Relationship Between Leukotriene Receptor Antagonists on Cancer Development in Patients With Bronchial Asthma: A Retrospective Analysis.

BACKGROUND/AIM: An association between leukotriene receptor antagonists (LTRA) and cancer has been previously reported, but the relationship between LTRA use and cancer prevention remains controversial. This study aimed to clarify the cancer-preventive effect of LTRA in Japanese patients with bronchial asthma. PATIENTS AND METHODS: We obtained information from a large populationbased medical information database to analyze data on patients who were newly diagnosed with bronchial asthma between 2006 and 2015. Eligible participants were patients who were prescribed an LTRA for at least 30 days (LTRA users) and those who were not using LTRA (LTRA non-users) during the objective period. LTRA users and LTRA non-users were matched 1:1 using propensity scores. RESULTS: The 1:1 propensity score matching of LTRA users and LTRA nonusers facilitated the inclusion of 3,744 participants each, in these two subgroups. The results of the Cox proportional hazards model after adjustment for covariates showed no significant difference in the cancer risk between LTRA users and non-users [adjusted hazard ratio (HR)=0.83, 95% confidence interval (CI)=0.59-1.16]. The subgroup analysis showed no significant difference in the cancer risk between the LTRA low-cumulative dose group and LTRA non-users, or between the LTRA medium-cumulative dose group and LTRA non-users. In contrast, the LTRA high-cumulative dose group had a significantly lower risk of developing cancer compared with LTRA non-users (adjusted HR=0.57, 95% CI=0.33-0.98). CONCLUSION: LTRA use may prevent cancer in patients with bronchial asthma.

Leukotriene Receptor Antagonist Use and Dementia Risk in Patients With Asthma: A Retrospective Cohort Study.

BACKGROUND/AIM: Recent experimental studies have reported that leukotriene receptor antagonists (LTRAs) might protect against dementia. However, few clinical studies have examined this in humans. This study assessed whether the use of LTRAs can prevent the onset of dementia in humans. PATIENTS AND METHODS: A large population-based retrospective cohort study was conducted using a health insurance claims database in Japan, which included patients newly diagnosed with bronchial asthma between 2006 and 2015. Each of these patients that was LTRA user was matched with a randomly selected LTRA non-user according to age, sex, and bronchial asthma diagnostic year. RESULTS: There were 10,471 patients in both the LTRA user and the LTRA non-user group. Using Cox proportional hazards models, a significant reduction in the risk of developing dementia was observed in the LTRA user group compared to the non-user group (adjusted hazard ratio=0.42, 95% confidence interval=0.20-0.87, p=0.019). CONCLUSION: Our data suggest that the use of LTRAs may prevent the onset of dementia in asthmatic patients.

Nrf2 Lowers the Risk of Lung Injury via Modulating the Airway Innate Immune Response Induced by Diesel Exhaust in Mice.

In the present study, we investigated the role of Nrf2 in airway immune responses induced by diesel exhaust (DE) inhalation in mice. C57BL/6J Nrf2+/+ and Nrf2-/- mice were exposed to DE or clean air for 8 h/day and 6 days/week for 4 weeks. After DE exposure, the number of neutrophils and macrophage inflammatory protein (MIP)-2 level in bronchoalveolar lavage fluid (BALF) and interleukin (IL)-17 level in the lung tissue increased in Nrf2-/- mice compared with Nrf2+/+ mice; however, the lack of an increase in the level of tumor necrosis factor (TNF)-α in the lung tissue in Nrf2+/+ mice and mild suppression of the level of TNF-α in Nrf2-/- mice were observed; the level of granulocyte macrophage colony-stimulating factor (GM-CSF) in the lung tissue decreased in Nrf2-/- mice than in Nrf2+/+ mice; the number of DE particle-laden alveolar macrophages in BALF were larger in Nrf2-/- mice than in Nrf2+/+ mice. The results of electron microscope observations showed alveolar type II cell injury and degeneration of the lamellar body after DE exposure in Nrf2-/- mice. Antioxidant enzyme NAD(P)H quinone dehydrogenase (NQO)1 mRNA expression level was higher in Nrf2+/+ mice than in Nrf2-/- mice after DE exposure. Our results suggested that Nrf2 reduces the risk of pulmonary disease via modulating the airway innate immune response caused by DE in mice.

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.

Effect of aerosol particles generated by ultrasonic humidifiers on the lung in mouse.

BACKGROUND: Ultrasonic humidifiers silently generate water droplets as a cool fog and produce most of the dissolved minerals in the fog in the form of an aerosolized "white dust." However, the health effect of these airborne particles is largely unknown. This study aimed to characterize the aerosol particles generated by ultrasonic humidifiers and to investigate their effect on the lung tissue of mice. METHODS: An ultrasonic humidifier was operated with tap water, high-silica water, ultrapure water, or other water types. In a chamber (0.765 m3, ventilation ratio 11.5 m3/hr), male ICR mice (10-week-old) were exposed by inhalation to an aerosol-containing vapor generated by the humidifier. After exposure for 7 or 14 days, lung tissues and bronchoalveolar lavage fluid (BALF) were collected from each mouse and examined by microarray, quantitative reverse transcription-polymerase chain reaction, and light and electron microscopy. RESULTS: Particles generated from the humidifier operated with tap water had a mass concentration of 0.46 ± 0.03 mg/m3, number concentration of (5.0 ± 1.1) × 10(4)/cm3, and peak size distribution of 183 nm. The particles were phagocytosed by alveolar macrophages in the lung of mice. Inhalation of particles caused dysregulation of genes related to mitosis, cell adhesion molecules, MHC molecules and endocytosis, but did not induce any signs of inflammation or tissue injury in the lung. CONCLUSION: These results indicate that aerosol particles released from ultrasonic humidifiers operated with tap water initiated a cellular response but did not cause severe acute inflammation in pulmonary tissue. Additionally, high mineral content tap water is not recommended and de-mineralized water should be recommended in order to exclude any adverse effects.

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.

Maternal exposure to carbon black nanoparticle increases collagen type VIII expression in the kidney of offspring.

The potential health risks of inhaling nanomaterials are of great concern because of their high specific activity and their unique property of translocation. Earlier studies showed that exposure to nanoparticles through the airway affects both respiratory and extrapulmonary organs. When pregnant mice were exposed to nanoparticles, the respiratory system, the central nervous system and the reproductive system of their offspring were affected. The aim of this study was to assess the effect of maternal exposure to nanoparticles on the offspring, particularly on the kidney. Pregnant ICR mice were exposed to a total of 100 µg of carbon black nanoparticle on the fifth and the ninth days of pregnancy. Samples of blood and kidney tissue were collected from 3-week-old and 12-week-old male offspring mice. Collagen expression was examined by quantitative RT-PCR and immunohistochemistry. Serum levels of creatinine and blood urea nitrogen were examined. Exposure of pregnant ICR mice to carbon black resulted in increased expression of Collagen, type VIII, a1 (Col8a1) in the tubular cells in the kidney of 12-week-old offspring mice but not in 3-week-old ones. The levels of serum creatinine and blood urea nitrogen, indices of renal function, were not different between the groups. These observations were similar to those of tubulointerstitial fibrosis in diabetic nephropathy. These results suggest that maternal exposure to carbon black nanoparticle induces renal abnormalities similar to tubulointerstitial fibrosis in diabetic nephropathy are induced in the kidney of offspring.

Pathological study for the effects of in utero and postnatal exposure to diesel exhaust on a rat endometriosis model.

Previous studies have shown that prenatal and postnatal exposure to diesel exhaust (DE), which is known to be one of the main constituents of air pollution, enhances the persistence of endometriosis in a rat model. The aim of this study is to investigate the pathological changes induced by DE exposure in a rat model of endometriosis. Pregnant Sprague-Dawley rats were exposed to DE or clean air beginning on gestational day 2 and neonatal rats were persistently exposed to DE or clean air. Endometriosis was induced by autotransplantation of endometrium onto the peritoneum of eight-week-old female offspring. Endometriotic lesions were examined at 7 and 14 days post-transplantation. As a result, infiltration of activated mast cells remained in deeper area of peritoneal tissue around the endometriosis model compared to the control group at 14 days post-autotransplantation. In the DE exposure group, 14 days post-transplant, the remaining lesions contained fibroblasts and activated mast cells, which were surrounded by collagen fibers. The data showed that prenatal and postnatal DE exposure enhances the activation of mast cells and prolongs the persistence of collagen fibers in the induced rat model of endometriosis.

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.

[Health effects of nanomaterials on next generation].

In order to discuss the health effects of nanomaterials, we cannot disregard the research on the health effects of airborne particulates. It is said that many of the fine or ultrafine particles in airborne particulates originate from diesel vehicles in metropolitan areas. The results of not only animal experiments but many epidemiologic surveys and volunteer intervention experiments in humans are reported on the health effects of particles. Although the health effects of the particulate matter particle sizes below 10 µm (PM10) were investigated in the initial studies, recently even smaller particles have come to be regarded as questionable and research of the health effects of the minute particulate matter below 2.5 µm (PM2.5) has been done. However, our recent study about maternal exposure to diesel exhaust suggests that health effect study of PM0.1, particles below 0.1 µm (100 nm), namely nanoparticles, is necessary from now on. We are proceeding with the study of the health effects of various types of intentionally produced nanomaterials such as carbon black, carbon nanotube, fullerene and titanium dioxide, examining in particular their influence on next generation. Although there are differences in the sites affected and the seriousness of the damage, basically similar findings to DEPs mentioned above are being discovered in research on nanomaterials. Regardless of dosage and administration method, such as inhalation, endotracheal administration, nasal drip and subcutaneous administration, once nanomaterials enter the bloodstream of a pregnant mother mouse, they move to the offspring and have effects on them. The effects may appear as various symptoms in the process of growth after birth, and can sometimes lead to the onset and aggravation of serious diseases.

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.

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.

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.

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. METHOD OF STUDY: In this study, the morphological and numeric comparisons of degranulated mast cells were performed between endometriotic lesions and comparative regions (eutopic endometrium and normal uterine serosa) of patients with and without endometriosis. RESULTS: In cases of endometriosis, diffuse infiltration of numerous mast cells was observed throughout the stromal lesions. These mast cells exhibited degranulation, and scattered granules were also observed. In the eutopic endometrium and normal uterine serosa of both the endometriosis patients and the controls, mast cells were rarely detected. CONCLUSIONS: These results suggest that an abnormal immune response, specifically a hypersensitivity reaction, is strongly related to endometriosis; our findings will be helpful in the development of methods for the treatment of endometriosis.

Localization of mast cells in endometrial cysts.

PROBLEM: Although the presence of mast cells in endometriosis lesions has been reported, there have been no reports of their pathological localization in ovarian endometrial cysts. METHODS OF STUDY: The localization of mast cells in ovarian endometrial cysts were investigated using hematoxyline and eosin and toluidine blue staining with light microscopy, immunohistochemical c-kits, and electron microscopy. RESULTS: Mast cells were identified in endometrial cyst tissues. Few mast cells were localized in the endometrial stroma, which is characteristic of endometrial gland-like regions. Many mast cells were noted around blood vessels and the interstitium with fibrosis, that is, the fibrotic interstitium of endometrial cysts. CONCLUSIONS: Mast cells may be involved in the development and progression of endometriosis. Localization of mast cells suggests a particularly close relationship with fibrosis and adhesion.

Light and electron microscopic evaluation of antileukotriene therapy for experimental rat endometriosis.

OBJECTIVE: To evaluate the effect of antileukotriene therapy, we examined the morphologic alteration in experimental rat endometriosis both with and without leukotriene receptor antagonist treatment. DESIGN: Light and electron microscopic analysis. SETTING: Tochigi Institute of Clinical Pathology, Japan. ANIMAL(S): Female Sprague-Dawley 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. After autotransplantation, the rats were given daily administration of leukotriene receptor antagonist, and the lesions were examined at 4 and 7 days after autotransplantation. MAIN OUTCOME MEASURE(S): The morphologic comparison of rat endometriosis with and without antileukotriene therapy. RESULT(S): In the rats treated with leukotriene receptor antagonist, a significant decrease in stromal proliferation was observed when compared with nontreated rats. The treated rats showed not only the suppression of infiltration and activation of mast cells but also widespread apoptosis of proliferative fibroblasts in the lesions. CONCLUSION(S): Our results reveal that a leukotriene receptor antagonist has significant therapeutic value for the treatment of rat endometriosis. It is likely that antileukotriene therapy would be efficacious in the treatment and prevention of human endometriosis.

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

STUDY OBJECTIVES: To investigate the pathophysiology of human endometriosis, we examined by morphological and molecular biological methods. METHODS: Samples 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. RESULTS: Infiltration 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 FCER1G 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, COL4A2, and COL5A2) were also higher in endometriotic lesions than in the normal ovarian tissue. CONCLUSIONS: Thus it is likely that mast cells and their related inflammatory immunoreactions via chemokines play important roles in producing fibrosis and adhesions in endometriotic lesions.

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.

[Therapeutic effect of roxithromycin on chronic sinusitis with nasal -- polyps clinical, computed tomography, and electron microscopy analysis].

To evaluate the therapeutic effect of Roxithromycin (RXM), we studied 56 chronic sinusitis patients with nasal polyps using computed tomography (CT) and electron microscopy in addition to conventional clinical assessment. The paranasal sinus of subjects was observed clinically before and after daily administration of RXM at 300 mg for 3 months all underwent allergy testing for possible complications of allergic rhinitis based on subjective symptoms and objective findings. Improvement after RXM treatment was seen in 50.3% based on subjective symptoms and 59.1% based on objective findings. Overall improvement was seen in 53.6%. In 41 cases (73.2%) of all patients with chronic sinusitis and complications of allergic rhinitis, no significant difference was seen between patients with and without complications (53.7% in those with complications and 53.3% in those without). In CT analysis the paranasal sinus in 51.8% of all posttreated patients showed obvious improvement. In electron microscopy in chronic sinusitis patients with complications of allergic rhinitis, pretreated ethmoidal sinus tissues showed high mucous epithelial cell apoptosis in addition to common histological lesions, while posttreatment patients showed only eosinophil apoptosis in the interstitium and no apoptotic epithelial cells. We divided ethmoidal sinus lesions in patients without complications into 3 types and evaluated them as follows: In type 1, pretreated ethmoidal sinus tissues showed plasma cell infiltration and posttreatment cell apoptosis. In type 2, pretreated tissues showed lymphocyte and plasma cell infiltration and posttreated showed only some lymphocytes and no plasma cells. In type 3, proliferation of fibroblasts, most of which showed apoptosis, was seen in addition to apoptotic epithelial cells before treatment, while after treatment, these lesions remained with some apoptotic bodies phagocytosed by macrophages. In type 3 patients relapsed after surgery. Our findings indicate that RXM treatment had a significant therapeutic effect on chronic sinusitis with nasal polyps with and without complications of allergic rhinitis. We clarify the morphological mechanism of therapeutic effect of RXM on each type of ethmoidal sinus lesion divided by light and electron microscopy.