Early Detrusor Application of Botulinum Toxin A Results in Reduced Bladder Hypertrophy and Fibrosis after Spinal Cord Injury in a Rodent Model

Abstract

Following spinal cord injury (SCI), pathological reflexes develop that result in altered bladder function and sphincter dis-coordination, with accompanying changes in the detrusor. Bladder chemodenervation is known to ablate the pathological reflexes, but the resultant effects on the bladder tissue are poorly defined. In a rodent model of contusion SCI, we examined the effect of early bladder chemodenervation with botulinum toxin A (BoNT-A) on bladder histopathology and collagen deposition. Adult female Long Evans rats were given a severe contusion SCI at spinal level T9. The SCI rats immediately underwent open laparotomy and received detrusor injections of either BoNT-A (10 U/animal) or saline. At eight weeks post injury, the bladders were collected, weighed, and examined histologically. BoNT-A injected bladders of SCI rats (SCI + BoNT-A) weighed significantly less than saline injected bladders of SCI rats (SCI + saline) (241 ± 25 mg vs. 183 ± 42 mg; p < 0.05). Histological analyses showed that SCI resulted in significantly thicker bladder walls due to detrusor hypertrophy and fibrosis compared to bladders from uninjured animals (339 ± 89.0 μm vs. 193 ± 47.9 μm; p < 0.0001). SCI + BoNT-A animals had significantly thinner bladder walls compared to SCI + saline animals (202 ± 55.4 μm vs. 339 ± 89.0 μm; p < 0.0001). SCI + BoNT-A animals had collagen organization in the bladder walls similar to that of uninjured animals. Detrusor chemodenervation soon after SCI appears to preserve bladder tissue integrity by reducing the development of detrusor fibrosis and hypertrophy associated with SCI.

Keywords: bladder hypertrophy; botulinum toxin; spinal cord injury.

Figure 1

Acute detrusor BoNT-A injections after SCI resulted in lighter bladders. (A) Detrusor BoNT-A injections results in significantly lighter bladders after SCI. (* p < 0.05, ** p < 0.01). (B) Representative photos bladders from the spinal intact and experimental groups showing visible differences between bladder sizes.

Figure 2

Acute detrusor BoNT-A injections after SCI resulted in reduced fibrosis and bladder wall thickening. Representative photomicrographs of Hematoxylin and Eosin (H&E) staining of bladder sections from spinal intact Sham (n = 5), SCI + Saline (n = 8) and SCI + BoNT-A (n= 8) groups at 8 wpi. Asterisk (*) in histology images denotes bladder lumen. SCI₊ Saline group had significantly thicker bladder walls compared to spinal intact animals (****, p < 0.0001). SCI + BoNT-A group had significantly thinner bladders compared to the bladders of SCI + Saline animals (****, p < 0.0001). Scale bar = 100 micrometer.

Figure 3

Acute detrusor BoNT-A injections after SCI resulted in ordered collagen directionality in the detrusor. Mason Trichrome staining showed keratin/muscle fibers in red, and collagen in blue. The left column shows representative photomicrographs from each of the experimental and control groups. The right column shows directionality histograms that are standard for their corresponding group. Relatively organized collagen has a single defined peak whereas unorganized collagen has no distinct peak. Directionality of collagen deposition was disorganized in the bladders of SCI + Saline animals (row 2). In the bladders of SCI + BoNT-A animals (row 3), the histogram showed a preferred orientation and organization similar to that of spinal intact animals (row 1). Scale bar = 100 micrometer.

Figure 4

Acute detrusor BoNT-A injections after SCI resulted in faster return of voluntary micturition. Bladder volumes were recorded daily in the AM for 10 days post injury. The SCI + Saline group (n = 8) had significantly higher bladder volume than the SCI + BoNT-A group (n = 8 **** p < 0.0001). *** p < 0.001, * p < 0.05.

Figure 5

Functional deficit and lesion size was similar between SCI + Saline and SCI + BoNT-A groups: (A) weekly BBB scores showed that there were no significant behavioral differences in the locomotor recovery for up to 8 wpi between the SCI + Saline group and the SCI + BoNT-A group (p > 0.05); (B) Representative photomicrographs of Nissl stained 20 μm thick sections of spinal intact, SCI + Saline, and SCI + BoNT-A spinal cords are shown. Scale bar = 200 μm; and (C) Nissl and myelin-stained spinal cord sections within 1 mm of the injury center were analyzed for percentage of spared tissue. There were no statistical differences between the SCI + Saline and SCI + BoNT-A group (86.16 ± 0.86% vs. 86.25 ± 0.96%; p > 0.05).