Chronic Intermittent Hypoxia Mice Research Articles

Postinspiratory and preBötzinger complexes contribute to respiratory-sympathetic coupling in mice before and after chronic intermittent hypoxia.

The sympathetic nervous system modulates arterial blood pressure. Individuals with obstructive sleep apnea (OSA) experience numerous nightly hypoxic episodes and exhibit elevated sympathetic activity to the cardiovascular system leading to hypertension. This suggests that OSA disrupts normal respiratory-sympathetic coupling. This study investigates the role of the postinspiratory complex (PiCo) and preBötzinger complex (preBötC) in respiratory-sympathetic coupling under control conditions and following exposure to chronic intermittent hypoxia (CIH) for 21 days (5% O2-80 bouts/day). The surface of the ventral brainstem was exposed in urethane (1.5 g/kg) anesthetized, spontaneously breathing adult mice. Cholinergic (ChAT), glutamatergic (Vglut2), and neurons that co-express ChAT and Vglut2 at PiCo, as well as Dbx1 and Vglut2 neurons at preBötC, were optogenetically stimulated while recording activity from the diaphragm (DIA), vagus nerve (cVN), and cervical sympathetic nerve (cSN). Following CIH exposure, baseline cSN activity increased, breathing frequency increased, and expiratory time decreased. In control mice, stimulating PiCo specific cholinergic-glutamatergic neurons caused a sympathetic burst during all phases of the respiratory cycle, whereas optogenetic activation of cholinergic-glutamatergic PiCo neurons in CIH mice increased sympathetic activity only during postinspiration and late expiration. Stimulation of glutamatergic PiCo neurons increased cSN activity during the postinspiratory phase in control and CIH mice. Optogenetic stimulation of ChAT containing neurons in the PiCo area did not affect sympathetic activity under control or CIH conditions. Stimulating Dbx1 or Vglut2 neurons in preBötC evoked an inspiration and a concomitant cSN burst under control and CIH conditions. Taken together, these results suggest that PiCo and preBötC contribute to respiratory-sympathetic coupling, which is altered by CIH, and may contribute to the hypertension observed in patients with OSA.

Remodeling of Ventricular Catecholaminergic Axons Following Chronic Intermittent Hypoxia in Mice

Chronic intermittent hypoxia (CIH) is a widely employed model for sleep apnea. The imbalance of autonomic control may contribute to CIH-induced cardiovascular diseases. Previously, we demonstrated that CIH increases sympathetic innervation of the atria. In this study, we determined whether CIH remodels peripheral cardiac tyrosine hydroxylase (TH, a marker for sympathetic efferent axons) innervation in the whole ventricles. The two challenges here were: 1) labeling the entire TH-IR axon innervation in the full thickness (~500 μm) of whole flat-mount ventricles. 2) Quantifying the topographical distribution of TH-IR axons through the thick ventricular walls at the single axon/varicosity scale. In this study, C57BL/6J mice (male, 2 months old, n=7/group) were exposed to room air (RA, 21% O2) or CIH (alternating between 21% and 5.7% O2 every 6 minutes for 10 hours/day) for 8-10 weeks. We prepared flat-mounts of the whole ventricles and septum and processed them with immunohistochemical labeling for TH. Using the Zeiss M2 Imager, confocal microscope, Zeiss Arivis Vision 4D, and Neurolucida system, we traced and digitized the TH-IR axons. We found that: 1) In RA and CIH mice, several large TH-immunoreactive (TH-IR) bundles from the base area entered the ventricles and septum. These large bundles branched into numerous smaller bundles as they traveled downward toward the apex and finally developed into fine varicose axons and terminals in the epicardial and myocardial layers. 2) Using Vision 4D, we were able to accurately detect, trace, and digitize all TH-IR axons in the ventricle walls with high accuracy. 3) CIH increased overall TH-IR axon density and network complexity of the whole ventricles and septum. 3) Abnormal CIH TH-IR axon terminal structures were frequently seen. 4) We geometrically and precisely registered the tracing data into a scaffold map for presentation and comparison in 3D. Altogether, CIH increased sympathetic innervation of the heart which may contribute to the augmented sympathetic drive. Our success in mapping data in the 3D heart scaffold will establish a heart-brain connectome/atlas. Supported by NIH 1 U01 NS113867-01 NIH R15 1R15HL137143-01A1, 2R01HL137832-05 and Carl Zeiss. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Identification of novel biomarkers in obstructive sleep apnea via integrated bioinformatics analysis and experimental validation

Background Obstructive sleep apnea (OSA) is a complex and multi-gene inherited disease caused by both genetic and environmental factors. However, due to the high cost of diagnosis and complex operation, its clinical application is limited. This study aims to explore potential target genes associated with OSA and establish a corresponding diagnostic model. Methods This study used microarray datasets from the Gene Expression Omnibus (GEO) database to identify differentially expressed genes (DEGs) related to OSA and perform functional annotation and pathway analysis. The study employed multi-scale embedded gene co-expression network analysis (MEGENA) combined with least absolute shrinkage and selection operator (LASSO) regression analysis to select hub genes and construct a diagnostic model for OSA. In addition, the study conducted correlation analysis between hub genes and OSA-related genes, immunoinfiltration, gene set enrichment analysis (GSEA), miRNA network analysis, and identified potential transcription factors (TFs) and targeted drugs for hub genes. Finally, the study used chronic intermittent hypoxia (CIH) mouse model to simulate OSA hypoxic conditions and verify the expression of hub genes in CIH mice. Results In this study, a total of 401 upregulated genes and 275 downregulated genes were identified, and enrichment analysis revealed that these differentially expressed genes may be associated with pathways such as vasculature development, cellular response to cytokine stimulus, and negative regulation of cell population proliferation. Through MEGENA combined with LASSO regression, seven OSA hub genes were identified, including C12orf54, FOS, GPR1, OR9A4, MYO5B, RAB39B, and KLHL4. The diagnostic model constructed based on these genes showed strong stability. The expression levels of hub genes were significantly correlated with the expression levels of OSA-related genes and mainly acted on pathways such as the JAK/STAT signaling pathway and the cytosolic DNA-sensing pathway. Drug-target predictions for hub genes were made using the Connectivity Map (CMap) database and the Drug-Gene Interaction database (Dgidb), which identified targeted therapeutic drugs for the hub genes. In vivo experiments showed that the hub genes were all decreasing in the OSA mouse model. Conclusions This study identified novel biomarkers for OSA and established a reliable diagnostic model. The transcriptional changes identified may help to reveal the pathogenesis, mechanisms, and sequelae of OSA.

Pseudoginsenoside GQ mitigates chronic intermittent hypoxia-induced cognitive damage by modulating microglia polarization

Obstructive sleep apnea (OSA), a state of sleep disruption, is characterized by recurrent apnea, chronic intermittent hypoxia (CIH) and hypercapnia. Previous studies have showed that CIH-induced neuroinflammatory plays a crucial role in cognitive deficits. Pseudoginsenoside GQ (PGQ) is a new oxytetracycline-type saponin formed by the oxidation and cyclization of the 20(S) Rg3 side chain. Rg3 has been found to afford anti-inflammatory effects, while whether PGQ plays a role of anti-neuroinflammatory remains unclear. The purpose of this study was to investigate whether PGQ attenuates CIH-induced neuroinflammatory and cognitive impairment and the possible mechanism it involves. We found that PGQ significantly ameliorated CIH-induced spatial learning deficits, and inhibited microglial activation, pro-inflammatory cytokine release, and neuronal apoptosis in the hippocampus of CIH mice. In addition, PGQ pretreatment promoted microglial M1 to M2 phenotypic transition in IH-induced BV-2 microglial, as well as indirectly inhibited IH-induced neuronal injury via modulation of microglia polarization. Furthermore, we noted that activation of HMGB1/TLR4/NF-κB signaling pathway induced by IH was inhibited by PGQ. Molecular docking results revealed that PGQ could bind to the active sites of HMGB1 and TLR4. Taken together, this work supports that PGQ inhibits M1 microglial polarization via the HMGB1/TLR4/NF-κB signaling pathway, and indirectly exerts neuroprotective effects, suggesting that PGQ may be a potential therapeutic strategy for cognitive impairment accompanied OSA.

Blockage of calcium-sensing receptor improves chronic intermittent hypoxia-induced cognitive impairment by PERK-ATF4-CHOP pathway

Obstructive sleep apnea-hypopnea syndrome (OSAHS) is involved in cognitive impairment of children. Chronic intermittent hypoxia (CIH) is considered as the critical pathophysiological mechanism of OSAHS. Calcium sensitive receptor (CaSR) mediated apoptosis in many neurological disease models by endoplasmic reticulum stress (ERS)-related pathway. However, little is known about the role of CaSR in OSAHS-induced cognitive dysfunction. In this study, we explored the effect of CaSR on CIH-induced cognitive impairment and possible mechanisms on regulation of PERK-ATF4-CHOP pathway in vivo and in vitro. CIH exposed for 9 h in PC12 cells and resulted in the cell apoptosis, simulating OSAHS-induced neuronal injury. CIH upregulated the level of CaSR, p-PERK, ATF4 and CHOP, contributing to the cell apoptosis. Treated with CaSR inhibitor (NPS-2143) or p-PERK inhibitor (GSK2656157) before CIH exposure, CIH-induced PC12 cell apoptosis was alleviated via inhibition of CaSR by downregulating p-PERK, ATF4 and CHOP. In addition, we established CIH mice model. With CIH exposure for 4 weeks in mice, more spatial memory errors were observed during 8-arm radial maze test. CIH significantly increased apoptotic cells in hippocampus via upregulating cleaved Caspase-3 and downregulating ratio of Bcl-2 to Bax. Besides, treatment of CaSR inhibitor alleviated the hippocampal neuronal apoptosis following CIH with downregulated p-PERK, ATF4 and CHOP, suggesting that CaSR contributed to CIH-induced neuronal apoptosis in hippocampus via ERS pathway. Sum up, our results demonstrated that CaSR accelerated hippocampal apoptosis via PERK-ATF4-CHOP pathway, holding a critical function on CIH-mediated cognitive impairment. Conversely, inhibition of CaSR suppressed PERK-ATF4-CHOP pathway and alleviated cognitive impairment.

Pterostilbene mediates glial and immune responses to alleviate chronic intermittent hypoxia-induced oxidative stress in nerve cells.

Chronic intermittent hypoxia (CIH) induces oxidative stress in the brain, causing sleep disorders. Herein, we investigated the role of pterostilbene (Pte) in CIH-mediated oxidative stress in the brain tissue. A CIH mouse model was constructed by alternately reducing and increasing oxygen concentration in a sealed box containing the mouse; brain tissue and serum were then collected after intragastric administration of Pte. Neurological function was evaluated through field experiments. The trajectory of the CIH mice to the central region initially decreased and then increased after Pte intervention. Pte increased the number of neuronal Nissl bodies in the hippocampus of CIH mice, upregulated the protein levels of Bcl-2, occludin, and ZO-1 as well as the mRNA and protein levels of cAMP-response element binding protein (CREB) and p-BDNF, and reduced the number of neuronal apoptotic cells, Bax protein levels, IBA-1, and GFAP levels. Simultaneously, Pte reversed the decreased levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), and BDNF and increased levels of malondialdehyde (MDA) in the serum of CIH mice. Pte increased Th2 cells, Treg cells, IL-4, IL-10, and TGF-β1 levels and decreased Th1 cells, Th17 cells, IFN-γ, IL-6, and IL- 17A levels in activated BV2 cells and hippocampus in CIH mice. The protein levels of p-ERK1/2, TLR4, p-p38, p-p65, and Bax, apoptosis rate, MDA concentration, Bcl-2 protein level, cell viability, and SOD and GSH-PX concentrations decreased after the activation of BV2 cells. Pte inhibited gliocytes from activating T-cell immune imbalance through p-ERK signaling to alleviate oxidative stress injury in nerve cells.

The protective role of Nrf2 on cognitive impairment in chronic intermittent hypoxia and sleep fragmentation mice

ObjectiveObstructive Sleep Apnea Hypopnea Syndrome (OSAHS) is a sleep respiratory disease associated with cognitive impairment, The nuclear factor erythroid 2 related factor 2 (Nrf2) plays a neuroprotective role. This study was designed to investigate the mechanism of Nrf2 protecting neural cells from endoplasmic reticulum stress (ERS), induced by chronic intermittent hypoxia (CIH) and sleep fragmentation (SF) which caused cognitive impairment in mice. MethodsEstablishment of CIH and SF mice to simulate OSAHS mouse model. An eight-arm maze behavior test measured the cognitive function of mice, and Nissl staining and TUNEL staining were used to detect pathological changes in hippocampal neurons. The expression of ERS and Nrf2 and its downstream related mRNAs and proteins were detected by qRT-PCR and Western blotting. ResultsCIH and SF lead to cognitive impairment in mice, and Sulforaphane (SFN, Nrf2 agonist) plays a protective role, while Nrf2-KO aggravates the cognitive impairment. CIH and SF reduced the number of Nissl bodies in neurons and induced apoptosis. The mRNA levels of BiP, CHOP, Nrf2, GCLC and Prdx1 in CIH, SF and CIH + SF groups were increased (p = 0.001), whereas the mRNA levels of BiP and CHOP in the CIH + SF + SFN group were decreased (p = 0.02) while those of Nrf2 and Prdx1 were increased (p = 0.005). The CIH + SF + Nrf2-KO group, the mRNA levels of CHOP were increased (p = 0.001) while Nrf2, GCLC and Prdx1 were decreased (p = 0.001). The protein levels of CHOP and active Caspase-12 in CIH, SF, CIH + SF and CIH + SF + Nrf2-KO groups were increased (p = 0.03), while those of Prdx1 and Nrf2 were increased (p = 0.03) in the CIH + SF + SFN group, while decreased (p = 0.02) in the Nrf2-KO group. ConclusionsChronic intermittent hypoxia(CIH) and sleep fragmentation(SF) could aggravate the inflammatory response of nerve cells through endoplasmic reticulum stress, leading to apoptosis of nerve cells, and causing cognitive impairment in mice.Nrf2 alleviates cognitive impairment induced by chronic intermittent hypoxia and sleep fragmentation by modulating endoplasmic reticulum stress. Activation of Nrf2 protects cognitive impairment through the Nrf2-Prdx1 signaling pathway.

Huperzine A-Liposomes Efficiently Improve Neural Injury in the Hippocampus of Mice with Chronic Intermittent Hypoxia.

Chronic intermittent hypoxia (CIH) could cause neuronal damage, accelerating the progression of dementia. However, safe and effective therapeutic drugs and delivery are needed for successful CIH therapy. To investigate the neuroprotective effect of Huperzine A (HuA) packaged with nanoliposomes (HuA-LIP) on neuronal damage induced by CIH. The stability and release of HuA-LIP in vitro were identified. Mice were randomly divided into the Control, CIH, HuA-LIP, and HuA groups. The mice in the HuA and HuA-LIP groups received HuA (0.1 mg/kg, i.p.), and HuA-LIP was administered during CIH exposure for 21 days. HuA-LIP contains the equivalent content of HuA. We prepared a novel formulation of HuA-LIP that had good stability and controlled release. First, HuA-LIP significantly ameliorated cognitive dysfunction and neuronal damage in CIH mice. Second, HuA-LIP elevated T-SOD and GSH-Px abilities and decreased MDA content to resist oxidative stress damage induced by CIH. Furthermore, HuA-LIP reduced brain iron levels by downregulating TfR1, hepcidin, and FTL expression. In addition, HuA-LIP activated the PKAα/Erk/CREB/BDNF signaling pathway and elevated MAP2, PSD95, and synaptophysin to improve synaptic plasticity. Most importantly, compared with HuA, HuA-LIP showed a superior performance against neuronal damage induced by CIH. HuA-LIP has a good sustained-release effect and targeting ability and efficiently protects against neural injury caused by CIH.

Inhibition of Calcium-Sensing Receptor Alleviates Chronic Intermittent Hypoxia-Induced Cognitive Dysfunction via CaSR-PKC-ERK1/2 Pathway.

Obstructive sleep apnea-hypopnea syndrome (OSAHS) is typically characterized by chronic intermittent hypoxia (CIH), associated with cognitive dysfunction in children. Calcium-sensing receptor (CaSR) mediates the apoptosis of hippocampal neurons in various diseases. However, the effect of CaSR on OSAHS remains elusive. In the present study, we investigated the role of CaSR in CIH-induced memory dysfunction and underlying mechanisms on regulation of PKC-ERK1/2 signaling pathway in vivo and in vitro. CIH exposures for 4weeks in mice, modeling OSAHS, contributed to cognitive dysfunction. CIH accelerated apoptosis of hippocampal neurons and resulted in the synaptic plasticity deficit via downregulated synaptophysin (Syn) protein level. The mice were intraperitoneally injected with CaSR inhibitor (NPS2143) 30min before CIH exposure and the results demonstrated CaSR inhibitoralleviated the apoptosis and synaptic plasticity deficit in the hippocampus of CIH mice. We established intermittent hypoxia PC12 cell model and found that the activation of CaSR accelerated CIH-induced PC12 apoptosis and synaptic plasticity deficit by upregulated p-ERK1/2 and PKC. Overall, our findings revealed that CaSR held a critical function on CIH-induced cognitive dysfunction in mice by accelerating hippocampal neuronal apoptosis and reducing synaptic plasticity via augmenting CaSR-PKC-ERK1/2 pathway; otherwise, inhibition of CaSR alleviated CIH-induced cognitive dysfunction.

Chronic intermittent hypoxia impaired collagen synthesis in mouse genioglossus via ROS accumulation: A transcriptomic analysis

Obstructive sleep apnea (OSA) is a sleep-related breathing disorder characterized by intermittent and recurrent upper airway collapse during sleep that leads to chronic intermittent hypoxia (CIH). The genioglossus (GG) is the largest dilator muscle, which controls the upper airway and plays an important role in OSA pathology. Elucidating its genetic alterations may help identify potential targets for OSA. However, the genetic aspects of the GG in CIH mice remain unclear. Here, we have conducted an RNA sequencing (RNA-Seq) analysis to assess the differentially expressed genes (DEGs) in the GG between CIH mice and normoxia (NOR) mice. A total of 637 DEGs were identified to be dysregulated in CIH mice compared with control mice. Bioinformatics analysis showed that the DEGs were related to various physiological processes, such as the endogenous stimulus responses, cellular component organization and metabolic processes. Extracellular matrix (ECM)–receptor interaction was the top KEGG pathway in the environmental information processing category with high significance and large fold changes. From the gene weight distributions of collagen (Col)-related biological processes (BPs), we found several significant DEGs, such as Col1a1, Col1a2, Mmp2, Col3a1, Col5a1, Fmod, and Col5a2. A PPI network showed that Col1a1 was linked to ECM–receptor interactions, responses to reactive oxygen species (ROS) and Col-related BPs. It was verified in vivo and in vitro that hypoxia can induce excess ROS and reduce Col expression levels. Moreover, we found NAC can effectively scavenge ROS and restore collagen synthesis. These findings contribute to a better understanding of the mechanisms linking OSA and upper airway muscle injury and may help identify potential therapeutic targets.

Effects of Bu Zhong Yi Qi decoction on CIH-induced interstitial lung fibrosis in mice

To investigate the effects of chronic intermittent hypoxia (CIH) on the expression of transforming growth factor-β (TGF-β), P-samd3, serum laminin (LN) and hyaluronidase (HA) in mouse lung tissues and the protective effects of Bu Zhong Yi Qi decoction on lung interstitial deposition damage in CIH mice. Fifty SPF-grade C57BL mice were randomly divided into five groups (n=10): blank control group, CIH model group, and CIH+ low, medium and high doses of Bu Zhong Yi Qi decoction group. Mice were placed under normoxia or CIH conditions, respectively. The Chinese medicine group was given the corresponding doses of drugs. HE staining was performed to assess pathological changes and Masson staining was performed to assess collagen deposition. Western blot was performed to detect the expressions of channel proteins such as TGF-β1, P-smad3 and down stream α-SMA and Collagen I. ELISA was performed to detect the serum levels of TGF-β1, LN and HA. HE staining showed alveolar collapse, septal thickening and epithelial cell necrosis in CIH mice, Masson showed massive collagen fiber proliferation and deposition in lung interstitium, while the above changes in lung tissues were significantly improved in the CIH + Bu Zhong Yi Qi decoction groups compared with the CIH group. TGF-β1, P-smad3 and Collagen I, Collagen Ⅲ, and α-SMA expression levels were increased compared with the blank control group (P＜0.05), and the expressions of TGF-β1 and LN in serum were upregulated (P＜0.05). The expressions of TGF-β1, P-smad3, Collagen I protein and SMA-α in the lung tissues of the CIH+ Bu Zhong Yi Qi decoction groups were downregulated significantly compared with those of the CIH group (P＜0.05), and the improvement of multiple indexes in the CIH+high-dose CIH intervention group was better than those of the low-dose group (P＜0.05). Bu Zhong Yi Qi decoction can inhibit alveolar structural changes and excessive collagen deposition in the interstitium of CIH mice, and then improve lung function in CIH mice. The mechanism may be related to the down-regulation of protein expression related to TGF-β/smads signaling pathway by Bu Zhong Yi Qi decoction.

Effects of NLRP3/Caspase-1 pathway on Banxia Houpu decoction in the intervention of chronic intermittent hypoxia in mice with renal inflammatory injury

Objective: To investigate the effects of Banxia Houpo decoction on the renal NLRP3/Caspase-1/IL-1β signaling pathway in chronic intermittent hypoxia mice. Methods: C57BL/6 mice were randomly divided into 3 groups, normal control group (Control), chronic intermittent hypoxia group (CIH), and Banxia Houpo decoction treatment group (BHD), with 10 mice in each group. Mice in the CIH group and BHD group were placed in a hypoxic chamber. The oxygen volume fraction in the cabin was decreased from 21% to 9% in 90 s, and then oxygen was filled in 90 s to gradually increase the oxygen volume fraction in the cabin to 21%, while the mice in the control group were placed in the cabin and filled with normal air, processing 8 hours per day for 21 days. The mice in BHD group were treated with Banxia Houpu decoction by gavage before entering the cabin every day, and the control group and CIH group were given an equal volume of normal saline. After modeling, the changes of renal function indexes in each group were detected; HE and Masson staining were used to observe the pathological conditions of the kidney; Western blot and immunohistochemical staining were used to detect the protein expression levels of the nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3), aspartate-specific cysteine protein 1(Caspase-1) and interleukine-1beta(IL-1β). Results: Compared with control group, the contents of serum renal functional indexes UA, BUN and SCr in CIH group were increased significantly (P<0.01), and after BHD treatment, they all were decreased significantly compared with CIH group (P<0.01). Compared with control group, the results of HE staining showed that in the CIH group, glomerular endothelial cells were degenerated and necrotic, and vacuoles of different sizes appeared in renal tubular epithelial cells, and a small amount of renal tubular epithelial cells fell off and died. The pathological condition of the BHD group was improved compared with CIH group, the glomerular morphology gradually returned to normal, and a small amount of renal tubular epithelial cells fell off and died. Compared with control group, Masson staining results showed that there was obvious fibrosis around the glomeruli in the CIH group, the fibrosis was significantly reduced in the BHD group. The expression levels of NLRP3, Caspase-1, IL-1β and IL-18 were increased significantly compared with control group (P<0.05 or P<0.01), and immunohistochemical staining showed that NLRP3 was mainly expressed in renal tubular epithelial cells and interstitial macrophages, caspase-1 and IL-1β were mainly found in the cytoplasm of renal tubular epithelial cells. After BHD treatment, the expression levels of each protein were decreased compared with CIH group (P<0.05). Conclusion: Banxia Houpu decoction can reduce the kidney damage by inhibiting the expression of related molecules in the NLRP3/Casapse-1/IL-1β signaling pathway.

Danggui Buxue Decoction improves heart function of chronic intermittent hypoxia mice by promoting mitochondrial autophagy and inhibiting cardiomyocyte apoptosis

The study established a chronic intermittent hypoxia(CIH) model in mice to investigate the effects of Danggui Buxue Decoction(DBD) on mitochondrial autophagy and cardiomyocyte apoptosis and explore its protective effect and mechanism on cardiac function of CIH mice. Forty C57 BL/6 N male mice were randomly divided into the control(CON) group, CIH group, CIH+DBD group, and DBD group, with 10 mice in each group. CIH was induced by filling the hypoxic chamber with N_2(90 s) to reduce the O_2 concentration to 5% and then filling the hypoxic chamber with O_2(90 s) to restore O_2 concentration to 21%, 3 min per cycle, and the CIH treatment continued for 35 d, 8 h per day. Mice in the CIH+DBD and DBD groups were treated with intragastric administration of DBD every day, while those in the CON and CIH groups with the same volume of normal saline. The cardiac function of mice was measured by echocardiography. The pathological changes in myocardium were observed after HE staining, followed by the observation of cardiomyocyte apoptosis by Tunel staining. The expression of apoptosis-related proteins pro-caspase-3, caspase-3, Bcl-2, and Bax and autophagy-related proteins LC3Ⅱ, LC3Ⅰ, P62, parkin, and cytochrome C(Cytc) was detected by Western blot. The mitochondrial membrane potential was observed using JC-1 fluorescent probe. Compared with the CON group, the CIH group exhibited remar-kably lowered left ventricular ejection fraction(LVEF) and left ventricular fractional shortening(LVFS), elevated left ventricular end-systolic volume(LVESV) and end-diastolic volume(LVEDV), disordered myocardial fiber arrangement, increased number of TUNEL-positive cells, decreased pro-caspase-3/caspase-3, Bcl-2/Bax, and LC3Ⅱ/LC3Ⅰ ratios, parkin, mitochondrial Cytc expression, and mitochondrial membrane potential, and up-regulated P62 and Cytc expression. Compared with the CIH group, DBD increased LVEF, LVFS, pro-caspase-3/caspase-3, Bcl-2/Bax, and LC3Ⅱ/LC3Ⅰ ratios, and parkin expression, as well as mitochond-rial Cytc expression, and mitochondrial membrane potential, decreased LVESV, LVEDV, and the number of Tunel-positive cells, and improved the myocardial fiber arrangement. DBD has a protective effect on the heart function of CIH mice. It improves the heart function possibly by promoting mitochondrial autophagy to ameliorate mitochondrial function and inhibiting the cardiomyocyte apoptosis.

Optogenetic stimulation of PICo neurons and the respiratory‐sympathetic coupling in in vivoCIH and control mice

The resting respiratory activity is generated by synchronized activity of a complex located within the brainstem. This contains a primary oscillator essential for the respiratory rhythmicity and is composed of pacemaker inspiratory neurons. However, recent studies suggest the postinspiratory complex (PiCo) is a novel respiratory oscillator which is critical for the postinspiratory activity in vivo and contains cholinergic and glutamatergic neurons. In addition to the generation of the respiration, studies suggest that the respiratory neurons also interact with the presympathetic neurons involved with the generation of sympathetic activity, contributing to the excitation of sympathetic activity in chronic intermittent hypoxia (CIH) conditions and promoting changes inthe respiratory‐sympathetic coupling. To investigate the contribution of PiCo in the respiratory‐sympathetic coupling, we used an optogenetic in vivo mouse preparation previously exposed to CIH for 21 days (5% O2 – 80 bouts/day). In the end of the protocol, the ventral surface of the brainstem in spontaneously breathing, anaesthetized (Urethane 1.5mg/kg) ChATCre/Ai32 adult and Vglut2cre/Ai32 mice was exposed, and an optical fiber positioned bilaterally over PiCo, while recording the diaphragm muscle (DIA), hypoglossal (HN), cervical vagus (cVN) and cervical sympathetic nerves (cSN). Our data showed that CIH protocol increased the respiratory frequency, post‐I vagus activity and sympathetic activity in the baseline conditions. Random light stimulation (10s) of PiCo ChAT neurons (~ 9mV, 447nm) in significantly increased the cSN discharge during postinspiration in control group. Although the sympathetic activity was increased during all respiratory phases in CIH mice, the stimulation of cholinergic neurons at PiCo did not change the correlation between post‐I and E2 phase. The stimulation of Vglut2 neurons at PiCo area evoked post‐inspiratory discharge of cSN in both groups. Our data demonstrates that during resting conditions, mice exposed to CIH for 3 weeks increased the baseline respiratory frequency and sympathetic activity in vivo and the optogenetic stimulation of PiCo neurons blunts the phasic respiratory –sympathetic coupling in CIH mice, increasing the post‐inspiratory discharge of the sympathetic nerve.

Sulforaphane Attenuates Chronic Intermittent Hypoxia-Induced Brain Damage in Mice via Augmenting Nrf2 Nuclear Translocation and Autophagy.

Obstructive sleep apnea–hypopnea syndrome (OSAHS), typically characterized by chronic intermittent hypoxia (CIH), is associated with neurocognitive dysfunction in children. Sulforaphane (SFN), an activator of nuclear factor E2-related factor 2 (Nrf2), has been demonstrated to protect against oxidative stress in various diseases. However, the effect of SFN on OSAHS remains elusive. In this research, we investigated the neuroprotective role of SFN in CIH-induced cognitive dysfunction and underlying mechanisms of regulation of Nrf2 signaling pathway and autophagy. CIH exposures for 4 weeks in mice, modeling OSAHS, contributed to neurocognitive dysfunction, manifested as increased working memory errors (WMEs), reference memory errors (RMEs) and total memory errors (TEs) in the 8-arm radial maze test. The mice were intraperitoneally injected with SFN (0.5 mg/kg) 30 min before CIH exposure everyday. SFN treatment ameliorated neurocognitive dysfunction in CIH mice, which demonstrates less RME, WME, and TE. Also, SFN effectively alleviated apoptosis of hippocampal neurons following CIH by decreased TUNEL-positive cells, downregulated cleaved PARP, cleaved caspase 3, and upregulated Bcl-2. SFN protects hippocampal tissue from CIH-induced oxidative stress as evidenced by elevated superoxide dismutase (SOD) activities and reduced malondialdehyde (MDA). In addition, we found that SFN enhanced Nrf2 nuclear translocation to hold an antioxidative function on CIH-induced neuronal apoptosis in hippocampus. Meanwhile, SFN promoted autophagy activation, as shown by increased Beclin1, ATG5, and LC3II/LC3I. Overall, our findings indicated that SFN reduced the apoptosis of hippocampal neurons through antioxidant effect of Nrf2 and autophagy in CIH-induced brain damage, which highlights the potential of SFN as a novel therapy for OSAHS-related neurocognitive dysfunction.

Abnormal lipid droplets accumulation induced cognitive deficits in obstructive sleep apnea syndrome mice via JNK/SREBP/ACC pathway but not through PDP1/PDC pathway

The mechanisms of chronic intermittent hypoxia (CIH)-induced cognitive deficits remain unclear. Here, our study found that about 3 months CIH treatment induced lipid droplets (LDs) accumulation in hippocampal nerve and glia cells of C57BL/6 mice, and caused severe neuro damage including neuron lesions, neuroblast (NB) apoptosis and abnormal glial activation. Studies have shown that the neuronal metabolism disorders might contribute to the CIH induced-hippocampal impairment. Mechanistically, the results showed that pyruvate dehydrogenase complex E1ɑ subunit (PDHA1) and the pyruvate dehydrogenase complex (PDC) activator pyruvate dehydrogenase phosphatase 1 (PDP1) did not noticeable change after intermittent hypoxia. Consistent with those results, the level of Acetyl-CoA in hippocampus did not significantly change after CIH exposure. Interestingly, we found that CIH produced large quantities of ROS, which activated the JNK/SREBP/ACC pathway in nerve and glia cells. ACC catalyzed the carboxylation of Acetyl-CoA to malonyl-CoA and then more lipid acids were synthesized, which finally caused aberrant LDs accumulation. Therefore, the JNK/SREBP/ACC pathway played a crucial role in the cognitive deficits caused by LDs accumulation after CIH exposure. Additionally, LDs were peroxidized by the high level of ROS under CIH conditions. Together, lipid metabolic disorders contributed to nerve and glia cells damage, which ultimately caused behavioral dysfunction. An active component of Salvia miltiorrhiza, SMND-309, dramatically alleviated these injuries and improved cognitive deficits of CIH mice.

Orexin A improves the cognitive impairment induced by chronic intermittent hypoxia in mice

The orexin neuron in lateral hypothalamus (LH) was involved in the regulation of sleep-wake cycle. However, the effect of orexin A (OXA) on cognitive impairment resulting from diverse diseases remains controversial. In this study, we investigated the effect of OXA on cognitive impairment induced by chronic intermittent hypoxia (CIH) in mice. Adult (10 weeks old) male C57BL/6 mice were randomly divided into the following four groups: normoxia control (NC)+normal saline (NS), NC + OXA, CIH + NS and CIH + OXA group. Following the CIH mice models establishment, OXA was injected into the right lateral ventricles of mice by a micro-injection system. Water maze test was used to assess spatial memory abilities of the mice. The expression of OXA and c-Fos in LH were analyzed by immunofluorescence staining. Apoptotic cell death and oxidative stress in hippocampus were evaluated using multiple methods including TUNEL, western blot and biochemical analysis. Behavioral tests revealed that CIH significantly increased the escape latency and time of arriving platform, of which were markedly decreased by OXA treatment. Similarly, the CIH + NS group was worse than NC + NS group in terms of the number of platform crossing and time in the target quadrant, of which were also significantly improved by OXA treatment. The number of OXA + neuron in LH was decreased, but the percentage of c-Fos+/OXA + neuron in LH was remarkably increased by CIH. Furthermore, we found that micro-injection of OXA attenuated CIH-induced apoptotic cell death and oxidative stress in the hippocampus. Our results suggested that OXA might improve cognitive impairment induced by CIH through inhibiting hippocampal apoptosis and oxidative stress.

RNA Bulk Sequencing Analysis and Differential Gene Expression of Multiple Myeloma Susceptibility Strains: KaLwRij and CIH

Multiple myeloma is a terminal cancer of bone marrow plasma cells whose pathophysiological mechanisms remain poorly understood. Similar genetic mutations are shared between myeloma and its requisite pre-malignant phase MGUS, suggesting that other factors are required for disease progression. There are two mouse strains that are susceptible to the lethal engraftment of malignant plasma cells. KaLwRij is a tumor-receptive, spontaneous mutant of the control mouse C57BL/6J and has a high rate of benign idiopathic paraproteinemia, analogous to human MGUS. Secondarily, we recently demonstrated that chronic intermittent hypoxia (CIH), a feature of sleep apnea, allows for the engraftment of malignant plasma cells in disease resistant C57BL6/J mice. To identify potential susceptibility pathways in the two models, we used bulk RNA-sequencing to quantify differential gene expression in KaLwRij vs. C57BLJ/6 and CIH-exposed C57BL6/J vs. normoxic C57BL6/J. Genes were considered significant if they had an adjusted p-value of less than 0.05 and a log2-fold change of less than -1 or greater than 1. We identified 400 significant genes for KaLwRij and 69 significant genes for CIH. Significant genes were assessed for overlap, resulting in 31 duplicates. The KaLwRij and CIH mice share 31 genes that are upregulated in comparison to normoxic C57BL6/J control mice. These results provide potential mechanistic targets to prevent the terminal engraftment of malignant plasma cells in the bone marrow. In future studies, we will use single-cell RNA sequencing data to determine which cell populations reflect the upregulation of the 31 shared genes to understand how the bone marrow microenvironment is altered in the KaLwRij and CIH mice to enhance MM proliferation.

Optogenetic stimulation of Dbx1 neurons enhances the respiratory‐sympathetic coupling in in vivo CIH mice

Under resting conditions, breathing is generated by synchronized activity of a complex respiratory network located in the ventral respiratory column in the brainstem. The preBötzinger Complex (preBötC), critical for generating the inspiratory phase of breathing, contains glutamatergic neurons derived from progenitor cells that are characterized by the transcription factor “developing brain homeobox (Dbx1)”. These neurons are thought to be important for inspiratory rhythmogenesis. Excitatory and inhibitory synaptic inputs from the respiratory networks modulate the sympathetic outflow which is generated by presympathetic neurons from the rostroventrolateral medulla (RVLM). Changes in the respiratory-sympathetic coupling contribute to imbalances as demonstrated in chronic intermittent hypoxia (CIH). To investigate the contribution of Dbx1 neurons in the respiratory-sympathetic coupling, we used an optogenetic in vivo mouse preparation previously exposed to CIH for 10 -12 days (5% O2 – 80 bouts/day). The ventral surface of the brainstem in spontaneously breathing, anaesthetized (Urethane 1.5mg/kg) Dbx1-ERT2Cre/Ai32 adult mice was exposed and an optical fiber positioned bilaterally over the preBötC, while recording the hypoglossal (HN), cervical vagus (cVN) and cervical sympathetic nerves (cSN). Our preliminary data indicate that CIH exposure increased the respiratory frequency and mean sympathetic activity in the baseline conditions. Random light stimulation (200ms) of preBötC Dbx1 neurons (~ 9mV, 447nm) significantly promoted inspiratory activity, increasing the respiratory frequency and amplitude of HN and cSN in relation to baseline in both groups. During optogenetic stimulation of Dbx1 neurons, the respiratory-sympathetic coupling was enhanced in both groups, without significant difference in variability of cSN amplitude. This is possibly due to the increase in mean cSN activity under baseline conditions in CIH mice. Our data demonstrates that stimulation of preBötC Dbx1 neurons aids in regulation of autonomic control modulating the cSN activity, leading to increased sympathetic drive and hypertension in the CIH.

Medical Image Analysis of Telmisartan-Induced Protection in Hippocampal CA1 After Chronic Intermittent Hypoxia

Objective: In this paper, we evaluate medical image analysis in hippocampus, immunohistochemistry analyzed and oxidative stress-mediated injury induced by chronic intermittent hypoxia on the mouse hippocampal CA1 area and the protective effects of telmisartan (TEL) on oxidative stress-mediated injury. Methodology: C57B6J mice have been randomly categorized as chronic intermittent hypoxia (CIH) group, CIH + telmisartan (CIH + TEL) group, the room air control group and blank control group, and examined during a period of 12 weeks. Brain images were obtained by Computed tomography scan, spatial learning and memory abilities were tested, and p47 NADPH oxidase subunit (p47PHOX) and 8-hydroxy-guanosine (8-OHG) were measured in the hippocampal CA1 area by immunohistochemistry and analyzed by Image-Pro Plus 6.0. Results: (1) After 12 weeks, there were no abnormalities in mice brain by CT scan. (2) Spatial learning ability measured by escape latency from the Morris water maze in the hypoxia group decreased, while intervention with telmisartan improved the hypoxiainduced spatial learning deficit (F = 3.04, P = 0.045). There exist no differences in spatial memory among any groups. (3) p47PHOX immunostaining significantly increased in hippocampal CA1 area after CIH, which was reduced by TEL intervention. (4) CIH significantly increased 8-OHG immunostaining in the hippocampal CA1 area, while intervention with TEL significantly reduced 8-OHG immunostaining. Conclusions: Telmisartan significantly reduced expression of NADPH oxidase p47PHOX and 8-OHG in CIH mice, and improved hippocampusdependent learning, suggesting that telmisartan can reduce CIH-induced neuronal oxidative stress-mediated injury.