不同频率间歇性低氧刺激对HT22细胞活性和Hif-1α及p-NF-κB表达的影响

间歇性低氧(intermittent hypoxia,IH)可通过氧化应激和炎症反应诱导认知障碍,而不同频率的IH刺激可对特定类型的细胞引起不同结果.因此本研究旨在比较3种频率IH刺激对海马神经元HT22细胞活性的影响,探讨IH刺激损伤神经元的相关分子机制.体外培养HT22细胞,分为对照组和3个不同频率IH刺激组(舱内...     

Poly (ADP-ribose) polymerase plays an important role in intermittent hypoxia-induced cell death in rat cerebellar granule cells

Background

Episodic cessation of airflow during sleep in patients with sleep apnea syndrome results in intermittent hypoxia (IH). Our aim was to investigate the effects of IH on cerebellar granule cells and to identify the mechanism of IH-induced cell death.

Methods

Cerebellar granule cells were freshly prepared from neonatal Sprague-Dawley rats. IH was created by culturing the cerebellar granule cells in the incubators with oscillating O₂ concentration at 20% and 5% every 30 min for 1-4 days. The results of this study are based on image analysis using a confocal microscope and associated software. Cellular oxidative stress increased with increase in IH. In addition, the occurrence of cell death (apoptosis and necrosis) increased as the duration of IH increased, but decreased in the presence of an iron chelator (phenanthroline) or poly (ADP-ribose) polymerase (PARP) inhibitors [3-aminobenzamide (3-AB) and DPQ]. The fluorescence of caspase-3 remained the same regardless of the duration of IH, and Western blots did not detect activation of caspase-3. However, IH increased the ratio of apoptosis-inducing factor (AIF) translocation to the nucleus, while PARP inhibitors (3-AB) reduced this ratio.

Results

According to our findings, IH increased oxidative stress and subsequently leading to cell death. This effect was at least partially mediated by PARP activation, resulting in ATP depletion, calpain activation leading to AIF translocation to the nucleus.

Conclusions

We suggest that IH induces cell death in rat primary cerebellar granule cells by stimulating oxidative stress PARP-mediated calpain and AIF activation.     

Intermittent hypoxia upregulates hepatic heme oxygenase-1 and ferritin-1, thereby limiting hepatic pathogenesis in rats fed a high-fat diet

Non-alcoholic fatty liver disease (NAFLD) is prevalent in patients with sleep apnea syndrome (SAS). Intermittent hypoxia (IH) and a high-fat diet (HFD) reproduce SAS and NAFLD, respectively, in rodents. In this study, rats were fed either an HFD or a standard diet (SD) for 2 weeks, and breathed either IH air or normoxic air for 4 days (early phase) or 6 weeks (late phase), with the same diets maintained during the exposure. HFD increased hepatic lipid accumulation, as detected by oil-red staining and triglyceride content. However, IH exposure reversed the hepatic steatosis at the late phase in these HFD-rats. IH exposure also increased hepatic expression of HO-1 and iron-binding protein ferritin-1 at the late phase, in association with increase in serum iron, bilirubin, and hepatic levels of lipid peroxides, such as 4-hydroxy-2-nonenal (HNE). IH exposure increased serum levels of hemoglobin (Hb) at the early phase and immunofluorescence of Hb and HO-1 in CD68-positive Kupffer cells (KCs) at the late phase. These findings support that IH induces erythrocytosis, erythro-phagocytosis, and generation of Hb in the KCs. The Hb promotes HO-1 expression in KCs, thereby produces iron, bilirubin, and carbon monoxide (CO). The iron would be either sequestrated by ferritin-1, transferred to the bone marrow for erythropoiesis, or would produce hydroxyradicals and HNE in the liver of rats fed an HFD. HNE might also contribute to the upregulation of HO-1, transferrin-1, and IκB, thereby limiting hepatic steatosis and inflammation via inhibition of nuclear factor κB (NFκB) activation.     

Intermittent hypoxia has organ-specific effects on oxidative stress

Obstructive sleep apnea is characterized by upper airway collapse, leading to intermittent hypoxia (IH). It has been postulated that IH-induced oxidative stress may contribute to several chronic diseases associated with obstructive sleep apnea. We hypothesize that IH induces systemic oxidative stress by upregulating NADPH oxidase, a superoxide-generating enzyme. NADPH oxidase is regulated by a cytosolic p47(phox) subunit, which becomes phosphorylated during enzyme activation. Male C57BL/6J mice were exposed to IH with an inspired O(2) fraction nadir of 5% 60 times/h during the 12-h light phase (9 AM-9 PM) for 1 or 4 wk. In the aorta and heart, IH did not affect lipid peroxidation [malondialdehyde (MDA) level], nitrotyrosine level, or p47(phox) expression and phosphorylation. In contrast, in the liver, exposure to IH for 1 wk resulted in a trend to an increase in MDA levels, whereas IH for 4 wk resulted in a 38% increase in MDA levels accompanied by upregulation of p47(phox) expression and phosphorylation. Administration of an NADPH oxidase inhibitor, apocynin, during IH exposure attenuated IH-induced increases in hepatic MDA. In p47(phox)-deficient mice, MDA levels were higher at baseline and, unexpectedly, decreased during IH. In conclusion, oxidative stress levels and pathways under IH conditions are organ and duration specific.     

Intermittent hypoxia treatments cause cellular priming in human microglia

Obstructive sleep apnoea syndrome (OSAS) is a sleep-disordered breathing characterized by nocturnal collapses of the upper airway resulting in cycles of blood oxygen partial pressure oscillations, which lead to tissue and cell damage due to intermittent hypoxia (IH) episodes. Since OSAS-derived IH may lead to cognitive impairment through not fully cleared mechanisms, herein we developed a new in vitro model mimicking IH conditions to shed light on its molecular effects on microglial cells, with particular attention to the inflammatory response. The in vitro model was set-up and validated by measuring the hypoxic state, HIF-1α levels, oxidative stress by ROS production and mitochondrial activity by MTS assay. Then, the mRNA and protein levels of certain inflammatory markers (NF-κB and interleukin 6 (IL-6)) after different IH treatment protocols were investigated. The IH treatments followed by a normoxic period were not able to produce a high inflammatory state in human microglial cells. Nevertheless, microglia appeared to be in a state characterized by increased expression of NF-κB and markers related to a primed phenotype. The microglia exposed to IH cycles and stimulated with exogenous IL-1β resulted in an exaggerated inflammatory response with increased NF-κB and IL-6 expression, suggesting a role for primed microglia in OSAS-driven neuroinflammation.     

Human adipocytes are highly sensitive to intermittent hypoxia induced NF-kappaB activity and subsequent inflammatory gene expression

Introduction

Intermittent hypoxia (IH)-induced activation of pro-inflammatory pathways is a major contributing factor to the cardiovascular pathophysiology associated with obstructive sleep apnea (OSA). Obesity is commonly associated with OSA although it remains unknown whether adipose tissue is a major source of inflammatory mediators in response to IH. The aim of this study was to test the hypothesis that IH leads to augmented inflammatory responses in human adipocytes when compared to cells of non-adipocyte lineages.

Methods and results

Human primary subcutaneous and visceral adipocytes, human primary microvascular pulmonary endothelial cells (HUMEC-L) and human primary small airway epithelial cells (SAEC) were exposed to 0, 6 or 12 cycles of IH or stimulated with tumor necrosis factor (TNF)-α. IH led to a robust increase in NF-κB DNA-binding activity in adipocytes compared with normoxic controls regardless of whether the source of adipocytes was visceral or subcutaneous. Notably, the NF-κB response of adipocytes to both IH and TNF-α was significantly greater than that in HUMEC-L and SAEC. Western blotting confirmed enhanced nuclear translocation of p65 in adipocytes in response to IH, accompanied by phosphorylation of I-κB. Parallel to p65 activation, we observed a significant increase in secretion of the adipokines interleukin (IL)-8, IL-6 and TNF-α with IH in adipocytes accompanied by significant upregulation of mRNA expression. PCR-array suggested profound influence of IH on pro-inflammatory gene expression in adipocytes.

Conclusion

Human adipocytes demonstrate strong sensitivity to inflammatory gene expression in response to acute IH and hence, adipose tissue may be a key source of inflammatory mediators in OSA.     

Intermittent hypoxia and activation of inflammatory molecular pathways in OSAS

Obstructive sleep apnoea syndrome (OSAS) represents a highly prevalent disease and is recognized as a risk factor for the development of various cardiovascular disorders. The pathogenesis of cardiovascular complications in OSAS is not completely understood, but the unique form of hypoxia with repetitive short cycles of desaturation followed by rapid reoxygenation termed intermittent hypoxia (IH) is likely to play a significant role. There is increasing evidence that IH leads to a preferential activation of inflammatory over adaptive pathways. This promotes activation of various inflammatory cells, particularly lymphocytes and monocytes, with the downstream consequence of expression of pro-inflammatory cytokines, chemokines and adhesion molecules that may contribute to endothelial dysfunction. This review provides a critical analysis of the current evidence of inflammatory mechanisms initiated by IH that may contribute to the cardiovascular pathogenesis in OSAS.     

Differential expression of ABH histo-blood group antigens and LAMPs in infantile hemangioma

Summary Although infantile hemangioma (IH) are the most common tumors of infancy, the mechanism of their proliferation and involution remains vague. Proliferation, differentiation and death of endothelial cells are the basic processes involved in their pathobiology. Here we hypothesize that the glycoconjugates ABH histo-blood group antigens (HBGA) and lysosome-associated membrane proteins (LAMPs) might be implied in both the differentiation and death of endothelial cells during vascular remodeling in IH. Proliferating and involuting IH were examined immunohistochemically for HGBA and LAMP expression together with vWF and CD31. Proliferative and apoptotic indices were determined. LAMPs were found in immature endothelium of proliferating IH. In involution an increased number of immunopositive cells stained with higher intensity was detected. The enhanced expression might be associated with augmented autophagy required for tissue remodeling during tumor involution. HBGA presented an opposite pattern of expression – they stained intensely the endothelium of mature capillaries, while the immature ones were positive for vWF. The presence of HBGA in endothelial cells of IH may be related to the differentiation process only, as well as to endothelial adhesion and angiogenesis. Novel evidence for differential expression of HBGA and LAMPs in proliferative and involutive phases of IH is presented.     

Roles for rice membrane dynamics and plasmodesmata during biotrophic invasion by the blast fungus

Rice blast disease is caused by the hemibiotrophic fungus Magnaporthe oryzae, which invades living plant cells using intracellular invasive hyphae (IH) that grow from one cell to the next. The cellular and molecular processes by which this occurs are not understood. We applied live-cell imaging to characterize the spatial and temporal development of IH and plant responses inside successively invaded rice (Oryza sativa) cells. Loading experiments with the endocytotic tracker FM4-64 showed dynamic plant membranes around IH. IH were sealed in a plant membrane, termed the extra-invasive hyphal membrane (EIHM), which showed multiple connections to peripheral rice cell membranes. The IH switched between pseudohyphal and filamentous growth. Successive cell invasions were biotrophic, although each invaded cell appeared to have lost viability when the fungus moved into adjacent cells. EIHM formed distinct membrane caps at the tips of IH that initially grew in neighboring cells. Time-lapse imaging showed IH scanning plant cell walls before crossing, and transmission electron microscopy showed IH preferentially contacting or crossing cell walls at pit fields. This and additional evidence strongly suggest that IH co-opt plasmodesmata for cell-to-cell movement. Analysis of biotrophic blast invasion will significantly contribute to our understanding of normal plant processes and allow the characterization of secreted fungal effectors that affect these processes.     

Chronic intermittent hypoxia induces lung growth in adult mice

Obstructive sleep apnea (OSA) increases cardiovascular morbidity and mortality, which have been attributed to intermittent hypoxia (IH). The effects of IH on lung structure and function are unknown. We used a mouse model of chronic IH, which mimics the O(2) profile in patients with OSA. We exposed adult C57BL/6J mice to 3 mo of IH with a fraction of inspired oxygen (F(I)(O(2))) nadir of 5% 60 times/h during the 12-h light phase. Control mice were exposed to room air. Lung volumes were measured by quasistatic pressure-volume (PV) curves under anesthesia and by water displacement postmortem. Lungs were processed for morphometry, and the mean airspace chord length (Lm) and alveolar surface area were determined. Lung tissue was stained for markers of proliferation (proliferating cell nuclear antigen), apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labeling), and type II alveolar epithelial cells (surfactant protein C). Gene microarrays were performed, and results were validated by real-time PCR. IH increased lung volumes by both PV curves (air vs. IH, 1.16 vs. 1.44 ml, P < 0.0001) and water displacement (P < 0.01) without changes in Lm, suggesting that IH increased the alveolar surface area. IH induced a 60% increase in cellular proliferation, but the number of proliferating type II alveolocytes tripled. There was no increase in apoptosis. IH upregulated pathways of cellular movement and cellular growth and development, including key developmental genes vascular endothelial growth factor A and platelet-derived growth factor B. We conclude that IH increases alveolar surface area by stimulating lung growth in adult mice.     

ALKBH5-m6A-FOXM1 signaling axis promotes proliferation and invasion of lung adenocarcinoma cells under intermittent hypoxia

Obstructive sleep apnea (OSA) is closely associated with cancer progression and cancer-related mortality. N6-methyladenosine (m⁶A) is involved in the process of intermittent hypoxia (IH) promoting tumor progression. However, it is unclear how m⁶A regulates the development of lung adenocarcinoma under IH. In this study, we found that ALKBH5 was elevated in lung adenocarcinoma cells and subcutaneous tumors in mice under IH, which was associated with decreased m⁶A levels in these cells and tissues. Next, we knocked out ALKBH5 in a human lung adenocarcinoma cell line under IH, and we found that the proliferation and invasion of these cells were significantly inhibited. Mechanistic analysis showed that under IH, knockout of ALKBH5 in lung adenocarcinoma cells upregulated the level of m⁶A in Forkhead box M1 (FOXM1) mRNA and decreased the translation efficiency of FOXM1 mRNA, resulting in downregulation of the FOXM1 protein. The FOXM1 protein is elevated in lung adenocarcinoma cells and subcutaneous tumor tissues of mice under IH. By knocking out FOXM1 in lung adenocarcinoma cells under IH, proliferation and invasion of these cells were inhibited, and overexpression of FOXM1 partially restored the inhibition of growth and invasion of lung adenocarcinoma cells due to ALKBH5 knockout. Collectively, our findings demonstrate that the m⁶A demethylase ALKBH5 affects the proliferation and invasion of lung adenocarcinoma cells under IH by downregulating m⁶A modification on FOXM1 mRNA and by promoting FOXM1 expression.     

Foetal hepatocyte transplantation in a vascularized AV-Loop transplantation model in the rat

The use of foetal liver cells (FLC) in the context of hepatic tissue engineering might permit efficient in vitro expansion and cryopreservation in a cell bank. A prerequisite for successful application of bioartificial liver tissue is sufficient initial vascularization. In this study, we evaluated the transplantation of fibrin gel-immobilized FLC in a vascularized arterio-veno-venous (AV)-loop model. FLC were isolated from embryonic/foetal (ED 16) rat livers and were enriched by using magnetic cell sorting (MACS). After cryopreservation, FLC were labelled by pkh-26. Cells were transplanted in a fibrin matrix into a subcutaneous chamber containing a microsurgically created AV-loop in the femoral region of the recipient rat. The chambers were explanted after 14 days. Subcutaneous implants without an AV-loop and cell-free implants served as controls. Fluorescence microscopy of the constructs was used to identify pkh-26⁺- donor cells. Characterization was performed by RT-PCR and immunhistology (IH) for CK-18 and CD31. Transplantation of FLC using the AV-loop permitted a neo -tissue formation in the fibrin matrix. A high-density vascularization was observed in the AV-loop constructs as shown by CD31 IH. Viable foetal donor cells were detected which expressed CK-18. FLC can be successfully used for heterotopic transplantation. Fibrin matrix permits rapid blood vessel ingrowth from the AV-loop and supports engraftment of FLC. It is therefore an appropriate environment for hepatocyte transplantation in combination with microsurgical vascularization strategies. Transplantation of fibrin gel-immobilized FLC may be a promising approach for the development of highly vascularized in vivo tissue-engineering-based liver support systems.     

细胞外信号调节激酶在丹参单体IH764-3诱导肝星状细胞凋亡中的作用

目的：探讨丹参单体1H764-3对H202刺激的肝星状细胞（HSCs）增殖、凋亡等细胞行为的影响及细胞外信号调节激酶,（ERKt）在其中的调节作用。方法：应用体外细胞培养技术,采用直接细胞计数法、0H．胸腺嘧啶核苷（0H-TdR）掺入法测定HSCs增殖;透射电镜、膜联蛋白（Annexin-V）／磺化丙啶（PI）双标记流式细胞术测定nscs凋亡;分别应用Western blot和逆转录-聚合酶链反应（RT-PCR）技术测定ERK1蛋白及其mRNA的表达。结果：①H202具有刺激HSC8增殖的作用;②丹参单体IH764-3剂量依赖性抑制也02刺激的HSCs增殖;③Annexin-V／PI检测显示,10mg／L,20mg／L,30mg／L及40mg／LIH764-3干预48h后各组凋亡率分别为6．35％、9．28％、15．10％、19．69％,而H2O2组为2．30％;30ms／L IH764-3干预HSCs不同时间（12h、24h、48h）的凋亡率分剐是6．73％、10．34％、15．10％,呈时间依赖性;④丹参单体IH764-3干预组,HSCs的ERK1蛋白及其mRNA表达下调。结论：丹参单体IH764-3可以抑制HSCs增殖并诱导其凋亡;这种作用与其抑制ERK1蛋白和ERK1 mRNA表达有关。     

Interactions between electron and proton currents in excised patches from human eosinophils

The NADPH-oxidase is a plasma membrane enzyme complex that enables phagocytes to generate superoxide in order to kill invading pathogens, a critical step in the host defense against infections. The oxidase transfers electrons from cytosolic NADPH to extracellular oxygen, a process that requires concomitant H+ extrusion through depolarization-activated H+ channels. Whether H+ fluxes are mediated by the oxidase itself is controversial, but there is a general agreement that the oxidase and H+ channel are intimately connected. Oxidase activation evokes profound changes in whole-cell H+ current (IH), causing an approximately -40-mV shift in the activation threshold that leads to the appearance of inward IH. To further explore the relationship between the oxidase and proton channel, we performed voltage-clamp experiments on inside-out patches from both resting and phorbol-12-myristate-13-acetate (PMA)-activated human eosinophils. Proton currents from resting cells displayed slow voltage-dependent activation, long-term stability, and were blocked by micromolar internal [Zn2+]. IH from PMA-treated cells activated faster and at lower voltages, enabling sustained H+ influx, but ran down within minutes, regaining the current properties of nonactivated cells. Bath application of NADPH to patches excised from PMA-treated cells evoked electron currents (Ie), which also ran down within minutes and were blocked by diphenylene iodonium (DPI). Run-down of both IH and Ie was delayed, and sometimes prevented, by cytosolic ATP and GTP-gamma-S. A good correlation was observed between the amplitude of Ie and both inward and outward IH when a stable driving force for e- was imposed. Combined application of NADPH and DPI reduced the inward IH amplitude, even in the absence of concomitant oxidase activity. The strict correlation between Ie and IH amplitudes and the sensitivity of IH to oxidase-specific agents suggest that the proton channel is either part of the oxidase complex or linked by a membrane-limited mediator.     

The role of β-adrenergic receptor signaling in the proliferation of hemangioma-derived endothelial cells

Background

Infantile hemangioma (IH) is a benign vascular neoplasm that arises from the abnormal proliferation of endothelial cells and enhanced angiogenesis. Recently, propranolol has been found to be effective in the management of IH, suggesting that β-adrenergic receptors (β-ARs) may play an important role in the pathogenesis of IH.

Results

In the present study, we investigated the β-adrenergic signaling that is associated with hemangioma-derived endothelial cell (HemEC) proliferation. The results showed that both β₁- and β₂-ARs were expressed in HemECs. Stimulation of the β-ARs by isoprenaline induced cell proliferation and elevation of second messenger cAMP levels. The proliferation-promoting action of isoprenaline was abolished by a β₁-selective antagonist and was more effectively abolished by a β₂-selective antagonist; the mechanism for the action of the antagonists was a G₀/G₁ phase cell cycle arrest which was associated with decreased cyclin D1, CDK-4, CDK-6 and phospho-Rb expression. Pre-treatment of the cells with VEGFR-2 or ERK inhibitors also prevented the isoprenaline-mediated proliferation of cells. In agreement with the involvement of β-ARs and VEGFR-2 in the HemEC response, β-AR antagonists and the VEGFR-2 inhibitor significantly attenuated isoprenaline-induced ERK phosphorylation. Moreover, treating the cells with isoprenaline markedly increased VEGF-A expression and VEGFR-2 activity in a β₂-AR-dependent manner.

Conclusions

We have demonstrated that the activation of the β-ARs in the ERK pathway may be important mechanisms in promoting HemEC growth. Furthermore, stimulation of the β-AR may transactivate VEGFR-2 signaling and further increase HemEC proliferation.     

Expression and localization of extracellular matrix metalloproteinase inducer in giant cell tumor of bone

Matrix metalloproteinases (MMPs) are regarded as a significant regulator in tumor invasion and metastasis. Previous studies have shown that extracellular matrix metalloproteinase inducer (EMMPRIN) in tumor cells induces the synthesis of MMPs. EMMPRIN is abundantly present on the surface of tumor cells and stimulate adjacent stromal cells to synthesize MMPs to induce tumor progression. Giant cell tumor (GCT) of bone is a benign but locally aggressive primary neoplasm of bone. The spindle-shaped mononuclear stromal cells are considered to be the tumor components of GCT, which are capable of inducing osteoclast formation by recruiting the circulating monocyte and macrophage. In this study, we proposed that EMMPRIN is associated with the biological progression and aggressiveness of GCT. We have conducted semi-quantitative RT-PCR to determine the correlation of EMMPRIN expression with the clinical stage of GCT. We have also examined the cellular localization of EMMPRIN in GCT using in-situ hybridization (ISH) and Immunohistochemistry (IH). The results showed that EMMPRIN was present in GCT and its mRNA levels were associated with the clinical stage of GCT. Higher expression level of EMMPRIN was observed in GCT with advanced stage (stage III). There was a great significance (P < 0.05) of EMMPRIN expression between stage I & II and stage III GCTs. Both ISH and IH demonstrated that EMMPRIN is present at the multinuclear osteoclast-like giant cells of GCT, with strong immunostaining on the cell membrane. The stromal-like tumor cells were also positively stained but the intensity was weaker. Interestingly, the production of EMMPRIN in osteoclast-like cells of GCT seems to be regulated by stromal-like tumor cells. Receptor activator of NF-kappaB ligand (RANKL), which has been previously shown to be produced by the stromal-like tumor cells for the recruitment of osteoclast-like giant cells in GCT, enhanced the expression of EMMPRIN mRNA during the differentiation of macrophage-like RAW(264.7) cells into osteoclasts. In short, our studies suggest that EMMPRIN may be an important regulatory factor involved in the biological behaviors of GCT.     

Activation of tyrosine hydroxylase by intermittent hypoxia: involvement of serine phosphorylation.

Regulation of tyrosine hydroxylase (TH) by intermittent hypoxia (IH) was investigated in rat pheochromocytoma 12 (PC-12) cells by exposing them to alternating cycles of hypoxia (1% O2, 15 s) and normoxia (21% O2, 3 min) for up to 60 cycles; controls were exposed to normoxia for a similar duration. IH exposure increased dopamine content and TH activity by approximately 42 and approximately 56%, respectively. Immunoblot analysis revealed that comparable levels of TH protein were expressed in normoxic and IH cells. Removal of TH-bound catecholamines and in vitro phosphorylation of TH in cell-free extracts by the catalytic subunit of protein kinase A (PKA) increased TH activity in normoxic but not in IH cells, suggesting possible induction of TH phosphorylation and removal of endogenous inhibition of TH by IH. To assess the role of serine phosphorylation in IH-induced TH activation, TH immunoprecipitates and extracts derived from normoxic and IH cells were probed with anti-phosphoserine and anti-phospho-TH (Ser-40) antibody, respectively. Compared with normoxic cells, total serine and Ser-40-specific phosphorylation of TH were increased in IH cells. IH-induced activation of TH and the increase in total serine and Ser-40-specific phosphorylation of TH were inhibited by Ca2+/calmodulin-dependent protein kinase (CaMK) and PKA-specific inhibitors but not by inhibitors of the extracellular signal-regulated protein kinase pathway, suggesting that IH activates TH in PC-12 cells via phosphorylation of serine residues including Ser-40, in part, by CaMK and PKA. Our results also suggest that IH-induced phosphorylation of TH facilitates the removal of endogenous inhibition of TH, leading to increased synthesis of dopamine.     

Chronic Intermittent Hypoxia Exerts CNS Region-Specific Effects on Rat Microglial Inflammatory and TLR4 Gene Expression

Intermittent hypoxia (IH) during sleep is a hallmark of sleep apnea, causing significant neuronal apoptosis, and cognitive and behavioral deficits in CNS regions underlying memory processing and executive functions. IH-induced neuroinflammation is thought to contribute to cognitive deficits after IH. In the present studies, we tested the hypothesis that IH would differentially induce inflammatory factor gene expression in microglia in a CNS region-dependent manner, and that the effects of IH would differ temporally. To test this hypothesis, adult rats were exposed to intermittent hypoxia (2 min intervals of 10.5% O₂) for 8 hours/day during their respective sleep cycles for 1, 3 or 14 days. Cortex, medulla and spinal cord tissues were dissected, microglia were immunomagnetically isolated and mRNA levels of the inflammatory genes iNOS, COX-2, TNFα, IL-1β and IL-6 and the innate immune receptor TLR4 were compared to levels in normoxia. Inflammatory gene expression was also assessed in tissue homogenates (containing all CNS cells). We found that microglia from different CNS regions responded to IH differently. Cortical microglia had longer lasting inflammatory gene expression whereas spinal microglial gene expression was rapid and transient. We also observed that inflammatory gene expression in microglia frequently differed from that in tissue homogenates from the same region, indicating that cells other than microglia also contribute to IH-induced neuroinflammation. Lastly, microglial TLR4 mRNA levels were strongly upregulated by IH in a region- and time-dependent manner, and the increase in TLR4 expression appeared to coincide with timing of peak inflammatory gene expression, suggesting that TLR4 may play a role in IH-induced neuroinflammation. Together, these data indicate that microglial-specific neuroinflammation may play distinct roles in the effects of intermittent hypoxia in different CNS regions.