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.     

Multiple inert gas elimination technique for determining ventilation/perfusion distributions in rat during normoxia, hypoxia and hyperoxia

1. The use of the multiple inert gas elimination technique (MIGET) in quantifying ventilation/perfusion distributions (V*A/Q*) in small animals, such as the rat, may cause results to be biased due to haemodilution produced by the large volume of liquid infused intravenously. 2. We tested two methods of administering inert gases in rats using the MIGET: (i) standard continuous intravenous administration of inert gases (method A); and (ii) a new method based on the physicochemical properties of each inert gas (method B). This method included acute simultaneous inert gas administration using three pathways: inhalation, intravenous infusion and rectal infusion. Both MIGET methods were applied to obtain data while breathing three different inspiratory fractions of oxygen (FIO2): normoxia, hypoxia and hyperoxia. 3. Inert gas levels obtained from blood or expired air samples were sufficient for chromatographic measurement, at least during a 2 h period. The V*A/Q* distributions reported using both methods were acceptable for all the physiological conditions studied; therefore, the alternative method used here may be useful in further MIGET studies in rats because haemodilution resulting from continuous intravenous infusion of less-soluble gases can be avoided. 4. Normoxic rats showed lower mean values of the V*A/Q* ratio of ventilation distribution and higher mean values of the V*A/Q* ratio of perfusion distribution with the usual method of inert gas administration (method A). These non-significant differences were observed under almost all physiological conditions studied and they could be caused by haemodilution. Nevertheless, the effect of interindividual differences cannot be discarded. An additional effect of the low haematocrit on cardiovascular changes due to low FIO2, such as pulmonary vasoconstriction or increased cardiac output, may explain the lower dispersion of perfusion distributions found in group A during hypoxia.     

不同来源骨髓间充质干细胞对新生大鼠高氧肺损伤的影响

目的 研究大鼠及人骨髓来源的问充质干细胞(mesenchymal stem cells,MSCs)对高氧所致新生大鼠肺组织损伤的干预作用.方法 采用贴壁选择法分离、培养、扩增大鼠及人骨髓来源MSCs,以PBS按要求稀释供注射用;3日龄清洁级SD新生大鼠24只,随机分为高氧 人MSCs组(A组)、高氧 鼠MSCs(B组)、高氧对照组(C组)三组;A、B、C三组均先将大鼠置于95%氧环境下7 d后,A组每只大鼠腹腔注射5×104的人MSCs,B组每只大鼠腹腔注射5×104的鼠MSCs,C组仅注射PBS,注射后72 h(13日龄)处死全部动物,肺组织病理学检测辐射状肺泡计数(radical alveolar eounts,RAC);ELASA法检测肺组织匀浆TNFα、IL-1β含量.结果 与对照组相比,二个注射MSCs组(A、B组)RAC值显著增高,TNFα、IL-1β显著降低.A、B组间差异也有统计学意义.结论 给新生大鼠腹腔注射人MSCs及鼠MSCs均可对高氧损伤的肺损伤起保护作用,其机制可能与MSCs影响损伤部位细胞因子的表达有关.     

高氧致慢性肺疾病早产鼠肺组织水通道蛋白-5及表面活性蛋白-A、B基因表达的意义

目的 探讨慢性肺疾病(CLD)早产鼠肺上皮细胞特异性标志物表面活性蛋白A、B(SP-A、SP-B)通道蛋白-5(AQP5)基因表达规律及其意义.方法 将80只早产鼠随机分为模型和对照组(每组40只),采用高体积分数氧诱导慢性肺疾病模型,于实验后1、3、7、14、21 d应用反转录聚合酶链式反应(RT-PCR)检测其肺组织SP-A、SP-B及AQP5 mRNA表达.结果 生后初期2组SP-A、SP-B及AQP5均无统计学差异(Pa＞0.05),7 d实验组SP-A及SP-B明显高于对照组(Pa＜0.05),14 d时显著高于对照组(Pa＜0.01),21 d时仍明显高于对照组(Pa＜0.05);同时间点与对照组比较,AQP5均维持在较低水平(Pa＜0.05).结论 高体积分数氧诱导早产鼠的Ⅱ型肺泡上皮细胞(AEC-Ⅱ)向Ⅰ型肺泡上皮细胞(AEC-Ⅰ)分化障碍可能是CLD肺上皮修复异常的重要机制.     

MMP-9和TIMP-1在新生大鼠持续高氧暴露后肺组织中的动态表达

目的:探讨基质金属蛋白酶9(MMP-9)和金属蛋白酶组织抑制剂1(TIMP-1)在高氧致慢性肺疾病(CLD)新生大鼠肺组织中的动态变化和意义。方法:足月新生大鼠生后12h分别持续吸入0.90～0.95的高氧和空气,于1,3,7,14,21d应用免疫组化和RT-PCR方法分别检测肺组织MMP-9和TIMP-1蛋白及mRNA表达。结果:MMP-9在高氧3d时蛋白和mRNA表达均较空气组增强(P<0.05,P<0.01);TIMP-1蛋白在高氧组表达高于空气组,3d和7d比较P<0.05,14d和21d比较P<0.01;TIMP-1 mRNA水平高氧组高于空气组,3d和7d比较P<0.05,和14d比较P<0.01,和21d比较无差异。结论:高氧暴露后MMP-9和TIMP-1的表达在不同阶段的变化不一致,MMP-9/TIMP-1平衡状态遭到破坏,可能是高氧后胶原异常沉积以及正常肺泡化过程受阻的重要因素。