Epothilone B impairs functional recovery after spinal cord injury by increasing secretion of macrophage colony-stimulating factor
Citation: Cell Death and Disease
Epothilone B impairs functional recovery after spinal cord injury by increasing secretion of macrophage colony-stimulating factor
Liang Mao 1 2
Wei Gao 0
Shurui Chen 6
Ying Song 1
Changwei Song 1 5
Zipeng Zhou 4
Haosen Zhao 4
Kang Zhou 4
Wei Wang 4
Kunming Zhu 4
Chang Liu 3
Xifan Mei 1 4
0 Department of Basic Medical Sciences, Jinzhou Medical University , Jinzhou 121000 , People's Republic of China
1 Key Laboratory of Medical Tissue Engineering of Liaoning Province, The First Affiliated Hospital of Jinzhou Medical University , Jinzhou 121000 , People's Republic of China
2 Department of Oncology, The First Affiliated Hospital of Jinzhou Medical University , Jinzhou 121000 , People's Republic of China
3 Department of Endocrinology, The First Affiliated Hospital of Jinzhou Medical University , Jinzhou 121000 , People's Republic of China
4 Department of Orthopedic Surgery, The First Affiliated Hospital of Jinzhou Medical University , Jinzhou 121000 , People's Republic of China
5 Department of Hand Surgery, The First Affiliated Hospital of Jinzhou Medical University , Jinzhou 121000 , People's Republic of China
6 Jinzhou Medical University , Jinzhou 121000 , People's Republic of China
The microtubule-stabilizing drug epothilone B (epoB) has shown potential value in the treatment of spinal cord injury (SCI) through diverse mechanisms. However, it remains elusive why a limited overall effect was observed. We aim to investigate the limiting factors underlying functional recovery promoted by epoB. The same SCI model treated by epoB was established as discussed previously. We used a cerebrospinal fluid (CSF) sample to assess the changes in cytokines in milieu of the SCI lesion site after epoB treatment. We then analyzed the source of cytokines, the state of microglia/macrophages/monocytes (M/Ms), and the recruitment of neutrophil in the lesion site by using the results of antibody array. Following these findings, we further evaluated the motor functional recovery caused by the reshaped microenvironment. Systemic administration of epoB significantly increased levels of several cytokines in the CSF of the rat SCI model; macrophage colony-stimulating factor (M-CSF) secreted by intact central nervous system (CNS) cells was one of the cytokines with increased levels. Along with epoB and other cytokines, M-CSF reshapes the SCI milieu by activating the microglias, killing bone marrow-derived macrophages, polarizing the M/M to M1 phenotype, and activating downstream cytokines to exacerbate the SCI injury, but it also increases the expression of neurotrophic factors. Anti-inflammatory therapy using a neutralizing antibody mix shows encouraging results. Using in vivo experiments, our findings indicate that epoB inhibits the SCI functional recovery in many ways by reshaping the milieu, which counteracts the therapeutic efficacy that led to the limited overall effectiveness. Cell Death and Disease (2017) 8, e3162; doi:10.1038/cddis.2017.542; published online 2 November 2017
Epothilone B (epoB) is an ideal drug for the treatment of spinal
cord injury (SCI), which can reduce scar formation in the lesion
site, reactivate the axons’ regeneration potential, and is
convenient for operation.1 In theory, epoB has a therapeutic
role in SCI by divergent mechanisms. First, epoB is a blood–
brain barrier-permeable microtubule-stabilizing drug that is
absorbed in the central nervous system (CNS) after being
administered.1 Second, through neuron-specific protein Tau
expression, epoB promotes axon elongation and reduces
fibrotic scarring simultaneously.1 Third, epoB reduces axonal
dieback and promotes serotonergic axon regrowth. Thus, the
rat SCI model exhibits functional recovery after epoB
administration.1 It seems epoB matches every step of SCI
However, the overall efficiency of epoB is not quite so
satisfactory. As a matter of fact, the number of footfall errors
was reduced ~ 50% on the horizontal ladder compared with
the control. These effects could be counteracted by ablation of
5-hydroxytryptamine.1 Furthermore, epoB does not appear
superior to any other monotherapies.2–4 To further evaluate
the therapeutic limitation of epoB, we can refer to the similar
microtubule-stabilizing drug taxol, which was tested as a
treatment for SCI for many years.5 Hellal et al.6 showed that
taxol also possessed multitargeted abilities in treatment of SCI
similar to epoB. Nevertheless, recently an independent
evaluation reported that taxol is able to reduce the scar
formation in the SCI lesion site; however, it does not possess
the ability to promote serotonergic axon growth and protect
neurons from damage.7 It is still unclear why these
A fundamental question is whether epoB is beneficial for the
SCI functional recovery overall and it also consists of some
detrimental factors. Here, we found the changes of cytokines in
the cerebrospinal fluid (CSF) instead of lesion site milieu. We
investigated the source and function of elevated macrophage
colony-stimulating factor (M-CSF) focus on the microglia/
macrophages/monocytes (M/Ms) in the SCI lesion site after
epoB treatment. We further evaluated the downstream cytokine
LIX (CXCL5) focused on the effects of neutrophil recruitment.
These results indicate that combined with elevated cytokines,
epoB suppresses SCI functional recovery by killing bone
marrow-derived macrophages (BMDMs), activating microglia,
polarizing M/M to M1 phenotype, and recruiting neutrophils to
increased lesion inflammation burden. Unlike previous reports,
these effects retarded the functional recovery after SCI.
to assume that the elevated M-CSF may be the key point for
interpreting the limited efficacy of epoB administration in SCI.
To clarify the biological effects of elevated M-CSF in milieu,
we need to identify the value, duration, and cell origin of
elevated M-CSF at first.
EpoB increases levels of several cytokines in CSF after
SCI. Because biologically active cytokines of SCI milieu exist
in the SCI lesion mainly, and the cytokines of SCI lesion differ
very little with CSF, we assessed the profiles of expression of
cytokines by cytokine protein array with CSF from the SCI
model treated with epoB (n = 3) or solvent control (n = 3).
Interestingly, epoB-treated SCI rats were only M-CSF
elevated compared with the control at 1 day post epoB
treatment (DPE) (Figures 1a and e); however, both anti- and
proinflammatory molecules including interleukin-1α (IL-1α),
IL-4, M-CSF, monocyte chemoattractant protein-1 (MCP-1),
tumor necrosis factor-α (TNF-α), and transforming growth
factor-β (TGF-β) were elevated in the CSF of epoB-treated
SCI models at 3 DPE (Figures 1b and f). In addition to IL-1α
and IL-4, insulin-like growth factor-1 (IGF-1) and LIX were
elevated 7 days after epoB treatment (Figures 1c and g) and
only IGF-1 increased 14 DPE in CSF (Figures 1d and h).
According to the CSF cytokine profiling data, M-CSF is the
earliest rise in CSF. Based on these results we have reason
Elevated M-CSF derived from intact CNS astrocytes,
T cells, M/Ms, and primarily ependymal cells lasting
4 days. We next performed the CSF cytokine profile using
the epoB-treated SCI model (n = 3) or intact model (n = 3).
Interestingly, the elevated six cytokines 3 DPE of
epoBtreated SCI model shows no obvious difference compared
with the epoB-treated intact model (Figures 2a and b). These
data suggest that the upregulated six cytokines of 3 DPE did
not generate from the injured CNS. For further evaluating the
cellular sources of elevated M-CSF, we conducted
immunohistofluorescence double staining (n = 6) using M-CSF and
specific antigen of CNS cells. It is shown that astrocytes,
T cells, M/Ms, ependymal cells, and neurons were the main
sources of elevated M-CSF at 3 DPE (Figures 2c, e–g, and i).
Meanwhile, enzyme-linked immunosorbent assay (ELISA)
experiments reveal that levels of M-CSF rose 1 DPE and
lasted up to 4 days at least (n = 3); the concentration of
M-CSF peaked at ~ 1.6 ng/ml at 4 DPE (Figure 2j).
EpoB and elevated M-CSF exhibit cytotoxic effects rather
than proliferation in SCI lesion M/Ms. M-CSF is
responsible for the survival, differentiation, activation, proliferation of
M/Ms, and recruitment of M/Ms, mainly to lesions.8
Accordingly, we next evaluated the biological effects of elevated
M-CSF on the above aspects caused by epoB in SCI. For
some experiments requiring cultured purified M/Ms, we used
the rat alveolar macrophage cell line NR8383 to evaluate the
proliferation of M/Ms. First, we determined if elevated M-CSF
caused expansion in the M/Ms. NR8383 cells were stained by
carboxyfluorescein diacetate succinimidyl ester (CFSE)
followed by 2 ng/ml M-CSF plus PBS and 1 nM, or 5 nM
epoB treatment in vitro. Either 3 or 5 days after stimulating,
the NR8383 cells were detected by flow cytometry (FC) for
cell proliferation. As a matter of fact, even at the highest level
and longest stimulation used, M-CSF did not significantly
stimulate M/M division compared with controls; on the
contrary, in epoB-treated NR8383 cells, division reduced
(Figures 3a–c). To clarify the reason for reduction in growth
rate, we further performed the cell viability experiment in vitro.
As Figure 3d shows, more than half of NR8383 cells were
killed by 1 or 5 nM epoB.
Following this route, we further assessed the cytotoxic effect
of epoB in SCI lesion M/M in vivo. We separated SCI lesion M/
M using CD11b antibody by FC (n = 6) (Figure 3e). Next, we
distinguished BMDMs from resident microglias using CD45
antibody. As expected, systemic administration of epoB
reduced SCI lesion BMDMs (Figures 3f and h). Unexpectedly,
this effect was not found in resident microglias (Figures 3g
and i). Immunofluorescence staining (n = 6) further shows a
decrease in BMDMs but not microglias at 3 and 7 DPE
(Figures 3j and k). Furthermore, this effect in BMDMs was
reinforced by APC-labeled peripheral monocytes homing to
the SCI lesion (n = 3) (Figure 3l). The next fundamental
question is whether the SCI milieu is cytotoxic to BMDMs after
epoB treatment or whether epoB kills peripheral monocytes,
thereby resulting in reduced recruitment of M/M into the SCI
The elevated M-CSF and MCP-1 promote recruitment of
M/M into the SCI lesion. M-CSF and MCP-1 recruit two
different types of M/M that go through two different paths to
the SCI site.9 MCP-1, also known as CCL2, is a chemotactic
factor that attracts M1 to the SCI lesion site through spinal
cord (SC) leptomeninges.9 The corresponding cytokine is
M-CSF, which recruits M2 to the SCI lesion site through
choroid plexus. In our research, both M1 and M2 chemotactic
factors elevated at 3 DPE. We therefore tested whether the
elevated two factors promote the homing process of M/M to
the SCI lesion. Immunofluorescent staining of the third
ventricle choroid plexus (n = 6) shows an increase of homing
M/M in SCI-epoB compared with SCI-vehicle. On the
contrary, these recruitment effects could be reduced by
M-CSF-neutralizing antibody treatment (Figure 3m). Similar
to M-CSF, Figure 3n shows that M/Ms are enriched by SC
leptomeninges (n = 6) of the SCI-epoB group; these effects
could be weakened by corresponding neutralizing antibody
treatment. Quantification analysis shows that the elevated
two factors M-CSF and MCP-1 contribute to ~ 2- and 2.5-fold
increase in homing M/M, respectively. These findings
suggested that both increased M1 and M2 cells infiltrated in
the SCI microenvironment after epoB administration, and the
homing M/Ms were killed by epoB mainly in the CNS.
EpoB plus elevated M-CSF activate microglias and
suppress microglia phagocytosis in CNS. To investigate
the active effect promoted by elevated M-CSF in M/Ms, we
used the CD11b, CD68, and Iba-1 staining to identify the
activated state. CD11b is a pan-marker of M/Ms, CD68 is a
macrophage marker related to its phagocytic function, and
Iba-1 represents the M/M activation. Meanwhile, they are all
affected by total amount of M/Ms. As a matter of fact, FC
(n = 6) shows that phagocytic markers of M/M CD68 in SCI
lesions were not changed significantly at 3 or 7 DPE between
the SCI-epoB- and vehicle-treated groups (Figures 3p and
3q). However, phagocytic markers could be reduced by the
administration of M-CSF-neutralizing antibody. The above
results led us to consider morphological changes in M/Ms; we
next focused on the immunostaining (n = 6) for CD68 and
Iba-1. Interestingly, in the SCI-vehicle group, CNS M/M
phagocytic phenotypes were altered to turgid and smooth.
By contrast, phagocytic M/M experience shrinkage and size
reduction in the SCI-epoB group (Figure 3r). Furthermore,
SCI-vehicle-group M/M tend to be in a resting state mainly; on
the contrary, M/M in the SCI-epoB group become active.
Typical representation of the resting state of M/M is slender
and thin; nevertheless, the M/M active state is rounded and
the tail is spread, which could be transformed by
M-CSFneutralizing antibody partly (n = 6) (Figure 3s). These results
reveal that the elevated M-CSF plus epoB changed the active
and phagocytic function via altering cell morphology rather
than changing its number. By reason, BMDMs’ phagocytic
and active states could not be distinguished by cellular
morphology.10 We have reason to believe that changes in
phagocytic and active morphology occur mainly in microglias.
The remodeled microenvironment polarized homing and
resident M/M to M1 phenotype. The previous results raised
another interesting question: the infiltrating and resident M/
Ms are exposed to the anti-inflammatory as well as the
proinflammatory milieu simultaneously, so how does the
remodeled milieu re-educate M/M phenotype? Is the
reeducated M/Ms detrimental or beneficial for SCI recovery?
These questions led us to detect a series of M1 and M2
phenotypes in homing and resident M/Ms in SCI lesions 3 or
7 DPE. Based on previous literature, we chose CD16/32,
CD86, and TNF-α as M1 markers and CD206, CD163, and
TGF-β as M2 markers.11 Cells isolated from SCI lesions were
triple-labeled M/M pan-marker CD11b and M/M phenotype
markers for FC analysis (n = 6). Figures 4b–d showed that
CD16/32, CD86, and TNF-α expressed an M/M increase and
was associated with decreasing M/M with CD206 expression
3 DPE. However, CD163- and TGF-β-expressed M/M was not
detected. It is worth noting that using all six neutralizing
antibodies, M/Ms tend to possess less M1 phenotype
compared with SCI-epoB group 3 DPE. The findings indicate
that beyond our antibody-array-detected cytokines, the rest of
the cytokines tend to be in the anti-inflammatory state. The
polarization is weakened; meanwhile, the neutralizing
antibody mix tends to be unable to counteract the inflammatory
milieu 7 DPE (Figures 4e–g).
Elevated LIX mediates neutrophil recruitment and aggra
vates SCI. According to the cytokine antibody-array results,
IGF-1 and LIX were both upregulated 7 DPE. IGF-1 is a
cytokine that shows neuroprotective effects and promotes
recovery effects in SCI.12–14 Nevertheless, LIX, also known
as CXCL5, exhibits potent chemoattractivity for neutrophils in
inflammatory microenvironments, which has never been
investigated by previous SCI research. To follow the route
of epoB exacerbating SCI recovery, we focused on biological
effects of elevated LIX after epoB exposure. Figure 5a shows
that the MPO assay (n = 6) illustrated that myeloperoxidase
activity obtained from SCI lesions of the SCI-epoB group
increased 1.6-fold compared with the SCI-vehicle 10 DPE.
Similarly, the infiltrating neutrophils rose in the SCI-epoB
group compared with the vehicle control, this effect could be
neutralized on different levels by LIX-neutralizing antibody
administration 10 DPE (n = 6) (Figure 5b). BBB scores (n = 9)
show that compared with SCI-epoB, the SCI-epoB-NAM
group recovers the best among the three groups from 7 DPE.
This score was followed by the SCI-epoB-M-CSF Ab group; it
recovers better than SCI-epoB from 21 DPE. At last,
SCIepoB-LIX Ab group presents superior to SCI-epoB at the
beginning of 42 DPE (Figure 5d).
Recent data indicate that epoB is a promising strategy for SCI
treatment.1 On the contrary, in the current study we found that
epoB is detrimental for SCI recovery via killing BMDMs,
activating microglias, skewing M/M to M1 phenotype in CNS,
and recruiting neutrophils to aggravating SCI lesion
inflammation in the acute phase, as well as therapeutic benefits for SCI.
These findings reveal that epoB could be a double-edged
sword for SCI recovery. These adverse effects are mainly due
to epoB reshaping the SCI microenvironment by increasing
the cytokine secretion in CSF. Actually, cytokines existing in
intercellular washing fluid of lesion site cells modulate the
reshaping of the microenvironment.15 However, it is very hard
to perform the experiments technically by using the
intercellular washing fluid of the lesion site. CSF-brain and SC
barrier are the weakest in the three parts of blood–brain
barriers and the majority of CSF cytokines produced by CNS
cells.16–18 As a result, we evaluated the cytokines changing by
CSF sample to instead of intercellular washing fluid of
Generally speaking, in inflammatory cascade reactions,
initially elevated cytokines reach peak within 12–48 h after
SCI.9 After that, initial cytokines stimulate other cells to secrete
downstream cytokines in the second or third cascade
reactions in CNS.19 However, we found that only M-CSF
shows 1.5-fold increase in CSF of SCI-epoB 24 h later, while
six kinds of cytokines rose ~ 3-fold in CSF 72 h after epoB
treatment. This phenomenon suggests that the six elevated
cytokines may be the second or third cascade reaction after
epoB administration. On the other hand, previous research
reported that M-CSF could increase expression level of many
cytokines, which include TNF-α, IL-1, TGF-β, and MCP-1.8,20
These results strongly suggest that except for IL-4, M-CSF
may be the central and initial cytokine in the SCI superacute
stage after epoB administration.
In the CNS, M-CSF modulates the survival, proliferation,
maturation, differentiation, recruitment, and activation of M/
Ms.20,21 In fact, M-CSF is a milieu-dependent cytokine that
exhibits both neuroprotective and/or neurotoxic effects in CNS
diseases.20 Our study revealed that treating SCI models with
epoB generates M-CSF increases in CSF; M/Ms of CNS
exposed to both elevated M-CSF and epoB showed
contradictory responses. Results showed that the number of CD45+
M/Ms in SCI lesion sites decreased as microglias (CD45−
M/Ms) of milieu exhibited active states. In other words, the SCI
milieu presents cytotoxic effects to homing M/Ms, which are
sensitive to microtubule-stabilizing drugs, while resident
microglias show activated states, which are sensitive to
M-CSF even when they are both exposed to the same
microenvironment. How could this dichotomy happen? First of
all, one of the most common side effects of epoB is
myelosuppression; meanwhile, epoB shows condensed forms
in CNS after administration.1,22,23 Second, recruited M/Ms via
ventricle choroid plexus and adjacent SC leptomeninges were
still increased because of the elevated M-CSF and MCP-1.
Third, microglias are more stable than homing M/Ms in
CNS.24,25 It is reasonable to infer that the main reason for
this contradiction is that the absorbed epoB exhibit cytotoxicity
for homing M/Ms and nonkilling effects for microglias. On the
other hand, elevated M-CSF activates microglia rather than
dying BMDMs. Then, activated microglia exhibits a variety of
neurotoxic effects, including secreting reactive oxygen
species, nitric oxide, TNF-α, excitatory amino acids, and so on,
and also promotes neuroprotection by secreting IGF-1 and
increasing macrophage phagocytosis, which is crucial for
debris removal.20,25 Unexpectedly, there was no significant
change in the M/M number of phagocytic markers of CD68
expression in the SCI-epoB and SCI-vehicle groups.
Nevertheless, CD68+ M/M shrink and become crenulated
morphologically after epoB treatment, which reminds us that epoB
shows quite a bit of cytotoxic effects to phagocytic M/Ms and
elevated M-CSF stimulated the M/M phagocytosis
Beyond M-CSF, there are five other cytokines elevated in
CSF at the same time, which contains three proinflammatory
cytokines (IL-1α, MCP-1, and TNF-α) and two
antiinflammatory cytokines (IL-4 and TGF-β).10,18,19,26 Many
studies reported that M-CSF-treated M/Ms changed to M2
phenotypes.9,21,27 Interestingly, which phenotypes are lesion
site M/Ms tend to when exposing well-balanced anti- and
proinflammatory cytokines in CSF? These questions guide us
to evaluate the M/M phenotype conversion after epoB
The M1 phenotype increased in the SCI-epoB lesion site
compared with the SCI-vehicle model 3 DPE. It is noteworthy
that the M1 phenotype in the SCI-epoB-NAM group is in
between; nevertheless, few M2 phenotypes were detected in
every group. But over time, phenotype conversion in the M/M
substantially weakened in every group 7 DPE. Previous
research reported that the M1 phenotype accounted for the
overwhelming majority, whereas M2 is a transient and minority
polarization, which homes into the lesion site through the
second wave after 1 DPI in the acute stage.9,25,28 It is
reasonable to believe that epoB administration induced an M1
phenotype temporary conversion in SCI lesions in the acute
SCI injury phase. Moreover, other studies have shown that
treatment by both M2-primed cytokine IL-4 and M1-primed
cytokines IL-1, MCP-1, and TNF-α induced polarization of M/M
to M1 phenotype.26 Based on our results, both M1 and M2
recruitment increases through SC leptomeninges and choroid
plexus. These results strongly suggest that the elevated
proinflammatory cytokines IL-1, MCP-1, and TNF-α polarized
homing monocytes and resident microglias to the M1
phenotype predominantly in the acute SCI injury phase
temporarily, while the elevated inflammatory cytokines
declined gradually leaving the difference weakened as time
goes on. As a matter of fact, only 24 cytokines were evaluated
in CSF after epoB treatment, with hundreds of cytokine
changes unknown, thus we conducted all six inflammatory
cytokines neutralizing in vivo. Most noteworthy are the SCI
microenvironment polarized M/M into less M1 numbers
compared with SCI-epoB, which provides a referable strategy
for alleviating the epoB side effects. Locomotion recovery
assay agrees with the M/M conversion effects in the SCI
Besides IL-1α and IL-4, there are two other cytokines, IGF-1
and LIX, that are both elevated 7 DPE. Hamilton and
Achuthan21 reported that IGF-1 is produced primarily by the
activated M/Ms when stimulated by M-CSF in the CNS.12
Moreover, LIX was secreted by cells after stimulated by IL-1 or
TNF-α.29 We can infer that the elevated IGF-1 and LIX are the
next cascade stage of upregulation of the six cytokines 3 DPE.
It has been verified that IGF-1 exhibits both neuroprotective
and neurotrophic effects in CNS injury.30,31 We found elevated
IGF-1 lasting for more than 7 days after epoB treatment, which
may partly explain axon regeneration phenomenon of epoB in
SCI. In contrast, LIX is a milieu-dependent cytokine that
exhibits the strong attracting effect for neutrophils.32,33
Furthermore, the expression of LIX correlated with the lesion
burden and poor prognosis in multiple sclerosis and
experimental autoimmune encephalomyelitis.34 To our knowledge,
little attention has been focused on the role of LIX in SCI. For
the above reason, we explored LIX’s effect in SCI. In
accordance with the previous report, the level of LIX is
proportional to locomotive disability mainly by attracting
neutrophils. However, no neutrophil decreasing was observed
in the SCI-epoB group, even though neutrophils are the most
sensitive hemocytes to epoB. It is mainly due to the time
disparity between the attracting neutrophils and epoB
Like a plethora of SCI research, we only used female
rodents as our experimental subjects.11,35,36 However, its
limitation is also obvious, as sex-specific factors bias could
lead to inaccurate results. One thing for sure is that estrous
cycle affects immunological responses by enhancing or
impairing specific responses of immunocompetent cells.37
Another sex-specific factor needs to take into account is
estrogen. Previous studies reported estrogen is a protective
factor by limiting tissue damage and improving functional
recovery after rodent SCI.38 However researches on human
show gender-related differences in recovery after SCI have
not been found.39,40 The evidence of estrogen effects in SCI
remains inconclusive. Therefore, the finding of our work tends
to fit female rodents. Further researches should eliminate the
effects due to sex-specific factors in this specific SCI model
treated by epoB.
The epoB promotes functional recovery of SCI in many
ways; however, current studies show many opposite events,
which may counteract the therapy effect. In addition, a few
questions are still unclear, such as whether M-CSF is the first
inflammatory cascade reactions factor of elevated cytokines?
Beyond the elevated cytokines we found, are there any other
crucial cytokine changes that influence the prognosis? How do
we turn the harmful context into helpful status for functional
recovery after epoB administration efficiently? Except for
CNS, do any other cells contribute to cytokine sources? Are
there any other factors that modulate the protein Tau to
facilitate SCI recovery? These findings remind us that the
mechanism underlying epoB for SCI treatment is not fully
investigated. Further research should focus on the
mechanism mentioned above.
The present study demonstrates that administration of epoB
not only has many positive roles in functional recovery but also
exhibits many unwanted adverse effects, mainly due to
changing the milieu after SCI. Further research should focus
on alleviating the side effects.
Materials and Methods
Animals, SCI surgery, and drug administration. Adult female
Sprague–Dawley rats (220 ± 20.0 g, aged 8–10 weeks; Capital Medical University,
Beijing, China) were used. All rats were housed in stainless-steel cages (six per
cage) in a room kept at 22 ± 1 °C on a 12–12 h light–dark cycle. All rats were
acclimatized to their environment for 1 week and free access to food and water. A
total of 198 animals were used for all the experiments (Table 1). The study was
approved by the Animal Care and Use Committee of Liaoning Medical University in
accordance with the Guidelines for the Care and Use of Laboratory Animals
published by the US National Institutes of Health.
SCI models were prepared as described previously.41 Briefly, the animals were
anesthetized with an intraperitoneal injection of chloral hydrate (0.33 ml/kg), and the
intact dorsal cord surface was exposed by laminectomy at T9. Subsequently,
moderate contusive injury was conducted by impounder striking (2 mm diameter,
12.5 g, 5 cm height) at the surface of T9 SC. The SCI models were maintained on
twice-daily bladder evacuation manually until the bladder function of the rat was
reestablished. After injury, the rats were randomly assigned to various groups. First, in
accordance with previous literature, SCI models were treated with epoB (2x0.75 mg/
kg; Sellek, Houston, TX, USA) or vehicle (30% PEG400, 5% Ppopylene glycol, 0.5%
Tween-80, 0.2% dimethylsulfoxide (DMSO) and 64.3% saline) via intraperitoneal 1
DPI systemically, which is referred to as SCI-epoB or SCI-vehicle. Second, SCI-epoB
were randomly administered corresponding neutralizing antibodies according to the
experiment design. Rat did not undergo SCI surgery, which was treated with epoB
(0.75 mg/kg; Sellek) is referred to as intact-epoB. Rats used for histological analysis
were killed by intracardiac perfusion with 0.9% NaCl, followed by 4%
paraformaldehyde after anesthesia. Rats used for others assays were killed with
an overdose of anesthetic.
CSF collection. Rats were fixed on a stereotactic instrument after anesthesia
by chloral hydrate. The rat’s head was hyperextended at a 135° angle to the body,
and the skin around the incision was prepared and sterilized. We then performed a
longitudinal incision in the occipital shin and blunt separated the subcutaneous
tissue and muscle to expose the foramen magnum region. Next, we inserted the
25 G needle into the cisterna magna and extracted ~ 100 μl CSF from each rat
without blood contamination. Finally, we sutured the skin and muscle, sterilized the
incision, and recuperated the rats at 37 °C. CSF was sampled one time a rat for
array (n = 3) and once every other day in experiment groups (animals used for
immunofluorescence staining and FC of 3 and 7 DPE) for ELISA (n = 3) ( Table 1).
Specifically, we collected CSF sample for ELISA in SCI-vehicle group for
immunofluorescence staining of 3 DPE (sample used for ELISA of days 1 and 3);
SCI-vehicle group for FC of 3 DPE (day 2); SCI-vehicle group for
immunofluorescence staining of 7 DPE (days 4 and 7); SCI-vehicle group for FC of 7 DPE (day
5). SCI-vehicle group of 14 DPE was collected for CSF sample only for ELISA. In
the same way, the CSF sample of SCI-epoB group was collected. The M-CSF
concentration was measured by ELISA on CSF sample. We analyzed 42 CSF
samples without blood contamination for ELISA.
Cytokine antibody array. RayBiotech Neuro Discovery Array C Series
(Raybiotech, Norcross, GA, USA) was used for detecting changes of CSF cytokine
levels after epoB or vehicle treatment (n = 3) and total 30 CSF samples were
analyzed. We centrifuged the CSF samples at 1000 × g for 5 min after thawing to
remove any particulates, aspirated 100 μl CSF samples, and diluted them to 1 ml
for all later array procedures. The manufacturer’s instructions were followed for all
steps involving the cytokine array. We briefly placed each membrane into a well of
the incubation tray and incubated in blocking buffer for 30 min at room temperature.
After washing, we added diluted CSF into each well and incubated it overnight at
4 °C. After consecutive washes, we pipetted 1 ml of the prepared detection antibody
cocktail into each well and incubated it overnight at 4 °C. With the same washes,
2 ml of HRP-streptavidin was added into each well and incubated overnight at 4 °C.
After consecutive washes, we then added 500 μl of the detection buffer mixture onto
each membrane and incubated them for 2 min at room temperature. Afterwards, we
transferred the membranes to the CCD camera and exposed them. The intensity of
the positive control signal was used to normalize the cytokine signal between the
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Enzyme-linked immunosorbent assay. The rats’ CSF of the SCI-vehicle
and SCI-epoB groups were collected as described above in consecutive time. Level
and duration of the increasing M-CSF was measured using ELISA (n = 3) (Sangon
Biotech, Shanghai, China) according to the manufacturer's instructions.
Administration of neutralizing antibodies. All neutralizing antibodies
were intravenously injected into the tail vein every other day after epoB or vehicle
administration. The injection doses of neutralizing antibodies are specified
according to the published studies, as follows: goat anti-IL-1α (200 μg per rat;
Peprotech, Rocky Hill, NJ, USA), goat anti-IL-4 (250 μg per rat; R&D, Minneapolis,
MN, USA), mouse anti-M-CSF (750 μg per rat; R&D), rabbit anti-MCP-1 (500 μg
per rat; Peprotech), rabbit anti-TNF-α (100 μg/rat; Peprotech), mouse anti-TGF-β
(250 μg/rat; Abcam, Cambridge, UK).9,42–46 A mix of isotype antibody served as the
control. The number and start time of neutralizing antibodies injection was based on
results of cytokines antibody array. SCI-epoB injected with M-CSF antibody or
MCP-1 antibody from 0 DPE until 7 DPE or killing are referred to as
SCI-epoB-MCSF Ab or SCI-epoB-MCP-1 Ab, respectively. SCI-epoB injected with neutralizing
antibody mix (IL-1α, IL-4, M-CSF, MCP-1, TNF-α, and TGF-β) from 0 DPE until 7
DPE or killing is referred to as SCI-epoB-NAM, and SCI-epoB injected with LIX (C–
X–C motif chemokine 5) antibody from 3 DPE until 14 DPE or killing is referred to as
Immunofluorescence staining. For in vivo experiments, SCI models were
anesthetized and killed via intracardiac perfusion with 0.9% NaCl, followed by 4%
paraformaldehyde 3, 7 or 10 DPE. Then SC and brain samples were removed and
fixed in 4% paraformaldehyde. After 3 days fixation, tissues were equilibrated in
paraformaldehyde supplemented with 30% sucrose. After that, the samples were
cut into 10-μm sections horizontally, transversely or sagittally, and the slides were
kept in a cryoprotective solution at − 80 °C.
At the beginning, we warmed the frozen slides to room temperature and washed
them once gently in phosphate-buffered solution (PBS), and then we blocked the
slides in blocking buffer (PBS supplementary with 1% BSA+5% goat serum). After
washing two times gently, the following primary anti-rat antibodies were incubated
with the sections in the blocking buffer at 4 °C overnight: APC-conjugated anti-CD11b
(1 : 100; Miltenyi, Hamburg Germany), chicken anti-GFAP (1:1000; Abcam), rabbit
anti-NeuN (1 : 200; Abcam), rabbit anti-CD45 (1 : 100; Abcam), mouse anti-CD31
(1 : 100; Novus, Littleton, CO, USA), chicken anti-vimentin (1 : 5000; Novus), mouse
anti-CD8 (1:100; Abcam), rabbit anti-Iba-1 (1:100; Abcam), rabbit
antimyeloperoxidase (MPO) (1:100; Abcam), mouse anti-CD68 (1 μg/ml; Abcam),
mouse anti-M-CSF (1:100; R&D), rabbit anti-M-CSF (1 : 100; Abcam) and mouse
anti-O4 (1 : 200; R&D). After overnight incubation, primary antibodies were discarded
and slides were rinsed gently in PBS three times. Afterwards, the fluorescent
secondary antibodies were added to sections for single or double staining:
FITCconjugated goat anti-rabbit (1 : 500; Bioss, Beijing, China), PE-conjugated goat
antimouse (1 : 500; Bioss), FITC-conjugated goat anti-mouse (1 : 500; Bioss),
PEconjugated goat anti-rabbit (1 : 500; Bioss), and CY3-conjugated goat anti-chicken
(1 : 500; Bioss) for 2 h at room temperature. Finally, nuclei were stained with Prolong
Gold Antifade reagent with DAPI (Invitrogen, Carlsbad, CA, USA). Sections were
captured with a fluorescent microscope (Olympus, Tokyo, Japan) at equal
Cell culture and viability assays. NR8383 cells (Procell, Wuhan, China)
were cultured in DMEM/F12K medium (Gibco-BRL, Grand Island, NY, USA)
supplement with 20% heat-inactivated fetal bovine serum (Gibco, Scoresby, VIC,
Australia), 50 U/ml penicillin, and 50 μg/ml streptomycin at 37 °C supplied with 5%
CO2. Cell viability was evaluated by monotetrazolium (MTT) assay and trypan blue
staining. In brief, NR8383 cells were planted at a density of 1x104 per well in 96-well
plates. After incubation for 24 h, the cells were treated by medium or 2 ng/ml
M-CSF, and meanwhile they were also exposed to epoB at the final concentration of
1 or 5 nM. After 3 or 5 days of stimulation, 20 μl MTT (5 mg/ml) was added to each
well, and 4 h later, cells were lysed by 100 μl of DMSO. The absorbance was
determined at 570 nm on a scanning multiwell spectrophotometer (Tecan,
Morrisville, NC, USA). For further assessing the cytotoxic effects of epoB on M/
Ms, trypan blue dye technique was used for distinguishing the dead cells. Briefly,
NR8383 was plated in 8-well chamber slides (BD-Falcon, Franklin Lakes, NJ, USA)
and treated by M-CSF plus epoB as above. Three days after stimulation, slides
were centrifuged to sedimentate suspension cells and stained with trypan blue at
the final concentration of 0.04% for 3 min at 37 °C. Data were collected as
Flow cytometry. For assessing the quantitative, phenotypic, polarized, and
functional status of M/Ms, the SCI-vehicle, SCI-epoB-M-CSF, SCI-epoB, and
SCIepoB-NAM group (n = 6) of rats were killed for FC 3 or 7 DPE. For the groups,
SCIepoB-M-CSF group serves as a positive control because only 24 cytokines were
detected by our antibody array, and six of these cytokines were elevated in the CSF.
Hundreds of cytokines changing were still unknown, so SCI-epoB-NAM was used
for detecting the combined effect of all unknown cytokines in the SCI milieu. Sample
collection was conducted by dissecting out the pieces of 0.5 cm length SC using the
lesion site as a center. We transferred the samples into cold Dulbecco's
phosphatebuffered saline (D-PBS) immediately after resection, and then washed the samples
two times with cold D-PBS gently to remove blood cells from the surface of the SC.
After that, we cut the sample into multiple small segments, and they were digested
by trypsin for 30 min at 37 °C. After digestion, trypsin was removed by centrifugation
and cells were resuspended in staining buffer (PBS with 2%FBS). Single-cell
suspension was obtained from filtering through the 48μm mesh. Samples were
stained according to the manufacturer’s instructions in the subsequent steps. Briefly,
1 × 106 isolated cells were stained in 100 μl staining buffer with the following primary
antibodies: APC-conjugated anti-CD11b (10 μl/106 cells; Miltenyi), rabbit
antiCD16/32 (2 μg/106 cells; Bioss, Woburn, MA, USA), mouse anti-CD206 (0.2 μg/106
cells; Rosemont, IL, USA), PE-conjugated anti-CD163 (10 μl/106 cells; GeneTex,
Irvine, USA), FITC-conjugated anti-CD86 (1 μg/106 cells; BioLegend, San Diego,
CA), rabbit anti-TNF-α (2 μg/106 cells; Peprotech, Rocky Hill, NJ, USA), mouse
antiTGF-β (2.5 μg/106 cells; Abcam, Cambridge, UK), mouse anti-CD68 (1 μg/106 cells;
Abcam, Cambridge, MA, USA), and rabbit anti-CD45(1 μg/106 cells; Abcam,
Cambridge, MA, USA). After incubation for 30 min at 4 °C with the first antibodies,
and washing twice by staining buffer, the following fluorescent-labeled secondary
antibodies were added to bind corresponding primary non-fluorescent antibodies:
FITC-conjugated goat anti-rabbit (0.5 μl/106 cells; Bioss, China) and PE-conjugated
goat anti-mouse (0.5 μl/106 cells; Bioss, China). After incubating and washing the
same as the last step, cells were fixed with 1% paraformaldehyde before analysis by
FC. A total of 50 000 events were collected and analyzed on FACSCalibur Flow
Cytometer (BD Biosciences, San Jose, CA, USA) using the FlowJo Software (Tree
Star, Inc., Ashland, OR, USA). Isotype-matched antibody-stained cells, positive-stained
cells, and unstained cells were used as controls to gate the cell populations of interest.
To detect the elevated M-CSF’s proliferative effect on M/Ms, the NR8383 cell line
and CFSE cell proliferation assay were used in vitro. NR8383 cells were stained
following the manufacturer’s instructions (Beyotime, Hangzhou, Zhejiang, China). In
brief, cultured NR8383 was digested and resuspended in CFSE staining buffer
without FBS as 2 × 106 cells/ml for 10 min at 37 °C. After incubation, adding FBS,
washing, and labeling, cells were seeded in six-well plates as 5 × 105 cells/well.
Subsequently, the cells were treated with medium or 2 ng/ml rat recombinant M-CSF
while being exposed to 1 nM/5 nM epoB or not. Either 3 or 5 days after labeling, cells
were analyzed on FACSCalibur flow cytometer as described above.
M/M labeling in vivo. For assessing the cytotoxic effect of epoB in SCI milieu
M/Ms, fluorophore-conjugated primary antibody was used for labeling the M/Ms as
described previously.47 In short, 1 ml saline containing 50 μl APC-conjugated
antiCD11b (Miltenyi) was injected via the coccygeal vein in SCI model 1 DPE. SC
tissues were taken for visualization of recruited M/Ms with immunofluorescence
staining (n = 3) 3 DPE.
MPO assay. MPO activity in the SC of the SCI-vehicle, SCI-epoB, and
SCIepoB-LIX Ab group 10 DPE were determined by MPO Detection Kit (n = 6)
(Jiancheng Bioengineering Institute, Nanjing, China). The experiment was
conducted according to the instructions. Briefly, SC tissue was washed by cold
PBS to remove blood cells from the surface. Then tissues were homogenized in
0.5% hexadecyltrimethylammonium hydroxide PBS solution and centrifuged. After
centrifugation, the supernatant was transferred into PBS (pH 6.0) containing
0.17 mg/ml 3,3′-dimethoxybenzidine and 0.0005% H2O2. Afterwards, supernatant
MPO catalyzed H2O2-dependent oxidation of 3,3′-dimethoxybenzidine to produce
yellow compound, which could be measured by 460 nm.
Quantification of immunostaining. Quantification of immunolabeling was
estimated by unbiased observers after removing the background threshold level
using the Image-Pro Plus Software 5.1 (Media Cybernetics Inc., Atlanta, GA, USA)
as described previously.12 Every group contained six or three (M/M labeling in vivo)
animals, and the SCs from rats were cut into five 10-μm-thick sections spaced
20 μm apart serially through the entire injury site. The target area was a range of
± 1000 μm surrounding the lesion epicenter, and the counting frame size was
550 μm × 550 μm. The injury size was detected by Luxol/Nissel staining, and the
lesion epicenter was identified as the unlabeled region by Luxol. Quantifications of
the M/M and neutrophils numbers, density per counting frame, and density per cell
were performed using specific tools of the Image-Pro Plus Software (Media
Cybernetics, Inc., Silver Spring, MD, USA).
Assessment of locomotion recovery. Locomotion recovery was
evaluated by the Basso, Beattie, and Bresnahan (BBB) open-field locomotor rating
scale (n = 9) as described by Basso et al.48 Briefly, two investigators, who were not
aware of the experiment design, operated the behavioral tests. BBB scores were
observed and recorded at 1, 3, 5, and 7 DPE and then weekly until 7 weeks.
Statistical analysis. Data were analyzed using SPSS Software, version 11.0
(Chicago, IL, USA) and expressed as the mean ± S.D. The differences among groups
were compared and analyzed by one-way ANOVA, followed by the Fisher's LSD
procedure. The rate of the group was analyzed by χ2 test or Fisher's exact test.
Heteroscedastic data was performed with the Mann–Whitney U-test to evaluate differences
among the experimental groups. Po0.05 was considered statistically significant.
Conflict of Interest
The authors declare no conflict of interest.
Acknowledgements. We are indebted to Dr. Guang Yu (Immunology
Laboratory, Jinzhou Medical University) for excellent technical assistance with the
flow cytometry. We also thank Zhimin Qi for her technical assistance on animal
experiments. This work was supported by the General Program of the National
Natural Science Foundation of China (No. 81671907) and the General Program of the
National Natural Science Foundation of China (No. 81471854).
Springer Nature remains neutral with regard to jurisdictional claims in published maps
and institutional affiliations.
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