The Influence of Laboratory Procedures and Environments on Experimental Results

Laboratory procedures and conditions exert influences on animals’ physiology and behaviors that are difficult to control and that can ultimately impact research outcomes. Animals in laboratories are involuntarily placed in artificial environments, usually in windowless rooms, for the duration of their lives. Captivity and the common features of biomedical laboratories—such as artificial lighting, human-produced noises, and restricted housing environments—can prevent species-typical behaviors, causing distress and abnormal behaviors among animals.^{Footnote 8} Among the types of laboratory-generated distress is the phenomenon of contagious anxiety.^{Footnote 9} Cortisone levels rise in monkeys watching other monkeys being restrained for blood collection.^{Footnote 10} Blood pressure and heart rates elevate in rats watching other rats being decapitated.^{Footnote 11} Routine laboratory procedures, such as catching an animal and removing him or her from the cage, in addition to the experimental procedures, cause significant and prolonged elevations in animals’ stress markers.^{Footnote 12} These stress-related changes in physiological parameters caused by the laboratory procedures and environments can have significant effects on test results.^{Footnote 13} Stressed rats, for example, develop chronic inflammatory conditions and intestinal leakage, which add variables that can confound data.^{Footnote 14}

A variety of conditions in the laboratory cause changes in neurochemistry, genetic expression, and nerve regeneration.^{Footnote 15} In one study, for example, mice were genetically altered to develop aortic defects. Yet, when the mice were housed in larger cages, those defects almost completely disappeared.^{Footnote 16} Providing further examples, typical noise levels in laboratories can damage blood vessels in animals, and even the type of flooring on which animals are tested in spinal cord injury experiments can affect whether a drug shows a benefit.^{Footnote 17}

In order to control for potential confounders, some investigators have called for standardization of laboratory settings and procedures.^{Footnote 18} One notable effort was made by Crabbe et al. in their investigation of the potential confounding influences of the laboratory environment on six mouse behaviors that are commonly studied in neurobehavioral experiments. Despite their “extraordinary lengths to equate test apparatus, testing protocols, and all possible features of animal husbandry” across three laboratories, there were systematic differences in test results in these labs.^{Footnote 19} Additionally, different mouse strains varied markedly in all behavioral tests, and for some tests the magnitude of genetic differences depended on the specific testing laboratory. The results suggest that there are important influences of environmental conditions and procedures specific to individual laboratories that can be difficult—perhaps even impossible—to eliminate. These influences can confound research results and impede extrapolation to humans.

The Discordance between Human Diseases and Animal Models of Diseases

The lack of sufficient congruence between animal models and human diseases is another significant obstacle to translational reliability. Human diseases are typically artificially induced in animals, but the enormous difficulty of reproducing anything approaching the complexity of human diseases in animal models limits their usefulness.^{Footnote 20} Even if the design and conduct of an animal experiment are sound and standardized, the translation of its results to the clinic may fail because of disparities between the animal experimental model and the human condition.^{Footnote 21}

Stroke research presents one salient example of the difficulties in modeling human diseases in animals. Stroke is relatively well understood in its underlying pathology. Yet accurately modeling the disease in animals has proven to be an exercise in futility. To address the inability to replicate human stroke in animals, many assert the need to use more standardized animal study design protocols. This includes the use of animals who represent both genders and wide age ranges, who have comorbidities and preexisting conditions that occur naturally in humans, and who are consequently given medications that are indicated for human patients.^{Footnote 22} In fact, a set of guidelines, named STAIR, was implemented by a stroke roundtable in 1999 (and updated in 2009) to standardize protocols, limit the discrepancies, and improve the applicability of animal stroke experiments to humans.^{Footnote 23} One of the most promising stroke treatments later to emerge was NXY-059, which proved effective in animal experiments. However, the drug failed in clinical trials, despite the fact that the set of animal experiments on this drug was considered the poster child for the new experimental standards.^{Footnote 24} Despite such vigorous efforts, the development of STAIR and other criteria has yet to make a recognizable impact in clinical translation.^{Footnote 25}

Under closer scrutiny, it is not difficult to surmise why animal stroke experiments fail to successfully translate to humans even with new guidelines. Standard stroke medications will likely affect different species differently. There is little evidence to suggest that a female rat, dog, or monkey sufficiently reproduces the physiology of a human female. Perhaps most importantly, reproducing the preexisting conditions of stroke in animals proves just as difficult as reproducing stroke pathology and outcomes. For example, most animals don’t naturally develop significant atherosclerosis, a leading contributor to ischemic stroke. In order to reproduce the effects of atherosclerosis in animals, researchers clamp their blood vessels or artificially insert blood clots. These interventions, however, do not replicate the elaborate pathology of atherosclerosis and its underlying causes. Reproducing human diseases in animals requires reproducing the predisposing diseases, also a formidable challenge. The inability to reproduce the disease in animals so that it is congruent in relevant respects with human stroke has contributed to a high failure rate in drug development. More than 114 potential therapies initially tested in animals failed in human trials.^{Footnote 26}

Further examples of repeated failures based on animal models include drug development in cancer, amyotrophic lateral sclerosis (ALS), traumatic brain injury (TBI), Alzheimer’s disease (AD), and inflammatory conditions. Animal cancer models in which tumors are artificially induced have been the basic translational model used to study key physiological and biochemical properties in cancer onset and propagation and to evaluate novel treatments. Nevertheless, significant limitations exist in the models’ ability to faithfully mirror the complex process of human carcinogenesis.^{Footnote 27} These limitations are evidenced by the high (among the highest of any disease category) clinical failure rate of cancer drugs.^{Footnote 28} Analyses of common mice ALS models demonstrate significant differences from human ALS.^{Footnote 29} The inability of animal ALS models to predict beneficial effects in humans with ALS is recognized.^{Footnote 30} More than twenty drugs have failed in clinical trials, and the only U.S. Food and Drug Administration (FDA)–approved drug to treat ALS is Riluzole, which shows notably marginal benefit on patient survival.^{Footnote 31} Animal models have also been unable to reproduce the complexities of human TBI.^{Footnote 32} In 2010, Maas et al. reported on 27 large Phase 3 clinical trials and 6 unpublished trials in TBI that all failed to show human benefit after showing benefit in animals.^{Footnote 33} Additionally, even after success in animals, around 172 and 150 drug development failures have been identified in the treatment of human AD^{Footnote 34} and inflammatory diseases,^{Footnote 35} respectively.

The high clinical failure rate in drug development across all disease categories is based, at least in part, on the inability to adequately model human diseases in animals and the poor predictability of animal models.^{Footnote 36} A notable systematic review, published in 2007, compared animal experimentation results with clinical trial findings across interventions aimed at the treatment of head injury, respiratory distress syndrome, osteoporosis, stroke, and hemorrhage.^{Footnote 37} The study found that the human and animal results were in accordance only half of the time. In other words, the animal experiments were no more likely than a flip of the coin to predict whether those interventions would benefit humans.

In 2004, the FDA estimated that 92 percent of drugs that pass preclinical tests, including “pivotal” animal tests, fail to proceed to the market.^{Footnote 38} More recent analysis suggests that, despite efforts to improve the predictability of animal testing, the failure rate has actually increased and is now closer to 96 percent.^{Footnote 39} The main causes of failure are lack of effectiveness and safety problems that were not predicted by animal tests.^{Footnote 40}

Usually, when an animal model is found wanting, various reasons are proffered to explain what went wrong—poor methodology, publication bias, lack of preexisting disease and medications, wrong gender or age, and so on. These factors certainly require consideration, and recognition of each potential difference between the animal model and the human disease motivates renewed efforts to eliminate these differences. As a result, scientific progress is sometimes made by such efforts. However, the high failure rate in drug testing and development, despite attempts to improve animal testing, suggests that these efforts remain insufficient to overcome the obstacles to successful translation that are inherent to the use of animals. Too often ignored is the well-substantiated idea that these models are, for reasons summarized here, intrinsically lacking in relevance to, and thus highly unlikely to yield useful information about, human diseases.^{Footnote 41}

Interspecies Differences in Physiology and Genetics

Ultimately, even if considerable congruence were shown between an animal model and its corresponding human disease, interspecies differences in physiology, behavior, pharmacokinetics, and genetics would significantly limit the reliability of animal studies, even after a substantial investment to improve such studies. In spinal cord injury, for example, drug testing results vary according to which species and even which strain within a species is used, because of numerous interspecies and interstrain differences in neurophysiology, anatomy, and behavior.^{Footnote 42} The micropathology of spinal cord injury, injury repair mechanisms, and recovery from injury varies greatly among different strains of rats and mice. A systematic review found that even among the most standardized and methodologically superior animal experiments, testing results assessing the effectiveness of methylprednisolone for spinal cord injury treatment varied considerably among species.^{Footnote 43} This suggests that factors inherent to the use of animals account for some of the major differences in results.

Even rats from the same strain but purchased from different suppliers produce different test results.^{Footnote 44} In one study, responses to 12 different behavioral measures of pain sensitivity, which are important markers of spinal cord injury, varied among 11 strains of mice, with no clear-cut patterns that allowed prediction of how each strain would respond.^{Footnote 45} These differences influenced how the animals responded to the injury and to experimental therapies. A drug might be shown to help one strain of mice recover but not another. Despite decades of using animal models, not a single neuroprotective agent that ameliorated spinal cord injury in animal tests has proven efficacious in clinical trials to date.^{Footnote 46}

Further exemplifying the importance of physiological differences among species, a 2013 study reported that the mouse models used extensively to study human inflammatory diseases (in sepsis, burns, infection, and trauma) have been misleading. The study found that mice differ greatly from humans in their responses to inflammatory conditions. Mice differed from humans in what genes were turned on and off and in the timing and duration of gene expression. The mouse models even differed from one another in their responses. The investigators concluded that “our study supports higher priority to focus on the more complex human conditions rather than relying on mouse models to study human inflammatory disease.”^{Footnote 47} The different genetic responses between mice and humans are likely responsible, at least in part, for the high drug failure rate. The authors stated that every one of almost 150 clinical trials that tested candidate agents’ ability to block inflammatory responses in critically ill patients failed.

Wide differences have also become apparent in the regulation of the same genes, a point that is readily seen when observing differences between human and mouse livers.^{Footnote 48} Consistent phenotypes (observable physical or biochemical characteristics) are rarely obtained by modification of the same gene, even among different strains of mice.^{Footnote 49} Gene regulation can substantially differ among species and may be as important as the presence or absence of a specific gene. Despite the high degree of genome conservation, there are critical differences in the order and function of genes among species. To use an analogy: as pianos have the same keys, humans and other animals share (largely) the same genes. Where we mostly differ is in the way the genes or keys are expressed. For example, if we play the keys in a certain order, we hear Chopin; in a different order, we hear Ray Charles; and in yet a different order, it’s Jerry Lee Lewis. In other words, the same keys or genes are expressed, but their different orders result in markedly different outcomes.

Recognizing the inherent genetic differences among species as a barrier to translation, researches have expressed considerable enthusiasm for genetically modified (GM) animals, including transgenic mice models, wherein human genes are inserted into the mouse genome. However, if a human gene is expressed in mice, it will likely function differently from the way it functions in humans, being affected by physiological mechanisms that are unique in mice. For example, a crucial protein that controls blood sugar in humans is missing in mice.^{Footnote 50} When the human gene that makes this protein was expressed in genetically altered mice, it had the opposite effect from that in humans: it caused loss of blood sugar control in mice. Use of GM mice has failed to successfully model human diseases and to translate into clinical benefit across many disease categories.^{Footnote 51} Perhaps the primary reason why GM animals are unlikely to be much more successful than other animal models in translational medicine is the fact that the “humanized” or altered genes are still in nonhuman animals.

In many instances, nonhuman primates (NHPs) are used instead of mice or other animals, with the expectation that NHPs will better mimic human results. However, there have been sufficient failures in translation to undermine this optimism. For example, NHP models have failed to reproduce key features of Parkinson’s disease, both in function and in pathology.^{Footnote 52} Several therapies that appeared promising in both NHPs and rat models of Parkinson’s disease showed disappointing results in humans.^{Footnote 53} The campaign to prescribe hormone replacement therapy (HRT) in millions of women to prevent cardiovascular disease was based in large part on experiments on NHPs. HRT is now known to increase the risk of these diseases in women.^{Footnote 54}

HIV/AIDS vaccine research using NHPs represents one of the most notable failures in animal experimentation translation. Immense resources and decades of time have been devoted to creating NHP (including chimpanzee) models of HIV. Yet all of about 90 HIV vaccines that succeeded in animals failed in humans.^{Footnote 55} After HIV vaccine gp120 failed in clinical trials, despite positive outcomes in chimpanzees, a BMJ article commented that important differences between NHPs and humans with HIV misled researchers, taking them down unproductive experimental paths.^{Footnote 56} Gp120 failed to neutralize HIV grown and tested in cell culture. However, because the serum protected chimpanzees from HIV infection, two Phase 3 clinical trials were undertaken^{Footnote 57}—a clear example of how expectations that NHP data are more predictive than data from other (in this case, cell culture) testing methods are unproductive and harmful. Despite the repeated failures, NHPs (though not chimpanzees or other great apes) remain widely used for HIV research.

The implicit assumption that NHP (and indeed any animal) data are reliable has also led to significant and unjustifiable human suffering. For example, clinical trial volunteers for gp120 were placed at unnecessary risk of harm because of unfounded confidence in NHP experiments. Two landmark studies involving thousands of menopausal women being treated with HRT were terminated early because of increased stroke and breast cancer risk.^{Footnote 58} In 2003, Elan Pharmaceuticals was forced to prematurely terminate a Phase 2 clinical trial when an investigational AD vaccine was found to cause brain swelling in human subjects. No significant adverse effects were detected in GM mice or NHPs.^{Footnote 59}

In another example of human suffering resulting from animal experimentation, six human volunteers were injected with an immunomodulatory drug, TGN 1412, in 2006.^{Footnote 60} Within minutes of receiving the experimental drug, all volunteers suffered a severe adverse reaction resulting from a life-threatening cytokine storm that led to catastrophic systemic organ failure. The compound was designed to dampen the immune system, but it had the opposite effect in humans. Prior to this first human trial, TGN 1412 was tested in mice, rabbits, rats, and NHPs with no ill effects. NHPs also underwent repeat-dose toxicity studies and were given 500 times the human dose for at least four consecutive weeks.^{Footnote 61} None of the NHPs manifested the ill effects that humans showed almost immediately after receiving minute amounts of the test drug. Cynomolgus and rhesus monkeys were specifically chosen because their CD28 receptors demonstrated similar affinity to TGN 1412 as human CD28 receptors. Based on such data as these, it was confidently concluded that results obtained from these NHPs would most reliably predict drug responses in humans—a conclusion that proved devastatingly wrong.

As exemplified by the study of HIV/AIDS, TGN 1412, and other experiences,^{Footnote 62} experiments with NHPs are not necessarily any more predictive of human responses than experiments with other animals. The repeated failures in translation from studies with NHPs belie arguments favoring use of any nonhuman species to study human physiology and diseases and to test potential treatments. If experimentation using chimpanzees and other NHPs, our closest genetic cousins, are unreliable, how can we expect research using other animals to be reliable? The bottom line is that animal experiments, no matter the species used or the type of disease research undertaken, are highly unreliable—and they have too little predictive value to justify the resultant risks of harms for humans, for reasons I now explain.