/. Embryol. exp. Morph. Vol. 39, pp. 261-266, 1977
Printed in Great Britain
261
SHORT PAPERS
A technique for introducing localized long-lasting
implants in the chick embryo
By MARIETA B. HEATON 1
From the Department ofNeuroscience,
University of Florida College of Medicine
SUMMARY
A technique is described for making chronic, localized implants of drug-impregnated
Silastic in early chick embryos. This silicone material has been shown to gradually and
uniformly release its impregnated contents under such conditions. Following implantation
of pieces of Silastic treated with the metaphase-blocking drugs colchicine and vinblastine into
the wing-buds of 4|-5 day chick embryos, 53-75% survived 4-5 days, at which time they
were sacrificed. Non-impregnated (control) Silastic was also implanted in a group of embryos.
In this group, 90% survived 4-5 days. The antimitotic effects of the drugs were very well
localized to implanted wings, which were reduced in size by approximately 55 % at the time
of sacrifice (day 8-9 of incubation). In control embryos, the implanted wings were normal in
size and general morphology. It is suggested that the technique could have numerous applications for the study of neuroembryology or other aspects of embryogenesis.
INTRODUCTION
The use of the chick embryo as a developmental model has been widespread
for many years. Much of the popularity of this preparation can be attributed
to its ready accessibility during the entirety of the embryonic period and to
the numerous techniques that have been perfected for making a variety of
manipulations during this period. One persistent problem, particularly in studies
of nervous system and behavioral development, however, has been the difficulty
in making chronic manipulations. It is often desirable, for example, to study
the effects of the prolonged, constant presence of certain drugs on behavioral
and/or nervous system development. Two techniques are currently available
to make such chronic manipulations: the infusion pumping method (e.g.
Drachman & Coulombre, 1962) or simply repeated injections of the agent of
interest, across days of incubation. Although these methods are effective, they
have a number of disadvantages.
Infusion pumping, for example, can usually only be applied to a few eggs at
a time and must be somehow installed into or used in conjunction with an
incubator or environmentally controlled chamber, a rather troublesome
1
Authors address: Department ofNeuroscience, University of Florida College of Medicine,
Gainesville, Florida 32610, U.S.A.
262
M. B. HEATON
procedure. The repeated injections technique has additional disadvantages: the
integrity of the egg must be repeatedly disturbed, probably lowering the survival
rate, and more importantly, the level of the agent administered is inconstant,
peaking and troughing cyclically, with the injections.
A further limitation of these traditional methods for introducing foreign
substances into eggs during development is that they allow only for systemic
applications, usually accomplished by (1) injecting the substances through an
opening in the egg onto the inner shell or vascularized chorioallantoic membrane
(e.g. Oppenheim & Reitzel, 1975); (2) injecting directly into the embryonic
circulation via microcatheters inserted into the chorioallantoic vessels (e.g.
Drachman & Coulombre, 1962); or (3) injecting into the amnionic fluid or the
yolk-sac (e.g. Kuo, 1939). Multiple injections directly into the embryo would
probably prove fatal within a short time and a chronic infusion into the embryo
proper would be difficult to maintain. Even if technically possible, either of
these methods would produce a widely diffuse application of the agent, and
while such widespread application might sometimes be desirable, for some
experimental applications, localization of the drug would be necessary.
The following description is of a technique that has proved useful in which
discretely localized, chronic application of drugs may be made in chick embryos,
throughout incubation, without repeated disruptions of the embryonic environment. This is made possible by localized implantation of small pieces of a
silicone rubber compound, Silastic. This substance, as has previously been
demonstrated (e.g. Gottlieb, Gerall & Thiel, 1974; Cull, 1975; Purves &Nja,
1976) when impregnated with a given agent, slowly and uniformly releases the
agent across time, thus providing a constant and relatively stable presence.
MATERIALS AND METHODS
Subjects
White Leghorn chicken eggs (N = 89) were incubated in forced draft
incubators at 37-5 °C, relative humidity 60-70 %. The operations were
performed at Hamburger & Hamilton (1951) stages 24-26 (4f-5 days of
incubation).
Procedure
Impregnation of the Silastic. The Silastic (Dow Corning) was impregnated
with the metaphase-blocking drugs, colchicine and vinblastine sulfate, in
concentrations of 0-1 %, 0-05 % and 0-025 %. These drugs were chosen for two
reasons: first, they are highly toxic to developing embryos when administered
systemically, usually causing death within a short time (Oppenheim, Heaton &
Chu-Wang, 1977). Thus, the survival following implantation of these agents
would serve as something of an index for the restriction of the effect to
a localized area. Secondly, the well known antimitotic effects of these drugs
Localized long-lasting implants in chick embryo
263
Fig. 1. Schematic representation of Silastic implantation procedure, in
5-day chick embryo.
would provide further evidence for their effectiveness as released, and the
localization of that effect.
The general impregnation procedure was as follows: the appropriate amount
of the drug (depending on the desired concentrations) was weighed on an
analytical balance and added to 1-0 ml Silastic in a 40 mm watch glass (0-1 % =
1 mg, 0-05 % = 0-5 mg, 0-025 % = 0-25 mg). The drug was thoroughly mixed
into the uncured Silastic using a small spatula. After 5-10 minutes of mixing,
5 jil of catalyst was added and thoroughly mixed. The Silastic was then allowed
to cure for 24 h.
Operative technique. The eggs were removed from the incubator and the
position of the embryos detected by transillumination and marked. The egg
was swabbed with 70 % alcohol and a small hole was made in the shell overlying
the embryo. Sterilized scissors and fine forceps were then used to made a small
cut in the chorioallantoic and amnionic membranes so as to expose the right
wing-bud.
A longitudinal slit was then made in the wing-bud, using a modification of
the vibrating needle of Wenger (1968). A small rectangular piece of drugimpregnated or non-impregnated (control) Silastic was then cut, approximately
1 mm long, 0-5 mm wide and 0-2 mm thick. This piece was placed in the egg
with forceps and manipulated into the slit with a fine probe (Fig. 1). The eggs
were sealed with parafilm and returned to the incubator. They were candled
daily for mortality and were sacrificed after 4-5 days to assess the effects of
the implantation.
RESULTS
Viability. As Table 1 shows, a total of 53-75 % of the implanted embryos
survived 4-5 days until sacrificed, or until they reached an age of 9-10 days of
264
M. B. HEATON
Table 1. Percent of embryos receiving drug-impregnated and control Silastic
implants which survived until sacrificed (4 or 5 days after implantation)
Drug concentration i n implant
A
o-i%
Vinblastine
0-05%
0025%
50%
(7V = 23)*
50%
50%
CO /o
(iV=16)*
Colchicine
—
0%
—
(N= 10)*
Control
—
—
—
90%
(iV=10)*
* N represents total number of embryos per group.
Table 2. Mean length of experimental and control wings following
Silastic implantation*
Vinblastine, all concentrations (N= 39)
Colchicine, all concentrations (N= 40)
Control (N= 10)
Right
(Experimental)
wing (mm)
Left
(Control)
wing (mm)
Mean age at
time of
sacrifice (days)
4-59
5-67
9 04
8-44
9-37
8-90
9-36
900
8-89
* Length measured from tip of first joint.
incubation. This percentage was similar across groups, regardless of drug or
concentration. Most of the control embryos survived until sacrificed (90%).
Effects of the implants. A number of test embryos were examined at intervals
following the implantation. This examination revealed that the wing healed
around the Silastic very quickly, usually being totally healed and enclosing the
implant within 24 h of the operation. In Table 2, the mean elbow-tip wing
lengths for the three groups are given. In both the colchicine and vinblastine
groups, the wing in which the implant was placed was approximately 55 % the
size of the control wings, a result of the antimitotic effects of the drugs. In
embryos in which non-impregnated Silastic was implanted, the two wing
measurements were similar, indicating that the implant per se was not responsible
for the size differential in the experimental group.
Localization of the drug effect. The drug effect in the experimental embryos
was extremely well localized to the wings in which the implant was placed, with
the remainder of the embryos being morphologically normal (Fig. 2). This
localization was found in every case but one, from a total of 79 cases (experimental group).
Localized long-lasting implants in chick embryo
265
Fig. 2. (A) Experimental embryo S-34, sacrificed at 8i days of incubation, after
implantation of Silastic impregnated with 0025% vinblastine in right wing-bud at
A\ days. Note reduced size of right wing. (B) Control embryo S-39, sacrificed at 9
days of incubation, after implantation of non-impregnated Silastic in right wing-bud
at 4£ days. Right and left wings are of similar size. (C) Control embryo S-56,
sacrificed at 9 days of incubation. (D) Experimental embryo S-80, sacrificed at 9 days
of incubation, after implantation of Silastic impregnated with 005% colchicinein
right wing-bud at 4£ days. Note reduced size of right wing. All x 2-3.
DISCUSSION
The use of drug-impregnated Silastic has had considerable success in studies
in a variety of organisms, with applications ranging from induced blockage
of axoplasmic flow in peripheral nerves (e.g. Cull, 1975) to chronic hormonal
administration (e.g. Gottlieb et ah 1974). The present study has extended the
use of this material to the embryonic period, and has demonstrated the efficacy of
266
M. B. HEATON
Silasticas a vehicle for introducing localized implants during embryonic development. As the results indicate, these implants are tolerated extremely well, even
when the impregnating agents are as toxic as colchicine and vinblastine. And the
excellent survival in the embryos with control implants suggests that in using
less toxic agents, the developmental progress should be minimally affected.
The technique also offers a considerable degree of control over the extent of
the drug effect, as illustrated by the extremely well localized antimitotic effects.
There seems to be no particular limit either to the time of implantation during
embryonic development or to the site of implantation. We have placed implants
in embryos as late as midway through incubation with excellent results and
feel confident that still later successful implantation is possible. In addition to
limb-buds, different parts of the developing brain are quite amenable to
implantation. We have made such implantations, for example in the optic
tectum, and found results quite similar to those reported above, although
mortality was higher when the metaphase-blocking drugs were used.
The Silastic implantation technique holds particular advantages for the
student of the developing nervous system. Within this realm, the technique
enables one to alter some specific aspect of the embryonic milieu within
particular, well defined areas, so that the totality of the innervation to that
area, as it arises, will be similarly affected. This sort of control, of both present
and future conditions, would seem to have numerous applications in the study
of neuroembryology as well as in other aspects of embryogenesis.
This research was supported by NIMH grant MH27677. The author would like to thank
Dr J. B. Munson for introducing her to Silastic and Dr W. G. Luttge for providing the
Silastic for the experiment. Appreciation also goes to Dr L. P. Lanier and Ms M. P. Braithwaite for technical assistance.
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CULL,
(Received 20 August 1976)