Recombinant insulin-like growth factor-1 activates satellite cells in the mouse urethral rhabdosphincter

Abstract

Background: The goal of this study is to demonstrate the efficacy of a new method for the treatment of urinary incontinence by stimulation of urethral rhabdosphincter satellite cells. We show that satellite cells do exist in the sphincter muscle of retired male mice breeders by staining for c-Met, a satellite cell specific protein. Once activated by recombinant mouse Insulin-like Growth Factor-1(rIgf-1), the satellite cells develop into muscle cells within the rhabdosphincter thereby potentially strengthening it.

Methods: 20 μl (1 μg/μl) of rIgf-1 was surgically injected directly into the urethral wall of retired male mouse breeders. Mice injected with phosphate buffered saline (PBS) were used as controls. 4 weeks later, urethras were harvested and serially-sectioned through the sphincter for routine hematoxylin-eosin staining as well as immunohistochemical staining with satellite cell specific anti-c-Met antibody and proliferation specific anti-Ki-67 antibody.

Results: Anti-c-Met antibody positive cells (c-Met+) were identified in the rhabdosphincter. c-Met+ cells increased by 161.8% relative to controls four weeks after rIGF-1 injection. Anti- Ki-67 antibody positive cells were identified and characterized as cells with centrally located nuclei in striated muscle bundles of rIGF-1 treated animals.

Conclusions: Satellite cells in the mouse rhabdosphincter can be activated by rIGF-1 treatment, which subsequently are incorporated into existing skeletal muscle bundles. Using this approach, the rhabdosphincter can be induced to regenerate and potentially strengthen via satellite cell activation and likely improve urinary continence.

Figure 1

Anatomy and surgical exposure. A: Male mouse urethra comprises 3 parts, an anatomy which is similar to that found in humans: the prostatic urethra, the membrane urethra and the spongy urethra. The membrane urethra is the section between the prostate and diagram, it is approximately 0.8 to 1.2 cm in length and it can be easily surgically exposed for local injections. B: After a low abdominal mid-line incision was made, the male mouse urethra was exposed for injection in the lateral aspects of the urethra.

Figure 2

Locally injected rIGF-1 accessibility to satellite cells in male mouse urethra rhabdosphincter. A, B: Cross section of the male membrane urethra. The inner layer consists of mucosa and sub-mucosa, the outer layer is a serosa and the middle layer is muscle layer contains the rhabdosphincter muscle goes along and surrounds the urethral lumen. C, D: Injection of India ink into the urethral rhabdosphincter. The rhabdosphincter was infiltrated by locally injected India ink, demonstrating potential accessibility of injections to satellite cells within the muscle.

Figure 3

Satellite cells (c-Met⁺) exist in retired male mouse urethral rhabdosphincter. There is positive staining with anti-c-Met antibody at the muscle periphery bundle demonstrating the presence of satellite cells (red). Nuclei were stained with chromomycin A₃ (green). Merged images show co-localization (yellow). A non-operated control; B: rIGF-1 Injected urethral rhabdosphincter.

Figure 4

Anti-c-Met positive satellite cells in animals treated with rIGF-1. In urethras injected with rIGF-1, the number of c-Met + satellite cells increased significantly versus PBS-injected or non-operated controls. Four weeks after treatment with rIGF-1, c-Met⁺ satellite cells increased by 161.8% (41.1 ± 5.4 verse 15.7 ± 1.3, p = 0.012), while the sham operated (PBS) group showed no significant change (20.4 ± 3.9 verse 15.7 ± 1.3, p = 0.316).

Figure 5

Proliferation of satellite cells after rIGF-1 injection. Cells within the rhabdosphincter stained with anti-ki67 antibody (red) indicating cell proliferation. Nuclei were stained with chromomycin A₃ (green). Note the co-localization (yellow) in the merged photograph.

Figure 6

New muscle cells were incorporated into muscle bundles after rIGF-1 injection. Skeletal muscle cells with central nuclei are present in the rhabdosphincter muscle bundles (arrows) which have differentiated from satellite cells. Cells with central nuclei were not detected in the controls.