CHAPTER 6
SELECTED CASE STUDIES: INSIGHTS AND ANALYSES
Print
The following 13 Case Studies are only a small selection of
research papers detailing experiments in France using dogs and
cats, in the period 1999 to 2002. It is notable that many of
the papers failed to provide adequate details of the handling
and housing of animals; their sources of supply; and whether
and what analgesics were provided after the immediate post-operative
period. Some papers even omitted to state how many animals were
used in the experiments.
Case Studies using Dogs
Dogs, usually beagles, who are relatively small and good-natured
and therefore easy to handle, are the favourite species for
a variety of research purposes. However, mongrels and greyhounds
are also used. Reading the research literature it is sometimes
difficult to know where mongrel dogs are obtained. Dogs are
used in a variety of research applications, of an applied and
fundamental kind, including to study the circulation of the
blood and the function of the heart, the nervous system, the
kidney and the digestive system, and the ways in which drugs
act.
Case Study One -- Factors influencing coronary
blood vessels in a dog ‘model’ of heart failure
Location: INSERM U400, Creteil
Funding: INSERM
Purpose of the research
Using dogs as a ‘model’ of heart failure in order
to characterise the role of local substances such as calcium
and transmitters such as nitric oxide (NO) which potentially
influence the variation in size of blood vessels in the heart.
No direct claims are made for the medical relevance of the study.
The experiments
Arteries are able to change their diameters in order to respond
to both local and distant need for increased blood flow and
delivery. It has become apparent that local chemicals play a
significant part in this dilation and constriction capability.
There are other nervous and hormonal factors that play a role
in these events but the authors were interested in local factors
especially nitric oxide (NO), involved in arterial dilation
(vasodilation) in an animal ‘model’ of heart failure.
Hypertension and angina pectoris involve a closing of the diameter
of blood vessels, especially those supplying the heart (the
coronary vessels). Various drug treatments have attempted to
reverse this effect by either inhibiting constriction or producing
dilation locally, by influencing the muscles in the walls of
the coronary arteries.
One class of drugs increases blood flow to the heart by blocking
calcium channels in the arterial muscle walls and the authors
used a new generation of such calcium channel blockers -- amlodipine
-- to investigate the role of a local transmitter, the gas nitric
oxide, in the vessel wall. In addition the researchers wished
to see if heart failure alters the response to amlodipine.
Nine mongrel (mixed-breed) dogs (presumably not purpose bred)
weighing between 25 and 35 kg were used. The sexes of the dogs
was not disclosed. Housing and handling of the dogs was not
described. Major chest surgery was carried out to insert various
instrumentation in the animals. This included a catheter in
the aorta, electrical pacing leads into the right ventricle
of the heart. Pressure measuring devices were also placed in
the left ventricle, a small diameter catheter was inserted into
the circumflex coronary artery and a flow probe was also placed
in this artery. In addition, the changes in size of the coronary
vessel were also monitored by means of crystals placed in the
lower section of the coronary artery. Analgesia was given after
surgery was completed but the authors do not mention if this
was maintained in the subsequent period of experimentation.
Antibiotics were administered daily for two weeks post-operatively,
presumably to prevent infections.
The results
Various test solutions were directly injected into the indwelling
catheters and the coronary blood vessel responses were captured
by recording with the various probes. After a series of tests
were carried out in order to assess ‘baseline’ responses
and characteristics of the coronary vessels in groups of dogs,
the heart rate was altered by a miniature pacemaker device linked
to the pacing leads which were inserted in the ventricle. The
dogs’ hearts were paced at 240-250 beats per minute (the
normal range for the size of dog described is 100-120 beats
per minute) continuously for three weeks. This increase in the
pace of the heart rate led, at the end of three weeks, to a
state of heart failure. At this time various measurements which
were undertaken to assess baseline conditions of the animal’s
hearts were repeated in order to measure the effect of the artificially-induced
heart failure.
Impact on the dogs
The animals described are not only subjected to major chest
surgery and heavy instrumentation which will be stressful and
possibly painful (no analgesia is described as being given after
the immediate post-operative period) but also the induced heart
failure will have a significant effect upon the dogs’
well being.
Chronic ventricular pacing will produce many of the signs and
symptoms of human dilated cardiomyopathy. These include exertional
breathing difficulties (dyspnoea) and ascites (retention of
fluid within the body -- this can be massive and lead to severe
pain and discomfort) as well as what the authors refer to as
‘haemodynamic changes’.
Critique of the research
Various test chemicals were directly infused into the blood
vessels supplying the heart -- this is highly artificial and
has little bearing upon normal or disease processes. As a result
of the various experiments the authors point out that NO appears
to impact on the blood vessels in a different manner in the
heart failure dogs as against normal animals, but how or if
this translates to clinical treatment is far from clear. There
are also species differences in the way that blood vessels and
cells comprising the heart respond to changes in the composition
of the blood (110).
There also exists immense potential for the use of fully informed
human volunteers in testing local effects of drugs and other
molecules within various blood vessels. A workshop held in 1993
discussed a number of direct, local intra-arterial methods to
understand the role of endothelial cells and the diameter of
vessels (111), especially in response to NO. Infusions of the
NO synthase inhibitor NG-monomethyl-L-arginine into the brachial
artery of volunteers in low and safe doses have facilitated
the precise exploration of the L-arginine pathway in humans.
By the use of angiography it has become possible to study coronary
vessel size and response to potential local substances in healthy
humans (112). Human patients can also participate to give clinically
relevant data.
In addition, the authors point out that they used the direct
method of applying test chemicals to the heart in order to assess
local effects rather than systemic -- it would have therefore
been more appropriate to use an in vitro method which is more
humane and allows local effects to be assessed directly.
In vitro preparations of isolated coronary microvessels have
been used to follow the responses to various calcium channel
blockers including amlodipine (113 - 114). In addition there
are a significant number of publications from groups in Europe
who are using endothelial cells from blood vessels and also
cardiac muscle cells to understand local effects, in particular
those of NO, and the role of genetic factors in various kinds
of heart disease (115- 116- 117- 118).
S Champagne et al [2002] Reduced coronary vasodilator responses
to amlodipine in pacing-induced heart failure in conscious dogs:
role of nitric oxide, British Journal of Pharmacology, 136,
264-270
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Case Study Two
-- The role of peptides in the changes of the diameter of coronary
blood vessels
Location: INSERM U400 and the Departement de Pharmacologie,
Hôpital Kremlin-Bicentre, Kremlin-Bicentre
Funding: INSERM and Zeneca Pharma, Cergy
Purpose of the research
To investigate the role of specific peptides -- the kinins and
their receptors -- on the diameter of coronary vessels. Use
was made of a known agonist and antagonist of kinin receptors
and other local hormones to characterise kinin receptors’
role in the coronary circulation in the dog. No medical relevance
was described for the study.
The experiments
The kinins, bradykinin and Lys-bradykinin (also known as kallidin),
play a wide range of roles in the body, including the contraction
of smooth muscle, pain initiation in some situations, and they
are important mediators of the inflammatory response. Kinins
are also known to increase the permeability of small blood vessels
-- the microvasculature -- induce vasodilation (widening of
the diameter of blood vessels) and to stimulate the release
of nitric oxide (NO), an important transmitter involved in blood
vessel diameter change. The study intended to look at the effects
at the local level, as in Case Study One, of ‘local hormones’
which have been shown to influence the change in size of the
diameter of blood vessels which supply the heart.
Receptors to the kinins are classified as being of two kinds,
according to their sensitivity to various agonists or antagonists
(119). B1 receptors respond to des-Arg(9)-bradykinin and Lys-des-Arg(9)-bradykinin
which are natural metabolites of Lys-bradykinin and bradykinin;
whilst B2 receptors are sensitive to bradykinin and kallidin.
Eleven mongrel dogs were used. Animals were heavily instrumented.
Under anaesthesia the chest was opened and a pressure transducer
was implanted in the chest cavity, plastic catheters were placed
in descending aorta and the left atrium of the heart. A further
plastic catheter was inserted in the circumflex coronary artery
and a flow probe was implanted around the circumflex coronary
artery. Two ultrasonic crystals were placed around the coronary
artery in order to measure coronary blood flow velocity and
coronary vessel diameter. Finally, an inflatable cuff was placed
around the coronary artery, away from the catheters and crystals,
in order to alter the blood flow through the artery.
Wires and catheters from the heart were passed under the skin
to the shoulder region. Morphine was given post-operatively
but no indication of long term analgesia is provided in the
publication. The authors refer to “…post-operative
care being given daily” but it is not clear what this
comprised. The antibiotic ampicillin was provided daily due
to risk of infection following the extensive surgery. Experiments
were carried out three to six weeks after surgery. In six dogs
a blood sample from the aortic catheter was taken before drug
administration to assess the chemical and cellular composition
of the blood. Then the test chemicals were delivered by syringe
to the coronary blood vessels. In these animals des-Arg(9)-bradykinin
and bradykinin were given in a range of doses in order to provide
a standardised set of dose-response recordings.
A second set of experiments were undertaken in six dogs -- it
is not stated if these were the same dogs as those used for
base-line drug responses -- in order to establish the specificity
of the coronary response to bradykinin and des-Arg(9)-bradykinin,
by infusion of a bradykinin-B2 antagonist.
A third set of experiments, also using six dogs assessed the
flow-dependent nature of the transmitters des-Arg(9)-bradykinin
and bradykinin by using the cuff-occluder. A fourth set of experiments
addressed the potentiating effect of bradykinin which angiotensin-converting
enzyme (ACE) has by use of the ACE inhibitor lisinopril which
was infused into the coronary vessels. Finally six dogs were
used to assess the role of nitric oxide (NO) in the effects
of the stimulation of the B1 receptor. NO activity was blocked
by the infusion of the NO synthase-inhibitor LNA.
Five dogs who were not instrumented had their hearts removed
under anaesthesia and the circumflex artery was dissected and
rings of the vessel were cut and after preparation were placed
in an organ chamber with either a control solution or one containing
the potent B1 receptor antagonist des-Arg(9)-[leu(8)]-bradykinin
in order to measure vessel diameter responses.
From the description in the text of the publication it is apparent
that the same six dogs were used for all the experimental tests.
All animals that were instrumented were killed at the end of
the experiments and the position and orientation of the flow
crystals were examined. The coronary artery and left ventricle
were also examined in order to find if any damage followed from
the instrumentation.
The results
The injection, directly into the coronary vessels, of des-Arg(9)-bradykinin
(3 to 100 ng/kg) and bradykinin (0.1 to 10 ng/kg) did not modify
blood vessels in the body outside the circulation to the heart.
In the heart these chemicals caused an increase, in a dose-dependent
way, in the coronary blood flow velocity and the diameter of
the coronary vessels. Bradykinin was found to be more potent
than des-Arg(9)-bradykinin.
The B2 receptor antagonist used (Hoe 140) at a concentration
of 10 mg/kg of body weight abolished the effects of bradykinin
but did not influence the effects of des-Arg(9)-bradykinin.
Using the cuff occluder the researchers found that although
the increase in blood velocity brought about by both des-Arg(9)-bradykinin
and bradykinin was stopped the coronary diameter increase still
occurred.
Use of intracoronary lisinopril did not alter bradykinin responses.
In the dogs, used in the study the B1 receptors were present
in the coronary vessels and when stimulated caused vasodilation
of the coronary vessels. This change in the diameter of the
coronary vessels is mediated by NO and is not modified by ACE.
It appears that the B1 receptor stimulation causes less marked
vessel change than that brought about by B2 receptor stimulation.
Impact on the dogs
As in Case Study One, the dogs in this research were subjected
to major chest surgery and then heavily instrumented. There
was no mention of analgesia being administered after the immediate
post-operative period. Although the drugs used in the study
were applied locally, their effects may be noticed by the animals
in other areas of their body. It is well known that the kinins
have a number of effects including the production of pain (120
- 121). There are no details given as to how the dogs were killed
and autopsied at the end of the experiments and this might be
an area of concern.
Critique of the research
The principal finding of the research in this Case Study is
that intracoronary injection of des-Arg(9)-bradykinin, a bradykinin
B1 receptor agonist produces a dose-dependent vasodilation in
the coronary blood vessels in dogs. But it is known that the
B1 receptor is expressed in various ways depending on both the
species used and the tissues examined (122). It is therefore
debatable if the present study has any bearing on human patients.
In addition it is already known that human coronary vessels
contain B1 receptors which can be upregulated (increased by
cellular signals), and that they can alter their diameter in
in vitro experiments (123-124). Furthermore, there are known
to be both species and age-dependent variation in the expression
of certain bradykinin receptors (125).
A number of in vitro studies have indicated that such non-animal
methods of investigation throw important light on the role of
B1 receptors and may have important consequences for patient
care (126). Studies by Plendl and colleagues (127) have shown
that there are increased levels of kinin and kinin-forming enzyme
receptors in angiogenic endothelial cells derived from corpora
lutea, suggesting that the role of kinins is more complex in
disease than that assumed by this Case Study. A combination
of in vitro studies of human tissues and clinical investigation
obviates the need for the use of dogs and other species with
the attendant variation in response.
J B Suet al [1999] Stimulation of bradykinin B1 receptors induces
vasodilation in conductance and resistance coronary vessels
in conscious dogs: comparison with B2 receptor stimulation,
Circulation, 101, 1848-1853
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Case Study Three -- Induced kidney failure and
its impact on gut function
Location: Unite Mixte de Recherche INRA de Physiologie et Toxicologie
Experimentalis, Toulouse; The University of Sydney, Australia
and University of Georgia, USA
Funding: Grant from the Scientific Committee of the National
Veterinary School of Toulouse
Purpose of the research
To assess the influence of ‘moderate’ kidney failure
on the function of the small intestine (especially the electrical
signals) and the time taken for food to pass through the digestive
tract. The authors claim that the research described will shed
light on human kidney disease and how it might influence gut
function and “encourage further assessment of gastrointestinal
function in patients”.
The experiments
The authors of the study -- from France, Australia and the USA
-- use dogs in these experiments as surrogates for humans in
order to understand how kidney damage potentially alters a number
of aspects of gastrointestinal function. The publication cites
a number of clinical studies which suggest a relationship between
kidney disease (chronic renal failure and uraemia) and dysfunctions
of various parts of the gastrointestinal system. The authors
suggest that conservative management of gastrointestinal problems
in patients with kidney disease would benefit the long-term
health of those patients.
The study intended to evaluate the physiology of the gut (electrical
signals and transit times) in dogs who had had experimentally
induced renal failure.
An unspecified number of beagle dogs were used, supplied by
Harlan (France). In 1998-1999, the British Union for the Abolition
of Vivisection (BUAV) carried out an undercover investigation
of Harlan (UK). Their investigator worked at the company for
10 months, and the BUAV’s report revealed poor standards
of routine husbandry and care for beagle dogs. Individual dogs
were not checked daily and infections and injuries were often
diagnosed belatedly. Routine medication of dogs to prevent infections
was sometimes behind schedule by several months.
Record-keeping was inadequate in several areas. Some dog pens
were chronically infested with mice, some drains were frequently
blocked and standards of hygiene were criticised. Bad teeth,
poor coats, abscesses and overgrown claws were common in the
dogs, and staffing levels were sometimes inadequate. Some dogs
were kept in pens providing less than the official minimum space,
and environmental enrichment was substandard. Thus, even in
authorised breeding and supplying establishments animals experience
miserable lives and poor standards of care.
In this study each dog was housed individually prior to the
start of the experiments, which itself can cause distress.
Renal failure was induced by surgical removal of the right kidney
and subsequent damage to the left kidney by up to 120 puncture
wounds under general anaesthesia. Although morphine was administered
at induction of anaesthesia and immediately following surgery
there is no information in the publication about long-term analgesia.
Each dog was further fitted with electrodes one week after surgery
in order to record electrical activity from the gastrointestinal
(GI) tract. Four electrodes were fitted in the stomach and at
regions throughout the intestine (ileum, jejunum and duodenum).
Recording was carried out over four days and continuously for
22 hours. Dogs were fasted on two out of the four days. Dogs
were allowed two hours of exercise daily in an outside run during
this time.
Blood sampling was also undertaken in each animal. Six dogs
were tested for the effects of renal failure on GI electrical
activity and also for digestive function, but it was not stated
whether the same dogs were used for each of these studies. At
the end of the recording and sampling period, the dogs were
killed by intravenous injection of sodium pentobarbital and
their kidneys, stomach, small and large intestine were examined
histopathologically.
The results
All dogs were reported to have lost weight during the course
of the study although the only signs of histopathological damage
in those organs examined was reported for the remaining kidney
which showed necrosis, blood cell infiltration and damage to
the filtration mechanism. Various alterations to the electrical
activity along the GI tract were noted. Food was stored in the
colon for longer periods in the dogs with induced kidney failure
than in unoperated animals.
Impact on the dogs
The authors point out that animals suffering from kidney failure
continued to eat during the course of the study but exhibited
excessive thirst and a marked desire to urinate. They also lost
weight as a result of the surgery. Patients who have kidney
failure show symptoms of shock -- pallor, vomiting and low pulse
rate and will collapse and die without treatment. Pain might
also be experienced if analgesia was not used after the immediate
post-surgery period.
Critique of the research
The induction of so-called moderate renal failure reported by
the authors is wholly artificial -- human kidney disease is
caused by a variety of conditions which will themselves also
possibly influence the gastrointestinal tract. Ravelli reported
in 1985 that patients with chronic renal failure showed complex
changes in their gut motility, using various methods (128).
Intestinal changes in renal failure patients was also reported
in 1982 and traced to poor fat absorption (129). Using a variety
of non-invasive techniques such as the recovery of marker molecules
from urine and faeces, the 13C octanoic acid breath test and
electrogastrography, disordered gut activity and changes in
blood chemical profiles of immediate relevance to clinical treatment
of kidney failure patients, have been found in human volunteers
(130 - 131 - 132 - 133).
The authors point out that the study may have been too short
to allow an understanding of the possible interactions between
GI tract and the kidneys. Furthermore, it is not clear how the
data from this study will be able to help design treatment regimes
for patients with renal failure, where pathologies build up
over time and may be the result of many different factors including
genetic susceptibilities. Indeed the authors comment: “The
underlying mechanisms of renal failure-induced gastrointestinal
dysfunction remain unknown and may involve various neurohormonal
and luminal factors. It is not clear whether these finding are
transferable from dogs to human patients”.
Alternative, clinically based research, as mentioned above,
not involving animals would provide directly relevant data on
the role of kidney failure and gastrointestinal dysfunction
on patient health and morbidity.
H P Lefebvre et al [2001] Small bowel motility and colonic transit
times are altered in dogs with moderate renal failure, American
Journal of Physiology: Integrative and Comparative Physiology,
281, R230-R238
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Case Study Four -- To elucidate the effect of
a novel formulation of an anticoagulant on clotting parameters
and the drug’s clearance from the body
Location: Sanofi-Synthelabo, Chilly Mazarin
Funding: Sanofi-Synthelabo
Purpose of the research
To investigate the properties of an anticoagulant drug (argatroban),
in a novel formulation, when this is administered to animals
by a sub-cutaneous injected route. Indices of clotting were
measured including thrombin time and activated partial prothrombin
time as well as plasma levels in dog and primate subjects. Clinical
and other data on the broad effects of this drug on clotting
has been assembled since the 1980s (134).
Blood clotting is a complex event involving chemicals, such
as thrombin, prothrombin and a cascade of other blood-derived
molecules, together with an involvement of cells such as platelets
and neutrophils. Disorders of clotting play a significant role
in heart disease. A number of human drugs have been developed
which intervene at some point in the sequence of clotting events
in order to reduce the incidence of thromboses and hence stroke,
blood vessel and heart disease.
The experiments
Thrombin is an important part of the clotting mechanism -- it
is a serine protease enzyme responsible for the last step in
the coagulation event which forms the fibrin molecule -- integral
to the clot formation. Thrombin also has cell directed activities
and blood vessel constriction roles in the body -- all these
play major roles in wound healing and clotting. Argatroban is
a synthetic thrombin inhibitor and acts specifically against
clot-associated thrombin. This study investigates, in rats,
rabbits, beagle dogs and the primate Macaca mulatta (rhesus
monkey), the effects of a novel mixture of argatroban on a range
of clotting indices as well as its clearance from the blood
plasma and hence the body.
Female beagles were used (number not specified but the text
indicates five or more). Each dog had a surgically implanted
catheter in either the jugular or femoral vein for blood sample
collection. Antibiotics were routinely given for ten days after
the surgery. The dogs were allowed 14 days recovery time before
the experiments began. Sub-cutaneous injections comprised 14
mg argatroban in a solvent mixture which delivered the drug
as a suspension (micelle). Doses of 1 and 2 mg/kg body weight
were given.
The results
Blood samples were collected and the time taken for clotting
to occur, with and without argatroban administration, were measured.
The four species showed marked differences in biochemical responses
to the anticoagulant activities of the drug. The duration of
action of the drug on clotting in each of the species tested
depended upon the dose given, and varied from three hours in
the rat to six hours in the dog. In the dog the drug appeared
to remain in the plasma for increased periods in comparison
with other species. In the dog and the primate there was a direct
relationship between the drug concentration and the increase
in the time taken for the blood to clot. The rhesus monkey in
the study was far more sensitive to the anticoagulant effects
of argatroban than the dog.
Impact on the dogs
No details were provided by the authors as to housing or handling
of the dogs or the other species. The use of an indwelling catheter
for repeat blood sample collection does reduce the levels of
stress experienced by the dogs but it is unclear how the drug
affected well-being. Reported side effects of the injected drug
in patients (not in this specific formulation) include nausea,
pain, heart irregularities including cardiac arrest, major haemorrhage
and stroke, and obviously some of these symptoms may well be
experienced by the dogs.
Critique of the research
Argatroban is a synthetic thrombin inhibitor and has been available
in Japan since the early 1980s (135- 136). In the USA the Food
and Drug Administration recently gave permission for argatroban
to be used as an effective treatment for heparin-induced thrombocytopaenia
and thrombosis syndrome. The drug has already been subjected
to pre-clinical and clinical safety and efficacy trials using
both animals and human volunteers. Although the study was primarily
directed at deep-vein thrombosis, this could have been undertaken
in volunteer human subjects, taking account of the toxicity
and other safety data already available.
The way in which a drug is prepared -- the use of various solvents
and their delivery method -- will have variable effects in different
species. Simple extrapolation to humans from other species is
fraught with problems.
The authors draw attention to the biochemical and physiological
variability between the four species used in the study -- including
clotting time and the rate of elimination of the drug from the
body. Such variation will make translation to the human difficult.
As the authors point out: “There are few reports available
which enable calibration of anti-coagulant data obtained in
animals with human volunteers or patients”. Data from
humans, some of which will have been collected during the clinical
trials of the drug in the 1980s, are far more reliable even
though it relates to a different formulation of the drug, than
reliance on information from other species, even other primates.
It is not clear how the studies would lead to reliable information
and justify the use of four species when clearly the human is
the preferred experimental choice -- fully consenting human
volunteers could be used to follow the effects of the drug on
the course of deep-vein thrombosis (137 - 138).
C N Berry et al [2000] Anticoagulant activity and pharmacokinetic
properties of a sub-cutaneously administered mixed micellar
formulation of argatroban in experimental animals, Thrombosis
& Haemostasis, 84, 278-285
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Case Study Five -- Calcium-binding sites in
the heart: the use of calcium channel-labelling chemicals in
PET studies
Location: Services Hospitalier Frederic Joliot and the French
Atomic Agency Orsay, and Institut de Recherche Servier, Suresnes
Funding: not disclosed in the paper
Purpose of the research
To identify and count calcium ion-binding sites in heart tissue
using Positron Emission Tomography (PET) imaging together with
a calcium-specific radiolabelled binding molecule, called S12968.
The authors claim that the methodology could be useful in investigations
of human disease.
The experiments
Calcium plays a vital role in a number of basic physiological
processes in humans and other animals. The heart uses calcium
in order to carry out its pumping function. Electrically charged
molecules such as calcium enter various tissues like muscle
(including the heart) and nerve by means of specific channels,
and it is known that the density and distribution of calcium
channels is changed in disease states. Each calcium channel
can bind certain kinds of molecules at defined sites which then
block the channel and thus stop the admission of calcium ions.
In congestive heart disease, one of the channel-blocking agent
binding sites -- that for 1,4-dihydropyridine (DHP) -- is altered
such that the molecule cannot bind. This feature can be used
in conjunction with an imaging system such as PET to map the
distribution of calcium binding sites and hence the disease
state of the heart.
The authors sought to quantify DHP binding sites, using the
label S12968, in the dog heart using a multi-injection approach
together with a mathematical treatment of how such sites are
distributed through the heart.
The results
Five female beagle dogs were used (mean weight 10 kg). The dogs’
housing and handling is not described. Scans were made on a
PET scanner able to measure heart events in small animals. Radiolabelled
reagents were prepared which could identify DHP binding sites
in PET scans. Dogs had either two or three injections of the
label and were anaesthetised and ventilated whilst undergoing
the scanning procedure. Blood samples were collected from the
femoral artery during the scanning in order to assess the tissue
binding of the molecule and its clearance from the organ of
the molecule S12968.
Impact on the dogs
Although not described in detail, the animals had to be rendered
unconscious in order to allow movement-free PET scanning of
their bodies. No information was supplied on what effects, if
any, the radiolabel material had on the well being of the dogs.
Critique of the research
Two of the authors have undertaken several calcium channel-specific
labelling investigations which have appeared in print over the
past ten years. Using beagles, Crouzel and Valette reported
as long ago as 1994 that the label molecule S11568 could be
used to assess the density of DHP-binding sites . (139) The
binding of this label to DHP sites had been reported using an
in vitro approach (140). It is not clear what further insights
will be obtained using the label S12968 rather than S11568 (141).
A study in anaesthetised pigs indicated that low doses of S12968
exerted a negative inotropic effect -- that is, weakened the
muscular action and contraction of the heart -- when certain
doses were used. It is difficult to evaluate how this finding
might influence the choice of this label for use in clinical
studies of patients. Few others than the Valette group have
used these particular chemical labels so it is difficult to
judge their potential clinical value.
In addition, there are marked species variations in calcium-binding
and heart biochemistry, with rats for instance showing a profound
kidney response to S12968 at low concentrations and a marked
hypotensive effect at higher doses (142)-- neither of these
responses was reported in the present study on dogs.
Given the obvious species differences, it is therefore unclear
what value repeating in dogs studies which were earlier carried
out in pigs, has for demonstrating clinical usefulness of this
chemical label.
H Valette et al [2002] In vivo quantification of myocardial
dihydropyridine binding sites: A PET study in dogs, Journal
of Nuclear Medicine, 43, 1227-33
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Case Study Six -- The effects of exercise on
muscle metabolism in untrained dogs
Location: Unite Mixte de Recherche INRA de Physiologie et Toxicologie
Experimentalis, Toulouse
Funding: not disclosed in the paper
Purpose of the research
To understand the possible effects of exercise in untrained
dogs on muscle and its metabolites and their presence in the
blood. This is basic research of little, if any, relevance to
human medicine.
The experiments
Various changes occur to the body when exercise is undertaken.
Such changes occur in the circulation, respiration and the biochemistry
of the muscle. Human athletes have been studied for many years
and their biochemical and physiological responses to their training
monitored. In this Case Study Six healthy but sedentary beagles
(comprising four females and two males) were studied to ascertain
their response to exercise. Each dog was housed individually
prior to and after testing. The dogs were expected to race on
a track for 60 minutes with a handler at a speed of 9 km/hour.
Twelve blood samples were taken before, during and after the
race.
Blood samples were analysed for various chemicals which are
well known to be produced from the muscles during and to remain
after exercise. In addition certain blood cells and their densities
in the samples were also analysed. All the animals in the study
were found to have recovered within three hours of being tested
-- their heart rates, breathing and general physical state having
all returned to their pre-exercising levels.
The results
The experiments showed that even in ‘sedentary’
dogs the effects of vigorous running on the composition of the
blood was very mild, only producing a decrease in a muscle metabolic
substance (plasma creatinine) which had been previously reported
in 1981. Animals that are trained, such as working dogs -- huskies
and greyhounds -- tend to show increases in this substance.
It is unclear from the publication of the research what particular
value is to be gained from using ‘sedentary’ dogs
to assess changes in blood composition as a result of vigorous
exercise.
Impact on the dogs
All the dogs were untrained and therefore not used to the stresses
and unpleasantness of being exercised to exhaustion. In fact
one of the animals had to be withdrawn from study because he
or she was completely exhausted after 52 minutes of running.
It is likely that taking the blood samples (not fully described)
will cause distress to the animals. They will have experienced
sore muscles over the subsequent few days.
Critique of the research
The authors have already published data from dogs to indicate
that the exercise described in the present paper resulted in
little change in specified blood chemistry (143). It is unclear
why further experiments were undertaken which have no importance
for human or animal health.
More valuable data on the effects of exercise of relevance to
human health is to be gained from studies on trained and untrained
humans, for which there is already a rich literature. This is
especially the case where Magnetic Resonance Spectroscopy can
provide detailed information on cellular events in human volunteers
(144 - 145 - 146) with little need for invasive investigations.
Small needle biopsies can be taken from fully consenting volunteers
before and after exercise sessions which is able to shed light
on events which occur at the muscle and cellular levels. In
addition the presence of metabolites, during and after exercise
can be assessed by taking small blood samples over the duration
of and in the period of the recovery from exercise.
G P Chanoit et al [2002] Exercise does not induce major changes
in plasma muscle enzymes, creatinine, glucose and total protein
concentrations in untrained beagle dogs, Journal of Veterinary
Medicine A, 49, 222-224
------------------------------------------------
Case Studies
using Cats
Case Studies Seven to Nine concern research into the sense of
balance and the central nervous system. Cats have been used
for a number of years to establish how the sense organ involved
with balance -- the vestibular apparatus located in the inner
ear -- and the brain are able to maintain the cat’s sense
of balance and how, despite alterations to the animal’s
orientation, a stable visual field is maintained. This permits
various complex activities to be planned and executed by the
animal despite changes in body and head orientation. Part of
the interest in the cat’s sense of balance may be traced
to their remarkable ability in righting themselves when falling.
Cats always land on their feet regardless of their original
orientation.
Case Studies Ten to Thirteen involve research on vision and
sleep and the role of various brain neurotransmitters.
Case Study Seven -- Behavioural recovery of
balance after surgical damage: the role of histamine
Location: UMR 6562, University of Provence
Funding: not disclosed in the publication
Both this study and the following are by two researchers interested
in the role of brain chemicals (neurotransmitters) and distinct
areas of the brain potentially implicated in the sense of balance.
In both papers the cats are subjected to severe surgery resulting
in profound disruption to the balance sense. Animals who are
surgically damaged are allowed to survive up to one year after
the surgery during which time they show compensation in their
sense of balance and this is regarded as a ‘recovery period’.
During this time posture and movement is achieved despite the
profound damage to one side of the animal -- this might involve
other areas of the central nervous system taking over function
from the damaged side. One of the authors (Lacour) has been
using this technique of disturbing the sense of balance since
the 1980s. Neither of these two Case Studies purports to have
any medical application.
Purpose of the research
To assess the role of a particular neurotransmitter -- histamine
-- in the behavioural ‘recovery’ of a sense of balance
in cats who have had the vestibular nerve and surrounding area
of the left side of the head surgically damaged.
In cats and other mammals including humans a key role in the
sense of balance is played by the bony labyrinths -- inner areas
of the ear. Output from the bony labyrinths is sent through
the vestibular nerve to specific locations in the brain. Within
the bony labyrinths are the semi-circular canals, and when tiny
hairs within the canals are stimulated by movements of the head
this sends signals via the vestibular nerve to the brain. The
brain acts to co-ordinate signals from various regions to give
an overall sense of balance. The vestibular machinery works
together with the muscles and the other senses to allow the
animal to walk, maintain posture and perform various activities
such as hunting, feeding and responding to changes in the environment.
The experiments
An undisclosed number of adult cats were used. No details of
anaesthesia used were given. Their handling, housing and analgesia
administration were not described. A profoundly damaged sense
of balance on one side of the animal which involved postural,
walking and visual components was brought about by cutting the
vestibular branches of the eighth cranial nerve, by partial
destruction of the bony labyrinth and exposure of the internal
auditory canal of the inner ear. This damage was assessed by
testing the ability of the cat to stand on a level surface,
and also by using a rotating platform to find both movement
and postural dysfunction.
The damaged cats were divided into three groups -- numbers in
each group were undisclosed -- according to the treatment they
received after surgery. It was not clear how long the animals
were allowed to recover nor whether they received any analgesia
for what had been extensive and invasive manipulation before
this stage of the experiment. Group one comprised animals receiving
no drug; group two received orally the drug betahistine hydrochloride
(50 mg/kg body weight); the third group were cats having betahistine
hydrochloride at 100 mg/kg body weight. Betahistine is a histamine-like
drug and has been used previously to treat balance disorders
and Meniere’s Disease. Research has indicated that betahistine
and histamine are able to exhibit effects on the vestibular
centres (nuclei) of the brain.
A second set of experiments were undertaken in order to evaluate
the role of histamine-containing regions of the brain in the
time-dependent ‘recovery’ of the sense of balance.
In these experiments cats were treated with thioperamide and
betahistine hydrochloride. These drugs act on different histamine
receptors and so can show which specific receptor sites are
implicated in the ‘recovery’ process. The cats used
in these drug tests were either operated on to destroy their
balance sense on one side or not operated on at all -- this
group was used as ‘controls’.
The results
Betahistine hydrochloride treatment was associated with a faster
rate of ‘recovery’ of balance function (described
as locomotor and postural recovery) than was found in the control
group -- those not receiving the drug.
Once it was established that betahistine hydrochloride was related
to ‘recovery’ the authors looked at the distribution
of histamine-containing nerve processes in the operated cat’s
brains, in those areas known to be involved with balance --
the vestibular nuclei. Previous studies going back over twenty
years ago have shown that histamine is a neurotransmitter having
a role in balance amongst other physiological processes (147
- 148) and the authors have also used betahistine hydrochloride
previously to disclose its role in vestibular-damage recovery
in cats (149) reported almost 20 years ago. In the present study
histamine-containing nerve processes and cells were identified
using an antibody marker to histamine. The results confirmed
research published previously about the location and projection
of histamine-containing areas of the brain but also showed,
the authors declare for the first time, novel distribution patterns.
Use of betahistine hydrochloride resulted in a marked increase
in histamine synthesis and release from nerve processes.
Impact on the cats
Damage to the sense of balance produces profound disturbances
to movement and posture -- this will cause significant levels
of stress and suffering. It is not described what anaesthesia
was used nor is analgesia mentioned for the deep surgical procedures
undertaken. Drug treatment used will also have effects other
than those described. The so-called recovery of vestibular function
which takes weeks to take effect is not described in any detail.
The test procedures will also cause stress. There is no claim
that the suffering endured by the cats will have any impact
on treatment of humans suffering from balance problems.
Critique of the research
The paper in which the research is described is lacking in information
on detailed methodology, size of groups used and after-surgery
care. The exact nature of the process of recovery of vestibular
function is nowhere described in detail and the rationale for
the experiments is lacking.
The use of a highly invasive and artificial disturbance to vestibular
function does not mimic disorders of balance. Histamine has
been known to be implicated in vestibular function in various
species for almost twenty years (150), and many of the research
findings reported in this paper merely confirm the authors’
own work or those of other investigators. Species differences
in brain function and plasticity would anyway complicate any
attempt to extrapolate results to humans. In summary, there
is nothing to justify the degree of severity suffered with the
outcome in terms of medical importance. Such fundamental research
on cats is indefensible.M Lacour and B Tighilet [2000] Vestibular
compensation in the cat: the role of histaminergic system, Acta
Laryngologica, Suppl 544, 15-18
------------------------------------------------
Case Study Eight -- The possible role
of the neurotransmitter GABA in normal and vestibular-damaged
cats
Location: UMR 6562, University of Provence and CNRS, Marseille
Funding: CNRS and Ministère de l’Enseignement Supérieur
et de la Recherche
Purpose of the research
Using the same surgically-induced vestibular damage as is described
in Case Study Seven the authors proposed to investigate another
brain chemical and its location -- gamma amino butyric acid
(GABA) -- and its role in balance. It was also claimed that
this study might be a useful means of following plasticity within
the brain -- how function is overtaken by various areas of the
brain following damage to a one particular region and the possible
involvement of GABA.
The experiments
As described in Case Study Seven, again cats had regions of
their vestibular apparatus surgically damaged -- this resulted
in animals with severe deficits in their postural and walking
capacities. In this study fluothene was used for anaesthesia
in the surgical stages of the experiments and ketamine was used
to kill the animal prior to tissue preparation for neurotransmitter
tracing.
Twenty adult cats were used (sexes not described) each weighing
3-4 kg. Twelve animals had their sense of balance on the left
side surgically damaged and were divided into three groups depending
on how long after surgery they were killed: one week, three
weeks and one year. Four cats were used as the “sham operated
controls” -- an area of the left side of their heads was
opened but no vestibular damage was carried out. The four remaining
cats were the control group -- their vestibular regions were
undamaged and thus used to compare the effects of damage and
the various subsequent treatment regimes. Post-operative antibiotics
and analgesia (unnamed) were given to all animals.
The brains of all cats were removed after deep anaesthesia and
perfusion of the body with a fixative. Slices of the brain were
examined histologically to locate the distribution and density
of GABA sites.
The results
The authors point out that the data provided in this paper supports
previous research on GABA distribution and vestibular function.
In addition, some cells in the vestibular regions of the brain
were found to be GABA-containing which is a novel finding. Damage
to the vestibular sense results in alterations to the GABA-containing
areas of the brain implicated in the sense of balance. There
is no medical benefit claimed by the research or the usefulness
of the surgically damaged cat as a surrogate for vestibular
disorders in humans.
GABA distribution, indicated by an immune reaction to the neurotransmitter,
in unoperated cats confirmed previous studies (151 - 152 - 153).
Increases in GABA-rich areas were found after a week and three
weeks in various regions of the vestibular nuclei in the brain.
After one year GABA appeared to increase in some areas and decrease
in others -- this, the authors suggested might be due to active
re-organisation which was coincident with the ‘recovery’
of balance described previously.
Impact on the cats
This is a long-term experiment in which vestibular damage will
cause the problems described in Case Study Seven, including
profound disturbances to movement and posture -- this will cause
significant levels of stress and suffering. It is not described
what steps were taken to reduce deep pain and wound-edge pain,
after the immediate post-operative period. There is no claim
that the suffering endured by the cats will have any impact
on patient treatment of humans.
As this study is primarily histological there are no stresses
incurred by testing regimes.
Critique of the research
Much of the work described either confirms previous studies
using the vestibular damage described in the cat or other animals
(such as the guinea pig). Where data is novel this is used to
provide hypotheses which will call for further inhumane experiments
-- none of which has any bearing on the human situation because
of the artificial nature of the induction of vestibular damage
and the many species differences.
The authors claim that the vestibular compensation which follows
from the surgical insult to the cat is a good model for understanding
brain plasticity -- how function after damage is resumed. However,
the technique is wholly artificial and there are a number of
systems being used to address neuronal plasticity at a cellular
level, which do not involve the level of pain and suffering
that are found in these two Case Studies. Examples include in
vitro approaches described by Smith and Jiang (154), and more
recently in vitro methods have been used to address plasticity
in the brain and the spinal cord (155 - 156).
Additionally, other scientists are studying brain plasticity
using Transcranial Magnetic Stimulation, sometimes combined
with PET scans, in human volunteers who have experienced strokes.
The authors are principally interested in the fundamental aspects
of vestibular function and its manipulation, and the use of
cats in such research is morally indefensible. B Tighilet and
M Lacour [2001] Gamma amino butyric acid (GABA) immunoreactivity
in the vestibular nuclei of normal and unilateral vestibular
neurectomized cats, European Journal of Neuroscience, 13, 2255-2267
------------------------------------------------
Case Study Nine -- Visual responses to changes
in body orientation
Location: INSERM U534, Bron, Centre de Recherche Cerveau et
Cognition, Toulouse & Institut Federatif des Neurosciences,
Lyon
Funding: Institut de la Santé et de la Recherche Médicale,
the CNRS & Conseil Regional Midi-Pyrenees
Purpose of the research
To compare the effects on the change of head orientation-induced
visual compensation mechanism (the vestibulo-ocular reflex --
VOR) by either abrupt step or smooth sinusoidal changes in the
position of the body in the dark It is claimed that the outcome
of the study will have benefits to designing effective clinical
tests for disturbances of balance in humans.
During changes in the orientation of the head the vestibular
apparatus provides information to the eye position controlling
areas of the brain about the speed of head movement. The signals
from the vestibular apparatus are conveyed to the brain centres
which control eye movement and there is a compensatory change
in the eye so as maintain a stable field of vision whilst the
head moves in space. This activity constitutes the VOR. So,
for instance, when the head turns to the right, the eyes rotate
to the left by an equal amount. During this movement the image
of the external world remains stationary on the retina. Past
research has shown that the VOR is capable of being ‘trained’
by different kinds of head orientation.
The experiments
The authors used 15 cats (sexes not described) with implanted
electrodes to record eye movements in the horizontal plane.
A head fixation device was also clamped to the skull and the
test situation consisted in either smooth changes or abrupt
steps of angular movement to the animal whilst the cat’s
head was fixated in the horizontal plane.
Cats were placed on a moveable turntable, surrounded by a circular
screen which could be lit in ways which stimulated the cat.
Alertness was maintained throughout the course of the experiments
by means of “acoustic and tactile stimuli”. There
were no details of post-operative analgesia. Experiments began
four days after surgery. Animals were tested for periods of
one hour in the morning and the afternoon over a period of five
consecutive days -- no details were given of handling procedures.
Four cats were subjected to repeated angular velocity step changes
and 11 cats were tested with sinusoidal oscillations of various
frequencies.
The results
The results indicated that sinusoidal training has a weak effect
upon VOR and that this does not remain from one test period
to the next. Whereas the changes to VOR brought about by abrupt
velocity changes does show retention over time and also transfers
to subsequent testing by sinusoidal changes in body and head
position. Abrupt, step change induced a compensation of the
visual sweeping which was retained over time.
Impact on the cats
Animals in these experiments will undoubtedly experience stress
and significant levels of distress not only because of the slow
or abrupt changes in body orientation over extended periods
but also because their heads remain fixed and they are unable
to move voluntarily. There may also be painful or frightening
aspects to the stimuli which are used to retain the cats’
alertness. In humans, motion sickness brought about by various
changes in body position is extremely unpleasant and similar
symptoms may have been experienced by the cats.
Critique of the research
Non-invasive studies in humans have been used in both the clinical
situation and for research purposes for at least thirty years
in order to understand the interaction between eye movement
and the sense of balance (157 - 158 - 159). Indeed one of the
authors (Clement) has published research into changes in orientation
and balance and subsequent visual modification in humans (160).
The declared objective of the study was to compare the effects
of repeated exposure to angular velocity steps and to continuous
exposure to different frequencies of orientation change in the
VOR in cats but this information can be easily obtained from
human volunteers.
Cats have been used extensively, since the 1970s, in experiments
on the role of the vestibular apparatus and the work is fundamental
rather than of medical value. Non-human primates have also been
used for at least 30 years for research into balance (161 -
162). It is unclear what further information, relevant to humans,
could be gained by carrying out identical experiments in cats
and other species, especially with the level of suffering inflicted.G
Clement et al [2002] Comparison between habituation of the cat
vestibulo-ocular reflex by velocity steps and sinusoidal vestibular
stimulation in the dark, Experimental Brain Research, 142, 259-267
------------------------------------------------
Case Study Ten -- The development of
brain cells which use 5-hydroxytryptamine as a neurotransmitter,
in the developing kitten
Location: Université Claude Bernard, Lyon; Shiga University
of Medical Science, Otsu, Japan and Fujita Health University,
Aichi, Japan
Funding: INSERM, France and the Ministry of Education, Science,
Sport and Culture, Japan
Purpose of the research
To locate, within particular areas of the brain of kittens,
certain classes of nerve cell which can produce a neurotransmitter,
5-hydroxytryptamine (5-HT) from a precursor substance, 5-hydroxytryptophan,
and to chart these cells during the kittens’ development.
The experiments
The cells within the brain and nerve fibres throughout the body
transmit messages and control various functions by means of
chemicals called neurotransmitters. Nerve cells containing the
neurotransmitter 5-hydroxytryptamine (5-HT) have been implicated
in a variety of processes, including sleep and anxiety.
The authors examined the time-course of the formation of the
biochemical processes which produce 5-HT in certain locations
of the brains of kittens aged between one and 30 days. Forty
kittens of both sexes were used and were divided into four groups.
The first group had a dose of tryptophan (100 mg/kg body weight),
which is a molecule used by the body in the production of 5-HT,
together with a monoamine oxidase-inhibitor -- pargyline --
(MAOI, 20 mg/kg) injected into the body cavity.
MAO has a role in the breakdown of active substances like 5-HT,
so pargyline will slow down the action of naturally-produced
MAO. The second group had 5-HTP (20 mg/kg), (this substance
is what tryptophan is converted into in the brain) plus MAOI
at 20 mg/kg injected. The third group had MAOI (20 mg/kg) only
injected. The fourth group had saline injected: these animals
were used as a non-experimental control group, not receiving
any of the molecules considered important in the nerve cells
being studied. Each of the four groups comprised kittens of
1, 3, 5, 7, 15 and 30 days of age.
Ninety minutes after the four different treatments were given
the kittens were killed by deep anaesthesia and prepared for
removal of the brains. The brain of each animal was then cut
and stained in order to locate nerve cells and their constituent
molecules of interest.
The results
Kittens of one to seven days of age had evidence of 5-HT containing
nerve cells in areas of the brain which was described in cats
twenty years ago (163). The injection of 5-HTP and MAOI increased
the number of 5-HT containing cells in all areas of the brain
-- this was not found in kittens older than one month. Certain
areas of the brain showed more 5-HT containing cells after 15
days of age than at younger ages. The injection of tryptophan
plus MAOI did not result in this 5-HT distribution pattern.
The authors point out that the pattern of 5-HT is species specific
and that the kitten shows a very similar distribution of such
cells as the adult cat (164). Rats show a similar developmental
progression where young animals (after stage 19) have a distribution
of 5-HT cells which can be found in adult animals.
Impact on the cats
There are no details provided on the housing of the kittens
or what effects the various injections had on the animal’s
well-being. The research clearly would have caused stress if
the kittens were separated from their mothers for extended periods.
The use of 40 kittens in a fundamental study is both excessive
and unjustifiable.
Critique of the research
The results of this research are not of medical interest. The
authors point out that the pattern of neurotransmitter location
is species-dependent adding: “In the adult cat and kitten,
we could not find 5-HT-immunoreactive cells in DMH cell groups.
This difference is probably attributable to the species difference
in free 5-HT uptake systems of these cells”. To obtain
medically-relevant data, human post-mortem tissue can be used
and the biochemical changes in neurotransmitter locations charted
over time from birth.
K Kitahama et al [2002] 5-Hydroxytryptophan (5-HTP) uptake and
decarboxylation in the kitten brain, Journal of Neural Transmission,
109, 683-689
------------------------------------------------
Case Study Eleven -- Eye movements and changes
within the visual field
Location: CNRS-College de France, Paris
Funding: Human Capital & Mobility Grant from Commission
of the European Union
Purpose of the research
To understand the ways in which cats visually pursue rapidly
moving objects presented to them. Untrained and head-restrained
cats had their eyeball movements recorded using implanted stainless
steel coils. No claims were made for the medical relevance of
the experiments.
The experiments
Animals who hunt require the ability to undertake complex visual
tracking of prey and other relevant objects in their environment.
Eye movements need to be able to respond to actual and likely
changes of parts of the visual field -- cats and other intelligent
animals can predict where prey species, for instance, might
appear or move towards and this activity involves the eyeball
in scanning, targeting, and predictive movements.
The authors used four adult cats. Implantation of eyeball-recording
wire and the head-restraining device is not fully described
in the paper. Ketamine and propofol were the anaesthetics used
for surgery, but there are no details of post-operative analgesia.
Up to two weeks before recording sessions, each animal had stainless
steel wires passed under the muscles of the eyeball and formed
into a coil inserted around the eyeball to record the movements
of the eye in response to visual stimulation. The free ends
of the wires were passed under the skin to a connector. At the
same time three bolts were embedded in a crown of dental cement
placed over the animal’s exposed skull. These bolts allowed
the experimenter to fix the head of the cat for recording purposes.
The body and limbs of each animal was enclosed in a cloth bag
closed with a loose elastic bandage. Each experiment lasted
up to two hours and alertness was maintained by feeding.
Data from the eye coils were collected by a magnetic searching
device placed over the cat’s head. Changes in the movement
of the eyeball were picked up as changes in the magnetic field
of the cat’s head.
The results
The experiments lead to two observations -- firstly, untrained
cats made either an interception sweep of the eye (saccade)
in the direction of the target but opposite to its motion, or
they tracked it in the direction of motion. The authors point
out that the eye movements in these untrained animals were complex
and often consisted of a number of saccades to direct the cat’s
gaze to follow the moving object. Such sweeps of the eyes were
either single or in the form of steps each of which were separated
by a smooth sweep.
All the cats also showed marked predictive eye searching movements
-- they were able to make choices which indicated a computation
by the brain of where an object might appear in the visual field.
Such computation depended upon the initial position of the object
and the initial position of the eye. The authors relate the
various measurements of the eye movement observed to likely
areas of the brain involved, dependent upon previous work on
cats, primates and human volunteers.
Impact on the cats
Stress is highly likely despite the claim made by the researchers
that the operated animals “easily adapted to the restraint”.
Cats will remain subdued and often quiet when in pain or faced
with continuous levels of stress. Furthermore placing steel
coils around the eye will be unpleasant and quite possibly cause
pain. Animals restrained for periods of up to two hours will
also be stressed and experience significant levels of anxiety.
Pain would also arise from wound edges and calls for analgesia
-- the paper supplies no information about pain control.
Critique of the research
The authors did not claim any medical or human relevance for
the studies undertaken. The experiments reported simply add
to the fundamental information that has been gathered over the
past thirty years on the cat visual system. It is already known
from work on human volunteers that there are highly specialised
eye movements which capture details of the visual space and
that these eye movements involve predictive elements -- indeed
one of the authors, S Ron, has been involved in studies of human
volunteers to gain such information (165 - 166).
It is increasingly possible to use sophisticated video-recording
and computation as well as scanning of brain function during
visual tasks in humans to derive data of genuine value and interest,
without the invasive and stressful use of animals. For example,
Jampel and Shi used a headband-mounted mini-video camera and
fibre-optic light source, together with finely tuned computer
algorithms, to assess human eye compensatory movement in response
to changes to head orientation (167).
Movement-dependent eye orientation and the brain structures
responsible can be investigated in humans using powerful computation
and scanning techniques (168 - 169). In addition there are a
number of species differences in the way in which visual information
is collected and computed, for instance it has been known since
the 1970s that cats show certain clear differences in their
eye movements and target tracking from primates (170). Obviously,
therefore, in order to understand human visual perception the
best experimental animal is the human.F Klam et al [2001] Predictive
elements in ocular interception and tracking of a moving target
by untrained cats, Experimental Brain Research, 139, 233-247
------------------------------------------------
The next two Case Studies concern research into sleep. Many
animals show that they, like humans, sleep. In the most widely
studied insect, the honeybee, there are distinct signs that
sleep is to be found in such animals who are not evolutionarily
close to humans. The honeybee rests at night for six to eight
hours, and will often roll over on to its side and become unresponsive
(171). We all know that companion animals such as cats and dogs
sleep. In France, despite the assistance of human volunteers
to understand sleep and the involvement of the brain, the cat
remains the favoured species in which to carry out painful and
highly stressful experiments. Unfortunately, the University
of Claude Bernard has maintained the tradition of such approaches
over many years.
Two of the authors of the following Case Studies have used highly
stressful methods since the 1970s to induce long periods of
sleep deprivation, and also inject drugs and chemicals of putative
transmitter function in order to manipulate the sleep and wakefulness
of cats. The supposed benefit to human sleep disorders is highly
dubious.
Case Study Twelve -- Effects of drugs on sleep
patterns in sleep-deprived animals
Location: INSERM U480, Université Claude Bernard, Lyon
and Laboratoire L Lafon, Maisons Alfort
Funding: Laboratoire L Lafon; Institut National de la Santé
et de la Recherche Médicale (Unite 52 & 480); and
Université Claude Bernard, Faculté de Médecine
Purpose of the research
Using substantial periods of sleep deprivation, the effects
of amphetamine and modafinil on the sleep/wake cycle of cats
was investigated. Claims were made that the study would disclose
more about the effects of the two drugs on the maintenance of
wakefulness in sleep-deprived animals.
The experiments
Six cats of either sex were used. Animals had electrodes surgically
implanted in order to record from two regions of the brain --
long associated with various aspects of sleep and wakefulness.
They also had electrodes implanted to record muscle and eye
activity, which varies during different phases of the sleep/wake
cycle. In addition cats had a temperature-recording device implanted
in another brain region. Ten days after this surgery, undertaken
with pentobarbital anaesthesia, animals were housed in a low
noise dimly lit cage at 24-26 degrees Celsius and were fed daily.
Recordings from the electrodes were made continuously over four
days.
Sleep deprivation was accomplished by a technique used by the
authors over a period of at least thirty years (172 - 173).
It consists of a shallow tank of water with a small island,
the surface of which is almost level with the surface of the
water; the cat is placed on the island. When the cat then attempts
to sleep it relaxes and so touches the water. This awakens it
and each time it attempts to sleep it is thus awakened. The
authors used this technique to deprive cats of sleep for periods
of 18 hours. This state of sleep deprivation was referred to
in the study as providing ‘sleep pressure’. Cats
deprived of sleep for long periods of time tend to sleep more
afterwards.
Three experimental groups of cats comprised one group receiving
modafinil (5 mg/kg body weight), amphetamine (1 mg/kg body weight)
or a placebo. Modafinil is a drug used in clinical practice
over the last 15 years, and it induces wakefulness in humans
as well as the cat (174). Similarly, amphetamine is a drug which
has been used for many years as a psychostimulant and is able
to reduce the urge to sleep in sleep-deprived animals including
humans. The intention was to see if either drug kept sleepy
cats awake, and to study what brain activity accompanied the
treatment and recovery from sleep deprivation.
Brain, muscle and eye movement recordings were made of cats
in the three groups.
The results
Unsurprisingly, sleep-deprived animals showed a range of brain
activity related to decreased arousal and the desire to sleep
-- this was recorded in the cat by one of the authors twenty
years ago (175). Modafinil was able to keep sleep-deprived cats
awake: they were reported as being alert and attentive (this
was directly observable in the animals’ appearance and
also shown by the recording of specific kinds of brain waves).
This is hardly surprising given that the drug has been developed
and tested in various species, including humans as a means of
alleviating narcolepsy (a clinical condition of chronic sleepiness).
Amphetamine, as is well known, induced wakefulness but increased
the level of arousal, and the treated cats showed “signs
of behavioural excitation”. Cats receiving this drug also
needed longer periods of sleep after the experiments in order
to recover from their sleep deprivation.
Impact on the cats
Cats deprived of sleep are obviously stressed as the method
used to create sleep deprivation involves water contact which
is not tolerated by these animals during periods when individuals
were trying to sleep. Cats underwent brain surgery and there
were no details of any analgesia used on wound edges. All cats
received the two active drugs which have reported side effects
-- those for modafinil include, in humans, nausea, abdominal
pain, gastrointestinal disturbances and heart disturbances (176).
The authors point out that amphetamines cause marked behavioural
excitation in cats, rats and humans, and are also known to produce
psychotic states (in humans and other animals).
Critique of the research
As the authors point out much of the data assembled in the study
is confirmatory and where new material is presented it is of
a trivial nature. The authors claim that the results “…indicate
that modafinil is effective against somnolence and hypersomnia
and does not produce a subsequent increase in sleep and suggest
that the pharmacological profile of modafinil is different from
that of amphetamines”. But this drug has been prescribed
for sleep disorders for many years and one of the authors (Lin)
published data in cats supporting this view ten years ago (177).
Other workers (including one of the authors of this study) have
also come to similar conclusions using other species (178 -
179). There is no scientific justification for repeating experiments
in a range of species when the data exist for humans -- this
is particularly the case for the drugs used in this study.
J S Lin et al [2000] Effects of amphetamine and modafinil on
the sleep/wake cycle during experimental hypersomnia induced
by sleep deprivation in the cat, Journal of Sleep Research,
9, 89-96
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Case Study Thirteen -- Activity of identified
cells in the brain and their role in sleep-wake cycles
Location: INSERM U480, Université Claude Bernard, Lyon
and University School of Dentistry, Tokushima, Japan
Funding: INSERM U480
Purpose of the research
To identify certain populations of nerve cells in the brains
of cats and to describe the activity of such cells during sleep
and wakefulness in freely moving animals. No medical relevance
is claimed for the research.
The experiments
Certain regions of the brain show characteristic electrical
patterns -- either being active or silent -- during either sleep
or wakefulness. Such phenomena have been known in cats since
the 1970s largely as a result of the work of one of the author’s
groups.
Eight adult cats were used in the experiments described in the
study. Whether both sexes were used is not mentioned. Electrodes,
consisting of steel wires arranged in bundles, were implanted
in the brains of the cats to record electrical activity. A further
two steel-wire electrodes were positioned to stimulate electrically
certain areas of the brain. Recording and stimulating electrodes
were positioned in different areas of the cat’s brain
-- locations known from previous studies to be implicated in
various aspects of sleep and wakefulness.
Previous research has suggested that cells in particular regions
of the cat brain that seem to be linked with sleep and wakefulness
comprise two populations distinguished by their electrical activity
and the neurotransmitter that they use -- either acetyl choline
(called cholinergic cells) or 5-HT, noradrenaline, histamine
or adrenaline (non-cholinergic cells). Animals were unrestrained
during the experiments.
Cats had one of two drugs administered by injection into the
bloodstream. The drugs were 5-methoxy-N,N-dimethyltryptamine
(5-MeODMT) and clonidine hydrochloride. These drugs were used
because 5-MeODMT is a 5-HT1A agonist (5-HT is a brain chemical
known to be involved in sleep and wakefulness and 5-MeODMT appears
to amplify the responses which are caused by 5-HT in cells having
one specific kind of 5-HT receptor). Clonidine is an alpha2
adrenergic agonist and is thus able to amplify the responses
in those nerve cells which use adrenaline as a neurotransmitter.
At the end of the experiments the cats were killed (the method
was not described) and the tracks and final location of the
various electrodes were followed in sections of each animal’s
brain. In addition, stained sections of the brain were made
in order to identify the neurotransmitters that were contained
in the various groups of nerve cells from which recordings were
made.
The results
Data were obtained using electrical recordings from the 84 cells
in the lower areas of the brain i.e. the medulla. Recordings
were made of cells which were silent (no electrical discharges)
during the so-called paradoxical sleep phase (these are termed
the PS-off neurons). These PS-off cells become active when the
cat is awake and have different kinds of electrical activity
in light and paradoxical sleep.
The authors found a number of PS-off neurons which appeared
to have a different kind of neurotransmitter sensitivity from
that previously described. The authors describe some of the
electrical patterns shown by this set of nerve cells which varies
according to the kind of sleep (light or paradoxical) that the
cat is in.
Impact on the cats
No details of housing or handling of the cats was provided.
Nor was post-operative analgesia mentioned. The two drugs used
are likely to have side effects which go unrecorded in the publication.
The authors pointed out that 8-OH-DPAT, a selective 5-HT1A agonist,
was not used in the present study since the “…preliminary
study showed that 8-OH-DPAT induced marked behavioural agitation
in the animals when administered”. Clonidine, a drug prescribed
for humans for a variety of disorders, has a number of side
effects recorded, these include depression, fluid retention,
dizziness, headache, nocturnal unrest and nausea.
Critique of the research
Both this and the twelfth Case Study describe research that
has been underway since the 1970s (180 - 181). A great deal
of data has been assembled over that time on the identity of
brain regions involved in sleep and wakefulness, the kinds of
electrical characteristics exhibited by nerve cells in those
areas and what potential neurotransmitter molecules are involved
(182).
Once a set of cells has been characterised, there remain possibilities
for yet more research to understand how such cells communicate
with one another and how this fits into any kind of understanding
of the biology of sleep. Despite thirty years of research, primarily
on rats and cats, the scope for using sentient creatures to
disclose what happens during sleep appears limitless. As the
authors point out, “The activity of both adrenergic and
noradrenergic medullary neurons during the sleep-waking cycle
is not yet known and will be an important subject for future
studies”. They add that the research reported, “…opens
up the possibility of multiple interactions between PS-on and
monoaminergic and non-monoaminergic PS-off neurons”. But
what, if any, is the value to humans of this ever-more detailed
work?
As was pointed out in the critique to Case Study Twelve, it
is now possible to use non-invasive and powerful imaging techniques
to discover information about sleep in human volunteers. Species
differences ( 183) as well as humane considerations make the
use of humans the animal of choice for work to
have any relevance to understanding normal or clinical aspects
of sleep in humans (184 - 185 - 186).
K Sakai and N Kanamori [1999] Are there non-monoaminergic
paradoxical sleep-off neurons in the brainstem? Sleep Research
Online, 2, 57-63
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Chiens & Chats
Cosmétiques
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