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Cytoplasm Became Hyalinized. in Contrast to This Remarkable Cyto- Plasmic Transformation, the Nuclei Evidenced No Stigmas of Degenera- Tion

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Cytoplasm Became Hyalinized. in Contrast to This Remarkable Cyto- Plasmic Transformation, the Nuclei Evidenced No Stigmas of Degenera- Tion ANTERIOR PITUITARY GLANDS IN PATIENTS TREATED WITH CORTISONE AND CORTICOTROPIN * RALPH A. KILBY, M.D.; WARREN A. BENNETT, M.D., and RANDALL G. SPRAGUE, M.D. From the Sections of Pathologic Anatomy and Medicine, Mayo Clinic and Mayo Foundation,t Rochester, Minn. In a notable contribution to knowledge of the pathology of Cush- ing's syndrome, Crooke,' in 1935, described a characteristic alteration in the cytoplasm of the pituitary basophil cells. In each of his original I2 cases he observed a replacement of the basophilic granules by a homogeneous substance of similar staining qualities, a process which he termed hyaline change. The almost constant appearance of this phenomenon received early confirmation2-6 and it became of impor- tance as establishing a common morphologic denominator for widely divergent etiologic concepts. In the initial phases the hyaline material was noted either in the peripheral areas of the cells, or, more frequently, in a crescent midway between the nucleus and the cell membrane. Eventually the entire cytoplasm became hyalinized. In contrast to this remarkable cyto- plasmic transformation, the nuclei evidenced no stigmas of degenera- tion. Other authors soon described nuclear ballooning, nucleolar hypertrophy, multinucleation, and generalized enlargement of the basophils.79 Severinghaus'0 described cytoplasmic degranulation and perinuclear blistering vesiculation in the pituitary basophils of the case of Cushing's syndrome on which data were given by Graef and asso- ciates."1 However, the perinuclear changes were stressed in this single observation, and the hyaline changes which were present were not emphasized. McLetchie8 pointed out the coexistence of normal granularity and the encroaching hyaline material, and elaborated on the increased inci- dence of abnormal vacuolization within these cells. Transitional baso- phils and large amphophilic cells with foamy cytoplasm, large vesicular nuclei, and ill-defined cell borders were thought by Gellerstedt7 and by Mellgren9 to represent a phase in the evolution of hyalinized cells. Although Crooke"2 has denied the common occurrence of hyaline * Presented at the Fiftieth Annual Meeting of the American Association of Patholo- gists and Bacteriologists, St. Louis, April 2, I953. Abridgment of thesis submitted by Dr. Kilby to the Faculty of the Graduate School of the University of Minnesota in partial fulfillment of the requirements for the degree of Master of Science in Medicine. Received for publication, April 27, I956. t The Mayo Foundation, Rochester, Minnesota, is a part of the Graduate School of the University of Minnesota. I55 KILBY, BENNETT, AND SPRAGUE VoI. 33, No. z basophils in states of adrenal cortical hyperfunction other than Cush- ing's syndrome, some investigators have described these cells in cases of adrenal virilism5"3'l5 and virilizing ovarian tumor.'6 Hyalinized basophils have been observed infrequently in a variety of non-endocrine diseases and in normal persons. Crooke found mini- mal hyaline changes in only 9 of 350 non-Cushing pituitary bodies.' Ecker17 discovered that only 8 of 72I pituitary glands of patients not afflicted with Cushing's syndrome contained hyalinized basophils, and emphasized the normal size of these cells and the lack of nuclear en- largement and multinucleation. Mellgren5 regarded a small number of hyaline cells as normal, with varying increases in several conditions, Cushing's syndrome and virilizing syndromes exhibiting the greater numbers. Crooke' expressed the belief that this hyaline change represents "an expression of altered physiological activity" and concluded that it "is the alteration of fundamental significance in the causation of the syn- drome." This view was not inconsistent with Cushing's concepts of etiology'8 based on pituitary hyperfunction, and acquired some ad- herents.5'7'8 Two schools were thus established, the first led by Crooke, the second or adrenogenic school included Bauer,'9 Albright,20 and Kepler and associates.2",22 The latter investigators were early critics of the hypothesis of a pituitary origin of the syndrome, and Kepler developed the thesis that the hyaline changes are a result rather than the cause of certain hyper- adrenal states. He pointed out the paucity of the change in basophil adenomas,22 and the apparent cure by subtotal adrenalectomy of some cases of Cushing's syndrome.2' He commented that hyaline cytoplasm implies a retrograde if not a degenerative change.22 Despite the accumulation of extensive data, the etiology of Cush- ing's syndrome has not yet been fully elucidated. For the cases in which the condition has been successfully treated in recent years, the therapy in the great majority has consisted of surgical removal of hyperfunctioning adrenal cortices,2'23 while a lesser number have responded to roentgen irradiation of the pituitary gland.24'25 The recent widespread use of corticotropin (ACTH) and I7 hydroxy-i i-dehydro-corticosterone (cortisone) in various non-endo- crine diseases has frequently resulted in the production of hyperadrenal states indistinguishable from spontaneous Cushing's syndrome.26'27 The occurrence of hyaline changes in the pituitary basophils of these hormone-treated patients would be telling evidence against the hypoth- esis that such changes are indicative of pituitary hyperfunction. Such Jan,-Feb., 1957 HYPOPHYSIS AND STEROID THERAPY I 57 was suspected when Golden and associates28 examined the hypophyses of two patients with chronic glomerulonephritis who received corti- cotropin prior to death. The first patient, who demonstrated "a distinct increase in basophilic cells, which displayed striking hyaline cyto- plasmic changes as described by Crooke" had received 400 mg. of corticotropin intramuscularly during a 5-day period before death. The second patient was given 490 mg. of corticotropin in 8 days, treatment terminating 7 days before death. Hyaline material was present in a few scattered basophilic cells. Statistically significant basophilia oc- curred in these cases at the expense of the chromophobes, Rasmussen's differential counting method being used. The pituitary body of a third patient with myasthenia gravis who had received two courses of corti- cotropin of 975 mg. and 500 mg., respectively, 9 months and 6 months before death, was examined. The paucity of Crooke's cells in this pituitary gland suggested reversibility of the phenomenon. In the pituitary body of a patient with Cushing's syndrome who survived 66 days after subtotal adrenalectomy, Kepler29 recorded that 40 per cent of the extra-adenomatous basophils were hyalinized. Luft24 found only small numbers of Crooke's cells in a patient with Cushing's syndrome who committed suicide almost 2 years after considerable im- provement had followed pituitary irradiation. Luft stated that the predominant pathologic cell in this case was the hypertrophic am- phophil of Gellerstedt7 and Mellgren.5 Laqueur's30 study of 8 patients treated with corticotropin, cortisone, or both substances confirmed the induction of hyaline changes in the basophils but indicated no correlation between the degree of change and the time and amount of hormone employed. No alterations were noted in one patient with acute leukemia who received 7.35 gm. of cortisone in 35 days. Advanced hyalinized basophils with hypertrophic nuclei were found by Gilbert-Dreyfus and Zara3' in the pituitary body of a female patient with disseminated lupus erythematosus who had received io gm. of cortisone in 2 months. Long before the advent of purified corticotropin and adrenal steroid preparations, Severinghaus and Thompson,32 by prolonged injection of extracts of sheep's anterior pituitary gland, had produced basophil alterations in dogs which strongly resembled Crooke's changes. Or- ganization of the basophilic granules into spherical masses and the eventual formation of a basophilic amorphous colloid-like substance had been described. Later, Heinbecker33 described almost identical changes in the basophils of dogs whose supra-optic and paraventricular nuclei of the hypothalamus had been destroyed. These alterations I58 KILBY, BENNETT, AND SPRAGUE VoI. 33, No. z closely resemble the early cytoplasmic changes in the basophils which we will describe. By the use of corticotropin and cortisone, degranula- tion of the pituitary basophils has been induced experimentally in dogs, guinea-pigs,34 and rats.35 On the basis of the foregoing observations, it is reasonable to suppose that the hyaline change of Crooke is a result of the exposure of pituitary basophils to excessive amounts of II-oxygenated adrenal steroids. Be- cause of the small number of cases reported and the apparent similarity, if not identity, between these induced hyaline cells and the Crooke cells of Cushing's syndrome, this study was undertaken. MATERIAL AND METHODS The pituitary glands of 76 patients who had received therapeutic cortisone, corticotropin, or both substances before death were studied. Following standard fixation in Io per cent formaldehyde and imbed- ding in paraffin, four or more midhorizontal sections were made. Al- though Crooke and Russell's modification36 of Mallory's acid-fuchsin aniline-blue method is tinctorially good and widely employed, there is great variation in the times of washing and differentiating for different pituitary bodies. By reversing the order of application of the acid and basic dyes, that is, staining first with aniline blue, then with acid- fuchsin, these difficulties were minimized. The clear gray-blue refractile appearance
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