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Animal experimentation in forensic sciences: How far have we come?
Accepted Manuscript Title: Animal experimentation in forensic sciences: how far have we come? Author: C. Cattaneo E. Maderna A. Rendinelli D. Gibelli PII: DOI: Reference:
S0379-0738(15)00259-5 http://dx.doi.org/doi:10.1016/j.forsciint.2015.06.024 FSI 8049
To appear in:
FSI
Received date: Revised date: Accepted date:
19-8-2014 24-6-2015 25-6-2015
Please cite this article as: C. Cattaneo, E. Maderna, A. Rendinelli, D. Gibelli, Animal experimentation in forensic sciences: how far have we come?, Forensic Science International (2015), http://dx.doi.org/10.1016/j.forsciint.2015.06.024 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Animal experimentation in forensic sciences: how far have we come?
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Prof. Cristina Cattaneo LABANOF, Laboratorio di Antropologia e Odontologia Forense Sezione di Medicina Legale
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Corresponding author:
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LABANOF, Laboratorio di Antropologia e Odontologia Forense Sezione di Medicina Legale Dipartimento di Scienze Biomediche per la Salute Università degli Studi di Milano
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C Cattaneo, E Maderna, A Rendinelli, D Gibelli
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Dipartimento di Scienze Biomediche per la Salute Università degli Studi di Milano V. Mangiagalli 37, Milan - Italy Phone number: +39-02-50315679 Fax number: +39-02-50315724 [email protected]
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Abstract
In the third millennium where ethical, ethological and cultural evolution seem to be leading more
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and more towards an inter-species society, the issue of animal experimentation is a moral dilemma. Speaking from a self-interested human perspective, avoiding all animal testing where
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human disease and therapy are concerned may be very difficult or even impossible; such testing may not be so easily justifiable when suffering – or killing – of non human animals is inflicted for
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forensic research.
In order to verify how forensic scientists are evolving in this ethical issue, we undertook a systematic review of the current literature. We investigated the frequency of animal
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experimentation in forensic studies in the past 15 years and trends in publication in the main forensic science journals.
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Types of species, lesions inflicted, manner of sedation or anesthesia and euthanasia were examined in a total of 404 articles reviewed, among which 279 (69.1%) concerned studies
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involving animals sacrificed exclusively for the sake of the experiment. Killing still frequently includes painful methods such as blunt trauma, electrocution, mechanical asphyxia, hypothermia,
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and even exsanguination; of all these animals, apparently only 60.8% were anesthetized. The most recent call for a severe reduction if not a total halt to the use of animals in forensic sciences was made by Bernard Knight in 1992. In fact the principle of reduction and replacement, frequently respected in clinical research, must be considered the basis for forensic science research needing animals.
Keywords: ethics, forensic pathology, forensic sciences, animal experimentation
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Introduction
Hundreds of millions of animals are used every year in laboratories all over the world, with a
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disproportionately small number of 11.5 million used in the European Union [1, 2]. The need for fast, scientific improvement has spread this pandemic to virtually every field of science, apparently fuelled by the illusion that the use of animals may quickly lead to sound and useful results.
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However, if in clinical research the sacrifice and suffering of non-human animals may be
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considered by a majority as secondary to the therapeutic advantages which may be derived, in forensic sciences the use of animal models can, in most instances, be questionable. In 1992
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Bernard Knight had already focused on this issue, and recognized that the forensic field had to face the thorny topic of animal rights [3]. Interestingly, very relevant arguments against the use of animals in forensic experimentation have to do not only with ethics but also with practical limits. Knight in fact had already observed that “a vast amount of published material using animal experimentation seems to have little practical relevance, other than to expand the curriculum vitae and the career prospects of the researcher” [3]. This point of view has been more generally stressed by Pound et al. who in 2004 shed doubts on the presumed validity of animal experimentation even in the clinical field [4]. Knight raised in 1992 some additional issues which need to be reconsidered, especially now, after more than 20 years, that the use of animals in forensic sciences is indeed continuing if not increasing: “painful, sometimes mutilating experiments on conscious animals” in order to obtain “tenuous potential benefit to some medico-legal problem” cannot be condoned, particularly if one considers that such works “are not regularly used in routine forensic practice” and just “gather dust in university libraries”. Furthermore, the pitfalls encountered when extrapolating results obtained on animals to the human situation have to be considered, particularly in the forensic 3 Page 3 of 21
field. Knight’s conclusion was not to accept such works unless there was a compelling reason and the results provided important advances in the practice of forensic science [3]. In his 1992 article Knight describes some cases of studies performed on animals. The infliction of wounds and burns for dating lesions by histological and histochemical techniques, and the
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“classical research” on drowning unanaesthetized dogs are given as examples of rather dubious correspondence between scientific data on animals and humans [3]. In 1992 the pointless abundance of such experimentation had led the Editor of one of the most important forensic
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journals to take a stance on this issue, discouraging animal testing; but what has happened since? Was the call taken/listened to? Has the forensic scientific world modified and evolved its attitude
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towards animal experimentation?
Our review shows that it has not. Literature still provides plenty of such cases, for example administration of pesticides [5,6,7], ante mortem production of traumatic brain injury [8,9,10],
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blunt injury to the precordial regions in dogs [11,12] air embolism in rabbits [13], gunshot or stab wounds to pigs [14,15]. In addition several experimental protocols concerning the assessment of the effects of hanging [16], electrocution [17,18,19], myocardial ischemia [20], asphyxia [21,22]
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trauma [23,24], drowning [25,26], hypovolemic shock [27] in mice, rabbits and dogs can still be
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found.
This short review briefly aims at illustrating how little has changed in the past 15 years; in addition
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to what had been anticipated by Knight, we wish to stress the exact same point, this time with numbers and details at hand.
Materials and methods
A meta-analysis of scientific articles published between 2000 and 2014 was undertaken on Pubmed and Medline databases. Articles published in the main forensic medicine and forensic science journals which referred to animal experimentation were selected. The journals considered were “American Journal of Forensic Medicine and Pathology”, “Forensic Science International”, “International Journal of Legal Medicine”, “Journal of Forensic and Legal Medicine”, “Journal of
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Forensic Sciences”, “Legal Medicine”, “Medicine, Science and Law”, “Science and Justice”, “Forensic Science, Medicine and Pathology”. The use of animals in general was the main criterion for selecting the article. This enabled the authors to discover how much research in forensics is actually being done using animal models, uses animals which have died for reasons not related to the
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how much of this research
experiment (for example pigs accidently crushed by other pigs [28]) compared to those who have
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been used and killed for specific forensic purposes.
The different kinds of species, types of lesions, manner of sedation and killing were also taken into
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account, as well as, in the case of animals selected and sacrificed for forensic purposes, whether the experiment took place ante mortem (eg. wound healing) or post mortem (eg. taphonomy),
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and with what anaesthesia.
Results
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The review yielded 404 articles. This is a probable underestimate since some articles do not bear
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“animal” or animal experimentation among the keywords, abstracts or titles. Of these, 279 (69.1%) represent experiments where the animals were specifically killed for the purpose of the
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forensic experiment, versus a more modest 105 (25.9%) where animals which had died from other causes were used. In 20 (4.9 %) of cases the specific reason why the animal was killed and how was not specified. Among the cases where animals were sacrificed for the purpose of the experiment, in 84.6% the test was performed antemortem sometimes with an unclear description of the manner of sedation (Table 1).
As can be seen in Table 2 a total of 8203 animals were sacrificed for the research: 81.3% (approx.) were rats and mice, the remaining 18.7% being rabbits, pigs, dogs, sheep, calves and monkeys (Fig. 1).
In relation to the different types of studies, 30.2% of all animals were killed for toxicological tests ranging down to 9.5% for the study of perimortem trauma, and to 3.5% for electrocution research (Fig. 2). Other areas concerned wound healing processes (14.6%), estimation of the post mortem interval (23.4%), hypothermia and hyperthermia (4.9%) and mechanical asphyxia (5.1%) (Fig. 2). Figure 3 shows the apparent trend in the number of articles reporting the use of animals. Table 2 shows the number of animals sacrificed, of which species and whether anesthesia was used for the experiment or if information of this sort was not given, as well as the area of forensic science 5 Page 5 of 21
the article involved. Mice, rats and rabbits were the most frequent animals used; however there is still a surprising use of dogs, rabbits and monkeys.
Research areas are still quite varied and
include experiments on wound healing (which involves inflicting lacerations or bone fractures on live animals and sacrificing them at set intervals), thermal injury, electrocution, various types of mechanical trauma and drugs and toxicology. Only in 4984 (60.8%) cases were the animals
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clearly anesthetized.
“Euthanasia” methods included the “humane” injection of sodium pentobarbital, but also
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exsanguination and blunt trauma [29,30,31]. In some cases, the type of trauma examined or the actual experiment ended up being the cause of death [32,33,34]. It is surprising how many details
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are omitted by various authors, even the number of animals used, and whether the animal was anesthetized and if so how it was euthanized. Table 2 shows the correlation between the species involved in the experiments, specific fields of research and type of anesthesia: experiments where is
not
used
or
mentioned
concern
wound
assessment
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anesthesia
[35],
toxicology
[36,37,38,39,40,41,42] and sometimes taphonomy/post mortem interval [43,44,45]; experiments as well as painful procedures involve traumatic injuries [46,47,48], asphyxia [49], hypothermia
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[50,51] and even psychological stress [52,53]) where the authors provide no specific information on treatment of the animals. Moreover the numbers reported in Table 2 are underestimated since
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the exact number of animals recruited for some studies is sometimes not mentioned.
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Approval by various ethics committees is stated by many authors. Rarely do journals request proof of that approval. The large differences in regulations across the world make local ethics approval a
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subjective standard. Journals should develop their own standards to such research as well and apply it .
Cruel experiments are still inflicted on animals in forensic research, the following being just a handful of examples. Some authors in studying erythrocytes and endothelial cells of electrocuted dead rabbits report that “one group of rabbits as a normal control was put to death by dislocating the cervical vertebra; model 1, 2, 3, 4 were electrocuted; the rabbits of model 5 were put into a bag and allowed to fall from a 40 m height, one by one; model 6: rabbits were burned to death using ethanol; model 7: poisoning with sodium nitrite; model 8: the cause of death of this group was asphyxia; rabbits were put into a small container with carbon dioxide” [19]: no mention of anaesthesia is made nor was it requested by the journal. In another toxicological study, the authors investigated the effects of sarin-like and soman-like compounds – highly toxic organophosphorus agents used in chemical warfare – by intravenous injection in rats [42]. To investigate mRNA expression of IL-10 during fracture healing for wound age estimation Takamiya and colleagues [48] reproduced an animal model of fracture in mice by sacrificing the 6 Page 6 of 21
last experimental group after 240 hours without specifying if the animals are kept under anesthetic for the duration of the experiment. In a further experiment mice were restrained in a polypropylene centrifuge tube with air holes for 1h/daily from 1 day to 4 weeks - as a model of psychological stress [53]. However as previously mentioned, only in 60.8% of cases is there clear mention that the animals
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have been anesthetized; in a disturbing 20.7% the presence of anesthesia was not specified: this information is worrying, since it suggests that in some experiments, the animals were clearly not
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anesthetized, although they were subjected to painful procedures.
Along with the actual pain the animals would certainly undergo for most experiments if no
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anesthetic were given, one should also bear in mind the strong ethological and neurological evidence that non human animals can anticipate suffering and can experience fear [54,55,56].
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Discussion
Animal experimentation for medical and scientific purposes has been a major topic in research
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ethics and the subject of general community debate and concern for at least 50 years [4,57,58,59]. In 1992 a discussion concerning the use of animals in forensic research was opened
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by Bernard Knight. He suggested that although the forensic context could not be considered detached from other fields of scientific research, the use of animals should be more strictly
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controlled, since forensic research does not lead to improvement of life conditions and health. In the case of clinical experiments, the use of animals has been widely analyzed and discussed by
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ethical and scientific authors. Russel and Burch (1959) suggested a compromise encompassed by the 3Rs principle of: refinement, reduction and replacement. This focused on the improvement of experimental techniques, limitation of the number of animals used and the application of alternative methods [57]. The models more commonly used for research into human disease usually must meet these three criteria, and face validity, predict validity and construct validity [60,61,62]. This means that, for animals to be a good model for investigating human disease, the animal has to show the same symptoms, the same reactions to treatment and the same aetiology as the human being. From this point of view, given the previously stated criteria, the animal model in the clinical context should provide a feasible and easily modifiable model for researchers in order to test different variables. Thus for some people (probably still a majority), there is acceptance of the sacrifice of animals for clinical trials because the results may improve human health. Forensic research does not provide advantages for health, but possible assistance in understanding criminalistic issues (eg. estimation of post mortem interval, assessment of injuries, etc.). As a consequence, the sacrifice of animals is more questionable, as are the results obtained by the studies, given the biological differences 7 Page 7 of 21
between humans and non humans. This limitation has been already faced by clinical researchers [63]. Some literature has observed that many clinical trials involving the use of animals have not been published [64] or that results are not usable [65]. In the forensic context the study becomes reliable if the data are systematically verified and if they proved to be useful in practical cases [4]. From this point of view, the history of forensic sciences has provided us with much evidence of the
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inapplicability of data obtained from studies performed on animal models, including the Daubert sentence [66] and several recent studies have again stressed the limits of this approach in
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different fields of forensic sciences, from the estimation of post mortem interval to genetics [6775]. In an article concerning the effectiveness of triphenyltetrazolium chloride to detect acute
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myocardial infarction, the authors state that “it is not realistic to apply methods confirmed in animals” [68]. Stokes et al. observed contrasting dynamics of mammalian analogues in experimental taphonomy, and state that “the results of this study showed many differences
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between porcine, bovine, and ovine SMTs (skeletal muscular tissues) when considering them as analogues for human SMT2 in decomposition soil studies”[69]. As another example there is the genomic responses in mouse models which poorly mimic human inflammatory disease, as shown
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by Seok et al. who state that “there are multiple considerations in our findings that the transcriptional response in mouse models reflects human diseases poorly including the evolutional
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distance between mice and humans, the complexity of human disease, the inbred nature of the mouse model” [70]. Even the microscopic anatomical basis of bones [76] and brain [74] show
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relevant differences between pigs and rodents and human beings and it has been shown that
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biomechanics as studied in non human animals may be useless for humans [63]. On the other hand, applying alternative methods rather than using animals has provided, in the forensic field, important and reproducible results, as presented in Table 3 [74,77-88]. And for many less elaborate projects, animals who have died from other causes can be used. In the clinical scenario, systematic reviews and meta-analyses of previous animal experiments in fact may ensure that new experiments will not be conducted in order to answer questions already contemplated making it thus possible to reduce the number of animals sacrificed. Reviews on this topic however are worryingly uncommon - only two studies in the past twelve years [71,72]. None, to our knowledge, have been performed for the forensic scenario.
If the avoidance of animal testing when disease and therapy are concerned is a difficult and unresolved issue, it should not be so when suffering – or in fact killing - is inflicted on living animals for questionable forensic purposes. The forensic context has been considered for a long 8 Page 8 of 21
time a “no-man’s land” where animal ethical issues somehow seem to be less “interesting”. This point was also made in 1992 by Knight who stated that when he had made enquiries concerning ethical issues the authors often seemed “genuinely astonished and even uncomprehending that such a trivial matter should be raised” [3]. The present study shows that little has changed since 1992, as highlighted by the numerous articles still dealing with experiments in vivo and the scarce
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attention given to animal sacrifice and suffering. In fact, numbers have increased. And even if anesthesia is applied, we need to ask: is painless killing of animals justified (assuming for the
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moment that ‘painless’ is possible) in this scenario?
Most scientists realize that some animal experimentation must be performed and/or tolerated (two of the authors have in the past directly or indirectly worked on animals for scientific purposes). No
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scientific researcher is in fact immune from using some material that involved the sacrifice and/or suffering of an animal, even if only indirectly (eg. the use of monoclonal antibodies in biomolecular testing). The 3 R rationale however should be strongly enforced, particularly in the clinical
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scenario. In forensic research one should question if animal experimentation is necessary or acceptable at all.
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From an ethical point of view, two aspects should be taken into consideration. First, the protection of animals has a central position as a moral achievement of civilization, since, as Immanuel Kant
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stated, “he who is cruel to animals becomes hard also in his dealings with men” [89]. This goes hand in hand with the novel advances of neuroethology and bioethics. Secondly, the forensic
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scientist, whose background is frequently biological or biomedical, should participate in the general understanding of all animals, which is a natural consequence of co-evolution. This was eloquently described by Charles Darwin in 1871 with the phrase “sympathy beyond the confines of man” [90]. Darwin also wrote that the feeling “that is humanity to the lower animals, seems to be one of the latest moral acquisitions…this virtue [concern for lower animals], one of the noblest with which man is endowed, seems to arise incidentally from our sympathies, becoming more tender and more widely diffused, until it extends to all sentient beings” [90]. Furthermore, as already mentioned, ethology and neurobiology are showing us how non-human brains can be extremely aware of and sensitive to pain, fear and suffering. Thus, human beings, who are endowed with “intelligence” so that they can verify scientifically the suffering of other species, would seem to have a scientifically based responsibility to take care of other animal species. There is therefore a moral obligation to pursue and respect this, especially where mankind’s actual survival is not at risk. This more ethical issue, combined with the practical problems associated with animal experimentation in forensic sciences, must constantly inform the undertaking of all scientific
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research. And scientific journals have a responsibility to ensure adequate surveillance of these issues.
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[50] N. Arican, M. Kaya, R. Kalayci, M. Kucuk, V, Cimen, I. Elmas, Effect of acute cold exposure on blood-brain barrier permeability in acute and chronic hyperglycemic rats. Forensic Sci Int 125
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[52] T. Gos, R.Hauser, M. Krzyzanowski, The post-mortem concentration of glutamate in the structure of rat brain as an exponent of short aversive sensory stimulation preceding death. Forensic Sci Int 123 (2001) 130-134.
[53] T.Hayashi, K. Ikematsu, Y. Abe, Y. Ihama, K. Ago, M. Ago, T.Miyazaki, M. Ogata, Temporal changes of the adrenal endocrine system in a restraint stressed mouse and possibility of postmortem indicators of prolonged psychological stress. Legal Medicine 16 (2014) 193-196 [54] N.S. Canteras, S.R. Mota-Ortiz and S.C. Motta, What ethologically based models have taught us about the neural systems underlying fear and anxiety. Braz J Med Biol Res 45(4) (2012) 321327. [55] H. Ferdowsian and D. Merskin, Parallels in Sources of Trauma, Pain, Distress, and Suffering in Humans and Nonhuman Animals. Journal of Trauma & Dissociation 13:4 (2012) 448-468.
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[56] P. Veinante, I. Yalcin and M.Barrot, The amygdala between sensation and affect: a role in pain. Journal of Molecular Psychiatry (2013) 1:9. [57] W.M.S. Russell, R.L. Burch, The principles of humane experimental techniques, London: Methuen, 1959
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[58] V. Baumans, Use of animals in experimental research: an ethical dilemma? Gene Therapy (2004) 11, S64- S66
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[59] N.H.Franco. Animal Experiments in Biomedical Research: A Historical Perspective. Animals
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[60] P. Willner, P.J. Mitchell, The validity of animal models of predisposition to depression, Behav
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[61] H. Anisman, K. Matheson, Stress, depression, and ahedonia: caveats concerning animal models, Neurosci Biobehav Rev 29 (2005) 525-46
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[62] B. Vollmayr, M.M. Mahlstedt, F.A. Henn, Neurogenesis and depression: what animal models tell us about the link, Eur Arch Psychiatry Clin Neurosci 257 (2007) 300-3
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[63] F.A. Bandak, Shaken baby syndrome: a biomechanics analysis of injury mechanisms, Forensic
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[64] R. Lehman, E. Loder, Missing clinical trial data, BMJ 344 (2012) d8158 [65] G.L. Francione, The use of nonhuman animals in biomedical research: necessity and justification, J Law Med Ethics 35 (2007) 241-8 [66] Daubert v. Merrell Dow Pharmaceuticals, Inc., 509 US 579. (1993). [67] R. Munro and H. M. C. Munro, Some Challenges in Forensic Veterinary Pathology: A Review. J. Comp. Path. 149 (2013) 57-73.
[68] Y. Kakimoto, T. Tsuruyama, M. Miyao, H. Abiru, S. Sumiyoshi, H. Kotani, H. Haga, K. Tamaki, The effectiveness and limitations of triphenyltetrazolium chloride to detect acute myocardial infarction at forensic autopsy, Am J Forensic Med Pathol 34 (2013) 242-7 [69] K.L. Stokes, S.L. Forbes, M. Tibbett, Human versus animal: contrasting decomposition dynamics of mammalian analogues in experimental taphonomy, J Forensic Sci 58 (2013) 583-91 [70] J. Seok et al., Genomic responses in mouse models poorly mimic human inflammatory diseases, Proc Natl Acad Sci USA 110 (2013) 3507-12 15 Page 15 of 21
[71] E. Boghossian, R. Clement, M. Redpath, A. Sauvageau, Respiratory, circulatory and neurological responses to hanging: a review of animal models, J Forensic Sci 55 (2010) 1272-7 [72] J.R. Jauchem, Repeated or long-duration TASER® electronic control device exposures: academia and lack of respiration, Forensic Sci Med Pathol 6 (2010) 46-53
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[73] E.M. Schotsmans, J. Denton, J. Dekeirsschieter, T. Ivaneanu, S. Leentjes, R.C. Janaway, A.S. Wilson, Effects of hydrated lime and quicklime on the decay of buried human remains using pig
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[74] M.C. Morganti-Kossmann, E. Yan, N. Bye, Animal models of traumatic brain injury: is there an
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optimal model to reproduce human brain injury in the laboratory? Injury 41 (2010) S10-3 [75] D. Cerretani, I. Riezzo, A.I. Fiaschi, F. Centini, G. Giorgi, S. D’Errico, C. Fiore, S.B. Karsch, M.
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Neri, C. Pomara, E. Turillazzi, V. Fineschi, Cardiac oxidative stress determination and myocardial morphology after a single ecstasy (MDMA) administration in a rat model, Int J Legal Med 122
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(2008) 461-9
[76] L. Harsanyi, Differential diagnosis of human and animal bone, in: Histology of
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Ancient Human Bone: Methods and Diagnosis, Springer-Verlag, Berlin (1993) pp.
[77] D. Raymond, C. Van Ee, G. Crawford, C. Bir, Tolerance of the skull to blunt ballistic temporo-
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parietal impact, J Biomech 42 (2009) 2479-85 [78] N. Yoganandan, F.A. Pintar, Biomechanics of temporo-parietal skull fracture, Clin Biomech 19 (2004) 225-39
[79] D.J. Carr, A. Wainwright, Variability of simulants used in recreating stab events, Forensic Sci Int 210 (2011) 42-6
[80] M.G. Perdekamp, B.P. Kneubuhel, T. Ishikawa, H. Nadjem, J. Kromeier, S. Pollak, A. Thierauf, Secondary skull fractures in head wounds inflicted by captive bolt guns: autopsy findings and experimental simulation, Int J Legal Med 124 (2010) 605-12 [81] E.J. Sharkey, M. Cassidy, J. Brady, M.D. Gilchrist, N. NicDaeid, Investigation of the force associated with the formation of lacerations and skull fractures, Int J Legal Med 126 (2012) 835-44
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[82] Z. Asgharpour, P. Zioupos, M. Graw, S. Peldschus, Development of a strain rate independent material model of human cortical bone for computer-aided reconstruction of injury mechanisms, Forensic Sci Int 236 (2014) 109-16 [83] M.J. Thali, B.P. Kneubuhel, U. Zollinger, R. Dirnhofer, The “skin-skull-brain model”: a new
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instrument for the study of gunshot effects, Forensic Sci Int 125 (2002) 178-89 [84] S.N. Kunz, J. Adamec, B. Zinka, D. Munzel, P.B. Noel, S. Eichner, A. Manthei, N. Grove, M.
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Graw, P. Peschel, Wound ballistic evaluation of the Taser ® XREP ammunition, Int J Legal Med 127 (2013) 119-26
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[85] M. Takamiya, N. Nakayashiki, K. Dewa, Hypothalamic transcript profiling in hypothermia using SuperSAGE, J Forensic Leg Med 19 (2012) 396-401
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[86] Y. Maeno, M. Isawa, H. Inoue, Direct effects of methamphetamine on hypertrophy and microtubules in cultured adult rat ventricular myocytes, Forensic Sci Int 113 (2000) 239-43
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[87] B.P. Kneubuehl, M.J. Thali, The evaluation of a synthetic long bone structure as a substitute for human tissue in gunshot experiments. Forensic Sci Int 138 (2003) 44-49.
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[88] E.M. Hassler, K. Ogris, A. Petroch, B. Neumayer, T. Widek, K. Yen, E. Scheurer, Contrast of
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artificial subcutaneous hematomas in MRI over time. Int J Legal Med (2014) [Epub ahead of print]. [89] I. Kant, 1785 [LE] 212 (27:459), Lecture on Ethics translated and edited by P. Health and J.B.
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Schneewind,Cambridge: Cambridge University Press, 1997 [90] C. Darwin, The descent of man, and selection in relation to sex, 1871
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Legends to figures
Fig. 1: species and number of animals sacrificed for the purpose of the experiment
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Fig. 3: trend of animals sacrificed in 15 years of forensic research
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Fig. 2: Percentage of animals sacrificed for the different types of studies
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unspecified/unknown relation of death of the animal to the experiment
1
ante-mortem experiment
post-mortem experiment
29
15
2
11
121
63
16
29
78
47
11
7
4
1
75
35
5
Legal Medicine Medicine Science & Law
70
57
7
3
2
Science & Justice Forensic Science Med and Path
8
3
13
10
TOTAL
404
236
13
17
3
1
1
35
0
4
2
1
0
0
5
0
1
2
0
105
20
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an
0
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death related to the experiment
death unrelated to the experiment (post mortem)
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American Journal of Forensic Medicine & Pathology Forensic Science International International Journal of Legal Medicine Journal of Forensic and Legal Medicine Journal of Forensic Science
N° of publications
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Review of years 2000-2014
43
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Table 1: journals, number of articles involving animal experimentation and type
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unspecified)
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of experimental project (death related or unrelated to the experiment or
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Toxicology PMI
Wound
Thermal
wounds
healing
injury
Mice
65
43
70
334
Rat
229
132
438
521
Rabbit
136
60
129
6
Pig
128
160
24
1
NA
NS
Mechanical asphyxia
Other
48
NA
NS
NA
45
30
NS
NA
NS
2
NA
87
12
26
NS
NA
NS
40
12
46
217
86
229
40
19
9
d
26
Monkey 10
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Macaques
NS
te
8
NA
49
1
Calves
sharp
us
NS
6
and
an
NA
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NS
Dog
Electricity
wound
NA
Sheep
Gunshot
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ANIMAL
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Blunt
Taphonomy
10
Baboons TOTAL
582
422
661
862
9
0
49
78
0
26
45
2
0
41
87
40
Table 2: number of animals sacrificed in the different areas of research, and non use (NA) or non specified (NS) use of anaesthesia
Field of research
Alternative techniques
Assessment of injuries/biomechanical studies
Biomechanical simulators [77,81]
Stab injuries
Simulating/backing materials [79]
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Analysis of cranial fracture
Artificial models (gelatin balls and skull-brain model) [78,80]
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Bio fidelic finite elements [82]
Artificial “skin-skull-brain” model [83]
Gunshot
Surrogate materials (synthetic foam, ballistic
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Wound ballistic impact
gelatin and soap) [84]
In vitro cell cultures [74, 86]
Hypothermia
Methods of serial analysis of gene expression [85]
Gunshot experiment
Non-biological bone model (polyurethane) [88]
Subcutaneous hematoma
MRI of artificial subcutaneous hematoma [87]
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Traumatic brain injury
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Table 3: alternative methods to the use of animals in forensic research
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S0379-0738(15)00259-5 http://dx.doi.org/doi:10.1016/j.forsciint.2015.06.024 FSI 8049
To appear in:
FSI
Received date: Revised date: Accepted date:
19-8-2014 24-6-2015 25-6-2015
Please cite this article as: C. Cattaneo, E. Maderna, A. Rendinelli, D. Gibelli, Animal experimentation in forensic sciences: how far have we come?, Forensic Science International (2015), http://dx.doi.org/10.1016/j.forsciint.2015.06.024 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Animal experimentation in forensic sciences: how far have we come?
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Prof. Cristina Cattaneo LABANOF, Laboratorio di Antropologia e Odontologia Forense Sezione di Medicina Legale
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Corresponding author:
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LABANOF, Laboratorio di Antropologia e Odontologia Forense Sezione di Medicina Legale Dipartimento di Scienze Biomediche per la Salute Università degli Studi di Milano
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C Cattaneo, E Maderna, A Rendinelli, D Gibelli
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Dipartimento di Scienze Biomediche per la Salute Università degli Studi di Milano V. Mangiagalli 37, Milan - Italy Phone number: +39-02-50315679 Fax number: +39-02-50315724 [email protected]
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Abstract
In the third millennium where ethical, ethological and cultural evolution seem to be leading more
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and more towards an inter-species society, the issue of animal experimentation is a moral dilemma. Speaking from a self-interested human perspective, avoiding all animal testing where
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human disease and therapy are concerned may be very difficult or even impossible; such testing may not be so easily justifiable when suffering – or killing – of non human animals is inflicted for
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forensic research.
In order to verify how forensic scientists are evolving in this ethical issue, we undertook a systematic review of the current literature. We investigated the frequency of animal
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experimentation in forensic studies in the past 15 years and trends in publication in the main forensic science journals.
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Types of species, lesions inflicted, manner of sedation or anesthesia and euthanasia were examined in a total of 404 articles reviewed, among which 279 (69.1%) concerned studies
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involving animals sacrificed exclusively for the sake of the experiment. Killing still frequently includes painful methods such as blunt trauma, electrocution, mechanical asphyxia, hypothermia,
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and even exsanguination; of all these animals, apparently only 60.8% were anesthetized. The most recent call for a severe reduction if not a total halt to the use of animals in forensic sciences was made by Bernard Knight in 1992. In fact the principle of reduction and replacement, frequently respected in clinical research, must be considered the basis for forensic science research needing animals.
Keywords: ethics, forensic pathology, forensic sciences, animal experimentation
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Introduction
Hundreds of millions of animals are used every year in laboratories all over the world, with a
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disproportionately small number of 11.5 million used in the European Union [1, 2]. The need for fast, scientific improvement has spread this pandemic to virtually every field of science, apparently fuelled by the illusion that the use of animals may quickly lead to sound and useful results.
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However, if in clinical research the sacrifice and suffering of non-human animals may be
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considered by a majority as secondary to the therapeutic advantages which may be derived, in forensic sciences the use of animal models can, in most instances, be questionable. In 1992
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Bernard Knight had already focused on this issue, and recognized that the forensic field had to face the thorny topic of animal rights [3]. Interestingly, very relevant arguments against the use of animals in forensic experimentation have to do not only with ethics but also with practical limits. Knight in fact had already observed that “a vast amount of published material using animal experimentation seems to have little practical relevance, other than to expand the curriculum vitae and the career prospects of the researcher” [3]. This point of view has been more generally stressed by Pound et al. who in 2004 shed doubts on the presumed validity of animal experimentation even in the clinical field [4]. Knight raised in 1992 some additional issues which need to be reconsidered, especially now, after more than 20 years, that the use of animals in forensic sciences is indeed continuing if not increasing: “painful, sometimes mutilating experiments on conscious animals” in order to obtain “tenuous potential benefit to some medico-legal problem” cannot be condoned, particularly if one considers that such works “are not regularly used in routine forensic practice” and just “gather dust in university libraries”. Furthermore, the pitfalls encountered when extrapolating results obtained on animals to the human situation have to be considered, particularly in the forensic 3 Page 3 of 21
field. Knight’s conclusion was not to accept such works unless there was a compelling reason and the results provided important advances in the practice of forensic science [3]. In his 1992 article Knight describes some cases of studies performed on animals. The infliction of wounds and burns for dating lesions by histological and histochemical techniques, and the
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“classical research” on drowning unanaesthetized dogs are given as examples of rather dubious correspondence between scientific data on animals and humans [3]. In 1992 the pointless abundance of such experimentation had led the Editor of one of the most important forensic
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journals to take a stance on this issue, discouraging animal testing; but what has happened since? Was the call taken/listened to? Has the forensic scientific world modified and evolved its attitude
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towards animal experimentation?
Our review shows that it has not. Literature still provides plenty of such cases, for example administration of pesticides [5,6,7], ante mortem production of traumatic brain injury [8,9,10],
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blunt injury to the precordial regions in dogs [11,12] air embolism in rabbits [13], gunshot or stab wounds to pigs [14,15]. In addition several experimental protocols concerning the assessment of the effects of hanging [16], electrocution [17,18,19], myocardial ischemia [20], asphyxia [21,22]
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trauma [23,24], drowning [25,26], hypovolemic shock [27] in mice, rabbits and dogs can still be
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found.
This short review briefly aims at illustrating how little has changed in the past 15 years; in addition
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to what had been anticipated by Knight, we wish to stress the exact same point, this time with numbers and details at hand.
Materials and methods
A meta-analysis of scientific articles published between 2000 and 2014 was undertaken on Pubmed and Medline databases. Articles published in the main forensic medicine and forensic science journals which referred to animal experimentation were selected. The journals considered were “American Journal of Forensic Medicine and Pathology”, “Forensic Science International”, “International Journal of Legal Medicine”, “Journal of Forensic and Legal Medicine”, “Journal of
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Forensic Sciences”, “Legal Medicine”, “Medicine, Science and Law”, “Science and Justice”, “Forensic Science, Medicine and Pathology”. The use of animals in general was the main criterion for selecting the article. This enabled the authors to discover how much research in forensics is actually being done using animal models, uses animals which have died for reasons not related to the
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how much of this research
experiment (for example pigs accidently crushed by other pigs [28]) compared to those who have
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been used and killed for specific forensic purposes.
The different kinds of species, types of lesions, manner of sedation and killing were also taken into
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account, as well as, in the case of animals selected and sacrificed for forensic purposes, whether the experiment took place ante mortem (eg. wound healing) or post mortem (eg. taphonomy),
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and with what anaesthesia.
Results
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The review yielded 404 articles. This is a probable underestimate since some articles do not bear
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“animal” or animal experimentation among the keywords, abstracts or titles. Of these, 279 (69.1%) represent experiments where the animals were specifically killed for the purpose of the
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forensic experiment, versus a more modest 105 (25.9%) where animals which had died from other causes were used. In 20 (4.9 %) of cases the specific reason why the animal was killed and how was not specified. Among the cases where animals were sacrificed for the purpose of the experiment, in 84.6% the test was performed antemortem sometimes with an unclear description of the manner of sedation (Table 1).
As can be seen in Table 2 a total of 8203 animals were sacrificed for the research: 81.3% (approx.) were rats and mice, the remaining 18.7% being rabbits, pigs, dogs, sheep, calves and monkeys (Fig. 1).
In relation to the different types of studies, 30.2% of all animals were killed for toxicological tests ranging down to 9.5% for the study of perimortem trauma, and to 3.5% for electrocution research (Fig. 2). Other areas concerned wound healing processes (14.6%), estimation of the post mortem interval (23.4%), hypothermia and hyperthermia (4.9%) and mechanical asphyxia (5.1%) (Fig. 2). Figure 3 shows the apparent trend in the number of articles reporting the use of animals. Table 2 shows the number of animals sacrificed, of which species and whether anesthesia was used for the experiment or if information of this sort was not given, as well as the area of forensic science 5 Page 5 of 21
the article involved. Mice, rats and rabbits were the most frequent animals used; however there is still a surprising use of dogs, rabbits and monkeys.
Research areas are still quite varied and
include experiments on wound healing (which involves inflicting lacerations or bone fractures on live animals and sacrificing them at set intervals), thermal injury, electrocution, various types of mechanical trauma and drugs and toxicology. Only in 4984 (60.8%) cases were the animals
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clearly anesthetized.
“Euthanasia” methods included the “humane” injection of sodium pentobarbital, but also
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exsanguination and blunt trauma [29,30,31]. In some cases, the type of trauma examined or the actual experiment ended up being the cause of death [32,33,34]. It is surprising how many details
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are omitted by various authors, even the number of animals used, and whether the animal was anesthetized and if so how it was euthanized. Table 2 shows the correlation between the species involved in the experiments, specific fields of research and type of anesthesia: experiments where is
not
used
or
mentioned
concern
wound
assessment
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anesthesia
[35],
toxicology
[36,37,38,39,40,41,42] and sometimes taphonomy/post mortem interval [43,44,45]; experiments as well as painful procedures involve traumatic injuries [46,47,48], asphyxia [49], hypothermia
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[50,51] and even psychological stress [52,53]) where the authors provide no specific information on treatment of the animals. Moreover the numbers reported in Table 2 are underestimated since
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the exact number of animals recruited for some studies is sometimes not mentioned.
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Approval by various ethics committees is stated by many authors. Rarely do journals request proof of that approval. The large differences in regulations across the world make local ethics approval a
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subjective standard. Journals should develop their own standards to such research as well and apply it .
Cruel experiments are still inflicted on animals in forensic research, the following being just a handful of examples. Some authors in studying erythrocytes and endothelial cells of electrocuted dead rabbits report that “one group of rabbits as a normal control was put to death by dislocating the cervical vertebra; model 1, 2, 3, 4 were electrocuted; the rabbits of model 5 were put into a bag and allowed to fall from a 40 m height, one by one; model 6: rabbits were burned to death using ethanol; model 7: poisoning with sodium nitrite; model 8: the cause of death of this group was asphyxia; rabbits were put into a small container with carbon dioxide” [19]: no mention of anaesthesia is made nor was it requested by the journal. In another toxicological study, the authors investigated the effects of sarin-like and soman-like compounds – highly toxic organophosphorus agents used in chemical warfare – by intravenous injection in rats [42]. To investigate mRNA expression of IL-10 during fracture healing for wound age estimation Takamiya and colleagues [48] reproduced an animal model of fracture in mice by sacrificing the 6 Page 6 of 21
last experimental group after 240 hours without specifying if the animals are kept under anesthetic for the duration of the experiment. In a further experiment mice were restrained in a polypropylene centrifuge tube with air holes for 1h/daily from 1 day to 4 weeks - as a model of psychological stress [53]. However as previously mentioned, only in 60.8% of cases is there clear mention that the animals
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have been anesthetized; in a disturbing 20.7% the presence of anesthesia was not specified: this information is worrying, since it suggests that in some experiments, the animals were clearly not
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anesthetized, although they were subjected to painful procedures.
Along with the actual pain the animals would certainly undergo for most experiments if no
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anesthetic were given, one should also bear in mind the strong ethological and neurological evidence that non human animals can anticipate suffering and can experience fear [54,55,56].
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Discussion
Animal experimentation for medical and scientific purposes has been a major topic in research
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ethics and the subject of general community debate and concern for at least 50 years [4,57,58,59]. In 1992 a discussion concerning the use of animals in forensic research was opened
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by Bernard Knight. He suggested that although the forensic context could not be considered detached from other fields of scientific research, the use of animals should be more strictly
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controlled, since forensic research does not lead to improvement of life conditions and health. In the case of clinical experiments, the use of animals has been widely analyzed and discussed by
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ethical and scientific authors. Russel and Burch (1959) suggested a compromise encompassed by the 3Rs principle of: refinement, reduction and replacement. This focused on the improvement of experimental techniques, limitation of the number of animals used and the application of alternative methods [57]. The models more commonly used for research into human disease usually must meet these three criteria, and face validity, predict validity and construct validity [60,61,62]. This means that, for animals to be a good model for investigating human disease, the animal has to show the same symptoms, the same reactions to treatment and the same aetiology as the human being. From this point of view, given the previously stated criteria, the animal model in the clinical context should provide a feasible and easily modifiable model for researchers in order to test different variables. Thus for some people (probably still a majority), there is acceptance of the sacrifice of animals for clinical trials because the results may improve human health. Forensic research does not provide advantages for health, but possible assistance in understanding criminalistic issues (eg. estimation of post mortem interval, assessment of injuries, etc.). As a consequence, the sacrifice of animals is more questionable, as are the results obtained by the studies, given the biological differences 7 Page 7 of 21
between humans and non humans. This limitation has been already faced by clinical researchers [63]. Some literature has observed that many clinical trials involving the use of animals have not been published [64] or that results are not usable [65]. In the forensic context the study becomes reliable if the data are systematically verified and if they proved to be useful in practical cases [4]. From this point of view, the history of forensic sciences has provided us with much evidence of the
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inapplicability of data obtained from studies performed on animal models, including the Daubert sentence [66] and several recent studies have again stressed the limits of this approach in
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different fields of forensic sciences, from the estimation of post mortem interval to genetics [6775]. In an article concerning the effectiveness of triphenyltetrazolium chloride to detect acute
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myocardial infarction, the authors state that “it is not realistic to apply methods confirmed in animals” [68]. Stokes et al. observed contrasting dynamics of mammalian analogues in experimental taphonomy, and state that “the results of this study showed many differences
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between porcine, bovine, and ovine SMTs (skeletal muscular tissues) when considering them as analogues for human SMT2 in decomposition soil studies”[69]. As another example there is the genomic responses in mouse models which poorly mimic human inflammatory disease, as shown
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by Seok et al. who state that “there are multiple considerations in our findings that the transcriptional response in mouse models reflects human diseases poorly including the evolutional
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distance between mice and humans, the complexity of human disease, the inbred nature of the mouse model” [70]. Even the microscopic anatomical basis of bones [76] and brain [74] show
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relevant differences between pigs and rodents and human beings and it has been shown that
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biomechanics as studied in non human animals may be useless for humans [63]. On the other hand, applying alternative methods rather than using animals has provided, in the forensic field, important and reproducible results, as presented in Table 3 [74,77-88]. And for many less elaborate projects, animals who have died from other causes can be used. In the clinical scenario, systematic reviews and meta-analyses of previous animal experiments in fact may ensure that new experiments will not be conducted in order to answer questions already contemplated making it thus possible to reduce the number of animals sacrificed. Reviews on this topic however are worryingly uncommon - only two studies in the past twelve years [71,72]. None, to our knowledge, have been performed for the forensic scenario.
If the avoidance of animal testing when disease and therapy are concerned is a difficult and unresolved issue, it should not be so when suffering – or in fact killing - is inflicted on living animals for questionable forensic purposes. The forensic context has been considered for a long 8 Page 8 of 21
time a “no-man’s land” where animal ethical issues somehow seem to be less “interesting”. This point was also made in 1992 by Knight who stated that when he had made enquiries concerning ethical issues the authors often seemed “genuinely astonished and even uncomprehending that such a trivial matter should be raised” [3]. The present study shows that little has changed since 1992, as highlighted by the numerous articles still dealing with experiments in vivo and the scarce
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attention given to animal sacrifice and suffering. In fact, numbers have increased. And even if anesthesia is applied, we need to ask: is painless killing of animals justified (assuming for the
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moment that ‘painless’ is possible) in this scenario?
Most scientists realize that some animal experimentation must be performed and/or tolerated (two of the authors have in the past directly or indirectly worked on animals for scientific purposes). No
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scientific researcher is in fact immune from using some material that involved the sacrifice and/or suffering of an animal, even if only indirectly (eg. the use of monoclonal antibodies in biomolecular testing). The 3 R rationale however should be strongly enforced, particularly in the clinical
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scenario. In forensic research one should question if animal experimentation is necessary or acceptable at all.
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From an ethical point of view, two aspects should be taken into consideration. First, the protection of animals has a central position as a moral achievement of civilization, since, as Immanuel Kant
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stated, “he who is cruel to animals becomes hard also in his dealings with men” [89]. This goes hand in hand with the novel advances of neuroethology and bioethics. Secondly, the forensic
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scientist, whose background is frequently biological or biomedical, should participate in the general understanding of all animals, which is a natural consequence of co-evolution. This was eloquently described by Charles Darwin in 1871 with the phrase “sympathy beyond the confines of man” [90]. Darwin also wrote that the feeling “that is humanity to the lower animals, seems to be one of the latest moral acquisitions…this virtue [concern for lower animals], one of the noblest with which man is endowed, seems to arise incidentally from our sympathies, becoming more tender and more widely diffused, until it extends to all sentient beings” [90]. Furthermore, as already mentioned, ethology and neurobiology are showing us how non-human brains can be extremely aware of and sensitive to pain, fear and suffering. Thus, human beings, who are endowed with “intelligence” so that they can verify scientifically the suffering of other species, would seem to have a scientifically based responsibility to take care of other animal species. There is therefore a moral obligation to pursue and respect this, especially where mankind’s actual survival is not at risk. This more ethical issue, combined with the practical problems associated with animal experimentation in forensic sciences, must constantly inform the undertaking of all scientific
9 Page 9 of 21
research. And scientific journals have a responsibility to ensure adequate surveillance of these issues.
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Legends to figures
Fig. 1: species and number of animals sacrificed for the purpose of the experiment
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te
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Fig. 3: trend of animals sacrificed in 15 years of forensic research
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Fig. 2: Percentage of animals sacrificed for the different types of studies
18 Page 18 of 21
unspecified/unknown relation of death of the animal to the experiment
1
ante-mortem experiment
post-mortem experiment
29
15
2
11
121
63
16
29
78
47
11
7
4
1
75
35
5
Legal Medicine Medicine Science & Law
70
57
7
3
2
Science & Justice Forensic Science Med and Path
8
3
13
10
TOTAL
404
236
13
17
3
1
1
35
0
4
2
1
0
0
5
0
1
2
0
105
20
M
an
0
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death related to the experiment
death unrelated to the experiment (post mortem)
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American Journal of Forensic Medicine & Pathology Forensic Science International International Journal of Legal Medicine Journal of Forensic and Legal Medicine Journal of Forensic Science
N° of publications
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Review of years 2000-2014
43
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Table 1: journals, number of articles involving animal experimentation and type
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unspecified)
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of experimental project (death related or unrelated to the experiment or
19 Page 19 of 21
Toxicology PMI
Wound
Thermal
wounds
healing
injury
Mice
65
43
70
334
Rat
229
132
438
521
Rabbit
136
60
129
6
Pig
128
160
24
1
NA
NS
Mechanical asphyxia
Other
48
NA
NS
NA
45
30
NS
NA
NS
2
NA
87
12
26
NS
NA
NS
40
12
46
217
86
229
40
19
9
d
26
Monkey 10
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Macaques
NS
te
8
NA
49
1
Calves
sharp
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NS
6
and
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NA
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NS
Dog
Electricity
wound
NA
Sheep
Gunshot
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ANIMAL
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Blunt
Taphonomy
10
Baboons TOTAL
582
422
661
862
9
0
49
78
0
26
45
2
0
41
87
40
Table 2: number of animals sacrificed in the different areas of research, and non use (NA) or non specified (NS) use of anaesthesia
Field of research
Alternative techniques
Assessment of injuries/biomechanical studies
Biomechanical simulators [77,81]
Stab injuries
Simulating/backing materials [79]
20 Page 20 of 21
Analysis of cranial fracture
Artificial models (gelatin balls and skull-brain model) [78,80]
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Bio fidelic finite elements [82]
Artificial “skin-skull-brain” model [83]
Gunshot
Surrogate materials (synthetic foam, ballistic
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Wound ballistic impact
gelatin and soap) [84]
In vitro cell cultures [74, 86]
Hypothermia
Methods of serial analysis of gene expression [85]
Gunshot experiment
Non-biological bone model (polyurethane) [88]
Subcutaneous hematoma
MRI of artificial subcutaneous hematoma [87]
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an
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Traumatic brain injury
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Table 3: alternative methods to the use of animals in forensic research
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