The overall goal of this procedure is to develop and characterize a metabolic syndrome model in rabbits using a high fat, high sucrose diet, which is of great importance when associating a phenotype with organ remodeling. This method can help answer key questions in the pathophysiology field, such as understanding organ remodeling and the progression of the disease, the detection of paraclinical and clinical markers to identify patients at risk, or testing new therapeutic approaches. The rabbit can provide many advantages over other animal models, including murine, canine, or porcine, given its similarity to human physiology, and the affordability of use in chronic protocols and measurements.
Demonstrating this procedure will be Oscar Arias, a PhD student from our laboratory. To acclimate the animals to their diet, feed each animal in the control group 120 grams of control diet daily for four weeks, and provide water ad libitum. Feed the animals in the MetS group 250 grams of chow starting with 50%control and 50%high fat chow.
Then, progressively increase the diet to 100%high fat chow by the end of week four. In addition, give the animals in the MetS group water with 5%sucrose at the start, and increase the sucrose concentration to 15%by the end of the fourth week. Register the daily intake of chow and sucrose solution to calculate the caloric intake.
Once the animals have been acclimated to their diets, feed each animal in the control group 120 grams of control chow, and water ad libitum daily. Feed the animals in the MetS group 250 grams of high-fat chow, and 15%sucrose in water. Replace the chow daily, and the sucrose solution every third day.
Weigh the remaining chow and water daily to estimate daily intake. To carry out morphological assessment, weigh each animal on a weekly basis. Before administration of the experimental diet and at weeks 14 and 28, anesthetize the animal by using a sterile disposable catheter to cannulate the right ear marginal vein.
Then, inject eight milligrams per kilogram of propofol, followed by 1.5 ml of 0.9%sodium chloride solution. Once anesthetization has been confirmed, place the animal in the lateral decubitus position and use a measuring tape to measure the distance from the nose to the heel. Record this measurement as the length.
With the animal in the same position, measure the distance from the acromion in the shoulder to the tip of the paw and record this as the height. Now, with the animal with a supine position, gently place the measuring tape around the abdominal contour and take a measurement. Then measure tibial length from the lower part of the knee joint to the insertion of the Achilles tendon.
Prepare a 60%glucose stock solution with 60 grams of glucose in 100 milliliters of 0.9%sodium chloride. Allow the animals to fast with only water for seven hours. Then, to determine fasting glycemia, place a conscious rabbit in a restrainer in the prone position.
Insert a new strip into a glucose meter, then, use a lancet on the left marginal ear vein to get a drop of blood and touch the drop to the test strip. Use the glucose meter to measure the blood glucose level. With a disposable catheter, cannulate the right ear marginal vein, and inject a bolus of a 60%glucose solution.
Use the lancet as before to take blood samples at 15, 30, 60, 90, 120, and 180 minutes after glucose injection and use the glucose meter to analyze them. Remove the disposable catheter, and use gauze to pinch the site of catheter insertion. Once blood is coagulated, remove the gauze and check the status of the animal.
For blood pressure recording, prepare the acquisition system that includes a pressure transducer, a 10 milliliter syringe with 0.9%sodium chloride, a three-way stopcock, an amplifier, and a PC laptop with the acquisition software. To set up the equipment, place the three-way stopcock and the syringe in the pressure transducer between the transducer in the catheter, and connect the pressure transducer to the amplifier. Then, connect the amplifier to the PC laptop.
Place the conscious animal that a rabbit restrainer in the prone position. Warm up the ear before cannulation, then topically apply a local anesthetic in the ear around the site of insertion. Gently tap the area where the vascular package runs to easily identify the artery.
Then insert a sterile catheter in the left ear central artery. Now, loosen the restraints, and allow the animal to stay quiet for 30 minutes. Position the pressure transducer next to the animal at heart level, and record the blood pressure continuously for 20 minutes, directly from the arterial catheter.
Once the recording is finished, remove the catheter and use gauze to pinch the catheter insertion site to stop blood loss. Once the blood is coagulated, remove the gauze and check the status of the animal. To take plasma measurements, allow the animals to fast with water only for seven hours.
Then place a conscious animal in a restrainer in the prone position, and insert a sterile 21 gauge needle into the left ear marginal vein. Once blood begins to drip, discard the first drop and collect the blood samples in EDTA tubes up to the level indicated in the tube. Then store the samples on ice.
Centrifuge the blood samples at 1500 G and four degrees Celsius for 15 minutes. Following centrifugation, use a pipette to collect the plasma and prepare 250 microliter aliquots. Immediately analyze the fresh samples for total cholesterol, HDL, LDL, and triglycerides.
As shown in this table, the weight of the rabbits in the MetS group increased progressively on the high-fat diet until the end of the experimental protocol. The behavior of energy intake and its fluctuations is illustrated in this graph, ranging from 250 to 815 kilocalories in the MetS group. In this graph, the relative contribution of the different sources of energy is depicted.
There are critical periods in weeks 14 and 28, because, given the stress produced by the experimental procedures, rabbits might decrease chow and water intake. Mild hyperglycemia is observed at week 14, which reaches a plateau, and maintains similar values at week 28. The area under the curve also increases in the MedS group.
Hypertension is closely and directly related to the severity of obesity. Rabbits fed a high fat, high sucrose diet for 28 weeks showed an increase in systolic, diastolic, and mean blood pressure already at week 14, and this increase in blood pressure is maintained at week 28. Finally, in this chronic model, an alteration in the lipid profile was observed as early as week 14, and this alteration remained stable until week 28.
This included an increase in triglycerides and LDL, a decrease in HDL, and no changes in total cholesterol in MedS animals versus controls at both time points. Using this chronic model, other studies could be performed to answer additional questions like how the pathology are morphing in a similar way in human metabolic syndrome, allowing new perspectives in the pathophysiological mechanisms involved in the progression of the disease. This experimental model paved the way for researchers in the field of interactive physiology and pathophysiology to explore the mechanisms underlying organ remodeling.
The detection of biomarkers to identify patients at risk, or testing new therapeutic approaches for the treatment of metabolic syndrome.
