STUDIES ON THE PAROTID AND THYROID GLANDS OF CARIES-SUSCEPTIBLE AND CARIES-RESISTANT STRAINS OF RATS (RATTUS NORVEGICUS, BERKENHAUT) By Roger F. Keller, Jr. A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Zoology 19 53 ProQ uest Number: 10008347 All rights reserved INFO RM ATIO N TO ALL USERS The quality o f this reproduction is dependent upon the quality of the copy subm itted. In the unlikely event that the author did not send a com plete m anuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQ uest 10008347 Published by ProQ uest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This w ork is protected against unauthorized copying under Title 17, United States Code M icroform Edition © ProQ uest LLC. ProQ uest LLC. 789 East Eisenhow er Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346 STUDIES ON THE PAROTID AND THYROID GLANDS OF CARIES-SUSCEPTIBLE AND CARIES-RESISTANT STRAINS OF RATS (RATTUS NQ RVEGICUS, BERKENHAUT) By Roger F. Keller, Jr. AN ABSTRACT Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Zoology Year Approved / .a .£ j . ~o 1953 Roger F. Keller, Jr. Abstract Hunt and Hoppert (1944) produced two strains of albino rats, one of which is extremely resistant to dental caries in the molar teeth and the other of which is susceptible to dental caries. This differentiation was accomplished by progeny test­ ing, close inbreeding and the use of a caries-producing diet. The present researches were conducted to ascertain whether these hereditary differences are due to either the secretions of the parotid gland or to the function of the thyroid gland. The parotid duct was severed and a section of it removed bilaterally from 35- to 40-day-old rats of both strains. These rats were then observed at bi-weekly intervals for the appear­ ance of macroscopic dental caries. The experimental procedure did not markedly alter the caries production of either strain of rats and the gros3 differences between the strains of rats persisted. It was concluded that the secretions of the parotid gland are not of great importance in the caries process of these animals. The thyroid glands of both strains of rats were examined in histological sections from tissues which were prepared with Bouin's fixative, mounted in paraffin, cut at seven microns thickness and stained with hematoxylin and eosin. It was found that caries-susceptible rats had larger colloid areas and that the mean height of their acinar epithelium was less than that of the caries-resistant rats. Roger F. Keller, Jr. It was observed that the thyroid glands of the rats in the susceptible series were about 43 percent heavier (on a mg. per 100 gm. body weight basis) than were the glands of the resistant series. There was shown to be a 42 percent faster turnover of intraperitoneally injected radioactive iodine in the thyroid glands of the resistant strain than in the susceptible strain. There were no demonstrable differences in oxygen consumption when the two strains were compared in the closed circuit type of unit. Therefore, it may be assumed that the thyroid secre­ tion rate is about the same in both strains of rats. It would appear that there is a compensatory mechanism in the susceptible strain's thyroid glands so that a large gland which has the appearance of an underactive gland, and which has a slower turnover of iodine, actually is metabolically equivalent to that of the smaller, more active gland of the resistant series. The use of thyroidally active protamone which raises the metabolic level and thiouracil, a goitrogen , failed to signif­ icantly alter the production of dental caries in the suscep­ tible series rats. Inbreeding tends to produce stocks which differ from one another in a number of traits. Each stock may approach homo­ zygosity for the traits which characterize it. The diverse traits in a stock are associated as a consequence of the chance recombination of genes in inbreeding, cause of another one. one trait need not be a It would appear that the thyroid traits Roger F. Keller, Jr. in these rats are associated with susceptibility or resistance to caries but that the former are not the cause of the latter. Hunt, H e R. and G. A. Hopp er t . Inheritance of susceptibility and resistance to caries in albino rats (Mus norvegicus) . J. Am. Col. Dentists. 11: 33-3?, 1944. i TABLE OF CONTENTS Acknowledgments ........................................... V i t a .................................................. Page iii iv Ligt of T a b l e s ...................................... List of Figures and P l a t e s ............................. . vi Introduction .......................................... 1 I. The selection which has been practiced II. v ........... 3 Is each line a homozygous p o p u l a t i o n ............. 3 Genic fixation within lines 4 ........................ Elimination of the secretion of the parotid gland and its relation to dental caries .................. 6 Cause of dental c a r i e s ............................. 6 Role of salivary secretions ........................ 9 The relationship of diet to tooth d e c a y ........... 11 The caries-producing diet used in the present s t u d y .......................................... 13 . ......... 13 ............................. 14 R e s u l t s .......................................... 1? C o n c l u s i o n s ...................................... 19 Dietary supplement of trace minerals Experimental procedure III. Experimental studies of the thyroid gland . . . . 30 The recovery of radioactive iodine from the thyroid gland following the intraperitoneal injection of tracer quantities of I1^ ! ........................ 30 Experimental study 22 ................................. 338664 Weight of the thyroid and pituitary glands . . . . Weight of the thyroid g l a n d s .................... 26 .................. 28 ............................. 29 Weight of the pituitary glands Respiratory metabolism Measurement of oxygen consumption in normal r a t s .................... 34 Induced hyper- and hypothyroidism . 38 Protamone fed to breeding rats . ............. 43 Thiouracil fed to breeding r a t s ............... 43 Secretion rate a s s a y ............................... 45 Biological assay of the thyroid secretion rate . 45 Experimental results ...................... 49 ... Histological examination of thyroid epithelium Experimental procedures . . ........................ Experimental r e s u l t s .......................... IV. ii Page 36 54 60 . 62 C o n c l u s i o n s ..................................... 71 S u m m a r y ............................................... 73 Physiology of the t h y r o i d ........................... 73 G e n e t i c s ............... 74 Parotid gland ........................................ 74 A p p e n d i x ................................................... 76 Literature cited .......................................... 90 i ii Acknowledgments The author wishes to express his most sincere thanks to Dr. H. R. Hunt, under whose supervision these investigations were conducted and who furnished the parents of the experi­ mental animals used. The author also wishes to thank Dr. E. P. Reineke for guidance in the thyroid studies and for providing the crystalline thyroxine used, as well as providing the use of the respiratory metabolism units. He is also greatly indebted to Dr. L. F. Wolterink for guidance in the radioactive studies and to Dr. B. V. Alfredson for making available the use of the facilities of the Physiology Department laboratories. Grateful acknowledgment is also due to Dr. C. A. Hoppert for assistance and instruction in checking r a t s ’ teeth for the presence of dental caries, to Mr. P. G. Coleman for the photo­ micrographs, and to Mr. E. Harrison for instructions in the care of the experimental animals and for assisting at the time of checking animals for the presence of dental caries. Special thanks are due also to Mrs. B, M. Henderson and to many other persons in the Zoology and Physiology Departments for helpful suggestions and assistance in many ways. Final thanks are due to his wife, Arline, for assistance in the experimental work and in the preparation of the manu­ script . Roger Folke Keller, Jr. Candidate for the degree of Doctor of Philosophy Final examination, May 22, 1953, Room 404, Natural Science Bldg. 8:30 A.M. Dr. H. R, Hunt, chairman Dr. E. P. Reineke Dr. R. H. Nelson Dr. H. J. Stafseth, Graduate Council representative Dissertation: Studies on the Parotid and Thyroid Glands of Caries=Susceptible and Caries=Resistant Strains of Rats (Rattus norvegicus, Berkenhaut) Outline of Studies: Major subject ~ Zoology Minor subjects - Physiology, Genetics Biographical Items: Born July 24, 1923, Manchester, New Hampshire. Undergraduate studies: University of New Hampshire, 1940-1943 George Washington University, 1946 Michigan State College, 1947 Graduate Studies: Michigan State College, 1947-1953* Experience: Graduate Assistant, Michigan State College, 1948-1952; Instructor in Zoology, Michigan State College, 19 52-19 53; Member, Army of United States, 1943=1946. Arts Lett ers for Advancement Sigma X i . List of Tables Number 1 Title Caries time for parotidectomized and colony controls. 2 Page 18 Average values of percent recovery of ll31# 24 3 Average weights of the thyroid glands. 28 4 Average weights of the pituitary glands. 29 5 Oxygen consumption of untreated rats of both strains. 6 Hyper- and hypothyroidism and dental caries. 7 Oxygen consumption of rats with induced hyper- and hypothyroidism. 8 Thyroid, secretion rate assay data. 9 Average height of acinar epithelial cells, (microns). 38 40 43 51 65 vi List Number I II III IV V of Figures and. Plates Title Facing Page Retention of radioactive iodide by the thyroid gland. 25 Method of plotting data to determine thyroid secretion rate of resistant series male rats. 52 Method of plotting data to determine thyroid secretion rate of resistant series female rats. 53 Relative numbers of acinar cells of various heights. Group I, (females) 69 Relative numbers of acinar cells of various heights. Group II, (males) 70 Photomicrographs of Histological Sections of Thyroid Glands Plate 1 66 Plate 2 67 Plate 3 68 I. Hunt and Hoppert INTRODUCTION (1944) proved that heredity is an important factor in the etiology of dental decay in the albino rat. This was accomplished by producing two decidedly dif­ ferent Btrains of rats, one of which is susceptible to dental caries in the lower molar teeth and the other which is very resistant. This differentiation was brought about by pheno­ typic selection, progeny testing, close inbreeding and the use of a caries producing diet devised by Hoppert, Webber and Caniff (1933). Continued selection and brother-sister inbreeding since the initiation of this project in 193? increased the differences in resistance to dental caries between these two strains of rats. The studies described in this paper were made on the seventeenth generation of the caries-resistant series and the twentieth generation of the caries-susceptible rats of these s trains. One of the objectives in the project of Hunt and Hoppert was to determine the mechanisms of the inheritance of these hereditary differences which were isolated. Before this may be determined, however, the physical and physiological factors which are involved in the hereditary aspects of dental caries must be determined. It was with this view in mind that the present researches were conducted. 2 The Selection Which Hag Been Practiced Phenotypic and genotypic selection have been practiced to produce caries-3usceptible and caries-resistant lines of rats. Those animals which developed caries soonest in the first generation were mated with each other, then the most susceptible progeny of such crosses were mated by the brother x sister system. Thereafter the most susceptible animals within each sibship were mated with each other. The most resistant rats in the first generation were crossed with one another, then the most resistant animals within each sibship were mated together. B y brother x sister matings a resistant strain was created. The performance of an animal was not an adequate index of the genes it carried (Hunt, Hoppert, and Erwin, 1944). Breeders were selected, not only on the basis of the time required to produce dental caries in themselves, but also according to the average time at which caries appeared in the sibship to which they belonged. The caries records of the females and their siblings were the only criteria for selecting female breeders. The weight of a prospective male breeder at 70 days of age was also considered, the heaviest male with the best caries record being used. Such a process of selection may be illustrated by the following diagram: Numbers Caries Production plus General Health 3 The entire population is represented by the area bounded by the larger curve and the animals selected for breeding by the shaded area. This situation would, in general, be true for each genera­ tion and for each strain of rats. Is Each Line a Homozygous Population? Inbreeding tends to produce homozygosity with respect to all the genes in a population. family within the resistant (or susceptible) series would be homozygous for the same gene 3 . ance One might think that every Intense selection for resist­ (or susceptibility) might be expected to lead to such a result. It seems likely, however, that different families with­ in the resistant strain are not homozygous with respect to alleles responsible for resistance. The resistants still show great variability, which decidedly discounts the notion that the stock is homozygous. On the other hand, the relative u n i ­ formity of the susceptibles suggests that there is a high degree of homozygosity within it. The possible contributions of dominance, epistasis, linkage, a n d their relations to the environment in these experimental animals have not been explored. tained as uniform as possible. The environment has been main­ However, inasmuch as the genetic factors may be varied, their respective interactions with the environment may vary also. The fact that selection is practised might very well be contributing to heterozygosity. This would be true if any of 4 the several criteria on which selection is based were due, even in part, to heterosis. Thus the heterozygous individuals pro­ duced wou l d be selected for parents of more heterozygous indi­ viduals and the less desirable homozygotes produced would be discarded (Haldane, 1936). Linkage could also be contributing to other forces which prevent a complete homozygosity of these two lines. The desired genes might conceivably be located close to undesired genes on the same chromosome and thus be discarded from the genic pool in the stocks. The problem is, of course, considerably more complicated than simple mono-factorial inheritance. multiple factors are involved. Obviously The genetics of such a problem must be analyzed from a biometrical point of view in order to utilize fully the tremendous wealth of information in the data. Such a task has not been undertaken as yet on a sufficiently extensive scale. Genic Fixation Within the Lines The method of selection of breeders and the breeding pro= gram being used may be contributing to the lack of uniform resistance to dental caries shown by the resistant line. It is undoubtedly true that in many cases desired genes have been discarded from the "genic pool” of these rats along with undesired genes. The mating of related individuals is, of course, the only known method of producing homozygosity for desired genic 5 characters where polygenes are present. The more intense the inbreeding, the more rapid is the approach to the genic fixa­ tion in the homozygous state. however, In the work of Hunt and Hoppert, it has been the purpose to produce the two lines of albino rats, one susceptible and the second resistant to dental caries. This has been very successful. This isolation of the desired genic conditions has been accomplished by using rigid selection in combination with intense inbreeding. Perhaps if the inbreeding had been less intense the approach to genic fixation would have been less rapid. A slow approach has the advantage that the genic complements present may be examined before they are discarded or kept in the "genic pool" repre- sented by the parental stocks used for the next generation. In a breeding system usi ng less intense inbreeding and thus pro­ ducing a slow fixation there is less likelihood that undesired genes would be fixed along with desired genes. It might prove desirable also to cross some of the present lines and thus obtain recombinations of desired genes. These new combinations could again be intensified and made homozygous if desired. The probability exists that each of the lines con­ tain some genes which may be lacking in other lines, and that such recombinations as suggested would prove to be better than any of the present line.3. 6 XI. ELIMINATION OF THE SECRETION OF THE PAROTID GLAND AND ITS RELATION TO DENTAL CARIES Causes of Dental Caries Caries is defined in Webster's New International Dietionary as ” ... the decay of animal tissues, ...n . especially of bony tissue Dental caries is defined as "...localized destruction of tooth tissues by micro-organisms...". The classical theory of the cause of dental caries is the so-called acid theory of Miller (1890), who contended that acids produced by oral micro­ organisms acting on dietary carbohydrate particles in the oral cavity dissolved the mineral salts from the teeth. It was also thought that proteolytic enzymes contributed to the process by dissolving the organic matrix of the teeth. A large proportion of the dental research since Miller's original hypothesis has been devoted to the removal of the mouth acids by various means. (1937) Bunting, Nickerson, and Hard observed that the bacterium Bacillus acidophilus^ was found in the mouths of persons having dental caries, that the presence of these bacteria tended to be localized in areas undergoing active caries, and that they were absent, or present in small numbers, where no active caries were found. Hawkins (1929) concluded that bacteria were able to break down carbohydrates, forming a concentration of acid sufficient to decalcify teeth. ^ Now known as Lactobacillus acidophilus 7 In summarizing a number of studies from his laboratory, (1938) Jay concluded that there existed a diagnostic relationship between oral lactobacilli and the incidence of dental caries. Neuwirth and Klosterman (1940) demonstrated that there was a rapid production of lactic acid in the mouth when carbohy­ drates were allowed to ferment there. It was demonstrated that this acid formation was probably due to the action of oral microorganisms. Several other microorganisms have also been isolated from mouths an d many of these have been considered as factors in the production of acids or of enzymes in the saliva. like organisms, Yeast­ (Candida al b ic a ns ), were isolated from mouths by Lilienthal (1950), who abserved a synergistic relationship between the yeast and lactobacilli which was responsible for more than normal amounts of lactic acid. Neither organism alone was able to produce the amount of acid which the two to­ gether generated. Streptococci were regarded by Bibby ejt al_. (1942) as important acid producers in the mouth and Shiere, Georgi and Ireland (1951)' observed that streptocci were able to convert fermentable sugars to lactic acid. Boyd, Cheyne and Wessels (1949) confirmed a very common observation that the lactobacillus count increases with mount­ ing caries activity. They found, however, that the high acidophilus counts were not necessarily a cause of dental caries per se , but that they might simply be an accompanying 8 phenomenon. Hugill and Box (1950) were able to produce experi- mental caries in the absence of bacterial activity, and the carious lesions resembled the dental pathological lesion. Hartles and MacDonald (i960) conclude that acid production per se is evidently not a sufficient cause for dental caries observed in v i v o . A new concept of the dental caries mechanism has been advanced by Csernyei (1950). Dental lymph, with its content of phosphatase, dissolved magnesium and fluorine, passes through the ultracapillaries of the tooth into the enamel. As soon as the physiological equilibrium between the dissolved magnesium and fluorine is altered, the phosphatase may hydrolyze enamel an d produce a carious lesion. The carious lesion is the first clinical sign of dental caries and would be initiated inside the tooth rather than on the outside, as the acid theory demands. The post-eruptive tooth has recently been shown to be more active metabolically than was first thought. It has been known for some time that there was an appreciable ionic exchange between bone and the circulating fluids. Manley and Bale (1939) observed that rat molars showed a slow, but measurable turnover of phosphorus. The turnover was found to be in the inorganic portion of the molars, and this finding invalidated the concept that there is no metabolism in teeth subsequent to the initial deposition of the inorganic salts. It has been shown that there is a metabolic relationship between the saliva and teeth with reference to calcium, 9 phosphorus, proteolytic enzymes as well as acids in the mouth. Dentay and Rae (1949) demonstrated that the saliva after filter­ ing contains no phosphatase. This enzyme then seems to come fr om cellular debris and from oral microorganisms. Thus, the importance of the phosphatase enzyme in the saliva is being recognized as at least a component in the complex etiology of dental caries. The Role of Salivary Secretions The role of salivary secretions determine accurately. is a difficult one to It has been shown that desalivation of rats hastened the onset of dental caries, and that the severity was also increased. Ginn and Volker (1941) desalivated a group of rats and compared them with nsham-surgery" controls and intact controls. The results of the sham-surgery, in which all procedures were duplicated except that the salivary glands were not removed, were the same as for the intact controls. The existence of an endocrine-like relationship between the salivary glands and the sex organs is indicated by the work of Higashizo (1941), who found that the removal of the sub- maxillary gland3 and ligation of the parotid duct in young rats resulted in hypertrophy of the uterus and atrophy of the t es t es . Hukusima (1941) removed both the parotid and submaxillary glands of young rats and markedly increased the incidence of molar caries. 10 Kite, Shaw and Sogannes desalivated rats. (i960) tube fed both normal and Their results showed that tooth decay is prevented in caries susceptible rats when the direct effects of food in the oral cavity are eliminated. They confirmed the fact that the salivary secretions are important in the etiology of tooth decay. Weisberger, Nelson and Boyle (1940) observed that the acceleration of dental caries activity following the extirpation of the salivary glands was due primarily to changes in the cementum of the teeth. Cheyne (1939b) removed various combinations of the major salivary glands of the rat. The greatest amount of caries followed the removal of the parotid and the submaxillary glands. The extirpation of the parotids alone produced only slightly more dental caries than in controls. Manhold and Manhold (1949) observed a significant corre­ lation between psychological factors and dental decay. Pre­ sumably excessive amounts of saliva would have the effect of washing the teeth, neutralizing acids, and thus reducing dental c ar i es . An unidentified factor in some salivas was reported by Hill (1939). There was a variation in intensity of this factor and its presence or absence was found to be consistent with the presence or absence of dental caries in rats. tigations have not identified this factor. Subsequent Hill inves­ (1953) was not able to determine any great differences between any inhibitory fractions in the susceptible and resistant strains of the Hunt- Hoppert rats. 11 The Relationship of Diet to Tooth Decay The deficiency of necessary dietary elements during the period of tooth formation may have a deleterious influence on deciduous tooth formation and structure (Howe, 1923). Thus the metabolic processes influenced by diet are also important com­ ponents in the etiology of dental caries. The major metabolic effects of diet upon tooth decay seem to operate prior to the time when the teeth have developed (Shaw, 1949). Shaw (1950), applying the Sogannes (1948) prenatal and preweaning cariogenic feeding techniques, rendered his cariesresistant rats susceptible to tooth decay. Bunting (1935) believed that the dietary factors influence dental caries through the determination of the environment of the teeth rather than through changes in the resistance of the tooth itself. Hoppert, Webber and Canniff (1933) were successful in pro­ ducing experimental dental caries in rats by the inclusion of coarse particles of grain in their diets. resulted when fine particles were used. Less dental caries Liberal amounts of vitamins A and D, as well as supplements of calcium and phosphorus did not appreciably retard the tooth decay resulting from the inclusion of coarse particles in the diet. confirmed by Rosebury and Karshan These findings were (1935) who prevented experi­ mental caries by using rice ground to flour fineness. Sogannes (1948) believed that the experimental caries produced in this manner were due to mechanical breakages and subsequent 13 enlargement of the lesion produced. Van Huysen (1950), however, observed that while some minor fracturing was present on a diet containing.large particles, the teeth did not fracture unless the cusps were first undermined by dental caries. Nakfoor, Hunt, and Hoppert (1952) tested the Hunt-Hoppert rats for resistance to breakage of the cusps of the lower molar teeth. It was found that the caries-resistant rats showed a greater resistance to breakage than did the caries-susceptible rats. Fracturing, however, was not found to be an important factor in initiating dental caries in these rats. These investigators were not able to duplicate the results of Braunschneider, Hunt and Hoppert (1948) in preventing dental caries by using flour-fine rice in the diets of the Hunt-Hoppert strains. In view of the fact that Nizel and Harris (1951) observed that foods grown on different soils had cariogenic properties, it may well be that these conflicting results were due to different origins of the foodstuffs. The rice used could have been nutritionally quite different though ground to flour fineness in both cases. Kifer (1953) believes that the sulci of the molar teeth of the Hunt-Hoppert susceptible and resistant rats differ somewhat. The sulci in the molar teeth of the caries- susceptible rats are wider than those in the caries-resistant strain. These differences would presumably be important in the impaction of food within the sulci. 13 Clise and Hunt (1953) observed that the rates of growth of the susceptible and resistant strains were essentially the same for the first 21 weeks in males and 40 weeks in females, after which differences in growth appeared. At one year of age susceptible males and females weighed significantly less than the resistant males and females. The causes of this deviation are unknown. The caries-producing diet used in the present stu dy . The composition of this diet, by weight, was 66 percent ground polished rice, 30 percent whole milk powder, 3 percent of alfalfa leaf meal and 1 percent of sodium chloride. The rice u s e d was passed through a precision grinder, adjusted so that about 3 or 3 percent of it would be retained on a 20-mesh screen .when sifted. This ration is used in the colony of Hunt and Hoppert as a standard diet. Dietary supplement of trace minerals. In order to deter­ mine whether or not a dietary supplement of vitamins and minerals would alter the response to dental caries in suscep­ tible series rats, a commercial product 11Vi ta-D-Mineral Sup­ pl em ent” , manufactured by Vitamineral Products Company, Peoria, Illinois, was added to the standard caries-producing ration in a preliminary study (unpublished data). The ingredients of this supplement were stated by the manufacturer to be as follows: ground limestone, defluorinated phosphate, dicalcium phosphate, steamed bone meal, vitamin D, 14 brewers yeast and trace minerals (Appendix VI). The supplement was added to the standard diet in the ratio of four parts per hundred an d fed to two litters of susceptible series rats (pre- a nd postnatally). The results were not conclusive but it was evident that this dietary supplement did have some effect on the incidence of dental caries in the Hunt-Hoppert rats. The rats themselves exhibited good growth and their incidence of dental caries appeared to be reduced. This study was not continued but the results are suggestive of a possible line of investigation for the f u t u r e . Experimental Procedure The lower counts of lactobacilli in the mouths of cariesresistant rats observed by Jay, Hunt and Hoppert (1944) suggest that the salivas of the two strains are chemically different. If that is true, the elimination of the secretions from one or more of the salivary glands might, strains of rats to develop caries conceivably, cause the two in about the same length of time if the differences in the strains were due to salivary secretions. It is a simple procedure to sever the parotid duct, so this operation was carried out. All animals used were produced according to the standard procedures of the project. The controls were from rats pro­ duced by Hunt and Hoppert in the prosecution of their experiment on the hereditary factors. After one or more litters of young had been produced for Hunt and Hoppert*s work, certain of the 15 breeding females were used to raise young for the parotid gland experiment. Full sibs produced in earlier litters were u se d as controls. All animals were fed the standard diet of the colony. When the experimental animals were thirty-five to forty days old they were anesthetized with ether, the hair covering the sides of the head removed and the exposed skin cleaned with alcohol. The rat was held on its back and an incision made in the skin of the head anterior and ventral to the ear on the side to be operated on. and a section of it removed. The parotid duct was located Care was taken not to injure the facial nerve which courses parallel to the parotid duct in this area (Greene, 1935). This was desirable since Jarbak (1950) showed that resection of this nerve in the rat caused paralysis in the facial muscles. This paralysis resulted in an increased incidence of dental caries, presumably due to a lack of buc­ cinator function which resulted in increased deposits of food along the side of the third and second molar teeth. A portion of the parotid gland was removed along with the duct. It was not considered advisable to remove all of the gland because its extirpation severs several fibers of the great auricular nerve derived from the cervical and brachial plexuses which emerge from the shoulder and penetrate the paro­ tid (Cheyne, very rich. 1939a). The blood supply to the parotid gland is The parotid covers the posterior auricular artery which may be broken by operative procedures for the complete removal of the parotid gland. 16 The exposed area was sprinkled with veterinary uride powder to prevent infection and to promote healing. was closed with wound clips. The incision As soon as the surgical procedures were completed on one side, they were repeated on the other side. Each animal was then ear-notched for identification purposes and placed in a clean cage. When the wound had healed suffi­ ciently the clip was removed. There was no case of serious inf ect i o n . All animals were kept in cages made of galvanized sheet steel closed on all sides except the front and top, which were covered with one quarter inch galvanized steel mesh. measured twelve inches in height, twenty inches in length. in a cage at one time. Cages fourteen inches in width and From one to five animals were kept The number of rats usually did not exceed four, however, and all were of the same sex. ings were used as litter. Wood shav­ Water was available at all times from a drip bottle. The animals were kept in a large, well lighted room main­ tained at a temperature of approximately 78° F. There were no means of preventing higher temperatures during the summer, when,occasionally, higher readings were noted. The experimental and the control rats were inspected at bi-weekly intervals for the presence of macroscopic dental caries. This inspection was made with the unaided eye. The tongue was pushed aside and the jaws held apart by a nasal speculum. Light was supplied from an ordinary desk lamp. The 17 animal to be examined was held firrnly by an assistant who grasped it by the loose skin at the back of the neck with one hand and prevented bodily movements with the other hand. When macroscopic dental caries were observed for the first time, this fact and the a n i m a l ’s age at that observation were recorded. This age in days minus a constant factor of thirty- five days was called the wcaries time” of a rat. At the outset of Hunt and H oppert * 3 experiment the ground rice in the ration contained about seventy percent of particles that would be retained on a 20=*mesh screen. This diet was introduced when the rats were thirty-five days old. This ration mechanically injured the upper molars so it was exchanged for a diet, present from birth, which contained rice having two or three percent of the coarse rice particles. Since that exchange, thirty- five days have been subtracted from the age at which caries first appeared to make the earlier and the later data comparable. All experimental rats were autopsied after they developed dental caries to verify the removal of the parotid duct. R esults. The removal of parotid secretion did not markedly alter the time of onset of the first carious lesion. The sus­ ceptible series rat3 developed caries twelve days later, average, than the susceptible controls. on the This difference, how­ ever, was les3 than the number of days between observation periods for carious cavities (Table 1). The ”t ” test (Snedecor, 1946) was used to test this difference for significance. 18 TABLE 1 CARIES TIME FOR PAROTIDECTOMIZED AND COLONY CONTROLS Susceptible Ser ies Treatment No. Avg. of rats caries time Avg. periods Intact Parotidectomized 85 55 Resistant Intact 160 39*3* 512 3* 479*15* 2.8=4.96 3. 5* .09 3 4*1 "t n 13 Avg. of sibship means 40*3 60 436*30* 31*2 0 .10 0. 78 No. of sibships Parot idectomized 11 551 8 28 487*63 12 452*35 * 4- Standard error of the mean. Since t “ 0.78, the difference was not significant and could be attributed to chance alone. The resistant series parotidec- tomized rats required forty-three days less, on the average, for the first appearance of dental caries than the colony controls. This difference is approximately three observation periods. The resistants were extremely variable so that this difference was not significant (t " 0.10). The mean caries time within each sibship was also computed and these means were averaged for a value, the average of the sibship mean. These averages for both series of rats are also presented in table 1. are presented in Appendix I. Complete caries times for all rats 19 Co nclusions. Cheyne (1939b) showed that parotidectomized rats had slightly more carious teeth on a diet containing coarse particles. Once initiated, caries were said to be more rapid in the rate of development in parotidectomized rats. the onset of dental caries was altered, however, Whether is not stated. If there was any increase in the rate of development of the carious lesion subsequent to its initiation, the method of observation in the present investigation would not bring it out. It has been demonstrated that the inherited difference between the caries susceptible and caries resistant HuntHoppert strains is not to any considerably degree due to the secretions of the parotid gland. The behavior of rats of the two strains remains essentially the same with respect to the development of dental caries when they are parotidectomized as when they are intact. 20 III. EXPERIMENTAL STUDIES OF THE THYROID GLAND Recovery of Radioactive Iodine from the Thyroid Gland following the Intraperitoneal Injection of Tracer Quantities of 1^*^ Iodine has been demonstrated to be a very essential element in vertebrate metabolism. The relationships between the func= tion of the thyroid gland in vertebrates and the metabolism of iodine have been the subject of numerous investigations, parti- cularly since the advent of radioactive tracer techniques. Iodine has a number of radioactive isotopes. Currently which has a half life of eight days, is used almost exclusively. 131 Iodine produces both gamma and beta radiation but the bio­ logical effect of the gamma rays is usually negligible because of the very small absorption of these rays by the thyroid (Chapman and Evans, 19 46). The administration of large quantities of the radioactive iodine will damage the thyroid gland by internal beta radiation. Since the parathyroid gland in the rat is largely within the thyroid gland, (Gorbman, it too becomes damaged by the internal radiation 131 1947). Sufficiently high doses of I will cau3e curtailment of growth, failure of functional and regenerative processes of the thyroid gland and parathyroid injury, as well as recurrent nerve injury, presumably due to the large quanti­ ties accumulated in the thyroid gland (Gorbman, 1950). Winchester, Comar and Davis (1949) completely eliminated 131 the thyroids of young chickens by I irradiation apparently 21 without damage to organs or tissues other than the thyroid and parathyroid glands. Replacement therapy with d,l-thyroxine in a water suspension allowed the birds to grow normally. On the other hand, however, with radioactive iodine in the quantities ordinarily used in tracer studies there is no apparent injury to the thyroid gland (Gorbman, 1950, and Pearlmaru et a l . 1941). When iodine is administered to normal animals, accumulated and deposited in the thyroid gland. it is The rate at which this occurs is dependent upon the amount, the form and manner in which it is administered as well as the amount present in the animal's body and in its diet. In addition, it has been shown that the thyroid gland of the rat is dependent on the presence of thyrotropic hormone for its capacity to concentrate iodine from the serum, to bind iodine to protein and to dis­ charge thyroid hormone (Vanderlaan and Greer, 1950). Adrainis131 tered doses of I enter the thyroid gland as inorganic iodine and this is rapidly transformed into diiodotyrosine a n d as such may be deposited in the gland (Leblond _et, al. , 1946). The presence of radioactivity in many but not all follicles of the gland as early as one hour after injection shows that follicles differ in their ability to fix iodine actively. In general, the .less active follicles are found under the capsule and in the isthmus of the gland (Leblond and Gross, 1948). 23 Experimental Study A standardized tracer dose of carrier-free radioactive . _ /T1 3 1 x iodine vi ; was injected intraperitoneally into a total of 184 rats of the two strains. zation at Rats were sacrificed by etheri- intervals of 18, 68 and 148 hours after injection. The thyroid glands were immediately removed, cleaned of con­ nective tissue and weighed on a Roller-Smith balance. The thyroids were then placed on small copper discs and allowed to dry, after which they were counted with a thin end window G.M. tube. At the time of administration, a measured amount of the injection solution was placed on similar discs and dried. These standard discs were counted at the same time as those containing the thyroid glands, thus eliminating the necessity of correcting arithmetically for the physical decay. The 131 thyroid I present at the times of sacrifice are summarized in table 2 and appendix table I I . The standard error accom­ panies each mean. At 18 hours after injection an average of 4.65 percent of the injected dose was recovered in the thyroids of the suscep­ tible series. In the resistant series the comparable recovery was 4.35 percent, which was not significantly different. At 68 hours the percent of administered dose present was determined in four separate groups of animals from three experi­ ments. Inspection of the values within strains shows that the groups were essentially similar and that they might then be grouped into a single larger category for comparison between strains. The thyroids of the resistant strain contained an 23 average of 3.55 percent of the injected dose. Those of the susceptible strain contained 5.57 percent of the injected dos a The difference between these means is highly significant. At 143 hours the thyroid I 131 activity was determined in three groups of rat3 of each strain. Since these three groups were also found to be essentially similar they too were grouped into larger categories for comparison between strains. The thyroids of the resistant strain contained an average of 2.69 percent of the injected dose. The comparable recovery was 4.53 percent from the susceptible strain. significantly different. These means are The mean values at each of the three times of sacrifice have been plotted in figure I. The 1^*31 retention values found in these rats are somewhat lower than those reported for the Sherman Strain of rats by Meite3 and Wolterink (1950) who obtained about 7.9 percent retention at eight hours. Cortell and Rawson (1944) report uptake values at four hours between four and thirteen percent, also with the Sherman Strain of rats. Other strains of rats on other diets have considerably higher uptake values, however. mined I u p t a k e Keating ejt al. to be about 17 percent, while Morton et. al. X31 (1943) found uptake values of I four hours. (1945) deter­ Pearlman ejt al. (1941) found that up to 65 percent 131 of an administered dose of I ° between 25 and 50 hours. to be over 60 percent at was taken up by the thyroid McGinty (1949) and Jones (1951) showed that the amount of iodine retained by the thyroid gland 34 may vary with the degree of hyperplasia and with the dietary levels of iodide. different Differences which have been reported from strains of rats could be due in part to differences in either of these variables as well as to functional differ­ ences. In the present study all rats were fed ad l i b , the standard caries diet of Hunt and Hoppert which contains 1.0 percent salt to which has been added 0.01 percent potassium iodide. Since this level is present, the thyroid glands of the experimental animals were saturated with dietary iodide. Thus there was a smaller percentage uptake of the injected dose of radioactive iodine which may be attributed to this fact. The differences in the uptake-retention curves of the susceptible and resistant strains then represent a faster 131 turnover rate of I in the resistant strain. From the data of this investigation, it appears that the turnover rate of the resistant strain is about 43 percent greater than that of the susceptible strain. TABLE 2 AVERAGE VALUES OF PERCENT RECOVERY OF I131 Time (Hours) Res istant________ ll31 sem* No. of rats Susceptible______ i!3i sem* N o . of rats 18 4.25*0.28 9 4.65*0.62 5 68 3.55*0.16 39 5.57*0.25 50 148 3.69+0.11 32 4.58+0.15 49 * Standard error of mean 25 FIGURE I Retention of Radioactive Iodide by the Thyroid Gland Mean values, plus and minus twice the standard error, are plotted. The retention curve is drawn from the mean values. 00 in Ul CSl m oo oo CM oo in ro CM ±N3S3Ud |£|X a 3 1 0 3 r N I 30 ±N30U3d HOURS oo INJECTION in 36 Weight of the Thyroid and Pituitary Glands Weight of the Thyroid G l a n d s . When differences in the gross thyroid weight of a number of rat 3 of the two strains were first noted, it was considered advisable to treat all available data statistically to determine whether or not these variations were the result of chance observations or whether true differences did exist. Among normal rats under standard environmental conditions the thyroid gland is nearly constant in size. Thyroxine is produced in response to the stimulus of the thyrotropic hormone of the anterior pituitary gland. When the thyroxine level drops below the required levels for an individual animal, the thyrotropic hormone of the anterior pituitary causes enlargeraent of the gland and increased production of thyroxine. Con- versely, when a thyroid gland produces an excess amount of thyroxine, this excess causes a reduced amount of thyrotropic hormone to be produced, which in turn causes a reduction in the production of thyroxine by the thyroid gland. A fine recipro­ cal control is thus exhibited by the thyrotropic hormone of the anterior pituitary gland and the thyroxine of the thyroid gland. This control is responsible for the constancy of thyroid gland weights usually observed among experimental an imals. Hyperemia and enlargement of the thyroid gland may be produced under experimental conditions as is stated below, but as previously mentioned the gland-body weight relationships are considered to remain fairly constant under normal conditions. 27 The thyroid glands of all rats of the investigations cont131 cermng I uptake and the assay of the rate of secretion of the thyroid hormone, were dissected from the animals. The glands were weighed to the nearest 0.01 milligram on a BoilerSmith precision balance. nearest gram. The body weight was taken to the All gland weights were expressed as gland weight in milligrams per one hundred grams body weight. The difference in thyroid weight per 100 g m s . body weight among the various groups was examined statistically by means of the "tn test (Snedecor, 1946). The difference between the males and females of each strain was found to be non-significant resistant series t = (susceptible series t - 1.12, .50, Appendix III). On this basis, then, the males and females of each strain were combined for a compar­ ison between the strains. Table 3 shows that the average weight of the thyroid gland expressed as milligrams per one hundred grams body weight in the susceptible series was 8.03*0.13 and the corresponding value for the resistant series was 5.62^0.15. The difference of 2.41 is highly significant, (t - 12 .2 ). thyroid glands of the susceptible series rats are about 43 percent larger than are those of the resistant series rats, when they are compared in this manner. The 28 TABLE 3 AVERAGE WEIGHTS OF THE THYROID GLAND Series No. of rats Mg. 100 Gm. B.W. Standard Deviat ion Suscept ible 118 8.03*0.13* 1.43 Resistant 114 5.63*0.15* 1. 58 Total 232 t “ 13.2, highly significant * Standard error of the mean. Weight of the Pituitary G l a n d s . The pituitary glands were removed from twenty-two susceptible series rats and seventeen resistant series rats. These glands were weighed on a Roller- Smith torsion scale and the weights compared on a gland weight basis as well as a gland weight per one hundred grams body weight basis. The results are summarized in table 4. The average gland weight from susceptible animals was 6.0*0,41 and that of resistant animals, The difference of 1.0 mg. tistically significant 5.0*0.19 milligrams. between the two values is not sta­ ("tn = 0.6). The average gland weight expressed as milligrams per 100 grams body weight, for the susceptible series animals, was 3.4*0.34 and for the resistant series was 3.9*0.17. ference between these two values of 0.5 is again non­ significant (Mt n - 0.3). The dif* 39 Thus there was no apparent difference in gross weights of the pituitary glands between the two strains of rats* TABLE 4 AVERAGE WEIGHT OF THE PITUITARY GLANDS Strain Humber W t . in milligrams W t . in m g . per 100 gm. B.W. Susceptible 22 6. Ot-O. 41* 3.4+0.34* Resistant 17 5.0+0.19* 3.9*0.17* "tw value * Standard error of the mean. 0.6 0.3 29 Respiratory Metabolism When apparent differences in iodine metabolism and gross size differences in the thyroid gland were noted, it was con­ sidered advisable to determine whether or not there were differences rats. in gross energy metabolism between the two strains of Metabolism will vary according to a large number of con­ ditions. Among these may be mentioned season, activity, and temperature variants. Seasonal rhythms of one sort or another are present most animals. in It is known that thyroid activity is sensitive to changes in temperature and light irradiation, and this factor along with anterior pituitary and gonadal factors may be at least partly responsible for some variations. (1936) observed that normal adult Sherwood rats exhibited up to 26 per­ cent diminution in metabolism during summer months. The larger animals showed the decrease to a lesser extent than did smaller animals. Benedict and MacLeod (1929) observed that heat pro­ duction as measured in gaseous metabolic studies is lower in the summer, and their values ranged to a 12 percent decline. It is well known that an animal working, or at exercise, consumes more oxygen than does an animal at rest. For this reason studies of basal metabolic levels must be made with the animal at rest. Benedict and MacLeod (1939) found that very mild activity in rats did not alter oxygen consumption values, and they concluded that ocular observations are sufficient to estimate activity. Observations on animals which are excess­ ively active would not be considered as basal but would be 30 considered as representing values obtained under conditions of exercise. The influence of age and size of animals are of importance in such determinations. Brody and Procter (1932) measured metabolic level and size in a large series of animals and found that basal energy varies as the. (0.73) power of body They suggested that this oorrection be used in making comparisons among animals of different body weights. Belasco and Murlin (1941) proved that the basal metabolism of normal rats is higher in young animals than in adults. The level declines rapidly during the first few months, then becomes rather constant. As mentioned in the discussion of thyroid secretion rates, there may be some differences between the sexes in this respect. Several investigations, however, no differences in gaseous metabolism. indicate little or Davis and Hastings (1934) failed to find any differences in rats up to four months of age in a series of 136 animals. Barker (1945), likewise, found no differences between the sexes. On the other hand, Benedict and ftfacLeod (1929) observed that male rats have a higher metabolism until an age of 14 months. Soliman (1952) observed that female rats in estrus con­ sumed more oxygen than did these rats in other phases of the estrus cycle. Lee (1928), however, in a similar study found that there was a significant increase in metabolism of rats during the last ten hours of diestrus and the first six hours of proestrus. There was no change in metabolic level in the 31 other phases of the estrus cycle. Some of the variation observed in female rats would seem to be due to the fact that there is some variation in metabolic rates during the several phases of the estrus cycle. The nature of the food which an animal has been eating has some effect on gaseous metabolism. Benedict (1934) studied this influence. Horst, Mendel and Many types of balanced rations in rats gave essentially uniform results. A diminished protein intake prior to the determination, however, generally caused a reduced metabolism in the animal. These investigators studied the effects of prolonged fasting and found the oxygen consumption dropped steadily and rapidly until the animal died. Benedict and MacLeod (1939) observed that the metabolism of the rat is depressed ten percent by twenty-four hours fasting. They suggest that in rats a seventeen hour period of fastingprior to gaseous metabolism checks is adequate to eliminate the complications of diet, so that the animal is at a basal level. Cori and Cori (1934) showed that the metabolism of a fasting rat is 90 percent fat oxidation, and hence because of this fact as well as the difficulty in making urinary collections, it is not considered necessary to make corrections for the nitrogen content of urine in determinations involving fasted rats. Horst, Mendel and Benedict is subject to diurnal variation. (1934) observed that the rat It was observed by these workers that oxygen consumption in the rat is high early in 32 the morning and in the late afternoon. tion, Because of this varia­ it is suggested by them that all determinations be made after 10 o'clock in the morning and before 4 o'clock in the afternoon. where In addition to this, it has been suggested else­ (Benedict and MacLeod, 1939) that determinations involv= ing rats be made in a well-lighted room and that the unit chamber be lighted so that the rat will be quieter. Since the rat is a homeothermic animal, any gross changes in environmental temperature will necessarily involve internal metabolic changes which counteract these external changes. The temperatures at which metabolic studies are made influence the net results. Using several environmental temperatures, Benedict and MacLeod (1929) found that 28° C. was the optimal temperature. Values obtained below 28° C. were not found to be as valid as those at 28° C. or slightly above. Rats maintained in an environment of 16° C. may consume up to twice as much oxygen as rat3 at 28° C. (Horst, Mendel, and Benedict, 1930). Certain exogenous substances have a marked influence on bodily metabolism. Since the work of Mackenzie and Mackenzie (1943) and others, goitrogens have been used widely to lower metabolic levels. It was found that the basal metabolic rate could be lowered as much as twenty percent in ten to fourteen days. Meyer and Ransom (1945) reported values as much as forty percent below normal by the use of goitrogens. Potent goitrogens have the same metabolic effect as does thyroidectomy in that the animal is deprived of the hormone 33 thyroxine and the metabolic level drops (Astwood ef, al,. > 1943). Rats receiving thiouracil show a slower decline in metabolic rate and heart rate than do thyroidectomized rats but they eventually reach the same level (Meyer and Ransom, 1945). Presumably this is because thiouracil inhibits the production of new thyroxine but it does not interfere with the use of any which is stored and thus is available (Halpert, Cavanaugh a nd Keltz, 1946). As an experimental tool in lowering the metabolism, a potent antithyroid drug has some advantages over thyroidectomy insofar as the parathyroids are not damaged and any scattered thyroid tissue is reached by the drug. When exogenous thyroxine is supplied to thyroidectomized animals or to thiouracil-treated animals the metabolism may be restored to nearly any desired level. Individual varia­ tions in thiouracil-treated rats are considerably larger than in thyroidectomized animals, and their response to ingested or injected standard doses of thyroid substances are somewhat erratic and irregular (Meyer and Ransom, 1945). The administration of exogenous thyroxine to normal animals does not necessarily raise their metabolic level. It may simply set in motion mechanisms which serve to suppress the bodily production of thyroxine. To raise the metabolic level the administered thyroxine must be in excess of that which the animal would normally produce. 34 Meyer and Wertz (1939) increased oxygen consumption 2.5 to 30 percent with the administration of thyroxine. In addition to raising metabolic levels as evidenced by increased oxygen consumption, thyroxine administration also causes body weight losses in mature animals or reduced rates of gain in growing animal3 (Belasco and Murlin, 1941). Reisfield (1950) found that exogenous thyroidally active substances caused decreased resistance to anoxia as evidenced by survival time in closed jars. This was because metabolic levels were raised and oxygen requirements were increased. Greenberg (1952) found that vitamin has no appreciable effect in counteracting the growth retarding action of dietary desiccated thyroid powder in rats while methyl linoleate plus cottonseed oil administration was found to protect the rat against such retardation. The health and general condition of animals in metabolic studies are important inasmuch as poor health may seriously alter such levels and serve to confuse the investigator or even invalidate his results. Physiologically abnormal rats show extreme variations in metabolic levels MacLeod, (Benedict and 1929). The measurement of oxygen consumption in normal ra t s . The oxygen consumption of male rats of both strains was checked in c lose d circuit metabolism units after the method of Mac Lagan an d Sheahan (1950). The rats were placed in calibrated desic­ cators filled with oxygen and which contained a pan of soda 35 lime. The air was partially removed from the desiccator and system w ith a vacuum pump and oxygen was added from a Douglas bag. This system was connected to a U tube filled with mercury. Pressure readings were made at fifteen-rainute intervals for a period of an hour and a half or more. The carbon dioxide pro- duced by the respiring rats was absorbed by the soda lime. The amount of oxygen used by the rat then was determined by con­ verting the pressure changes in the system by the use of the following formula: x 373 T x Where V^ s Volume of desiccator V r = Volume of the rat assuming 1 gm. = 1 oo. Vol. p s Fall in pressure in mmHg per hour T = Absolute temperature inside the desiccator Og = Oxygen consumption per unit of body weight adjusted to the .73 power The rat weight was adjusted by using the .73 power because the range in body weight was too great to use a simple correc­ tion. Body weights ranged from 195 to 408 gras. Procter Brody and (1933) suggest using this correction when comparing animals of different body weights because they find that the metabolism of mammals varies with the .73 power of body weight. The determinations were made in an air conditioned room maintained at a constant temperature (24>1° C.) and constant 36 humidity. Prior to the test the rats were maintained on the standard caries-producing diet of Hunt and Hoppert. Food was withheld for at least 17 hours preceding the tests, but water was avilable except during the actual oxygen determination. The units were arranged in a bank of twelve. Thus it was pos­ sible to run determinations on six rats of each strain at one time. The purpose was to compare the two strains with each other. The rat was placed in the desiccator and the unit sealed. Air was evacuated by means of a vacuum pump which produced a pressure change of about 150 mm. Hg. and oxygen w a 3 added from a Douglas bag. The unit was allowed to reach an equilibrium temperature during which time the rat also became quiescent. This preliminary period lasted at least one-half hour after which the pressure inside the unit was adjusted to equal the atmospheric pressure by adding oxygen. After an additional period of five minutes the readings were begun on a fifteen minute schedule for at least six uniform readings. Oxygen consumption measurements were not begun until the animal had been in the chamber for one half hour in order to (l) accustom the animal to the chamber, to equilibrium temperature, (2) bring the system (3 ) establish an equilibrium between the absorbing rate of the soda lime and the carbon dioxide production rateii 26*0.5° C. The temperature within each unit was 37 Table 5 shows the average oxygen consumption for the several groups of rats of each strain. Oxygen consumption is expressed both as c c . used per 100 grams of body weight of the animal and a3 c c . per 100 g m . e^3. Data on the age and body weights of these rats is given in appendix IV. The consumption of oxygen of the two strains was compared by the use of the method of analysis of variance of the original data. of such computations are shown in appendix IV. The results The caries" susceptible rats used slightly more oxygen per unit of time than did the caries resistants. Because of the variations present, however, this greater amount is not statistically significant. This is the case whether the oxygen consumption is determined on a 100 gm. body weight basis or adjusted to the .73 power of body weight. The individual chambers used in the oxygen consumption determinations were not always used for the same strain of rats. The strains were alternated after each run, thus it was possible to compare chambers in the various runs to determine whether or not these chambers were concealing any biological variation. The comparison was made using the method of analysis of variance (appendix IV). The values F for chambers of 0.75 and F for runs of 1.41 indicate that there were no significant differences in the individual chambers or in the successive runs which would conceal biological variation if any were present in sufficient quantity to be measured with the method employed here. 38 TABLE 5 OXYGEN CONSUMPTION OF UNTREATED RATS OF BOTH STRAINS Strain Run c c - per ( i o o ‘) ’73 c c . per 100 gm. Suscept ible 1 124.82+3.99* Suscept i ble 2 136.94+6 .96 100.7+4.81 Suscept ible 3 146.14+4.81 113.3+5.38 135.96+3. 70 101.2+3.47 Susceptible Av. 89.2+3.24* Resistant 1 128.45+4.20 89. 8+3.27 Resistant 2 135.08+4.90 96.0+4.06 Resistant 3 121.17+3.26 94.0+3.75 128.12+2.74 93.2+1.94 Resistant Av. B.W. * Standard error of the m e a n . Induced hyper- and hypothyroidism. Hyperthyroidism wa3 induced by oral administration of protamone, an iodinated casein product having thyroxine activity. This thyroactive iodinated protein is effective when given orally (Reineke, 1949). The lot of protamone used in this work was assayed by means of the isotope dilution method. These assays indicate that the thyroxine activity was probably about 1.0 to 1.2 percent (Reineke, 19 53). H y p o t h y r o i d i s m was induced by using thiouracil administered as 0.2% of the standard caries producing diet. Protamone was 39 administered in three dosage levels. These were 0.02, 0.05 a n d 0.1 percent of the standard caries producing diet. Con­ trols were maintained on the standard diet alone. All the rats used in the experiment were produced by brother-sister mating of animals of the caries-susceptible strain of Hunt and Hoppert. Up until the time of weaning, the animals were kept with the parental female and the standard caries producing diet was available. At 25 days of age the young were separated from their mother and fed the modified diets as indicated. Each rat was weighed once a week and it was examined for the presence of dental caries, according to the method described above. A 3 soon as at least one carious lesion appeared in any lower molar tooth, the rat was con­ sidered as showing dental caries. (The age of the rat when caries first appeared in any of its lower molars minus a fac­ tor of 35 days is considered as the rat's caries time.) Protamone in the quantities given caused the rats to show a reduced rate of growth and a higher rate of metabolism than the controls. The level of 0.1 percent proved to be above the tolerance dose in these rats and caused death. The rats treated with thiouracil were 30 to 60 percent below normal in weight. There was no great effect on caries time in the rats, although rats on 0.02 percent protamone did show a somewhat increased resistance. of rats, Table 6 shows the dosage levels, number sex, and average caries times for the various groups. 40 These data were treated using the method of analysis of variance with corrections for disproportionate sub-class numbers (Sneciecor, 1946). The completed analysis of variance gave the results shown in appendix IV. The caries times were not significantly altered by the treatments of hypo- and hyperthyroidism. TABLE 6 HYPER- AND HYPOTHYROIDISM AND DENTAL CARIES Females Caries t irae Rats Treatment Males Caries time Rats Control 4 24.0 4 21. 7 0.2% thiouracil 6 20. 8 4 27.3 0. 02 % pro tamone 5 48. 0 5 34.2 0.05% protamone 6 25. 7 4 15. 4 F sexes — 1.25 F treatments - 0.11 F interaction s 0.99 This analysis shows that in the numbers used here the treatments had no significant effect on caries time of the susceptible series rats. It also shows that there was no dif­ ference in reaction of the two sexes to the treatments, nor was there any significant interaction between sexes and treatments. 41 The oxygen consumptions of a few of rats in each group were measured in a closed circuit type of metabolism unitunit use d was modified after the Regnault-Reiset by Reineke (1953). In this unit chamber with a water sealrat, The (1890) method the rat was placed in a closed As oxygen was used by the respiring it was replaced from a graduated cylinder so that the quantity used could be measured. The carbon dioxide produced was removed from the system by a saturated barium hydroxide solution. Gases from the rat chamber were removed passed over the alkali and then returned to the chamber by means of a rocking mechanism. All rats were fasted prior to making the determination of oxygen consumption. The unit used had four chambers so four rats could be run at each trial. Readings of oxygen consump­ tion were made at intervals of fifteen minutes over a period of at least two hours, and all volumes were corrected to standard conditions of temperature and pressure. rats were fairly uniform in body size, Because all of the the computations of oxygen consumption were made on a basis of one hundred grams body weight in each case. in table 7. The results of this check are shown 42 TABLE 7 OXYGEN CONSUMPTION OF PATS WITH INDUCED HYPEP- AND HYPOTHYROIDISM T reatment No. of rats Op per s 100 Control 9 206.4 0.2 thiouracil 9 184.6 0.02 io protamone 3 236.0 0.05$ protamone 3 372. 5 The numbers of rats used in this check were very small. The purpose was to determine whether or not protamone in the diet raised oxygen consumption and whether or not thiouracil lowered oxygen consumption. The data were not examined sta­ tistically but is it obvious that, on the average, thiouracil- fe d rats used less oxygen than normal controls or the protamonef e d rats. The protamone-fed rats used more oxygen than did the normal rats or the thiouracil-fed rats. Within the protamone- f ed rats, the rats receiving the higher percentage of this thyroactive substance required the greater amount of oxygen. At the time the oxygen determinations were made the rats weighed 60-75 grams. For this reason these values are higher than those reported above for more mature animals of the strain since young growing rats have a higher consumption of oxygen than do adults. 43 Protamone fed to breeding, r a t s . of the susceptible series rats Several breeding females (20th generation) were fed the standard diet to which had been added protamone. Protamone was added as 0.025 and 0.013 percent of the diet a n d several females were kept with one male litter mate accord­ ing to standard practice in this laboratory for the production of rats. One litter was born to a female on the 0.025 percent protamone treatment but the young (three) all died three days later. These were the only young known to have been born to any of the eight females in this series of trials. There were no young known to have been born to any of the eight females on the 0.013 percent protamone diet. There was a high mortality of both males and females and these trials were abandoned after sixty days. It was not considered possible to raise young on these dietary levels of protamone. Thiouracil fed to breeding r a t s . In order to determine what effect dietary thiouracil would have on the incidence of dental caries in the molar teeth of growing resistant series rats, several were raised on a standard caries-producing diet to which had been added 0.2 percent thiouracil. It has been demonstrated by Jones et. all. (1946) that it is possible to raise young on a diet containing thiouracil provided the female parents were not on this diet too long prior to the onset of pregnancy. Complete sterility was not found but there was an altered estrus and a high incidence of resorption of embryos present. 44 Barker (1949) found, also that it was possible to raise young from adult female rats under thiouracil treatment, but in this case the females were mated and fertilized while they were on a standard non-goitrogenic diet, and then they were switched to the goitrogenic diet after the onset of pregnancy. Thiouracil was continued in the diet of the female and then in the diet of her litter. It was found possible to raise cretinoid young until they were sixteen months of age, but their metabolism was reduced markedly and they showed lowered growth curves. In the present study three resistant females were placed on a diet of the standard caries-producing ration containing O.S percent thiouracil seven days after mating with a littermate male. cage. The male was left with the females in the breeding The females were observed every four days for signs of pregnancy. Twenty-one days after the thiouracil was added to the diet one female died, at which time she was obviously pregnant. An autopsy showed that there were nine large fetuses present in the uterus. birth to occur. It appeared that it was impossible for Sixteen days later a second female died and an autopsy showed that young were also present in her uterus. The third female and the male were continued on a diet containing 0.2 percent thiouracil for sixty days without the female ever showing signs of pregnancy, and then regular feed was substituted. Approximately sixty days later, produced and raised a normal litter. this female She was returned to the cage with the male and examined periodically for evidence of 45 pregnancy. The next time she showed signs of pregnancy she was placed on the diet containing thiouracil and a litter of five young was born five days later. w eane d by the female These young were raised and in a nearly normal fashion. The five young all showed reduced vigor and lowered growth curves. were observed periodically for dental caries. They All five young died at an age of seventy days and their molar teeth did not show visible signs of carious lesions. It appeared then that making resistant rats hypothyroid with thiouracil did not grossly alter the incidence of dental caries. For this reason no further attempts were made to raise rats with the thiouracil treatment. Secretion Hate Assay Active colloid appears in the thyroid gland of the rat after about 16 days of gestation, but 18 days marks the threshold of thyroid activity (Hall and Kaan, 1943). From this time on the rat thyroid secretes thyroxine which is necessary for its well-being and which affects nearly all tissues and organs of the body. The presence of thyroxine in the body of the rat is essential to growth, reproduction, and normal homeostasis (Jones, Delfs and Foote, 1946, and many others). Biological assay of the thyroid secretion r a t e . The rate of secretion of thyroid substance by the thyroid gland has been the subject of many investigations and many methods have been suggested for this determination. based on the observation of changes The earlier methods were in the thyroid gland which 46 accompanied induced changes in its activity. The mitotic behavior of thyroid cells and the ocular measurement of thyroid follicles or of follicular epithelium were taken as indexes of thyroidal activity. Such indexes are useful for measuring the response of the thyroid gland to stimuli such as temperature shocks and the presence of thyrotropic hormone but they are not satisfactory for the determination of the actual rate of thyroid secretion. They are also time consuming and difficult to sta n da r d i z e . The methods most frequently employed for estimating the rate of thyroid secretion have been based on the replacement of circulating thyroid hormone by crystalline thyroxine. The amount of exogenous thyroidally active substance or of crystalline thyroxine which is required to maintain thyroidectomized animals in a normal state is taken as representing the normal level of thyroidal secretion of that animal. The thyroidal sub­ stance is usually standardized on a basis of crystalline thyrox­ ine. Thyroxine as produced in the laboratory consists of the racemic mixture of optically active dextro- and levo- forms. Biological assays of this racemic mixture and of the 1 -form show that l=*thyroxine uniformly has about twice the potency of the mixture, hence it is concluded that the activity of the mixture may be accounted for by the l~component and Turner, present 1945). (Reineke It has also been shown that the thyroxine in the thyroid is the levo-rotatory form (Harington an d Salter, 1930). Thus, when biological assays of thyroidal 47 material or of secretion rates are determined, these facts must be considered. In the assay based on replacement therapy, the best criteria of normalcy are basal metabolic rate and heart rate. The growth rate of rats has also been used but this is an extremely laborious procedure and subject to errors. Subsequent to the work of Mackenzie and Mackenzie (1943) most assays of thyroidal substances and of thyroid secretion rates have been done following the method of Dempsey and Astwood (1943). Test animals are made effectively athyroid by the use of a potent goitrogenic drug and graded doses of thyroxine or thyroidally active material are given simultaneously for a suitable period. The minimum amount of administered thyroxine which maintains a normal thyroid weight balance in the presence of the pituitary gland is considered the normal secretion rate in thyroxine equivalents. In the presence of the goitrogen, if too little exogenous thyroxine is supplied, the thyroid weight is greater than normal. reduces the gland weight. Increasing the dosage of thyroxine It has been demonstrated that this decrease in thyroid weight with increasing dosage of thyroxine compares closely with the increase in metabolic rate which also occurs. This was shown by Reineke, Mixner and Turner (1945), who concluded on this basis that thyroidal assays or measure­ ments of thyroidal function by the goitrogenic technique were directly comparable with results obtained by the older and more laborious standard metabolic method. 48 Taurog and Chaikoff (1947) studied the rate of turnover 131 of I in the thyroid gland with the method proposed by Zilversmit ejt_ al. content (1943). The turnover rate times the iodine is assumed to be equal to the thyroid output. This would not distinguish between the output of hormonal and nonhormonal iodine, however. Several investigators have used the method of Dempsey and Astwood (1943) to determine the thyroid secretion rates in thyroxine equivalents. Purves (1943) using this technique and rape seed as the goitrogen found that 2.0 to 3.0 micrograms of thyroxine per hundred grams body weight would maintain the thy­ roids of male rats at a normal size. Turner Reineke, Mixner and (1945) found that approximately 4.8 micrograms of d, l~thyroxine were required to return thyroid weights of 140gram rats to normal. micrograms of Dempsey and Astwood (1943) found that 5.2 1 -thyroxine were required to maintain the thyroids at a normal level, but as pointed out elsewhere Turner, 1945), this 1 (Reineke and -thyroxine may have also had d-thyroxine present, which would account for the rather large value obtained. Monroe and Turner (1946) investigated the thyroid secretion rate of rats during several phases of activity. These investi­ gators found that the thyroid secretion rate in growing female r a t 3 ranged from 4.63 micrograms of d, 1-thyroxine per hundred grams body weight for 50 to 99 grams body weight rats to 2.82 micrograms per hundred grams body weight for 2 50 to 300 gram body weight rats. 49 Griesbach and Purves (1943) employed subtotally thyroid- ectomized rats to estimate the normal rate of thyroxine secreted a nd found that a daily supply of 2.25 micrograms d> 1 -thyroxine per hundred grams body weight prevented hypertrophy or atrophy of the remaining thyroid fragment. quantity They than assumed that this (2.25 micrograins per hundred grams body weight) may be taken as equivalent to the normal secretion rate of the hormone in rats. Monroe and Turner (1946) conclude that the normal secretion rate of thyroidal substance by the thyroid gland of the rat appears to be equivalent to two to five micrograms of d, 1- thyroxine per hundred grams body weight per day. Experimental results. Using the method of Dempsey and Astwood (1943), the rate of secretion of the thyroid gland expressed in equivalent amounts of crystalline d, l=thyroxine was determined for rat3 of the strain resistant to dental caries. The rats were kept in an air conditioned room maintained at 34*1° C. and fed the standard caries-producing diet described above. Thiouracil was mixed in the feed (0 . 2 percent by weight), which was fed ad l i b . Rats were separated by sex and divided into five lots for each sex and graded doses of thyroxine were administered daily by intraperitoneal injections of a water suspension of the sodium salt. One group of each sex on the standard diet with thiouracil added received no thyroxine. In addition to these groups, a number of rats of each sex were maintained as a control with neither thiouracil in the diet nor 50 exogenous thyroxine being supplied. The treatment continued for two weeks, after which all animals were sacrificed. Animals were killed with ether anaesthesia, and the body weight of each animal w a 3 taken along with its respective thyroid weight. From these data the average thyroid weight per hundred grams body weight was calculated for each of the lots receiving the respec­ tive doses of exogenous thyroxine. These weights were then compared graphically with the values for the normal controls. The point of intersection of the curves of thiouracil-thyroxinetreated aniraal3 and the control weight line is taken as repre­ senting the estimated secretion rate of the rats expressed in equivalent amounts of crystalline d, 1 -thyroxine per hundred grams of body weight. These data are summarized in table 8 . The estimated secretion rate of male rats of the resistant strain in this study is 1.95 to 2.02 micrograms d, 1-thyroxine per 100 grams body weight. female rats is weight. 2.1 to 2.2 The estimated secretion rate of micrograms per hundred grams body In figures II and III thyroid weights are plotted using the average weight and showing plus and minus one standard error from this mean to indicate the levels of confidence provided by these data. The secretion rates of the resistant series rats indicated by these assays are lower than values obtained by other workers usi ng other rats. They are in agreement with those obtained by Griesbach and Purves (1943), however, who obtained values 51 indicating that the quantity 3.35 micrograms d, l=thyroxine per grams body weight was equivalent to the normal rate of 100 secretion. Sufficient numbers of rats of the caries-susceptible strain wer e not available for a similar determination of their thyroid secretion rate. TABLE 8 PATS' THYROID SECRETION PATE ASSAY DATA Thiouracil in feed, % N o . of rats Thyroxine /100 g. B.W. Avg. Body Weight Thyroid Wt /100 g. B.W. Males 10 0 163 6.08+0.50* 0.2 5 0 196 16.75+2.22 0.2 5 0. 57 176 15.7 0±0.43 0.2 5 1.15 173 13.85+0.56 0.3 5 1.71 175 10.43+0.91 0.2 4 3.09 191 4.52+0.33 13 0 132 5. 35+0. 58 0.2 5 0 150 22.73+1.15 O • CO 0 6 0. 76 139 32.90+0.90 0.2 7 1.44 131 15.30+1.13 0.2 6 3.10 143 5.58+1.51 0.2 7 3 .0 1 133 5.04+0.83 F eraales 0 * Standard error of mean. 52 FIG URE II Method of Plotting Data to Determine Thyroid Secretion Rate of Resistant Series Male Rats WT 16 WE I GH T PER 100 GM.BODY MALE RATS 16.75 THYROID 10.4 3 MILLIGRAMS 6.08 4.52 0 0.5 1.0 1.5 2.0 A D M I N I S T E R E D DOSE. M I C R O G R A M S PER 100 G R A M S BODY WEI G H T E S T . S E C R E T I O N RAT E , I. 9 5 - 2 . 0 2 * y . / l 0 0 G R A M S B OD Y W E I G H T 53 FIGURE III Method of Plotting Data to Determine Thyroid Secretion Rate of Resistant Series Female Rats 2 2.90 RATS E S T. S E C R E T I O N 2.1- 2 . 2 JULG./ GRAMS BODY 100 G M . BODY WEIGHT FEMALE RATE 10 0 W EIGHT MILLIGRAMS THYROID WEIGHT PER I 5.30 5.35 3 2 ADMINISTERED PER 100 GRAMS DOSE. BODY MICROGRAMS WEIGHT. 54 Histological Examination of Thyroid Epithelium When differences were observed in the gross weight of the thyroid glands, it was considered advisable to examine histo~ logical preparations of these glands. The thyroid gland is composed of lobules within which are the follicles. The follicles are the structural units of the thyroid gland. When seen in histological sections, the follicles are nearly always circular, but they may appear very irregular. The wall of the follicle is a layer of epithelial cells which vary from nearly flat to high columnar. There is no basement membrane and the cells rest directly on reticular tissue. There have been two types of epithelial cells described, ’’chief" cells and "colloid" cells, but it is thought that both of these merely represent different phases of secretory activities of the same type of cell. Within the lumen of the follicle, there is a hyaline material which stains deeply with eosin and which is called the colloid of the thyroid. ordinary fixatives, In preparations made by the use of (e.g. Bouins, formalin, etc.) the colloid is separated from the epithelium by peripheral vacuoles, which have been created by a contraction of the colloid in fixation. Colloid in sections prepared by the freezing-drying technique is free from such vacuoles. It is thus assumed that they are artifacts of preparation and that they are not present in the normal living gland (DeRobertis, 1941). 55 When a thyroid, gland is called upon to produce larger amounts of thyroxine than it doe 3 in normal circumstances, it is considered an "activated" gland and the normal morpho­ logical histology is altered. A thyroid gland may be experi­ mentally activated by exposure to cold or by the administra­ tion of thyrotropic hormone of the anterior pituitary gland. In such cases the colloid is diminished and there is an increase in the number of follicular cells by mitosis, as well as an increase in cell height. activation. New follicles are not found during The unity of the follicle seems to be determined by the connective tissue network surrounding it (DeRobertis, 1941). The central follicles contain the most active cells and the least active are the peripheral cells and those in the isthmus connecting the paired lobes. The non-secreting gland is characterized by follicles distended with colloid which has been accumulated and stored. The epithelial cells become markedly flattened. Of the number of common fixatives which are available for histological examination of the thyroid epithelium, B ou i n 's seems to be the most preferred. The staining method used most frequently is the hematoxylin-eosin combination which has the advantage of being both satisfactory and convenient. Other stains are, of course, available for special investigations, but those mentioned have the most favor for examination of the thyroid gland to determine qualitatively the state of activity based on height of the cells. The paraffin method is used almost exclusively as an embedding procedure. 56 There appears to be a good correlation between colloid volume and body size, and the colloid potency depends on the volume present, at least, as determined by a staining reaction (Hall and Kaan, 1942). Turner and Turner (1944) suggest a method of determining colloid volume by photographing sections of the thyroid gland and measuring follicular walls with a planimeter which eliminates the subjective selection of repre­ sentative cells. Readings are obtained at once in terms of area, and these may be transferred to volume with the proper methods of computation involving the theory of sampling and known constants. The method of measuring the height of a number of repre­ sentative acinar cells with an ocular micrometer and determining an average, common use. called the cell height "index” , has come into fairly Of the various techniques which have been intro­ duced for this estimation, that of Rawson and Starr (1938) has been most widely employed. The height of a cell of average size in the wall of two hundred successive distinct acini is determined, the interacinar cells being neglected. These measurements are tabulated and a graph is made of the frequency curve and the mean and standard error of the mean are given. Using this tecynique, Uotila (1940) made one hundred measure­ ments while Gorbman (1949) and others have used the height of fifty cells with satisfactory results. Dvoskin (1347) used the height of twenty-five successive follicular cells and found that the results were consistent within each group observed. 57 Griesbach and Purve 3 (1943) suggested a modification of the above method in which a representative section from the center of the gland is projected onto a screen and thirty acinar cells were measured by means of a standardized celluloid scale prepared for that purpose. and Purves, These workers (Griesbach 1943) demonstrated that thirty measurements are all that are required for statistical accuracy and that addi­ tional measurements would be a waste of effort, for they would contribute no additional accuracy or reliability to the average value obtained for a gland. The heights of cells which have been reported as normal in different strains of rats vary considerably. It is to be expected that there would exist differences among rats subjected to various external conditions, but the extremes of "normalcy" are surprising. The mean acinar cell height of the Sherman strain of rats reported by Cortell and Rawson (1944) was 3.86^.47 microns. Dvoskin (1948) reported that the average cell height in his normal control animals was range of 7.8 to 8.6 8.0 with a microns, while the average height in thio­ uracil- treated animals was 13.5 microns. Mackenzie and Mackenzie (1943) found that the hyperemia and enlargement of the thyroid gland in rats being fed goitrogens was accompanied by histologically observable reduction in colloid and an increase in height of the thyroid epithelium. These workers found that these changes could be prevented by the administration of exogenous thyroxine, and they suggested 58 that the thyroid enlargement was probably mediated through a hormone from the anterior pituitary body. This has been proved to be the thyrotropic hormone by a number of other researches. Uotila (1940) observed that the regulation of t h 3 thyrotropic function of the anterior pituitary depends primarily on humoral pathways with variation in the organism's thyroxine level as the most important factor, but that under certain conditions, such as exposure to cold, stimuli from the hypothalmus are transmitted through the pituitary stalk modifying the basal secretory rhythm of thyrotropin. The morphological activation of the thyroid gland by a goitrogen and by exogenous thyrotropic hormone are identical in appearance. Since the activation of the thyroid by a goitrogen does not take place in the absence of the pituitary gland, it is then assumed that this activation is caused by thyrotropic action. Thyrotropic hormone thus appears to be the most conspicuous endocrine factor in the control of thy­ roxine, and it stimulates the thyroid gland to make its own hormone (Means, 1943). When potent goitrogens are present a n d sufficient exogenous thyroxine is not present for homeo­ stasis, this morphological activation is futile since it cannot overcome the functional inactivation induced. Thus the thyroid gland becomes larger and has the histological appear­ ance of an activated gland. The ability of thyroxine to lower the height of the acinar epithelium has been repeatedly confirmed (Cortell and R&wson, 59 1944, and others). Thus it has been established that the functional level of the individual thyroid gland is indicated by its morpho-histological structure and that the height of the acinar epithelium is a means of estimating this level. This is valid except when an antithyroid substance is present w h ic h prevents the thyroid from manufacturing the thyroidally active substance. When sufficient amounts of iodine are lacking for the gla nd to produce the thyroidally active substance, the effects are the same as when an antithyroid drug is present when hyper­ plasia and hypertrophy of the thyroid gland occurs as a result of thyrotropic stimulation (Astwood ejfc aJL. , 1943). In this case also the morphological activation is futile since it can­ not overcome the functional inactivation in the absence of the required iodine. It has been demonstrated that the acinar cell height is inversely proportional to experimentally altered environmental temperatures. Both the thyroid gland and the adrenal glands are in some manner concerned with the maintenance of a constant body temperature and these may operate in conjunction with the neural thermoregulatory mechanisms of the animal body. Adrenalectomized rats are unable to withstand the stress of low environmental temperatures and death occurs several hours post operatively due to decreased heat production (Bernstein, 1941). The thyroid glands of rats kept in the cold develop hyper­ plasia, thickened acinar epithelium and the colloid loss 60 characteristic of a hyperactive thyroid (Bailif, 1937). This condition may be prevented, or at least lessened, by increasing the level of iodide in the diet (Kenyon, 1933) or by the admin­ istration of exogenous thyroxine Experimental procedures. (Turner, 1946). Histological examination of the thyroid epithelium was made of normal and cold-treated rats of both sexes and strains. The rats were ra,ised from parents which came from the colony of Hunt and Hoppert. These parents were mated and the young were born and cared for in a manner similar to those in the colony and under the same environmental conditions as to diet ana general care. All rats were Killed by ether anaesthesia and their thyroids removed, cleaned of excess connective tissue and immediately placed in Bouin's solution for fixation. The paraffin tech­ nique was used, sections were cut at a thickness of seven microns and stained with Ehrlich’s hematoxylin and eosin. Examinations were made with the oil immersion lens of a standard microscope, and the height of the acinar epithelium was measured with the aid of an ocular micrometer. Twenty- five measurements, made in a systematic manner, were taken for the determination of the average height of the cells of each gland. Sections from the central portion of each gland were examined. The instrumental measurements were used in the statistical analysis without converting them to microns. analysis The method of of variance was employed in analyzing the data. 61 For individual comparisons, rats of approximately equal age and sex were paired and then the resistant strain was compared with the susceptible strain. In addition, there was a comparison made on the basis of measurements of the indi­ vidual cells since there were twenty-five such measurements made to determine a value for an individual rat. Group I consisted of three female rats of each strain. The susceptible rats averagedl 8 8 grams in weight and the resistant rats averagedl 8 3 grams. These six animals were killed in the month of August. Group II comprised seven males of each strain. ceptible series rats had an average weight of 308 The sus­ grams while the average weight of the resistant series rats was 395 grams. These fourteen rats were killed in the month of November. Group III was made up of two females from each strain and they were killed in the month of May. These rats were kept alive in a refrigerator which was maintained at about 40° F. for six days prior to sacrifice. The thyroid weights of these rats, as well as body weights, were taken. The susceptible series body weights averaged 219 grams and the resistants 23? grams. The thyroid weights per hundred grams body weight for the four rats were slightly above the averages previously given for each series. The actual values of milligrams of thyroid weight per hundred grams body weight were as follows: susceptible series: and 7.54. 9.03 and 8.81 and resistant series*. The body weights of all four rats remained sub­ stantially unchanged during the cold treatment. 6.02 62 E x p e r imental r e s ul t s. resistant rats are, The thyroid follicles of the caries- in general, smaller than are those of the caries-susceptible strain. Quantitative determinations were not made but this condition was evident from visual examination of histological sections prepared from the respective tissues. Photomicrographs of representative sections of thyroid tissue are reproduced in plates 1 , sections from Group I females. Figures 2 and 3 1 , . 2 Plate 1 and are sections 3 shows from the thyroid glands of susceptible series rats, and Figures 4, 5 and ferences 6 are from resistant series rats. The dif­ in the size of the follicles are obvious by inspection. Plate 2 , Figures 1=7 inclusive, are photomicrographs of normal group II susceptible series males. Figures 8 and 9 of this plate are from representative sections of cold-treated Group III susceptible series females. These figures may be compared with those of plate 3 where Figures 1, and 9 2 , 3, 5, 6 , 8 are from representative sections of group II resistant series males. Figures 4 and 7 of this plate are from cold- treated group III females. The differences in follicle size among the females of group I were also evident among the males of group II. follicles The of the susceptible series thyroid glands tended to be larger than did those of the resistant series glands. Exami- nation of the thyroids of cold-treated animals of group III, however, strains. shows little apparent difference between the two The colloid area of the follicles of the susceptible 63 serie3 t h y r o i d glands appears to have been reduced as a result of the col d treatment. This suggests that there were different p h y s i o l o g i c a l responses in the two strains. The trequency polygons of Figures IV and V show the distribut ions of the heights of acinar cells in each of the two strains. It is apparent from Figure IV, group I that the acinar epithelium of the resistant series rats was, on the average, slightly higher than was that of the susceptible series animals. While many of the cells were of about the same height, there were more high cells in the thyroid glands of the resistant series and more low cells in the susceptible series glands. This was also true in Group II as shown in Figure V. The mean acinar heights for the three groups of rats of the resistant and susceptible strains are given in table 9. The standard error accompanies each mean. The mean height for each rat. in each group with its standard error is also given. The standard error of the group means were calculated from the individual cell heights. The mean acinar height for the three females of the sus­ ceptible series was 8.11+0.24 microns; the comparable value for the resistant series was 10.22+0.3 microns. 2.11 microns The difference of is not statistically significant when it is com­ pared on the basis of the three r a t s 1 averages. There is evi­ dence that group I contains more than one type of cell, however. When the six females are tested for homogeneity by the method of analysis of variance, the F value is 16.8 showing that there 64 is more than one type present. nifioant This F value is highly sig— (Appendix V). Since season and group I and II rats were killed at a different since they were notsubjected to the stress of cold as were those of group III, they were maintained separately for statistical analysis. The data for group II rats are sub- stantially in agreement with group I rats in the matter of dif­ ferences between the two strains represented. The mean values representing the cell heights of both strains of rats are higher in this group than in group I. This confirms the observation made by Bernstein (1941) that the acinar cell height was greater in winter than in summer. The mean acinar height of the susceptible series thyroids in group II was 11.19^0.25 microns and that for the resistant glands was 12.87^0.25 microns. heights of individual rats gave the results shown in Appendix V. The mean values of acinar cell The analysis shows that the difference of 1.68 microns is significant at the five percent level. As would be expected, a statistical treatment of the 350 individual cell heights in group II shows that there were two types of acinar cells (Appendix V ). The data of the cold-treated rats were analyzed in a similar manner (Appendix V). Since the difference between the two group means was only 0.35 microns (Table 9), which is a value close to the standard error of each mean, nificant. it is not sig­ This shows that among the cold-treated rats no indications of differences were found. 65 TABLE 9 AVERAGE HEIGHT OF ACINAR EPITHELIAL CELLS (MICRONS) Paired rat no. an d group no. I Sex Strain Susceptible Resistant 1 F 8.37+0.50* 10.83*0.40 2 F 7.21+0 . 2 1 11.78+0.18 3 F 8 .74+0.39 8.05+0.32 Average F 8.11+0.24 10.22+0.31 1 M 12.61+0.56 12 2 M 12.99+0.53 12.49+0.47 3 M 9 .96+0. 53 12.77+0.50 4 M 10.65+0.50 11.70+0.50 5 M 10.26+0.47 12 .86+0.42 6 M 12.03+0.24 15.52+0.78 7 M 9.90+0.19 12.63+0.74 Average M 11.19+0.25 12.87+0.25 III Cold treated 1 F 11.63+0.62 11.88+0.44 2 F 10.18+0.45 9.23+0.25 Average F 10.91*0.39 10.56+0.32 II * + Standard error of the mean. «1 0 + 0.48 66 PLATE 1 Photomicrographs of Histological Sections of Thyroid Glands Group I (females) Figures 1, 2, 3 Susceptible Series Figures 4, 5, 6 Resistant Series The scale is shown in figure 6 where the vertical lines are 10 microns a p a r t . 67 PLATE 3 Photomicrographs of Histological Sections of ThyroicL Glands Groups II and III Susceptible Series Figures 1-7 Group II males Figures 8^9 Group III females The scale is shown in figure 9 where the vertical lines are 10 microns a p a r t . 68 PLATE 3 Photomicrographs of Histological Sections of Thyroid Glands Groups II and III, Pesistant Series Figures 1 , 2, Figures 4, 7 3 , 5, 6 , 8 , 9 Group II males Group III females The scale is shown in figure 9 where the vertical lines are 10 microns a p a r t . 69 FIGURE IV Relative Numbers of Acinar Cells of Various Heights Group I (females) - RESISTANT FREQUENCY -SUSCEPTIBLE 6 ~\ 6 8 10 12 14 MICRONS 16 18 20 70 FIGURE V Relative Numbers of Acinar Cells of Various Heights Group II (males) - -RESISTANT — SUSCEPTIBLE FREQUENCY 2 0 71 p9.^clusA?HfL• The thyroid follicles are larger in the sus­ ceptible series rats than are those of the resistant series. This could account, in part at least, for the heavier thyroid gland which is found in the caries-susceptible rats of the Hunt-Hoppert strains. Measurements made of the height of the acinar epithelium of the thyroid glands of these strains show that the resistant series cells are higher than are those of the susceptible series. The higher cells indicate a greater function per cell in the resistant glands. ceptible series glands The larger colloid area of the sus­ indicates that there is more storage of colloid substance in these thyroid glands. The thyroid gland of an animal which is exposed to cold becomes activated. This activation results in a diminution of colloid and an increase in height of the follicular cells (Monroe an d Turner, 1946). This response appeared to have been obtained in the susceptible series females where the normal cell height was 8 . 1 1 aa and the cold-treated thyroid cell height was 10.91 Jjl . The amount of the colloid was also diminished as is shown by comparing plates 1 and 2 and 3. responses, Neither of these however, appear evident in the thyroids of the resistant series rats where there was little change either in colloid or cell height. It would seem then, that the resistant series rat was able tc adjust its metabolism in response to the cold treatment without cytological alteration of the thyroid gland. The thyroid gland of the susceptible series rat, however, became activated in response to the cold treatment. 72 The threshold of response to cold treatment seems to have ✓ been reached in the susceptible series rats but not in those of the resistant series. 73 IV. SUMMARY Physiology of the thyroid. there are significant differences It has been demonstrated that in the weight, in the uptake of radioactive iodine, as well as in the normal histological appearance of the thyroid gland3 of the Hunt-Hoppert strains of rats. It was not possible to demonstrate any significant dif­ ferences in gaseous metabolism. If there were any thyroid secretion rate differences, they would be expected to be shown in metabolism variations* Since there were none, apparently the thyroid secretion rats is about the same in both strains of rats. The thyroid glands of the caries-susceptible rats are approximately 43 percent larger and show about 42 percent 131 slower turnover of I . If the thyroid iodine content of the susceptible rats is increased in proportion to their weight, the net output daily would be equal in the two strains of rats. Unfortunately iodine analysis of the thyroids are not available. It appears, however, that the thyroid mechanism has been com­ pensated in the susceptible strain so that the net output metabolically equivalent to that of the resistant strain. is This compensation resulted in a larger gland with more colloid. The relatively low total uptake of radioiodine by the thyroid glands may be attributed to the fact that the rats were being fed an iodine-rich diet. It has been shown that the thyroid secretion rate of the resistant series rats is somewhat lower than values reported by other investigators using other strains of rats. 74 There appeared to be no difference in the weight of the pituitary glands of rats of the two strains. Genet i c s . There are genes for caries resistance and caries susceptibility. There are also genes for the various character^ istics involving the thyroid which have been studied here. weight of the gland, The thickness of the epithelium and the amounts of colloid were different in the two strains. differences in retention of radioactive iodine. of rats were fairly uniform in these respects. ences were present between the strains. There were also The strains Gross differ- Perhaps the same genes responsible for thyroid characteristics also affect caries, or perhaps the thyroid genes may be linked with the caries genes. Selection has been attempted to sort out and fix the caries genes, but no conscious effort has been made to do this for the genes effecting the thyroid gland. The selection preferences for good fertility, rapid growth and generally good condition were about equal for the two strains of rats. This selection would not tend to sort the genes for caries and those for thyroid physiology into separate lines as has been done. It would thus seem logical to conclude that the inherited differences in thyroid physiology are either closely linked to or share common genes with the inherited factors in tooth decay. Parotid gland. It has been demonstrated that the inherited differences for the production of caries between the cariessusceptible and caries-resistant strains of rats are not due to 75 the secretions of the parotid gland. Caries were neither inhibited nor were they markedly accelerated in the absence of secretions of the parotid gland. 76 APPENDIX I. Caries times for parotidectomized rats and their intact sibs . II. III. IV. 131 Tables of I recovery. Average weights of the thyroid glands among the gr oup3. Tables of age and analysis of variance of respiratory metabolism. V. VI. Tables of analysis of variance of acinar cells. Vitamineral supplement analysis. 77 APPENDIX I CARIES TIME FOR PAROTIDECTOMIZED RATS AND THEIR INTACT SIBS Susceptible Series Cross Rats 532 360 Colony Exp. Colony Exp. 37 43 27 37 27 37 46 47 47 90 none 43 28 28 38 28 43 Caries time Cross Rats 358 Colony Exp. 75 47 47 47 91 35 35 63 49 49 Colony 39 39 39 39 39 39 39 Rats Colony Caries time 31 43 57 31 31 31 57 31 77 63 77 49 49 49 26 36 36 36 26 26 36 Exp. 67 67 39 Colony 43 43 29 43 43 43 367 Exp. 54 40 60 55 55 55 67 55 Colony 38 38 50 22 36 52 36 36 29 29 29 39 43 59 Exp. 35 91 373 371 . 369 Cross 41 37 41 27 83 27 365 S&2 Caries time Colony 361 Exp. Colony Exp. Colony Exp. 43 43 44 44 72 58 24 24 24 24 24 24 24 33 33 19 46 33 33 46 46 39 39 53 39 39 39 39 71 140 99 71 29 29 29 29 Exp 41 44 40 54 368 Colony Exp. 38 53 41 41 56 41 56 78 CARIES TIME FOP PAROTIDECTOMIZED RATS AND THEIP INTACT SIRS (cont.) Resistant Series Cross 501 Rats Colony Car ies t ime 203 420 119 466 613 480 480 480 201 Cross 346 301 170 325 436 386 321 196 506 Exp. 119 163 105 105 163 189 119 163 163 510 Rats Colony Caries time 516 377 88 421 290 290 305 305 465 501 674 32 5 289 355 547 543 381 338 471 353 472 443 505 344 268 719 574 558 473 Rats Colony Car ies time 703 214 437 584 626 774 569 783 704 486 783 734 691 232 110 544 477 647 709 385 665 681 518 546 616 Exp. 393 703 326 483 552 Colony 223 397 291 249 291 220 700 279 159 685 355 438 Exp. 243 119 154 304 Colony 677 795 592 419 755 207 549 710 Exp. 601 631 858 858 641 Colony 477 619 682 69 5 563 774 774 619 517 134 813 619 Colony 431 234 2 79 320 270 417 Exp. 221 283 221 415 542 Colony 718 388 264 566 482 614 482 496 446 614 538 477 752 646 660 477 Exp. 511 384 436 529 535 560 451 Colony 487 377 563 390 430 318 239 304 621 199 318 507 Exp. 120 120 523 Exp. 876 700 477 508 Colony Exp. 853 281 808 712 476 566 824 887 606 387 238 668 749 491 304 435 421 543 534 533 Exp. 509 521 520 525 Cross Colony 508 Exp. 134 232 548 490 233 Colony 420 897 685 789 441 441 480 698 612 689 554 701 52 7 743 126 Exp. 516 821 669 526 516 79 APPENDIX II COUNTING RECORDS OF I131 RECOVERY Trial I 18 hr. no. and series 68 hr. no. and series (5)* susc. (9) res. (6) susc. (9) res. 34.53 45. 37 24.87 Counts per sec. per thyroid 38.97* * 5,18 7.67 3.00 3.67 Counts per sec. per 100 gm. B.W. 27.91 27.96 34.65 17.74 4.61 5.63 3.10 3.13 Counts per sec. per mg. thyroid 3.84 3.48 4.21 3.04 1.56 0.47 0.26 0.37 Percent of administered dose present at sacrifice 4.65 4.25 5.41 2.97 0.62 0.28 0.36 0.44 Trial II 68 hr. sacrifice, sex series and, no. of rats (12) susc.F (20)susc.M (15) res.F (7) res.M Counts per sec. per thyroid 55.88 46.44 33.60 33.50 4.68 2 .79 1.83 2.97 Counts per sec. per 100 gm. B.W. 41.92 46.44 18.04 8.30 1.87 0.64 1.16 1.12 Counts per sec. per mg. thyroid 4.07 2.52 3.28 2.45 0.29 0.28 0.17 0.72 Percent of administered dose present 6.34 5.27 3. 82 3.80 0.53 0.31 0.23 0.37 60 APPENDIX II (cont.) Trial II (cont.) 148 hr. sacrifice, sex series and no. of rats (13) susc.F (24)s usc .M Counts per sec. per thyroid 43 .96 40.07 24.20 25.15 3.06 1.62 1.72 1 .18 Counts per sec. per 100 gm. B.W. 30. 51 16.28 13.24 8.87 2.00 0.94 0. 86 0.43 3. 47 2.01 2.22 1.82 0.20 0.13 0.17 0.13 4.99 4.50 2.75 2. 85 0. 35 0.18 0.19 0.12 Counts per sec. per mg. thyroid Percent of administered dose present (16)res .F (7) r e s .M Trial III 68 hr,sex series, and no. 148 hr,sex series, no. (l2)susc.F (8) r e s . M (l3)susc. F (9) re3.M Counts per sec. per thyroid 51.95 33.41 41.96 23.37 5.80 2.65 1.74 1.13 Counts per sec. per 100 gm. B.W. 32.70 10.74 26.25 8.03 4.20 1.02 1.68 0.38 3.83 1,95 3.01 1.54 0.34 0.18 0.18 0.06 5. 39 3.46 4.35 2 .53 0.61 0.33 0.61 0.11 Counts per sec. per mg. thyroid Percent of administered dose present * Number of rats ** Each value shown with its standard error. 81 APPENDIX III Average Weight of the Thyroid Gland among the Groups Susceptible Series (males, M; females, F) Group Sex 1 F 12 8.84^.32* 2 F 13 8.81 ♦ .36 3 F 13 8.15^.49 4 F 13 8 . 79+.38 Sum F 49 8. 58+ .19 1 M 20 7.06*.25 3 M 24 8.15^.25 44 7.65*.20 Sum No. of rats Thyroid W t . per 100 gm. B.W. t sexes “ 1.13 Resistant Series Thyroid W t . per 100 gm. B.W. Sex 1 F 15 5.54^.20 2 F 17 6.04*.18 Sum F 32 5.625.33 1 M 7 5.005.43 2 M 7 4.935.25 3 M 8 5.545.30 4 M 9 5.225*31 31 5.195.16 Sum ♦ct- No. of rats Group t sexes “ O.50 * Standard error of the m e a n . 82 APPENDIX IV Mean Age and Body Weights of Rats Used for Respiratory Metabolism Determinations St rain Suscept ible Res istant Run Age, days B.W. gm. 1 347+0* 348+9* 2 292+8 308+13 3 133+1 258+18 1 330+3 374+7 2 334+7 358+15 3 105+0 255+9 * Standard error of the mean. f 83 BW \ • .73 23L\ Analysis of Variance, cc. Og per 1 ^ 100 J Source S.S. D.F. 1 553.43 Individual rats 34 6.514.88 Total 35 7,068.31 Strains . M.S. 553.43 191.614 F. “ 2.89, t * 1.70 (not significant) Analysis of Variance, cc. Op. Consumption per 100 g. B.W. Strains 1 576.8 567.8 143.1 Individuals ]$4, 4.865.5 Total 35 5,442.83 F # s' 3,949, t * 1.98 (not signiileant; Analysis of Variance of the Runs and the Chambers f BW ^ .73 values) vua a ^ 151.20 1,663.20 11 Chambers 282.36 564.73 2 Runs 200.17 4 .840.38 22 Discrepance Total --------------------- --- T?r‘ — 7,068.31 35 — r\ n a ( o+■. a icrrti f leant ) Runs, F. = 1.416' (not significant) - 84 Analysis of Variance of Induced Hypo— and Hyperthyroidism Source Degrees of Freedom Sexes 1 Sum of Squares Mean Square 2 50.0 250.0 Treatments 3 63.10 21.03 Interaction 3 592.68 197.56 * Individuals 30 * From an analysis of the original data F. sexes - 1.25 F. treatments s .11 F. interaction “ .99 194.44 85 APPENDIX V Analysis of Variance of Acinar Cells of Group I (Measurements) Source Total Degrees of freedom Mean square 149 126,417.3 1 20,230.4 20,230.4 148 106,186.9 717.5 Groups Measurements Sum of squares F = 28.2 (highly significant) t = 5.3 (highly significant) Analysis of Variance of Acinar Cells of Group I (Mean Values, Paired) Source Degrees of freedom Sum of squares Mean square F Pairs 2 2.15 1.08 .26 Strains 1 8.09 8.09 1.92 Discrepance 2 8.43 4.22 Total 5 18.67 F pairs - 0.26 (not significant) F strains " 1.92 (not significant) (The discrepance term was used as the error term.) 86 Analysis of Variance of Acinar Cells of Group I (Mean Values, not Paired) Source Sum of squares Mean square 5 46,650.3 9,330.1 Measurements 144 79.767.0 553.9 Total 149 136,417.3 Degrees of freedom Means F s 16.844 (highly significant) The value F = 16.8 indicates that there i3 significant variation present and that the cells do not constitute a single population. 87 Analysis of Variance of Acinar Cells of Group II (Mean Values, P a i r e d ) Source Degrees of freedom Sum of squares Pairs 6 136,067.4 33,677.9 Strains 1 117,394.5 117,394.5 Discrepance J£ 99.281.5 16,546.9 Total 13 352,743.4 F strains ‘= 7.09 Mean square (significant) Analysis of Variance of Acinar Cells of Group II (Measurements) Mean square Degrees of f reedom Sum of squares Hats 6 34,394.5 5,732.4 Strains 1 29,679.3 39,679.3 Interaction 6 24,691.1 4,115.18 Measurements 336 331.491.3 986.58 Total 349 420,356.2 Source (a) On the basis of measurements as the error terms F rats - 5 . 8 1 F .strains “ 30.08 (highly significant) F interaction “ 4.17 (b) On the basis of interaction as the error term: F rats “ 1.39 F strains = 7.21 . (significant at .05 level) 88 Analysis of Variance of Acinar Cells of Cold-Treated Rats (Mean Values Paired) Source Degrees of freedom Sum of squares Mean square Pairs 1 509.15 509.15 Strains 1 15.21 15.21 _1 71.08 71.08 3 565.02 Discrepance Total F strains s 0.21 (not signif icant) Analysis of Variance of Acinar Cells of Cold^Treated Rats (Measurements ) Mean square Degrees of f reedom Sum of squares Rats 1 12,633.8 12,633.8 Strain 1 376.4 376.4 Interaction 1 1,102.3 1,102.2 Measurements 96 61.696.6 Total 99 75,809.0 Source 643.67 (a) On the basis of measurements as the error term: F rats - 19.66 F strains “ .586 F interaction “ 1.71 (b) On the basis of interaction as the error term: F rats = 1 1 . 4 6 . F strains = .34 The F values are not significant. 89 APPENDIX VI Vitamineral Supplement Analysis not more than not less than 30.0000% Phosphorous not less than 5 .0000% Manganese not less than 0.1400% Iodine not less than 0.0435% Iron not less than 0.1700% Calc ium Salt, sodium chloride 3 8 .0000 % none 90 LITERATURE CITED Astwood, E. B. , J. Sullivan, A. Bissell, and R. Tyslowitz. Action of* certain sulfonamides and of thiourea on the thyroid gland of the rat. Endocrinology, 32: 310-225, 19 43. Ba i l l i f , R. N. Cytological changes in the rat thyroid follow­ ing exposure to heat and cold and their relationship to the physiology of secretion. American J. of Anat. 61: 1-19, 1937. Barker, S. B. Effects of thyrotrophin on metabolism of thiouracil-treated rats. Endocrinology. 37: 230-336, 1945. Barker, S. B. The influence of thiouracil on reproduction and growth in the rat. J. of Endocrinology. 6: 137-144, 1949. Belasco, I. J. , and J. R. Murlin. The effect of thyroxin and thy­ rotropic hormone on the basal metabolism and thyroid tissue respiration of rats at various ages. Endocrinology. 28: 145-153, 1941. Benedict, F. G . , and G. MacLeod. The heat production of the albino rat. II. Influence of environmental temperature, age and sex; comparison with the basal metabolism of man. Jour. Nutrition. l: 367-398, 1929. Bernstein, J. G. The effect of thermal environment on the morphology of the thyroid and adrenal cortical glands in the albino rat. Endocrinology. 28: 985-997, 1941. Bibby, B. G., J. F. Volker, and M. Van Kesteren. Acid pro­ duction and tooth decalcification by oral bacteria. Jour. Dent. Res. 21: 61, 1942. Boyd, J. D., V. D. Cheyne, and K. E. Wessels, Is the salivary lactobacillus count a valid index of activity of dental caries? Proc. S o c . Exp. Biol, and Med. 71: 535-537, 1949. Braunschneider, G. E. , H. R. Hunt, and C. A. Hoppert. The influence of age in the development of dental caries in the rat (R. norvegicus). Jour. Dental Res. 27: 154-160, 1948. Brody, S. Bioenergetics and Growth. Reinhold, New York, 1945. 91 Brody, S. , and R. C. Procter. Relation between basal metabo— ■ lism a n d mature body weight in different species of mammals and birds. Univ. Missouri Agr. EX p. Sta. Res. Bull. 166: 89-101, 1933. Bunting, R. W. Assoc. 22: Diet and dental caries. 114-122, 1935. Jour. Amer. Dental Bunting, R. W., G. Nickerson, and D. G. Hard. Further studies Bacillus acidophilus in the relation to dental caries. Jour. Amer. Dental Assoc. 14: 416-417, 1927. Chapman, E. M . , and R. D. Evans. The treatment of hyperthyroid­ ism with radioactive iodine. Jour. Amer. Med. Assoc. 131: 86-91, 1946. Cheyne, V. D. A description of the salivary glands of the rat and a procedure for their extirpation. Jour. Dental Res. 18: 457-468, 1939. Cheyne, V. D. Effects of selective salivary gland extirpation upon experimental dental caries in the rat. Proc. Soc. Exp. Biol, and Med. 42: 587-590, 1939. Clise, R. L . , and H. R. Hunt. Growth rate and pilosity in caries-resistant and caries-susceptible albino rats (Rattus norvegicus ?. Jour. Dental Res. (in press) 1953. Cori, C. F. and G. T. Cori. Changes in liver glycogen. Annual Review of Biochemistry, Vol. III. 151-174. Stanford Univ. P r e s s . 1934. Cortell, R., and R. W. Rawson. The effect of thyroxin on the response of the thyroid gland to thyrotropic hormone. Endocrinology. 35: 488-498, 1944. Csernyei, J. Dental caries. A biochemical process. State Dental Jour. 16: 241-245, 1950. Davis, J. E. and A. B. Hastings. consumption of immature rats. 683-687, 1934. New York The measurement of the oxygen Amer. Jour. Physiol. 109: Dempsey, E. W. and E. B. Astwood. Determination of the rate of thyroid hormone secretion at various environmental tempera­ tures. Endocrinology. 33: 509-518, 1943. Dentay, J. T. and J. J. Rae.Phosphatase in saliva. Dental Res. 38: 68— 71, 1949. Jour. DeRobertis, E. The intracellular colloid of the normal and activated thyroid gland of the rat studied by the freezingdrying method. Am. J. A n a t . 68: '317-337, 1941. 93 Dvoskin, S. The thyroid— like action of elemental iodine in the rat and chick. Endocrinology. 40: 334-351, 1947. Dvoskin, S. The effects of pituitary and non— pituitary gland factors on the formation of intracellular colloid droplets in the thyroid epithelium of hypophysectomized rats. Endocrinology. 43: 52-70, 1948. Ginn, J. rats. T., and Volker, J. F. Rusting in desalivated albino Endocrinology. 31: 382-283, 1941. Gorbman, A. Effects of radiotoxic dosages of 1 ^ 1 U p Cn thyroid and contiguous tissues in mice. S o c . Exptl. Biol, and Med. Proc. 66: 313-213, 1947. Gorbman, A. Reactions of thyroid glands to juxtathyroidal implants of thyrotropic agents. Endocrinology. 46: 397402, 1949. Gorbman, A. Functional and structural changes consequent to high dosages of radioactive iodine. Jour. Clin. Endo­ crinology. 10: 1177-1191, 1950. Greenberg, S. M. The protective effect of dietary fat on immature rats fed thyroid. Jour. Nutrition. 47: 31-39, 1952. Greene, E. G. Anatomy of the Rat. - 27 ns. 1-370, 1935. Trans. Amer. Philos. Soc. Greisbach, W. E. and H. D. Purves. The assay of thyrotropic activity by the cell height response in guinea pigs. British Jour. Exp. Path. 24: 185-192, 1943. Haldane, J. B. S. The amount of heterozygosis to be expected in an approximately pure line. Jour. Genetics. 32: 375391, 1936. Hall, A. R . and H. W. Kaan. Anatomical and physiological studies on the thyroid gland of the albino rat. Anatomical Record. 84: 231-339, 1942. Ha l p e r t , B. , Cavanaugh, J. W. , and B. F. Keltz. Structural changes in the thyroid glands of patients treated with thiouracil. Arch. Path. 41: 155-165, 1946. Harington, C. R. and W. T. Salter. The isolation of L. thyrox­ ine from the thyroid gland by the action of proteolytic enzymes. Biochem. Jour. 24: 456-471, 1930. 93 H a r t l e s , R. L. and N. D. MacDonald. The metabolism of the oral flora. I. The oxygen uptake and acid production by mixed human saliva in the presence and absence of glucose. Biochem. Jour. 47: 60-64, 1950. Hawkins, H. F. Dental decay and what it is and means for its control. Jour, of Amer. Dental Assoc. 16: 781=795, 1929. Higashijo, T. Histologische studien ueber die einfluss der speichel — druesen - extirpation auf die wirkune: des geschlechtshormones. Jour. Amer. Dental Assoc.' 28: 862, 1941.(Abst. from Soc. Path. Jap. Tr. 30: 252, 1940). Hill, T. J. A salivary factory which influences the growth of L. acidophilus and is an expression of susceptibility or resistance to dental caries. Jour. Amer. Dental Assoc. 26: 239=244, 193?. Hill, T. J., personal communication, 1953. Hoppert, C. A., P. A. Webber, and T. L. Caniff. The produc­ tion of dental caries in rats fed on an adequate diet. Jour. Dental Res. 12: 161-173, 1932. Horst, K . , L. B. Mendel and F. G. Benedict. The influence of previous diet, growth and age upon the metabolism of the rat. Jour. Nutrition. 8: 139“ 163, 1934. Howe, P. R. Further studies of the effect of diet upon the teeth and bones. Jour. Amer. Dental Assoc. 10: 201-212, 1933. Hugill, R. A. and H. K. Box. Experimental artificial produc­ tion of dental caries in vitro. Jour. Dental Res. 29: 669, 1950. Bukusima, M. Ueber den einflus3 der innern sekretion der speicheldruese auf der zahns. Jour. Amer. Dental Assoc. 38: 863-844, 1941 (Abst. from Soc. Path. Jap. Tr. 30: 345, 1940). Hunt, H. R., and C. A. Hoppert. Inheritance of susceptibility and resistance to caries in albino rats Mus norvegicua. Jour. Am. Col. Dentists. 11: 33-37, 1944. Hunt, H. R., C. A. Hoppert, and W. G. Erwin. Inheritance of susceptibility to caries in albino rats (Mus noryegicus,). Jour. Dental Res. 33: 385=401, 1944. Jarabak, J. R. Caries in the molars of the rat following resection of facial nerve. Jour. Dental Res. 29. 692, 1950. 94 J a y A P ’ ,,^ ctQbacilIus acidophilus and dental caries. Pub. Health. 28: 759=761, '1938. Amer. J. Jay, P., H. R. Hunt and C. A. Hoppert. The incidence of oral h* acidophilus in Hunt=Hoppert resistant and susceptible rats. Jour. Dental Res. 33: 205, 1944. Jones, G.E.S., E. Delfs, and E. C. Foote. The effect of thio= uracil hypothyroidism on reproduction in the rat. Endocrinology. 38: 337=344, 1946. Jones, H. B. Molecular exchange and blood perfusion through tissue regions. Advances in Biological and Medical Physics, Vol. II. 53=75. Academic Press, New York, 1951. Keating, F. R. Jr., R. W. Rawson, W. Peacock and R. D. Evans. The collection and loss of radioactive iodine compared with the anatomic changes induced in the thyroid of the chick by the injection of thyrotropic hormone. Endocrinology. 36: 137=148, 1945. Kenyon, A. T. The histological changes in the thyroid gland of the white rat exposed to cold. Am. J. Path. 9: 347= 369, 1933. Kifer, P. E. personal communication, 1953. Kite, 0. W . , J. H. Shaw, and R. F. Sogannes. An influence on dental caries incidence produced in rats by tube feeding. Jour. Nutrition. 43: 89=3 05, 1950. Lee, M. 0. Basal metabolism during estrus cycle. Physiol. 86: 694-705, 1928. Amer. Jour. Lilienthal, B, Studies of the flora of the mouth. IV. Some observations on acid production by lactobacilli and Candida a l b i c a n s : a preliminary report. Australian Jour. Exptl. Biol, and Med. Sci. 38: 387=290, 1950. McGinty, D. A. Iodine absorption and utilization under the influence of certain goitrogens. Annals of the New York Acad. Sci. 50: 403=418, 1949. Mackenzie, C. G. , and J. B. Mackenzie. Effect of sulfonamides and thioureas on the thyroid gland and basal metabolism. Endocrinology. 33: 185=209, 1943. MacLagan, N. F. , and M. M . Sheahan. The measurement of oxygen consumption in small animals by a closed circuit method. The Journal of Endocrinology. 6: 456=462, 1950. 95 anhold, J. H. and V. W. Manhold. A preliminary report on the study of the relationship of psychomatics to oral con­ ditions ^relationship of personality to dental caries. Science. 110: 585. 1949. Manly, M. L, , and W. F„ Bale. The metabolism of inorganic phosphorous of rat bones and teeth as indicated by the radioactive isotope. Jour. Biol. Chem. 139: 125-134, 1939. Means, H. J. Some new approaches to the physiology of the thyroid. Annals Int. Med. 19: 567-586, 1943. Meites, J., and L. F. Wolterink. Uptake of radioactive iodine by the thyroids of underfed rats. Science. Ill: 175-176, 1950. Meyer, A. E . , and G. V. Ransom. The metabolism of rats after thyroidectomy or during thiouracil treatment and the effect of thyroid feeding. Endocrinology. 36: 259-365, 1945. Meyer, A. E., and A. Wertz. The calorigenic efficiency of thyroid material in relation to thyroxine and to iodine content. ,Endocrinology. 24: 683-692, 1939. Miller, W. D. Microorganisms of the human mo uth . Dental Mfg. £T6~.T^^hiladelphia. S. S. White Monroe, R. A., and C. W. Turner. Thyroid secretion rate of albino rats during growth, pregnancy and lactation. Mo. Agr. Sta. Res. Bui. 403. 1946. Morton, M. E. , I. Pearlraan, E. Anderson, and I. L. Chaikoff. Radioactive iodine as an indicator of the metabolism of iodine. Endocrinology. 30: 495-510, 1942. Nakfoor, E. C., H. R. Hunt, and C. A. Hoppert. Fracturing of the molar teeth in caries— susceptible and caries-resistant albino rats (Rattus norvegicus). Jour. Dental Res. 31: 143-150, 1952. Neuwirth, I., and J. A. Klosterman. Demonstration of rapid production of lactic acid in oral cavity. Proc. Soc. Exptl. Biol, and Med. 45: 464-467, 1940. Nizel, Abraham E . , and R. S. Harris. The caries-producing effect of similar foods grown in different soil areas. New England Jour. Med. 244: 361-363, 1951. beblond, C. P., I. D. Puppel, E. Riley, M. Radike and G. M. Curtis. Radioiodine and iodine fractionation studies of human goitrous thyroids. J. Biol. Chem. 162: 275-285, 1946. 96 Leblond, C. P., and J. Gross. Thyroglobulin formation in the thyroid follicle visualized by the coated autograph tech­ nique. Endocrinology. 43: 306-324, 1946. Perlman, I., I. L. Chaikoff, and M. E. Morton. Radioactive iodine as an indicator of the metabolism of iodine. J. Biol. Chem. 139: 433-447, 1941. Purves, H. D. Studies on experimental goitre. IV. The effect of diiodotyrosine and thyroxine on the goitrogenic action of Brassica seeds. Brit. J. Exp. Path. 24: 171=173, 1943. Rawaon, R. W., and P. Starr. thyroid epithelium. Arch. Direct measurement of height of Int. Med. 61: 726-738, 1938. Regnault, V. et Reiset J. Recherches chimiques sur la respira­ tion des animaux des diverses classes. Ann. Chim. et de Physique. 26: 229, 1849. (Quoted by Brody, p. 319, 1945). Reineke, E. P. The formation of thyroxine in iodinated pro­ teins. Annals N. Y. Acad. Sci. 50: 450-465, 1949. Reineke, E. P. oral communication, 1953. Reineke, E. P., and C. W. Turner. The relative thyroidal potency of 1= and d, l=thyroxine. Endocrinology. 36: 200=206, 1945. Reineke, E. P., J. P. Mixner, and C. W. Turner. Effect of graded doses of thyroxine on metabolism and thyroid weight of rats treated with thiouracil. Endocrinology. 36: 64= 67, 1945. Reisfield, D. R . , and J. H. Leathern. The closed vessel technic for testing thyroid activity in mice. Endocrinology. 46: 122-124, 1950. Rosebury, T., and M. Karshan. Susceptibility to dental caries in the rat, V. Influence of calcium, phosphorous, and vitamin D, and corn oil. Archives of Path. 20: 697=707, 1935. Shaw, J. H. Ineffectiveness of certain essential nutrients in prevention of tooth decay in cotton rat molars. Proc. Soc. Exptl. Biol, and Med. 70: 479=483, 1949. Shaw, J. H. Effects of dietary composition on tooth decay in the albino rat. Jour, of Nutrition. 41: 13-24, 1950. Sherwood, T. C. The relation of season, sex and weight to the basal metabolism of the albino rat. Jour. Nutrition. 12: 223-236, 1936. 97 Shiere, F. R. , C. E. Georgi, and R. L. Ireland. A study of StreptQCOCCU3 3 a 1 ivarius and its relationship to the dental caries process" " T o u r . Cental Res. 30116-125, 1951. Snedecor, G. w. Press, Ames. Statistical M e t h o d s . 1946. Iowa State College Sogannes, R. F. Caries conducive effect of a purified diet when fed to rodents during tooth development. Jour. Amer. Dental Agsoc. 37: 676-692, 1948. Soliman, F. A. Cyclic variation in thyroid functions of mature female rats and mice. Thesis for the degree of Ph.D. Michigan State College, East Lansing, Michigan, 1952. Taurog, A., and I. L. Chaikoff. The metabolic interrelations of thyroxine and diiodotyroxine in the thyroid gland as shown by a study of their specific activity time relation­ ships in rata injected with radioactive iodine. J. Biol. Chem. 169: 49, 1947. Turner, M. L. The effect of thyroxin and dinitrophenol on the thermal responses to cold. Endocrinology. 3 8 : 233269, 1946. Turner, R. S., and M. L. Turner. The oxygen consumption and histology of the thyroid gland in v i t r o . Endocri nol ogy . 38: 32-40, 1944. Uotila, V. V. The regulation of thyrotropic function by thyroxin after pituitary stalk section. Endocrinology. 26: 129-135, 1940. Van Huysen, G. Microscopical study of caries of rats' teeth. Jour. Dental Res. 29: 809-814, 1950. Vanderlaan, W. P., and M. A. Greer. Some effects of the hypophysis on iodine metabolism by the thyroid gland of the rat. Endocrinology. 47: 36-47, 1950. W e i 3 b e r g e r , D., C. T, Nelson and P. E. Boyle. The development of caries in the teeth of albino rats following extirpa­ tion of the salivary gland. Am. J. Orthodontics Oral Sure. 2 6 : 8 8 , 1940. Winchester, C. F . , Q*,L. Comar, and G. K. Davis. destruction by I13i and replacement therapy. 302-304, 1949. Thyroid Science 110: Zilversmit, D. B . , C. Entenman, and M. C. Fishier. On the cal­ culation of "turnover time" and "turnover rate" from experi­ ments involving the use of labeling agents. J. Gen. Phvsiol 2 6 : 325-331, 1943. y