Functional and histologic imaging of urinary bladder wall after exposure to psychological stress and protamine sulfate

Abstract

To quantify the urinary bladder wall T₁ relaxation time (T₁) before and after the instillation contrast mixture in rats previously subjected to water avoidance stress (WAS) and/or acute exposure to protamine sulfate (PS). Female Wistar rats were randomized to receive either sham (control) or 1 h of WAS for ten consecutive days before the evaluation of nocturnal urination pattern in metabolic cages. T₁ mapping of urinary bladder wall at 9.4 T was performed pre- and post- instillation of 4 mM Gadobutrol in a mixture with 5 mM Ferumoxytol. Subsequently, either T₁ mapping was repeated after brief intravesical PS exposure or the animals were sacrificed for histology and analyzing the mucosal levels of mRNA. Compared to the control group, WAS exposure decreased the single void urine volume and shortened the post-contrast T₁ relaxation time of mucosa- used to compute relatively higher ingress of instilled Gadobutrol. Compromised permeability in WAS group was corroborated by the urothelial denudation, edema and ZO-1 downregulation. PS exposure doubled the baseline ingress of Gadobutrol in both groups. These findings confirm that psychological stress compromises the paracellular permeability of bladder mucosa and its non-invasive assay with MRI was validated by PS exposure.

Figure 1

Effect of WAS and PS on voxel-wise T₁ mapping- T₁ weighted images (TR/TE 1410/7ms) acquired before the instillation of CM (pre-contrast), after the instillation of CM but prior to the exposure to protamine sulfate PS (post-contrast) and after PS exposure in the rat bladder from control (A–C) and WAS group (D–F). To derive the functional meaning of a slight increase in the signal intensity of WAS bladder wall, color coded, voxel-wise T₁ maps with volumetric coverage were constructed from the mono exponential fit of signal in T₁ weighted images acquired at six different TR values of 400–10,000 ms using aRARE factor of 2 and signal averages of 2. Just as the traditional histochemical stains reveal the biochemical differences between tissue layers, the color-coded T₁ maps with the resolution of 50 µm for each pixel after zero filling displays the three individual layers of mucosa (U), lamina propria (L) and detrusor (D) in bladder wall differentiated by the physical parameter of T₁ relaxation time mapped to voxels sized 0.1 × 0.1 × 0.7 mm³. The relative ingress of extracellular T₁ shortening agent, Gadobutrol in the post-contrast T₁ maps expanded the spatial separation between pixels displaying the layers of U and L (Eii vs Bii) and predicted an expansion of extracellular space in L of WAS group compared to controls. The spatial separation between U and L of WAS group increased further after a brief PS exposure (Fii vs Cii).

Figure 2

Gadobutrol Permeability Derived from Voxel-wise T₁ mapping: (A) To derive the Gadobutrol concentration [Gd] in the bladder mucosa using the Eq. (1), we first estimated the T₁ relaxation rate constant or relaxivity (r₁) of Gadobutrol at 9.4 T by the linear fitting of the reciprocal of the water T₁ relaxation time or T₁ water relaxation rate (1/T₁) measured in the serial two fold dilutions of [Gd] 2 mM at 37 °C. Linear fitting generated the regression equation: y = 3.576x + 0.1975 with the coefficient of determination (R²) of 0.999 and r₁ value of 3.576 L/mmol/s for Gadobutrol at 9.4 T. In contrast to the linear relationship between T₁ water relaxation rate of the phosphate buffered saline (PBS) vial (blank) supplemented with ascending concentrations of Gadobutrol from 0.1-2 mM [Gd], the non-linearity between [Gd] and signal intensity is illustrated by the doubling of [Gd] from 1 to 2 mM producing only a minor change in the signal intensity of T₁ weighted images acquired at TR/TE of 640/14 ms. (B) Compared to control group, the post-contrast T₁ relaxation time computed from 20 pixels in ROI of mucosa* was significantly shorter in WAS group (*p < 0.005, two-way ANOVA followed by Sidak’s test) and T₁ relaxation time was further shortened upon PS exposure (0.5 mL of 1%w/v) for 30 min. (C) Higher mucosal permeability in WAS group is indexed by a significantly higher ingress of Gadobutrol derived from the non-invasive, quantitative measurement of increased T₁ water relaxation rates (n = 5; *p < 0.001, two-way ANOVA and Sidak’s test) which is doubled from the respective pre-PS levels by PS exposure in both groups. (D) Bladder wall depth of instilled Gadobutrol penetration was evaluated by measuring the spatial separation between U and L on post-contrast and post-protamine T₁ maps. The penetration depth of Gadobutrol after PS in WAS group was significantly larger than in the control group *p < 0.05. All values are expressed as Mean ± SD.

Figure 3

MRI successfully visualizes the sandwich of lamina propria (L) layer between the tissue layers of mucosa (U) and detrusor (D) and spatial separation of layers on MRI predicted the histological differences noted on H&E staining of rat bladder harvested prior to PS exposure from both groups. Compared to the histology of control rats (A–C), tissue sections from WAS group (B–D) exhibited a relative thinning of mucosa (U) with the decrease in the number of cell layers, expansion of lamina propria (L) from edema as evident from the relative expansion of L and D layers of WAS group compared to the control group (bladder wall thickening), focal areas of mononuclear inflammatory cells (*), venous congestion and elongated blood vessels (telangiectasia ∇) together supporting a prominent role for inflammation in WAS group even without PS exposure (D). (E) A significant elevation in the median semi-quantitative score of inflammation in WAS group relative to the control group (plot of median and interquartile range as error bars, n = 4, *p < 0.0001, Mann–Whitney Test) reflects the conspicuous absence of inflammatory cells in control group (A–C), a circular shape (x) instead of elongated blood vessels and mucosa with multiple layers and thinner bladder wall signifying a relative absence of edema compared to WAS group (B–D). (F) WAS exposure evokes barrier dysfunction per se even in the absence of PS exposure as evident from histological analysis and the thinning of mucosal layers is corroborated by the downregulation of tight junction protein ZO-1 in the mucosa of WAS group. Values are Mean ± SD; *p < 0.05, unpaired t test.