SCIREQ SOFTRESTRAINT FOR EXPOSURE STUDIES
EVALUATING THE STRESS RELATED EFFECTS OF VARIOUS RESTRAINT METHODS IN RODENTS
Any restraining device used in an inhalation exposure set-up will be, by definition, restricting the subject’s ability to move. For nose-only inhalation set-ups, typical restraints are rigid, fully enclosed and include a metal or acrylic plunger forcing the subject forward and preventing the subject from retreating. Example of such restraints are shown below:
As evidenced in the above images, the subjects are heavily restricted in movement and do not have any ability to regulate their heat output. Furthermore, when not properly secured, the subject may be able to rotate within the restraint, this has resulted in subjects breaking their necks and suffocating.
SCIREQ’s SoftRestraints are designed to minimize some of these important effects typical restraints have on the subject. Firstly, since it is not fully enclosed, it allows some airflow around the skin and tail thereby enabling the subjects to naturally regulate their body temperature. In addition, the mesh gently restrains the subjects while imposing little-to-no compression or restriction around the thorax. Because of this, chest movements are not impeded and the subject’s ability to adjust to environmental cues is maintained. This can be observed during hyperventilation periods as the subjects are able to regulate their breathing patterns in response to a given exposure environment. Finally, a clip can be added at the back of SCIREQ’s SoftRestraints to maintain the subject’s position. This lock mechanism is optional and is a gentle alternative to a classic metal or acrylic backstop, which can compress the thoracic cavity and impede breathing and/or breathing regulation.
WORKING WITH THE SOFTRESTRAINTS
The use of SCIREQ’s SoftRestraints, like any other restraining devices, requires an acclimation period. Because of its design, this process is likely to be better tolerated by the subjects in comparison to typical restriction devices and acclimation time is reduced.
SCIREQ’s standard protocol recommendations for acclimatization to SoftRestraints are listed below:
» Place the subjects in the restraints and back into their cage for 5 minutes a day, and slowly increase the time each day. This will take place over a period of 1 – 2 weeks, until reaching the desired exposure time dictated by the protocol.
» Place the restrained subjects in the exposure system with only a bias flow for the duration of the experimental period in sequential days.
The proper placement of the subject within the restraint is important to its intended use. SCIREQ’s SoftRestraints are designed to hold the subject firmly but with enough flexibility to prevent undue constriction. It is particularly important that no bulbous shape in the mesh should be allow around the subject’s neck as this would allow for excessive movement and potential injury should the subject becomes caught at the junction of the mesh and the plastic ring. Please refer to the following images below for reference, noting that the subject is not anaesthetized and merely acclimated to the restraint.
The inExpose system is a computer-controlled exposure system that can reproducibly generate exposure conditions. Once the subject is properly positioned and acclimated to the SoftRestraint, the exposures will be consistent across subjects within a group and experimental days.
Many SCIREQ customers utilize the SoftRestraints in their daily experiments. Some of their feedback and corresponding contact information is listed below for easy reference:
» Dr. McHowat notes that once the mice were acclimatized (always using the same animal handler), they actually just walk right inside the SoftRestraints. The mice do not require any further locking mechanism and they accepted the exposures without difficulty.
» Dr. Crotty-Alexander regularly uses these restraints and found that if the mice are handled gently and properly, they typically just walk right in since mice are naturally curious and like small spaces.
Validation Study
Drs. Tate and Mansell at the Hudson Institute of Medical Research performed an internal preliminary assessment regarding the comparative stress levels in mice when restrained using the inExpose SoftRestraints.
They investigate the levels of corticosterone in serum in mice, comparing the following groups:
» Group 1: Subjected to isoflurane anaesthetisation (Anaesthesia).
» Group 2: Restrained in SoftRestraint for 15 minutes (Restraint).
They also analysed serum previously collected during their standard experimental procedure in mice to examine their stress levels for comparison:
» Group 3: Serum from mice 3 days after isoflurane anaesthetisation and intranasal delivery of PBS (i.n. PBS).
» Group 4: Serum from mice 3 days after isoflurane anaesthetisation and intranasal inoculation with influenza A virus (i.n. IAV).
» Group 5: Serum from mice 2 hours after intraperitoneal injection of PBS (i.p. PBS).
» Group 6: serum from mice 2 hours after intraperitoneal injection of LPS (i.p. LPS).
Drs. Tate and Mansell found no significant difference in stress using the SoftRestraint compared to their anesthetised (p=0.0967), instranasal PBS (p=0.0967) and intraperitoneal injected PBS (p=0.0923) mice (using One-way ANOVA).
Figure 1 – Unpublished serum corticosterone levels in mice comparing different restraint and treatment methods.
Taken together, these data demonstrate restraining mice in the SoftRestraints induces no more stress than current methods of immobilising and treating mice such as isoflurane anesthetisation.
CONCLUSION
In summary, any restraint will produce stress on the subjects. As outlined above, through its design, the SoftRestraint serves to minimize the impact to the subject. Listed below are some scientific publications using the nose-only configuration of SCIREQ’s inExpose system.
» Chronic Inhalation of E-Cigarette Vapor Containing Nicotine Disrupts Airway Barrier Function and Induces Systemic Inflammation and Multi-Organ Fibrosis in Mice. Crotty Alexander LE., et al. (2018). Am J Physiol Integr Comp Physiol. ajpregu.00270.
» Cigarette Smoke Triggers IL-33–associated Inflammation in a Model of Late-Stage Chronic Obstructive Pulmonary Disease. Lee, J.H., et al. (2019). American Journal of Respiratory Cell and Molecular Biology, 61(5)
» Comparison of airway responses induced in a mouse model by the gas and particulate fractions of gasoline direct injection engine exhaust. Maikawa, C. L., et al. International Journal of Environmental Research and Public Health, 15(3). 2018.
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