A comparison of normalization approaches for in vitro contraction experiments of urinary bladder

Erdogan B R1, Yesilyurt Z E1, Karaomerlioglu I1, Muderrisoglu E1, Arioglu-Inan E1, Michel M C2

Research Type

Basic Science / Translational

Abstract Category

Pharmacology

Abstract 725
Non Discussion Abstracts
Scientific Non Discussion Abstract Session 36
Basic Science Pharmacology Animal Study Overactive Bladder Bladder Outlet Obstruction
1. Ankara University, 2. Johannes Gutenberg University
Links

Abstract

Hypothesis / aims of study
Organ bath experiments with isolated tissue strips are a cornerstone for the in vitro assessment of urinary bladder contractility and its possible alterations in disease and/or upon treatment. It appears obvious that a larger strip can generate a greater force of contraction. As it is close to impossible to cut bladder strips that have an identical size, investigators typically normalize force of contraction based on strip size. However, there is disagreement in the community whether size should be defined by strip weight, length or cross-sectional area, and all three approaches are used (1-3). Comprehensive comparisons of normalization approaches have not been reported. Moreover, it remains unclear whether a given normalization approach is equally applicable for contraction induced by different stimuli. Therefore, we have analyzed data from an ongoing study to compare these three normalization approaches for three different contraction parameters, peak and plateau contraction by a muscarinic receptor agonist and peak contraction by the receptor-independent stimulus KCl.
Study design, materials and methods
The present study has used animals from an ongoing study related to type 2 diabetes and its treatment. Male Sprague-Dawley (4 weeks of age) rats were obtained from an external animal breeding facility. At 6 weeks of age, some rats were put on a high-fat diet, whereas others continued on a standard diet. Four weeks later, the rats on the high-fat diet received an intra-peritoneal injection of streptozotocin (25 mg/kg); rats on standard chow received a vehicle injection. At 12 weeks of age, one group each on high-fat and standard diet started receiving the SGLT2 inhibitor dapagliflozin (1 mg/kg once daily by oral gavage). At 24 weeks of age, animals were sacrificed under isoflurane anesthesia, and urinary bladder and other tissues were harvested. In total, 4 groups of animals were studied: A) control, B) control + dapagliflozin, C) high-fat diet + streptozotocin and D) high-fat diet + streptozotocin + dapagliflozin.

The bladder was cleaned from surrounding adipose and connective tissue, the trigone and he uppermost part of the dome, and 4 strips were cut from each bladder body. They were mounted in 10 ml organ bath in Krebs-Henseleit buffer and connected to a force transducer for isometric tension recording under a resting tension of 10 mN. Following 75 min of equilibration with regular freshening of buffer, strips were exposed twice to 50 mM KCl. The peak tension recorded after the second KCl addition was used to define “KCl peak”. After washing and an additional 45 min of equilibration, a cumulative carbachol concentration-response curve was generated. Based on peak contraction response to all concentrations, the maximum response (Emax of peak carbachol response) was calculated by fitting a sigmoidal curve to the experimental data. After another wash and 45 min of equilibration, 1 µM carbachol was added and the plateau tension was recorded (plateau carbachol response). 

At the end of the experiment, strips were taken from the organ bath, strip weight and length were measured, and cross-sectional area calculated (weight/(length*1.05)). For each of the three contractile responses (KCl peak, carbachol peak, carbachol plateau), a correlation analysis against weight, length and cross-sectional area was performed. A greater r2 of the correlation analysis was defined as a more suitable form of normalization. The investigators performing the organ bath experiments and those doing the data analysis were blinded to group allocation of experimental animals.
Results
95 strips were available for analysis. Correlation coefficients are shown in Table 1 and correlations for peak carbachol as an example are shown in Figure 1. In contrast to reported findings from a smaller series of strips (2), length correlated only poorly if at all with any of the three contraction responses. Cross-sectional area exhibited a very moderate correlation, whereas the correlation with strip weight was tightest but still only moderate.

Table 1: Coefficient of correlation (r2) for the three contractile responses and the three potential normalization parameters.
	                                      Peak KCl	Peak carbachol	Plateau carbachol
Weight	                                 0.2908	      0.4022	                 0.2967
Length	                                 0.0026	      0.0343	                 0.0321
Cross-sectional area	         0.2164	      0.1942	                 0.1356

Figure 1: Correlation of peak carbachol response with potential normalization parameters.
Interpretation of results
Our data are based on a considerably larger number of strips than previous validation attempts. In contrast to previously reported smaller data sets (2), they suggest that contraction is only poorly correlated with strip length; therefore, normalization for length does not appear useful. In confirmation of a previously reported small data set (1), contraction was correlated to strip weight, but the tightness of correlation was only moderate (r2 ≈ 0.3-0.4). Cross-sectional area exhibited a tighter correlation than strip length, but a poorer than strip weight. However, all three parameters explained only up to about 40% of variability. Accordingly, comparison of contraction across the 4 experimental groups was similar regardless which, if any, normalization was applied.
Concluding message
While it is intuitive to normalize force of contraction for strip size, none of the three chosen parameters yielded a tight correlation, i.e. could explain more than 50% of inter-strip variability in contraction. Among the tested parameters, strip weight appears most appropriate if normalization for strip size is desired. Given the exploratory character of our analysis, a confirmatory study on an independent data set appears desirable.
Figure 1
References
  1. 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: 524-531
  2. Schneider T, Hein P, Bai J, Michel MC (2005) A role for muscarinic receptors or rho-kinase in hypertension associated rat bladder dysfunction? J Urol 173: 2178-2181
  3. Longhurst PA, Levendusky MC, Bezuijen MWF (2004) Diabetes mellitus increases the rate of development of decompensation in rats with outlet obstruction. J Urol 171: 933-937
Disclosures
Funding Intramural grant Ankara University (BAP-16L0237006) and Deutsche Forschungsgemeinschaft (Mi 294/8-1) Clinical Trial No Subjects Animal Species Rat Ethics Committee Ankara University Rectorate Animal Experiments Local Ethics Committee, approval 2015-4-82