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Introduction
Traumatic spinal cord injury (SCI) can result in severe
damage, leading to paraplegia, tetraplegia, or worse. Many
strategies, including surgical, pharmacological,
neurophysiol-ogical, and technological approaches, have been used to
allow patients to regain the use of their paralyzed limbs. One
such strategy is cell transplantation into the damaged spinal
cord. Cell transplantation not only has the ability to provide
therapeutic agents, it also has the potential to repair and
replace neuronal tissue. Stem cells have greatly increased
the hope that treatments may be available for once untreatable
central nervous system (CNS) diseases and injures, which
has captured the attention of both the scientific community
and the general public. Mesenchymal stem cells (MSC), a
type of adult stem cell, can be easily acquired, maintained,
expanded quickly, and differentiated into neural cell
types in vitro and in vivo. It was reported that transplanted MSC
could promote the functional recovery of spinal cord injury
in animals, including primates and other
mammals[1-3].
However, the adult CNS, especially the injured CNS, may
provide a relatively non-permissive environment for
transplanted MSC and other stem cells. Even under the best
circumstances, cell survival in injured CNS has been
estimated to be near 10%[4-6], and few cells differentiate into
mature neuronal phenotypes[7-10]. One of the most important
reasons why those cells could not survive in the injured
CNS is probably due to the sustained inflammation in the
injury sites or the immediate inflammatory response elicited
by surgery and injection
manipulation[8,11]. Methods to provide neuroprotection support to the transplanted cells, by
suppressing the inflammation in the injured spinal cord and
brain, may benefit long-term behavioral outcomes and the
grafted cell survival.
Salvianolic acid B (Sal B) is a water-soluble compound
extracted from Salvia miltiorrhiza (SM), a herb that was
clinically used for thousands of years in traditional Chinese
medicine. SM has been used as a common treatment
medicine for stroke, and is now widely used for the treatment of
cardiovascular diseases. Previous studies have
demonstrated that SM is an anti-inflammatory, antioxidative, and
apoptosis-inducing plant[12,13]. Also, Sal B has been reported
to exert protective action against TNF-α injury in human
aortic vascular endothelial cells[14]. Sal B offers an
interesting possible approach for SCI treatment, because it plays an
important cytoprotective role and reduces inflammatory
responses, thereby affecting a variety of processes that
contribute to secondary degeneration. Indeed, the
neuropro-tective effects of Sal B have been demonstrated in some
experimental models of cerebral ischemia and brain
injury[15-17].
Thus, the aim of the present study was to investigate the
effect of Sal B on the recovery of motor function of SCI rats
and whether Sal B could enhance MSC survival in an
in vitro environment with TNF-α or in SCI rats.
Materials and methods
MSC isolation and culture MSC were collected from
femurs of adolescent, male Sprague_Dawley (SD) rats
(60_80g) as reported[18]. Briefly, the bilateral femurs and tibias
were harvested and the marrow was flushed out by using a
syringe filled with Dulbecco's modified Eagle's medium/F12
containing 10% fetal bovine serum (FBS). The bone marrow
was plated in 25 cm2 culture flasks. The flasks were
incubated at 37 °C with 5% CO2. Forty eight hours after plating,
the supernatant containing non-adherent cells was removed
and fresh medium was added. After growing near 80%_90%
confluency, the cells were passaged 2 or 3 times by
detachment (0.25% trypsin/0.02% EDTA for 2_3 min) and replated.
The cells were then frozen in aliquots and stored in liquid
nitrogen.
Analysis of the protective function of Sal B on MSC
in vitro Sal B (molecular formula:
C36H30O16, molecular weight:
746, purity: 98.5%, Green-Valley, Shanghai, China) was kept
from direct exposure to light and air during the experiments.
Sal B and recombinant rat TNF-α (PeproTechEC, London,
UK) were dissolved in phosphate buffered saline (PBS).
Cell viability was determined by counting the number of
attached cells on a culture plate. MSC (at passages 4_8),
cultured in 12-well plates at 1×105
/plate for 2 d, were treated as follows. MSC were treated with 10 ng/mL
TNF-α for 24 h. Sal B 100 ng/mL was added, along with the
TNF-α, to protect MSC from injury resulting from10 ng/mL
TNF-α in one group, while no Sal B was used in the other group. Cell death was
observed under a microscope and characterized by
rounding up the cells and detaching from the plates. After treatment,
each well was photographed at least 5 times by a digital
camera. The attached cell numbers were counted from 3
randomly-selected pictures.
Animal group design A total of 42 adult, female SD rats
(weight 220_250 g) were used. The experimental rats were
divided depending on the treatment of rats after thoracic
SCI: Sal B group, Sal B injected intraperitoneally on d 0, 1, 2,
and 3, post-injury, n=6; PBS group, PBS injected
intraperitoneally on d 0, 1, 2, and 3, post-injury,
n=6; MSC group, MSC transplanted in
situ on d 7, post-injury, and PBS injected intraperitoneally at d 7, 8, and 9, post-injury,
n=9; MSC plus Sal B group, MSC transplanted
in situ on d 7, post-injury, and Sal B injected intraperitoneally on d 7, 8, and 9,
post-injury, n=9; and the PBS control group, PBS transplanted
in situ on d 7, post-injury, n=6.
Thoracic SCI Laminectomy was performed at
T8-T10 under 10% chloral hydrate anesthesia. The impact rod,
weighing 10 g, was centered above T9 and dropped from a height
of 25 mm to induce a consistent partial, incomplete SCI.
Dural tear was not observed following the impact. Muscle and
skin were sutured by layer after injury. Gentamicin (8
mg·kg-1·d-1, ip) was given to prevent urinary tract infection for 3 d,
and lactated Ringer's solution was administered to avoid
dehydration (2 mL, ip, after surgery). Urinary bladders were
emptied manually 2 times per day until recovery of urinary
function. In the present study, the animal experiments were
performed in accordance with the National Institute of Health
animal care and use guidelines.
Sal B treatment and MSC transplantation For the Sal B
and PBS groups, the rats were given Sal B (8 mg/kg) or PBS
only, intraperitoneally, at different time points as mentioned
earlier.
For the MSC group and the MSC plus Sal B group, the
rats were treated with Sal B or MSC from d 7, post-injury as
mentioned. Before the experimental manipulations, MSC were
labeled with bromodeoxyuridine (BrdU; 100 µmol/L, Boster,
Wuhan, China) for 48 h to permit later identification. This
procedure led to 80%_90% BrdU labeling of the cultures.
The flasks were washed and the cells were resuspended in
PBS. Before transplantation, MSC were spun down and
resuspended in a microcentrifuge tube in PBS, approximately
100 000 cells/µL, and kept on ice. A 25 gauge Hamilton
syringe was lowered into the upper region central of the injured site
at approximately 5 mm. MSC suspension or only PBS filled in
the syringe in 10 µL was injected at rate of 2.5 µL/min. The
syringe was allowed to remain in the position for several
minutes after the injection to allow the cells to settle. The
incision was then closed with nylon suture. Animal care was
carried out until the rats were killed.
Tissue harvest and histological evaluation
Histological evaluation was performed at the endpoints of the different
groups in the experiment. The animals were anesthetized
with 10% chloral hydrate and perfused transcardially with
4% paraformaldehyde. The T8-10 portion of the spinal cord
was removed from the vertebral column and was then
immersed for 24 h in the same fixative. The tissue was
embedded in paraffin and sectioned sagittally (6 µm thickness).
For the Sal B and PBS groups, sections were collected on
microscope slides, the paraffin was removed in xylene, and
the slides were rehydrated through graded ethanol. All
sections were stained with hematoxylin_eosin (HE) for general
histology. The area of cavitation was measured by setting
the optical density threshold to delineate regions lacking
cellular components with the outlined lesion.
For the MSC group, MSC plus Sal B group, and the PBS
control group, the paraffin of sections was removed in xylene,
and the slides were rehydrated through graded ethanol. Half
the sections were used for immunohistochemical staining to
determine the survival of grafted MSC. Other sections were
stained with HE for general histology.
Immunohistochemical detection of the survival of grafted
MSC and quantification analysis After rinsing in PBS, the
sections were exposed to 3%
H2O2 for 30 min to quench endogenous peroxidase activity. Before the incubation of
the primary antibodies, non-specific binding was blocked
for 1 h with 3% normal serum from the species in which the
secondary antibody was raised with 0.25% Triton X-100 in
PBS. The primary antibodies used were directed against the
rabbit anti-BrdU monoclonal antibody (1:100, Boster, China).
The sections were incubated with secondary antibodies
conjugated to biotin (1:100, Boster, China). Subsequently, the
sections incubated with secondary antibodies conjugated
to biotin were washed in PBS and incubated with a
avidin-biotin-horseradish peroxidase complex (1:100, Boster, China).
3',3'-Diaminobenzidine (Boster, China) was used as a
chromogen. Control slides lacking primary or secondary
antibodies were analyzed with each series. The sections
were studied using light microscopy.
Behavioral testing The rats were tested behaviorally to
evaluate the effect of MSC and Sal B on the recovery of
motor function of the injured rats. Behavioral testing was
performed for each hind limb using the
Basso-Beatie-Bresnahan (BBB) scale at different time points (before injury
and on d 1, 7, 14, 21, 28, and 35,
post-injury)[9]. BBB scores categorize combinations of rat hind limb movements, trunk
position and stability, stepping, coordination, paw placement,
toe clearance, and tail position; a 0 score
represents no locomotion and a 21 score represents normal motor function.
The test was conducted by 2 independent observers, and
the scores for the left and right leg were averaged to
generate the actual score for each trial.
Statistical analysis The statistical analysis was
calculated using SPSS software (SPSS, Chicago, IL, USA). All
values are expressed as the mean±SEM. The results were
analyzed by one-way ANOVA followed by a Bonferroni
post-hoc test for multiple comparisons. A
P-value of <0.05 was considered significant.
Results
Effects of Sal B on rats with spinal cord
injuries A representative bright-field photomicrograph of the spinal cord
sections following PBS control or Sal B treatments were
selected to express the effect of Sal B treatment on SCI (Figure
1A,1B). For the measurement of cavities, sagittal sections
were stained with HE, and the area of the cavity in the spinal
cord was measured. The areas of the cavities were measured
from 20 sections in every rat in the Sal B and PBS groups.
The cavity area was then calculated by the average area in
the 2 groups (n=6 each group). Sal B treatment significantly
reduced the cavity area from 0.26±0.05
mm2 to 0.15±0.03 mm2, compared with the control groups
(P<0.01, Figure 1C).
Performance in locomotor function was enhanced in the
Sal B-treated animals. In contrast to the inability of the
control animals to support their weight with their hind limbs, the
Sal B-treated rats demonstrated partial weight-supported
ambulation. A statistical difference in the BBB scores was
achieved 2 weeks after injection. After 4 weeks, there was a
significant difference of approximately 2 points on the BBB
scale between the Sal B and PBS groups (Figure 5A).
Sal B protects MSC from the injury of TNF-α
in vitro The effect of Sal B on the cell death of MSC induced by
TNF-α was investigated. MSC were killed with
TNF-α, and cell death was determined by a microscopic observation of
cell shrinkage and detachment from the culture plates. The
cells began to shrink and detach from the culture plates 3 h
after incubation with TNF-α. Within 24 h, a large number of
cells were detached from the culture plates. However,
TNF-a had no effect on MSC treated with Sal B (Figure 2A_2C).
Cell viability (%) was determined by comparing the cell
numbers of the control with those of the treated cells. Data
are presented as mean±SEM from 3 separate experiments.
According to the counting result, TNF-a significantly
decreased the cell viability by 65% compared with the control
level. In contrast, Sal B plus TNF-α treatment had no
significant effect on the viability of the cells (Figure 2D).
Sal B promotes the survival of MSC in spinal
cord-injured rats On d 7, post-transplantation, 4 animals from each
group were used, and 5 animals from each group were used
on d 28, post-transplantation. For the evaluation of the number
of the grafted MSC, 2 representative sections at the center
of the lesion were selected from each MSC-grafted animal in
the MSC group and the MSC plus Sal B group. In the MSC
group, few surviving MSC with intact BrdU nuclei were found
and these were scattered throughout the graft at d 28,
post-transplantation (Figure 3A). In contrast, in the MSC plus Sal B
group, many more surviving MSC (dark brown or black) were
densely packed throughout the transplantation site (Figure
3B).
BrdU-labeled MSC were quantified from slides from 3
sections with the center of the lesion in each group (Figure
3C). The MSC plus Sal B group contained significantly more
positive cells (1143.3±195.6) than the MSC group
(569.3±72.3; (P<0.05), at d 7, post-transplantation. The quantification
of MSC in the 2 groups at d 28, post-transplantation was
also done. A greater number of surviving MSC (764.0±81.3)
in the MSC plus Sal B group was found than that in the MSC
group (237.0±61.3). The number of surviving MSC at d 28
was fewer than that at d 7 in the MSC plus Sal B group, but
more MSC were still found in the MSC plus Sal B group
compared with MSC in the MSC group (P<0.05).
The anatomical integrity of injured spinal cords in the
PBS control group, MSC group, and MSC plus Sal B group
were evaluated. Large cavities were found in the spinal cord
of rats in the PBS control group (Figure 4A). Fewer cavities
were found in the injured spinal cord of rats in the MSC
group (Figure 4B), but tissue structure seemed to be porous.
The anatomical integrity of the injured spinal cord of the rats
in the MSC plus Sal B group (Figure 4C) was better than
those in the MSC group, which suggested that the
enhancement of MSC survival could promote the restoration of
anatomical integrity of injured spinal cords.
The effect of MSC with or without Sal B treatment on
motor function recovery was assessed by the BBB scale
(Figure 5B). Treated animals generally showed better
performance of gait than the PBS control animals. MSC plus Sal
B-treated rats showed remarkable recovery (weight
supporting plantar steps and consistent coordination between the
forelimbs and hind limbs) on d 35, post-operation (BBB score
12.6±2.0). In contrast, MSC- treated rats had a BBB score of
10.1±1.8. From d 28, post-operation, there were significant
differences between the MSC-treated animals and the MSC
plus Sal B-treated animals (P<0.05).
Discussion
The present study provides evidence that Sal B improves
the recovery of motor function after contusive spinal cord
injury in rats, and significantly reduces the cavity area. The
rats that received Sal B at a dose of 8 mg/kg displayed a more
rapid recovery of BBB assessment performance than the
control rats receiving PBS only. The ability of Sal B to
improve motor function is probably partially due to its
protective effect on neural cells injured by contusion. This could
be because more neurons were left in the rats treated with
Sal B than in the control rats. The study also showed that
MSC were protected from TNF-α damage by the use of Sal B
in vitro, and Sal B promoted the survival of MSC
in vivo. The results in these studies suggested that Sal B may
provide neuroprotection to the transplanted MSC in SCI rats.
Sal B treatment may inhibit the increase of the level of
TNF-α in the damaged spinal cord after injury.
TNF-α directly activates neutrophils and increases the expression of
E-selectin, which contribute to the formation of cavities and
tissue injury[20,21]. Whether Sal B treatment reduces the
production of TNF-α should be detected by RT_PCR and
Western blotting in future studies.
One possibility of the benefits of Sal B treatment is that
Sal B induced the expression of a particular set of genes.
Neuroprotective genes, such as brain-derived neurotrophic
factors, are induced in a variety of conditions, such as brain
injury, cerebral ischemia, and electrical
stimulation[22,23]. Hence, if neuroprotective genes are directly induced by Sal
B, the factors that mediate that induction remain to be defined.
Any of these alterations in gene expression could play a role
in enhancing locomotor behavior and protecting spinal cords
against a progressive series of necrosis and cavitation after
injury.
It is known that the non-permissive environment caused
by immediate injury and second injury may have an adverse
effect on MSC transplantation to the injured spinal cord.
Because of this, many researchers selected d 7_10,
post-injury, as the transplantation time, however, MSC survival
was still not sufficient[6]. One of likely reasons for this, as
discussed by Coyne et al[11], was that injections may elicit
dangerous inflammatory reactions, which may cause the
transplanted cells to die. In our study, few transplanted
MSC survived on d 14, post-injury. Sal B treatment was
conducted when MSC were transplanted into the injured
rats in the present study. The results suggest Sal B
promotes MSC survival on d 35, post-injury. It was estimated
that injections may cause the production of TNF-α, and Sal
B might protect MSC in vivo.
In conclusion, the study suggests that Sal B provides
neuroprotection to SCI and can promote the survival of MSC
in vitro and after their transplantation to the injured spinal
cord. These findings suggest that a combination of Sal B
and MSC may be an effective way in the treatment of SCI.
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