- Email: [email protected]
The role of gonadal hormones in the hypoglossal discharge activity of rats exposed to chronic intermittent hypoxia
Brain Research Bulletin 149 (2019) 175–183
Contents lists available at ScienceDirect
Brain Research Bulletin journal homepage: www.elsevier.com/locate/brainresbull
Research report
The role of gonadal hormones in the hypoglossal discharge activity of rats exposed to chronic intermittent hypoxia
T
⁎
Si Tang, Xiufang Zhou, Ke Hu , Pei Liu, Mengqing Xiong, Huimin Li Division of Respiratory Disease, Renmin Hospital of Wuhan University, Zhangzhidong Road No. 99, Wuhan 430060, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Chronic intermittent hypoxia Neuroplasticity Sex hormone Hypoglossal nerve
Objective: The role of gonadal hormones in chronic intermittent hypoxia (CIH)-evoked hypoglossal nerve (XII) neuroplasticity has not been thoroughly studied. The purpose of this study was to reveal the effects of gonadal hormone concentration variations on the XII discharge activity of rats exposed to CIH and the corresponding relationship with 5-hydroxytryptamine (5-HT). Methods: This study employed five groups of female rats and six groups of male rats. Gonadal hormone levels were modified through gonadal resection and daily supplementation with gonadal hormones in rats of both sexes. Rats in the CIH groups were exposed to an additional 4 weeks of CIH once the operative incision for gonadectomy had healed. Finally, XII spontaneous discharge activities were recorded, and serum estradiol, testosterone and 5-HT concentrations were detected by ELISA. Results: Among the female rats, the normal estradiol level groups expressed XII neuroplasticity, while the low estradiol level group failed to express this phenomenon. XII neuroplasticity was related to the serum estradiol concentration. In the male rats, XII neuroplasticity was successfully evoked in the normal testosterone level group but was suppressed in the low testosterone level group and aromatase inhibitor group. XII neuroplasticity was not significantly related to serum testosterone concentrations. Both estradiol and testosterone concentrations were related to 5-HT concentrations. Conclusions: This is the first study to analyze the effects of gonadal hormones on XII neuroplasticity in both female and male rats. The results suggest that the estradiol level is related to XII neuroplasticity rather than the testosterone level, and testosterone may indirectly adjust XII neuroplasticity by converting to estradiol. Estradiol and testosterone levels are related to 5-HT levels in the respective genders.
1. Introduction Obstructive sleep apnea syndrome (OSAS) is a common sleep-related breathing disorder that affects nearly 5–14% of middle-aged adults (Peppard et al., 2013). One of the main pathogenic factors of OSAS is periodic collapse of the upper airway induced by upper airway neuromuscular dysfunction. Repeated hypoxia and hypercapnia can lead to dysfunction in neurocognition (Lal et al., 2012), endocrine disorder (Seif et al., 2013) and hemodynamic changes (Wilcox and Semsarian, 2009), ultimately resulting in multisystem damage, especially cardiovascular diseases (Buchner et al., 2007; Marin et al., 2005; Parati et al., 2007). Nevertheless, continuous positive airway pressure (CPAP), the “gold standard” for treatment, has limited effectiveness because of poor compliance (17–54%) (Weaver and Grunstein, 2008). Therefore, a drug therapy that will facilitate better compliance is
urgently need. Epidemiological data show that middle-aged men account for the dominant OSAS incidence population; the number of male patients is 3.3 times higher than the number of women, and the incidence among postmenopausal women is ˜4.5 times higher than that among premenopausal women (Bixler et al., 2001). In addition, studies have discovered that testosterone levels were lower in OSAS patients compared to those in average persons (Luboshitzky et al., 2002), and women with low estrogen and progesterone levels(Netzer et al., 2003) are more likely to suffer from OSAS. However, whether CPAP therapy rescued gonadal hormone levels was ambiguous as conclusions are conflicting in different studies (Celec et al., 2014; Li et al., 2016; Zhang et al., 2016). As the incidence of OSAS is characteristically related to age and gender, the occurrence of OSAS is evidently associated with declining gonadal hormone concentrations; however, the mechanism
⁎
Corresponding author. E-mail addresses: [email protected] (S. Tang), [email protected] (X. Zhou), [email protected] (K. Hu), [email protected] (P. Liu), [email protected] (M. Xiong), [email protected] (H. Li). https://doi.org/10.1016/j.brainresbull.2019.04.017 Received 11 January 2019; Received in revised form 15 March 2019; Accepted 18 April 2019 Available online 22 April 2019 0361-9230/ © 2019 Elsevier Inc. All rights reserved.
Brain Research Bulletin 149 (2019) 175–183
S. Tang, et al.
remains unclear. The genioglossus muscle, which is innervated by the hypoglossal nerve (XII), is the main respiratory muscle maintaining expansion of the upper airway. Motoneurons in XII nuclei of both female and male rats have been found to express estrogen receptors (α, β) and androgen receptors (Behan and Thomas, 2005). Estrogen-related receptor α can regulate muscle fiber differentiation in the upper airway of OSAS patients (Chen et al., 2016), and estrogen can increase the anti-fatigue ability of the genioglossus in chronic intermittent hypoxia (CIH)-exposed rats (Huang and Liu, 2011). Although both testosterone and estrogens influence muscle physiology and metabolism (Carson and Manolagas, 2015), testosterone directly stimulates myotube hypertrophy but not differentiation(Hughes et al., 2016). In addition to the genioglossus, gonadal hormones can also adjust ventilatory function through the XII. Zabka et al. (2005, 2006) and Nelson et al. (2011) partially revealed the effect of gonadal hormones on respiratory plasticity in male and female (Dougherty et al., 2017) rats via acute intermittent hypoxia (AIH) but not CIH. Respiratory plasticity is a kind of serotonin-dependent hypoglossal or phrenic nerve neuroplasticity that is expressed as enhanced breathing motor output that can be sustained for up to an hour after intermittent hypoxia (Mahamed and Mitchell, 2007). Respiratory plasticity in OSAS patients helps counter airway occlusion during sleep, thus maintaining the stability of upper airway dilatation and expansion of the airway (Mateika and Narwani, 2009). CIH models better simulate the actual intermittent hypoxia course of OSAS patients, and CIH can induce more prominent XII neuroplasticity compared to AIH models (Tu et al., 2013). Many reports show that the central and peripheral serotonergic system is involved in XII neuroplasticity (Neuzeret et al., 2009; Tu et al., 2015; Wu et al., 2017). Moreover, numerous serotonergic neurons express estrogen receptors (α, β) and androgen receptors in macaques (Bethea et al., 2015), mice and rats (Nikmahzar et al., 2016; Sheng et al., 2004), and 5-hydroxytryptamine (5-HT) in hypoglossal nuclei is affected by hormone levels, raising the question of whether serum gonadal hormones can influence the 5-HT level. This is the first study to analyze the effects of serum gonadal hormones on CIH-induced XII neuroplasticity and the corresponding relationship with 5-HT levels in both genders.
Table 2 Serum gonadal hormone and 5-HT concentrations in male rats.
NC SO CIHC OV OV + E2
991.02 982.97 918.61 339.48 921.01
1.02 1.12 0.99 0.98 1.01
39.57 ± 4.20 38.64 ± 6.06 37.21 ± 3.85 27.95 ± 2.72# 39.428 ± 3.78
± ± ± ± ±
67.91 89.10 103.20 39.17* 42.76
± ± ± ± ±
0.23 0.26 0.07 0.07 0.05
NC SO CIHC OR OR + T OR + T+AI
11.45 ± 0.96 12.02 ± 1.17 9.81 ± 1.43* 1.76 ± 0.65*# 10.17 ± 1.29 9.78 ± 1.43*
128.17 160.28 167.45 157.72 174.68 148.95
39.58 37.95 36.94 27.17 36.99 30.43
± ± ± ± ± ±
38.54 39.45 30.52 26.51 37.64 40.80
± ± ± ± ± ±
4.77 5.82 4.02 4.11 & 4.46 5.21&
Serum testosterone, estradiol and 5-HT concentrations in male rats are presented in Table 2. Serum testosterone concentrations were not significantly different between the NC (11.45 ± 0.96 ng/ml) and SO groups (12.02 ± 1.17 ng/ml) but were lower in the CIHC group (9.81 ± 1.43 ng/ml), CIH with orchiectomy group (OR group, 1.76 ± 0.65 ng/ml) and testosterone and aromatase inhibitor supplementation group (OR + T + AI group, 9.78 ± 1.43 ng/ml) (P < 0.05), revealing that surgical stimulation did not change the serum testosterone level, but 4 weeks of CIH exposure resulted in decreased levels. Compared with the CIHC group, the serum testosterone concentration in the OR group (1.76 ± 0.65 ng/ml) decreased significantly (P < 0.001) but did not evidently change in the OR + T group (10.17 ± 1.29 ng/ml) and OR+T+AI group (9.78 ± 1.43 ng/ ml), showing that orchiectomy could significantly reduce the serum testosterone level, that testosterone supplementation could effectively restore the serum testosterone level, and that aromatase inhibitors seemed to have no distinct effect on the serum testosterone level. The serum 5-HT concentration was significantly reduced in the OR (27.17 ± 4.11 ng/ml) and OR + T + AI groups (29.23 ± 4.17 ng/ml) compared with that in the CIHC group (35.74 ± 5.44 ng/ml), whereas the serum 5-HT concentration in the OR + T group (36.79 ± 2.79 ng/ ml) was not significantly changed. No differences in serum estradiol levels were found among the groups.
Table 1 Serum gonadal hormone and 5-HT concentrations in female rats. 5-HT (ng/ml)
5-HT (ng/ml)
2.2. Serum gonadal hormone and 5-HT concentrations in male rats
Serum estradiol, testosterone and 5-HT concentrations in female rats are summarized in Table 1. No significant differences in the serum estradiol concentration were found among the normoxia control group (NC group, 991.02 ± 67.91 pg/ml), normoxia sham operation group (SO group, 982.97 ± 89.10 pg/ml), CIH control group (CIHC group, 918.61 ± 103.20 pg/ml) and CIH with ovariectomy and estrogen supplementation group (OV + E2 group, 921.01 ± 42.76 pg/ml), whereas the concentration in the CIH with ovariectomy group (OV
Testosterone (ng/ml)
Estradiol (pg/ml)
group, 339.48 ± 39.17 pg/ml) decreased significantly (P < 0.001), indicating that surgical stimulation or 4 weeks of CIH exposure may not notably affect estradiol levels, that ovariectomy can reduce these levels significantly, and that these levels can be effectively restored through estradiol supplementation. The serum 5-HT concentration in the OV group (27.95 ± 2.72 ng/ml) was markedly decreased compared with those in the other groups. On the other hand, we did not observe significant variations in serum testosterone levels among the groups.
2.1. Serum gonadal hormone and 5-HT concentrations in female rats
Estradiol (pg/ml)
Testosterone (ng/ml)
Serum testosterone concentration (ng/ml) in the CIHC (CIH control) group, OR group and OR + T + AI group were lower than that in the NC (Normal control) group (*P ＜ 0.05), and was decreased significantly in the OR group compared with the CIHC group (#P < 0.001). Serum 5-HT concentration (ng/ml) in the OR and OR + T+AI groups was significantly reduced compared with that in the CIHC group (&P ＜ 0.05). No significant differences in serum estradiol levels among groups were detected.
2. Results
Group
Group
2.3. Changes in XII spontaneous discharge activity in female rats Fig. 1 shows the 60-min integrated traces of XII spontaneous discharge in female rats after normoxia or CIH exposure. The normoxic group was represented by one trace because no differences were observed between the NC and SO groups. The XII discharge amplitudes (Fig. 3A) in the CIHC and OV + E2 groups were significantly greater than those in the normoxic group at each time point post-CIH (P < 0.001) and increased only within 30 min (P < 0.05) in the OV group. That is, the CIHC and OV+E2 groups successfully presented XII neuroplasticity, while the OV group failed to demonstrate XII neuroplasticity. No remarkable differences in XII neuroplasticity were found between the CIHC and OV + E2 groups (P > 0.05). No significant
Serum estradiol concentration (pg/ml) in the OV group decreased significantly compared with that of the CIHC (CIH control) group (*P ＜ 0.001). Serum 5-HT concentration (ng/ml) in the OV group decreased markedly compared with those of the other groups (#P ＜ 0.05). No significant variation in serum testosterone level was observed among the groups. 176
Brain Research Bulletin 149 (2019) 175–183
S. Tang, et al.
Fig. 1. Representative integrated XII discharge traces of female rats. Fig. 1 shows 60 min integrated traces of XII spontaneous discharge in female rats after normoxic or CIH exposure. The normoxic group was represented by one trace because no evident differences were observed between the normoxic control and SO groups.
percentage from baseline at 60 min: △%BL. In addition, only the CIH individuals were included. After drawing a scatter diagram (Fig. 5) of serum estradiol concentrations and XII neuroplasticity in female rats, we found a positive linear correlation. The regression equation was Y = −23.77 + 0.054 × X (Y = XII neuroplasticity X = estradiol concentration); R2 = 0.922, P < 0.001. Further correlation analysis showed a Pearson correlation coefficient of r = 0.960, P < 0.001. The results indicate that XII neuroplasticity in female rats was closely correlated with the estradiol level.
differences in the XII discharge frequency were observed among the groups (Fig. 3B) 2.4. Changes in XII spontaneous discharge activity in male rats Fig. 2 displays the 60-min integrated traces of XII spontaneous discharge in male rats after normoxia or CIH exposure. The normoxic group was represented by one trace as no differences were observed between the NC and SO groups. The XII discharge amplitudes (Fig. 4A) in the CIHC and OR + T groups were significantly greater than those in the normoxic group at each time point post-CIH (P < 0.001) and increased only within 30 min (P < 0.05) in the OR and OR+T+AI groups. Specifically, XII neuroplasticity occurred in the CIHC and OR +T groups but not in the OR and OR+T+AI groups. XII neuroplasticity in the CIHC and OR+T groups did not show any differences (P > 0.05). No significant differences in the XII discharge frequency were observed among the different groups (Fig. 4B)
2.6. Relationship between the serum testosterone concentration and XII neuroplasticity in male rats XII neuroplasticity was represented as the △%BL, and only CIH groups were analyzed. After drawing a scatter diagram (Fig. 6) of serum testosterone concentrations and XII neuroplasticity in male rats, we found a positive correlation. Further regression analysis yielded R2 = 0.391, P > 0.05, indicating that XII neuroplasticity in male rats was weakly positive and non-significantly correlated with the testosterone level.
2.5. Relationship between the serum estradiol concentration and XII neuroplasticity in female rats XII neuroplasticity was represented as the XII amplitude change 177
Brain Research Bulletin 149 (2019) 175–183
S. Tang, et al.
Fig. 2. Representative integrated XII discharge traces in male CIH rats. Fig. 2 shows 60 min integrated traces of XII spontaneous discharge in male rats after normoxic or CIH exposure. The normoxic group was represented by one trace because no evident differences were observed between the normoxic control and SO groups.
2.7. Relationship between serum 5-HT and estradiol concentrations in female rats
2.8. Relationship between serum 5-HT and testosterone concentrations in male rats
After drawing a scatter diagram (Fig. 7) of serum estradiol and 5-HT concentrations in female rats, we found a positive linear correlation. The regression equation was Y = 20.78 + 0.02 × X (Y = 5-HT concentration X = estradiol concentration); R2 = 0.699. Further correlation analysis showed a Pearson correlation coefficient of r = 0.836; P < 0.001. The results indicate that the serum 5-HT concentration in female rats was closely correlated with the estradiol level.
From the scatter diagram (Fig. 8) of serum testosterone and 5-HT concentrations in male rats, we found a positive linear correlation. The regression equation was Y = 22.95 + 1.3 × X (Y = 5-HT concentration, X = testosterone concentration); R2 = 0.564. Further correlation analysis showed a Pearson correlation coefficient of r = 0.751; P < 0.001. The results indicate that the serum concentrations of testosterone and 5-HT in male rats were closely correlated.
178
Brain Research Bulletin 149 (2019) 175–183
S. Tang, et al.
Fig. 3. Comparison of XII discharge amplitude and frequency in female rats. The symbols at the broken lines represent discrete data at every 15-min interval. A, The XII discharge amplitudes in the CIHC and OV + E2 groups were significantly greater than those of the normoxia group at 15, 30, 45 and 60 min (*P < 0.001) and increased within only 30 min in the OV group (#P < 0.05). Compared with the CIHC group, the XII discharge amplitudes in the OV group reduced within 60 min (&P ＜ 0.05). B, No significant differences in XII discharge frequency were observed among groups.
3. Discussion This article was designed to clarify the effect of gonadal hormones on CIH-induced XII neuroplasticity in male and female rats and their influence on the 5-HT concentration. Through gonadal resection and daily supplementation with gonadal hormone in both female and male rats, we managed the circulating levels of estradiol and testosterone, respectively. Meanwhile, 4 weeks of CIH exposure elicited enhanced continuous XII discharge activity in rats of both sexes, namely, XII neuroplasticity. In female rats, ovariectomy could counteract this respiratory plasticity manifestation, which was restored through estradiol supplementation. XII neuroplasticity was closely correlated with the estradiol level. In male rats, orchiectomy counteracted XII neuroplasticity, which was restored through testosterone supplementation, whereas supplementation with an aromatase inhibitor, which restricts conversion of testosterone to estradiol, could weaken XII neuroplasticity. XII neuroplasticity was not significantly correlated with the testosterone level. In addition, the serum 5-HT level was linked to the estradiol and testosterone levels in the respective genders.
Fig. 5. Scatter plot of serum estradiol concentration and XII neuroplasticity in female rats. XII neuroplasticity was represented as the XII amplitude change percentage from baseline at 60 min: △%BL. There was a positive linear correlation between serum estradiol concentration and XII neuroplasticity in female rats (R2 = 0.922, P < 0.001).
3.1. Changes in serum gonadal hormone concentrations We successfully modified the serum concentrations of sex hormones in rats by gonadal resection and daily supplementation with gonadal hormones. Female rats in the OV group had lower serum estradiol concentrations compared to the CIHC group, which were restored to the CIHC group level in the OV + E2 group, suggesting that the serum estradiol concentration in female rats was significantly altered through ovariectomy and daily estrogen supplementation. The serum testosterone level in male rats in the OR group was lower than that in the CIHC group but was restored to the CIHC group level in the OR + T and OR + T + AI groups, demonstrating that the serum testosterone concentration in male rats could be altered by orchiectomy and
testosterone supplementation. Nevertheless, aromatase inhibitors, which can inhibit the conversion of testosterone to estradiol, did not alter the serum testosterone level in this study. We also found that the serum testosterone concentration in the male rats in the CIHC group was significantly reduced compared to that in the normoxia group, showing that the testosterone concentration in male rats decreased after 4 weeks of CIH exposure. This outcome is in accordance with clinical studies: testosterone levels in male OSAS patients were significantly decreased compared to those in control
Fig. 4. Comparison of XII discharge amplitude and frequency in male rats. The symbols at the broken lines represent discrete data at every 15-min interval. A, Compared with the normoxic group, the XII discharge amplitudes in the CIHC and OR + T groups were significantly greater at each time point post-CIH (*P < 0.001) but increased within only 30 min in the OR and OR+T+AI groups (#P < 0.05). The XII discharge amplitudes in the CIHC and OR + T groups did not show any differences (P ＞ 0.05). Compared with the CIHC group, the XII discharge amplitudes in the OR and OR + T+AI groups reduced within 60 min (&P ＜ 0.05). B, There were no significant differences in XII discharge frequency among the different groups. 179
Brain Research Bulletin 149 (2019) 175–183
S. Tang, et al.
Fig. 6. Scatter plot of serum testosterone concentration and XII neuroplasticity in female rats. XII neuroplasticity was represented as the XII amplitude change percentage from baseline at 60 min: △%BL. There was a weak and non-significant correlation between XII neuroplasticity and testosterone concentration in male rats (R2 = 0.391, P > 0.05).
Fig. 8. Scatter plot of serum testosterone and 5-HT concentrations in male rats. There is a positive linear correlation between serum testosterone and 5-HT concentration in male rats (R2 = 0.564, P＜0.001).
3.2. Effects of gonadal hormone concentration variations on XII discharge activity The results indicated that 4 weeks of CIH exposure could result in enhanced continuous XII discharge activity in rats of both sexes, namely, XII neuroplasticity. Persistent elevation of XII activity contributes to the maintenance of genioglossus excitability and upper airway patency. In female rats, ovariectomy can counteract this neuroplasticity manifestation, which can be restored through estradiol supplementation, and XII neuroplasticity is significantly related to serum estradiol concentrations. In male rats, orchiectomy also counteracts XII neuroplasticity, which can be restored through testosterone supplementation, and supplementation with an aromatase inhibitor, which restricts the conversion of testosterone to estradiol, could weaken XII neuroplasticity, although the testosterone concentration was not notably changed; thus, XII neuroplasticity does not seem to be related to serum testosterone concentrations. These findings suggest that estradiol and testosterone can adjust CIH-evoked XII neuroplasticity in female and male rats and that XII neuroplasticity is closely related to estradiol rather than testosterone concentrations. Testosterone may indirectly alter XII neuroplasticity through conversion to estradiol. The role of gonadal hormones in CIH-evoked XII neuroplasticity has not been thoroughly studied. Other studies involving gonadal hormones have emphasized AIH-induced XII neuroplasticity, discovering that estradiol (Dougherty et al., 2017) and testosterone (Nelson et al., 2011; Zabka et al., 2005) may play important roles in modulating the neuroplasticity of upper airway motor nerves and that conversion of testosterone to estradiol modulates this neuroplasticity (Nelson et al., 2011; Zabka et al., 2006), which is consistent our outcomes. Unlike these AIH models, our outcomes revealed no correlation between testosterone concentrations and XII neuroplasticity. Behan et al. showed that respiratory motoneurons express gonadal hormone receptors (Behan and Thomas, 2005), suggesting that gonadal hormones may regulate respiration plasticity through the respiratory center.
Fig. 7. Scatter plot of serum estradiol and 5-HT concentrations in female rats. There is a positive linear correlation between serum estradiol and 5-HT concentration in female rats (R2 = 0.699, P＜0.001).
individuals(Bercea et al., 2013, 2015; Luboshitzky et al., 2005), and the decrease in the testosterone level was associated with AHI, the oxygen desaturation index (ODI), and nadir oxygen saturation(Hammoud et al., 2011). The low testosterone concentration among middle-aged men, a consistent finding among OSAS patients, can be explained by following causes: hypoxia, sleep deprivation, obesity and aging(Luboshitzky et al., 2005; Hammoud et al., 2011). On the other hand, we failed to detect significant differences in serum testosterone concentrations among female rats and serum estradiol concentrations among male rats. However, the serum gonadal hormone concentrations that we studied here simply represent circulating hormone density, which is not equivalent to concentrations in other parts of the body, such as respiratory-related areas in the central nervous system (tails of the raphe, phrenic and hypoglossal nerves). Therefore, further investigation of the concentrations of testosterone and estradiol in the hypoglossal nerve nuclei may provide more information.
3.3. Influence of gonadal hormone concentrations on serum 5-HT Through additional detection of the serum 5-HT concentration, the serum 5-HT concentration was found to be reduced in ovariectomized rats (the OV group) and restored through estradiol supplementation (the OV + E2 group), and the serum 5-HT level in female rats was strongly linked to the estradiol level. Among the male rats, the serum 5180
Brain Research Bulletin 149 (2019) 175–183
S. Tang, et al.
Aladdin (Shanghai, China). The reagents were dissolved in absolute ethyl alcohol to serve as mother liquor and then diluted with injectable soybean oil to the requisite concentrations. The final concentrations of E2, T and AI were 20 μg/ml, 5 mg/ml and 100 μg/ml, respectively. The ELISA kits for detecting serum estradiol, testosterone and 5-HT levels were supplied by Elabscience (Wuhan, China).
HT concentration was decreased in orchiectomized rats (the OR group) and testosterone and aromatase inhibitor supplementation rats (the OR + T+AI group) and increased through testosterone supplementation (the OR + T group). The serum 5-HT and testosterone concentrations were also closely correlated. Thus, serum 5-HT concentrations in rats were indirectly reformed by gonadal hormone concentration modification. As an important neurotransmitter in the brain, 5-HT is involved in multiple functions, including respiratory plasticity. Recent studies have explored the relationship between gonadal hormones and 5-HT, mostly in Parkinson’s disease (Sanchez et al., 2011) and aggression (Kuepper et al., 2010). Gonadal hormone-induced changes in the 5HT level may be significant in sleep-disordered breathing disorders such as OSAS. Researchers have found that serotonergic neurons express gonadal hormone receptors, including estrogen receptors (α, β) and androgen receptors, in macaques (Bethea et al., 2015) and rodents (Nikmahzar et al., 2016; Sheng et al., 2004). Estradiol increases serotonin reuptake transporter (SERT), 5-HT and serotonin metabolite 5HIAA concentrations (Matragrano et al., 2012; Sanchez et al., 2013a, b) and modulates serotonin receptors (5-HT1A and 5-HT2A) (Sanchez et al., 2011). Testosterone stimulates tryptophan hydroxylase 2 (TPH2) and SERT gene expression. Aromatase activity contributes to increased serotonin axon density (Bethea et al., 2014), and aromatase inhibitors decrease 5-HT production and release (Bethea et al., 2013). Thus, gonadal hormones affect the synthesis, release, reuse, and degradation of 5-HT, which may explain how gonadal hormones transform the 5-HT concentration. In a prior study, we demonstrated that increasing peripheral 5-HT levels contributed to inducing or enhancing XII neuroplasticity following CIH exposure in rats, which is regulated by the 5-HT2A receptor and activated through the PKC pathway (Tu et al., 2015). Other kinds of respiratory plasticity, including glossopharyngeal plasticity (Cao et al., 2010) and phrenic plasticity (Hoffman and Mitchell, 2013), also require the serotonergic system. As serum gonadal hormones can influence the level of 5-HT as stated above, gonadal hormones may also indirectly modulate XII neuroplasticity through the 5-HT system.
4.2. Experimental protocol Five groups of female rats were used in this study: (1) the normoxia control group (NC, n = 5), (2) the normoxia sham operation group (SO, n = 5), (3) the CIH control group (CIHC, n = 5), (4) the CIH with ovariectomy group (OV, n = 5), and (5) the CIH with ovariectomy and estrogen supplementation group (OV + E2, n = 5). Six groups of male rats were used: (1) the normoxia control group (NC, n = 5), (2) the normoxia sham operation group (SO, n = 5), (3) the CIH control group (CIHC, n = 5), (4) the CIH with orchiectomy group (OR), (5) the CIH with orchiectomy and testosterone supplementation group (OR + T, n = 5), and (6) the CIH with orchiectomy and testosterone plus aromatase inhibitor supplementation group (OR + T+AI, n = 5). The rats in the CIH groups were exposed to 4 continuous weeks of CIH after the operative incision for gonadectomy had healed. Finally, hypoglossal nerve spontaneous discharge activities were recorded, and serum estradiol, testosterone and 5-HT concentrations were detected by ELISA. 4.3. Gonadectomy and hormone replacement In this experiment, we adopted a gonadectomy and hormone replacement protocol to alter gonadal hormone levels. Female rats in the OV and OV + E2 groups underwent ovariectomy, namely, the bilateral ovaries were removed, while only a small amount of adipose tissue around the ovaries was removed in the SO group. Subsequently, the OV + E2 group received a daily estradiol supplement (1 ml/kg/d, intraperitoneal injection) at a defined time (8:00 am), whereas the OV group received an equivalent amount of injectable soybean oil. The male rats in the OR, OR + T and OR + T + AI groups underwent orchiectomy, namely, the bilateral testicles were extirpated, while only a small amount of adipose tissue around the spermatic cords was removed in the SO group. Thereafter, the OR + T and OR + T + AI groups received daily testosterone supplements (1 ml/kg/d, intraperitoneal injection) at a defined time (8:00 am). The latter group simultaneously received extra aromatase inhibitor (1 ml/kg/d, intraperitoneal injection), and the OR group received an equivalent amount of injectable soybean oil. No antibiotic was administered to prevent infection after surgery, and all rats recovered well within 2 weeks.
3.4. Conclusions This study reveals that serum gonadal hormone concentration variations can modulate XII neuroplasticity and affect the serum 5-HT level. Gonadal hormone reduction is commonly associated with OSAS in both sexes; nevertheless, hormone replacement therapy is not widely employed in clinical practice. Estradiol replacement therapy for highrisk postmenopausal women is limited considering the associated side effects, such as an enhanced risk for endometrial and breast cancer (Grady et al., 1992). Testosterone replacement therapy without adequate treatment of OSAS may further compromise respiratory and polysomnographic parameters (Burschtin and Wang, 2016). As we creatively combine gonadal hormones and 5-HT in OSAS models and demonstrate the effects of gonadal hormones on 5-HT, 5-HT transformation may be another factor to consider.
4.4. CIH exposure All animals in the CIH groups received CIH treatment according to the method that we previously established. Briefly, the rats were placed in animal cabins with constant atmospheric pressure and intermittent hypoxia conditions for 8 h (9:00 a.m.–5:00 p.m.) per day for four consecutive weeks. The oxygen concentration in the cabins was automatically controlled to alternate between 30 s of hypoxia (FiO2 10%) and 60 s of reoxygenation (FiO2 21%), simulating an apnea hypopnea index (AHI) of 40 times per minute. The rats in the normoxia groups were placed in identical animal cabins with continual circulation of normal air over the same time frame. At other times, the rats were fed in an animal breeding device with free access to food and water. Before initiating formal CIH processing, the rats were maintained under a reversed-phase day-night cycle with artificial light (darkness from 6:00 to 18:00, illumination from 18:00 to 6:00) to transform their circadian rhythms. According to our previous experience and studies, AIH (5 min/time, 3 times) processing was conducted to enhance hypoglossal nerve LTF before recording discharge activity.
4. Experimental procedure 4.1. Animals and reagents A total of 25 female rats and 30 male rats (Sprague–Dawley, 4 weeks old) were acquired from Hubei Research Center of Laboratory Animals. The rats were raised in a pathogen-free barrier animal breeding device under a 12:12-h light-dark cycle at a constant temperature of 25 °C with free access to food and water. The work in our article was carried out in accordance with EC Directive 86/609/EEC for animal experiments and was subject to approval by the Ethics Committee of Renmin Hospital of Wuhan University. All efforts were made to minimize the number of animals used and their suffering. Estradiol (E2), testosterone (T) and 4androsten-4-ol-3,17-dione (aromatase inhibitor, AI) were obtained from 181
Brain Research Bulletin 149 (2019) 175–183
S. Tang, et al.
4.5. XII spontaneous discharge recording
doi.org/10.1016/j.neuroscience.2004.09.058. Bercea, R.M., Patacchioli, F.R., Ghiciuc, C.M., Cojocaru, E., Mihaescu, T., 2013. Serum testosterone and depressive symptoms in severe OSA patients. Andrologia 45, 345–350. https://doi.org/10.1111/and.12022. Bercea, R.M., Mihaescu, T., Cojocaru, C., Bjorvatn, B., 2015. Fatigue and serum testosterone in obstructive sleep apnea patients. Clin. Respir. J. 9, 342–349. https://doi. org/10.1111/crj.12150. Bethea, C.L., Reddy, A.P., Robertson, N., Coleman, K., 2013. Effects of aromatase inhibition and androgen activity on serotonin and behavior in male macaques. Behav. Neurosci. 127, 400–414. https://doi.org/10.1037/a0032016. Bethea, C.L., Coleman, K., Phu, K., Reddy, A.P., Phu, A., 2014. Relationships between androgens, serotonin gene expression and innervation in male macaques. Neuroscience 274, 341–356. https://doi.org/10.1016/j.neuroscience.2014.05.056. Bethea, C.L., Phu, K., Belikova, Y., Bethea, S.C., 2015. Localization and regulation of reproductive steroid receptors in the raphe serotonin system of male macaques. J. Chem. Neuroanat. 66–67, 19–27. https://doi.org/10.1016/j.jchemneu.2015.04.001. Bixler, E.O., Vgontzas, A.N., Lin, H.M., Ten, H.T., Rein, J., Vela-Bueno, A., Kales, A., 2001. Prevalence of sleep-disordered breathing in women: effects of gender. Am. J. Respir. Crit. Care Med. 163, 608–613. https://doi.org/10.1164/ajrccm.163.3.9911064. Buchner, N.J., Sanner, B.M., Borgel, J., Rump, L.C., 2007. Continuous positive airway pressure treatment of mild to moderate obstructive sleep apnea reduces cardiovascular risk. Am. J. Respir. Crit. Care Med. 176, 1274–1280. https://doi.org/10.1164/ rccm.200611-1588OC. Burschtin, O., Wang, J., 2016. Testosterone deficiency and sleep apnea. Sleep Med. Clin. 11, 525–529. https://doi.org/10.1016/j.jsmc.2016.08.003. Cao, Y., Liu, C., Ling, L., 2010. Glossopharyngeal long-term facilitation requires serotonin 5-HT2 and NMDA receptors in rats. Respir. Physiol. Neurobiol. 170, 164–172. https://doi.org/10.1016/j.resp.2009.12.005. Carson, J.A., Manolagas, S.C., 2015. Effects of sex steroids on bones and muscles: similarities, parallels, and putative interactions in health and disease. Bone 80, 67–78. https://doi.org/10.1016/j.bone.2015.04.015. Celec, P., Mucska, I., Ostatnikova, D., Hodosy, J., 2014. Testosterone and estradiol are not affected in male and female patients with obstructive sleep apnea treated with continuous positive airway pressure. J. Endocrinol. Invest. 37, 9–12. https://doi.org/10. 1007/s40618-013-0003-3. Chen, H.H., Lu, J., Guan, Y.F., Li, S.J., Hu, T.T., Xie, Z.S., Wang, F., Peng, X.H., Liu, X., Xu, X., Zhao, F.P., Yu, B.L., Li, X.P., 2016. Estrogen/ERR-alpha signaling axis is associated with fiber-type conversion of upper airway muscles in patients with obstructive sleep apnea hypopnea syndrome. Sci. Rep. 6, 27088. https://doi.org/10.1038/srep27088. Dougherty, B.J., Kopp, E.S., Watters, J.J., 2017. Nongenomic actions of 17-beta estradiol restore respiratory neuroplasticity in young ovariectomized female rats. J. Neurosci. 37, 6648–6660. https://doi.org/10.1523/JNEUROSCI.0433-17.2017. Grady, D., Rubin, S.M., Petitti, D.B., Fox, C.S., Black, D., Ettinger, B., Ernster, V.L., Cummings, S.R., 1992. Hormone therapy to prevent disease and prolong life in postmenopausal women. Ann. Intern. Med. 117, 1016–1037. https://doi.org/10. 1016/0020-7292(93)90679-Q. Hammoud, A.O., Walker, J.M., Gibson, M., Cloward, T.V., Hunt, S.C., Kolotkin, R.L., Adams, T.D., Meikle, A.W., 2011. Sleep apnea, reproductive hormones and quality of sexual life in severely obese men. Obesity (Silver Spring) 19, 1118–1123. https://doi. org/10.1038/oby.2010.344. Hoffman, M.S., Mitchell, G.S., 2013. Spinal 5-HT7 receptors and protein kinase A constrain intermittent hypoxia-induced phrenic long-term facilitation. Neuroscience 250, 632–643. https://doi.org/10.1016/j.neuroscience.2013.06.068. Huang, Y., Liu, Y.H., 2011. Effects of phytoestrogens on genioglossus contractile properties in ovariectomized rats exposed to chronic intermittent hypoxia may be independent of their estrogenicity. Eur. J. Oral Sci. 119, 128–135. https://doi.org/10. 1111/j.1600-0722.2011.00815.x. Hughes, D.C., Stewart, C.E., Sculthorpe, N., Dugdale, H.F., Yousefian, F., Lewis, M.P., Sharples, A.P., 2016. Testosterone enables growth and hypertrophy in fusion impaired myoblasts that display myotube atrophy: deciphering the role of androgen and IGF-I receptors. Biogerontology 17, 619–639. https://doi.org/10.1007/s10522-0159621-9. Kuepper, Y., Alexander, N., Osinsky, R., Mueller, E., Schmitz, A., Netter, P., Hennig, J., 2010. Aggression–interactions of serotonin and testosterone in healthy men and women. Behav. Brain Res. 206, 93–100. https://doi.org/10.1016/j.bbr.2009.09.006. Lal, C., Strange, C., Bachman, D., 2012. Neurocognitive impairment in obstructive sleep apnea. Chest 141, 1601–1610. https://doi.org/10.1378/chest.11-2214. Li, Z., Tang, T., Wu, W., Gu, L., Du, J., Zhao, T., Zhou, X., Wu, H., Qin, G., 2016. Efficacy of nasal continuous positive airway pressure on patients with OSA with erectile dysfunction and low sex hormone levels. Respir. Med. 119, 130–134. https://doi.org/ 10.1016/j.rmed.2016.09.001. Luboshitzky, R., Aviv, A., Hefetz, A., Herer, P., Shen-Orr, Z., Lavie, L., Lavie, P., 2002. Decreased pituitary-gonadal secretion in men with obstructive sleep apnea. J. Clin. Endocrinol. Metab. 87, 3394–3398. https://doi.org/10.1210/jcem.87.7.8663. Luboshitzky, R., Lavie, L., Shen-Orr, Z., Herer, P., 2005. Altered luteinizing hormone and testosterone secretion in middle-aged obese men with obstructive sleep apnea. Obes. Res. 13, 780–786. https://doi.org/10.1038/oby.2005.88. Mahamed, S., Mitchell, G.S., 2007. Is there a link between intermittent hypoxia-induced respiratory plasticity and obstructive sleep apnoea? Exp. Physiol. 92, 27–37. https:// doi.org/10.1113/expphysiol.2006.033720. Marin, J.M., Carrizo, S.J., Vicente, E., Agusti, A.G., 2005. Long-term cardiovascular outcomes in men with obstructive sleep apnoea-hypopnoea with or without treatment with continuous positive airway pressure: an observational study. Lancet 365, 1046–1053. https://doi.org/10.1016/S0140-6736(05)71141-7. Mateika, J.H., Narwani, G., 2009. Intermittent hypoxia and respiratory plasticity in humans and other animals: does exposure to intermittent hypoxia promote or mitigate
The rats were anesthetized with intraperitoneal injections of urethane (1.0–1.2 g/kg) until corneal reflexes weakened and were fixed on a heating pad in the supine position. Then, an endotracheal tube was placed to maintain unobstructed breathing and bilateral vagotomy was performed. The hypoglossal nerve was dissociated from surrounding tissues by a glass needle with blunt ends and immediately immersed in tepid mineral oil. Then, a bipolar silver electrode was gently suspended onto the hypoglossal nerve without contacting other tissues to record spontaneous discharge. The nerve discharge signal was captured and analyzed by a biological medical signal processor (Power Lab, ADInstruments, Australia) with the bandpass filtered at 100–10,000 Hz and a time constant of 50 ms. All procedures were conducted such that unnecessary stimulation of the rats was minimized. 4.6. Detection of hormone and 5-HT levels Approximately 3 ml of blood was collected from each rat as soon as XII spontaneous discharge was recorded. The blood samples were labeled for the various rat groups and centrifuged (3000 rpm, 10 min) to obtain serum. Serum samples were labeled and used to detect testosterone, estradiol and 5-HT levels using competitive ELISA. According to the kit instructions, diluted serum samples and reagents were sequentially added to the ELISA plate. After the color reaction was terminated, optical density (OD) values at 450 nm were calculated by a microplate reader (TECAN, Switzerland). A standard curve was drawn utilizing the concentrations of the standard substances and OD values. Testosterone, estradiol and 5-hydroxytryptamine (5-HT) concentrations were calculated according to the standard curve. 4.7. Statistical analysis Statistical analysis was performed using IBM SPSS Statistics 22.0. The discharge amplitude and frequency of the hypoglossal nerve were recorded and analyzed at 0 (baseline), 15, 30, 45, and 60 min after normoxia or CIH exposure. All data in this study are absolute values and are expressed as the mean ± SD. Averages were compared by repeated-measures ANOVA to reveal significant differences among groups. Post hoc multivariate ANOVA was used to determine differences between groups. An LSD t-test was performed when the variance was the same; otherwise, Dunnett’s C test was used. For bivariate correlation analysis, we used the Pearson correlation coefficient. P values < 0.05 were considered statistically significant. Competing interests None declarations of interest. Funding This work was supported by grants from the National Natural Science Foundation of China (Nos. 81770089 and 81370181). Acknowledgments We thank Dr. Jing Feng and Prof. Baoyuan Chen, Respiratory Department, Tianjin Medical University General Hospital, China, for their support with the intermittent hypoxia chamber and the gas control delivery system used in this study. References Behan, M., Thomas, C.F., 2005. Sex hormone receptors are expressed in identified respiratory motoneurons in male and female rats. Neuroscience 130, 725–734. https://
182
Brain Research Bulletin 149 (2019) 175–183
S. Tang, et al.
Seif, F., Patel, S.R., Walia, H., Rueschman, M., Bhatt, D.L., Gottlieb, D.J., Lewis, E.F., Patil, S.P., Punjabi, N.M., Babineau, D.C., Redline, S., Mehra, R., 2013. Association between obstructive sleep apnea severity and endothelial dysfunction in an increased background of cardiovascular burden. J. Sleep Res. 22, 443–451. https://doi.org/10. 1111/jsr.12026. Sheng, Z., Kawano, J., Yanai, A., Fujinaga, R., Tanaka, M., Watanabe, Y., Shinoda, K., 2004. Expression of estrogen receptors (alpha, beta) and androgen receptor in serotonin neurons of the rat and mouse dorsal raphe nuclei; sex and species differences. Neurosci. Res. 49, 185–196. https://doi.org/10.1016/j.neures.2004.02.011. Tu, X., Wang, N., Hu, K., Xianyu, Y., Cao, X., Chen, H., Kang, J., 2013. The influence of intermittent hypoxia on long-term facilitation of hypoglossal nerve discharge in spontaneously breathing rats. Chin. J. Tuberc. Respir. Dis. 36, 437–440. https://doi. org/10.3760/cma.j.issn.1001-0939.2013.06.009. Tu, X., Zuo, J., Hu, K., Kang, J., Mei, Y., Wang, N., 2015. Effect of systemic application of 5-Hydroxytryptamine on hypoglossal nerve discharge in anesthetized rats. J. Mol. Neurosci. 57, 435–445. https://doi.org/10.1007/s12031-015-0590-x. Weaver, T.E., Grunstein, R.R., 2008. Adherence to continuous positive airway pressure therapy: the challenge to effective treatment. Proc. Am. Thorac. Soc. 5, 173–178. https://doi.org/10.1513/pats.200708-119MG. Wilcox, I., Semsarian, C., 2009. Obstructive sleep apnea a respiratory syndrome with protean cardiovascular manifestations. J. Am. Coll. Cardiol. 54, 1810–1812. https:// doi.org/10.1016/j.jacc.2009.06.039. Wu, X., Lu, H., Hu, L., Gong, W., Wang, J., Fu, C., Liu, Z., Li, S., 2017. Chronic intermittent hypoxia affects endogenous serotonergic inputs and expression of synaptic proteins in rat hypoglossal nucleus. Am. J. Transl. Res. 9, 546–557. http://www.ncbi. nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_ uids=28337282&query_hl=1. Zabka, A.G., Mitchell, G.S., Behan, M., 2005. Ageing and gonadectomy have similar effects on hypoglossal long-term facilitation in male Fischer rats. J. Physiol. 563, 557–568. https://doi.org/10.1113/jphysiol.2004.077511. Zabka, A.G., Mitchell, G.S., Behan, M., 2006. Conversion from testosterone to oestradiol is required to modulate respiratory long-term facilitation in male rats. J. Physiol. 576, 903–912. https://doi.org/10.1113/jphysiol.2006.114850. Zhang, X.B., Lin, Q.C., Zeng, H.Q., Jiang, X.T., Chen, B., Chen, X., 2016. Erectile dysfunction and sexual hormone levels in men with obstructive sleep apnea: efficacy of continuous positive airway pressure. Arch. Sex. Behav. 45, 235–240. https://doi.org/ 10.1007/s10508-015-0593-2.
sleep apnoea? Exp. Physiol. 94, 279–296. https://doi.org/10.1113/expphysiol.2008. 045153. Matragrano, L.L., Sanford, S.E., Salvante, K.G., Beaulieu, M., Sockman, K.W., Maney, D.L., 2012. Estradiol-dependent modulation of serotonergic markers in auditory areas of a seasonally breeding songbird. Behav. Neurosci. 126, 110–122. https://doi.org/10. 1037/a0025586. Nelson, N.R., Bird, I.M., Behan, M., 2011. Testosterone restores respiratory long term facilitation in old male rats by an aromatase-dependent mechanism. J. Physiol. 589, 409–421. https://doi.org/10.1113/jphysiol.2010.198200. Netzer, N.C., Eliasson, A.H., Strohl, K.P., 2003. Women with sleep apnea have lower levels of sex hormones. Sleep Breath. 7, 25–29. https://doi.org/10.1007/s11325-0030025-8. Neuzeret, P.C., Sakai, K., Gormand, F., Petitjean, T., Buda, C., Sastre, J.P., Parrot, S., Guidon, G., Lin, J.S., 2009. Application of histamine or serotonin to the hypoglossal nucleus increases genioglossus muscle activity across the wake-sleep cycle. J. Sleep Res. 18, 113–121. https://doi.org/10.1111/j.1365-2869.2008.00708.x. Nikmahzar, E., Jahanshahi, M., Ghaemi, A., Naseri, G.R., Moharreri, A.R., Lotfinia, A.A., 2016. Hippocampal serotonin-2A receptor-immunoreactive neurons density increases after testosterone therapy in the gonadectomized male mice. Anat. Cell Biol. 49, 259–272. https://doi.org/10.5115/acb.2016.49.4.259. Parati, G., Lombardi, C., Narkiewicz, K., 2007. Sleep apnea: epidemiology, pathophysiology, and relation to cardiovascular risk. Am. J. Physiol. Regul. Integr. Comp. Physiol. 293, R1671–83. https://doi.org/10.1152/ajpregu.00400.2007. Peppard, P.E., Young, T., Barnet, J.H., Palta, M., Hagen, E.W., Hla, K.M., 2013. Increased prevalence of sleep-disordered breathing in adults. Am. J. Epidemiol. 177. https:// doi.org/10.1093/aje/kws342. Sanchez, M.G., Estrada-Camarena, E., Belanger, N., Morissette, M., Di Paolo, T., 2011. Estradiol modulation of cortical, striatal and raphe nucleus 5-HT1A and 5-HT2A receptors of female hemiparkinsonian monkeys after long-term ovariectomy. Neuropharmacology 60, 642–652. https://doi.org/10.1016/j.neuropharm.2010.11. 024. Sanchez, M.G., Morissette, M., Di Paolo, T., 2013a. Oestradiol modulation of serotonin reuptake transporter and serotonin metabolism in the brain of monkeys. J. Neuroendocrinol. 25, 560–569. https://doi.org/10.1111/jne.12034. Sanchez, M.G., Morissette, M., Di Paolo, T., 2013b. Estradiol and brain serotonin reuptake transporter in long-term ovariectomized parkinsonian monkeys. Prog. Neuropsychopharmacol. Biol. Psychiatry 45, 170–177. https://doi.org/10.1016/j. pnpbp.2013.05.008.
183
Contents lists available at ScienceDirect
Brain Research Bulletin journal homepage: www.elsevier.com/locate/brainresbull
Research report
The role of gonadal hormones in the hypoglossal discharge activity of rats exposed to chronic intermittent hypoxia
T
⁎
Si Tang, Xiufang Zhou, Ke Hu , Pei Liu, Mengqing Xiong, Huimin Li Division of Respiratory Disease, Renmin Hospital of Wuhan University, Zhangzhidong Road No. 99, Wuhan 430060, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Chronic intermittent hypoxia Neuroplasticity Sex hormone Hypoglossal nerve
Objective: The role of gonadal hormones in chronic intermittent hypoxia (CIH)-evoked hypoglossal nerve (XII) neuroplasticity has not been thoroughly studied. The purpose of this study was to reveal the effects of gonadal hormone concentration variations on the XII discharge activity of rats exposed to CIH and the corresponding relationship with 5-hydroxytryptamine (5-HT). Methods: This study employed five groups of female rats and six groups of male rats. Gonadal hormone levels were modified through gonadal resection and daily supplementation with gonadal hormones in rats of both sexes. Rats in the CIH groups were exposed to an additional 4 weeks of CIH once the operative incision for gonadectomy had healed. Finally, XII spontaneous discharge activities were recorded, and serum estradiol, testosterone and 5-HT concentrations were detected by ELISA. Results: Among the female rats, the normal estradiol level groups expressed XII neuroplasticity, while the low estradiol level group failed to express this phenomenon. XII neuroplasticity was related to the serum estradiol concentration. In the male rats, XII neuroplasticity was successfully evoked in the normal testosterone level group but was suppressed in the low testosterone level group and aromatase inhibitor group. XII neuroplasticity was not significantly related to serum testosterone concentrations. Both estradiol and testosterone concentrations were related to 5-HT concentrations. Conclusions: This is the first study to analyze the effects of gonadal hormones on XII neuroplasticity in both female and male rats. The results suggest that the estradiol level is related to XII neuroplasticity rather than the testosterone level, and testosterone may indirectly adjust XII neuroplasticity by converting to estradiol. Estradiol and testosterone levels are related to 5-HT levels in the respective genders.
1. Introduction Obstructive sleep apnea syndrome (OSAS) is a common sleep-related breathing disorder that affects nearly 5–14% of middle-aged adults (Peppard et al., 2013). One of the main pathogenic factors of OSAS is periodic collapse of the upper airway induced by upper airway neuromuscular dysfunction. Repeated hypoxia and hypercapnia can lead to dysfunction in neurocognition (Lal et al., 2012), endocrine disorder (Seif et al., 2013) and hemodynamic changes (Wilcox and Semsarian, 2009), ultimately resulting in multisystem damage, especially cardiovascular diseases (Buchner et al., 2007; Marin et al., 2005; Parati et al., 2007). Nevertheless, continuous positive airway pressure (CPAP), the “gold standard” for treatment, has limited effectiveness because of poor compliance (17–54%) (Weaver and Grunstein, 2008). Therefore, a drug therapy that will facilitate better compliance is
urgently need. Epidemiological data show that middle-aged men account for the dominant OSAS incidence population; the number of male patients is 3.3 times higher than the number of women, and the incidence among postmenopausal women is ˜4.5 times higher than that among premenopausal women (Bixler et al., 2001). In addition, studies have discovered that testosterone levels were lower in OSAS patients compared to those in average persons (Luboshitzky et al., 2002), and women with low estrogen and progesterone levels(Netzer et al., 2003) are more likely to suffer from OSAS. However, whether CPAP therapy rescued gonadal hormone levels was ambiguous as conclusions are conflicting in different studies (Celec et al., 2014; Li et al., 2016; Zhang et al., 2016). As the incidence of OSAS is characteristically related to age and gender, the occurrence of OSAS is evidently associated with declining gonadal hormone concentrations; however, the mechanism
⁎
Corresponding author. E-mail addresses: [email protected] (S. Tang), [email protected] (X. Zhou), [email protected] (K. Hu), [email protected] (P. Liu), [email protected] (M. Xiong), [email protected] (H. Li). https://doi.org/10.1016/j.brainresbull.2019.04.017 Received 11 January 2019; Received in revised form 15 March 2019; Accepted 18 April 2019 Available online 22 April 2019 0361-9230/ © 2019 Elsevier Inc. All rights reserved.
Brain Research Bulletin 149 (2019) 175–183
S. Tang, et al.
remains unclear. The genioglossus muscle, which is innervated by the hypoglossal nerve (XII), is the main respiratory muscle maintaining expansion of the upper airway. Motoneurons in XII nuclei of both female and male rats have been found to express estrogen receptors (α, β) and androgen receptors (Behan and Thomas, 2005). Estrogen-related receptor α can regulate muscle fiber differentiation in the upper airway of OSAS patients (Chen et al., 2016), and estrogen can increase the anti-fatigue ability of the genioglossus in chronic intermittent hypoxia (CIH)-exposed rats (Huang and Liu, 2011). Although both testosterone and estrogens influence muscle physiology and metabolism (Carson and Manolagas, 2015), testosterone directly stimulates myotube hypertrophy but not differentiation(Hughes et al., 2016). In addition to the genioglossus, gonadal hormones can also adjust ventilatory function through the XII. Zabka et al. (2005, 2006) and Nelson et al. (2011) partially revealed the effect of gonadal hormones on respiratory plasticity in male and female (Dougherty et al., 2017) rats via acute intermittent hypoxia (AIH) but not CIH. Respiratory plasticity is a kind of serotonin-dependent hypoglossal or phrenic nerve neuroplasticity that is expressed as enhanced breathing motor output that can be sustained for up to an hour after intermittent hypoxia (Mahamed and Mitchell, 2007). Respiratory plasticity in OSAS patients helps counter airway occlusion during sleep, thus maintaining the stability of upper airway dilatation and expansion of the airway (Mateika and Narwani, 2009). CIH models better simulate the actual intermittent hypoxia course of OSAS patients, and CIH can induce more prominent XII neuroplasticity compared to AIH models (Tu et al., 2013). Many reports show that the central and peripheral serotonergic system is involved in XII neuroplasticity (Neuzeret et al., 2009; Tu et al., 2015; Wu et al., 2017). Moreover, numerous serotonergic neurons express estrogen receptors (α, β) and androgen receptors in macaques (Bethea et al., 2015), mice and rats (Nikmahzar et al., 2016; Sheng et al., 2004), and 5-hydroxytryptamine (5-HT) in hypoglossal nuclei is affected by hormone levels, raising the question of whether serum gonadal hormones can influence the 5-HT level. This is the first study to analyze the effects of serum gonadal hormones on CIH-induced XII neuroplasticity and the corresponding relationship with 5-HT levels in both genders.
Table 2 Serum gonadal hormone and 5-HT concentrations in male rats.
NC SO CIHC OV OV + E2
991.02 982.97 918.61 339.48 921.01
1.02 1.12 0.99 0.98 1.01
39.57 ± 4.20 38.64 ± 6.06 37.21 ± 3.85 27.95 ± 2.72# 39.428 ± 3.78
± ± ± ± ±
67.91 89.10 103.20 39.17* 42.76
± ± ± ± ±
0.23 0.26 0.07 0.07 0.05
NC SO CIHC OR OR + T OR + T+AI
11.45 ± 0.96 12.02 ± 1.17 9.81 ± 1.43* 1.76 ± 0.65*# 10.17 ± 1.29 9.78 ± 1.43*
128.17 160.28 167.45 157.72 174.68 148.95
39.58 37.95 36.94 27.17 36.99 30.43
± ± ± ± ± ±
38.54 39.45 30.52 26.51 37.64 40.80
± ± ± ± ± ±
4.77 5.82 4.02 4.11 & 4.46 5.21&
Serum testosterone, estradiol and 5-HT concentrations in male rats are presented in Table 2. Serum testosterone concentrations were not significantly different between the NC (11.45 ± 0.96 ng/ml) and SO groups (12.02 ± 1.17 ng/ml) but were lower in the CIHC group (9.81 ± 1.43 ng/ml), CIH with orchiectomy group (OR group, 1.76 ± 0.65 ng/ml) and testosterone and aromatase inhibitor supplementation group (OR + T + AI group, 9.78 ± 1.43 ng/ml) (P < 0.05), revealing that surgical stimulation did not change the serum testosterone level, but 4 weeks of CIH exposure resulted in decreased levels. Compared with the CIHC group, the serum testosterone concentration in the OR group (1.76 ± 0.65 ng/ml) decreased significantly (P < 0.001) but did not evidently change in the OR + T group (10.17 ± 1.29 ng/ml) and OR+T+AI group (9.78 ± 1.43 ng/ ml), showing that orchiectomy could significantly reduce the serum testosterone level, that testosterone supplementation could effectively restore the serum testosterone level, and that aromatase inhibitors seemed to have no distinct effect on the serum testosterone level. The serum 5-HT concentration was significantly reduced in the OR (27.17 ± 4.11 ng/ml) and OR + T + AI groups (29.23 ± 4.17 ng/ml) compared with that in the CIHC group (35.74 ± 5.44 ng/ml), whereas the serum 5-HT concentration in the OR + T group (36.79 ± 2.79 ng/ ml) was not significantly changed. No differences in serum estradiol levels were found among the groups.
Table 1 Serum gonadal hormone and 5-HT concentrations in female rats. 5-HT (ng/ml)
5-HT (ng/ml)
2.2. Serum gonadal hormone and 5-HT concentrations in male rats
Serum estradiol, testosterone and 5-HT concentrations in female rats are summarized in Table 1. No significant differences in the serum estradiol concentration were found among the normoxia control group (NC group, 991.02 ± 67.91 pg/ml), normoxia sham operation group (SO group, 982.97 ± 89.10 pg/ml), CIH control group (CIHC group, 918.61 ± 103.20 pg/ml) and CIH with ovariectomy and estrogen supplementation group (OV + E2 group, 921.01 ± 42.76 pg/ml), whereas the concentration in the CIH with ovariectomy group (OV
Testosterone (ng/ml)
Estradiol (pg/ml)
group, 339.48 ± 39.17 pg/ml) decreased significantly (P < 0.001), indicating that surgical stimulation or 4 weeks of CIH exposure may not notably affect estradiol levels, that ovariectomy can reduce these levels significantly, and that these levels can be effectively restored through estradiol supplementation. The serum 5-HT concentration in the OV group (27.95 ± 2.72 ng/ml) was markedly decreased compared with those in the other groups. On the other hand, we did not observe significant variations in serum testosterone levels among the groups.
2.1. Serum gonadal hormone and 5-HT concentrations in female rats
Estradiol (pg/ml)
Testosterone (ng/ml)
Serum testosterone concentration (ng/ml) in the CIHC (CIH control) group, OR group and OR + T + AI group were lower than that in the NC (Normal control) group (*P ＜ 0.05), and was decreased significantly in the OR group compared with the CIHC group (#P < 0.001). Serum 5-HT concentration (ng/ml) in the OR and OR + T+AI groups was significantly reduced compared with that in the CIHC group (&P ＜ 0.05). No significant differences in serum estradiol levels among groups were detected.
2. Results
Group
Group
2.3. Changes in XII spontaneous discharge activity in female rats Fig. 1 shows the 60-min integrated traces of XII spontaneous discharge in female rats after normoxia or CIH exposure. The normoxic group was represented by one trace because no differences were observed between the NC and SO groups. The XII discharge amplitudes (Fig. 3A) in the CIHC and OV + E2 groups were significantly greater than those in the normoxic group at each time point post-CIH (P < 0.001) and increased only within 30 min (P < 0.05) in the OV group. That is, the CIHC and OV+E2 groups successfully presented XII neuroplasticity, while the OV group failed to demonstrate XII neuroplasticity. No remarkable differences in XII neuroplasticity were found between the CIHC and OV + E2 groups (P > 0.05). No significant
Serum estradiol concentration (pg/ml) in the OV group decreased significantly compared with that of the CIHC (CIH control) group (*P ＜ 0.001). Serum 5-HT concentration (ng/ml) in the OV group decreased markedly compared with those of the other groups (#P ＜ 0.05). No significant variation in serum testosterone level was observed among the groups. 176
Brain Research Bulletin 149 (2019) 175–183
S. Tang, et al.
Fig. 1. Representative integrated XII discharge traces of female rats. Fig. 1 shows 60 min integrated traces of XII spontaneous discharge in female rats after normoxic or CIH exposure. The normoxic group was represented by one trace because no evident differences were observed between the normoxic control and SO groups.
percentage from baseline at 60 min: △%BL. In addition, only the CIH individuals were included. After drawing a scatter diagram (Fig. 5) of serum estradiol concentrations and XII neuroplasticity in female rats, we found a positive linear correlation. The regression equation was Y = −23.77 + 0.054 × X (Y = XII neuroplasticity X = estradiol concentration); R2 = 0.922, P < 0.001. Further correlation analysis showed a Pearson correlation coefficient of r = 0.960, P < 0.001. The results indicate that XII neuroplasticity in female rats was closely correlated with the estradiol level.
differences in the XII discharge frequency were observed among the groups (Fig. 3B) 2.4. Changes in XII spontaneous discharge activity in male rats Fig. 2 displays the 60-min integrated traces of XII spontaneous discharge in male rats after normoxia or CIH exposure. The normoxic group was represented by one trace as no differences were observed between the NC and SO groups. The XII discharge amplitudes (Fig. 4A) in the CIHC and OR + T groups were significantly greater than those in the normoxic group at each time point post-CIH (P < 0.001) and increased only within 30 min (P < 0.05) in the OR and OR+T+AI groups. Specifically, XII neuroplasticity occurred in the CIHC and OR +T groups but not in the OR and OR+T+AI groups. XII neuroplasticity in the CIHC and OR+T groups did not show any differences (P > 0.05). No significant differences in the XII discharge frequency were observed among the different groups (Fig. 4B)
2.6. Relationship between the serum testosterone concentration and XII neuroplasticity in male rats XII neuroplasticity was represented as the △%BL, and only CIH groups were analyzed. After drawing a scatter diagram (Fig. 6) of serum testosterone concentrations and XII neuroplasticity in male rats, we found a positive correlation. Further regression analysis yielded R2 = 0.391, P > 0.05, indicating that XII neuroplasticity in male rats was weakly positive and non-significantly correlated with the testosterone level.
2.5. Relationship between the serum estradiol concentration and XII neuroplasticity in female rats XII neuroplasticity was represented as the XII amplitude change 177
Brain Research Bulletin 149 (2019) 175–183
S. Tang, et al.
Fig. 2. Representative integrated XII discharge traces in male CIH rats. Fig. 2 shows 60 min integrated traces of XII spontaneous discharge in male rats after normoxic or CIH exposure. The normoxic group was represented by one trace because no evident differences were observed between the normoxic control and SO groups.
2.7. Relationship between serum 5-HT and estradiol concentrations in female rats
2.8. Relationship between serum 5-HT and testosterone concentrations in male rats
After drawing a scatter diagram (Fig. 7) of serum estradiol and 5-HT concentrations in female rats, we found a positive linear correlation. The regression equation was Y = 20.78 + 0.02 × X (Y = 5-HT concentration X = estradiol concentration); R2 = 0.699. Further correlation analysis showed a Pearson correlation coefficient of r = 0.836; P < 0.001. The results indicate that the serum 5-HT concentration in female rats was closely correlated with the estradiol level.
From the scatter diagram (Fig. 8) of serum testosterone and 5-HT concentrations in male rats, we found a positive linear correlation. The regression equation was Y = 22.95 + 1.3 × X (Y = 5-HT concentration, X = testosterone concentration); R2 = 0.564. Further correlation analysis showed a Pearson correlation coefficient of r = 0.751; P < 0.001. The results indicate that the serum concentrations of testosterone and 5-HT in male rats were closely correlated.
178
Brain Research Bulletin 149 (2019) 175–183
S. Tang, et al.
Fig. 3. Comparison of XII discharge amplitude and frequency in female rats. The symbols at the broken lines represent discrete data at every 15-min interval. A, The XII discharge amplitudes in the CIHC and OV + E2 groups were significantly greater than those of the normoxia group at 15, 30, 45 and 60 min (*P < 0.001) and increased within only 30 min in the OV group (#P < 0.05). Compared with the CIHC group, the XII discharge amplitudes in the OV group reduced within 60 min (&P ＜ 0.05). B, No significant differences in XII discharge frequency were observed among groups.
3. Discussion This article was designed to clarify the effect of gonadal hormones on CIH-induced XII neuroplasticity in male and female rats and their influence on the 5-HT concentration. Through gonadal resection and daily supplementation with gonadal hormone in both female and male rats, we managed the circulating levels of estradiol and testosterone, respectively. Meanwhile, 4 weeks of CIH exposure elicited enhanced continuous XII discharge activity in rats of both sexes, namely, XII neuroplasticity. In female rats, ovariectomy could counteract this respiratory plasticity manifestation, which was restored through estradiol supplementation. XII neuroplasticity was closely correlated with the estradiol level. In male rats, orchiectomy counteracted XII neuroplasticity, which was restored through testosterone supplementation, whereas supplementation with an aromatase inhibitor, which restricts conversion of testosterone to estradiol, could weaken XII neuroplasticity. XII neuroplasticity was not significantly correlated with the testosterone level. In addition, the serum 5-HT level was linked to the estradiol and testosterone levels in the respective genders.
Fig. 5. Scatter plot of serum estradiol concentration and XII neuroplasticity in female rats. XII neuroplasticity was represented as the XII amplitude change percentage from baseline at 60 min: △%BL. There was a positive linear correlation between serum estradiol concentration and XII neuroplasticity in female rats (R2 = 0.922, P < 0.001).
3.1. Changes in serum gonadal hormone concentrations We successfully modified the serum concentrations of sex hormones in rats by gonadal resection and daily supplementation with gonadal hormones. Female rats in the OV group had lower serum estradiol concentrations compared to the CIHC group, which were restored to the CIHC group level in the OV + E2 group, suggesting that the serum estradiol concentration in female rats was significantly altered through ovariectomy and daily estrogen supplementation. The serum testosterone level in male rats in the OR group was lower than that in the CIHC group but was restored to the CIHC group level in the OR + T and OR + T + AI groups, demonstrating that the serum testosterone concentration in male rats could be altered by orchiectomy and
testosterone supplementation. Nevertheless, aromatase inhibitors, which can inhibit the conversion of testosterone to estradiol, did not alter the serum testosterone level in this study. We also found that the serum testosterone concentration in the male rats in the CIHC group was significantly reduced compared to that in the normoxia group, showing that the testosterone concentration in male rats decreased after 4 weeks of CIH exposure. This outcome is in accordance with clinical studies: testosterone levels in male OSAS patients were significantly decreased compared to those in control
Fig. 4. Comparison of XII discharge amplitude and frequency in male rats. The symbols at the broken lines represent discrete data at every 15-min interval. A, Compared with the normoxic group, the XII discharge amplitudes in the CIHC and OR + T groups were significantly greater at each time point post-CIH (*P < 0.001) but increased within only 30 min in the OR and OR+T+AI groups (#P < 0.05). The XII discharge amplitudes in the CIHC and OR + T groups did not show any differences (P ＞ 0.05). Compared with the CIHC group, the XII discharge amplitudes in the OR and OR + T+AI groups reduced within 60 min (&P ＜ 0.05). B, There were no significant differences in XII discharge frequency among the different groups. 179
Brain Research Bulletin 149 (2019) 175–183
S. Tang, et al.
Fig. 6. Scatter plot of serum testosterone concentration and XII neuroplasticity in female rats. XII neuroplasticity was represented as the XII amplitude change percentage from baseline at 60 min: △%BL. There was a weak and non-significant correlation between XII neuroplasticity and testosterone concentration in male rats (R2 = 0.391, P > 0.05).
Fig. 8. Scatter plot of serum testosterone and 5-HT concentrations in male rats. There is a positive linear correlation between serum testosterone and 5-HT concentration in male rats (R2 = 0.564, P＜0.001).
3.2. Effects of gonadal hormone concentration variations on XII discharge activity The results indicated that 4 weeks of CIH exposure could result in enhanced continuous XII discharge activity in rats of both sexes, namely, XII neuroplasticity. Persistent elevation of XII activity contributes to the maintenance of genioglossus excitability and upper airway patency. In female rats, ovariectomy can counteract this neuroplasticity manifestation, which can be restored through estradiol supplementation, and XII neuroplasticity is significantly related to serum estradiol concentrations. In male rats, orchiectomy also counteracts XII neuroplasticity, which can be restored through testosterone supplementation, and supplementation with an aromatase inhibitor, which restricts the conversion of testosterone to estradiol, could weaken XII neuroplasticity, although the testosterone concentration was not notably changed; thus, XII neuroplasticity does not seem to be related to serum testosterone concentrations. These findings suggest that estradiol and testosterone can adjust CIH-evoked XII neuroplasticity in female and male rats and that XII neuroplasticity is closely related to estradiol rather than testosterone concentrations. Testosterone may indirectly alter XII neuroplasticity through conversion to estradiol. The role of gonadal hormones in CIH-evoked XII neuroplasticity has not been thoroughly studied. Other studies involving gonadal hormones have emphasized AIH-induced XII neuroplasticity, discovering that estradiol (Dougherty et al., 2017) and testosterone (Nelson et al., 2011; Zabka et al., 2005) may play important roles in modulating the neuroplasticity of upper airway motor nerves and that conversion of testosterone to estradiol modulates this neuroplasticity (Nelson et al., 2011; Zabka et al., 2006), which is consistent our outcomes. Unlike these AIH models, our outcomes revealed no correlation between testosterone concentrations and XII neuroplasticity. Behan et al. showed that respiratory motoneurons express gonadal hormone receptors (Behan and Thomas, 2005), suggesting that gonadal hormones may regulate respiration plasticity through the respiratory center.
Fig. 7. Scatter plot of serum estradiol and 5-HT concentrations in female rats. There is a positive linear correlation between serum estradiol and 5-HT concentration in female rats (R2 = 0.699, P＜0.001).
individuals(Bercea et al., 2013, 2015; Luboshitzky et al., 2005), and the decrease in the testosterone level was associated with AHI, the oxygen desaturation index (ODI), and nadir oxygen saturation(Hammoud et al., 2011). The low testosterone concentration among middle-aged men, a consistent finding among OSAS patients, can be explained by following causes: hypoxia, sleep deprivation, obesity and aging(Luboshitzky et al., 2005; Hammoud et al., 2011). On the other hand, we failed to detect significant differences in serum testosterone concentrations among female rats and serum estradiol concentrations among male rats. However, the serum gonadal hormone concentrations that we studied here simply represent circulating hormone density, which is not equivalent to concentrations in other parts of the body, such as respiratory-related areas in the central nervous system (tails of the raphe, phrenic and hypoglossal nerves). Therefore, further investigation of the concentrations of testosterone and estradiol in the hypoglossal nerve nuclei may provide more information.
3.3. Influence of gonadal hormone concentrations on serum 5-HT Through additional detection of the serum 5-HT concentration, the serum 5-HT concentration was found to be reduced in ovariectomized rats (the OV group) and restored through estradiol supplementation (the OV + E2 group), and the serum 5-HT level in female rats was strongly linked to the estradiol level. Among the male rats, the serum 5180
Brain Research Bulletin 149 (2019) 175–183
S. Tang, et al.
Aladdin (Shanghai, China). The reagents were dissolved in absolute ethyl alcohol to serve as mother liquor and then diluted with injectable soybean oil to the requisite concentrations. The final concentrations of E2, T and AI were 20 μg/ml, 5 mg/ml and 100 μg/ml, respectively. The ELISA kits for detecting serum estradiol, testosterone and 5-HT levels were supplied by Elabscience (Wuhan, China).
HT concentration was decreased in orchiectomized rats (the OR group) and testosterone and aromatase inhibitor supplementation rats (the OR + T+AI group) and increased through testosterone supplementation (the OR + T group). The serum 5-HT and testosterone concentrations were also closely correlated. Thus, serum 5-HT concentrations in rats were indirectly reformed by gonadal hormone concentration modification. As an important neurotransmitter in the brain, 5-HT is involved in multiple functions, including respiratory plasticity. Recent studies have explored the relationship between gonadal hormones and 5-HT, mostly in Parkinson’s disease (Sanchez et al., 2011) and aggression (Kuepper et al., 2010). Gonadal hormone-induced changes in the 5HT level may be significant in sleep-disordered breathing disorders such as OSAS. Researchers have found that serotonergic neurons express gonadal hormone receptors, including estrogen receptors (α, β) and androgen receptors, in macaques (Bethea et al., 2015) and rodents (Nikmahzar et al., 2016; Sheng et al., 2004). Estradiol increases serotonin reuptake transporter (SERT), 5-HT and serotonin metabolite 5HIAA concentrations (Matragrano et al., 2012; Sanchez et al., 2013a, b) and modulates serotonin receptors (5-HT1A and 5-HT2A) (Sanchez et al., 2011). Testosterone stimulates tryptophan hydroxylase 2 (TPH2) and SERT gene expression. Aromatase activity contributes to increased serotonin axon density (Bethea et al., 2014), and aromatase inhibitors decrease 5-HT production and release (Bethea et al., 2013). Thus, gonadal hormones affect the synthesis, release, reuse, and degradation of 5-HT, which may explain how gonadal hormones transform the 5-HT concentration. In a prior study, we demonstrated that increasing peripheral 5-HT levels contributed to inducing or enhancing XII neuroplasticity following CIH exposure in rats, which is regulated by the 5-HT2A receptor and activated through the PKC pathway (Tu et al., 2015). Other kinds of respiratory plasticity, including glossopharyngeal plasticity (Cao et al., 2010) and phrenic plasticity (Hoffman and Mitchell, 2013), also require the serotonergic system. As serum gonadal hormones can influence the level of 5-HT as stated above, gonadal hormones may also indirectly modulate XII neuroplasticity through the 5-HT system.
4.2. Experimental protocol Five groups of female rats were used in this study: (1) the normoxia control group (NC, n = 5), (2) the normoxia sham operation group (SO, n = 5), (3) the CIH control group (CIHC, n = 5), (4) the CIH with ovariectomy group (OV, n = 5), and (5) the CIH with ovariectomy and estrogen supplementation group (OV + E2, n = 5). Six groups of male rats were used: (1) the normoxia control group (NC, n = 5), (2) the normoxia sham operation group (SO, n = 5), (3) the CIH control group (CIHC, n = 5), (4) the CIH with orchiectomy group (OR), (5) the CIH with orchiectomy and testosterone supplementation group (OR + T, n = 5), and (6) the CIH with orchiectomy and testosterone plus aromatase inhibitor supplementation group (OR + T+AI, n = 5). The rats in the CIH groups were exposed to 4 continuous weeks of CIH after the operative incision for gonadectomy had healed. Finally, hypoglossal nerve spontaneous discharge activities were recorded, and serum estradiol, testosterone and 5-HT concentrations were detected by ELISA. 4.3. Gonadectomy and hormone replacement In this experiment, we adopted a gonadectomy and hormone replacement protocol to alter gonadal hormone levels. Female rats in the OV and OV + E2 groups underwent ovariectomy, namely, the bilateral ovaries were removed, while only a small amount of adipose tissue around the ovaries was removed in the SO group. Subsequently, the OV + E2 group received a daily estradiol supplement (1 ml/kg/d, intraperitoneal injection) at a defined time (8:00 am), whereas the OV group received an equivalent amount of injectable soybean oil. The male rats in the OR, OR + T and OR + T + AI groups underwent orchiectomy, namely, the bilateral testicles were extirpated, while only a small amount of adipose tissue around the spermatic cords was removed in the SO group. Thereafter, the OR + T and OR + T + AI groups received daily testosterone supplements (1 ml/kg/d, intraperitoneal injection) at a defined time (8:00 am). The latter group simultaneously received extra aromatase inhibitor (1 ml/kg/d, intraperitoneal injection), and the OR group received an equivalent amount of injectable soybean oil. No antibiotic was administered to prevent infection after surgery, and all rats recovered well within 2 weeks.
3.4. Conclusions This study reveals that serum gonadal hormone concentration variations can modulate XII neuroplasticity and affect the serum 5-HT level. Gonadal hormone reduction is commonly associated with OSAS in both sexes; nevertheless, hormone replacement therapy is not widely employed in clinical practice. Estradiol replacement therapy for highrisk postmenopausal women is limited considering the associated side effects, such as an enhanced risk for endometrial and breast cancer (Grady et al., 1992). Testosterone replacement therapy without adequate treatment of OSAS may further compromise respiratory and polysomnographic parameters (Burschtin and Wang, 2016). As we creatively combine gonadal hormones and 5-HT in OSAS models and demonstrate the effects of gonadal hormones on 5-HT, 5-HT transformation may be another factor to consider.
4.4. CIH exposure All animals in the CIH groups received CIH treatment according to the method that we previously established. Briefly, the rats were placed in animal cabins with constant atmospheric pressure and intermittent hypoxia conditions for 8 h (9:00 a.m.–5:00 p.m.) per day for four consecutive weeks. The oxygen concentration in the cabins was automatically controlled to alternate between 30 s of hypoxia (FiO2 10%) and 60 s of reoxygenation (FiO2 21%), simulating an apnea hypopnea index (AHI) of 40 times per minute. The rats in the normoxia groups were placed in identical animal cabins with continual circulation of normal air over the same time frame. At other times, the rats were fed in an animal breeding device with free access to food and water. Before initiating formal CIH processing, the rats were maintained under a reversed-phase day-night cycle with artificial light (darkness from 6:00 to 18:00, illumination from 18:00 to 6:00) to transform their circadian rhythms. According to our previous experience and studies, AIH (5 min/time, 3 times) processing was conducted to enhance hypoglossal nerve LTF before recording discharge activity.
4. Experimental procedure 4.1. Animals and reagents A total of 25 female rats and 30 male rats (Sprague–Dawley, 4 weeks old) were acquired from Hubei Research Center of Laboratory Animals. The rats were raised in a pathogen-free barrier animal breeding device under a 12:12-h light-dark cycle at a constant temperature of 25 °C with free access to food and water. The work in our article was carried out in accordance with EC Directive 86/609/EEC for animal experiments and was subject to approval by the Ethics Committee of Renmin Hospital of Wuhan University. All efforts were made to minimize the number of animals used and their suffering. Estradiol (E2), testosterone (T) and 4androsten-4-ol-3,17-dione (aromatase inhibitor, AI) were obtained from 181
Brain Research Bulletin 149 (2019) 175–183
S. Tang, et al.
4.5. XII spontaneous discharge recording
doi.org/10.1016/j.neuroscience.2004.09.058. Bercea, R.M., Patacchioli, F.R., Ghiciuc, C.M., Cojocaru, E., Mihaescu, T., 2013. Serum testosterone and depressive symptoms in severe OSA patients. Andrologia 45, 345–350. https://doi.org/10.1111/and.12022. Bercea, R.M., Mihaescu, T., Cojocaru, C., Bjorvatn, B., 2015. Fatigue and serum testosterone in obstructive sleep apnea patients. Clin. Respir. J. 9, 342–349. https://doi. org/10.1111/crj.12150. Bethea, C.L., Reddy, A.P., Robertson, N., Coleman, K., 2013. Effects of aromatase inhibition and androgen activity on serotonin and behavior in male macaques. Behav. Neurosci. 127, 400–414. https://doi.org/10.1037/a0032016. Bethea, C.L., Coleman, K., Phu, K., Reddy, A.P., Phu, A., 2014. Relationships between androgens, serotonin gene expression and innervation in male macaques. Neuroscience 274, 341–356. https://doi.org/10.1016/j.neuroscience.2014.05.056. Bethea, C.L., Phu, K., Belikova, Y., Bethea, S.C., 2015. Localization and regulation of reproductive steroid receptors in the raphe serotonin system of male macaques. J. Chem. Neuroanat. 66–67, 19–27. https://doi.org/10.1016/j.jchemneu.2015.04.001. Bixler, E.O., Vgontzas, A.N., Lin, H.M., Ten, H.T., Rein, J., Vela-Bueno, A., Kales, A., 2001. Prevalence of sleep-disordered breathing in women: effects of gender. Am. J. Respir. Crit. Care Med. 163, 608–613. https://doi.org/10.1164/ajrccm.163.3.9911064. Buchner, N.J., Sanner, B.M., Borgel, J., Rump, L.C., 2007. Continuous positive airway pressure treatment of mild to moderate obstructive sleep apnea reduces cardiovascular risk. Am. J. Respir. Crit. Care Med. 176, 1274–1280. https://doi.org/10.1164/ rccm.200611-1588OC. Burschtin, O., Wang, J., 2016. Testosterone deficiency and sleep apnea. Sleep Med. Clin. 11, 525–529. https://doi.org/10.1016/j.jsmc.2016.08.003. Cao, Y., Liu, C., Ling, L., 2010. Glossopharyngeal long-term facilitation requires serotonin 5-HT2 and NMDA receptors in rats. Respir. Physiol. Neurobiol. 170, 164–172. https://doi.org/10.1016/j.resp.2009.12.005. Carson, J.A., Manolagas, S.C., 2015. Effects of sex steroids on bones and muscles: similarities, parallels, and putative interactions in health and disease. Bone 80, 67–78. https://doi.org/10.1016/j.bone.2015.04.015. Celec, P., Mucska, I., Ostatnikova, D., Hodosy, J., 2014. Testosterone and estradiol are not affected in male and female patients with obstructive sleep apnea treated with continuous positive airway pressure. J. Endocrinol. Invest. 37, 9–12. https://doi.org/10. 1007/s40618-013-0003-3. Chen, H.H., Lu, J., Guan, Y.F., Li, S.J., Hu, T.T., Xie, Z.S., Wang, F., Peng, X.H., Liu, X., Xu, X., Zhao, F.P., Yu, B.L., Li, X.P., 2016. Estrogen/ERR-alpha signaling axis is associated with fiber-type conversion of upper airway muscles in patients with obstructive sleep apnea hypopnea syndrome. Sci. Rep. 6, 27088. https://doi.org/10.1038/srep27088. Dougherty, B.J., Kopp, E.S., Watters, J.J., 2017. Nongenomic actions of 17-beta estradiol restore respiratory neuroplasticity in young ovariectomized female rats. J. Neurosci. 37, 6648–6660. https://doi.org/10.1523/JNEUROSCI.0433-17.2017. Grady, D., Rubin, S.M., Petitti, D.B., Fox, C.S., Black, D., Ettinger, B., Ernster, V.L., Cummings, S.R., 1992. Hormone therapy to prevent disease and prolong life in postmenopausal women. Ann. Intern. Med. 117, 1016–1037. https://doi.org/10. 1016/0020-7292(93)90679-Q. Hammoud, A.O., Walker, J.M., Gibson, M., Cloward, T.V., Hunt, S.C., Kolotkin, R.L., Adams, T.D., Meikle, A.W., 2011. Sleep apnea, reproductive hormones and quality of sexual life in severely obese men. Obesity (Silver Spring) 19, 1118–1123. https://doi. org/10.1038/oby.2010.344. Hoffman, M.S., Mitchell, G.S., 2013. Spinal 5-HT7 receptors and protein kinase A constrain intermittent hypoxia-induced phrenic long-term facilitation. Neuroscience 250, 632–643. https://doi.org/10.1016/j.neuroscience.2013.06.068. Huang, Y., Liu, Y.H., 2011. Effects of phytoestrogens on genioglossus contractile properties in ovariectomized rats exposed to chronic intermittent hypoxia may be independent of their estrogenicity. Eur. J. Oral Sci. 119, 128–135. https://doi.org/10. 1111/j.1600-0722.2011.00815.x. Hughes, D.C., Stewart, C.E., Sculthorpe, N., Dugdale, H.F., Yousefian, F., Lewis, M.P., Sharples, A.P., 2016. Testosterone enables growth and hypertrophy in fusion impaired myoblasts that display myotube atrophy: deciphering the role of androgen and IGF-I receptors. Biogerontology 17, 619–639. https://doi.org/10.1007/s10522-0159621-9. Kuepper, Y., Alexander, N., Osinsky, R., Mueller, E., Schmitz, A., Netter, P., Hennig, J., 2010. Aggression–interactions of serotonin and testosterone in healthy men and women. Behav. Brain Res. 206, 93–100. https://doi.org/10.1016/j.bbr.2009.09.006. Lal, C., Strange, C., Bachman, D., 2012. Neurocognitive impairment in obstructive sleep apnea. Chest 141, 1601–1610. https://doi.org/10.1378/chest.11-2214. Li, Z., Tang, T., Wu, W., Gu, L., Du, J., Zhao, T., Zhou, X., Wu, H., Qin, G., 2016. Efficacy of nasal continuous positive airway pressure on patients with OSA with erectile dysfunction and low sex hormone levels. Respir. Med. 119, 130–134. https://doi.org/ 10.1016/j.rmed.2016.09.001. Luboshitzky, R., Aviv, A., Hefetz, A., Herer, P., Shen-Orr, Z., Lavie, L., Lavie, P., 2002. Decreased pituitary-gonadal secretion in men with obstructive sleep apnea. J. Clin. Endocrinol. Metab. 87, 3394–3398. https://doi.org/10.1210/jcem.87.7.8663. Luboshitzky, R., Lavie, L., Shen-Orr, Z., Herer, P., 2005. Altered luteinizing hormone and testosterone secretion in middle-aged obese men with obstructive sleep apnea. Obes. Res. 13, 780–786. https://doi.org/10.1038/oby.2005.88. Mahamed, S., Mitchell, G.S., 2007. Is there a link between intermittent hypoxia-induced respiratory plasticity and obstructive sleep apnoea? Exp. Physiol. 92, 27–37. https:// doi.org/10.1113/expphysiol.2006.033720. Marin, J.M., Carrizo, S.J., Vicente, E., Agusti, A.G., 2005. Long-term cardiovascular outcomes in men with obstructive sleep apnoea-hypopnoea with or without treatment with continuous positive airway pressure: an observational study. Lancet 365, 1046–1053. https://doi.org/10.1016/S0140-6736(05)71141-7. Mateika, J.H., Narwani, G., 2009. Intermittent hypoxia and respiratory plasticity in humans and other animals: does exposure to intermittent hypoxia promote or mitigate
The rats were anesthetized with intraperitoneal injections of urethane (1.0–1.2 g/kg) until corneal reflexes weakened and were fixed on a heating pad in the supine position. Then, an endotracheal tube was placed to maintain unobstructed breathing and bilateral vagotomy was performed. The hypoglossal nerve was dissociated from surrounding tissues by a glass needle with blunt ends and immediately immersed in tepid mineral oil. Then, a bipolar silver electrode was gently suspended onto the hypoglossal nerve without contacting other tissues to record spontaneous discharge. The nerve discharge signal was captured and analyzed by a biological medical signal processor (Power Lab, ADInstruments, Australia) with the bandpass filtered at 100–10,000 Hz and a time constant of 50 ms. All procedures were conducted such that unnecessary stimulation of the rats was minimized. 4.6. Detection of hormone and 5-HT levels Approximately 3 ml of blood was collected from each rat as soon as XII spontaneous discharge was recorded. The blood samples were labeled for the various rat groups and centrifuged (3000 rpm, 10 min) to obtain serum. Serum samples were labeled and used to detect testosterone, estradiol and 5-HT levels using competitive ELISA. According to the kit instructions, diluted serum samples and reagents were sequentially added to the ELISA plate. After the color reaction was terminated, optical density (OD) values at 450 nm were calculated by a microplate reader (TECAN, Switzerland). A standard curve was drawn utilizing the concentrations of the standard substances and OD values. Testosterone, estradiol and 5-hydroxytryptamine (5-HT) concentrations were calculated according to the standard curve. 4.7. Statistical analysis Statistical analysis was performed using IBM SPSS Statistics 22.0. The discharge amplitude and frequency of the hypoglossal nerve were recorded and analyzed at 0 (baseline), 15, 30, 45, and 60 min after normoxia or CIH exposure. All data in this study are absolute values and are expressed as the mean ± SD. Averages were compared by repeated-measures ANOVA to reveal significant differences among groups. Post hoc multivariate ANOVA was used to determine differences between groups. An LSD t-test was performed when the variance was the same; otherwise, Dunnett’s C test was used. For bivariate correlation analysis, we used the Pearson correlation coefficient. P values < 0.05 were considered statistically significant. Competing interests None declarations of interest. Funding This work was supported by grants from the National Natural Science Foundation of China (Nos. 81770089 and 81370181). Acknowledgments We thank Dr. Jing Feng and Prof. Baoyuan Chen, Respiratory Department, Tianjin Medical University General Hospital, China, for their support with the intermittent hypoxia chamber and the gas control delivery system used in this study. References Behan, M., Thomas, C.F., 2005. Sex hormone receptors are expressed in identified respiratory motoneurons in male and female rats. Neuroscience 130, 725–734. https://
182
Brain Research Bulletin 149 (2019) 175–183
S. Tang, et al.
Seif, F., Patel, S.R., Walia, H., Rueschman, M., Bhatt, D.L., Gottlieb, D.J., Lewis, E.F., Patil, S.P., Punjabi, N.M., Babineau, D.C., Redline, S., Mehra, R., 2013. Association between obstructive sleep apnea severity and endothelial dysfunction in an increased background of cardiovascular burden. J. Sleep Res. 22, 443–451. https://doi.org/10. 1111/jsr.12026. Sheng, Z., Kawano, J., Yanai, A., Fujinaga, R., Tanaka, M., Watanabe, Y., Shinoda, K., 2004. Expression of estrogen receptors (alpha, beta) and androgen receptor in serotonin neurons of the rat and mouse dorsal raphe nuclei; sex and species differences. Neurosci. Res. 49, 185–196. https://doi.org/10.1016/j.neures.2004.02.011. Tu, X., Wang, N., Hu, K., Xianyu, Y., Cao, X., Chen, H., Kang, J., 2013. The influence of intermittent hypoxia on long-term facilitation of hypoglossal nerve discharge in spontaneously breathing rats. Chin. J. Tuberc. Respir. Dis. 36, 437–440. https://doi. org/10.3760/cma.j.issn.1001-0939.2013.06.009. Tu, X., Zuo, J., Hu, K., Kang, J., Mei, Y., Wang, N., 2015. Effect of systemic application of 5-Hydroxytryptamine on hypoglossal nerve discharge in anesthetized rats. J. Mol. Neurosci. 57, 435–445. https://doi.org/10.1007/s12031-015-0590-x. Weaver, T.E., Grunstein, R.R., 2008. Adherence to continuous positive airway pressure therapy: the challenge to effective treatment. Proc. Am. Thorac. Soc. 5, 173–178. https://doi.org/10.1513/pats.200708-119MG. Wilcox, I., Semsarian, C., 2009. Obstructive sleep apnea a respiratory syndrome with protean cardiovascular manifestations. J. Am. Coll. Cardiol. 54, 1810–1812. https:// doi.org/10.1016/j.jacc.2009.06.039. Wu, X., Lu, H., Hu, L., Gong, W., Wang, J., Fu, C., Liu, Z., Li, S., 2017. Chronic intermittent hypoxia affects endogenous serotonergic inputs and expression of synaptic proteins in rat hypoglossal nucleus. Am. J. Transl. Res. 9, 546–557. http://www.ncbi. nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_ uids=28337282&query_hl=1. Zabka, A.G., Mitchell, G.S., Behan, M., 2005. Ageing and gonadectomy have similar effects on hypoglossal long-term facilitation in male Fischer rats. J. Physiol. 563, 557–568. https://doi.org/10.1113/jphysiol.2004.077511. Zabka, A.G., Mitchell, G.S., Behan, M., 2006. Conversion from testosterone to oestradiol is required to modulate respiratory long-term facilitation in male rats. J. Physiol. 576, 903–912. https://doi.org/10.1113/jphysiol.2006.114850. Zhang, X.B., Lin, Q.C., Zeng, H.Q., Jiang, X.T., Chen, B., Chen, X., 2016. Erectile dysfunction and sexual hormone levels in men with obstructive sleep apnea: efficacy of continuous positive airway pressure. Arch. Sex. Behav. 45, 235–240. https://doi.org/ 10.1007/s10508-015-0593-2.
sleep apnoea? Exp. Physiol. 94, 279–296. https://doi.org/10.1113/expphysiol.2008. 045153. Matragrano, L.L., Sanford, S.E., Salvante, K.G., Beaulieu, M., Sockman, K.W., Maney, D.L., 2012. Estradiol-dependent modulation of serotonergic markers in auditory areas of a seasonally breeding songbird. Behav. Neurosci. 126, 110–122. https://doi.org/10. 1037/a0025586. Nelson, N.R., Bird, I.M., Behan, M., 2011. Testosterone restores respiratory long term facilitation in old male rats by an aromatase-dependent mechanism. J. Physiol. 589, 409–421. https://doi.org/10.1113/jphysiol.2010.198200. Netzer, N.C., Eliasson, A.H., Strohl, K.P., 2003. Women with sleep apnea have lower levels of sex hormones. Sleep Breath. 7, 25–29. https://doi.org/10.1007/s11325-0030025-8. Neuzeret, P.C., Sakai, K., Gormand, F., Petitjean, T., Buda, C., Sastre, J.P., Parrot, S., Guidon, G., Lin, J.S., 2009. Application of histamine or serotonin to the hypoglossal nucleus increases genioglossus muscle activity across the wake-sleep cycle. J. Sleep Res. 18, 113–121. https://doi.org/10.1111/j.1365-2869.2008.00708.x. Nikmahzar, E., Jahanshahi, M., Ghaemi, A., Naseri, G.R., Moharreri, A.R., Lotfinia, A.A., 2016. Hippocampal serotonin-2A receptor-immunoreactive neurons density increases after testosterone therapy in the gonadectomized male mice. Anat. Cell Biol. 49, 259–272. https://doi.org/10.5115/acb.2016.49.4.259. Parati, G., Lombardi, C., Narkiewicz, K., 2007. Sleep apnea: epidemiology, pathophysiology, and relation to cardiovascular risk. Am. J. Physiol. Regul. Integr. Comp. Physiol. 293, R1671–83. https://doi.org/10.1152/ajpregu.00400.2007. Peppard, P.E., Young, T., Barnet, J.H., Palta, M., Hagen, E.W., Hla, K.M., 2013. Increased prevalence of sleep-disordered breathing in adults. Am. J. Epidemiol. 177. https:// doi.org/10.1093/aje/kws342. Sanchez, M.G., Estrada-Camarena, E., Belanger, N., Morissette, M., Di Paolo, T., 2011. Estradiol modulation of cortical, striatal and raphe nucleus 5-HT1A and 5-HT2A receptors of female hemiparkinsonian monkeys after long-term ovariectomy. Neuropharmacology 60, 642–652. https://doi.org/10.1016/j.neuropharm.2010.11. 024. Sanchez, M.G., Morissette, M., Di Paolo, T., 2013a. Oestradiol modulation of serotonin reuptake transporter and serotonin metabolism in the brain of monkeys. J. Neuroendocrinol. 25, 560–569. https://doi.org/10.1111/jne.12034. Sanchez, M.G., Morissette, M., Di Paolo, T., 2013b. Estradiol and brain serotonin reuptake transporter in long-term ovariectomized parkinsonian monkeys. Prog. Neuropsychopharmacol. Biol. Psychiatry 45, 170–177. https://doi.org/10.1016/j. pnpbp.2013.05.008.
183