Normalization of organ bath contraction data for tissue specimen size: does one approach fit all?
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Organ bath experiments are a key technology to assess contractility of smooth muscle. Despite efforts to standardize tissue specimen sizes, they vary to a certain degree. As it appears obvious that a larger piece of tissue should develop greater force, most investigators normalize contraction data for specimen size. However, they lack agreement which parameter should be used as denominator for normalization. A pre-planned analysis of data from a recent study was used to compare denominators used for normalization, i.e., weight, length, and cross-sectional area. To increase robustness, we compared force with denominator in correlation analysis and also coefficient of variation with different denominators. This was done concomitantly with urinary bladder strips and aortic rings and with multiple contractile stimuli. Our urinary bladder data show that normalization for strip weight yielded the tightest but still only moderate correlation (e.g., r2 = 0.3582 for peak carbachol responses based on 188 strips). In aorta, correlations were even weaker (e.g., r2 = 0.0511 for plateau phenylephrine responses normalized for weight based on 200 rings). Normalization for strip size is less effective in reducing data variability than previously assumed; the normalization denominator of choice must be identified separately for each preparation.
KeywordsNormalization Urinary bladder Aorta Weight Length Cross-sectional area
MCM and EAI conceived and planned the study. BRE, IK, ZEY, NO, and AEM carried out the experiments. BRE and AEM processed the experimental data. BRE and MCM performed the analysis and designed the figures. BRE and MCM drafted the manuscript. All authors have read the manuscript, provided input for finalization, and approved the final version.
This study was supported by in part by grants from Ankara University (BAP-16L0237006), the Scientific and Technological Research Council of Turkey (TUBITAK SBAG-115S564), Deutsche Forschungsgemeinschaft (Mi 294/8-1), and the Innovative Medicines Initiative 2 Joint Undertaking (grant agreement no. 777364); this Joint Undertaking receives support from the European Union’s Horizon 2020 research and innovation program and EFPIA). BRE is a PhD student supported by Scientific and Technical Research Council of Turkey (TUBITAK-2211/A) and by a grant from ERASMUS+ Program of the European Union.
Compliance with ethical standards
The study protocol had been approved by the animal welfare committee of Ankara University (permit 2015-4-82, 2017-10-92) and was in line with NIH Guidelines for Care and Use of Laboratory Animals.
Conflict of interest
The authors report no conflict of interest related to this paper.
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