Thomas M. Monticello

Digital Microscopy Imaging and New Approaches in Toxicologic Pathology

Digital microscopy, a comprehensive integration of digital imaging and light microscopy, can assist the pathologist to observe, acquire, record, share, analyze, and manage pathology image data. To lead the activity for establishing new generation digital microscopy capacity, novel concepts and strategies of digital pathology information flow and digital pathology platform were designed to integrate personal digital pathology microscopy workstations and other pathology imaging modalities with centralized data storage/management. In addition, a strategy for Web-enabled interactive telepathology that would permit global capacity was designed. A novel concept of high content pathology was also created to develop an automated tissue microscopy imaging and screening approach. These new concepts, strategies, and approaches guided the development and implementation of a digital pathology platform, a telepathology platform, and automated tissue slide imaging capacity. Digital microscopy photography is now able to replace photographic film in toxicologic pathology. Digital pathology and telepathology platforms can provide a networked environment for multisite, global team participation. Our practice also ascertained the central value of digital microscopy which can provide innovative quantitative pathology information and data mining capability with various imaging biomarkers via advanced digital image processing and pathology informatics; these are now the focus of ongoing development.

Amelioration of ischemia/reperfusion injury in isolated rat hearts by the ATP-sensitive potassium channel opener BMS-180448

OBJECTIVE Aims of this study were: (1) Evaluate by morphology and specific physiological and biochemical parameters, the protective effects of the cardioselective ATP-sensitive potassium channel opener BMS-180448 on ischemic/reperfused isolated rat heart, and (2) Determine the earliest time point ischemia-induced myocardial injury is observed by light microscopy. METHODS Hearts from Sprague-Dawley rats were perfused on a Langendorff apparatus. After equilibration, hearts were treated with BMS-180448 (10 micro M) or vehicle (0.04% DMSO) for 10 min before the onset of ischemia. Four hearts/group were collected following 10, 18, or 25 min of ischemia. A nonischemic control group was also evaluated. Following 25 min of ischemia, another set of hearts was reperfused with oxygenated Krebs-Hensleit solution and allowed to recover for 30 min. Light and electron microscopic changes of the myocardium were semi-quantitatively evaluated together with physiological (i.e., heart rate, left ventricular diastolic pressure, time to contracture formation) and biochemical (i.e., lactate dehydrogenase, LDH, release) endpoints. RESULTS Cardioprotective effects of BMS-180448 following ischemia/reperfusion consisted of a reduced rate of contracture formation, reduced LDH release, and enhanced recovery of contractile function during reperfusion (P < 0.05). Light microscopic evidence of myocardial damage was detected following 18 min of ischemia. Morphological changes in ischemic/reperfused hearts included interstitial edema, myofiber degeneration, and hypercontraction band formation. Ultrastructurally, swollen myofibrils, swollen mitochondria with disrupted cristae and electron-dense deposits, myofibrillar lysis, and contraction bands, were observed. Light and electron microscopic severity scores were significantly less (P < 0.05) in BMS-180448-treated hearts at the 25 min ischemic time point and in reperfused hearts, as compared to similarly-treated vehicle hearts. CONCLUSIONS BMS-180448 ameliorates morphological evidence of ischemia/reperfusion myocardial damage in the isolated rat heart model, in agreement with physiological and biochemical parameters.

Utilization of Electron Probe Microanalysis in Gadolinium-Treated Mice

Gadolinium is used as a contrast media for magnetic resonance imaging and, experimentally, to block Kupffer cell phagocytosis. In this study, we utilize electron probe microanalysis to determine the subcellular localization of gadolinium chloride (GdCl 3) administered to mice in a short-term toxicology study. Male CD-1 mice were administered 0.0, 2.5, or 8.0 mg/kg GdCl3 iv for 14 consecutive weekdays. Liver-associated enzymes were significantly elevated in high-dose animals only and correlated histologically with multifocal, hepatocellular degeneration associated with a neutrophilic infiltrate. Morphological investigations were performed on high-dose animals. Hepatocytes and Kupffer cells had morphologic features of cellular injury consisting of swollen mitochondria and vesiculated profiles of endoplasmic reticulum. Hepatocytes, Kupffer cells, bile canaliculi, and neutrophils in the liver contained discrete aggregates of electron-dense granular material, as did pulmonary interstitial macrophages, splenic macrophages, and mesangial cells of the renal glomerulus. The intracellular granular material in the liver, lung, spleen, and kidney was confirmed as gadolinium by qualitative electron probe microanalysis. These results document both hepatic and extra-hepatic accumulation of gadolinium in cells of the mononuclear phagocytic system and highlight the importance of electron probe microanalysis in toxicologic assessment.

Comparison of Acute Hepatocellular Proliferating Cell Nuclear Antigen Labeling Indices and Growth Fractions, p34cdc2 Kinases, and Serum Enzymes in Carbon Tetrachloride-Treated Rats

We evaluated various biomarkers associated with cell proliferation immediately following insult with the classic hepatotoxicant carbon tetrachloride (CCl4). Rats were administered a single necrogenic dose of CCl4 and euthanized at either t = 4, 8, 12, 16, or 24 hr postdose. Parameters evaluated included the following: immunohistochemical detection of hepatocellular proliferating cell nuclear antigen labeling indices (PCNA-LIs; percentage of cells in S phase) and growth fractions (PCNA-GFs; percentage of cells in the cell cycle); PCNA and the cyclin-dependent kinase p34cdc2 (CDK) protein in S-9 fractions by Western blot and enzyme-linked immunosorbent assay (ELISA); and liver-related serum enzymes. An increase in PCNA-GF was observed at t = 4 hr, concomitant with elevations in CDK and PCNA protein (Western blot). PCNA-LIs were increased by t = 24 hr, as were CDK and PCNA by ELISA. Sorbitol dehydrogenase was the most sensitive enzyme, with increases observed at t = 4 hr. Our results indicate that PCNA-GF, CDK, and PCNA levels reflect hepatocellular regeneration as early as 4 hr following CCl4 insult. We conclude that these assays are early and sensitive indicators of acute hepatotoxicity that may be advantageous to evaluate in the early stages of exploratory studies.

LARGE AREA SECTIONING FOR MORPHOLOGIC STUDIES OF NONHUMAN PRIMATE NASAL CAVITIES

The nasal region is important for studies in inhalation toxicology but is difficult to prepare for histological examination, especially in species as large as primates. A method for the histologic preparation of undecalcified, complete transverse sections of the nonhuman primate nasal cavity is summarized as follows. After removal of excess soft tissue, mandible and calvaria, the head is fixed in 10% neutral buffered formalin. The nasal region is transversely sectioned into serial 3-mm-thick blocks from the nares to the posterior aspect of the soft palate using a low speed saw with a water-cooled diamond-coated blade. The blocks are embedded in a mixture of glycol and methyl methacrylates, with polyethylene glycol-1500 and dibutylphthalate as plasticizers. The plastic blocks average 5.0 x 5.0 x 1.5 cm; 2-4 microns sections are cut on an automated sliding microtome. In spite of the size of the blocks, this technique yields complete transverse sections of the nasal cavities with excellent morphologic detail. The sections are amenable to a wide range of staining procedures. The procedure lends itself to autoradiographic studies. The embedding mixture is ideally suited for studies of undecalcified bone and teeth.