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'TI'I' II'I‘ITjI‘: III II' III'I'I' T'I‘I'I IIIITII 'I" III. I . "III" .I '3" ,1. T .IIIIIIIII. . IIII.I IE‘II IIIII "IIII II'IT,II I IITTI;. .l.!,..z,:I.II...I 35 It;- I I I. I II .I'IITIIT‘T II'III IIIII' 'ITI' IT": II'I" III 151'. ‘I' '"TTTTT II-III '5 IIIT' I T _ I IT'II'“ I". I I 'ITI'III T'II' III: 'Il'TI 'T'TITIIIIY/ IIIIIIIIIIT'T 'TT' 3 I T‘ 'll' IT'IIITI'TIT'I'I'I"IHT"TIIIIII "II. I "III ..I" I I .. '-.I. IT‘;".‘T'T T. IT!” I" ,f'IIIszIIII.‘IT I I' I ’ .1 ’IT‘ .TI- ,g. .I12‘.IIII1’TI I‘II' I II ITT 'I 'T ‘ITT‘JI I .II I» TIIITIII II... ' 'I III lI'lIT'III TITJ'. I I H T II. ',, I IT TITT I II I I‘ 'III 'I TI I ‘l‘ I ‘1 ‘IO' II I II I 'I I. I Tl I 'II 'I I II I T I\. H I In 'I T ' III . ._ II I I, ' " I III III I; . II. N LIBRARY meme- ’ m This is to certify that the thesis entitled Morphology, Distribution, and Histochemical Characteristics of Fluorescent (5-hydroxytryptamine-Containing) Cells in Tracheal Epithelium of Adult Rabbits presented by Richard D. Dey has been accepted towards fulfillment of the requirements for Ph . D. degree in Anatomy %/4 Afl/L—a Robert Echt, Ph.D. Major professor Date February IO. I979 0-7 639 OVERDUE FINES ARE 25¢ PER DAY PER ITEM Return to book drop to remove this checkout from your record. MORPHOLOGY, DISTRIBUTION, AND HISTOCHEMICAL CHARACTERISTICS OF FLUORESCENT (5-HYDROXYTRYPTAMINE-CONTAINING) CELLS IN TRACHEAL EPITHELIUM OF ADULT RABBITS BY Richard Dennis Dey A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Anatomy 1979 ABSTRACT MORPHOLOGY, DISTRIBUTION, AND HISTOCHEMICAL CHARACTERISTICS OF FLUORESCENT (5-HYDROXYTRYPTAMINE-CONTAINING) CELLS IN TRACHEAL EPITHELIUM OF ADULT RABBITS BY Richard Dennis Dey Amine-containing endocrine-like cells in the tracheal epithelium of adult male New Zealand White rabbits were studied by morphologic, morphometric, and histochemical techniques. Tracheas were freeze-dried and treated with formaldehyde vapor to convert cellular amines to fluorescent substances. Numerous fluorescent cells were identified within the tracheal epithelium of formal- dehyde treated specimens, but they were not seen in untreated samples. Distribution of the fluorescent epi- thelial cells was evaluated by counting the number of cells per section in six tracheal regions. Cell counting was also used to evaluate changes in amine-containing cells after pretreatment with L-dihydroxyphenylalanine (L-DOPA)cu' reserpine. Ventral aspects of the tracheal epithelium from control rabbits contained significantly more fluorescent cells than the dorsal aspects. The number of fluorescent cells Richard Dennis Dey was not affected by treatment with L-DOPA, but tracheas from reserpine treated rabbits contained significantly fewer cells than controls. Identification of the cellular amine in control and L-DOPA treated rabbits was accomplished by micro- spectrofluorometric and histochemical techniques. Excitation and emission peaks of fluorescent cells from controls indicated that the cellular amine was SHT. This finding was supported by positive staining with diazo- safranin and ferric-ferricyanide. The emission peak of fluorescent cells from L-DOPA treated rabbits was shifted to shorter wavelengths indicating uptake of L-DOPA in the SHT-containing cell. Histochemical reactions were cor- related with the SHT-containing cell by photographing a tracheal section by fluorescent microscopy, then staining and rephotographing the same section by light microscopy. In addition to positive reactions with ferric- ferricyanide and diazosafranin, the fluorescent cells were also argyrophilic. However, no alcian blue reaction was detectable in the fluorescent cells. Serotonin-containing cells in extrapulmonary airway epithelium may regulate mucus secretion, ciliary motion, blood flow or smooth muscle tone either by direct action within the trachea or throughout the respiratory system by reflex nervous pathways. The possibility that these cells contain an as yet Richard Dennis Dey unidentified bioactive peptide is suggested by APUD characteristics and argyrophilia. To my loving wife, Midge. ii ACKNOWLEDGMENTS I would like to acknowledge and extend my sincere appreciation to Dr. Robert Echt for his guidance, encouragement, and advice throughout my graduate program and for unlimited use of laboratory space and facilities. My thanks are also due to Drs. Marek Pienkowski, Lawerence Ross, David Reinke, and Jon Kabara for their many helpful comments and valuable advice while serving on my guidance committee. Special thanks to Dr. James Bennett for his help with freeze-drying and fluorescence microscopy and to Dr. Robert Dinerstein for providing the microspectro— fluorometric data. I gratefully acknowledge the Department of Anatomy, Michigan State University, for their generous support throughout my graduate program. My sincere thanks to all the faculty, staff, and students in the Anatomy Department for their friendship and help. iii TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . Chapter 1. INTRODUCTION . . . . . . . . . . Statement of the Problem . . . . . Research Plan and Rationale . . . . Significance . . . . . . . . . Limitations . . . . . . . . . . 2. LITERATURE REVIEW . . . . . . . . Endocrine-like Cells in Tracheobronchial Epithelium . . . . . . . Neuroepithelial Bodies . . . . . Unified Endocrine Cell Concept (APUD Series) . . . . . . . . 3. MATERIAL AND METHODS . . . . . . . Experimental Animals . . . . . . . Specimen Removal . . . . . . . . Freezing and Freeze Drying . . . . . Formaldehyde Vapor Treatment . . . Paraffin Embedding and Sectioning . . Microscopy and Photography . . . . . Specificity of the FluorOphore and Formaldehyde-induced Fluorescence . . Microspectrofluorometry . . . . . . Histochemical Procedures . . . . . Combined Fluorescence and Light Microscopy . . . . . . . . Distribution of Fluorescent Tracheal Epithelial Cells . . . . . Effects of L-DOPA and Reserpine on the Numbers of Fluorescent Cells . . . Statistical Analysis . . . . . . . iv Page vi vii axons-w \l 28 31 31 31 32 36 37 38 39 40 41 44 45 46 47 Chapter Page 4. RESULTS 0 o o o o o o o o o o o o 48 Morphologic Description of the Rabbit Trachea . . . . . 48 Morphology of Fluorescent Cells . . . . 49 Microspectrofluorometric Data . . . . . 49 Specificity of the Fluorophore . . . . 52 Distribution of Fluorescent Cells in Control Tracheas . . . . 6l Effects of Intraperitoneal L-DOPA or Reserpine Injections . . . . . . . 61 Histochemical Results . . . . . . . 69 5. DISCUSSION . . . . . . . . . . . . 84 Morphologic Characteristics . . . . . 84 Identity of the Fluorophore . . . . . 85 Functional Significance of SHT in Tracheal Epithelium . . . . . . . 89 Effect of Injecting L-DOPA . . . . . . 91 Argyrophilic Reaction . . . . . 93 Peptide-producing Cells in Tracheal Epithelium . . . . . . . . . . 93 6. SUMMARY, CONCLUSIONS, AND RECOMMENDATIONS . 95 Summary . . . . . . . . . . . . 95 Conclusions . . . . . . . . . . . 96 Recommendations . . . . . . . . . 97 Appendix A. DATA AND STATISTICAL TABLES . . . . . . 100 B. HISTOCHEMICAL TECHNIQUES . . . . . . . 105 REFERENCES . . . . . . . . . . . . . 114 Table 1. LIST OF TABLES Spectral data of fluorescent cells from control and L-DOPA injected animals and serotonin standards . . . . . Mean and standard error of fluorescent cells in a 15 um section from six regions. Compared by SNK with a = 0.05 (n = 6) O O O C O O O O O O 0 Actual range and least significant range (in parentheses) used in SNK multiple comparisons test. a = 0.05. (n = 6) Mean and standard error of number of fluorescent cells in 30 tracheal sections from control, L-DOPA, and reserpine groups. Compared by SNK with a = 0.05. (n = 9) . . . . . Significance and location of selected histochemical reactions . . . . . vi Page 53 64 65 66 70 LIST OF FIGURES Figure Page 1. Freeze—drying apparatus: (a) vacuum evaporator, (b) vapor trap, (c) refrigeration unit . . . . . . . 33 2. Vapor trap: (a) chemical trap, (b) cold trap, (c) Dewar for liquid nitrogen . . 33 3. Inside the freezing chamber: (a) refrig- eration coils, (b) container for acetone and dry ice, (c) vacuum flask containing P205 . . . . . . . . . . . . . 33 4. Freeze-dried formaldehyde vapor treated tracheal section. Fluorescent cells present in epithelium. 175x . . . . . 50 5. Freeze-dried formaldehyde vapor treated tracheal section. High magnification of fluorescent epithelial cells. 715x . 50 6. Excitation and emission curves of a fluorescent cell from control rabbit . . 54 7. Excitation and emission curves of a fluorescent cell from L-DOPA injected rabbit . . . . . . . . . 56 8. Freeze-dried tracheal section not treated with formaldehyde vapor. No fluorescent cells appear in the epithelium. This section was taken from the same rabbit as the tracheal section in Figure 4. 175x . . . . . . . . . . . . . 58 9. Distribution of fluorescent cells in six regions of rabbit trachea. The values are expressed as the average number of cells in one 15 um tracheal section. n = 6 . . . . . . . . . . . . 62 vii Figure 10. 11. 12. 13. 14. 15. 16. 17. Average number of fluorescent cells in ‘30 tracheal sections from control, L-DOPA, and reserpine treated rabbits. n = 9 . . . . . . . . . Freeze-dried formaldehyde vapor (FD-FV) treated tracheal section stained with alcian blue and safranin 0. 80X . . . FD-FV treated tracheal section demonstrating two argyrophilic cells. 715x . . . . . . . . . . The same tracheal section photographed first by fluorescence microscopy (lower panel), then stained for argyrophilia and rephotographed by light microscopy (upper panel). 715 X . Alcian blue and argyrophilic stains applied to the same tracheal section. 715x 0 O O O O O I I O O O FD-FV treated tracheal section stained with ferric-ferricyanide. 715x . . The same tracheal section photographed first by fluorescence microscopy (upper panel) and then stained with ferric-ferricyanide and rephotographed by light microscopy (lower panel). 288x . . . . . . . . . . . . FD-FV treated tracheal section stained with diazosafranin. 715x . . . viii Page 67 71 71 74 76 76 79 81 CHAPTER 1 INTRODUCTION The respiratory epithelium is composed of several cell types distinguishable by morphological and cyto- chemical characteristics (Rhodin and Dahlman, 1956; Jeffery and Reid, 1975). One cell type, the endocrine- like cell, is characterized by dense-cored secretory granules (Bensch gt 31., 1965), argyrophilic reaction (Tateishi, 1973), and either endogenous amine content (Lauweryns and Peuskens, 1969) or induced amine content after administration of an amine precursor (Hage, 1971). The same characteristics are associated with peptide- producing endocrine cells in the gastrointestinal tract, pancreas, thyroid and other endocrine organs (Pearse, 1977). It has been hypothesized that all peptide-producing endocrine cells share a common embryo- logic origin (APUD concept, Pearse, 1969). Endocrine-like cells reveal regional variations along the respiratory tract. Studies of intrapulmonary epithelium demonstrated that the cells occurred either in groups (Lauweryns and Peuskens, 1972; Lauweryns and Godderis, 1975) or singly (Hage, 1972; Cutz and Conen, 1972), while endocrine-like cells in extrapulmonary airways always occurred singly (Ericson gt_gt., 1972; Ewen gt gt., 1972; Cutz gt gt., 1975). Endogenous amine-containing cells have been found within intra- pulmonary epithelium (Lauweryns and Peuskens, 1969), but have not been reported in the extrapulmonary airways. Age and species also influence the appearance of endocrine-like cells. Differences in morphology and type of secretory granules, histochemical profiles, and amine content (Hage, 1976; Cutz gt gt., 1975) have been reported. Endocrine-like cells in lungs from fetal, newborn and adult humans each demonstrated unique ultra- structural, cytochemical, and fluorescent profiles (Hage, 1973 a,b; Lauweryns 23.2l'r 1970; Hage gt gt., 1977). In one study, differences in ultrastructural features of endocrine-like cells were reported in tracheas from different animal species (Cutz gt gt., 1975). Endocrine-like cells in lungs of fetal animals have been extensively studied by Hage (1974) and Cutz gt gl.,(1974), but investigations of newborn and adult animal lungs are incomplete. Because several functional types of endocrine- like cells may be present, correlation of specific morphologic and cytochemical characteristics in individual cells is essential for their complete identifi- cation. For example, in the gastrointestinal tract, eight different endocrine-like cell types are each associated with a different peptide hormone. The signif- icance of unique cytochemical and morphological character- istics in different endocrine-like cells of respiratory epithelium has been postulated (Hage, 1976) but not proven. The importance of direct correlation of these characteristics has been stressed in two recent reviews (Solcia gt gt., 1975; Fujita and Kobayashi, 1977). The distribution of argyrophilic cells in human adult (Tateishi, 1973) and newborn (Lauweryns gt gt., 1970) lungs have been qualitatively described. However, detailed quantitative analyses of specific amine- containing cells have not been reported. Quantitative evaluation of cell distributions have been used to assess dynamic properties of amine-containing cells (Hakanson gt gt., 1970) and peptide-containing cells (Polak gt gt., 1975) of the gastrointestinal tract. Statement of Problem Pilot studies conducted by the author showed that the tracheal epithelium of adult rabbits contained formaldehyde-induced fluorescent cells. This type of fluorescence indicated the presence of endogenous amines. The present study was undertaken because of the lack of any reports identifying endogenous amine-containing cells in extrapulmonary airways, increasing evidence for the existence of endocrine-like cells in respiratory epithelium, and the potential biological importance of cells which synthesize and secret biogenic amines or peptide hormones. The purpose of this study was to identify and characterize the tracheal amine-containing cells using morphologic, morphometric, and cytochemical techniques. Research Plan and Rationale The study will describe certain morphologic and cytochemical characteristics of endocrine-like cells in the tracheal epithelium of adult male New Zealand White rabbits. Identification of the amine contained in these cells is essential in order to postulate their functional roles. Endogenous biogenic amines will be demonstrated by treating freeze-dried tracheas with formaldehyde vapor. The identity of the amine will be evaluated by histochemical and microspectrofluorometric techniques. Histochemical characteristics will provide a basis for functional descriptions of endocrine-like cell populations in general, and direct correlation of these characteristics in individual cells will determine if different cell types are present. Argyrophilia and lead- hematoxylin positive cells will imply peptide production. Argentaffin and ferric-ferricyanide positive cells will indicate the presence of amines, while diazosafranin will be used to indicate serotonin. Alcian blue will be used to detect mucopolysacchrides. Cytochemical profiles will be correlated directly by applying these histochemical reactions to identical tracheal sections. Quantitative evaluation of the distribution of amine-containing cells in the tracheal epithelium will also provide a basis for postulating their functional roles. Distribution will be evaluated by counting the number of fluorescent cells in sections taken from dorsal and ventral aspects of cranial, middle, and caudal tracheal segments. The same counting technique will be used to com- pare the number of fluorescent cells in tracheas from L-dihydroxyphenylalanine (L-DOPA) or reserpine injected animals to controls. In this way it will be possible to determine: (1) if L-DOPA reveals the presence of endocrine-like cells in the tracheal epithelium which do not contain endogenous amines, (2) if the endogenous amines are stored by a reserpine sensitive mechanism, and (3) the efficacy of cell counting as a technique to evaluate dynamic changes in endocrine-like cell populations. Because amine precursor uptake is characteristic of APUD cells, the effect of injecting L-DOPA will be evaluated by microspectrofluormetry in amine-containing cells. Significance This study will extend the existing knowledge about endocrine-like cells in the respiratory epithelium, especially the extrapulmonary airways. By affecting integrated pulmonary function, these cells may play important roles in health and disease of the respiratory system. Defining the exact role of this possibly diverse population of cells can begin only after complete morphologic and cytochemical identification. Limitations 1. This study is restricted to the tracheal epithelium of adult male New Zealand White rabbits. 2. Selected histochemical techniques were used to describe particular facets of intracellular chemistry and do not necessarily describe the full chemical potential of the tissue. 3. The observations are restricted to the limitations of light and fluorescence microscopy. CHAPTER 2 LITERATURE REVIEW Tracheobronchial epitehlial cells have been clas- sified anatomically according to structural and histo- chemical differences. The cell type of primary interest in this study is characterized by dense-cored granules, argyrophilic staining, and amine content or ability to incorporate and decarboxylate amine precursors and store the resulting monoamine. These cells are commonly named Kultschitzky-like, Argyrophil Fluorescent and Granulated (AFG), or endocrine-like. Neuroepithelial bodies (NEBs) are specialized groups of endocrine-like cells and will be presented separately. The presence of endocrine cells in tracheobron- chial epithelium was postulated first by Feyrter (1934). According to Bensch gt gl., (1965), Feyrter originally described clear cells in gastrointestinal tract, pan- creatic ducts and gall bladder of several mannals including human, and hypothesized that the clear cells comprised a "diffuse endocrine epithelial organ." Later, Feyrter (1953) identified endocrine cells in bronchi, gen- itourinary tract, nasal mucosa, and sweat and salivary glands. Bensch gt gt" (1965) cited the work of Froelich (1949) as the first reported histologic evidence of clear cells in the lung. Froelich also reported argyrophilic cells in bronchial epithelium. Endocrine-like Cells in Tracheobronchial Epithelium Intrapulmonary Airways Adult Human Lung Several investigations have described endocrine- like cells in the adult human lung. Using the Bodian argyrophil technique, Tateishi (1973) estimated that 98% of lung autopsy specimens from Japanese adults contained oval or triangular shaped argyrophilic cells. Of the 45 specimens, 91% had agryrophilic cells present in bronchioles, 19-33% had them present in segmental bronchi, and 6-9% had them present in lobar bronchi. One specimen had a cell in the terminal bronchiole, and one had a cell in a glandular duct. The cells were usually in clusters but single cells were also seen. Cytoplasmic processes which contained argyrophilic material were also observed. The investigator noted that the cells were frequently associated with goblet cells. Argentaffin silver methods were negative. Argyrophilic cells in EurOpean adults were observed by Hage gt_gl,, (1977) using Grimelius and Sevier-Munger reactions. The triangular or flask—shaped cells were near the basement membrane but did not reach the lumen. In contrast to Tateishi's findings, Hage gt gt,,(1977) believed the cells were more numerous in lobar and proximal segmental bronchi; however, their method of quantitation was not described. In addition, the cells were seen more frequently as single cells than in groups. Hage gt gl.,(l977) and Hage (1973a) have incubated pieces of adult human lung tissue in L-DOPA or 5- hydroxytryptophan (5HTP) and detected fluorescent cells with morphology and distribution similar to argyrophilic cells in freeze-dried formaldehyde vapor treated speci- mens. Fluorescence was not present in unincubated samples. The L-DOPA fluorophore appeared green and the 5HTP fluorophore was a weak yellow color. Diazonium Fast Black K, lead—hematoxylin, and argentaffin histochemical reactions were all negative. The first ultrastructural account of pulmonary endocrine—like cells was reported by Bensch gt gt., (1965L Adult human submucosal glands fixed with gluteraldehyde, post-fixed in osmium, and stained with lead, contained cells with secretory granules 1400 A in diameter. The granules had dense cores separated from limiting mem- branes by electron-lucent halos. Pseudopod-like extensions of the cytoplasm penetrated the intercellular spaces and some extended 12 um from the cell body. Other ultrastructural characteristics included electron-lucent 10 cytoplasm, abundant mitochondria and free ribosomes, a prominent smooth endoplasmic reticulum, and an inconspic- uous Golgi complex. The cells were often situated at junctions between mucous and serous cells. Endocrine-like cells were also found in adult human bronchioles by Gmelich gt gt., (1967). The cells contained dense-cored granules which were 1200 to 1500 A in diameter. Apical ends of the cells did not extend to the airway lumen. Clusters of granulated cells were also observed. The ultrastructural studies of Hage gt gt., (1977) revealed a continuum of shapes ranging from pyramidal cells which extended processes along the basement membrane to columnar-shaped cells. Cytoplasmic processes were seen but the apical extensions never reached the airway lumen. Cells in the lobar and proximal segmental bronchi were usually single while those in the distal bronchi and bron- chioles were mostly in groups. Dark cytoplasm, many mito- chondria, a prominent Golgi complex, free ribosomes, and microfilaments characterized the cytoplasmic ultrastruc— ture. Also present were dense-cored secretory granules 1100 to 1400 X in diameter. A few intraepithelial nerves were seen but none formed synapses with the granulated cells. Terzakis gt gt., (1972) found endocrine-like cells 0 with dense-cored secretory granules 800 to 1700 A in dia- meter in segmental bronchi. In addition, dense-cored 11 secretory granules identical to the ones seen in granu- lated cells were also observed in goblet cells. Neonatal Human Lung A few reports have described endocrine-like cells in lungs from newborn human lungs. The name Argyrophil, Fluorescent, and Granulated (AFG) was used by Lauweryns and coworkers to describe the endocrine-like cells in lungs from term newborns (Lauweryns and Peuskens, 1969; Lauweryns gt gt., 1970). Triangular or pyramidal-shaped argyrophilic cells with oval nuclei were situated within the epithelium near the basement membrane. Stains for argentaffinity, chromaffinity, and lipofuchsin were nega- tive. In freeze-dried formaldehyde vapor treated sec- tions, fluorescent bright green cells morphologically similar to the argyrophilic cells were observed. The argerphilic cells were randomly distributed in bronchi and bronchioles. About one third of the large bronchi contained an average of three cells, one fourth of the small bronchi and bronchioles an average of two cells. The respiratory regions also contained scattered argyro- philic cells. Specimens observed by electron microscopy revealed cells with granules 800 to 1500 A in diameter 0 and 100 to 150 A translucent halos between the dense cores and the limiting membranes (Lauweryns gt 31., 1970). 12 The cell membrane was modified to form desmosomes with adjacent epithelial cells. A small Golgi apparatus, smooth endoplasmic reticulum, glycogen and many mito- chondria were present in the cytoplasm. Intraepithelial nerve fibers formed "direct contacts" with granulated cells. The nerve endings contained both granular and agranular vesicles and were separated from the granulated 0 cell by a 200 A gap. Thickenings of the axon membrane and granular cell membranes were seen. Fetal Human Lung Endocrine-like cells have also been studied in the human fetal lung. Hage (1971, 1972) showed that, although a few cells with green formaldehyde-induced fluorescence were normally present in bronchi, £2 ztttg incubation with L-DOPA increased the number and intensity of the cytoplasmic fluorescence. Cells which showed positive staining with Grimelius argyrophilia, HCl-basic dyes, and lead-hematoxylin corresponded in number to the fluorescent cells from control lungs. The cells were triangular or bottle shaped and found in main and intra- pulmonary bronchi only. They occurred either in groups or as single cells and were mostly seen at divisions of bronchi. Argentaffin and diazonium staining reactions were negative. 13 The amine-handling properties were further investigated in subsequent work by Hage (1973a). Freeze- dried formaldehyde vapor treated lungs from 18 fetuses (35 to 140 mm crown-rump_length) were studied after: (1) incubation in pargyline, a monoamine oxidase inhibitor; (2) incubation in L-DOPA, L-S-HTP, D-DOPA, or D-S-HTP; (3) incubation in L-DOPA or L-S-HTP after treatment with Ro4-4602, an aromatic amino acid decarboxylase inhibitor; (4) dopamine or 5-hydroxytryptamine incubation; or (5) control. Yellow to green fluorescence was observed in bronchial epithelium of a few cells from controls. After treatment with pargyline, there was a slight increase in intensity of the fluorescence but no change was observed in the number of cells. After incubation in the L-isomers of DOPA or 5-HTP a large increase in the number of cells occurred. Fluorescence after DOPA treatment was green and distributed over both cytoplasm and nucleus, while yellow fluorescence restricted to the cytoplasm developed after 5-HTP. With the D-isomers, the number of fluorescent cells and the fluorescence intensity was greater than the controls but not as great as with L-isomers. The decarboxylase inhibitor did not affect fluorescent cells in controls. However, the fluorescence intensity was lower after Ro4-4602 plus L-DOPA or S-HTP than after L-DOPA or 5-HTP alone even though the number of cells seen was similar (greater than controls). 14 Treatment with amines did not change the appearance or numbers of fluorescent cells above those of control tissues. Ultrastructural features of endocrine-like cells from human fetuses have been reported by Hage (1973b) and Cutz and Conen (1972). Cells appeared singly or in groups near the basement membrane and contained electron dense granules. Smooth endoplasmic reticulum was present in large amounts. Also seen was a small amount of rough endOplasmic reticulum, a small Golgi complex, and many mitochrondria and microfibrils. Glycogen accumulations made the cytoplasm less electron dense than that reported in older animals. These cells were well differentiated compared to the neighboring epithelial cells which con- tained glycogen and a few organelles. Contact with nerves was not observed. The endocrine-like cells were classified by Hage (1973b) into three types based on the appearance of the secretory granules. Type I cells were columnar, poly- gonal, or horizontally elongated with pseudopod processes extending along the basement membrane below as many as six epithelial cells. The granules averaged 110 nm in diameter and contained dense cores and narrow halos. A larger more osmiophilic granule was also seen which did not contain a dense core. The large granule was argen- taffin positive and both large and small granules were 15 argyr0philic. Type I cells were found at all levels of airway as far as terminal bronchioles. Type II cells were mostly pyramidal and occasionally had processes. They sometimes contacted the airway lumen and had microvillus borders. The dense-cored granules were 140 nm in diameter, argyrophilic, but negative with argen- taffin staining. Type II cells were found at all levels of the bronchial tree and in the alveolar regions where cytoplasmic extensions penetrated between the alveolar epithelium and basement membrane near capillaries. Type III cells were oval or pyramidal with-solid granules 190 nm in diameter. The granules were argyrophilic but not argentaffin. Hage (1976) has speculated that Type I cells correspond to cells with endogenous fluorescence and Type II cells corresponded to cells that were seen after precursor injection. Rosan and Lauweryns (1971) found granulated cells in lungs of two prematurely born infants (.600 and 1600 grams). Granulated cells in the younger infant were seen more frequently and had more granules per cell than the older infant. The cells of both were often associated with capillaries. In a more extensive study using prematurely born infants, Rosan and Lauweryns (1972) confirmed their earlier findings. 16 Fetal Animal Lungs Endocrine-like cells in lungs of fetal rats were studied by Cutz gt gl., (1974). By light microscopy, triangular-shaped cells were seen near the basement membrane at all levels of the bronchial tree. They were seen singly and in groups of 3 to 6 cells. Formaldehyde- induced fluorescence produced a few weakly fluorescent cells in control animals, but after injection of L-DOPA or 5-HTP, increased numbers and intensity of fluorescent cells were observed. The number and distribution of fluorescent cells corresponded to the argyrophilic cells. Argentaffin and diazonium stains were negative, but some weakly metachromatic cells were observed after HCl- toluidine or lead-hematoxylin. Hage (1974) described endocrine-like cells in lungs of fetal rabbit, mouse, and guinea pig. Only the fetal rabbit lungs contained formaldehyde-induced fluorescent cells without precursor administration. After L-DOPA was administered to the fetal animals through the maternal circulation, the number of cells in fetal rabbit lungs was unchanged, but the fluorescence intensity was increased. In the mouse, groups of pyramidal shaped fluorescent cells were seen only in the large bronchial tubes after administration of precursor to the mother. Guinea pig fetuses contained a few scattered fluorescent cells after the L-DOPA admin- istration. Argerphilic cells in rabbit corresponded in 17 number and distribution to fluorescent cells. Argyro- philic cells were detected in neither fetal mouse nor guinea pig lungs. Lead-hematoxylin, HCl-toluidine blue, argentaffin, and diazonium reactions were negative in all the species studied. The general ultrastructural characteristics of all the endocrine-like cells found in fetal lungs of rat (Cutz gt gt., 1974), rabbit, mouse, and guinea pig (Hage, 1974) were similar. Both single and grouped cells had cytoplasmic process extending along the basement membrane. The cytoplasm contained many mitochondria, microtubules, free ribosomes, a small Golgi complex, and small amounts of rough endoplasmic reticulum. Desmosomes were seen in the species studied by Hage. Cutz gt gl., (1974) described unmyelinated nerve endings in contact with the grouped cells but not with the single ones. The dense-cored granules of rat were 700 to 1200 A in diameter, in rabbit they were 1420 A, and 1070 A in mouse. Only one cell was observed by electron microscopy in guinea pig. The granules of rabbit were both argentaffin and argyrophilic, and those of mouse were slightly argyrophilic. Two cell shapes were observed in rabbit. One was polygonal, often contacted the lumen with a microvillus border, and had a diffuse distribution of dense-cored granules. The other was columnar, did not contact the lumen, and the secretory granules were distributed in the basal part of the cell. 18 Newborn Animal Lungs Moosavi, Smith and Heath (1975) investigated the effects of age on endocrine-like cells in lungs from new- born rats. The total number of argyrophilic cells per linear centimeter of bronchi decreased from birth to thirty days of age. The percentage of bronchi with argyrophilic cells also decreased in the same period. These investiga— tors subjected four newborns to hypobaric pressure in an attempt to create a hypoxic condition. Neither numbers of cells per length of bronchial epithelium nor percentage of bronchi with endocrine—like cells was affected by 20 hours of living in an atmosphere of 380 mm Hg. Adult Animal Lungs Using perfusion-fixation rather than freeze-drying, Eaton and Fedde (1977) have reported two types of fluores- cent cells in lungs from adult mice without prior adminis- tration of amine precursor. Both yellow and yellow-green cells were seen in the same section. Spectral analysis of the fluorophores was not done, and therefore the authors' assumption that two different amines were demonstrated is questionable. Jeffery and Reid (1975) were unable to identify any endocrine-like cells in the lungs of male rats by electron microscopy. 19 Trachea Human Tracheal epithelium from newborn and young (up to 15 years) humans were investigated as part of a larger study by Cutz gt gt., 1975. Formaldehyde-induced fluorescent cells were not seen in control tracheas. However, after 30 minutes of 12.21EEE incubation in L-DOPA, formaldehyde-induced fluorescence was observed in cells which corresponded in shape and position to argyrophilic cells from control tissues. Argyrophilic cells were triangular and cellular extensions sometimes reached the lumen, while their bases rested upon the basement membrane. The cells in trachea always occurred singly. Argentaffin reactions were negative. Ultra- structural studies revealed granules within the cells which averaged 1150 : in diameter with a halo 160 to 180 A wide. The cell membranes formed desmosomes and inter- digitations with adjacent epithelial cells. Unmyelinated axons were seen in the epithelium but synaptic contacts were not observed. Endocrine—like cells in tracheal epithelium from human fetuses were found by Cutz gt gt., (1975). Fetal tracheas contained yellow-green fluorescent cells after freeze-drying and treatment with formaldehyde vapor. Incubation in L-DOPA increased the number and intensity of cells in fetal tracheas. The ultrastructural 20 appearance of granulated cells was identical to the granulated cells described in newborns and children cited above. Animal Tracheal epithelium from lamb and rabbit fetus, newborn and adult rabbit, and adult armadillo (Cutz gt gt., 1975) and adult mouse (Ericson gt gl., 1972) contain endocrine-like cells. All of the species revealed formaldehyde-induced fluorescence only after injection of L-DOPA or L-S-HTP, which produced green and yellow fluorescent cells respectively. The cells were on or near the basement membrane, and, in mouse trachea, some- times extended to the lumen. Morphologically similar cells were argyrophilic, and in mouse, subsequent silver impregnation and rephotographing sections photographed first by fluorescence showed that the endocrine-like cells observed after precursor injection were also argyrophilic. Adult rat trachea also contained cells which became fluorescent after injecting L-DOPA (Dey and Echt, 1976). Ericson gt gt., (1972) reported the effects of several pharmacologic agents in adult mouse trachea. Thirty minutes after injecting L-DOPA, a diffuse green formaldehyde-induced fluorescence was observed in endocrine-like cells. After 120 minutes, the fluorescence 21 became more intense, localized in the basal cytoplasm, but was also present in the apical portion of the cell. After six hours, the fluorescence was faint, and at 20 hours fluorescent cells could no longer be detected. If the decarboxylase inhibitor Ro4-4602 was administered 2 hours prior to precursor injection, only a very faint fluorescence was observed. Treating control animals with the monoamine oxidase inhibitor nialamide did not result in the appearance of any fluorescent cells in the trachea, nor did administration of dopamine. Only very low levels of fluorescence were produced if the D—isomers of precursors were used. Pretreatment with reserpine 4 hours prior to L-DOPA injection resulted in lower fluorescence than with L-DOPA alone. The ultrastructural appearance of granulated cells in the tracheas of several species has been des- cribed. A consistent feature was the presence of dense- cored secretory granules. In mouse, the granules were 800 to 1000 A in diameter and were in an infranuclear position (Ericson gt gt., 1972). These granules were argyrophilic and after precursor were also argentaffin. In lamb fetuses, two sizes of granules were seen; one 1680 A and the other 1120 A in diameter. Rabbits of all ages con- tained vesicles 1400 A, and the granules from armadillo o 0 were two sizes, 1750 A and 1250 A (Cutz gt gt., 1975). 22 Jeffery and Reid (1975) reported granulated cells with dense-cored granules 130 nm in rat tracheal epithelium. After injecting radiolabled amine precursors into mice, Ericson gt gl., (1972) reported that the ultra- structural localization of silver grains was much heavier over the granules of endocrine-like cells than other epithelial cells. Larynx Larynx of adult rat (Ewen gt gt., 1972), mouse (Ericson gt gt., 1972), and guinea pig (Kirkeby and Romert, 1977) contain endocrine-like cells. In rat, the cells become fluorescent after injecting L-DOPA or 5-HTP. This fluorescence was much lower if the rats were pre- treated with Ro4-4602. However, argyrophilic cells were not detected and the diameter of cytoplasmic dense-cored granules in cells thought to be ultrastructural correlates of the fluorescent cells was 2000 nm. In mouse larynx, cells with formaldehyde-induced fluorescence after pre- cursor injections were most numerous near the base of the epiglottis and below the cricoid cartilage. The guinea pig larynx contained argerphilic cells in the mucosa and in glands. They were observed either singly or in groups of up to 15 to 20 and were present throughout the larynx except the most superior part. The argyrophilic cells were near the basement membrane and often reached the 23 airway lumen by apical processes. Fluorescence investiga- tions were not conducted. Extrapulmonary airways of domestic chickens also contain endocrine-like cells (Cook and King, 1969; Walsh and McLelland, 1974). Formaldehyde-induced fluorescence was observed after precursor injection but not in control birds. The dense-cored granules were 750 to 1300 A or 1400 A respectively in the two reports. Both reports described intraepithelial nerves but Cook and King (1969) found evidence of synaptic contacts between granulated cells and unmyelinated nerves with clear granules. Walsh and McLelland (1974) did not see synaptic contacts, and the unmyelinated nerves contained both dense and clear granules. Neuroepithelial Bodies Groups of endocrine-like cells have been reported by several investigators and are generally not distin- guished from similar cells which exist singly. However, Lauweryns and his coworkers have studied groups of endocrine-like cells and claimed that they were distinct morphologic structures and have called them Neuroepithelial Bodies (NEBs). NEBs were first detected with light microscopy in human newborns (Lauweryns and Peuskens, 1972). The NEBs were composed of inner corpuscular cells and outer lining cells. In hematoxylin-eosin stained 24 sections, the inner cells had clear cytoplasm and formed intercalated cone-shaped corpuscles. The base of the cone rested on the basement membrane sometimes displacing it into the lamina propria, and the apex of the cone contacted and sometimes bulged into the lumen. The out— side layer of cells were smaller and flatter than the inner cells. The corpuscular cells were argyrophilic but the lining cells were not. Formaldehyde-induced fluorescence was seen in cells similar to the argyro- philic areas. The NEBs were seen at all levels of the pulmonary epithelium including respiratory bronchioles and alveoli. Nerve fibers were seen entering the cor— puscular areas of the NEBs. Lungs from fetal, neonate, and adult rabbits (Lauweryns gt gt., 1972, 1973) were investigated by both light and electron microscopy. With hematoxylin-eosin, up to 5 NEBs could be seen in a single bronchus of neo- nates and fetuses, but fewer were seen in adults. The NEBs measured 5 to 25 um high and 10 to 65 um at the base with 10 to 30 cells per NEB in neonates and fetuses, and 3 to 10 in adults. They were periodic acid-Schiff positive and argyrophil positive, slightly argentaffin, and intensely yellow in freeze-dried formaldehyde treated sections. The corrected emission maximum of the yellow fluorophore was 530 nm leading the authors to conclude that the corpuscular cells contained serotonin. 25 Ultrastructurally, the apices of the cells contacted the lumen by a microvillus border. Cell mem- branes interdigitated with their neighbors forming inter— cellular gaps and desmosomes. Fenestrated capillaries were often located beneath the basement membrane. The apical cytoplasm contained mitochondria, a well developed Golgi complex and rough endoplasmic reticulum, and numerous free ribosomes. Microtubules and fibrils were rarely seen. The most characteristic feature of NEBs was the presence of large quantities of dense-cored granules throughout the cytoplasm but especially in the basal region. With gluteraldehyde-osmium fixation and lead citrate-uranyl acetate staining, 70% of the granules measured 1340 by 1021 A with dense cores close to the limiting membranes thus showing no or only very narrow halos (Type I granules). The remaining granules measured 1121 by 989 A with distinct halos between the dense cores and the limiting membranes (Type II granules). Using the formaldehyde-gluteraldehyde-dichromate fixation technique, electron-dense deposits indicating serotonin (Jaim- Etcheverry and Zieher, 1968) were observed in granules presumed to be the Type I granules. Applying a histo- chemical test for acetylcholinesterase (AChE) to thin sections for electron microscopy, Lauweryns and Cokelaere (1973a) identified AChE-positive material in the halos between the dense cores and the limiting membranes of the 26 large Type I granules. In the same study, both NEBs and intraepithelial nerves were AChE-positive by light microscopy. The NEBs were also positive for o-glycerophosphate dehydrogenase and lead-hematoxylin. Unmyelinated nerves were observed within the mucosa and direct contacts with granulated cells were seen. The contacts were characterized by 200 A gaps at sites of membrane thickenings suggesting synaptic con- nections. The nerve endings contained numerous small mitochondria, clear granules 500 A in diameter, and, occasionally, large 800-900 A dense-cored granules. The effects of hypoxia and reserpine on NEBs were studied in neonatal rabbits (Lauweryns and Cokelaere, 1972a,b). By light microscopy, no differences were seen between controls and animals breathing 5, 10, or 15 per cent oxygen for 2, 10, or 20 minutes. Fluorescence intensity of NEBs in the experimental groups was similar to controls. However, by electron microscopy, marked exocytosis of dense-cored granules was observed in the experimental groups. Exocytosis was rarely observed in the controls. The authors theorized that NEBs were chemosensitive organelles which sampled airway oxygen concentration and regulated local ventilation-perfusion by releasing serotonin near the fenestrated capillaries located in the submucosa. 27 Reserpine treatment resulted in no changes in the light microscopic appearance of the NEBs (Lauweryns and Cokelaera, 1973a). However, a marked decrease in cyto- plasmic fluorescence did occur. Electron microscopy revealed that only a few dense-cored granules remained in the cytoplasm. Granules presumed to have had dense cores were now empty and disintegrated. NEBs were found as far as terminal bronchioles in neonatal mouse lung by Hung and Loosli (1974). With light microscopy, groups of cells lined by normal or modified Clara cells were observed. Portions of the apical surface were exposed to the lumen. Electron microscopy revealed dense-cored granules 800 to 1000 A in diameter distributed mostly in the basal half of the cytoplasm. Golgi complex, rough endOplasmic reticulum and mitochondria were also observed. Nerve fibers, that had originated from nonmyelinated nerves in the sub- mucosa, were seen branching among the granulated cells. Numerous mitochondria, a few neurotubules and microfila- ments were in the nerve endings. No synaptic vesicles were reported, and synaptic contacts were not observed. The occurrence of NEBs in 5 children (21 months to 12 years) and 12 adults (31 to 61 years) was reported by Lauweryns and Goddeeris (1975). Groups of four to ten cells were found which measured 20 to 40 um wide and 6 to 15 um tall. The number of cells per NEB and the size 28 of individual NEBs progressively decreased from bronchi to alveoli. An argyrophil reaction was present in the NEBs. Capillaries were identified beneath the basement membrane directly opposite the NEBs. Unified Endocrine Cell Concept APUD Series Similar cytochemical and morphologic character— istics in cells known to secrete peptide hormones have stimulated attempts to create a conceptual framework which describes an endocrine cell system. It was originally proposed that peptide-producing endocrine cells shared common embryologic origins, migrating to peripheral organs from the neural crest or neural ectoderm during embryogenesis (Pearse, 1968a; Polak gt gt., 1971). Neural crest origins have been established for thyroid C cells, carotid body Type I cells, and adrenomedullary cells (Pearse and Takor, 1977). Later, because of the inability to prove (LeDouarin and Teillet, 1973) and evidence disproving (Andrew, 1974) neural crest origin of gastrointestinal endocrine cells, the concept was modified to state that, irregardless of origin, the peptide-producing endocrine cells shared a common "neuroendocrine programme" (Pearse, 1977). However, the nature of the program has not been described. Although there is a lack of evidence favoring a common embryologic origin, there are data showing that 29 endocrine cells share certain morphologic and histo- chemical characteristics. Thus, the APUD concept was proposed by Pearse (1968a) after the following charac- teristic of thyroid C cells had been reported: (a) high levels of a-glycerophosphate dehydrogenase detected by histochemical methods (Foster gt gt., 1964); (b) amine precursor uptake and decarboxylation (APUD is an acronym to identify the system, Pearse, 1966a); (c) secretory granules 1000 to 2000 A in diameter (Pearse, 1966b); and (4) calcitonin immunofluorescence (Bussolati and Pearse, 1967). The APUD concept has been extended by Pearse (1969, 1977) and now includes: thyroid C cells, ultimobranchial C cells, carotid body Type I cells, ACTH- and MSH-secreting cells of pituitary, endocrine cells in gastrointestinal tract and pancreas, and pos- sibly cells in pulmonary and genitourinary tracts. APUD cells share some or all of the following characteristics: (a) amine content or amine precursor uptake and decarb- oxylation; (b) high levels of a—glycerophosphate dehydrogenase and/or non-specific esterases or cholines- terase histochemical activity; (c) masked metachromasia; and (d) endocrine granules. However, the primary function and essential requirement to include any cell in the APUD series is synthesis, storage, and secretion of specific peptide hormones. The significance of neuroectodermal 30 origin or common "neuroendocrine programme" is not clear at this time. CHAPTER 3 MATERIALS AND METHODS Experimental Animals Adult male albino New Zealand White rabbits weigh- ing 3 to 4 Kg were maintained in individual cages with water and standard laboratory rabbit chow gg libitum. All rabbits had been living in the Michigan State Univer- sity Laboratory Animal Care facility at least two weeks prior to killing. Specimen Removal The animals were killed by rapidly injecting 200 mg of sodium penobarbital (Nembutal, Jensal) in the marginal ear vein. With the rabbit in a supine position, a ventral midline incision was made from the level of the mandible to the xiphoid process. The cranial trachea was freed by clamping a hemostat above the larynx and gently teasing the trachea from surrounding tissues. Cutting and spreading the ribs along one side of the sternum exposed the intrathoracic portion of the trachea. After the carina was identified, the trachea was removed by cutting immediately cranial to the bifurcation. Three pieces, approximately 2 cm long, were cut from cranial, 31 32 middle and caudal segments and immediately frozen. The length of time from injection to freezing was approxi- mately three minutes. Pieces of stomach were removed and used as controls for the various staining procedures. Freezing and Freeze-drying Tracheal segments were frozen by immersion in isopentane (Eastman) cooled in liquid nitrogen. Tempera- ture of the isopentane was near its melting point (-160°C) judged by the presence of frozen areas inside the con- tainer. Tissues were left in isopentane for 3 minutes, transferred to cold metal tissue containers and stored in liquid nitrogen for subsequent freeze-drying. The freeze-dryer used in this study was con- structed from existing laboratory equipment. A refrigera- tion unit (Edwards) contained a 1000 ml flask (Virtis) attached to a vacuum generated from the mechanical and oil diffusion pumps of a vacuum evaporator (Kinney) (Figure 1L A cold trap which was immersed in liquid nitrogen, and a chemical dessicant trap protected the pump system from corrosive gases, and also maintained an effective vapor gradient (Figure 2). The flask, which held the tissue and phosphorous pentoxide (P205, Fisher), was placed in a -80°C slush of acetone and dry ice for one day (Figure 3). On the second day, the temperature was increased to -30°C and maintained by the refrigeration unit. Flask 33 Figure l.--Freeze-drying apparatus: (a) vacuum evaporator, (b) vapor trap, (c) refrigeration unit. Figure 2.--Vapor trap: (a) chemical trap, (b) cold trap, (c) Dewar flask for liquid nitrogen. Figure 3.--Inside the freezing chamber: (a) refrigera- tion coils, (b) container for acetone and dry ice, (c) vacuum flask containing P205. 35 temperature was raised to 25°C on the third day and fin- ally heated to +80°C in a water bath for three hours at the end of the fourth day. Increasing the temperature to 80°C helped reduce condensation of water vapor onto the tissue surfaces when dried tissues were removed from the freeze-dryer. To preserve morphologic and histochemical detail in histologic sections, the original samples were frozen rapidly and dried thoroughly. Rapid freezing was accom— plished by immersing the tissue in isopentane cooled in liquid nitrogen. Isopentane provided a high rate of heat transfer, and did not form bubbles upon contacting the tissues. Excessive bubbling impedes heat transfer and is the reason liquid nitrogen alone is not suitable as a freezing medium. Slow freezing promotes ice crystal forma- tions which disrupt the histologic features of tissue sections. Propane and freon are also useful freezing media. They can be liquified by passage through copper condensation coils immersed in liquid nitrogen. Drying was accomplished by sublimation. Sublima- tion is vaporization of a solid substance (in this case water) to a gas without passing through a liquid state. Vacuum was applied to reduce the energy necessary for vaporization. Water vapor must be removed from the space surrounding the drying tissue. Thus, a vapor gradient was established by adding chemical (P205) and physical (liquid 36 nitrogen) traps in the vacuum line. During the drying procedure, tissue temperatures must remain low enough to prevent ice crystal formation but high enough to provide an adequate rate of sublimation. Temperatures in the range of -40°C to -80°C seemed to satisfy these require- ments. Discussions of freezing and freeze-drying principles have been provided by Pearse (1968b, Chapter 3) and VanOrden (1975) and technical aspects are reviewed by Bjorklund gt gt., (1972), Falck, (1962), and Falck and mean, (1965). Formaldehyde Vapor Treatment The most important variables to control during the formaldehyde vapor treatment are humidity of the gas, environmental temperature, and time of treatment (Jonsson, 1971). Low humidity will reduce fluorescent yield, but excesses will cause diffusion of the fluoro- phore. To insure proper humidity, paraformaldehyde powder (Electron Microscopy Sciences) was equilibrated for at least 10 days in a closed dessicator in which a 60% relative humidity had been generated from a mixture of sulfuric acid and water (Hamberger gt gl., 1965; Pearse, 1972, Appendix 27). Freeze-dried tissues were treated with formalde- hyde vapor in the following way. A tightly sealed 500 ml. glass jar containing five grams of equilibrated 37 paraformaldehyde powder was placed in an 80°C oven one hour before the tissues were removed from the freeze- dryer. After removal, the dried tissues were sealed in the glass jar at 80°C for 90 minutes and then embedded in paraf- fin. A few small pieces of trachea embedded directly into paraffin without the gas treatment provided controls for each experiment. Paraffin Embedding and Sectioning The tracheal segments were vacuum impregnated with melted paraffin (Tissue Prep, Fisher) at 65°C for one hour. Each segment was embedded in a metal tissue mold, fitted with a plastic chuck holder, and labelled for future reference. After allowing the mold to solidify at room temperature overnight, the blocks were stored at 0°C until sectioning. Fresh paraffin was used for each embed- ding because formaldehyde contamination would produce fluorescence in tracheas not exposed to the vapor treatment. Sectioning of cold trimmed blocks was done with cold steel microtome knives. All sections were cut 15 mm thick. Sections used for routine staining were mounted from a 50°C water bath onto acid-cleaned albuminized slides. For fluorescence microscopy, sections were mounted by melting the paraffin ribbons on warm glass slides. After cooling, the sections were deparaffinized with xylene and a glass cover slip was applied with xylene as the mounting medium. 38 In a few cases, the slides were intended for fluorescence microscopy and subsequent staining. These sections were floated from a warm 1% gelatin solution onto clean glass slides. Although this procedure com- promised fluorescent quality and the staining potential of the sections, it was a necessary step to avoid dif- fusion of the fluorophore and insure adequate adhesion of the sections to the slide. Microscopy and Photography Fluorescence was observed and photographed on either a Leitz Ortholux II or a Zeiss Standard micro- scope. In either case, the fluorescence was observed in transmitted light from a 200 watt mercury vapor lamp using BG12 and BG38 excitation filters and a 470 mm barrier filter. Kodak Ektachrome film (Tungsten, ASA 160, ET 135) was used for photography. Vertical and horizontal coordinates were recorded if the sections being photographed were intended for subsequent staining and light microscopy. A Zeiss Universal microsc0pe with automatic exposure was used for light microscopy. Photographs were recorded on Kodak Professional Ektachrome (Tungsten, ASASO, EPY135) with No. 81A filter. 39 Speciticity of the Fluorophore and Formgldehyde-Induced Fluorescence To evaluate specificity of the fluorescence, three tests were applied to sections of trachea. The first test showed whether the fluorophore resulted from treatment with formaldehyde vapor. Tracheas that were freeze-dried but not treated with formaldehyde vapor were compared to treated samples. The second test determined whether the fluorophore was quenched by water. Paraffin sections of formaldehyde treated tracheas mounted from a water bath were compared to sections mounted by melting. In the third test, sodium borohydride in 80% ethanol was used to reduce the fluorescent 3,4- dihydro-B-carboline to the nonfluorescent 1,2,3,4- tetrahydro-B-carboline (Corrodi gt gt., 1964). The crucial point of the test was regenerating the fluoro- phore by a second exposure to hot formaldehyde gas. Thus, sections were observed by fluorescence microscopy to confirm the presence of fluorescent cells, treated with fresh 0.5% sodium borohydride (Sigma) in 80% ethanol, observed a second time, treated with formalde- hyde gas, and observed a third time. 40 Microgpectrofluorometry Excitation and emission characteristics of the fluorophore were determined with a microspectrofluoro- meter housed in the Department of Pharmacology, University of Chicago, Chicago, Illinois. Light from a mercury vapor lamp was passed through the excitation monochrometer to a glass slide on the stage of a Leitz Ortholux Fluorescence Microscope. Fluorescence was excited by incident light from a Ploem illuminator. After passing an emission monochrometer,intensity of emitted fluorescence was recorded by photon counting in a photomultiplier tube. Excitation and emission characteristics were measured separately. Emission curves were obtained by measuring light intensity at wavelengths from 450 to 560 nm in 5 nm steps with unfiltered light from the mercury vapor lamp. Then, the excitation curve was found by measuring inten- sities of the emission peak while excitation wavelengths were increased from 310 to 440 nm in Snm steps. Other than during periods of measurement, the exciting light was interrupted by a shutter to avoid bleaching the cellular fluorophore. Data points were corrected for wavelength- dependent sensitivities of the microspectrofluorometer. Spectra were obtained from tracheas of three control rabbits and two L-DOPA injected rabbits (see section on effects of DOPA and reserpine). One cell was evaluated from each trachea. The values were obtained by 41 positioning fluorescent cytoplasmic areas over a measure- ment window and subracting that value from.mea$urements of nonfluorescent areas in adjacent epithelium. The spectra from cells were compared with spectra from formaldehyde treated models of serotonin dissolved in 5% sucrose -0.1% glycine matrix (Bjorklund gt gt., 1968). Histochemical Procedures Freeze-dried formaldehyde vapor treated tracheas from the control groups were used to investigate histo- chemical characteristics of the tracheal epithelium. All the procedures were applied to paraffin embedded sections mounted from a water bath. The sections were deparaffin- ized in xylene and hydrated through graded alcohols. After staining was complete, the sections were dehydrated through graded alcohols, cleared in xylene, and mounted in Permount (Fisher). Pieces of stomach were used as control tissues. Details of the staining procedures appear in Appendix B. Grimelius Silver Nitrate (Grimelius, 1968) In this procedure,tracheas were treated with 1% silver nitrate at pH 5.4 for 6 hours. Ionic silver becomes bound to cytoplasmic components of certain endo- crine cethypes. Subsequent treatment in]% hydroquinone 42 reduces the ionic silver to metalic silver which appears as a black deposit. The specificity and mechanisms of staining for this reaction has not been established. However, Solcia gt gt., (1976) have shown that argyro- philia is localized in the halos of dense-cored cytOplasmic vesicles and that the deposits do not appear if sections are treated with proteolytic enzymes. The argyrophilic reaction is important because it demon- strates peptide-producing endocrine cells--e.g., thyroid C cell (Carvalheira gt gt., 1968), A cells of pancreas (Grimelius, 1968) and several gastrointestinal endocrine cells (Solcia gt gl., 1975). Fontana's Arggntaffin Silver (Cullin , 1974) The sections were incubated in a mixture of silver nitrate and dilute ammonium hydroxide for 24 to 48 hours. Unlike the argyrophil reaction described above, the argentaffin reaction does not require extraneous reducing agents such as hydroquinone. Silver deposits accumulate in cells which contain reducing substances such as catecholamines and indoleamines. Ferric-Ferricyanide (Lillie and Burtner, 1953), Sections were stained in a mixture of FeCl3 and K3Fe(CN)6. When dissociated ferric ion (Fe+3) is reduced to ferrous ion (Fe+2) by reducing substances 43 contained in certain cells, the ferrous ion reacts with 3 to form a blue precipitate (Turnbull's blue). Fe(CN)6- Sections are incubated for not more than 20 minutes. This reaction is more sensitive than argentaffinity for detecting the presence of indoles and phenols (Lillie and Fullmer, 1976). Diazosafranin (Lillie, Burtner and Henson, I953) Fresh stable diazotates were prepared by reacting acid safranin with sodium nitrite at 4°C for 10 minutes. Tissue sections were incubated in a dilute solution of diazotates at pH 7.7 for 5 minutes and differentiated in acid-alcohol. Aromatic diazonium salts will couple with - phenols and indoles present in tissues and produce colored products. In the case of diazosafranin, specific coupling reactions with indole substances have been dem- onstrated (Lillie gt gt., 1973). Coupling causes the red color of diazosafranin to shift to blue or violet. Alcian Blue (Luna, 1968) Sections were prepared for staining by a mordant with 3% acetic acid for three minutes. Treating for 30 minutes in 0.1% alcian blue in 3% acetic acid stained glycoprotein containing cells light blue while other epithelial cells remained unstained. To add contrast,tflma alcian blue sections were counterstained with nuclear 44 fast red which stains the cytoplasm pink. Alcian blue combined with periodic acid—Schiff at different pH levels has provided information about the types of mucous- containing cells present in the respiratory system (Jones and Reid, 1973a). Lead-Hematoxylin (Solcia, Capella, and Vassallo, 1969) Hematoxylin was dissolved in a solution of 5% lead nitrate in saturated ammonium acetate. The sections were stained in this solution for 1 to 2 hours at 45°C. Reactive structures stained blue-black. This technique has been used to demonstrate endocrine cells in thyroid, pituitary, pancreas, and gastric mucosa. The mechanism and significance of staining is not understood, but probably reflects a common chemical property in the secretory granules of endocrine cells. Combined Fluorescence and Light Microscgpy In order to directly establish that fluorescent cells were identical to cells seen by the staining techniques described above, sections photographed by fluorescence microscopy were subsequently stained with ferric-ferricyanide or Grimelius silver nitrate and rephotographed by light microscopy. Special section mounting techniques were required for two reasons: (1) sections mounted by melting were not sufficiently 45 adherent and would float off the glass slide during the staining procedure, and (2) sections mounted by floating from a water bath resulted in total loss of fluorescence. However, mounting slides from 1% gelatin provided adequate adhesion and did not affect the cellular fluorophore. A special procedure was applied to water mounted sections to determine if argyrophilic cells were the same as goblet cells. Sections were first stained in Grimelius silver nitrate for argyrophilia and then restained in alcian blue. Argyrophilic reactions would not develop when the staining order was reversed. Distribution of Fluorescent Tracheal Epithelial Cells The distribution of fluorescent cells was evaluated by counting the number of cells in six regions of trachea. One centimeter long segments were taken from cranial (nearest the larynx), middle, and caudal (nearest the carina) trachea. Freeze-dried, formaldehyde vapor treated, paraffin embedded segments were serial sectioned at 15 um and mounted on glass slides by melting. The total number of fluorescent cells in each section was counted during observation by fluores- cence microscopy. The numbers of fluorescent cells in dorsal (membranous) and ventral (cartilagenous) portions of each section were recorded separately so that the dorsal-ventral distribution could also be evaluated. Ten 46 sections at least 150 um apart were counted from each region. Only cells which contained distinct nuclei were counted. Means and standard errors were determined for each of the six regions and expressed as the number of cells in one 15 um section. A total of six rabbit tracheas were evaluated in this manner. Effects of L-DOPA and Resegpine on the Numbers of Fluorescent Cells In this experiment, the effects of L-DOPA and reserpine on the number of detectable fluorescent cells were evaluated. A total of nine rabbits provided tracheas for three identical experiments. Each experiment con- tained three rabbits: one control, one injected with L-DOPA, and one injected with reserpine. Data from the three control rabbits in this experiment were also used to evaluate the distribution of fluorescent cells described in the previous section. A dosage of 250 mg/kg of L-DOPA (Aldrich) prepared as a suspension in 10 ml of distilled water was injected intraperitoneally (IP) into rabbits in the L-DOPA group. Reserpine solution was prepared by dissolving 300 mg of reserpine (United States Biochemical) and 375 mg of citric acid (Mallinckrodt) in 6 m1 benzyl alcohol by gentle heating (Bennett, 1967). This solution was diluted to 100 ml with 15 ml Tween 80 (Sigma) and 79 ml distilled water for a final reserpine concentration 47 of 3 mg/ml. Three rabbits received 6 mg/kg reserpine by IP injection (Hakanson gt gt., 1970). The L-DOPA and reserpine injected rabbits were killed 90 minutes after the time of injection and the tracheas were prepared as previously described. The number of fluorescent cells was evaluated in the same way described in the previous section. However, the data from different regions were combined to estimate the average number of cells in 30 sections. Statistical Analysis Data from the distribution evaluation, and the effects of L-DOPA and reserpine injections were analyzed in the same way. A one-way analysis of variance was used to determine if all the sample means were derived from the same or different populations. The critical region of rejection was set at a = 0.01 prior to applying the test. If the analysis of variance detected significantly different populations, a Student—Newman-Keuls g posteriori multiple comparison test (SNK) was used to compare individual means (Sokal and Rohlf, 1969). The critical region of rejection for the SNK test was set at a = 0.05 prior to making the comparisons. The raw data and statistical tables appear in Appendix A. CHAPTER 4 RESULTS Morphologic Description of the Rabbit Trachea In freeze-dried formaldehyde treated tracheas, the lumen was lined by epithelium which contained periodic fluorescent cells and small round fluorescent structures located close to the basement membrane. The latter were approximately 10 to 15 um in diameter with a round central mass 5 to 8 pm in diameter which was more intense than the peripheral area. These structures were easily distin— guished from the fluorescent cells by their round shape, different emission characteristics, and intensely fluores- cent central mass. They also appeared in specimens not exposed to formaldehyde. The nature of these structures was not determined; however, they did not appear to represent cellular components. The epithelium was adja- cent to an intensely autofluorescent lamina propria which contained a few formaldehyde-induced fluorescent nerve fibers. The submucosa contained loose connective tissue and large venous plexuses. The plexuses were distributed in the submucosa adjacent to the cartilagenous portions of 48 49 the trachea, but were not present in the submucosa of the membranous portions. Fluorescent nerves were present in the walls of the plexuses. C-shaped cartilages opened toward the dorsal (membranous) surface. The trachealis muscle extended between the internal surfaces of the dor— sal ends of the cartilage. Fluorescent nerve fibers were observed within the muscle. Morphology of Fluorescent Cells In tracheas treated with formaldehyde vapor (Figure 4), numerous intensely yellow fluorescent epithelial cells were ovserved. The apical ends of the cells tapered toward the lumen and their wider rounded bases, which contained nonfluorescent nuclei, were near the basement membrane but usually separated from it by a narrow space. The cells were approximately 30 um tall and 10 um wide at the base. Many cells extended narrow processes toward the lumen and/or along the basement membrane (Figure 5). Cytoplasmic fluorescence was most abundant in the apical regions. Lesser amounts of intense fluorescent material were also usually present beside and below the nucleus. Microspectrofluorometric Data These data were collected to establish the spec- tral properties of the cellular fluorophore. Excitation and emission curves were determined for one cell from each 50 Figure 4.—-Freeze-dried formaldehyde vapor treated tracheal section. Fluorescent cells present in epithelium. 175 x. Figure 5.--Freeze-dried formaldehyde vapor treated tracheal section. High magnification of two fluorescent epithelial cells. 715x. , ... at" ..“ '\ “19,9441" 52 of three control rabbits and one cell from each of two L—DOPA injected rabbits. The excitation and emission peaks are reported in Table 1 along with values of standards which contained different concentrations of serotonin. The actual spectral curves of one cell from a control rabbit and one cell from an L—DOPA injected rabbit are seen in Figures 6 and 7 respectively. The excitation peaks of the three cells from control rabbits averaged 400 nm and the emission peaks averaged 521 nm. For the L—DOPA injected animals, the excitation peaks averaged 413 nm and the emission peaks averaged 505 nm. The excitation and emission peaks of the different serotonin concentrations were 413 nm and 525 nm respectively. Specificity of the Fluorophore Three tests were used to evaluate the nature of the cellular fluorophore. The first test determined whether the fluorophore was the result of a reaction with formaldehyde vapor. The section seen in Figure 8 was not exposed to formaldehyde vapor and is from the same rabbit as the tracheal section shown in Figure 4. Figure 8 does not contain endogenous fluorescent epithelial cells. This indicated that the fluorophore in the cell was the result of a reaction with formaldehyde. Fluorescent nerve fibers 53 mmm mmm mam mmw mam omm cam mmm omv mHv mow Gav mHv mmm mmm 0N¢ 9mm Hs\ms o.H 9mm Hs\me m.o 9mm H5\ms H.o N* dmOQIA H¢ fimODIQ ma Houucou N* Houucou Ha Houucoo Aficvxmmm :onmem Aficvxowm cowumuwoxm .mpumccmum cacououwm can mansion cmuomflcw moon—lg Unmaouucoo Eoum maaoo ucoomwuosHm mo rump Hmuuummmlié mam—<8 54 Figure 6.--Excitation and emission curves of a fluorescent cell from control rabbit. 55 Ikozw4m><3 0.00h 0.000 0.000 0.00¢ 0.00m 0.00N q . _ d _ 0 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 1.0.mN _ _ _ . _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ n u n 1 0.2. _ _ _ _ _ _ _ _ _ _ L 0.00. AllSNBlNI 56 Figure 7.--Excitation and emission curves of a fluorescent cell from L-DOPA injected rabbit. 57 Ikozm4m><3 0.02. 0.000 0.00... 0.000 0.00» 0.00m 1 _ _ _ _ O _ _ _ _ _ _ _ _ _ _ _ _ _ _ 0.8 u _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 0.0m. _ _ _ _ _ _ _ _ _ _ _ _ u u u n 0.2. _ _ _ _ _ _ _ _ _ _ 0.00. AllSNBlNI 58 Figure 8.--Freeze-dried tracheal section not treated with formaldehyde vapor. This section was taken from the same rabbit as the tracheal section in Figure 4. 175x. 59 O I l‘ ‘0 .83: ‘0‘ ' _ ‘1‘," ‘i‘ffl-a' 0 "0,44 ' ‘0‘ ‘. $.O“ .. s. ‘7 0 '0'.» w. 60 were not observed in smooth muscle around the venous plexuses nor in the trachealis muscle. The second test determined whether the fluorophore was affected by treatment with water. If sections of formaldehyde vapor treated tracheas were mounted from water, fluorescent cells were never present. The fluores- cence was not regenerated by subsequent exposure to formaldehyde vapor. Sections of the same trachea that were mounted by melting always contained fluorescent cells. Also, fluorescent nerve endings were not present in water treated sections. This demonstrated that the fluorophore was quenched by water and could not be regenerated by subsequent formaldehyde treatment. The third test investigated the effects of sodium borohydride on cellular fluorescence. Sections of formaldehyde treated tracheas were observed by fluores- cence microscopy to establish that fluorescent cells were present in the epithelium. Treating these sections with 0.5% sodium borohydride in 80% ethanol caused complete disappearance of cellular fluorescence. However, treat- ing only with 80% ethanol did not cause any change in the appearance of the fluorophore. Exposing the sodium boro- hydride treated sections to formaldehyde vapor at 80°C for one hour regenerated the fluorescence. 61 Distribution of Flgorescent Cells in Control Tracheas The regional distribution of fluorescent cells in the rabbit trachea was determined by counting the number of cells in dorsal and ventral aspects of sections from cranial, middle, and caudal segments. The average number of cells in one 15 um section from each region is graph- ically presented in Figure 9. One-way analysis of variance indicated that the counts were derived from different populations of cells. The SNK g posteriori test detected no differences between any of the dorsal regions. However, the dorsal regions contained signif- icantly fewer cells than the ventral regions. There were also significantly more cells in the ventral cranial region than any other region. Results of the multiple comparisons test are summarized in Table 2. Means that were not significantly different are connected by under- lining. Table 3 indicates the actual range between each comparison and the least significant range (in parentheseQ estimated by the SNK test. Effects of Intrapgtitoneal L-DOPA or Resetpine Injections The effects of injecting the catecholamine precursor L-DOPA or the amine-depleting agent reserpine were investigated by counting the number of fluorescent epithelial cells in cranial, middle and caudal tracheal 62 Figure 9.—-Distribution of fluorescent cells in six regions of rabbit trachea. The values are expressed as the average number of cells in one 15 um tracheal section. n=6. 63 440 :40 449.00 L'IIVQ 20.0wm U400:2 44.2410 440040 M4095. 44.24mu 44mm00 449.00 445.20) 44¢hzm> 44¢th> ”7 .5. 6 6 m. .6 I. I “I . n... .+ I. I. Z .l .0 c. 2 I. 6 L o 00‘ .v c. 81130 :lO HBBWON 64 .ucmummmac haucoowmwcmflm 902 can wcwcflaumccs an concoccoo mace: « h.H H ¢.m H.H H N.v 0.0 H 5.0 h.m M H.m v.N H H.m m.m H N.ma mm H cows Hmcsmo manna: Hmwcmuo Amparo manna: Haficmuo codwm Hmmuoo Homuoo Homuoa Hmuucm> Hmuucm> Hmuucm> . m :ofluomm 8: .Am n :0 mo.o u 0 sues MZm >0 poucmfiou .mcoflmwu xwm Scum ma 0 ca mHHmo uncommuosam mo Hones: mo Houum cumccmum can cmmzll.~ mqm4e 65 Hmesmo Hanson Amm.m0 manna: -.H Hmmuon Awh.mv 40>.NV Hmwcmuo 05.0 ~m.0 acmuoo 40>.N0 400.m0 Amm.m0 Hocsmu «mm.m «mm.¢ «mm.v HMHUGO> Amm.m0 400.m0 400.mv 400.N0 canvas «m0.m «mm.v «mm.0 .0 Hmuucw> Amw.mv Ama.vv Avm.mv Amm.mv A00.N0 Hmwcmuo «00.0 «00.0H «00.0H «ma.0 ama.0 Hmuucw> Hmcsmu canvas Hmwcmuo ampsmu macpwz HMHCMHU Hmmuoo acumen Hmmuoo kuucw> Hmuucw> Hmuuco> .mocmowmacoflm n « Am :0 m0.0 u .ummu mGOmflHmmEoo mamwuase Mzm :H cums “mononucmumm nay momma unmowmwcmflm Umoma can mmcmn Hmnuu4il.m mam4a 66 segments from nine rabbits. Three treatment groups were compared: control, L—DOPA, and reserpine. The average number of cells counted per rabbit in each group is represented graphically in Figure 10. The control group averaged 676 cells in 30 sections. The L-DOPA injected group averaged 603 cells per 30 sections. The reserpine injected group averaged only 301 cells per 30 sections, less than half the number counted in controls. Analysis of variance indicated that the data were obtained from significantly different populations of cells. Subsequent SNK g posteriori comparisons indicated that the number of cells counted in tracheas from control and L-DOPA injected rabbits were not significantly different. How- ever, significantly fewer fluorescent cells were present in tracheas from reserpine injected rabbits. The group means and results of the SNK comparison are summarized in Table 4. The two means connected by an underline were not significantly different. TABLE 4.--Mean and standard error of number of fluorescent cells in 30 tracheal sections from control, L-DOPA, and reserpine groups. Compared by SNK with a = 0.05. (n = 9.) Treatment Control L-DOPA Reserpine Mean 1 SE 676 r 176 603 1 210 301 i 16 * * Means connected by underlining are NOT signif- icantly different. 67 Figure 10.--Average number of fluorescent cells in 30 tracheal sections from control, L-DOPA, and reserpine treated rabbits. n = 9. 68 szmmmmm 9|:IOQ 030mm 0.29204me 4000 I4 OIZIEOS 40m...200 91!;919 00. 00m 00m 00¢ 000 000 00k 000 000 $1130 :IO HBBWON 69 Histochemical Results The histochemical stains were applied to freeze- dried formaldehyde vapor treated paraffin sections. Sec- tions of trachea and stomach were stained in parallel. A chart summarizing the staining reactions appears in Table 5. Alcian Blue The alcian blue stain reacts with mucopolysac- charides leaving a blue reaction product indicating site of mucus storage. Goblet cells in the tracheal epithelium are vividly demonstrated in Figure 11. Higher magnifica- tions not shown here demonstrated that goblet cells occurred in groups and were often in contact with the lumen. Their bases were in contact with the basement membrane and the lateral borders were parallel as they approached the lumen. Other features demonstrated in Figure 11 include the large venous plexuses in the submucosa and the blue staining cartilage indicating the presence of glycoprotein. Grimelius Silver Nitrate Reaction for Argerphilia The Grimelius argyrophilic reaction has been assoc- iated with secretory granulesixiseveral types of peptide- producing endocrine cells. An argyrophilic cell in the trachea epithelium is demonstrated in Figure 12. An intensely reactive area is seen above the nucleus and 70 moascwuw cwaaxouofimm + I mcwuooccm Ipqu I I figummflomousz 25m. c0492 mmascmuw + + mcwuooccm oflafiamoummu4 I + cacououmm :wcmummeNMHQ moocmumnsm mcwcmmoauumm I + mcflospmm Iownuwm I mmucoumasm I mcaospmm unficfimmmucmmu4 “nonhumwum ocean £443 +0 mocfleoahnum mocmommnonam I + Iaaconm 00050:“ no ImaoocH IwcmnmnamEHom Haou mu GE 4400 4400 oxen HHmU cacououmm um» um: on camum umanou IcwmmmEousooumucm cwummeounooumucm wmmnumua mucoquSm . .mgOfiflumwh HMUflEGSUOHmflfi UwflumHmm MO COHflMDOH UCM mUCMOflMfiGOHmlIom 3:0... 71 Figure ll.--Freeze-dried formaldehyde vapor (FD-FV) treated tracheal section stained with alcian blue and safranin 0.80X. Figure 12.--FD-FV treated tracheal section demonstrating two argyrophilic cells. 715x. 72 ‘:*V- m.“ ( no. If! ,.. . n 1'3“"... . . I 94...? «MW-v-23»; ~. 12cm 1.94“?!" I. ,' O . .. V i I .' L a ‘ I . ,. 73 less dense deposits are present in the apical cytOplasmic region which tapers toward and approaches the lumen. A second argyrophilic cell is seen at the right edge of the photomicrograph. As expected, argyrophilic cells were numerous in the stomach epithelium. The question of whether argyrophilic cells were the same as fluorescent cells was answered by photograph- ing a section by fluorescence, staining for argyrophilia, and rephotographing by light microscopy. The result of this procedure clearly demonstrated that fluorescent cells were argyrophilic. The five fluorescent cells seen in the lower panel of Figure 13 are identical to the five argyrophilic cells in the upper panel. The possibility that the argyrophilic-fluorescent cell represented a goblet cell was excluded by applying both the argyrophilic stain and alcian blue to the same section. The middle part of the epithelium in Figure 14 contains two light blue goblet cells flanked on each side by a dark staining argyrophil positive cell. These find- ings clearly indicate that goblet cells and fluorescent- argyrophilic cells are two distinct cell types in the tracheal epithelium of adult rabbits. Ferric-ferricyanide The ferric-ferricyanide reaction indicates the presence of reducing substance often associated with 74 Figure 13.-—The same tracheal section photographed first by fluorescence microscopy (lower panel) then stained for argyrophilia and rephotographed by light microscopy (upper panel). Identical numbers in the two panels identify the same cells. 715x. 75 “Ii-"1;,” “m 21‘ ’«FWV; ; W m 76 Figure 14.--Alcian blue and argyrophilic stains applied to the same tracheal section. 715x. Figure 15.--FD-FV tracheal section stained with ferric- ferricyanide. 715x. 78 phenols or indoles. The solitary blue cell seen in Figure 15 reflects the morphOlogic profile deScribed for the fluorescent-argyrophil cell. An intense blue deposit is present directly above the nucleus. The deposit in the apical cytoplasm is less intense but clearly demon- strates the tapering apical pole of the cell. Blue deposits are also present in the basal region and the cell seems to be in direct contact with the basement membrance. Numerous ferric-ferricyanide positive cells were also present in the stomach epithelium. The results of restaining the same section with ferric-ferricyanide after fluorescence microscopy clearly demonstrated that the ferric-ferricyanide positive cells are identical to fluorescent cells. The three fluorescent cells in the upper panel of Figure 16 correspond exactly to the morphology and localization of the blue ferric-ferricyanide positive cells in the lower lower panel. Diazosafranin Diazosafranin was used as a specific histochemical technique to demonstrate the presence of serotonin (or closely related indoles). Figure 17 demonstrates a blue- black deposit in an epithelial cell of a tracheal section stained with diazosafranin. The dense deposit above the nucleus and tapering apical pole which contains less dense 79 Figure l6.--The same tracheal section photographed first by fluorescence microscoPy (upper panel) and then stained with ferric-ferricyanide and rephotographed by light microsc0py (lower panel). Identical numbers in the two panels identify the same cell. 288x. '.' I‘I "~"I .‘o I.“ ‘ ’ I ‘2‘ " i‘ " run-I" \.II:T'?V::.I ' - 1‘94“” yg' “ .0. 'y.‘:‘ '. ".‘ 1“ “LINK 81 Figure l7.--FD-FV treated tracheal section stained with diazosafranin. 715x. 82 83 but distinct deposits is consistent with descriptions of the fluorescent, argyrophilic, and ferric-ferricyanide positive cell. Unfortunately, the diazosafranin reaction was not present in sections mounted from 1% gelatin. Numerous diazosafranin positive cells were present in the stomach epithelium. Lead-Hematoxylin and Argentaffin Reactions Neither argentaffin positive nor lead-hematoxylin positive cells were present in the tracheal epithelium. Sections of stomach epithelium stained in parallel with the tracheal sections contained numerous positive cells with both reactions. CHAPTER 5 DISCUSSION This investigation has described morphologic, cytochemical, and distribution properties of a specific cell type in the adult rabbit tracheal epithelium. The presence of an endogenous substance that fluoresced after treatment with formaldehyde vapor was a unique feature of this cell type. Anesthesia, dissection, and freezing procedures were performed as quickly as possible to pre- vent metabolic destruction of the endogenous substance. Rigorous freeze-drying conditions preserved morphological and cytochemical integrity of the tracheal epithelium. Morphological Characteristics Tracheal epithelium of formaldehyde vapor treated tissues contained numerous pyramidal or teardrop-shaped cells when observed by fluorescence or after histochemical staining. Tapering apical extensions appeared to contact the luminal surface. Supranuclear portions of the cells were more intensely fluorescent than infranuclear regions. Bases of the cells seldom contacted the basement membrane except by extensions which were also slightly fluorescent. This description is very similar to granulated cells 84 85 described in tracheas of adult New Zealand White rabbits by Cutz gt gt., (1975) using electron microscopy. In their study, however, endogenous formaldehyde-induced fluorescence was not detected without injecting amine precursors. This contrast with the present investigation may reflect age, dietary, or technical differences. This study also demonstrated that fluorescent cells were more numerous in the ventral aspects of the tracheal epithelium. The specific importance of this finding cannot be determined at this time. However, localization of an extensive submucosal venous plexuses along the ventral aspects of the trachea may reflect a functional association between the plexuses and fluores- cent cells. Identity of the Fluorophore The specific nature of the fluorophore was evaluated to more clearly identify the cellular substance responsible for producing the fluorescence. The first test established that the fluorescence was the result of formaldehyde vapor treatment. Fluorescent cells were not demonstrated in tracheal sections untreated with formaldehyde vapor. The Picket-Spengler reaction (Jonsson, 1971) occurs between an amine and an aldehyde by condensation to form heterocyclic compounds which spontaneously oxidize yielding fluorescent 86 dihydro-B-carboline or dihydroisoquinoline depending on whether the amine contains an indole or catechol ring structure respectively (Corrodi and Jonsson, 1967). Production of fluorescence after treatment with formalde- hyde is accepted as evidence that an amine is present. Quenching of an amine fluorophore will occur after exposure to water (Ritzen, 1966). In this study, mounting formaldehyde vapor treated sections from water extinguished specific cellular fluorescence. Although this cannot be considered an amine-specific reaction, it supports the hypothesis that an amine-fluorophore is responsible for the fluorescence. Fluorescence quenching after treatment with sodium borohydride is considered a specific test of formaldehyde-induced fluorophores (Corrodi gt gt., 1964). Fluorescent dihydro-compounds are reduced to nonfluores- cent tetrahydro-compounds during exposure to sodium borohydride. Reoxidation to fluorescent dehydro- compounds will occur after subsequent exposure to hot formaldehyde gas. This study confirmed that the cellular fluorophore was quenched by sodium borohydride and regen- erated subsequent to formaldehyde treatment. Therefore, this test was consistent with the hypothesis that the fluorophore is an amine-aldehyde reaction product. Spectral properties of the fluorophore were determined by microspectrofluorometry. Fluorophores can 87 be identified by determining the excitation and emission wavelengths. Formaldehyde-induced amine fluorophores have been extensively studied (Bjorkland 23.21:! 1968; Ewen and Rost, 1972). The averaged excitation (400 nm) and emission (521 nm) peaks of the cellular fluorophore observed in the present study closely agree with those reported for S—hydroxytryptamine (SHT) and 5-hydroxytryptophan (5HTP). Spectral properties of formaldehyde treated SHT models (excitation = 413 nm, emission = 525 nm) recorded with the same microspectro- fluorometer used in this study closely paralleled the spectral peaks recorded for the cellular fluorophore. Spectra of other indole- or catehol-derivatives which have been reported are not sufficiently similar to implicate identity with this cellular fluorophore (Bjorkland and Falck, 1973). Although spectral data of 5-HTP were not gathered, the published spectra of this substance is indistinguish- able from SHT. However, it seems unlikely that the fluorescence resulted from 5HTP for two reasons. First, 5HTP fluorescence intensity is ten times less than SHT (Bjorklund and Falck, 1973). Intensity of the cellular fluorophore described here was nearly as great as that observed in stomach enterochromaffin cells. If the epithelial cell fluorophore did represent 5HTP, it should have demonstrated a much lower fluorescence intensity 88 (Bjorklund and Falck, 1973). Second, 5HTP added to organ homogenates is rapidly converted to SHT (Gaddum and Giarman, 1956). Therefore, because 5HTP has low fluorescence yield and is rapidly converted to SHT, it is unlikely that this substance is responsible for the cellular fluorescence. Amine content in these fluorescent cells is also supported by histochemical findings that ferric- ferricyanide positive cells are identical to the fluores- cent cells and that diazosafranin positive cells are present in the tracheal epithelium. Although the ferric- ferricyanide reaction is not specific for amines, model experiments indicate that several types of indole sub- stances will cause the appearance of blue precipitate (Lillie and Burtner, 1953). According to Lillie gt gt., (1972), positive reactions with diazosafranin specifically reflect the presence of SHT or 5HTP. A decrease in the number of fluorescent cells after IP injections of reserpine also indicated that an amine was present in the fluorescent tracheal epithelial cell type. Hakanson gt_gt., (1970) have demonstrated decreased numbers of fluorescent epithelial cells in the rabbit stomach after reserpine administration. Therefore, it is reasonable to conclude that the fluorophore described in this study represents an amine for the following reasons: (1) fluorescence was 89 present only after formaldehyde vapor treatment, (2) the fluorophore was quenched by water, (3) fluorescence was quenched by sodium borohydride but regenerated by sub- sequent formaldehyde treatment, (4) the fluorescent cell was ferric-ferricyanide positive, and (5) reserpine decreased the number of detectable fluorescent cells. It is also valid to conclude that either SHT or 5HTP was responsible for the fluorescence because (1) the excitation and emission characteristics were similar to SHT and 5HTP models but different from any other amines normally present in animal tissues, and (2) the cells demonstrated a positive reaction with diazosafranin. Lower fluorescent yield of 5HTP and its rapid conversion to SHT tg’gtgg lead to the conclusion that the substance contained in formaldehyde-induced fluorescent cells of adult rabbit tracheal epithelium was SHT. Functional Significance of SHT in the Tracheal Epithelium Localization of SHT-containing cells in the trachea suggest that this substance may act within the mucosa. Afferent intraepithelial nerves have been reported in adult rabbit (Cutz gt gt., 1975) and rat (Jeffery and Reid, 1975) trachea. Irritation of tracheal mucosa has been shown to stimulate both afferent and efferent vagal nerve fibers (Widdicombe, 1954, 1966). In addition, receptors which mediate respiratory and cardiovascular 90 reflexes are sensitive to SHT (Doughas and Toh, 1953; Ginzel, 1957; Paintal, 1955). Lauweryns and Cokelaera (1973) have presented evidence of SHT release from NEBs in response to hypoxia. Thus, mechanical, chemical or other stimuli might cause SHT release which would mediate reflex activity originating from sensory nerves within the tracheal mucosa. SHT may also affect tracheal mucous production either directly, which is believed to occur in salivary glands (Berridge, 1970), or indirectly by affecting autonomic innervation of secretory units (Gallagher gt gt., 1975; Fozard and Mobarok A11, 1978). The sections stained with both silver nitrate and alcian blue indicate that goblet cells and serotonin-containing cells are closely associated. Nerve fibers have been demonstrated near goblet cells in tracheal epithelium of rat (Jeffery and Reid, 1975) and in human submucosal glands (Bensch gt gt., 1965). Apical processes were only suggested in this study, but Cutz gt gt., (1975) have demonstrated granu- lated cells with apical extensions contacting the tracheal lumen. These processes may represent a sensory link to intralumenal events such as mechanical, chemical or environmental changes. Lumenal contacts may also be sites for SHT secretion onto the epithelial surface. 91 Beating frequency of cilia and rate of mucus transport are affected by SHT (Tsuchiya and Kensler, 1959; Dadaian gt gt., 1971). Venous plexuses have been observed in tracheas of several species (Hughes, 1969). In the adult rabbit, the plexuses are located within the ventral cartilaginous regions. Ventral aspects of the tracheal wall contain more than twice as many fluorescent cells as the dorsal aspects. This distribution of fluorescent cells is con- sistent with the suggestion that a functional link exists between the amine-containing cells and the plexuses. Formaldehyde-induced fluorescence indicates that adrenergic nerve fibers are present in the smooth muscle around the plexus. Diffusion of SHT across the basement membrane and lamina prOpria could affect endothelial permeability (Majno gt gt., 1967) and/or vascular smooth muscle, either directly (Aviado, 1960) or by modulating synaptic neurotransmission (Fozard and Mobarok Ali, 1978). Effect of Injecting L-DOPA Injections of L-DOPA did not affect the numbers of cells detectable by fluorescence microscopy. This indi- cates that endocrine-like cells other than the 5HT- containing cell were not present. Previous studies of L-DOPA administration have shown either that the numbers of fluorescent cells in respiratory epithelium were 92 increased (Hage, 1974; Cutz gt gt., 1974) or that fluorescent cells appeared where none was previously detected (Ericson 2E.2l" 1972; Hage gt gt., 1977). An investigation of tracheal epithelium from adult New Zea- land White rabbits demonstrated formaldehyde-induced fluorescence in cells after injections of L—DOPA but no endogenous fluorescence in control (noninjected) animals (Cutz gt gl., 1975). The significance of these varied reports is not known at this time, but may reflect age, dietary, or other metabolic differences. However, the present study also showed that the emission peak of the cellular fluorophore had shifted from 520 nm in controls to 505 nm in L-DOPA injected animals. The emission peak of catecholamines is approxi- mately 480 nm (Bjorklund gt gt., 1972). Shifts to shorter wavelengths after injecting a catecholamine precursor may reflect a mixture of SHT and catecholamine in the same cell and thus indicate that L-DOPA uptake had occurred in the endogenous (SHT-containing) fluorescent cell. This supports the findings of Cutz gt gt., (1975) who have reported uptake of L-DOPA in rabbit tracheal epithelium. Uptake of L-DOPA is a characteristic of peptide-producing APUD cells (Pearse, 1969). 93 ArgyroPhilicvReaction The present study demonstrated argyrophilic cells in rabbit tracheal epithelium. This finding is consistent with several reports which have found argyrophilic cells throughout the respiratory epithelium (Tateishi, 1973; Cutz gt_gl., 1975). However, direct evidence that argyrophilia occurs in amine-containing cells or in cells which take up amine precursors has not been previously reported in the respiratory system. The present study successfully demonstrated that argyrophilic cells were identical to SHT-containing cells. This was accomplished by observing the same section initially with fluorescence microscopy and then with light microscopy after staining for argyrophilia. The nature of argyrophilic staining has been investigated by Solcia gt gl., (1976). Although no con- clusions about the actual chemistry of the reaction were reached, it was established that argyrophilia is assoc- iated with the dense-cored granules in known peptide- producing cells. These types of granules are believed to represent intracellular organelles which store and release peptide hormones (Trifaro, 1977). Peptidefiproduging Cells in TracheaIYEpithelium Cells in the rabbit tracheal epithelium which contain SHT may be peptide-producing APUD cells for the 94 following reasons: (1) the cells contain an amine, (2) the cells can take up and store amine precursors, and (3) the cells are argyrophilic. Amine content and amine precursor uptake are APUD characteristics defined by Pearse (1969). The argyrophilic reaction has been associated with several known peptide-producing cells located in gastrointestinal tract (Solcia gt 30" 1975), thyroid (Solcia gt gt., 1976), and pancreas (Grimelius, 1968). However, identification and localization of specific peptide hormones is necessary to fully qualify the SHT-containing tracheal epithelial cell as a peptide- producing APUD cell. CHAPTER 6 SUMMARY, CONCLUSIONS AND RECOMMENDATIONS Summary Amine-containing endocrine-like cells in the tracheal epithelium of adult male New Zealand White rabbits were studied by morphorphologic, morphometric, and histo- chemical techniques. Tracheas were freeze-dried and treated with formaldehyde vapor to convert cellular amines fim:fluorescence microscopy. Numerous fluorescent cells were identified within the tracheal epithelium of formal- dehyde treated specimens, but they were not seen in untreated samples. Distribution of the fluorescent epithelial cells was evaluated by counting the number of cells per section in six tracheal regions. Cell counting was also used to evaluate changes in amine-containing cells after pretreatment with L-DOPA or reserpine. Ventral aspects of the tracheal epithelium from control rabbits contain significantly more fluorescent cells than the dorsal aspects. The number of fluorescent cells was not affected by treatment with L-DOPA, but tracheas from reserpine treated rabbits contained significantly fewer cells than controls. 95 96 Identification of the cellular amine in control and L-DOPA treated rabbits was accomplished by micro- spectrofluorometric and histochemical techniques. Excitation and emission peaks of fluorescent cells from controls indicated that the cellular amine was SHT. This finding was supported by positive staining with diazo- safranin and ferric-ferricyanide. The emission peak of fluorescent cells from L-DOPA treated rabbits was shifted to shorter wavelengths indicating uptake of L-DOPA in the SHT-containing cell. Histochemical reactions were correlated with the SHT-containing cell by photographing tracheal sections with fluorescent microscopy, subsequent histochemical staining, and rephotographing the same section by light microscopy. In addition to positive reactions with ferric-ferricyanide and diazosafranin, the fluorescent cells were also argyrophilic. However, no alcian blue reaction for mucopolysacchrides was detected in the fluorescent cells. Conclusions l. Histochemical investigations of the adult rabbit tracheal epithelium demonstrate the presence of a single endocrine-like cell population which contained SHT (or a closely related indole substance). 97 2. SHT-containing cells were more numerous in the ventral aspects of the trachea than in the dorsal aspects. 3. The number of fluorescent cells in tracheal epithelium was not affected by L-DOPA pretreatment. 4. The number of fluorescent cells in tracheal epithelium was decreased by reserpine. 5. SHT-containing cells are argyrophilic. 6. SHT-containing cells do not contain mucus (mucopolysacchride). Recommendations l. Amine-containing cells should be evaluated in animals subjected to experimental conditions affecting respiratory function. 2. An electron microsc0pic study describing the specific ultrastructural features of SHT-containing cells should be undertaken. 3. Additional microspectrofluorometric evalua- tions, especially after L-DOPA treatment, should be made in view of the small sample size used here. 4. Tracheobronchial and alveolar epithelium of humans and animals should be surveyed with immunochemical techniques for the presence of peptide-producing endocrine cells. 98 5. A quantitative study of the distribution of endocrine-like cells in the lungs should be undertaken to provide a baseline from which the effects of various experimental conditions could be evaluated. 6. The effect of age (fetal through adult) and species on endocrine-like cells should be systematically evaluated in a single study. may APPENDICES 99 APPENDIX A DATA AND STATISTICAL TABLES 100 APPENDIX A1 Average number of fluorescent cells per 15 um section from the six different regions of individual control rabbit tracheas. Each value is the average of ten sections. Animal Ventral Ventral Ventral Dorsal Dorsal Dorsal Number Cranial Middle Caudal Cranial Middle Caudal 1 25.5 11.7 15.9 6.7 3.9 11.1 2 25.3 19.8 24.1 7.6 9.0 10.0 3 9.7 7.1 3.0 2.6 2.6 2.1 4 12.6 5.5 2.8 3.7 2.3 2.0 5 9.2 4.6 2.6 2.5 2.1 2.9 6 8.8 5.6 5.9 5.2 5.3 4.4 101 APPENDIX A 2 Analysis of variance table for the number of cells per 15 um section from the six regions of control rabbit tracheas. Source of Degrees of Sum of Mean Variation Freedom Squares Square F Among levels 5 1148.91 229.78 6.66* Within levels 30 1034.55 34.48 Total 35 2183.46 * = F(0.01)5’ 3'70 102 APPENDIX A3 Number of fluorescent cells in individual tracheas from control, L-DOPA, and reserpine groups. Each value is the total number of fluorescent cells in 30 tracheal sec- tions. Experiment # CON L-DOPA Reserpine 83 718 427 316 76 958 1022 318 89 352 362 269 103 APPENDIX A 4 Analysis of variance table for the number of fluorescent cells in control, L-DOPA, and reserpine groups. Source of Degrees of Sum of Mean F Var1at1on Freedom Squares Square Among levels 2 237463 118732 6.74* Within levels 24 422418 17601 Total 26 659881 *F(0.01)2’24 = 5'61 104 APPENDIX B HISTOCHEMICAL TECHNIQUES 105 APPENDI X B 1 Grimelius Silver Nitrate (Grimelius, 1968) Stock Solutions 1. Sodium acetate, 0.2 M 5.44 9 Na acetate (Baker) 200.0 ml distilled water* *All water in this procedure is demineralized and glass distilled. 2. Acetic acid, 0.2 M 2.3 m1 glacial acetic acid (Mallinckrodt) 197.7 ml distilled H20 3. 1% silver nitrate l g AgNO (Polysciences) 3 100 ml distilled water Staining Solutions 1. Staining solution 1 ml 0.2 M acetic acid 9 ml 0.2 M sodium acetate 3 ml 1% silver nitrate 87 ml distilled water 106 107 2. Reducing Solution 1 g hydroquinone (Polysciences) 5 g sodium sulfite (Mallinckrodt) 100 ml distilled water. StainingtProcedure 1. Bring sections to water. 2. Immerse in staining solution (60°C) for 6 hours in the dark. 3. Drain briefly and immerse in reducing solution (45°C) for 2 minutes in the dark. 4. Sodium thiosulfate 5%: 5 minutes. 5. Rinse in distilled water. 6. Dehydrate, clear, and mount. APPENDI X B 2 Dizaosaffranin (Lillie, Burtner, and Henson, 1973) Stock Solutions 1. Acid safranin (stable for weeks) 3.6 g safranin 0 (Allied Chemical) 60.0 ml distilled water 30.0 ml 1 N hydrochloric acid 2. Sodium nitrite, 1 N (stable for 3 months at 4°C) 6.9 g sodium nitrite (Baker) 100.0 ml distilled water 3. Disodium phosphate, 0.1 M 14.2 g disodium phosphate (anhydrous) 1000.0 ml distilled water Staining Solution 1. Add 0.5 ml of sodium nitrite solution to 4.5 m1 of cold safranin solution. Keep cold for 15 minutes. 2. Dilute 1 ml of this solution with 40 ml disodium phosphate solution. Use immediately. Procedure 1. Bring sections to water. 108 109 Place slides in cold c0p1in jar and pour in the fresh staining solution: 5 minutes. Decant stain and wash slides in 3 changes of 0.1N HCl: 10-13 seconds total. Wash in distilled water. Dehydrate, clear, and mount. APPENDI X B 3 Ferric-Ferrigyanide (Lillie and Burtner, 1953) Stock Solutions 1. 1% potassium ferricyanide (Baker) 1 g potassium ferricyanide 100 ml distilled water 1% ferric chloride (Baker) 1 g ferric chloride 100 ml distilled water Staining Solution 10 m1 1% potassium ferricyanide 75 ml 1% ferric chloride 15 ml distilled water Procedure 1. 2. Bring sections to water. Stain in freshly prepared staining solution: 15 minutes. Rinse in distilled water. Dehydrate, clear, and mount. 110 APPENDIX B4 Fontana Argentaffin Silver (Culling, 1957) Stock Solutions 1. 10% silver nitrate 10 9 silver nitrate (Polysciences) 100 ml distilled water (all water for this technique should be demineralized and glass distilled). Staining Solution 1. Silver solution 25 ml 10% silver nitrate Ammonium hydroxide (Mallinckrodt) added drop by drop until the precipitate which first forms almost disappears. Then add 25 ml distilled water. Procedure 1. 2. Bring sections to water. Transfer to silver solution for 18-48 hours in the dark at room temperature. Rinse in distilled water. Fix in 3% sodium thiosulfate: 2 minutes. Wash in tap water. Counterstain in 1% safranin: 1 minute. Wash in tap water. Dehydrate, clear, and mount. 111 APPENDIX B5 Alcian Blue (Luna, 1968) Stock Solution l. 3% acetic acid 3.0 ml glacial acetic acid (Mallinckrodt) 97.0 ml distilled water Staining Solution 1. 1% alcian blue 1.0 g alcian blue (Allied Chemical) 100.0 ml 3% glacial acetic acid Procedure 1. Bring sections to water. 2. Mordant in 3% acetic acid for 3 minutes. 3. 1% alcian blue solution for 30 seconds. 4. Wash in running water for 10 minutes. 5. Rinse in distilled water. 6. Dehydrate, clear, and mount. 112 APPENDIX B 6 Lead-Hematoxylin (Solcia, Capella, and Vassallo, 1969) Stock Solutions 1. 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