Intrathecal decompression versus epidural decompression in the treatment of severe spinal cord injury in rat model: a randomized, controlled preclinical research
Zhang et al. Journal of Orthopaedic Surgery and Research
Intrathecal decompression versus epidural decompression in the treatment of severe spinal cord injury in rat model: a randomized, controlled preclinical research
Jian Zhang 0
Huili Wang 0
0 No.1 Department of Orthopedic Surgery, Tianjin Baodi Hospital , No.8, Guangchuan Road, Baodi District, Tianjin 301800 , China
Background: In the setting of severe spinal cord injury (SCI), there is no markedly efficacious clinical therapeutic regimen to improve neurological function. After epidural decompression, as is shown in animal models, the swollen cord against non-elastic dura and elevation of intrathecal pressure may be the main causes of aggravated neurologic function. We performed an intrathecal decompression by longitudinal durotomy to evaluate the neuroprotective effect after severe SCI by comparing with epidural decompression. Methods: Eighty-four adult male Sprague-Dawley rats were assigned to three groups: sham group (group S), epidural decompression (group C), and intrathecal decompression group (group D). A weight-drop model was performed at T9. The Basso-Beattie-Bresnahan (BBB) score was used to evaluate neurological function. Animals were sacrificed at corresponding time points, and we performed pathohistological examinations including HE staining and immunohistochemical staining (IHC) of glial fibrillary acidic protein (GFAP), neurocan, and ED1 at the epicenter of injured cords. Finally, the lesions were quantitatively analyzed by SPSS 22.0. Results: The mortality rates were, respectively, 5.55 % (2/36) and 13.9 % (5/36) in groups C and D, and there was no significant difference between groups C and D (P = 0.214). Compared with epidural decompression, intrathecal decompression could obviously improve BBB scores after SCI. HE staining indicated that more white matter was spared, and fewer vacuoles and less axon degradation were observed. The expression peak of GFAP, neurocan, and ED1 occurred at an earlier time and was down-regulated in group D compared to group C. Conclusions: Our findings based on rat SCI model suggest that intrathecal decompression by longitudinal durotomy can prompt recovery of neurological function, and this neuroprotective mechanism may be related to the down-regulation of GFAP, neurocan, and ED1.
Spinal cord injury; Basso-Beattie-Bresnahan score; Decompression; Animal model; Pathophysiology
Traumatic spinal cord injury (SCI) is a disastrous event for
patients and families; the global incidence rate is estimated
at 23 cases per million individuals [
]. Although prominent
improvements have been made in acute medicine including
surgical management and rehabilitation, which have
significantly elevated survival rate and decreased long-term
complications for individuals with SCI, significant
neurological recovery has not been clinically obtained.
In the setting of severe SCI, multiple preclinical studies
aimed at improving neurological recovery by attenuating
the process of secondary injury have been conducted.
These studies have also shown that pharmacological
therapy and early surgical decompression could improve the
neurological outcome. Unfortunately, clinical studies have
not currently demonstrated markedly efficacious
therapeutic regimens that improve neurological function for
paraplegic or tetraplegic individuals using techniques that
include early surgical decompression, local hypothermia,
drugs, and electrical stimuli [
]. The improved degree
of neurological function in complete SCI patients is
relatively low; the proportion that improve from the American
Spinal Injury Association (ASIA) A to ASIA B or C is less
than 10 %, and there are nearly no patients that undergo
neurological recovery from ASIA A to D or E [
Although decompressive surgery is a very important
strategy for maintaining adequate blood flow and perfusion
to prompt neurologic recovery, the ensuing edema and
hemorrhage may lead to expansion of the injured cord and
increase spinal cord interstitial pressure (CIP) against the
relatively non-elastic dura mater [
]. Jones et al. found
cerebrospinal fluid (CSF) pressure differential cranial-caudal to
injured site increased after SCI [
], and they also found
swollen cord immediately occluded subarachnoid space in
severe SCI [
These observations suggested that decompression only
by removal of osseous and soft-tissue elements might not
be adequate and should be further improved to restore
normal CIP. Waleed et al. found a durotomy could lead to
a dramatic CIP drop in a distraction SCI model in vitro [
To date, only one preclinical study has evaluated the
effect of durotomy in mild SCI in vivo [
]. Here, we
hypothesized that severe thoracic SCI in rats would imitate
paraplegia, and a durotomy might show some efficacy in
relieving the injury. To elucidate the neuroprotective
mechanism of durotomy, parameters including
Basso-BeattieBresnahan (BBB) scores, HE staining, and the expression of
glial fibrillary acidic protein (GFAP), neurocan, and ED1
Animals and allocation
Eighty-four adult, male Sprague-Dawley rats (200–250 g,
8 weeks) were used in this study and provided by the
Experimental Animal Center of Academy of Military
Medical Sciences (Beijing, China). All animals were maintained
for 5–7 days before surgery in a temperature-regulated
room (22–25 °C) on a 12-h light/dark cycle with free
access to food and water. After surgery, each rat was
The rats were randomly assigned to three groups:
sham group (group S, N = 12), only laminectomy;
epidural decompression group (group C, N = 36), only
laminectomy and SCI, which is deemed as epidural
decompression; and intrathecal decompression group
(group D, N = 36), longitudinal durotomy after SCI. The
rats were evaluated before surgery and at 4 h and 1, 3, 7,
14, and 28 days post injury (DPI).
All experiments were conducted with approval of the
Institutional Animal Care and Use Committee of Beijing
Institute of Radiation Medicine and adhered strictly to
the NIH Guide for the Care and Use of Laboratory
Animals. All surgery was performed under chloral hydrate
anesthesia, and all efforts were made to minimize
suffering. We certify that all applicable institutional and
governmental regulations concerning the ethical use of animals
were followed during the course of this research.
Surgical procedure for animal model
All rats were anesthetized with chloral hydrate (300 mg/kg,
i.p.). After successful anesthesia, the rats were situated in
prone position on the operating table. A 5-cm posterior
midline incision was made, and the paravertebral
musculature was separated from the lamina of T7–11. The spinous
processes of T8–10 were removed, and then a laminectomy
was performed at T8–10. After the cord was exposed, the
spinal column was rigidly immobilized. A calibrated glass
guide tube was positioned perpendicular above the cord
center. The cord was subjected to a collision of a 10 g
stainless steel rod from 50 mm height, described by Allen [
Rats in group D immediately received a longitudinal
durotomy 10 mm in length (approximately two levels)
cephalic-caudal to the injured cord by microsurgical
apparatus under a microscope; gelfoam was overlaid onto
dural incision. The muscle and skin were sutured in layers.
All rats were injected daily for three consecutive days with
physiological saline (2 ml/100 g, i.p.) to prevent
hypovolemia. Their bladders were manually pressed twice daily
for urination until they re-established bladder reflexes.
Locomotor function assessment
The BBB scores were used to evaluate movement, body
support, and coordination ability. Three days before
surgery, all the animals were trained, and animals with
intrinsic motor dysfunction were excluded. All results
were recorded by a video camera for subsequent
blinded examination by two scorers.
The rats were sacrificed at corresponding times (N = 5 in
groups C and D) with an overdose of chloral hydrate
(500 mg/kg, i.p.) and transcardially perfused with saline
solution (0.9 %) followed by 4 % cold paraformaldehyde.
The injured cords were carefully removed en bloc, then
fixed in 4 % paraformaldehyde at 4 °C for 24 h, and then
embedded in paraffin after dehydration.
Four consecutive transverse sections (5-μm thickness)
were prepared in all rats with a Leica microtome (Leica
RM2035, Germany) at epicenter. One section was stained
with HE, and the other sections were used for
immunohistochemical staining (IHC) analysis.
The expression levels of proteins were detected by IHC
using monoclonal mouse anti-rat GFAP (1:400; Sigma,
St. Louis, MO), monoclonal mouse anti-rat neurocan
(1:200; Sigma, St. Louis, MO), monoclonal mouse
antirat ED1 (1:200; Abcam, New Territories, Hong Kong), as
described by Cattoretti et al. [
deparaffinization, hydrogen peroxide incubation, microwave antigen
retrieval, and incubation in 2 % goat serum (SP9001 kit,
Zhongshan Golden Bridge, Beijing, China), the sections
were separately incubated in corresponding primary
antibodies overnight at 4 °C. On the following day, the
sections were incubated with a biotinylated secondary
antibody at room temperature for 1 h and then
incubated in avidin-biotin peroxidase complex (SP9001 kit,
Zhongshan Golden Bridge, Beijing, China) for 30 min.
The sections were visualized by 0.025 %
diaminobenzidine (SP9001 kit, Zhongshan Golden Bridge, Beijing,
China). For negative controls, PBS was used instead of
All sections were examined under a light microscope
(Olympus BH-2, Tokyo, Japan) and photographed (Sony,
CCD-IRISI, Tokyo, Japan) by a pathologist who was
blinded to experimental conditions. IHC-positive cell
counts were performed in four obviously expressed,
highly magnified fields, and averages were determined in
The statistical analyses were performed by SPSS 22.0. All
data were reported as means ± standard deviation. Values
of P less than 0.05 were considered statistically significant.
Observations from the operations
An enormous contraction of the hind limbs and tail was
observed after collision, and an intrathecal hematoma
occurred immediately; the spinal cord swelled and
obstructed the intradural cavity. After durotomy was
performed, the contusive, hemorrhagic spinal cord with
a dark purple appearance herniated more than half of
the intradural cavity.
Differences in mortality rate
The mortality rates were 8.33 % (1/12), 5.55 % (2/36),
and 13.9 % (5/36) in groups S, C, and D, respectively,
and there was no significant difference between groups
C and D (Table 1). For mortality reason, one died due to
unrecovery after anesthesia, five died from pulmonary
bleeding, and two died from bladder bleeding by
Changes of neurological function
The BBB scores in each group all consisted of a value of
21 before the surgery (BBB scores in group S gradually
recovered to normal levels at 1 DPI, not shown). The
scores were sharply increased within 14 DPI in group D,
but in group C, the scores were lower at 1 DPI,
indicating neurological function was aggravated (Fig. 1).
To analyze histological changes after SCI, HE staining was
applied. No lesion was detected in group S, while irregular
hemorrhage, neuron loss, vacuoles, axon degradation, and
cavitation were observed in groups C and D (Fig. 2). More
white matter was spared, and fewer vacuoles and less axon
degradation were observed in group D (Fig. 2).
We performed IHC to elucidate the expression of GFAP,
ED1, and neurocan. In group S, GFAP+ astrocytes without
hypertrophy were observed, and there was no expression
of neurocan and ED1. No staining was observed in
The number of astrocytes did not increase in groups C
and D compared to group S (Fig. 3), but the volume of
astrocytes was significantly increased. GFAP+ astrocytes
were detected mainly in gray matter. GFAP expression
in group D increased sharply, reached peak values at 3
and 7 DPI, and then gradually declined from 14 to 28
DPI. However, GFAP expression in group C
continuously increased from 3 to 28 DPI and reached peak
values at 14 and 28 DPI.
Neurocan was mainly located in the white matter
(Fig. 4). A two-peak expression pattern of neurocan
occurred separately at 4 h and 7 DPI in group C, while in
group D, neurocan expression reached a peak value at 1
DPI and then continuously decreased.
ED1 was extensively distributed in the gray and white
matter (Fig. 5). The expression peak of ED1 occurred at
an earlier time and was much lower in group D than
that in group C.
Epidural decompressive surgery is recognized as an
important intervention and a general trend for treating
SCI. Epidural decompression seems adequate during
operation; however, postoperative magnetic resonance
imaging often reveals that the swollen cord has filled
subarachnoid space [
]. Jones et al. found CSF
pressure was only partially decreased by epidural
decompressive surgery , and the enlarged cord immediately
occluded subarachnoid space in severe SCI [
]. Smith et al.
found the swollen cord partially herniated through incision
site following durotomy [
]. These observations indicate
that epidural decompression may not be adequate, and it is
often necessary to perform further decompression after
Secondary injuries including edema and hemorrhage
lead to an expanded volume of the injured cord against
the non-elastic dura mater [
], leading to a circumstance
similar to compartment syndrome, and ultimately
worsening ischemia in the injured cord. Elevation of CSF
pressure may worsen ischemia at the injured cord [
and reduction of CSF pressure can enhance blood flow
and improve microcirculation [
]. Obvious elevation of
CSF pressure has been shown after SCI in preclinical
and clinical studies [
Only a few studies have examined durotomy use for
the treatment of SCI. The possible mechanisms of the
neuroprotective effect of a durotomy are as follows: a
decrease in CIP [
], cavitation, scar formation, and lesional
]; relief of congestive epidural veins; and
restoration of CSF flow [
Due to the differences in injury types, severity, and
levels, clinical manifestation of SCI is diverse, and none
of the animal SCI models available can completely simulate
the clinical situation, which may include fractures and
dislocation. Furthermore, due to transportation, physical
and radiographic examination, neurological function
assessment, and the determination to perform surgery, patients
cannot be surgically treated immediately after SCI
]. This study was an ideal situation in which a
durotomy was performed immediately to observe the
Our SCI model imitated severe SCI, in which the
obviously swollen injured cords against the dura were identified
after collision. The neurological function was significantly
improved after intrathecal decompression by longitudinal
durotomy, and neurological recovery was in agreement
with the results of pathohistological analysis in which more
white matter was spared and fewer vacuoles and less axon
degradation were observed. Our findings based on rat SCI
model suggest that intrathecal decompression may be
useful as a promising therapeutic regimen for SCI.
Damage to the spinal cord can result in a glial
reaction and eventually glial scar formation, in which GFAP
+ astrocytes and neurocan are important components
in inhibiting neurological repair. In early stage, reactive
astrocytes may exert beneficial effects by regulating
local immune responses and promoting tissue repair;
however, these beneficial effects arise at the expense of
inhibiting damaged axon regeneration [
is an inhibitor of central nervous system regeneration.
Inhibition of neurocan expression can improve neurological
]. In this study, GFAP+ astrocytes were
hypertrophic with richly branched processes at the epicenter.
However, after durotomy, GFAP expression peak occurred
at an earlier time after SCI and was obviously
downregulated when comparing with epidural decompression.
Neurocan was also down-regulated after durotomy. These
results suggest that the neuroprotective mechanism of
durotomy might be related to inhibit glial scar formation
by down-regulating the expression of GFAP and neurocan
at the injured site.
Because activated macrophages can release cytotoxic
substances that aggravate inflammatory reaction in
secondary injury [
], ED1 is a specific marker for
macrophages. A key intervention is to control
inflammatory reactions following SCI. We found that ED1
expression was down-regulated in group D compared
to group C. Thereby, the durotomy might inhibit
inflammatory reactions by suppressing the expression of
We also found that in the early stage after SCI, the
expression of GFAP, neurocan, and ED1 were supppressed
in group C. It is likely that the injured cord was
functionally depressed and did not express corresponding
proteins due to the compression from intact dura which
possibly causes ischemia. However, we did not have the
direct evidence which can show some degree of
restoration of blood flow to the injured site after durotomy.
Further studies need to be conducted to confirm whether
blood flow can be restored by durotomy [
Maintenance of the dural integrity can inhibit
inflammatory reaction and reduce scar formation [
9, 23, 25
However, there is no consensus on how to cover the neural
tissues after durotomy. In studies by Smith [
] and Iannotti
], they used a fibrin sealant to fix an allograft onto the
incised dura. In a large animal model, Neulen et al. found
that collagen matrix was an attractive alternative in
duraplasty due to its easy handling, lower surgical time, and
high biocompatibility [
]. However, when performing a
decompressive craniectomy, the neurosurgeon usually
applies nothing to cover cerebral tissue [
]. In this study, we
applied gelfoam to cover the injured cord and did not find
exacerbation of the inflammatory reaction or adhesive scar
after a durotomy.
Although these results are encouraging, there are some
limitations in this study. The mortality rate was increased
after intrathecal decompression, which indicated that
systematic pathophysiological reactions after durotomy
were aggravated. We did not perform CIP measurements
due to the small animal model. Further research upon
durotomy need to be conducted to acquire more
comprehensive data, for example, performing durotomy in large
animal SCI model can better imitate the situation in
human and evaluate the safety and efficacy of this
procedure, investigation on microcirculation at the injured site
can better understand the pathophysiological process of
SCI, and suitable dural substitute can inhibit inflammatory
reaction and reduce adhesive scar.
Our findings based on rat SCI model suggest that
intrathecal decompression can prompt recovery of neurological
function which was in accordance with the
pathohistological process, and this neuroprotective mechanism may be
related to the down-regulation of GFAP, neurocan, and
ED1. Therefore, intrathecal decompression may be useful
as a promising therapeutic regimen for SCI.
ASIA: American Spinal Injury Association; BBB: Basso-Beattie-Bresnahan;
CIP: spinal cord interstitial pressure; CSF: cerebrospinal fluid; DPI: days post
injury; GFAP: glial fibrillary acidic protein; IHC: immunohistochemical staining;
SCI: spinal cord injury.
The authors declare that they have no competing interests.
JZ, HLW, and WGL carried out all the research work. JZ and CGZ carried out
the statistical analysis. JZ and CGZ drafted the manuscript. All authors read
and approved the final manuscript.
This research was performed at Institute of Radiation and Radiation Medicine,
Academy of Military Medical Sciences, Beijing, China, and we would like to
thank Liang Yan and Wanxia Niu for the help in BBB testing and thank Zhihui Li
and Yan Gao for the help in immunohistochemistry analysis.
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