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The development and differentiation of the fetal adrenal cortex was studied in the rat by lipid and enzyme histochemistry.The adrenal cortex began to have small amounts of lipid droplets at 171/2 days by oil red O stain and the amounts increased gradually. Cholesterol by Schultz reaction became clear at 201/2 days. At 151/2 days weak activity of glucose 6-phosphate dehydrogenase (G-6-PDH) was already present in the inner cortex, which was followed a day later by weakly positive reaction of 3β-hydroxysteroid dehydrogenase (3β-HSD). At 181/2 days positive activity of secondary alcohol dehydrogenase (SAD) was obvious in the inner cortex. Activity of alkaline phosphatase (Al-Pase) was observed in the subcapsular zone from day 181/2 on. Enzymatic activities of the cortex increased gradually after their appearance as development proceeded. These findings show that histochemical activities of such principal enzymes as G-6-PDH and 3β-HSD precede appearance of lipid droplets.As a conclusion, zona reticularis is not distinguishable from zona fasciculata during the fetal stage, zona fasciculo-reticularis differentiates histochemically in a 171/2 day old fetus, and finally differentiation of zona glomerulosa occurs at 201/2 days in gestation.

An autopsy case of polysplenia with absence of the hepatic segment of the inferior vena cava in a 53-year-old female is reported. The venous blood from the lower parts of the body was drained through the azygos vein and the superior vena cava into the right atrium. Other than acquired pathological change, the heart was found to be normal. Except for the partial absence of the inferior vena cava, isomerism of asymmetric organs, and heterotaxia of abdominal organs, characteristic of typical polysplenia, were not present. This case of atypical polysplenia suggests the presence of a transitional form in the complex.

Three-dimensional culture with collagen gel, developed recently for the in vitro study of some mammalian cells in a more physiological condition than a monolayer culture, was applied for a biological study of unilocular fat cells. Successfully embedded in the gel, the unilocular fat cells were shown to be able to keep their cellular function and actively proliferate. These findings confirm that unilocular fat cells do undergo proliferation under in vitro conditions as demonstrated in monolayer culture.

A case of malignant cystosarcoma phyllodes with metastasis in the interpectoral lymph node (Rotter's) is presented in this paper. To the author's best knowledge, this is the first case in Japan on this disease, with lymph node metastasis. Although the surgical management of the disease has not yet been standardized, radical or modified radical mastectomy is thought to be the appropriate procedure.

An autoradiographic method was applied in the study of esterase localization in the ultrastructure of rat adrenal glands. Reaction with 3H-diisopropyl fluorophosphate was used to demonstrate esterase activity in various intracellular compartments. The silver grains were observed over lipid granules, mitochondria and smooth-surfaced endoplasmic reticulum, each of which is envolved in steroidogenesis. No silver grains could be detected over the nuclei or over lysosomes. The results are in agreement with those obtained by an ultrahistochemical heavy metal method.

Fetuses with cystic hygroma or loose skin of the neck were studied chromosomally and phenotypically to clarify the relation between neck abnormality and cardiovascular malformations. Of 12 fetuses, 9 had chromosome abnormalities: 4 with 45,X, 3 with trisomy 21, one each with trisomy 13, dup 6q. One had normal chromosomes. Two cases, in which chromosome analysis was unsuccessful, were morphologically suspected to be trisomy 13. Nine of the 12 fetuses had either bilateral cystic hygroma of the neck (7 cases) or nuchal bleb (2 cases: trisomy 13 and dup 6q). Two of the 3 remaining cases (trisomy 21) had loose skin of the neck, and one had edematous swelling of the skin of the neck. Except for the last case of trisomy 21, 11 fetuses (91.7%) had severe and/or rare cardiovascular malformations. They were divided into 3 major groups: a) spectrum of hypoplastic left heart syndrome (45,X and dup 6q), b) double outlet right ventricle, agenesis of semilunar valve (trisomy 13), and c) abnormality of atrioventricular orifice or valves (trisomy 21). One fetus with normal chromosomes had persistent left superior vena cava instead of absent right one and calcification of myocardium. Histological observation of edematous skin demonstrated the abnormal distribution of lymph vessels, including their absence. Some cases showed hypoplastic thymus. To integrate the findings of the present study and the descriptions in the literature, a pathogenesis is hypothesized in relation to migration of neural crest cells and extracellular matrix.

Effects of several conditions known to stimulate the zona glomerulosa of the adrenal cortex in the adult were examined on the fetal adrenal cortex of rats histochemically and electron microscopically. The stimulating conditions were the following; administration of Na restricted diet to the mother, administration of KCl or angiotensin II to the fetus, spironolactone or insulin to the mother. In these, only an intraperitoneal injection of KCl solution to the fetus affected the fetal zona glomerulosa, in which increased activity of glucose-6-phosphate dehydrogenase and hyperfunctional alteration of the mitochondria and the smooth endoplasmic reticulum were observed. This fact shows the fetal glomerulosa cells can react to orae of the specific stimuli on the adult glomerulosa cells., Acta Histochemica et Cytochemica, 13(4), pp.324-329; 1980

Mouse trisomy 16, which was induced by mating males of Rb(16.17)7 Bnr/Rb(9.16)9Rma with three different strains of female, C57BL/6, C57BL/10 and C3H/He, was examined in fetuses and newborns in terms of incidence of occurrence, malformations and sex ratio. The incidence of trisomies tended to be higher in C3H/He than in C57BL/6 and C57BL/10. Trisomies could not survive beyond term in C57BL/6, which was most thoroughly examined in the three strains. Trisomy-associated generalized edema, beginning to appear on day 14 of pregnancy on fetus's back, reached the maximum on day 16 in all strains. On cardiovascular anomalies, persistent common atrioventricular canal was observed in virtually all trisomies from C57BL/10 and C57BL/6, while in about 80% from C3H/He. In conotruncal region, all trisomies from former two crossing exhibited double outlet right ventricle or persistent truncus arteriosus. In addition to these anomalies, riding aorta was found in C3H/He. Hydroureter and hydronephrosis developed from day 17 of pregnancy in trisomies other than those in C57BL/10. The histological study of gonads revealed that trisomy 16 tended to be predominant in males of each crossing. The anomalies encountered in mouse trisomy 16, such as edema, persistent common atrioventricular canal, and hydroureter and hydronephrosis appeared to be in common with human trisomy 21 embryos and fetuses. The tendency of predominance of mouse trisomies in males may provide further homology to human trisomy 21. It is supposed that genetic background by different female strains may affect phenotypic expression of malformations. The extremely high incidence of cardiovascular anomalies is regarded as suitable material for elucidation of pathogenesis.

Pigmented lipid histiocytes (PLH) of chronic granulomatous disease (CGD) were examined by histochemistry and electron microscopy. The yellowish brown pigments were stainable with Sudan III and IV even in paraffin sections, and the sudanophilia was kept after oxidation with peracetic acid followed by methylation. In addition, the granules were positive to Gomori's chromium hematoxylin stain, 0.02% Nile blue sulfate stain and leuco-malachite green stain. Ultrastructural observation showed numerous intracytoplasmic granules in variable shapes and sizes measuring up to 4.8μ in diameter, with various electron densities. From these results, the pigment in PLH of CGD is comparable to a ceroid-like substance. The nature of the pigment and the pathogenesis of PLH are also discussed., Acta Histochemica et Cytochemica, 10(4), pp.500-507; 1977

Cytoplasmic microtubules of fat-laden cells containing fine lipid droplets, such as brown fat cells and adrenocortical cells, were studied in relation to the metabolism of intracellular lipid. In these cells, the amount and distribution of lipid droplets reflect the state of inherent cellular function. Materials used were primary monolayer culture of fetal rat brown fat cells and that of bovine adrenocortical cells. The method was the immunocytochemistry with anti-tubulin antibody. When brown fat cells were being lipolyzed or the steroidogenesis of adrenocortical cells were being stimulated, the cytoplasmic microtubules in the cells were organized in a radial pattern in response to the behavior of the lipid droplets. It is assumed that the microtubules were in the regulation of cellular function in terms of the metabolism of lipid droplets in these cells. We have devised, in the course of the current study, a double fluorescence technique as an observational method whereby microtubules were observed immunocytochemically and lipid droplets by a secondary fluorescence with phosphine E staining.

Two cases of membranous lipodystrophy (T. Nasu) were reported in 24- and 32-year-old men. The roentgenogram showed multiple symmetrical radiolucencies of limb bones where the marrow fat was replaced by numerous characteristic undulating membranes. These membranes consisted of complicated membrane structures, the inner side of which proved to be smooth membrane structures under the electron microscope. Numerous minute tubular structures projected perpendicular to the interstitium from the inner smooth membrane structures. The microscopical findings of subcutaneous fat tissue were described, and the histogenesis of membrane structures under the electron microscope was reported.

Rats were fed on low potassium diets in order to observe the effect of dietary low potassium on the adrenal cortex. The authors clarified morphological changes of the hypofunctional glomerular zone and compared these changes with those of the hyperfunctional glomerular zone. Three weeks after or 2 months after the start of a low potassium diet, slight narrowing of the glomerular zone of the adrenal cortex was observed followed by miniaturization of cells, presence of binuclear cells and an increase of lipid with enlarged lipid drops. Electron microscope mainly disclosed changes of mitochondrial cristae consisting of markedly reduced, enlarged and irregularly dilated cristae with shortening or elongation. Granules appeared in mitochondria. Lysosomes or dense bodies were enlarged. The Golgi's apparatus was atrophied but endoplasmic reticulum did not show remarkable changes. These changes were directly opposite to those of the hyperfunctional glomerular zone noted after a potassium load or seen in sodium deficiency. Consequently, these changes were considered to be the changes of the hypofunctional glomerular zone associated with decrease of aldosterone production. ACTA PATH. SAP. 29: 351–362, 1979.

Recently it has been stressed that catecholamines play a significant role in the pathogenesis of hypertension in Spontaneously Hypertensive Rats (Okamoto and Aoki) (SHR). In this report electron microscopical observation in male SHR was made on the adrenal medulla through the processes of the hypertension. The mild hyperfunctional state of norepinephrine-secreting cells in the initial stage of hypertension was expected on the feature of increased population of norepinephrine-secreting cells, its prominent Golgi complex and rough-surfaced endoplasmic reticulum and increased degranulation in the cell. Moreover, it may be assumed a certain functional change of epinephrine-secreting cell system on the base of increased population of dark epinephrine-secreting cells which are regarded as premature cells., Acta Histochemica et Cytochemica, 5(1), pp.39-44; 1972

This report consists of two cases who died after a neurological disorder of the central nervous system consistent with Behcet's disease clinically and pathologically. Post-mortem examination of the central nervous system revealed mild diffuse meningo- encephalitis with multiple foci of softening, demyelination and glial cell accumulation in the pons, midbrain, cerebral peduncle and internal capsule. The significance of these morphological findings has been discussed from an etiological viewpoint of Neuro-Behcet's syndrome.

In the site of steroid production, the histochemical activity of secondary alcohol dehydrogenase on isopropanol as its substrate with NAD as its coenzyme will reflect the inversion of 20α-22R-dihydroxycholesterol in the biosynthesis of steroid hormone, We applied this method on organs from all parts of the body of Wistar strain rats and observed positive reaction in the adrenal cortex, ovary and testis. In the adrenal cortex of mature rats, the zona glomerulosa exhibited a weakly positive activity, the outer to middle portions of the zona fasciculata an intensely positive activity, the inner portion of the zona fasciculata a weakly positive or negative activity, and the zona reticularis a positive activity. A proportional relationship between secondary alcohol dehydrogenase activity and the amount of lipid was present in the adrenal cortex of mature rats. In addition, the specific distribution of young and old rats was described., Acta Histochemica et Cytochemica, 2(3), pp.119-123; 1969

Intracortical nodules in the zona fasciculata of rat adrenal glands were studied electron microscopically and histochemically. The nodules were classified into the following three types. A) Hyperplastic small cell type having numerous elongated mitochondria with vesicular cristae. Alkaline phosphatase activity was increased in this type of nodule. B) Hypertrophic large cell type having well developed smooth-surfaced endoplasmic reticulum, and occasionally forming a characteristic feature of fenestrated cisterna. Histochemical activity of steroid 3/3-ol dehydrogenase was increased. These morphological appearances suggest that this type of nodule has a greater capacity for steroid synthesis. C) Lipid droplet type having many mitochondria containing lamellar cristae and intramltochondrial deposits often found near large lipid vacuoles. The activity of secondary alcohol dehydrogenase was generally increased. Structural and cellular relationships among these three types of nodule and the pathogenesis of cortical adenomatous nodule were also discussed.

Cancer cell-stromal cell interaction plays a crucial role in the malignant growth of cancer cells. In the skin, the main stromal cell types consist of dermal fibroblasts and subcutaneous adipocytes. Fibroblasts are shown to promote the invasive growth of various cancer cell types. The interaction between cancer cells and stromal adipocytes, however, has not been sufficiently studied even in cutaneous carcinoma. To address the effects of adipocytes on the biologic behavior of cancer cells, we examined the growth and differentiation of a squamous cell carcinoma cell line of the skin (DJM-1), using a three-dimensional collagen gel matrix culture with a cutaneous environmental factor, air exposure. The growth was estimated by the uptake of bromodeoxy-uridine (BrdU) for 24 h. The BrdU indices of DJM-1 cells in stromal-cell-free, fibroblast-containing, and adipocyte- containing conditions were 19.7 +/- 1.9%, 19.8 +/- 2.8%, and 4.7 +/- 1.4%, respectively, whereas the BrdU index on the gel containing both fibroblasts and adipocytes was 10.4 +/- 3.3%. In terms of differentiation, DJM-1 cells cocultured with adipocytes constructed the best-organized stratified layer with a cornified-like structure in all conditions above. The differentiation markers involucrin and cytokeratin 10 were immunohistochemically detected in this structure of DJM-1 cells. Adipocyte-induced phenomena were not affected distinctively by air exposure. These results indicate that adipocytes, but not fibroblasts, promote the differentiation of squamous cell carcinoma cells (DJM-1) and inhibit their growth. These adipocyte-induced phenomena were not completely inhibited by fibroblasts. In conclusion, we suggest that stromal adipocytes may be involved in the differentiating mechanisms of cutaneous carcinoma cells.

A case of Myxo-papillary ependymoma of the cauda equina is reported herein.The patient is a ten-year-old girl who complained of coccyalgia, low back pain and urinary disturbance.In physical examination, muscle weakness of lower extremities and positive stretch leg raising test were observed, but sensibility and tendon reflexes were normal.There were observed xanthochromy and an increase of protein in the celebro-spinal fluid.By myelogram, complete block at the L4 level was seen. A diagnosis of an intradural tumor, laminectomy and removal of the tumor were performed. The tumor in th intradural space was dark-red and gelatious.Histologically the tumor was diagnosed as myxo-papillary ependymoma which is very rare in childhood. After operation the symptoms and pathologic disappeared quickly.

The tissue differentiation of the zona glomerulosa of the fetal adrenal cortex of rats was studied by giving experimental treatments to the fetus in vivo. A low-glucocorticoid-condition was given to the fetus by bilateral adrenalectomy of pregnant rats for removing exogenous glucocorticoids from the fetus, and by brain aspiration of the fetuses for removing the fetal pituitary gland (ACTH) and endogenous glucocorticoids. When the fetus was placed under a low-glucocorticoid-condition for the last couple of days of gestation, poor differentiation of the zona glomerulosa occurred specifically in the fetal adrenal cortex. The degree of the poor differentiation seemed to be proportional to the duration of the low-glucocorticoid-condition. Supplemental administration of glucocorticoids could prevent this poor differentiation of the zona glomerulosa. These results indicate that the tissue differentiation of the zona glomerulosa of the fetal adrenal cortex depends much on glucocorticoids.

ACTH was administered subcutaneously to rat fetus directly at the late stage of fetal development and acute reaction on the fetal adrenal cortex was observed histochemically and electron microscopically. By administration of ACTH the adrenal cortex became remarkably hyperemic and there were swollen cells in all layers, particularly in the middle and inner layers (corresponding to the zona fasciculata and reticularis in adult rat). Marked reduction of lipid, enlarged mitochondria with increased vesicular cristae and increased smooth-surfaced endoplasmic reticulum (SER) were characteristic. The alterations of mitochondria preceded the change of SER, and thereafter mitochondria showed rapid degeneration. The outer layer (corresponding to the zona glomerulosa in adult rat) also showed similar changes by ACTH to those of the other two layers. These results indicated that the fetal adrenal cortex of rats was exogenous ACTH-reactive and its reaction which was different from that of adult cortical cells, seemed to be speciflcally related to the development and differentiation of the cells. ACTA PATH. JAP. 27: 477–484, 1977.

Freezing has been shown to damage pancreatic islets and to disrupt their insulin release, probably because of intracellular ice formation. We compared frozen islets with fresh ones and with others stored at temperatures above freezing from a standpoint of insulin release response to glucose and transplantation. Group A islets, isolated from rats and immersed in 10% dimethyl sulfoxide in RPMI 1640, were stored at -2 degrees C, and group B islets at -196 degrees C, for 7 days. As for group B, the islets were cooled at 1 degree C/min from room temperature to -40 degrees C, subsequently at 3 degrees C/min to -80 degrees C and then put into liquid nitrogen to be rapidly frozen to -196 degrees C. The control islets were fresh. In vitro, basal release at 3.3 mM glucose was similar in group A to that in the controls, but was higher in group B than group A. Stimulated release against 16.7 mM glucose was lower in group A than group B. However, insulin responsiveness, i.e., the ratio of insulin release at 16.7 mM glucose to that at 3.3 mM glucose, was lost in group B. Freezing also caused damage to the group B cells visible under the light and electron microscopes, while group A islets were largely intact. In vivo, after 600 islets were transplanted into streptozotocin-induced diabetic rats, group A was better able to lower fasting blood glucose than was group B, and remained so for 4 weeks. Above sub-zero preservation in the non-frozen state thus seems adequate for the short-term storage for 7 days.

Mature white fat cells (unilocular fat cells) have generally been considered to be in terminal differentiation and, hence, to have no proliferative ability. A new method, referred to as ''ceiling culture,'' has been devised in our laboratory to culture unilocular fat cells in vitro. Under such culture conditions, the fat cells continue to exhibit specific functions of lipid metabolism and proliferate extensively. Intracytoplasmic lipid droplets did not inhibit division of the cells. There were two modes of proliferation of unilocular fat cells: ''loculus-dividing'' cell division, in which the single loculus of fat in the dividing cell was broken down into multiple droplets and distributed evenly between the daughter cells, and ''loculus-preserving'' cell division, in which the loculus in the dividing cell was minimally broken down and inherited with its shape preserved by one of the daughter cells with the other getting only a small number of fine lipid droplets. Such findings suggest that unilocular fat cells in mature fat tissue in vivo are probably capable of proliferation in such modes under some conditions.

Mature white fat tissue consists primarily of unilocular fat cells. Clearly, the study of the biology of these cells would be most helpful for the elucidation of the mechanism of obesity. We describe a new method termed 'ceiling culture' for culturing in vitro unilocular fat cells obtained from humans and rats. These cells can be maintained in culture for long periods of time and, under such conditions, continue to exhibit specific functions such as lipogenesis and lipolysis. Under the culture conditions described, unilocular fat cells change into multilocular fat cells or cells with a fibroblast-like appearance. These cells then proliferate, form a cell monolayer attached to the substratum, and after becoming confluent, exhibit accumulations of intracytoplasmic lipid droplets. These attached dedifferentiated cells continue to exhibit lipogenesis and lipolysis.

This report consists of one case who died after a neuropsychological disorder of the central nervous system consistent with Behcet's syndrome clinically and pathologically. Post-mortem examination of the central nervous system revealed disseminated meningo-encephalitis with multiple foci of softening, demyelinization and glial accumulation throughout the brain, especially in the thalamus, midbrain and pons. Review of the literature revealed about 30 cases including 17 cases in Japan.

The effects of SU-4885 on the rat adrenal cortex by oral administration of 50mg/kg, single or daily for 10 days, were morphologically studied particularly with histochemical methods for the enzyme and lipid. Lipid depletion with fragmentation of droplets throughout the zona fasciculata in rats with single administration conformed to the pattern of adreno-cortical response to an acute moderate stimulus. Reduced activity of secondary alcohol dehydrogenase, fragmentation of lipid droplets, and acidophilic and trabecular changes in the cell cords were observed in the inner fasciculata following a single or daily administration for 10 days. The medication apparently brought about hypersecretion of adrenocortical steroids, which was similar to the effect of ACTH administration. Reduced activity of secondary alcohol dehydrogenase, which reflected the transformation of cholesterol in the steroid biosynthesis, was always observed in the zona fasciculata following the administration of SU-4885 or ACTH. The result might indicate that the administration inhibits some decrease in the steroid biosynthesis from cholesterol possibly through the ‘short route’.

Observations were made in dogs to determine whether X-irradiation affects the secretion of androgens by the testes, and their responses to exogenous gonadotrophin. The secretory activity was, under pentobarbital anesthesia, determined by measuring 17-oxosteroids in spermatic vein blood. The radiation was applied with 200 or 2, 000 R only to the testes, but it failed to stimulate or significantly alter the secretion of 17-oxosteroids in a period of up to 4 hr after exposure. However, at 200 R an increased secretion of testicular 17-oxosteroids into the spermatic vein became evident within 1-6 days, whereas after 2, 000 R it was observed within 13 days only in a few cases. During the post-irradiation period of 30 days, irradiated testes responded to pregnant mare serum in doses of 40iu/kg by producing larger amounts of 17-oxosteroids than normal, the maximum response occurring within a few days following irradiation. In connection with these experiments, enzyme histochemical studies were carried out on the activity of secondary alcohol dehydrogenase in the testicular tissue. At 200 R, the enzyme activity in the Leydig cell areas was increased considerably and attained to the maximum in a faw days after irradiation, and was then progressively reduced.

A 72-year-old patient with von Recklinghausen's disease showed akinetic mutism within 6 months of the onset of dementia. The findings of diffuse cerebral atrophy on CT and periodic synchronous discharges (PSDs) in EEG suggested Creutzfeldt-Jakob disease. However, autopsy findings of diffuse softening of the subcortical white matter and marked arteriosclerotic changes of the subcortical arterioles with sparing of the cortex in the cerebrum confirmed a diagnosis of Binswanger's disease. Binswanger's disease should be included in the differential diagnosis of dementia showing PSDs in EEG.

Histological findings of the thyroid gland in SHR-control showed that the process from synthesis of thyroglobulin to pinocytosis of iodinated thyroglobulin was actively metabolized under the increased stimulation of TSH. Therefore, it was suggested that a decreased secretion rate of thyroxine was mainly resulted from an impairment of hydrolysis of iodinated thyroglobulin or that of following metabolic process.By administration of thyroxine, the thyroid gland of SHR showed less atrophic changes. From the morphological finding of TSH secreting cells of the anterior pituitary gland in SHR, this result would be due to less suppressed TSH secretion.