Androgen Supplementation in Rats Increases the Inflammatory Response and Prolongs Urethral Healing

doi:10.1016/j.urology.2014.11.025

Urology

Volume 85, Issue 3, March 2015, Pages 691-697

https://doi.org/10.1016/j.urology.2014.11.025 Get rights and content

Objective

To describe the effects of androgens on urethral wound healing, we compared the urethral healing process in castrated Sprague-Dawley rats with and without testosterone supplementation.

Methods

Of 30 castrated male Sprague-Dawley rats, 15 received testosterone cypionate (3 mg/kg; T+ rats). All rats underwent an urethroplasty procedure and were sacrificed at postoperative days 5, 10, and 20. Neutrophils, macrophages, vessels, myofibroblasts, Ki67+ cells, collagen, and cytokines were quantified with immunofluorescence and real-time polymerase chain reaction.

Results

Penile length was significantly increased in T+ rats (21.8 vs 13.25 mm; P <.001) and operative time decreased (20.8 vs 23.3 minutes; P <.017). On day 5, T+ rats showed elevated neutrophil (727.4 vs 30.75 per high power field; P = .051) and macrophage counts (1295.8 vs 481.5 per high power field; P = .051) compared with those of T− rats. This elevation persisted throughout day 10 (291.7 vs 34; P = .002 and 1283.7 vs 110.2; P = .005) and day 20 (252.7 vs 12.2; P <.001 and 1672.7 vs 115.2; P <.001) reflecting increased and prolonged inflammation. Myofibroblasts were decreased in T+ rats on day 5 (215.7 vs 808.3; P <.001) and increased by day 10 (1490.1 vs 263.0; P = .001) and day 20 (1964.0 vs 210.0; P <.001) consistent with a delayed onset but with prolongation of the proliferative phase. Limitations include the use of castrated rats, which may have been exposed to androgens before castration.

Conclusion

Testosterone supplementation leads to an increased inflammatory response and myofibroblast proliferation accompanied by prolonged inflammatory and proliferative phases. These novel findings suggest a delayed and possibly impaired urethral healing in the presence of excessive androgens.

Section snippets

Animals

The Institutional Animal Care and Use Committee of the Ann & Robert H. Lurie Children's Research Center, Chicago, IL, approved this study. Thirty castrated male Sprague-Dawley rats (∼250 g) were obtained from Charles River Laboratories (Wilmington, MA). Fifteen T+ rats received 3 mg/kg testosterone cypionate intramuscularly and given 2 weeks to equilibrate. Testosterone levels were checked on arrival and after resupplementation with an enzyme-linked immunosorbent assay (Calbiotech, Spring

Androgen Supplementation Is Associated With Increased Penile Length

After resupplementation, we observed significantly elevated testosterone levels in T+ rats (mean, 1.25 ± 0.41 vs 0.28 ± 0.23 ng/mL; P <.001) and those levels corresponded to the physiological levels reported for male Sprague-Dawley rats.¹³ T+ rats had a significantly increased mean penile length compared with those of the T− rats (21.8 ± 1.69 vs 13.25 ± 2.34 mm; P <.001; Supplementary Fig. 1) and significantly decreased operative time (20.8 ± 1.69 minutes [median, 21.0 minutes] vs 23.33 ± 2.66

Comment

We have used a previously reported urethroplasty model¹¹ in castrated rats with and without androgen supplementation to identify the effects of androgens on the process of urethral healing. We found that castrated rats receiving androgens preoperatively show an increase in the inflammatory response and a prolongation of the inflammatory phase during urethral healing. Also, the onset of the proliferative phase was delayed and associated with an increase in myofibroblast proliferation and

Conclusion

We found that testosterone supplementation was advantageous for the surgical procedure because of the increased penile length that facilitated it. The presence of testosterone during the urethral wound healing process, however, was associated with an increased and prolonged inflammatory phase followed by a delayed and prolonged proliferative phase. This may predispose the healing urethra to complications, and specifically, excessive or aberrant inflammatory response may impair urethral healing.

References (26)

A. Springer et al.
Trends in hypospadias surgery: results of a worldwide survey
Eur Urol
(2011)
R.D. Malik et al.
Survey of pediatric urologists on the preoperative use of testosterone in the surgical correction of hypospadias
J Pediatr Urol
(2014)
J.M. Netto et al.
Hormone therapy in hypospadias surgery: a systematic review
J Pediatr Urol
(2013)
J.P. Gearhart et al.
The use of parenteral testosterone therapy in genital reconstructive surgery
J Urol
(1987)
C. Kaya et al.
The efficacy of dihydrotestosterone transdermal gel before primary hypospadias surgery: a prospective, controlled, randomized study
J Urol
(2008)
D.B. Gorduza et al.
Does androgen stimulation prior to hypospadias surgery increase the rate of healing complications? A preliminary report
J Pediatr Urol
(2011)
S.C. Gilliver et al.
Androgens influence expression of matrix proteins and proteolytic factors during cutaneous wound healing
Lab Invest
(2007)
M.D. Hofer et al.
Analysis of primary urethral wound healing in the rat
Urology
(2014)
K.J. Livak et al.
Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method
Methods
(2001)
L.S. Inamdar Doddamani et al.
Acceleration of neutrophil precursors' maturation and immunostimulation of CD3+, CD4+ lymphocytes by stanozolol in mice
J Steroid Biochem Mol Biol
(2012)

S. Salmi et al.

Co-localization of androgen receptor with estrogen receptor beta in the lower urinary tract of the male rat

J Urol

(2001)

S.C. Gilliver et al.

The hormonal regulation of cutaneous wound healing

Clin Dermatol

(2007)

A.A. Donjacour et al.

FGF-10 plays an essential role in the growth of the fetal prostate

Dev Biol

(2003)

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: Financial Disclosure: The authors declare that they have no relevant financial interests.

: Funding Support: This study was supported by the Ann and Robert H. Lurie Children's Hospital of Chicago faculty practice plan.

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Reconstructive UrologyAndrogen Supplementation in Rats Increases the Inflammatory Response and Prolongs Urethral Healing

Objective

Methods

Results

Conclusion

Section snippets

Animals

Androgen Supplementation Is Associated With Increased Penile Length

Comment

Conclusion

Eur Urol

J Pediatr Urol

J Pediatr Urol

J Urol

J Urol

J Pediatr Urol

Lab Invest

Urology

Methods

J Steroid Biochem Mol Biol

J Urol

Clin Dermatol

Dev Biol

Reconstructive Urology
Androgen Supplementation in Rats Increases the Inflammatory Response and Prolongs Urethral Healing