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Johns Hopkins Bloomberg School of Public HealthCAAT

Research Grants 2004-2005

Summary of Research Grants

Organotypic Culture of Cerebellar Slice for Studies of Developmental Neurotoxicity of Methylmercury
William D. Atchison, Ph.D.
Michigan State University, East Lansing, Michigan
Methylmercury (MeHg) is extremely toxic and among the most common forms of mercury found in the environment. MeHg causes prominent neurotoxicicity. The developing brain is especially sensitive to MeHg. Our goal is to understand the mechanisms responsible for this selective vulnerability of certain cells in the brain to MeHg. During development, neurons migrate from one region of the brain to another. This process, can be studied using cultures of brain slices of neonatal rat. Migration of these neurons involves the complex interactions between chemical messengers known as neurotransmitters which either excite, or inhibit electrical activity in the neurons. We propose to develop this system, in our lab for studies of developmental neurotoxicity with MeHg. This preparation can be used for studies of acute and semichronic exposure to MeHg, thus reducing the need for semichronic injections of neonatal animals with MeHg. Furthermore, more than one preparation can be made from a single rat brain, animal, again reducing the overall number of animals needed for a series of studies. Because this preparation maintains the normal intact and developing synaptic connections, it offers a greater advantage over cell culture systems in which the normal connections between neurons (known as circuits) are lost. Movement of cerebellar neurons (in particular, a group called the "granule cells") and concurrent measures of changes in calcium within the cell will be studied using confocal laser scanning microscopic measures in response to acute and sernichronic exposure to MeHg. These studies will be an important contribution to the understanding of developmental neurotoxicity with MeHg.
Assessment of Heart Rate Variability as a Measure of Postoperative Pain in Dogs
John Dodam, DVM, MS, PhD
University of Missouri College of Veterinary Medicine, Columbia, Missouri
Invasive diagnostic procedures or surgery bave the potential to result in significant pain in research animals. Unfortunately, animals are unable to cotmnunicate this discomfort effectively to caregivers. Thus, a noninvasive and reliable metbod of asmssment of pain would benefit animals used in research. The purpose of this investigation is to evaluate heart rate variability analysis as a way to assess postoperative pain in dogs. We hope that this noninvasive monitoring tool can be used to develop a reliable method of pain assessment in research animals, and that the use of heart rate variability analysis will lead to the development of effective and safe analgesic protocols for use in research and clinical patients. This study is co-funded by the Morris Animal Foundation and the Center for Alternatives to Animal Testing.
Development of an In Vitro System to Test the Effects and Toxicity of Anti-Inflammatory and Anti-Fibrotic Drugs in the Human Liver
Geny Groothuis, PhD
University of Groningen, Groningen, The Netherlands
Liver cirrhosis is a fatal liver disease and belongs to the top ten causes of death in the Western World. Cirrhosis is characterized by, among others, excessive deposition of connective tissue (fibrosis) leading to loss of liver function. To date, no adequate drug therapy is available due to the fact that the potential drugs show either lack of effect or serious side effects. The only effective treatment is liver transplantation. In addition, the pathogenesis of the disease process, being the onset and progression of fibrosis and its reversibility, are only partly understood. Research concerning the pathogenesis and the development of drugs is usually performed in animal experiments, which is accompanied by relatively high discomfort for the animals. In this project an in vitro system using liver slices will be developed in order to be able to investigate the pathogenesis of the disease without treatment of the animals, thereby reducing the number and the discomfort of the animals to be used. Moreover, the system will allow us to test drugs, developed in our institute for efficacy and toxicity, in vitro instead of in vivo, as is currently the case. In addition, such an in vitro system can be applied to human liver tissue, enabling us to study human specific features of the pathogenesis of liver fibrosis and cirrhosis. This will contribute to a safer first administration of newly developed drugs in man and to a reduction, replacement and refinement of animal experiments.
Reducing Postoperative Pain and Distress in Mice
Alicia Z. Karas, DVM
Tufts University, Boston, Massachusetts
Scientists are asked to describe how they will care for mice after procedures such as surgery, in order to minimize pain and distress. A major obstacle is that relatively little is known about drugs and techniques that are effective in mice. There is a critical need to discover and publish information regarding the extent of postsurgical pain in the mouse, and techniques that reduce pain or distress.

We study the effects of pain-relieving drugs, nutritional support, anesthetic techniques and environment on mice recovering from surgery. Our model uses bodyweight monitoring and behavioral observation using a video camera system that films in both day and night lighting conditions. We demonstrated that following surgery, mice lose a significant percentage of body weight and also show marked reductions in activities such as climbing on the cage top, rearing, eating and exploring. When a single dose of a pain-reliever is given prior to surgery, weight loss is minimized and behavior patterns become more normal. We believe that this indicates dug weight loss and reduced activity may be in part due to pain. We are now examining whether giving more than one pain relieving drug or dose, adding intensive fluid and nutritional support; or altering the environment can further improve well-being. We are also looking to see whether the type of anesthesia used contributes to improved outcome. The goal is to improve the well-being of mice used in science, increase the overall awareness of rodent procedural pain in science, and make the task of ensuring minimization of pain and distress a realistic objective for scientists.
Genomic Approaches to Defining Pain and Distress in Mice
Norman C. Peterson, DVM, PhD
Johns Hopkins University, Baltimore, Maryland
This project will utilize micro-chip technology to simultaneously monitor the expression of thousands of genes in mice brains in response to inflammatory pain delivered to a hind paw. By using this technology to develop a "pain stimulus gene expression map", we will be able to better characterize pain perception in mice. Ultimately, this will provide us with a means to more objectively identify conditions that cause pain and distress in laboratory animals, and to develop specific methods/reagents that inhibit or alleviate it in animals and people.
Measures to Reduce Stress Caused by Experimental Procedures
Bert van Zutphen, PhD, Vera Baumans, DVM, PhD, Margot Meijer, MSc
University of Utrecht, Utrecht, The Netherlands
Animals that are used in biomedical research are often subjected to routine experimental procedures such as injections or withdrawal of blood samples. These procedures may cause an imbalance of the animal's homeostasis ('stress'). Stress can have an effect on the well-being of the animals and is likely to influence study results, leading to less reliable results and an increase the number of animals needed. The aim of the present project is to measure the animal's stress response to experimental procedures and to investigate the options for refinement. It is hypothesized that the stress response not only depends on the experimental procedure per se but also on the animal's environment (e.g. social vs. individual housing; sawdust bedding vs. cage enrichment).

In this project, the impact of injection techniques in mice is studied. Parameters for measuring the stress response include heart rate, body temperature, body weight, behavior and corticosteroids. Radio-telemetry is used for stress-free collection of data on heart rate and body temperature.

In the first experiment performed in this project, we investigated the influence of cage enrichment, handling and social housing. The results of this experiment showed that individual housing may increase the stress response after injections, based on the slower recovery of tachycardia. Mice are social living animals and individual housing should therefore not be recommended. However, individual housing cannot always be avoided in experimental situations, and therefore we will now focus on the possibilities to reduce the stress response in individually housed mice. What will be the effect of cage enrichment in these animals? Would an increase in the predictability of the procedures by means of conditioning have a positive effect? Can the stress response be reduced when the experimental procedure is followed by a reward? Besides these questions, we will compare different injection techniques as to the degree of discomfort provoked by each of the techniques.
Use of Ultrasonic Vocalizations in Pain Assessment for Laboratory Rats
Daniel M. Weary
University of British Columbia, Vancouver, Canada
Laboratory rats are routinely subjected to surgical procedures, and it is important that they are provided with appropriate pain relief. However, in order to mitigate post-surgical pain in rats, we must first develop reliable methods of pain assessment. Promising pain assessment methods are currently under development for use with abdominal surgeries, but it may not be possible to generalize these methods to other types of surgeries.

Rats are known to emit high-frequency vocalizations in response to painful stimuli, and these vocalizations, can be reduced with the provision of analgesics. We will therefore investigate whether detailed vocalization analysis can be used as a general tool to assess post-surgical pain in rats. In our first experiment we will examine how rats' vocalizations change following a routine surgical procedure. In our second experiment we will provide animals with two dosages of analgesics to determine whether changes in vocalizations can be attributed specifically to pain. These studies will thus establish if rat ultrasonic vocalizations can be used as a reliable method of pain assessment in support of developing appropriate pain management regimes.