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Microcirculation

Introduction:

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The most important process associated with the circulatory system is the exchange of substances between the blood and the interstitial fluid. The collection of vessels that is involved in this exchange is often referred to as the MICROCIRCULATION and consists of arterioles, met-arterioles, capillaries and venules. A conceptual diagram of a typical capillary bed is shown below.

The ARTERIOLES are surrounded by a layer of smooth muscle that is controlled by both the autonomic nervous system (ANS) and circulating hormones. Contraction of this smooth muscle causes the lumen of the arteriole to become narrower (constrict) and thereby decrease the blood flow to that region. Relaxation of the smooth muscle causes the vessel to dilate. In this way the ANS and the circulating hormones can divert the blood in the body from one area to another in response to the overall needs of the body areas for blood. The VENULES also have a smooth muscle layer in their walls, but it is not as extensive as that surrounding the arterioles. As a result, these vessels function little in the control of blood flow. The METARTERIOLES are direct channels that interconnect the arterioles and venules. Their walls contain scattered amounts of smooth muscle and hence can constrict or dilate actively. The actual exchange of substances between the blood and the interstitial fluid takes place across the walls of the TRUE CAPILLARIES. The true capillaries are offshoots of the met-arterioles and have walls consisting of a single layer of epithelial cells, but no smooth muscle. Their constriction or dilation is a purely passive process, depending on whether blood is flowing through them or not. At the entrance to each true capillary is a ring of smooth muscle called a PRECAPILLARY SPHINCTER. Its constriction or dilation controls the entrance of blood into the true capillaries. The precapillary sphincters are influenced largely by local factors resulting from tissue metabolism. Factors such as increase [H+] or low pH, elevated pCO2, decreased pO2, increased temperature, histamine, nitric oxide and various other metabolites resulting from tissue activity cause the sphincter to dilate, thus allowing blood to enter the capillaries. When the tissues are inactive and these products are not produced, the sphincters constrict and thereby shut off the blood flow through the capillaries. In this way the tissues control their own blood flow locally - a phenomenon that is called AUTOREGULATION.

In this exercise you will observe the microcirculation and its response to some of the stimuli that operate in the body to ensure that blood is delivered at adequate rates to the tissues and the organs. The microcirculation can be observed in a number of places such as, the tongue of a frog, the webbing in a frog’s foot, the tail of a goldfish and the wing of a bat. In this experiment we will use the tail of the goldfish because of its availability and ease of preparation.

C. Goldfish Tail (Carassius auratus)

1. Anesthetize goldfish by immersing it in a solution of 1MS222 (tricaine methane sulfonate). Dose at 50-100mg/L. Induction in 1-5 minutes, recovery in 3-15 minutes in clean water.  Wear gloves at all times while handling the goldfish.

2. When anesthetized, wrap the goldfish in a wet Kim Wipe to aid its respiration. Leave the tail exposed. Place the goldfish on its side on a large petri dish with its tail covering the condenser light at the center of the stage. Spread the tail over and if necessary hold it down with a cover slip. Make sure the tail is over the aperture in the stage. Keep the tail and the fish wet with tap water. If the goldfish tail moves too much use a disposable pipet and add anesthetic to gill area until the tail stops moving. Apply as needed.

II. Observation of the Microcirculation

1. Adjust the microscope as needed and select a view of the fish tail that includes vessels of many sizes. Examine first under low power and then high power.

2. Identify the different types of vessels. Compare the rate and manner of blood flow in the various vessel types. Note how the rate of flow in the capillaries fluctuates. This is known as VASOMOTION (smooth muscle contraction and relaxation). Blood flows in a pulsatile manner in some vessels and smoothly in others. Try to estimate the dimensions of the different vessels by comparing them to the red blood cells which are approximately 10 µm in diameter. Note the single file progress of red cells through the capillaries. Also take notice of how the red cells change shape as they are squeezed through the narrowest capillaries. Look for leukocytes.

a. How can the different types of vessels in the microcirculation be distinguished?

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b. In which vessels is the blood flow slowest?

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c. In which vessels does the blood pulsate? In which does it flow smoothly? How can this difference in flow be explained?

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d. Why does the rate of blood flow in the capillaries fluctuate (Vasomotion)? How can this be explained?

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e. In which vessels do the red cells travel in single file?

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f. Given that the average diameter of a goldfish red blood cell (RBC) is 10 μm,

estimate the approximate diameters of all three blood vessel types.

Arteriole diameter _______ Capillary diameter ______ Venule diameter ________

g. Calculate the heart rate of your fish. ________________

III. Vasoactive Agents

1. Using a disposable pipet apply several drops of each of the substances listed on the table below to the field of view and note whether they cause vasoconstriction or vasodilation. The application of the substances will disrupt your view of the vessels temporarily so you will have to remember how the normal flow looks and quickly compare it with the post-experimental flow. BE SURE TO REMOVE EACH SUBSTANCE BY WASHING THE PREPARATION THOROUGHLY WITH WATER BEFORE A NEW SUBSTANCE IS APPLIED. Fill in the chart that follows.

|EXPERIMENTAL PROCEDURE |VASOCONSTRICTION |VASODILATION |CAN’T TELL |

|Epinephrine | | | |

|Acetylcholine | | | |

|Cold Water | | | |

|Hot Water | | | |

2. Return the goldfish to the recovery tank (clean water) at the completion of the exercise. If necessary, euthanize using 1MS222 (300mg/L) mg for 10 minutes or more.  Always wait 10 minutes or more before disposing of the fish.

3. Answer the following questions:

a. What local factors are responsible for increasing the blood flow through the skeletal muscles during exercise?

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b. Explain the action of the vasoactive agents used in this experiment.

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1MS-222 is a valuable tool for aquatic animal keepers. It provides a high degree of safety and reliability. It has proven its efficacy in laboratory, aquaculture, fishery, and veterinary settings.

Helpful sources of information on the use of MS-222

Ross, Linda G., BSc, PhD and Barbera Ross, BSc, PhD. Anesthetic and Sedative Techniques for Aquatic Animals. 2nd ed. Malden, MA: Blackwell Science, 1999.

Gourdon, J. "CARE 110.01 Fish and Aquatic Amphibian Anesthesia." Cornell Center for Animal Resources and Education, Cornell University, 2003.

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