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Johns Hopkins Bloomberg School of Public HealthCAAT

Animals and Alternatives in Testing: History, Science, and Ethics

Joanne Zurlo, Deborah Rudacille, and Alan M. Goldberg

Chapter 2

The Eye of Science

If I have seen further than other men, it is by standing on the shoulders of giants. -Sir Isaac Newton

The 18th and 19th century physiologists who vivisected animals in order to understand the structure and function of various organs were succeeded by the pioneers of immunology, who used in vitro techniques to create vaccines for anthrax, cholera, and tuberculosis. Many early vaccines were developed using primitive in vitro methodologies and were then tested in animals, so it is difficult to draw a line between what has come to be called in vitro research and that which utilized whole animals, when discussing past achievements (see Appendix A: Methodologies of Vaccine Development).

In fact, in vitro studies have played a key function in increasing biomedical knowledge since Anton von Leeuwenhoek first observed protozoa, sperm, and bacteria under the lens of his self-made microscope in the 17th century. The work of Marcello Malpighi (who in 1660 viewed capillaries through a microscope, thus confirming Harvey's prediction of a system of tiny vessels carrying blood to the lungs) is an early example of the way in vitro studies have complemented and completed other forms of experimentation and observation throughout modern biomedical history. The development of cell theory and germ theory in the late 19th century greatly expanded the role of nonwhole-animal methodologies.

In 1665, Robert Hooke called the tiny chambers in a piece of thinly sliced cork he viewed under a microscope "cells" due to their resemblance to monks' rooms in monasteries. One hundred and sixty-six years later, Robert Brown observed that all plant cells contain a small body, which he called "little nut" or nucleus, and by 1835, Jan Purkinje had confirmed that animal tissues are also composed of cells. However, German scientists Schleiden and Schwann were the first to realize the significance of the cell as the basic unit of living organisms, with Schleiden proposing in 1838 that all plant tissues are made from cells, and Schwann suggesting one year later that eggs are cells, that animal tissues are also made from cells, and that life begins with a single cell. Rudolph Virchow, who insisted that "all cells arise from cells," augmented the cell theory of Schleiden and Schwann in the 1850s.

Meanwhile, Robert Koch and Louis Pasteur were laying the foundations of germ theory, with Koch discovering in 1876 that the microorganism responsible for anthrax in cattle could be grown in culture, and Pasteur learning in 1879 that weakened cholera bacteria would immunize chickens against its more virulent strain. In 1877, Koch developed a method for obtaining pure cultures of bacteria. By 1880, he was using solid cultures of gelatin or agar to grow microbes. In that same year, Pasteur published On the Extension of the Germ Theory to the Etiology of Certain Common Diseases and presented his findings on vaccination to the French Academy of Medicine.

In reviewing the tremendous progress made in the biomedical sciences over the past 100 years, it is clear that the tripartite approach, incorporating clinical (human) studies, animal experimentation, and in vitro studies, has been an extremely fruitful mode of inquiry. The discovery of a vaccine for polio is a classic example of the success of this approach, in which each of the three methodologies played a key role at different stages of research.

"When the work began, little was known about polio transmission or the mechanism of viral spread through the body. There was no means of prevention; after a bad summer, polio left thousands of children dead or paralyzed" (Fee, 1992). Researchers first attempted to study the route of viral infection using rhesus monkeys, "but the rhesus monkey proved to be a poor model for investigating the human disease" (Fee, 1992). Chimpanzees were used next and they proved a much better model, leading researchers to the key discovery that the initial route of infection was the digestive system, with the virus then spreading through the bloodstream into the nervous system. The work of teams headed by David Bodian at Johns Hopkins and Dorothy Horstman at Yale University led to the development of a killed-polio vaccine, which was tested in 12 children. The inactive vaccine was successful. However, a safe and easy method of growing the virus in large quantities was needed.

"In 1949, John Enders, Thomas Weller, and Frederick Robbins at Harvard were able to culture the polio virus in a variety of tissues and to develop a simple method of identifying the virus in culture" (Fee, 1992). Jonas Salk grew all three types of the polio virus in vitro, and Albert Sabin developed a live vaccine by growing viruses on monkey kidney tissue. The University of Michigan School of Public Health designed large-scale human trials of the vaccine in 1954, and one year later it was clear that the vaccine was a success. Research on the etiology and course of the disease and the development of a safe and effective vaccine required the use of an integrated approach, incorporating clinical, animal, and in vitro studies.

This three-pronged approach to the prevention and treatment of disease remains the standard methodology in biomedical research, as a more recent example illustrates. One of the more promising avenues in current cancer research involves the search for chemoprotectors. Epidemiological studies have indicated that people who eat plenty of green and yellow vegetables are less likely to develop cancer. Paul Talalay, a molecular pharmacologist at Johns Hopkins, has been studying the role of dietary chemicals in cancer protection since the late 1970s. Animal studies illustrated the mechanism by which certain food additives and other chemicals stimulated the production of protective enzymes in cells and demonstrated that increased enzyme activity resulted in higher resistance to cancer-causing chemicals, thus decreasing the incidence of cancer in the animals. This permitted the researchers to develop a quantitative tool to measure enzyme activity.

By 1985, Dr. Talalay and his team had begun to experiment with cell culture systems, using microtiter plates to automate the testing process. Researchers found that a component in broccoli proved most efficient in boosting enzymes that detoxify carcinogens. However, it took several high-tech spectroscopic methods to identify the chemical. Mass spectrometry, nuclear magnetic resonance, and infrared and ultraviolet spectroscopy revealed the structure of the chemoprotector sulforaphane. It will now be necessary to use animal studies to map the metabolic effects of sulforaphane and to correlate sulforaphane's effects in vitro with an ability to block the action of carcinogens in vivo. Human studies will inevitably follow.

As these examples indicate, it is not completely correct to say that progress in the biomedical sciences is wholly the result of animal research, nor is it correct to say that animal research has contributed nothing of value and that improvements in human health are solely the result of better diets and hygiene. The truth is that in vivo, in vitro, and clinical studies each provide pieces of the research puzzle and each contributes important information (Stephens, 1987). Progress depends on these three strands of investigation flowing into and feeding each other in an infinity circle of inquiry and information (Fig. 2).

Figure 2. Infinity circle of research