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PubMed1983

Microdissection of the mouse egg.

A rapid and efficient method for microdissection of the mouse egg is described. The dissection is carried out in hanging drops of medium surrounded by heavy liquid paraffin oil at room temperature. Eggs are first deformed into a cylindrical shape and then dissected at a predetermined site with a glass needle on a Leitz micromanipulator. The survival rate of the dissected fragments is 75-90% and between 20 and 30 eggs can be dissected in an hour. Development of the dissected eggs is at least as good as that described after other types of manipulation. Cytoplasts and karyoplasts of various sizes can be prepared, as well as gynogenetic and androgenetic eggs with different amounts of cytoplasm. This procedure may help to examine nuclear-cytoplasmic interactions in eggs reconstituted from a variety of fragments.

Animals↗
PubMed1988

Vitamin E distribution in ocular tissues following long-term dietary depletion and supplementation as determined by microdissection and gas chromatography-mass spectrometry.

Vitamin E is thought to be important for protection of polyunsaturated fatty acids (PUFA) from oxidative damage. A microbiochemical procedure using microdissection and gas chromatography-mass spectrometry was developed to determine vitamin E distribution in ocular tissues in a rodent model, with the eventual goal of using it in a study of phototoxic degeneration of the retina, where PUFA oxidation is potentially the causal mechanism. Sample preparation was achieved by freeze-drying the retina followed by micro-dissection to obtain the desired structures for analysis. A deuterated alpha-tocopherol internal standard is added to the tissue sample before extraction and derivatization which are achieved in a single step. The data presented show the vitamin-E content in various structures of the retina, particularly the outer segments and retinal pigment epithelium (RPE); however, the vitamin E content of other ocular tissues is also included. Data were obtained from albino and pigmented rats receiving vitamin E-depleted, supplemented, and regular chow diets, and from rabbits and cats receiving regular chow diets formulated for each species. Within all dietary groups the highest concentration of vitamin E was located in the RPE followed by the outer segments of the photoreceptor cells. Other ocular tissues consistently contained lower amounts of vitamin E. Different tissues were depleted of vitamin E at different rates and this points out the importance of determining vitamin E levels in tissues of interest in studies on the consequences of dietary depletion.

Animals↗
PubMed1990

Measurement of immunoreactive angiotensin II levels in microdissected brain nuclei from developing spontaneously hypertensive and Wistar Kyoto rats.

Levels of immunoreactive angiotensin II (ANG II) were measured in specific microdissected nuclei from the brains of newborn (NB; less than 1 week of age), 4-, 8-, and 12-week-old spontaneously hypertensive rats (SHR) and their age-matched normotensive controls, Wistar Kyoto (WKY) rats, using a sensitive radioimmunoassay. The structures investigated included the paraventricular nucleus of the hypothalamus (PVH), the nucleus of the solitary tract (NTS), the dorsal motor nucleus of the vagus (DMN of X), the locus coeruleus (LC), and the A1 region of the medulla. A section of cerebellar cortex was used as a control. Although ANG II was detected in each of the nuclei examined, there were no differences in the ANG II contents of any of these structures between young (NB and 4 week old) SH and WKY rats. However, by 8 weeks of age, the SHR had significantly higher ANG II levels in the PVH, NTS, and DMN of X than its normotensive control, and at 12 weeks of age, significantly higher ANG II levels were observed in the PVH, NTS, DMN of X, and LC of the SHR compared to those in the WKY. During the developmental period under investigation, both strains revealed increases in the ANG II content of all nuclei except for the LC, where the ANG II levels decreased with age. No detectable ANG II was found in the cerebellar cortex of either strain at any age.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II↗
PubMed1995

Immune responses of sheep to microdissected parts of Haemonchus contortus.

Adult specimens of Haemonchus contortus were microdissected into four body fragments: oesophagus, cuticle of oesophagus, gut and cuticle with adjacent muscle layer. The antigenicity of these different body fragments was analysed in comparison to total (whole) worm extracts with immunoblotting and ELISA using sera of H. contortus-infected sheep. In particular, oesophagus-derived antigens appeared to be specifically recognized and may prove valuable in diagnostic assays.

Animals↗
PubMed1989

Characterization of the long terminal repeats of micropia elements microdissected from the Y-chromosomal lampbrush loops "threads" of Drosophila hydei.

Four micropia elements from Drosophila melanogaster and D. hydei have been analysed by sequencing. Two elements, from D. hydei, micropia-DhMiF8 and -DhMiF2, were recovered by cloning microdissected Y-chromosomal lampbrush loops "threads". This method allows isolation of repetitive sequences from defined chromosomal positions, but recovery of large and overlapping inserts is difficult. In case of the Y-chromosomal micropia elements it was not possible to define the endpoints of their long terminal repeat sequences precisely. Comparison of these locus-defined micropia elements to complete micropia elements isolated from D. melanogaster allowed identification of micropia-DhMiF8 and micropia-DhMiF2 long terminal repeats (LTRs). LTR sequences from the two Drosophila species are not conserved except for a few short sequences found at comparable positions that are believed to have functional significance. In contrast, the Leu-tRNA primer binding site and plus strand primer binding site are conserved between D. melanogaster and D. hydei.

Animals↗
PubMed1986

The structure of the wall of the rat intraacinar pulmonary artery: an electron microscopic study of microdissected preparations.

Rat intraacinar arterial segments that by light microscopy lack a medial muscle layer are capable of constriction and, during pulmonary hypertension, acquire morphologically differentiated smooth muscle. These facts suggest that effector cells of smooth muscle type are present in the normal vessel wall. Studies in the literature, however, fail to agree on their location or even existence. By combining microdissection, step sectioning, and electron microscopy, we have now performed a serial study of six arterial pathways. At its proximal end, the artery has a circumferentially continuous single layer of smooth muscle cells, separated from the endothelium by a fenestrated internal elastic lamina. Myoendothelial junctions are frequent and incorporate basal laminae of both cell types. More distally, the internal elastic lamina is discontinuous and the smooth muscle cells lose myofilaments and dense bodies so as to resemble intermediate cells. They still form a continuous layer, however. At mid-alveolar-duct level, this layer is discontinuous, and in the most distal arteries investigated, the cells are often solitary. They lie close to the endothelial cell, but, except for localized regions of contact, are separated from it by a single basal lamina that is continuous with one covering their abluminal surface.

Animals↗
PubMed1991

Nuclear estrogen receptor binding in microdissected brain regions of female rats during pregnancy: implications for maternal and sexual behavior.

Previously, we showed that changes in nuclear estrogen receptor (NER) concentrations within large dissections of the preoptic area and hypothalamus of pregnant rats might underlie changes in both maternal and sexual behaviors. To more precisely localize these biochemical changes, NER concentrations were measured in microdissected nuclei within the preoptic area and hypothalamus of pregnant rats on days 8, 16 and 22. Results indicated that NER levels changed significantly throughout pregnancy and that the magnitude and direction of change was site specific. Although a different pattern of change was found in each nucleus, NER levels in most preoptic area nuclei were significantly higher by the 16th day of pregnancy compared to those on day 8, while receptor levels in most hypothalamic nuclei did not increase until day 22 of pregnancy. The higher NER levels found in preoptic area nuclei of female rats as pregnancy advances can be correlated with growing maternal responsiveness during pregnancy, while elevated concentrations of NER in hypothalamic nuclei at the end of pregnancy may indicate preparation for postpartum estrus.

Amygdala↗
PubMed1986

A tissue culture model for the study of peptide synthesis and secretion from microdissected hypothalamic explants.

Using established tissue culture methodology, a protocol has been developed for the culture of specific hypothalamic regions. A microdissection technique is used to remove the paraventricular and supraoptic nuclear regions from fresh tissue. These explants are maintained in culture and appear to be viable on the basis of several criteria: the continuing presence of peptides in both tissue and media, the ability to incorporate a labeled amino acid precursor into proteins and peptides, the histological appearance using phase contrast microscopy, and the immunocytochemical identification of peptide neurons. This tissue culture model may prove useful in the study of neuroendocrine mechanisms in specific brain regions.

Catecholamines↗
PubMed1976

Cystic changes in adult human kidneys after ureteral obstruction: microdissection studies.

Five kidneys from patients suffering from ureteral obstruction were selected for the study; from each, 100 nephrons and 20 collecting ducts were studied using Darmady's microdissection technique. Whereas three kidneys showed macroscopic cysts, microscopically, all kidneys displayed dilation and diverticulation of the nephrons particularly of the loop of Henlé and of distal convoluted tubules. Some dilatation of the collecting ducts was also noticed.

Aged↗
PubMed1978

Microdissection findings of medullary sponge kidney.

Findings on microdissection of specimens of medullary sponge kidney showed uniform enlargement of collecting tubules in part of the papillae. Nephrons were normal in fragments without calculi and infection.

Dissection↗
PubMed1984

Molecular clones of the mouse t complex derived from microdissected metaphase chromosomes.

Fragments of the proximal half of mouse chromosome 17 including the t-complex region were microdissected from metaphase spreads. DNA was isolated from a pool of such fragments, and was cloned on microscale. Individual clones were used to probe genomic digests of DNA from a pair of Chinese hamster cell lines with or without mouse chromosome 17, and livers of congenic inbred lines of mice carrying wild-type and/or t-haplotype forms of chromosome 17. The data obtained indicate that 95% of the low copy number microclone inserts recognize DNA sequences present on mouse chromosome 17. It has been possible to use one-third of these clones to identify restriction-fragment-length polymorphisms between wild-type and t-haplotype DNA on a congenic background. These results demonstrate that these clones have been derived from the t-complex or regions closely linked to it. Clones of this type should provide starting points for a molecular analysis of this region of the mouse genome.

Animals↗
PubMed1989

Neurotensin in microdissected brain nuclei and in the pituitary of the lean and obese Zucker rats.

Neurotensin (NT) is a tridecapeptide common to the gastrointestinal tract and central nervous system which suppresses food intake when centrally injected in various regions of the hypothalamus. We measured neurotensin levels in several microdissected brain nuclei as well as in the pituitary in 10 obese hyperphagic Zucker (fa/fa) rats, 9 heterozygous Fa/fa and 5 Fa/Fa lean rats. The greatest NT concentration and content were observed in the anterior lobe of the pituitary in the median eminence and in the lateral preoptic area (500 to 1000 pg/area, 3 to 5 ng/mg protein). NT was also detected in the median preoptic area, paraventricular (PVN), supraoptic, ventromedian nuclei (VMN) (about 250 pg/nucleus, 1.5 to 2 ng/mg protein). The smallest amounts were found in the suprachiasmatic (SCH) and accumbens nucleus (about 100 pg/nucleus, 1 ng/mg protein) and the peptide was absent in the cortex. NT content in the obese rat was significantly lower in all brain nuclei examined except the accumbens nucleus. This was most evident in the three nuclei involved in the regulation of feeding behaviour: PVN (276 +/- 38 (Fa/Fa) vs 188 +/- 15 (fa/fa) pg/nucleus, P less than 0.05), VMN (226, +/- 21 (Fa/Fa) vs 75 +/- 22 (fa/fa) pg/nucleus, P less than 0.001), and SCH (98 +/- 14 (Fa/Fa) vs 52 +/- 11 (fa/fa) pg/nucleus, P less than 0.05). There was no difference in the pituitary lobes between lean and obese rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1996

RT-PCR microlocalization of bradykinin B2 receptor mRNA in microdissected rat nephron segments.

Microlocalization of mRNA coding for the bradykinin 2 (BK2) receptor was carried out in the rat kidney. We combined reverse transcription and polymerase chain reaction (RT-PCR) with microdissection of individual renal tubule segments. Relative quantitation of the resulting amplified cDNA utilized densitometry of autoradiograms from Southern blots probed with a specific 32P labeled probe. The largest signals for BK2 receptor PCR product were detected in the cortical collecting tubule (CCT) and the proximal straight tubule (PST). BK2 receptor mRNA expression was also detected in the glomerulus, inner medullary thin limb (IMTL) and in the distal tubule (DT). Small but detectable signals were founded in the medullary thick ascending limb (MTAL). This distribution is consistent with multiple sites of BK action to account for different renal effects such as hemodynamics, water and electrolytic balance regulations.

Acid-Base Equilibrium↗
PubMed1983

Application of high-performance liquid chromatography with electrochemical detection to the determination of catecholamines in microdissected regions of the rat brain.

High-pressure liquid chromatography (HPLC) with electrochemical detection was applied for measurement of catecholamines in microdissected tissues from discrete brain regions. Brain areas were dissected, extracted and processed for HPLC assay without further purification. The advantage of this method is its simplicity and relatively low cost and may therefore be applied to the measurement of amines in brain micropunches.

Animals↗
PubMed1996

Partial deletion of the long arm of chromosome 5: a fluorescence in situ hybridization study using band-specific painting probes generated by chromosome microdissection.

A series of 17 patients with myeloid disorders and partial deletions of the long arm of chromosome 5 were investigated using fluorescence in situ hybridization with 5q34-q35 region-specific painting probes generated by chromosome microdissection. This approach confirmed that partial 5q deletions are interstitial in all the cases studied. In addition no translocation involving chromosome 5 was detected.

Adult↗
PubMedDate not supplied

Chromosome microtechnology: microdissection and microcloning.

The physical microdissection of chromosomes and subsequent microcloning of dissected fragments is enabling the generation of very large numbers of cloned unique sequences from defined chromosomal regions. In addition to use in constructing region-specific libraries of the entire human genome and providing probes for mapping and sequencing purposes, such chromosome microtechnology should facilitate the search for disease-associated genes in defined chromosome regions.

Chromosomes↗
PubMed1988

Quantitation of N-acetyl-aspartyl-glutamate in microdissected rat brain nuclei and peripheral tissues: findings with a novel liquid phase radioimmunoassay.

Antibodies were raised in rabbits against the neuropeptide N-acetyl-L-aspartyl-L-glutamate (NAAG) coupled to bovine serum albumin via a carbodiimide linkage. One of these rabbit antisera, which preferentially recognizes coupled NAAG-like immunoreactivity (LIR), has been previously used to immunocytochemically localize NAAG-LIR. We have now employed a second of these antisera, which preferentially recognizes free NAAG, to develop a competitive liquid phase radioimmunoassay (RIA). Using this assay, we were able to detect picomole amounts of NAAG in rat tissue extracts. The specificity of the assay revealed a 60-fold greater affinity of the antibody for NAAG over N-acetyl-aspartate (NAA) and greater than one million-fold specificity for NAAG over both aspartate and glutamate. High-pressure liquid chromatographic (HPLC) separation of tissue extracts yielded only two detectable peaks of NAAG-LIR in collected fractions and these co-chromatographed with NAAG and NAA. NAAG levels determined by this liquid phase RIA and by HPLC were essentially identical after correction for the presence of NAA crossreactivity. The antibody that preferentially recognizes coupled NAAG was used to immunocytochemically localize NAAG-LIR to the red nucleus, the facial nucleus, the dorsal raphe, and the locus coeruleus. To further confirm this localization of NAAG, these and other nuclei were microdissected and levels of NAAG were determined by liquid phase RIA. Nuclei which stained intensely were found to contain high levels of NAAG by RIA and between 60 and 100% of this NAAG-LIR co-chromatographed with NAAG. These results support our previous conclusion that NAAG is co-localized in noradrenergic, serotonergic and cholinergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Microdissection--elemental analysis of the mineralizing growth cartilage of the normal and rachitic chick.

The concentrations of elements in avian growth cartilage were studied by electron probe x-ray emission microanalysis (EDX). The cartilage was prepared for analysis by freezing, freeze-fracturing, freeze-drying, and carbon coating techniques. Cells and matrix fragments were removed from the tissue by microdissection with a tungsten needle in a scanning electron microscope (SEM) equipped with a real-time stereoscopic viewing facility. The samples were analyzed in the same SEM by EDX. Elemental analyses were performed on each fragment at a distance from the tissue sample, and hence background radiation due to the sample was eliminated. An important finding was that the intracellular potassium concentration of chondrocytes in calcified cartilage was similar to the levels in the premineralized zones. This observation supports the view that chondrocytes do not die in the process of, or as a consequence of, mineralization of the surrounding matrix. Calcium peaks were seen in the matrix at all levels and in chondrocytes immediately prior to mineralization. In contrast, phosphorus levels were always high in cells and low or absent from the premineralized matrix. At the mineralization front the appearance of a phosphorus peak in the matrix just preceded the deposition of mineral. We propose that the transfer of phosphorus from cell to matrix is a rate-limiting step in mineralization. Finally, when rachitic and normal cartilage were compared, little difference was seen in the profile of either intracellular or extracellular elements. However, in rickets the mineralized matrix remained soft in consistency. We suggest that this may reflect a phosphorus-related calcification defect that prevents growth and interlocking of the apatite crystallites.

Animals↗