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
M. SMEDLEY AND M. STANISSTREET
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
(<200jum)
Larger 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 (<200ium) wounds had started to close; cells at the
ends of the wounds appeared elongated radial to the wound and cells at the
lateral margins appeared to be curling under (Fig. 4). Larger (>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
SEM of wound healing in rat embryos
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
M. SMEDLEY AND M. STANISSTREET
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.
SEM of wound healing in rat embryos
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
M. SMEDLEY AND M. STANISSTREET
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.
SEM of wound healing in rat embryos
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)