Involvement of Rho-Kinase/LIM Kinase/Cofilin Signaling Pathway in Corporal Fibrosis after Cavernous Nerve Injury in Male Rats

Introduction: The molecular mechanism of corporal fibrosis leading to erectile dysfunction (ED) following cavernous nerve (CN) injury is poorly understood.

Aim: To determine whether the LIMK2/cofilin pathway, the downstream effectors of ROCK1, was involved in ED and corporal fibrosis following bilateral CN injury in male rats.

Methods: Forty-eight 10-week-old male Sprague-Dawley rats were equally divided into three groups: sham surgery (S); bilateral CN crush injury (I); and bilateral CN resection (R). Within each groups, two subgroups were analyzed at 1 and 4 weeks postoperatively.

Main outcome measures: Electrostimulation was performed to assess erectile function by the ratio of maximal intracavernous pressure to mean arterial pressure (ICP/MAP) and areas under the ICP curve to MAP (AUC/MAP). Penile tissue was processed for Masson's trichrome staining, Western blot (ROCK1, total LIMK2, phospho-LIMK2, total cofilin, phospho-cofilin), immunohistochemistry (alpha-SM actin [α-SMA]), and double immunofluorescent staining (ROCK1, phospho-LIMK2, vimentin).

Results: At each time point, both I and R groups showed a significantly lower percent of ICP/MAP and AUC, and decreased SM cell/collagen ratio and expression of α-SMA than S group. Densitometry revealed a significantly higher expression of ROCK1 in I and R groups compared with S group at all time points. The LIMK2 phosphorylation in I and R groups significantly increased at 1 week, but not at 4 weeks. The cofilin phosphorylation in R group significantly increased to that in S group starting at 1 week, while that in I group was increased significantly at 4 weeks. The double immunofluorescent staining noted that coexpression of vimentin with ROCK1 or phospho-LIMK2 in I and R groups was significantly increased mainly in the subtunical area at 1 week but not at 4 weeks.

Conclusions: The ROCK1/LIMK2/cofilin pathway may be involved in ED related to corporal fibrosis, and it appears to be functional particularly in the early period after CN injury.