The work is supported by the Ministry of Science and Higher Education Republic of Poland, Diamond grant no: DI2017 009247.

The experiments were performed on male Wistar Kyoto rats according to Directive 2010/63 EU on the protection of animals used for scientific purposes and were approved by the I Local Bioethical Committee in Warsaw.
1. Insertion of the Line for Intraintestinal Administration
NOTE: Here we propose intracolonic administration of a marker using a catheter. It may be modified by oral administration or gavage at various levels of the digestive tract e.g. stomach or duodenum. Remember to use disposable surgical clothing, including surgical gown, hood and gloves, and ensure to follow the safety precautions related to the sharp tools used in surgery (needles, etc.) during procedures 1-6.
<ol>
	<li>Fast animals overnight before the procedure. Perform all procedures during general anesthesia, i.e., obtained by injection of urethane 1.5 g/kg bw i.p. Assess proper anesthetization by the lack of palpebral and corneal reflexes, and by toe-pinch and tail-pinch method.</li>
	<li>Use a pediatric Foley catheter (10F or 8F) as a colonic catheter. Mark the catheter to indicate the part that will be inserted into the colon (approximately 8 cm).</li>
	<li>Check the anal region and the stool content in the rectum before inserting the catheter into the colon. If stool is present, empty the rectum by massaging the rectal area.</li>
	<li>Put a lubricant (e.g. glycerin or petrolatum) along the catheter. Moisten the anus and its surroundings with the lubricant.</li>
	<li>Insert the catheter with a guide wire approximately 8 cm through the external anal sphincter. Make slow forward-backward and circular movements. 
	NOTE: Keep on checking the location of the catheter by abdominal palpation while inserting the catheter.</li>
</ol>
2. Inferior Vena Cava Catheterization
<ol>
	<li>Shave fur in the groin. Alternately disinfect the skin with alcohol and povidone iodine 3 times and cover the groin area with surgical drapes.</li>
	<li>Try to feel the pulse on the femoral artery and cut the skin longitudinally for the length of about 2.0 cm in the place where the pulse is palpable.</li>
	<li>Dissect the fascia and muscles to visualize the neurovascular bundle.</li>
	<li>Dissect the femoral vein from the neurovascular bundle: first nerves, then the femoral artery, and then the vein. 
	NOTE: Be careful during the dissection of the neurovascular bundle, since tiny branches of the femoral vein may easily be damaged, producing bleeding.</li>
	<li>Put two ligatures on the femoral vein. Do not tie&#160;the knots yet. Catch the ends of the proximal ligature with a needle holder.</li>
	<li>Carefully pull the ligature ends with the holder upwards to close the proximal part of the vein. Wait until the vein is filled with blood and tie&#160;the distal knot.</li>
	<li>Make a small incision (ca. 1 mm) on the vein between the knot and proximal ligature, using microsurgical scissors. Insert the catheter using tweezers or the needle with the curved end. 
	NOTE: Puncture the vein and use the bended tip of the needle as a guide for the catheter. Loosen the proximal ligature while inserting the catheter. Insert the catheter for 6-7.0 cm.</li>
	<li>Secure the catheter in the femoral vein with two single surgical knots. Tie&#160;the proximal ligature as well.</li>
	<li>Check the patency of the catheter by attempting to draw blood with a syringe. Rinse the catheter with 0.3 mL of the heparinized saline (100 units/mL).</li>
	<li>Close the surgical wound with two layers of single stitches.</li>
</ol>
3. Portal Vein Catheterization
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58456/58456fig1.jpg" /> 
Figure 1: Portal catheter. The portal catheter consists of a needle OD: 0.9 mm with a length of about 25.0 mm [A], a flexible polyurethane catheter OD: 0.025&#34;, length about 100.0 mm [B], a flexible polyethylene tip of the catheter OD: 0.040&#34;, approximately 15.0 mm long [C], a plug [D], and a ligature 3/0 with a length of 100.0 mm [E]. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58456/58456fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Prepare the portal catheter according to Figure 1.
	<ol>
		<li>Insert the cut end of the needle (OD: 9 mm) into the polyurethane catheter OD: 0.025&#34;.</li>
		<li>Tie&#160;the ligature 3/0 at the junction of the needle and catheter. 
		NOTE: Ensure that the longer part of the ligature is at least 6 cm long.</li>
		<li>Insert the end of the catheter OD: 0.025&#34; into the polyethylene catheter OD: 0.040&#34;.</li>
		<li>Close the catheter with a metal or plastic plug.</li>
	</ol>
	</li>
	<li>Midline laparotomy
	<ol>
		<li>Shave fur in the abdomen, alternately disinfect the skin with alcohol and povidone iodine 3 times, and cover the area with surgical drapes.</li>
		<li>Cut the skin longitudinally from the xiphoid of the sternum to the navel.</li>
		<li>Cut the muscles of the abdominal wall along the white line.</li>
		<li>Expand the cut rostrally in the Y shape so that the xiphoid cartilage is between two cuts.</li>
	</ol>
	</li>
	<li>Portal vein dissection
	<ol>
		<li>Moisten the surgical swabs with saline.</li>
		<li>Exteriorize the cecum, ascending and transverse colon, and small intestine loop. Put the intestines on the left side to expose the root of the mesentery. 
		NOTE: Cover the intestines with gauze moistened with a physiological saline to protect the intestines from drying.</li>
		<li>To expose the portal vein, carefully move the hepatic lobes to the sides or upwards towards the diaphragm with the moistened swabs.</li>
		<li>Localize the part of the portal vein that is not covered with the mesentery (in the hepatic hilum, about 5 mm long) and pass the ligature 3/0 (15 cm long) under the portal vein. 
		NOTE: To protect tissues from damage while placing the ligature, moisten the ligature with a physiological saline solution.</li>
		<li>Clamp the ends of ligature with forceps and tighten it gently to stabilize the vessel.</li>
	</ol>
	</li>
	<li>Insertion and stabilization of the catheter
	<ol>
		<li>Pass the longer part of the portal catheter&#39;s ligature under the free part of the portal vein and pull it so that the catheter is located just next to the portal vein.</li>
		<li>Insert the needle into the upper mesenteric vein 3 mm below the junction of upper mesenteric vein and the portal vein. Hold the needle at a 30&#176; angle and, after entering into the vein, reduce the angle and advance the needle almost horizontally, in parallel to the portal vein. 
		NOTE: Insert the needle for a length of approximately 6-7 mm. The stabilizing ligature should gently tighten the portal vein while inserting the catheter.</li>
		<li>Apply 1-2 drops of tissue glue at the place where the needle is inserted. Remove the swabs that cover the liver.</li>
		<li>Put the intestines back into the abdominal cavity.</li>
		<li>Moisten the intestines with a warmed up saline solution and cover it with moistened sterile gauze.</li>
		<li>Check the patency of the catheter and rinse the catheter with 0.3 mL of the heparinized saline (100 units/mL). 
		NOTE: Venous blood spontaneously backflows in the catheter.</li>
	</ol>
	</li>
	<li>Ending of the surgery
	<ol>
		<li>After 5 minutes, check the color of the intestines and peristaltic movements, make sure that the proper mesenteric blood flow is maintained.</li>
		<li>Close the abdominal cavity with 3 stitches: wall peritoneum with the inner layer of the abdominal wall muscles - a continuous, absorbable suture; remaining muscles of the abdominal wall - a continuous, absorbable suture; skin and subcutaneous tissue - single, non-absorbable sutures. 
		NOTE: Exteriorize the distal part of the catheter around the navel.</li>
	</ol>
	</li>
</ol>
4. Portal Vein Blood Sampling
<ol>
	<li>Sample portal vein blood at times according to the specific testing protocol used; see Table 1.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Short protocol</td>
			<td>Long protocol</td>
		</tr>
		<tr>
			<td>t0 &#8211; baseline (before intracolonic administration)</td>
			<td>t0 &#8211; baseline (before intracolonic administration)</td>
		</tr>
		<tr>
			<td>t1 &#8211; 5 min after intracolonic administration</td>
			<td>t1 &#8211; 30 min after intracolonic administration</td>
		</tr>
		<tr>
			<td>t2 &#8211; 30 min after intracolonic administration</td>
			<td>t2 &#8211; 60 min after intracolonic administration</td>
		</tr>
	</tbody>
</table>
Table 1: Portal blood sampling protocols for gut permeability assessment.
NOTE: The time between consecutive blood sampling depends mainly on the bioavailability of the tested substances and the site of administration (colon, stomach, etc.).
<ol start="2">
	<li>Open the portal catheter plug and let the blood flow freely.</li>
	<li>Use syringe (vol. 2 mL) and blunt needle OD: 0.9 mm. Collect no more than 0.7 mL of blood.</li>
	<li>Rinse the catheter with 0.3 mL of heparinized saline (100 units/mL) and close the catheter plug.</li>
</ol>
5. Inferior Vena Cava Blood Sampling
<ol>
	<li>Sample inferior vena cava blood at times according to the specific testing protocol used; see Table 2.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Portal vein</td>
			<td>Inferior vena cava</td>
		</tr>
		<tr>
			<td>t0 &#8211; baseline (before intracolonic administration)</td>
			<td>t0 &#8211; baseline (before intracolonic administration)</td>
		</tr>
		<tr>
			<td>t1 &#8211; 30 min after intracolonic administration</td>
			<td>t1 &#8211; 30 min after intracolonic administration</td>
		</tr>
	</tbody>
</table>
Table 2: Protocol of blood sampling for liver clearance measurement and tracking the gut-portal blood-liver-systemic blood pathway.
<ol start="2">
	<li>Open the inferior vena cava catheter plug and let the blood flow freely.</li>
	<li>Collect no more than 0.7 mL of blood using syringe (vol. 2 mL) and broken needle OD: 0.9mm.</li>
	<li>Rinse the catheter with 0.2-0.3 mL of heparinized saline (100 units/mL) and close the catheter plug.</li>
</ol>
6. Administration of a Gut Permeability Marker
<ol>
	<li>Remove the guide wire and inflate the colonic catheter balloon, using adequate volume of sterile water (usually 1 mL but check actual balloon size before insertion). 
	NOTE: The balloon diameter should not exceed 1 cm.</li>
	<li>Place the rat head down (inclination about 15%) to minimize the risk of the outflow of the administered solution from the colon.</li>
	<li>Slowly administer the tested substance (e.g. trimethylamine, 100 mg/kg bw) using a drainage port in colonic catheter. 
	NOTE: Do not exceed the volume of 0.75 mL of the administered solution and the feeding speed of 0.5 mL/min to prevent the outflow of the administered solution from the anus.</li>
	<li>After 10 min deflate the catheter balloon.</li>
	<li>Sample blood from the inferior vena cava and the portal vein according to the specific testing protocol used; see Table 1 and Table 2.</li>
	<li>Euthanize animal via approved method.</li>
</ol>
7. Calculation of Liver Clearance
<ol>
	<li>Express liver clearance, understood as hepatic extraction, by the difference between portal blood concentration and inferior vena cava blood concentration or by the ratio of inferior vena cava to portal blood concentration, (1 - (inferior vena cava concentration/portal vein concentration)).</li>
</ol>
8. Evaluation of the Test Substance Concentration n Blood Samples
<ol>
	<li>Depending on the test substance and test methodology, subject the sample to appropriate laboratory procedures (centrifugation, etc.). In the proposed protocols, we evaluate TMA/TMAO and SCFA concentration using liquid chromatography coupled with triple-quadrupole mass spectrometry. Please find a detailed description of the method in Supplemental Material.</li>
</ol>

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The authors have nothing to disclose.

The described direct, in vivo, method of measuring the GBB permeability maintains closetophysiological conditions in the gastrointestinal system (preserves the intestinal blood flow), and is virtually not influenced by liver and kidney function.
The critical step of this technique is the insertion of the portal catheter. This must be done gently and decisively at the same time. A mild, short bleeding may occur from the correctly performed puncture of the portal vein; however, it stops when the needle is inserted into the vessel. Persistent bleeding indicates that the portal vein is perforated. To facilitate the catheter insertion, the portal vein should be well exposed. After exteriorizing the intestines, when the mesenteric root is well exposed, the upper mesenteric vein should also be visible (mesenteric vein enters cranially into the portal vein). The portal vein is usually covered by the hepatic lobes, which have to be moved to the sides. Also, the proper stabilization of the portal catheter is crucial for a successful procedure, since the catheter&#39;s movement may produce portal vein rupture and bleeding, especially in longer experiments. Additional stabilization of the catheter may be achieved by attaching the catheter to mesentery by sticking it to a mesentery with tissue glue or by applying two single stitches (thread 6/0). After closing the abdominal cavity to secure the placement of the catheter, a purse-string suture may be applied on the catheter.
There are several minor difficulties that may occur during the experiment. After catheterization of the femoral vein, if the venous blood does not backflow in the catheter, try the following solutions: flush the catheter with heparinized saline, gently pull the catheter 1-2 mm from the vein, remove the surgical knots, and tie&#160;a new one, pull the catheter out and reinsert, or replace with a new catheter. Remember to confirm the proper placement of the catheter after the experiment. The catheter should be inserted for 6-7 cm, depending on the size of the animal, to place the proximal tip of the catheter in the inferior vena cava just above the hepatic vein confluence. When it comes to colon catheterization, if you have problems with advancing the catheter you may inject 0.3-0.5 mL of saline or leave the catheter in the colon for 5-10 minutes, and try again. Do not use force while inserting a catheter to avoid perforation of the intestine.
In our studies, we used a gut bacteria-derived molecule, trimethylamine (TMA), as a marker of the colon GBB permeability, as TMA is produced mostly by colonic bacteria. However, many other substances, including classic permeability markers like FITC-dextran or sugars, may be used as well (see Table 4). When preparing a solution of the test substance, take into account its irritating effect on the intestinal mucosa and appropriately choose the concentration of the substance. Further laboratory procedures of the blood samples must be adjusted to the selected marker.
In our protocol, we propose intracolonic administration of a marker; however, it may be modified by oral administration or gavage at various levels of the digestive tract. The variable speed of peristalsis and possible interactions with enzymes and gastric acid should be taken into account while administering a marker into upper parts of the gastrointestinal tract e.g. stomach or duodenum. Accordingly, time of blood sampling after administration of a marker needs to be adjusted.
There are several limitations of the presented method, including adverse effects of anesthesia and fasting overnight, that may both influence GBB function. It should be taken into account as the procedure is terminal and involves blood sampling during not fully physiological conditions. However, as mentioned before, it has still many advantages over other experimental methods assessing GBB permeability, especially performed in vitro11. For example, an Ussing chamber measures the conductance and particle flux through the intestinal epithelial cells. The main weakness of this technique lies in its excessive simplification. It is difficult to describe the complex physiological system of the intestinal mucosa using a small number of measurements on epithelial cell layer alone. Some researchers use whole-thickness intestine for Ussing chamber studies, but this procedure is accompanied by several methodological complications15. Furthermore, the accuracy of the method is compromised by a limited viability of tissues isolated from the organism. Some in vitro methods used in pharmacokinetic studies use artificial membranes as a model of the intestinal barrier12. However, those methods, similarly to the Ussing chamber, do not reflect the complexity of the GBB structure and functions.
There are also in vivo permeability assays available in experimental and clinical studies. They are mostly based on urine or peripheral blood sampling after oral or colonic administration of various markers13. The widely used sugar test involves oral intake of mono- and oligosaccharides, which are not metabolized in mammalian organism, e.g. mannitol and lactulose. The method is non-invasive and may be employed in both experimental and clinical use16,17; however, the results are affected by first-pass liver metabolism and kidney function, which may differ significantly between the study subjects. In contrast to the above mentioned indirect methods, collecting blood from the portal vein allows direct evaluation of the GBB permeability12. This method is not dependent on liver and kidney function and virtually preserves physiological conditions in the intestines which is an important advantage over ex vivo or in vitro methods.
The techniques described in this paper also allow for a relatively accurate liver clearance evaluation, as the blood is collected from the portal vein and inferior vena cava just above the hepatic vein confluence. The representative results for the hepatic extraction are presented in Figure 3 (for TMA) and Table 3 (for SCFA). Our data suggest that three main SCFA, acetate, propionate, and butyrate, are characterized by different hepatic clearance, which is supported by previous studies, where Bloemen et al. shown that intestinal release of butyrate and propionate, but not acetate, is almost equaled by hepatic uptake18. Therefore, the presented protocol is suitable for tracking intestinal absorption and liver metabolism of drugs, which can be used in pharmacokinetic studies.
The techniques may also be adjusted to other experimental purposes. Catheterization of the portal vein may be used to measure portal blood pressure or for administration of drugs directly to the portal vein, in order to study hepatic circulation. For instance, in our previous work, we administered hydrogen sulfide donors to the portal vein to assess its influence on hepatic circulation and portal pressure19.

The gut-blood barrier (GBB), also known as the intestinal barrier, is a complex multilayer system that separates the gut lumen from the bloodstream in order to limit the passage of harmful compounds while allowing the absorption of nutrients1. It consists of the three main layers: the mucus layer, epithelium and lamina propria.
Numerous factors may affect the GBB integrity and function2. It has been shown that GBB is disturbed in both gastrointestinal and extraintestinal diseases, including cardiovascular and metabolic diseases3, which may lead to an increased passage of gut bacterial metabolites to the bloodstream4. An increased penetration of gut bacterial metabolites may affect the functioning of the entire organism. For example, recent studies show a significant impact of bacterial metabolites, such as indoles, H2S, short-chain fatty acids (SCFA), and trimethylamine N-oxide, on the circulatory system functions5,6,7,8,9. Finally, it has been proposed that an increased GBB permeability may serve as a marker of cardiovascular and metabolic diseases which are associated with morphological and functional alterations in the intestines10. Therefore, tracking the gut-portal blood-liver-systemic blood pathway of bacterial metabolites may be of interest for both basic and clinical sciences.
Commonly utilized experimental methods for the evaluation of GBB permeability are performed in vitro using resected intestinal segments, fragments of mucosa, or artificial membranes11,12. The accuracy of those methods is compromised by the fact that proper functioning of the GBB requires constant intestinal blood flow. On the other hand, the available in vivo methods are based on the evaluation of urine or peripheral blood concentrations of exogenous markers13. However, peripheral blood and urine concentration of exogenous compounds is influenced by kidney function, i.e., glomerular filtration rate and tubular excretion, as well as by liver metabolism, i.e., first pass metabolism. Both parameters may differ significantly between study subjects independently of the GBB function.
This paper describes a direct measurement of the GBB permeability in rats using portal blood sampling. This in vivo method preserves the intestinal blood flow and is virtually not influenced by liver and kidney function. The described approach is not commonly used, possibly because of some methodological difficulties. We describe in detail the catheterization of the portal vein and inferior vena cava just above the hepatic vein confluence. Blood sampling from the portal vein and inferior vena cava allows evaluation of the GBB permeability and liver clearance as well as tracking of gut-portal blood-liver-systemic blood pathway of molecules of interest, such as gut bacterial metabolites or medicines. We also present several applications of the method that were tested in our laboratory. These include the evaluation of the colon permeability to TMA, a gut bacterial metabolite, evaluation of liver clearance of TMA, and evaluation of a gut-portal blood-liver-systemic blood pathway of SCFA.
To evaluate gut-blood barrier permeability, the following protocol steps should be followed, in order: 1 (insertion of the line for intraintestinal administrations), 3 (portal vein catheterization), 4 (portal vein blood sampling), 6 (administration of a gut permeability marker), 4.
To evaluate liver clearance and a gut-portal blood-liver-systemic blood pathway, the following protocol steps should be followed, in order: 1 (insertion of the line for intraintestinal administrations), 2 (inferior vena cava catheterization), 3 (portal vein catheterization), 4 (portal vein blood sampling), 5 (inferior vena cava blood sampling), 6 (administration of a gut permeability marker), 4, 5, 7 (calculation of liver clearance).

<table><tbody><tr><td>Needle OD: 9 mm</td><td>Becton Dickinson&amp;nbsp; S.A.</td><td>301300</td><td></td></tr><tr><td>Polyethylene catheter ID: 0.025&quot;, OD: 0.040&quot;&amp;nbsp;</td><td>Scientific Commodities, Inc.</td><td>#BB520-40</td><td></td></tr><tr><td>Polyethylene catheter ID: 0.012&quot;, OD: 0.025&quot;&amp;nbsp;</td><td>Scientific Commodities, Inc.</td><td>#BB520-25</td><td></td></tr><tr><td>C-Flex Tubing,Opaque White 1/50&quot;ID x 1/12 &quot; OD&amp;nbsp;&amp;nbsp;</td><td>Cole-Parmer Instrument Co.</td><td>06424-59</td><td></td></tr><tr><td>Pediatric Foley catheter (size 10F or 8F)&amp;nbsp;</td><td>Sigmed</td><td>0000 80305</td><td></td></tr><tr><td>Surgical ligatures 3/0</td><td>Yavo Sp. Z o.o.&amp;nbsp;</td><td>P48JE</td><td></td></tr><tr><td>Absorbable surgical sutures - Polyglactine 910 4/0</td><td>KRUUSE Polska Sp. Zo.o.</td><td>152336</td><td></td></tr><tr><td>Tissue glue - Loctite 454Cyanoacrylate Adhesive</td><td>Loctite&amp;nbsp;</td><td>1370127</td><td></td></tr><tr><td>Povidone iodine</td><td>EGIS Pharmaceuticals PLC</td><td>4449 11</td><td></td></tr><tr><td>Heparin - Heparinium WZF&amp;nbsp;</td><td>WZF Polfa S.A.</td><td>02BK0417</td><td>Dilute 10 times with physiological saline</td></tr><tr><td>Glycerin 86%</td><td>Laboratorium Farmaceutyczne Avena</td><td>5.90999E+12</td><td>Serves as a lubricant in colon catheterization</td></tr><tr><td>Xylocaine 2%</td><td>AstraZenca&amp;nbsp;</td><td>9941342</td><td></td></tr><tr><td>Urethane</td><td>Sigma-Aldrich (Merck)&amp;nbsp;</td><td>U2500-500G</td><td></td></tr><tr><td>Trimethylamine solution 45%</td><td>Sigma-Aldrich (Merck)&amp;nbsp;</td><td>92262-1L</td><td></td></tr><tr><td>Syringes 2 mL</td><td>B.Braun Melsungen AG</td><td>4606027V</td><td></td></tr><tr><td>Saline 250 mL</td><td>Fraesenius Kabi Polska Sp. Z o.o.</td><td>15LL707WL</td><td></td></tr><tr><td>Surgical scissors, straight, length 115 mm, 4 1/2 &quot;blunt ends</td><td>Braun</td><td>NS-010-115-PKM</td><td></td></tr><tr><td>Artery forceps type Micro-Adson bent, length 140 mm 5 1/2 &quot;</td><td>Braun</td><td>KN-008-140-ZMK</td><td></td></tr><tr><td>Anatomic forceps, lenght 95 mm, 3 3/4&quot; sharp 0.7x0.55</td><td>Braun</td><td>PO-001-007-ZMK</td><td></td></tr><tr><td>Micro Scissors type Vannas,&amp;nbsp; straight, lenght 85 mm, 3 3/8 &quot; the length of the blades 6 mm</td><td>Braun&amp;nbsp;</td><td>NO-010-085-PMK</td><td></td></tr><tr><td>Towel clamps type Backhouse, lenght 130 mm, 5 1/8&quot;&amp;nbsp;</td><td>Braun</td><td>HO-128-130-PMK</td><td></td></tr><tr><td>Needle holders, lenght 150 mm, 6&quot; t=0.4 1/2&amp;nbsp;</td><td>Braun&amp;nbsp;</td><td>IM-927-150-PZMK</td><td></td></tr><tr><td>Delicate Scissors, lenght 110 mm , straight, 4 3/8&amp;rdquo; sharp&amp;nbsp;</td><td>Braun</td><td>NO-052-110-PMK</td><td></td></tr><tr><td>Anatomic forceps, lenght 95 mm, 3 3/4&quot; sharp</td><td>Braun</td><td>PO-022-001-PMK</td><td></td></tr></tbody></table>

an in vivo method for evaluating the gut-blood barrier and liver metabolism of microbiota products

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity is disturbed in gastrointestinal, cardiovascular and metabolic diseases, which may result in easier access of biologically active compounds, such as gut bacterial metabolites, to the bloodstream. Thus, the permeability of the GBB may be a marker of both intestinal and extraintestinal diseases. Furthermore, the increased penetration of bacterial metabolites may affect the functioning of the entire organism.
Commonly used methods for studying the GBB permeability are performed ex vivo. The accuracy of those methods is limited, because the functioning of the GBB depends on intestinal blood flow. On the other hand, commonly used in vivo methods may be biased by liver and kidney performance, as those methods are based on evaluation of urine or/and peripheral blood concentrations of exogenous markers. Here, we present a direct measurement of GBB permeability in rats using an in vivo method based on portal blood sampling, which preserves intestinal blood flow and is virtually not affected by the liver and kidney function. 
Polyurethane catheters are inserted into the portal vein and inferior vena cava just above the hepatic veins confluence. Blood is sampled at baseline and after administration of a selected marker into a desired part of the gastrointestinal tract. Here, we present several applications of the method including (1) evaluation of the colon permeability to TMA, a gut bacterial metabolite, (2) evaluation of liver clearance of TMA, and (3) evaluation of a gut-portal blood-liver-peripheral blood pathway of gut bacteria-derived short-chain fatty acids. Furthermore, the protocol may also be used for tracking intestinal absorption and liver metabolism of drugs or for measurements of portal blood pressure.

We have successfully measured the GBB permeability and liver clearance of TMA in rats. We have demonstrated that hypertensive rats have an increased colon permeability to TMA in comparison to normotensive rats (Figure 2)4. In another study we found that high salt intake does not affect the GBB permeability and liver clearance of TMA (Figure 3)14.
Measuring the concentration of SCFA in stools, portal blood, and peripheral blood, we traced the path of the molecules from the intestine to the peripheral blood. The exemplary results for those experiments are presented in Table 3.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58456/58456fig2.jpg" /> 
Figure 2: Hypertension-associated changes in gut-blood barrier permeability. Intracolonic administration of TMA produced a significant increase in portal blood TMA in each group (n=12 for each group). The increase in portal blood TMA in the hypertensive (SHR) group was significantly higher than in normotensive (WKY) group. We used the long protocol consisting of blood sampling 30 min and 60 min after TMA administration (IC TMA). Values are means, + SE, *p &#60; 0.05 vs baseline, #p &#60; 0.05 WKY vs SHR. This figure has been modified from Jaworska et al.4 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58456/58456fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58456/58456fig3.jpg" /> 
Figure 3: Gut-blood barrier permeability and liver clearance after high salt intake. (A) Intracolonic administration of TMA produced a significant increase in portal blood TMA. The size of the increase was similar between the groups (n=7 for each group). We used a simplified protocol, taking blood samples at baseline (0) and 15 min after administration of TMA (IC TMA). (B) TMA liver clearance was similar between the groups at baseline, and 15 min after the intracolonic administration of TMA. Values are means, + SE. *p &#60; 0.05 vs baseline. This figure has been modified from Bielinska et al.14 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58456/58456fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>SCFA</td>
			<td>Stool concentration (&#181;M)</td>
			<td>Portal blood concentration (&#181;M)</td>
			<td>Peripheral blood concentration (&#181;M)</td>
		</tr>
		<tr>
			<td>AA- acetic acid (C2)&#160;</td>
			<td>15998.40 &#177; 4317.58</td>
			<td>564.22 &#177; 155.34</td>
			<td>149.89 &#177; 31.74</td>
		</tr>
		<tr>
			<td>IPA- propionic acid (C3)</td>
			<td>5390.70 &#177; 1016.19</td>
			<td>138.25 &#177; 55.50</td>
			<td>5.36 &#177; 3.25</td>
		</tr>
		<tr>
			<td>IBA- isobutyric acid (C4)</td>
			<td>191.20 &#177; 123.87</td>
			<td>4.51 &#177; 1.60</td>
			<td>1.14 &#177; 1.16</td>
		</tr>
		<tr>
			<td>BA- butyric acid (C4)</td>
			<td>4159.80 &#177; 3141.68</td>
			<td>143.14 &#177; 68.42</td>
			<td>6.43 &#177; 4.18</td>
		</tr>
		<tr>
			<td>2MeB- 2 methylbutyric acid (C5)</td>
			<td>80.90 &#177; 59.86</td>
			<td>2.02 &#177; 0.88</td>
			<td>1.14 &#177; 1.42</td>
		</tr>
		<tr>
			<td>IVA- isovaleric acid (C5)</td>
			<td>109.10 &#177; 56.05</td>
			<td>2.59 &#177; 1.07</td>
			<td>0.90 &#177; 1.22</td>
		</tr>
		<tr>
			<td>VA- valeric acid (C5)</td>
			<td>281.9 &#177; 158.20</td>
			<td>8.55 &#177; 3.56</td>
			<td>0.72 &#177; 1.02</td>
		</tr>
		<tr>
			<td>ICA- isocaproic acid/ 4-methylvaleric acid (C6)</td>
			<td>5.9 &#177; 2.95</td>
			<td>0.61 &#177; 0.15</td>
			<td>1.76 &#177; 0.87</td>
		</tr>
		<tr>
			<td>CA- caproic acid (C6)</td>
			<td>287.00 &#177; 309.68</td>
			<td>11.19 &#177; 4.94</td>
			<td>1.12 &#177; 0.93</td>
		</tr>
	</tbody>
</table>
Table 3:&#160;SCFA concentration in stool, portal blood, and peripheral blood (n=7).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Test substance</td>
			<td>Possible application</td>
		</tr>
		<tr>
			<td>Bacterial metabolites: 
			trimethylamine (TMA), short chain fatty acids (SCFA), hydrogen sulfide, etc.</td>
			<td>GBB permeability studies 
			Tracking a gut-portal blood-liver-systemic blood pathway 
			Hepatic clearance studies</td>
		</tr>
		<tr>
			<td>Classic permeability markers: 
			FITC-dextran, polysaccharides, PEG, etc.</td>
			<td>GBB permeability studies</td>
		</tr>
		<tr>
			<td>Drugs</td>
			<td>absorption and hepatic clearance studies</td>
		</tr>
	</tbody>
</table>
Table 4: Exemplary test substances with possible applications.

Watch this Scientific Journal Video about An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products at JoVE.com

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The access of nutrients, microbiota metabolites and medicines to the circulation is controlled by the gut-blood barrier (GBB). We describe a direct method for measuring the GBB permeability in vivo, which, in contrast to commonly used indirect methods, is virtually not affected by liver and kidney functions.

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity ...

an-vivo-method-for-evaluating-gut-blood-barrier-liver-metabolism

Nanyang Technological University

Research

JoVE Journal

Medicine

8.4K Views.  Medical University of Warsaw. De darm-bloedbarrière is een meerlaags systeem dat de passage van voedingsstoffen, bacteriële metabolieten, drugs, en andere exogene verbindingen van darmlumen naar de bloedstroom regelt. Zoals we weten, kan de integriteit van de barrière worden aangetast in het maagdarmstelsel en in cardiovasculaire en sommige stofwisselingsziekten die kunnen leiden tot gemakkelijkere toegang tot biologisch actieve verbindingen zoals darmbacteriële metabolieten tot de bloedstroom en kan de werking van he...

Video: An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin synthesis. They also supply the host with a considerable amount of nutrients, making this ecosystem an essential metabolic organ. In the context of increasing evidence of the link between the gut flora and the metabolic syndrome, understanding the metabolic interaction between the host and its gut microbiota is becoming an important challenge of modern biology.1-4
 Colonization (also referred to as normalization process) designates the establishment of micro-organisms in a former germ-free animal. While it is a natural process occurring at birth, it is also used in adult germ-free animals to control the gut floral ecosystem and further determine its impact on the host metabolism. A common procedure to control the colonization process is to use the gavage method with a single or a mixture of micro-organisms. This method results in a very quick colonization and presents the disadvantage of being extremely stressful5. It is therefore useful to minimize the stress and to obtain a slower colonization process to observe gradually the impact of bacterial establishment on the host metabolism.
 In this manuscript, we describe a procedure to assess the modification of hepatic metabolism during a gradual colonization process using a non-destructive metabolic profiling technique. We propose to monitor gut microbial colonization by assessing the gut microbial metabolic activity reflected by the urinary excretion of microbial co-metabolites by 1H NMR-based metabolic profiling. This allows an appreciation of the stability of gut microbial activity beyond the stable establishment of the gut microbial ecosystem usually assessed by monitoring fecal bacteria by DGGE (denaturing gradient gel electrophoresis).6 The colonization takes place in a conventional open environment and is initiated by a dirty litter soiled by conventional animals, which will serve as controls. Rodents being coprophagous animals, this ensures a homogenous colonization as previously described.7
 Hepatic metabolic profiling is measured directly from an intact liver biopsy using 1H High Resolution Magic Angle Spinning NMR spectroscopy. This semi-quantitative technique offers a quick way to assess, without damaging the cell structure, the major metabolites such as triglycerides, glucose and glycogen in order to further estimate the complex interaction between the colonization process and the hepatic metabolism7-10. This method can also be applied to any tissue biopsy11,12.

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

A progressive colonization procedure is described to further assess its impact on the host hepatic metabolism. Colonization is monitored non invasively by evaluating the urinary excretion of microbial co-metabolites by NMR-based metabolic profiling while hepatic metabolism is assessed by High Resolution Magic Angle Spinning (HR MAS) NMR profiling of intact biopsy.

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin ...

Immunology and Infection

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the gastrointestinal tract, as resident microbiota occurs and prevents colonisation by exogenous bacteria. Indeed, more than 600 species of bacteria are found in the oral cavity, and a single individual may carry around 100 different at any time. Oral bacteria possess the ability to adhere to the various niches in the oral ecosystem, thus becoming integrated within the resident microbial communities, and favouring growth and survival. However, the flow of bacteria into the gut during swallowing has been proposed to disturb the balance of the gut microbiota. In fact, oral administration of P. gingivalis shifted bacterial composition in the ileal microflora. We used a synthetic community as a simplified representation of the natural oral ecosystem, to elucidate the survival and viability of oral bacteria subjected to simulated gastrointestinal transit conditions. Fourteen species were selected, subjected to in vitro salivary, gastric, and intestinal digestion processes, and presented to a multicompartment cell model containing Caco-2 and HT29-MTX cells to simulate the gut mucosal epithelium. This model served to unravel the impact of swallowed bacteria on cells involved in the enterohepatic circulation. Using synthetic communities allows for controllability and reproducibility. Thus, this methodology can be adapted to assess pathogen viability and subsequent inflammation-associated changes, colonization capacity of probiotic mixtures, and ultimately, potential bacterial impact on the presystemic circulation.

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Gut host-microbe interactions were assessed using a novel approach combining a synthetic oral community, in vitro gastrointestinal digestion, and a model of the small intestine epithelium. We present a method that can be adapted to evaluate cell invasion of pathogens and multi-species biofilms, or even to test probiotic formulations&#39; survivability.

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the ...

Biochemistry

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by ...

Developmental Biology

An Intestine/Liver Microphysiological System for Drug Pharmacokinetic and Toxicological Assessment

The recently introduced microphysiological systems (MPS) cultivating human organoids are expected to perform better than animals in the preclinical tests phase of drug developing process because they are genetically human and recapitulate the interplay among tissues. In this study, the human intestinal barrier (emulated by a co-culture of Caco-2 and HT-29 cells) and the liver equivalent (emulated by spheroids made of differentiated HepaRG cells and human hepatic stellate cells) were integrated into a two-organ chip (2-OC) microfluidic device to assess some acetaminophen (APAP) pharmacokinetic (PK) and toxicological properties. The MPS had three assemblies: Intestine only 2-OC, Liver only 2-OC, and Intestine/Liver 2-OC with the same media perfusing both organoids. For PK assessments, we dosed the APAP in the media at preset timepoints after administering it either over the intestinal barrier (emulating the oral route) or in the media (emulating the intravenous route), at 12 &#181;M and 2 &#181;M respectively. The media samples were analyzed by reversed-phase high-pressure liquid chromatography (HPLC). Organoids were analyzed for gene expression, for TEER values, for protein expression and activity, and then collected, fixed, and submitted to a set of morphological evaluations. The MTT technique performed well in assessing the organoid viability, but the high content analyses (HCA) were able to detect very early toxic events in response to APAP treatment. We verified that the media flow does not significantly affect the APAP absorption whereas it significantly improves the liver equivalent functionality. The APAP human intestinal absorption and hepatic metabolism could be emulated in the MPS. The association between MPS data and in silico modeling has great potential to improve the predictability of the in vitro methods and provide better accuracy than animal models in pharmacokinetic and toxicological studies.

We exposed a microphysiological system (MPS) with intestine and liver organoids to acetaminophen (APAP). This article describes the methods for organoid production and APAP pharmacokinetic and toxicological property assessments in the MPS. It also describes the tissue functionality analyses necessary to validate the results.

The recently introduced microphysiological systems (MPS) cultivating human organoids are expected to perform better than animals in the preclinical tests ...

Chemistry

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

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying tissue compartments, restricting the transport of smaller molecules across the epithelium and almost completely prohibiting epithelial macromolecular transport. This selectivity is determined by the mucous gel layer, which limits the transport of lipophilic molecules and both the apical receptors and tight junctional protein complexes of the epithelium. In vitro cell culture models of the epithelium are convenient, but as a model, they lack the complexity of interactions between the microbiota, mucous-gel, epithelium and immune system. On the other hand, in vivo assessment of intestinal absorption or permeability may be performed, but these assays measure overall gastrointestinal absorption, with no indication of site specificity. Ex vivo permeability assays using &#34;intestinal sacs&#34; are a rapid and sensitive method of measuring either overall intestinal integrity or comparative transport of a specific molecule, with the added advantage of intestinal site specificity. Here we describe the preparation of intestinal sacs for permeability studies and the calculation of the apparent permeability (Papp) of a molecule across the intestinal barrier. This technique may be used as a method of assessing drug absorption, or to examine regional epithelial barrier dysfunction in animal models of gastrointestinal disease.

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

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.

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

Applying Advanced In Vitro Culturing Technology to Study the Human Gut Microbiota

The human gut microbiota plays a vital role in both human health and disease. Studying the gut microbiota using an in vivo model, is difficult due to its complex nature, and its diverse association with mammalian components. The goal of this protocol is to culture the gut microbiota in vitro, which allows for the study of the gut microbiota dynamics, without having to consider the contribution of the mammalian milieu. Using in vitro culturing technology, the physiological conditions of the gastro intestinal tract are simulated, including parameters such as pH, temperature, anaerobiosis, and transit time. The intestinal surface of the colon is simulated by adding mucin-coated carriers, creating a mucosal phase, and adding further dimension. The gut microbiota is introduced by inoculating with the human fecal material. Upon inoculation with this complex mixture of bacteria, specific microbes are enriched in the different longitudinal (ascending, transverse and descending colons) and transversal (luminal and mucosal) environments of the in vitro model. It is crucial to allow the system to reach a steady state, in which the community and the metabolites produced remain stable. The experimental results in this manuscript demonstrate how the inoculated gut microbiota community develops into a stable community over time. Once steady state is achieved, the system can be used to analyze bacterial interactions and community functions or to test the effects of any additives on the gut microbiota, such as food, food components, or pharmaceuticals.

Here, we present a protocol for culturing the gut microbiota of the colon in vitro, using a series of bioreactors that simulate the physiological conditions of the gastro intestinal tract.

The human gut microbiota plays a vital role in both human health and disease. Studying the gut microbiota using an in vivo model, is difficult due to its ...

Bioengineering

An In Vitro Batch-culture Model to Estimate the Effects of Interventional Regimens on Human Fecal Microbiota

The emerging role of the gut microbiome in several human diseases demands a breakthrough of new tools, techniques and technologies. Such improvements are needed to decipher the utilization of microbiome modulators for human health benefits. However, the large-scale screening and optimization of modulators to validate microbiome modulation and predict related health benefits may be practically difficult due to the need for large number of animals and/or human subjects. To this end, in vitro or ex vivo models can facilitate preliminary screening of microbiome modulators. Herein, it is optimized and demonstrated an ex vivo fecal microbiota culture system that can be used for examining the effects of various interventions of gut microbiome modulators including probiotics, prebiotics and other food ingredients, aside from nutraceuticals and drugs, on the diversity and composition of the human gut microbiota. Inulin, one of the most widely studied prebiotic compounds and microbiome modulators, is used as an example here to examine its effect on the healthy fecal microbiota composition and its metabolic activities, such as fecal pH and the fecal levels of organic acids including lactate and short-chain fatty acids (SCFAs). The protocol may be useful for studies aimed at estimating the effects of different interventions of modulators on fecal microbiota profiles and at predicting their health impacts.

This protocol describes an in vitro batch-culture fermentation system of human fecal microbiota, using inulin (a well-known prebiotic and one of the most widely studied microbiota modulators) to demonstrate the use of this system in estimating effects of specific interventions on fecal microbiota composition and metabolic activities.

The emerging role of the gut microbiome in several human diseases demands a breakthrough of new tools, techniques and technologies. Such improvements are ...

An Intestinal Gut Organ Culture System for Analyzing Host-Microbiota Interactions

The structure of the gut tissue facilitates close and mutualistic interactions between the host and the gut microbiota. These cross-talks are crucial for maintaining local and systemic homeostasis; changes to gut microbiota composition (dysbiosis) associate with a wide array of human diseases. Methods for dissecting host-microbiota interactions encompass an inherent tradeoff among preservation of physiological tissue structure (when using in vivo animal models) and the level of control over the experiment factors (as in simple in vitro cell culture systems). To address this tradeoff, Yissachar et al. recently developed an intestinal organ culture system. The system preserves a naive colon tissue construction and cellular mechanisms and it also permits tight experimental control, facilitating experimentations that cannot be readily performed in vivo. It is optimal for dissecting short-term responses of various gut components (such as epithelial, immunological and neuronal elements) to luminal perturbations (including anaerobic or aerobic microbes, whole microbiota samples from mice or humans, drugs and metabolites). Here, we present a detailed description of an optimized protocol for organ culture of multiple gut fragments using a custom-made gut culture device. Host responses to luminal perturbations can be visualized by immunofluorescence staining of tissue sections or whole-mount tissue fragments, fluorescence in-situ hybridization (FISH), or time-lapse imaging. This system supports a wide array of readouts, including next-generation sequencing, flow cytometry, and various cellular and biochemical assays. Overall, this three-dimensional organ culture system supports the culture of large, intact intestinal tissues and has broad applications for high-resolution analysis and visualization of host-microbiota interactions in the local gut environment.

This article presents a unique method for analyzing host-microbiome interactions using a novel gut organ culture system for ex vivo experiments.

The structure of the gut tissue facilitates close and mutualistic interactions between the host and the gut microbiota. These cross-talks are crucial for ...

Investigating Intestinal Barrier Breakdown in Living Organoids

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was not possible in classical cell culture monolayers. Moreover, monolayer cell cultures are good in vitro model systems but do not represent the complex cellular differentiation processes and functions that rely on 3D structure. This has so far only been possible in animal experiments, which are laborious, time consuming, and hard to assess by optical techniques. Here we describe an assay to quantitatively determine the barrier integrity over time in living small intestinal mouse organoids. To validate our model, we applied interferon gamma (IFN-&#947;) as a positive control for barrier destruction and organoids derived from IFN-&#947; receptor 2 knock out mice as a negative control. The assay allowed us to determine the impact of IFN-&#947; on the intestinal barrier integrity and the IFN-&#947; induced degradation of the tight junction proteins claudin-2, -7, and -15. This assay could also be used to investigate the impact of chemical compounds, proteins, toxins, bacteria, or patient-derived probes on the intestinal barrier integrity.

Here we describe a technique to quantify the barrier integrity of small intestinal organoids. The fact that the method is based on living organoids enables the sequential investigation of different barrier integrity modulating substances or combinations thereof in a time-resolved manner.

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

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