EFFECT OF THYMECTOMY IN

RATS AND CALVES

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By

EUGENE PAPP, D.V.M., M.Sc.

The Ohio State University I960

Approved by

Advise] Department of Veterinary Anatomy ACKNOWLEDGMENTS

The author wishes to thank his adviser, Dr. W. G.

Venzke, for his great aid and indispensable supervision, and Dean W. R. Krill of the College of Veterinary Medicine for his continuing interest and encouragement in this work.

He is grateful to Dr. E. H. Bohl for his participation in the work with tissue cxilture. Thanks are due Drs. A. A.

Gabel, R. L. Rudy, and W. J. Roenigk for their technical assistance in the field of surgery and to Dr. R. 0. Moore for his aid in the biochemical aspects of the problem.

Gratitude is extended to Dr. M. Y . Andres for his technical assistance, to Mr. Bud Kramer for many of the excellent illustrations which appear in this dissertation, and to Mr. D. 0. Oliver for his aid in the preparation of the manuscript. Gratitude is extended to contributors to the Development Fund of Ohio State University. Finally, the author thanks his wife, Mrs. Helen Papp, both for her technical help and her forbearance.

ii TABLE OP CONTENTS

Chapter Page Introduction ...... 1

Review of Literatiire...... h. Developmental A;natomy...... 1+ Gross Anatomy...... 5 Histology ...... 6 Cytology ...... 11 Reticulum C e l l s ...... 11 Thymic Lymphocytes...... 13 Myoid C e l l s ...... 16 Hassall's Bodies ...... 17 Plasma Cells, Granulocytes, and Mast C e l l s ...... 19 Biochemistry of the Thymus ...... 20 Hi stones...... 20 Protamines...... 20 Lipoproteins ...... 21 E n z y m e s ...... 21 Peptide ...... 22 Cholesterol...... 23 Vitamins...... 2lp Involution of the T h y m u s ...... 25 Hypertrophy and Hyperplasia ...... 31 Thymectomy...... 32 Irradiation...... 31+ Augmentation and Substitution .... 36

Experiment 1 ...... 1+1 Part 1 ...... !).l Part 2 ...... 1+6

Experiment 2 ...... 51+

Experiment 3 ...... 69 Part 1 ...... 69 Part 2 ...... 73

Experiment 1+...... 87

iii Chapter Page

Experiment 5>...... 93 Part 1 ...... 93 Part 2 ...... 100 Part 3 ...... 10l|. Part ij...... Ill Part 5 > ...... 116 Part 6 ...... ,...... 13£

Experiment 6 ...... lJ+7

Experiment 7 ...... 15>6 Experiment 8 ...... 161

D i scus sion ...... 170

S u m m a r y ...... 183

Bibliography...... 187

Autobiography ...... 201

iv LIST OP TABLES

Table Page

1 Hemogram in Intact Rats as a Function of A g e ...... £8

2 Weekly Concentrations of Certain Blood Constituents in Intact R a t s ...... $9 3 Hemogram in Intact Calves as a Function of Age '...... 60

I). Weekly Concentrations of Certain Blood Constituents in Intact Calves ...... 61

5 Weekly Concentrations of Certain Ions in the Blood of Intact C a l v e s ...... 62

6 Effect of Thymectomy on Numbers of Leuco­ cytes Per Cubic Millimeter of Blood Before and After Splenectomy ...... 96

7 Effect of Thymectomy on Numbers of Lympho- cytes Per -Cubic Millimeter Before and After Splenectomy ...... 97

8 Effect of Thymectomy on Numbers of Segmented Neutrophiles Before and After Splenectomy...... 98

9 Effect of Thymectomy on the Hematocrit Value in Rats Before and After Splenectomy...... 99

10 Effect of Splenectomy on Numbers of Leuco­ cytes Per Cubic Millimeter of Blood in Thymectomized and Control Rats ...... 101

11 Effect of Splenectomy on Numbers of Lympho­ cytes Per Cubic Millimeter of Blood in Thymectomized and Control Rats ...... 102

v Table rage 12 Effect of Splenectomy on Numbers of Segmented Neutrophiles per Cubic Millimeter of Blood in Thymectomized and Control R a t s ...... 103

13 Effect of Hypophysectomy With or ’Without Thymectomy on Numbers of Leucocytes Per Cubic Millimeter of Blood on R a t s ...... 106

lk Effect of Hypophysectomy With or Without Thymectomy on Numbers of Lymphocytes Per Cubic Millimeter of Blood in Rats ...... 107

15 Effect of Hypophysectomy With or Without Thymectomy on Numbers of Segmented Neutrophiles Per Cubic Millimeter of Blood in R a t s ...... 108

16 Effect of Hypophysectomy ‘With or Without Thymectomy on Hematocrit of R a t s ...... 109

17 Effect of Hypophysectomy With or Without Thymectomy on Body Weight of Ra t s ...... 110

18 Organ Weights in Thymectomized and Intact Calves on January 8, i9 6 0 ...... lklj.

vi LIST OP ILLUSTRATIONS

Figure Page

1 Transverse serial sections of thymus of three day old rat H and E. x L|7£...... 50

2 Transverse serial sections of .thymus of three day old rat H and E. x l\.j5 .... 5>2

3 Parabolic regression of second degree based on blood samples drawn three times weekly from rats of group A . . . . 63

k Parabolic regression of second degree based on blood samples drawn two times weekly from rats of group B . . . . 6£

5 Parabolic regression 'of second degree based on blood samples drawn once weekly from rats of group C ...... 67

6 Thymectomy of the rat. Splitting of the sternum before the removal of the thymus gland ...... 77

7 Split sternum up to the third sternal cartilage. Exposed thymus gland in the r a t ...... 79

8 Removal of the thymus gland in the rat with the help of blended scissors and forceps ...... 81

9 Exposed mediastinal region in the rats after thymus gland was re m o v e d ...... 83

10 Cross suture with cat-gut closing the split sternum and the mediastinum ....

11 Testicular and body weights of thy­ mectomized and intact rats as a function of a g e ...... 112

vii Figure Page

12 Spleen and body weights of thymectoralzed and intact rats as a function of age . . . 112 13 Adrenals and body weights of thymecto­ mized and intact rats as a function of a g e ...... lli(.

llj. Cervical lymph node of a thymectomized littermate rat 22 days following operation H and E. x 1 0 0 ...... 118'

15 Cervical lymph node of an intact litter- mate of the same age as above. H and E. x 1 0 0 ...... 118

16 Bone marrow of an thymectomized rat 22 days following operation. H and E. x 1x7 i? ...... 1 2 0

17 Bone marroxtf of the above intact litter- mate of the same a g e ...... 120

18 Adrenal cortex of a thymectomized rat 22 days following operation. H and E. x h i S ...... 123 19 Adrenal cortex of an intact littermate of the same age as above. H and E. x hlS ...... 123

20 Testis of a thymectomized rat SO days following operation. H and E. x [(.75 . • • 126

21 Testis of an intact littermate rat of the same age as above. H and E. x h7S • • 126

22 Kidney of a thymectomized rat of SS days following operation. H and E. x hlS • • • 128

23 Kidney of an intact littermate ra.t of the same age as above. H and E. x hlS .... 128

2l| Spleen of a thymectomized rat 28 day^s following operation. H and E. x hlS . • . 130

25 Spleen of an intact littermate rat of the same age as above. H and E. x hlS .... 130

viii Figure Page

26 Liver of a thymectomized rat 28 days following operation. H and E. x 1+75 . . • 132

27 Liver of our intact littermate rat of the same age as above. Ii and E. x i+75? • • 132

28 Testis of the thvmectomized calf Sq_ born M a y 2 k , 1959 li-j.l

29 Testis of the intact calf S>2 Lorn June 17, 1959...... • li*l

30 Comparison of weights between calves 115 and 111+; Hi and H2; Si and S2; ?i and ?2 .’ 11+5

31 Thymus of the rat exposed to., whole-body irradiation of 150 r and removed on fifth day following tumor implantation H and E. x 1350 ...... 165

32 Thymus of an other rat exposed to whole- body irradiation of 150 r and removed on fifth day following tumor implantation. H and E. x 1350 ...... 165

ix INTRODUCTION

Jacob Berengar of Carpi (died l£50, quoted after Singer)

(li^.6 ) was the first to give a clear account of the thymus.

Since that time the thymus has attracted increasing interest among many investigators, and the majority of them have left the problems without’ determining its function. A solution to the problem of function of the thymus must be considered far from being solved.

Some authors have ascribed an endocrine function to the thymus (e.g. Bomskov, (II4.), Comsa (33), Schmincke (llf.2)

Lowenthal (101 )3 , while others do not regard this organ as a hormone-producing gland (e.g. Bargmann (9), Hammar (63)J.

Metcalf (110), Bomskov (168), Metcalf and Buffett (ill), and

Comsa (32) have suggested that the thymus exerts a remote influence upon various organs, including stimulation of lymphocytopoiesis.

In the literature there are many reports of cyst forma­ tions in the thymus of various species of animals. Authors frequently regard these structures as actively secreting units. Others view these structures as degenerative states of the epithelium. 1 Brolin (25) et, al. regard the thymus as a reacting

organ, or as some workers call it, a target organ for vari­

ous steroids. Van Allen (165) states that the effect of

surgical removal of the thymus in animals is still somewhat uncertain because of the difficulty in distinguishing between

effects of thymoprivia and complications arising during or

after the operation.

Comsa (3 2 ) and. Bomskov (17) found that there is a r e ­

lationship between the thymus and the growth hormone of the

adenohypophysis. It is known that Injections of this hor­ mone result in an enlargement of the thymus.

The hypothesis of an association between the function

of the thymus and the pathogenesis of leukemia, at least in mice, has attracted ever-increasing interest since the dis­

covery by Furth (5l) that thymectomy in young AK mice reduced

the incidence of spontaneous leukemia from a very high to a low level.

Comsa (33) says that thymus preparations injected into

the animals were able to prevent or mitigate all the conse­

quences of thymectomy. Molnar (115) et. al. found that thymic

extract has the tendency to Increase the malignancy of the

Brown-Pearce-Carcinoma of rabbits.

All works reported were performed on guinea pigs, mice,

rabbits, and/or very few on rats, and none on domesticated

animals. All conclusions were made from results obtained by histological or hematological methods and statistical evalua- was not conducted. The present work is intended to increase the knowledge of the effects of thymectomy in two little-

studied species: rats and cattle. Where possible, data will be compiled so as to permit statistical analysis. In

addition to the study of the effects of thymectomy in other­ wise untreated rats, the influence of thymectomy on rats

irradiated or implanted with transmissible carcinoma will

also be investigated. If significant changes are shown to follow thymectomy,

a further question arises about the neture of the substance

or substances whose removal by thymectomy produced the

changes. REVIEW OP LITERATURE

Developmental Anatomy

Despite the wealth of information in the literature on pharyngeal derivatives in animals, few references pertain to these structures in the calf. Bargmann (9) states that as a rule the body of the thymus in vertebrates develops from the entodermal epithelium, principally from branchial pouches-,

III and IV as well as others. In the case of the calf

Ellenberger and Baum (J4I1.) also include branchial pouches, II.

Hagstrom (59), Anderson (Ij.), and Fedolfi ( I 4 . 7 ) estab­ lished that the thymus of the calf developed only from branchial pouches,III, and that the vesicula cervicalis had no connection with the thoracic complex. Anderson ( k) divides the thymus III into head, Intermediary cord, mid- cervical, cervical thoracic, and thoracic segments. Kingsbury (91) reports that pouches IV In the calf do not make contact x^Ith the surface ectoderm to establish a branchial membrane as it does in man, and as pouches III apparently always do. There seems to be no justification for considering the epithelium of pouches IV of the pharyngeal complex as a thymic primordium. When a thymus IV appears, it is developed on the stalk and rather intimately associated with the parathyroid glands. Compared with the definitive thymus, or thymus III, the differentiation of a thymus in the fourth pouch complex is not only inconstant but relatively late.

In the rat it has been shown by Godwin (£li) that fourth pouches IV do not form at all, but large u.ltimobranchial bodies alone shift caudad and fuse with the thyroid just as in the pig. Here, also as in the pig no thymus is ever found with the thyroid, thus eliminating a thymic transforma­ tion of the ultimobranchial body.

Gross Anatomy

Hammar (63) divides the mammals into three groups on the basis of the position of the thymus: (l) those having cervical and thoracic thymic tissue, (2) those having only thoracic thymic tissue, and (3) those in x^hich the thymus is present only in the cervical region. Of the animals included in this study, the calf (Bos taurus) falls into the first group, and the white rat (Hus rattus) into the second.

Sisson and Grossman (llj-7) state that at the period of its greatest development the thymus in the calf occupies the greater part of the anterior mediastinal space, extending caudad to the pericardium, pulmonary artery and aortic arch. It Is pale and distinctly lobulated. Its median (left) face covered by the mediastinal pleura, is in contact with the thoracic wall and the apical lobe of the left lung as far back as the third rib. The right and left cervical lobes form the bulk of the organ, extending along the trachea and the esophagus from the thoracic inlet to the thyroid gland.

The two lobes are large at the base of. the neck, where they are in apposition and cover the trachea, esophagus, carotid arteries, and vagosympathetic trunks. Cephalically they gradually diminish in size and diverge to the side of the trachea. They are related superficially to the sterno- cephalicus and the stemo thyro-hyoideus muscles and the ex­ ternal jugular veins. The thymus involutes slowly with time but remnants of the thoracic lobe often remain even at advanced ages.

Hammar (63 ) describes the thymus in the rat as a two- lobed organ having two ventral, two medial, and two dorsal surfaces. It is the only organ in the anterior mediastinal region and is separated from the heart by the pericardium.

Histology

Hammar (70) states that In the mammalian thymus each lobe is surrounded by a thin capsule of connective tissue.

This extends into the substance of each lobe to form septa and to divide the lobes into lobules, which are usually from one to two millimeters in width. The septa, on penetrating into the substance of each lobe, meet and fuse with other septa, but not extensively enough to siirround the thymic tissue of each lobe completely; hence, the thymic tissue of each lobule is connected with that of other lobules.

Trautmann and Fiebiger (163) state that the thymus in domestic animals is composed of pyramidal or polygonal lobules which average five to 13 millimeters in size. The superficial portion of the lobule is often notched and sub­ divided into smaller compartments, which are separated by connective tissue. These subdividions have a diameter of about one millimeter and consist mostly of cortical sub­ stance. More centrally, they blend with the undivided medulla. The medullary substance of the lobules differs from the cortical substance in that the reticular cells have more cytoplasm, the thymocytes are fex-jer in number and show no mitotic figure or Hassall's bodies.

Trautmann and Fiebiger (163) describe the vasculature and innervation of the thymus in domestic animals with these words: "The blood bessels of the thymus are ensheathed in lymphatic tissue, which takes an active part in the produc­ tion of lymphocytes. The arteries lie in the medulla near the cortex and supply capillary nets, which become especially dense toward the surface of the cortex. These are drained by veins which course around the lobules on their surface. The lymphatics encircle the medulla and are continued as net-like lymph channels, which lie in the periphery of the lobules and are drained by interlobular vessels. The nerve fibers pre­ sent are mainly vasomotor. Only a few have free endings in the medulla."

Renaud (133), Monroy (117), and Tondo (l6l) found an accessory arterial supply to the thymus from the surfaces of the lobules in the rat and cat. His (76) and Jolly (83) describe vessel arches on the border between the cortex and medulla, whereas a capillary network spread up to the surface of the lobule in the thymus of the calf and cat. The medulla is practically without capillaries. Sobotta (15>3) stated that the medulla has essentially only venous capillaries,

Salkind (13&) and Cortivo (37) found in the rat that the capillary network is equally distributed in the medulla and in the cortex of the thymus. Hammar (69), Monroy (I17).and Smith (lIj-9) observed that obliteration of arteries, veins, and capillaries occurs in involuted thymic tissue. Hart (72) and Herxheimer (7^) ob­ served fat deposition in the endothelial cells of larger vessels and also in the adventitia and endothelium of thymus capillaries and called this "fatty degeneration." Matsunaga

(IOI4.) in the calf and Hoepke and Spanier (79) in the rat have shown that the veins are accompanied by interlobular lymph vessels and that these lymph vessels are connected to the 9 lymph vessel network on the surface of the lobules very

similar to that found in lymph nodes. Hoepke and Spanier

also found lymph vessels in the thymic medulla of the rat. Smith (lip9) reported that lymphatic channels filled xvith

deeply stained lymphocytes are seen as well-formed tubes accompanying the. arteries and veins with anastomoses appear­ ing in sections as partially or wholly encircling sheaths.

In the regions of the forking of the smaller arteries and veins, the lymph vessels may enlarge slightly and In the angles of the larger ones the dilations are frequently large and complex. Congested lymphatics have been seen only associated with the main arteries and their branches in the medulla or in the cortical-medullary zone.

When the lymphatics reach the hilus, they either accomp-

any the blood vessels for some distance, become smaller and

disappear in the adventitia of the arteries and veins, or

they fade out almost immediately into the septal adipose or loose connective tissue. Even outside the thymus the lympha­

tics increase and decrease in diameter. In sections impregnated xvith silver, those vessels packed with lymphocytes are outlined by fine reticular fibers which are continuous with the reticular fibers surrounding

the arteries and veins. In these preparations the framework

of the endothelium-lined lymphatic vessels appears as if 10 differentiated from and continuous with the reticular sheaths

of the blood vessels.

In the thymus from animals injected with trypan blue and in those injected and irradiated; spaces lined with endothel­ ium and emptied of cells are seen in the perivascular network.

These spaces are interpreted as sections of lymphatic vessels.

In these thymus tissues the extra-thymic lymphatic vessels are usually much smaller in diameter than the intrathymic vessels, and are difficult to follow any distance from the thymus. The presence of intrathymic lynphatic vessels was made known by earlier workers (Hammar, 5k.)*

Braeuker (23), Riegele (l3lp)} and Terni (l£8) found branches of the vagus and sympathetic nerves following blood vessels entering the thymus. Pines (128) found these branches connected to the medial plexus of arteries and veins.

Bargmann (9) believed the thymus parenchyma completely innervated with nerve fibrils but it was not possible to estimate the extent of the innervation or nerve architecture because of difficulties of insufficient silver impregnation.

Hammar (66, 61) stated the function of the vagus innerva­ tion was to regulate the output of Vitamin B. Sunder-

Plassmann (l£6) believed that the parasympathetic innervation served to connect the thymus with the thyroid gland in a complex mechanism. Hallion and Morel (60) and Hammar (6£, 11

6ii) believe the sympathetic nerve supnly has to do with vasoconstriction in the thymus.

Cytology

Heticulum Cells

Since it has been thoroughly established that the thymus begins as an epithelial structure, the concept is prevalent that it is basically an epithelial gland. The histological character of the thymus transformation has been rather well established particularly through the investigations of

Hammar (?0), Maximow (106), and Kingsbury (92).' The epi­ thelial stage differentiation is of brief duration, and with the loss of a free surface, it is succeeded by what might be termed an epithelioid stage. This in turn is followed by a period of thymic transformation through lymphocytic infiltra­ tion. With the conversion of the epithelioid mass into a cytoreticulum whose meshes are occupied by cells of the lymphocytic series, the essential features of the thymus structure are established. It is evident from these state­ ments that Kingsbury (92) holds the opinion that the epithelium which constituted the foundation of the thymus still retains, in the differentiated state, potentialty for growth and differentiation so characteristic of the pharyn­ geal epithelium from which it was derived, even though it is profoundly altered. 12

Harland (71) confirmed the fact that vacnolation was the

beginning of reticulum formation in the rat. Godwin (5 5 )

believed thst even before the lymphocytes appear, the epi­

thelium itself becomes somewhat reticulated in appearance.

Maximow and Bloom (107) describe the reticular cells as

elongated in form, with pale, round or oval nuclei. In most

cases the nuclear membrane is smooth. The nucleus contains

a few small nucleoli. In the cortex it is difficult to

follow the outlines of the cytoplasm, since the surrounding

lymphocytes are so closely packed. However, in the medulla,

where the lymphocytes are less numerous, it can be seen that

the reticular cells form a network whose meshes are filled

with lymphocytes. In the embryo the epithelial nature of many of the reticular cells is quite obvious; but as the

organ becomes more and more heavily infiltrated with lympho­

cytes, these epithelial cells become flattened and it is

difficult to distinguish them from the nuclei of connective

tissue reticular cells.

They further state that most of the reticular cells are

of entodermal origin, although there are a few reticular

cells of mesenchymal origin around the blood vessels. Con­

siderable controversy based upon functional observations

CSchridde (II4.3), Tschassownikow (16I[.), Deanesly, (I4.0), and

Terni (l58)J Has centered around the origin of these cells, but Bargmann (9) states that it is impossible to make any decision about the genesis of cells on the basis of their function alone.

Thymic Lymphocytes

Yoffey and Hanks (172) state that although lymphocyte production has been studied for many years, it still presents a number of unsolved problems. One of the first, perhaps, is to define what a lymphocyte is. Having done this, it would be desirable to know (l) where lymphocytes are produced and (2) in what numbers. Whether the lymphocytes are medium­ sized or small, a very distinctive feature is the high ratio of nuclear material to cytoplasmic material, so that the cytoplasm may form only a small tuft at one pole of the cell.

This is especially marked in the thymus and the bone marrow, and in fact this type of cell is now designated the "polar" lymphocyte. The presence of a nucleolus in lymphocytes has been dis­ puted by many. The absence of a nucleolus has been thought by Yoffey and Hanks (172) to fit in -with the concept that the small lymphocyte is a mature cell, whereas the presence of a nucleolus has been regarded as evidence of immaturity. Nucleoli tend to be obscured in air dried smears, although they can often be seen surrounded by an irregular coating of

DNA, In supravital preparations the nucleoli stain readily with brilliant cresol-blue and frequently appear to be H l perfectly spherical. Pulvertaft and Jayne (132) as well as

Ackerman and Bellios (l) have observed nucleoli in living lymphocytes b;/ means of phase contrast microscopy. Nucleoli have also been seen in lymphocytes examined with the electron microscope (Bernard £t al, (10); Pease (127).

Gyllesten (57) and Gyllesten and Ringerts (58) declare that it is clear that germinal centers are not essential for lymphocyte formation and that this formation can occur actively in the absence of these centers. Even in the adult there may be a considerable amount of lymphoid tissue, devoid of germinal centers, in x^hich lymphocyte formation neverthe­ less is proceeding actively.

Since the germinal centers are so readily damaged by noxious substances, Heilman (73) describes these as reaction

"centers.” However, they undergo cyclic changes even in absolutely healthy animals, as first described by Maximow

(105) and Conway (38). At the most active phase of the cycle there is great mitotic activity in the germinal centers, so that it is difficult to regard them as other than centers of lymphocytopoiesis.

Sainte-Marie and Leblond (135) believed that mitoses of reticular cells x^ould yield an equal number of large lympho­ cytes would then begin a series of successive divisions so that a total of eight generations of smaller and smaller lymphocytes would be produced and thus each initial large 15 lymphocyte would yield 128 "mature" small lymphocytes.

Hammar (23?), Maximow (105), Salkind (136), Kingsbury

(91), Tobari (160), Godwin (55), and Venzke (166) stated

that the migration of lymphocytes into the epithelio-thymic primordium occurs in animals and lymphocyte production

commences.

Stohr (155) found through observations in the human, mouse, bovine, sheep, cat, and pig that thymic lymphocytes

developed from epithelial cells in the thymic anlage and that they are able to transform into epithelioid cells again.

This is called the pseudotransformation theory. Similar ob­

servation were made by Schval, Marcus, Schridde, Basch, and

Dustin,

Most histologist agree that there are no significant differences between lymphocytes in the thymus and those in

the lymph nodes (123, I6I4-, 130, 89, 90, 171, 122, and 121). Uncertainty, however, has arisen as to whether thymocytes

are indeed lymphocytes, since the thymus sometimes reacts

differently than lymphatic tissues. A direct comparison between the autoradiographs after transfusion of thymic and lymph node lymphocytes shows quantitative differences. The autoradiographs of the hepatic lymph node in both .series are blackened to about the same degree during photographic reproduction. It then 16 appears that the spleen shows a stronger blackening through­ out after an injection of thymic cells than after an injec­ tion of lymph node cells. Transfusion of lymph node cells, on the other hand, causes blackening of the intestinal lymph node, which is not blackened when thymic cells are injected. The only conclusion one can draw from this com­ parison is that thymus lymphocytes and lymph node lymphocytes are treated in different ways by the recipient, a fact that has been shown by Fichtelius (1|8).

Myoid Cells

Mayer (108), Hammar (70), and Teichmann (157) found fibrils with cross striations very similar to the skeletal muscle fibers in the retioular cell cytoplasm. On this basis Hammar (70) named these cells "myoid cells." Dustin

(J4.3) found them in goats and 3 months old cats. These cells were seen by Hammar (70) in dogs and cattle and by G-amburzew

(52) in pig embryos, Terni and Teichmann usually found these cells around the vessels, separately or in groups. Hammar is certain that these cells have nothing to do with the mesen­ chymal cells or vessels in the thymus anlage and that striations by themselves cannot serve as a basis for the conclusion that these cells are of mesenchymal origin. 17 Hassall1s Bodies

Maximow and Bloom (107), describing the thymus in the

human, state that the medulla contains the bodies of the

Has sail, which are characteristic of the thymus. They are rounded, acidophilic structures which vary from 30 "to over

100 microns in diameter. They are composed of concentrically

arranged cells, many of which show evidences of degeneration and hyalinization. Reticular cells are encountered at one or more places within the periphery of each Hassail's body.

The cells of the central part of a Hassall's body may degen­

erate completely, so that small cysts may develop in the

center. In other cases calcium may be deposited in them.

The medulla begins atrophy involution at puberty. This process continues throughout life. The last elements to be replaced are the Hassall's bodies, but even in very old persons there are scattered Hassall's bodies surrounded by

a few reticular cells and lymphocytes.

Jordan and Horsley (8I4.) state that the attachment of

concentric corpuscles to the wall of blood vessels follows from the fact that some arise as areas of luminal occlusion at the point where an arteriole or precapillary branches into

smaller vessels. They interpret the corpuscle proper as representing the locus of occlusion of the blood vessel lumen following hypertrophy of the lining endothelial cells. It is 18 their opinion that a satisfactory explanation of Hassall's corpuscles has been delayed largely through neglect of their earliest stages, and especially through failure to arrange a genetic series between the simpler one-cell types and the larger, more complex follictilar varieties.

Kingsbury (92) believed the Hassall's corpuscle was an expression of (1) growth in a confined space (a result of absence of a free surface) and (2) a disjunctive growth differentiation due to the extreme reticulation that the epithelium has to undergo. Had the epithelium not lost its surface relations and had it not undergone reticulation with the a.ssociated lymphocytic invasion from the mesenchyme, growth, differentiation, and desquamation would have pro­ ceeded typically. In the case of larger, typical thymic lobules, it is possible to trace the continuity of "duct" epithelium and that lining the centra.1 thymic canals within such lobules. The cavity is occupied by desquamated and disintegrating epithelial cells. The continuity of this epithelium with the thymic corpuscles is quite apparent in such regions.

Trautman and Fiebiger (163) theorize that the Hassailfe corpuscles are transitory structures which originate by hypertrophy of isolated medullary reticular cells, followed by the apposition of more reticular cells. During the 19 growth of the corpuscle the center undergoes degenerative changes, i.e., karyolysis, hyaline degeneration, fatty changes, calcification, and invasion of leucocytes.

Plasma Cells, G-ranulocytes, and Mast Cells

Weill (169) and Hoepke and Peter (78) observed typical plasma cells in the cortical portion of the human and other mammalian thymi. Maximow and Bloom (107) state that the lymphocytes of the thymus are morphologically identical with the small lymphocytes in the lymph node and other lymphatic tissue and that the transformation of the small thymocytes into plasma cells and eosinophic myelocytes is generally admitted. In addition to lymphocytes and reticular cells, eosinophile myelocytes and plasma cells occur not infrequ­ ently In the medulla.

Maximow (106) observed neutropliiles In the thymus of rat embryos. On the basis of his remarks, all three types of granulocytes are present, and even mast cells from sur­ rounding connective tissue may migrate into thymic tissue.

Weill (169) observed younger mast cell forms in the cortical portion of the thymus, but because it was not possible to find mitotic division in basophilic, granulated cells, he believed that these cells originated from non-granulated cells. 20 Biochemistry of the Thymus

Histones

Calf thymus histone has been partially resolved by

column chromatography on Amberlite IRC-^O into four princi­ pal fractions, each of which is still heterogeneous.

Resolution was achieved by elution with quanidimium chloride in a suitable concentration gradient at a pH of 6.8.

Fraction I, although heterogeneous, contains valine or

valyllysine or both in the N-terminal position and lysine

in the G-terminal position. Fraction II contains alanine or

proline or both as the N-terminal residues and glycine as

the C-terminal residue (15>).

In other experiments, rat thymus was frozen in liquid

nitrogen immediately after extirpation and powdered in the

frozen state. Chromatography of the acid-soluble extract

of this tissue gave results very similar to those reported

in the calf (I3l)»

Protamines

Mirsky and Pollister (112) found that the two main

characteristics of the thymic protamines are low molecular weight and basicity. They are also characterized by a

peculiar amino acid composition, for they are exceedingly

rich in arginine and Litterly lacking in aromatic amino

acids, such as tyrosine and tryptophan. The small size of 21 the protamines is made evident by the fact that they will diffuse through a cellophane membrane. The characteristic basicity of these proteins is the result of a predominance of one or more of the basic amino acids arginine, histidine, and lysine.

Lipoproteins

Zbarskij and Debov (17^), and Wang, Mayer, and Thomas (I67) have described the preparation from isolated nuclei and thymus chromosomes of protein fractions which are soluble in dilute alkaline solutions and which precipitate at about pH 6.0 when these proteins are acidified. It Is probable that such procedures represent rather drastic fractionation of the residual chromosome. A recent study of proteins pre­ pared in this way shows that they contain approximately 10 per cent of firmly bound lipid, which gives positive tests for phospholipid and cholesterol. Such lipoprotein fractions have been studied electrophoretically. Those obtained from calf and rat liver nuclei seem electrophoretically homo­ geneous, while that obtained from thymus nuclei has various components, as described by Wang, et..al. (I67K

Enzymes

Smith, Wharton and Gerhardt (l5l) made histo-chemical studies of the thymus in comparison to the spleen and lymph node in normal and irradiated mice. In the irradiated 22 animals the following changes were observed: (a) During the degenerative phase, a reduction in activity of succinic de­ hydrogenase and esterase occurred. An increase in activity in the periphery of the splenic white pulp with a strong reaction in the endothelium of the large medullary veins in the thymus for non-specific alkaline phosphatase and adeno- sine-5>-phosphatase was observed. An increased number of cells reactive for adenosine triphosphatase in the thymic cortex and in the splenic red pulp was recorded. (b) During the regenerative phase (3 through 9 days), marked increase in activity of succinic dehydrogenase with a peak at 5> days and a gradual return to normal activity of all enzymes by about 9 days after treatment was noted.

Peptide

Many investigators have succeeded in extracting anti­ microbial substances from various normal tissues during the past several deca.des. Some of these factors have been shown to be basic proteins or peptides. Bloom et_ al. (12) obtained .an anthracidal substance from calf thynus and other tissues.

This material was found to be stable to acid and heat, of polypeptide nature, and alkaline because of the presence of a large amount of lysine. Weissman and Graf (170) suggested that this antibacterial peptide was derived from a histone. 23 Dubos and Hirsch (1^.2) described a mycobactericidal thymus peptide in a series of papers.

Skarnes and Watson (li).8) found that the in vitro anti­

bacterial potency of the thymus peptide was quite high.

The growth of a staphylococcus x^as inhibited for 8 hours by 5 mg of thymus peptide per ml of broth. The peptide was

active against staphylococci, group A streptococci and an­ thrax bacilli, but not type II pneumococci. While

Escherichia coli was Inhibited, Salmonella typhosa and

Vibrio comma were not affected markedly by its antibacterial

action. It was quite potent against a strain of Micrococcus pyogenes, var. aureus (5-10 mg per ml.), in nutrient broth, but in more complex media its activity was decreased some­ what.

Cholesterol

Means and Andrews (109) beginning with the well-known

facts that hypothyroidism is associated with hypercholestero­

lemia In children and that partial thyroidectomy is followed by an increase in blood cholesterol level in man, decided to compare the blood cholesterol values of normal and dvmrf

cattle. They found that plasma cholesterol levels of normal, nonpregnant heifers and of normal steers showed relatively little individual variation, the extreme range being 96.7 to

107*8 mg per 100 ml. of serum. There vras no significant 2l± difference between heifers and steers. If thyroid activity is reduced during the summer, it would be expected that plasma cholesterol levels ivould rise. These authors found instead that the cholesterol values were somewhat higher in the December to February interval than In June, July, and

August.

Albritton (2 ) reports a mean plasma cholesterol value of 110 mg per 100 ml. of serum in beef cattle. Terri, Keener and Morrow (159) found in dairy calves from 9 to 2J+ weeks of age the range of plasma cholesterol levels was 71 to 186 mg/

100 ml. of serum and that the mean value was 108 mg/100 ml.

Brody ( ) found Jersey and Holstein cows kept in a controlled climatic chamber show a marked reduction in total blood cholesterol as ambient temperature is increased from JO to

100° F.

Vitamins

Schaffer, Ziegler and Rowntree (IJ4.0) found in a large number of experiments that the rat thymus contains vitamins

B and G. G-limstedt (53) made a similar observation in guinea pigs. Sanchez-Rodriguez (137) states that at the time of thymus involution due to age, the control of the production of ascorbic acid is taken over by the gonads.

Bargmann (9 ) indicates the proof that the thymus possesses large quantities of vitamin B lends credence to the experiments which have shown the thymus promotes grox-fth.

On the basis that the involuted thymus has a lesser biolog­

ical effect upon growth, Bargmann believed that the thymic

lymphocytes are the carriers of vitamin B.

Euler (45) was able to cure avitaminosis B of rats by

feeding fresh calf thymic tissue. Hirota (75) was also able

to eliminate avitaminosis B in rats by feeding thymic sub­

stance. Glimstedt (53) and Laquer (96 ) declare on the basis of such observations that the thymus is a vitamin carrier

organ and not a hormone producer.

Involution of the Thymus

In 1659 Charleton (27) described the phenomenon of thymic involution, a process which proceeds gradually and

continuously throughout life under normal conditions. Hammar

(70) found that in relation to body weight, the thymus is largest during embryonic life and in childhood up to the period of puberty. After this It begins to involute and on this basis he calls this normally occurring phenomenon

"puberty involution.” He found that separation of the thymus into a cortex and medulla occurs normally in the later em­ bryonic period or post-natally. Normally, involution begins as a gradual thinning out of the lymphoid cells of the cortex, the reticular cells become compressed, and are gradually re­ placed by adipose tissue. The medulla beings to atrophy at puberty. 26

Plagge (129) found thymic involution starts after puberty in rats, but the weight starts to decrease after 60 days of age. The author states that the male rat reaches puberty at $0 days and the female at days of age.

Hammar (63) and Plagge (129) found a correlation exists between gonad development and thymus involution. These workers found that if the animals were castrated after puberty, there were no changes in the thymus. On the other hand, if animals were castrated before puberty, hypertrophy and hyperplasia of the thymus developed and some reduction of the cortex ensued. These observations are in agreement with those of Knipping and Reider (93) and others. Buhler

(26) claims that early castration of rats causes enlargement of the thymus as a result of deposition of lipids and not hypertrophy or hyperplasia.

Plagge (129) found in rats that there is only a one­ sided action between interstitial cells of the gonads and the thymus, because after thymectomy there were no changes in the interstitial cells. Bomskov (16), on the other hand, found that in thymectomized guinea pigs the interstitial cells of the testis hypertrophied, and on this basis con­ cluded that there exists an antagonistic action between the thymus and the sex hormones,

Chiodi (28) expresses the opinion that the lymphocyte- depressing substance is very possibly identical with the 27 sex hormones, because if injected into castrated or non­

castrated animals, the latter cause thymic involution.

Fiore and Franchetti (ij.9) and Hammar (68) found that the in­ jection of blood from sexually mature rats and rabbits

caused thymic involution in young rats and rabbits before puberty. These authors believe that this involution is the result of the sex hormones.

Krupski (95) observed in calves that changes in feeding from milk to grass, poisoning, and x-rays caused thymic in­ volution and very peculiar histological changes in this very sensitive organ. Infectious diseases, neoplasms, experi­ mental avitaminosis, and pregnancy, as has been found by the work of Leblond and Segal (98) and Chodkowski (29) will also cause thymic involution.

Selye (lL|-5>) found that in addition to various infectious diseases, drugs could cause a sudden so-called ''accidental" involution, and that in a few preliminary experiments atro­ pine, morphine, and formaldehyde were particularly effective in producing rapid thymic involution. The author observed

that drugs and operative procedures which produce thymic involution almost invariably lead to hypertrophy of the

adrenals and that the size of these two glands is in inverse

proportion. It appears that in the absence of the adrenals none of the drugs and injuries which would lead to thymic 28 involution in normal animals will have any effect on the thymus. A secretion of the adrenal gland must therefore be considered essential for the ability of the thymus to undergo sudden involution.

Selye (ll^5>), on the basis of known fact that adrenal activity is largely dependent upon pituitary function, per­ formed hypophysectomy and shoi'red that the removal of the hypophysis in itself has little effect on the size of the thymus, but a certain degree of thymus involution can occur under the influence of external stimuli immediately after hypophysectomy. It seems that the secretion by the adrenal of the hormones causing thymus involution is inhibited, but not completely abolished, in the absence of the hypophysis.

Santisteban and Dougherty (138), working with adrenal- ectomized mice, reported that a ketone or an hydroxyl group at the steroid site Cpp was essential for thymolytic acti­ vity. An hydroxyl group at C17 increased the activity, and maximum thymus atrophy was achieved when both Cpp and C17 had hydroxyl groups. The relative potency of the adrenal corticoids was reported to be, in descending order: hydro­ cortisone, cortisone, corticosterone, and 11-dehydrocorti- costerone, according to Stephenson (l^li). Stephenson (l^JLp) is of the opinion that the molecular configuration which is responsible for the atrophy of the thymus gland also stimu­ lates gluconeogenesis in the liver and increases the ability 29

of the muscle to do work. This author also observed that

the potency of 11-deoxycorticosteroids relative to naturally

occurring corticoids is significantly greater When the

steroids are injected in an aqueous medium than when they

are given in corn oil. In the same study, the author found

that the involuting activity of prednisone and prednisolone

relative to hydrocortisone was significantly higher when the

corticoids were given in an aqueous solution.

Brolin (25), analyzing experimental inducement of acute thymic involution in rats, found that injections of estradiol

and hexestrol resulted in a marked thymic involution. In

sections, the medulla was predominant and irregular cells were hypertrophied and resembled epitheloid cells. None of

the adrenalectomized rats when Injected with estradiol and hexestrol showed signs of thymus involution, the microscopic

appearance being similar to that of control animals. Selye

(II4.5), however, states that thymic involution can be brought

about by administering estradiol to adrenalectomized rats.

Honey et_ al. (116) observed progesterone was the only com­ pound which significantly increased the size of the thymus.

Selye (ll}.5) Has shown that the adrenal medulla, xfhich devel­

ops from nerve tissue, is necessary for the appearance of an

acute involution.

Involution of the thymus has also been noted after thy­

roidectomy (Marine, (103)), and the lymphatic tissue 30 hyperplasia seen in toxic human hyperthyroidism is believed to be due to the overactivity of the thyroid gland (Selye

(lij.5) )• Weaver (168) found that both the administration of thyroid substance and thyroidectomy caused involution, which differed in degree from animal to animal.

Partial removal of the dog’s pancreas results in hyper­ trophy of the thymus (Scapaticci (139)). Bargmann (9) obser­ ved that thymus involution occurred after total pancreas extirpation, but in his opinion this accidental involution is not the result of endocrine correlation between the islet tissue and the thymus. Bomskov (l£), on the other hand, expressed the opinion that the increased number of pancreatic islets in thymectomized guinea pigs indicates an antagonism between the panscreas and the thymus. Padda (i|6) did not observe changes in the pancreatic islets of thymectomized rabbits. Bomskov (13) found that the so-called thymic hormone has a diabetogenic effect and causes a decrease in liver glycogen and an increase in blood sugar. Epiphysectomy in rats and cats did not cause structural changes in the thymus as Hoffman (80) and Davis and Martin

(39) stated, but Lindenberg (100) observed epiphyseal atrophy after thymectomy in immature rats. Vecchi (as quoted by Bargmann (9)) observed inhibition in thymic devel­ opment in epiphysectomized young male rats. 31 Hypertrophy and Hyperplasia

Parabutschew (126) suggested that delayed involution of the thymus occurs because the sex organs are not developed and are not producing hormones, but on the other hand

Bargmann (9) has noted the consistent occurrence of this condition in chronic adrenal deficiency. In the literature two correlations stand out with rather impressive consist­ ency, namely: thymus enlargement following castration and a converse hypertrophy under conditions in which a deviation of adrenal cortical function occurs. The thymus is influenced by several endocrine glands.

Comsa (32) expressed the belief that a relation between the thymus and the somatotrophic hormone of the adenohypophysis exists. This observation has been assumed by many authors.

Houssay (82) remarks that the adeno-hypophysis has a direct stimulating effect on the thymus due to the somatotrophic hormone.

Hammar (62), giving criteria for hyperplasia of the thymus, noted that the thymus can be said to be hyperplastic if the amount of the parenchyma significantly increases

(i.e., that the cortex is increased in size and number of thymocytes and the number of small Hassall's bodies. Hammar

(62) indicates that a true hyperplasia is one which persists and does not give way to involution after puberty. Joest

(175) found there are many papers in the literature abovit 32 hyperplasia of the thymus which in reality are describing tumors of the thymus or inflammatory diseases.

Thymectomy

Anderson (I}.) established that thymectomy does not affect the age or weight at which female rats reach puberty, when the cirterion for the latter is the opening of the vagina. This is true even if the operation is performed at the age of one day. The author found that thymectomy does not affect the age at onset of estrus circles, and does not affect the growth, appearance, or behavior of young rats, even when the operation is performed at the age of one day.

Asher (7) notes that the works of the physiology institute in Bern (Switzerland) showed that a material was prepared named ’’thymocrescinir from the thymus which when in­

jected daily (l mg per rat subcutaneously or intramuscular) had a growth-promoting effect on young animals. There was an increase in total body weight and length compared to con­ trols the same age, and it was observed that the growth of sex organs was enhanced to an even greater extent than body weight. The same institute established that this material was a peptide and was not identical with the growth-promoting vitamin B.

Bomskov et al. (16) showed in an extensive work that the thymus gland is important for life. The author thymectomized 33 approximately 200 guinea pigs at the age of 1-2 days and

showed that total thymectomy leads to death. Bomskov et al (16) proved the thymus has enormous vitality and that complete

regeneration is possible in 10-lii. days. In the authors

opinion, the negative results published in the literature are due to rapid regeneration, improper selection of animals,

and Incomplete removal of the thymus tissue. The animal used was the guinea pig because an easily accessible cervical

thymus is present.

Comsa (30) thymectomized l$0-gm guinea pigs to observe

changes on the circulating blood and hemopoietic organs. It was found that during the first few days following thymec­ tomy the animals behaved quite normally. At about 10 days

after the operation, they gradually deteriorated. The weight increase was slowed down or completely stopped, and

the animals became drowsy and flabby. The lymphocytes were

decreased in number in the spleen, lymph nodes, and the

circulating blood. The eosinophils were decreased in number

in the spleen and in the bone marrow. The eosinophil count

increased In the circulating blood. These observations seem

to show that thymectomy results in a decreased production of

lymphocytes and a decreased production, as well as an in­

creased elimination, of eosinophils. The results of Gross (£6) and Levinthal (99) are very similar, and Gross (5>6) concluded that thymectomy, either preceding or following inoculation of leukemic passage A filtrate, inhibits development of leukemia in otherwise susceptible G^H mice. Furth (5l) and his associates foimd that the incidence of spontaneous lymphomas in a suscept­ ible strain was sharply reduced after thymectomy. Subse­ quently, Kaplan (85?) and Law and Miller (97) reported a similar influence of thymectomy or lymphoma development under other experimental conditions. Furth (51) and Kaplan

(85) attributed this phenomenon simply to removal of the tissue of maximal susceptibility. However, Law and Miller

(97) reported that the incidence of lymphoid tumors could be restored to essentially normal levels in thymectomized mice by autologous or homologous thymic implants, suggesting that the thymus may be involved in the pathogenesis of the disease in an additional role, possibly of endocrine nature.

Irradiation

Many investigators have emphasized the damage done to the thymic cells in terms of injuries to the nuclei as the effects of irradiation. Smith ejb al. (1.5l) found in mice

that lipids accumulated as soon as four hours after exposure to x-rays and decreased in amounts after five days. This author found in young, irradiated mice that there were'lipids in the small reticular cells and in granular leucocytes of the thymi, the most striking reactions in the irradiated thymi being the presence of macrophages loaded with cells.

Eighteen hours after irradiation, the epithelial reticular cells begin to be noticeable, and after 32 and 72 hours following exposure to x-rays they are hypertrophied and con­ spicuous .

Miya (113) ejfc al. reported that the post radiation ad­ ministration of cell-free mouse spleen extract to whole body x-irradiated mice did not result in protection against post­ radiation death. The treated animals died at an accelerated rate when compared to the controls.

Kaplan _et al_. (86) worked on the basis of the observa­ tions of McEndy, Law, and Miller, who established that lymphoid tumors and lymphatic leukemias arising either spontaneously or in response to exogenous agents (x-rays, estrogen, etc.) tended to originate in the thymus in several mouse strains, and their incidence could be drastically reduced by thymectomy. The effect of thymectomy was not due simply to removal of potentially malignant cells. Kaplan et al. (86) implanted thymic tissue into irradiated animals and produced lymphoid tumors. In some strains, although not in all, the thymus is the site of origin of the malignant lymphoid cells, and in all strains thus far studied it appears to contribute an influence necessary for the develop­ ment of lymphomas and lymphatic leukemias, even when it is not involved in the tumor process. 36 Augmentation and Substitution

Bomskov and Kuckes (19) reported the injection or feed­ ing of thymic hormone ("thymic oil") in tadpoles increased

the weight and length and changed the form of these animals.

Karras (88) found the feeding or injection of thymic substance to female mice inhibited estrus and that in infant

females the development of the ovaries was retarded.

Bomskov and Sladovic (22) perceives a thymotrophic

effect in the actions of the growth hormone, diabetogenic hormone, or the hormone of carbohydrate metabolism of the

anterior pituitary because these actions are completed only

through the intact thymus gland. These authors further

found that one dose of thymic hormone is able to depress liver glycogen in experimental animals (rat, guinea pigs, pigeon) to 3>0 per cent of the normal value. The standard unit is 10 mg of thymic oil. Bomskov and Kreft (18) made

further observations that even a small amount of this hor­ mone ca.uses lymphocytosis and leucocytosis, but does not affect the red blood cell count. It was noted that the lymphocytosis present during the age of growth is an ex­ pression of greater thymic function. Bomskov and Lipps (20) found that thymic hormone is

able to inhibit the development of the gonads. After many weeks of injection of this hormone into male rats, atrophy

of the testis developed. Bomskov (22) remarks that the 37 action of the thymic hormone is nothing more than an ex­

pression of an infantilism, because increased growth,

lymphocytosis, inhibited development of the gonads, and

decreased liver glycogen are common physiological phenomena

in young individuals. On this basis the thymic hormones

were specific only and exclusively for the age of infancy,

Bomskov (22) reported that the largest amount of this hormone is present in the thymus of young animals. It was

found that the thymic hormone, in contrast to other hormones,

did not exist in the blood serum but was attached to the small thymocytes and stored in the cortical portion of the

thymus and at the time of need, it was transported by these cells.

Bomskov (ll|.) noted that the thymic hormone had no

effect on the basal metabolism, but slightly increased the weight of the thyroid gland in the guinea pig. Histological studies showed an accumulation of colloid material in the

thyroids. There is an antagonism between the thymus and the

thyroid, Bomskov and Kaulla (17) found that the thymus hormone had a strong action on carbohydrate metabolism and had the ability to decrease liver and heart glycogen in

experimental animals, whereas non-specific animal and vege­ table oils and fats were without action on the liver glycogen in the same amounts. Gomsa and Gross (3E>) found daily doses of thymic ex­

tract prepared according to the Berssonoff and Gomsa protocol decreased mortality in irradiated infant guinea pigs. Less

than 10 per cent of the animals so treated died, but nearly

half of the untreated died. Examination of the pituitary, the thyroid gland, the thymus gland, the bone marrow, the

testes, and the adrenal glands showed that sequelae of the

irradiation were present in these organs, but that healing

took place much sooner under treatment with the thymus extract.

Comsa (31) found that highly purified thymic extract in

in vitro experiments exerted a co-catalytic influence upon

the denaturation of thyroxin added to liver brei. The

changes in .the thyroid, induced by thymus extract may re­

flect both an antithyroid and an antithyrotrophic influence

on the thymus.

Gomsa (33) giving thymus extract daily to guinea pigs,

showed that growth increased significantly. The rate of

sexual development was inhibited or slowed. Thyroid atrophy

occurred after 120 days of treatment.

Comsa (32) observed that separate doses of growth hor­ mone or thymus extract did not increase growth in hypophysec-

tomized rats, whereas simultaneous injections of thymus

extract and (2.0 Evans units) of growth hormone resulted in

a significant enhancement of the influence growth hormone 39 upon the growth rate. It was concluded that the thymus is

a significant factor in the growth effect of anterior pitui­

tary growth hormone.

Comsa and Bezssonoff (3^4-) state that their extract is

able to prevent the disturbances Induced by thymectomy in

Infantile guinea pigs. These thymic extracts are specific

in their action, since the extracts of liver, lungs and muscle, prepared by the same method, produced negative re­

sults and show no detectable activity in any of the bioassay tests.

Nakamoto (118) observed that an acid extract of the thymus of the young calf caused an increase in lymphocytes and mitochondria in the lymphocytes within ij.8 hours follow­ ing injection into young rabbits. This was observed only

following injections of lipoid fractions of the thymus, arid no such effects were obtained with acid extracts made from alcohol-insoluble thymus substance or extracts prepared

from thymi which showed fatty degeneration.

Nakamoto (119) found that injection of the lipoid frac­ tion of the spleen produced low counts of both lymphocytes and mitochondria in the peripheral blood. Therefore, the

stimulating action on lymphocyte formation is specific for

the thymus and is not a common property of lymphatic tissues.

Metcalf (110) reported the thymus has been shown to pro­

duce a lymphocytosis-stimulating factor (LSP) and that this factor is involved in the maintenance of normal lymphocyte homeostasis, both by its direct stimulation of lymphopoetic cells and indirectly through the suppression of lymphocyte production by adrenal corticoids. The author believes the

LSP is a true hormone which is detectable only in the human and mouse thymus.

Molnar hb al. (llip) found that thymic extract increases the growth and malignancy of implanted Brown-Pearce carcinoma in rabbits. It was observed that an activation of the hypophyseal-adreno-cortical mechanism occurred. Molnar et al. (ll£) verified these results in further experiments.

These authors also found that extracts prepared in the same manner from liver, spleen, and kidney and even from the same tumor had some effect on growth. It was believed that this increase in malignancy was due to changes in the ability of the host to react, rather than to an action on the tumor. EXPERIMENT NO. 1

Part 1

The objective of this portion of the study was to observe the histological changes occurring in the rapidly developing thymus of the rat.

Materials and Methods The rats used were Sprague Dai-jley strain obtained from

Madison, Wisconsin, when 21 days of age and bred in the Department of Veterinary Anatomy at the age of 80 da7/s.

They were kept in metal cages, four to each cage, and were fed Purina Dog Chow ad libidum; the supply of drinking water was also unrestricted. Thirty-three rats were used for this experiment, and only one animal was sacrificed on each day as required. On this basis, five thymi of 1-day-old rats; five thymi of 2-day-old rats: four thymi of 3-d.ay-old rats; five thymi of lj_-day-old rats; three thymi of 5>-day-

After proper fixation, they were embedded in paraffin and

1+1 sectioned transversely at b/* . The serial sections were mounted and stained with hematoxylin and eosin. The trachea,

esophagus, cervical lymph nodes, vessels, and vagus nerves

were removed and sectioned together with the thymus. Each

section was observed and each lobe was examined from the

ventral surface: first the capsule, the subcapsular region,

the cortex, and then the medulla.

Results

In one-day-old rats the thymic capsule was composed of

large, oval, pale-nucleated connective tissue cells 8-A*- to

1 0 / in diameter in a single layer. In a few areas beloitf

this fine capsule, histiocytes, plasma cells, reticular

cells, and eosinophils were present, particularly in the

areas giving rise to the interlobular septa. Some small

vessels were present in the interlobular spaces. These

spaces were large (30 to !+£/*' ) when compared, to those in

older thymi.

There was no difference between the cortex and the

medulla on the first day. Reticular cells of three differ­

ent sizes (5>.8, 6.8, and in diameter) formed the basic

tissue. Between these reticular cells large numbers of

thymocytes (thymic lymphocytes) ranging from l-Sy^ in

diameter were present. The nuclei of the cells were very

pecularly lobulated. These nuclei were strongly basophilic. 43 There was no evidence of mitotic division, and no true

Hassail's bodies were present in the thyrni. All of the

cells were loosely arranged, but compared to those of the

cervical lymph nodes present in the same sections they were more basophilically stained and more tightly packed together.

In some sections of the one-day-old thymus, enlarged, indi­ vidual epitheloid cells resembling small Hassall's bodies

10/<. in diameter were present.

Not until the sections from 2-day-old rats and then

particularly in the cortex were a few mitotic figures pres­

ent. The lobules were more organized and the medulla was

distinguishable from the cortex. The predominant cells in

the medulla were large reticular cells which were loosely

connected with each other. Small thymocytes were scattered between them. The vessels were developed in the interlobular

spaces, running transversely or longitudinally and branching,

A remnant of the third pharyngeal pouch composed of cuboidal

cells forming a cyst was seen in all cases and in succeeding

sections branching tubuli were observed from it to various

lobes in the medulla. The impression was gained that in the

absence of these cysts typical Hassall's bodies were missing.

In subsequent sections the vascular pattern became more and more evident. Because these were cross sections, only the

openings of the vessels or, if branching, only a small part

thereof could be seen. Extracellular colloidal material, PAS positive, mucopolysaccharide was present in the medulla around the reticular cells. A few Hassall's bodies approxi­ mately 1 !]./*> in diameter composed of one or two reticular cells, were present.

A few mitotic figures were present in the cortex, and medulla of 3-day-old rat thyrni. The capsule appears histo­ logically similar as in the previous thyrni, but in this case the subcapsular area had started to develop large, oval, pale-nucleated reticular cells. These were loosely connected to each other in a manner very similar to those in the medulla, The remainder of the cortex was densely packed with small and medium-sized thymocytes and had few visible reticu­ lar cells. In all sections, clearly visible cysts could be seen in that part of the thymus facing the trachea, and branches from these could be followed to the medullary por­ tions of each of the lobules. Prom this stage of development the Hassall's bodies 10-25./*' in diameter appear in the medulla. Another layer of flat, pale-nucleated cells had been added to the capsule in the l|-day-01d rats. The thymic cor­ tex contained a larger number of mitotic figures than did the medulla. The medulla was more extensive than previously, and the exact demarcation between cortex and medulla was not obvious. The subcapsular area, with its large number of mitotic figures and few large cells was still differentiable hs from the dense, closely packed portion of the cortex. This dense area extends on both sides of the septa and encloses the medulla on three sides, although the medulla is continu­ ous with those of the adjacent lobules. Small cyst formed by few low columnar cells were present in the medullae of the lobules. The capsule was thickened by many layers of flat cells in ^-day-old rat thyrni. The cortex was enlarged, and the medulla reduced, as compared with k-day-old rat thymus.

The number of mitotic figures appeared to be increased in all of the cortical portion, and the numbers of small and medium sized thymocytes were also greater. It appeared that the medulla was reduced in area because of the increased number of cells on the border between the cortex and the medulla. The cysts were larger, and occasionally a lympho­ cyte or thymocyte was present in the lumen in addition to the PAS or mucopolysaccharides material.

In 6-day-old rat thyrni there are less mitotic figures in the medulla than in the cortex. Only four mitotic figures as compared to ll[. could be seen under oil immersion in a field of these two zones, respectively. The medulla was composed of a large number of medium sized thymocytes and some visible reticular cells. Hassall's bodies were not observable in 6-day-old thyrni. In 7“day-o.ld rat thyrni, as many as 37 mitotic figures were counted in a single cortical field under oil immersion, whereas in the medulla only five were seen. Cysts were present. The interlobular soaces were very small and the left and right lobes were in close apposition on the ventral portion of the trachea. The thymus was closer to the vagus nerves, which were enclosed in the common connect­ ive tissue capsule. The Hassall's bodies were present. The medulla x^as small and not greatly extended. The cortex was enlarged, the subcapsular area diminished, and the number of small thymocytes increased as compared to those in the thymi of younger rats,

Part 2

The objective of this portion of the experiment was to observe the cytological changes occurring In the rapidly developing thymus of the rat.

Histological observation of the thymus at various ages in various species and under different conditions did not completely settle the question of the origin of the thymo­ cytes and the reticular cells of this organ. Seeking a technique which would better elucidate the biological pro­ cesses in the thymus, cell culture wTas employed.

Materials and Methods With the aid and collaboration of the Department of Bacteriology, a procedure was worked out which made these observations possible. Thymic tissue was obtained for cul­ ture from sexually mature and immature white rats. For each experiment the thyrni from two rats were used to provide sufficient tissue for approximately culture tubes.

Immediately after the thymus was removed from the rat, it was placed in a petri dish containing a small amount of

Hanks’ balanced salt solution. Two sharp scalpels were used to cut the thymus into minute pieces, which were placed on sterile coverslips by means of a capillary tube. These coverslips were placed in culture tubes without medium. The culture tubes were held at Jp0 C for 30 minutes. After the culture tubes were removed from refrigeration, 2 cc of Hanks' balanced salt solution containing 20 per cent lamb serum and one per cent of an antibiotic solution were added for nutri­ tion and control of bacterial growth. The culture tribes were placed in an incubator at 37° 0. The pH of the culture medium was approximately 7*2. Five per cent pig embryo extract was added to some of the culture tubes, but did not enhance the growth of thymic tissue.

Thymus explants containing blood appeared to grow most rapidly in culture. Thymic tissue obtained from sexually mature rats showed' more connective tissue growth in cell culture than that of younger rats. The first observation on the culture growth was made after hour of incubation. Four coverslips containing the cell growth were removed at each kB observation interval and stained by the May-Grunwald-Giemsa staining technic.

Results

Detailed results are described by i'app and Venzke (l2l|.). Cyclic production of thymocytes and reticular cells was ob­ served. The duration of this cycle was usually between four and six days. This procedure did not provide sufficient evidence that thymocytes are produced by reticular cells, so it was decided to add various quantities of cortisone while maintaining the pH constant at 7*2.

The culture tubes used in the experiment were divided into five groups, each of which consisted of lp8 tube cul­ tures which were used as described above. To the medium in group A (k8 tubes) 100 mg of cortisone acetate in addition to the 20 per cent serum and 1 per cent antibiotics were added. To group B, 75 mg of cortisone were added; to group 0, 50 mg; to group D, 25 mg xtfhile group E served as a control and received no cortisone. The control group consisted of lhl\. tubes. Three times daily four coverslips were stained from each of the five groups.

It was demonstrated that the rate and extent of the effect was proportionate to the cortisone dosage. At the end of the second day only reticular cells i-jere present in the tubes of group A, whereas in groups B, C, and D, medium and small thymocytes were present. The medium, was changed when the pH declined, usually every three or four days. The cul­ tures in all tubes stained during the first ten days showed only reticular cells. At that time the medium was removed and replaced with medium which did not contain cortisone, and the staining was continued three times daily. It was shown that the reticular cells started to divide and that thymocytes were present in small numbers after 32 hours from the time the medium was changed. The reticular cells gave rise to ever-increasing numbers of thymocytes in all groups.

The previous!?/- described experiment was repeated with the same results. The most obvious changes were seen in the group of cell culture tubes which contained 5>0 mg of cortisone per 100 cc of medium. By this procedure it was established that thymocytes were formed from reticular cells. After longer periods of culture, the reticular cells were converted into fibroblasts. It was observed that the fibroblasts in thymic cell culture formed a circular pattern but that no Hassall’s bodies resulted. The cell cultures were maintained for three months. There was no formation observed of plasma cells or of types of cells other than various sizes of thymocytes and reticular cells, and later, of fibroblasts. 5 0

Pig. 1 Transverse serial sections of thymus of three day old rat H and E. X I(.75»

52

Fig. 2 Transverse serial sections of thymus of three day old rat. H and E. X I|_75« hi lW M EXPERIMENT NO. 2

Albino rats of the Sprague-Dawley strain were selected as experimental animals primarily on the basis of economy and ease of operation. Some of the most important observa­ tions were to be based on the hemograms of these animals, so it was important to establish the extent to which these measurements were reliable.

Creskoff ejt al. (38) have stated that the rat, when compared with the human and most other mammalian species, is microcytic, hypochromic, and polycythemic. In adult rats, a relative reticulocytosis, thrombocytosis, leucocytosis, gran­ ulocytopenia, and relative or absolute lymphocytosis are the rule. The normal range of variation in the hemogram of this species is also wider than in most other mammals.

It was also necessary to establish a standard and valid blood sampling technique. In this connection, Creskoff et al.

(38) declared, "The total blood volume of the rat is relative­ ly small and the accessible areas for obtaining blood samples are limited. Because of these facts even small frequent bleedings induce anemia and reticulocytosis, and the trauma of repeated sampling produces local inflammation and leukocy­ tosis. " 5 k &

This statement indicated a need for two important in­ vestigations: (a) to establish standards for the hemogram in

Sprague-Dawley rats as a function of age and weight, and

(b) to test the hypothesis that frequent bleeding induces leucocytosis. It was established that none of these objec­ tions was apt to apply to the other experimental animals in these studies, the calf.

No references were found giving detailed weekly or daily hemograms for rats; therefore, weekly hemograms were com­ piled over a period of two years for rats of both sexes up to 16 weeks of age. The means of these values appear in

Table 1.

The blood samples upon which this table is based were initiated when the rats were 28 days of age and weighed 5>0 gms and were continued for 12 weeks. The cell volume

(hematocrit) during this period ranged from lj.l-5>0 cc per 100 cc of blood and was in very close agreement xwith reports in the literature (38)* It remained at the lower level until the 10th week and thereafter was approximately constant at the upper limit of the quoted range.

The white blood cell count was made on the standard

clinical basis. It has been shown statistically that there

are no important sex differences in total or differential

leucocyte counts in rats (38). The table presents numbers per cubic millimeter rather than percentages for the various types of leucocytes because the former are better suited to

statistical analysis.

The same was done with calves. The data in Table 3

represent averages of three calves for a period of 28 weeks

at approximately the same ages but not the same weights.

In this case the hemoglobin concentration was also determined.

The average values are more constant in calves.

In order to determine whether frequency of blood samp­ ling had an effect on the hemogram, three groups of five rats each (A, B, and C) of both sexes were bled at frequencies

of one, tx^o and three times a week respectively. The numbers of leucocytes per cubic millimeter of blood and the per­

centages of segmented neutrophiles and lymphocytes were measured and statistically analyzed. Attempts were made to fit these data to parabolic regressions on age. As might have been expected, only the leucocyte counts were found to vary as a parabolic function of age, and the regression equations derived for the data from the three groups did not differ significantly. Graphs for these three equations, showing the actual in comparison to the predicted values of the observations, are presented in Figures 3, If., and £.

Neither the percentage of segmented neutrophiles nor of lymphocytes could be fitted to a parabolic equation, nor were

the differences between groups with respect to these measure­ ments significant. These findings may be taken to indicate that there were no significant quantitative nor qualitative differences in the leucocyte contents of the blood of rats due to differences in frequency of bleeding.

Since other observations on the blood were also to be made in the studies of the effects of thymectomy, it was necessary to establish some standards for the levels of other blood constituents as a function of age. Such standards were ascertained for the blood of rats in the case of carbon dioxide by the method of Natelson (1 2 0 ); chloride with a

Cotlove chloridometer; sodium and potassium with a Baird flame photometer; cholesterol, by the method of Zak (173)J and calcium, by the method of Bachra, et al. (8 ). The results of these determinations are presented in Table 2.

Similar standards were sought for calves, but due to the larger volumes of the blood samples, it was not necessary to use microdeterminations. Therefore, calcium was determined by the method of Kramer and Tisdall (9l+)j sodium and potass­ ium with a Coleman flame photometer, chloride by the method of Schales and Schales (UL|^l), blood urea nitrogen by the method of Karr (87), glucose by the method of Folin and Wu (5>0), and cholesterol by the method of Sackett (176). The findings with respect to the electrolyte levels in the blood of calves are reported in Table £. The levels of the remaining blood constituents determined in calves are reported in Table I4.. 58

Table 1

Hemogram in Intact Rats as a Function of Age

VJks. Hct WBG Seg.N. N . Seg.]1. Lym. E o s . B a s . Mon. wt. % mm3 mm3 mm3 mm3 mm3 mm3 mm3 gm.

1+. 1+5 7.210 1.053 189 5.720 11+0 1+8 60 50 < l+o 9.651 1.707 ISO 7-631+ 123 5 2 63 6 I4.1 11.738 2.31+1+ 23U- 8.995 81+ 36 1+5 81+ 7 k k 17.607 3.765 296 13-1+17 56 1+3 30 86 8 1+1+ 17.599 k .1 8 6 390 1 2 .8 9 6 112 15 131+ 9 1+1 1 6 .9 6 8 3.237 173 i3 .Ii.ll4. 51+ 90 152 10 1+1+ 19.753 2 .655 11+9 1 6 .6 9 2 97 160 179 11 1+9 1 8 .8 2 0 2.977 1+1 1 5 .61+0 162 193 12 1+3 15.665 2 .1 1 0 20 13.1+1+1+ 82 9 220 13 1+8 1 8 .6 0 0 2.756 30 15.11-67 30 319 235 15 1+8 1I+.6 I+3 1.890 96 12.1+85 21+3 16 5o 11+. 022 2 .101+ 17 11.853 1+8 227 59

TaTSe 2

Weekly Concentrations of Certain Blood Constituents in Intact Bats

Weeks Carbon Chloride Sodium Potassium Cholesterol Calciur Dioxide Vol. % mEq/L. mEq/L mEq/L mg 5 mg i o

9 23.67 95.00 1^0.1 5.3 61].. 38 8.73

10 2U..67 96.1)5 153.3^ - 81+.1+5 9.77

11 25.78 91+.00 138.56 8.65 87.89 9.59

12 2J4.. 89 100.22 139.67 6.79 63.78 9.87

13 2^.67 87.56 138.31+ 8.03 56.78 9.79 60

Table 3

Hemogram in Intact Calves as a Function of Age

Wks. Hb. HOT. WBC. Seg.N. N.Seg.N. Lym. Eos. Mon. mg % % ram3 mm3 mm3 mm3 mm 3 mm3

3 35 12,003 3.096 163 8,325 I83 161 k 11.18 32 8,833 2.539 6 , Olli 2 0 k 76 5 10.6 31 8,703 2.625 111+ 5,693 163 108 6 9.65 28 7 , 1 1 k 1.890 232 5,518 68 66 7 9.8 27 8 ,lL|ii 2.070 178 5,525 65 63 8 10.0 28 7,676 1.706 125 5,530 131 l Q k 9 10.6 28 7,816 1.811 228 5*58o 95 102 10 9.5 29 8,771 1.995 282 6,136 160 198 11 io.5 32 9,971 1.1+1+3 113 8,162 153 100 12 10. k 31 8,929 2.503 111 6,229 86 13 1 2 . 8 33 9,019 1.233 139 7,61|-7 I k 11.1 32 9,067 1.799 122 7,11+6 15 ii.5 33 9,833 2.160 81 7,592 16 10.95 31 9,537 2.593 162 6,762 20 17 n.95 3 k 12,357 3.771 121*. 8,326 136 18 11.25 3 k 12,006 3.915 1+21 7,670 19 11.0 31 10,125 2.1+57 21 7,597 1+9 20 10.8 31 9,925 2.367 253 7,186 88 21 H . 5 32 10,601 3.159 200 7,0^0 82 120 22 12.6 3 k 13,71+3 3.567 205 9,971 23 11.8 37 11,261 3.638 7,380 151 92 2 k 12.2 35 11,106 3.310 185 7,261 350 25 11.75 32 11,036 2.730 8,021 285 27 10.95 31 13,92li. 3,777 9,1+27 3 W 28 11.1+2 35 11,1l79 1,977 9,011+ lt-68 6 1

Table k

Weekly Concentrations of Certain Blood Constituents in Intact Calves

Blood Urea Glucose Cholesterol Nitrogen mg fo______mg; c/o______mg %

8 2 0 . 9 k 9 6 . 6 7 6 5 .67 9 1 9 . 7 0 85.50 5 5 . 3 k 10 1 6.27 9 8 . 0 0 6 0 . 6 7 11 20.70 6 7 . 0 0 1 0 8 . 6 7 12 16. 6k 83.67 8k. 67 13 1 6 . o5 97-00 1 1 0 . 3 k lli. 25.71- 113.31- 1 0 0 . 3 k 15 22. kk 102.67 120.67 16 2 5.37 89.67 1 0 k .67 17 21}.. 10 98.31- i k o . o o 18 3 0 . 5 k 79.67 208.3k 19 26.37 6 7 . 2 5 1 3 0 . 2 5 20 2 3 . 0 2 86.00 1 5 5 . 7 5 21 11.1-3 9 0 . 0 0 1 8 3 . 2 5 22 1 0 . 1 7 95.50 1 0 1 . 7 5 23 1 1 . 1 2 92.00 1 6 9 . 2 5 2k 1 1 . 7 2 1 0 5 . 5 0 1 1 8 . 7 5 2.9 11-. 92 8 6 . 7 5 8 9 . 0 0 26 12 • lk 7 8 .00 8 5 . 2 5 27 8.35 61.75 8 3 . 5 0 28 15.30 75.50 102.50 29 16. kO 73.50 1 2 6 . 0 0 30 1 8 . 2 0 85.25 9 9 . 7 5 62

Table 5

Weekly Concentrations of Certain Ions in the Blood of intact Calves

Weeks Calcium Sodium Potassium Chlorid< mg fo mEq/L mEq/L mEq/L

18 9.70 ii+5.31+ 5.07 101.25

19 9.80 ii+5.50 5.25 100.25

20 10.13 1!l7. 25 li.95 10J+.00

21 10.80 151+.00 5.05 10k.75

22 10.13 153.25 5.83 10k.50

23 10.50 153.00 5.70 106.00

2 k 10.78 152.50 5.53 102.25

2 5 10.k3 11+9.25 5.io 106.50

26 10.65 153.50 l|.85 106.75

2 7 10.55 114.8.25 JLf.,85 105.00

28 10.65 ll±9 .25 k.65 103.75

29 10.73 150.00 1+.98 102.75

30 10.38 11^9.50 U.85 105.00 63

Pig. 3 Parabolic regression of second degree based on blood samples drawn three times weekly from rats of group A. Control Group'A" ^W BC

l ©

/ \

o\i

^ ~ " Curves for Group A

* • - - + Data for Group A ® ® Data forGroup B ® ® Data for Group C 65

Pig. It Parabolic regression of second degree based on blood samples drawn two times weekly from rats of group B. 66 A W.B.C Control Group"B’

p>9f 1

Curves for Group B + + Da+a for Grou p A ■■® Da+a for G roup B Da+a for Group G 67

Fig. £ Parabolic regression of second degree based on blood samples drawn once weekly from rats of group G. A W.B.C Control G roup "C EXPERIMENT NO. 3

Part 1

Despite extensive investigations, the effect of surgi­

cal removal of the thymus in animals is still somewhat uncertain. This uncertainty is due, in part, to the diffi­ culty that has been experienced in distinguishing between effects of thymoprivia and effects that might be attributable to other causes, especially complications arising during or subsequent to operation, postoperative care of animals, and the use of methods that give varying degrees of success with respect to operative injury and complete removal of the gland.

In the course of studies that were being carried out, it became desirable to compare the behavior of thymectomized rats and calves with that of normal rats and calves from which other glands or organs had been partially or completely removed. This necessitated an investigation of the operation that could be depended upon for complete removal of the thy­ mus with a minimum loss of animals, and second, a determina­ tion of whether the thymus could be removed from rats and

calves of certain ages without producing any serious

69 70 impairment of health, within specified periods of time.

Procedure

On the basis of approximately 237 rat thymectomies of both sexes and various weights, the best and most successful time for operation is the 19 to 21 days. Losses of males in

surgery were relatively greater than those for females.

Sprague-Dawley strain ranging rats between 27 and 200 gms in weight were thymectomized. Older rats have developed more connective tissue, which is more friable, and also the heavier fascia connecting the thymus gland to surrounding tissues made the removal more difficult. The thymi of 19 to 21-day-old rats were relatively larger and more easily removed than those of older rats.

Anesthesia

On the suggestion of Dr. 0. R. Smith, 2^-30 mg Nembutal

I.P. were used in the preliminary work. Even 100 gm rats remain in deep anesthesia for many hours, and recovery is very slow, or death results. Nembutal I.P. and other bar­ biturates induce rapid anesthesia, but they complicate the state of postoperative shock or post operative respiratory difficulties.

Moderate depth ether anesthesia was found to be most satisfactory as an anesthetic. With deep ether anesthesia 71 more animals died from the operation proper than when moderate depth ether anesthesia was used.

Surgical Procedure

An ether-anesthetized rat was placed on its back on a softwood board, with the head toward the operator. The legs were pinned down, the front legs with palms up and the hind legs with the palms down on the board. One pin was used to secure the head in position. A rubber band was passed behind the upper incisor teeth and around the pin, thus stretching the head and neck, for removal of the hair from the region of the thorax and cervical region soap was applied. The area x^as shaved with a razor and disinfected with JO per cent alcohol. Soap does not help much, and the alcohol acted as an anti-coagulant in case of hemorrhages. Using only mer- thiolate before shaving has been of help, making the hair

\ wet and at the same time disinfecting the operation area.

The skin incision was made with pointed scissors from the level of the third rib to a point just beyond the manubrium sterni. The skin incision should not exceed 1-2 cm in length if clips are used. Small pointless scissors were used for the longitudinal incision which was made through the manubrium sterni and the sternum to the level of the second or third rib. This incision should not exceed

3/lj. cm in length, and it is essential to make the incision with one rapid cut having the scissors directed against the dorsal aspect of the sternum. The thymus cannot be seen through this incision. The pointless scissors was used to split the ribs and sternohyoid musculature horizontally.

The scissors were held in the left hand of the operator and the thymus gland was grasped firmly with moderately blunt curved forceps in the right hand of the operator. The assistant with straight forceps helps to pull gently and slowly. It was possible to remove both lobes at once in small animals. If both lobes are not removed in a single attempt the operator has to find the second lobe and proceed in a manner previously described. Changing forceps behind forceps and pulling carefully has produced good results.

Sometimes the thymus tissue is very soft and has to be taken out in parts, which makes the operation complicated and dangerous because the negative pressure Is lessened.

The remainder of the thymus may be drawn deep into the thoracic cavity. Deep exploration may result in rupture of the great vessels, causing the animal to expire.

The pectoralis musculature in young animals Is very easily damaged. Therefore, a 00 size gut attached to a half inch, round, atraumatic, gastrointestinal needle was employed.

The suture was passed between the first and second ribs. If the incision is larger, a second suture is passed between the second and third ribs, and by cross closing, the split 73 sternum is unified. By this method close apposition of the sectioned surfaces of the sternum was reached, and the bone healed rapidly. A disadvantage of this method is that the operator may puncture the superior vena cava with the needle.

Some animals survived if the thorax was closed rapidly even though the vena cava received damage.

After removal of the thymus gland some respiratory difficulty was exhibited as a result of the loss of negative pressure in the thoracic cavity. The operator or the assist­ ant closed the chest immediately with forceps or hemostat and sutured rapidly. Prompt closure of the thoracic wall was very important and considered a determining factor in the siiccess of the operation. Skin can be sutured with the same gut or silk or closed with wound clips. Surgical auto-clips are preferred. Usxially the animals exhibited Cheyne-Stokes respiration, but it passed in a short time and the animals walked immediately after the operation. The mortality rate depends upon the operator and the anesthesia. Rapid closure of the thoracic cavity, and the avoidance of rupture of the great vessels was necessary for complete success.

Part 2

Because it was desired to determine the changes in the hemogram, electrolytes, sugar, cholesterol, and CO2 resulting from thymectomy it was necessary to demonstrate that this surgical procedure resulted in complete wound healing. Necropsy was performed on all thymectomized animals, for two very important reasons: (l) to determine whether thymic tissue was left and whether thymic tissue had regenerated since thymectomy. (2) to establish that wound healing was good without pus formation. If the gross determination was dubious, histological slides were prepared; and if thymus tissues were present, the animal was eliminated from expert ment. In three cases out of 100 thymectomized rats there developed small, dry absesses. These animals were eliminated from the experiment. It was impossible in our experiment to use antibiotics or drugs, and they were not used, because of changes in the hemogram.

When blood was taken from the femoral vein a separate sterile needle was not used for each puncture, and the use of 70 per cent alcohol had also to be avoided. In the ab­ sence of these precautions, the sites of venipuncture in all control animals in the early trials developed abcesses the size of a pea, whereas no such complications developed in the thymectomized animals.

Materials

The operation described above has been performed on 237 male and female rats to date. Of these, 23 were 21 days of age, and the remainder ranged in age from 19 to 53 days and in weight from 3h- to 279 gms. The operations were performed, during every season of the year. The longest period of observation on thymectomized animals was 8 to 9 months.

Results

Operative Results - Among the first groups upon which

thymectomy was performed, there were 27-36 per cent deaths related immediately or remotely to the operative procedure.

Upon modification of the surgical procedure with improvement in the anesthesia and the use of younger animals, there were

2 per cent or less deaths following surgery. The causes of death included injuries to vessels, excess anesthesia, air embolism, and punctured thorax. In the early trials post­ operative complications occurred, for example wound infections, usually an abscess developed within the medi­ astinum. These infections became encapsulated and pursued a chronic, idolent course which did not appear to affect the general health of the animals. There were no pulmonary com­ plications, except in the preliminary operations in which several of these animals developed pneumonia, atelectasis, or empyema.

Recovery from the operation was prompt and the operated rats showed a gain in weight or a maintenance of weight com­ parable in all respects to intact normal rats. The chest wall incision united firmly in all of the animals that

survived the operation with very little deformity of the 76 thoracic framework and no interference with the function of the fore legs.

Postmortem examination usually showed that removal of the thymus was complete. If some thymic tissue was left, the animal was eliminated from the observations or data collections. If any fragments were left, they showed no appreciable tendency of hyperplasia in rats operated at 19 days of age or later. The thymic bed was obliterated by firm, fibrous adhesions between the pericardium and the thoracic wall. The lungs were expanded to their normal proportions. Pig. 6 Thymectomy of the rat. Splitting of the sternum before the removal of the thymus gland. «/»(/ Fig. 7 Split sternum up to the third sternal cartilage. Exposed thymus gland in the rat. \

'H...

?*•> t V 81

Pig, 8 Removal of th.e thymus gland in the rat with the help of blended scissors and forceps. \ 82 83

Fig. 9 Exposed mediastinal region in the rats after thymus gland was removed. /#« 8S

Fit?. 10 Gross suture with eat-p;ut closing the split sternum and the mediastinum. 86

K

Y \

St,lit* EXPERIMENT NO. I4.

Hammar (63) divided the mammals into three groups, on the basis of the position of the thymus: group I are those having a cervical and thoracic thymus, group II are those having only a thoracic thymus, and group III are those which possess only a cervical thymus. The purpose of this experi­ ment was to choose a domesticated animal in which thymectomy had not previously been performed, and from xfnich the thymus could be completely removed. It was also desired to employ an animal in which leukemia or leukemic-like disease frequ­ ently occurs. There is no domesticated animal which possesses only a cervical thymus. In cattle the thymus is present on the left side of the thorax and cervical region. Lymphomas are frequently observed. Further it was necessary that the species selected for the experiment possess a readily accessable vein for frequ­ ent peripherally located blood letting. It was also desired that the species possess lymph nodes for palpation. Finally, it was desired that the selected species be of economic im­ portance and of significance to the veterinary profession.

87 88 These criteria were more nearly met in all respects by Bos taurus than by any other species. Following selection of the species to be employed, it became necessary to devise a technique for thymectomy.

Thymectomy of the Calf

In August, 1958, six dairy calves ten to fourteen days of age and weighing fifty to sixty pounds were purchased with the intention of thymectomizing three and using the remaining three as controls. Prior to the operations, an eight-month- old fetus was dissected to serve as a guide. It was found that the thymus could be expected to extend from the mandible well into the thorax, being present on both sides of the neck but only to the left of the mediastinum in the thorax. In view of the large area involved, it was decided that only a cervical incision would be made and that the thoracic thymus would be withdrawn through the thoracic inlet.

Anesthesia presented a problem, since no anesthetic has been demonstrated to be ideal for cattle. Nembutal was chosen for the first surgical anesthesia.

A 53-pound calf (designated number l) was selected, and food was withheld for twelve to twenty-four hours before the operation. The ventral and lateral aspects of the neck from the mandible caudad and the ventral aspect of the anterior thorax to the third rib were shaved. Seven milliliters of 89 nembutal were injected intravenously, and the calf was posi­ tioned supine on the operating table with all four limbs stretched caudad and the head extended craniad. A median incision was made through the skin from the level of the angle of the mandible to the manubrium sterni. The sterno­ hyoid and sternomastoid muscles were separated along the median line. These muscles were dissected away from the trachea to expose the thymus. Pour more milliliters of nem­ butal were administered in the course of the operation. The thymus was separated from the surrounding fascia and connective tissue by blunt dissection. The bleeding vessels were tied off with absorbable gut. The cervical thymus was dissected free starting at the cranial end and was withdrawn gently, so as to include as much as possible of the thoracic thymus. A total of sixty grams of the thymic tissue was removed.

Closure was effected by continuous suture of the sterno­ hyoid and sternomastoid muscles along the median line with

#1 chromic gut and interrupted silk suture of the skin. No provision was made for drainage. The calf remained in deep anesthesia for tx-jenty-four hours and had to be turned period­ ically. Body temperatures as low as 9f?° ^ were recorded. Twenty-eight grams of thoracic thymus was recovered at necropsy two months later. The surgical technique was un­ satisfactory. 90 The second animal (designated number 2) was anesthetized with eight milliliters of nembutal and positioned as described previously. In this case the median skin incision was con­ tinued caudad 'to the level of the third rib. The cervical region was treated as described previously, but the left pectoral muscles were separated, a portion being left attached to the sternum, and the left first and second ribs were de­ tached at the costosternal articulation.

Both the cervical and thoracic thymus (a total of£8 gm of tissue) was removed. The cervical muscles were sutured as previously described. The ribs were placed in position and the pectoral muscles joined with a continuous #2 chromic gut suture. The skin was closed as previously described.

The animal expired at 1:00 A.M. the morning following the operation, presumably from the effect of anesthesia.

A third animal (designated number 6 ) was anesthetized with six milliliters of nembutal intravenous and thymecto­ mized as described for number 1 . Sixty-five grams of thymic tissue were removed, and no thymic tissue was found at necropsy two months later. Although the anesthesia was so light that the calf had to be retrained during the operation, it remained unconscious for nearly twenty-four hours and required constant attention during the recovery period.

A fourth calf (designated number 3) was also thymecto­ mized by the same procedure. Only eleven grams of thymic 91 tissue were removed, another seventy-two grams were found in

the thorax at necropsy two months later. The same anes­

thetic procedure was used as for number 6 , and the same

comments apply. Calves numbers Ij. and 5 remained as controls,

A 17-day old Angus calf designated Kq was anesthetized

with lf?0 ml. of 7 pe^* cent chloral hydrate solution and posi­ tioned as previously described. Tracheal intubation was per­ formed with a firm rubber tube, the end of which was surrounded

by an inflatable rtibber bag. The bag was inflated in the

trachea with a syringe and closed off to effect a seal around

the main tube. The main tube was connected to a resuscitator which inflated the lungs intermittently with a mixture of 9£ per cent oxygen and 5 per cent carbon dioxide.

The skin was opened as described for number 6, and the jugular vein was cannulated and attached to a syringe of pentothol sodium for use if deeper anesthesia was required.

No such requirement occurred. After dissection of the cervi­

cal muscles was completed, the sternum was split medially from the manubrium sterni caudad to the level of the third rib with a human dissecting saw. The point of the saw was

directed ventrocaudad. The sternum was then spread forcibly

to expose the thoracic viscera. Shortly after this was com­ pleted an unexplained failure in the trachea tube or resusci­

tator resulted in complete pneumothorax, and the animal

expired. Calf K2 (Hereford), 2.1\. days of age, was also brought 92 to this stage of the operation by similar steps. However, the anterior vena cava was accidentally sectioned in the course of the intrathoracic dissection, and death resulted. Calf 115 (Holstein), 80 days of age was operated upon in the same x^ay, both the cervical and thoracic thymus being removed. Two transverse holes were drilled in the bony sternum in each of the first and second intercostal spaces.

A platinum x^ire was passed from right to. left through one of the holes, back through the other, twisted, and cut. The same was done in the other intercostal space. The continu­ ous suture of the cervical muscles was laid running caudad from the cranial end of the incision. Before the final stitch x^as emplaced, a tube was passed into the thorax through the thoracic inlet and the resuscitator was stopped.

The thorax was compressed manually to express any air en­ trapped within, and the suture was completed and tied. If the animal was respiring satisfactorily, the resuscitator was disconnected and the skin closed as described previously. If not, resuscitation was maintained as indicated. Recovery from anesthesia x^ras rapid and complete.

Animals Hp (Hereford), 129 days of age; Sp (Shorthorn),

£3 days of age; and Pp (Holstein, 12 days of age, were also operated upon successfully by this technique. In the latter two cases the dose of 7 per cent chloral hydrate was reduced to 120 ml. because the animals were small. EXPERIMENT NO. £

Part 1

The objective of this experiment was to determine the

effects of thymectomy upon the hemogram in rats, alone or in

combination with splenectomy or hypophysectomy, and of thy­ mectomy alone in calves.

Preliminary observations on a group of six rats, three

of which were thymectomized according to the method of

Segaloff (liuip) with Nembutal anesthesia, indicated that significant changes in the hemogram might result from thy­ mectomy. In order to investigate the nature of these changes more rigorously, a larger group of rats of both sexes were

subjected to the same treatment. Thirty-seven rats were

thymectomized at 21 days of age, of which 3k- survived the arbitrary 15>-day postoperative standardization period.

Eight more animals were randomly selected from the same lot

and were left intact as controls. Blood vras withdrawn from

the femoral vein thrice weekly. Bleeding from the tail had been used in the preliminary trial, but observations in keeping with those in the literature (8l) that leucocyte

counts in tail blood were higher than in other portions of the blood led to the change to femoral vein blood letting.

93 9k Each blood sample was observed for hematocrit value, total white blood cell content per cubic millimeter, and percentage

concentration of each major type of leucocyte in the white blood cell population. The observations during the first 15

days after the date of thymectomy in both the operated animals and the intact controls were excluded from the analy­

sis to insure as nearly as possible that any changes observed were the result of thymoprivia rather than surgical stress.

The same animals were splenectomized 2-§- months after

the date of thymectomy. The results of the- analyses of all data collected in the course of this trial are presented in

Table 6 , 7» and 9. The findings' for the post splenectomy period will be discussed later. Attention at this time is being directed to the effects of thymectomy alone as evidenced by the comparisons of treatment for groups 1 and 2, which

includes blood samples from the thyme ctomi zed and control animals, respectively, prior to splenectomy. Observations on the concentrations of white cells other than lymphocytes

and segmented neutrophiles were excluded from analysis for the reason that their relative concentrations x^ere too low

to permit of statistically reliable measurement. On the basis of these tables, it appears that thymectomy under the conditions described resulted in a highly signi­

ficant (p< 0.01) leucopenia (Table 6 ) and a highly signi­

ficant (p< 0.01) lymphopenia (Table 7) with no significant differences in the segmented neutrophile determinations

(Table 8 ), or hematocrit values (Table 9 ). Since the lymphocytes comprise the majority of the leucocytes, the two differences reported here as significant are probably inter­ dependant. The lack of significance in the remaining two comparisons was taken to indicate that no prolonged, non­ specific effects of surgery per se need be considered. 96

Table 6 Effect of Thymectomy on Numbers of Leucocytes Per Cubic Millimeter of Blood Before and ifter Splenectomy

Group Treatment Y

1 Presplenectomy thymectomized 196.7 (WBC x : mm 2 Presplenectomy control 222.1 ii ii 3 Postsplenectomy thymectomized 262.8

it k Postsplenectomy control 295.1

Source of Variation D.P. S.S. M.S. P.

Among Groups 3 563120 187707 28. 78k-::--* Grps. 1/2 vs. 3/k- 1 520610 520610 65.967-:;--

Grp. 1 vs . 2 1 39665 39665 5 .026-;:-::- Grp. 3 v s . ij. 1 2835 2835 0.359

Within Grps. i+Ol 3I6I1615 7892

Total Ij.Ok 3727735

““'Significant at 1 per cent level

"Significant at 5 per cent level Table 7

Effect of Thymectomy on Numbers of Lymphocytes Per Cubic Millimeter Before and After Splenectomy

Group Treatment Y 1 Presplenectomy thymectomized 162.1 (Lymphocytes x 10~^) mm 2 Presplenectomy control 189.0 "

3 Postsplenectomy thymectomized 205. k ii Postsplenectomy control 237.2 "

Source of Variation D.F. S.S. M.S. F.

Among Groups 3 208593 69531 12.357 Grps. 1/2 vs. 3/k 1 11+7182 11+7182 26.156

Grp. 1 vs. 2 1 1+21+31 1+21+31 7.51+1

Grp. 3 vs. k 1 18980 18980 3.373 Within Groups l_i_00 2250752 5627

Total J/0 3 21+5931+5

'“"'“'Significant at 1 per cent level

’“Significant at 5 P©r cent level 98

Table 8 Effect of Thymectomy on Numbers of Segmented Neutrophiles Before and After Splenectomy

Group Treatment Y

1 Presplenectomy thymectomized 31.2 (Seg.Neutr. x 10~*~) mm

2 Presplenectomy control 32.0 ir It 3 Postsplenectomy thymectomized 72.2

1+ Postsplenectomy control £2.0

Source of Variation D.F. S.S. M.S. E.

Among Groups 3 101+390 31+797 29.290-::-* Grps l/2 vs. 3/I4. 1 96702 96702 81.399-”-*

Grp. 1 vs. 2. 1 3k 3k 0.029

Grp. 3 vs. I|. 1 765k 7651+ 6.1+k3*

Within Grps. 1+01 k76353 1188

Tot al l+ol+ 58071+3

'“''“'Significant at 1 per cent level

’“"Significant at 5 per cent level 99

Table 9

Effect of Thymectomy on the Hematocrit Value in Rats Before and After Splenectomy

Group Treatment Y

1 Presplenectomy thymectomized k-7 »k

2 Presplenectomy control lp7.1

3 Postsplenectomy thymectomized 1|.6.8

ij. Postsplenectomy control i+5.5

Source of Variation D.P. S.S. M.S. P.

Among Groups 3 28 1.333

Within Groups 355 7386 21 Total 358 7^-50

“’“Significant at 1 per cent level

“Significant at 5 per cent level 100 Part 2

The effects of splenectomy with, or without thymectomy were studied using the same groups of animals reported in

Tables 6, 7, 8, and 9. It was felt that these effects could better be detected if the comparisons in the analyses of variance were rearranged. The restrlts of this treatment of the data are reported in Tables 10, 11, and 12. Since the among groups mean square for hematocrit value (Table 9) showed no significance, no further treatment of these data was indicated.

As can be seen from these tables, splenectomy was follow­ ed by a highly significant (p< 0 .01) increase in total leuco­ cytes (Table 10) and lymphocytes (Table 11) in all animals and in total segmented neutrophiles in thymectomized animals

(Table 12). Table 12 also shows a postsplenectomy increase in the segmented neutrophile counts of nonthymectomized rats which was significant at the £ per cent level. 101

Table 10 Effect of Splenectomy on Numbers of Leucocytes Per Cubic Millimeter of Blood in Thymectomized and Control Rats

Group Treatment Y

1 Presplenectomy \-2 thymectomized 196.7 (w b c x : mmb

2 Presplenectomy control 222.1 i i

3 Postsplenectomy thymectomized 282.8 it

k Postsplenec t omy control 295.1 ii

Source of Variation D.F. S.S. M.S. F.

Among Groups 3 563110 187707 2 8.781+*-* Grps. 1/3 vs. 2/1+ 1 35508 35508 1+.1+99*' Grp. 1 vs. 3 1 U 2 6 H 9 i+26119 53.99I+-::--* Grp. 2 vs. 1+ 1 ioil+93 1011l93 12.81+0-::-- Within Groups 1+01 3161+615 7892

Total l+ol+ 3727735

‘"'Significant at 1 per cent level

""Significant at 5 per cent level 102

Table 11

Effect of Splenectomy on Numbers of Lymphocytes Per Cubic Millimeter of Blood in Thymectomized and Control Rats

Group Treatment Y

1 Presplenectomy 162.1 (Lymphocytes x 10~2) thymectomized mm3

2 Presplenectomy control 189.0 11

Postsplenectomy 3 11 thymectomized 205.4

k Postsplenectomy control 237.2 11

Source of Variation D.F. S.S. M.S. F.

Among Groups 3 208593 69531 12.357** Grps. 1/3 vs. 2/Ll 1 58002 58002 10.308** Grp. 1 vs. 3 1 106293 106293 18.890** Grp. 2 vs, i). 1 1+1+298 141-298 7 .872-::--::- Within Groups I4.OO 2250752 5627

Total 1+03 21+59314-5

'"'“'Significant at 1 per cent level

“Significant at 5 per cent level Table 12

Effect of Splenectomy on Numbers of Segmented Neutrophiles Per Cubic Millimeter of Blood in Thymectomized and Control Rats

Group Treatment Y

1 Presplenectomy thymectomized 31.2 (Seg.Neutr. x 10~2 mm3

2 Presplenectomy 11 control 32.0

3 Postsplenectomy thyme c t omi z e d 72.2 11

Postsplenectomy k 11 control 52.0

Source of Variation D.F. s.S. M.S. F.

Among Groups 3 10^390 31+797 29.290 Grps. 1/3 vs. 2/1+ 1 1583 1583 1.332 Grp. 1 vs. 3 1 95169 95169 80.109' Grp. 2 vs, | ‘ 1 7638 7638 6.1+29' Within Grps. 1+01 1+76353 1188

Total koi\. 58071+3

Significant at 1 per cent level

'“'Significant at 5 per cent level Part 3

The effect on the hemogram and body weight of thymectomy

in combination x^ith hypophysectomy was compared to the effect

of Ir/pophysectomy using five groups of rats. Group HP con­

sisted of 10 female rats hypophysectomized at 21 days of age

by Hormone Assay Laboratories, Inc., and thymectomized at

38 days of age. Group HM consisted of 10 male rats treated

in the same manner. Group CP consisted of five intact female

rats the same age as those of group HF. Group CM consisted

of five intact male rats of the same age as those in group

HM. Group HC consisted of 10 male rats hypophysectoraized at

21 days of age. Observations for statistical analysis were

compiled from the age of 53 days.

Table 13 reports the results of the statistical analysis

performed on the numbers of leucocytes per cubic millimeter

of blood obtained by sampling all rats every third day during

the period of the experiment. The most important finding in

this case is that thymectomy caused a highly significant

(p<,0.0l) decrease in the total leucocyte counts of hypo-

physectomized animals.

Table lip reports the results of the statistical analysis

performed on the estimated number of lymphocytes per cubic

millimeter of blood in these same experimental groups. It

appears from these findings that thymectomy more than re­ versed the lymphocytosis resulting from hypophysectomy. io£ Table 1^presents the results of the statistical analysis performed on the estimated numbers of segmented neutrophiles per cubic millimeter of blood in these samples. All hypo­ physectomized animals were lower in this respect than the

controls, and the effect of thymectomy on the segmented neu-

trophile count was not significant. Table 16 demonstrates that thymectomy did not significantly alter the decrease in hematocrit value which followed hypophysectomy. The results of the statistical analysis performed on the mean body weights of the animals in the various groups during

the period of the experiment appear in Table 17. Thymectomy was not found to alter the highly significant (p< 0.01) inhibition of grox^th which followed hypophysectomy.

Nine intact male rats and nine rats which were selected randomly from the same tx^o litters and thymectomized at 25>

days of age x^ere used to study the effects of thymectomy on

the blood levels of calcium, cholesterol, potassium, sodium,

chloride, and carbon dioxide. A statistical analysis was performed by the Statistical Laboratory on the levels of

these substances in blood obtained weekly from the femoral veins of these animals after 6£ days of age. The thymecto­ mized animals were found to be significantly lower in blood

calcium and sodium and higher in blood cholesterol. The levels of the other substances did not differ significantly betx*ieen the tx^o groups. 106

Table 13 Effect of Hypophysectomy With Or Without Thymectomy on Numbers of Leucocytes Per Cubic Millimeter of Blood, on Rats

Group Treatment Y

HP Hypophysex / Thymectomized (WBC x : Females 200.0 mm

HM Hypophysex / Thymectomized Males 167.6 ii

CF Intact Females 176.7 ii n CM Intact Males 221.1 n HC Hypophysectomized Males 2i|,0.i>

Source of Variation D.P. s.s. M.S. F.

Among Groups k 98416 2l(.6o6 Il.l81p«:- H vs. C Hypophysex vs. Intact 1 5163 5163 2.347 HF / HM vs. HC 1 I16683 14.6683 21 . 220'*-* HF vs. HM 1 11511 11511 5.232-* CF vs. CM 1 35059 35059 15.936-*-* Within Groups 153 336524 2200

Total 157 434940

'“'"'Significant at 1 per cent level "Significant at 5 pe^ cent level 107

Table llj.

Effect of Hypophysectomy with or Without Thymectomy on Numbers of Lymphocytes Per Cubic Millimeter of Blood in Rats

Group Treatment Y

HP Hypophysex / Thymectomized „ Females 179.7 (Lymphocytes x 10 ) mm'3

HM Hypophysex / Thymectomized . u Males lit-9 • 2 tt CP Intact Females llj-8.0 n CM Intact Males 195.9

HC Hypophysex Males 213.8 tt

Source of Variation D.P. S.S. M.S. P.

Among Groups k 9551i2 23886 15.612-** H vs. C Hypophysex vs. Intact 1 9739 9739 6.365* HP / HM vs. HC 1 35I|-71 35V71 23.189** HP vs. HM 1 9639 9639 6.300-* CP vs. CM Within Groups 152 23251P- 1530 Total 156 328083

'"“Significant at 1 per cent level

'^Significant at 5 per cent level 108

Table 1$

Effect of Hypophysectomy With or Without Thymectomy on Numbers of Segmented Neutrophils Per Oubic Millimeter of Blood in Hats

Group Treatment Y HP Hypophysex / Thymectomized Females 17«1+ (Seg.Heutr. x 10~2) mm3

HM Hypophysex / Thymectomized Males 11+.]+ it

CF Intact Females 26.3 tt

CM Intact Males 21+.9 tt HC Hypophysectomized Males 20.3 n

Source of Variation D.F. S.S. M.S. F.

Among Groups k 2665 666 3.361+- H vs. C Hypophysex vs. Intact 1 2255 2255 11.389' HF / HM v s. HC 1 278 278 l.j+Oli HF vs. HM 1 95 95 0 .1+80 CF vs. CM 1 37 37 0.187 Within Groups 1^2 30081 198

Total 156 3271+6

'"'"'Significant at 1 per cent level

•V. “Significant at 5 pe** cent level 109

Table 16

Effect of Hypophysectomy With or Without Thymectomy on Hematocrit of Rats

Group Treatment Y

HP Hypophysex / Thymectomized Females 1+3.1+ (fo)

II HM Hypophysex / Thymectomized Males kk.Q CP Intact Females 1+6.6 R

II 0M Intact Males 1+7.1

II HC Hypophysectomized Males 1+5.1

Source of Variation D.F. S.S. 1. s. P.

Among Groups If 317 79 3.160* H vs. C Hypophysex vs. Intact 1 259 259 10.360'::": HP / HM vs. HC 1 33 33 1.320 HP vs. HM 1 20 20 .800 CP vs. CM 1 £ 5 .200 Within Groups 151+ 3875 25

Total 158 1+192

‘“"“'Significant at 1 per cent level

'“'Significant at 5 Per cent level 110

Table 17

Effect of Hypophysectomy With or Without Thymectomy on Body Weight of Rats

Group Treatment Y

HP Hypophysex / Thymectomized Females 82.1 (grams)

HM Hypophysex / Thymectomized Males 81.2 I!

OP Intact Females 193.9 !t CM Intact Males 272.1+ it HC Hypophysectomized Males 81+. 8 IT

Source of Variation D.F. S.S. M.S. P.

Among Groups 1+ 9^8690 239672 35>.671- H vs. C Hypophysex vs. Intact 1 8£o£67 8£o£67 126.912' HP / HM vs. HC 1 l£8 l£8 .021+ HP vs. HM 1 8 8 .001 CF vs. CM 1 1079^7 1079^7 16.108 Within Groups lh$ 971789 6702

Total 11+9 19302+^9

’"""'Significant at 1 per cent level

’"'Significant at per cent level Ill

Part L(.

The testes, spleens, and adrenals of the animals killed for histological observation were weighed after formalin fixation. Figure 11 shows the testicular and body weights of the animals in each group as a function of age. In these animals the weights of the testes were consistently higher in the thymectomized than the control rats during the period from [j.0 to 55 days of age. Figure 12 compares the weights of the spleens of the animals in these groups, indicating a brief but sharp in­ crease in splenic weight in the thymectomized animals immediately after thymectomy, after which the two groups were virtually identical. Figure 13 illustrates the comparisons made between the adrenal weights and body weights of the txzo groups of rats.

In this case a decline in adrenal and body weights was noted in the thymectomized animals immediately after the operation, after which the patterns of adrenal weights roughly followed those of the body weights throughout the period of the experiment. 112

Pig. 11 Testicular and body weights of thymectomized and intact rats as a function of age

Pig. 12 Spleen and body weights of thymectomized and intact rats as a function of age TESTES WEIGHT TESTES WEIGHT 1 3 0 5 TESTES WEIGHT vs BODY WEIGHT 1500 1500 A 180 200 THYMECTOMIZED / THYMECTOMIZED 1600 - CONTROL '/ 190 14002! l«n CONTROL / 1400fi BODY WEIGHT / 170 . _■ UJ - BODY WEIGHT i 15001- 180. 1 In 13002 / i 16Q i ««i /I ll= v I M00{= 17Q I /: W 1200 ^ raooS V■ A / /: 150. 1 in 1 5 1 .--V / i i w- 1.5005 // 16Q 1.100 < iio o 2 14Q // /\/ 12Col ISO I V 1® 1JOOO 1000 I3Q 1 : 1300 I4Q / 1 // .900 / // 900 / 1 / 120 r/ 1 / 1 / 1000 \ ' I 130 \\ I' \ / V 6 0 0 / s IIO. 1 1/ goo 1 2 0 TOO / \/ IOQ 1 1 600 11Q I/ \l "T 600 S > <5Q 1 V *? 5 2 300 ./ JOO SCO \t ■* 500X f • 0 u*.60 1 .// 2 2 /*•: 1 .600 v GI (6 9Q 400 t 1 ' i I I 'I ■ lr> I1* I...... 400 DAYS 7Q 1 4 36 38 40 42 44 46 46 50 52 54 56 500 80 34 36 38 40 42 44 46 40 50 52 54 56 1 400 DAYS 70 i I ■ I ■ I ■ I : "I ■ I ' I ■ I ■ I ■ I 1 I 34 56 38 40 42 44 46 48 50 52 54 5* 900 SPLEEN WEIGHT 1COO S 7 2 . 0 1 ••• THYMECTOMIZED 190 SPLEEN WEIGHT v» BO D Y W EIGH T / 190 650$ -CONTROL **?z. i SPLEEN WEIGHTv» BODYWEIGHT ■ <0 1 CONTROL «JS THYMECTOMIZED 16Q 600 BODYWEIGHT 18Q ■?ii 19sH - BODYWEIGHT k q K: 750^/: # / * / 170 TSol / I7Q I - 1 TOO ; 16Q *»s> I6Q 1 I llT) 650 : 'SI / \ I 150 I5Q 1 : 1 * v * r 600 | 600 I 140 600$ j4Q 1 : 1 I 550 / 150 550 130 *?/ ,/ft Ml 1 ? 500: 1 2 0 A-, 120. *W 1 i 450/ 450/ tp n o 450 / / /V i a O 1; / / 1! o / / s / 4 0 | IOQ 400 / IOQ ■*¥ 5 5 V 350 / v 9 0 351? / / 9Q 1 / I / 2 2 1 / iiGi U> 300 \ / 6 0 6Q 113 1 T M ^ / / X , y _ 250 DAYS 3S 7a 250 DAYS H 7 0 250 W- V T" I "fi I I M 94363840424446 48 50 5254 56 56 34 36 36 40 42 44 46 46 50 52 545658 34 36 36 40 42 44 46 48 50 52 54 56 56 Fig. 13 Adrenals and body weights of thymectomized and intact rats as a function of age 024 ADRENAL WEIGHT 023 THYMECTOMIZED h CONTROL S ~ \ Ii 022 // \i\ Hi 021 < Z I i / 020 UJ a I i i / 019 o il ;/ < I 025- ADRENAL WEIGHT v s . BODYWEIGHT b 210 CIS n v " .. 024- THYMECTOMIZED 200 017 < jl ------BODYWEIGHT ct / 023 190 016 o / V 022 - 160 015 i I OH 021 - < 170 Z /i!S♦ ® 11 0 2 0 - UJ : » i: 160 013 cc IS'! 019- a : ^: 130 025 ADRENAL WEIGHT vs. BODY WEIGHT 2 1 0 012 < s * : 018 in 140 024------CONTROL 200 011 i i i i™ i i i i i i‘ -i 5 BODYWEIGHT { 34 36 50 40 4Z 44 46 -46 30 52. 34 56 3B01T- a< : 130 023- 190 o 016- 120 0 2 2 - / 160 015 110 D21' / 170 / 014-1 1O0 020 / "1 160 / 013 9 0 019 / I 150 012 80 Ol© "V / 140 / \J 011 7 0 017 \ _/ 130 31 36 3© 40 42 44 46 48 50 52 34 5657 / r 016- / V 120 015- 9o no 014 100 013 H * 2 9 0 012 Ciu> 8 0 3 Oil —I------r — I- 11 i I i i i i i 70 34 36 30 40 4 2 44 46 48 50 S2 51 56 50 t-* H VA 116 Part 5

It should be included in these observations that an in­ creased number of eosinophiles and a temporary appearance of plasma cells and of nucleated red blood cells were seen in the blood samples from thymectomized rats. These findings were not treated statistically because the observations were rather qualitative than quantitative in nature. In the thymectomized rats it appeared that the percent­ age of eosinophiles in the blood was higher compared to that In the intact and sham operated animals. On the other hand, plasma cells were present beginning 18 to 22 days after thy­ mectomy and ranged between 1 and 6 per cent. In some individ­ ual cases these cells comprised 10 per cent of the hemogram.

These observations are in agreement with the reports of

Bomskov (16) In guinea pigs and rat-s and of Comsa (30) in guinea pigs.

A group of 72 male rats of approximately the same weight and age were used for histological observations and organ xtfeight determinations. Half of these, or 36 rats, were thy­ mectomized at the age of 21 days; and 36 rats served as con­ trols. The animals were killed with ether and the spleen, bone marrow, cervical lymph node, adrenal, kidney, liver, heart, and thyroid were fixed in Bouin's fixative cut at 6 micra, and stained with hematoxylin and eosin at 3, 5 , 10,

12, 1^, 18, 20, 21, 22, 23, 2^, 28, 30, 33, 35, i|-0, Ll5 and 117 00 days after the operation (2 thymectomized rats and 2 con- tols on each occasion).

Results

During the first few days following thymectomy the lymphocytes increased in number in the spleen and in the lymph nodes, but the increase was slight, if present, in the bone marrow.

On the fifth day and to a greater extent after the tenth day post-thymectomy, the number of lymphocytes had decreased slowly in the red pulp of the spleen, particularly in the perifollicular regions. The follicles were somewhat smaller compared to the controls. The eosinophils were also decreased in number in the spleen. Megakaryocytes were few in the spleens of thymectomized animals compared to those of controls.

The numbers and sizes of the cortical nodules in the lymph nodes of the newly thymectomized rats were increased compared to those in the controls. The nodules of the corti­ cal substance were often very close together or even fused, increased in size up to approximately 2 to 3 and at times were seen close to the medulla; whereas in the controls the nodules were few and widely separated and did not exceed 1 mm in diameter. Mitotic figures in the lymphocytopoetic centers of the thymectomized rats were relatively numerous during 118 '

Pig. II4. Cervical lymph, node of a thymectomized littermate rat 22 days following operation H and E. x 100

Pig. 15 Cervical lymph node of an intact littermate of the same age as above, H and E. x 100 » W ^ c , i §

& * & $ & & l&flUiffiSRt* U * s i l !&V$\V £« W ?~ P i3fc 120

Fig. 16 Bone marrow of an thymectomized rat 22 days following operation. H and E. x li75>

Fig. 17 Bone marrow of the above intact littermate of the same age

122 the first few days and accumulation of small lymphocytes around these centers was heavy. The lymph node medulla in thymectomized animals was packed with large lymphocytes and plasma cells, a phenomenon not observed in the controls.

The numbers and the sizes of the nodules were altered remark­ ably 10 days after thymectomy. The mitotic activity of the centers decreased, as well as the accumulation of small lymphocytes around the centers. The medulla began to show a depletion of lymphocytes. The vessels were more prominent, and plasma cells were observed between the large lymphocytes.

The hypertrophy and hyperplasia of the cortex persisted in most of the thymectomized animals for 10 to 13 days compared to the controls, and after this period a gradual diminution was observed. This decrease might result from a retardation of the production of lymphocytes and eosinophiles.

The influence of the adrenal cortex upon the thymus is well known, but little has been reported about the influence of the thymus upon the adrenal cortex. Thymectomy resulted in significant changes of the adrenal. These changes are expressed in the hypertrophy of the zona fasciculate and atrophy of the zona glomerulosa. The differences were great­ est between 20 days and 21+ days after the operation and gradually became less marked thereafter. They provide evi­ dence of a transitory stimulation of the adrenal cortex follov/ing thymectomy. 123

Pig. 18 Adrenal cortex of a thymectomized rat 22 days following operation. H and E. x ilj.7^

Pig. 19 Adrenal cortex of an intact littermate of the same age as above. H and E. x I4.7!?

125 In the liver no changes were observed other than a periportal infiltration of lymphocytes and plasmacytes and in some cases liver cell degeneration in thymectomized rats

compared to intact animals.

In the kidneys infiltration of lymphocytes and plasma ce.lls was observed near the afferent arteries and inside

capsular epithelium of the glomeruli. .In some cases degen­ eration of the renal corpuscles was also observed.

The histological appearance of the thyroid showed a slight stimulation of the gland within five days after thy­ mectomy. In the long-term experiments, a somexvhat puzzling histological picture was found. As a rule, the appearance was that of complete functional rest. In single follicles, however, signs of stimulation still persisted. Elsewhere, degenerative changes could be observed, such as desquamation of the epithelium into the follicular lumina or small process­ es growing from the follicular cell walls.

Thymectomy in the young rat has not been shown to effect the maturation of the testes (129). In these studies, how­ ever, it was found that the diameter of the seminiferous tubules in the 5>0-day-old thymectomized male rat measured 122 to 155>* and spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids, and spermia were present.

The diameters of the seminiferous tubules of a littermate control ranged from 102 to ll).6, and spermatogenesis had 126

Fig. 20 Testis of a thymectomized. rat £0 hays following operation. H and E. x [{.75

Fig. 21 Testis of an intact littermate rat of the same age as above. H and E. x 1|_75>

128

Fig. 22 Kidney of a thymectomized rat of 55 days following operation. H and E. x i_j-7

Fig. 23 Kidney of an intact littermate rat of the same age as above. H and E. x 1+75

Pig. 2I4. Spleen of a thymectomized rat 28 days following operation. H and E. x Lj_75>

Pig. 2J? Spleen of an intact littermate rat of the same age as above. H and E. x Lj.75 m

J&<%^v^LfJSJ l*Sfe *3p X

L. « 13 132

Pig. 26 Liver of a thymectomized rat 28 days following operation. H and E. x

Pig. 27 Liver of our intact littermate rat of the same age as above. H and E. x ij.75

13k proceeded only to the spermatid stage. The interstitial cells and connective tissue were both more abundant in the sections from the thymectomized animals. In previous experiments, it was observed that thymecto­ mized female rats had litters at 82 to 85 days of age, and appropriate controls only at 106 to 110 days of age when continuously housed with males. It was also observed that the period from first to second parturition was from lj.0 to

60 days longer in the controls than in the thymectomized females. The litter size varied between 9 and 19 in the thymectomized and between 10 and 16 in the control animals.

No histological differences following splenectomy or hypophysectomy were observed between rats which were and were not thymectomized. 135 Part 6

It was Indicated, in the literature review that changes were observed in the endocrine system after thymectomy.

Bomskov and Sladovic (21 ) found that these disturbances are specially pronounced in carbohydrate metabolism but that the general metabolism is changed too. Comsa (33) observed an increased activity of the thyroid gland in thymectomized infantile guinea pigs. Comsa (33)> Bomskov and Holscher

(16), Asher ejt aJL. (7 ) found that thymectomy caused Imbal­ ances in calcium metabolism and development of the skeleton.

On the basis of these references and of histological observations, it was important to check such blood compon­ ents as glucose, urea nitrogen, cholesterol, calcium, sodium, potassium, and chloride in calves.

Pour calves of various breeds were operated upon at three months of age, and each was paired with another calf of the same breed and age which had not been operated upon. Each pair was reared together under as nearly identical conditions as possible. Blood samples were collected from all animals twice weekly for hemograms studies, and once weekly for the determination of blood glucose, urea nitrogen, and choles­ terol by the methods quoted in Experiment 2. A statistical analysis performed on the data compiled during the first three months after thymectomy indicated that there were no 136 significant differences between the groups in total leuco­

cyte, lymphocyte, or segmented neutrophile counts per cubic

millimeter of blood, nor in the blood glucose, urea nitrogen,

or cholesterol. A significantly higher mean hematocrit value

was found in the case of one control animal when compared to its thymectomized counterpart, but this was probably due

to hemoconcentration because of dehydration from chronic

diarrhea.

The same observations were continued for another three months, with additional determinations being made of the blood levels of total protein, calcium, potassium, sodium,

and chloride. For this period, the thymectomized animals were consistently lower than the control calves in total

white blood cells and lymphocytes per cubic millimeter of

blood and in blood urea nitrogen. The variables studied did

not differ consistently between the two groups.

Calves Hp and S-^, described in experiment Ip, and Hp* and

Sg, intact control animals paired by breed and age with the

former two animals, were killed when 312, 229, 3^4-9, and 20$ days of age respectively. The adrenals, cervical lymph node,

spleen, lungs, liver, testes, ovary and thymus, when present,

were fixed in Bouin's fixative and cut at Two slides

were stained with hematoxylin and eosin and two in Crossman’s

Modification of Mallory's Triple Stain. 137 The organs were compared on the same basis as the calves were paired in the experiment. The adrenal of -Calf Hp was contrasted with the adrenal of Calf Hp, and it was found that the three layers of the cortex were different in width. The zona glomerulosa in Hp was approximately 220/*-, as opposed to

300^ in B-2 , and consisted of short columnar cells forming arches. The nuclei were stained deeply and basophilic granu­ les were pronounced in the cytoplasm. In H2 the columnar cells were somewhat taller and ovoid in configuration rather than in columns. These cells did not show granulation in the cytoplasm. The zona fasciculeta in Calf Hp was approximately 18^0/^ in width and the cells appeared to be larger. The sinuses between them i^rere extended. The radial orientation of these cords was not as clear as in Calf H2. The zona fasciculata in Calf H2 was approximately 1700/* in width. The width of the zona reticularis in Calf Hp was 68/* , whereas in Calf H2 it ^^as 75>/4' • The medulla of Hp was 2800/*- in width, compared to 315>0A in Calf H2. In H2, the vascularization was more prominent than in the thymectomized Calf Hp,

The same measurements were made and compared in Calf Si and S2 , and it was found that zona glomerulosa was 150/*- in thymectomized Si and 178/* in Calf S2 . The zona fasciculata was 15>00./*- in Sp and 13^0/*- in S2 . The zona reticularis in

Sp -was ^6y/*" a nd 60/4* in Calf S£, There were also small 138 differences between the medulla of Sp, which was 110CL/*'1 , and

S2 which was 1200*/* in diameter.

Observations were also made on the cervical lymph nodes, and it was found that in Calf Hp numerous lymphocytopoietic centers 675-975v/4‘ in diameter were found, with cells of 7*5>- to 8. and only a few mitotic figures present. No ring of small lymphocytes was found accummulated around these centers. In the control calf, H2, the lymphocytopoietic centers were few, and they were of 330 to 6l£/*~ in diameter.

They were composed of smaller cells of 3>k to 5.6/^ in size and more frequent mitotic figures. Small lymphocytes were accumulated, forming a many-layered circle around the centers. Observation of the thymus showed that the 7 gin- of thymic tissue in the neck region after operation in Calf Hp did not differ to any great extent histologically from the thymus of the control. The cortex was in breadth and the medulla

£90/*, whereas in Calf H2, the cortex was 600/4 and the medulla in diameter. There were small differences observed in the number and size of so-called "myoid" cells present in the thymic medulla of both calves. The size was

12.8 to 17.0/ in Hp and 12.3 to l£.3/* in ^2* Hassail's bodies were more numerous in Calf Hp than in H2 . The eosinophiles around the vessels and in the septa and medulla

In Hp, were less numerous than in calf H2. 139 The findings in the cervical lymph nodes were very

similar in Calf Si to those in Hi and in S2 to those in H2 . In comparing the spleens, special attention was directed to

the relative volumes of the white pulp and red pulp. It was

found that the white pulp in Hi was 278 S*- in diameter and

227/^ in H2 as an average. The cell components x^ere between

6.8 and 10.2 S’ in diameter and only a few mitotic figures were observed. Small lymphocytes x^ere accumulated round the

lymphocytopoietic centers in the control animal but not in the thymectomized animal. The red pulp in Calf H2 was less

dense as compared to that in the thymectomized calf. Mega­ karyocytes were more frequent in the thymectomized calf and rarer in the control. The findings in Calves Sp and S2 were

very similar to those just described for their older counter­ parts .

No differences were observed between the thymectomized and control animals comparing the histological findings in

the lungs, liver and kidneys. The most pronounced differ­

ence was observed between the testes of calves Sp (thymec­

tomized) and S2 (control). The diameters of the seminiferous

tubules in the Sp were between 136 and 170/*" and spermato­ gonia as well as primary spermatocytes were present in large

numbers. The sizes of these primary spermatocytes were

between 6.8 and 10. 2r . The lxmens of the seminiferous l[|-0 tubules were open and interstitial cells were present, im­ bedded in the loose connective'tissue around the tubules.

In the control Calf S2 the tubules were imbedded in more connective tissue and fewer interstitial cells x^ere seen. The lumens of the seminiferous tubules were closed, and only some spermatogenisis was observed.

The ovaries of Calves H]_ and H2 were also compared.

It was found in both that the secondary follicles varied between 136 and ij.76/4- in diameter and that primary follicles were present. Corpora lutea were observed in the ovaries of both of these animals. Several organs of these four animals were weighed at necropsy. The findings are reported in Tables 18 and 20.

Since the body weight of each of the thymectomized animals is lower than .that of its intact counterpart, it is diffi­ cult to attach any significance to the observation that all of the organ weights recorded are also loiter in the thymec­ tomized animals. hone of the differences in organ weights appear strikingly disproportionate to the differences in body weights. The body weights of all of the thymectomized and control calves as a function of time are shown In Figure 30. It can be seen that in calves ll£ and Hi)., which were very nearly the same age, the thymectomized calf Ilf? was consistently Ikl

Pig. 28 Testis of the thymectomized Calf horn May 2li, 19^9 •

Pig. 29 Testis of the intact Galf Sp born June 17, 19£9 lower in body weight. In the Hi - S2 pair, interpretation is raade difficult by the persistence of a small quantity of cervical thymic tissue in Hp after operation. When adjusted for age, the differences in the x^eights of these animals are probably not significant. Calf Sp which was completely thymectomized, was lower in body weight than the intact S2 to an extent which was probably greater than could be explain­ ed on the basis of the 2ii-day difference in age. Thyme c tom- ized calf Pp, was higher in body weight than its intact counterpart, P2. The intact animal may have been stunted by chronic diarrhea and an umbilical Infection. Table 18

Organ Weights .n Thymectomized and Intact Calves On January 8, I960

Hl, Lorn 3/2/59, Hp, born l/2k/59, (thymect.) (control)

Kidney (right) 272.1 gms 3l|0.2 gms Kidney (left) 226.8 " 338.0 ;; Spleen 317.5 " 5 ^ . 3 Liver 3,265.9 " 3 ,7 1 9 . 5 " Ovaries 5.1 " 5.5 " Adrenals 5.2 " Thymus 7.0 " 1+53 Total Body Weight 552 lb 6k2 lb

Sp, born 5/2k/59, S2, born 6/17/59 (thymect.) (control)

Kidney (right) 226.8 gms 272.1 gms Kidney (left) l8l.k " 267.0 ” Spleen 228.8 " 317.5 ” Liver 2,268.0 " 2,9k8.1j_ n Adrenals k.ij. '' 6.1 ” Testis 58.2 " 60.1 Thymus 1+78.3

Total Body Weight 297 lb 1+65 lb 145

Pig. 30 Comparison of weights between calves ll£ and 111}.; % and H2 ; Sp and S2 J Pi and P2 ...... Mr I .• ...... ■■■■■ - . - i p o u n d s i i rr* \\ p o u n d s . : _y>*-THYMECTOMY I I / \ I ! -THYMECTOMY

M V A A 3 s ss

§

POUNDS ■THYMECTOMY

868886 40 40 VV-THYMECTOMY POUNDS j \ s ! \\ J>-3D ,£> A A 5;' 9 CP o o !!'*£ JP u, i3 •o A S i «

91]! EXPERIMENT NO. 6

It has been established (f. Experiment No. 5) that thymectomy causes leukopenia and. lymphopenia in rats and calves. Consequently a properly prepared thymic extract may have the ability to reverse these conditions. The ob­ jective of this experiment was to prepare homologous and heterologous thymic extracts and to discover whether or not the extracts are active, with the hemogram as the criterion. It was also desired to ascertain the smallest dose which had sufficient potency to cause detectable changes in the hemogram.

Material and Methods

With the help of Dr. R.. 0. Moore, of the Department of

Agricultural Biochemistry, thymic tissue of rats and calves was fractionated with Spinco Ultra centrifuge Model L., and the fractions were injected into thymectomized and intact rats.

Extract I_ - Thymi were removed aseptically from 80 young rats, and on the same day the tissue was dispersed in 0.15 M

lij-7 li±8

Phosphate Buffer, 2 ml. per gram of tissue, using the Waring

Blendor. It was stirred and centrifuged at 30,000 r.p.m. for 30 minutes. A lipid fraction was separated from the aqueous fraction by this method; and the clearly different­ iated sediment, which contained the mitochondria and micro- somes, was discarded. The lipid fraction was homogenized with 5 ml. of 95 pe*5 cent ethyl alcohol per 100 ml. of lipid, and the aqueous fraction was to be used as separated.

Because of the small quantity available, 0.5 ml. of the lipid extract was injected into only three animals. None of the aqueous extract was employed. No conclusions could be draxwn on the basis of results from only three animals, so it x-ras decided to repeat the extraction with 120 rat thymi and to prepare similar extracts of calf thymus. The relative number of lymphocytes and reticular cells in the thymus varies with the age and condition of the animal. Calf thymus was procured from commercial slaughter houses. It was found that variations in this tissue effected the yield of the ex­ tract. In general, a larger quantity of extract is obtained per unit quantity of tissue from the thymi of young, weanling calves than can be obtained from the thymi of older cattle.

The thymi of three young calves were collected and freed of blood, fat, connective tissue and vessels as completely as possible; and extracts were prepared by the same method as was described for extraction of rat thymi. 124-9 Bjp-as say The aqueous and lipid calf thymus extracts described, as well as a buffer control, were injected into four 117 day-old rats each in a balanced design which provided that each substance would be injected into one or more rats which had been thymectomized 80 days previously and one or more rats which had been sham-operated on the date of thymectomy

These animals were injected xnith 0.25 ml. of aqueous extract,

0 .2^ ml. of lipid extract, or 0.25 ml. of saline as required by the experimental design and were bled thrice weekly.

Observations of the total numbers of leucocytes per cubic millimeter and the percentages thereof of lymphocytes and segmented neutrophiles were recorded for each sample. A preliminary analysis was performed by the Statistics

Laboratory of the percentages of lymphocytes and segmented neutrophiles. The results were taken to indicate that the blood of all thymectomized rats had contained a lower per­ centage of lymphocytes and a higher percentage of segmented ne^^trophiles than that of the sham-operated animals, both before and after the injection. These findings vjere inter­ preted to reflect the lymphopenia already reported as an effect of thymectomy, as well as an absence of significant effect of the injected extracts. By x>ray of confirmation, the percentage of lymphocytes was multiplied by the number of leucocytes per cubic millimeter in each blood sample and was i£o recorded as an estimate of the number of lymphocytes per cubic millimeter of blood. The results of the analysis of these data indicated that the blood samples from the sham- operated animals prior to the injections contained more total lymphocytes per cubic millimeter than those of the thymecto­ mized rats, that both extracts had further increased the lymphocyte concentrations in the blood of the sham-operated animals, and -that the lipid extract had somewhat enhanced the differences in this respect between the sham-operated and thymectomized rats into which it was injected.

Bioassay of the rat thymic extract was carried out using the same experimental design as that described for the calf thymic extract. The first analysis of the data was performed by the Statistics Laboratory on the basis of the percentages of segmented neutrophiles and lymphocytes in the blood samples. These figures were not found to differ significant­ ly between groups during any stage of the experiment. Since it was thought that the use of percentages in the first analy­ sis might have masked real differences, a further analysis was performed, based on the estimated numbers of lymphocytes per cubic millimeter in the blood samples. The pattern of significant (p< 0.05) differences found indicated that the aqueous fraction may have caused increased lymphocyte counts in sham-operated rats and decreased lymphocyte counts in thymectomized rats. The apparent effect of the lipid frac­ tion was not consistent.

Preparation and Partial Identification of Thymus Extracts

Two thymic extracts were to be prepared which would be more highly purified than those described previously. The extractions were performed s.o as to obtain by means of one a portion of the tissue components soluble in aqueous solvents and from the other a part of the moiety soluble in organic solvents. One-dimensional paper electrophoresis was selected as a means of determining the homogeneity of these extracts, a complete qualitative analysis of their composition being beyond the scope of this study.

Extract II was prepared by the method of Cornsa and

Bezssonoff (3^)» Tb-e preparation includes three main steps: isoelectric precipitation, dialysis, and extraction. The method of preparation of the extract resembles that of other investigators (77, 115>) though the product is dissimilar in its behavior. Most of these investigators state that their extracts are peptides, perhaps products of the hydrolysis of thymus histone. Dubos and Hirsch (1|_2) reported an isoelec­ tric point between 10 and 11 for their extract, while that of the extract prepared for this study was between 6.1 and

6.5. Ihe method of preparation of extract III would appear to result In a higher degree of purification than those 152 reported elsewhere. The ammonia content was totally washed out. The extract was electrophoretically homogeneous within the pH-range tested, and was soluble in water at acid and neutral reactions. No phosphorous could be detected by the method of Beveridge and Johnson (11), indicating that there were no nucleic acids present.

No precipitate was formed with phosphotungstic acid or x*jith trichloracetic acid, showing that no whole protein mole­ cules remained. The non-ammonia nitrogen content of the extract xvas 13.51 per cent. The carbohydrate content of the extract was not determined.

A number of electrophoretic measurements were made with a Heco E-800-2 electrophoresis apparatus. In all cases only one fraction \-j s l s observed, which was compared on one occasion with thymine hydrochloride.

Two strips of paper were placed on the electrophoresis apparatus. On one of them the extract and the other one thymine hydrochloride was placed. The Merck thymine hydro­ chloride did not appear to be as homogeneous electrophoret­ ically as the extract. Three determinations were made, one each at pH 2.15, pH 3.7, ard pH 8.6. The different pH's were used because L. Hnilica and S. Ilupka (77) encountered difficulties at certain pH values, while analyzing thymus historic derivatives electrophoretically. In all three determinations only one fraction was observed. 153 Bjpas say

The solid, purified extract in powdered form was in­ corporated into paraffin pellets in the amount of g per pellet. These pellets were implanted subcutaneously into ten of eighteen male rats. Five rats with and five without pellets had been thymectomized 16 days previously, at 25 days of age. The results of three bleedings over a period of eight days indicated that the extract might have depressed the numbers of lymphocytes per cubic millimeter of blood of thymectomized animals to an even greater extent than thymec­ tomy did. Other differences observed were typical of the results of thymectomy as reported previously.

Extract III

Five hundred gms of calf thymic tissue were ground and made into a suspension in a Waring Blendor. The suspension was mixed with a 1000 mis. of 2:1 mixture of chloroform and methanol. The container was shaken for 30 minutes, and the two layers were separated. There was an interfacial layer, possibly composed of lipoprotein. The tissue residue was extracted with 1000 mis. of 2:1:1 ether; ethanol: n-hexane.

The second extract was mixed with the first one and concen­ trated by vacuum evaporation at 30° 0. The concentration was

10-fold. The concentrate was made up to 75>0 ml. with chloro­ form and precipitated with s.cetone, the precipitation being 15k repeated twice. After the last precipitation, the residue was dissolved in 250 ml. of ether and held at 3°^ overnight.

There was no precipitation. The ether was then evaporated and the residue stored in a closed container at -5°C.

Two electromatograms were prepared, one of the extract before and one,after the precipitation. The first electro- matogram (before acetone precipitation) contained seven distinguishable ninhydrin-reactive fractions at pH 8.6. In the second electromatogram most of the residue had dis­ appeared. Fraction No. 5 had been concentrated, as had parts of No. 7, No. 1, and No. 2. No. 1± was completely eliminated, showing that some of the neutral (acetone-soluble) lipids contained amino nitrogen, although the major portion consisted of phospholipids.

The final extract contained 5.12 per cent phosphorus and

1.56 per cent nitrogen Cphosphorus was determined according to Beveridge and Johnson (11)D. A 1-gram portion of the extract was hydrolized with potassium hydroxide. After hy-. drolysis 16.2 per cent of the extract was insoluble in water. All above experiments and the method of preparation indicate that the major constituents of the extract are phospholipids.

Bioassay

The extract was prepared for administration in Bacto-

Adjuvant Incomplete (Freund) at a concentration of 1.5 raicrograms per 0.1 ml. iSS Bioassay of the calf thymic extract described was -under­ taker; usin^ three groups of four male littermate rats, two in

each group having been thymectomized six days prior to treat­ ment. In each group one thymectomized and one control rat

rtfere injected with thymic extract, the levels of administra­

tion being 0.2, 0.3* and 0.1). ml. per animal per injection, respectively. Injections were made at 6, 8, and 10 days

after thymectomy, and a total of eight sets of blood samples

were taken In the course of the study. The data for total

leucocytes, lymphocytes, and segmented neutrophiles per cubic millimeter of blood were compared but not analyzed statist­

ically, since inspection indicated the observations only

tended to confirm previous findings on the effects of thymec­ tomy, without'any predictable or consistent pattern of find­

ings with respect to the extract injections. EXPERIMENT NO. 7

Objective

The objectives of this experiment vxere to determine the effect of thymectomy upon irradiated rats through: (a) esta­ blishing the time at which thymectomy may have an effect, (b) use of 100 gm rats receiving 800 r, (c) use of 50 gm rats receiving 900 r.

Part I

The thymus reacts very sharply to various stimuli, in­ cluding toxin and starvation, and loses its lymphocytes

(Selye (ll+5)). Kaplan ejt al. (86) have noted a loss in size or weight of the thymus as an effect of x-irradiation. In­ vestigators are agreed that this loss in x-zeight of the organ may result primarily from the death of the cortical thymo­ cytes. Dubois and Petersen (L}.l) showed that the alteration in enzyme activity was reversible x^ith the rate of reversal depending upon the dose of x-ray. Within the dose range of

25-1+00 r the amount of Increase in the adenosine triphospha­ tase, the activity of the thymus gland of rats was directly related to the dose of x-ray administered. Smith (l5l) showed that in the normal thymus only a few reticular cells 156 15>7 gave an acid phosphatase positive reaction, whereas after two hours of irradiation the reticular cells stained deeply with

the acid phosphatase technic.

The present study is primarily concerned with discover­ ing what effect thymectomy has in the rat after whole body exposure to massive doses of radiation.

Material and Methods

Sixteen Sprague-Daitfley r*ats of both sexes, weighing

approximately 90-100 gm each were irradiated with 800 r for 20 minutes using a G.E. Maximar 200 kvp unit. HVL was Lj_.0 mm Al; Ivvp. was ll(.0; Ma 10; and dose rate was i^l.O r/min. This equipment was used and the same procedure followed in

all irradiations performed. During x-ray exposure the rats were placed in individual square-shaped plastic holders. The usual procedure was to expose four experimental and four con­

trol rats simultaneously. After treatment, the rats were hovised in metal cages, four to a cage. On the fifth day

after irradiation, two animals from each cage were thymec­ tomized. The rats were observed and weighed at weekly

intervals. Tap water and Purina Laboratory Chow were avail­ able ad libidum. A thirty-day mortality was used as a

criterion for evaluation. 158

Results Of the 16 rats, the first to die were the eight controls two on 5th, three on 7th, three on 12th day whereas four thymectomized rats died on 21, 26, 28, and 30 days. Pour thymectomized rats are still living and in good health up to the present time. There are three males and one female.

All were placed in a single cage. There is no litter from this female up to the present time. After observing these results two groups of twenty male rats each were exposed. Each rat weighing approximately 50 gms was exposed to 900 r of x-ray irradiation for 20 minutes.

All the control rats died before the thymectomized animals.

Twenty-five per cent of the thymectomized rats survived more than 30 days; they are still living. Twenty rats were thy­ mectomized on the fifth day and 20 served as controls. All the animals were treated in the same manner as the first group of 16 rats.

Part II

Among the measures currently available for the treatment of animals exposed to lethal whole body x-irradiation the only injectable preparations which afford post-protection against mortality appear to be bone marroxtf suspensions, spleen homogenates and antibiotics. In the present experiment 1S9 the objective was to discover the effects of partially puri­ fied calf thymic extracts.

Materials and Methods

There were four groups of rats employed in this study.

Each rat weighed approximately £0 gms. The first three groups were of the Sprague-Dawley strain bred in the Depart­ ment of Veterinary Anatomy: one group had seven littermates of both sexes; one had nine littermates of both sexes; and one had 12 littermates of both sexes. The fourth group was a Hooded strain and had four littermates of both sexes.

All the rats ivere irradiated with 900 r for 20 minutes.

After treatment- the rats were housed in metal cages. The first two groups were left with their mothers; the last two groups were separated immediately after irradiation and divided four to a cage.

In the first group of seven animals two rats were in­ jected with daily doses of 0.1 cc (15/ig) of water soluble calf thymic extract (described in the next experiment) subcutaneously for five days; two were injected with daily doses of 0.1 cc (l5/*g) of a lipid fraction of thymic extract intramuscularly for five days; and three animals served as controls.

Using the same doses, a second group of nine animals received water soluble calf thymic extract; three animals 160 received a lipid fraction and three animals served as con­ trols. The last two groups were similarly divided and treated.

Results The animals injected with the lipid fraction all died starting nine days after irradiation. The rats injected with water soluble calf thymic extract died on 10, 11, 12,

and 16 days following treatment. The control animals also

died on 10, 11, 12, and 16 days. None of the animals

survived for 30 days. EXPERIMENT NO. 8

Objective

This experiment was designed to establish if thymectomy has any effect on implanted epitheloid carcinoma in rats, and to establish if thymic extracts have any effect on these tumors.

Part I

Toolan (162) reported that human neoplasm, implanted in the form of cell suspension from five mice, proliferated extensively for 8-10 days in the subcutaneous tissues of young irradiated rats (heterologous hosts) before changes associated with regression occurred. Human neoplasms did not survive in non-irradiated animals. The percentage of

"takes" and proliferation of cancer cells was considerably greater in x-irradiated weanling rats treated with cortisone

as compared to rats not treated with cortisone.

Histological examination has established that after whole body irradiation of rats, the dissolution of the lymphocytes commences in the thymus within a few hours. The decrease in the mass of the thymus is due almost entirely to the destruction of lymphocytes, and few lymphocytes remain

161 162 in involuted thymus J4.8 hours after irradiation. The reticu­ lar cells predominate in involuted thymus. The action of cortisone on the lymphatic tissue and on thymus is well known. X-irradiation and cortisone treated rats show more involution of the thymus than rats treated with only cortisone (Toolan (162)). The objective of this experiment was to observe the effect of thymectomy on the growth of Toolan's H Ep.-3 tumor.

Materials and Methods

This experiment was conducted in Dr. J. H. Holzaepfel's laboratory where weekly transplantations of a strain of human carcinoma, designated PI. Ep,-3 originally received from Helen

Toolan, are taking place. Female albino rats (Wistar strain) and of weanling age weighing approximately £0 gm each were used for these experiments. These animals were exposed to whole-body irradiation of 15>0 r from a 2f>0 kv. tube. The rats were given four subcutaneous injections of 3 mg/dose of cortisone. The initial dose was on the day of tumor implan­ tation, with subsequent injections on following alternate days. Tumors were obtained as fresh and as sterile as poss­ ible from previously implanted animals, and were finely minced with scalpels so that the minced cancer tissue could be implanted by inject,ions. Ringer's solution, buffered to a pH of approximately 7.3 and with 6 mg of glucose and 200 163 units of penicillin added/cc, was used as the suspending medium. The implantations were made in the subcutaneous flank tissue of the treated rats. On the fifth day after tumor implantation, seven rats were thymectomized. Another group of seven rats served as controls. A total of 28 rats were thymectomized in four groups, one group each week for four weeks. A total of five rats died during thymectomy; thymi were approximately 1/3 the size of that in normal animals of the same age and weight. Thymus removalwas difficult because primarily fibrous connective tissue was all that remained.

Results

During the first few days following thymectomy, the rats were in critical condition, but after several days their re­ cuperation was rapid and weight gain was more obvious than that in the control animals. The tumors grew in both the thymectomized and control animals at the same rate. After thymectomy the tumor growth was more pronounced in the con­ trols than in the thymectomized animals. A group of five thymectomized and five control animals was killed 17 days after treatment and it was found that the tumors in the con­ trol animals weighed approximately 1J? gm compared to those in the thymectomized animals which weighed J-8 gms. The thymectomized rats’ spleens were 2 or 3 times larger (1.133V gm average) than the control rats’ spleens (,5>220 gm average). 161|

The livers of the thymectomized rats weighed 15>.Oli-9 mg while those of the control rats weighed on the average

10.179 mg. The thymectomized rats’ adrenals weighed .0392 mg compared with .0227 mg on the average in the control rats. The body weight of thymectomized rats was 63 gm as compared to control rats ’ body weight of 78. gms on the average.

Comparisons made in the following three groups showed similar results. Histological observations were made on the extirpated thymi at time of thymectomy and on the liver, spleen, adrenals, lymph nodes and tumors at time of death or when the surviving rats were sacrificed 18 days after implan­ tation. In the thymi only some small thymocytes were left; the remainder of the cells were hypertrophied reticular cells showing darkly stained clumped nuclei and large amounts transparent cytoplasm.

There xwas considerable variation in the microscopic characteristics of the spleen in effected rats. The folli­ cles often appeared small and increased in number and sometimes increased in size. The germinal centers usually were not present. Necrotic foci of varying size originating in the white pulp were frequently present. Erythropoietic foci were sometimes present or sometimes missing in the red pulp. There were massive accumulations of immature lympho­ cytic-type cells with many mitotic figures present. 165

Pig. 31 Thymus of the rat exposed to whole-body irradiation of 150 r and removed on fifth day following tumor implantation H and E. x 1350

Pig. 32 Thymus of another rat exposed to wholebody irradiation of 150 r and removed on fifth day following tumor implantation. H and E. x 1350

167 Adrenal enlargement was almost always noted in thymecto- mized animals. The enlargement of the adrenals apparently was caused by the thickening of the zona fasciculata and zona reticularis. The sinusoids were often dilated. The lymph nodes in thymectomized rats were somewhat enlarged but the degree of enlargement showed variations. The number of germinal centers increased in thymectomized rats. Plasma cells were present in the medulla and in the cortex.

The tumor cells in the control animals showed very frequent mitotic figures. Vascularization was very exten­ sive. Small amounts of connective tissue and few lympho­ cytes or leucocytes were observable. The tumor cells in the thymectomized animals showed only a few mitotic figures.

The nuclei were pyknotic and evidenced karyolysis. Increased connective tissue; infiltration of lymphocytes and plasma cells; and vacuolization of the large reticular cells were also observed.

Part II

Molnar (III4.) et al. found that thymic extracts increased the growth of Brown-Pearce carcinoma, malignancy and also changed the localization of the metastasis.

The objective of this portion of the experiment was to determine if calf thymic aqueous extract or the lipid ex­ tract has any effect on the tumor growth, and the localiza­ tion of metastasis. 168

Material and Methods

Nine rats previously implanted with human H. Ep.-3 tumors were divided into three groups, each containing three

animals. On the second day after implantation, 0.1 cc (15

/Vg) of the aqueous fraction was injected subcutaneously into the first three rats. The second group received 0.1 cc (if?

of the lipid fraction injected intramuscularly. The last three rats served as controls. There was no observable

difference; all the animals died between 13 and 17 days

following treatment. Only macroscopic observation was made.

On the basis of these results, nine other rats were di­

vided into three groups. Daily doses of 0.1 cc (l£/*eg) of

the aqueous fraction were subcutaneously administered to the

first three rats; the same amount of the lipid fraction was injected intramuscularly to the next three rats; and the last

three rats served as controls.

Results

Those rats injected with the lipid fraction died on 5,

6, and 8 days following treatment. The remainder of the rats

were killed on the llpth day. There was no difference in

tumor size between aqueous fraction treated and the control

rats. Metastasis was found in the lungs and livers of the

aqueous fraction treated rats but not in the control animals. Enlarged spleens and lymph nodes, were noted in the rats injected with the aqueous fraction but very small spleens and small variations in the size of the lymph nodes were found in the control animals. DISCUSSION

The development of the thymus from the pharyngeal

pouches in the rat and calf is presented. It was observed that In calf embryos up to 60 cm, the two lobes in the mediastinum were separated and each connected separately to

the same side of the cervical lobes by separate stalks. In

the foetus and new-born, the two lobes in the mediastinum are pushed to the left side and are only partially separable.

The vagus nerve is embedded in the connective tissue between the two lobes. Involution is slow in cattle, and it has been found through the years in the Department of Veterinary

Anatomy that a large thymus can be found in older animals and is present as such in all calves, heifers, and bulls.

The observations of Papp and Venzke (12Il) pertaining to

thymic cell culture are partially in agreement with the obser­ vations of Sainte-Marie and Leblond (135). Both reports agree that lymphocytes or thymocytes are produced by reti­

cular cells, but there is a disagreement as to how this is accomplished. Sainte-Marie and Leblond (135) state that the reticular cells undergo mitosis and yield an equal number of large lymphocytes and reticular cells and that the large

170 171 lymphocytes then begin a series of successive mitotic divi­ sions producing smaller and smaller lymphocytes. It was

observed in cell culture, however, that first basophilicly stained cells 1 to in diameter appear in large numbers,

and later larger and larger cells are present. In cultures treated with cortisone, the small thymocytes were found to

be very sensitive and disappeared completely. The small

thymocytes begin to reappear only after the medium was

changed and the cortisone washed out.

These thymic cell cultures showed cyclic production of thymocytes. These observations are in agreement with the

findings of Yoffey and Hanks (172), who reported that lympho­ cyte production as measured In the thoracic lymph duct Is cyclic in nature. The observations on cultured thymocytes

are also in agreement xxrith those of Yoffey and Hanks (172)

as to the validity of using the presence or absence of a nucleolus in small lymphocytes as an indication of the maturity of the cell. It appears that these small thymo­

cytes have the potency to develop into medium-sized lympho­

cytes and reticular cells and are not degenerating cells.

Stohr (155) states that thymic lymphocytes develop from

epithelial cells and are able to transform into epitheloid

cells. Prom the experiment reported here, it was not poss­

ible to make any statement concerning the difference between lymph node lymphocytes and thymocytes in this respect. 172 It is usually stated in text books of histology that

the presence of Hassall's bodies is a basic and exclusive

property of thymic tissue. However, different authors have found similar structures in dog palatine tonsils, pig sub- maxillary lymph nodes, and squamous cell carcinomas of the skin. Jordan and Horsley (8I4.) believe that Hassall's bodies

arise .from occluded arterioles or precapillary branches as

they join with smaller vessels.

On the other hand, in Kingsbury's (92) opinion, Hassail's bodies are epithelial cells degenerating as a result of growth in a confined space (a result of the absence of a free sur­

face). He observed in the case of larger, typical thymic lobules that it was possible to trace the continuity of the

"duct" epithelium and that lining the central thymic canals within such lobules. The observations made on the serial

sections of the nev/born rat from 1 day to 7 or even to 21

days and described in Exp. No. 1 seem to be in ragreement with Kingsbury's conclusions on the origin of Hassall's bodies in the rat. In the calf it was difficult to make serial section of the post-natal whole thymus but it is

very likely that the same is true for the calf.

Hammar (63), Mayer (108), Dustin (I13) an8- G-amburzew

(5>2) observed in the thymus of the dog, goat, cat, and pig

respectively, large cells with striations in the cytoplasm,

and Hammar named them "myoid" cells. These cells were 173 found very frequently in sections of calf and sheep thymi.

Thyme ctomies performed on rats and calves were described in Experiments No. 3 and I4.. The modified technique for thy­ mectomy of the rat at the age of 19 to 21 days seems to be successful and not overly complicated. Thymectomy in one- and two-day-old rats was unsuccessful. If the thymus was removed completely, the animal died on the second or third day, but if a portion of the thymus was left, the animal survived, and the thymus regenerated completely to the size of a normal thymus but was present as a single lobe occupy­ ing the entire mediastinal region.

In the calf a technique was developed which permitted the complete removal of the thymus. It was established that there were no surgical complications in calves as indicated by the normality of the levels of segmented neutrophiles, blood chlorides, and hematocrit after operation. The same was true in rats. These conclusions are based upon statist­ ically analyzed data from blood samples.

In the earlier studies with blood samples from rats, the tail was cut and approximately two drops of blood xwere ex­ pressed for white blood count. It was only possible to follow this procedure for a short time, and the white blood

cell counts were very high. Changing from tail blood sampl­ ing to the femoral vein resulted in a significant drop in white blood cell content. Seeking an explanation for this, 17!|. it was found that Hollingsworth et_ aJL. had reported that there is a difference of approximately 5000 white cells per cubic millimeter of blood the same animal between tail and femoral vein samples. The portion of Experiment No. 2 con­ cerned with the frequency of blood sampling was very import­ ant, in that it confirmed that there was no difference between the groups bled once, twice, or thrice weekly.

It is generally believed that the thymus gland is an organ of lymphocyte production or a large lymph node which loses its function after puberty. If this is true, and if the removal of a lymph node does not significantly effect an animal, then thymectomy ought not result in profound changes either.

Some authors have ascribed an endocrine function to the thymus C(Schmincke (ll|.2), Lowenthal (101), Corns a (32),

Bomskov (lLi)D, while others do not regard this organ as a hormone-producing gland (Bargmann, 9). Recent experimental data tend to support the hypothesis advocated by many workers that the thymus exerts a remote influence upon lymphatic organs with stimulation of lymphocytopoiesis (Metcalf (110)

Metcalf and Buffett (111), Cornsa (30), Selye (li|3>) studied stress reactions in female rats afber the administration of various conrpounds which led to rapid involution of the thymus. In the involuted organ tubular epithelial structures were seen which contained an 175 eosinophilic colloid and resembled thyroid tissue. Intact rats did not show similar■structures. Bargmann (9) also emphasized that intrathymic cysts are often seen during periods of thymus involution. Tschassownikow (I61j-) studied the thymi of rabbits and did not mention cysts, tubules, or alveolar formations, but they found a hypertrophic medullary epithelium, including Hassall’s bodies with mitochondria and golci apparatus, indicating an active secretory phase of the cells.

In serial sections of rat thymi to seven, and at times 21 days of age PAS-positive material was seen in alveoli, tubuli, and cysts, which was assumed to be mucopolysaccha­ rides. The appearance of ciliated cells with a distinct cuticular border favors the concept of an active, specific function rather than degeneration of the cells, Bomskov (16), Comsa (30), and many other authors have found that thymectomy in the guinea pig and rabbit causes a decrease in lymphocyte numbers in the spleen, the lymph nodes, and the circulating blood, but these observations were not statistically supported. Experiment Ho. 5 showed that in the rat immediately after thymectomy there was lymphocytosis and leukocytosis, which later changed to a lymphopenia and leucopenia persisting for 2-§- months or even longer. The lymphopenia and leucopenia vjere found to be statistically significant. It was also found that plasma cells were present 176 in the blood, as well as an increased number of eosinophiles. Histological observations showed that the splenic red pulp first showed hypertrophy and hyperplasia and did not differ greatly from the white pulp, but in a later stage there was a decrease in numbers of lymphocytes in the red and white pulp of the spleen and in the 17/mph nodes (especially in the medulla) and an increased number of plasma cells. Infiltra­ tions of lymphocyte and plasma cells in the liver, kidney and lungs were also observed.

It is noteworthy that Maximow and Bloom (107) state that plasma cells are of common occurrence in the lymphatic tissue, especially in the medullary cords of the lymp>h nodes; their number Is subject to marked variation, particularly under pathological conditions. In some animals (rat, mouse) plasma cells are especially numerous. In the experiments performed, it was observed that comjjared with the controls, plasma cell were found markedly increased in the lymphatic tissues and. blood and their infiltration into the liver and kidney was also noted in thymectomized animals.

When splenectomy was performed 2h months after thymec­ tomy, lymphocytosis and leucocytosis resulted in both thymectomized and control rats. The infiltrations were more marked and at this time lymphocytes were predominant but the plasma cells were also present. The bone marrow showed hypertrophy and hyperplasia. After thymectomy the spleen 177 and lymph nodes showed compensatory activity, but the bone marrow did not. After splenectomy the lymph nodes and the bone marrow acted as compensatory organs in thymectomized animals. In the controls, the thymus, lymph nodes, and to a lesser extent the bone marrow apparently performed the same function. It appeared that after thymectomy the adreno- hypophyseal mechanism was disturbed. This inference is based upon the histological changes seen in the adrenals; hyper­ trophy of the zona fasciculata and reduction of the zona glomerulosa and reticularis. Further evidence that hormonal disturbances occurred in the rats is provided by the electro­ lyte determinations which indicated that blood sodium and

calcium were significantly lower as determined by statistical

analysis. Blood cholesterol was also increased in thymec­ tomized rats, while blood levels of urea nitrogen in calves were significantly lower in calves during the period beginning

60 days after thymectomy.

Since it is well known that hypophysectomy results in lymphocytosis and leucocytosis, it was of interest to learn whether thymectomy would alter this chronic condition in hypophyzectomized rats. It was shown statistically that

thymectomy reverses the lymphocytosis of hypophysectomized

animals.

The comments of Dr. Knouff and Dr. Wiseman were of

value, in that they questioned whether the changes found to occur in rats might not hold for other mammals and should be investigated in other species. A technique for thymectomy

in calves was successfully developed for the first time and

the hemograms and blood levels of electrolytes, cholesterol, urea nitrogen and glucose determined for these animals.

During the first three months there were no statistically

significant changes detectable, but after that period

lymphopenia and leucopenia developed and persisted until the end of the period of observation at six months after thymec­

tomy. Blood urea nitrogen was lower in thymectomized calves

and cholesterol possibly higher compared to the controls. There were other changes observed also in electrolytes and

blood constituents but these were either not statistically

significant or consistent. In addition to the changes in

hemogram, electrolytes, and other blood constituents, there

were other changes observed:

1. In both thymectomized male rats and calves, the sex­

ual libido was increased. Rats were seen to copulate on the

table after they were bled, which normally Is not observed.

The thymectomized calves would mount their stall-mate con­ trols while the latter were being restrained for bleeding,

even at an early age, whereas the control animal did not show this behavior. For a short period the testes of thymec­

tomized Calf No. ll£ appeared relatively smaller than those

of its larger control, after which the proportion was 179 reversed. This precocious testicular development was con­ firmed by observations on the young, totally thymectomized

Calf, Sp which was compared histologically to the control S2 .

A similar contrast was observed after thymectomy in male litter mate rats.

Andersen {3 ) has said, "the findings of Calzolari (1868) suggested the possibility of a reciprocal relationship of some kind between the thymus and testes. There have been reports of precocious development, of delayed development, and of no change at all in the ovaries and testes following thymectomy, but in each instance the data have been too scanty to permit statistical analysis. In most of the ex­ periments in which delayed sexual development was reported, the animals were underweight or suffered from infections."

It seems that the observations in rats and calves are in agreement with those of Andersen, but two thymectomized and two control male calves have been retained for further study.

Andersen (3) has also performed experiments with female rats in which she concluded that thymectomy does not affect the age or weight at which female rats reach puberty when the criterion for the latter is the opening of the vagina, and that thymectomy does not affect the age at onset of estrus cycles. This is in contradiction to our observations, because thymectomized females had pups earlier and more frequently. l8o Based on literature and textbook readings, the impres­ sion was gained that thymectomy would cause a decrease in resistance against infections, irradiations, and tumor groitfth. Frequent blood sampling resulted in the control rats developing infections in the vicinity of the femoral vein. In the preliminary trial with calves, diarrhea devel­ oped, which was stopped completely after thymectomy and persisted for weeks in the controls. In the last trial one of the control animals, Pp. developed diarrhea and later a navel infection whereas the thymectomized animals of approxi­ mately the same age in the same box stall did not become ill at any time. In the case of the Up and H2 pair, the control animal died from enterotoxemia, as diagnosed by Department of Veterinary Pathology. These two animals were constantly together, both before and after the operation. Other observations are reported in Experiments 7 &nd 8 which suggest that involuted thymic tissue may produce some substance or substances which support tumor growth and potentiate the lethal effects of irradiation. There is evidence that the 11 take" of experimental tumor transplants is only possible if the involution of the thymus has pre­ viously been brought about by irradiation and cortisone treatment CToolan .(162D . The presence of the involuted thy­ mus prevents hypertrophy of the spleen and other lymphatic tissues. Removal of the involuted thymus reversed this 181 condition and increased the quantities of lymphocytes and plasma cells produced for a short time. This inference is based on the study of rats killed on the fourteenth day after thymectomy on the fifth day after implantation of a human buccal carcinoma. The spleens in thymectomized animals were three times larger than those of the controls and of dark brown color, whereas in controls the spleen was small and of light pink color. Enlargement of the adrenals, lymph nodes and liver were observed in thymectomized, but. not in control rats.

It is possible that this mechanism is also active in the case of irradiation. These observations suggest that the thy­ mus functions not only as a lymphocyte-producing, but as a lymphocyte- and plasma cell-controlling organ.

Recent experiments in mice by Kaplan et al. (85) have shown that lymphatic leukemia and lympho-sarcoma develop spontaneously or may be induced by exogenous agents in many strains of mice. The tumors usually arise in the thymus gland, and their incidence may be drastically reduced by thy­ mectomy. On the other hand, when isologous thymus glands were implanted into previously thymectomized, irradiated hosts, a partial restoration of the incidence of tumors re­ sulted. It seems possible that involuted thymic tissue plays an important part in the development of neoplasms. 182 Molnar ejt al. (115) found that thymic extracts increased malignancy of Brown-Pearce Carcinoma in the rabbit and changed the localization of metastasis. This observation appears to be in agreement with the results here reported with a single injection of calf thymic extract prepared by the method of

Bezsonoff-Comsa which apparently caused the localized implant of buccal carcinoma to spread to the lungs, liver, and spleen, whereas the tumor remained localized in the controls.

Thymic extracts (aqueous and lipid) from young calves were prepared by various methods and bio-assays were per­ formed to find the single smallest dose which would have a detectable effect on the hemogram. Comsa and Bomskov (33) describe this extract as effective in small doses with fre­ quent administration. There may have been some indication of an effect in these experiments also, but it was not statistically significant, perhaps because only l5/*'g of peptide fraction or only 0.2 to 0.Ip cc of lipid fraction were injected, or the method of preparation ma?/- have been unsuitable although resulting in a high degree of purity indicated by electrophoresis. It would be of great interest to study the effects of extracts prepared from calf thymi when injected into thymectomized and control calves. SUMMARY

Experiment #1, The morphogenesis of the thymi of 33 rats from one to seven days of age was studied by means of transverse serial sections. During this ueriod cysts, tubuli, and alveoli were formed leading from the main duct in the form of finger-like projections to the medullae of each of the thymic lobules. Individual epithelial cells and

Hassall's corpuscles originated from these epithelial struc­ tures .

Indirect evidence is presented by means of tissue cul­ ture that thymocytes are produced on a cyclic basis.

Treatment of these cultures with various dosage levels of

cortisone showed that reticular cells are the precursors of the thymocytes.

Experiment #2. Standard curves in more than 20 albino rats of the Sprague-Dawley strain for total and differential leucocyte counts and hematocrit, as well as for blood levels

of carbon dioxide, chloride, sodium, potassium, cholesterol, and calcium as a function of age were established for com­ parative purposes. Also, standard curves in three untreated

calves for total and differential white blood cell content 183 l8i(. per cubic millimeter, hemoglobin, hematocrit, calcium, sodium, chloride, blood urea nitrogen, glucose, and cholesterol were prepared.

From 15 rats of both sexes, blood samples were taken once, twice, and thrice weekly for a period of three months, and it was statistically established that there were no significant quantitative nor qualitative differences in the leucocyte contents of the blood of rats due to differences in frequency of bleeding. Experiment #3. Thymectomy was performed on 237 rats at various ages. The modified technique for thymectomy of the rat at the age of 19 to 21 days was found to be successful, simple, and virtually free of surgical complications. Experiment #h. Thymectomy was performed on ten calves.

A technique was developed which permitted the complete re­ moval of the thymus. On the basis of statistical analyses, it was established that there were no surgical complications due to this operation in calves, as indicated by the normal­ ity of the levels of segmented neutrophiles, blood chlorides, and hematocrit following the operation. Experiment #5. In rats and calves, thymectomy resulted in a statistically significant lymphopenia and leucopenia and also in an increase in plasma cells and eosinophiles. Histologically, hypertrophy and hyperplasia of the spleen and lymph nodes were observed which later were reversed. 185 Infiltration of lymphocytes and plasma cells into the liver, kidney, and lungs were observed.

Splenectomy performed on thymectomized and control rats

2|,- months after thymectomy resulted both in lymphocytosis and leucocytosis. Thymectomy performed on ten hypophysec- tomized female and ten hypophyctomized male rats appeared to reverse the lymphocytosis resulting from hypophysectomy.

Thymectomy performed at 25 days of age in nine male rats resulted after 65 days of age in statistically signifi­ cantly lower blood calcium and sodium levels and higher blood cholesterol levels than those of nine male litter- mate controls.

In four calves of various breeds, statistically significant lymphopenia, leucopenia, and low blood urea developed three months after thymectomy. Precocious testi­ cular development as evidenced by behavior and histological observation was found in rats and calves. Also, greater reproductive efficiency was noted in thymectomized female rats than in intact controls. Hypertrophy of the zona fasciculata, and hypertrophy of red and white pulp of spleen followed by depletion were the regular findings after thy­ mectomy in both calves and rats.

Experiment #6. Thymic extracts prepared by various methods are described. The effects of these extracts were 186 tested on littermate groups of rats, and it was found that a water-soluble thymic homogenate and electrophoretically homogeneous peptide extract lowered the blood lymphocyte contents in thymectomized rats and that a lipid thymic homogenate increased the blood lymphocyte content in sham- operated rats.

Experiment #7» Thymectomy performed on the fifth day after irradiation of 100 gm rats with 800 r and of £0 gm littermate rats with 900 r resulted in prolonged survival after more than the LDpoo irradiation. Lipid calf thymic extract, and to a lesser degree water-soluble calf thymic extract (peptide), shortened survival after 2 LDpgo of irradiation.

Experiment #8, Thymectomy performed on the fifth day after implantation of H. Ep. -3 -human buccal carcinoma cells resulted in retardation of tumor growth, regression, and necrosis of tumors in rats. Implanted paraffin pellets containing calf thymic extract (p>eptide) in rats with groxtf- ing human buccal carcinoma resiilted In the spreading of malignancy and metastasis. BIBLIOGRAPHY

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17l+. Zbarskij, I. B. and Debov, S. S.: Proteins of cell nuclei. Doklady Akad, Nauk, SSSR. 63 (191-1-8) 796-798. AUTOBIOGRAPHY

I, Eugene Papp, was born in Budapest, Hungary, October

6, 1919. I received my secondary school education at

I'iukacevo High School, Mukacevo, Czechoslovakia, and my professional training at the Hungarian University of Agri­ cultural Science, College of Veterinary Medicine, which granted me the degree of Doctor of Veterinary Medicine in

19lp3 and the Master's Degree in 19^Ul-. 1 was appointed

Assistant Professor in the Department of Obstetrics and

Sterility in June, 19L3» and held that position for two years while completing the requirements for the Master's

Degree. I was appointed as a Specialist in Artificial In­ semination and Sterility by the West-German government in

March, 191D?, and held that position for five years.

I was employed by the Colombian Ministry of Agriculture as a specialist in Artificial Insemination and Sterility in

April, 195>0, and held that position for five years. From

April, 195>£» until July, 195>6, I worked In partnership with a general practicioner In Akron, Ohio. I accepted a posi­ tion as a technician in the Department of Veterinary Anatomy of the Ohio State University in June, 195>6, and held this

201 202 position for one year, after which I was appointed as an instructor in the same department and held this position to the present while completing the requirements for the degree of Doctor of Philosophy. I have completed the veterinary state board examination and have been certified by the Ohio State Board of Veterinary Examiners to practice

Veterinary Medicine in the state of Ohio.