The cytology of adrenocortical regeneration in the rat and the effects of x-irradiation
Brenner, Robert Murray
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The results presented in this dissertation were obtained from three related studies of adrenocortical regeneration which may be characterized as follows: the cytology of (a) adrenocortical regeneration after immediate autotransplantation, (b) adrenocortical regeneration after in vitro x-irradiation of the adrenal gland and (c) the response of young autotransplants to a lethal dose of whole body x-irradiation. Experimental procedures a. Immediate autotransplantation. Sixty-four male albino rats of the Wistar strain weighing between ninety and one hundred grams were bilaterally adrenalectomized by the dorsal route under ether anesthesia. Both adrenals were bisected and immediately autotransplanted to pockets in the dorsal musculature. All animals were given one per cent saline drinking water for the entire experimental period and Purina Laboratory Chow was fed ad libitum. These animals were sacrificed and their transplants removed for study over a twenty-one day period as follows: three rats on each of days one, two, four, five, seven, eight, ten and eleven after operation; and five rats on each of days three, six, nine, twelve, fourteen, sixteen, eighteen and twenty-one. In addition, transplants were removed from two animals killed at six hours and two at twelve hours after operation. Ten normal animals in the same weight range were killed and their adrenal glands removed for comparison with regenerating transplants. All tissues were fixed in ten per cent neutral formalin for a minimum of two weeks. After fixation the tissues were embedded in gelatin, sectioned five microns in thickness by the freezing method, mounted on albuminized slides and stained first for three minutes in Mayer's hemalum and then for four minutes in a filtered, saturated solution of Sudan Black B in seventy per cent alcohol. The tissues were analyzed for cell depth, cell type and per cent take (Wyman et al, 1954). b. X-irradiation of the adrenal gland in vitro. One hundred and ten animals were adrenalectomized and the left adrenal gland of each was placed in mammalian Ringer's and either held there for sixteen minutes at room temperature or irradiated with two thousand roentgens given at a dose rate of one hundred and twenty-five roentgens per minute (the x-ray source was run at one hundred and forty-five kilovolts and six and four tenths milliamperes with a target distance of nine inches). After the irradiation period or the control period the adrenal gland was bisected and autotransplanted to the dorsal musculature. Forty-five animals bearing in vitro control transplants and sixty-five animals bearing in vitro irradiated transplants were sacrificed according to the following schedule: five control and five experimental animals on each of days two, three, four, seven, eight, nine, fourteen, twenty-one and thirty. Ten additional animals with irradiated transplants were killed on each of days two and three after transplantation. The transplants were removed, processed and stained as described (v.s.) and analyzed for cell depth, cell type and per cent take. c. Whole body x-irradiation. Thirty-four rats with two day old transplants and the same number of rats with four day old transplants were placed in small confining boxes and either held there for sixteen minutes as controls or given a total body x-ray dose of two thousand roentgens. All animals were sacrificed twenty-four hours after the control or experimental period and their transplants removed for study. Tissues were processed and analyzed as described above. Observations a. Regeneration after immediate transplantation. The cells of the zona glomerulosa survived after implantation and underwent a series of transformations which resulted in the production of a fully differentiated cortex by the twenty-first day. The transformations involved characteristic changes in the nuclei and the distribution of lipid so that a sequence of five cell types could be recognized, including, in order, "static" glomerulosa cells (one to two days after implantation), "transitional" glomerulosa cells (three to six days), "empty" cortical cells (seven to ten days), "granular" cortical cells (eleven to fourteen days) and mature cortical cells (sixteen to twenty-one days). Immediately after implantation the lipid droplets of glomerulosa cells enlarged and crowded together to such an extent that nuclei were partially obscured. The hemalum staining of those nuclei which were visible was characteristic of cells that are viable. These cells were generally arranged in small patches around blood vessels in the subcapsular area. They were termed static glomerulosa cells because they appeared to be in a resting or static state prior to growth and differentiation. Three days after implantation, coincident with increased blood vessel growth in the capsular area, the amount of lipid in groups of static cells nearest the capsule decreased. Their nuclei were no longer obscured by a mass of lipid but a few large intensely stained droplets were seen against a gray-green background of cytoplasm. The nuclei were spherical and each contained a single, enlarged nucleolus. These cells were termed transitional glomerulosa cells since they represent a stage through which static cells pass preparatory to mitosis and centripetal migration. At seven days, groups of transitional cells directly beneath the capsule lost all their cytoplasmic lipid and became empty cortical cells. The nuclei remained large and spherical with a single large nucleolus. These cells had the highest rate of mitosis and the total depth of regenerated cortex increased rapidly after empty cortical cells appeared. Lipid droplets and spheroid complexes reappeared in the regenerating tissue between the eleventh and the fourteenth day, but since these lipid forms were finer and more sparsely distributed than those of adult glands, the cells in which they appeared were termed granular cortical cells. Granular cells were found in a spatial arrangement similar to that of adult glomerulosa and fasciculata. Increasing numbers of cortical cells became mature between sixteen and twenty-one days. Mature glomerulosa cells had lipid droplets grouped in clusters, mature fasciculata cells had spheroid complexes of adult size and distribution, and mature reticularis cells contained very large spheroid complexes. Empty cortical cells were assumed to be secreting adrenal hormones at a maximal rate and the appearance of lipid in granular cells was considered an indication of decrease in the secretory rate. Mature cortical cells were assumed to be secreting at normal rates. b. The effect of in vitro x-irradiation. The total depth of cortical tissue regenerated by adrenal glands which had been x-irradiated in vitro was significantly reduced as compared with the in vitro controls from the ninth through the thirtieth day after implantation. Accompanying this reduction in growth rate was a retardation in the development of the sequence of cell types. Transitional and empty cells remained in the slowly growing parenchyma of irradiated glands until the fourteenth day, whereas in the in vitro controls these cells had all been transformed into granular cortical cells in the same time. Granular cells became predominant in the regenerated parenchyma of irradiated glands by the twenty-first day but attained a smaller depth than those of in vitro controls. They remained predonunant through the thirtieth day. A few mature cortical cells were present at that time but they represented only a small fraction of the total mass and had attained far less depth than those of the in vitro controls. This presumably indicates that the secretory rates of granular cells in transplants of irradiated glands are higher than those of in vitro controls on the thirtieth day. Comparison of the sequence of cell types of in vitro controls with that of the immediate transplants revealed that the in vitro control procedure itself caused a delay in the maturation of granular cells. In immediate transplants the depth of granular cortical cells attained a maximum on the fourteenth day and then diminished. Mature cortical cells were the predominant type on the twenty-first day. In the in vitro controls, however, granular cells persisted as the predominant type through the thirtieth day and far fewer mature cells were present at that time than in immediate transplants at twenty-one days. This delay was not accompanied by a retardation in grovJth rate of in vitro controls and was therefore considered a direct effect of the control procedure. c. The effect of whole body x-irradiation on immature transplants. Seventy per cent of the animals subjected to total body x-irradiation on the fourth day after implantation had empty cells in their transplants twenty-four hours after the time of x-irradiation, but these cells were present in only thirty per cent of the controls at that time. Also, the depth of empty cells was approximately doubled while the depth of transitional cells was diminished in the transplants of the animals irradiated on the fourth day. These changes did not occur in the transplants of the animals irradiated on the second day. This seemed to indicate that either the two day old transitional cells were not sufficiently differentiated to respond to ACTH or, that the areas where two day old transitional cells occur were less effectively vascularized than at four days. Static cells in animals irradiated on the second or the fourth day after implantation were not affected by the stress of x-irradiation. This seemed to indicate that static cells were not responsive to ACTH at any time.
Thesis (Ph.D.)--Boston University