Evaluation of endothelium-derived nitric oxide mediated vasodilation utilizing ex vivo perfusion of an intact vessel.
Evaluation of endothelium-derived nitric oxide mediated vasodilation utilizing ex vivo perfusion of an intact vessel.
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利用完整血管的离体灌注评估内皮衍生的一氧化氮介导的血管舒张。
DOI：
10.1016/0022-4804(92)90305-j
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发表时间：
1992
期刊：
影响因子：
--
通讯作者：
Borovetz,HS
中科院分区：
文献类型：
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作者：
LigushJr,J;Labadie,RF;Berceli,SA;Ochoa,JB;Borovetz,HS
内皮源性一氧化氮（EDNO）通路的传统评价涉及带有应变片的离体主动脉环。该模型是非生理性的，不允许研究持续超过几个小时。我们的目的是克服这些"传统"方法的局限性，利用生理学的全血管模型作为EDNO的可重复测定。将犬颈动脉（n = 4）取出（体内动脉几何形状），固定在专门设计的连续流回路中，并以100 ml/min，80 mm Hg的速度用中等浓度-199/10%犬血清灌注。生理pH、pCO2、pO2和温度被精确调节。将非接触式氦氖激光测微计与当前系统连接，以提供血管外径的连续测量，并定量血管壁几何形状对肾上腺素（EPI; 2 × 10 − 5至2 × 10 − 3mg/ml）和乙酰胆碱（ACh; 0.1至100 μ M）激发的响应变化。灌注系统的进一步表征包括使用EDNO产生的竞争性抑制剂，NG-单甲基-L-精氨酸（1-NMMA），以及该化合物对ACh诱导的血管舒张的作用。这种阻断的可逆性通过依次加入l-精氨酸（l-ARG; 0至3mM）来验证。数据表示为血管活性物质给药后稳态血管横截面积（CSA）与血管活性激发前CSA的比值。结果表明，EPI和ACh分别产生明显的剂量依赖性血管收缩和舒张反应（P <0.001，ANOVA）。向灌注液中加入2 × 10 − 5、2 × 10 − 4和2 × 10 − 3mg/ml EPI浓度，导致标准化CSA降低0.94 ± 0.01、0.87 ± 0.01和0.80 ± 0.04。向灌注液中添加0.1、1.0、10和100 μ MACh分别使标准化CSA增加1.003 ± 0.005、1.09 ± 0.04、1.21 ± 0.07和1.27 ± 0.07。1mMl-NMMA可阻断ACh反应，即使在最高ACh剂量100 μ M时也能阻断（P <0.001，双因素方差分析）。添加1-ARG（1、2和3 mM）可引起1-NMMA效应的剂量依赖性逆转，在1 mMl-ARG时观察到血管舒张开始，在3 mMl-ARG时几乎完全逆转。我们的研究结果表明，这novelex vivomodel提供了一个可靠和有效的检测进一步调查的机制内皮和非内皮依赖性血管活性介质。
The traditional evaluation of the endothelium-derived nitric oxide (EDNO) pathway involves isolated aortic rings with attached strain gauges. This model is nonphysiologic and does not permit studies lasting longer than several hours. Our objective was to overcome the limitations of these “traditional” methods utilizing a physiologic, whole vessel model as a reproducible assay of EDNO. Canine carotid arteries (n= 4) were removed (maintainingin vivoarterial geometry), mounted in a specially designed, continuous-flow circuit, and perfused at 100 ml/min, 80 mm Hg with Medium-199/10% canine serum. Physiologic pH, pCO2, pO2, and temperature were precisely regulated. A non-contacting, helium-neon laser micrometer was interfaced with the current system to provide continuous measurement of vessel external diameter and to quantitate changes in vessel wall geometry in response to epinephrine (EPI; 2 × 10−5to 2 × 10−3mg/ml) and acetylcholine (ACh; 0.1 to 100 μM) challenge. Further characterization of the perfusion system included the use of a competitive inhibitor to EDNO production,NG-monomethyl-l-arginine (l-NMMA), and the effect of this compound on ACh-induced vasodilation. The reversibility of this blockade was verified via the sequential addition ofl-arginine (l-ARG; 0 to 3 mM). Data are expressed as the ratio of steady-state vessel cross-sectional area (CSA) following administration of vasoactive substance to the CSA prior to vasoactive challenge. Our results indicate that EPI and ACh produced significant dose-dependent vasoconstrictive and vasodilatory responses, respectively (P< 0.001, ANOVA). The addition of 2 × 10−5, 2 × 10−4, and 2 × 10−3mg/ml EPI concentrations to the perfusate caused 0.94 ± 0.01, 0.87 ± 0.01, and 0.80 ± 0.04 decrease in normalized CSA. The addition of 0.1, 1.0, 10, and 100 μMACh to the perfusate produced a 1.003 ± 0.005, 1.09 ± 0.04, 1.21 ± 0.07, and 1.27 ± 0.07 increase in the normalized CSA, respectively. The ACh response could be blocked by 1 mMl-NMMA, even at the highest ACh dosage—100 μM(P< 0.001, two-way ANOVA). The addition ofl-ARG (1, 2, and 3 mM) caused a dose-dependent reversal of the effects ofl-NMMA, with initiation of vasodilation noted at 1 mMl-ARG and near complete reversal at 3 mMl-ARG. Our findings demonstrate that this novelex vivomodel provides a reliable and valid assay for further investigation into the mechanisms of endothelium- and nonendothelium-dependent vasoactive mediators.