Experimental Biology and Medicine

Since potassium cyanide inhibits certain oxidative processes in living matter such as respiration in plants and animals, the segmentation of fertilized egg cells, etc., behaving in this regard like an atmosphere of hydrogen, it might be expected that the destructive action of light upon cells suspended in fluorescent solutions, an action that is in part at least one of oxidation, would be diminished or obliterated by the addition of potassium cyanide to such solutions. On the contrary, the addition of very small quantities of potassium cyanide to sea water containing fluorescent substances (eosin, methylene blue, neutral red and combinations of these) causes a marked acceleration of the destructive action of visible light rays upon star-fish eggs immersed in these solutions. The chemical character of the fluorescent substances being different, it is evident that the chemical character of the fluorescent substance does not influence the action of potassium cyanide. But if starfish eggs in such solutions be deprived of oxygen, by substituting for air an atmosphere of hydrogen, they no longer undergo deterioration under the action of light and this result remains uninfluenced by the addition of potassium cyanide to the solution. It asserts this accelerating action only if added to eggs in fluorescent solutions exposed to light. If the cells were first immersed in a solution of potassium cyanide in the dark and afterwards transferred to the fluorescent solution in the light, potassium cyanide did not show any effect. Also control experiments in which small amounts of acid, alkali or certain alkaloids took the place of potassium cyanide showed no such results.

It would seem therefore that the oxidative processes, which perhaps in combination with other chemical processes, take place through the action of light upon cells in fluorescent solution, must be of a different character from the oxidative processes taking place in respiration arid in cell division, since the former are accelerated instead of, like the latter, being inhibited by the action of potassium cyanide.

The term physiological age refers to the stage of development in contradistinction to age in years and months, which is the usual method of designating age. Various evidences of physiological age are tooth appearance, pubescence, change of voice, menstruation, menopause, etc.

Pubescence is an evidence of sexual ripening and the beginning of adolescence. For the purpose of record, three physiological groups are distinguished corresponding to three successive stages of development (1) prepubescence, (2) pubescence, (3) post-pubescence. The first group is characterized by an absence of hair upon the pubis, the second is an intermediate stage, the third group have hair upon the pubis. The data of the results below are taken from 4,500 New York High School boys, and are divided into half year age groups designated by the middle age value. The following table shows the percentage composition of each chronological age:

It gives also the relative size of the subgroups. These facts have been hitherto disregarded and the chronological age group treated as if it were homogeneous.

These subgroups in each age exhibit characteristic differences in physical measurements which differences are far greater than the difference between contiguous year groups.

At the age of 15.75 the postpubescent group (85 per cent. of all) are 34 per cent. heavier, 32 per cent. stronger and 9 per cent. taller than the prepubescent group (forming 5 per cent, of all at the same age) as indicated on the next page:

The differences in weight, height and strength between prepubescents and postpubescents of the same age are equal to the differences between age groups that are 6 to 8 years apart.

Conclusion. — Age groups are heterogeneous and cannot serve as a unit for reference and experiment. We must substitute groups based upon physiological age.

Some time ago I described a method by means of which gastric peristalsis in the rabbit could be studied under normal conditions, without any operative interference whatsoever. The method is briefly as follows: If the hair of the epigastrium of a rabbit, fed well two hours before, is cut short, the stomach is largely outlined through the abdominal wall, and the peristaltic waves may be studied by inspection or by registering the waves with a tambour.

As the stomach is provided with extrinsic nerves (vagi and splanchnic) and with an intrinsic system (plexuses of Meissner and Auerbach) it was of interest to study the mobility of the stomach when deprived partly or entirely of its extrinsic innervation. This question has been studied by a number of observers, most recently and thoroughly by Cannon who used the X-ray method on cats. By means of the simple and physiological method outlined above the effects of sectioning the vagi or the splanchnics or both were studied. The vagi were, in this series, invariably cut below the diaphragm in order to preserve the vagus innervation of the thoracic viscera.

First series. Subdiaphragmatic section of the vagi only.—Two hours after operation the tambour registered only very slight volume changes of the stomach. Inspection showed no visible peristalsis. After some days peristalsis was again normal.

Reflex stoppage of peristalsis resulted from strong odors, sudden movements before the animal's eyes, loud noises, pain, just as in normal animals.

Second Series. Section of the splanchnic nerves only— As a rule good, effective peristalsis occurred less than one hour after the operation. The waves, however, are slower, and the period of contraction seems to be of longer deviation than normally.

Regarding the reflex effect of sensory stimuli, the data are as yet too few to permit a definite statement, but it seems as if the ordinary stimuli employed exerted no marked effect on the course of stomach peristalsis.

Stimulation of motor nerves hastens the onset of rigor mortis in the corresponding skeletal muscles. Cutting the nerves retards it, the retardation being due, it is believed, to the elimination of subminimal nerve impulses. Would the retardation of the rigor be still greater if inhibitory nerves could be stimulated? This question is not applicable to skeletal muscles, but it is a definite problem with reference to the onset of rigor in the heart muscle. Would a prolonged effective stimulation of the vagus nerves retard the onset of cardiac rigor? There were reasons to expect that the effect of such a stimulation would be indeed a retardation. The action of the cardiac vagus is inhibitory and the reverse of the action of a motor nerve; we might then expect that the effect of its stimulation upon cardiac rigor would also be the reverse of the effect of stimulation of a motor nerve upon the skeletal muscles, that is, retardation instead of hastening. Furthermore, increased muscular activity hastens the onset of rigor; it seemed reasonable to anticipate that the diminished activity, such as frequent standstill or slowing of the heart, would retard the onset of its rigor.

We have studied this question in 42 dogs, 16 cats and 10 rabbits. Both vagi were stimulated for half an hour before death and frequently also after death. Death was caused uniformly by bleeding and opening of the thorax. The outcome was a surprise; the obtained results were just the reverse of what was expected. But the results were uniform and unmistakable, We shall state them very briefly. They are as follows :

In all animals in which the vagi were stimulated, left and right ventricles stopped beating after death sooner than in the controls. The interval between the time of death and the beginning of the rigor in the left as well as in the right ventricle is in the experimented animals shorter than in the controls.

Calcium and magnesium are chemically closely related elements. They are also close companions in the tissues of the animal body. It is the prevailing view that the physiological effect of both elements is similar in character. Many physiologists are at present of the opinion that calcium as well as magnesium exerts an inhibitory influence in the functions of the animal body. Loeb published in 1899 his observations of the inhibitory action of calcium upon the twitchings of frog muscles brought on by solutions of sodium chloride. It was then assumed by Loeb that all the members of the group of alkali earths possess inhibitory properties including at first even barium. In the numerous subsequent papers by Loeb and his pupils the discussion turned, however, essentially around the inhibitory effect of calcium. Outside of the effect of calcium upon the heart, with reference to which Howell and his followers insist, contrary to the teachings of Loeb and his school, that calcium is an exciting and not an inhibiting agent, the opinion is now widely accepted that calcium is an inhibiting factor in the animal organism. It found its way also into pathology. For instance, a number of German and Italian writers hold the view that tetany of children is due to a diminution of the calcium content of the brain.

As to magnesium we have within the last few years published several studies in support of the hypothesis that magnesium salts favor inhibitory processes. The first fact which gave rise to that hypothesis was demonstrated in 1899 to the American Physiological Society when an intracerebral injection of a few drops of a solution of magnesium sulphate caused a state of paralysis in a rabbit while the injection of other solutions brought on convulsions.

I have shown that a segment of carotid artery of a dog transplanted onto the aorta of a cat can act as artery for one year at least. In order to ascertain whether the same result is obtainable when the animals are zoologically more distinct than cat and dog, segments of human arteries have been transplanted in dogs. One of these animals was shown to the Society.

Seven months and twelve days ago, the abdominal aorta of the bitch shown to the society was severed, and a short segment of human popliteal artery was sutured to its cut ends. This popliteal artery belonged to a young man whose thigh was amputated by Dr. Ellsworth Eliot for an osteosarcoma. The vessel had been preserved in Locke's solution and kept in cold storage during the twenty four days which elapsed between the amputation and the transplantation.

After the transplantation, the bitch remained in excellent health and the pulse of the femoral arteries was normal. Five months and twelve days after the operation, an exploratory laparotomy was performed. The circulation of the aorta was found normal and the popliteal artery in about the same condition as at the time of the operation. Seven months and twelve days after the operation, the pulse of the femoral arteries is still normal.

This experiment shows that an artery, transplanted under certain conditions from man to dog, can act as artery for seven months at least.

The etiological factors or conditions concerned in the production of thrombosis may be briefly summarized as:

(1) Those due to central or peripheral slowing of the blood stream.

(2) Those associated with lesions in the walls of the blood vessels.

(3) Alterations in the blood itself, such as tend to favor coagulation.

In actual cases, it appears without exception that two or more of these factors are associated in the production of thrombosis. Although the etiological agents mentioned above are generally accepted as the correct interpretation of thrombosis, it must be remembered that experimentally, as well as clinically, very discordant results are reported as to the causation of thrombosis, notably such as occurs in clinical phlebitis. Until more certain data are secured in regard to the process, but little can be expected in the way of successful prophylactic treatment or in the certain prognosis of those instances in which this lesion is to be feared.

The object of this brief study has been an attempt to show to what extent increased and decreased coagulability of the blood, artificially induced, may play a part in the production of thrombosis, or, expressed, in other words, whether in conditions productive of phlebitis, thrombosis is more likely to occur when the coagulation time of the blood has been lowered artificially or less apt to take place when analogous artificial means have been employed to prolong the coagulation time of the blood.

At the November meeting of the New York Pathological Society, 1 I reported briefly on a series of experiments I have undertaken on white rats, with the aim to investigate whether the great success attained lately in the transplantation of malignant growths from one rat into another is not due, in part at least, to the different behavior of the organism of the white rat to tissue implantation, as compared with other laboratory animals.

The majority of the workers who have experimented with the tumors of the white mouse or rat seem to be of the opinion that the success in the transplantations of these tumors is due to the great intrinsic power of limitless proliferation of the cells of these tumors, and they ignore the possible cellular reaction of the organism of the host to the implanted tissue. But the fact that pieces of the same tumor from the same rat or mouse grow readily on white mice of one race, and not at all on the white mice of another race; the fact, further, that while the original tumor, when implanted, grows in only about 10 per cent. of animals used, and, when re-implanted, it grows in 90 per cent., and some other similar instances, seem to indicate, a priori, that the white mouse or rat reacts differently to implantation of tumor, as well as of normal tissue, than the other laboratory animals.

My first set of experiments consisted in the implantation of normal tissue from one animal into another. Pieces of skin, liver, spleen, testicle, or mammary gland are implanted under the skin or in the peritoneal cavity of another animal, In the great majority of the experiments (about 60 to 75 per cent.) the pieces remained unabsorbed for as long as three weeks, even when, as in the peritoneum, they do not become attached anywhere, but float like a foreign body.

Dogs with lympho-sarcoma in every stage of growth were made use of, including those in which growth was active and progressive, those in which the tumor was quiescent, those in which the growth was regressing, and those in which recovery with complete absorption of the tumor had taken place. Some of the dogs were in good general condition, others were cachectic. Controls were also made use of, including dogs in poor and in good condition. In all, the material comprised 34 dogs, 18 with a tumor history, and 16 without. These were all bled from the femoral artery or jugular vein. In all of these dogs, blood was obtained for serum, and for a 1 per cent. suspension of corpuscles. The serum of each animal was tested on the corpuscles of a number of other animals in order to determine its hæmolytic power. Up to the present time over 300 such tests have been made. The serum obtained from tumor dogs is almost without exception possessed of hemolytic power. This is least marked in the early and active stages of tumor growth, more so in the broken down and softened tumors. It persists even in the dogs which have recovered of their tumors. The corpuscles of the tumor dogs manifest a much greater resistance to this hemolytic activity of the serum than do the corpuscles of other dogs.

The characteristics of this hemolytic substance in the serum of tumor dogs have been the subject of further study. The serum loses some, or only little of its power, by being passed through a Rerkefeld filter. Heating it to 85° for one hour does not destroy its activity. It differs markedly from the immune bodies known as amboceptors. It resembles in certain respects the hemolytic substances derived by extraction from necrotic tumors.

The experiments embodied in this abstract deal quantitatively with the renal blood flow, under different experimental conditions. They were performed upon dogs with the stromuhr described by Burton-Opitz. 1 The right and the left renal veins were used.

Besides the quantitative data, the authors succeeded in obtaining vaso-motor effects on stimulation of the præ, as well as post ganglionic fibers, the constrictory effects being in both cases the most prominent. The constriction of the blood vessels of the kidney was betrayed by a decrease in the venous return from this organ and a fall in venous blood pressure, this change being preceded by a brief increase of flow.

Among the post ganglionic fibers (renal plexus) a nerve was isolated which gave decided vaso-constrictory results.

In another series of experiments the pressure in the ureter was increased while the blood-flow in the corresponding renal vein was being recorded. The pressure was increased by means of air led into the ureter in the immediate vicinity of the kidney and of the bladder. Every increase in pressure from 20-120 mm. Hg resulted in a decrease in the venous return from the kidney and a fall in venous pressure, the decrease being in harmony with the height of the ureter pressure. A pressure of 20 mm. Hg or less remained ineffective when introduced next to the bladder, but produced a weak retardation of the venous flow when led into the upper portion of the ureter.

The stromuhr referred to in the preceding abstract was used in obtaining quantitative data regarding the portal circulation. The experiments so far performed have given an average flow of 1.56 c.c. per second in the splenic vein (weight of spleen 78 grams) and a flow of 1.05 c.c. per second in the mesenteric.

In the case of the spleen, the veins draining the pyloric end of the stomach and fatty tissue of this region were ligated. The stromuhr was then inserted centrally to the last branch draining this organ. The venous pressure was recorded centrally to the instrument. In the case of the mesenteric the stromuhr was inserted distally to the point of entrance of the vena gastrolienalis.

The nerves innervating the spleen and intestines were stimulated electrically while the blood flow in the veins was being measured. For the stimulation were selected first of all the entire bulk of the præ or post ganglionic fibers, and later on the most prominent fibers of the post-ganglionic paths.

On stimulation of the post-ganglionic fibers innervating the spleen (splenic plexus) vaso-constrictory effects of a very pronounced character were obtained. Immediately on stimulation the flow in the splenic vein showed an extraordinary increase which soon gave way to an equally pronounced decrease. The flow regained its normal value sometime after the cessation of the stimulation.

The primary increase in the flow is not due to a vaso-dilation of the splenic blood vessels, but to a squeezing out of the blood “resident” in the spleen. Not until this amount of blood has been expelled by the constricting blood-vessels, can the decrease become evident. Thus, it appears that constrictory effects are obtained from the first when the nerves aforesaid are stimulated.

Although I have tested the vaso-motor power of several of the fibers composing the plexus, I have succeeded in obtaining only vaso-constrictory results.

A pipette is used having a length of 20 cm. and a diameter of about 0.7 mm. The capacity of the tube, measured from its tip to a point above its enlarged middle portion, amounts to 1 c.c. 1 The tube is connected with a suction-pump developing from 50—100 mm. H₂O; at tube is interposed so that the pressure can be measured by means of a water manometer.

The fluid, the viscosity of which is to be determined, is drawn into the pipette from a receptacle. By means of the usual method the time is recorded which elapses between the dipping of the pipette into the liquid and the moment when the fluid passes the mark above the bulb. A comparison is then made between this value and the value previously obtained for distilled water, the latter being regarded as 1. The experiments are performed at room temperature.

Some time ago we reported to this society¹ that the intravenous injection of nicotin produced in the aorta of rabbits lesions practically identical with those obtained by Josue and others with intravenous injections of adrenalin.

The following is a brief summary of our further experiments along those lines. Many observers have laid particular stress upon the fact that where macroscopic lesions are not found, minute microscopic changes, stretching of the elastica and the like, could be recognized. We have counted as positive only such where there were distinctly visible macroscopic lesions. All observers agree that the best results can be obtained with older animals weighing over 2,000 grams. It was our misfortune that the great majority of rabbits obtainable by us were young and under the weight mentioned.

Twenty-four rabbits were treated with intravenous injections of nicotin, the dose being 5 drops of a 1-200 solution of pure nicotin. 7 died before they had had a sufficient number of injections. Of the remaining 17, results were positive in 7 after 18 to 77 injections. In 10 there were no macroscopic lesions after 20 to 117 injections.

In 6 rabbits nicotin and euphthalmin were used. Five were negative but one which had received alternate nicotin and euphthalmin injections 69 times showed aneurysm and calcification. The euphthalmin did not alter the typical nicotin reaction, such as convulsions, etc.

Nine rabbits received intravenous injections of nicotin and subcutaneous injections of iodipin. All were negative.

Two rabbits received their nicotin subcutaneously, the dose being double that given intravenously. One died too soon; the other was killed after 25 injections. Neither showed any arterial lesions.

One rabbit was given nicotin simultaneously with 1/10 grain atropin. It died after 14 injections; negative.

One nicotin and amyl nitrite; negative. Convulsions in no way influenced by these drugs.

Recent experiments by Maxwell 1 have shown that the nerve cells in the cerebral cortex are not stimulated by the ordinary nerve stimulants, such as oxalates, citrates, tartrates, etc. On the other hand we are in the possession of the well-known fact that an increase in the CO₂ tension of the blood supplied to the brain at first accelerates and later, if the increase in CO₂ tension be sufficiently great, inhibits the rhythmic discharge of impulses from the respiratory center or centers. It appeared possible that although the ordinary nerve stimulants do not excite nerve cells yet some other group of substances might be found which does so. Accordingly the following experiments were undertaken.

The roof of the skull, in frogs, was removed by means of a fine pair of bone forceps as far down as the tip of the fourth ventricle; in some instances the membrane covering the floor of the fourth ventricle was removed, in others not. The cerebrum was removed by cutting across the thalamencephalon just in front of the optic lobes; by this means it was found that a more regular respiratory rhythm was obtained, the frog was quieter, and the results were more uniform. The cavity left in the skull by the removal of the cerebrum was then plugged with absorbent cotton and absorbent cotton was placed all round the wound so as to absorb any fluid which might escape from the depression left by the removal of the roof of the skull. With practice the operation could be performed in 3 to 4 minutes, and, if successful, within 3 to 5 minutes after the operation respiration, which at first is suspended, is resumed. After about 5 to 10 minutes, the rhythm of respiration usually becomes constant and, if the frog be left untouched, remains so for over two hours.

We reported to this society on several previous occasions some facts regarding a tumor of the rat which we have propagated for more than two years. This tumor is now in its twelfth generation. The original tumor was of complex histological structure and has been regarded by us as consisting predominantly of a tissue of sarcomatous nature and to a less extent of irregular tubular formations resembling structures seen in certain endotheliomata. In the fifth generation of one series of transplantations, metastases were first noted in the regional lymphatic glands. In all previous generations the metastases were to the lungs and the kidneys; and in the other series this change in properties has not been observed. In the succeeding generations between the fifth and the eleventh, of the particular series mentioned, the lymphatic metastases have grown more common and more widely disseminated. Coincident with this change in properties of the tumor, a modification in the histological structure has been noted; the tumor has become progressively more and more adenomatous in appearance until now it has entirely cleared itself of that part which had been taken to resemble sarcoma. Hence nietaplasia of the tumor in the direction of carcinoma has taken place, with which change is associated the acquisition of the property of involving the nearby and distant lymphatic glands in its growth.