Paul H. Pevsner

A photothrombotic model of small early ischemic infarcts in the rat brain with histologic and MRI correlation.

Over the last two decades several studies have suggested the role of photothrombotic occlusion of cerebral microvessels using rose bengal, resulting in small strokes in rodents that resemble those in humans. This paper describes such a photothrombotic method of acute small stroke induction in rats with histopathologic and in vivo magnetic resonance imaging (MRI) observations from 3 to 6 h after irradiation, which is homologous to a human autopsy specimen. Utilizing 30 min of irradiation with minimal beam intensity (0.1 W/cm(2)) cold white light in conjunction with 20 mg of intravenous (iv) rose bengal as a rapid infusion, small infarcts were induced photochemically in the frontal lobes of six rats. The infarcts showed a consistent pattern on histologic and in vivo MR sections when examined within 7 h or less of irradiation. Both MRI and histologic sections were comprised of (a) a superior zone of infarcted neurons, (b) a middle curvilinear transition zone of edema on MRI and histologically vacuolated neuropil, and (c) an inferior zone of normal neurons. Shorter duration water-sensitive (T2)- and postgadolinium longer duration (T1)-weighted signal decay images both showed a curvilinear hyperintense transition zone of edema. The mean infarct and transition zone areas measured from the histologic sections were comparable to those measured on the MRI. The infarct model described above allows in vivo observations using MRI with the potential for use in testing putative neuroprotective agents. As demonstrated by a comparison with the histologic features of such infarcts in surgical and autopsy brain specimens, the model is relevant to acute human ischemic infarcts.

A murine photochemical stroke model with histologic correlates of apoptotic and nonapoptotic mechanisms

INTRODUCTION The neuronal cell death that occurs after ischemia-induced cerebral infarction (stroke) contains elements of apoptosis and necrosis, an intermediary form of the two, and a distinct excitotoxic process. We previously developed a photochemical model of stroke in the rat. We have now adapted this model for use in the mouse. The present manuscript describes the mouse model. METHODS Minimal beam intensity (0.1 W/cm(2)) cold white light (8 min exposure) was used to evoke discrete infarcts in the parietal lobes of 11 mice sensitized by the administration of fresh Rose Bengal (10 mg/kg by rapid iv infusion). RESULTS At 2 h, five out of five mice and at 6 h, six out of six mice demonstrated light microscopic histologic features like those in the rat model. These included a superior ischemic zone with shrunken and pyknotic nuclei, a middle transition zone of edematous vacuolated neuropil but normal neurons with open chromatin and retained Nissl granules, and an inferior zone with normal neurons. There was widespread nuclear terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) in the superior infarct zone in 11/11 mice. However, in the edematous vacuolated transition zone, 11/11 mice had TUNEL positive and negative nuclei randomly mixed. Light microscopic analysis of that same transition zone showed no pyknosis or chromatin bodies in the TUNEL positive or negative cells. DISCUSSION In mice, photoactivation of Rose Bengal evoked similar infarct and transition zone patterns found previously in rats, with TUNEL evidence of apoptotic and nonapoptotic events. Thus, it will be possible to use this model for further quantitative study of apoptotic and excitotoxic events in wild and transgenic mice.

Mass spectrometry MALDI imaging of colon cancer biomarkers: a new diagnostic paradigm

Colorectal cancer (CRC), is the second-leading cause of cancer-related deaths in the USA, affecting both men and women. Current projections show little or no change since the publication of a morbidity and mortality study in 2005. The projected number of new cases for 2008 is 154,000, and the projected number of CRC cancer deaths for 2008 is 53,000. The standard diagnostic paradigm is based on histopathology of either biopsy or surgical specimens. This article suggests a new paradigm for colon cancer diagnosis and staging using matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS or IMS). IMS may identify potential tumors in normal tissue of cancer patients and predict those cancer patients who are at risk for recurrent cancer.

Minimally invasive method for murine brain fixation

Complete brain fixation can be achieved with transthoracic cardiac infusion without thoracotomy. Light and electron microscopy tissue sections reveal preservation of cytoplasmic and nuclear structure at all magnification levels. Punched samples were obtained from the fixed tissue specimens in precisely localized areas for study using electron microscopy. This perfusion fixation technique provides both faster tissue harvesting capability and higher quality tissue preservation, without the artifacts of brain swelling and ventricular dilation observed in direct cardiac perfusion. Acute, discrete change in brain tissue can be studied.

Micropipette tips – the unsung heroes of mass spectrometry

Micropipette tip (tip) pipetting accuracy and precision are functions of tip manufacture consistency, morphological variation, and retained inner tip wall particulate matter. Irregular tip inner wall surfaces and retained particulate matter cause pipetting inaccuracy. Washed and unwashed tips from seven manufacturers were compared using weight by difference (six), microscopic Luxol Fast Blue staining (seven), and matrix-assisted laser desorption/onization time-of-flight (MALDI-TOF) mass spectrometry (six). Photomicrographs revealed tip irregularity and inner wall retained particulate matter. Computer model analysis was used to identify tip irregularity and retained matter. These tests established the utility of a pipette solvent wash to increase the performance and the accuracy of tips and, thus, improve the MALDI mass spectra obtained.

Computational Analysis of Mass Spectrometry Data Using Novel Combinatorial Methods

The analysis of proteome profiles offers a new approach to understanding how cellular machinery functions and responds under certain conditions. By combining twodimensional electrophoresis with mass spectrometry (MS), a snapshot of the cells protein expression status and quantitative proteome profiling can be provided. As the cells proteome becomes defined in normal and altered states, possible utilization of MS proteome profiling as a diagnostic tool becomes a reality. The ability of Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) to generate a spectrum with thousands of data points, necessitate the development of sophisticated analytical algorithms. In this paper, we describe how MALDI-MS can be used in monitoring proteomic profile in patients before and after treatment using a non- invasive sampling method. Because data analysis in this process possesses a challenge, we present a novel mathematical approach for analyzing data produced by MALDI MS, and discuss current applications of mass spectrometry in clinical medicine as well as challenges faced during procedures and experimentation. As a case study, we analyze protein expression patterns in premenopausal versus postmenopausal women. We also provide a proteomic profiling of premenopausal women versus postmenopausal women treated with estrogen as a hormone replacement therapy.

Crossing the Blood-Brain Barrier with Antibody-Labeled, Gold-Coated Nanoconjugates: A Preliminary Step in Targeting and Eradicating Brain Tumors

Crossing the Blood-Brain Barrier with Antibody-Labeled, Gold-Coated Nanoconjugates: A Preliminary Step in Targeting and Eradicating Brain Tumors This paper shows ultrastructural proof of principle that antibodylabeled nanoconjugates can cross the blood-brain barrier and bind to their targeted antigens. Gold-coated nanoconjugates labeled with monoclonal antibodies targeting brain antigens-specifically, the synaptophysin and glial fibrillary acidic proteins-were injected intravascularly into 45 Balb/c mice. Through electron microscopy a portion of the nanoconjugates were shown to have crossed the blood-brain barrier and become localized either on the intermediate filaments in the astrocytic cytoplasm (anti-glial fibrillary acidic proteins) or presynaptic membranes (anti-synaptophysin). Three of the authors have already reported on a similar and successful use of radioactive nanoconjugates to target antigens in a syngeneic murine lung tumor model using CMT-6 breast cancer cells. This proof of principle supported by concrete evidence does not include an analysis of the mechanism by which the nanoconjugates crossed the blood-brain barrier. The success of targeting proteins in the brain and, by implication, antigens in brain tumors, has immense potential for the treatment of a wide variety of metastases and tumors, including brain cancers such as glioblastoma.

S1967 Mass Spectrometry MALDI Imaging and LCMS Identification of Colon Cancer Proteins in Benign Polyps

Introduction: Recently soma-wide monoallelic germline epigenetic inactivation of MLH1 has been proposed as a potential mechanism for DNA mismatch repair (MMR) gene inactivation in some individuals with colorectal cancer (CRC). The present study aimed to investigate whether this methylation defect would be limited to the MLH1 promoter, or represents a broader epigenetic defect involving multiple genes. Methods: A 20 year old woman presented with a sigmoid colon cancer, and a negative family history for any cancer. Her tumor was evaluated for DNA mismatch repair (MMR) gene defects, and she was tested for germline mutations in MMR genes. Quantitative pyrosequencing and bisulfite sequencing of cloned PCR products was used to ascertain MLH1 methylation status in all three germ-layers (blood, hair and buccal tissues), as well as in her Epstein-Barr virus (EBV)-transformed lymphoblastoid cells. Genome wide/global methylation status for ~1500 genes/methylation loci was determined in the patients blood and controls (which included both parents, her sibling, and 3 unrelated controls) using Illumina GoldenGate methylation microarrays. Results: The patients tumor showed MSI and loss of expression of MLH1, PMS2 and MSH6 proteins. However, no germline mutations were detected in MLH1, MSH2 or MSH6. A single nucleotide polymorphism (SNP) permitted allele identification; 14%monoallelicMLH1 methylation was found in the tumor, but there was no allelic imbalance at MLH1 in the tumor. MLH1 methylation was observed in the patients blood (10%), EBV transformed lymphocytes (10%), buccal mucosa (22%) and hair follicles (48%). No evidence for MLH1 methylation was present in patients family members or any healthy control (all <1%). Interestingly, MLH1 methylation increased to 24% in the patients blood after 5-FU based chemotherapy treatment. Genome wide methylation analyses showed that aberrant methylation in the blood was not limited to MLH1, but included 43 additional hypermethylated genes (including RUNX3, IGF1, PPARγ and NOTCH4) and another 34 genes which were hypomethylated (including CDK2, STAT5 and IL10). Conclusions: This study reports the youngest patient with soma-wide MLH1 hypermethylation and CRC. The increase in MLH1 methylation following chemotherapy suggests that cells with methylated MLH1 promoters are relatively resistant to the toxic effects of this drug. More importantly, our observation that the epigenetic defects were not restricted to the MLH1 promoter suggests the possibility of a broader epigenetic defect in those individuals with soma-wide methylation of MLH1.