Oral Gavage in Neonatal Mouse Pups and Functional Assessment of Gut Barrier Integrity Using Ussing Chambers

We studied the role of microRNAs in host microbial interaction and explore their potential as new therapeutics in IBD. Current methods lack reliable tools to assess early life gut barrier integrity. This protocol addresses that gap by enabling accurate investigation of developmental factors underlying chronic diseases.

To begin, place clean paper towels on a disinfected heating pad set to approximately 38 degrees Celsius. Wash gloves with 70%ethanol prior to opening the animal cages to reduce odor transfer to the pups and limit the risk of cannibalization. Now, transfer the dam to a separate cage and relocate it to another room to minimize stress caused by the pup's vocalizations.

Rub cleaned gloves with nesting materials to prevent the transfer of external odors to the pups during gavage. Attach the head of the feeding needle to a syringe using sterile technique. Measure the distance from the xiphoid process to the snout of a mouse pup aged six days or more to mark the maximum insertion length on the feeding needle.

Draw more than the desired volume of gavage solution to avoid air bubble administration. Using the thumb and index finger of the non-dominant hand, gently pinch the skin between the scapulas to lift the pup. After lubricating, hold the pup in a near horizontal position and insert the feeding needle into the oral cavity, advancing it perpendicularly toward the pharynx until it reaches the back of the throat.

Then, slide the needle to either side of the oral cavity to avoid the tongue while maintaining the same angle in depth. Slowly tilt the pup's head backward while adjusting the needle angle until the syringe, head, and back are aligned with the head slightly inclined toward the back. Then, let the syringe descend slowly under its own weight without applying pressure until the insertion mark approaches or reaches the level of the snout.

Quickly dispense the desired volume of gavage solution once the insertion mark is reached or nearly reached. Next, gently withdraw the needle, maintaining the same angle as during insertion. Place the pup on the paper towel previously laid on the heating pad and monitor for recovery, ensuring normal breathing resumes within 20 seconds.

Once the pup has recovered, place it back into the nest and regroup it with the dam. Place the euthanized pup aged 10 to 20 days previously subjected to oral gavage under a dissection binocular, and apply 70%ethanol to the abdomen of the animal. Using forceps, pinch the abdominal skin and peritoneum, then make a large transverse incision with fine scissors oriented perpendicular to the rostral-caudal axis, without cutting into the intestine.

Now, gently insert the fine scissors into the incision and extend the cut laterally through the peritoneum on both sides. Using forceps and fine scissors, open the superior and inferior parts of the peritoneum by cutting from the thoracic region to the lower pelvic area. Then, identify the distal colon and make an incision parallel to its axis, moving toward the rectum.

Now, make an incision in the terminal portion of the colon to mobilize it, and gently pull the colon to proceed toward the cecum, removing any connective or fat tissue encountered. Free the colon from the abdomen by cutting at the ileocecal junction just above the cecum. Discard the cecum and the initial few millimeters of the proximal colon, then collect one or two colon samples that are larger than the aperture of the Ussing slider.

With the scissors closed, apply light pressure and slide from the proximal to distal end to remove fecal content from the flattened biopsy. Next, place the emptied piece of colon into ice cold Krebs-Ringer Bicarbonate Buffer. Lightly dry the cleaned colon sample using a paper towel to remove any excess moisture.

Then, place the colon sample under a magnifying glass, and carefully insert one blade of fine scissors into the luminal part to cut it open. Using fine forceps, mount the tissue on the white part of the slider with the luminal side facing upward toward the operator. Finally, close the slider under a binocular microscope and visually confirm that the slider is tightly closed.

While maintaining gentle pressure to keep the slider closed, insert it into the Ussing chamber with the lumen, marked by the transparent part of the slider, facing to the left. Perform colonic permeability assay using FITC-dextran and HRP. Collect flux samples over time to assess barrier function.

Colonic permeability was quantified by calculating time-dependent flux of FITC-dextran from Ussing chamber measurements for each experimental group. The flux values significantly increased in biopsies from animals treated with microRNA-B, but not microRNA-A, when compared to controls. Flux evolution of individual samples was verified to identify potential anomalies, as flux values were expected to remain stable across time points.

Addition of 10 millimolar EDTA at time zero resulted in a marked increase in flux compared with control samples, while maintaining a similar overall flux evolution pattern. This protocol is a powerful strategy to study how specific molecules impact neonatal gut barrier function. Alongside training, swiftness and precision are required to ensure correct handling of the animals and optimal conditions for a permeability assay.

Colonic permeability changes can be validated using independent assays, such as qPCR quantification of tangentially related transcripts, to confirm barrier modulation.