Normalization of organ bath contraction data for tissue specimen size: does one approach fit all?

  • Betul R. Erdogan
  • Irem Karaomerlioglu
  • Zeynep E. Yesilyurt
  • Nihal Ozturk
  • A. Elif Muderrisoglu
  • Martin C. MichelEmail author
  • Ebru Arioglu-Inan
Original Article


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.


Normalization Urinary bladder Aorta Weight Length Cross-sectional area 


Authors’ contributions

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.

Funding information

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.

Supplementary material

210_2019_1727_MOESM1_ESM.docx (20 kb)
ESM 1 (DOCX 19 kb)


  1. Abebe W, Harris KH, MacLeod KM (1990) Enhanced contractile responses of arteries from diabetic rats to α1-adrenoceptor stimulation in the absence and presence of extracellular calcium. J Cardiovasc Pharmacol 16:239–248CrossRefGoogle Scholar
  2. Braverman AS, Ruggieri MR Sr (2003) Hypertrophy changes the muscarinic receptor subtype mediating bladder contraction from M3 toward M2. Am J Phys 285:R701–R708Google Scholar
  3. Cameron NE, Cotter MA (1992) Impaired contraction and relaxation in aorta from streptozotocin-diabetic rats: role of polyol pathway. Diabetologia 35:1011–1019CrossRefGoogle Scholar
  4. Conklin DJ, Boor PJ (1998) Allylamine cardiovascular toxicity: evidence for aberrant vasoreactivity in rats. Toxicol Appl Pharmacol 148:245–251CrossRefGoogle Scholar
  5. Conklin DJ, Cowley HR, Wiechmann RJ, Johnson GH, Trent MB, Boor PJ (2004) Vasoactive effects of methylamine in isolated human blood vessels: role of semicarbazide-sensitive amine oxidase, formaldehyde, and hydrogen peroxide. Am J Phys 286:H667–H676Google Scholar
  6. Conklin DJ, Haberzettl P, Prough RA, Bhatnagar A (2009) Glutathione-S-transferase P protects against endothelial dysfunction induced by exposure to tobacco smoke. Am J Phys 296:H1586–H1597Google Scholar
  7. Frazier EP, Braverman AS, Peters SLM, Michel MC, Ruggieri MR Sr (2007) Does phospholipase C mediate muscarinic receptor-induced rat urinary bladder contraction? J Pharmacol Exp Ther 322:998–1002CrossRefGoogle Scholar
  8. Fujishige A, Takahashi K, Tsuchiya T (2002) Altered mechanical properties in smooth muscle of mice with a mutated calponin locus. Zool Sci 19:167–174CrossRefGoogle Scholar
  9. Halsey LG, Curran-Everett D, Vowler SL, Drummond GB (2015) The fickle P value generates irreproducible results. Nat Med 12:179–185Google Scholar
  10. Jin L, Lipinski A, Conklin DJ (2018) A simple method for normalization of aortic contractility. J Vasc Res 55:177–186CrossRefGoogle Scholar
  11. Kershaw JDB, Misfield M, Sievers H-H, Yacoub MH, Chester AH (2004) Specific regional and directional contractile responses of aortic cup tissue. J Heart Valve Dis 13:798.803Google Scholar
  12. Kilkenny C, Browne W, Cuthill IC, Emerson M, Altman DG (2010) Animal research: reporting in vivo experiments: the ARRIVE guidelines. Br J Pharmacol 160:1577–1579CrossRefGoogle Scholar
  13. Klausner AP, Rourke KF, Miner AS, Ratz PH (2009) Potentiation of carbachol-induced detrusor smooth muscle contractions by ß-adrenoceptor activation. Eur J Pharmacol 606:191–198CrossRefGoogle Scholar
  14. Kories C, Czyborra C, Fetscher C, Schneider T, Krege S, Michel MC (2003) Gender comparison of muscarinic receptor expression and function in rat and human urinary bladder: differential regulation of M2 and M3? Naunyn Schmiedeberg's Arch Pharmacol 367:524–531CrossRefGoogle Scholar
  15. Kristek F, Cacanyiova S, Gerova M (2009) Hypotrophic effect of long-term neuronal NO-synthase inhibition on heart and conduit arteries of the Wistar rats. J Physiol Pharmacol 60:21–27Google Scholar
  16. Kunert MP, Dwinell MR, Lombard JH (2010) Vascular responses in aortic rings of a consomic rat panel derived from the fawn hooded hypertensive strain. Physiol Genomics 42A:244–258CrossRefGoogle Scholar
  17. Longhurst PA, Kang J, Wein AJ, Levin RM (1990) Length-tension relationship of urinary bladder strips from streptozotocin-diabetic rats. Pharmacology 40:110–121CrossRefGoogle Scholar
  18. Meini S, Lecci A, Cucchi P, Catalioto RM, Criscuoli M, Maggi CA (1998) Inflammation modifies the role of cyclooxygenases in the contractile responses of the rat detrusor smooth muscle to kinin agonists. J Pharmacol Exp Ther 287:137–143Google Scholar
  19. Michel MC (2014) Do ß-adrenoceptor agonists induce homologous or heterologous desensitization in rat urinary bladder? Naunyn Schmiedeberg's Arch Pharmacol 387:215–224CrossRefGoogle Scholar
  20. Michel-Reher MB, Michel MC (2015) Regulation of GAPDH expression by treatment with the ß-adrenoceptor agonist isoprenaline - is GAPDH a suitable loading control in immunoblot experiments? Naunyn Schmiedeberg's Arch Pharmacol 388:1119–1120CrossRefGoogle Scholar
  21. Ozcelikay AT, Tay A, Güner S, Tasyaran V, Yildizoglu-Ari N, Dincer UD, Altan VM (2000) Reversal effects of L-arginine treatment on blood pressure and vascular responsiveness of streptozotocin-diabetic rats. Pharmacol Res 41:201–209Google Scholar
  22. Paro M, Italiano G, Travagli RA, Petrelli L, Zanoni R, Prosdocimi M, Fiori MG (1990) Cystometric changes in alloxan diabetic rat: evidence for functional and structural correlates of diabetic autonomic neuropathy. J Auton Nerv Syst 30:1–11CrossRefGoogle Scholar
  23. Pieper GM, Langenstroer P, Siebeneich W (1997) Diabetic-induced endothelial dysfunction in rat aorta: role of hydroxyl radicals. Cardiovasc Res 34:145–156CrossRefGoogle Scholar
  24. Sand C, Michel MC (2014) Bradykinin contracts rat urinary blader largely independent of phospholipase C. J Pharmacol Exp Ther 348:25–31CrossRefGoogle Scholar
  25. Schneider T, Fetscher C, Krege S, Michel MC (2004a) Signal transduction underlying carbachol-induced contraction of human urinary bladder. J Pharmacol Exp Ther 309:1148–1153CrossRefGoogle Scholar
  26. Schneider T, Hein P, Michel MC (2004b) Signal transduction underlying carbachol-induced contraction of rat urinary bladder. I. Phospholipases and Ca2+ sources. J Pharmacol Exp Ther 308:47–53CrossRefGoogle Scholar
  27. Schneider T, Hein P, Bai J, Michel MC (2005a) A role for muscarinic receptors or rho-kinase in hypertension associated rat bladder dysfunction? J Urol 173:2178–2181CrossRefGoogle Scholar
  28. Schneider T, Hein P, Michel-Reher M, Michel MC (2005b) Effects of ageing on muscarinic receptor subtypes and function in rat urinary bladder. Naunyn Schmiedeberg's Arch Pharmacol 372:71–78CrossRefGoogle Scholar
  29. Schneider T, Fetscher C, Michel MC (2011) Human urinary bladder strip relaxation by the ß-adrenoceptor agonist isoprenaline: methodological considerations and effects of gender and age. Front Pharmacol 2:11CrossRefGoogle Scholar
  30. Stevens LA, Sellers DJ, McKay NG, Chapple CR, Chess-Williams R (2006) Muscarinic receptor function, density and G-protein coupling in the overactive diabetic rat bladder. Auton Autacoid Pharmacol 26:303–309CrossRefGoogle Scholar
  31. Török J, Zemancikova A, Kocianova Z (2016) Interaction of perivascular adipose tissue and sympathetic nerves in arteries from normotensive and hypertensive rats. Phys Rep 65(Suppl. 3):S391–S399Google Scholar
  32. Weber LP, Chow WL, Abebe W, MacLeod KM (1996) Enhanced contractile responses of arteries from streptozotocin diabetic rats to sodium fluoride. Br J Pharmacol 118:115–122CrossRefGoogle Scholar
  33. Wyse DG (1980) On the “normalization” of active developed force of isolated helical strips of muscular and elastic arteries for variation in wall thickness. J Pharmacol Methods 4:313–326CrossRefGoogle Scholar
  34. Yang Q, Fujii W, Kaji N, Kakuta S, Kada K, Kuwahara M, Tsubone H, Ozaki H, Hori M (2018) The essential role of phospho-T38 CPI-17 in the maintenance of physiological blood pressure using genetically modified mice. FASEB J 32:2095–2109CrossRefGoogle Scholar
  35. Yesilyurt ZE, Erdogan BR, Karaomerlioglu I, Müderrisoglu AE, Michel MC, Arioglu Inan E (2019) Urinary bladder weight and function in a rat model of mild hyperglycemia and its treatment with dapagliflozin. Front Pharmacol 10:911CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Pharmacology, Faculty of PharmacyAnkara UniversityAnkaraTurkey
  2. 2.Department of PharmacologyJohannes Gutenberg UniversityMainzGermany

Personalised recommendations