seems more relevant for the study of SCI
(Filoni et al. 1984; Lee-Liu et al. 2013). Spinal cord transection consists of a transverse cut that completely severs the
spinal cord at the thoracic level, and spinal cord resection
consists of two transverse cuts followed by the removal of
the spinal cord segment, leaving a gap between the rostral
and caudal stumps (Edwards-Faret et al. 2017; Slater and
Larraín 2021). Of note, these injury paradigms are surgically very simple, can be performed in R- and NR-stages,
and are very reproducible across animals. Although these
experimental approaches are still not identical to SCI in
humans, they resemble the models of injury used in rodents,
one of the favorite models to study SCI, allowing the comparative investigation of the mechanisms involved in this
process in X. laevis and other model organisms.
Furthermore, sensory and motor functional recovery
can be evaluated using simple behavioral tests. For this,
tadpole swimming behavior can be classif ed qualitatively
into paraplegia, partial locomotor recovery, and coordinated
swimming phenotypes (Gaete et al. 2012). Additionally, the
free swimming distance can be quantifed using a semiautomatized video-tracking system (Edwards-Faret et al.
2017; Muñoz et al. 2015). More recently, a new method was
developed using kinematic technology to allow the detection
of slight improvements in NR-stage swimming recovery that
could be helpful to identify compounds that improve spinal
cord regeneration (De Vidts et al. 2019). This method consists of determining kinematic features during swimming,
including synchronization and symmetry between the right
and left hindlimb and the right and left foot range movement.
20.3.2. CELLULAR RESPONSE TO SPINAL CORD INJURY
1. Histological differences in R- and NR-stage spinal cord: R-stage animals have a spinal cord of
400–600 μm diameter, with a cell rich ventricular layer lacking a complex stratifcation. In contrast, NR-stage animals have a spinal cord with a
diameter of 800–1600 μm, with a more complex
stratifcation and cellular organization in the ventricular zone (Edwards-Faret et al. 2018; Muñoz
et al. 2015). The cells lining the central canal are
heterogeneous, encompassing fve different cells
types, with fundamental differences between
R- and NR-stages (Edwards-Faret et al. 2018).
R-stage cells are uniciliated, exhibit a radial morphology and elongated nuclei with lax chromatin,
and thus resemble mammalian radial glial cells.
Nevertheless, in NR-stages, the most abundant
cells are multiciliated and reveal extensive changes
in the maturation and differentiation state, including round-oval shaped nucleus, with deep invaginations and clumped chromatin (Edwards-Faret et
al. 2018). R-stages contain a signifcant number of
proliferative cells, whereas NR-stages have a lower
proportion of these cells (Edwards-Faret et al.
2018; Thuret et al. 2015), which correlates with the
more differentiated profle of their cells.
2. Early cellular response: In R-stage animals, a rapid
sealing of the injured stumps is observed at 2 days
post-transection (dpt). The cells that accomplish
sealing resemble the ones lining the central canal
but with a few new features such as a fusiform shape,
lack of cilia, and lack of cell junctions between
them, suggesting they are migratory. Additionally,
macrophage infltration in the ablated gap leads to
an early immune response by phagocytosing cell
debris (Figure 20.1, Table 20.1) (Edwards-Faret et
al. 2021). In contrast, in NR-stages, the cells lining the central canal appear necrotic and therefore
are not able to seal the ablated stumps, leading to a
disorganized central canal. There is also an abundant infltration of red blood cells (RBCs) that f ll
the ablated gap (Figure 20.1, Table 20.1) (EdwardsFaret et al. 2021).
3. Intermediate cellular response: In R-stages, at
6 dpt, Sox2/3 expressing cells self-organize into
rosettes or neural tube-like structures in the ablation gap (Figure 20.1), and cells lining the central
canal differentiate into neurons, which extend their
axons into the ablation gap (Edwards-Faret et al.
2018; Muñoz et al. 2015). Abundant unmyelinated
bundles of axons populate the lumen of the central
canal caudal to the gap at 10 dpt (Figure 20.1, Table
20.1) and are in close contact with neuronal somas,
postsynaptic densities, and synaptic vesicles, suggesting that active synaptic processes occurred in
the lumen of the caudal central canal (EdwardsFaret et al. 2021). Importantly, a transient increase
of Fibronectin and Collagen is detected, probably
serving as supportive extracellular matrix (ECM)
for the advancing axons to cross through the ablation gap. In contrast, in the NR-stage, massive disorganization of the central canal persists at 6 dpt,
with extracellular spaces and vacuolated cells lining the central canal. Glial processes are observed
surrounding the borders of both stumps, and RBC
are accompanied by a massive macrophage inf ltration in the injury site. Moreover, abundant ECM
Understanding Spinal Cord Regeneration
to identify the signals and genetic mechanisms involved
in tissue regeneration. BMP, FGF, Wnt, and Notch signaling play an important role in tail regeneration (Beck et al.
2003). Furthermore, the Amaya lab has reported a key role
for reactive oxygen species (ROSs) in the initiation of the
regenerative response that seems to be evolutionarily conserved among many species (Love et al. 2011; Love et al.
2013; Phipps et al. 2020).
Although fruitful to understand the basic and general
mechanism of tissue regeneration, the tail amputation
paradigm presents caveats for SCI studies. On one hand,
it involves regeneration of multiple tissues, not just of the
spinal cord, and on the other hand, it is different from the
situation that occurs in humans who suffer SCI. For these
reasons, the second model of injury, spinal cord transection and resection, seems more relevant for the study of SCI
(Filoni et al. 1984; Lee-Liu et al. 2013). Spinal cord transection consists of a transverse cut that completely severs the
spinal cord at the thoracic level, and spinal cord resection
consists of two transverse cuts followed by the removal of
the spinal cord segment, leaving a gap between the rostral
and caudal stumps (Edwards-Faret et al. 2017; Slater and
Larraín 2021). Of note, these injury paradigms are surgically very simple, can be performed in R- and NR-stages,
and are very reproducible across animals. Although these
experimental approaches are still not identical to SCI in
humans, they resemble the models of injury used in rodents,
one of the favorite models to study SCI, allowing the comparative investigation of the mechanisms involved in this
process in X. laevis and other model organisms.
Furthermore, sensory and motor functional recovery
can be evaluated using simple behavioral tests. For this,
tadpole swimming behavior can be classif ed qualitatively
into paraplegia, partial locomotor recovery, and coordinated
swimming phenotypes (Gaete et al. 2012). Additionally, the
free swimming distance can be quantifed using a semiautomatized video-tracking system (Edwards-Faret et al.
2017; Muñoz et al. 2015). More recently, a new method was
developed using kinematic technology to allow the detection
of slight improvements in NR-stage swimming recovery that
could be helpful to identify compounds that improve spinal
cord regeneration (De Vidts et al. 2019). This method consists of determining kinematic features during swimming,
including synchronization and symmetry between the right
and left hindlimb and the right and left foot range movement.
20.3.2. CELLULAR RESPONSE TO SPINAL CORD INJURY
1. Histological differences in R- and NR-stage spinal cord: R-stage animals have a spinal cord of
400–600 μm diameter, with a cell rich ventricular layer lacking a complex stratifcation. In contrast, NR-stage animals have a spinal cord with a
diameter of 800–1600 μm, with a more complex
stratifcation and cellular organization in the ventricular zone (Edwards-Faret et al. 2018; Muñoz
et al. 2015). The cells lining the central canal are
heterogeneous, encompassing fve different cells
types, with fundamental differences between
R- and NR-stages (Edwards-Faret et al. 2018).
R-stage cells are uniciliated, exhibit a radial morphology and elongated nuclei with lax chromatin,
and thus resemble mammalian radial glial cells.
Nevertheless, in NR-stages, the most abundant
cells are multiciliated and reveal extensive changes
in the maturation and differentiation state, including round-oval shaped nucleus, with deep invaginations and clumped chromatin (Edwards-Faret et
al. 2018). R-stages contain a signifcant number of
proliferative cells, whereas NR-stages have a lower
proportion of these cells (Edwards-Faret et al.
2018; Thuret et al. 2015), which correlates with the
more differentiated profle of their cells.
2. Early cellular response: In R-stage animals, a rapid
sealing of the injured stumps is observed at 2 days
post-transection (dpt). The cells that accomplish
sealing resemble the ones lining the central canal
but with a few new features such as a fusiform shape,
lack of cilia, and lack of cell junctions between
them, suggesting they are migratory. Additionally,
macrophage infltration in the ablated gap leads to
an early immune response by phagocytosing cell
debris (Figure 20.1, Table 20.1) (Edwards-Faret et
al. 2021). In contrast, in NR-stages, the cells lining the central canal appear necrotic and therefore
are not able to seal the ablated stumps, leading to a
disorganized central canal. There is also an abundant infltration of red blood cells (RBCs) that f ll
the ablated gap (Figure 20.1, Table 20.1) (EdwardsFaret et al. 2021).
3. Intermediate cellular response: In R-stages, at
6 dpt, Sox2/3 expressing cells self-organize into
rosettes or neural tube-like structures in the ablation gap (Figure 20.1), and cells lining the central
canal differentiate into neurons, which extend their
axons into the ablation gap (Edwards-Faret et al.
2018; Muñoz et al. 2015). Abundant unmyelinated
bundles of axons populate the lumen of the central
canal caudal to the gap at 10 dpt (Figure 20.1, Table
20.1) and are in close contact with neuronal somas,
postsynaptic densities, and synaptic vesicles, suggesting that active synaptic processes occurred in
the lumen of the caudal central canal (EdwardsFaret et al. 2021). Importantly, a transient increase
of Fibronectin and Collagen is detected, probably
serving as supportive extracellular matrix (ECM)
for the advancing axons to cross through the ablation gap. In contrast, in the NR-stage, massive disorganization of the central canal persists at 6 dpt,
with extracellular spaces and vacuolated cells lining the central canal. Glial processes are observed
surrounding the borders of both stumps, and RBC
are accompanied by a massive macrophage inf ltration in the injury site. Moreover, abundant ECM