Thirty
30 Wistar adult male rats weighing 200-230 g were
obtained from the Experimental Research Section of
Zonguldak Karaelmas University. Animals were maintained
in their cages at a constant room temperature using a 12 h: 12
h light/dark cycle and provided with commercially available
rat chow and top water ad libitum. All experimental
procedures described below were approved by the Ethical
Committee of the University.
On day before the surgical procedures, the animals were
fasted overnight but allowed ad libitum access to water.
Animals were anesthetized with sodium thiopenthal (50 mg/kg) followed by a midline incision made into the
peritoneal cavity. The small bowel was exteriorized gently to
the left onto moist gauze; then, the SMA was carefully
isolated and ligated by using a non-traumatic microvascular
clamp. Following 30 min of occlusion time, the clamp was
gently removed and the intestine inspected for proper
reperfusion. During the surgical procedure, the animal was
positioned under a heating lamp so that the body temperature
was kept at constant level (i.e. 37 ºC). Animals were
assigned to five groups: i) sham-operated control, subjected
to laparatomy without performing the clamping of SMA; ii)
I/R control, subjected to the clamping followed by
reperfusion; iii) adenosine + I/R; iv) A1AR agonist CPA +
I/R; and v) A1AR antagonist DPCPX + adenosine + I/R. In
each treatment, 10 ml of warmed (37 ºC) saline solution
containing either adenosine (10 nM) or A1AR agonist CPA
(10 nM) was placed into abdominal cavity 5 min before
inducing ischemia. To determine the adenosine receptor
subtype involved in adenosine-induced intestinal protection,
selective A1AR antagonist DPCPX (10 nM) was also
administered topically 15 min before adenosine treatment.
For the control groups, only saline solution was placed into
the abdominal cavity. The solution with or without any
treatment was let to stay in the abdominal cavity for 5 min
followed by gentile soacking the solution with absorbent
sterile gause. Dose regimen for adenosine and for specific
agonist or antagonist was based on the literature20,37,38.
Upon completing the 180 min of reperfusion period
followed by exanguination of abdominal aorta, a segment
(1 cm long) of terminal ileum 10 cm proximal to the
ileocecal area was rapidly excised and transferred into a Petri
dish containing Krebs solution (in mM: NaCl 118, NaHCO3
24.88, KH2PO4 1.18, KCl 4.7, MgSO4 1.16, CaCl2 2.52 and
glucose 11.1). Then, the strip was suspended in a standard
organ chamber which was continuously perfused with
preoxygenated Krebs solution (pH 7,4), which was bubbled
constantly with a mixture of %95 O2 and %5 CO2 and
maintained at a temperature of 37º C. One end of the strips
was tied to a fixed post and other attached to an isometric
force transducer under a resting tension of 2 g. The isometric
responses were recorded on the computer using MP 30 data
acquisition and analysis system (Biopac Systems Inc., CA,
USA). In organ bath, each strip was allowed to equilibrate
for 1 h with intervening washings at every 15 min before
adding any compound. Additional tissue samples were also
taken from the same region and stored at - 40 ºC for
biochemical measurements.
The contractile responses to various final doses of
carbachol ranging from 10-9 M through 10-2 M were
measured as it was pipetted into the organ bath in a
cumulative fashion at equal intervals. The amplitude of
contraction is expressed as grams per gram of tissue weigth.
Each experiment was performed with a tissue sample taken
from one animal.
Adenosine, carbachol, CPA, and DPCPX were
purchased from Sigma (St. Louis, MO, USA). They were
dissolved in double distilled water, except for CPA and
DPCPX which were initially prepared in DMSO and then
diluted in physiological saline. Adenosine, CPA, and
DPCPX were prepared freshly just before usage. Carbachol
was made up at different concentrations and kept frozen in
aliquots. Compounds for Krebs solution were purchased from Merck (Merck KGaA, Darmstadt, Germany). All other
reagents were obtained from Sigma.
Tissue MDA was measured to estimate the extent of
lipid peroxidation in the tissues. Tissue samples were
washed in ice-cold Krebs solution, blotted on absorbent paper
and weight. Afterwards, each sample was minced followed
by homogenizing with 10% TCA in ratio 1:10 w/v, using
motor-driven homogenizer. Then, the tissue MDA levels
were measured spectrophotometrically according to a method
described by Casini et al39 and expressed as nmol/g of
tissue. The GSH content, expressed as μmol/g of tissue, was
measured using a modified Ellman method40.
Data are presented as the mean ± standard error (SE) of
the mean. All statistical procedure was performed using SPSS
statistical software (11.0, SPSS Inc., Chicago, IL, USA)
package program. Kruskal-Wallis H test was applied for
statistical comparison of groups, followed by analysis with
Bonferroni-corrected Mann-Whitney test so as to determine
the different groups. Probability values of 0.05 or less were
considered statistically meaningful.
)
)