THE
`
`ANATOMICAL RECORD
`
`MANAGING EDITOR
`
`CHARLES M. Gross
`Louisiana State University
`
`ASSOCIATE EDITORS
`
`JOHN W. EVERETT
`Duke University
`
`WILLIAM U. GARDNER
`Yale University
`
`Roy Gr. WILLIAMS
`THOMAS E. HUNT
`University of Alabama University of Pennsylvania
`
`NEWTON“ B. EVERETT WILLIAM W. G-REULICE
`University of Washington
`Stanford University
`
`OLIVER P. JONES
`University of Bufialo
`
`W. LANE WILLIAMS
`University of Mississippi
`
`VOLUME 138
`
`SEPTEMBER, OCTOBER, NOVEMBER, DECEMBER 1960
`
`IN MEMORIAL TO
`
`NORMAND LOUIS HOERR
`
`PUBLISHED BY
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`THE VVISTAR INSTITUTE OF ANATOMY AND BIOLOGY
`
`PHILADELPHIA, PA.
`
`Astrazeneca Ex. 2122 p. 1
`Mylan Pharms. Inc. V. Astrazeneca AB IPR2016-01326
`
`

`
`The oflicial organ of the
`
`AMERICAN ASSOCIATION OF ANATOMISTS
`
`Printed in
`The United States of America at the
`PRESS OF
`THE WISTAR |N$‘E‘iTU“'f‘E.
`OF-' ANATOMY AND BIOLOGY
`PHILADELPHIA
`
`Astrazeneca Ex. 2122 p. 2
`
`

`
`CONTENTS
`
`No. 1 SEPTEMBER 1960
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`ARNOLD LAZAROW. Foreword .
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`GORDON H. Scorr. Normand Louis Hoerr: The Man and His
`Achievements .
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`ARNOLD LAZAROW. Normand Louis Hoerr: His Contributions to
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`Cytochemistry and His Role as a Teacher .
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`\ C. H. U. CHU. A Study of the Subcutaneous Connective Tissue
`2‘
`of the Mouse, with Special Reference to Nuclear Type,
`Nuclear Division and Mitotic Rhythm .
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`11
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`PAUL R. PATEK AND SOL BERNICK. Time Sequence Studies of
`Reticuloendothelial Cell Responses to Foreign Particles .
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`SYLVIA H. BENSLEY. Microscopic Studies of the Living Iris .
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`S. J. COOPERSTEIN, P. K. Dxxrr AND ARNOLD Leznuow. Studies on
`the Mechanismof Janus Grreen B Staining of Mitochondria.
`IV. Reduction of Janus Green B by Isolated Cell Fractions
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`MARY ELLEN HARTMAN. The Relationship of Grlomerular Plasma
`Flow to the Etiology and Progression of Hypertension .
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`Books. Reviews by Charles M. Goss .
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`NO. 2 OCTOBER 1960
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`GARMAN HARLOW DARON. Morphology of the Cerebellar Dentate
`Nucleus in a Chimpanzee .
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`WELIAM L. STRAUS, JR. The Subarcuate Fossa in Primates .
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`27
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`39
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`49
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`67
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`73
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`81
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`93
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`OLIVER P. JONES. Origin of Megakaryocyte Granules from Golgi
`Vesicles .
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`105
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`ANTHONY A. PEARSON AND ARTHUR L. ECKHARDT. Observations
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`on the Gray and White Rami Communicantes in Human
`Embryos .
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`115
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`AU‘Binderyspggaggf
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`Astrazeneca Ex. 2122 p. 3
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`

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`CONTENTS
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`SYDNEY M. FRIEDMAN AND CONSTANCE L. FRIEDMAN. The Use of
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`Ion—Speeific Glasses in Biological Systems .
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`M. NEIL MACINTYRE, JAMES E. HUNTER AND ALICE H. MoReAN.
`' Spatial Limits of Activity of Fetal Gonadal Inductors in
`the Rat
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`JOHN D. HARTMAN AND DENNIS M. GORETSKY. Changes in the
`Glyeolytic Activity of Blood and Exudate Leukocytes dur-
`ing an Inflammatory Reaction .
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`PAUL GIRRONS ROOFE. The Rate -of Flow of Blood through
`Capillaries in the Olfactory Lobe of the Brain ‘of Ambigu-
`toma tigr/mum .
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`CHARLEs N. LOESER, SEYMOUR S. WEsT AND MELVIN D. SoHoEN-
`BERG. Absorption and Fluorescence Studies on Biological
`Systems: Nucleic Acid-Dye Complexes .
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`STUART W. SMITH AND PAUL N. ANDERSON. Qualitative Cyto~
`chemical Localization of Pyrimidine Deoxyribonucleotide
`Residues in Fixed Tissue Sections .
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`129
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`137
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`149
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`159
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`163
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`179
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`No. 3 NOVEMBER 1960
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`WILLIAM L. SIMPSON AND YUTAKA HAYASHI. Distribution of
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`Mast Cells in the Skin and Mesentery of BALB/c and
`C57BL Mice .
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`193
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`EDWARD R. ARQUILLA AND CHARLOTTE GORLENOE. The Isolation
`oi Rabbit Insulin Antibodies
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`RAYMOND G. MURRAY AND AssIA MURRAY. The Fine Structure
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`of the Taste Buds of Rhesus and Cynomalgns Monkeys .
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`IVORY AND J. W. PATTERs0N. Glucose
`T. G. FARKAS, R. F.
`Uptake and Utilization of Isolated Lenses from Normal and
`Diabetic Rats Following Insulin Injection .
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`JAE NAM KIM, WALTER RUNGE, LEMEN J. WELLS AND ARNOLD
`LAZAROW. Pancreatic Islets and Blood Sugars in Prenatal
`and Postnatal Offspring from Diabetic Rats: Beta Granula-
`tion and Glycogen Infiltration .
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`EDWARD H. BLOOH AND ROBERT E. HAss. The Thermal Control
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`of Tissue Used in Quartz-Rod Transillumination .
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`211
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`235
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`2-39
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`261
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`Astrazeneca Ex. 2122 p. 4
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`

`
`CONTENTS
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`GEORGE W. BARTELMEZ. Neural Crest from the Forebrain in
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`Mammals
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`MARION HINES. A Histochemical Study of the Localization of
`Cholinesterase in Skeletal Muscle during its Regeneration
`
`AMERICAN SOCIETY or ZOOLOGISTS. Annual Meeting; New York
`City, New York, December 28 through 30, 1960. Officers,
`Proceedings, Program and List of Titles, Abstracts of
`Papers, Author Index .
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`269
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`283
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`299
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`‘ No. 4 DECEMBER 1960
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`J OHN W. PATTERSON. Effects of Galactosemia .
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`LOUISE WARNER AND MURRAY 0. BROWN. Techniques for Study-
`ing Microcirculation in the Eye .
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`399
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`SANFORD L. PALAY. The Fine Structure of Secretory Neurons
`in the Preoptic Nucleus of the Goldfish (flarassius cmmtus)
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`417 .
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`Ismoes GERSH, JUAN VERGARA AND GIOVANNI L. Rossx. Use of
`Anhydrous Vapors in "Post-Fixation and in Staining of
`Reactive Groups of Proteins in Frozen-Dried Specimens for
`Electron Microscopic Studies .
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`SARAH A. LUSE. The Ultrastructure of Normal and Abnormal
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`44:5
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`461
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`Oligodendroglia .
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`Books :
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`Reviews '
`Received .
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`493
`495
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`Astrazeneca Ex. 2122 p. 5
`
`

`
`A Study of the Subcutaneous Connective Tissue of the
`Mouse, with Special Reference to Nucleotr Type,
`Nuclear Division and Mitotic Rhythm‘
`
`C. H. U. CI-IU
`
`Department of Anatomy, School of Medicine, Western Reserve University,
`Cleveland, Ohio
`
`
`
`Connective tissue may be looked upon
`as a culture medium, on which epithelial
`cells survive. Between any epithelium and
`the underlying blood vessels, lymph vessels,
`and nerves, a certain amount of connective
`tissue is always interposed. Whatever is
`brought to or from the epithelium, must
`first pass through the connective tissue.
`Any change in the connective tissue, be it
`physiological or pathological, affects the
`epithelial cells. The well-being of epithe-
`lium depends upon the healthiness of con-
`nective tissue. To understand the behavior
`of an organ, a knowledge of the form and
`function of its connective tissue is as im-
`portant as that of its epithelium. In recent
`years, the significance of connective tissue
`is commanding increasingly attention of
`many investigators in different fields.
`Mitotic rhythm has been observed in
`both plant and animal cells (Hughes, ’52).
`The animal cells studied include only the
`epithelia of the frog (Mollerberg, ’48), rat
`(Blumenfeld,
`’38), mouse (Cooper and
`Franklin,
`’-40), and man (Cooper and
`Schiff, ’38). Such a rhythm has not been
`reported in connective tissue. This prompt-
`ed the present investigation. However, cer-
`tain problems must be resolved before it is
`possible
`to determine whether mitotic
`rhythm is observable in connective tissue.
`First,
`to estimate the rate of mitosis in-
`volves the counting of dividing and non-
`dividing cells.
`If the estimation is to be
`Valid, a large number of cells must be
`counted. This can hardly be done in tissue
`sections or tissue spreads. Secondly, for ac-
`curate evaluation,
`it is essential that the
`original architecture of the tissue be as lit-
`tle distorted as possible.
`In this respect,
`tissue - spreads are useless. The teasing
`and tearing in preparing the spreads may
`
`push or pull a number of cells from one
`area of
`the tissue to another. Results
`based on such specimens cannot be ac-
`curate. Finally, there are many kinds of
`cells described in connective tissue. These
`cells must be made easily recognizable in
`order
`to ascertain which ones undergo
`division by mitosis. Tissue sections do not
`show the characteristics of these cells dis-
`tinctly enough to be practical. For the
`present study,
`the difficulty lies in the
`search for a method, other than tissue sec-
`tions and tissue spreads,
`to satisfy the '
`above requirements. The lack of a satis-
`factory method may account for the fact
`that mitotic rhythm in connective tissue
`cells -has not been reported previously.
`After considerable trial and error, a
`method of preparing connective tissue for
`quantitative studies has been found.
`In
`brief, a complete layer of subcutaneous
`connective tissue is obtained by separating
`it from the skin specimen that has been
`fixed already. Details of this method are
`presented later. For the first
`time it is
`possible to count a large number of cells
`in a single specimen. The cells can be
`identified with more certainty. The origi-
`nal architecture of the connective tissue is
`fairly well preserved.
`Results of this study indicate there are
`only three types of nuclei in connective tis-
`sue cells;
`lymphocytic,
`rnonocytic
`and
`fibrocytic. All three types of nuclei under-
`go division by mitosis. Furthermore, ami-
`totic division is not an uncommon feature
`of all three types of nuclei. By counting
`
`1 This study has been supported by a research
`grant (C-4-023) from the National Cancer Insti-
`tute of the National Institutes of Health, United
`States Public Health Service.
`
`11
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`Astrazeneca Ex. 2122 p. 6
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`

`
`12.
`
`C. H. U. CHU
`
`and comparing the number of dividing and
`non—dividing nuclei, a mitotic rhythm is
`just as demonstrable as in the epithelial
`cells.
`
`Throughout this study, I have paid spe-
`cial attention to the internal structures of
`the nuclei of connective tissue cells.
`It is
`felt that nuclear structures remain more
`
`constant than that of the cytoplasm which,
`in connective tissue cells, is known to be
`easily modified. The differential identifi-
`cation of the several kinds of cells based
`
`therefore, more
`on nuclear structures is,
`consistent. Also, I have limited my study
`to subcutaneous connective tissue, mainly
`because the skin is best suited to the tech-
`
`nic adopted in this study. Although speci-
`mens from different
`laboratory animals
`have been prepared and studied, only the
`subcutaneous
`connective
`tissue of
`the
`
`mouse is described in this report. A com-
`parative study of connective tissue is de-
`ferred to a later date. Whether connective
`
`tissue differs among animals from various
`classes and phyla remains to be seen.
`
`The “tissue-split method”
`
`‘ For microscopic observations, connec-
`tive tissue is usually prepared either by
`sectioning fixed tissue or by spreading
`fresh tissue. Neither of these methods is
`
`In sec-
`suitable for quantitative studies.
`tions, even when cut serially, the observa-
`tion is restricted to a small area of the tis-
`
`sue and limited to the cut surface of cer-
`tain cellular elements. Tissue spreads may
`overcome such limitations, yet in spread-
`ing fresh tissue onto a slide, the normal
`pattern of the tissue is so unduly distorted
`by teasing and tearing that cell counts
`and other computations are rendered in-
`valid.
`V
`
`it has been found
`In this laboratory,
`that connective tissue, even after it has
`been fixed, can be easily removed by blunt
`dissection from certain organs
`(skin,
`artery, etc.).
`I have succeeded in obtain-
`ing from fixed mouse skin a complete sheet
`of subcutaneous connective tissue of one
`
`or two cells in thickness, covering an area
`of 5 cm in length and 4 cm in width. The
`advantages of this over the sectioning and
`spreading methods are obvious. First, it
`permits observation of cells in surface
`
`‘view. This is more informative, in view
`of the fact that since most connective fig.-
`sue cells are flat in form, little can be re.
`vealed by sectioning them into thinner
`slices. Secondly, it offers a large area fa;
`study. This is a necessity when a large
`number of cells is to be counted. Lastly,
`tissue has been fixed before separation,
`whereby its normal pattern is little dis.
`turbed; such, of course,
`is not
`the Case
`with the tissue-spread method.
`Many specimens have been prepared
`with this method. Our experience in the;
`separation of subcutaneous connective tig.
`sue is summarized here in detail. After a
`piece of skin is excised from the back of
`an animal,
`it is attached to a piece of
`Schleicher and Schuell no. 470 filter paper,
`This paper is chosen to keep the skin flat
`and to allow easy penetration of fixing
`fluids. Either aqueous or alcoholic fixative
`may be used. Formalin, Bouin’s, Zenker’s,
`Carnoy’s and Rossman’s fluids have been
`tried with equal success. After fixation,
`it is advisable to trim off the rolled edges
`of the skin. The skin is then removed from
`the filter paper and pinned to a dis-
`secting dish with the furred side down.
`Water is added to the dissecting dish to
`keep the skin from drying. With a Bard-
`Parker no. 15 blade and a pair of fine for-
`ceps, the subcutaneous connective tissue is
`separated into complete layers under a
`binocular dissecting microscope.
`Very
`little cutting action is required and un-
`necessary cutting is to be avoided. After
`separation,
`the connective tissue is flat-
`The
`tened out on a 2" X 3" glass slide.
`slide is left in the air to dry. The connec--
`tive tissue adheres firmly to the slide. No
`adhesive is needed. After drying, the slide
`is ready for further processing. As
`an
`alternative, separation may be done after
`the specimen has been stained. In stained
`specimens,
`the individual
`layers of
`the
`connective tissue can be recognized more
`easily.
`a
`This method is extremely simple. All it
`requires is a little practice and patience.
`To distinguish this from spreading and,
`sectioning, I propose to call it the “tissue-.
`split method.” The word “split” used here
`has the same meaning as in the jargon of
`leather manufacturers.
`
`Astrazeneca Ex. 2122 p. 7
`
`

`
`
`
`MITOSIS or CONNECTIVE TISSUE CELLS
`
`13
`
`The architecture of subcutaneous
`connective tissue
`
`When the skin from the dorsum of a
`mouse or other rodents is removed,
`the
`excision usually takes place between the
`panniculus carnosus and the rest of the
`body. Between the panniculus carnosus
`and the underlying structures, a consider-
`able amount of loose connective tissue is
`present to hold the ‘skin to the body.
`It 1S
`this loose connective tissue that
`forms
`mainly the material for the present study.
`In the fresh state the subcutaneous con—
`nective tissue of the mouse appears whit-
`ish in color, soft in consistency, slightly
`translucent and moist. It is very loose; so
`loose that a whole piece of skin can be
`pulled away easily from the body. Blood
`vessels, lymph vessels and nerves traverse
`the subcutaneonus connective tissue to
`supply the various structures of the skin.
`The vessels and nerves are held together
`by the connective tissue. One bundle of
`vessels and nerves emerges from each in-
`tervertebral space. The bundles are ar-
`ranged in linear order along both sides of
`the vertebral column about 1.5 cm later-
`ally from the dorso-median line. The loose
`connective tissue does not show any defi-
`nite arrangement in the fresh state.
`It is
`generally classified as “irregular connec-
`tive tissue.”
`In the fixed state, the subcutaneous con-
`nective tissue can be separated into a num-
`ber of layers by the tissue—split method.
`The total number of layers separable varies
`in different animals; from 6 to 8 in the
`rabbit, 4 to 6 in the rat and two to 4 in the
`mouse. As has been mentioned above,
`each layer can be separated from the other
`by blunt dissection without much cutting.
`Whatever cutting is needed is to sever the
`vessels, nerves and connective tissue fibers
`here and there that interconnect the layers
`with each other.
`.
`
`After preparing a large number of speci-
`mens, I am led to believe, contrary to cur-
`rent consensus, that loose connective tissue
`definitely has a regular arrangement. The
`arrangement
`in loose connective tissue
`may be compared with that of the compact
`bone, which is a modified form of connec-
`tive tissue. The connective tissue elements
`are arranged in the form of lamellae.
`In
`subcutaneous connective tissue, the lamel—
`
`lae are parallel to the surface of the skin,
`whereas in compact bone, the lamellae are
`arranged circumferentially or they form
`the Haversian system. Each lamella of
`the subcutaneous connective tissue is con—
`
`nected with the other by fibers running
`more or less perpendicularly to the larnel-
`lae. Similar
`fibers
`interconnecting the
`larnellae in Compact bone are known as
`“Sharpey’s fibers.”
`After separation, each lamella of sub-
`cutaneous connective tissue of the mouse
`
`looks like a piece of onionskin. The dorsa-
`median line can be seen as a thin white
`streak similar to the linea alba at the ven-
`tral side of the animal. About 1.5 cm
`
`line,
`dorso—median
`laterally from the
`stumps of vessels and nerves appear as
`white spots arranged in linear order. The
`connective tissue, extending about 3 cm
`between these white spots and about 5 cm
`antero—posteriorly, is entirely free from any
`vessels and nerves. This connective tis-
`
`sue, within an area of about 15 cm”, pro-
`vides an excellent field for cell studies and
`cell counts.
`
`The nuclei of connective tissue cells
`
`Many kinds of cells have been described
`in loose connective tissue. The literature
`
`on this subject is filled with confusion and
`controversy. This is due, at least in part,
`to differences in the method of preparing
`the tissue and to differences in the condi-
`tions of the animals at
`the moment
`the
`
`tissue is excised. Just how many different
`kinds of cells there are in loose connective
`
`tissue, probably no two authors agree.
`In the subcutaneous connective tissue
`
`of the mouse, prepared by the tissue-split
`method, stained with Harris’ hematoxylin
`and counter-stained with eosin, the follow-
`ing kinds of cells can be identified; (1)
`flbrocytes,
`(2) monocytes,
`(3) lympho-
`cytes, (4) mast cells, (5) eosinophils and
`(6) fat cells. Plasma cells, pigment cells,
`macrophages
`and lymphoid Wandering
`cells cannot be identified. Plasma cells
`are extremely rare in animals not affected
`with chronic inflammation. Pigment cells
`are not found in subcutaneous connective
`
`tissue of the mouse. Macrophages must
`show signs of phagocytosis. A cell cannot
`be identified as a macrophage unless one
`sees in its cytoplasm some ingested mate-
`rial, such as hemosiderin, bacteria or col-
`
`Astrazeneca Ex. 2122 p. 8
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`

`
`14
`
`‘
`
`C. H. U. CI-IU
`
`loidal dye—stuff. Lymphoid wandering cells
`must be able to wander.
`In fixed tissues,
`such a faculty no longer exists.
`While no difficulty has been experienced
`in identifying eosinophils, mast cells and
`fat cells, each shows its characteristic cyto-
`plasmic granules or globules;
`the same,
`however, cannot be said about cells in
`which the cytoplasm does not contain any
`inclusion.
`In such cases,
`identification
`will have to rely upon the differences in
`their nuclear structures. Moreover, nu-
`clear structures are known to undergo
`changes less readily than those of
`the
`cytoplasm, therefore, they serve as more
`dependable criteria in differentiating the
`cells.
`
`than
`After having examined more
`300,000 individual nuclei in loose connec-
`tive tissue, insofar as their intranuclear
`structures are concerned, only _three types
`can be differentiated. These are the lym-
`phocytic type, the monocytic type and the
`fibrocytic
`type.
`In the
`following de-
`scription, each type of nucleus is described
`with regard to its nuclear membrane, nu-
`clear sap (enchylema), nuclear framework
`(linin), karyosome (chromatin—nucleolus),
`and plasmosome (true nucleolus). These
`terms are used here with exactly the same
`meaning as defined by Wilson (’25).
`A. Nucleus of
`the lymphocytic type.
`In most lymphocytes,
`the nuclear mem-
`brane is smooth all over (figs. 6, 11, 19).
`In some lymphocytes, frequently the nu-
`clear membrane invaginates toward the
`center of the nucleus (figs. 13, 23). By
`focusing the microscope at various planes,
`the nuclear membrane at the invagination
`can be seen to fold into 4 layers. The
`situation here is not unlike that of an
`
`optic vesicle developing into an optic cup.
`In lymphocytes, the space inside the cup
`is filled with some substance, a part of
`which protrudes into the cytoplasm, form-
`ing a spherical body. This substance is
`colorless in specimens stained with hemat-
`oxyljn and eosin. With the uranyl acetate
`and silver method, the substance is stained
`black.
`It looks like a black ball, part of
`- Which is inside the cup, and part in the
`cytoplasm. Occasionally, many such in-
`vaginations may occur on a single nucleus,
`which now appears with a scalloped bor-
`der. The nuclear sap in lymphocytic nu-
`clei is stained with hematoxylin, instead
`
`of being colorless as in other nuclear types,
`The staining is so intense in most lympho.
`cytes that other nuclear structures are
`obscured. Linin threads can be seen only
`with difficulty. There are usually three tg
`6 karycsomes. The karyosomes are Spher.
`ical bodies, arranged concentrically and
`stained more deeply than the nuclear sap,
`No karyosome has been observed to attach
`to the nuclear membrane. A typical plag.
`mosome cannot be observed.
`In
`B. Nucleus of the monocytic type.
`monocytes, the nuclear membrane may be
`smooth (figs. 18, 24) or wrinkled (figs. 8,
`25). Often the nuclear membrane shows
`an indentation at one side of the nucleus."
`The indentation may be shallow, in which
`case, the nucleus appears to be reniform;
`or the indentation may be deep, in which
`case, the nucleus looks like a curved saus-
`age. The indentation is quite different
`from the invagination described above in
`lymphocytes. At the indentation, the nu-
`clear membrane never shows the folds in
`4 layers. The. nuclear sap is colorless;
`because of
`this,
`the linin threads are
`brought clearly into view. The number of
`karyosomes Varies from two to 4. Some
`of the karyosomes, instead of being more
`or less spherical as in lymphocytes and
`fibrocytes, seem to spread over the linin
`threads thus assuming a somewhat angu-
`lar shape. Occasionally, a karyosome may
`be seen to attach to the nuclear membrane.
`Usually, a typical plasmosome can be seen
`clearly.
`The_
`C. Nucleus of the fibrocytic type.
`nuclei of fibrocytes seem to contain very
`little material
`that stains with hemat-
`oxylin. The nuclear membrane can be
`seen more distinctly than that of lympho-
`cytes and monocytes. The nuclear mem-
`brane often Shows a fold (fig. 29). The
`fold may be parallel to the long or the
`short axis of the nucleus. The nuclear
`
`sap is entirely colorless. The linin is not
`uniformly stained with hematoxylin as in
`the monocytes, but appears to be Varie-
`gated. The number of karyosomes varies
`from 4 to 6 or more. As many as 12
`karyosomes have been seen.
`In fibrocytes,
`the karyosomes have a tendency to attach
`to the nuclear membrane, where they of-
`ten flatten out into plates (fig. 5). Usu-
`ally one but rarely two plasmosomes are
`present. Sometimes,
`the periphery of a‘
`
`Astrazeneca Ex. 2122 p. 9
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`

`
`MITOSIS OF CONNECTIVE TISSUE CELLS
`
`15
`
`lasmosome is stained with hematoxylin,
`fO1v1'nj11g an amphinucleolus.
`The above description may be stated
`differently if the word “chromatin” is used
`to represent
`the intranuclear substance
`that stains with hematoxylin. Nuclei of
`the lymphocytic type contain the largest
`amount of chromatin.
`It fills the entire
`It obscures all other nuclear
`nucleus.
`structures except
`the karyosomes. The
`jg deeply stained’ lymphocytic nucleus,
`to-
`gether with its invagination,
`looks very
`much like the nucleus and the acrosome
`of a human spermatozoon. Nuclei of the
`monocytic type do not contain as much
`chromatin. The chromatin in monocytic
`nuclei seems to concentrate on the karyo-
`sornes and the linin. Both are clearly vis-
`ible against
`the colorless nuclear
`sap.
`Moreover, some chromatin material seems
`to form bridges connecting the linin with
`the karyosomes, which now appear angu-
`lar instead of spherical. Nuclei of the fi-
`brocytic type contain the least amount of
`chromatin. Some minute chromatin gran-
`ules or dustlike chromatin are visible on
`the linin, giving it a variegated appear-
`ance. Most chromatin is located on the
`karyosomes.
`In some fibrocytes,
`l<aryo—
`somes are the only visible structures in
`the nucleus.
`
`‘‘
`
`In differentiating the nuclei of the con-
`nective tissue cells, I have followed a very
`simple rule. When few structures but the
`karyosomes are stained,
`the nucleus is
`classified as of the fibrocytic type. When
`the linin threads are stained, the nucleus
`is of the monocytic type. When the nu-
`clear sap is stained, the nucleus is of the
`lymphocytic type.
`I have found this sim-
`ple rule quite helpful in classifying many
`border-line cases.
`i
`-
`In the subcutaneous connective tissue
`of the mouse, 6 kinds of cells can beidenti-
`fied. When their intranuclear structures
`
`are compared, however, only three types
`of nuclei are distinguishable.
`I have seen
`cells in which the cytoplasm contains base-
`philic granules identical to those of mast
`cells, yet their nuclei may either be that
`5 -of a lymphocyte (fig. 7), a rnonocyte (fig.
`12), or a fibrocyte (fig. 17). In the mouse,
`the nuclei of eosinophils are ring shaped.
`Again, I have seen cells with eosinophilic
`granules in the cytoplasm, but their intra-
`nuclear structures are either of the lym-
`
`
`
`phocytic type (fig. 6), the monocytic type
`(fig. 11) or the fibrocytic type (fig. 16).
`Fat cells are not present in the connective
`tissue within the vicinity of
`the dorsa-
`median line. They are grouped into lob-
`ules along blood vessels, lymph vessels and
`nerves. Each fat cell contains a large fat
`droplet. The nucleus is pushed to the pe-
`riphery of the cell. The nuclei of fat cells,
`again, may look like those of lymphocytes,
`monocytes or fibrocytes. From these ob-
`servations, it is conceivable that in loose
`connective tissue there are only three kinds
`of cells; lymphocytes, monocytes and fibro-
`cytes. The cytoplasm of these cells may
`be modified to become mast cells, eosino-
`phils or fat cells, but the nucleus of these
`cells remains as that of a lymphocyte,
`monocyte or fibrocyte.
`
`Amitotic nuclear division of
`connective tissue cells
`
`While searching for nuclei undergoing
`mitotic division, I have noticed many cells
`in which the nuclei divide without any
`evidence of formation of spireme or chro-
`mosomes. Each of these nuclei may divide
`into two portions, yet
`its
`intranuclear
`structures remain the same as those of a
`
`non-dividing nucleus. A more thorough
`search reveals that this mode of division
`
`is observable in all three types of nuclei.
`No difficulty has been experienced in dif-
`ferentiating the nuclear
`types of cells
`undergoing amitosis.
`Figure 4 shows a lymphocyte in which
`the nucleus assumes an hourglass shape.
`The whole nucleus is deeply stained; The
`karyosomes can be seen clearly. One por-
`tion of the nucleus still retains its invagi-
`nation. Two fibrocytic nuclei overlapping
`one another are included in the figure for
`comparison. Figure 5 shows another lym-
`phocytic nucleus at a later stage of amito-
`sis. The two portions of the nucleus seem
`to have drawn farther apart. A bridge
`connecting the two portions can be seen.
`The invaginations are still present. The
`intranuclear structures remain the same
`
`as in a non-dividing lymphocytic nucleus.
`Figure 9 shows a monocytic nucleus under-
`going amitotic division. Figure 10 shows
`a monocytic nucleus at a later stage of
`amitosis. Figure 14 shows an hourglass
`shaped nucleus of the fibrocytic type. Fig-
`ure 15 shows another fibrocytic nucleus
`
`Astrazeneca EX. 2122 p. 10
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`

`
`16
`
`c. H. U. CHU
`
`nearly divided into two, but the two por-
`tions are still connected by a narrow
`bridge. Nuclei that have completed amit—
`otic division are not illustrated, because
`they look exactly like non—dividing nuclei.
`Amitosis probably is a very common
`phenomenon
`among connective
`tissue
`cells. Amitosis of connective tissue cells
`
`has been observed by Nowikoif as early
`as 1909
`(see Wilson,
`’25). Although
`many theories have been suggested, none
`of them explains satisfactorily why cells
`divide by amitosis. The whole process of
`arnitosis deserves a more thorough study.
`For our purpose,
`it suffices to note that
`connective tissue cells not only divide by
`mitosis, but also by amitosis.
`In estimat-
`ing the rate of Cellular multiplication, nu-
`clei undergoing amitotic division must also
`be taken into consideration.
`
`Mitotic nuclear dz'm'sio'n. of
`connective tissue cells
`
`In tissue splits, numerous mitotic figures
`have been observed. The number of con-
`
`nective tissue cells undergoing mitosis is
`much greater than one ordinarily suspects.
`The question about the precursors of these
`dividing nuclei naturally arises. Thus far,
`I have relied upon intranuclear structures
`in differentiating the nuclei of connective
`tissue cells. These criteria, however, are
`applicable only to the intermitotic or inter-
`phasic nuclei. When a cell undergoes mi-
`totic division, especially when it reaches
`the metaphase and anaphase, such criteria
`are totally obliterated.
`In our specimens,
`it is easy to recognize mitotic figures, but
`to determine to which nuclear type a cer—
`tain mitotic figure belongs requires a dif~
`ferent standard of judgement.
`When tissue splits are examined under
`a microscope, usually all
`three types of
`nuclei are present within a certain field.
`With the aid of a net micrometer reticule
`
`(American Optical Company, no. 1408)
`inserted in the ocular, one can measure
`and compare a dividing nucleus with that
`of
`a nearby non—dividing lymphocyte,
`monocyte or fibrocyte. Even when a nu~
`cleus is in the metaphase or anaphase,
`such measurements and comparisons are
`feasible. A set of chromosomes may be
`visible either from the polar or equatorial
`view.
`In either case,
`the extent of the
`equatorial plate where the chromosomes
`
`are gathered is more or less equivalent to_'
`the diameter of the short axis of a non—_
`dividing nucleus. An equatorial plate
`measuring 15 to 1'7 it probably is derived-
`from a fibrocytic nucleus, Whose short
`axis has about
`the same measurement."
`One that measures 12 to 14 it probably is
`derived from a monocytic nucleus. Sim-
`ilarly, an equatorial plate of 6 to 8 u is
`derived from a lymphocytic nucleus.
`It
`is very unlikely for a lymphocytic nucleus,
`the diameter of which measures from 6 to
`8 u, to give rise to an equatorial plate as '
`extensive as 15 to 17 u.
`Figure 20 shows a lymphocytic nucleus in
`in early prophase and two lyrnphocyticl
`nuclei in late prophase. Figure 21 shows
`a dividing lymphocytic nucleus in meta-
`phase and a non-dividing lymphocytic nu-'
`cleus slightly out of
`focus.
`Figure 22 j
`shows a lymphocytic nucleus in anaphase.
`Figure 23 shows a lymphocytic nucleus in '3
`telophase and a non-dividing lymphocytic I
`nucleus. With a compass, one can actually '-
`measure and compare the equatorial plates
`in figures 20, 21,22 and 23, with the
`diameter of the non—dividing lymphocytic
`nuclei
`in figures 19 and 23. They all
`measure about 6 to 8 u. Monocytic nuclei-
`in various phases of mitosis are shown
`in figures 25, 26, 27 and 28. Fibrocytic-
`nuclei
`in various phases of mitosis are
`shown in figures 30, 31, 32 and 33. The
`equatorial plates of monocytic nuclei are
`from 12 to 14 1.: and those of
`fibro—
`cytic nuclei 15 to 17 u. Figures 19, 24
`and 29 are included in the plate for com— o
`parison.
`It is easy to see the differences
`when the dividing nuclei are brought to-
`gether side by side as in the illustrations, 5
`but the origin of a single mitotic figure
`Within a certain microscopic field can be Q
`determined only by comparing it with.
`nearby non-dividing nuclei.
`
`Mitotic rhythm of connective
`tissue cells
`
`the
`Twenty—four C3H female _mice of
`same age (two months) were used for
`the study of mitotic rhythm. These mice
`were collected from several
`litters born
`on the same day. One mouse was sacri-
`ficed by ce