Myometrial wound healing post-Cesarean delivery in the MRL/MpJ mouse model of uterine scarring

Abstract

There is little known about healing of the uterus after Cesarean delivery (CD). Uterine wound repair was studied by using two strains of mice with different wound healing characteristics: MRL/MpJ(+/+) (MRL: "high-healer" phenotype) and C57Bl/6 ("low-healer" phenotype). First, we examined the morphology and histology of the uterine wall repair. We identified wound granulation tissue 3 days post-CD in both strains, albeit less in the MRL strain. Macroscopically, no scar could be identified either in MRL or C57Bl/6 mice on day 60 post-CD. However, histologically, we found significant differences in wound integration, inflammation, and collagen birefringence between the two strains of mice. Using a histological index, we provided evidence for significant differences in mitotic activity in the initial phases of uterine healing among strains. Functional behavior of the uterine scar also was analyzed by using biomechanical parameters such as slope (measure of stiffness), yield point (measure of elasticity), and break point (measure of strength). There were significant differences in stiffness of the scarred myometrium between the two phenotypes. MRL mice displayed a significantly lower yield point compared with C57Bl/6. The break point was reached faster on days 15 and 60 in both C57Bl/6 and MRL strains compared with day 3 post-CD. Our findings indicate that differences in regenerative ability translate in histological, mitotic, and functional differences in biomechanical properties of the scarred myometrium after CD.

Figure 1

Concurrent macroscopic evaluation of the ear and uterine healing in the MRL/MpJ^+/+ (MRL) and C57Bl/6 strains of mice. Representative pictures of the ears 3 days after surgery in the C57Bl/6 “low healer” (A) and MRL “high healer” (B) mouse are shown. Representative pictures of the ears 60 days after surgery in the C57Bl/6 (C) and MRL (D) mouse are shown. The arrow in D points to the remaining lumen of the ear-hole in an MRL mouse at 60 days after puncture. E displays the percent change in ear-hole area postinjury. Statistical analysis: two-way analysis of variance followed by posthoc Student-Newman-Keuls tests; **P < 0.01 versus initial ear punch area (day 0); *P < 0.01 versus C57Bl/6 phenotype. Representative pictures of the uterus three days post cesarean in C57Bl/6 (F) and MRL (G) mouse are shown. Representative pictures of the uterus 60 days postcesarean in the C57Bl/6 (H) and MRL (I) mouse are shown.

Figure 2

Representative histological sections of the uterine scar site on days three and five post-Cesarean delivery in C57Bl/6 and MRL/MpJ^+/+ (MRL) mice are shown. Adjacent sections were stained with either H&E [C57Bl/6: day three (A) and day five (C) versus MRL: day three (B) and day five (D)] and Sirius red visualized in either white light [C57Bl/6: day three (E) and day five (G) versus MRL: day three (F) and day five (H)] or polarized light [C57Bl/6: day three (I) and day five5 (K) versus MRL: day three (J) and day five (L)]. The green arrowheads in the insets mark PMNs. The green arrows outside the insets indicate the site of the uterine scar as marked with tissue dye (green or black). The round intensely birefringent multilocular structures are the Vicryl multifilament surgical sutures (Su; marked only on the top panels). Lu marks the uterine lumen. Original magnification, ×100 (Scale bar = 200 μm). The small squares on the H&E panels mark the area shown magnified (Scale bar = 40 μm) in the inset of the corresponding panel.

Figure 3

Representative histological sections of the uterine scar site on days 15 and 60 post-Cesarean delivery in C57Bl/6 and MRL/MpJ^+/+ (MRL) mice are shown. Adjacent sections were stained with either H&E [C57Bl/6: day 15 (A) and day 60 (C) versus MRL: day 15 (B) and day 60 (D)] and Sirius red and visualized in either white light [C57Bl/6: day 15 (E) and day 60 (G) versus MRL: day 15 (F) and day 60 (H)] or polarized light [C57Bl/6: day 15 (I) and day 60 (K) versus MRL: day 15 (J) and day 60 (L)]. The green arrowheads in the insets mark PMNs. The green arrows outside the insets indicate the site of the uterine scar as marked with tissue dye (green or black). The white arrows in B point to hemosiderin deposits in the area of integration. The round intensely birefringent multilocular structures are the Vicryl multifilament surgical sutures (Su; marked only on the top panels). Lu marks the uterine lumen. Original magnification, ×100 (Scale bar = 200 μm). The small squares on the H&E panels mark the area shown magnified (Scale bar = 40 μm) in the inset of the corresponding panel.

Figure 4

BrdUrd nuclear labeling of the uterine scar 3 days post-Cesarean delivery in C57Bl/6 and MRL/MpJ^+/+ (MRL) mice. BrdUrd labeled nuclei (brown) could be identified into the wound site of both C57Bl/6 (A) and MRL (C) mice. Original magnification, ×100. The green dye marks the site of uterine wound at the time of tissue collection and is indicated by the black arrows. B and D show the respective boxed areas at higher magnification (×400). White arrows mark BrdUrd labeled nuclei. E: Mitotic index (ratio of BrdUrd labeled nuclei-to-total counted nuclei). Statistical analysis: two-way analysis of variance followed by posthoc Student-Newman-Keuls tests: **P < 0.05 versus day three of same strain. *P < 0.05 versus C57Bl/6 phenotype. Ser, uterine serosa; Lu, uterine lumen.

Figure 5

Topographical co-localization of BrdUrd and smooth muscle actin post-Cesarean delivery in C57Bl/6 and MRL/MpJ^+/+ (MRL) mice. Cells with BrdUrd labeled nuclei (purple black) could be identified between the red muscle bundles in C57Bl/6 [day 3 (A) and day 5 (C)] and MRL [MRL: day 3 (B) and day five (D)] mice. At the later times, cells positive for both BrdUrd and smooth muscle actin could be observed in proximity to serosa in the scar area both in C57Bl/6 [day 15 (G) and day 60 (I)] and MRL [MRL: day 15 (H) and day 60 (J)] mice. Original magnification, ×100 (Scale bar = 100 μm). The areas delineated by the squares are further shown in the left lower corner of each panel as higher magnification captions of the boxed areas (×640; Scale bar = 33 μm). E and K show representative areas at ×1000 magnification of D (day five) and J (day 60) in MRL mice. F shows a section from an MRL animal (day five) processed identically but omitting the two primary antibodies (Scale bar = 100 μm). The green arrows indicate the site of the uterine scar as marked with tissue dye (green or black). The white arrow in K points to a BrdUrd⁺/α-SMA⁺ cell in the subserosal area of regenerated uterine wall. Su, suture material; Lu, uterine lumen; Bv, blood vessel (arrowhead).

Figure 6

Analysis of the load-displacement relationships of scarred uterine rings in C57Bl/6 and MRL/MpJ^+/+ (MRL) mice. Uterine rings were collected at 3, 15, and 60 days post-Cesarean delivery and demonstrated differences in the slope (A: measure of stiffness), yield point (YP, B: measure of plasticity), and break point (BP, C: measure of strength). Statistical analysis: two-way analysis of variance followed by posthoc Student-Newman-Keuls tests. **P < 0.05 versus day 3 of same strain. *P < 0.05 versus C57Bl/6 phenotype.