Comparison of Histopathological and Molecular Pathological Phenotypes in Mouse Models of Intrauterine Adhesions Induced by Two Concentrations of Ethanol Perfusion

doi:10.12300/j.issn.1674-5817.2024.183

Abstract

Abstract:

Objective To construct intrauterine adhesion (IUA) mouse models induced by two different concentrations of ethanol injury, compare the phenotypes, and optimize a more stable IUA modeling method. Methods Twenty 8-week-old female C57BL/6N mice were randomly divided into two groups: the 95% ethanol injury group and the 50% ethanol injury group. Using a self-control method, the left uterine horn was infused with ethanol to establish the IUA model, while the right uterine horn was infused with saline as the sham operation. Five mice from each group were euthanized on day 7 and 15 after modeling, and uterine tissues were collected. Hematoxylin-eosin (HE) staining was used to observe the endometrial pathology, and Masson staining was used to assess the degree of endometrial fibrosis. Quantitative real-time PCR was employed to detect the expression levels of fibrosis markers and pro-inflammatory factors in the uterine tissues. Results Compared to the sham operation, these two ethanol injury led to a significant reduction in elasticity of the uterus, an increase in inflammatory infiltration, and a marked increase in the degree of fibrosis on day 7 after modeling (P<0.05). The 95% ethanol injury group showed a significant decrease in endometrial thickness (P<0.05), whereas no significant change was observed in the 50% ethanol injury group when compared to the sham operation (P>0.05). The expression levels of fibrotic marker molecules collagen type Ⅳ alpha 1 chain (Col4A1), α-smooth muscle actin (α-SMA), transforming growth factor-β (TGF-β), and pro-inflammatory factors tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were significantly elevated in the 50% ethanol injury group when compared to the sham operation (P<0.05), although there was an increasing trend of the same markers in the 95% ethanol injury group, the differences were not statistically significant (P>0.05). On day 15 after modeling, the histopathological changes in both ethanol injury groups were not significant when compared to the sham operation, the expression levels of Col4A1, TGF-β, TNF-α and IL-1β remained significantly higher in the 50% ethanol injury group (P<0.05), while only IL-1β was significantly elevated in the 95% ethanol injury group (P<0.05). Conclusion Uterine infusion with 95% ethanol results in more marked histopathological changes in the IUA mouse model compared to the 50% ethanol injury group. The 95% ethanol injury model is suitable for histopathological studies. However, the 50% ethanol injury group shows higher expression levels of fibrosis markers and pro-inflammatory factors compared to the 95% ethanol injury group, suggesting that the 50% ethanol injury model is more suitable for molecular pathological study.

Key words: Intrauterine adhesions, Mouse model, Inflammatory factors, Fibrosis, Ethanol

CLC Number:

Q95-33

JIANG Juan,SONG Ning,LIAN Wenbo,et al. Comparison of Histopathological and Molecular Pathological Phenotypes in Mouse Models of Intrauterine Adhesions Induced by Two Concentrations of Ethanol Perfusion[J]. Laboratory Animal and Comparative Medicine, 2025, 45(4): 393-402. DOI: 10.12300/j.issn.1674-5817.2024.183.

Figures/Tables 4

Figure 1 Construction of mouse intrauterine adhesion models induced by two concentrations of ethanol injury and HE staining results of uterine tissues collected one day 7 after injuryNote： A， Schematic diagram of the surgical process for establishing the ethanol perfusion model， Sham represents the sham operation group， and IUA represents intrauterine adhesions （IUA）model group（the ethanol injury group）. B， On day 7 after modeling， images of uterine tissues of mice with two types of ethanol injury. In each image， the left side of the uterine tissue is the sham operation group， and the right side of uterine tissue is the injury group. The scale bar indicates 5 mm. C， HE staining results of uterine tissues on day 7 after modeling. In the upper images， the scale bar indicates 100 μm （×400）； in the lower images， the scale bar indicates 500 μm （×100）； the red arrow indicates inflammatory cell infiltration；the black line indicates the direction for measuring the thickness of endometrium. D， Statistical results of endometrial thickness in sham operation group and IUA model group， ?P<0.05， nsP>0.05.

Figure 2 Construction of mouse intrauterine adhesion models induced by two concentrations of ethanol injury and Masson staining results of uterine tissues collected one day 7 after injuryNote： A， On day 7 after modeling， Masson staining was used to assess the content of collagen fibers. In the upper images， the scale bar indicates 250 μm （×400）； in the lower images， the scale bar indicates 1 000 μm （×100）. B， On day 7 after modeling， statistical results of the percentage of fibrotic area in the bilateral uterine tissues of mice， ?P<0.05. C， On day 15 after modeling， statistical results of the percentage of fibrotic area in the bilateral uterine tissues of mice， nsP>0.05.

Figure 3 qPCR results of expression levels of fibrosis and inflammation markers in uterine tissues of IUA model mice on day 7 after modelingNote： A， The mRNA expression levels of Col4A1， α-SMA， TNF-α， IL-1β and TGF-β in uterine tissues one day 7 after modeling with 95% ethanol injury. B， The mRNA expression levels of Col4A1， α-SMA， TNF-α， IL-1β and TGF-β in uterine tissues one day 7 after modeling with 50% ethanol injury. nsP>0.05， ?P<0.05， ??P<0.01.

Figure 4 Detection results of fibrosis and inflammatory response in mouse uterine tissues on day 15 after IUA modelingNote： A， The mRNA expression levels of Col4A1， α-SMA， TNF-α， IL-1β and TGF-β in uterine tissues one day 15 after modeling with 95% ethanol injury. B， The mRNA expression levels of Col4A1， α-SMA， TNF-α， IL-1β and TGF-β in uterine tissues one day 15 after modeling with 50% ethanol injury. C， On day 15 after modeling， HE staining results of uterine tissues. In the upper images， the scale bar indicates 100 μm （×400）； in the lower images， the scale bar indicates 500 μm （×100）. The red arrow indicates inflammatory cell infiltration. D， On day 15 after modeling， statistical results of endometrial thickness in sham operation group and IUA model group. nsP>0.05， ?P<0.05.

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