Wiener Tierärztliche Monatsschrift – Veterinary Medicine Austria
101 (2014)
From the Department for Nuclear Medicine1, University Medical Center Freiburg, Freiburg, Germany, and the
Clinical Unit of Anaesthesiology and perioperative Intensive-Care Medicine2, Department/Clinic for Small Animals
and Horses, University of Veterinary Medicine Vienna, Austria
Considerations for anaesthesia of experimental animals
for neuromolecular imaging by means of positron emission tomography (PET) or single photon emission computed tomography (SPECT) with a focus on tree shrews
S. GEHRIG1* and Y. MOENS2
received Mai 14, 2013
accepted October 7, 2013
Keywors: PET/SPECT, lab animals, tupaia, anaesthesia, brain receptor, quantification.
Summary
Anaesthesia in the context of preclinical neurological PET/SPECT studies is challenging because tracers and anaesthetics access the same cerebral region and often the same molecular structures.
Unspecific effects of the anaesthetics influence cerebral receptor systems, perfusion and metabolism and
hamper the design of an appropriate protocol. Furthermore a commonly used animal model - the tree
shrew - seems to show severe side effects after the
application of many anaesthetics that are commonly
used in other species. The information in the present
article should provide the basis for the design of a
well tolerated anaesthesia protocol allowing accurate
and reproducible neurological PET/SPECT studies,
especially in tree shrews.
Abbreviations: Cc(t) = time-dependent radiopharmaceutical concentration in the region of interest; Cp(t) = timedependent radiopharmaceutical concentration in the plasma; CNS = central nervous system; DAT = dopamine transporter; D receptor = Dopamine receptor; 18F-FDG = 18Ffluordeoxyglucose; GABA = gamma amino butric acid; HCN
receptor = hyperpolarization-activated cyclic nucleotidegated cation receptor; 5-HT = 5-hydroxytryptamin; k1 = plasma cerebral tracer transfer rate; nACh receptor = nicotinic acetylcholine receptor; NMDA receptor = N-methyl-Daspartate receptor; PET = positron emission tomography;
ROI = region of interest; SPECT = single photon emission
computed tomography; TCM = tissue compartment model
Schlüsselwörter: PET/SPECT, Versuchstier, Spitzhörnchen, Narkose, Gehirnrezeptor, Quantifizierung.
Zusammenfassung
Überlegungen hinsichtlich der Anästhesie von
Labortieren für die neurologische molekulare
Bildgebung mittels Positronen Emissions Tomographie (PET) bzw. Single Photon Computertomographie (SPECT) mit besonderem Augenmerk
auf Spitzhörnchen
Die Anästhesie im Rahmen von präklinischen neurologischen PET/SPECT Studien stellt aufgrund der
Tatsache, dass Anästhetika und Radiotracer dieselben Gehirnregionen und oft dieselben molekularen
Zielstrukturen ansteuern, eine besondere Herausforderung dar. Unspezifische Effekte der Anästhetika beeinflussen zudem zerebrale Rezeptorsysteme,
Perfusion und Metabolismus und erschweren das
Design eines passenden Anästhesieprotokolls. Ein
in der neurologischen Forschung verwendetes Versuchstier – das Spitzhörnchen (Tupaia) – reagiert mit
ausgeprägten Nebenwirkungen auf viele Anästhetika, die regelmäßig bei anderen Versuchstieren angewendet werden, was das Design eines passenden
Protokolls zudem erschwert. Basierend auf aktueller
Literatur hinsichtlich neurologischer molekularer
Bildgebung sowie Literatur hinsichtlich der Narkose
von Spitzhörnchen wird eine Empfehlung als Grundlage für das Design eines gut verträglichen und reproduzierbaren Anästhesie-Protokolls gegeben.
Introduction
Neurological molecular imaging using positron
emission tomography (PET) and single photon emission
computed tomography (SPECT) have become
important tools in the clinical diagnosis of human
movement disorders, dementia and brain tumours
(HEISS and HERHOLZ, 2006). These techniques use
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radiopharmaceuticals, which are radioactively labeled receptor ligands and enzyme substrates that selectively interact with molecular targets such as receptors (e.g. Dopamine D2 receptors), transporters
(e.g. Dopamine Transporter - DAT) and enzymes (e.g.
acetylcholine esterase) (BARTENSTEIN et al., 2002;
TATSCH et al., 2002a,b,c; VANDER BORGHT et al.,
2006; HEISS and HERHOLZ, 2006). It is thereby possible to follow and to quantify in vivo cerebral and
neuronal processes (e.g. glucose metabolism) and
structures (e.g. receptor density) by means of threedimensional cross-sectional imaging methods. Today
several small animal PET/SPECT systems are commercially available and enable longitudinal non-invasive studies of several neurological pathologies, such
as age-dependent brain alterations in various small
animal models (MYERS and HUME, 2002; ROWLAND
and CHERRY, 2008). Of small animal species, mainly
rodents serve as models for human brain disorders
such as Parkinson’s, Alzheimer’s and Huntington’s diseases. An example is the 6-hydroxydopamine
(6-OHDA)-lesioned rat that is used to study
Parkinson’s disease (STROME and DOUDET, 2007).
Apart from the well known rodent models, tree
shrews are emerging as an interesting small animal
species for the study of specific brain disorders, e.g.
stress and resulting depression (OHL et al., 1999;
FUCHS and FLUEGGE, 2002,2003). Moreover, a high
genetic compliance between tree shrews and primates with regard to the expression of neuronal receptor
proteins and the amyloid beta precursor protein
(YAMASHITA et al., 2010) allows the study of age-dependent brain alterations in genetically and socially homologous cohorts (FUCHS and CORBACH-SÖHLE,
2010). The use of tree shrews for neurological studies
by PET/SPECT is highly promising due to the relatively larger size of the tree shrew brain compared to
that of mice and rats, which enables cerebral structures to be imaged with a much higher resolution than
is possible in these species. As an example, the tree
shrew striatum is already divided into nucleus caudatus and putamen and represents an important target
structure for the investigation of movement disorders
(e.g. Parkinson’s disease) (RICE et al., 2011).
In contrast to human patients, laboratory animals
must be anaesthetized to provide the immobilization
that is essential during PET/SPECT image acquisition.
However, anaesthesia might alter central neuromolecular mechanisms. As a result, PET findings obtained in anesthetized animals may not correctly represent normal properties of the awake brain (ALSTRUP
and SMITH, 2013). Besides the inevitable influence of
anaesthetics on cerebral (glucose) metabolism and
perfusion (SOKOLOFF, 1981), which must be kept in
mind when interpreting PET/SPECT studies, the interaction of the anaesthetics with cerebral target structures might represent a major issue. While the radiopharmaceutical is administered in an amount that is high
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enough to enable it to be tracked in vivo due to its
radioactivity but low enough not to mediate pharmacological effects (ZANZONICO, 2012), a pharmacological effect is explicitly desired following the administration
of an anaesthetic. If the radiopharmaceutical and an
anaesthetic drug interact at the same cerebral structure
(receptor), the results of a PET/SPECT scan may be significantly influenced due to a competitive situation, as
recently shown by WAELBERS et al. (2013) in cats.
Specific requirements for experimental anaesthesia protocols in
neurological PET/SPECT imaging
To allow meaningful PET and SPECT imaging of experimental animals, general anaesthesia is essential.
Especially when recovery is planned, any anaesthetic
protocol must be well tolerated and safe. The
following additional requirements need to be met.
The animals must be properly immobilized during
the scan.
This is a prerequisite for avoiding imaging artefacts
as well as for the correct calculation of tracer kinetics
and local perfusion in cerebral areas.
Specific and unspecific influence of target structures by anaesthetic drugs should be avoided.
Anaesthetics mediate their pharmacological effect
through binding to cerebral receptors and/or ligandgated ion-channels (FRANKS and LIEB, 1994;
KRASOWSKI and HARRISON, 1999). Alterations of
these cerebral receptor systems are involved in many
neurological pathologies (HEISS and HERHOLZ,
2006). During neuromolecular PET/SPECT analysis of
these receptor systems in anesthetized animals, the
receptors represent the target structures for both the
radiopharmaceuticals and the anaesthetics. HEISS
and HERHOLZ (2006) reviewed cerebral molecular
structures that are involved in common human brain
disorders and that therefore represent possible molecular targets for neurological molecular imaging. The
dopaminergic, the serotoninergic, the cholinergic and
the GABAergic (gamma aminobutric acid) systems
are of interest for the diagnosis of cerebral pathologies. Furthermore, adenosine and opioid receptors
have gained importance as cerebral target structures.
Among these, the dopaminergic, the GABAergic, the
opioidergic and the serotoninergic systems also represent molecular target structures for a multiplicity of
anaesthetics, as summarized by MEYER and FISH
(2008). The dopaminergic system has an important role
in various cerebral functions. A degeneration of the
presynaptic nigrostriatal dopamine system results in
Parkinson’s disease. A functional alteration in the postsynaptic dopamine receptors can be observed inpatients suffering from schizophrenia. Dopamine D1 and D2
Wiener Tierärztliche Monatsschrift – Veterinary Medicine Austria
receptors are of interest for PET/SPECT studies but the
D2 receptor also represents the main target for a commonly used neuroleptic sedative drug, acepromazine.
Amongst others, the GABAergic neurotransmission is
altered in patients suffering from epilepsy and anxiety.
Benzodiazipines, barbiturates and alphaxalone mediate
their anaesthetic effect through binding to the GABA A
receptor. Radioactively labelled benzodiazepines are
used as tracers in neuromolecular imaging of this system.
The opioidergic receptor system plays a major part in
the emotional processing of pain and radioactively
labelled opioids such as diprenorphine (agonistic and
antagonistic properties) or carfentanyl (opioid agonist)
are used to study the system. Opioids are frequently
used in clinics for their sedative and analgesic properties.
Malfunctioning of serotoninergic neurotransmission
can be seen in depression and anankastic personality
disorder, as well as in patients suffering from
Alzheimer’s disease, Parkinson’s disease, autism and
schizophrenia. For the diagnosis of these diseases,
radiolabelled 5-hy-droxytrpyptamin receptor ligands
(5-HT1a and 5-HT2a) are used. Although they do not
represent its main site of action, ketamine may access
serotonin receptors. In a SPECT study in cats,
WAELBERS et al. (2013) demonstrated an interaction
between ketamine and cerebral 5-HT2a receptors resulting in decreased binding of the tracer used in the study.
The central acetylcholine receptor system also represents an important target for the diagnosis of
Alzheimer’s disease. Diagnosis is possible by
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evaluating the acetylcholine esterase activity using
radioactively labelled acetylcholine analogues. The
nicotinic acetylcholine (nACh) receptor is another interesting target (HALLDIN et al., 1992; GOULD et al.,
2013). Cerebral nACh receptors modulate the release
of neurotransmitters such as GABA, dopamine and
5-hydroxytrpyptamin and are targeted by halogenated
ethers and alkanes (KRASOWSKI and HARRISON,
1999). Table 1 shows the molecular targets involved in
human brain alterations and anaesthetics that mediate
their effect through binding at these targets.
As shown in various publications for the cerebral
dopamine system, certain anaesthetics have not only
specific effects (mediated through binding to a cerebral receptor) but also unspecific effects on cerebral
receptor systems, influencing transporter availability
and ligand release. This might interfere with neuromolecular imaging. Halothane and isoflurane cause
inhibition of the dopamine transporter (DAT) and increase extracellular dopamine concentrations during
anaesthesia (EL-MAGHRABI and ECKENHOFF, 1993).
Another theory is that volatile anaesthetics cause
trafficking of the DAT into the cytoplasm. VOTAW et
al. (2003) showed in rhesus monkeys that under isoflurane anaesthesia the DAT, which is usually located
at the plasmalemma, is internalized into the neuron.
Ketamine anaesthesia also significantly altered the
availability of DAT, leading to an increased binding of
radioactively labelled dopamine receptor ligands
in rhesus monkeys (TSUKADA et al., 2001). In rats
Tab. 1: Cerebral molecular targets involved in common brain disorders. Radiotracers and anaesthetics respectively anaesthesia related
drugs targeting these molecular structures. Derived from HALLDIN (1992), KRASOWSKI and HARRISON (1999), HEISS and HERRHOLZ
(2006) and MEYER and FISH (2008).
Molecular target
Pathologies
Tracers
Anaesthetics
GABA A- receptor
Anxiety
Epilepsy
Radiolabelled
benzodiazipines
Alfaxalone
Benzodiazipines
Barbiturates
Opioid receptors
Emotional processing
of pain
11
D1/2-receptors
Parkinson
Schizophrenia
11
C-Diprenorphine
C-Carfentanyl
Opioids
18
C-Raclopride
F-Fallypride
Acepromazine
5HT1/2-receptors
Depression
Anxiety
Parkinson
Schizophrenia
WAY-100635
family
Ketamine
Halogenated ethers
Nicotinic acetylcholine
receptor
Cocaine dependence
11
C-nicotin
Halogenated ethers
Alkanes
Acetylcholine
-esterase
Alzheimer’s disease
Radiolabelled acetylcholine analogues
Atropine
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ketamine has been shown to lead to an increased
dopamine release in the pre-frontal cortex (VERMA
and MOGHADDAM, 1996).
These effects make it obvious that when anaesthetics are used in neuromolecular imaging procedures
it is important to know the target structures of the
anaesthetics as well as possible unspecific effects on
cerebral receptor systems; there should be no interference with the target structures of the radiopharmaceutical. The anaesthesia protocols should
use as few components as possible to limit factors
that influence the CNS. In theory it would be beneficial to use a single component anaesthesia during the
scan, targeting only one specific type of receptors.
Unspecific effects on cerebral metabolism and
regional perfusion should be kept constant
For accurate quantification in PET/SPECT studies,
mathematical models (first-order differential equations)
are used that contain the time-dependent cerebral
radiopharmaceutical concentration Cc(t) in the region
of interest (ROI) and the time-dependent radiopharmaceutical concentration in the plasma Cp(t) (SCHMIDT
and TURKHEIMER, 2002). In addition to the presence
or the activity of the cerebral target parameter, accumulation of a tracer is also determined by unpredictable factors such as unspecific binding and regional
perfusion, which is directly influenced by anaesthetic
drugs (SCHMIDT and TURKHEIMER, 2002). The regional
cerebral radiopharmaceutical concentration is therefore not necessarily equivalent to the quantity of the
target parameter. Using a model of the in vivo kinetics
of any radiopharmaceutical makes the influence of unspecific effects of anaesthetics on the quantification of
cerebral molecular targets obvious. The three- or twotissue compartment model (TCM) shows the influence
of regional perfusion on the quantification of cerebral
structures (Fig. 1). The rate constant k1 (Fig. 1) describes
the net transfer rate of tracer from plasma to cerebral
tissue and is directly proportional to the regional perfusion. A change in the regional perfusion will affect
the amount of tracer delivered to the cerebral tissue
per time. Furthermore, as illustrated in Fig. 1, plasma
tracer concentration contributes only a small amount
to the radioactivity in the ROI. Anaesthetics influence
regional perfusion by causing a change (generally a
decrease) in neuronal activity and thus a change in cerebral metabolism, which in turn causes vasoconstriction
or vasodilation (flow-metabolism coupling; SOKOLOFF,
1981). For some anaesthetics, additional intrinsic vascular effects have been described (MATTA et al., 1999).
To obtain measurements that are comparable and
reproducible between animals and time points, the
variable ‘anaesthesia‘, i.e. the unavoidable unspecific effects, must theoretically become a constant.
Every variation in this variable can be considered as
a systematic error that influences the interpretation
of the results. The anaesthetic protocol must be
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designed such that at the start of the PET/SPECT examination the concentration of anaesthetic drug in
the CNS is at a steady state. This means that equilibrium must be reached between plasma and CNS
concentration of the drug. To facilitate the realization
of this condition it is practical to divide anaesthesia
into two phases, strictly separated by a transition
phase (Fig. 3). Phase I consists of premedication (if
necessary) and induction of anaesthesia and potentially a combination of several anaesthetic drugs is
needed. The plasma elimination half-life of centrally
acting drugs used in phase II should be as short as
possible. The subsequent transition phase should allow for the complete excretion of the drugs that are
not needed for the maintenance of anaesthesia and
the plasma-cerebral equilibrium should be realized.
This is easily done by the use of only volatile anaesthetic. However, when an injectable anaesthetic drug
is preferred, the amount that is metabolized and excreted per time unit must be redelivered intravenously to establish steady state after initial saturation of
anaesthetic target receptors by a bolus injection. A
bolus/infusion technique described by CARSON
Fig. 1: Three (A) and two (B) compartment models by SLIFSTEIN
and LAURELLE (2001): Abbreviations: C: Concentration, p: plasma, f: free ligand; ns: nonspecific binding; s: specific binding. ki
(i=1...6 (A) , respectively i=1...4 (B)) rate constants describing the
transfer of the tracers between compartments. As illustrated in
(A) by the PET ROI (Region of Interest), radioactive tracer and its
metabolites in the plasma account for a small amount to the measured radioactivity in the ROI. To simplify matters in most cases a
2 TCM (B) is considered. Figure taken from MEYER (2009).
Fig. 2: Influence of anaesthesia on regional cerebral metabolism,
blood flow and subsequently plasma cerebral tracer transfer rate
derived from SOKOLOF (1981), WAHL et al. (1992), MATTA et al.
(1999), SLIFSTEIN and LAURELLE (2001) and MEYER and FISH
(2008). The term plasma cerebral tracer transfer rate equates the
rate constant k1 described in Fig 1.
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(2000) for neurological PET tracers can be applied,
with injectable anaesthetics that have a short plasma
elimination time. Further necessary preparations
such as the introduction of intravenous and intra-arterial catheters can be performed in the transition
phase. Phase II starts immediately before tracer injection and continues until the end of the scanning
procedure. The number of components acting on the
CNS should now be reduced to an absolute minimum.
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associated with anaesthesia should be avoided. If it is
necessary to perform a static scan, the tracer should
theoretically be injected in a conscious state, as
cerebral glucose metabolism is not altered by anaesthetics in this situation. Whether this is relevant in
practice depends on the sensitivity of the species
and the individual animal to stress. Because of
metabolic trapping, it is not necessary to establish
steady state and unspecific effects of the anaesthetics
can be neglected.
Anaesthetic considerations for
tree shrews in biomedical research
Anaesthesia of tree shrews in the scientific
literature
Fig 3: Theoretical approach to set up plasma cerebral equilibrium for anaesthetics, used to provide unconsciousness and
immobility, and to excrete CNS active anaesthetics respectively
anaesthesia related drugs before start of a PET/SPECT measurement. In clinical context, anaesthesia is divided in premedication,
induction, maintenance and recovery respectively euthanasia. In
the context of PET/SPECT measurements phase I is related to the
premedication and induction phase, while the transition phase
and phase II is related to maintenance phase. After completion of
the measurement the animal will be recovered or euthanized. It is
obvious that this figure represents ideal circumstances and unexpected events are not taken into account. The transition phase is
determined based on plasma excretion time of the drugs intended
to be removed before start of the measurement. For some species
used in neuromolecular imaging studies there is no data available
and the plasma excretion time must be estimated.
The effects of anaesthetics on the blood glucose
level should also be considered. The radioactively
labelled glucose analogue 18F-fluordeoxyglucose
(18F-FDG) is an important tool for the evaluation of
cerebral glucose metabolism in certain areas of the
brain (BARTENSTEIN et al., 2002). Similar to glucose,
18
F-FDG is internalized into cells, by means of the
GLUT1 and GLUT4 glucose transporters, and subsequently phosphorylated. However, unlike glucose it
is not further metabolized and the phosphorylated
metabolite is not able to leave the cell (metabolic
trapping). The accumulation can be measured using
PET and is proportional to neuronal (metabolic)
activity (GALLAGHER et al., 1978). In addition to the
inevitable down-regulation of the cerebral glucose
metabolism linked to anaesthetic state, an increase of
blood glucose levels following injection of some
anaesthetics, such as α 2-agonists or ketamine, has
been reported (MEYER and FISH, 2008). Pre-anaesthetic handling and subsequent agitated induction
phases are further factors that may increase blood
glucose levels (MEIJER et al., 2006). If 18F-FDG is
used as a tracer, normo-glycemic conditions should
exist at the time of injection and during uptake
(WAHL et al., 1992). This means that the anaesthetics
should not affect the blood glucose level and stress
SCHWAIER (1978) performed a systematic clinical
assessment of a large number of injectable anaesthetics and sedatives in tree shrews, using them singly or
in combination. The tolerance of the initial dose, the
quality of anaesthesia and recovery, surgical tolerance
and the duration of anaesthesia were assessed.
Barbiturates, thiazines, benzodiazipines and ketamine
were evaluated alone or in combination with one
another or with a non-depolarizing muscle relaxant
(gallamine triethiodide, Flaxedil) or the opioid derivative
fentanyl. All components and combinations caused
remarkable side effects such as improper immobilization, salivation, dyspnoea and insufficient surgical
tolerance. SCHWAIER (1978) also evaluated the steroid anaesthetic saffan. Saffan is a 3/1 mixture of the
two pregnandiones alphaxalone and alphadolon.
Anaesthesia is largely mediated through binding
of alphaxalone to the GABA A-receptor. To date,
Alfaxan® (Vetoquinol GmbH, Ravensburg, Germany)
is the only available product and contains only alphaxalone. Alphaxalone-based anaesthetics generally have a wide therapeutic range, with a fast onset
of anaesthesia and a fast recovery (NOLAN et al.,
1997). At doses between 12 mg/kg and 36 mg/kg saffan produced a well tolerated anaesthesia without
particular side effects and with no negative influence
on cardiovascular parameters in tree shrews. The
duration of anaesthesia depended on the dose administered. Animals could be kept anaesthetized for up
to 8 h with a continuous rate of infusion. MCQUEEN
et al. (1984) provide evidence that anaesthetics containing alphaxalone are suitable for studies of regional cerebral glucose metabolism involving 18F-FDG.
Due to the selective action on the GABA A receptor, the
excellent tolerance, the wide therapeutic range and the
fast onset of anaesthesia, alphaxalone-containing products represent promising candidates for anaesthesia
in the context of neurological molecular imaging. This
notion is supported by our own observations.
Other protocols, of greater or lesser complexity, for
the anaesthesia of tree shrews have been published
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but their suitability for use with neuromolecular
imaging has not been critically addressed. Some
groups use ketamine combined with either xylazine
or sodium-pentobarbital administered i.p. or i.m
(MURRAY et al., 1982). In these non-survival studies,
remarkably high doses of ketamine were used, ranging from 90 mg/kg up to 450 mg/kg combined with
10 mg/kg xylazine or 10 mg/kg sodium-pentobarbital
(MURRAY et al., 1982; PHILLIPS et al., 2000).
MURRAY et al. (1982) also described a combination of
ether inhalation (via a nosecone) and sodiumpentobarbital (40 mg/kg) injection. Furthermore,
mono-anaesthetic protocols using ketamine (130 mg/kg)
(SESMA et al., 1984), nembutal (100 mg/kg)
(WALLETSCHEK
and
RAAB,
1982),
ether
(BAMROONGWONG et al., 1975) and halothane
(ROCKLAND et al., 1982) have been described. More
complex protocols have also been suggested, in
which multiple components are used for induction, to
avoid side effects and to maintain anaesthesia.
OHL et al. (1999) used xylazine (2 mg/kg), ketamine
(10 mg/kg) and atropine (0.02 mg/kg) followed by intubation and maintenance of anaesthesia with
halothane (max. 2%) in a 60/40 mixture of oxygen and
nitrous oxide. In more recent publications isoflurane
has replaced halothane. The anticholinergic agent atropine is believed to be necessary to avoid salivation.
LU and PETRY (2003) and VEIT et al. (2011) used similar protocols with varying doses and occasionally
neuromuscular blocking agents such as pancuronium and gallamine (especially when xylazine was not
used) and an anti-cholinergic compound.
Molecular properties of the anaesthetics and
related drugs and their suitability for use with
neurological molecular imaging
Ketamine acts largely as an antagonist of the
N-methyl-D-aspartate (NMDA) receptor but it also
has effects on GABA A receptors, acetylcholine receptors, serotonin receptors and opioid receptors.
An increase in blood glucose levels can be observed
following the i.v. administration of ketamine (MEYER
and FISH 2008). The direct and indirect influence of
many different cerebral receptors makes ketamine’s
effects on the CNS unpredictable and the possibility
of interactions with the target receptors of the PET
tracers cannot be excluded. Furthermore, ketamine
produces typical side effects, which can be observed
during anaesthesia of tree shrews with many compounds (SCHWAIER, 1978). Due to the variety of
target receptors, the influence of various neurotransmission systems, the increase of blood glucose levels
and the necessity of adding further compounds to
avoid side effects, ketamine is not the compound of
choice for neurological PET scans.
Xylazine is an α 2-receptor agonist that acts both in
the CNS and peripherally. The sedative effect is
due to a decrease in central noradrenergic
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neurotransmission. Its high selectivity for α1- and
α 2-adrenoreceptor represents a big advantage.
Furthermore, α 2-receptor agonists have muscle-relaxant properties that are useful to enhance immobilization (MEYER and FISH, 2008). Due to its lack of
hypnotic properties, xylazine can only be considered
as a supplementary drug to facilitate the induction of
anaesthesia and to reduce the required dosage of the
main anaesthetic component. The concomitant
elevation of blood glucose level makes xylazine unsuitable for studies employing 18F-FDG.
Isoflurane and halothane are inhalable volatile
anaesthetics, as are enflurane, sevoflurane and
desflurane. The mechanism of their anaesthetic action is not fully understood, although it seems to be
mediated by a number of molecular targets in various
receptor systems. The GABA A receptor in the brain
and the spinal cord is likely to be one of the main
targets, with further targets including the two pore
potassium channels, the NMDA receptor and HCN(hyperpolarization-activated, cyclic nucleotide-gated
cation) and sodium channels (FRANKS, 2006). An
advantage of volatile anaesthetics is the ease of
establishing a steady state and equilibrium between
body compartments. The limited knowledge of their
molecular targets can be considered a disadvantage.
Furthermore, the publication by TER LAAK et al.
(1975) indicates that the use in tree shrews of volatile
agents such as halothane, isoflurane and sevoflurane
alone causes severe side effects, as also described
by SCHWAIER (1978). This means that supplementary drugs are needed, which are likely to influence
further cerebral molecular structures.
Nitrous oxide mediates its anaesthetic action by
competitive and non-competitive inhibition of the
NMDA receptor (JEVTOVIĆ-TODOROVIĆ et al., 1998).
However, when used alone it is incapable of producing reliable anaesthesia. Nitrous oxide can only be
considered as a supplementary component in an
anaesthesia regime (BECKER and ROSENBERG,
2008) and supplements should generally be avoided
to keep the protocol as simple as possible.
Atropine is used in veterinary anaesthesia for the
treatment of anaesthesia-induced side effects such as
excessive salivation or cholinergic bradycardia (LEMKE
2007). Its effects are mediated through the competitive
inhibition of muscarinic acetylcholine receptors. It has
also been described to exert a minimal effect on
nicotinic receptors. Therapeutic doses of atropine are
primarily active peripherally but as the blood/brain barrier is incompletely passed there are also central antimuscarinic effects (ROBENSHTOK et al., 2002). Atropine can be a useful component of some anaesthesia
protocols but its use may be disadvantageous in PET/
SPECT studies that quantify acetylcholine receptors or
acetylcholinesterase activity.
Pancuronium bromide is a neuromuscular blocking
agent that acts as a competitive inhibitor of the
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acetylcholine receptor on the post-junctional membrane of the neuromuscular junction (AGLAN and
POLLARD, 1995). Because it acts strictly in the periphery it can be useful to ensure complete immobilization. However, the need to intubate and ventilate the
animals is a disadvantage and animal welfare considerations make it necessary to ensure that the animals are rendered unconscious by other anaesthetic
drugs.
Conclusion
Based on the literature and our own unpublished
experience with a limited number of animals, the following approaches should be considered for anaesthesia in neuromolecular imaging studies in Tupaia.
As the publication by SCHWAIER et al. (1978) shows,
the use of alfaxalone-containing formulations alone
provides a well tolerated anaesthesia. Our own unpublished experience indicates that induction of anaesthesia is possible by i.m. injection of alfaxan (7.5 mg/
kg) and that anaesthesia can be satisfactorily maintained by a constant infusion of this drug.
Although the sole use of halogenated ethers such
as halothane and isoflurane is not recommended due
to their severe side effects and the improper immobilization (SCHWAIER et al., 1978; TER LAAK et al.,
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1975), complicated protocols based on halogenated
ethers have been described (OHL et al., 1999; LU and
PETRY, 2003; VEIT et al., 2011). The widespread use
and the easy application and set-up of steady state
justify the search for simplified anaesthetic protocols
that make volatile anaesthetics suitable for neuromolecular imaging studies. Our observations indicate
that a supplementary i.m. injection of 0.4 mg/kg butorphanol either before or immediately after induction
with isoflurane can prevent the side effects that have
been described and ensure a proper immobilization.
Satisfactory prolongation of anaesthesia can be obtained by intramuscular reinjection of half the initial
dose every 30 min. It is conceivable that this approach might be refined by administering butorphanol via an i.v. continuous rate infusion; such protocols
should be investigated.
A further approach, based on publications by LU
and PETRY (2003) and VEIT et al. (2011), that is worth
considering is to relax the animals using neuromuscular blocking agents following induction of anaesthesia with halogenated ethers. Proper immobilization
can be expected but intubation and mechanical ventilation are mandatory. The protocol might work well
and while intubation protects against problems linked
with excessive salivation, animal welfare considerations make management more demanding.
References
AGLAN, M.Y., POLLARD, B.J. (1995): Molecular mechanisms of neuromuscular blocking agents: Is the increased understanding of importance to the practising
anaesthetist? Pharmacol Ther 68, 365–383.
ALSTRUP, A.K., SMITH, D.F. (2013): Anesthesia for positron emission tomography scanning of animal brains.
Lab Anim (UK) 47, 12–18.
BAMROONGWONG, S., SOMONA, R., ROJANANEUNGNIT,
S., CHUNHABUNDIT, P., RATTANACHAIKUNSOPON,
P. (1991): Scanning electron microscopic study of the
splenic vascular casts in common tree shrew (Tupaia
glis). Anat Embryol (Berl) 184, 301–304.
BARTENSTEIN, P., ASENBAUM, S., CATAFAU, A., HALLDIN,
C., PILOWSKI, L., PUPI, A., TATSCH, K. (2002): European
Association of Nuclear Medicine. European Association of Nuclear Medicine procedure guidelines for brain
imaging using [(18)F]FDG. Eur J Nucl Med Mol Imaging
29, 43–48.
BECKER, D.E., ROSENBERG, M. (2008): Nitrous oxide and
the inhalation anesthetics. Anesth Prog 55, 124–130.
CARSON, R. (2000): PET physiological measurements
using constant infusion. Nucl Med Biol 27, 657–660.
EL-MAGHRABI, E.A., ECKENHOFF, R.G. (1993): Inhibition of dopamine transport in rat brain synaptosomes by
volatile anesthetics. Anesthesiology 78, 750–756.
FRANKS, N.P. (2006): Molecular targets underlying general
anesthesia. Br J Pharmacol 147, 72–81.
FRANKS, N.P., LIEB, W.R. (1994): Molecular and cellular mechanisms of general anaesthesia. Nature 367,
607–614
FUCHS, E., FLÜGGE, G. (2002): Social stress in tree
shrews: effects on physiology, brain function and
behavior of subordinate individuals. Pharmacol Biochem
Behav 73, 247–258.
FUCHS, E., FLÜGGE, G. (2003): Chronic social stress:
effects on limbic brain structures. Physiol Behav 79,
417–427.
FUCHS, E., CORBACH-SÖHLE, S. (2010): Tree Shrews. In:
HUBRECHT, R., KIRKWOOD, J. (Eds): The UFAW handbook on the care and management of laboratory and other
research animals. Vol. 8, Wiley-Blackwell, Oxford, UK.
262–271.
GALLAGHER, B.M., FOWLER, J.S., GUTTERSON, N.I.,
MACGREGOR, R.R., WAN C.N., WOLF, A.P. (1978):
Metabolic trapping as a principle of radiopharmaceutical
design: some factors responsible for the biodistribution of [18F] 2-deoxy-2-fluoro-D-glucose. J Nucl Med 19,
1154–1161.
GOULD, R.W., GARG, P.K., GARG, S., NADER, M.A.
(2013): Effects of nicotinic acetylcholine receptor agonists on cognition in rhesus monkeys with a chronic cocaine self-administration history. Neuropharmacology
64, 479–488.
17
Wiener Tierärztliche Monatsschrift – Veterinary Medicine Austria
HALLDIN, C., NAGREN, G., SWAHN, C.G., LANGSTROM,
B., NYBACK, H. (1992): (S)- and (R)-[11C]nicotine and the
metabolite (R/S)-[11C]cotinine. Preparation, metabolite
studies and in vivo distribution in the human brain using
PET. Int J Rad Appl Instrum 19, 871–880.
HEISS, W.D., HERHOLZ, K. (2006): Brain receptor imaging.
J Nucl Med 47, 302–312.
JEVTOVIĆ-TODOROVIĆ,
V.,
TODOROVIĆ,
S.M.,
MENNERICK, S., POWELL, S., DIKRANIAN, K.,
BENSHOFF, N., ZORUMSKI, C.F., OLNEY, J.W. (1998):
Nitrous oxide (laughing gas) is an NMDA antagonist, neuroprotectant and neurotoxin. Nat Med 4, 460–463.
KRASOWSKI, M.D., HARRISON, N.L. (1999): General
anesthetic actions on ligand-gated ion channels. Cell
Mol Life Sci 55, 1278–1303
LEMKE, K.A. (2007) Anticholinergics and Sedatives. In:
THURMON, J.C., TRANQUILLI, W.J., GRIMM, K.A., (Eds)
Lumb and Jones‘: Veterinary anesthesia and analgesia.
Vol. 4, Wiley-Blackwell Oxford, UK. 203–207.
LU, H.D., PETRY., H.M. (2003): Temporal modulation
sensitivity of tree shrew retinal ganglion cells. Vis Neurosci 20, 363–372.
MATTA, B.F., HEATH, K.J., TIPPING, K., SUMMORS, A.C.
(1999): Direct cerebral vasodilator effects of sevoflurane
and isoflurane. Anesthesiology 91, 677–680.
MCQUEEN, J.K., MARTIN, M.J., HARMAR, A.J. (1984):
Local changes in cerebral 2-deoxyglucose uptake during alphaxalone anaesthesia with special reference to
the habenulo-interpeduncular system. Brain Res 300,
19–26.
MEIJER, M.K., SPRUIJT, B.M., VAN ZUTPHEN, L.F.,
BAUMANS, V. (2006): Effect of restraint and injection methods on heart rate and body temperature in mice. Lab
Anim 40, 382–391.
MEYER, P.T. (2009): Evaluation von Radiopharmaka für
die molekulare Neurobildgebung am Kleintiermodel und
Menschen: Pharmakokinetische Modellierung und Einflussfaktoren auf die Quantifizierung. Professorial Dissertation, Medical Faculty of the University of Freiburg,
Germany.
MEYER, R.E., FISH, R.E. (2008): Pharmacology of injectable
anesthetics, sedatives and tranquilizers. In: FISH, R.E.,
BROWN, M.J., DANNEMANN, P.J., KARAS, A.Z. (Eds.):
Anesthesia and Analgesia in Laboratory animals. Vol. 2,
Elsevier, London, 27–67.
MURRAY, H.M., DOMINGUEZ, W.F., MARTINEZ, J.E.
(1982): Catecholamine neurons in the brain stem of tree
shrew (Tupaia). Brain Res Bull 9, 205–215.
MYERS, R., HUME, S. (2002): Small animal PET. Eur Neuropsychopharmacol 12, 545–555.
NOLAN, M.F., GIBSON, I.C., LOGAN, S.D. (1997): Actions of the anaesthetic Saffan on rat sympathetic
preganglionic neurones in vitro. Br J Pharmacol 121,
324–330.
OHL, F., MICHAELIS, T., FUJIMORI, H., FRAHM, J., RENSING, S., FUCHS, E. (1999): Volumetric MRI measurements of the tree shrew hippocampus. J Neurosci Methods 88, 189–193.
18
101 (2014)
PHILLIPS, J.R., KHALAJ, M., MCBRIEN, N.A. (2000):
Induced myopia associated with increased scleral creep
in chick and tree shrew eyes. Invest Ophthalmol Vis Sci
41, 2028–2034.
RICE, M.W., ROBERTS, R.C., MELENDEZ-FERRO, M., PEREZ-COSTAS, E. (2011): Neurochemical characterization
of the tree shrew dorsal striatum. Front Neuroanat 5:53,
doi: 10.3389/fnana.2011.00053
ROBENSHTOK, E., LURIA, S., TASHMA, Z., HOURVITZ, A.
(2002): Adverse reaction to atropine and the treatment
of organophosphate intoxication. Isr Med Assoc J 4,
535–539.
ROCKLAND, K.S., LUND, J.S., HUMPHREY, A.L. (1982):
Anatomical binding of intrinsic connections in striate
cortex of tree shrews (Tupaia glis). J Comp Neurol 209,
41–58.
ROWLAND, D.J., CHERRY, S.R. (2008): Small-animal preclinical nuclear medicine instrumentation and methodology. Semin Nucl Med 38, 209–222.
SCHMIDT, K.C., TURKHEIMER, F.E. (2002): Kinetic modeling in positron emission tomography. QJ Nucl Med 46,
70–85.
SCHWAIER, A. (1978): Anaesthesia of Tupaias (tree shrews).
Z Versuchstierkd 20, 216–221.
SESMA, M.A., CASAGRANDE, V.A., KAAS, J.H. (1984):
Cortical connections of area 17 in tree shrews. J Comp
Neurol 230, 337–351.
SLIFSTEIN, M., LARUELLE, M. (2001): Models and methods for derivation of in vivo neuroreceptor parameters
with PET and SPECT reversible radiotracers. Nucl Med
Bio 28, 595–608.
SOKOLOFF, L. (1981): Relationships among local
functional activity, energy metabolism, and blood flow in
the central nervous system. Fed Proc 40, 2311–2316
STROME, E.M., DOUDET, D.J. (2007): Animal models of
neurodegenerative disease: insights from in vivo imaging
studies. Mol Imaging Biol 9, 186–195
TATSCH, K., ASENBAUM, S., BARTENSTEIN, P., CATAFAU,
A., HALLDIN, C., PILOWSKY, L.S., PUPI, A. (2002a): European association of nuclear medicine. European association of nuclear medicine procedure guidelines for
brain neurotransmission SPET using (123)I-labelled dopamine D(2) transporter ligands. Eur J Nucl Med Mol
Imaging 29, 30–35.
TATSCH, K., ASENBAUM, S., BARTENSTEIN, P., CATAFAU,
A., HALLDIN, C., PILOWSKY, L.S., PUPI, A. (2002b): European association of nuclear medicine. European association of nuclear medicine procedure guidelines for
brain neurotransmission SPET using (123)I-labelled dopamine D(2) receptor ligands. Eur J Nucl Med Mol Imaging 29, 23–29.
TATSCH, K., ASENBAUM, S., BARTENSTEIN, P.,
CATAFAU, A., HALLDIN, C., PILOWSKY, L.S., PUPI,
A. (2002c): European association of nuclear medicine.
European
association
of
nuclear
medicine
procedure guidelines for brain perfusion SPET using
(99m)Tc-labelled radiopharmaceuticals. Eur J Nucl Med
Mol Imaging 29 36–42.
Wiener Tierärztliche Monatsschrift – Veterinary Medicine Austria
TER LAAK, H.J., THIJSSEN, J.M., VENDRIK, A.J. (1975): A
method for prolonged electrophysiological experiments
with the tree shew (Tupaia chinensis). Z Versuchstierkd
17, 195–204.
TSUKADA, H., NISHIYAMA, S., KAKIUCHI, T., OHBA, H.,
SATO, K., HARADA, N. (2001): Ketamine alters the availability of striatal dopamine transporter as measured by
[(11)C]beta-CFT and [(11)C]beta-CIT-FE in the monkey
brain. Synapse 42, 273–280.
VANDER BORGHT, T., ASENBAUM, S., BARTENSTEIN, P.,
HALLDIN, C., KAPUCU, O., VAN LAERE, K., VARRONE,
A., TATSCH, K. (2006): European association of nuclear
medicine (EANM). EANM procedure guidelines for brain
tumour imaging using labelled amino acid analogues. Eur
J Nucl Med Mol Imaging 33, 1374–1380.
VEIT, J., BHATTACHARYYA, A., KRETZ, R., RAINER, G.
(2011): Neural response dynamics of spiking and local
field potential activity depend on CRT monitor refresh
rate in the tree shrew primary visual cortex. J Neurophysiol 106, 2303–2313.
VERMA, A., MOGHADDAM, B. (1996): NMDA receptor antagonists impair prefrontal cortex function as assessed
via spatial delayed alternation performance in rats: modulation by dopamine. J Neurosci 16, 373–379.
VOTAW, J., BYAS-SMITH, M., HUA, J., VOLL, R. (2003):
Interaction of isoflurane with the dopamine transporter.
Anesthesiology 98, 404–411.
101 (2014)
WAELBERS, T., POLIS, I., VERMEIRE, S., DOBBELEIR,
A., EERSELS, J., DE SPIEGELEER, B., AUDENAERT, K.,
SLEGERS, G., PEREMANS, K. (2013): 5-HT2A receptors
in the feline brain: 123I-5-I-R91150 kinetics and the influence of ketamine measured with micro-SPECT. J Nucl
Med 54, 1428–1433
WAHL, R.L., HENRY, C.A, ETHIER, S.P. (1992): Serum glucose: effects on tumor and normal tissue accumulation
of 2-[F-18]-fluoro-2-deoxy-D-glucose in rodents with
mammary carcinoma. Radiology 183, 643–647.
WALLETSCHEK, H., RAAB, A. (1982): Spontaneous activity
of dorsal raphe neurons during defensive and offensive
encounters in the tree-shrew. Physiol Behav 28, 697–705.
YAMASHITA, A., FUCHS, E., TAIRA, M., HAYASCHI, M.
(2010): Amyloid beta (Aβ) protein- and amyloid precursor
protein (APP)-immunoreactive structures in the brains of
aged tree shrews. Curr Aging Sci 3, 230–238.
ZANZONICO, P. (2012): Principles of nuclear medicine imaging: planar, SPECT, PET, multi-modality, and autoradiography systems. Radiat Res 177, 349–364.
*Corresponding author’s address:
Sebastian Gehrig,
Department for Nuclear Medicine,
University Medical Center Freiburg,
Hugstetter Strasse 55, 79106 Freiburg, Germany
e-mail: [email protected]
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