Nuffield Council on Bioethics

Ethics of Research involving animals

Physiological studies

5.5 We include here experiments involving surgical, dietary or drug treatment of animals that are directed at understanding biological processes at the physiological, cellular or molecular levels. Better understanding of these processes has historically contributed to the body of scientific knowledge on animal and human biology. It has played an important role in the discovery of treatments for diseases, usually as a result of systematic methodological enquiry, and in some cases serendipitously (see Box 5.2).

Box 5.1: Example of research – Manipulation of circadian rhythms and comparison of gene expression in the liver and heart of mice

Storch KF, Lipan O, Leykin I et al. (2002) Extensive and divergent circadian gene expression in liver and heart Nature 417: 78–83.*
In many mammalian tissues, the expression of genes that are responsible for the daily timing of physiological processes is controlled by biological timing mechanisms called circadian clocks. In this study, researchers used mice to compare gene expression in the liver and heart. They found that many of the genes expressed were under circadian control, although there were substantial differences between the two organs with regard to the kinds of genes affected. The authors hypothesised from their results that circadian clocks have a specialised role in each tissue, and that the extent of circadian gene regulation meant that it influences many different processes. They concluded that their work addressed important aspects of circadian gene regulation that applied to all mammals and made comparisons between the genes in mice and those in plants and fruit flies.
The following methods were used: mice were synchronised to a 12-hour light/dark cycle for at least two weeks and then placed in constant dim light for at least 42 hours. The mice were subsequently killed at various intervals of a light/dark cycle and their tissues collected and analysed. The mice would have experienced mental and physical disruption in their daily rhythms for the period that they were kept in constant dim light.

* This is an example of animal research that has been carried out in the UK and published in a peer-reviewed journal. Details relate to this specific example and should not be taken to represent a ‘typical’ animal experiment. It is important to note that individually published experiments usually form one part of a continuing area of research, and the significance of the results may therefore be difficult to interpret.

Box 5.2: Examples of how basic research has lead to unexpected clinical benefit

Narcolepsy
Narcolepsy is a disabling sleep disorder estimated to affect between three and five people per 10,000 in European populations.
* Affected individuals have overwhelming feelings of sleepiness and fatigue. They may also experience dream-like hallucinations and the sudden onset of paralysis lasting for a few seconds, usually brought on by strong emotion. The cause and nature of narcolepsy were unknown until recently. In 1998 two groups, neither of which was working on narcolepsy, independently identified a neurotransmitter made by the hypothalamus in the brain; one group called it hypocretin and the other called it orexin. When the gene encoding the neurotransmitter was experimentally inactivated in mice, the mice developed narcolepsy.† The following year, a group studying an inherited form of narcolepsy in dogs isolated a defective gene, and found that it encoded a membrane receptor for one of the two forms of orexin/hypocretin.‡ Based on the evidence that defects in the orexin/hypocretin signalling system caused narcolepsy in mice and dogs, two research groups examined the brains of deceased humans who had suffered from narcolepsy. They found that orexin/hypocretin-producing cells in the hypothalamus were greatly decreased or absent.∫ It is now thought that narcolepsy in humans is usually caused by the autoimmune destruction of these cells in the brain, much as type I diabetes is usually caused by the autoimmune destruction of the cells that produce insulin in the pancreas. Identification of the biological basis of narcolepsy is thus a significant step in developing more effective ways of treating the disorder.

Myasthenia gravis

Myasthenia gravis is a life-threatening disease in which muscles become progressively weaker with exercise. The annual incidence of new people diagnosed with the disease is between 0.25 and two per 100,000.
** A crucial discovery relevant to the pathology of this disease was made in 1973 by researchers who were studying the structure and function of receptors of the chemical transmitter acetylcholine. They isolated and purified the receptors from the electric organ of electric fish (eels, skates and rays) and injected them into rabbits to raise antibodies against them for use in their research (see paragraphs 5.24–5.25). Unexpectedly, the rabbits developed what was identified to be myasthenia gravis.†† It was found that patients with myasthenia gravis make antibodies against their own acetylcholine receptors and that these ‘auto-antibodies’ are usually causally linked to weakening of their muscles. The receptors are normally on the surface of muscle cells and are activated when motor nerves release acetylcholine to stimulate the muscle to contract. In patients with myasthenia gravis, the antireceptor antibodies inactivate the receptors so that acetylcholine is relatively ineffective. The presence of anti-acetylcholine receptor auto-antibodies is now widely used in the diagnosis of myasthenia gravis, and treatment is directed at removing or inhibiting the production of the antibodies. As a result of these pioneering studies, a number of other muscle and neurological diseases, such as Lambert–Eaton myasthenic syndrome and acquired neuromyotonia, were also found to be caused by the inactivation of receptors and channels by auto-antibodies.

* Zeman A, Britton T, Douglas N et al. (2004) Narcolepsy and
excessive daytime sleepiness BMJ 329: 724–8.
† Sakurai T, Amemiya A, Ishii M et al. (1998) Orexins and
orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behaviour Cell 92: 573–85; De Lecea L, Kilduff TS, Peyron C et al. (1998) The hypocretins: hypothalamus-specific peptides with neuroexcitatory activity Proc Natl Acad Sci USA 95: 322–7.
‡ Lin L, Faraco J, Li R et al. (1999) The sleep disorder canine narcolepsy is caused by a mutation in the hypocretin (orexin) receptor 2 gene Cell 98: 365–76. ∫ Peyron C, Faraco J, Rogers W et al. (2000) A mutation in a case of early onset narcolepsy and a generalized absence of hypocretin peptides in human narcoleptic brains Nat Med 6: 991–7; Thannickal TC, Moore RY, Nienhuis R et al. (2000) Reduced number of hypocretin neurons in human narcolepsy Neuron 27: 469–74.
** Vincent A, Palace J and Hilton-Jones D (2001) Myasthenia gravis Lancet 357: 2122–8.
†† Patrick J and Lindstrom J (1973) Autoimmune response to
acetylcholine receptor Science 180: 871–2; See also pages of the Myasthenia Gravis Association website, including http://www.mgauk.org/mganews/0203-01.htm. Accessed on:
23 Apr 2005.

Study of the endocrine system

5.6 Most of what we know about the endocrine system (which produces and releases hormones), has resulted from studies involving animals. Typically, hormone-producing endocrine glands, such as the thyroid, were surgically removed or chemically inactivated in adult animals. The effects of this treatment on the behaviour and physiology of the animals were analysed, and attempts were made to reverse them by administering extracts of the gland. If successful, the next step was to purify the active hormone(s) from the extracts. Most of the known hormones in humans were discovered in this way. Even today, newly discovered molecules that are thought to be responsible for signalling between cells are often tested by injecting them into a living animal (usually a rodent). This is because those who undertake such research believe that this procedure is the most scientifically valid, and often the only way of determining hormone function in physiology and development. The welfare implications for the animals involved will vary depending on the kind of hormone and the dose administered. In humans, hormonal imbalances can cause unpleasant symptoms, including lethargy and headaches.

Box 5.3: Example of research – How do monkeys view faces?

Guo K, Robertson RG, Mahmoodi S et al. (2003) How do monkeys view faces? – a study of eye movements Exp Brain Res 150: 363–74.*
Perception of faces plays a crucial role in social communication. The aim of this research was to study accurately how faces are viewed by primates. The researchers investigated the organisation of eye movements in two adult male rhesus macaque monkeys in response to facial images. Previous studies had suggested similarities between humans and monkeys in the neural mechanisms responsible for the perception of faces. Thus, it was concluded that the results of this study could be compared to findings obtained from humans by less invasive means.
The monkeys underwent an operation under anaesthesia to implant a head-restraint device (see paragraph 4.47). Coils were then surgically implanted into the white, outer layer of the eyeball (the sclera) so that eye movements could be recorded. During experiments, the monkeys were seated in ‘primate chairs’ (see also Box 5.5), which enable the head of the monkey to be fixed. The monkeys’ eye positions were recorded while images of monkey and human faces were presented on a computer screen.
It was already known that when monkeys are shown faces of other monkeys, their eyes fix on the eyes in the image. This particular experiment investigated the visual process that occurs when the faces were unfamiliar to the monkeys, and when the images were inverted or scrambled. Differences in perceptual processing when either a monkey or a human face was shown were also assessed. It was found that the monkeys exhibited similar eye scan patterns while viewing both familiar and unfamiliar monkey faces, or while viewing monkey and human faces. There was a greater incidence of fixation of the eye region of all the face images, and particularly re-fixation of the eyes of unfamiliar faces during the first few seconds, confirming that the eyes are important for initial identification. However, it was found that the eyes in the scrambled face images were much less of a focus than those in the upright or inverted faces. The researchers concluded that, while viewing faces, the eye movements in non-human primates are controlled by more than one level of perceptual processing; i.e. that the targeting of the eye region may occur at a relatively low level of visual processing (before identification of the object) and that the probability that the eyes will become the eventual target in the image is affected by higher levels.
With regard to welfare implications, the implants could have caused discomfort; the monkeys would also have needed to be carefully trained to avoid psychological distress caused by the restraint during the experiment. No reinforcements in the form of ‘rewards’ or ‘punishments’ were given during this procedure.

Study of the immune system

5.7 Many studies on living animals, involving mainly mice and rats, have been conducted to examine the vertebrate immune system, and most current knowledge is based on this research. The immune systems of animals and humans protect them from infection. If the adaptive immune system is challenged by a particular infectious agent that it has previously overcome, it is able to do so on subsequent occasions much more quickly and effectively.
Research on the adaptive immune system usually involves an initial immunisation of animals with foreign (from another animal) biological molecules or cells or microorganisms such as bacteria. Immune responses are characterised by the production of immune cells and antibodies, which specifically recognise and help eliminate the foreign molecules, cells or microorganisms (all referred to as antigens). Experiments of this kind provided the first evidence that the cells responsible for adaptive immune responses were a class of white blood cells called lymphocytes. In these experiments, rats or mice were irradiated with X-rays to kill most of their white blood cells, including lymphocytes, rendering them unable to make adaptive immune responses. When different cell types were transferred into these animals, only lymphocytes were found to reverse this deficiency. The welfare of the animals was usually affected because of increased susceptibility to infections, particularly in the gut, due to the destruction of the lining of mucosal cells caused by the irradiation. These infections were usually treated with antibiotics. In the first series of experiments of this kind, significant numbers of animals died, most likely due to diarrhoea. In general, it can be assumed that the experiments entailed at least some malaise for the animals involved.

5.8 These irradiation experiments depended on the availability of inbred strains of rats and mice, which are produced by repeated rounds of inbreeding until the animals within each strain are nearly genetically homogeneous. The use of these strains allows cells to be transferred between animals of the same strain without the problems of immunological rejection. If cell transfers are attempted between animals of different strains or species, the transferred cells are recognised as foreign by the immune system and the body mounts a reaction and tries to destroy them. Experiments in which skin grafts were transplanted between mice of different strains established that graft rejection is an immunological response. Studies of these immune responses, and the development of medicines that are able to overcome them, eventually facilitated organ transplantation in humans. Transplantation experiments cause some distress to the animals involved, partly because of the anaesthesia used and partly because bandaging the grafts may cause irritation.

5.9 The approach of transferring lymphocytes into the same inbred strain of irradiated mice or rats has also been used to show that different classes of lymphocyte mediate different types of immune responses. New subclasses of lymphocyte and response are still being discovered in this way. Since immune responses in mice and rats are remarkably similar to those in humans, many researchers have applied the knowledge gained from research in rodents to humans. It is also possible to transfer human lymphocytes to immunodeficient mice to enable the study of ‘human’ immune responses using mice. Such ‘humanised’ mice have been important in understanding the function of a range of viruses, including how HIV/AIDS destroys the human immune system and eventually causes the death of the patient. Since mice without a functioning immune system are highly susceptible to infections, they are usually kept in sterile environments, and enrichments are not commonly provided.

Study of cell differentiation

5.10 Similar experiments involving cell transfer in mice are currently being carried out to study the potential of unspecialised stem cells to develop into various specialised cell types. Stem cells isolated from adult organs are called adult stem cells, whereas those isolated from early embryos are called embryonic stem (ES) cells. Experiments involving the transfer of mouse stem cells into irradiated, or otherwise injured, mice have contributed to knowledge about the potential of using human stem cells to treat conditions in which cells die, such as strokes, heart attacks, diabetes and Parkinson’s disease (see paragraph 5.26).4 Blood-forming stem cells from bone marrow have long been used to treat patients whose own blood cells had been destroyed by disease, irradiation or anti-cancer medicines. Welfare problems for the animals used in the experiments referred to above could result from the underlying disease, as well as from the cell transplantation procedure itself, which involves an injection of cells through the lining of the abdominal cavity or into the bloodstream or an organ.

Study of the nervous system

5.11 Much of our knowledge about the functioning of the central nervous system (CNS) has come from invasive animal experiments in which parts of the nervous system are electrically monitored, stimulated or destroyed. Many studies have been undertaken in primates, as the cerebral cortex, which is responsible for most higher brain functions such as thought and speech, is very poorly developed in animals other than primates. For example, individual nerve cells or groups of cells in the cortex of a conscious monkey that are involved in anticipating a movement before it occurs can be distinguished from those cells that send the signal for the movement itself. In a similar way, it is possible to distinguish areas of the cortex involved in recognising the colour of an object from those involved in recognising motion of that object. Although non-invasive imaging techniques such as magnetic resonance imaging (MRI) and positron emission tomography (PET) now allow the physiological activity of large groups of nerve cells in the human brain to be studied, the resolution of these methods is still too poor to study individual nerve cells, or even small groups of nerve cells. Currently, therefore, the only way in which individual or small groups of cells can be studied is by inserting needle electrodes into the brain (see Box 5.4). Nonetheless, imaging techniques are rapidly improving and are likely to provide increasingly powerful alternatives to invasive animal research of this type (see Box 11.1).

Box 5.4: Example of research – Studying control and function of the hand using primates

This is an example of animal research witnessed by some members of the Working Party during a visit to a research establishment.
The objective of this research involving macaque monkeys was to increase understanding of how a stroke can impair use of the hand in humans. It sought to investigate how activity in groups of brain cells in a part of the brain called the motor cortex controlled specific hand and finger movements. Primates were used because only these animals have a sufficiently similar brain structure, function and cognitive ability to ensure that the results were relevant to humans. Research of this type has recently made significant contributions to the diagnosis and therapy of movement disorders and has been crucial to the development of deep brain stimulation (DBS), a new treatment for Parkinson’s disease.*
The monkeys were procured from a breeding colony in the UK where normal practice was to rear them in groups of 16–18 animals and accustom them to contact with humans. In the laboratory, the animals were housed in pairs from the age of 18 months. The cages measured approximately 2.40x1.80x1.20m (width/height/depth) and contained objects for enrichment such as toys, mirrors, puzzle boxes and swings. Foraging material was provided in one part of the cage. The room was lit with natural daylight through windows on two walls. In winter, the light was regulated on a 12-hour scheme with fading transitions. Researchers reported that they maintained frequent social contact with the monkeys.
This project and the procedures were classified as ‘moderate’ by the Home Office. The first procedure was usually an MRI scan. Under anaesthesia, threedimensional scans of the monkey’s skull and brain were taken. These pictures aid the accurate targeting of areas of the brain from which recordings are made.
The animals then underwent a period of training and learned that they would be rewarded with treats such as fruit, nuts and biscuits when they performed certain tasks correctly. Over a period of time, which varied between six and 12 months, they were also trained to remain still while the research was being carried out, which required the use of some degree of restraint to which they became accustomed.
The primary surgical intervention was the implanting of devices necessary to record specific nerve-cell and muscle activity. Under general anaesthesia, a headrestraint device and recording chamber were fitted. The implants weighed 150g and consisted of a metal ring of approximately 10cm in diameter and 1mm in thickness, which was attached to the monkey’s head by means of four bone screws of about 3mm diameter. The screws were inserted through holes made in the skull and were fixed on the inside. These screws were subsequently used to attach the head of the monkey to a specially designed primate chair during an experimental procedure. During surgery, electrodes were also implanted to record the activity of the various nerve cells and muscles that are involved in moving the hand and arm.
After surgery, monkeys received post-operative care including pain relieving medicines and antibiotics and were monitored according to a regime approved by the named veterinary surgeon (NVS).
The average recovery time to normal behaviour was two to three days. The recording procedure itself, which involved introducing very fine microelectrodes into the brain, is not painful, because the brain itself has no pain receptors. With regard to the psychological effects on the animals, there was usually a period of two to three days during recovery from surgery when the monkeys touched the implant. They then became accustomed to it and stopped doing so. In order to allow for the recording of neural and muscular activity, the monkey was placed in a primate chair. This is a steel device, measuring approximately 70x30x30cm. Once the monkey was seated in the chair, a metal disk was put over the ring attached to its skull, thereby immobilising the head by connecting it to the chair. This is required to allow for the stable recording of the activity of single neurones. The monkey remained able to move its jaw and chew, and the rest of

Continued

the body was free to move. The monkey appeared not to resist this procedure (see paragraph 3.34). The multiple electrodes inserted through the implanted recording chamber into the monkey’s brain were connected with wires to a computer, and to devices recording the activity of muscles in the arm and hand. With regard to the experimental procedure itself, the standard task required the monkey to perform a highly skilled hand movement, using its thumb and index finger to squeeze two levers into precise target zones. Each time it squeezed the levers successfully, it was given a food reward by an animal technician sitting next to the monkey. Once researchers had obtained sufficient data on the connection between certain neural areas of the motor cortex and hand movements, the electrodes were inserted into a new area of the brain. There were typically three to five sessions per week, with regular breaks of three to four weeks. Each session lasted approximately three hours, during which a monkey received around 600 food rewards. On average, each monkey provided 100–200 fully analysed neurones over 18 months. Animals were killed at the end of this period by administering deep general anaesthesia from which they did not recover. This allowed electrophysiological and neuroanatomical investigations of brain pathways involved in hand control which enabled the scientists to verify the anatomical position of the electrodes that had been inserted during the research. At this particular laboratory, approximately one monkey per year was used for this type of research.

* DBS involves the implantation of small stimulating electrodes of approximately 1x3mm in the brain circuits of patients suffering from Parkinson’s disease. The electrodes are connected with wires to a unit implanted close to the collar bone. This unit generates electrical impulses in a method similar to pacemakers. To date, approximately 22,000 patients have been treated with DBS. The technique helps to reduce dramatically the manifestation of tremors, episodes of spasticity and other forms of abnormal movement typically experienced by sufferers of Parkinson’s disease. See Rodriguez-Oroz MC, Zamarbide I, Guridi J, Palmero MR and Obeso JA (2004) Efficacy of deep brain stimulation of the subthalamic nucleus in Parkinson’s disease four years after surgery: double blind and open label evaluation J Neurol Neurosurg Psychiatry 75: 1382–5; Kumar R, Lozano AM, Kim YJ et al. (1998) Double-blind evaluation of subthalamic nucleus deep brain stimulation in advanced Parkinson’s disease Neurology 51: 850–5.

Study of gene function in embryos

5.13 Developmental biologists often seek to determine the roles of single genes in animal development. A useful way of doing this is to create GM animals in which the expression of a specific gene is increased or decreased (see paragraphs 5.19–5.20). For example, in some experiments, molecules of ribonucleic acid (RNA, an intermediary involved in the transfer of genetic information between DNA and proteins) are injected into early frog or fish embryos.
This will transiently increase or decrease the expression of a specific protein, thereby helping to determine how that protein (and thereby the gene that codes for it) normally functions in early development. The welfare implications of such experiments are difficult to predict and, depending on the gene, could range from no adverse affects to severe developmental abnormalities and disability (see paragraph 4.57). It is for this reason that in this, and similar types of genetic research, endpoints are defined in licence applications and research should be stopped humanely if they are exceeded (see paragraphs 5.22 and 12.21).5

5.14 Embryologists who study early development in mice sometimes mix cells from embryos of two different mouse strains to form a mouse that is made up of cells from the two strains. If a specific gene in one of the sets of cells is altered before mixing them, the influence of that gene on the development of the altered cells (and the cells that derive from them) can be determined in an embryo in which many of the cells are unaltered. The mixed embryos need to be implanted into the uterus of a surrogate mother in order to develop. The mothers may then be killed in order to obtain the embryo at different stages of development. The welfare implications for the animals relate to the anaesthesia and implantation procedure for the surrogate mother and to any developmental abnormalities in the chimeric offspring.6

Study of development after birth in mammals

5.15 Since development continues after birth in mammals, many studies in this area involve research on animals after they are born. Neurophysiologists, for example, first demonstrated the importance of a critical period in visual development by patching one eye of newborn cats and monkeys.7 If this is done for one week during the first six months after birth, the covered eye becomes permanently blind as a result of alterations in the way in which nerve cells are interconnected in the brain. Patching after this time does not produce the same effect. The same phenomenon was later found in children with one lazy eye. These children are now treated with alternating left and right eye patching to maintain vision in the affected eye until after the critical period, as first demonstrated in kittens.

Footnotes

4 Nuffield Council on Bioethics (2000) Stem cell therapy: the ethical issues (London: NCOB).
5 See Wolfensohn S and Lloyd M (2003) Handbook of Laboratory Animal Management and Welfare, 3rd Edition (Oxford:
Blackwell Publishing Limited), Chapter 4.
6 See Morton DB and Hau J (2002) Welfare assessment and humane endpoints, in Handbook of Laboratory Animal Science: Essential
principles and practices, Volume I, 2nd Edition, Hau J and Van Hoosier GL (Editors) (Seattle, WA: CRC Press), Chapter 18, pp457–86.
7 Early work includes, for example, Wiesel TN and Hubel DH (1965) Comparison of the effects of unilateral and bilateral eye closure on cortical unit responses in kittens J Neurophysiol 28: 1029–40; Wiesel TN and Hubel DH (1965) Extent of recovery
from the effects of visual deprivation in kittens J Neurophysiol 28: 1060–72.
8 Many of those opposed to animal research are concerned about certain types of basic research, and argue that the alleged benefits cannot justify the suffering involved (see paragraphs 3.52–3.55).

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Ethics of Research involving animals

Physiological studies

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