MICROCIRCULATION IN FROG TISSUE AND LOCAL BLOOD FLOW CONTROL IN

RESPONSE TO CHEMICAL AND THERMAL STIMULI

Carandang | Cruz | Pasumbal | Salem | Tolentino
Institute of Biology, University of the Philippines Diliman, Diliman, Quezon City

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ABSTRACT

Microcirculation and the effect of different vasoconstrictors, vasodilators, temperature, and pain to the
blood flow within the capillary system were observed in frog tissue using a light microscope. A frog
specimen was fixed in place and the foot was held securely under a light microscope. The thin webbed
tissue between the phalanges was spread over a glass slide to monitor the blood flow inside the
capillaries. Each of the reagents were added alternately between vasoconstrictors and vasodilators and the
diameter of the capillaries and blood flow were monitored. Infliction of pain, subjecting the tissue to
colder Ringer’s solution, and treatment with nicotine and adrenaline resulted in constriction of the
vessels. Exposure to histamine acid phosphate, acetylcholine, ethanol, lactic acid, sodium bromide and
warm Ringer’s solution displayed dilatory effects to the vessel.
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INTRODUCTION
In animals larger than 1mm in diameter, a
circulatory system for the material transport is
necessary. This system has evolved to transport
respiratory gases, nutrients, waste products,
hormones, antibodies, salts and other materials
among various regions of the body. The basic
plan for this system consists of a main
propulsive organ, the heart in most cases, which
propels blood to various parts of the body via an
arterial system and back to the heart via a
venous system (Randall et al., 2002).
Material transfer among tissues are facilitated by
capillaries, these are fed by arterioles which
branch from arteries and feed into venules which
subsequently flow into veins. The arterioles,
capillaries, and venules are collectively referred
to as the microcirculation because they are only
visible microscopically (Sherwood et al., 2013).
The walls of these vessels are thin, thus allowing
high rates of transfer between tissues by
diffusion, transport or filtration (Randall et al.,
2002).
Microcirculation blood flow is regulated by
vasoconstriction or vasodilation in response to
stimuli. Electrical signals initiated on
microvascular smooth muscle and endothelial
cells can travel rapidly for millimeters through
cell-to-cell conduction via gap junction
channels, rapidly coordinating vasodilator
responses that govern the distribution and
magnitude of blood flow (Segal, 2005).
This experiment aims to observe
microcirculation by observing frog webs under
varying conditions microscopically. These
conditions are created by exposing frog skin to
solutions containing different compounds and
observing the frog's reaction to the stimuli.
MATERIAL AND METHODS
To observe the effect of the different
vasoconstrictor and vasodilator on the capillaries
of the frog sample using a light microscope, it
was prepared by restraining the sample with a
plastic rope.

The vasodilators which are composed of
histamine, acetylcholine, ethanol, lactic acid
sodium bromide and warm ringer solution were
tested in sequential tandem with its
vasoconstrictor counterparts which are Nicotine,
Adrenaline, pain infliction and cold ringer
solution. The effect on the blood flow, the
volume of blood that passes and the diameter of
the vessel were then observed using a light
microscope.
RESULTS
When viewed using a light microscope under
low power objective (LPO), the transparent
blood vessels were distinguished as
anastomosing pathways with transparent disc-
shaped translucent erythrocytes. Capillaries
were observed as very thin pathways with a very
small diameter that only one cell of erythrocyte
can pass through. Arterioles and venules were
much larger than capillaries and have a faint red
translucent color. The erythrocytes flowing
through the vessels were much harder to monitor
since three to four cells can pass through the
diameter. Venules were detected based on the
flow of blood cells - the venules receive the
blood from numerous branches of capillaries.
The arterioles were indicated as the sources of
the blood cells. The blood flow originated from
arterioles to capillaries and then to venules.
The blood flow was observed to be fastest in the
arterioles. When the capillaries receive the blood
cells from the arterioles, the rate of blood flow is
greatly reduced. The blood collected in the
venules flowed in a very slow rate.
The flow of the blood cells were generally
smooth, but cycles of fast flow followed by a
slower flow were observed. The cycle of
differing rates were observed to be pulsating.
The erythrocytes were generally flexible and can
rotate, slightly bend, and can stack with other
cells in a slanted manner as the cells passed
through the small capillaries. Only erythrocytes
were observed in the blood vessels since the
cells have a faint red color especially when in
larger blood vessels; no leukocytes were located.
Leukocytes may be possibly located in sites of
inflammation or lymphatic tissues. Vasomotion
was not observed.
Blood vessel diameter and blood flow rate
changes were observed when different
substances were added to the tissue as shown by
Table 1. The general trend observed was that an
increase in blood vessel diameter also increase
blood flow rate in the capillaries. Acetylcholine,
histamine, lactic acid, ethanol, and sodium
bromide induced an increase in blood vessel
diameter and blood flow rate. Warm
temperatures also caused the dilation of
capillaries. Other substances such as adrenaline
and nicotine caused the constriction of
capillaries. Cold temperatures and infliction of
pain also caused the constriction of capillaries.
Table 1. Substances immersed to the web foot
tissue of a frog sample and the effect on blood
flow rate and blood vessel diameter.
Effect on the
Substance or Effect on blood
diameter of
stimulus flow rate
blood vessel
Nicotine slower smaller
Adrenaline slower smaller
Pain infliction slower smaller
Cold Ringer’s temporarily
smaller
solution stopped, slow
 capillary wall, which tends to increase towards
Histamine acid
faster larger the venous end. The net loss of fluid at the
phosphate
arterial end is somewhat greater than the net
uptake at the venous end of the capillary. In the
Acetylcholine faster larger
arterial end of the capillary bed, the hydrostatic
pressure is stronger than the colloid osmotic
temporarily
pressure. This leads to a net loss of fluid as it
Ethanol stopped, larger
passes through the capillary. However, the
slightly faster
efflux of fluid leads to a loss in hydrostatic
slow, then pressure. On the venous end, the decrease in
Lactic Acid larger
normal hydrostatic pressure becomes too great such that
Sodium temporarily osmotic colloid pressure draws fluid back in.
larger Thus, the accumulation of fluid in the
Bromide stopped, faster
extracellular matrix is reduced. (Randall et al.,
Warm Ringer’s temporarily 2002).
larger
solution stopped, faster
Blood pressure is one of the factors that affect
the vasomotion of blood vessels. The other two
DISCUSSION are blood flow rate and resistance. Flow rate is
the measurement of how far blood can go over a
Blood vessels are composed of a single layer of certain period of time. This is directly
flat, epithelial cells known as the endothelium. proportional to blood pressure wherein the
This layer is present in arteries, veins, and higher the pressure the faster blood will flow.
capillaries, each of them having lumen wherein Both are inversely proportional to volume where
blood flows through. The diameter of the lumen the larger the volume the lower the flow rate and
in a capillary is small enough to arrange the pressure would be. This applies when blood
blood cells that pass through it in a single line. moves from artery to arteriole to capillary
This is necessary in order to effectively provide because the cross-sectional area of the capillary
exchange between the red blood cells and the bed is much larger than that of the arteriole,
somatic cells. Arteries have thicker walls to be causing blood to move slower. It will speed up
able to withstand high blood pressure coming again when crossing the venules since they
from the aorta, in which it transports oxygenated would have less cross-sectional area than the
blood to the organs. Veins possess thinner walls capillaries (Reece et al., 2014).
and larger diameters compared to arteries
because there is less blood pressure when Resistance is defined as any factor that can slow
delivering deoxygenated blood from the organs the flow of blood, therefore it is said to be
back to the heart. They also have valves to inversely proportional to flow rate. Viscosity
prevent backflow of blood (Reece et al., 2014). and friction are some factors than can be
counted as resistance. In order to lessen
The Starling Hypothesis states that the net resistance, the inner lining of the endothelium is
movement of fluid at any point along the usually smooth to avoid friction between the
capillary is determined by two factors: the cells and the wall (Reece et al., 2014).
difference between blood pressure and colloidal
osmotic pressure, and the permeability of the
The arterial muscle contraction can be explained
by vasomotion which is the response to different
stimuli. Examination of the response of the
microcirculatory system of the frog’s webbing to
different reagents showed that temperature, and
certain vasomotion stimulants can alter the rate
of blood flow, diameter of the vessels, or the
direction of blood flow.
Two sets of reagents were used to determine
their respective effects on blood flow and
vasomotion. Group A is composed of nicotine,
adrenaline, cold Ringer’s solution and pain
infliction, while acetylcholine chloride,
histamine, sodium bromide, ethanol, lactic acid,
and warm Ringer’s solution were classified
under group B. Theoretically, group A are
considered as vasoconstrictors while those in
group B are classified as vasodilators, and these
coincide with the experimental data gathered
where slower blood flow and smaller blood
vessels was observed in the former while the
reverse was seen in the latter.
Vasoconstrictors
Nicotine is usually known as a stimulant that
causes the heart to pump faster, leading to an
increase in blood pressure and vasoconstriction.
However, it is observed in some cases that it
also induces vasodilation in skeletal muscles.
Therefore, it can be said that nicotine has both
cholinergic and adrenergic in nature depending
on the dosage and the environment. Higher
concentrations would lead to vasodilation while
lower amounts would cause vasoconstriction
(Hilton, 1954; Black et al., 2001). For this
experiment the compound induced
vasoconstriction in the capillaries of the frog
which led to slower blood flow.
Adrenaline is a hormone produced by the
adrenal medulla and functions primarily by
eliciting a sympathetic response to the body by
constriction of minute blood vessels by binding
with ⍺-adrenergic receptors. It can also cause
vasodilation in other blood vessels like those in
the liver and the heart by binding with another
type of receptors, the 𝛽-adrenergic receptors.
(Rogers, 2015). Vasoconstriction was observed
in this experiment since the capillaries are the
ones subjected to this hormone.
Infliction of pain will cause the body to elicit a
fight or flight response, which is under the
sympathetic nervous system (Chawla & Kochar,
1999). Epinergic hormones would increase the
blood pressure and lead to vasoconstriction in
the capillaries in order to let more nutrients enter
the cells which is needed to have energy to
either fight the threat or flee from the scene as
seen in nature.
Vasodilators
Histamine is a hormone that elicits an
inflammatory response when the body gets
injured by an external force. It results to the
vasodilation of blood vessels, making their
endothelium more porous for the leukocytes and
components of the blood to flow to the site of
injury, preventing bacteria and other unwanted
materials from infecting the cells. This response
often causes swelling and redness since the
blood would be stuck or clumped together due to
the dilation of the blood vessel (Suzuki et al.,
1999).
Acetylcholine is the primary transmitter emitted
by cholinergic nerves which leads to the
constriction of the blood vessels. Sympathetic
cholinergic fibers would induce vasoconstriction
of coronary vessels in the heart while
parasympathetic cholinergic nerves would
innervate skeletal muscles in dogs and cats to
constrict in order to decrease blood pressure
(Klabunde, 2016).
Ethanol when initially taken would cause the
constriction of blood vessels, but would
eventually result to vasodilation. Previous
assumptions said that ethanol induces
vasodilation by increasing the bioavailability of
nitric oxide, however this was disputed by recent
findings in research (Tawakol et al., 2004).
Nitric oxide can either cause direct vasodilation
wherein it binds to receptors on the blood vessel,
or by indirect means by inhibiting compounds
that promote vasoconstriction like angiotensin II
(Klabunde, 2016).
A muscle at work would need oxygen to
continuously function aerobically. When this
activity is prolonged in that anaerobic
respiration took over, lactic acid would build up.
This would result to a decrease in pH which
would elicit the blood vessel to dilate in order to
increase its blood flow. This effect would
increase the amount of oxygen that would be
transported along the bloodstream, which is
needed to pay for the oxygen debt to convert
lactic acid produced from anaerobic respiration
to pyruvate (Klabunde, 2007).
Osmolarity also plays an important role in blood
vessels. If the blood vessel osmolarity is greater
than that of the interstitial fluid, water would
normally flow into the cells since the blood is
hypertonic compared to the outside fluid. In
order to prevent the further increase of blood
osmolarity, the body would secrete hormones in
order to promote vasodilation that would
increase the permeability of the endothelium,
thus letting some of the water exit the
bloodstream and into the cell. This mechanism is
usually seen when urine is concentrated with
salts such as sodium bromide, wherein the body
would promote vasodilation by secreting a
hormone called the anti-diuretic hormone that
would reabsorb water into the cells (Reece et al.,
2014).
Effect of Temperature
Higher temperatures would result to the
vasodilation of blood vessels while
vasoconstriction would occur in colder
temperatures. The former is for the dissipation
of heat due to the larger surface area of the
endothelium, while the latter is for conservation
of heat by lowering its surface area (Li et al.,
2009).
CONCLUSION
Microcirculation of the blood in the vessels with
respect to the addition of vasoconstrictor and
vasodilator significantly changed the blood flow,
diameter of the blood vessel and the volume of
blood that passes to it. Results obtained adhered
to the theoretical expectations where the group
of the vasoconstrictor decreases the diameter of
the vessels, blood flow and blood volume.
Similarly, vasodilators increases the diameter of
the blood vessels, the blood flowing and the
volume that passes. It should be noted however
that among the vasoconstrictors added, the warm
ringer solution temporarily stopped blood flow
while for the vasodilators, ethanol, warm ringer
solution and sodium bromide momentarily
stopped the blood flow.
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