MATERIALS AND METHODS
This protocol was approved by the Keio University School of Medicine Council on
Animal Care in accordance with the guideline of National Institute of Health.
Preparatory surgery
Thirty-two healthy rabbits (New Zealand White, male, SEASCO, Saitama, Japan),
weighing 2.0-2.5 Kg (average 2.3 Kg) and fasted for 24 hours, underwent the
instrumentation under general anesthesia. With sevoflurane 3-4% inhalation in oxygen
(3-4 L/min) via facemask, the animals underwent tracheostomy and intravenous line
access on the marginal ear vein. The animals were then mechanically ventilated to
maintain normocapnia (fraction of inspiratory oxygen 0.3, inspiratory pressure 12-15
cmH2O and 10-12 breaths/min) using intensive care unit type ventilator (New Port
E-100, New Port Medical Inc., CA). An indwelling arterial catheter (22 gauge) was
inserted into the right carotid artery.
Following a midline abdominal incision, a
perivascular probe was attached around portal vein for measurement of portal blood
flow (Transit-Time Ultrasound Flowmeter, T206; Transomic System Inc., Ithaca, NY)
(15). A flexible catheter was inserted through mesenteric vein to the distal portion of
the portal vein for subsequent blood sampling.
A sigmoid tonometer catheter
(Tonometrics, Worcester, MA) was surgically inserted into the terminal ileum via
ilieocecal portion as described previously (15).
To obviate the effects of inhalational
anesthesia and simultaneously to mimic the animals to critically ill patients in intensive
care unit, inhalational anesthesia was discontinued after the closure of laparotomy and a
mixture of buprenorphine (0.1 mg/mL), midazolam (2 mg/mL) and pancuronium (1
mg/mL) was continuously infused at a rate of 1 mL/kg/hr as described previously (16).
Rectal temperature was monitored and maintained at approximately 37ºC to 38ºC.
Study Protocol
After the preparatory surgery and equilibration period for 45 min, the rabbits were
randomly assigned to two groups, normocapnia (n = 17) and hypercapnia (n = 15)
groups, using computer-generated random numbers.
The latter group received FICO2
5% under mechanical ventilation to achieve hypercapnia throughout the study periods,
whereas the former with FICO2 0%.
In our pilot study, 45 min was long enough to
stabilize systemic hemodynamic parameters, arterial blood gas and lactate level after the
preparatory surgery. The baseline measurements described in the Measurement of
intramucosal pH (pHi), gut mucosal permeability and biochemical analyses were
performed (baseline) (Figure 1).
Another 45min later, lipopolysaccharide (LPS from
Escherichia coli serotype 055:55B5; Sigma Chemical, St Louis, MO) infusion at the
rate of 15 g/kg/min was initiated to all animals, accompanied by Ringer’s acetate
infusion (15mL/kg/hr) throughout the study periods.
The measurements were repeated
at 0, 2 and 4 hr time periods during LPS infusion.
After the 4 hr study, we determined the changes of gut mucosal permeability
using fluorescein isothiocyanate-conjugated dextran with a molecular weight of 4,000
Da (FD4) and an in situ loop of gut as described below.
After tissue sampling of lung
and ileum, the animals were sacrificed with intravenous pentobarbital overdose.
Additionally, to determine the time-course effects of hypercapnic acidosis on normal
rabbits in our experimental setting, we studied 8 sham animals (normocapnia, n = 4 or
hypercapnia, n = 4) which received the same catheterization without LPS infusion under
the same Study Protocol.
Measurement of intramucosal pH (pHi), gut mucosal permeability and biochemical
analyses
Gut intramucosal pH (pHi) was monitored using an automated air tonometry (Tonocap,
Datex Ohmeda, Helsinki, Finland) (16).
The measured regional PCO2 (PrCO2),
together with simultaneously obtained arterial [HCO3-], were applied in the
Henderson-Hasselbalch equation for calculation of pHi according to the manufacture’s
instruction:
pHi = 6.1 + log[HCO3-] / 0.03 x PrCO2
[HCO3-] being the arterial bicarbonate concentration, 6.1 the dissociation constant of
HCO3- and 0.03 the solubility of CO2 in plasma.
Previous studies demonstrated that
air tonometry exhibited good correlation with saline tonometry, with particular
advantages on shorter equilibration times (17).
To examine the effects of systemic
acidosis on pHi over the hypercapnic and endotoxemic stages, we further examined the
PCO2 gap defined as the difference between measured PrCO2 and PaCO2.
Gut mucosal permeability was measured by using an in situ loop preparation as
described previously (16,18).
Briefly, double ligature at both ends were made on the
10cm-length of terminal ileum.
Through a cannula placed into this segment of
terminal ileum, fluorescein isothiocyanate-conjugated dextran (FD4) (50mg) was
injected.
After 30 min, blood samples from portal vein were obtained and then
centrifuged, and plasma FD4 concentrations were measured using fluorescence
spectrometry (Spectrofluorophotometer:
RF-1500, Shimadzu, Kyoto, Japan).
Since
plasma protein concentration in this animal model varied between individual animals
possibly depending on the severity of increased vascular permeability and aggressive
fluid resuscitation, the results were corrected for the plasma protein contents measured
by the Lowry method (19).
Arterial blood samples were used to determine white blood cell (WBC) by an
analyzer (Celltac, Nihon Kohden, Tokyo, Japan), and blood gas and lactate by a
standard blood gas analyzer (ABL 700 series, Radiometer Trading, Copenhagen).
Residual arterial blood and portal blood samples were centrifuged at 2,500rpm for 10
min at 4°C, and then stored at -80°C for measurements of lactate dehydrogenase (LDH)
activity which was measured by spectrophtometric assay (Cayman, Detroit, MI) as
described previously (16).
As an index of leukocyte sequestration, MPO activity of
ileal wall was assayed as reported previously with minor modification (20). Briefly, a
small portion of the terminal ileum was frozen in liquid nitrogen and stored -80 °C for
subsequent assay.
The samples were homogenized at 4°C and placed into 0.5%
hexyldecyltrimethyl ammonium bromide in 50mmol/L potassium phosphate solution
(pH 6.0; 1mL/100mg-ileum).
Tissue was sonicated on ice and underwent three
freeze-thaw cycles (liquid nitrogen bath/ 37°C water bath).
The solution was
centrifuged at 18,000g for 20 min at 4°C. Aliquots (0.04 mL) of supernatant were
added to 0.96 mL of assay buffer (0.17 mg/mL) and measured after 5 min of incubation
by spectrophotometry at 492nm (Spectrofluorophotometer: RF-1500, Shimazu, Kyoto,
Japan).
MPO activity was expressed as units per milligram of protein.
Measurements of all parameters were performed in duplicate and mean values were
used for results.
Analyses of BALF and wet-to-dry weight ratio of lung and terminal ileum
At the completion of experiments, a randomly selected left or right lung was lavaged to
measure protein concentration of bronchoalveolar lavage fluid (BALF) in duplicate as
described elsewhere (21).
curve.
Bovine serum albumin was used to construct the standard
Five 2-cm fragment of the lung contralateral to that used for BALF collection,
as well as ileal tissues located 10 cm proximal to in situ loop preparation were harvested
and set aside for the measurement of wet to dry weight ratio.
Lungs and ileal tissues
were weighed before and after drying up in a vacuum oven (DP22; Yamato Scientific,
Tokyo, Japan) at 95°C and -20 cm H2O for 48 h to calculate the wet-to-dry weight ratio.
These data were not corrected for residual blood in lungs and iluem.
Statistical analysis
Data are expressed as mean ± standard deviation unless otherwise specified.
Analysis
of variance with repeated measures was employed to evaluate the differences using
SPSS/12.0J for Windows (SPSS Inc., Chicago, IL).
if the interaction was statistically significant.
Separate analysis was performed
When P<0.05, the Scheffe multiple
comparison test was applied to distinguish differences between measurement variables.
If the data were not normally distributed, the Friedman test was used to evaluate
pair-wise comparisons.
Since the sham animals were not randomized with the others,
statistical analyses versus the endotoxemic rabbits were not applied.
considered statistically significant if P < 0.05.
Differences were