Title: Cellular mechanisms of beta radiation inhibition of corneal wound healing /
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Permanent Link: http://ufdc.ufl.edu/UF00097731/00001
 Material Information
Title: Cellular mechanisms of beta radiation inhibition of corneal wound healing /
Physical Description: 243 leaves : ill. ; 28 cm.
Language: English
Creator: Morrison, Dennis Robert
Publication Date: 1970
Copyright Date: 1970
Subject: Beta rays -- Physiological effect   ( lcsh )
Animal Science thesis Ph. D
Dissertations, Academic -- Animal Science -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
Thesis: Thesis (Ph. D.)--University of Florida, 1970.
Bibliography: Bibliography: leaves 231-240.
Additional Physical Form: Also available on World Wide Web
General Note: Typescript.
General Note: Vita.
Statement of Responsibility: by Dennis Robert Morrison.
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Bibliographic ID: UF00097731
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000437276
oclc - 24663988
notis - ACJ7352


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To my pa-ents,

Mr. and !rs. M ':: .. M.r rison,

for 23 -e3ars

of in piratei n, cncour2ee.e:: and g id lnce.

The auhnor would like to express his gratitude for the valuable

assista-ic ad-. guidance given him by his c'lairman, Dr. George '. Davis,

his co-chair-..an, Dr. Herbert E. iauf.an and the other members of his

supervisory C-:r'.citee, Dr. Paul R. Jllioct, Dr. R.E. Shirley, Cr. John

Feaater .nd Dr. Fhillip Achev.

He is sincercil grateful for the council .nd guidance given him by

his chair!in, -r. K'eorge K. Davis, during his tenure in the Cellular -nd

MLolecular Biolog.' doctoral program.

The aut-Uir is especially indebted co his co-chairman Dr. Herbert E.

Kaufman, of the Denartment of Cplithalnolog', v:ho provided the facilities

and encouragement that made chis research possible. In addition, a most

sincere thank you is given to Dr. Kaufman for his inexhaustible enthusias-m,

suggestions, insight and compassion that gave direction and inspiration

to the author during the period of this research. He indeed has been the

author's men.or.

The author also expresses his deep appreciation to Dr. Antonio Casset

for his invaluable assistance, instruction, enthusiasm and indoctrination

in Dr. Gasser's dynamic method of research.

He is most indebted to Dr. Atsushi Kanai for his generous efforts in

tutoring the author and helpii1 hi- decipher the intricate intracellular

world observed through the electron microscope.

The author would like especially to express his deep graditude to

Miss Carcle Anes of the Veterans Administration Hospital researchh unic

for her invaluable assistance in the preparation of the iler.se number

of histologica] s .eciens used throughout this research.. Her boundless

patience- and abili't to do such crenendous wor'. while enduring che

daily -:i.tic is- of the author, has r.ade chis research :-sible.

A m.st ap':-reciative than!,s is given co "'rs. E nily Ellison for her

prepDration of che gra-h illustrcrions used herein ard for her technical

assistance a.~nd end.ess compass on in the day to day trials of this


The author would like to e:x:ress his deeo appreciation co his

brother, Jarry, f-r hi.3 in"2luable help in the e::ecution of .anv. of

the e::uperiencs, in collecting and tabulating much of che daca, and

in the compilation of this dissertation.

He would like to thank Mr. J.esoph Capella for his insight sr.d

thoughtful suggestion., and Mrs. Claire Chiappini of the Departoent of

Ophthalmology for her concern, help, and friendship.

He also e:%pressae his gratitude to the staff physicians, residents,

and others of the Department of Ophthalmology for cheir intellectual

and moral support.

Finally, the author would like to e::press his thanks co his

parents for their enccuragem:nt during chis study, and Miss Elaine

Las-ola for her tireless efforts in the preparation of this disser-



ACKNO;.-L E DGME S . . . . . . . . . . .

LIST OF T.'iLES . . . . . . . . . ii

LIST OF FIGURES- . . . . . .... .. . . . .. viii

LIST OF .'.B 'I TIO S . . . . . . ... . . . ..:iii

ABSPTRACT. . . . . . . .... ... .... . .. xiv


1. GEN1EPRA I!-TRODUCTION:. . . . . . . . . 1

Wound Healing .. . . . . . ..... 1
Corneal Wound Healing ..... . . . .
Corneal Metabolism . . . . . . . ... 13
Beta Radiation in Ophthalmciogy . . . . . 15
Inhibition of Wound Healing . . . . . .. 19
Radiation Inhibition of Wound Healing ...... 22



The Clinical Course of Corneal Healing . . . 34
Tensile Strength of Corneal Wounds . . . ... 39
Methods . . . . . . . . . . 41
Results . . . . . . . .. . . . 42
Com..ent . . . . . . . . ... .. A5


Methods . . . . . . . . . . . 67
Results . . . . . . . . .. . . 68
Comment . . . . . . . . .. . . 7S


5. E.ZY 7 HISTOCH7 '.ITSTRY . . . . . . . . 1'3

Enzn-ze Accivit: in Corneal Healinr . . . . 143
Ma ods . . . . . . . . . . 145
Resu . . . . . . . . . . 151
Co ent . . . . . . . . . . 153


e h ds . . . . . . . . . . 163
Results . . . . . . . . . . 175
Com e . . . . . . . . . 189

7. GE ':EFr'.L DISCUSSION' . . . . . . . ... 220

3. CO:CLUSIO:'S . . . . . . . . . . .. 23

BIBLIOGRAPHY . . . . . . . . ... . . . 231

BIOGR PHICAL SK TCH . . . . . . . . ... 2.1


1. Tensile Scrength of Corneal Wounds
Irradiated at Surger; . . . . . . . . . 43

2. Tensile Scrength of Cornedl Wounds
in Eyes Irtadiated Three Months Prior to Surgery . . .. 43

3. Empirical Evaluacion of SDH Accivicy . . . . . ... 152

4. Labeled Cell Counts for D:A Snchesis . . . . ... 179

5. Relative nuclear DNA Content in Healing Cornea . . . . 181

6. Labeled Cell Counts for R:.\ Synthesis . . . . . . 183


1. technique or surgical wounding, graefe knife
ccMpletinr sec ion . . . . . . . . 33

2. oun.ding co:.plece, incision sutured . . . . . 33

3. Control healing, after nine days of healing . . .. 49

4. Healing at nine days in cornea that was irrldiated
two and one-half months before wounding . . ... 49

5. Healinr; in rin-irradiaced eye at day 20 . . ... 51

6. Cornela healing in contralateral eye (right eye of
same anL.al as above) at day 20, following beca
radia i i e iacel after surgery . . . . . 51

7. Healina ar day 20 in non-irradiated cornea
(left eye) ...... . . . . . . . 53

8. Corneal healing in con:ralateral eye (right eye
of sane animal in :!7) at day 20, following beta
radiation ir~mediately after surgery . ... . .. 53

9. Control eye healing at nine months . . . ... 55

10. Healirn after nine months in contralaLeral eye thae
had bean irradiated two and one-haif months before
wounding . .. . . . . . . . . 55

11. Healing in non-irradiated cornea at day 11 . . .. 57

12. Cornea ::ound after 11 days of healing, in cornea chat
received 10,000 rads of beta radiacion ten months
prior to surgical wounding . . . . . . 57

13. Healing at 21 days in sane control as shot.-n in
Fig. 11 . . . . .... . . . . . . . 59

14. Healing in same irradiated eve as sho.:r in Fig. 12
after 21 days of repair . . . . . . . . 59

15. Control cor.-ea after nine weeks of healin, . . .. 61


16. Contralateral eye, irradiated ten months before
wouni-.n, shown at nine weeks after wounding .... 61

17. Development of complications during inhibited healing 63

18. Histological section of control corneal woun ac
three weeks of healing . . . . . . . . 84

19. Section of wound in concralateral irradiated eye
ac day 21 . . . . . . . . .. . . 84

20. Electron photomicrograph of basal epithelial cell
in control corneal wound, three weeks after wounding 86

21. ElectroLn phocomicrograph of basal epithelial cell in
irradiated corneal wound, three weeks after wounding 88

22. Eleccron photomicrog--aph of basal epichelium near
periphery of wound in irradiated cornea . . ... 90

23. Electron plocomicrograph of basal epithelial cell in
center of irradiated corneal wound . . . ... 92

24. Electron photomicrograph of stromal fibroblastic
cells in anterior stroma of corneal wound ...... 94

25. Electron photomicrograph of fibroblast-like cells in
irradiated corneal wound . . . . . . ... 96

26. Electron photomicrograph of fibroblast-like cell in
anterior stroma of non-irradiated corneal wound . . 98

27. Electron photomicrograph of fibroblast in posterior
stroma amid collagen . . . . . . . ... 100

28. Electron photomicrograph, middle stroma of
irradiated strcca at three weeks . . . . .. 102

29. Electron photomicrograph of newly synthesized
collagen at three weeks of healing (PTA stain) . .. 104

30. Electron photonicrograph of central wound only
isolated collagen fibril amid fibrin filaments . . 106

31. Electron photomicrograph of endothelium in control
cornea at three weeks . . . . . . ... 108

32. Electron photomicrograph of posterior stroma at three
weeks of healing showing fibroblas: anid collagen ... 110

33. Electron phoconicrograph of detailed fine
structure of endo:haliur- ............. 112

34. Eleecron photomicrograph of E.-ochelial cell
junction at three weeks s of healing in normal cornea . 114

35. Electron phocomicrograph of posterior wound in
irr3diSted cornea after three weekss of healing . . 116

36. Electroi ohocoricrograph of fibrir filament detail in
irradiated corneal wound at three weeks of healing . 113

37. Photoricro6raph of anterior corneal 1 found after nine
weeks cf healing in cornea irradiated cen months
before wori ning . . . . . . . . 120

38. Phocomicro2raph of posterior irradiated wound
after nine i :eks of healing . . . . . . . 120

39. Eleccrcn photomicrograph of irradiated corneal
epiitheliu- covering nine-ueek wound . . . . . 122

40. Electron photomicrograph of anterior stroma in
irradiated cornea after nine weeks of healing . . 124

41. Electron photomicrograph of stroral cells in nine-
week wound in cornea irradiated ten months
before wounding .. . . . . . . . .. 126

42. Electron photomicrograph of collagen and fibrin in
nine-week ..'ound of irradiated cornea . . . .. 12S

43. Electron photomicrograph showing detailed fine
structure of endothelium at nine weeks of healing
in irradiated wound . . . . . . . . . 130

44. Electron phoconicrograph of endotheliu-n and
regenerating Descemet's membrane in nine-week wound
of control cornea . . . . . . . . . 132

45. Electron photomicrograph of endotheliuiL covering
nine-wsek wound of irradiated cornea . . . ... 134

46. Photomicrograph of control wound after nine months
of healin . . . . . . . . ... . .... 136

47. Photomicrograph of ni.e month wound in cornea
irradiated three -.onths before surgery . . . 136

48. Electron photomicrograph of stroma after nine
months of healing in pre-irradi3ted cornea
(PTA stain) . . . . . . . . . . 138

49. Electron photomicrograph of endotheliun and e'escemc's
membrane covering nine-mon.h '.:ouna in pre-
irradiated cornea (PTA stain) . . . . . . 140

50. Electron photonicrograph of endothelial ulcrascructure
after nine months of healing in irradiated cornea . 142

51. Phocomicrograph of non-irradiated rou-d shoving
Succinic Dehydrogenase activity (Nictro-Er Ctain) . 163

52. Photonicrcgraph of non-irradiated corneal wound
deor.scrating Lactic Dehydrogen.se activity
(:itro-Br stain) . . . . . . . . ... 163

53. Photomicrograph illustrating histochemical stai',in'g
of 5-nucleocidase in non-irradiated corneal wound . 164

54. Autoradiograph of Thynidine-3H labeled cells in non-
irradiated wound after 68 hours of healing (DN.) . 195

55. Autoradiograph of 68-hour wound in irradiated
cornea (DNA) . . . . . . . . . . . 195

56. Autoradiograph of Thymidine- H labeled cells in
non-irradiaced wound after 91 hours of healing (DNA) 197

57. Autoradiograph of 91-hour wound in irradiated
corn a (D A) . . . . . . . . . 197

58. Autoradiozraph of DNA synthesis in 120-hour
control wound . . . . . . . . . . . 199

59. Autoradicogr2ph of DNjA synthesis in 120-hour irradiated
wound . . . . . . . . ... .. . .. 199

60. Autoradiograph of Th.yidine-3H uptake in
corneal epitheliumn at 95 hours of healing ...... 201

61. .Aucoradio-raph of Uridine- H uptake in corneal
apithelium at 95 hours of healing . . . . .. 201

62. Graph of labeled cell counts for DNA synchecis rin
corneas irradiated at surgery . . . . . . 203

63. Graph of labeled cell counts for CNA synthesis in
corneas irradiated two and one-half months
before surgery . . . . .... . ... 205

64. Graph of labeled cell counts for D'NA synthesi: in
corneas irradiated ten months before surgery . .. .207

65. AutoradioLrarh of Uridine- H uptake in nrn-irradiaced
corneal wound after 4S hours of healing ...... .209

66. Autoradiosraph ofL Uridine- H upoake in irradiated
corneal wound afler AS hours of healing. ...... 209

67. Autoradiograph of Uridine- H uptake in anterior woucind
of control cornea at 96 hours . . . . . ... ?11

6S. Au'oradiograph of Uridine- H uptake in anterior .wound
of irradiated cornea at 96 hours . . . . .. 211

69. Autoradiograph of 96 hour control corneal wound
illustrating stroal uptaee of Uridine-3; ....... .213

70. Autoradiograph of control corneal wound illustrating
Uridine-3H uptake in endotheliu.- at day 20 .. .... 2.13

71. Autoradiograph of five-day control wound sho.:ing
RNA synthesis at endotheliu. . . . . . . 215

72. Autoradicgraph of five-day irradiated wound sho-:ir.
RNA synthesis ac endotheliu- . . . . . . . 215

73. Graph of labeled cell c-n c :s for ?tA s-,r.:.esis in
corneas irradiated at two and one-half months
before surgery . . . . . . . . .. 217
74. Autoradiogr'.ph of 35S incorporation into sulfate
mucopoly --cchnrides in 20-day ..ound of non-
irradiated corneas . . . . . . . ... 219


1. DNA .......................

2 EA ........................

. AP .... ...................

7. LE -DH .....................

6 LDH .......................
7. LET .......................

80. NAAD .......................
9 NAD ,......................

11. NADPHI-.....................

12. P m .I .......................

13 P IA .......................

14. SD .........................

deoxvribonuc]eic acid

endoolasfic retici t-lum

flav.in adenine dinucleocide

.g] yceraldeh-de-3-hoas3hace


la:cic deh;ydrogenase

linear energy crnn.ser

.nicotiranide adenlne dinucl

.reduced nicocinanide adenin

nicotinanide adenine dinucl
phace (TPN)

.reduced nicocinamide adenirn
CIhos h';

polymorphortuclear leukocyte

ribonucleicc acid

succinicc dehydrogenase


eotide (DPN)

e dinucleotide

eocide phos-

e dinucleotide


Abstract of Dissertation Presented to the
Graduate Council of the Universit.- of Floridz in Partcial 7lfi!l:.:ent
of the Requ'iirer.nnts for :he Degree of Doctor of Philo3sphy



Dennis P.oberc :lorrison

August 1970

Chair.;an: Dr. GCorte Dav'is
Co-Chairnan: Dr. Herbert Kaufmran
Major Department: Division of Biological Sciences
Cellular and :;olecular Bioilgy Program
Department of Animal Science

Rabbit corneas were treated with 10,000 rads of beta radiation

from a Stron;tiuL-90 ophthalmic applicator up to cen months prior to or

immediately after surgical wounding. The clinical ;cc:rse of healing in

the irradiated corneas was contrasted ;.ith the nr-'al healing observed

in the non-irradiated contralateral corneal wound. Tensile screnoth

measurenrrnts ..ere correlated with light and trans-ission electron i-icro-

scopic s-_ici- of the corneal wounds. Histocher-ical enz,-ie studies were

carried ouL to evaluate the radiation effects on enzyme activities induced

by wounding the cornea. Isotopically labeled precursor studies using

autoradiograiphic :ec:.i ues were used to determine the njuber of cells

synthesizing D''A and RE:A in the wound area. Mlicrophotor,-etric measurements

of Feulgen stained nuclear DNA were used to quantify the total D:.N content

per nucleus. Statistical interpretation of aucoradiographic grain counts

per cell :;cre used to approxji.'ate the rate of RN'A synthesis of the involved


The inhibition of corneal '.wound healing by beta radiation was shown

to involve many incracellular changes which had profound effects on the

efficiency of the normal healing process. Beta radiation effects were

found to be separate and different for the three najor layers of the cornea.

A lack of censile strength development in the irradiated corneal

wounds azcpared to be a result of a lack of synthetically competent cells,

correlated i.-th the absence of collagen synthesis in the wound area. In-

duced succinic dehydrogenase activity was reduced by the radiation effect,

while 5-nucleotidase, ;ADH.., diaphorase, and lactic dehydrogenase activities

were unchanged.

ITlcrastructural alterations resulting from the beta radiation treat-

ments were correlated with inhibition of stromal collagen synthesis, and

a decrease in RNA synthesis in all cells involved in the immediate wound.

These findings together with the lack of regeneration of epithelial base-

ment membrar.e and Descenet's rnembrane suggested that the protein synthesis

capability of the cells had been inhibited by the beta radiation.

A reduction in fibroblastic proliferation was notably consistent with

a reduction in cells synthesizing DNA and the total D;IA content per cell


Partial recovery of the healing capability was demonstrated when

a ten month recovery period elapsed between the irradiation treatment

and the subsequent surgical wounding.

The many couple:: effects of the radiation inhibition of the cor-

neal healing phenomenon suggest that some comprehensive control mechanism

was inhibited by the radiation, rather than inhibition of a single criti-

cal metabolic or functional scep in the norr'a! repair process. Th: pos-

sibility of radiation interference with the mechanism t.hac injLia-cs :;.cnd

repair and the potential damage to Di.A s.nchesis and coding are discusseJ.



Wound Healing

There is scarcely any question that wound healing and regenera-

tion are important in the life and survival of a species. What con-

stitutes the difference in connective tissues response between species

which results in full regeneration of a limb for one species and

cicatrication of an amputation in another species? This perennial

questioil remains as a challenge since Eighteenth Century scientists

first proposed to methodically characterize the schematic process of

tissue regeneration in mammalian species.

A vast diversity of scientific knowledge has been accumulated

to describe the co.nple: phenomenon of i:ound healing. Various reactions

between cells guide the process of healing. Many mechanisms are inter-

woven to control the course of movement and mitosis of involved cells,

to eliminate the damaged cells through degeneration, and to determine

from moment to moment the quantity and kind of intercellular material

secreted and destroyed. The final result is manifested in the modulation

of cell form, collagen and extracellular fabric, and eventually the

process of wound contraction.

In skin w'ou;ds, epidermal regeneration begins fi-mediately and

progresses chrou.!i chree st res in the reconstruction of c;he corium (1).

The first s age i .'c -,-called "latent" period in whir.-, fi rcblascs

proliferate, migrace, and assemble. It is characterized by a low,

collagen ccntent n.d low tensile stren.ch of the ~:ound during the

first fe': days after injury. This latent phase, later renamed the "sub-

strate" phase, has also been sho.-n to be a period of intense biochemical

activity (2). The second stage is characterized by fibrcplasia which

is indexed by a decline in the proliferation of fibroblasEs, intense

collagen synchesis, and the fornacion of fibers conco-.itanc with

increases in che tensile strength of the .:ound. The third stage

involves macuration of these fibers, aggregacion into bundles, and

eventually contraction of the wound.

As yet the delineation and explanation of the incerrelationship

of these sequences of skin healing remain unfinished and challenging.

Direct correlacives in the healing of the cornea provide ar. e::cellent

opportunity to study the comple:-ity of both normal and abnormal corneal

repair, as well as the influence of various agents on the processes.

Corneal Wound Healin?

The cornea is a unique tissue for the studies of wound repair as

it is avascular and transparent. Anatomically it is arranged in several

layers: (1) a stratified columnar epichelium, (2) an amorphous membrane

(Bow.man's), (3) a stromal layer, composed of collagen fibers imbedded

in a mucopolysaccharide ground substance chat includes a space. but

evenly distributcd, complement of fibrocyces (keratocytes), (.) a

second r:.bci-rane (Descemont's), and (5) an endothelial cell lavet.

The architecture and physiology that determines the transparency

of the cornea also dra:c.-ically influences the repair processes that

occur in. this structure. These parameters include the functional

integrity and capability of the endothelium. the screngch. regularity,

and arrangemnen of collagen fibrilS of the scrona, the nature of the

mucopolysaccharide ground substance, the integrity and physiology of

the endothelial layer, and ultimately the state of decurgescence of

these tissues.

Fu:ctionally, the three najor layers of the cornea have separate

and distinctly different roles in the process of healing. Briefly, the

epithelial cells expand and slide to recover a defect in the epichelium.

They music then multiply to re-establish the normal multiple cell-

layered barrier to the en'.ironrment. The stroma is important in pro-

viding strength co the cornea; therefore, during :ound repair, stromal

cell activity is predominantly thac of synthesizing new protein and

collagen. This requires morplhological transformations of keratocyces

and other invading cells into fibroblasts which svnchesize new materials

and proliferate. The endothelium is a highly differentiated cell

layer who.e prime imncrran:e is that of a selective barrier and pump

that mainLains the state of deturgescence of the cornea. The endo-

thelial cells, therefore, are not required to multiply following injury,

but only to expand, slide over the defect, and re-escablish the selective

regulation of fluid flow through the cornea.

Considering :he different functions of these r.3jor layers in the

normal homeostasis of the cornea, it is appropriate to discuss the de-

tails of the healing process categorically by chese layers.

Corneal Enitheliu.

The enpiCheii'j covers the corneal strona acting primarily as a

regulator of fluid and electrolyte e::chan2es and as a barrier to loss of


Renewa a

Cells of the basal layer of epitheliu. divide by micosis about

once weekly (3). These basal cells are the stem cells for the stratified

squanous epithelial cells. migrationn of the basal epithelial cells

outward into the spinous layer occurs independently from the mitosis

giving rise to the cell (4). The migration to che superficial layers

follows miuosis. The epithelial cell turnover terminates ultimately in

the superficial keratinized layers where the cells are finally de-

squa.aced inte :.e tear fil.

Repair of the epithelium

Defects in the epithelial layers are first covered by a process

of spreading and mizration by pseudcDcdial e::ctension from the surrounding

intact epithelial cells. Each cell can cover several times its normal

area by expanding" and flattening out. Recovering by lateral migration

begins approximacely one hour after injury and concludes .;ith complete

covering of the strc.na by a: least one layer of epichelial cells. The

initiation of lazeral call slidin3 apparently depends upon a breach of

cell-to-cell contact. producing a loss of normal "contact inhibition"

to cell migration (3).

Epithelial r..tosis is depressed during the sliding and covering

process due to ocme inhibitory effect of the mechanical insult. 'litotic

activity returns aftEr appro:-:imately si:: hours and accounts for the

eventual replacement of the normal five to si..: cell layers several days,

or e.'en !eaeks, lacer (6).

The: successful rener:al of the epithelium and permanent recovering

of the injury depends both on formation of an intact layer of epithelial

cells and the re-establishmenc of a cight adhesion to the underlying

stroma. Tight adhesion is dependent upon the integrity of the basement

membrane and re-ascablishment often requires several days (7).

The ultrastruccure of the epithelial cells changes during the

lateral sliding and recovering process. Most prominent is the initial

lack of a basement mem.brane in the basal cells during the first 36 hours

after injury. At this time surface cells possess villus-like processes

and cells in the depth of the wound incerdigitace. The nucleoplasm of

chose cells ::iEh intact nuclei is irregular and irregularly shaped

mitochondria appear as long, simple, organelles containing sparse in-

ternal menbranas and almost no cristae. After 36 hours the mitochondria

are increased in number, size, and complexity indicative of increased

metabolic activity (3).

Importance of Enichelium in Stronal Repair

The successful recovering of the anterior corneal surface by

epithelium hs other i-mlications in the normal physiology and repair

process; that occur in che adjacent stroma. Optimum increases in

tensile s:rength cf corneal wounds has been sho%.- to be dependent uDon

the pres-nce of epicheliu-i (9). Epitheliu- is necessary for the

efficient incorporation of sulfate into .mucopolysaccharides of the

stroma (10).

Weimar has ncced that the early stages of stroial healiE.e re

dependent upon the presence of epithelium. Apparently. a reconstituted

epitheliu-. is necessary for normal poly, orphonuclear leukocyte (P'2:)

invasion from the periphery and keratocyte transformation into fibro-

blasts. This epichelial influence is thought to be mediated by a

chemotactic substance released from the corneal epithelium upon injury


Bo.wman's Mem-brane

Along the posterior of the basal epithelial cell layer of the

cornea there is a basement membrane which separates the epithelium from

the stroma. In certain species, in addition to the basement membrane,

there is a region in which the collagen fibrils are randomly oriented

(12). These two basic layers make up the Bo%.an's membrane.

In the posterior layer the random orientation is contrasted with

the bundled laalle.r architecture of the collagen fibril network found

in the underlying stroza. Healing of Bci.-.oan's nc.mbrane is normally

considered part of the healing of the stroca since both layers are

composed of co-rparable auntsns of collagen. There is some question,

however, as to w:he'-h.r the complete structure of Bo:.:n's rmembrane ever

regeneraces. ow-rjan' s membrane is found in the normal human cornea,

but not in the cornea- of the rabbit (13).

Corneal S erc-na

The subs:a":'is propia, or stroma, of the cornea represents about

95 percent of :he total corneal thickness. Its main function is to

provide Frimary strength and body to the cornea structure. Fibrocytes

(keratoc.tes) constitute about 95 percent of the cells of the stronal

layer of the cornea. Cell division is very infrequent and keratocvte

turnover is quite slow. Transplanted keratocytes labeled with radio-

isotopes have been shown to persist in the strona for a year or more (14)

Collagen turnover in the corneal scroma is extremely slow with a

probable half-life of several years (15). Mucopolysaccharide turnover

is much faster, however, with a biological half-life of approximately

one month (16, 17).

Scro-al repair

The first resp-ise to injury of the stroma is pollymorphonuclear

leukocyte and mon:cy:te invasion within five to six hours (18). Morpho-

logical transformations of h-eratocytas adjacent to the wound edge begin

within 24 hours. After some 60 hours, approximately 25 percent of the

observed fibroblasts have been derived from the transformations of

keratecytes, 10 percent have been produced by cell division of these

fibroblasts, and about 53 percan: have. been transformed Ft :- invading

monocytes (19).

In healing of skin wounds, perivascular fibroblasts are thought

to be responsible for che eventual replacement of tissue losses.

Apparently, the major source of these fibroblasts is the proliferation

and transformation of local connective tissue cells (20). Once mitotic

division of the-e fibroblasts declines and fib:oplasia begins, their

role beco-es one of synthesizing new collagen for replacement of the

losses incurred in .- he wound. Intact fibrablascs appear necessary for

the synthesis of new collagen although poly-.erization ma.y ctually take

place e:xtracellularly (21). Fibroblastic synthesis of collagen is

indexed by: (1) uptake and subsequent release of sulfate compounds,

(2) uptake and subsequent release of proline which later appears as

hydroxyproline in the collagen fibers, and (3) the appearance of

tropocoilagen and the subsequent aggregation of tropocollagen into

collagen fibrils in the extracellular substance adjacent to the call

surfaces (22).

Fibrocyte als: ihave a rapid turnover of sulfate compunds;

however, their role in the rebuilding of net: collagen fibers and bundles

is unknow'.-n (23).

Fibroplasia in the first t-:o wee':s of corneal healing is indexed

by new collagen synthesis by the fibroblasts in the stromal area of the

wound. .':e.: collagen fibrils are first found in a disorganized arrange-

ment, but by three i.eeks they are organized in a parallel fashion

typical of the nor:.;al lamellar type architecture.

In severe screnal injury perilimbal vessels will invade the stroma

when severe stromal edema is present. It is thought that the edema

causes separation of the stro.al fibers, reducing the normal compactness

of the tissue and changing the state of deturgescence. Presumably,

the reduction of tissue pressure and the action of some cheaotaccic

factor then invites neovascularization (24). Stromal regeneration and

restoration of the normal stromal thickness and state of deturgescence

generally results in regression of these vessels, leaving only faint

tracts observable upon bionicroscopic e:-:amination.

Endothelial lined lymphatic channels also develop in severely

damaged stromas, connecting the more central stroma with the limbus (25).

These lymphatic channels persist for some time after inflammation has

subsided; however, they are only demonstrable with special histochemical


Scar formation in the cornea results in a concomitant loss of

transparency in the areas adjacent co the scar. Electron microscope

studies have sho'.-n 'tat the normal coll.-c-an fibrils are replaced by

fibrils w-ith greater variation of diameters (12). Changes in both the

normal architecture of the collagen fibers and in the amounts of extra-

cellular mucoFoly.sa:charides during the healing process are responsible

for the loss of transparency in the wound area. Restoration of the

normal lamellar arrangement, normal diameter of the collagen fibrils,


and normal mucocclysaccharide ground substance is required for rere':al

of corneal transparency.

Descemec's 'Mebrane

An amorphous layer, called Descemet's membrane, secreted by che

corneal endothelii': lies between the posterior stroma and the endothelial

layer. This n3mbrane has scronal collagen fibrils embedded in the anterior

portion adjacent co the scroma. Below the anterior ccrtion is an organ-

ized portion, different fro= the lae2llar structure of the stroma, and

below the organized portion in the human cornea, is the oosterior-most

portion which has no regular organization (26).

Descelret's rembrana contracts and rolls up at the free edges when

it is torn. A new membrane is secreted slowly by the endochelium, re-

quiring three months to attain one-half of the normal thickness (27).

Corneal Endothelium


2itotic figures are never seen ir. normal adulc corneal endotheliun.

Regeneratic.n cf the endothelial cell layer apparent: occurs b amitotic

division at a slow cuirn;.'er race (28). Anitotic cell division requires

approximately one hour and mitosis occurs only under unusual circum-

stances. The average life span of the corneal endochelium has been

calculated to be 344 da-:s in the rabbit.

Endothelial repair

Defects in the corneal endothelium are covered by endothelial cell

migration beginning within 12 hours after injury (28, 29). Some mitotic

divisions occur during the period required to cover the defect; however,

the predominant activity is that of cell enlargement, spreading, and

amitotic division persisting for several weeks.

Extensive damage to the endothelium may never be completely re-

paired resulting in an atea of severely edematous stroma directly anterior

to the area of incomplete endothelium and Descemet's membrane. A long-

term swollen condition of the cornea invites complications such as neo-

vascularization and non-specific inflammatory responses that may increase

the severity of symptoms of the endothelial defect.

Biochemical Synthesis in the Healing Corhea

A variety of biochemical syntheses occur during the initial phase

of corneal wound repair. Morphological transformations of keratocytes

into fibroblasts can occur from one to two hours after injury, followed

by dramatic increases in succinic dehydrogenase and 5'-nucleotidase

activity in keratocytes and fibroblasts beginning approximately six

hours after wounding (29). By two days an area approximately 200 mp

wide on either side of the wound edge contains fibroblasts and white

blood cells intensely rich in both enzymes. Although cytochrome

oxidase is not demonstrable in corneal wounds, another oxidase has

been found to be active within the first 24 to 48 hours after injury

and disappears between four and seven days (30).


Oxidativ.' enz'y.es normally present in the cornea increase

substantially after wcundin; (31). 'icocina.nide adenrine dinucleocide

(NAD) and nicocinn-ide adenine diroucleocide phosphate (:;AF) dia-

phorases both increase in the stromal and endothelial tissues fc'llcwin-

wounding of the cornea. Lactic dehydrogenase (LDM), malic dehydrogenase

and alpha glycerophosphate dehvdrogenase also increase follcwir.n injury.

Apparently, these increases are required as a result of the increased

metabolic;: acccTaanying wound healing.

Protein and ribonucleic acid (RA:) synthesis begir: i:ithin six

hours after wounding while deoxyribonucleic acid (D:.A) synthesis is

not apparent until appro.:i-ately 12 hours after injury (32). Leucine

incorporation into protein synthesis is especially high from 2-i to 72

hours after wounding. Uridine uptake and incorporation into RN.:.A syn-

thesis peaks at about 12 hours and remains intense throughout 72 hours.

Thymidine incorporation into DNA synthesis peaks around three to four

days after wounding; however, protein, RN.A, and D:A syntheses are all

reduced by eight days of healing.

Mucopolysaccharide synthesis is elevated in the wour.d area begin-

ning within n 2! re A3 hours after- ounoing (33). ::,r-al c-orned stroa-

is complemented by at least three different types of pclysaccharides:

(1) chondroitin, (2) chondroitin-4-sulfate, and (3) kerato sulfate

(34, 35). Kerato sulfate is thought to be specific for the cornea arnd

has not been found in other tissues of thhe body. Although polysaccharide

turnover is .ore rapid for the first one or two months of healing, the

total stromal content of sulfated polysaccharides remains subnormal

throughout this period (36). Particularly noticeable is the gradual

disappearance of kerazo sulface following wounding. The kerato sulfate

reappear.- by approximately 30 days of healing, but is not present in

normal quantities until after three months (37).

Collagen turiio.'er in the cornea is extremely slow (38) and it is

thought that ne'- collagen is typically a soluble form which later

thickens and becrzes insoluble. ::ew fibers are deposited over a period

of months, but the exact rate of collagen synthesis is not well

documented for the healing cornea. Corneal collagen is somewhat

different from normal skin collagen in that the former contains more

lysine and less _hreonine, serine, methionine, tryosine, and hydro:.:y-

lysine than skin collagen (39).

Corneal Metabolisn


Enzye activity determinations show that the Emden-Meyerhof path-

way of glycolysis, the tricarboxylic-acid cycle, and the hexose mono-

phosphate: shunt (pentose shunt) are all present in the cornea (40).

In the corneal epithelium, 65 percent of the glucose is metabolized

by the glycolytic pathway and 35 percent by the pentose shunt (41). In

the stror.al layer, however, oxidation of glucose appears to be entirely

confined to the tricarboxylic-acid cylic pathway. Glucose exchange

across the different cell layers of the cornea shows that the source

of glucose for corneal metabcolls- is predominantly through e:x:charge

across the endotheli_:. Estimates of glucose consurPtion b:. the cornea

indicate a rate of 9C 'g. per square centimeter per hour (40).

The he::-ose monophosphate shunt, which accounts for 35 percent of

glucose metabolisn in the corneal epithelium is thought to be linked to

aerobic glycolysis via the reo:,idation of the reduced form of :IADP

(e.g. N.-DPH) by lactic acid dehydrogenase (LDH) (-i).

Other Metabolites

Lactic acid production in the complete absence of oxygen is about

10 pg. per hour per milligram of dry weight in the rabbit cornea (42).

The lactic acid concentration in the stroa. is appro:-imately one-tenth

that found in the endothelium and epithelium. It is thought that the

epithelium probably generates and utilizes more lactate than the stroma,

thus offsetting an activity level of LDH in the epithelium 200 times

greater than the LDH activity in the stroma.

Glycogen is metabolized at a rate of 25 ug. per hour per milligram

in excised cornea whenever the endogenous supply of glucose is depleted

(43). Restricting the air supply to the cornea causes selling anJ]

edema which a-pear to be related to glycogen depletion.


The cornea respires only across the epithelial and endothelial

surfaces with very little gaseous exchange with the limbal blood vessels.

The oxygen flu:: from the at-.c-phere across the epithelium has been

calculated to be 7 ul. per square centimeter per hour (44). As one

moves posteL'iorly thc ou::ygen tension decreases steadily to a minimal

level at the endothelium iwherer the flu:: into the anterior chamber is

quite small. This flux across the endothelium into the anterior

chamber has been calculated to be only one-tenth that of the flux

across the epithelium. The oxygen tension in the aqueous humor has

been measured to be about 55 mr.. Hg in the normal eve.

Oxygen utilization in the intact rabbit cornea has been measured

at a normal rate of S ul. per hour per milligram dry weight (45). The

rate of utilization is apparently independent of the oxygen tension in

levels above that of normal air. Of this consumption the epithelium

uses about 6 ul. per hour per milligram dry weight and the stroma and

endothelium use the remainder.

The respiratory quotient of the cornea has been estimated to be

unity; hence, the amount of carbon dio::ide production in the cornea will

be about the same as the oxygen consumption (45). Carbon dio:.ide is

removed almost entirely by way of exchange across the epithelium and

efflux into the air.

Beta Radiation in Ophthal:olovg

Beta Radiation Therapy

Beta radiation has been used in ophthalmology for over 50 years.

Despite difficulties with depth dose dosimetry, construction and calibra-

tion of various applicators, and other technical problems, beta radia-

tion is used for therapeutic treatment of many ocular diseases (46).

In a clinical stud: of 69- patients treated :'.ith beta radiation,

'0 percent :wre treated with Scrontium-90 applicators (-6). Inprcve-

ments in design and construction methods (47) have made Strontium-90

applicators the major source of beta radiation used in ocular therapy


Amcng the diseases of the eye treated with Strontium-90 beta

radiation are: .Looren's ulcer, rosacea keratitis, ptery.giu::i, .erral

catarrh, and vascularizacion of corneal grafts (S4, 19). it has also

been used for treatalenc of limbal tumors and malignant epibulbar

melanonma (50). Therapeutic doses for these diseases range from 500 rads

of beta radiation ro 12,000 ra-s, v'ith an average of 500 to 1,000 rads

given four cines at weekly intervals.

Ophthalmic Sources of Beta Radiation

Several sources of beta radiation have been used for ophthalmic

applicators. These include: Radiu-,, Radon. Radium D-E, and Strontium-90.

Strontium-90 emits pure beta particles1 with an energy of 0.54 M ev, and

decays to a daughter nuclide Ytteriun-90 that also emits a beta particle

(energy o 2.2 Me'.) S.tbh RadiuL and Radon ophthalmic applicators have

a gamma ray component in their radiation emission spectrum.

'Radiological "eal:h Handbook', U.S. Department of Health, Education,
and Welfare: Office of Technical Services, September, 1960.

The advantage of using the pure beta emissions of Strontium-90

is based on the doFsia.-ry and therapeutic inde:.: of the beta particles'

shallow penetration of the ocular tissues. Depth dose determinations

of beta radiation in ocular tissues have indicated that the dose at a

depth of 1 rm. is only 50 percent of the surface dose, whereas the dose

at 0.5 mm. is appro:-:irately 75 percent of the surface dose (47). Since

the human cornea has an average thickness of approximately 0.5 to 0.6 mm.,

and the rabbit corner? has an average thickness of appro::imatel.y 0.- mm.,

a majority of the surface dose of Strontium-90 beta particles will

penetrate beyond 2 rn,. of ocular tissue.

Increasin: the source strength of beta radiation applicators,

giving higher surface dose rates, does not significantly raise the depth

dose distribution of the beta radiation; instead, it effects the lateral

dose distribution (51). Therefore, higher surface dose rates of a

particular beta source result in isodose distribution curves that

are larger in the area of tissue irradiated, but not significantly

different in the Jose race at a specific depth in the tissue (52).

Dosicairy of beta particles in the presence of gamma rays becomes

very com-licated and less accurate than simple beta particle dosimetry.

In addition, the greater penetration of the gammza ray component of nany

isotopic beta sources due to a lower Linear Energy Transfer (LET) makes

undesirable -::rosure of the lens and other deeper ocular structures a

therapeutic liability.

Sealed Strontium-90 applicators are, therefore, the most efficient

isocopic sources for bet? radiation therapy of the eve.

Beta Radiation Fatholc.gy of the Cornea

The effects of cets particle irradiation of the cornea have been

classified as a function of the does delivered to the tissue, subsequent

changes occurring in the epithelium and stroma, followed by infla-nmatorv

reactions (53). The effects are cumulative and always become evident

after a latent period which is inversely proportional to the dose re-

ceived. Eventually, regenerative changes occur in all corneal structures;

however, these never appear until after all the pathological changes are


The time sequence of the appearance of the various pathological

effects cf beta radiation on the cornea have been classified as follows:

(1) early effects -- ex::eplified by edem. and opacity, (2) delayed

changes -- usually characterized by ulceration, perforation, and vascular-

ization, and (3) late changes -- usually appear as re-occurrences of the

original inflaimmatory lesions (54).

The threshold inflammOatory dose of beta particle irradiation of

the rabbit cornea (46) has been estimated at 22,500 rep,I however, the

threshold dose in the hu:ran cornea has been documented to be 33,000 rep

(53). Although ulceration -.av occur at 35,000 rep, consistent production

of such lesions usually requires approximately 70,000 rep (5-, 55).

The unit roentgen equivalent physical (rep) is an obsolete unit of
absorbed dose, equal to 93 ergs per gram. This unit has been replaced by
the "rad," whichh is defined as 100 ergs per grarri of absorbed radiation.
N.B.S. Handbook No. 66, Safe Design and Use of Industrial Beta-Rav Sources,
U.S. Department of Commerce, 1958.

Re-occurrences of infla._atory lesions after several weeks or months

is more frequency ac the higher dose levels (usually above 50,000 rep).

These late effects of beta radiation on the cornea often require several

weeks or months to subside and many cases result in permanent scarring

of the cornea.

Inhibition of l'ound Healing

The co-nple:: phenomenon of .:ound healing involves so many diversi-

fied interactions of cells, morphological transformations, and new bio-

chemical syntheses chat the delicate balance of these interactions can

be upsec by a variety of agents and circumstances. The result is a

delay or partial inhibition of the normal time sequencing of the healing

process, and often complete inhibition of the repair process resulting

in permanent rnd se-ere cicatrization and possible necrosis of the

damaged issue.

Scorbutic iound Healing

Ascorbic acid (vitamin C) is known to be necessary for both the

healing of :wund tissue and the subsequent maintenance of the wound

(56, 57). Three rajor changes are found in scorbutic wounds: (1) che

endoplasmic reticulum is no longer found as elongated, flat, inter-

connecting channels, but rather becomes separate, vacuolated ciscernae,

(2) lipid accumulates in distinct deposits within the fibroblasts, and

(3) non-banded filamencous material appears in place of the normal

collagen fibrils in the extracellular spaces (s5). In addition, the

rates of uptake and release of tritiated proii:ie are slo;wd in the

scorbutic W.LInds (59), indicating that the rates of synthesis of pco-

tein collagen is chan-ed. Ic is thought that the scorbutic condition

causes an alteration in the ultrastructural detail of the microsomes

and endoplasmic reticulum that are responsible for hydroxylation of

proline to hydrovyproline and subsequent s-nthesis of collagen proLein.

Corticosteroid Inhibition of Wound Healing

Corticosteroid treatment of skin wounds produces a delay in the

normal secuenca of healing (60). The action of cortisone derivatives

depresses fibroblastic proliferating, va-cularirticr.: and deposi:ion

of extracellular ground substances.

Similar observations in corneal wounds indicate that cortisone

reduces fibroblastic activity (61), effects mitosis of epithelium (62),

and reduces the tensile stren-th of corneal .wunds when applied topically

(63). Prednisolone reduced the development of tensile strength of

corneal wounds when applied topically for two weeks following penetrating

surgery (64); however, there was no effect on tensile strength if the

prednisolone was withheld for the first ten post-operative days. De:a-

methasone also retarded the development of tensile strength (652 and

decreased the D'0A s.nthesis in connective tissue cells (66) of the

wounded cornea when applied during the first few:. days of healing. The

effect of steroids on corneal wound repair appears to be proportional to

the dose, the frequency of application, and may well be cumulative since

the effect w.as demonstrable when the steroid was applied two days prior

to wounding.

Antimetacolices and' Corneal Healinz

Antimitotic drugs such as azathioprine (66) and 5-iodo-l-deoxy-

uridine (67, 68) ha.e been sho.n to inhibit corneal wounds made by

freezing. In these studies a correlation was noted between a low number

of regenerating :erarocytes and the extend of delay in healing. The

mechani-ns are similar in blocking chymidine incorporation into DMA

synthesis. This is accomplished with 5-iodo-2-deox;yuridine by selective-

ly blocking D'A polymerase through competitive inhibition of ch,iLidine

uptake and incorporation into D':A (69, 70).

Surgical Procedures Following i1ounding

Central corneal wounds do not heal as fast as peripheral wounds

because the more p:riohe-cl poundss are vascula:ized and invaded by

cellular components of wound repair more easily than central wounds (71).

Closure of the eyelids following surgical injury to the cornea

decreases the oxidative enzymes in the anterior cornea and delays cell

division, but does not restrict the oxygenation of the repairing cornea

severely enough to reduce the development of tensile strength of the

wound (71, 31).

Covering of corneal wounds by a forni::-based conjuncti'.al flap

does not influence the tensile strength of the wound; ho:v.'er, removal

of the epithelium drastically inhibits che corneal repair process (71).

Radiation Inhibition of u.'ond Healing

For cver 35 yesrs scientific reports have documented evidence of

radiation inhibition of wound healing. Radium treatment of carcinoma

of the cervix has caused problems with local healing of bladder fistulas

(72) and irradiation of tissue prior to mastectomy has resulted in

difficult healing and late degeneration of the irradiated tissues (73).

Although it has been suggested that .ery small loses of radiation

may stimulate healing of animal skin wounds (7L), it is well established

that larger doses of 1,000 rads or more of x-rays will definitely inhibit

skin foundd healing (75). Even doses of 465 rads of x-radiation decrease

the closure of skin wounds (76).

Radiation Effects on Corneal Healing

Various types of radiation have been shown co influence wound

healing in corneal epithelium. .tonr chese difference radiations are

Grenz rays, ultraviolet and x-radiation and beta particles.

Grenz rays are soft electromagnetic radiations ';ith wavelengths

of 1 to 4 A, which is shorter than ultraviolet radiation but longer

than comcn gamma radiation. Grenz rays have been used in ophthalmology

for superficial therapy because of their shallow penetration into

eve tissues.


Grenz rays cause a temporary inhibition of mitosis at threshold

dose levels (77). This inhibition of citosis is followed by a rebound

excess of epithelial cell mitosis.

Healing of corneal epichelium is similarly altered following

exposures to ultraviolet anJ :x-radiation (78) and threshold doses of

beta radiation (79). Epithelial cell migration was not inhibited, un-

less doses reached levels high enough to cause nuclear fragmentation

and indiscriminate destruction of epithelial cells.

Beta radiation effects on corneal healing

Doses as low as 2500 rep or approximately 23,000 rads of beta

radiation have drastically inhibited penetrating corn-eal wounds (SG).

In these studies, 5,000 rep of beta radiation similarly inhibited

corneal healing even when administered three months prior to wounding.

Apparently, the beta radiation causes a delay in fibroblastic prolifera-

tion in the stromal portion of the wound as well as extensive strom.al

edema in the adjacent areas. The delay of fibroblastic activity and

delay of scromal regeneration was evident six months later, concomitant

with a residual edema surrounding the irradiated wound area. At higher

doses of 27,000 rep fibrcblastic proliferation was decreased and healing

inhibited in corneas that remained normal in appearance for two years

between the radiation treatments and surgical wounding.

The inhibitory effect of the beta radiation treatments apparently

began to subside or decrease toward the end of these si:x month studies

as healing eventually occurred. Thus, short term inhibition of epithelial

mitosis and prolonged inhibition of fibroblastic proliferation appear

to be manife- cautions of a radiacion-induced delay in the healing process

and not a complete and permanent inhibition of corneal repair.

Recent studies of the effects of beta radiation on normal corneal

endothelium (81), D':A synthesis during the first three days of corneal

healing (82), anJ other observations of problems in corneal repair

following combined therapeutic procedures involving local beta radiation

have renewed interest in the radiation induced delay of corneal repair.

In addition, investigations of beta radiation suppression of the

immune response to corneal xenografcs have raised questions concerning

effects on corneal healing of graft recipients (33). Complete inhibition

of the normal irLrnune rejection of intralamellar corneal :enografts :.as

complicated by a residual edema and changes in mucopolysaccharije chat

were shown to be independent of any direct radiation pathology. Rather,

the edema and mucopolysaccharide changes were thought to represent a

problem in healing of the recipient's cornea (following the combined

procedure of beta irradiation and surgery) and not a manifestation of

immune rejection of the xenograft.

These studies su.I:st that more extensive .:.r.: needs to be done

on the site of beta radiation influence on the comple:- phenomenon of

corneal wound repair. Specifically, more detailed studies are needed

on the effects of beta radiation on cellular interactions, essential

enzymatic activities, and macromolecular synthetic processes involved in

corneal .wound healing.


The following research was undertaken to delineate the sites of

beta radiation influence on corneal heali.n. and to gain insight inco

possible .echanisms of radiation inhibition of normal corneal wound repair.



The object of this research was co scudy the effects of beta

radiation on corneal wound healing. Previous reports mencicned in

the incrG.uction have noted some inhibitiory effect of beta radiation

on the process of healing of corneal wounds; however, no hypctheses

have been established for the mechanism of chis radiation induced


Gross morphological and hiscological observations, along w'ich

tensile strentr.h neasuren.ins of healing correal wounds ere used

to escimace the magnitude and duration of the radiation inhibition.

The initial experimental design also included biochemical observations

and electron microscope examination of the fine strac:ure of the

cells involved in normal and irradiated healing corneal wounds. It

has been dccuranced that certain enz, e activities (29,30), nucleic

acid, protein, and nucopolysaccharide syntheses (32) and ocher bio-

chemical processes are increased dramatically in the fundeded cornea.

The cells of the different corneal layers, having separate functions,

undergo different alterations in their ultrastructural organization

during corneal wound repair. Such drastic changes in ncr-al bio-

chemistry and structure of these cells, observed only during -wund

healing, suggested that these physiological changes were essential to

the success of the healing process in corneal uiur.cs. It was reasoned


that the radiation would probably have some effect on one or more

of these essential physiological processes if it inhibited and delayed

the corneal healing phenomenon.

Beta radiation effeccs on the ultrastructure of cells involved

in the healing ccrr.ea were correlated with the observed alterations

in gross and microscopic morphology. Based on these findings,

specific enzyme systems were evaluated by histochemical techniques

to determine any radiation effects on critical metabolism in the

healing cornea. These alterations further suggested that investiga-

tions into the different biochemical synthetic processes should empha-

size evaluations of DNA and ?JA. synthesis. Radioaucographic techniques

were used to determine the effects of beta radiation on DMA and R:IA

syntheses in the repairing cornea.

The methods, execution, and results of these experiments are

individually documented in the succeeding sections; however, the

general materials and methods cor--on to all these experiments are

described belo..

General Naterials and methods s

Experimental Ani'nalc

One hundred si::ty-five albino rabbits (Orlt:'a.s *2nic.4us),

weighing between 3 and 5 kg., were used in These experiments. The

animals ware purchased through the Animal Department of the J. Hillis

Miller Health *.nter, CGinesville, Florida, fromn the Blueberry Rabbit

Farm., ::- Port 'ich:.e, Florida. All the animals were rnia.1tained in

the animal quarters at the J. Hillis millerr Health Center during the

experiments and all animals received identical care, feed, and treat-

men .

Fa cilities

All facilities, costs of materials, animals, and technical

assistance for these experiments were provided by the Department of

Ophth.llr.olo'., College of medicine, Uni'versity of Florida, Gainesville.

Animal surgery facilities were provided by the Animal Department of the

J. Hillis Miller Health Center, Gainesville, Florida. Histological

facilities and the electron microscope were made available by the

Research Service Unit of the Veterans Administration Hospital,

Gainesville, Florida.

Surgical 'ounding of the Cornea

Each animal received bilateral, penetrating surgical wounds in

the central cornea. Preoperatively, 4 percent atropine sulfate oph-

thalmic drops were used to dilate the pupils. Intramuscular injections

of pentobarbital sodium were administered 20 to 30 ninuzes prior to

surgery. Four (4) to 3 cc. of 50 rg. per cc. aqueous solution of

pentobarbital sodium was used as an anesthetic dose depending on the

size and weight of each animal.

Using standard lid retractors for exposure, an 8 am., non-

penetrating incision was made in the center of the corner to a depth

cf approximately one-half of the total thickness of the cornea. The

anterior edges of the incision were disecied along the stromal lamellar

plane, back a'wa from the wound edge scme 1 to 2 rn. providing a flap

of corneal tissue on either side of the wound. Two 7-0 sutures were

placed 5 nm. apart and evenly spaced from the center of the incision.

Each suture was placed through both corneal flaps, perpendicular to

and across the wound. The portion of the untied suture that bridged

the wound was looped out of the way and the section was completed by

insertion of a cataract knife at one end of the original incision,

through the anterior chamber, and out at the far end of the original

cut. A single upward movement of the cataract knife completed the

section with a snooth cut that created a full penetrating wound at

least 6 to 8 C-n. in length. The sutures were then tied and the

juxtaposition of the wound edges checked to insure uniform contact

between the two edges of the closed wound.

Postooerative Care

Topical administration of 4 percent atropine sulfate and Neo-

sporin ophthalmic ointment was maintained on a daily basis through-

out the first threee days following surgery. Thereafter, 'ieosporinR

was administered topically each day for the next four days of healing.

Cases of infection or anterior synechiae were excluded from the


Irradiation Treatr-ents

All aim-iarl received bilateral -:ounds, hc:, ver, cnly one cornea

in each animal received beta radiation. Each animal served as its

o'n control since the observations made on each irradiated corneal

wound ware cormparAd with the normal foundn d healing in the non-

irradiated contra.lateral cornea. The records of the identity of the

irradiated and non-irradiated eves in each animal and the dose

administered were coded and all observations were made on a double

blind basis.

Doses of 10,000 reds .f beta radiation were selected, as this

dose level w7as considerably higher than the doses previously re-

ported to cause inhibition of cornaal healin; (30). Also, this dose

is less than one-half the threshold dose (35,000 rep) of beca radia-

tion required to produce observable sy-.ptoms of radiation pathology

of the cornea (53).

Of the 165 animals used in these experiments, 60 animals

received 10,000 rads of beta radiation izimediacely after surgery,

60 animals received the radiation treatments two and one-half months

before surgery, and 45 animals received the beta radiation ten

months prior to surgical wounding.

Irradiation procedure

The eyelids -.ere he-ld open ::ith standard retractors with the

animals under pentobarbital sodium anesthesia and a drop of pro-

paracaine hydrochloride ophthalmic anesthetic was administered

topic-lly. The beta radiation :.a.s adm-inistered by corneal contact

with a Strontium-90 ophthalmic applicator having a surface dose rate

of 104 rads of beta radiation per second (Atlantic Research Serial

No. 232-Eye The-.y Applicator). Dre.es fi 10,000 reds of beta

radiation required a 96 second exposure to the source. Other doses

were calculated irnividually fro-, the specified dose race with a

standard deviation of 6 percent.

Clinical Observations

The clinical course of the corneal healing was followed in all

of the e::perieents by both gross observations and detail studies using

the bionicroscopic slic lamp. Photographs were caken with a 35 mm.

camera apparatus designed for close-up pictures of the single eve.

Pictures '..ere taken at appropriate intervals to illustrate the pro-

gression of the healing process in the non-irradiated, control eye,

as co:.mpared wich the irradiated, contralateral eye in the same animal.

Figure 1. Technique of surgical wounding, graefe knife completing
section. Note sutures placed superficially and looped
out of the way.

Figure 2. Wounding complete, incision sutured.

.......... .... ......... ........ .. .



The Cl.i.ical Course of Corneal Hezlin2z

Healing iof he penetrating surgic. -l woun-ds in the non-i:-radia:ed

control eyes progre.s-ed normall:. WiThin an hour after surgery ,

aqueocs humor, lea'.ing out, had farned i;i fibrin clot in the wourj,

sealing off the anterior chamber. By six to twel'e hcLrs later,

edema was noted in the local area around the u.cu:d. This produced

an area of haziness along the wound perimeter; howe.'er, the remainder

of the cornea remained clear.

Twelve to thirty-six hours was required in most cases for the

epithelium to co-pletel.' recover the wound. At this :Aie the wound

appeared as a narrow band of hazirness :ich a hairline depression in

the center, overgrown with epichelium. The control corneas rea-ined

the same throughout :he first postoperative week except that the band

of hazinessz and local corneal edema became slightly wi,:ar and more

diffuse. By six or seven days the depression disappeared from rhe

center of the narrow opaque band and the entire corneal surface was

smooth. e::ce'r at the points where the sutures entered the tissue. The

band of haziness was also enlarged at these points to encompass an

area approxicrltel-- 0.5 rm.. around the suture.

Fourteen to sixteen days after wounding the band of haziness

was more restricted to the :wou-nd and shorter as the more peripheral

ends of the incision became less obvious. The rcst of the cornea was

clear. By 21 days of healing the scar appeared less distinct and

smaller, suggesting chac wound contraction had begun.

Healing of Irradiated Corneas

In all of cte eyes irradiated with doses of 10,000 rads of beta

radiation, either prior to or at the time of surgery, the healing

process was similar. Uichin two to three days after wounding, the

central cornea ;:as chara.trerized by a diffuse haziness and edema, not

confined to the found d peri:.eter as in the control corneas. Although

epithelial cell migration recovered the incision, the wound began to

gape open anteriorly before the end of seven days of healing. By nine

or ten days the gaping was more severe, edema and opacity was more

extensive, and sone superficial degeneration of cih cornea was noted

along the wound edge and around the sutures. The anterior chamber

was maintained only by the fibrin clot and the epithelial overgrowth.

Some cases of neovascularizat ion we:re noted as the jerilim-bal vessels

began cc extend into the cornea toward the nearest portion of the


After 14 to 16 days of healing, the irradiated corneas gaped

anteriorly some 1 to 2 rIm., and superficial degeneration of the cornea

around the sutures resulted in some of the sutures pulling loose

from C-the tissue. o'.'cl.ariztinr. '-.s praent in mo-: cases, cften

extending. 2 =m. or more to the wound perimeter. Corneal opacicy was

so severe chat the pupil was often obscure.

By the tcentieth postoperative day, 85 percent of all irradiated

corneas were characterized by gaping wounds, swollen scromal areas

involving che wound and adjacent to the wound eJge, severe corneal

edema and corneal opacity in the central half, and neovascularization

extending to the wound edge. More than 90 percent of all irradiated

corneas had some anterior degeneration directl- adjacen_ o cho

perimeter of the incision. This superficial de"enerarion,or corall

melcin';' was apparently a manifestation of the lack of tissue regenera-

tion at the wound, complicated by further de-eneracion alone; :.

perimeter of the wound and tension produced by the closing sj.ures.

SConjunctivitis and blephritis also accompanied the delayed

healing of the cornea. The irradiated corneas were more prone co

infection than the control corneas foilo'.:ing cer-inatin of Lhe

topical antibiotic postoperative treatments. It is interesting to note

that corneal edae:a is a relative measure of endothelial disfunction,

and histological findings chat the endothelium had not recovered the

wound at this :ine e:.:erli fied this correl'-ton.

Of the remaining eyes that were not q-.ite o severe, all had

retarded regeneration of the stromal connective tissue, some raping of

the wound, edena and a;.'llir.g, haziness of the central cornea, and

conjunctivitis, hut not all had extensive neov-ascularicat ion. In

spite of sc..e superficial degeneration, some retained the --tures in

place which h-lK-ad to .3m 't'in. clcs~re cf the ..:ound.

Gross examination ard bionicroscopic observations indicated that

the predo.minanc characteristic of the delayed healing :as the lack of

connective tissue regeneration. The second most obvious problem was

superficial degeneration and severe corneal edema which invited

neova.3cularization front the perilimbal vessels. Even though the

degree of inhibition varied somewhat from animal to animal, the dif-

ference beti.een the non-irradiated control wound and the concralateral-

irradiated wound was so obvious by the end of the first week that the

code ..'as not needed to identify the irradiated eye in a particular


Some anicmls were followed for nine months after wounding. Heal-

ing was extremely slow and appeared to require at least three times

longer for substantial connective tissue regeneration to occur. When

stromal regeneration finally filled in the gaping wound the overall

state of healing began to approximate normal healing characteristics

found at one or cwo weeks after wounding. Edema and haziness persisted

for several months, slowly decreasing until by eight or nine months

the irradiated wounds appeared comparable to non-irradiated wounds of

two or three '-onths healing. Regression of the edema was the last

symptom of delayed healing to disappear. Scar contraction had occurred

and the residual remaining scar was as faint as controls and only about

one-third the size of the original wound. Apparently, between six and

nine months, the inhibited wound healing process began to achicve

enough progress to insure eventual complete healing.

In contrast to the eyes that w-ere irradiated either cwo and one-

half months prior to wounding or at surgery, those eyes th.t had re-

ceived 10,000 rads of beta radiation ten months prior to woundin'

did n:t exhibit as severe an inhibition and delay of normal healing.

Apparently the lascin; effect of the beta radiation, on the intact

cells prior to injury, had be3un to decrease during the cen monch

period between irradiation a-d noundin2.

Throughout the entire ten month period between irradiation and

surgery, the treated eves were indistinguishable frca che non-

irradiated control eyes. .:o evidence of radiation danace was observed

in any of the animals prior to the surgical ~ oundin2.

Six co seven days of healing were required to sho-. a.v difference

between the control wounds and the irradiated wounds in an' animals

created ten months previously. At this time, however, more extensive

haziness and edemaa ere noted in the areas adjacent to the irradiated

wounds. Gaping of the irradiated wounds was present, but not sev.ere.

By comparison to the other irradiated wounds it was apparent that sone

connective tissue regeneration had begun, although not as much as was

noted as in the control eyes at a comparable healing time.

Throughout the second and third weeks the mild inhibition of

healing. in the irradiated corneas was characterized by edena and

haziness of the central one-third of the cornea and s::.e evidence of

a partial lack of connective tissue regeneration in Che wound.

Three of these animals were obser-ed until nine weeks of healing.

At this time the corneal hazine.s w.as confined to the irr-ediate area

of the ucund, and wound shrinkage had begun; however, the :.ouna edges

gaped open slightly and remained swollen. Normal healing was still

inhibited throughout the nine ueeks. the animals were sacrificed at

this ti-e for hisroloeg and electron microscopic scudy.

It vas apparent that, although the inhibitory effect of the prior

irradiation treatment was wearing off, sufficient cellular damage

remained to cause marked delay of normal corneal healing. Obviously,

the beta radiation had affected more than just the epichelial cell

layers as norral regeneration of the epithelial cells would have

involved many complete turnover cycles. Thus, any epithelial cells

present at the time of the irradiation would have been long since

replaced before the cornea was wounded either zwo and one-half or

ten months later. The characteristic lack of stromal connective

tissue regeneration even in those animals irradiated ten months

before wounding would suggest that the scromal keratocytes were

affected by the beta radiation.

Tensile Strength of Corneal pounds s

Tensile strength measurements have been used as a measure of

tissue repair for several years. Since the application of this method

to corne s wounds (84), tensile strength measurements have been used

as a simple tool to test the effect of many agents and circumstances

on the corneal repair process. This method has served as a measure

of corneal collag3en production and, therefore, repair of the corneal


Experiments designed to evaluate various surgical parameters

that influence ensilee strength of corneal wounds have established

that che greatest rate of tensile strength dev'elopmer.n occurs in

corneal wounds between one and four weeks of healing (71). During

this time, tensile strength of the corneal wound increases from about

5 to 30 percent of the strength of the intact tissue. This period of

ma.xial increase in wound tensile strength is preceded by a lag

period of iix days, during which censile streng h is not dececcable,

and is followed by a period of several months wherein the increase of

strength of the wound is very slow. More than three months is re-

quired to achieve a tensile strength of 50 percent of that of normal

intact cornea.

The healing sequence of the cornea is apparently considerably

slower than the same sequence in skin wounds, as the latter require

only three weeks of healing tcie to regain the full tensile strength

of intact skin (35).

Corneal tensile strength measurements have been used t3 )test the

effects cf drugs such as topical steroids and idoxuridine on corneal

wound healing (36). To date, this system has not been used to evalu-

ate the effect of beta radiation on corneal healing.


Two groups of fifteen rabbits each were used to correlate the

observed beta radiatiCci induced delay in healing with the subnormal

levels of tensilescrength. One group received 10,000 rads of beta

radiation immaediately after surgical wounding, the other group was

irradiaced two and one-half months prior to injury. The limited

number of animals that had been irradiated ten months before wounding

precluded tensile strength measurements following wounding in this


All animals received identical, bilateral corneal wounds and

postoperative treatment of atropine sulface and ::eosporinR ophthalmic


A corneal strip 5 r-n. wide was cut from each cornea, perpendicular

to the wound, with a double-bladed, parallel razor blade knife. Sutures

were removed and the strip was disecced from the sclera at each end,

leaving a narrow; border of scleral tissue to aid in clamp fixation.

The corneal strip was carefully fastened in special clamps and suspended

on the censiometer. A preweighed plastic bottle was hung from the

corneal strip at the lover clamp and water was allowed to flow into

the bottle at a constant rate. When the increasing weight was

sufficient to break the wound, the bottle fell, stopping the addition

of water automatically. The bottle and its contents were then weighed

to decermine the load required to break the wound.


The tensile streng.:h of the irradiated and non-irradiate1

cornel wounds, after "2 days of healing, are compared in Tables 1

and 2. The mear. and standard deviation of the control wounds were

within the ranges previously described by. Gasset an, Dohlman (71).

In the group that had bean irradiated immediately, after: surgery, the

mean of the irradiated poundss was only one-third thac of the control

wound tensile strength mean. In those e'es that had received beta

radiation two and one-half months prior to injury, the mean tensile

strength of the irradiated wounds i;as only 23 percent: of the mean

tensile strength of the non-irradiated control wounds.

Paired statistics showed a significant difference between the

tensile strength of the control wounds and the contralateral wounds

irradiated at the time of surgery, with 99 percent confidence (p =

0.005). Correspondingly, with 93 percent confidence, there .'as a

significant difference between the tensile strengths of the non-

irradiated and the irradiated corneal wounds in the group that were

treated two and one-half months prior to injury (p = 0.01).

It should be noted that all of the non-irradiated control wounds

demonstrated ireasureable tensile strength. Several wounds from both

of the irradiated grouTs,'however, pulled aparc with just the weight of

the 10 gm. clamps used to secure the corneal strip to the tensiometer.

Snedecor, G.W.: Statistical methods s Aoolied to Experiments in
Agriculture --d SBilo'~ 5th ed., Iowa State -Un-iv'erzity Press, Ames,
Iowa, 1965, p. -,9.



No. of Control Mean
Rabbits (non-irradiated)
g /5 mr.i.

Irradiated MIean

Paired Statistics**

gt./5 r 0. alc.
gm./5 cn. 8:0.005 calc.

9 307 103 3.35 4.23

*Animals sacrificed 20 days post-op
**t-values calculated at 99% confidence




;>o. of Control :ean
Rabbits (non-irradiated)
gm./5 mm.

Irradiated Mean

gjn./5 mm.

Paired Statistics'"

9:0.01 calc.

10 260 60 2.82 2.87

*Animals sacrificed 20 days post-op
*^t-values calculated at 98% confidence

~~~I~ ~ ~~ _

In contrast to the control wound tensile strengths at three weeks of

healing, these several i-radiated -ounds '.,ere a :,_ak as those

normally found in the first six days, i.e., the lag period of corneal

healing (71).

The tensile screncth measurements ::ere well correlated with the

clinical observations of delayed corneal healing in irradiated ::ounds

following surgery. After three weeks of healing the apparent la-c of

stromal connZctive tissue regeneration noted in che biomicroscopic

evaluations were confirmed by the significant lack of tensile strength.


Six of the animals from each group were sacrificed, the eyes

were-nucleared, fixed in formalin, embedded in paraffin, and stained

with hematovylin and eosin.

Light microscopic e::amination showed striking differences be-

tween the irradiated rounds and the non-irradiated controls. Epithelial

migration and overgrowth of the incision had occurred, as indicated

by the biomicroscopic slit lanp inspections; however, the epithelial

cells in the irradiated :.cunds w:ere enlarged and aborr:'al in -ross

morphology even when compared to more peripheral epicheliun in the

same irradiated eye. The epithelium adjacent to and covering the

incision i:as only three to four cells thick by the third w.ee of

healing. Invariably, some polynorphonuclear leukocytes (P'.;) were

present in the anterior stroma, aligned along the basal epithelial

cell layer. In contrast, the epithelial cells overlying the incision

in the control eyes were eight to ten cell layers thick.

Notable in all the irradiated wounds was the complete absence

of endothelial cells covering the posterior of the wound, and the

lack of stromal tissue regeneration. Formation of the anterior

char:-ter was achieved only through closure of the incision by the fibrin

clot remaining in the wound. Fibroblascic proliferation was sub-

stantially less ,n the irradiated eyes than in the controls.

In control wounds, after three weeks of healing, sufficient

stronml tissue regeneration had occurred to give an overall thickness

at the wound that approximated the thickness of a normal cornea, and

endothelium had completely covered the wound posteriorly.

SA consistent observation in the irradiated wounds was a marked

thinning of the stroma at the wound site, concomitant with severe

swelling of the scroma directly adjacent to the wound edges. The

actual stromal portion of the irradiated wounds were only about one-

third the thickness of the control wounds.

Corn. ant

Simple tensile strength measurements of healing corneal wounds

have shown that dcses of 10,000 rads of beta radiation cause a signi-

ficant inhibition of normal repair. The lack of tensile strength

development is well correlated with clinical and microscopic observa-

tions comparing the irradiated wounds with the non-irradiated controls.

These radiation effects are quially demonstrable either when the beta

radiation is administered prior to, or immediately after, penetrating

surgery. Paired sample statistics illustrate the high significance

of these data when comparing the mean tensile strengths and standard

deviations of the irradiated and the non-irradiacej corneal wounds.

The observed lack of tensile strength in the irradiatej corneal

wounds appears to be a result of an inhibition of connective tissue

replacemLent, perhaps a lack of collagen synthesis, and complications

of normal corneal hydration that apparently result from the failure

of the endothelium to close the wound posteriorly.

Epithelial migration occurs almost normally, even though

the cells are enlarged and somewhat abnormal in morphology. Epi-

thelial metabolism may be inhibited thereby inhibiting normal

stromal regeneration and tensile strength development, however,

this is not yet established.

The absence of endothelial migration and recovering the

posterior wound would be expected to have a drastic effect on the

normal stromal metabolism and maintenance of normal corneal

hydracion. The clinical observations and tensile strength measure-

ments indicated chat the abnormal edema and the lack of scromal

regeneration might well be related to endothelial disfunction.

The inhibitory effect on corneal healing caused by beta radia-

tion seos to be a long lasting one. Eet.;een two and one-half and

ten months are required to begin recovery of normal corneal healing.


Even when ten months elapse between the irradiation and wounding,

penetrating surgical wounds are characterized by a definite inhibi-

tion of connective issue replacement, inhibition of fibroblascic

proliferation, a lack of endothelial migration and a lack of normal


Figure 3. Control healing after nine days of healing. Noce
edema around incision.

Figure 4. Healing at nine days in cornea irradiated tuo and
one half months before wounding. Wound is gaping
severely. Note extensive edema and superior neo-


Figure 5. Healing in non-irradiated eye at day 20. Iote good
scar formation and limited edema.

Figure 6. Healing in contralateral eye, irradiated two and one
half months before surgery, at day 20. Severe wound
gaping, neovascularization, and severe edema are vis-
able. Anterior chamber is maintained only by fibrin
clot in the wound.



.... ....................
...... ...........
..................... ... .....
.......................... ..... ....::: ; ... ... .

... ... ... ....

!!" .i'iiiiE ''i :" ,..... .... i" ," ..

Figure 7. Healing at day 20 in non-irradiaced cornea (lefc
eye). ioce quiet conjuncc i'v, well defined scar
indicati'.e of good healing.

Figure 8. Corneal healing in contralateral eye (right eye
of same animal as above) at day 20, follov'iNg beca
radiation immediately after surgery. Conjuncti'.icis,
neovascularizacion, severe edema and anterior corneal
melting characte-ize inhibicion of healing.

:- ::::::: ................ i iiiiiiiiiiii iiiiiiiiiiii iiiiiiiiiil

I I i
`- -*-

Figure 9. Control eye healing at nine m.oncths. Wound
contraction is notable and scar is disappearing.

Figure 10. Healing after nine months in contralaceral ev' that
had been irradiated two and one-half months before
wounding. Inhibition of healing has dimini='ed during
the nine-month healing period.


.... ...... .. . . .

......... ...EEE:::EEEEEEEEEEEEEE ........ ~ EE :EEE ... .. ......... ..... .........
......... ...........
.... .... .... ... .... .. ...

.............. .............. .......
...... ..........

... ...........

Figure 11. Healing in non-irradiated cornea at da' 11.

Figure 12. Corneal wound after 11 days of healing, in cornea
that relieved 10,000 rods of beta radiation 10 months
prior to surgical wounding. Inhibition of healing
is not as drastic. Edema is limited and sore tissue
regeneration is noted in the margins of the wound.


..... ",...... ",,m ~ m m ................iii

........ ...................


........................... .......... .... .............. .........................

........................ ...... ........ ...................... ,... : :::::::: r '
.................. .. ... .. .. .........

......... ...

Figure 13. Healing at 21 days in same control eye as shown in
Figure 11.

Figure 14. Healing in same irradiated eye as shown. in Figure 12
after 21 days of repair. Edema and wound gaping have
increased slightly since day 11 (see Figure 12).
Inhibition of healing is considerably less than ob-
served in corneas irradiated closer to wounding.


Figure 15. Control cornea after nine weeks of healing. Sub-
stantial wound contraction has occurred.

Figure 16. Contralateral eye, irradiated 10 months before wound-
ing, shown at nine weeks after wounding. Although
gaping is apparent some regeneration within the wound
is noted. Persistant edema has attracted superior
and inferior neovascularization.


S .................. i

. .. .... ........... ................i: .....,EiiiE....: :..:,,,,EE~ iiiiE



.... .. ... ...... ..... .......... .

.............. ~ ~ ~ ~ ~ ~ ... ......;!i~iE: .'EEEEE ...iEEEE 'iEH ii

::iidiii ..... ........ .. ...... ....... i. .iii iiiiiiii .........: ......... ...

...... ......

Figure 1-. Develow-.ent of co-.-licaticIs during inhibiced healing.
The severity of healing co-.licai ions is e:.:pressed
in te-.s of an e-.irical sradina scale of 0 co 4.
Graph illustrates the typical difference found between
irradiated and non-irradiated corneas in the sa-ie
anima l.


C 4


2 i it
0n Nc n

n. -'
1 -0

s::'Olivjiju 2, 1 T'mi j iH n. I A? oII V1'd
Cn N i 1 '

v: ^ ^s

-r r- -J_

SOlV ncn0 ::n 'H^ \.h/S3IVT



Morphological cransformacions, cell migration, cell divisions,

and changes in intercellular organization, as described by light

microscopic studies, are only a part of the extensive changes ti

occur in normal corneal healing. The ultrastruccural changes thac

occur within a particular cell during the repair process are r=laced

to the functional capability of che cell co meet the dT-anJs sec

forth by the cell's eventual role in the couple:: scheme of healing.

In normal corneal healing of surgical incisions, epithelial

migration recovers the wound by 24 to 36 hours after injury. E:xten-

sive ultrastruccural changes are noced in che epithelial cells during

the period of lateral migration and for several months afcer the

wound has been recovered by epithelium. At the \wound site the basal

cells of the epichelium lack a basement membrane and che hemidesma-

somes which form the attachments between the basal cells and che

basement membrane. Epithelial cells incerdigicate a.d in che depth

of the wound, the cells are joined by desmosomes. Epichelial mico-

chondria, in the intact cornea and in early corneal healing, appear

as long,slender organelles with very sparce internal membranes and

cristae. By- 24 hours of healing, the epichelial micochondria are

more numerous, larger, and possess a more complex internal structure

(8). Frequently thee mitochondria are found constricted at the

center, and multilobed suggesting mitochondrial division and an in-

creased metabolic activity. Even after two months of healing many

alterations in epithelial ultrastrucutre still persist. The base-

ment membrane is incomplete with large gaps that have not been filled

in at the wound site (12). The basement membrane, where present,

is often abnormally varied in thickness. WVhere large gaps exisc,

short segments of basement membrane, complete with basal desmosomes,

can be seen witL-in the gaps, suggesting that reconstruction of the

basement inr.bran-e does not take place simultaneously amonS the basal

epithelial cells that have recovered the wound.

The first stages of stromal healing are indexed by polymorpho-

nuclear leukocyte invasion within 21. hours. Keratocvtes are un-

changed at this time and often mononucleated cells begin to appear near

the wound. The mononucleated cells are characterized by short,

pseudopod-like processes of the cytoplasm, yet they contain few

endoplasmic reticula, mitochondria or ribonuclear protein (F!IP)

particles (3).

After 36 hours of healing, other mononucleated cells appear at

the wound and keracocytes away from the wound undergo changes. These

mononucleated cells contain extensive endoplasmic reciculum, larger

and more numerous mitochondria, numerous R'P particles and long,

chin cytoplasnic processes, typical of nmtGre fibroblascs. In the

keratoc.yces away frcm the '.:uund, rough endeplasmic reticulum appears

as numerous free RNP particles and numerous v'esicles.

Once the various cell types appear there is a migration and

aligrment within the wound. Presumably, collagen synthesis is initiated

by the fibroblasts associated '.ith the wound and the long term pro-

cess of stromal reconscruccion begins. Two months lacer, the fibro-

blasts are scill present in the scrom-a, amid e:tcensi'.ve collagen fibers

with an abnorial -:ariecy of diameters. Collagen fibril orientation

is random with occasional layering in the deeper stroma.

Corneal endothelium changes within 2i' hours after corneal

surgery. :uclei become lobular, rough er.doplasmic reticulum. increases,

and mitochondria are larger and more numerous. The endothelial

changes are characteristic of an increase in metabolic activity of

the cell and are consistent with the eventual regeneration of

Descemets' membrane.

Descenets' membrane is choughr to be secreted by the endoche-

lium. It may be produced as the basement membrane of this cell

layer (26). Regeneration occurs slcwlv after injury and after two

months the new Des:eme.es' membrane is still abnormrally thin and

irregularly layered.

The clinical observations, hiscology,and tensile strength

measurements that were carried out earlier, all suggested thac the

lack of normal connective tissue regeneration and the absence of

endothelial recovering of the posterior wound were a direct result of

the beta radiation treatments. The radiation m.-st also h'.ve caused

some alterations of the normal patterns of changes in ulcrastructure

that occur in corneal healing. Ic w'as hoped that any radiation-in-

duced intracellular change mighe provide some index to the nature

of radiation effects on normal cell function in corneal healing.


Immediacel; prior to enuclea:ion, two drops of cold, 2 percent

osmium tetroxide in phosphate buffer were placed on each cornea. The

animals were sacrificed, the eyes enucleated and the cornea carefully

dissected out rich a 1 mm. rim of sclera attached. Separation of the

cornea from the aqueous, iris,and lens capsule was carefully performed

with forceps, taking care not to touch the anterior or posterior

corneal surfaces near the control wounds.

The whole cornea was then fixed in cold 2 percent osmium

tetroxide with phosphate buffer for two hours. The specimens were

cut into small sections wich a razor, dehydrated with alcohol, and

embedded in Zpon. Thick sections (1 co 2 u) were stained with tolui-

dine blue and inspected with the light microscope for electron

microscopy orientation. E-.I sections (500 A) were cut with a diamond

knife on a Porter-Blum microtor.e and stained with uranyl acetate

and lead citrate. Specific collagen staining was carried out using

phosphotungstic acid (PTA). Electron micrographs were taken with

a Hitachi-II Type C electron microscope.

Corneal wondJ ultrastructure was examined after three weeks

of healing to correlate with the observed lack of nor=_al tensile

strength of the wound following beta radiation. Electron -icroscopic

studies were carried out on corneas that had received beta radiation

immediately after surgery, or two and one-half or :ren .rotha prior

to surgical wounding. Some specimens of corneas irradiated either

two and one-half months or ten months before voundine- were e::a-iined

at nine weekss and nine months of healing.


General Effects of Beta Radiation on Fine Structure of HealinT Cornea

The effects of beta radiation on the intracellular changes that

take place during corneal healing were essentially the same whether

the radiation was administered immediately after or two and one-half

months before wounding. If surgical wounding ':was delayed for ten

months after beta irradiation, the magnitude of the radiation inhibi-

tion of healing was reduced and the ultrastructure of the in'.ol.-ed

cells was more normal.

In all of the irradiated corneas, if th'-e foundd ade-z-.:ely.

resisted infection during the period of radiation delay of normal

healing, eventually the delay subsided and a normal healing pattern

was established. The inhibition of normal healing was proniin.ent at

three and nine weeks cf healing, fibroblasts were inhibited, and

abnormal ultrastructure was noted. Collagen synthesis was drastically

reta-rce .an endorheliu-. did not recover the posterior -:ound in

those corneas irradiated either two and cne-half monthss before or

immediately after wounding. By three weeks, in those corneas that

received beta radiation ten montlis before wounding, the endotheliu-.

had spread to recover the posterior wound. After nine weeks, the

fine structure of the involved fibroblasts appeared Lmore normal,

with a greater complement of intracellular organelles, and some new

collagen synthesis was evident. Epithelial ultrastructure also

appeared more normal.

After nine months of healing, chose irradiated corneas that

had remained free of infection had satisfactorily healed and appeared

comparable to the non-irradiated, contralateral, wounded corneas.

Light microscopic examinations showed only slight differences between

the wounds in the irradiated and non-irradiated controls. Differences

in fine structure were minimal, suggesting that the delay in healing

was almost over. The details of these findings are described below.

Three Week Wounds

Ultrastruccure of epithelium

Three weeks after surgery, non-irradiated cocneal epithelium

was characterized by some interdigitacion, enlarge-ent of intercellular

spaces, a layering of some eight to ten cell layers deep, formation

of some hemi-desmosomes, and the presence of portions of basement

membrane where the opichelium attached to the stroma. The basal cell

cytoplasm was esenctilly normal; however, clumps of ribosonal granules

were scattered thrcughouc, and tonofilaments were dispersed at random

throughout the cytplasm., with occasional bunilin3 of tonofilaments

in some scattered areas.

In contrast, the irradiated corneal epithelium was only one-

fourth to one-fifth as thick as normal wounded epithelium. It con-

sisted of two or three cell layers with some abnormally large cells

near the wound edge. The epithelial cells stained much lighter with

uranyl acetate-lead citrate than the non-irradiated cells, especially

basal epithelial cells. Epithelial cells were interconnected by a

few desmosomes and had distended intercellular spaces. There was a

complete absence of any basement membrane along the stromal border,

although many cytoplasmic processes were extended into the stroma.

The epithelial cytoplasm contained very few endoplasmic reticula,

only scattered ribosomal granules, and tonofilaments that were found

only in bundles. Hitochondria and other cellular organelles were

poorly developed and few in number. No hemi-desmosomes connected

the basal epithelial cells in the wound area. Basement membrane was

found only in the basal epithelial layers peripheral from the wound area.

Free RNP particles, Golgi apparatus, in lamellar form, and vesciles

appeared normally distributed in the cytoplasm.

Ultrastructure of the stroma

Proliferative collagen fiber formation was evident in the control

stromal wound areas immediately adjacent to the epithelial layers

after the three week healing period. There was a large number of

fibroblasts in the anterior stroma, but very few polymorphonuclear


The fibroblastic cells observed in these investigations were

grouped into two different classifications based on distinct morpho-

logical differences. The first type, which will be henceforth referred

co simply as a true fibroblasc. w.as characterized by extensive,

distended endoplasorjic reticula with extensive ribosomal grannies

lining the ER membrane, large numerous mitochondria, a well-

developed Golgi complex, and increased flocculent filaments aggre-

gated near the cell membrane. The second type, which h shall be

referred to as a fibroblast-like cell, was characterized by poorly

developed cellular organelles, a small amoui c of endoDlamric

reticulum, few ribosomal granules associated with the endoplazric

reticular membrane, few mitochondria, and yet an extensive ap-

pearance of intracellular flocculent filaments aggregated near the

cell mermbrane, quice similar to the uell-developed fibrcbla-c.

The fibroblasts found in the anterior stromal areas of the

control wounds were elongated, flattened, and adjoining each other,

almost connected in an interlacing pattern, with collagen fibers

filling the intercellular spaces. Some fibroblast-like cells were

noted but more were apparent in the deeper portions of the wound.

In the central and posterior stromal areas of the control

wounds collagen fibers were dispersed more randomly in a fibrin

macrix z id scattered fibroblasts and fibroblast-like cells. Under

higher magnification the collagen fibers were found to have a large

variation in the diameter of separate fibers. Also, the collagen

fibers in the posterior stroma of the wound were consistently found

to be aggregated near the fibroblasts and fibroblast-like cells

rather then scattered randomly throughout the intercellular epjces.

Irradiated stromal wound areas were characterized by very few

cells a: all in the anterior stroia, almost all of the fibroblast-

like type, and no cells in the posterior stromal portion of the

wound. The fibroblast-like cells that were present %were not elongated

nor flattened, but were roughly triangular in shape and quite dif-

ferent from the fibroblasts found in the control wounds. Collagen

fibers were conspicuously absent from all areas of the wound. Only

an occasional coliagen fiber was found among the dense a.2'reates

of fibrin that extended from the anterior chamber up to che epithe-

lium. The wound matrix: was composed of a low density cementing

substance, and numerous fine filaments that were not banded and were

occasionally found grouped in bundles. These bundles of fibrin

filaments were often closely entwined and almost run together. This

dense interweaving pattern beca-e more compact as one scanned more

posteriorly in the wound; however, collagen fibers '.ere conspicuously

absent even in deep layers.

Fine strucLure of endotheliun

The control corneal endothelium had recovered the wound

posteriorly very early in the three w:ee' healing period. The endo-

theliu- at thr-e weeks was characterized by large intercellular

spaces, which w-er rather straight passages between the cells rather

than interdigitated. Terminal bars appeared at the posterior surface

of the endothelial layer. The endothelial cell cytoplasm possessed

normal Colgiapparatus and normal sized mirocbondria; however, the

cells were thickened in overall dir.cter. There was an increased amount

of endoplasmic reticulum chat was notably distended, typical of

some synthetic funztionin:. The cell membrane of the endothelial

cells was undulated posteriorly along the anterior chamber. Al-

though Dececet's membrane had not been reformed, there was a dense

amorphous substance that had been formed along the anterior endo-

thelial cell borders, suggesting that synthesis of the membrane

was in progress.

The endotheliu-. in the irradiated corneas had not recovered

the posterior wound at this time. The endochelium peripheral to

the wound area appeared normal all the w:ay ouc to the limbus.

Again, the anterior chamrbar as apparently maintained only by the

epithelium anteriorly and the fibrin clock which closed the wound


All the radiation induced changes were equally demonstrable

in three week wounds when the beta radiation was administered either

immediately after or two and one-half months prior to wounding. In

those corneas irradiated ten months before surgery, howe.-er, some

differences were noted. By three weeks the endothelium had recovered

the wound posteriorly, more fibroblasts were involved in the wound

site, and some suggestion of connective tissue regeneration was


Nine-Week !Wounds

In those corneas irradiated two and one-half months before

wcundin", anterior src:nal deene.eration was still evident after nine

weeks of healing. In the corneas chat received beca radiation'-ten

months prior to surgery, the inhibition of healing was considerably

less. Apparent.:1; some reco-vry v f the radiation damage had begun

by ten months after irradiation.

Light microscopic examinations of che nine-week wounds in

corneas irradiated czn months before wcunding sho:-ed thac endothelium

covered the wound posteriorly and that some arorphous substance was

being laid dcl.n adjacent to the endochelium. Stromal regeneration

was noted in the anterior one-thirdof the wound and scr.e fibrin had

disappeared from the posterior portion of the wound.

Electron microscope studies showed that, although the epithe-

lium 'was much less abnorr.l than in the corneas irradiated t:.o and

one-half months before surgery, so-e epithelial abnormal iies ware

apparent. Superficial epithelial cells had less well developed and

less numerous organelles. The mitochondria were small, simple, and

few in number. The epithelial cytoplasm ::as still less electron

dense than the cytoplasm found in the contralateral, non-irradiated

cornea. Desmosomes were present at cell junctions, but intercellular

spaces between the epithelial cells remained abnormally large.

Basal epithelial cells hd a greater co-zpl--rnt' of mitochondria

and other organelles than were found in the basal epithelial cells

of the other irradiated groups. Basem-enc membrane was still totally

absent from the wound area, in contrast to the non-irradiated corneas,

wherein segments of basement membrane were found along the basal

epithelium covering the wound.

The anterior stroma of the irradiated corneas, at nine weeks of

healing, showed a notable amount of newly synthesized collagen adjacent

to numerous fibroblasc-like cells and some few scattered fibroblasts.

Most of the cells involved in the immediate wound area were similar

to those previously described as fibroblast-like cells; however, the

majority of these cells contained a substantial complement of smooth

endoplasmic reticulun and more numerous, better defined, organelles

than the fibroblast-like cells noted in the more severely inhibited

corneas. These fibroblast-like cells noted at this time appeared

more like inactive or non-synrhesizing fibroblasts, rather than some

morphologically different cell type or a primordial form of a develop-

ing fibroblast. A considerable quantity of fibrin remained scattered

throughout the middle and posterior stromal portion of the wound.

Cells were less numerous as one scanned posteriorly toward the endo-

theliun. In the posterior of the stroma near the endothelium, more

of the cells observed were identical to the fibroblast-like cells,

lacking prominent organelles, found in the more damaged corneas which

were irradiated at surgery.

Phosphotungstic -cid (PTA) staining for collagen showed normal

banded collagen fibers in the anterior stroma at nine weeks of healing.

The concentration of collagen fibers was less in the corneas irradiated

ten months before wounding than in the non-irradiated corneas.

Near the endothelial layer of the corneas irradiated ten

months before wounding, sparse collagen fibers were found amid fibrin

filaments. Along the endothelini cells an electron dense amorphous

substance was found, alr.ost adhering to and lining the endothelial

surface facing the strona. This apparently was an initial sta;e in

the regeneration of Descemet's memb-.rane by the endochelium. The

endothelium at nine weeks of healing was characterized by increased

ribosomal-lined endoplasmic reciculum, increased nitochondria and

vehicles, and enlarged intercellular spaces. The tenrinel bars at

the endothelial surface junccion; and the desmosomes w..ere normal.

In surmiry, the ten monch period between irradiation and

wounding seemed to be sufficient to allo,. some recovery of che

damage to the corneal cells and partial recovery of the normal

repair capability.

Nine-Monch t.ounds

Corneal wounds after nine months of healing appeared clinically

to be almost completely healed, with che exception of a superficial

hay- scar, smaller chan the original incision. Light microscopy

shows that the basal cell epithelium in the wound area is not com-

pletely normal, and epithelium is thickened in the ic-mediate area.

Anterior strc-a is somewhat disorganized and in the area of the scar

the lamellar architecture has not been reestablished. The posterior

half of the stroma has the lamellar collagen arrangement and appears

normal e::cept for greater than normal numbers of stromal connective

tissue cells. Descemet's membrane has been reformed, the curled

ends of the original, severed, Descemet's membrane are present, and

the endothelium is normal. If it had not been for the presence of

severed ends of the original Descemet's membrane, it would have

appeared that the :ound had only been superficial arid rnt penetrating.

After nine months of healing the irradiated wounds appear

similar to uhe non-irradiated control wounds; however, the dis-

organized state of the stromal collagen arrangement is found at all

depths of the stroma. The scar is much deeper, involving all of the

stroma, and the stromal wound area has far fewer connective tissue

cells than the non-irradiated. Descemer's membrane has been reformed

and endothelium is intact.

Electron micrcs:opic studies demonstrated that the epithelium

at the wound was similar between the irradiated and the non-irradiated

corneas. The stroia of the irradiated corneas was characterized by

collagen bundles, organized in uneven lamellae-like layers, surrounded

by mucopolysaccharide ground substance, and sparsely populated by

normal keratocytes. Only rarely w-ere fibroblasz-like cells present

and only a rare ?P;C or mononuclear leukocyce w.as found in the entire

corneal wound.

Descemet's membrane, although thinner than normal, was com-

pletely reformed, but the surface of Descemet's membrane adjoining

the stroma was not evenly nor clearly demarcated.

The endothelial layer of the irradiated corneas appeared normal.

Endothelial cells had lost the increased endoplasmic reticulum found

earlier in the delayed healing corneas. The mitochondria, Golgi

complex, ER, and other cellular organelles appeared normal. Tight

junctions between cells formed normal compact, intercellular spaces

ending with normal terminal bars at the anterior chamber side of the

cell layer. The endothelium appeared viable and functioning, based

on its complement of internal structures within each call and -he

normal interdigitacion of adjacent cells.


Transmission electron microscopic studies have shown that

changes in the fine structure of epichelial cells, fibroblastic cells,

and endothelial cells are part of the beta radiation inhibition of

normal corneal repair. Poorly developed intracellular organelles and

a conspicuous lack of collagen synthesis :'ere consistent '..ith an ap-

parent decrease in cellular activity and the lack' of tensile strength

found in the proceeding experiment.

The most prominent radiation induced changes observed in the

basal epithelial cells that recover the wound were: (1) few and

poorly developed mitochondria, endoplasnic reticulum (ER), and

other cellular organelles, (2) a notable lack of hemi-desmosomncs and

enlarged intercellular spaces, and (3) a lack of basement membrane

regenerct ion.

Fibroblasts and fibroblast-like cells were found in the normal

healing stroca; however, fewer cells were found in the anterior portions

of the wound and no cells were found in the posterior wound. In the

irradiated corneas, among what few cells were involved in the wound,

fewer fibroblasts and a higher proportion of fibroblast-like cells

contrasted with the high ra:io of fibroblasts to fibroblast-like

cells found in the non-irradiated corneas. The fibroblast-liko cells

lacked well-developed cellular organelles, and distended, ribosomal

granule-lined endoplasmic ret.iculum found normally in synthesizing

fibrobiasts as described by Ross (37 ). In addition, the fibroblast-

like cells were not flattened, elongated, nor functionally arranged

(in almost interconnecting fashion) as were mature fibroblasts found

in the normal hearing corneal wounds. Whether these two types of

fibroblasric cells are intermediate forms of developing fibroblasts,

as described previously in healing corneas (S3 ), or whether they

are functionally different fibroblasts as found in tendon regenera-

tion (39 ) is not well understood.

The beta radiation treatments apparently prohibited the

appearance of mature, synthesizing, fibroblasts in the stromal

areas of the wound. Collagen synthesis was also inhibited by the

radiation, either as a result of the fibroblast inhibition, or con-

comitant with the lack of mature fibroblasts normally found in the

healing cornea.

It has been reported earlier ( ') that the most distinctive

intracellular feature of the mature fibroblast, i.e., extensive,

highly-developed rough endoplasmic reticulum (ER), is the organelle

coi-only associated with e::tracellular protein synthesis, and pre-

sumably responsible for collagen synthesis. The most obvious dif-

ference between the fine structure of the fibroblast-like cells and

the mature fibroblasts was the absence of extensive rough endoplasmic

reticulum in the former cell. In the irradiated corneas where the

proportion of fibroblast-like cells made up almost the entire

population of cells involved in the wound, collagen synthesis was

conspicuously absent even after three weeks of healing. Only after

nine weeks, when the proportion of mature fibroblasts had increased,

was some minimal collagen synthesis observed in the corneas that had

been allowed the longest interval between irradiation and surgery.

The presence of the mature fibroblast seemed necessary for

collagen synthesis in the healing cornea. This observation is

consistent with the current concepts of the functional capability

of the fibroblast and synthesis of collagen precursors with fibro-

blasts (90, 91).

The fibroblast-like cells that well out-numbered the mature

fibroblasts in the irradiated corneas were morphologically, and

probably functionally, different from connective tissue cells

capable of collagen synthesis. This observation was further sup-

ported by the conspicuous absence of any tropocollagen or other pre-

collagen macromolecular aggregates near the fibroblast-like cells.

If the cells were capable of intracellular synthesis of collagen

precursors, which is dubious in the absence of extensive rough ER,

some indication of these macromolecules should have been demonstrable

either within the cell's Golgi, or cytoplasm, or extracellularly

adjoining the cell. Therefore, the lack of collagen synthesis in

the irradiated corneal wounds would seem to be correlated with the

lack of cells that are capable of synthesis, rather than some inter-
fil .....

ference with the extracellular aggregation of cropocollagen into

the classical, banded collagen fibrils.

The radiation inhibition of endothelial migration emphasized

the difference between the relative radiosensitivities of the epi-

thelial and endothelial cell layers. Epithelial cells received

higher doses of beta radiation than did endothelial cells, yet,

epithelial migration was not inhibited. This observation is in-

consistent with classical guidelines of radiosensitivity of dif-

ferent cells based on their degree of differentiation and frequency

of cell division (92). Apparently, radiation damage to the ability

of a cell to migrate and the overall radiosensitivity of the cell

are two separate parameters of radiation effects on cells.

It was interesting that the intracellular characteristics of

the intact peripheral endothelium in the irradiated corneas at

three weeks of healing, seemed normal even when the endothelium did

not spread and cover the wound posteriorly. Vhen the radiation

inhibition decreased and endochelial migration was observed, the

fine structure of the endothelial cells was characterized by in-

creased organelles and particularly rough ER, normally associated

with increased protein synthesis. Increased protein synthesis would

be expected in an expanding, spreading cell. At later stages of

healing the intracellular fine structure of endothelial cells showed

only minimal amounts of ER, indicating that the need for increased

protein synthesis had diminished. Although current concepts would

suggest that endothelial protein synthesis was ctill needed for

secretion of Desrce=t' re=r r n- it see=s reasonable cha: once the

total demands for protein (n eded for bocth ct.-:co as-ic e::a-:.sion

and e::tracellular produc-sz) decreased, by the amount recuir;: for

cell ex.panion, che nec for such an e:::ensive rough ER would like-

wise decrease. Hence, the disappearance cf e::tensi.ve rough ER

following cessation of endothelial ,iarac:ion was not totally


It was hoepd ChaCt he sAcceeing e-:7.rie.ens, wh.:ich 'ere

designed to examine the enz'-Me activity and nuclaic acid synthesis

in b.th nrmasl and irradiated corneal wounds, would give a brter

understan:-in0 of the iz-prtance and i.cerrelationships of :he

ultrastrucrural differences nced in the irrdiatd, ':oundced


Figure 18. Histological section of control corneal wound at three
weeks of healing. Showing: thickness of regenerated
wound, epithelium (ep), severed Descemet's membrane (Dm),
fibroblasts in stroma (St), and endothelium (en) recov-
ering the posterior wound. Epon, toluidine blue, x200.

Figure 19. Section of wound in contralateral irradiated eye, at
day 21. Demonstrating: abnormal, enlarged epithelial
cells (ep), lack of stromal regeneration (St). and
absence of endothelium. Epon, toluidine blue, x200.

S I u .: .........'

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