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Johns Hopkins Bloomberg School of Public HealthCAAT

Research Grants 2003-2004

Summary of Research Grants

Telemetric Measurements of Effects of Post-Operative Analgesics
Gregory P. Boivin, DVM
University of Cincinnati, Cincinnati, Ohio
An essential aspect of a quality animal care program is the provision for post-operative analgesia. We now have the ability to determine evidence of pain directly in rodents by the use of radio-telemetry. Evidence of pain can be demonstrated by telemetric changes in blood pressure, heart rate, core body temperature, electrocardiogram and locomotor activity levels. In this application we have devised a series of studies utilizing radio-telemetry to help determine whether post-operative analgesia improves the well being of the mouse.

We have 2 Specific Alms in this application. The first aim is to determine whether preemptive analgesia (the administration of analgesics before pain starts) improves analgesic activity over post-operative administration of analgesics. To examine this we will test the hypothesis that a single dose of flunixin meglumine or buprenorphine given prior to surgery will provide better post-operative analgesia than a dose given post-operatively. The second aim is to deterrnine whether multiple doses of buprenorphine improves analgesia compared with a single dose. Because buprenorphine provides analgesia in the tail flick test for only 35 hours in mice, we believe that post-operative pain will only be partially relieved by a single dose of buprenorphine, and thus can alter biological function in the rodent. We thus hypothesize that multiple dosages of buprenorphine given at 4 hour intervals will provide prolonged and improved post-operative analgesia compared with a single dose of buprenorphine.

Results of this research will provide information for optimization of the use of postoperative analgesics in mice. This will be very beneficial in preventing pain in numerous mouse surgical protocols used nationally and intemationally.
Development and Use of an Immortalized Human Goblet Cell Line for the Use in Ocular Toxicity Testing
Darlene Dartt, PhD
The Schepens Eye Research Institute, Harvard University, Boston, Massachusetts
The conjunctival epithelium along with the cornea makes up the ocular surface. The conjunctiva has long been used as a component of the Draize test and mucin secretion from this tissue as one of its endpoints. To replace the Draize test, many laboratories have developed cell lines and primary cultures of corneal epithelial cells and conjunctival stratified squamous cells. No one, however, has been able to culture conjunctival goblet cells. The longterm objective of the present application is to develop a cell line of human conjunctival goblet cells with an extended life span and subsequently to use these cells for ocular irritancy testing. The specific aims are:

  1. Develop a cell line with and extended life span from human goblet cells in primary culture;
  2. Characterize the new cell line; and
  3. Determine the effect of neurotransmitters and growth factors on mucin and protein secretion from the cell line and the effect of ocular irritants on this secretion.

We recently established and characterized primary cultures of human conjunctival goblet cells from explants of surgically removed conjunctiva. For specific aim 1, we will attempt to transfect these cells with SV40 and telomerase. We will select the surviving colonies. After selection of telomerase positive cells, they will be expanded into cell line(s) and frozen down.

For specific aim 2, we will characterize the colonies of cells. We will first determine if the surviving cells retain their goblet cell characteristics by western blotting and immunofluorescence, microscopy. We will investigate whether these cells contain the intermediate filament keratin 7, the secretory mucin muc 5ac, terminal sugars on the secretory mucins that recognize the lectin Helix pornatia agglutinnin (HPA), and secretory product that stains with alcian blue/periodic acid Schiffs reagent. These are all specific markers of goblet cells. We will next determine if these cells contain keratin 4 and terminal sugars on the mucin secretory product that recognize the lectin Banderia, both of which are specific for the stratified squamous cells of the conjunctiva. We will select colonies that contain goblet cell markers. Colonies of cells will also be analyzed by electron microscopy to investigate if they contain large numbers of secretory vesicles, also indicative of goblet cells. Finally, we will determine the population doubling time of these cells with the goal of obtaining a 20 fold or more doubling capacity. For specific aim 3, we will use the newly created cell line of conjunctival goblet cells to determine the effect of neurotransmitters and growth factors on their secretion of mucin. Cells will be grown on glass coverslips in 6 well tissue culture plates. To measure secretion coverslips containing cells will be incubated with agonists for varying times from 15 to 120 minutes. The medium will be removed and the cells scraped and homogenized. Medium and cell homogenate will be analyzed for the amount of secretory product using an enzyme linked lectin assay with HPA lectin. We will test the neurotransmitters and growth factors that we have previously found to stimulate rat conjunctival goblet cell secretion. These results will be used as a baseline to then determine if known ocular irritants effect basal, neurotransmitter, or growth factor stimulated secretion.

The development of a goblet cell line with an expanded life span that secretes mucins and other antibacterial proteins is a useful model that could be potentially used as an alternative to the Draize test.
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 partially 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 used. Moreover, the system will allow us to test drugs, developed in our institute for efficacy and toxicity in vitro and 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
A major problem of humane experimental use of animals becomes evident as IACUCS review mouse use Protocols. Investigators who are asked, to provide post surgical supportive care after painful procedures, such as surgery, have relatively little information regarding doses, techniques; and effects Of supportive therapy. Unfortunately, evidence-based studies are comparatively rare. Our group has been studying postoperative behavior and weight changes in mice following surgery, and has developed a model to study interventions that might improve postoperative welfare. Briefly, we videotape singly housed mice in a standard setting, for 5 minute intervals every 3 hours for 48 hours. Using a camera that produces images during the mouse's active period (darknesss), we have demonstrated that typical mouse behaviors; such as reaching for the top of the cage, climbing on the roof of the cage and eating or drinking are markedly attenuated compared to control groups who have had anesthesia only. In addition, they spend significantly more time sleeping and experience weight loss of 8-12% over the initial 24 hours following surgery. We hypothesize that these aberrations result from pain or metabolic stress or both. Furthermore we hypothesize that interventions that reduce pain and that minimize metabolic stress will improve animal well-being as well as the validity of experimental models. Specifically, we hypothesize that the abnormal behavior and weight loss experienced by mice after surgery can be:

  1. rectified by analgesics, supportive therapies, or anesthetic regime and
  2. used as a tool to decipher the amount of stress caused by various experimental procedures performed routinely on mice, and thus to evaluate different techniques.

We will test these hypotheses by

  1. continuing the current work in order to identify one or more analgesic regimens that will reduce postoperative derangements in activity and weight in mouse models of laparotomy and or ovariectomy, and
  2. study of supportive therapies (early postoperative thermal support, fluid and nutritional support) to determine if the weight loss is responsive to non-drug therapies, and
  3. begin to study of five commonly used anesthetic regimens for mice (pentobarbitol, avertin (tribroinoethariol), ketamine-xylazine, isoflurane, and ketamine-isoflurane) in order to evaluate their effect on the postoperative course.

Further evaluation of combination techniques (i.e. anesthetic regimen plus analgesia plus supportive care) will determine optimal postoperative care of mice.
Genomic Approaches to Defining Pain and Distress in Mice
Norman C. Peterson, DVM, PhD
Johns Hopkins University, Baltimore, Maryland
A major objective of performing humane research on animals is to minimize or eliminate the painful experience of the subjects under study. In order to accomplish this objective, it is necessary to be able to identify if the animal is actually experiencing pain. This is often difficult in rodents as they have adapted protective strategies to hide potential weaknesses, and their normal activity levels are low during daylight hours when humans are working. Pain perception is influence by multiple genes, and if those genes (or responding genes) can be specifically identified, and their protein products quantified, an efficient analytical system of pain/distress detection in mice could be developed. Here, we propose to utilize high density microarray technology to identify those specific gene clusters in the mouse brain that are influenced by noxious stimuli. The results of these studies will then be used in other studies to further characterize different types of pain, to identify mouse strain differences in pain perception, and to characterize selected potential biomarkers for their application in the diagnosis of pain in mice.
Measures to Reduce Stress Caused by Experimental Procedures
Bert van Zutphen, PhD and Vera Baumans, DVM
University of Utrecht, Utrecht, The Netherlands
Stress can be described as the inability of an individual to maintain homeostasis, which can occur as a reaction to a (negative) physical or mental stimulus. Laboratory animals are frequently subjected to routine experimental procedures such as injections or withdrawal of blood samples. Each of these treatments influences the horneostasis of the experimental animal to a certain extent, and can, therefore, have an effect on the well-being of the animals and, as a consequence, on the study results. The aim of the four-year PhD-project is to investigate the possibilities of environmental measures to reduce the degree of stress as experienced by the animals (mice) subjected to routine experimental procedures. It is anticipated that relative simple measures like cage enrichment, social housing and conditioning of the animals will have a substantial impact on the stress response of the animals. If so, the implementation of such measures would be a refinement in the use of laboratory animals.

In this research project, the following questions will be tackled:

  1. What is the effect of the method of injection (e.g. subcutaneous vs. intraperitoneal) on the stress response (level and duration) of the animal?
  2. What is the effect of different physical and social environmental factors such as cage enrichment or group housing, on the stress response (level and duration) in an experimental procedure?
  3. What is the effect of the strain (genetic background) on the stress response of animals?
  4. Can the stress response be reduced by conditioning the animals (by means of a stimulus like a noise, an air-puff or a light flash) on the experimental procedure and/or by combination of the procedure with a reward?
  5. What is the effect of a stress response in an animal on its conspecifics; i.e. can stress in one animal induce stress in another animal that does not undergo any procedure but is present in the treatment room?

In the first year, an experiment has been performed that focused specifically on questions numbers two and three. In the second year, the effects of cage enrichment, handling, individual housing and strain will be investigated in more detail. Furthermore, we will focus on question four. It is hypothesized that an unpleasant, stressful procedure such as an injection will be less stressful when the animal can predict when it will happen (conditioning) and/or when it has a pleasant experience quickly afterwards (reward). In order to measure the stress experienced by the animals in this experiment, we will use radio-telemetry techniques for the stress-free measurement of heart rate and body temperature. Although radio-telemetry requires a surgical procedure to implant the transmitter, it is generally accepted as a refinement of animal experimentation. Furthermore, it reduces the number of animals used, as animals can serve as their own controls.

As far as we know, there has been no experience so far in using radio-telemetry to measure anticipation and/or stress during classic Pavlovian conditioning paradigms. Behavioral observations, however, have been extensively used to this purpose. We therefore want to support and validate the physiological parameters by behavioral observations using LABORAS, a device for the automatic registration of behavior of mice or rats. A combined device of LABORAS with the radio-telemetry system of DataSciences International is currently being developed and will be available in the near future.

As it will not be necessary to obtain organs for post-mortem examination after the conditioning experiments, the transmitter-implanted animals will not have to be euthanized. Therefore, the implanted animals can also be used in another experiment focusing on question number one. Experiments will be performed that compare different injection techniques to see if these techniques cause different levels of stress responses. If so, the less stressful methods can be advised to animal experimenters if both more stressful and less stressful methods are possible options.
Bovine Corneal Organ Culture: An ex vivo Model for Chemical Toxicity Tests
Keping Xu, PhD
Medical College of Georgia, Augusta, Georgia
The long-term goal has been to develop an ex vivo assay system to predict ocular irritation potential of test chemicals and consumer products. The objective of this proposal is to understand the role of growth factors released upon corneal injury in regulation of wound healing. We hypothesize that chemical or other injury induce the release of growth factors such as heparin-binding EGF-like growth factor (HB-EGF) from epithelial and hepatocyte growth factor (HGF) from stroma keratocytes and continue release of these factors is required for the recovery of corneal injury. In this study we will use porcine corneal organ culture as an experimental model (Xu KP, Li XF, Yu FX. Corneal organ culture model for assessing epithelia[ responses to surfactants. Toxicological Sciences 2000;58:306-314). The specific aims are:

  1. To determine if and at what concentrations a chemical cause the release of H13-EGF from epithelia[ cell and activation of c-MET (HGF receptor) in epithelial cells released. A variety of chemicals including detergents, alcohols, oxidants and solvents (such as propylene glycol) will be tested. We speculate that exposure of cultured corneas to a chemical causing minimal to mild ocular irritation should lead the release of H13-EGF and phosphorylation of c-MET while a chemical causing severe ocular irritancy will result in destruction of the cultured corneas.
  2. To determine if release of H13-EGF and HGF is require for the recovery using reagents specifically block HB-EGF and HGF activity.
  3. To determine whether exogenous HB-EGF and HGF prevent chemical from inducing cell death and enhance corneal recovery after chemical exposure. Our previous studies have proved the usefulness of corneal organ culture in assessing chemical toxicity.

The proposed study will provide a better understanding of molecular bases for corneal response to and recovery from chemical injury and several measurable ex vivo parameters much needed for developing an ex vivo model for evaluating the ocular initancy potential of chemicals and consumer products.