Identifying the clinical presentations, progression, and sequela of pelvic inflammatory disease through physiological, histological and ultrastructural evaluation of a rat animal model

Abstract

Background: Patients with pelvic inflammatory disease (PID) are at an increased risk of ectopic pregnancy, infertility, and varying degrees of chronic pelvic pain. The aims of this study were to establish a rat model of PID and characterize its progression in order to assist in the study of pathophysiological mechanisms and to provide animal model for future studies of PID treatments.

Methods: Fifty Sprague-Dawley rats (female, 6-weeks-old) were divided into a model group (n=28) and a control group (n=22). The rat endometrium was mechanically injured by a needle which moved back and forth 3 times on the endometrial tissue, and a mixed bacterial solution (6×10⁸ CFU) of equal concentrations of Escherichia coli and Staphylococcus aureus was injected into both horns of the rat uterus. Physiological characteristics including weight, temperature, blood, and inflammatory factors were compared, and immunohistochemistry and transmission electron microscopy were used to evaluate the progress and sequela of PID.

Results: The model rats experienced acute PID in the first 14 days and exhibited higher body temperatures and decreased body weight. Infection-related factors in the blood were also significantly changed compared with the normal group, with obviously increased serum levels of C-reactive protein (CRP), interferon gamma (IFN-γ), and interleukin-4 (IL-4). Congestion and edema were observed in the uteri of the model rats, followed by infiltration of numerous inflammatory cells and ultrastructural morphology changes. Histological examination of the uterus showed that adhesion initially appeared at approximately 21 days. In addition to the increased collagen fibers biomass, the expression of transforming growth factor-beta 1 (TGF-β1) was elevated, which might have contributed to pelvic tissue adhesion formation in the PID sequela.

Conclusions: This study clearly described the characteristics and progression of PID in a rat model. The detailed evidence increased our understanding of the pathogenesis and progression of PID and may be useful for future studies of PID treatments.

Keywords: Pelvic inflammatory disease (PID); rat model; tissue adhesion; transforming growth factor-beta 1 (TGF-β1).

Figure 1

Schematic diagram of the experimental and sample collection.

Figure 2

Changes of rectal temperature (A) and weight (B) of rat in acute PID period. The sham group was the control and model group was the PID group. All data were expressed as the mean ± SD (n=15). *P<0.05, **P<0.01, ***P<0.001. PID, pelvic inflammatory disease; SD, standard deviation.

Figure 3

Changes in infection indices in the blood of acute PID rats. The routine blood test was performed to evaluate the level of WBC (A), PLT (B), NE% (C), MO% (D), LY% (E) and HGB (F). All data were expressed as the mean ± SD (n=15). *P<0.05, **P<0.01, ***P<0.001. WBC, white blood cells; PLT, platelet count; NE%, neutrophil ratio; MO%, monocyte ratio; LY%, lymphocyte ratio; HGB, hemoglobin; PID, pelvic inflammatory disease; SD, standard deviation.

Figure 4

The expression of serum inflammatory factors CRP (A), IFN-γ (B) and IL-4 (C) were tested by ELISA analysis on D3 and D7. All data were expressed as the mean ± SD (n=15). *P<0.05, **P<0.01. ELISA, enzyme-linked immunosorbent assay; SD, standard deviation; CRP, C-reactive protein; IFN-γ, interferon gamma; IL-4, interleukin-4.

Figure 5

A noticeable tissue inflammatory response appeared in acute PID rats. Morphology of the pelvic tissue was assessed after dissection on D14 (A). Dissection congestion were evident in the uterine tissue (A, model 1), hyperemia leads considerable darker color of the uterus, allopian tubes, and ovarian tissue (A, model 2), and edema formed and filled the uterine cavity (A, model 3). The degree of inflammation in the uterine tissue was assessed by HE staining analyses (B,C). The infiltration of massive numbers of lymphocytes, neutrophils, and plasma cells were observed and concentrated in the mucosal layer (B, D4model). On D14, some epithelial cells appeared degenerated and undergoing necrosis, inflammatory cells extended to the muscular layer (C, D14 model). Scale bars: 100 µm. PID, pelvic inflammatory disease; HE, hematoxylin and eosin.

Figure 6

Ultrastructural morphology changes in the uterus epithelium of rats on D14 was observed by transmission electron microscopy. The sham group showed normal uterus morphology with an obvious intercellular adhesion (white arrow) and few lipid drop (yellow arrow) in simple columnar epithelium cells, which has normal mitochondria morphology (red arrow) and rich intact microvilli (black arrow) (A). The uterus mucosa of model group were more infiltrated by mononuclear inflammatory cells (blue arrow) and numerous lipid drop. The microvilli of the model group were disrupted and glycocalyx was reduced sharply or even disappeared. The mitochondria were observed with ruptured membranes, swelling, and low cell matrix density (B).

Figure 7

The pelvic tissue adhesion of PID sequela rats on D21. The morphology of the pelvic tissue was assessed after dissection. In the model group, adhesion appeared around the uterine tissue, which was closely related to the surrounding pelvic tissue (A, model 1, model 2, model 3). The adhesion degree of uterine tissue was evaluated after HE staining (B,C). The uterine cavity was reduced and endometrial adhesions which areas had filtration of inflammatory cells were observed in the model group (B). On D 60, overall, the uterine adhesions were extensive, and the infiltration of lymphocytes and macrophages could be seen throughout the entire tissue samples (C). Scale bars: 100 µm. PID, pelvic inflammatory disease; HE, hematoxylin and eosin.

Figure 8

Changes of the expression of TGF-β1 and collagen fibers in adherent uterine tissue. The expression of TGF-β1 in adhesion uterine tissue of PID sequela (D21, D60) was detected by immunohistochemistry (A), Scale bars: 100 µm. The collagen fibers expression in uterine tissue was detected by Masson’s trichrome staining and was stained blue (B). Image-Pro Plus 6.0 analysis software was used for Masson’s trichrome staining analysis and pixel area was used as the standard unit (C). Each group choose three tissues, and each tissue section choose five visual fields. IOD, integrated optical density; average optical density (AOD) = IOD/Area(collagen positive pixel area); collagen fiber area (%) = collagen positive pixel area/tissue pixel area × 100. *P<0.05. TGF-β1, transforming growth factor-beta 1; PID, pelvic inflammatory disease.