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The effect of various substances on the microcirculation of blood in Rana sp. CENA, MJ., DIMABAYAO, BT., GARCERA, AAC., GUILLERMO, MLB., OMAR, AT.

Institute of Biology, College of Science, University of the Philippines, Diliman, Quezon City, 1101

ABSTRACT

Different factors have different effects on the vasomotion and bloodflow in the capillaries. In this experiment, the capillary blood flow in the hind foot of a live frog (Rana sp.) was observed under a dissecting microscope. The effect of temperature, and two groups (Group A and Group B) of substances were investigated. Group A substances included nicotine, adrenaline chloride, and pain, while Group B included lactic acid, histamine acid phosphate, acetylcholine bromide, absolute or 95%, ethanol and sodium bromide. Cold temperature as well as the addition of Group A substances caused vasoconstriction (slower blood flow) while the converse (increased blood flow) occurred with the application of warm temperature and Group B substances. Vasomotion coupled with a change in the rate of blood flow occurs in response to an intrinsic control from tissues and organs, such in the presence of stimulants (nicotine), hormones (adrenaline chloride), or in response to differing temperatures, as a means to conserve (during cold temperatures) or dissipate (during warm temperatures) heat. It may also occur in response to allergic reactions (histamine), metabolic needs (lactic acid), mechanical and chemical stimuli (ethanol), and changing osmolarities (NaBr). Also, it may be triggered by sympathetic and parasympathetic stimuli (acetylcholine bromide; pain). INTRODUCTION Unlike most invertebrates, higher animals rely on a closed circulatory system to facilitate the transport of substances over large distances, through many layers of cells. In these systems, blood is confined to vessels and is distinct from interstitial fluid (Campbell, 1996).

Different blood vessels are employed in shuttling cells and other substances through the body. Both arteries and veins are composed of three basic layers: the outermost tunica externa composed of loosely woven collagen fibers; the middle tunica media composed of circularly arranged smooth muscle and elastin fibers; and the innermost tunica intima consisting of a single layer of flattened endothelial cells (Porth, 2007). Arteries deliver blood to tissues and by way of a thickened smooth muscle layer are designed to withstand the pressure of cardiac systole (Kent, 1992). Towards the receiving tissue, main arteries subdivide into smaller vessels called arterioles, which in turn branch into smaller metarterioles that deliver blood to the capillary bed (Randall, 2002). After exchanging its contents with that of

interstitial fluid, blood passes on to venules and larger veins which direct the blood back to the heart. Veins need not cope with as much blood pressure as arteries; hence contain less smooth muscle and elastic fibers (Kent, 1992).

Capillaries are essential blood vessels that collectively offer a tremendous surface area for the exchange of gases, nutrients, wastes, hormones and other substances between blood and tissue (Starr, 2005). Capillaries are arranged into extensive networks called capillary beds that infiltrate each tissue (Campbell, 1996). At this level, the exchange of material between capillary and the surrounding cells occurs mainly through diffusion. However, because of the slow nature of this process, almost all living cells must be in close proximity to at least one capillary (Starr, 2005). Unlike arteries and veins, capillaries lack smooth muscle and connective tissue. To facilitate diffusion, capillary walls are thin, consisting of a single layer of endothelial cells surrounded by a collagenous basement membrane. (Randall, 2002).

Capillaries are capable of carrying up to 14% of an animal’s total blood volume. However, it is not practical for all capillaries to

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be open all at once. Instead, animals adjust capillary blood flow according to the demands of their tissues (Randall, 2002). This regulation of microcirculation can either be under neuronal or local control. Often, the purpose of neuronal control is to maintain circulation in organs that require a constant supply of blood such as the brain and heart. This is accomplished by reducing blood flow to less vital tissues. Most arterioles are innervated by sympathetic or parasympathetic nerves. Stimulation of these nerves causes them to release substances like norepinephrine or acetylcholine that lead to the contraction (vasoconstriction) or expansion (vasodilation) of blood vessels (Randall, 2002). The local control of blood flow is equally important in servicing the demands of tissues. Indeed, vascular smooth muscles respond to numerous stimuli applied in the vicinity of tissues in question. For instance, vasoconstriction or vasodilation may be induced by mechanical stress or temperature flux. Changes in the chemical environment due to metabolism of tissue, release of catecholamines and inflammatory mediators, as well material transfer across the endothelium also serve to affect capillary blood flow (Fung, 1997; Randall, 2002). In this study, the thin webbing between the toes of frogs was chosen for ease of observing the blood vessels within this tissue.

The objectives of this study are to observe blood flow through the capillaries present in the webs of frogs, as well as to determine whether various chemical, physical, and temperature stimuli elicit constriction or dilation in these vessels.

MATERIALS AND METHODS A live frog (Rana sp.) was used to determine the effects of different test solutions on the vasomotion of blood capillaries. The frog was wrapped with a damp cloth and was secured in a frog board with its ventral side down using strings. The toes of one hindfoot

were spread across one of the holes in the frog board, in order to allow the passage of light through the thin webbing between the toes, and was pegged with pins. Blood flow was observed in the foot using a dissecting microscope. One to two drops of different substances were dropped onto the foot where subsequent changes in blood flow and capillary activity were recorded. Solutions used were warm Ringer’s solution at 40°C, cold Ringer’s at 8°C, Group A substances (nicotine, adrenaline chloride, pain) and Group B substances (lactic acid, histamine acid phosphate, acetylcholine bromide, 95% ethanol, sodium bromide). Pain was applied by lightly scratching the membrane of the foot being observed using a needle. Observation was done over a period of 3-5 minutes after exposure to a test substance to allow reagent to diffuse thoroughly within the tissues. Test solutions from Group A and B were applied alternately. In between applications, the foot was rinsed and flushed with Ringers’ solution. RESULTS

Table 1 shows the changes observed on the frog capillary blood flow upon application of various test solutions. Group A test solutions had a general effect of vasoconstriction on the capillary which was manifested by the slowing down of blood flow. On the other hand, Group B solutions caused vasodilation wherein an increase in the speed of blood flow was seen. Similarly, the application of warm Ringer’s solution resulted to vasodilation while the cold Ringer’s showed an opposite effect. Table 1. Observed effects of test solutions to blood flow.

Experimental Procedure

Observations

Warm Ringer’s at 40°C

Vasodilation, fast blood flow

Cold Ringer’s at 8°C

Vasoconstriction, slow blood flow

GROUP A

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Nicotine Vasoconstriction, slow blood flow

Adrenaline Chloride

Vasoconstriction, slow blood flow

Pain Vasoconstriction, slow blood flow during first few seconds; Blood flow eventually stopped

GROUP B Lactic Acid Vasodilation, fast blood

flow Histamine Acid Phosphate

Vasodilation, fast blood flow

Acetylcholine Bromide

Vasodilation, fast blood flow

Absolute or 95% Ethanol

Vasodilation, fast blood flow

Sodium Bromide Vasodilation, fast blood flow

DISCUSSION The endothelial lining of small blood vessels is free from any adhering blood cells. It very small diameter allows only a single blood cell to pass though at any given time. Arterioles, capillaries and venules may be hard to distinguish from each other but by looking closely at the rate of blood flow in them, the differences can be easily established. The aorta pumps blood with a very high pressure resulting to a fast flow of blood. But blood vessels progressively decrease in size so eventually the fast velocity of blood in the arteries slows down as brought about by the limited space by which they can move in to. This slowing down is progressively increased as they enter the capillary network. However, the total resistance of all the capillaries is less than that of the arterioles since though they are very narrow (about 5-10µ in diameter), they have a huge cross-sectional area. As the blood enters the venules and eventually the veins, its velocity increases due to the decreasing cross sectional area. The small diameter of capillaries do not allow for any blood cell to adhere to its endothelium. Capillaries can also be easily distinguished since most appear as “ghost

cells” wherein there are no blood cells flowing through. The precapillary sphincter in the arterioles is able to regulate whether blood should flow through the capillary or not. Most of the time, blood flows to the organs where it is most needed. For example, blood flow is directed more to the digestive system after a meal (Vander, et al. 2001; Abramson 1962). Starling Forces The processes occurring across the capillaries are not just diffusion of gases or nutrient but also the bulk-flow of protein plasma which functions for the distribution of extracellular fluid comprising the blood plasma and interstitial fluid. This movement is important to the capillaries for it maintains the continuous exchange of nutrients and gases particularly the transfer of O2 and CO2. In 1896, Ernest Starling hypothesized that this fluid movement across the capillary wall is dependent on the balance of two gradients: hydrostatic pressure gradient and colloid pressure gradient. Hydrostatic pressure, the pressure exerted by a fluid in a confined space causes the blood fluid to exert a force on the capillary wall resulting to the filtration of the fluid from the blood plasma into the interstitial space. Colloid osmotic pressure is the reverse – it causes the fluid from the interstitial space to move to the blood plasma. Since the blood plasma has a higher protein concentration, the fluid moves from the interstitial fluid into the blood plasma. At the arterial end of the capillaries, hydrostatic pressure is greater than colloidal osmotic pressure. This decreases as blood flows towards the venous end and more fluid is transferred from the blood plasma to the interstitial fluid. Towards the venous end, colloidal osmotic pressure overcomes hydrostatic pressure and this condition remains constant throughout (Vander, et al. 2001; Cliff 1976). Factors that Affect Vasomotion The three main factors that affect the movement of blood across the circulatory

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system are (1) pressure, (2) flow, and (3) resistance. In all cases, blood flows from a region of higher pressure to an area of lower pressure. The hydrostatic pressure of the cardiovascular system denotes the force exerted by the blood at the point in the system; this force being generated by the contraction of the muscular heart and varying in magnitude at different points in the circulatory system. Rather than the absolute pressure at any point, it is the difference in pressure between two points that determines the flow rate of blood (Vander et al. 2001; Mader 2004). While pressure difference is directly proportional to flow rate, resistance is inversely proportional to flow rate. Resistance is the measure of the friction that impedes blood flow and is determined by: (1) viscosity (η) – the friction between adjacent layers of a flowing fluid; (2) length (L); and (3) inner radius (r) of the tube through which the fluid is flowing. Equation 1 shows the relationship among the three factors affecting vasomotion while Equation 2 shows the contributions of various determinants to fluid resistance (Vander et al. 2001).

In Equation 2, it could be seen that resistance is directly proportional to both viscosity and the tube length; while it is inversely proportional to the fourth power of the tube radius. Under most physiological conditions, blood viscosity and tube length remain constant. On the other hand, tube radius is typically regulated to ultimately affect blood flow rate (Vander et al. 2001). Local Regulation of Blood Flow Other than the neurohumoral mechanisms that affect blood flow, tissues and organs have the intrinsic ability to regulate their own blood flow. This is called local regulation (Klabunde 2004).

Factors which affect blood flow in local regulation may originate from within the blood vessels (endothelial factors) or from the surrounding tissues (tissue factors). Moreover, mechanical factors (such as compressive forces during muscle contraction) can also influence vascular resistance to ultimately affect blood flow (Vander et al. 2001; Ganong 2003; Klabunde 2004) Effects of Temperature Studies have shown that responses to local heat is to dilate blood vessels so this excess heat can be dissipated to the environment while the reverse happens during times of cold temperature – blood vessels contract to minimize heat loss. A study on bat wings have shown that local heat causes endothelial shear stress leading to vasodilation (Widemer, et al., 2005). Vasodilators In this experiment, the action of vasodilator metabolites – lactic acid, acetylcholine, ethanol, and sodium bromide – have been experimentally demonstrated. The metabolic theory of blood flow regulation proposes that actively metabolizing cells surrounding arterioles release vasoactive substances which cause vasodilation. In this manner, it is ensured that the tissue is supplied by enough oxygen while by-products of metabolism are sufficiently removed. Lactic acid is one such metabolic by-product. During periods of intense metabolic activity, anaerobic respiration is activated to supplement aerobic metabolism; while producing lactic acid as a by-product. Lactic acid effectively increases H+ concentration. It is this low pH which causes vasodilation, particularly in the cerebral circulation (Klabunde 2004). Other tissue factors affecting local regulation of blood flow are not coupled to metabolism. Such factors include bradykinin, eicosanoids, and histamine. Histamine is released by tissue mast cells in response to injury, inflammation and allergic responses (Klabunder 2004). It causes local vasodilation

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to increase blood flow to the affected area; this increased blood flow recruits cells of the immune system to the site of infection to deal with infectious agents (Goldsby et al. 2000).

It has been experimentally found that ethanol induces vasodilation in frogs. In a study of the effect of ethanol on human vascular function, on the other hand, responses elicited by ethanol application varied. Apparently, alcohol induces vasoconstriction at rest, while it augments endothelium-dependent vasodilation via a mechanism that does not involve nitric oxide. Ethanol has been found to decrease the synthesis of endothelin-1 (Creager 2004). Endothelin-1 (ET-1) is an important vasoconstrictor paracrine agent released by endothelial cells in response to certain mechanical and chemical stimuli. In some cases, ET-1 may be present in high enough concentrations, to serve as a hormone, causing widespread arteriolar vasoconstriction (Vander et al. 2001).

Osmolarity changes in the blood have also been implicated in vasodilation. Evidently, intra-arterial infusions of hyperosmolar solutions induced vasodilation; even if the molecules making up the solution were not by themselves vasoactive. The salt NaBr used in this experiment may perhaps have caused vasodilation via increasing the osmolarity of the tissue interstitium. This vasodilatory response to osmolarity may have arisen from the fact that during metabolic activity, the osmolarity of interstitial fluid and venous blood is significantly increased.

Tissue injury also causes vasodilation since a variety of substances that makes the arteriolar smooth muscle relax are released locally from the cells or generated by plasma precursors. These mediators also cause local capillaries and venules to become permeable to protein by contracting their endothelial walls thus allowing proteins to move through the open spaces (Vander et al., 2001). The constriction observed in the experiment resulted from excessive damage to the frog foot web leading to eventual blood clotting.

Besides the intrinsic control initiated by tissues and organs, blood flow is also regulated by extrinsic controls such as the sympathetic and the parasympathetic nervous system. Most arterioles are innervated by sympathetic nerves whose terminals release norepinephrine. This metabolite binds to a-adrenoreceptors in the smooth muscle of arterioles to cause vasoconstriction. Some arterioles, on the other hand, are innervated by parasympathetic nerves whose terminals release acetylcholine. Acetylcholine has been found to activate nitric oxide synthase and prostaglandin production – metabolites acting as potent vasodilators (Kellogg et al. 2005). Both parasympathetic and sympathetic systems act to ensure that not all capillaries are open at a particular time; otherwise, a drop in arterial pressure results in reduced blood flow to the brain (Randall et al. 2001). Vasocontrictors

Vasoconstriction is brought about by the binding of α-adrenergic receptors to norepinephrine, which is released by the sympathetic nerve fibers innervating most of the arterioles. Epinephrine, angiotensin II and vasopressin also cause the constriction of the smooth muscles of blood vessels (Vander, et al., 2001).

In the experiment, group A – nicotine, adrenaline chloride and a painful stimulus was found to cause vasoconstriction in the microcirculatory network observed under the microscope. Nicotine is a known vasoconstrictor but several studies have shown conflicting results. Nicotine stimulates the sympathetic ganglia, adrenal medulla, sympathetic nerve endings and chromaffin tissues to release catecholamines. Various organs respond differently to nicotine – sometimes in a paradoxical manner reflecting the relative importance of constrictor and dilator receptors, their sensitivity to nicotine and the amount of pre-existing muscle tone. Some tissues respond in divergent manners depending on the route of nicotine administration and dose (Johnson et al., 1991).

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Gastrocnemius muscle in cat tested for the vasomotor effects of nicotine reflected a biphasic response wherein high doses resulted to vasoconstriction but small doses resulted to vasodilation (Hilton 1954). There were also cases wherein nicotine caused an increase in blood flow to the tissue being studied (Johnson et al., 1991). But in the human skin nicotine has been found to amplify norepinephrine(NE)-induced vasoconstriction and impair endothelium-dependent vasorelaxation (Black 2001). Johnson et al. (1991) speculated that nicotine causes irritation to the tissue in small doses and thus leads to an inflammatory response (Johnson, et al., 1991).

Another factor observed was adrenaline, also known as epinephrine. Epinephrine is released from the sympathetic nerves and binds to α-adrenergic receptors in arteriolar smooth muscles in as much the same manner as norepinephrine. However, there are also β-adrenergic receptors in the smooth muscle cells to which epinephrine could bind to leading to vasodilation. But since there are more α-adrenergic receptors, vasoconstriction is most readily observed. The case is different for the arterioles in skeletal muscles which experiences more vasodilation since β-adrenergic receptors are more numerous than α-adrenergic receptors (Vander, et al. 2001). Literature Cited

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