The Role of CCL2 Chemoattractants in Lumber Myeloid Cell Trafficking and
Locomotor Recovery Following Thoracic Spinal Cord Injury
Undergraduate Research Thesis
Kimberly J.J. Berry1, 4
Tyler T. Thaxton1,2, Timothy D. Faw1,2,3, Diana Norden1,2, Rochelle J Deibert1,2
Lesley C. Fisher1,2, and D. Michele Basso1,2
1School
of Health and Rehabilitation Services, 2Center for Brain and Spinal Cord
Repair, 3Neuroscience Graduate Program, 4Department of Neuroscience
1
Approximately 282,000 people in the United States live with Spinal Cord Injury (SCI), a
lifelong debilitating injury without a cure (NSCISC). Spinal Cord Injury develops into a chronic
injury caused by damage to the spinal cord or nerves that often results in sensory processing and
motor learning deficits, including increased spasticity, chronic pain, or paralysis. (Hagen, 2015).
At the sight of injury, the lesion epicenter, neuronal communication via axons between the brain
and areas of spinal cord below the lesion are reduced or lost completely, depending on the
severity of the injury. Lesions that occur in the upper thoracic regions of the cord, the site of
nerves that control the chest and abdomen, typically result in greater deficits than lesions in the
lower lumbar regions, which are responsible for hip and leg movements. Spinal cord injury can
be termed as complete or incomplete, depending on the severity of the lesion and the amount of
spared healthy tissue after injury that can aid in the generation or continuation of neural signals.
For complete SCI the brain is unable to send or receive signals from the spinal cord, a necessary
function for voluntary movement or perception of touch, pressure, and pain. Incomplete SCI has
some communication between the brain and spinal cord, but may have reduced sensation or
range of voluntary movement in the affected regions below the lesion site (Villenes, Z, 2015).
Microenvironment
Scientific and medical fields have worked together to identify effective approaches to
alleviate symptoms and promote neuronal plasticity in the spinal cord after injury. Inflammation
plays a key role in tissue response to injury, often leading to neuronal damage and apoptosis, or
cell death (Zhang et al., 2012). Our study focuses on the inflammatory response of peripheral
nervous system (PNS) blood-derived monocytes, and central nervous system (CNS) resident
microglia, two cell types that activate and contribute to macrophage-mediated release of
proinflammatory cytokines, reactive oxygen species, nitric oxide, and proteases (Kigerl et al.,
2009). Macrophages express phagocytic activity after injury that, initially, mediate cell
maintenance and recovery.
The blood-spinal-cord-barrier (BSCB) is made up of endothelial cells and tight junctions,
and regulates the trafficking of cells from the periphery into the CNS. Blood-spinal-cord-barrier
permeability is altered in SCI, allowing the entrance of monocytes and other peripherally-derived
cells into the spinal cord and contributing to a compromised microenvironment (Hansen et al.,
2013). A multitude of studies have characterized the microenvironment at the lesion epicenter,
reporting both resident microglia and hematogenous cells as contributors to acute macrophage
activation (Popovich & Hickey, 2001). In turn, macrophage activation aggravates the immune
response and effects secondary degradative influencers of axon myelination, which hinder
locomotor recovery (Wang et al., 2015). Below the lesion, immunohistochemical studies show
microglial activation in the lumbar cord is increased at 24hr and 7 days after injury compared to
microglial activation in the cervical cord in mice. Chimeric green fluorescent protein (GFP)
studies indicate continued presence of bone-marrow derived myeloid cells at 14d (Norden et al.,
2017). Less is known about chronic macrophage activation and its role in cell maintenance or
recovery at later time points.
Trafficking
The first main objective of our study is to assess the chronic immune response in regions
distal to the epicenter, specifically the lumbar cord. We have previously shown increases in
2
matrix metalloproteinase-9 (MMP-9), a protein that is thought to increase vascular permeability,
at the lumbar level of the cord after thoracic injury (Hansen et al., 2013). Macrophage infiltration
does not occur in the absence of MMP-9 (Hansen et al., 2016). Cell trafficking is mediated by
chemoattractants, cytokines that attract white blood cells to sites of infection, and intracellular
cell adhesion molecules (ICAM) that aid in binding monocyte cells to endothelial cells of the
BSCB (Furie, 2014). We are interested in the role of the CCL2 chemoattractant and ICAM in
cell trafficking. We have previously shown levels of CCL2 and ICAM are increased in lumbar
locomotor networks 24h and 7 days post injury (dpi) when compared to WT mice. The
upregulation of CCL2 and ICAM is concurrent with an increase in myeloid cell infiltration,
supporting our hypothesis that these cells are being actively trafficked into the lumbar spinal
cord in response to thoracic injury (Hansen et al., 2016). To test the role of CCL2 in the
trafficking of monocytes, we use a CCL2-null transgenic model to observe the
microenvironment of the lumbar cord after thoracic injury.
Training
It is imperative to identify an optimal time window during which the induction of
neurorehabilitation approaches like treadmill training promotes a microenvironment conducive
to improved recovery and functional outcomes. The intermediate grey matter of the lumbar
spinal cord is of particular importance when discussing recovery after injury, as it contains
locomotor central pattern generators (CPGs). Central pattern generators are neuronal circuits that
are activated and induce specific movement patterns for locomotion. After SCI, activation of the
correct CPGs could initiate patterns of walking (Kjaerulff & Kiehn, 1996; Marder & Bucher,
2001). We use treadmill training to target CPGs and improve locomotor recovery.
Previous studies conclude that after acute 2-9dpi training, monocyte levels increased in
the peripheral circulation (blood), while levels of inflammatory monocytes in the blood was
reduced. Green fluorescent protein cell counts reveal reduced amounts of parenchymal blood
myeloid (BM) cells at 7d (Ghasemlou et al., 2005). This study will provide further insight into
the microenvironment at later time points. The second objective is to investigate the effects of
rehabilitation intervention at a sub-acute time point (14-21dpi), and compare the inflammatory
profiles that training induces in a CCL2-deficient and WT model.
Hypothesis
In the absence of CCL2 via genetic knockout (KO), we hypothesize that fewer myeloid
cells will traffic into the lumbar cord after SCI. Fewer myeloid cells will create a more
permissive microenvironment for sub-acute rehabilitation interventions when compared to wild
type SCI.
Materials and Methods
Mice
Experiments conducted here adhered to The Ohio State University Institutional
Laboratory Animal Care and Use Committee. A total of 25 mice were used for this study, n=14
CCL2KO and n=11 WT. Adult (12-9 weeks of age; 17-23g) female C57BL/6J WT mice were
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obtained from Jackson Laboratories. WT mice were assigned to either the treadmill (TM) trained
or untrained (NoEx) groups. The CCL2KO mice were engineered at Jackson Laboratories in
accordance with MCP-1(-/-) processes. CCL2 KO mice were also assigned to a TM trained or
NoEx group, and all were used for histology measures. Mice were housed 2-5 per cage with a
12-hour light-dark cycle and provided food and water ad libitum.
Spinal Cord Injury
Mice were anesthetized using ketamine (138mg/kg) and xylazine (20mg/kg) and given
prophylactic antibiotics (gentocin, 1 mg/kg). Taking precautions to avoid sepsis, the lamina of
T9 and surrounding tissue were removed to expose the dura. The vertebral column was stabilized
and an Infinite Horizon (IH) device was used to create a moderate/severe contusion (75
kilodynes), according to the force and displacement trends determined by Ghasemlou et al.,
(2013) (mean force: 78.33 ± 0.78; mean displacement: 548.7 ± 35.50). Biomechanics of the
injury were examined on day 0 to rule out aberrant mechanics. The incision was closed in 2cm
intervals and sterile saline was applied subcutaneously to prevent dehydration. Mice were given
antibiotics (1 mg/kg gentocin, s.q.) and saline once a day for 5 days. Bladders were manually
expressed twice per day every day for the duration of the study (Hoschouer, Basso, & Jakeman,
2010).
Behavioral Testing: Basso Mouse Scale (BMS)
We used the BMS scale to track locomotor hind limb function and recovery over time:
pre-injury, 1d, 3d, 7d, 14d, 21d, 28d, and 35dpi (17). During the 4-minute test, mice were placed
in a plastic open field, 100cm in diameter, with clear plastic walls in a quiet room and normal
lighting. Two raters blind to group assignment trained in BMS assessment assigned scores for
each hind limb from (0-9), with a 0 score indicating no hind limb movement and 9 being normal
walking.
Group Assignment
Animals were excluded from group assignment if the BMS score was determined
abnormally high or outside the typical range for a 70 kilodyne IH impact at any time point up
to14dpi, when compared to animals of the same genetic strain (n=2). n=1 animal was excluded
from the study due to perfusion complications, and n=1 was excluded from the study due to
accidental lethal saline injection during animal care. Additionally, n=1 animal was excluded due
to both eyes being sunken in, which may have hindered sight and confounded training
interventions. This study had four groups: WT TM (treadmill trained) (n=6), WT NoEx (n=5),
CCL2KO TM (n=5), and CCL2KO NoEx. (n=4). Mice were assigned to groups at 14dpi, just
prior to beginning treadmill training, and were balanced based on their 14d BMS score to ensure
all groups had similar locomotor abilities (WT TM: mean BMS 6 ± 0.365 SEM; WT No Ex:
mean BMS 4.6 ± 0.447 SEM; CCL2KO TM: mean BMS 4.6 ± 0.4 SEM; CCL2KO NoEx: mean
BMS 4.625 ± 0.375 SEM.
Training Intervention
4
We used downhill (DH) treadmill walking as out neurorehabilitation intervention for both
WT and CCL2KO groups. A custom-built treadmill angled at a 6 degree, 10% grade, located in a
quiet room with normal lighting was used for all treadmill training sessions. Training occurred
sub-acutely from 14d-21dpi, at the same time each day (±2 hrs). The bladders of animals were
expressed prior to training. Each training session consisted of two 10min bouts of walking
separated by a 20 min rest break in the home cage. Starting at a speed of 7 M/min at 14d postinjury, speeds were gradually increased each training day based on the following criteria:
increased speed does not worsen stepping, cause undue fatigue to the point that stepping
degraded.
Tissue Collection
At 35dpi, mice were transcardially perfused with 0.1 M phosphate buffered saline (PBS;
pH 7.4) followed by 4% paraformaldehyde (PFA, pH 7.2). The cervical enlargement, T9 lesion
epicenter (1cm), and lumbar (L1-L6) spinal cord of animals were then immediately removed and
kept in PFA for one hour at 4°C, rinsed overnight in 0.2M phosphate buffer (PB; pH7.4), and
kept in 30% sucrose for 3-5d. Spinal cord segments were then embedded in Optimal Cutting
Temperature (OCT) Compound (Tissue-Tek) and stored frozen at -80°F.
Histology
Epicenter and lumbar transverse sections of the spinal cord were cut into 20 μm sections
on a Microm HM505E cryostat. Fluorescent immunohistochemistry (IHC) was used to label the
presence of monocytes and resident microglia, and ICAM in the lumbar segments of the spinal
cord. To examine monocytes/non-resident microglia and resident microglia, a co-stain of 1:400
dilution was used for rat anti-mouse CD45 (Bio Rad Abd Serotec: MCA1388) and a 1:200
dilution was used for rabbit anti IBA-1 (Wako: 019-19741), respectively. The antibody was
prepared in a blocking solution of 1% BSA, 0.1% FG, 10% NGS, 0.2% Tx-100 in PBS.
Incubation of the primary antibody occurred overnight at 4°C. A donkey anti-rabbit secondary
antibody was used at 1:400 dilution (abcam: ab96892). An IBA-1 goat anti-rat antibody was
used at (1:1000) dilution (LifeTech A11006). To identify the presence of ICAM, a 1:250 dilution
was used for goat anti-ICAM (R&D AF796). The antibody was prepared in a blocking solution
of 3% NDS, 1%BSA in PBS, with a washing solution of 1% BSA in PBS. Incubation of the
primary antibody occurred overnight at 4°C. A donkey anti-goat secondary antibody was used at
1:250 (Abcam ab96935).
For all IHC staining, positive control sections were processed by omitting the primary
antibody and substituting with blocking solution to ensure positive labeling. Negative control
sections were also processed by omitting the secondary antibody and replacing with blocking
solution.
Cell Quantification
Tissue sections were imaged using a Nikon Eclipse E800 fluorescence microscope.
Anatomical maps were used to identify intermediate laminae V, VI, and VII. Quantification of
CD45 (+)/IBA-1(-) co-labeling and cell morphology were performed by one rater who was
5
blinded to group assignment. This rater demonstrated strong interrater reliability against an
expert in the lab prior to quantification. Thresholds for positive staining were monitored by the
blinded rater (Basso et al., 2006). A total of six images of lumbar tissue sections per animal were
collected for monocyte counts (24 to 36 per group). The cell counts were averaged per group. To
quantify ICAM labeling, we used ImageJ software to demarcate an area of analysis (726lx272h
um) for the intermediate laminae of the lumbar gray matter where CPG interneurons reside. The
proportional area was calculated as the percent area of positive staining within the designated
boundaries for each section of tissue captured digitally. N=1 animal from the CCL2KO TM
group was excluded from cell quantification of CD45 (+)/IBA-1(-) and proportional area of
ICAM due to perfusion complications that rendered the tissue inadequate for IHC staining.
Kinematic Assessment of Locomotion
Kinematic assessment of gait during walking on a flat treadmill occurred before injury
(pre) and at the end of training (35dpi). Using the Peak Motus Motion Measurement System, 2dimensional analysis of the coordinates of the pelvis, hip, knee, ankle, and toe were collected.
Strict criteria for the acceptance of steps for kinematic assessment included taking at least 5
successive left hind-limb steps on the treadmill at a steady speed, without appearing to lunging
forward or drifting backward. A total of 20 steps of steady state locomotion per animal, collected
in single or multiple bouts, were analyzed. In this study, trunk instability, hip excursion, and toe
dragging were measured to identify an effect of genetic strain or treadmill training on gait. Trunk
instability was determined by the peak pelvis height during each step, which was averaged across
all steps to yield a single trunk instability score per animal. Hip excursion was calculated as the
minimum degree of hip angular motion subtracted from the maximum hip angle for each step.
Toe dragging was measured as the percentage of time that the left-hind toe contacted the ground
during forward limb advancement.
White Matter Sparing (WMS)
The degree of lesion size and white matter spared following SCI is a predictor of
locomotor recovery (Scholtes et al., 2011). Sections of 35dpi lumbar tissue 20um thick were cut
transversely. Using immunohistochemical procedure the tissue was stained with eriochrome
cyanine (EC) to examine the lesion area and white matter spared of the lumbar cord. In addition
to EC, a 5% iron aluminum and borax ferricyanide cocktail was used for differentiation. Light
microscopy measures were used to evaluate the tissue, and tissue images were converted to
computer images (MCID-Elite, Imaging Research, Ontario). Using a Cavalerri estimator, a grid
was positioned over the tissue at random, and each point within the grid was designated as white
matter or grey matter (Fig B). Per each image area, point totals were combined and calculated to
area as follows: estimated area = (ΣP) * (a/p), where ΣP is the total points per section and a/p is
the total area per point. Average white matter spared was compared between groups to identify
differences.
Statistics
Group differences for injury biomechanics (displacement and force), white matter sparing
6
(WMS), histology cell counts, proportional area, and all kinematic measures were compared
using one-way analysis of variance (ANOVA) with a Tukey’s post-hoc analysis. The BMS
scores were analyzed using a two-way repeated measures ANOVA (group x time) with a
Tukey’s post-hoc analysis with the harmonic mean to account for variability in group size. The
relationships between genetic strain or treadmill training and tissue level variables were
determined by the Pearson product moment correlation. The null hypothesis was rejected at the
p≤0.05 level. All statistical analyses utilized the IBM SPSS program, SPSS Inc. Mean and
standard error of the mean (SEM) are shown throughout.
Results
White Matter Sparing Differs between CCL2KO and WT models
All study animals received an impact injury that was within the parameters of acceptable
force and displacement ranges (mean force: 78.33 ± 0.78 SEM; mean displacement: 548.7 ±
35.50 SEM) (Ghasemlou et al., 2005). Despite equal injury biomechanics across groups, there
were significant group differences in the size of the lesion. The WMS had a significant main
effect (p<0.05), with the mean area of each group as follows: (CCL2KO TM mean 2.46 ± 0.2
SEM; CCL2KO NoEx mean 2.78 ± 0.43 SEM; WT TM mean 3.67 ± 0.55 SEM; WT NoEx
mean 3.03 ± 0.48 SEM). Interestingly, the CCL2KO groups (regardless of training condition)
had significantly less white matter spared when compared to the WT groups (p<0.01; Figure 1).
Figure 1. Effect of CCL2 on white
matter sparing. A mouse lacking
CCL2 (1A) recovers differently
than a WT mouse (1B) The
CCL2KO groups had significantly
less WMS when compared to the
WT groups (p<0.05)(1C).
BMS Open-Field Locomotion Reveals Group Difference
We measured the recovery of locomotor function over time to monitor the influence of
7
both CCL2-deficiency and/or sub-acute treadmill training on hind limb motor recovery. This
measure is of value to our experiment because if a significant difference between the motor
performances of certain groups occurs, we are able to identify the time period during which the
groups began to recover differently after injury. At 21 days, the CCL2KO TM (mean BMS 4.9 ±
0.4 SEM) (p<.05) and CCL2KO NoEx (mean BMS 4.25 ± 0.48 SEM) (p<.01) groups performed
significantly worse than the WT TM (mean BMS 7.25 ± 0.31 SEM) and WT NoEx (mean BMS
7.3 ± 0.46 SEM) groups. This significant BMS deficit continued for the CCL2KO NoEx group at
28dpi (mean BMS 4.5 ± 0.29 SEM) (p<.05), and 35dpi (mean BMS 4.38 ± 0.38 SEM) (p<.01),
when compared to both the WT TM (28dpi mean BMS 6.83 ± 0.46 SEM; 35dpi mean BMS 7.17
± 0.31 SEM) and WT NoEx (28dpi mean BMS 7 ± 0.27 SEM; 35dpi BMS 7.2 ± 0.37 SEM)
groups. (Fig2). There was no effect of treadmill training at any time point in WT SCI animals.
BMS
10
9
BMS SCORE + SEM
8
7
6
*
5
CCL2KO TM
CCL2KO no ex
4
3
**
*
**
21D
28D
35D
2
WT TM
WT no ex
1
0
PRE
1D
3D
7D
14D
DPI
Figure 2. WT groups show increased locomotor mobility compared to CCL2KO groups. The
CCL2KO groups displayed lower locomotor abilities at late time points when compared to the
WT groups, with the CCL2KO NoEx group performing worse at 21 (p<0.01), 28 (p<0.05), and
35dpi (p<0.01). The CCL2KO TM group had poor locomotor outcomes at 21dpi (p<0.05).
* = significantly different from WT groups (p<0.05)
** = significantly different from WT groups (p<0.01)
The Role of CCL2 in Cell Trafficking of Monocytes into the Intermediate Lamina
Little is known about the role of CCL2 in myeloid cell trafficking after SCI. Previously,
we showed that the presence of inflammatory cell types, such as bone-marrow derived myeloid
cells, contributes to tissue damage and may therefore impede recovery within the lumbar
locomotor networks after SCI. This is relevant because CCL2 and ICAM in the lumbar cord are
upregulated at 7dpi, along with an increased presence of myeloid cells (10). Hence, the purpose
of this study was to observe cell trafficking in a CCL2-deficient model, in hopes of identifying a
direct causal relationship between CCL2 and cell trafficking levels. The presence of monocytes
in the intermediate lamina of the lumbar cord at 35dpi was identified in all groups (Fig 3B).
8
Figure 3A displays the labeling of cells (CD45+/IBA-1−) by group. There were no significant
differences across groups in the number of monocytes within the intermediate lamina. Neither
genetic strain nor treadmill training altered monocyte trafficking when measured at 35dpi, with
the average number of cells in each group as follows: CCL2KO TM mean 4 ± 1.7 SEM;
CCL2KO NoEx mean 2.75 ± 1.03 SEM; WT TM mean 2.17 ± 0.70 SEM; WT NoEx mean 4 ±
1.05 SEM.
A
B
9
Figure 3. No detected group difference of monocyte cell counts in the lumbar spinal cord. A
double stain of IBA-1 (green) and CD45 (red) was used to quantify the presence of myeloidderived monocytes in the lumbar spinal cord. Scale bars: 10x, 250 um. (3A). Cells labeled with
IBA-1 only were counted as monocytes. Scale bars: 10x, 250um and 60x, 50um. (3B). CD45 (-)
/ IBA-1(+) labeled cell counts did not reveal a significant group difference in the number of
monocytes present in the lumbar spinal cord at 35dpi (3C).
The Relationship between CCL2 and ICAM in Response to Injury.
Cell adhesion molecules (ICAM) assist chemoattractants in the facilitation of cell
trafficking (Furie, 2014). In this study, we assessed ICAM expression in CCL2KO model. The
answer to this question would provide us with mechanistic insight on chemoattractant and cell
adhesion interactions. We hypothesized that both CCL2KO groups, regardless of training, would
have decreased expression of ICAM when compared to the WT groups. Fig4 shows the
proportional area of ICAM in the lumbar intermediate lamina of each group. These data did not
show a significant difference between groups in the amount of ICAM present in the intermediate
laminae of the lumbar cord, with the average ICAM proportional area of each strain as follows:
CCL2KO mean 25.24 ± 0.4 SEM; WT mean 6.71 ± 0.37 SEM.
10
Fig4. No detected relationship
between CCL2 deficiency and the
presence of ICAM: Labeling of
ICAM protein in the lumbar spinal
cord does not reveal significant
differences between presence of
cell adhesion molecules in WT vs
CCL2-deficient models.
Kinematic Analysis of Gait
Utilizing a mathematical approach to analyze gait of walking allows for a thorough
diagnostic of hind limb position, and provides insight into the effect of genetic strain and training
on locomotion. In this study we analyzed the pelvis instability, hip excursions, and toe drags of
each animal at 35dpi. We theorized that the introduction of downhill treadmill training at a subacute time point would encourage locomotor plasticity and recovery in the WT TM and
CCL2KO TM groups, more so than in the NoEx groups. Analysis of the trunk instability reveals
a significant main effect of group (p=0.05), with the CCL2NoEx (mean 2.05 ± 0.09 SEM) and
WT NoEx (mean 2.48 ± 0.09 SEM) groups as the main contrasting groups in the Tukey’s HSD
post-hoc analysis. The analysis showed that significantly greater trunk instability occurred in
CCL2KO NoEx to the WT groups (p<.05) (Figure 5A). A wave graph representative of the most
stable stepper of each group shows that these specific CCL2KO TM and CCL2KO NoEx
animals walk at a lower height than the selected WT TM and WT NoEx animals (Figure 5B).
The hip excursion and toe drag data do not reveal significance between groups, with mean data
for hip excursion from 20.32°-32.6° (CCL2 TM mean 29.92° ± 4.74 SEM; WT TM mean 31.86°
± 4.25 SEM; CCL2KO NoEx mean 20.32° ± 2.61 SEM; WT NoEx mean 32.6° ± 2.63 SEM),
and mean data for time of toe drag between 59.80s and 72.07s (CCL2KO TM mean 69.35 ± 6.24
SEM; WT TM mean 60.12 ± 4.0 SEM; CCL2KO NoEx mean 72.07 ± 8.04 SEM; WT NoEx
mean 59.81 ± 2.74 SEM) (Figure 5C,5D).
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Figure 5. Kinematic Analysis of Gait Reveals Disparities in Pelvis Instability. There was a
significant main effect across all groups when analyzing pelvis stability (p<0.05), with the
CCL2KO NoEx and WT NoEx groups exhibiting the most differences (5A). A representative
wave graph of the most stable animal of each group reveals that these specific CCL2KO TM and
CCL2KO NoEx animals walk at a lower height than the WT TM and WT NoEx groups.
Statistical outputs of the hip flexion (5C) and toe drag (5D) do not reveal significance between
groups.
Behavioral Correlations (𝑅𝑅2 )
BMS
WMS
CD45/IBA
Monocyte
Cell Count
0.037
0.0316
ICAM
Proportional
Area
0.032
0.0082
Pelvis
Instability
Hip
Excursion
% Toe
Drag
0.4198**
0.1369
0.2842*
0.1274
0.5654*
0.2081*
Figure 6. Behavioral Correlations reveal relationships between BMS, WMS, and measured
outcomes. BMS scores are strongly related to the pelvis instability (p<0.01), hip excursion
(p<0.05), and toe drag (p<0.05) outcomes. The WMS data has a relationship with the toe drag
outputs (p<0.05).
(* = p<0.05) (** = p<0.01)
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Discussion
Open Field Locomotion and White Matter Sparing
Open-field BMS scores show an insignificant difference between groups at 14dpi. Thus,
we are confident that our group assignment of TM vs NoEx was not behaviorally skewed prior to
training, which began at 14dpi. The emergence of significant findings in locomotor performance
between CCL2KOTM (p<.05), and CCL2KO NoEx (p<.01), when compared to both the WT
TM and NoEx groups at 21dpi. In the CCL2KO NoEx model, the continuation of this deficit in
training continues out to 28d (p<.05), and 35dpi (p<0.01). The disparities that surface at later
time points were not a result of training, but may be influenced by genetic strain. Some
researchers suggest that increased levels of infiltrating monocytes play an anti-inflammatory role
when compared to resident microglia, which would support the idea that the microenvironment
of the cord may need CCL2 chemoattractants during subacute immune response for exercise to
promote functional recovery (Shechter et al., 2009). The lack of sub-acute training effect on
locomotor recovery in a CCL2KO contrasts with the improved locomotor recovery that results
from acute (2-9dpi) training intervention in an MMP9KO model (10). It is possible that the
subacute training intervention in this study was too late, and unable to produce improved
recovery rates because it could not utilize a CCL2-mediated inflammatory response. Hansen et.
al. also reported an increase in the WMS of MMP9KO groups. The significantly less white
matter spared in the CCL2KO groups at 35dpi correlated with BMS scores (p<0.05, r = 0.752)
and provide possible reasoning for the lower BMS scores of the CCL2KO groups. Compared to a
CCL2-deficient model, microenvironments lacking MMP9 salvage more white matter at the
epicenter and may be more responsive to rehabilitative interventions.
Training Intervention: Kinematics Outcomes
We use a mathematical approach to assess the kinematics of mouse locomotion because it
is a sensitive and reliable measure to supplement the open field BMS scores. Kinematics tell us
the precise coordinates of the leg joints as the animal walks, and BMS scores track the hind limb
paw position, coordination, and trunk instability. Although these two behavioral outcomes
measure different aspects of hind limb function, we expected kinematics and BMS outcomes to
reveal similar patterns when assessing the effect of genetic strain and training intervention on
rehabilitative recovery. It is therefore paradoxical that the BMS scores revealed marked deficits
in gross locomotor function with CCL2KO but kinematics failed to show differences induced by
treadmill training. This causes us to examine differences between these two tests that may
partially explain the locomotor outcomes. First, kinematic data were collected on the treadmill
where the direction and speed of locomotion were carefully controlled. In the open field,
locomotion is multidirectional at highly variable speeds so the range of locomotor behavior is
greater in the open field. Second, the 20 total steps selected for kinematic analysis were
reflective of “best stepping” (Basso et al., 2006). Plantar steps, defined as initial contact of
bottom of the foot with the floor, were prioritized over dorsal steps whereby the top of the foot
contacts the floor. Perhaps best stepping is comparable across our groups but worst stepping may
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be a better indicator of genetic and treadmill training effects. BMS and kinematics do not analyze
the same steps, which may lead to discrepancies when comparing behavioral outcomes. In an
ideal design, the kinematics of stepping for each animal will analyze steps that are representative
of the same steps assessed during BMS, but this is currently impossible.
Detection of Cell Trafficking
A main objective of the study was to examine the effects of training on cell trafficking in
response in injury. After thoracic SCI, we detected CD45+ bone-marrow derived monocytes in
the lumbar cord where CPG interneurons are located. This means that the infiltration of cells
during immune response has potential to hinder or facilitate the activation of these neural
networks that initiate movement. We can confirm that the removal of CCL2 results in worse
subacute functional outcomes. However, we did not see significant differences due to treadmill
training. The removal of CCL2 may render the inflammatory environment refractory to CPG
regeneration.
We must also consider the efficiency of our chosen staining antibodies at the late time
point. Monocytes migrate into the spinal cord after injury and, over time, begin to express
microglial proteins (Hansen et al., 2016; Strauss-Ayali et al., 2007). Additionally, CD45
expression is down regulated after infiltrating into the CNS. These two conditions make it
difficult to differentiate bone-marrow derived monocytes from resident microglia at later time
points. It is possible that we underestimated CD45+ bone-marrow derived monocytes by waiting
until 35dpi to collect tissue samples. Our next approach to quantifying monocytes in the spinal
cord will be to use a p2ry12 (-)/IBA-1(+) IHC stain. P2ry12 is an antibody that will only label
resident microglia (Butovsky et al., 2014). Combined with IBA-1, which labels all cells
expressing microglial IBA protein, this double stain will allow us to count the number of
p2ry12(-)/IBA-1(+) labeled-cells as monocytes that migrated into the cord during immune
response, and may be able to better quantify monocytes expressing microglial proteins at late
time points.
Compensatory Reactions after Injury
The CCL2 chemoattractant belongs to a family of key chemokines that facilitate
chemotaxis of immunocompetent cells into the central nervous system (Popovich & Hickey,
2001). It is a ligand that, when bound to the receptor (CCR2), allows for the conformational
change of the receptor transmembrane into a functional site of cell transport (Zheng et al., 2016).
Studies of experimental autoimmune encephalomyelitis (EAE), a demyelinating disease
characterized by aggressive onset of inflammation, using CCR2-deficient mice provide evidence
that CCR2 is a strong mediator of monocyte trafficking in the early stages of EAE (Gaupp et al.,
2003). This same study also reports that in mouse model lacking CCR2, late-onset of EAE still
occurred. In the context of our CCL2KO model, it is possible that the deletion of CCL2 is
sufficient to delay the trafficking of inflammatory cell types at early time points, but may be
more adaptive to change at a late 35dpi time point.
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On a broader scope, CCL2 is just one of several chemokines with an affinity for
monocytes (Zlotnik & Yoshie, 2000). Various studies have shown compensatory inflammatory
responses in conditional knock-out models, essentially an upregulation of one cell type in the
absence of another (Mietto, Mostacada, & Martinez, 2015; Stirling, Liu, Kubes, & Yong, 2009).
Neutrophils are deleterious white-blood cells that are recruited into the lesion epicenter within
hours of SCI (Hansen et al., 2016; Neirinckx et al., 2014). The deletion of CCL2 may cause the
upregulation of neutrophils, or other compensatory cell types, that create a greater lesion and
greater behavioral deficits. The late-onset behavioral deficits seen in the CCL2KO models may
also indicate that there is a delay in inflammatory cell infiltration, which peaks at subacute time
points and interferes with locomotor recovery.
Conclusion
Appropriate rehabilitation interventions are able to aid in creating a more permissible
microenvironment for locomotor recovery after SCI. The characterization of cell trafficking
during immune response will provide greater insight into the components that influence motor
outcomes. We hypothesized that the removal of CCL2 will prevent the trafficking of myeloid
cells into the lumbar spinal cord after injury, allowing for a more favorable microenvironment
for subacute rehabilitation interventions when compared to WT SCI. Results show that subacute
training interventions did not have a significant effect on locomotor outcomes. The CCL2KO
groups displayed lower locomotor abilities at late time points when compared to the WT groups,
with the CCL2KO NoEx group performing worse at 21 (p<0.01), 28 (p<0.05), and 35dpi
(p<0.01). The CCL2KO TM group had poor locomotor outcomes at 21dpi (p<0.05). The deletion
of CCL2 resulted in less white matter spared when compared to WT groups (p<0.05). Due to
insufficient measures, we cannot confirm the effect of CCL2 deficiencies on the trafficking of
blood-derived monocytes into the lumbar spinal cord after injury. The outcomes of this study
provide argument for CCL2 as a necessary component for functional recovery and rehabilitation
after injury.
15
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