Stimulatory effect of methylglyoxal on capsaicin-sensitive lung vagal afferents in rats: role of TRPA1.

Methylglyoxal (MG), a reactive metabolic byproduct of glycolysis, is a causative of painful diabetic neuropathy. Patients with diabetes are associated with more frequent severe asthma exacerbation. Stimulation of capsaicin-sensitive lung vagal (CSLV) afferents may contribute to the pathogenesis of hyperreactive airway diseases such as asthma. However, the possibility of the stimulatory effect of MG on CSLV afferents and the underlying mechanisms remain unknown. Our results showed that intravenous injection of MG (25 mg/kg, MG25) in anesthetized, spontaneously breathing rats elicited pulmonary chemoreflexes characterized by apnea, bradycardia, and hypotension. The MG-induced apneic response was reproducible and dose dependent. MG25 no longer evoked these reflex responses after perineural capsaicin treatment of both cervical vagi to block C-fibers' conduction, suggesting that the reflexes were mediated through the stimulation of CSLV afferents. Pretreatment with HC030031 [an antagonist of transient receptor potential ankyrin subtype 1 protein (TRPA1)] or AP18 (another TRPA1 antagonist), but not their vehicle, markedly attenuated the apneic response induced by MG25. Consistently, electrophysiological results showed that pretreatment with HC030031 largely attenuated the intense discharge in CSLV afferents induced by injection of MG25 in open-chest and artificially ventilated rats. In isolated CSLV neurons, the perfusion of MG evoked an abrupt and pronounced increase in calcium transients in a concentration-dependent manner. This stimulatory effect on CSLV neurons was also abolished by HC030031 treatment but not by its vehicle. In conclusion, these results suggest that MG exerts a stimulatory effect on CSLV afferents, inducing pulmonary chemoreflexes, and such stimulation is mediated through the TRPA1 activation.NEW & NOTEWORTHY Methylglyoxal (MG) is implicated in the development of painful diabetic neuropathy. A retrospective cohort study revealed an increased incidence of asthma exacerbations in patients with diabetes. This study demonstrated that elevated circulating MG levels stimulate capsaicin-sensitive lung vagal afferents via activation of TRPA1, which in turn triggers respiratory reflexes. These findings provide new information for understanding the pathogenic mechanism of diabetes-associated hyperreactive airway diseases and potential therapy.

The arginine methyltransferase PRMT5 promotes mucosal defense in the intestine.

PRMT5 is a type II arginine methyltransferase abundantly expressed in the colonic epithelium. It is up-regulated in inflammatory bowel disease and colorectal cancer. However, its role in mucosal defense against enteric infection has not been studied. Here, we report that Prmt5 in the murine colon is up-regulated in response to Citrobacter rodentium infection. Pathogen clearance in mice with haploinsufficient expression of Prmt5 is significantly delayed compared with wildtype littermate controls. Transcriptomic analyses further reveal that PRMT5 regulates the expression of canonical crypt goblet cell genes involved in mucus production, assembly, and anti-microbial responses via methyltransferase activity-dependent and -independent mechanisms. Together, these findings uncover PRMT5 as a novel regulator of mucosal defense and a potential therapeutic target for treating intestinal diseases.

Effect of prolonged mechanical ventilation on cough function and TRPV1 expression.

Cough is a pivotal airway protective reflex, yet the effects of prolonged mechanical ventilation (PMV) on cough function are unknown. This study compared the cough function in subjects with PMV (≥ 21 days, n = 29) and those with short-term mechanical ventilation (SMV, ≤ 7 days, n = 27). Cough reflex sensitivity was measured by capsaicin provocation concentrations after extubation. The cough strength of respiratory muscles was assessed by involuntary cough peak expiratory flow (iCPEF). The mRNA expression of transient receptor potential vanilloid 1 (TRPV1), a cough sensor activated by capsaicin, in tracheal tissues was determined. We found that cough reflex sensitivity and iCPEF were significantly lower in the PMV group than in the SMV group. The tracheal expression of TRPV1 was similar in both groups, suggesting that changes in TRPV1 expression may not be a contributing factor. Our finding regarding the cough dysfunction after PMV highlights the need to implement effective airway clearance management and rehabilitation in this population.

Airway hyperresponsiveness induced by intermittent hypoxia in rats.

This study investigated whether intermittent hypoxia (IH) induces airway hyperresponsiveness (AHR) and associated with lung inflammation. Male Brown Norway rats were exposed to 14-day IH or room air (RA) for 6 h/day. One day after the last exposure, total lung resistance to various doses of methacholine was measured as an index of bronchoconstrictive responses. Compared with RA controls, methacholine significantly induced an augmented bronchoconstriction in IH-exposed rats. Moreover, IH exposure evoked lung inflammation which was reflected by increased inflammatory cell infiltration, concentrations of interleukin-6 and prostaglandin E2 in bronchoalveolar lavage fluid, and lung lipid peroxidation. IH-induced AHR and lung inflammation were completely abolished by daily intraperitoneal injection of N-acetylcysteine (an antioxidant) or ibuprofen (a cyclooxygenase inhibitor), but not by apocynin (an inhibitor of NADPH oxidase) or vehicle. In conclusion, AHR and lung inflammation occur after 14-day IH exposure, with endogenous reactive oxygen species and cyclooxygenase metabolites being responsible for these responses.

HIF-1α-Mediated, NADPH Oxidase-Derived ROS Contributes to Laryngeal Airway Hyperreactivity Induced by Intermittent Hypoxia in Rats.

Obstructive sleep apnea, similar to intermittent hypoxia (IH) during sleep, is associated with laryngeal airway hyperreactivity (LAH). IH-induced laryngeal oxidative stress may contribute to LAH, but the underlying mechanism remains unknown. Conscious rats were subjected to repetitive 75 s cycles of IH for 7 or 14 consecutive days. Reflex apneic responses to laryngeal provocations with chemical stimulants were measured to reflect laryngeal reflex reactivity. Compared with control rats, rats exposed to IH for 14 days, but not for 7 days, displayed enhanced apneic response to laryngeal chemical stimulants. The apneic response to chemical stimulants, but not to mechanical stimulation, was totally abolished by perineural capsaicin treatment of superior laryngeal nerves (SLNs) or by the sectioning of the SLNs, suggesting that the reflex was mediated through capsaicin-sensitive SLNs. Daily intraperitoneal administration of N-acetyl-L-cysteine [NAC, a reactive oxygen species (ROS) scavenger], apocynin (an inhibitor of NADPH oxidase) or YC-1 (an inhibitor of HIF-1α), but not their vehicles, largely attenuated this augmented apneic response in 14 days IH rats. Laryngeal lipid peroxidation (an index of oxidative stress) was elevated in 7 days IH rats and 14 days IH rats, and was abolished by any of these three pharmacologic interventions. The protein expression of HIF-1α (an index of HIF-1 activation) and p47phox subunit in the membrane fraction (an index of NADPH oxidase activation) in the laryngeal tissues increased in 14 days IH rats; the former was reduced by NAC, whereas the latter was inhibited by YC-1. These results suggest that 14 days of IH exposure may sensitize capsaicin-sensitive SLNs and result in exaggerated apneic reflex response to laryngeal chemical stimulants. This phenomenon depends on the action of HIF-1α-mediated, NADPH oxidase-derived ROS.

Estrogen Modulates the Sensitivity of Lung Vagal C Fibers in Female Rats Exposed to Intermittent Hypoxia.

Obstructive sleep apnea is mainly characterized by intermittent hypoxia (IH), which is associated with hyperreactive airway diseases and lung inflammation. Sensitization of lung vagal C fibers (LVCFs) induced by inflammatory mediators may play a central role in the pathogenesis of airway hypersensitivity. In females, estrogen interferes with inflammatory signaling pathways that may modulate airway hyperreactivity. In this study, we investigated the effects of IH on the reflex and afferent responses of LVCFs to chemical stimulants and lung inflammation in adult female rats, as well as the role of estrogen in these responses. Intact and ovariectomized (OVX) female rats were exposed to room air (RA) or IH for 14 consecutive days. On day 15, IH enhanced apneic responses to right atrial injection of chemical stimulants of LVCFs (e.g., capsaicin, phenylbiguanide, and α,ß-methylene-ATP) in intact anesthetized females. Rats subjected to OVX prior to IH exposure exhibited an augmented apneic response to the same dose of stimulants compared with rats subjected to other treatments. Apneic responses to the stimulants were completely abrogated by bilateral vagotomy or perivagal capsaicin treatment, which blocked the neural conduction of LVCFs. Electrophysiological experiments revealed that in IH-exposed rats, OVX potentiated the excitability of LVCFs to stimulants. Moreover, LVCF hypersensitivity in rats subjected to OVX prior to IH exposure was accompanied by enhanced lung inflammation, which was reflected by elevated inflammatory cell infiltration in bronchoalveolar lavage fluid, lung lipid peroxidation, and protein expression of inflammatory cytokines. Supplementation with 17ß-estradiol (E2) at a low concentration (30 µg/ml) but not at high concentrations (50 and 150 µg/ml) prevented the augmenting effects of OVX on LVCF sensitivity and lung inflammation caused by IH. These results suggest that ovarian hormones prevent the enhancement of LVCF sensitivity and lung inflammation by IH in female rats, which are related to the effect of low-dose estrogen.

Inflammatory Role of ROS-Sensitive AMP-Activated Protein Kinase in the Hypersensitivity of Lung Vagal C Fibers Induced by Intermittent Hypoxia in Rats.

Obstructive sleep apnea (OSA), manifested by airway exposure to intermittent hypoxia (IH), is associated with excess reactive oxygen species (ROS) production in airways, airway inflammation, and hyperreactive airway diseases. The cause-effect relationship for these events remains unclear. We investigated the inflammatory role of ROS-sensitive AMP-activated protein kinase (AMPK) in IH-induced airway hypersensitivity mediated by lung vagal C fibers (LVCFs) in rats. Conscious rats were exposed to room air (RA) or IH with or without treatment with N-acetyl-L-cysteine (NAC, an antioxidant), Compound C (an AMPK inhibitor), ibuprofen (a cyclooxygenase inhibitor), or their vehicles. Immediately after exposure (24 h), we found that intravenous capsaicin, phenylbiguanide, or α,ß-methylene-ATP evoked augmented LVCF-mediated apneic responses and LVCF afferent responses in rats subjected to IH exposure in comparison with those in RA rats. The potentiating effect of IH on LVCF responses decreased at 6 h after and vanished at 12 h after the termination of IH exposure. The potentiating effect of IH on LVCF-mediated apneic and LVCF afferent responses was significantly attenuated by treatment with NAC, compound C, or ibuprofen, but not by their vehicles. Further biochemical analysis revealed that rats exposed to IH displayed increased lung levels of lipid peroxidation (an index of oxidative stress), AMPK phosphorylation (an index of AMPK activation), and prostaglandin E2 (a cyclooxygenase metabolite), compared with those exposed to RA. IH-induced increase in lipid peroxidation was considerably suppressed by treatment with NAC but not by compound C or ibuprofen. IH-induced increase in AMPK phosphorylation was totally abolished by NAC or compound C but not by ibuprofen. IH-induced increase in prostaglandin E2 was considerably prevented by any of these three inhibitor treatments. The vehicles of these inhibitors exerted no significant effect on the three IH-induced responses. These results suggest that 24-h IH exposure sensitizes LVCFs, leading to an exaggerated reflex and afferent responses to chemical stimulants in rats. Moreover, this IH-induced LVCF sensitization is mediated through a cascade of inflammatory responses in the airways involving increases in ROS, AMPK activation, and cyclooxygenase metabolite release.

NADPH Oxidase-Derived ROS Induced by Chronic Intermittent Hypoxia Mediates Hypersensitivity of Lung Vagal C Fibers in Rats.

Obstructive sleep apnea (OSA), manifested by exposure to chronic intermittent hypoxia (CIH) and excess production of reactive oxygen species (ROS) in the airways, is associated with hyperreactive airway diseases. ROS, particularly when created by NADPH oxidase, are known to sensitize lung vagal C fibers (LVCFs), which may contribute to airway hypersensitivity pathogenesis. We investigated whether CIH augments the reflex and afferent responses of LVCFs to chemical stimulants and the roles of ROS and NADPH oxidase in such airway hypersensitivity. Rats were exposed to room air (RA) or CIH with/without daily treatment with MnTMPyP (a superoxide anion scavenger), apocynin (an NADPH oxidase inhibitor), or vehicle. At 16 h after their last exposure, intravenous capsaicin, adenosine, or α,ß-methylene-ATP evoked an augmented apneic response in anesthetized rats with 14-days CIH exposure, compared to anesthetized rats with 14-days RA exposure. The augmented apneic responses to these LVCF stimulants were abolished by bilateral vagotomy or perivagal capsaicin treatment, which block LVCFs neural conduction and were significantly suppressed by treatment with MnTMPyP or apocynin, but not vehicle. Electrophysiological studies revealed that 14-days CIH exposure potentiated the responses of LVCFs to these stimulants. This effect was inhibited by treatment with MnTMPyP or apocynin treatment and was not seen in rats who received 7-days of CIH exposure. Biochemical analysis indicated that 14-days CIH exposure increased both lung lipid peroxidation, which is indicative of oxidative stress, and expression of the p47(phox) subunit in the membrane fraction of lung tissue, which is an index of NADPH oxidase activation. The former was prevented by treatment with either MnTMPyP or apocynin, while the later was prevented by treatment with apocynin only. These results suggest that 14-days CIH exposure sensitizes LVCFs in rats, leading to an exaggerated reflex and afferent responses to stimulants and that this sensitization is mediated via ROS generated by NADPH oxidase.

Effect of superoxide anion scavenger on rat hearts with chronic intermittent hypoxia.

Only very limited information regarding the protective effects of the superoxide anion scavenger on chronic intermittent hypoxia-induced cardiac apoptosis is available. The purpose of this study is to evaluate the effects of the superoxide anion scavenger on cardiac apoptotic and prosurvival pathways in rats with sleep apnea. Forty-two Sprague-Dawley rats were divided into three groups, rats with normoxic exposure (Control, 21% O2, 1 mo), rats with chronic intermittent hypoxia exposure (Hypoxia, 3-7% O2vs. 21% O2per 40 s cycle, 8 h per day, 1 mo), and rats with pretreatment of the superoxide anion scavenger and chronic intermittent hypoxia exposure (Hypoxia-O2 (-)-Scavenger, MnTMPyP pentachloride, 1 mg/kg ip per day; 3-7% O2vs. 21% O2per 40 s cycle, 8 h per day, 1 mo) at 5-6 mo of age. After 1 mo, the protein levels and apoptotic cells of excised hearts from three groups were measured by Western blotting and terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) assay. The superoxide anion scavenger decreased hypoxia-induced myocardial architecture abnormalities, left ventricular hypertrophy, and TUNEL-positive apoptosis. The superoxide anion scavenger decreased hypoxia-induced Fas ligand, Fas death receptors, Fas-associated death domain (FADD), activated caspase-8, and activated caspase-3 (Fas-dependent apoptotic pathway) as well as Bad, activated caspase-9 and activated caspase-3 (mitochondria-dependent apoptotic pathway), endonuclease G (EndoG), apoptosis-inducing factor (AIF), and TUNEL-positive apoptosis. The superoxide anion scavenger increased IGF-1, IGF-1R, p-PI3k, p-Akt, p-Bad, Bcl-2, and Bcl-xL (survival pathway). Our findings imply that the superoxide anion scavenger might prevent cardiac Fas-mediated and mitochondrial-mediated apoptosis and enhance the IGF-1-related survival pathway in chronic intermittent hypoxia. The superoxide anion scavenger may prevent chronic sleep apnea-enhanced cardiac apoptotic pathways and enhances cardiac survival pathways.

Role of TRPA1 and TRPV1 in the ROS-dependent sensory irritation of superior laryngeal capsaicin-sensitive afferents by cigarette smoke in anesthetized rats.

BACKGROUND: Laryngeal exposure to cigarette smoke (CS) evokes sensory irritation, but the mechanisms are largely unclear. The TRPA1 and TRPV1 receptors are two types of Ca(2+)-permeant channels located at the terminals of airway capsaicin-sensitive afferents. We investigated the mechanisms underlying the airway reflex evoked by laryngeal CS exposure in anesthetized rats. METHODS: CS (7 ml) was delivered into a functionally isolated larynx, while the animals (n = 201) breathed spontaneously. Respiratory parameters were measured. All use of pharmacological agents involved pretreatment by laryngeal application. RESULTS: Laryngeal CS exposure immediately evoked a concentration-dependant apneic response that was unrelated to the nicotine content of the CS. This inhibition of breathing was abolished by bilateral sectioning of the superior laryngeal nerves (SLNs) or by perineural capsaicin treatment of the SLNs (selective blocking of capsaicin-sensitive afferent neural conduction), suggesting the involvement of superior laryngeal capsaicin-sensitive afferents in the reflex. The reflex apnea was significantly attenuated by N-acetyl-l-cysteine (antioxidant), EGTA (extracellular Ca(2+) chelator) and BAPTA-AM (intracellular Ca(2+) chelator), indicating the importance of reactive oxygen species (ROS) and Ca(2+). This reflex apnea was also partially reduced by HC030031 (TRPA1 receptor antagonist) and capsazepine (TRPV1 receptor antagonist), and was nearly abolished by a combination of these two antagonists, suggesting a central role for the TRPA1 and TRPV1 receptors. Furthermore, the reflex apnea was attenuated by indomethacin (cyclooxygenase inhibitor); however, the attenuation by indomethacin was not increased by pretreatment with HC030031 or capsazepine, indicating that TRPA1 and TRPV1 receptor functionality is, at least in part, linked to cyclooxygenase metabolites. CONCLUSIONS: The reflex apnea evoked by laryngeal CS requires activation of both TRPA1 and TRPV1 receptors, which are likely to be located at the terminals of superior laryngeal capsaicin-sensitive afferents. Laryngeal sensory irritation by CS seems to depend on the actions of ROS and cyclooxygenase metabolites on these two types of receptors.

Perivagal antagonist treatment in rats selectively blocks the reflex and afferent responses of vagal lung C fibers to intravenous agonists.

The terminals of vagal lung C fibers (VLCFs) express various types of pharmacological receptors that are important to the elicitation of airway reflexes and the development of airway hypersensitivity. We investigated the blockade of the reflex and afferent responses of VLCFs to intravenous injections of agonists using perivagal treatment with antagonists (PAT) targeting the transient receptor potential vanilloid 1, P2X, and 5-HT(3) receptors in anesthetized rats. Blockading these responses via perivagal capsaicin treatment (PCT), which blocks the neural conduction of C fibers, was also studied. We used capsaicin, α,ß-methylene-ATP, and phenylbiguanide as the agonists, and capsazepine, iso-pyridoxalphosphate-6-azophenyl-2',5'-disulfonate, and tropisetron as the antagonists of transient receptor potential vanilloid 1, P2X, and 5-HT(3) receptors, respectively. We found that each of the PATs abolished the VLCF-mediated reflex apnea evoked by the corresponding agonist, while having no effect on the response to other agonists. Perivagal vehicle treatment failed to produce any such blockade. These blockades had partially recovered at 3 h after removal of the PATs. In contrast, PCT abolished the reflex apneic response to all three agonists. Both PATs and PCT did not affect the myelinated afferent-mediated apneic response to lung inflation. Consistently, our electrophysiological studies revealed that each of the PATs prevented the VLCF responses to the corresponding agonist, but not to any other agonist. PCT inevitably prevented the VLCF responses to all three agonists. Thus these PATs selectively blocked the stimulatory action of corresponding agonists on the VLCF terminals via mechanisms that are distinct from those of PCT. PAT may become a novel intervention for studying the pharmacological modulation of VLCFs.

Hypersensitivity of lung vagal C fibers induced by acute intermittent hypoxia in rats: role of reactive oxygen species and TRPA1.

Obstructive sleep apnea, manifested by intermittent hypoxia and excess production of reactive oxygen species (ROS) in airways, is associated with hyperreactive airway diseases, but the mechanism remains unclear. Sensitization of lung vagal C fibers (LVCFs) contributes to the airway hypersensitivity. We investigated the mechanisms underlying the sensitization of LVCFs with acute intermittent hypoxia (AIH), by 10 episodes of exposure to 30 s of hypoxic air (0%, 5%, or 10% O(2)) followed by 30 s of room air in anesthetized, open-chest, and artificially ventilated rats. Reflex apneic response to intravenous capsaicin (an LVCF stimulant), as measured by phrenic nerve activity, was concentration dependently augmented by AIH. Similarly, reflex apneic response to intravenous α,ß-methylene-ATP (another LVCF stimulant) was augmented by AIH (0% O(2)). The reflex apnea evoked by these two stimulants was abolished by bilateral vagotomy, which suggests the involvement of lung vagal afferents. The AIH-augmented apneic response to these two stimulants was prevented by pretreatment with dimethylthiourea (a hydroxyl radical scavenger), N-acetyl-l-cysteine (an antioxidant) and HC-030031 [a transient receptor potential ankyrin 1 (TRPA1) receptor antagonist]. Consistently, electrophysiological study revealed the afferent responses of LVCFs to capsaicin or α,ß-methylene-ATP were augmented by AIH, and this sensitization of LVCFs was prevented by dimethylthiourea, N-acetyl-l-cysteine, and HC-030031. In contrast, AIH did not alter the afferent response of LVCFs to mechanical stimulation by lung hyperinflation. We concluded that AIH sensitizes LVCFs in rats, thus resulting in exaggerated airway reflexogenic responses to chemical stimulants, possibly by ROS action and activation of TRPA1 receptors.

Influence of antipsychotic agents on heart rate variability in male WKY rats: implications for cardiovascular safety.

BACKGROUND: Sudden cardiac death is higher among schizophrenic patients and is associated with parasympathetic hypoactivity. Antipsychotic agents are highly suspected to be a precipitating factor. Thus, we aimed to test if the antipsychotics haloperidol, risperidone and clozapine affect cardiac autonomic function, excluding the confounding effect of altered sleep structure by the drugs. METHODS: In this study, haloperidol, risperidone and clozapine were given separately by intraperitoneal injection to male Wistar-Kyoto rats for 5 days. Electroencephalogram (EEG), electromyogram (EMG) and electrocardiographic signals were recorded at baseline and 5 days after drug treatments. Sleep scoring was based on EEG and EMG signals. Cardiac autonomic function was assessed using heart rate variability analysis. RESULTS: Clozapine increased heart rate and suppressed cardiac sympathetic and parasympathetic activity. Cardiac acceleration was more severe during sleep. Haloperidol tended to decrease heart rate while risperidone mildly increased heart rate; however, their effects were less obvious than those of clozapine. There was a significant drug-by-stage interaction on several heart rate variability measures. CONCLUSION: Taking this evidence as a whole, we conclude that haloperidol has a better level of cardiovascular safety than either risperidone or clozapine. Application of this approach to other psychotropic agents in the future will be a useful and helpful way to evaluate the cardiovascular safety of the various psychotropic medications that are in clinical use.

Ceftriaxone attenuates hypoxic-ischemic brain injury in neonatal rats.

BACKGROUND: Perinatal brain injury is the leading cause of subsequent neurological disability in both term and preterm baby. Glutamate excitotoxicity is one of the major factors involved in perinatal hypoxic-ischemic encephalopathy (HIE). Glutamate transporter GLT1, expressed mainly in mature astrocytes, is the major glutamate transporter in the brain. HIE induced excessive glutamate release which is not reuptaked by immature astrocytes may induce neuronal damage. Compounds, such as ceftriaxone, that enhance the expression of GLT1 may exert neuroprotective effect in HIE. METHODS: We used a neonatal rat model of HIE by unilateral ligation of carotid artery and subsequent exposure to 8% oxygen for 2 hrs on postnatal day 7 (P7) rats. Neonatal rats were administered three dosages of an antibiotic, ceftriaxone, 48 hrs prior to experimental HIE. Neurobehavioral tests of treated rats were assessed. Brain sections from P14 rats were examined with Nissl and immunohistochemical stain, and TUNEL assay. GLT1 protein expression was evaluated by Western blot and immunohistochemistry. RESULTS: Pre-treatment with 200 mg/kg ceftriaxone significantly reduced the brain injury scores and apoptotic cells in the hippocampus, restored myelination in the external capsule of P14 rats, and improved the hypoxia-ischemia induced learning and memory deficit of P23-24 rats. GLT1 expression was observed in the cortical neurons of ceftriaxone treated rats. CONCLUSION: These results suggest that pre-treatment of infants at risk for HIE with ceftriaxone may reduce subsequent brain injury.

Differences in left ventricular cardiomyocyte loss induced by chronic intermittent hypoxia between spontaneously hypertensive and Wistar-Kyoto rats.

RATIONALE: Chronic intermittent hypoxia (CIH) is thought to induce several cardiovascular effects in patients with obstructive sleep apnoea (OSA). However, the effects of CIH on patients with long-standing hypertension are unknown. PURPOSE: This prospective study aimed to investigate the influence of combined OSA and hypertension on cardiomyocyte death. METHODS: Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) were exposed to repetitive hypoxia-reoxygenation cycles (30 s of 5% O(2); 45 s of 21% O(2)) or room air for 6 h/day during the light phase (10 a.m.-4 p.m.) for 10, 20, or 30 days, and the levels of necrosis and apoptosis induced in their left ventricular cardiomyocyte were examined. RESULTS: CIH increased the accumulation of reactive oxygen species, which induced cardiomyocyte necrosis in WKY and SHR (both p < 0.05). Cardiomyocyte oxidative stress levels by CIH were higher in SHR than in WKY (p < 0.05); therefore, cardiomyocyte necrosis was amplified (p < 0.05). Notably, if a superoxide-scavenging agent is injected beforehand, cardiomyocyte necrosis can be effectively inhibited (p < 0.05). When WKY and SHR are exposed to CIH, increases in mitochondria-released cytochrome c and activation of caspase-3 are found in the cytosolic fraction only in WKY. CONCLUSIONS: CIH causes cardiomyocyte loss in SHR mainly through cardiomyocyte necrosis. In WKY however, CIH simultaneously induces apoptosis and necrosis of cardiomyocytes.

Reactive oxygen species are the cause of the enhanced cardiorespiratory response induced by intermittent hypoxia in conscious rats.

This study was carried out to investigate the role of reactive oxygen species (ROS) in the elevation of cardiorespiratory responses during the development of intermittent hypoxia (IH)-induced hypertension. Rats were exposed to either 30 days of IH [(30s N2)+(45 s room air (RA)] or RA for 6 h/day. After 5 days of exposure, stable mean arterial pressure, normalized low-frequency power of pulses interval spectrogram (a marker of cardiac sympathetic outflow), and minute ventilation (an index for arterial chemoreflex activation) were significantly increased throughout the observation period in IH-exposed rats, but not in RA-exposed rats. FosB expression in rostral ventrolateral medulla was elevated after IH exposure for 5 days. Intraperitoneal injection of MnTMPyP (a superoxide scavenger) or N-acetylcysteine (an antioxidant) prevented IH-induced elevation of the cardiorespiratory responses and lipid peroxidation of lung tissues. These results suggest that ROS are essential for IH-induced elevation of arterial chemoreflex activation and sympathetic outflow, which may, in turn, contribute to IH-induced hypertension.

Involvement of sympathetic function in the sleep-related change of gastric myoelectrical activity in rats.

The gastric myoelectrical activity (GMA) fluctuates across sleep-wake states as a result of modulation by the brain-gut axis. The role of the autonomic nervous system in this phenomenon, however, was not elucidated fully. Through simultaneous recording and subsequent continuous power spectral analysis of electroencephalogram, electromyogram, electrocardiogram and electrogastromyogram (EGMG) in 16 freely moving Wistar rats, the sleep-wake states of the animals were defined and indices of cardiac autonomic regulation and GMA were calculated. We found that both cardiac autonomic regulation and GMA fluctuated through sleep-wake cycles. Correlation analysis further revealed significant correlations between EGMG power and each of the R-R interval, high-frequency power, low-frequency power, very-low-frequency power, low-frequency power to high-frequency power ratio and normalized low-frequency power of heart rate variability with respect to their trend of change across different sleep-wake states. These results suggest that the sleep-wake-related change of GMA was related to sympathovagal balance. The sympathetic nerve may play a more important role in the central modulation of GMA than perceived previously.

Time course of cardiovascular neural regulation during programmed 20-sec apnea in rats.

OBJECTIVES: Long-term hypoxia results in hemodynamic breakdown in patients in the intensive care unit; however, intermittent hypoxia causes hypertension in individuals with sleep apnea. The objective of this study was to explore the sequential cardiovascular neural alterations in response to acute hypoxic apnea. DESIGN: The authors conducted a prospective, randomized animal study. SETTING: The study was conducted in a university animal laboratory. METHODS: A programmable apnea model was developed in anesthetized rats, in which a 20-sec period of apnea was produced and subsequently divided into the early (2.0 to 7.9 secs), middle (8.0 to 13.9 secs), and late apneic phases (14.0 to 19.9 secs) and immediate reventilatory phase (20.0 to 25.9 secs). RESULTS: Evident hypoxia developed during 20-sec apnea. Arterial pressure increased in the early apneic phase and returned to control level in the middle phase. Significant hypotension developed in the late apneic phase and deteriorated in the reventilatory phase. Interbeat interval increased mildly along the apneic event. The increase of pressure in the early and middle phases was inhibited by propranolol (1.0 mg/kg intravenously [iv]) but was provoked by phentolamine (2.5 mg/kg iv). The decrease of pressure in the late and reventilatory phases was reversed, at least in part, by phentolamine. Atropine (0.5 mg/kg iv) did not produce discernible effects in the arterial pressure. The increase of interbeat interval was suppressed by propranolol. Power spectral analysis of arterial pressure variability demonstrated significant increases of the low-frequency (sympathetic vasomotor activity) and normalized high-frequency (cardiac sympathetic modulation) power after reventilation. CONCLUSION: Although the sympathetic activity is excited during and after a hypoxic apnea, the immediate pressor effect is related to an inotropic response of cardiac sympathetic regulation, whereas the negative chronotropic and subsequent depressor effect is associated with a failure in the cardiovascular response to sympathetic excitation.

The involvement of hydroxyl radical and cyclooxygenase metabolites in the activation of lung vagal sensory receptors by circulatory endotoxin in rats.

Circulatory endotoxin can stimulate vagal pulmonary C fibers and rapidly adapting receptors (RARs) in rats, but the underlying mechanisms are not clear. We investigated the involvement of hydroxyl radicals and cyclooxygenase metabolites in the stimulation of C fibers and RARs by circulatory endotoxin (50 mg/kg) in 112 anesthetized, paralyzed, and artificially ventilated rats. In rats pretreated with the vehicle, endotoxin stimulated C fibers and RARs and caused a slight increase in total lung resistance (Rl) and a decrease in dynamic lung compliance. In rats pretreated with dimethylthiourea (a hydroxyl radical scavenger) alone, indomethacin (a cyclooxygenase inhibitor) alone, or a combination of the two, C-fiber and RAR responses [C fiber: change (Delta) = -62, -79, and -85%; RAR: Delta = -80, -84, and -84%, respectively] were reduced, and the Rl response was prevented. The suppressive effects of a combination of dimethylthiourea and indomethacin on the C-fiber and RAR responses were not superior to indomethacin alone. In rats pretreated with isoproterenol (a bronchodilator), the C-fiber response was not significantly affected (Delta = -26%), the RAR response was reduced (Delta = -88%), and the Rl response was prevented. None of these pretreatments affected the dynamic lung compliance response. These results suggest that 1) both hydroxyl radicals and cyclooxygenase metabolites are involved in the endotoxin-induced stimulation of C fibers and RARs, and 2) the involvement of these two metabolites in the C-fiber stimulation may be due to their chemical effects, whereas that in the RAR stimulation may be due to their bronchoconstrictive effects.

Activation of lung vagal sensory receptors by circulatory endotoxin in rats.

Although endotoxin is known to induce various pulmonary responses that are linked to the function of lung vagal sensory receptors, its effects on these pulmonary receptors are still not clear. This study investigated the effects of circulatory endotoxin on the afferent activity of lung vagal sensory receptors in rats. We recorded afferent activity arising from vagal pulmonary C fibers (CFs), rapidly adapting receptors (RARs), tonic pulmonary stretch receptors (T-PSRs), and phasic pulmonary stretch receptors (P-PSRs) in 64 anesthetized, paralyzed, and artificially ventilated rats. Intravenous injection of endotoxin (50 mg/kg; lipopolysaccharide) stimulated 7 of the 8 CFs, 8 of the 8 RARs, and 4 of the 8 T-PSRs studied, while having no effect on the 8 P-PSRs tested. The stimulation started 3-16 min after endotoxin injection and lasted until the end of the 90-min observation period. The evoked discharge of either CFs or RARs was not in phase with the ventilatory cycle, whereas that of T-PSRs showed a respiratory modulation. Injection of a saline vehicle caused no significant change in the discharge of these receptors. Additionally, endotoxin significantly produced an increase in total lung resistance, and decreases in dynamic lung compliance and arterial blood pressure. Our results demonstrate that a majority of lung vagal sensory receptors are activated following intravenous injection of endotoxin, and support the notion that these pulmonary receptors may function as an important afferent system during endotoxemia.