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Studies on wound healing in the neuroepithelium of the chick embryo.

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THE ANATOMICAL RECORD 233:291-300 (1992)
Studies on Wound Healing in the Neuroepithelium of the
Chick Embryo
Department of Anatomy, University of Ghana Medical School, Accra, Ghana (A.L.),and
Department of Anatomy, The Medical School, University of Leicester,
Leicester, U.K. (M.A.E.)
Wound healing has been studied by light microscopy, SEM, and
TEM in the neuroepithelium of the early neurula (stages 6 and 8) and advanced
neurula (stages 10 and 12) chick embryos. Healing involves two major events: (1)
apposition of the wound edges and (2) restitution of the neuroepithelium at the
wound site (i.e., restoration of the epithelial integrity of neuroepithelium). Apposition of the wound edges occurs within the first 15 minutes of re-incubation and
involves the entire length of the wound. The main event during restoration is a
change in the shapes of the rounded cells to elongated forms (i.e., spindle, wedge,
and inverted wedge shapes). Wounds of younger embryos heal faster than those of
older ones. o 1992 Wiley-Liss, Inc
Wound healing is a series of processes that lead to
the restoration of tissue integrity. These processes
have been investigated by light microscopy, transmission, and scanning electron microscopy and also by immunofluorescence and cytohistochemical techniques in
a number of embryonic and adult epithelia (Croft and
Tarin, 1970; Pfister, 1975; Takeuchi, 1976; England
and Cowper, 1977; Stanisstreet and Panayi, 1980;
Stanisstreet et al., 1980, 1985).
In studies of embryonic systems, two layers that
have been investigated in detail are the ectoderm,
which will later form the neuroectoderm and surface
epithelium of the embryo, and the endoderm, which
contributes to the gut. In the chick and amphibian embryos studied, early stages were used, i.e., stages 3-5
(Hamburger and Hamilton, 1951) in the chick and 1415 (Nieuwkoop and Faber, 1956) in Xenopus laeuis. In
the early neurula stages, these embryonic layers provided a simple model for studying the process of wound
healing (England and Cowper, 1977; Mareel and Vakaet, 1977; Stanisstreet et al., 1980). In these simple
epithelia, the primary mode of healing for the endoderm layer is cell rearrangement and migration. A
similar method of healing occurs in a variety of adult
systems including the skin and cornea (Gabbiani et al.,
1978; Pang et al., 1978). The ectodermal layer has a
more complicated structure than the endodermal layer,
as the cells are more elongated and have lots of extracellular spaces. Wounds made in this layer are closed
by changes in cell shape in both chick and amphibian
embryos. Additionally, some cell proliferation is apparent in the avian embryo. Increased mitosis does not
appear to play a crucial role in wound healing in the
Xenopus, as embryos treated with colchicine and
wounded still possess the ability to heal (Stanisstreet
and Panayi, 1980).
Wound healing in the neuroepithelium has received
relatively little attention. As naturally occurring neural tube defects are believed to be the result of failure
of the initial fusion of the neural folds or of a re0 1992 WILEY-LISS, INC
opening of the neural tube later in development, the
ability of the neural tube t o heal is of great interest.
Further, as the neuroepithelium is not fully differentiated in the early embryo, it is likely that the processes
involved in both neural tube formation and wound
healing may be directed by similar cellular behaviours.
Neural tube wound healing may therefore offer a useful model system to study aspects of neurulation that
are still not fully understood. Clark and Scothorne
(1988,1990) report that in chick embryos in which the
closed neural tube roof plate is experimentally incised,
there is a sharp decline in healing capacity between
stages 14 and 15. In younger chick embryos (stages 12
and 13) similar wounds in the roof plate heal completely. Even when treated in ovo with Streptomyces
hyaluronidase immediately after incision of the roof
plate, healing occurs completely (Clark, 1987) in this
age group. Some early studies (Waddington and Cohen,
1936; Watterson and Fowler, 1953; Kallen, 1955) reported that wounded neural tubes are capable of healing at somite stages but not a t presomite stages. Others
(Spratt, 1940; Yntema and Hammond, 1945, Wenger,
1950; Birge and Hilleman, 1953) reported that some
healing can occur a t neural plate stages, but none at
early somite stages.
The present study re-examines the exact process of
neuroepithelial wound healing, not only as a sequence
of cellular stages in time but also as a means for examining normal neural tube formation. This work incorporates studies of early neurula stages (6 and 8) and
advanced neurula stages (10 and 12) in the chick embryo as studied by light microscopy, scanning electron
microscopy (SEM), and transmission electron microscopy (TEM).
Received July 2, 1991; accepted October 29, 1991.
Address reprint requests to Dr. Aaron Lawson, Department of
Anatomy, University of Ghana Medical School, P.O. Box 4236, Accra,
Ghana, West Africa.
Wound Healing Studies
finally examined with a JEOL lOOCX transmission
electron microscope.
Fertilised eggs from White Leghorn hens were
incubated at 38°C to obtain embryos at stages 6, 8, 10,
and 12 (Hamburger and Hamilton, 1951). These were
mounted by New Culture (New, 1955) and then
prepared for wounding as follows. The well, formed by
the glass ring, was flooded with saline and the cranial
edge of the blastoderm was carefully detached from
the vitelline membrane using jeweller’s forceps. The
detached part was folded onto the caudal half of the
blastoderm to expose the neural plate, groove, or tube.
Special care was taken to avoid damage to the
underlying vitelline membrane. A single, straight,
longitudinal, or transverse wound, approximately
0.2-0.5 mm long, was made on one side of the neural
fold or tube in the midbrain region with a cactus
needle (England, 1981). For stage 6 and 8 embryos in
which neural folds were present, the rostra1 end of the
notochord served as a guide t o the midbrain region and
wounds were made in the neural folds about halfway
between the region of the notochord and the edge of
the neural fold. This involved the full thickness of the
neuroepithelium (not mesoderm and endoderm as
well). In contrast, in stages 10 and 12 embryos in
which the neural tube has formed in the midbrain
region, the wounds were made through the surface
ectoderm into the neural tube. The folded part of the
blastoderm was then reflected back onto the vitelline
membrane. Control embryos were similarly treated,
but their neural folds or tubes were left intact.
The wounded embryos were either fixed immediately
in half-strength Karnovsky’s (1965) fixative or re-incubated for 15 minutes, 1 hour, 2 hours, or 5 hours
(some stage 10 and 12 embryos were re-incubated for 8
hours) before fixation. Following fixation for 2-24
hours, they were washed in 0.2 M sodium cacodylate
buffer (pH 7.4) (Plumel, 1948) for an equal period of
time, postfixed in 2% cacodylate-buffered osmium
tetroxide, and dehydrated in graded ascending series of
ethanouwater up to 100%ethanol. All microdissection
to expose the neural tube surfaces of control or experimental embryos was done in 70% ethanol.
Specimens for light microscopy, which consisted of 50
embryos for each stage, were embedded in araldite and
sectioned serially a t 2 pm along the length of the
wound. They were then stained with 1%toluidine blue
in 1% borax. Those for scanning electron microscopy,
which were of the same number for each stage, were
transferred from 100%ethanol to 100% acetone, critical point dried by acetone replacement with liquid carbon dioxide, mounted on stubs, and coated with 20 nm
of gold. They were finally examined with International
Scientific Instruments IS1 60 and DS 130 scanning
electron microscopes at 15kv. Forty stage 6 embryos
were wounded and processed for transmission electron
microscopy. They were processed like those for light
microscopy but were fixed with half-strength Karnovsky’s fixative to which had been added l%tannic
acid to prevent the leakage of glycosaminoglycans
(GAGS)from the extracellular matrix. Following postfixation and dehydration, they were embedded in epon.
Ultrathin sections were cut with a diamond knife and
stained with uranyl acetate and lead citrate. They were
Morphometric Analysis
Slides were selected randomly from a collection of
serial sections of wounds of four stage 10 embryos for
each of the following re-incubation periods: 0 minute,
15 minutes, 1 hour, 2 hours, or 5 hours. They were
coded and the experimenter conducting the analysis
was unaware of the significance of the codes. For each
embryo and for each re-incubation period, four sections
were randomly selected and analyzed. Rounded cells
were counted within a 1 mm2 area on each side of the
wound using an eye piece graticule a t a total magnification of 400. The criterion used for selecting rounded
cells was as follows: the presence of a rounded nucleus
with a well-defined nuclear membrane and nucleolus.
By this method of selection, rounded cells undergoing
mitosis were avoided. Other cells within the area (spindle, wedge, and inverted wedge shape; Schoenwolf and
Franks, 1984) were also counted in order to obtain the
total number of cells within the area. The numerical
density of rounded cells for each treatment group Ke.,
number of rounded cells per total number of cells
counted) was calculated as well as the mean density
and standard error of the mean for each time of reincubation. A one-factor analysis of variance (ANOVA)
was done using Scheffe F-test at a 95%significance.
The rate of healing varied from one stage to the next
and even among embryos of the same stage. The sequence of events during healing, however, remained
the same. Furthermore, within certain limitations the
size of the wound did not affect the rate of healing.
Wound healing did not alter the pattern of neurulation
in the wounded embryos probably because of the rapidity of healing, but the rate at which the latter occurred
was slightly slower in some wounded embryos of
younger stages than in control embryos of the same
stages. The detached anterior edge of the blastoderm,
when reflected back onto the vitelline membrane,
healed within an hour of re-incubation.
Wound Healing Studies
Early neurulae-stages 6 and 8
Control. In control embryos, the neural folds formed
a thickened pseudostratified columnar epithelium,
comprising closely packed neuroepithelial cells. These
were elongated apicobasally. The TEM appearance of
an unwounded neuroepithelium was similar to that de-
basal lamina
cell protrusion
cell debris
elongated cell
extracellular material
lateral side of neural fold
neural fold lumen
medial side of neural fold
rounded cell
Fig. 1. A freshly made stage 6 wound (arrows)in transverse section.
x 100.
Fig.2. SEM of the apical surface of a freshly made stage 6 wound. x
Fig.3.(a)TEM of the edge of a freshly made stage 6 wound. x 1900.
(b)Higher magnification of the rounded cell in Figure 3. Note the
well-defined nuclear membrane and the intense tannic acid staining
of the nuclear envelope. x 14,000.
scribed by Schoenwolf and Franks (1984). Briefly, they
reported the presence of wedge-shape cells in the supranotochordal region with their nuclei close to the
basal lamina. In other regions, spindle-shape cells were
Zero hour (fresh wounds). A freshly made wound involved the full thickness of the right neural fold and a
gap was present between the wound edges (Fig. 1). The
wound appeared slit-like or gaped very slightly when
viewed from the basal surface of the neural folds by
SEM. The basal lamina appeared as a cut sheet at its
edges. On the apical surfaces, there were rounded cells
a t the wound edges and numerous blebs on the edge
cells (Fig. 2). Cell debris was also present. The cells a t
the wound edges appeared to have lost their normal
configuration in the neuroepithelial sheet and were
loose at the edge. Transmission electron microscopy
confirmed the presence of rounded cells in the immediate vicinity of the wound and elongated cells away
from the wound (Fig. 3a). The rounded cells had welldefined and rounded nuclei with a distinct nuclear
membrane and nucleolus. They also had blebs projecting into the wound (Fig. 3b). Extracellular materials
were present in the wound as short fibrils and tufts of
punctate deposits. The latter also coated the surfaces of
the rounded cells and blebs immediately bordering the
wound (Fig. 3b).
Fifteen minutes. The wound edges became apposed to
each other near the apical surface and a depression was
present near the basal side (Fig. 4). The full thickness
of the neuroepithelium in the wound was decreased
when compared with the opposite side. Whereas cells a t
the wound edges were rounded, those away from the
wound were elongated and showed a normal orientation. By SEM, it was observed that apposition of the
wound edges had occurred along the whole length of
the wound. The basal surface of the wound was curled
inwardly and showed that the depression observed by
LM was present along the length of the wound. This
made it difficult to identify the cut edges of the basal
lamina. The apical surface showed the wound plugged
with cell debris, rounded cells and blebs (Fig. 5a,b).
Some of the rounded cells were large and projected
from the surface. Also, some were wedge shaped with
their apices pointing in the direction of the wound. Cell
protrusions were seen by TEM at the apical surface.
Some of these comprised mainly cell debris with disintegrating cytoplasm and cell membrane, whereas oth-
Fig. 4. A stage 6 wound in transverse section, re-incubated for 15
minutes. Note triangular-shape depression (*) a t the basal end of the
wound. x 200.
Fig. 5. (a) SEM of the apical surface of a stage 6 wound re-incubated
for 15 minutes. Most of the cell debris has been removed to show
apposed wound edges. x 1000.(b)Higher magnification of the wound
in (a) showing wedge-shape cells (*). x 2500.
Fig. 6. TEM of the apical end of a stage 6 wound re-incubated for 15
minutes. Intercellular spaces are absent. x 2900.
Fig. 7. TEM of a stage 6 wound re-incubated for 15 minutes showing
a rounded cell adjacent to a depression (*). x 7200.
ers had contact with the wound and it appeared they
were being extruded from it (Fig. 6). The rounded cells
in the region where the wound edges were apposed to
each other and those adjacent to the depression had
rounded nuclei with clearly defined nuclear membranes and nucleoli and the cytoplasm around them
was uniform (Figs. 6,7). There was a marked reduction
of intercellular spaces around the rounded cells. Cells
undergoing mitosis were seen very rarely at the wound
Fig. 8. TEM of a stage 6 wound re-incubated for 15 minutes showing cell processes in contact with each other at
discrete points (thick arrow) within a depression. X 5800.
Fig. 9. TEM of a stage 6 wound re-incubated for 15 minutes showing interlocking cell processes (long arrows).
x 14,000.
Fig. 10. A stage 6 wound in transverse section re-incubated for 1 hour. Arrows indicate wound site. x 200.
site. The depression on the basal margin was filled
with extracellular materials in the form of short fibrils
and granular material. In some regions of the wound,
the edges of the depression ran almost parallel with
each other and in other regions, cell processes approached each other across the depression (Figs. 7, 8).
The cell processes were especially numerous in regions
devoid of a basal lamina. When those from the opposite
sides came into contact with each other, fusion of the
wound occurred across the depression. This process occurred first at discrete points with intervening oval
spaces filled with extracellular materials (Fig. 8). The
cell processes often seemed to interlock with each other
(Fig. 9).
One hour. It became more difficult to identify the
wound by light microscopy after 1 hour of re-incubation. Either a shallow depression present on its basal
surface or cell debris on its apical surface often helped
to identify it. In these sections, most of the cells in the
wound had become elongated and displayed an apicobasal orientation (Fig. 10). The basal surface of the
wound, by SEM, showed ones where the depression previously seen had disappeared (Fig. 11)and these were
interspersed among areas where the depression was
still present. In the former, there was a uniform basal
lamina over the basal surface of the wound implying
that healing was completed in these areas. It was normal in appearance when compared with that in control
embryos. At the apical surface, the wound edges were
more apposed to each other and appeared to be fusing
together. Blebs and cell debris were fewer in number
and rounded cells were sometimes seen in some areas
(Fig. 12a,b). There were other parts of the apical surface that showed complete healing with a restoration of
the normal architecture of the neuroepithelium. Sections examined by TEM showed that although a basal
lamina being formed over the wound comprised laminae densa and rara, these could not be clearly distinguished from each other.
Two hours. Two hours after re-incubation, the wound
examined by LM showed the features of that a t 1 hour,
but in addition to these the neural folds had become
Fig. 11. SEM of the basal surface of a partially healed stage 6
wound, re-incubated for 1 hour. Arrowheads indicate healed areas
and long arrows, unhealed areas. x 1000. Inset shows whole wound.
x 300.
Fig. 12. (a) SEM of the apical surface of a stage 6 wound re-incubated for 1 hour. x 1000. (b) shows a fracture through the wound.
Note the large rounded cells in the wound and the elongated cells
adjacent to them. x 4000.
Fig. 13. TEM of the basal end of a healed stage 6 wound re-incubated for 2 hours. Wound site (arrowheads). x 19000.
more elevated, thus deepening the neural groove further, and there was convergence of the folds in the
midbrain region. The shallow depression could sometimes be seen on the basal surface. Scanning electron
microscopy of the whole extent of the wound at this
time revealed parts of the basal surface that still had
the depression (i.e., parts that were not completely
healed). On the apical surface, however, all parts of the
wound had healed and the integrity of this surface of
the neuroepithelium had been restored. Transmission
electron microscopy also revealed that the elongated
cells in the wound had characteristics similar to those
away from the wound. Cell debris was scanty a t this
time and in some parts of the wound a very shallow
depression was present. A uniform basal lamina covered the basal surface of the wound and this was similar to that in adjacent parts of the neuroepithelium.
The lamina rara was interposed between the cell membrane and the lamina densa and extracellular materials were present outside the latter (Fig. 13).
Five hours. The wound healed completely within 5
hours of re-incubation, and no depression was seen on
its basal surface by SEM (Figs. 14, 15).
Advanced neurulae-stages 10 and 12
Wound healing. This followed a pattern similar to that
of the early neurula stages, but the rate of healing was
slower in the advanced embryos than in the younger
ones (Fig. 16a-d). Nevertheless, wounds in these embryos often healed by eight hours of re-incubation.
Statistical analysis
The bar chart (Fig. 17) illustrates the numerical densities of rounded cells in the wound per time of reincubation. By our criteria, no rounded cells were
present in the control unwounded neuroepithelium.
The densities of these cells increased sharply to about
72% immediately following injury (i.e., in a freshly
Fig. 14. SEM of the basal surface of a completely healed stage 6
wound. &-incubation for 5 hours. Arrowheads indicate wound site. x
Fig. 15. SEM of the apical surface of a healed stage 6 wound reincubated for 5 hours. x 1000.
made wound) and this level was maintained with some
variability by 15mins of re-incubation. By 1 hour the
density had significantly decreased to about 26%.Densities thereafter fluctuated insignificantly with respect
to that a t 1 hour, although levels were always significantly lower than that in the freshly made wound.
The present study demonstrates that wound healing
in the neuroepithelium of the chick embryo involves
two events. The first comprises apposition and fusion of
the wound edges, and this is followed by a second event
during which there is restitution of the neuroepithelium in the wound.
Our study shows that apposition of the wound edges
is: (1) a rapid process that occurs within the first 15
minutes following wounding, and (2) involves the
whole length of the wound simultaneously. Healing
therefore commences along the whole length of the
wound. The wound at this time is "plugged" by rounded
cells, and this results in a decrease in the thickness of
the neuroepithelium in the wound area. The depression
on the basal surface of the wound may be a reflection of
Fig. 16. LM of stage 10 wounds in transverse section. (a)Freshly made wound; (b)re-incubated for 15
minutes; ( c ) re-incubated for 1 hour; (d) re-incubated for 5 hours. x 150.
this. This pattern of healing clearly differs from that in
the endoderm or ectoderm of chick and Xenopus embryos where the wound edges are not apposed to each
other at the start of healing (England and Cowper,
1977;Stanisstreet et al., 1980).Wound healing in these
tissues, therefore, starts from the ends of the wound,
and the cells migrate t o close it. Adult epidermal
wounds similarly heal by cell migration (Odland and
Ross, 1968; Croft and Tarin, 1970; Krawczyk, 1971;
Winter, 1972; Repesh and Oberpriller, 1980). This
study also demonstrates that wound fusion immediately follows apposition of its edges. At the start of
healing when the wound edges first meet at its apical
end, the cell processes from the rounded cells become
interlocked with each other. This stage of the healing
process simulates a stage in the morphogenesis of the
neural tube when there is an apposition followed by a
fusion of the neural folds (Gouda, 1974; Bancroft and
Bellairs, 1975; Waterman, 1975: Santander and Cuadradro, 1976; Silver and Kerns, 1978). We suggest here,
as has been suggested for neurulation (Waterman,
1975, 19761, that the cell Processes might guide the
edges of the wound together for fusion to occur.
60 -
Time of healing
Fig. 17. A bar chart illustrating densities (mean + standard error)
of rounded cells per time of re-incubation. ANOVA: F (4, 15) = 43.5;
p = 0.0001. * rounded cells significantly different from 0 min (p <
Our study further demonstrates that restitution of
the neuroepithelium in the wound (i.e., restoration of
the full thickness of the neuroepithelium in the wound
area) involves a change in the shapes of the rounded
cells to elongated forms. We have shown that rounded
cells with markedly reduced intercellular spaces are
present a t the edges of freshly made wounds and also
during the initial stages of healing. Although these
cells were closely apposed to each other, no junctional
complexes were established a t this time as these could
hinder a change in their shapes (Stanisstreet et al.,
1980). Schoenwolf (1982) has described four classical
cell types that characterize a normal pseudostratified
neuroepithelium; spindle, wedge, inverted wedge, and
globular (rounded). The globular cells are rarely seen
and only when they are undergoing mitosis. Moreover,
they are only seen at the apical surface. Our study has
identified another variety of globular (rounded) cells
present during neuroepithelial wound healing. These
were evidently not undergoing mitosis as they possessed definite and rounded nuclei with well-defined
nuclear envelope. Further, these cells lacked mitotic
figures and they were present both near the apical and
basal sides of the wound.
The appearance of rounded cells during neuroepithelial wound healing, however, is only a transient phenomenon. Indeed this study reports a very high percentage (about 72% of the total cell population near the
wound) immediately following wounding. The density
remains around this level through the first 15 minutes
of healing, but by about 1 hour, there is a significant
decrease to about 26%. This latter event coincides with
the time of appearance of more elongated cells in the
wound with a concomitant re-establishment of intercellular spaces. It is at this time that the full thickness
of the neuroepithelium is established in the wound.
That the neurulating chick embryo is capable of undergoing changes in the shapes of the neuroepithelial cells
has been demonstrated during neural plate shaping
and bending in previous studies (Schoenwolf and
Franks, 1984), and it is, therefore, not surprising that
it utilizes one of the same mechanisms during wound
healing. The cytoskeleton has long been known to be
associated with changes that occur in cell shape in the
neuroepithelium and in other embryonic organ systems during morphogenesis. Previous experimental
studies that have used colchicine and cytochalasin B to
disrupt microtubules and microfilaments, respectively,
in the neuroepithelium have suggested that these cytoskeletal elements may direct the observed cell shape
changes during neurulation (Handel and Roth, 1971;
Karfunkel, 1971,1972; Fernandez et al., 1987; Schoenwolf and Powers, 1987; Schoenwolf et al., 1988). Studies are currently being undertaken to determine the
role microtubules and microfilaments play in wound
healing in the neuroepithelium.
Our study revealed a close association between extracellular materials (specifically Glycosaminoglycans-GAGS) and the healing process. Although this
may suggest their active involvement in healing, it
was not clear what precise role they played. However,
in view of their known ability to modulate cell shape
(Van Hoof et al., 1986), we suggest that they might act
in concert with the cytoskeleton to perform this function during neuroepithelial wound healing. Also, they
are likely t o provide part of the “building blocks” for a
new basal lamina over the basal surface of the wound.
Further experiments are needed to determine precisely
the role GAGS play in neuroepithelial wound healing
in the chick embryo.
It is intriguing that different parts of the neural tube
of the chick embryo display varied mechanisms of
wound healing. In contrast to our results, Clark and
Scothorne (1990) reported that cell migration was the
main mechanism for healing of the roof plates of spinal
cords of stages 12 to 18 chick embryos and also that
there was a 95% failure of healing at stages 17 and 18.
It is yet to be determined, however, whether the failure
of healing in the older age group is due purely to mechanical factors in the neural tube or to a loss of healing
capabilities (i.e., loss of regulation). If the latter situation is true, it will be interesting to find out whether the
loss of regulation is localized to the spinal cord alone or
generalized, involving the entire CNS, and also
whether there is a craniocaudal timing of onset.
We thank Darko Farms, Ghana, for donating eggs
for part of the study. We are grateful to Drs. Gary C.
Schoenwolf, C.N.B. Tagoe, and R.S.K. Apatu for their
useful comments on the manuscript. We are also grateful to Mr. G.L.C. McTurk for operating the IS1 60 and
DS 130 scanning electron microscopes. Dr. I-Li Chen
helped with the TEM. Miss M. Reeve did an excellent
and rapid job of typing the manuscript. A.L. was supported by the British Council and the Association of
Commonwealth Universities as a Medical Fellow while
at Leicester University, Great Britain. The DS 130
scanning electron microscope was obtained through a
Medical Research Council grant to M.A.E.
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