Scanning electron microscopy of wound healing in rat ... - Development

J. Embryol. exp. Morph. 83, 109-117 (1984) Printed in Great Britain © The Company of Biologists Limited 1984

Scanning electron microscopy of wound healing in rat embryos By MARTIN J. SMEDLEY AND MARTIN STANISSTREET Department of Zoology, University of Liverpool, Liverpool, Merseyside, L69 3BX, U.K.

SUMMARY Wound healing in rat early embryos has been studied by scanning electron microscopy. Initially the wound gapes slightly and cells peripheral to the wound assume a cobble-stone appearance. Wound closure is quite rapid; some small wounds are almost closed within 10 min of incision. Wound closure is accompanied by the appearance of some elongated cells at the wound edge. These features are similar to, although less pronounced than, those which have been observed to accompany wound closure in amphibian and avian embryos. Healing of wounds made in the amnion is also accompanied by changes in the shapes of cells at the wound margins. Wound healing in embryos cultured in Hank's saline is similar to wound healing in embryos cultured in serum, suggesting that the macromolecular components of serum are not essential to wound healing. Cytochalasin B, which inhibits wound closure in amphibian embryos, does not inhibit wound healing in rat early embryos unless used at a concentration high enough to cause cell dissociation. Similarly chelation of the free calcium in the medium, which also prevents wound closure in amphibian embryos, does not inhibit wound closure unless the embryo is dissociating. Removal of free calcium does however cause collapse of the elevated neural folds. These observations suggest that the cellular mechanisms involved in wound healing are different in mammalian and amphibian embryos. INTRODUCTION

Early embryos of all vertebrate species which have been studied show a remarkable ability to heal following the wounding incurred during experimental manipulation (Deuchar, 1975). It is unlikely that early embryos, especially mammalian embryos, would be subject to precise wounding under natural conditions. In addition, the mechanisms of wound healing in early embryos appear to be different from those employed in adult organisms (Stanisstreet, Wakely & England, 1980). Thus it is probable that wound healing is effected by those properties of cells normally employed in morphogenesis rather than by specialized mechanisms, and wound healing in early embryos can be used as a model for morphogenesis (Stanisstreet & Jumah, 1983). Consequently analysis of the factors which effect, control and co-ordinate wound healing might yield useful information about the mechanisms of morphogenesis. However only recently has the process of wound healing in embryos been studied. Scanning electron microscopical observations of healing wounds in

110

M. SMEDLEY AND M. STANISSTREET

avian and amphibian embryos have shown that wound healing is accompanied by changes in the shapes of the cells at the wound margin (Stanisstreet et al. 1980). Mammalian embryos too possess the ability to heal following wounding; for example, Deuchar (1969) found that in many cases rat embryos which had been completely transected had healed after 24 h of culture in vitro. Similarly, wounds in mammalian extraembryonic membranes are also capable of repair; for example, the amnion heals quickly following wounding incurred during the manipulation of embryonic tissues (Deuchar, 1969, 1971). However until now, observations on wounded mammalian embryos have been made to assess the eventual effect of wounding on developmental processes, rather than to elucidate the cellular changes which accompany and perhaps effect wound closure. Inhibitor studies have suggested that in amphibian embryos the changes in cell shape which accompany wound closure are effected by calcium-activated micron"laments, since they are inhibited by cytochalasin B (Stanisstreet & Panayi, 1980) or by removal of calcium from the culture medium (Stanisstreet, 1982). Analogous cellular mechanisms are thought to be responsible for at least some aspects of morphogenesis (Stanisstreet & Jumah, 1983). In the present work the process of wound closure in rat embryos and embryonic membranes has been studied by scanning electron microscopy. In addition the effects on wound healing in rat embryos of cytochalasin B (CCB), which inhibits microfilament function, and ethylenediaminetetra-acetic acid (EDTA) and ethyleneglycol-bis (/3-aminoethyl ether) N,N'-tetra acetic acid (EGTA), which chelate calcium ions, have been assessed to attempt to gain information about the cellular mechanisms involved in wound healing in mammalian early embryos.

MATERIALS AND METHODS

Rat embryos at the neural fold stage were obtained from white Wistar rats at 10-5 days of gestation, timed from midnight preceding the morning on which vaginal plugs were observed (New, 1978). Embryos were explanted in Hank's balanced saline containing 4-2 x 10~3M-sodium bicarbonate (Flow Laboratories Ltd.). Embryos were wounded through the yolk sac and amnion using an electrolytically sharpened tungsten needle; a small incision was made in the lateral region of the mesencephalon. Wounds in the yolk sac and amnion were produced in a similar way. Following wounding, embryos were cultured at 37 °C in rotating bottles according to the method of New, Coppola & Terry (1973). Unless otherwise stated the culture medium was pooled rat serum obtained from blood centrifuged immediately after withdrawal from the dorsal aorta. Streptomycin and penicillin were added to final concentrations of lOO/zg/ml and lOOi.u./ml respectively. The serum was stored at — 20 °C for a maximum of 28 days until required. Immediately before use the thawed serum was inactivated

SEM of wound healing in rat embryos

111

at 56 °C for 30min. The culture bottles were equilibrated with a 20 % O2, 5 % CO2,75 % N2 gas mixture (British Oxygen Co. Ltd.) to provide the appropriate oxygen tension for embryos of this stage (New, Coppola & Cockroft, 1976). Embryos were fixed for scanning electron microscopy at 0,5,10,15,30 or 60 min after wounding. In addition some intact embryos were fixed to serve as controls. In some experiments wounded embryos were cultured in Hank's saline in which the bicarbonate had been supplemented to a total concentration of 1-6 x 10~ 2 M to provide a pH of 7-3 when equilibrated with the gas mixture containing 5 % CO2. In addition the effects on wound healing of cytochalasin B (CCB) or calcium chelators were studied. Cytochalasin B (Sigma Ltd.) was added to the culture serum to final concentrations of 10,5,2-5,1,0-5 or 0-1 jug/ml from a stock solution in dimethyl sulphoxide (DMSO). Controls contained DMSO at a concentration equivalent to that required for 5/ig/ml cytochalasin B. Ethylenediaminetetra-acetic acid, EDTA, (B.D.H. Ltd.) was used at 2-5 x 10" 3 M either alone or in the presence of additional 2-5 x 10~3 M-calcium chloride. Ethyleneglycol-bis (/3-aminoethyl ether) N,N'-tetra-acetic acid, EGTA, (Sigma Ltd.) was used at 5 x 10"3, 2-5 x 10~3,10~3 or 5 x 10~ 4 M. Embryos were preincubated in the test serum for 30 min (for cytochalasin B) or 60 min (for EDTA or EGTA) before wounding and were fixed for scanning electron microscopy 60 min after wounding. Embryos were rinsed briefly in Hank's saline and were then fixed overnight in 2-5 % glutaraldehyde in 2 x 10"1 M-cacodylate buffer pH7-2 (Karnovsky, 1965). Embryos were then washed in changes of buffer. At this stage the embryonic membranes were removed using watchmakers' forceps. The embryos were then dehydrated in a graded ethanol series, the absolute alcohol was replaced with liquid CO2 and the embryos were dried using the critical-point method. The embryos were affixed to stubs, coated with gold-palladium and observed and photographed using a Philips 501B scanning electron microscope. RESULTS

Unlike previous observations of wound healing in amphibian embryos (Stanisstreet et al. 1980; Stanisstreet, 1982) it was not possible to make preliminary light microscopical observations of wounds in rat embryos due to the opacity of the embryonic membranes and the translucency of the embryonic tissues. Hence the observations reported here depend upon scanning electron microscopy. For the same reasons it was not possible to standardize the size of the wound to the same extent that it had been in previous experiments with amphibian embryos, although in most cases scanning electron microscopical observations enabled the sizes of wounds to be estimated in retrospect. Embryonic tissues For the initial experiments early 10-5-day embryos, in which the neural folds

112


Table 1. Number of wounds in different states of closure in rat embryos cultured in serum or Hank's saline for various times after wounding and observed by scanning electron microscopy Time in No. of culture embryos culture medium (min) (n) Rat serum

0 5 10 15 30 60

Hank's saline with 16 rriM NaHCO3

15 30 60

(19) (13) (11) (13) (11) (16) (12) (19) (13)

Smaller wounds (200]um)

open

closing

closed

open

closing

closed

7 5 2 1 0 0

0 5 1 2 3 4

0 0 4 5 5 6

12 2 1 2 0 0

0 1 3 3 2 4

0 0 0 0 1 2

6 3 0

4 4 2

2 9 10

0 1 0

0 2 1

0 0 0

had elevated but not fused, were used (Fig. 1). The results of observations on wounds in embryonic tissue are summarized in Table 1 and examples of the appearances of individual wounds are shown in Figs 3 to 6. Observations on unwounded control embryos showed that the cells of the lateral surface of the mesencephalon had polyhedral shapes with raised borders. The surface of the tissue was not smooth but undulate (Fig. 2). Immediately after incision the wounds were seen to gape slightly and the cells around the lateral edges of the wound had a slight cobble-stone appearance (Fig. 3). After 5 min the smaller (200 pm) wounds Fig. 1. Scanning electron micrograph of early 10-5-day rat embryo, before the neural folds have fused. x70. Fig. 2. Scanning electron micrograph of lateral surface of mesencephalon of 10-5day rat embryo. Surface is undulate and cells have polyhedral outlines with raised borders. X1020. Fig. 3. Scanning electron micrograph of wound in 10-5-day rat embryo immediately after incision. Wound is gaping slightly and cells peripheral to the wound are cobblestoned. x450. Fig. 4. Scanning electron micrograph of wound in 10-5-day rat embryo after 5 min culture in serum. Cells at the end of the wound are elongated and cells at the edges are curling under. X825. Fig. 5. Scanning electron micrograph of wound in 10-5-day rat embryo after 10 min culture in serum. Wound appears almost closed and is filled with cellular debris. X750. Fig. 6. Scanning electron micrograph of wound in 10-5-day rat embryo after 60 min culture in serum. Cells around the wound are elongated radial to the wound. X520.

.< I

*>_

13

^ , J ^

8


113

appeared not to have started to heal. After 10 min there was again some variation between wounds of different sizes (Table 1). Smaller wounds appeared almost closed and the wounds were filled with exuded cells and cellular debris (Fig. 5); larger wounds were still open, although cells around these wounds now appeared to be changing shape. By 30 min all wounds showed evidence of healing and many of the smaller wounds had healed completely. After 60 min in culture all of the wounds had either closed or had started to close (Fig. 6). Extraembryonic membranes The surface of the wounded yolk sac observed by scanning electron microscopy appeared convoluted or brain-like. Individual cells were detectable by their slightly sunken margins. Upon wounding the yolk sac collapsed onto the underlying intact amnion. Wound healing appeared to proceed more slowly than with embryonic tissue; after 60 min culture in serum the wounds had narrowed and the tissue was curling under at the wound edge (Fig. 7). However it was difficult to detect changes in the shapes of individual cells. The surface of amnion was smoother than that of the yolk sac and the boundaries of the cells could more readily be distinguished by their sunken borders. Upon wounding the amnion collapsed and became flaccid; the cobble-stone effect observed peripheral to wounds in embryos was not observed. After 15 min the amnion was tightening onto the underlying intact embryo. After 60 min culture in serum most of the wounds were closing. As the wounds became smaller, elongated cells radial to the wound could be observed (Fig. 8).

Fig. 7. Scanning electron micrograph of wound in yolk-sac of 10-5-day rat embryo after 60 min culture in serum. Tissue is curling under at the wound edge. x280. Fig. 8. Scanning electron micrograph of wound in amnion of 10-5-day rat embryo after 60 min culture in serum. Cells are elongated radial to the wound. x925. Fig. 9. Scanning electron micrograph of wound in 10-5-day rat embryo after 30 min culture in Hank's saline. Cells at the end of the wound are elongated and cells at the edges are curling under. X510. Fig. 10. Scanning electron micrograph of wound in 10-5-day rat embryo after 60 min culture in Hank's saline. Wound has closed and isfilledwith cellular debris. x560. Fig. 11. Scanning electron micrograph of wound in 10-5-day rat embryo after 60 min culture in serum containing lO/ig/ml cytochalasin B. The wound is gaping and the cells are dissociating. X340. Fig. 12. Scanning electron micrograph of early 10-5-day rat embryo, in which the neural folds had elevated but not fused, after 60 min culture in serum containing 2-5 x 10" 3 M EDTA. Neural folds have collapsed. x85. Fig. 13. Scanning electron micrograph of late 10-5-day rat embryo, in which neural folds had fused, after 60 min culture in serum containing 2-5 x 10"3 M EGTA. Neural folds have not collapsed. x70. Fig. 14. Scanning electron micrograph of wound in 10-5-day rat embryo after 60 min culture in serum containing 2-5 x 10~ 3 M EGTA. Wound is closing. x560.

114


Table 2. Effects of cytochalasin-B (CCB) and ethyleneglycol-bis-(fi-amino ethyl ether) N,N'-tetra acetic acid (EGTA) on closure of wounds in 10-5-day rat embryos Culture medium Rat serum + CCB (jUg/ml)

Rat serum + EGTA (HIM)

Concentration of inhibitors

No. of embryos

0-1 0-5 1-0 2-5 5-0

10-0

(7) (7) (7) (7) (6) (6)

0-5

(3)

1-0 2-5 5-0

(4) (3) (5)

Wound state

Tissue dissociation

healing healing healing/open open open gaping healing healing healing healing

intact intact intact/slight slight/intermediate intermediate severe intact intact intermediate intermediate

Embryos were pre-incubated for 30 min (CCB) or 60 min (EGTA) before wounding and observed by scanning electron microscopy 60 min after wounding.

Effects of Hank's saline The medium normally used for the in vitro culture of rat embryos is rat serum. In order to test whether serum is required for wound healing also, embryos were wounded and subsequently cultured in Hank's balanced saline with the bicarbonate supplemented to 1-6 x 10~ 2 M. The results of these experiments are summarized in Table 1. After 15 min culture in Hank's saline many of the wounds were seen to be still open with no obvious signs of healing. After 30 min most of the smaller wounds were closed or closing and appeared similar to the wounds of embryos after 10 or 15 min culture in serum (Fig. 9). After 60 min a similar situation obtained; most of the smaller wounds had now closed (Fig. 10). Thus wound healing does occur in rat embryos cultured in saline, but appears to be delayed in comparison with embryos cultured in serum. Effects of cytochalasin B The results of the experiments to determine the effects of cytochalasin B on wound healing are summarized in Table 2. Wounds in control embryos cultured in serum plus DMSO healed normally. At the lower concentrations used, 0-1 or 0-5/ig/ml, cytochalasin B did not affect wound healing. At 2-5 or 5/ig/ml cytochalasin B inhibited wound healing, but it also caused dissociation of the embryonic tissue. At lO/ig/ml cytochalasin B caused the wound to gape, but it also produced severe dissociation of the embryo (Fig. 11). Thus, unlike with amphibian embryos, cytochalasin B did not inhibit wound healing unless used at a concentration which provoked cell dissociation.


115

Effect of calcium ion removal EDTA at 2-5 x 10~ M caused little or no dissociation of the embryonic tissue, but it did cause collapse of the elevated neural folds, thus obscuring the wound site (Fig. 12). Attempts to remove the collapsed neural folds in fixed specimens to reveal the wound site were unsuccessful. The collapse of the neural folds was prevented by the addition of 2-5 x 10~3 M-calcium chloride; in such embryos wounds were healing normally. In an attempt to prevent, without replacement of calcium, the neural folds collapsing and thus obscuring the wound, the more specific chelator of calcium, EGTA, was employed and slightly older embryos in which the neural folds had fused were used (Fig. 13). The results of these experiments are summarized in Table 2. In embryos in the lower concentrations of EGTA, 0-5 x 10~3 or 10~ 3 M, wounds were healing normally and no tissue dissociation was observed. In the higher concentrations of EGTA, 2-5 x 10~3 or 3 x 10~ 3 M, although the embryos showed some dissociation, the wounds appeared to be closing (Fig. 14). Thus, unlike with amphibian embryos, divalent cation chelators did not appear to prevent wound closure in rat embryos. 3

DISCUSSION

The present results show that wound healing in rat early embryos, like that in amphibian and avian embryos (England & Cowper, 1977; Stanisstreet et al. 1980) is rapid; even quite large wounds may close within 30min. The scanning electron microscopical observations also suggest that wound healing is accompanied by similar changes in cell shape in all three types of embryo, although the changes in cell shape are less pronounced in mammalian embryos. Initially the wound gapes and the cells peripheral to the wound bulge to give a cobble-stoned appearance. Gaping of incisions made in the ectoderm of amphibian embryos has been considered to be the result of the release of a pre-existing lateral tension in the ectoderm (Lewis, 1947; Jacobson, 1970; Karfunkel, 1974). Both gaping of the wound and bulging of the cells were less pronounced in rat embryos than in amphibian embryos, suggesting that the tissue of mammalian embryos is under less tension than that of amphibian embryos. Following this initial reaction in amphibian and chick embryos there is a narrowing of the wound which is accompanied by changes in the shapes of the cells at the ends of the wound to become elongated radial to the wound (Stanisstreet etal. 1980). Elongated cells were also observed in the present experiments with rat embryos, although less frequently than in amphibian embryos. Such observations are compatible with the suggestion that wound healing is effected, at least in part, by coordinated changes in the shapes of cells at the wound margin. Mammalian embryos require serum for normal development in culture. The process of wound closure, however, continues in embryos cultured in saline,

116


suggesting that the macromolecular components of serum are not essential to the cellular mechanisms involved. In non-mammalian embryos an indication of the mechanisms involved has been gained from studies of the effects of inhibitors on wound healing. In amphibian embryos, for example, wound closure is inhibited by 1-25 /ig/ml cytochalasin B, suggesting that microfilaments are essential to this process (Stanisstreet & Panayi, 1980). The present results suggest that the cells of mammalian embryos are more sensitive to cytochalasin B than are those of amphibian embryos; a concentration of 1/ig/ml cytochalasin B causes dissociation of rat embryos whereas 5 /ig/ml are required to dissociate amphibian embryos. Thus it was not possible to dissect the inhibition of wound healing from cell dissociation; in rat embryos wound healing is inhibited by cytochalasin B only at concentrations which also cause cell dissociation. Calcium ions are important to a number of morphogenetic movements which are effected by changes in the shapes of cells (Stanisstreet & Jumah, 1983). For example drugs which prevent or perturb calcium fluxes inhibit neurulation in amphibian (Moran, 1976) and mammalian (O'Shea, 1982) embryos. In amphibian embryos wound healing too requires calcium ions since conditions which effectively remove free calcium ions from the culture medium, or addition of drugs which block calcium channels inhibit both wound healing and the concomitant changes in cell shape (Stanisstreet, 1982). The present results indicate that removal of divalent cations from the culture medium causes collapse of the neural folds. Thus these ions are required for the maintenance of the elevation of the folds. Removal of divalent cations, however, does not appear to inhibit wound closure. Wound closure in adult and later embryonic systems appears to be accomplished by mechanisms other than changes in cell shape, such as cell proliferation or cell migration. In the epithelium of mouse lens, for example, cell proliferation appears to be the major method of repair (Rafferty & Smith, 1976). In contrast, closure of the wounds in the cornea of avian late embryos (Takeuchi, 1975) and in the epidermis of amphibian tadpoles (Derby, 1978) is accompanied by active ingrowth of cells at the wound edge. In the case of tadpole epidermis, the cells form lamellipodia which spread over the underlying cells (Radice, 1977), but neither lamellipodia nor filopodia were seen in the present experiments. Early morphogenetic movements, such as gastrulation and neurulation, are accompanied and probably effected by changes in the shapes of cells. The morphological features of wound healing in early embryos are more akin to those seen during morphogenetic movements than to those seen in wound healing in later embryos and adults. Thus it is likely that the cellular mechanisms utilised for wound closure in early embryos are the same as those normally employed for morphogenesis, rather than specialized mechanisms. We wish to thank Mr C. J. Veltkamp and Mr B. Lewis for their expert help with the scanning electron microscopy and photography, Mrs E. A. Sheehan for helpful advice and Miss A. Callaghan who prepared the manuscript.


111

REFERENCES (1978). Wound healing in tadpole tailfin pieces in vitro. J. exp. Zool. 205,277-284. E. M. (1969). Effects of transecting early rat embryos on axial movements and differentiation in culture. Ada Embryol. exp. 2, 157-167. DEUCHAR, E. M. (1971). The mechanisms of axial rotation in the rat embryo: an experimental study in vitro. J. Embryol. exp. Morph. 25, 189-201. DEUCHAR, E. M. (1975). The course of rotation in rat embryos after bisection of the axis. Ada. Embryol. exp. 3, 265-271. ENGLAND, M. A. & COWPER, S. V. (1977). Wound healing in the early chick embryo studied by scanning electron microscopy. Anat. Embryol. 152, 1-14. JACOBSON, C. D. (1970). Experiment on /3-mercaptoethanol as an inhibitor of neurulation movements in amphibian larvae. J. Embryol. exp. Morph. 23, 463-471. KARFUNKEL, P. (1974). The mechanisms of neural tube formation. Int. Rev. Cytol. 38, 245-271. KARNOVSKY, M. J. (1965). A formaldehyde/glutaraldehyde fixative of high osmolarity for use in electron microscopy. /. Cell Biol. 27, 137-138A. LEWIS, W. H. (1947). Mechanics of invagination. Anat. Rec. 97, 139-156. MORAN, D. J. (1976). A scanning electron microscopical andflamespectrometry study of the role of Ca ++ in amphibian neurulation using papaverme inhibition and ionophore induction of morphogenetic movement. /. exp. Zool. 198, 409-416. NEW, D. A. T. (1978). Whole embryo culture and the study of mammalian embryos during organogenesis. Biol. Rev. 53, 81-122. NEW, D. A. T. & COPPOLA, P. T. (1970). Effects of different oxygen concentrations on development of rat embryos in culture. /. Reprod. Fert. 21, 109-118. NEW, D. A. T., COPPOLA, P. T. & COCKROFT, D. L. (1976). Improved development of headfold rat embryos in culture resulting from low oxygen and modifications of culture serum. /. Reprod. Fert. 48, 219-222. NEW, D. A. T., COPPOLA, P. T. & TERRY, S. (1973). Culture of explanted rat embryos in rotating tubes. /. Reprod. Fert. 35, 135-138. O'SHEA, K. S. (1982). Calcium and neural tube closure defects: an in vitro study. Birth Defeds 18, 95-106. RADICE, G. P. (1977). Active and passive cellular movements during wound closure in vivo. J. Cell Biol. 75, 441a. RAFFERTY, N. S. & SMITH, R. (1976). Analysis of cell populations of normal and injured mouse lens epithelium. Anat. Rec. 186, 105-114. STANISSTREET, M. (1982). Calcium and wound healing in Xenopus early embryos. J. Embryol. exp. Morph. 67, 195-205. STANISSTREET, M. & JUMAH, H. (1983). Calcium, microfilaments and morphogenesis. Life Sciences 33, 1433-1441. STANISSTREET, M. & PANAYI, M. (1980). Effects of colchicine, cytochalasin-B and papaverine on wound healing in Xenopus early embryos. Experientia 36, 1110-1111. STANISSTREET, M., WAKELY, J. & ENGLAND, M. A. (1980). Scanning electron microscopy of wound healing in Xenopus and chicken embryos. /. Embryol. exp. Morph. 59, 341-353. TAKEUCHI, S. (1976). Wound healing in the cornea of the chick embryo. III. The influence of pore size of millipore filters on the migration of isolated epithelial sheets in culture. Devi Biol. 51, 49-62. DERBY, A. DEUCHAR,

(Accepted 8 May 1984)