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Figure 1. Experimental
design. In the first phase, the N-glycome of the adult
female rat spinal cord was characterized. Phase II examined how this
profile changes in response to spinal cord transection, and treatment
with collagen hydrogel in either a random or aligned orientation,
at 7 and 14 days post-injury (dpi). The third phase employed X. laevis to model injury and compared the glycosylation
response in the regenerative premetamorphic stage and the nonregenerative
postmetamorphic stage, at 1 and 7 dpi. UPLC, ultrahigh-performance
liquid chromatography; WAX-HPLC, weak anion exchange high-performance
liquid chromatography; LC-MS, liquid chromatography coupled mass spectrometry.
Created with BioRender.com.
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Figure 2. Main panel
of exoglycosidase digestions profiled using HILIC-UPLC.
ABS removes α(2–3, 6, 8)-sialic acids, BTG removes β(1–3,
4) galactose, BKF removes core α(1–6)-fucose and outer-arm
α(1–2)-fucose, AMF removes α(1–3, 4)-fucose,
GUH removes β-GlcNAc, and JBM removes mannose. Peaks are labeled
with GU values. EU, emission units.
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Figure 3. Sialic acids of rat spinal cord. WAX-HPLC
separates glycans on
the basis of charge, and fetuin-N was used as a reference
standard. Neutral N-glycans (77%); mono-charged (12%); di-charged
(4%); tri-charged (1%); tetra-charged (1%); unidentified highly charged
N-glycan species (5%); 2AB, excess 2AB label; (A) UND, undigested;
(B) ABS, ABS (sialidase) digested. The dashed line indicates the zoomed
region. (C) Reference standard for DMB analysis. (D) Neu5Ac is the
most common type of sialic acid in the rat spinal cord, with minor
amounts of Neu5Gc- and Neu5xAc2-type sialic acids. Sialic acids were
released from spinal cord glycans by hydrolysis and separated on the
basis of chemical structure. Peak 1, Neu5Gc; peak 2, Neu5Ac; peak
3, Neu5,7Ac2; peak 4, Neu5Gc9Ac; peak 5, Neu5,8Ac2; peak 6, Neu5,9Ac2;
and peak 7, Neu5,x,xAc3, where x is an unknown position; * contaminant
peak. Note that DMB analysis was performed on total spinal cord glycans,
and WAX-HPLC on N-glycans.
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Figure 4. Summary of the types of N-glycan identified in the healthy adult
rat spinal cord. (A) All of the major classes of N-glycan can be found
in the spinal cord, oligomannose, hybrid, and branched. Branched glycans
also occur with a bisecting GlcNAc β(1–4) linked to the
central mannose. Some more unusual features such as acetylated sialic
acids, sulfate groups, and α-linked galactose can also be found
on complex or hybrid glycans. (B) Number of branches on complex glycans,
some also feature a bisecting GlcNAc β(1–4) linked to
the central mannose. A2 and A3 structures are most common. (C) Charge
abundance as calculated using WAX-HPLC. (D) Extent of branch elongation
with galactose on each class of N-glycan. (E) Proportion of total
glycans decorated with both core and outer-arm fucose. (F) Distribution
of fucosylation across the main N-glycan classes.
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Figure 5. Differences in N-glycan features between sham and transection-only
groups, at 7 and 14 dpi in the rat spinal cord. (A) Lesion epicenter,
(B) rostral to injury, and (C) caudal to injury. Asterisks indicate
a statistically significant difference between sham and transected
groups, using the t-test, n = 3, p < 0.05.
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Figure 6. Changes in
N-glycans in the lesion epicenter, rostral and caudal
to injury of rat spinal cord with collagen hydrogel treatment. Individual
GPs were grouped according to whether they were high-mannose, hybrid,
complex, or complex with bisect, and the type of fucose (F) they were
decorated with. Data presented here are expressed as the percentage
of the sham group for the appropriate time point, n = 3. Variance within groups was low, with RSD being ≤10%
in most cases and ≤20% in the remainder. Blue squares indicate
a reduction compared to sham, with the greatest reduction shown in
dark blue at 59.26%. Red squares indicate an increase compared to
sham, with the greatest increase shown in dark red at 152.02%.
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Figure 7. Distribution of sialic
acid labeled with SNA-I lectin and its relationship
to CD11b-positive microglia and macrophages in the rat spinal cord.
(A–C) Sham (D–F) transection-only, (G–I) random
collagen hydrogel-treated, and (J–L) aligned collagen hydrogel-treated.
SNA-I single-channel images are shown for each group in (B, E, H,
K); CD11b single-channel images are in (C, F, I, L). Images from injured
animals (D–L) were captured at the borders of the injury. All
images are from the spinal cord at 7 dpi. Scale bar is 20 μm.
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Figure 8. Changes in GlcNAc following
SCI in pre-and postmetamorphic X. laevis. Graphs show DSA lectin staining in spinal
cord tissue in tadpole lesion epicenter (A) and in tadpole and froglet
lesion borders rostral (B) and caudal (C) and in intact tissue rostral
(D) and caudal (E) at 1 and 7 dpi. Data were normalized to sham before
analysis with two-way ANOVA and Bonferroni’s post hoc test,
and are presented as mean ± SEM. A value of p < 0.05 was considered significant. Groups that differ significantly
are indicated with an asterisk.
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Figure 9. Changes in sialic acid following SCI in pre- and postmetamorphic X. laevis. Graphs show SNA-I lectin staining in spinal
cord tissue in tadpole lesion epicenter (A) and in tadpole and froglet
lesion borders rostral (B) and caudal (C) and in intact tissue rostral
(D) and caudal (E) at 1 and 7 dpi. Data were normalized to sham before
analysis with two-way ANOVA and Bonferroni’s post hoc test,
and are presented as mean ± SEM. A value of p < 0.05 was considered significant. Groups that differ significantly
are indicated with an asterisk.
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Figure 10. Changes in GalNAc following
SCI in pre- and postmetamorphic X. laevis. Graphs show WFA lectin staining in spinal
cord tissue in tadpole lesion epicenter (A) and in tadpole and froglet
lesion borders rostral (B) and caudal (C) and in intact tissue rostral
(D) and caudal (E) at 1 and 7 dpi. Data were normalized to sham before
analysis with two-way ANOVA and Bonferroni’s post hoc test,
and are presented as mean ± SEM. A value of p < 0.05 was considered significant. Groups that differ significantly
are indicated with an asterisk.
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Figure 11. Changes in monosaccharide abundance following
sham injury to the
spinal cord in pre- and postmetamorphic X. laevis at 1 and 7 dpi. Graphs show lectin staining in sham injured tissue
at 1 and 7 dpi in tadpole and froglet. (A) GlcNAc, (B) sialic acid,
and (C) GalNAc. Data were analyzed by two-way ANOVA and Bonferroni’s
post hoc test, and are presented as mean ± SEM. For all tests,
a value of p < 0.05 was considered significant.
Groups that differ significantly are indicated with an asterisk.
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Figure 12. Summary and future perspectives. (A)
Between rat and Xenopus, pre- and post-metamorphosis,
there were distinct glycosylation
responses to SCI. In the regenerative tadpole, there was a moderate
increase in sialylation, a transient increase in GlcNAc in close proximity
to the injury, and a continued increase in GlcNAc distant from the
injury. (B) Glyco-enzymes of the Golgi could be targeted, ideally
in a spatiotemporal manner, to create a more proregenerative environment
in the injured spinal cord which, when combined with the structural
cues and guidance provided by an aligned collagen hydrogel, should
encourage repair of the injured spinal cord. Created with BioRender.com.
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