Chronic Intermittent Hypoxia Rats Research Articles

Chronic intermittent hypoxia-induced oxidative stress activates TRB3 and phosphorylated JNK to mediate insulin resistance and cell apoptosis in the pancreas.

This study explores the potential mechanisms of obstructive sleep apnoea (OSA) complicates type 2 diabetes mellitus (T2DM) by which chronic intermittent hypoxia (CIH) induces insulin resistance and cell apoptosis in the pancreas through oxidative stress. Four- and eight-week CIH rat models were established, and Tempol (100 mg/kg/d), was used as an oxidative stress inhibitor. This study included five groups: 4-week CIH, 4-week CIH-Tempol, 8-week CIH, 8-week CIH-Tempol and normal control (NC) groups. Fasting blood glucose and insulin levels were measured in the serum. The expression levels of 8-hidroxy-2-deoxyguanosine (8-OHdG), tribbles homologue 3 (TRB3), c-Jun N-terminal kinase (JNK), phosphorylated JNK (p-JNK), insulin receptor substrate-1 (IRS-1), phosphorylated IRS-1 (Ser307) (p-IRS-1ser307 ), protein kinase B (AKT), phosphorylated AKT (Ser473) (p-AKTser473 ), B cell lymphoma protein-2 (Bcl-2), cleaved-caspase-3 (Cl-caspase-3), and the islet cell apoptosis were detected in the pancreas. CIH induced oxidative stress in the pancreas. Compared with that in the NC group and CIH-Tempol groups individually, the homeostasis model assessment of insulin resistance (HOMA-IR) and apoptosis of islet cells was increased in the CIH groups. CIH-induced oxidative stress increased the expression of p-IRS-1Ser307 and decreased the expression of p-AKTSer473 . The expression levels of TRB3 and p-JNK were higher in the CIH groups than in both the CIH-Tempol and NC groups. Meanwhile, the expressions of Cl-caspase-3 and Bcl-2 were upregulated and downregulated, respectively, in the CIH groups. Hence, the present study demonstrated that CIH-induced oxidative stress might not only induce insulin resistance but also islet cell apoptosis in the pancreas through TRB3 and p-JNK.

Effect of chronic intermittent hypoxia on ocular and intraoral mechanical allodynia mediated via the calcitonin gene-related peptide in a rat.

Obstructive sleep apnea, a significant hypoxic condition, may exacerbate several orofacial pain conditions. The study aims to define the involvement of calcitonin gene-related peptide (CGRP) in peripheral and central sensitization and in evoking orofacial mechanical allodynia under chronic intermittent hypoxia (CIH). Male rats were exposed to CIH. Orofacial mechanical allodynia was assessed using the eyeblink test and the two-bottle preference drinking test. The CGRP-immunoreactive neurons in the trigeminal ganglion (TG), CGRP-positive primary afferents projecting to laminae I-II of the trigeminal spinal subnucleus caudalis (Vc), and neural responses in the second-order neurons of the Vc were determined by immunohistochemistry. CGRP receptor antagonist was administrated in the TG. CIH-induced ocular and intraoral mechanical allodynia. CGRP-immunoreactive neurons and activated satellite glial cells (SGCs) were significantly increased in the TG and the number of cFos-immunoreactive cells in laminae I-II of the Vc were significantly higher in CIH rats compared to normoxic rats. Local administration of the CGRP receptor antagonist in the TG of CIH rats attenuated orofacial mechanical allodynia; the number of CGRP-immunoreactive neurons and activated SGCs in the TG, and the density of CGRP-positive primary afferent terminals and the number of cFos-immunoreactive cells in laminae I-II of the Vc were significantly lower compared to vehicle-administrated CIH rats. An increase in CGRP in the TG induced by CIH, as well as orofacial mechanical allodynia and central sensitization of second-order neurons in the Vc, supported the notion that CGRP plays a critical role in CIH-induced orofacial mechanical allodynia.

Characterization of metabotropic glutamate receptor 7 and 8 in rat superior cervical ganglion and their changes following chronic intermittent hypoxia

To investigate the expression and localization of metabotropic glutamate receptors 7 and 8 (mGluR7/8) in rat superior cervical ganglion (SCG) and their changes in response to chronic intermittent hypoxia (CIH). We detected the expressions of mGluR7 and mGluR8 in the SCG of 8-week-old male SD rats using immunohistochemistry and characterized their distribution with immunofluorescence staining. The expression of mGluR7 and mGluR8 in the cytoplasm and nucleus was detected using Western blotting. A 6-week CIH rat model was established by exposure to intermittent hypoxia (6% oxygen for 30 s followed by normoxia for 4 min) for 8 h daily, and the changes in systolic blood pressure, diastolic blood pressure and mean arterial pressure were measured. The effect of CIH on expression levels of mGluR7 and mGluR8 in the SCG was analyzed using Western blotting. Positive expressions of mGluR7 and mGluR8 were detected in rat SCG. mGluR7 was distributed in the neurons and small fluorescent (SIF) cells with positive staining in both the cytoplasm and nuclei, but not expressed in satellite glial cells (SGCs), nerve fibers or blood vessels; mGluR8 was localized in the cytoplasm of neurons and SIF cells, but not expressed in SGCs, nerve fibers, or blood vessels. Western blotting of the nuclear and cytoplasmic fractions of rat SCG further confirmed that mGluR7 was expressed in both the cytoplasm and the nucleus, while mGluR8 exists only in the cytoplasm. Exposure to CIH significantly increased systolic blood pressure, diastolic blood pressure and mean arterial pressure of the rats (all P < 0.001) and augmented the protein expressions of mGluR7 and mGluR8 in the SCG (P < 0.05). mGluR7 and mGluR8 are present in rat SCG but with different localization patterns. CIH increases blood pressure of rats and enhanced protein expressions of mGluR7 and mGluR8 in rat SCG.

Long noncoding RNA HOXA-AS2 ameliorates chronic intermittent hypoxia-induced lung inflammation by regulating miR-17-5p/tipe2 axis.

The purpose is to confirm whether long noncoding RNA HOXA-AS2 relieves chronic intermittent hypoxia (CIH)-induced lung inflammation. Male Sprague Dawley rats were used to establisha CIH rat model. Hematoxylin and Eosin staining was used on the lung tissue injury to determine the successful construction of CIH animal model. Arterial partial pressure of oxygen (PaO2) and carbon dioxide (PaCO2) were measured. HOXA-AS2 was overexpressed to evaluate its role in the progression and development of CIH. T cell differentiation and cytokine production were determined using flow cytometry. Cell apoptosis was determined using terminal deoxynucleotidyl transferase dUTP nick end labelling assay kit. The target of HOXA-AS2 and miR-17-5p was predicted by the Encyclopedia of RNA Interactomes (ENCORI) and confirmed using luciferase assay. HOXA-AS2 was downregulated in CIH rat models. Lung tissue injury was observed in CIH rats, and the injury was attenuated by the overexpression of HOXA-AS2. PaO2 was reduced and PaCO2 was induced in CIH rats, which was reversed by the overexpression of HOXA-AS2. The overexpression of HOXA-AS2 inhibited CIH-induced cell apoptosis. It also reversed alterations in the levels of interferon gamma (IFNγ), interleukin (IL)-2, IL-6, IL-1β, tumor necrosis factor alpha (TNF-α), and transforming growth factor beta1 (TGF-β1) in rats caused by CIH. The overexpression of HOXA-AS2 prevented the induction in CD4+ IFN-γ+ T cells and reduction in CD4+TGF-β1+ T cells. The overexpression of HOXA-AS2 upregulated tumor necrosis factor-alpha-induced protein 8-like 2 (tipe2) key regulator through directly targeting miR-17-5p. Further experiments proved that tipe2 was the direct target of miR-17-5p. This study manifested that HOXA-AS2 acted as an anti-inflammatory regulator and protected lung tissue injury from CIH in the rat model; this was mediated by upregulation of tipe2 through directly targeting miR-17-5p. HOXA-AS2 upregulated the expression of tipe2, providing new understanding and therapeutic target for CIH.

Chronic intermittent hypoxia-induced BNIP3 expression mitigates contractile dysfunction and myocardial injury in animal and cell model via modulating autophagy.

Obstructive sleep apnea syndrome is generally associated with multiple cardiovascular disorders, such as myocardial hypertrophy. Autophagy is strictly modulated to maintain cardiac homeostasis. Post-injury autophagy is closely associated with pathological cardiac hypertrophy. BCL2 interacting protein 3 (BNIP3) and BNIP3-like protein (BNIP3L) can cause cell death and are important for hypoxia-elicited autophagy. Here, we evaluated whether BNIP3 could mitigate functional remodeling and cardiac hypertrophy through regulation of autophagy. Male WT rats or rats with BNIP3 knockout were subjected to chronic intermittent hypoxia (CIH) for 8h/day over 5weeks. Echocardiography and morphology were employed to assess the cardioprotective effects. Autophagy was assessed via transmission electron microscopy and detection of LC3, p62, and Beclin-1. Terminal deoxynucleotidyl transferase dUTP nick end labeling and the Bax/Bcl2 ratio were used to monitor apoptosis. Biochemical evaluations were performed to assess oxidative stress. Additionally, BNIP3-knockdown H9c2 cells that were subjected to CIH were used to examine autophagy and apoptosis to confirm the findings of the animal study. The CIH group showed elevated heart weight/body weight and left ventricle weight/body weight proportions, along with left ventricular remodeling. CIH-exposed rats exhibited dramatically higher fractional shortening and ejection fractions than the controls. In addition, the levels of autophagy markers Beclin-1 and LC3-II/I were increased, whereas the level of p62 was reduced by CIH treatment. The oxidative marker levels and the apoptosis index in the CIH group were markedly increased. Knockout of BNIP3 significantly aggravated the impairment in cardiac function, apoptosis, oxidative stress, and hypertrophy of CIH rats, while significantly reducing autophagy. The autophagy-associated PI3K/Akt/mTOR pathway was also deactivated by BNIP3 knockout. At the cellular level, CIH treatment significantly upregulated autophagy and apoptosis; however, BNIP3 silencing reduced autophagy and promoted apoptosis. CIH treatment-mediated upregulation of BNIP3 expression plays a crucial role in autophagy by targeting the PI3K/Akt/mTOR pathway, alleviating cardiac hypertrophy.

The role of ferroptosis in chronic intermittent hypoxia-induced cognitive impairment.

Obstructive sleep apnea (OSA) is a sleep disorder that may lead to cognitive impairment. The primary pathophysiological feature of OSA is chronic intermittent hypoxia (CIH), but the underlying mechanisms of CIH are not known. There have been few studies on the role of ferroptosis, a novel form of programmedcell death, during CIH-induced cognitive impairment. Therefore, this paper examined ferroptosis' effect on CIH-mediated cognitive impairment. The study randomized twenty-four Sprague-Dawley (SD) male rats to control or CIH group. CIH rats were subjected to intermittent hypoxia for 4weeks. Rat learning and memory were analyzed by the Morris water maze (MWM) test. Alterations of hippocampal neuronal ultrastructure were observed by transmission electron microscopy (TEM). Malondialdehyde (MDA) and ferrous iron (Fe2+) levels and superoxide dismutase (SOD) and reduced glutathione (GSH) contents were determined. Ferroptosis-associated protein levels were examined by Western blotting. The MWM test indicated that rats in the CIH group exhibited neurocognitive impairment. TEM showed that CIH induced mitochondrial damage. Significant increases in Fe2+ and MDA levels were observed in the CIH group, and GSH and SOD levels were decreased. Expression of Acyl-CoA synthetase long-chain family member 4 (ACSL4) increased, and glutathione peroxidase 4 (GPX4) protein levels were decreased, suggesting that ferroptosis was induced in CIH model rats. The NF-E2-related factor 2 (Nrf2) protein level in the CIH group was decreased. Ferroptosis had an essential effect on CIH-mediated cognitive impairment, and it may occur via Nrf2 dysregulation. These findings lay a solid foundation for the subsequent study of OSA-associated cognitiveimpairment offering potential evidence for the development of therapeutic strategies.

PS-B08-4: CHRONIC INTERMITTENT HYPOXIA INDUCES RENAL FIBROSIS BY MR ACTIVATION

Purpose: Obstructive sleep apnea is mainly characterized by chronic intermittent hypoxia (CIH), which is associated with chronic kidney disease. Macrophage is known to play an important role in renal fibrosis. However, little is known of the potential role of macrophage-to-myofibroblast transition (MMT) in hypoxic conditions. The aim of this study was to understand how infiltrating macrophages may contribute to renal fibrosis in hypoxic conditions. Methods: In this study, 30 adult male sprague dawley (SD) rats were randomly divided into normoxic control(NC), chronic intermittent hypoxia(CIH), and eplerenone(EPL) groups-each containing ten rats. The CIH and EPL groups were exposed to hypoxia in a commercial hypoxic chamber for 5 weeks. Rats in the EPL group were administered EPL orally via diet for 5 weeks. All rats were then sacrificed and serum, 24-hour urine and tissue specimens were collected for renal function, histopathology, western blot, immunohistochemistry. The RAW264.7 macrophages were subjected to 24 hours of hypoxia and 12 hours of reoxygenation. Cells were collected for immunofluorescence staining. Results: In addition to blood pressure, renal aldosterone level was also increased in CIH rats. Massons trichrome and Sirius red staining showed significantly higher collagen deposition and fibrosis in CIH rats. A markedly increased co-expression of α-SMA and CD68 in vitro and in vivo after CIH exposure. MR nuclear translocation was seen in hypoxic macrophages. Western blot showed that the expression of SGK-1 and p-SGK-1 was upregulated. These effects were reversed by MR blocker (EPL). Conclusions: CIH-induced macrophage infiltration, an important source of myofibroblasts, might participate in the development of renal interstitial fibrosis. These effects were reversed by EPL. Therefore, we speculate MR activation may be related to CIH-induced renal fibrosis.

The Beneficial Effect of the Blockade of Stim-Activated TRPC-ORAI Channels on Vascular Remodeling and Pulmonary Hypertension Induced byIntermittent Hypoxia Is Independent of Oxidative Stress.

Obstructive sleep apnea (OSA), a sleep breathing disorder featured by chronic intermittent hypoxia (CIH), is associated with pulmonary hypertension (PH). Rats exposed to CIH develop systemic and lung oxidative stress, pulmonary vascular remodeling, and PH and overexpress Stim-activated TRPC-ORAI channels (STOC) in the lung. Previously, we demonstrated that 2-aminoethyl-diphenylborinate (2-APB)-treatment, a STOC-blocker, prevents PH and the overexpression of STOC induced by CIH. However, 2-APB did not prevent systemic and pulmonary oxidative stress. Accordingly, we hypothesize that the contribution of STOC in the development of PH induced by CIH is independent of oxidative stress. We measured the correlation between right ventricular systolic pressure (RVSP) and lung malondialdehyde (MDA) with the gene expression of STOC and morphological parameters in the lung from control, CIH-treated, and 2-APB-treated rats. We found correlations between RVSP and increased medial layer and STOC pulmonary levels. 2-APB-treated rats showed a correlation between RVSP and the medial layer thickness, α-actin-ir, and STOC, whereas RVSP did not correlate with MDA levels in CIH and 2-APB-treated rats. CIH rats showed correlations between lung MDA levels and the gene expression of TRPC1 and TRPC4. These results suggest that STOC channels play a key role in developing CIH-induced PH that is independent from lung oxidative stress.

Contrast Enhanced Ultrasonography of Kidney in Chronic Intermittent Hypoxia Rat Model.

The objective of our study was to assess the ability of contrast-enhanced ultrasound (CEUS) in evaluating renal microperfusion in an animal model. Twenty Sprague-Dawley rats were subdivided into two groups: the normal and chronic intermittent hypoxia (CIH) groups. In the CIH model, 10 Sprague-Dawley rats were exposed to CIH for 8 weeks to mimic obstructive sleep apnea (OSA). The CEUS parameters of the renal cortex and medulla were obtained and compared between groups. The pathological changes of the kidney tissues were examined by histological staining such as hematoxylin and eosin (H&E) and Masson's trichrome. CIH caused morphological damage to kidneys. In the cortex, the peak intensity (PI) (P=.009) was significantly lower and time to peak (Ttop) (P=.019) was significantly prolonged in the CIH group compared with the controls. The area under ascending curve (WiAUC) in the medulla and cortex were both significantly lower in the CIH group than those in the control group (P both <.05). CEUS parameters (including PI and WiAUC of the cortex and WiAUC of the medulla) were negatively correlated with serum creatinine (P all <.05). In the medulla, the area under descending curve (WoAUC) was positively correlated with serum creatinine (P=.027), PI was negatively correlated with uric acid (P=.034). CEUS parameters (including Ttop, PI, WoAUC, and WiAUC) reflect renal microvascular changes in CIH. CEUS could be a safe and accurate imaging method to assess renal microvascular damage in CIH rats.

Effects of chronic intermittent hypoxia on atrial electrical remodeling in rats

The aim of this study was to investigate the effects of chronic intermittent hypoxia (CIH) on atrial electrical remodeling in Sprague-Dawley (SD) rats, which provide the explication for the mechanisms of CIH promoting atrial fibrillation (AF). Eighty SD rats were randomly divided into 2 groups: control group and CIH group (n=40). CIH rats were subjected to CIH 8 h/d for 30 days. After the echocardiography and hemodynamics examination, cardiac electrophysiological experiments, histological experiments, and molecular biological experiments were executed. AF susceptibility was measured by isolated heart electrophysiological experiments. Masson's trichrome stain was used to assess the degree of atrial fibrosis. The protein expression levels of sodium voltage-gated channel alpha subunit 5 (SCN5A/Nav1.5), calcium voltage-gated channel subunit alpha1 C (CACNA1C/Cav1.2) and potassium voltage-gated channel subfamily D member 3 (KCND3/Kv4.3) were measured by Western blot. In whole-cell patch clamp experiments, current clamp mode was used to record AP, and APD90 and APD50 were analyzed and compared between the two groups. In voltage clamp mode, INa, ICa-L, Ito and their kinetic parameters were recorded and compared between the two groups. Compared to the control rats, atrial interstitial collagen deposition (P＜0.01) and AF inducibility (P＜0.05) were increased in CIH rats, whereas the expression levels of Nav1.5, Cav1.2 and Kv4.3 were decreased (P＜0.05). APD90 and APD50 in CIH rats' atrial myocytes were longer than those of control rats, and CIH rats showed decreased current density of INa, ICa-L(P＜0.01) and Ito(P＜0.01). CIH-induced changes in the protein expression levels of ion channel subunits, current intensity, APD, and AF susceptibility, which may be the mechanisms of CIH promoting AF.

Inhibition of STAT3 signal pathway recovers postsynaptic plasticity to improve cognitive impairment caused by chronic intermittent hypoxia.

Chronic intermittent hypoxia (CIH) is a major cause of cognitive dysfunction in people with obstructive sleep apnea syndrome (OSAS), as it damages synapse structure, and function. This study aimed to investigate the potential mechanisms resulting in cognitive impairment caused by CIH in patients with OSAS. Healthy adult SD male rats (n = 36) were randomly divided into four groups: control, CIH, WP1066, and dimethyl sulfoxide (DMSO). The CIH, WP1066, and DMSO groups were exposed to intermittent hypoxic environments for 8h per day for 28 d. The WP1066 group received intraperitoneal injection of WP1066, a selective signal transducer and activator of transcription-3 (STAT3) inhibitor. All the experimental rats were subjected to the Morris water maze. Hippocampal tissue samples (n = 6 per group) were used for western blot analysis, and brain tissue samples (n = 3 per group) were used for immunohistochemistry and hematoxylin and eosin staining. The cognition of rats exposed to prolonged CIH was impaired. P-STAT3 expression was found to be higher in CIH rats than in control rats. Postsynaptic density95 (PSD95) expression was significantly reduced in rats with CIH-induced learning and memory impairment, but it significantly increased after the STAT3 signaling pathway was blocked, which improved learning and memory ability. However, inhibition of the STAT3 signaling pathway failed to improve the decline of synaptophysin (SYP) protein caused by CIH. When rats are exposed to CIH, STAT3 in the brain is activated, PSD95 and SYP levels decrease, and cognition is impaired. Inhibition of the STAT3 signaling pathway increases PSD95 to recover postsynaptic plasticity, thereby improving cognitive dysfunction.

Chronic intermittent hypoxia induces renal fibrosis through MR activation.

Obstructive sleep apnea syndrome (OSAS) is a disorder characterized by recurrent arousal from sleep and chronic intermittent hypoxia (CIH). OSAS-associated chronic kidney disease is mainly caused by CIH-induced tissue damage. Therefore, an OSAS model was established by CIH exposure in a hypoxic chamber for five weeks. In our study, macrophage infiltration and macrophage-myofibroblast transition (MMT) were observed in the kidneys of CIH rats and appeared to contribute to the development of renal fibrosis. However, the underlying mechanisms are not well defined. We also found that upon binding to the mineralocorticoid receptor (MR), aldosterone stimulated MMT and consequently led to renal fibrosis under hypoxic conditions. Additionally, an in vitro study of RAW264.7 macrophages demonstrated that MR activation may contribute to MMT, which resulted in a predominant M1 phenotype under hypoxic conditions. These effects were reversed by the MR blocker eplerenone. These results provide preliminary evidence that MR activation might be involved in the transdifferentiation of macrophages into myofibroblasts in the CIH model. The attenuation of renal injury demonstrates a protective role of MR blockade in CIH-induced renal disease.

Improving effects of eplerenone on atrial remodeling induced by chronic intermittent hypoxia in rats

ObjectiveAtrial fibrillation (AF) is closely associated with the overactivation of the renin-angiotensin-aldosterone system. Large cohort studies and recent meta-analyses have shown that the utilization of mineralocorticoid receptor antagonists has positive effects on the prevention and development of AF. This study aimed to investigate the effects of eplerenone on atrial remodeling in AF model rats and elucidate its intrinsic mechanism. MethodsNinety male Sprague-Dawley rats were randomly divided into the control group, chronic intermittent hypoxia (CIH) group, and CIH-eplerenone intervention (CIH-E) group. Rats in the CIH and CIH-E groups received CIH for 6 weeks, and rats in the CIH-E group were additionally administered eplerenone gavage (10 mg/kg/d). After modeling, the baseline parameters of each group were examined. Histopathology, molecular biology, isolated electrophysiology, and patch clamp experiments were performed after sampling. ResultsCompared with the control group, rats in the CIH group showed atrial enlargement, significant aggravated fibrosis, upregulated JAK/STAT3 pathway, shortened effective refractory period (ERP), increased AF inducibility, and decreased peak current density of characteristic voltage-gated ion channels in atrial myocytes. After eplerenone intervention, rats in the CIH-E group had a smaller atrial diameter than those in the CIH group. Furthermore, downregulated JAK/STAT3 pathway, prolonged ERP, decreased AF inducibility, and increased peak current density of characteristic ion channels were also observed in the CIH-E group. ConclusionsCIH induced significant atrial remodeling in rats and eplerenone significantly ameliorated the atrial remodeling caused by CIH. This could be attributed to the downregulation of the JAK/STAT3 pathway and the increase in the characteristic ion current density of atrial myocytes.

Renal Sympathetic Nerve Activity Responses to Hypoxia and Hypercapnia in Rats Exposed to Chronic Intermittent Hypoxia

Increased responsiveness of the carotid bodies to acute hypoxia was shown in animals exposed to chronic intermittent hypoxia (CIH). The kidney is sensitive to hypoxia and decreased cortical partial pressure of oxygen (PO2) levels was revealed in rats exposed to moderate CIH. We hypothesized that the response of the renal sympathetic nerve activity (RSNA) to acute hypoxia and hypercapnia is enhanced in rats exposed to CIH. Wistar rats were exposed to 96 cycles of CIH (6% O2, 8 hr/day, n=6) or to normoxia (sham, 21% O2, n=9) for 14 days. On day 15, rats were anesthetized (induction: urethane, α‐chloralose and sodium pentobarbitone mixture i.p. (416, 27 and 33mg/kg), maintenance: urethane and α‐chloralose mixture i.v. (62 and 4mg/kg)) to record cardiovascular parameters and RSNA. Rats were provided with supplementary oxygen to maintain PO2 levels &gt; 84 mmHg. During hypoxia, rats breathed 10% O2for two minutes. This was followed with a hypercapnic challenge during which rats breathed 10% CO2 for 5 minutes. Following a 90‐minute recovery period, capsaizepine (CPZ, 5µg/ml/hr), a TRPV1 blocker, was infused into the right kidney. Then, hypoxic and hypercapnic challenges were repeated following the intra‐renal infusion of CPZ. Data are expressed as mean±SD and were analyzed using t‐test or ANOVA, where relevant. CIH rats were hypertensive (130±9 vs. 118±9mmHg, p=0.039) with comparable basal HR (425±38 vs. 407±34bpm), and RSNA (% of max, 33±12 vs. 25±12) compared with sham rats. In response to acute hypoxia, RSNA was increased to an equivalent extent in CIH‐exposed and sham rats (Maximum change in RSNA of baseline, 128±23 vs. 125±16%; RSNA (% of max), 36±7 vs. 44±22%). Additionally, the increase in HR in response to acute hypoxia was similar between CIH‐exposed and sham rats (∆HR, +42±29 vs. +30±27bpm). Mean arterial blood pressure (MAP) was similarly decreased in CIH‐exposed and sham rats during acute hypoxia (Maximum change in MAP, ‐33±24 vs. ‐27±17mmHg). The decrease in PaO2 levels during hypoxia was comparable between exposure groups (∆PaO2, ‐48±15 vs. ‐47±10mmHg). Hypercapnia induced a comparable increase in the RSNA in CIH‐exposed and sham rats (Maximum change in RSNA of baseline, +121±15 vs. +113±8%). The analysis of absolute RSNA when normalized to maximum RSNA showed that CIH‐exposed rats had increased RSNA compared with sham rats before and after exposure to hypercapnia (RSNA (% of max), 37±8 vs. 26±10%, p=0.037). The increase in PaCO2 levels during hypercapnia was similar between CIH and sham groups (∆PaCO2, +24±8 vs. +19±6mmHg). Intra‐renal CPZ did not alter the increase in RSNA and HR in response to hypoxia and hypercapnia in CIH‐exposed and sham rats. Protein expression of TRPV1 receptors in kidney tissue was equivalent in both exposure groups (1.88±0.23 vs. 1.68±0.26ng/mg). Our results indicate that the responsiveness of RSNA to acute hypoxia and hypercapnia is not altered following exposure to moderate CIH sufficient to increase RSNA and blood pressure.

Oxytocin and corticotropin‐releasing hormone augment synaptic activity in the nTS of rats subjected to chronic intermittent hypoxia

Individuals with obstructive sleep apnea often develop hypertension. Chronic intermittent hypoxia (CIH) in rodents mimics the hypoxia‐induced hypertension seen in these individuals. Alterations in synaptic and neuronal activity within the nucleus tractus solitarii (nTS), the central integration site of sensory afferents, are involved in the CIH‐induced hypertension. The paraventricular nucleus of the hypothalamus (PVN) has a modulating role in controlling nTS activity to influence blood pressure. The PVN sends direct neuronal projections containing oxytocin (OT) and/or corticotropin‐releasing hormone (CRH) to the nTS, and these neuropeptides alter nTS synaptic signaling. During hypoxia, there is an increase in the production of these neuropeptides, yet their contribution to nTS activity after CIH is not understood. We hypothesized OT and CRH will individually increase nTS synaptic activity, their influence will be enhanced after CIH, and co‐application of OT and CRH will further magnify synaptic transmission in CIH. Male Sprague‐Dawley rats (3‐4 weeks) were exposed to either 10 days normoxia (21% O2) or CIH (30‐40 sec of 6% oxygen, 10 episodes/h, 8 h/day). Horizontal brainstem slices were generated, and using whole‐cell patch‐clamp recordings, we examined synaptic neurotransmission in monosynaptic nTS neurons. nTS neurons were also acutely dissociated and calcium (Ca2+) influx was examined via Fura‐2 imaging to isolate postsynaptic effects. Spontaneous (s) and afferent (TS)‐evoked excitatory postsynaptic currents (EPSCs) in slices or Ca2+ fluorescence in isolated cells were examined during sequential aCSF control, OT (600 nM) alone, CRH (300 nM) alone, and the combination of OT (600 nM) and CRH (300 nM). Vehicle (aCSF) application during the equivalent time period served as controls. In nTS normoxic slices, vehicle application (1 hr) did not change sEPSC frequency or amplitude, or TS‐evoked EPSC amplitude. During neuropeptide application (n=9), OT alone did not alter s‐ or TS‐EPSCs. CRH alone increased sEPSC and TS‐EPSC amplitude, and elevated overall currents in response to 20 Hz stimulation. Co‐application of OT and CRH did not further alter TS‐ or sEPSCs. After CIH, as in normoxia rats, vehicle did not alter synaptic currents. OT and CRH elevated TS‐EPSC amplitude after CIH (n=9), although sEPSCs were unaffected. Co‐application of OT and CRH significantly augmented TS‐EPSC amplitude evoked in response to 0.5 and 20Hz stimulation. Comparing the OT+CRH responses in both groups demonstrated the relative increase in current amplitude during 20 Hz stimulation was greater in CIH than Norm. In isolated nTS neurons, individual OT and CRH application increased Ca2+ relative to baseline and vehicle in normoxia and CIH. The co‐application of OT and CRH enhanced the increase of cytosolic Ca2+ in CIH rats compared to their normoxic controls. In summary, these data show that following CIH, OT and CRH enhanced afferent‐driven neurotransmission to nTS neurons via an increase in postsynaptic responses. The data predicate an increase in their receptors and a shift in CRH receptors after CIH.

Chronic intermittent hypoxia induces the pyroptosis of renal tubular epithelial cells by activating the NLRP3 inflammasome

ABSTRACT Obstructive sleep apnea syndrome (OSAS) is a respiratory disorder and chronic intermittent hypoxia (CIH) is an important pathological characteristic of OSAS. Injuries on renal tubular epithelial cells were observed under the condition of CIH. Pyroptosis is a programmed mode of cell death following cell apoptosis and cell necrosis, which is mediated by NLRP3 signaling. The present study aims to investigate the effects of CIH on the pyroptosis of renal tubular epithelial cells and the underlying mechanism. Firstly, CIH was induced in two renal tubular epithelial cell lines, HK-2 cells and TCMK-1 cells. As the aggravation of hypoxia, an increasing trend of elevated apoptotic rate was observed in HK-2 cells and TCMK-1 cells, accompanied by the excessive release of ROS and LDH, and upregulation of NLRP3. Subsequently, the CIH model was established on rats. The pathological analysis results indicated that in CIH rats, the glomerular bottom membrane and mesangium were slightly thickened and edema was observed in the renal tubule epithelium. More serious injury was observed in the moderate intermittent hypoxia group. The expression level of IL-1β and IL-18 was promoted as the aggravation of hypoxia, accompanied by the elevated production of LDH and ROS. The expression level of cleaved Caspase-1, Caspase-1, GSDMD, TLR4, MyD88, NF-κB, p-NF-κB, and NLRP3 was found significantly upregulated as the aggravation of hypoxia. Lastly, the pathological changes in rats induced by CIH were dramatically abolished by MCC950, a specific inhibitor of NLRP3. Collectively, CIH triggered the pyroptosis of renal tubular epithelial cells by activating the NLRP3 inflammasome.

Enhanced Ocular Surface and Intraoral Nociception via a Transient Receptor Potential Vanilloid 1 Mechanism in a Rat Model of Obstructive Sleep Apnea

Obstructive sleep apnea (OSA), characterized by low arterial oxygen saturation during sleep, is associated with an increased risk of orofacial pain. In this study, we simulated chronic intermittent hypoxia (CIH) during the sleep/rest phase (light phase) to determine the role of transient receptor potential vanilloid 1 (TRPV1) in mediating enhanced orofacial nocifensive behavior and trigeminal spinal subnucleus caudalis (Vc) neuronal responses to capsaicin (a TRPV1 agonist) stimulation in a rat model of OSA. Rats were subjected to CIH (nadir O2, 5%) during the light phase for 8 or 16 consecutive days. CIH yielded enhanced behavioral responses to capsaicin after application to the ocular surface and intraoral mucosa, which was reversed under normoxic conditions. The percentage of TRPV1-immunoreactive trigeminal ganglion neurons was greater in CIH rats than in normoxic rats and recovered under normoxic conditions after CIH. The ratio of large-sized TRPV1-immunoreactive trigeminal ganglion neurons increased in CIH rats. The density of TRPV1 positive primary afferent terminals in the superficial laminae of Vc was higher in CIH rats. Phosphorylated extracellular signal-regulated kinase (pERK)-immunoreactive cells intermingled with the central terminal of TRPV1-positive afferents in the Vc. The number of pERK-immunoreactive cells following low-dose capsaicin (0.33 µM) application to the tongue was significantly greater in the middle portion of the Vc of CIH rats than of normoxic rats and recovered under normoxic conditions after CIH. These data suggest that CIH during the sleep (light) phase is sufficient to transiently enhance pain on the ocular surface and intraoral mucosa via TRPV1-dependent mechanisms.

Daily acute intermittent hypoxia enhances serotonergic innervation of hypoglossal motor nuclei in rats with and without cervical spinal injury

Intermittent hypoxia elicits protocol-dependent effects on hypoglossal (XII) motor plasticity. Whereas low-dose, acute intermittent hypoxia (AIH) elicits serotonin-dependent plasticity in XII motor neurons, high-dose, chronic intermittent hypoxia (CIH) elicits neuroinflammation that undermines AIH-induced plasticity. Preconditioning with repeated AIH and mild CIH enhance AIH-induced XII motor plasticity. Since intermittent hypoxia pre-conditioning could enhance serotonin-dependent XII motor plasticity by increasing serotonergic innervation density of the XII motor nuclei, we tested the hypothesis that 3 distinct intermittent hypoxia protocols commonly studied to elicit plasticity (AIH) or simulate aspects of sleep apnea (CIH) differentially affect XII serotonergic innervation. Sleep apnea and associated CIH are common in people with cervical spinal injuries and, since repetitive AIH is emerging as a promising therapeutic strategy to improve respiratory and non-respiratory motor function after spinal injury, we also tested the hypotheses that XII serotonergic innervation is increased by repetitive AIH and/or CIH in rats with cervical C2 hemisections (C2Hx). Serotonergic innervation was assessed via immunofluorescence in male Sprague Dawley rats, with and without C2Hx (beginning 8 weeks post-injury) exposed to 28 days of: 1) normoxia; 2) daily AIH (10, 5-min 10.5% O2 episodes per day; 5-min normoxic intervals); 3) mild CIH (5-min 10.5% O2 episodes; 5-min intervals; 8 h/day); and 4) moderate CIH (2-min 10.5% O2 episodes; 2-min intervals; 8 h/day). Daily AIH, but neither CIH protocol, increased the area of serotonergic immunolabeling in the XII motor nuclei in both intact and injured rats. C2Hx per se had no effect on XII serotonergic innervation density. Thus, daily AIH may increases XII serotonergic innervation and function, enhancing the capacity for serotonin-dependent, AIH-induced plasticity in upper airway motor neurons. Such effects may preserve upper airway patency and/or swallowing ability in people with cervical spinal cord injuries and other clinical disorders that compromise breathing and airway defense.

Adiponectin Ameliorated Pancreatic Islet Injury Induced by Chronic Intermittent Hypoxia through Inhibiting the Imbalance in Mitochondrial Fusion and Division

Objective This study aimed to assess the protective value of adiponectin (APN) in pancreatic islet injury induced by chronic intermittent hypoxia (CIH). Methods Sixty rats were randomly divided into three groups: normal control (NC) group, CIH group, and CIH with APN supplement (CIH+APN) group. After 5 weeks of CIH exposure, we conducted oral glucose tolerance tests (OGTT) and insulin released test (IRT), examined and compared the adenosine triphosphate (ATP) levels, mitochondrial membrane potential (MMP) levels, reactive oxygen species (ROS) levels, enzymes gene expression levels of Ant1, Cs, Hmox1, and Cox4i1 which represented mitochondrial tricarboxylic acid cycle function, the protein and gene expression levels of DRP1, FIS1, MFN1, and OPA1 which represented mitochondrial fusion and division, and the protein expression levels of BAX, BCL-2, cleaved Caspase-3, and cleaved PARP which represented mitochondrial associated apoptosis pathway of pancreatic islet. Results OGTT and IRT showed blood glucose and insulin levels had no differences among the NC, CIH and CIH+APN groups (both P>0.05) at 0 min, 20 min, 30 min, 60 min, 120 min. However, we found that compared to NC group, CIH increased the ROS level, reduced ATP level and MMP level. The islets of CIH exposed rats showed reduced gene expression levels of Ant1, Cs, Hmox1, and Cox4i1, decreased protein and gene expression levels of MFN1 and OPA1, increased protein and gene expression levels of DRP1 and FIS1, increased protein expression levels of cleaved Caspase-3 and cleaved PARP, with lower ratio of BCL-2/BAX at protein expression level. All the differences among three groups were statistically significant. APN treated CIH rats showed mitigated changes in the above measurements associated with islet injuries. Conclusion APN may ameliorate the pancreatic islet injury induced by CIH via inhibiting the imbalance in mitochondrial fusion and division.

O‑GlcNAcylation contributes to intermittent hypoxia‑associated vascular dysfunction via modulation of MAPKs but not CaMKII pathways.

Intermittent hypoxia (IH) leads to vascular dysfunction, and O-linked-β-N-acetylglucosamine (O-GlcNAc)ylation may regulate vascular reactivity through the modulation of intracellular signaling. The present study hypothesized that O-GlcNAc modifications contributed to the vascular effects of acute IH (AIH) and chronic IH (CIH) through the MAPK and Ca2+/calmodulin-dependent kinase II (CaMKII) pathways. Rat aortic and mesenteric segments were incubated with DMSO, O-GlcNAcase (OGA) or O-GlcNAc transferase (OGT) inhibitor under either normoxic or AIH conditions for 3 h, and arterial function was then assessed. Meanwhile, arteries isolated from control and CIH rats were exposed to 3 h of incubation under normoxic conditions using DMSO, OGA or OGT as an inhibitor, before assessing arterial reactivity. CIH was found to increase the expression of vascular O-GlcNAc protein and OGT, phosphorylate p38 MAPK and ERK1/2, and decrease OGA levels, but it had no effects on phosphorylated CaMKII levels. OGA inhibition increased global O-GlcNAcylation and the phosphorylation of p38 MAPK, ERK1/2 and CaMKII, whereas OGT blockade had the opposite effects. OGA inhibition preserved acetylcholine-induced relaxation in AIH arteries, whereas OGT blockade attenuated the relaxation responses of arteries under normoxic conditions or undergoing AIH treatments. However, the impairment of acetylcholine dilation in CIH mesenteric arteries was improved. CIH artery contraction was increased following angiotensin II (Ang II) exposure. Blockade of p38 MAPK and ERK1/2, but not CaMKII, attenuated Ang II-induced contractile responses in CIH arteries isolated from the non-OGT inhibitor-treated groups. OGT inhibition significantly blocked contractile responses to Ang II and abolished the inhibitory effects of MAPK inhibitors. These findings indicated that O-GlcNAcylation regulates IH-induced vascular dysfunction, at least partly by modulating MAPK, but not CaMKII, signaling pathways.