Neutrophil elastase inhibition effectively rescued angiopoietin-1 decrease and inhibits glial scar after spinal cord injury

Abstract

After spinal cord injury (SCI), neutrophil elastase (NE) released at injury site disrupts vascular endothelium integrity and stabilization. Angiopoietins (ANGPTs) are vascular growth factors that play an important role in vascular stabilization. We hypothesized that neutrophil elastase is one of the key determinants of vascular endothelium disruption/destabilization and affects angiopoietins expression after spinal cord injury. To test this, tubule formation and angiopoietins expression were assessed in endothelial cells exposed to different concentrations of recombinant neutropil elastase. Then, the expression of angiopoietin-1, angiopoietin-2, and neutrophil elastase was determined at 3 h and at 1, 3, 5, 7, 14, 21, and 28 days in a clinically relevant model of moderate compression (35 g for 5 min at T10) spinal cord injury. A dichotomy between the levels of angiopoietin-1 and angiopoietin-2 was observed; thus, we utilized a specific neutrophil elastase inhibitor (sivelestat sodium; 30 mg/kg, i.p., b.i.d.) after spinal cord injury. The expression levels of neutropil elastase and angiopoietin-2 increased, and that of angiopoietin-1 decreased after spinal cord injury in rats. The sivelestat regimen, optimized via a pharmacokinetics study, had potent effects on vascular stabilization by upregulating angiopoietin-1 via the AKT pathway and preventing tight junction protein degradation. Moreover, sivelestat attenuated the levels of inflammatory cytokines and chemokines after spinal cord injury and hence subsequently alleviated secondary damage observed as a reduction in glial scar formation and the promotion of blood vessel formation and stabilization. As a result, hindlimb locomotor function significantly recovered in the sivelestat-treated animals as determined by the Basso, Beattie, and Bresnahan scale and footprint analyses. Furthermore, sivelestat treatment attenuated neuropathic pain as assessed by responses to von Frey filaments after spinal cord injury. Thus, our result suggests that inhibiting neutropil elastase by administration of sivelestat is a promising therapeutic strategy to inhibit glial scar and promote functional recovery by upregulating angiopoietin-1 after spinal cord injury.

Electronic supplementary material
The online version of this article (10.1186/s40478-018-0576-3) contains supplementary material, which is available to authorized users.

Full text

R E S E A R C H Open Access
Neutrophil elastase inhibition effectively
rescued angiopoietin-1 decrease and
inhibits glial scar after spinal cord injury
Hemant Kumar
1
, Hyemin Choi
1
, Min-Jae Jo
1
, Hari Prasad Joshi
1
, Manjunatha Muttigi
2
, Dario Bonanomi
3
,
Sung Bum Kim
4
, Eunmi Ban
5
, Aeri Kim
5
, Soo-Hong Lee
2
, Kyoung-Tae Kim
6,7
, Seil Sohn
1
, Xiang Zeng
8*
and Inbo Han
1*
Abstract
After spinal cord injury (SCI), neutrophil elastase (NE) released at injury site disrupts vascular endothelium integrity
and stabilization. Angiopoietins (ANGPTs) are vascular growth factors that play an important role in vascular
stabilization. We hypothesized that neutrophil elastase is one of the key determinants of vascular endothelium
disruption/destabilization and affects angiopoietins expression after spinal cord injury. To test this, tubule formation
and angiopoietins expression were assessed in endothelial cells exposed to different concentrations of recombinant
neutropil elastase. Then, the expression of angiopoietin-1, angiopoietin-2, and neutrophil elastase was determined
at 3 h and at 1, 3, 5, 7, 14, 21, and 28 days in a clinically relevant model of moderate compression (35 g for 5 min
at T10) spinal cord injury. A dichotomy between the levels of angiopoietin-1 and angiopoietin-2 was observed;
thus, we utilized a specific neutrophil elastase inhibitor (sivelestat sodium; 30 mg/kg, i.p., b.i.d.) after spinal cord
injury. The expression levels of neutropil elastase and angiopoietin-2 increased, and that of angiopoietin-
1 decreased after spinal cord injury in rats. The sivelestat regimen, optimized via a pharmacokinetics study, had
potent effects on vascular stabilization by upregulating angiopoietin-1 via the AKT pathway and preventing tight
junction protein degradation. Moreover, sivelestat attenuated the levels of inflammatory cytokines and chemokines
after spinal cord injury and hence subsequently alleviated secondary damage observed as a reduction in glial scar
formation and the promotion of blood vessel formation and stabilization. As a result, hindlimb locomotor function
significantly recovered in the sivelestat-treated animals as determined by the Basso, Beattie, and Bresnahan scale
and footprint analyses. Furthermore, sivelestat treatment attenuated neuropathic pain as assessed by responses to
von Frey filaments after spinal cord injury. Thus, our result suggests that inhibiting neutropil elastase by
administration of sivelestat is a promising therapeutic strategy to inhibit glial scar and promote functional recovery
by upregulating angiopoietin-1 after spinal cord injury.
Keywords: Neutrophil elastase, Spinal cord injury, Glial scar, Angiopoietins, Functional recovery, Neuropathic pain
Introduction
Spinal cord injury (SCI) is a clinically devastating condi-
tion that can cause either temporary or permanent disabil-
ity in young adults [49,76]. SCI pathology is multifaceted.
It involves several major biological cascades [48,54,55]
and results in expeditious and enduring changes to the
structure and function of microvessels [27,56,85], such
as a loss of structural organization and microcirculation, a
disruption of the blood-spinal cord barrier (BSCB), and
endothelial cell (EC) and vascular remodelling [49,85].
Vascular damage following SCI augments secondary
damage, and vascular protection or the maintenance of
vascular integrity mitigates this damage [30,35,49]. ECs
participate in all facets of vascular homeostasis and play a
variety of critical roles in the control of vascular functions,
including in thrombosis, inflammation, and vascular wall
* Correspondence: zengx33@mail.sysu.edu.cn;hanib@cha.ac.kr
8
Department of Histology and Embryology, Zhongshan School of Medicine,
Sun Yat-sen University, Guangzhou 510080, Guangdong Province, China
1
Department of Neurosurgery, CHA University School of Medicine, CHA
Bundang Medical Center, Seongnam-si, Gyeonggi-do 13496, Republic of
Korea
Full list of author information is available at the end of the article
© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Kumar et al. Acta Neuropathologica Communications (2018) 6:73
https://doi.org/10.1186/s40478-018-0576-3
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

remodeling. The death of ECs disengages the vascular net-
work, and ischemia results in apoptosis and cell death of
central nervous system (CNS) cells due to the lack of
blood supply [23,54,55].
Recovery from SCI is preceded by angiogenesis, the extent
of which correlates with neural regeneration, suggesting that
angiogenesis may play a significant role in repair. Angiopoie-
tins (ANGPTs) are vascular growth factors involved in blood
vessel formation and maturation, as well as in EC survival
[31,86], and are critical regulators of vascular functions in
the brain [46,98]andspinalcord[30,35,74]. There are four
members of the ANGPT family: ANGPT-1, −2, and −4are
expressed in humans, and ANGPT-3, an ortholog of
ANGPT-4, is expressed in mice [31,92]. ANGPT-1 and
ANGPT-2 are released from ECs, with ANGPT-1 constitu-
tively expressed in normal CNS vasculature [61]and
ANGPT-2 weakly expressed under homeostatic conditions
and increased during hypoxia, inflammation, and vascular
remodeling [86]. ANGPT-1 exerts anti-inflammatory effects,
reduces vessel permeability, and protects against plasma
leakage in the adult vasculature [28,87]. These effects are
opposed by the actions of ANGPT-2. Therefore, it is not
surprising that ANGPT-2 can antagonize the benefits to vas-
cular integrity from endogenous or exogenous ANGPT-1
after SCI.
Neuropathological changes in spinal cord tissue result
from acute inflammatory reactions that can involve
elastases derived from neutrophils. Neutrophils also play a
critical role in the initial events in demyelinating neuroin-
flammatory diseases and are intimately linked with the sta-
tus of the blood-brain barrier/BSCB [7]. For example,
neutrophils release a destructive proteolytic enzyme called
neutrophil elastase (NE) [44]. At the vascular interface, NE
induces cellular damage and dysfunction, degradation of
the extracellular matrix, and pathways leading to cell death
[44]. In response to proinflammatory stimuli, NE regulates
the adhesion of leukocytes, clears their path for diapedesis/
transmigration [62,83,97], and mediates the degradation
of endothelial junction proteins [34,39]. Furthermore, NE
can induce apoptosis of ECs [96] and has a broad substrate
specificity [69,89]. These complex secondary pathome-
chanisms are responsible for extending spinal cord damage
into previously uncompromised segments [66,67,73]. NE
can induce vascular damage leading to spinal cord ischemia
[84] and is also a determinant of long-term functional re-
covery after traumatic brain injury [81].
We hypothesized that NE might be a key determinant
for the disruption/destabilization of the vascular endo-
thelium and alter ANGPT expression after SCI. To test
this, we utilized a selective NE inhibitor (sivelestat
sodium; 30 mg/kg, i.p.,b.i.d.) in a rat model of moderate
compression (35 g for 5 min at T10) SCI. Sivelestat
attenuates NE-induced pathologies and is approved for
use in patients with acute lung injury in Japan and the
Republic of Korea [5,90], and attenuates the periopera-
tive inflammatory response in pediatric patients under-
going cardiopulmonary bypass surgery [38]. Moreover,
administration of sivelestat attenuated the ischemia [41],
and the chemo-attractant mRNA and protein [88]inan
experimental model of SCI. However, the effect of NE
inhibition on the glial scar, secondary damage, vascular
stabilization, ANGPTs, ECs survival and angiogenesis
after SCI remains to be determined. In the current
study, we ascertain the role of NE with ANGPTs after
SCI and suggest that NE inhibition endows multidimen-
sional therapeutic strategy in tissue protection and glial
scar inhibition in treating SCI.
Material and methods
Cell culture and treatment
In an attempt to understand the biological role of NE in
ECs, we used HUVEC (ATCC) cells. HUVECs were cul-
tured in fully supplemented endothelial growth medium
as per the manufacturer instructions. Recombinant hu-
man NE protein (R&D Systems, Minneapolis, USA) was
activated with 50 μg/ml Cathepsin C in assay buffer be-
fore use as per manufacturer instruction and was used at
a functional concentration of 100 ng/ml, 250 ng/ml and
500 ng/ml and 1000 ng/ml, in ECs. Corning matrigel
matrix was used for the tubule formation assay as per
the manufacturer recommendations. Briefly, matrigel
matrix was polymerized at 37 °C in a 24 well plate and
HUVEC cells (passage 3) at a seeding density of 1.2 × 10
5
. The EGM-2 bullet kit medium were supplemented
with human NE at a concentration of 100 ng/ml (group
2), 250 ng/ml (group 3), 500 ng/ml (group 4), and
1000 ng/ml (group 5). HUVEC supplemented with the
only medium served as control (group 1). After 18 h,
capillary-like tubules was stained with calcein AM fluor-
escent dye on the matrilgel. Images were randomly ac-
quired using Cytation 3 Cell Imaging Multi-Mode
Reader (Biotek Instruments,Inc., Winooski, VT, USA).
Subjects and surgical procedures
Total 146 adult female Sprague-Dawley (SD) rats were
used in the study. Rats (220–240 g) for this study were
purchased from Orient Bio Inc. (Seongnam, Korea),
housed in a facility at 55–65% humidity and controlled
temperature of 24 ± 3 °C with light / dark cycle of 12 h,
and had free access to food and water. All animal proce-
dures were performed according to the approved proto-
col by the Institutional Animal Care and Use Committee
(IACUC) of CHA University (IACUC160076) and Principles
of laboratory animal care [63]. The animals were anesthe-
tized with Zoletil® (50 mg/kg, Virbac Laboratories, France) /
Rompun® (10 mg/kg, Bayer, Korea) solution administered in-
traperitoneally. Complete anesthesia was assessed using
hindlimb withdrawal in response to a noxious foot pinch.
Kumar et al. Acta Neuropathologica Communications (2018) 6:73 Page 2 of 18
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

After skin preparation and precise positioning of anesthe-
tized rats, a laminectomy was performed to expose T10
spinal cord. The vertebral column was supported and stabi-
lized by Allis clamps at T8 and T12 spinous processes as de-
scribed previously [48,75]. A metal impounder (35 g ×
5 min) was then gently applied on T10 dura, resulting in
moderate standing weight compression. Following compres-
sion injury, the surgical site was closed by suturing the
muscle and fascia and suturing the skin; followed by external
povidone-iodine application. Animals were kept on a heat-
ing pad to maintain body temperature, and then 5 mL of
0.9% sterile saline injected subcutaneously. Manual bladder
expression of urine was performed twice daily until a
bladder reflex was established.
Drugs and treatments
Sivelestat sodium (Dong-A-pharma, Seoul, Korea) was
dissolved in distilled water and administered twice daily
intraperitoneally at the dose of 30 mg/kg 1 h after SCI;
Animals were sacrificed at different time point’s day
post-injury (DPI) after vehicle or sivelestat treatment.
The total number of injection(s) varied based on the effi-
cacy parameter and time points. DPI-1 (2 injections of
sivelestat were given), DPI-7 (14 injections of sivelestat
were given), DPI-14 (28 injections of sivelestat were
given), DPI-28 (28 injections of sivelestat were given till
day 14 and animals were observed till day 28 for behav-
ioral studies and efficacy experiments).
Pharmacokinetic study
We used SCI-injured animals for a pharmacokinetic
study to correlate PK-PD. Sivelestat was treated at 1 h
after SCI, The blood, brain and spinal cord samples were
collected at 15 min, 30 min, 60 min, 90 min, 120 min,
150 min, 180 min after a single dose of sivelestat. For
1D, 7D, and 14 D samples, the blood, brain and spinal
cord were collected after two, 14 and 28 doses of sivele-
stat respectively. Plasma was separated from blood using
centrifugation and stored at −80 °C until analysis. Spinal
cord tissues were collected and washed with PBS and
homogenized in PBS. Analysis of sivelestat in samples
were performed as described in the Additional file 1.
Behavioral assessment
Footprint analysis
Gait behavior and motor coordination were evaluated on
1, 7, 14, 21 and 28 days following injury, and after sivele-
stat treatment using the manual method as described
previously with some modifications [80]. Right fore and
hind paws, left fore and hind limb was painted with dyes
of different colors and animals were placed over an ab-
sorbent paper surrounded by cage border. The animals
were encouraged to walk in a straight line by putting a
clue at the finish line. The footprint pattern was then
digitalized and representative pictures were shown to as-
sess coordination.
Hindlimb locomotor score
Hindlimb motor function was evaluated using the
open-field Basso, Beattie, and Bresnahan (BBB) [10]loco-
motor test on 1, 7, 14, 21 and 28 days following injury,
and after sivelestat treatment. The animals hindlimb loco-
motor score were evaluated by two experienced investiga-
tors who were blinded to treatment group.
Test for nociception
Nociception was checked using Von Frey filaments (Bioseb)
as per the reported method [17]. Animals were acclimatized
in rat chambers (Ugo Basile), and filaments probing were
done when the animals were calm and not moving. The
simplifiedup-downmethod(startedwith2g)wasusedto
determine the mechanosensitivity/paw withdrawal thresh-
old (PWT) with Von Frey filaments on 1, 7, 14, 21 and
28 days following injury, and after sivelestat treatment. A
positive response includes flinching, licking, vocalization, or
overt behavioral cue corresponding to discomfort. A total
of five stimuli per test were recorded and average of three
readings (lowest and highest removed) was used to deter-
mine the average PWT in Sham and after injury or treat-
ment with sivelestat.
qRT-PCR
Quantitative real-time PCR was carried out using an
SYBR Green Master Mix and the mRNA detection was
analyzed using an ABI StepOne Real-time PCR System
(Applied Biosystems, Foster City, CA, USA). Primer se-
quences for the genes of interest and the reference gene
18S or GAPDH were as given in Additional file 2:TableS1:
Typical profile times were the initial step, 95 °C for 10 min
followed by a second phase at 95 °C for 15 s and 60 °C for
30 s for 40 cycles with a melting curve analysis. The target
mRNA level was normalized with the level of the 18S or
GAPDH and compared with the control. Data were ana-
lyzed using the ΔΔCT method.
Western blot analysis
Spinal cord tissues were collected and washed with PBS,
placed at 4
∘
C, and homogenized using T 25 digital
homogenizer (IKA, Seoul, Korea) in lysis buffer (1× RIPA
lysis buffer) and then finally passed through a 31
1/2
gauge
syringe needle and centrifuged at 14,000 rpm at 4
∘
Cfor
15 min. Protein concentration was determined in superna-
tants using Bio-Rad DC Protein Assay (Hercules, CA,
USA). Equal amounts (40 μg) of protein were separated
electrophoretically by 10% SDS-PAGE electrophoresis,
and the resolved proteins were transferred to PVDF mem-
branes (Millipore, Bedford, MA, USA). The membranes
were incubated for 1 h with 5% non-fat skim milk
Kumar et al. Acta Neuropathologica Communications (2018) 6:73 Page 3 of 18
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

prepared in TBS buffer to block nonspecific binding. The
membranes were then incubated overnight with primary
antibodies to ANGPT-1 (1:10000, Abcam, Cambridge,
UK), ANGPT-2 (1:1000, Abcam), NE (1;1000), ZO-1
(1:500, Invitrogen, California, USA), AKT (1:1000, Cell
Signaling Technology, Danvers, MA, USA), pAKT
(1:1000, Cell Signaling Technology,) LC3B (1:1000, Cell
Signaling Technology,), and Actin (1:10000, ABM). After
1-h incubation with corresponding secondary antibodies,
The blots were visualized with a PowerOpti-ECL (Animal
Genetics Inc., Gainesville, FL, USA) detection system, ac-
cording to the recommended procedure. Immunoreactivity
was detected using the BIORAD ChemiDoc™XRS.
Immunohistochemistry and immunofluorescence
At 1, 7, 14 and 28 days after compression of the spinal cord
at T10, animals were anesthetized with mixture of Zoletil®
(50 mg/kg, Virbac Laboratories, France) / Rompun®
(10 mg/kg, Bayer, Korea) solution administered intraperito-
neally and perfused with 0.9% saline followed by 4% para-
formaldehyde for tissue fixation. The spinal cord at the
compression site was removed, and immersed in 4% para-
formaldehyde for 1 day, and then embedded in paraffin,
sectioned at 5 or 10 μm, dewaxed, and stained with anti-
bodies against ANGPT-1 (1:500, Abcam, Cambridge, UK),
ANGPT-2 (1:200, Abcam), N.E. (1;50, Abcam), GFAP
(1:1000, Abcam), GFAP (1:200, Sigma), Iba-1 (1:200,
Abcam) ZO-1 (1:50, Invitrogen, California, USA), Occludin
(1:50, Invitrogen), RECA (1:100, Abcam), TGF-β1 (1:100,
Abcam), NG-2 (1:500, Millipore), α-SMA (1:500, Abcam)-
PECAM(1:100, Abcam), Caspase-3 (1:50, Abcam), CD68
(1:200, Abcam), Arginase (1:100, Abcam), Laminin (1:50,
Abcam), BDNF (1:200, Alomone Lab), NT-3 (1:200, Alo-
mone Lab), NT-4 (1:200, Alomone Lab), vWF (1:50,
Abcam), NF-200 (1:1000, Abcam), Tuj-1 (1:200, Abcam),
VEGF (1:100, Abcam) TNF-α(1:50, Abcam), Fibronectin
(1:50, Abcam), RECA-1 (1:100, Abcam), IL-6 (1:50,
Abcam). Secondary antibodies (1:200 or 1:500) were goat
anti-rabbit Alexa Fluor
®
488 (Abcam), goat anti-rabbit
Alexa Fluor
®
647 (Abcam), goat anti-rabbit Alexa Fluor
®
568
(Abcam), goat anti-rabbit Alexa Fluor
®
555 (Abcam), goat
anti-mouse Alexa Fluor
®
488 (Abcam), goat anti-mouse
Alexa Fluor
®
568 (Abcam), chicken-anti-goat 647 (Invitro-
gen).Following washing after secondary antibody incuba-
tion DAPI (1:500) was incubated for 10 min. Sections were
mounted and examined using a fluorescence microscope
(Zeiss, Oberkochen, Germany or Leica, Germany).
Statistical analysis
All data were analyzed using Graph Pad Prism ver. 5.01
(Graph Pad, Inc., La Jolla, CA, USA). All data are
expressed as mean ± SEM. One-way ANOVA was per-
formed and Dunnett’s posthoc test was used to analyze
the in-vitro data. The in-vivo PCR data was analyzed using
One-way ANOVA followed by Tukey’stest.TheBBB
scores were analyzed statistically with Kruskal-Wallis test
at each time point. The nociception data were analyzed
using Two-way ANOVA followed by Bonferroni test.
P-values < 0.05 were considered statistically significant.
Results
NE suppresses capillary-like tubule formation and ANGPT
expression in ECs, whereas proinflammatory factors
differentially modulate ANGPT expression
We used various strategies to elucidate the effects of NE
and inflammation on ANGPT expression in ECs. First, we
determined the effects of recombinant human NE protein
(100, 250, 500, and 1000 ng/ml) on human umbilical vein
ECs (HUVECs); HUVECs treated with medium only served
as the control. The results of a tubule formation assay
showed a dose-dependent decrease in the tubule-covered
area, total tube length, and total numbers of tubes, with sig-
nificant effects observed at NE concentrations of 500 ng/ml
and 1000 ng/ml (Fig. 1a–d).The expression of ANGPT-1
and ANGPT-2in HUVECs treated with NE for 24 h was
determined by RT-PCR and immunocytochemistry. The
expression of ANGPT-1 was dose-dependently decreased
following the addition of NE (Fig. 1e and f), and ANGPT-2
expression decreased at higher doses (500 ng/ml and
1000 ng/ml) (Fig. 1g and h). Additionally, the effect of in-
flammation on ANGPT expression was assessed by treating
HUVECs with lipopolysaccharide ([LPS] 2 μg/ml) and
tumornecrosisfactoralpha([TNF-α] 100 ng/ml).
ANGPT-1 expression increased at 0.5 and 3 h and
decreased at 6, 9, and 12 h after the addition of LPS to the
medium (Fig. 1i), whereas expression was significantly
decreased by TNF-αat 3–12 h (Fig. 1j). By contrast,
ANGPT-2 expression increased at 3, 6, and 9 h after the
addition of LPS (Fig. 1k) and increased at 3 and 9 h after
TNF-αwas added (Fig. 1l). These results suggest that NE
and inflammation differentially modulate the expression of
ANGPTs in ECs.
SCI disrupts vascular endothelial integrity and alters NE
and ANGPT expression
We characterized the time course of NE, ANGPT-1, and
ANGPT-2 mRNA and protein expression at the epicen-
ter of the damaged spinal cords in rats at 3 h and 1, 3, 5,
7, 14, 21, and 28 days after moderate compression injury
(35 g for 5 min) (Fig. 2a).There was a significant increase in
NE expression from 3 h to 5 days after SCI, with maximum
expression observed at 1 day after SCI (Fig. 2b and ei). A
dichotomy was observed between ANGPT-1 and ANGPT-2
expression patterns. The expression of ANGPT-1 was ini-
tially drastically reduced (the maximum decrease was ob-
served 1 day after SCI when NE expression was maximal)
and then increased at 7 days after SCI and remained ele-
vated (Fig. 2c and eii), whereas the expression of ANGPT-2
Kumar et al. Acta Neuropathologica Communications (2018) 6:73 Page 4 of 18
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Fig. 1 Neutrophil elastase (NE) impedes tubule formation and decreases angiopoietin (ANGPT) expression, whereas inflammatory factors
differentially modulate ANGPT expression, in human umbilical vein endothelial cells (HUVECs). A tubule formation assay was performed as
described in the Methods section. Recombinant human NE was added at concentrations of 100, 250, 500, and 1000 ng/ml (HUVECs exposed only
to medium served as the control) to determine tubule formation (a), the percentage of covered area (b), total tube length (c), and total numbers
of tubes (d). eand gTotal RNA was prepared from HUVECs exposed to various concentrations of NE for 24 h to determine the expression of
ANGPT1 and ANGPT2.fand hANGPT-1 and ANGPT-2 immunocytochemistry was performed on fixed HUVECs as described in the Methods
sections. i–lANGPT1 and ANGPT2 mRNA expression was determined by real-time quantitative reverse transcription-polymerase chain reaction in
HUVECs collected 0.5, 1, 3, 6, 9, and 12 h after treatment with lipopolysaccharide ([LPS] 2 μg/ml) and tumor necrosis factor alpha ([TNF-α] 100 ng/ml).
18S was used as the internal control. Data represent means ± SEMs (n=2–3/group performed in triplicate). *p<0.05, **p< 0.01, ***p< 0.001 vs. control
Kumar et al. Acta Neuropathologica Communications (2018) 6:73 Page 5 of 18
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

continuously increased through 5 days after SCI and unex-
pectedly decreased at 7 days, when the ANGPT-1 expres-
sion was increased, and consequently returned to normal
(Fig. 2d and eiii). As ANGPTs are expressed primarily by
ECs, we determined the integrity of the vascular endothe-
lium using immunohistochemistry (IHC) with rat EC anti-
gen ([RECA-1] Fig. 2eii), which revealed progressive damage
after SCI. RECA-1-stained vessels were readily identified
within the injured spinal cord.
Sivelestat increases ANGPT-1 and decreases ANGPT-2 and
NE expression after SCI
The data above indicated that peak expression of NE
occurred 1 day after SCI, accompanied by increased
ANGPT-2 and decreased ANGPT-1 expression. There-
fore, we determined the effect of inhibiting NE on
ANGPT expression at DPI-1. One group of animals was
treated with sivelestat (30 mg/kg, i.p., b.i.d.), a specific
inhibitor of NE, and the concentrations in plasma, brain,
and spinal cord were monitored over 14 days (Fig. 3a).
Samples from sham, injured untreated, and injured
sivelestat-treated (two doses) animals were prepared on
DPI-1. Interestingly, sivelestat treatment prevented the
SCI-induced decrease in ANGPT-1 expression (Fig. 3b–d)
and attenuated the SCI-induced increase in ANGPT-2
at DPI-1 (Fig. 3e and f). Treatment with sivelestat
also significantly reduced NE expression at DPI-1
(Fig. 3e and g). Additionally, the phosphorylation of
Fig. 2 Neutrophil elastase (NE) and angiopoietin-2 (ANGPT-2) expression increases and angiopoietin-1 (ANGPT-1) and rat endothelial cell antigen
(RECA-1) expression decreases after spinal cord injury (SCI) at the epicenter of the damage. aSchematic showing SCI method. Total RNA was
prepared from spinal cord tissues at the epicenter of the damage collected 3 h and 1, 3, 5, 7, 14, 21, and 28 days after SCI to determine the
expression of NE (b), ANGPT-1 (c), and ANGPT-2 (d). eRepresentative images of immunohistochemistry performed on longitudinal sections for NE
(i), ANGPT-1 and RECA-1(ii), and ANGPT-2 (iii) at different time points after SCI [3 fields/slide, n=2–3/group (sham = 2, and injury = 3)]. GAPDH was
used as internal controls for real-time quantitative reverse transcription–polymerase chain reaction. Data represent means ± S.E.M. [n=2–3/group
(sham = 2, and injury = 3) performed in triplicates]. *p < 0.05, **p< 0.01, ***p< 0.001 compared with Sham group
Kumar et al. Acta Neuropathologica Communications (2018) 6:73 Page 6 of 18
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Fig. 3 (See legend on next page.)
Kumar et al. Acta Neuropathologica Communications (2018) 6:73 Page 7 of 18
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

AKT (p-AKT) was reduced in the spinal cord follow-
ing SCI, which was prevented by treatment with sive-
lestat (Fig. 3c).
NE inhibition attenuates the expression of inflammatory
cytokines and chemokines after SCI
We and others have shown that the peak expression of in-
flammatory markers (cytokines and chemokines) occurs in
the acute phase of SCI [48]. Therefore, we determined the
effect of NE inhibition on inflammatory parameters using
spinal cord samples from sham, injured untreated (Injury),
and injured sivelestat-treated (two doses) animals prepared
on DPI-1. Sivelestat treatment significantly attenuated the
SCI-induced expression of TNF-α(Fig. 4a and c)andinter-
leukin (IL)-6 (Fig. 4b and d).Similarly, the induction of the
inflammatory mediators inducible nitric oxide synthase
(iNOS) and IL-1βwas also significantly decreased with sive-
lestat (Fig. 4e and f, respectively). Interestingly, sivelestat re-
versed the suppression of the anti-inflammatory cytokine
IL-10 (Fig. 4g) and significantly reduced the SCI induction
of C-C motif chemokine ligand (CCL)-2 and CCL-3
(Fig. 4h and i, respectively) and TGF-β(Fig. 4j).
NE inhibition attenuates TJ disruption and blood-spinal
cord permeability after SCI
The disruption of the BSCB and subsequent blood infil-
tration after SCI initiates a secondary injury cascade via
the production of inflammatory mediators, such as IL-6,
TNF-α, and iNOS. To establish the role of NE in BSCB
disruption, we examined samples from sham, injured
untreated, and injured sivelestat-treated (two doses) ani-
mals prepared DPI-1. NE inhibition via sivelestat admin-
istration significantly inhibited the loss of the tight
junction (TJ) proteins occludin and zonula occludens-1
(ZO-1) after injury (Fig. 5a–d). SCI induced haemor-
rhage which was reduced by sivelestat treatment, and ex-
pression of cleaved PARP and LC3B was also decreased
following treatment (Fig. 5b).
NE inhibition attenuates secondary damage and prevents
glial scar formation after SCI
Fibrotic scar tissue is rich in microglia, astroglia, and
laminin, and fibronectin forms at the lesion site after
SCI in rodents and humans [77]. Several axon-inhibitory
molecules present at this scar tissue facade hinder axon
regeneration. To evaluate the role of NE in secondary dam-
age and glial scar formation, we examined samples from
sham, injured untreated, and injured sivelestat-treated (for
14 days) animals prepared 28 days after SCI (DPI-28). We
observed decreases in secondary damage and glial scar for-
mation in animals treated with sivelestat, which were associ-
ated with substantial inhibitory effects observed via IHC for
microglia (Iba-1), astroglia (GFAP), and fibronectin (Fig. 6a)
at the injury site. The decreases in astroglial and microglial
activation, as well as macrophage activation, were confirmed
at the transcriptional level via quantitative reverse
transcription-PCR analysis (Fig. 6bi–iii). IHC for laminin
showed a J- or T-shaped morphology under normal condi-
tions, whereas fibrotic scars rich in laminin formed at the
epicenter after SCI (Fig. 6c). Treatment with sivelestat
attenuated the laminin expression at DPI-28 as well as
SCI-induced transforming growth factor beta (TGF-β)
expression (Fig. 6d). IHC for the neuronal marker TuJ1
and the axonal marker neurofilament (NF) revealed sig-
nificant regeneration in the lesions of sivelestat-treated
rats (Fig. 6e).
NE inhibition stabilizes vascular endothelium formation
after SCI
SCI substantially disrupts the vasculature, especially at
the epicenter of the injury, where blood vessels undergo
remodeling and structural changes before they become
functional. Therefore, we delineated the changes associ-
ated with vascular-related proteins and factors after SCI
in untreated and sivelestat-treated animals. SCI-induced
increases in TGF-β, platelet-derived growth factor beta
(PDGF-β), neuropilin1, and platelet-endothelial cell
adhesion molecule (PECAM), indicative of vascular
changes/damage and vascular remodeling. In contrast to
those of untreated (vehicle-injected) animals, the spinal
cords of sivelestat-treated animals at 7 and 14 days after
SCI exhibited an attenuation of these increases in
vascular-related factors and a stabilization of the vascu-
lature (Fig. 7a and b). IHC of spinal cord microvessels
also revealed a change in the expression of neural/glial
(See figure on previous page.)
Fig. 3 Neutrophil elastase (NE) inhibition via sivelestat prevented the spinal cord injury (SCI)-induced modulation of angiopoietins (ANGPTs) and
inhibited the expression of NE. aMolecular structure of sivelestat (i) and concentrations determined in plasma (ii), brain (iii), and spinal cord (iv) at
several time points as described in the Methods section (n= 2/timepoint). Samples from sham, injured untreated, and injured sivelestat-treated
animals were prepared DPI-1 as described in the Methods section. Representative images of immunohistochemistry for ANGPT-1 and rat
endothelial cell antigen (RECA-1) (b) and ANGPT-2 and NE (e) at 1 day after SCI [3 fields/slide, n=2–3/group (sham = 2, injury = 3 and sivelestat = 3)]. c
Western blots of ANGPT-1, p-AKT, and AKT expression at 1 day after injury. Actin was used as internal controls for western blot [n=2–3/group (sham = 2,
injury = 3 and sivelestat = 3)]. Total RNA and spinal extracts from sham or injured untreated (Injury) or after sivelestat treatment were prepared 1 day after
the injury. RT-PCR results of Ang-1 (d), Ang-2 (f)andN.E(g) expression 1 day after injury [n=2–3/group (sham = 2, injury = 3 and sivelestat = 3) performed
in triplicates]. GAPDH was used as internal controls for real-time quantitative reverse transcription–polymerase chain reaction. Data represent means ± S.E.M.
###
p<0.001vs.shamgroup.*p< 0.05, **p< 0.01, ***p < 0.001 vs. Injury group
Kumar et al. Acta Neuropathologica Communications (2018) 6:73 Page 8 of 18
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

antigen2 (NG-2), alpha-smooth muscle actin (α-SMA)
(Fig. 7c), and von Willebrand factor (vWF) (Fig. 7d).
Treatment with sivelestat increases the expression of
NG-2, α-SMA and vWF.
NE inhibition improves functional recovery, attenuates
nociception, and provides neuroprotection after SCI
We performed behavioral tests at 1, 7, 14, 21, and
28 days following SCI on untreated animals and animals
treated for 14 days with sivelestat. Specifically, walking
patterns (gait) were evaluated via manual analyses of
footprints. After SCI, all animals showed distinct reduc-
tions in motor coordination in forepaw-hindpaw step-
ping. Those treated with sivelestat showed a marked
recovery of gait and improved motor coordination in
comparison with untreated animals (Fig. 8a). The per-
formance of sham rats remained unchanged throughout
the testing period. Functional recovery was also assessed
in open-field testing using the 21-point Basso, Beattie,
and Bresnahan (BBB) locomotor test [10]. Animals de-
veloped paraplegia after SCI, corresponding to a low
BBB score, but showed evidence of modest improve-
ments of motor function as early as 3–4 days after injury
(Fig. 8b). Recovery then continued reasonably faster for
another 1–2 weeks and then subsequently proceeded at
a slower rate. The recovery in motor function was sig-
nificantly faster in animals treated with sivelestat than in
untreated animals. Interestingly, BBB scores were signifi-
cantly higher in the sivelestat group even after the treat-
ment had stopped (i.e., after 14 days). As nociception is
one of the most common devastating conditions after
SCI, we evaluated the effect of sivelestat on SCI-induced
pain using vonFrey filaments. After SCI, animals exhib-
ited tactile hypersensitivity (decrease in withdrawal
threshold) on days 7, 14, 21, and 28, but not at day 1
(Fig. 8c). Treatment with sivelestat significantly attenu-
ated the SCI-induced hypersensitivity.
Fig. 4 Neutrophil elastase (NE) inhibition reduces the expression and production of inflammatory mediators (cytokines and chemokines) after
spinal cord injury (SCI). Samples from sham or injured untreated (Injury) or after sivelestat treatment were prepared 1 day after injury as described
in the Methods section. Representative section of tumor necrosis factor-alpha [TNF-α(a)], Interleukin [IL-6 (b)] immunofluorescence 1 day after SCI
[3 fields/slide, n=2–3/group (sham = 2, injury = 3 and sivelestat = 3)]. Total RNA from sham, vehicle (injury)- or sivelestat-treated samples were
prepared DPI-1 after injury as described in the Methods section. RT-PCR results showing relative expression levels of TNF-α(c), IL-6 (d), inducible
nitric oxide synthase [iNOS (e)], IL-1β(f), IL-10 (g), chemokine (C-C motif) ligand 2 [CCL-2 (h)], chemokine (C-C motif) ligand 3 [CCL-3 (i)] and TGF-β(j)
after injury/treatment [n=2–3/group (sham = 2, injury = 3 and sivelestat = 3) performed in triplicates]. GAPDH was used as internal controls for real-time
quantitative reverse transcription–polymerase chain reaction. Data represent means ± S.E.M.
##
p< 0.01,
###
p<0.001 vs. sham group.*p<0.05, **p<0.01,
***p<0.001 vs. Injurygroup
Kumar et al. Acta Neuropathologica Communications (2018) 6:73 Page 9 of 18
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Finally, the expression of neurotrophic factors, which
are mediators of neuronal and axonal plasticity and regen-
eration after SCI, was examined by IHC. The expression
of brain-derived neurotrophic factor (BDNF), ANGPT-1,
neurotrophin3 (NT-3), and neurotrophin4 (NT-4) de-
clined, whereas the expression of GFAP increased, after
SCI (Fig. 8d,gand f). Consistent with the other findings
in this study, sivelestat treatment prevented the decrease
in BDNF, ANGPT-1, NT-3, and NT-4 and attenuated the
increase in GFAP expression.
Discussion
In the present study, we demonstrate that NE is a key deter-
minant of vascular endothelium disruption/destabilization
and affects ANGPT expression in vitro (ECs) and in a rat
model of SCI. First, the effects of various concentrations of
recombinant NE were assessed on the most-characterized
type of ECs, HUVECs. ECs form capillary-like structures in
response to angiogenic factors present in the growth
medium; however, the addition of NE recombinant protein
dose-dependently prevented the formation of capillary-like
tubules, reducing the total length and numbers of tubes.
These findings suggest that NE influences a required step in
the process of angiogenesis. NE also suppressed the expres-
sion of ANGPT-1 and ANGPT-2 in ECs. The reduced ex-
pression of these factors may explain the decreased tubule
formation in ECs. We also found that factors known to in-
duce inflammation (i.e., LPS and TNF-α), which impact SCI
and ANGPT in ECs [26,48,49], decreased the expression of
ANGPT-1 and increased that of ANGPT-2, suggesting that
NE and inflammation differentially modulate the expression
of ANGPTs in ECs.
The expression patterns of NE and ANGPTs and the
roles of NE inhibition in neuroinflammation, BSCB disrup-
tion, vascular damage, functional recovery, and neuropro-
tection after SCI were also examined. The compression
injury used in this study results in damage to both the
Fig. 5 Neutrophil elastase inhibition prevented the spinal cord injury (SCI)-induced disruption of tight junction protein (ZO-1 and Occludin) in a rat
model. Samples from sham, injured untreated, and injured sivelestat-treated animals were prepared DPI-1 as described in the Methods section. a
Representative section of zonula occludens (ZO)-1 and occludin immunofluorescence, at DPI-1[3 fields/slide, n=2–3/gro up (sham = 2, injury = 3 and
sivelestat = 3)]. bWestern blots of ZO-1, cleaved PARP, and LC3B expression at 1 day after injury. Actin was used as internal controls for western blot.
Total RNA and spinal extracts from sham or injured untreated (Injury) or after sivelestat treatment were prepared DPI-1 as described in the Methods
section. RT-PCR results of occludin and zonula occludens (ZO)-1 expression 1 day after injury [n=2–3/group (sham = 2, injury = 3 and sivelestat = 3)
performed in triplicates] (cand d). GAPDH was used as internal controls for real-time quantitative reverse transcription–polymerase chain reaction.
Data represent means ± S.E.M.
#
p<0.05,
###
p< 0.001 vs. sham group. **p< 0.01, ***p< 0.001 vs. Injury group
Kumar et al. Acta Neuropathologica Communications (2018) 6:73 Page 10 of 18
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

dorsal and ventral spinal cord, transient ischemia, and im-
paired blood flow, which act synergistically to promote sec-
ondary pathology as typically observed in SCI in humans.
Neutrophil infiltration is linked to progressive damage to
the BSCB after SCI, and the prevention of this promotes
functional recovery [52,64]. In the present study, the ex-
pression of NE increased significantly from 3 h to 5 days,
with maximal expression observed at DPI-1. We also ob-
served damage to the vascular endothelium at this time
point via IHC for RECA-1. Since the release of NE from
Fig. 6 Neutrophil elastase inhibition attenuates inhibitory glial/fibrotic scarring and secondary damage after spinal cord injury (SCI). Samples from
sham or injured untreated (Injury) or after sivelestat treatment were prepared at DPI-28 as described in the Methods section. Sivelestat (30 mg/kg, i.p.)
was given for 14 days (28 doses) and animals were sacrificed at day 28. Zeiss confocal microscope was used to evaluate the Immunofluorescence after
IHC. The merge function of Zeiss microscope was used and 16 areas were evaluated. aRepresentative merges images (longitudinal) for Iba-1 (green),
GFAP (red), fibronectin (yellow) at DPI-28. RT-PCR results of GFAP (b-i), Iba-1 (b-ii), and Mac-1 (b-iii), expression at DPI-28 (n= 3/group). Representative
images of laminin (green), rat endothelial cell antigen (RECA-1; yellow) (c), TGF-β1 (green) (d) and neurofilaments (N.F) and Tuj-1 (e), at DPI-28 (3 fields/
slide, n= 3/group). GAPDH was used as internal controls for real-time quantitative reverse transcription–polymerase chain reaction. Data represent
means ± S.E.M.
###
p< 0.001 vs. sham group. **p<0.01, ***p< 0.001 vs. Injury group
Kumar et al. Acta Neuropathologica Communications (2018) 6:73 Page 11 of 18
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

neutrophils can damage ECs, we surmised that NE can also
affect the proteins released from ECs. Interestingly,
ANGPT-1 expression was downregulated when the expres-
sion of NE was maximal and corresponded to the initial
progressivedamagetotheendothelium. From day 7 on,
ANGPT-1 expression increased, which may be due to ex-
pression in a subset of glial and neuronal cells [13].
ANGPT-2 sensitizes ECs to inflammation, enhancing vas-
cular responsiveness to proinflammatory cytokines [26].
ANGPT-1 and ANGPT-2 have similar affinities for the
same receptor but produce opposing effects on blood
vessels. SCI induces a lasting decrease in ANGPT-1 levels
[14,35,58,74] but a persistent upregulation of ANGPT-2
[22]. Indeed, we observed an increase in ANGPT-2 ex-
pression that may counterbalance the ANGPT-1 decrease
after SCI. These findings suggest that SCI-induced NE
expression can cause endothelium damage and modulate
the activity of ANGPT-1 activity after SCI.
To examine this, we used sivelestat, a specific inhibitor
of NE. The pharmacokinetic results in the present study
show that the concentration of sivelestat in blood peaks
at around 30 min, with no accumulation, observed even
after 28 doses, indicating that the drug is cleared from
the body. Sivelestat treatment significantly attenuated
the SCI-induced NE and ANGPT-2 expression, reduc-
tion in ANGPT-1, and endothelial damage. These results
suggest that specific inhibition of NE prevents the
modulation of ANGPT, restores SCI-induced endothelial
damage, and prevents vascular disruptions.
SCI is accompanied by a series of intense immune re-
sponses, including inflammation, the synthesis of che-
mokines and cytokines, and the infiltration of peripheral
Fig. 7 Neutrophil elastase inhibition stabilizes vascular endothelium after spinal cord injury (SCI). Samples from sham or injured untreated (Injury)
or after sivelestat treatment were prepared 7 or 14 or 28 day after injury as described in the Methods section. Total RNA from sham, vehicle
(injury) - or sivelestat-treated samples were prepared 7 and 14 day after injury as described in the Methods section. RT-PCR results showing
relative expression levels of Transforming growth factor-β1[TGF-β1; aand b(i)], Platelet-derived growth factor [PDGF; aand b(ii)], Neuropilin-1 [aand
B(iii)], and platelet endothelial cell adhesion molecule [PECAM; aand b(iv)] [n=2–3/group (sham = 2, injury = 3 and sivelestat = 3) performed in
triplicates]. Representative section of neural/glial antigen [NG-2 (c); green], alpha-smooth muscle actin [α-SMA(c) red]; Von Willebrand factor [vWF (d)
orange] after SCI. Bar charts show the percentage of NG2 or α-SMA positive area per randomly selected field (3 fields/slide, n=3/group) (c). GAPDH
was used as internal controls for real-time quantitative reverse transcription–polymerase chain reaction. Data represent means ± S.E.M.
#
p< 0.05,
##
p<0.01,
###
p<0.001vs.shamgroup.**p<0.01, ***p< 0.001 vs. Injury group
Kumar et al. Acta Neuropathologica Communications (2018) 6:73 Page 12 of 18
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Fig. 8 Sivelestat treatment improved motor functions and attenuated neuropathic pain after spinal cord injury (SCI). aRepresentative footprints
of animal walking 1, 7, 14, 21 and 28 days after SCI. Black: Left forepaw footprints; Blue: Right forepaw footprints; Purple: left hind paw footprints;
Red: Right hind paw footprints. bFunctional recovery was then assessed in open-field testing by using the 21-point Basso, Beattie, and Bresnahan
(BBB) locomotor test at 1, 7, 14, 21 and 28 days after SCI. cNociception was evaluated using Von Frey-filaments; SCI-induced hypersensitivity
(decrease in withdrawal threshold) was assessed at 1, 7, 14, 21 and 28 days after SCI (sham = 6, injury = 16 and sivelestat = 12 for behavioral experiments).
Representative sections of ANGPT-1 and PECAM (d), neurotrophin-3 (NT-3) and Glial fibrillary acidic protein (GFAP) (e), and neurotrophin-4
(NT-4) and Brain-derived neurotrophic factor (BDNF) (f), at DPI-28. Data represent means ± S.E.M.
#
p< 0.05,
###
p< 0.001 vs. sham group.
***p< 0.001 vs. Injury group
Kumar et al. Acta Neuropathologica Communications (2018) 6:73 Page 13 of 18
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

leukocytes to the site of damage. Sivelestat was shown to
prevent neutrophil infiltration in a rat model of SCI [88].
In addition to their production of NE [6,48], infiltrating
neutrophils contribute to inflammation via their produc-
tion of IL-6 and TNF-α[42]. Systemic inflammation
promotes vascular endothelial injury and results in organ
dysfunction [60,91]. Inflammatory cells and ECs express
ANGPTs [4,25,53], suggesting that ANGPT signaling
plays a central role in commencing and continuing the in-
flammatory response. Of note, ANGPT-1 and ANGPT-2
show dichotomous pro- and anti-inflammatory properties,
with ANGPT-1 primarily considered anti-inflammatory
and ANGPT-2 proinflammatory, which are influenced by
other inflammatory mediators and proteolytic enzymes,
such as NE [2,9,33,44]. NE can regulate acute as well as
chronic inflammation [21]. The data presented here dem-
onstrate that SCI induces the expression of cytokines
(TNF-α, IL-6, iNOS, and IL-1β) and chemokines (CCL-2
and CCL-3); the expression of the anti-inflammatory cyto-
kine IL-10 decreased. The inhibition of NE with sivelestat
treatment decreased these proinflammatory factors,
including the expression of TNF-α,suggestingthebenefi-
cial role of NE inhibition on the ANGPT pathway after
SCI. A previous report suggested that ANGPT-1 inhibits
TNF-α-stimulated leukocyte transmigration [28], and
ANGPT-2 sensitizes ECs to TNF-αand plays a crucial role
in the induction of inflammation [26]. IL-6 is a proinflam-
matory cytokine that sharply increases in the acute phase
after SCI and not only downregulates ANGPT-1 signaling
[45] but also stimulates defective angiogenesis [29]. In the
present study, treatment with sivelestat attenuated the
SCI-induced IL-6 expression as well as that of iNOS,
which is implicated in immune responses, inflammation,
and apoptosis following SCI [79]. ANGPTs regulate vascu-
lar reactivity after hemorrhagic shock in rats through the
Tie-2-nitric oxide pathway [95]. ANGPT-1 neutralizes the
activity of proinflammatory factors on ECs, suppressing EC
permeability induced by vascular endothelial growth factor,
thrombin, bradykinin, and histamine [70,72]. In addition,
we observed that sivelestat augmented anti-inflammatory
IL-10 while attenuating SCI-induced proinflammatory
IL-1β, CCL-2, and CCL-3. Altogether, the evidence from
this study supports the notion that acute sivelestat inhib-
ition prevents the decrease in ANGPT-1 and acts as an
anti-inflammatory in an experimental model of SCI.
NE is downstream in the humoral mediator network
and is essential in vascular endothelial injury and
increased permeability [78]. By contrast, ANGPT-1 sup-
presses vascular leakage/inflammation and expedites
angiogenesis [51]. Previous studies showed that ANGPT-1
lowers vascular leakage by strengthening related endothe-
lial molecules and regulating interendothelial adhesion.
However, NE can protect the blood-brain barrier by in-
creasing ANGPT-1 expression and EC survival in an
animal model of focal ischemia [37]. BSCB disruption fol-
lowing SCI enables leukocytes, including neutrophils, to
infiltrate the injured parenchyma, contributing to second-
ary injury [1,32,99]. As TJs provide a “barrier”or “fence”
to regulate permeability and endothelial dysfunction [12],
we speculated that NE is involved in the degradation of TJ
proteins after SCI. We previously showed that the expres-
sion of the TJ proteins occludin and ZO-1 is decreased after
moderate compression injury [48], suggesting a disruption
of the BSCB. The decline in ANGPT-1 and increase in
ANGPT-2 correspond with marked blood-brain barrier
breakdown after brain injury [68]. Similarly, ANGPT-1
treatment was shown to attenuate BSCB permeability in an
animal model of SCI [30,35]. In the present study, the ex-
pression of the TJ proteins occludin and ZO-1 was reduced
after SCI, suggesting BSCB disruption, which was pre-
vented by treatment with sivelestat. Thus, specific inhib-
ition of NE effectively prevented ANGPT-1 disruption and
increased TJ protein expression after SCI.
After SCI, fibrotic scar tissue at the lesion site be-
comes rich in microglia, astroglia, and laminin and
fibronectin forms in rodents and humans [77], which
impedes axonal regeneration [50,82]. NE damages fibro-
nectins, laminins, and other matrix proteins, resulting in
increased vascular permeability and haemorrhaging in
tissues [36,40]. Damaged ECs and the basal lamina
deposited at the epicenter of the lesion diminish angio-
genesis and are concurrent with cystic cavity formation.
Sivelestat treatment reduced glial scar formation and
secondary damage, facilitating neuronal regeneration at
DPI-28. This was associated with a strong reduction in
the amounts of microglia and astroglia at the injury site,
as observed by IHC. However, SCI also induces inflam-
mation, which contributes to fibrosis scarring in part via
TGF-βsignaling. Indeed, we observed a significant in-
crease in TGF-βexpression after SCI, which was attenu-
ated in animals treated with sivelestat in accordance
with the reduction in scar tissue formation.
Blood vessel density correlates with improved func-
tional outcomes; hence sparing or regenerating the vas-
culature postinjury is desirable [43]. Although blood
vessels tend to grow rapidly into the lesion site after
SCI, there is substantial regression around 14 days post-
injury [18,56]. After nerve injury, PDGF-βexpression
increases [71], which leads to increased EC proliferation
[11]. In the present study, sivelestat attenuated the in-
crease in PDGF-βexpression at the transcriptional level,
as well as that for neuropilin1, whose expression in the
spinal cord is normally low but is also upregulated after
hemisection and dorsal column crush where it acts as an
inhibitory molecule in regulating the organization of the
sensory network [3]. Similarly, sivelestat attenuated the
SCI-induced increase in PECAM-1, which acts as a
mechanosensor in ECs [65] and whose localization to
Kumar et al. Acta Neuropathologica Communications (2018) 6:73 Page 14 of 18
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

cell junctions is regulated by ANGPT-1 to maintain cel-
lular integrity [28]. Another study found a significant
loss of PECAM-1-positive cells at early time points
(until day 3) after SCI, with a significant increase from
7 days onwards [94]. SCI also causes a robust decrease
in vWF [59], which was observed in the present study.
However, RECA-1 stained vessels could be readily iden-
tified within the injured spinal cord, suggesting that, al-
though ECs are present, there is altered angiogenesis,
which is prevented with NE inhibition.
As SCI can cause paralysis, altered motor coordi-
nation, and even neuropathic pain, we assessed these via
behavioral tests in the animal model. A footprint analysis
revealed reduced motor coordination in forepaw-
hindpaw stepping after SCI. The functional impairment
is likely influenced by the decrease in ANGPT-1 and re-
lated vascular dysfunction [15,49,73,93], as exogenous
administration of ANGPT-1 has shown favorable effects
on both functional and vascular recovery [30,35]. Ac-
cordingly, animals treated with sivelestat showed an
increased ANGPT-1, marked recovery of gait and im-
proved motor coordination compared with that of un-
treated injured animals. This functional recovery was
also reflected by the increase in the BBB score in
sivelestat-treated animals. The functional improvements
were also accompanied by a reduction in SCI-induced
hypersensitivity, an indicator of neuropathy, as assessed
by hindpaw responses to stimulation with von Frey fila-
ments. Thus functional recovery is a reflection of the in-
crease in the regenerated area of the lesion. Previous
reports suggest that enhanced axon regeneration corre-
lates with functional recovery after SCI [19,24]. Interest-
ingly, intravenous injection of ANGPT-1 and αvβ3
integrin peptide results in almost complete recovery
after SCI [30]. In the current study, the regeneration
may have been facilitated by the increase in ANGPT-1,
which promotes neurite outgrowth [47] and supports
the differentiation of neural progenitor cells via the AKT
pathway [8],asevidencedinthepresentstudybythe
increase in AKT phosphorylation in sivelestat-treated
animals. Furthermore, sivelestat treatment also main-
tained the levels of several neurotrophins (BDNF,
NT-3, and NT-4) that are linked with EC survival [20]
and are dramatically reduced in the adult spinal cord
[16,57]. In the current study, we treated sivelestat 1 h
after injury. However additional studies are required to
see whether sivelestat also work if treatment is delayed
till 3~ 4 h after injury simulating the clinical settings.
Secondly, owing to the short half-life of the sivelestat
we treated it twice a day; it would be interesting to ob-
serve the effect of sivelestat as continuous infusion
with a lower dose or increase the half-life or directly
delivering the sivelestat into the spinal cord by several
available approaches.
Conclusions
In conclusion, our results indicate that NE expres-
sion is increased after SCI, resulting in a dissociation
of ECs from microvessels, reduced ANGPT-1 expres-
sion, decreased angiogenesis, tissue damage, vascular
destabilization, BSCB breakdown, and cell injury.
The inhibition of NE via treatment with sivelestat
significantly attenuated SCI-induced inflammation,
prevented the decrease in ANGPT-1 expression, and
attenuated the increase in ANGPT-2, BSCB break-
down,andcellinjury.Asaresult,secondarydamage,
functional impairment, and neuropathic pain were
reduced while vascular stabilization was promoted.
Thus, NE inhibition could serve as a promising
therapeutic strategy after SCI.
Additional files
Additional file 1: Method for pharmacokinetic study. (DOCX 14 kb)
Additional file 2: Primer sequences for the genes of interest and the
reference genes. (DOCX 16 kb)
Acknowledgments
This work was supported by a grant of the National Research Foundation of
Korea (NRF) (NRF-2015H1D3A1066543 and NRF-2017R1C1B2011772), Korea
Healthcare Technology Research & Development Project, Ministry for Health
& Welfare Affairs, Republic of Korea (HI16C1559) and the National Natural
Science Foundation of China (31600780).
Availability of data and materials
Data and material used in the current study can be requested from the
corresponding author.Ethics approval: All animal procedures were performed
according to the approved protocol by the Institutional Animal Care and Use
Committee (IACUC) of CHA University (IACUC160076).
Authors’contributions
HK and KK carried out the molecular genetic studies, participated in the
sequence alignment and drafted the manuscript. HC,MJ, HJ,MM, and SK
carried out the immunoassays. EB and AK participated in pharmacokinetic
study. IH, XZ, and SL participated in the design of the study and performed
the statistical analysis. DB conceived of the study and participated in its
design and coordination. All authors read and approved the final manuscript.
Ethics approval and consent to participate
All animal procedures were performed according to the approved protocol
by the Institutional Animal Care and Use Committee (IACUC) of CHA
University (IACUC160076).
Competing interests
The authors declare that they have no competing interests.
Publisher’sNote
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Author details
1
Department of Neurosurgery, CHA University School of Medicine, CHA
Bundang Medical Center, Seongnam-si, Gyeonggi-do 13496, Republic of
Korea.
2
Department of Biomedical Science, CHA University, Seongnam-si,
Gyeonggi-do, Republic of Korea.
3
Molecular Neurobiology Laboratory,
Division of Neuroscience, San Raffaele Scientific Institute, Milan, Italy.
4
Department of Neurosurgery, Kyung Hee University, Dongdaemun-gu, Seoul
02447, Republic of Korea.
5
College of Pharmacy, CHA University,
Seongnam-si, Gyeonggi-do, Republic of Korea.
6
Department of Neurosurgery,
Kumar et al. Acta Neuropathologica Communications (2018) 6:73 Page 15 of 18
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Kyungpook National University Hospital, Kyungpook National University, 130,
Dongdeok-ro, Jung-gu, Daegu 41944, Republic of Korea.
7
Department of
Neurosurgery, School of Medicine,Kyungpook National University, 130,
Dongdeok-ro, Jung-gu, Daegu 41944, Republic of Korea.
8
Department of
Histology and Embryology, Zhongshan School of Medicine, Sun Yat-sen
University, Guangzhou 510080, Guangdong Province, China.
Received: 25 June 2018 Accepted: 23 July 2018
References
1. Abbott NJ, Ronnback L, Hansson E (2006) Astrocyte-endothelial interactions
at the blood-brain barrier. Nat Rev Neurosci 7:41–53. https://doi.org/10.
1038/nrn1824
2. Acarin L, Gonzalez B, Castellano B (2000) Neuronal, astroglial and microglial
cytokine expression after an excitotoxic lesion in the immature rat brain. Eur
J Neurosci 12:3505–3520
3. Agudo M, Robinson M, Cafferty W, Bradbury EJ, Kilkenny C, Hunt SP,
McMahon SB (2005) Regulation of neuropilin 1 by spinal cord injury in adult
rats. Mol Cell Neurosci 28:475–484. https://doi.org/10.1016/j.mcn.2004.10.008
4. Ahmad S, Cudmore MJ, Wang K, Hewett P, Potluri R, Fujisawa T, Ahmed A
(2010) Angiopoietin-1 induces migration of monocytes in a tie-2 and
integrin-independent manner. Hypertension 56:477–483. https://doi.org/10.
1161/HYPERTENSIONAHA.110.155556
5. Aikawa N, Kawasaki Y (2014) Clinical utility of the neutrophil elastase
inhibitor sivelestat for the treatment of acute respiratory distress syndrome.
Ther Clin Risk Manag 10:621–629. https://doi.org/10.2147/TCRM.S65066
6. Ankeny DP, Popovich PG (2009) Mechanisms and implications of adaptive
immune responses after traumatic spinal cord injury. Neuroscience 158:
1112–1121. https://doi.org/10.1016/j.neuroscience.2008.07.001
7. Aube B, Levesque SA, Pare A, Chamma E, Kebir H, Gorina R, Lecuyer MA,
Alvarez JI, De Koninck Y, Engelhardt B, Prat A, Cote D, Lacroix S (2014)
Neutrophils mediate blood-spinal cord barrier disruption in demyelinating
neuroinflammatory diseases. J Immunol 193:2438–2454. https://doi.org/10.
4049/jimmunol.1400401
8. Bai Y, Cui M, Meng Z, Shen L, He Q, Zhang X, Chen F, Xiao J (2009) Ectopic
expression of angiopoietin-1 promotes neuronal differentiation in neural
progenitor cells through the Akt pathway. Biochem Biophys Res Commun
378:296–301
9. Bartholdi D, Schwab ME (1997) Expression of pro-inflammatory cytokine and
chemokine mRNA upon experimental spinal cord injury in mouse: an in situ
hybridization study. Eur J Neurosci 9:1422–1438
10. Basso DM, Beattie MS, Bresnahan JC (1995) A sensitive and reliable
locomotor rating scale for open field testing in rats. J Neurotrauma 12:1–21.
https://doi.org/10.1089/neu.1995.12.1
11. Battegay EJ, Rupp J, Iruela-Arispe L, Sage EH, Pech M (1994) PDGF-BB
modulates endothelial proliferation and angiogenesis in vitro via PDGF
beta-receptors. J Cell Biol 125:917–928
12. Bazzoni G, Dejana E (2004) Endothelial cell-to-cell junctions: molecular
organization and role in vascular homeostasis. Physiol Rev 84:869–901.
https://doi.org/10.1152/physrev.00035.2003
13. Beck H, Acker T, Wiessner C, Allegrini PR, Plate KH (2000) Expression of
angiopoietin-1, angiopoietin-2, and tie receptors after middle cerebral artery
occlusion in the rat. Am J Pathol 157:1473–1483
14. Belaaouaj A, McCarthy R, Baumann M, Gao Z, Ley TJ, Abraham SN, Shapiro
SD (1998) Mice lacking neutrophil elastase reveal impaired host defense
against gram negative bacterial sepsis. Nat Med 4:615–618
15. Benton RL, Maddie MA, Worth CA, Mahoney ET, Hagg T, Whittemore SR (2008)
Transcriptomic screening of microvascular endothelial cells implicates novel
molecular regulators of vascular dysfunction after spinal cord injury. J Cereb
Blood Flow Metab 28:1771–1785. https://doi.org/10.1038/jcbfm.2008.76
16. Blesch A, Yang H, Weidner N, Hoang A, Otero D (2004) Axonal responses to
cellularly delivered NT-4/5 after spinal cord injury. Mol Cell Neurosci 27:190–201
17. Bonin RP, Bories C, De Koninck Y (2014) A simplified up-down method
(SUDO) for measuring mechanical nociception in rodents using von Frey
filaments. Mol Pain 10:26. https://doi.org/10.1186/1744-8069-10-26
18. Casella GT, Marcillo A, Bunge MB, Wood PM (2002) New vascular tissue
rapidly replaces neural parenchyma and vessels destroyed by a contusion
injury to the rat spinal cord. Exp Neurol 173:63–76
19. Coumans JV, Lin TT-S, Dai HN, MacArthur L, McAtee M, Nash C, Bregman BS
(2001) Axonal regeneration and functional recovery after complete spinal
cord transection in rats by delayed treatment with transplants and
neurotrophins. J Neurosci 21:9334–9344
20. Donovan MJ, Lin MI, Wiegn P, Ringstedt T, Kraemer R, Hahn R, Wang S,
Ibañez CF, Rafii S, Hempstead BL (2000) Brain derived neurotrophic factor is
an endothelial cell survival factor required for intramyocardial vessel
stabilization. Development 127:4531–4540
21. Doring G (1994) The role of neutrophil elastase in chronic inflammation. Am
J Respir Crit Care Med 150:S114–S117. https://doi.org/10.1164/ajrccm/150.6_
Pt_2.S114
22. Durham-Lee JC, Wu Y, Mokkapati VU, Paulucci-Holthauzen AA, Nesic O
(2012) Induction of angiopoietin-2 after spinal cord injury. Neuroscience
202:454–464. https://doi.org/10.1016/j.neuroscience.2011.09.058
23. Engelhardt B, Coisne C (2011) Fluids and barriers of the CNS establish
immune privilege by confining immune surveillance to a two-walled castle
moat surrounding the CNS castle. Fluids Barriers CNS 8:4. https://doi.org/10.
1186/2045-8118-8-4
24. Fawcett JW (2009) Recovery from spinal cord injury: regeneration, plasticity
and rehabilitation. Brain 132:1417–1418
25. Feistritzer C, Mosheimer BA, Sturn DH, Bijuklic K, Patsch JR, Wiedermann CJ
(2004) Expression and function of the angiopoietin receptor Tie-2 in human
eosinophils. J Allergy Clin Immunol 114:1077–1084. https://doi.org/10.1016/j.
jaci.2004.06.045
26. Fiedler U, Reiss Y, Scharpfenecker M, Grunow V, Koidl S, Thurston G, Gale
NW, Witzenrath M, Rosseau S, Suttorp N (2006) Angiopoietin-2 sensitizes
endothelial cells to TNF-αand has a crucial role in the induction of
inflammation. Nat Med 12:235
27. Figley SA, Khosravi R, Legasto JM, Tseng YF, Fehlings MG (2014)
Characterization of vascular disruption and blood-spinal cord barrier
permeability following traumatic spinal cord injury. J Neurotrauma 31:541–
552. https://doi.org/10.1089/neu.2013.3034
28. Gamble JR, Drew J, Trezise L, Underwood A, Parsons M, Kasminkas L, Rudge
J, Yancopoulos G, Vadas MA (2000) Angiopoietin-1 is an antipermeability
and anti-inflammatory agent in vitro and targets cell junctions. Circ Res 87:
603–607
29. Gopinathan G, Milagre C, Pearce OM, Reynolds LE, Hodivala-Dilke K, Leinster
DA, Zhong H, Hollingsworth RE, Thompson R, Whiteford JR (2015)
Interleukin-6 stimulates defective angiogenesis. Cancer Res 75:3098–3107
30. Han S, Arnold SA, Sithu SD, Mahoney ET, Geralds JT, Tran P, Benton RL,
Maddie MA, D'Souza SE, Whittemore SR, Hagg T (2010) Rescuing
vasculature with intravenous angiopoietin-1 and alpha v beta 3 integrin
peptide is protective after spinal cord injury. Brain 133:1026–1042. https://
doi.org/10.1093/brain/awq034
31. Hansen TM, Moss AJ, Brindle NP (2008) Vascular endothelial growth factor
and angiopoietins in neurovascular regeneration and protection following
stroke. Curr Neurovasc Res 5:236–245
32. Hawkins BT, Davis TP (2005) The blood-brain barrier/neurovascular unit in health
and disease. Pharmacol Rev 57:173–185. https://doi.org/10.1124/pr.57.2.4
33. Hayashi M, Ueyama T, Nemoto K, Tamaki T, Senba E (2000) Sequential
mRNA expression for immediate early genes, cytokines, and neurotrophins
in spinal cord injury. J Neurotrauma 17:203–218
34. Hermant B, Bibert S, Concord E, Dublet B, Weidenhaupt M, Vernet T, Gulino-
Debrac D (2003) Identification of proteases involved in the proteolysis of
vascular endothelium cadherin during neutrophil transmigration. J Biol
Chem 278:14002–14012. https://doi.org/10.1074/jbc.M300351200
35. Herrera JJ, Sundberg LM, Zentilin L, Giacca M, Narayana PA (2010) Sustained
expression of vascular endothelial growth factor and angiopoietin-1
improves blood-spinal cord barrier integrity and functional recovery after
spinal cord injury. J Neurotrauma 27:2067–2076. https://doi.org/10.1089/neu.
2010.1403
36. Houtz PK, Jones PD, Aronson NE, Richardson LM, Lai-Fook SJ (2004) Effect of
pancreatic and leukocyte elastase on hydraulic conductivity in lung
interstitial segments. J Appl Physiol (1985) 97:2139–2147. https://doi.org/10.
1152/japplphysiol.00567.2004
37. Ikegame Y, Yamashita K, Hayashi S, Yoshimura S, Nakashima S, Iwama T
(2010) Neutrophil elastase inhibitor prevents ischemic brain damage via
reduction of vasogenic edema. Hypertens Res 33:703–707. https://doi.org/
10.1038/hr.2010.58
38. Inoue N, Oka N, Kitamura T, Shibata K, Itatani K, Tomoyasu T, Miyaji K (2013)
Neutrophil elastase inhibitor sivelestat attenuates perioperative
inflammatory response in pediatric heart surgery with cardiopulmonary
bypass. Int Heart J 54:149–153
Kumar et al. Acta Neuropathologica Communications (2018) 6:73 Page 16 of 18
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

39. Ionescu CV, Cepinskas G, Savickiene J, Sandig M, Kvietys PR (2003)
Neutrophils induce sequential focal changes in endothelial adherens
junction components: role of elastase. Microcirculation 10:205–220. https://
doi.org/10.1038/sj.mn.7800185
40. Ishikawa N, Oda M, Kawaguchi M, Tsunezuka Y, Watanabe G (2003) The
effects of a specific neutrophil elastase inhibitor (ONO-5046) in pulmonary
ischemia-reperfusion injury. Transpl Int 16:341–346. https://doi.org/10.1007/
s00147-003-0556-8
41. Iwamoto S, Higashi A, Ueno T, Goto M, Iguro Y, Sakata R (2009) Protective
effect of sivelestat sodium hydrate (ONO-5046) on ischemic spinal cord
injury. Interact Cardiovasc Thorac Surg 8:606–609
42. Jablonska E, Kiluk M, Markiewicz W, Piotrowski L, Grabowska Z, Jablonski J
(2001) TNF-alpha, IL-6 and their soluble receptor serum levels and secretion
by neutrophils in cancer patients. Arch Immunol Ther Exp (Warsz) 49:63–69
43. Kaneko S, Iwanami A, Nakamura M, Kishino A, Kikuchi K, Shibata S, Okano
HJ, Ikegami T, Moriya A, Konishi O (2006) A selective Sema3A inhibitor
enhances regenerative responses and functional recovery of the injured
spinal cord. Nat Med 12:1380
44. Kawabata K, Hagio T, Matsuoka S (2002) The role of neutrophil elastase in
acute lung injury. Eur J Pharmacol 451:1–10
45. Kayakabe K, Kuroiwa T, Sakurai N, Ikeuchi H, Kadiombo AT, Sakairi T,
Matsumoto T, Maeshima A, Hiromura K, Nojima Y (2012) Interleukin-6
promotes destabilized angiogenesis by modulating angiopoietin expression
in rheumatoid arthritis. Rheumatology. https://doi.org/10.1093/
rheumatology/kes093.
46. Kim H, Lee JM, Park JS, Jo SA, Kim YO, Kim CW, Jo I (2008) Dexamethasone
coordinately regulates angiopoietin-1 and VEGF: a mechanism of
glucocorticoid-induced stabilization of blood-brain barrier. Biochem Biophys
Res Commun 372:243–248. https://doi.org/10.1016/j.bbrc.2008.05.025
47. Kosacka J, Figiel M, Engele J, Hilbig H, Majewski M, Spanel-Borowski K (2005)
Angiopoietin-1 promotes neurite outgrowth from dorsal root ganglion cells
positive for Tie-2 receptor. Cell Tissue Res 320:11–19
48. Kumar H, Jo MJ, Choi H, Muttigi MS, Shon S, Kim BJ, Lee SH, Han IB (2017)
Matrix Metalloproteinase-8 Inhibition Prevents Disruption of Blood-Spinal
Cord Barrier and Attenuates Inflammation in Rat Model of Spinal Cord
Injury. Mol Neurobiol. https://doi.org/10.1007/s12035-017-0509-3
49. Kumar H, Ropper AE, Lee S-H, Han I (2017) Propitious Therapeutic Modulators
to Prevent Blood-Spinal Cord Barrier Disruption in Spinal Cord Injury. Mol
Neurobiol 54(5):3578–3590. https://doi.org/10.1007/s12035-016-9910-6
50. Leal-Filho MB (2011) Spinal cord injury: From inflammation to glial scar.
Surg Neurol Int 2:112. https://doi.org/10.4103/2152-7806.83732
51. Lee HS, Han J, Bai HJ, Kim KW (2009) Brain angiogenesis in developmental
and pathological processes: regulation, molecular and cellular
communication at the neurovascular interface. FEBS J 276:4622–4635.
https://doi.org/10.1111/j.1742-4658.2009.07174.x
52. Lee SM, Rosen S, Weinstein P, van Rooijen N, Noble-Haeusslein LJ (2011)
Prevention of both neutrophil and monocyte recruitment promotes
recovery after spinal cord injury. J Neurotrauma 28:1893–1907. https://doi.
org/10.1089/neu.2011.1860
53. Lemieux C, Maliba R, Favier J, Theoret JF, Merhi Y, Sirois MG (2005)
Angiopoietins can directly activate endothelial cells and neutrophils to
promote proinflammatory responses. Blood 105:1523–1530. https://doi.org/
10.1182/blood-2004-09-3531
54. Ling X, Liu D (2007) Temporal and spatial profiles of cell loss after spinal
cord injury: Reduction by a metalloporphyrin. J Neurosci Res 85:2175–2185.
https://doi.org/10.1002/jnr.21362
55. Liu XZ, Xu XM, Hu R, Du C, Zhang SX, McDonald JW, Dong HX, Wu YJ, Fan
GS, Jacquin MF, Hsu CY, Choi DW (1997) Neuronal and glial apoptosis after
traumatic spinal cord injury. J Neurosci 17:5395–5406
56. Loy DN, Crawford CH, Darnall JB, Burke DA, Onifer SM, Whittemore SR (2002)
Temporal progression of angiogenesis and basal lamina deposition after
contusive spinal cord injury in the adult rat. J Comp Neurol 445:308–324
57. Maisonpierre PC, Belluscio L, Friedman B, Alderson RF, Wiegand SJ, Furth
ME, Lindsay RM, Yancopoulos GD (1990) NT-3, BDNF, and NGF in the
developing rat nervous system: parallel as well as reciprocal patterns of
expression. Neuron 5:501–509
58. Man S, Ubogu EE, Ransohoff RM (2007) Inflammatory cell migration into the
central nervous system: a few new twists on an old tale. Brain Pathol 17:
243–250. https://doi.org/10.1111/j.1750-3639.2007.00067.x
59. Matsushita T, Lankford KL, Arroyo EJ, Sasaki M, Neyazi M, Radtke C, Kocsis JD
(2015) Diffuse and persistent blood–spinal cord barrier disruption after
contusive spinal cord injury rapidly recovers following intravenous infusion
of bone marrow mesenchymal stem cells. Exp Neurol 267:152–164
60. McGill SN, Ahmed NA, Christou NV (1998) Endothelial cells: role in infection
and inflammation. World J Surg 22:171–178
61. Nag S, Papneja T, Venugopalan R, Stewart DJ (2005) Increased
angiopoietin2 expression is associated with endothelial apoptosis and
blood-brain barrier breakdown. Lab Invest 85:1189–1198. https://doi.org/10.
1038/labinvest.3700325
62. Nakatani K, Takeshita S, Tsujimoto H, Kawamura Y, Sekine I (2001) Inhibitory
effect of serine protease inhibitors on neutrophil-mediated endothelial cell
injury. J Leukoc Biol 69:241–247
63. National Research Council (2011) Guide for the care and use of laboratory
animals. Eighth Edition. Washington, DC: National Academies Press.
64. Neirinckx V, Coste C, Franzen R, Gothot A, Rogister B, Wislet S (2014)
Neutrophil contribution to spinal cord injury and repair. J
Neuroinflammation 11:150
65. Newman PJ (1999) Switched at birth: a new family for PECAM-1. J Clin
Invest 103:5–9
66. Noble LJ, Mautes AE, Hall JJ (1996) Characterization of the microvascular
glycocalyx in normal and injured spinal cord in the rat. J Comp Neurol 376:
542–556. https://doi.org/10.1002/(SICI)1096-9861(19961223)376:4<542::AID-
CNE4>3.0.CO;2-1
67. Noble LJ, Wrathall JR (1989) Distribution and time course of protein
extravasation in the rat spinal cord after contusive injury. Brain Res 482:57–66
68. Nourhaghighi N, Teichert-Kuliszewska K, Davis J, Stewart DJ, Nag S (2003)
Altered expression of angiopoietins during blood-brain barrier breakdown
and angiogenesis. Lab Invest 83:1211–1222
69. Okajima K, Harada N, Uchiba M, Mori M (2004) Neutrophil elastase
contributes to the development of ischemia-reperfusion-induced liver injury
by decreasing endothelial production of prostacyclin in rats. Am J Physiol
Gastrointest Liver Physiol 287:G1116–G1123. https://doi.org/10.1152/ajpgi.
00061.2004
70. Oubaha M, Gratton JP (2009) Phosphorylation of endothelial nitric oxide
synthase by atypical PKC zeta contributes to angiopoietin-1-dependent
inhibition of VEGF-induced endothelial permeability in vitro. Blood 114:
3343–3351. https://doi.org/10.1182/blood-2008-12-196584
71. Oya T, Zhao YL, Takagawa K, Kawaguchi M, Shirakawa K, Yamauchi T,
Sasahara M (2002) Platelet-derived growth factor-b expression induced after
rat peripheral nerve injuries. Glia 38:303–312
72. Pizurki L, Zhou Z, Glynos K, Roussos C, Papapetropoulos A (2003)
Angiopoietin-1 inhibits endothelial permeability, neutrophil adherence and
IL-8 production. Br J Pharmacol 139:329–336. https://doi.org/10.1038/sj.bjp.
0705259
73. Popovich PG, Horner PJ, Mullin BB, Stokes BT (1996) A quantitative spatial
analysis of the blood-spinal cord barrier. I. Permeability changes after
experimental spinal contusion injury. Exp Neurol 142:258–275. https://doi.
org/10.1006/exnr.1996.0196
74. Ritz MF, Graumann U, Gutierrez B, Hausmann O (2010) Traumatic spinal
cord injury alters angiogenic factors and TGF-beta1 that may affect vascular
recovery. Curr Neurovasc Res 7:301–310
75. Ropper AE, Zeng X, Anderson JE, Yu D, Han I, Haragopal H, Teng YD (2015)
An efficient device to experimentally model compression injury of
mammalian spinal cord. Exp Neurol 271:515–523. https://doi.org/10.1016/j.
expneurol.2015.07.012
76. Rossignol S, Schwab M, Schwartz M, Fehlings MG (2007) Spinal cord injury:
time to move? J Neurosci 27:11782–11792. https://doi.org/10.1523/
JNEUROSCI.3444-07.2007
77. Ruschel J, Hellal F, Flynn KC, Dupraz S, Elliott DA, Tedeschi A, Bates M, Sliwinski
C, Brook G, Dobrindt K, Peitz M, Brustle O, Norenberg MD, Blesch A, Weidner N,
Bunge MB, Bixby JL, Bradke F (2015) Axonal regeneration. Systemic
administration of epothilone B promotes axon regeneration after spinal cord
injury. Science 348:347–352. https://doi.org/10.1126/science.aaa2958
78. Russell JA (2006) Management of sepsis. N Engl J Med 355:1699–1713.
https://doi.org/10.1056/NEJMra043632
79. Satake K, Matsuyama Y, Kamiya M, Kawakami H, Iwata H, Adachi K, Kiuchi K
(2000) Nitric oxide via macrophage iNOS induces apoptosis following
traumatic spinal cord injury. Brain Res Mol Brain Res 85:114–122
80. Scali M, Begenisic T, Mainardi M, Milanese M, Bonifacino T, Bonanno G, Sale
A, Maffei L (2013) Fluoxetine treatment promotes functional recovery in a
rat model of cervical spinal cord injury. Sci Rep 3:2217. https://doi.org/10.
1038/srep02217
Kumar et al. Acta Neuropathologica Communications (2018) 6:73 Page 17 of 18
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

81. Semple BD, Trivedi A, Gimlin K, Noble-Haeusslein LJ (2015) Neutrophil
elastase mediates acute pathogenesis and is a determinant of long-term
behavioral recovery after traumatic injury to the immature brain. Neurobiol
Dis 74:263–280. https://doi.org/10.1016/j.nbd.2014.12.003
82. Silver J, Miller JH (2004) Regeneration beyond the glial scar. Nat Rev
Neurosci 5:146–156. https://doi.org/10.1038/nrn1326
83. Smedly LA, Tonnesen MG, Sandhaus RA, Haslett C, Guthrie LA, Johnston RB
Jr, Henson PM, Worthen GS (1986) Neutrophil-mediated injury to
endothelial cells. Enhancement by endotoxin and essential role of
neutrophil elastase. J Clin Invest 77:1233–1243. https://doi.org/10.1172/
JCI112426
84. Taoka Y, Okajima K, Murakami K, Johno M, Naruo M (1998) Role of
neutrophil elastase in compression-induced spinal cord injury in rats. Brain
Res 799:264–269
85. Tator CH, Koyanagi I (1997) Vascular mechanisms in the pathophysiology of
human spinal cord injury. J Neurosurg 86:483–492. https://doi.org/10.3171/
jns.1997.86.3.0483
86. Thomas M, Augustin HG (2009) The role of the Angiopoietins in vascular
morphogenesis. Angiogenesis 12:125–137. https://doi.org/10.1007/s10456-
009-9147-3
87. Thurston G, Rudge JS, Ioffe E, Zhou H, Ross L, Croll SD, Glazer N, Holash J,
McDonald DM, Yancopoulos GD (2000) Angiopoietin-1 protects the adult
vasculature against plasma leakage. Nat Med 6:460–463. https://doi.org/10.
1038/74725
88. Tonai T, Shiba K, Taketani Y, Ohmoto Y, Murata K, Muraguchi M, Ohsaki H,
Takeda E, Nishisho T (2001) A neutrophil elastase inhibitor (ONO-5046)
reduces neurologic damage after spinal cord injury in rats. J Neurochem 78:
1064–1072
89. Travis J (1988) Structure, function, and control of neutrophil proteinases. Am
J Med 84:37–42
90. Tsuboko Y, Takeda S, Mii S, Nakazato K, Tanaka K, Uchida E, Sakamoto A
(2012) Clinical evaluation of sivelestat for acute lung injury/acute respiratory
distress syndrome following surgery for abdominal sepsis. Drug Des Devel
Ther 6:273
91. Ueno H, Hirasawa H, Oda S, Shiga H, Nakanishi K, Matsuda K (2002)
Coagulation/fibrinolysis abnormality and vascular endothelial damage in the
pathogenesis of thrombocytopenic multiple organ failure. Crit Care Med 30:
2242–2248. https://doi.org/10.1097/01.CCM.0000030445.64104.E0
92. Valenzuela DM, Griffiths JA, Rojas J, Aldrich TH, Jones PF, Zhou H, McClain J,
Copeland NG, Gilbert DJ, Jenkins NA, Huang T, Papadopoulos N,
Maisonpierre PC, Davis S, Yancopoulos GD (1999) Angiopoietins 3 and 4:
diverging gene counterparts in mice and humans. Proc Natl Acad Sci U S A
96:1904–1909
93. Whetstone WD, Hsu JY, Eisenberg M, Werb Z, Noble-Haeusslein LJ (2003) Blood-
spinal cord barrier after spinal cord injury: relation to revascularization and wound
healing. J Neurosci Res 74:227–239. https://doi.org/10.1002/jnr.10759
94. Whetstone WD, Hsu JYC, Eisenberg M, Werb Z, Noble-Haeusslein LJ (2003)
Blood-spinal cord barrier after spinal cord injury: Relation to
revascularization and wound healing. J Neurosci Res 74:227–239
95. Xu J, Lan D, Li T, Yang G, Liu L (2012) Angiopoietins regulate vascular
reactivity after haemorrhagic shock in rats through the Tie2-nitric oxide
pathway. Cardiovasc Res 96:308–319. https://doi.org/10.1093/cvr/cvs254
96. Yang JJ, Kettritz R, Falk RJ, Jennette JC, Gaido ML (1996) Apoptosis of
endothelial cells induced by the neutrophil serine proteases proteinase 3
and elastase. Am J Pathol 149:1617–1626
97. Young RE, Thompson RD, Larbi KY, La M, Roberts CE, Shapiro SD, Perretti M,
Nourshargh S (2004) Neutrophil elastase (NE)-deficient mice demonstrate a
nonredundant role for NE in neutrophil migration, generation of
proinflammatory mediators, and phagocytosis in response to zymosan
particles in vivo. J Immunol 172:4493–4502
98. Zacharek A, Chen J, Cui X, Li A, Li Y, Roberts C, Feng Y, Gao Q, Chopp M
(2007) Angiopoietin1/Tie2 and VEGF/Flk1 induced by MSC treatment
amplifies angiogenesis and vascular stabilization after stroke. J Cereb Blood
Flow Metab 27:1684–1691. https://doi.org/10.1038/sj.jcbfm.9600475
99. Zlokovic BV (2008) The blood-brain barrier in health and chronic
neurodegenerative disorders. Neuron 57:178–201. https://doi.org/10.1016/j.
neuron.2008.01.003
Kumar et al. Acta Neuropathologica Communications (2018) 6:73 Page 18 of 18
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

1.
2.
3.
4.
5.
6.
Terms and Conditions
Springer Nature journal content, brought to you courtesy of Springer Nature Customer Service Center GmbH (“Springer Nature”).
Springer Nature supports a reasonable amount of sharing of research papers by authors, subscribers and authorised users (“Users”), for small-
scale personal, non-commercial use provided that all copyright, trade and service marks and other proprietary notices are maintained. By
accessing, sharing, receiving or otherwise using the Springer Nature journal content you agree to these terms of use (“Terms”). For these
purposes, Springer Nature considers academic use (by researchers and students) to be non-commercial.
These Terms are supplementary and will apply in addition to any applicable website terms and conditions, a relevant site licence or a personal
subscription. These Terms will prevail over any conflict or ambiguity with regards to the relevant terms, a site licence or a personal subscription
(to the extent of the conflict or ambiguity only). For Creative Commons-licensed articles, the terms of the Creative Commons license used will
apply.
We collect and use personal data to provide access to the Springer Nature journal content. We may also use these personal data internally within
ResearchGate and Springer Nature and as agreed share it, in an anonymised way, for purposes of tracking, analysis and reporting. We will not
otherwise disclose your personal data outside the ResearchGate or the Springer Nature group of companies unless we have your permission as
detailed in the Privacy Policy.
While Users may use the Springer Nature journal content for small scale, personal non-commercial use, it is important to note that Users may
not:
use such content for the purpose of providing other users with access on a regular or large scale basis or as a means to circumvent access
control;
use such content where to do so would be considered a criminal or statutory offence in any jurisdiction, or gives rise to civil liability, or is
otherwise unlawful;
falsely or misleadingly imply or suggest endorsement, approval , sponsorship, or association unless explicitly agreed to by Springer Nature in
writing;
use bots or other automated methods to access the content or redirect messages
override any security feature or exclusionary protocol; or
share the content in order to create substitute for Springer Nature products or services or a systematic database of Springer Nature journal
content.
In line with the restriction against commercial use, Springer Nature does not permit the creation of a product or service that creates revenue,
royalties, rent or income from our content or its inclusion as part of a paid for service or for other commercial gain. Springer Nature journal
content cannot be used for inter-library loans and librarians may not upload Springer Nature journal content on a large scale into their, or any
other, institutional repository.
These terms of use are reviewed regularly and may be amended at any time. Springer Nature is not obligated to publish any information or
content on this website and may remove it or features or functionality at our sole discretion, at any time with or without notice. Springer Nature
may revoke this licence to you at any time and remove access to any copies of the Springer Nature journal content which have been saved.
To the fullest extent permitted by law, Springer Nature makes no warranties, representations or guarantees to Users, either express or implied
with respect to the Springer nature journal content and all parties disclaim and waive any implied warranties or warranties imposed by law,
including merchantability or fitness for any particular purpose.
Please note that these rights do not automatically extend to content, data or other material published by Springer Nature that may be licensed
from third parties.
If you would like to use or distribute our Springer Nature journal content to a wider audience or on a regular basis or in any other manner not
expressly permitted by these Terms, please contact Springer Nature at
onlineservice@springernature.com