Adult male Sprague-Dawley rats (Harlan, Madison, WI) were used for all experiments. They had ad libitum access to water and standard chow (Purina) during exposure to either CIH or normoxia (NORM). Room temperature and relative humidity were maintained at 24±1° C and 20–70%. Rats were housed in accordance with recommendations set forth in the National Institutes of Health Guide for the Care of Laboratory Animals (NIH Pub. No. 85–23, Revised 1985). All protocols were approved by the University of Wisconsin-Madison School of Medicine and Public Health’s Institutional Animal Care and Use Committee.
In addition to CIH and NORM rats (n=9 per group) two additional groups of rats were treated with losartan in the drinking water (25–30 mg/kg/day) for 7 days prior to and during the 28-day exposure period (CIH-Los and NORM-Los, n=8 per group). A subset of rats from each group (n=4–5) was instrumented with indwelling femoral catheters to allow measurement of blood gases in the unanesthetized state on the 27th day of the exposure period. Body weight before exposure was not significantly different between groups (249±10, 239±4, 246±6, and 236±3 g, for NORM, CIH, NORM-Los, and CIH-Los respectively, p=0.463 by ANOVA).
2.2 Chronic intermittent hypoxia (CIH) and normoxia (NORM) exposures
The CIH protocol was identical to one we previously used to demonstrate impaired endothelium-dependent vasodilation (Phillips et al., 2004
) and increased arterial pressure (10–15 mmHg) (Marcus et al., 2009
). Briefly, rats in their home cages were placed into a Plexiglas chamber and exposed to intermittent hypoxia for 12 hours per day (from 18:00 hours to 06:00 hours) for 28 days. Oxygen concentration in the chamber was monitored using a heated zirconium sensor (Fujikura America, Pittsburgh, PA). A microprocessor-controlled timer was used to operate solenoid valves that controlled the flow of oxygen and nitrogen into the chamber to provide hypoxic exposures at 4-minute intervals. During the first minute of each cycle, nitrogen was introduced at a rate sufficient to achieve a fraction of inspired oxygen (FIO2
) of 0.10 within 45 seconds and to maintain this level of FIO2
for an additional 60 seconds. Then, oxygen was introduced at a rate sufficient to achieve an FIO2
of 0.21 within 30 seconds and to maintain this level of FIO2
for the remainder of the 4-minute cycle. Control rats were housed under normoxic conditions adjacent to the hypoxia chamber for 28 days. There they were subjected to light, noise, and temperature stimuli similar to those experienced by the CIH rats.
2.3 Arterial blood gases in unanesthetized rats
After 14 days of CIH or NORM exposure, catheters were placed into the abdominal aorta distal to the renal arteries via the femoral artery. The catheter tubing was exteriorized by tunneling it beneath the skin to an opening between the scapulae. On day 27 of CIH or NORM exposure, rats were removed from their home cages and placed in a Plexiglas animal carrier for sampling of arterial blood under normoxic conditions. Rats were allowed to acclimatize to the surroundings for at least 30 minutes and care was taken to insure that rats were not sniffing or exploring when blood was drawn. Blood samples (0.2ml) were analyzed (Radiometer Copenhagen ABL505) and corrected for body temperature.
2.4 Surgical preparation
Within 4 hours of the end of the final exposure period on day 28, rats were anesthetized for study of chemoreflex function. Anesthesia was induced with isoflurane (5% in O2) and maintained with 2.5–3% isoflurane in O2. The right femoral artery and vein were cannulated for measurement of arterial pressure and blood gases and infusion of fluids and drugs. Rats were tracheotomized, paralyzed (pancuronium bromide, 3.5 mg/kg, i.v.), and mechanically ventilated (Rodent Ventilator 683, Harvard Apparatus, Holliston, MA, USA), and subsequently converted from isoflurane to urethane anesthesia (1.6–1.8 g/kg, i.v.) over the course of 20–25 minutes. An intravenous infusion of lactated Ringer’s solution (0.5–1 ml/hour) was maintained throughout the surgery and experiment. Rectal temperature was maintained at 37±1° C. using a heat lamp and heating pad.
2.5 Recording of sympathetic nerve activity
The lumbar sympathetic chain between the renal and inferior mesenteric arteries was exposed through a midline abdominal incision. The nerves were dissected free, placed on bipolar platinum electrodes, and covered with silicone elastomer (Kwik-Cast, World Precision Instruments, Sarasota, FL). The electrode wires were connected to a high impedance probe (Grass HIP511). Action potentials were amplified 20 thousand-fold by a Grass P511 AC Amplifier and bandpass filtered with a bandwidth of 100–1000 Hz. The filtered neurogram was routed through a storage oscilloscope (Gould Model 420, Cleveland, OH) and an amplitude discriminator to an audio amplifier. For permanent recording and analysis, the filtered neurogram was routed though a nerve-traffic analyzer (University of Iowa Bioengineering Model 66C-2), which counts nerve spikes exceeding a threshold voltage level (time constant, 0.5 sec), to a Grass Model 7 polygraph and Windaq computer data acquisition system (Dataq Instruments, Akron, OH). At the beginning of each experiment the threshold voltage level was set using a window discriminator to a level just above the background noise as determined during phenylephrine-induced sympathoinhibition. The window remained constant throughout the experiment.
We used the scaling method to quantify LSNA (Guild et al., 2009
). At the end of each experiment, the 0% LSNA voltage level was measured during ganglionic blockade with pentolinium tartrate (10 mg/kg, i.v.). The maximal (or 100%) calibration voltage was determined in each rat by passing ammonia vapor over the nasal mucosa (Dorward et al., 1985
). Absolute values for maximal voltage elicited by this stimulus were comparable in all rats (0.17±0.02, 0.18±0.03, 0.18±0.02, and 0.17±0.05 arbitrary units for NORM, CIH, NORM-Los, and CIH-Los respectively). For all analyses, LSNA was expressed as a percentage of the maximal LSNA during the nasopharyngeal response.
2.6 Physiological measurements
Blood pressure, arterial oxygen saturation (SaO2; MouseOx, Starr Life Sciences, Oakmont, PA), and end-tidal CO2 (PETCO2; Capnogard, Respironics Novametrix, Wallingford, CT) were measured continuously during the experiment. Arterial blood gases were measured periodically from 0.2 ml samples (Radiometer Copenhagen ABL505). We monitored depth of anesthesia by assessing changes in blood pressure in response to a foot pinch. Urethane was supplemented as necessary to maintain a surgical plane of anesthesia.
2.7 Chemoreflex testing protocol
Response to apneas
After surgical procedures were completed, arterial blood gases were measured. Using these measurements, ventilatory rate and tidal volume were adjusted until blood gases matched the mean values measured in unanesthetized rats from the same experimental group. After a stable baseline had been established, we began a series of six 20-second apneas produced by turning off the ventilator.
In the first set of three apneas, LSNA, mean arterial pressure (MAP), and arterial oxygen saturation were measured. The first three apneas were separated by 30 minutes each, because we were concerned that more frequent apneas would elicit long-term facilitation of LSNA as has been demonstrated for phrenic nerve discharge (Bach et al., 1999
). During the second set of apneas, 0.2 ml of blood was drawn at end-apnea for determination of arterial blood gases. After each blood draw, 0.5 ml of lactated Ringer’s solution was infused to flush the catheter and replace volume. Because we considered it unlikely that long-term facilitation could affect arterial blood gases in paralyzed, mechanically ventilated rats, the second set of three apneas were separated by 10 minutes in the interest of time. At least 10 minutes after the final apnea, another arterial blood sample was taken to verify that the preparation remained stable during the experimental period.
Response to isocapnic hypoxia
We exposed a subset of rats from each group to three 2-minute periods of isocapnic hypoxia (FIO2, 0.12). The LSNA response to hypoxia was quantified by calculating the area under the LSNA curve (see below) for the first 30 seconds during hypoxia exposure and for the final 30 seconds of the pre-hypoxia baseline. We chose to analyze only the first 30 seconds of hypoxia exposure because longer periods of hypoxia caused decreases in blood pressure. The mean value for change in area under the curve (Δ AUC) from baseline to hypoxia in the three trials was used as an index of chemoreflex sensitivity.
Response to cyanide
Potassium cyanide was administered in three successive injections (30 μg/kg, i.v.) separated by 5 minutes Because of the rapid, transient LSNA responses to bolus injections of cyanide, we measured initial peak responses (averaged over 0.5 seconds) within 4 sec of injection. For each trial, the LSNA response was calculated by subtracting the peak LSNA value during cyanide (expressed as % of ammonia maximum) minus the value for the 60-sec baseline immediately prior to injection (also expressed as % of ammonia maximum). LSNA response values were averaged across the three trials.
2.8 Stimulation of cutaneous afferents
To determine the effect of CIH on sympathetic responses to non-chemoreflex stimuli, we immersed the rat’s tail in hot water (55o C.) for 60 seconds, an intervention that produced transient increases in sympathetic activity and blood pressure. To quantify this response we subtracted the peak LSNA value during stimulation (expressed as % of ammonia maximum) minus the value for the 60-sec baseline immediately prior to immersion (also expressed as % of ammonia maximum).
2.9 Pressor response to angiotensin II
Following chemoreflex testing, we determined the efficacy of AT1R blockade by measuring the peak increase in mean arterial pressure elicited by intravenous injection of Ang II (25 ng/kg). Pressor responses were averaged across the three trials.
2.10 Carotid body superoxide production
Both carotid bodies were removed, flash frozen in liquid nitrogen, and stored at −80° C. until analyzed. Superoxide anion production was measured using the lucigenin chemiluminescence method. Carotid body samples were homogenized and centrifuged (3000 rpm) at 4° C. for 5 minutes. The supernatant was placed in 0.5 ml microfuge containing dark adapted lucigenin (5 μM), then read in a TD-20/20 Luminometer (Turner Designs, Sunnyvale, CA). Light emission was recorded for 5 minutes and expressed as mean light units (MLU)/min/100 μg protein.
2.11 Western blot analysis for carotid body AT1R, nNOS, and NADPH oxidase content
Carotid bodies were harvested (as described above) and pooled (two carotid bodies from each rat) to yield 25–30 μg protein/rat. We performed 4–6 blots in each experimental group, for which protein samples were obtained from 4–6 rats. In addition, using a stripping buffer, we used one membrane to measure different target proteins. The procedures for Western blot analysis have previously been described (Ding, Y. et al., 2008
) and are briefly summarized here. The protein was extracted with lysing buffer (10 mM PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1% SDS) plus protease inhibitor cocktail (100 μl/ml) and centrifuged at 12.000 g for 20 minutes at 4o C. Five μg of protein (as determined by BCA protein assay kit from Pierce Chemical, Rockford, IL) was mixed with loading buffer containing mercaptoethanol, heated at 100° C. for 5 minutes, separated in a 10% polyacrylamide gel along with molecular weight standards, and transferred to a PVDF membrane. For AT1
R and nNOS determinations, the membrane was probed with a mouse anti-AT1
R and mouse monoclonal anti-nNOS (Santa Cruz Biotechnology, Santa Cruz, CA) and a peroxidase-conjugated goat anti-mouse IgG (Pierce Chemical, Rockford, IL). For NADPH oxidase components the membrane was probed with goat anti gp91phox
antibodies (Santa Cruz, CA, USA) and a peroxidase-conjugated rabbit anti-goat IgG (Santa Cruz, CA, USA). The signals were detected using enhanced chemiluminescence substrate (Pierce) and the bands were analyzed using UVP BioImaging Systems. Protein loading was controlled by probing all Western blots with mouse anti-GAPDH antibody (Santa Cruz) or β-actin (NADPH oxidase components) and normalizing AT1R, nNOS, gp91phox
protein intensity to that of GAPDH or β-actin as appropriate.
2.12 Data analysis
During apneas, LSNA (% maximum) and arterial oxygen saturation were averaged in 2.5-second bins to produce stimulus response curves. For each apnea, the stimulus response curve was quantified using the following logistic expression: R = Rmin + (Rmax−Rmin)/(1+(D/EC50)^S), where R=LSNA response, Rmin=baseline LSNA, Rmax=peak LSNA, D=arterial oxygen saturation, and S= Hill slope coefficient determining the steepness of each curve. If the R2 value for the curve fitting was less than 0.7, the data were not used. Slope coefficients were used to compare chemoreflex sensitivity between groups.
To quantify LSNA responses to isocapnic hypoxia, the area under the curve was calculated by graphing all data points within the time periods of interest (Sigmaplot software, version 8.02, Systat software, Chicago IL) and using the ‘area below curves’ function to calculate an AUC for each exposure period and pre-exposure baseline.
All data are presented as means±SEM. Animal weight and age at the beginning of the experimental protocol was analyzed by 1-way ANOVA. Within-group determinations of change in LSNA, PETCO2
, and SaO2
relative to zero were made using t-tests. Unpaired t-tests were used to compare all other variables between exposure group (i.e.
CIH vs. NORM and CIH-Los vs. NORM-Los). We did not make comparisons across treatment groups (losartan-treated vs. untreated) in order to guard against the possibility that the drug would affect our tests of chemoreflex responsiveness in a non-specific way (e.g.
via blood pressure lowering or its effect on central sympathetic outflow, plasma renin activity, plasma or tissue Ang II) (Gradman et al., 2008
; Li et al., 1996
). Statistical significance was accepted when p<0.05.