Establishment and Evaluation of a Rat Model of Non-Puerperal Mastitis

doi:10.12300/j.issn.1674-5817.2024.065

Abstract

Abstract:

Objective This study aims to establish a non-puerperal mastitis (NPM) rat model by simulating hyperprolactinemia and immune-inflammatory states, and to evaluate its local inflammatory characteristics in the mammary gland, thereby laying the foundation for research on the diagnosis and treatment of this clinically challenging disease. Methods Twelve SPF-grade Wistar female rats were evenly divided into a control group and a model group. During the experiment, the control group received no experimental treatment or medication. The model group received daily subcutaneous injections of 100 mg/kg metoclopramide hydrochloride for 7 consecutive days. Serum prolactin (PRL) levels were measured using ELISA on the 10th, 20th, and 30th days after the first injection. After 7 days of injections, 200 μL of lactating SD rat milk was mixed with 200 μL of complete Freund's adjuvant to prepare an oil-in-water emulsion, which was administered by multiple subcutaneous injections into the back of the Wistar rats for the initial immunization. Seven days after the initial immunization, the emulsion was injected subcutaneously into the third, fourth, and fifth mammary glands for the final immunization. After the final immunization, the rats were observed for 28 days for changes in mammary gland appearance, and the size of mammary nodules was calculated. On the 3rd, 7th, 14th, and 28th days, hematoxylin-eosin (HE) staining was used to analyze mammary tissue morphology. Immunohistochemistry was employed to detect CD138 expression levels. ELISA was used to measure the levels of interleukin (IL)-6, IL-1β, tumor necrosis factor (TNF)-α, and inducible nitric oxide synthase (iNOS) in mammary tissue to comprehensively assess the model. Results Rats in the model group exhibited mammary skin ulceration and foul odor at the ulcer sites. Palpation and ultrasound revealed the formation of mammary nodules. HE staining showed that on the 3rd day after the final immunization, normal ductal and lobular structures in the mammary glands disappeared, with significant infiltration of plasma cells. On the 7th day, ductal dilation, epithelial necrosis and detachment, and pronounced periductal plasma cell and lymphocyte (predominantly T-lymphocytes) infiltration were observed. On the 14th day, there was a proliferation of fibrofatty tissue, small blood vessels, and granulation tissue, with scattered plasma cells in the interstitium. By the 28th day, inflammatory cell infiltration and fibrous tissue proliferation were reduced, with granuloma formation. Serum PRL levels in the model group were significantly increased on the 10th day (P<0.05) and the 20th day (P<0.001). IL-6 and TNF-α levels in mammary tissue were higher in the model group compared to the control group on the 3rd, 7th, 14th, and 28th days (P<0.05). IL-1β levels were higher on the 3rd, 7th, and 14th days compared with the control group (P<0.01) but lower than the control group on the 28th day (P>0.05). iNOS levels were significantly higher on the 7th day after the final immunization (P<0.001). Conclusion A successful NPM model was established in rats, which exhibited typical pathological features such as local mammary masses, abscesses, ulcers, ductal dilation and plasma cell infiltration. This model can serve as a foundation for further research into the diagnosis and treatment of this clinically challenging disease.

Key words: Non-puerperal mastitis, Wistar rats, Prolactin, Immune inflammation

CLC Number:

S857.26

YIN Yulian,MA Lina,TU Siyuan,et al. Establishment and Evaluation of a Rat Model of Non-Puerperal Mastitis[J]. Laboratory Animal and Comparative Medicine, 2024, 44(6): 587-596. DOI: 10.12300/j.issn.1674-5817.2024.065.

Figures/Tables 4

Figure 1 Comparison of local mammary changes and nodule size in control and model groups of ratsNote：A represents the appearance of the mammary gland in the model group after successful modeling， where multiple nodules are observed， with local redness and swelling， and some ulcerations with purulent discharge. B and C represent the ultrasonography results of the mammary gland in the model group， where B shows multiple dilated mammary ducts （indicated by arrows）， and C displays inflammatory nodules within the mammary tissue （indicated by arrows）. D represents the normal mammary tissue appearance in the control group. E represents the pathological mammary nodule manifestation in the model group. F represents the trend of changes in the size of mammary nodules over 28 days of observation after the final immunization for both the control group （n=6） and model group （n=6）. Compared with the control group， d1-d8， ***P<0.001； d9-d14， **P<0.01； d15-d28， *P<0.05.

Figure 2 Comparison of prolactin levels in the two groups measured by ELISANote： The prolactin levels were detected by ELISA method on the 10th， 20th， and 30th days after metoclopramide hydrochloride injection in the control group （n=4）. Compared with the control group （n=4）， *P＜0.05， ***P＜0.001.

Table 1 Comparison of IL-6, IL-1β, TNF-α, and iNOS levels between the control group and the model group at four observation time points after the final immunization

时间节点 Time point	组别 Group	IL-6 ρ/(pg·mL^-1)	IL-1β ρ/(pg·mL^-1)	TNF-α ρ/(pg·mL^-1)	iNOS c/(μmol·L^-1)
第3天 Day 3	对照组 Control group	65.10±15.51	34.41±1.97	235.39±23.00	8.74±0.87
第3天 Day 3	模型组 Model group	91.42±5.64^***	71.45±25.20^**	332.05±16.48^***	9.04±0.85
第7天 Day 7	对照组 Control group	43.18±16.58	34.56±2.18	324.54±7.83	9.53±1.87
第7天 Day 7	模型组 Model group	68.92±17.70^**	52.44±1.85^***	364.07±27.15^**	13.95±0.92^***
第14天 Day 14	对照组 Control group	59.00±6.71	23.56±4.10	258.08±34.30	9.13±1.67
第14天 Day 14	模型组 Model group	71.14±4.95^***	43.71±10.05^**	348.70±36.87^**	11.13±1.63
第28天 Day 28	对照组 Control group	51.15±7.70	44.66±2.64	256.21±39.02	11.33±0.84
第28天 Day 28	模型组 Model group	84.76±18.18^***	40.92±3.41	303.89±33.40^*	12.20±0.55

Figure 3 HE and CD138 staining results of mammary tissue in the control group and the model group at different time points after the final immunization （×400）

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