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PubMed2000

Oligoclonality of early lesions of the urothelium as determined by microdissection-supported genetic analysis.

AIM: To contribute to the ongoing discussion of clonality of human urothelial cancer it was considered a valuable approach to analyze multiple areas from cystectomy specimens for deletions of chromosomes known to be involved early in bladder cancer development. MATERIAL AND METHODS: Thus, in 86 biopsies of 4 human cystectomies with different histological findings (maximal diagnosis: pT1G2, pTaG3, pT2G2, normal) loss of heterozygosity (LOH) was investigated as a deletion marker using markers of chromosomes 8p, 9p, 9q and 17p. Findings were compared to histology of the lesion. RESULTS: Findings indicate: (1) no changes in the markers investigated in the bladder with histologically normal urothelium in contrast to detection of LOH in normal urothelium of tumour-bearing bladders; (2) an accumulation of the number of LOH with increasing malignancy of lesions within one bladder, and (3) indications of oligoclonal neoplastic lesions in two of the urinary bladders investigated. CONCLUSIONS: The investigation of multiple lesions within one bladder presents a snapshot of genetic changes in differently advanced tumour stages. The hypotheses of tumour evolution and oligoclonality as derived from our LOH data need to be supported by deletion-independent clonality studies as X-chromosomal inactivation analysis.

Carcinoma in Situ↗
PubMed1989

Corticosteroid receptors and glucocorticoid content in microdissected brain regions: correlative aspects.

Stereoselective competition was used to determine (3H)-aldosterone binding to type I corticosteroid receptors, and (3H)-dexamethasone binding to type II receptors in punches obtained from 11 brain regions of short-term adrenalectomized (ADX) rats. It was observed that type I receptor binding was almost exclusive of the hippocampus (HIPPO), while type II receptor binding was more generally distributed among HIPPO, cerebral cortex, lateral septum, ventromedial and arcuate hypothalamic nuclei, with lower levels in 6 additional regions studies. We determined corticosterone (CORT) in brain punches from ADX rats, ADX rats receiving CORT for 5 days, intact rats and intact rats receiving ACTH for 5 days. We correlated (3H)-ligand binding with CORT content in punches obtained from identical brain regions and showed a significant positive correlation in the case of the ADX plus CORT group, for type II corticosteroid receptors. Similarly, a significant correlation emerged with type II sites, when binding capacity was correlated with percentage increases of CORT in brain areas of rats receiving ACTH. It is suggested that in situations where CORT levels are elevated, changes in CORT retention throughout the brain occur as a function of the type II glucocorticoid receptor, although at the level of the HIPPO, both receptors may provide appropriate control of the CNS-pituitary-adrenal axis, according to the physiological or stress levels of circulating hormone.

Adrenalectomy↗
PubMed1994

Development of the atherosclerotic core region. Chemical and ultrastructural analysis of microdissected atherosclerotic lesions from human aorta.

Lipid deposits in human atherosclerotic fibrous plaques exhibit marked differences in chemistry and ultrastructure from lipid deposits in fatty streaks, leading some investigators to question whether fibrous plaques originate from fatty streaks. To examine lesion transition, we employed lipid microanalysis, electron microscopy, and immunohistochemistry on fatty streaks, fibrolipid lesions (small raised lesions), and fibrous plaques from human aorta. Both fatty streaks and caps of fibrolipid lesions were high in esterified cholesterol content (mean, 62% of total cholesterol) and high in cholesteryl oleate content compared with cholesteryl linoleate content. Fatty streaks and fibrolipid lesion caps also showed similar morphology, characterized mostly by macrophage-derived foam cells in the superficial intima. Core lipids in both small and large raised lesions differed markedly from this pattern. Fibrolipid lesion cores showed mostly vesicular extracellular deposits, sometimes accompanied by cholesterol clefts, while fibrous plaque core deposits were also extracellular but had a variable appearance. Compared with fatty streaks, fibrolipid lesion cores showed significantly increased free/total cholesterol fractions (63%) and decreased fractional contents of cholesteryl oleate. Fibrous plaque cores had variable distributions of free and esterified cholesterol but significantly decreased cholesteryl oleate fractions compared with fatty streaks. The results support the concept of lesion transition, which is marked by deep intimal, extracellular deposition of cholesterol-rich, vesicular lipid deposits in small raised lesions. In the core region of larger raised lesions, both cholesterol-rich and cholesteryl ester-rich lipid deposits appear to form in the extracellular space.

Aorta↗
PubMed1983

Lung structure as revealed by microdissection. Positional morphology of human lung.

A technique of lung structure analysis is presented that allows visualization of the airway in its conceptual form. The lung is dissected such that the bronchial tree appears as a branching, continuous surface whose fine structure varies as a function of position along the airway. In these preparations, single loci of interest can be studied sequentially with any optical or electron microscope. This provides precise correlation of surface morphology and intracellular structure. Microstructural information can be indexed by either branch order or linear distance along the air path. Morphometry can be performed on surface features of the airway imaged with the scanning electron microscope using this indexing system. The anatomy of a human airway is presented.

Aged↗
PubMed1993

Different localization and regulation of two types of vasopressin receptor messenger RNA in microdissected rat nephron segments using reverse transcription polymerase chain reaction.

Recent studies have revealed that arginine vasopressin (AVP) has at least two types of receptors in the kidney: V1a receptor and V2 receptor. In this study, microlocalization of mRNA coding for V1a and V2 receptors was carried out in the rat kidney using a reverse transcription and polymerase chain reaction. Large signals for V1a receptor PCR product were detected in the glomerulus, initial cortical collecting duct, cortical collecting duct, outer medullary collecting duct, inner medullary collecting duct, and arcuate artery. Small but detectable signals were found in proximal convoluted and straight tubules, inner medullary thin limbs, and medullary thick ascending limbs. Large signals for V2 receptor mRNA were detected in the cortical collecting duct, outer medullary collecting duct, and inner medullary collecting duct. Small signals for V2 receptor were found in the inner medullary thick limbs, medullary thick ascending limbs, and initial cortical collecting duct. Next, we investigated V1a and V2 receptor mRNA regulation in the dehydrated state. During a 72-h water restriction state, the plasma AVP level increased and V2 receptor mRNA decreased in collecting ducts. In contrast, V1a receptor mRNA did not change significantly. Thus, the two AVP receptor subtypes are distributed differently along the nephron, and these mRNAs are regulated differently in the dehydrated state.

Animals↗
PubMed1986

Effects of hyperprolactinemia on estrous cyclicity, serum luteinizing hormone, prolactin, estradiol, and progesterone concentrations, and catecholamine activity in microdissected brain areas.

The purpose of the present study was to determine the duration of PRL treatment required to suppress estrous cyclicity and preovulatory LH surges and to ascertain whether such treatment affects cyclicity by altering catecholamine activity and/or ovarian steroid secretion. Rats exhibiting 4-day estrous cycles were treated with ovine PRL (oPRL) or vehicle beginning on diestrous day 1 at 0900 h, diestrous day 2 at 2400 h, proestrus at 0600 h, or proestrus at 1200 h. Jugular veins of rats were cannulated to the level of the right atrium on diestrous day 2. Unrestrained rats were bled on proestrus. Preovulatory LH surges and ovulation were completely blocked, and vaginal cytology remained leukocytic on the expected day of proestrus and estrus when oPRL treatment was begun on diestrous day 1. Such treatment elevated progesterone levels beginning on diestrous day 2 and suppressed the preovulatory rise in estradiol observed on proestrous morning and afternoon in control rats. To determine the effect of oPRL on catecholamine activity, alpha-methylparatyrosine was administered to groups of oPRL- or vehicle-treated rats at 0900 or 1500 h on diestrous day 1, diestrous day 2, or proestrus. Animals were killed 0, 45, or 90 min later. Norepinephrine and dopamine concentrations were measured in the median eminence, suprachiasmatic nucleus, medial preoptic nucleus, striatum, ventral aspect of the stria terminalis, and posterior pituitary gland by radioenzymatic assay. Controls exhibited increased norepinephrine turnover rates in the median eminence, suprachiasmatic nucleus, and medial preoptic nucleus on proestrous afternoon concomitant with preovulatory LH surges. In contrast, oPRL-treated rats showed no such increase. In addition, median eminence dopamine turnover rates were elevated beginning on the afternoon of diestrous day 1 in oPRL-treated rats compared to control values. No other differences in norepinephrine and dopamine turnover rates were observed in oPRL-treated rats compared with controls in any other brain area on any day examined. Thus, the data indicate that elevated PRL concentrations have profound effects on reproductive cyclicity by disrupting ovarian steroid secretion and essential preovulatory neurochemical events in selected brain areas involved in the regulation of LHRH.

Animals↗
PubMed1987

The quantitative distribution of cytosolic androgen receptors in microdissected areas of the male rat brain: effects of estrogen treatment.

Estrogen and androgen synergize in the regulation of various neuroendocrine functions. To determine a potential cellular basis of this synergism, we measured androgen receptor (AR) in the cytosol of 16 hypothalamic and limbic nuclei and subregions in castrated male rats and castrated rats treated with estradiol. Androgen receptor was measured by a previously validated in vitro binding assay using the synthetic androgen methyltrienolone [( 3H]R1881). Male Sprague-Dawley rats (250-350 g) were castrated 2 weeks before the implantation of a 2.5-cm Silastic capsule filled with crystalline 17 beta-estradiol. Control rats were sham implanted. Estrogen treatment lasted for 1 week, after which time the animals were killed, their brains were frozen and sectioned, and individual nuclei and subregions were removed by a tissue punch technique. Tissue from six rats were combined for each determination. The highest levels of AR were found in the ventromedial nucleus (16.5 +/- 1.4 fmol/mg protein), medial preoptic area (12.1 +/- 1.4 fmol/mg protein), bed nucleus of the stria terminalis (11.6 +/- 1.4 fmol/mg protein), lateral septum (11.4 +/- 1.4 fmol/mg protein), arcuate nucleus-median eminence (10.9 +/- 2.1 fmol/mg protein), and medial amygdala (10.3 +/- 0.9 fmol/mg protein). Estrogen treatment resulted in significant increases in AR in medial preoptic area (14.8 +/- 0.6 fmol/mg protein; P less than 0.05) and medial amygdala (14.6 +/- 1.2 fmol/mg protein; P less than 0.02). Subsequent studies using block-dissected hypothalamus-preoptic area, anterior pituitary, and prostate revealed significant estrogen-mediated elevations in AR in anterior pituitary cytosol [42.2 +/- 3.0 vs. 26.4 +/- 1.6 fmol/mg protein (control); P less than 0.01], but not in hypothalamus-preoptic area or prostate cytosols. Estrogen treatment had no effect on AR affinity. The binding of [3H]R1881 was specific for AR and was not affected by the addition of radioinert progesterone to the incubation tube. Estimates of AR concentration were similar regardless of whether [3H]R1881 or [3H]dihydrotestosterone was used as the ligand. In this study, we describe the distribution of AR throughout the hypothalamus and limbic areas using biochemical techniques. In addition, we have identified some cellular events that may mediate the synergistic actions of estrogen and androgen on the neuroendocrine system.

Animals↗
PubMed1980

Ultrastructure of polytene chromosomes of Drosophila isolated by microdissection.

Drosophila polytene chromosomes prepared by a new micromanipulative procedure, which avoids acid squashing, have been examined at the ultrastructural level in the electron microscope. Puffs at 2B, 68C, 74EF, 75B and 85EF, have been examined in some detail, along with the chromocentre and various interbands. The ultrastructure of these chromosomes, which have never been exposed to acid protein denaturants, compares favourably with that of classical acid-fixed specimens. Ribonucleoprotein particles in puffs are seen to be organized in linear arrays and evidence is adduced for looped transcription units. Particles with characteristic sizes and morphologies are observed near the chromocentre, in puffs and in interbands. In interbands RNP particles and 'superbead'-like chromatin particles may be distinguished. Drosophila polytene chromosomes isolated by micro-manipulation should prove useful for the localization of native chromosomal proteins at an ultrastructural level.

Animals↗