LAT Chapter 12 Research Methodology PDF

LAT Chapter 12 Research Methodology Any animal model has the potential to result in pain or distress. Remember that it’s your responsibility to advocate for the animals, while keeping research goals in mind. Report sick, painful or injured animals to the veterinary staff. Animal research encompass...

LAT Chapter 12 Research Methodology Any animal model has the potential to result in pain or distress. Remember that it’s your responsibility to advocate for the animals, while keeping research goals in mind. Report sick, painful or injured animals to the veterinary staff. Animal research encompasses many kinds of studies that aim to find new cures or verify product safety. Laboratory animal technicians benefit in having a basic knowledge of common research methods to better support the often-specialized husbandry procedures necessitated by the research, to monitor and anticipate health changes in the animals caused by a study, and to communicate with investigators about their animals. Laboratory animal technicians often participate in the conduct of a study or test, assisting with technical procedures and the collection of data. Toxicology • In the broadest sense, toxicology is the science of poisons, and the harmful or noxious effects these substances have on living organisms. Studies are designed to obtain data by assessing the toxicity of substances administered to animals. • This data helps toxicologists make predictions about the hazardous nature of the substances tested and their potential impact on the environment and on human and animal populations. Toxicologists are multidisciplinary scientists • who are familiar with pharmacology, biochemistry, pharmacokinetics, physiology, inorganic and organic chemistry, and cellular and molecular biology. • Toxicology tests can be categorized in multiple ways. One way is by the timeframe of the exposure to the toxic substance:     Acute Subacute Subchronic Chronic • Acute test is the administration of a single dose of the toxic substance. • Subacute, subchronic, and chronic tests: the substance is administered in multiple doses, or as an extended exposure for increasing durations. • Toxicology categorization: Another way to categorize toxicity tests is by the functions or organs targeted within the body where the toxic substance exerts its effect. • Examples are: • Mutagenesis, referring to the induction of mutations in the DNA • Developmental, referring to the damage of a growing fetus • Carcinogenesis, referring to the cause of cancer • Some toxicology tests focus on a type of tissue, such as the eye or the respiratory tract. • Each type of test uses specific animal models and experimental criteria that are based on standards in the field, the needs of the research, and the regulatory requirements overseen by the Food and Drug Administration or the Environmental Protection Agency. These tests are useful in classifying toxic agents, protecting people and animals by identifying safe exposure levels, and selecting appropriate doses for longer term toxicity testing. Acute Toxicity Studies • Acute toxicity studies involve exposure to a toxicant, often in a single dose, for a short period, usually less than 24 hours. • Most acute toxicity studies for drugs require rodents or rabbits, but fish are often used for determining chemical safety of substances such as pesticides and industrial chemicals. • The route of exposure is usually chosen to reflect conditions under which humans may encounter the test substance. • Historically, acute toxicity was related to the LD50, which means the median lethal dose of a substance, which is the amount of a substance required to kill 50 percent of a test population. In recent years, acute toxicity testing has undergone significant design changes. The Organization for Economic Co-operation and Development (OECD), of which the United States is a member, codified regulatory guidelines for acute toxicity testing. The OECD guidelines replaced the LD50 test with alternative methods that use fewer animals of only one sex (usually females) and provided toxicity information to estimate the LD50. The OECD methods determine exposure ranges for a test substance in which lethality is likely. These alternative methods were inspired by the 3Rs – to reduce the number of animals used and to refine methods for improvement of animal welfare in toxicity testing. Acute Toxicity Studies Acute studies may be used to determine the irritancy and cytotoxicity of a test substance involving a brief exposure. • Irritancy: refers to causing irritation, typically of the skin and eye. • Cytotoxic: means toxic to cells. • Draize Test: the application of the test substance to the epidermis or the corneal surface of an animal, most commonly a rabbit. On the skin, the timeframe may be 24 hours. On • the eye, the exposure is for seconds. • Draize Test Controversy: due to the perceived cruelty of this test, and studies demonstrating its subjectivity and lack of reproducibility, the Draize test has become less frequently used. • Alternatives to the Draize Test: A variety of tests have been established that assess the same criteria as the Draize test – cytotoxicity, skin and corneal irritation, corrosivity, and inflammation. • Ex vivo (meaning “outside an animal”) testing of isolated rabbit and cattle eyes - useful, but these models lack important factors like tear production and endogenous inflammatory mediators. • In vitro testing, Three-dimensional models of the cornea and the skin, have correlated well with the Draize test and have been extensively validated. • Despite the success of these ex vivo and in vitro tests, the complexity of both the skin and eye make developing models of acute toxicity challenging. Subacute, Subchronic, and Chronic Toxicity Studies • Subacute, subchronic, and chronic studies are performed to investigate the effects of repeated administrations or prolonged exposure to a substance. • Subacute studies are often 28 days (1 month) in duration. • Subchronic studies typically last 13 weeks (3 months), approximating 10 percent of the life span of a rodent. • Chronic studies are designed to provide information on the effects of repeated administration of a substance over a larger portion of an animal’s lifespan and may run 26 weeks (6 months), 38 weeks (9 months), or 52 weeks (1 year). • The species and study methods required are determined according to the type of substance tested. Global regulatory testing guidelines generally require studies in one rodent and one nonrodent species. Rats, dogs, or nonhuman primates are the preferred non-rodent models. However, these species may not be appropriate for some biotechnology products. In these cases, the appropriate species must be selected based on the specific drug action or tissue specificity. • Whenever possible, the route of administration, the formulation type, and the dose regimen should mimic the intended or expected route of administration in humans. These studies are lengthy, complex to perform, and require much time to generate the data and review the results. Subacute, Subchronic, and Chronic Toxicity Studies • The number of animals included in subacute and subchronic studies is based on the need for a statistically relevant population size, as well as the ethical motivation to minimize the number of animals. • Groups. • In these studies, there is one group of animals per dose level, plus one group for controls. • Often there are 4 test groups in a study. • The number of animals per sex in a subacute study group is typically 10 rodents or 2 to 3 non-rodents. • Satellite and Recovery Animals. Additional animals per group may be needed for necropsies midway through the study, as satellite animals, or for evaluation of a recovery phase, as recovery animals. • Satellite animals (4 to 5 per sex, per dose) are administered the substance solely for the blood samples needed to determine the toxicokinetics of the substance— that is, how it is absorbed, distributed in the body, metabolized, and excreted. • In some studies, recovery animals are used to assess the reversibility of any changes from toxic effects after dosing is completed. Often these animals are used to evaluate recovery from the effects of a high dose. The use of recovery animals adds 5 rodents or 1 to 2 nonrodents research animals per sex to the study. Toxicology – Subchronic & Chronic studies • Subchronic studies typically require 15 rodents or 3 non-rodents per sex per group. • Chronic studies require 20 rodents or 4 nonrodents per sex per group. • In both subchronic and chronic studies, satellite groups may be needed, which would further increase the number of animals used. Altogether, a series of toxicity tests, from acute to chronic, may use thousands of animals to test a single substance. • Given how so many animals are needed, much attention has been given to alternative methods to reduce the number of animals used in toxicity studies, again inspired by the 3Rs. • For example, in some studies, blood microsampling techniques can eliminate the use of satellite animals for the purpose of toxicokinetic analyses. Microsampling techniques for blood sampling would eliminate all the satellite animals – a potential reduction of 24 rats. Toxicology - Dosing Phase • During the dosing phase of these studies, data are collected from clinical observations, including animal body weights and food consumption. • At the end of a study’s dosing regimen, study animals are euthanized, necropsies are conducted on the test groups, and subsequently, on recovery animals. • Organ weights are determined, and tissues are evaluated for evidence of toxicity using histopathology, ophthalmoscopic exams, and clinical pathology (hematology, clinical chemistry, urinalysis). Other tests may be included if warranted. Reproductive & Developmental Studies • Reproductive and developmental toxicity studies are performed to investigate the effect of a test substance on the fetus at various stages of the reproductive cycle and gestation. • Teratogen: A substance that damages a developing fetus. • In these studies, the substance’s formulation and its route of administration are selected based on the expected exposure in humans. Multiple doses are commonly given in these studies, or exposure extends over multiple days. The study protocol accounts for existing data on toxicity, pharmacodynamics, kinetics, and the chemistry of similar substances Reproductive & Developmental Studies • Protocols designed to evaluate these areas of interest are generally divided into three types:  Segment I (reproductive toxicity in male and female animals)  Segment II (fetal toxicity)  Segment III (toxicity in newborn pups) • Each segment overlaps another in time so that some processes, like fetal organ development, can be evaluated in more than one study. • Segment I studies are typically conducted in rodents but can be conducted in rabbits, minipigs, or nonhuman primates. These studies are designed to detect changes in male and female gametes, mating performance, and the fertilized ova until implantation. The substance is administered to cycling females and sexually mature males. The time frame for administration starts two weeks prior to mating for both sexes and continues in the female until ova implantation is complete. • Segment II studies are performed to evaluate fetal toxicity, typically in a rodent and a non-rodent species. Pregnant females are administered the compound throughout the period of fetal organ development, which is generally defined as the interval of time between ova implantation and closure of the fetus’ palate (roof of the mouth). • Segment III studies are designed to evaluate the effect of a substance when administered through the stages of gestation, parturition, and lactation. The substance is administered to females, typically rodents, from late pregnancy through parturition and lactation. Observations include gestational changes, dystocia, postnatal mortality, and impairments in maternal behavior and pup development. At weaning, one male and one female pup are selected from each litter to mate and produce an F1 generation. These F1 pups are followed up to sexual maturity with assessments including behavioral testing. At sexual maturity, they are mated to evaluate their fertility. Pyrogen Tests • Pyrogen: A substance that produces a fever, such as bacterial toxins. • A pyrogen test is used to detect bacterial toxins in medical products, like intravenous fluids, that are administered by injection. • Historically: The test involved administering the product intravenously to several animals, such as rabbits or guinea pigs, then recording their body temperature periodically for several hours. A rise in body temperature in one or more animals indicates the presence of a pyrogen in the sample. • Alternative methods have largely replaced the use of mammals in testing for pyrogens. • Limulus amebocyte lysate (LAL) test : Uses the hemolymph of the horseshoe crab (Limulus). Hemolymph is a fluid in invertebrates that is equivalent to blood. • • • • The hemolymph of this species contains immune cells that are extremely sensitive to the presence of bacterial toxins. These primitive immune cells, called amebocytes, are taken from the horseshoe crab and mixed with the test substance. These cells clump together in the presence of a bacterial toxin. The Limulus amebocyte lysate (LAL) test, serves as an alternative to the use of rabbits for the detection of some types of pyrogens in medical products, although it is sometimes used in combination with rabbit assays. • Monocyte activation test: Uses a cell line of immortalized immune cells. These cell line tests provide a refinement to either in vivo pyrogen tests or the LAL test, as they can detect both nonbacterial pyrogens and a wider array of bacterial toxins. Immunodeficiency Models • Immunodeficient animals lack one or more cell types of a normal immune system, giving them a deficiency in immune function. • Eg: RAG-1 deficient mice lack both mature B and T cells. • Immunodeficient animals may make good research models of spontaneous or infectious diseases, such as AIDS in humans. These animals are also used as models in immunology and cancer research because these defects have contributed to an understanding of how the entire immune system functions. • Immunodeficient animals provide a convenient method of keeping various tumor cell lines alive because they can serve as living hosts for the cell lines. • Why? No immune system = no transplant/tissue rejection • Special Housing: Immunodeficient animals are incapable of producing a normal immune response to microbes. Housing these animals in barrier caging is necessary to protect them from infectious microorganisms. Immunodeficiency Models Spontaneous Immunodeficiency Mice are commercially available with a variety of spontaneous immune defects, such as deficiencies in lymphocytes, macrophages, or hematologic factors. Animals are also available with autoimmune diseases caused by their genetic makeup. Immunodeficiency Models – Nude Mice • Nude mice are a widely used genetically immunodeficient animals. Genetic T cell deficiencies also have been found in rats and hamsters, and these animals are used for similar studies of immune function and disease. • In mice, the nude mutation causes both hairlessness and the lack of a thymus gland. Other animals lack T cells but can have fur • No thymus = no development of T cells, a type of lymphocyte responsible for attacking viruses and tumor cells and for helping other lymphocytes react to antigens. The role of T cells in triggering the response of other immune cells is known as cell-mediated immunity. • No immune system = more susceptible to infections that normal mice could easily resist. “nude” mice are homozygous for the Foxn1nu, or “nude,” mutation. Foxn1 encodes a transcription factor required for both hair follicle and thymic development. In its absence, mice are both hairless and athymic. Because the thymus fails to form, there is no place for CD4+ and CD8+ T cells to differentiate and mature, making nude homozygotes T celldeficient. Immunodeficiency Models - SCID Mice • Several strains of mice have been produced that carry a mutation resulting in severe combined immunodeficiency disorder (SCID). • Originally a spontaneous mutation in mice of the BALB/c strain, this inherited genetic defect causes both T cells and B cells to be absent. • These animals cannot mount either a cell-mediated or an antibody response to infection. • These animals can produce other immune system cells, like monocytes and granulocytes, but the lack of T- and B cells leaves them extremely susceptible to infection. • Lessened immune response = an excellent model for investigating cancers. Cancer cells can be implanted in SCID mice without being rejected and destroyed by the immune system. Immunodeficiency Models - Others • Hereditary B cell defects occur in CBA/N mice, and various strains of asplenic mice (mice that genetically lack a spleen). • Beige mice lack natural killer (NK) cells. Like T cells, NK cells are cytotoxic and kill other cells. • Other genetic immunodeficiencies include defects in macrophages in mice and in the complement proteins (small proteins in blood that play a role in the antibody-mediated immune response)of guinea pigs, mice, and rabbits. Induced Immunodeficiency • In addition to genetic manipulation, immunodeficiencies can be introduced into animal models by means of surgery, exposure to chemicals, irradiation, and through induced tolerance. Induced tolerance will not be discussed here. Induced Immunodeficiency Surgically Induced Immunodeficiency • The thymus gland can be removed surgically from newborn mice and rats, thus depriving them of T cells. • This operation may be performed by skilled laboratory animal technicians. Induced Immunodeficiency Chemically Induced Immunodeficiency Chemotherapy • A variety of agents can be used to suppress immunity, including 6mercaptopurine, cyclophosphamide, 5-fluoro- 2-deoxyuridine (FUDR), and actinomycin-D. • They interfere with protein production by interrupting DNA or RNA synthesis. Thus, they suppress antibody production and functions of cell mediated immunity. • These drugs are all relatively toxic, so proper training in working safely with hazardous chemicals is essential. Technicians should wear gloves and respiratory protection if warranted. • Animal deaths can be expected when immunosuppressant drugs are administered, so the animals must be carefully monitored for any signs of health problems. Induced Immunodeficiency Irradiation-induced Immunodeficiency Irradiation provides a method of inducing immunosuppression that is faster and more easily controlled in comparison with other methods. • • High-energy radiation suppresses cell division. Physiological functions that involve cell division, like antibody formation, are easily damaged by radiation. • Gamma Irradiator: The most common irradiation equipment for experimental purposes is a gamma irradiator which uses cobalt or cesium isotopes as the source of radiation. • X-ray source may also be used. • Specially trained operators are necessary to handle irradiation equipment, and a radiation safety program is essential for these procedures. • Three types of radiation exposure are used to induce immunodeficiencies: • • • Single exposure radiation: Total body radiation in a single exposure is the easiest method to define and interpret. Extensive literature is available detailing the effects of various levels of radiation, up to lethal doses. Low-dose semi-continuous radiation: Exposures are intermittent, which permits cage cleaning and animal feeding. Partial-body radiation: Partial-body radiation is accomplished using lead shields to protect certain parts of the body, limiting the animal’s radiation exposure. Induced Immunodeficiency Transgenic and Knockout Technologies • The ability to produce animals with genetic immune deficiencies is a valuable research tool. • Genetic engineering has enabled researchers to create mice with specific immune system genes either turned off, overexpressed, induced, or conditionally expressed. • These models are essential in understanding disease related to hereditary and acquired immune disorders. Caring for Immunocompromised Animals • Immunocompromised animals present special challenges for the laboratory animal technician. • Structural and procedural barriers, such as positive pressure rooms and the use of PPE and cage-change cabinets, must be in place to help prevent the transmission of infectious agents to these animals. • Microisolation-type cages or individually ventilated cages are frequently used. • Water is either acidified at pH 2.4 to 2.8, or chlorinated to suppress bacterial growth and prevent infection. • Feed, bedding, and cages must all be sterilized before use. • Animal biosecurity and Bioexclusion procedures must be carefully followed, both for the sake of the experiment and for the welfare of the animals. • The animals themselves frequently require extra attention. They may have a poor appetite and have difficulty eating and drinking. They usually take much longer to recover from wounds or ailments than normal animals. Antibody Production • One major area of study with laboratory animals is the production of antibodies and other components of the immune system. Antibodies can be used for basic research studies and to study diseases such as cancer. • Antibodies: Proteins that are produced by lymphocytes in response to exposure to foreign substances • Antigens: Substances foreign to the body, such as bacteria, viruses, plant pollen. • • • Bacteria, viruses, plant pollen, and toxins are substances that can produce an antibody response, so can be considered antigenic. In simple terms, antigen X stimulates the production of anti- X antibody; antigen Y stimulates the production of anti-Y antibody, and so on. Anti-X antibody reacts with antigen X, and likewise, anti-Y antibody reacts with antigen Y. Neither antibody reacts with the other antigen. • Immunized animals are used to evaluate the effectiveness of specific vaccines. • Cells of the immune system, such as lymphocytes and macrophages, are routinely collected and studied to investigate their role in defense against infections, disease, and tumors. • The antibody produced in response to immunization is often used as a critical component of a non-animal laboratory test, such as in the immunoserology tests used to diagnose many diseases in humans. Antibody Production Polyclonal Antibodies Polyclonal antibodies (pAbs) are antibodies that are secreted by different B cell lineages within the body (whereas monoclonal antibodies come from a single cell lineage). They are a collection of immunoglobulin molecules that react against a specific antigen, each identifying a different epitope. • Antigens have multiple unique regions on their surface that stimulate the production of different antibodies. When an antigen is injected into an animal, the immune response generates multiple antibodies; each recognizing a different area on the surface of the antigen. Cells that produce antibodies are known as plasma cells, which are derived from B cells. Each plasma cell and all its progeny, which are called its clones” produce a single type of antibody. The multiple types of antibody molecules that make up the response to the antigen are from several (hence “poly”) clones. Thus, serum from animals making a typical antibody response to an antigen contains polyclonal antibodies—derived from clones of many different antibody-forming cells. • Virtually any laboratory animal species can be used to produce antibodies. Because of their larger size and the ease with which their blood may be collected, rabbits, sheep, and goats are commonly used for antibody production. • How? • • • • • First, a pre-immunization blood sample is collected to ensure the animal does not already have antibodies that may complicate the study. Animals are then given a series of injections of an antigen preparation. In all cases, maintaining the sterility of the solutions and injection materials is essential. About 3 weeks after the series of injections, blood is again collected to test the serum for the presence of antibodies. This procedure is known as a test bleed. The amount of antibody in the blood is called a titer. {tie-tr) If the titer is not adequate for research purposes, then more antigen injections are given. After the first antigen injection, successive administrations are known as boosters. • If an animal’s antibody response is exceptional, it may be kept at the animal facility for a long period of time, sometimes years. During this time, the animal will receive occasional booster immunizations and have its blood collected periodically. This will provide the investigator with a continuous supply of an essential antibody. Antibody Production Polyclonal Antibodies • Some antigens are poor stimulators of antibody, and occasionally some animals do not respond well to antigen stimulation. • To stimulate a stronger antibody response, substances called adjuvants are mixed with the antigen before administration into the animal. • Adjuvant: a substance that enhances the body's immune response to an antigen, Adjuvants directly stimulate immune cells to produce antibodies, and they prolong the absorption of the antigen from the injection site. Antibody Production Polyclonal Antibodies • Several adjuvants are commercially available. Common ones are: o Freund’s complete adjuvant (FCA), o Freund’s incomplete adjuvant (FIA), o TiterMaxR, o TiterMaxR Gold, o MPLR, o AlhydrogelR. Polyclonal antibodies (pAbs) are antibodies that are secreted by different B cell lineages within the body (whereas monoclonal antibodies come from a single cell lineage). They are a collection of immunoglobulin molecules that react against a specific antigen, each identifying a different epitope. • Freund’s complete adjuvant (FCA) has gained the reputation for producing antibodies the most consistently, but it causes side effects in animals, such as granulomas and ulcerations at the injection site. The formulation of FCA includes killed Mycobacteria (the bacterial agent that causes tuberculosis), so primates injected with FCA will develop a positive reaction to subsequent TB tests. The USDA lists the administration of FCA as a painful procedure and gives IACUCs the responsibility to minimize animal pain. Therefore, institutions typically have guidelines for the use of FCA in animal studies. • Technicians handling these products, particularly FCA, should be very careful not to accidentally inject themselves or splash the material into their eyes, since exposure to the adjuvant can elicit a severe allergic response. • Freund’s incomplete adjuvant (FIA) does not contain Mycobacteria cells and, therefore, causes fewer and milder side effects than does FCA. For this reason, the usual immunization schedule for adjuvant administration involves giving the first injection using FCA and subsequent immunizations using FIA. • Most adjuvants are locally irritating, and some can cause open sores at the sites of injection. • It is generally considered unacceptable technique to inject adjuvants intraperitoneally, in weight-bearing areas such as foot pads, or in any location in volumes larger than 0.5 mL. Even smaller injection volumes are frequently suggested for most laboratory species. • Small, multiple-site injections are ideal for producing an adequate antibody response and may be better for the health of the animal than larger single-site injections. • After a series of immunizations, animals immunized with antigen/adjuvant mixtures may show signs of illness, such as reduced feed intake. Antibody Production Monoclonal Antibodies • Unlike polyclonal antibodies, monoclonal antibody production yields antibodies that recognize only one specific portion of the antigen. • The procedure consists of • immunizing mice (or sometimes rats) with an antigen. • Following immunization, the animal is test bled, as previously described. • If the animal is producing the desired antibody, the immunized animal is then euthanized, and the plasma cells from its spleen are isolated and maintained in small individual growth chambers. • Collecting the plasma cells that produce the antibody of interest, and then culturing a single clone means a large amount of the antibody can be produced. • Each plasma cell produces just one specific antibody. By isolating and reproducing the selected plasma cell, scientists have a source of antibody that is uncontaminated with other antibodies or serum components. • Usually only about 2% of the cells cultured will be the ones that produce the antibody of choice, so the culture must be purified using a series of techniques to isolate the correct clone. Antibody Production - Hybridomas • The goal of monoclonal antibody production is to expand and maintain the cell clone and collect its antibodies. Unfortunately, plasma cells do not live long enough in culture to produce antibodies for research. To solve this problem, a type of cancerous B cell is fused with a plasma cell to create an immortal cell line. • Cancer cells reproduce easily both in vitro (in cell culture) and in vivo (in an animal). When the cancer cell is fused with a plasma cell, the hybrid cell or “hybridoma” has the properties of both parent cells: good growth and survival rates from the cancer cell, plus production of the desired antibody from the plasma cell. • Large quantities of these hybridomas can be grown both in vitro and in vivo and may be maintained indefinitely. • The in vitro technique involves growing large numbers of hybridoma cells in a special nutrient fluid called a medium. As these cells grow, they secrete antibodies into the medium; the antibodies are then purified from the medium, which may then be collected and purified. • The in vivo technique involves injecting the hybridoma cells into the peritoneal cavity of mice, where the cells grow rapidly. Inside the abdominal cavity, the hybridomas grow rapidly, producing antibodies in the fluids that accumulate, which are called ascites. • • • • • • • The antibody-rich ascites fluid can be collected by inserting a needle into the abdomen and allowing the fluid to drip into a collection tube, or by aspirating it with a syringe. To prevent them from suffering due to excess accumulation of fluid in the abdomen, animals used in ascites production must be monitored frequently, up to several times daily once fluid accumulation begins. Fluid should be aspirated before abdominal distention interferes with respiration. Research facilities have guidelines for how many times fluid can be collected from the mouse prior to euthanasia. In addition, institutions should follow the Report of the Committee on Methods of Producing Monoclonal Antibodies, published by the Institute for Laboratory Animal Research. This report has recommendations that address the health and welfare of mice used in producing monoclonal antibodies. The development of ascites in this procedure is considered painful and distressing, so this use of mice must be scientifically justified to the IACUC if the procedure is permitted by your institution at all. Not all antibodies can be produced in sufficient quantities by the in vitro method, but new technology may increase the yield and reduce the need for the more invasive in vivo method. As the hybridoma is a type of cancer, animals may become sick as the disease spreads via tumor metastasis. Since metastasis is undesirable for the procedure, as well as for animal wellbeing, sick animals should be euthanized. Cancer Models • There are many types of cancers that affect humans and other species. Acquiring knowledge about the cause, progression, diagnosis, and treatment of these diseases is a common research goal. • Animals are frequently necessary for such studies, especially when causes, diagnostic procedures, and new treatment methods are being evaluated. • Animal models of cancer are generally classified into two types: induced and spontaneous. I • n both spontaneous and induced cancer studies, close observation of the animals is necessary, so the size of the tumor or the extent of its spread through the body (metastasis) can be evaluated. • Frequent monitoring is important in these studies to ensure that the extent of tumor burden does not cause unnecessary pain or distress and does not exceed an established humane endpoint. • Humane endpoints are an integral part of cancer models and are defined in either the animal use protocol or institutional policy. Typically, humane endpoints are specified by clinical signs, such as listlessness, loss of appetite, weight loss, or when tumors reach a predetermined size threshold or form open sores on the skin. When humane endpoints are reached, affected animals must be euthanized. Induced Cancer Models • Cancer can be induced in a group of laboratory animals to track the progression of the disease. • The method of induction depends on the type of cancer being studied. • For example, cells from immortalized cancer lines, or those derived from human tumors may be injected into the animal's body, or a cancer-causing chemical may be applied onto its skin or mucous membranes. • When evaluating new treatments or preventive drugs using research animals, untreated (control) animals are maintained throughout the study for comparing data between test and control animals. • Technicians involved in the handling of cancer cells or carcinogenic chemicals must be aware of the potential dangers and be appropriately trained. Always follow proper safety precautions to avoid contamination of the workplace, which could expose yourself and others to the carcinogens. Spontaneous Cancer Models • Spontaneous models are useful for evaluating diagnostic and preventive treatments in a large population known to be susceptible to this cancer. Some strains of inbred rodents have an unusually high incidence of naturally occurring cancers. • More than 80 percent of AKR mice develop leukemia before the age of one year. • Spontaneous tumors can also be transplanted from one inbred animal to another of the same strain. In these animals, the cancer reproduces without the immunologic rejection of the tumor that would occur in outbred animals. Behavioral Studies • What animals are used? • • • • Rats are the most frequently studied laboratory animal in behavioral research because of its convenient size, ready availability, low cost, and the vast amount of literature available on its biology and behavior. Mice are used for in neurobiology and neurobehavioral research due to the ability to manipulate their genome Cats are sometimes chosen for neurophysiological experiments because of their size and their well-mapped neuroanatomy, although their use has greatly declined in recent years. NHPs are used due to their close phylogenetic relationship to humans. Data obtained from these animals is valuable for human neurobiology and health. Rhesus monkeys (Macaca mulatta) are the most commonly investigated Old World monkeys in behavioral research, although the cynomolgus (M. fascicularis) and other macaque species (M. nemestrina and M. arctoides) are also studied. Of the New World species, the squirrel monkey (Saimiri sciureus) is the most common model for behavioral research. • Often in behavioral studies, the researcher teaches an animal to respond to a stimulus or to solve a problem for a reward. The reward is something received as a result of behavior—often a food treat, water, or juice. If an animal repeats a desired behavior to acquire a reward, the process is known as positive reinforcement. The reward reinforces the behavior. • Motivation may also result from the animal’s desire to avoid an unpleasant stimulus, such as an electric shock. In such studies, the shock is called an aversive stimulus. If the animal learns to avoid the aversive stimulus, the process is known as negative reinforcement. To further motivate the animal to perform a task and obtain a reward, feed or water may be restricted prior to testing. The extent of feed or water restriction in such experiments must be carefully outlined in the approved animal use protocol and strictly monitored by research or animal care staff. Animals may be on a continuous restriction of food or water, in which they receive a slightly reduced portion of a typical ration. Alternatively, feed or water is withheld for 12–24 hours prior to behavioral testing or training, then offered as the reward during the session. A careful record is kept of the amount of feed or water taken during the training session. If the amount taken during training is less than the calculated daily need, the animal is provided the difference at the conclusion of the training session. Careful records are kept of the animal’s weight and are analyzed by veterinarians at regular intervals. Animals are often given “vacations” from feed and water restriction several times per year. • An animal learns constantly, not just when under observation by an investigator. Most animals spend more than 90 percent of their time unobserved. During this time, many extraneous variables could be introduced that might interfere with the experimental results. Any type of stress, including noise, improper handling, abrupt changes in feeding, or the introduction of new animals, should be avoided for animals in behavioral research studies. Dietary Studies • Dietary changes can induce changes in health, growth, behavior, body conformation, and reproduction. • Various assays, diagnostic clinical chemistry, histology, and other measurements are used to evaluate the animals’ response to these dietary changes. • Dietary studies may be performed using any animal species, including humans. The choice of species depends on the purpose of the study and the resources available. Studies of the dietary needs of dogs, cats, and rodents commonly use the species for which the information is desired in order to avoid errors in cross-species interpretation. • Some dietary studies are performed to gain knowledge applicable to many species. For these purposes, the rat is the most frequent subject animal because of its ready availability, economy, and ease of handling. More significant, however, is its long growth period after weaning and its rapid weight gain during that period. • In general, the more rapidly an animal develops, the greater its nutritive requirements are during the development period, and the more critical those requirements are to the animal’s health. Nutritional deficiencies in growing animals are thus more easily detectable. Dietary Studies • Laboratory animal technicians must be aware of the change in feeding procedures for animals that are being used in dietary studies. • The most common feeding method is ad libitum, in which the feed is available at all times. This term is often shortened to “ad lib.” • In a typical study with ad lib feeding, two groups of animals are fed diets that differ only by the single substance being studied. • In other studies, the diet may be the same for both groups, but the test substance is introduced into the water. • Ad libitum feeding and watering may not be appropriate for some studies. For example, if the test diet has an unpleasant flavor, the experimental animals may eat less than the control animals. Likewise, substances added to the drinking water may cause animals to drink more or less water than the controls, thus introducing an additional variable into the study. One method of circumventing this problem is pair-feeding. • Pair-feeding is a method of ensuring that the control group and the experimental group receive the same amount of feed. • The simplest way to accomplish this is to start the control group on the study one day later than the test group. • The amount of test diet consumed is measured daily, and then the control animals are fed an equivalent amount of control diet. This way, the amount of food provided to the control animals each day is determined by how much test diet was consumed by the study group on the previous day. • If the test substance is provided in water, pairwatering may be used to ensure equivalent consumption. When using pairwatering, the volume of water provided to the control group is determined by the water consumption of the test cohort. Dietary Studies • An apparatus frequently used in dietary studies is a metabolism cage. These cages allow measurement of food and water intake and feces and urine output. More sophisticated metabolism cages facilitate the measurement of gases (such as oxygen and carbon dioxide) entering and leaving the cage. • Before working with animals kept in metabolism cages, technicians should study the construction of the cage thoroughly and should practice taking it apart and reassembling it. While there are many different types of metabolism cages, all share certain features. • Feed is placed into a recessed container so that the animal cannot readily remove and waste large quantities. • The water bottle is also recessed in such a way that any extra drops of water will be caught in a special trough rather than mixing with the animal’s urine. • The bottom of the cage is wire mesh and allows the animal’s feces to immediately fall through to a chamber for collection. • A double inverted funnel arrangement separates urine from feces so that each accumulates in a separate receptacle. • Food and water consumption by animals housed in metabolism cages are measured at regular intervals. The difference between two sequential measurements minus the amount collected in the food and water trough is the amount the animal has ingested. Feces and urine production are measured directly. Stereotaxis/Neurosurgical Research • Research projects studying the brain frequently involve the placement of needles to inject drugs, cannulas to collect fluids, or electrodes to measure electrical activity in specific areas of the brain. • A stereotaxic apparatus is used to stabilize the head and precisely measure its dimensions from anterior to posterior, and lateral to medial. These measurements allow for • correct placement of the implants, needles, or electrodes and the appropriate tissue depth. • Stereotaxic apparatus is most often used for the surgical placement of implants, although it may also be used to ensure the accuracy of intracranial injections. Bioimaging • Bioimaging uses various types of imaging technologies to gather data from both large and small animal models. • Types of imaging procedures include ultrasound, magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), computed tomography (CT), and fluorescent and bioluminescence systems. • What can they do? • Offer opportunities for dynamic studies of disease progression or drug effect in a group of animals over time. • Provide the means to examine biological and chemical processes in the animals’ bodies at the level of molecules, tissues, and organ systems, reducing the number of animals needed in a study since the animals can be imaged and not euthanized. . • Imaging technologies offer high resolution images and detailed analysis of those images, so large amounts of data are gathered from a relatively small number of animal subjects. • All precautions associated with the use of these technologies must be followed. • Bioimaging studies involve restraint, administration of substances by intravenous catheters or other means, anesthetic regimens of varying lengths, and a monitored recovery period. • Documentation of the animals’ imaging sessions is required and includes regular monitoring according to standards and SOPs. Imaging studies vary in duration from a few minutes to several hours, depending on the purpose of the study, the distribution and half-life of any injected substances, and the instrumentation used. Bioimaging • Various kinds of probes are used in these technologies to detect specific molecules and products of chemical processes in the imaged tissue. • The probes both localize where the molecules are in the body and indicate how much of these molecules are present. • These probes cause areas of the image to become bright or colored, depending on the imaging modality. • In PET and SPECT, probes are radioactive atoms that are often called radiopharmaceuticals. Some of these radiopharmaceuticals are shortlived (minutes to hours) and are used for brief imaging studies. Others are long-lived (hours to days) and can be used for investigating slower metabolic processes. • Because all are radioactive, be sure to adhere to all radiation safety procedures and principles. • The ALARA principle refers to keeping your radiation exposure to As Low As Reasonably Achievable by means of time, distance, and shielding. To do so, personnel working with radioactivity should: • Minimize the time spent in the presence of radioactive materials. • Double your distance between yourself and a radiation source. • Place shielding between you and the radiation source. • Animals imaged with PET or SPECT may be given these radioactive probes while in the imaging equipment. The animals, their feces, and their urine may remain radioactive for hours or days, depending on the radioisotope. Your radiation safety office will set up practices and equipment for safely providing animal care during transport and husbandry and for disposing of wastes. Positron emission tomography (PET), single-photon emission computed tomography (SPECT) use radioactive probes…remember animals are ‘hot’ for a period of time after the procedure is complete Bioimaging • Other imaging probes use bioluminescence to visualize cells, molecules, or processes. • This bioluminescence signals a gene’s expression. • The probe is called a reporter protein because it can be detected easily but is not present normally in the animal. • For example, an enzyme luciferase, found in fireflies, is the basis for bioluminescence imaging. By genetic manipulation, the luciferase gene is incorporated into the genome of an animal, such as a zebrafish or mouse. • When the gene sequence integrated with the luciferase gene is activated and when the enzyme’s substrate luciferin is administered to the animal, the enzyme acts on the luciferin and emits light from the chemical reaction. • The light’s intensity is measured by optical imaging equipment and related to the activity level of the gene. Bioimaging • In other imaging modalities, such as MRI, X-rays, computed tomography (CT), and fluoroscopy, drugs known as contrast agents may be administered to the animal to enhance the contrast between anatomical structures. Different families of chemicals are used as contrast agents, depending on the type of imaging modality. • Some imaging equipment is small and can be kept in labs and procedure rooms near animal housing areas. • Other equipment, like MRI machines, are very large, and require a separate room or building. • Biohazards: No additional safety precautions are needed for transporting or handling the animals or disposing of their wastes when these contrast agents are used. • Safe Transport: When imaging equipment is in remote locations, safe and secure transport of animal subjects to and from these sites must be arranged. • Shared Equipment: Cleaning SOPs should be followed for situations in which human patient equipment is shared with laboratory animals. Summary • Animal research methodologies are diverse, spanning studies in toxicology, cancer, genetics, immunology, behavior, neuroscience, and diet, to name a few. • It is important for laboratory animal technicians to understand how each type of study may affect the animals under their care. • For example, what health changes might be expected in an animal undergoing toxicity testing? • What husbandry practices are vital to protect the health of animals with an immunodeficiency? • In addition to roles in husbandry and animal monitoring, laboratory animal technicians may have responsibilities in conducting research procedures, such as measuring the amount of food consumed daily or injecting a test substance.