Novel erectile analyses revealed augmentable penile Lyve-1, the lymphatic marker, expression

Abstract

Purpose: The pathophysiology of penis extends to erectile dysfunction (ED) to conditions including sexually transmitted diseases (STDs) and cancer. To date, there has been little research evaluating vascular drainage from the penis. We aimed to evaluate penile blood flow in vivo and analyze its possible relationship with the lymphatic maker.

Materials and methods: We established an in vivo system designed to assess the dynamic blood outflow from the corpus cavernosum (CC) by dye injection. To analyze lymphatic characteristics in the CC, the expression of Lyve-1, the key lymphatic endothelium marker, was examined by the in vitro system and lipopolysaccharide (LPS) injection to mimic the inflammatory conditions.

Results: A novel cavernography methods enable high-resolution morphological and functional blood drainage analysis. The expression of Lyve-1 was detected along the sinusoids. Furthermore, its prominent expression was also observed after penile LPS injection and in the erectile condition.

Conclusions: The current in vivo system will potentially contribute to the assessment of penile pathology from a novel viewpoint. In addition, current analyses revealed inducible Lyve-1 expression for LPS injection and the erection state, which requires further analyses on penile lymphatic system.

Keywords: Lyve‐1; corpus cavernosum; erectile dysfunction; lymphatic vessels; sinusoid.

FIGURE 1

A novel in vivo cavernography system utilizing FITC‐dextran. (A) An illustration showing the experimental diagram. Procedures and subsequent time‐lapse analysis of fluorencet status are shown. (B) After general anesthesia, the mouse was placed in the supine position and the root of the penis was occluded by the polyurethane tube. FITC‐dextran was injected through a 29G needle into the left CC under the tube. (C, C′, C″) After releasing the obstruction, we performed time‐lapse images of the fluorescence signal changes in the central region of the right corpus cavernosum under a stereomicroscope. Corpus cavernosum (CC), corpus cavernosum glandis (CCG). Scale bar (B, C, C′) 1000 μm, (C″) 200 μm.

FIGURE 2

Demonstration of dynamic FITC‐dextran signal alternation in the CC. (A) The width of the penis with digital calipers was measured before and after injection. After injection, the width of the penis (2.52 ± 0.06[SD] mm) was increased compared to that before injection (2.08 ± 0.03[SD] mm). (B) The signal of FITC in the right CC after injection of FITC‐dextran into the left CC. The FITC‐dextran injection filled the sinusoidal spaces. (C) The occlusion was released and the fluorescence intensity (FI) of the right CC was observed. Time‐lapse images were obtained every 10 s and the dynamic change of FI was analyzed under a stereomicroscope. From the sequential images, the right side of CC was marked and the alternation of FI was calculated. After 10 min, the FI disappeared in wild‐type mice. On the other hand, the FI remained in the priapism model mice. A time‐lapse video analysis was performed by ImageJ software. CC, corpus cavernosum, SS, sinusoidal space, scale bar (B)100 μm, (C) 200 μm.

FIGURE 3

The characteristic mode of expression of Lyve‐1 in the CC. (A) The expression of Lyve‐1 in the DAB‐stained CC sections. The yellow dotted line indicates the circumference of the CC and the area between the yellow and orange dotted lines represents the tunica albuginea. Green dotted line indicates the outer region of the CC. Lyve‐1 was expressed along sinusoids of the outer CC adjacent to the tunica albuginea. (Blue arrowheads). The urethra is generally located in the ventral (lower) region of the entire external genitalia. Red dotted line indicates the CC around the dorsal (upper) region of the urethra. The expression of Lyve‐1 was also located in the sinusoids of the CC adjacent to the dorsal part of urethra (blue arrows). (B) The expression of Lyve‐1 was colocalized with CD34‐positive cells in sinusoids of the outer CC adjacent to the tunica albuginea. (C) The expression of Lyve‐1 was located in the sinusoids of the CC adjacent to the dorsal part of urethra. The colocalized expression of Lyve‐1 and CD34 in the sinusoids surrounding the dorsal part of urethra was less prominent compared to that in the sinusoids of the outer CC adjacent to the tunica albuginea. CC, corpus cavernosum, sinusoidal space (SS), dorsal vein (DV), dorsal artery (DA), Scale bar (A) 100 μm, (B, C) 20 μm.

FIGURE 4

The expression of Lyve‐1 after the lipopolysaccharide (LPS) injection. Mice were administrated with LPS in saline at the dose of 25 mg/kg. After the general anesthesia, the LPS solution through the corpus cavernosum glandis (CCG) using a 29‐gauge needle was slowly administered left intracavernously. (A–C) The expression of Lyve‐1, located in the CC adjacent to the dorsal part of urethra, showed an increase at 1 day post injection. By 7 days post‐injection, the expression returned to the original level of the pre‐injection. The yellow dotted line indicates the circumference of the CC and the area between the yellow and orange dotted lines represents the tunica albuginea. (D) An Illustration showing the possible link of the external urethral, external micro‐organisms and CC. CC, corpus cavernosum. Scale bar 50 μm.

FIGURE 5

The expression of Lyve‐1 in erection/flaccid state. We induced the CC into erection/flaccid states utilizing the in vitro CC culture system that was previously established., (A, B) The expression of Lyve‐1 was located in the outer CC adjacent to the tunica albuginea and was upregulated in erection compared to the flaccid state. (C) After inducing erection/flaccid state utilizing in vitro CC culture system, mRNA was extracted from CC explants. The augmented expression of Lyve‐1 in the erection state compared to the flaccid state. (D) An Illustration suggesting the possible relationship between the CC and Lyve‐1. SS, sinusoidal space. Scale bar 20 μm.