Central neural plasticity in respiratory control has been a subject of increasing interest in recent years (
Feldman et al., 2003;
Huey et al., 2003;
Mitchell and Johnson, 2003;
Mahamed and Mitchell, 2006; Prahbakar et al., 2007;
Zimmer et al., 2007). One of the most extensively studied examples of respiratory plasticity is respiratory long-term facilitation (LTF), a prolonged increase in respiratory motor output that cannot be accounted for by changes in chemoreceptor stimuli (
Baker et al., 2001;
Mitchell et al., 2001a;
Feldman et al., 2003;
Mahamed and Mitchell, 2006). Millhorn and colleagues were the first to describe LTF, although it was not referred to as LTF at that time (
Millhorn et al., 1980a,
b;
Eldridge and Millhorn, 1986). In their pioneering studies on anesthetized and vagotomized cats, Millhorn and colleagues electrically stimulated the carotid sinus nerve in an episodic pattern, and observed that inspiratory phrenic nerve activity remained increased above baseline levels for at least 90 min post-stimulation (
Millhorn et al., 1980a). Although this prolonged enhancement of phrenic activity was primarily expressed as increased phrenic burst amplitude (the neural equivalent of tidal volume), lesser increases in phrenic burst frequency were also observed (the neural equivalent of breathing frequency). Similar LTF is also observed following acute intermittent hypoxia (AIH) (
Hayashi et al., 1993;
Bach and Mitchell, 1996; for review, see:
Powell et al., 1998;
Mitchell et al., 2001a).
LTF has now been reported in many species under different experimental conditions (for review, see:
Fuller et al., 2000;
Baker et al., 2001,
Mitchell et al., 2001a;
Feldman et al., 2003;
Mahamed and Mitchell, 2006). In general, studies on anesthetized animals report that LTF is expressed as increased burst amplitude from respiratory-related nerves, such as the phrenic or hypoglossal nerve (for review see
Mitchell et al., 2001a;
Feldman et al., 2003), with inconsistent changes in burst frequency. By contrast, studies on awake animals generally report that LTF is associated with increased breathing frequency, with small and inconsistent changes in tidal volume (
Turner and Mitchell 1997;
Mitchell et al., 2001b;
Olson et al., 2001;
McGuire et al., 2002,
2003,
2004;
Kline et al., 2002;
McGuire and Ling 2005;
Terada et al., 2008). LTF in awake animals also tends to be of smaller magnitude and shorter duration than in anesthetized preparations (
Mitchell et al., 2001a). In reduced, but unanesthetized preparations, such as the working heart brainstem preparation (
Tadjalli et al., 2007) or the rhythmogenic brainstem slice (
Blitz and Ramirez, 2002), LTF is also expressed largely as increased respiration-related nerve burst frequency (but note that
in vitro LTF induced with neuromodulators and not hypoxia primarily involves amplitude changes;
Lovett-Barr et al., 2006,
Bocchiaro and Feldman, 2004,
Neverova et al., 2007). On the other hand, in documented slow wave sleep, AIH-induced ventilatory LTF is expressed as increased tidal volume (
Nakamura et al., 2006;
Pierchala et al., 2007;
Terada et al., 2008), as well as increased breathing frequency (
Nakamura et al., 2006;
Terada et al., 2008). The significance of differences in frequency versus amplitude responses under different experimental conditions is unclear, but may suggest that different neural mechanisms underlie prolonged increases in ventilation (or its neural analog) in different experimental conditions.
In order to better understand factors that influence frequency LTF, we performed a meta-analysis on an extensive data set collected in our laboratory by different investigators using similar equipment and experimental protocols on anesthetized and vagotomized male rats, the most extensively used model for studies of cellular/synaptic mechanisms of LTF (
Mitchell et al., 2001a;
Feldman et al., 2003;
Mahamed and Mitchell, 2006). These data were taken largely from published studies (
Baker and Mitchell, 2000;
Zabka et al., 2001a,
2006;
Fuller et al., 2001a,
b;
Baker-Herman and Mitchell, 2002;
Bavis and Mitchell, 2003;
Behan et al., 2003;
Baker-Herman et al., 2004;
Golder and Mitchell 2005;
Golder et al., 2008;
Wilkerson et al., 2008;
MacFarlane and Mitchell, 2007a,
b;
Mahamed and Mitchell, 2008). All data were collected since our previous meta-analysis in 2000 (
Fuller et al., 2000), which focused exclusively on phrenic amplitude LTF without consideration of changes in phrenic burst frequency.
Our meta-analysis confirmed significant AIH-induced phrenic amplitude and frequency LTF in anesthetized, vagotomized rats; however, frequency LTF in this preparation is considerably smaller than phrenic amplitude LTF. We also report a strong correlation of frequency LTF with the initial, pre-hypoxia baseline burst frequency. This relationship may account for considerable variation in reported values of frequency LTF, even by the same investigator using the same experimental protocol, and may underlie the apparent effects of certain drugs. Thus, our analysis reveals that frequency LTF in anesthetized rats is small and variable, and cautions that loss of frequency LTF following certain experimental manipulations must be interpreted with consideration of this fact. Anesthetized, vagotomized rat preparations are more suited to studies of phrenic amplitude LTF, where the results are robust and repeatable.
2.1. Methods
302 male Sasco/Charles River and Harlan Sprague Dawley rats from 18 different studies were used in our analysis. Animal husbandry and all procedures were approved by the Institutional Animal Care and Use Committee of the School of Veterinary Medicine at the University of Wisconsin, Madison.
2.2. Surgical preparation
All rats used in the analysis were prepared similarly, although there are undoubtedly subtle investigator differences in how the protocols were performed. Rats were anesthetized initially with isoflurane in 50% O2 (balance N2) and then placed on a custom-designed heated table to maintain body temperature at 37–38°C. The rats were tracheostomized, vagotomized and pump-ventilated (2.0–2.5 ml, rodent respirator model 683; Harvard Apparatus, South Natick, MA). The femoral artery was cannulated to sample blood gases (ABL-500; Radiometer, Copenhagen, Denmark) and monitor blood pressure, and either the femoral or a tail vein was cannulated to deliver drugs and fluids (5 ml/kg/h, lactated Ringers with 0.8% sodium bicarbonate i.v.). The left phrenic nerve was isolated via a dorsal approach, desheathed, placed on bipolar silver recording electrodes and submerged in mineral oil. End-tidal PCO2 was measured continuously using a CO2 monitor (Capnogard; Novametrix Medical Systems, Wallingford, CT) with sufficient response time to measure end-tidal CO2 in anesthetized rats. Following surgery, the rats were converted to urethane anesthesia (1.6–1.7 g/kg i.v.) while isoflurane was gradually discontinued over a 10–20 min period. Rats received pancuronium bromide (1 mg/kg i.v.) for neuro-muscular paralysis (supplemented as necessary to prevent spontaneous breathing movements).
In some protocols, rats received additional “sham surgeries” that were not considered relevant to the analysis. For example, some rats were prepared for an intrathecal catheter as described in
Baker-Herman and Mitchell (2002), but only those receiving vehicle treatments were included in the analysis. Others received sham surgery for gonadectomy (
Behan et al., 2003), spinal hemisection (
Golder and Mitchell, 2005) or carotid sinus nerve transection (
Bavis and Mitchell, 2003). No differences were noted between these experimental groups and those that did not receive sham surgery.
Phrenic nerve activity was amplified (gain, 10,000; A-M Systems, Everett, WA), band-passed filtered (100 Hz to 10 kHz), rectified, and processed with a moving averager (CWE 821 filter; Paynter, Ardmore, PA; time constant 50 ms). The signal was digitized, recorded, and analyzed using the WINDAQ data-acquisition system (DATAQ Instruments, Akron, OH). Blood pressure and phrenic nerve activity were allowed to stabilize for at least 1 hour following urethane administration before the CO2 recruitment threshold was determined to allow establishment of standardized baseline conditions.
2.3. Experimental protocol
The apneic and recruitment thresholds for CO2 were measured in each rat. Briefly, end-tidal PCO2 was decreased by altering ventilator frequency and/or inspired CO2 until phrenic inspiratory activity ceased, and then slowly increased until phrenic inspiratory activity resumed. The end-tidal PCO2 in which phrenic inspiratory activity resumed was deemed to be the recruitment threshold. Baseline PCO2 was set at 2–3 mmHg above this threshold, and then the integrated phrenic neurogram was recorded for 20–30 min to establish baseline values of respiratory activity.
Following establishment of baseline conditions, rats were exposed to one of three protocols: acute intermittent hypoxia (AIH), acute sustained hypoxia (ASH) or no hypoxia (sham/controls). Hypoxia was created by changing the inspired gas mixture from 50% O2 to 11% O2 (balance N2). Intermittent hypoxia consisted of 3 hypoxic episodes separated by a 5 min return to baseline conditions. The duration of the hypoxic episodes varied from 3–5 min, depending on the study. Sustained hypoxia consisted of one 9–25 min episode of hypoxia. Finally, a subgroup of rats were maintained for an equivalent duration at baseline conditions and were not exposed to hypoxia to control for time dependent changes in phrenic activity unrelated to hypoxia (time controls). In all rats, the inspired gas mixture was returned to baseline conditions following the hypoxic exposures (or equivalent duration in time controls) and maintained at this level for at least 60 min.
Arterial blood (0.4 ml) was sampled during baseline conditions immediately before treatment, during the first hypoxic episode, and 15, 30 and 60 min following hypoxia (or equivalent duration in time controls not receiving hypoxia) to ensure that arterial PO2 and PCO2 met defined criteria. Briefly, these criteria included: PO2 > 120 mmHg before and after hypoxia, PO2 between 35–45 mmHg during hypoxic exposures and arterial PCO2 within 1 mmHg of baseline following hypoxia.
2.4. Analysis
Phrenic burst frequency and amplitude were analyzed in 30 sec bins immediately prior to blood gas samples that met the criteria outlined above. Frequency LTF was determined by calculating the absolute change in phrenic burst frequency 60 min following hypoxia from burst frequency prior to hypoxia (i.e., baseline). Phrenic (burst amplitude) LTF was determined as the percentage change in amplitude 60 min following hypoxia from the pre-hypoxia (baseline) levels.
Two-way ANOVA with a repeated measures design was used to determine significant differences in PaCO2, body temperature and breathing frequency (raw values) before and after AIH, ASH and equivalent duration in time controls. Individual comparisons were made using the Student-Neuman-Keuls post hoc test. A t-test was used to determine if the percentage change in phrenic amplitude or burst frequency 60 min post-hypoxia (i.e., phrenic amplitude LTF and frequency LTF, respectively) were significantly different than zero. One-way ANOVA was used to determine significant differences between rats exposed to AIH, ASH and control conditions in frequency LTF and phrenic amplitude LTF (expressed as % changes from baseline).
Multiple regression analysis with backwards selection was used to determine factors significantly correlated with frequency LTF and phrenic amplitude LTF. The factors identified as significant predictors were then subjected to a simple linear regression analysis. Differences were considered significant at p<0.05. Reported variances represent +/− 1 standard error.
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