Name: functional assessment of intestinal tight junction barrier and ion permeability in native tissue by ussing chamber technique
Uploaded: 2021-05-26T14:30:00
Description: Watch this Scientific Journal Video about Functional Assessment of Intestinal Tight Junction Barrier and Ion Permeability in Native Tissue by Ussing Chamber Technique at JoVE.com

This work is supported by 17K00860 (to HH) and 19K20152 (to NI). WH would like to acknowledge the Otsuka Toshimi Scholarship Foundation for their financial support from 2018-2021.

All animals used in these experiments were maintained in the animal care facility at the University of Shizuoka and the experiments were conducted according to the guidelines for animal research set out by the University of Shizuoka. All experiments were carried out with approval from the Animal Care and Use Committee at the University of Shizuoka (Permits #205272 and #656-2303).
1. Preparation of NaCl electrodes
NOTE: The electrodes used in these experiments consist ...

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+Ussing's+chambers:+contemporary+thoughts+on+integration+of+transepithelial+transport.">Beyond Ussing's chambers: contemporary thoughts on integration of transepithelial transport.</a> American Journal of Physiology - Cell Physiology. 310, 423-431 (2015).</li><li>Ishizuka, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Luminal+Na+++homeostasis+has+an+important+role+in+intestinal+peptide+absorption+in+vivo.">Luminal Na + homeostasis has an important role in intestinal peptide absorption in vivo.</a> American Journal of Physiology - Gastorintestinal and Liver Physiology. 315, 799-809 (2018).</li><li>Ikehara, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subepithelial+trypsin+induces+enteric+nerve-mediated+anion+secretion+by+activating+proteinase-activated+receptor+1+in+the+mouse+cecum.">Subepithelial trypsin induces enteric nerve-mediated anion secretion by activating proteinase-activated receptor 1 in the mouse cecum.</a> Journal of Physiological Sciences. 62, 211-219 (2012).</li><li>Furuse, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+the+core+structure+of+tight+junctions.">Molecular basis of the core structure of tight junctions.</a> Cold Spring Harbor Perspectives in Biology. 2, 002907 (2010).</li><li>Tsukita, S., Tanaka, H., Tamura, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+claudins:+From+tight+junctions+to+biological+systems.">The claudins: From tight junctions to biological systems.</a> Trends in Biochemical Sciences. 44, 141-152 (2019).</li><li>Li, B. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+Ussing+chamber+studies+of+mouse+intestine.">A guide to Ussing chamber studies of mouse intestine.</a> American Journal of Physiology - Gastrointestinal and Liver Physiology. 296, (2009).</li><li>Dobson, J. G., Kidder, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Edge+damage+effect+in+in+vitro+frog+skin+preparations.">Edge damage effect in in vitro frog skin preparations.</a> American Journal of Physiology. 214, 719-724 (1968).</li><li>Corman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streaming+potentials+and+diffusion+potentials+across+rabbit+proximal+convoluted+tubule.">Streaming potentials and diffusion potentials across rabbit proximal convoluted tubule.</a> Pfl&#252;gers Archiv: European Journal of Physiology. 403, 156-163 (1985).</li><li>Shen, L., Weber, C. R., Raleigh, D. R., Yu, D., Turner, J. 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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ionic+conductances+of+extracellular+shunt+pathway+in+rabbit+ileum.">Ionic conductances of extracellular shunt pathway in rabbit ileum.</a> Journal of General Physiology. 59, 318-346 (1972).</li><li>Otani, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Claudins+and+JAM-A+coordinately+regulate+tight+junction+formation+and+epithelial+polarity.">Claudins and JAM-A coordinately regulate tight junction formation and epithelial polarity.</a> Journal of Cell Biology. 218, 3372-3396 (2019).</li></ol>

In this experiment, Ussing chambers were used to measure the baseline electrical parameters and the dilution potential of NaCl in the small intestine of Cldn15-/- and WT mice. It is very important when doing Ussing chamber experiments to verify that the membrane preparation used in the experiments is viable. This is usually done by adding glucose or the adenylate cyclase activator forskolin and seeing whether there is an appropriate rise in Isc (100-300 &#181;A/cm2 in mi...

The Ussing chamber method was first developed by the Danish scientist Hans Ussing. Ussing first used it to measure the short-circuit current of sodium transport across frog skin after it was observed that NaCl could be transported across the skin against a steep concentration gradient1. His system consisted of the frog skin mounted between two chambers with access to either side of the skin. Each chamber contained Ringer&#39;s solution which was circulated and aerated. Two narrow agar ringer bridges situated near the skin and connected to saturated KCl-calomel electrodes measured the potential difference as read by a potentiator. A second pair of agar ringer bridges were situated at the opposite end of each chamber connected to beakers with saturated KCl saturated with AgCl to apply an electromotive force provided by a battery. A potential divider was used to adjust the voltage so that the potential difference across the skin remained zero, thus creating short-circuit conditions. A microampere meter was also connected to read the current passing through the skin (see the figure in ref.1 for original chamber design).
Over the past 70 years, this technique has been applied to many different tissues, particularly intestinal tissue, to study nutrient and ion transport. For example, the mechanism of cholera-induced diarrhea was studied by mounting rabbit ileum in these chambers, and it was found that cholera toxin-induced diarrhea is mediated by cAMP2. In addition, these chambers were also used to study the mechanism underlying glucose transport via Na+-Glucose cotransporter 1 (SGLT1)3. Our lab focuses on transcellular and paracellular transport in intestinal epithelial cells. Using the Ussing chamber method, peptide transport was assessed in Claudin 15 knockout mice, which have impaired paracellular sodium transport, using Ussing chambers to measure the absorption of the nonhydrolyzable dipeptide glycylsarcosine. It was found that luminal Na+ homeostasis is important for proton-coupled peptide transport4. In addition, these chambers were also used to investigate anion secretion in the murine cecum in response to submucosal activation of proteinase activated receptor 1 by the serine protease trypsin5.
Ussing chambers have also recently been used to assess the paracellular pathways in epithelial tissue. Paracellular pathways are regulated by tight junctions, which are complexes of proteins that form at the point where two or more cells meet6. The barrier function and ion selectivity (whether anions or cations are selectively able to pass through the tight junction) is determined by the presence of claudin family proteins; some of which act as barriers (claudin 3 and 7), anion pores (claudin 10a), or cation pores (claudin 2, 10b, and 15)7. Other methods have been used to assess the paracellular pathway, such as oral gavage of FITC accompanied by blood plasma FITC concentration8, or EDTA-Cr9; however, these techniques are of lower resolution and cannot assess ion selectivity or a specific section of the sections of the intestinal tract. Ussing chambers, however, can be used to assess the dilution potential of target ions, and, therefore, determine the ion selectivity of the tight junctions. For example, with NaCl, the selectivity of the tight junctions for Na+ and Cl- can be calculated by diluting one side of the membrane (usually the mucosal side) and measuring the change in transepithelial potential difference. The relative permeabilities of Na+ and Cl- can be estimated by the Goldman-Hodgkin-Katz equation10 and the selectivity of the tight junction can be estimated using the Kimizuka-Koketsu equation11. These chambers, therefore, have the advantage of measuring the electrophysiological parameters of tissue and as a result provide more information about the passage of ions through the tight junctions than other lower resolution methods.
The Ussing chamber method is not only limited to the intestinal tract, although it is widely used in studies concerning the intestine, it has many other applications as well. For example, these chambers have been used to study Cystic Fibrosis, and specifically the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR)12. Cystic Fibrosis is caused by a mutation in CFTR13, which results in impaired chloride secretion and fluid transport by respiratory epithelial cells, and a resulting thicker, drier mucous layer14. Study of airway epithelial CFTR has been performed with these chambers to not only understand the disease, but to discover ways to treat the disease. For example, in patients with rare mutations causing Cystic Fibrosis, analysis of patient respiratory epithelial cells has been used to test therapies such as Orkambi and an amplifier co-therapy15.
Ussing chambers have also been used to study routes of drug delivery, such as with human biopsy tissue to study drug uptake and pharmacokinetics16. Intestinal uptake is not the only route of drug delivery. These chambers have also been used to study nasal drug delivery systems17. Drug delivery studies with Ussing chambers have also been performed for the eye. In the rabbit cornea, permeability and uptake studies were conducted with Labrasol, a drug that is designed to increase the absorption of drugs across tissues18. Another study examined the effect of benzylalkonium chloride on transscleral drug delivery in the rabbit sclera19.
The Ussing chamber method is useful because native tissue can be used. As such, it is preferable over in vitro models such as Caco-2 cell lines. However, the technique requires skill and time to prepare specimens, so it is not suitable for high throughput applications. The electrophysiological properties of cell monolayers can be studied using cell culture inserts in these chambers. Recent discoveries have allowed for the culture of organoids which are mini-organs grown in culture from the harvest of epithelial or endothelial stem cells20. Organoid culture can be manipulated to be grown in a monolayer, thereby making it possible to mount organoids in an Ussing chamber21. Organoids of various epithelial and endothelial tissues can be studied, lowering the number of animals required, as organoid culture can be maintained long term. This will also increase the throughput since time consuming and laborious tissue dissection and preparation steps will not be needed. In the future, Ussing chamber studies will continue to be very useful for studying tissue transport and they will be especially important in the field of personalized medicine.
The following protocol demonstrates the application of the Ussing chamber method to assess the permselectivity and barrier function of the tight junctions in the small intestine of Claudin 15 knockout (Cldn15-/-) mice and wild type (WT) controls by measuring the dilution potential of NaCl. Tight junctions (TJ) are formed at the point where two or more cells meet in epithelial and endothelial tissue. Bicellular tight junctions (bTJ), particularly the claudin family proteins found within the bTJ, are thought to determine the barrier function and permselectivity of TJ7. Cldn15-/- mice have a mega small intestine22 and reduced nutrient uptake capability due to the loss of intestinal Na+ recycling that occurs via claudin 154,23,24. Cldn15-/-&#160;mice have impaired Na+ homeostasis, which makes them an interesting model for studying the permselectivity of the TJ. The following protocol assesses the permeability of the TJ to NaCl by measuring the dilution potential of NaCl (PNa/PCl) in the middle small intestine. Briefly, the change in membrane potential difference that occurs by diluting one side of the membrane (M side or S side, both are measured in the below protocol) can be used to calculate the permeability of Na+ (PNa) and Cl- (PCl), and the dilution potential (PNa/PCl) will show whether the tight junction has a cationic or anionic selectivity.
The experiments in this protocol were conducted using a customized Ussing chamber (Figure 1A), which consists of two halves, between which the intestinal preparation is mounted vertically, voltage clamp amplifier, electrical recorder, electrodes, salt bridges, Ringer&#39;s solution, HEPES buffer (150 mM NaCl), diluted HEPES buffer (75 mM NaCl), intestinal preparation (for details about equipment see the Table of Materials).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>#3 polyethyl tubing</td>
			<td>Hibiki</td>
			<td></td>
			<td>outer diameter 1.0 mm; inner diameter 0.5 mm</td>
		</tr>
		<tr>
			<td>#7 polyethyl tubing</td>
			<td>Hibiki</td>
			<td></td>
			<td>outer diameter 2.3 mm; inner diameter 1.3 mm</td>
		</tr>
		<tr>
			<td>10 mL locking syringe</td>
			<td>Terumo</td>
			<td>SS-10LZ</td>
			<td>Locking syringes are necessary to prevent the needle from dislodging during filling</td>
		</tr>
		<tr>
			<td>19 g needle</td>
			<td>Terumo</td>
			<td>NN-1938R</td>
			<td>Please use caution when working with needles and dispose of in sharps container</td>
		</tr>
		<tr>
			<td>23 g needle</td>
			<td>Terumo</td>
			<td>NN-2332R</td>
			<td>Please use caution when working with needles and dispose of in sharps container</td>
		</tr>
		<tr>
			<td>5 mm punch</td>
			<td>NA</td>
			<td>NA</td>
			<td>Use to punch holes in filter paper and parafilm</td>
		</tr>
		<tr>
			<td>acupuncture needles</td>
			<td>Seirin</td>
			<td>NS</td>
			<td>Used as dissection pins to pin tissue to dissection plate</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Fujifilm Wako</td>
			<td>010-15815</td>
			<td></td>
		</tr>
		<tr>
			<td>Alligator clips</td>
			<td>NA</td>
			<td>NA</td>
			<td>Connects the electrode to the amplifier</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Fujifilm Wako</td>
			<td>038-00445</td>
			<td></td>
		</tr>
		<tr>
			<td>D(-)-Mannitol</td>
			<td>Fujifilm Wako</td>
			<td>133-00845</td>
			<td>This is used to correct for the osmolality difference in dilution HEPES buffer</td>
		</tr>
		<tr>
			<td>D(+)-Glucose</td>
			<td>Fujifilm Wako</td>
			<td>049-31165</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection kit</td>
			<td></td>
			<td></td>
			<td>You will need, scissors and curved forceps</td>
		</tr>
		<tr>
			<td>Dissection plates</td>
			<td></td>
			<td></td>
			<td>We used 10 cm cell culture plates and covered with silicon rubber</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>472301-500ML</td>
			<td>For making forskolin stock</td>
		</tr>
		<tr>
			<td>Electrical recorder</td>
			<td>TOA Electronics</td>
			<td>PRR-5041</td>
			<td>Other equivalent electrical recorders are available commercially</td>
		</tr>
		<tr>
			<td>Epithelial voltage clamp amplifier</td>
			<td>Nihon Kohden</td>
			<td>CEZ9100</td>
			<td>Other equivalent amplifiers are available commerically</td>
		</tr>
		<tr>
			<td>filter paper, cut into squares</td>
			<td>NA</td>
			<td>NA</td>
			<td>Punched with a 5 mm punch, used to hold intestinal preparation</td>
		</tr>
		<tr>
			<td>fine forceps</td>
			<td>Fast Gene</td>
			<td>FG-B50476</td>
			<td>For blunt dissection of the muscle layer</td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Alomone Labs</td>
			<td>F-500</td>
			<td>Make 10 mM stock in DMSO, final concentration will be 10 &#181;M</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H4034-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Indomethacin</td>
			<td>Sigma</td>
			<td>I7338-5G</td>
			<td>Make a 1 mM stock in 21 mM NaHCO3, final concentration is 10 &#181;M</td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Fujifilm Wako</td>
			<td>164-04295</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fujifilm Wako</td>
			<td>163-03545</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl/calomel electrode</td>
			<td>Asch Japan Co.</td>
			<td>SCE-100</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Kanto chemical</td>
			<td>32379-00</td>
			<td></td>
		</tr>
		<tr>
			<td>L(+)-Glutamine</td>
			<td>Fujifilm Wako</td>
			<td>074-00522</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Fujifilm Wako</td>
			<td>135-00165</td>
			<td></td>
		</tr>
		<tr>
			<td>Mixed Gas (95% O2/5% CO2)</td>
			<td>Shizuoka Oxygen Company</td>
			<td></td>
			<td>Used for bubbling Ringer solution and chambers when using Ringer solution</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fujifilm Wako</td>
			<td>191-01665</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl electrode</td>
			<td>NA</td>
			<td>NA</td>
			<td>Handmade electrodes which require concentrated NaCl and Silver wire</td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Fujifilm Wako</td>
			<td>191-01305</td>
			<td></td>
		</tr>
		<tr>
			<td>O2 Gas</td>
			<td>Shizuoka Oxygen Company</td>
			<td></td>
			<td>Used for bubbling chambers when using HEPES buffer</td>
		</tr>
		<tr>
			<td>parafilm</td>
			<td>Bemis</td>
			<td>PM-996</td>
			<td>Used to help seal Ussing chambers</td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>DKK-TOA Corp</td>
			<td>HM-305</td>
			<td>HEPES buffer needs to be adjusted to pH 7.4 at 37 &#176;C</td>
		</tr>
		<tr>
			<td>pH meter electrode</td>
			<td>DKK-TOA Corp</td>
			<td>GST-5311C</td>
			<td></td>
		</tr>
		<tr>
			<td>silicone rubber</td>
			<td>Shinetsu Chemical</td>
			<td>KE-12</td>
			<td>Used to fill dissection plates</td>
		</tr>
		<tr>
			<td>silver wire</td>
			<td></td>
			<td></td>
			<td>Used for making NaCl electrodes</td>
		</tr>
		<tr>
			<td>Small jars w/ plastic lids</td>
			<td>NA</td>
			<td>NA</td>
			<td>Use for NaCl electrodes</td>
		</tr>
		<tr>
			<td>stereomicroscope</td>
			<td>Zeiss</td>
			<td>Stemi 305</td>
			<td>A stereomicroscope allows you to see depth, so you can dissect the tissue more easily</td>
		</tr>
		<tr>
			<td>Tris (Trizma base)</td>
			<td>Sigma</td>
			<td>T1503-1KG</td>
			<td>Make a 1M solution to adjust pH of HEPES buffers</td>
		</tr>
		<tr>
			<td>Ussing chambers</td>
			<td>Sanki Kagaku Kougei</td>
			<td></td>
			<td>These chambers are custom made continuous perfusion Ussing chambers with a window diameter of 5 mm</td>
		</tr>
		<tr>
			<td>Water pump and heating system</td>
			<td>Tokyo Rikakikai Co. Ltd.</td>
			<td>NTT-110</td>
			<td></td>
		</tr>
	</tbody>
</table>

functional assessment of intestinal tight junction barrier and ion permeability in native tissue by ussing chamber technique

The Ussing chamber technique was first invented by the Danish scientist Hans Ussing in 1951 to study the transcellular transport of sodium across frog skin. Since then, this technique has been applied to many different tissues to study the physiological parameters of transport across membranes. The Ussing chamber method is preferable to other methods because native tissue can be used, making it more applicable to what is happening in vivo. However, because native tissue is used, throughput is low, time is limited, and tissue preparation requires skill and training. These chambers have been used to study specific transporter proteins in various tissues, understand disease pathophysiology such as in Cystic Fibrosis, study drug transport and uptake, and especially contributed to the understanding of nutrient transport in the intestine. Given the whole epithelial transport process of a tissue, not only transepithelial pathways, but also paracellular pathways are important. Tight junctions are a key determinant of tissue specific paracellular permeability across the intestine. In this article, the Ussing chamber technique will be used to assess paracellular permselectivity of ions by measuring transepithelial conductance and dilution potentials.

The results shown in this paper are results that were part of larger project that has been completed (see ref.4,23,24).
Transepithelial electrical conductance of the small intestine is decreased in Cldn15-/- mice. 
The baseline transmucosal conductance (under short circuit conditions) of the middle small intes...

Watch this Scientific Journal Video about Functional Assessment of Intestinal Tight Junction Barrier and Ion Permeability in Native Tissue by Ussing Chamber Technique at JoVE.com

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

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.

The Ussing chamber technique was first invented by the Danish scientist Hans Ussing in 1951 to study the transcellular transport of sodium across frog ...

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