Pneumadin in the ventral prostate of rats during postnatal development: a radioimmunological and immunocytochemical study

We have recently demonstrated that the ventral prostate of adult rats contains high levels of pneumadin (PNM), a decapeptide originally isolated from mammalian lung, and that testosterone is needed for the maintenance of a normal level of the peptide in the prostate. Hence, we have investigated, by radioimmunoassay (RIA) and light ultrastructural immunocytochemistry (ICC), PNM concentration and localization in the rat ventral prostate during postnatal development. RIA showed that PNM content increased steadily from day 20 to day 90 of postnatal life, parallel to the increase in the prostate weight. In contrast, PNM concentration remained rather stable, although it showed a marked rise at day 40 when rat testes are known to reach their full maturation. ICC demonstrated that PNM immunoreactivity was mainly located in the apical pole of epithelial cells of rat ventral prostate, especially in the subcellular organelles involved in protein secretion, i.e. rough endoplasmic reticulum cisternae, vacuoles and granules. Taken together our study suggests the involvement of PNM in the functional control of rat prostate during postnatal maturation, although its exact role remains to be elucidated.     

Densitometric and morphometric study of immunocytochemical estrogen receptors detection in breast carcinomas.

Immunohistochemical quantitative evaluation of estrogen receptors (ER) detected in tissue sections from 30 breast tumors by monoclonal antibody was performed using a densitometric method. In particular, ER concentration was calculated by nuclear mean optical density (nMOD), while heterogeneity in ER content was calculated by the coefficient of variation (CV) of the nuclear optical density histogram. Tumors which showed more than 60% of positive cells had a mean value of ER-nMOD of 0.116 +/- 0.002 a.u. and of ER-CV of 33.74 +/- 0.68. Tumors which showed 30% to 60% of positive cells had a mean value of ER-nMOD of 0.082 +/- 0.006 a.u. (arbitrary units) and of ER-CV of 36.25 +/- 3.44. Tumors showing less than 30% of positive cells had ER-nMOD of 0.052 +/- 0.009 a.u. and ER-CV of 48.49 +/- 5.61. These results indicate that the greater the concentration the lower the ER heterogeneity within the tumor sample. No significant differences between ER-ICA results, nuclear size and form factors were found.     

The sinusoidal barrier in rats with portacaval anastomosis: A morphometric study

Summary Shunting of portal blood in the rat leads to liver atrophy and to an increase in arterial blood flow with microcirculatory disturbances. The aim of this study was to investigate the effects of these disturbances on the liver sinusoidal barrier (endothelial and perisinusoidal cells) using morphometric techniques. Rats with portacaval anastomosis (PCA) and sham operated pair-fed controls were studied 3 months after the shunt. Sinusoidal volume density in PCA increased but not significantly and the volume density (V_v) of total endothelial (EC) and perisinusoidal cells (PSC) increased by 104.54% compared to sham operated pair-fed rats. The increase of EC V_v was not associated with an increase in surface density (S_v) suggesting a fall in the number of small fenestrations and an increase in cell thickness. This interpretation supports the morphological observations. The increase of PSC V_v was mainly related to the increase in their subendothelial processes V_v and not to that of the cell body V_v. Lipids V_v and RER S_v expressed per sinusoidal cells remained unchanged suggesting that the balance between the 2 hypothetical functions of the PSC, namely fibrogenesis and storage of vitamin A, was maintained.In conclusion, changes of EC and PSC after PCA result mainly in thickening of the sinusoidal barrier. This increase may impair exchanges between the sinusoidal lumen and Disse space and contribute to functional abnormalities.This work was supported by a grant from INSERM CRL n^o 807003     

Stromal cells in cerebellar haemangioblastomas: an immunocytochemical study

The nature of the stromal cells in formalin-fixed paraffin-embedded material from 23 cerebellar haemangioblastomas was investigated using antisera to intermediate filaments (glial fibrillary acidic protein, vimentin and desmin), histiocytic markers (alpha 1-antitrypsin, alpha 1-antichymotrypsin and lysozyme), glycolytic enzymes (alpha and gamma enolase and aldolase C4) and the endothelial markers, factor VIII related antigen and Ulex europaeus I lectin. Most stromal cells stained positively for vimentin and the glycolytic enzymes. Occasional process-bearing cells within the stroma stained strongly for glial fibrillary acidic protein, alpha 1-antitrypsin and alpha 1-antichymotrypsin. No stromal cell staining for desmin, lysozyme or the endothelial markers was observed, although the latter stained the vascular endothelium within all neoplasms. The findings do not support previous suggestions of an endothelial or histiocytic origin for the stromal cells. They appear to be a heterogeneous population including entrapped reactive astrocytes and locally-derived non-angiogenic cells of neuroectodermal (pial) origin.     

Nuclear iron deposits in hepatocytes of iron-loaded HFE-knock-out mice: a morphometric and immunocytochemical analysis

Nuclear deposits of stainable iron in hepatocytes are a sign of liver iron overload in mice. Animals with no, partial or total knock-out of the HFE alleles, the deletion of which is responsible for hereditary haemochromatosis, were given different forms of dietary iron to measure nuclear iron deposits which were then related to cytoplasmic iron load. Wild type and heterozygous HFE-knock-out mice kept for 52 weeks on a standard diet showed no such deposits. These were, however, demonstrated in low numbers and with small diameters in homozygous HFE-knock-out mice kept on this diet. Nuclear iron deposits were most abundant in all type of mice fed carbonyl iron (2.5% w/w) for 52 weeks almost irrespective of their genetic background. The diameter of these deposits increased with the genetically conditioned extent of hepatocellular iron overload. Mice that were fed a diet containing TMH-ferrocene for 4 weeks showed amounts of hepatic iron that were comparable to those in the carbonyl iron-fed group but nuclear deposits were small and present in only 0.3% of the hepatocytes. While surrounding karyoplasm was immunostained for H- and L-ferritin, the nuclear iron deposits were not. As the nuclear iron deposits corresponded electron microscopically to aggregated ferritin molecules, they represent a non-immunoreactive form of presumably denatured ferritin.     

Ultrastructure of neurons in the auditory cortex of ageing rats: a morphometric study

Summary The cell bodies of pyramidal cells in layers II and V of rat auditory cortex were quantitatively examined in groups of rats 3, 6, 15, 23, 27 and 34–36 months of age. The mean diameters of cell bodies of both layer II and layer V neurons, as measured in 1 m plastic sections, increased between 3 and 15 months of age, then decreased to a diameter that was less in the 36-month-old than in the 3-month-old rats. Morphometry of the nuclei of the cells was done by measuring nuclear area and nuclear envelope length directly on electron micrographs. In the layer II cells, neither parameter changed with advancing age. In the layer V cells, the mean nuclear area decreased significantly in the old animals and the mean envelope length increased. Point-counting techniques were applied to electron micrographs of cell bodies to determine the relative volume of selected organelles, inclusions and ground substance in the perikaryal cytoplasm. In this part of the study the chronological pattern of change in layer II and layer V pyramidal cells was similar. The relative volume of dense bodies increased linearly with advancing age, with a slightly more accelerated rate in layer II cells. The relative volume of ground substance remained essentially constant through 27 months, and then at 34–36 months decreased to 83% and 89% of the three-month level in layer V and layer II, respectively. The relative volume of the rough endoplasmic reticulum (RER) did not change significantly until after 15 months, at which time it began to occupy increasingly a larger fraction of the perikaryal cytoplasm. Finally, the relative volumes of mitochondria, multivesicular bodies and Golgi apparatus did not show clear trends of change during the 33-month period.     

Intact and sympathectomized carotid bodies of long-term hypoxic rats: a morphometric ultrastructural study

Summary The ultrastructure of the carotid body after exposure to hypoxia (10% O₂) for one, two or three weeks was investigated morphometrically. The study was performed on rats after unilateral removal of the superior cervical ganglion. The normally occurring bimodal distribution of type I cells, representing cells with small vesicle profile diameters (SVC) and large vesicle profile diameters (LVC) respectively, changed after one week of hypoxia into a unimodal population. After one or two weeks of hypoxia the diameter range of dense-cored vesicle (DCV) profiles in type I cells was not different from that of DCV profiles in control LVC. After three weeks of hypoxia the DCV vesicle size was intermediate between those of control SVC and LVC. The volume density of DCV decreased after one week but returned to initial values after two and three weeks of hypoxia. At two or three weeks of hypoxia, however, the total cell volume was increased about 1.4 times which should reflect an increase of the total content of DCV at these times of exposure to hypoxia. An increased mean area of cell profiles indicates a hypertrophy of the type I cells, but no signs of hyperplasia could be detected. The ganglionectomy did not cause any remarkable changes compared to the intact carotid body except for a higher volume density of DCV during the early periods of hypoxia.It is inferred from the study that the increased total mass of type I cell tissue during long-term hypoxia is due to a hypertrophy of the cells. Furthermore, the type I cells can increase their storage capacity for catecholamines during hypoxia by an increase in the size and number of DCV.     

Pregnancy and dexamethasone: effects on morphometric parameters of gonadotropic cells in rats

The effects of pregnancy and multiple dexamethasone (Dx) treatment on morphometric parameters of adenohypophyseal gonadotropic cells that produce follicle stimulating hormone (FSH) and luteinizing hormone (LH) were investigated in female Wistar rats. The rats in the experimental group received injections of 1.0, 0.5 and 0.5mg Dx/kg b.w. on days 16-18 of pregnancy, while the control group received equal volumes of saline. There was also an age-matched adult virgin control group. The experimental and control animals were sacrificed 24 and 72h after the last injection. Using the peroxidase-anti-peroxidase immunohistochemical labeling procedure, morphometric analyses showed that cell volume and volume density of FSH and LH cells on day 19 of pregnancy, as well as the number of LH cells, were significantly decreased compared to the virgin control values. On day 21 of gestation, the volume of FSH and LH cells remained smaller than in the virgin controls. Moreover, FSH and LH cell volume was significantly decreased 24h after multiple Dx treatment in comparison with the pregnant controls. Thus, during the last days of pregnancy, the morphometric parameters of gonadotropic cells decreased in comparison with the control virgin rats, but Dx treatment of pregnant rats had an inhibitory influence on FSH and LH cell size only 24h after the last dose.     

Membranous glomerulonephritis: a morphometric study

Archival material from 45 renal biopsies with a diagnosis of idiopathic membranous glomerulonephritis (MGN) were studied by computer-aided image analysis in order to evaluate the prognostic significance of glomerular and interstitial morphometry in MGN. The control group consisted of thirty seven normal renal biopsy specimens. The surface area, the perimeter, the major axis length and the shape factor of renal glomeruli as well as the percentage of the interstitial fibrosis were measured. All the morphometric parameters related to the size of glomeruli had significantly higher values in the patient group (p = 0.000 for all the parameters). However, no significant difference of the glomerular size between different stages of MGN was observed. In contrast, the percentage of interstitial fibrosis increased as the MGN stage rose (median values: 10.3% in stage 1, 14.2% in stage II, 26.9% in stage III, 28.9% in stage IV and 34.2% in stage V, Kruskal-Wallis ANOVA H = 37.645, p = 0.000). In the multivariate analysis the percentage of interstitial fibrosis was the only independent prognostic factor (p = 0.013). Our findings suggest that, in membraneous glomerulonephritis, the interstitial fibrosis increases as the MGN stage progresses, while the size of renal glomeruli has increased at a very early stage of the disease. This fact may indicate that interstitial fibrosis, not glomerular lesions, is mainly responsible for the reduction of renal function.     

Changes in nucleolar morphology during macrophage development in non-Hodgkin's lymphoma: a morphometric study

Nucleolar ultrastructure of macrophages developing in suspension culture from monocytes was studied in 20 patients with non-Hodgkin's lymphoma and 20 normal subjects. Morphometric measurements of nucleolar volume, surface area, volume fraction, surface-to-volume ratio and number of profiles per section were made over a 6 day period. Multivariate analysis of variance showed that culture time and subject group had significant effects: changes during macrophage development were less marked in the patient group, nucleoli were fewer, rounder and possibly smaller than normal. The results indicate disturbed macrophage development in non-Hodgkin's lymphoma and may be related to mononuclear phagocyte dysfunction.     

The supraoptic nucleus in hypothyroid and undernourished rats: an experimental morphometric study

The supraoptic nuclei of both male and female 30-day-old rats rendered hypothyroid by daily subcutaneous injection of propylthiouracil were studied and the results were compared with age- and sex-matched rats fed ad libitum (control rats) and with undernourished rats. Morphometric methods were used to evaluate the volume of the supraoptic nucleus and the areal and numerical densities of its neurons. These parameters allowed us to estimate the total number of neurons of this nucleus. In addition, the mean cross-sectioned area and mean nuclear diameter of the same neurons were also evaluated. The volume of the supraoptic nucleus was reduced both in hypothyroid and undernourished animals when compared with normal controls. The areal and numerical densities of neurons from the former groups were increased and the volume density of the neuropil was reduced. As a consequence, the total number of neurons was found to be identical in all groups studied. Furthermore, the mean nuclear diameters and cross-sectioned areas of the supraoptic neurons were similar in all groups. The volumetric differences and the increased packing found were always more marked for hypothyroid than for undernourished rats. Differences were not detected between male and female groups. The present results support the view that the increased cell packing in hypothyroid animals depends upon a reduction in the neuropil of the nucleus, as has been described under similar conditions in other central nervous system areas displaying identical patterns of neurogenesis. In addition, it was found that the effects of undernourishment cannot be discriminated from those dependent on neonatal hypothyroidism.     

G-cell hyperplasia in chronic hypercalcemia. An immunocytochemical and morphometric analysis

The distribution of antropyloric G cells was mapped immunocytochemically and quantitated morphometrically in chronically hypercalcemic and normocalcemic patients. In the normocalcemic (control) patients, the G-cells were sparse in number and confined to the lower third of the antropyloric mucosa, where they were distributed singly or in small clusters within the glands. The absolute G cell (AGC) counts were 5.8 +/- 0.26 (mean +/- SE) per 0.25 sq mm of mucosa. The hypercalcemic patients showed a marked increase in their antropyloric G-cell population. The cells were uniformly distributed throughout the lower two-thirds of the mucosal thickness and were present in large numbers in practically every gland. The AGC counts in these hypercalcemic patients were 48.2 +/- 13.0, a statistically significant increase. These observations indicate that in man chronic hypercalcemia of diverse etiology is associated with antropyloric G-cell hyperplasia. The physiologic significance of this finding and its role in the pathogenesis of peptic ulcer disease needs to be elucidated.     

Effect of a GABA agonist on the expression and distribution of GABAA receptors in the plasma membrane of cultured cerebellar granule cells: an immunocytochemical study.

The effect of the gamma-aminobutyric acid (GABA) agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, 150 microM) on the localization and density of GABAA receptors in the plasma membrane of rat cerebellar granule cells in primary cultures was studied at the electron microscope (EM) level by preembedding immunogold staining using the monoclonal antibody bd-17 directed against the beta-subunit of the GABAA receptor complex. In THIP-treated as well as untreated control cultures, GABAA receptors were found to be evenly distributed in the plasma membrane of cell bodies as well as processes. However, the density of the GABAA receptors was significantly increased in the THIP-treated cultures as compared to the control cultures and this effect of THIP was particularly pronounced in the processes. GABAA receptors were occasionally observed to form 'hot spots' in process-like structures and again the frequency of these areas with an extremely high density of GABAA receptors was greatly increased in the THIP-treated cultures compared to the controls. It has thus been demonstrated that the ability of GABA agonists to induce formation of low-affinity GABA receptors can be directly visualized and quantified at the EM level using the preembedding immunogold technique. It is likely that low-affinity GABAA receptors are preferentially located in the cell processes and to a considerable extent in the form of 'hot spots'. However, these 'hot spots' also contain high-affinity receptors.     

Histologic,morphometric, and immunocytochemical analysis of myometrial development in rats and mice: II. Effects of DES on development

The effects of diethylstilbestrol (DES) treatment on myometrial development from the prenatal to adult period were examined in rats and mice by histologic and immunocytochemical methods using anti-actin, -vimentin, and -laminin to assess cytodifferentiation of smooth muscle and fibroblastic cells, and by morphometric procedures to assess quantitatively the effect of DES on the expression of cellular orientation in the emerging inner circular myometrial layer. Neonatal rats and mice were treated with DES from day 0 (day of birth) to day 2 with dosages known to perturb myometrial development. Neonatal treatment with DES increased the degree of circular orientation within the uterine mesenchyme, an effect detectable following as little as 24 hr of DES treatment. This effect on spatial organization of the mesenchyme was followed by an increase in the thickness of the actin-positive middle layer (prospective circular myometrium) of uterine mesenchyme during days 3–15; from day 15 onward, however, the circular myometrial layer began to fragment into irregular bundles of smooth muscle, and the longitudinal myometrial layer became thinner and more irregularly organized than controls. Vimentin localization in rats treated with DES neonatally was more intense than in controls within the circularly orientated uterine mesenchyme at 5 days. By 60 days the circular and longitudinal myometrial layers of DES-treated animals showed strands and bundles of vimentin-positive cells, which were not present in controls. Both rats and mice show comparable effects of DES treatment.     

Repopulation of guinea-pig skin by melanocytes during wound healing: a morphometric study

Epidermal keratinocytes and melanocytes have a close functional interrelationship. In order to study this relationship we used computer-assisted three-dimensional morphometry (CAM) to investigate the shape and size changes of the cutaneous melanocyte in healing guinea-pig skin. The combination of CAM with osmium iodide staining and resin embedding of tissue gave excellent results and allowed qualitative and quantitative morphometric assessment of melanocytes in vertical epidermal sections. The changes in melanocytes and keratinocytes during healing of a standard 1 cm full thickness wound in the guinea-pig were studied. After an initial decrease, more melanocytes per mm2 of epidermis were seen (from 36 days). These were smaller in volume with shorter, less branched dendrites compared to controls. An unexpected finding was a late phase of melanocyte proliferation, at the end of our study period (99 days). Clearly, the complex changes in the melanocyte-keratinocyte relationship during wound healing continue throughout and beyond the period of our study.     

The endometrial capillaries during the normal menstrual cycle: a morphometric study.

The areas of the capillary lumen, the entire capillary, the endothelial cells and the adventitia, as well as the thickness of the endothelial cell layer and the adventitia were studied using morphometric methods in endometrial samples from 34 fertile women who had a hormonal profile compatible with normal ovarian function. The biopsies were grouped around the luteinizing hormone surge. The results were calculated as mean values of 72-h periods and related to the mean levels of oestradiol and progesterone circulating in plasma 72 h prior to the biopsy. The results indicated that the sub-epithelial capillary plexus of the human endometrium undergoes dynamic changes during the normal menstrual cycle with a significant dilatation of the vessels during the post-ovulatory phase. A significant correlation was found between the area of the capillary lumen and the mean level of progesterone circulating in the plasma 72 h prior to the biopsy (P = 0.037). We conclude that the ovarian steroids produced during the normal menstrual cycle are likely to influence sub-epithelial vascularization causing dilatation in the post-ovulatory phase. This dilatation of the sub-epithelial capillaries may be related to the development of oedema appearing in the stroma at the time of the expected implantation. The possible functional significance of the capillary dilatation in terms of implantation, however, needs to be further investigated.     

Chromogranin as a marker of neuroendocrine cells in cytologic material--an immunocytochemical study

Cytologic material, including fine-needle aspirations, bronchial brushings, body cavity fluids, and tissue imprints from 39 neuroendocrine and 26 nonneuroendocrine tumors, was stained for chromogranin by the immunoperoxidase technic. Our results suggest that chromogranin is useful in identifying primary as well as metastatic neuroendocrine lesions from a variety of body sites. All lymphoid proliferations and carcinomas, including small-cell anaplastic carcinomas of the lung (oat-cell carcinomas), were negative. Chromogranin appears to be a useful marker in diagnostic cytology. This technic can be applied to routinely prepared cytologic material.