A technique for introducing localized long-lasting implants in the chick embryo

Transcription

1 /. Embryol. exp. Morph. Vol. 39, pp , Printed in Great Britain 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.

2 262 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 %. The operations were performed at Hamburger & Hamilton (1951) stages (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 %. 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

3 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, % = 0-25 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 % of the implanted embryos survived 4-5 days until sacrificed, or until they reached an age of 9-10 days of

4 264 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) Vinblastine Colchicine Control o-i% 50% (iv=16)* Drug concentration in implant 0-05% 50% (N= 10)* * N represents total number of embryos per group. A 0025% CO /o (7V = 23)* 50% 0% 90% (iv=10)* Table 2. Mean length of experimental and control wings following Silastic implantation* Right Left Mean age at (Experimental) (Control) time of wing (mm) wing (mm) sacrifice (days) Vinblastine, all concentrations (N= 39) Colchicine, all concentrations (N= 40) Control (N= 10) * 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).

5 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

6 266 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 MH 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. REFERENCES CULL, R. E. (1975). Role of axonal transport in maintaining central synaptic connections. Expl Brain Res. 1A, DRACHMAN, D. B. & COULOMBRE, A. J. (1962). A method for continuous infusion of fluids into the chorioallantoic circulation of the chick embryo. Science, N.Y. 138, GOTTLIEB, H., GERALL, A. A. & THIEL, A. (1974). Receptivity in female hamsters following neonatal testosterone, testosterone propionate, and MER-25. Physiol. Behav. 12, HAMBURGER, V. & HAMILTON, H. L. (1951). A series of normal stages in the development of the chick. J. Morph. 88, Kuo, Z. Y. (1939). Studies in the physiology of the embryonic nervous system. I. Effect of curare on motor activity of the chick embryo. /. exp. Zool. 82, OPPENHEIM, R. W., HEATON, M. B. & CHU-WANG, I. W. (1977). Further observations on the retrograde transport mechanism in the chick embryo. Manuscript in preparation for submission to Brain Res. OPPENHEIM, R. W. & REITZEL, J. (1975). Ontogeny of behavioral sensitivity to strychnine in the chick embryo: Evidence for the early onset of CNS inhibition. Brain, Behav. Evol. 11, PURVES, D. & NJA, A. (1976). Effect of nerve growth factor on synaptic depression after axotomy. Nature, Lond. 260, WENGER, B. S. (1968). Construction and use of the vibrating needle for embryonic operations. Biosci. 18, (Received 20 August 1976)