Experimental Biology and Medicine

The problem of regeneration can also be stated in a negative way, namely: Why do tissues and dormant anlagen of organs not grow in an intact organism, while we know that they grow when the buds or tissues are isolated? The laws of inhibition were studied by a quantitative method.

No notch of a leaf of Bryophyllum will grow while the leaf is a part of the whole plant but the notches will grow when the leaf is isolated. When a leaf is subdivided into as many pieces as there are notches, each notch will give rise to a shoot; but if the leaf remains intact only few will grow out. The writer concluded that this again is a phenomenon of inhibition.

This inhibition he had explained in former papers 1 as being due to the fact that the inhibiting organ takes away the material required for the growth of the inhibited organ. If this were the case, we should expect that the total mass of shoots produced by a leaf in a certain time is approximately the same no matter whether the leaf produces few or numerous shoots. This is true to a surprising degree of exactness and the following numerical relations were established :

I. If a pair of sister leaves (of equal size) are isolated both will produce under equal conditions and in equal time approximately the same mass of shoots, although the number of shoots may differ considerably in the two sets of leaves.

2. If the mass of one of the two sets of sister leaves is diminished (by cutting out pieces of the leaves), the mass of shoots produced in the two sets is in proportion to the masses of the two sets of leaves.

I wish to demonstrate a new method of obtaining a sample of air from the lungs, bronchi and trachea of the dog. Chloretone, dissolved in olive oil, is given intraperitoneally. The animal is tracheotomized and a T-shaped glass cannula is introduced. A Meltzer pleural cannula (new form) is placed in each pleural cavity. The intrathoracic negative pressure is then restored and the pleural spaces tested for air-tightness. It should be possible to maintain the negative pressure indefinitely. If any change occurs, it must be in the direction of an increase owing to the absorption of gases through the pleuræ.

Both pleural cannulæ are now connected with a source of air pressure. A small-sized rubber bag is then attached to the horizontal portion of the tracheal cannula. The bag is thoroughly exhausted. The cocks of the pleural cannulæ are now opened. At the end of an expiration, the upright portion of the tracheal cannula is quickly clamped, the air pressure is turned on and the bag opened. A mercury valve is provided so that the desired pressure cannot be exceeded. The lungs collapse. Their contents are forced into the trachea and rubber bag. A portion of the air remains in the bronchi and trachea. The sample therefore approximates the total air.

It has been found that when no precaution is observed to maintain the body temperature, very uniform percentages of carbon dioxid may be obtained. Five experiments have been made thus far. In each experiment the carbon dioxid exhibited uniformity, either immediately or after a preliminary period of fluctuation. The periods of constancy ranged from one and a half to three and a half hours. In three experiments in each of which 8 determinations were made, the maximum deviation from the average was ± 3.0-3.5 per cent.; in one experiment, in which 7 determinations were made, the maximum deviation from the average was ± 2.4 per cent.; in another experiment, in which 5 determinations were made, the maximum minus deviation was 0.5 per cent. and the maximum plus deviation 1.4 per cent.

The disappearance of the ocular and the equilibrium disturbances following unilateral removal of the labyrinth is attributed to the activity of a compensatory mechanism.

As the labyrinthine symptoms recur after complete decerebration, the nervous paths concerned in the process of compensation may be roughly localized in the cerebrum above the level of the corpora quadrigemina.

The knowledge concerning the diuretic action of caffeine and theobromin has been obtained almost entirely through work on rabbits. It is from this work that the many explanations of how these drugs act in producing diuresis have arisen. The dog was early recognized as being somewhat refractory or uncertain in his response, and consequently but little work has been done on this animal. It has seemed to us however that the dog's urinary function is much more comparable to that of man than is the rabbits, and we have accordingly carried out a series of diuretic experiments using dogs as the experimental animals.

The dogs used were placed on a fixed diet, with a fixed daily intake of water. We have collected and analyzed the urine in twenty-four periods in order to avoid the frequent and unexplained variations which occur in shorter periods. Drugs were withheld until a comparatively constant daily output of urine both in quantity and composition was obtained. Caffeine, theobromin or theobromin sodio-salicylate was then given in dosage varying from 0.05 to 0.2 g. three times a day, for periods of from one to five or more days. They were given with the food and in capsules.

The results may be summarized as follows: With none of these drugs, in the dosage employed have we seen any appreciable increase in the urine output. On the contrary there has been almost invariably a decrease. When the drug is given for one day only, the decrease may occur on that day or the one following. When given for several days the decrease usually continues during the entire period, although in some instances the urine output may return to the normal level on the fourth or fifth day.

In the last few years a number of workers in this country have reported favorably on the use of cholesterinized antigens for Wassermann reaction. According to some reports, these so-called reinforced antigens gave even more satisfactory results than the acetone-insoluble fraction of tissue lipoids advocated by Noguchi. 1 In studying the rôle of cholesterin in the preparation of antigen we came to the following conclusions:

The apparent superiority of reinforced antigens is not due to the heightened specificity of these antigens, but to a heightened anticomplementary power of the same.

In addition, cholesterol retards and, if used in sufficient connection, greatly protects the blood cells from the action of hemolytic agents, thus further increasing the tendency towards a higher percentage of positive reactions.

That this influence of cholesterol is not specific is evidenced by the fact that more recently differeht authors noted the occurrence of nonspecific fixations obtained with reinforced antigen in a high percentage of normal cases.

The erroneous procedure recommended by some of the recent texts of using 1/2 to 1/3 of anticomplementary dose of reinforced antigen in the test may increase the occurrence of nonspecific fixations up to 40 per cent. of normal cases, as reported by some of the investigators.

On the other hand we found that removing all the cholesterol from the tissue extracts actually improves them for the use in the complement fixation test.

By redissolving and reprecipitating the acetone insoluble fraction of tissue lipoids one does not diminish its antigenic value, while one very markedly decreases its anticomplementary power.

The antigen prepared in this way can be used in the amount 10 or even 20 times smaller than the dose fixing I H E unit 1 of complement, and yet even such minute amounts of antigen may contain considerably more than 10 antigenic units.

Suitable temperature is one of the most important prerequisites to the proper progress of biological reactions. Since the complement fixation reaction is used so widely at present and the relation between the temperature and rate of fixation has not been studied systematically thus far, we thought it advisable to study this question. This was especially necessary, since many authors have varied the technique of the routine complement fixation test in this respect. The practical points brought out in this study are as follows:

1. If a rapid fixation of complement is desired we found that a temperature of 37° C. is the best. For diagnosis one-half-hour incubation at this temperature is the most efficient. We found, however, that, if sufficient antibody is present in the serum (3-5 units or more), fixation of two units of complement already takes place within the first five minutes, provided the amount of antigen used contains several antigenic units. We find it possible to use this procedure for presumptive elimination of strongly positive sera from a large series of cases. One places in a tube 0.05 c.c. of the patient's serum, adds the proper amount of antigen and salt solution and incubates at 37° C. in the water bath for five minutes and then adds sensitized cells to test for free complement. 1

2. If the time element is not so important, but complete fixation of the complement is desired, then we find that incubation in the ice box for 8-10 hours is best, These fixations on ice, however, may not be specific, for the reaction of fixation is so complete under these conditions that even traces of secondary circulating antigens and their corresponding antibodies may cause fixation of complement. 1 The ice-box fixation can therefore be used only as a presumptive test to eliminate the negative cases. 2

1. The acute kidney injury which develops late in poisoning by bichloride of mercury does not show any dependence in the frequency with which it occurs with the severity of the mercury enteritis.

2. The delayed kidney injury in acute bichloride intoxications has been constantly associated with the development of an acid intoxication. The severity of the damage to the cells of the convoluted tubules and the extent of fatty infiltration in the cells of the ascending limb of Henle's loop show a correlation with the degree of acid intoxication.

1. In acute experiments on cats, anesthetized with urethane, section of the spinal cord in the cervical region caused no demonstrable diminution in the rate of liberation of epinephrin from the adrenals as tested by allowing the adrenal blood collected in a cava pocket to elicit eye-reactions (after preliminary excision of the superior cervical ganglion). Four such experiments were made. The cord section in one of these was between the third and fourth cervical vertebræ, in another opposite the body of the fourth cervical vertebrae, in a third just below the body of the fifth cervical vertebra, and in a fourth cat between the fifth and sixth cervical vertebræ. In the second of these animals the blood was collected from the adrenal vein before and after section of the cord. The epinephrin assay (by rabbit intestine segments) gave the same output of epinephrin per minute after as before the section. The blood flow from the adrenals was much slower after the section, but was correspondingly richer in epinephrin.

2. In two survival experiments the cord was cut in the cervical region (in one just above the body of the last cervical vertebra, in the other at the level of the body of that vertebra). In the first cat, the superior cervical ganglion had been previously excised. Two days after the cord section adrenal blood was tested by the cava pocket method and gave good eye reactions, indicating a fairly good liberation of epinephrin (at least 0.0004 mgm. per minute). In the second experiment blood was drawn three days after the cord section and tested on rabbit intestine and uterus segments. Good concentrations of epinephrin were found (I : I, 500, 000 in the fourth adrenal sample, more than I : 2, 500, 000 and less than I : 1, 500, 000 in the second adrenal sample).

1. We showed in a previous paper 1 by the blood pressure and eye reactions that after section of the nerve supply of the adrenal no demonstrable liberation of epinephrin was present in cats as long as five weeks after the nerve section.

2. As it is easier to detect very small concentrations of epinephrin by the rabbit intestine and uterus segments, we have made a series of experiments (on 7 cats) in which these tests were used to supplement the eye reactions. In all the animals one adrenal was excised and the nerves of the other cut.

In a cat tested two weeks after the operation, it was shown that the adrenal blood serum could not have contained I : 300,000,000, or the blood I : 400,000,000, of epinephrin; and that the rate of liberation of epinephrin could not have been at most O.OOOOOI mgm. per minute for one adrenal. In another cat three weeks after the operation 2 the serum of the adrenal blood was proved to contain less than I : 400,000,000, and the blood less than I : 700,000,000 epinephrin. The output of epinephrin per minute could not have been as much as o.oooooog mgm. per minute, for one adrenal. The segments used for the tests in these experiments were extremely sensitive, and the limits of adrenalin concentrations which could be detected with certainty were carefully determined. The eye reactions were negative. In these two cats the rate of liberation of epinephrin, if any liberation whatever was going on, must have been several hundred times less than the rate in normal animals under the same experimental conditions.

It is scarcely necessary to point out that experiments yielding completely negative results indicating the absence of epinephrin with very sensitive test objects are much more important for the questions studied than experiments in which small amounts of epinephrin can still be detected.

Rabbits were injected subcutaneously with uranium nitrate in doses which varied from those which produced marked anatomical and functional changes to those which led to no certain effect.

A strain was placed on the urea-excreting function of the kidneys by the administration of urea by stomach tube.

While the kidneys were under the influence of this strain, the volume of urine, the rate of urea excretion, and the concentration of urea in the urine and blood were determined before and after the injection of uranium.

The most marked and constant functional change produced by the uranium was found to be a depression of the ratio between the rate of urea excretion and the concentration of urea in the blood. The degree of depression in this ratio was fairly closely parallel to the degree of anatomical damage, as judged from the extent and intensity of the necrosis or of the degenerative changes found in the terminal portion of the proximal convoluted tubule.