Regression of endometrial autografts in a rat model of endometriosis treated with etanercept

Objective

To determine the efficacy of anti-tumor necrosis factor therapy (etanercept) for treating endometriosis in the rat endometriosis model.

Study design

A randomized, placebo-controlled, blinded study using rat endometriosis model. After the peritoneal implantation of endometrial tissue, twenty-eight Wistar female rats were randomized to two equal intervention groups: the control group and the etanercept-treated group. After measuring implant volume, pretreatment blood and peritoneal fluid samples were obtained. A vehicle treatment of 2 mL saline to the rats in control group and 0. 4 mg/kg etanercept SC once weekly were administered in the etanercept-treated group. After four weeks treatment period, the volumes and histopathological properties of the implants were evaluated. A scoring system was used to evaluate preservation of epithelia. Endometrial explants were evaluated immunohistochemically for tumor necrosis factor receptor type 2 (TNFR2). A scoring system was used to evaluate expression grade of TNFR2.

Results

There was not a significant difference in spherical volume between control (131.0 (60.3-501.2)) and treatment groups (72.8 (31.2-149.6)) (p > 0.025). There was a significant change in between the volumes of implants before and after treatment in etanercept group (p < 0.05). At the end of the treatment significant differences among the groups were found in histopathological and immunohistochemical parameters (p < 0.05) also histologic scores and HSCORES were decreased in the treatment group significantly (p < 0.05).

Conclusion

These results indicate that etanercept was found to effectively reduce the development of endometriosis in this experimental rat model.     

Effectiveness of photodynamic ablation for destruction of endometrial explants in a rat endometriosis model

OBJECTIVE: To evaluate the potential of photodynamic therapy with aminolevulinic acid (ALA-PDT) for ablation of endometrial explants in a rat endometriosis model and to compare the effect of ALA-PDT, electrosurgery, and surgical resection on normal peritoneum. DESIGN: Prospective controlled experimental trial. SETTING: University medical center. ANIMAL(S): Mature Sprague-Dawley female rats. INTERVENTION(S): Induction of endometriosis and subsequent treatment with ALA-PDT; electrosurgery, and simple resection, and ALA-PDT of normal peritoneum. MAIN OUTCOME MEASURE(S): Histopathological assessment.RESULT(S): Systemic ALA followed by exposure to photoactivating light for 10 or 15 minutes resulted in ablation of all explants harvested 3-4 days after treatment. Permanent destruction was confirmed by absence of regrowth by week 3. Exposure of normal peritoneum to ALA-PDT resulted in initial necrosis, with complete recovery by day 16. Adhesions were present on day 16 in 50% of cases after electrosurgery and in 100% of cases after resection. No adhesions were present in ALA-PDT-treated animals. CONCLUSION(S): Systemic ALA followed by exposure to photoactivating light at relatively low power densities for periods as brief as 10 minutes resulted in ablation of endometriotic explants. Exposure of normal peritoneum to ALA-PDT resulted in complete resurfacing. Both electrosurgery and surgical resection resulted in a greater incidence of surface adhesions.     

Effects of pinealectomy and melatonin supplementation on endometrial explants in a rat model

Objective

To determine the effects of pinealectomy on endometrial explants in rats and evaluate the activity of superoxide dismutase (SOD) and catalase (CAT) and the levels of malondialdehyde (MDA) in the rat endometriosis model.

Study design

Rats with experimentally induced endometriosis were randomly divided into three groups after second-look laparotomies. Group 1 (pinealectomy, n = 8) and Group 2 (pinealectomy + melatonin, n = 8) underwent pinealectomies after the second-look laparotomies. Group 3 was presented as control group (vehicle solution + without pinealectomy (n = 6)). Melatonin was administered intraperitoneally for 4 weeks in Group 2, whereas an equal volume of vehicle solution was given to Groups 1 and 3. Evaluation of the volume of the endometrial explants, histopathological examination and preservation of explant epitheliums according to the scoring system were undertaken.

Results

There was a statistically significant increase in spherical explant volumes of Group 1 compared to Groups 2 and 3. In Group 1, the level of MDA was significantly higher and SOD and CAT activity was significantly lower compared to Groups 2 and 3. A statistically significant increase in the epithelial lining scores of explants was noted in Group 1 compared to Groups 2 and 3.

Conclusion

The effects of pinealectomy on the progression of endometriosis explants were reversed by melatonin.     

Three-dimensional in vitro culture of endometrial explants mimics the early stages of endometriosis

OBJECTIVE: To reproduce the earliest phases of endometriosis using a new in vitro model in which cells from a cultured endometrial fragment can proliferate, invade, reconstitute new endometrial-like tissue, and generate blood vessels. DESIGN: Experimental in vitro study. SETTING: A hospital-based academic research institute. PATIENT(S): Five normal ovulating women undergoing surgery for various benign gynecological indications. INTERVENTION(S): Endometrial samples obtained from the fundus of the uterine cavity were placed in a three-dimensional fibrin matrix culture system. MAIN OUTCOME MEASURE(S): Degree of proliferation of stromal cells and invasion of the fibrin matrix, gland, and stroma formation, vessel sprouting, and immunohistochemical characterization of various cellular components. RESULT(S): During the first week of culture, an endometrial cell outgrowth was observed from the original fragments in 120 of 144 wells (83.3%). Subsequently, cell outgrowths could be quantified in 132 (91.6%), 129 (89.5%), and 127 (88.1%) of the wells after 15, 60, and 90 days, respectively. An invasion of the matrix by the human endometrial cells led to the formation of tubular structures that coalesced into tissue, architecturally resembling endometrium and in which the glands were immunohistochemically positive for cytokeratin. New capillaries, immunohistochemically positive for CD31 and vimentin, sprouted from the endometrial outgrowths at the beginning of the fifth week of culture. CONCLUSION(S): These data show that cells from endometrial explants can proliferate and invade a fibrin matrix in vitro generating new glands, stroma, and vessels consistent with endometriosis. The three-dimensional fibrin matrix used in the present study provides an opportunity to observe the earliest biological events of endometriosis in a quantifiable way.     

子宫内膜异位症动物模型红细胞免疫功能变化及中药内异方的影响

目的 :探讨红细胞免疫功能在子宫内膜异位症 (内异症 )发病中的作用及中药内异方对其的影响。方法 :建立子宫内膜异位症大鼠动物模型 ,随机分为内异症非治疗组、中药内异方治疗组及对照组 ,应用红细胞C3b受体花环试验 (RC3bR)、红细胞免疫复合物花环试验 (RIcR)及红细胞免疫粘附调节因子活性测定方法 (RFER及RFIR)检测各组红细胞免疫功能的变化。结果 :非治疗组RC3bR、RFER明显低于对照组及治疗组 (P<0 .0 1) ,而RIcR、RFIR则高于对照组及治疗组 (P <0 .0 5 )。结论 :内异症红细胞免疫功能降低可能与该病的发生发展有关 ;中药内异方对内异症大鼠红细胞免疫功能有一定的促进作用     

Effect of caffeic acid phenethyl ester on the regression of endometrial explants in an experimental rat model

The objective of this study is to determine the effects of antioxidant and anti-inflammatory caffeic acid phenethyl ester (CAPE) on experimental endometriosis, peritoneal superoxide dismutase (SOD) and catalase (CAT) activities, and malondialdehyde (MDA) levels in the rat endometriosis model. Thirty rats with experimentally induced endometriosis were randomly divided into 2 groups and treated for 4 weeks with intraperitoneal CAPE (CAPE-treated group; 10 micromol/kg/d, n = 13) or vehicle (control group; n = 13). The volume and weight changes of the implants were calculated. Immunohistochemical and histologic examinations of endometriotic explants by semiquantitative analysis and measurements of peritoneal SOD, CAT, and MDA levels were made. Following 4 weeks of treatment with CAPE, there were significant differences in posttreatment spherical volumes (37.4 +/- 14.7 mm(3) vs 147.5 +/- 41.2 mm(3)) and explant weights (49.1 +/- 28.5 mg vs 158.9 +/- 50.3 mg) between the CAPE-treated groups and controls. The mean evaluation nomogram levels in glandular epithelium for COX-2 positivity by scoring system were 2.1 +/- 0.3 in the CAPE-treated group and 3.9 +/- 0.3 in the control group. In the CAPE-treated group, peritoneal levels of MDA and activities of SOD and CAT significantly decreased when compared with the control group (P < .01). Histologic analysis of the explants demonstrated mostly atrophy and regression in the treatment group, and semiquantitative analysis showed significantly lower scores in rats treated with CAPE compared with the control group. CAPE appeared to cause regression of experimental endometriosis.     

FOXM1在大鼠子宫内膜异位症中的表达

目的:研究FOXM1在子宫内膜异位症大鼠模型中的表达,探讨其在内异症中的作用。方法:以雌性未孕Wistar大鼠自身作为供体,取其子宫内膜,采用自体皮下移植法建立内异症大鼠模型。假手术组作为对照组。术后观察28天,测量异位病灶体积。取建模成功的大鼠异位病灶与正常对照组大鼠的子宫内膜,HE染色确定建模成功。免疫组织化学法和Western blot法检测异位病灶与正常对照组大鼠子宫内膜中FOXM1表达。结果:大体观,异位病灶呈椭圆形囊泡,内含清亮液体,表面有新生血管。组织学证实为异位内膜。免疫组织化学染色显示,异位内膜中FOXM1表达于细胞核和细胞质中,主要表达于子宫腔上皮细胞和间质细胞。对照组中,FOXM1在在位内膜中几乎不表达。内异症大鼠异位病灶和在位内膜中FOXM1表达量分别为1.439±0.267和0.886±0.286,差异有统计学意义(P0.05)。结论:异位病灶中的FOXM1表达明显高于在位内膜,其表达增高可能与异位内膜的恶性增殖有关。     

Pathological evaluation of the rat endometriosis model

OBJECTIVE: To observe in detail the morphology of experimental rat endometriosis, specifically in peritoneum adjacent to uterine transplants attached via autotransplantation. DESIGN: Light and electron microscopic study. SETTING: Tochigi Institute of Clinical Pathology, Japan. ANIMAL(S): Female-SD rats maintained on a schedule of 12 hours of light and 12 hours of dark for 2 weeks. INTERVENTION(S): Uterine transplants were attached to rat peritoneum via the surgical autotransplantation technique. The implanted area of peritoneum, including abdominal muscle, were excised from anesthetized rats at four (n = 10), seven (n = 10), and 14 (n = 10) days after uterine autotransplantation. The mesenteries were autotransplanted as a comparative control. MAIN OUTCOME MEASURE(S): We examined the morphologic alterations of uterus-attached peritoneum following the time interval after the implantation. RESULT(S): In rat endometriosis models, the stromal tissue of uterus-attached peritoneum showed proliferation and infiltration of mast cells, eosinophils, plasma cells, lymphocytes, and macrophages. These lesions increased with time after implantation; however, ultimately these infiltrating cells disappeared and proliferation declined. CONCLUSION(S): Our findings suggest that uterine autotransplantation induces the infiltration of allergic inflammatory-related cells and proliferative lesions in peritoneal stroma attached endometrium. These data should prove useful for investigations of human endometriosis.     

Photodynamic therapy of rabbit endometrial transplants: a model for treatment of endometriosis

The potential of photodynamic therapy for endometriosis was evaluated by autotransplantation of endometrial tissue in 15 female virgin New Zealand white rabbits. After maturation, 14 animals were injected intravenously with 10 mg/kg of dihematoporphyrin ether (DHE) and, 24 hours later, transplants were exposed to 630 nm light at 100 to 210 mW/cm2. Sixteen transplants received 100 J/cm2, 10 transplants received 50 J/cm2; 13 untreated transplants served as controls. In the remaining animal that did not receive DHE, 3 transplants were irradiated with 100 J/cm2. Six days after treatment, transplants were harvested with the underlying musculature, and multiple sections were examined histopathologically. Complete endometrial epithelial destruction was seen in 13 of 16 transplants (81%) in the 100 J/cm2 group and in 6 of 10 transplants (60%) in the 50-J/cm2 group. Nearly complete endometrial epithelial destruction was seen in 2 other transplants in each group. No damage occurred in either the 3 transplants that received 100 J/cm2 without prior DHE or in the 13 transplants with DHE and no irradiation. The sensitivity of ectopic rabbit endometrial tissue encourages further evaluation of photodynamic therapy for the treatment of human endometrial disorders.     

Evaluation of endometrial tissue specific complement activation in women with endometriosis.

OBJECTIVE: To determine if endometrial tissue specific complement (C) activation occurs in patients with endometriosis. DESIGN: Prospective. SETTING: University of Oklahoma Health Sciences Center, a tertiary care referral center. PATIENTS: Twenty-six women, 9 with endometriosis and 17 without endometriosis. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Uterine and ectopic endometria were evaluated by the immunofluorescence assay for the presence of activated products of the initial (C3d) and terminal (C5b-9) C pathway, C3 regulatory proteins (decay-accelerating factor and membrane cofactor protein), and terminal C regulatory proteins (clusterin and vitronectin). RESULTS: The initial (C3d) and terminal (C5b-9) C components were deposited on blood vessel walls in uterine and/or ectopic endometria of women with and without endometriosis. In the stromal compartment at both sites, C deposition was colocalized with terminal C inhibitors/cell-cell attachment factors, clusterin and vitronectin on elastic fibers. No specific staining for C proteins was detected on the glandular epithelium of uterine and ectopic endometrial tissues examined. Furthermore, intense staining of endometrial epithelium for C3 regulatory proteins, decay-accelerating factor, and membrane cofactor protein was noted on endometrial glands from women with and without endometriosis. CONCLUSIONS: Our findings demonstrate a lack of specific deposition of C in the glandular epithelial cells of endometria of women with endometriosis. The localization of C3 regulatory proteins, decay-accelerating factor, and membrane cofactor protein on glandular epithelium may suggest the involvement of intrinsic protective mechanisms on these cells from autologous C attack in vivo.     

Effects of peritoneal macrophages from women with endometriosis on endometrial cellular proliferation in an in vitro coculture model.

OBJECTIVE: To study the effects of peritoneal macrophages on endometrial cellular proliferation in an in vitro coculture model and to compare the magnitude of these effects between macrophages from women with endometriosis and normal women. DESIGN: Controlled study of peritoneal macrophage function. SETTING: University hospital. PATIENT(S): Patients with a normal peritoneal cavity (n = 15) and with pelvic endometriosis (n = 20) undergoing laparoscopy. INTERVENTION(S): Peritoneal macrophages were cocultured with endometrial epithelial and stromal cells; endometrial cell cultures without macrophage coculture acted as controls. MAIN OUTCOME MEASURE(S): Endometrial cellular proliferation measured by 3H-thymidine incorporation. RESULT(S): Endometrial epithelial cells cocultured with peritoneal macrophages from women with endometriosis showed significantly increased proliferation compared with cocultures using macrophages from normal women when assessed at 24 hours (1.56 versus 1.03 times, respectively, over control) and at 72 hours (1.55 versus 1.10 times over control). Endometrial stromal cells cocultured with peritoneal macrophages from women with endometriosis similarly exhibited increased proliferation compared with cocultures using macrophages from normal women when assessed at 24 hours (1.65 versus 1.17 times over control) and at 72 hours (1.65 versus 1.21 times over control). CONCLUSION(S): Peritoneal macrophages of patients with endometriosis stimulate cellular proliferation of endometrial epithelial and stromal cells in vitro.