Operating and Biocontainment Procedures of a Facility for Laboratory Mice with a Natural Microbiome: Immunophenotyping Procedure

We investigate the effects of nutrition on the immune system. And we are particularly interested in the anti-aging and anti-inflammatory effects of calorie restriction and fasting regimens and the role of the microbiota in these processes. Wildling mice combine the well-characterized genetic background of laboratory mouse strains and the natural microbiome of free-roaming mice.

However, the natural microbiome of these wildling mice contains viruses, bacterias, and also parasites, and poses, therefore, a challenge to animal husbandry facilities. The contribution of the QUEST Center for Responsible Research was development of the six R framework, which enhances the three Rs that we know from Russell and Burch, replacement, refinement, and reduction, by incorporating three additional Rs robustness, registration, and reporting. This framework aims at enhancing the reproducibility and transparency of pre-clinical research.

And the idea is that experimental findings are more reliable, trustworthy, and ultimately translatable to clinical contexts. Over the past four years, we have developed the concept of a facility for wildling mice with distinct biocontainment procedures. We would like to share our expertise with researchers to enable them to include mice with a natural microbiome into their work.

Stroke are most common in aging people. The immune system has memory cells from fighting infectious particles and living with heterogeneous microbiota. Thus, modeling stroke in wildling mice is a refined model in rodents that better reflects patients'conditions.

After changing into area pants, cossack, and disposable shoe covers, remove shoes and socks in the anteroom of the air shower. Put on area socks and full-body personal protective equipment, including Tyvek overalls, hairnet, cotton gloves, nitrile gloves, and face masks. Enter the air shower wearing area socks via the adhesive floor covering.

While air showering, raise your arms and perform a 360-degree turn. Exit the air shower and put on area shoes on the other side. Transfer the mouse from their room in a closed, individually ventilated cage to the procedure room.

Position the anesthetized mouse prone on the disinfected table and grip the scruff of its neck to compress the neck veins. Using one hand, secure the mouse's head with the thumb and forefinger. Insert a fresh capillary into the medial canthus of the eye under the nictitating membrane at a 45-degree angle.

Axially rotate the capillary through the sinus membrane at the back of the orbit until blood begins to flow. Collect a minimum of 15 microliters of blood in a blood collection tube containing a drop of EDTA. Loosen the grip on the scruff of the neck before removing the capillary to minimize bleeding into the tissue.

Use a clean gauze pad or swab to stop the bleeding and ensure hemostasis. Slightly swivel the tube to mix the blood and EDTA without shaking it and store the blood sample on ice. After completing blood sampling, change gloves and disinfect all materials and surfaces.

Put on new protective clothing in the access laminar air flow, or LAF, located in front of the LAF cabin. Disinfect the sample containers and remove them via the sliding door in the glass wall of the LAF cabin. After transferring the sample containers to the person two, disinfect all surfaces.

Receive disinfected sample containers from the wildling area without direct contact through the sliding door in the glass wall of the LAF cabin. Then, leave disposable overalls and area shoes inside the designated area. Dispose of the hairnet, rubber gloves, and face mask.

Enter the air shower immediately in area socks. Raise your arms and perform a 360-degree turn while air showering. Leave the air shower and place area socks in the laundry collector.

Put on personal socks, shoes, and disposable shoe covers. Deposit area clothing in the changing room laundry collector and change into regular clothes.