18 - World Journal of Gastroenterology

2576 ISSN 1007-9327 CN 14-1219/R World J Gastroenterol May 14, 2007 Volume 13 Number 18
which exists extensively in genomes of eukaryotes. It
participates in normal cell growth and proliferation and
regulates message transfer in cells. Most previous studies
have focused on c-Fos expression that is induced by the
controlled and natural irritations [2-4] Recently, it has been
proven that the normal motion of the digestive tube relies
on reflex activity controlled by extrinsic nerves and enteric
nervous system (ENS). c-Fos as the third messenger,
which regulates the target gene, provides a reliable and
direct method to study the mechanism of functional
gastrointestinal diseases [5] . Our previous study [6] reported
that the c-Fos expression in the gastric myenteric plexus
was dramatically associated with c-Fos expression of the
spinal cord in the rats with cervical spondylosis. It is the
sympathetic nerve that results in the c-Fos expression
both in the spinal cord and the gastric myenteric plexus
in cervical spondylosis and this suggests that the
gastrointestinal function may be affected by cervical
spondylosis. To provide further evidence, c-Fos, caspase-3
and IL-1β were detected in the cord and stomach. The
rationale being that caspase-3 is a potential mediator of
apoptosis after central nerve system (CNS) injury [7,8] and
its activation may be used as a marker of apoptotic cell
death. Several studies have provided evidence that cell
death from moderately severe spinal cord injury (SCI) is
regulated, in part, by apoptosis that involves the caspase
family of cysteine proteases [8,9] . In the hippocampus of
aged rats, the concentration of IL-1β is increased and this
increase is accompanied by enhanced caspase-3 activity
indicative of cell death [10] . These findings suggest that
neuronal apoptosis in the CNS is induced by increased
IL-1β through the activity of the caspase-3 apoptotic
pathway. IL-1β induced apoptosis in neurons in vitro [11]
and in cultured human astrocytes [12] and oligodendrocytes
in vivo [13] .
In the present study, after establishing the cervical
spondylosis model of rats according to a previously
described method [6] , the cord and stomach were collected
at 3 mo and 5 mo to determine the expression of c-Fos,
caspase-3 and IL-1β in the cervical cord and gastric
antrum by immunohistochemistry and/or Western blot.
MATERIALS AND METHODS
Animal models
Ninety-six four-month-old Sprague Dawley rats (provided
by the Experimental Animal Center of Shantou University
Medical College, Shantou, China), weighting 250 g (range,
220-280 g), were used in this study. The rats were randomly
divided into model and control groups and fed a normal
diet, with eight in one big cage, and kept for 3 mo and 5
mo, respectively, after the experimental or sham operations
as described below. Each group consisted of 12 male and
12 female rats at each time point.
The rats in the model group were anesthetized
by intraperitoneal (ip) injection of 40 mg/kg sodium
pentobarbital . The dorsal neck was shaved and a
longitudinal incision about 2.5 cm was made. The dorsal
muscles were reserved, the spinal processes were removed,
as well as the inter-spinal ligaments, the capsule of articular
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processes and part of the superior and inferior articular
processes between C3-7 levels were removed till the
movement between the neighboring superior and inferior
laminae was obviously increased after the operation, and
the incision was closed. Three and five months after
the operation, the models were confirmed by evaluating
X-ray films and the motion function with oblique board
test according to the previous studies [14,15] . X-ray films
showed disappeared or stiff nature cervical curve, stenosis
of the vertebral space and osteosis spur in the model
groups compared with the control groups. To test motion
function, the rats were put on a tilted board, and the angle
between the board and horizontal plane was recorded,
which showed a significant difference in control groups
and model groups. The rats in the control group (sham
operation group) had only a longitudinal incision on the
dorsal neck which was closed without further intervention.
Immunohistochemistry
The rats were euthanized with a lethal dose of
pentobarbital sodium (100 mg/kg) and perfused via
cardiac puncture with 0.1 mol/L phosphate-buffered
saline (PBS) (pH 7.4; 150 mL) and subsequently with 40%
paraformaldehyde in 0.1 mol/L PBS (250 mL). The cord
and gastric antrum tissues were dissected out and postfixed
by immersion in 40 mL/L paraformaldehyde for 3 h
and then cryoprotected by immersion in 200 g/L sucrose
(in PBS) overnight. Tissue segments were embedded in
Tissue-Tek O.C.T. (Lab-Tek Division, Miles Lab, Inc.) and
frozen as previously described [16] . Free-floating transverse
sections (10 μm) were cut with a cryostat.
Immunohistochemical staining was carried out
using the avidin–biotin complex method as previously
described [6,17] . The concentrations for rabbit anti-IL-
1β, rabbit anti-caspase-3 and rabbit polyclonal anti-c-fos
primary antibodies were 1:200, 1:200 (Sigma, Genetimes
Technology Inc.) and 1:100 000 (diluted in NGS-T-
PBS). Positive immunoreactive labeling was observed
qualitatively.
The method for gastric neuronal counterstaining was
adapted from previous studies [6,18,19] . c-Fos cells were
counted under microscopy in 25 ganglia from each antral
preparation and expressed as a mean percentage count per
myenteric ganglion. Myenteric ganglia were recognized as
clearly delineated groups of neurons separated by welldefined
internodal fiber tracts. The mean from all animals
in each group was used to calculate the group mean. Data
were expressed as mean ± SD of the number of cells
or neurons per ganglion. Buffy spots or particles in cells
were regarded as positive expression of caspase-3 and IL-
1β. The random sections were observed under the optical
microscope at 200 × magnification, the positive cells were
counted in ten random visual fields, and then the mean in
each group was calculated.
Western blot analysis
At 3 mo and 5 mo after the destabilizing operation,
an 8 mm spinal cord segment was dissected 4 mm
rostral and 4 mm caudal from the center of the C3-7
cord from each group. Five millimeters of the gastric