Tomomi Ihara

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.

Increase of Activated Mast Cells in Human Endometriosis

Problem:  The proliferation of stromal cells in endometriosis promotes extensive adhesion; therefore, the morphological analysis of stromal lesions is important in the investigation of the pathogenesis of endometriosis.

Cytokine and chemokine expression in a rat endometriosis is similar to that in human endometriosis.

The pathogenesis of endometriosis, a gynecologic disorder associated with infertility, appears to involve immune responses. However, the details involved have not been clarified. In this study, we analyzed expression levels of interleukin (IL)-6, IL-10, monocyte chemoattractant protein-1, eosinophil chemotactic protein, macrophage inflammatory protein-1alpha, and regulated on activation normal T cell expressed and secreted (RANTES) and CC chemokine receptor 1 in endometriotic lesions in a rat model in which endometrium is autotransplanted onto peritoneal tissue and found that they were remarkably increased, while those of IL-2, IL-4, and interferon-gamma were not. These results were obtained in a rat model induced by autologous, not allogeneic, transplantation of endometrial epithelium to the peritoneum. Expression of these factors is consistent with that of endometriosis in humans. Therefore, this model may be useful in the investigation of the pathogenesis and treatment of endometriosis.

Role of immunoreactions and mast cells in pathogenesis of human endometriosis-morphologic study and gene expression analysis-

Study objectivesTo investigate the pathophysiology of human endometriosis, we examined by morphological and molecular biological methods.MethodsSamples of ovarian endometriosis and normal ovarian tissues were obtained laparoscopically after informed consent. A morphological study by toluidine blue staining, immunohistochemistry of c-kit and electron microscopy demonstrated the localization of mast cells in the stromal lesions of endometriosis. Oligonucleotide microarrays were used for gene expression analysis.ResultsInfiltration of numerous mast cells and development of fibrosis was observed throughout the stromal lesions. Gene expression analysis by oligonucleotide microarrays indicated inflammatory immunoreactions in the lesions. Expressions of the FCERlG and PGDS, which are considered to be mast cell-specific genes, were upregulated in the ovarian endometriotic lesions as compared to the normal ovarian tissues. Furthermore, expressions of genes associated with immunological inflammation, such as IL-8, GRO1, GRO2, CXCR4, MCP1, and those related to tissue remodeling (MMP, COLAA2, and COL5A2) were also higher in endometriotic lesions than in the normal ovarian tissue.ConclusionsThus it is likely that mast cells and their related inflammatory immunoreactions via chemokines play important roles in producing fibrosis and adhesions in endometriotic lesions.

Effects of Maternal Exposure to Ultrafine Carbon Black on Brain Perivascular Macrophages and Surrounding Astrocytes in Offspring Mice

Perivascular macrophages (PVMs) constitute a subpopulation of resident macrophages in the central nervous system (CNS). They are located at the blood-brain barrier and can contribute to maintenance of brain functions in both health and disease conditions. PVMs have been shown to respond to particle substances administered during the prenatal period, which may alter their phenotype over a long period. We aimed to investigate the effects of maternal exposure to ultrafine carbon black (UfCB) on PVMs and astrocytes close to the blood vessels in offspring mice. Pregnant mice were exposed to UfCB suspension by intranasal instillation on gestational days 5 and 9. Brains were collected from their offspring at 6 and 12 weeks after birth. PVM and astrocyte phenotypes were examined by Periodic Acid Schiff (PAS) staining, transmission electron microscopy and PAS-glial fibrillary acidic protein (GFAP) double staining. PVM granules were found to be enlarged and the number of PAS-positive PVMs was decreased in UfCB-exposed offspring. These results suggested that in offspring, “normal” PVMs decreased in a wide area of the CNS through maternal UfCB exposure. The increase in astrocytic GFAP expression level was closely related to the enlargement of granules in the attached PVMs in offspring. Honeycomb-like structures in some PVM granules and swelling of astrocytic end-foot were observed under electron microscopy in the UfCB group. The phenotypic changes in PVMs and astrocytes indicate that maternal UfCB exposure may result in changes to brain blood vessels and be associated with increased risk of dysfunction and disorder in the offspring brain.

Microarray analysis provides insight into the early steps of pathophysiology of mouse endometriosis model induced by autotransplantation of endometrium

AIMS To characterize the biochemical alterations that occur in the peritoneal tissue of the mouse endometriosis model during early development of the lesion using microarray analysis. MAIN METHODS The endometriosis model was induced by autotransplantation of endometrium in 8-week-old female ICR mice. Peritoneum only (excluding the transplant) was obtained 24, 48, and 96 h after the autotransplantation and subjected to microarray analysis. To interpret the large amounts of data generated and to enable a functional analysis, genes were classified using Gene Ontology (GO) and Medical Subject Heading (MeSH) terms, and the results were compared with previous reports on endometriosis. KEY FINDINGS Of the upregulated genes, those involved in the inflammatory response, cell adhesion, extracellular matrix, wound healing, hormones, and leukocytes were significantly enriched 24 and 48 h after autotransplantation. Those of cytokines, antibody-producing cells, dendritic cells, inflammation, and infertility were enriched after 96 h. Analysis using GO and MeSH provided different information. Particularly, MeSH showed a link between an anatomical and diseased phenotype with common genes found to be upregulated. SIGNIFICANCE The factors occurring during early development of endometriosis induced by endometrium autotransplantation are increase in adhesion molecules and inflammatory responses rather than angiogenesis. Data presented herein may reveal a novel therapeutic gene targets and will contribute to knowledge for the treatment of this currently incurable disease.

Expression Profile of Extracellular Matrix and Adhesion Molecules in the Development of Endometriosis in a Mouse Model

Ectopic endometrial tissue induces various reactions in surrounding tissues, such as the surface of the ovary and peritoneal cavity, leading to endometriosis. The aim of this study is to investigate the expression profile of extracellular matrix (ECM) and adhesion molecules in the early steps of development of experimental mouse endometriosis, specifically in peritoneum adjacent to endometrium transplants attached via autotransplantation. The endometriosis model was induced by autotransplantation of endometrium to peritoneal tissue. Peritoneal tissues adjacent to the transplant were obtained at 1, 4, and 7 days posttransplantation. The results showed that messenger RNA expression levels of most of the integrins, collagens, and other ECM reached a peak at 7 days posttransplantation. Uniquely, Lamc2 was significantly increased to its maximum level within 24 hours posttransplantation and may be strongly associated with initiation of the development of endometriosis. These data will be helpful in further investigations of the treatment of endometriosis.

Mouse Colitis Induced by Escherichia coli Producing Yersinia enterocolitica 60-Kilodalton Heat-Shock Protein (Light and Electron Microscope Study)

In patients with inflammatory bowel disease(IBD), including ulcerative colitis (UC), heatshockprotein (Hsp) 60 has been detected in serum and theintestinal tract. Our mouse colitis model wasestablished using Escherichia coli transformed withYersinia enterocolitica Hsp60 gene as an immunizingantigen, and examined light and electron microscopicallyas compared with lesions of UC. The large intestine of mice injected with Hsp60 antigen showed swollengoblet cells, glandular dilation, erosion, ulceration,and infiltration of inflammatory cells. The change likecrypt abscesses was also found, and various phases of inflammation were observedsimultaneously in individual mice. In addition, thereticulum fibers were absent ultrastructurally in thesubepithelial reticular layer. Hyperplasia of the thymuswas found in antigentreated mice. These lesionswere similar to those of UC. These results suggest thatUC-like enteritis in mice was induced by using Hsp60,considered as one of the pathogens for UC.

Clarithromycin and telithromycin increases interleukin-10 expression in the rat endometriosis model

Endometriosis is a common gynecological disorder associated with infertility. However, treatment options remain limited at present. Since the pathogenesis involves immune responses, the immunomodulatory effect of macrolide on endometriosis has been the focus of much research. A previous study showed that clarithromycin decreased stromal proliferation and promoted apoptosis of fibroblasts in an endometriosis model in rats; however, the mechanism of the effect remains unknown. The aim of this study is to investigate the effect of clarithromycin, one of the major macrolides, and telithromycin, one of the antibiotics belonging to a macrolide group (ketolide), on IL6, IL10 and Ccl2 expression in a rat endometriosis model induced by the surgical transplantation of endometrium onto the peritoneum in 8-week-old female Sprague-Dawley rats. After autotransplantation, the rats were given daily administration of clarithromycin (16 mg/kg/day or telithromycin (12 mg/kg/day) for 3 days. The induced lesions were examined 4 days after autotransplantation. After treatment, IL10 expression in the lesions was increased in rats treated with clarithromycin (1.70-fold) and telithromycin (2.88-fold). The drugs attenuated proliferative stromal lesion of the endometriosis model. The results showed that in the endometriosis model, the drugs enhanced expression of IL10, which may play a role in inhibiting excess inflammatory reaction with its therapeutic effect on the lesion. Macrolide and ketolide therapy may have significant value for the treatment of human endometriosis.

An ultrastructural study on the ligamentum flavum of the cervical spine in patients with ossification of the posterior longitudinal ligament

Some histological analyses of the ossification of the posterior longitudinal ligament (OPLL) have been reported, but no ultrastructural studies of the ligamentum flavum (LF) in patients with OPLL have been published to date. To understand the pathology of the ossification of the spinal ligament, we examined, by electron microscopy, ultrastructural changes in the LF in cases of OPLL and made a comparison with the LF in cervical spondylotic myelopathy (CSM). Subjects were three men and two women with cervical OPLL who underwent longitudinal spinous process-splitting laminoplasty. During surgery, a small piece of the LF was collected from C2–C3 to C7–T1 and was then analyzed by light and electron microscopy. We observed atrophic elastic bundles with a two-layer structure and disarrangement, a partially torn area, the disappearance of microfibrils, and an enlarged interstitium with an irregular alignment of collagen fibrils. We observed some properties of a cell preceding its death: the initial phase may be the disappearance of the plasma-membrane, followed by the scattering of many organellae around its degenerated nucleus. Finally, many extracellular plasma membrane-invested particles that resemble matrix vesicles remain there without phagocytosis. These results suggest that ultrastructural abnormalities exist in the spinal ligament in cases of ossification of the spinal ligament.