The demonstration of a secretory material and cycle in the parenchymal cells of the mammalian pineal organ.

THE function of the pineal organ in man and other mammals is still an enigma, although gradually accumulating evidence suggests a relation to antigonadotrophic activity [3]. The state of uncertainty regarding the functional significance of the pineal organ is at least partly due to the lack of evidence clearly demonstrating secretory activity within the pineal. Previously described pineal parenchymal cytoplasmic granulations and staining reactions are not clearly indicative of secretory activity and a cytological secretory cycle has not been successfully demonstrated [l]. A cytological technique for showing the specific activity of pineal parenchymal cells should aid greatly in analyses of the factors regulating the pineal and in the standardization of pineal preparations used in experimental therapy. It was discovered in the study reported herein that the parenchymal cells of rat (Sprague-Dawley strain) pineals fixed for 16-24 hours in 10 per cent formalin buffered to neutrality with monobasic and dibasic sodium phosphate display an intensive and specific staining reaction with the chrome alum hematoxylin and phloxine technique of Gomori [2] for staining pancreatic islets. After fixation the pineals were washed in distilled water for 24 hours, dehydrated through ethanol solutions, cleared in benzene and embedded in paraffin. Sections seven microns in thickness were then stained according to Gomori’s technique [2]. It was found that initial fixation in Bouin’s fluid is unsatisfactory and that the step entailing refixation of the deparaffinized sections in Bouin’s fluid for 24 hours is necessary for successful results with the pineal. In order to differentiate the cytoplasmic material of the pineal chromophils from the other phloxinophilic structures such as the erythrocytes, the following procedure was added after the phloxine (National Aniline Div., dye content 83 per cent, C.I. No. 778, Certification No. NPh-14) staining, acidification and washing steps in the Gomori procedure: (1) stain for about 1 minute in a solution containing 2 g orange G (National Aniline Div., dye content 93 per cent, C.I. No. 27, Certification No. NO-14), 0.5 g aniline blue (National Anillhe Div., C.I. No. 707, Certification No. NK-8) and 1.0 g of phosphomolybdic acid per 100 cc. distilled water; (2) wash rapidly