Increased extracellular matrix stiffness accompanies compromised bladder function in a murine model of radiation cystitis

Abstract

Radiation cystitis, a long-term bladder defect due to pelvic radiation therapy, results in lower urinary tract symptoms, such as urinary frequency and nocturia, suggestive of compromised bladder compliance. The goal of this study was to identify alterations to the mechanical behavior of the urinary bladder extracellular matrix of a murine model of radiation cystitis, at 3 and 6 months after radiation exposure. The results of this study demonstrated that the extracellular matrix of irradiated bladders was significantly less distensible when compared to age matching controls. These findings coincided with functional bladder changes, including increased number of voids and decreased voided volume. Both mechanical and functional changes were apparent at 3 months post-irradiation and were statistically significant at 6 months, demonstrating the progressive nature of radiation cystitis. Overall, the results of this study indicate that irradiation exposure changes both the mechanical and physiological properties of the bladder.

Graphical Abstract

In humans, radiation cystitis results in lower urinary tract symptoms, such as urinary frequency and nocturia, suggestive of compromised bladder compliance. This pathology can significantly affect recovery and quality of life for cancer survivors. Gaining knowledge about how alterations to the mechanical behavior of the urinary bladder extracellular matrix can affect urinary function will have a significant impact on this population. The results of this study demonstrated that the extracellular matrix of irradiated bladders was significantly less distensible when compared to age matching controls, in a mouse model of radiation cystitis. These findings were accompanied by functional voiding changes, including increased number of voids and decreased voided volume. The results of this study uncovered that irradiation exposure changes the mechanical and physiological properties of the bladder.

INTRODUCTION

Radiation cystitis (RC) is a chronic bladder condition characterized by frequent urination, nocturia, urinary incontinence, pelvic pain, and hematuria. RC is the result of radiation therapy used to treat pelvic cancers such as prostate, cervical, colorectal, and ovarian cancer. Approximately 10% of cancer survivors treated with pelvic radiation therapy will develop RC [1]. Current treatment options are focused on arresting bleeding; in mild cases, these include rest, hydration, and bladder irrigation; in severe cases, these include instillation with astringent agents or formalin and bladder cystectomy. However, no therapies are currently available to treat or reverse other symptoms of RC such as urinary frequency and nocturia [2–4].

We previously developed a preclinical RC model that mimics the human condition [5,6]. In this model, we have shown that radiation exposure results in increased deposition of collagen (i.e., fibrosis) within the bladder detrusor muscle. In irradiated bladders, the extracellular matrix (ECM) consists of a higher concentration of collagen type I and III fibers when compared to controls. These are two types of collagen fibers that play an important role in tissue remodeling in response to disease, not just in the bladder but in several soft tissues [7]. By 12 months post-irradiation, radiation exposure still resulted in increased expression of collagen I in comparison to littermate controls, supporting the notion that RC is a progressive condition [5].

It has been suggested that bladder fibrosis alters bladder compliance, resulting in lower urinary tract symptoms such as urinary frequency and nocturia[8]. However, no study has been done to investigate changes in mechanical properties of the bladder in response to radiation. This study aims to identify alterations to the mechanical behavior of urinary bladder tissue following radiation exposure in a murine model. Specifically, we aim to characterize these changes through mechanical testing and analysis, by estimating linearized tissue stiffness at several points of interest along the stress-stretch curve. We also employed a continuum mechanics model to estimate material parameters. This model has been employed to describe several soft tissues in the past [9–11], including the urinary bladder [12]. To our knowledge, this study is the first-of-its-kind to present mechanical data on bladder tissue after radiation exposure.

METHODS

Bladder radiation treatment

This study was performed with full approval from the Beaumont Institutional Animal Care and Use Committee (AL-20-04), in compliance with the NIH Guide for the Care and Use of Laboratory Animals. Animals were housed, treated, and cared for in an AAALAC accredited facility. RC was induced in the mouse bladder as previously described [5]. Forty 8-week old female C57Bl/6 mice were purchased from Charles River (Wilmington, MA). Mice were randomly assigned to an irradiated or control treatment group (n = 20/group). Radiation was delivered to the mouse bladder using the Small Animal Radiation Research Platform (SARRP; Xstrahl, Suwanee, GA) using a two-beam approach. For radiation treatment, anesthesia was induced using 2.5–3% isoflurane through inhalation and maintained at 1.5–2% throughout the procedure (30–45 minutes). Mice were placed on the SARRP platform and a CT image was taken to localize the bladder. The CT image was used to determine the placement of the two beams and assure the whole bladder was irradiated. All care was taken to avoid the spinal cord, the long bones, colon and overlap of entrance and exit beams to help minimize damage to other organs such as the skin. Mice received a single dose of 40 Gy, evenly divided over the two beams, using a 5 × 5 collimator. Mouse breathing and heart rate was continuously monitored during the procedure. After radiation treatment, mice were placed in a heated recovery cage and returned to regular housing when fully recovered, where they received mash and hydrogel for 7 days. Untreated mice were anesthetized for the same duration of time as their irradiated littermate controls.

Void Spot Assay

Bladder function was assessed at baseline, and 3- and 6 months post irradiation using the Void Spot Assay (VSA) as previously described [5,13]. In short, mice were singly housed for 4 hours in a cage lined with thick filter paper. During the assay, mice had access to food, but water was withheld. To minimize destruction of the filter paper, mice were given a small rounded and open bottom house, as well as a short piece of string paper. After 4 hours, mice were returned to regular housing, filter paper was placed on an XcitaBlue Conversion Screen (BioRad Cat# 1708182), and urine spots were visualized under TransUV light. Using Fuji Software, urine spots were counted, and total and average void volumes were calculated.

Tissue preparation

Three- and six-months post-irradiation, animals were euthanized, and bladders collected. Ten irradiated and ten age-matched control animals were sacrificed at each time point. Bladders were placed in transportation media (Krebs-Hensleit buffer) and shipped on ice overnight to Michigan State University. The calcium-free Krebs-Hensleit buffer used in this study contains (in g/L distilled water): 2 C6H12O6, 0.141 MgSO4, 0.16 KH2PO4, 0.35 KCl, 6.9 NaCl, 2.1 NaHCO3. Upon arrival, each bladder was cut into ring-shaped samples by removing the dome and trigone, leaving behind a lateral ring. The rings were decellularized, as previously described [12,14]. This approach has been used in the past to isolate the contribution of specific constituents to the mechanics of the tissue overall [12,15]. Briefly, rings were soaked in phosphate-buffered saline containing heparin sodium salt for 15 minutes, 1% sodium dodecyl sulfate for 48 hours, deionized water for 15 minutes, and 1% Triton X-100 for 30 minutes. Samples were decellularized for two reasons: (1) to focus on the altered mechanical behavior of the remodeled ECM, and (2) to remove interference of possible contracting smooth muscle cells during mechanical testing.

Bladder ring radius measurement

An effective radius was measured for each tissue ring, before and after decellularization. A photograph of the tissue was taken from above including also measuring tape for scaling. The photo was then converted to greyscale in post-processing. The inner and outer circumference of each sample was traced (NeuronJ plugin for ImageJ), and the length of each tracing was measured in pixels and converted to millimeters. The inner and outer radius of each ring was calculated from its circumference, and the average of these two radii was calculated (i.e., the effective radius). An effective radius is used rather than the inner and outer radii to negate the effect of bladder wall folding into itself in photographs.

Mechanical testing

All mechanical testing was performed in a custom-built saline bath containing calcium-free 300m Osm Krebs-Hensleit buffer to ensure proper hydration of the tissue. The samples were mounted onto a uniaxial machine using black cotton thread to secure the sample to the saline bath/uniaxial machine on the bottom and to a load cell (FUTEK, LSB200, 250g capacity) at the top. Cameras were mounted facing the front (Hitachi KP-M2A) and side (iPhone, 12MP) of the sample to capture the front and side profile of the sample. The uniaxial machine’s DC motor, along with the cameras and load cell were controlled through LabView. For a schematic of the experimental set-up, see Fig. 1.

Figure 1: Experimental set-up for uniaxial testing.

On the left, schematic of the custom-built mechanical stretcher. On the right, representative set of front and side view of the sample during testing. Also highlighted are the geomteric charcateristics quantified before each loading cycle, i.e., (all measure in pre-loaded samples). The initial cross-sectional area is evaluated as the width multiplied by the depth of the sample.

A mechanical testing protocol loading, adapted from a previous work [12], was applied to the samples as follows; first, 10 loading-unloading cycles (preconditioning) to a maximum stretch of 1.05 (i.e., 5% elongation), followed by three cyclical loading protocols. Each cyclical loading protocol consisted of 5 cycles of loading-unloading to maximum stretch of 1.1, 1.15, and 1.2, respectively (i.e., 10%, 15%, and 20% elongation). All loading and unloading were performed at a rate of 0.01 s⁻¹ [16,17]. Before each set of cycles, a pre-load of 2 ± 0.25g was placed onto the sample and the reference length (L₀) was estimated. This length was used to estimate the displacement to be applied to each sample to achieve the testing maximum stretch for each loading protocol. During the loading cycles, the height and axial force were continuously measured via the front-facing camera and the load cell, respectively. Pictures were taken of the sample from the front and side before each set of cycles (after application of the pre-load) to measure the initial width and depth of the samples to estimate the initial cross-sectional area.

Samples were assumed to be incompressible and isotropic, resulting in a deformation gradient of $\mathit{F}=diag\left[\lambda ;\raisebox{1ex}{$1$}\!\left/ \!\raisebox{-1ex}{$\sqrt{\lambda }$}\right.;\raisebox{1ex}{$1$}\!\left/ \!\raisebox{-1ex}{$\sqrt{\lambda }$}\right.\right]$ where λ is the stretch in the direction of testing (i.e., circumferential). Following the classic theory of continuum mechanics in large deformations [18–21], we define stretch as $\lambda =\frac{l}{L_0}$, where L₀ represents the reference length appropriate for the loading cycle, and l represents the deformed length throughout the loading cycle. Due to the hypothesis of incompressibility, it follows that the width and depth of the sample scale respectively throughout the test by $\raisebox{1ex}{$1$}\!\left/ \!\raisebox{-1ex}{$\sqrt{\lambda }$}\right.$. The designated reference configuration was based on the geometry (initial cross-sectional area A₀ and initial length L₀) measured before each set of cycles, after pre-load. For more details on the identification of the appropriate reference geometry and an in-depth discussion of the repercussions of this choice on the calculated mechanical properties, see our previous work [12]. Then, the Cauchy stress in the uniaxial direction is evaluated as the ratio between the axial force, read by the load cell, and the current cross-sectional area (i.e., A = A₀/λ) throughout the loading test.

Constitutive modeling framework

For each tested sample, the final loading curve of the last loading cycle (i.e., target stretch of 1.2) was used for mechanical analysis. We described the tissue employing a two-parameter, Demiray constitutive model [9,10,12],

$$W(\mathit{C})=\frac{c_1}{2c_2}\left(e^{c_2(\mathrm{tr}(\mathit{C})-3)}-1\right)$$ (1)

where c₁ and c₂ are material parameters (c₁ has the dimension of a stress and c₂ is nondimensional), C is the right Cauchy-Green deformation tensor defined as C = F^TF, and I₁(C) = tr(C) is C first invariant. Following, the definition of Cauchy stress for an incompressible material is:

$$\mathit{T}=-p\mathit{I}+2\mathit{F}\frac{\partial W}{\partial \mathit{C}}{\mathit{F}}^{\mathit{T}}$$ (2)

where p is a Lagrange multiplier and I is the identity tensor. For a uniaxial ring test accompanied by appropriate no-load boundary conditions in the transverse directions, the Cauchy stress in the circumferential direction (i.e., direction of testing) is:

$$T=c_1\left({\lambda }^2-\raisebox{1ex}{$1$}\!\left/ \!\raisebox{-1ex}{$\lambda $}\right.\right)\left[exp\left(c_2\left({\lambda }^2+\raisebox{1ex}{$2$}\!\left/ \!\raisebox{-1ex}{$\lambda $}\right.-3\right)\right)\right].$$ (3)

Finally, optimal values for parameters c₁ and c₂ were estimated for each sample by minimizing the normalized root mean square deviation between the behavior predicted using Equation (3) and the data collected experimentally for the final loading curve of the last loading cycle. The minimization was performed through the MATLAB function ‘lsqnonlin,’ as previously described by Chen et. al. [22]. The constitutive model parameters estimation resulted in values of root mean square deviations between model prediction and experimental data included in the interval 2–11% for all samples.

Mechanical parameters and stiffness calculations

To characterize how the mechanical behavior of the ECM may be changing with respect to RC, we also measured several discrete characteristics of the stress-stretch curves. These characteristics were measured using a method adapted from a previously published work [17], wherein the model curves for all (produced by Equation 3) are simulated from zero stress to 100kPa, as this is the physiological maximum the bladder may experience [23]. These curves were then separated into three regions: low-stiffness and high-stiffness linear regions and transition region. The low-stiffness linear region was defined as the region between the points λ = 1 and λ = λ₁ The value of the stretch λ₁ is estimated by, first, fitting a straight line between the first and second points of the stress-stretch curve (i.e., the first point is represented by λ = 1; t = 0). Then, increasing values of stretch (and corresponding Cauchy stress) are added incrementally until the linear fit produces a R² value smaller than 0.999. This defines the upper boundary of the low-stiffness linear region, λ₁. The high-stiffness linear region is defined following a similar process, starting from λ_max (defined as the stretch corresponding to a Cauchy stress of 100 kPa) and incrementally adding points for decreasing values of stretch until the linear fit produces a R² value smaller than 0.999. This defines the lower boundary of the high-stiffness linear region, λ₂. The Cauchy stresses for stretch values of λ₁ and λ₂, namely t₁ and t₂, were also calculated. Lower and upper stiffnesses of the tissue, namely k_low and k_high, were estimated as the slopes of these two semi-linear regions, respectively. Additionally, to characterize the non-linearity of the stress-stretch curve, we also located the point at which the two linear portions of the curves would intersect, i.e., λ_int and t_int. For a schematic representation of these parameters on a representative curve see Fig. 2.

Figure 2: Schematic of mechanical parameters, low- and high-stiffness regions and transition region boundaries.

Identified in the schematic are: a) low-stiffness linear region’s stress and stretch boundary and lower stiffness parameter (i.e., λ₁, t₁, and k_low); b) high-stiffness linear region’s stress and stretch boundary and upper stiffness parameter (i.e., λ₂, t₂, and k_high); c) the transition region (shaded area); d) the linear intersection point’s stretch and stress (i.e., λ_int and t_int; identified as a star). All mechanical parameters considered are identified on a representative stress-stretch curve from a control animal 3-months post-sham treatment.

Statistical analysis

Statistical analysis was performed using GraphPad Prism version 9.0.2. Two-way ANOVA was used to determine time (3 months versus 6 months) or treatment (control versus irradiated) dependent changes. Significant P-values (P < 0.05) obtained with two-way ANOVA were further analyzed using the Bonferroni’s post-hoc multiple comparison test.

RESULTS

Radiation treatment was well-tolerated by all animals

All animals were closely monitored during and following radiation treatment for any acute side-effects. No change in body weight or behavior was noted after radiation exposure. The irradiated mice developed skin discoloration at beam entrance and exit sites by 10 weeks after irradiation, as shown previously [5]. Two mice, 1 irradiated and 1 untreated, were lost prior to the 6-months time point due to complications unrelated to irradiation. Approximately 30% of mice developed alopecia, which is inherent to the inbred C57BL/6 strain [24, 25].

Decellularization increases bladder wall radius

The process of decellularization significantly increased the bladder wall radius for both untreated and irradiated animals at all times (Fig. 3A–B, 2-way ANOVA P < 0.001 for all groups). This result is to be expected, since smooth muscle cells and collagen fibers are physically intertwined in the intact tissue [26]. Decellularization has been reported to alter the zero-load reference state of the bladder ECM in rats before [12]. We observed no changes in radius related to time or treatment for all comparisons.

Figure 3: Decellularization increases effective radius.

(A) Calculation of effective radius (r_eff) was performed using the NeuronJ plugin for ImageJ. (B) Average effective radius for bladders for the untreated (empty bars) and irradiated (filled bars) groups at 3-months (left) and 6-months (right) post-irradiation. Results shown for both intact tissue and isolated ECM. Two-way ANOVA was performed followed by Bonferroni’s post-hoc test correcting for multiple comparisons. ***P < 0.001 comparing intact to decellularized tissue.

Irradiation induces decreased distensibility of bladder ECM

We sought to determine the changes to bladder ECM mechanics in terms of a constitutive model. For each sample, the final loading curve for the last loading cycle curve was interpolated into 10 points within the highest common range of stretch, to allow for the evaluation of an average curve for each group. At 3 months post-radiation, there were no significant differences between the untreated and irradiated tissues (Fig. 4A). At 6 months, for larger values of stretch, the irradiated tissue showed a significantly higher value of stress for the same value of stretch than the untreated tissue, indicating a decrease in distensibility of the ECM induced by the radiation treatment (Fig. 4B). There were no changes in either the untreated or the irradiated groups related to time.

Figure 4: Irradiation induces increased stress-stretch curves.

Average Cauchy stress-stretch curves for decellularized bladders from untreated (empty circles) and irradiated (filled circles) animals collected 3-months (A) and 6-months (B) post-treatment. Error bars represent standard error mean. Two-way ANOVA was performed followed by Bonferroni’s post-hoc test correcting for multiple comparisons. *P < 0.05, ***P < 0.001 when comparing untreated and treated animals at each stretch.

The material parameters estimated support this finding. While there were no changes to parameter c₁ both in regard to time and radiation treatment (Fig. 5A), parameter c₂ was consistently higher in the irradiated tissue compared to untreated tissue (Fig. 5B; 2-way ANOVA P = 0.0015). This increase was not significant at the 3 months timepoint (P = 0.15) and significant at the 6 months timepoint (P = 0.0088). This supports the time dependent nature of RC that has been reported in the past [5]. In the model considered here, an increase in c₂ is consistent with an increase in tissue stiffness for larger values of stretch/stress.

Figure 5: Constitutive model parameter affecting mechanical behavior at high-stretch is increased in response to irradiation.

Average constitutive model parameters, c (kPa) and k (−), estimated for tissue from irradiated animals (filled bars) and for untreated animals (empty bars) collected 3-months (A) and 6-months (B) post-treatment. Error bars represent standard error mean. Two-way ANOVA was performed followed by Bonferroni’s post-hoc test correcting for multiple comparisons. **P < 0.01 when comparing untreated and irradiated animals at each time point.

Shifted linear and transition regimes and increased stiffness in response to irradiation

The analysis of the mechanical parameters considered (Fig. 2) can shed light on what are the areas of the ECM stretch-stress curves that are mostly altered by irradiation. In general, for all stretch parameters calculated (i.e., λ₁, λ₂, λ_int and λ_max) irradiation significantly lowered values independent of timepoint (Fig. 6A; 2-way ANOVA p < 0.05 for each stretch parameter). Bonferoni’s post hoc test correcting for multiple comparisons revealed no statistical difference between treatment groups at 3 months and were trending towards significance at 6 months (0.1 > P > 0.05). The corresponding stress values for these points (t₁, t₂, t_int, and t_max) showed no significant changes with radiation treatment (Fig. 6B). For all parameters, there were no significant changes between the 3-month and 6-months timepoints. Finally, both low-stress and high-stress stiffnesses assumed larger values on average in the irradiated groups compared to the untreated (Fig. 6C). This increase in stiffness, however, was only significant for the high-stress region stiffness parameter (2-way ANOVA irradiation effect P = 0.0042), and specifically at 6-months post-treatment (Bonferoni’s posthoc test P = 0.0156).

Figure 6: Irradiation induces a left-word shift of the linear and transition regimes and increases upper stiffness.

Average mechanical parameters (see points of interest definition in Fig 3.) estimated for tissue from irradiated animals (darker bars) and for untreated animals (lighter bars) collected 3-months (filled bars) and 6-months (shaded bars) post-treatment. A) Stretch limits for low- and high-stiffness linear regions (λ₁, λ₂), intersection point (λ_int), and for the maximum value of stress (λ_max). B) Cauchy stress limits for low- and high-stiffness linear regions (t₁,t₂), intersection point (t_int), and maximum value of stress (t_max). Note that t_max is set to 100kPa for all samples. Error bars represent standard error mean. Statistical analysis was performed with 2-way ANOVA followed by Bonferroni’s post-hoc test correcting for multiple comparisons. P-values: ° 0.1 > p > 0.05, * p < 0.05 when comparing untreated and irradiated animals at each time point.

Radiation exposure induces increased voiding frequency and decreased voiding size

To determine if irradiation-induced bladder stiffness alters bladder function, we performed VSAs on untreated and irradiated mice. The VSA is a non-invasive commonly used test to assess bladder function in rodents [13, 27]. Over a four-hours period, mice exposed to irradiation had an increased number of urine spots in comparison to littermate controls (Fig. 7A). In addition, irradiated mice voided smaller volumes as noted by the average voided volume, without significantly changing the total voided volume during the assay (Fig. 7B–C). The change in micturition behavior started at 3-months post-irradiation and became more apparent and significant by 6-months.

Figure 7: Irradiation exposure to the bladder induces long-term urinary frequency and decreased void volume.

Untreated (empty bars) and irradiated (filled bars) animals were subjected to void spot assay, and (A) number of voids, (B) average void volume, and (C) total void volume were quantified before treatment (BL) and 3-months and 6-months post-treatment. Error bars represent standard error mean. Two-way ANOVA was performed followed by Bonferroni’s post-hoc test correcting for multiple comparisons. *P < 0.05, *** P < 0.001 when comparing untreated and irradiated animals at each time point.

DISCUSSION

Radiation treatment can impact bladder function and induce lower urinary tract dysfunction in pelvic cancer survivors. We sought to determine if radiation altered the mechanical properties of the bladder in a murine model. This study specifically examined ECM remodeling and mechanical behavior of the bladder ECM. To do so, we measured several mechanical and physiological parameters. Overall, we found that radiation causes an increase in ECM stiffness (Fig. 5 and 6C) and impaired bladder function as measured by increased urinary frequency and decreased void volume (Fig. 7). Early changes were detectable at 3-months post-treatment, and reached statistical significance by 6-months, indicating the progressive nature of RC.

Average stress-stretch curves of the irradiated animals showed overall lower distensibility as compared to untreated (Fig. 4). This was accompanied by the constitutive model parameters analysis, which showed no changes to parameter c₁, which dominates mechanical behavior in the low-stiffness region, and at the same time highlighted an increase of the parameter c₂ in irradiated tissues, which dominates behavior in the high-stiffness region (Fig. 2 and 6C). Overall, these results indicate that RC causes a progressive increase in stiffness in the ECM of the bladder that is focused on the upper-end of the stress-stretch curve, when the bladder would be nearing maximum distension from urine filling.

The concept of radiation-induced stiffening at in the high stress/stretch region is reinforced by the boundaries and stiffnesses of the regimes measured from the stress-stretch curves (Fig. 6A and B). While the stress-value of the boundaries is conserved across treatment and recovery time (i.e., low- and high-stiffness linear regions, intersection, and maximum stress), each point of interest considered is characterized by a value of stretch that is decreased by radiation treatment by the 6-months timepoint. Additionally, the stiffnesses of both quasi-linear regions is increased with radiation, yet this difference is only significant in the high-stiffness linear region at 6-months post-treatment. This type of alteration to the stress-stretch behavior of the ECM could be indicative of a decreased bladder capacity, a common hallmark of RC [28], as the tissue is reaching maximum in vivo stress at a much lower stretch. This is supported in that the irradiated mice also voided smaller volumes of urine per void event, without significantly changing their total urine output (Fig. 7B). This is not reflected in the estimation of the effective radius reported here, when comparing intact tissue between treated and untreated mice at the 6 months timepoint. This measure, however, only reflects changes in the circumferential direction and does not necessarily reflect the overall change in volume.

Moreover, the result of the VSA also confirmed that radiation results in a compromised bladder capacity. Irradiated animals had an increased number of voids that were smaller in size than untreated animals (Fig. 7A). Decreased bladder capacity after irradiation has been described using several different models [5, 6, 8, 29] and is a known complication in pelvic cancer survivors suffering from RC [28]. Loss of functional bladder capacity, as indicated by decreased flow rate, voided volume, and bladder capacity, is also common in the aging bladder and is attributed to an accumulation of ECM in the bladder wall [30, 31]. In a previous study, using a preclinical model of RC, we demonstrated that irradiated bladders are enriched in collagen I and III [5]. Even more so, collagen I expression was still elevated 1 year after radiation exposure, indicating the gradual accumulation of fibrosis over time [5]. Similarly, in a study done by Ikeda and colleagues, bladder irradiation resulted in chronic fibrosis, which was associated with decreased bladder capacity, decreased detrusor contractility and overflow incontinence in mice [8]. Treatment with relaxin-2 for 2 weeks brought collagen content back to baseline levels, and restored functional bladder capacity and detrusor contractility. Thus, these studies support the correlation between irradiation, fibrosis accumulation, and compromised bladder capacity.

One limitation of this study is that both the discrete stiffness parameters (i.e., k_high and k_low) as well as the material parameters from the continuum model (i.e., c and k), quantify the bladder’s behavior exclusively in the circumferential direction. Moreover, the continuum model is based on an assumption of the tissue behaving in an isotropic manner. Several studies have, however, reported before on how bladder tissue behaves in an anisotropic manner and how the anisotropy may be affected by tissue remodeling in disease [32–37]. Nonetheless, isotropic models similar to the one used here have been often used in prior studies to describe the bladder mechanical behavior [17, 38].

Another possible limitation stems from the variability associated with measuring bladder function using the VSA. A multitude of factors impact voiding functions and consequently the results of this assay, such as time of day, water consumption, hormone levels, any stresses from outside [39, 40]. That’s why it is important that these studies are controlled and that the assay is run at same time of day. We also make sure we have mixed cages (i.e., mice that are treated and untreated mixed together in each cage) as there can be hierarchy issues between mice. So, while we recognize there is an inherent variability from assay to assay, we have done our upmost best to remove as many variables as possible.

CONCLUSIONS

This study is the first to show the mechanical properties of the bladder, as well as bladder function, are altered after irradiation. Radiation, and subsequent changes in ECM content, significantly increases the stress on bladder tissue as the bladder fills with urine and subsequently stretches. These changes are accompanied by reduced functional bladder capacity and more frequent urination.