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PubMed1985

A 43 kilobase cosmid P transposon rescues the fs(1)K10 morphogenetic locus and three adjacent Drosophila developmental mutants.

The K10 female sterility locus involved in establishment of the embryonic dorsoventral axis maps genetically to the 2E2-2F1 interval of the Drosophila X chromosome. We microdissected the 2E2-2F3 region from salivary gland chromosomes and used clones obtained from the microdissected fragments to establish a chromosomal walk covering more than 200 kb. To identify the K10 gene we used P-mediated transformation with cosmid clones constructed in cos-P, a cosmid vector incorporating the terminal repeats of the P element. Clone cos9, containing a 43 kb insert, transformed the germ line of homozygous K10 females and allowed production of normal progeny. It also rescued three genes, crooked neck, pecanex, and kurz, which map genetically near K10. Transformation experiments using smaller fragments of cos9 localize the K10+ function within 11 kb. Northern blots hybridized with probes from this region indicate the presence of several mRNA species. Each transcript has been assigned to a complementation group.

Alleles↗
PubMed1991

Pathogenesis of various forms of double outlet right ventricle in mouse fetal trisomy 13.

The pathogenesis of double outlet right ventricle with or without pulmonary infundibular atresia in mouse fetal trisomy 13 was studied at the organ level using microdissection and scanning electron microscopy. Altogether, 394 karyotyped trisomic embryos were collected between 11 days and 16 hours of gestation (presence of a vaginal plug = day 1) and 15 days of gestation at intervals of 8 hours, and at 16 days of gestation. The hearts were perfusion-fixed, microdissected, and prepared to be observed in scanning electron microscope in the following standardized orientations: frontal, right or left profile, septal and parietal halves of the right ventricle and outflow tract (conotruncus). Comparison of 276 trisomic hearts with their normal counterparts described previously has shown that: the first pathognomonic feature is the abnormal anterior position of the proximal part of the parietal outflow tract ridge or of both ridges (at 12 days and 16 hours of gestation); the abnormal anterior fusion of these ridges ("coalescence") results in a mesenchymal mass behind which is deviated the pulmonary part of the outflow tract lumen; from 14 days and 16 hours of gestation on, this lumen is either obstructed, resulting in a supravalvar stenosis of the pulmonary trunk and subsequently evolving into double outlet right ventricle with pulmonary infundibular atresia; or, in a minority of cases, this lumen is not obstructed and the heart develops into double outlet right ventricle without pulmonary infundibular atresia. The pathogenesis of these malformations differs from most of the known hypotheses based on deductions from human malformed hearts, as well as from observations of the pathogenesis of similar outflow tract malformations, such as those found in the Keeshond dog or rats treated with trimethadione.

Animals↗
PubMed1994

Encapsulated genetically engineered fibroblasts: release of nerve growth factor and effects in vivo on recovery of cholinergic markers after devascularizing cortical lesions.

Genetically engineered rat fibroblasts producing nerve growth factor (NGF) were encapsulated in alginate-polylysine-alginate gels with the objective to produce viable "minifactories" continuously producing and secreting NGF into the rat brain. Microencapsulated fibroblasts (NGF secretors and NGF non-secretors) were placed onto the surface of the lesioned rat cortex (unilateral devascularizing lesion) and animals were sacrificed 30 days after surgery. Fibroblasts NGF-non secreters normally produce tumors after implantation, therefore, they were irradiated prior to encapsulation. Three other experimental groups were studied in parallel: non-lesioned (controls), lesioned rats receiving "empty" alginate spheres and lesioned animals without treatment and microspheres. Biochemical analysis of microdissected brain tissues of lesioned animals treated with encapsulated NGF-secretor fibroblasts showed a significant increase in choline acetyltransferase (ChAT) activity in cortices adjacent to the lesion but not far from it (entorhinal cortex). This may indicate a gradient of concentration of the released NGF and/or differential responsivity of lesioned vs non-lesioned target tissue. ChAT enzymatic activity in the microdissected nucleus basalis magnocellularis (NBM) was significantly decreased (P < 0.05) in all lesioned animals treated with spheres without fibroblasts and those with fibroblasts not secreting NGF. Morphometric analysis of ChAT-IR and low affinity NGF-receptor IR cholinergic neurons in the middle portion of the NBM shows a prevention of neuronal shrinkage and extensive neuropil in animals treated with microencapsulated NGF-secretor fibroblasts. The results of this study demonstrate that NGF from encapsulated genetically engineered fibroblasts can be secreted for at least long enough to prevent degenerative changes of cholinergic neurons in the NBM.

Alginates↗
PubMed1986

Three-dimensional visualization of the intraganglionic structures of the rat myenteric plexus: scanning electron microscopy with the connective tissue digestion method.

The rat myenteric plexus was chemically microdissected and the internal structures of the ganglia were demonstrated under a scanning electron microscope. The present preparation offers a view of the three-dimensional features of ganglion cells and permits their surface structures and the size and pattern of varicosities to be observed. Numerous finger-like processes were observed on the cell bodies of the studded neurons in close relationship with the varicose axons. The intramuscular branches of the plexus were also microdissected and their running pattern was observed.

Animals↗
PubMed1996

Dentate granule cell layer collagen explant cultures: spontaneous axonal growth and induction by brain-derived neurotrophic factor or basic fibroblast growth factor.

The molecular mechanisms that underlie dentate granule cell axon (i.e., mossy fiber) growth during development and following seizure-induced hippocampal injury remain unknown. Part of this process may involve specific factors that support dentate granule cells during differentiation, and molecular cues that allow the appropriate growth of mossy fiber axons toward their targets. To study this process, we developed an in vitro assay system to measure the activity of putative trophic, chemoattractant and chemorepulsive factors. Two-hundred-micrometer-thick transverse hippocampal sections were prepared from neonatal rats and microdissected to isolate the middle one-third of the superior blade of the dentate granule cell layer. These were embedded in a three-dimensional collagen matrix either alone or with microdissected regions of the CA2 pyramidal cell layer. Cultures were maintained in a defined medium and grown for two to three days in a standard culture environment. Results showed that numerous processes grew primarily from the hilar side of explants into the collagen matrix, often in excess of 500 microns in length. These were determined to be axons based on: (i) morphological criteria including size and presence of growth cones, (ii) synaptophysin and growth-associated protein-43 immunoreactivity, (iii) lack of glial fibrillary acidic protein immunoreactivity and (iv) contiguity of biocytin-filled processes with neuronal soma within the explant. Treatment of cultures with brain-derived neurotrophic factor caused a significant increase in axon number and length, and this effect was partially reversed by the addition of a trkB-immunoglobulin fusion protein that blocks the activity of brain-derived neurotrophic factor and neurotrophin-4/5. Basic fibroblast growth factor also caused a marked increase in axon number and length, and caused a migration of neuron-like cells out of the explant into the collagen. These results show that cultured dentate granule cell layer explants are capable of growing mossy fibers into a neutral collagen matrix, and the growth of axons can be modified by the addition of exogenous growth factors. Furthermore, since target tissue and point sources of purified factors can easily be co-cultured with the explants, this new system provides a direct means for testing the molecular cues that influence mossy fiber growth.

Animals↗
PubMedDate not supplied

Distribution of norepinephrine and dopamine in cerebral cortical areas of the rat.

Concentrations of norepinephrine and dopamine were determined using enzyme isotope assay in 27 microdissected cerebral cortical areas of the rat. A detailed map is presented for microdissection of rat cerebral cortex. Norepinephrine was found in low but still measurable quantities throughout the cortex. Differences between cortical areas are also low. Relatively highest levels were demonstrated in the pyriform, insular and entorhinal cortices. The distribution of dopamine was found to be uneven with a maximal regional difference of 1:24. Concentration of dopamine was in all areas lower than that of norepinephrine. The highest dopamine concentration (2,4 ng/mg protein) was measured in the rostral pyriform cortex but other mesocortical (cingulate, frontal, insular and entorhinal) dopaminergic areas also contained relatively high amounts. Except for the caudal occipital and caudal entorhinal cortices all regions studied contained measurable quantities of dopamine. Its low concentration relative to norepinephrine (below 15%) suggests that in the cortical areas studied dopamine is present as the precursor of norepinephrine.

Animals↗
PubMed1987

A model to describe the size distribution of mammalian genomic fragments recovered by microcloning.

Experiments involving the use of microdissection and microcloning of mammalian chromosomes to obtain genomic clones from individual chromosome regions have demonstrated an aberrant clone recovery. The number average microclone size is well below the empirically observed number average size of genomic restriction fragments. A model is proposed that describes the distribution of microclone sizes by the use of two parameters: (1) the frequency of restriction-enzyme cleavage per bp and (2) the frequency of a second DNA event per bp. The model assumes that any DNA fragment subject to this second event is unclonable. The model shows good fit to the observed microclone size distribution from various experiments when the frequency of the second event is of the order of 0.01. The nature of the second event is unknown but it likely represents a hydrolytic event on the DNA caused by acid fixation of metaphase chromosomes prior to microdissection and microcloning.

Animals↗
PubMed1992

11p15.5-specific libraries for identification of potential gene sequences involved in Beckwith-Wiedemann syndrome and tumorigenesis.

Constitutional and somatic chromosomal abnormalities of the chromosome 11p15 region are involved in an overgrowth malformation syndrome, the Beckwith-Wiedemann syndrome (BWS), and in several types of associated tumors. The bias in parental origin for the different etiologic forms of this syndrome and for loss of heterozygosity in the tumors suggests that a gene (or genes) mapping to this region undergoes genomic imprinting. However, the precise localization of the locus (or loci) for the BWS and associated tumors is still unknown and more markers are required. We therefore isolated 11p15 markers from two libraries: the first one obtained by microdissection of the chromosome 11p15.5 region and the second one, a phage library, constructed from a hybrid cell line containing this region as its sole human DNA. Of 19 microclones isolated from the microdissection library, 11 were evolutionarily conserved. Four phage clones were isolated; one (D11S774) detected a highly informative variable number of tandem repeats (VNTR) and another (D11S773) a biallelic polymorphism. These clones were sublocalized using a panel of somatic cell hybrids that defines eight physical intervals in 11p15.5. Twenty-one clones map to the distal interval that harbors the BWS locus.

Animals↗
PubMed2004

Gene expression in precursor cells of prostate cancer associated with activin by combination of subtractive hybridization and microarray technologies.

Prostatic intraepithelial neoplasia (PIN) is considered the pre-malignant stage of prostate carcinoma, but little is known of its initiation and evolution. The identification of genes associated with these precursors of prostate cancer may elucidate the pathways of the early oncogenesis of this disease. Previously, we have reported that activin, a member of the TGFbeta superfamily, acted as an inhibitory growth factor in prostate cancer. We used laser capture microdissection, mRNA-library amplification (RNA-PCR), subtractive hybridization, and complementary DNA microarray to examine gene expression profiles in activin-positive PIN, compared with activin-negative PIN. Subtractive hybridization showed that 28 genes were differentially expressed (13 and 15 genes were up- and down-regulated, respectively). Microarray analysis identified 29 and 56 more genes (4 times) up- and down-regulated, respectively, suggesting that DNA microarray is a more effective method in screening gene profiles. We have validated the known genes identified by both subtractive hybridization and microarray technologies, using Northern blot analysis in the mRNA libraries generated from cells microdissected from pathological slides. We have successfully showed that at least 13 genes are involved in activin-associated PIN. The evaluation of candidate genes that emerge from these experiments provides a rational approach to investigate those genes significant in evolution from PIN to prostate carcinoma.

Activins↗
PubMed2006

'Functional' neuroanatomical tract tracing: analysis of changes in gene expression of brain circuits of interest.

Neuroanatomical tracing when considered as an isolated method produces relatively straightforward answers. Although single-, double- or even triple-tracing paradigms produce valuable data on the organization of brain circuits, the final outcome often is too simplistic since it is not possible to elucidate the activity of these circuits. In this regard, emerging technologies contribute with additional information about the status of neuronal circuits. The laser-guided capture microdissection microscope (LCM) allows the accurate dissection of small brain areas under the microscope that could be further analyzed for gene expression or proteomics. In order to elucidate the gene expression of a given circuit of interest, we have developed a combination of methods comprising (i) fluorescent non-radioactive in situ hybridization for the detection of vGLUT2 mRNA expression combined with retrograde tracing with Fluoro-Gold (FG; analysis performed under the confocal microscope) and (ii) laser-guided capture microdissection of brain areas containing neurons retrogradely labeled with FG followed by the measurement of changes in mRNA levels encoding for vGLUT2 by real-time PCR. Our goal was to detect changes in gene expression of the thalamostriatal pathway in unilaterally 6-OHDA lesioned rats. Taking advantage of this procedure, we found a three-fold increase in vGLUT2 mRNA expression within thalamic neurons projecting to the dopamine-depleted striatum when compared with the activity of the thalamic neurons innervating the control striatum.

Animals↗
PubMed2005

Analysis by comparative genomic hybridization of gastric cancer with peritoneal dissemination and/or positive peritoneal cytology.

Peritoneal metastasis is an important prognostic factor in cases of gastric cancer. Although studies on comparative genomic hybridization (CGH) in gastric cancer have been reported, there are few reports on the peritoneal metastasis (P) and peritoneal cytology (CY) factors in this cancer. In this study, we analyzed the chromosomal changes in the primary tumor with a combination of laser microdissection analysis and CGH in an attempt to detect the unknown abnormal chromosomal regions. We analyzed 34 primary tumors, including 13 primary tumors with peritoneal metastasis (P1) and/or positive peritoneal cytology (CY1) using a combination of laser microdissection and CGH. The minimal overlapping regions in gains were assigned to 5p14 (46.2%), 7q21.3 (61.5%), 7q31 (46.2%), 7q36 (46.2%), 8q23 (53.8%), 15q26 (46.2%), 20q12 (61.5%), 20q13.1 (53.8%), and 20q13.2 (53.8%) in primary tumors with P1 and/or CY1. The minimal regions of losses that occurred most frequently were 4q34-q35 (23.1%) and 22q11.2 (23.1%). There were significant differences in the minimal regions of 5p14 (P=0.033), 7q21.3 (P < 0.0001), 7q31 (P=0.013), 7q36 (P=0.033), and 22q11.2 (P=0.048) between primary tumors with and without P1 and/or CY1. In this study, gain/amplification of 5p14, 7q21.3, 7q31, and 7q36, and loss of 22q11.2 were significant in gastric cancer cases with peritoneal dissemination and/or positive peritoneal cytology.

Aged↗
PubMedDate not supplied

Tetrasomy 12pter-12p13.31 in a girl with partial Pallister-Killian syndrome phenotype.

A dysmorphic patient was shown to carry a small supernumerary marker chromosome. Multicolor, centromere-multicolor and regular FISH experiments proved the marker to be an analphoid 12pter derived isochromosome. Microdissection of the marker followed by reverse painting and array CGH analysis showed that the isochromosome contains approximately 6 Mb of 12pter-12p13.31 derived sequence. This is only the second report of a marker with a neocentromere 12pter and the molecular fine mapping of the duplicated region further refines the 12p region defining the Pallister-Killian syndrome phenotype. In addition, we show the feasibility of using microdissected chromosomes or chromosomal fragments to molecularly map the chromosomal breakpoints on array CGH. This technology may aid in the identification of chromosomal translocation breakpoints.

Abnormalities, Multiple↗
PubMed2006

Multiple, parallel cellular suicide mechanisms participate in photoreceptor cell death.

Photoreceptor degeneration in human photoreceptor dystrophies and in the relevant animal models has been thought to be executed by one common mechanism -- caspase-mediated apoptosis. However, recent experiments have challenged this concept. In previous experiments, analyzing gene expression in the degenerating rd/rd mouse retina, we have suggested that the gene defect leads to oxidative stress and altered metabolism, which may induce caspase-dependent and caspase-independent cell death mechanisms such as the activation of cystein-proteases, lysosomal proteases, autophagy and complement-mediated lysis. In this study we asked two questions. First, whether a temporal analysis of these different mechanisms during the course of degeneration would enable us to establish a causal relationship between these events; and second, whether photoreceptor degeneration in different models of photoreceptor dystrophies occurs by activating the same mechanisms. Three models of photoreceptor degeneration were chosen in which photoreceptor degeneration is caused by different events: the rd/rd mouse (calcium overload); the rds/rds mouse (structural defect); and light-damage (LD; oxidative stress). Marker genes were selected for the identified processes. PCR-analysis on laser capture microdissection samples was used to verify the expression of these genes in the rod photoreceptor layer. A temporal relationship between the processes was established at the mRNA level, using quantitative RT-PCR. The time course of gene expression was compared to that of cell loss (loss of rows of photoreceptor nuclei) and apoptosis (TUNEL labeling). Apoptosis and autophagy was analyzed using enzymatic assays. The time course of apoptosis and TUNEL labeling coincide in all three models. Complement-activated lysis was found to either parallel (rd/rd and rds/rds) or precede (LD) the development of TUNEL-positive cells. Autophagy was determined to parallel (rd/rd and LD) or lag (rds/rds) behind the development of TUNEL-positive cells. In all three models, glucose metabolism was found to be increased significantly prior to the onset of cell death, but then dropped in parallel with the loss of cells. The presence of the marker genes was verified by laser capture microdissection, and apoptosis (caspase activity) and autophagy (lysozyme and cathepsin activity) were verified in retina extracts. These results provide evidence that irrespective of whether photoreceptor degeneration is triggered by gene defects (lack of beta-PDE or rds/peripherin) or environmental stress (light-damage), a number of pro-apoptotic mechanisms are triggered leading to the degeneration of the photoreceptor cells. The temporal pattern of the different pathways suggests that the non-caspase-dependent mechanisms may actively participate in the demise of the photoreceptors, rather than represent a passive response of the retina to the presence of dying cells. Thus, unless the common upstream initiator for a given photoreceptor dystrophy is found, multiple rescue paradigms need to be used to target all active pathways.

Animals↗
PubMed2004

Serum testosterone levels in patients with nonmosaic Klinefelter syndrome after testicular sperm extraction for intracytoplasmic sperm injection.

We measured testosterone levels in 24 patients with nonmosaic Klinefelter syndrome before and at 6 and 12 months after conventional or microdissection testicular sperm extraction. Testosterone levels decreased after surgery by either technique, and they did not recover to baseline concentrations, even when using less invasive microdissection techniques.

Humans↗
PubMed2005

Paracentric inversion of chromosome 7(q22-31) associated with nonobstructive azoospermia.

OBJECTIVE: To examine an unusual paracentric inversion of chromosome 7 found in an azoospermic man. DESIGN: Case report. SETTING: Infertility clinic at a private hospital. PATIENT(S): An azoospermic but otherwise apparently healthy man. INTERVENTION(S): Karyotyping with peripheral blood lymphocytes, microdissection testicular sperm extraction (TESE), and intracytoplasmic sperm injection (ICSI). MAIN OUTCOME MEASURE(S): Sperm count and GBG banding. RESULTS: A karyogram revealed paracentric inversion of chromosome 7 with breakpoints at 7q22 and 7q31. Testicular histopathology confirmed the Sertoli cell-only syndrome. Successful pregnancy and delivery were achieved with microdissection TESE and ICSI. CONCLUSION(S): Paracentric inversion of chromosome 7(q22-31) associated with nonobstructive azoospermia is rare. Chromosome 7q22-31 may be responsible for impaired spermatogenesis.

Adult↗
PubMed2005

Four pregnancies in nonmosaic Klinefelter's syndrome using cryopreserved-thawed testicular spermatozoa.

OBJECTIVE: To investigate feasibility of using cryopreserved-thawed testicular spermatozoa from patients with nonmosaic Klinefelter's syndrome for intracytoplasmic sperm injection (ICSI). DESIGN: Case report. SETTING: University-based hospital and IVF clinic. PATIENT(S): Six patients with nonmosaic Klinefelter's syndrome who underwent testicular sperm extraction for ICSI. INTERVENTION(S): Microdissection testicular sperm extraction (TESE) and ICSI. MAIN OUTCOME MEASURE(S): We compared results of ICSI using cryopreserved testicular spermatozoa with those previously reported in Klinefelter's syndrome and those in nonobstructive azoospermia patients using cryopreserved testicular spermatozoa at our institution with respect to embryo cleavage rate, implantation rate, and pregnancy outcome. RESULT(S): Four of six patient couples with successful microdissection TESE achieved pregnancy using cryopreserved-thawed testicular spermatozoa. One pregnancy resulted in early-pregnancy abortion, two in delivery of healthy singleton girls, and one delivery of a healthy singleton boy. CONCLUSION(S): Cryopreserved-thawed testicular spermatozoa can be used successfully for ICSI in patients with nonmosaic Klinefelter's syndrome.

Adult↗
PubMed2006

Adult onset of declining spermatogenesis in a man with nonmosaic Klinefelter's syndrome.

OBJECTIVE: To examine an unusual case of Klinefelter's syndrome. DESIGN: Case report. SETTING: An infertility clinic at a private hospital. PATIENT(S): A patient with nonmosaic Klinefelter's syndrome who had had a child with his ex-wife without artificial reproductive technologies. INTERVENTION(S): Karyotyping with peripheral blood lymphocytes, microdissection testicular sperm extraction, and intracytoplasmic sperm injection (ICSI). MAIN OUTCOME MEASURE(S): Sperm count and testicular histopathology. RESULT(S): Semen analysis revealed severe oligoasthenozoospermia. Intracytoplasmic sperm injection using ejaculated spermatozoa was performed, and successful pregnancy and delivery were achieved. Two years later, multiple semen analyses revealed azoospermia. Microdissection testicular sperm extraction was performed, and motile sperm were successfully retrieved. Unfortunately, the first cycle of ICSI using testicular sperm failed. Testicular histopathology was compatible with decreased spermatogenesis. CONCLUSION(S): This is a case of a man with nonmosaic Klinefelter's syndrome who exhibited progressive decline of spermatogenesis. This case suggests the merit of early sperm retrieval and cryopreservation for future management of infertility in Klinefelter's syndrome.

Adult↗
PubMed2006

Genomic alteration is not associated with fatty and clear cell change in hepatocellular carcinomas and its precursor nodular lesions in cirrhotic liver.

BACKGROUND: The aim of this study was to investigate whether fatty and clear cell areas in large regenerative nodules (LRN), dysplastic nodules (DN), and hepatocellular carcinoma (HCC) show higher degree of genomic mutation compared to non-fatty/clear cell area in the same nodule or non-lesional tissue. METHODS: We examined 22 nodular lesions (9 HCC, 5 DN and 8 LRN) from seven cirrhotic livers removed at transplantation. Frozen sections were used for manual microdissection of areas with fatty/clear cell change. DNA from microdissected tissue was amplified using arbitrarily primed polymerase chain reaction (AP-PCR), and PCR products were run on polyacrilamide gel generating a "fingerprint" band pattern. Autoradiographs were analysed using Adobe Photoshop version 6.0. Fingerprints from lesional tissue were compared to reference tissue and the total number of bands in excess or defect was calculated and divided by the total number of bands identified, obtaining the genomic damage fraction (GDF). RESULTS: Increasing GDF average values were seen from cirrhotic liver (0.13+/-0.04), to LRN (0.16+/-0.1), DN (0.28+/-0.08) and HCC (0.30+/-0.07). A statistically significant difference in GDF values was documented between cirrhotic liver and DN (p=0.008) and HCC (p=0.005) and between HCC and LRN (p=0.02). No significant difference was documented between DN and HCC, and between LRN and cirrhotic liver. Eleven nodules containing fat/clear cell areas were compared to the other 11 nodules without fat/clear cell areas. The GDF was not different between the two groups: 0.29+/-0.11 versus 0.25+/-0.12; p=0.5. The average value of genomic damage fraction between fat/clear cell areas (0.29+/-0.11) and no fat/clear cell areas (0.25+/-0.1) within the same nodules were not significantly different (p=0.11). CONCLUSION: Fatty and clear cell change in nodular lesions in cirrhotic liver may be an epigenetic phenotypic modification caused by microenvironmental factors such as ischaemia rather than indicating areas of increased malignant potential per se.

Journal Article↗