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Results for “Microdissection”

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PubMed1986

Microdissection studies on the polarity of unequal division in grasshopper neuroblasts. I. Subsequent divisions in neuroblast-type cells produced against the polarity by micromanipulation.

Equal or unequal division against the polarity of normal division was induced in grasshopper neuroblasts by means of a microdissection technique. The subsequent cell divisions were traced in order to analyse the factors that determine the polarity. Daughter cells of two types (neuroblast-type and ganglion cell type) were produced by operations in which the mitotic apparatus was rotated or shifted. Cell types were classified by such characteristics as nuclear shape, mitotic activity, and inequality or equality of the subsequent cytokinesis. It became evident that the fate of daughter cells was determined simply by the cytoplasmic volume. In 27 cases out of 40 microdissecting operations, both sister cells were recognized as of neuroblast type. Mitosis of these neuroblast-type sister cells proceeded asynchronously. The time required for neuroblast-type cells to reach metaphase of the second division depended on their volume. It is considered that the polarity of unequal division in grasshopper neuroblasts may be maintained by a joint action of the cap cells attaching to one of the polar regions of the cell and the cortex differentiated in the previous cell division.

Animals↗

PubMed1976

Microdissection of peripheral nerve: collagen and lipid distribution with morphological correlation.

Peripheral nerve is a complex tissue composed of endoneurial fascicles surrounded by perineurium and epineurium. We separated endoneurium from peri- and epineurium in human sural nerves by "endoneurial plucking", a method of microdissection. Endoneurial contents (axons, myelin sheaths, Schwann cells, vessels, and interstitial collagen) were cleanly separated in high yield from enveloping connective tissue, by both microscopic and biochemical criteria. Most of the nerve sulfatide and unesterified sterol was found in the endoneurial fraction while most of the collagen was in the peri-epineurial fraction. This microdissection method should prove useful in biochemical investigations of peripheral nerve.

Adult↗

PubMed1995

Gene amplification elucidated by combined chromosomal microdissection and comparative genomic hybridization.

Gene amplification is an important manifestation of genetic instability in cancer. Recently, the study of gene amplification has been greatly facilitated by the development of the molecular cytogenetic techniques of comparative genomic hybridization and chromosome microdissection. We describe in this brief overview a combined approach using both techniques, which allows the identification of chromosomal regions of gene amplification and provides entry point clones for target gene identification. This molecular/cytogenetic approach consisting of chromosome microdissection and comparative genomic hybridization should be valuable in identifying novel amplified genes important in neoplastic development and progression.

Chromosomes↗

PubMed1991

Laser microdissection of the fragile X region: identification of cosmid clones and of conserved sequences in this region.

Laser microdissection has been used to dissect material from the X-chromosome region involved in fragile-X-linked mental retardation. After dissection, single chromosome slices corresponding to this fragile site were subjected to DNA amplification using either a vector ligation method (to provide known anchor sequences) or primer oligonucleotides corresponding to the ubiquitous Alu sequences. Amplified material was then cloned or, alternately, used to screen a gridded cosmid library. Eight cosmid clones identified in this way were regionally mapped using a panel of hybrid cell lines and shown to originate from a narrow interval centered on the fragile X site. Two clones are included in the approximately 6-cM interval defined by probes RNI (DXS369, 5 cM proximal) and VK21 (DXS 296, 1-2 cM distal) and which includes the fragile site, and at least one clone contains sequences conserved across species suggestive of a gene. This method combines the focused approach of microdissection and the convenience of obtaining cosmid (rather than small-insert) clones; it may be useful for studies of other defined chromosomal regions.

Animals↗

PubMed1991

New markers for the neurofibromatosis-2 region generated by microdissection of chromosome 22.

To identify new DNA markers around the neurofibromatosis-2 gene on human chromosome 22, the critical region (22q12-q13.1) was microdissected and microcloned from GTG-banded metaphase chromosomes. Eighteen thousand recombinant clones were obtained. Twenty-seven of 55 clones tested (50%) detected single-copy DNA sequences. Nine of nine clones analyzed in detail were found to map to chromosome 22. Interestingly one clone (EAN04) is part of the leukemia inhibitory factor gene which has previously been mapped to 22q11.2-q13.1. Four clones (EAN01, EAN47, EAN57, and EAN68) detect DNA polymorphisms. These probes were used to compare constitutional and tumor genotypes of 41 patients with acoustic neurinoma. Loss of constitutional heterozygosity was identified in 17 of 31 informative cases (55%). From our data we conclude that the microdissection library is a valuable resource for physical and genetic mapping studies in neurofibromatosis-2.

Chromosome Mapping↗

PubMed1992

Microdissection of human chromosomal regions 8q23.3-q24.11 and 2q33-qter: construction of DNA libraries and isolation of their clones.

Human chromosomal regions 8q23.3-q24.11 and 2q33-qter were microdissected, DNAs from the regions were amplified with the primer-linker method of polymerase chain reaction (PCR), and their DNA libraries were constructed by cloning into pUC19. The primer-linker PCR involved Sau3AI digestion of microdissected chromosomal DNAs, ligation of the digests to a 10mer DNA linker and 24mer primer, filling the recessed 3' ends, and PCR amplification using the 24mer DNA as a primer. A total of 3.5 x 10(4) pUC19 recombinants (8q library) from the 8q region and 5.0 x 10(4) pUC clones (2q library) from the 2q region were obtained. From the 8q library, 60 pUC clones were selected, while 88 pUC-clones were selected from the 2q library. These clones were Southern blot analyzed on hybrid cell panels with or without human chromosome 8 or 2. Twelve (20%) of the 60 8q-derived clones were unique DNA sequences, and 9 were subjected to deletion analysis in the genomic DNA of two patients, one with trichorhino-phalangeal syndrome (TRPS) type I and the other with TRPS type II, both with del(8) (q23.3q24.13). Five of the 9 pUC clones tested showed a one-copy density in both patients, an indication that the clones map to the region deleted in both patients. Screening a genomic DNA library constructed in the phage revealed a clone with a 9.4-kb insert and a one-copy density in both patients. From the 2q library, 15 (17%) of the 88 pUC clones obtained were unique sequences. When a phage library was screened, 8 clones were obtained: 4 were identical and 2 were overlapping sequences.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormalities, Multiple↗

PubMed1995

Region-specific cosmids and STRPs identified by chromosome microdissection and FISH.

A strategy for identifying short tandem repeat (STR)-containing cosmid clones from a specific chromosomal region is described. The approach is based on the use of uncloned, PCR-amplified DNA derived from chromosome microdissection and pooled groups of STR sequences as hybridization probes to screen a cosmid library. Cosmid clones that display a positive signal common to both hybridizations are then characterized for repeat length polymorphisms. This method has been applied to chromosome bands 17q12-q21, a region that includes a gene (BRCA1) involved in early onset familial breast and ovarian cancer. Of 1536 chromosome 17-specific cosmid clones tested, 38 were identified by the dual screening procedure. Fluorescence in situ hybridization revealed that 19 cosmids originated from the microdissected target region. Thirteen of the 19 cosmids were mapped between markers flanking the BRCA1 region and selected for further characterization. Tetranucleotide repeats were identified in 10 of these 13 cosmids. Primers designed for each marker were tested on a panel of 80 CEPH parents for allele sizes, frequencies, and observed heterozygosities. From these studies six polymorphic and one nonpolymorphic STRs were identified. A similar approach should be applicable for screening whole genomic or chromosome-specific cosmid libraries in efforts to isolate new polymorphic markers from any chromosomal region of interest.

Base Sequence↗

PubMed2005

Genetic tumor archeology: microdissection and genetic heterogeneity in squamous and basal cell carcinoma.

Carcinogenesis is a multi-step series of somatic genetic events. The complexity of this multi-hit process makes it difficult to determine each single event and the definitive outcome of such events. To investigate the genetic alterations in cancer-related genes, sensitive and reliable detection methods are of major importance for generating relevant results. Another critical issue is the quality of starting material which largely affects the outcome of the analysis. Microdissection of cells defined under the microscope ensures a selection of representative material for subsequent genetic analysis. Skin cancer provides an advantageous model for studying the development of cancer. Detectable lesions occur early during tumor progression, facilitating molecular analysis of the cell populations from both preneoplastic and neoplastic lesions. Alterations of the p53 tumor suppressor gene are very common in non-melanoma skin cancer, and dysregulation of p53 pathways appear to be an early event in the tumor development. A high frequency of epidermal p53 clones has been detected in chronically sun-exposed skin. The abundance of clones containing p53 mutated keratinocytes adjacent to basal cell (BCC) and squamous cell carcinoma (SCC) suggests a role in human skin carcinogenesis. Studies using p53 mutations as a clonality marker have suggested a direct link between actinic keratosis, SCC in situ and invasive SCC. Microdissection-based studies have also shown that different parts of individual BCC tumors can share a common p53 mutation yet differ with respect to additional alterations within the p53 gene, consistent with subclonal development within tumors. Here, we present examples of using well-defined cell populations, including single cells, from complex tissue in combination with molecular tools to reveal features involved in skin carcinogenesis.

Basal Cell Carcinoma↗

PubMed2004

Laser microdissection microscopy in parasitology: microscopes meet thermocyclers.

The new methods of laser microdissection microscopy have received wide acceptance in biology and have been applied in a small number of parasitology investigations. Here, the techniques and applications of laser microdissection microscopy are reviewed with suggestions of how the systems might be used to explore applied questions in parasite molecular biology and host-parasite interactions.

Animals↗

PubMed2005

Targeting pancreatic islets with phage display assisted by laser pressure catapult microdissection.

Heterogeneity of the microvasculature in different organs has been well documented by multiple methods including in vivo phage display. However, less is known about the diversity of blood vessels within functionally distinct regions of organs. Here, we combined in vivo phage display with laser pressure catapult microdissection to identify peptide ligands for vascular receptors in the islets of Langerhans in the murine pancreas. Protein database analyses of the peptides, CVSNPRWKC and CHVLWSTRC, showed sequence identity to two ephrin A-type ligand homologues, A2 and A4. Confocal microscopy confirmed that most immunoreactivity of CVSNPRWKC and CHVLWSTRC phage was associated with blood vessels in pancreatic islets. Antibodies recognizing EphA4, a receptor for ephrin-A ligands, were similarly associated with islet blood vessels. Importantly, binding of both islet-homing phage and anti-EphA4 antibody was strikingly increased in blood vessels of pancreatic islet tumors in RIP-Tag2 transgenic mice. These results indicate that endothelial cells of blood vessels in pancreatic islets preferentially express EphA4 receptors, and this expression is increased in tumors. Our findings show in vivo phage display and laser pressure catapult microdissection can be combined to reveal endothelial cell specialization within focal regions of the microvasculature.

Amino Acid Sequence↗

PubMed2003

Hypermethylation of the human glutathione S-transferase-pi gene (GSTP1) CpG island is present in a subset of proliferative inflammatory atrophy lesions but not in normal or hyperplastic epithelium of the prostate: a detailed study using laser-capture microdissection.

Somatic inactivation of the glutathione S-transferase-pi gene (GSTP1) via CpG island hypermethylation occurs early during prostate carcinogenesis, present in approximately 70% of high-grade prostatic intraepithelial neoplasia (high-grade PIN) lesions and more than 90% of adenocarcinomas. Recently, there has been a resurgence of the concept that foci of prostatic atrophy (referred to as proliferative inflammatory atrophy or PIA) may be precursor lesions for the development of prostate cancer and/or high-grade PIN. Many of the cells within PIA lesions contain elevated levels of GSTP1, glutathione S-transferase-alpha (GSTA1), and cyclooxygenase-II proteins, suggesting a stress response. Because not all PIA cells are positive for GSTP1 protein, we hypothesized that some of the cells within these regions acquire GSTP1 CpG island hypermethylation, increasing the chance of progression to high-grade PIN and/or adenocarcinoma. Separate regions (n =199) from 27 formalin-fixed paraffin-embedded prostates were microdissected by laser-capture microdissection (Arcturus PixCell II). These regions included normal epithelium (n = 48), hyperplasticepithelium from benign prostatic hyperplasia nodules (n = 22), PIA (n = 64), high-grade PIN (n = 32), and adenocarcinoma (n = 33). Genomic DNA was isolated and assessed for GSTP1 CpG island hypermethylation by methylation-specific polymerase chain reaction. GSTP1 CpG island hypermethylation was not detected in normal epithelium (0 of 48) or in hyperplastic epithelium (0 of 22), but was found in 4 of 64 (6.3%) PIA lesions. The difference in the frequency of GSTP1 CpG island hypermethylation between normal or hyperplastic epithelium and PIA was statistically significant (P = 0.049). Similar to studies using nonmicrodissected cases, hypermethylation was found in 22 of 32 (68.8%) high-grade PIN lesions and in 30 of 33 (90.9%) adenocarcinoma lesions. Unlike normal or hyperplastic epithelium, GSTP1 CpG island hypermethylation can be detected in some PIA lesions. These data support the hypothesis that atrophic epithelium in a subset of PIA lesions may lead to high-grade PIN and/or adenocarcinoma. Because these atrophic lesions are so prevalent and extensive, even though only a small subset contains this somatic DNA alteration, the clinical impact may be substantial.

Atrophy↗

PubMed2003

Expression profiling of mouse endometrial cancers microdissected from ethanol-fixed, paraffin-embedded tissues.

Expression-profiling studies have helped define genetic changes associated with carcinogenesis. Determining which alterations in gene expression are causally associated with cancer and which result from the general dysregulation in gene expression that is characteristic of malignancies remains a problem. Transcriptional profiling of early lesions (small cancers or precancers) holds promise for identifying biologically important changes in gene expression. There are, however, technical barriers to the study of small tumors. The total number of cells available for analysis is limiting. It is also often difficult to distinguish cancer cells from normal proliferating cells in frozen sections that are typically used as a source of RNA. Here we describe an ethanol fixation and paraffin-embedding protocol that preserves tissue architecture and cellular morphology of the mouse endometrium, and allows for the recovery of high-quality RNA from microdissected cells. We performed GeneChip expression profiling using RNA from 800 to 4400 cells microdissected from ethanol-fixed, paraffin-embedded uteri. Endometrial adenocarcinomas exhibited changes in the levels of a number of messages known to be abnormally expressed in cancer, and differential expression of additional transcripts not previously implicated in carcinogenesis. We confirmed increased Amd1 expression in RNAs from mouse endometrial carcinomas that were hybridized to GeneChips and validated overexpression of this transcript in additional tumors.

Animals↗

PubMed2002

mRNA expression profiling of laser microbeam microdissected cells from slender embryonic structures.

Microarray hybridization has rapidly evolved as an important tool for genomic studies and studies of gene regulation at the transcriptome level. Expression profiles from homogenous samples such as yeast and mammalian cell cultures are currently extending our understanding of biology, whereas analyses of multicellular organisms are more difficult because of tissue complexity. The combination of laser microdissection, RNA amplification, and microarray hybridization has the potential to provide expression profiles from selected populations of cells in vivo. In this article, we present and evaluate an experimental procedure for global gene expression analysis of slender embryonic structures using laser microbeam microdissection and laser pressure catapulting. As a proof of principle, expression profiles from 1000 cells in the mouse embryonic (E9.5) dorsal aorta were generated and compared with profiles for captured mesenchymal cells located one cell diameter further away from the aortic lumen. A number of genes were overexpressed in the aorta, including 11 previously known markers for blood vessels. Among the blood vessel markers were endoglin, tie-2, PDGFB, and integrin-beta1, that are important regulators of blood vessel formation. This demonstrates that microarray analysis of laser microbeam micro-dissected cells is sufficiently sensitive for identifying genes with regulative functions.

Animals↗

PubMed1999

11q13 allelic imbalance discriminates pulmonary carcinoids from tumorlets. A microdissection-based genotyping approach useful in clinical practice.

Pulmonary tumorlets are minute neuroendocrine cell proliferations believed to be precursor lesions to pulmonary carcinoids. Little is known of their molecular pathogenesis because of their small size. Using tissue microdissection, we evaluated 11q13 region allelic imbalance in the pathogenesis of pulmonary tumorlet/carcinoid lesions. The int-2 gene was selected because of its chromosomal location at 11q13 in close proximity to MEN1, a tumor suppressor gene frequently mutated in familial forms of neuroendocrine cancer. Three cohorts of patients were studied: subjects with typical carcinoid tumors and coexisting tumorlets (n = 5), typical carcinoids without tumorlets (n = 6), and tumorlets alone without carcinoid lesions (n = 5). A total of 11 carcinoids and 11 tumorlets were microdissected from 4-micrometer-thick histological sections. Genotyping was designed to detect allelic imbalance of the int-2 gene and involved DNA sequencing of two closely spaced deoxynucleotide polymorphisms. Subjects shown to be informative were evaluated for allelic imbalance in tumorlet/carcinoid tissue. Eight of 11 (73%) carcinoids manifested allelic, in contrast to only one of 11 (9%) of tumorlets. Int-2 allelic imbalance was significantly associated with carcinoid tumor formation (P < 0.01). In patients having both carcinoid tumors and tumorlets, the latter showed allelic balance and were thus discordant in genotype with coexisting carcinoid excluding pathogenesis of tumorlets from intramucosal spread from carcinoid tumors. Int-2 allelic imbalance was shown to be an early event in carcinoid tumor formation by virtue of the absence of allelic imbalance for other common cancer-related gene disturbances involving 11p13 (Wilms' tumor), 3p25 (von-Hippel-Lindau), and 17p13 (p53). Demonstration of 11q13 allelic imbalance by microdissection/genotyping may be a useful discriminatory marker for pulmonary neuroendocrine neoplasia.

Alleles↗

PubMed1999

Microdissection-based analysis of mature ovarian teratoma.

The genotypic features of mature ovarian teratomas (MOTs) are controversial. Early studies detected a homozygous genotype in MOTs suggesting that these tumors are composed of germ cells that have undergone meiosis I. Other studies, however, revealed a heterozygous genotype in a substantial proportion of MOTs suggesting an origin either from premeiotic germ cells or from a somatic cell line. In view of the complex morphology of MOTs and to increase the sensitivity of teratoma genotyping, we applied tissue microdissection before genetic analysis of teratomatous tissue. This approach allowed selective analysis of different heterotopic tissue elements as well as the lymphoid tissues within MOTs the origin of which is unknown. After DNA extraction, the tissue samples were polymerase chain reaction amplified using a random panel of highly informative genetic markers for different chromosomes to evaluate heterozygosity versus homozygosity. In all seven cases that were analyzed, heterotopic tissues consistently revealed a homozygous genotype with several markers; in two cases, heterozygosity was detected with a single marker, indicating a meiotic recombination event. Lymphoid aggregates within MOTs were heterozygous and derived from host tissue rather than from teratomatous growth. However, well differentiated thymic tissue was consistently homozygous, suggesting lymphoid differentiation capability of MOTs. We conclude that potential pitfalls in genotyping of teratomas including meiotic recombination and host cell participation can be avoided by a microdissection-based approach in combination with a panel of genetic markers.

Adolescent↗

PubMed1999

Composite low grade B-cell lymphomas with two immunophenotypically distinct cell populations are true biclonal lymphomas. A molecular analysis using laser capture microdissection.

Low grade B-cell lymphomas comprise several well defined, clinically and immunophenotypically distinct disease entities. Composite lymphomas showing phenotypic characteristics of more than one of these tumor subtypes in the same site are rare, and both common and separate clonal origins of the two tumor parts have been reported for cases studied by molecular methods. We describe the detailed immunohistochemical and molecular findings in three cases with features of composite low grade B-cell non-Hodgkin's lymphoma (B-NHL). All three neoplasms contained morphologically distinct but interwoven compartments of different cell types, which exhibited discordant expression of several markers, including CD5, CD10, CD43, and cyclin D1. According to their morphology and phenotypes, they were classified as mantle cell lymphoma and follicular lymphoma (Case 1), follicular lymphoma and small lymphocytic lymphoma (Case 2), and mantle cell lymphoma and chronic lymphocytic leukemia/small lymphocytic lymphoma (Case 3). PCR analysis of DNA obtained from whole tissue sections failed to reveal evidence for biclonality in any of the cases. We therefore isolated cell populations with different antigen expression patterns by laser capture microdissection and analyzed them by polymerase chain reaction amplification and sequencing of clonal immunoglobulin heavy chain gene rearrangements and oncogene rearrangements. Sequence analysis revealed unrelated clonal rearrangements in each of the two tumor parts in all three cases, suggesting distinct clonal origins. In addition, Case 1 showed a bcl-2 rearrangement present only in the follicular lymphoma part. Our findings suggest that low grade B-NHL with two distinct morphological and immunophenotypic patterns in the same anatomical site are frequently biclonal. This is in keeping with current classification schemes, which recognize subtypes of low grade B-NHL as separate disease entities. Furthermore, our analysis demonstrates the power of laser capture microdissection in revealing molecular microheterogeneity in complex neoplasms.

Aged↗

PubMed2004

Laser-assisted microdissection: methods for the molecular analysis of psychiatric disorders at a cellular resolution.

Gene expression arrays and proteomics together provide a great opportunity to reveal the molecular pathophysiology of psychiatric disorders; however, their potential will not be realized unless due attention is paid to the cellular heterogeneity of the brain and the likely differential neuropathological involvement of specific neuronal and glial cell types. Hence, methods are needed which can procure homogeneous populations of cells as a source of messenger RNA, protein, or DNA. Laser-assisted microdissection techniques provide such a tool. Here we briefly discuss the principles, applications, and limitations of laser-assisted microdissection in psychiatric research.

Animals↗

PubMed1995

Microdissection of mouse and human zona pellucida using a 1.48-microns diode laser beam: efficacy and safety of the procedure.

OBJECTIVE: To investigate the efficacy and safety of a small and affordable 1.48-microns continuous wave diode laser for zona pellucida (ZP) microdissection. DESIGN: Mouse and human oocytes and zygotes were submitted to ZP drilling. The hole characteristics and possible laser-induced structural alterations of the neighboring cytoplasm were investigated with scanning and transmission electron microscopy. The safety of the procedure was checked on control and drilled zygotes by determining their ability to develop in vitro and in vivo. SETTING: Collaborative study between three Swiss academic centers. INTERVENTIONS: The collimated diode laser beam was delivered through a 45x objective of an inverted microscope and focused through the culture dish and culture medium in 1- to 3-microns spots. MAIN OUTCOME MEASURE: Safety assessment of the laser drilling procedure. RESULTS: The 1.48-microns radiation achieves a rapid, precise, and easily controlled lysis of the ZP without any micromanipulative handling of the eggs. Different shapes of holes can be produced by varying the laser beam intersection site on the ZP, laser power, and irradiation time. The energy needed to drill holes of a given diameter is greater for zygotes than for oocytes. Safety of the drilling procedure is confirmed by the lack of damage at the ultrastructural and biologic levels. CONCLUSIONS: The low-cost 1.48-microns diode laser allows an easy, objective-driven, nontouch microdissection of the ZP. The procedure is safe, as drilled embryos give rise to normal and fertile offspring.

Animals↗