Isolated Organs

ISOLATED ORGANS. Organs are called isolated when their vital activity is artificially maintained outside the organism. The method of isolated organs allows for the examination of their function outside the complex conditions existing in the whole organism and makes it possible to resolve a whole series of problems in physiology and related disciplines. The possibility of the functioning of isolated organs is based on the automatism inherent in the activity of most organs. The task of maintaining the vital activity of an isolated organ reduces to the question of creating conditions that replace blood circulation, and for the organs of warm-blooded animals, also body temperature. The ideal nutrient fluid for isolated organs is the normal, non-coagulated blood of an animal of the same species. Using normal blood for an isolated organ is possible only by directly connecting the organ to the circulatory system of the animal from which the blood is supplied. Such an experimental setup, apart from purely technical difficulties, has the disadvantage of the isolated organ's dependence on changes in the blood and circulation of the animal at the expense of which the organ is nourished. Numerous studies, the beginning of which dates back to the 1880s (Kobert and others), have been conducted on isolated organs supplied with defibrinated blood, which was collected in sufficient quantity from a number of animals. However, defibrinated blood contains poisonous substances that form during blood coagulation. In particular, these substances possess vasoconstrictive properties, as a result of which defibrinated blood, when passed through the vessels of isolated organs, causes a strong spasm of the vessels, which hinders proper circulation. The use of blood with the addition of substances that delay its coagulation also proved to be of little use; such substances are partly not indifferent to tissues themselves (citrated blood), and partly the appearance in the released blood of certain substances analogous in action to serum is not fully eliminated (hirudinized blood). Recently, as a substance preventing blood coagulation when working with isolated organs, the so-called Germanin (Bayer 205) has been proposed (Bryukhonenko). According to the report of Bryukhonenko, who worked mainly with the isolated head of a dog, he obtained good results with this method. Salt solutions have great advantages as agents for maintaining the vital activity of isolated organs due to their simplicity, constancy of composition, and absence of toxic properties. Even a physiological solution of table salt is capable of maintaining organ function for some, albeit very short, time, and a frog heart irrigated with a physiological salt solution beats for a long time. However, for a longer survival of tissues and their normal function, the presence of other cations in the environment besides Na is necessary. The credit for introducing Ca and K ions into the physiological solution belongs to Ringer (Ringer, 1882), and solutions containing a combination of sodium, potassium, and calcium chlorides in amounts most suitable for maintaining tissue viability are called Ringer's solutions after him. There are many variants of balanced solutions proposed by various authors and used by various laboratories. The most significant additions were made by Locke, who introduced glucose into the solution as a nutrient material for the tissues of surviving organs, which is especially important for the organs of warm-blooded animals. (The commonly used composition of Ringer's fluid: NaCl-9.0; NaHCO₃-0.2; KCl-0.2; CaCl₂-0.2; distilled water-1,000.0.) Further improvements consisted of ensuring greater buffering of the nutrient fluid; a more constant pH of the medium can be created by introducing a mixture of phosphates into the fluid (Tyrode's fluid). The same author (Tyrode) proposed adding a certain amount of magnesium chloride to the solution, which gives good results when working with an isolated intestine. A general rule of the isolated organ method, when dealing with any object of significant size, is the establishment of washing (perfusion) of its vascular system with nutrient fluid. This washing is achieved by inserting cannulas of the appropriate diameter into the artery supplying the given organ, or into the arteries if there are several, through which the nutrient fluid enters. Since one of the conditions for the vital activity of the tissues of warm-blooded animals is a specific and sufficient temperature, the isolated organ itself is placed in a thermostat. To deliver nutrient fluid to the isolated organ, saturated with oxygen, heated to body temperature, and under sufficient and constant pressure, there are apparatuses of various designs. A very simple and convenient one is the model from the laboratory of Professor N. P. Kravkov (described in the dissertations of Kurdinovsky and Zakusov). It consists of a Mariotte bottle located at the height necessary to maintain pressure. The Mariotte vessel communicates with a burette, where the column of nutrient fluid is saturated with oxygen coming out in bubbles from a tube lowered to the bottom of the burette. From the burette, the fluid enters a coil located in a water bath, where it is heated to body temperature; from the coil, the heated and oxygen-saturated fluid goes to the isolated organ. In order to be able to change the pure nutrient fluid for a fluid containing the test substance without changing other experimental conditions (pressure, temperature), the apparatus consists of two identical systems of a Mariotte vessel, a burette, and a coil—with one system serving for the pure fluid and the other for the fluid containing the test substance. When perfusing the vessels of any isolated organ with nutrient fluid, the question of the survival and vital activity of the vascular wall itself arises first. We owe comprehensive research in this area under the conditions of nutrition with Ringer-Locke fluid to N. P. Kravkov and his school; in his laboratory, appropriate methods were developed for a whole series of organs of both warm-blooded and cold-blooded animals. The vital activity of vessels manifests itself in their ability to contract and dilate, which, under the conditions of isolated organs, can be revealed by passing one or another vasomotor poison through the vessels. As an object for studying vessels, the isolated rabbit ear (Kravkov-Pisemsky) presents special advantages, a method adopted by pharmacological and physiological laboratories all over the world. The rabbit ear, which in life was subjected to the influence of various temperatures, does not require heating even under experimental conditions; its vessels, washed with Ringer-Locke fluid at room temperature, and even without additional oxygen supply, manifest vital activity, reacting to the passage of the most insignificant concentrations of vasoconstrictive substances (adrenaline 1 : 100,000,000 - 1 : 1,000,000,000). The vital resistance and ability to survive of the vessels of the isolated ear, as apparently of other peripheral vessels, are extremely great, and provided they are kept in the cold, the vessels retain the ability to react to passed poisons for several days. This ability is preserved to a certain extent even after drying the ear and then washing it with Locke's fluid (Kravkov). The constriction or dilation of the vessels of an isolated organ is usually judged by the change in the amount of fluid flowing out of the veins, which, under the condition of constant pressure, depends on the lumen of the vessels. Just like the vessels of animals, with the help of the isolated organ method, the vessels of humans can be studied on organs taken from a cadaver shortly after the moment of death or during amputations (Anichkov, Shkavera, and others). This method opened up wide possibilities both in the field of human vascular pharmacology and their pathology. In view of the discovered viability of vessels even several hours after the death of the subject (the vessels of the fingers proved to be especially resistant to post-mortem changes), the isolated organ method allows for the determination of those functional changes that occurred while still alive. Thus, it turned out that vessels, especially of the abdominal organs, lose their contractile ability in some infectious diseases (typhus, relapsing fever) and are no longer able to react to vasoconstrictive poisons. This method thus allows for the investigation of the action of medicinal substances not only on the vessels of normal but also of pathologically altered organs of both animals and humans. One of the rewarding objects for research is the isolated heart. For working with the isolated heart of warm-blooded animals, it is required to establish the circulation of nutrient fluid in the coronary vessels; on the small hearts of common laboratory animals (rabbit, cat), this is achieved by inserting a cannula that delivers fluid into the ascending arch of the aorta in the direction of the semilunar valves (Langendorff's method): the pressure of the fluid closes the aortic valves, and the fluid rushes into the coronary vessels. With this method, the heart contracts as if "in vain," since its left cavities do not fill. The rabbit heart isolated according to Langendorff, bathed in Locke's fluid, contracts for several hours and makes it possible to perform all kinds of both physiological and pharmacological experiments on it.

The isolated heart of a rabbit exhibits the greatest vital resistance under these conditions; however, the cat's heart is distinguished by greater sensitivity to most poisons. It has also been possible to observe the contractions of an isolated human heart. Such experiments were first performed by Kulyabko (1902), who, using Locke's solution, "revived" the heart of a child who had died of diphtheria, taking it from the corpse 20 hours after death. Isolated hearts of adults are less capable of survival; conversely, experiments with the hearts of newborns and premature infants are most successful. Smooth-muscle organs are extremely viable in conditions of artificial nutrient fluids. Thus, an isolated rabbit uterus (Kurdinovsky) produces its characteristic automatic contractions and is capable of performing the entire act of labor. If one takes a small segment of a smooth-muscle organ, for example, a segment of the small intestine of a rabbit or cat, its survival and characteristic rhythmic contractions can be obtained without perfusion through the vessels, but by simple immersion in a small beaker with nutrient fluid (Magnus's method). In this case, to record the contractions, one end of the segment is fixed to a glass hook immersed in the fluid, and the other is connected by a thread to the arm of an Engelmann writing lever. Particularly regular and uniform curves are produced by the contractions of a rabbit intestinal segment; a similar method can be used for work on segments of uterine musculature, for example, the uterine horn of a guinea pig, which serves as an object for the biological testing of uterine agents—pituitrin and ergot. The same method is suitable for studying the contractile activity and reaction to poisons of pieces of uterine musculature of larger animals as well, including the human uterus. Also, segments of other smooth-muscle organs, such as bronchi, ureters, the gallbladder, and ducts, exhibit their contractile capacity under similar conditions. In this case, the beaker with nutrient fluid, into which the segment of the smooth-muscle organ is immersed, is placed in a water bath heated to body temperature to maintain the temperature, and air bubbles are constantly passed through the nutrient fluid. In 1924, based on an idea by N. P. Kravkov, the method of isolated organs, nourished by Ringer-Locke solution, was used to study endocrine glands (Shkavera, Kuznetsov, et al.). This idea was based on the fact that endocrine organs, which during life secrete the products of their vital activity (hormones) into the blood, would, under conditions of nourishment with Ringer-Locke solution, secrete them into the fluid flowing through the vessels of the organ. This idea, implemented even earlier at the Physiological Institute in Bern, found full confirmation in studies on isolated adrenal glands of cattle. The fluid flowing out of the veins of an isolated adrenal gland yields biological and chemical reactions for adrenaline, and the amount of it released over several hours is greater than the amount that can be obtained by simple extraction. In view of this, the release of adrenaline from the tissue of an isolated adrenal gland cannot be considered as a simple "washing out"; the viability of the medullary layer of the isolated adrenal gland is also confirmed by the fact that it reacts to minute concentrations of poisons that selectively act on the adrenal gland. Thus, nicotine in a dilution of 1:1,000,000 and even 1:100,000,000 significantly increases the concentration of adrenaline in the Ringer-Locke solution flowing out of the isolated adrenal gland ("adrenal fluid"). The sensitivity of the isolated adrenal gland to poisons passed through its vessels has made it possible to widely use this method for pharmacological research. Experiments with other endocrine glands have yielded less fruitful results, as it has not been possible to detect the hormones secreted by them in the outflowing fluid as consistently and with such clarity as in the isolated adrenal gland. Despite the great advantages that Ringer's solutions, in particular Ringer-Locke solution, have due to their simplicity and constancy of composition, they are by no means ideal nutrient fluids, and the conditions in which an organ irrigated by them is located can in no way be considered identical to those present in the organism when tissues are irrigated by blood. First of all, Ringer's solution is a solution of crystalloids and is completely devoid of the colloidal properties of blood. Ringer's solution lacks the sum of nutrients, enzymes, and hormones necessary for long-term vital activity that blood carries. Despite special oxygen saturation, the possible amount of it in Ringer's solution is far inferior to the supply available in arterial blood, and thus an organ nourished by Ringer's solution is in a state of oxygen starvation. As a consequence, the vital activity of an isolated organ is limited in time; consuming its existing reserves, it does not replenish them, and dissimilation processes are not compensated by assimilation processes, which as a result inevitably leads to the death of the tissue. The absence of colloidal properties in the nutrient fluid affects the water balance of the tissues; an isolated organ nourished by Ringer-Locke fluid gradually becomes edematous. During the perfusion of organs with external secretion (stomach, intestine), abundant transudation of fluid by the glands occurs. For studying the urinary function of isolated kidneys, their perfusion with Ringer-Locke solution is completely unsuitable. The central nervous system of warm-blooded animals also ceases its vital activity when irrigated with Ringer's solutions. Hence, the unceasing attempts to use blood preparations for nourishing isolated organs are understandable. Defibrinated blood, as stated above, turns out to be poorly suited for the perfusion of isolated organs due to its toxicity. Starling showed that if defibrinated blood is passed through the vessels of an isolated lung, it loses its poisonous properties and can successfully serve for nourishing isolated organs. By replacing the systemic circulation with a system of tubes with artificial resistance and leaving the natural pulmonary circulation, Starling developed the heart-lung preparation method, which served him for solving a number of fundamental problems of heart physiology, as a result of which the so-called "law of the heart" was formulated by Starling. By including various isolated organs in the artificial systemic circulation, Starling used his method for studying their function. In particular, by this method, Starling and his students studied the physiology and pharmacology of urinary secretion in an isolated kidney; the same method proved to be very suitable for the survival of the centers of an isolated dog's head. By including an adrenal gland in the heart-lung preparation (Anichkov and Kuznetsov), one can study the secretion of adrenaline, and the vital function of the adrenal medullary tissue is preserved more fully than during its perfusion with Ringer-Locke solution. The combination of several isolated organs in Starling's heart-lung preparation represents a synthesis of individual physiological elements, which individual isolated organs are. One of the ways to combine the method of isolated organs with the study of the organism as a whole is the artificial perfusion with nutrient fluid of an organ whose circulatory network is separated from the general circulation, while the nerve connections of the given organ with the central nervous system are preserved. This method was used by Sollman, who studied the vessels of the kidney by means of their artificial perfusion, with the kidney remaining in situ and its innervation remaining intact. The same principle was used by M. P. Nikolaev in relation to a rabbit's ear. With such a method, organs that are isolated but connected by nerve pathways to the organism can serve as indicators of the state of the vasomotor center and are suitable, in particular, for studying the pharmacology of the vasomotor center. Despite all the value of the method of isolated organs as a means for physiological and pharmacological analysis, it is necessary to remember that artificial nourishment of an organ, no matter how perfect the nutrient fluid may be, creates conditions different from normal ones and in one way or another alters the vital activity of the tissues of isolated organs. The absence of central innervation, while on the one hand providing the opportunity to investigate the organ's own automatism, on the other hand places it in completely special conditions. In particular, it should be noted that significant fluctuations in sensitivity to pharmacological agents are observed in isolated organs, within limits far exceeding those changes in sensitivity that occur in the whole organism. As an indispensable rule, data obtained on isolated organs completely separated from the organism require control experiments in vivo for transfer to the whole organism.