Wade H. Brown

Animal resistance and the endocrine system of the rabbit in experimental syphilis

In the course of the work that has been carried out in this laboratory during the past 8 years, it has become more and more apparent that of the several factors concerned in determining the general course and severity of syphilitic infections, the spirochete is of minor importance as compared with the number of other influences, especially the factor of animal resistance. This is strikingly illustrated by the experimental infection in the rabbit which, as a rule, is comparatively mild but in exceptional instances may assume a more malignant form or persist in an active state with severe constitutional symptoms for months or even years and in the end cause the death of the animal. Little has been known as to the nature or source of the forces employed by the animal in combating this disease but there is a great deal of evidence to indicate that resistance to this infection is a function of animal economy and is subject to the same influences as are concerned in the regulation of growth and development, and the maintenance of general metabolic activities. Moreover, there have been specific indications that resistance was influenced to some extent by the endocrine system. This possibility was first suggested to us by the occurrence of marked seasonal changes in the severity of the disease but was most forcibly impressed upon us by the abnormal resistance displayed at times by pregnant and lactating females.

Neoplasia in experimental syphilis

Neoplasia as a sequel to syphilitic infection is not uncommon in man but has never been recorded in an experimental animal. Recently, we have observed an atypical growth arising from the scar of an old syphilitic lesion in the scrotum of a rabbit which may prove to be a neoplasm. The animal was inoculated in the scrotum, June 16, 1916. Small chancres developed and then underwent spontaneous regression. Several months later, there was a recurrence and the lesion in the left scrotum persisted for some time. In October, 1920, there was a slight diffuse infiltration of the left scrotum and a small nodule appeared at the site of the old chancre. Although it was known that the animal still harbored spirochetes, 1 none could be demonstrated by dark-field examination of material from the nodule and it was excised for histological examination. There was a prompt recurrence and with the growth of the second cutaneous lesion, the left inguinal glands became markedly enlarged and indurated. Again no spirochetes could be demonstrated and the lesion with one of the adjacent glands was excised under ether anesthesia. Histological examination of the cutaneous nodules and gland showed a growth which presented more the appearance of a neoplasm than of a syphilitic lesion. It was composed for the most part of atypical epithelioid cells undergoing active proliferation and exhibiting marked invasive tendencies. Meantime there was a second recurrence and extension of the skin lesion over the mid line at the pubis with enlargement and induration of the right inguinal nodes. A deterioration in the physical condition of the animal was then apparent and progressed very rapidly, culminating in emaciation, weakness, severe anemia, loss of sphincter control with some spasticity of the hind legs and the formation of trophic ulcers about the anus and sheath.

Seasonal changes in organ weights and their relation to meteorological conditions.

It is well known that many of the endocrine glands of normal animals undergo rhythmic changes in weight per unit of body weight which conform, in general, with seasonal conditions. Still, the cause of these changes is not clearly understood nor is it generally known that other organs and tissues such as the heart, the liver, the kidneys, the spleen, the thymus, the lymphoid tissues, and even the brain, undergo changes of a similar character. In November, 1921, we began a series of investigations which included among other things a study of the ratio of the weights of various organs and tissues to the body weight of normal rabbits and the limits of variation that might be encountered, with especial reference to the causes of such variations. The factors studied included age, breed, length of caging, time of killing (with reference to the regular system of feeding), and meteorological conditions. All of the animals used were males, and an effort was made to minimize the influence of dietary factors by maintaining a uniform system of feeding and a uniform diet consisting of hay, oats, and cabbage, which is the regular diet given all rabbits in these laboratories. The plan followed was to kill and examine two groups of rabbits each month selected from stocks that were being used for experimental work. All animals were well nourished and free from any external evidence of disease. During the first few months the groups were small and intentionally of a mixed character, but beginning with June, 1922, the monthly groups consisted of from ten to twenty rabbits. The animals were killed prior to the daily feeding; they were anesthetized with ether and bled from the inferior vena cava while the heart was still beating. The organs were removed, freed of all adventitious material, and weighed on suitable balances.

The reaction of the endocrine system of the rabbit to tumor inoculation and the relation of this reaction to malignancy

Within recent years, a great deal of evidence has been accumulated from both clinical and laboratory sources which has tended to show that some kind of connection exists between the occurrence and growth of tumors and the system of organs regulating animal economy. For the most part, the evidence bearing upon this problem has been circumstantial. In a few instances a definite relationship appears to have been established as in the case of the experiments reported by Loeb 1 concerning the effects of castration on the incidence of mammary tumors in mice and those of Rohdenburg, Bullock, and Johnson 2 on the effects of operative removal of various organs upon the growth of transplanted tumors and the immunity of tumor animals. There are also therapeutic observations on the use of thyroid and of thymic products alone or combined with castration 3 which might be regarded as equally suggestive were it not for the fact that similar results have been obtained by the use of a variety of means. 4 Two years ago, we reported the occurrence of a malignant tumor in the scrotum of a rabbit infected with Treponcma pallidum. 5 Since that time, a careful study has been made of the conditions presented by this animal 6 and a long series of investigations based upon the behavior of transplanted tumors derived from this stock has been carried out which have shown that there is an essential connection between the growth of transplanted tumors and certain members of the endocrine system on the one hand, and the mechanism of animal defense on the other. In the animal with the spontaneous tumor, there was a notable tendency to atypical epithelial proliferation in many parts of the body associated with a deficiency in the reaction of the surrounding connective tissues and a widespread tendency to degeneration. There were also very striking alterations in such organs as the thyroid the thymus, the suprarenals and the lymphoid tissues, which might have been regarded as part of a general organic deterioration attributable to one or both of the diseases present.

The penetration of normal mucous membranes of the rabbit by Treponema pallidum

The fact may be recorded that highly virulent strains of Treponema pallidum are capable of penetrating some portion of the genital mucosa of normal rabbits and setting up an infection without necessarily producing the first gross lesion at the portal of entry. This fact was recently determined in 9 rabbits and with two highly virulent strains of Treponema pallidum. The experimental method employed was as follows: The sheath of the animal was drawn forward to form a pouch into which was instilled 0.05 c.c. of a testicular emulsion rich in spirochetes. About 30 seconds were allowed for the emulsion to spread before releasing the sheath. Most of the fluid then ran out and between 0.04 and 0.05 c.c. could be recovered showing that only a thin film of the emulsion was retained. Infection was first indicated by enlargement and induration of the inguinal lymphnodes and later by the development of a general lymphadenitis with syphilitic lesions in other parts of the body. All animals thus far inoculated by this method have become infected. In some of them, enlargement and induration of the inguinal nodes was clearly recognizable within 24 hours after the application of the emulsion. Thus far (5 weeks) only one of the animals has developed a visible lesion on either the penis or the sheath, although several of them have characteristic lesions in the testicles and scrotum. The observations on these animals are not yet complete and the full significance of the experiments cannot be ascertained until the course of the infection has been followed much longer. Similar experiments with other mucous membranes are in progress.

On the generalization of treponema pallidum in the rabbit following local inoculation

That a widespread dissemination of Treponema pallidum may be produced in the rabbit by local inoculation has been shown by the recovery of the organisms in isolated instances from the blood, lymphnodes or other organs as well as by the occasional occurrence of generalized lesions in infected animals. However, there is no evidence to show either the time or frequency with which this dissemination occurs or whether the organisms thus distributed over the body are capable of sustaining the infection in these animals. With these questions in mind, a series of experiments was undertaken, the object of which was to determine the frequency of invasion of the regional lymphatics and the general circulation following inoculation in the scrotum or testicles and how soon a self-sustaining generalized infection might be established. Time and Frequency of Invasion of Regional Lymphatics.- An examination was made of the inguinal lymphnodes in a series of 29 rabbits which had been inoculated by the introduction of a bit of infected tissue beneath the skin of the scrotum. The nodes were excised under ether anesthesia at intervals of from 61 days down to 48 hours after inoculation and the presence or absence of Treponema pallidurn determined by dark field examination or by animal inoculation. The first group of nodes studied included those showing well marked enlargement and induration and these gave positive results in all cases. Nodes were then taken 5 days after inoculation and after the lapse of only 48 hours. Positive results were again obtained in all cases.

Effects of sunlight on the malignancy of a transplanted tumor of the rabbit.

Since the tumor upon which this report is based was first transplanted in the spring of 1921, 1 we have been engaged in a study of the problem of animal resistance as related to the growth and malignancy of the tumor with especial reference to factors of constitutional resistance and conditions that might cause an increase or decrease in animal resistance. Several reports have been published dealing with some of the results of this series of investigations. 2 Among other things, it has been noted that although a good primary growth can be obtained in practically all rabbits by intratesticular inoculation, the ultimate malignancy of the tumor is subject to wide variations and that, from the beginning, there has been a striking tendency to the occurrence of periods of excessive malignancy during the spring and fall of each year. This focused our attention on the possible influence of meteorological conditions and led to a study of phenomena of resistance and of tumor growth from the point of view of meteorological influences. The results of these investigations contain many points of interest, but the present report will be limited to a consideration of sunlight. The method employed involved the inoculation of a group of 5 to 10 rabbits each month. Inoculations were made by injecting 0.2 to 0.3 cc. of a cell emulsion into one testicle. The animals were then kept under observation for a period of two months, at which time the surviving animals were killed. Records were kept covering the clinical course of the disease and all animals were subjected to a careful postmortem examination which included the weighing of organs, noting the number and distribution of metastases, and the extent and condition of the growth in all parts of the body. The data thus obtained were reduced to a form that would permit of a quantitative expression.

On the production of generalized syphilis in the rabbit by local inoculation

Two of the most striking features of the infection usually produced in rabbits by testicular or scrotal inoculations of well-adapted strains of Treponema pallidum are the marked reaction at the site of inoculation and the total absence of generalized lesions. In fact, these features of the reaction to infection are so conspicuous as to suggest a casual connection between the two, especially when it has been shown that the failure to produce generalized lesions can in no wise be attributed to the absence of a generalized infection or to an insusceptibility on the part of the animals tissues to react to such organisms. Specifically, it appeared to us that in all probability, the failure to produce generalized lesions was due in a large measure to an inhibitory influence arising from the reaction at the primary focus of infection and that the reduction or suppression of this reaction might be sufficient in itself to permit the development of generalized lesions. In order to test this hypothesis, three types of experiments were carried out which were intended to compare the effects produced by unilateral and bilateral inoculations, the effects of castration and the effect of suppression of the primary lesions by the use of therapeutic agents. The castrations were done under ether anesthesia. Effects of Unilateral and Bilateral Inoculation and of Castration. -In the first series of experiments, there were 27 rabbits inoculated in one testicle and 20 inoculated in both testicles, giving a total of 47 rabbits. These were divided into two groups, one of which was castrated soon after the appearance of the primary lesion and the other held as controls. Both groups were kept under observation for a period of 4 months after inoculation.