Wound healing responses of urinary extravasation after urethral injury

Abstract

The post-surgical fluid leakage from the tubular tissues is a critical symptom after gastrointestinal or urinary tract surgeries. Elucidating the mechanism for such abnormalities is vital in surgical and medical science. The exposure of the fluid such as peritonitis due to urinary or gastrointestinal perforation has been reported to induce severe inflammation to the surrounding tissue. However, there have been no reports for the tissue responses by fluid extravasation and assessment of post-surgical and injury complication processes is therefore vital. The current model mouse study aims to investigate the effect of the urinary extravasation of the urethral injuries. Analyses on the urinary extravasation affecting both urethral mesenchyme and epithelium and the resultant spongio-fibrosis/urethral stricture were performed. The urine was injected from the lumen of urethra exposing the surrounding mesenchyme after the injury. The wound healing responses with urinary extravasation were shown as severe edematous mesenchymal lesions with the narrow urethral lumen. The epithelial cell proliferation was significantly increased in the wide layers. The mesenchymal spongio-fibrosis was induced by urethral injury with subsequent extravasation. The current report thus offers a novel research tool for surgical sciences on the urinary tract.

Figure 1

The schema of the murine urethral injury model is shown. The urethral epithelium is formed by pseudostratified columnar epithelium (shown by purple-colored epithelial cells). The urethra was separated from the corpus cavernosum, and the compression injury using mosquito forceps was suffered (right bottom rows). The isolated 0.2 ml urine was injected from the lumen of urethra using 29 gauge needle and the urethral mesenchyme was thus exposed to urine (urinary extravasation).

Figure 2

The urethral statuses for POD 3 were shown in the Hematoxylin–Eosin staining. The second row is a magnification of the first row. Severe edematous lesions of urethral mesenchyme and the narrow urethral lumen in Group1 (urethral injury with urinary extravasation) is compared with those of Group 2 (urethral injury without urinary extravasation), Group 3 (sham operation) (Scale bar: 100 µm).

Figure 3

The time courses of the urethral status after urethral injury with urinary extravasation (Group1) were shown by Hematoxylin Eosin staining (Scale bar: 100 µm). The second row is a magnification of the first row. Mild inflammatory responses of urethral mesenchyme were shown at POD1 (A). Severe urethral mesenchymal edematous lesions continued from POD3 to POD7 (A, B). Urethral lumen has narrowed from POD 3 to POD14 (A, B). White squared regions are enlarged in the lower rows.

Figure 4

Post-surgical epithelial and mesenchymal histological structures were investigated by the expression of E-Cadherin (green) and Vimentin (red) in immune-fluorescence staining (Scale bar: 100 µm). The epithelial E-Cadherin expressions disappeared in both Group1 (urethral injury with urinary extravasation) and Group 2 (urethral injury without urinary extravasation) at POD1. As for mesenchymal histology of the Group1, the Vimentin expression disappeared at POD3 (white arrow in the second image from the left of the upper row). However, such mesenchymal expression was already recovered in POD3 in Group2 (yellow arrow in the second image from the left of the middle row). In Group1, the Vimentin expressions were gradually recovered in POD7-14, albeit the loss of mesenchymal sinusoidal structures (the third and fourth images from the left of the upper row). The urethral epithelium was thickened from POD3 (white arrowhead) in Group1.

Figure 5

Cell proliferation in Group1 was analyzed by immunohistochemistry of anti-Ki-67 on POD3. The second row is a magnification of the first row (A). The significantly increased cell proliferation in Group1 was compared with that of Group2 (B; white arrowheads in the dot box region). The positive cells of anti-Ki-67 were positioned in the wide layers in Group 1 and those of Group 2 were positioned only in the basal side. The time course of cell proliferation in Group 1 was shown. The second row is a magnification of the first row (C; POD1-14) (Scale bar: 100 µm).

Figure 6

The time course of expression of α smooth muscle actin for Group1 was indicated (A). Its prominent expression (at POD7) and remaining expression (at POD14) were shown in red box (A). Post-surgical time course of expression of α smooth muscle actin (green) and Collagen I (red) was shown by immune-fluorescence staining (B). The prominent Collagen I expression (at POD14) and its low level of expression (at POD7) were shown in red box (B). “U” indicates the location of urethral lumen (Scale bar: 100 µm). The second row is a magnification of the first row.

Figure 7

The schema of the urethral stricture in murine urethral injury model (Group 1) is shown (upper). The antegrade urethrography images in POD 3 were shown (lower). The lower row represented the magnified view of the upper row. The black arrow showed the site of urethral stricture.

Figure 8

The model schema shows the status after urethral injury with urinary extravasation. The severe edematous lesions of urethral mesenchyme in post-operative days 3 are represented as the pink spread lesion (the upper right image). Red sinusoidal or dot lesions are represented by the positive lesion of anti-Vimentin staining. The blue sharp rod lesions were represented as the positive fibrous Collagen I expression (the images of lower row). The status and the time course of spongio-fibrosis are indicated (the images toward the left low one).