Synopsis
stress level of the
animals influences most of the study designs where preclinical MRI is utilized.
Furthermore, anesthesia has profound
effect on fMRI, and extreme care has to be taken while choosing the type of
anesthesia and for monitoring the anesthesia level and physiological state of
the animal in order to obtain reliable and reproducible results. Protocols for
scanning awake animals have been around for two decades but only recently have
become more popular due to increased awareness on importance of physiological
factors for (f)MRI results.
TARGET AUDIENCE
Technicians and investigators performing and/or
designing preclinical MRI/fMRI experimentsOBJECTIVES
To understand limitations coming from social
isolation, stress and use of anesthetized animals in preclinical imaging and
what can be done to control or reduce the effects of these factors in order to increase the
reliability, repeatability, and comparability of the experiments and data?ANIMAL HANDLING, STRESS AND SOCIAL ISOLATION
Stress level significantly affects animal behavior and
metabolism and thus is an important factor in experimental designs, including preclinical
MRI. Housing and animal handling conditions in a typical modern animal facility
are highly standardized and can be assumed to cause rather small variation in
the baseline stress level of the animals. Prior to MRI experiments animals often
undergo surgery either to generate pathological condition or to chronically
implant an invasive manipulation or recording device. It is common practice to
house animals in individual cages after the surgery. However, rodents are
social animals, and social isolation is a significant stress factor that has
shown to cause behavioral changes and oxidative stress, increase the production
of stress hormones and inflammatory cytokines (Möller et al., 2013; Krügel et
al., 2014; Shao et al., 2015; Butler et al., 2016) and even significantly
modulate progression of epileptogenesis (Manouze et al. 2019). Perhaps the
simplest way to circumvent social isolation during post-operative phase is to
use so-called double decker cages where two rats are separated by a grid that
prevents physical contact but allows other social interaction.ANESTHESIA AND PHYSIOLOGICAL MONITORING
During preclinical MRI experiments, animals are
typically anesthetized to prevent excessive movement and stress (Tremoleda et
al., 2018). It is well known that anesthesia affects metabolism, brain function,
hemodynamic response (Martin et al., 2006) and
also causes long-term effects lasting up to several weeks (Colon et al., 2017). The significance of these confounding factors depends
on the type of experiment. In endpoint structural imaging, the effects of
anesthesia on results can be considered to be relatively minor, and only basic
level physiological monitoring and control is required. This typically consists
of feedback-controlled body temperature control and pneumatic breathing
monitoring system. In addition, liquid has to be administered during long scans
(>1 h) to prevent dehydration, typically via subcutaneous or intravenous cannula.
In functional MRI, the choice and level of anesthesia
becomes a major factor in experimental design and can have profound effects on
results (Paasonen et al 2017, Paasonen et al 2018). Currently, popular choices
for terminal rodent fMRI experiments are alpha-chloralose and urethane, while medetomidine,
isoflurane/medetomidine, propofol, or ketamine/xylazine are used for recovery
experiments. However, responses to stimuli and strength and pattern of
functional connectivity has been found to differ remarkably between anesthetics
(Grandjean 2014, Paasonen et al 2018) It should also be noted that anesthetics might
create dynamic state variations of baseline brain activity, which should be
taken into account in the data analysis (Zhurakovskaya et al 2019). In functional
MRI, more rigorous physiological monitoring is required than in structural
imaging. Many anesthetics suppress breathing, and in spontaneously breathing
animals elevated arterial pCO2 levels are often found. This is a serious
confounding factor for fMRI as increased CO2 causes vasodilatation
thus affecting basal blood flow and hemodynamic contrast. Mechanical
ventilation with muscle relaxant together with monitoring of arterial blood
gases from blood samples should be used to obtain reliable and consistent
results. There are a few exceptions to this, including the use of anesthetics
such urethane that preserves rather normal breathing rate, and some
longitudinal study designs where invasive monitoring can be replaced by the use
of capnograph and pulse oximetry. The indirect measures require, however, very
careful instrumentation and calibration for reliable results.AWAKE fMRI
MRI protocols in awake animals have been introduced to
circumvent the adverse effects of anesthesia (King et al 2005, Stenroos et al
2018, Gao et al 2017). Typically, head and body restrainer is used to prevent movement
during the scanning. Animals are habituated to restrainer in a mock scanner,
and it has been shown that as short as 4-day training period can be sufficient
to reduce stress level of the animals to the level that is comparable to stress
level in anesthetized animals (Steenroos et al 2018 ). Occasional movement of
the animals during the scanning may still be a challenge, but can be typically taken
into account in data analysis pipeline. Nevertheless, highly consistent results
in large number of animals have been obtained in awake fMRI studies (Ma et al
2018) and results have significantly advanced our understanding of the rat
brain functional organization. Furthermore, novel fMRI scanning approaches,
which are more quiet and less sensitive
to movement than echo planar imaging (EPI), may provide more robust approach for
awake fMRI (Paasonen et al 2020).CONCLUSIONS
Rodents are social animals and stress level of the
animals influences most of the study designs where preclinical MRI is utilized.
Furthermore, anesthesia has profound
effect on fMRI, and extreme care has to be taken while choosing the type of
anesthesia and for monitoring the anesthesia level and physiological state of
the animal in order to obtain reliable and reproducible results. Protocols for
scanning awake animals have been around for two decades but only recently have
become more popular due to increased awareness on importance of physiological
factors for (f)MRI results.Acknowledgements
Academy of Finland, Erkko Foundation, NIH U01-NS103569References
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