TABLE 2:

TROUBLESHOOTING

Cognitive function
Step	Problem	Possible reason	Possible Solution
6	Some animals might spend most of the time in a corner, immobile and with limited exploring	Stress	This could be related to stress and if occurring in individual animals only, they should be removed from the analysis. If this is a feature seen in many experimental animals, consider eliminating possible stressors present in the behavioural room or the animal facility. It may also be necessary to increase handling and/or habituation time.
6	Some objects stimulate the animals to “play” with the object including climbing on top of the object and remaining there for a while.	This is most often due to mobile pieces in the object or a big surface on top of the object	Select a different pair of objects
7	Lack of increased exploration time of novel vs. familiar object	Stress Insufficient training Lack of interest in objects	Identify and eliminate possible stressors in the behavioural testing or animal housing environment, increase handling and/or habituation time Increase training time Pre-select a different pair of objects
Muscle strength and neuromuscular function
Grid hanging test
Step	Problem	Possible reason	Possible Solution
4	Mice let go very quickly	Grid not high enough/loss of fear of falling	Place the grid slightly higher
Grip strength
Step	Problem	Possible reason	Possible Solution
4	Very variable readings	Operator holding the mouse at an angle	Ensure the mouse is kept horizontally when pulling and the force applied is proportionate
4	Very variable readings	Claws are too long	Claw length may be affected by activity or environment (e.g. bedding). Change bedding or clip claws
Hanging bar
Step	Problem	Possible reason	Possible Solution
2906313	2906313	2906313	2906313
1	Mouse let go quickly even if they are young	Insufficient fear of falling	Position the bar higher
Rotarod
Step	Problem	Possible reason	Possible Solution
8	Mouse change position on the rotarod.	Insufficient training	Gently reposition the mouse.
8	Mice fall from the rotarod too quickly.	Old, frail mice, background noise.	Gently position mouse on the track again. Reduce exposure to possible distractions. Remove extremely frail mice from the assay.
8	Mouse turns around in the middle of the experiment and start walking in the opposite direction	Insufficient training	Gently reposition the mouse until they have learnt
Metabolic Health: Body Composition
Step	Problem	Possible reason	Possible Solution
2	Machine unable to calibrate, takes an unusually long time to calibrate, or takes an unusually long time to read a single animal.	Too much metal mass within a 6-foot radius of the MRI.	The MRI machine must be positioned further away from large metal objects, including heavy metal tables, chairs, and gas cylinders.
Metabolic Health: Energy Balance
Step	Problem	Possible reason	Possible Solution
8	RER results are non-physiological (continuously > 1.0 or < 0.7) and the O2 in and CO2 in readings are constant.	If the reference cage is only read at the beginning of each experimental run, but room air O2/CO2 concentrations change, the delta O2 and delta CO2 will be calculated incorrectly as the software will be using incorrect O2 in and CO2 in readings.	Set the system to read the reference cage (and update O2 in and CO2 in values) every time it cycles through the experimental chambers.
8	RER results are non-physiological (continuously > 1.0 or < 0.7)	Valves on one or more of the calibration air tanks may have been shut during the calibration procedure; alternatively, the calibration air may have settled or the tank may be empty.	Recalibrate system, checking that there is adequate gas flow from the calibration tanks.
8	The gas values read from all of the chambers (O2 out/CO2 out) becomes “stuck” on the same value	A bubble may have formed in the ammonia traps.	Replace the ammonia traps.
Data analysis	Food consumption values are negative or absurdly high.	Mice may have greater than intended access to the feeder, and either deposit waste products or remove food without eating it; or the scale may be defective.	Discard data from cages with negative food consumption values, and inspect the cage bottom to determine if food was removed from the feeder and not consumed. The cage floor may be able to be reset to limit the access of small/lean mice. Verify that the scale is functioning properly and replace/repair as needed.
Metabolic Health: Glucose tolerance test
Step	Problem	Possible reason	Possible Solution
8	Blood glucose reading seems extraordinarily high or low based on reading at prior time point.	Approximately 2% of reads are unusual, due to causes that include defective or contaminated glucose test strips or samples, or samples that are too small or partially clotted.	Wipe the tail, and with a fresh drop of blood immediately repeat the reading with a fresh test strip. If value differs by more than 10% from the first reading, use the data from this second reading; otherwise, keep the first reading.
8	Blood glucose monitor says all or most glucose test strips are defective.	Monitor failure.	Replace blood glucose monitor. As many monitors are inexpensive, spares can often be kept on hand.
Metabolic Health: Insulin tolerance test
Step	Problem	Possible reason	Possible Solution
9	Blood glucose reading seems extraordinarily high or low based on reading at prior time point.	Approximately 2% of reads are unusual, due to causes that include defective or contaminated glucose test strips or samples, or samples that are too small or partially clotted.	Wipe the tail, and with a fresh drop of blood repeat the reading with a fresh test strip. If value differs by more than 10% from the first reading, use the data from this second reading; otherwise, keep the first reading.
9	Blood glucose monitor says all or most glucose test strips are defective.	Monitor failure.	Replace blood glucose monitor. As many monitors are inexpensive, spares can often be kept on hand.
Cardiac function
Step	Problem	Possible reason	Possible Solution
5	No heart rate/ECG signal	Poor contact with platform, lack of signal conduction	Check foot placement and use electroconductive gel if needed, check connections of platform to system
11	Initial poor image quality	Insufficient skin preparation or insufficient ultrasonic gel for contact	Check skin for effective and thorough hair removal, add additional warmed gel.
11	Excess LV dilation/poor systolic function with slow, gradual recovery is observed	Excess anaesthesia upon induction resulting in anaesthesia-induced cardiodepression	Practice rapidly placing the animal on the platform, inducing anaesthesia with the lowest level of isoflurane possible, and rapidly lowering to a minimal plane of anaesthesia. Avoid use of an anaesthesia box for induction when possible (particularly with obese mice),
5	Significant bradycardia (HR<450) and poor LV function	Excess administration of anaesthesia, excess pressure on chest	Lower anaesthesia concentration; ensure negligible pressure is applied to the chest while imaging
5	Profound bradycardia (HR<250), LV dilation, and arrhythmias	Adverse response to anaesthesia	Remove animal from nose cone and allow to recover fully. Attempt imaging again after full recovery or on another day.