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PubMed2001

Colonic crypt cell proliferation state assessed by whole crypt microdissection in sporadic neoplasia and familial adenomatous polyposis.

BACKGROUND: It has yet to be established whether proliferative activity in the macroscopically normal colonic mucosa is causally correlated with neoplastic risk. Measurement of proliferative activity in human subjects is of necessity usually undertaken using indirect methods with inherent limitations, and relatively little has been published on the effect of normal biological variables on such indices. AIMS: To establish the validity of mitosis counts following whole crypt microdissection as an index of the crypt cell proliferative state (CCPS) and to examine the effect of normal biological variables (age, sex, and colonic site) and colonic neoplasia on the mitotic index in macroscopically normal human colon. SUBJECTS: Mucosal samples were obtained at colectomy or colonoscopy from 107 individuals (24 controls, 23 sporadic adenoma patients, 31 sporadic carcinoma patients, and 29 patients with familial adenomatous polyposis (FAP)). METHODS: Mucosal specimens were hydrated, hydrolysed, and small groups of crypts separated from the main specimen under a dissecting microscope. The total number of mitoses/crypt were counted by one observer for each of 10 complete crypts. RESULTS: Validation work established that whole crypt mitoses counts were reliable and reproducible. There was no relation between age and mean mitoses/crypt (Pearson correlation coefficient -0.1). The CCPS count was higher for males than for females (difference in means 2.8 (95% confidence interval 0.80-4.66)) among controls but there was no gender difference in the three disease groups. For all disease groups and controls, the crypt mitotic count showed a significant linear increase (p=0.004) from the rectum to the caecum. Biopsies from within 5 cm of the macroscopic margin of a carcinoma (near) gave a mean mitosis count of 12.6 while those from more than 10 cm (far) were lower but not significantly so (p=0.12) with a count of 9.0. The mean mitoses/crypt were similar for the controls and adenomas (5.6 and 4.7, respectively) but greater for the cancers and especially for FAP (8.3 and 14.2, respectively). Statistical analysis confirmed that there were significant differences (p<0.05) between controls and all disease groups together, between sporadic disease and FAP, and between adenoma and carcinoma subjects at each of the four colonic sites. Post hoc comparison by t test showed significantly greater CCPS for FAP compared with controls (p<0.001) and for sporadic cancer versus controls (p=0.04). CONCLUSIONS: Whole crypt microdissection and mitosis counting is a reliable, reproducible, and robust technique for assessing CCPS in the human colon. CCPS is unaffected by age but increases from the distal to the proximal colon. CCPS is increased if a sporadic cancer is present and markedly increased in FAP. However, the precise relation of an increased CCPS to the neoplastic process remains uncertain.

Adenoma↗
PubMed1998

A simple and efficient method for microdissection and microFISH.

A simple and efficient method for the dissection of (marker) chromosomes, (micro)nuclei, and chromosome regions is presented. Before microdissection, metaphases are overlaid with milli-Q water to rehydrate the chromosomes, which makes them soft and sticky. The dissected chromosome fragments are dissolved without proteinase-K or topoisomerase treatment and directly amplified using a degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR). The advantages of this microFISH method over previously reported methods are: (1) microdissection in this way is very fast; (2) a chromosome, marker, (micro)nucleus, or chromosome region is collected as a whole using only one microneedle; (3) the dissected material sticks tightly to the needle without the risk of getting lost; (4) no Sequenase is used in the DOP-PCR reaction which reduces the risk of contamination.

Humans↗
PubMed2000

Detection of SYT-SSX fusion transcripts in both epithelial and spindle cell areas of biphasic synovial sarcoma using laser capture microdissection.

To investigate the distribution of tumour cells expressing the SYT-SSX fusion gene in biphasic synovial sarcoma, modified reverse transcription polymerase chain reaction (RT-PCR) analysis was performed using microdissected specimens from haematoxylin and eosin stained sections of archival paraffin wax embedded tissues. This modified RT-PCR included a stage with degenerate oligonucleotide primed (DOP) PCR, which randomly amplified cDNA after reverse transcription. SYT-SSX fusion transcripts were detected in both epithelial and spindle cell areas of all three biphasic synovial sarcomas examined. Subsequent sequence analysis confirmed that the detected messages were derived from the SYT-SSX1 fusion gene in two cases and from SYT-SSX2 in one. These results indicate that SYT-SSX fusion transcripts are found in both epithelial and spindle cell areas of biphasic synovial sarcoma, and RT-DOP-PCR-PCR analysis is a useful method for detection of extremely small amounts of mRNA in microdissected samples from archival formalin fixed, paraffin wax embedded tumour tissues.

Adenocarcinoma↗
PubMed2000

Detection of c-kit mutation Asp 816 to Val in microdissected bone marrow infiltrates in a case of systemic mastocytosis associated with chronic myelomonocytic leukaemia.

BACKGROUND/AIMS: The occurrence of myeloid leukaemia in patients with systemic mastocytosis is a well recognised phenomenon. However, the pathophysiological basis of such a coevolution has not been clarified. Recent data have shown that the c-kit mutation Asp 816 to Val is detectable in neoplastic mast cells in most patients with systemic mastocytosis, including those who have associated haematological disorders. The aim of this study was to study clonal disease evolution by analysing bone marrow cells from a patient with systemic mastocytosis and associated chronic myelomonocytic leukaemia (CMML) for the presence of this mutation. METHODS: The DNA of microdissected bone marrow cells from a patient with systemic mastocytosis and associated CMML was analysed for the presence of the c-kit mutation Asp 816 to Val by means of HinfI digestion and direct sequencing of semi-nested polymerase chain reaction (PCR) products. RESULTS: The two neoplasms could easily be identified and discriminated in paraffin wax embedded bone marrow sections by tryptase and chloroacetate esterase staining. A total number of 10 tryptase positive systemic mastocytosis infiltrates and 10 tryptase negative CMML infiltrates were removed by microdissection. As assessed by HinfI digestion and direct sequencing of semi-nested PCR products, the c-kit mutation Asp 816 to Val was detected in five of seven systemic mastocytosis infiltrates and four of six CMML infiltrates. By contrast, no c-kit mutation Asp 816 to Val was found in bone marrow infiltrates in patients with CMML without associated systemic mastocytosis (n = 20). CONCLUSION: These data support a monoclonal evolution of systemic mastocytosis and concurrent CMML in the patient studied.

Aged↗
PubMed1988

In situ phosphorylation of proteins in MCTs microdissected from rat kidney: effect of AVP.

Adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein phosphorylation is considered a key step in the cellular action of vasopressin (AVP) to regulate water permeability in collecting tubules. However, the proteins serving as a substrate(s) for phosphorylation in undisrupted cells have not yet been identified. In the present study, we developed a method for investigation of in situ phosphorylation of microdissected segments of medullary collecting tubules (MCT) from rat kidney. Incubation of microdissected MCT segments with low concentrations of saponin, "semipermeabilization," increased permeability of the membrane for ATP but did not allow leakage of macromolecules such as lactate dehydrogenase. This treatment also did not cause major disruption of cell structure, or impairment of AVP-sensitive adenylate cyclase. Incubation of semipermeabilized MCT with gamma-[32P]ATP resulted in incorporation of 32Pi into two major protein bands [band "A" of apparent molecular mass (Mr) approximately equal to 66 kDa, and band "B" of Mr approximately equal to 45 kDa] detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequent autoradiography. Similar incubation of tubules disrupted by hyposmotic solutions and a stronger detergent Triton X-100 resulted in 32Pi incorporation into multiple protein bands. Incubation of MCT with 1 microM AVP resulted in increased 32Pi radioactivity in band A and decreased 32Pi radioactivity in band B. These findings demonstrate a novel method for identification of endogenous protein substrate(s) for cAMP-dependent protein kinase and other protein kinases and phosphatases that are probably involved in post-cAMP steps in the cellular action of AVP in the intact cells of collecting tubules.

Adenosine Triphosphate↗
PubMed1999

Quantification of nitric oxide synthase activity in microdissected segments of the rat kidney.

This study was designed to quantify nitric oxide synthase (NOS) activity in microdissected glomeruli (Glm), pars convoluta, pars recta, cortical collecting duct, cortical thick ascending limb, outer medullary collecting duct, medullary thick ascending limb and thin limb, inner medullary collecting duct (IMCD) and thin limb, and vasa recta (VR). Total protein from microdissected segments was incubated with L-[3H]arginine and appropriate cofactors, and the L-arginine and converted L-citrulline were separated by reverse-phase HPLC and radiochemically quantitated. NOS activity was found to be greatest in IMCD (11.5 +/- 1.0 fmol citrulline. mm-1. h-1) and moderate in Glm (1.9 +/- 0.3 fmol. glomerulus-1. h-1) and VR (3.2 +/- 0.8 fmol. mm-1. h-1). All other renal structures studied exhibited significantly less NOS activity. The mRNA for NOS isoforms in the NOS activity-positive segments was then identified by RT-PCR. The IMCD contained mRNA for neuronal (nNOS), endothelial (eNOS), and inducible NOS (iNOS), but Glm and VR only expressed the mRNA for nNOS and eNOS. These experiments demonstrate that the greatest enzymatic activity for NO production in the kidney is in the IMCD, three- to sixfold less activity is present in the Glm and VR, and minimal NOS activity is found in other segments studied.

Animals↗
PubMed1999

Micro- and macrochromosome paints generated by flow cytometry and microdissection: tools for mapping the chicken genome.

Despite the chicken being one of the most genetically mapped of all animals, its karyotype remains poorly defined. This is primarily due to microchromosomes that belie assignment by conventional methods. To address this problem, we have developed chromosome-specific paints using flow cytometry and microdissection. For the microchromosomes it was necessary to amplify and label DNA from single microdissected chromosomes.

Animals↗
PubMed2000

FISH-microdissection (FISH-MD) analysis of complex chromosome rearrangements.

We combined the techniques of fluorescence in situ hybridization (FISH) and chromosomal microdissection in one experiment (FISH-MD). This novel method permits rapid identification of the composition, origin, and breakpoints of rearranged chromosomes. Rearranged chromosomes are first identified by multicolor-FISH, then the fluorophore-labeled derivative chromosomes are directly isolated by microdissection and reverse painted to identify the breakpoints.

Chromosome Aberrations↗
PubMedDate not supplied

A comparative kinetic RT/-PCR strategy for the quantitation of mRNAs in microdissected human renal biopsy specimens.

Molecular biology techniques, to be applicable to a diagnostic renal biopsy specimen, should (1) be highly sensitive to be performed on a very small quantity of tissue; (2) be quantitative because they have to analyze genes normally expressed in the tissue and (3) allow the analysis of as large a number of genes as possible. Among different methods, only the reverse-transcriptase polymerase chain reaction (RT/-PCR) might comply with previous requisites, but the few RT/-PCR examples on renal biopsies in the literature do not allow starting RNA quantification and quality control; furthermore they have the drawback of analyzing only few genes. In an ongoing study to assess the expression of a number of genes in glomeruli and in tubulointerstitium of patients with different nephropathies, we developed a comparative RT/-PCR kinetic strategy based on the purification and quantification of total glomerular and tubulointerstitial RNA and on the use of an internal standard, the housekeeping gene G3PDH. We demonstrate that in microdissected diagnostic renal biopsies (1) glomerular and interstitial starting RNA can be quantified; (2) the G3PDH gene may be used both as an internal standard and as an indirect marker of RNA integrity; (3) as low as 28 ng of total RNA is sufficient to obtain PCR products of eight genes, and (4) it is worth to operate on microdissected biopsy specimens because of the different expression of genes in the two renal compartments.

Actins↗
PubMed1991

Painting of defined chromosomal regions by in situ suppression hybridization of libraries from laser-microdissected chromosomes.

"Painting" of defined chromosomal regions provides a powerful tool for cytogenetic analyses. Here, we demonstrate that chromosomal in situ suppression (CISS)-hybridization of DNA libraries derived by microcloning laser-microdissected chromosomal regions can be applied to achieve this goal. As an example, we used unbanded metaphase spreads from a female patient carrying a balanced translocation. t(1;7)(1qter----1p36::7q11----7qter). Fragments from the long arms of 130 translocation chromosomes were microdissected. After microcloning, human inserts with an average size of about 3 kb were pooled from 400 recombinant bacteriophage DNA clones and used as a complex probe set in CISS-hybridization experiments. This resulted in painting of the translocation chromosome along the region 7q35 to 1p31. Painted chromosomal subregions in normal chromosomes 1 and 7 were consistent with this finding. This approach may be used to perform painting of any chromosome regions for which microlibraries can be established. Possible applications include the definition of marker chromosomes in clinical and tumor cytogenetics and studies of chromosomal evolution, as well as studies of nuclear chromosome topography in animal and plant species.

Cells, Cultured↗
PubMed1993

Detailed characterization of a human 8q24.1 microdissection library and generation of "sequence-tagged sites".

A total of 533 clones from a human 8q24.1 microdissection library was analyzed by automatic DNA sequencing. Three hundred and thirty-seven different insert sequences were found. The insert size ranged from 45 to 376 bp (mean, 170 bp). Eighty-six percent (291/337) of these sequences were free of repetitive DNA. Each of 19 clones tested was successfully translated into a sequence-tagged site. We conclude that the microdissection library is a rich source of DNA markers for 8q24.1.

Base Sequence↗
PubMed1995

Direct microdissection and microcloning of a translocation breakpoint region, t(1;11)(q42.2;q21), associated with schizophrenia.

We describe the generation of large-fragment microclone libraries from the chromosomal breakpoint of a reciprocal balanced translocation linked to schizophrenia. The abnormality was visible under the phase-contrast microscope, allowing direct dissection from unstained, unbanded metaphases. Two separate microdissection experiments yielded 443 and 672 recombinants, respectively. Following complete EcoRI digestion, inserts with an average size of 0.3 kb (range, 0.2-3 kb) were obtained in the first experiment and 1.5 kb (range, 0.15-6.5 kb) in the second. FISH analysis of pooled clones "painted" back onto the derivative chromosome and assignment of microclones to somatic cell hybrids confirmed the fidelity of the method. Microdissection of chromosome regions identified by karyotype rearrangements in unstained, unbanded metaphases is a potentially powerful tool for positional cloning.

Base Sequence↗
PubMed1996

Generation and mapping of human chromosome 2 microdissection clone-derived STSs.

Twenty-nine new sequence-tagged sites (STSs) were derived from DNA sequences of clones from two human chromosome 2 microdissection libraries. The specificity of the STSs for human chromosome 2 was first demonstrated by PCR amplification of DNA from genomic human and hamster cells and a human chromosome 2-containing human x hamster hybrid cell line. The STSs were then mapped to chromosome 2 by two different approaches. In the first attempt, 12 of the STSs were shown to PCR amplify YAC clones associated with genetic markers on the chromosome. In the second approach, 27 of the STSs were localized to chromosome bands by FISH using cosmid or PAC clones encoding the STSs. The specific STSs mapped to chromosome 2 by these two approaches tie together the genetic and cytogenetic maps of the chromosome at the two termini. The distribution of these STSs further defines the region of the chromosome present in the two microdissection libraries.

Chromosome Banding↗
PubMed1997

Comparative analysis of Y chromosome structure in Bos taurus and B. indicus by FISH using region-specific, microdissected, and locus-specific DNA probes.

Results of fluorescence in situ hybridization (FISH) of Bos taurus and B. indicus Y chromosomes using the bovine locus-specific Y probes BC1.2 and lambda ES6.0 and region-specific probes of B. indicus and B. taurus Y chromosomes, which were generated by microdissection and DOP-PCR, indicate that the Y chromosomes of B. indicus (BIN Y) and B. taurus (BTA Y) differ by a pericentric inversion. Parts of the short and long arms of the Y chromosome in B. taurus and the distal half of the Y chromosome in B. indicus were microdissected, amplified by DOP-PCR, biotinylated, and rehybridized in situ to the corresponding metaphase chromosomes to test the chromosome fragment specificity of the DNA probes. The region-specific painting probes were used for hybridization to metaphase chromosomes of the other species. The DNA painting probes BTA Yp12 and BTA Yq12.1-ter derived from BTA Y hybridized to the distal and proximal halves of BIN Y, respectively. Complex hybridization signals on BTA Yq12.1-->qter were generated with the DNA probe BIN Yqcen-centr (centromere-central) after FISH. The results demonstrate that BTA Yp is homologous to the distal half of BIN Y and that BTA Yq corresponds to the proximal part of BIN Yq. Hybridization of the Y chromosome-specific DNA probes lambda ES6.0 to BTA Yp12-->p11 and near to the telomere of BIN Y and BC1.2 to BTA Yq12-->q13 and to the telomere of BIN Y indicate an opposite orientation of the homologous chromosome fragments BTA Yp and of the distal half of BIN Yq.

Animals↗
PubMedDate not supplied

Detection of heterozygosity loss in microdissected fine needle aspiration specimens of breast carcinoma.

OBJECTIVE: To use the polymerase chain reaction (PCR) to detect loss of heterozygosity (LOH) in microdissected cells form cytologic smears obtained by fine needle aspiration (FNA) from 20 cases of invasive breast carcinoma. STUDY DESIGN: In each case, histologic sections of the primary tumor were also available. Tumor and nontumor cells were dissected from both the cytologic smear and tissue section in all cases except in three smears that showed only tumor cells. RESULTS: LOH was identified in 10 of 19 informative cases using two polymorphic DNA markers at chromosome 11q13 (INT-2, PYGM). The same results were obtained in both the cytologic and histologic specimens, including three cases that had hypocellular cytologic smears. CONCLUSION: FNA of breast lesions provides adequate samples for direct microdissection of the cytologic smear to detect LOH using PCR amplification.

Biopsy, Needle↗
PubMed1996

Repair of naphthalene-injured microdissected airways in vitro.

Nonciliated bronchiolar epithelial (Clara) cells, as both the primary target for metabolically activated pulmonary toxicants and the progenitor cell for repair after bronchiolar injury, are critical for distal airway epithelial function and regeneration. Previously, we described a model system whereby differentiated Clara cells can be maintained in culture using explants of microdissected distal airways. The purpose of this study is to establish whether distal airway explants can be used to study bronchiolar epithelial repair in vitro. Lungs from adult mice treated with naphthalene, a metabolically activated Clara cell cytotoxicant, or vehicle were inflated with agarose and distal airways were microdissected. Distal airway explants were cultured for up to 7 days in serum-free medium. Clara cells in explants from naphthalene-treated mice exhibited the characteristic cytotoxic responses previously reported in vivo when maintained in vitro: cell swelling, formation of cytoplasmic vacuoles, and exfoliation of injured cells into the airway lumen 1 to 2 days after injury (DAI). Epithelial cells squamated to cover the injured area 2 to 4 DAI. At 7 DAI, the epithelium generally consisted of cuboidal cells. Proliferating cells and marker proteins for differentiated Clara cells (Clara cell 10 kD secretory protein, or CC10, and cytochrome P450 monooxygenase isozyme 2B, or CYP2B) were detected immunochemically and their pattern of distribution during the injury and repair response in vitro paralleled the pattern of cell regeneration seen previously in vivo. We conclude that Clara cells in explants from defined regions of murine tracheobronchial airways can be used to study the early phases of the repair response to naphthalene injury, including differentiation and proliferation, in vitro.

Animals↗
PubMed1984

Estradiol-induced daily luteinizing hormone and prolactin surges in young and middle-aged rats: correlations with age-related changes in pituitary responsiveness and catecholamine turnover rates in microdissected brain areas.

These studies assessed the effects of age on the ability of estradiol-17 beta (E2) to induce LH and PRL surges in ovariectomized young and middle-aged rats that previously had normal estrous cycles. We determined whether any changes in the timing or amplitude of these surges could be correlated with changes in pituitary responsiveness to GnRH or with changes in norepinephrine (NE) and dopamine (DA) turnover rates in microdissected brain regions involved in cyclic gonadotropin release. Young (3-4 months old) and middle-aged (9-12 months old) rats were ovariectomized. One week later (day 0), they received Silastic capsules containing E2 which produced physiological serum concentrations of E2. Groups of rats were bled sequentially via indwelling right atrial cannulae 1-4 days after capsule implantation (days 1-4). All young rats displayed maximal LH surges by day 2 and exhibited equivalent surges on days 3 and 4. Middle-aged rats required the presence of E2 for at least 3 days before a maximal positive feedback response was achieved. Even at these times the timing of the LH rise was delayed by 1 h and peak concentrations were lower in middle-aged rats. E2-induced PRL surges did not exhibit any age-related differences. Pituitary responsiveness to GnRH was tested by administering two injections of GnRH to pentobarbital-blocked young and middle-aged rats on days 2 and 4. Pituitary responsiveness to the first injection on day 2 was blunted in middle-aged rats; however, the LH response at all other times was normal. Catecholamine turnover rates were examined on days 2 and 4 by giving alpha-methyl-para-tyrosine at 1000 or 1500 h and killing rats 45 or 90 min later. Resting initial catecholamine concentrations were assessed in untreated rats killed at 1000 h or 1500 h. The medial preoptic nucleus, suprachiasmatic nucleus, and median eminence were microdissected and assayed for NE and DA by radioenzymatic assay. In young rats, NE turnover rates increased during the afternoon in all brain areas on both days. In contrast, in middle-aged rats, no increase in NE turnover rates was observed during the afternoon of day 2. By day 4, the delayed and attenuated LH surge was accompanied by increased turnover rates in the median eminence only; no change occurred in the suprachiasmatic nucleus or medial preoptic nucleus. No age-related differences were observed in DA turnover rates.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗
PubMed1990

Neuropeptide-Y concentration in microdissected hypothalamic regions and in vitro release from the medial basal hypothalamus-preoptic area of streptozotocin-diabetic rats with and without insulin substitution therapy.

Experimental diabetes adversely affects hypothalamic control of gonadotropin secretion and sex behavior and induces hyperphagia accompanied by severe body weight loss. Neuropeptide-Y (NPY) stimulates pituitary gonadotropin release, inhibits sexual behavior, and stimulates robust feeding in rats by acting at different sites in the hypothalamus. Therefore, we tested the hypothesis that altered hypothalamic NPY neurosecretion may mediate the constellation of effects observed in streptozotocin-induced diabetic (STZ-D) rats. Adult male rats were made diabetic by a single injection of STZ (50 mg/kg). Five months later, in vitro NPY release from the hypothalamic fragment encompassing the medial basal hypothalamus and preoptic area and NPY concentrations in seven hypothalamic sites were assessed. Basal NPY release was not significantly changed after STZ treatment. However, in response to a 30-min pulse of KCl (45 mM), NPY release from the medial basal hypothalamus-preoptic area of STZ-D rats was significantly increased compared to that in age-matched controls. In the STZ-D rats, NPY concentrations in six of the seven microdissected nuclei, including those mediating control of pituitary gonadotropin, sexual, and feeding behaviors, were increased compared to control values. In an additional study similar increments in NPY concentrations in the hypothalamic sites were observed 6 months after STZ treatment. The effects of insulin on NPY levels in microdissected hypothalamic sites in STZ-treated and BB diabetic rats was next assessed. One group of rats was treated with STZ, and the other group of rats was additionally treated with insulin (6 U/kg.day) for 3 months after development of diabetes with STZ. Again, STZ treatment alone, even for 3 months, increased NPY levels in all seven nuclei, including the suprachiasmatic nuclei. Insulin therapy completely prevented the STZ-induced increments in NPY levels in all hypothalamic sites, and the blood glucose level was 233 +/- 22 mg/dl in insulin-treated STZ-D rats and 496 +/- 6 mg/dl in untreated STZ-D rats. Similarly, NPY concentrations in five of the seven nuclei were unchanged in spontaneously diabetic BB rats (blood glucose, 435 +/- 67 mg/dl) maintained on insulin (5-8 U/kg.day). These results demonstrate that STZ-D rats have a widespread increase in NPY levels in hypothalamic sites, and there is an increase in the evoked release of NPY from the hypothalamus.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗