Effects of Temperature on Tadpole Hearts in Vitro
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/. Embryol. exp. Morph., Vol. 17, 1, pp. 147-159, February 1967 147 With 2 plates Printed in Great Britain Effects of temperature on tadpole hearts in vitro By ELSIE M. STEPHENSON1 From the School of Biological Sciences, University of New South Wales, Sydney INTRODUCTION The temperatures currently used for amphibian cell and tissue culture appear to range from 26 °C (Seto, 1964) to 18 °C (Shah, 1964). The experiments of N. G. Stephenson & Tomkins (1964), in which Pseudophryne tadpole humeri and femora transplanted to chick chorioallantoic membranes at 38 °C showed definite growth, suggested the possible advantage of culturing other amphibian tissues at temperatures above 26 °C. The present study has therefore been carried out in an attempt to establish more precisely the optimal and maximal conditions for amphibian cell and organ culture. Whole hearts of tadpoles of the Leptodactylid frog, Limnody- nastes peroni (Dumeril & Bibron) were cultured at a series of temperatures ranging from 5 to 37°C for a period of at least 1 week. Although the short-term behaviour of an isolated, adult frog's heart in Ringer's solution in relation to increased temperature is known (Mitchell, 1956), only incidental observations have been recorded with regard to temperature effects on rate of beat of cultured amphibian heart fragments (Johnson, 1915; Morosow, 1929) or embryonic heart rudiments (Stohr, 1924). By using whole tadpole hearts in culture it has been possible to observe the effects of different temperatures on organ function and maintenance, as indicated by heart beat, as well as on cell outgrowth. MATERIAL AND METHODS The tadpoles were identified by their labial teeth arrangement, lateral-line patterns and other features recorded by Phillis (1958), but some specimens were kept alive until metamorphosis as a further taxonomic check. Four main experimental series of hearts were cultured (Table 1), each series containing thirty-six hearts. Additional hearts, including those of an incomplete series C, were grown for special purposes such as transfers from one temperature to another. All tadpoles except those of series E came from eggs collected from a single locality at the same time. Each tadpole was rinsed in tap water and immobilized in MS 222. The rate 1 Author's address: School of Biological Sciences, The University of New South Wales, P.P. Box 1, Kensington, New South Wales, Australia. 148 E. M. STEPHENSON of heart beat per minute in vivo of every fourth or fifth animal was recorded. Each tadpole was then washed in detergent (7 X) and immersed briefly in 95 % alcohol before being passed through two changes of Pannett and Compton's saline containing antibiotics. For series A and B, only penicillin sodium G (40 i.u./ml) and streptomycin sulphate (50/*g/ml) were used. As one culture in series A and two in series B later showed fungal contamination, mycostatin (20/tg/ml) was added to all subsequent series and effectively prevented any further infection. Table 1. Identification of main experimental series Mean room temperature during ex- Mean heart perimental Approximate age rates/min Experimental temperatures periods Series of tadpoles in vivo used (°C) (°C) A 3 weeks 97 37, 30, 25, 20, 15, 5 23 B 7 weeks 96 As above, but 8° sub- 23 stituted for 5° D 4^- months 84 As for series B 19 E 18-19 days 105 As for series A 23 Each animal was handled individually with a perforated metal spoon which prevented breakage of skin surface. Removal of the hearts was carried out in saline under a binocular dissecting microscope, using iridectomy scissors and jeweller's forceps. Each heart was later checked for signs of damage and any adhering tissue was removed. When all hearts for a complete series had been prepared, clots of equal quantities of cockerel plasma and chick embryo extract (50 % in Hanks saline) were made on small coverslips adhering to larger ones, according to the Maximow technique (see Paul, 1960). While each clot was still semi-liquid, a heart was dropped lightly on to its surface so that it was held in position when the clot set. A drop of liquid medium was added. When sealed to cavity slides the coverslips were inverted to produce lying-drop cultures. The liquid medium consisted of Medium 199 (single strength), 7 parts; Hanks BSS, 7 parts; cockerel serum, 4 parts; chick embryo extract (50 % in Hanks BSS), 1 part. Antibiotics were added in the same concentrations as for the washing saline. The medium was adjusted to a pH of 7-4. The cultures in each series were allocated at random in batches of six to each of six different temperatures (Table 1). The hearts in each batch were numbered in sequence and their rates of beat were recorded before transfer to the incubation temperatures took place. The incubators used varied in type but were all thermostatically controlled and fitted with accurate thermometers. In general, Tadpole hearts in vitro 149 the amount of temperature fluctuation was negligible. The 15 °C incubator used for series A and B was found to be faulty and was replaced for all later experiments. Although most cultures were maintained for at least 9-10 days, the main experimental period was taken as 1 week. The cultures were usually removed daily from their incubators and examined for a very brief period at room temperature (Table 1). Recording of heart-beat rates was carried out with maximum speed and always preceded any examination and estimation of cell outgrowth. Counting of beats followed a standardized routine which made use of a stop-watch and telecounter. The process was simplified by the fact that, although variation in intensity of beat occurred, relatively little irregularity of pulsation or asynchrony of different regions were evident. So-called subculturing took place usually every second day. This was un- necessarily frequent for hearts at lower temperatures but was standardized to meet the probable demands of cultures in the upper part of the temperature range. The small coverslips and cultures were washed in saline and were then provided with fresh liquid medium and sealed in a new culture chamber. The clots were not usually added to or changed but in the rare cases where a heart became loose a very small drop of fresh plasma was provided. All hearts were washed individually in watch-glasses to prevent any possible cross transfer of dissolved substances or of contaminants. Heart-beat records were made after each subculture. When harvested, the cultures were either fixed in formol-saline and stained in Mallory's aqueous haematoxylin, or fixed in absolute methanol and stained in Jenner-Giemsa (see Paul, 1960). In some cases, treatment with silver nitrate (1:400) preceded fixation. Selected cultures were treated with colchicine and hypotonic saline for karyotype analysis (Robinson & Stephenson, 1967). All photographs were taken with a Zeiss photomicroscope. For series A-D, estimations of outgrowth were made visually in relation to a standard microscope field. For series E, drawings of all cultures were made at regular intervals, using a Zeiss projection drawing apparatus and paper of standard weight. Mean weights of explants and outgrowths at each temperature were later obtained (see Paul, 1960). RESULTS (1) Maintenance of heart beat In general, allowing for occasional temporary cessation, total maintenance of heart beat occurred at all temperatures from 30 to 5 °C inclusive. The only exceptions to this occurred in the first two series A and B in which at 15 and 20 °C not more than one-third of the hearts in culture were beating at the end of a week. The 15 °C results were apparently caused by an incubator fault but those at 20 °C, though obviously anomalous, are less easy to explain. However, 150 E. M. STEPHENSON in all subsequent series and in additional cultures kept at 15 and 20 °C, 100 % maintenance of beat was recorded. At 37 °C all hearts continued to beat for at least 1-2 days. In series B, D and E all hearts had stopped permanently by the fifth day in culture but in series A three out of six hearts were still beating on the sixth day. Only one long-term observation was made on heart-beat maintenance. This is discussed under section 5 below. The immediate effects of subculturing on heart-beat rate were unpredictable. In most cases the beat was maintained but occasionally it stopped for a tem- porary period. At times, where a beat had stopped before subculturing took place, it began again immediately afterwards. Series D 1 2 3 4 5 6 7 Days in culture Text-fig. 1. Series D. Graphical illustration of the influence of different temperatures on rate of heart beat during 1 week in culture. Except at 37 °C, where figures in parentheses indicate the number of hearts still beating, each point indicates the mean rate/minute for six hearts. Means have been calculated only from hearts actually beating. (2) Rate of heart beat Comparison of mean rates of heart beat per minute in vivo (Table 1) with recordings made before the extirpated hearts in culture chambers were trans- ferred to their incubation temperatures showed that a very marked drop in rate occurred in each series. The factors causing this must have included general operational effects and reactions to a new environmental medium. Throughout the period of culture, mean heart-beat rates in all series and at all temperatures remained well below the mean rate in vivo, except in series E at 30 °C, where by the end of a week the hearts had almost regained their in vivo average. In order to compare information relating to heart-beat maintenance, heart- beat rate and cell outgrowth, it was necessary to use the same hearts throughout the full term of any one experimental series.