Cardiovascular and respiratory responses to chronic intermittent ...

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Research Paper

Cardiovascular and respiratory responses to chronic intermittent hypoxia in adult female rats George Miguel P. R. Souza, Leni G. H. Bonagamba, Mateus R. Amorim, Davi J. A. Moraes and Benedito H. Machado

Experimental Physiology

Department of Physiology, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, 14049-900, SP, Brazil

New Findings r What is the central question of this study? Chronic intermittent hypoxia (CIH) induces hypertension in male rats. There is evidence that the development of high blood pressure in females is attenuated in other models of hypertension. Due to the lack of information about the cardiovascular effect of CIH in female rats, we set out to determine whether female rats develop hypertension after CIH. r What is the main finding and its importance? Different from other experimental models of hypertension, adult female rats develop high blood pressure after CIH. These findings provide new perspectives for a better understanding of the neural mechanisms underlying the development of hypertension in females. Adult male rats develop hypertension in response to chronic intermittent hypoxia (CIH). Female rats are known to be protected against the development of hypertension in several experimental models. In this study, we aimed to verify whether the development of hypertension was also prevented in female rats exposed to CIH. Adult female rats were submitted to 35 days of CIH, 8 h per day. At the end of the CIH protocol, the rats were anaesthetized for the implantation of an arterial catheter and the next day the mean arterial pressure and heart rate were recorded in conscious rats. Considering that changes in the respiratory pattern have been associated with the development of hypertension in the CIH model, the respiratory pattern of adult female rats was also evaluated after CIH exposure using whole-body plethysmography. Adult female rats submitted to CIH (n = 27) presented a significant increase in mean arterial pressure when compared with the control group (n = 26). Moreover, CIH-exposed female rats presented an increase in the frequency and duration of apnoeas when compared with control rats. These data show that adult female rats develop changes in the respiratory pattern and high blood pressure in response to CIH. (Received 15 September 2014; accepted after revision 12 December 2014; first published online 16 December 2014) Corresponding author B. H. Machado: Department of Physiology, School of Medicine of Ribeirão Preto, University of São Paulo, 14049-900, Ribeirão Preto, SP, Brazil. Email: [email protected]

Introduction There is evidence that men are more susceptible to the development of hypertension than premenopausal women (Lima et al. 2012). After the onset of menopause, the risk of developing hypertension rises, probably due to changes in the circulating levels of sexual hormones (Reckelhoff & Fortepiani, 2004). Likewise, in different animal models

of hypertension, such as spontaneously hypertensive rats (Reckelhoff, 2001), Dahl salt-sensitive rats (Rowland & Fregly, 1992), angiontensin II infusion in mice (Xue et al. 2005) and the deoxicorticosterone model (Ouchi et al. 1987), the observed increase in blood pressure of female rats was lower than in male rats, indicating that in these experimental models females are less susceptible

C 2014 The Authors. Experimental Physiology C 2014 The Physiological Society

DOI: 10.1113/expphysiol.2014.082990

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G. M. P. R. Souza and others

to the development of hypertension than males. It is well accepted that hormonal (Reckelhoff, 2001), neural (Wang et al. 2008) and genetic differences (Ely & Turner, 1990) between female and male animals may explain, at least in part, the protective mechanisms developed by females to attenuate the development of hypertension. Hypertension is the most common comorbidity in patients with obstructive sleep apnoea (OSA; Somers et al. 1995), which is a disturbance of breathing in humans characterized by episodes of intermittent hypoxia during sleep (Caples et al. 2005). There is a well-known sexual dimorphism in the prevalence of OSA, with a higher incidence in men than in women (Wolk et al. 2003). However, there are opposing findings in the literature regarding sex differences and the prevalence of hypertension in OSA patients. Studies by Hermans et al. (2014), for example, found no sexual dimorphism in the prevalence of hypertension in diabetic patients with OSA, while Laaban et al. (2010) observed that hypertension is more prevalent in women than in men with OSA (Laaban et al. 2010). On the contrary, Huang et al. (2008) documented a lower prevalence of hypertension in female OSA patients compared with men. Therefore, the prevalence of cardiovascular disease in men and women with OSA remains unsettled, most probably due to different characteristics of the populations studied, age and confounding comorbidities, such as obesity and diabetes. The prevalence of OSA increases among women with the onset of menopause and it is equivalent to the prevalence in men (Bixler et al. 2001). In postmenopausal women, hormonal therapy is associated with a lower risk for the development of OSA (Bixler et al. 2001). For a better evaluation of the effects of intermittent hypoxia on the cardiovascular system, Fletcher et al. (1992) developed an experimental model, in which rats are chronically exposed to intermittent hypoxia (CIH). Previous studies from our laboratory have shown that adult male rats submitted to CIH develop a significant increase in arterial pressure and heart rate (Zoccal et al. 2007, 2008). The aim of the present study was to investigate whether the development of hypertension was prevented or blunted in adult female rats submitted to CIH. Considering that changes in the respiratory pattern are an important mechanism underpinning hypertension in male rats exposed to CIH (Zoccal et al. 2008; Moraes et al. 2012, 2013), respiratory parameters in conscious adult female rats submitted to CIH were also evaluated during quiet rest.

Methods In this study, Wistar adult female rats (55–60 days olds) were housed in Plexiglass chambers for the control (n = 26) and intermittent hypoxia exposures (n = 27);

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the control group was maintained in normoxia and the experimental group was exposed to a CIH protocol for 35 consecutive days. The rats were provided by the Animal Care Facility of the University of São Paulo, campus of Ribeirão Preto, Brazil. All experimental protocols were approved by the Institutional Ethical Committee (CETEA #188/2011). Chronic intermittent hypoxia

Nitrogen was infused into the chamber to reduce the fraction of inspired oxygen (F I,O2 ) from 20.8 to 6%. The infusion of gases was controlled by software that regulated the flow of O2 and N2 through the valves of the Oxycycler system (Biospherix, Redfield, NY, USA). Every 9 min, the rats were submitted to episodes of hypoxia, in which the F I,O2 was maintained at 6.0% for 40 s and then O2 was infused into the chamber to bring F I,O2 back to normoxia (20.8%) for the next 9 min, when a new episode of hypoxia started. The rats were submitted to this protocol for 8 h per day from 08.00 to 16.00 h. The experimental group was submitted to CIH (n = 27) for 35 consecutive days and the control group (n = 26) was maintained in normoxia (F I,O2 = 20.8%) for the same period, as previously described by Zoccal et al. (2007). Evaluation of oestrous cycle

The oestrous cycle of all female rats used in this study was monitored by taking a vaginal smear 4 days before the end of the 35 day protocol for the CIH and control groups and on the day of cardiovascular and respiratory recordings in order to verify whether or not female rats exposed to CIH preserve the normal oestrous cycle. Cardiovascular recordings

After 35 days in the control or CIH protocols, the rats were anaesthetized with tribromoethanol (250 mg kg−1 , I.P.; Aldrich, Milwaukee, WI, USA) and a catheter was inserted into the femoral artery in the direction of the abdominal aorta. The level of anaesthesia during the surgery was monitored by the lack of reflex responses to frequent tail pinching. The anti-inflammatory and analgesic flunixin meglumine (1 mg kg−1 , Banamine; Mantecorp Ind. Qu´ım. e Farm. Ltda, Rio de Janeiro, Brazil) was injected I.M. at the end of surgery. After a recovery period of 24 h, the pulsatile arterial pressure was recorded in conscious rats for periods lasting 30–40 min. To perform the recording of pulsatile arterial pressure, we used a pressure transducer (MLT0380; ADInstruments, Bella Vista, NSW, Australia) connected to an amplifier (Bridge Amp, ML221; ADInstruments) and an acquisition C 2014 The Authors. Experimental Physiology C 2014 The Physiological Society

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Chronic intermittent hypoxia in female rats

system (PowerLab 4/25, ML845; ADInstruments), and the signals were digitized with appropriate software (Chart Pro; ADInstruments). Heart rate (HR) and systolic arterial pressure (SAP) variability were analysed using appropriate software (CardioSeries v2.4; http://www.danielpenteado.com). The variability of SAP and HR series were calculated in the time domain and expressed as the variance (σ). The analysis was also performed in the frequency domain using the spectral analysis as described by Tezini et al. (2013). In brief, SAP and HR spectra were calculated using fast Fourier transform and were integrated in two frequency bands, i.e. low frequency (LF, 0.2–0.75 Hz) and high frequency (HF, 0.75–3.0 Hz). The LF/HF ratio was used as an index of sympathovagal balance.

Respiratory recordings

The whole-body plethysmographic method (Malan, 1973; Barros et al. 2002) was used to record respiratory variables. The respiratory pattern and arterial pressure were recorded simultaneously. These recordings were performed in the morning (08.00–12.00 h), and typically, included one control and one CIH rat studied simultaneously in adjacent plethysmographic chambers. With this approach, cardiorespiratory variables from both groups of rats were recorded in similar environmental conditions. Each rat was placed inside an acrylic plethysmographic chamber (6 litres) and had a settling period of 30 min before commencing the cardiorespiratory recordings. After the baseline recording of blood pressure, the plethysmographic chamber was closed and the respiratory variables were recorded for periods of 5–10 min. The arterial catheter was passed through a small hole in the upper part of the chamber to allow simultaneous recording of cardiovascular parameters. During the cardiorespiratory recording, unanaesthetized rats of both groups remained quiet most of the time, with no evident behavioural differences between the two groups. An injection of 1 ml of air was used for volume calibration, and the temperature inside and outside the chamber was continuously monitored. The oscillations in pressure inside the chamber produced by breathing were detected by a pressure transducer (ML141, Spirometer, PowerLab; ADInstruments). The signal was amplified and recorded. Tidal volume (VT ) and respiratory frequency (fR ) were calculated as described by Malan (1973), and ventilation (V˙ E ) was obtained as the product of VT and fR . These parameters were calculated using a period of 2 min of respiratory recordings in conscious rats when they were quiet and presenting no body movements. The data recorded when the rats were moving inside the chamber were excluded from analysis because the respiratory activity was contaminated by larger oscillations C 2014 The Authors. Experimental Physiology C 2014 The Physiological Society

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in the pressure inside the chamber. The rats were weighed on the day of recordings to determine the body weight, which was used to correct the ventilation index for each animal. In order to assess the respiratory variability, the breath-to-breath (BBn ) and subsequent interval (BBn+1 ) of 200 breaths were analysed (Peng et al. 2011). Short-term variability (SD1) and long-term variability (SD2) were calculated as an index of breathing variability for control and CIH-exposed rats. Poincaré plots (BBn versus BBn+1 ) for 200 breaths were used to represent this analysis. Evaluation of respiratory deep breaths and apnoeas

The magnitude of the decrease in mean arterial pressure (MAP) during episodes of deep breaths observed in control and CIH-exposed female rats was evaluated. A deep breath was considered as an increase in the tidal volume to at least twice the amplitude observed in a regular respiratory cycle. To quantify the magnitude of changes in arterial blood pressure, we took the peak fall in MAP during the episode of deep breathing in relationship to the baseline MAP (20 s before the event). All deep-breath events observed in the period of respiratory recording (5–10 min) in each rat were analysed. Respiratory events affected by any body movement of the rats during the recording were not considered for data analysis. The rats considered for this analysis presented at least two events of deep breaths within the time window of 5–10 min of continuous recordings. Rats that did not meet these criteria were not considered for this evaluation. An apnoea event was characterized as periods of breathing cessation of at least two missed breaths (Edge et al. 2012); the proportion of deep breaths followed by apnoeas in each rat was calculated. The duration of apnoea observed after deep-breath events was also evaluated in control (n = 13) and CIH rats (n = 22). Statistical analysis

Data were expressed as mean values ± SEM. Differences between control and CIH-exposed adult female rats were determined by Student’s unpaired t test or two-way ANOVA as appropriate, and differences were considered significant at P < 0.05. Results Body weight before and after CIH protocol

Adult female rats exposed to CIH (n = 27) and normoxia (n = 26) presented similar body weights on the day before the protocol (244 ± 3 versus 242 ± 2 g, respectively). After 35 days, adult female rats exposed to CIH presented a lower body weight when compared with control rats maintained in normoxia (273 ± 3 versus 347 ± 5 g, respectively).

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Table 1. Number of adult female rats in each phase of the oestrous cycle on the day of cardiovascular and respiratory recording after 35 days in control or experimental chronic intermittent hypoxia (CIH) protocols

n Metoestrus Dioestrus Pro-oestrus Oestrus

Control

CIH

26 11 5 2 8

27 11 9 0 7

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versus 104 ± 1 mmHg), systolic arterial pressure (136 ± 1 versus 129 ± 2 mmHg), diastolic arterial pressure (92 ± 2 versus 86 ± 1 mmHg) and heart rate (400 ± 6 versus 376 ± 5 beats min−1 ) when compared with the control group (n = 26; Fig. 1). Regarding the SAP and HR variability, we verified that adult female rats exposed to CIH presented higher variance of SAP (26.1 ± 2 versus 19.3 ± 1 mmHg2 ) compared with control rats but no changes in the variance of HR [226 ± 29 versus 194 ± 25 (beats min−1 )2 ]. These data are presented in Table 2.

Oestrous cycle after CIH protocol

On the day of cardiovascular and respiratory recordings, 11 control and 11 CIH-exposed female rats were in the metoestrous phase, five control and nine CIH-exposed females were in the dioestrous phase, eight control and seven CIH-exposed female rats were in the oestrous phase, and two control and no CIH-exposed female rats were in the pro-oestrous phase (Table 1). Cardiovascular parameters

Adult female rats submitted to CIH (n = 27) presented a significant increase in mean arterial pressure (111 ± 1

Respiratory parameters

Adult female rats exposed to CIH presented no significant changes in VT (8.2 ± 0.3 versus 7.8 ± 0.4 ml kg−1 ), V˙ E (779 ± 44 versus 796 ± 47 ml kg−1 min−1 ) or fR (94 ± 3 versus 102 ± 3 breaths min−1 ) compared with control rats (Table 3). The short-term variability of breathing (SD1) in CIH-exposed female rats was higher when compared with control rats (158 ± 19 versus 98 ± 11 ms), as was the long-term variability of breathing (SD2, 203 ± 19 versus 142 ± 12 ms; Fig. 2). The increase in tidal volume during deep breaths was similar in both groups [323 ± 15 (CIH) versus 301 ± 18%

Figure 1. Cardiovascular parameters of adult female rats submitted to chronic intermittent hypoxia (CIH; n = 27) and control protocols (n = 26) The parameters analysed were mean arterial pressure (MAP; A), systolic arterial pressure (SAP; B), diastolic arterial pressure (DAP; C) and heart rate (D). Note that all cardiovascular parameters were significantly increased in adult female rats exposed to CIH (∗ P < 0.05).


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Table 2. Variability in heart rate and systolic arterial pressure in female rats exposed to CIH Parameter Systolic arterial pressure

Heart rate

n LF (mmHg2 ) HF (mmHg2 ) σ (mmHg2 ) LF/HF σ [(beats min−1 )2 ]

Control

CIH

P Value

26 4.37 ± 0.5 1.68 ± 0.2 19.3 ± 1.0 0.40 ± 0.06 194 ± 25

27 3.98 ± 0.3 2.17 ± 0.2 26.1 ± 2.0 0.32 ± 0.03 226 ± 29

— n.s. n.s. 0.0073 n.s. n.s.

Abbreviations: HF, high frequency; LF, low frequency; and σ, variance. Values are means ± SEM.

Table 3. Respiratory parameters in control and CIH-exposed female rats evaluated by whole-body plethysmography

n VT (ml kg−1 ) fR (breaths min−1 ) V˙ E (ml kg−1 min−1 )

Control

CIH

26 7.8 ± 0.4 102 ± 3 796 ± 47

27 8.2 ± 0.3 94 ± 3 779 ± 44

Values are means ± SEM. Abbreviations: fR , respiratory frequency; VT , tidal volume; and V˙ E , minute ventilation.

(Control) of increase from the baseline]. The fall in MAP during the deep breaths was significantly higher in CIH-exposed female rats (change, −13 ± 1 versus −9 ± 1 mmHg from the baseline; Fig. 3B). Control and CIH-exposed female rats presented deep breaths followed by apnoea and deep breaths without apnoea. The proportion of deep breaths followed by apnoea was higher in CIH-exposed female rats than in control animals (59 ± 7 versus 29 ± 7% of deep breaths followed by apnoea; Fig. 4A). When an apnoea event was present, the duration

Figure 2. Variability of breathing in control and CIH-exposed adult female rats A, Poincare´ plot of breath-to-breath (BBn ) and next breath-to-breath interval (BBn+1 ) of control female rats. B, Poincare´ plot of BBn and BBn+1 of CIH-exposed female rats. C, average SD1 (short-term variability) of breathing in control (n = 26) and CIH-exposed female rats (n = 27). D, average SD2 (long-term variability) of breathing in control and CIH-exposed female rats (∗ P < 0.05). Note the higher variability of breathing in adult female rats submitted to CIH compared with the control group.


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of apnoea was significantly longer in CIH-exposed female rats (n = 22; 2.36 ± 0.19 s) than in control animals (n = 13; 1.60 ± 0.15 s; Fig. 4B). Discussion The CIH-exposed adult female rats presented reduced body weight gain during the 35 days of this protocol in comparison to control rats, indicating important metabolic changes. Adult female rats preserved the oestrous cycle during CIH exposure, because they presented variations in the oestrous cycle phase, and the distribution of animals in each phase of the oestrous cycle was similar in CIH and control female rats, indicating that the intermittent hypoxia protocol produced no major changes in oestrous cycle. Cardiovascular changes in adult female rats exposed to CIH

The mechanism involved in the sexual hormonedependent protection of female rats against the development of hypertension is not completely understood, but oestrogens and androgens may have opposing roles. The blockade of androgenic receptors, for example, reduces the arterial blood pressure in adult male spontaneously hypertensive rats, whereas testosterone treatment in ovariectomized female spontaneously hypertensive rats facilitates the development of hypertension, suggesting a critical role for testosterone in this cardiovascular dysfunction (Reckelhoff

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et al. 1999). Ovariectomy does not affect the level of blood pressure in female spontaneously hypertensive rats (Brinson et al. 2014). Nevertheless, in the experimental model of infusion of angiotensin II in mice (Xue et al. 2005) and in Dahl female rats submitted to salt-sensitive hypertension (Brinson et al. 2014), ovariectomy facilitates the development of hypertension, suggesting a protective role for female sexual hormones in the development of high levels of blood pressure. Taken together, these studies show the complexity of the mechanisms involved in the development of hypertension in females. In the present study, we evaluated the cardiovascular parameters of adult female rats following 35 days of intermittent hypoxia in order to verify whether adult female rats were also protected against the development of hypertension in response to this protocol. Our data show that adult female rats presented a significant increase in all cardiovascular parameters evaluated (MAP, SAP, DAP and HR). The increase in these cardiovascular parameters was similar to those observed previously in adult male rats in equivalent experimental conditions (Zoccal et al. 2007), showing that adult female rats also develop high blood pressure after exposure to CIH when compared to the normoxia group. In contrast, studies by Hinojosa-Laborde & Mifflin (2005) showed that adult female rats present a smaller increase in MAP and no change in HR when compared with male rats exposed to a different protocol of CIH (F I,O2 = 10%, for 7 days). Our findings differ from those of Hinojosa-Laborde & Mifflin (2005), who suggested that sex hormones play a key role in protecting female rats against the development

Figure 3. Changes in mean arterial pressure (MAP) after deep breaths in adult female rats exposed to CIH and normoxia A, simultaneous cardiovascular and respiratory recordings were made in conscious rats, and this shows representative traces from one female rat exposed to normoxia and another exposed to CIH. Abbreviation: PAP, pulsatile arterial pressure. B, average reduction in MAP during deep breaths in adult CIH-exposed (n = 25) and control female rats (n = 22). Note that adult female CIH-exposed rats present a longer time of apnoea after a deep breath and that the reduction in MAP is greater than in the normoxia group (∗ P < 0.05).


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of high blood pressure during CIH. In this case, we must consider the different protocol of CIH used in each study. For instance, Hinojosa-Laborde & Mifflin (2005) exposed adult female rats to a CIH protocol that consisted of 10% of F I,O2 for 3 min, during 8 h per day for 7 days, which is different from our protocol, which lasted 35 days, and in each episode of hypoxia the F I,O2 was as low as 6%. Therefore, differences in the intensity and duration of challenges produced by the CIH protocol appear to be an important variable contributing to the observed significant increases in arterial pressure, as previously described by Fletcher et al. (1992). It is interesting to note that the diaphragmatic muscle dysfunction following exposure to CIH in adult male rats is also dependent on the duration and intensity of the CIH protocol (Shortt et al. 2014). Although spectral analysis indicates an increase in the variance of SAP, the LF component and the LF/HF ratio were not altered (Table 2), suggesting no major changes in sympathetic activity in CIH-exposed female rats. Studies by Lucking et al. (2014) found that the increase in cardiac output in rats exposed to CIH for 2 weeks is a possible factor that contributes to CIH-induced hypertension in adult male rat. In addition, evidence indicates that sympathetic overactivity may underlie the high blood pressure in male rats exposed to CIH (Zoccal et al. 2007). In this context, the findings of the present study provide new perspectives for studies on the mechanisms underlying hypertension in female rats exposed to CIH, as well on the sex differences observed.

Respiratory changes in adult female rats exposed to CIH

Studies from our laboratory using the working heart– brainstem preparation showed that the respiratory activity

in CIH-treated juvenile male rats is increased in the late-expiratory phase of the respiratory cycle (Zoccal et al. 2008). In a recent study from our laboratory, we observed that the activity of the abdominal muscle in the late-expiratory phase is also increased in conscious, freely moving rats (Moraes et al. 2013). Furthermore, this enhanced late-expiratory activity is correlated with increased sympathetic activity in male rats exposed to CIH, suggesting that alterations in the respiratory pattern contribute to increases in the sympathetic outflow, which explains, at least in part, the high blood pressure observed in male rats exposed to CIH (Zoccal et al. 2008; Moraes et al. 2012, 2013). Considering that changes in respiratory–sympathetic coupling are an important mechanism of hypertension in juvenile male rats exposed to CIH, we also evaluated the respiratory pattern of conscious adult female rats after exposure to CIH. It is important to note that whole-body plethysmography is less precise than respiratory nerve recording, but it is a suitable approach to evaluation of the respiratory pattern in conscious rats because it is non-invasive. We observed no major changes in the baseline respiratory parameters of adult female rats after CIH. These findings are in agreement with those described by Skelly et al. (2012) in conscious rats, in which the authors documented that adult female rats submitted to CIH do not develop changes in VT , V˙ E or fR when evaluated in normoxic conditions. The frequency of episodes of apnoea following deep breaths was higher in CIH-treated female rats than in control animals. Moreover, the duration of the apnoeas following deep breaths was also longer in CIH-exposed female rats. These findings suggest that CIH-exposed female rats develop a delayed ability to initiate the regular respiratory cycles after deep breaths compared with rats in the control group, indicating that CIH promotes changes in the rhythm and pattern of respiration in adult female

Figure 4. Apnoea following deep breaths in adult female rats submitted to control and CIH protocols A, proportion of deep-breath (DB) events followed by apnoeas in the control (n = 22) and CIH groups (n = 25). B, average duration of the apnoea events in control (n = 13) and CIH-exposed female rats (n = 22). Note that the proportion of deep breaths followed by apnoeas and the duration of these events are higher in CIH-exposed female rats than in control animals (∗ P < 0.05).


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rats. Studies by Edge et al. (2012) also documented that 7 days of CIH produced an increase in the number of episodes of apnoea in adult male rats, including spontaneous apnoea and apnoea following deep breaths. Additional evidence that CIH produces changes in the female respiratory pattern is the variability of breathing, which was increased in CIH-exposed female rats compared with control rats, indicating instability of breathing in these animals. Studies by Peng et al. (2011) showed that mice heterozygous for hypoxia-inducible factor-2α develop increased variability of breathing as well as increased incidence of postsigh apnoeas. Moreover, the deficiency of hypoxia-inducible factor-2α induces hypertension and sympathetic overactivity in mice (Peng et al. 2011). In this context, the observed increase in the breath-to-breath variability provides new perspectives for studies of regulatory factors involved in the respiratory and autonomic dysfunction in female rats submitted to CIH. Respiratory neurons at specific brainstem sites, such as the inspiratory neurons of the pre-Bötzinger complex, are believed to be involved with the generation of respiratory rhythm, and their intrinsic electrophysiological properties could be altered after exposure to CIH in males, as proposed by Moraes et al. (2013). Studies by McKay & Feldman (2008) showed that unilateral ablation of the pre-Bötzinger complex at the brainstem increases the number of apnoeas during the natural sleep cycle in rats. The increased number of apnoeas and the delayed time to recover the normal respiratory cycle in adult female rats exposed to CIH are probably related to changes in the activity of the inspiratory neurons located in the pre-Bötzinger complex. These findings are evidence of a possible dysfunction in the neural respiratory network in adult female rats exposed to CIH. Moreover, the results indicate that intermittent episodes of hypoxia, as in obstructive sleep apnoea, should be considered to be a possible contributory factor in the development of apnoeas in female OSA patients.

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is elevated in a specific phase of the respiratory cycle (late expiration) during regular breathing in juvenile CIH-exposed male rats (Zoccal et al. 2008), indicating that the respiratory network is contributing to this increase (Moraes et al. 2013). Moreover, this increased sympathetic activity may contribute to the high levels of blood pressure in CIH-exposed male rats. However, during deep breaths, the respiratory network activity is probably different from that generating regular respiratory cycles because it results in a large increase in tidal volume. Considering that different neurons of the respiratory network are activated during deep breaths, the respiratory modulation of presympathetic neurons is probably different in relationship to regular breaths and may produce a reduction in blood pressure instead of an increase. The physiological role of deep breaths and simultaneous cardiovascular adjustments is probably related to an increase in gas exchange efficiency and increased lung perfusion. The physiological role of deep breaths, their haemodynamic consequences and the corresponding neural mechanisms in CIH-exposed rats are a matter for further investigation.

Conclusion

We conclude that adult female rats develop high blood pressure when submitted to a CIH protocol lasting 35 days, indicating that adult female rats are not protected from the development of hypertension in response to CIH. In humans, OSA is more prevalent in men than in women (Bixler et al. 2001; Wolk et al. 2003; Tufik et al. 2010); however, the experimental data of the present study suggest that, at least in rats, CIH represents a similar cardiovascular risk for both sexes. Our data also show that CIH produces changes in the respiratory pattern, which may be a factor that contributes to high blood pressure in adult CIH-exposed female rats.

The impact of deep breaths on arterial blood pressure

Perspectives

The impact of deep breaths on arterial pressure was greater in CIH-exposed female rats than in control animals because the fall in MAP during deep breaths was greater. Mechanical and haemodynamic effects may play a role in the reduction of MAP after augmented inspiration, in which a deep breath reduces the intrathoracic pressure and may activate a cardiopulmonary reflex due to an increase in venous return (Olsen et al. 1985; Machado et al. 1992). Changes in the blood pressure due to respiratory events could also be explained by central mechanisms. There is experimental evidence that the sympathetic activity

In contrast to other experimental models, the data obtained in CIH-exposed female rats show that in this experimental model, sex does not play a critical role in the development of hypertension. The data of the present study provide interesting perspectives for studies on the mechanisms underlying CIH-induced hypertension in female rats. Electrophysiological experiments on the neurons of the respiratory network and their interaction with presympathetic neurons will be critical for a better understanding of these complex neurophysiological phenomena.


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Additional information Competing interests None declared. Author contributions G.M.P.R.S., M.R.A. and L.G.H.B. performed the experiments. G.M.P.R.S., M.R.A., L.G.H.B. and D.J.A.M. analysed the data. G.M.P.R.S. and B.H.M. designed the research. All authors approved the final version of the manuscript. Funding This work was funded by the Fundaçaõ de Amparo a` Pesquisa e Desenvolvimento do Estado de São Paulo (FAPESP-2013/ 06077-7) and Coordenaçaõ de Aperfeiçoamento de Pessoal de N´ıvel Superior (CAPES; fellowship to G.M.P.R.S.).
