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PubMed2005

Branching ducts similar to mesonephric ducts or ureteric buds in teratomas originating from mouse embryonic stem cells.

Ureteric bud epithelial cells and metanephric mesenchymal cells that comprise the metanephric kidney primordium are capable of producing nephrons and collecting ducts through reciprocal inductive interaction. Once these cells are induced from pluripotent embryonic stem (ES) cells, they have the potential to become powerful tools in the regeneration of kidney tissues. In this study, we investigated these renal primordial cells and structures in mouse ES cell outgrowths and their transplants. Gene expression essential for early kidney development was examined by RT-PCR in embryoid body (EB) outgrowths and their transplants in adult mice. Histochemical detection of kidney primordial structures and gene expression analysis coupled with laser microdissection were performed in transplant tissues. RT-PCR analysis detected gene expression of Pax-2, Lim-1, c-Ret, Emx2, Sall1, WT-1, Eya-1, GDNF, and Wnt-4 in the EB outgrowths from days 6-9 of expansion onward, and also in the teratoma tissues 14 and 28 days after transplantation. Histochemical analysis 14 days after transplantation showed that some ducts were positive for Pax-2, endo A cytokeratin, kidney-specific cadherin, and Dolichos biflorus agglutinin and that dichotomous branching of these ducts had occurred. These staining patterns and morphological features are intrinsic for mesonephric ducts and ureteric buds. In long-term survival of 28 days, Pax-2-immunoreactivity disappeared in some renal primordia-like structures, indicating their differentiation. Some ducts were accompanied by mesonephric nephron-like convoluted tubules. RT-PCR analysis of those structures collected by microdissection confirmed that they expressed kidney development-related genes. In conclusion, these data suggest the potential of ES cells to produce renal primordial duct structures and provides an insight into the regeneration of kidney tissues.

Animals↗
PubMed2005

Laser pressure catapulting (LPC): optimization LPC-system and genotyping of colorectal carcinomas.

Genotype analysis is becoming more and more useful in clinical practice, since specific mutations in tumors often correlate with prognosis and/or therapeutic response. Unfortunately, current molecular analytical techniques often require time-consuming and costly steps of analysis, thus making their routine clinical use difficult. Moreover, one of the most difficult problems arising during tumor research is that of their cell heterogeneity, which depends on their clear molecular heterogeneity. SSCP analysis discriminates by means of aberrant electrophoresis migration bands, mutated alleles which may represent as little as 15-20% of their total number. Nevertheless, in order to identify by sequencing the type of alteration revealed by this technique, only the mutated allele must be isolated. The advent of laser microdissection is a procedure which easily solves these problems of accuracy, costs, and time. The aims of this study were to perfect the system of laser pressure catapulting (LPC) laser microdissection for the assessment of the mutational status of p53 and k-ras genes in a consecutive series of 67 patients with colorectal carcinomas (CRC), in order to compare this technique with that involving hand-dissection and to demonstrate that since the LPC system guarantees more accurate biomolecular analyses, it should become part of clinical routine in this field. The LPC-system was perfected with the use of mineral oil and the LPC-membrane. To compare the techniques of hand- and LPC-microdissection, alcohol-fixed, paraffin-embedded tissue from 67 cases of CRC were both hand- and laser-microdissected. In either case, dissected samples were analyzed by SSCP/sequencing and direct sequencing for k-ras and p53 gene mutations. LPC-microdissection made it possible to pick up mutations by direct sequencing or SSCP/sequencing, whereas hand-microdissection mutations were identified only by means of SSCP followed by sequencing; direct sequencing did not reveal any mutation. In the 67 patients examined by either method, 36% (24/67) showed p53 mutations, 32 of which identified. Seventy-eight percent (25/32) were found in the conserved areas of the gene, while 12% (4/32) were in the L2 loop, 50% (16/32) were in the L3 loop, and 12% (4/32) in the LSH motif of the protein. Moreover, of the 67 cases examined, 40% (27/67) showed mutations in k-ras, with a total of 29 mutations identified. Of these, 14 (48%) were found in codon 12 and 15 (52%) in codon 13. The modifications which we brought to the LPC system led to a vast improvement of the technique, making it an ideal substitution for hand-microdissection and guaranteeing a considerable number of advantages regarding facility, accuracy, time, and cost. Furthermore, the data obtained from the mutational analyses performed confirm that the LPC system is more efficient and rapid than hand-microdissection for acquiring useful information regarding molecular profile and can therefore be used with success in clinical routine.

Carcinoma↗
PubMed2001

Breast cancer after mantle irradiation for Hodgkin's disease: correlation of clinical, pathologic, and molecular features including loss of heterozygosity at BRCA1 and BRCA2.

PURPOSE: Hodgkin's disease patients who receive mantle irradiation have an age-dependent increased risk of developing breast cancer. To determine if genetic factors predispose these patients to develop breast cancer, we evaluated breast cancer specimens for loss of heterozygosity (LOH) at regions where BRCA1 and BRCA2, two breast cancer tumor suppressor genes, are located. We also evaluated whether breast cancers in patients who were previously treated with radiation have a more aggressive phenotype, and whether the clinical course differed from a sporadic group of breast cancer patients. METHODS AND MATERIALS: All females with Hodgkin's disease who were subsequently diagnosed with breast cancer and for whom tissue blocks were available were included. Using a case-control design, case patients (previously treated with radiation therapy) were matched with sporadic control breast cancer patients for age, breast cancer stage, and date of breast cancer diagnosis. After microdissection of tumor and normal tissue from paraffin-embedded tissue blocks, DNA was extracted and samples were examined for LOH at chromosomal segments encompassing BRCA1 and BRCA2. Breast cancer specimens were also evaluated in a blinded fashion for tumor grade and immunoreactivity to estrogen and progesterone receptors, p53, her2-neu, and topoisomerase II alpha. Comparisons were made between the case and control populations using chi2 analysis, and a paired Student's t test. Survival differences were evaluated using a log-rank test. RESULTS: From January 1960 to December 1983, 917 patients were diagnosed with Hodgkin's disease. Twelve patients were subsequently diagnosed with breast cancer and tissue blocks were available on 10 cases. No statistical difference was observed between the case and control populations for LOH at BRCA1 or BRCA2. In the Hodgkin's disease group, LOH was observed in 30% of tumors at BRCA1 and 10% of tumors at BRCA2 vs. 10% and 0% of tumors in the control group at BRCA1 and BRCA2, respectively. Breast tumors from patients who received radiation therapy for Hodgkin's disease displayed greater nuclear pleomorphism (p < 0.02), and an increase in topoisomerase II alpha expression (p < 0.05) vs. the control population. Five of 10 patients were pregnant at the time of their Hodgkin's treatment, and those patients had a shorter time interval to the development of breast cancer compared with the patients who were not pregnant (12.4 years compared with 18.6 years). There was no significant difference in disease-free survival; however, overall survival was inferior in the population previously treated with radiation therapy for Hodgkin's disease (p = 0.01). 80% of patients with a previous Hodgkin's diagnosis died of breast cancer or treatment related effects vs. 30% in the control group. CONCLUSION: We were unable to find statistical evidence for LOH at BRCA1 and BRCA2 in breast cancers from patients previously irradiated for Hodgkin's disease. Breast cancer diagnosed after mantle irradiation may be more biologically aggressive based on the greater nuclear pleomorphism and increase in topoisomerase II alpha staining. This did not translate into a statistical difference in breast cancer disease-free survival; however, overall survival was significantly inferior in the Hodgkin's disease patients.

Adolescent↗
PubMed2002

Identification of nitric oxide synthase neurons for laser capture microdissection and mRNA quantification.

An immunohistochemical technique was developed to visualize nitric oxide synthase (NOS)-immunopositive neurons in fresh-frozen tissue sections of rat brain for laser capture microdissection (LCM) and mRNA analysis. The effect of tissue fixation and the choice of fluorophore were investigated. Here we describe a rapid immunofluorescence protocol that allows the processing of fresh-frozen tissue sections within eight minutes and subsequent mRNA extraction and real-time PCR from pools of 20 NOS-immunopositive LCM neurons. The cellular complement of a subset of ionotropic glutamate receptors, specifically N-methyl-D-aspartate receptor subunit mRNAs, was examined because these receptor complexes are thought to mediate the effects of fast and slow glutamate excitotoxicity. Real-time PCR data revealed that striatal NOS interneurons express the mRNAs encoding NR1, NR2A, NR2B, and NR2D but not NR2C. These LCM mRNA data are consistent with previous in situ hybridization studies and demonstrate the utility of rapid immuno-LCM with real-time quantitative PCR for the study of mRNA abundance in discrete populations of neurons within the mammalian brain.

Animals↗
PubMed1991

Three-dimensional architecture of the human myosalpinx isthmus. Scanning electron microscopy after NaOH digestion and ultrasonic microdissection.

The three-dimensional architecture of the human isthmic myosalpinx is directly visualized by means of scanning electron microscopy after removal of interstitial connective tissue through NaOH maceration and ultrasound microdissection. These investigations show that the myosalpinx is composed of irregularly running bundles of smooth muscle cells, changing their orientation within the myosalpinx and displaying longitudinal, oblique and circular directions. The muscular bundles anastomose and intermingle with other bundles running at different levels in the oviduct wall, and actually give rise to a wide and complex muscular network in which no distinct layers are readily discernible. These morphological data are consistent with the physiological findings that the transport of gametes and embryo in very early stages in the isthmic portion of the oviduct tube is the result of a discontinuous pattern of forward and backward movements.

Animals↗
PubMed2004

A new method for imaging and 3D reconstruction of mammalian cochlea by fluorescent confocal microscopy.

Traditional methods for anatomical and morphometric studies of cochlear tissues have relied upon either microdissection of the organ of Corti or the generation of serial sections of the cochlea. Such methods are time-consuming, disruptive to three-dimensional relationships and often restrict sampling to very limited numbers of cells. We have found that cells and tissue components of the cochlear duct may be labelled by fluorescent markers within intact cochleae, which are then embedded in epoxy resin for subsequent viewing by fluorescent microscopy methods. This approach allows imaging through thick optical volumes with preservation of three-dimensional relationships. Unlike sectioned tissue, alignment of the sample relative to the focal axis may be easily corrected by re-orientation of the optical volume with common image processing software. Fluorescently labelled cochleae embedded in epoxy can be viewed by most fluorescent microscopy methods including laser scanning confocal microscopy, multi-photon confocal microscopy and widefield epi-fluorescence microscopy with deconvolution. Furthermore, semi-thin sections made from these preparations are compatible with traditional histological stains, as well as allowing brightly labelled epi-fluorescent images.

Animals↗
PubMed2004

Identification of proteins in laser-microdissected small cell numbers by SELDI-TOF and Tandem MS.

BACKGROUND: Laser microdissection allows precise isolation of specific cell types and compartments from complex tissues. To analyse proteins from small cell numbers, we combine laser-microdissection and manipulation (LMM) with mass spectrometry techniques. RESULTS: Hemalaun stained mouse lung sections were used to isolate 500-2,000 cells, enough material for complex protein profiles by SELDI-TOF MS (surface enhanced laser desorption and ionization/time of flight mass spectrometry), employing different chromatographic ProteinChip Arrays. Initially, to establish the principle, we identified specific protein peaks from 20,000 laser-microdissected cells, combining column chromatography, SDS-PAGE, tryptic digestion, SELDI technology and Tandem MS/MS using a ProteinChip Tandem MS Interface. Secondly, our aim was to reduce the labour requirements of microdissecting several thousand cells. Therefore, we first defined target proteins in a few microdissected cells, then recovered in whole tissue section homogenates from the same lung and applied to these analytical techniques. Both approaches resulted in a successful identification of the selected peaks. CONCLUSION: Laser-microdissection may thus be combined with SELDI-TOF MS for generation of protein marker profiles in a cell-type- or compartment-specific manner in complex tissues, linked with mass fingerprinting and peptide sequencing by Tandem MS/MS for definite characterization.

Animals↗
PubMed2004

Impact of fixative on recovery of mRNA from paraffin-embedded tissue.

Due to the evolution of advanced tissue-analysis tools, such as proteomics and functional and structural genomics, the demands for handling and preserving samples are changing. For gene expression analysis, the presence of intact and extractable messenger RNA in the test material is mandatory. To find an optimal fixative for tissues aimed for such analyses, we evaluated the morphology-, protein antigen-, and RNA-maintaining abilities of 2 precipitating tissue fixatives, methanol-acetone and Carnoy's. Both fixatives preserved the morphology and protein epitopes of tissues and allowed extraction of total RNA that was of significantly higher quality than RNA extracted from formalin-fixed tissue. Carnoy's fixative performed better than methanol-acetone in maintaining the integrity of RNA, especially when the fixed, paraffin-embedded tissue blocks were stored at room temperature for more than 3 months. Total RNA extracted from epithelial cells microdissected from Carnoy's-fixed tissue samples contained intact template for up to a 977-base pair (bp) amplicon for beta-actin. Because of the emerging role of gene expression analyses in research, and in clinical work in the near future, an RNA-preserving fixative should replace formalin as the primary human tissue fixative. According to our data, Carnoy's fixative is an excellent candidate for a new primary fixing reagent for human tissue samples.

Acetic Acid↗
PubMed1995

Increased p53 protein does not correlate to p53 gene mutations in microdissected human testicular germ cell tumors.

PURPOSE: To determine if primary testicular germ cell tumors that overexpress p53 tumor suppressor gene protein have p53 gene mutations. MATERIALS AND METHODS: We examined 30 primary testicular tissues from 26 patients representing two groups. Group one consisted of eleven cases (6 nonseminomatous germ cell tumors and 5 seminomas) in which tissue samples for DNA analysis were microdissected from paraffin block regions with elevated immunohistochemical staining for p53 protein. Group two consisted of 19 testis tumor tissues which had been fresh frozen and were chosen to correspond to archival tissue specimens exhibiting elevated levels of p53 protein. The DNA was extracted from these tissues and subjected to exon specific amplification by polymerase chain reaction (PCR) and cold single-strand conformation polymorphism (Cold SSCP) analysis. RESULTS: In these cases with elevated p53 protein, no p53 gene exon 5-8 mutations were detected except 1 seminoma with a codon 140 silent mutation (no protein alteration). CONCLUSIONS: Testicular tumors appear to exhibit elevated levels of wild-type p53 protein, the significance of which is yet to be elucidated.

Base Sequence↗
PubMed2004

Use of microsatellite marker loss of heterozygosity in accurate diagnosis of pancreaticobiliary malignancy from brush cytology samples.

BACKGROUND: Brush cytology of biliary strictures to diagnose pancreaticobiliary malignancy suffers from poor sensitivity. AIM: To improve the diagnostic yield of pancreaticobiliary brush cytology through analysis of tumour suppressor gene linked microsatellite marker loss of heterozygosity (LOH) and k-ras codon 12 mutation detection. METHODS: Twenty six patients with biliary strictures underwent endoscopic retrograde cholangiography with brush cytology. A panel of 12 polymorphic microsatellite markers linked to six tumour suppressor genes was developed. Genomic DNA from cell clusters acquired from brush cytology specimens and microdissected surgical malignant and normal tissue underwent polymerase chain amplification reaction (PCR). PCR products were compared for LOH and k-ras codon 12 mutations. RESULTS: Seventeen patients were confirmed to have pancreaticobiliary adenocarcinoma. Nine patients had benign strictures (eight proven surgically, one by follow up). Cytomorphological interpretation was positive for malignancy (n = 8), indeterminate (n = 10), and negative for malignancy (n = 8). Selected malignant appearing cytological cell clusters and microdissected histological samples from cancer showed abundant LOH characteristic of malignancy while brushings from nine cases without cancer carried no LOH (p<0.001). LOH and k-ras mutations profile of the cytological specimens was almost always concordant with the tissue samples. Presence of k-ras mutation predicted malignancy of pancreatic origin (p<0.001). CONCLUSION: LOH and k-ras codon 12 mutation analysis of PCR amplified DNA from biliary brush cytology discriminates reactive from malignant cells, with 100% sensitivity, specificity, and accuracy. Minor variations in LOH in brushings and in different sites within the same tumour likely reflect intratumoral mutational heterogeneity during clonal expansion of pre- and neoplastic lineages.

Adenocarcinoma↗
PubMed2004

A comprehensive nonredundant expressed sequence tag collection for the developing Rattus norvegicus heart.

Congenital heart defects affect approximately 1,000,000 people in the United States, with 40,000 new births contributing to that number every year. A large percentage of these defects can be attributed to septal defects. We assembled a nonredundant collection of over 12,000 expressed sequence tags (ESTs) from a total of 30,000 ESTs, with the ultimate goal of identifying spatially and/or temporally regulated genes during heart septation. These ESTs were compiled from nonnormalized, normalized, and serially subtracted cDNA libraries derived from two sets of tissue samples. The first includes microdissected rat hearts from embryonic (E) days E13, E15, and E16.5-E18.5 and adult heart. The second includes hearts from embryonic days E17, E19, and E21 and postnatal (P) days P1, P12, P74, and P200. Over 6,000 novel ESTs were identified in the libraries derived from these two sets of tissues, all of which have been contributed to the NCBI rat UniGene collection. It is anticipated that such EST and cDNA clone resources will prove invaluable to gene expression studies aimed at the understanding of the molecular mechanisms underlying heart septation defects.

Animals↗
PubMed2006

Laser-microdissection for cell type- and compartment-specific analyses on genomic and proteomic level.

Morphological study and identification of cell differentiation within tissues are the basis for assessment of physiological and pathological processes. Applying molecular techniques, the analysis of tissue homogenates may lead to masking of genetic deviations or expression changes of an individual cell type by the bulk of surrounding cells. To overcome the tissue heterogeneity, cells have to be isolated selectively. Therefore, microdissection techniques were developed making retrieval of target cells simple, rapid and precise. Presenting an overview of our approaches, it is demonstrated that single cell isolation is often preconditional for the investigation of splicing isoform expression. To reveal gene regulation combining microdissection of few cells and real-time RT-PCR allows to determine relative mRNA expression in a cell type-specific manner, even after immunofluorescence staining of target cells. Combination with RNA preamplification techniques and micro arrays results in cell type- or compartment-specific expression profiles that especially differ from those of tissue homogenates when minor represented cell types are investigated. For proteomic biomarker screening, the application of mass spectrometry techniques (SELDI/MALDI TOF) turned out to be feasible in combination with microdissected minute amounts of tissues. However, further identification of marker peaks still needs a remarkable effort. Strategies to deal with this problem are presented. In consequence, the isolation of cells or cell types allows a more accurate investigation of complex tissues and gives deeper insight to regulation processes and crosstalk of the respective cells.

Animals↗
PubMed2001

Monoclonality in normal epithelium and in hyperplastic and neoplastic lesions of the breast.

The clonal nature of neoplastic lesions such as invasive breast cancer and ductal carcinoma in situ (DCIS) has been widely proven by several proliferative, genetic or other malignancy-associated markers. The aim of this study is to clarify whether benign hyperplastic lesions such as ductal hyperplasia of usual type (DH) and papilloma can be distinguished from neoplastic lesions such as DCIS by X-chromosome inactivation analysis. Clonal analysis was performed using a polymerase chain reaction-based assay for non-random X-chromosome inactivation of the human androgen receptor gene (HUMARA). Formalin-fixed and paraffin-embedded archival tissue of ten DCIS, sixteen DH, nine papillomas, and seven normal terminal ductal lobular units (TDLUs) was laser-microdissected to avoid contamination with surrounding tissue. All of the cases analysed revealed a monoclonal origin. Furthermore, in one of these cases, opposite X chromosomes were inactivated within the same breast. X-linked inactivation analysis clearly demonstrates that, at least in the breast, monoclonality is not restricted to neoplastic processes. The data support the hypothesis that the mammary gland is organized into distinct stem cell-derived monoclonal patches and that TDLUs are monoclonal in origin. Any proliferative lesion arising within such a pre-existing clonal patch should therefore be clonal, irrespective of whether it originates from one or more patch cells. Thus, X-chromosome inactivation analysis cannot be considered a valid method for distinguishing between neoplastic and hyperplastic breast lesions.

Breast↗
PubMed2006

Laser microdissection of fungal septa as visualised by scanning electron microscopy.

Laser microdissection has been proven a successful technique to isolate single cells or groups of cells from animal and plant tissue. Here, we demonstrate that laser microdissection is suitable to isolate subcellular parts of fungal hyphae. Dolipore septa of Rhizoctonia solani containing septal pore caps were cut by laser microdissection from sections of mycelium and collected by laser pressure catapulting. Subsequently, microdissected septa were visualised using a wheat germ agglutinin labelling of cell walls, septa and septal pore caps and scanning electron microscopy. The use of laser microdissection on fungal cells opens new ways to study subcellular fungal structures and the biochemical composition of hyphal cells.

Cell Separation↗
PubMed2005

DNA topoisomerase IIalpha and Her-2/neu gene dosages in pediatric malignant gliomas.

Pediatric malignant non-brainstem glioma (PMNBG) is a rare tumor that accounts for only about five percent of childhood intracranial neoplasms. DNA topoisomerase IIalpha (TIIalpha) is a novel marker of cell-cycle turnover and a target of high-risk chemotherapy in PMNBG. We have shown that TIIalpha protein expression strongly correlates with event-free and overall survival in these malignancies. The molecular mechanism causing the varying TIIalpha protein expression in PMNBG remains unknown. Utilizing a combined approach of immunocytochemistry-based morphology guidance, laser-assisted microdissection and quantitative real-time PCR, we report a low-level co-amplification of the neighboring TIIalpha and Her-2/neu gene loci on chromosome 17q11-q22 in one of seventeen examined PMNBGs. Analysis of both genes by real-time PCR in the crude tumor samples without prior tissue heterogeneity reduction via laser microdissection, resulted in loss of detection of amplification of the syngeneic Her-2/neu locus. Gene dosage assessment in a microscopically distant tumor area revealed no amplification of either gene. Our results suggest that low-level amplification of the TIIalpha gene locus may be a sporadic mechanism of increased TIIalpha protein expression in PMNBG, which can coincide with low-level amplification of Her-2/neu. The observed intratumor genetic heterogeneity for TIIalpha in PMNBGs may have an impact on the relevance of TIIalpha as a biological constituent of outcome in these neoplasms.

Antigens, Neoplasm↗
PubMed2006

Signatures from tissue-specific MPSS libraries identify transcripts preferentially expressed in the mouse inner ear.

Specialization in cell function and morphology is influenced by the differential expression of mRNAs, many of which are expressed at low abundance and restricted to certain cell types. Detecting such transcripts in cDNA libraries may require sequencing millions of clones. Massively parallel signature sequencing (MPSS) is well suited to identifying transcripts that are expressed in discrete cell types and in low abundance. We have made MPSS libraries from microdissections of three inner ear tissues. By comparing these MPSS libraries to those of 87 other tissues included in the Mouse Reference Transcriptome online resource, we have identified genes that are highly enriched in, or specific to, the inner ear. We show by RT-PCR and in situ hybridization that signatures unique to the inner ear libraries identify transcripts with highly specific cell-type localizations. These transcripts serve to illustrate the utility of a resource that is available to the research community. Utilization of these resources will increase the number of known transcription units and expand our knowledge of the tissue-specific regulation of the transcriptome.

Animals↗
PubMed2001

[Promoter hypermethylation during tumor progression: quantitative analysis employing kinetic PCR].

The methylation of cytosine residues in the promoter region of tumour-suppressor genes is now widely recognised as an alternative mechanism of gene inactivation in cancer. To address the question whether promoter methylation is an early event in the process of malignant transformation and whether quantitative changes are occurring during clonal evolution we developed a new assay for the measurement of cytosine methylation, which could also be applied to laser-microdissected archival specimens. We first improved the bisulfite conversion of genomic DNA in order to analyse cells isolated from archival tissue sections by laser-assisted microdissection. Analysing the methylation of key regulatory genes in archival specimens (n = 38) we could demonstrate that aberrant promoter hypermethylation is an early event during breast cancer development and changes quantitatively and in a gene-specific manner during progression. This novel methodological approach allows the morphology-guided quantitative analysis of changes in the methylation pattern during the clonal evolution of tumour cells. Quantitative differences detected by this assay will offer new insights into early steps in carcinogenesis and tumour cell heterogeneity.

DNA Methylation↗
PubMed2003

Discrimination of components in atherosclerotic plaques from human carotid endarterectomy specimens by magnetic resonance imaging ex vivo.

Specific MRI techniques have been used to determine the dimensional and compositional properties of atherosclerotic lesions in carotid endarterectomy tissues. A quantitative comparison of areas of specific features in typical tissue segments was performed using MR images and histologic images. The mean difference for the measurements by the two methods was 4.5% for the total vessel, 5.3% for the internal carotid artery lumen, and 5.0% for the external carotid lumen. For other less abundant components, the mean difference was 14.2%. For direct characterization, individual tissue components were isolated by microdissection and their T1 and T2 relaxation times measured. Highly calcified areas typically had rather short T1 (452-837 ms) and short T2 (10.4-18.4 ms). In contrast, regions enriched in lipid had much longer T1 (1,380-1,480 ms) and longer T2 (35.3-49.0 ms). Other components such as thrombus had intermediate T1 (1,180 ms) and short T2 (15.4 ms). T2 parametric imaging was used as a complementary approach for segmentation and quantitation of tissue components. In fresh tissue, several different components exhibited different T2 ranges: calcified/solid lipid (13-18 ms). cellular/ECM (9-30 ms), fluid lipid (35-40 ms): fibrous (50-60 ms). These results demonstrate the utility of MRI for identifying and quantifying specific components of atherosclerotic plaque ex vivo, and suggest its value for these measurements in vivo as well.

Carotid Artery Diseases↗