Stability of the acetic acid-induced bladder irritation model in alpha chloralose-anesthetized female cats

Abstract

Time- and vehicle-related variability of bladder and urethral rhabdosphincter (URS) activity as well as cardiorespiratory and blood chemistry values were examined in the acetic acid-induced bladder irritation model in α-chloralose-anesthetized female cats. Additionally, bladder and urethra were evaluated histologically using Mason trichrome and toluidine blue staining. Urodynamic, cardiovascular and respiratory parameters were collected during intravesical saline infusion followed by acetic acid (0.5%) to irritate the bladder. One hour after starting acetic acid infusion, a protocol consisting of a cystometrogram, continuous infusion-induced rhythmic voiding contractions, and a 5 min "quiet period" (bladder emptied without infusion) was precisely repeated every 30 minutes. Administration of vehicle (saline i.v.) occurred 15 minutes after starting each of the first 7 cystometrograms and duloxetine (1mg/kg i.v.) after the 8(th). Acetic acid infusion into the bladder increased URS-EMG activity, bladder contraction frequency, and decreased contraction amplitude and capacity, compared to saline. Bladder activity and URS activity stabilized within 1 and 2 hours, respectively. Duloxetine administration significantly decreased bladder contraction frequency and increased URS-EMG activity to levels similar to previous reports. Cardiorespiratory parameters and blood gas levels remained consistent throughout the experiment. The epithelium of the bladder and urethra were greatly damaged and edema and infiltration of neutrophils in the lamina propria of urethra were observed. These data provide an ample evaluation of the health of the animals, stability of voiding function and appropriateness of the model for testing drugs designed to evaluate lower urinary tract as well as cardiovascular and respiratory systems function.

Figure 1. Experimental protocol.

A. Schematic of the protocol design. The bladder was infused with saline for 1h, followed by saline with 0.5% acetic acid for the remaining of the experiment. V1-V7 indicate vehicle (saline 0.5mg/kg) given i.v. every 30 minutes. D indicates duloxetine, 1mg/kg i.v. Numbers under grey boxes show time in hours from the beginning of the recordings. B. Inset illustrates the 30 minute four-step protocol, which is repeated throughout the experiment following each vehicle or drug (V/D) delivery. The steps are: post drug (PD), quiet period (QP), cystometrogram (CMG) and free run cystometry (FR). PD is a period of 10 minutes of continuous cystometry immediately following drug injection. After this period, the infusion pump is stopped and the bladder is drained and allowed to rest for 5 minutes, which defines the QP. At the end of the QP the bladder is drained again, the infusion pump turned on and a CMG is started. This period is followed by continuous cystometry (FR). CMG and FR periods total 15 minutes. # indicates bladder emptying. ▼ indicates time points for drawing blood samples (0.5ml) for blood gas analysis.

Figure 2. Typical recording traces.

A. Bladder pressure, URS-EMG activity and voided volume recorded while bladder was infused with saline followed by acetic acid. Grey boxes underneath bladder pressure trace indicate quiet periods. Letters inside grey boxes correspond to insets enlarged in B. Calibration bars are 10 mmHg for bladder pressure, 1 µV for URS-EMG activity, 2 ml for voided volume and 30 min for time scale. B. Enlargement of traces illustrating bladder and URS-EMG activity during QP and CMG periods at different time points during the experiment: a) in saline after 0.5 h; b, c) after 2.5 and 5h of acetic acid irritation, respectively; d) after duloxetine 1mg/kg. Scales for bladder pressure, voided volume and time are the same for all insets, 10 mmHg, 10 ml, 2 min, respectively, and are illustrated in inset a. The scale for URS-EMG is 0.1 µV and 0.5 µV in insets a, b-d respectively. Vehicle is saline (0.5 ml/kg i.v.). ^ indicates the time point when bladder infusion syringes were replenished. & indicates resetting the balance collecting the volume voided.

Figure 3. Summary of bladder parameters throughout the experiment.

A. Voiding frequency. B. Bladder contraction amplitude. C. Bladder capacity. Data included are from 12 cats. Vehicle is saline (0.5 ml/kg i.v.). Asterisks (*) represent statistically significant differences relative to saline in A and relative to V7 in B, tested with ANOVA followed by Dunn’s posthoc.

Figure 4. Summary of URS-EMG parameters.

A–D. URS-EMG expressed as RMS during QP (A, B) and CMG periods (C, D). A, C show averaged data from 12 cats and B, D show data from individual cats. E–H. URS-EMG expressed as spikes/s during QP (E,F) and CMG periods (G,H). E, G show averaged data from 12 cats and F, H show data from individual cats. I,J. Summary of URS-EMG normalized to V3 to illustrate stability from V3 to V7. Vehicle is saline (0.5 ml/kg i.v.). Asterisks (*) represent statistical significant values relative to S (saline) tested with ANOVA followed by Dunn’s posthoc test.

Figure 5. Time course of cardiovascular and respiratory parameters.

Raw values from individual cats (each line represents data from an animal) are shown for: . A. Mean arterial pressure (MAP). Data included are from 12 cats. B. Heart rate (HR). Data included are from 12 cats. C. Respiratory rate (RR). Data included are from 11 cats. Averaged values are shown in table 1.

Figure 6. Histological evaluation of bladder tissue.

A. Schematic of the preparation of bladder and urethra tissue for cryostat blocking and cutting. Bladder was cut in the longitudinal orientation. Urethra was cut in 4 blocks of 10 mm each, indicated by Proximal 1, Proximal 2, Mid and Vaginal, and transverse sections were cut from each block. B-E. Masson’s trichrome examples from a saline (B, C) and 0.5% acetic acid (D, E) infused bladder. Muscle tissue stains red/pink, collagen fibers stain blue, and nuclei stain black. B. 10x montage image of saline infused bladder tissue. Black box is enlarged in C. C. 40x image illustrating intact urothelium, sub-urothelium and lamina propria. D. 10x image montage of 0.5% acetic acid infused bladder tissue. Black box is enlarged in E. E. 40x image illustrating the lack of urothelium and edema in the sub-urothelium and lamina propria areas. F–G. Toluidine blue examples from a saline (F,G) and 0.5% acetic acid (H,I) infused bladder. F. 10x image montage of saline infused bladder tissue. Black box is enlarged in G. G. 40x image illustrating intact urothelium, sub-urothelium and lamina propria. H. 10x image montage of 0.5% acetic acid infused bladder tissue. Black box is enlarged in I. I. 40x image illustrating the lack of urothelium, edema and infiltration of presumed neutrophils and other cell types in the sub-urothelium and lamina propria areas. Scale bars are 1000 µm for B and D, 500 µm for F, H and 50 µm for C, E, G, I. Images in F, H are from adjacent sections of B, D respectively. Data included are from 2 cats with bladder irritation and 2 controls.

Figure 7. Histological evaluation of urethral tissue.

A-D. Masson’s trichrome examples from a saline (A, B) and 0.5% acetic acid (C, D) infused urethra. Sections shown are from mid part of the urethra. A. 10x image montage of saline infused urethral tissue. Black box is enlarged in B. B. 10x image illustrating intact epithelium, lamina propria/smooth muscle. C. 10x image montage of 0.5% acetic acid infused bladder tissue. Black box is enlarged in D. D. 10x image illustrating the lack of epithelium and edema in the lamina propria/smooth muscle area. E–J. Toluidine blue image montage examples from a saline (E–G) and 0.5% acetic acid (H–J) infused urethra. E. 10x image montage of saline infused urethral tissue. Black box is enlarged in F. F. 10x image illustrating intact epithelium, lamina propria/smooth muscle. G. 60x enlargement illustrating a presumed group of neutrophils. H. 10x image of 0.5% acetic acid infused bladder tissue. Black box is enlarged in I. I. 40x image illustrating the lack of epithelium, edema and infiltration of presumed neutrophils and other cell types in lamina propria/smooth muscle area. Note the increased number of presumed neutrophils. J. 60x enlargement illustrating a presumed group of neutrophils. Scale bars are 1000 µm for E, H, 500 µm for A, C, 200 µm for B, D, F, I and 20 µm for G, J Images in E, H are from adjacent sections of A, C respectively. Data included are from 2 cats with bladder irritation and 2 controls.