Micro-mechanical fingerprints of the rat bladder change in actinic cystitis and tumor presence

Abstract

Tissue mechanics determines tissue homeostasis, disease development and progression. Bladder strongly relies on its mechanical properties to perform its physiological function, but these are poorly unveiled under normal and pathological conditions. Here we characterize the mechanical fingerprints at the micro-scale level of the three tissue layers which compose the healthy bladder wall, and identify modifications associated with the onset and progression of pathological conditions (i.e., actinic cystitis and bladder cancer). We use two indentation-based instruments (an Atomic Force Microscope and a nanoindenter) and compare the micromechanical maps with a comprehensive histological analysis. We find that the healthy bladder wall is a mechanically inhomogeneous tissue, with a gradient of increasing stiffness from the urothelium to the lamina propria, which gradually decreases when reaching the muscle outer layer. Stiffening in fibrotic tissues correlate with increased deposition of dense extracellular matrix in the lamina propria. An increase in tissue compliance is observed before the onset and invasion of the tumor. By providing high resolution micromechanical investigation of each tissue layer of the bladder, we depict the intrinsic mechanical heterogeneity of the layers of a healthy bladder as compared with the mechanical properties alterations associated with either actinic cystitis or bladder tumor.

Fig. 1. Micromechanical map of healthy bladder wall and aging effect on stiffness.

a YM by AFM represented in logarithm in base 10 (log10) in Pa for the different bladder tissue layers. b YM values obtained with the nanoindenter. The mechanical heterogeneity of the bladder correlates with its anatomical distribution: there is a gradient of YMs, being the urothelium the softest layer, with increasing stiffness when reaching the lamina propria, and then decreasing over the muscle layers. The association of stiffness gradients with the anatomical bladder tissue layers is shown by overlay of the mechanical map with hematoxylin-eosin staining. Blue pixels indicate rejected measurements. c Mean of the median values of a healthy rat bladder wall ± SEM, extracted by the two instruments. AFM and nanoindenter provide comparable results (2-way Anova test showed no statistically significant differences). For a complete comparison of between nanoindenter and AFM, the reader is referred to the Supplementary Fig. 4. d Evolution of the stiffness of the different bladder tissue layers (urothelium, lamina propria and muscle) with aging of the adult animal (AFM and nanoindenter data pulled together). The distribution of YM values for the whole tissue (i.e. all layers together) is also shown. N = 3 rats per time point, each tissue layer of each single rat is characterized by over 3000 force curve measurements. Data shown are logarithm in base 10 of YM’s (in Pa). e Mean of the median values of each rat ± SEM. Nonparametric Mann-Whitney test showed no statistical significant differences when comparing the different tissue layers at the studied time points. f Quantification of the bladder area that expresses collagen from control rats at different time points; each symbol represents the measurement in a tissue slice, with multiple slices measured for each bladder, in three animals. Source data behind graphs and charts can be found in https://figshare.com/projects/Micro-mechanical_fingerprints_of_the_rat_bladder_change_in_actinic_cystitis_and_tumor_presence/158222.

Fig. 2. Micromechanics of murine bladder in a model of actinic cystitis (X ray radiation).

a Schematic representation of the experiment: X-ray radiotherapy is used to induce actinic cystitis on the bladder. Rats are sacrificed at different time points ant tissue elasticity is assessed. b Representative bladder wall stiffness gradient collected with the nanoindenter at month 4: X-ray causes a stiffening of the whole bladder wall compared to non-treated healthy animals. Mechanical spatial differences within the fibrotic bladder are maintained and associated to the different tissue layers (U: urothelium, L: lamina propria, M: muscle). c Kinetics of YM from X-ray radiated bladders at different time points (red) and comparison to healthy animals of the same age (grey) measured both with nanoindenter and AFM. N = 3 rats per time point and condition, each tissue layer of each single rat was characterized by over 3000 force curve measurements. Data shown are logarithm in base 10 of YM’s (in Pa). d Fold of change of mean of the median log10 YM values of tissue layers of treated rats respect to age-matched control rats. T-test showed statistically significant stiffening with respect to the control animals (mean ± standard deviation with propagated error are shown. ns=not significant, *=p value < 0.05, **=p value < 0.005). e Mean of the median log10 YM values of tissue layers of each rat ± SEM. 2-way Anova showed no statistical significant differences in kinetics of fibrosis development. f) Quantification of the bladder area that expressed collagen from control rats and X ray-irradiated animals at different time points; each symbol represents the measurement in a tissue slice, with multiple slices measured for each bladder, in three animals. Source data behind graphs and charts can be found in https://figshare.com/projects/Micro-mechanical_fingerprints_of_the_rat_bladder_change_in_actinic_cystitis_and_tumor_presence/158222.

Fig. 3. Animal with no effect by X-ray treatment.

a The micromechanical profile (blue) was comparable to those of control healthy tissues of the same age (4 months after irradiation) (grey). b The histological analysis showed denser deposition of ECM (*) in an X-ray-responding animal (left) accompanied by increasing of stiffness. Histology on the bladder with no stiffening (right) revealed absence of fibrotic damage (4 months after irradiation). Source data behind graphs and charts can be found in https://figshare.com/projects/Micro-mechanical_fingerprints_of_the_rat_bladder_change_in_actinic_cystitis_and_tumor_presence/158222.

Fig. 4. Micromechanics of murine bladder in a model of bladder cancer (BBN).

a) Animal model establishment. Representative bladder wall stiffness gradients collected with nanoindenter at (b) 2 months of BBN treatment, where urothelium dysplasia is marked by *; (c) 4 months of BBN treatment, where pTa tumor limited to the urothelium without invading the lamina propria is marked by **. pT1 tumors in which urothelial tumor cells break the basal membrane and invade the lamina propria below at 4 and (d) 6 months BBN treatment are marked by ***. U: urothelium, L: lamina propria, M: muscle. YM’s from BBN treated bladders (black) at (e) 2 months, (f) 4 months and (g) 6 months of BBN treatment; and comparison to healthy animals of the same age (grey) measured both with nanoindenter and AFM. N = 3 rats per time point and condition, each tissue layer of each single rat is characterized by over 3000 force curve measurements. Data shown are logarithm in base 10 of YM’s (in Pa). h Fold of change of mean of the median log10 YM values of treated rats ± standard deviation (with propagated error) respect to control rats. T-test showed statistically significant softening with respect to the control animals (mean ± standard deviation with propagated error are shown. ns=not significant, *=p value < 0.05, **=p value < 0.005, ***=p value < 0.0005). i Mean of the median values of each rat. 2-way Anova showed that statistically significant differences in the BBN model were observed in the urothelium from month 2 to month 4, and from month 4 to month 6; and for the lamina propria from month 4 to month 6. Source data behind graphs and charts can be found in https://figshare.com/projects/Micro-mechanical_fingerprints_of_the_rat_bladder_change_in_actinic_cystitis_and_tumor_presence/158222.

Fig. 5. Micromechanics of high grade human bladder cancer.

a Hematoxylin-eosin staining of muscle invasive bladder cancer and paired non-neoplastic muscle tissue from a patient with high-grade urothelial carcinoma. b Mechanical profile of neoplastic (orange) and non-neoplastic (blue) muscle tissue from the same patient (n = 1). Data collected with the nanoindenter. Data are shown as logarithm in base 10 of YM’s (in Pa). Source data behind graphs and charts can be found in https://figshare.com/projects/Micro-mechanical_fingerprints_of_the_rat_bladder_change_in_actinic_cystitis_and_tumor_presence/158222.

Fig. 6. Methodological approach for bladder stiffness characterization.

Sample preparation protocol and pipeline of experiments. First, ultrasound (US) imaging is performed in order to determine the physiological OCT cryoprotectant bladder instillation volume. Animals are then sacrificed, the cryoprotectant is instilled into the bladder through a catheter and the bladder is then explanted and frozen. Cryosections are prepared and microindentation experiments are performed on the tissue slides both by AFM and nanoindenter. Afterwards, histology analysis was performed to confirm the location of the bladder anatomical layers.

Fig. 7. Indentation techniques. AFM (left) and Chiaro nanoindenter (right).

The main difference between both indentation instruments are the z piezo range, the detection of cantilever deflection and the fixed parameter during measurements.