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Corneal scar formation
8xp. Bye Res. (1973) 17, 251-259
Corned Scar Formation CHARLES
~ep~~t~le~~t
CISTRON,
HAROLD
of Comea Research, Rrtina
SCHNEIDER
Foundation,
AXD
CLAIRE
KUBLIN
Boston, Mass. 0.2114, U.S.A.
(Received 30 May 1973, and in revised fomx 5 July 1973, Boston) The present study is concerned with scar formation in rabbit cornea after removal of a centrally-located 2.0 mm full thickness cornea1 “button”. Simplicity in wounding, reproducibility in healing with minimum complications, and ease of recovery of adequate amounts of “pure” scar tissue make this experimental model suitable for quantitative biochemical analyses. Quantification of hexosamine in purified glycosaminoglycan fractions from cornea1 excision wounds indicates that the amount of glycosaminoglycan progressively increases to 74% of that in the normal stroma by the fourth week of healing. This observation is not consistent with previous findings in cornea1 incision wounds. Hydroxyproline determinations in cornea1 wounds strongly suggest that collagen accumulation is very rapid during the first two weeks of healing. This is followed by a gradual increase in collagen, approaching the level in normal cornea1 stroma by the nmth week of healing.
1. Introduction -4 variety of wounding techniques has been used to enable investigators to study various aspects of cornea1repair. Among the most common methods used are penetrating and non-penetrating linear scalpel incision with or without sutures (Robb and Kuwabara, 1962; G-assetand Dohlman, 1968; Kitano, and Goldman, 1966). Other types of wounds have involved scraping endothelium and Descemet’s membrane (,4nseth and Fransson, 1969), cautery (Robb and Kuwabara, 1964), freezing (Baum, 19711, and intrastromal injection of carrageenin (Garg and McCandless, 1968). A perforating t,rephination excision wound in rabbit cornea wa,s used to study the effects of various drugs on the regeneration of the stroma using clinical and histological observations (Heydenreich, 1962). The present report describes the use of the experimental model developed by Heydenreich (1962), with some modifications: to study the biochemistry of scar formation. Simplicity in wounding, reproducibility in healing with minimum complications: and ea.seof recovery of adequate amounts of “pure” scar tissue make this experimental model suitable for qualitative and quantitative analyzes. Measurements of dry weight, water? hydroxyproline content and quantity of hexosamine in purified glycosal~liizoglycan fractions from the developing scar tissue are determined LOillustrate the use of the model for quantitative biochemical studiee. 2. Materials and Methods Albino rabbits, weighing 2-3 kg, were given 4% atropine drops to each eye prior to anesthesiawith intravenous sodiumpentobarbital. Anesthetized animals were then given topical proparacaine and in some cases a retrobular injection of 2% lidocaine. A 2%mm diameter full thickness “button” was excised from the center of each cornea with an * Reprint 20 Staniford
requests to: Charles Cintron, Street, Boston, Mass. 02114,
Ph.D., U.S.A.
Retina 261
Foundation,
Department
of Cornea
Research,
252
C. CIPL’TRON,
H.
SCHNEIDER
AND
C. KUBLIN
Elliot trephine. Pieces of Descemet’s membrane remaining fully dissected away with Vannas scissors. Wounded eyes myxin B-bacitracin-neomycin) ointment only immediately of 44 eyes with cornea1 wounds were examined periodically the progress of wound healing.
in the were after with
wound area wTere caregiven Neosporin (polythe operation. A total a slit lamp to evaluate
selection and preparation 0Jpsumples front healing corneas for analysis A scar tissue sample from an eye was used for biochemical analysis if the following criteria were met: (1) normal anterior chamber depth by the seventh day; (2) surface of healing wound not cratered; (3) no infection, vascularization, or iris incarceration; (4) decreased i&is by the seventh day. Epithelia of scarred corneas of various ages, and normal corneas, were scraped with a scalpel before removal of the wound area or stroma with a 2-O-mm Elliot trephine. Scarred and normal stromal “buttons” used for water and hexosamine determinations were pooled (9-26 buttons), blotted on Whatman no. 1 filter paper and weighed on a Mettler balance. Tissues were then lyophilized and weighed on a Cahn electrobalance. Hydroxyproline was det,ermined in individual “buttons” without pooling. Histology Cornea,1 excision wounds of various a,ges were processed for histology by fixing the whole cornea in 10% neutral formalin. Tissues were dehydrated, embedded in paraffin and 5 pm sections were made on an A0 Spencer microtome. Sections were stained with Hematoxylin and Eosin or Van Giessen’s acid fuehsin. Biochemical
analyses
The details of the method used for isolation of total glycosaminoglycan were similar to that described by Praus and Dohlman (1969). The procedure involves enzymaic degradation of the tissue with papain, followed by trichloroacetic acid (TCA) precipitation of proteins and alcohol precipitation of total glycosaminoglycans. The alcohol-precipitated material was tested for the presence of protein using the Lowry procedure (Lowry, Rosebrough, Farr and Randall, 1951). Hexosamine was determined with the method of Elson and Morgan (1933) as modified by Swann and Balazs (1966). Separation of glycosaminoglycans was accomplished with the microcellulose column technique of Antonopoulos, Gardell, Szirmai and De Tyssonsk (1964). Isolated glycosaminoglycan was dissolved in 50 ~1 of water and applied to a column (3.0x60.0 mm) of chloride (CPC). cellulose previously equilibrated with 4.0 ml of 1% eetylpyridinium The sample was then washed into the column with 50 ~1 of water. Glycosaminoglycans were separated into two fractions by addition of 1.5 ml of 1% cetylpyridinium chloride (CPC) which elutes CPC soluble components (CPC soluble fraction) followed by 1.5 ml of 6 N-IICl which removes all other glycosaminoglycans from the column (CPC insoluble fraction). All fractions were hydrolyzed in 8 N-HCI at 95°C in sealed tubes for 3 hr. Hydrolysates were then dried under a flow of air in a 40°C water bath. Hexosamines from CPC soluble and CPC insoluble fractions of normal stroma were identified by paper chromatography after ninhydrin degradation of hexosamines to pentoses (Stoffyn and Jeanloz, 1954). The quantity of hexosamine from CPC soluble and CPC insoluble fractions of scarred cornea1 tissue was determined, although the individual hexosamines were not identified. (2) Collagen. Lyophilized scarred and normal stromal “buttons” were hydrolyzed with 10 N-HCl for 5 hr at 95°C and dried down under a current of air. The method of Prockop and Undenfriend, (1960) as modified by Switzer and Summer (1971) was used to determine the quantity of hydroxyproline in the hydrolysates. (1)
Glycos~~n~nog~~ca~s.
CORNEAL
SCAR
253
FORMATION
Quantitative biochemical data in this section are expressed in two ways; (a) micrograms of hexosamine or hydroxyproline per 2.0 mm trephined “button” of normal or scar tissue and (b) micrograms per milligram dry weight of tissue. The latter allows one to compare the present data with that in the literature, while the former makes clear any quantitative changes obscured by concomitant changes in dry weight. 3. Results
Xlit lamp und histological
descriptio9z
Upon removal of a central 2.0~mm cornea1 button, the anterior chamber collapsed and the edge of the wound immediately became opaque [Fig. l(a)]. Since the pupil was greatly dilated, iris was not pulled into the gaping wound. In approximately 10 min the aqueous humor, filling the wound area, became viscous. Within 15-30 min the anterior chamber began to reform as the wound took on an opaque fibrous appearance. This opacity became quite pronounced in a few hours [Fig. l(b)].
FIG.
‘button”.
1. Cornea1 wound healing after removal of a centrally-located (a) 5 min, (b) 3 hr, (c) 3 days, (d) 1 week, and (e) 2 weeks
after
2+mm full wounding.
thickness
cornea1
254
C. CINTRON,
H.
SCHNEIDER
AND
C. KUBLIN
By the third day after wounding, the conjunctiva often appearedinflamed. Although the wound edge remained opaque, the central region became translucent [Fig. l(c)]. The rest of the cornea remained transparent. In 55% of the cases (24 out of 44 wounds), a coagulum extended down from the wound into the anterior chamber and often attached to the lens. Although iris incarceration was rarely found, iritis was noted in 45% of the cases and hyphema could sometimes be seen. In 45% of the cases the anterior chamber was shallow at this stage.
2.0.mm full thickness corneal FIG. 2. Cornea1 wound healing after removal of a centrally-located “button”. (a) 3 days, (b) 1 week, (c) 2 weeks after wounding. (a) and (b) stained with Van Giessen’s acid fuchsin; (c) stained with Hematorylin and Eosin. ( x 56)
CORNEAL
SCAR
FORMATION
255
Histologically, the regenerating cornea1 epithelium on the third day was continuous across the wound although only one cell thick in most places [Fig. 2(a)]. The stromal area of the wound was occupied primarily by an acellular fibrous coagulum. Fibroblast-like cells were located in the peripheral and anterior segment of the wound under the regenerating epithelium. By t,he seventh day the conjunctival inflammation seemed to have decreased. The wound was opaque except for a small central portion which was still translucent [Fig. l(d)]. There was less coagulum in the anterior chamber and the number of eyes with iritis had decreased. Approximately 25% of the eyes still exhibited a shallow anterior chamber. Vascularization of the cornea was noted in 7% of the eyes. Infeection, characterized by con.junctival inflammation along with the appearance of mucous on the eye and lids was found in 4:/, of the eyes. Cell number in both the epithelial and etromal area of the wound was greatly increased by the seventh day [Fig. 2(b)]. Extracellular material was evident between the fibroblast-like cells in the peripheral portion of the wound. AIthough most fibroblast-like cells were Iocated under the regenerating epithelium, some cells were found under the fibrous coagulum forming an apparently sepnrate population of fibroblast-like cells continuous with the posterior surface of the cornea. The wound was completely opaque by the second week [Fig. l(e)]. The frequency of occurrence of iritis and opaque coagulum in the anterior chamber was decreased further to approximately 13% of all eyes. Those eyes which exhibited shallow anterior chambers after one week remained the same two weeks after wounding. In only one out of 44 cases did the wound surface appear cratered. By the second week the wound area was filled with fibroblast-like cells [Fig. 2(c)]. A remnant of fibrous coagulum in the center of the stromal area was still present at this time. The epithelium wa,s variable in thickness over the wound area, 4-11 cells thick. The scar progressively increases in opacity and appears less edematous during the third and fourth week after wounding. Larger trephination wounds (e.g. 3-O mm in diameter), healed as described above. However, more than 50% of the wounds exhibited complications (i.e. vascularization, infection, cratered wound surface, and iris incarceration). With 2.0 mm excision wounds approximately 75% of the total number of eyes operated on were considered usable for biochemical analysis.
Approximately 72% of the wet weight of the normal rabbit cornea1 stroma was due to wa,ter. One week after removal of a 2.0 mm irephined cornea1 “button” the wound area was 94% water by weight (Fig. 3). B? the second week the wet weight of the healing wound increased due to an increase m both water and dry matter. At this time the dry weight of the scar was 80% of that of the normal stroma. A gradual increase in dry weight of the scar was noted in t.he succeeding weeks of healing. The water content, on the other hand, progressively decreased after the second week approaching that of the normal cornea1 stroma by t’he eleventh week after wounding. B~ioclienhxd
analyses
(1) Glycosaminoglycans. During the isolation of total glycosaminoglycans, samine was not detected in TCA precipitable material after papain treatment
hexoof the
256
C. CINTRON,
H.
SCHNEIDER
AND
C. KUBLIN
/$, II/ \ b‘a--we,----__ wt. di 4 ‘a-J-l
L!.
FIG. 3. Changes in dry and wet weight of healing cornea1 wounds after removal of it centrally-located 2-O-mm full thickness cornea1 “button”. 0-0, dry weight of tissue; O-..--0, wet weight; A-- --A, water weight (arithmetic difference between wet weight and dry weight of tissue).
normal cornea1 stroma. In addition, protein was not detected in the alcohol precepitated fraction containing glycosaminoglycans. Glycosaminoglycans fram normal rabbit cornea1 stroma were separated with a CPC-cellulose column into a 1% CPC soluble fraction and a lo/, CPC insoluble fraction which was subsequently released from the column with 6 N-HCl. Paper chromatography of hydrolysates from each of these two fractions demonstrated the presence of glucosamine in the CPC soluble fraction and galactosamine in the CPC insoluble fraction. Total hexosamine content of extracted, purified and fractionated glycosaminoglycans from normal stroma was 1.43% of the dry tissue weight. The ratio of hexosamine between the CPC soluble fraction and CPC insoluble fraction was 1.64 (Table I). TABLE
Hexosamine
Weeks after wounding
NOl-llldl
1 2 3 4
* 2.0-mm
Xumber of “buttons” pooled
17 26 20 I6 11
diameter
content of normal
cornea1 stroma and scar tissue
ug Hexosamine/button * ’ 1% CPC 1% CPC soluble insoluble fraction fraction
2.68 0.23 0.64 i.64 1.68
full thickness
I
pg Hexosamine/mg tissue wt. 1% CPC soluble fraction
1% CPC insoluble fraction
5.90 1.45 2.62 7.03 6.18
5.40 2.43 3,34 6.23 5.80
1.63 0.39 0.81 i.45 1.50
excised
“buttons”
dry
from
normal
stroma
Ratio (CPC sol: CPC insol.)
and sear tissue.
1.64 0.60 0.79 1.13 1.23
CORNEAL
SCAR
257
FORMATION
There was a progressive increase in the hexosamine content of the CPC soluble and CPC insoluble fractions obtained from developing cornea1 scar tissue between one and three weeks after wounding (Table I). However, no apparent accumulation of hexosamine was noted between the third and fourth week of healing in either the @PC soluble or CPC insoluble fractions. The quantity of hexosamine in the CPC soluble fraction of the four-week-old wound reached 63% of that in the normal tissue. Hexosamine content from the CPC insoluble fraction, on the other hand, approximated that of the normal stroma. The ratio of hexosamine between the CPC soluble fraction and CPC insoluble fraction increased to 75% of the normal stroma by t,he fourth week of healing. TABLE
Hydroxyproline after wounding
Weeks
Normal 1 2 3 4 5 9
* 2.0-mm diameter t S.E. of the mean.
II
content of normal corned stroma and scar tissue Number of determinations
10 9 9 9 10 9 10
full thickness
Pg Hypro/button
19.60&0.69 3G36*0.32 9+33&0~70 11-03fl~07 12.52kO.68 13.60&1.00 19.16f1.25
excised
“buttons”
from
*
t
dry tissue
wt
60.03 & 1.96 24.81 f 1.78 40.14*1.64 43.12&2-78 41.92,t4+37 5O.OS&2.63 58.31&1.56
normal
and scar tissues.
(2) Collagen. The quantity of hydroxyproline in rabbit cornea1 stroma was 60.0 pg/ mg of dry tissue or 19.6 pgj2.0 mm trephined stromal “button” (Table II). During the first two weeks of healing, hydroxyproline rapidly accumulated in the wound area to a.pproximately half of that in the normal stromal “buttons”. This was followed by a more gradual accumulation of hydroxyproline in the scar, approaching that of t,he normal stroma by the ninth week. 4. Discussion T’he e~per~ment~~ model Removal of a centrally-located 2.0 mm full thickness cornea1 “button” is easily and quickly accomplished in normal rabbits to elicit cornea1 repair and in healing corneas to sample “pure” scar tissue for analysis. Slit lamp observations have demonstrated the reproducibility of wound-healing in this system (75% of all eyes operated on healed in a similar manner) along with a minimum of complications (i.e. infection, vascularization, and iris incarceration). Centrally-located 3.0 mm full thickness cornea1 excision wounds, similar to that used by Heydenreich (1962), resulted in a 50% recovery of usable tissue for biochemical analysis. The present observations on the healing of excised cornea1 wounds are consistent with those described by McDonald (1957, 1961), for penetrating linear wounds of the rabbit cornea. This would suggest that the healing of excision and penetrating incision wounds of the cornea differ only in the resulting size and shape of the scar. D
258
C. CINTRON,
H.
SCHNEIDER
AND
C. KUBLIN
Glycosaminoglycans There are basically two types of glycosaminoglycans in the rabbit cornea1 stroma : keratin sulfate and chondroitin sulfate (Anseth, 1961b; Bleckmann and Buddecke, 1972). The method of Antonopoulos et al. (1954); based on the elution of cetylpyridinium. complexes of the glycosaminoglycans from a cellulose column, has been used to separate keratin sulfate from chondroitin sulfate of rabbit cornea (Praus and Dohlman, 1969). The same technique has been used in this investigation to measure the accumulation of glycosaminoglycans in 2.0-mm full thickness excision wounds of the rabbit cornea. The hexosamine content of glycosaminoglycans from normal rabbit cornea1 stroma and the ratio of hexosamine between CPC soluble and CPC insoluble glycosaminoglycans, determined in this study, are consistent with those reported by Anseth (1961b). Furthermore, the presence of glucosamine in the CPC soluble fraction and galactosamine in the CPC insoluble fraction strongly suggest that the former fraction contains keratin sulfate while the latter is primarily chondroitin sulfate. In this study, the CPC soluble and CPC insoluble glycosaminoglycans from healing cornea1 excision wounds have not yet been identified. CPC solubility characteristics for normal stromal glycosaminoglycans would suggest that the two fractions of concern probably contain keratin sulfate in the 1 O’ lo CPC soluble fraction and chondroitin sulfate and perhaps hyaluronic acid in the 1% CPC insoluble fraction. The present investigation indicated that glycosaminoglycans accumulate in the wound during the first four weeks of healing. Other investigators dealing with corn.eal incisions have demonstrated the accumulation of glycosaminoglycan in the developing scar (Anseth, 1961a; Kitano and Goldman, 1966; Praus and Dohlman, 1969). Although Anseth (1961a) showed a slight increase in hexosamine from 0.02 M- and 2-O M-HCl fractions eluted from ECTEOLA columns during the first month of healing, total hexosamine values actually decreased during this time. Dohlman and Praus (1968), on the other hand, found very little change in total hexosamine content during a similar period. Discrepancies in the changes of hexosamine content may be due to the inclusion by previous investigators of tissue adjacent to the developing scar. The wound healing tissue used in this investigation does not include adjacent normal cornea1 tissue. Collagen
The present study strongly suggests that collagen accumulation is very rapid during the first two weeks of healing, but becomesgradual after the second week. This may reflect a decreasedrate of collagen synthesis and/or increasedbreakdown. Similar accumulation curves were found in open full-thickness skin wounds in guinea pigs (Grillo, 1968; Gri‘11o et al., 1955). Recently, Madden and Peacock (1971) have shown an abrupt levelling off of the scar collagen accumulation curve in rat linear skin wounds at a time when the rate of new collagen deposition is high, Grill0 and Gross (1967) have demonstrated lysis of reconstituted collagen gels in cultures of 15-day guinea pig open skin wounds. Brown and Weller (1970) have shown similar lysis with linear full thickness cornea1 wounds cultured between the third and fifteenth day after wounding. Thus there is evidence that suggestsa collagenolytic system playing a role in cornea1wound repair.
CORNEAL
SCAR
FORMATION
259
ACKNOWLEDGMENTS
The authors are great,ly for their valuable advice Yagoda for the histological This investigation was Grant EY-00043; Special National Eye Institute; Inc.
indebted to Drs B. J. Jackobson, M. Refojo, and C. H. Dohlma,n and criticism. We wish to express our thanks to Mrs Dorothy preparations. supported in part by Research Grant EY 00208 and Training Fellowship II?03 EY 5032501 VSN (Dr Schneider), from the and in part by the Massachusetts Lions Eye Research &ad, REFERENCES
Eye Res. 1, 122. Eye Res. 1, 196. Anseth, A. and Fransson, L.-A. (1969). Exp. Eye Res. 8, 302. Sntonopoulos, C. A., Garde& S., Szirmai, J. A. and De Tyssonsk, E. R. (1964). Biochim. Biophys. Acta 83, 1. Baum: J. L. (1971). Arch. Ophth.aZmoZ. 85, 473. Bleckmann, H. and Buddecke, E. (1972). Ezp. Eye Res. 13, 248. Brown, S. I. and Weller, C. A. (1970). Arch. Ophthalmol. 83, 74. Cifonelli, J. A., Saunders, A. and Gross, J. I. (1967). Carbohydrate Res. 3, 478. Dohlman, C. H. and Praus, R. (1968). In Biochemistry of the Eye, Xymp. Tutzing Caz?tZe (Ed. Dardenne, M. U. and Nordmann, J.). P. 120. S. Karger-Base& New York. Elson, L. A. and Morgan, W. T. J. (1923). Biochem. J. 27, 1824. Garg. B. D. and McCandless, E. L. (1968). Anat. Rec. 162, 33. Gasset, A. R. and Dohlman, C. H. (1968). Arch. Ophthalmol. 79, 595. Grille, H. C. (1968). Quart. J. Swg. L&i. 4, 50. GriIIo, H. C. and Gross, J. (1967). Develop. Biol. 15, 300. Grillo, H. C., Watts, G. I. and Gross, J. (1958). Ann. Surg. 148, 145. Heydenreich, Von A. (1962). Klin. Monatsbl. Augenheilk. 141,365. Kitano, S. and Goldman, J. N. (1966). Arch. Ophthalmol. 76, 345. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). J. Biol. Chem. 193, 265. Xadden, J. W. and Peacock, E. E. Jr. (1971). Amer. Surg. 174, 511. McDonald, J. E. (1957). Arch. Ophthalmol. 58, 202. McDonald, J. E. (1961). Amer. J. Ophthalrnol. 51, 930. Praus, R. and Dohlman, C. H. (1969). Exp. Eye Res. 8, 69. Prockop, D. J. and Udenfriend, S. (1960). Anal. Biochem. 1, 225. Robb, R. M. and Kuwabara, ‘I’. (1962). Arch. O~hth~ZmoZ. 68, 636. Robb, R. M. and Kuwabara, T. (1964). Arch. Ophthalmol. 72, 40. Stoffyn, P. J. and Jeanloz, R. W. (1954). Arch. Biochem. Biophys. 62, 373. Swann, D. A. and Balazs, E. A. (1966). Biochim. Biophys. Acta 130, 112. Switzer, B. R. and Summer, G. K. (1971). Anal. &o&em. 39, 487.
Xnseth, A. (1961a). Exp. Snseth, A. (1961b). Exp.
Corned Scar Formation CHARLES
~ep~~t~le~~t
CISTRON,
HAROLD
of Comea Research, Rrtina
SCHNEIDER
Foundation,
AXD
CLAIRE
KUBLIN
Boston, Mass. 0.2114, U.S.A.
(Received 30 May 1973, and in revised fomx 5 July 1973, Boston) The present study is concerned with scar formation in rabbit cornea after removal of a centrally-located 2.0 mm full thickness cornea1 “button”. Simplicity in wounding, reproducibility in healing with minimum complications, and ease of recovery of adequate amounts of “pure” scar tissue make this experimental model suitable for quantitative biochemical analyses. Quantification of hexosamine in purified glycosaminoglycan fractions from cornea1 excision wounds indicates that the amount of glycosaminoglycan progressively increases to 74% of that in the normal stroma by the fourth week of healing. This observation is not consistent with previous findings in cornea1 incision wounds. Hydroxyproline determinations in cornea1 wounds strongly suggest that collagen accumulation is very rapid during the first two weeks of healing. This is followed by a gradual increase in collagen, approaching the level in normal cornea1 stroma by the nmth week of healing.
1. Introduction -4 variety of wounding techniques has been used to enable investigators to study various aspects of cornea1repair. Among the most common methods used are penetrating and non-penetrating linear scalpel incision with or without sutures (Robb and Kuwabara, 1962; G-assetand Dohlman, 1968; Kitano, and Goldman, 1966). Other types of wounds have involved scraping endothelium and Descemet’s membrane (,4nseth and Fransson, 1969), cautery (Robb and Kuwabara, 1964), freezing (Baum, 19711, and intrastromal injection of carrageenin (Garg and McCandless, 1968). A perforating t,rephination excision wound in rabbit cornea wa,s used to study the effects of various drugs on the regeneration of the stroma using clinical and histological observations (Heydenreich, 1962). The present report describes the use of the experimental model developed by Heydenreich (1962), with some modifications: to study the biochemistry of scar formation. Simplicity in wounding, reproducibility in healing with minimum complications: and ea.seof recovery of adequate amounts of “pure” scar tissue make this experimental model suitable for qualitative and quantitative analyzes. Measurements of dry weight, water? hydroxyproline content and quantity of hexosamine in purified glycosal~liizoglycan fractions from the developing scar tissue are determined LOillustrate the use of the model for quantitative biochemical studiee. 2. Materials and Methods Albino rabbits, weighing 2-3 kg, were given 4% atropine drops to each eye prior to anesthesiawith intravenous sodiumpentobarbital. Anesthetized animals were then given topical proparacaine and in some cases a retrobular injection of 2% lidocaine. A 2%mm diameter full thickness “button” was excised from the center of each cornea with an * Reprint 20 Staniford
requests to: Charles Cintron, Street, Boston, Mass. 02114,
Ph.D., U.S.A.
Retina 261
Foundation,
Department
of Cornea
Research,
252
C. CIPL’TRON,
H.
SCHNEIDER
AND
C. KUBLIN
Elliot trephine. Pieces of Descemet’s membrane remaining fully dissected away with Vannas scissors. Wounded eyes myxin B-bacitracin-neomycin) ointment only immediately of 44 eyes with cornea1 wounds were examined periodically the progress of wound healing.
in the were after with
wound area wTere caregiven Neosporin (polythe operation. A total a slit lamp to evaluate
selection and preparation 0Jpsumples front healing corneas for analysis A scar tissue sample from an eye was used for biochemical analysis if the following criteria were met: (1) normal anterior chamber depth by the seventh day; (2) surface of healing wound not cratered; (3) no infection, vascularization, or iris incarceration; (4) decreased i&is by the seventh day. Epithelia of scarred corneas of various ages, and normal corneas, were scraped with a scalpel before removal of the wound area or stroma with a 2-O-mm Elliot trephine. Scarred and normal stromal “buttons” used for water and hexosamine determinations were pooled (9-26 buttons), blotted on Whatman no. 1 filter paper and weighed on a Mettler balance. Tissues were then lyophilized and weighed on a Cahn electrobalance. Hydroxyproline was det,ermined in individual “buttons” without pooling. Histology Cornea,1 excision wounds of various a,ges were processed for histology by fixing the whole cornea in 10% neutral formalin. Tissues were dehydrated, embedded in paraffin and 5 pm sections were made on an A0 Spencer microtome. Sections were stained with Hematoxylin and Eosin or Van Giessen’s acid fuehsin. Biochemical
analyses
The details of the method used for isolation of total glycosaminoglycan were similar to that described by Praus and Dohlman (1969). The procedure involves enzymaic degradation of the tissue with papain, followed by trichloroacetic acid (TCA) precipitation of proteins and alcohol precipitation of total glycosaminoglycans. The alcohol-precipitated material was tested for the presence of protein using the Lowry procedure (Lowry, Rosebrough, Farr and Randall, 1951). Hexosamine was determined with the method of Elson and Morgan (1933) as modified by Swann and Balazs (1966). Separation of glycosaminoglycans was accomplished with the microcellulose column technique of Antonopoulos, Gardell, Szirmai and De Tyssonsk (1964). Isolated glycosaminoglycan was dissolved in 50 ~1 of water and applied to a column (3.0x60.0 mm) of chloride (CPC). cellulose previously equilibrated with 4.0 ml of 1% eetylpyridinium The sample was then washed into the column with 50 ~1 of water. Glycosaminoglycans were separated into two fractions by addition of 1.5 ml of 1% cetylpyridinium chloride (CPC) which elutes CPC soluble components (CPC soluble fraction) followed by 1.5 ml of 6 N-IICl which removes all other glycosaminoglycans from the column (CPC insoluble fraction). All fractions were hydrolyzed in 8 N-HCI at 95°C in sealed tubes for 3 hr. Hydrolysates were then dried under a flow of air in a 40°C water bath. Hexosamines from CPC soluble and CPC insoluble fractions of normal stroma were identified by paper chromatography after ninhydrin degradation of hexosamines to pentoses (Stoffyn and Jeanloz, 1954). The quantity of hexosamine from CPC soluble and CPC insoluble fractions of scarred cornea1 tissue was determined, although the individual hexosamines were not identified. (2) Collagen. Lyophilized scarred and normal stromal “buttons” were hydrolyzed with 10 N-HCl for 5 hr at 95°C and dried down under a current of air. The method of Prockop and Undenfriend, (1960) as modified by Switzer and Summer (1971) was used to determine the quantity of hydroxyproline in the hydrolysates. (1)
Glycos~~n~nog~~ca~s.
CORNEAL
SCAR
253
FORMATION
Quantitative biochemical data in this section are expressed in two ways; (a) micrograms of hexosamine or hydroxyproline per 2.0 mm trephined “button” of normal or scar tissue and (b) micrograms per milligram dry weight of tissue. The latter allows one to compare the present data with that in the literature, while the former makes clear any quantitative changes obscured by concomitant changes in dry weight. 3. Results
Xlit lamp und histological
descriptio9z
Upon removal of a central 2.0~mm cornea1 button, the anterior chamber collapsed and the edge of the wound immediately became opaque [Fig. l(a)]. Since the pupil was greatly dilated, iris was not pulled into the gaping wound. In approximately 10 min the aqueous humor, filling the wound area, became viscous. Within 15-30 min the anterior chamber began to reform as the wound took on an opaque fibrous appearance. This opacity became quite pronounced in a few hours [Fig. l(b)].
FIG.
‘button”.
1. Cornea1 wound healing after removal of a centrally-located (a) 5 min, (b) 3 hr, (c) 3 days, (d) 1 week, and (e) 2 weeks
after
2+mm full wounding.
thickness
cornea1
254
C. CINTRON,
H.
SCHNEIDER
AND
C. KUBLIN
By the third day after wounding, the conjunctiva often appearedinflamed. Although the wound edge remained opaque, the central region became translucent [Fig. l(c)]. The rest of the cornea remained transparent. In 55% of the cases (24 out of 44 wounds), a coagulum extended down from the wound into the anterior chamber and often attached to the lens. Although iris incarceration was rarely found, iritis was noted in 45% of the cases and hyphema could sometimes be seen. In 45% of the cases the anterior chamber was shallow at this stage.
2.0.mm full thickness corneal FIG. 2. Cornea1 wound healing after removal of a centrally-located “button”. (a) 3 days, (b) 1 week, (c) 2 weeks after wounding. (a) and (b) stained with Van Giessen’s acid fuchsin; (c) stained with Hematorylin and Eosin. ( x 56)
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Histologically, the regenerating cornea1 epithelium on the third day was continuous across the wound although only one cell thick in most places [Fig. 2(a)]. The stromal area of the wound was occupied primarily by an acellular fibrous coagulum. Fibroblast-like cells were located in the peripheral and anterior segment of the wound under the regenerating epithelium. By t,he seventh day the conjunctival inflammation seemed to have decreased. The wound was opaque except for a small central portion which was still translucent [Fig. l(d)]. There was less coagulum in the anterior chamber and the number of eyes with iritis had decreased. Approximately 25% of the eyes still exhibited a shallow anterior chamber. Vascularization of the cornea was noted in 7% of the eyes. Infeection, characterized by con.junctival inflammation along with the appearance of mucous on the eye and lids was found in 4:/, of the eyes. Cell number in both the epithelial and etromal area of the wound was greatly increased by the seventh day [Fig. 2(b)]. Extracellular material was evident between the fibroblast-like cells in the peripheral portion of the wound. AIthough most fibroblast-like cells were Iocated under the regenerating epithelium, some cells were found under the fibrous coagulum forming an apparently sepnrate population of fibroblast-like cells continuous with the posterior surface of the cornea. The wound was completely opaque by the second week [Fig. l(e)]. The frequency of occurrence of iritis and opaque coagulum in the anterior chamber was decreased further to approximately 13% of all eyes. Those eyes which exhibited shallow anterior chambers after one week remained the same two weeks after wounding. In only one out of 44 cases did the wound surface appear cratered. By the second week the wound area was filled with fibroblast-like cells [Fig. 2(c)]. A remnant of fibrous coagulum in the center of the stromal area was still present at this time. The epithelium wa,s variable in thickness over the wound area, 4-11 cells thick. The scar progressively increases in opacity and appears less edematous during the third and fourth week after wounding. Larger trephination wounds (e.g. 3-O mm in diameter), healed as described above. However, more than 50% of the wounds exhibited complications (i.e. vascularization, infection, cratered wound surface, and iris incarceration). With 2.0 mm excision wounds approximately 75% of the total number of eyes operated on were considered usable for biochemical analysis.
Approximately 72% of the wet weight of the normal rabbit cornea1 stroma was due to wa,ter. One week after removal of a 2.0 mm irephined cornea1 “button” the wound area was 94% water by weight (Fig. 3). B? the second week the wet weight of the healing wound increased due to an increase m both water and dry matter. At this time the dry weight of the scar was 80% of that of the normal stroma. A gradual increase in dry weight of the scar was noted in t.he succeeding weeks of healing. The water content, on the other hand, progressively decreased after the second week approaching that of the normal cornea1 stroma by t’he eleventh week after wounding. B~ioclienhxd
analyses
(1) Glycosaminoglycans. During the isolation of total glycosaminoglycans, samine was not detected in TCA precipitable material after papain treatment
hexoof the
256
C. CINTRON,
H.
SCHNEIDER
AND
C. KUBLIN
/$, II/ \ b‘a--we,----__ wt. di 4 ‘a-J-l
L!.
FIG. 3. Changes in dry and wet weight of healing cornea1 wounds after removal of it centrally-located 2-O-mm full thickness cornea1 “button”. 0-0, dry weight of tissue; O-..--0, wet weight; A-- --A, water weight (arithmetic difference between wet weight and dry weight of tissue).
normal cornea1 stroma. In addition, protein was not detected in the alcohol precepitated fraction containing glycosaminoglycans. Glycosaminoglycans fram normal rabbit cornea1 stroma were separated with a CPC-cellulose column into a 1% CPC soluble fraction and a lo/, CPC insoluble fraction which was subsequently released from the column with 6 N-HCl. Paper chromatography of hydrolysates from each of these two fractions demonstrated the presence of glucosamine in the CPC soluble fraction and galactosamine in the CPC insoluble fraction. Total hexosamine content of extracted, purified and fractionated glycosaminoglycans from normal stroma was 1.43% of the dry tissue weight. The ratio of hexosamine between the CPC soluble fraction and CPC insoluble fraction was 1.64 (Table I). TABLE
Hexosamine
Weeks after wounding
NOl-llldl
1 2 3 4
* 2.0-mm
Xumber of “buttons” pooled
17 26 20 I6 11
diameter
content of normal
cornea1 stroma and scar tissue
ug Hexosamine/button * ’ 1% CPC 1% CPC soluble insoluble fraction fraction
2.68 0.23 0.64 i.64 1.68
full thickness
I
pg Hexosamine/mg tissue wt. 1% CPC soluble fraction
1% CPC insoluble fraction
5.90 1.45 2.62 7.03 6.18
5.40 2.43 3,34 6.23 5.80
1.63 0.39 0.81 i.45 1.50
excised
“buttons”
dry
from
normal
stroma
Ratio (CPC sol: CPC insol.)
and sear tissue.
1.64 0.60 0.79 1.13 1.23
CORNEAL
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257
FORMATION
There was a progressive increase in the hexosamine content of the CPC soluble and CPC insoluble fractions obtained from developing cornea1 scar tissue between one and three weeks after wounding (Table I). However, no apparent accumulation of hexosamine was noted between the third and fourth week of healing in either the @PC soluble or CPC insoluble fractions. The quantity of hexosamine in the CPC soluble fraction of the four-week-old wound reached 63% of that in the normal tissue. Hexosamine content from the CPC insoluble fraction, on the other hand, approximated that of the normal stroma. The ratio of hexosamine between the CPC soluble fraction and CPC insoluble fraction increased to 75% of the normal stroma by t,he fourth week of healing. TABLE
Hydroxyproline after wounding
Weeks
Normal 1 2 3 4 5 9
* 2.0-mm diameter t S.E. of the mean.
II
content of normal corned stroma and scar tissue Number of determinations
10 9 9 9 10 9 10
full thickness
Pg Hypro/button
19.60&0.69 3G36*0.32 9+33&0~70 11-03fl~07 12.52kO.68 13.60&1.00 19.16f1.25
excised
“buttons”
from
*
t
dry tissue
wt
60.03 & 1.96 24.81 f 1.78 40.14*1.64 43.12&2-78 41.92,t4+37 5O.OS&2.63 58.31&1.56
normal
and scar tissues.
(2) Collagen. The quantity of hydroxyproline in rabbit cornea1 stroma was 60.0 pg/ mg of dry tissue or 19.6 pgj2.0 mm trephined stromal “button” (Table II). During the first two weeks of healing, hydroxyproline rapidly accumulated in the wound area to a.pproximately half of that in the normal stromal “buttons”. This was followed by a more gradual accumulation of hydroxyproline in the scar, approaching that of t,he normal stroma by the ninth week. 4. Discussion T’he e~per~ment~~ model Removal of a centrally-located 2.0 mm full thickness cornea1 “button” is easily and quickly accomplished in normal rabbits to elicit cornea1 repair and in healing corneas to sample “pure” scar tissue for analysis. Slit lamp observations have demonstrated the reproducibility of wound-healing in this system (75% of all eyes operated on healed in a similar manner) along with a minimum of complications (i.e. infection, vascularization, and iris incarceration). Centrally-located 3.0 mm full thickness cornea1 excision wounds, similar to that used by Heydenreich (1962), resulted in a 50% recovery of usable tissue for biochemical analysis. The present observations on the healing of excised cornea1 wounds are consistent with those described by McDonald (1957, 1961), for penetrating linear wounds of the rabbit cornea. This would suggest that the healing of excision and penetrating incision wounds of the cornea differ only in the resulting size and shape of the scar. D
258
C. CINTRON,
H.
SCHNEIDER
AND
C. KUBLIN
Glycosaminoglycans There are basically two types of glycosaminoglycans in the rabbit cornea1 stroma : keratin sulfate and chondroitin sulfate (Anseth, 1961b; Bleckmann and Buddecke, 1972). The method of Antonopoulos et al. (1954); based on the elution of cetylpyridinium. complexes of the glycosaminoglycans from a cellulose column, has been used to separate keratin sulfate from chondroitin sulfate of rabbit cornea (Praus and Dohlman, 1969). The same technique has been used in this investigation to measure the accumulation of glycosaminoglycans in 2.0-mm full thickness excision wounds of the rabbit cornea. The hexosamine content of glycosaminoglycans from normal rabbit cornea1 stroma and the ratio of hexosamine between CPC soluble and CPC insoluble glycosaminoglycans, determined in this study, are consistent with those reported by Anseth (1961b). Furthermore, the presence of glucosamine in the CPC soluble fraction and galactosamine in the CPC insoluble fraction strongly suggest that the former fraction contains keratin sulfate while the latter is primarily chondroitin sulfate. In this study, the CPC soluble and CPC insoluble glycosaminoglycans from healing cornea1 excision wounds have not yet been identified. CPC solubility characteristics for normal stromal glycosaminoglycans would suggest that the two fractions of concern probably contain keratin sulfate in the 1 O’ lo CPC soluble fraction and chondroitin sulfate and perhaps hyaluronic acid in the 1% CPC insoluble fraction. The present investigation indicated that glycosaminoglycans accumulate in the wound during the first four weeks of healing. Other investigators dealing with corn.eal incisions have demonstrated the accumulation of glycosaminoglycan in the developing scar (Anseth, 1961a; Kitano and Goldman, 1966; Praus and Dohlman, 1969). Although Anseth (1961a) showed a slight increase in hexosamine from 0.02 M- and 2-O M-HCl fractions eluted from ECTEOLA columns during the first month of healing, total hexosamine values actually decreased during this time. Dohlman and Praus (1968), on the other hand, found very little change in total hexosamine content during a similar period. Discrepancies in the changes of hexosamine content may be due to the inclusion by previous investigators of tissue adjacent to the developing scar. The wound healing tissue used in this investigation does not include adjacent normal cornea1 tissue. Collagen
The present study strongly suggests that collagen accumulation is very rapid during the first two weeks of healing, but becomesgradual after the second week. This may reflect a decreasedrate of collagen synthesis and/or increasedbreakdown. Similar accumulation curves were found in open full-thickness skin wounds in guinea pigs (Grillo, 1968; Gri‘11o et al., 1955). Recently, Madden and Peacock (1971) have shown an abrupt levelling off of the scar collagen accumulation curve in rat linear skin wounds at a time when the rate of new collagen deposition is high, Grill0 and Gross (1967) have demonstrated lysis of reconstituted collagen gels in cultures of 15-day guinea pig open skin wounds. Brown and Weller (1970) have shown similar lysis with linear full thickness cornea1 wounds cultured between the third and fifteenth day after wounding. Thus there is evidence that suggestsa collagenolytic system playing a role in cornea1wound repair.
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ACKNOWLEDGMENTS
The authors are great,ly for their valuable advice Yagoda for the histological This investigation was Grant EY-00043; Special National Eye Institute; Inc.
indebted to Drs B. J. Jackobson, M. Refojo, and C. H. Dohlma,n and criticism. We wish to express our thanks to Mrs Dorothy preparations. supported in part by Research Grant EY 00208 and Training Fellowship II?03 EY 5032501 VSN (Dr Schneider), from the and in part by the Massachusetts Lions Eye Research &ad, REFERENCES
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Xnseth, A. (1961a). Exp. Snseth, A. (1961b). Exp.