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Proliferation and Migration of Epithelial Cells*
PROLIFERATION AND MIGRATION O F E P I T H E L I A L CELLS* DURING CORNEAL WOUND REPAIR IN THE RABBIT AND THE RAT CALVIN H A N N A , P H . D .
Little Rock, Arkansas Corneal wound healing is characterized by an early migration of neighboring epi thelial cells over the denuded area. Later the thinned epithelium is repaired by an in crease in cell division. This movement of epithelial cells was observed in vivo by Buschke1 in the frog cornea after pin prick and small linear injuries. He found that epi thelial repair in the pin-prick injury was in itiated by the epithelial cells throwing out cytoplasmic pseudopodial extensions into the denuded area. The linear injuries were repaired by a spread and flattening of epi thelial cells to seal the wound margin. Arey and Covode2 observed that many basal epithelial cells were undergoing mito sis in the cornea. After the wounding, the number of mitotic cells in the basal epitheli al cell layer over the entire cornea markedly decreased. In these wounds the epithelial cells covered the denuded area in one-half day while the epithelial cell mitotic rate did not go above the prewound value until after the fourth day. This dissociation of cell mi gration and cell proliferation in the corneal wound healing process is well documented and carefully reviewed by Duke-Elder.3
tion at one day after the wounding migrated toward the wound crater.4 Wound healing of the skin and tongue studied by Block, Seiter, and Oehlert6 also showed a dissociation between the initial mi gration and the later proliferation of epi thelial cells. However, details of the process differ from those reported for the corneal healing by Arey and Covode2 and Hara. 4 Block, Seiter and Oehlert found, after skin and tongue wounding, an initial cessa tion of the normal thymidine-H 3 uptake by epithelial cells. This was followed by an early increase in the number of thymidineH 3 incorporating epithelial cells at some dis tance from the wound. As the skin and tongue wound repair proceeded a wave of thymidine-H 3 incorporating cells moved to the wound crater with time. A preliminary investigation showed that a similar phenomenon in basal epithelial cells of the cornea occurred following corneal wounding. This report details the changes that take place in the proliferation and mi gration of epithelial cells following corneal wounding in the rabbit and rat.
Hara 4 studied a variety of corneal wounds in the rabbit utilizing thymidine-tritium (-H 3 ) using the method of Hanna and O'Brien. 5 After wounding there was a ces sation of the incorporation of thymidine-H 3 into basal epithelial cells at the wound edge. The number of cells utilizing thymidine-H 3 gradually returned to normal over a five-day period.4 Also, they found that those epitheli al cells undergoing thymidine-H 3 incorpora-
METHODS AND MATERIALS
Adult albino rabbits (3 to 4 kg) were an esthetized with intravenous thiopental sodi um ( 2 % ) . The cornea was treated with lidocaine (2%) and injured by making an open ing into the stroma about 1.5 mm from the limbus, using a keratome and inserting a cyclodialysis spatula into the half depth of the cornea. The spatula was pushed across the cornea and then the spatula was cut out using two razor blade cuts to give an initial wound of about 1-mm wide and 5-mm long. At various periods before and after wounding the rabbits were anesthetized and 5 μc of thymidine-H 3 (3 and 13 c/mmole;
* From the Department of Pharmacology, Uni versity of Arkansas Medical Center. This investi gation was supported by PHS research grants NB-04024 and NB-05076 from the National In stitue of Neurological Diseases and Blindness, US Public Health Service. 55
CALVIN HANNA
56
Schwarz BioResearch, Inc., Orangeburg, New Jersey) in 0.04 ml of TC-199 tissue culture media (Difco Laboratories, Inc., Detroit, Michigan) was injected into the an terior chamber of the eye using a 33-gauge needle. At the completion of the individual experiments, the rabbits were killed and the corneas removed and fixed in 10% formalin, and embedded in paraffin. Sections 5-μ thick were cut and covered with Kodak N T B 3 liquid emulsion. 7 After exposure for two weeks, the film was developed in Kodak D19 solution for 4 min, fixed, and the sec tions were stained with hematoxylin and eosin. 7 Selected sections were treated with deoxyribonuclease ( D N a s e , Worthington Biochemicals, Freehold, New J e r s e y ) 8 be fore autoradiography to remove tritium that was incorporated into D N A . Similar studies were carried out using the rat except that a cautery needle was used to produce the wound. The wounds were both superficial involving only the epithelium and deep involving the epithelium and the stroma. RESULTS THYMIDINE-H
3
UPTAKE I N T H E E P I T H E L I A L
CELLS
Rabbits were given a nonpenetrating corneal wound and then killed in pairs 6, 12, 15, 2 1 , 24, 30, 36, 42 hours and 2, 3, 4, 6, 10 days later. T w o hours before death the eye of each animal was injected with thymidineH 3 . During the first 12 to 15 hours of the healing process, the epithelial cell migrated into the wound crater and the epithelial layer near the wound thinned. During this time and up to about one day, there was a progressive decrease in the number of basal epithelial cells incorporat ing thymidine-H 3 which extended to about 120 epithelial cell lengths from the wound. Basal cells in a narrow band just beyond the edge of the zone of decreased thymidine-H 3 incorporation began a marked increase in the number of cells incorporating thymi dine-H 3 and undergoing mitosis.
T h e zone of decreased thymidine-H 3 in corporation and the following zone of in creased thymidine-H 3 uptake and mitosis moved to the wound edge in four to six days (fig. 1 ) . T h e zone of the decreased thymidine-H 3 incorporation in some wounds contained an occasional basal epithelial cell with a near normal incorporation of thymi dine-H 3 and a few cells with a very low in corporation of thymidine-H 3 . These latter cells were detected by using the high specific activity thymidine-H 3 . W h e n the wound crater was repaired the epithelial cells in the center of the wound underwent thymidine-H 3 uptake. T h e rate at which the zone of dividing (thymidineH 3 uptake and mitotic figures) epithelial cells reached the wound crater depended on the size of the wound. W h e n the wound cra ter was small, as illustrated in Figure 2, the wave of dividing cells reached the wound edge in three days whereas when the crater was larger this took about six days (figs. 3 and 4 ) . Several rabbits were studied at 2, 4, 6, and 10 days after the cornea was given a small penetrating wound with a razor blade. T h e healing process involving thymidine-H 3 uptake by and mitosis of epithelial cells
f
CELLS/30 MM epithelium
6-4
3 2 DAYS stroma
/12 o
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1
Fig. 1 (Hanna). Graphic representation of changes in cell division of corneal epithelial cells at various times following wounding. There is an initial decrease in the number of cells dividing (DNA synthesis and mitosis) in the cornea ex tending from the wound edge. In the healing process at one day at an average of 120 cells from the wound edge, there is a marked increase in cell division in a narrow band of basal cells. This band of cells undergoing division migrates to and includes the basal cells in the wound.
THYMIDINE UPTAKE DURING CORNEAL REPAIR
57
Fig. 2 (Hanna). Graphic representation of movement of corneal epithelial cells into the wound crater. Each cell type, that is, basal, wingtype and superficial, migrates into the wound ac cording to its initial position as indicated by ar rows. TJ7n
r
around the perforating wound was similar to that found in the nonperforating wound. In initial rabbit studies on nonperforating corneal wounding, the edges of the wound were left with jagged edges. W h e n the heal ing process began, epithelial cells migrated over the jagged edges and into the fissures in the stroma. These epithelial cells deep in
the stroma continued to undergo thymidineH 3 uptake at a moderate rate (fig. 4 and 5 ) . Small superficial corneal burns were pro duced in rats using a cautery. In those burns where only the epithelial cells were killed the adjacent basal epithelial cells un-
Fig. 3 (Hanna). Autoradiograph of a corneal section from a rabbit killed three days after the wound ing. Thymidine-Ha was injected into the anterior chamber two hours before the animal was killed. This section was taken near one end of the wound where the crater was small. Those epithelial and stromal cells incorporating thymidine-H3 are indicated by a large black dot covering each cell nuclei and by an arrow. (Restained with Geimsa, χ400.)
CALVIN HANNA
58
derwent thymidine-H 3 incorporation be tween eight and 24 hours. In these superficial burns there was no zone of ces sation of thymidine-H 3 incorporation ex tending from the burned area. CORNEAL
EPITHELIAL
CELL
MIGRATION
IN
WOUND
After a thymidine-H 3 injection, the tri tium label appears in the various layers of the corneal epithelium with time. I n this way the basal, the wing-shaped and the superficial squamous cells were shown to contain the tritium label by killing the rab
bits 1, 4 and 10 days later, respectively. T h e migration of the various epithelial cell layers into the wound crater was studied by inject ing thymidine-H 3 into the anterior chamber of rabbits and then wounding at the ap propriate time later. T h e rabbits were then killed 1, 2, 3, and 6 days later. W h e n the cornea was wounded one day after the thymidine-H 3 administration and the animal killed one or two days later, the following was found: Outside the wound the tritium-labeled ep ithelial cells were in the basal layer; at the wound edge there were relatively fewer tri
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Fig. 4 (Hanna). Autoradiograph of a corneal section from a rabbit killed four days after wounding. The thymidine-H3 was injected two hours before the animal was killed. The presence of tritium-labeled epithelial and stromal cells are indicated by arrows. (Hematoxylin-eosin, X320.)
THYMIDINE UPTAKE DURING CORNEAL REPAIR
59
Fig. 5 (Hanna). Autoradiograph of a corneal section from a rabbit killed six days after wounding. The thymidine-H3 was injected two hours before the rabbit was killed. In this section note the group of tritium-labeled epithelial cells near the wound edge and a tritium-labeled epithelial cell in a wound fissure, as indicated by arrows. (Hematoxylin-eosin, X350.)
tium-labeled basal cells ; in the wound crater the tritium-labeled epithelial cells were in the innermost cell layer. In the corneas wounded four days after tritium labeling and killed one to six days after wounding the tritium label was in the wing-shaped cells outside the wound and in the rounded cells in the middle layer of the epithelial cells located in the wound crater. Corneas that were wounded 11 days be fore tritium labeling and the animals killed one to two days later showed tritium-labeled superficial epithelial cells over all parts of the cornea (fig. 6). When this was repeated and the animals killed three to six days later, the tritium-labeled squamous epitheli al cells were found only over the wound cra ter region. These results indicate that each epithelial cell layer migrated into the wound while generally maintaining its initial rela tive position (fig. 7). Also, the epithelial cells in the wound were sloughed off at a slow rate, depending
on the speed of healing of the stromal layer and the return of cell division to cells in the wound crater. Results of studies on the wounded rat cornea were similar except the time intervals between thymidine-H 3 admin istration and the wounding was shorter in the rat because the turnover time of the rat corneal epithelium is shorter (about seven days) than that of the rabbit (about 12 days). TWO-HOUR THYMIDINE-H 3 UPTAKE IN STROMAL CELLS
Between 15 and 48 hours after penetrat ing or nonpenetrating corneal wounding, cells near the wound edge incorporated thy midine-H 3 . These cells, either on tangential or saggital plane sectioning and hematoxylin and eosin staining, had the shape and stain ing characteristics of fixed stromal cells. The tritium-labeled cells in the stroma were usually found in a band around the wound crater at about two cell lengths from the
60
CALVIN HANNA
tions indicating that the thymidine-H 3 was incorporated into DNA. DISCUSSION
It is generally accepted that the initial re sponse in corneal wound healing is the mi gration of epithelial cells into the defect. The way the epithelial cells migrate depends to some extent on the type of wound. When a fine-pointed needle is used to produce the wound in the frog cornea, the marginal basal cells elongate with pseudopodial exten sions reaching into the defect.1·9 These studies have been taken to indicate that basal epithelial cells migrate into and fill the crater by an ameboidlike movement. This evidence has been extrapolated to indicate that all epithelial cells in the wound crater are basal cells in origin and evidence for this has been that the epithelial cells in the crater are spherical in shape and not unlike basal cells in appearance.3 Evidence from the present study suggests that each epitheli al cell layer participates in supplying cells to cover a moderately sized defect, possibly by sliding into the wound. Fig. 6 (Hanna). Autoradiograph of a corneal section from a rabbit killed six days after wounding and two hours after the injection of thymidine-H3. This section was taken through a fissure in the ηorneal stroma. The cells underly ing the black areas in the center of the picture are epithelial cells. (Hematoxylin-eosin, original magnification X600.)
wound edge and extending outward about 10 cell lengths (figs. 4 and 5). Occasionally a tritium-labeled cell in the stroma was found at the wound edge in contact with an epithelial cell or at some distance from the wound edge. When the wound healing was slowed due to the presence of a large jagged wound the uptake of thymidine-H 3 by stromal cells continued until the crater healed. Similar results were found in the rat. Representative corneal wound sections were treated with DNase before autoradiographic analysis. The treatment with DNase removed the tritium from the sec
The second generally accepted response in corneal wound healing is that of cell divi sion to replenish the cells lost. Arey and Covode2 observed that mitosis in the basal epithelial layer ceased for several days fol lowed by a temporary increase above normal in the mitotic rate. Hara 4 observed a similar cessation in the number of epithelial cells of the wounded rabbit cornea undergoing thymidine-H 3 in corporation for several days. However, Smelser10 found that the total number of mitotic figures in the rat cornea following wounding was about the same as the num ber of figures found before wounding. The results of Arey and Covode2 and Hara 4 indicate that the corneal epithelial cells cease cell division several days after corneal wounding. These results are not consistent with the mitotic changes observed by Smelser.10 However, the results obtained in the present study indicate that those epi-
THYMIDINE UPTAKE DURING CORNEAL REPAIR
61
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Fig. 7 (Hanna). Autoradiograph of a corneal section from a rabbit killed 10 days after the injection of thymidine-H3 and two days after wounding. The tritium-labeled epithelial cells were in the superficial layers of the cornea at the time of wounding and this section shows the migration of these cells into the wound crater. (Restained with Geimsa, original magnification X300.)
thelial cells near the wound cease to divide incorporation in the basal epithelial cells of while those cells at some distance from the the skin and tongue.6 wound epithelial were undergoing an in The environmental conditions for the creased rate of cell division. As a result, the maintenance of cell division are not well un number of epithelial cells dividing over the derstood. Further, the various stages of cell entire cornea a day after a small wound division apparently need not be completed. would not be greatly different from that of When the corneal stroma lamella are sepa the nonwounded cornea. rated using a blunt spatula the stromal cells Thymidine-H 3 is an especially helpful adjacent to the injury undergo DNA method in these studies because there are synthesis.11'12 Some of these stromal cells do several times more cells incorporating thy not appear to undergo a mitotic change with midine-H 3 than the number of mitotic time.11 Freezing of cells in the cornea pro figures in the cornea at any one time. There duced stimulation of DNA synthesis and fore, it was relatively easy to locate the zone thymidine-H 3 incorporation in various cells of cells in mitosis when sagittal plane cuts and these corneal cells retain the tritium-la were used. The sequence of events in cor beled DNA for long periods.13 Lens wound neal wound healing reported here resembles ing also initiated a wave of cell division very closely the sequence of thymidine-H 3 starting near the wound edge,14 and in ga-
CALVIN HANNA
62
lactose cataracts the wave of D N A synthesis starts at the equator region and moves to ward the anterior pole. 5 In most of these ex amples the cells adjacent to the swelling and / o r edema intiate the cell division. With the corneal wound edema is present around the wound crater. T h e cells in the stroma undergoing D N A synthesis are located ad jacent to this area and not usually in the edematous portion. T h e cessation of D N A synthesis in the basal epithelial cells and the continued D N A synthesis of these same epithelial cells in a fissure following corneal wounding is in some ways a perplexing phenomenon for several reasons. Basal epithelial cells not undergoing D N A synthesis in the wound crater can be found adjacent to an occassional cell in the stroma that is undergoing D N A synthesis. T h e basal epithelial cells normally undergo D N A synthesis whereas the stromal cells in the adult eye do not nor mally undergo D N A synthesis. 15 T h e epi thelial cells that migrated into the open wound crater do not undergo D N A synthe sis until the crater is repaired but those epi thelial cells that migrated into a fissure con tinue to undergo D N A synthesis. It is pos sible that the release of tension of anterior epithelial cells on the basal epithelial layer produced by the wound may cause a cessa tion of D N A synthesis in the basal cells. The epithelial cells in the wound crater be come spherical, indicating a release of ten sion by the superficial cell layers. Those epi thelial cells that migrated into stromal fis sures would be expected to be under tension and these epithelial cells do continue D N A synthesis. SUMMARY
Corneal wounds were produced in rabbits and rats and the changes in D N A synthesis were estimated utilizing thymidine-tritium and autoradiography. E a r l y in the healing process each layer of the epithelium slides into the wound crater. After this there is a
cessation of D N A synthesis and mitosis in the basal epithelial cells reaching some dis tance from the wound. By one day D N A synthesis in the stromal cells near the wound edge begins and this continues until the wound is healed. Also there is a wave of cell division that moves toward the wound crater and finally involves the basal epitheli al cells above the healed wound crater be fore returning to normal. These changes in cell division in corneal cells closely resemble those changes observed in the healing of skin wounds. University of Arkansas Medical Center. REFERENCES
1. Buschke, W. H : Morphlogic changes in cells of corneal epithelium in wound healing. Arch. Ophth., 41:306-316, 1949. 2. Arey, L. B., and Covode, W. M. : The method of repair in epithelial wounds of the cornea. Anat. Rec, 86:75-86, 1943. 3. Duke-Elder, S. : Textbook of Ophthalmol ogy. St. Louis, Mosby, 1958, v. 6, p. 5964. 4. Hara, J. : An autoradiographic study of re generation of the corneal epithelium. Folia Ophth. Jap., 13:443-466, 1962. 5. Hanna, C, and O'Brien, J. E. : Cell produc tion and migration in the epithelial layer of the cornea. Arch. Ophth., 64:536-539, 1960. 6. Block, P., Seiter, I., and Oehlert, W. : Autora diographic studies of the initial cellular response to injury. Exper. Cell. Res., 30:311-323, 1963. 7. Hanna, C, and O'Brien, J. E. : Lens epitheli al cell proliferation and migration in radiation cataracts. Radiβt. Res., 19:1-11, 1963. 8. Amano, M. : Improved techniqques of the en zymatic extraction of nucleic acids from tissue sections. J. Histochem. Cytochem, 10 :204, 1962. 9. Friedenwald, J. S., and Buschke, W. : Influence of some experimental variables on the epithelial movements in the healing of corneal wounds. J. cell. Comp. Physiol., 23:95-107, 1944. 10. Smelser, G., and Ozanics, V. : Effeffct of chemotherapeutic agents on cell division and heal ing of corneal burns and abrasions in the rat. Am. J. Ophth., 27:1063, 1944. 11. Hanna, C, and O'Brien, J. E. : Thymidinetritium labeling of the cellular elements of the corneal stroma. Arch. Ophth., 66:362-365, 1961. 12. Hanna, C, and Irwin, E. S. : Fate of cells in the corneal graft. Arch. Ophth., 68:810-817, 1962. 13. Polack, F. M., Smelser, G. K., and Rose, J. : Long-term survival of isotopically labeled stromal and endothelial cells in the corneal homografts. Am. J. Ophth., 57:67, 1964. 14. Harding, C. V., Donn, A., and Srinivasan,
THYMIDINE UPTAKE DURING CORNEAL REPAIR B. D. : Incorporation of thymidine by injured lens epithelium. Exper. Cell Res. 18:582-585 (Nov.) 1959.
IS. Hanna, G, and O'Brien, J. E. : Cell turnover in the adult human eye. Arch. Ophth., 65:695-698, 1961.
THE MECHANISM OF EXTERNAL M I L E S A. GALiN.t M.D.,
63
IRVING BARAS, M.D.,
FILTRATION*
AND J O H N M. M C L E A N ,
M.D.
New York
External filtration is the operative proce present data concerning the mechanics of dure most commonly used for the control of outflow in eyes which have been subjected glaucoma. However, the mechanism by to cautery sclerostomy with peripheral which this form of surgery exerts its effect iridectomy.10 Also described will be a tech has not been completely elucidated. Initially, nique whereby aqueous may be collected it was believed that neurohumoral factors across a filtering bleb in sufficient quantities reduced intraocular pressure after surgical to permit detailed analysis. intervention. Presently prevalent is a more METHODS mechanistic view which holds that aqueous actually leaves the anterior chamber. Of the A group of successive glaucoma patients physical pathways available for the egress operated upon at The New York Hospitalof aqueous after external filtration, two Cornell Medical Center by the technique of major routes seem most logical : cautery sclerostomy were chosen for this 1. Transconjunctival flow, in which study. In all cases a wide limbus-based conaqueous actually passes through the epitheli junctival flap had been utilized, the initial um of the bleb and mixes with the tears, incision being well above the insertion of was proposed by Verhoeff,1 Seidel2 and the superior rectus tendon, at least 10 mm from the limbus. After reflection of the flap, others.3"6 2. By the second path, championed by the the Hildreth cautery was applied to the ap early advocates of the iridencleisis opera propriate perilimbal area and cauterization tion,7'8 aqueous may pass into the subcon- carried out. The anterior chamber was en junctival tissues and thence into the venous tered only with the cautery tip and the pe ripheral iridectomy then performed. Closure system. It is at once apparent that both of these was carried out in layers. mechamisms may be operative in any single Postoperatively all patients were observed eye after antiglaucoma surgery, though one carefully, and particular attention was focu or the other may dominate, depending on sed on the type of bleb that formed. the type of operative procedure per In addition, the Seidel2 fluorescein test* 9 13 formed. ' * In this test, the eye must first be anesthetized It is the purpose of this communication to . to prevent blinking and squeezing due to stinging From the Department of Surgery (Ophthal mology), New York Hospital-Cornell Medical Center. This paper was presented in part at the meeting of the American Academy of Ophthalmololgy and Otolaryngology in Chicago, October, 1964. This study was aided by grants from the United States Public Health Service, the Nation al Council to Combat Blindness, and the National Society for the Prevention of Blindness. t Career scientist, Health Research Council, New York.
by fluorescein. The patient is seated at the biomicroscope and instructed to maintain fixation downward. The upper lid is immobilized by the observer. A moistened Fluori-strip slid across the bleb area thin-coats the bleb with an orange layer of concentrated fluorescein. A positive test consists of a greenish discolora tion in the center of the orange-stained area as the fluorescein becomes diluted. This can be ob served with white light or through the blue filter used for applanation tonometry. As the area is kept under observation, the lighter green areas
Little Rock, Arkansas Corneal wound healing is characterized by an early migration of neighboring epi thelial cells over the denuded area. Later the thinned epithelium is repaired by an in crease in cell division. This movement of epithelial cells was observed in vivo by Buschke1 in the frog cornea after pin prick and small linear injuries. He found that epi thelial repair in the pin-prick injury was in itiated by the epithelial cells throwing out cytoplasmic pseudopodial extensions into the denuded area. The linear injuries were repaired by a spread and flattening of epi thelial cells to seal the wound margin. Arey and Covode2 observed that many basal epithelial cells were undergoing mito sis in the cornea. After the wounding, the number of mitotic cells in the basal epitheli al cell layer over the entire cornea markedly decreased. In these wounds the epithelial cells covered the denuded area in one-half day while the epithelial cell mitotic rate did not go above the prewound value until after the fourth day. This dissociation of cell mi gration and cell proliferation in the corneal wound healing process is well documented and carefully reviewed by Duke-Elder.3
tion at one day after the wounding migrated toward the wound crater.4 Wound healing of the skin and tongue studied by Block, Seiter, and Oehlert6 also showed a dissociation between the initial mi gration and the later proliferation of epi thelial cells. However, details of the process differ from those reported for the corneal healing by Arey and Covode2 and Hara. 4 Block, Seiter and Oehlert found, after skin and tongue wounding, an initial cessa tion of the normal thymidine-H 3 uptake by epithelial cells. This was followed by an early increase in the number of thymidineH 3 incorporating epithelial cells at some dis tance from the wound. As the skin and tongue wound repair proceeded a wave of thymidine-H 3 incorporating cells moved to the wound crater with time. A preliminary investigation showed that a similar phenomenon in basal epithelial cells of the cornea occurred following corneal wounding. This report details the changes that take place in the proliferation and mi gration of epithelial cells following corneal wounding in the rabbit and rat.
Hara 4 studied a variety of corneal wounds in the rabbit utilizing thymidine-tritium (-H 3 ) using the method of Hanna and O'Brien. 5 After wounding there was a ces sation of the incorporation of thymidine-H 3 into basal epithelial cells at the wound edge. The number of cells utilizing thymidine-H 3 gradually returned to normal over a five-day period.4 Also, they found that those epitheli al cells undergoing thymidine-H 3 incorpora-
METHODS AND MATERIALS
Adult albino rabbits (3 to 4 kg) were an esthetized with intravenous thiopental sodi um ( 2 % ) . The cornea was treated with lidocaine (2%) and injured by making an open ing into the stroma about 1.5 mm from the limbus, using a keratome and inserting a cyclodialysis spatula into the half depth of the cornea. The spatula was pushed across the cornea and then the spatula was cut out using two razor blade cuts to give an initial wound of about 1-mm wide and 5-mm long. At various periods before and after wounding the rabbits were anesthetized and 5 μc of thymidine-H 3 (3 and 13 c/mmole;
* From the Department of Pharmacology, Uni versity of Arkansas Medical Center. This investi gation was supported by PHS research grants NB-04024 and NB-05076 from the National In stitue of Neurological Diseases and Blindness, US Public Health Service. 55
CALVIN HANNA
56
Schwarz BioResearch, Inc., Orangeburg, New Jersey) in 0.04 ml of TC-199 tissue culture media (Difco Laboratories, Inc., Detroit, Michigan) was injected into the an terior chamber of the eye using a 33-gauge needle. At the completion of the individual experiments, the rabbits were killed and the corneas removed and fixed in 10% formalin, and embedded in paraffin. Sections 5-μ thick were cut and covered with Kodak N T B 3 liquid emulsion. 7 After exposure for two weeks, the film was developed in Kodak D19 solution for 4 min, fixed, and the sec tions were stained with hematoxylin and eosin. 7 Selected sections were treated with deoxyribonuclease ( D N a s e , Worthington Biochemicals, Freehold, New J e r s e y ) 8 be fore autoradiography to remove tritium that was incorporated into D N A . Similar studies were carried out using the rat except that a cautery needle was used to produce the wound. The wounds were both superficial involving only the epithelium and deep involving the epithelium and the stroma. RESULTS THYMIDINE-H
3
UPTAKE I N T H E E P I T H E L I A L
CELLS
Rabbits were given a nonpenetrating corneal wound and then killed in pairs 6, 12, 15, 2 1 , 24, 30, 36, 42 hours and 2, 3, 4, 6, 10 days later. T w o hours before death the eye of each animal was injected with thymidineH 3 . During the first 12 to 15 hours of the healing process, the epithelial cell migrated into the wound crater and the epithelial layer near the wound thinned. During this time and up to about one day, there was a progressive decrease in the number of basal epithelial cells incorporat ing thymidine-H 3 which extended to about 120 epithelial cell lengths from the wound. Basal cells in a narrow band just beyond the edge of the zone of decreased thymidine-H 3 incorporation began a marked increase in the number of cells incorporating thymi dine-H 3 and undergoing mitosis.
T h e zone of decreased thymidine-H 3 in corporation and the following zone of in creased thymidine-H 3 uptake and mitosis moved to the wound edge in four to six days (fig. 1 ) . T h e zone of the decreased thymidine-H 3 incorporation in some wounds contained an occasional basal epithelial cell with a near normal incorporation of thymi dine-H 3 and a few cells with a very low in corporation of thymidine-H 3 . These latter cells were detected by using the high specific activity thymidine-H 3 . W h e n the wound crater was repaired the epithelial cells in the center of the wound underwent thymidine-H 3 uptake. T h e rate at which the zone of dividing (thymidineH 3 uptake and mitotic figures) epithelial cells reached the wound crater depended on the size of the wound. W h e n the wound cra ter was small, as illustrated in Figure 2, the wave of dividing cells reached the wound edge in three days whereas when the crater was larger this took about six days (figs. 3 and 4 ) . Several rabbits were studied at 2, 4, 6, and 10 days after the cornea was given a small penetrating wound with a razor blade. T h e healing process involving thymidine-H 3 uptake by and mitosis of epithelial cells
f
CELLS/30 MM epithelium
6-4
3 2 DAYS stroma
/12 o
Ä .
P * '
1
Fig. 1 (Hanna). Graphic representation of changes in cell division of corneal epithelial cells at various times following wounding. There is an initial decrease in the number of cells dividing (DNA synthesis and mitosis) in the cornea ex tending from the wound edge. In the healing process at one day at an average of 120 cells from the wound edge, there is a marked increase in cell division in a narrow band of basal cells. This band of cells undergoing division migrates to and includes the basal cells in the wound.
THYMIDINE UPTAKE DURING CORNEAL REPAIR
57
Fig. 2 (Hanna). Graphic representation of movement of corneal epithelial cells into the wound crater. Each cell type, that is, basal, wingtype and superficial, migrates into the wound ac cording to its initial position as indicated by ar rows. TJ7n
r
around the perforating wound was similar to that found in the nonperforating wound. In initial rabbit studies on nonperforating corneal wounding, the edges of the wound were left with jagged edges. W h e n the heal ing process began, epithelial cells migrated over the jagged edges and into the fissures in the stroma. These epithelial cells deep in
the stroma continued to undergo thymidineH 3 uptake at a moderate rate (fig. 4 and 5 ) . Small superficial corneal burns were pro duced in rats using a cautery. In those burns where only the epithelial cells were killed the adjacent basal epithelial cells un-
Fig. 3 (Hanna). Autoradiograph of a corneal section from a rabbit killed three days after the wound ing. Thymidine-Ha was injected into the anterior chamber two hours before the animal was killed. This section was taken near one end of the wound where the crater was small. Those epithelial and stromal cells incorporating thymidine-H3 are indicated by a large black dot covering each cell nuclei and by an arrow. (Restained with Geimsa, χ400.)
CALVIN HANNA
58
derwent thymidine-H 3 incorporation be tween eight and 24 hours. In these superficial burns there was no zone of ces sation of thymidine-H 3 incorporation ex tending from the burned area. CORNEAL
EPITHELIAL
CELL
MIGRATION
IN
WOUND
After a thymidine-H 3 injection, the tri tium label appears in the various layers of the corneal epithelium with time. I n this way the basal, the wing-shaped and the superficial squamous cells were shown to contain the tritium label by killing the rab
bits 1, 4 and 10 days later, respectively. T h e migration of the various epithelial cell layers into the wound crater was studied by inject ing thymidine-H 3 into the anterior chamber of rabbits and then wounding at the ap propriate time later. T h e rabbits were then killed 1, 2, 3, and 6 days later. W h e n the cornea was wounded one day after the thymidine-H 3 administration and the animal killed one or two days later, the following was found: Outside the wound the tritium-labeled ep ithelial cells were in the basal layer; at the wound edge there were relatively fewer tri
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Fig. 4 (Hanna). Autoradiograph of a corneal section from a rabbit killed four days after wounding. The thymidine-H3 was injected two hours before the animal was killed. The presence of tritium-labeled epithelial and stromal cells are indicated by arrows. (Hematoxylin-eosin, X320.)
THYMIDINE UPTAKE DURING CORNEAL REPAIR
59
Fig. 5 (Hanna). Autoradiograph of a corneal section from a rabbit killed six days after wounding. The thymidine-H3 was injected two hours before the rabbit was killed. In this section note the group of tritium-labeled epithelial cells near the wound edge and a tritium-labeled epithelial cell in a wound fissure, as indicated by arrows. (Hematoxylin-eosin, X350.)
tium-labeled basal cells ; in the wound crater the tritium-labeled epithelial cells were in the innermost cell layer. In the corneas wounded four days after tritium labeling and killed one to six days after wounding the tritium label was in the wing-shaped cells outside the wound and in the rounded cells in the middle layer of the epithelial cells located in the wound crater. Corneas that were wounded 11 days be fore tritium labeling and the animals killed one to two days later showed tritium-labeled superficial epithelial cells over all parts of the cornea (fig. 6). When this was repeated and the animals killed three to six days later, the tritium-labeled squamous epitheli al cells were found only over the wound cra ter region. These results indicate that each epithelial cell layer migrated into the wound while generally maintaining its initial rela tive position (fig. 7). Also, the epithelial cells in the wound were sloughed off at a slow rate, depending
on the speed of healing of the stromal layer and the return of cell division to cells in the wound crater. Results of studies on the wounded rat cornea were similar except the time intervals between thymidine-H 3 admin istration and the wounding was shorter in the rat because the turnover time of the rat corneal epithelium is shorter (about seven days) than that of the rabbit (about 12 days). TWO-HOUR THYMIDINE-H 3 UPTAKE IN STROMAL CELLS
Between 15 and 48 hours after penetrat ing or nonpenetrating corneal wounding, cells near the wound edge incorporated thy midine-H 3 . These cells, either on tangential or saggital plane sectioning and hematoxylin and eosin staining, had the shape and stain ing characteristics of fixed stromal cells. The tritium-labeled cells in the stroma were usually found in a band around the wound crater at about two cell lengths from the
60
CALVIN HANNA
tions indicating that the thymidine-H 3 was incorporated into DNA. DISCUSSION
It is generally accepted that the initial re sponse in corneal wound healing is the mi gration of epithelial cells into the defect. The way the epithelial cells migrate depends to some extent on the type of wound. When a fine-pointed needle is used to produce the wound in the frog cornea, the marginal basal cells elongate with pseudopodial exten sions reaching into the defect.1·9 These studies have been taken to indicate that basal epithelial cells migrate into and fill the crater by an ameboidlike movement. This evidence has been extrapolated to indicate that all epithelial cells in the wound crater are basal cells in origin and evidence for this has been that the epithelial cells in the crater are spherical in shape and not unlike basal cells in appearance.3 Evidence from the present study suggests that each epitheli al cell layer participates in supplying cells to cover a moderately sized defect, possibly by sliding into the wound. Fig. 6 (Hanna). Autoradiograph of a corneal section from a rabbit killed six days after wounding and two hours after the injection of thymidine-H3. This section was taken through a fissure in the ηorneal stroma. The cells underly ing the black areas in the center of the picture are epithelial cells. (Hematoxylin-eosin, original magnification X600.)
wound edge and extending outward about 10 cell lengths (figs. 4 and 5). Occasionally a tritium-labeled cell in the stroma was found at the wound edge in contact with an epithelial cell or at some distance from the wound edge. When the wound healing was slowed due to the presence of a large jagged wound the uptake of thymidine-H 3 by stromal cells continued until the crater healed. Similar results were found in the rat. Representative corneal wound sections were treated with DNase before autoradiographic analysis. The treatment with DNase removed the tritium from the sec
The second generally accepted response in corneal wound healing is that of cell divi sion to replenish the cells lost. Arey and Covode2 observed that mitosis in the basal epithelial layer ceased for several days fol lowed by a temporary increase above normal in the mitotic rate. Hara 4 observed a similar cessation in the number of epithelial cells of the wounded rabbit cornea undergoing thymidine-H 3 in corporation for several days. However, Smelser10 found that the total number of mitotic figures in the rat cornea following wounding was about the same as the num ber of figures found before wounding. The results of Arey and Covode2 and Hara 4 indicate that the corneal epithelial cells cease cell division several days after corneal wounding. These results are not consistent with the mitotic changes observed by Smelser.10 However, the results obtained in the present study indicate that those epi-
THYMIDINE UPTAKE DURING CORNEAL REPAIR
61
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Fig. 7 (Hanna). Autoradiograph of a corneal section from a rabbit killed 10 days after the injection of thymidine-H3 and two days after wounding. The tritium-labeled epithelial cells were in the superficial layers of the cornea at the time of wounding and this section shows the migration of these cells into the wound crater. (Restained with Geimsa, original magnification X300.)
thelial cells near the wound cease to divide incorporation in the basal epithelial cells of while those cells at some distance from the the skin and tongue.6 wound epithelial were undergoing an in The environmental conditions for the creased rate of cell division. As a result, the maintenance of cell division are not well un number of epithelial cells dividing over the derstood. Further, the various stages of cell entire cornea a day after a small wound division apparently need not be completed. would not be greatly different from that of When the corneal stroma lamella are sepa the nonwounded cornea. rated using a blunt spatula the stromal cells Thymidine-H 3 is an especially helpful adjacent to the injury undergo DNA method in these studies because there are synthesis.11'12 Some of these stromal cells do several times more cells incorporating thy not appear to undergo a mitotic change with midine-H 3 than the number of mitotic time.11 Freezing of cells in the cornea pro figures in the cornea at any one time. There duced stimulation of DNA synthesis and fore, it was relatively easy to locate the zone thymidine-H 3 incorporation in various cells of cells in mitosis when sagittal plane cuts and these corneal cells retain the tritium-la were used. The sequence of events in cor beled DNA for long periods.13 Lens wound neal wound healing reported here resembles ing also initiated a wave of cell division very closely the sequence of thymidine-H 3 starting near the wound edge,14 and in ga-
CALVIN HANNA
62
lactose cataracts the wave of D N A synthesis starts at the equator region and moves to ward the anterior pole. 5 In most of these ex amples the cells adjacent to the swelling and / o r edema intiate the cell division. With the corneal wound edema is present around the wound crater. T h e cells in the stroma undergoing D N A synthesis are located ad jacent to this area and not usually in the edematous portion. T h e cessation of D N A synthesis in the basal epithelial cells and the continued D N A synthesis of these same epithelial cells in a fissure following corneal wounding is in some ways a perplexing phenomenon for several reasons. Basal epithelial cells not undergoing D N A synthesis in the wound crater can be found adjacent to an occassional cell in the stroma that is undergoing D N A synthesis. T h e basal epithelial cells normally undergo D N A synthesis whereas the stromal cells in the adult eye do not nor mally undergo D N A synthesis. 15 T h e epi thelial cells that migrated into the open wound crater do not undergo D N A synthe sis until the crater is repaired but those epi thelial cells that migrated into a fissure con tinue to undergo D N A synthesis. It is pos sible that the release of tension of anterior epithelial cells on the basal epithelial layer produced by the wound may cause a cessa tion of D N A synthesis in the basal cells. The epithelial cells in the wound crater be come spherical, indicating a release of ten sion by the superficial cell layers. Those epi thelial cells that migrated into stromal fis sures would be expected to be under tension and these epithelial cells do continue D N A synthesis. SUMMARY
Corneal wounds were produced in rabbits and rats and the changes in D N A synthesis were estimated utilizing thymidine-tritium and autoradiography. E a r l y in the healing process each layer of the epithelium slides into the wound crater. After this there is a
cessation of D N A synthesis and mitosis in the basal epithelial cells reaching some dis tance from the wound. By one day D N A synthesis in the stromal cells near the wound edge begins and this continues until the wound is healed. Also there is a wave of cell division that moves toward the wound crater and finally involves the basal epitheli al cells above the healed wound crater be fore returning to normal. These changes in cell division in corneal cells closely resemble those changes observed in the healing of skin wounds. University of Arkansas Medical Center. REFERENCES
1. Buschke, W. H : Morphlogic changes in cells of corneal epithelium in wound healing. Arch. Ophth., 41:306-316, 1949. 2. Arey, L. B., and Covode, W. M. : The method of repair in epithelial wounds of the cornea. Anat. Rec, 86:75-86, 1943. 3. Duke-Elder, S. : Textbook of Ophthalmol ogy. St. Louis, Mosby, 1958, v. 6, p. 5964. 4. Hara, J. : An autoradiographic study of re generation of the corneal epithelium. Folia Ophth. Jap., 13:443-466, 1962. 5. Hanna, C, and O'Brien, J. E. : Cell produc tion and migration in the epithelial layer of the cornea. Arch. Ophth., 64:536-539, 1960. 6. Block, P., Seiter, I., and Oehlert, W. : Autora diographic studies of the initial cellular response to injury. Exper. Cell. Res., 30:311-323, 1963. 7. Hanna, C, and O'Brien, J. E. : Lens epitheli al cell proliferation and migration in radiation cataracts. Radiβt. Res., 19:1-11, 1963. 8. Amano, M. : Improved techniqques of the en zymatic extraction of nucleic acids from tissue sections. J. Histochem. Cytochem, 10 :204, 1962. 9. Friedenwald, J. S., and Buschke, W. : Influence of some experimental variables on the epithelial movements in the healing of corneal wounds. J. cell. Comp. Physiol., 23:95-107, 1944. 10. Smelser, G., and Ozanics, V. : Effeffct of chemotherapeutic agents on cell division and heal ing of corneal burns and abrasions in the rat. Am. J. Ophth., 27:1063, 1944. 11. Hanna, C, and O'Brien, J. E. : Thymidinetritium labeling of the cellular elements of the corneal stroma. Arch. Ophth., 66:362-365, 1961. 12. Hanna, C, and Irwin, E. S. : Fate of cells in the corneal graft. Arch. Ophth., 68:810-817, 1962. 13. Polack, F. M., Smelser, G. K., and Rose, J. : Long-term survival of isotopically labeled stromal and endothelial cells in the corneal homografts. Am. J. Ophth., 57:67, 1964. 14. Harding, C. V., Donn, A., and Srinivasan,
THYMIDINE UPTAKE DURING CORNEAL REPAIR B. D. : Incorporation of thymidine by injured lens epithelium. Exper. Cell Res. 18:582-585 (Nov.) 1959.
IS. Hanna, G, and O'Brien, J. E. : Cell turnover in the adult human eye. Arch. Ophth., 65:695-698, 1961.
THE MECHANISM OF EXTERNAL M I L E S A. GALiN.t M.D.,
63
IRVING BARAS, M.D.,
FILTRATION*
AND J O H N M. M C L E A N ,
M.D.
New York
External filtration is the operative proce present data concerning the mechanics of dure most commonly used for the control of outflow in eyes which have been subjected glaucoma. However, the mechanism by to cautery sclerostomy with peripheral which this form of surgery exerts its effect iridectomy.10 Also described will be a tech has not been completely elucidated. Initially, nique whereby aqueous may be collected it was believed that neurohumoral factors across a filtering bleb in sufficient quantities reduced intraocular pressure after surgical to permit detailed analysis. intervention. Presently prevalent is a more METHODS mechanistic view which holds that aqueous actually leaves the anterior chamber. Of the A group of successive glaucoma patients physical pathways available for the egress operated upon at The New York Hospitalof aqueous after external filtration, two Cornell Medical Center by the technique of major routes seem most logical : cautery sclerostomy were chosen for this 1. Transconjunctival flow, in which study. In all cases a wide limbus-based conaqueous actually passes through the epitheli junctival flap had been utilized, the initial um of the bleb and mixes with the tears, incision being well above the insertion of was proposed by Verhoeff,1 Seidel2 and the superior rectus tendon, at least 10 mm from the limbus. After reflection of the flap, others.3"6 2. By the second path, championed by the the Hildreth cautery was applied to the ap early advocates of the iridencleisis opera propriate perilimbal area and cauterization tion,7'8 aqueous may pass into the subcon- carried out. The anterior chamber was en junctival tissues and thence into the venous tered only with the cautery tip and the pe ripheral iridectomy then performed. Closure system. It is at once apparent that both of these was carried out in layers. mechamisms may be operative in any single Postoperatively all patients were observed eye after antiglaucoma surgery, though one carefully, and particular attention was focu or the other may dominate, depending on sed on the type of bleb that formed. the type of operative procedure per In addition, the Seidel2 fluorescein test* 9 13 formed. ' * In this test, the eye must first be anesthetized It is the purpose of this communication to . to prevent blinking and squeezing due to stinging From the Department of Surgery (Ophthal mology), New York Hospital-Cornell Medical Center. This paper was presented in part at the meeting of the American Academy of Ophthalmololgy and Otolaryngology in Chicago, October, 1964. This study was aided by grants from the United States Public Health Service, the Nation al Council to Combat Blindness, and the National Society for the Prevention of Blindness. t Career scientist, Health Research Council, New York.
by fluorescein. The patient is seated at the biomicroscope and instructed to maintain fixation downward. The upper lid is immobilized by the observer. A moistened Fluori-strip slid across the bleb area thin-coats the bleb with an orange layer of concentrated fluorescein. A positive test consists of a greenish discolora tion in the center of the orange-stained area as the fluorescein becomes diluted. This can be ob served with white light or through the blue filter used for applanation tonometry. As the area is kept under observation, the lighter green areas