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Abstract
Purpose
There has been a scarcity of integrated, longterm (＞4 week) studies on structural and functional alterations in the penis according to the period following cavernous nerve (CN) injury. The aim of this study was to investigate time-dependent structural and functional changes in the corpus cavernosum following CN injury in a rat model.
Materials and Methods
Ninety male Sprague-Dawley rats (10 weeks old) were divided into 4 groups: normal control (C), sham (S), bilateral CN resection (R), and bilateral CN crush injury (I) groups. At 1, 4, and 12 weeks after the procedure, erectile function was assessed by electrostimulation. The terminal deoxynucleotidyl transferase-mediated 2’-deoxyuridine 5’-triphosphate nick end labeling (TUNEL) assay was performed for detection of apoptosis. Masson's trichrome staining and immunohistochemistry were performed for detection of alpha smooth muscle actin (α-SMA). Western blot analysis was then performed.
Results
The R and I groups showed persistent impairment of erectile function at all three points in time. Apoptosis peaked at 1 week after resection or crush injury and then gradually subsided. The smooth muscle cell/collagen ratio and expression of α-SMA gradually decreased over time after CN resection or crush injury. Myosin phosphatase target subunit 1 phosphorylation progressively increased over time after CN resection or crush injury. On the other hand, expression of phospho-protein kinase B, phospho-endothelial nitric oxide synthase, and neuronal nitric oxide synthase transiently decreased at 1 week after resection or crush injury and then recovered to the control values.
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Fig. 1.
Comparison of erectile function. Results of cavernous electrostimulation in the four experimental groups are expressed as percent ICP/MAP. Each bar represents the mean± standard error of the means. ∗p＜0.05 vs. control group. ICP: intracavernous pressure, MAP: mean arterial pressure, C: control group (n=5 in each subgroup), S: sham operation group (n=5 in each subgroup), R: CN resection group (n=10 in each of the 1-, 4-, and 12-week subgroups), I: CN crush injury group (n=10 in each of the 1-, 4-, and 12-week subgroups).
Fig. 2.
Comparison of mean apoptosis index. Representative micrographs show apoptotic cells stained black-brown by the TUNEL method in rat penile cavernosum (magnification 400×). Bar graphs represent quantitative image analysis. The apoptotic index was defined as the percentage of apoptotic cells within the total number of cells in a given area. ∗p＜0.05 vs. control group. TUNEL: terminal deoxynucleotidyl transferase-mediated 2’-deoxyuridine 5’-triphosphate (dUTP) nick end labeling, C: control group (n=5 in each subgroup), S: sham operation group (n=5 in each subgroup), R: CN resection group (n=10 in each of the 1-, 4-, and 12-week subgroups), I: CN crush injury group (n=10 in each of the 1-, 4-, and 12-week subgroups).
Fig. 3.
Masson's trichrome staining and immunohistochemical analysis of smooth muscle content in rat penile sections. Smooth muscle and collagen fibers were stained in red and blue, respectively (magnification 40×). (A) Representative images for Masson's trichrome staining. (B) Comparison of Masson's trichrome staining results among four experimental groups. The results are presented as the smooth muscle cell (SMC)/collagen ratio (mean±standard error of the means). Smooth muscle cells in the endothelial lining of sinusoids (black arrows), SMCs in the cavernous tissue beneath the tunica albuginea (black-head arrows), collagen fibers in the peri-sinusoidal area or the cavernous tissue beneath the tunica albuginea (asterisks). The smooth muscle component is shown as brown areas (magnification 100×). (C) Representative images for immune-stained alpha smooth muscle actin (α-SMA). (D) Comparison of the expression of α-SMA among four experimental groups. The results are presented as the percentage of smooth muscle fibers in a given area. ∗p＜0.05 vs. control group, †p＜0.05 vs. crush injury group. C: control group (n=5 in each subgroup), S: sham operation group (n=5 in each subgroup), R: cavernous nerve (CN) resection group (n=10 in each of the 1-, 4-, and 12-week subgroups), I: CN crush injury group (n=10 in each of the 1-, 4-, and 12-week subgroups).
Fig. 4.
Western blot analysis demonstrating corporal expression of phospho-myosin phosphatase target subunit 1 (MYPT1), phospho-protein kinase B (Akt), phospho-endothelial nitric oxide synthase (eNOS), and neuronal nitric oxide synthase (nNOS). (A) Representative immunoblots show expression of phospho-MYPT1, phospho-Akt, total Akt, phospho-eNOS, nNOS, and β-actin from the corporal tissues of the four groups (according to period). (B) Bar graphs demonstrating the comparison of phospho-MYPT1 protein expression among the four experimental groups using densitometry. Results were normalized by β-actin expression. (C) Bar graphs demonstrating the comparison of phospho-Akt protein expression among the four experimental groups using densitometry. Results were normalized by total Akt expression. (D) Bar graphs demonstrating the comparison of phospho-eNOS protein expression among the four experimental groups using densitometry. Results were normalized by β-actin expression. (E) Bar graphs demonstrating the comparison of nNOS protein expression among the four experimental groups using densitometry. Results were normalized by β-actin expression. Results were presented as fold changes over controls. ∗p＜0.05 vs control group. C: control group (n=5 in each subgroup), S: sham operation group (n=5 in each subgroup), R: cavernous nerve (CN) resection group (n=10 in each of the 1-, 4-, and 12-week subgroups), I: CN crush injury group (n=10 in each of the 1-, 4-, and 12-week subgroups, respectively).
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