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HO
CH 2OH
O
OH OH
OCH 2CH 2 OH
Figure 1. The chemical structure of salidroside.
In our present work, we established rat models of SCI
by hemisecting the spinal cord at the T8 vertebra and
attempted to study the effects of salidroside on Bcl-2/Bax
protein levels in rats with hemisection-induced SCI. The
Bcl-2/Bax protein levels were evaluated by
immunohistochemistry methods.
MATERIALS AND METHODS
Salidroside was purchased from China pharmaceutical and
biological products inspection (Lot: 110818-201005). MPSS was
obtained from Pharmacia and Upjohn Company (Belgium).
Animals
A total of 54 healthy, female, Sprague Dawley rats, aged 2 months,
weighing 180 to 220 g, were purchased from the Laboratory Animal
Centre of Zhejiang University, China. Animal care and experiments
were performed in accordance with the Guidelines for the Care and
Use of Laboratory Animals of Zhejiang University.
Establishment of SCI models
The SD rats were anesthetized with chloral hydrate, and placed in a
prone position on a heating pad to maintain a constant body
temperature. A longitudinal incision was made at the midline of the
back and the paravertebral muscles were exposed. These muscles
were dissected and thoracic level 7 to 11 (T7 to 11) vertebrae were
exposed. A laminectomy at T7 to 11 was performed. Acute SCI was
induced by hemisection at the T8 vertebra to a depth of 0.5 cm. The
wound was sutured layer-by-layer (Kim et al., 2009). Rats in the
Sham operation group experienced the same procedures, while
without the hemisection was at the T8 vertebra. Following spinal
cord hemisection, the rat tails swung spastically, and the affected
hind limbs exhibited flaccid paralysis after several spastic seizures.
Rats were sacrificed 24 h after administration of salidroside or 0.9%
normal saline (NS).
Drug treatment and sample preparation
The sham operation group underwent laminectomy to expose the
spinal cord without hemisection, and received 0.9% NS (2 ml/kg).
The SCI model group underwent laminectomy followed by SCI, and
received 0.9% NS (2 ml/kg). The MPSS treatment group, positive
control group underwent laminectomy followed by SCI, and was
administered 100 mg/kg single dose of MPSS (2 ml/kg, i.p.) 5 min
after hemisection. The 25, 50, and 100 mg/kg salidroside groups
Wang et al. 1939
underwent laminectomy followed by SCI, and were given a single
dose of 25, 50 or 100 mg/kg of salidroside (dissolved in 0.9% NS, 2
ml/kg, i.p.) 5 min after hemisection. 24 h after administration, the
rats were anesthetized with chloral hydrate (0.3 g/kg) transcardially
perfused with 150 ml of 0.9% NS and 200 ml of 4%
paraformaldehyde in 0.1 M PBS. Approximately 2 cm of spinal cord
segments between the T7 and T11 levels were obtained and
cryopreserved at -70°C for measurements of Bcl-2/Bax protein
levels.
Immunohistochemistry (ICH)
The spinal cord segments were cut on a freezing microtome into six
adjacent series of 4 μm-thick coronal sections. The sections were
dehydrated through an alcohol series. Prior to immunohistochemical
processing, sections were rinsed in 2% PBS-Triton X-100
and mounted onto gelatine-coated slides. Immunohistochemistry
was performed on slide-mounted sections utilizing the following
antibodies: Bax or Bcl-2 (dilution 1:100). The sections were
incubated overnight at room temperature with the primary antibody
diluted in PBS-bovine serum albumin (BSA). After rinsing, sections
were incubated for 1 h at room temperature in biotinylated goat
antimouse serum (1:500), sections were incubated for 1 h in avidin–
biotin–horseradish peroxidase complex (1:200). Following rinses,
sections were placed for 30 min in chromagen solution consisting of
0.05% diaminobenzidine and 0.01% H2O2. The reaction was
monitored visually and stopped by rinses of 0.1 M PBS. In order to
minimize variability, sections from all animals were stained
simultaneously. Cell counts were performed blindly in all sections
using a Nikon Eclipse E800 microscope. Counts were made in six
randomly selected optical fields under 400× magnification by
individuals who were blinded to diagnosis. Bcl-2 or Bax
immunoreactivity were assessed semi-quantitatively using Image
Pro Plus software Version 4.5.129 (Media Cybernetics). The
percentage area covered by immunoreactivity was measured and
the mean value taken.
Statistical analysis
Data were expressed as Mean ± SD, and analyzed by one-way
analysis of variance followed by Least Significance Difference
multiple comparison or Dunnett’s multiple comparison tests using
SPSS 16.0 software. Multiple comparison tests were used when
appropriate. A P-value of 0.05 was considered statistically
significant.
RESULTS
Quantitative analysis of experimental animals
Of the 72 selected adult SD rats, 60 were used to
establish the model of spinal cord hemisection and were
assigned to 5 groups (n=12), which were intraperitoneally
injected, respectively, with 25, 50 and 100 mg/kg of
salidroside, 100 mg/kg of MPSS, and 2 ml/kg of NS. The
remaining 12 rats served as the Sham operation group. A
total of 60 rats were finally included in the final analysis.
Bcl-2 expression following SCI
As shown in Figure 2 and Table 1, the protein levels of