Animal Physiology
Sonoma State University

Introduction:

The real business of the circulatory system takes place in the exchange of substances between the interstitial fluid and the small blood vessels. The collection of vessels through which this exchange occurs is often referred to as the microcirculation and consists of blood flow through the capillaries, arterioles, and venules. The microcirculation may be examined using several preparations , for example, the tongue, mesentery, or foot web of the frog. Because it is a simple preparation (no need to sacrifice the animal), you will use the web of the hind foot of the bull frog, Rana catesbiana, in this experiment.

Purpose: To examine the microcirculation in the hind foot web, and study the effects certain vasoactive agents on this phenomenon.

Procedure:

A) Examination of Microcirculation

Step 1. Anesthetize the frog by immersing in one gallon of tap water containing Tricane (1 gm/ L). It might take about 15 min. for the animal to slow down.

Step 2. After anesthetizing, place the animal ventral side down on a frog board having a hole at one end. Spread the toes of one hind foot over the hole and gently fasten them in place loosely with pins. Do not stretch the foot too tightly or you will shut off the circulation. Keep the web moist with wqter and cover the frog with a wet paper towel to aid its respiration. Place the board on the microscope stage. Stay tuned for a demonstration of all these!

Step 3: Examine the field under low and high power.

While observing the microcirculation, try to answer the following Questions?

a) How can you distinguish arterioles, venules, and capillaries?

b) Does the blood flow in a pulsating manner or in a smooth fashion?

c) Estimate the diameter of each type of blood vessel using the red blood cell size (~10 um) in diameter as a measuring device

d) Notice the pliability of the red blood cells as they move through the small capillaries.

e) Look for the alteration of dilation and constriction of the capillaries over period of several minutes. This process is called vasomotion

f) What causes vasomotion?

B) Effect of vasoactive agents on microcirculation:

Apply 4 to 5 drops of each of the solutions in the following list to the frog web using a plastic transfer pipette. Make sure you wash off the web with Ringer's between different solutions. The application of the solutions will disrupt your view of the web temporarily, so you will have to remember how the normal flow looks and quickly compare it with the post-experimental flow.

Record your observation, in a tabular form, in your laboratory notebook in the following order.

Vasoactive agents:

1) Cool Water (10^o C)

2) Warm Water (40^o C)

3) Vasopressin or epinephrine (1: 1000)

4) Lactic acid or Acetic acid (0.5%)

5) Histamine (1:10,000). If you see no response try pricking a hole in the skin and then adding the histamine.

Recovery: After investigating the effects of different vasoactive agents listed above, carefully remove the animal from the frog board and immerse it in a bucket containing fresh tap water (2-3 gallons). Make sure the animal is completely recovered before you leave the lab; it normally takes about 15 min.

 C) Draw and label an artery, vein and capillary bed.

Conclusions:

Questions:

1) Describe the factors that determine the capillary blood flow.

2) Describe the ways in which substances are transferred between blood and tissues across capillary walls.

3) Describe the ways in which vasopressin or epinephrine may affect capillary function.

NOTES

Materials & Supplies:

6 healthy frogs per lab
6 frog boards
20 large T-pins
4 L of1 g/L Tricaine in water

Solutions in dropper bottles:

6 Vasopressin or epinephrine (1: 1000)
6 Histamine (1:10,000)
6 Lactic acid or Acetic acid (0.5%)
6 Cool Water (10^o C)
6 Warm Water (40^o C)