Scientific uses of animals: harm-benefit analysis and complementary approaches to implementing the three Rs.

The principles of humane experimental technique, first described by Russell and Burch in 1959, focus on minimising suffering to animals used for scientific purposes. Internationally, as these principles became embedded in the various systems of oversight for the use of animals in science, attention focused on how to minimise pain, distress and lasting harm to animals while maximising the benefits to be obtained from the work. Suffering can arise from the experimental procedures, but it can also arise from the manner in which the animals are housed and cared for. Increased attention is therefore being paid to the entire lifetime experience of an animal, in order to afford it as good a quality of life as possible. Russell and Burch were also concerned that animals should not be used if alternatives to such use were available, and that animals were not wasted through poor-quality science. This concept is being revisited through new efforts to ensure that experiments are well designed and properly reported in the literature, that all results--positive, negative or neutral--are made available to ensure a complete research record, and that animal models are properly evaluated through periodic systematic reviews. These efforts should ensure that animal use is truly reduced as far as possible and that the benefits derived through the use of animals truly outweigh the harms.

Report on the ILAR International Workshop on the Development of Science-based Guidelines for Laboratory Animal Care.

The Institute for Laboratory Animal Research of the National Academies hosted a meeting in November 2003 in Washington, DC, titled "International Workshop on the Development of Science-based Guidelines for Laboratory Animal Care." The purpose of the workshop was to bring together experts from around the world to assess the available scientific knowledge that can have an impact on the current and pending guidelines for laboratory animal care. Platform presentations focused on a variety of issues, from information exchange on mechanisms for the development of regulations across different countries and cultures, to data-based scientific studies on the effects of environmental enrichment on research outcomes. In the discussion sessions, participants were tasked with addressing the current scientific literature on the specific session topics; identifying gaps in the current knowledge in order to encourage future research endeavors; and assessing the effects of current and proposed regulations on facilities, research, and animal welfare. Participants had ample opportunities to share research outcomes and viewpoints in the multiple breakout sessions. Summaries of all breakout sessions were presented in the general session. On the final day of the workshop during the point/counterpoint session, a diverse group of speakers presented their cases for and against harmonization of standards. Although some of the speakers had serious reservations about harmonization, most of the panel members supported some form of harmonization. A positive outcome of the workshop was the opportunity for scientists and veterinarians from many countries to begin a dialogue with a goal of understanding the basis for the differences in regulatory approaches in laboratory animal care and the hope of continuing discussions on ways to work together toward some type of harmonization.

TestSmart-high production volume chemicals: an approach to implementing alternatives into regulatory toxicology.

This article examines the status and application of alternatives defined as replacements, refinements, and reduction for screening high production volume (HPV) chemicals. It specifically focuses on the Screening Information Data Set (SIDS), a series of toxicological tests recommended by the Organization for Economic Cooperation and Development to screen such chemicals. Alternative tests associated with acute, repeat-dose, genetic, and reproductive and developmental toxicity were examined at 2 meetings of academic, industry, and regulatory scientists and their status determined. Tests were placed in 1 of 3 categories: ready for immediate use, in need of or currently undergoing validation, or needing research/developmental work. With respect to traditional acute toxicity testing, the basal cytotoxicity approach was placed in the category of research with the up-and-down, fixed-dose, limit test, and the acute toxic class categorized as available for immediate use and the neutral red assay under validation. Cell culture methods that could provide information on acute target organ toxicity were all categorized in the research stage. Studies of the Ah receptor were placed under validation. All alternative tests for repeat-dose toxicity were placed in the category of research. With regard to genetic toxicity, the Ames, mouse lymphoma, and Chinese hamster ovary methods were considered ready for immediate use, while the in vitro micronucleus and Syrian hamster ovary assays were placed in the validation category. All alternatives for developmental toxicity, with the exception of gene chip technology, were placed in the category of validation. Gene chip technology is considered to be in the research stage. For reproductive toxicity, sperm motility and morphology were considered as ready for immediate use, with the other assays categorized as needing validation or in the research stage. Follow-up to these results is obvious. Work needs to be conducted to move those tests from the research stage to the validation and use stage. This is one approach to the development of alternatives to SIDS. Progress along these lines would apply not only to SIDS but also to toxicology in general.

Anterior approach for knee arthrography.

OBJECTIVE: To develop a new method of magnetic resonance arthrography (MRA) of the knee using an anterior approach analogous to the portals used for knee arthroscopy. DESIGN: An anterior approach to the knee joint was devised mimicking anterior portals used for knee arthroscopy. Seven patients scheduled for routine knee MRA were placed in a decubitus position and under fluoroscopic guidance a needle was advanced from a position adjacent to the patellar tendon into the knee joint. After confirmation of the needle tip location, a dilute gadolinium solution was injected. RESULTS AND CONCLUSION: All the arthrograms were technically successful. The anterior approach to knee MRA has greater technical ease than the traditional approach with little patient discomfort.

The TestSmart-HPV Program--development of an integrated approach for testing high production volume chemicals.

The TestSmart program was developed in response to the High Production Volume Chemical Challenge, a voluntary initiative under which chemical producers provide basic toxicity data on chemicals produced in greater than one million pounds annually. Specifically, under the Challenge, chemical producers will generate data as needed to complete the Screening Information Data Set (SIDS), as defined by the Organization for Economic Cooperation and Development (OECD). The TestSmart program is a collaborative effort of the Johns Hopkins Center for Alternatives to Animal Testing, the Environmental Defense Fund, Carnegie-Mellon University, and the University of Pittsburgh. The goal of the TestSmart program is to provide a humane and efficient approach to collecting SIDS data. The program has two objectives, one immediate and the other more long term. The immediate objective has been to make recommendations to reduce the number of animals used in collecting SIDS data under the Challenge. This was accomplished, through a group process, by examining the current status of alternative methods for SIDS endpoints and by providing an assessment of the "state of readiness" of current and potential future alternatives. The long-term objective is to provide a model for other programs to follow the TestSmart concept of a more efficient and humane approach to obtain toxicological data of interest to regulators and the public.

Mycobacterium chelonae: nonhealing leg ulcers treated successfully with an oral antibiotic.

BACKGROUND: Mycobacterium chelonae is an important human pathogen and should be considered when a physician is faced with nonhealing cutaneous wounds, including ulcers of the lower leg. METHODS: The medical literature was searched from 1965 to the present using the key words "Mycobacterium chelonae" and "leg ulcers." A case of Mycobacterium chelonae infection is reported. RESULTS AND CONCLUSION: Clarithromycin as single-agent oral therapy has been effective in treating these infections once the proper diagnosis is established. Diagnosis of M. chelonae infection requires being alert to this infectious agent and obtaining cultures for mycobacteria. Aggressive surgical debridement with direct excision of the wound might now be unnecessary because of the effectiveness of oral clarithromycin administered as a single oral agent.

The influence of flip angle on the magic angle effect.

OBJECTIVE: To assess the impact of flip angle with gradient sequences on the "magic angle effect". We characterized the magic angle effect in various gradient echo sequences and compared the signal-to-noise ratios present on these sequences with the signal-to-noise ratios of spin echo sequences. DESIGN: Ten normal healthy volunteers were positioned such that the flexor hallucis longus tendon remained at approximately at 55 degrees to the main magnetic field (the magic angle). The tendon was imaged by a conventional spin echo T1- and T2-weighted techniques and by a series of gradient techniques. Gradient sequences were altered by both TE and flip angle. Signal-to-noise measurements were obtained at segments of the flexor hallucis longus tendon demonstrating the magic angle effect to quantify the artifact. Signal-to-noise measurements were compared and statistical analysis performed. Similar measurements were taken of the anterior tibialis tendon as an internal control. RESULTS AND CONCLUSIONS: We demonstrated the magic angle effect on all the gradient sequences. The intensity of the artifact was affected by both the TE and flip angle. Low TE values and a high flip angle demonstrated the greatest magic angle effect. At TE values less than 30 ms, a high flip angle will markedly increase the magic angle effect.

Encephalocoele as a complication of intranasal sinus surgery: optimal evaluation with magnetic resonance imaging.

We report a case of post-operative frontal basal encephalocoele evaluated using a new magnetic resonance imaging (MRI) sequence, fast inversion recovery for myelin suppression (FIRMS). FIRMS was developed to enhance the differentiation between grey and white matter. In this case, the sequence was beneficial in distinguishing the encephalocoele from adjacent nasal mucosa and secretions.

Internal derangement of the knee after ipsilateral femoral shaft fracture: MR imaging findings.

OBJECTIVE: This study uses magnetic resonance (MR) imaging to delineate the types and frequencies of injuries seen in the knee after ipsilateral femoral shaft fracture. We also compare the results of the orthopedic knee examination with the MR findings. DESIGN AND PATIENTS: MR imaging of the ipsilateral knee was performed on 34 patients with closed femoral shaft fractures. Indications for knee MR imaging included knee pain at the time of fracture, soft tissue swelling or an effusion of the knee, or a positive knee examination under anesthesia. The patients had a mean age of 27 years and all were stabilized with intramedullary nails. Imaging was performed a mean time of 2.5 days after surgery. All patients had knee examinations done under anesthesia, and the MR results were compiled and compared with the clinical examinations. RESULTS: Ninety-seven percent of patients demonstrated knee effusions. Twenty-seven percent of patients demonstrated meniscal tears, with the posterior horn of the medial meniscus most frequently torn. The medial collateral ligament was the most frequent site of ligamentous injury (38%) followed by the posterior cruciate ligament (21%). Fifty percent of patients had injuries of the extensor mechanism. Bone bruises were noted in 32% of patients. Articular cartilage injuries were confined to the patella in four cases. One occult tibial plateau fracture and one meniscocapsular separation were seen. CONCLUSIONS: There is a common incidence of both ligamentous and meniscal injury to the knee after ipsilateral femoral shaft fracture. MR imaging can be useful in assessing the extent of injury, and may reveal findings unsuspected after clinical examination of the knee.

Cytochrome P450 isoenzyme activities in cultured rat and mouse liver slices.

1. The objective of this study was to determine the basal and inducible activities of several cytochrome P450 (CYP) isozymes and monitor the acinar and hepatocyte morphology in precision cut, cultured rat and mouse liver slices. 2. The slices were cultured up to 96 h in Chee's essential medium supplemented with insulin, transferrin, selenium, DMSO, dexamethasone and epidermal growth factor. A dynamic roller system was used to incubate the slices at 37 degrees C in an atmosphere of 95% O2:5% CO2. 3. Histopathology of the liver slices revealed maintenance of normal hepatic lobular architecture with time in culture. 4. CYP isozyme activities were measured at various times of culture. In rat liver slices, at 72 h, CYP1A1/1A2 activity was induced 4-fold by beta NF and 37-fold by dioxin (TCDD) whereas in mouse liver slices, 1A1/1A2 activity was not inducible by beta NF but was induced 19-fold by TCDD. At 72 h, CYP2A5 (coumarin-7-hydroxylase) activity was not detected in rat liver slices but in mouse liver slices, 2A5 was induced 2-fold by beta NF, 11-fold by phenobarbital (PB) and 3-fold by TCDD. 5. Hydroxylation of testosterone at specific positions was used as an indication of the activities of various P450 isoforms. Testosterone was added to the cultures at 0 and 72 h and the metabolites were measured at 24 and 96 h respectively by hplc analysis. Depending upon the species, the treatment and the time in culture, CYP1A, 2A, 3A, 2B and 2C activities were detectable. 3A activity was highly induced by PB in both rat and mouse liver slices. These results demonstrate that this culture system can be used to assess and compare xenobiotic metabolism in liver slices from rodent species.

Asbestos plaques in a typical Veteran's hospital population.

Previous authors have described several pleural abnormalities on chest radiology as being pathognomonic for asbestos exposure. We sought to determine the percentage of admissions and outpatients at a typical Veteran's Affairs hospital with these findings, and researched medical records to verify the frequency at which patients having positive radiographs were suspected either by clinical/occupational history or radiologically to have had prior significant exposure to asbestos. Radiographs of 1,212 consecutive patients were evaluated by a certified B reader, and the medical records as well as previous radiology reports of all positive patients were reviewed. Twenty-eight (2.3%t) of the radiographs had pleural abnormalities consistent with asbestos exposure, with the patients, all male, ranging in age from 50 to 98 years (mean 75.6). Radiology reports described pleural plaques in only 12 of the 21 (57%) cases with prior exams available; in only seven (33%) was an asbestos etiology considered by the interpreting radiologist. The plaques were misdiagnosed in four instances as being indicative of other, unrelated pathology. A history of known dust exposure was expressed by only five patients (18%). Eleven described working in occupations now known to have a high incidence of exposure, but neither patient nor examining physician expressed consideration of dust inhalation. In conclusion, we have found that a significant percentage of patients in certain subpopulations show radiographic evidence of asbestos exposure that may be a harbinger of related pathology. Unfortunately, because of a low index of suspicion, thorough environmental histories are often deferred, many radiographic changes are either not recognized or are misdiagnosed, and these patients are not followed with the stringent protocols they deserve.

Characterization of a primary hepatocyte culture system for toxicological studies.

An hepatocyte culture system was developed for potential use in toxicological studies in vitro. Rat hepatocytes were isolated by two-step collagenase perfusion and cultured on Vitrogen-coated Permanox dishes in a modified Chee's medium containing 1 microM dexamethasone and 1% dimethylsulfoxide. The cells remained highly viable for at least 10 d as determined by lactate dehydrogenase release and total protein levels. Albumin secretion into the medium, as a measure of differentiated function, was maintained at elevated levels over the course of 10 d in culture. A number of CYP activities were determined by the analysis of testosterone metabolism in freeze-thawed cells, diazepam metabolism in live cells, and specific assays for CYP 1A1/2, 2B1/2, 2E1, and 3A. Results of these assays indicated that a wide range of CYP isozymes were maintained, some activities were enhanced under the conditions of culture and some activities were inducible. Activities of the phase II enzymes, glutathione S-transferase and UDP-glucuronosyltransferase, and glutathione levels were also maintained in the cultured hepatocytes for at least 6 d. These results strongly support the use of this hepatocyte culture system for in vitro toxicological studies.

Rat hepatocyte morphology and function on lactose-derivatized polystyrene surfaces.

Hepatocytes isolated from male Fisher 344VF rats were cultured on two substrates, collagen I and a lactose-derivatized polystyrene (PS-lactose), to compare morphological and functional differences. Hepatocyte morphology changed dramatically depending upon the substrate, shown through actin cytoskeletal staining and scanning electron microscopy. Functional assays performed included albumin secretion, reduced glutathione content, UDP-glucuronosyl transferase, and cytochrome P4501A1 activity. The presence of dexamethasone and dimethylsulfoxide (DMSO) in the media was required for the maintenance of several differentiated functions for cells cultured on collagen. In general, cells cultured on the PS-lactose substrate showed a much slower loss of function over the same period of time. The maintenance of differentiated function of cells on PS-lactose was enhanced with the addition of dexamethasone and DMSO. This is the first report of a culture system in which hepatocytes, cultured on a polymer substrate without additional protein coatings or media additives, have been able to maintain differentiated functions for up to 1 week. (c) 1996 John Wiley & Sons, Inc.