Muscle Fragments Research Articles

A photoelectric recording set for pulsation curves of heart muscle cultures in vitro

The fact that the light quantity passing through hanging drop cultures of beating heart muscle fragments varies during the pulsations, offers the possibility of recording contraction curves by the photoelectric method. These curves, which are informative not only with regard to the frequency but also to the amplitude of the pulsations, show us immediately, when registered by a directly inscribing electrocardiograph, the result of an experimental influence and can be evaluated quantitatively. Such a photoelectric recording set, as it has been developed by the author, is described in this paper.

The pathogenesis of bronchiectasis; a roentgen contribution.

This essay is presented in an attempt to harvest for the pathology of bronchiectasis knowledge gained since the advent of bronchography twenty-five years ago. “There appears to be no unanimity of opinion regarding anything about bronchiectasis with exception of the definition. All seem agreed that bronchiectasis is nothing more than a condition in which the bronchi show an abnormal dilatation,” was the conclusion in a recent paper on bronchiectasis (21). Even that statement is inaccurate, however, for it has been declared by others that “a bronchiectatic cavity is not a dilated bronchus, but an excavation in the lung substance, starting in a bronchus” (24). It has been widely contended that inflammatory damage to the structures of the bronchial wall precedes dilatation of the bronchi. On the other hand, in surgical specimens, dilated bronchi are often found to have grossly intact walls. In view of such basic misunderstandings, the present analysis must start with an examination of the terminology. It should be stated at the outset that this discussion will deal with bronchiectasis and not with chronic bronchitis. It is generally accepted that irreversible dilatation of the bronchi makes for persistence of infection. Perry and King (29) believe that chronic infection, particularly in such dilated bronchi, determines the serious prognosis as to health and longevity. It seems worth while, therefore, to understand why bronchi become dilated, with the hope that we may counteract this development. Definitions Etymologically, bronchiectasis represents a combination of the words bronchus and ectasis, i.e., dilatation of bronchi. The term is commonly used for the disease entity; occasionally “a bronchiectasis” (plural: bronchiectases) stands for the individual dilated bronchus. The pathologist defines bronchiectasis as a condition characterized by dilatation and usually infection of the bronchi. He recognizes a pulmonary cavity as a dilated bronchus by certain criteria. A cavity in communication with the bronchial tree with a wall composed of bronchial elements might be expected to be a dilated bronchus. Most or all of these bronchial parietal elements, such as mucous membrane, cartilage, smooth muscle, elastic tissue, and mucous glands, must be present in an orderly arrangement. It is not enough to find fragments of muscle or cartilage somewhere in the neighborhood of a cavity; such debris is often encountered in cases of suppurative pulmonary disease and the resultant fibrosis, even though the disease did not originate from bronchi or did not dilate them. By itself, the epithelial lining of a cavity obviously does not characterize it as a dilated bronchus. The following definition is suggested: Bronchiectasis is a condition characterized by dilatation of bronchi.

Experimental Serum Studies: Part II: Effect of Experimental Serum on Cell Respiration in vitro of Sarcoma 180

Two highly sensitive and precise methods are available for determining the effect of any substance on the physiology of living cells. One, the cultivation of tissues in vitro as developed by Harrison, Carrel and others, permits a careful study of cell proliferation; the other, the manometric method originated by Barcroft and perfected by Warburg, provides an accurate method for studying cell respiration. A previous communication (1) described the preparation and the use of an experimental serum obtained from sheep which had been previously injected with defibrinated blood of patients suffering from malignant disease, or with emulsions of human malignant cells. This experimental serum was found to exert an inhibitory effect on growth in vitro of mouse sarcoma 180, mouse spleen and kidney, embryo chick heart, human tumors, and human normal thyroid. It was also shown that this serum had no apparent toxic effect, since chick heart muscle fragments, though they produced no proliferating cells in its presence, still continued to beat. Normal proliferation of these heart muscle fragments, as well as of other tissues, took place upon transfer to normal media. It was considered of interest to make a study of the effect of this experimental serum on cell respiration in vitro. Experimental Procedure Mice with sixteen- to eighteen-day-old tumors of sarcoma 180 were selected. Throughout the experiment these animals received a normal diet consisting of bread, vegetables, and water ad libitum. The kidney of the same tumor-bearing animal, the organ having the most stable and even respiration as reported by Warburg (2), Richardson, Loebel and Shorr (3) and others, was used as a control. Experimental serum was either injected into the animal or added to the Ringer's phosphate medium used during the investigation. The animal was killed by pitting the neck. Under aseptic precautions the tumor and kidney were promptly removed and immediately used for the determination of respiration. Since Boyland and Boyland (4) have shown that with this type of tissue more uniform results were achieved by using minced tissue than sliced, we have also used this method and found it satisfactory. Because kidney tissue has a higher oxygen uptake than tumor tissue, approximately 35–40 mg. of minced kidney tissue and 70–80 mg. of tumor tissue were found most satisfactory. For the determination of the respiratory quotients, paired amounts of each type of tissue, whose difference in weight never exceeded 1 mg., were weighed on a torsion balance.

THE CULTIVATION OF TISSUES FOR PROLONGED PERIODS IN SINGLE FLASKS

1. Fragments of breast muscle from a 12 day old chick embryo have been kept alive in single flasks for an entire year without being transferred. The nutrient materials were supplied by frequent applications of adult fowl serum diluted with Tyrode solution. 2. When fragments of fixed tissues are cultivated in serum, cell multiplication and cell death are both reduced to an extremely low level. 3. The presence of a plasma coagulum is not essential to the continued survival and further development of tissues cultivated inserum. 4. The fibrinogen, prothrombin, and fibrin of coagulated plasma are not essential to the development of connective tissue fibers in vitro.

Influence of the Temperature upon the Life Duration and the Growth of the Fibroblast Cultivated in vitro

(1) The fragments of heart muscle of the chicken embryo incubated at 39° for 8 or 9 days were embedded in the culture medium, composed of equal volumes of the hen plasma and the embryonic tissue juice. All the cultures underwent 5 or 7 passages and then were kept in the incubator 39° for one day. Then the plates with the tissue fragments were placed in the incubators at various temperatures, i. e. 45°, 39°, 30°, 20°, 12° and 5°. In every medium one fragment was cultivated or two together. The outline of the total area of the new cells was traced every day, thus the degree of growth at various temperatures was determined. After the lapse of a certain length of time the culture was transferred into a fresh medium and put into an incubator at 39°, to determine whether the tissue was living or not. (2) The degree of growth was largest at 39°, the next at 30° and the smallest one was found at 45°. The tissue kept at 20°, 12° and 5° did not grow at all. (3) In the cases where one tissue fragment was cultivated in one medium, the temperature coefficients of the degree of growth at 39° and 30° were 2.0-2.4 in the interval of a few days, during which the tissues continued to grow both at 39° and 30°. The Q10 values between 45°and 39° were smaller than unity (0.5 to 0.2) and diminished as the life duration in vitro advanced. (4) In the cases with two fragments in each medium the Q10 values were smaller than those of the above between 39° and 30° and sometimes after a few days' life in vitro it became smaller than unity. Between 45° and 39° it was the same as that with one fragment in each medium. (5) The maximum interval, in which all the fragments were kept alive, when the medium remained unaltered, was estimated as 1 day at 45°, 7 days at 39°, 12 days at 30°, 11 days at 20°, 6 days at 12° and 3 days at 5°. The longest life duration for one fragment in each medium was noted as 5 days at 45°, 11 days at 39°, 20 days at 30°, 14 days at 20°, 11 days 12° and 8 days at 5°, and that in the cases with two fragments 5 days at 45°, 9 days at 39°, 16 days at 30°, 12 days at 20°, 7 days at 12° and 7 days at 5°. (6) The temperature coefficients of the number of days of life duration were the highest at low temperature (2.0 between 20° and 12°) and the more temperature was heightened, the smaller they became.

PHYSIOLOGICAL ONTOGENY

1. The Arrhenius equation giving the relationship between the velocity of chemical reaction and temperature, was found suitable for the special case of the contraction rate of embryonic heart muscle fragments. 2. There was no constancy in the values of micro for the rate of contraction in culture, nor was the scattering evenly distributed around certain (more than one) points. 3. There seemed to be no correlation between micro and other functions such as the contraction rate, the site from which the piece was removed, the age of the embryo, etc.

The reserve energy of actively growing embryonic tissues

In 1902 Fletcher1 showed that the sartorius muscle of a frog will give a maximum contraction when stimulated every five minutes over a period of two hours in an atmosphere of pure nitrogen. In an atmosphere of oxygen, the same muscle will contract for a much longer time and the fatigue developing in an atmosphere of nitrogen may be removed by placing the muscle again in an atmosphere of oxygen.If differentiated muscle has sufficient reserve energy to allow it to contract for a period without oxygen, the question arises, will not the same reserve be found in an actively growing tissue.In a previous article in 19172 the author described a method which allows one to study quantitatively the effect of various concentrations of oxygen on the growth of cells in vitro. In that paper the main point studied was the relative effect of pure oxygen and various dilutions of oxygen in nitrogen on the rate and extent of the growth. The tissues used were fragments of heart muscle and other tissue of chick-embryo of vario...

Studies on the metabolism of cells in vitro. The toxicity of dipeptids for embryonic chicken cells

In a previous paper we have shown that peptone prepared from the yolk of egg is non-toxic for growing chicken cells even when added in considerable concentration to the medium. Egg yolk digested to the point of crystallizing out an a-amino acid is toxic. We tested a large number of a-amino acids and have found that all are toxic for the cells. In low dilution they stimulate the contraction of heart muscle fragments but did not affect the growth. In higher dilution they inhibit the growth of cells completely and killed. Having established this fact it became of interest to study the effect of dipeptids in a similar manner. Leucyl-glycin and carnosin have been tested. The leucyl-glycin was a synthetic product. The carnosin was obtained from muscle. It was prepared by Dr. D. W. Wilson. Both of these substances are toxic in large doses. Their toxicity is not as marked, however, as is that of the α-amino acids. The leucylglycin is more toxic than the carnosin. During the course of these experiments we prepared and tested peptones prepared from beef. These were found not to affect in any way the growth of the cells.

IDENTITY OF THE TOXINS OF DIFFERENT STRAINS OF BACILLUS WELCHII AND FACTORS INFLUENCING THEIR PRODUCTION IN VITRO

Twenty-two additional strains of Bacillus welchii have been collected from widely different sources and tested with regard to toxin production. Each strain produces a toxin which, on animal inoculation, gives rise to lesions comparable in every respect to those produced by the toxins previously reported on, and each toxin was neutralized by an immune (antitoxic) serum produced with one of the former toxins. The toxins obtained from the several individual strains varied in potency, the lethal dose ranging from 0.3 to 3 cc. Experiments have been made to determine the influence of fresh muscle and glucose on toxin production and the relation of acidity to toxicity in the filtrates. It has been found that the addition of fresh muscle to the medium increases the potency of the toxin fivefold. Autoclaved muscle is without effect. Beef infusion broth containing 0.2 to 1 per cent glucose gives a more potent product than sugar-free broth, while when higher percentages are employed the toxin production is lowered. There is no direct relation between acidity and toxicity, the most acid products manifesting little or no toxic action. In every medium used for culture the potency of the filtrates rapidly diminished after 24 hours' incubation, while the acidity increased or remained constant. The exception to this rule has been pointed out. The most active toxin is obtained by growing a virulent strain of the bacilli in a 0.2 or 0.3 per cent glucose broth to which fragments of fresh muscle have been added, and collecting the filtrate after from 18 to 24 hours' incubation. A review of the literature on the pathogenic effects and toxic products of Bacillus welchii and on the results of immunization of animals with the bacilli or toxic products does not indicate that the exotoxic nature of Bacillus welchii had been previously determined or an antitoxic serum in the true sense produced. The antitoxin for Bacillus welchii toxin can apparently be prepared from a single strain of the organism which yields under the conditions described a high titer of toxin, and this antitoxin can be employed to combat infection with or prevent infection by any strain whatever of the bacillus.