Ethical and moral considerations in the design of experiments

Neuroscience Vol. 57, No. 1, pp. 183-189, 1993 Printed in Great Britain

0306-4522/93 $6.00 + 0.00 Pergamon Press Ltd

IBRO

ETHICAL AND MORAL CONSIDERATIONS IN THE DESIGN OF EXPERIMENTS

R. NAQUET

Laboratory de Physiologic Nerveuse, CNRS, BPI, F-91190 Gif-sur-Yvette, France

Biomedical research, and above all fundamental research in biology necessitating the use of in vivo experiments on mammals ranging from mice to monkeys, poses and will continue to pose in the next few decades, ethical problems confronting researchers.

Already in the twentieth century Claude Bernard in his Introduction h la M@decine experimentale re- marked "Les pr6jug6s qui se sont attach6s au respect des cadavres ont pendant longtemps arr~t6 le progr6s de l 'anatomie. De m6me la vivisection a rencontr6 dans tous les temps des pr6jug6s et des d6tracteurs."* A little further on he posed the following question: "A-t -on le droit de faire des exp6riences et des vivisections sur les animaux? Q u a n t a moi, je pense qu 'on a ce droit d 'une mani6re enti6re et absolue. I1 serait bien 6trange, en effet, qu 'on reconnfit que l 'homme a le droit de se servir des animaux pour tous les usages de la vie, pour ses services domestiques, pour son alimentation, et qu 'on iui d6fendit de s'en servir pour s'instruire dans une des sciences les plus utiles ~i l 'humanit6. II n'y a pas fi h6siter; la science de la vie ne peut se constituer que par des exp6riences, et I 'on ne peut sauver de la mort des 6tres vivants qu'apr6s en avoir sacrifi6 d'autres. II faut faire les exp6riences sur l 'homme ou sur les animaux. Or je trouve que les m6decins font dej~i trop d'exp6riences dangereuses sur l 'homme avant de les avoir &udi6es soigneusement sur les an imaux ." t These are notions full of humanistic truths from which modern ethical committees could well take inspiration. 7

* "Prejudices about the use of cadavers have for a long time halted the progress of anatomy. Similarly vivisection has at all times encountered prejudice and detractors".

t"Does one have the right to undertake experiments and vivisections on animals? Myself l believe that one has this right completely and absolutely. It would, indeed, be strange if one recognized that man has the right to use animals for all purposes in life, for their domestic services, or as food, but denied the right to use them for instruction in one of the sciences of most use to human- ity. There is no reason for hesitating; the life sciences can only be established by experiments, and one can only save some living creatures from death by sacrificing others. It is necessary to undertake experiments either on people or on animals. It seems to me that doctors already undertake too many dangerous experiments on people without first having carefully studied animals".

Abbreviation: MPTP, l-methyl-4-phenyl-l,2,3,6-tetrahydropyridine.

Claude Bernard also based the fight of vivisection on a physiological point of view which A. Prochiantz qualified as "materialist ic" as opposed to the "vitalis- tic" theories which were still fully in fashion in the nineteenth century. One can understand the reasons proposed by certain militants of animal spiritualism who expressed their hostility toward Claude Bernard and to physiologists for whom he represented (and continues to represent) a symbol.

At the end of the twentieth century, despite numerous reservations issued by the antivivisectionists, when biomedical research with clear health impli- cations requires it, the use of cats, dogs or monkeys should cause fewer reactions. Non-specialists should be able to understand the practical interests better and thus be more capable of understanding the necessity of such use.

Fundamental research is more difficult for non- specialists to accept. They reproach the researcher's natural tendencies to be curious and to seek knowledge; indeed they do not accept the character of such work, the immediate interest of which they misunder- stand.

Unfortunately, all good biomedical research is inseparable from good fundamental research and all true scientific progress is produced by a fundamental approach. In addition, when a fundamental research study is "finalized" it then is no longer a question of true research but of applications. If certain of these "finalized" studies are considered, everyone, even some antivivisectionists, will find them to be useful and even necessary (medical, antibacterial, antiviral, anticancerous and other therapies) whereas others, such as those which concern cosmetics, are found to be less so.

Researchers, philosophers, lawyers and some antivivisectionists have established, in some countries, documents and laws that demonstrate and justify the view that animals cannot be treated as an object and that undertake the defence of animal rights in a broad sense. Recently Goodwin and Gordis, 4 in a letter following a commentary of Arluke, expressed their feeling about the new position taken by philosophers who support "animal rights" (in a more restricted sense as employed by antivivisectionist movements in some countries such as the U.S.A.): "The new philos- ophy of animal rights and the tactics of its adherents

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caught society off guard. Because the use of animals in research is based on millennia of Western and Middle Eastern philosophical and religious tradition, the analysis of this moral basis had not been an important topic of inquiry until the animal rights movement thrust it upon us. Indeed, the moral bases for using animals had become so ingrained that they had become for most scientists unquestioned assump- tions. What we seem to have forgotten, however, is that there was a good reason why the scientific community had internalized the propriety of using animals in research--to protect human rights. The Nazi atrocities of World War II led the nations of the world to formulate the Nuremberg code and the Helsinki accords. These explicitly state that new medical treatments must be tested on animals before being tested on humans. Scientists were therefore under a moral compulsion to behave as they did. No wonder we were so confused and disarmed when the animal activists began to call our behaviour immoral. We knew we were doing the right thing, but we could not remember why."

Despite the publication in 1978 of a "Dfclaration universelle de Droits de i 'Aninial",: the "rights of the animals" are not, at the present time, judicially defendable and should not be compared with "human rights". The authors of this "Declaration" emphasized that man, and particularly the scientist, has responsibilities with regard to animals. They also admit that experimentation on live animals remains necessary for the development of science, but they recommend as much as possible the use of cell cultures and models supplied by computers in order to replace animals.

Even if researchers consider that the moment has not yet arrived when experimentation on live animals is no longer necessary, no self-respecting researcher should undertake useless experiments. In addition, experiments lacking scientific rigor should be elimi- nated, pain and anxiety of the animal should be minimized by using anesthesia, animals should be maintained in sanitary conditions and transported as comfortably as possible, and animals that are used should come from breeding colonies and not from endangered species.

Such ethical concerns of scientists approach the principle defined by Russel and Burch 8 known as the principle of the three Rs: reduction, refinement and replacement. In other words, reduce the number of animals used, refine the experimental protocols so as to obtain those which are most efficient, least painful and use fewest animals, and seek techniques of replacement.

The problem has currently reached such a level that when in June of 1991 Barbara Culliton published a courageous article in Nature on animal experimentation entitled "Can reason defeat unreason?", for several months thereafter this same journal published numerous letters to the editor in reply.

In her article several phrases catch one's attention such as: "the animal rights people 'go for the heart', the biologists for the head"; and "the animal rights movement would like to eliminate the use of animals in research". This point of view is shared with that of Goodwin and Gordis 4 as to whether "the debate inspired now by animal rightists is about whether research on animals should be done at all".

The letters that followed (and perhaps will continue to do so in the time to come) are very interesting since some even use the themes and terms of the antivivisectionists when they attack directly or in- directly the people who do research on living animals. These letters demonstrate that there seems to be no incompatibility in doing research in such fields as physics, sociology, etc. and sharing the idea of antivivisectionists, a situation that is already well known in France.

Those who support the themes expressed in Culli- ton's article are those who work with living animals but a number of critical points about particular types of experiment are expressed. It is of interest to quote some of the different positions expressed in this correspondence.

- - " F o r many years the general principle of "keep your head down and it will go away" dominated the thinking on how to deal with the problem. But now there is a growing conviction in the scientific and medical community that the problem has become too serious to ignore". (Matfield M., July 1991).

- - " A sociologist might suspect that the vital organ for vivisectionists is in fact the wallet. (No other members of the scientific community are nearly as incensed by the animal rights movement as those whose careers it threatens.)" (Wolpert D., August 1991).

- - "Wolper t himself has benefited from experiments on animals, dating from his receipt of vac- cines, developed in animals, against diseases that killed earlier generations . . . . Most of our colleagues in the field of biomedical research are concerned with the prevention and cure of disease, many of them entering the field because of first-hand knowledge of the effects of disease and injury on family and friends" (Ralston H. J. II, D. D. Ralston, October 1991).

- - " W e who work with animals find the animal rights organizations irritating, illogical and sometimes dangerous. If we do not defend our research, who will? Theories, mathematical models and tissue cultures are a good start to understanding the cause and (possible) cure(s) for disease and dysfunction. However, at some point an animal is required to address the question "does this work?" I have been fortunate not to have had to resort to the use of apes in my research . . . . The thought of chimps behind bars with electrodes in their heads disturbs m e . . . " (Love J., October 1991).

- - " I n the past few years some major research organizations in the United States have decided that

Ethical and moral considerations in experiment design 185

the animal rights community needs to be put back in their proper place. As a result, they have started to tout the benefits of animal research much more aggressively, using high profile spokespersons. Also they have portrayed "animal rights" activists as fringe types who endanger American health standards . . . . The public is uneasy about the per- ceived treatment of animals in laboratories and consequent animal suffering . . . . Scientists brake to accentuate the "pet-like" nature of laboratory animals rather than talking of scientifically sound standards of nutrition and the best animal care programme that money can buy" (Rawan A. N., November 1991).

These different examples show that when working with living animals, no matter what scientific work is being done or what precautions taken, the scientist may come under attack and should be forewarned. It is necessary to work under conditions which are defendable from an ethical point of view. This is a notion which is fully accepted by the scientific community (see bibliography in Buserl), but one has to be careful not to be too limiting in one's restrictions. Some positions already taken by scientists may prove to have perverse effects:

- -Some scientists today hesitate to use the cat to do experiments in neurobiology although the animal is known to be well suited to this field. The reasons often cited include price of animals from breeding colonies, the risk of using animals from other origins, the fact that it is considered to be a pet; in addition, it is one of the animals most protected by the antivivisectionists.

---Others will not use chimpanzees for AIDS research even though it is the best animal model for this disease. They are influenced by the fact that it is a protected species. At the same time they admit that the basic research for understanding and treating this modern "plague" is not advancing as rapidly as it should.

Experimentation in living animals remains necessary today. One need not feel guilty when carrying out an experiment to increase knowledge and which, above all, can help not only the human condition but also that of animals.

To illustrate this position, three examples will be chosen from problems which arise particularly in neuroscience research. Everyone knows that brain research often requires working not just on animals but also on awake animals if one wishes to study behaviour (control of movement, communication, memory, perception, emotion) and/or pain. In addition, at times the animals must be immobilized without anaesthesia with electrodes in their heads (this is not painful if the electrodes are implanted under anaesthesia and is widely used in humans). Such procedures can cause additional problems. The three examples are: epilepsy and animal models of epilepsy; neurodegenerative diseases; and transgenic animals.

Epilepsy and animal models of epilepsy

Epilepsy currently affects about 0.8% of the French population (approximately 400,000 cases). This corresponds to the level in developed countries; in developing countries it is around 1.5-2%. In the U.S.A. at least 150,000 new cases are diagnosed each year, of which only 70% can be controlled by an appropriate medical therapy.

Characterized by repetitive seizures resulting from an excessive discharge of nerve cells of the brain, epilepsy is a chronic affection of which the origin, in the majority of cases, is unknown. "Partial" epilepsy is generally the consequence of an acquired focal cerebral lesion (following cranial trauma, cerebral tumors or encephalitic complications particularly of infectious diseases of newborn babies or children), whereas "generalized" epilepsies can have a familial origin. In fact, most often it is more of a passing on of a predisposition to epilepsy than of a transmission of a particular type. These "generalized" types show up in three forms: Grand Mal (generalized convul- sions), Petit Mal absence (brief loss of consciousness with an interruption of mental activity) and Petit Mal myoclonic (sudden contraction of muscles of one or both arms and occasionally the trunk). Both forms of Petit Mal are frequent in children and are generally well controlled by certain medications known to be efficient, such as sodium valproate or benzodiazepines. Although benzodiazepines are very pow- erful they are not always long-lasting. In addition, most of these drugs cause a decrease of vigilance and produce learning problems in children. More than 10% of these epileptics, no matter what the treatment, remain subject to seizures and this poses enor- mous problems in daily life. This is true in generalized epilepsies but certainly more in partial complex epilepsies. These are most often due to a lesion of the frontotemporal region, particularly the infero-in- ternal parts, and the limbic system. It is thus necessary to continue research on the mechanisms which lead to the epileptic discharge and also to understand the long-term effects of treatments on the seizures, on vigilance, memory and learning as well as the toxic side-effects of the treatments (notably in pregnant women). These studies are necessary if one wishes to discover new and more specific anti-epileptic drugs.

These studies must generally be carried out in living animals without anaesthesia, like many other experiments in neurobiology, but for different reasons. Generalized anaesthesia blocks most of the electrographic electrical discharges, raising the threshold and modifying or completely blocking the clinical symptoms. It is a method used in humans to stop some forms of status epilepticus which are intractable with customary anticonvulsants.

Certain fundamental mechanisms can be studied in vitro on tissue slices coming from lesioned human or animal brains or on cultured cells or on normal

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tissue slices maintained in a solution containing a convulsant. These methods, however, are insufficient alone because they only provide partial information that comes from cerebral tissue cut from its natural environment.

This limitation is even more important when cultured cells are used since an epileptic discharge brings into play a large number of synaptically intercon- nected cells organized as networks through which the discharge is propagated. Although slices permit the study of such propagation, the brain as a whole is necessary to study other types of propagation that may occur with an epileptic discharge, for example propagation in "the oil spot" manner. The onset of seizures is in addition regulated by sleep, stress, hormonal secretions and sensory information such as noise and intermittent light stimulation, and all these need to be studied in living animals.

It is thus indispensable to continue efforts not only on animal models (chicken, mouse, rat, dog and primate) which present a natural form of epilepsy that may appear spontaneously or be brought on by sensory stimulation, but also to study in normal animals, including, for example, cats in which seizures may be induced by electrical stimulation of the brain, or by the local application or systemic injection of convulsants. These experiments are necessary in order to describe in a precise manner the causes of epilepsy as a disease, the different symptoms which characterize epileptic manifestations, and the consequences of such seizures. It is necessary to establish the correlations between clinical, electrographic, metabolic, neuropathologic, and immuno- histochemic data, to judge, in each model, the effect of an anticonvulsant drug and to appreciate the effect of surgical treatment where this proves necessary.

Fortunately, studies have been carried out on photosensitive myoclonic Petit Mal of Papio papio, a Senegalese baboon (species not protected) which pre- sents a genetic epilepsy; in fact not all Papio papio baboons are sensitive to intermittent light. They have to be captured in Casamance in order to obtain the 60% of the population that is photosensitive. In this form of epilepsy which is very close to myoclonic Petit Mal in man, researchers have already succeeded in defining some significant results. One may mention the importance of the cerebral cortex, and notably of the occipital and frontorolandic regions, the modula- tory role of different neurotransmitters and the fundamental importance of excitatory and inhibitory amino acids. Further, the model has served to help in the choice of new anti-epileptic drugs and in forecast- ing the favourable effects provoked by the section of the corpus callosum in certain severe forms found in children. All of these experiments must be carried out in vivo and one can hardly imagine at the present time an in vitro preparation in a mammal using the eye, the entire visual pathway and the cortical cerebral mantle.

Other models of genetic epilepsy may provide very valuable and different information: one may mention an audiogenic form found in the mouse and rat and a photosensitive one in the Fayoumi chicken.

Experimentally created models of generalized or focalized seizure discharges may be very useful: for example the one induced by kainate injected intra- cerebrally in the limbic system or systemically. This toxic amino acid provokes lesions in the cerebral territory where it is injected and produces status epilepticus which may or may not be followed by seizures occurring chronically. Lesions can be produced at a distance in susceptible territories such as the hippocampus and these may be responsible for recurrent seizures. These types of lesion are analogous to those which one finds in human partial complex epilepsy. This is considered to be the least easily treated form and the experimental lesions may serve to explain the mechanism of induction and as a consequence the prevention of the epilepsy. One may also mention focal status epilepticus induced by cessation of intracortical injection of the inhibitory amino acid, GABA (GABA withdrawal syndrome). This status has the property of remaining very localized, comparable to the Kojevnikov syndrome in man, but it also remains invariable for many hours and is easily reproducible in different rats. Once it is induced one can carry out in vitro studies on slices passing through the lesion.

In vitro experiments in general permit a better understanding of elementary mechanisms at the origin of "epileptic" discharges as well as providing insights into their maintenance and local propagation. Such experiments will in the near future permit the elucidation and discovery of new anti-epileptic drugs having more specific actions.

But the development of research of new drugs also requires a confirmation by in vivo experiments to evaluate the long-term effects of the compound and its toxicity. In vivo experiments are also necessary to test the effects of implantation of neuronal stem cells. These cells may be transformed in such a way so as to secrete inhibitory substances in certain cerebral regions considered to control the excitability of the nervous system (e.g. substantia nigra). The secretion of GABA, for example, should be sufficient to diminish the hyperexcitability which accompanies an epileptogenic lesion but not too great if one considers that stopping an intracortical injection of this same compound may itself be epileptogenic (GABA withdrawal syndrome).

These experiments which are currently under way need to be evaluated over a long period in in vivo experiments in animals presenting recurring seizures. These stem cell implantations started to be used in kindled rats. They are not yet utilized in baboons. Their use in primates would be necessary before being tested in man since one can imagine that they raise numerous ethical problems.


From these studies on epilepsy which, as can be seen, require alternatively in vitro and in vivo experiments, one can expect to acquire knowledge of the epileptic discharge, of the epileptic disease and of its treatment in man.

Other than their human applications, these studies will also benefit animals in the sense that a number of them are naturally, due to genetic reasons, or secondarily, due to lesions (e.g. encephalitis) that produce epileptic seizures. Current treatments, in particular those for dogs and horses, have undesir- able side effects (reduced efficiency for the horse, loss of the sense of smell for hunting dogs, etc.) which is unfortunately still currently dealt with by eu- thanasia, which is regrettable and ethically barely defensible.

Neurodegenerative diseases

The experimental study of certain neurodegenerative diseases has developed considerably over the past ten years. The possibility of creating models that are close, if not analogous, to the human disease has opened the way to a whole series of studies in the rat and more recently in subhuman primates. These studies, in which the lesioned animals are followed over a period of time, have permitted not only a better understanding of the symptoms and the evolution of the disease but also in some instances have allowed an evaluation of a deficit of some neuro- mediators. Finally, they have allowed an appreciation of the more or less total and long lasting recovery possible with pharmacological treatment or after grafts.

The contributions of animal experimentation and the problems posed will be considered for three such diseases. They are the object of very important studies because of the importance they are assuming with the lengthening of the human life- span.

Alzheimer's disease certainly poses the most problems to researchers. It is one of the most frightening and devastating of all neurological disorders. The dementia caused by it affects 4,000,000 Americans. Forgetfulness and memory loss, time or place disori- entation, and difficulty with concentrating or calcu- lating are the earliest symptoms which usually begin during the patient's mid-60's. Final stages leave the victim incapable of self-care.

Alzheimer's disease is a form of localized amy- loidosis characterized by cerebral cortical amyloid plaques, neurofibrillary tangles and amyloid deposits within the walls of the leptomeningeal vessels. Although most causes of Alzheimer's disease are sporadic, kindreds with autosomal-dominant in- heritance of the syndrome suggest that a single mutation may be important in its pathogenesis. Some single amino acid substitutions may correlate with the presence of Alzheimer's disease in patients and may be the inherited factor causing both amyloid fibril formation and dementia; but it has to be

recognized that in such a disease different pathogenic processes may lead to a common clinical presen- tation.

For these reasons it is difficult to find or create the best animal model. Those that are being used reproduce only a part of the symptoms. They have already produced some interesting results for which living animals and particularly monkeys are necessary. Alzheimer's brains have lower than normal levels of acetylcholine, somatostatin and mono- amines due to the loss of the specific cell groups that synthesize these transmitters in the brainstem. The acetylcholine loss can be mimicked by alteration of the cholinergic pathway which originates in Meynert's nucleus and which projects onto the frontal lobe of the rat and monkey and controls its metabolism. It is possible in monkeys to visualize with a PET scan after a lesion of one of the Meynert nuclei produced by the cytotoxic amino acid, ibotenic acid, that there is a metabolic lessening at the level of the frontal lobe homolateral to the lesion.

Parkinson's disease lends itself much more readily to an appropriate animal model. At present it is known that this disease is the result of a deficit of the dopaminergic nigrostriatal system and that in man, as was demonstrated ten years ago, it may have a toxic origin as demonstrated with 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine (MPTP). Lesions of the substantia nigra in mice and more particularly in monkeys can make symptoms appear that are very close to those of human Parkinsonism. The use of 6-hydroxydopamine or ibotenic acid injected locally allows the production of a syndrome of the parkinsonian type but it is difficult to destroy all of the substantia nigra by such a method in monkeys. The utilization of MPTP permits the specific destruction by systemic injection of all the dopaminergic cells of the substantia nigra. In the monkey one can reproduce the most typical and long-lasting parkinsonian syndrome (movement disorders). Parkinsonian monkeys allow one to follow the evolution of the clinical symptoms and to compare them with those provided by the PET scan of the dopamine receptors. Further, it becomes possible to study the effects of the appropriate medical therapies such as L-DOPA or other more recent compounds which can improve the animals or slow the evolution of their disease.

Finally, such models allow the study of short- and long-term effects of cerebral grafts. The transplantation of embryonic neural cells into diverse regions of the brain is one technique widely used in animals at the present time. These cells can develop, differen- tiate and grow so as to insert themselves into the host's nervous system. The use of cyclosporine has revolutionized the prognosis of these grafts. But while there are numerous data in the rat there are not yet many in the monkey in which implants of embryonic cells from a rat fetus have been placed in the striatum.

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To obtain the survival without rejection of functional dopaminergic cells, to study the duration of this survival, the reality of the clinical improvement and its evolution, necessitate carrying out such experiments. One also anticipates modifications of the genome of the cells to allow a better secretion of dopamine (as is proposed for GABA cells in epilepsy) or a procedure of encapsulation to make the cells less likely to suffer rejection.

Huntington's chorea is a monogenic disease (affect- ing 25,000 Americans, for example) which appears in humans between the ages of 30 and 45 years and which evolves during a period of 10-15 years. It leads patients to a progressive insanity with uncontrollable movements. The discovery of the gene raises in man considerable ethical problems for the future which we will not discuss; but while waiting for an efficient genetic therapy to be proposed (which is not for tomorrow), one envisions turning to cerebral grafts to alleviate the clinical symptoms and above all the abnormal movements.

Cerebral lesions at the level of the striatum created by ibotenic acid bring on the motor symptomatology. The first results obtained in the monkey led to the acceptance of the validity of this model. Grafts of embryonic cells are being carried out and the analysis of the data will, within a few years, let us know to what extent such embryonic tissue grafts are to be recommended in man.

The study of these models of neurodegenerative diseases in the subhuman primate is for many a necessity. Everyone who is doing research in this field considers that the comparison with human disease is much more valid than when the work is carried out in a rodent. For some, however, these types of experiment in monkeys raise some ethical problems. They were recently well expressed by Hoffer and OlsonS: "Yet another requirement of the Nuremburg Code is that a sufficient number of animal studies in vivo or in vitro, or both, should have been carried out to justify extension of any research to humans. This is also an area of ethical controversy with respect to neural grafting. A large number of studies that document graft efficacy have been carried out in rodent models of Parkinson's disease, but far fewer studies have been conducted in non-human primates. Some people believe it is unethical to proceed to studies of humans. Others believe it is equally unethical to produce debilitating brain lesions in monkeys when human clinical trials are already under way. The situation is even more equivocal for other neural disorders, such as Alzheimer's disease and Huntington's disease, for which less animal work has been carried out with respect to graft function".

One may answer that results obtained in man are still too controversial to discourage the pursuit of animal experimentation, in particular in monkeys. If some consider that research on infra-human primates is ethically debatable, what would be the position of

the scientific community and the different ethical committees if the grafts in man were permitted without discrimination at the present time? One has already seen the poor results obtained after intracere- bral grafts of the suprarenal gland.

Transgenic animals

No one any longer denies the interest of the techniques of transgenesis. According to Houdebine 6 they can be classified into five categories:

(1) The fundamental study of the functioning of genes and their role during embryonic development and in adult life. (2) Obtaining pedigrees for clinical study: diabetic animals or those developing tumors of selected organs, or animals that have become sensitive to human viruses. The possibility of generating transgenic animals with which to study neurodegenerative events has become ever greater for neuroscientists. Different approaches have been successfully employed to generate such changes as spongiosis (reproducing scrapie), amyloid deposits (as with Alzheimer's disease), and neural degener- ation (in animals expressing a transcriptionally activated mos-proto-oncogene).

"Promotors are continuously being identified, the activity of which is restricted to specific populations of cells within the CNS. Using these promotors it will become possible to target gene expression to very precisely defined groups of cells. For example, cells can already be targeted not only on the basis of expression of global neural or glial markers, but also on the basis of their expression of particular peptides, receptors or regulatory proteins". Transgenic animals may help to provide a greater understanding of neurodegenerative diseases and their causes. (3) In the production of recombinant proteins by transgenic animals: one already knows that such proteins are successfully produced in culture by bac- teria, yeasts, mushrooms and eukaryotic cells. A certain number are already on the market: for example, insulin, growth stimulating hormone and interferon. For others with a complex structure, it is necessary to use eukaryotic cells. But these cultures are costly and require a delicate maintenance. Trans- genic animals can represent an interesting alternative if one can recover the proteins from their blood or milk. Animals of a reasonable size (rabbits, pigs, sheep, goats) already produce important quantities of human proteins. Currently 35 proteins are good candidates to be thus produced. It is not impossible that in the future the antivivisectionists could be against such practices which attempt to diminish the use of cell cultures for industrial purposes: they could react in this way if the animal in question were not a pig or a sheep but a dog. (4) In the protection of animals against diseases: a certain number of genes provide the animals which possess them with a resistance against one or another


illness. It is conceivable to transfer such genes to other animals. (5) In the modifications of animal physiology: The most profound changes are those which introduce new biological properties of interest to breeders, consumers or scientists (high proliferation, acceler- ated body or wool growth, resistance to stress, etc.).

These five categories bring certain reflections. The production of such transgenic animals is not without the creation of certain ethical problems or biological risks.

Some people react against the principle of changing animals for man's benefit, and others speak of raping the fundamental laws of creation. One could reply that the creation is in a perpetual evolution; in fact, it is thought that 80% of the species which existed have disappeared naturally or disappeared due to cataclysms.

Certain transgenic animals, showing a cancer gene introduced by transfer, present tumors, the painful consequences of which one can imagine. This leads one to reflect about the use of such animals. How- ever, such tumors are comparable to tumors provoked by different pathogenic agents which have proved to be necessary for cancer research. In addition, transgenic mice expressing a human cancer offer immense perspectives for the study of different stages of development of a cancer and the understanding of the genetic predisposition to a cancer. Transgenic animals can also entail a risk for the environment. The release of genetically modified animals into nature may constitute a real danger for the environment. Although in principle there are few risks for domestic animals, this is not the case for other species.

Concluding remarks

These few examples, taken from the recent litera- ture and dealing with subjects tied more or less directly to future research in neuroscience, demonstrate the difficulty which exists from an ethical point of view of having too rigid a position. It is evident that what one considers a necessity today may no longer be so in a few years. Could it be that the cell

cultures for production for industrial purposes, still recommended today by the "antivivisectionists" could rapidly be abandoned to transgenic animals. In contrast, the development of the patch-clamp technique and its interest for the study of the functioning of ionic channels could allow in pharmacology an in vivo cost limitation to the benefit of the research on in vitro cell cultures. The use of the patch-clamp should in fact help, by its molecular electrophysio- logical approach, to provide better comprehension of the action of a new compound, to find its more localized action and thus to discover compounds which are more therapeutically efficient. Early gener- alizations are always dangerous in science. If it is necessary to inculcate young generations of researchers with ethical notions, which have been ab- sent for preceding generations, it is also important to not "demotivate", to not "charge them with guilt" and to allow them to carry out their research free from "limitations" dictated by their peers. (Unfortu- nately some of these peers sometimes go beyond their rights under political pressure or in a particular cultural environment!). If an animal has the right to be respected, the scientist should also. But the scientist should not despair without trying to understand the reasonable positions of those who are criticizing him. Perhaps he considers, and in this he is incorrect, that he must not lose time in trying to explain to others the fundamental importance and necessity of his experiment on an in vivo animal. To explain on the basis of solid facts, to help others to understand a profound motivation is never time lost and should allow the bridging of differences. It should help the difficulties to disappear between self-respecting scientists and intelligent people who are sometimes too sensitive and at the same time misinformed. The comprehension and support of these people are necessary in the face of hard-line antivivisectionists. Finally, the researcher who has been confronted by attacks from these antivivisectionists must not capit- ulate but rather must take some distance, and without passion, and using all means available, try to con- vince others not of his innocence (which would be going too far) but of the merits of his intellectual and humanitarian position.

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Ethical and moral considerations in the design of experiments