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'1 11"1111111111111111111”, 1-1 11 1 '1111111 1111111'1'111 11111 1.11119 1': 1-111:- 111 1111 111 111111111 111111111 '11" ‘1: 11111111313121.11111 1111 11111111111 ,11] 1'1 1 1111111 1111' 111111111111 h.———-h — I- -__ _-_—_--——-—O-——-——.. \ lig- rll'..;.;.--I l , L g LE :- 1“ ‘ “t5 -'%i "“5 . aw u. ma st. 31% 51' l ‘1‘?! q.- ar‘A" - ~- .. . -fi r": .’ .7 p. E“ hrs-zv.,. Lafi- $63 This is to certify that the dissertation entitled Fluorescent, Cytochemical, and Ultrastructural CharacteristiCSCHFAmine Containing Cells in the Laryngeal and Tracheal Epithelium of Adult Albino Rats presented by Allan Dorland Pearsall III has been accepted towards fulfillment of the requirements for Ph . D. degree in Anatomy Robert Echt, Ph.D. Major professor Date 12-31-82 MS U i: an Affirmative Action/Equal Opportunity Institution 0— 12771 lViESI_J RETURNING MATERIALS: Place in book drop to LIBRARIES remove this checkout from “ your record. FINES will be charged if book is returned after the date stamped below. ‘1 .' ‘Q If. . 3"“ “j " ’ I! f3 "’1. gnu .. " " w t - . 1- a: -.-_ i b ‘i’lz- J ‘4: :1. .2! iii 1’,“ "Fm-v, ‘ ’ 3‘ ’3‘.- r._ ‘ , '3. l 3' 3. III)” III? I: r“ rad if I’d" 4: R0 /i/I:9-./ FLUORESCENT, CYTOCHEMICAL, AND ULTRASTRUCTURAL CHARACTERISTICS OF AMINE CONTAINING CELLS IN THE LARYNGEAL AND TRACHEAL EPITHELIUM OF ADULT ALBINO RATS BY Allan Dorland Pearsall III A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Anatomy 1982 ABSTRACT FLUORESCENT, CYTOCHEMICAL, AND ULTRASTRUCTURAL CHARACTERISTICS OF AMINE CONTAINING CELLS IN THE LARYNGEAL AND TRACHEAL EPITHELIUM OF ADULT ALBINO RATS BY Allan Dorland Pearsall The extrapulmonary epithelium of adult Sprague Dawley rats contain numerous endogenously fluorescent (weak) cells. These cells are present following the classic histofluorescent method (FIF) of Falck and Hillarp (1962). Cytospectrofluorimetry revealed an emission maximum of 493nm. The excitation maximum could not be calculated but appeared to be below 350nm, which requires a special quartz optical system. In agreement with Hoyt gt El. (1979), ,aqueous formaldehyde fixation and embedding in glycol methacrylate failed to identify these cells even though mast cells (excitation and emission maxima: 40l/515nm) were evident. Treatment of the sections with formaldehyde and HCl vapors (LFIF-HCl) (Bjorklund gt 31., 1968) resulted in the identification of a new population of intensely fluore- scent cells (455/537nm) throughout the epithelial lining of the extrapulmonary airways. This spectral shift and increase in fluorescent intensity following acidification is similar to reported values for tryptamine, tryptophan, Allan Dorland Pearsall and peptides with NH -terminal tryptophan (Bjorklund gt 31., 2 1968 and Larsson 33 31., 1975) markedly different from those reported for the phenylethylamines or serotonin (Bjorklund 35 31., 1968, 70 and 71) duodenal enterochromaffin cells, prepared according to the LFIF-HCl method, displayed the characteristic Spectra for the presence of serotonin (425/513nm). The morpholoqy of the fluorescent cells was similar when prepared according to the FIF or LFIF-HCl techniques. Conjunctive light microscopy demonstrated that the fluorescent granules were methylene blue, alcian blue, periodic-acid Schiff and ferric-ferricyanide positive. Fresh cryostat sections an intense monoamine oxidase activity throughout the epithelial lining. These cells were similar to globule leukocytes which previously have been identified in the mucus membranes of the urinary (Cantlin gt 31., 1972), intestinal (Murrey 23 al., 1968), and respiratory (Kent, 1966) systems. These results were supported through the conjunctive use of fluorescence and electron microscopy which demonstrated that the fluorescent epithelial cell was indeed a globule leukocyte. Addi- tionally, microspectrofluorimetric recordings and electron microscopic investigation further support Hoyt gt El- (1979) findings that aqueous formaldehyde fixed tissues embedded in glycol methacrylate are useful for the histo- chemical identification of serotonin containing cells. Allan Dorland Pearsall Furthermore, acid hydrolysis may extend the usefulness of this technique by identifying other closely related B-(3-indolyl) ethylamines. This work is dedicated to four people who have given me love, support, and encouragement throughout all of my life. To my parents, Ruth and Allan, for providing me with an appreciation for higher education and the means to attain it, and to my sisters, Karen and Sandy, for their invaluable support and understanding, I dedicate this work. ii AC KNOWLEDGEMENT S As with most complex and involved research projects, many individuals have made significant contributions to this work. I would like to express my appreciation to the following persons for their support in this endeavor. I would like to express my deepest appreciation to Dr. Robert Echt, Associate Chairman of the Department of Anatomy for his constant help and guidance throughout the formulation and executive of the present thesis. His deep knowledge, as well as his personal friendship made these five years of my life a most valuable and unforgettable experience. The research presented in this dissertation was con- ducted under the direction of Dr. Lawrence Ross, Dr. Robert Echt, Dr. Robert Roth, Dr. Jerry B. Scott, and Dr. C. Wayne Smith. I am extremely grateful and indebted to them for their encouragement and aid during the course of this research. Their knowledge and constant understanding were an important part of this thesis, without which it would have been impossible to complete. Tragically, Dr. Jerry B. Scott has since passed away. The memory of this great scientist and good friend will always be remembered. I am also grateful to Dr. Stephen T. Kitai, Professor and Chairman of the Department of Anatomy, for his support iii throughout my tenure at Michigan State University. I would also like to express my sincere thanks to Dr. Arthur L. Foley and the other members of the Department of Anatomy for their interest and assistance and for making my time at Michigan State University both educational and enjoyable. Special thanks to Patricia Patterson and Esther Roege for the endless help in the use of the transmission electron microscope and to whom time was never a problem. My appreciation to Dr. Robert Dinerstein for his generous help and time in the use of the microspectro- fluorimeter. Finally, I would like to acknowledge the able assistance of Judy James in the preparation and typing of this dissertation. iv TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . . . . . . . . . Chapter 1. INTRODUCTION . . . . . . . . . . . . . . . . . . State of the Problem . . . . . . . Research Plan and Rationale . . . Significance . . . . . . . . . . . Limitations . . . . . . . . . . . 2. LITERATURE REVIEW . . . . . . . . . . . . . . . Trachea and Extrapulmonary Bronchial Respiratory Epithelium of Humans . . . . . . Intrapulmonary Single Endocrine-like Cells and Neuroepithelial Bodies in Humans . . . . . . Laryngeal, Tracheal, and Extrapulmonary Bronchial Respiratory Epithelium of Animals Intrapulmonary Single, Endocrine-like Cells and Neuroepithelial Bodies in Animals . . . Physiologic and Pharmacologic Stimulation of Single Respiratory Endocrine-like Cells and Neuroepithelial Bodies . . . . . . . . . . . 3. MATERIAL AND METHODS . . . . . . . . . . . . . . Animal Care . . . . . . . . . . . . . . . . . Specimen Removal . . . . . . . . . . . . . . . Fluorescence Histochemistry, Freeze-Dried Formaldehyde Induced Histofluorescence . . . Freezing and Freeze-Drying . . . . . . . . Formaldehyde Vapor Treatment . . . . . . . Plastic Embedding and Sectioning . . . . . Liquid Formaldehyde Induced Histofluores- cence . . . . . . . . . . . . . . . . . . Drug Treatment . . . . . . . . . . . . . . Surgical Procedure . . . . . . . . . . . . Fixation and Dehydration . . . . . . . . . V Page vii ix H \D\O\IO\ ll 12 14 35 45 64 80 Chapter Page Plastic Infiltration, Embedding and Sectioning . . . . . . . . . . . Formaldehyde Vapor and HCl Treatment . . . 93 Specificity of the Fluorophore . . . . . . . 93 Microspectrofluoreometric Analysis . . . . . 94 Histochemical Procedures . . . . . . . . . . 97 Lee's Methylene Blue-Basic Fuchsin Stain . 98 Ferric-Ferricyanide . . . . . . . . . . . 99 Alcian Blue . . . . . . . . . . . . . . . 100 PAS—Lead Hematoxylin . . . . . . . . . . . 100 Acid Phosphatase . . . . . . . . . . . . . 101 Cryostat Histochemistry . . . . . . . . . . 101 Monoamine Oxidase . . . . . . . . . . . . 102 O- -Phtha1adehyde . . . . . . . . . . . . . 103 Formaldehyde-Ozone . . . . . . . . 103 Combined Fluorescence and Light Microscopy . 104 Electron Microscopy . . . . . . . . . . . . 104 Microscopy and Photography . . . . . . . . . 105 Statistical Analysis of Excitation and Emission Maxima . . . . . . . . . . . . . 105 4. RESULTS 0 O O O O O O O I O O O O O O O O O O O 107 Distribution of Fluorescent Tracheal Epithe- lial Cells . . . . . . . . . . . . . . 107 Morphology of the Fluorescent Epithelial Cells . . . . . . . . . . . . . . . . . . . 108 Microscpectrofluorometric Data . . . . . . . . 114 Specificity of the Fluorophore . . . . . . . . 125 Histochemical Results . . . . . . . . . . . . 128 Electron Microscopy . . . . . . . . . . . . . 141 5. DISCUSSION . . . . . . . . . . . . . . . . . . . 159 Secretory Granules of the Cell . . . . . . . . 159 Content of the Cell . . . . . . . . . . . . . 161 Nature of the Fluorophore . . . . . . . . . 163 Microscpectrofluorimetry . . . . . . . . . . 167 Stimulus-Secretion Coupling . . . . . . . . . 171 Secondary Characteristics . . . . . . . . . . 172 Histochemistry . . . . . . . . . . . . . . . 173 6. CONCLUSIONS AND RECOMMENDATIONS . . . . . . . . 176 Conclusions . . . . . . . . . . . . . . . . . 176 Recommendations . . . . . . . . . . . . . . . 180 APPENDICES O O O O O I O O O O O O O O O O O O O O O O 181 REFERENCES 0 C O O O O O O O O O O O O O O O O O O O C 191 Table 1. LIST OF TABLES Single (+) and groups (***) of endocrine-like cells which have been demonstrated in extra- pulmonary airways at various ages in animals and man. Note: (?) indicates that the author did not specify if single or groups of endocrine-like cells were observed . . . Single (+) and groups (***) of endocrine-like cells which have been demonstrated in intra- pulmonary airways at various ages in animals. Note: (?) indicates that the author did not specify if single or groups of endocrine- 1ike cells were observed . . . . . . . . . Relative fluorescence intensity of cells in the respiratory and gastrointestinal tracts of adult rats after various histochemical procedures . . . . . . . . . . . . . . . . Spectral properties of tracheal (TRACH) and duodenal (GI) fluorescent cells from formal- dehyde-HCl treated sections. (Liquid formal- dehyde fixed tissue embedded in Spurr's resin, 6mu thick). Mean and standard deviation of the excitation and emission maxima are also presented 0 O O O O O O O O O O O O O O O 0 Spectral properties of fluorescent tracheal epithelial cells (EPITHELIAL) and fluorescent connective tissue mast cells (MAST) from freeze-dried paraffin embedded sections, 15mu thick. Mean and standard deviation of the excitation and emission maxima are also presented 0 O O O C O O O O I O C C O O O 0 vii Page 15 65 113 115 116 Table Page 6. Mean and standard error of emission maxima (in nm) of fluorescent cells in a 6mu section from four cell types. The tissues were either fixed with liquid formaldehyde, embedded in plastic and treated with HCl vapor (LFPHCL) or freeze-dried and embedded in paraffin (FDP). The emission maxima were first analyzed with analysis of variance and when significance was noted the means were compared by Scheffé and Duncan a_posteriori tests with a = .05. . . . . 126 7. Mean and standard error of excitation maxima (in nm) of fluorescent cells in a 6mu section from three cell types. The tissues were either fixed with liquid formaldehyde, embedded in plastic and treated with HCl vapor (LFPHCL) or freeze-dried and embedded in paraffin (FDP). The excitation maxima were first analyzed with analysis of variance and when significance was noted the means were compared by Scheffé and Duncan a posteriori tests with a = .05. . . . . 127 8. Significance and location of selected histo- chemical reactions . . . . . . . . . . . . . . 142 viii LIST OF FIGURES Figure Page 1. Freeze-Drying Apparatus . . . . . . . . . . . . . 83 2. Picket-Spengler reaction of an amine with an aldehyde by condensation to form heterocyclic compounds which spontaneously oxidize yielding fluorescent dihydroisoquinoline or dihydro-B- carboline depending on whether the amine contains a catechol or indole ring structure respectively (Corrodi and Jonsson, 1967; and Bjorklund gt 31., 1973) . . . . . . . . . . . . 86 3. Plastic embedding appratus; (A) vacuum chamber, (B) chemical traps; (C) vacuum pump . . . . . . 89 4. Microspectrofluorometer; (A) illuminating light (———+); (B) fluorescing light (---+) . . . . . 96 5. Freeze-dried formaldehyde vapor treated and glycol methacrylate embedded tracheal section (4mu). Yellow fluorescent cells are present in the respiratory epithelium (2,000x) . . . . 109 6. Liquid formaldehyde fixed formaldehyde—HCl induced fluorescent tracheal epithelial cells (lmu), embedded in Spurr's resin (1,000x) . . . 109 7. Freeze-dried, formaldehyde vapor treated, and glycol methacrylate embedded tracheal section (4mu). Yellow fluorescent mast cells are present in the submucosa (2,000x) . . . . . . . 111 8. Liquid formaldehyde fixed, Spurr embedded, formaldehyde-HC1 vapor treated duodenal section. Fluorescent enterochromaffin and mast cells are present in mucosa (1,000x) . . . . . . . . . . 111 9. Excitation (---) and emission (———) curves of a fluorescent tracheal epithelial cell, liquid formaldehyde-fixed, embedded in Spurr's resin, and treated with formaldehyde-HCl vapor . . . . . . . . . . . . . . . . . . . . . 117 ix Figure 10. 11. 12. 13. 14. 15. 16. 17. 18. Page Excitation (---) and emission (———) curves of a liquid formaldehyde fixed, Spurr embedded, formaldehyde-HCl vapor induced fluorescent gastrointestinal (duodenum) cell . . . . . . . . . . . . . . . . . . . . . Emission (———) curve of a fluorescent tracheal epithelial cell, freeze-dried and paraffin embedded O O O O O O O O I O O O O O O I O O O Excitation (---) and emission (———) curves of a fluorescent tracheal connective tissue mast cell, freeze-dried and paraffin embedded . . . The same tracheal section (6mu) photographed first during fluorescence microscopy (upper panel), and then stained with methylene blue and rephotographed during ordinary light microscopy (lower panel) (2,000x) . . . . . . The same tracheal section (6mu) photographed first during fluorescence microscopy (upper panel), and then stained with alcian blue and rephotographed during ordinary light microscopy (lower panel) (2,000x) . . . . . . The same 6mu tracheal section photographed first during fluorescence microscopy (upper panel) and then rephotographed by ordinary light microscopy after staining with ferric- ferricyanide (lower panel) (2,500x) . . . . . A 6mu tracheal section photographed after staining with methylene blue (1,000x) . . . . The same 6mu tracheal section (Figure 16) after washing with tap water, and restaining with PAS and photographing again (1,000x) . . . . . Globular leukocyte in tracheal respiratory epi- thelium of adult rat showing nucleus (N); large granules of uniform electron density, type I granules (l); or dense with small rims of less electron dense matrix, type II granules (2). Rough endoplasmic reticulum (arrowhead) appears around the type I granule. The cell does not extend to the tracheal lumen. Junctional complexes (arrows) were noted between other tracheal epithelial cells (E) lumen (L). Formaldehyde (6 percent), uranyl acetate, and lead citrate. 7,300x . . . . . . X 119 121 123 131 133 136 139 139 145 Figure Page 19. Globular leukocyte within the tracheal respiratory epithelium of an adult rat. Type I granules (1), type II granules (2), nucleus (N), the cell membrane (arrow) and rough endoplasmic reticulum (R). Formal- dehyde (6%), uranyl acetate and lead citrate. 16,600x . . . . . . . . . . . . . . . . . . . 147 20. Globular leukocyte in the tracheal respiratory epithelium of adult rats. The cell cyto- plasm contains granules of uniform density (1) and a type IV granule (4) with light and dark matrix areas. Nucleus (N) and epithe- lial junctional complexes (arrows) can be seen. Formaldehyde (6 percent), uranyl ace- tate, and lead citrate. 10,760x . . . . . . 149 21. Globular leukocyte in tracheal respiratory epi- thelium of adult rats showing a variety of granules: (1) electron dense matrix; (2) small rims of less electron dense matrix surrounding a denser core; (3) light and dark matrix areas; (4) light matrix with para- crystalline structures (arrow). Formaldehyde (6 percent), uranyl acetate, and lead citrate. 11,390x . . . . . . . . . . . . . . . . . . . 151 22. Globular leukocyte in the tracheal epithelium of an adult rat showing granules of various sizes within the same cell. Nucleus (N), homogenous electron-dense granules (1), portion of a unit membrane around granule (arrow), junctional complexes (arrowheads), lumen (L), and ribosomes (R). Formaldehyde (6 percent), uranyl acetate, and lead citrate. 9,080x . . . . . . . . . . . . . . 153 23. Liquid formaldehyde fixed (6 percent), Spurr embedded HCl vapor treated tracheal section, lmu. The two fluorescent epithelial cells (a and b) are identical to the cells demon- strated at the ultrastructural level in Figure 24. Lumen (L), Basement membrane (BM) (2,000x) . . . . . . . . . . . . . . . . 155 24. Globular leukocyte within the tracheal epithe- lium of the adult rat. The cells observed at the ultrastructural level are identical to those observed at the light microscopic level. Formaldehyde (6 percent), uranyl acetate and lead citrate. 7,500x . . . . . 155 xi Figure Page 25. Two fluorescent enterochromaffin cells (a and b) from liquid formaldehyde fixed (6%), Spurr embedded, HCl-vapor treated duo- denal section (upper panel), lmu thick (2,000x). Lumen (L), nucleus (N), basement membrane (BM). The same enterochromaffin cell (b) at the ultrastructural level (lower panel). 6,400x . . . . . . . . . . . . . . . 157 xii CHAPTER I INTRODUCTION The epithelium lining the lumen of the respiratory tract is composed of many cell types distinguishable by morphologic and cytochemical characteristics (Rhodin, 1956; 1966; Jeffery and Reid, 1975; and Breeze gt 51., 1977). Three cell types which contain biogenic monoamines have been described in the respiratory epithelium. Histochemical studies by Pearse (1969) demonstrated that many kinds of polypeptide hormone-producing cells were able to take up amine precursors, decarboxylate them and produce biogenic amines. In an attempt to provide a conceptual framework for the diffuse endocrine-like cell system, Pearse (1969) proposed the Amine Precursor Uptake and Decarboxylation concept (APUD). APUD cells share some or all of the follow- ing characteristics: (a) amine content or amine precursor uptake and decarboxylation; (b) high levels of a-glycero- phosphate dehydrogenase and or non—specific esterases or cholinesterase histochemical activity; (c) masked metachro- masia; and (d) endocrine-like granules. In an attempt to extend this conceptual framework, Fujita (1976) proposed the paraneuron concept which was based on morphologic and 2 physiologic characteristics. Paraneurons are cells which traditionally have not been directly associated with the nervous system but because of their similar structure, metabolism, and function should be regarded as closely related. Paraneurons share to some degree all of the following characteristics: (a) contain neurosecretion-like and/or synaptic vesicle-like granules; (b) contain or produce substances identical with, or related to neurosecre- tions or neurotransmitters; (c) possess receptosecretory function; and (d) are embryologically derived from neuro- ectoderm. Fujita and Kobayashi (1979) have subsequently eliminated the neuroectoderm origin characteristic. In the respiratory system, three cell types have been described which fulfill the paraneuron concept. One of these cell types, the globular leukocyte, is present in the respiratory epithelium of larynx, trachea, and bronchioles (Kent, 1966). This cell is characterized by large electron-dense granules closely encircled by a unit membrane (Kent, 1966; Ewen gt gt., 1972; and Jeffery and Reid, 1975) and a lack of morphologic attachments to adjacent epithelial cells (Kent, 1966; Cantin gt gt., 1972). Both endogenous amine content (Murray gt_gt., 1968; Ewen gt gl., 1972) or induced amine content after administration of an amine precursor (Ewen gt gt., 1972) have been demon- strated in globular leukocytes. Based on subjective observa- tions of cytofluorescence, the endogenous amine has been suggested to be dopamine (Murray gt 21°! 1968; Ewen gt gt., 1972; and Breeze gt gt., 1977). The exact function of the globular leukocyte in the tracheobronchial epithelium remains unclear. In the gastrointestinal tract, they have been investigated in relation to parasitic infections (Murray gt gt., 1968; and Miller, 1971). It has been suggested that the biogenic monoamine contained with the globular leukocyte is released to increase the permeability of the gastro- intestinal wall and to allow circulating or locally produced antibodies to pass through the epithelium and gain access to invading antigens (Miller, 1971). Light and electron microscopy (Kent, 1966 and Jeffery gt gt., 1975) have demonstrated that the endocrine granules vary in size and shape even within the same cell. Despite this wide range of granule diameter, all of the granules are well above the optical limit of resolution, 2,000 A (Kent, 1966). The second type of biogenic amine containing cell occurs singly in the extra and intrapulmonary airways of animals (Ericson, gt gt., 1972; Cutz gt gt., 1975; and Dey gt gt., 1976, 1980) and humans (Cutz gt gl., 1972 and 1975; and Hage gt_gl., 1977). They are endogenously fluorescent for serotonin (Dey gt gt., 1976; 1980) and are able to take up amino acid precursors (Hage, 1973a; and Hoyt gt gt., 1979). The single cells are triangular or flasked shaped (Tateishi 4 gt gt., 1973; and Hage gt gt., 1977), with the base resting along the basement membrane (Cutz gt gt., 1972, 1974; and Hage gt gt., 1974). The most characteristic feature of these cells at the ultrastructural level was the presence of small dense-core vesicles (approximately 1,200 A) surrounded by a clear area and limited by a single unit membrane (Gmelich gt gt., 1967; Cutz gt gt., 1972; and Hage 1973b). Recently Echt gt gt., (1980), reported a significant decrease in the number of detectable tracheal fluorescent cells in neonatal rabbits after exposure to hypoxic hypoxia or carbon monoxide induced hypoxia. The functional role of these cells remains to be determined. The third cell type containing biogenic amines exists in a closely associated group of similarly fluorescent cells. Because of their unique structural relationships and endocrine-like features, Lauweryns and Peuskens (1972a) called these cell clusters neuroepithelial bodies (NEBs). Endogenous fluorogenic amines have been demonstrated in NEBs of both animals and humans (Hage gt gt., 1974; and Lauweryns gt g;., 1972b and 1975). NEBs also have been demonstrated to take up amino acid precursors (Hage 1974; and Cutz gt gl., 1974). With spectrofluorimetry, the excitation and emission spectra of NEBs were found to be identical with serotonin standards (Lauweryns, gt gt., 1973a, 1977a). The NEBs appear to be well innervated by both afferent and efferent nerve fibers (Lauweryns gt gt., 1973b). 5 However, direct synaptic contacts have not been observed (Lauweryns gt gt., 1973a). The neuroepithelial bodies are composed of both granulated cells and modified Clara cells (Hung gt gt., 1979). Lauweryns gt gt. (1973a; 1973b; 1977d; 1978) have investigated their possible functional roles. In these studies, it was demonstrated that the NEBs responded to reduced alveolar oxygen content by degranulation and therefore were probably intrapulmonary chemoreceptors. Palisano and Kleinerman (1980) have also investigated the response of clusters as well as singly occurring pulmonary endocrine-like cells in rats exposed to NO Using silver 2. staining it was determined that the number of arygyrophilic cells decreased following exposure to N02. Whether singly occurring endocrine-like cells or neuroepithelial bodies respond to the same stimuli remains to be determined. In addition, any relationship with other epithelial cell types such as ciliated or goblet cells has not been investigated. Further investigations will be necessary to confirm and extend the functional roles of these and other intrapulmonary endocrine-like Cells. The classification of endocrine-like cells present many difficulties. These difficulties increase as our knowledge of structure, function and species differences expands. A single staining reaction and/or morphologic description of endocrine-like cells cannot fully characterize the functional significance of these cells. For example, numerous endocrine-like cells in various endocrine glands, and the 6 gastrointestinal and respiratory system give a positive chromaffin reaction. However, the biochemical content and function of these cells are different. On the basis of morphologic and cytochemical criteria, five uniquely different endocrine-like cells have recently been demon- strated in the intrapulmonary airways of adult hamster (Hoyt, personal communication). In view of these observa- tions, it is imperative that these endocrine-like cells be fully characterized by morphologic and histochemical methods in order to support effective investigations of their functional roles. Statement of the Problem The recent discovery of endogenous amine-containing cells in the extrapulmonary respiratory epithelium of adult rats (Ewen gt gl., 1972), rabbits (Cutz, gt gt., 1975; Day 23.31-r 1980), and human fetuses (Cutz gt gt., 1975), has generated great interest in the morphological and histo- chemical characterization of pulmonary amine-containing cells. With the Falck-Hillarp (1962) formaldehyde-induced fluorescent technique, the laryngeal and tracheal respiratory epithelium of albino rats has been found to contain endogenously (weak) fluorescent cells (Ewen, gt gl., 1972). These cells also demonstrated the ability to take up amino acid precursors (L-dihydroxyphenylaline and 5-hydroxytryptophan). Amino acid uptake and decarboxylation 7 is a characteristic feature of cells which belong to the APUD system of endocrine polypeptide cells (Pearse, 1969). Despite the ability of Ewen gt gt. (1972) to demonstrate the uptake of amino acid precursors, the morphological and cytochemical characteristics of these cells were very different from those of the APUD series (Pearse, 1969), as well as the tracheal amine-containing cells described by previous investigators. Therefore, the purpose of this study will be to investigate the morphologic and histochemical characteristics of amine-containing cells in the laryngeal and tracheal respiratory epithelium of adult rats. Research Plan and Rationale The present investigation will further elucidate certain morphologic and cytochemical characteristics of cells in adult rat laryngeal and tracheal respiratory epithelium which are believed to contain an endogenous biogenic monoamine. The fluorescent histochemical demonstration of endo- genous biogenic amines will be accomplished by two methods. Freeze-dried extrapulmonary airways will be treated with formaldehyde vapor and embedded in glycol methacrylate. The second method will involve the fixation of extrapulmonary airways with 6% liquid formaldehyde, embedding in Spurr's resin and subsequently exposing sections to hydrochloric acid vapor. The identity of the HCl-induced fluorophore will be evaluated by microspectrofluorimetric analysis. The 8 amine handling properties of rat extrapulmonary epithelial cells will be evaluated by administration of the amine precursors L-dihydroxyphenylalanine (L-DOPA) and S-hydroxy- tryptophan (S-HTP). The histochemical fluorescent method of Falck and Hillarp (1962) demonstrates the presence of monoamines at the cellular level. In an attempt to correlate directly fluorescence microscopy with subsequent electron microscopic observations of the same cells, tissues will be fixed with liquid formaldehyde, embedded in Spurr's resin and exposed to formaldehyde and HCl-vapors. Ultrathin sections will then be cut for electron microscopy. This will determine if the endogenous biogenic amine containing cell is a small granule cell (less than 2,000 A in diameter) or a globular leukocyte with large granules (greater than 2,000 A in diameter). Cytochemical methods will be used to elicit further information concerning the nature of the fluorescent epithe- lial cells. The cytochemical procedures will be carried out on freeze-dried, formaldehyde vapor-treated plastic embedded tissue sections so that direct correlation between fluorescent cells and their cytochemical characteristics may be demonstrated. PAS-lead hematoxylin positive cells will demonstrate the presence of endocrine-like cells with small granules. Methylene blue and alcian blue positive cells suggest the presence of acidic proteins and or acid 9 mucopolysaccharides. Ferric-ferricyanide positive cells will demonstrate the presence of amines. Acid phosphatase positive cells will demonstrate the presence of hydrolytic enzymes which are frequently associ- ated with lysosomes. Additional cytochemical procedures will also be carried out on cryostat sections of fresh frozen tissues for the demonstration of monoamine oxidase (MAO), and freeze dried cryostat sections for the demonstration of histamine (O-phthalaldehyde method) and tryptamine (ozone method). Significance This investigation will further elucidate the current knowledge concerning extrapulmonary endocrine-like cell structure. At present, the potential biological importance of these cells which synthesize and secrete biogenic amines and/or peptide hormones, can only be surmised. The knowledge of how these cells fit into the function of the extra- pulmonary airways and the organism as a whole is, as yet, unknown. A morphologic and cytochemical characterization of endocrine-like cells will allow future studies of the function and dysfunction of these cells. Limitations 1. This study is restricted to the laryngeal and tracheal epithelium of adult albino rats. 2. The fluorescent and light microscopic histochemical characterization of these extrapulmonary epithelial cells is 10 restricted to freeze-dried and liquid formaldehyde fixed tissues. 3. The cytochemical characterization of the endocrine- like cells in extrapulmonary airways is restricted only to the methods employed in this study. No attempt was made to characterize the full cytochemical potential of these cells. 4. Electron microscopic observation and correlation is restricted to formaldehyde fixed tissue. 5. The microspectrofluorimetric analysis of the fluorophore is restricted tofkeeze dried or liquid formal— dehyde, HCl-induced fluorescence. 6. The microspectrofluorimetric standards used in this study were the fluorescent cells of the duodenum. These cells have been reported to contain serotonin. CHAPTER II LITERATURE REVIEW Feyrter (1938) described a system of epithelial cells in the gastrointestinal tract with characteristic morpho- logic and histochemical pr0perties. Because of their almost water-clear cytoplasm after hematoxylin and eosin staining, he called them 'Helle-Zellin' cells. He postulated that these clear cells were part of a "diffuse endocrine epithe- lial organ" and suggested the possibility of finding these cells in the respiratory epithelium. The first description of these clear-cells was reported by Fr6hlich (1949) in the bronchial epithelium of man as well as several animal species. Pearse (1966) investigated the cytochemical properties of different endocrine-like cell types in several animals. In an attempt to provide a conceptual framework for the diffuse endocrine-like cell system, Pearse gt gt. (1968) proposed the APUD (Amine Precursor Uptake and Decarboxyla- tion) concept. The APUD series of endocrine-like cells described by Pearse and the Helle-Zellin System of endocrine cells described by Feyrter are almost identical. The major difference between these two theories is the embryologic 11 12 origin of the endocrine-like cells. Feyrter (1953) suggested that the Helle-Zellin cells arose from epithelial cells. Pearse gt gt. (1976) demonstrated a neural crestorigin of some endocrine-like cells, however, Andrew (1974) presented evidence disproving the neural crest origin of gastrointesti- nal endocrine cells. Despite the differences regarding origin, the morpho- logic, physiologic, and pharmacologic characterization of respiratory endocrine-like cells has continued. The ultra- structural characteristics of a specific type of endocrine- like cell was first described in human bronchial glands by Bensch gt gt.(l965) and in the bronchial epithelium by Gmelick gt gt. (1967). Since this time, numerous studies have investigated the histochemical (Hage 1973a; Dey gt gt., 1980), ultrastructural (Kent gt gt., 1966; Lauweryns gt gt., 1972b); and Cutz gt gt., 1975), and physiologic (Lauweryns gt gt., 1977b, 1978; Echt gt gt., 1980) functions of endocrine-like cells in respiratory epithelium. Trachea and Extrgpulmonary Bronchial Respiratory_§pithelium of Humans Fetal and Infant The presence of endocrine-like cells in the tracheal respiratory epithelium of human fetuses, infants, and children was reported first by Cutz gt gt. (1975). The histochemical fluorescent method of Falck and mean (1965) 13 demonstrated only a few weakly fluorescent yellow-green cells. However, after tg ttttg incubation with L-DOPA, the numbers of fluorescent cells and the intensity of fluores- cence increased. Argyrophilic positive cells were scattered throughout the epithelium. The cells were predominantly single, but occasionally groups of two or three cells were observed. Neuroepithelial bodies (Lauweryns gt gt., 1972b) were never observed in the tracheal epithelium. However, it should be noted that a detailed morphometric study was not performed. The fluorescent and argyrophilic cells were triangularly shaped, with the base resting on the basement membrane and the apex projecting towards the lumen. Argen- taffin positive cells were not observed. Fetal and Infant Ultrastructure Cutz gt gt. (1975) also investigated the ultrastruc- tural characteristics of endocrine-like tracheal cells in human fetuses, newborn infants, and children. Granulated basal cells were found with an electron-lucent cytoplasm containing numerous dense-core, membrane-bound vesicles of the neurosecretory type. The vesicles had a mean diameter of 1150 A, while the space surrounding the dense-core was 160-180 A. Additional cytoplasmic features common to the granulated cells were: small mitochondria, moderate amounts of smooth and rough endoplasmic reticulum, and Golgi complexes. 14 M .The presence of endocrine-like cells in the tracheal respiratory epithelium of human adults has not been reported. In summary, the tracheal respiratory epithelium of human fetuses, infants, and children contain single occurring fluorescent and argyrophilic positive Cells (Cutz gt gt., 1975). Only a very weak endogenous fluorescence was reported. However, an intense fluorescence was noted after incubation with L—DOPA. Microspectrofluorimetric analysis has not been performed on the extrapulmonary endocrine-like cell of fetal, infant, or adult humans. The most charac- teristic feature of these cells at the ultrastructural level was the presence of dense-core vesicles (approximately 1150 A) surrounded by a clear area and limited by a single unit membrane. The cytoplasmic matrix contained small mitochon- dria, moderate amounts of smooth and rough endoplasmic reticulum and a prominent Golgi complex. Intrapulmonary Single Endocrine-like Cells and Neuroepithelial Bodies in Humans m Endocrine-like cells in the bronchial respiratory epithelium of human fetuses were studied by Hage (1971, 1972). With light microscopy the cells appeared pyramidal or bottle-shaped and were located close to the basement membrane. Formaldehyde induced fluorescence produced a few +1aov .mm mm xooo +1vac .mm mm swam: Iavac .mm mm swam: saxowsu almsc .mm mm man «Ame. mcam uao «Ache .mm mm 6mmmwwmcom IAHmI .mm mm :wmnmmemaum II II Ilam.om.aav hHaIWm awn +1~m.omc .Hm ww ucom +1~m1 .Hmluwlauuam +1mac .Hm um mono Ixmav .H um muse +1mn. .Hm um "use banana ...Ilvac .mm mm snaxuflx +Am>NI.Hw mm uao: «Amnv .mm mm cwxouom .Am>.mhv .Hm Wm huouwon «Ammv .mm mm mmxwumm mam mmcwsu +1~av .Ha mm cmzm Ixmav .Ha ow usom yam «Amp. .Hw mm saxouom +A~hv .Hm um cowoaum I mmsoz +Amnv .mm mm nusu +Amnv .MM mm nuuo an: I 3: .m Mm 35o gang—.2 +A~mv .Hm no :flxouom csoun manuaq m0>¢ «Hagan: uaav< wumcooz wsuom wwd3mH€ rmdzozqamdmfixm .oo>ummno ouo3 mHHmo oxwauocwuoocco mo mmsouo no mamcflm ma mwwooom uo: cap wonusm ecu umnu mwumOAocH Amv "ouoz .cmE can «unseen cw moon m50wum> um mansuwo >um:0&asmmuuxo ca omumuum:OEmo coon m>mc Lodz3 mHHoo mwaImCMHUOpcm wo Aacav monoum can A+v wamcfimll.a ounce 16 weakly fluorescent cells (control), but after incubation with L-DOPA the number of fluorescent cells increased. The number and distribution of control fluorescent cells was similar to the number and distribution of cells that were stained with lead-hematoxylin, HCl-toluidine blue and silver techniques for argyrophilia. Hage (1973a) investigated the amine handling properties of endocrine-like cells in the bronchial epithelium of human fetuses. Lung segments were incubated at 37°C in Tyrodes solution containing the following agents: pargyline (an irreversible inhibitor of monoamine oxidase); D- or L-3,4- dihydroxyphenylalanine (D- or L-DOPA); D- or L-5-hydroxy- tryptophan (D- or l-S-HTP); Ro4-4602 (an inhibitor of decarboxylating enzyme) followed by L-DOPA or l-S-HTP; dopamine; and 5-hydroxytryptamine (5-HT). Control, formal- dehyde vapor-treated tissues demonstrated a few greenish- yellow fluorescent cells in the bronchial epithelium. The cells were pyramidal or oval shaped and the fluorescence was concentrated in the basal or paranuclear cytoplasm. With the administration of pargyline, the fluorescence intensity increased slightly. After incubation with D-DOPA or S-HTP, the number of fluorescent cells also increased. Tissues incubated with L—DOPA or 5-HTP resulted in an increased number of green or yellow fluorescent epithelial cells. The L-DOPA incubated specimens emitted the most intense fluorescence and frequently, both nucleus and 17 cytoplasm appeared to be fluorescent. After L-S-HTP incubation, the fluorescence of most respiratory epithelial cells was restricted to the cytOplasm. Specimens pretreated with Ro4-4602, followed by L-DOPA or S-HTP produced a weaker fluorescence than those incubated with precursors alone. Incubation with dopamine or S-HTP produced a weak fluorescence, and the number of cells was smaller than after incubation with precursor amino acid. Wharton gt gt.(l978) reported the presence of bobesin-like immunoreactivity in human fetal lung segments. Single cells as well as groups of cells with bombesin-like immunoreactivity were identified in the bronchial and bron- chiolar epithelium. Fetal Ultrastructure The ultrastructural appearance of granulated cells in the pulmonary epithelium of human fetuses was studied by Cutz gt gt.(l972). Lung samples were fixed with 5% glutaral- dehyde in cacodylate buffer (pH 7.4) for two hours at room temperature and then post-fixed in 1% 0504. Endocrine- like cells were found in all lungs and appeared as single cells or in groups. The cells were always located along the basement membrane and frequently formed pseudopod-like processes which extended between adjacent epithelial cells. A consistent feature was the presence of cytoplasmic elec- O tron-dense granules (dense-core vesicles) 1000-3000 A in 18 diameter, surrounded by a single limiting membrane. The number and size of the granules varied. In some endocrine- 1ike cells, the dense-core vesicles were randomly distributed while in other cells the granules were concentrated at the base close to the basement membrane. Other cytoplasmic organelles included: numerous vesicles of smooth and rough endoplasmic reticulum, many mitochondria, microfibrillar material, and a small Golgi apparatus. Hage (1973b) also investigated the ultrastructural characteristics of pulmonary endocrine-like cells of human fetuses. All endocrine-like respiratory epithelial cells were adjacent to or resting on the basement membrane with pseudopod—like extensions penetrating deeply into the mesenchymal tissues. The cell shape was variable while the nucleus was round or oval and basally located. Smooth endoplasmic reticulum was present along with some rough endoplasmic reticulum, mitochondria, and a prominent Golgi complex. Contacts between nerve fibers and endocrine-like cells were never observed. On the basis of the morphology of the secretory granules three types of endocrine-like cells were postulated. Type I cells were extremely variable in shape. The shape ranged from polygonal to narrow columnar or horizontally elongated. The cell seldom reached the luminal surface and the cytoplasm appeared more pale than other endocrine-like cells. Randomly distributed secretory granules were few in number but two 19 distinct populations of granules could be identified. Some granules were small (approximately 1100 A) while other gran- ules were considerably larger. The smaller granules contained dense-cores which were separated from their surrounding mem- branes by narrow, clear spaces (approximately 180 A). The small granules were also argyrophilic positive. The larger granules did not contain a dense-core and were both argen- taffin positive and argyrophilic. This cell type was located individually throughout the bronchial tree or in groups of up to five cells. Type II cells were pryamidal or polygonal in shape and occasionally reached the luminal surface. Cytoplasmic extensions were also observed. The cytoplasm appeared much darker than other endocrine-like cells due to the presence of thick bundles of microfilaments and a greater number of cytoplasmic granules. The dense-core vesicles were uniformly round with little variability in the electron density of their cores. The granules were confined to the basal part of the cells with a mean diameter of 1400 A. These granules were argentaffin negative and argyrophilic positive. Type II cells were also located throughout the bronchial tree. In the larger bronchi they were found in groups along with endocrine-like cells of the other types. Only a few of the Type III cells were observed. This cell type was oval or pyramidal with a high nucleus to cytoplasm ratio and few cytoplasmic organelles. The cell was never observed to make contact with the lumen of the 20 bronchi. The secretory granules were round, about 1900 A in diameter, and were nearly always located in the basal part of the cell. The electron dense-core was homogenous and surrounded by a membrane. The argyrophilic reaction was positive while the argentaffin reaction was negative. These cells were only observed in the larger bronchi. A fourth type, Type IV, of endocrine-like cell was also identified in the pulmonary epithelium of fetuses with a crown-rump length between 20 and 54 mm. These cells differed from the Type III cell by having a higher content of cytoplasmic glycogen and by the appearance of the secretory granules. The granules were more variable in both size, shape, and electron density. This cell type was only found in the larger bronchial tubules. No endocrine cells were identified in a fetus with a crown rump length of 20 mm or less. Infant Lauweryns gt gt.(l969 and 1970) reported the presence of endocrine cells, in the bronchial and bronchiolar respir- atory epithelium of newborn human infants. The histochemical fluorescent technique according to Falck and mean (1965) demonstrated the presence of amine containing cells. Argyrophilic staining was also positive. The cells were triangular to pyramidal in shape, with their bases resting on the basement membrane. A predilection in the distribution of the argyrophilic positive cells was not observed. These 21 investigators were unable to demonstrate argentaffin or chromaffin positive cells. Many clusters of argyrophilic positive cells in human infant bronchial and bronchiolar epithelium were observed by Lauweryns and Peuskens (1972a). Silver impregnation and Falck's histochemical amine technique were utilized to demonstrate and characterize these groups of cells. Because of the unique structural relationship of these endocrine- 1ike cells, Lauweryns called them neuroepithelial bodies (NEBs). The spherical to ovoid NEBs were located periodically along the entire respiratory bronchial and bronchiolar tree and usually protruded slightly into the lumen. The height of the NEBs varied from 19 to 74 mu with an average of 35 mu. In hematoxylin-eosin stained sections, the more basal cells had clear cytoplasm and formed intercalated cone-shaped corpuscles. The apical part of the NEB protruded into the lumen above the level of the ciliated epithelial cells and was composed of non-ciliated cuboidal cells. The base of the cone rested on the basement membrane and at times displaced it into the lamina propria. The cells in the peripheral layer were smaller and flatter than the inner cells. The corpuscular cells were both fluorescent and argyrophilic positive but the peripheral cells were not. Silver impregnation demonstrated numerous nerve fibers ramifying into the NEBs. Fluorescent histochemistry revealed 22 nerve fibers with a distinct green fluorescence in the immediate vicinity of these corpuscles and thus, suggested adrenergic innervation. Single cells and groups of cells with bobesin-like immunoreactively have also been observed in the bronchial and bronchiolar epithelium of neonatal human lung (Wharten gt gt., 1978) . Infant Ultrastructure The ultrastructural appearance of granulated respiratory epithelial cells in the lungs of human infants has been described by several investigators. A consistent feature was the presence of dense-core secretory granules. Lauweryns gt gt.(1970) using glutaraldehyde fixed lungs, noted that the endocrine-like cell membrane had typical desmosomal attachments with its neighboring cells. The granulated vesicles ranged from 800 to 1500 A in diameter. The cyto- plasm contained many mitochondria, filaments, free ribosomes, a small Golgi-complex, small amounts of rough and smooth endoplasmic reticulum, and some glycogen. The most striking discovery was the presence of nerve endings in close association with the groups of granulated cells. The intra- mucosal nerve fibers, which lacked Schwann cell envelopment, were characterized by an electron transparent axoplasm and several beaded enlargements along the course of the axon. The enlargements contained numerous small granular and agranular vesicles. Lauweryns believed that these variosities 23 were highly suggestive of "direct contacts" between the granulated cells and the nervous system. These nerve fibers were located deep to the basement membrane and in intimate relationship to the granulated cells, being separated by about 200 i. Rosan and Lauweryns (1971) noted granulated respiratory epithelial cells in the lungs of prematurely born infants. Lung samples from two infants, 600 and 1,600 g, were fixed in phosphate buffered glutaraldehyde (pH 7.2), post:fixed in osmium and embedded in Epon. The endocrine-like cells were: cuboidal; protruded into the submucosa; closely associated with fenestrated capillaries; not in contact with the lumen; and devoid of long narrow protoplasmic processes between or around cells. The presence of cytoplasmic elec- tron-dense granules (800 to 1000 A in diameter), surrounded by a clear area and a unit membrane (1100 to 1300 A in diameter) were the most characteristic features of these endocrine-like cells. The relative frequency of cells was greater in the 600 9 infant. In the smaller infant the granules were more numerous, denser, and more uniform in size than in the 1,600 9 infant. Adult Hage (1973a) investigated the amine-handling properties of adult human main bronchial epithelium. Human bronchial segments were incubated in L-DOPA or dopamine. In both 24 control and dopamine treated animals, fluorogenic amines were not demonstrated with the formaldehyde—induced fluorescent technique. However, an intense green fluorescence with an exclusively cytoplasmic localization was noted after treatment with the precursor amino acid L-DOPA. Tateishi gt gt. (1973) reported that the epithelium of bronchi and bronchioles of forty-five human, Mongolian adults (23 to 71 years of age) contained a number of distinctly argyrophilic cells. The cells occurred singly or in clusters; the cell clusters tended to occur more frequently in the smaller bronchi or in the bronchioles. By light microscopy, triangularly—shaped cells were seen near the basement membrane. Both single and grouped cells had cytoplasmic processes extending along the basement membrane. The number of argyrophilic cells tended to increase as the caliber of bronchi decreased in size. However, the number of cells in the terminal bronchioles was small. The cells frequently occurred in close association with goblet cells. There appeared to be no relationship between age of the subjects and the number of cells present, however, women tended to have more argyrophilic cells, particularly in the larger bronchi. Hage (1973c) also examined the histological and histo- chemical features of human surgical lung specimens from six bronchial carcinoid tumor cases. Lung samples were fixed with a variety of fixing agents and subsequently embedded 25 in paraffin. Sections were stained by lead-hematoxylin, HCl-toluidine blue, Masson-Hamperl's and Grimelius's silver method, zanthydrol, and diazonium reaction to demonstrate endocrine cell granules. Her previous classification (Type I, II, III, and VI) of endocrine cell granules was utilized to characterize the endocrine cells (Hage, 1973b). Cells of the Type II category were unreactive to all staining methods except for the agyrophilic silver method. Type III cells did not conclusively react with any of the stains, however, electron microscopy did reveal their presence. The polymorphic granules, observed only in tumors, were argentaffin, diazonium, argyrophilic, lead-hematoxylin, and HCl-toluidine blue positive. In 1975, Lauweryns gt gt. identified NEBs in lung tissue biopsed from children and adults. Lung segments were removed and fixed in Bouin's fluid. Hematoxylin and eosin in conjunction with silver staining revealed clusters of 4 to 10 argyrophilic positive cells in the bronchial, bronchiolar, and alveolar respiratory epithelial lining. The cellular morphology was similar to that previously described by Lauweryns gt gt. (1972a) for human fetal lung tissue. At the level of the bronchi, the NEBs were 20 to 40 mu wide and 15 mu high; the alveolar NEBs were about the same width (30 to 36 mu) but were shorter (6 mu). The individual cells comprising the bronchiolar NEBs were 4 to 8 mu wide and 15 mu high. The cells of the alveolar NEBs were the same width but only 6 mu high. 26 Adult human lungs, prepared for routine light micro- scopy, were studied by Hage gt gt. (1977) for the presence of endocrine-like respiratory epithelial cells. In the lobar and proximal segmental bronchi, a few singly placed cells were reactive to the argyrophil methods of Grimelius (1968). Argyrophilic cells were rarely present in the bronchiolar epithelium, alveolar lining cells, or in the acini and ducts of the parabronchial glands. The cells were triangular or flask shaped, located at or near the basal lamina, without luminal contact, and only occasionally were observed in groups. No positive reaction was obtained by methods demonstrating biogenic amines (diazonium nor argentaffin stains) or by methods staining endocrine cell granules (lead hematoxylin or HCl-toluidine blue). No endogenous fluorescent cells were demonstrated by the amine formaldehyde-induced fluorescent technique. However, incubation of lung segments with L-DOPA or S-HTP produced an intense green or weak yellow fluorescence respectively in singly occurring cells. Fluorescent cells in groups or clusters were not observed. Adult Ultrastructure Bensch gt gt. (1964) investigated the ultrastructural characteristics of endocrine-like cells in adult human bronchial glands. Segments of normal human bronchus were 27 fixed by immersion in glutaraldehyde and subsequently in osmium. A rare cell type was frequently observed at the junction between mucus and serious cells. The characteristic feature of this cell was the presence of electron opaque cytoplasmic granules of the neurosecretory type. The granules were of uniform appearance and size, averaging 1400 A in diameter. The core was filled with a homogenous electron-opaque substance surrounded by a clear area encircled by a membrane. No pattern of distribution was obvious, but the majority of these granules appeared to be located between the nucleus and the basal surface of the cell. At the base of these cells the cytoplasm frequently formed pseudopod-like processes which extended from the cell body. These cell processes contained granules (1400 A in diameter) of the neurosecretory type. Similar cell processes were also found in the intercellular space between serous cells. The granules in these cell processes were different from those previously described in that: the neurosecretory granules were smaller (900—1000 A); and there were large numbers of empty vesicles (500-600 A diameter) resembling pinocytotic or synaptic vesicles. Synapses of the outer cell membrane were not observed. The ultrastructural characteristics of small granule endocrine-like cells in adult human bronchiolar surface respiratory epithelium was first reported by Gmelich gt gt. 28 (1967). Lung tissue from a 68 year old woman was surgically removed and processed for routine light and electron microscopic observations. Lung segments prepared for electron microscopy were fixed for 24 hours in a 3.5% buf- fered glutaraldehyde solution. The tissue studied consisted of normal areas and also several tumor nodules. Light micro- scopy revealed that one nodule appeared to be a mucus- producing adenocarcinoma while the other nodules resembled a peripheral bronchial carcinoma. Electron microscopy of the normal bronchioles revealed cuboidal ciliated epithelial cells which alternated with cells with relatively short plump pseudopods on a luminal surface. Scattered sparsely throughout the bronchiolar epithelial lining were a number of electron-translucent cells. These cells never reached the luminal surface. The most conspicuous cytologic feature was the presence of great numbers of intracytoplasmic granules, 1200 to 1500 A in diameter. The cores of these granules were homogeneously electron opaque and surrounded by a zone of electron transparency and encircled by a unit membrane. Single cells, as well as clusters were present above the bronchiolar basement membrane. The carcinoid tumor consisted of all of the normally observed cell types, including the dense-core endocrine-like cell. Terzakis gt gt. (1972) investigated the ultrastructural characteristics of endocrine-like epithelial cells in adult 29 human segmental bronchi. Segments of human bronchi were removed from 14 surgical cases. Four of the cases had no neoplastic disease while ten of the cases had lung carcinoma. The tissues were immersion fixed for at least 24 hours in 0.075M cacodylate-buffered glutaraldehyde followed by 2 hours in veronal acetate-buffered osmium tetroxide, dehy- drated in alcohols, and embedded in Epon. The bronchial respiratory epithelium consisted of ciliated, basal, goblet, and irregularly shaped cells which were more electron translucent than the surrounding cells. These cells of low cytoplasmic density, usually occurred singly but groups of two to twelve were also observed. The cells were located along the basement membrane and had small oval nuclei. The cytoplasm contained numerous electron- dense granules 800 to 1700 g in diameter. These granules were surrounded by a clear area and a single trilaminar membrane. Other cytoplasmic organelles of these endocrine- like cells included: numerous vesicles of smooth and rough endoplasmic reticulum, mitochondria, Golgi apparatus, free ribosomes, fine filaments, and rare lipid and lipofuchsin granules. No evidence of any synapse was observed in relation to the granule-containing cells. The cell surface interdigitated with neighboring cells but no desmosomes nor terminal bars were noted. However, several goblet cells contained small, dense, membrane-bound granules which appeared identical to those observed in the endocrine-like epithelial cells. 30 Since carcinoid tumors of the gastrointestinal tract are derived from endocrine cells in the intestinal mucosa, Hage (1973c) investigated the ultrastructural similarities between endocrine-like cells in the pulmonary respiratory epithelium of human fetuses and the cells of bronchial carcinoid tumors. Lung tumors from six adults were fixed in cacodylate buffered glutaraldehyde (3%), post-osmicated (1%), and embedded in Epon. Nearly all of the tumor cells contained secretory granules. The number and appearance of these granules were variable. Three categories of secretory granules were postulated according to her previous classi- fication of endocrine-like cells in the pulmonary epithelium of human fetuses (Hage 1973b). Type I cells were not observed in any tumor material. Cells which contained small, round, membrane-bound granules of uniform shape and size, characteristic of Type II cells, were observed in all carcinoid tumors. The secretory granules were concentrated in the cytoplasmic extensions. In two cases, a few cells contained larger, round granules of uniform size and bound by a discontinous membrane. The granules of these cells (Type III) were evenly distributed in the cell body. A cell type not observed in the bronchial epithelium of human fetuses was observed in three tumor cases. These cells contained polymorphic granules. She concluded that these cells were very similar to enterochromaffin cells of the human gastrointestinal tract. 31 Electron microscopy examination of normal, adult pulmonary epithelium was also performed by Hage gt a1. (1977). Four adult lung samples from areas with no obvious macroscopic lesions, were fixed in 3% glutaraldehyde buffered cacodylate (pH 7.4), post-fixed in osmium, and stained with lead-citrate and uranyl-acetate. The charac- teristic neuroepithelial bodies (Lauweryns 33 31., 1972a), were only rarely observed. Endocrine-like cells occurred singly in the respiratory epithelium of lobar bronchi, segmental bronchi, acini, and ducts of the parabronchial glands, but more often in clusters of three to six in the epithelium of the lower segmental bronchi and bronchioles. The characteristic feature of the endocrine-like cell was the presence of small round cytoplasmic secretory granules. These granules, 1100-1400 g in diameter, were enclosed by unit membranes and contained dense-cores surrounded by narrow, clear spaces. The cytoplasm was rich in mitochon- dria, microfilaments, a prominent Golgi complex as well as smooth and rough endoplasmic reticulum. The lateral cell membranes were straight with a few infoldings into adjacent cells. Several desmosomal structures were noted. The endocrine-like cells observed in the adult lungs were similar to the endocrine-like cells of Type II described previously in the human fetal lung (Hage 1973b). It has been demonstrated that the intrapulmonary airways of fetal, infant, and adult human lung contain numerous 32 groups (Hage 1973a; Lauweryns gt gt., 1972a, 1975) as well as single endocrine-like cells (Cutz gt gt., 1971; and Tateishi gt gt., 1973). The single, argerphilic cells are triangular or flask shaped, located at or near the basal lamina and are without luminal contact (Tateishi gt gt., 1973; Hage gt gt., 1977). With the amine tracing fluores- cence technique (Falck gt gt., 1965), no endogenously fluorescent cells have been demonstrated in adult human lung. Incubation of lung segments with L-DOPA or L-HTP, however, has revealed green and yellow fluorescent cells respectively (Hage gt gt., 1977). In fetal human lung, some singly occurring cells emit an endogenous green fluores- cence (Hage, 1973a). An increase in the number of green fluorescent cells has also been demonstrated after incuba- tion with L-DOPA (Hage, 1973a). Groups of argyrophilic and fluorescent cells have also been identified in infant and adult human lung (Lauweryns gt gt., 1969, 1970, 1972a, 1975). Because of the unique structural relationship of these endocrine-like cells, Lauweryns gt gt. (1972a) called them neuroepithelial bodies (NEBs). In infant and adult, the NEBs were: spherical to ovoid, periodically located along the entire respiratory bronchial, bronchiolar and alveolar epithelium, and usually protruded slightly into the lumen (Lauweryns gt gt., 1972a). Silver impregnation demonstrated numerous nerve fibers ramifying into the NEBs. Fluorescent histochemistry 33 suggested an adrenergic component in the nerve fibers (Lauweryns gt gt., 1972a). The presence of single and groups of endocrine-like cells with bombesin-like (polypep- tide-like) immunoreactivity has also been demonstrated in the bronchial and bronchiolar epithelium in fetal and neonatal human lung (Wharton gt gt., 1978). Microspectro- fluorimetric analysis of single and groups of fluorescent cells in human intrapulmonary airways has not been reported. The fine structure of single (Gmelich gt gt., 1967; Cutz gt gt., 1971; Hage 1973b) and groups (Cutz gt gt., 1971; Hage 1973; and Lauweryns gt gt., 1970) of endocrine- like cells in respiratory epithelium has been described in fetal, infant, and adult lung tissue. Single cells were always located along the basement membrane (Cutz gt gt., 1972; and Hage 1973b). In addition, pseudopod-like processes were frequently observed to extend between adjacent epithelial cells (Gmelich gt gt., 1967; Cutz gt_gl., 1971). No relationship between single occurring endocrine- like cells and nerve fibers has been reported. The ultra- structure of groups of endocrine-like cells has been extensively studied by Lauweryns gt gt. (1970) and Rosan gt gt. (1971 and 1972). Lauweryns EE.ELr(l970) noted numerous varicose nerve endings in close association with the neuroepithelial bodies, separated by approximately 200 g. From these observations, it was suggested that "direct 34 contacts" were present between the granulated cells and the nervous system. The most characteristic cytoplasmic feature of these endocrine-like cells in respiratory epithelium is the presence of dense-core granules (Cutz gt gt., 1972; Hage gt gt., 1973b). Several studies have attempted to classify these granulated cells according to the morphology of their secretory granules (Hage gt gt., 1973b). However, the size, shape, and distribution of the secretory granules is quite variable. One possible explanation for the variability could be the many different methods of tissue fixation. For example, it has been demonstrated that the distribution pattern and structure of vesicles within the carotid body show specific changes according to the method of tissue preparation for microscopy (Hans, 1977). The diameters of the dense-cored vesicles ranged from 800 A in the infant (Rosan gt gt., 1971) to 1900 i (Hage, 1973b) in the fetus. Additional cytoplasmic organelles included: many small mitochondria, moderate amounts of smooth and rough endo— plasmic reticulum, many free ribosomes, filaments, and a small Golgi complex (Lauweryns gt gt., 1970; Hage gt gt., 1977). 35 Laryngeal, Tracheal, and Extrgpulmonaty Bronchial Respiratory Epithelium of Animals 23a; Cutz gt gt.(l975) investigated the endocrine-like cells of fetal lambs and rabbits as well as infant rabbit and armadillo. In all animals studied, singly occurring argyro- philic cells were observed. The cells were triangular with their bases resting on the basement membrane. Frequently, a thin apical extension was observed to reach the lumen. Argentaffin positive cells were not observed. In control animals, only a few weakly fluorescent cells were demon- strated by the formaldehyde induced—fluorescence technique. However, following injection or 12 ytttg incubation with L-DOPA, a significant number of fluorescent cells were observed in all animals studied. Fetal Ultrastructure Cutz gt gt.(1975) also investigated the ultrastruc- tural characteristics of endocrine-like respiratory epithe- lial cells in fetal lambs and rabbits, and infant. rabbits and armadillo. The general appearance of the cytoplasmic organelles in these cells was similar to that of humans (Cutz gt gt., 1975). Two distinctively different endocrine- like cells in both lamb and armadillo were distinguished on the basis of their dense-core vesicle size and morphology. The dense-core vesicles in granulated cells designated 36 Type I were large (1700 A in diameter) and had a homo- genously dense central core. In Type II cells the DCVs were generally smaller, 1200 A in diameter. In rabbit only one type of DCV was identified which tended to be more pleomorphic with a mean diameter of 1400 A. Neonatal and Adult Ericson gt gt. (1972) reported the presence of endo- crine-like cells in the tracheo-bronchial mucosa of the mouse. Fluorogenic amines were not demonstrated with the formaldehyde-induced fluorescent technique in the epithelium of control mice. However, after the administration of DOPA or S-HTP, an intense formaldehyde-induced fluorescence selectively accumulated in cells. Subsequently, these same cells were found to be argyrophilic. The endocrine-like cells were most numerous in the epithelium of the trachea just below the cricoid cartilage. In the lower parts of the trachea and main bronchi the number of fluorescent cells decreased. In the bronchi, fluorescent cells were mainly in clusters (5 to 20 cells). The cells were triangular with their basal portions resting on the basement membrane. Endocrine-like cells were found in the laryngeal epithelium of rats (Ewen gt gt., 1972) and adult guinea-pigs (Kirkeby gt gt., 1977). Ewen gt gt. (1972) were unable to demonstrate argyrophilic cells in the larynx, but did report that these cells were eosinophilic, PAS positive, and showed 37 a slight metachromasia with toluidine blue. The granules were intensely fluorescent in the animals treated with L-DOPA and L-S-HTP. Without pretreatment, only weakly fluorescent granules could be seen. Pretreatment of animals with Ro4-4602, an L-amino acid decarboxylase inhibitor, prevented the appearance of intense fluorescence in cells when the animal was subsequently injected with L-DOPA. In contrast, Kirkeby gt gt. (1977) was able to identify argyrophilic cells in both the lining epithelium and in the small glands of the guinea pig larynx. The cells were either in groups of up to 15 to 20 cells, or single. The cells were pyramidally shaped with their bases located on the basement membrane. Argyrophilic cells were not present in the superior portion of the larynx but were found mainly in the middle third. Serotonin containing endocrine-like cells in the trachea and lung of adult rats were identified by fluorescent histochemistry as well as routine light microscopy (Sorokin gt gt., 1978; Hoyt gt gt., 1979; and Echt gt gt., 1980). Tissue segments were fixed with buffered formaldehyde and embedded in glycol methacrylate. Endogenous fluorogenic amines were not demonstrated in any tracheal epithelial cells. but were evident in L-DOPA treated animals. In control ani- mals, however, fluorescence was present in a varity of cells known to contain serotonin (mast cell and neuroepithelial 38 bodies). Following administration of 5-HTP an intense yellow fluorescence was observed in scattered single tracheal epithelial cells. In control animals, fluorescence was not observed in plastic embedded sections of adrenal medulla nor thyroid "C" cells. Because of these results, it was suggested that this method selectively demonstrates the monoamine, S-HT. Complementary monoamine fluorescence techniques on plastic sections of trachea from control animals prior to additional histologic staining demonstrated that fluores- cent cells were identical to those stained by PAS-lead hematoxylin (Sorokin EE.El-r 1978). The morphologic, morphometric, and histochemical characteristics of endocrine-like cells in the tracheal epithelium of adult rabbits were studied by Dey and Echt (1976), Dey (1979), and Dey gt gt. (1980). Endogenously fluorescent cells were found to be both ferric-ferricyanide and argyrophilic positive. Single, triangularly-shaped cells were located throughout the entire trachea. Micro- spectrofluorimetric analysis of the fluorescent cytoplasm in individual cells, and sodium borohydride treatment (Corrodi gt gt., 1964) demonstrated that the fluorescent cells con- tained serotonin. The ventral aspects of the tracheal epithelium contained significantly more fluorescent cells than the dorsal region. In addition, the ventral cranial region had significantly more cells than any other region. The number of fluorescent cells was not affected by 39 pretreatment with L-DOPA. However, pretreatment with reserpine resulted in a significant decrease in cell numbers when compared with control rabbits. Neonatal and Adult Ultrastructure The ultrastructure of the rat laryngeal granulated cells was also studied by Ewen gt gt. (1972). The laryngeal mucosa contained numerous cells with large, 5,000 to 20,000 A in diameter, pleomorphic granules. These granules were densely osmiophilic and randomly dispersed throughout the cytoplasm, though mainly on the luminal side of the nucleus. The granulated cells seldom reached the luminal surface. Mitochondria and smooth endoplasmic reticulum were present in the cytoplasm of all granulated cells. Tracheal epithelium from adult mice (Ericson gt gt., 1972) and adult rabbit (Cutz gt gt., 1975) contain numerous granulated cells. In adult mice the greatest number of endocrine-like cells were found in the upper trachea. The cells were variable in shape and rested on the basement membrane. The cytoplasm contained spherical secretory vesicles 800—1000 A in diameter and the space surrounding the dense-core was 100-200 A. The outer limiting membrane was 70-80 A thick. In addition, the cytoplasm contained: microfibrillar material, smooth and rough endoplasmic reticulum, mitochondria, and Golgi complexes. Storage of amine was suggested by the intense staining using the argentaffin silver method of cytoplasmic granules 40 in DOPA treated animals. Only a very weak argyrophilic reaction was observed. After administration of 3H-DOPA or 3H-S-HTP, autoradiographic silver grains selectively appeared over all tracheal endocrine-like cells. Cutz gt gt. (1975) also noted similar cytoplasmic organelles in adult rabbits. However, the DCV tended to be more pleomorphic and larger (1400 A in diameter). The core was uniformly homogenous and the halo almost indistinguishable. Studies of adult rat (Jeffery gt gt., 1973) and adult cat (Das gt gt., 1978, Hung gt gt., 1975) also demonstrated endocrine-like cells in the trachea. However, the endocrine-like cells were only rarely found in association with nerve fibers. The ultrastructural studies of Jeffery gt gt. (1975) revealed the presence of dense-core secretory vesicles in the tracheal cells of adult rat. Tracheas were removed and fixed by immersion in 1% buffered glutaraldehyde and post- fixed in osmium. Occasionally, granulated cells with numerous dense-core, membrane bound vesicle (1300 A in dia- meter) were observed. A significant number of globular leukocytes were also noted. This cell was characterized by the presence of very large, homogenous, electron dense granules (6,250 A mean diameter). Cell projections were numerous and filiform. The granules of the globular leuko- cyte were significantly larger than the granules of the connective tissue mast cell. The concentration of globular leukocytes was greatest in the upper trachea. 41 Walsh and McLelland (1974), investigating the innerva- tion of the tracheal epithelium of domestic chickens, noted the presence of endocrine-like cells. The granulated cells contained DCVs which were 1400 A in diameter. These granules were surrounded by a clear area and followed by a single membrane. Two types of vesicles, agranular and granular, were present in the axon fibers. Evidence for synaptic contact between intraepithelial axons and epithe- lial cells was not observed. Globular Leukocytes Globular leukocytes are found within the epithelium of mucous surfaces and most typically within the inter- cellular spaces of the epithelium itself (Kent gt gt., 1966; Breeze gt gt., 1977). Kent gt gt. (1954) investigated the effects of corticotropin, cortisone, and adrenalectomy on the number of globular leukocytes in rat tracheal epithelium. Following administration of cortisone or corticotropin, a significant reduction in the number of globular leukocytes was observed. Adrenalectomy, however, exerted no consistent effect on the number of globular leukocytes in the tracheal epithelium. For each animal, the numbers of globular leukocytes per five histologic sections of trachea were counted and averaged. The sections were stained with Masson's trichrome technique or phosphotungstic acid hematoxylin and basic fuchsin. The 42 staining technique used for quantifying cell numbers was not identified by these investigators. The effect of irradiation and hypophysectomy on globular leukocytes in rat intestine were investigated by Kent gt gt. (1956). After four days of whole body irradia- tion (650r) the number of globular leukocytes were reduced to 1-2% of their normal number. The effect appeared to be locally mediated due to the fact that lead shielding prevented any decrease in the number of globular leukocytes in the intestine. However, after 30 days of x-irradiation (800r) there was a significant decrease in the number of globular leukocytes in lead-shielded intestinal segments. Hypophysectomy prevented this decrease and, therefore, it was concluded that the decrease was not directly related to the x-irradiation but rather to stress. The number of globular leukocytes was also significantly decreased after hypophysectomy. Kent gt gt. (1966) also investigated the histochemical and ultrastructural characteristics of globular leukocytes in the respiratory epithelium of bronchioles, bronchi, trachea, and larynges of adult rats. Numerous globular leukocytes were present in almost every intercellular space. The spherical cytoplasmic inclusions (globules) are acido- philic. These globules are phosphotungstic acid, hema- toxylin and basic fuchsin positive. The globules also stain metachromatically with toluidine blue and azure. 43 The fine structure of the globular leukocyte was also investigated. Tissue segments were fixed in aldehyde followed by postfixation with osmium. The cytoplasmic matrix contained small amounts of rough endoplasmic reticu- lum, free ribosomes and a well-developed Golgi complex. The most characteristic feature was the presence of globules. No predilection in the distribution of these globules was noted. The globules were finely granular and electron dense. There were however variations, even in globules within the same cell. The globules were frequently bounded by a typical unit membrane. Rounded vacuoles were occasionally observed within a globule and the cell nucleus was frequently eccentrically located. Most studies of the substance contained within these globules have been carried out on the gastrointestinal globular leukocyte. Murray gt gt, (1968) demonstrated a greenish fluorescence in the gastrointestinal globular leukocyte. It was suggested that a monoamine may be stored within these globules. In summary, of all of the species studied, only the fetal (Cutz gt gt., 1975), infant (Echt gt gt., 1982), and adult rabbit trachea (Dey gt gt., 1976 and 1979) demonstrated endogenous fluorescent cells. Microspectrofluorimetric analysis of these fluorescent cells demonstrated the pre- sence of serotonin (Dey 23.2l-v 1980). Following admini- stration of 5-HTP Hoyt gt gt. (1979) also demonstrated 44 the presence of serotonin in single endocrine-like cells of the neonatal rabbit trachea. Following administration of L-DOPA or 5-HTP, an intense formaldehyde-induced fluores- cence. selectively accumulated in the extrapulmonary airways of all animal species at all ages studied (Erickson gt gt., 1972; Ewen gt gt., 1972; and Cutz gt gt., 1974). In addition, argyrophilic cells were also present in all animal extrapulmonary airways (Kirkeby gt gt., 1977), except for adult rat (Ewen gt gt., 1972). The morphology of the fluorescent and argerphilic cells was similar. The cells were always observed singly along the basement membrane and were triangularly shaped with a thin apical cytoplasmic projection. Argentaffin positive cells were not observed in any animal species. The fine structure of endocrine-like cells in extra- pulmonary respiratory epithelium has also been investigated. The most characteristic feature in the cytoplasmic matrix is the presence of dense-core granules. These dense-core granules ranged from 800 to 1700 A in diameter in most species studied (Cutz gt gt., 1974; Ericson gt gt., 1972). In adult rat (Ewen gt gt., 1972), the granules ranged from 5,000 to 20,000 A in diameter. At the ultrastructural level the tracheal endocrine-like cells were also identified by their ability to accumulate radioactivity after 3 3 administration of H-DOPA and H-5-HTP(Ericson gt gt., 1972). In addition to dense-core granules in the cytOplasmic 45 matrix, the cytoplasm contains moderate amounts of smooth and rough endoplasmic reticulum, small mitochondria, micro- fibrillar material, and a prominant Golgi complex (Cutz gt gt., 1974). Endocrine-like cells of adult rat (Jeffery gt gt., 1973) and adult cat (Hung gt gt., 1975; and Das gt gt., 1978) were rarely found in association with nerve fibers. Evidence for synaptic contacts between intraepithelial axons and granulated epithelial cells have not been observed (Jeffery gt gt., 1973; Walsh gt gt., 1974; and Das gt gt., 1978). Intrapulmonarnyingle, Endocrine-like Cells and Neuroepithelial Bodies in Animals Fetal Hage (1974) investigated the morphologic and histochem- ical characteristics of endocrine-like cells in lungs of fetal rabbit, mouse and guinea-pig. Injection of the mother or incubation of fetal lungs were performed with D- or L- DOPA, L-S-HTP, dopamine, pargyline, and Ro4-4602 (a decarboxylase inhibitor) in order to investigate the amine handling mechanisms of endocrine-like cells in respiratory epithelium. In samples of fetal lung from control rabbits groups of 2 to 10 cells as well as single cells exhibited a weak yellow fluorescence. The cells were round to cylindrical 46 and occasionally reached the luminal surface of the epithe- lium. Injection or incubation with L-DOPA or L-S-HTP resulted in an increased fluorescence intensity. The number and distribution of fluorescent cells were similar to control animals. Following pretreatment with L-5-HTP the fluorophore was strictly localized to the cytoplasm. After pretreatment with L-DOPA, the florophore could be found over the nucleus. Fluorogenic amines were not demonstrated in the bronchial epithelium of untreated fetal mice or guinea-pigs. After administration of L-DOPA or L-5-HTP groups of fluorescent cells were observed in the mouse but only rarely in the guinea-pig. No epithelial cells reacted to HCl-toluidine blue or lead-hematoxylin staining in any of the animal species examined. However, argyrophilic cells were demonstrated in fetal rabbit bronchi. No argyrophilia was observed in the bronchial epithelium of fetal mice or guinea-pigs. Intraepithelial nerve endings were not in contact with the fetal argyrophilic cells. Clusters of fluorescent, argentaffin, and argyrophilic positive cells were observed by Lauweryns 23.2l- (1974) in fetal rabbit lung. The cellular morphology was similar to that previously described by Lauweryns EE.El' (1972a) for human fetal lung. The neuroepithelial bodies were 15 to 25 mu high and 25 to 35 mu wide (at the base). The number of cells per NEB ranged between 10 and 30. Silver impreg- nation as well as formaldehyde-induced fluorescence 47 demonstrated numerous nerve fibers which were in close association with the NEB. However, no direct contacts between neuroepithelial bodies and axons were noted. Cutz gt gt. (1974) reported the presence of amines along with some of the amine handling properties of endo- crine-like cells in rat fetal lungs. With light micro- scopy the argyrophilic cells appeared singly as well as in clusters (3-6 cells). At all levels of the bronchial respiratory epithelium the single cells were triangular with the base resting on the basement membrane. In contrast, groups of endocrine-like cells were most numerous in large or medium sized bronchi. Serial sectioning revealed that the transition of single cells into groups did not occur. Only a few cells exhibited a slight metachromasia with HCl~toluidine blue staining. Control formaldehyde vapor treated lung segments demonstrated only a few weakly fluorescent cells. Following pretreatment both tg zttg and tg ttttg) with L-DOPA or S-HTP, the number of cells and intensity of fluorescence increased. In samples treated with 5-HTP the color of fluorescence was yellow-green and with L-DOPA more green. The general size, shape and distribution of fluorescent cells were similar to the argyrophilic cells. Morikawa gt_gt.(1978) noted the occurrence of acetylcholinesterase (AChE) positive cells in the intra- pulmonary airway epithelium of fetal rats. Neuroepithelial- like cells with lightly staining cytoplasmic 48 acetylcholinesterase positive granules were first noticed at 17 days of fetal life. Numerous AChE positive cells were identified throughout the tracheobronchial epithelium from 17 to 21 days of gestation. Hung (1978, 1979a, and 1979b) investigated the innervation of pre and post-natal mouse trachea and lungs. Acetylcholinesterase positive cells were not observed in the epithelial lining. A few single nerve fibers were observed to innervate the neuroepithelial bodies. These nerves were neither acetylcholinesterase positive nor fluorescent. It was inferred from these results that these nerve fibers were sensory in function. Lung explants (7 to 21 days) from fetal rabbits were examined by light and fluorescent microscopy to determine if endocrine-like cells could maintain their structural integrity (Sonstegard gt gt., 1979). Single endocrine-like cells and neuroepithelial bodies were identified by formal- dehyde induced fluorescence and argyrophilia in the bronchial epithelium of control and cultured lung explants. Fetal Ultrastructure The ultrastructural appearance of neuroepithelial bodies in the pulmonary respiratory epithelium of rabbit fetuses was investigated by Lauweryns EE.El° (1972b). Lung segments were fixed by four different methods: (1) 2.5% phosphate buffered glutaraldehyde for two hours at 4°C 49 followed by post fixation in osmium for one hour at 4°C; (2) 1% osmium; (3) 3% glutaraldehyde for four hours at 4°C followed by a 2.5% solution of potassium-dichromate con- taining 1% sodium phosphate (GD technique); and (4) prefixed in 8% formaldehyde for 24 hours followed by the GD techni- que. The last technique described is a specific technique (Jaim-Etcheverry gt gt., 1968) for the demonstration of serotonin granules at the ultrastructural level. The cyto- plasmic matrix of the endocrine-like cells contained: many free ribosomes; elongated mitochondria mostly in a supranuclear position; a well developed rough endoplasmic reticulum; a few glycogen particles; and many dense-core vesicles. After glutaraldehyde fixation, two types of DCVs were observed within the same cell. The Type I granules repre— sented 70% of the entire population of dense—core vesicles. These granules were pleomorphic (1340 A by 1021 A), and usually no halo was observed between the outer limiting membrane and the dense-core. Within a single dense-core two subpopulations of course granules were described. The smaller granules were 30 A in diameter and were suggested to consist of a carrier substance. Such a carrier sub- stance has been demonstrated in the adrenal medulla and composed of ATP, proteins, lipids and calcium ions (Kirshner gt gt., 1967). The larger granule, 90 to 100 A in diameter, was suggested to be composed of the active ingredient 50 (serotonin) bound to the carrier substance. The second type of DCV, which represented 30% of the vesicle population, was more circular (1121 A by 989 A). An obvious halo of 150-200 A was always present. The small granules within the DCV were 30 A in diameter. With both glutaraldehyde-dichromate and formalin- glutara1dehyde-dichromate fixations, the two populations of DCVs were again identified. The Type I granules were identifiable without any contrast staining. In contrast, the Type II granules were only visualized after uranyl acetate staining. According to Jaim-Etcheverry gt gt. (1968) only sites which contain serotonin will react to form an electron dense precipitate. After contrast staining with uranyl acetate the type II DCVs were also visualized. From these results, it was concluded that the Type I vesi- cles contain serotonin. Ultrastructurally, several unmyelinated nerve fibers were observed within the epithelium and demonstrated a "direct contact" with the grouped endocrine-like cells. The nerve endings were separated from the granulated Cells by a 200 A gap. Local thickenings of both the axon and granulated cells were also noted. The axoplasm contained numerous small mitochondria and granular vesicles of about 0 500 A in diameter. 51 Hage gt gt.(l974) reported the ultrastructural characteristics of endocrine-like cells in respiratory epithelium of fetal rabbit, guinea pig, and mouse. Lung segments were fixed by immersion in buffered glutaraldehyde or formaldehyde. Groups of columnar endocrine-like cells were most frequently observed. Desmosomes were occasionally noted. The cytoplasmic matrix was composed of: many free ribosomes; scant amounts of rough endoplasmic reticulum; mitochondria; a Golgi complex; and numerous dense-core vesicles. The granulated cells of fetal rabbits were columnar and seldom reached the lumens of bronchi. A discontinuous membrane surrounded all of the secretory granules. The average secretory granule diameter of 1420 A. The shape of endocrine-like cells of the mouse fetuses were columnar or polygonal. Occasionally, cells with their long axis parallel to the basement membrane were observed. Frequently, the luminal surface of the cell was covered by extensions from surrounding epithelial cells. Only rarely were they observed reaching the bronchial lumen. The secretory granules were: surrounded by a discontinuous membrane; uniformly round; and approximately 1070 A in diameter. Only one cell could be demonstrated in one bronchial tubule from the lungs of guinea-pig fetuses. The ultrastructural appearance of granulated cells in the respiratory epithelium of rat fetuses was studied by 52 Cutz gt gt.(1974). Granulated cells, singly or in groups, were present within the bronchial epithelium. Lung samples were fixed by immersion in phosphate buffered glutaralde- hyde, post-fixed in osmium, and embedded in Epon. The single cells were triangular with the base close to the basement membrane. The apical portion of the cell nerve reached the bronchial lumen. Cytoplasmic extensions between adjacent epithelial cells were frequently observed. These endocrine-like cells contained intracytoplasmic granules (700 to 1200 A in diameter) with a central dense-core surrounded by a single limiting membrane. The granules were randomly distributed throughout the cell. Other numerous cytoplasmic organelles were microfibrils, micro- tubules, and free ribosomes. Less conspicuous organelles were smooth and rough endoplasmic reticulum and Golgi apparatus. The group endocrine-like cells bulged slightly into the lumen. These granulated cells were frequently covered by other epithelial cells but occasionally reached the lumen. The cytoplasmic organelles were similar to those of the single granulated cells. In contrast to the single cells, intraepithelial nerve endings were found in contact with the endocrine-like cells. The ultrastructural features of the intraepithelial nerve endings were: neurotubules, small mitochondria, and occasional groups of empty vesicles 0 400 to 500 A in diameter. 53 Sonstegard gt_gt, (1979) demonstrated that the ultra? structural characteristics of cells within the neuroepithe- lial bodies could be maintained in short-term organ cultures. Lung explants from fetal rabbits were maintained in organ culture for up to 22 days. Tissue segments were fixed in phosphate buffered glutaraldehyde, post-fixed in osmium, and embedded in Epon. The ultrastructure of NEB cells in organ cultures was different from that of uncultured controls. In lung explants the DCVs in NEB cells: appear more pleomorphic; are significantly larger; appear to be redistributed throughout the cytoplasm (from an infranuclear position to a supranuclear position); and a few have a clear space within the matrix. Neonatal The histological features of bronchial endocrine-like cells of the neonatal rat were investigated by Moosavi gt gt. (1973). Argyrophilic cells were present singly as well as in clusters. These cells were: cuboidal or colum- nar, located on the basement membrane, and usually exposed to the lumen of the airway. Lauweryns gt gt. (1973a) reported the presence of argyrophilic, argentaffin, yellow fluorescent cells in the intrapulmonary epithelium of neonatal rabbits. Micro— spectrographically, the emission spectrum (maximum 540- 550 nm) of the neuroepithelial bodies (Lauweryns gt gt., 1972b) corresponded to the emission spectrum of serotonin 54 in bovine albumin standards. In addition, the excitation spectra (460 nm) of the NEBs was identical to that of serotonin standards and colonic enterochromaffin cells. Sorokin, and Hoyt (1978) and Hoyt gt gt. (1979) investigated the staining characteristics of endocrine-like cells in tracheas and lungs of neonatal rabbits. Tissue segments were fixed in 6% formaldehyde and subsequently embedded in glycol methacrylate. Single cells as well as clusters of cells emitted a weak yellow fluorescence. This endogenous fluorescence was intensified following administration of 5-HTP (30 minutes prior to sacrifice). Tissue sections stained with PAS-lead hematoxylin demon- strated that the fluorescent cells corresponded exactly with the PAS-lead positive cells. Utilizing the histochemical fluorescent method of Falck-Hillarp (1962), Wasans gt gt. (1979) demonstrated the presence of endogenous amine-containing cells in the primary and secondary bronchi of infant chickens. Numerous triangularly shaped, basally located, yellow, fluorescent cells were observed throughout the epithelial lining. These cells occurred singly as well as in clusters. Neonatal Ultrastructure Lauweryns and Cokelaire (1973b) investigated the ultrastructural characteristics of the intrapulmonary neuroepithelial bodies in neonatal rabbits and mice. Following reserpine pretreatment the cores of the DCVs were 55 cleared in about one third of the vesicle population, leaving only a half moon-like or a completely empty vesicle. Numerous unmyelinated nerve fibers were present within the NEBs. Two types of varicose nerve endings were reported to be in contact with the corpuscular cells. Type 1a (afferent-like nerve fibers) nerve endings were characterized by: many mitochondria; small agranular synap- tic vesicles (300-500 A diameter); and larger dense-core vesicles (800-900 A). The second type (type 2) were like those previously described (Lauweryns gt gt., 1972b). These afferent-like varicose nerve endings were similar to the type la fibers except they never, revealed varicosities filled with mitochondria. Afferent and efferent-like nerve endings revealed synaptical end formations making "direct contacts" with granulated cells. Occasionally, an exocytosis of corpuscular DCVs was observed at these areas of direct contact. In rabbit NEBs, after applying the ultrastructural acetylcholinesterase (AChE) technique of Bloom gt gt. (1966). a very fine, dense AChE-positive precipitate was noted exclusively in the halos of the DCVs (type 1) while the cores remained unstained. The type 2 DCV, as well as other cytoplasmic organelles, remained unstained. Cytochemically the cells of the NEBs revealed a positive reaction for acetylcholinesterase, alpha-glycero- phosphate, and Solica's lead-hematoxylin stain for endocrine 56 cells producing peptides and amines. In view of the fluorescent, histochemical, and ultrastructural results it was concluded that NEBs contain serotonin and probably a related amine or polypeptide. The possible occurrence of an additional substance other than serotonin in the corpuscular cells of neuro- epithelial bodies of neonatal rabbits (Lauweryns gt gt., 1972b) was investigated using fluorescamine by Lauweryns and Liebens (1977a). Fluorescamine is a reagent reported to stain selectively cells containing and secreting polypeptides (Larsson gt gt., 1975). Unfixed lung sections freeze—dried and treated with fluorescamine demonstrated an intense fluorescamine-induced fluorescence in groups of cells. The average emission maximum for the control group, was 510-520 nm. Lung tissue fixed with gaseous formaldehyde and post-treated with fluorescamine also demonstrated an intense and selective fluorescamine induced fluorescence in clusters of cells. The average emission maximum was 550-560 nm (510-520 corrected). From these results it was suggested that neonatal rabbit neuropeithelial bodies probably contain a polypeptide substance. Scanning and transmission electron microscopy were used to demonstrate the presence of bronchiolar neuroepithelial bodied in neonatal mouse lungs (Hung gt gt., 1974 and 1979). Lungs were fixed by immersion in phosphate buffered glutaral- dehyde for two hours, post-fixed in osmium for one hour, and 57 embedded in Epon. Those lung segments to be used for scanning electron microscopy were fixed in glutaraldehyde, dehydrated and then critical point dried. Two cell types (Clara, and specialized granular) were observed to compriSe. the neuroepithelial bodies. They were most numerous at the bifurcation points of the bronchioles. The specialized granular cells contained many dense cored granules (800 to 1000 A in diameter) surrounded by a clear area and followed by a unit membrane. These granules were located mainly in the basal half of the cytoplasm. Golgi apparatus, mito- chondria, and rough endoplasmic reticulum were also present in the cytoplasm. Intraepithelial axons were found to originate from non-myelinated axons in the lamina propria. These axons penetrated the epithelial basal lamina and ramified among the granular cells and the Clara cells. No specific synaptic contacts were noted between the axons and any of the epithelial cells. Scanning electron microsc0py demonstrated that the NEBs bulged into the bronchiolar lumen and that both cell types were in contact with the lumen. The boundaries of the NEBs were outlined by both the ciliated and Clara cells. Clara cells covered most of the surface of the NEB leaving only small oval areas for the exposed surfaces of the specialized granulated cells. Microvilli were noted to project from both Clara and granulated cells. 58 Intrapulmonary airways of chickens (3to 5 days old),also contain endocrine-like cells (Wasano gt gt., 1979). Dis- tinctive granulated cells were identified in both the primary and secondary bronchi. These cells were located along the basement membrane and appeared as single cells or in groups (3-5 cells). Infrequently, a single cilium was observed projecting from the exposed surface of the cell into the lumen. Close to the luminal surface of the epithelium these granulated cells were joined to the adja- cent epithelial cells by junctional complexes. The cytoplasm contained numerous dense-core vesicles whose average diameter was 1400 A. The dense-core was followed by a clear area (140 A) and surrounded by a unit membrane. At the ultrastructural level both the argentaffin and chro- maffin reactions produced electron dense precipitates in the cytoplasm of the granulated cells. Three of the fifty four cells studied were closely associated with intra- epithelial nerve endings. The interspace between nerve ending and the cell membrane was 200 A. Membrane thickenings of both nerve ending and cell membrane, suggesting a synapse, were noted. 50112.12 Two populations of biogenic amine-containing cells in the mouse lung were identified by Eaton and Fedde (1977). Mice were simultaneously perfused fixed via the circulatory and respiratory systems with a phosphate buffered 59 formaldehyde solution. Lung segments were removed and processed according to the amine histochemical method of Falck and mean (1965). An intense yellow-green fluores- cence was emitted from one population of cells. The second group of cells produced a bright yellow fluorescence. Microspectrofluorimetric analysis demonstrated a different emission.‘wavelength in these two groups of fluorescent cells. Spectral analysis of the fluorophores was not reported and, therefore, their conclusion that two different amines were present was questionable. Adult Ultrastructure Nerves and their associated cells in the bronchiolar epithelium of mice (Hung gt gt., 1973) and in the primary and secondary bronchi of avian lung (Cook and King gt gt., 1969; and King gt gt., 1974) were studied with the electron microscope. Axons, devoid of Schwann cells, were noted between the epithelial cells in the basal zone of the epithelium. Circular or oval intraepithelial axons were frequently associated with epithelial cells which contained many dense-cored granules. The diameters of the epithelial cell dense-core granules ranged from 750 to 1300 A. King gt gt. (1974) noted that the nerve endings were of the afferent type because they contained: many narrow mito- chondria; a few granular vesicles 600-1000 A in diameter; 60 and numerous agranular vesicles 300-1000 A in diameter. In all three reports evidence of synaptic contacts between small clusters of granulated cells and unmyelinated nerve endings were observed. Hung gt gt. (1973) reported that the endocrine-like cells appeared in groups. These cell clusters were similar to those described as neuroepithelial bodies by Lauweryns gt gt (1972b). Summary In summary, using the histochemical method of Falck and mean (1965), endogenous fluorogenic amines were demon- strated in the intrapulmonary airways of fetal (Hage gt gt., 1974; Lauweryns gt gt., 1974), and infant rabbiu3(Lauweryns gt gt., 1973a. The number and intensity of fluorescent cells in fetal animals increased significantly in animals pretreated with L-DOPA or S-HTP (Hage, 1974; Cutz gt gt., 1974). The fluorescent cells occurred singly (Hage gt gt., 1974) as well as in groups (Lauweryns gt gt., 1973a and 19730). Endogenously fluorescent cells were also demonstrated in mouse lungs which were liquid fixed, freeze-dried, and exposed to formaldehyde vapor (Eaton gt gt., 1977). Two populations of fluorogenic amine-containing cells were demonstrated with this method. One population of cells emitted an intense yellow fluorescence while the other emitted a yellow-green fluorescence. Spectral analysis of the fluorophore was not done, and therefore their conclusion 61 that two different amines were present in mouse intrapul- monary airways was questionable. In the intrapulmonary airways of all animal species and age groups studied argyrophilic cells were observed (Cutz gt gt., 1974 and Lauweryns gt gt., 1974 and 1976). Argentaffin positive cells were rarely present. However, Hage (1974) and Lauweryns gt_gt. (1973b and 1974) reported the presence of argentaffin positive cells in fetal and infant rabbit lungs. Several investigators (Mossavi gt gt., 1973; Palisano gt gt., 1980; Keith gt gt., 1981; and Redick gt gt., 1982) have attempted to quantify neuroendocrine cells in the intrapulmonary airways of animals at various stages of development. Despite the numerous efforts to quantify the number of neuroendocrine cells in fetal, neonatal, and adult lungs the results vary and are confusing at best. The general impression is that neuroendocrine cells increase in number until just prior to birth and then decrease (are not demonstrated using fluorescent microscopy) just prior to birth and then reappear following birth (Redick gt gt., 1982). After birth the number of neuroendocrine cells then decrease (Keith gt gt., 1981), remain constant (Palisano gt gt., 1980), or increase (Moosavi gt gt., 1973). The method of fixation, identification, quantification, as well as species differences have not been universally addressed. 62 Only a very detailed morphometric analysis (perhaps serial reconstruction) will provide an understanding of these neuroendocrine cells and their developmental pattern. The ultrastructure of single (Hage 1974) and groups (Lauweryns gt gt., 1973b, 1974) of respiratory endocrine- like cells has been described in fetal, infant, and adult animal intrapulmonary airways. Single cells were most frequently triangular or oval with the base close to the basement membrane (Cutz gt gt., 1974). The thin apical projections seldom reached the lumen (Hage EE.El-r 1974 and Cutz gt gt., 1974). Intraepithelial nerve fibers were frequently in close association with the single cells but no synaptic contacts were observed (Cutz gt gt., 1974). Nerve fibers were frequently observed to penetrate and ramify among the groups of granular cells (Hung gt gt., 1979). Evidence of synaptic contacts between groups of granulated cells and unmyelinated nerve endings have been demonstrated (Lauweryns gt gt., 1972b; Hung, 1973; Cook gt gt., 1969; and King gt gt., 1974). Hung (1978 and 1979) demonstrated that these nerve fibers were neither acetyl- cholinesterase positive nor fluorescent. It was inferred from these results that the nerves were sensory. The morphology of these nerve fibers at the ultrastructural level suggested to Lauweryns gt gt.(l973b) that both afferent and efferent nerve fibers were in "direct contact" with the neuroepithelial bodies. 63 The most characteristic cytoplasmic feature of these endocrine-like cells is the presence of dense-core vesicles surrounded by a clear area and followed by a single unit membrane (Cutz gt gt., 1972; Hage, 1974; and Lauweryns gt gt., 1972b). The diameter of the dense-core vesicles ranged from 700 A in the rat fetus (Cutz gt gt., 1972) to 1420 i in fetal rabbits (Hage gt gt., 1974). Additional cytoplasmic organelles include: many free ribosomes (Hage gt gt., 1974); a well developed rough endOplasmic reticulum; many small mitochondria; and a Golgi complex (Lauweryns gt gt., 1972b). Several studies have attempted to characterize the contents of these respiratory endocrine-like cells. Micro- spectrof1uorimetric analysis of groups of endogenously fluorescent cells of neonatal rabbits (Lauweryns gt gt., 1973a, 1977b) demonstrated the presence of serotonin. Ultrastructurally, two populations of dense-core vesicles have been demonstrated in fetal and infant rabbit endocrine- like cells (Lauweryns gt gt., 1972b). Using formalin glutaraldehyde-dicromate fixation, a specific stain for serotonin (Jaim-Etcheverry gt gt., 1968), the DCV, formed an electron dense precipitate (Type I) and therefore contained serotonin (Lauweryns gt gt., 1972b). After contrast staining with uranyl acetate the Type II DCVs were visualized. This suggested that an additional sub- stance was present within fetal and infant rabbit 64 neuroepithelial bodies. NEBs have also demonstrated a positive reaction for a-glycerophosphate dehydrogenase and acetylcholinesterase. This is indicative of polypeptide secreting cells (Lauweryns gt gt., 1973b). In addition, using fluorescamine, reported to be a specific stain for polypeptides, a distinct fluorescence in groups of endocrine-like cells (NEBs) was also observed in neonatal rabbits (Lauweryns gt gt., 1977a). Sorokin gt gt. (1978) have also demonstrated groups of fluorescent cells in neonatal rabbit and hamster after liquid fixation. This technique is reportedly specific for serotonin containing cells (Hoyt gt gt., 1979). In summary, it appears that fetal and neonatal rabbit neuroepi- thelial bodies contain serotonin as well as an additional substance which is probably a polypeptide. At this time one cannot extrapolate these results to other animal species or to single occurring endocrine-like cells. Physiologic and Pharmacologic Stimulation of Single Respiratory Endocrine-like Cells and Neuroepithelial Bodies The endocrine-like cells of both the extrapulmonary (Cutz gt gt., 1975; Dey, 1979; Echt gt_gl., 1980, 1982; and Friedenbach gt gt., 1980) and intrapulmonary (Lauweryns gt gt., 1972a; Cutz EE.§l°r 1974; and Hage, 1974) epithelium of various mammals, including man (Hage, 1973a; and Lauweryns gt gt., 1975, 1976), have been investigated with 65 «caaoa.onv .«q no caused ccuAQNV .Mm mm as...) «canuov .x-aounqcoo cacaowv nuuiouaucou cucaouv .Mu.mfl ocean: cacnoov .mfl II cannon «:33 .fl I «agacfiv .mfl mu mama cccAaov .m. ya 1000 H guano: «- cecAnnnv .‘I mm n:>h0):14 2.133 mm mm 33253 :2. 4|. mm .3 «a. 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Each technique has revealed the presence of endocrine-like cells throughout the airways. Despite the extensive morphologic characterization of these cells little is known about their functional roles. The effects of various pharmacologic agents on the amine handling properties of endocrine-like cells were investigated by Ericson gt gt. (1972). The tracheal epithelium of untreated control mice contained no cells with formaldehyde-induced fluorescence. Thirty minutes after administration of L-DOPA, however, an intense green formaldehyde-induced fluorescence was observed in a large number of endocrine like cells. No fluorescence was visible after inhibition of monoamine breakdown by nialamide. Pretreatment of the mice with an inhibitor of the decarboxylating enzyme, Ro4-4602, one hour prior to L-DOPA- injection resulted in only a very faint greenish fluores- cence. These results suggested that the amino acid itself did not accumulate in the cells and therefore the fluorescent cells contained the decarboxylation product. This conclusion was also supported by the fact that administration of D-DOPA, a poor substrate for the decarboxylating enzyme, usually did not result in any visible fluorescence. A cellular accumulation of dopamine 67 did not occur, even when the amine was administered in high doses. Pretreatment of the animals with reserpine 4 hours prior to L—DOPA injection resulted in a lower fluorescence intensity than after L-DOPA injection alone. From these results it was concluded that the amine was stored in the cytoplasm by a reserpine-sensitive mechanism. Moosavi gt gt. (1973) was one of the first to study lung endocrine-like cells and their possible physiological role during chronic hypoxia. Four day old rats and their mothers were placed in a pressurized oxygen chamber. The atmospheric pressure was gradually reduced to 380 mm Hg, stimulating an altitude of 18,000 feet. The animals were maintained at this pressure until their sacrifice at 20 hours to 1 month. Lung samples were removed and fixed for light and electron microscopy. Lung segments, studied by electron microscopy, were rapidly fixed in ice cold glutaraldehyde. Silver staining revealed argyrophilic granules con- taining cells in lung epithelium. These cells occurred singly as well as in clusters. Electron microscopy of control animals demonstrated a number of endocrine-like cells with dense-core vesicles, 1200 to 1730 A in diameter. The central dense-core was surrounded by a narrow, clear halo and limited externally by a single unit membrane. The remaining cytoplasm contained mitochondria, smooth and rough endoplasmic reticulum, central nucleus, 68 microfibrilis and a Golgi apparatus. Light microscopy of hypoxic animals, did not reveal any detectable histologic changes. Pronounced alterations were observed in the ultra- structure of dense-core secretory vesicles in endocrine-like cells from hypoxic animals. The peripheral halos were wider while the central cores were smaller. The diameters of the altered osmiophilic bodies ranged from 1270 to 2070 A; some as large as 3730 A. The ratio of the area of the central core to that of the whole granule was significantly decreased in the hypoxic rats. At four days of age, the number of endocrine-like cells per cm of bronchial epithe- lium during hypoxia was not significantly different from control animals. No other age groups were studied. Age related differences were observed in control rats. There was a significant decrease in the number of endocrine- like cells with age (70 cells/cm at 7 days and 16 cells/cm at 31 days). The percentage of bronchi and bronchioles containing endocrine-like cells decreased from 89% at the age of 7 days to 45% at the age of 31 days. It was noted that the fine structure of the respiratory endocrine-like cells was similar to that of the chief cell of the carotid body. Edwards 22,2l- (1972) demonstrated that the chief cells of the carotid body responded, at the ultrastructural level, to chronic hypoxia and therefore were chemoreceptors. It was concluded that the ultrastructural changes in the respiratory endocrine-like cells of animals 69 exposed to hypoxia were very similar to those reported by Edwards gt gt. (1972) in the chief cells. Because of their similarity in structure and response to hypoxia it was suggested that the respiratory endocrine-like cell may serve as a chemoreceptor function. Lauweryns and Cokelaire (1973b, 1973c) suggested at the ultrastructural level that the neuroepithelial bodies of rabbits released the contents of their dense-core vesicles at their basal vascular pole after exposure to hypoxia. Neonatal rabbits were exposed for 2,10 or 20 minutes to a low oxygen atmosphere (5,10 or 15%). Lung samples were removed and prepared for routine staining procedures or prepared for electron microscopy. Material for electron microscopy was fixed in phosphate buffered, 2.5% glutaraldehyde (pH 7.2) for 2 hours at 4°C, post-fixed in osmium (1 hour at 4°C), and embedded in Epon. Light microscopy of hypoxic animals did not reveal any detectable histologic changes of the neuroepithelial bodies. Ultra- structurally, however, numerous alterations were observed between the NEBs of control and hypoxic animals. All animals exposed to hypoxia revealed a distinct exocytosis of the corpuscular dense-core vesicles along the basement membrane. All classical morphologic phases of the exocytotic cycle (Hubbard, 1971) were noted in hypoxia exposed rabbits. 70 In contrast to the control animals, the hypoxia exposed rabbit dense-core granules were: more numerous in the basal area of the cell; apparently fused with the cell membrane itself; were fragmented; many were entirely empty; and some were smaller than the classically described DCV. In addition, Lauweryns noted occasional mitochondrial lysis and a prounounced development of the Golgi complex in hypoxia exposed animals. No attempt to quantitate differ- ences in control and experimental animals was reported. Neonatal rabbit neuroepithelial bodies were studied under various experimental conditions by Lauweryns gt gt. (1977b). The reaction of neuroepithelial bodies to hypoxia (10% oxygen for 20 minutes), hyperoxia (30% oxygen for 20 minutes), and hypercapnia (5% C02) , were investigated by microspectrofluorimetric and morphometric methods. Lung samples were processed according to previously described techniques (Lauweryns, 1973b). The effect of several phar- macologic agents (nicotine, L-DOPA, S-HTP, reserpine, and L-DOPA followed by hypoxia) on neuroepithelial bodies were also investigated. As defined previously (Lauweryns gt gt., 1972b) the dense-core vesicles (DCV), with a dense amorphous depot in their granules were considered DCVi, while those without a dense depot in their core were classified as DCVZ. Morphometric studies were carried out at the ultra- structural level to determine significant alterations in the shape and location of the dense-core secretory vesicles. 71 An increase in the number of DCVs contacting the basal cell membrane per unit length, in conjunction with a decrease in the infranuclear cytoplasmic volume percent occupied by the dense-core vesicles were being released from the cell. Hypoxia and hypercapnia caused a significant decrease in the peak fluorescence intensity emitted by the NEB at the emission wavelength of serotonin (550 nm). No spectral changes were observed following hyperoxic exposure. Morphometric measurements, of hypoxic rabbits, indicated a significant decrease of the infranuclear cytoplasmic volume percent occupied by the dense-core vesicles (DCV and DCV2) l and an increase number of DCVs contacting the basal cell mem- brane per unit length, thus, suggesting a release of the dense-core vesicles. In hypercapnic animals, an increase number of DCVs contacting the basal cell membrane per unit length were noted. There was no decrease of the infranuclear cytoplasmic volume percent occupied by the dense-core vesi- cles (DCV2). However, there was a significant decrease of the infranuclear cytoplasmic volume percent occupied by the dense-cored vesicles (DCVl). Hypercapnia resulted in a frag- mentation of the dense-core vesicles (a change from DCV1 to DCVZ). The total number of DCVs contacting the basal cell membrane per unit length, of hyperoxia treated animals, were significantly decreased from controls. Despite this significant treatment effect, the authors concluded that hyperoxia did not significantly alter the ultrastructure 72 of the DCVs in the neuroepithelial bodies. However, it was suggested that the NEBs responded to hypoxia and hypercapnia as intrapulmonary chemoreceptors. Nicotine, reserpine, 5-HTP, and L-DOPA all induced ultrastructural changes of DCVs. Nicotine was a strong stimulus for the exocytosis of DCVs, an increase in DCVs contacting the basal cell membrane per unit length, and a decrease in the infranuclear cytoplasmic volume percent occupied by the dense-core vesicles. Reserpine pretreatment had no effect on the infranuclear cytoplasmic volume percent occupied by the dense-core vesicles but there was a distinct depletion of DCV1 altering their morphology to DCV Ultra- 2. structurally, the injection of 5-HTP and L-DOPA resulted in an increased electron density of the dense-core vesicles. The total volume percent occupied by the DCVs remained unchanged when compared to control animals. Microspectro- fluorimetric analysis revealed a decreased fluorescence intensity following administration of nicotine or reserpine. Sonstegard gt gt. (1979) studied the effects of reserpine and a Ca++ ionophore (A23187) on the ultrastructure of NEBs in lung explants cultured for seven days. The primary changes were in the dense-core granules. The Ca++ ionophore produced a coarsely granular and slightly less electron-dense DCV. The perigranular halo was also wider than control. Reserpine pretreatment produced clearing and fine granularity of the matrix. The electron-dense core 73 appeared more granular and the perigranular halo was larger in some DCVs. The effect of reserpine on NEBs was similar to that reported by Lauweryns gt gt. (1977b). The number of DCVs affected by reserpine was dose-related (l to 10 ug/ ml), but not with the Ca++ ionophore, A23187. By means of cross-circulation experiments, Lauweryns EE.El- (1978) investigated the response of neuroepithelial bodies to: (1) alveolar hypoxia and arterial normoxic blood; and (2) alveolar normoxia and arterial hypoxic blood. Pre- viously described (Lauweryns gt gt., 1977b) techniques for. analysis of the dense-core vesicles were used. Fluorescent and light microscopic techniques revealed no differences between the neuroepithelial bodies of either group. Lung neuroepithelial bodies from young rabbits which received hypoxemic blood did not exhibit, at the ultrastructural level, an increased exocytosis of their dense-core secretory vesicles. Rabbits exposed to normoxic atmospheres containing lowered p02 but receiving normoxic blood, did demonstrate significant ultrastructural differences from control animals. Morphometric analysis revealed a signi— ficant increase in the number of DCVs in contact with the basal cell membrane per unit lenth as well as a decreased infranuclear cytoplasmic volume percent occupied by the dense-core vesicles, suggesting exocytosis of these vesicles. Keith and Will (1981) have also investigated the poten- tial relationship between neuroendocrine cells and medial 74 thickness of pulmonary arteries in neonatal rabbits following acute and chronic hypoxia. New Leland White does were maintained in a hypobaric (520 mm Hg) chamber from the twentieth day of gestation. The neonates were born and raised in the hypobaric chamber until sacrifice (one, three or five days after birth). All hypoxic neonates were sacrificed within 15 minutes after the pressure chamber was normalized. Neonates that died during the course of the experiment were also included in the study. However, no information was provided regarding the time sequence between death and fixation. In addition, several 4 day old hypoxic neonates were allowed to recover from hypoxia for one day prior to sacrifice. Grimelius's silver method in conjunction with Miller's elastic VanGiesan's stain were used for morphometric analyses. Although the exact method of analysis was not defined, the authors stated that an average surface area of 1.95 cm2 per animal was examined. From these experiments, it was concluded that hypoxia significantly increased the number of neuroendocrine cells and pulmonary artery medial thick- ness in S-day old neonates. In addition, one and three day old rabbits that died during hypoxia exposure also had significantly higher numbensof neuroendocrine cells than the hypoxic survivors. This result is difficult to accept due to the lack of information regarding the determination of death and subsequent handling of the 75 lung tissue. Hypoxic rabbits four days postpartum that were returned to normoxic conditions for 24 hours prior to sacrifice demonstrated a significant decrease in the number of neuroendocrine cells and in medial thickness when compared to the 5 day hypoxic neonates. Echt gt gt. (1980 and 1982) and Friedenbach gt gt. (1980) investigated the effects of age, hypoxic hypoxia, and carbon monoxide on endogenous serotonin containing cells in tracheal respiratory epithelium of young rabbits (3,10 and 28 days old). Echt gt gt. (1982) employed the histochemical fluorescent technique according to Falck (1962) to demonstrate the presence of amine containing cells. Rabbits were exposed for 6 hours to an atmosphere of 13% oxygen (alveolar and arterial hypoxia) or to a concentration of carbon monoxide which produced a decrease in arterial oxyhemoglobin saturation equivalent to 13 percent oxygen (tissue hypoxia). Exposure of animals to atmospheres containing lowered p02 results in an airway or arterial hypoxia. In contrast, exposure of animals to normoxic atmospheres containing carbon monoxide only produces a tissue hypoxia without affecting airway or arterial p02 (Roth and Rubin, 1976). Quantification of the fluorescent tracheal epithelial cells was accomplished by calculating the number of fluorescent cells per centimeter of epithelium. Compared to their respective controls, there was a significant decrease in the number of 76 fluorescent cells per cm of epithelium in 10 day old hypoxic hypoxia exposed rabbits. The overall effect of hypoxic hypoxia (all ages considered) demonstrated a significant decrease in the number of fluorescent cells compared to controls. When all control animals (3,10 and 28 day animals) were compared to all treated (carbon monoxide) age groups there was a significant decrease in the overall number of fluorescent cells. It was concluded that in rabbits the amine-containing epithelial cells of the trachea respond to tissue hypoxia and a decreased airway or arterial p02 is not necessary to elicit a response. These results were different than Lauweryns gt gt. (1978) regarding the response of intrapulmonary NEBs in rabbits. They demon- strated that NEBs in rabbit lungs respond directly to lowered oxygen content of inhaled air and not to decreased oxygen of pulmonary blood. Echt 23.3l- (1982) also investigated the effect of age on the number of fluorescent cells. There was a significantly greater number of fluores- cent. epithelial cells in 28 day old compared to 3 day old rabbits. Ultrastructural changes of tracheal, endocrine-like cells in young rabbits after 6 hours exposure to carbon monoxide was reported by Friedenbach gt_gt.(l980). Animals were perfused via the vasculature at systolic pressures, with buffered aldehydes and routinely prepared for electron microscopy. The size and shape of the granules were not 77 altered after exposure. Infranuclear granules were smaller (1300 A) than supranuclear granules (1410 A). Those granules infranuclear were also more circular than supra- nuclear., When compared to controls, the number of granules per cell decreased from 38 to 26; the number of granules per unit cell area decreased from 45 to 34 per um; the granule area decreased from 67 to 52%; and the percentage of granules infranuclear increased from 37 to 58%. It was concluded that these data supported a basal movement of granules which may be released from the cell following exposure to carbon monoxide. In summary, the existence of intrapulmonary sensory receptors reactive to changes in the composition of gases in the lung has been postulated by several authors (Fishman, 1960; Dejours, 1962). Lauweryns and Purskens (1972a) identified intrapulmonary corpuscles (neuroepithe- lial bodies) in the bronchial and bronchiolar epithelium of human infants. It was concluded that these unique histologic structures were probably intrapulmonary chemo-, stretch-, and/or tactile neuroreceptor organs. The influence of acute (Lauweryns gt gt., 1973b and 1973c) and chronic (Moosavi gt_gt., 1973) hypoxia on fetal rat and neonatal rabbit suggested numerous ultrastructural alterations of NEBs cells in both instances. It was concluded from these results that the NEBs were chemo- receptors and did respond to alveolar oxygen content. 78 Lauweryns gt gt. (1977b) further investigated the possible chemoreceptor function of the NEBs. The reaction of neo- natal rabbit neuroepithelial bodies to hypoxia, hyperoxia, and hypercapnia, were investigated by microspectrofluori- metric and morphometric methods. From the results obtained it was suggested that the cells of the NEBs responded to hypoxia and hypercapnia and thus were intra- pulmonary chemoreceptors. Despite a significant decrease in the number of DCVs touching the basement cell membrane, it was concluded that hyperoxia did not significantly alter the ultrastructure of NEBs. By means of cross-circulation experiments Lauweryns gt gt. (1978) investigated the response of NEBs to alveolar hypoxia and/or arterial hy- poxia. Since the NEBs responded directly to alveolar hyposiz, but not to arterial hypoxia it was concluded that NEBs were chemoreceptors which responded directly to the gaseous composition of the inhaled air. The cells of neuroepithelial bodies have some resemblance to single endocrine-like cells. Both cell types are argyrophilic, fluorescent and ultrastructurally granulated. However, NEBs appear to be innervated (Lauweryns gt gt., 1973b) whereas single endocrine-like cells have not been reported to have any association with the nervous system (Cutz gt gt., 1975). All of the physiologic studies regarding the possible functional roles of intrapulmonary respiratory endocrine-like cells have 79 been evaluated from groups of cells (NEBs). The only physiologic investigations on the functional roles of single endocrine-like cells during hypoxia have been performed on the extrapulmonary airways of neonatal rabbits (Echt gt gt., 1980 and 1982; Friedenbach gt gt,, 1980). Neonatal rabbits exposed to both hypoxic hypoxia or carbon monoxide-induced hypoxia demonstrated a significant decrease in the number of detectable tracheal fluorescent cells when compared to controls (Echt gt_gt., 1980 and 1982). At the ultrastructural level Friedenbach gt gt. (1980) reported significant alterations of the cytoplasmic location and number of dense-core secretory vesicles after exposure to carbon monoxide. CHAPTER III MATERIALS AND METHODS Animal Care Adult rats, bred and maintained in a colony in the Anatomy Department at Michigan State University, were housed in cages (36 cm x 31 cm x 16 cm) in a relatively constant environment. The animals had access to food (Wayne Laboratory Blox) and water gg libitum. Specimen Removal Forty-eight adult male and female rats (150 to 450 g) were anesthetized with sodium pentobarbital (Nembutal, Abbott), 50 mg/Kg, i.p. The rat was placed in a supine position and a ventral transverse incision was made below the level of twelfth rib. The diaphragm was dissected away from its reflection on the anterior abdominal wall and followed by a midline incision through the sternum up to the level of the mandible. The larynx and trachea were exposed and a hemostat was clamped around the oropharynx just beyond the epiglottis. The trachea was freed by gently teasing it away from the esophagus and surrounding connective tissue. The larynx, trachea, lungs, and heart were then removed 22 bloc. The trachea was cut from the larynx at the cricoid 80 81 cartilage and from the mainstem bronchi of the carcina. A small piece of duodenum, approximately 1 cm below the level of the pyloric valve, was used as a positive control for the fluorescent procedures. The length of time from the first incision to tissue processing was about two minutes. Fluorescence Histochemistty,Freeze-Dried Formaldehyde Induced Histofluorescence Freezing and Freeze-drying The Falck-Hillarp (1962) fluorescence histochemical technique involves treatment of freeze-dried tissues with formaldehyde gas. The principles of freezing and freeze- drying have been described by VanOrden (1975), while the technical aspects of this procedure have been reviewed by Falck (1962), Falck _e_t_ a_1. (1965), and Bjorklund gt gt. (1972). Tracheal segments were placed on small pieces of aluminum foil and immersed in 2-methy1butane (Eastman) cooled by liquid nitrogen for quick freezing. The quick freezing prevented significant ice crystal formation and also preserved morphology. The temperature of the 2-methyl- butane was near its melting point (-160°C) judged by the presence of frozen areas within the container. The tissue segments remained in the 2-methylbutane for about two minutes and then were transferred into liquid nitrogen for storage and subsequent freeze-drying. 82 The freeze-dryer utilized in this study was constructed from existing laboratory equipment (Figure l; Dey gt gt., 1980). Tissue segments were placed in a 2,500 m1 flask (Virtis) which had been pre-cooled to -80°C by an acetone and dry ice slush. The flask, which contained phosphorous pentoxide (P20 Fisher) to enhance moisture absorption, was 5: then connected to a vacuum system (Kinney) which generated a vacuum of 10-5 torr. The mechanical and oil diffusion pumps were protected from corrosive gases by cold and chemical traps. Drying of the tissue segments was accomplished by sublimation (vaporization of a solid substance, water, to a gas without passing through a liquid state). The vacuum system reduced the energy necessary for vaporization while the cold trap in conjunction with the phosphorous pentoxide provided an effective vapor gradient for the removal of moisture from the tissue. To prevent ice crystal formation and still allow an adequate rate of sublimation the temper- ature of the tissue segments was maintained by a refriger- ation unit (Edwards). The flask was placed in the acetone and dry ice slush and maintained at - 80°C for one day (24 hours). During the second day, the dry ice was removed and the temperature was gradually raised to -30°C and maintained by the refrig- erator. On the third day, the temperature was again raised, this time to room temperature (25°C). At the end of the 83 Figure l.--Freeze-drying apparatus; (a) main valve; (b) roughing-backing valve; (c) air inlet valve; (d) pump air inlet valve; (e) mech. pump switch; (f) diff. pump switch; (9) main switch; (h) thermocouple gauge; (i) thermo- couple gauge switch; (j) base plate Opening; (k) adapter block; (1) cold trap; (m) N2(l) trap; (n) refrigeration switch; (0) vacuum jar; (p) vacuum jar cap; (r) refrigera- tion chamber. 84 FREE ZE-DRYING APPARATUS Refrigeration Unit Mechanical and Diffusion Pumps 85 third day, the tissues were heated to 80°C for three hours in a water bath to prevent condensation of water, and then removed from the freeze-dryer for subsequent formaldehyde vapor treatment and/or embedding. Formaldehyde Vapor Treatment The fluorescence histochemical demonstration of cate- cholamines, tyramines, tryptamines and related substances depends upon condensation reactions with formaldehyde vapor leading to ring closure and the formation of fluorescent isoquinoline, quinoid or B-carboline (Figure 2). This reaction only takes place when the amines are associated with the tissue protein. The most important factors affec- ting the quality and intensity of formaldehyde gas induced fluorescence are; humidity, temperature, pH or acidity of the protein environment, monoamine concentration, and reaction time (Corrodi 23 gl-, 1967). In order to obtain optimal fluorescence intensity 5 grams of paraformaldehyde powder (Electron Microscopy Sciences) was equilibrated for at least ten days in an atmosphere of 60% relatively humidity (Hamberger.gt.gt., 1965; Pearse, 1972, Appendix 27), attained from a 10 molar aquaeous solution of sulfuric acid. The sulfuric acid was removed and the closed container with paraformaldehyde powder was preheated to 80°C for one hour to generate an 86 Figure 2.--Picket-Spengler reaction of an amine with an aldehyde by condensation to form heterocyclic compounds which spontaneously oxidize yielding fluorescent dehydroiso- quinoline or dihydro-B-carboline depending on whether the amine contains a catechol or indole ring structure respec- tively (Corrodi and Jonsson, 1967; and Bj5rklund_gt_gt., 1973). 87 m>wum>wnm© Edwcflaonnmo nanoucssaona.muasnuwsum Acmnmoummuu HmGHEHmu mammamumo mm>Hum>Hum© ocflaonumo Iocflfim nuwz mmoaummm omamv wflo< muoncmnmuuwulv.m.m.a msHEmumwuu umusuflquSmla N m m m z a o m mz\o 2 Oman mmz osflaonumolmnoownflolv.m swag U/mu . + 0 1,0 0 mm xlmzoo mm xlmzou coflumpaxousd xImZOU ACHIW may AGIH mmv Show Hmoflocfldq moaaocaquma mcflaosflsvowaouomnmuuwu Amswfimmoo .mmv OHHmEousmu Iowoanwo.v.m v.m.m.anmxouoanwolh.m mowemaonomumv . uwmm camp N m m / oTl' z\ miou @686 m om c ma :2 on .ATIIIII: IQWIIIHI / ‘llll mNI uh HA +3 O 0: oz 03 88 optimum formaldehyde vapor concentration. Freeze-dried tissue segments were then immediately removed from the freeze dryer, placed in the closed container with para- formaldehyde powder for 90 minutes and subsequently embedded in glycol methacrylate. Control tissues were not exposed to formaldehyde vapor prior to being embedded. Plastic Embedding and Sectioning The tissue segments were infiltrated and embedded in glycol methacrylate (Polysciences, Inc.) according to the method of Hoyt gt gt. (1979). Trachea, larynx, and small intestine were placed in small vials containing embedding solution A (which included catalyst). The vials were put in a dissicator and exposed to a mild vacuum (for 2 hours) containing on line chemical traps (Figure 3) and then allowed to stand overnight at atmospheric pressure. Tissue samples were then placed in the complete glycol methacrylate medium consisting of embedding solution A, solution B, plus accelerator and allowed to polymerize. Details of the embedding procedure appear in Appendix A. Sections (1-10 um) were cut on an ultramicrotome (Sorvall JB-4) with glass knives, placed on a drop of water on acid cleaned glass slides, briefly dried on a warming tray at 40°C and mounted in Entellan (EM Laboratories, Inc.). 89 Figure 3.--Plastic embedding apparatus; (a) vacuum chamber; (b) chemical traps, and (c) vacuum pump. 91 Liquid Formaldehyde Induced Histofluorescence Drug Treatment Two groups of animals (four animals per group) were. given a single intraperitoneal (i.p.) injection of 100 mg/Kg body weight of either L-DOPA (Sigma) or L-S-HTP (Sigma) twenty five to thirty minutes perior to sacrifice. The amine precursors were dissolved in distilled water and a total volume of 1 ml was delivered per animal. Four control animals received i.p. injections of distilled water only. Surgical Procedure Twenty five minutes after administration of the amine precursor the animals were anesthetized with sodium pento- barbital, 50 mg/Kg, i.p. The rats were then placed in a supine position and a midline incision was made at the level of the xiphoid process and extended up to the level of the first rib. The left ventricle was cannulated with an 18 gauge sterile needle and the animals were perfused at systolic pressures. Immediately following the cannulation of the left ventricle, the right atrium was incised to provide an outflow of the perfusion medium. Fixation and Dehydration The animals were initially perfused with a cold (4°C) buffered (0.1M phosphate, pH 7.2) solution of heparin (Nutritional Biochemicals Corporation) for 15 seconds. 92 Following the prewash the rats were perfused with a 6% formaldehyde (Ted Pella, Inc.) solution buffered at pH 7.2- 7.3 with 0.1 M phosphate. The formaldehyde solution was always freshly prepared by dissolving paraformaldehyde powder to warm (90°C) phosphate buffer. The formaldehyde solution was immediately filtered and the pH corrected to 7.2 to 7.3 when necessary. The fixative solution was then cooled to 4°C for subsequent infusion via the left ventricle. The animals were perfused for 3 to 5 minutes until the vas- cular outflow was clear. Removal of the larynx, trachea, and duodenum was similar to that previously described for freeze-drying. The tissue segments were then immersed in fresh 6% formaldehyde solution for 48 hours at 4°C. Follow- ing fixation, the specimens were washed for two minutes in ice cold distilled water and then dehydrated in increasing concentrations of ethyl alcohol (25,50,75,95,100%) for ten minutes each at 4°C. The final dehydrating medium of 100% ethyl alcohol was allowed to come to room temperature over a 30 minute time interval. Electron Microscopic Infiltration, Embedding and Sectioning Tissue samples were infiltrated for one hour each in a 2:1, 1:1, 1:2 mixture of ethyl alcohol and Spurr's resin (Polysciences, Inc.). The final infiltration medium which consisted of pure plastic was allowed to infiltrate the tissues overnight at room temperature. Tissue segments 93 were then transferred to fresh Spurr's resin for embedding by polymerization in an oven for twelve hours at 65°C. After embedding in plastic, 1-6 mu thick sections were cut on a ultramicrotome (Sorvall Mt-Z). Each section was then placed on a drop of water on a glass slide, dried on a warming tray at 55°C and then mounted with a coverslip in non-fluorescent immersion oil (R.P. Cargille Laboratories, Inc.). Formaldehyde Vapor and HCl Treatment Formaldehyde vapor was generated from paraformaldehyde powder as previously described. Sections (1-6 mu) were transferred to the closed vessel and exposed to formaldehyde vapor for one hour at 80°C. The sections were then immediately transferred to 2.51 jar which contained a small beaker with 60 m1 of concentrated (37%) analytical grade hydrochloric acid (Mallincrodt Chemical Works). Sec- tions were exposed for one hour to the HCl vapor generated at room temperature in the closed vessel. Specificity of the Fluorgphore Although the histochemical fluorescence method of Falck and Hillarp (1962) for the demonstration of biogenic amines has a very high specificity, it is important to determine the specificity of the fluorophore. A number of histochemical criteria have been proposed (Corrodi and Jonsson, 1967) to determine if the observed fluorescence is due to the presence of a monoamine. The first criterion 94 demonstrated whether the fluorophore resulted from the formaldehyde vapor or was simply autofluorescence. Tracheal and gastrointestinal tissue segments that were freeze-dried but not formaldehyde vapor treated were compared to exposed tissue samples. The second test determined if the fluorophore was sensitive to irradiation with ultraviolet light. Following exposure to ultraviolet light the fluorescent compounds of the monoamines demonstrate photodecomposition and loss of fluorescence intensity, whereas autofluorescent structures and proteins do not. This was accomplished by observing the fluorophore after exposure to ultraviolet light. Corrodi_et_al. (1964) demonstrated that sodium boro- hydride in ethanol reduced monoamine fluorescent compounds. In the presence of sodium borohydride (NaBHé) the carbolines are reduced to their corresponding nonfluorescent l,2,3,4- tetrahydroderivatives. The proof of specificity however is the regeneration of fluorescence after NaBH4 reduction. Fluorescent cells were first identified, then the tissue sections were exposed to 0.5% sodium borohydride (Sigma) in 80% ethanol, observed a second time, and finally treated again with formaldehyde HCl vapor and observed a third time. Microspectrofluorimetric Analysis Fluorescence excitation and emission spectra were measured with a microspectrofluorimeter (Figure 4) housed 95 Figure 4.--Microspectrofluorimeter; (a) illuminating light (-——»), and (b) fluorescing light (---+). 96 unmfla mcflomwuosHm "" pswfla mcflumsflgaafl 4| uwuomfioo umpuoomm _ kumfiouonm mumuaam . Hofluumm l MHQEMU HoumEonnoocoz coammflfim was» HOHHQ Ifluasfiouonm mMBMZHmODAhomeummmomUHz mousom fir unmaq HOUMEOHSUOGOZ coflumuwoxm 97 in the laboratory of Dr. Robert Dinerstein, Department of Physiology and Pharmacology, University of Chicago, Chicago, Illinois (Dinerstein gt gt., 1979). Fluorescence microscopy was carried out with a Leitz Orthoflux Fluorescent Micro- scope using light from a 200 watt mercury vapor lamp. Light from the lamp was passed through an excitation monochromator, objective, and glass coverslip to the tissue section. Fluorescent light was then directed through an emission monochromator and then emitted fluorescence intensity from each cell was recorded by photon counting. In all instances measurements were made in sections mounted in immersion oil. Both excitation and emission spectra were measured indepen- dently from the same cell. The emission curves were first determined by measuring light intensities at wavelengths from 500 to 600 nm in 5 nm steps with unfiltered light from the mercury vapor lamp. The excitation peak was calculated by measuring the intensities of emission peak while excita- tion wavelengths were increased from 350 to 500 nm in 5 nm steps. The excitation and emission spectra were directly recorded as quanta per unit of wavelength by an on line computer. The excitation and emission spectra were both corrected for the spectral sensitivity of the microspectro- fluorimeter. Histochemical Procedures Freeze-dried, formaldehyde vapor-treated, tracheal and gastrointestinal segments (control) embedded in glycol 98 methacrylate were utilized to investigate the cytochemical staining characteristics of adult rat tracheal epithelium. Sections (1-6 mu) were placed on a drop of water on a glass slide, dried on a warming tray at 37°C, and subsequently stained by various histochemical procedures: methylene blue, ferric ferricyanide, alcian blue, periodic acid Schiff (PAS), PAS-lead hematoxylin; details of the histochemical proce- dures appear in Appendix A. Lee's Methylene Blue-Basic Fuchsin Stain (Bennett gt gt., 1976) Methylene blue, along with many other basic dyes, has been employed in the staining of endocrine tissues. Manocchio (1964) reported the metachromatic staining of pancreatic D cells with toluidine blue. It was concluded that toluidine blue reacted with carboxyl groups in the endocrine cell granules. Solcia gt gt. (1968) reported the selective staining of endocrine cells with basic dyes after acid hydrolysis. These investigators found that pancreatic A cells, which normally are not baSOphilic, showed a distinct metachromatic reaction. It was concluded that the mechanism of the metachromasia related to an unmasking of acid groups which were "blocked in fixed tissues". Fujita gt gt. (1968) reported that aldehyde-fuchsin and pseudoisocyanin stained the pancreatic B-cell granules. It was concluded that these stains did not stain the insulin but rather the acidic protein of a large molecule, presum- ably a glycolipoprotein, contained within the granules. 99 Fujita gt gt. (1977) suggested that the masked metachromatic staining may have been due to the blocking of acidic groups in the hormone's carrier protein by basic amines and/or peptides. They believed that the carrier protein's acidic groups became exposed or unmasked after acidic hydration of the hormone and, are thus, available for staining by basic dyes. Sections were stained according to the method of Bennett gt gt. (1976) for the demonstration of endocrine cell granules. Ferric Ferricyanide (Lillie and Burtner, 1953) Plastic sections were stained in a solution of ferric chloride (FeCl3) and potassium ferricyanide (KFe(CN)6), for thirty minutes. The solution formed, ferric-ferricyanide, [Fe4+3(Fe+3(CN-16)]r is promptly reduced by various tissue components (phenols, indoles, etc.) to form ferric-ferrocya- nide [Fe4+3(Fe+2(CN-l)6)§ which is Prussian blue. Weak reducing agents or low concentrations of strong reducing agents are thought to reduce ferric-ferricyanide to the intermediate product ferroso-ferric ferricyanide +3Fe+2 [Fe4 (Fe+3(CN which is Prussian green. The 6)6]I ferric-ferricyanide solution is not stable and must be made up freshly every fifteen minutes to avoid precipitation on the tissue. 100 Alcian Blue (Luna, 1968) Alcian blue is a highly selective stain for acid mucopolysaccharides. The basis of its staining activity is not fully understood but it has been suggested that the acidic groups of the carbohydrate may form salt linkages with the dye. Plastic sections were prepared for staining by a mordant with 3% acetic acid for 30 minutes. Sections were then placed in 1% alcian blue in 3% acetic acid for five hours at 60°C. The method described by Luna (1968) for paraffin sections was modified for plastic sections. PAS-Lead Hematogylin (Sorokin and Hoyt, 1978) One of the most widely employed methods for carbohydrate staining is the periodic acid Schiff reaction. The periodic acid breaks carbon chains ofgmflysaccharidescontaining 1,2 glycol groups and then oxidizes the broken ends into aldehyde groups. The exact mechanism by which Schiff's reagent colors the aldehyde groups is not well understood. Periodic acid-Schiff in combination with lead hematoxylin allows for the rapid identification of small-granule endocrine cell populations in the lung and gastrointestinal tract (Sorokin and Hoyt, 1978). Sections were immersed in periodic acid for 3 hours and then were stained with Schiff's reagent for 15 minutes. The sections were subsequently allowed to dry overnight and then stained with lead hematoxylin (l to 3 hours). The mechanism for lead hematoxylin reactivity in tissues is not well understood. 101 Acid Phogphatase (Higuchi gt gt., 1979) Acid phosphatase appears to be associated almost exclusively with lysosomes (Humason, 1972). This enzyme exhibits optimal activity at a pH between 3.8 and 6.0 (H-mason, 1972). A20 dye methods are frequently used for the demonstration of acid and alkaline phosphatases, nonspecific esterases, B-glucuronidase, B-glucosaminidase and aminopeptidases (Lillie and Fullmer, 1976). Naphthol AS phosphates (substrate) are phosphate esters which release highly insoluble napthols following enzymatic hydrolysis. These napthols subsequently couple with diazonium salts (e.g. fast red violet) to form insoluble azo dyes at tissue sites of acid phosphatase activity. Using a modification of Burstone's procedure, Higuchi gt gt. (1972) developed a method for demonstrating acid phosphatase in glycol methacrylate embedded tissue. Plastic embedded laryngeal and tracheal sections were incubated for twenty four hours, rinsed in running tap water, counter- stained with methyl green and mounted in Entellan. Cryostat Histochemistty The histochemical procedures described in this section were carried out on either cryostat sections (monoamine oxidase) or freeze-dried cryostat sections (histamine or tryptamine). Laryngeal and tracheal segments were quickly frozen as described previously. The frozen tissue segments 102 were rapidly transferred to an International Cryostat and allowed to equilbrate to -20°C thin sections fifteen microns thick were collected on glass slides without thawing. The sections were then transferred to a freeze-dryer and dried overnight (-30°C). The following day, the temperature was allowed to rise to 24°C and evacuation was continued for another 6 hours. The sections were then removed and immediately processed for the histochemical demonstration of histamine or tryptamine. Monoamine Oxidase (Glenner gt gt., 1957) The histochemical demonstration of monoamine oxidase (MAO) by the use of tryptamine as substrate and tetrazolium salts as indicators was reported by Glenner gt gt. (1957). Tissue sites which contain monoamine oxidase deaminate tryptamine or serotonin and result in the formation of an aldehyde. The aldehyde formed subsequently reduces the tetrazolium to an insoluble colored formazan (Glenner gt gt., 1960). Fresh laryngeal, tracheal, and duodenal tissue sections (15 mu) were incubated in a solution containing tryptamine or serotonin and nitro-blue tetrazolium for 30 minutes at 37°C. Additional sections were also incubated in substrate free incubation medium for controls. 103 O-Phthalaldehyde Technigue (OPT) (BjOrklund at al., 1972) Bjorklund gt gt,- (1972) described a fluorescent histo- chemical method for the demonstration of histamine. A closed vessel (150 ml) containing lg of o-phthalaldehyde (OPT) was placed in an oven at 100°C for 10 minutes to generate OPT vapors. The vessel was then transferred to room temperature and the freeze-dried tissue sections were placed in the vessel for 90 seconds. Laryngeal and tracheal sections were then removed and exposed to the steam of boiling water for 5 sec. Tissue sections were finally transferred to a 100°C oven, dried for 5 minutes, and mounted in Entellan. Formaldehyde-Ozone Treatment @jorklund gt a1., 1972) Simultaneous exposure of freeze-dried cryostat sections to formaldehyde and ozone vapor was described by Bjorklund gt gt.. (1972) for the fluorescent histochemical demonstration of tryptamine. In this procedure freeze-dried cryostat sections were placed in a 1 liter jar which contained 59 of paraformaldehyde and a Tesla coil which was attached to two metal electrodes. The jar was sealed, exposed to ozone vapor for thirty minutes, and finally transferred to an oven and heated at 80°C for 1 hour. Tissue sections were removed and immediately mounted in Entellan. 104 Combined Fluorescence and Light Microscopy Freeze-dried formaldehyde vapor treated tracheas were used to determine if fluorescent cells were identical to cells seen by the staining techniques described above. Sections were first observed under the fluorescent microscope, photographed, and then were stained with methylene blue and rephotographed. Methylene blue staining was reversed by gently washing the sections in running water for up to ten minutes. Subsequently, the sections were stained with either ferric-ferricyanide, PAS-lead hematoxylin or alcian blue. Sections were always photographed without a coverslip. Electron Microscopy The investigation of tracheal tissue ultrastructure was carried out with the same tissue which had been fixed with 6% formaldehyde and embedded in Spurr's resin. After fixation, dehydration and embedding, l mu sections were treated with formaldehyde and HCl vapors as described earlier. When the fluorescent tracheal cells were distinctly visible in such sections, a correlated electron microsc0pic investigation was carried out. The blocks from which the sections were taken for fluorescence microscopy as described above were subsequently trimmed for thin sectioning with an Sorvall MT-2 ultramicrotome. The thin sections were stained with 1% alcoholic uranyl acetate and lead citrate (Reynolds, 1963) and examined with the electron microscope (EM Philips 200). 105 Microscopy and Photography Fluorescence was observed and photographed with an Olympus BH-RFL epifluorescence microscope. The mounted sec- tion was observed by epifluorescence from a 200 watt mercury vapor lamp. A BG 12 excitation filter and a 470 nm barrier filter were used to visualize formaldehyde-induced, monoamine fluorescence in tracheal and gastrointestinal tissues. Photographs were recorded on Kodak Ektachrome Film (Daylight, ASA 400, DIN 27) and push processed one f stop. If the sections photographed were intended for subsequent staining and light microscopy, the appropriate horizontal and vertical coordinates were recorded from the microscope stage. Light microscopy was observed and photographed by use of a Zeiss Universal microscope equipped with automatic exposure camera. Photographs were captured on Kodak Ektachrome Film (Daylight, ASA64, DIN19) with an 80B filter. Statistical Anatysis of Excitation Emission Maxima The excitation and emission maxima of fluorescent cells in the rat trachea_and duodenum were evaluated by‘a one-way analysis of variance (AOV), to determine if the spectral means were derived from the same or different populations. AOV assumptions of randomization, normality, equality of variances and independence were met. The critical region of rejection was set at a = .01. If the AOV detected signifi- cantly different populations, a Duncan and Scheffe‘ 106 g posteriori test were used to compare individual means. The critical region of rejection for the Duncan and Scheffé g posteriori test were set at a = .05. CHAPTER IV RESULTS Distribution of Fluorescent Tracheal Epithelial Cells Application of the standard Falck-Hillarp technique followed by embedding in glycol methacrylate revealed the presence of numerous cells which were intensely fluorescent in the respiratory epithelial lining of the extrapulmonary airways (Figure 5). Yellow fluorescent mast cells and green fluorescent nerve fibers were evident in mucosal and submu- cosal connective tissues. Yellow fluorescent mast cells (Figure 7) and enterochrOmaffin cells were also seen in sections from duodenum and stomach. The fluorescent extra- pulmonary cells in respiratory epithelium were most numerous in the larynx and upper trachea. However, they were seldom observed in the true vocal folds. Liquid formaldehyde fixed tissues did not reveal any endogenous fluorescent epithelial cells in the larynx or trachea. Weakly fluorescent cells however, were present in the mucosa and submucosa of the extrapulmonary airways and gastrointestinal tract. In the epithelial lining of the duodenum numerous yellow fluorescent triangular cells with their base resting on the underlying basement membrane, 107 108 and thin apical processes projecting towards the lumen were present. Following formaldehyde vapor treatment, the intensity of fluorescence increased in mast cells and entero- chromaffin cells. No visible endogenous fluorescence was observed in the epithelium of the extrapulmonary airways. Subsequent treatment of the plastic embedded section with fumes of concentrated HCl (37%) for one hour resulted in the demonstration of numerous intensely fluorescent cells in the epithelium of the larynx and trachea (Figure 6). The fluorescent intensity of respiratory mast cells as well as gastrointestinal mast and enterochromaffin cells (Figure 8) also increased. A summary of relative fluorescent inten- sities of the various cells studied is included in Table 3. Pretreatment with the amino acid precursors L-DOPA or L-S-HTP did not result in the demonstration of any fluores- cent cells in the tracheal epithelium fixed with liquid formaldehyde. Following exposure of the sections to HCl vapor the number, distribution, color, and intensity of the fluorescent cells appeared similar to control animals. Morphology of the Fluorescent Epithelial Cells In both freeze-dried (treated with formaldehyde vapor) and liquid formaldehyde fixed tissues (treated with formal- dehyde and HCl vapor) the morphology of the fluorescent epithelial cells was similar. The diameter of the fluores- cent epithelial cells ranged from 4 to 8 mu. The cells 109 Figure 5.--Freeze-dried formaldehyde vapor treated and glycol methacrylate embedded tracheal section (4 mu). Yellow fluorescent cells are present in the respiratory epithelium (2,000x). Figure 6.--Liquid formaldehyde fixed, formaldehyde-HCl induced fluorescent tracheal epithelial cells (1 mu), embedded in Spurr's resin (1,000x). 111 Figure 7.--Freeze-dried, formaldehyde vapor treated, and glycol methacrylate embedded tracheal section (4 mu). Yellow fluorescent mast cells are present in the submucosa (2,000x). Figure 8.--Liquid formaldehyde fixed, Spurr embedded, formaldehyde-HCl vapor treated duodenal section. Fluorescent enterochromaffin and mast cells are present in mucosa (1,000x). 113 .moamommuooam mcouum u A+++v .mocmomouosfim muwumooa n A++V .wocmommnosaw usmflam u A+V .mosoomwnooam on u AJV mummcmusw mosmommuooam +++ +++ +++ +++ Aoavm .u: av Mom Homm> Hum com Aooom .Hn Hy Homm> mcmnw©HMEHom ou musmomxmlmm I I I I A.cHE NV usofiummuu mpflnomnouom Eoflpom +++ +++ +++ +++ Aoovm .un av uoom> Hum pmumuucmosoo ++ ++ ++ I Aooom .u: av Homm>lm©>nmcamsuom ++ + + I mmommflu coxfim mcasmoameuom aw :HMmmEounoonwusm HHmO ummz HHmU ummz Hamo Hawamnuflmm ucmfiummue Hmcfiummucflouummw Hmwnowne .mmuspmooum HMUHEanODmH: msowum> Hmumm mum“ “Homo mo muomuu Hmswu Imoucflonummm pom huoumnflmmmu man :H maawo mo muflmsmucw wocmommuosam m>fiumammII. m magma 114 were characterized by conspicuous fluorescent cytoplasmic granules. The number of granules per cell varied from 1 to 15. The granules also varied in size in different cells and even within the same cell. The fluorescence was always confined to the granules within the cytoplasm. The granules appeared to be randomly distributed throughout the cyto- plasm. The nucleus was seldom observed by fluorescence microscopy. Occasionally, a dark area was observed between the fluorescent granules which appeared to be the nucleus of the cell. Fluorescent cells were located at all levels of the epithelium, but most frequently they were located lightly apical to the more basal, non-fluorescing epithelial cells. Microspectrofluorimetric Data The fluorescence excitation and emission maxima of tracheal epithelial cells and gastrointestinal entero- chromaffin cells fixed with liquid formaldehyde, embedded in plastic and treated with formaldehyde and HCl vapors are reported in Table 4. Spectral analyses of seven tracheal fluorescent cells from four animals demonstrated an average maximum excitation of 456 nm : 5 and a maximum emission at 535 nm : 13. The actual spectral curves from one represen- tative is demonstrated in Figure 9. The average excitation and emission peaks of five gastrointestinal enterochromaffin cells were 425 nm : 0 and 513 nm : 3 respectively. An example of the actual spectral curve from one cell is presented in Figure 10. 115 Excitation and emission maxima of fluorescent tracheal epithelial cells and connective tissue mast cells which were prepared according to the standard Falck Hillarp method are reported in Table 5. Spectral analysis of three tracheal epithelial fluorescent cells demonstrated an average maximum emission of 493 nm : 3. The excitation maximum was not demonstrated between 350 nm and 450 nm. The actual emission spectrum from one cell is demonstrated in Figure 11. In addition, the average excitation and emission maxima of three fluorescent tracheal connective tissue mast cells were 401 nm 1 11 and 515 nm : 5 respectively. An example of the actual spectra from one cell is presented in Figure 12. Table 4.--Spectral properties of tracheal (TRACHEAL) and duodenal (GI) fluorescent cells from formaldehyde-HCl treated sections. (Liquid formaldehyde fixed tissue embedded in Spurr's resin, 6 mu thick). Mean and standard deviation of the excitation and emission maxima are also presented. Excitation Peak (nm) Emission Peak (nm) Tracheal l 455 540 2 455 530 3 460 550 4 465 545 5 455 530 6 450 540 7 450 510 Mean 456 i 5 535 i 13 gt 1 425 515 2 425 515 3 425 510 4 425 510 5 425 515 Mean 425 i 0 513 i 3 116 Table 5.--Spectral properties of fluorescent tracheal epi- thelial cells (EPITHELIAL) and fluorescent connective tissue mast cells (MAST) from freeze-dried paraffin embedded sections, 15 mu thick. Mean and standard deviation of the excitation and emission maxima are also presented. Tracheal Fluorescent Excitation Emission Cell Peak (nm) Peak (nm) EPITHELIAL l --- 490 2 --- 495 3 --- 495 Mean 493 i 3 MAST l 415 515 2 395 520 3 395 510 Mean 401 i 11 515 i 5 Statistical Analysis of Excitation and Emission Maxima The excitation and emission maxima of fluorescent cells in the rat tracheal epithelium and duodenum were evaluated by a one-way analysis of variance (AOV). All AOV assumptions of randomization, normality, equality of variances, and independence were met. One way analysis of variance indi- cated that excitation and emission maxima from the different cells evaluated were derived from different populations of fluorogenic substances at 5:01. In order to determine the source of significance, a Scheffe and Duncan g posteriori testswere calculated. The critical region of rejection for the g posteriori testswere set at a=.05. 117 Figure 9.--Excitation (—--) and emission (———) curves of a fluorescent tracheal epithelial cell, liquid formaldehyde fixed, embedded in Spurr's resin, and treated with formal- dehyde-HCl vapor. 118 .55 155.5%; 08 of. Rx. Tom 1mh I96] ALISNBLNI 119 Figure 10.--Excitation (---) and emission (———) curves of a liquid formaldehyde fixed, Spurr embedded, formaldehyde-HCl vapor induced fluorescent gastrointestinal (duodenum) cell. 120 _E:_ IPOZmAm><>> CON 0 m 3 3 8. a 1. 13 l 10m . l- ? In” as I x \x l n b F '00—. l%l AlISNalNI 121 Figure ll.—-Excitation (---) and emission (———) curve of a fluorescent tracheal epithelial cell, freeze-dried and paraffin embedded. 122 80 .55 155.323 0?, 9.x. 0?. tom l%l AllSNalNl 123 Figure 12.--Excitation (----) and emission ( ) curves of a fluorescent tracheal connective tissue mast cell, freeze- dried and paraffin embedded. 124 .55 IP02m4m><>> as 3. as 8. as. 0 1| In" I Ion \I :l s Um“ \ x \ \ x \ l ,(\/\ I96] AllSNBlNI 125 The results from the Scheffe and the Duncan multiple comparisons tests were identical. They demonstrated a significant difference between the emission maxima of the tracheal epithelial cells when prepared by different methods. However, in cells known to contain serotonin (tracheal mast cells and duodenal enterochromaffin cells) the method of preparation had no effect on the emission maxima. In addition, regardless of the method of preparation a signi- ficant difference in the emission maxima was noted between tracheal epithelial cells and cells known to contain serotonin. Results of both multiple comparisons tests are summarized in Table 6. The excitation maxima of all the cells studied were significantly different from each other. However, excitation maxima from freeze-dried, formaldehyde vapor treated, paraffin embedded fluorescent epithelial cells were not recorded. Results of both multiple comparisons tests are presented in Table 7. Specificity of the Fluorgphore Freeze-dried and liquid formaldehyde fixed tissues, embedded in glycol methacrylate and Spurr's resin were subjected to three histochemical tests to determine if the fluorophores were due to the presence of a monoamine. The first histochemical test on freeze-dried tissue demonstrated that the cellular fluorescence resulted from the formaldehyde vapor treatment. Fluorescent cells in respiratory and 126 .usmnwmmaw wauamoflmasmflm Boz mum msflcwaumoso an omuomccoo mammz« as; H mama N; H CAR m.~ H 0.3m o.m H mem cams mug mug mug F": Amnmv Anomach Amomc Aqommmqv Hamo Hamo Hamo Hawo HMflHmnuHmm sawmmeonnoouwucm ummz Hafiamzuwmm Hmmnomna amsflumoucfiou0mmw Hmmnomua Hmmnomus mmwa HHmU .mo.ua sufl3 mummy HHoHHmumom m cmosso can mmmonom an poummfioo muo3 mamme on» @muos mm3 mocmoflmacmflm sons can mustanm> mo mflmwamcw :DHS vmuwamsm umuflm mums maflxmfi :oflmmflEo was .Amomv sflmmmumm ca wwocwnfiw can owwuoImnmmum Ho Aqommmqv uomm> Hum cues @mummuu paw oaummHm ca cmoomnfim .mohnmo IHmEuom pflswfla nufl3 pmxflm nonuflm mum3 mwsmmau one .mmmhu Hamo Moon Eoum coauowm DE m 6 ca mHHmo unmommnosam mo “E: Gav meme coflmmfifim mo Honum oumnsmum can smozII.m manna 127 .ucmummmap waucmoflmwsmwm Boz mum mcficwaumvso ho pmuomscoo mammza am.m + m.Hov o + o.mmv o.~ + h.mm¢ cmmz mug mug Fug Amomv xqommmqv “dormant Hawo Haoo HHmO ummz cflmmmeonnooumucm HMflHmnuHmm Hmmnomne Hmsflumousflouummw Hmmnomne omha Hawo .mo.ua sues mummy HuoflumumomIm cmosza pom wmwonom >9 UmHmmEoo wum3 msmofi map owuoc mm3 mosmoHMHcmHm c033 can OOQMHHM> mo mflmwamsm nufl3 cmuwamcm umuflm mum3 mEflme coflumufloxm 039 .AQOV cwmmmumm cw omowonfiw new amaHaImammum no Aqomamac uoam> Hum spa; noummuu 6cm oaummaa an amacmnsm .mcssmo Iamfinom weaved £0fl3 omxflm nonuwo mumz mmSmmwu was .mmmmu Haoo moan» Eonm coauowm 58 m m CH mHHmo unmommuosam mo AB: say MEmeE cofiumuwoxm mo Honum cumocmum can :mmSII.n manna 128 gastrointestional epithelia were absent in tissue samples not exposed to formaldehyde vapor. The second test demonstrated that irradiation with ultraviolet light produced a marked photodecomposition and loss of fluorescent intensity in fluorescent cells in the laryngeal and tracheal respiratory epithelium. This was demonstrated in both freeze-dried and liquid formaldehyde- HCl vapor treated samples. The third test investigated the reducing capabilities of sodium borohydride on cellular fluorescence. Freeze-dried and liquid formaldehyde fixed HCl vapor treated tracheal sections embedded in plastic were observed with fluorescence microscopy to establish that the fluorescent cells were present in the epithelium. The specific fluorescence disappeared completely within one or two minutes after the sections were treated with a 0.5% solution of sodium borohydride in 80% ethanol. Sections treated only with 80% ethanol did not cause a change in the appearance of the fluorophore. Following exposure to sodium borohydride, the cellular fluorescence was regenerated after renewed formal- dehyde vapor or formaldehyde-HCl vapor treatment. A summary of the results are presented in Table 3. Histochemical Results Nearly all of the histochemical procedures described in the literature were accomplished with paraffin embedded sections. These results, however, concern the use of 129 histochemical techniques on tissue sections embedded in plastic. It has been reported (Bennett gt gt., 1976) that many of the conventional histologic staining methods used for paraffin can be applied to tissues embedded in glycol methacrylate. Freeze-dried, formaldehyde vapor treated plastic sections from control animals were used to investi- gate the histochemical characteristics of rat tracheal epithelium. A chart summarizing the staining reactions used appears in Table 8. Methylene Blue The methylene blue stain has been shown to be associated with the secretory granules of endocrine cells storing biogenic amines (Solcia gt gt., 1968; and Bennett gt gt., 1976). Methylene blue staining demonstrated numerous brilliant blue cells in the respiratory epithelial lining while leaving the remaining epithelial surface a pale blue. Mast cells, tracheal cartilage matrix, and nerve bundles also demonstrated a strong affinity for methylene blue. The staining of these various tissue components was reversed by washing the section in running tap water. The question of whether methylene blue positive cells were the same as fluorescent cells in tracheal respiratory epithelium was answered by photographing a fluorescent section, subsequently staining with methylene blue, and then rephotographing the same area when viewed with ordinary light microscopy. The results of this procedure clearly 130 demonstrated that the methylene blue positive cells were identical to the fluorescent cells. The three fluores- cent cells in the upper panel of Figure 13 were also methylene blue positive (lower panel). Alcian Blue Alcian blue has been used to demonstrate the presence of acid mucopolysaccharides in tissue sections (Luna, 1968). Alcian blue stained tracheal epithelial cells, cartilage, mast cells, and some cells in the submucosal glands. The staining in the epithelium suggested two different cell populations. One population of cells appeared to stain a very faint blue. These cells were often in contact with the lumen and never appeared granular. The second population of cells stained much darker, seldom reached the lumen and always appeared to be very granular. The results of restaining the same section with alcian blue after fluorescence microscopy demonstrated that the granular alcian blue positive cells were identical to fluorescent positive cells. The intensely fluorescent cell shown in the upper panel of Figure 14 is identical with the alcian blue positive cell in the lower panel. Several weakly fluorescent cells appearing in this figure were also intensely alcian blue positive. Ferric-ferricyanide Ferric-ferricyanide reacts with various tissue compo- nents which are capable of reducing ferric-ferricyanide to 131 Figure 13.--The same tracheal section (6mu) photographed first during fluorescence microscopy (upper panel), and then stained with methylene blue and rephotographed during ordinary light microscopy (lower panel) (2,000x). 133 Figure l4.--The same tracheal section (6 mu) photographed first during fluorescence microscopy (upper panel), and then stained with alcian blue and rephotographed during ordinary light microsc0py (lower panel) (2,000x). 135 ferric-ferricyanide (Lillie and Burtner, 1953). This reaction is often associated with phenols or indoles (Lillie and Fullmer, 1976). One distinct ferric-ferricyanide positive cell can clearly be identified in the lower panel of Figure 15. Three faintly positive cells are also 4 demonstrated in the same picture. The results of observing the same section after two different treatments is presented in Figure 15. The fluorescent epithelial cells demonstrated in the upper panel are also ferric ferricyanide positive (lower panel). This series of photographs demonstrated that the fluorescent and ferric-ferricyanide positive cells were identical. PAS-Lead Hematotylin The periodic acid Schiff (PAS) reaction has been associated with the presence of carbohydrates, neutral mucopolysaccharides, mucoproteins, and lipids (Sheehan, gt gt., 1973). PAS positive cells in the tracheal epithe- lium are demonstrated in Figure 1?. Numerous PAS positive cells were observed to contact the luminal surface and a few positive cells were closer to the basement membrane. The question of whether PAS positive cells were the same as fluorescent and methylene blue positive cells was answered by photographing fluorescent epithelial cells, staining the same tissue section with methylene blue, rephotographing during observations with ordinary light 136 Figure 15.--The same 6 mu tracheal section photographed first during fluorescence microsc0py (upper panel) and then rephotographed by ordinary light microscopy after staining with ferric-ferricyanide (lower panel) (2,500x). 138 microscopy, subsequently washing the section with running tap water, and then restaining with PAS and photographing again. The results of methylene blue staining are demonstrated in Figure 16. The methylene blue positive cells in Figure 16 also were PAS positive (Figure 17). In addition, a second population of PAS positive but methylene blue negative cells was also demonstrated. Subsequent staining of the same section with lead hematoxylin resulted in a darkening of all of the PAS positive cells. Definitive selective staining of the fluorescent and methylene blue positive cells could not be demonstrated with lead hema- toxylin. Acid Phosphatase The acid phosphatase stains react almost exclusively with lysosomes (Humason, 1972). A20 dye granules were pre- sent in cells located in the lamina propria of the duodenum and the submucosa of the esophagus. However, acid phosphatase positive cells were not observed in the epithe- lial lining of the larynx, trachea, or duodenum. Monoamine Oxidase Monoamine oxidase acts on long and short-chain mono- amines Chayen gt gt. (1973). Glenner gt gt. (1960) suggested that monoamine oxidase activity could be demonstrated by the reduction of tetrazolium salts. In Glenner's scheme the product of monoamine oxidase, indolyl-3-acetyldehyde, 139 Figure 16.—-A 6 mu tracheal section photographed after staining with methylene blue (1,000x). Figure l7.--The same 6 mu tracheal section (Figure 16) after washing with tap water and restaining with PAS and photographing again (1,000x). 140 \ u‘l I I -‘ I ‘ — Q \ k S p. P' ' ' u “ .. T f\ l o ' . Q. ) ' ‘ 1 l ‘s‘ ' ' ‘ ‘ ,. /' ~ ' olfit 3.. - _ . ‘ 1 ~ '. . - a, "’ Ear {(521}? in J .I ~ ‘: a & :‘I ‘ t ’1 .1. 141 reacts spontaneously with nitro-blue tetrazolium to yield a colored formazan. A very intense monoamine oxidase activity was observed in fresh cryostat sections of larynx, trachea, and kidney epithelium incubated with serotonin. In contrast, sections that were incubated without the substrate serotonin, did not result in the formation of formazan granules. O-Phthaladehyde and Formaldehyde- Ozone Reactions The fluorescent histochemical demonstration of hista- mine or tryptamine was not observed in the laryngeal or tracheal epithelium of freeze-dried cryostat sections. Electron Microscopy One micron thick sections were cut from liquid formal- dehyde fixed (6%) Spurr embedded material. Tracheal and gastrointestinal sections were then exposed to formaldehyde and HCl vapor and observed for cellular fluorescence. Numerous duodenal mast cells and enterochromaffin cells as well as tracheal mast cells and epithelial cells were distinctly fluorescent. Following identification of fluor- escent epithelial cells, the blocks were carefully trimmed and a correlated ultrastructural investigation was carried out on immediately adjacent ultrathin sections. Two fluorescent cells shown in Figure 23 are also observed at the ultrastructural level in Figure 24. 142 I I . I mmEOmOmhq m+ m+ m+ moascmuw oanUOUcm mooflumnoommhaomoosz Hmuuomz I I + mmumuohsonumu I I + moosmumnsm mcfloswmm meH—CMHU wGHHOOUCW mmmumsmmonm oflod :aaxxoumfimm Iwmmq mdm m