INTRODUCTION

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The high prevalence of hypertension and the clinical significance of hypertension-associated morbidity and mortality are well recognized (1). Increasingly, the high prevalence of sleep-related breathing disorders (SRBD) in adult populations is also being recognized. A laboratory finding of significant obstructive sleep apnea (OSA), defined by the presence of repetitive upper airway obstruction during sleep, has been identified in as many as 24% of working adult men and 9% of similar women (2). Central sleep apnea (CSA) is less prevalent as a syndrome than OSA but can be identified in a wide spectrum of patients with medical, neurological, and neuromuscular disorders (3). Furthermore, CSA and OSA may share some common pathophysiological mechanisms (3, 4).

It has been suggested that SRBD may predispose to hypertension, and also that hypertension may predispose to SRDB. With respect to the latter possibility, hypertensive patients demonstrate an increased prevalence of sleep apnea in some studies (5, 6) but not others (7). Also, pharmacological treatment of hypertension in patients with SRDB may improve sleep-related respiratory function (8). The mechanisms underlying the connection between hypertension and SRBD have not been defined. The difficulty stems in part from a lack of appropriate animal models to investigate such mechanisms.

We and others have described and characterized sleep-disordered respiration in the rat. Rats of various strains demonstrate spontaneous central apnea during all sleep states, but with the greatest frequency during rapid eye movement (REM) sleep (9). These apneas are also significantly associated with various cardiac arrhythmias (10). Apnea in the rat is a robust phenomenon; all rats express this behavior during sleep, although the interanimal variability is significant, as it is in man.

Spontaneously hypertensive rats (SHR), a well-characterized substrain of Wistar Kyoto (WKY) rats, has proven to be a useful model of genetic hypertension. We demonstrated that SHR exhibited nearly 10-fold more apneas than WKY rats (14). Moreover, when blood pressure was acutely normalized by systemic administration of hydralazine, apnea expression fell to the level of normotensive WKY animals (14). This previous study, however, did not demonstrate the effects of sustained normotension on sleep-related breathing in SHR.

Berecek and colleagues (15) have shown that lifelong treatment of SHR by an angiotensin-converting enzyme inhibitor, captopril, completely blocks the development of hypertension and related cardiovascular derangements in adult animals. Even when treatment is discontinued at age 8 wk, captopril-treated SHR (cap-SHR) indefinitely maintain normal blood pressure and heart rate (15), as well as an absence of the vascular changes and cardiac hypertrophy typically observed in SHR (19).

In this study, we report that significant sleep disordered respiration persists in cap-SHR despite complete and sustained normalization of blood pressure. The implications of this finding and the promise of the model system for investigating the pathogenic mechanisms of sleep-related breathing disorders are discussed.

	METHODS

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Implantation of EEG and EMG Recording Electrodes

The surgical procedures employed for electrode implantation have been previously described and are outlined below (12, 13). Groups of 10 male SHR, cap-SHR, and WKY rats aged 3 mo were individually housed under optimum conditions of temperature and humidity and a 12 h/12 h light/dark cycle with free access to food and water throughout the experiments. Animals were anesthetized with a combination of 80 mg/kg ketamine and 5 mg/kg xylazine by intraperitoneal injection. Rats were mounted in a stereotaxic instrument to immobilize the skull. Stainless-steel electroencephalogram (EEG) screw electrodes were bilaterally threaded into the frontal and parietal bones of the skull and electromyogram (EMG) wire electrodes were implanted in the dorsal nuchal musculature. All EEG and EMG electrode leads were soldered to a miniature connector plug and the assembly was affixed to the screw electrodes and skull using acrylic dental cement. Scalp wounds were closed with wound clips and rats were returned to their home cages for at least 7 d of recovery.

Blood Pressure Monitoring

One week after the surgical implantation of EEG and EMG electrodes, three animals from each group underwent a second surgery. In these animals, a small self-contained blood pressure transducer and radio transmitter (TL11M2-C50-PXT; Data Sciences, St. Paul, MN) was implanted under aseptic conditions via a midline laparotomy. This device was fitted with a gel-tipped fluid-filled catheter introduced through a small longitudinal incision into the abdominal aorta 1.5 cm proximal to its bifurcation. The incision was closed around the catheter using tissue adhesive and the transmitter was sutured to the abdominal wall. The skin incision was closed in layers and the animal was allowed a minimum of 7 d postsurgical recovery before recording. This technique has been used by many investigators to reliably obtain chronic blood pressure recordings from rats for periods of months (cf. 22-25).

Polygraphic Recording

Following the surgical recovery period, rats were attached to recording cables 24 h prior to the experimental session to permit adaptation to the recording system. All experimental recording sessions began at 10:00 and lasted 6 h. The recording apparatus comprised a mercury-filled slip ring commutator from which a flexible, insulated cable extended and attached to the miniature connector implanted in the skull of the rat. During all recording sessions rats were individually housed in plexiglass single-chamber plethysmographs allowing unrestricted movement while attached to the recording cables. All signals were simultaneously displayed and digitized on an IBM compatible computer.

Respiratory Measurements

For recording, each rat was placed in a bias-flow-ventilated whole-body plethysmograph (Buxco Electronics, Inc., Sharon, CT) where changes in box pressure represented the difference between the thoracic expansion/contraction and the tidal volume. The bias flow of room air (2 L/min) was more than one order of magnitude greater than the alveolar ventilation of the rat, ensuring that no rebreathing occurred. The dimensions of the chamber were approximately 6 in. wide × 10 in. long × 6 in. high, allowing the animal free movement within this space. Access to water was provided by a bottle passed through a pressure tight seal.

To minimize the influence of thermal and convective transients in room pressure, the box pressure was referenced to a low-pass filtered (with a time constant of approximately 5 s) version of itself. In this way, low-frequency trends or drift in box pressure represented a common-mode signal and were rejected by the differential pressure transducer (Model DP45-14; Validyne Engineering, Northridge, CA). Prior to each experimental study, the plethysmograph was calibrated using the method described by Epstein and coworkers (26).

Lifetime Captopril Treatment

Lifetime captopril treatment was used to produce normotensive SHR (cap-SHR) with normal chronotropic baroreflexes as previously described (15, 16). Cap-SHR used in this study were the offspring of captopril treated mares. These breeders were given captopril, 100 mg/kg/d in their drinking water beginning at the time of mating. Captopril treatment of the breeders continued throughout pregnancy and lactation. After weaning, the dosage of captopril in the drinking water of the pups was 50 mg/kg/d and was maintained until 8 wk of age. Thus, the cap-SHR used in the present study were removed from captopril treatment at least 5 wk prior to their recording.

Data Reduction

The amount of time spent in wakefulness (W), non-rapid eye movement (NREM) sleep, and rapid eye movement (REM) sleep were determined from the digitized data on 10-s epochs using software developed by Benington and coworkers (27), which we have previously employed in several investigations (14, 23, 28). This software discriminated wakefulness as a high-frequency low-amplitude EEG with a concomitant high EMG tone, NREM sleep by increased spindle and theta activity together with decreased EMG tone, and REM sleep by a low ratio of delta to theta activity and an absence of EMG tone.

The timing and volume of each breath were scored by automated analysis (Experimenter's Workbench; Datawave Technologies, Longmont, CO). For each animal the mean respiratory rate (RR) and inspiratory minute ventilation (MV) were computed for each sleep/wake state throughout the 6 h of the recording. Normalized RR (NRR) and MV (NMV) were computed by dividing the appropriate value for each breath by the mean value recorded during W for that animal. These normalized values facilitated evaluation of the effects of sleep and animal group on respiratory pattern.

As in previous investigations (14, 23, 28), sleep apneas, defined as cessations of respiratory effort for at least 2.5 s, were scored for each recording session and were associated with the stage in which they occurred: NREM or REM sleep. The duration requirement of 2.5 s represented at least two "missed" breaths, and was therefore analogous to a 10-s apnea duration requirement in humans. These events represented central apneas because decreased ventilation associated with obstructed or occluded airways would generate an increased plethysmographic signal, rather than a pause. In addition to apneas, all sighs (tidal volume at least 150% larger than the average tidal volume during regular breathing [11]) were detected. As in previous reports, apneas were characterized as "postsigh" (PS) or spontaneous (SP) according to the presence or absence, respectively, of a preceding sigh. Apnea index (AI), defined as apneas per hour in a stage, was separately determined for NREM and REM sleep.

Similar software was employed to analyze the blood pressure waveform; for each beat of each recording, systolic (SBP) and diastolic (DBP) blood pressures and pulse interval were measured for each animal instrumented by telemetry. The pulse interval provided a beat by beat estimate of heart period (HP). Mean BP (MBP) was estimated according to the weighted average of SBP and DBP for each beat: MBP = DBP + (SBPDBP)/3. The parameters for each beat were also classified according to the sleep/wake state during which they occurred.

Statistical Treatment of Results

To test for differences in apnea index, respiratory rate, minute ventilation, blood pressure, heart period, and sleep/wake architecture among WKY, cap-SHR, and SHR, we employed parametric and nonparametric analysis of variance (ANOVA). Post hoc pairwise comparisons were controlled using Fisher's protected least significant difference (parametric ANOVA) or Bonferonni correction (nonparametric ANOVA). To examine interactive effects of animal group and sleep state on respiratory variables including apnea index, we used two-way ANOVA. In all cases, p < 0.05 was considered significant.

	RESULTS

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Table 1 summarizes the relationships of cardiovascular parameters among the three animal groups. As expected, cap-SHR maintained blood pressure and heart period values that were equivalent to WKY animals during wakefulness and sleep. In contrast, untreated SHR exhibited elevated mean blood pressure and heart period (p < 0.05 for each versus WKY during all behavioral states). Also, mean blood pressure fell slightly but consistently during sleep in all animal groups (p < 0.05 by ANOVA with repeated measures). Heart period displayed a significant sleep-related increase (p < 0.05 by ANOVA with repeated measures).

As depicted by Figure 1, there were no significant differences in sleep architecture among the three groups of animals (p = 0.59 for group effect; p = 0.29 for group × state interaction). Numerically, cap-SHR exhibited slightly higher sleep efficiency whereas SHR had slightly lower sleep efficiency than did WKY animals, but these trends were not statistically significant.

View larger version (33K): [in this window] [in a new window]   Figure 1.   Distribution of behavioral states during 6 h recordings in WKY, SHR, and cap-SHR. Each bar represents the mean ± standard error of 10 animals. The ordinate indicates the percentage of the 6 h recording spent in the behavioral state specified by the abscissa. There were no significant differences in the distribution of states among any of the three animal groups (p = 0.59 for group effect; p = 0.29 for group × state interaction).

Figures 2 and 3 present the sleep-related changes in respiratory rate and minute ventilation, respectively. For each animal, mean values during sleep were normalized to the mean value during wakefulness as defined above. Respiratory rate decreased significantly during sleep in all animal groups (p < 0.0001 by ANOVA). There were no significant differences in this effect among the groups (group by state interaction term not significant, p = 0.26). Normalized respiratory rates during NREM and REM sleep were equivalent in all animals groups. Sleep exerted a similar impact on inspiratory minute ventilation (Figure 3). Normalized inspiratory minute ventilation decreased significantly and equivalently during NREM and REM sleep versus wake in all groups (p < 0.0001 by ANOVA; group by state interaction not significant, p = 0.48).

View larger version (48K): [in this window] [in a new window]   Figure 2.   Normalized respiratory rate, determined as mean respiratory rate in a given behavioral state divided by the mean respiratory rate during wakefulness in the same animal. The normalized respiratory rate during wakefulness was therefore 1.0 in each animal, by definition (see text for details). Respiratory rate decreased significantly during NREM and REM sleep versus wake (p < 0.0001 by ANOVA with repeated measures). There were no significant differences in this state effect among the animal groups (group × state interaction term not significant, p = 0.26).

View larger version (46K): [in this window] [in a new window]   Figure 3.   Normalized inspiratory minute ventilation (NVI), determined as mean inspiratory minute ventilation in a given behavioral state divided by the mean inspiratory minute ventilation during wakefulness in the same animal. The NVI during wakefulness was therefore 1.0 in each animal, by definition (see text for details). NVI decreased significantly during NREM and REM sleep versus wake (p < 0.0001 by ANOVA with repeated measures) with no significant differences in this effect among the animal groups (group × state interaction term not significant, p = 0.48).

Figures 4 and 5 illustrate the differences in apnea expression among the three animal groups. During NREM sleep (Figure 4), spontaneous-apnea index was significantly and equivalently elevated in SHR and cap-SHR versus WKY animals. Postsigh apnea index was also significantly elevated in SHR and cap-SHR versus WKY animals, with the index for cap-SHR being intermediate between WKY animals and SHR.

View larger version (45K): [in this window] [in a new window]   Figure 4.   Apnea index during NREM sleep. Ordinate shows apneas per hour of NREM sleep for each animal group. Each bar represents mean ± standard error for 10 animals with SP and PS apneas depicted separately. SP apnea index was significantly and equivalently elevated in SHR and cap-SHR versus WKY animals. PS apnea index was significantly elevated in SHR versus WKY animals, with the apnea index for cap-SHR being intermediate between WKY animals and SHR.

View larger version (35K): [in this window] [in a new window]   Figure 5.   Apnea index during REM sleep. Ordinate shows apneas per hour of REM sleep for each animal group. Each bar represents mean ± standard error for 10 animals with SP and PS apneas depicted separately. SP apnea index was significantly elevated in SHR versus WKY animals, with the apnea index for cap-SHR being intermediate between WKY animals and SHR. PS apnea index during REM sleep was elevated in SHR versus WKY, but the WKY and cap-SHR indexes were equivalent.

During REM sleep (Figure 5) spontaneous-apnea index was significantly elevated in SHR and cap-SHR versus WKY animals, with the apnea index for cap-SHR being intermediate between WKY animals and SHR. Postsigh apnea index during REM sleep was elevated in SHR versus WKY, but the WKY and cap-SHR indexes were equivalent.

	DISCUSSION

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The present study demonstrates for the first time that sleep-related respiratory abnormalities persist in genetically hypertensive rats rendered normotensive by continuous captopril treatment until age 8 wk. In untreated SHR, the rate of spontaneous apneas versus normotensive WKY animals was elevated 15-fold during NREM sleep and 10-fold during REM sleep. Captopril treatment, despite normalizing blood pressure and heart period, yielded no reduction in NREM apnea expression and the REM-related spontaneous apnea index remained elevated by 500%. Despite the significant sleep-related respiratory disorder exhibited by treated and untreated SHR, mean respiratory rate and minute ventilation were equivalent among all three animal groups in all behavioral states. The gross distribution of behavioral states was also similar across groups.

The SHR rat substrain of WKY rats was developed and described by Okamoto and colleagues in the early 1960s (31). Hypertension develops, typically by the fifth week, and is maintained throughout adulthood in 100% of this substrain population (32). SHR rats exhibit hypertensive complications similar to man, such as cerebral infarction or hemorrhage, myocardial infarction and nephrosclerosis (32). The average life span of SHR animals is approximately 18 mo; Wistar animals typically live at least 30-36 mo.

Previously, Berecek and colleagues (15) have shown that lifelong treatment of SHR by captopril completely blocks the development of hypertension in adult animals. Even when treatment is discontinued as early as age 8 wk, cap-SHR animals indefinitely maintain normal blood pressure and heart rate (15), as well as an absence of the vascular changes and cardiac hypertrophy typically observed in SHR animals (19). The findings presented above confirm the persistence of normal heart rate and blood pressure in the cap-SHR group, although captopril treatment had been discontinued at least 5 wk prior to the study.

It has been hypothesized that aberrant cardiovascular homeostasis leading to hypertension might predispose patients to SRBD. Some studies have found an increased prevalence of SRBD in hypertensive patients (5, 6) and treatment of hypertension may ameliorate the SRBD (8). We previously demonstrated the increased sleep-disordered respiration in SHR with respect to the normotensive WKY strain (14). Moreover, when blood pressure in SHR was acutely normalized by 2 mg/kg hydralazine, apnea expression fell to the level of WKY animals for a period of at least 6 h (14). This earlier finding contrasts with the present study, in which sustained control of hypertension in SHR does not correspond to a sustained reduction in apnea expression.

It is worth mentioning that most studies associating hypertension with SRBD in humans are based on patients with primarily obstructive sleep apnea. In contrast, SRBD in the rat is expressed as central apnea. It is our view that both central and obstructive apnea reflect, at least in part, dysregulation of central neural motor output patterning to the respiratory system. In humans with upper airways predisposed to collapse by anatomical, mechanical, or muscular factors, this dysregulation may be manifest primarily by obstructive apneas. In humans or rats with mechanically stable upper airways, dysregulation of respiratory motor output patterning may be expressed primarily by central apneas or hypopneas. Thus, investigating the mechanisms of unstable respiratory motor patterning in the rat may yield relevant insights for the pathogenesis of obstructive sleep apnea in patients.

Indirect support for this view comes from several lines of investigation. Most patients with sleep apnea syndrome, including hypertensive patients, exhibit a combination of central, mixed, and obstructive apneas in a single sleep period, leading to the suggestion that any factor that destabilizes respiratory drive during sleep promotes apnea genesis. Önal and Lopata (4) demonstrated that patients with sleep apnea exhibited obstructive apneas when breathing through their own upper airways, but central apneas when breathing through a tracheostomy. These authors concluded that obstructive apnea reflects unstable central respiratory drive in individuals with upper airways predisposed to collapse by anatomical or neuromuscular defects. Furthermore, in some cases continuous positive airway pressure converts obstructive apneas to central apneas, again supporting the conclusion that unstable central respiratory motor patterning contributes to the pathogenesis of obstructive sleep apnea syndrome. Still the relationship between mechanisms of central apnea and obstructive apnea remains uncertain.

It is possible that the acute suppression of apnea in the rat by hydralazine bolus injection reflected a transient reduction in baroreceptor feedback to the brainstem. Even a slight lowering of blood pressure can disinhibit respiratory drive, yielding significantly increased ventilation in conscious animals (33). In support of this possibility, we demonstrated that hydralazine-induced hypotension produced respiratory stimulation and suppressed apnea in Sprague-Dawley (24) and Zucker (29) rats. We did not examine the effects of long-term hydralazine administration. It is possible that baroreflex resetting, alterations in receptor expression or localization, or other factors may result in a loss of apnea suppression during sustained pharmacological management of hypertension. Further, we cannot exclude the possibility that the acute suppression of apnea by hydralazine resulted from nonspecific circulatory alterations or from a direct influence of hydralazine on respiratory control, rather than from reducing blood pressure.

In the present study, differences in respiratory or cardiovascular behaviors between SHR and cap-SHR presumably reflect differences in the developmental course of the nervous system induced by administration of captopril from conception to postnatal age 8 wk. Untreated SHR demonstrate clear cardiovascular and respiratory derangements with respect to the normotensive WKY control strain. However, captopril treatment dissociates these derangements: cardiovascular parameters remain normal throughout the adult life of the animal (15- 21), whereas sleep-related respiratory disorder persists. The mechanisms underlying persistent sleep-disordered respiration in cap-SHR cannot be determined from the present data.

It is interesting to speculate, however, that the respiratory disorder in cap-SHR may reflect "gene trapping." The SHR strain was developed by selective breeding of WKY animals with spontaneous hypertension due to random mutation (31). The only phenotype used to control breeding protocols was hypertension, which is completely conserved in SHR. It is possible that other gene mutations were also "captured" by this breeding process. Because respiration was not monitored when the SHR strain was developed, it is possible that one or more abnormal genes with direct influence on respiratory control have been conserved in the SHR genome. Captopril treatment may minimize the impact of the abnormal gene(s) on cardiovascular development without significantly altering the influence of other abnormal gene(s) on respiratory development.

Another possibility is pleiotropy; the same abnormal gene(s) may have both direct cardiovascular and direct respiratory effects. In this view, the effects of captopril treatment may vary according to the anatomical or functional target within the central and peripheral nervous systems. For example, although many other possibilities exist, abnormalities in angiotensin receptor expression and localization have been suggested to contribute to the cardiovascular derangements in SHR (16, 18, 20). However, activation of angiotensin receptors also produces direct respiratory responses (34). Thus, abnormalities in brain angiotensin receptor expression or receptor-mediated effects in SHR may independently contribute to both the hypertension and the sleep-disordered respiration characteristic of this strain. Treatment with captopril during maturation may effectively normalize development of the cardiovascular control system but not the respiratory control system. This possibility is made more credible by growing evidence that angiotensin type 2 receptors play an important role in the development, differentiation, connectivity, electrophysiology, and apoptosis of neurons (37). Weichler and coworkers (8) demonstrated that treatment of hypertension by the angiotensin-converting enzyme inhibitor cilazipril improved sleep apnea, providing indirect evidence of angiotensin-mediated effects in human sleep-related breathing disorders.

It is also possible that there is a "dose dependence" to the captopril effect, with blood pressure normalization occurring at a lower dose than respiratory normalization. The partial normalization apnea index in REM sleep with captopril treatment is consistent with this dose-dependent effect interpretation.

It is also possible that nongenetic factors may contribute to the observed differences in respiratory behavior among the experimental groups. For example, we cannot rule out possible maternal exposures in both treated and untreated SHR groups that were identical for respiratory effects but not cardiovascular effects. This seems unlikely, however, because all animals were raised under identical kenneling and handling conditions within the laboratory of one of the investigators. It is also of note that the effect of captopril treatment on postsigh apnea index was strongly state dependent, increasing apnea expression in NREM sleep but decreasing it in REM sleep. This finding could be consistent with either genetic or nongenetic differences among groups. There is no way to discriminate between these possibilities from the present data.

The surprising finding of this study, that phenotypically normotensive but genetically hypertensive rats exhibit striking sleep-disordered breathing similar to untreated SHR, merits further investigation. The model system described above may allow identification of genetic, cellular, and molecular determinants of disordered respiratory control. The possible direct involvement of angiotensin receptor systems in sleep-disordered breathing suggests that investigations of sleep and breathing in genetically altered strains of rat or mouse, such as the angiotensin type 2 receptor knock-out mouse, will be important. Also, animal and human pharmacological studies of sleep and breathing using specific agents, such as the angiotensin type 1 receptor antagonist losartan, may prove fruitful.

In summary, this study demonstrates that phenotypically normotensive but genetically hypertensive rats exhibit striking sleep-disordered respiration equivalent to untreated hypertensive animals, with a mean apnea index of more than 20 per hour. This elevation in apnea genesis occurs without alteration in sleep architecture, respiratory rate, or inspiratory minute ventilation, using normotensive Wistar Kyoto animals as a control. These findings strongly argue that sleep-disordered respiration in SHR is genetically determined and not secondary to hypertension or other cardiovascular derangement.