Practical Physiology Gm 2023 1 Часть ! -12 30 Марта-1

FEDERAL STATE BUDGETARY EDUCATIONAL INSTITUTION OF HIGHER
EDUCATION
«BASHKIR STATE MEDICAL UNIVERSITY»
OF THE MINISTRY OF HEALTHCARE OF RUSSIA
DEPARTMENT OF NORMAL PHYSIOLOGY
PRACTICAL PHYSIOLOGY
Education Guidance for Students of General Medicine Faculty
Part I
Physiology of Blood, Excitable Tissues, Muscles, CNS, Cardio-Vascular System
A.F. Kayumova, O.S. Kiseleva, K.R. Ziyakaeva, I.R. Gabdulkhakova
UFA 2023
УДК 612.8
ББК 28.903я73
G 36
Printed by the decision of ―Federal State Budgetary Educational Institution of Higher Education
BSMU‖ (document № ______ 2023)
Practical Physiology. Part I. Physiology of blood, excitable tissues, muscles, CNS, cardio-
vascular system: education guidance for students of General Medicine faculty /A.F. Kayumova,
O.S. Kiseleva, K.R. Ziyakaeva., I.R. Gabdulkhakova- Ufa: BSMU, 2023. 118 pages.
The Practical Physiology was compiled on the basis of the Federal State Educational Standard of
Higher Education 3 ++, in the specialty 31.05.01.–General Medicine (2021), Main Educational
Program and in accordance with the requirements of the work program on discipline Normal
Physiology (2021), the current curriculum of students of the faculty of General Medicine.
The Practical Physiology contains the techniques and theory to master universal competencies UC-1,
general professional competencies GPC-4, GPC-5. It presents the physiology of blood, excitable
tissues, muscles, CNS, cardio-vascular system. The manual contains the techniques and theory
necessary for the study of students of the faculty of General Medicine.
Illustrations are taken from open Internet resources.
For English speaking students of General Medicine faculty.
Recommended for publication by the Coordination Scientific and Methodological Council and
approved by the decision of the Editorial and Publishing Council of the FSBEI HE BSMU of the
Ministry of Health of Russia work.
УДК 612.8
ББК 28.903я73
G 36
© Kayumova A.F., Kiseleva O.S.,
Ziyakaeva K.R, Gabdulkhakova I.R.
FSBE I of HE
© Bashkir State Medical University, 2023
2
CONTENTS
Introduction………………………………………………………….………..…………...……6
THEME 1. Physiology of Blood.………………………………….…………………….…...…8
Lesson 1. Composition of the Blood. The Formed Elements of Blood. Hemoglobin. Estimation of
hemoglobin content. Color index. Reproduction of haemolysis. Erythrocyte sedimentation
rate…………………………………………….…………………………………………..…….8
Exercise 1.Sampling of Blood.………………………………….………..…………..……..…18
Exercise 2.Hematocrit Value.………………………………………..………..…..…………...19
Exercise 3. Color Index.………………………………………………..…….…………….….21
Exercise 4.Hemolysis.…………………………………………………....……………………..21
Exercise 5.Erythrocyte Sedimentation Rate.……………………………….…………………..23
Lesson 2. Blood groups. Rhesus factor. Thrombocytes. Retraction and Fibrinolysis. Blood
Coagulation Regulation. Anti-coagulation System.…………..………………………….…..…25
Exercise 1. ABO Blood Group. ………………………………………………………………..30
Exercise 2.Rh factor.……………………………………………………….…………………...32
Exercise 3. Bleeding Time .……………………………………………………………….……33
Exercise 4.Coagulation Time.…………………………….………………..……………….. …34
THEME2. Neuro-muscular Physiology...................................................................................35
Lesson 1. The Basic Physiological Properties of Living Tissues. Action
of Stimulus on Tissues. Laws of Irritation. .................................................................................35
Exercise 1.Stimulus Action on Neuro-muscular Preparation. ....................................................38
Exercise 2.Direct and Indirect Irritation of Muscles. Comparison of Excitability of Nerve and
Muscle. ....................................................................................................................................... 39
Lesson 2. Bioelectrical Phenomenon of Excitation Tissues. Membrane Potential.
Action Potential. .........................................................................................................................40
Exercise1. Galvani’s Test of Contracting with Metal. ...............................................................44
Exercise2. Galvani’s Test of Contracting without Metal .......................................................... 45
Exercise3. The Matteuchee’s Test (Secondary Muscle Contracting-
Secondary Tetanus ..................................................................................................................... 45
Lesson 3. Physiology of Nerve Fibres and Synapses. ............................................................... 46
Exercise 1. Electrical Phenomena in Nerve Fibres. ................................................................... 49
Exercise 2. Impulse is Propagated Along a Nerve in Both Directions. ..................................... 50
Exercise3. Physiological Entirety of Nervous Fiber. Parabiosis ................................................51
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Lesson 4. Physiology of Muscles. ............................................................................................. 52
Exercise 1. Kinds of Muscular Contraction. .............................................................................54
1.1. Record and the analysis of single contraction and tetanus of a muscle ………………….54
1.2. Theoretical calculation of frequency of irritations for reception of various kinds of muscular
contractions ............................................................................................................................... 56
Exercise 2. Optimum and Pessimum of Frequencies of Irritation. ........................................... 57
Exercise 3. Dynamometry. ........................................................................................................58
Exercise 3.1 Determination of Muscle Force ............................................................................58
THEME 3. Common Physiology of Central Nervous System ............................................. 60
Lesson 1.General Characteristics of Reflex Action. ................................................................ 60
Exercise 1. Tendon Reflexes ......................................................................................................62
Exercise 2.Analysis of the Spinal Shock in a Frog. .................................................................. 65
Exercise 3.Analysis of the Spinal Reflexes in a Frog. .............................................................. 66
Exercise 4. Analysis of the Arch Reflex in a Frog .....................................................................66
Lesson 2.Properties of Nerve Centers. Inhibition…...................................................................67
Exercise 1.Temporal Summation of EPSP in Motoneurons. .....................................................71
Exercise 2.Mesurement of Reflex Time by Turk’s Method. .....................................................71
Exercise 3.Irradiation of Excitation. .................................................................…………….....72
Exercise 4.Sechenov’s Inhibition of Reflex. ............................................................................ 72
Exercise 5.Golz’ Reciprocal Inhibition of Spinal Reflex. .........................................................74
THEME4. Cardio-Vascular System.………………………..………..………………….……...75
Lesson 1. Physiology Properties of Cardiac Muscle.…………………..………………..……..75
Exercise 1. Study of Cardiac Cycle and Autonomy of Different
Parts of Frog’s Heart. Stannius’s Ligatures. ………………….……………………………....79
Exercise 2. Determination of Duration of a Cardiac Cycle…….…………………..…………81
Exercise 3.Features of excitability of the heart. Extrasystole……………………….…………81
Lesson 2. Regulation of heart activity……………………………………………..…..….……82
Exercise 1. The Effect of Vagosympathetic Trunk Stimulation on
Frog's Heart and Phenomenon of Vagal Escape. ……………………….………………….….87
Exercise 2.Reflexes of Heart (Golz’s Reflexes). ………………………………………..……89
Exercise 3.Danyny-Ashner’sOculocardiac Reflex. ………………….……………………….90
Exercise 4. Effect of Some Factors (Temperature,
Electrolytes, Hormones, Mediators) on Frog's Heart. ………………………….…………….91
4
Lesson 3. Modern Investigation Methods of Heart Work. …………………………………...92
Exercise 1. Electrocardiography …………………………………….……………..…….…..99
Exercise 2.Phonocardiography …………………………………….……………………......101
Lesson 4. Physiology of Circulation. Arterial pressure. Arterial Pulse...................................101
Exercise 1. Recording of Blood Pressure. …………………………………………………...108
Exercise 2. Examination of Arterial Pulse. …………………………….……………….……111
Test Tasks…………………………………………………………………………….……….114
Standard Answers…………………………………………………………………………….116
Recommended Literature…………………………………………………………….……….117
5
Introduction
The Practical Physiology was compiled on the basis of the Federal State Educational Standard of
Higher Education 3 ++, in the specialty 31.05.01.–General Medicine (2021), Main Educational
Program and in accordance with the requirements of the work program on discipline Normal
Physiology (2021), the current curriculum of students of the faculty of General Medicine.
The Practical Physiology contains the techniques and theory to master universal competencies UC-1,
general professional competencies - ability to carry out a critical analysis of problem situations
based on a systematic approach, to develop an action strategy; GPC-4 - ability to use medical
devices provided for by the procedure for the provision of medical care, as well as conduct
examinations of a patient in order to establish a diagnosis; GPC-5 - the ability to assess
morphological ad functional states and pathological processes in the human body for solving
professional problems.
The aim of the course is to develop basic understanding of the functions of the body and their
applications in management of patients and to develop skills in assessing the functions of systems of
the body and basic clinical examination. At the end of the course the students should be able to:
- Describe the basic principles of homeostasis, water and electrolyte balance, acid base balance, and
temperature regulation.
- Describe the role of various systems of the body, how they function, the mechanisms that regulate
them and the factors that alter the functions.
- Describe the physiological basis of various tests used to assess the functions of these systems and
interpret the results obtained.
- Investigate blood for hemoglobin concentration, red blood cell count, white blood cell count,
bleeding time, clotting time, blood groups and Rh factor.
- Measure blood pressure, arterial pulse and recognize rate, regularity and volume of the pulse,
identify normal heart sounds, identify waves and intervals in normal ECG, record respiratory
movements.
- Having attained the knowledge and skills mentioned above, the student should view man as a
whole organism and not a collection of systems, apply the knowledge and skills in understanding and
managing patient problems and keep on continued study of physiology.
The teaching learning activities include lecture discussions, practical classes and tutorials
demonstrations. Lecture discussions will be delivered by the departmental staff where students are
informed of the topics well in time and are expected to read up based on the objectives given to them
at the beginning of the course as a book. Practical classes will be conducted in the laboratory with
6
the aim of developing basic clinical skills related to physiology and to demonstrate important
physiological principles. Tutorials will be in different forms such as free oral question-answer
sessions, answer writing sessions, sessions for students to clear their doubts and so on as requested
by the students. All these activities will be interactive encouraging student participation and
performance instead of simple delivery of information. In addition, video shows on functions of
various systems are shown to illustrate their structure and function.
Further, there will be formative evaluations at the end of or during the course of each section or
system. The marks of in-course assessments conducted at the end of each term will be given to
students and the answers will be discussed with the students. The students are given detailed
objectives for the course in physiology and guides for each practical class developed by the
department as teaching material.
7
THEME 1. PHYSIOLOGY OF BLOOD
Lesson 1.Composition of Blood. The Formed Elements of Blood. Hemoglobin. Estimation of
hemoglobin content. Color index. Reproduction of haemolysis. Erythrocyte sedimentation rate.
The questions for studying:

1. Composition of the Blood. Blood system and its parts. The functions of blood.
2. Blood plasma, its composition. Osmotic and oncotic pressure of blood. Acid-base
balance, its regulation. Specific gravity of blood. Blood viscosity.
3. Uniform elements of blood. Erythrocytes.
4. Leucocytes, kinds of leucocytes. Functions. Differential count of leucocytes.
5. Hemoglobin: structure, functions, types. Color index (C.I.)
6. Hemolysis. The types of hemolysis. Reproduction of hemolysis.
7. Erythrocyte sedimentation rate (ESR).Factors influence on erythrocyte sedimentation rate.
Blood is a specialized connective tissue in which there is liquid intercellular substance known as
plasma and formed elements suspended in plasma. The specific gravity of whole blood varies from
1055 to 1060.
The total blood volume in the average-sized adult is about 5 liters, constituting about 6-8% of
the total body weight. Blood leaving the heart is referred to as arterial blood. Arterial blood is bright
red because of high concentration of oxyhemoglobin (the combination of oxygen and hemoglobin)
in red blood cells. Venous blood is blood returning to the heart. Except for the venous blood from
the lungs, it contains less oxygen, and venous blood is therefore darker red than the oxygen-rich
arterial blood.
Blood is composed of a cellular portion, called as formed elements, and a fluid portion, called as
plasma. When a blood sample is centrifuged, the heavier formed elements are packed on the bottom
of the tube, leaving plasma at the top (fig. 1). The formed elements constitute approximately 45%
of the total blood volume (a measurement is called the hematocrit), and the plasma is 55%.
The cellular elements of the blood have a short life span and must be continuously replaced.
The formation of red blood cells, white blood cells, and platelets, collectively, is referred to as
hematopoiesis. This process takes place in the red bone marrow. In adults, red bone marrow is
found in the pelvis, ribs, and sternum.
Plasma is a straw-colored liquid consisting of water and dissolved solutes. The major solute of
the plasma in terms of its concentration is Na+. In addition to Na+, plasma contains many other ions, as
well as organic molecules such as metabolites, hormones, enzymes, antibodies, and other proteins.
8
Plasma Proteins.
Plasma proteins constitute 7% to 9%of the plasma. The three types of proteins are albumins,
globulins, and fibrinogen.
Albumins account for most (60% to 80%) of the plasma proteins. An important function of
albumins is to bind with various molecules in the blood and serve as a carrier protein , transporting
these substances throughout the circulation. Substances that bind with albumin include hormones;
amino acids; fatty acids; bile salts; and vitamins. Albumin also serves as an osmotic regulator.
Because capillary walls are impermeable to plasma proteins, these molecules exert a powerful
osmotic force on water in the blood. In fact, the plasma colloid osmotic pressure exerted by plasma
proteins is the only force that retains water within the vascular compartment and therefore maintains
blood volume. Albumin is synthesized in the liver. Globulins account for about 38% of plasma
proteins. The three types of globulins are alpha (), beta (), and gamma ().The alpha and beta
globulins are produced by the liver and involved with several activities. They transport substances
in the blood (hormones, cholesterol, iron), function as clotting factors, and serve as precursor
molecules (angiotensinogen). The gamma globulins function as antibodies, which play an important
role in the immune response. Alpha and beta globulins are synthesized in the liver; the gamma
globulins are made by the lymphocytes (a type of white blood cell).
Fibrinogen, which accounts for only about 4% of the total plasma proteins, is an important clotting
factor produced by the liver. During the process of clot formation, fibrinogen is converted into
insoluble threads of fibrin. Thus, the fluid from clotted blood, called serum, does not contain
fibrinogen, but it is otherwise identical to plasma (see Lesson 2).
Plasma Volume
A number of regulatory mechanisms in the body maintain homeostasis of the plasma volume. If the
body should lose water, the remaining plasma becomes excessively concentrated, its osmolality
increases. This is detected by osmoreceptors in the hypothalamus, resulting in a sensation of thirst
and the release of antidiuretic hormone (ADH) from the posterior pituitary. This hormone promotes
water retention by the kidneys, which together with increased intake of fluids helps to compensate for
the dehydration and lowered blood volume.
The formed elements of blood
The formed elements of blood include three types of blood cells: erythrocytes, or red blood
cells (RBC),leucocytes, or white blood cells (WBC) and thrombocytes or platelets (PLT) (fig. 1).
Blood contains of 4,5 - 5,5x1012 erythrocytes per liter in males and 3,8 - 4,5x1012 erythrocytes per
9
liter in females. The same volume of blood, by contrast, contains only 4 – 9x109 leucocytes per
liter. The average number of platelets is about 200 - 400x109 platelets per liter.
Figure 1.The composition of blood. Blood cells become packed at the bottom of the test tube when
whole blood is centrifuged, leaving the fluid plasma at the top of the tube. Red blood cells are the
most abundant of the blood cells, white blood cells and platelets form only a thin, light-colored
"buffy coat" at the interface between the packed red blood cells and the plasma.
Erythrocytes
Erythrocytes or red blood cells (RBC) are flattened, biconcave discs, about 7 µm in diameter and 2,2
µm in thick. This shape maximizes the surface area of the cell and facilitates the diffusion of oxygen
across the cell membrane. Furthermore, red blood cells are very flexible and easily change their
shape. This feature allows them to squeeze through capillaries as narrow as 3min diameter.
However, as the red blood cells age, their membranes become quite fragile and the cells are prone to
rupture. Each erythrocyte consists of 95%hemoglobin, approximately 280 million hemoglobin
molecules, which give blood its red color. Each hemoglobin molecule consists of four protein chains
called globins, each of which is bound to one hem, a red-pigmented molecule that contains iron.
The iron group of hem is able to combine with oxygen in the lungs and bring oxygen to the tissues.
This hemoglobin/oxygen-carrying capacity of the red blood cell is facilitated by lack of a nucleus
and any other membranous organelles within these cells.
Unique shape of RBCs relates to their function of transporting oxygen; it provides an increased
surface area through which gas can diffuse. Erythrocytes do not have nucleus and mitochondria’s
(they obtain energy through anaerobic respiration). Partly because of these deficiencies, erythrocytes
have a relatively short circulating life span on the average 60-90 days, maximum 120 days. Older
erythrocytes are removed from the circulation by phagocytic cells in the liver, spleen, and bone
10
marrow. As such, red blood cells must be replaced at a rate of 2 to 3 million cells per second.
Erythrocyte production is regulated by the hormone erythropoietin. Low levels of oxygen stimulate
release of erythropoietin from the kidneys into the blood.
Reticulocytes are newly produced red blood cells. They are slightly larger than totally mature
red blood cells, and have some residual ribosomal RNA. The presence of RNA allows a visible blue
stain to bind or, in the case of fluorescent dye, result in a different brightness. This allows them to be
detected and counted as a distinct population. The normal fraction of reticulocytes in the blood
depends on the clinical situation but is usually 0.5% to 2.5% in adults and 2% to 6% in infants.
Functions of erythrocytes.
1. Respiratory. Erythrocytes carry oxygen and carbon dioxide.
2. Acid – base balance. Erythrocytes help to maintain acid – base balance. It carried out by the
buffering action of hemoglobin and other intracellular buffers.
3. Ion balance. By the special permeability of the cell membrane, the red cells help to maintain
balance of positive and negative ions in the blood.
4. Erythrocytes help to maintain the viscosity of blood.
5. Various pigments are derived from hemoglobin after the disintegration of the red cells (bilirubin,
biliverdin, etc.).
Leucocytes
Leucocytes or white blood cells (WBCs) differ from erythrocytes in several aspects. Leucocytes
contain nuclei and mitochondria and can move in an amoeboid fashion. Because of their amoeboid
ability, leucocytes can squeeze through pores in capillary walls and move to a place of infection,
whereas erythrocytes usually remain confined within blood vessels. The movement of leucocytes
through capillary walls is referred to as diapedesis or extravasation.
White blood cells are classified according to their staining properties and morphology. The
leucocytes that have granules in their cytoplasm are called granular leucocytes; the leucocytes
without clearly visible granules are called agranular (no granular) leucocytes (fig. 2).
Granular leucocytes are formed in the bone marrow from the time of birth onwards. The
granules may take three different stains: neutral, acid and basic. Neutrophils have nucleus with 2-7
lobes and granules in the cytoplasm take neutral stain. After formation in the red bone marrow for
8-10 days, they enter the bloodstream, where they stay for 3-5 days and then are transferred to the
tissues, where they turn into microphages and, having performed their functions, die. Neutrophils
are the most important elements of nonspecific blood protection! The main functions of neutrophils:
1) phagocytosis
2) intracellular digestion
11
3) cytotoxic action
4) degranulation with the release of lysosomal enzymes
Basophils’ nucleus is lobed, the cytoplasm contains granules of various sizes and it takes deep
basic stain. After creation for 36 hours and leaving the bone marrow, basophils circulate in the
blood for only 6 hours, after which they migrate to tissues. There are 2 types of basophils:
circulating and tissue basophils or mast cells. The functions of basophils:
1) the formation of allergic reactions
2) maintaining blood flow and the growth of new capillaries
3) ensuring the migration of other leukocytes in tissues
4) phagocytosis. Basophils synthesize histamine, heparin, "platelet activating factor",
"eosinophilic anaphylaxis factor", which promotes the release of eosinophils from the vessels to the
place of accumulation of basophils. With an increase in the body's sensitivity to allergens, a "slow-
reacting substance of anaphylaxis" is formed in basophils, causing a spasm of smooth muscles.
Eosinophils’ nucleus is commonly 2-3 lobed. The granules are coarse and stain with acid
dyes. Eosinophils are formed in the red bone marrow for 34 hours, after which they enter the
bloodstream for 2-4 hours, from where they are sent to peripheral tissues: skin, mucous membranes
of the gastrointestinal tract, urinary tract. The functions of eosinophils:
1) reduce allergic reactions
2) create antiparasitic immunity
3) prevent the penetration of foreign antigens into the bloodstream.
In allergic reactions, eosinophils secrete substances - antagonists of heparin, histamine and
basophil anaphylaxis substances. They are able to phagocytose granules secreted by basophils. So,
eosinophils produce the enzyme histaminase, which destroys the substance histamine.
There are two types of agranular leucocytes: lymphocytes and monocytes. Monocytes are the
largest cells of leucocytes, diameter is 12-20 µm and generally have kidney- or horseshoe-shaped
nuclei. Monocytes are formed in the bone marrow and come out incompletely matured. The
average residence time in the blood is 36-104 hours. From the blood, monocytes are released into
tissues where they grow. Having reached maturity, monocytes turn into immobile cells -
histiocytes or tissue macrophages. Monocytes are the most important cellular factors of
nonspecific cell protection, due to their phagocytic and bactericidal activity. Functions of
monocytes:
1) phagocytic protection against microbial infection
2) antiparasitic protection
3) participation in the immune response and inflammation
12
4) tissue regeneration and antitumor protection
5) phagocytosis of old blood cells
The maximum activity of macrophages is manifested in an acidic environment, where
neutrophils lose their activity.
Lymphocytes are usually the second most numerous type of leucocytes; they are small cells
with round nuclei and little cytoplasm, their average size is 6-14 µm. Lymphocytes are the central
link of the immune system responsible for the formation of specific immunity. Lymphocytes are
able to distinguish between "self" and "foreign" in the body. Functions of lymphocytes:
1) synthesis of protective antibodies
2) lysis of foreign cells
3) provide a transplant rejection reaction
4) provide immune memory
5) destroy their own mutant cells
All lymphocytes are divided into T-lymphocytes, B-lymphocytes and O-lymphocytes. T-
lymphocytes from the bone marrow enter the thymus, where they are trained under the influence
of its hormones. Among T-lymphocytes, several types are distinguished. T-helpers - stimulate the
differentiation of B-lymphocytes, carrying out delayed-type hypersensitivity reactions. T-killers -
lyse foreign cells, participate in transplant rejection. T-suppressors - prevent the development of
autoimmune reactions by suppressing lymphocytes that can respond to the body's own antigens.
Immune memory T-cells - store information about all antigenic effects on the body, providing an
immune response upon repeated contact with the antigen. T-lymphocytes provide cellular
immunity!
B-lymphocytes are formed in the bone marrow, and differentiation takes place in the
lymphoid tissue of the intestine, appendix, palatine and pharyngeal tonsils. The main function of
lymphocytes is to create humoral immunity by producing antibodies.
O-lymphocytes do not undergo differentiation, but, if necessary, can turn into B- or T-
lymphocytes.
Determination of percentage of different kinds of leucocytes is known as the differential
count of white blood cells - leucoformula (tab. 1, fig. 2):
I. Granular leucocytes:
1. Neutrophils (polymorphonuclear) – 45-75 % (young forms 0 – 1 %, band neutrophils – 1 – 5
%, segmented – 50-65 %).
2. Eosinophils – 1 – 5 %.
3. Basophiles – 0 – 1 %.
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II. Agranular leucocytes:
1. Lymphocytes – 20- 40 %.
2. Monocytes – 2 – 10 %.
Table 1.
Significance of high and low white blood cells count
WBC type High count may indicate Low count may indicate
Neutrophils bacterial infection, burns, stress, radiation exposure, drug toxicity,
inflammation vitamin B12 deficiency, systemic lupus
erythematosus (SLE)
Eosinophils allergic reactions, parasitic infections, drugtoxicity, stress
autoimmune diseases
Basophils allergic reactions, leukemias, cancers, pregnancy, ovulation, stress,
hypothyroidism hyperthyroidism
Lymphocytes Viral infections, some leukemias prolonged illness, immunosuppression,
treatment with cortisol
Monocytes viral or fungal infections, tuberculosis, bone marrow suppression, treatment
some leukemias, other chronic with cortisol
diseases
a b
Figure 2.The formed elements of blood. a- The white blood cells depicted are granular leucocytes
(above); the lymphocytes and monocytes are no granular leucocytes. b -The reticulocytes are the cells
with the dark blue dots and curved linear structures (reticulum) in the cytoplasm.
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Automated hematology analyzer
Modern hematology analyzers are capable of counting as well as determining the size of
various circulating blood cells in blood, including RBCs, WBCs, and platelets (PLT). One such
instrument, the Beckman–Coulter analyzer, generates an electrical pulse when a blood cell passes
through the analyzer channel, which consists of a small aperture surrounded by electrodes. Each
electrical pulse represents an individual cell, and pulse height indicates the cell volume. Modern
hematology analyzers are also capable of multimodal assessment of cell size and cell count, thus
providing additional information regarding various categories of WBCs, such as neutrophils,
lymphocytes, monocytes, eosinophils, and basophils.
The following are examples of various channels in a hematology analyzer:
1) Channel for red cells (and also platelets): This channel is capable of analyzing red blood cells and
platelets.
2) Channel for WBC and hemoglobin measurement: Lytic agents lyse red cells first before analysis.
3) Channel for WBC differential count.
4) Channel for reticulocyte count.
5) Other channels: nucleated red blood cell (NRBC) channel, separate hemoglobin (HGB) channel,
WBC/basophil channel, and immature granulocyte channel.
Different hematology analyzers may use different methods for counting, including the
following (one analyzer may employ multiple methods):
1) Impedance
2) Conductivity measurements with high-frequency electromagnetic current (depends on the internal
structure, including nuclear cytoplasmic ratio and nuclear density to granularity ratio)
3) Light scatter
4) Fluorescence-based methods.
Nowadays different automated hematology analyzers provide reported results for blood cells
(tab. 2).
RBC Count and Hemoglobin Measurement
Typically, one channel is used to detect RBCs and platelets. The detector is set such that any
cell between 2 and 30 fL will be counted as a platelet, and any cell between 40 and 250 fL will be
counted as a red blood cell. If there are large platelets, these will be counted as red cells and will also
result in a falsely low platelet count. Similarly, if there are fragmented red cells, these smaller red
cells will be counted as platelets.
The mean cell (corpuscular) volume (MCV) indicates the volume of the ―average‖ red blood
cells (RBC) in a sample. This value is measured in microns cubic (µm²) or femtoliters (fL), where 1
15
µm² = 1 fL = 10-15L. MCV is increased in pernicious anemia and megaloblastic anemia. MCV is
decreased in iron deficiency anemia.
Present-day automated hematology analyzers provide a more accurate, direct measure of
MCV, based on the actual volume of the cell as it passes through a laser (laser-based hematologic
analyzers) or an electronic beam (―impedance‖ methods). The amount of laser light scattered in a
forward direction or the amplitude of pulses created in the electronic field as the cells through the
detector is equivalent to the cell volume, which is averaged based on the number of cells analyzed by
the instrument.
RBCs with the MCV above the reference interval are termed macrocytic. Conversely, red
blood cell populations with the MCV below the reference interval are termed microcytic. The RBC
in a blood smear may not be obviously macrocytic or microcytic, particularly if all RBC are affected
by the change.
Table 2
Normal values of formed elements.
Result Derivation Normal blood values
Hematocrit, HCT (%) Calculated Male: 40-48
HCT = (MCVxRBC)÷10 Female: 36-42%
Red blood cells concentration, Direct measurement Events that Male: 4,5-5,5 /L
RBC ( /L) pass through a laser beam Female:4,0-5,0 /L
Hemoglobin, HGB (g/L) Direct measurement: RBCs are Male: 130-160 g/L
lysed and hemoglobin is measured Female:
at a specific wavelength (540 nm) 120-150 g/L
Mean Cell Volume, MCV (fL) Calculated: 80-100 fL
MCV = (HCT ÷ RBC) x 10
Mean cell hemoglobin, MCH (pg) Calculated:MCH = 26-34 pg
(HGB x 10)÷RBC
Mean cell hemoglobin Calculated: 30-370 g/L
concentration, MCHC (g/L) MCHC = (HGB÷HCT) x 100
Red blood cell distribution width, Calculated:(Standard deviation ÷ 11,5-14,5%
RDW (%) mean) cell volume x 100
Platelet concentration Direct measurement 180-400 /L
(thrombocytes), PLT ( /L)
16
Mean Platelet Volume, MPV (fL) Direct measurement 7,5-12fL
White blood cells, WBC ( Direct measurement 4,0-9,0 /L
/L)
Granulocyte concentration, Direct measurement 1,2-6,8 /L
Gran /L)
Granulocyte concentration, Gran Direct measurement 47-72%
(%)
Neutrophils, NEUT (%) Direct measurement 50-65%
Eosinophil, EOS ( /L) Direct measurement 0,1-0,3 /L
Eosinophil, EOS (%) Direct measurement 1-4%
Basophils, BAS ( /L) Direct measurement 0,01-0,08 /L
Basophils, BAS (%) Direct measurement 0-1%
Lymphocyte concentration, LYM Direct measurement 1,2-3,0 /L
( /L)
Lymphocyte concentration, LYM Direct measurement 25-40%
(%)
Monocyte concentration, MONO Direct measurement 0,1-0,7 /L
( /L)
Monocyte concentration, MONO Direct measurement 2-8%
(%)
Reticulocytes, RTC (%) Direct measurement 0,5-2,5%
Erythrocyte sedimentation rate Direct measurement Male: 1-10
ESR (mm per hour) Female: 2-15 mm per
hour
Formula used for calculating corrected reticulocyte count

Corrected reticulocyte count=reticulocyte count x patient Hct / normal Hct
Mean Cell Hemoglobin (MCH) results in blood analysis and it is expressed in piсogram,
marked pg. 1 pg = 10-12g) (fig. 3).
17
a b
c
Figure 3. Different automated hematology analyzers (a,b) with reported results for bloodcells (c).
Exercise 1.Sampling of Blood.

Patients prefer the finger prick method of taking blood samples for testing.
Equipment.
1. Pipette for collection and mix of blood samples.
2. 70% alcohol, ether, cotton.
3. Rubber gloves.
4. Sterile scarifies.
Procedure.
Warning: the volunteer’s and patient’s blood must be regarded as a potential source of infection and
should not be touched.
18
1. Use the rubber gloves for collection of blood.
2. Clean the thick of the 4th finger tip of the left hand with cotton swab dipped in alcohol, wait
for the skin dry completely. Press on the sides of finger thick and prick it with a sterile
scarifier (fig. 4).
3. Wipe away the first 2 or 3 drops of blood from the skin puncture with dry sterile cotton swab.
The first drops of blood usually are mixed with lymph. If this blood is drawn the blood test
result will be wrong.
4. Draw gently blood into the pipette.
Figure 4.Erythrocyte parameters MCH, MCV and MCHC
Exercise 2.Hematocrit Value.

Purpose. When blood sample is centrifuged, the heavier blood cells are setting to the bottom of the
tube, leaving plasma at the top(fig. 5).
Equipment.
1. Hematocrit graduated pipettes.
2. 5% sodium citrate (anticoagulant).
Procedure.
1. Irrigate the pipette with solution of anticoagulant (to prevent the blood clotting). Then allow
the pipette dry completely.
2. Draw the blood into the hematocrit pipette.
3. Close the apex of pipette with wax plug.
4. Centrifuge pipette at speed of 3 000 revolution per minute for 30 minutes, until plasma and
formed blood cells will be completely separated.
19
5. The proportion of plasma and corpuscles may be known by the graduations of the pipette.
Normal hematocrit value.
Normal hematocrit value varies between 38 - 40 % for females and between 40 –
45 % for males.
Figure 5. Hematocrit graduated pipettes.
Observations, results and conclusion.
Exercise 3. ColorIndex (CI).

Purpose. Color index of blood — laboratory setting of the saturation of the red blood cells by the
pigment hemoglobin. Color Index (CI) is the ratio between the percentage of hemoglobin and the
percentage of red blood cells in the blood.
CI is calculated to identify different types of anemia: Hypo-, normo-, hyperchromic anemia. At the
moment the CI is not calculated, since replaced by other indicators due to the automation process of
laboratory research.
Observations, results and conclusion
CI is calculated by a simple formula: the amount of hemoglobin in g/l is multiplied by 3, then the
resulting figure is divided by the first three digits of the number of red blood cells without a comma.
3 x Hb (g/L)
CI= ------------------------------
the first three digits of the number of red blood cells
20
For example, if the content of erythrocytes of 5.0*1012/L, and the amount of hemoglobin 120 g/l,
CI = 120*3/500 = 0,72.
In essence, this ratio, which gives an idea about the content of hemoglobin in erythrocytes.
Norm CI is 0,8–1,1. If the CI is within these limits, the condition is called normochromic
and pathology of erythrocytes and hemoglobin is not excluded. If the overall analysis shows that the
CI usage is lowered, then this is evidence of hypochromia, i.e. a low content of pigment in
erythrocytes. CI over 1,1 means hyperchromia or macrocytic anaemia.
Exercise 4. Hemolysis.
The membrane of red blood cells is very fragile. The suitable solution for red blood cells is a
physiological solution or isotonic solution of NaCl (concentration 0,9%). When red blood cells are
placed in the hypotonic solution (for example 0,3%), they gain water and may burst with releasing
hemoglobin into the saline (fig. 6). This process is called osmosis hemolysis. There are some another
types of hemolysis – chemical, mechanical, thermic and biological. Hemolysis can also result by the
action of certain drugs and infections.
Figure 6.RBCs in different NaCl solutions.
Equipment.
1. 5 glass tubes.
2. 0,9% NaCl, 0,7% NaCl, 0,48% NaCl, 0,28% NaCl solution.
3. Distilled water.
4. Sample of blood.
21
Procedure.
1. List 5glass test-tubes.
2. Add to the first tube 5 ml of physiological solution and 1-2 drops of blood.
3. Add to the second tube 5 ml of NaCl solution (concentration 0,7%) and 1-2 drops of blood.
4. Add to the third tube 5 ml of NaCl solution (concentration 0,48%) and 1-2 drops of blood.
5. Add to the fourth tube 5 ml of NaCl solution (concentration 0,28%) and 1-2 drops of blood.
6. Add to the fifth 5 ml distilled water and 1-2 drops of blood.
7. Note the presence or absence of hemolysis in each tube after an hour(fig. 7).
Figure 7.Osmotic hemolysis.
If there is no hemolysis, the red blood cells will be found at the bottom of the tube. If some
hemolysis has occurred, the investigated solution will be slightly coloured with hemoglobin in red. If
some hemolysis has occured, the investigated solution will be slightly red color because of
hemoglobin of destroyed erythrocytes. If hemolysis is complete, the solution will be uniformly
coloured and transparent (so-called ―varnished‖ blood).
Measurement of osmotic stability (resistance) of erythrocytes is concentration of sodium
chloride at which the hemolysis begins. At the person it occurs in a solution of 0,48 % of
concentration (the top limit of resistance), in a solution of 0,28 % there is a full hemolysis (the lower
limit of resistance).
Write down, the tube and the concentration of NaCl solution with the beginning of hemolysis,
then the tube and the concentration of NaCl solution with completed hemolysis. Define limits of
osmotic erythrocyte resistance and compare results to norm.
22
Exercise 5. Erythrocyte Sedimentation Rate (ESR).
Purpose. If blood containing an anticoagulant is allowed to stand in a tube placed vertically, the red
cells settle down gradually to the bottom since their specific gravity is greater than gravity of plasma.
Red blood cells are negatively charged. If red blood cells are in a test tube, different substances are
deposited on the red blood cells, reducing this charge. As a result, erythrocytes begin to stick together
and settle to the bottom of the tube. Thus, the rate of ESR depends on the presence and amount of
various substances in the blood plasma.
Equipment.
1. Punchenkov’s stand (sedimentation tubes holder) with graduated glass tubes.
2. Glass ware.
3. 5% sodium citrate solution.
4. Sample of blood.
4. Cotton swab.
Procedure:
1. Draw the sodium citrate solution into the special tube to mark P and blow it at the glass ware.
2. Suck blood into the same tube up to mark K twice and also blow it at the glass ware. Mix the
content.
3. Fill the tube again with citrate blood and, keeping the finger over the upper open end, transfer the
tube to the Punchenkov’s stand (fig. 8).
4. Place the lower end of the tube firmly over the rubber cork and fix its upper end in vertical
position.
Figure8.Punchenkov’s stand.
5. Allow the tube to remain in this position for one hour at the end of which take the reading in
millimeters of the clear plasma column above red cells (fig. 9).
23
1. Read the length of the clear plasma on top of the tubes in mm (ESR).
2. Describe the factors that affect the sedimentation of the erythrocyte.
Normal values of ESR: males: 1-10 mm per hour; females: 2-15 mm per hour, pregnant women:
40-45 mm per hour.
Figure 9.Punchenkov’s stand with determined blood samples.

Clinical Significance of ESR
The erythrocyte sedimentation rate (ESR) is a non-specific test. It is raised in a wide range of
infectious, inflammatory, degenerative, and malignant conditions associated with changes in plasma
proteins, particularly increases in fibrinogen, immunoglobulins, and C-reactive protein. The ESR is
also affected by many other factors including anaemia, pregnancy, haemoglobino pathies, haemo
concentration and treatment with anti-inflammatory drugs.
Factors affecting ESR:
1. The number of red blood cells. The fewer erythrocytes, the faster they stick together and settle,
therefore the ESR will be higher. And if there are a lot of erythrocytes, they stick together slowly
and settle to the bottom of the tube for a longer time.
2. Shape of erythrocytes. Biconcave erythrocytes stick together and settle faster than spherrocytes
(round cells).
3. Plasma proteins: albumin slows down ESR; globulins and fibrinogen ESR accelerate.
4. Аntigens, antibodies (immunoglobulins);cholesterol accelerate ESR.
5. Alkalosis accelerates, and acidosis slows down ESR.
24
6. Calcium ion salts. The less calcium in the blood plasma, the higher the ESR and vice versa.
Lesson 2. Blood Groups. Rhesus-factor. Thrombocytes. Retraction and Fibrinolysis. Blood

Coagulation Regulation. Anti-coagulation System.
1. Thrombocytes and their functions.
2. Thrombocyte’s blood coagulation factors.
3. Plasma coagulation factors.
4. The phases of blood coagulation.
5. Retraction and fibrinolysis.
6. Blood coagulation regulation.
7. Anti-coagulation system.
8. Blood groups. Rhesus-factor.
There are certain molecules on the surfaces of all cells in the body that can be recognized as
foreign by the immune system of another individual. These molecules are known as antigens. As
part of the immune response, particular lymphocytes secrete a class of proteins called antibodies that
bond in a specific manner with antigens. The specificity of antibodies for antigens is analogous to
the specificity of enzymes for their substrates, and of receptor proteins for neurotransmitters and
hormones.
ABO System
Antigens (agglutinogens) A and are polysaccharides, they are located in the membrane of erythrocytes
and are covered with proteins and lipids. In addition to reducing agglutinogens in erythrocytes, antigen 0
can be detected, in which antigenic properties are poorly expressed and there are no high levels of
agglutinins in the blood. Antibodies (agglutinins) α and β are present in the blood plasma. Agglutinogens
and agglutinins of the same name do not occur in the blood of the same person under special conditions.
If, in an experiment in a test tube, blood is mixed with elevated agglutinogens and agglutinins, then
agglutination will occur. It is accompanied by agglutination and destruction (hemolysis) of erythrocytes.
A similar condition in the return line is very severe and is called hemotransfusion shock. It is
accompanied by deliveries of presentations and can end fatally.
The division of people into blood groups in the AB0 system is based on various combinations of
agglutinogens, erythrocytes and plasma agglutinins.
25
Thus, agglutinogens and agglutinins (A and α), (B and β) of the same name cannot be present in the
blood at the same time. It is between them that an agglutination reaction can occur during blood
transfusions. In addition, varieties of agglutinogens A and B were found in erythrocytes: agglutinogens
A1-7 and B1-6. These agglutinogens differ in their antigenic properties.
Figure 10.Agglutination reaction. People with type A blood have type A antigens on their red blood
cells membrane and antibodies against the type В antigen in their plasma. People with type В blood have
type В antigens on their red blood cells membrane and antibodies against the type A antigen in their
plasma. Therefore, if red blood cells from one blood type are mixed with antibodies from the plasma of
the other blood type, reaction of agglutination occurs. In this reaction, red blood cells stick together
because of antigen-antibody binding.
Currently, according to the ABO system, 4 blood groups are distinguished (tab.3):
Table 3
Containing of agglutinins and agglutinogens in different blood groups.
Blood group Agglutinogen Agglutinin

I O αβ
II A β
III B α
IV AB _
26
Rh Factor
Another group of antigens found on the red blood cells of most people is the Rh factor (named
for the rhesus monkey, in which these antigens were first discovered). There are a number of different
antigens in this group, but one stands out because of its medical significance. This Rh antigen is termed
D, and is often indicated as Rh (D). If this Rh antigen is present on a person's red blood cells, the person
is Rh positive; if it is absent, the person is Rh negative. The Rh-positive condition is by far the more
common (with a frequency of 85% in the Caucasian population, for example)(fig. 11).
Figure 11. How Rh hemolytic disease develops
The Rh factor is of particular significance when Rh-negative mothers give birth to Rh-positive
babies. Since the fetal and maternal blood is normally kept separate across the placenta, the Rh-
negative mother is not usually exposed to the Rh antigen of the fetus during the pregnancy. At the
time of birth, however, a variable degree of exposure may occur, and the mother's immune system
may become sensitized and produce antibodies against the Rh antigen. This does not always occur,
however, because the exposure may be minimal and because Rh-negative women vary in their
sensitivity to the Rh factor. If the woman does produce antibodies against the Rh factor, these
antibodies could cross the placenta in subsequent pregnancies and cause hemolysis of the Rh-
positive red blood cells of the fetus. Therefore, the baby could be born anemic, with a condition called
erythroblastosis fetalis, or hemolytic disease of the newborn.
Thrombocytes or Platelets
Thrombocytes, platelets are irregularly disc-shaped, 2-4 µm diameter cellular fragments of
megakaryocytes. They have a short life span (4-9 days) in the circulation and then are removed by
fixed macrophages in the spleen or liver. They have no nuclei but contain many vesicles which
27
promote blood clotting upon the release of their content. Platelets additionally help stop blood loss
by forming a platelet plug in the damaged vessels. Functions of platelets:
Platelets are non-nucleated round or oval, biconvex discs having various sizes and unit membrane.
Characteristic properties are:
1. Sticking to water - wet table surface or otherwise rough surface (injured or
diseased endothelium).
2. Easy clumping.
3. Easy disintegration and thus liberation of thrombokinase.
4. Total number of platelets is about 200 – 400x109 cells/l .
Functions of platelets.
1. Initiate blood clotting.
2. Repair capillary endothelium.
3. Hemostatic mechanism.
4. Hasten clot retraction.
5.Regulation of the tone of the vascular wall due to serotoning, thromboxane A2, formed from
arachidonic acid. Platelets have the following properties: amoeboid mobility, secret activity,
adhesion - the ability to stick to the surface of the vessel, aggregation - crowding, i.e. ability to stick
together
Hemostasis.
When blood is shed it loses its fluidity in a few minutes and sets into a semisolid jelly. Thus
phenomenon is called coagulation or clotting. On further keeping, the clot retracts to a smaller
volume and presses out a clear straw – colored fluid, called the serum. Serum will not clot any
more.
Hemostasis is defined as the arrest of bleeding. The following three processes may act to stem the
flow of blood: vasoconstriction, platelet aggregation and blood coagulation. The velocity of blood
flow is inversely related to the total cross – sectional area of the vasculature at any downstream point
align the vascular system.
The platelet plug is strengthened by a meshwork of insoluble protein fibers known as fibrin. Blood
clots therefore contain platelets and fibrin, and they usually contain trapped red blood cells that give the
clot a red color (clots formed in arteries, where the blood flow is more rapid, generally lack red
blood cells and thus appear gray). Finally, contraction of the platelet mass in the process of clot
retraction forms a more compact and effective plug. Fluid squeezed from the clot as it retracts is called
serum, which is plasma without fibrinogen, the soluble precursor of fibrin. (Serum is obtained in
28
laboratories by allowing blood to clot in a test tube and then centrifuging the tube so that the clot and
blood cells become packed at the bottom of the tube.) The conversion of fibrinogen into fibrin may
occur via either of two pathways. Blood left in a test tube will clot without the addition of any
external chemicals; the pathway that produces this clot is thus called the intrinsic pathway. The
intrinsic pathway also produces clots in damaged blood vessels when collagen is exposed to plasma.
Damaged tissues, however, release a chemical that initiates a "shortcut" to the formation of fibrin.
Since this chemical is not part of blood, the shorter pathway is called the extrinsic pathway (fig 12).
Figure 12.The extrinsic and intrinsic clotting pathways. Both pathways lead to the formation of
insoluble threads of fibrin polymers.
The intrinsic pathway is initiated by the exposure of plasma to a negatively charged surface, such
as that provided by collagen at the site of a wound or by the glass of a test tube. This activates a plasma
protein called factor XII, which is a protein-digesting enzyme (a protease). Active factor XII in turn
activates another clotting factor, which activates yet another. The plasma clotting factors are numbered
in order of their discovery, which does not reflect the actual sequence of reactions. The next steps in
the sequence require the presence of Ca2+ and phospholipids, the latter provided by platelets. These steps
result in the conversion of an inactive glycoprotein, called pro-thrombin, into the active enzyme
thrombin. Thrombin converts the soluble protein fibrinogen into fibrin monomers. These
monomers are joined together to produce the insoluble fibrin polymers that form a meshwork
supporting the platelet plug. The intrinsic clotting sequence is shown on the right side of figure 12.
The formation of fibrin can occur more rapidly as a result of the release of tissue
thromboplastin from damaged tissue cells. This extrinsic clotting pathway is shown on the
29
left side of figure 12. Note that the intrinsic and extrinsic clotting pathways eventually merge
to form a final common pathway that results in the formation of insoluble fibrin polymers.
Exercise 1. ABO Blood Group.

A. Serum Method.
Purpose. The red blood cells membrane contains different types of agglutinogens and plasma
contains agglutinins. In order to determine the blood group of a subject, the red cells are allowed to
react with sera, containing known agglutinins.
Equipment.
1. Glass slides.
2. Droppers, glass sticks.
3. Standard serum of O (I), A (II), B (III) blood groups.
4. Sample of blood.
Procedure.
1. Take a white palette which contains upper and lower rows with 3 holes or a glass slide.
5. Place subsequently drops of serum of the I, II and III blood groups in the 3 holes of the upper
line.
2. Place subsequently drops of serum of the I, II and III blood groups in the 3 holes of the lower
line.
3. Add 1-2 drops of blood to each hole of the upper row and 1-2 drops of blood sample to each hole
of the lower row.
4. Mix the content of each hole with a glass stick.
5. Wait for 6-8 minutes and examine all holes whether any agglutination has taken place or not.
6. Make conclusions (tab. 4).

Serum of I blood group that contains -and -agglutinins, is obtained from a subject with blood
group O, serum of II blood group that contains - agglutinins, is obtained from a subject with blood
group A, and serum of the III blood group that contains - agglutinins, is obtained from a subject
with blood group B.
30
Table 4
The ABO blood group. Serum method.
Agglutinogens 1st hole (serum 2nd hole (serum of 3d hole (serum
(antigens) in red cells of I blood group) II blood group) of III blood group)
,  agglutinins  agglutinin  agglutinin
I (O)   
II (A)   
III (B)   
IV (AB)   
Symbol: reaction of agglutination.
B. Antibodies Method.
Equipment.
1. Glass slides.
2. Droppers, glass sticks.
3. Anti-A reagent (contains anti-A antibodies), anti-B reagent (contains anti-B antibodies) and anti-
AB reagent (contains anti-A and anti-B antibodies).
4. Sample of blood.
Procedure.
1. Place 1 drop of anti-A, anti-B and anti- AB reagent separately on a glass slide.
2. Add 1-2 drops of the blood to the each drop of reagent.
3. Mix the contents using the individual glass sticks.
4. Wait for 1-2 minutes and examine all drops with the naked eye to see whether any agglutination
has taken place or not (fig 13), (tab. 5).
5. Make the conclusions.
The main reason of agglutination is the combination of the factors under the same name, for
example, the agglutinogen A and agglutinin , or agglutinogen B and agglutinin . The
agglutinogens are antigens (passive factors), while the agglutinins are the antibodies (active factors).
If any agglutination has occurred, it is usually visible with the naked eye as dark reddish clumps of
different sizes. If the number of RBC is very high, they may simply give an impression of
agglutination, that is why microscopic confirmation is essential.
31
Figure 13.Blood typing. Agglutination (clumping) of red blood cells occurs when cells with A-type
antigens are mixed with anti-A antibodies and when cells with B-type antigens are mixed with anti-B
antibodies. No agglutination would occur with type О blood (not shown).
The agglutinated red cells form clumps of different sizes and acquire a dark reddish color due to
hemolysis of RBC. If there is no agglutination, the RBC appear grouped together in masses but
without any clumping, agglutination or hemoglobin liberation.
Table 5
The ABO blood group. Antibodies method.
Agglutinogens anti-A reagent anti-B reagent anti-AB reagent
(antigens) in red cells (with anti-A antibodies)
(with anti-B (with anti-AB antibodies)
antibodies)
I (O)   
II (A)   
III (B)   
IV (AB)   
Exercise 2.Rh Factor.

The main reason of agglutination is the reaction between the antigen D of Rh Factor and
special anti D antibody. If any agglutination has occurred, it is usually visible with the naked eye as
dark reddish clumps of different sizes. If there is no agglutination, the RBCs appear grouped
together in masses but without any clumping.
32
Purpose . Another group of antigens found on the red blood cells of most people is the Rh factor In
order to determine the Rh Factor of a subject, the red cells are allowed to react with
antirhesusserum.
Equipment.
1. Glass slides.
2. Droppers, physiological solution.
3. Standard antirhesus serum.
4. Sample of blood.
Procedure.
1. Take a white palette.
2. Place subsequently drops of antirhesus (anti-D) serum.
3. Add 1-2 drops of blood to the hole.
4. Mix the contents by use of the individual glass stick.
5. Wait for 5 minutes and inspect hole with the naked eye to see whether any
agglutination has taken place or not.
Exercise 3.BleedingTime.
Purpose. If a prick is given in the skin with a needle, bleeding occurs and continues for some time
then stops. The time elapse between skin puncture and the arrest of bleeding is called bleeding time.
Equipment.
1. Pipette for collection and mix of blood samples.
3. Rubber gloves.
4. Filter paper.
Procedure.
1. Get a deep skin puncture on the tip of the finger under sterile conditions and note the time.
2. Remove the drops of blood every 30 seconds by touching the skin gently with a filter paper,
absorbing the drops of blood along its edge.
3. Note the time when there is no trace of blood on the filter paper, another words, when the bleeding
has stopped.
4. Count the spots of blood, divide this number by 2 and express the bleeding time in minutes.
33
Normal bleeding time = 2-4 minutes.
Exercise 4.Coagulation Time.
Principle If the blood is removed from the body and kept in a glass capillary tube or a test tube, it
coagulates, forming a jelly-like mass. Time elapse between the withdrawal of blood and clot
formation is a coagulation time.
Equipment.
1. Glass capillary tube.
3. Rubber gloves.
4. Filter paper.
Procedure.
1. Get a deep skin puncture on the fingertip and discard the first 2-3 drops of blood. It is essential
to prevent admixture of blood with tissue fluids.
2. Fill a chemically clean and dry glass capillary tube by dipping one end in the drop of blood.
(Blood will fill the tube by capillary action). Note the time.
3. Rock carefully the capillary tube from one end to another end. Note the time when the blood
stops its movement inside of capillary tube.
Normal coagulation time is from 2 to 5 minutes.
34
THEME 2.NEURO-MUSCULAR PHYSIOLOGY
Lesson 1. The Basic Physiological Properties of Living Tissues. Action
of Stimulus on Tissues. Laws of Irritation.

1. The General properties of excitable tissues: excitability, conductivity, lability.
2. Irritants (definition and classification). Concepts of irritation and irritability.
3. Change of excitability at excitation.
4. Laws of irritation tissues:
- The law of force; a threshold of irritation as a measure of measurement of excitability;
- The law « all or none» or ―all-or-nothing‖;
- The law « forces - time » (a parity between force of irritation and time of its action);
- The law of a gradient of irritation (increase of force of a current), the phenomenon of
accommodation.
5. Methods of studying the excitability of nerves and muscles. Chronaximentry
The concept of excitability.

Excitable tissues in response to the action of stimuli are able to pass from a state of rest to a state of
excitement. In this way, cells generate an electrical impulse called an action potential. Excitability is
the ability of excitable tissues (nerve, muscle, glandular) to form excitation - action potential on the
membrane in response to a stimulus. Stimulus is environmental factors that cause the transition from
a state of rest to a state of activity.
Excitability indicators:
- Threshold of stimulation. It is the minimum power of intensive at which action on a membrane
action potential is formed (these is an excitement). Stimuli happen to be threshold, subthreshold and
superthreshold
- Rheobase – it is a threshold for electric current.
- Chronaxia – this is time during which the incentive of two rheobases will cause excitement
- Accommodation is accustoming of tissue to slowly increasing stimulus. Thus the irritation
threshold grows.
- Lability is functional mobility.
Classical object of biological researches is a frog. The international rules of protection of animals
provide experiments on them with the educational and research purpose, categorically forbidding to
operations on alive animals. All experiences on them spend after a narcosis (ether) and the
35
subsequent destruction back and a brain. In experimental physiology traditional object for studying
physiological excitability of fabrics is a neuromuscular preparation of a frog. It is necessary to learn
how to prepare frog’s neuromuscular preparation before starting to carry out of experimental work
on neuro-muscular physiology.
Technique of making of a neuromuscular preparation.
Principle. Much of the knowledge in the physiology of excitable cells has been derived from the
experiments on the classic gastrocnemius sciatic muscle preparation. The gastrocnemius muscle is
easily prepared and has been extensively studied. It originates from the femur from above and behind
the knee joint and terminates in a long tendon- the tendon of Achilles (fig.14, 15).
Figure 14. Make the frog’s neuromuscular preparation (an explanation in the text).
Purpose. To dissect the frog sciatic nerve - gastrocnemius muscle preparation. Equipment. A frog,
preparation set, Ringer’s solution, ether.
Procedure:
1. Stun, pith and fix the frog on the board.
2. Cut the spinal column in the middle of the body, and remove the upper body part.
3. Remove the viscera using the scissors and forceps.
4. Take off the skin from the lower body part and hind legs.
5. Remove the muscles of back by blunt dissection and expose the sciatic nerve roots – you get
the two hind limb preparation.
6. Gently slit the preparation at midline, and get the one hind limb preparation.
7. Isolate the vertebral segment above and below the origin of sciatic nerve roots.
36
Figure 15.The frog’s sciatic nerve – gastrocnemius muscle preparation, 1-8 –steps of dissection.
8. Using a glass rod isolate the sciatic nerve in the thigh.

9. Cut away the thigh muscles leaving the nerve exposed.
10. Pass the thread around the Achilles tendon at the heel and tie it tightly around the tendon.
11. Sever the tendon below the tie and pull the muscle away from the leg.
12. Cut away the bone just below the knee.
Observations, Figures and Conclusion.

All excitable tissues (muscular, nervous) are characterized both general properties, and specific
properties which are inherent only in the given kind of tissues. Each of properties has the certain
criteria of an estimation that allows not only to estimate, but also to compare excitable tissues (tab.
6).
Table 6
Properties and parameters of excitable tissues.
Properties Parameters
Excitability - ability to answer action Threshold of irritation, rheobase,
stimulus process of excitation, i.e. to chronaxie, duration of absolute
generate action potential (AP) refractory period, speed of
accommodation
Conductivity - ability to spend excitation on Speed of carrying out of action potential
a membrane of cells
Lability (or functional mobility) - ability to The maximal number APin unit of time,
rhythmic activity reproduced by a tissue
37
Exercise 1.Stimulusaction on neuro-muscular preparation.
Principle. Transition from a condition of rest to a condition of activity occurs under influence of
irritants. Irritant is a factor of the external or internal environment that, affecting different fabrics,
causes response of a live organism. By the form of influence all irritants can be divided into:
biological, chemical, physical (mechanical, thermal, light, sound, etc.) and physical-chemical. On
biological value all irritants are divided into adequate and inadequate.
Purpose. To compare features of action of mechanical, chemical, thermal and electric irritants on
excitable tissues and to estimate their properties.
Equipment. A frog, preparation set, Ringer’s solution, a glass stick, a spirit-lamp, salt, an electro
stimulator, 0,9% NaCl (physiological solution, isotonic sodium chloride solution).
Procedure:
1. Prepare the sciatic nerve – gastrocnemius muscle preparation.
2. Mechanical irritation.
Pinch a nerve with tweezers or slightly press on a nerve with an edge of the closed scissors. Note,
whether response is kept at recurrence of irritation.
3. Thermal irritation.
Heat a glass stick with a spirit-lamp, touch a nerve with heated glass stick, then touch a nerve
with a cold glass, and compare results.
4. Chemical irritation.
Put on a nerve some crystal of salt. Measure the latent period of reaction. When the muscle will
start to contract, take away these crystals of salt; wash a nerve with 0,9% NaCl carefully. After
that measure the aftereffect - time when muscles tops to contract.
5. Electric irritation.
Put electrodes of electrostimulator on a nerve. Put super threshold irritation. Note, how the
response of a muscle to electric irritant differs from reaction to other kinds of irritants.
6. Write down the results into the table 7.
Table 7
Effects of different irritants on excitable tissues
Type of irritants Latent period Aftereffect Damaging effect
Mechanical
Thermal
Chemical
Electric
38
Exercise 2. Direct and indirect irritation of a muscle. Comparison of excitability of a nerve and
a muscle.
Principle. Excitable fibres differ from each other with degree of excitability. The excitability of
different tissues varies widely. Some tissues are highly excitable whereas others are dull. A measure
of excitability is the threshold of irritation, the minimal strength of irritant which causes the
response. A parameter of the excitation which has arisen in a muscle is its contraction. If the
irritation is put directly on a muscle fiber,it names as a direct irritation and its minimal strength
characterizes excitability of a muscle. If irritation of a muscle is put from a motor nerve, it names as
an indirect irritation, and the size of electric current which causes the minimal contraction, reflects
nerve excitability.
Purpose. To define rheobase size of a nerve and a muscle and to compare excitability of these
fibres.
Equipment. A frog, preparation set, Ringer’s solution, an electro stimulator.
Procedure. Make neuromuscular preparation. Place the electrodes of electric stimulator under a
nerve. Put the frequency selection switch at frequency «1» Hz; and the handle of the amplitude
selector at «0» V (a range of pressure is 0,01V). Define a threshold of irritation at indirect irritation
of a muscle. For this purpose, moving the handle of the amplitude selector on 1-2 points, find out the
minimal strength of irritation causing contraction of a muscle. The determined value of electric
current defines a threshold of irritation of a nerve, rheobase of a nerve. Not changing value of
irritation, place electrodes directly on a muscle; it is direct irritation of muscle. If contraction is not
present, add strength of irritant, changing amplitude from 0,1V to 1V. Note strength of irritation at
occurrence of contraction of a muscle. The determined value of electric current characterizes
rheobase of a muscle.
Write down the results into the table 8.
Table 8
Comparison of threshold of irritation
Type of a tissue Threshold of irritation (V)
Nerve
Muscle
39
Lesson 2. Bioelectrical Phenomenon of Excitation Tissues. Membrane Potential. Action
Potential.

1. The membrane structure of the cell. Transport through the cell membrane. Basic mechanisms of
passive and active transport (the concentration difference, diffusion, ions pump, and secondary
active transport).
2. Membrane potential. Measurement and calculation of the membrane potential. The value of
membrane potential in different cells.
3. Action potential. The phases of action potential. Changes in sodium and potassium conductance
during the course of the action potential.
4. The threshold for excitation. The refractory period. The excitability curve (Absolutely refractory
period, relative refractory period, supernormal and subnormal periods.).
Principle. The Membrane Potential
As a result of the permeability properties of the plasma membrane, the presence of non
diffusible negatively charged molecules inside the cell, and the action of the Na+/K+ pumps, there is
an unequal distribution of charges across the membrane. As a result, the inside of the cell is
negatively charged compared to the outside. This difference in charge, or potential difference, is
known as the membrane potential (fig. 16).
Figure 16. The effect of fixed anions on the distribution of cations. Proteins, organic phosphates,
and other organic anions that cannot leave the cell create a fixed negative charge on the inside of
the membrane. This negative charge attracts positively charged inorganic ions (cations), which
therefore accumulate within the cell at a higher concentration than is found in the extracellular
40
fluid. The amount of cations that accumulates within the cell is limited by the fact that a
concentration gradient builds up, which favors the diffusion of the cations out of the cell.
Resting Membrane Potential

Intracellular and extracellular environments contain ions. All cells at rest have different charges on
a membrane. The cellular membrane is polarized. The difference of charges between internal and
external membrane surfaces is called the membrane potential (resting membrane potential). In
excitable cells concentration of ions of potassium in a cell is more than out of a cell. Potassium ions
pass freely through a cellular membrane.
The actual value of the resting membrane potential depends on two factors:
1. The ratio of the concentrations of each ion on the two sides of the plasma membrane.
2. The specific permeability of the membrane to each different ion.
3. Working of the sodium - potassium pump.
Many ions—including K+, Na+, Ca2+, and Cl¯ contribute to the resting membrane potential.
Their individual contributions are determined by (a) the differences in concentration of the ions
across the membrane (fig. 17), and (b) by their membrane permeability.
Figure 17. Concentrations of ions in the intracellular and extracellular fluids. This distribution of
ions, and the different permeability of the plasma membrane to these ions, affects the membrane
potential and other physiological processes.
The resting membrane potential of most cells in the body ranges from -65 mV to -85 mV (in
neurons it averages -70 mV).
41
Local response and its characteristic.
Subthreshold irritant doesn`t cause critical level of depolarization on a membrane. There is a local
respond (local potential) (tab. 9). It is under the law of the power relations, doesn`t extend on long
distances and is capable of being summarized.
Table 9
Comparison of actions and local potential capacities
Action Potential Local potential
Spread Spreads without attenuation over Distributed by1-2mm from the

long distances along the entire point of irritation with
length of the nerve fiber damping
The dependence of the Doesn’t depend (subject to the law Increases with the strength of
strength of the stimulus "all or nothing") stimulus
The phenomenon of It does not summate It is summates

summation
Excitable tissue in the Decreases until the completed on Increasing
event of potential excitability (refractory)
Action Potential
Sodium channels are open as a result of an action of some irritant. When the axon membrane
has been depolarized to a threshold level, the Na+ gates open and the membrane becomes permeable
to Na+. It permits Na+ to enter the axon by diffusion, which further depolarizes the membrane (makes
the inside less negative or more positive). Since the gates for the Na+ channels of the axon membrane
are voltage regulated, this additional depolarization opens more Na+ channels and makes the
membrane even more permeable to Na+. As a result, more Na+ can enter the cell and induce
depolarization that opens even more voltage-regulated Na+ gates (fig. 18).
Figure 18. Action potential 1- resting state; 2 – phase of depolarization
42
The explosive increase in Na+ permeability results in a rapid reversal of the membrane potential
in that region from -70 mV to +30 mV. At that point in time, the channels for Na+ close, causing a
rapid decrease in Na+ permeability. Also at this time, as a result of a time-delayed effect of the
depolarization, voltage-gated K+ channels open and K+ diffuses rapidly out of the cell.
Since K+ is positively charged, the diffusion of K+ out of the cell makes the inside of the cell
less positive, or more negative, and acts to restore the original resting membrane potential of -70
mV. This process is called repolarization and represents the completion of a negative feedback loop
(fig. 19).
After that undershoot processes begin, during which the charge slowly comes to its initial
position.
Figure 19. Action potential 3- phase of repolarization; 4 – undershoot
All changes in Na+ and K+ diffusion and the resulting changes in the membrane potential they
produce constitute an event called the action potential, or nerve impulse(fig. 20).
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Figure 20. A - Action potential: 1- phase of depolarization, 2 - phase of repolarization, 3- phase of
after depolarization, 4 - phase of hyper polarization.
Б–Phases of excitability: 1-absolutely refractory period, 2-relative refractory period, 3 -
supernormal period, 4 – subnormal period.
Exercise 1. Galvani’s Test of Contracting with Metal.

This Galvani’s experience with a copper-zink plate proved for the first time existence of "animal"
electricity.
Purpose. Studying of contraction of a muscle of nerve-muscle preparation under the influence of
Galvani's forceps.
Equipment. Zinc plate with a copper hook, scissors, tweezers, Ringer’s solution, ether, gauze,
electro stimulator, glass-plate.
Procedure.
1. Destroy the frog’s spinal cord and brain.
2. Cut the chest part of the vertebral column.
3. Take off the frog’s upper part.
4. Take off the skin from lower limbs. You can see the nervous trunks.
5. Carefully hang the lower limbs on a cooper hook of a zinc (or iron) plate. You can watch the
nervous trunks (fig. 21).
6. Sketch the scheme of experiment and explain the observed effect.
Figure 21.Galvanic (metallic) tweezers.
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Exercise 2. Galvani’s Test of Contracting without Metal.
Purpose. Studying the frog’s muscle contraction without metal influence. Determination of currents
of "rest".
Equipment. A frog, preparation set, Ringer’s solution, ether, glass hook.
Procedure.
1. Prepare the nerve-muscle preparation.
2. Cut with scissors the part of muscle near the articulation.
3. Put the nerve over both parts of the muscle (on damaged and not damaged parts). At the time the
nerve contacts damaged and intact sites of a muscle, the muscle contracts.
4. Sketch the scheme of experiment (fig. 22) and explain the observed effect.
Figure 22. Galvani’s test of contracting without metal.

This experiment confirmed existence of "animal" electricity and the bioelectric phenomena in living
tissues.
Exercise 3. The Matteuchee’s Test (Secondary Muscle Contracting – Secondary Tetanus).
Italian physicist and physiologist Carlo Matteuchee proved that the action potential arising in a
muscle at excitement is rather strong to cause excitement of a nerve which attached to the first
muscle, and it involves contraction of the second muscle.
Purpose. Studying the frog’s muscle contraction and the bioelectric phenomena in living tissues.
Equipment. Nerve-muscle preparation, electro stimulator, scissors, tweezers, Ringer’s solution,
ether, glass hook, gauze, glass-plate.
Procedure.
1. Take two nerve-muscle preparations and put them on glass-plate.
2. Put a nerve of the first nerve-muscle preparation on the second nerve-muscle preparation.
3. Put a nerve of the second nerve-muscle preparation on electrodes of electro stimulator (fig. 23).
45
Figure 23.The Matteuchee’s test. 1- nerves, 2 – electrodes.
4. Turn on the electro stimulator. Put the frequency selection switch at frequency «50» Hz, and the
handle of the amplitude selector at «15» V. Observe the tetanus contraction of both muscles.
5. Sketch the scheme of experiment; in conclusion explain the cause of secondary tetanus.
Lesson 3. Physiology of Nerve Fibers and Synapses.
The questions for studying.

1. The nerve fibres – classification, properties and function of nerve fibres.
2. Mechanism of nerve impulse conduction. Saltatory conduction in the myelinated nerve fibre.
3. Conductivity.
4. Parabiosis (N.E. Vvedensky).
5. Synapse: structure, classification, mechanism of synaptic transmission.
6. Transmitter substances: mechanism of synthesis, releasing to the synaptic cleft and
combining with receptors of the postsynaptic membrane.
7. Receptors: classification, properties.
8. Specialty of structure and function of neuromuscular synapse (junction). Mechanism of
postsynaptic potential (end-plate potential).
9. The basic principles of synaptic transmission blockade in neuromuscular junction.
The nervous fiber has the following physiological properties: excitability, conduction and
lability. Nerve fibres are classified in different features: histological – medullated and non-
medullated; functional – motor (efferent) and sensory (afferent); chemical- adrenergic and
cholinergic; according to diameter and conduction velocity Erlanger and Gasser divided the nerve
fibres into A, B and C groups (tab. 10).
46
Table 10
Classification of nerve fibers.
Types of fibre Diameter of Velocity of Function
fibre in conduction in
m/sec.
A-α 12-20 70-120 Proprioreception; somatic motor
A-β 5-12 40-70 Touch, pressure
A-γ 3-6 15-40 Motor to muscle spindle
A-δ 2-5 12-30 Pain, temperature
B Less than 3 3-15 Preganglionic sympathetics
C 0,3-1,3 0,5-3 Postganglionic sympathetics
The nerve impulse is propagated with definite speed. The conducting velocity depends on a
diameter of the nerve fibres, the thicker fibres the higher the velocity. The conducting velocity also
depends on the presence or absence of myelination. The conduction velocity of myelinated nerve
fibres is much higher than the conduction velocity of unmyelinated nerve fibres. It increases with the
diameter of nerve fibres. The nerve impulse is conducted along the nerve fibre.
Mechanism of the nerve impulse conduction.

According to the membrane theory, the nerve impulse is a propagated wave of depolarization.
The nerve fibre remains in polarized state, with positive charge lined up along the outside of
membrane and negative charge along the inside. As soon as the fibre is excited a point the polarity is
changed, it is actually reversed. This depolarization wave is developed. A local circuit current flows
between the depolarized membrane and the resting membrane areas. Positive current flows inward
through the depolarized membrane and outward through the resting membrane and in this way circuit
is completed. This local depolarization current then excites the adjacent portion of the membrane
producing more and more depolarization. The depolarization wave travels in all directions along the
entire length of the nerve fibre. This type of conduction is observed in non medullated nerve fibers
(fig. 24).
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Figure 24. Conduction of action potential in non medullated nervous fiber
Saltatory conduction in the myelinated nerve fibres.

Myelin sheath is an effective insulator. Ions cannot pass through the myelin sheath and nodes
of Ranvier permeate ions to pass though it more easily. For this reason the impulse is transmitted
from node to node rather than continuously along the entire length of the fibre. The depolarization in
myelinated axon jumps from one node of Ranvier to next. This type of jumping is known as saltatory
conduction. The myelin increases the velocity of conduction(fig. 25).
Figure 25. Conduction of action potential in medullated nervous fiber
The conductivity shows the following characteristics:

The first law of conductivity: impulse is propagated along a nerve in both directions.
The second law of conductivity: anatomical and physiological entirety.
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The third law: the isolated conduction of irritation.
Synapse. Neuro-muscular junction.

The specialized region between nerve and muscle is called the neuromuscular junction or synapse.
The synapse consists of a presynaptic membrane, a synaptic cleft and a postsynaptic membrane (fig.
26).
Figure26. Structure of synapse. Neuro-muscular junction.
One neuromuscular junction includes axon terminal with 1) presynaptic membrane; the junctional
folds on the muscle -2) postsynaptic membrane or the end plate; and gap between the axon
membrane and the muscle membrane – 3) synaptic cleft.
The nerve impulse opens Ca2+ channels on a presynaptic membrane. Calcium ions arrive into
terminal. As a result, a transmitter –acetylcholine (ACh)- is released into the synaptic cleft.
Molecules of ACh contact with receptors on a postsynaptic membrane. Opening of Na+ channels
initiate depolarization and formation the end plate potential on a postsynaptic membrane. This
potential initiates propagation of muscle action potential along the membrane and as the result – the
muscle contraction.
Exercise 1. Nerve Conduction Velocity.

Purpose. To study velocity of sciatic nerve.
Procedure.
1. Draw the scheme of this experiment (fig. 27).
2. Prepare the nerve-muscle preparation.
49
3. Put the sciatic nerve on a stimulating and an exploring electrodes as it is shown on the
scheme. The distance S equals 45 mm.
4. Set up and switch on the apparatus which will record and stimulate the preparation and watch
an action potential on the device screen.
Figure 27. The observation of excitation along the nerve
5. Measure and record the distance between the artifact and the beginning of the stimulation.
Knowing the speed of development of a beam on the device screen, we define latent time
(sec). It equals about 0,002 sec. The conducting velocity of a nerve fibre bundle can be
studied by stimulating the nerve bundle at one end and recording an action potential at other
end. Then we have to analyze and make calculation.
6. Calculate the conducting velocity of the nerve:
V=S/T,
Where:
V – Velocity of conduction in m/sec.
S - distance between stimulating and a exploring electrodes (m).
T –latent time (sec).
Exercise 2.The Impulse is Propagated Along a Nerve in Both Directions.

Procedure.
1. Draw the scheme of this experiment (fig. 28).
2. Prepare the nerve-muscle preparation and place it into the humid camera of the apparatus on
stimulating and exploring electrodes.
3. Set up and switch on the apparatus which will record and stimulate the preparation.
50
4. Impulse is propagated along a nerve in both directions (in the experiment).
You can see two actions potential on the cathode ray oscillograph. The excitation takes place at the 1
canal from the first exploring electrode. The excitation takes place at the 2 canal from the second
exploring electrode.
Figure 28. The observation of the bilateral excitation along the nerve.
Exercise 3. Physiological entirety of Nervous Fiber. Parabiosis.

Principle. To understand the law of anatomical and physiological nerve integrity it is necessary to
know the parabiosis phenomenon. This phenomenon was discovered by the famous physiologist
N.E. Vvedensky in 1901. He gave the following scheme of consecutive conditions of the alternating
locus: rest -excitement - inhibition - death. Therefore, the parabiosis is a state, boundary between life
and death.
Parabiosis is a define condition of the excited tissue caused by alterative actions such as cooling,
traumatic hurt or anesthesia and other factors. These alterative actions change nerve excitation,
conducting characteristics and lability. Parabiosis develops gradually and has 3 phases:
1. equalizing
2. paradoxical
3. inhibitory.
1. Equalizing phase. Fabric answers with identical responses (contraction of muscle) on irritation of
any power.
51
2. Paradoxical phase. On strong irritation there is a weak reaction of fabric, and on weak irritation
there can be very rough (inadequate) reaction of fabric.
3. Inhibitory phase. Fabric doesn't answer on any irritants (actually a parabiosis or an anesthesia).
After cut off an irritant the lability of a nerve is restored, passing the same phases in upside-
down.
Procedure.
1. Prepare the nerve-muscle preparation and place it into the humid camera of the apparatus on
stimulating and exploring electrodes.
2. Place the ligature under the nerve. Then put a cotton ball wetted with Novocain under the
ligature.
3. Set up and switch on the apparatus which will record and stimulate the preparation.
4. Every 30 second stimulate this part of the nerve, which is beyond Novocain action.
5. This procedure is supposed to cause the nerve impulse blockade along the sciatic nerve.
This experiment causes a blockade of nerve impulse conduction along the sciatic nerve. You
see that stimulation does not happen in the blocked part as impulse on a nervous fiber isn't carried
out (parabiosis) and the muscle doesn’t contract.
Conclusions:
The conduction of impulse along the nerve has the following characteristics:
1. The impulse is propagated along a nerve in both directions (but only experimentally).
2. The nerve impulse is propagated at a definite velocity.
3. The nerve impulse is propagated along the nerve fibre in case the nerve preserves its anatomical
and physiological integrity.
Lesson 4. Physiology of Muscles

The questions for studying.
1. Structurally functional features of skeletal muscles. Concept about motor units. The basic
differences in a structure and function of smooth muscles.
2. Modern representation about mechanisms of muscular contraction and a relaxation.
Bioelectric, chemical and structural changes in a muscular fiber at the moment of contraction.
3. Kinds of muscular contraction. Single muscular contraction, its phases.
4. Tetanus, its kinds, the mechanism of formation (summation full and incomplete).Optimum
and pessimum frequencies of irritation.
5. Force of muscles, factors it’s determining. Methods of estimation (dynamometry).
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6. Work done by the muscle, its size. Methods of an estimation of physical efficiency. Fatigue
of muscles.
There are three kinds of muscles at vertebrate animals and the person: striated muscles, which
include skeletal and cardiac muscle, and smooth muscles of internal organs and vessels (fig. 29). All
of them differ according to structure and physiological properties. Skeletal muscles are an active part
of the musculoskeletal system. As a result of their contractile activity is the moving of body in space;
moving of parts of the body relatively each other; maintenance of a position. Smooth muscles of
internal organs and blood vessels are responsible for the involuntary vegetative reactions directed on
maintenance condition of metabolism, of homeostasis parameters. The cardiac muscle - unique both
on the structure, and on functions, works as the pump on own automatism.
Figure 29.Different types of muscle tissue.
All kinds of muscles possess the general physiological properties: excitability, conductivity,
contractility and lability. The physical properties of skeletal muscles are: an extensibility, elasticity,
work and force. The important property of smooth muscles is their plasticity, i.e. ability to keep the
form after stretching or deformation of the form, and also ability to spontaneous automatic activity
that distinguishes them from skeletal muscles.
Modern theory of muscular contraction and relaxation.

The impulse spreads along a membrane of muscle fiber and is carried into the sarcoplasm into
the structures of a sarcoplasmic reticulum. Calcium channels open and Ca2+ ions release from the
sarcoplasmic reticulum and diffuse to the contraction system. Ca2+ ions contact with Troponin, which
cause a shift in Tropomyosin and opening active centers of actin for connection myosin filaments.
Process of formation of action-myosin complex takes place (fig. 30) with wasting energy of ATP.
53
Energy is also used for separation of myosin heads from threads of actin. The muscle is shortened as
a result of sliding of threads of myosin along threads of actin.
Figure 30. Muscular contraction
Exercise 1. Kinds of Muscular Contraction.

Principle. Single muscular contraction is a reaction of a muscle in response to single threshold or
super threshold stimulus (fig. 31). In an organism muscles are contracted under influence of the
rhythmic impulses from the central nervous system. Impulses come to muscles with a bigger
frequency, than the period of muscle single contraction. The muscle gets the next following impulse
when its relaxation phase isn’t finished. In this case muscle comes in a condition of long contraction
which is named tetanus. It is result of summation of contractions. The given phenomenon is
connected with very short refractory period of skeletal muscle (depending on frequency of the
impulses coming from CNS). There are various kinds of tetanus. So, if impulses come in a phase of
muscle's relaxation there will be incomplete summation of contractions. Gear tetanus develops. If
each following impulse (at the great frequency of impulses from CNS) comes in a phase of muscle's
shortening the relaxation does not occur. Result is long contraction, i. e. smooth tetanus. Thus there
is full summation of contractions and its amplitude is more, than amplitude of single contraction or
gear tetanus.
1.1. Record and the analysis of single contraction and tetanus of a muscle
There are three phases of a curve of muscle's single contraction:
54
Figure 31. The single muscle contraction
1 The latent period - time from the moment of irritation till the beginning of muscle
contraction (0,01 sec.);
2 Phase of contraction - an ascending part of a curve - from the beginning of contraction up
to its maximum (0,04 sec.);
3 Phase of a relaxation - a descending part of a curve - from a maximum of contraction up
to the end of a relaxation (0,05 sec.).
Equipment: vertical myograph, kymograph, an electrostimulator, a frog, surgical instruments,

Ringer’s solution.
Procedure.
1. Prepare neuromuscular preparation and strengthen it in myograph.
2. Put electrodes directly on muscle and find the strength of irritation causing strong contraction
of muscle.
3. Record a curve consisting of 3-5 single contractions of a muscle with frequency of impulses
approximately in 0,5Hz.
4. After record of single contraction of a muscle start to increase gradually frequency of irritation
(up to 10-20Hz.) and lead to it to sizes when the muscle will start to be contracted as gear
tetanus, i.e. impulses will act in a phase of relaxation (fig. 32).
5. Continue to increase frequency of stimulation and record smooth tetanus. Duration of irritation
of a nerve in each case should not exceed 3-4 sec.
Compare the height of single and tetanic contractions.
6.Sketch the curve of single and tetanic kinds of muscle contractions.
7. Specify, at what frequency of irritation single contractions pass to gear, and
then in smooth tetanus.
55
Figure 32. Various kinds of muscular contractions;.
1-singlecontractions; 2 - gear tetanus; 3 -smooth tetanus
1.2. Theoretical Calculation of Frequency of Irritations for Reception of Various Kinds of

Muscular Contractions.
Knowing the duration of phases of single muscular contraction, it is possible to calculate
theoretically in experiment frequency of irritations for reception of various kinds of contraction-
single, gear and smooth tetanus.
For sural muscles of a frog the whole single cycle of contraction lasts 0, 1 sec. Thus duration of
the latent period is made 0,01 sec, a phase of contraction - 0,04 sec, a phase of a relaxation - 0,05
sec(fig. 31).
Use the formula for calculation of stimulus frequency:
V = 1/Т,
where V - frequency of stimulus (Hz), Т - the period of duration of each phase.
For example, it is necessary to calculate frequency of irritation for reception gear tetanus. We
choose a time interval from duration of phase relaxation, for example 0, 03 sec. We summarize time
of the latent period, a phase of contraction and the chosen time interval of relaxation phase -
0,01+0,04+0,05=0,1sec. We substitute the given data in the formula and calculate the frequency of
stimulus:
1/0,05=20 Hz.
Thus, at the given frequency of stimulus it is possible to observe muscle contractions as gear
tetanus.
56
Exercise 2. Optimum and Pessimum of Frequencies of Irritation.
It has been shown by N.E. Vvedensky in 1886 that the amplitude of tetanic contractions may be
various depending on frequency of irritations. There are optimum conditions at which the amplitude
of smooth tetanus is the greatest than it would be expected. Usually the force and frequency of
stimulus are moderate. But at excessively strong and frequent stimulus the effect provided to be
weakened - pessimal. It is connected with change of muscle’s functional condition (excitability)
under the influence of stimulus acting on the muscle depending on its frequency. If each subsequent
impulse acts with such interval when it reaches muscle in a condition of heightened (supernormal)
irritability, the amplitude of contraction will be maximal. If the impulses follow with such frequency
when each subsequent impulse reaches the muscle in a condition of depressed irritability(relative
refractory phase) the amplitude of muscle contraction will be minimal –pessimal. Thus, muscle’s
response to stimulation - optimal or pessimal response - depends on functional condition of muscles.
Values of stimulus frequency cause the maximal amplitude of tetanus, is named an optimum of
stimulus frequency. Values of stimulus frequency above optimum cause the minimal amplitude of
tetanus, is named a pessimum of stimulus frequency.
Equipment. Vertical myograph, kymograph, an electro stimulator, a frog, surgical instruments,

Ringer’s solution.
Procedure.
1. Prepare neuromuscular preparation and fasten to myograph.
2. Put electrodes directly on muscle and find the strength of irritation causing strong contraction of
muscle.
3. Find out such frequency of stimulation when smooth tetanus will be recorded.
4. Increase frequency of stimulation and record the highest amplitude of muscle contraction. Each
irritation should last for 3-4 sec, intervals between irritations lasts for of 1-2 minutes. Usually
frequency is 40-50 Hz (fig.21,2).It is an optimal frequency.
5. Then sharply increase frequency of stimulation above an optimal level and decrease the
amplitude of muscle`s contraction is observed. This frequency of stimulation is pessimal
frequency (fig. 33).
6. To prove, that pessimal contraction is not connected with muscle’s fatigue, quickly switch a
stimulator from pessimal to optimal value of frequency - the muscle contraction will be maximum
again.
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Figure 33. Various kinds of muscle contraction:1- single muscle contraction, 2- gear tetanus, 3-
smooth tetanus; 4-optimum; 5-pessimum.
Exercise 3. Dynamometry.
Principle. Each muscle by certain functional status has the ability of raising of weight of maximum
quality. This ability characterizes muscle strength.
It may be determined also by maximum strain which the muscle develops in condition of isometric
contraction. Muscle strength may be measured by load weight applied to the muscle. Absolute
muscle strength is one of characteristics of motor apparatus.
Various factors influence muscle strength: level of training, sex, age, etc. By determining the muscle
strength we can estimate physical development of the man. One of the known methods of muscle
force measuring is force of compression using manual Colin’s dynamometer.
Exercise3.1 Determination of Muscle Force.

Equipment. Carpal Colin’s dynamometer, stop-watch timer.
Procedure.
1. In vertical position extend an arm aside and press with a hand the carpal Colin’s dynamometer
with maximum possible force (fig. 34).
2. The arrow of dynamometer moves from left to right (from «0»). The level of its maximum
deviation is registered.
3.Repeat the test three times.
4. Find out the average of results obtained.
5. Determine the muscle strengths of both hands.
The hand muscle strength may determine by a formula:
Х= (F/ P) · 100%, where
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X – index of the muscle strength (%),
F – muscle strength of hand (kg),
P – body weight (kg).
This index equals for male - 60%, for female – 45% in normal.
Figure 34. Hand dynamometer
Average normal hand strength in men – 40-50 kg, among women – 25-35 kg.
Compare findings results with normal ones.
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THEME 3. COMMON PHYSIOLOGY OF CENTRAL NERVOUS SYSTEM
Lesson 1. General Characteristics of Reflex Action.
Questions for studying.
1. The functions of the central nervous system.
2. The morphological and functional properties of neuron. Classification.
3. Neuroglia. Functions.
4. Generation of action potentials in neurons and conduction of nerve impulses.
5. The synapses in central nervous system (CNS) (electrical and chemical). Classification of
synapses. Neurotransmitters in CNS. Synaptic potentials (EPSP and IPSP). Excitatory and
inhibitory neurotransmitters in CNS.
6. A reflex is as a main mechanism of central nervous system activity. Classification of reflexes.
7. Reflex arc. The main units of reflex arc. Varieties of reflex arc.
The central nervous system is a collection of nerve structures in the spinal cord and brain. CNS
receives information about changes in the external and internal environment and adapts physiological
systems to them. In the central nervous system, two types of cells are distinguished - neurons and
neuroglia. The main function of neurons is to generate an action potential. There are 10-50 times
more neuroglial cells than neurons (fig.35). They have basic and trophic functions.
Reflex is the basic principle of the central nervous system. A reflex is the body's response to changes
in the external and internal environment with the participation of the central nervous system.
Figure 35. Neurons and neuroglial cells.
The impulses are delivered to the body and dendrites of the neuron along the axons of other neurons.
Synapses are located between these axons and the body and dendrites of the neuron. Excitatory
postsynaptic potentials are formed on the body and dendrites of the neuron as a result of the
passage of impulses through synapses. Postsynaptic potentials are summed up on the axon of the
axon hillock. As a result of this summation, the membrane of the axon hillock is depolarized to a
critical level. This is excitement (action potential). Thus, the achievement of a critical level of
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depolarization on the membrane of the axon tubercle is a condition for the addition of EPSPs in the
neuron (fig. 36).
Figure 36. Excitatory postsynaptic potential
The morphological and physiological substrate of reflex is a reflex arc (fig.37).
A B
Figure 37. The reflex arc of somatic (A) and vegetative reflex (B).
Somatic reflex arc consists of 5 parts (fig.37, A):

I-Receptors .
II- Afferent part – sensory neuron (1) and his nerve fibers, which pass the excitation from periphery
to spinal cord.
III - Central part (gray substance of spinal cord) with interneuron (2), and motoneuron (3) connected
by synapses.
IV -Efferent part -motor fibers, which propagate the excitation from spinal cord
to effectors.
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V- Effector – the skeletal muscle.
Vegetative reflex arc consists of 5 parts(fig.37, B):
I- Receptors
II - Afferent part –sensory neuron(1) and his nerve fibers, which pass the excitation from periphery
to spinal cord.
III - Central part (gray substance of spinal cord) with vegetative neuron (4).
IV -Efferent part - vegetative fibers, which propagate the excitation from spinal cord to vegetative
ganglion (5) and then to effectors (V).
V- Effector – any target organ, which is innervated by vegetative fibers.
Exercise 1. Tendon Reflexes.

Principle. Tendon reflexes are deep proprioceptive (kinesthetic) reflexes and should be regarded as
stretch reflexes. A sharp tap on the slightly stretched tendon, will elicit an equally sharp contraction
of the corresponding muscles. These muscles show characteristic variations in many diseases and are
of great diagnostic value. Knee jerk, ankle jerk, biceps jerk, triceps jerk, etc., are the examples.
The muscle jerks are used by neurologists to assess the degree of facilitation of spinal cord centers.
When large numbers of facilitatory impulses are being transmitted from the upper regions of the
central nervous system into the cord, the muscle jerks are greatly exacerbated. On the other hand, if
the facilitatory impulses are depressed or abrogated, the muscle jerks are considerably weakened or
completely absent. These reflexes are used most frequently to determine the presence or absence of
muscle spasticity following lesions in the motor areas of the brain. Ordinarily, large lesions in the
contralateral motor areas of the cerebral cortex, especially those caused by strokes or brain tumors,
cause greatly exacerbated muscle jerks.
Patellar tendon reflex or knee jerk. Knee being semiflexed, a sharp tap on the patellar tendon with a
percussion hammer causes contraction of the quadriceps and the leg jerks up.
Reflex path. The sharp tapering on the patellar tendon causes stretch on the muscle spindle of the
extensor muscle and afferent impulses pass through the primary afferent fibres to the spinal
motoneuron via dorsal root ganglion. This afferent fiber synapses with the alpha motoneuron and the
contraction of extensor extrafusal muscle fibres occurs. With this a short jerky forward movement of
the leg occurs. It is a monosynaptic reflex. For this jerky movement the flexor muscle relax,
simultaneously with the contraction of the extensor, due to inhibitory motor influences exerted on the
antagonistic muscle group during reflex.
Purposes. To observe the myotatic reflex in a relaxed volunteer, and observe the effects of other
activity on the reflex.
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Equipment. Patellar hammer.
Knee-jerk (myotatic) reflex.

Procedure.
1. The volunteer sits in a chair and crosses the right leg over the left, the right foot swings backwards
and forwards freely.
2. The observer firmly taps the right patellar tendon (just below the knee cap) and watches the knee
jerk (fig. 38).
3. The volunteer cups and links the fingers of both hands, then pulls outwards very strongly, it is
called Jendrassik’s test.
4. The observer repeats step 3 while the volunteer continues to do this.
The briskness of knee jerk varies in different individuals. A pendular knee-jerk is seen in cerebellar
disease.
Figure 38. Knee-jerk (myotatic) reflex
Ankle (Achille's)jerk.
Procedure.
1. Place the lower limb on the bed, so that it lies averted and slightly flexed. With one hand,
dorsiflex the foot, so that the Achille's tendon, is stretched.
2. Now with the other hand strike the patellar hammer on the posterior surface of the tendon. A
sharp contraction of the calf muscles results (fig. 39).
3. The reflex can also be conveniently elicited when the patient is kneeling on a chair. Reflex
depends upon 1st and 2nd sacral segments.
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Figure 39. Ankle (Achille's) jerk
Biceps jerk.
Procedure.
1. The elbow is flexed at a right angle and forearm is placed in a semipronated
position.
2. The observer places his thumb or index finger on the volunteer’s biceps tendon
and strikes it with the patellar hammer. The biceps muscle contracts. Reflex
depends upon the 5th and 6th cervical segments of the cord (fig.40, a).
Triceps jerk.
Procedure.
1. Flex the elbow of the patient and allow the forearm to rest along his chest.
2. Tap the triceps tendon, just above the olecranon. The triceps muscle contracts.
Reflex depends upon the 6th and 7th cervical segments (fig.40, b).
Information about some important tendon reflexes is based in table 11.
Figure 40. a- Biceps jerk, b- Triceps jerk.
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Table11.
Brief details about some important tendon reflexes.
Tendon reflexes Method of eliciting Response Centre
Knee jerk or Tapping patellar tendon Jerking forward of Lumbar (L) 2 to 4
Patellar tendon leg
reflex
Ankle jerk or Tapping Achilles Plantar flexion of Sacral (S)2 to 4
Achilles tendon tendon foot
reflex
Biceps jerk Tapping biceps tendon Flexion of forearm Cervical (C) 5 to 6
Triceps jerk Tapping triceps tendon Extension of forearm Cervical (C) 7 to 8
The clinical significance of tendon reflexes.

1. Tendon reflexes may become stronger. Cause of this is lesion of pyramidal tract. The
pyramidal tract is the system through which the cerebral cortex transmits inhibitory impulses
to the spinal centers.
2. Tendon reflexes may become low or may be absent due to changes of reflex arc`s integrity
and conductivity.
3. Tendon reflexes may be irregular (anisoreflexia) due to some organic diseases.
Exercise 2. Analysis of the Spinal Shock in a Frog.

Purpose. To observe the spinal shock in a frog after spinal cord was destroyed.
Equipment. Stand with hook, scissors, tweezers (pincers), filter paper, Ringer’s
solution, H2 SO4, gauze, ether, cotton wool, stop-watch timer.
Procedure.
1. Observe the behavior of the intact frog placed on a board. The head is well raised on the forelimbs,
the hind limbs are flexed, and respiratory movements are present.
2. Give the frog a slight ether anesthesia.
3. Then cut off the upper mandible by scissors just behind its eyes. Any animal without brain is
named spinal animal.
4. Hang the spinal frog on its lower jaw at the tripod and note that a frog does not do any
movements. Pinch the skin with forceps - there is no reflex response. This state of activity
depression, accompanied by complete paralysis, is called spinal shock.
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5. The state of spinal shock disappears soon (the duration of spinal shock varies –from a few minutes
in a frog to a few months in a human). The legs are now drawn well up to the body. However, the
posture is quite different from normal. The frog does not make any ―spontaneous‖ movements- crawl
or jump. Some reflexes can, however, be demonstrated.
Exercise 3.Analysis of the Spinal Reflexes in a Frog.

Purpose. Study different spinal reflexes in a frog.
Equipment. Tripod with hook, scissors, tweezers (pincers), filter paper, Ringer’s solution, H2 SO4,
gauze, ether.
Procedure.
1. Hang the spinal frog on the tripod by its lower jaw.
2. Pinch one toe gently. Than a flexion of the limb occurs. This is a flexion reflex of the limb.
3. Put the piece of filter paper wetted with sulphuric acid solution on the limb and observe how the
frog begins to throw off the piece of filter paper. This is a withdrawal reflex.
Exercise 4. Analysis of the Reflex Arch in a Frog.

Principle. The main mechanism of activity of central nerve system (CNS) is a reflex. Reflex it is a
response of living organism to any stimulus (as external or as internal ones) by using of CNS. The
morphological and physiological substrate of reflex is a reflex arc.
Purposes. To learn the structure of reflex arc and to prove the importance of all parts of reflex arc.
Equipment. Tripod with hook, shears, tweezers (pincers), filter paper, ligatures, Ringer’s solution,
H2 SO4, gauze, ether, Novocain solution – 2%, cotton wool.
Procedure.
1. Prepare a spinal frog.
2. Hang the frog on the tripod by its lower jaw.
3. Immerse any hind limb up to knee joint of frog into the glass with 0,5 % sulphuric acid solution
and observe flexion reflex of the limb.
4. Then make a ring incision of skin around the hip and strip the skin from leg. Stimulate this leg by
sulphuric acid again and observe the absence of withdrawal reflex.
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5. Prepare the sciatic nerve at the frog hip at the distance of 1,5-2 centimeter and place the ligature
under the nerve. Then put the cotton wool soaked with Novocain under the ligature. This
procedure must evoke a blockade of nerve impulse conduction along the sciatic nerve. After the
certain period the defense reflex disappeared.
6. Destroy the spinal cord and repeat the experiment. Observe the disappearance of all reflexes.
Lesson 2. Properties of Nerve Centers. Central Inhibition.

1. The coordination of spinal reflex circuits (the standard reflex, the phenomenon of dominant).
2. Synaptic connections: the divergence and the convergence. The occurrence of decrement. The
long-term depression and long-term potentiation.
3. Plasticity of central synapses: facilitation, potentiation.
4. Spatial and temporal summation of EPSP.
5. The occurrence of occlusion. The transformation of rhythm impulses. The reciprocal
communication.
6. The central inhibition by Sechenov. Classification. Synaptic potential – IPSP (inhibitory
postsynaptic potential). Inhibitory neurotransmitters in CNS: GABA and glycine. Inhibitory
interneurons (Renshaw cells).
7. Classification of central inhibition.
8. Presynaptic inhibition. Mechanism.
9. Postsynaptic inhibition (recurrent, reciprocal, lateral inhibition). Mechanisms.
10. Varieties of secondary inhibition. Mechanisms.
The nerve center is a group of neurons at different levels of the central nervous system that regulate
one function or some activity of the body. The nerve centers have the following properties:
- The impulse spreads only in one direction from the receptor to the working organ.
- The center delay. The impulse quickly spreads along the nerve and is delayed in the nerve center,
because of the chemical synapses.
- Summation. A reflex reaction occurs to several subminimal stimuli. In this case, postsynaptic
potentials are summed on the axonal hillock. Summation can be temporal(fig.41, a) and spatial
(fig.41, b).
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a b
Figure 41. Summation a- temporal, b - spatial
- Rhythm transformation. An irritant acts on the receptor at one frequency, but the impulse leaves the
nerve center at a different frequency.
- Aftereffect. The stimulus has already stopped to act on the receptor, but the impulses continue to go
out to the working organ.
- The tone of the nervous center is a state of increased excitability. This state is maintained by
impulses from the overlying parts of the central nervous system and impulses from peripheral
receptors.
General principles of coordination in the central nervous system.

Reflex coordination is the interaction of the processes of excitation and inhibition in the central
nervous system, which ensures the adaptation of the organism to environmental conditions. Approval
principles:
- Divergence of signals. The impulse comes to CNS` from the periphery and disperses on branches
of an axon or through inserted neurons. Many central neurons are excited in result (fig. 42 a).
- Convergence of signals. Multiple paths end (converge) on one central neuron. These neurons
receive impulses from many other neurons (fig. 42 b).
- The principle of dominance. Discovered by A.A. Ukhtomsky. Dominance is a group of nerve
centers with increased excitability. The dominant possesses the properties of hyperexcitability, the
ability to summarize (sums up the impulses coming from other receptive fields) is inertial (once it
has arisen, it does not disappear completely).
- Reverberation - circulation of impulses through closed circuits of neurons (fig.42 c).
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a b c
Figure 42.Principles of coordination. a –divergence, b – convergence, c – reverberation.
Irradiation -the impulse spreads to many neighboring centers and causes their activation.
Final common path - many nerve pathways converge to one motor neuron.
Central Inhibition.
Inhibition in the central nervous system is an active process that occurs as a result of one excitation
and is aimed at weakening another excitement. There are two types of inhibition in the central
nervous system: postsynaptic and presynaptic.
Postsynaptic inhibition occurs on the postsynaptic membrane of the inhibitory synapse (fig. 43).
The pulse comes to the terminal. The Ca2+ channels are open. The transmitter exits through the
presynaptic membrane, diffuses through the synaptic cleft, and binds to receptors on the postsynaptic
membrane. Channels for K+ and Cl- open and elicit appearance of inhibitory postsynaptic potential.
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Figure 43.Inhibitory synapse, inhibitory postsynaptic potential.
Presynaptic inhibition.
Presynaptic inhibition occurs at the axo-axonal synapse (fig. 44). This synapse is formed by the axon
of excitatory neuron (neuron I) and the axon of the Renshaw cell (inhibitory neuron). The impulse
propagates along the axon of neuron I and causes the formation of the excitatory postsynaptic
potential (EPSP) of neuron II. This impulse fades away and does not cause activation of neuron II if
the impulse on the axon of the Renshaw cell simultaneously enters the axo-axonal synapse. Thus, the
excitatory postsynaptic potential on neuron II does not arise.
Neuron II
Figure 44.Presynaptic axo-axonal inhibition.
I.M. Sechenov made an experiment in 1863. He exposed a section of thalamus in a frog and
determined the time of the reflex according to Turk. This time lasts from the moment the frog's hind
leg is lowered into acid until the frog's leg is raised. Then Sechenov put salt crystals on the thalamus
and again measured the time of the reflex according to Turk.
The second time was longer than the first time. Sechenov made 2 conclusions.
- There is an inhibitory process in the central nervous system;
- The brain has an inhibitory effect on the spinal cord.
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Exercise 1. Temporal summation EPSP in the Motoneurones in a frog.
Principle. If a number of subminimal stimuli are applied, their effects will be summated together
and the excitatory postsynaptic potential (EPSP) will be sufficient to induce the motoneurons to
discharge impulses and produce the reflex response – the frog makes jump.
Equipment. Stand with hook, a thalamic frog, shears, tweezers (pincers), ligatures, Ringer’s
solution, gauze, ether, cotton wool, electric stimulator.
Procedure.
1. Prepare at thalamic frog, make incision behind frog's eyes, and then hang it on the tripod by its
lower jaw.
2. To a back paw of a frog bring electrodes from an electric stimulator. Establish frequency of
irritation of 1 Hz and, gradually increasing force of a current, observe occurrence of weak
contractions of muscles of a shin.
3. Find out threshold force of a current which establishes frequency of stimulation of 20-50 Hz and
irritates a back leg of a frog. The frog makes a jump on stimulus of high frequency.
Exercise 2. Measurement of reflex time by Turk’s method.

Principle. The reflex time by Turk’s method is the time from the beginning of action of stimulus
before occurrence of the reflex response which measure a metronome with frequency of 100 impacts
in a minute.
Purposes. To observe the defense reflex in spinal frog in response to the stimulation by different
concentrations of sulphuric acid, and to measure a time of defense reflex with each acid
concentrations.
Equipment. Stand with hook, shears, tweezers (pincers), filter paper, Ringer’s solution, 0,1%,
0,25%, 0,5% and 1,0% H2 SO4 (sulphuric acid), gauze, ether, cotton wool, water, metronome (stop
watch).
Procedure.
2. Hang the spinal frog on the cupric hook.
3. Immerse any hind limb suspended frog into the glass with sulphuric acid solution in
concentration 0,1 % and simultaneously switch on the stop-watch (metronome) to measure the
duration of reflex response – that is time between the moment of immersing the limb into acid
71
and withdraw this limb from the acid. This time is so-called flexion reflex or defence reflex
time.
4. After each contact of a frog’s back paw with sulphuric acid solution you should wash it in
water.
5. Wait for 1-2 minutes for recovery nerves and muscle functions.
6. Repeat step 3 of procedure with 0,25%, 0,5 % and 1,0% sulphuric acid solution.
Note that the more concentration of sulphuric acid the shorter reflex time. The reflex time depends
on force of irritation. If force of irritation increases, then time of a reflex decreases.
Exercise 3. Irradiation of Excitation.

Equipment. Stand with hook, shears, tweezers (pincers), filter paper, Ringer’s solution, gauze, ether.
Principle. The spinal frog at weak irritation of toes of its back paw has a weak reflex movement of
limb. At gradual strengthening irritation observe involving in reflex reaction of other limbs of a frog.
Procedure.
2. Hang the spinal frog on the cupric hook.
3. Pinch one toe gently. There is only a slight reflex movement of that limb.
4. Pinch the toe with greater force, the movements increase accordingly and the other limb, and
even the whole animal, may exhibit movements. This state is called irradiation of excitation
or irradiation of reflexes.
If the sensory stimulus is too strong, the impulse will spread on many neighboring neurons in the
center and produce a wider response. A strong stimulus will cause reflex contraction of the extensor
muscles of the opposite limb.
Exercise 4. Sechenov’sInhibition Reflex.

Principle. Sechenov inhibitory mechanism. The bodies of the neurons of the reticular formation lie
in the region of the thalamus. The axons of these neurons descend into the spinal cord and connect
with Renshaw's inhibitory cells. The Renshaw cell axon forms inhibitory synapses on motor neurons.
The impulse along the axons of the neurons of the reticular formation in the spinal cord decreases
and excites Renshaw cells when salt is applied to the thalamus. The impulse travels along the axon of
the Renshaw cell to the motor neuron and causes the formation of IPSP.
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The spinal reflex is inhibited (fig 45).
Equipment. Stand with hook, shears, tweezers (pincers), filter paper, Ringer’s solution, salt
crystals,0,1%, 0,25%, 0,5% and 1,0% H2 SO4(sulphuric acid), gauze, ether, cotton wool, water.
Procedure.
Experiment is carried out on a thalamic frog.
1. You have to make incision behind frog's eyes, and then hang it on the tripod by its lower jaw.
Figure 45.Mechanism of Sechenov’s inhibition

2. Define reflex time by Turk (2-3 times).
3. After that take the frog away from the tripod, dry the surface of the incision with the cotton
wool.
4. Carefully uncover thalamus by scissors. Dry the incision by cotton wool and hang up the frog
on a tripod again.
5. Put a salt crystal on the incision, try to identify reflex time by Turk immediately. Note if the
time of reflex would get longer.
6. Take away the salt crystal; wash the surface of thalamus with a physiological solution. After
5 minutes try to determine the reflex time once again. Check, if it will take the same time.
7. Pay attention if convulsive contraction occurs after putting the salt crystals. It means that the
brain surface was dried badly; salt was dissolved and permeated into lower brain sections. In
this case
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you have to wash the brain surface again, dry it carefully and repeat this work from step1
(fig.46).
Figure 46. The central inhibition (by Sechenov).

Exercise 5. Golz’ Reciprocal Inhibition of the Spinal Reflexes.
Equipment. Stand with hook, shears, tweezers (pincers), filter paper, Ringer’s solution, 0,5% H2
SO4(sulphuric acid), gauze, ether, cotton wool, water, metronome (stop watch).
Procedure.
2. Hang up the spinal frog on a tripod.
3. Put its leg into the 0,5% sulphuric acid solution and observe the reflex of flexion.
4. Determine reflex time by Turk.
5. After that, during putting the frog’s leg into the sulphuric acid, you have to press another leg
with the Pean’s squeezer. The reflex of flexion won’t appear, or the reflex time become much
longer.
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THEME 4. CARDIO-VASCULAR SYSTEM.
Lesson 1. Physiology Properties of Cardiac Muscle.

1. Structure of the heart.
2. Physiology of cardiac muscle, its basis properties (excitability, automatism,
conductivity, contractility).
3. Action potentials of non pacemaker cells and pacemaker cells. Pacemaker
potential. Refractory period of cardiac muscle.
4. The cardiac cycle. Pressure changes during the cardiac cycle. Function of the
atrioventricular and semilunar valves.
5. Conduction of the impulse. Conducting tissues of the heart. Experiment by
Stannius.
6. Contraction of cardiac muscle. Mechanism of contraction.
The heart is a generator of pressure in the vascular system, that is, it creates a driving force for the
movement of blood in the large and small circle of blood circulation (fig.47).
Figure 47. The phases of cardiac cycle.
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The pumping function is provided during the cardiac cycle, which is understood as one complete
contraction and relaxation of the atria and ventricles of the heart. There
are 3 phases of the cycle: atrial systole, ventricular systole, total diastole
Indicators of the hemodynamic function of the heart are: stroke, or systolic volume (SV), minute
volume (MV).
The minute volume is determined by the following formula:
MV = SV (60-70 ml) • HR (60-80 beats / min) = 4.5-5 liters
I. Automaticity
Heart automaticity (a specific property of the heart muscle) is the ability of the heart to be excited
under the influence of impulses that arise in it without external stimuli. The rhythmic activity of the
heart occurs due to the presence in the right atrium of the center of automation - this pacemaker
region is called the sinoatrial node (SA node). The SA node is located in the rite atrium, near the
opening of the superior vena cava. It provides the heart rate 60-80/min. If this node is damaged for
some reason the second pacemaker will generate impulses –atrioventricular node (AV node),
which is located on the inferior portion of the interatrial septum. It is called the pacemaker of second
order. The frequency of impulse generation of AV node is about 40-45/min. The next department of
heart conducting system – is a bundle of His (pronounced ―hiss‖) and left and right branches,
beginning at the top of interventricular septum. They provide heart’s rate30-35 /min. Finally, within
the ventricular walls the Purkinje fibers are located –the last part of heart conducting system with
heart rate only about 20/min (fig. 48).
Figure 48. Cardiac conductive system.
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Physiological properties of the heart muscle.
Normally, the pacemaker of the heart is the sinoatrial node. On the outer surface of the membrane of
these cells, a decrease in resting membrane potential occurs, called slow diastolic depolarization
(SDD).
There are several reasons for SDD:
- During diastole, there is a decrease in membrane permeability for potassium ions.
- High permeability to incoming sodium and calcium ions. Therefore, the value of the membrane
potential of the cells of the sinoatrial node is 60 mV.
- During diastole, the activity of Na + –K + - ATPase decreases.
When the level of the resting potential decreases to the threshold, a sharp increase in membrane
permeability occurs, first for Na +, and later for Ca2 +, an action potential (AP) arises - the true
pacemaker potential (fig. 49).
Figure 49. Pacemaker potential.
Action potential arrives in sino-atrial node (the true pacemaker)earlier than in latent pacemakers their
own SDD. The automatism of the other pacemaker will manifest itself only in those cases when it
does not receive impulses from the sinus node.
Gaskell established the law of the automatic gradient of the heart, according to which the less
automatic ability the closer the pacemaker is to the apex of the heart.
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II. Conductivity:
1. The excitation propagates at a speed of 4 m/s on the conducting system of the heart.
2. The presence of an atrioventricular delay, the duration of which is 0,1 s.
3. Slower conduction of excitation along the working myocardium - up to 1 m / s.
III. Excitement and excitability.
Excitability of a working cardiac muscle is characterized by a fast phase of depolarization and two-
stage phase of repolarization: slow initial repolarization, plateau and fast repolarization. During this
stages it is observed long period of absolute refractivity (fig. 50). This period lasts for the whole
phase of depolarization, the whole phase of slow repolarization, plateau and about a third of a phase
of fast repolarization. The long period of absolute refractivity of a working cardiac muscle makes the
work of heart possible only in the mode of single muscular contraction (not tetanus) and makes
impossible retrograde distribution of excitement.
Figure 50. Excitation and contractility curves of working myocardium.
IV. Contractility of a cardiac muscle

Contractility of a working cardiac muscle submits to the law ―all or nothing‖ – the muscle doesn`t
contract by subthreshold incentives, and superthreshold incentives with the maximum amplitude.
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Exercise 1. Study of Cardiac Cycle and Autonomy of Different Parts of Frog’s
Heart. Stannius’s Ligatures.
Principle. The cardiac cycle is a repeating pattern of contraction and relaxation of the heart muscle.
The phase of contraction is called systole and the phase of relaxation is called diastole. Usually this
terms are referred to contraction of ventricles, however the atria also contract and relax. Two atria
are filled with blood and then contract simultaneously. This is followed by simultaneous contraction
of both ventricles, which sends blood through the pulmonary and systemic circulations. Contraction
of the ventricles closes the atrioventricular (AV) valves and opens the semilunar valves; relaxation of
the ventricles causes the closing the semilunar valves. The blood from systemic circulation hits to
aortic valve and again return in systemic circulation vessels. Theme an duration of whole cardiac
cycle (at heart rate 75 /min) is 0,8 seconds. Then the heart pause occurs, which lasts about 0,4
seconds. The main properties of the heart – is an automaticity. The automaticity – is a heart ability
to contract under the influence of impulses, arising in heart itself. If the heart of frog (or human) is
removed from the body and neural innervation is severed, it will still continue to beat as long as the
myocardial cells remain alive.
In frog’s heart the following parts of conducting system are distinguished: 1) Remak bundle, located
between venous sinus and atria, 2) Bidder bundle in the interatrial septum, 3) Purkinje fibers, which
coming from Bidder bundle.
Purposes. Observation of the hearts’ activity and the automaticity phenomenon.

To investigate the conductive system of the frog heart by Stannius’s ligatures.
Figure 51. Application of Stannius’ ligatures: 1,2,3, - 1st, 2nd, 3rd Stannius’ ligatures, respectively.
Contracting parts of heart are shown in dark.
79
Procedure.
Exposure of heart.
1. Give a frog a slight ether anesthesia.
2. Cut off the upper mandible by shears just behind its eyes and destroy quickly the spinal cord by
cupric probe.
3. Make the mid-line incision though the skin over a sternal region from the xiphisternum
(cartilaginea pars of sternum) to lower jaw.
4. Remove the triangular pieces of skin on both sites of incision and anterior chest
wall is now exposed. Rise the xiphisternum by forceps and make a horizontal cut through the muscle
and remove the anterior chest wall.
5. The heart is now revealed beating within the pericardial sac. Remove the parietal pericardium.
Analysis of automatic contraction of the whole heart.
1. Count the rate of frog heart in normal condition – it will be a control mean.
2. Separate the venous sinus from two atria by 1-st Stannius ligature – that is tie a ligature at the level
of white crescentic line (between the sinus and atrium of the frog heart). The venous sinus is seen to
beat at the previous rate, but the heart stops after a few beats (fig.51,1).
3. Count the rate of the sinus and compare it with that of the other portion of the heart. The heart may
resume beating after some minutes, but it will be better to tie a 2nd ligature at the atrio-ventricular
groove. This ligature stimulates the second node –Bidder bundle of frog heart (or atrioventricular
node of human heart). Note, that the heart rhythm is decreased – about 2 times. The second ligature
is applied in the same heart in between the atrium and ventricle(fig.51,2).
4. The third ligature is applied on apex of the heart. Note, that the apex of the heart of a frog is not
contraction. If to irritate a apex of heart, then the myocardial tissue will be contracted (fig.51,3).
Results of supervision write down in the table 12.
Table 12
The results after applying ligatures on the heart
Kind of activity The venous sinus The atria The Apex of the heart
ventricle
The heart rate in normal
condition
First Stannius lugature
Second Stannius ligature
Third Stannius ligature
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1. The heart will continue to beat outside the animal’s body. This phenomenon is called automaticity.
2. The heart rhythm is maximal in the sinus, less in the atria and least in the ventricle. In the intact
heart atrial and ventricular activity is subordinated to the sinus. This is called the sinus rhythm.
3. In normal heart the SA node controls the rest of heart muscle by its higher rhythmical activities.
Exercise 2. Determination of Duration of a Cardiac Cycle.
Principle. The cardiac cycle consists of a systole of atria, a systole of ventricles and the general
diastolic pause of heart. The resting heart is 60-80 beats per minute. At the healthy person in a
condition of rest duration of a cardiac cycle makes in norm 0,6-1,0 sec.
Duration of the cardiac cycle count under the formula: T= 60 seconds: frequency of contraction of
heart in a minute (Т-duration of the cardiac cycle).
Procedure. Ask the patient to be seated comfortably and fully relaxed. Note the presence of the
main arterial radial pulses by palpating this vessel on one side. Examine the radial pulse at the wrist
with tips of the first three fingers. Count up pulse within a minute. Calculate duration the cardiac
cycle under the formula:
T= 60 seconds / frequency of contraction of heart in a minute
Compare result to norm (60-80 beats per minute).
Duration of a cycle increases during a dream and in a condition of functional rest, the cycle
decreases during physical or emotional loading.
Exercise 3. Features of excitability of the heart. Extrasystole.

Purpose: to investigate the excitability of the ventricles of the heart in different phases of its activity.
Procedure: immobilize the frog and prepare it for cardiography. Attach one of the stimulator
electrodes to the surf in, the other - under the frog's back. The initial contractions of the heart are
recorded. The optimal voltage for electrostimulation is selected. While recording the contractions of
the heart, single stimuli are applied in the following phases of the cardiac cycle: in the middle of the
contraction of the ventricle, at the height of contraction of the ventricle, in the middle of its
relaxation and during diastole. The appearance of extra systoles is observed. At the same time,
attention is paid to the increase in the pause after the extrasystole (compensatory pause) (fig. 52).
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Figure 52. Extrasystole and compensatory pause.
A - I - contraction (arrows show irritations applied in different phases of the cardiac cycle, triangles -
impulses from the sinus node); II - excitability. B - contraction curve.

Lesson 2. Regulation of Heart Activity.
1. Heart's innervations by wandering and sympathetic nerves.
2. Influence of wandering nerve on heart. Meaning of tone of a wandering nerve in
regulation of heart activity.
3. Influence on heart of sympathetic nerves. Strengthening nerve by Pavlov.
Trophic character of its action.
4. Reflex regulation of activity of heart. Vagal and sympathetic reflexes.
5. The mechanism of transfer of nervous impulses on heart (experience by Levy), mediators, choline
-and adrenoreactive structures of heart.
6. Influence of cerebral cortex on work of heart.
7. Hetero-and homeometric self-control of hear beat. «The law of heart» by Franc-Starling. A
phenomenon of staircase by Bowditch. Effect by Anrep.
8. Intracardial mechanisms of regulation of heart activity.
9. Influence on activity of frog's isolated heart of temperature, electrolytes, mediators, hormones and
other substances.
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Regulation of heart work.
Pump function of heart changes at transition from a condition of functional rest to active (for
example, orthostatic, psycho-emotional effort, physical work, extreme situations). These changes are
carried out due three mechanisms of regulation of heart activity:
1. intracellular mechanism;
2. intracardial mechanism;
3. extracardial mechanism.
Intracellular mechanism.
They are caused by properties of cardiac muscle, therefore they are named myogenic
mechanisms. Two kinds of such regulation are distinguished:
1. Heterometric self-control mechanism.
Force of cardiac muscle's contraction depends on length of fibers of myocardium. This
mechanism is known as «the law of heart» by Franc-Starling (fig.53).
Basically, the Frank-Starling mechanism means that the greater the heart muscle is stretched
during filling, the greater is the force of contraction and the greater is the quantity of blood
pumped into the aorta. This is a heterometric autoregulation.
Fig. 53. Frak-Starling Law of the heart
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2. Homeometric self-control mechanism of heart activity. Changes of force of cardiac muscle’s
contraction occur at not changing length of myocardium fibers. Force of myocardium'
contraction grows due the following factors:
 As response to increasing of exiting frequency. It's called phenomenon of staircase by
Bowditch.
 As response to increasing of resistance to ejection of blood from left ventricle into aorta. It's
called effect Anrep. The more is the pressure in the blood vessels leaving the heart (aorta) the
greater is the force of contraction of the cardiac muscle.
Intracardial mechanisms.
They are realized due to so-called peripheral reflexes which reflex arches are located within the
myocardium. These reflexes provide stability blood filling of arterial system (fig.54).
Figure 54. Structure of intracardial nervous system (by G.I.Kositskiy).
Extracardial Mechanisms. Reflex (nervous) mechanisms are caused by influence of vegetative

system (on sympathetic and parasympathetic nerves). These mechanisms are expressed in change of
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frequency of heart contractions, excitability, velocity of carrying out of excitation and force of heart
contraction.
The parasympathetic nerves are distributed mainly to the S-A and A-V nodes and to the muscle of
the two atria. The sympathetic nerves, conversely, are distributed to all parts of the heart. If you raise
the tone of these nerves four tropic effects arises (tab.13).
Table 13.
Effects on the heart of the vagus and sympathetic nerves
Tropic effects Sympathetic Parasympathetic
nervous system nervous system
I. Chronotropic – the change in a heart rate + -
II. Inotropic – the change in the force of cardiac + -

contractions
III. Dromotropic – the change of conductivity + -
IV. Batmotropic – the change in excitability + -
Parasympathetic influences are carried out by a vagus nerve. Inhibitory influence is typical for it's
steady (the nucleus of vagus nerve is in a tone which is supported by afferent impulses from an arch
of aorta and from carotid sinus). The mechanism of inhibitory influence is as follow:
1. Acetylcholine escapes in the ends of postganglionic fibers of vagus nerves; Acetylcholine
acts with M-cholinergic receptors of postsynaptic membranes;
2. The permeability of cellular membrane for K-ions increases; hyperpolarization of cellular
membrane occurs;
3. Therefore excitability and conductivity of myocardium decrease (due to increase a threshold
depolarization);
4. Velocity of SDD in pacemakers is decreased and so frequency of contractions decreases.
5. Besides increasing of permeability of membrane for K-ions prevents from coming into the
cell Ca-ions that leads decreasing of contraction's force.
Sympathetic influences are follow:

1. Noradrenalin is secreted by sympathetic postganglionic fibers.
2. Noradrenalin interacts with β-adrenoreceptors of postsynaptic membrane of cardiac myocytes
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3. Thus their permeability for Na⁺ and Ca²⁺ ions increases, that leads increasing of excitability
and conductivity of myocardium (due to its depolarization) and of force of contraction.
4. Increasing of velocity of SDD under influence noradrenalin leads to increasing of frequency
of heart beat. (fig.55).
Figure 55. Sympathetic and parasympatic innervation of heart:

VMC - vasomotor center; Sc- a spinal cord; Sg- sympathetic ganglion, Th- thoracic segments, Ach –
Acetylcholine, NA - Noradrenaline .
Two kinds of proper cardiac reflexes are distinguished:
1. Vagal reflexes (Danyny-Ashner’s reflex, Golz’ reflex, etc.). They decrease frequency and force
of heart contractions.
2. Sympathetic reflexes are expressed in development of tachycardia and increasing force of
contraction.
Vagal and sympathetic reflexes are usually combined with change of a tone of vessels and breathe.
Humoral influences. Calcium, potassium ions, hormones are transported by blood. And these
factors act on heart (fig.56).
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Figure 56.Character of influence of some humoral factors of heart activity (pointers show the
beginning of irritation).
1. Ca²⁺ions are necessary for electromechanical coupling. Under influence of action potential they
leave sarcoplasmic network and incorporate with regulatory protein – troponin. Therefore active
centers of actin open, that provides formation of actomyosin complex and contraction of a muscle.
So increasing of Ca²⁺ ion’s concentration in blood causes increasing the force and frequency of heart
contraction.
2. Excess of K⁺ ions leads to inhibition of heart beats till its stop in diastole phase. It is caused with
decreasing or even disappearance of concentration gradient. It leads to decreasing or stop potassium
outflow from the cell and the level of resting potential is decreased. So excitability downs to total no
energized condition.
3. Adrenaline (a hormone of adrenal glands) has a specific effect on heart. It causes increasing of
force and frequency of heart beats.
4. Glucagon and corticosteroids cause increasing of force of heart contractions. Iodine-containing
hormones of thyroid gland increase frequency of heart contractions.
5. Acetylcholine has braking influences on activity of heart and renders as inhibitory factor.
Exercise 1. The effect of Vagosympathetic Trunk Stimulation on the Frog's Heart and
Phenomenon of Vagal Escape.
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Principle. The frog's heart is supplied by both sympathetic and parasympathetic divisions of the
autonomic nervous system. The first neurons, which axons are the sympathetic (accelerator) and
parasympathetic vagal (depressor) fibers, are located in the central nerve system. The frog's mixed
vagosympathetic trunk consists of both types of autonomic nerve fibers; therefore it contains
postganglionic sympathetic and preganglionic fibers.
Purposes: to study features of vagosympathetic trunk stimulation's effect on the frog's heart.
Procedure.
1. Destroy a frog's cerebrum and spinal cord in the bloodless way.
2. Fix the frog on preparation board.
3. Expose the frog's heart and then find a neurovascular bunch (it consists wandering, sympathetic,
guttural nerves, and also a skin artery). Hypoglossal, glossopharyngeal and humeral nerves serve as a
reference point for finding vagosympathetic trunk (fig. 57). The neurovascular bunch goes outside
obliquely and down wards.
4. Prepare a neurovascular bunch by means of a glass hook and count the frequency of heart beat.
5. Impose ligature also probably further from heart.
6. Carefully raise a neurovascular bunch by thread, bring under it electrodes.
7. For irritation use an electro stimulator. Stimulate a neurovascular bunch with impulses with
frequency of 50 Hz and amplitude - 20-30 V during 5-10 sec. Count the frequency of heart beat after
stimulation and after10 min.
Figure 57. Topography of vagosympathetic trunk of frog: 1 - glossopharyngeal nerve, 2 -

hypoglossal nerve; 3 - ligature;4-neurovascular bunch; 5-humeral nerve.
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Delay and weakening of cardiac activity are recorded (even stoppage of heart)just after
stimulation vagosympathetic bunch (which are caused by action of wandering nerve). After the
termination of stimulation force and frequency of reductions of heart come back to norm, and then
increase (fig. 58 a). It occurs due to excitation of sympathetic nerves which were stimulated at the
same time with parasympathetic nerve. But sympathetic nerves have longer latent period and a long
aftereffect. So the effect from their irritation occurs later and lasts longer. Preliminary atropinization
of heart ―switches off‖ wandering nerve's influence (fig. 58b).
Figure 58. Change of frog's heart activity during stimulation of vagosympathetic trunk:
a -vagal inhibition and subsequent sympathetic aftereffect; b – recording of heart rate in 10 min after
application of Atropine.

1. The parasympathetic nerve stimulation has an inhibitory affects on the heart.
2. The stimulation of sympathetic nerve fibers lead to the increasing of muscle contraction amplitude
and rate of heart beats.
3. Strong stimulation of the vagus stops the heart beats, however if the stimulation is continued the
heart escapes from the inhibitory effect and starts beating again.
Exercise 2. Reflexes of Heart (Golz’ reflex).

Purpose: to observe Golz’ reflex in frog’s heart.
Procedure.
1. Eliminate cerebral hemispheres making incision behind frog’s eyes.
2. Fix the frog on preparation board with abdomen up.
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3. Expose heart with small window in a chest wall. Count the frequency of heart beat.
4. Wait for 10-15 minutes, then give a slap in the stomach with the handle of a tweezers vigorously.
Heart stops for some min.
5. After contractions of heart will be restored, destroy a spinal cord and repeat experience again.
Cardiac arrest does not occur.
6. The reflex arch of this reflex is shown on figure 59.
Figure 59. Holtz’ reflex. 1 -receptors of intestines; 2 - afferent pathway; 3 - a spinal cord; 4 -
medulla;5 - a wandering nerve; 6 – effector’s organ (heart).
Exercise 3. Eyeball-cardiac Reflex (Danyny-Ashner’s reflex).

Purpose: to examine Danyny-Ashner’s reflex at the person.
Procedure.
1. Count the patient’s pulse frequency.
2. Then ask him to close his eyes. Put four fingers to temporal surface of patient’s head and press on
his eyes with thumbs slowly during 10-20 s. Do not press on both closed eyes strongly, then quickly
stop press. Again count up frequency of intimate reductions and compare to initial size. Pulse is
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counted up right after with pressings on eyeballs and through 5 mines after the termination of
influence(fig.60).
Figure 60. Danyny-Ashner’sReflex.
Exercise 4. The effect of some factors (temperature, electrolytes, hormones, mediators) on

frog's heart.
Purpose: To obtain and investigate the effects of some factors on the frog's heart activity.
Procedure.
1. Expose the frog's heart as describe on exercise 1.
2. Tie up both arches of aorta; do not cutting off ligature (because it is convenient to hold).
3. Pass ligature below place of bandaging of the left arch of aorta and do a small loop. Then make
an incision in the left arch of aorta with thin scissors. Advance the end of Shtraub’s cannula into
ventricle through an aperture with a probe.
4. Tight a loop that was imposed on the left arch of aorta, strengthen heart on cannula. After that
quickly fill cannula with solution by Ringer. Raise cannula together with heart, cut both arches of
aorta and then make the second cut below vena cava, cut out heart from frog’s body, do not
damaging venous sinus.
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5. Cannula with isolated heart is connected to Mariot’s vessel (there is warm Ringer’s solution, (t =
36-37C)in it) using rubber tubule.
6. Perfuse heart with Ringer’s solution. Count initial frequency of heart contraction for a minute,
then examine the influence on isolated heart cold (t = 8-10°C) and warm (t = 36-37°C) Ringer’s
solutions, excess of potassium and calcium ions, adrenaline and acetylcholine. Achieve recovery
of frequency of heart contraction each time after action of given factors.
7. Write down the results into table 14.
Table 14
Effects of influence of different factors on cardiac muscle

Factors Effects of influence on Conclusions
cardiac muscle
Warm Ringer’s solution
Cold Ringer’s solution

Excess calcium ions
Excess of potassium ions
Adrenaline
Acetylcholine
Lesson 3. The Modern Investigation Methods of Heart Work.

1. Modern methods of heart investigation: electrocardiography, phonocardiography.
General analysis of their benefits.
2. Electrocardiography, methods of monitoring.
3. Analysis of electrocardiographic curve, genesis of specific waves and intervals.
Their importance for the clinical picture.
4. Vector theory of ECG formation. Electric axis of heart and importance of its
determination.
5. Cardiac tones, their origin.
6. Phonocardiography, methods of monitoring, analysis of phonocardiographic curve.
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Presently the practice cardiology applies electrocardiography and phonocardiography
methods most often. These methods allow giving the unbiased information about affectability and
conductibility transformation during of heart work. All of these methods are free and handy for
speedy checkup of cases.
Electrocardiography.
The ECG is a record of the electrical activity of the heart. The body is a good conductor of
electricity because tissue fluids have a high concentration of ions that move in response to potential
differences. Potential differences generated by the heart are thus conducted through the body surface,
where they can be recorded by surface electrodes placed, on the skin.
The recording of small extracellular signals produced by the conduction of action potentials
through cardiac myocytes is called an electrocardiography and obtained record -electrocardiogram
(ECG). The recording device (apparatus) is called an electrocardiograph. Note that the ECG is a
result of generation and conduction of action potentials in the heart, but is not a registration of heart’s
contraction.
The cardiac impulse (wave of depolarization) passes quickly through the conductive system
from muscle cell to muscle cell by syncytium. There is a slight pause between the atrial and
ventricular contractions due to normal partial block to the passage of excitatory wave through the
atrio-ventricular node.
There are two rules of typing of ECG recording electrodes or ―leads‖: according to order of
electrodes – the limb and chest, according to physical principle - the bipolar and unipolar.
The conventional leads. The bipolar leads record the voltage between electrodes placed on
the wrists and legs. These leads were requested by Einthoven and they were indicated Roman
numerals I, II, III:
The lead I - right arm to left arm.
The lead II - right arm to left leg.
The lead III – left arm to left leg.
The right leg is used as ground lead (electrode). The left arm electrode marks letter L, the
right arm electrode marks - R, the left leg electrode marks - F.
There is equilateral triangle by connecting of all these points (fig.61).
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Figure 61.Einthoven’striangle, different R waves in conventional leads (I, II, III).
The unipolar limb leads.

In the unipolar leads voltage is recorded between a single ―exploratory or active electrode‖
placed on the body and an electrode that is built into the electrocardiograph and maintained at zero
potential (ground). The unipolar limb leads are placed on the right arm is abbreviated aVR, left arm -
aVLand left leg (foot) – aVF (fig. 62)
Figure 62. The unipolar limb leads and ECG.
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There are also unipolar chest leads (by Wilson, 1946), which labeled 1 to 6 – see fig. 63.The
active electrode sets on the chest, the indifferent electrodes connect with ―ground‖.
There are 6 positions of the chest electrode:
1.The right edge of 4th intercostal space.
2. The left edge of 4th intercostal space.
3. The midline between the left edge of 4th intercostal space and the apex of heart.
4. The nipple line of 5th intercostal space.
5. The left frontal axillaries lineof 5th intercostal space.
6. The left middle axillaries lineof 5th intercostal space.
There are thus a total a twelve standard ECG leads that ―view‖ the perpetually changing pattern of
the heart’s electrical activity from different perspectives.
Figure 63. The chest leads.
The nature of electrocardiogram waves

There are three positive P, R, T waves on ECG and two negative Q and S waves. The
conductivity process reflects by intervals (fig.64).
P wave represents depolarization of atrial muscle. The peak is 0,25 mV. The width is 0,06 -
0,1 sec.
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Q wave reflects excitement of intraventricular septum, right mammilla muscle, basis of right
ventricle, apex of heart. The peak is 0,25 mV. The width is 0,03 sec.
R wave shows the spread of excitation on the lateral wall of both ventricles. It consists of
ascending and descending elbows. The high of R wave in lead II is 0,6 -1,6 mV.
S wave conforms of excitation time of both ventricles and basis of left ventricle, usually
negative.Thepeakis0 - 0,6 mV.
Figure 64. The structure of ECG.

S-T segment is the segment from the end of the S wave to the beginning of the T wave,
represents the period when the entire ventricle is depolarized. S-T segment displacement is no more1
mm.
T wave is ventricular repolarization. The peak is 0,25-0,6 mV. The width is 0,06 - 0,25 sec.
Sometime U wave records after T wave in 0,02-0,04 sec. The peak is no more 0,1 mV. The
width is 0,09 – 0,16 sec.
P-Q interval is the interval from the first atrial depolarization to the beginning of the Q wave,
reflects the period of conduction in atrioventricular node, His band, Purkinje’s fibers. The width is
0,12 – 0,18 sec.
QRS complex represents depolarization of ventricles. The width is 0,06 – 0,1 sec.
Q-T interval (QRST complex) conforms all of ventricles excitation period, i.e. electric
systole. Thewidthis0,32-0,37 sec. in male, 0,35-0,40 sec. in female if beat’s rate is 60 – 80 per
minute.
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Т-Рinterval (from the ending of the T wave to the beginning of the P wave) reflects the
electric diastole. The width of interval depends of heart rate (if the rate is slower, the T-P interval is
longer).
R-R interval is а distance between the near R waves. It complies to the whole cardiaccycle.
The width is 0,7-1,0 sec.
Electrocardiogram procedure analysis.

1. To definite of the heart’s rate correctness. P wave sites before QRS complex (sinus rhythm). The
width of the R-R interval is equal. The heart rate abnormalities take place if the width of R-R
intervals differ more than 0,1 sec.
2. To definite of the heart rate. To count of the R-R interval width per millimeter. Heart rate = 60
sec/(R-R).
3. To definite of the ECG wave speak (1 mV = 10 mm).
To carry out measurements of the waves (the width).1 mm conforms 0,04 sec. if the speed is 25
mm/sec. (0,02 sec. –if the speed is 50 mm/sec.).
4. Todefiniteofelectricalaxisofheartpositionbyventriclecomplexesintheconventional leads. The
connection between them reflects on Bailey six-axis coordinate system (fig. 65).
Figure 65. Definition of electrical axis of heart.
It framed by suggestion that the heart is in the center of equilateral triangle and on the ends of it is
the electrodes from the limbs. The electrical axis of heart is a relative line, connecting of two points
with most difference voltage. The electrical axis of heart definite by measuring of the QRS complex
range in leads I and III. The range of Q and S waves is negative, R wave is positive. Count of the
arithmetic sum. The high of the waves measure from the end of the wave to its basis. The range of
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summary of the QRS complex protracts in lead I on the upper side of triangle. If this range is
positive, it protracts to the left from the middle point (to the left arm), if it is negative – to the right
(to the right arm).Do it same with the vector of the QRS Шcomplex. If it is positive, it protracts
down from the middle point, if it is negative – to up. The perpendiculars draw to the ends till cross of
them. The crossing point connects with the center of the triangle, so we take the summary vector of
leads I and III, the direction of the electrical axis of heart. Then we draw the circle around the
triangle. (fig. 66).
Figure 66. Six-axis coordinate system by Bailey.
If the summary vector sets in the +30° до +70оsector – it is normal state of the electrical axis; from 0°
to -90° - it is deviation to the left; from +90° to 180° - it is deviation to the right.
Visual location determination of electrical axis for three standard leads
The method is based on two principles: 1) the maximum value of the algebraic sum of the waves Q,
R,S is in the lead where the electric axis of the heart is parallel to the axis of the lead. 2)waves R =S
in the lead, the axis of which is perpendicular to the electric axis of the heart.
Normogram - Normal position of the electrical axis. The amplitude of the R wave in lead II is the
highest. In turn, the R wave in the I standard lead exceeds RIII wave. Normal location of the
electrical axis of the heart drawn up by the following entry: RII> RI> RIII.R and S in leads III and
AVL are almost equal.
Horizontal position of the electrical axis. Deviation of the electrical axis of the heart to the left is
schematically written: RI> RII> RIII and SIII> RIII. In lead I and AVL there are high wave R, in
lead III there is a deep wave S.
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Vertical position of the electrical axis. Deviation of the electrical axis of the heart to the
right is schematically written: RII>RIII> RI and SI> RI. In lead III and AVR there are high teeth R,
and in leads I and AVL there is a deep tooth S.
Exercise 1.Electrocardiography
Purpose: to get acquainted with the principles and techniques of electrocardiography, to master
methods of electrocardiographic interpretation (reading).
Procedure. Put circuit cable into a wall outlet. Place electrodes on limbs and chest according to the
conventional guard lines. To improve contact put 5-10% saline gauze under the electrode. Fix
electrodes in accordance with their color: red – right arm, yellow – left arm, green – left leg, black –
right leg, white – chest (fig. 67).
Figure 67. Limb lids

Switch the device on; set the required speed on the tape - recording mechanism (lead switch in
position ―0‖); adjust amplification to correspond to the lead of ECG graph paper 1 mV: 10 mm.
Let the patient fully relax. Adjust the lead switch in position ―I‖, switch the tape - recording
mechanism on, record the electrocardiogram. To record standard lead III, lead IV, unipolar limb and
chest leads use the same principles.
Phonocardiography
Phonocardiography (PCG) allows investigating acoustic phenomena accompanying cardiac activity
in the range of frequencies which are not available for hearing perception; to make their qualitative
and quantitative analyses; to register changes; murmurs, additional sounds, tinges and everything that
is of great importance for the diagnosis of heart disease
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Cardiac activity is accompanied by tones: tone I – results from atrioventricular valves fluctuations
(oscillations), tendinous fibers vibration and ventricular muscles tension; tone II – results from
closing of semilunar valves cusps. Tone I and tone II are usually classified as the obligatory valve
tones, as they are constantly auscultated. Tone III and tone IV are called elective muscular tones.
They are not always revealed (even by phonocardiography). Tone III is caused by ventricular
response (generally the left one) to the rapid blood flow (passive filling phase); and tone IV can be
caused by systole (active filling phase).
In phonocardiographic analysis intervals measured by electrocardiographic waves are of great
importance, so phonocardiographic recording is accompanied by parallel registration of
electrocardiography (fig.68). In phonocardiographic analysis the microphone is used for recording
that is applied to the place where the corresponding tones are auscultated properly. Signals are
recorded in any electrocardiograph and phonocardiograph.
Figure 68.The relationship between changes in intraventricular pressure, the ECG, and the PCG.
Tone I refers to the second part of QRS complex, tone II coincides with the end of wave T in ECG.
Tone I is represented by 8 oscillations and fluctuations (usually 4-5 fluctuations). They are quite
large in their amplitude. The beginning of tone I refers to the second part of QRS complex in
electrocardiogram. Tone II is expressed by 2-3 fluctuations; the first one is the highest in the (PCG)
amplitude. The beginning of tone II coincides with the end - wave T in electrocardiogram. Tone III
and IV are low - amplitude fluctuations (1-2 oscillations).In healthy adult tones and pauses with 75
contractions per minute are of the following duration: tone I – 0,11 seconds, pause I – 0,2 sec.; tone
II – 0,07 sec.; pause II – 0,42 sec.
Murmurs occurring in valves defects are recorded as additional low - amplitude oscillations
accumulating on the available cardiac tones or appearing in intervals of the tones (it is determined by
the specific pathology). The presence of the murmurs is the symptom of the disease.
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Exercise 2. Phonocardiography
Purpose: to get acquainted with the principles of phonocardiography monitoring and to make
recordings with the analysis of phonocardiographic curve.
Procedure. Attach electrodes to the device and to the patient in accordance with the given
instructions. Fix the microphone with the rubber belt above heart area, and make phono-
electrocardiographic recordings adjusting phonocardiographic amplifier handle (fig. 69).
Figure 69. Phonocardiography
Lesson 4. Physiology of Circulation. Arterial pressure. Arterial Pulse.

Questions for studying:
1. Classification of blood vessels.
2. Factors maintaining the blood flow through the vessels with different pressure.
3. Blood pressure in different parts of circulatory system.
4. Arterial pressure: systolic, diastolic, pulse pressure and mean pressure, central and peripheral.
Venous pressure. Factors determining the level of blood pressure. Hypotension and
hypertension.
5. Arterial pulse. Genesis of arterial pulse. The main properties of pulse, methods of investigation.
Analysis of sphigmogramm.
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6. Blood vessels innervation: vasoconstrictors and vasodilatators.
7. Center of blood pressure regulation in medulla. The tone of this center, localization, efferent
influence on it.
8. Functional characteristics of baroreceptors of blood vessels. Importance of carotid and aortic
reflex zones in blood tone regulation. Influence of high centers of CNS on blood tone regulation
(hypothalamus, cerebral cortex)
9. Humoral factors influence on blood tone (adrenalin, vasopressin, rennin, histamine,
prostaglandins).
Physical characteristics of the blood circulatory system.
The circulation is divided into the systemic
circulation and the pulmonary circulation
(fig.70). The systemic circulation is called the
greater circulation or peripheral circulation.
1) Functional parts of the circulation.
Arteries. The function of the arteries is to
transport blood under high pressure to the
tissues. The arteries have strong vascular
walls. The arterioles are the last small
branches of the arterial system. They act as
control conduits through which blood is
Figure 70. Blood flows from the heart through released into the capillaries. The arteriole has
arteries and into capillaries a strong muscular wall that can close the
arteriole.
The function of the capillaries is to exchange fluid, nutrients, electrolytes, hormones, and other
substances between the blood and the interstitial fluid. The capillary walls are very thin and have
numerous minute capillary pores. The venules collect blood from the capillaries, and they gradually
coalesce into progressively larger veins.
Veins. The veins function as conduits for transport of blood from the venules back to the heart.
Because the pressure in the venous system is very low, the venous walls are thin. They are capable of
constricting and enlarging and storing small or large quantities of blood and making this blood
available when it is required by the remainder of the circulation. The peripheral veins can also propel
blood forward by means` of a so-called venous pump. Every time one moves the legs, one tightens
the muscles and compresses the veins in or adjacent to the muscles, and this squeezes the blood out
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of the veins. But the valves in the veins` are arranged so that the direction of venous blood flow can
be only toward the heart. This pumping system is known as the ―venous pump‖ or ―muscle pump‖.
Blood pressure.
1) Pressure in the various portions of the circulation (fig.71).
2) The arterial pressure alternates between a systolic pressure level of 100-139 mm Hg and a
diastolic pressure level of 60-84 mm Hg at normal.
Figure71. Pressure in the various portions of the circulation.
As the blood flows through the systemic circulation, blood pressure falls progressively. The pressure
in the systemic capillaries is about 17 mm Hg. The pressure of the venae cavae falls to about 0 mm
Hg. In the pulmonary arteries the pressure is pulsatile, just as in the aorta, but the pressure level is far
lower: pulmonary artery systolic pressure averages about 25 mm Hg and diastolic pressure 8 mm Hg.
The pulmonary capillary pressure averages only 7 mm Hg. It is necessary to perform gas exchange
function of the lungs.
Regulation of arterial pressure.
Arterial pressure is regulated not by a single pressure controlling system but by several interrelated
systems, each performing a specific function.
I. Autoregulation
When the pressure in the blood vessels becomes too high, they become stretched and keep on
stretching more and more for minutes or hours; as a result, the pressure in the vessels falls toward
normal. This is called stress-relaxation.
103
II. Nervous regulation of arterial pressure.
Nervous control of the circulation has more global functions, such as redistributing blood flow to
different areas of the body, increasing or decreasing pumping activity by the heart, and especially,
providing very rapid control of systemic arterial pressure. The nervous system controls the
circulation almost entirely through the automatic nervous system.
In most tissue all the vessels except the capillaries, precapillary sphincters and metarterioles are
innervated by sympathetic nerve fibers (sympathetic vasoconstrictors). Stimulation of sympathetic
nerves leads to increased vascular resistance. Parasympathetic control plays only a minor role in
regulation of the circulation.
Sympathetic vasoconstrictor system and its control by the central nervous system. Vasomotor
center (VMC) in the brain controls the vasoconstrictor system. It is located bilaterally mainly in the
reticular substance of the medulla and of the lower third of the pons. This center transmits
sympathetic impulses through the spinal cord and peripheral sympathetic nerves to virtually all
arteries, arterioles, and veins of the body. There important areas are identified at the center.
- A vasoconstrictor area. The neurons originating in this area distribute their fiber to all levels
of the spinal cord, where they excite preganglionic vasoconstrictor neurons of the
sympathetic nervous system.
- A vasodilator area. The fibers from these neurons project upward to the vasoconstrictor area
just described; they inhibit the vasoconstrictor activity in this area, thus causing vasodilation.
- A sensory area. Signals from this area then help to control activities of both the
vasoconstrictor and vasodilator areas.
Control of the vasomotor center by higher nervous centers.
The hypothalamus can exert either powerful excitatory or inhibitory effects on the vasomotor center.
The posterolateral portions of the hypothalamus cause mainly excitation, whereas the anterior
portion can cause either mild excitation or inhibition.
Stimulation of the motor cortex anterior temporal lobe, the orbital areas of the frontal cortex, the
anterior part of the cingulate gyrus, the amygdala, the septum, and the hippocampus can all either
excite or inhibit the vasomotor center, depending on the precise portions of these areas that are
stimulated and on the intensity of stimulus.
The baroreceptor arterial pressure control system – baroreceptor reflexes. By far one of the best
known nervous mechanisms for arterial pressure control is the baroreceptor reflex. This reflex is
initiated by baroreceptor or pressoreceptors, located at specific points in the walls of several large
systemic arteries. Baroreceptors are extremely abundant in the carotid sinus and the wall of the aortic
arch (fig. 72).
104
Figure72. Baroreceptors of the carotid sinus and the wall of the aortic arch.
Impulses from baroreceptors progressively increase when the pressure is high. The baroreceptor
signals inhibit the vasoconstrictor center of the medulla. The net effects are vasodilation of the veins
and arterioles throughout the peripheral circulatory system. Therefore, excitation of the baroreceptors
reflexly causes the arterial pressure to decrease because of a decrease in peripheral resistance.
Conversely, low pressure has opposite effects.
Humoral control of the arterial pressure.
Humoral control of the arterial pressure means control by substances secreted or absorbed into the
body fluids.
The renin-angiotensin system
Renin is a protein enzyme released by the kidneys. Renin is synthesized and stored in an inactive
form called prorenin in the juxtaglomerular cells (JG cells) of the kidneys. The JG cells are modified
smooth muscle cells located in the walls of the afferent arterioles immediately proximal to the
glomeruli. Renin acts enzymatically on another plasma protein, angiotensinogen, to release a 10-
amino acid peptide, angiotensin 1. Two amino acids are split from the angiotensin 1 to form the 8-
amino acid peptide angiotensin 2. Angiotensin 2 is an extremely powerful vasoconstrictor. It also
prolongs the action of the sympathetic agents and increases the production of aldosterone. This is a
hormone of the adrenal cortex. It enhances water reabsorption by the kidneys. All this leads to
pressure increasing (fig.73).
105
Figure 73. The renin-angiotensin system.
Vasoconstrictor agents.
Norepinephrine and epinephrine. Norepinephrine is an especially powerful vasoconstrictor hormone.
The effect of epinephrine depends on its concentration in the blood. Epinephrine in small
concentration causes the resistance of vessels extension in skeletal muscle (β-epinephrine effect). In
high concentration it increases the resistance of vessels extension.
Vasopressin, also called an antidiuretic hormone, is a vasoconstrictor. It is formed in nerve cells in
the hypothalamus of the brain but then is transported downward by nerve axons to the posterior
pituitary gland, where it is finally secreted into the blood. Vasopressin has a major function to greatly
increase water reabsorption from the renal tubules back into the blood. This leads to an increase in
circulating blood volume.
Endothelin – a powerful vasoconstrictor in damaged blood vessels. This substance is present in the
endothelial cells of all or most blood vessels. The usual stimulus for release is endothelium damage.
1. Arterial pressure pulsation.
Arterial pressure pulsation is recorded on all arterial vessels. The curve of the arterial pulse is
called sphygmogram. Figure 75 sharp upstroke and slow rise to peak.
Incisura and microtechnique rise on the curve of the period of exile and closing of the semilunar
valves. The decaying limb occurs at the end of the protodiastolic period (fig.74).
106
Figure74. Sphygmogram (ab – anacrota, bc – incisura, cd – microtechnique rise,
de – the decaying limb - katacrota)
2. Venous pressure pulsation.

The pressure in the right atrium is called the central venous pressure. Right atrial pressure is
regulated by a balance between (1) the ability of the heart to pump blood out of right atrium and
ventricle into the lungs and (2) the tendency for blood to flow from the peripheral veins into the right
atrium.
The normal right atrial pressure is about 0 mm Hg, which is equal to the atmospheric pressure around
the body. Venous pulse register on venae cavae. Curve venous pulse is called phlebogram (fig.75).
Figure75. Phlebogram.
On the curve venous pulse we distinguish three waves. Wave ―a‖ occurs in the systole of the
ventricles. Wave ―c‖ reflects fluctuations in the adjacent carotid artery. Wave ―v‖ occurs in diastole
when filling the heart with blood.
107
Exercise 1. Recording of blood pressure.
Principle The term ―blood pressure‖ refers to systemic arterial pressure. Its measurement in man is
an important clinical procedure, because it provides valuable information about the circulation under
normal and abnormal conditions.
A variety of factors like cardiac output, elasticity of blood vessels, vasomotor tone, viscosity of
blood and its volume operate to maintain the resting level of arterial blood. The effects of such
factors, as emotional stress, muscular exercises and posture, which produce transient changes in
blood pressure can, therefore, be excluded during its measurement.
Definitions: blood pressure is a lateral pressure, exerted by the moving column of blood on the
walls of the blood vessels. With the pumping action of the heart, the pressure rises to a maximum
level during systole and falls to a minimum level during diastole. The pressure in the arteries is thus
pulsatile.
There are 4 main values of blood pressure:

Systolic pressure: this is maximum pressure exerted on the walls of the vessels during systole of the
heart.
Diastolic pressure: it is the minimum pressure in the vessels during the diastole of the heart.
Pulse pressure: this is the difference between systolic and diastolic pressure.
Mean arterial pressure: it is average pressure throughout a cardiac cycle. The duration of systole
being less, than that of diastole, the mean pressure is slightly less than the average of systolic and
diastolic pressures. A more reasonable approximation is diastolic pressure plus one third of the pulse
pressure:
Pm = Pd +( Ps – Pd)/3 ( for small vessels)
Pm = Pd + (Ps – Pd)/2 (for large vessels)
Principle. In clinical practice the blood pressure is measured with an instrument called
sphygmomanometer (“sphygmo” means pulse, “manos” means thin and ―meter‖ refers to
measurement). A cloth-covered rubber bad (or sack) is wrapped around the upper arm and is inflated
with air, till the extra-arterial pressure occludes the brachial artery or till the sounds are disappeared.
Pressure in the bag is then gradually reduced and readings for systolic and diastolic pressures noted.
Stethoscop. Although the stethoscop (steth = chest) was introduced by Laennec in 1819, it was not
until 1905 than Korotkoff used it in the measurement of blood pressure. It has two end pieces, the
bell type and the diaphragm type (phonendoscope). The ear pieces are designed so as to conform to
108
the direction of the external auditory meatus in the ear. The diaphragm type chest piece is applied for
recording blood pressure.
Procedure.
The subject may be either lying down or sitting, but must be relaxed and free from excitement and
anticipation. The upper arm should be at the level of the heart. Lay the arm bare up to the shoulder
and ensure that no clothing is constricting the arm. There are two methods:
I. Palpatory Riva-Rocci’s method.

1. Wrap the armlet around the upper arm, keeping its lower edge about 2,5 cm above the bend of
the elbow. (The middle of the bag should lie over anterolateral aspect of the antecubital fossa).
2. Locate the radial artery at the wrist and place three fingers over it. Hold the rubber bulb in you
palm in such a way, that your thumb and index finger are free to manipulate the leak-valve
screw. Inflate the pneumatic bag and enhance the pressure to 30 mm Hg above the level, where
the pulse disappears.
3. Reduce the gradually (2 mm Hg per 1 sec). Note the reading, when the radial pulse reappears.
The first appearance of the pulse is the systolic pressure. It corresponds of the time, when the
blood stars flowing through the compressed segment of the artery during each systole. The first 2
or 3 beats, being thin, may be missed, so than actual systolic pressure is 6-8 mm higher than the
record value. Note: the disadvantage of this method is that the diastolic pressure cannot be
determined.
Figure 76. Registration of arterial pressure by Korotkov’s method
109
II. Auscultatory Korotkov’s method (fig. 76).No sounds are ordinarily heard if the stethoscope is
applied over the bifurcation of the brachial artery. If the cuff pressure is raised above the systolic
pressure and then gradually lowered, a series of sounds are heard as the smooth laminal flow
becomes turbulant and sets up vibrations. The cause of sounds is the small amounts of blood that are
flow at high velocity through the partially occluded artery, at the peak of each systole. Korotkov
described these sounds and correlated with systolic and diastolic pressure.
Procedure.
1. Locate the lower end of the brachial artery in the cubital space, just medial to the tendon of
biceps. Place the diaphragm of the stethoscope over this region and keep it in the position with
the fingers and thumb of a hand, ensuring that it does not touch the cuff or the tubes.
2. Inflate the cuff slowly and rise the pressure to about 30 mm above the systolic level.
3. Lower the cuff pressure gradually until the first sound is heard, usually as a clear sharp tap.
Record the reading at this point which marks the systolic pressure. Lower the pressure column
further, at the same time listening to the sounds carefully. The manometer readings at the instant,
when the sounds disappear completely, marks the diastolic pressure.
The blood pressure is usually expressed as Systolic/Diastolic. For example, 120/80 mm Hg

(newborns have a systolic pressure of between 20 - 60 mm Hg).
A. One student acts as observer and other the subject. Record the blood pressure thrice at intervals of
5 to 7 minutes and note down the readings as shown in table 15.
B. Record the blood pressure in the sitting, standing and supine positions, to determine the effect of
posture.
C. Record the blood pressure after muscular exercise of light and moderate intensity.
Table 15
Observer Self Pulse Systolic Diastolic Pulse

Subject rate pressure pressure Pressure
Reading N
1
2
3
Average
110
Discussion. Normal blood pressure: in most normal young adults the systolic and diastolic pressures
are – 100-140 for systolic and 60-86 for diastolic. There is no sharp demarcation line between
normal and abnormal values, but the farther away a reading lies from the average, the greater is the
probability of the presence of disease. For example, if the blood pressure is 150/90 there is greater
probability of disease than if the reading were 130/84. If the BP is 160/100 under resting conditions,
there is even a greater probability that a disease process is causing the elevated pressure. If the
pressure is consistently higher, then there is no doubt that the person is suffering from hypertension.
Exercise 2. Examination of arterial pulse.

Principle.
:
The ―pulse‖ is the wave of increasing pressure, propagated centrifugally with each ventricular
contraction and blood ejection, at an increasing velocity (from 4 to 10 m/sec). It is the artery wall
swing. The pulse does not occur synchronously in all arteries. It is important to distinguish
transmission of pressure wave (the pulse) and the velocity of blood flow. The velocity of blood
flow in the aorta, for example, is about 1 m/sec and in capillaries – 1mm/sec. much information
can be gained from the rate, quality of pulse and the shape of pulse curve particularly at patients
with disorders of the heart.
For recording the pulse the radial artery is generally chosen, since it is easily accessible and lies
]
against the bone. If it is aberrant, palpate the opposite radial artery. Whenever disease of the
cardiovascular system is suspected, both radial pulses should be felt simultaneously and carefully
i compared.
I
The pulse rate is not constant in any one subject; the pulse interval varying with a pulse rate
of 60/min might vary between 0.7 and 1.3 seconds. This regular inspiration and slows during
expiration; this is called sinus arrhythmia and is particularly noticeable in younger subjects.
Since examination of the pulse provides valuable information as to the state of the circulatory
system and the general condition of the patient, the conditions under which the pulse rate is
determined, must be recorded: whether it was under basal conditions - early in the morning, before
the patient has taken any meals or gets up out of the bed - or during the day time.
Purpose. To examine the arterial pulse in human.

Characteristics of pulse:
1. Pulse rate. In normal conditions varies from 60 to 90 per minute. Pulse rate above 90/min is
111
called tachycardia and below 60/min - bradycardya.
Physiological tachycardia is seen in:
- Emotional stress, nervousness (e.g. at the time of an interview);
- In newborns, the rate may be from 120/min to 150 /min;
- The rate is comparatively high in females;
- Diurnal variations: comparatively higher rates are seen in the late evening (above 100/min);
- Muscular exercise.
Pathological tachycardia is seen in:
- Fever due to any case (the raised temperature acts directly on the SA
node);
- Atrial flutter and fibrillation;
- Thyrotoxicosis;
- In haemorrhagic shock the pulse is fast and weak (―thready pulse‖).
Physiological bradycardia is present in:
- Athletes. The resulting pulse rate may be between 50-55/min and is due to increase to vagal
tone.
- During deep sleep and meditation.
Pathological bradycardia is seen in:
- Myxedema.
- Heart block: in complete heart block the ventricular rate may be around 40per minute.
- General weakness and debility following prolonged illness,
2. Rhythm. If the beats follow at regular intervals, the rhythm is regular. If the rhythm is irregular,
try to assess the nature of this irregularity - due to the extrasystole or partial heart block, or
atrial flutter and fibrillation.
3. Character and form: though the character of the pulse wave can be studied at the radial artery,
a better method is to palpate the carotid artery. Place your thumb on the level of thyroid
cartilage and press backwards at the medial border of the sternocleidomastoid muscle. Usually
it is possible to detect the waves of the normal pulse by palpation alone. In certain diseases
some abnormality can be detected.
(a) Corrigan or collapsing pulse (―water-hammer‖). The pulse wave tises and collapses rapidly.
This is seen in aortic regurgitation, when the leak from arterial system (for example,
arteriovenous fistula) is occurred. It is best appreciated when the patient’s arm is elevated.
(b) Anacrotic pulse. The pulse wave has a slow upstroke, and is usually of small volume. This
type is seen in aortic stenosis.
112
(c) Alternate pulse. Ventricular beats are, alternately, strong and weak. It indicates severe
damage to the myocardium.
4. Volume. Estimate, by gentle palpation, the amplitude of the movement or expansion of the
artery during passage of pulse wave. The volume depends upon the pulse pressure. The pulse
pressure depends upon the stroke volume and compliance or expansibility of the arteries. Thus,
if the vessel wall is normal, the pulse wave gives information about stroke volume. In older
persons the vessels are more rigid (less compliant), so the pulse pressure is widened for the
same stroke volume. The pulse volume may be small, medium or large.
5. Condition of the vessel wall. Put enough pressure on the brachial artery with a thumb to
abolish the pulsation in the radial artery and then try to roll the empty artery against to underlying
bone. In young subjects the vessel wall cannot be felt or is very soft. It is more easily palpable in
older persons. In arteriosclerosis it is felt as a thick, hard cord. Another way to determine the
condition of the vessel wall is to empty out the vessel by pressing hard on it with the proximal finger
(the finger towards the heart) and palpating and trying to roll the vessel against the bone by other two
fingers.
The typical arterial pulse of a normal adult subject should be described in the following terms:
the rate is about 75/min, the beats are regular in rhythm, normal in character and volume. The vessel
wall is not palpable.
Note. The thumb is not used for palpating the pulse, because the pulse in thumb of many
observers is sufficiently strong to be confused with the pulse of the subject. (You can test this in your
own case by slightly pressing your thumb in various positions against the surface of a table).
Procedure.
1. Ask the patient to be seated comfortably and fully relaxed, with eyes closed.
2. Note the presence of the main arterial pulses: the common carotid, radial, femoral, posterior
tibial and dorsalis pedis (it is important in peripheral vascular disease) by palpating these vessels on
both sides.
3. Examine the radial pulse at the wrist with tips of the first three fingers (index finger towards the
heart), keeping the patient’s forearm pronated and wrist slightly flexed.
4. Note the rate, rhythm, volume, character and condition of the vesselwall.
5. Count the number of beats for one minute. Make two more determinations at intervals of 5
minutes each and record the three values in your notebook.
113
TEST TASKS
TO DETERMINE BASE KNOWLEDGES
The solution of these test tasks is aimed at the formation of: UC-1, GPC-4, GPC-5.
Closed type tests. Choose one correct answer
1. (GPC-5).Where is the Rh factor located:

a) in red blood cells
b) in white blood cells
c) in blood plasma
d) in platelets
2. (GPC-4).What does the serum of the fourth blood group contain?

a) Alpha and beta agglutinins
b) Alpha agglutinins
c) Beta agglutinins
d) 0-agglutinins
3. (UC-1). Where is the swallowing center located?

a) in the cerebral cortex
b) in the hypotalamus
c) midbrain
d) in the medulla oblongata
Open type tests. Complete the sentences.
4.(GPC-5). The set of muscle fibers innervated by one motor neuron is called _________.
5. (UC-1). The volume of blood ejected by the left ventricle during the period of exile is
called________.
6. (GPC-4). Which of the plasma coagulation factors is involved in all phases of blood
coagulation?
7. (GPC-5). A monosynaptic reflex arc is an arc consisting of _____ neurons.

114
8. (GPC-4). The neurotransmitter at the neuromuscular junction is ______.
9. (UC-1). The mode of transmission of nerve impulses along the myelin fiber is called
_______.
10. Which part of the vascular system offers the greatest resistance to blood flow?
SITUATIONAL TASKS
TO CHECK THE FINAL LEVEL OF KNOWLEDGE
The solution of these test tasks is aimed at the formation of UC-1, GPC-4, GPC-5.
Task 1. (GPC-5).
A patient was admitted to the clinic for a tonsillectomy (removal of the tonsils). Before the
operation, he was anesthetized at the root of the tongue in the posterior pharyngeal wall.
Why did they do it? Explain the physiological meaning of this manipulation.
Task 2. (GPC-4).
Two athletes with the same external respiration data decided to organize competitions for
the duration of their stay under water. One of them jumped after a preliminary
hyperventilation, the other dived, taking a breath. Which of them will stay under water for
the longest time?
Task 3. (GPC-4).
The patient has an attack of tachycardia (rapid heartbeat). The doctor does not have the
necessary medicines at hand. However, he relieved the attack by pressing on the patient's
eyeballs. What is this effect based on?
115
STANDARDS OF ANSWERS
TO TESTS AND SITUATIONAL TASKS
STANDARDS OF ANSWERS TO CLOSED AND OPEN TYPE TESTS
Question Answer Question Answer Question Answer

number number number
1. a 4. Motor unit. 7. Two neurons
2. d 5. Systolic or 8. Acetylcholine
stroke volume
3. d 6. Factor IV - 9. Saltatory
calcium ions. conduction
10. Arteries and arterioles
STANDARDS OF ANSWERS TO SITUATIONAL TASKS

Task 1.
Anesthesia was performed to avoid involuntary swallowing. The root of the tongue and the
back wall of the pharynx are the reflexogenic zone of the pharyngeal involuntary phase of
swallowing. Mechanical stimulation of this area causes involuntary swallowing. Anesthesia of
this zone inhibits this reflex.
Task 2.
An athlete after voluntary hyperventilation will last longer under water because the partial
pressure of carbon dioxide in his blood, the main stimulator of the respiratory center, will
decrease.
Task 3.
The vagal reflex of Danini Ashner was used - with pressure on the eyes, the tone of the vagus
nerve increases, which slows down the heart rate.
116
RECOMMENDED LITERATURE
Main literature
Main literature
1. Zinchuk, V. V. Normal physiology- Normal physiology : textbook / V. V. Zinchuk, O. A.
Balbatun, S. D. Orekhov, and others; ed. prof. V. V. Zinchuk. - Minsk: Higher School, 2020.
- 496 p. - Text: electronic // EBS "Student Consultant": [website]. - URL
:https://www.studentlibrary.ru/book/ISBN9789850632456.html
2. Gening T.P., Abakumova T.V., Mikhailova N.L., Kadysheva E.N. /NORMAL
PHYSIOLOGY Part 1/ Ulyanovsk State University, 2018. – 74 p.: il.
3. Physiology Animation. Visible Body app. – on-line. - DB of scientific medical 3D
illustrations Visible Body Premium Package http://ovid.visiblebody.com/physiology/
Additional literature
4. Chatterjee, Chandi Charan. Human Physiology [Текст]: in 2 vol.: for preclinical medical and
degree courses in physiology / C. C. Chatterjee. - 11th ed., revised enlarged coloured reprint.
- New Delhi: CBS Publishers & Distributors. -Vol. 1. - 2017. - 704 p.: il.
5. Chatterjee, Chandi Chara. Human Physiology [Текст]: in 2 vol.: for preclinical medical and
degree courses in physiology / C. C. Chatterjee. - 11th ed., revised enlarged coloured reprint.
- New Delhi: CBS Publishers & Distributors, 2017. -Vol. 2. - 2017. - 612 p.: il.
6. Na, John. Practical Physiology for MBBS Students [Text]: test, Clinical Case Scenarios and
Viva Voce. Question and Answers / J. Na. - New Delhi: CBS publishers & distributors, 2016.
- 244 p.: il.
7. Mulroney S. Netter's Essential Physiology [Electronic resource] / S. Mulroney, A. Myers. -
Second edition.-Philadelphia, PA: Elsevier, 2016. -on-line:
https://search.ebscohost.com/login.aspx?direct=true&db=nlebk&AN=1287551&lang=ru&sit
e=eds-live&ebv=EB&ppid=pp_cover
117
Kayumova Aliya Faritovna
Kiseleva Olga Sergeevna
Ziyakaeva Klara Rashitovna
Gabdulkhakova Irina Rashidovna
PRACTICAL PHYSIOLOGY Part I

Physiology of Blood, Excitable Tissues, Muscles, CNS, Cardio-Vascular System
Education Guidance for Students of General Medicine
License No. 0177 dated 10.06.96

Signed for printing
Printed on digital equipment
from the finished original layout presented by the authors.
Format 60x84 1/16. Condition-print. l. 4.19.
Circulation copies. Order No.
450008, Ufa, St. Lenin, 3,

Phone: (347) 272-86-31, e-mail: izdat@bashgmu.ru
FSBEI of HE BSMU of the Ministry of Health of Russia
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FEDERAL STATE BUDGETARY EDUCATIONAL INSTITUTION OF HIGHER

DEPARTMENT OF NORMAL PHYSIOLOGY

Physiology of Blood, Excitable Tissues, Muscles, CNS, Cardio-Vascular System

A.F. Kayumova, O.S. Kiseleva, K.R. Ziyakaeva, I.R. Gabdulkhakova

The questions for studying:

Formula used for calculating corrected reticulocyte count

Exercise 1.Sampling of Blood.

Figure 4.Erythrocyte parameters MCH, MCV and MCHC

Exercise 2.Hematocrit Value.

Figure 5. Hematocrit graduated pipettes.

Observations, results and conclusion.

Exercise 3. ColorIndex (CI).

Figure 6.RBCs in different NaCl solutions.

Observations, results and conclusion.

Figure 7.Osmotic hemolysis.

Figure 9.Punchenkov’s stand with determined blood samples.

Lesson 2. Blood Groups. Rhesus-factor. Thrombocytes. Retraction and Fibrinolysis. Blood

Blood group Agglutinogen Agglutinin

Figure 11. How Rh hemolytic disease develops

Exercise 1. ABO Blood Group.

Observations, results and conclusion.

Observations, results and conclusion.

Exercise 2.Rh Factor.

Observations, results and conclusion.

Exercise 4.Coagulation Time.

Observations, results and conclusion.

The questions for studying:

The concept of excitability.

8. Using a glass rod isolate the sciatic nerve in the thigh.

Observations, Figures and Conclusion.

Observations, results and conclusion.

The questions for studying:

Principle. The Membrane Potential

Resting Membrane Potential

Spread Spreads without attenuation over Distributed by1-2mm from the

The phenomenon of It does not summate It is summates

Figure 18. Action potential 1- resting state; 2 – phase of depolarization

Figure 19. Action potential 3- phase of repolarization; 4 – undershoot

Exercise 1. Galvani’s Test of Contracting with Metal.

Figure 21.Galvanic (metallic) tweezers.

Observations, results and conclusion.

Figure 22. Galvani’s test of contracting without metal.

Observations, results and conclusion.

Exercise 3. The Matteuchee’s Test (Secondary Muscle Contracting – Secondary Tetanus).

Lesson 3. Physiology of Nerve Fibers and Synapses.

The questions for studying.

C 0,3-1,3 0,5-3 Postganglionic sympathetics

Mechanism of the nerve impulse conduction.

Saltatory conduction in the myelinated nerve fibres.

Figure 25. Conduction of action potential in medullated nervous fiber

The conductivity shows the following characteristics:

Synapse. Neuro-muscular junction.

Figure26. Structure of synapse. Neuro-muscular junction.

Exercise 1. Nerve Conduction Velocity.

Figure 27. The observation of excitation along the nerve

Exercise 2.The Impulse is Propagated Along a Nerve in Both Directions.

Observations, results and conclusion.

Exercise 3. Physiological entirety of Nervous Fiber. Parabiosis.

Lesson 4. Physiology of Muscles

Figure 29.Different types of muscle tissue.

Modern theory of muscular contraction and relaxation.

Figure 30. Muscular contraction

Exercise 1. Kinds of Muscular Contraction.

There are three phases of a curve of muscle's single contraction: