Loyola University ChicagoLoyola eCommons

Master's Theses Theses and Dissertations

1967

Age and Generation Cycle of Oral EpitheliumNabil John BarakatLoyola University Chicago

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Recommended CitationBarakat, Nabil John, "Age and Generation Cycle of Oral Epithelium" (1967). Master's Theses. Paper 2076.http://ecommons.luc.edu/luc_theses/2076

AGE AND GENBRA'1'ION CYCLE

OF ORAL EPITHELIUM

by

NuU J. Barakat.

A The.i. Sw.1tted to t.he Facult.y o~ the Graduate School

of Loyola Unl.erait.y in Partial Pulfl1lmant o~

the Requirement.. for the Degre. of

Ma.ter of Sclence

June 1961

- I UBRARY , l,UYOI:A UN1VERSlTY MEDICP.L a::r,,1"JLj1

LIFE

Nab11 John Barakat. was born on March 25 t 1940,

in <:a1ro, EtYpt.. He graduated :from HOly Fam11y·.

Je.uit coll.,. 1n 1958. From 1958 to 1959 be attended

t.he Faoulty of Scitance., Calro Un!verslt.y.

ae recelved hi. Bachelor of Dental surqery deqr ..

from the School of D6ftt1atry, Cairo Unlveralty, in 1963.

Upon graduatlon, he staned a rotat.ln9 int.ernship at.

ca1ro·a Un.lveralty heap1Ul.

In January 1,965, he b8CJan a two-year Graduate

pzoqram a1t Loyola unlvers1t.y le.dinq t.o a Mast.r of sclenee e

4eqr.. 1n Oral Biology. Clinical t.rUni.n9 waa clone in t.he

Oral Surgery t>ep&.rt.lIeJlt. u.n4er Dr. Niebolas ChOUkas.

During' his coura. of at.u41es, he inat.ructed Phyalolo;y

and PharmacolOCJY' at. th. achool.

ACKNOWLBDGBMENTS

The author would like t.o expre.s hi. appreciat.ion

t.o Dr. Patrick Toto for hi. encouragement, many augge.tlons,

and idea., during t.he pa8t year.

TO Dr. Harry Sierher and Dr. John O' Malley, lIf!;/

Sincere thank. for their helpful crlticiam.

I am lnd.beed to ~ faculty advisor, Dr. Nicholas

Cheukaa, for hi. guidance and the philosophy of the deaire

to learn.

Appreciat.ion 18 offered to Dr. Allen Goldberq,

Veterans Administration Hospital, Htnes, Illinois, for

hia aid in obtatniftf the photom1croc;rapha.

It baa b41en a, great opportunity and privileqe to

work under such celebrated and gifted teachers.

Finally I to Arqonne Nat.ional Laboratory I Lemont.,

Illinoi8, and Dr. samuel Lesher, for~. apectmana they

made available tor atudy, I am deeply thankful.

I.

II.

TABLE OF CONTBN'l'S

INTRODUCTION • • • • • .. .. • .. • • • • • .. •

RBVIBW OF LXTERA'l'URE. • . .. ,. . . • • • > ••

A. HISTOLOGY OF THE ORAL MUCOSA . .. . .. • •

1

3 , B. CBLL RBNBWAL SYSTEMS • .. • .. • • • .. II.. 6

C.

1. Renewal of Cell Popula~1on • .. .. .... 6

2. Cause. of Cell R8n ... l .. • • • . . ,. :I.. Method. of Det.8rm1na't1on. of t.he

Ga.nt.1oft Cycle of cells · . . .. . . ..

.. . . . . .

10

11

18

26

III. MATERIALS AND MB'l'HODS • • .. • • *' • • .. • ... 21

V.

FINDINGS ..

DISCUSSION

,"i · .. .. .. . . . .. .. .. . . .. .. . ..

· .. • • • .. . . .. . .. it • • . . .. 35

VI. StJMM.AR'!l AND CONCLUSIONS ,.....:..... •• :41

BIBLIOGRAPHY .. ,. .. .. ..... ,. •• .. • • ... 43

APPENDIX • • .. .. .. · .. .. .. .. A. Photomicroqraph. • .. • .. .. B. 'l'able • • .. • • .. • .. .. .. c. Gzoaph • • • • • • • • • •

.. " .. .. .

.. .. • • • .. j, • • .. .. • • .. ..

.. . • •

• •

.. •

4' 58

59

L

CHAPTER I

INTRODUCTION

1

In the adult mammal, larqe number of aells are

produaed and lost everyday. All aell populations increase

in number durinq the period of qrollth of the orqanism. In

some cell populations, the rate of proliferation is <Jreater

than that needed to sustain qrowt:h, this means that some

cells are lost and replaced by newly formed aells. Such

cell populations are aalled nCell renewal systems"

(Leblond 1956). However, upon aompletion of the qrowinq

period for a given renewal system a steady state is estab­

lished, Cell ~renewal> balance. cell los ••

The f~r.t author to rais. the question whether

physiologic regeneration of epithelia actually slows with

aqe was Ortiz Picon (1933), who studied the epidermis of

abdomen and baak of mice from birth to 33 months of ag8.

At 6 months the rate of division was twice as hiqh as at one

month and remained hiqher than at one month for the entire

period studied. In 1949, Bullouqh found the mitotic::

frequency in mouse epidermis to be considerably hiqher in

middle-aqed. than in younq animals even before they have

reachad full size. Marwah (1956) found the mitotiC frequency

in human qinq1va to be 50% h1qher 1n the older aqe qroups.

2

The.e tinding. contradict the common concept ot

aging, according to Which bodily proc •••••• low down and

the r8gen.rative oapacity ot the ti.sue. dimini.he. with

age. Th ••• observation. would point -to the po •• ibility

that the mechanisms ~ which normal cellular prolit.ration

i. controlled are reduced in the older indiVidual. The

incidenc. ot mali9ftaney i. known to incr .... with age.

Marwah state. that one might .uspect the tull emancipation

trom control ot the tull tledged malignant. c.ll i. prepared

tor ~ the partial 8mancU.pation t rom control Which oacur.

with age.

Th. pre.ent study will attempt to •• timate the

g.n.ration cycle ot the oral .pithelium in 600 day old miae.

In partiaular this .tudy employs the "direct graphic method"

to .tudy the renewal ot cell population.

CHAPTER II

REVIEW OF THE LITERATURE

A. HISTOLOGY OF THE ORAL MUCOSA

Kutuzov and Sieher (1953) described in detail

the dorsum of the mouse tonque.

Macr08copically, the dorsum of the tonque can be

divided into 3 reqion., eaoh oharacterized by a specific

type of papillae.

1. Anterior portion of 8imple conical papillae.

2. Intermolar em1nence characterized by the qiant

papillae.

3. Posterior portion beset with the filiform

papillae.

The stratified squamous epithelium of theae three areas is

keratinized.

Sonntag (1924) pointed out that thou9'h animals

compri8in9' the Order of Rodentia are very diversified the

lin9'U&l characters do not differ very much specifically.

In 1959, Medak qava a detailed aacount of the

oral epithelium of the mouse. ae described it as having a

lamina propria consistinq of connective tissue cells and

fibers, a basement membrane with collagenous and reticular

fibers and a stratified squamous epithelium.

3

4 The basal layer of the epithelium haa a sinq1e row

of columnar cella. They are perpendicular to the baaement

membrane. The cytopla.mic nucleic acid content. are hiqher

in the.e cells than the cella fOrming the rest of the epithe­

lium. The.e nucleic acid contents decrease peripheralward.

The strat.um apinosum bas larqe polygonal cella with

atar-ahaped outline. and intercellular connections. Kerato­

hyaline granule. are found in the cell. of the stratum

spinoaum in later staqe. of oellular differentiation. In

1967, CUtright stated that the keratohyaline granules, whlch

are eventually shed with maturing cells, do not conta1n any

DNA component.

Madak (1956) described the "ratum qranulosum as

havlng no residual cytopla.m and the unstained Clranule. are

of uniform 81ze only adjacent to the compact keratin areas.

There ls evidence of phosphamidaa. actlvity 1n the kerato­

hyaline granules. CUtright (1967) noted clear apaces in the

nuclei of oells approaohing the keratin layer and attrlbuted

lt to the normal loss of their nuclear DNA in the oour.. of

kerat.lnization.

Leblond (1956) described the morpholoq1cal change

in the ba.al cell layer. A. th... cella move outwards they

abu<,Je from columnar to polyhedral and finally to flattened,

granule containing c.lls, whlch then transform abruptly into

a homoqenoua qla.ay layer.

5 This, in turn, ohan~.8 into soft k.ratin.

In 1967, CUtright atated that the normal oral

.pith.lium haa the property to aohieve oytoplaam1c diff.ren­

tiation while und.rgoinq nucl.ar deg.n.ration coupl.d with •

mechanism by which the DNA rele.aed from k.ratinizing c.lls

i. reutiliz.d by newly proliferating cell ••

Normal hlstol9.9X Of aging oral soft tissue,

Wentz .1 Al (1952) founcS an absenc. of aqe ohanqes

in the thickne.. of epithelium and length and confiquratlon

of the epith.lial ridges of the gingiva. Rllnqaberg (1960)

found no major alterationa in the keratin lay.r and the

ov.rall .pith.lium with incre.sing a~e.

MArwah (1956) found 80me ag. Chang •• in human

qinglva aa followl

(1) a deore... in incidence of a granular layer (2) a decrease

in inoidence of parakeratosis and (3) a decrea •• in the

.. aociation of a granular layer with paraken'tosis, (4) an

incre .. e in cell density by a third. a. attributed that in­

cr .... in cell d.nsit.y t.o be due in pan. t.o lower wat..r

content of the t1.sue and in pan. to the smaller sl.e of cell ••

Burzinak1 at Al (1965) repon.ed:.::.t.hat there was no

aignificant difference in the palatal t.issues with ag1nq

between sax.. of',: the same age groups I and 1n palatal tissu ••

of l-year-old and 4-year-old animals in non-collagenous to

collagenoua ratiOS.

B. CELL RBNBWAL SYSTBMS

I. Renewal of Cell Population

6

In 1956, Leblond r.viewed in detail the proce ••

of the renewal of oell population. He attributed the lack

of in~reat of classical histologi.t. in the subject to

their belief that new cell. were proc!uae4 by "amito.1s" a.

well as by "m:Lt081." and therefore the true rate of pro­

liferation could not be estimated. However, in 1955, Ria

poin'ted out that amitosis consist. of a _re nuclear

fragmentation, a proce.. which venerally precludes further

proliferation. Thus mito.i. 1. the only means of cell

production, and the number of new cells formed in this

tissue.

Howard (1953) divided the "9eneration cycle" into

the followinq phas •• , S phase (DNA syntheSiS time) durinq

Which sufficient DNA is synthe.ized to form a new complement

of chromosomes, G2 ph .. e which ls an interval between S

Phase and beqinninv of mitoais, M pha •• in which mitOSis

occur. and a Gl phase (rest.ing- phas.) betwe.n termination

of mitosis and c:onmencement of the S ph .. e. The perlod

~ween the two mitosis ls called an .. int.rphaa .... - In 1963,

Patt. pointed out that. the duration of Gl (restlnq pha.e)

vari.s wid.ly, ln typical mammalian cell syetems, and is

malnly responsible for the very larqe difference. ln cell

cycle time amonq varioua tis.ues.

7 ~he time required for one generation cycle to occur is

referred to as the "qeneration Ume" which should be clearly

distingui.hed trom the "turnover time" which i. defined as

the time taken tor the replacement at a number of cells equal

to that in the whole population (Leblond 1956).

In 1960, Messier considered the stratified squamous

epithelium ot the tonque as an example ot renewing cell popu­

lation with a "restricted proliferation site." New cell.

are always for..ed in the stratum germ1nativum of the epithe­

lium, since only cell. in this layer are labeled (that i.,

synthesizing DNA prior to mitosis) one hour after isotope

injections. ae further stated that cell "miqration" i.

common in the tongue epithelium a. shown by the migration

of a labeled baaal cell to the upper strata. CUtright (1967)

concluded that the greater the speed ot epithelial cell

turnover, the more cells miqrate.

CO'Wdry (1942) reported that the dauqhter cells of

a cell dividing in the reprodUCing layer may either become

member. of the surtace layers in whiCh no mitosis occur

(Poat mitotic cell) or remain in the qerminative layer and

there undezvo further mitosis (veqatativ. intemitotics and

differentiating' interm1t~ic.) •

. In 1965, Leblond pointed out that two dauqbt.er cells

Of a mitosis coma out independently and at random, thus the

division of a bual cell may qive two basal, or one basal

8 and one spinous, or two spinous cells. He considered the

coming out ot the basal layer as the cri't.ical step I after

Which the evolu't.ion of the cell is irreversible up to

keratiniza'tion and sheddinq. He concluded, theretore, that

on the average halt ot the cell. ari8ing from mito.is diVide

again and half transtorm into spinous cell., thus, there is

no change in the number ot cells of the epithelium and the

steady state ot the cell population i8 maintained.

In 1961, CUtright demonstrated that the maintenance

ot epithelial homeosta.is i. helped by the reutilization of

the DNA, released fram keratinizing cells, by the newly

proliterating cells_ He poin~ed outt.hat this recycling

could be important when rapid regeneration is needed, since

it make. the epithelium le.. dependent on blood-born DNA

precursor. and provides a local pool of e •• ential DNA

metabolite ••

Henry (1952) used the "colchicine method" to

•• timate the rate ot regeneration ot the rabbit's oral

epithelium and tound it to be "S.5" days. Since then,

many workers have qathered "data" on the "turnovert.1me"

ot t.he oral epithelium. They estimated it to be between 3

days (Gott..sbe1'ge 1963) and 5.S days (Cutriqht 1961) tor

the various portions ot the oral mucosa. These values are

.ummarized in Table I.

Toto (1962) conducted an investigation of the

qeneration cyele of the epithelium of the tongue in mice

in which he concluded that the duplication staqe lasts for

ten hours. In 1964, Dhawan found that the synthesis time

9

1s eiqht hours for the palate and doraa1 surfaae of the

tonque, and 81qht and one-half hours for the ventral surfaae

of the tongue, in the 60 day old miae.

Leblond (1956) pointed out that variations in the

•• timation of the duration of mitosis were probably due to

observational difficu1ti.s, since prophase and telophase are

hard to delimit in livinq ae1ls. Henry (1952) concluded

that the mitosis required 64 minutes 1n the oral epithelium

of rabbit. In cue of mice's oral mUCOsa 1t was found to

require 40 minutes by Medak (1959). This findinq is in

agreement with tho.e of Toto (1962) and Dhawan (1964).

In 1908, Minot studied the epidermis of rabbit

embryos at varying age ranges, he found different mitotia

indiaes in various tissu.s, thus establishint evidence of

.differential rate. of q«."Owth. He further, described the

mitotia index (MI) as the number of cells to be found at

any given moment in the active proc... of division out of

a total of 1,000 cells. Mitotic index is used to measure

the renewal time. Messier (1960) pointed out that mitotic

index is not altogether satisfaatory to assess the rate of

10

cell proliferation in a tissue beaause the counts of mitosis

in sections are proportional not only to the number of cell.

entering mitosls, but a180 to the time taken by the.e cells

to complete vislble mitosi., also, mitotlc figure. are

difficult to reooqnlze in small and elongated cells.

II. Cause. of Cell Renewal

In 1963, Bertalantfy assumed that cell renewal is

a protective mechanism. ae stated that many epithelia are

almoat continuouslYf:·exposed to .tress of varlous kinds, e.g.,

mechanical (oral epithellum) I enzymatic (duodenal epithelium),

bacterial agents (respiratory epithelium), to site a few,

therefore, were there no cell renewal, aells of varlous

epit.helia might .oon become abraded by mechanical contact or

dige.ted by proteolytic enzyme.. Vulpe (1954) found that

irritation due to urine causes mitosis ln the transitional

epithelium of the bladder. Le.her (1964) estimated that the

turnover time Of duodenal epithelium of the germ-free mice

i. longer than that of conventional mice. ae postulated

that the germ-free envlronment reduce. attrition of cells

on the villi and the re.ulting decreased need for renewal 1.

recoqnized and responded to by decreased cell production.

In 1956, Leblond demonstrated that most renewal systems,

though responslve to extrinsic force., are capable of re­

newing in their ab.ence, e.g. , cells were renewed even in

isolated intestinal loops not exposed to the mechanical

.tres. that may be exerted by inqe.ted food. Accordinqly,

the ability to proliferate is an inherent property of the

cells of the renewal system.

11

Leblond (1965) pointed out 'that the addition of

new cells by mitosis tends to increase population pre.sure

in the basal layer .s shown, often, by the racket shape of

the cells leav1ng this layer, as if squeezed out by pressure.

Bierman (1955) sugyested that cell. may be el~

1nated by hormonal factors as s.en 1n caee of lymphocytes

by the adrenal cortical hormone (hydrocortisone).

Harvey (1963) stated that the production rate in

hematopoietic tissues is death-controlled. That is, ad­

Justed by variations 1n the mortality of newly formed cells.

Berta1anffy (1963) concluded that the component cells of

tis.ue. with cell renewal are replaced at constant rate.

governed by the steady state, whether they are stres •• d at

any particular time or not.

III. Methods of Determination of the aeneration cycle of

Cella

There are various methods for determination of the

rate of regeneration of cella preferred by different workers

for different reasons. some of the methods used will be

described.

12

,MitO!ig, ln~~ - is the simplest approach. Est.imation of M. I.

involves little interterence wit.h the tissue and therefore,

a minimum of artifacts. An important disadvantaqe of this

method is t.hat no measurement. of time 1s involved and

therefore t.he rat.e at Whlch t.he cells of a tissue are re­

placed cannot be calculat.ed. It. finds its main applicat.ion

when relatlve measurements are needed, for 1nst.ance, in

comparlng t.he effect of various aqenta on t.he renewal of

epldarmal cells. (Leblond 1956).

£o!c.hlg,l!!a_M.!t~ - Colchicina, a plant alkalOid, has tha

property of blOCking mitotic divisions at metaphase and

was used by Henry (1952) in oral mucosa of rabbits. It

has the main disadvantaqa of altaring the rate at. whlch cells

enter mitosls.

~-.la.x - Lesher (1961) sWigested the use Of x-ray irradiations

for this study of the reweneration of cells. It stops early

prophases 1n the material treated in this manner •

.£e!l_L,!b,!l!n,i - Cell. can be labeled with radioactive i.o­

topes, Vital dye, etc., and then traced through their life

cycle. The turnover time of cell population can then be

calculated by measuring either t.he rate at Which the labeled

cell. form, or the time they spend 1n a particular compart­

ment of t.heir migration path, or t.he rate at which they are

lost from the ti •• ue. (Leblond 1956)

13

Volkman (1950) aa8.ssed the renewal time of the

epidermia by meaaurinq the rate at. which India ink injected

into the deep layers was c:arried to the surface.

Leblond (1965) atate. that the advent of radio­

autoqraphy qave hi.tology a new dimenaion, time. When cella

double their DNA prior to mltosl., they take up varlous DNA

precursors. If labeled precursors are injected lnto animals,

they are incorporated ln divldinq cella, which are then

t.awed anet may be traaed in radioautographs.

Phosphate-p32 , Adanine-c14, and c14 formate have

been used ln thls manner (Lajtha 1954). However, the firat

one of the.e preaursora 1s taken up int.o a varlety of

substances besldea DNA, the second and th1rd lnto both RNA

and DNA and only "Thymidine" ia taken into DNA alone

(Relchard 1951). Amano (1959) ob •• rved that I after

thymidine-H3, radioautoqraphi<: reactiona were present only

over nuclei, and that pretreatment of the sections with the

enzyme Deoxyribonuoleaa. prevented the oacurrenoe of

reactions. It may, therefore, be atated that the only

raetioa=lve substance present. in •• ctiona after labeled

thymidine inject10n 1a DNA, and that any cell tound to be

labeled ls about to d1v1de by mitoaia. Thymidlne haa the

advant.aqe ot be1nq aval1able wlt.h a trltium (H3) label,

Whlch produce. Beta-ray wlt.h such a low energy that they are

recorded in t.he phot.ographic emulslon very Cl0S8 to their

site of emission, the re.ult is a f1ne radiographlc reso­

lution (Taylor 1951).

Me.sier (1960) used thymidine Hl for hi. studi ••

of the cell proliferation and mJ,gration ln male rat. and

mice. The frequencY of labeled cella ls proportional to

14

the duration of tbe duplicatlon lItaq_. A close relationship

exi.ts between the labeled thymidine uptake and mitosis ln

the tissues. He further .tates that the radioactlve index

(perc:entag'e labeled aells) i. a rOug'h lndex of the rate of

cell dlvislon, slnce it is lntluenaed by the rate of incor­

poration of the label into the inter,mediate substance leading'

t.o DNA synthesl. and by the duration of the perlod of DNA

synthesls. Neverthele.s, the radioactive 1rldax has two

advantaqes. (1) labeled cella are more readl1y ldentlfled

than mitotic flgures in many ti.sue. and are many times as

numerous, (2) the cells t.aqqe4 by t.hymidlne-H] could be

traced lon, after mito.ls had been accomplished.

There is a 41fference of opinion., among' sclentlst.,

a. to the extent of injury to chromosomes caused by

thymidine-H] • Schoenhe14er (1960) I reviewed the method of

autoradioqraphy, as.ume4 that the chemical behavlor of the

labeled substance 1s ldentical with that of lts stable

countexpart. It 1. llke a "tracer."

\

15

Leblond (1965) stated that the autoradiography 8hows movement

under physiological conditions, on the other hand Quaatler

(1959) pointed out that this method of inv •• tiqation is not

physiological, because of the qrowth factor in young' animal.,

the stres. of lnjec~lon and the injury to the chromosomes

caused by triated t.hymidine. In 1962, Toto concluded t.hat

there is no m1.n1mal threshold for triated thymicU.ne at which

injury t.o the chrOmosomes does not oc:c:ur.

Lesher (1961) u.ed both the graphic method and

the staqe durat.ion index one to determine t.he qeneration

t:Lme. He concluded that the qraphic method 1a superior to

t.he at.aqe durat.ion indek one because the lat.t.er require.

preci.. knowledge of the 81z. of the proliferative pool and

of the duration of a particular staqe of the qeneration

cycle which are difficult to obtain. Its only advantage is

t.hat it can be applied wit.hOut observing' the complete

qeDerat.ion ayele. Waher (1961), Toto (1962) and Dhawan

(1964) calculat.ed the 9eneration cycle of cells by a4dinq

the DNA synthesis per104 (s), a reet. period (G2), the dura­

tion of mitosi. eM) I and t.he interphase CG1 ).

Lesher (1961) reported t.hat. the qeneration time

computed by the staqe durat.ion index formula,

Number of Cells in Mito.1a = Duration of Mitosi. Number of Calla in Inte~ha.e = Duration of Interphase

i8 unreliable proportioned to the deqre. to which the ••

estimate. of the mitotic duration are unreliable.

In 1967, CUtriqht used a pure hiatoloqic and a

mathematical technique to determine the turnover time of

16

the Stratified squamous epithelium. The histoloqic method

expressed the renewal time as thet:..1me required for the

last initially labeled cell to pas. from the stratum qermi­

nativum to t.he kerat.in layer whioh was used as an end point

because it normally contains no living cells and t.hus no

labeled nuclei. On the other hand, the mathematical method

used the racU.oac:tlve index (percent.age labeled cells) of the

basal layer and calculation i. al.o baaed on the t.1me needed

for DNA aynt.hesis, 7.5 hours. The following formula was

used.

100 x 7.5 Radloac:t.i ve Index 24 hour.

He pointed out. t.hat t.hi. approach is based on the assumpt.ion

t.hat. all newly formed cell. derive f~ the basal layer and

that., therefore, the percentage of proliferat.ing' cella

within the baaal layer raflecrt the t1.tmover .tat.e. He

considered the mathematical method to be superior t.o the

histolOjJic one.

17

TABLE I

TURNOVER TIME OF ORAL EPITHELIUM

Author Source P'ind-and Method of lnqs In

Year Used Material Reqion Days

Henry Colchicine Rabbit Oral Mucosa 8.5 (1952)

Toto Autora41o- Mice Tongue Epithelium 4.1 (1962)

KoWrq Autoradio- Mice Hard Palate 4.4 (1962) grapby

;BJlu:talanffy Colcbicine Rat Buccal Mucosa 4.3 (1963) Tongue Epithelium 4.9

Gottesberqe Autoradio- Mice Ginqival Epithelium 3.5 (1964) qraphy

Ohawan Auto radio- Mice Palatal and Dorsal 4"""--(1964) qraphy Tongue Epithelium

Ventral Tonque 4.1 ....-Epithelium

cutright Autoradio- Rat Post Dorsal Tongue 3.2 ../ (1967) qraphy Epithelium

Anterior Tonque 3.5 Epithelium

Ventral Tonque 5.1 Epithelium

Hard palate-Ginqiva, 4.3 attac:hed

18

c. FACTORS AFFECTING MITOTIC ACTIVI'r'f

Wherever mitotic activity i. pre.ent in adult

mammalian ti •• u.. its rate is commonly variable from hour to

hour and from day to day. It is always evid.nt, however,

that there .xist strict limits to this variation and that

these limits are characteri.tic for each particular tis.ue.

Amonq the factors known to influence the daily variation in

mitotiC activity are.

Hormonal. Many workers in the past have d.termined

.. that endocrinological influenc.. are important to the rate of

mitotic activity. Earthly (1951) showed that "thyroxin." in

.mall do •• s d.pr ••••• mitotic activity and that Ute.to.t.rone"

in mal. enhanc.s the rate of cell division. In 1950, Bullouqh

suqq •• ted a po •• ibl. mechani.m for thyroxine's action. Energy

r.quired for mitosis is supplied by qlycogen. Now, thyroxine

has the well known ability to deplete tissues of qlycoqen,

and con.equently deerease mitotic activity. Leblond (1955)

stated that "Growth hormone" accelerates mitosis. Bullouqh

(1961) showed that mitotic activity is siqnificantly increased

in "adrenalectomized" or "hypophysectomized" animals.

Bullouqh (1946) pre.ented evidence that ·the "e.trone" level

in the female mouse affected the renewal of epidermal cell ••

However, in 1961, he stated that while the mitotic rate of

a tissue may be influenced by hormone., it is not controlled

by them. The findings for some of the investiqators are

summarized in Table II.

19

Physioal Stimulation I Carleton (1934) found that

continuous exposure of animals to light caused an alteration

in the rhythmical periodicity of mitosis. In 1955, Peters

suqqested that irritation causes cell proliferation, since

it i8 noticed that at the edqes of a wound the mitotic

activity may rise to more than ten times the normal rate.

Bullough (1960) found that even qentle massaqa causes a

marked increase in the epidermal mitotic rate both invitro

and invivo.

Temperature, BulloUg'h (1949) concluded that

mitotic activity is high during lowered temperatures and

low durinq higher temperature. Blumenfeld (1939), in a

study of mitosis 1n the epidermiS of the albino rat, spec­

ulatedthat the early morning increase was related to a

high body temperat.ure at that time • In 1950 I storey found

a rise of mitotio activity at a temperature of 25° - 30°C.

in rats epidermi8. However, Leblond (1956) is of the opinion

that in an integrated animal there is probably no effect

because of the internal temperature regulation mechanism.

Blood syqar Levels, Diller (1946) pointed out

that starvation lowers mitotic activity in the rat intestine.

In 1949, Bullough observed the existence of a direct relation­

ship between mitotiC activity and blood sugar levels.

,

20 He found that epidermal glycogen content increases durinq

sleep when the mitotic rate increases and considers the

glucose or glycogen to be the critical substance effecting

the mitotic aotivity 1n the epiderm1s of the adult mouse and

of man. In 1954, he reported evidenoe that a moderate in­

orea.e 1n insulin conoentration may lead to a raised epidermal

mitotio rate. O'Connor (1954) reported that the mitotic

activity of a tissue 1s dependent on the rate of carbohy­

drate metabolism.

Gluoose OXidation. Orban (1942) USing a repeated

application of 30% hydrogen peroxide whioh in turn produced

monomeriC oxygen, found the basal oell layer of human

gingival epithelium to form from four to six layers of oella

with a remarkable increa.e in mitosis. Xn (1961) Garqiulo

after .ingle exposure. of 30% hydrogen peroxide for a

thirty-day period, found an apparent prolongation of the

mitotic period in the attached interdental gingival epithe­

lium of humans. This seemect to be more specifio in prophase.

This apparently gives evidence that the mitotic process is

retarded by the colchicine-like effect of hydro;en peroxide.

Medawar (1947) found that anoerobic conditions prevent both

epidermal mitotic activity and epidermal migration. By

re-introducing oxygen it is possible to obtain a normal rate

of division in the same cells. In 1951, Bullough concluded

that mitotic activity could be increased by stimulating the

21

golucose OXidation. Th. number of mitoses can be increased

by stimulating ~. rat. of .nergy production from qlucoa.

oxidation. Thi. has it. .ffect immediately prior to the

prophase which appears to be a critical ttma in the

histophysiology of mito.is. However, GliCkman (1950)

discouraged the assumption that the artificial introduction

of oxyg.n would appreciably hasten the normal cellular

proce •• e. in the cour.e of gingival healing.

Str.... Bullough (1952) demon.trat.d that .tre ••

ia an influencinq factor in mitotic activity and that the

qlucocorticoid hormone. play a role in the antimitotic

mechanisma. In 1954, Maoapanpan found an increa.e in

activity of fibroblasts in the periodontal ligament space

When abnormal force was exerted on the teeth.

Radiation a Chase (1961) found that the mito.i.­

inhibition .ffects of ionizing radiation on the normal

oral epith.lium of the buccal mucosa in human caused a

reduction in the number of cella. In 1963, Patt stated

that different transitions in the proliferative cycle are

blocked for different lengths of time by a given dose of

ionizing radiation.

Chemical.. H.nry (1952) reported. that "colchicin."

arr.sts all m1to.~ •• special1y in the metaphase stag.. In

1958, Orr demonstrat.d an increase in the mitotic activity

of the epidermis and hair follicles by paintinq the skin

22 with "croton oil" or ItMethylocholanthrene."

Mitotic RhythmiCitYI Many investigators have

shown the existence of a diurnal mitotic: dycle in both

animals and man. As yet, however, there is no agreement as

to the timing of peak mitotic activity.

Bullough (1947) reported that there are hiqher

mitotic activity with sleep and lower mitotic activity with

those of wakefulness. Experimentally, the proliferation

could be either increased by inducing sleep with an anes­

thetic or decreased by having an animal run in a revolvinq

box to keep him awake. In 1961, he attributed this diurnal

mitotic cycle to the mitotic inhibition caused by adrenaline

Any mouse when awake is evidently under suffiCient stress to

cause an increased adrenal in output and consequently a

reduced epidermal mitotic rate, conversely a sleeping mouse

with less circulating adrena11n shows a raised epidermal

mitotic rate. on the other hand, Muhlemann (1954) found a

night low and day hiqh in retromolar epithelium and perio­

dontal membrane of five month old black male rats. Henry

(1952) found a maximal activity of 7.2 and a minimal activ­

ity of 3.8 mitOSis per 1.000 cells in the oral mucosa of

a 3~ month old rabbit at 1.00 P.M. and 10.00 P.M.,

respectively. A summary of these values is found in

Table III.

Other Factor.. In 1960, Marwah found that

I'chronic inflammation" increase. mit.otie activity in 50%

of the ea ••• ob.erved in both age group ••

Halberg (1954) showed an inhibition of mitotic

activity in the connective tissue of the int.rdental

papillae of rats which were treat.d with large dos.. of

.. corti.on ....

23

Thuring.r (1924, 1928) found reqional "differene.s. II

H. counted on. mitosi. for 2,414 cells on the saalp, for

378,325 ael1s on the leg, and 268,275 on the .ar.

storey (1949) described "seasonal variations"

in the mitotic activity of epidermis. He recorded the

turnover time of .pith.lium during summer (9.8 days), in

the autumn (16.3 days) in the wint.r (18.8 days).

Marwah (1956) reported that the pr.sence of a

"granular layer" and the presence ot "parakeratosis"

influ.nae mitotiC activity. The former markedly r.duced

the mitotiC rate, the latter increased it slightly.

24

TABLE II

HORMONES AND ITS EFFECT ON MITOTIC ACTIVITY

Hormone.

Author Year Stimulate Inhibit

Bullouqh 1946 Estrone

Earthly 1951 Testosterone Thyroxin

Bulloug'h 1952 Testosterone Glucocorticoid

Bulloug'h 1954 Insulin

Halberg- 1954 Cortisone

Leblond 1955 Growth Hormone

Bullough 1961 ACI rana 1 in Nor-ACIrenalin

25

TABLE III

MITOTIC RHYTHMICITY

Time of Source Mitotic Cyclea

Author and of MaXimal Minimal Year. Material Q~an Activity Activity

Carleton Miee Hair Follicle 8,00 P.M. 12,00 A.M '(1934)

Blumenfeld Rat Skin 8100 A.M. Early (1939) Evening'

Bullouqh Mice Skin 6,00 A.M. 10,00 A.M • (1947) 2.00 P.M. 8,00 1l.M •

Henry Rabbits Oral Buccal laOO P.M. 10.00 P.M • (1952) Mucosa

Halberq Rats Retromolar Niqht Morning (1954) Epithelium

. and Perio-dontal Membrane

Mul1 emann Rats Retromolar Day Night Pad

26

D. AGING AND MITOTIC ACTIVITY

In 1933, ortiz Plcon lnvestigated the rate of

division in the .pld.~s of abdomen and back of mice from

birth to 33 months of ag.. He found that at six months the

rate of divlsion was twlce as hlgh as at one month and

remalned higher than at one month for the entlre perlod.

Bullouqh (1949) reported that mitotlc frequenoy ln mouse

epidermis ls oonslderably hlgher ln mlddle-aged than ln

young anlmals even before they have reached. full slze. On

the other hand, K11junen (1956) found that the mltotic index

in the epldermis of young rats ls hlgher 'than that of older

ones.

In 1961, Lesher investlgated the effeat of age on

the generatlon tlme of the mouse duodenal eplthelial cell.

He found that the fraction of the crypt populatlon ln the

S-8tage ls practlcally independent of age.

The first mitotlc lndex of human oral mucous

membrane was publlshed by Marwah (1956). He reported that

the frequency of mitosis ln eplthelium of human glnglva ls

5~ hlgher 1n older age groups (SO to 78 years) than ln

younger age groups (25 to 55 years). A study on human

attached glnglval epithellum by Garglulo (1961) showed age

ohanges ln the same dlreetion. In 1956, Marwah stated that

thls flndlng would seem to contradlct the oommon conoept of

aging, according to which bodily processes slow down and

the regenerative capacity Of the tiS8ue diminishes with

age. He concluded that the concept of aging as a general

slowing down of physiologic regenerative process cannot

be maintained for epithelia.

27

In 1956, Falzone indicated that age changes which

are not demonstrable in resting tissue can be discerned

under condition of 8tress. Carter (1956) te8ted the

reaction8 of gingiva to a brushing stimulus at different

age8. He found that brushing led to a significant increase

in the thickness of cornified cella and the height of the

epithelial rete pegs. He, a180, noted that within the

range studied (early adult to middle age) aging per se

was without significant influence on the thickness of the

cornified layer or of the epithelium. On the other hand,

Klingsberg (1960) investigated the adaptive potentiality

of the oral epithelium, to an acute stress, with aging of

the individual. He nOted a significantly greater increase ~

in the adaptive capaCity on the part of the younger animal

a8 compared to the older one.

In 1961, Lesher reported that the generation time

and heterogenicity of the cell population is increased.

28

He concluded th~t whatever mechanism is responsible in old

age, the reproduction of epithelial cells in the intestinal

crypt ot mice is changed.

A review of the literature reveals no studies

reported on the effect of age on the generation eycle of

oral epitheliwn.

Materials

CHAPTER III

MATERIALS AND METHODS

Twenty-four 600 day old mice, a strain from C57,

Black and Albino, weighing an average of 37 grams, and

being fed a diet of Wayne Lab Blox for mouse or rat were

injected intraperitoneally with thymidine-H3 (specific

gravity 1.9 auriesper milli-mole) at a dose rate of one

miarocurie per gram of animal weight.

The time of injection was recorded for each

mouse to assure preaise timing at sacrifice. The mice

were sacrificed at intervals of a quarter of an hour, 1,

9, 10, 12, 14, 24, 30, 44, 54, and 100 hours.

29

The tongues were dissected out, cut mid-sagittally,

fixed in formalin, dehydrated with ascending series of

alcohol, and embedded in paraffin. Seetions of 3 micron

thickness were made.

Methods

Autoradioqrama were prepared by a modified

Fitzgerald's (1959) technique using Kodax NTB3 emulsion,

the exposure time of 30 days at 40 e was used. The slide.

were developed in KOdak 0-19 developer for 5 minute. at

190 e. The slides were rinsed in distilled water and fixed

in aci4 fixer with hardener (Kodak). The sli4.s are then

washed in runninq water, cleaned and the tissue sections

stained, throuqh the overlyinq emulsion layer, with

(1) Hematoxylin and eosin or (2) Nuclear-faat red and

indiqo carmine.

The times at which the first labeled prophase.

and telophases were .een were recorded. OWinq to the

difticulty ot identifyinq prophase. and telophases in

beavily labeled cells, only metaphases and anaphas.s were

scored. One hundred mitotic tiqures tor each int.rval of

time were observed tor each ot the specimens under pres.nt

investiqations and the percentaqe of labeled mitotic

tiqures was determined.

The staqes ot mitosis are identified accordinq

to DeRobertis (1954) standards, as tollows:

30

1. prophase is identified by an increased visibility

ot chromatin threads, and an increased basophilia

of nuclei.

2. Metaphas. is recognized by the absence ot a

nuclear membrane and the chromosomes arranqinq

themselves: along' the equatorial plate.

3. Anaphase i8 characterized by tbe separation ot

the dauqhter chromosomes in the undivided cell

body.

4. Telophase is identified by the division of the

cell body into two daughter cells.

A curve plottinq the percentage of labeled

mitotic fiqures as a function of time wasprapared. The

two coordinates representinq 50% labeling on the ascending

and descending limbs of the curve were located and the

time interval recorded a. the DNA synthesis time.

Radioactive index: Under high dry magnification

"400 basal cells" c:ontiquously were counted in the one

hour interval sections. The number of labeled nUClei

observed in each of the 400 cell. was then recorded. The

count of labeled cell. was divided by 4 to give the per­

centage of labeled cells.

The followinq formula was used to estimate the

turnover time I

100 Radioactive Index

DNA syn'th.sis time 24 hours (1 day)

Where t.he radioactive index i. the percentaqe

of labeled call. at 1 hour period. The DNA synthesiS

time is taken from the curve.

31

CHAPTER IV

FINDINGS

The stratified squamous epithelium on the dorsal

aurfaee of the tongue 1n 600 day old mice, at any given

aaorifice period showed always some oells in preparation

for mitOSis.

At the f1fteen minute interval, following in­

ject10n of the T_H3, some labeled cella are observed in

the basal oell layer of the epithelium. Theae labelad

cells are randomly distributed.

Within the first hour of injection, the time

that triated thymidine 1s available, it i8 bound in the

DNA of the oells 1n the S-phase fram whioh it oannot

escape during the period under consideration.

At one hour interval, the basal cell layer of

the dorsal surface of thetonque showed a radioactive

index (percentage of labeled cells) of 10 which represents

the percentage of the oell population in the S stage at

injec:tion.

Only cells in the stratum germ1nativum are

labeled one hour after isotope injection. Also nuclei

of fibroblasts and salivary glands oontained the isotopes.

(Figure 3)

32

33

In the course of 8 hours the number of labeled

mitotic figures rose rapidly from zero to 100 percent

(ascending limb), where it remained for about 4 hours

(plateau), then fell rapidly to 50% at 14 hours (descending

11mb). Graph 1

No labeled mitotiC figures were found either

at 24, 30, 44, 54, or at 100 hours following the injection

of the triated thymidine. (Table IV)

The first labeled prophase. are observed at 15

minute. and the first telophase. at one hour following

the injection with triated thymidine. (Figure 1, 2)

The time interval between the coordinate. extra­

polated on the curve a. repre.enting fifty percent labeling

is 12 hours (Graph I).

Between the 8 and 14 hour interval labeled cells

are observed in the stratum germinativum, often paired and

showing a reduction of grains over the nuclei, appearing

approximately half as many grains, as the originally

labeled cella. The paired cells were, uaually, arranged

as tollowwa 2 baaal, one baaal and ofta spinous or two

spinous. (Figure 4)

Many cells had migrated tZ"Om the stratum

germinativum to the upper layer of the .eratum spinosum

at the 24 hour period. (Fiqure 5) ~FGBOO~~\ .( 0 LOYOLA ·11

UNIVERSiTY 1/

Labeled cells are obaerved near the stratum

corneum at the 44-54 hour period. Clear apace. are noted

ln the nuclei Of oells approachinq the keratln layer.

Individual trltlum expo.ed .ilver grains are observed

out.alde the nuclel of the migratlftg' oella undel'901nq

ke rat 1nlzat. ion • (Flqure 6, 7 )

One hundred hours following the inJectlon of

TH3, a marked deerea.e ln number of heavily labeled Cells

le observed as compared t.o the 44-54 hour periods. Many

cel18 ln t.he stratum qerminatlvum contaln. nuclel d1s­

playinq 2-4 tritlum exposed .11ver qralna. (Flqure 8)

34

CHAPTBR V

DISCUSSION

~e stratified squamoua epithelium llninq the

dorsal surface of the tongue is constantly renewed by

multiplication of cells in the stratum qerm1nativum. This

findinq ia supported by Leblond (1965) who stated that

cell proliferation occurs within the basal layer of the

epithelium, since only cella in this layer of the stratum

qarm1nativum are labeled one hour af,ter iSotope injeat:.ion.

Med.ak (1959) a180 pointed out that in the oral epithelium

of mice, the baaal cella contributed 6~98% in the

renewal of the cell population.

At any qiven tim., there are alway. some cella

in the S phase. It i. durlnq this stag-e that the injected

triat.ed thymidine ent.ers the DNA molecule in place of the

naturally occurinq thymine. < The perc.nug'e of labeled

35

cells (radioactive index) found one hour followinq in­

jection represent. all .taga. 1n DNA synthesis aa determined

by the availability of the t.riate4 t.hymidine during this

time.. some labeled cells represented by the aacend1nq

11mb are in lat.e stag. of DNA synthesis period, those

represented by the 4e.eendlnq 11mb are 1n an early phase

of synthesis and thoae shown on the plateau are in some

interval in bet.ween.

...

Th. number of c.lla in the S stag. at the time

of inj.ction 18 10% of the cell. in the verminative lay.r.

It indicates the rate of homeo.tatic epithelial call re­

placement. Thi. ob.ervation i. very clo.. to the radio­

active index, at tha one hour perioct, estimated by Toto

(1962, 1966) and Cutright (1967) for the stratum vermi­

nativum of the dorsal epithelium of the tongue 1n the 300

and 400 days old mice anet 100 day old rat.. Therefore,

the fraction of the cell population 1n the S stage at the

time of injection is practJ.cally independent of &qe. It.

36

is concluded t.hat there ia no increa.. in mitotic activity

oocuinq wit.h aqing'. Furthermore, lAsher (1967) inv •• tiqated

the generation cycle of the mou.e duodenal .pithelium. ae

found out that the fraction of the crypt population in the

s atage at the time of injection 1. .mallet in the older

animal. than in the younqer on.s. The •• finding. dl.aqr.e

wit.h previOus reports of an increased mitotic actiVity

with age (Ortiz Picon) (1933), Bullough (1949), Marwah

(1956) and Gatgiulo (1961).

~The radioactive index increas.. .s the ttm. of

.acrifice inor.a.... Thi. i. due to the division of the

l.~led cell population a. indioated by the paired labeled

cell showinq approximat.ely balf as many qrains per nucleus

•• t.b. originally labeled cell.

37

Mitotic division, migration and maturation of

the epithelial cells occur completely at random to assure

the maintenance of a uniform eplthellum and a steady state

ot the cell population ls, consequently, aChieved.

Bertalantfy (1962) st.ated t.hat ln spite of mit.otic formation

of cell., in the adult, organs with cell renewal inorease

neither in siza nor weight. This ia because identioal

number of cel1111 ara oontinuously be1n9 extruded from the

o%9ana.

The fint labeled propha... are obaerved at

fifteen minut.. folloWing the injection of tr1&te4

thymidine. The intelVal of time between tthe end Of DNA

synthesis and prophase, which i. known as G 2' DlUS"t. be 1 •••

t.han 15 minut.s but. more than zero 1. e. I approximately

12 minutes. The first. labeled telopha.e. are •• an at one

how: I the period Of approximately 48 minute. is est.imat..d

1::.0 be required for mito.is. The •• findin'l. are close to

those caloula1::.ed by Toto (1962, 1966) and Dhawan (1964)

for the dorsal surface of ~e t.ongue epithelium of adult

anet yoUft9 m1ce. They 8IIIt1mat.ed a G2 IMtriod of twenty

minutes and forty minute. required by the cell division.

The DNA eyntheai. time tor the doraal surface

of the tongue is 12 hoU%'8 .s determined by extrapOlation

on the curve.

Toto (1962) found it to be 10 hours in the adult mice and

Dhawan (1964) found it to be 8 hours in the 60 days old

mice. It is concluded therefore that the DNA synthesis

time increases with age. This observation is supported

by Lesher (1961, 1967) who found out that the S phase is

longer in the older animals than in the younger ones.

Cutright (1967) u.ed the followinq formula

100 Radioactive Index (at 1 hour period)

to calculate 'turnover time.

)( DNA Synthesis Time 24 hours (1 day)

In the pre.ent studies, thfr

39

calculations based on such a method show that the qen.ra­

tion cycle for the dorsal surface of the tonque epithelium,

in the 600 days old mice, to be 5 days (120 hours). No

autoradiographic studie. have been published which could

be used for comparison. Toto (1962, 1965), Dhawan (1964)

and Cutriqht (1967) used the autoradi09'raphic technic to

estimate the turnover time of the dorsal surface of the

tongue epithelium in 300 days, 400 days, and 600 days old

mice and 100 days old rats. They found it to be 4.1 days,

3.7 days, 4 days and 3.2 days, respectively. It is

concluded that the physiologic regeneration of epithelia

slowed with aqe. This findinq agrees wi'th the common

concept of aginq, accordinq to which bodily processes slow

down and the regenerative capacity of the tissues diminishes

/with age.

Lesher (1961), Toto (1962, 1965) and Dhawan

(1964) calculated the generation cyole of oells by adding

;' the DNA synthesis (s), a rest period (G2), the duration

39

of mitosis (M), and the interphase (Gl ). Using this method,

the Gl phase is estimated to be 107 hours. The preparation

of the cells for DNA synthesis occurs in Gl , it is suggested

that this preparation period is prolonged.

Epithelial cells in the process of maturation

und.~o karyolysis, karyorhexis and the formation of

keratohyalin. granules. The loss of their nuolear DNA in

the course of keratinization is indicated by the scattering

of labeled particles of DNA around degenerating nuclei as

soon as karyolysis begins.

The labeled DNA particles, scattered in the inter-

cellular spaces, may be taken up by the "basal eells lt in

the S phase 1.e., the DNA extruded from maturing epithelial

oells may be reutilized. Cutright (1967) stated that the

reutilization of intra-epithelially released DNA is independent

of the Circulation, since, it occurs in tissue explants and

does not include the oells of the submueosa •.. tturthermore,

the labeled DNA is inoorporated first by those epithelial

cells which lie near DNA-releasing oells.

He concluded that squamous epithelium, with its unique

property of nuclear degeneration during physio1oqic

maturation, thus conserves the fragmented nuclear material

for the local use of newly formed cells. This recycling

may be important when rapid regeneration is needed, sinoe

it makes the epithelium less dependent on blood-borne

DNA precursors and provides a loeal pool of essential

DNA metabolites.

40

CHAPTER VI

SUMMARY AND CONCLUSIONS

Twenty-tour 600 day old mic., a strain ot CS7 '

were injected with triated thymidine intra peritoneally.

They were sacrificed at intervals ot trom lS

minutes throuqh 100 hours.

The tonques were dissected out and cut mid-

aag-itally.

'"" . Autoradioqrams were prepared and stained with

(1) Hematoxylin and Eosin or (2) Nuclear - fast red and

indigo carmine.

The number of labeled cells in the stratum ge~

inativum 1 hour after injection was determined.

One hundred mitotiC tigures tor each interval

of time were counted tor each ot the specimens under

investigations and the percentage of labeled mitotic

figures was determined.

A curve plotting the percentage ot labeled

mitotic tiqures as a tunction of time was prepared.

41

The DNA synthesis time is 12 hours, G2, the

interval between end DNA synthesis and prophase, 12 minutes1

mitotiC time 48 minutes, interphase, 107 hours. The esti­

mated generation cycle ot the dorsal surface ot the tongue

epithelium is 5 days (120 hours).

The stratified squamous epithelium lininq the

dorsal surface of the tonque is constantly renewed by

multiplication of cells in the stratum qerminativum.

At any qiven time, 10% of the epithelial cells,

of the dorsal surface of the tonque in 600 day old mice,

are in preparation for mitosis.

Mitotic division, miqration and maturation of

the epithelial cells occur completely at random to assure

a uniform epithelium and maintain a steady state of the

cell population.

There is no substantial difference in the

duration of G2 and Mitosis (M) in the old animals as

eompared to the younqer ones.

The synthesis time, interphase (Gl ) and the

qeneration time of the oral epithelium are increased with

aqe.

Epithelial cells in the proces. of maturation

loose its nuclear DNA into the surroundinq intercellular

spece ••

The DNA extruded from maturinq epithelial cell.

may be reutilized by the basal cells in the S phaae.

The physioloqle reqeneration of oral epithelia

slowa with aqe.

42

BIBLIOGRAPHY

Amano, M., Me.sier, B., and Leblond, C. P. "specificity ot Labeled Thymidine as a DNA Precursor in RadioautO<,1raphy, If J. Histoehem. Cytoehem., 7.153, 1959

Berta1anffy, F. D. llMitotic Rates and Renewal Ti.sues of the Diqestive Tract Epithelia in the Rat," Acta Anat., 40,130, 1960

Berta1anfty, F. D., and Lau, C. IICe11 Renewal, II International Review ot Cytology, 13, 357, 1962

Bertalanffy, F. D. "Cell Renewal aa the Ba.i. of Diaqno.tic Exfo1iat.ive Cytology,lI Am. J. Obat. and Gynec:., 85, 383, 1963

Bierman, H. R., Kelly, K. H., and Cordea, F. L. Ann. N. Y. Acad. sc., 59. 850, 1955

Blumenfeld, C. M. "Periodic Activity in Epidermis of .. Albinos Rat.," SCience, 90, 446, 1939

Bullough, W. S. "Mitot.ic ACt.ivity 1n t.he Adult Mous., tI

Philoa. Tran. B. 231. 452, 1946

Bullough, w. S. "Mitotic Activity in the Adult Male Mouse. The diurnal Cycle. and Their Relation to Wakinq and Sleepinq," 35. 212, 1941

Bul10ugh, W. S. "The Relations Between Epidermal Mitotic Activity and the Blood SUg'ar Level Of the Mouse Musculus," J. Exp. Biol., 26. 261, 1949

Bul10uqh, w. s., and Eiaa, E. A. J. Exp. Biol. 27: 275, 1950

Bul10Ug'h, W. S. Tension,"

"Bpidermal Mitotic Activity and OXyqen Nature 167. 488, 1951

Bul10U9h, W. S. "The Enel9Y Relat.ions of Mitotic Activity, .. Biol. Rev., Cam. Philos. Soc. 27. 133, 1952

Bu110uqh, W. S./ and Laurenee, B. B. ItTh. Control of Epidermal Mitotic Activity in the Mouse,·t ~. Roy. Soc. B., 151. 517, 1960

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44

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49

figure 1 At 15 minutes in'terval. (A ) the firs 't prophase is observed. (H & E X 750)

Figure 2 At 1 hour interval. is observed. (H & E X 750)

50

(A) 'the firs 't t :elophase

Figure 3 At 1 hour interval. All labeled cells are in the stratum germinativum. (H & E X 300)

51

«

Figure 4 At 12 hours in'terval. All mitotic figures are labeled.

52

Daughter cells can be seen: (A) 2 basal cells (B) 2 spinous cells (C) 1 basal and 1

spinous cell (H & E X 750)

Figure 5 At 24 hours interval. (A), (B) & (C) represent labeled cells migrating from the basal layer ,to the stratum spinosum. (H & E X 750)

53

. ,

Figure 6 At 44 hours interval. (A), (B) & (C) represent migrating labeled cells in the upper layers of the stratum spinosum. (H & E X 750)

54

«

55

Figure 7 At 54 hours interval. (A) & (C) represent labeled cells in the process of degeneration and showing scattering of labeled DNA particles outside 'their nuclei. (B) represents an unlabeled mitotio figure (early Telophase). (H & E X 750)

rt

Figure 8 At 100 hours interval. Scattered labeled DNA particles can be seen in the stratum germinativum. (A) represents a labeled (2 grains) mitotiC figqre (Metaphase). (H & E X 300)

56

•

58

TABLE IV

EPITHELIUM - DORSAL SURFACE OF THE TONGUE IN 600 DAY OLD MICE

THE NUMBER OF MITOTIC FIGURES COUNTED IN EACH INTERVAL OF TIME IS ONE HUNDRED.

Hours (Following the Percentaqe Injection of Mean Labeled Labeled Triated ThYmidinel Mitotic Figures Mitotic F1iures

~ 0 0 1 40 40 8 100 100

10 100 100 12 100 100 14 50 50 24 0 0 30 0 0 44 0 0 50 0 0

100 0 0

~ 0

1 ~ 0

~ ~ ~\ 0

~

~

z 0 , - ~ -~ 0 ~ -/

~ 0

~ 0

-o o

APPR.OVAL SHEET

The thesis submitted by Nabil J. Barakat has

been read and approved by the director of theth.sia.

FUrthermore, the final copies have been examined by the

director and the signature which appears below verifi.. the

fact that any neces.ary chanqes have been incorporated,

and that the t.he.is is now 9iven final approval with

reference to content and form.

The thesi. is therefore accepted in partial

fulfillment of the requirement. for the Deqre. of Master

of Science.

~d.i1/@,Z Date Siqnature of Advisor