the healing of rabbit corneal endothelium

A C T A O P H T H A L M O L O G I C A 62 (1984) 796-807

Uniiipr~iQ Eye Department (Head: Amid Anseth), and the Institute of Pathology,

Electron Microscopic Laboratory (Head: Torstein Hosig), Rikshospitalet, Unisertity of Oslo, N o m y

THE HEALING OF RABBIT CORNEAL ENDOTHELIUM

BY

ERLING GR0NVOLD OLSEN and MARTIN DAVANGER

A circular 4 mm endothelial defect was induced by transcorneal freezing. The experimental damage and the healing took place in the living rabbit in 15 eyes, and in the isolated cornea in organ culture in further 20 eyes. The reparative process was studied by SEM. and proved to be the same in vivo and in vitro. The defect was covered with endothelial cells after 3 days. The normal hexagonal pattern was regained after 3 weeks. Both cell migration and cell division were involved in the reparative process. Only cells recruited from a zone close to the defect were active; the cells situated more than a few cell diameters from the original edge maintained their form and size unchanged. T h e first phase of cell division was the formation of a spherical cell with numerous blehs on its surface.

Kry word,t: corneal endothelium - rabbit - regeneration - tissue culture - scanning electron microscopy.

T h e corneal endothelium is considered to be a stable cell group in which cell renewal does not take place under ordinary circumstances. The cells appear to be permanently stalled in the GI phase of the cell division cycle (Young 1982).

If some cells are destroyed so that the posterior surface of the cornea is no longer completely covered by the endothelium, a reparative process takes place. After a certain time the Descemet’s membrane is again covered by endothelial cells (Chi et al. 1960; Van Horn & Hyndiuk 1975; Yano 8cTanishima 1980).

Received on October 28th. 1SXS

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Olsen & Davanger Healing of corneal endothelial defect

Obviously one o r both of two different processes must be involved in the healing process, namely cell division and cell migration. If only cell migration was involved, the end situation would be a reduced number of cells covering the same surface; i.e. each cell would have to cover a larger area.

During recent years, it has been amply demonstrated that in many clinical situations, the density of endothelial cells is reduced from the normal. Further, it has been shown that if the cell density is much reduced, the cornea becomes oedematous (Mishima 1982a,b; Olsen 1982). The result is reduced transparency and damage to the epithelium.

On this background it is understandable that considerable interest has been focused on the corneal endothelium and its potentials for renewal and repair (Von Sallmann et al. 1961; Oh 1963; Alvarada 1981 ; Moor et al. 1981; Rahi 8c Robins 198 1 ; Renard et al. 198 1 ; Treffers 1982. Review in Mishirna 1982a).

The present paper deals with the healing of an experimentally induced standard defect of the rabbit corneal endothelium. The reparation has been scrutinized by the help of‘ scanning electron microscopy (SEM), an examination method which has proven to be well suited for this purpose. The repair process has taken place partly in the living rabbit, and partly in organ culture.

The immediate aim has been to examine the details of the healing. Emphasis has been put on the study of cell division and cell migration. A further purpose has been to develop models and methods, and to provide baseline information for studies of the effects of experimental influences on the repair process of the corneal endothelium.

Material and Methods

Eves from young adult rabbits have been iised for this study. In 15 eves, the experimental damage and the healing took place in living rabbits, after in in.jec.tion of 1 ml fluanisonum (0.1 T Hypnorm vet., Leo). I n further 20 eyes, all experimental proc-edures were performed after the sacrifice of the animal and enucleation of the eye. In both cases the standard defect ofthe endothelium was made on the intact, unopened eve by freezing writh a cryo probe made for retinal surgery. With a 3 mm probe the central cornea was indented to touch the lens. Then the temperature was brought down to -70°C. The ice formed stabilized in siLe about 4 mm diameter in few seconds. Then the switch to freeze was released, and the probe was removed when it no longer adhered. After its removal, the cornea was still adherent to the lens and was therefore indented centrally. By gentle pressure on the globe, this adherence was rather abruptly loosened. It was assumed that the cells damaged by the freezing and thawing to some extent adhered t o the anterior lens surface and thereby were removed from the Descemet’s membrane. The whole procedure was repeated 4 times. The positioning o f the prohe at the correct place was facilitated by the formation of a circular faint haze formed after the first freezing.

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In the in vitro experiments, the cornea was removed at this stage under sterile conditions b y a perforating incision at the limbus with a sralpel and continuing along the limbus with a pair of scissors. By this procedure, care was taken to avoid damage to the endothelium. Then the cornea was brought directly to the culture medium, the endothelial side facing upwards.

T h e organ culture was performed in tissue culture clusters containing 1 0 ml tissue culture medium: KPMI 1640, supplemented with penicillin (100 IU/ml), streptomycin (500 bg/rnl), and 1076 inactivated foetal calf serum (FCS).

At the termination of the in vivo experiments, the animal was sacrifized with an overdoseof pentoharhitone sodium intravenously. The corneas from both the in vivo and the in vitro experiments were fixed for 18-24 h in 2.5% glutaraldehyde huffered to pH 7.4 with 0.1 M phosphate and post-fixed for 2 h in 1% Os04 buffered with 0.1 M phosphate, pH 7.4. Thereafter, the specimens were dehydrated in graded acetone solutions, and dryed by the critical point niethod in fluid COZ. In some cases, the peripheral part of the cornea was examined by light microscopy. The dried cornea was mounted on specimen holders. By preliminary experiments, it was found that if the epithelium was defect and/or the cornea was oedematous. the solvent of the silver glue conventionally used for the mounting on specimen holders frequently penetrated through the cornea to the endothelial side. This caused ar t i fkts so that these specimens had to be discarded. To avoid this, a new method was developed. The specimen was glued to the holder by metacrylate glue (Araldit Rapid@) and care was taken to cover the whole epithelial side of the cornea with the glue. After polymerization, the surface of the metacrylate was covered with the silver glue to prevent charging during SEM.

T h e specimens were conventionally sputter coated with gold-palladium alloy. SEM wa5 performed with a Jeol JSM-50. In a few cases, the specimen was also photographed after the preparation, hy the help of an incident light microscope (Orthoplan, Ernst Ixi tz Wetzlar (;mhH, Germany).

Resu I ts

By SEM of specimens which were processed immediately after freezing, it could be seen that all endothelial cells on the frozen area were destroyed. Most of them were removed from Descemet’s membrane, which always was found to be completely intact, with no visible damage (Fig. la,b).

Twenty-four hours after freezing, the frozen area was oedematous, especially pronounced along the periphery of the frozen part. This caused the formation of a distinct mound delineating the circular area on which the endothelial cells had been destroyed (Fig. 2a). The healing process could be followed by noticing the position of the leading edge of proliferating endothelium in relation to this mound.

The reparative process was very similar in vivo and in vitro. This concerns both the appearance of the defect as a whole, the time taken for the different stages, and the morphology of the individual cells. The following description concerns both in vivo and in vitro experiments.

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FZg. I . a) Defect of the corneal endothelium immediately after freezing (15 x). b) The Descemet’s membrane is seen in the central part as an even surface, contrasting to the dull surface of the

cell-covered areas. Incident light microscopy (30 x).

The gross appearance and timing

After 24 h the cells bordering on the uncovered area had started to migrate over the denuded Descemet’s membrane. An approximately 0.5 mm broad brim of transformed endothelial cells surrounded the defect (Fig. 2b,c) Forty-eight hours after the freezing the defect was smaller (Fig. 3a), and after 3 days the endothelial cells covered the whole surface (Fig. 3b). The centre could still be easily recognized, indicated hy the radial pattern characterizing the damaged area.

In the course of the following days, the radial lines and the centre became less obvious. Three weeks after the start, the cells of the area had regained a hexagonal pattern; the cell surface and cell borders, however, being more uneven and less regular than normal.

The appearance of the cells

The advancing cells bordering on the defect had the same appearance as long as there was an uncovered area. They bore clear evidence of migration (Fig. 4a). Flat cytoplasmic processes stretched out from that part of the cell which was directed towards the centre of the defect. From these sheets, thin extensions stretched further out along Descemet’s membrane. The whole cell was elongated in a radial diretion. These cells were obviously very thin, each of them covering a large area.

Radial elongation was even more evident in the cells peripheral to the central, leading edge of cells. Most cells between this leading edge and the periphery of the damaged area were spindle-shaped, their transverse sections were nearly circular,

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Fig. 2. a) An oedematous mound (*) delineating the edge of the previously frozen area (200 x).

b) Transformed endothelial cells surrounding the defect, 24 h after freezing (230 x). c) Migrating endothelial cells bordering on the defect (780 x).

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Healinq of corneal endothelial defect Olsen & Davanger

Fig. 3. a) Endothelial defect 48 h after freezing (120 x). b) Endothelial cells covering the surface 3

days after freezing. Note the radial cellular pattern (120 x).

Healing of corneal endothelial defect Olsen & Davanger

Fig. 4 . a) Detail of a migrating endothelial cell 24 h after freezing (13 200 x). b) Spindle-shaped

endothelial cells in the area of repair (1300 x).


Fig. 5. a) Newly covered area. A few dark cells with an even surface are seen intermingled with the spindle-shaped cells (350 X). b) A narrow transitional zone between the hexagonal pattern of

the unfrozen area, and the irregular cell pattern of the repair zone (350 x).

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and thev had a number of filmientous processes covering a large part of their surflice (J:ig. 3b).

Scattered among the spindle-shaped cells, there were a few cells with another appearance (Fig. 521). They were tlat. had a highly irregular shape, and their surface was even. which made these cells appear darker by SEM.

The peripheral part of cornea. The border of the damaged area

Both in the in vivo arid the in vitro experiments the cells peripheral to the frozen central area developed an uneven surfacc with more microvilli than normal. However, these cells retained their normal sire and the regular hexagonal pattern. This concerns the whole period of observation, up to the total healing of the defect.

‘The transitional zone between the peripheral, unfrozen part, and the central, damaged area was narrow and sharply delineated (Fig. .5b). From the periphery, the cells were 1-egrilar hexagonal up to a borderline. Central to this circular border. the cells changed rather abruptly into the elongated cells which gave the impression ofa centrally directed migration.

By SEM and by light microscopy of sections, it could be seen that the keratocytes and the epithelium did not invade the posterior corneal surface of cultured corneas. This point has been evaluated by special experiments and details will be described in a following puhlicat ion.

Fzg. 6 . Stages of cell-division. a) Narrowing of a transversal belt. b) Narrow bridge between cells.

c) T w o separated spherical cells. (1600 x).

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Cell division

In the whole area which had been frozen, there were scattered cells which were obviously dividing. All stages of his process were found, from a certain cellular elongation with a slight narrowing at a transversal belt (Fig. cia), through the stage of two cells joined by a narrow bridge (Fig. fib), up to two separated spherical shaped cells lying close to each other (Fig. cic). The seemingly dividing cells were characterized by the presence of numerous blebs. Most ofthe blebs were localized at the part of the two spheres which was opposite to the bridge connecting them. On this bridge the surface was studded with long microvilli.

It was difficult to determine the number of dividing cells, because numerous more or less spherical shaped cells were present. Whether such cells were preparing to divide, o r alternatively, whether they were newly divided cells, could not be safely ascertained.

Discussion

The experiments have shown that the healing of defects of the corneal endothelium in rabbits takes place rapidly, and that the healing appears to be complete. A circular 4 mm defect made by freezing is covered by endothelium after 3 days. This is consistent with the findings of other investigators (Khodadoust 8c Green 1976; Van Horne t al. 1977; Staats &Van Horn 1980; Waller 1980).

By applying identical defects by in vivo and in vitro experiments, it has been possible to state that the healing process appears to be the same in the culture medium and in the living animal.

After the paper of Cogan (195 1) the general belief was that an endothelial defect was repaired by a re-arrangement of the remaining cells. The processes involved were migration and enlargement of the cells.

This concept has been revised after more recent works (Capella 1972; Treffers 1982). T h e present study points to cell division as the main factor in the healing process. Cell migration takes place only to the extent it is balanced by cell division.

A further main point in the present work is that only the cells adjoining to the edge of the original defect are active. Cell division and cell migration are restricted to this cell group. The area of the original defect is gradually covered with cells descending from the original edge cells.

It follows from this that the number of migrating and dividing cells increases during the process. The belt in which dividing and migrating cells are seen, increases in width during the 3 days until the whole damaged area is covered.

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The morpholoLgy o f t h e division process of the corneal endothelial cells is similar

to t h a t described for fibroblasts (Saxholm 8c Reith 1979). o u r knowledge, the

corneal endothelial cells have riot been seen by SEM d u r i n g the actual cell division

before . The process starts with the f<)rmation of a spherical cell with numerous

blebs o n its surface. While retaining its anchorage to the Descemet's membrane, the

cell becomes elongated, a n d a dividing furrow develops. Th i s furrow is studded

with microvilli.

T h e present s tudy has confirmed that both cell d27~2.520?2 a n d cell mzg-/.ation are

i m p o r t a n t for t h e healing of the corneal endothel ium in rabbits. In fu tu re studies

cellular mechanisms involved in the healing process will be investigated. Further,

t h e f indings of the present work will be used as a reference for a similar study of the

heal ing of t h e human corneal endothel ium.

Acknowledgment

Kind assistance of7'orolf Moen, M.D., Tissue Typing Laboratory, Rikshospitalet. is gratefully acknowledged.

References

Alvarada J A, <;ospodarowicz D & Greenburg G (198 1 ) : Corneal endorhelial replacement. I. In vitro formation o f an endothelial monolayer. Invest Ophthalmol21: 300-3 16.

Capella J A (1972): Regeneration of endothelium in diseased and injured corneas. Am J Ophthalmol74: 810-817.

Chi H H, l 'eng C C & Katzin H M (1960): Healing process in the mechanical denudation of the corneal endothelium. Am J Ophthalmol49: 693-703.

Cogan D G (1951): Applied anatomy and physiology o t the cornea. Trans Am Acad Ophthalmol Otolaryngol55: 329-359.

<;loor B P, Gloor M I., Marshall I & Meszaros I (198 1): Healing of mechanical wounds of the corneal endothelium of rhesus monkeys and rabbits. In: Trevor-Roper P D (ed). Vlth Congr Eur Sor Ophthalmol, Brighton, 1980. The cornea in Health and Disease. Grune SC Stratton, New York.

Khodadoust A A 8c Green K (1976): Physiological function of regenerating endothelium. Invest Ophthalmol 15: 96- 101.

Mishima S (1982a): Clinical investigations on the corneal endothelium. Am J Ophrhalmol93: 1-29.

Mishima S (1982b): Clinical investigations on the corneal endothelium. Ophthalmology 89: 525-530.

Oh J 0 (1963): Changes with age in the corneal endothelium of normal rabbits. Acta Ophthalmol (Copenh) 41: 568-573.

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Olsen 'I ( 1982) : Specular microscopic investigations on the corneal endothelium and its involvement in corneal oedema. l.hesis. hcta Ophthalmol ((hpenh). Suppl 155.

Rahi A H S & Rabins E (1981): Human corneal endothelial cell repair in health and disease. Trans 0phth;llmol Soc C!K 104: 30-34.

Kenard (;, Pouliqrien Y & Hirsch M (1981): Regeneration o f the human corneiil endothe- hum. Alhrecht von Graefes Arch Klin Bxp Ophthalmol2 1

Saxholm H I K & Keith A (1979): T'he surface structure of 7.12 - dimethylbenz (a) anthracene transformed ( 3 H I I o ' I cells. A cp~ntitative scanning electron microscopic-al study. F,ur,] Cancer 1.5: 84:1-8.55.

Staatz U' D & Van Horn (l98f)): 'The effects o f aging and inflammation on corneal endothelial wound healing in rabbits. Invest Ophthalmol 19: 983-986.

l'reffers W F (1982): Human corneal endothelial wound repair. I n vitro and i n vivo.

Van H o r n I) L &- Hvndiuk R iZ (1975): Endothelial wound repair in primate cornea. Exp Eye Res21: 113-124.

Van Horn I) I., Sendele D 11, Seideman S k Auco P S (1977): Regenerative capacity of the corneal enclothelium in rabbit and cat. Invest Ophthalmol 16: 597-6 13.

Von Sallnxin I-, Caravaggio I. I . & Grimes P (1961): Studies on the corneal endothelium o t the rabbit. I. Cell division and growth. Am.1 Ophthalmol 51

U'aller W K (1980): Die Regeneration des Hornhautendothels nach KSlteanwendung. In: Naumann 6 0 H & (;loor H P (eds). Wundheilung des Auges und ihre Komplikationen, p 227-2SO.J F Kergn'ann Verlag, Miinchen.

Yano M & Tanishima 1 (1980): Wound healing in rabbit corneal endothelium. Jpn 1 Ophthalmol24: 297 -309.

Young R W (1982): ~I'he Bowman I.ec.ture, 1982. Biological Renewal. Applications to the eve. Trans Ophthalmol Soc UK 102: 42-75.

O p h t h ; i l n ~ ~ l ~ g y 89: 605-6 13.

.4 ulho r\ ' adrltuws : Dr. Erling Gronvold Olsen, University Eye Department, Rikshospitalet, Oslo 1, Norway.

Dr. Martin Davanger, University Eye Department, Kikshospitalet, Oslo 1 , Norway.

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the healing of rabbit corneal endothelium

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