Physiological Changes Indicative of 'Nausea'
Abbreviated to "PCIN"…Thirty-Plus Years of Experience Using 7 Species

We have been actively involved in emesis research experimentation for more than thirty years. During this period, we have worked with over twenty pharmaceutical companies on discovery projects relating to emetic liability testing and anti-emetic discovery. We routinely use ferrets and Suncus murinusfor most experimentation, but also use rats, mice, pigeons, cats and zebrafish.
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• Nausea and vomiting can occur independently of one another - so we expect that different control mechanisms are involved.
• Animals are unable to communicate directly their emotions in a way that we can understand - so how can we detect that they are experiencing nausea, if indeed they do?
• We are the only group in the World that brings together a myriad of techniques to assess nausea; we have experience in ferrets, Suncus murinus, rats, mice, cats and pigeons.
• We have examined a wide variety of emetic challenge and collected a vast amount of data on behavioural profiles with radiotelemtry.
• We have profiled a number of successful anti-emetic drugs and understand the limitations of experimentation into mechanisms of nausea; side effect profiles of drugs often mask interpretations.

​Assessing Nausea in Animals​

• ​Assessing nausea in animals can be done objectively if the experiment is well-designed; there must be appropriate randomization and blinding of the investigators concerned.
• Which species is best for these purposes? The answer may depends on the emetogen being studied.
• We bring together several technologies to simultaneously assess physiological functions known to be disturbed during nausea.
• We have profiled our serval drug treatments that induce nausea in our models (e.g. apomorphine, morphine, cisplatin etc.) and assessed the profiles against what happens when ferrets have eaten a meal. This approach gives perspective and meaning to our experiments.
• We use advances analytical techniques to assess clusters of changes behaviour and physiological markers including GMA/BP/HRV/Respiration and temperature homeostasis.
• We also assess the profile of action of novel chemical entities against profiles obtained with the major classes of anti-emetics including 5-HT3, NK1, D2 and histamine receptor antagonists.

Behavioural Data

• As much behavioural data should be captured as possible, and it should have a temporal expression relative to the stimulus strength.
• The significance of the data may be skewed, if the stimulus also produces vomiting, since the act of vomiting itself would displace behaviour normally expressed during nausea.
• Relying on one behavioural marker is not wise – it is better to cluster behaviours together, but this is both species- and emetogen-specific/variable.
• Changes in feeding/drinking patterns or pica can be considered indicative of nausea but may also be species/stimulus/strain-specific.

Physiological Data

• Physiological data should be obtained, preferably by radiotelemetry and non-invasive methods of whole body plethysmography.
• There needs to be assessment of gastric function by electrogastrography to assess data on gastric slow waves, and an assessment of blood pressure and heart rate variability and temperature to assess imbalances in autonomic nervous system tone; these control systems are known to be disturbed during nausea in man.
• There need to be an assessment of respiratory function, since brainstem function controls normal respiration and pattern changes during nausea and emesis.
• Forebrain and brainstem activity should be assessed either in real-time (electrophysiological/microdialysis/fMRI) or using c-fos immunohistochemistry.
• We also use infrared imaging techniques to examine changes in thermoregulation.

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Plasma/Tissue Biomarker Data/C-Fos

• Biomarker data should be collected, but this is not always possibly if the animal would need to be restrained during the observation period to collect the sample.
• Example biomarkers include vasopressin, ghrelin, cortisol, 5-HT, substance P, glutamate; these biomarkers also change in situations not related to nausea, so great care is needed when interpreting changes.
• Forebrain and brainstem activity should be assessed either in real-time (electrophysiological/microdialysis/fMRI) or using c-fos immunohistochemistry; this could be misleading if the stimulus also has other actions not necessarily related to nausea and/or emesis.

Validation Relative to Off-Target Effects

• The magnitude of change in the behavioural/biomarker/physiological marker/fos pattern data should be reduced appropriately by drugs known to reduce nausea in man.
• For example the 5-HT3 receptor antagonists, ondansetron and palonosetron, would be active against the early nausea and emesis induced by cisplatin, but ineffective against the nausea and emesis induced by apomorphine or motion. Alternatively, the D2 dopamine receptor antagonist, domperidone, would reduce apomorphine- but not motion-induced emesis. In both examples, the anti-emetics have clean profiles as they have few side effects administered alone.
• Conversely, anti-histamines reduce emesis induced by motion, but used also cause sedation, affect thermoregulation and gastric motility and autonomic nervous system control - these off-target side effects obscure assessments made on nausea.
• New anti-emetic candidates therefore need rigorous profiling to evaluate their usefulness to reduce nausea.

Physiological Changes Indicative of 'Nausea': PCIN

Radiotelemetry

• Gastric Myoelectric Activity: We have identified the dominant frequency (DF) of slow waves and the percentages power ranges of bradygastria, normogastria, and tachygastria in four species. We monitor these variables during normal situations (e.g. feeding) versus drug treatments causing nausea and emesis; we also profile the action of anti-emetics. It is important to understand that emetic drugs themselves may have direct effects on the gastrointestinal tract that may be independent of the sensation of ‘nausea’. Knowing how to identify off-target effects is therefore critical to a correct interpretation of data.
• Cardiovascular: The heart is modulated by the autonomic nervous system and changes in blood pressure with, or without, changes in heart rate can occur during ‘nausea’ and episodes of emesis. Monitoring heart rate variability (HRV) is done to provide insight into alterations of autonomic outflow during experimentation. A decrease in HRV and the shift to “sympathetic dominance” can be visualised by an increase the Low Frequency:High Frequency ratio. Just like changes in GMA, these changes alone cannot be taken as indicative of nausea, since such effects may be seen following generalised stress, and HRV changes also occur normally during feeding. It is pertinent to measure cardiovascular parameters to also know if changes in c-Fos in the brainstem are independent of BP increases or decreases, since cardiovascular control and emetic mechanisms are both relayed through the brainstem. Again, without doing this, off target effects may complicate an interpretation of data relevant to PCIN.
• Temperature: Fever and hypothermia have both been associated with sickness behaviour; hypothermia is readily observed during motion sickness experiments. Therefore, it is difficult to ascribe thermoregulatory changes alone as resulting from a sensation of ‘nausea’. However, it is extremely important to access thermoregulatory data because temperature affects gastric myoelectric activity; hypothermia may cause bradygastria; some drugs can induce hypothermia without the sensation of nausea. Specialist knowledge is therefore required when assessing telemetry data collected from emesis experiments in animals.

c-Fos Patterns

• C-Fos immunohistochemistry has been used to map active brain areas in a variety of species following treatments that are known to induce nausea and emesis; anti-emetic drugs have been used to validate the relevance of brain nuclei but interpretation is subject to bias as data is not real-time; in most cases it is done without double staining for other markers of interest, such as those capable of identifying neuronal type, or a particular receptor.
• Nevertheless, several key brain areas have been identified as being involved across a variety of stimuli. Knowing which clusters of brain areas relate to inputs/outputs controlling somatic and autonomic control with respect to PCIN and emesis is key to accessing information on ‘nausea’.
• We have clustered changes of brain activity in the brainstem, midbrain and brainstem, to assess separate mechanisms of 'nausea', feeding and emesis, looking in particular at those animals that are resistant to emesis during emetic treatments.
•  We have performed c-Fos studies against a range of emetic stimuli. We performed an exemplar study with the GLP-1 agonist, exendin-4, in ferrets, showing it is possible to cluster brain areas involved in ‘nausea’ and emesis. We also performed studies in Suncus murinus revealing that some anti-emetics themselves paradoxically induce c-Fos.
• We have extensive knowledge on the effect of brain- and non-brain penetrating ghrelin mimetic to affect c-Fos expression in the brain of Suncus murinus.

Behaviour & Respiratory

• There have been several attempts to ascribe changes in behaviour, or patterns of behaviour, with ‘nausea’. However, it is apparent that different species have different patterns of behaviour to emetic challenges; just as increasing doses of emetogens can induce different patterns in the same species. We have assessed behavioural patterns in ferrets, Suncus murinus and rats. In ferrets, we focus on backing, burrowing, scratching and licking behaviour, relative to generalised locomotor activity; in Suncus murinus, we focus on sniffing, chin on the floor, scratching, licking, lying flat, relative to generalised locomotor activity.
• Not all species exhibit emesis, so why would we expect all species to exhibit nausea? Even feeding patterns following chemotherapy are different; for example, ferrets decrease food consumption dramatically, Suncus murinus eats less, but the effects are less pronounced, rat decrease food consumption but eat kaolin (at least some strains). Clearly food consumption during experimentation impacts on gastric myoelectric activity, thermoregulation and a host of autonomic measures which would ultimately affect c-Fos expression in the brain. Knowing about these differences associated with each emetic treatment is key to understanding the limit of interpretation of mechanisms relevant to nausea and emesis.
• We simultaneously assess respiratory function, which is disturbed during nausea and emesis, in Suncus murinus. This is key to our interpretation of PCIN, and lets us accurately record emetic events using burst analysis. Respiration and emesis involve the same sets of neurones in the brainstem that control muscle groups to facilitate breathing and retching and vomiting, so our hypothesis is when nausea almost translates to emesis, the pattern of breathing is altered as the brainstem processes information. Our ability to record respiratory activity also enables us to see if drugs cause sedation, as an off-target effect.
• We also simultaneously assess how the body thermoregulates in response to treatments inducing 'nausea' and emesis.