体外测量小鼠组织的耗氧量。

Measurement of oxygen consumption by murine tissues in vitro.

作者信息

Al Samri Mohammed T, Al Shamsi Mariam, Al-Salam Suhail, Marzouqi Farida, Al Mansouri Aysha, Al-Hammadi Suleiman, Balhaj Ghazala, Al Dawaar Shaikha K M, Al Hanjeri Ruqayya S M S, Benedict Sheela, Sudhadevi Manjusha, Conca Walter, Penefsky Harvey S, Souid Abdul-Kader

机构信息

Department of Pediatrics, Faculty of Medicine and Health Sciences, United Arab Emirates University, Al Ain, Abu Dhabi, United Arab Emirates 17666, UAE.

出版信息

J Pharmacol Toxicol Methods. 2011 Mar-Apr;63(2):196-204. doi: 10.1016/j.vascn.2010.10.002. Epub 2010 Oct 27.

DOI:10.1016/j.vascn.2010.10.002

PMID:21034836

Abstract

INTRODUCTION

A novel in vitro system was developed to measure O₂ consumption by murine tissues over several hours.

METHODS

Tissue specimens (7-35 mg) excised from male Balb/c mice were immediately immersed in ice-cold Krebs-Henseleit buffer, saturated with 95% O₂:5% CO₂. The specimens were incubated at 37 °C in the buffer, continuously gassed with O₂:CO₂ (95:5). [O₂] was determined as a function of time from the phosphorescence decay rates (1/τ) of Pd(II) meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin. The values of 1/τ were linear with [O₂]: 1/τ=1/τo + kq [O₂]; 1/τo=the decay rate for zero O₂, kq=the rate constant in s⁻¹ μM⁻¹.

RESULTS

NaCN inhibited O₂ consumption, confirming oxidation occurred in the mitochondrial respiratory chain. The rate of respiration in lung specimens incubated in vitro for 3.9≤t≤12.4 h was 0.24±0.03 μM O₂ min⁻¹ mg⁻¹ (mean±SD, n=28). The corresponding rate for the liver was 0.27±0.13 (n=11, t≤4.7 h), spleen 0.28± 0.07 (n=10, t≤5h), kidney 0.34±0.12 (n=7, t≤5h) and pancreas 0.35±0.09 (n=10, t≤4h). Normal tissue histology at hour 5 was confirmed by light and electron microscopy. There was negligible number of apoptotic cells by caspase 3 staining.

DISCUSSION

This approach allows accurate assessment of tissue bioenergetics in vitro.

摘要

引言

开发了一种新型体外系统，用于测量小鼠组织数小时内的氧气消耗量。

方法

从雄性Balb/c小鼠身上切除的组织标本（7 - 35毫克）立即浸入用95% O₂:5% CO₂饱和的冰冷Krebs - Henseleit缓冲液中。标本在37°C的缓冲液中孵育，持续用O₂:CO₂（95:5）通气。根据Pd(II) 中 - 四 - (4 - 磺基苯基) - 四苯并卟啉的磷光衰减率（1/τ）随时间测定[O₂]。1/τ的值与[O₂]呈线性关系：1/τ = 1/τo + kq [O₂]；1/τo = 零氧气时的衰减率，kq = 以s⁻¹ μM⁻¹为单位的速率常数。

结果

NaCN抑制氧气消耗，证实氧化发生在线粒体呼吸链中。体外孵育3.9≤t≤12.4小时的肺标本呼吸速率为0.24±0.03 μM O₂ min⁻¹ mg⁻¹（平均值±标准差，n = 28）。肝脏的相应速率为0.27±0.13（n = 11，t≤4.7小时），脾脏为0.28±0.07（n = 10，t≤5小时），肾脏为0.34±0.12（n = 7，t≤5小时），胰腺为0.35±0.09（n = 10，t≤4小时）。通过光镜和电镜确认5小时时组织组织学正常。通过半胱天冬酶3染色，凋亡细胞数量可忽略不计。