Probiotic studies, in neonatal mice using gavage. Introduction, this video protocol will detail the setup and procedure for gavaging probiotics to a neonatal mouse, on it's day of life two. The sterile collection of intestines, for intestinal microbiome analysis, is also detailed in this protocol.
Some representative results, are also included in this protocol, to introduce some of the types of data, that can be derived from following this procedure. Intra-esophageal gavage of neonatal mouse, day of life two. Place the dam's cage in the same hood where the, experimental cages is placed on the heating pad, the heating pad is set to medium level, which is approximately 38 degrees Celsius.
Remove some soil nesting from the home cage, rub the nesting with gloved hands to transfer the scents, of the nesting material onto the glove. This reduces the transfer of foreign scents, onto the pup when handled. This in turn reduces the risk, of abandonment or cannibalization, by the dam due to foreign scents.
When the pups are returned to the home cage, after the procedure. Create a conical shaped holding nest, and place it in the new and sterile experimental cage, which has been pre-warmed. Transfer all the pups from the home cage, into the experimental cage.
Move the pups into the conical nesting, in the experimental cage. Remove the dam's cage and place it, away from the gavaging area. This reduces the stressful conditions for the dam, by preventing the dam from hearing the pup, doing the procedure.
Open the syringe packaging for easy access. Remove the auto clave gavage feeding needle, in a sterile manner, and attach it to the head of the syringe. The needle is washed with 70%ethanol, and water, before autoclaving.
Different sets of feeding needles, are used between the treatment and control groups, to avoid cross contamination. Draw up the probiotic dye solution into the syringe, invert and flick with finger to remove bubbles, and empty space in the volume inside the syringe. Draw back on the plunger to clear liquid, from the needles dead space, and then depress the plunger, to expel air and air bubbles, this is important to ensure, that no air bubbles are gavaged, into the animal.
Adjust the desired volume in the syringe, for a day of life two mouse, no more than 30 microliters should be gavaged. The black marking that you see on the needle, indicates the maximum length, that the needle can be inserted into the mouse. This length is obtained by externally measuring, the length between the snout and the xiphoid process.
Place the syringe down on a sterile surface, remove the pup to be gavaged from the experimental cage, and observe for health signs. These health signs include, regular breathing and pink coloration of the skin. Place the pup on the sterile observant sheet, on the heating pad.
Lubricate the external surface of the feeding needle, using the solvent used to dissolve the probiotic. Here it is dextrose water, this facilities the smooth entry of the needle, into the esophagus of the animal. Scruff the pup by the skin on the neck, using your thumb and your index finger.
Grasp the syringe in your dominant hand, in a position that allows you to insert the needle, and depress the plunger without adjusting your fingers. Insert the bulb of the feeding needle at a 45 degree angle, until it reaches the back of the mouth. Pivot your hand with the needle away from you, while holding the bulb of the needle in place, once the needle is plain with the torso, it will start to slide.
Make sure no extra pressure is applied to the syringe, to stimulate movement. The process of swallowing will be slow, and patience is key. As the pup gets older the swallowing of the needle, will become faster and easier.
The feeding needle has been pre marked, to prevent the bulb from entering the stomach. Once the marking approaches the snout, arrest the movement of the needle, and slowly deliver the probiotic solution. Once the delivery is complete, remove the needle slowly.
Place the mouse on the heating pad and observe for recovery. A gasping reflex resolves and the heart rate rises, a healthy pink coloration reappears, and unresolved gasping reflex after 45 seconds, indicates a failed gavage and the mouse must be euthanized. The gavage dye should be visible through the pale skin, the dye should be only visible in the area, where the milk spot is usually found.
The confinement to the dye to the stomach, indicates a successful gavage. If the dye is found in the thoracic cavity, or any other cavity than the stomach, the mouse must be euthanized, as it indicates a failed gavage. Minimal pressure is needed, to scruff a day of life two mouse.
Signs of scruffing to hard can include, inability to breath, significant gasping, and sticking the tongue out for a long duration. A few suggestions for the gavaging procedure, rest the back of your scruffing hands finger, on the palm of the hand holding the syringe, this ensures the pups position is stable, during the procedure and the needle, is not moved around inside the animal. During the gavage, the weight of the needle, will facilitate the sliding, allow the needle to be swallowed by the pup, without exerting any external pressure.
Return the pups to the dam's cage, and follow up with monitoring as per your schedule. Collection of intestinal samples for colonization analysis. The animal was humanely euthanized, by methods approved in the Animal Care and Use Protocol.
The surgical table is lined with a sterile absorbent mat. The tools have been sterilized using 70 percentage ethanol, and a hot beat sterilization at 250 degrees Celsius. The external service of the animal, is disinfected with 70 percentage ethanol.
Cut the external skin using forceps and scissors, into four quadrants, while taking extra caution, to not lacerate the peritoneum. Tuck the skin to the side such that, the peritoneum is well exposed. Use different, new sterile tools, to cut the peritoneum open, and take extra caution not to lacerate, the internal organs in this process.
Locate the stomach of the pup, and clamp at the duodenum. Using two sterile forceps, locate the stomach, and locate the duodenal end of the stomach. Clamp right at the intersection, of the stomach and the duodenum.
Use two new sterile forceps to run the intestines, use one forcep to hold on the the duodenum, while using the other to rip away any connective tissue, holding the intestines intact. As you unravel, place the intestines, on the sterile absorbent pad covering the animal. If you happen to rip the intestines, during the unraveling procedure, mark down the area at which it ripped, so that the orientation of the intestine is intact.
Clamp at the rectal end, and make cuts at both the clamps. Place the intact intestine, on a sterile marked aluminum foil in the right orientation. Mark any sections of the intestine, necessary for your subsequent analysis.
DNA is extracted from these intestines, using an optimized extraction procedure, and QPCR analysis is done to quantify the probiotic. The DNA is extracted from fresh intestines, or the intestines can be stored, at negative 80 degrees Celsius using a freezer box. Representative results, lactobacillus plantarium, colonization with gavage everyday and every other day.
A higher LP signal was observed in intestines of mice, gavaged every other day, in comparison to mice gavaged everyday. Thus, are subsequent experiments were built, using gavages every other day. Lactobacillus plantarum gavaged mice, co-housed with ungavaged mice.
Variable signals of LP were observed in untreated mice, that were co-housed with LP gavaged mice. The colonization spread of LP, is possible with the litter mates, and thus the treatment conditions, were separated by cages for our subsequent experiments. Conclusion, this procedure can be used to deliver, precise amounts of any liquid to newborn mice.
The sampling of intestines can be extrapolated, to explore the changes in microbiome, through other methods of analysis. The scope of this video is to provide a platform, for researchers to use neonatal mouse gavage models, to understand the mechanisms behind the effects observed, with early administration of probiotics. A special thanks to the Kollmann Lab Team, The Animal Care Services of, University of British Colombia, for making this study possible.
