Probiotic Studies in Neonatal Mice Using Gavage

All procedures were carried out pertaining to the guidelines established by the support staff at the Animal Care Facility at the University of British Columbia and all procedures were approved by the UBC Animal Care Committee.

1.Quantification of probiotics administered

NOTE: This step is recommended to determine the exact amount of probiotic CFU that can be administered in a single dose. The quantity of probiotics and vehicle (FOS and maltodextrin) determine the saturation conditions of the solution. From experience, no more than 30 µL (~20 mL per kg) of fluid can be administered to mice on DOL 2 as any greater volume increases risk of aspiration.

1. Prepare six 1.5 mL microcentrifuge tubes for serial dilutions with each tube containing 180 µL of sterile 5% dextrose saline.
2. Weigh a 0.2 g aliquot of a probiotic-prebiotic mixture and dissolve it in 1 mL of 5% dextrose saline in a sterile manner.
3. Vortex for 30 seconds and pipette to break clumps. Repeat until no visible clumps are observed.
   NOTE: Maltodextrin makes the solution viscous and contribute to solution saturation.
4. Perform a serial dilution using tubes prepared in step 1.1. Vortex to mix.
5. Plate 40 µL of every dilution onto a labelled quadrant of the MRS agar plate. Plate each dilution in duplicate.
6. Incubate under anaerobic (or microaerophilic) conditions at 37 °C for 48 h in a vacuum jar using a gas pack.
7. Count each plate within a range of 20-70 colonies per quadrant. Average plate counts with the same dilution and calculate back to the desired units.

2. Preparation of probiotics and prebiotics for gavage

NOTE: The proper dissolution of probiotic and prebiotic is necessary to ensure the smooth injection of liquid through the feeding needle during gavage.

1. Combine the required amount of lyophilized probiotic organism with the desired amounts of prebiotics and vehicle in a sterile microcentrifuge tube.
2. Add appropriate amounts of solvent (5% dextrose saline) to dissolve the probiotic-prebiotic mixture.
   NOTE: The capacity of dissolution is limited by the prebiotic and vehicle used. From experience, the synbiotic combination (with FOS and maltodextrin) reached saturation at approximately 0.3 g/mL while dissolving in a 2 mL microcentrifuge tube using 1 mL of solvent.
3. Vortex to mix until all solids are dissolved. Use a pipet to break up globules of solid particles in the solvent by pipetting up and down.
4. Incubate the solution in a 37 °C water bath for 20 min.
   NOTE: This step can be skipped if the probiotic-prebiotic solution is created from a live culture.
5. Plate a five series 10-fold dilution on MRS agar plates before gavage to accurately quantify the probiotic administered to the pup. This step can be skipped if the accurate CFU count is not needed.

3. Preparation of the biosafety cabinet

1. Use a biosafety cabinet when working with probiotics to maintain aseptic technique. Set the cage with the dams and pups on a heating blanket (set to approximately 38 °C) on one half of the blanket. Place a clean, empty animal cage on the other half of the heating blanket.
2. Place a disinfected or sterile, absorbent pad on the heating blanket to tend to the mouse during gavage.
3. Collect nesting material for the pups from the top of the existing, dam-created nest and create a new conical nesting cup using gloved hands, disinfected using 70% ethanol and dried. Place this new nest in the clean, empty holding cage. This facilitates the transfer of the scent of the nest to the gloved hands and thus minimizes the introduction of other scents on the pup while handling them for the procedure, reducing the risk of cannibalization.
4. Move the pups into the conical nest holding cage and remove the cage with the dam from the cabinet. This decreases the stress for the dam by preventing it from hearing the pups during the procedure.
   NOTE: If the probiotic is a known colonizer of the murine intestines, the treatment conditions must be separated by cages or even different biosafety cabinets to avoid the possibility of cross colonization.

4. Intra-esophageal gavage of neonatal mouse

1. Open the syringe packaging for easy access. Open the packing of the needle in a sterile manner and attach it to the head of the syringe. Wash the needle with 70% ethanol and autoclave prior to the procedure. Use different sets of needles for the treatment and the control group to avoid contamination.
2. Draw a little more than the desired amount of probiotic-dye solution into the syringe. Hold the syringe facing up. Then pull down further and flick with finger to dislodge bubbles and push on the plunger to expel bubbles and the extra liquid volume until the desired volume is reached. This ensures that there is no air space in the needle. For DOL 2 mouse, the volume of gavage must not exceed 30 µL.
3. Place the pup onto the sterile absorbent pad on top of the heating pad. Use the feeding needle (24 Gauge, 1" needle length, 1.25 mm ball diameter) externally to measure the length of the esophagus by placing the ball of the needle just below the xiphoid process (bottom end of the sternum). Mark the needle at the level of the snout to note the limit of insertion of the needle (Figure 1). Observe the pup for health signs, which include regular breathing and pink coloration of the skin.
4. Dip the tip of the needle in the dipping solvent (5% dextrose saline or – the medium used to dissolve the probiotic and pre-biotic) to lubricate the external surfaces of the feeding needle. This facilitates the smooth entry of the needle into the esophagus of the mouse.
5. Lift the pup by the scruff or by gently holding the head and body between the thumb and index finger. Ensure the head, neck and body are held in a straight position. Do not hold the pup by the scruff for longer than 60 s as there is a risk of obstruction of the trachea leading to suffocation. Ensure the pup can breathe. Signs of scruffing too hard can include inability to breathe, significant gasping and the tongue extended out the mouth. Monitor the pup's color and breathing during the entire procedure.
6. Insert the bulb of the needle into the center of the mouth of the pup at a 45° angle to the plane of the torso until it reaches the back of the throat.
7. Gently change the angle of the needle by pivoting on the bulb of the needle and moving the syringe away from the gavaging person (towards the dorsal side of the pup) until it is parallel to the plane of the pup's vertebral column. Scruffing the pup helps keep the needle in place in the back of the throat, and also prevents the mouse from squirming. Make sure the ball of the needle does not advance or exert any pressure against the back of the throat during the angle change.
8. If the mouse attempts to swallow the needle, allow it to naturally slide downward and arrest the movement when the marking on the needle aligns with the snout. The syringe and needle are usually heavy enough to slide down the due to gravity. Support the weight of the needle at all times so the needle slides easily down the esophagus with no downward pressure from the person carrying out the gavage.
   1. If the needle meets resistance in the back of the throat, withdraw the gavage needle slightly to dislodge the ball of the needle and re-angle the needle inside the mouth towards left of the mouse (handler's right) slowly in small, 1 mm increments. The needle should start to slide easily down the esophagus.
   2. If the needle stops before the marking on needle reaches the mouth, do not inject the solution.
   3. Do not keep the needle inserted for more than 20 s. If this occurs, retract the needle slowly while keeping the syringe parallel to the torso and let the pup rest on the paper towel for 30 s to 1 min. Try gavaging again after lubricating the external surface of the needle with the solvent.
      NOTE: Anaesthesia is not used for the procedure as the mouse's response is necessary to gauge the success of gavage.
9. When the marking on the feeding needle is above the snout and aligned with the tip of the snout, do not let the needle move or advance any further. Slowly inject the desired volume of liquid. If the liquid is aspirated or observed to bubble through the nose, stop the injection immediately and slowly retract the needle.
   1. Place the pup upright on the paper towel on the heating pad to aid in its recovery. Monitor closely for continued breathing problems or change in color of the pup which indicates aspiration. Euthanize pups that have aspirated immediately.
10. Once the gavage is complete, gently withdraw the feeding needle at the same angle it was inserted. Place the pup on the paper towel on the warmed heating pad. Wait 10 s for the pup to regain normal activity and breathing pattern. A healthy pink hue should appear over the pup's body and the dye should only be visible in the stomach compartment. Move it back to the cage with the other pups.
    NOTE: Gavaging blue food coloring is an excellent way to practice the procedure outlined above. If the gavage is successful, the stomach of the mouse will be visible as a blue hue.If the blue dye is found outside the stomach of the pup (neck, chest or axillary region), the animal should be humanely euthanized (in accordance with the animal care rules), as this indicates a rupture of the esophagus or aspiration.

5. Collection of intestional samples for colonization analysis

1. During subsequent monitoring or gavaging, collect fecal microbiome samples from the pups.
   NOTE: The pup frequently urinates and defecates when gavaged and this time can be used as an opportunity to collect the fecal samples for microbiome analysis.
2. For termination of experiments, collect the intestines from duodenum to rectum after euthanasia of the pups. Pin the pup to a surgical board and disinfect the skin with 70% ethanol. Cut away the skin into four quadrants without damaging the peritoneal layer using tools sterilized with 70% ethanol and a hot bead sterilization at 250 °C.
3. Use a different set of sterile tools to cut the peritoneum into four quadrants and move it away from the center in a way that the visceral organs are exposed.
4. Locate the stomach and use a clamp to pinch below the pyloric sphincter and at the end of rectum. Run the length of the intestine using a blunt tool or forceps to streamline the intestine and free it from the connective tissue and mesenteric tissue. Once the entire length of the intestine has been freed of connective tissue, cut at the clamped ends.
5. Mark the aluminum foil with the orientation of the intestine, wrap in a secure manner and freeze at -80 °C.
   NOTE: The DNA extraction procedure can be carried out at this point without freezing. The blue dye was also seen to pass through the intestine over 24 h and collection of samples for colonization analysis is best when the intestines are collected at least 24 hours post the last gavage. Signals might be amplified before that timepoint by the non-adhered bacteria transiently passing through the gavage mixture.

6. DNA extraction from intestines for colonization analysis

NOTE: The DNA extraction is done using a commercial kit with optimizing modifications made to the protocol for the intestine DNA extraction. Ensure the heating apparatus is set to the desired temperature and the solutions that need alterations or pre-warming are prepared appropriately.

1. Prepare the Enzymatic Lysis Buffer (ELB) as follows: Make a solution with 20 mM Tris-Cl, 2 mM sodium EDTA and 1.2% Triton X-100. Adjust the pH to 8.0. Immediately before using ELB, add lysozyme to a final concentration of 20 mg/mL.
2. Pre-weigh the garnet bead tubes on analytical balance with the caps removed.
   NOTE: This is done so that if the required weight is overshot, it is easier to remove the intestinal contents.
3. Cut the intestines into small segments using a sterile disposable scalpel and scoop the desired segments into the pre-weighed garnet bead tubes.
   NOTE: Make sure to change scalpels between every sample as DNA is ubiquitous and can affect PCR results.
4. Add 1 mL of ELB with lysozyme (from step 6.1) to each tube, place on the vortexing bead beater and run at maximum setting (14) for 5 minutes.
5. Once the tissue is disrupted, transfer the tubes to the 37°C water bath and incubate for 30 minutes.
   NOTE: This step is done to activate lysozyme and induce the breakdown of cell wall peptidoglycan of gram-positive bacteria.
6. Prepare tubes with 20 µL of Proteinase K for every sample at a concentration of 600 mAU per mL.
7. Centrifuge the tubes at 400 x g for 10 minutes. The lysate should look clear with some tissue residue on top of the beads.
8. Transfer 180 µL of supernatant (the upper phase) into a tube containing Proteinase K and then add 200 µL of AL buffer to the tube. Vortex for 15 s to mix.
9. Place tubes on the heating block at 56 °C for 10 minutes.
10. Add 200 µL of 100% ethanol to the tube and mix by vortexing for 15 s.
11. Add approximately 600 µL of lysate to the spin column (from kit).
12. Centrifuge for 1 minute at 8,000 x g. Discard flow through.
13. Repeat step 6.12 until all the lysate has been drawn through the column.
14. Place the column in a new collection tube. Add 500 µL of AW1 buffer and centrifuge at 8,000 x g for 1 minute.
15. Discard the flow through. Add 500 µL of AW2 buffer and centrifuge at 8,000 x g for 3 min.
16. Discard the flow through and centrifuge the column in an empty collection tube at 8,000 x g for 3 min.
17. Transfer the column to DNA elution tube. Add 60 µL of PCR grade ultrapure water directly onto the membrane and incubate for 2 minutes at room temperature.
18. Use the pre-warmed elution water at 37 °C to elute. The elution can be done twice by using half the final elution volume and repeating step 6.15 twice to increase yield.
19. Centrifuge for 1 minute at 8,000 x g to elute DNA.
20. Measure the concentration of the DNA eluted using the desired quantification method. The extraction process yields are in the range of 10-40 ng/µL DNA.
21. Store the eluted DNA at -20 °C.

7. qPCR setup

1. PCR Conditions
   1. Turn on the machine and load the program in Table 1 into a real time qPCR machine.
   2. Loop steps 3 to 5 in Table 1 for 40 cycles and hold the sample at 4 °C at the end of the reaction.
2. PCR experimental setup
   1. Use the primers and temperature found in Table 2. Use the concentrations and reaction conditions found in Table 3. Set up each reaction in triplicate to control for procedural variation.
3. Place the PCR reaction tubes/plate in the qPCR system and the run the program loaded into the system from step 7.1.
4. Remove the tube at the end of the run, place it on 4 °C and prepare for gel loading.

8. Quantification of LP colonization

1. Prepare qPCR mixes for 10 µL or 20 µL reactions according to Table 3.
2. LP genomic DNA standard curve 10⁷ to 10¹ copies/µL
   NOTE: Since 4 µL of each dilution will be plated, 10⁷ copies in 4 µL or 2.5 x 10⁶ copies per µL is required in the starting stock. Use the same principle for the rest of the curve.
   1. Prepare a 1:4 dilution: 10⁷ copies per µL in 50 µL.
      3.147 µL of LPDNA + 46.85 µL of dH₂O = 2.5 x 10⁶ copies per µL
   2. Serially dilute 10-fold: add 5 µL to 45 µL dH₂O for 1.25 x 10⁵ copies/µL.
   3. Plate 4 µL per dilution per well.
3. Visualization of LP amplicons
   1. Use a 2% agarose gel to reach a clear separation of the ~ 197 bp LP amplified fragment.
   2. Load 9 µL of each PCR product in the gel.
   3. Run the gel for 30 minutes at 120 V.