A CYTOPHOTOMETRIC ANALYSIS OF ANTERIOR PITUITARY CHANGES IN RATS EXPOSED TO REDUCED PRESSURE RESEARCH REPORT Kenneth Rockwe 11 , Ph .D . Adam Anthony , Ph .D . Physiology-Biophysics Laboratories The Pennsylvania State University March 1967 Supported by Public Health Service Grant GM-05112 from the National Institute of General Medical Sciences, National Institutes of Health, and in part by NASA Grant NGR-39-009-015(2) ii FOREWORD The present investigation was supported by Public Health Service Research Grant No. GM-05112 from the National Institute of General Medical Sciences, National Institutes of Health, Bethesda, Maryland. This report was prepared by Kenneth Rockwell and was submitted to the Graduate School in partial fulfillment of the requirements for the degree of Doctor of Philosophy at The Pennsylvania State University. The research was conducted by Dr. Kenneth Rockwell as a subproject of a research project entitled "Biophysical and Endocrine Changes in Acclimated Rats" with Dr. Adam Anthony, Professor of Zoology acting as the project director and Dr. G; K. Strother, Associate Professor of Biophysics as co- investigator. This work was also aided in part by funds from NASA Grant NGR-39-009-015 (2) . The author wishes to thank Dr. Adam Anthony for his patient encouragement, guidance, and support throughout the conduct of this study. Thanks are also due to Dr. G. K. Strother for his helpful assistance. The experiments reported herein were conducted according to the principles outlined in the "Guide for Laboratory Animal Facilities and Care" and adopted by the Institute of Laboratory Animal Resources, National Academy of Sciences-National Research Council. TABLE OF CONTENTS Page ACKNOWLEDGMENTS ........................ ii LISTOFTABLES ........................ V LIST OF FIGURES ........................ vi LISTOF PLATES ........................ vii INTRODUC T ION ......................... 1 GENE RAL CON S IDERAT IONS .................... 2 A. Statement and Definition of Terms ........... 2 B. Adaptation to Simulated High Altitude ......... 3 1. General mechanisms .............. 3 2. Neural mechanisms ............... 4 3. Endocrine mechanisms ............. 4 C. The Histophysiology of the Anterior Pituitary ..... 6 1. Development and structure ........... 6 2. Cell types in the pars distalis ........ 7 3. Hormones ................... 8 4. The "one hormone-one cell type" theory .... 9 D. Effects of Altitude Exposure on Anterior Pituitary Function .............. 11 E. Microspectrophotometry ................ 13 F. Restatement of the Problem .............. 14 MATERIALS AND METHODS ..................... 16 A. Experimental Animals, the Decompression Chamber, and Histological Technique .......... 16 B. Cell Counting Procedure ................ 21 C. Microspectrophotometric Analysis ........... 23 D. Statistical Analysis of Data ............. 29 RESULTS,. .......................... 30 A. Identification of Anterior Pituitary Cells in Tissue Sections ............... 30 B. Changes in Rat Pituitary Weight in Response to Altitude Exposure ............. 33 C. Effect of Altitude upon Relative Abundance of Chromophobes, Acidophils,and Basophils ....... 38 1. Acidophil cells ................ 41 2. Gonadotropic and thyrotropic basophils .... 41 3. Chromophobes ................. 42 4. Additional Observations ............ 42 iv Page D. Altitude-Induced Changes in Anterior Pituitary Histophysiology Demonstrated by Cytophotometry ................... 43 1. Confirmation of instrument sensitivity and response ........... 43 2. Spectral characterization of stains and pituitary cell types ........... 43 3. Relative concentration of intracellular hormones during acclimation to hypoxia . e 53 DISCUSSION .......................... 59 SUMMARY ............................ 70 BIBLIOGRAPHY ......................... 72 APPENDIXA .......................... 81 APPENDIXB .......................... 84 LIST OF TABLES Table Page 1 Anterior pituitary staining: A comparison of reported and actual results from five common stain procedures ................ 31 2 Gross and relative pituitary gland weight changes and whole body weight adjustments in male rats exposed to simulated high altitude ....................... 37 3 Cell count summary: Changes in relative abundance of acidophil, basophil and chromo- phobe cells in the rat anterior pituitary in response to altitude exposure ............. 85 4 Cell count summary: Changes in relative abundance of gonadotropic (type A) and thyrotropic (type B) basophil cells in the rat anterior pituitary in response to altitude exposure ....................... 86 Absorption data for Corning CS1-60 didymium glass filter: Effect of monochromator exit slit width upon spectral resolution .......... 87 Spectral characterization of acidophil cells from ambient pressure control and altitude- exposed rats ..................... 88 Spectral characterization of gonadotropic (type A) basophil cells from ambient pressure control and altitude-exposed rats ........... 89 Spectral characterization of thyrotropic (type B) basophil cells from ambient pressure control and altitude-exposed rats ........... 90 Spectral quantification of intracellular hormone in chromophil cell types of ambient pressure control and altitude-exposed rats ........... 91 vi LIST OF FIGURES Figure Page 1 Placement and direction of cell count scans shown on a mid-coronal rat pituitary section ...... 22 2 Diagram of major components of the Leitz microspectrophotometer ................. 27 3 Actual and relative pituitary weight changes during altitude exposure ................ 36 4 Changes in relative number of anterior pituitary chromophil cells during altitude exposure.. ...................... 39 5 Changes in relative number of anterior pituitary chromophobe cells during altitude exposure ................... 40 6 Absorption spectrum of Corning CS1-60 didymium L glass filter obtained with the Leitz micro- spectrophotometer ................... 46 7 Absorption spectra of dye solutions used for differential pituitary staining ............ 47 8 Mean absorption spectrum for pituitary acidophils (alcian blue, P.A.S., picric acid stain) ...................... 49 9 Mean absorption spectrum for pituitary gonadotropic basophils (alcian blue, P.A.S., picric acid stain) ............... 51 10 Mean absorption spectrum for pituitary thyrotropic basophils (alcian blue, P.A.S., picric acid stain) ................... 52 11 Effect of altitude exposure upon intracellular hormone concentration in pituitary acidophils (optical density at 400 qt) .............. 56 12 Effect of altitude exposure upon intracellular hormone concentration in pituitary gonadotropic c basophils (optical density at 560 q~)...... * a 0 57 13 Effect of altitude exposure upon intracellular hormone concentration in pituitary thyrotropic basophils (optical density at 560 q~). e , 58 vii LIST OF PLATES Plate Page I External view of the decompression chamber . = a a 18 I1 The Leitz microspectrophotometer . a . ., . 25 I11 Cell types in the anterior pituitary of an ambient pressure control rat. Alcian blue, P.A.S., picric acid stain . , e * 35 IV Vacuolization in rat pituitary basophil cells following altitude exposure . - . 45 1 INTRODUCTION The altitude-exposed rat is uniquely suited for an investiga- tion of the pituitary during acclimation, since the endocrine state of hypoxia-exposed animals is well known from extensive studies of the thyroid, adrenal, and reproductive organs employing a multitude of biophysical and biochemical methods (34, 46, 54, 79). Investiga- tions of the functional state of the rat anterior pituitary during altitude exposure are rare, however, and there are no available data which relate pituitary cytochemical changes to the process of acclimation. Recent developments in the technique of visible cytophoto- metric analysis (62, 80, 81) permit, for the first time, a direct investigation of histochemically stained pituitary cell types and promise to further clarify the secretory response of individual cells during acclimation to environmental change. A major aim of the present study is to describe the adaptive cytochemical responses of the hormone-producing cells of the rat anterior pituitary following exposure of varying duration to simulated high altitude. All data are obtained by application of differential cell count and analytical cytophotometric techniques. A related and supplemental aim is to cytophotometrically characterize the various pituitary cell types on the basis of their absorption spectra. 2 GENERAL CONSIDERATIONS A. Statement and Definition of Terms. In those animal forms in which aerobic cellular respiration constitutes the chief biochemical source of metabolic energy, the environmental oxygen tension is the major parameter to which the organism must respond, if it is to survive. Homeostatic mechanisms have been demonstrated which enable the organism to adjust to changes in such environmental factors (7), and there is general agreement that those mechanisms reach their highest expression in species which maintain a fairly constant internal environment in the presence of great changes in external conditions (64). The French physiologist Bert was among the first to recognize the relationship which exists between altitude and the partial pres- sure of oxygen and was the pioneer in the systematic study of the effects of increased altitude upon the living organism. His observations of the external manifestations of altitude exposure demonstrated the physiological importance of adequate oxygen