Exp. Bye Res. (1979) 29, 595-600
The Reepithelialization of Rabbit Cornea Following Single and Multiple Denudation
(Received 16 Jwne 1978 and in revised forw, 22 December 1978, Zezo York) The reepithelialization of rabbit corneas completely denuded once by abrasion with a dental stone was compared with the rat,e of reepithelia,lization of the contralateral eye following repeated but gentle repeeling of the epithelium at Z-day intervals for 6 days after an initial complete denudation by abrasion. Two or three times repeeled corneas showed a more rapid initial reepithelialization than observed after the first denud.ation. Polymorphonuclear leukocytes (PMJSs) were found in the cornea1 stroma and tear fluid following both single and repeated denudation, but only up to 24 hours after the first, more traumatic, denudation were large numbers of PMNs found adherent to the exposed basement membrane of t,he cornea1 epithelium. It was hypothesized that PI;IXs on the corned surface may interfere with reepithelialization. cornea1 epithelium; polymorphonuclear leukocytes; k’ey lords : cornea1 denudation; inflammation; cornea1 reepithelialization.
1. Introduction In a previous study (Srinivasan, Worgul, Iwamoto and Eakins, 1977) incomplete or regressive healing patterns were observed following complete deepithelialization but not after partial deepithelialization of the cornea, regardless of whether the epithelium was removed by an abrasive dental stone tip or by surgical knife. These results mere thought to be due to the greater amount of inflammation produced by the complete removal of the cornea,1 epithelium. The inflammatory response was characterized by the presence of numerous polymorphonuclear leukocytes (PMNs) in the denuded corneas. To further define the role of inflammation and PMNs in the repair of cornea1 epithelial denudation, we compared healing after denudation by stone abrasion with healing following a less traumatic deepithelialization by taking advantage of t,he observation (Khoudadoust, 1967) that newly-migrated cornea1 epithelium does not adhere to the basement membrane until 6-7 days after reepithelialization of the cornea1 surface. In preliminary experiments, we found that up to 48 hr after deepithelialization the regenerating cornea1 epithelium coultl easily be removed as a sheet with minimal trauma to the eye. By utilizing this method of repeated deepithelialization at 48 hr intervals, we compared the healing characteristics of a single complete denudation by abrasion with deepithelialization accomplishecl by gentle repeeling of corneas in the ot,her eye at 3, 4 or 6 days after a complete denudation by abrasion. 2. Materials
and Methods
ilTew Zealand white rabbits (1.5-2.5 kg) were anesthetized with sodium pentobarbital (Diabutal, Diamond Labor&ories; 30 mg/kg iv.). Following topical application of 0.5°/0 proparacaine
(&on
00144835/79/12059Ei+06 B
Labora.tories),
one
cornea
$01.00/O
leas
abraded
with
0 1979 Academic 595
a round
dental
“green
Press Inc. (London)
Limited
596
B. D. SRINIVASAN
ANi)
K.
E. EAKINS
stone” attached to a dental drill until examination of the fluorescein-stained cornea under ultraviolet light revealed complete deepithelialization. Reepithelialization was measured daily by fluorescein staining and photography as previously described (Srinivasnn et al., 1977). Approximately 48 hr after unilateral deepitlle~alizatioll by abrasion, the migrating from the same eye with forceps cornea1 epithelium was gently but completely removed and knife blade (repeeled once). This procedure was repeated once more (repeeled t\\-ice) or two more times (repeeled three times) at 48 hr intervals. The contralateral (control) eye of each rabbit was subjected to a single denudation by abrasion with the dental stone eye. at the time of last repeeling of the epithelium of the experimental In order to quantitate the number of P~!FXs that were released from the conjunctiva following trauma, tear fluid of both eyes was sa,mpled at 1, 2, 4, 6, 24 and 48 hr after the last repeeling procedure. Since in these rabbits there was not sufficient tear flow to allow direct sampling, 100 ,~l of isotonic saline was instilled into the inferior forms and after into a heparin-coated Hamilton 15 see of gentle mixing 30 pl of this fluid was withdrawn syringe fitted with a soft plastic tip. An equal volume of Toisson’s fluid was mixed with the aspirated fluid and the PIINs were counted in the 25 central squares of an improved Keubauer hemocytometer.
Rabbits were killed by an overdose of Diabutal at 24, 48, 72 or 96 hr after the last repeeling of the cornea of the experimental eye and the simultaneous first denudat,ion by abrasion of the control eye. The eyes were enucleated and fixed in lOyo neutral buffered formalin. The corneas were excised, dehydrated through a graded alcohol series and em bedded in paraffin. Six micron sections were stained with hematoxylineosin.
3, Results At 1S hr after conqM,e deepithelializatior1; a larger area was reepithelialized in cmmeas which underwent multiple repeeling thalL in the contralateral control comeas that were denuded for the first time by abrasion (Big. 1). This difference was most 100 r
80
I-
6 c
i b
-
L---
18-----1 Hours
FIG. 1. The reepithelialization a single denudation by abrasion two (m), or three( q ) additional, intervals.
(in percent, (a)
( control consecutive
after
of
c
40--J lost
0
L---
b
c
90 -
deepithelioiizotion
total cornea1 area covered) eye) ; or a single denudation complete repeeling of the
of rabbit cornea, by abrasion plus epithelial regrowth
following one (0). at Z-day
REEPITHELIALIZATIOS
OF
RABBIT
CORNEA
597
marked between animals that had mdergone three comecutive deepithelializations ovev a 6-day period in one eye and a single cornea1 denudation by abrakon in the other eye. By 96 hr after deepithelialization, there was no appreciable difference in the area healed between three times repeeled and once denuded corneas.
PIG. 2. (a) Photomicrograph of light microscopic section of rabbit cornea 21 hr after a single complete denudation. Sate the presence of clumps of PMNs on the denuded surface (arrows) in front of the advancing edge of epithelium (E). x 175, H & E. There are PMKs in the stroma. (b) Photomicrograph of light microscopic section of rabbit cornea 24 hr after repeeling three times at Z-day intervals after an initial, complete denudation by abrasion. The advancing edge of epitheiium (E) is shown at the top left corner. Although PMNs are present in the anterior stroma, there are no PM,l’s on the cornea1 surface, (x 134.) (H & E.)
598
B. D. SRINIVASAN
AND
K. E. EAKINS
d comparison of light microscopic sections of rabbit corneas 24 hr after a singIe denudation and 24 hr after repeeling (three times) revealed that the number and distribution of PMNs in the central cornea1 stroma did not differ appreciably [Pig. 2(a), (b)]. However, a substantial number of PMNs were present on the surface of once denuded corneas 24 hr after abrasion. These PMNs were often present in clumps in front of the leading edge of the migrating epithelium and were also found on the surface of central regions of deepithelialized corneas. By 48 hr after injury PMNs were not observed on the cornea1 surface. In contrast, two or three times repeeled corneas demonstrated very few or no PMNs on the surface 24 hr after final repeeling. In some of the corneas that were repeeled only once, some adherent PMNs were found in a pattern similar to, but less dense than, that observed in corneas that were abraded but not repeeled.
2500
0
12
24 Time
FIG. 3. The number of PMNs per mm3 contained of the inferior conjunctival cul-de-sac with 100 pl by abrasion (O--O) and repeeling three times at 6 hr following single denudation, the number significantly increased.
36
48
(hr)
in a sample oftear fluid obtained after gentle washing of saline 1 to 48 hr after a single complete denudation (a- - -0). While the number of PNNs are increased of PMXs in the tear fluid following repeeling is not
The number of PMNs found in a sample of tear fluid obtained by gently washing the inferior conjunctival cul-de-sac with 100 ~1 of saline at various times following cornea1 denudation is shown in Fig. 3. PMNs arrived in the tear fluid 1 hr after a single complete denudation by abrasion and are found in increasing numbers at 6 hr after injury. The tear fluid sample of corneas repeeled three times in 2-day intervals after an initial denudation by abrasion similarly contained some PMNs 1 hr after injury, but there was no significant increase in the number of tear film PMNs at 6 hr following final repeeling. By 48 hr after injury, only a few PMNs were found in the tear fluid of corneas that were denuded either by abrasion or by repeeling. 4. Discussion The differences in the initial epithelial healing rate observed here after a single abrasion and after repeated repeeling may suggest that the conjunctival epithelium
REEPITHELIALIZATION
OF
RABBIT
CORNEA
599
requires some time to be “primed” in order to migrate after deepithelialization. With repeated deepithelialization, faster healing may be accomplished by epithelial cells that have already been primed to migrate. Another possible expIanation for this difference may be in the peripheral vascularization displayed only by many multiply denuded corneas. The presence of new blood vessels in this region may enhance reepithelialization at the periphery following repeeling. However, these differences may also be explained by the observed presence of large numbers of PMNs on the surfaces of abraded corneas but not on those corneas which were repeeled for a third time 6 days after the first denudation by abrasion. The absence of PMNs on the surface of corneas which were repeeled three times during a total of 6 days may allow a more rapid initial migration of epithelial cells than occurs after a much more extensive trauma of the first deepithelialization, after which PMNs lie on the epithelial basement membrane in front of the healing epithelium. The rate of migration as well as adhesion of the epithelium to the basement membrane could be adversely affected by the presence of PMNs or by chemical mediators released by the PMNs. PMNs arrive at the cornea1 wound site by migrating through the cornea1 stroma and also across the conjunctival epithelium (Robb and Kuwabara, 1962; Matsuda and Smelser, 1973; Pfister, 1975; Van Barsewisch, 1975; Shanklin, Sheppard and Burke, 1975). It is not clear whether any temporal or quantitative relationship exists between the PMNs migrating across the conjunctival epithelium into the tear film and the PMNs migrating into the stroma. Robb and Kuwabara (1962) observed that 5 hr after a central incision in the rabbit cornea PMNs were present in conjunctival epithelium, but no movement of PMNs into the peripheral cornea1 stroma was found. Shanklin, Sheppard and Burke (1975) noted PMNs in the stromal periphery 2 hr after complete deepithelialization of rabbit cornea, but made no mention of PMYNs on the denuded surface. In the present experiments, a large number of PMNs was found in the tear fluid 6 hr after the fust denudation but not after the third repeeling which took place 6 days after a similar denudation. This contrasts with the time course of PMN invasion of the stroma which is minimal at 6 hr and increases gradually to a maximum at about 24 hr after a single denudation by abrasion. PMNs lying on the cornea1 surfa,ce are attached either to the basement membrane or to epithelial cells by a fibrinous network and may remain along the wound edge of a 6 mm abrasion for up to 38 hr after injury (Pfister, 1975). In our experiments, PMNs were present on the cornea1 surface 24 hr after a single denudation by abrasion but were not present on the surface at 48 hr after injury. Appreciable numbers of PMNs were not- found on the basement membrane at any time after the third repeeling which occurred 6 days after the first abrasion. At the present time, two alternatives can be considered to explain why PMNs are found on the surface of corneas denuded once by abrasion but not on t’he surface of repeeled corneas which were denuded by abrasion 6 days previously. (1) The exposed basement membrane of repeatedly repeeled corneas is altered during the 6 days after initial abrasion in a manner that does not allow PMNs to attach to it. In this regard, it must be noted that corneas that were repeatedly repeeled received 1 or 2 drops of local anesthetic (0.5% proparaca,ine hydrochloride) prior to each repeeling. Whether or not this small amount of local anesthetic could affect the basement membrane to prevent PMNs from adhering to it rema,ins to be determined. {2) PMNs that are present in the tear film after repeated repeeling a,re different from those arriving in response to the initial denudation.
ACKXOWLEDGBIENTR
We thank BIs Melinda tance. This investigation EY 00457.
Chow and Mr Felix Zaragoza for their valuable teclmical assiswas supported by U.S.P.H.S. Research Grants EY 01977 and
REFERENCES Khodadoust, A. A. (1967). Techniques for the preparation of sheets of pure cornea1 epithelium. Am. J. Ophthulmol. 63, 942-6. Pfister, R. R. (1975). The healing of cornea1 epithelial abrasion in the rabbit: a scanning electron microscope study. Invest. Ophthnlmol. 14, 645-61. Robb, R. M. and Kuwabara, T. (1962). Cornea1 wound healing. 1. The movement of polymorphonuclear leukocytes into cornea1 wounds. Anz. J. Ophthalmol. 68, 63642. Shanklin, W. %I., Sheppard, L. B. and Burke, G. W., Jr. (1975). Pseudoeosinophilic leucocyte response to denuding the rabbit cornea. J. Arrat. 1, 97-104. Srinivasan, B. D., Worgnl, B. V., Iwamoto, T. and Eakins, K. E. (1977). The reepithelializat’ion of rabbit cornea following partial and complete epithelial denudation. Exp. Eye Res. 25, 343-51. Von Barsewisch, B. (1975). Die gerichtete Leukocyten. Emigration aus Conjunctival-Gefassen. Albrecht vu. Graefes Arch. Klin. ezp. Opkthnl. 193,197-204.
The Reepithelialization of Rabbit Cornea Following Single and Multiple Denudation
(Received 16 Jwne 1978 and in revised forw, 22 December 1978, Zezo York) The reepithelialization of rabbit corneas completely denuded once by abrasion with a dental stone was compared with the rat,e of reepithelia,lization of the contralateral eye following repeated but gentle repeeling of the epithelium at Z-day intervals for 6 days after an initial complete denudation by abrasion. Two or three times repeeled corneas showed a more rapid initial reepithelialization than observed after the first denud.ation. Polymorphonuclear leukocytes (PMJSs) were found in the cornea1 stroma and tear fluid following both single and repeated denudation, but only up to 24 hours after the first, more traumatic, denudation were large numbers of PMNs found adherent to the exposed basement membrane of t,he cornea1 epithelium. It was hypothesized that PI;IXs on the corned surface may interfere with reepithelialization. cornea1 epithelium; polymorphonuclear leukocytes; k’ey lords : cornea1 denudation; inflammation; cornea1 reepithelialization.
1. Introduction In a previous study (Srinivasan, Worgul, Iwamoto and Eakins, 1977) incomplete or regressive healing patterns were observed following complete deepithelialization but not after partial deepithelialization of the cornea, regardless of whether the epithelium was removed by an abrasive dental stone tip or by surgical knife. These results mere thought to be due to the greater amount of inflammation produced by the complete removal of the cornea,1 epithelium. The inflammatory response was characterized by the presence of numerous polymorphonuclear leukocytes (PMNs) in the denuded corneas. To further define the role of inflammation and PMNs in the repair of cornea1 epithelial denudation, we compared healing after denudation by stone abrasion with healing following a less traumatic deepithelialization by taking advantage of t,he observation (Khoudadoust, 1967) that newly-migrated cornea1 epithelium does not adhere to the basement membrane until 6-7 days after reepithelialization of the cornea1 surface. In preliminary experiments, we found that up to 48 hr after deepithelialization the regenerating cornea1 epithelium coultl easily be removed as a sheet with minimal trauma to the eye. By utilizing this method of repeated deepithelialization at 48 hr intervals, we compared the healing characteristics of a single complete denudation by abrasion with deepithelialization accomplishecl by gentle repeeling of corneas in the ot,her eye at 3, 4 or 6 days after a complete denudation by abrasion. 2. Materials
and Methods
ilTew Zealand white rabbits (1.5-2.5 kg) were anesthetized with sodium pentobarbital (Diabutal, Diamond Labor&ories; 30 mg/kg iv.). Following topical application of 0.5°/0 proparacaine
(&on
00144835/79/12059Ei+06 B
Labora.tories),
one
cornea
$01.00/O
leas
abraded
with
0 1979 Academic 595
a round
dental
“green
Press Inc. (London)
Limited
596
B. D. SRINIVASAN
ANi)
K.
E. EAKINS
stone” attached to a dental drill until examination of the fluorescein-stained cornea under ultraviolet light revealed complete deepithelialization. Reepithelialization was measured daily by fluorescein staining and photography as previously described (Srinivasnn et al., 1977). Approximately 48 hr after unilateral deepitlle~alizatioll by abrasion, the migrating from the same eye with forceps cornea1 epithelium was gently but completely removed and knife blade (repeeled once). This procedure was repeated once more (repeeled t\\-ice) or two more times (repeeled three times) at 48 hr intervals. The contralateral (control) eye of each rabbit was subjected to a single denudation by abrasion with the dental stone eye. at the time of last repeeling of the epithelium of the experimental In order to quantitate the number of P~!FXs that were released from the conjunctiva following trauma, tear fluid of both eyes was sa,mpled at 1, 2, 4, 6, 24 and 48 hr after the last repeeling procedure. Since in these rabbits there was not sufficient tear flow to allow direct sampling, 100 ,~l of isotonic saline was instilled into the inferior forms and after into a heparin-coated Hamilton 15 see of gentle mixing 30 pl of this fluid was withdrawn syringe fitted with a soft plastic tip. An equal volume of Toisson’s fluid was mixed with the aspirated fluid and the PIINs were counted in the 25 central squares of an improved Keubauer hemocytometer.
Rabbits were killed by an overdose of Diabutal at 24, 48, 72 or 96 hr after the last repeeling of the cornea of the experimental eye and the simultaneous first denudat,ion by abrasion of the control eye. The eyes were enucleated and fixed in lOyo neutral buffered formalin. The corneas were excised, dehydrated through a graded alcohol series and em bedded in paraffin. Six micron sections were stained with hematoxylineosin.
3, Results At 1S hr after conqM,e deepithelializatior1; a larger area was reepithelialized in cmmeas which underwent multiple repeeling thalL in the contralateral control comeas that were denuded for the first time by abrasion (Big. 1). This difference was most 100 r
80
I-
6 c
i b
-
L---
18-----1 Hours
FIG. 1. The reepithelialization a single denudation by abrasion two (m), or three( q ) additional, intervals.
(in percent, (a)
( control consecutive
after
of
c
40--J lost
0
L---
b
c
90 -
deepithelioiizotion
total cornea1 area covered) eye) ; or a single denudation complete repeeling of the
of rabbit cornea, by abrasion plus epithelial regrowth
following one (0). at Z-day
REEPITHELIALIZATIOS
OF
RABBIT
CORNEA
597
marked between animals that had mdergone three comecutive deepithelializations ovev a 6-day period in one eye and a single cornea1 denudation by abrakon in the other eye. By 96 hr after deepithelialization, there was no appreciable difference in the area healed between three times repeeled and once denuded corneas.
PIG. 2. (a) Photomicrograph of light microscopic section of rabbit cornea 21 hr after a single complete denudation. Sate the presence of clumps of PMNs on the denuded surface (arrows) in front of the advancing edge of epithelium (E). x 175, H & E. There are PMKs in the stroma. (b) Photomicrograph of light microscopic section of rabbit cornea 24 hr after repeeling three times at Z-day intervals after an initial, complete denudation by abrasion. The advancing edge of epitheiium (E) is shown at the top left corner. Although PMNs are present in the anterior stroma, there are no PM,l’s on the cornea1 surface, (x 134.) (H & E.)
598
B. D. SRINIVASAN
AND
K. E. EAKINS
d comparison of light microscopic sections of rabbit corneas 24 hr after a singIe denudation and 24 hr after repeeling (three times) revealed that the number and distribution of PMNs in the central cornea1 stroma did not differ appreciably [Pig. 2(a), (b)]. However, a substantial number of PMNs were present on the surface of once denuded corneas 24 hr after abrasion. These PMNs were often present in clumps in front of the leading edge of the migrating epithelium and were also found on the surface of central regions of deepithelialized corneas. By 48 hr after injury PMNs were not observed on the cornea1 surface. In contrast, two or three times repeeled corneas demonstrated very few or no PMNs on the surface 24 hr after final repeeling. In some of the corneas that were repeeled only once, some adherent PMNs were found in a pattern similar to, but less dense than, that observed in corneas that were abraded but not repeeled.
2500
0
12
24 Time
FIG. 3. The number of PMNs per mm3 contained of the inferior conjunctival cul-de-sac with 100 pl by abrasion (O--O) and repeeling three times at 6 hr following single denudation, the number significantly increased.
36
48
(hr)
in a sample oftear fluid obtained after gentle washing of saline 1 to 48 hr after a single complete denudation (a- - -0). While the number of PNNs are increased of PMXs in the tear fluid following repeeling is not
The number of PMNs found in a sample of tear fluid obtained by gently washing the inferior conjunctival cul-de-sac with 100 ~1 of saline at various times following cornea1 denudation is shown in Fig. 3. PMNs arrived in the tear fluid 1 hr after a single complete denudation by abrasion and are found in increasing numbers at 6 hr after injury. The tear fluid sample of corneas repeeled three times in 2-day intervals after an initial denudation by abrasion similarly contained some PMNs 1 hr after injury, but there was no significant increase in the number of tear film PMNs at 6 hr following final repeeling. By 48 hr after injury, only a few PMNs were found in the tear fluid of corneas that were denuded either by abrasion or by repeeling. 4. Discussion The differences in the initial epithelial healing rate observed here after a single abrasion and after repeated repeeling may suggest that the conjunctival epithelium
REEPITHELIALIZATION
OF
RABBIT
CORNEA
599
requires some time to be “primed” in order to migrate after deepithelialization. With repeated deepithelialization, faster healing may be accomplished by epithelial cells that have already been primed to migrate. Another possible expIanation for this difference may be in the peripheral vascularization displayed only by many multiply denuded corneas. The presence of new blood vessels in this region may enhance reepithelialization at the periphery following repeeling. However, these differences may also be explained by the observed presence of large numbers of PMNs on the surfaces of abraded corneas but not on those corneas which were repeeled for a third time 6 days after the first denudation by abrasion. The absence of PMNs on the surface of corneas which were repeeled three times during a total of 6 days may allow a more rapid initial migration of epithelial cells than occurs after a much more extensive trauma of the first deepithelialization, after which PMNs lie on the epithelial basement membrane in front of the healing epithelium. The rate of migration as well as adhesion of the epithelium to the basement membrane could be adversely affected by the presence of PMNs or by chemical mediators released by the PMNs. PMNs arrive at the cornea1 wound site by migrating through the cornea1 stroma and also across the conjunctival epithelium (Robb and Kuwabara, 1962; Matsuda and Smelser, 1973; Pfister, 1975; Van Barsewisch, 1975; Shanklin, Sheppard and Burke, 1975). It is not clear whether any temporal or quantitative relationship exists between the PMNs migrating across the conjunctival epithelium into the tear film and the PMNs migrating into the stroma. Robb and Kuwabara (1962) observed that 5 hr after a central incision in the rabbit cornea PMNs were present in conjunctival epithelium, but no movement of PMNs into the peripheral cornea1 stroma was found. Shanklin, Sheppard and Burke (1975) noted PMNs in the stromal periphery 2 hr after complete deepithelialization of rabbit cornea, but made no mention of PMYNs on the denuded surface. In the present experiments, a large number of PMNs was found in the tear fluid 6 hr after the fust denudation but not after the third repeeling which took place 6 days after a similar denudation. This contrasts with the time course of PMN invasion of the stroma which is minimal at 6 hr and increases gradually to a maximum at about 24 hr after a single denudation by abrasion. PMNs lying on the cornea1 surfa,ce are attached either to the basement membrane or to epithelial cells by a fibrinous network and may remain along the wound edge of a 6 mm abrasion for up to 38 hr after injury (Pfister, 1975). In our experiments, PMNs were present on the cornea1 surface 24 hr after a single denudation by abrasion but were not present on the surface at 48 hr after injury. Appreciable numbers of PMNs were not- found on the basement membrane at any time after the third repeeling which occurred 6 days after the first abrasion. At the present time, two alternatives can be considered to explain why PMNs are found on the surface of corneas denuded once by abrasion but not on t’he surface of repeeled corneas which were denuded by abrasion 6 days previously. (1) The exposed basement membrane of repeatedly repeeled corneas is altered during the 6 days after initial abrasion in a manner that does not allow PMNs to attach to it. In this regard, it must be noted that corneas that were repeatedly repeeled received 1 or 2 drops of local anesthetic (0.5% proparaca,ine hydrochloride) prior to each repeeling. Whether or not this small amount of local anesthetic could affect the basement membrane to prevent PMNs from adhering to it rema,ins to be determined. {2) PMNs that are present in the tear film after repeated repeeling a,re different from those arriving in response to the initial denudation.
ACKXOWLEDGBIENTR
We thank BIs Melinda tance. This investigation EY 00457.
Chow and Mr Felix Zaragoza for their valuable teclmical assiswas supported by U.S.P.H.S. Research Grants EY 01977 and
REFERENCES Khodadoust, A. A. (1967). Techniques for the preparation of sheets of pure cornea1 epithelium. Am. J. Ophthulmol. 63, 942-6. Pfister, R. R. (1975). The healing of cornea1 epithelial abrasion in the rabbit: a scanning electron microscope study. Invest. Ophthnlmol. 14, 645-61. Robb, R. M. and Kuwabara, T. (1962). Cornea1 wound healing. 1. The movement of polymorphonuclear leukocytes into cornea1 wounds. Anz. J. Ophthalmol. 68, 63642. Shanklin, W. %I., Sheppard, L. B. and Burke, G. W., Jr. (1975). Pseudoeosinophilic leucocyte response to denuding the rabbit cornea. J. Arrat. 1, 97-104. Srinivasan, B. D., Worgnl, B. V., Iwamoto, T. and Eakins, K. E. (1977). The reepithelializat’ion of rabbit cornea following partial and complete epithelial denudation. Exp. Eye Res. 25, 343-51. Von Barsewisch, B. (1975). Die gerichtete Leukocyten. Emigration aus Conjunctival-Gefassen. Albrecht vu. Graefes Arch. Klin. ezp. Opkthnl. 193,197-204.
