Chronic Intermittent Hypoxia Rats Research Articles

A study on rat cardiovascular injury induced by intermittent hypoxia and the protective role of adiponectin

To observe the effect of chronic intermittent hypoxia (CIH) on rat cardiac function and blood pressure, and the protective role of adiponectin (Ad). A total of 24 male Wistar rats were randomly and equally divided into 3 groups: normal control group (NC group), chronic intermittent hypoxygen group (CIH group) and CIH+Ad group. Normal air breathing for NC group and CHI for CIH and CIH+Ad groups were conducted for 28 days. In addition, rats in CIH+Ad group were given intravenous adiponectin at a dosage of 20 µg each time, once a week for successive 4 weeks. The results of echocardiography, blood pressure, plasma adiponectin, endothelin-1 (ET-1) and endothelial nitric oxide synthase (eNOS) were compared among the three groups after 28 days. HE stain showed that the myocardial cells of CIH rats were damaged by CIH. Compared with NC group, rats in CIH group presented with a greater heart/body weight ratio (0.070 ± 0.008 vs. 0.057 ± 0.009, P < 0.05) and systolic blood pressure [(132 ± 4) vs. (123 ± 6) mmHg(1 mmHg = 0.133 kPa), P < 0.05] and a lower LVEF [(70.3 ± 4.1)% vs. (84.1 ± 2.5)%, P < 0.05]. Plasma ET-1 was increased while NO(-)(2)/NO(-)(3) and eNOS decreased in CIH group, compared with NC group [(26.2 ± 6.9) ng/L vs. (7.7 ± 2.7) ng/L, (37 ± 9) µmol/L vs. (65 ± 10) µmol/L, (18 ± 5)µg/L vs. (27 ± 6) µg/L, respectively; P < 0.05]. The heart/body weight ratio, blood pressure and LVEF were improved in CIH+Ad group compared with those in CIH group [0.064 ± 0.009 vs. 0.070 ± 0.008, (127 ± 6) mmHg vs. (132 ± 4) mmHg, P > 0.05; (79 ± 7)% vs. (70 ± 4)%, P < 0.05; respectively]. Plasma ET-1 levels in CIH+Ad and CIH groups showed no significant difference, but were significantly lower in NC group. However, rats in CIH+Ad group had a higher NO(-)(2)/NO(-)(3) level than that in CIH group. Bivariate Correlations showed that NO(-)(2)/NO(-)(3) and eNOS were negatively correlated with systolic blood pressure while heart/body weight ratio, LVEDs, ET-1 and NO(-)(2)/NO(-)(3) were negatively correlated with left ventricular ejection fraction. Through xidative stress, ET-1 and NO imbalance and impaired vascular endothelial function, cardiac function could be damaged by CIH in rats, while supplement of extrinsic adiponectin could improve these damages.

Cardioprotective effects of melatonin against myocardial injuries induced by chronic intermittent hypoxia in rats.

Obstructive sleep apnea (OSA) associated with chronic intermittent hypoxia (CIH) increases the morbidity and mortality of ischemic heart disease in patients. Yet, there is a paucity of preventive measures targeting the pathogenesis of CIH-induced myocardial injury. We examined the cardioprotective effect of melatonin against the inflammation, fibrosis and the deteriorated sarcoplasmic reticulum (SR) Ca(2+) homeostasis, and ischemia/reperfusion (I/R)-induced injury exacerbated by CIH. Adult male Sprague Dawley rats that had received a daily injection of melatonin (10 mg/kg) or vehicle were exposed to CIH treatment mimicking a severe OSA condition for 4 wk. Systolic pressure, heart weights, and malondialdehyde were significantly increased in hypoxic rats but not in the melatonin-treated group, when compared with the normoxic control. Levels of the expression of inflammatory cytokines (TNF-α, IL-6, and COX-2) and fibrotic markers (PC1 and TGF-β) were significantly elevated in the hypoxic group but were normalized by melatonin. Additionally, infarct size of isolated hearts with regional I/R was substantial in the hypoxic group treated with vehicle but not in the melatonin-treated group. Moreover, melatonin treatment mitigated the SR-Ca(2+) homeostasis in the cardiomyocyte during I/R with (i) Ca(2+) overloading, (ii) decreased SR-Ca(2+) content, (iii) lowered expression and activity of Ca(2+) -handling proteins (SERCA2a and NCX1),and (iv) decreased expressions of CAMKII and phosphorylated eNOS(ser1177). Furthermore, melatonin ameliorated the level of expression of antioxidant enzymes (CAT and MnSOD) and NADPH oxidase (p22 and NOX2). Results support a prophylactic usage of melatonin in OSA patients, which protects against CIH-induced myocardial inflammation and fibrosis with impaired SR-Ca(2+) handling and exacerbated I/R injury.

Telmisartan attenuates myocardial apoptosis induced by chronic intermittent hypoxia in rats: modulation of nitric oxide metabolism and inflammatory mediators.

NO and NO synthase (NOS) are known to play key roles in the development of myocardial apoptosis induced by ischemia/hypoxia. Current evidence suggests that angiotensin II type 1 receptor blockers, such as telmisartan, lower blood pressure and produce beneficial regulatory effects on NO and NOS. Here, we examined the protective role of telmisartan in myocardial apoptosis induced by chronic intermittent hypoxia (CIH). Adult male Sprague-Dawley rats were subjected to 8h of intermittent hypoxia/day, with/without telmisartan for 8weeks. Myocardial apoptosis, NO and NOS activity, and levels of inflammatory mediators and radical oxygen species were determined. Treatment with telmisartan preserved endothelial NOS expression and inhibited inducible NOS and excessive NO generation, while reducing oxidation/nitration stress and inflammatory responses. Administration of telmisartan before CIH significantly ameliorated the CIH-induced myocardial apoptosis. This study show that pre-CIH telmisartan administration ameliorated myocardial injury following CIH by attenuating CIH-induced myocardial apoptosis via regulation of NOS activity and inhibition of excessive NO generation, oxidation/nitration stress, and inflammatory responses.

Cardiovascular responses to chronic intermittent hypoxia in rats are not affected by previous exposure to hypoxia (856.9)

Rats exposed to chronic intermittent hypoxia (CIH) develop hypertension. In the present study we evaluated the effect of a previous protocol of CIH on the cardiovascular responses produced by a new protocol of CIH. Male Wistar rats (45 ‐ 50 g) were divided in 3 groups: Control group (n=24), maintained under normoxia during 35 days; CIH group (n=18), maintained under normoxia during 25 days, followed by 10 days of CIH (6% O2 for 40 s every 9 min, 8 h/day); Pre‐CIH group (n=19), exposed to CIH for 10 days, followed by 15 days under normoxia and then a second exposure to CIH for additional 10 days. At the end of the protocol, rats were instrumented for arterial pressure recording. CIH and Pre‐CIH presented a significant increase in mean arterial pressure (108 ± 2 and 107 ± 2 mmHg, respectively) when compared to the control group (98 ± 1 mmHg), but the increase in both groups exposed to CIH was similar. We conclude that previous exposure to CIH produced no effects on the cardiovascular response to a new protocol of CIH.Grant Funding Source: FAPESP, CAPES, CNPq

Electrophysiological properties of RVLM pre‐sympathetic neurons modulated by the respiratory network in rats (875.11)

The respiratory pattern generator modulates the sympathetic outflow which strength is enhanced by hypoxia. This coupling is due to the respiratory‐modulated pre‐sympathetic neurons in the RVLM, but the underlining electrophysiological mechanisms remain unclear. For a better understanding of the central generation of respiratory‐sympathetic coupling, we combined immunofluorescence, single cell qRT‐PCR and whole cell patch clamp recordings of the pre‐sympathetic neurons in in situ preparations from normal and rats submitted to chronic intermittent hypoxia (CIH). Our results show that the spinally projected C1‐ and non‐C1 respiratory‐modulated pre‐sympathetic neurons (inspiratory‐modulated: 22; post‐I‐modulated: 23; inspiratory‐inhibited: 11; non‐modulated: 20) present distinct electrophysiological properties, synaptic inputs and expression of ionic currents, albeit all neurons presented iNaP‐dependent intrinsic pacemaker after synaptic blockade. Only the non‐C1 post‐I‐modulated neurons presented enhanced excitatory synaptic inputs from the respiratory network after CIH (n=14), which may contribute to the increased sympathetic activity observed in CIH rats. We conclude that the different respiratory‐modulated pre‐sympathetic neurons contribute to the central generation of respiratory‐sympathetic coupling and in response to the challenges produced by CIH may contribute to respiratory‐related increase in the sympathetic activity and hypertension observed in this experimental model.Grant Funding Source: Supported by CAPES and FAPESP

Evaluation of the cardiovascular changes in juvenile and adult female rats submitted to chronic intermittent hypoxia (680.3)

Chronic intermittent hypoxia (CIH) induces hypertension in juvenile and adult male rats due to an increase in sympathetic activity. There is experimental evidence that changes in respiratory network are driving the sympathetic overactivity in this model. Female rats are more resistant to the development of hypertension in several experimental models. In this study, we evaluated the cardiovascular changes in juvenile and adult female rats in response to CIH. Juvenile female rats were exposed to CIH for a 10 days protocol and adult female rats to a 35 days protocol and at the end of the protocols direct measurements of arterial pressure were performed in control and CIH rats. Juvenile female rats exposed to CIH (n=30) presented significant increase in arterial pressure compared to their respective control [n=34 (94±1 vs 86±1 mmHg, p&lt;0.05)], as well adult female rats exposed to CIH (n=27) when compared to their respective control [n=26 (111±1 vs 104±1 mmHg, p&lt;0.05)]. We concluded that juvenile and adult female rats submitted to CIH develop high arterial pressure similarly to male rats after CIH, suggesting that sexual characteristics and hormones are not playing a protective role in this experimental model of hypertension.Grant Funding Source: Supported by CAPES, CNPQ and FAPESP

Angiotensin II receptor type‐1a knockdown in subfornical organ prevents sustained increase in mean arterial pressure associated with chronic intermittent hypoxia (686.20)

Sleep apnea (SA) is associated with a sustained increase in mean arterial pressure (MAP) even during waking hours. Chronic intermittent hypoxia (CIH) models the hypoxemia associated with SA and produces elevated MAP during CIH and normoxia. Angiotensin II (Ang II) is implicated in the CIH associated increase in MAP. Subfornical organ (SFO), a forebrain circumventricular organ, lacks blood brain barrier and is a major site for the central effects of circulating Ang II. We investigated the effects of Ang II type 1a receptor knockdown (AT1aRKD) in the SFO on CIH hypertension in adult male rats. Adeno‐associated viral vectors carrying GFP and either AT1aR shRNA or scrambled shRNA (SCM) were injected in SFO. Using laser‐capture microdissection and qRT‐PCR of amino‐allyl RNA, AT1aRKD rats showed decreased SFO AT1aRmRNA in comparison with SCM rats (p&lt;0.05). During light phase, the AT1aRKD rats exposed to CIH(3 min 10% O2 and 3 min room air cycles for 8 h during light phase for 7 d) exhibited significant increases in MAP vs. AT1aRKD‐Normoxia group (p&lt;0.05). During the normoxic dark phase, there was no difference in MAP between CIH and normoxic AT1aRKD rats (p=0.69). SCM‐CIH group showed significant increase in MAP from SCM‐Normoxia group during light phase CIH (p&lt;0.001) and the normoxic dark phase (p&lt;0.001). Our data indicate that AT1aRs in SFO may play a role in the sustained increases in MAP during normoxia associated with CIH.Grant Funding Source: Supported by: P01HL088052‐05

Atorvastatin attenuates myocardial remodeling induced by chronic intermittent hypoxia in rats: Partly involvement of TLR-4/MYD88 pathway

Inflammatory processes and oxidative stress are known to play a key role in the development of cardiovascular complications such as cardiac hypertrophy induced by chronic intermittent hypoxia (CIH), the most characteristic pathophysiological change of obstructive sleep apnea syndrome (OSAS). Current evidence suggests that competitive inhibitors of 3-hydroxy-3-methylglutaryl-CoA coenzyme A reductase, such as atorvastatin, not only reduce blood lipids but also have anti-inflammatory and inhibit oxidative stress benefits. This study examined the protective role of atorvastatin in CIH-induced cardiac hypertrophy. Adult male wistar rats were subjected to 8h of intermittent hypoxia/day, with/without atorvastatin for 6weeks. Ventricular remodeling, toll-like receptor 4 (TLR-4), myeloid differentiation primary response protein 88 (MYD88), inflammatory agents and radical oxygen species were determined. As a result, we found that treatment with atorvastatin markedly inhibited the mRNA and protein expressions of TLR4, MYD88 and the downstream inflammatory agents and radical oxygen species. Administration of atorvastatin following CIH significantly ameliorated the myocardial injury, such as cardiac hypertrophy. In conclusion, Pre-CIH atorvastatin administration may attenuate TLR-4/MYD88 mediated inflammatory processes and oxidative stress in the injured rat myocardium, and this may be one mechanism by which atorvastatin ameliorated myocardial injury following CIH.

Electrophysiological Properties of Rostral Ventrolateral Medulla Presympathetic Neurons Modulated by the Respiratory Network in Rats

The respiratory pattern generator modulates the sympathetic outflow, the strength of which is enhanced by challenges produced by hypoxia. This coupling is due to the respiratory-modulated presympathetic neurons in the rostral ventrolateral medulla (RVLM), but the underlining electrophysiological mechanisms remain unclear. For a better understanding of the neural substrates responsible for generation of this respiratory-sympathetic coupling, we combined immunofluorescence, single cell qRT-pCR, and electrophysiological recordings of the RVLM presympathetic neurons in in situ preparations from normal rats and rats submitted to a metabolic challenge produced by chronic intermittent hypoxia (CIH). Our results show that the spinally projected cathecholaminergic C1 and non-C1 respiratory-modulated RVLM presympathetic neurons constitute a heterogeneous neuronal population regarding the intrinsic electrophysiological properties, respiratory synaptic inputs, and expression of ionic currents, albeit all neurons presented persistent sodium current-dependent intrinsic pacemaker properties after synaptic blockade. A specific subpopulation of non-C1 respiratory-modulated RVLM presympathetic neurons presented enhanced excitatory synaptic inputs from the respiratory network after CIH. This phenomenon may contribute to the increased sympathetic activity observed in CIH rats. We conclude that the different respiratory-modulated RVLM presympathetic neurons contribute to the central generation of respiratory-sympathetic coupling as part of a complex neuronal network, which in response to the challenges produced by CIH contribute to respiratory-related increase in the sympathetic activity.

Medullary respiratory network drives sympathetic overactivity and hypertension in rats submitted to chronic intermittent hypoxia

We hypothesized that hypertension following chronic intermittent hypoxia (CIH) exposure is, at least in part, dependent on alterations in the respiratory network and its interaction with the sympathetic nervous system. With simultaneous recordings of respiratory and sympathetic nerves and whole cell path-clamp of respiratory and pre-sympathetic neurons of ventrolateral medulla, we evaluated the mechanisms underlining CIH-induced hypertension in juvenile rats. In CIH rats, the decrease in the frequency discharge of post-inspiratory (post-I) neurons reduced the post-I activity of cervical vagus nerve.

ERK signaling mediates enhanced angiotensin II-induced rat aortic constriction following chronic intermittent hypoxia

Background Obstructive sleep apnea (OSA) has been recognized as an independent risk factor for systemic hypertension. The study investigated the functional consequences of chronic intermittent hypoxia (CIH) on aortic constriction induced by angiotensin II (Ang II) and the possible signaling involving ERK1/2 and contractile proteins such as myosin light chain kinase (MLCK), myosin phosphatase targeting subunit (MYPT1) and myosin light chain (MLC). Methods Male Wistar rats were randomly divided into CIH group and normoxia group and exposed to either CIH procedure or air-air cycles. Phosphorylation of ERK1/2, MYPT1 and MLC was assessed by Western blotting following constrictor studies in the presence or absence of PD98059 (10 μmol/L). Results CIH-exposure resulted in more body weight gain and elevated blood pressure, which could be attenuated by pretreatment with PD98059. Endothelium-removed aortic rings from CIH rats exhibited higher constrictor sensitivity to Ang II (Emax: (138.56±5.78)% versus (98.45±5.31)% of KCl; pD2: 7.98±0.14 versus 8.14±0.05, respectively). CIH procedure exerted complex effects on ERK expressions (total ERK1/2 decreased whereas the ratio of phosphorylated to total ERK1/2 increased). CIH aortas had higher MLCK mRNA and basal phosphorylation of MYPT1 and MLC. In parallel to greater increases in phosphorylation of ERK1/2, MYPT1 and MLC, Ang II-induced aortic constriction was significantly enhanced in CIH rats, which was largely reversed by PD98059. However vascular constriction of normoxia rats remained unchanged despite similar but smaller changing tendency of proteins phosphorylation. Conclusion These data suggest that CIH exposure results in aortic hyperresponsiveness to Ang II, presumably owing to more activated ERK1/2 signaling pathway.

Endarterium injury and the related pathway in chronic intermittent hypoxia rats

To examine the pathological change and intima thickness of thoracic aorta, detect the serum concentration of hypoxia-inducible factor-1α (HIF-1α), oxidized LDL (ox-LDL), and pentraxin 3 (PTX3) in the rat model of chronic intermittent hypoxia (CIH), and to determine the effect of CIH on endarterium injury and its possible pathway. Twenty-four male Sprague-Dawley (SD) rats were divided into 4 groups: a CIH+Nacetylcysteine (NAC) group, a CIH+normal saline (NS) group, a CIH control group and a control group. CIH rats were subjected to alternating cycles of hypoxia (6%-8% O2 in N2 for 20-25 s) and normoxia (21% O2 in N2 for 2 min) every 180 s for 7 h/d. Rats in the control group were not treated. Rats in the CIH+NAC group were treated with NAC [800 mL/(kg.d)] intraperitoneal injection, and rats in the CIH+NS group were treated with NS [5 mL/(kg.d)] intraperitoneal injection. After 42 day treatment, the rats were sacrificed, blood taken, and thoracic aorta cut off. The serum concentration of HIF-1α, ox-LDL, and PTX3 were detected by ELISA. The thickness of intima was taken by computer digital image analysis. Vascular endothelial cell injury and detachment were found in the thoracic aorta in the CIH and the CIH+NS group. The intima in the CIH and the CIH+NS group was thicker than that in the control and the CIH+NAC group (P<0.001). The serum concentration of HIF-1α, ox-LDL, and PTX3 in the CIH and the CIH+NS group was higher than that in the control and the CIH+NAC group (P<0.001). The serum concentration of HIF-1α, ox-LDL, and PTX3 was pairwise positive correlation, and the serum concentration of ox-LDL and PTX3 was positively correlated with the thickness of intina (P<0.001). The vascular endothelial cell injury and endarterium thickening can be induced by CIH. It is an important pathway that CIH activates oxidative stress and elevates the levels of HIF- 1α, ox-LDL, and PTX3.

Impaired hypoxic ventilatory response following neonatal sustained and subsequent chronic intermittent hypoxia in rats

Neonatal chronic intermittent hypoxia (CIH) enhances the ventilatory sensitivity to acute hypoxia (acute hypoxic ventilatory response, HVR), whereas sustained hypoxia (SH) can have the opposite effect. Therefore, we investigated whether neonatal rats pre-treated with SH prior to CIH exhibit a modified HVR. Rat pups were exposed to CIH (5% O2/5min, 8h/day) between 6 and 15 days of postnatal age (P6-15) after pre-treatment with either normoxia or SH (11% O2; P1-5). Using whole-body plethysmography, the acute (5min, 10% O2) HVR at P16 (1 day post-CIH) was unchanged following CIH (67.9±6.7% above baseline) and also SH (58.8±10.5%) compared to age-matched normoxic rats (54.7±6.3%). In contrast, the HVR was attenuated (16.5±6.0%) in CIH exposed rats pre-treated with SH. These data suggest that while neonatal SH and CIH alone have little effect on the magnitude of the acute HVR, their combined effects impose a synergistic disturbance to postnatal development of the HVR. These data could provide important insight into the consequences of not maintaining adequate levels of oxygen saturation during the early neonatal period, especially in vulnerable preterm infants susceptible to frequent bouts of hypoxemic events (CIH) that are commonly associated with apnea of prematurity.

Lycium Barbarum Polysaccharides Mitigate Chronic Intermittent Hypoxia Induced Hippocampal Oxidative Stress, Inflammation And Apoptosis In Rats

Chronic intermittent hypoxia (CIH) induces oxidative stress, inflammation and apoptosis in hippocampus. Lycium barbarum polysaccharides (LBP), bioactive ingredients of traditional Chinese herbal medicine Goji, were proposed to possess anti‐oxidative and anti‐inflammatory properties. Our study aims to investigate the neuroprotective effect of LBP in CIH rats. We hypothesize that LBP ameliorate oxidative stress, inflammation and apoptosis in the hippocampus of rats exposed to CIH. Malondialdehyde (MDA) assay was used to measure the lipid peroxidation extent. Western blot was employed to examine the expressions level of antioxidant enzymes (SOD‐1, SOD‐2); inflammatory mediators (IL‐1β, TNFα, COX‐2); caspase‐dependent extrinsic (FADD, cleaved caspase 8, Bid) and intrinsic apoptotic (Bax, Bcl2, cytochrome C) cell death (cleaved caspase 3) cascades. In situ cell death staining (TUNEL) was utilized to reveal the apoptotic status of hippocampal subfields (DG, CA1 and CA3). LBP administrations remarkably attenuated oxidative stress, inflammation and apoptosis in hypoxic group. In addition, LBP significantly mitigated caspase‐dependent apoptotic extrinsic and intrinsic cascades in hypoxic group. To conclude, LBP are neuroprotective against CIH‐induced hippocampal injuries. Studies supported by RGC research grants (HKU 7510/06M, HKU 766110M) and HKU funding (201007176007, SFPBR 200911159072).

Angiotensin and AMPA receptor subunit mRNA levels are reduced in NTS catecholaminergic neurons following chronic intermittent hypoxia (CIH)

Catecholaminergic A2 neurons in caudal NTS are activated during stressors such as hypoxia. The aim of this study was to assess the effect of CIH on the mRNA expression levels of AT1a, AT1b and AMPA and NMDA receptor subunits in the A2 neurons. AAV vector mediated delivery of green fluorescent protein (GFP) labeled tyrosine hydroxylase promoter (AAV‐GFP‐TH) was used to label A2 neurons. 7 virus injected rats were exposed to 7 days CIH (alternating 6 min periods of 10% O2 and 4 min of 21% O2 from 8am to 4pm; from 4pm to 8am rats were exposed to 21% O2). Laser capture microdissection was performed to capture A2 neurons from caudal NTS. Total RNA from these neurons was extracted and mRNA levels assessed by quantitative real time reverse transcription PCR and compared between the control and CIH rats using 2‐ÄÄct method. CIH decreased AT1a (p=0.002; control − 1.08 ± 0.13, n=7; CIH − 0.48 ± 0.07, n= 6) and AMPA receptor subunit GluR2 (p=0.03; control − 1.11 ± 0.24, n=7; CIH‐ 0.52 ± 0.12, n= 6) and increased transcription factor FosB (p=0.03; control − 1.14 ± 0.25, n=7; CIH‐ 1.97 ± 0.25, n= 5) mRNA levels in the A2 neurons. CIH did not alter levels of GluR1, NR1, NR2 or delta FosB. These results indicate that exposure to CIH selectively alters receptor subunit mRNAs in A2 neurons which could influence their responses to ligand. Supported by HL88052.

Changes in ionic currents of respiratory neurons produce sympathetic overactivity in chronic intermittent hypoxic rats

The coupling between respiratory and sympathetic neuronal activities is enhanced in rats submitted to chronic intermittent hypoxia (CIH) and contributes to the development of hypertension in this experimental model. We evaluated the mechanisms involved in the CIH‐induced changes in electrophysiological properties of medullary respiratory and pre‐sympathetic neurons. One day after the protocol of 10 days of CIH, using an in situ preparation we performed simultaneous recordings of respiratory and sympathetic nerves with blind whole cell path‐clamp of respiratory and pre‐sympathetic neurons of ventrolateral medulla. In CIH rats, the increased firing frequency of pre‐sympathetic neurons (n=27) and thoracic sympathetic activity (n=30), time‐locked with abdominal late‐expiratory activity (n=25), were dependent of synaptic inputs from respiratory neurons. Since medullary post‐ and pre‐inspiratory (pre‐I) neurons modulate the firing frequency of pre‐sympathetic neurons, we observed that the decreased and increased of intrinsic firing frequency of post‐I (n=28) and pre‐I neurons (n=28), respectively, were accompanied by changes in their input resistance and rheobase after CIH. These changes were due to increase in the inward riluzole‐sensitive persistent sodium current (iNaP; n=6) in pre‐I neurons and in the 4‐aminopyridine‐sensitive component of transient outward potassium current (TOC; n=9) in the post‐I neurons after CIH. These data describe the mechanisms underlying sympathetic overactivity, which critically depend on changes of ionic currents of medullary respiratory neurons of CIH rats.Support:FAPESP, CAPES and CNPQ.

Chronic intermittent hypoxia aggravates intrahepatic endothelial dysfunction in cirrhotic rats

Chronic intermittent hypoxia (CIH) occurs with obstructive sleep apnea syndrome (OSAS) and provokes systemic endothelial dysfunction, which is associated with oxidative stress and low nitric oxide (NO) bioavailability. Cirrhotic livers exhibit intrahepatic endothelial dysfunction, which is characterized by an impaired endothelium-dependent response to vasodilators and hyperresponse to vasoconstrictors. We hypothesized that CIH may also contribute to intrahepatic endothelial dysfunction in cirrhosis. Normal and cirrhotic rats were exposed for 14 days to repetitive cycles of CIH mimicking OSAS in humans, or caged with room air (handled controls [HC]). Hepatic endothelial function was assessed in isolated and perfused rat livers by dose-response curves to acetylcholine (ACh) and methoxamine (Mtx). In a group of cirrhotic rats, in vivo systemic and hepatic hemodynamic parameters were evaluated at baseline and after volume expansion. In addition, liver samples were obtained to assess endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (p-eNOS), NO bioavailability, and nitrotyrosinated proteins as a marker of oxidative stress. Cirrhotic rats exposed to CIH exhibited an attenuated vasodilatory response to ACh and hyperresponse to Mtx compared with HC rats. During volume expansion, similar portal pressure increases were observed in CIH and HC rats, although the mean arterial pressure increase was lower after CIH. These functional responses were associated with the presence of increased hepatic oxidative stress without changes in p-eNOS after CIH exposure. In normal rats, no hemodynamic changes were found. CIH exacerbates intrahepatic endothelial dysfunction in cirrhotic rats, which is associated with increased oxidative stress that may reduce NO bioavailability. Clinical studies are needed to assess whether OSAS contributes to endothelial impairment in human patients with cirrhosis.

Effects of genistein and estrogen on the genioglossus in rats exposed to chronic intermittent hypoxia may be HIF‐1α dependent

Chronic intermittent hypoxia (CIH) is a frequent feature of OSAHS. The present study was designed to evaluate the effects of genistein and estrogen on genioglossus contractile and regeneration properties in CIH rats and investigate the involvement of HIF-1α. Ovariectomized female rats were exposed to CIH for 5 weeks. Genistein and estrogen were administered by intraperitoneal injection. The genioglossus myoblasts of rat were also isolated and cultured in vitro, and the HIF-1α shRNA lentivirus was used. Muscle fatigue resistance and myogenic regeneration were significantly decreased after CIH but were partially reversed by estrogen and genistein treatment. The effect of estrogen was more powerful than that of genistein. Compared with control group, RT-PCR and western blotting showed higher levels of HIF-1α mRNA and protein in the CIH group, but estrogen and genistein treatment reduced the levels of HIF-1α mRNA and protein in rats exposed to CIH. In genioglossus myoblasts, the expression of HIF-1α was up-regulated under hypoxia rather than normoxia and decreased over time under both hypoxia and normoxia during myogenic differentiation. HIF-1α knockdown relieved myogenesis inhibition under hypoxia. We concluded that genistein and estrogen may inhibit the overexpression of HIF-1α induced by CIH and improve the endurance and regeneration of the genioglossus muscle.

Obstructive Sleep Apnoea, Intermittent Hypoxia and Respiratory Muscle Structure and Function

Obstructive sleep apnoea is a common condition associated with significant morbidity and increased mortality. During sleep, the sub atmospheric pressure developed in the extra thoracic airway by the contraction of the thoracic pump muscles during inspiration causes collapse of the compliant upper airway, resulting in an episode of apnoea. Normally, upper airway collapse is prevented by contraction of upper airway muscles and if the airway does collapse, it is re-opened by a reflex augmented neural drive and greater contraction of these muscles. Therefore, the function of these muscles plays a critical role in the pathogenesis of the obstructive sleep apnoea. The multiple episodes of apnoea that occur during the course of sleep cause intermittent hypoxia. We have proposed that chronic intermittent hypoxia causes adverse changes in structure and function of upper airway muscle that result in an exacerbation of the condition. We have shown that chronic intermittent hypoxia in rats causes greater fatigability of upper airway, diaphragm and limb muscles. In general, this appears to be true also for chronic continuous hypoxia, although the diaphragm muscle is relatively tolerant of sustained hypoxia compared to limb muscle. We propose that the chronic intermittent hypoxia of obstructive sleep apnoea causes a positive feedback that exacerbates the condition by reducing upper airway muscle endurance, thus making the upper airway more vulnerable to collapse. This effect involves the generation of reactive oxygen species and treatment with antioxidants either prevents or ameliorates the adverse effects on muscle function, suggesting that antioxidants could be used as an adjunct therapy for obstructive sleep apnoea. This may be true also for respiratory disease in general since chronic continuous hypoxia is also associated with increased muscle fatigue.

Chronic Intermittent Hypoxia Depresses Afferent Neurotransmission in NTS Neurons by a Reduction in the Number of Active Synapses

Long-term synaptic plasticity has been recently described in brainstem areas associated to visceral afferent sensory integration. Chronic intermittent hypoxia (CIH), an animal model for studying obstructive sleep apnea in humans, depresses the afferent neurotransmission in nucleus tractus solitarii (NTS) neurons, which affect respiratory and autonomic regulation. Here we identified the synaptic mechanisms of CIH-induced depression of the afferent neurotransmission in NTS neurons in juvenile rats. We verified that CIH reduced the amplitude of both NMDA and non-NMDA glutamatergic excitatory currents (eEPSCs) evoked by tractus solitarii stimulation (TS-eEPSC) of second-order neurons in the NTS. No changes were observed in release probability, evidenced by absence of any CIH-elicited effects on short-term depression and failures in EPSCs evoked in low calcium. CIH also produced no changes in TS-eEPSC quantal size, since the amplitudes of both low calcium-evoked EPSCs and asynchronous TS-eEPSCs (evoked in the presence of Sr(2+)) were unchanged. Using single TS afferent fiber stimulation in slices from control and CIH rats we clearly show that CIH reduced the quantal content of the TS-eEPSCs without affecting the quantal size or release probability, suggesting a reduction in the number of active synapses as the mechanism of CIH induced TS-eEPSC depression. In accordance with this concept, the input-output relationship of stimulus intensity and TS-eEPSC amplitude shows an early saturation in CIH animals. These findings open new perspectives for a better understanding of the mechanisms underlying the synaptic plasticity in the brainstem sensory neurons under challenges such as those produced by CIH in experimental and pathological conditions.