Патологическая физиология теоретический курс = Pathological physiology theoretical course. Учебное пособие

warm sterile saline for liquefaction of inflammatory exudate should be introduced into the abdominal cavity. As a control for another animal, inject warm sterile physiological solution in the same volume within the indicated time.

At the beginning of the lesson, slowly introduce 10-15 ml of defibrated blood heated to 38° C or 3% of a suspension of red blood cells of a bird or frog. Then, every 15-20 minutes, using a Pasteur pipette or a syringe, take 0.5-1.0 ml peritoneal fluid for microscopic examination.

The process of phagocytosis is studied with the help of stained smears of peritoneal fluid and the "hanging drop" method. For the preparation of the "hanging drop" sampling, 5 to 7 drops of peritoneal fluid should be applied to the watch glass and mixed with an equal number of drops of 0.1% neutral red prepared in physiological saline. Then take the glass with the hole and make a rim of vaseline on the edges of the hole. On a clean cover glass in the center, place a drop of stained neutral red peritoneal fluid the size of a pinhead and cover it with chambered slide so that the drop is projected into the middle of the well, but does not touch it. Then turn the sample over the cover glass up and examine it under a microscope.

With a minor increase in the microscope with a narrowed diaphragm and a slightly lowered condenser, find the edge of the drop. After discovering the phagocytic cell, place it in the center of the visionfield, then establish a large increase and analyze in detail the dynamics of phagocytosis,

In the "hanging drop" you can observe all the stages of phagocytosis. The process of intracellular digestion of a foreign cell (erythrocyte) with a phagocyte is determined by staining the protoplasm of the cell in pink, and then in a ruby red color, which indicates the transition of a neutral reaction to an acidic reaction (unoxidized metabolic products appear in the cell).

To prepare a smear, apply a drop of peritoneal liquid to the center of one end of a clean, degreased slide and make a long, wide smear using a polished rib of another slide. Dry the smear

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for 3-5 minutes in methyl alcohol or in a mixture of equal parts of ethyl alcohol-rectificate and ethyl ether for 30 minutes for fixation, then stain with azur-eosin according to RomanovskyGiemsa (6 ml of paint per 200 ml of distilled water) for 20-25 min. After staining, the smear is rinsed with tap water, dried with filter paper and examined under a microscope with an immersion system. Mark and sketch all the stages of phagocytosis.

Control questions

1.The concept of resistance. General nonspecific resistance of the organism and its main factors.

2.Barrier mechanisms of the body and their protective role,

3.The concept of immunity. Types of immunity.

4.The immune system of the body, its specific and nonspecific components.

5.Cellular immunity factors. The role of reticulum macrophage system and leukocytes in immune reactions.

6.Phagocytosis and its concept. Mechanisms of phagocytic process development.

7.Relationship of the phagocytic reaction with the reactivity of the organism.

8Features of phagocytosis at different stages of development of the pathological process.

T o p i c 7

HUMORAL IMMUNITY MECHANISMS: ROLE OF SPECIFIC AND NON-SPECIFIC FACTORS

IN IMMUNE REACTIONS OF THE ORGANISM

Purpose of the lesson. To study the mechanism of specific humoral immunity, the role of specific and nonspecific factors in immunobiological reactions.

Tasks.

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1.To determine whether the blood serum of a rabbit immunized with erythrocytes of a ram (hemolysin) acts on the erythrocytes of a ram and an animal of another species. Based on the results of the experiment, explain the cause of these phenomena and the mechanism of the reaction "antigen-antibody". Make a conclusion about the specific factors of humoral immunity.

2.Find out whether the destruction of the antigen (erythrocyte ram) is caused by specific antibodies (hemolytic blood serum of the rabbit) without the involvement of a complementary blood factor; whether a complement of another species of an animal (e.g. guinea pig) can participate in an immune response between said antigens and antibodies. To make a conclusion about the role of nonspecific factors in the reactions of humoral immunity.

Research Methodology

To solve the set tasks, prepare the working solutions of complement, hemolysin and a suspension of red blood cells. Complement, obtained from the serum of guinea pigs, is released by the biological industry in dried form. To establish the immune hemolysis reaction, the dry complement is dissolved and diluted to the necessary titers in physiological saline (1:30). Hemolysin (hemolytic amboceptor), obtained as a serum from the blood of rabbits immunized with erythrocyte ram, is also produced by biofactories. Working solution of hemolysin is prepared by diluting the drug with saline 1:50. A suspension of erythrocytes from a ram and some other kind of animal (pigs, rats, etc.) should be prepared from venous blood taken immediately before the reaction. To do this, freshly defeated blood, place in a centrifuge tube to half of its volume and add to physiological solution, mix, centrifuge for 10-15 minutes at 1500-2000 rpm, suck out the supernatant clear layer and add saline again to the precipitate, mixing it with erythrocytes. In this way, centrifuge up to three times until the blood plasma is washed from the red blood cells. After this, remove the upper layer of liquid and add saline solution to the

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erythrocyte sediment to half the volume of the test tube (initial volume of defibrinated blood), mix thoroughly, take 2.5 ml of the mixture and dilute in 97.5 ml of physiological solution, obtaining a 2.5% erythrocyte suspension, which can be stored in the refrigerator up to 3-5 days.

To solve 1st and 2nd tasks, number 5 clean dry tubes. In one of them, pour 0.5 ml of erythrocyte suspension of ram (2.5%), hemolysin (1:50), complement (1:30) and 1.0 ml of physiological solution, in the second – all these components, except hemolysin, in the third – the same, with the exception of complement, in the fourth – the same components, but instead of erythrocytes of the ram to take red blood cells of another kind of animal, in the fifth – only suspension of erythrocytes of ram and saline (control sample).

All tubes are placed in a thermostat or water bath for 20 minutes at 37-38 ° C, then record the results ("reading" the reaction). With a positive reaction, hemolysis occurs: the contents of the tube become transparent and acquires a pink lacquer shade. A negative reaction is characterized by the absence of hemolysis and a uniformly cloudy mixture.

Reaction Scheme

Control questions

1.Humoral immunity, its specific and nonspecific factors.

2.Interrelation of cellular and humoral factors of the body's immune defense.

3.The concept of antigens and antibodies, their properties and classification.

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4.Complement, its properties and role in specific immune reactions.

5.The mechanism of development of specific humoral im-

munity.

6.General principles of serological diagnosis and immuneprophylaxis of infectious and invasive diseases of farm animals.

T o p i c 8

ALLERGY. PATHOGENESIS OF ANAPHYLACTIC SHOCK. THE PHENOMENON OF ARTHUS

Purpose of the lesson. To study the mechanism and conditions for the occurrence of allergic reactions in the body, to become familiar with their common and local manifestations in anaphylaxis.

Tasks.

1.To determine whether any noticeable disorders occur in the body after administration to the intact animal parenterally (e.g. under the skin) of a small dose of foreign protein (normal horse blood serum).

2.Find out what is the reaction of the animal to repeated parenteral administration (after 2-3 weeks) of the same protein (serum) in a relatively small (desensitizing) and large (permissive) doses.

3.Find out which pathological changes at the injection site develop after the 1st, 2nd, 3rd, 4th, 5th and 6th injections under the skin of the animal in 5 ml of normal horse serum at intervals of 5- 6 days. Explain the cause of the phenomena noted, their originality and generality with the results of the previous experience.

Research Methodology

Among experimental animals, guinea pigs are most susceptible to anaphylaxis. To solve 1st and 2nd tasks, 3 clinically healthy intact animals are needed.

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1.Determine the pre-physiological state of the two guinea pigs. Observing the rules of aseptic and antiseptic, administer them 0.1 ml of normal horse serum subcutaneously, the third (control) – the same sterile isotonic sodium chloride solution. Then, for 10-15 minutes, monitor the condition of the animals.

2.2-3 weeks after the first injection, one of that received horse serum and the control animal was injected intravenously or into the heart cavity with 1.0 ml of the same blood serum, the second of the sensitized ones – 0.1 ml of serum subcutaneously. Immediately after repeated injections, carefully watching the animals, assess and record all changes in their clinical and physiological state, note the 20 most specific clinical signs of anaphylactic shock, explain their cause and mechanism.

After 5-6 h, the last of the guinea pigs that received serum under the skin, you can enter a resolving dose (1.0 ml) of the allergen to demonstrate the phenomenon of desensitization.

3.Local manifestations of anaphylaxis can be experimentally studied using the example of the Artyus-Sakharov phenomenon in an experiment on rabbits. A rabbit a month before classes (work is done by students of the group – members of the scientific club) inject 5 ml of normal horse blood serum. Subsequently, subcutaneous injections in the same dose should be repeated every 5 to 6 days. Simultaneously, as a control to another rabbit at the same time, inject 5 ml of sterile physiological saline solution subcutaneously.

During the experiment, daily monitor the clinical and physiological state of animals, carefully examining the skin and subcutaneous tissue at the injection site and noting the morphofunctional changes that have arisen.

Control questions

1.The concept of allergy, its types.

2.Allergens, their properties and classification.

3.Communication of allergy with immunity.

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4.General patterns of development of allergic conditions.

5.Anaphylaxis and its stages.

6.Sensitization. The mechanism of development of anaphylactic shock, its main clinical manifestations.

7.Desensitization and anti-anafilaxia.

8.Local anaphylaxis and its features.

9.Auto allergy. Nature of auto-allergens.

10.Allergic and auto-allergic diseases, features of their etiology and pathogenesis, clinical manifestations.

11.Features of the pathogenesis of allergic reactions of delayed type.

12.General principles of allergic diagnosis of infectious and invasive diseases of farm animals.

TYPICAL PATHOLOGICAL PROCESSES

T o p i c 9

DISORDERS OF PERIPHERAL CIRCULATION: HYPEREMIA, ISCHEMIA, THROMBOSIS. EMBOLISM

Purpose of the lesson. In the experiment on animals, to study the causes and mechanism of development, the main signs, the effects of arterial and venous hyperemia, local anemia, thrombosis and embolism.

Tasks.

1. To determine whether the general condition (color, local temperature, organ volume, etc.) and the intensity of blood supply to tissues and the state of blood vessels change and how:

a) the auricle in the rabbit after thermal (heating) or chemical (xylene lubrication), as well as after clamping the ear veins;

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b)tongue in the frog after mechanical (rubbing with a wet cotton ball) or chemical (turpentine, hydrochloric acid) stimulation, and also after vein ligation;

c)the membrane of the foot of the frog after denervation (transection of the sciatic nerve).

To establish whether the marked phenomena extend to the circulatory state in other parts of the body or are local, limited in nature.

2. Determine if (and if so, what, and in what order) changes in the functional state of the vessels, microcirculatory processes and blood supply of the swimming membrane of the frog's foot:

a)with irritation of the peripheral end of the cut sciatic

nerve;

b)when exposed to the study site solution of adrenaline;

c)after ligation of the femoral artery.

Explain the causes and mechanism of the development of these phenomena, determine their consequences.

3. To study under a microscope the process of formation of a white parietal thrombus in vessels of the mesentery of the intestine in a frog, (when exposed to a sodium chloride crystal), and a red obturating thrombus after vascular injury. Explain the mechanism of thrombus formation, its physiological and pathological role in the body.

4. Find out whether blood circulation in the vessels of the tongue (or other organs) is disturbed in the frog after introduction of fat particles (vaseline or vegetable oil, fat emulsion) into the circulatory channel.

5. To determine whether there are any kind of disorders in the animal's body when airenters the bloodstream.

Research Methodology

Objects that are convenient for studying by a microscope of peripheral circulation and experimental modeling of its disorders are the tongue, the swimming membrane of the paw and the mes-

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entery of the intestine in the frog. Local manifestations of circulatory disorders and changes in the functional state of tissues and organs in this case in warm-blooded animals can be reproduced on the ear of a rabbit (preferably white suits).

1. Before setting the experiments carefully consider in the transmitted light the initial state of the blood vessels of the auricle, the degree of their blood filling, determine the color, the local temperature (electrothermometer), the volume of the organ. Then one of the experimental rabbits lightly wipe the ear with cotton wool soaked in xylene. To the auricle of another animal, attach a test tube with warm water (45-50° C) for 0.5-1.0 minutes. After this, determine the changes in the general condition and circulation of the ear tissues.

To clamp the ear veins, beforehand (a few hours before the session), insert a cork stopper into the pinnae of the rabbit so that it falls on the lumen of the central artery of the ear. Then tighten the tube tightly to the tissues, pulling it together with the base of the auricle with a thick ligature. Identify changes in the blood supply and general condition of the organ, comparing it with the opposite ear. At the end of the experiment, remove the ligature, remove the plug and note the subsequent changes in the functional state of the ear and its vessels.

Immobilized with anesthesia or destruction of the spinal cord without decapitation, fix the frog back up on a special plank so that the front edge of its jaw protrudes above the rectangular opening of the plaque. Extract the tongue, straighten and, without allowing excessive stretching, to fix it in a fan-like manner with pins at the edges of the hole. The drug is placed under a microscope and observe a picture of normal blood circulation in the vessels of the tongue. Then, lifting the tube of the microscope and not changing the position of the preparation, lightly wipe the area of the tongue with a cotton swab soaked with physiological saline, or apply a drop of turpentine or 0.1% hydrochloric acid solution. Having lowered the tube of the microscope to its original position, register changes in the blood supply of tissues.

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Find the right and left venous trunks near the root of the tongue and bring the ligature with a needle through them. The drug should be placed under a microscope and follow the changes in blood circulation in the frog's tongue after dressing one, and then another vein.

The foot's swim membrane is immobilized and fixed on a frog plank to spread and fasten above the triangular hole. Under low magnification in the microscope, examine the state of the circulation in the tissues of the membrane before and after (within 10-12 min) cut of the sciatic nerve. The nerve should be cut carefully, not injuring nearby vessels, and as high as possible, closer to the hip joint.

2.With a low magnification, the microscope set the initial picture of blood circulation in the vessels of the frog's swimming membrane and, by stimulating the peripheral end of the cut sciatic nerve with an electric current from the stimulator or mechanically, observe the condition of the lumen of the vessels and the intensity of blood flow in the area under investigation.

Prepare for microscopy the swimming membrane of another frog's foot, determine the initial level of blood circulation, apply a drop of 0.1% solution of hydrochloric adrenaline to the site and continue the observation under a microscope. Prepare the femoral artery from the neurovascular bundle above the knee joint, tightly tie it with a thin ligature and trace it under a microscope for changes in the blood supply to the swollen membrane.

3.The immobilized frog should be placed on the dissection board with its back up so that its right side is at the edge of the round (lateral) opening of the plank. Open the abdominal cavity in the right side of the abdomen. Extract the loop of the small intestine, spread its mesentery over the hole and fix it with pins, making sure that the loop of the intestine is not twisted, and the mesentery is excessively stretched. The prepared sample is strengthened on the microscope table and under a lowmagnificationstudy the picture of normal blood circulation in the mesentery. After a while, when, due to the stretching and drying of the

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