Animal Model for In Vivo Evaluation of Cholesterol Reduction by Lactic Acid Bacteria

María Pía Taranto, Gabriela Perdigón, Marta Médici, and Graciela Font de Valdez

1. Introduction

For many years, it has been recognized that elevated serum cholesterol is a risk factor associated with atherosclerosis and coronary heart disease, the latter being a major cause of death in Western countries. Numerous drugs that lower cholesterol have been used to treat hypocholesterolemic individuals (1). However, the undesirable side effects of these compounds have caused concerns about their therapeutic use (2). Ingestion of probiotic (beneficial for health) lactic acid bacteria (LAB) would possibly be a more natural method to decrease serum cholesterol in humans (3), as has been was reported (4,5).

Previous studies (6,7) have demonstrated that Lactobacillus reuteri administered in low doses has a hypocholesterolemic effect both therapeutically and preventively. One of the key studies in the development of a probiotic is to determine the minimal effective dose of live microorganisms that might be ingested without producing adverse effects (i.e., translocation) in the host.

In this chapter, we describe an animal model that allows us to evaluate reduction in hypercholesterolemia by LAB and, also to determine the minimal dose of the microorganism, a critical step in the development of a safe probiotic product.

2. Materials

2.1. Establishment of a Hypercholesterolemia Animal Model (see Note 1)

2.1.1. Test Group

1. Ten male Swiss albino mice aged 4-6 wk and weighing 20-25 g were used (see Notes 25). They were kept under a 12-h light/dark cycle at 22-26°C and a relative humidity of 50%.

3. Fat-enriched liquid diet: 10% (w/v) sterile nonfat milk (NFM) supplemented with 10% cream. The composition is detailed in Table 1 (see Note 6).

4. Glass containers with adapters to facilitate consumption by the mice of the fat-enriched liquid diet (see Note 7).

5. Solid commercial conventional diet: 32% protein, 5% fat, 2% fiber, and 61% nitrogen-free extract.

6. Balance.

7. Pasteur pipet.

8. Scissors.

9. Clamps.

10. Enzymatic reagent kit (Sigma, St. Louis, MO).

2.1.2. Control Group

1. The same as items 1-2 of Subheading 2.1.1.

2. Tap water ad libitum.

3. The same as items 4-10 of Subheading 2.1.1.

2.2. Lactobacilli Growth Medium

1. de-Man-Rogosa-Sharpe (MRS) broth (8): 10 g/L polypeptone, 10 g/L meat extract, 5 g/L yeast extract, 20 g/L glucose, 5 g/L sodium acetate, 2 g/L ammonium citrate, 2 g/L KH2PO4, 0.25 g/L MgSO4-7H2O, 0.058 g/L MnSO4-4H2O, and 1.08 mL/L Tween-80, final pH 6.4 ± 0.2. Sterilize at 121°C for 20 min.

2.3. Media for Testing Bacterial Translocation

1. LBS agar broth.

2. MacConkey's agar: 20 g/L peptone from casein, 10 g/L lactose, 5 g/L Oxgall bile dried, and 0.01 g/L bromocresol purple.

3. Brain-heart infusion agar.

2.4. Testing Bacterial Translocation (see Note 8)

1. An active culture grown in MRS broth for about 16 h at 37°C (overnight culture).

2. Phosphate buffer.

a. Solution A (0.2 M NaH2PO4): weigh 27.6 g NaH2PO4-H2O and make to 100 mL with distilled water (dH2O).

b. Solution B (0.2 M Na2HPO4): weigh 53.05 g Na2HPO4-7 H2O and make to 100 mL with dH2O.

c. To prepare 1 L of 0.1 M sodium phosphate buffer, pH 7.0, mix 195 mL of solution A and 305 mL of solution B, and bring to 1000 mL with dH2O.

d. Sterilize at 121°C for 20 min.

3. 1 g/L Peptone water: to prepare 1 L, weigh 1 g peptone and bring it to 1000 mL with dH2O. Sterilize at 121°C for 20 min.

4. 8 g/L Saline solution: to prepare 1 L, weigh 8 g NaCl and bring it to 1000 mL with dH2O. Sterilize at 121°C for 20 min.

6. MacConkey's agar.

Table 1

Fat-Enriched Liquid Diet Composition

Table 1

Fat-Enriched Liquid Diet Composition

Component

Amount

Proteins

0.26 g

Carbohydrate

0.38 g

Lactic fat

0.26 g

Lecithine

0.002 g

Vitamin B2

0.014 mg

Vitamin B12

0.018 mg

Calcium

9.3 mg

Phosphorus

7.5 mg

Cholesterol

0.3 g

Lipids

0.2 g

Water

10 mL

Calories

10.5 kcal

7. Brain-heart infusion agar supplemented with blood.

2.5. Lactobacilli Treatment

2.5.1. Test Group

1. The same as items 1-5 of Subheading 2.1.1.

2. An active culture grown in MRS broth for about 16 h at 37°C (overnight culture).

3. Phosphate buffer.

4. Nonfat milk reconstituted at 10%.

5. The same as items 6-10 of Subheading 2.1.1.

2.5.2. Control Group

1. The same as items 1-6 of Subheading 2.1.2.

2. Nonfat milk reconstituted at 10%.

3. The same as items 6-10 of Subheading 2.1.1.

3. Methods

3.1. Establish of the Hypercholesterolemic Model

3.1.1. Test Group

1. Place the mice in separate cages.

2. Feed the animals 5 mL of the fat-enriched liquid diet (to induce hypercholesterolemia) and the solid conventional diet ad libitum for 15 consecutive days (see Note 9).

3. Determine the weight of the animals daily and observe the characteristics of the feces (see Note 10).

4. Take blood samples carefully from the retro-orbital venous plexus with a Pasteur pipet and place them in perfectly clean glass tubes (see Note 11).

5. Centrifuge the blood (6000g, 10 min, at room temperature), separate the serum (supernatant), and determine total cholesterol, HDL cholesterol, LDL cholesterol, and serum triglycerides with enzymatic reagent kits following the instructions of the suppliers. Express the results as mg/dL.

6. Dissect the animal at the level of the abdominal zone, cutting the skin and muscle with a scissors and avoiding damage to internal organs (see Note 12).

7. Remove the spleen and liver with clamps.

8. Weigh the removed organs and observe their anatomical characteristics (see Note 13).

3.1.2. Control Group

1. Place the mice in separate cages.

2. Feed the animals 5 mL of water and solid conventional diet ad libitum for 15 consecutive days.

3. Repeat steps 3-6 of Subheading 3.1.1.

3.2. Bacterial Translocation

Lactic acid bacteria preparation is performed as follows:

1. Grow the probiotic strain in MRS broth at 37°C for 16 h (see Note 14).

2. Harvest the cells by centrifugation at 5000g for 10 min.

3. Wash the pellet obtained twice with phosphate buffer.

4. Suspend the pellet in 5 mL sterile 10% NFM at the following concentrations: 102-108 colony-forming units (CFU)/mL.

5. Feed the mice with the cell suspensions in NFM at 20% (v/v) in the drinking water for 2, 5, and 7 d.

6. Kill the mice by cervical dislocation after feeding each microbial suspension.

7. Remove and weigh the spleen and liver under aseptic conditions and homogenize the organs with saline solution

8. Plate in the selective agarized media the proper dilutions of the organs from 10-fold serial dilutions prepared in 1 g/L peptone water (see Note 15).

9. Incubate the plates in anaerobic jars (Oxoid Systems) at 37°C for 48 h, and count the resulting colonies. Results are expressed as CFU per mL per gram of organ tissue.

10. Select the minimum dose that does not produce translocation.

3.3. Evaluation of the Hypocholesterolemic Effect

3.1.1. Test Group

1. Place the mice in separate cages.

2. Repeat steps 2-3 of Subheading 3.1.1.

3. Feed the animals 5 mL of the probiotic strain at the concentration established in step 10 of Subheading 3.2. diluted to 20% (v/v) in the drinking water for seven consecutive days.

4. Repeat steps 3-6 of Subheading 3.1.1.

3.1.2. Control Group

1. Place the mice in separate cages.

2. Repeat steps 2-3 of Subheading 3.1.1.

3. Feed the animals with 5 mL of NFM (10%) diluted to 20% (v/v) in the drinking water for 7 consecutive days.

4. Repeat steps 3-6 of Subheading 3.1.1.

4. Notes

1. Research using animal models should not be carried out until the protocol is reviewed by an appropriate animal care committee to ensure that the procedures are appropriate and humane.

2. Mice are selected because their physiological system is similar to that of humans and they are easy to obtain and handle. The animals used for this study belong to a closed colony. The Swiss albino mouse was chosen because it is extremely robust and is used for reproductive and physiological studies.

3. Young adult mice were chosen because they have an active cholesterol metabolism, guaranteeing a detectable reaction.

4. The sex of the mouse should be chosen with study aims in mind. In the hypercholester-olemic model, it is advisable to work with males because the hormonal system of the females can indirectly influence the lipid levels.

5. Each animal must be housed in a separate cage, for individual control of the normal consumption of food and other supplies corresponding to each treatment; it also allows evaluation of any secondary reaction. The number of mice for each group was established as 10 for statistical reasons. The experiment must be repeated at least three times.

6. Prepare the fat-enriched liquid diet using NFM sterilized at 115°C for 15 min and perfectly clean glass materials to ensure the best maintenance of the diet for long periods.

7. A suitable container was designed to administer both the liquid diet and the drinking water. This device ensures efficient food intake by the mice; being a closed container, it also allows better control of the amount ingested

8. It is very important to establish the minimum dose of probiotic bacterium that does not demonstrate undesirable effects such as a translocation (9). This phenomenon is known as the passage of the intestinal microbiota from the gut into such internal organs as the liver and spleen, which might later trigger septicemia. LAB are considered GRAS microorganisms (Generally Recognized as Safe); however, excessive proliferation of these bacteria in the gut may cause damage to the intestinal epithelial, generating cracks that favor the passage of undesirable enteric bacteria.

9. The fat-enriched liquid diet must be renewed every 24 h, and the containers must be perfectly washed before they are loaded with the recently prepared diet. These precautions will avoid diet spoilage owing to microbial contamination and prevent loss in cell viability of the probiotic strains during the treatment period.

10. Daily observation of the feces characteristics of each mouse during the test period is of utmost importance. This allows one to detect changes in color, consistency, and/or amount indicating either that the animal is not responding properly to the probiotic treatment or that it is necessary to reformulate the diet. Some mice within a treatment group may present negative symptoms and may need to be put aside. In case the number of mice discarded is above a significant value (more than three), it is advisable to reinitiate the experiments.

11. Harvesting of mouse blood must be made carefully to avoid hemolysis. This phenomenon releases the contents of the red cells, which may interfere with enzymatic determination of cholesterol and triglycerides, producing an overestimation.

12. Dissection procedure. For the dissection, perfectly clean and disinfected clamps and scissors as well as gloves are used. Kill the mouse, place it in a ventral position, and fix it with pins by the extremities. Cut the skin with the scissors, separate it carefully, and fix the skin with pins at both sides of the body. Cut the peritoneum, avoiding damage to the internal organs. Remove carefully the organs of interest (liver, spleen, and small and large intestines) using clamps and scissors. Place the removed organs into Petri dishes containing sterile saline solution to avoid dehydration.

13. The spleen and liver should be observed post mortim. This allows one to detect any change in color, consistency, and/or size, which, as explained in Note 10, might indicate that the animal is responding negatively to the fat-enriched liquid diet. If negative changes are seen, it is advisable to redesign the assay conditions, taking into account the amount and composition of the diet and the period of administration.

14. The probiotic strains should be selected on the basis of in vitro cholesterol reduction and the rate of tolerance to bile and gastric juice (10).

15. The selective media used (LBS agar for the enumeration of lactobacilli, MacConkey's agar for enterobacteria, and blood-suplemented brain-heart infusion broth for anaerobes) were appropriate for selective enumeration (lactobacilli and enterobacteria) and total counts (anaerobes).

References

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7 Taranto, M. P., Medici, M., Perdigón, G., Ruíz Holgado, A. P., and Valdez, G. F. (1998) Evidence of hypocholesterolemic effect of Lactobacillus reuteri in hypercholesterolemic mice. J. Dairy Sci. 81, 2336-2340

8. de Man, J. C., Rogosa, M., and Sharpe, M. E. (1960) A medium for the cultivation of lactobacilli. J. Appl. Bacteriol. 23, 130-135

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10. de Valdez, G. F. and Taranto, M. P. (2000) Probiotic properties of lactobacilli: cholesterol reduction and bile salt hydrolase activity. In: Food Microbiology Protocols. Methods in Molecular Biology (Walker, J., ed.). Humana, Totowa, NJ, pp. 173-181.

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