Name: Oral Gavage in Neonatal Mouse Pups and Functional Assessment of Gut Barrier Integrity Using Ussing Chambers (Video) | JoVE Journal
Uploaded: 2026-01-09T14:00:00.000+00:00
Duration: 7 min 18 s
Description: Learn oral gavage and Ussing chamber techniques to assess gut barrier integrity in neonatal mouse pups. This protocol enables precise measurement of paracellular and transcellular permeability for developmental biology research.

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This work was performed with financial support from ANR-11-IDEX-0005-02 Laboratoire d&#39;excellence INFLAMEX, an award from the Fondation de l&#39;avenir (2025, AP-RM-24-012), and ANR grants Diab1GUT (ANR-24-CE15-7152). The study was conducted within the context of the INFIBREX consortium and benefited from the support of the &#34;France 2030&#34; investment plan, launched by the French government and implemented by Universit&#233; Paris Cit&#233; under its &#34;Initiative of Excellence&#34; IdEx program (ANR-18-IDEX-0001). We gratefully acknowledge the Ussing Chamber Technical Platform at the Inflammation Research Center for their invaluable support, and extend special thanks to Alexandra Willemtz and Maude Le Gall for their expert assistance.We would also like to express our sincere gratitude to the Animal Facility Platform at the Inflammation Research Center for their essential support, with special thanks to Isabelle Renault and Guillaume Anselmet.

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<li>McClain, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neonatal+mouse+model%3A+review+of+methods+and+results%5BTitle%5D)+AND+%22Toxicol+Pathol%22%5BJournal%5D)">Neonatal mouse model: review of methods and results.</a> Toxicol Pathol. 29 (Suppl), 128-137 (2001).</li>
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<li>Clarke, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+guide+to+Ussing+chamber+studies+of+mouse+intestine%5BTitle%5D)+AND+%22Am+J+Physiol+Gastrointest+Liver+Physiol%22%5BJournal%5D)">A guide to Ussing chamber studies of mouse intestine.</a> Am J Physiol Gastrointest Liver Physiol. 296 (6), G1151-G1166 (2009).</li>
<li>Westerhout, J., Wortelboer, H., Verhoeckx, K. Ussing chamber. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aWesterhout%2C+J+%22The+Impact+of+Food+Bioactives+on+Health%3A+in+vitro+and+ex+vivo+models%22">The Impact of Food Bioactives on Health: in vitro and ex vivo models</a>. , (2015).</li>
<li>Hempstock, W., Ishizuka, N., Hayashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+assessment+of+intestinal+tight+junction+barrier+and+ion+permeability+in+native+tissue+by+Ussing+chamber+technique%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Functional assessment of intestinal tight junction barrier and ion permeability in native tissue by Ussing chamber technique.</a> J Vis Exp. (171), e62468(2021).</li>
<li>Sjögren, E., Eriksson, J., Vedin, C., Breitholtz, K., Hilgendorf, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Excised+segments+of+rat+small+intestine+in+Ussing+chamber+studies%3A+A+comparison+of+native+and+stripped+tissue+viability+and+permeability+to+drugs%5BTitle%5D)+AND+%22Int+J+Pharm%22%5BJournal%5D)">Excised segments of rat small intestine in Ussing chamber studies: A comparison of native and stripped tissue viability and permeability to drugs.</a> Int J Pharm. 505 (1), 361-368 (2016).</li>
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<li>Gleeson, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+enhanced+intestinal+permeability+of+infant+mice+enables+oral+protein+and+macromolecular+absorption+without+delivery+technology%5BTitle%5D)+AND+%22Int+J+Pharm%22%5BJournal%5D)">The enhanced intestinal permeability of infant mice enables oral protein and macromolecular absorption without delivery technology.</a> Int J Pharm. 593, 120120(2021).</li>
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The authors declare that they have no competing financial interests.

Oral gavage enables repeated administration of a precise, defined luminal dose to individual neonatal mice. In contrast, voluntary feeding, although a viable alternative, requires extended administration time per animal to deliver a lower compound quantity with reduced dosing precision12. Moreover, oral gavage accommodates larger volumes than those feasible via injectable routes, facilitating higher total doses at lower concentrations and thereby minimizing potential toxicity upon adminis...

Gut barrier integrity plays a fundamental role in maintaining overall health by coordinating mucosal immune response and regulating the selective permeability of the intestinal epithelium. This barrier system controls the passage of nutrients while preventing the translocation of pathogens, antigens, and toxins into systemic circulation1. Chronic disruption of this interface, associated with increased intestinal permeability, commonly referred to as &#34;leaky gut&#34;, is now identified not only as a consequence but also as a driver of chronic inflammatory states and a range of extra-intestinal pathologies2.
For instance, compromised gut barrier function has been shown to be a key component of autoimmune and metabolic disorders such as inflammatory bowel disease (IBD), juvenile idiopathic arthritis (JIA), and type 1 diabetes (T1D). This highlights the gut barrier state as a key biomarker, but also as a therapeutic target to prevent the initiation of pathophysiological processes as early as possible and to prevent the progression of acute inflammation to a chronic state3,4,5,6.
In this context, the neonatal period represents a unique and highly sensitive window of development during which the establishment and maturation of the gut-microbiota interface profoundly influence lifelong intestinal and systemic health7. At this early stage, the intestinal epithelium and immune system display heightened sensitivity to both endogenous and exogenous factors, undergoing rapid growth and differentiation while simultaneously adapting to microbial colonization and dietary changes. Disruptions impairing gut barrier integrity during this critical developmental window may play a pivotal role in the onset of a wide spectrum of acute and chronic disorders later in life. These conditions include immune-mediated diseases such as food allergy, and more severe chronic illnesses, such as IBD, JIA, and T1D7,8,9. These diseases underscore how pathophysiological processes often originate early, as the primary onset generally occurs between childhood and early adulthood10,11. Therefore, understanding the mechanisms that govern gut barrier function in neonates is essential for developing preventive and therapeutic strategies aimed at reducing the burden of many chronic diseases.
Given this context, there is a pressing need for reliable, physiologically relevant tools to study gut barrier integrity, chronic disease mechanisms, and inflammation specifically within the neonatal setting. Such tools must allow the replication of experimental assays already proven in the adult context without sacrificing precision and efficacy. Additionally, it is becoming increasingly urgent to investigate both short- and long-term effects of many molecular candidates of interest, which are expected to have beneficial or harmful effects on gut homeostasis in the neonatal context. Among the tools available, oral gavage administration in neonatal mouse pups represents a more than reliable method of delivering molecules of interest at a defined developmental stage. It allows for controlled investigations into the effects of various bioactive compounds, drugs, or microbial metabolites on gut maturation and barrier function.
The first part of this article describes a method for gavage of mouse pups as early as Day Of Life (DOL) 6, allowing the study of interventions before, during and after the massive expansion of the gut microbiota species numbers induced by food diversification. Thus, this technique enables longitudinal analyses of treatment effects on intestinal health as pups grow and develop.
In the second part of this article and complementing this approach, ex vivo assessment of colonic epithelial barrier function using Ussing chambers provides a sensitive and quantitative measure of permeability characteristics. Evaluation of both paracellular and transcellular permeability in colon biopsies from neonatal mice (e.g., DOL 10 pups) allows for precise determination of the impact of several key candidates on epithelial barrier integrity and function. Paracellular transport is an essentially passive mechanism occurring through the intercellular space of the epithelial layer. It allows for the passage of small solutes driven by electrochemical and osmotic gradients from the lumen to the basolateral side. This mechanism is mainly regulated by proteins of the tight junction family, such as claudins and occludin. Transcellular transport, in contrast, is an active mechanism, allowing for the absorption of bigger solutes and occurring through the epithelial cells via specific channels, carriers, and pumps.
Fluorescein isothiocyanate (FITC)-dextran (4 kDa) predominantly undergoes paracellular transport due to its size, whereas horseradish peroxidase (HRP; 44 kDa) is selectively conveyed to the basolateral side via transcellular routes. Quantification of these non-endogenous markers thus enables distinct assessment of paracellular and transcellular permeability, respectively. The Ussing chamber assay with FITC and HRP preserves tissue architecture and allows real-time measurement of respectively paracellular and transcellular permeability of any segment of the intestinal tract. This combination of neonatal gavage and Ussing chamber analysis offers a powerful strategy to study how specific molecules impact gut barrier function both immediately and over longer timeframes, informing strategies to mitigate inflammation and prevent chronic disease from the earliest stages of life.
In summary, this protocol describes two complementary experimental procedures allowing for: (1) administration of molecules of interest in the early neonatal period to mouse pups (step 1); (2) assessment of both para and transcellular permeability in intestinal tract segments from neonatal to weaning pups using Ussing chambers (steps 2-6).

<table><tbody><tr><td>10x DPBS W/O Ca &amp;amp; Mg</td><td>Dulbeccos</td><td>14200091</td><td></td></tr><tr><td>Anti-HRP antibody</td><td>Nordic Biosite</td><td>GTX10183</td><td>Monoclonal Mouse Horseradish Peroxidase IgG [2H11]</td></tr><tr><td>Binocular loupe</td><td>Olympus&amp;nbsp;</td><td>SZ61</td><td>No specification required.</td></tr><tr><td>Black 96-well plate flat-bottom</td><td>ThermoFisher Scientific</td><td>137101</td><td>Plates must be black and adapted to fluoresence readings. Lid must be taken off during reading.</td></tr><tr><td>BSA</td><td>Merck</td><td>A4503</td><td></td></tr><tr><td>C57BL/6J mice</td><td>Charles River</td><td>632C57BL/6J&amp;nbsp;</td><td></td></tr><tr><td>CaCl2</td><td>Merck</td><td>2382</td><td></td></tr><tr><td>Clear 96-well Clear Flat Bottom High Binding</td><td>Corning</td><td>9018</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Merck</td><td>G8270&amp;nbsp;</td><td></td></tr><tr><td>Distilled H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>D-Mannitol</td><td>Merck</td><td>M4125</td><td></td></tr><tr><td>Fine Forceps</td><td>FST</td><td>11445-12</td><td></td></tr><tr><td>Fine Scissors</td><td>FST</td><td>14058-09</td><td></td></tr><tr><td>FITC-Dextran 4Kd</td><td>FD4</td><td>TdB Labs</td><td>Sensitive to photodegradation.</td></tr><tr><td>Heating pad</td><td>Minerve</td><td>20 031 01</td><td></td></tr><tr><td>HRP</td><td>Merck</td><td>P8375</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;, 6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Merck</td><td>M2670&amp;nbsp;</td><td></td></tr><tr><td>Milli-Q H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>P2300 EasyMount Ussing System &amp;amp; Chambers</td><td>Physiological Instruments</td><td>EM6-C-CE</td><td>Systems, chambers and sliders must all be compatible.</td></tr><tr><td>P2306 Ussing Chamber Slider</td><td>Physiological Instruments</td><td>PI-P2306</td><td>Different models of sliders are available for different sizes and forms of tissues.</td></tr><tr><td>QuantaBlu Fluorogenic Peroxidase Substrate kit</td><td>ThermoFisher Scientific</td><td>15169</td><td></td></tr><tr><td>Reusable Feeding Needles Round Tip</td><td>FST</td><td>18061-24</td><td>24 Gauge, 1.25mm tip diameter, round tip. Must be washed with sterile water, ethanol 70% and be autoclaved before use.</td></tr><tr><td>Sodium bicarbonate</td><td>Merck</td><td>S6297</td><td></td></tr><tr><td>Sodium carbonate</td><td>Merck</td><td>S7795</td><td></td></tr><tr><td>Spectrophotometer</td><td>TECAN&amp;nbsp;</td><td>Spark 10M</td><td>A plate reader with top-acquisition is required if the 137101 black plates are used.</td></tr><tr><td>Tween-20</td><td>Merck</td><td>P7949</td><td></td></tr></tbody></table>

oral gavage in neonatal mouse pups and functional assessment of gut barrier integrity using ussing chambers

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

Growing evidence underscores the importance of the first 1,000 days of life in shaping the gut-microbiome axis. This early-life window is critical in the establishment of long-term physiological trajectories and immunological adaptations, potentially influencing susceptibility to diseases linked to dysbiosis and barrier dysfunction. A deeper understanding of the underlying mechanisms requires a comprehensive assessment of key physiological parameters, including paracellular and transcellular permeability of the neonatal gut. Evaluating these parameters is essential to elucidate how early exposures to exogenous molecules may influence gut integrity and long-term health outcomes. Therefore, the first part of this article describes the oral administration of molecules of interest in mouse pups as early as day of life 6, while minimizing stress, risk of injury, and cannibalism. Lubricated, rounded-tip 24-G feeding needles are used to gavage mouse pups weighing a minimum of 2.5 g. The second part outlines the ex vivo assessment of paracellular and transcellular permeability using Ussing chamber assays on colonic samples from pups between DOL-10 and weaning. Pinless sliders adapted for small biopsies are used in combination with Ussing chambers to mount neonatal colonic samples. The paracellular probe FITC-Dextran 4 kDa and the transcellular marker horseradish peroxidase 44 kDa Type VI are added to the apical compartment of the system at the start of the assay. Samples are collected in the basolateral compartment at 0 min, 30 min, 60 min, 90 min, and 120 min to quantify probe passage. The passage of both markers is quantified directly (FITC) and indirectly (HRP) by a plate reader, calculated using standard curves, and expressed as flux.

Standard curve and well concentration quantification 
The average value of blanks was subtracted from every well of the plate. With absorbance values on the Y axis and concentrations on the X axis, a standard curve was created using the "Interpolate a standard curve" function from GraphPad Prism. If all standard points are aligned, the "Line" model provides the best results. Alternatively, the "Sigmoidal, 4 PL, X is concentration" model offers great flexibility and accuracy with non-linear standard cu...

This article describes: (1) oral gavage delivery of test molecules to neonatal mice as early as DOL&#8239;6; and (2) ex vivo evaluation of colonic epithelial barrier function in biopsies from DOL 10 to weaning (DOL 20) pups by assessing paracellular and transcellular permeability with Ussing chambers.

Growing evidence underscores the importance of the first 1,000 days of life in shaping the gut-microbiome axis. This early-life window is critical in the ...

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.

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Research

JoVE Journal

Biology

Postoperative ileus (POI) is a complication of abdominal surgery leading to increased morbidity and a prolonged hospital stay. Because prophylactic or therapeutic strategies are lacking intensified research is necessary. Therefore we established a standardized and feasible mouse model to investigate the pathophysiology of POI and to study potential therapeutic options.

Functional Assessment of Intestinal Motility and Gut Wall Inflammation in Rodents: Analyses in a Standardized Model of Intestinal Manipulation

This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

This study details the process of gavaging precise amounts of probiotics to neonatal mice. The experimental set-up was optimized to include but is not limited to probiotic dosing, methods of administration, and quantification of bacteria in the intestines.

Probiotic Studies in Neonatal Mice Using Gavage

Taking advantage of intravital microscopy, the method presented here enables real-time visualization of intestinal epithelial cell shedding in living animals. Therefore, topically stained intestinal mucosa (acriflavine and rhodamineB-dextran) of anesthetized mice is imaged up to single-cell resolution using confocal microscopy.

An Intravital Microscopy-Based Approach to Assess Intestinal Permeability and Epithelial Cell Shedding Performance

Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

Intestinal epithelium confers not only nutrient absorption but protection against noxious substances. The apical-most epithelial intercellular junction, i.e., the tight junction, regulates paracellular solute and ion permeability. Here, a protocol for the preparation of mucosal sheets and assessment of the ion selectivity of tight junctions using Ussing chamber technique is described.

Functional Assessment of Intestinal Tight Junction Barrier and Ion Permeability in Native Tissue by Ussing Chamber Technique

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that...

A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis

To study how the small bowel handles particulates of varying sizes, we have modified an established in vivo method to determine small bowel transit.

Studying Murine Small Bowel Mechanosensing of Luminal Particulates

In the present study, fluorescein-isothiocyanate-labeled (FITC) dextran is administered to mice via oral gavage to evaluate intestinal permeability both in vivo and in plasma and fecal samples. As gut barrier function is affected in many disease processes, this direct and quantitative assay can be used in diverse research areas.

Assessment of Gut Barrier Integrity in Mice Using Fluorescein-Isothiocyanate-Labeled Dextran

This protocol outlines a neonatal mouse model of necrotizing enterocolitis (NEC) and subsequent isolation of lamina propria immune cells from the neonatal murine small intestine.

A Neonatal Mouse Model of Necrotizing Enterocolitis and Lamina Propria Isolation for Immune Cell Profiling

The goal of this protocol is to provide a standardized approach for quantitatively assessing the integrity of the murine gingival epithelial barrier by topically applying fluorescence-labeled dextrans of varying molecular sizes on the gingival surface in vivo, followed by ex vivo imaging using a single-photon microscopy system.

Functional Assessment of Mouse Gingival Epithelial Barrier Using Fluorescent Tracers

Learn oral gavage and Ussing chamber techniques to assess gut barrier integrity in neonatal mouse pups. This protocol enables precise measurement of paracellular and transcellular permeability for developmental biology research.

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

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