Partial urethral obstruction of rabbit urinary bladder: stereological evidence that the increase in muscle content is mostly driven by changes in number, rather than size, of smooth muscle cells

Abstract

The effects of partial urethral obstruction on the detrusor muscle of rabbit urinary bladder were investigated using stereological sampling and estimation tools. Twelve female Norfolk rabbits (2.5-3.0 kg body weight) were divided into four groups: 3, 7 and 12 weeks after surgical intervention to produce a standard partial obstruction and unobstructed controls. Following removal, bladder axes (craniocaudal, dorsoventral and laterolateral) and organ weights were recorded. Bladders were prepared for light microscopy by multistage random sampling procedures. Stereological methods were used to estimate the volume of muscle and the packing density and total number of myocyte nuclei in each bladder. We also estimated mean myocyte volume and the mean cross-sectional area and length of myocytes. Group comparisons were made by one-way analysis of variance. Changes in bladder axes were mainly laterolateral and craniocaudal. Mean bladder weight increased roughly six-fold by 3 weeks and 17-fold by 12 weeks and was accompanied, on average, by 12- and 33-fold increases in total muscle volume. These variables did not differ at 3 and 7 weeks post-obstruction. Increases in muscle content were not accompanied by changes in packing densities but were associated with increases in the total numbers of myocyte nuclei (13-fold by 3 weeks, 28-fold by 12 weeks). Mean myocyte volume did not vary significantly between groups but cells in obstructed groups were shorter and wider. These findings support the notion that partial outflow obstruction leads to an increase in the number, but not mean volume, of myocytes. If due solely to myocyte mitosis, the total of 43 x 10(8) cells found at 12 weeks could be generated by the original complement of 15 x 10(7) cells if an average of only 2.1 x 10(6) new cells was produced every hour. In reality, even this modest proliferation rate is unlikely to be achieved because myocyte proliferation rates are very low and it is possible that new myocytes can arise by differentiation of mesenchymal or other precursor cells.

Fig. 1

Schematic illustration of the operative procedure to induce partial urethral obstruction: (b) bladder, (u) urethra, (p) 3-mm Steinmann pin, (t) 2-0 nylon silk used to tie the ligature around the pin. Scale bar, 5 mm.

Fig. 2

Macroscopic appearance of rabbit urinary bladder. (A) unobstructed and (B) 12 weeks post-obstruction. Craniocaudal (CC) and laterolateral (LL) axes are indicated. In unobstructed bladders, group mean CC was 40.7 mm and LL was 23.0 mm. Corresponding values at 12 weeks post-obstruction were 78.8 mm and 85.7 mm, respectively (see text).

Fig. 3

Microscopical appearance of the detrusor muscle in resin sections: (A) unobstructed, (B) 3 weeks post-obstruction, (C) 7 weeks post-obstruction and (D) 12 weeks post-obstruction. Profiles of smooth muscle cells are indicated by arrows. Toluidine blue. All scale bars: 5 µm.

Fig. 4

Disectors (pairs of physical sections) used for counting myocyte nuclei in 7-week (A,B) and 12-week (C,D) obstructed bladders. The reference (A,C) and look-up (B,D) sections are separated by a spacing of 1 µm. Arrows show nuclei in reference sections which do not appear (arrowheads) in the partner look-up sections. Scale bars: 20 µm.