水通道蛋白5在高氧肺损伤中的表达及调节机制

目的探讨水通道蛋白5(AQP5)在高氧肺损伤中的表达及其地塞米松对AQP5的调节作用。方法2周左右Wistar大鼠64只,按随机数字表法分为空气对照组、高氧暴露3、7、14d组和相应的地塞米松干预组。高氧暴露组置于常压氧仓中(O2体积分数≥95%);空气对照组置于同室常压空气中(O2体积分数为21%);各地塞米松干预组在暴露于空气或高氧的同时,经腹腔注射地塞米松5mg·kg-1·d-1,连续3d。采用逆转录聚合酶链反应(RT PCR)和免疫组化方法观察AQP5的mRNA表达和分布变化,并与地塞米松干预后进行比较分析。结果AQP5主要表达在肺泡型上皮细胞及气道分泌上皮顶质膜;与空气对照组相比,高氧暴露不同时间后,AQP5特异性表达部位保持不变,但随暴露时间延长,AQP5表达呈逐渐减弱趋势,高氧暴露3、7和14d,AQP5mRNA较空气对照组均降低(P均<0.05)。与同期高氧暴露组比较,地塞米松干预后不同时间点AQP5mRNA表达均无明显变化(P均>0.05)。结论高氧肺损伤时AQP5表达降低,可能是高氧肺损伤肺水肿形成的原因之一;而未见地塞米松对高氧肺损伤AQP5的表达有调节作用。     

Bronchiolar Remodeling in Adult Mice Following Neonatal Exposure to Hyperoxia: Relation to Growth

Preterm infants who receive supplemental oxygen for prolonged periods are at increased risk of impaired lung function later in life. This suggests that neonatal hyperoxia induces persistent changes in small conducting airways (bronchioles). Although the effects of neonatal hyperoxia on alveolarization are well documented, little is known about its effects on developing bronchioles. We hypothesized that neonatal hyperoxia would remodel the bronchiolar walls, contributing to altered lung function in adulthood. We studied three groups of mice (C57BL/6J) to postnatal day 56 (P56; adulthood) when they either underwent lung function testing or necropsy for histological analysis of the bronchiolar wall. One group inhaled 65% O₂ from birth until P7, after which they breathed room air; this group experienced growth restriction (HE+GR group). We also used a group in which hyperoxia‐induced GR was prevented by dam rotation (HE group). A control group inhaled room air from birth. At P56, the bronchiolar epithelium of HE mice contained fewer Clara cells and more ciliated cells, and the bronchiolar wall contained ～25% less collagen than controls; in HE+GR mice the bronchiolar walls had ～13% more collagen than controls. Male HE and HE+GR mice had significantly thicker bronchiolar epithelium than control males and altered lung function (HE males: greater dynamic compliance; HE+GR males: lower dynamic compliance). We conclude that neonatal hyperoxia remodels the bronchiolar wall and, in adult males, affects lung function, but effects are altered by concomitant growth restriction. Our findings may partly explain the reports of poor lung function in ex‐preterm children and adults. Anat Rec, 297:758–769, 2014. © 2014 Wiley Periodicals, Inc.     

粘着斑激酶在早产大鼠高氧肺损伤肺组织中的表达变化和意义

目的探讨粘着斑激酶(FAK)与高氧肺损伤发生、发展的关系。方法剖宫术取出孕21 d大鼠作为早产鼠,分别置早产鼠于85％高氧环境下3、7和14 d,各组均以空气组早产鼠为对照,留取肺组织标本,采用免疫组织化学法和Western blot技术对高氧组和空气组肺组织FAK多肽表达进行定位、定量检测,采用RT-PCR方法对FAK mRNA表达水平进行半定量分析。结果 FAK mRNA和蛋白在空气组早产大鼠肺组织均有较高水平表达,高氧暴露3、7和14 d后,FAK mRNA和蛋白表达水平均呈不同程度的下降,尤以高氧14 d最明显。结论高氧抑制FAK表达是导致正常肺泡化过程受阻以及不成熟肺组织损伤后异常修复的重要因素,其机制可能与其抑制肺泡上皮细胞增殖、分化,以及毛细血管形成有关。     

The relationship between oxygen and adenosine in astrocytic cultures

Brain tissue oxygenation affects cerebral function and blood flow (CBF). Adenosine (Ado), a purine nucleoside, moderates neuronal activity, and arterial diameter. The cellular source of Ado in brain remains elusive; however, astrocytes are a logical site of production. Using astrocytic cultures, we tested the hypothesis that astrocytic derived Ado reflects cerebral oxygenation. We found that during alterations in pO₂, extracellular levels of Ado [Ado]_e changed rapidly. Graded reductions of oxygen tension revealed that[Ado]_e reached 10⁻⁷ M to 10⁻⁶ M with a pO₂ of 30–10mmHg, comparable with [Ado]_e and oxygen levels found in brain tissue during normoxemia. Higher O₂ levels were associated with a depression of [Ado]_e. Under conditions of low pO₂ (pO₂ ≤ 3 mmHg), inhibition of extracellular catabolism of adenosine monophosphate (AMP) prevented an increase of [Ado]_e and resulted in a rise in [AMP]_e. The rise in [AMP]_e preceded the increase in [Ado]_e. In the presence of nucleoside transporter inhibitors, accumulation of [Ado]_e persisted. On the basis of our studies in culture we conclude that astrocytes are a significant source of Ado and that during hypoxia, the changes in [Ado]_e are in a range to affect both neuronal activity as well as CBF. © 2010 Wiley‐Liss, Inc.     

慢性高氧肺损伤新生鼠肺微血管超微结构的动态变化

目的观察高氧对新生鼠肺微血管发育的影响。方法以新生鼠慢性高氧肺损伤28例和对照24例为研究对象,在实验3、7和14d观察肺组织病理、微血管超微结构变化,放射性肺泡计数(RAC)。结果高氧组3d微血管发生炎症反应,血管基底膜增厚、断裂;7d炎症反应明显,血管腔被炎细胞和坏死的内皮细胞堵塞,血管减少;14d毛细血管稀少,间质有胶原纤维沉积,气血屏障增厚。高氧组RAC值7天降低(6.87±1.11)vs(7.53±0.86),P<0.05,14d差异更显著(P<0.001)。结论炎症是高氧肺微血管损伤的一个始动环节,由此导致的毛细血管闭塞可能是微血管减少的一个重要原因。提示控制炎症反应可能是治疗和预防高氧肺损伤的有效手段。     

高氧吸入后新生鼠肺组织一氧化氮及氧自由基的动态变化

目的探讨持续吸入高氧后新生大鼠肺组织病理和一氧化氮(NO)及氧自由基的动态变化规律。方法足月新生鼠生后12h内分别持续吸入(90±5)%的高氧和空气,于1、3、7、14、21d,动态观察其肺组织病理学改变以及NO、MDA含量和SOD活性。结果肺形态学:吸高氧3d时炎性细胞渗出,7d时肺间隔增宽,终末气腔明显扩张,小肺泡数量减少,14和21d间质增生、肺泡化降低越来越明显;NO水平:在7、14和21d,高氧组水平高于空气组,数值分别为(99.38±7.80)vs(88.78±8.00),P<0.05;(128.18±33.78)vs(93.30±16.73),P<0.05;(170.66±34.00)vs(106.37±25.11),P<0.01;MDA含量:高氧组在3、7和14d高于空气组,数值分别为(28.10±2.03)vs(22.11±1.25),P<0.05;(30.82±4.17)vs(19.91±2.17),P<0.01;(26.27±3.78)vs(22.56±2.35),P<0.05;SOD的活性,在吸高氧7、14和21d时高于空气组(213.87±18.58)vs(185.55±18.79),P<0.05;(219.81±4.17)vs(19.91±2.71),P<0.01;(251.09±15.10)vs(194.56±8.12),P<0.01。结论持续吸入高氧可致新生大鼠发生与人类BPD类似的病理改变;肺组织的自由基损伤,可能在疾病发生、发展过程中起重要作用。     

血小板源性生长因子对新生大鼠高氧肺损伤的影响

目的　探讨血小板源性生长因子 (PDGF)在新生大鼠高氧肺损伤中时空作用机制。方法　应用免疫组织化学法定位PDGF表达 ,反转录 聚合酶链反应 (RT PCR)检测各时间点 (生后 4、7、10、14d)PDGFmRNA水平。结果　随日龄增加 ,PDGF A、 B含量发生不同的变化。高氧暴露下PDGF BmRNA含量及其表达均显著高于空气组 (P <0 .0 5 ,<0 .0 1)。而PDGF AmRNA水平及其表达却显著低于空气组 (P <0 .0 5 ,P <0 .0 1)。结论　高氧降低PDGF A水平 ,增加PDGF B含量。PDGF A、 B共同参与高氧暴露下肺组织损伤过程 ,是肺发育阻滞的重要因素。     

Ventilatory response to CO2 at rest and during positive and negative work in normoxia and hyperoxia

Summary Ventilation versus alveolar relationships were determined by the steady-state method in 6 normal male subjects at rest and during positive and negative work at one load in both normoxic and hyperoxic condition. In 5 subjects the slopes of the lines during positive and negative work increased in normoxia as compared with rest. This effect was less evident in hyperoxia. It was also found that the slopes of the lines in positive and in negative work were about the same in both normoxic and hyperoxic conditions. Oxygen uptake and CO₂ production during positive work is higher than during negative work.These results suggest that: 1) the disagreement between various authors on the change of the slope of the line may be due to the differences in the method of calculation of the slope or the method of the determination of lines; 2) the stimuli from the muscle spindles in the working muscle during exercise probably do not contribute to the increase in ventilatory response to CO₂; 3) the increased slope of the normoxic line during exercise may be due to the interaction of several factors such as impulses from working muscles, chemosensitivity of central or peripheral chemoreceptors, adrenal-sympathetic pathways or temperature; 4) respiratory oscillations of or do not seem to influence the respiratory response to CO₂.This study was supported in part by a grant from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.)     

Ageing and exposure to oxidative stress in vivo differentially affect cellular levels of PrP in mouse cerebral microvessels and brain parenchyma

The biological function of cellular prion protein PrPc has not been established, despite in vitro studies suggesting antioxidant activity or link to signal transduction pathways. In this study, mice were exposed to hyperoxia to establish whether oxidative stress affected prion expression in vivo. C57Bl/6J mice aged 6, 18, and 24 months, maintained under normoxic conditions, exhibited age-related increases in PrPc in both cerebral microvessels and in microvessel-depleted brain homogenate. We demonstrate that PrPc is differentially affected by exposure to hyperoxia in vivo for 1 (24 h) or 2 (48 h) days, or for 1 day hyperoxia, followed by 1 day normoxia. Brain parenchymal cells from 6-month-old mice exposed to 1 day hyperoxia showed elevation of a glycosylated approximately 36 kDa form, whereas in 24-month-old mice cellular prion level was substantially reduced. Extending hyperoxia from 1 to 2 days resulted in significantly reduced PrPc level, regardless of age. Parenchymal PrPc is substantially elevated in 6-month-old mice, but declines in 18- and 24-month-old animals following 1 day hyperoxia. By contrast, PrPc content in cerebral microvessels from 6-month-old mice declined after a 2 day exposure to hyperoxia, while microvessels from 24-month-old brains showed elevated prion levels 24 h after hyperoxia. Moreover, unglycosylated 25-30 kDa PrPc, and a previously undescribed 50-64 kDa band containing at least some glycosylated protein, predominated in microvessels with lesser content of the glycosylated approximately 36 kDa form. Cellular content of these unglycosylated forms was correlated with age, while the response to hyperoxia was evident in both unglycosylated and glycosylated forms of the protein following 1 and 2 day exposures. The observed elevation of the 25-30 and 50-64 kDa bands of microvessel PrPc is not sustainable following 1 day hyperoxia, but returns to near normoxic levels within 24 h after hyperoxia. We also show in a knockout mouse for methionine sulfoxide reductase (MsrA), the enzyme responsible for reducing methionine sulfoxide back to methionine, and a regulator of cellular antioxidant defence, that following hyperoxia brain PrPc in the null mutant is elevated relative to PrPc content in the parent strain. Our results show up-regulated PrPc expression or reduced turnover in response to age-related, and hyperoxia-induced oxidative stress.     

Role of arteries in oxygen induced vaso-obliteration

In mice the retinal vasculature develops in the first postnatal week by spreading from the optic nerve head towards the retinal periphery. During this growth period, exposure to hyperoxia causes vaso-obliteration of capillaries in the retinal center but not in peripheral regions. High oxygen levels lead to downregulation of vascular endothelial growth factor (VEGF), an important survival factor for vascular endothelial cells, which could explain the vaso-obliteration caused by hyperoxia. However, it is not clear why only capillaries in the center of the retina are affected. We therefore investigated how capillary obliteration correlates with VEGF mRNA distribution by in situ hybridization in retinal whole mount preparations. In mouse pups reared under normoxic conditions VEGF mRNA was detectable across the entire vascular network but was virtually absent in the immediate vicinity of arteries. This was true along developing retinal arteries but also around the optic nerve head through which the entire arterial blood supply for the retinal and hyaloid vasculature passes. In these areas capillaries were absent, resulting in so-called capillary free zones. Exposure to hyperoxia caused an expansion of areas with low VEGF mRNA which correlated with capillary obliteration in these regions. Combined capillary obliteration around the optic nerve head and along retinal arteries lead to a large capillary free zone in the center of the retina. Thus, our observations suggest that hyperoxia affects the retinal vasculature by reducing VEGF mRNA levels near arteries and causing a widening of capillary free zones.