Effects of the Grow Cultures Tolerant Methadone Th of Organ Prepared and Control Hydro Otypic from Rats Chloride on Cerebellar Methadone-

Abstract

WILLSON, N. J., J. F. SCHNEIDER, L. ROIzIN, J. F. FLEISS, W. RIVERS AND J. E. DEMARTINI: Effects of methadone hydrochloride on the growth of organotypic cerebellar cultures prepared from methadone-tolerant and control rats. J. Pharmacol. Exp. Then. 199: 368-374, 1976. Male and female Sprague-Dawley rats were given d!-methadone (5 mg/kg) for at least 3 months and then mated. The drug was continued throughout pregnancy and after delivery. The newly born pups were divided into two groups. One group was tested for in vivo methadone tolerance, while the animals in the other group were used to prepare organotypic cerebellar cultures. Various amounts of d!-methadone were added to the media of half of these cerebellum cultures. The effect of the drug in the medium was assessed by measuring explant outgrowth. Similar experiments were carried out with control animals. Statistical analysis of the data obtained in the in vivo portion of the experiment indicates that the pups of methadone-treated mothers tolerate methadone better than those of untreated mothers. The culture experiments revealed that the addition of methadone to the medium reduced explant outgrowth size and this was a dose-related effect. Also, there was significantly less outgrowth from explants prepared using pups of methadone-treated mothers as compared to the controls. There was no significant difference in the effect of methadone on the growth of cultures prepared from the methadone-tolerant and control animals. Methadone hydrochloride is a synthetic narcotic with multiple actions the most prominent of which involve the central nervous system and organs containing smooth muscle. Although the analgesic and sedative properties of narcotics usually receive most emphasis. cell culture Received for publication July 22, 1974. studies have shown that some are capable of retarding the growth of immature nervous system tissues (Ghadanian, 1969). Methadone crosses the placental barrier and accumulates in measurable amounts in fetal tissues, including brain (Peters et a!., 1972). Although abnormalities have been reported in the offspring of methadone-treated animals (Buchenauer et a!., 1974), they are not of the magnitude that would be expected if in vivo drug activity paralleled 1976 METHADONE EFFECT ON CNS CULTURE GROWTH 369 that in vitro. There are a number of plausible explanations for the greater in vitro toxicity of the drug, including the possibility that individual fetal cells develop tolerance to the growth retardant action of methadone. Tolerance may develop following the administration of a wide variety of agents and it has been shown that tolerance to narcotics can develop in utero (Johannesson and Becker, 1972). Tolerance sometimes appears after a single exposure but is generally most obvious clinically when an agent has been administered chronically. Its manifestations are quite vanable as are the means by which it is measured (Cochin, 1970). Some investigators (Richter and Goldstein, 1970; Kalant et at., 1971; Misra et at., 1973) have suggested that tolerance may be due to decreased sensitivity of individual cells because of prolonged occupation of specific cell receptor sites by agents which blockade the receptors or modify them biochemically. Others have postulated that altered drug metabolism (Sung et at., 1953; Olsen, 1972), particularly in the liver (Sung and Was’, 1950), could be a factor. Another interesting possibility is that neutralizing antibodies (Kornetsky and Cochin, 1964; Liu and Adler, 1973) play a role. The present study was designed to investigate the possibility that individual central nervous cells develop tolerance to the growth-inhibiting action of methadone in utero. To do this, cultured cerebellar cells from methadone-tolerant and control pups were maintained in a methadone-containing culture medium. The pharmacologic activity of the drug was assessed by measuring its influence on explant outgrowth. The use of organotypic cell cultures eliminates many of the systemic factors which complicate in vivo studies. Methods and Materials Young (100 g) male and female Sprague-Dawley rats were given daily subcutaneous injections of methadone hydrochloride (Dolophine hydrochloride, Eli Lilly and Company, Indianapolis, Ind.) (4,4-diphenyl-6 dimethylamino-heptanone-3 hydrochloride). The initial dose (2 mg/kg) was increased by increments (0.5 mg/kg) every 7 days until a maintenance level (5 mg/kg) was reached. The animals were kept at this dosage for 2 months and then mated. Methadone (5 mg/kg) was administered throughout pregnancy and on the day of delivery. Each litter of newborns was randomly divided into two groups. The pups ofthe first group (M,) were used for in vivo methadone tolerance tests. These animals were given a single subcutaneous injection of methadone (5-15 mg/kg) 12 to 24 hours after birth. The number of animals alive 6 hours later was recorded. Onganotypic cenebellar cultures were prepared from 12 to 24 and 48 to 72 hours old pups of the second group (M2) using standard techniques (Bonnstein and Murray, 1958). Following removal of the meninges, each cerebellum was dissected into 10 approximately equal pieces. The tapering end segments were discarded and the remaining eight fragments were explanted on collagen-coated coverslips mounted in a no. 1006 Falcon plastic Petni dish. Control explants were maintained in a solution composed of heat inactivated fetal calf serum (35%), Eagles minimum essential medium (42 ), Simm’s balanced salt solution (20%) and 20% glucose (3). Methadone, concentration 1 10 ” M in the total solution, was added to the medium of half of the cultures prepared from each litter of 12 to 24 hours old animals. Equal numbers of randomly selected explants from 48 to 72 hours old pups were maintamed in the following solutions: 1) standard medium; 2) standard medium + methadone (10”M); 3) standard medium + methadone (10#{176} M); 4) standand medium + methadone (10” M). All explants were kept in an incubator under the following conditions: atmosphere 95(7 air, 5 CO2; temperature 35#{176}C; relative humidity 98 (Schneider and Rue, 1974). The cultures were examined after 48 hours. Those that were excessively granular on from which the meninges had not been completely removed were discarded. The outgrowth zone of the remaining cultures was measured using a Zeiss net micrometer viewed at magnification 125 . The original explant was still well delineated at 48 hours so it was possible to measure the size of the explant and the outgrowth zone separately . The extent of the outgrowth, expressed in terms of the number of micrometer squares (1 square = 50 M2) which it covered, was determined and an average outgrowth area size was calculated for each litter subgroup (approximately 30

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@inproceedings{Willson2005EffectsOT, title={Effects of the Grow Cultures Tolerant Methadone Th of Organ Prepared and Control Hydro Otypic from Rats Chloride on Cerebellar Methadone-}, author={Nicholas Willson and James F. Schneider and L. Roizin and J{\"{u}rgen Flei\ss and Westcountry Rivers}, year={2005} }