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Histochemistry and Cell Biology

, Volume 149, Issue 5, pp 491–501 | Cite as

Comparative immunohistochemical characterization of interstitial cells in the urinary bladder of human, guinea pig and pig

  • Clara Steiner
  • Thomas Gevaert
  • Roman Ganzer
  • Dirk De Ridder
  • Jochen NeuhausEmail author
Original Paper

Abstract

Interstitial cells (ICs) are thought to play a functional role in urinary bladder. Animal models are commonly used to elucidate bladder physiology and pathophysiology. However, inter-species comparative studies on ICs are rare. We therefore analyzed ICs and their distribution in the upper lamina propria (ULP), the deeper lamina propria (DLP) and the detrusor muscular layer (DET) of human, guinea pig (GP) and pig. Paraffin slices were examined by immunohistochemistry and 3D confocal immunofluorescence of the mesenchymal intermediate filament vimentin (VIM), alpha-smooth muscle actin (αSMA), platelet-derived growth factor receptor alpha (PDGFRα) and transient receptor potential cation channel A1 (TRPA1). Image stacks were processed for analysis using Huygens software; quantitative analysis was performed with Fiji macros. ICs were identified by immunoreactivity for VIM (excluding blood vessels). In all species ≥ 75% of ULP ICs were VIM+/PDGFRα+ and ≥ 90% were VIM+/TRPA1+. In human and pig ≥ 74% of ULP ICs were VIM+/αSMA+, while in GP the percentage differed significantly with only 37% VIM+/αSMA+ ICs. Additionally, over 90% of αSMA+ ICs were also TRPA1+ and PDGFRα+ in human, GP and pig. In all three species, TRPA1+ and PDGFRα+ ICs point to an active role for these cells in bladder physiology, regarding afferent signaling processes and signal modification. We hypothesize that decline in αSMA-positivity in GP reflects adaptation of bladder histology to smaller bladder size. In our experiments, pig bladder proved to be highly comparable to human urinary bladder and seems to provide safer interpretation of experimental findings than GP.

Keywords

3D confocal laser scanning microscopy Quantitative analysis PDGFR-alpha TRPA1 Alpha-smooth muscle actin Vimentin 

Notes

Acknowledgements

The authors thank Mrs. Annett Weimann, Mrs. Mandy Bernd-Paetz (Leipzig) and Mrs. Nathalie Volders (Leuven) for the excellent technical assistance. We thank Dr. Thomas Pannicke (Paul-Flechsig Intitute of Brain Research, University of Leipzig) for providing guinea-pig tissue.

Funding

This study was in part funded by the Dr. Siegfried Krüger Stiftung Leipzig.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval/standards

All procedures were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. Human studies were approved by the Ethical Committee of the University of Leipzig (UKL8/2004) and informed consent was obtained from all patients included in this study.

Supplementary material

418_2018_1655_MOESM1_ESM.tif (3.5 mb)
Fig. S1. IF in GP supporting TRPA1-antibody validation data using a second antibody against TRPA1; upper panels (a - c) show main antibody from alomone (ACC-037), lower panels (d - f) show second antibody from Santa Cruz (sc-32353) in bladder and gut. Asterisks show TRPA1-IR in enteric ganglia. Bladder stains with blocking peptides show no IR for TRPA1. Channels: TRPA1 in red, VIM in green; nuclei in blue (DAPI); U = Urothelium readily discernible by characteristic arrangement of nuclei. Scale bar indicates 20 µm. (TIF 3615 KB)
418_2018_1655_MOESM2_ESM.tif (480 kb)
Fig. S2. Validation of alomone anti-TRPA1 antibody in WB; banding was closely resembling the WB reported in the datasheet. First four bands show housekeeping genes β-actin and GAPDH. Each antibody was used in two different GPs. (TIF 479 KB)
418_2018_1655_MOESM3_ESM.tif (415 kb)
Fig. S3. Parts-of-a-whole diagrams show analysis of αSMA/PDGFRα and αSMA/TRPA1 in human, GP and pig. Amount of PDGFRα- and TRPA1- ICs with positive αSMA-IR is <10% in all species. GP shows less αSMA+ cells than human and pig. (TIF 414 KB)
418_2018_1655_MOESM4_ESM.avi (684 kb)
Video S4. 360° Projection of a 45 optical slices confocal stack of human ICs (see figure 2a); Channels: PDGFRα in red, Vimentin in green and DAPI in blue. (AVI 684 KB)
418_2018_1655_MOESM5_ESM.avi (918 kb)
Video S5. 360° Projection of a 45 optical slices confocal stack of GP ICs (see figure 2b); Channels: PDGFRα in red, Vimentin in green and DAPI in blue. (AVI 917 KB)
418_2018_1655_MOESM6_ESM.avi (907 kb)
Video S6. 360° Projection of a 45 optical slices confocal stack of pig ICs (see figure 2c); Channels: PDGFRα in red, Vimentin in green and DAPI in blue. (AVI 906 KB)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Urology, Research LaboratoriesUniversity of LeipzigLeipzigGermany
  2. 2.Organ Systems, Laboratory of Experimental UrologyKU LeuvenLeuvenBelgium
  3. 3.Department of UrologyUZ LeuvenLeuvenBelgium
  4. 4.Department of UrologyUniversity Hospital Leipzig AöRLeipzigGermany
  5. 5.Department of UrologyAsklepios Hospital Bad TölzBad TölzGermany

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