Measurement of intestinal permeability during sepsis

Abstract

Gut barrier function has been hypothesized to play a critical role in the pathophysiology of sepsis. Measuring intestinal permeability allows for a determination of barrier dysfunction under conditions of health and disease. Fluorescent-conjugated dyes such as fluorescein isothiocyanate-4 kDa dextran (FD4) have been commonly used for evaluating hyperpermeability. Here we describe a common method to measure gut permeability in vivo, following gavage with different sized dyes. In addition, we describe an ex vivo everted gut sac model that allows for discrimination of permeability by segmental geographic location along the intestine.

1. Introduction

The gut has been hypothesized to be the “motor” of critical illness, both driving and then propagating dysregulated inflammation, in part via disruption of its barrier [1]. Intestinal hyperpermeability allows for translocation of microbes, microbial products and other molecules that ordinarily are restricted to the gut lumen [2]. Improving intestinal permeability by either pharmacologic intervention or genetic manipulation improves survival in murine sepsis, suggesting the potential importance of a leaky gut in human sepsis [3, 4]. Accurately assessing gut permeability allows for measurement of barrier function in pre-clinical models of sepsis.

The gut barrier is maintained in a semi-permeable state in part via expression of tight junction proteins that, depending on their expression and function, lead to increases or decreases in permeability [5]. Gut permeability is mediated through three pathways -- two tight junction-dependent (pore and leak) and one tight junction-independent (unrestricted). The pore pathway transports small molecules such as water, nutrients, and ions with a maximum size of 6Å in a charge-dependent manner [6, 7]. The leak pathway non-selectively transports substances <100Å such as antigens and lipopolysaccharide [5]. The unrestricted pathway allows free passage of macromolecules and microbes as a result of apoptosis and epithelial damage [8] (Fig.1).

Figure 1.

The pore, leak, and unrestricted pathways are responsible for determining the penetration of substances, depending on the molecular characteristics and pathological condition. The pore pathway (<6Å) transports small molecules such as water, nutrients, and ions in a charge and size selective manner. The pore pathway can be assayed by gavaging mice with creatinine (6Å) and measuring its appearance in the bloodstream, Macromolecules such as antigens and lipopolysaccharide non-selectively passed through the leak pathway (<100Å), which can be assayed with FD4 (28Å). Unlike the pore and leak pathways which are mediated by tight junctions, the unrestricted pathway is unrelated to tight junctions and is related to apoptosis or epithelial damage, allowing for passage of larger substances including intact bacteria. The unrestricted pathway can be assayed by rhodamine70 (120Å).

Oral gavage with fluorescein isothiocyanate-4 kDa dextran (FD4: 28Å) followed by measuring its appearance in the bloodstream is a commonly used method for assessing gut permeability, dominantly examining the leak pathway. Oral gavage of creatinine (6Å) and rhodamine B isothiocyanate-70 kDa dextran (rhodamine70: 120Å) provide additional information regarding the pore and unrestricted pathways [9]. The advantage of this approach is that it allows for an in vivo assessment of permeability. However, this does not allow one to discriminate which portions of the intestine exhibit alterations in permeability. The everted gut sac model is an ex vivo technique that allows for segmental specificity in determining the cause of intestinal hyperpermeability [10, 11]. Here we describe these two complementary methods for assessing gut barrier function.

2. Materials

2.1. Measurement of Intestinal Permeability (in vivo)

1. Fluorescent-conjugated dyes: FD4 (22 mg/mL) and rhodamine70 (16 mg/mL).

2. Creatinine (40 mg/mL)

3. Feeding needle

4. Blood collection tubes with ethylenediaminetetraacetic acid (EDTA)

5. 96-well plate

6. Phosphate-buffered saline (PBS)

7. Creatinine Serum Detection Kit

8. Fluorescence Plate Reader

2.2. Everted Gut Sac Model (ex vivo)

Overlapping materials from the previous section are not described.

1. Fluorescent-conjugated dye: FD4 (100 μg/mL)

2. Ice-cold Krebs-Henseleit Bicarbonate Buffer (KHBB): pH 7.4, 6.9 g/L sodium chloride, 2.1 g/L sodium bicarbonate, 0.35 g/L potassium chloride, 0.145 g/L magnesium sulfate, 0.145 g/L potassium phosphate monobasic, 0.175 g/L calcium chloride dihydrate, and 2 g/L glucose

3. Silk braid (4–0)

4. Surgery tools (forceps, scissors, and tweezers)

5. Water bath with temperature control

6. Oxygen

3. Methods

3.1. Measurement of Intestinal Permeability (in vivo)

1. Prepare 22 mg/ml of FD4, 16 mg/mL of rhodamine70, and 40 mg/mL of creatinine in sterile PBS. After preparation, wrap in foil (see Note 1).

2. Five hours prior to when permeability will be measured, gavage mice with 0.5 ml of creatinine, FD-4, and rhodamine70 dissolved in PBS using a 20-gauge feeding needle (Fig.2) (see Notes 2, 3 and 4).

3. At the time of sacrifice, collect blood in tubes with EDTA and mix well to prevent clots.

4. Spin down the blood at 10,000 rpm for 5 minutes at 4°C.

5. Collect plasma.

6. Dilute plasma 1:2 in PBS (50 μl plasma + 50 μl PBS) and keep on ice.

7. Prepare sequentially diluted dyes as standard.

8. Add 50 μl of diluted plasma samples and standards onto 96 well plates in duplicate.

9. Determine the concentration of FD4 and rhodamine70 using a Fluorescence Plate Reader with an excitation wavelength of 485 and 530 nm and an emission wavelength of 528 and 645 nm.

10. Measure the creatinine concentration with a Creatinine Serum Detection Kit following the manufacturer’s protocol (see Note 5).

Figure 2.

After appropriate anesthesia, the mouse is held with the neck straight (A) so that the feeding needle is easily inserted into the esophagus through the pharynx (B) Dyes are then gavaged into the stomach via a feeding needle (C).

3.2. Everted Gut Sac Model (ex vivo) (Fig. 3)

Figure 3.

One end of the segmental jejunum is ligated with 4–0 silk suture (A). After being flipped over using the feeding needle, another end of the everted gut sac is ligated so that molecules can be transferred through the mucosa during the incubation. The inflated gut sac is placed in the beaker containing FD4 dissolved in HBSS at 37 °C with 100% oxygen for 30 minutes (B).The schematic figure (C) displays the positional relationship between the gut sac, oxygen, and a water bath. Arrows show the expected direction that FD4 moves through the everted mucosa.

1. Prepare 100 μg/ml of FD4 in KHBB (see Note 6). After preparation, wrap in foil.

2. Remove the gut from the abdomen, flush the intestinal lumen with ice-cold KHBB, and submerge the tissue in the buffer (see Notes 7 and 8).

3. Ligate one end of the intestine with 4–0 silk suture.

4. Bring the tip of a feeding needle with a 1mL syringe close to the knot, holding the ligated intestine. Then, push the knot toward the opposite side to evert the intestine. After flipping over the intestine and keeping the needle inside the sac, fix the position of the everted gut sac over the needle with another silk suture (see Notes 9 and 10).

5. Inject either 1 mL (small intestine) or 0.5 mL (colon) of KHBB, to inflate the gut sac (see Notes 7, 8 and 9).

6. Prepare a water bath at 37 °C and gently aerate with 100% oxygen.

7. Place the everted gut sac in the beaker onto the bath with 100 μg/mL of FD4 in KHBB for 30 minutes.

8. Collect a 1mL sample of the buffer from the beaker at the beginning and end of the incubation to determine the initial and final mucosal FD4 concentration, respectively.

9. After 30 minutes, take the sac out and wash the outside of the gut sac to avoid contamination of beaker fluid on collected samples. After collecting the fluid inside the gut, measure the length and diameter of each sac to standardize the clearance later.

10. Centrifuge the samples at 10,000 rpm for 5 minutes and collect the supernatant (see Note 11).

11. Dilute the fluid 1:2 with PBS, and determine the concentration of FD4 with a Fluorescence Plate Reader, using the method described above (section 3.1, step 9).

12. Permeability is calculated as clearance of FD4 from mucosa to serosa in nL/min/cm² using the following formula:

    $$\text{Clearance}\text{of}\text{FD4}=\left(\text{serosa}\text{FD4}\text{concentration}\times \text{injected}\text{volume}\right)/\text{mucosal}\text{FD4}\text{concentration}\times \text{mucosal}\text{surface}\text{area}\times \text{30}\text{minutes}$$

    $$\text{Mucosal}\text{surface}\text{area}\text{=}\pi \times \text{length}\text{of}\text{the}\text{sac}\times \text{diameter}\text{of}\text{the}\text{sac}$$

4. Notes

1. FD40 (molecular weight 40 kDa) may be used instead of FD4. While larger than FD4, FD40 still measures the leak pathway.

2. Blunt handling of the needle during gavage may cause mucosal injury of the digestive tract, leading to absorption directly from vessels. To avoid this, mice should be anesthetized during the procedure. Because deep sedation decreases swallowing, which increases the possibility of regurgitation, careful adjustment of anesthesia is needed.

3. To optimize oral gavage, hold the neck of a mouse straight so that a feeding needle is easily inserted into the esophagus through the pharynx.

4. While five hours is recommended between gavage and blood collection for sepsis models, researchers have used three hours in a murine colitis model [9]. The rationale for increasing the time in sepsis relates to decreased gut motility in critical illness.

5. Creatinine concentration after gavage is significantly higher than endogenous creatinine concentration; therefore, endogenous creatinine has a negligible effect on the results obtained.

6. It is possible to measure FD4 and creatinine in an everted gut sac model at the same time.

7. The segment of the intestine examined should be determined depending on the research interest or the purpose of the project. We typically prepare 10 cm of jejunum, ileum, and colon when measuring permeability [11].

8. A hole in an everted gut sac reduces the accuracy of permeability measurement. When removing the intestine from surrounding fat and mesentery, meticulous attention must be paid to surgical technique so as not to injure the intestine.

9. If the everted gut sac leaks after injection of the fluid into the ligated gut lumen, the experiment should be terminated and a different gut sac should be examined in order to obtain consistent results.

10. Although the length of the intestine is accounted for in the formula to calculate the final result, it is more difficult to inflate the gut equally with KHBB when longer segments of intestine are obtained, affecting the accuracy of the measurement. Alternatively, when examining a short intestinal segment, you can use the actual volume injected to calculate permeability if unable to inject the entire volume that was planned.

11. To separate mucus and epithelial cells, the sample collected from an everted gut sac should be centrifuged prior to measuring fluorescent activity.