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The use of dextrans as particulate tracers in respiratory epithelial permeability studies Forster, Allan Bruce Burton 1981

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THE USE OF DEXTRANS AS PARTICULATE TRACERS IN RESPIRATORY EPITHELIAL PERMEABILITY STUDIES by ALLAN BRUCE BURTON FORSTER B.Sc, University of B r i t i s h Columbia, 1979 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Pathology) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA November, 1981 © Al l a n Bruce Burton Forster, 1981 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree a t the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or heir r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of PATHOLOGY The U n i v e r s i t y o f B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date November 5, 1981 / - i n \ - i i -ABSTRACT These experiments were designed to test the s u i t a b i l i t y of dextrans as alternate l i g h t and electron microscopic tracers to horseradish peroxidase (HRP) i n studies of respiratory e p i t h e l i a l permeability i n control and cig a r e t t e smoke-exposed guinea pigs. Nine animals were u t i l i z e d i n i t i a l l y to e s t a b l i s h the most e f f e c t i v e means of preserving highly water-soluble dextran while adequately maintaining airway c e l l u l t r a s t r u c t u r e . The perfusion-nebulization f i x a t i o n and carbohydrate retention techniques developed were then used on the twenty-six guinea pigs i n the permeability study. Five animals served as controls, while others were exposed to the dextran tracer, smoke from 10 c i g a r e t t e s , or both. Intratracheal tracer i n s t i l l a t i o n s of sonicated preparations of unlabelled or e i t h e r FITC- or i r o n - l a b e l l e d dextran TlO or T40 were carried out 1/2-hour a f t e r smoke exposure. A l l dextrans could be v i s u a l i z e d l i g h t -m icroscopically along the respiratory epithelium of airways and lung parenchyma by the Mowry-Millican technique, a modified a l c o h o l i c periodic a c i d - s c h i f f (PAS) s t a i n . As witnessed i n previous HRP studies, some post-mortem tracer d i f f u s i o n was seen, and no differe n c e i n dextran e p i t h e l i a l penetration was noted between smoked and sham-smoked animals. At the electron microscopic l e v e l , treatment of guinea p i g trachea and lung with aldehyde-OsO^-lead c i t r a t e f i x a t i v e c o c k t a i l - i i i -preserved the unlabelled dextran as i r r e g u l a r , highly electron-dense aggregates of p a r t i c l e s along the luminal surfaces only of airways of c o n t r o l guinea pigs. One-half of the animals i n the smoke-exposed group showed penetration of dextran between the tracheal e p i t h e l i a l c e l l s . Incomplete tracer d i s s o l u t i o n p r i o r to i n s t i l l a t i o n , as shown by negative s t a i n i n g of the i n s t i l l a t e s , i s thought to be respon-s i b l e f o r t h i s inconsistency. No intrapulmonary or alveolar trans-e p i t h e l i a l tracer transport could be detected. FITC-dextrans presented the a d d i t i o n a l advantages of q u a l i -t a t i v e detection by f l u o r e s c e n c e - d i f f e r e n t i a t e d interference contrast .(DIC) microscopy and quantitative detection by spectrophotofluoro-metric means. Iron-dextrans exhibited too large a molecular si z e f o r p r a c t i c a l use with smoke-exposed guinea pig tracheobronchial epithelium. We conclude that dextrans provide d i s t i n c t advantages over HRP i n terms of low p r i c e , inertness, and s i z e v e r s a t i l i t y , as l i g h t and electron microscopic tracers. - i v -TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGEMENTS x INTRODUCTION 1 A. Previous Tracheobronchial Mucosal Permeability Studies 1 B. The Use of Dextran as an Electron Microscopic Tracer 9 1. General Considerations 9 2. Q u a l i t a t i v e Determination of Dextran 11 3. Quantitative Determination of Dextran 12 C. Scope and Aim of the Present Investigation 14 MATERIALS AND METHODS 15 A. General Techniques 15 1. Cigarette Smoke Exposure 15 2. Surgery and Experimental Apparatus 15 3. Tracer Preparation and I n s t i l l a t i o n 16 4. Fi x a t i o n and Tissue Processing 19 5. Light Microscopic Demonstration of Dextran -The Mowry-Millican Method 22 - V -Page B. S p e c i f i c Experiments 24 1. Fix a t i o n Technique 24 2. Dextran Tracer Studies 25 RESULTS 27 A. Fix a t i o n Technique 27 B. Dextran Tracer Studies 29 1. Unlabelled Dextrans 29 a) Light microscopy 29 b) Electron microscopy 3 1 2. FITC-labelled Dextran 35 3. Iron-Dextran 38 TABLES 39 FIGURES 41 DISCUSSION 67 A. F i x a t i o n 67 B. Unlabelled Dextran Tracer Studies 69 1. Light Microscopy 69 2. Electron Microscopy 73 C. FITC-Dextran Tracer Studies 81 D. Iron-Dextran Tracer Studies 82 CONCLUSION 84 BIBLIOGRAPHY 87 - v i -LIST OF TABLES Experiments f o r determining the most e f f e c t i v e f i x a t i o n method for dextran s t a b i l i z a t i o n . Number of animals used i n each treatment f or dextran tracer studies. - v i i -LIST OF FIGURES Figure Page 1. Normal guinea p i g tracheal epithelium (TBO) 41 2. Normal guinea pig tracheal epithelium (Mowry-M i l l i c a n method) 41 3. Normal guinea pig lung parenchyma (TBO) 41 4. Dextran along the tracheal e p i t h e l i a l surface (Mowry-Millican method) • 43 5. Washout section of trachea from dextran-treated animal (Mowry-Millican method) 43 6. Dextran along the alveolar e p i t h e l i a l surface (Mowry-Millican method) 43 7. Washout section of lung from dextran-treated animal (Mowry-Millican method) 43 8,9 Negative-staining of dextran T40 45 10. Junctional complex between two tracheal e p i t h e l i a l c e l l s 45 11. Dextran T40 along the tracheal e p i t h e l i a l surface of non-smoke-exposed animals 47 12. Dextran TlO along the bronchial e p i t h e l i a l surface of non-smoke-exposed animals 47 - v i i i -Figure 13. Dextran T40 along the alv e o l a r e p i t h e l i a l surface 14. Dextran T40 i n the a p i c a l i n t e r c e l l u l a r space of the tracheal epithelium of a smoke-exposed animal 15. Dextran T40 i n the basal i n t e r c e l l u l a r space of the tracheal epithelium of a smoke-exposed animal (stained) 16. Dextran T40 i n the basal i n t e r c e l l u l a r space of the tracheal epithelium of a smoke-exposed animal (unstained) 17. Dextran T10 i n the a p i c a l i n t e r c e l l u l a r space of the tracheal epithelium of a smoke-exposed animal 18. Dextran T10 i n the basal i n t e r c e l l u l a r space of the tra c e a l epithelium of a smoke-exposed animal 19. Fluorescence microscopy of the tracheal epithelium of a smoke-exposed animal 20. Fluorescence microscopy of the lung parenchyma of a smoke-exposed animal 21. Fluorescence microscopy of FITC-dextran i n mucous layer of the tracheal epithelium of a smoke-exposed animal - ix -Figure Page 22. DIC microscopy of FITC-dextran i n mucous layer of the tracheal epithelium of a smoke-exposed animal 57 23. FITC-dextran i n mucous layer of tracheal epithelium of a smoke-exposed animal (PAS) 57 24. Composite of fluorescence/DIC micrographs of FITC-dextran i n mucous layer of the tracheal epithelium of a smoke-exposed animal 59 25. Fluorescence microscopy of FITC-dextran along the alv e o l a r epithelium of a smoke-exposed animal 61 26. DIC microscopy of FITC-dextran along the alveolar epithelium of a smoke-exposed animal 61 27. FITC-dextran along the alveolar epithelium of a smoke-exposed animal (PAS) 61 28. Composite of fluorescence/DIC micrographs of FITC-dextran along the al v e o l a r epithelium of a smoke-exposed animal 63 29. Fe^Ojj-dextran-TlO along the tracheal epithelium of a smoke-exposed animal 65 - X -ACKNOWLEDGEMENTS To my supervisor during the course of study, Dr. J.C. Hogg, I extend sincere thanks. His guidance and encouragement throughout the planning and execution of the experiments and during the prepar-ation of th i s manuscript were greatly appreciated. I also wish to thank Drs. Yeung, Ledsome, Reid and Pearce f o r t h e i r i n t e r e s t and suggestions throughout the programme, and Dr. R.S. Molday for supplying the FITC- and iron-dextrans. My thanks are also extended to the s t a f f of the Pulmonary Research. Laboratory f o r providing an environment that encourages productive research, and e s p e c i a l l y to Dr. W.C. Hulbert, without whose knowledge and patience these experiments could never have been undertaken. I am also indebted to the Medical Research Council f o r f i n a n c i a l support i n the form of a two-year Studentship. - x i -" I t i s nice to think that there are so many unsolved puzzles ahead fo r biology, although I wonder whether we w i l l ever f i n d enough graduate students." Lewis Thomas, "The Lives of a C e l l " - 1 -INTRODUCTION A. Previous Tracheobronchial Mucosal Permeability Studies The lung, i n addition to i t s function i n gas exchange and metabolism, provides a continuous c e l l u l a r b a r r i e r between man and his environment. Among the contaminants i n the 10,000 to 20,000 l i t e r s of a i r that adult humans breathe d a i l y (22) are spores, pollens, bacteria, infected droplets, smokes and nuclei from the combustion of coal and o i l (61). Yet so e f f i c i e n t are the airways and lung parenchyma i n blocking the entry of or removing i n j u r i o u s p a r t i c l e s that the lung i s normally s t e r i l e from the f i r s t bronchial d i v i s i o n to the terminal lung units (96). The mechanisms of respiratory defense are c l o s e l y associated with the s i z e of the inspired p a r t i c u l a t e material. Larger p a r t i c l e s (1-5 u) which are able to escape nasal f i l t r a t i o n deposit by impac-t i o n and sedimentation i n the conducting airways, and are c a r r i e d up the mucociliary escalator to the oropharynx, where they can be e l i m i -nated by expectoration or swallowing (138). Smaller p a r t i c l e s (less than 0.1 u), deposited i n the respiratory airways and lung parenchyma by d i f f u s i o n , are removed mainly by a l v e o l a r macrophagic phagocytosis and subsequent translocation to the pharynx v i a mucociliary transport (101). Lymphatic transport and uptake of p a r t i c l e s into the blood comprise subordinate a l v e o l a r clearance mechanisms (77). - 2 -Yet as a b a r r i e r i n preventing the movement of retained foreign p a r t i c l e s and resident macromolecules into the airway wall, the tracheobronchial epithelium i s of fundamental importance. A major s t r u c t u r a l aspect of t h i s b a r r i e r function are the zonulae occludentes or t i g h t junctions l i n k i n g the a p i c a l portions of the adjacent respiratory e p i t h e l i a l c e l l s . Early studies by Hogg and co-workers (66,67) showed that the width of such j u n c t i o n a l complexes and the number of sealing strands formed by the fusion of the outer membrane l e a f l e t s make the tracheobronchial mucosa one of the least permeable of the body's e p i t h e l i a l layers (30,31,126). It i s not impermeable, however, as the cytochemical tracer horseradish per-oxidase (HRP) has been demonstrated under normal p h y s i o l o g i c a l conditions to penetrate tracheal epithelium. This t r a n s e p i t h e l i a l transport has been shown by Boucher and associates (17,18), using excised canine trachea mounted i n Ussing chambers, to occur mainly vi a p a r a c e l l u l a r pathways, although i n separate studies, v e s i c u l a r uptake of HRP was occasionally seen (109,111). The i n t e r c e l l u l a r junctions thus represent the "pores" through which l i p i d insoluble molecules such as HRP move, calculated by Boucher (17) to be 15 nm i n diameter i n i n v i t r o e l e c t r o p h y s i o l o g i c a l studies of dog trachea. Consistent with t h i s i n t e r p r e t a t i o n of tracheal mucosal permeability are the experiments of Hogg and co-workers i n which guinea pigs exposed to a v a r i e t y of noxious inhalants, i n c l u d i n g c i g a r e t t e smoke (19,63,122), showed increased tracheal e p i t h e l i a l - 3 -permeability to HRP through increased movement of t h i s probe through p a r a c e l l u l a r paths. Under normal p h y s i o l o g i c a l conditions, there was no evidence of jun c t i o n a l penetration of t r a c h e a l l y - i n s t i l l e d HRP by transmission electron microscopic (T.E.M.) observation. Yet a f t e r exposure to c i g a r e t t e smoke "doses" ranging from 100 puffs (19) to 600 c i g a r e t t e s (122), marked entry of HRP i n t o the i n t e r -c e l l u l a r space of tracheal epithelium was seen. Following c i g a r e t t e smoke inhalation, then, carcinogens present i n the p a r t i c u l a t e and vapor phases of smoke would have increased access to the m i t o t i c a l l y -a c t i v e basal c e l l layer of the tracheobronchial epithelium, thereby creating optimum conditions f o r dy s p l a s t i c change. Recently, Boucher et a l (19) examined the morphology of the e p i t h e l i a l t i g h t junctions using the freeze-fracture technique, and concluded that s t r u c t u r a l damage to the sealing strands of the t i g h t junctions was responsible f o r t h i s increased t r a n s e p i t h e l i a l tracer transport. Richardson (68) observed a s i m i l a r fragmentation of junctional strands i n the freeze-fracture analysis of mouse tracheal epithelium a f t e r ether anaesthesia. This c i g a r e t t e smoke-induced permeability increase i s , how-ever, time-dependent as Hulbert and associates (63) demonstrated that the penetration r a t i o (number of t i g h t junctions penetrated/ t o t a l number tight junctions counted) was maximal (90%) 1/2-hour post-challenge with 100 puffs of ci g a r e t t e smoke and decreased to cont r o l l e v e l s (.0%) within 12 hours. S i m i l a r l y , the rate of appear-ance of HRP i n the blood of smoke-exposed animals as measured by an - 4 -ELISA plate assay (.027$ of delivered dose /min) was approximately seven times that of control l e v e l s at 1/2-hour post-challenge. Yet, c l e a r l y , measurement of plasma HRP i s a more s e n s i t i v e assessment of tracheobronchial mucosal permeability than i s l o c a l -i z a t i o n of the tracer by E.M.; i n two separate studies (19,20), control groups showed low but measurable blood HRP l e v e l s , yet no q u a l i t a t i v e evidence of e p i t h e l i a l HRP penetration. Conceivably, E.M. techniques are not s u f f i c i e n t l y s e n s i t i v e to detect minimal tracer transport v i a p i n o c y t i c v e s i c l e s to the l a t e r a l and basal areas of e p i t h e l i a l c e l l s (20). Sampling errors are also l i k e l y as l o c a l i z e d areas of c e l l - s l o u g h i n g or increased turnover may be missed (17), and the p r o b a b i l i t y of sampling the "pore area" required to accommodate the measured translocation of HRP across the tracheal epithelium ( .05% of the t o t a l tracheal surface area i n i n v i t r o studies (18)) i s very low. S i m i l a r acute a l t e r a t i o n s i n r e s p i r a t o r y mucosal permeability to HRP are seen following administration of a number of other i r r i -tants, including antigen i n a l l e r g i c bronchoconstriction (21), nitrogen dioxide (104), methacholine, histamine and ether (20) and l a r y n g o t r a c h e i t i s virus (110). The demonstration by several i n v e s t i -gators (19,60,63) of nerve endings i n the i n t e r c e l l u l a r spaces just below the tight junctions i n the guinea pig tracheobronchial mucosa i s therefore p a r t i c u l a r l y s i g n i f i c a n t . Thought to represent the i r r i t a n t receptors which i n i t i a t e non-specific r e a c t i v i t y of these - 5 -airways (141), these nerve endings may be more e a s i l y stimulated a f t e r mucosal damage. Furthermore, t i g h t junction disruption would allow inhaled antigens greater access to the larger number of mast c e l l s i n the submucosa (60) as well as serving the adaptive function of f a c i l i t a t i n g the egress of polymorphonuclear leukocytes (PMN's) and IgG into the airway lumen i n inflammatory states (20). Non-respi r a t o r y tissues also showing increased i n t e r c e l l u l a r penetration of HRP or a l t e r a t i o n s i n t i g h t junction morphology following experi-mental manipulation include small i n t e s t i n a l epithelium a f t e r s u r g i c a l trauma (107) and treatment with deconjugated b i l e s a l t s (40), proximal tubular epithelium following osmotic shock (65), uterine luminal epithelium a f t e r treatment with ovarian hormones (95), and pancreatic acinar c e l l s maintained i n C a + + - d e f i c i e n t media (44). The precise mechanisms involved i n respiratory e p i t h e l i a l tight junction disruption by c i g a r e t t e smoke remain unclear. A previous study (17) concluded that i t was the tars and nicotine i n the p a r t i c u l a t e phase of c i g a r e t t e smoke, and not the acroleines of the vapor phase which increased t r a n s e p i t h e l i a l probe movement. Conceivably, these agents could have a d i r e c t e f f e c t on the i n t e g r a l membrane proteins of the t i g h t j unctional sealing strands, "opening" the junction. However, using histamine, another agonist known to increase junctional permeability, Marin et a l (83) reported that i t s d i r e c t a p p l i c a t i o n to canine tracheal e p i t h e l i a l preparations mounted - 6 -i n v i t r o produced no s i g n i f i c a n t increase i n j u n c t i o n a l permeability, as assessed by monitoring transmembrane e l e c t r i c a l resistance. Vagal r e f l e x mechanisms may also p a r t i c i p a t e i n the response, for although there i s presently no d i r e c t evidence, an association between agents reported to stimulate i r r i t a n t receptor a c t i v i t y (including c i g a r e t t e smoke) and those enhancing HRP penetration into tracheal epithelium has been noted (20). A l t e r n a t i v e l y , systemic absorption of these agents, r e s u l t i n g i n increased microvascular permeability, increased i n t e r s t i t i a l hydrostatic pressure and junction disruption may occur. Yet Boucher and co-workers (20) observed no systemic hypotension i n guinea pigs challenged with histamine, methacholine or ether. Direct e f f e c t of these agents on l o c a l tracheobronchial microvasculature cannot, however, be ruled out. Recently, Hulbert et a l (63) correlated airway permeability to HRP i n guinea pigs i n the 24-hour period immediately following injury by cigarette smoke with morphologic assessment of airway c e l l population. Of i n t e r e s t was the apparent sharp reduction i n goblet c e l l number, s i g n i f y i n g increased mucin discharge, which occurred synchronously with the period of maximum e p i t h e l i a l permeability. S i g n i f i c a n t l y , Inoue and Hogg (67) previously reported a high degree of disarray i n junctional complexes between c i l i a t e d and discharged goblet c e l l s , a l t e r a t i o n s also observed i n osmotically-stressed epithelium (135). Goblet c e l l discharge and increased mucosal permeability may thus be l i n k e d . Simple mechanical disruption of - 7 -e p i t h e l i a l confluency by the thickened mucous layer i s possible, although d i f f i c u l t to prove, however, "st r e t c h i n g " of goblet c e l l junctions due to physical stress imposed by the mucous-packed goblet c e l l s seems more l i k e l y (67). Recent work from the laboratories of Bentzel (11) and Martinez-Palomo (89) suggests that tight junction permeability i s under cy t o s k e l e t a l control and i s subject to rapid r e v e r s i b l e modulation. The presence of micro-filament or micro-tubule-active agents i n cigarette smoke, goblet c e l l mucous or at some stage i n the inflammatory reaction which follows airway i n s u l t (63) have yet to be investigated. Tumour promoters capable of inducing dramatic increases i n tight junction permeability v i a cyt o s k e l e t a l mechanisms have, however, been i d e n t i f i e d (100). Another important component i n regulation of tight j u n c t i o n a l i n t e g r i t y i s e x t r a c e l l u l a r C a + + concentration. Martinez-Palomo and co-workers (85) have demonstrated that the removal of C a + + and the addition of EGTA to the bathing medium of cultered c e l l mono-layers increased t r a n s e p i t h e l i a l permeability and caused a " s i m p l i -f i c a t i o n " of junctional sealing strand patterns. Yamaguchi (144) was able to show a much more dramatic p r o l i f e r a t i o n of bronchiolar epithelium following i n t r a p e r i t o n e a l administration of high doses of Na^-EDTA to guinea pigs. The relevance of junctional s e n s i t i v i t y to e x t r a c e l l u l a r C a + + can best be appreciated i n l i g h t of Boat and Cheng's data (16), which emphasizes that 30% of C a + + from tracheal secretions - 8 -i s non-dialyzable, i n d i c a t i n g an extremely strong a f f i n i t y of airway mucous for t h i s ion. After periods of airway i n s u l t , then, the much thickened mucous layer might be expected to act as a C a + + sink, drawing ions away from the juxta-junctional region and perhaps a l t e r i n g junctional permeability. Yet, regardless of the precise e f f e c t by which hypersecretion of mucous by goblet c e l l s increases tracheobronchial mucosal perme-a b i l i t y , i t seems u n l i k e l y that a s i m i l a r mechanism would operate i n the intrapulmonary airways as no goblet c e l l discharge e f f e c t i s seen at t h i s l e v e l following smoke exposure i n r a t s (71). Much valuable data on respiratory mucosal permeability has been obtained using HRP; i t s propensity for r e l a t i v e l y easy q u a l i -t a t i v e and quantitative detection make i t an extremely e f f e c t i v e probe molecule. Yet as a tracer f o r bronchial mucosal permeability, HRP does exhibit one major deficiency. From a p r a c t i c a l standpoint, i t s high cost ($175.00/g f o r Sigma Type II) r e s t r i c t s i t s use on a large scale, and has forced most in v e s t i g a t o r s to r e l y on somewhat invasive tracheal i n s t i l l a t i o n s as modes of exposure (19,20,63,109, 110,122). F i n a n c i a l considerations also necessitate the use of small amounts of the tracer with each i n s t i l l a t i o n , thus l i m i t i n g perme-a b i l i t y studies to the more proximal airways. To date, research has concentrated on the trachea (19,20,63,109,110) due to i t s r e l a t i v e l y large s i z e and accessible p o s i t i o n f o r HRP a p p l i c a t i o n . Obviously a tr a c e r of lower cost i s required, as t h i s would enable a more physio-l o g i c a l l y r e a l i s t i c method of exposure (nebulization) r e s u l t i n g i n - 9 -deposition of the test substance at a l l airway le v e l s i n a s i n g l e run. Qu a l i t a t i v e comparison of airway' permeability at d i f f e r e n t l e v e l s under p h y s i o l o g i c a l exposure conditions would be p a r t i c u l a r l y relevant to hypotheses that small airways are more " s e n s i t i v e " to i r r i t a n t s such as tobacco smoke (33,59,98). In addition to the benefits of examination into small airways disease mechanisms, the use of alternate tracers would be highly desirable i n extending the dimensional spectrum of macromolecular probes and i n avoiding the p o t e n t i a l confusion i n HRP data i n t e r p r e -t a t i o n which could a r i s e with the recently demonstrated presence of endogenous peroxidase i n rat (143) and guinea p i g (28) re s p i r a t o r y epithelium. With these factors i n mind, dextran has been selected as an electron microscopic tracer f o r respiratory e p i t h e l i a l permeability a n a l y s i s . B. The Use of Dextran as an Electron Microscopic Tracer 1. General Considerations Dextrans are i n e r t , heterodisperse polysaccharides of b a c t e r i a l o r i g i n c o n s i s t i n g of chains of D-glucopyranose units linked mainly by«K-l:6 g l u c o s i d i c bonds (121). As they are commercially a v a i l a b l e f o r approximately 5(Wgram, dextrans can be nebulized as high concentration solutions and because of t h e i r i n e r t nature, do not induce tissue release of histamine i n experimental animals other - 10 -than i n the rat (5). Moreover, they do not bind appreciably to plasma proteins (1,136) and show no evidence of polymer degradation i n the blood f o r up to 4 hours a f t e r I . V . i n j e c t i o n (5), thereby allowing t h e i r d i r e c t quantitation i n plasma. In addition, as they have been used extensively i n p h y s i o l o g i c a l studies of vascular ( e s p e c i a l l y glomerular) permeability (27,45,72,137) the f i l t r a t i o n behaviour of dextrans i s well-known. That dextrans are commercially a v a i l a b l e i n a wide range of molecular s i z e s , from 4.0 to over 20 nm i n diameter (25), should also prove advantageous as q u a l i t a t i v e determination of i n t e r c e l l u l a r "gap s i z e " i n disrupted tracheobronchial epithelium i s possible. Over the past f i f t e e n years, i t has become apparent that smoking i n a dusty environment (eg, asbestos (13,119), coal (102), gold (125,142), or uranium mines (115a)), produces an adverse s y n e r g i s t i c e f f e c t demonstrated by pulmonary function t e s t s (77) and lung c e l l reactions (115), and increases the r i s k of developing the associated occupational lung disease (eg, bronchiogenic carcinoma, chronic b r o n c h i t i s ) . I n t e r c e l l u l a r gap measurements would help to i d e n t i f y which i n d u s t r i a l dusts, i n combination with c i g a r e t t e smoke, pose the greatest r i s k of increased retention and possible pulmonary disease. Extension of these respiratory e p i t h e l i a l permeability studies to human subjects about to undergo lung lobectomies i s also f e a s i b l e , owing to the inertness of dextran. - 11 -2. Q u a l i t a t i v e Determination of Dextran Light microscopically, the c l a s s i c technique f o r dextran demonstration i s the Mowry-Millican reaction (93), a modified a l c o -h o l i c periodic a c i d - s c h i f f (PAS) reaction designed to avoid the heavy tracer losses which occur when tissue containing highly water-soluble dextran i s treated with aqueous reagents. At the concentrations of dextran reached i n tissues i n two studies (64,123), however, p o s i t i v e Mowry-Millican reactions were not obtained, although u l t r a s t r u c t u r a l analysis disclosed substantial tracer to be present. Unlike HRP, which can be v i s u a l i z e d only through intermediacy of a reaction product (51), dextran molecules can be v i s u a l i z e d i n d i v i d u a l l y and d i r e c t l y by electron microscopy a f t e r aldehyde-OsO^-lead c i t r a t e f i x a t i o n (25,123,124). Although the precise mechanism by which f i x a t i o n and s t a i n i n g operates i s unknown, i t i s presumed that dextran i s retained i n tissues by f i x a t i o n of surroun-ding proteins, and that the lead c i t r a t e (or lead-OsO^ complex) binds s e l e c t i v e l y to the tracer, thus enhancing i t s contrast i n surrounding tissue (25). This technique, developed by Palade and associates (123,124), has been used su c c e s s f u l l y i n the study of c a p i l l a r y ( i n t e s t i n a l (123,124), glomerular (25,29), hamster cheek pouch (64)) and lymphatic (75) permeabilities, and i n i n t r a c e l l u l a r tracer studies of post-exocytosis luminal membrane r e t r i e v a l (58) and synaptic v e s i c l e turnover at neuromuscular junctions (26). In a l l cases, dextrans appear as i r r e g u l a r aggregates of highly e l e c t r o n -- 12 -dense p a r t i c l e s , presumably formed from aggregation of single dextran molecules during f i x a t i o n (25,124). Ainsworth (1) presents an a l t e r -nate method to u l t r a s t r u c t u r a l l y v i s u a l i z e dextran, based upon post-f i x a t i o n of tissue with OsO^ p a r t i a l l y reduced by potassium f e r r o -cyanide, however only one other i n v e s t i g a t o r (56) has u t i l i z e d t h i s technique. 3. Quantitative Determination of Dextran Unlabelled dextrans can be quantit'ated i n blood plasma and tissu e digests following t h e i r p r e c i p i t a t i o n with ethanol by the anthrone reaction (113,118). However, s e n s i t i v i t y with t h i s c o l o r i -metric assay i s only i n the range of 10 (113) to 50 (3) ug/ml. Preliminary c a l c u l a t i o n s based on HRP permeability data (19,20,63) indicate that quantitative methods f o r dextran detection i n guinea pig airway permeability analysis should have a minimal detection l i m i t 14 of below 1 ug/ml. To t h i s end, r a d i o l a b e l l e d dextrans ( C or t r i -t i a t e d ) , used extensively i n glomerular (27) and c a p i l l a r y (45) perme-a b i l i t y studies, would o f f e r the greatest s e n s i t i v i t y . A recent report by Davis and associates (36) describes the use of t r i t i a t e d dextran i n the quantitative assessment of respiratory e p i t h e l i a l permeability i n guinea pigs exposed to ozone. P a r t i c u l a r l y a t t r a c t i v e i s the ease of synthesis of t r i t i a t e d dextran v i a end-group reduction (D.E. Brooks, unpublished data), however some investigators have reported loss of attachment i n vivo of the r a d i o l a b e l l e d portions of the dextran molecules (J.C. Hogg, pers. comm.). Moreover, the use of - 13 -r a d i o l a b e l l e d dextran would necessitate extremely w e l l - c o n t r o l l e d nebulization conditions, and preclude the extension of these studies to humans. 14 Except for C-dextran, spectrophotofluorometric analysis of dextran coupled to f l u o r e s c e i n isothiocyanate (FITC) has proven more s e n s i t i v e than any other quantitative means (117). S e n s i t i v i t y i n the concentration range of 0.05-30 ug/ml of plasma has been reported (117). As the fluorescent l a b e l renders them d i r e c t l y v i s i b l e by fluorescent microscopy, FITC-dextran concentration deter-minations are also possible by micro-fluorometry (114), r e q u i r i n g only a few nanolitres of sample. Fluorescent dextrans, used exten-s i v e l y i n micro-circulatory perfusion studies (4,64,117) and, recently, i n quantitating alveolar e p i t h e l i a l permeability (42), are e a s i l y synthesized by one of two methods (37, R.S. Molday, unpublished data) are highly stable i n vivo, and exhibit physical (eg, f i l t r a t i o n ) and E.M. s t a i n i n g c h a r a c t e r i s t i c s i d e n t i c a l to t h e i r unlabelled counterparts (56,64). Another dextran analog, iron-dextran, has shown promise as a c e l l - s u r f a c e marker i n detecting and quantitating the u l t r a s t r u c t u r a l organization and biochemical properties of membrane glycoproteins (9,91). Based on c h a r a c t e r i s t i c s i z e and shape, these labels have been l o c a l i z e d on c e l l surfaces by transmission electron microscopy (T.E.M.), scanning electron microscopy (S.E.M.) using the secondary electron imaging mode, and, i f possessing a s u f f i c i e n t quantity of heavy metal, can also be detected by x-ray micro-analysis (92). The - 14 -inherent electron density of the i r o n core around which dextran i s coupled allows d i r e c t examination of unstained sections, as has been shown by Muir and Goldberg (94) i n a study of iron-dextran phago-cy t o s i s by macrophages. Quantitation of the tracer through c o l o r i -metric methods (57) or attachment of i r o n radioisotopes i s also f e a s i b l e . C. Scope and Aim of the Present Investigation Previous i n v e s t i g a t i o n s have well characterized, both q u a l i -t a t i v e l y and q u a n t i t a t i v e l y , the tracheal e p i t h e l i a l permeability changes seen a f t e r c i g a r e t t e smoking i n guinea pigs using the cyto-chemical tracer HRP. Yet, i n terms of cheapness, inertness, and probe si z e v e r s a t i l i t y , dextrans o f f e r considerable advantages as macromolecular probes, p a r t i c u l a r l y i n the study of small airways disease and i n airway permeability analysis i n humans. Yet, since the use of dextran as an E.M. tracer has been r e s t r i c t e d to studies of i n t e s t i n a l and glomerular c a p i l l a r y permeabilities, new f i x a t i o n and histochemical techniques are required for i t s l i g h t and electron microscopic demonstration and contrast enhancement i n r e s p i r a t o r y airways and lung parenchyma of guinea pigs. The present i n v e s t i -gation was concerned with optimizing those carbohydrate retention methods f o r the l o c a l i z a t i o n of unlabelled, FITC-labelled, and i r o n -l a b e l l e d dextrans. In addition, the u l t r a s t r u c t u r a l d i s t r i b u t i o n of dextrans along and within the airways of control and smoke-exposed animals was be compared with the published data using HRP as a t r a c e r . MATERIALS AND METHODS A. General Techniques 1. Cigarette Smoke Exposure T h i r t y - f i v e mixed sex Camm-Hartley guinea pigs (average weight 400 g) maintained on a diet ab l i b i t u m of guinea pig chow and water were used i n the following experiments. Those animals u t i l i z e d f o r permeability studies were exposed i n the awake, restrained p o s i t i o n to 200 puffs of e i t h e r a i r (sham-smoked) or smoke from reference c i g a r e t t e s (Canadian Tobacco Manufacturers* Council). F i f t e e n m i l l i t e r puffs of smoke were delivered every 20 seconds v i a syringes and two-way valves into perforated compartments surrounding the guinea pigs' mouths and noses (122). Each c i g a r e t t e was composed of approximately 20 puffs and lasted 7-8 minutes. Animals assigned to the f i x a t i o n experiment received neither smoke nor sham-smoke treatment. 2. Surgery and Experimental Apparatus As previous experiments (62,63) suggested that both vascular perfusion and nebulization of f i x a t i v e are necessary to adequately f i x guinea p i g respiratory epithelium and airway mucous layers f o r u l t r a s t r u c t u r a l study, ca r o t i d cannulation and tracheostomies were performed on most animals. Those animals used f o r immersion f i x a t i o n studies required no cannulation. Guinea pigs were anaesthetized - 16 -i n t r a p e r i t o n e a l l y with Nembutal^ (25 mg/kg) and subcutaneously i n (B) the neck region with Xylocaine (approximately 0.5 ml for 400 g animal). Following a v e n t r a l , mid-line i n c i s i o n i n the neck region, a tracheostomy tube (PE-10) was inserted just caudal to the larynx to avoid the r e f l e x sub-mucosal gland mucous release observed on laryngeal stimulation (47). A cannula (Jelco 22g radiopaque teflon) was then inserted into the r i g h t c a r o t i d with i t s other end connected to the f l u i d output l i n e (5/32 O.D. Tygon tubing). The l a t t e r was also equipped with a slide-clamp to prevent retrograde blood flow a f t e r cannulation, and a two-way valve to bleed out a i r before per-fu s i o n . This tubing was then connected to the two perfusion so l u t i o n l i n e s by means of a two-way valve. The solutions (saline and f i x -a tive) were housed i n two inverted 250 cc I.V. f l a s k s which were also connected by a T - f i t t i n g to a constant pressure apparatus. This apparatus consisted of a Baum Model 300 Manometer connected to a 2 L Erlenmeyer f l a s k which served as a pressure r e s e r v o i r . Each I.V. f l a s k also had i t s own i n d i v i d u a l pressure l i n e clamp. A l l (B) perfusion solutions were f i l t e r e d through M i l l i p o r e 5 u f i l t e r s p r i o r to perfusion to remove p a r t i c u l a t e matter that could obstruct c a p i l l a r i e s (140). Both I.V. flasks were than charged with a pressure of 130 mmHg. 3. Tracer Preparation and I n s t i l l a t i o n Each experimental animal received, v i a the tracheostomy tube, an i n s t i l l a t i o n of e i t h e r 0.3 ml of 20% dextran (Pharmacia Fine - 17 -Chemicals) ( l a b e l l e d or unlabelled) i n 0.9% s a l i n e , or an equal volume of saline alone delivered over 10 minutes. A l l i n s t i l l a t i o n s were performed 1/2-hour a f t e r smoke or sham-smoke exposure, the time period at which tracheal e p i t h e l i a l permeability to HRP has been shown to be maximal (63)- Prior to tracer i n s t i l l a t i o n , the dextran solutions were sonicated for 30 minutes i n a Bransonic 220 bath-type sonicator (123) or heated to 60°C f o r 20 minutes (10) to aid i n d i s s o l u t i o n . FITC- and i r o n - l a b e l l e d dextrans were only dispersed by sonication. Droplets of tracer solutions were then placed on formvar-coated grids , allowed to dry, and negatively stained with 2.5% uranyl acetate (pH 4.3). Iron-dextran solutions d i l u t e d to 0.05% with normal saline were observed by d i r e c t electron microscopy on coated grids, without negative s t a i n i n g (84). The preparations were examined, with minimum beam i n t e n s i t y to avoid melting (84), on a P h i l i p s EM-400 transmission electron microscope to assess p a r t i c l e s i z e and degree of aggregation. At t h i s point, a comment on the s i z e of dextran chosen f o r thi s i n v e s t i g a t i o n seems necessary. Since the tracer was i n t r a -t r a c h e a l l y i n s t i l l e d rather than nebulized, the e f f e c t of molecular siz e on deposition was minimal, e s p e c i a l l y i n the upper airways (22). Respiratory e p i t h e l i a l permeability studies using HRP (molecular weight 40,000) as a tra c e r (19,20,63,109,110,111,122) indic a t e that molecules of i t s dimensions (approximately 5.0 nm diameter) (51) w i l l indeed penetrate the i n t e r c e l l u l a r space of damaged tracheal - 18 -mucosa. As mentioned previously, Boucher (17) has estimated that the equivalent pore radius of normal canine tracheal epithelium i n  v i t r o through which HRP passes p a r a c e l l u l a r l y i s 7.5 nm. Based on exhaustive studies of the renal clearance of dextrans of varying molecular weight i n dogs and humans (6,7,8,54), several workers have concluded that smaller molecules of molecular weight 14,000 to 18,000 (approximately 3.5 nm i n diameter) (123) are quickly excreted i n the urine, having an intravascular h a l f - l i f e of only 15 minutes. Larger dextran molecules are more r e s t r i c t e d i n t h e i r passage across the glomerular f i l t e r , with molecules of 40,000 molecular weight e x h i b i t i n g very low clearance values (54). Cle a r l y , a dextran of approximately the same molecular dimensions as HRP would seem i d e a l . With these factors i n mind, dextran T40, with a molec-u l a r weight of 40,000 and molecular diameter of about 7.0 nm was chosen f o r the i n i t i a l tracheal permeability studies. As a p o s i t i v e c o n t r o l , dextran TlO (molecular weight 10,000, molecular diameter approximately 4.0 nm) was also i n t r a t r a c h e a l l y i n s t i l l e d into some animals to account f o r the possibly d i f f e r e n t f i l t r a t i o n character-i s t i c s of dextrans as opposed to HRP. It must be remembered, how-ever, that for subsequent quantitative studies of tracheobronchial mucosal permeability, dextran TlO i s unsuitable due to i t s extremely short h a l f - l i f e i n the c i r c u l a t i o n . One animal was also given dextran T2000, (molecular weight 2,000,000, molecular diameter 30 nm) for reasons discussed i n the "Results" section. - 19 -With respect to l a b e l l e d dextrans, FITC-dextran and i r o n -dextran were kindly provided by Dr. R.S. Molday, Department of Bio-chemistry, University of B r i t i s h Columbia. The former was a v a i l a b l e only as FITC-dextran-T40, and the l a t t e r only as Fe^O^-dextran-T10. Schroder and co-workers (117) have shown that FITC labels have n e g l i g i b l e e f f e c t s on the f i l t r a t i o n c h a r a c t e r i s t i c s of dextrans. I n i t i a l estimates of the diameter of Fe^O^-dextran-TlO molecules are approximately 9 nm (R.S. Molday, unpublished data). In a l l cases, dextrans were prepared as 20% solutions i n 0.9% s a l i n e , with 0.02$ azide added (118) to prevent b a c t e r i a l growth, and stored at 4°C. 4. Fixation and Tissue Processing Following tracer or saline i n s t i l l a t i o n , a time period of about 15 minutes was allowed before the onset of f i x a t i o n to allow f o r t r a c e r penetration. Fixation was then i n i t i a t e d , f i r s t by nebu-l i z i n g the f i x a t i v e (Hudson #1700 Nebulizer) down the airways of the guinea pigs during which the animals remained a l i v e . This step was accomplished by f i x i n g Tygon tubing to the e x i t port of the nebulizer and d i r e c t i n g the mist down the tracheostomy tubes. Approximately 10 ml of f i x a t i v e was placed inside the nebulizer f o r each animal and a e r o s o l i z a t i o n continued f o r 10 minutes at an outflow rate of 5 li t e r s / m i n u t e . The nebulizer generates 5 m i l l i o n p a r t i c l e s per f t . at t h i s rate, approximately 75$ of which are <5 u i n diameter - 20 -(P.D. Pare, pers. comm.), the p a r t i c l e s i z e most l i k e l y to deposit i n the conducting airways (23). Animals to be immersion-fixed were then k i l l e d with an i n j e c t i o n of saturated KCI administered by cardiac puncture, and the trachea and lungs were ra p i d l y removed f or f i x a t i o n . Tissues from some animals given FITC-dextrans as tracers were immersion-fixed i n 4% formaldehyde i n 70$ buffered ethanol (56) to avoid the quenching of fluorescence seen i n osmium-fixed t i s s u e s (64). In perfusion animals, i n order to i s o l a t e the cardio-thoracic vasculature p r i o r to perfusion, the l e f t c a r o t i d artery was li g a t e d just proximal to o c c i p i t a l artery b i f u r c a t i o n , and the l e f t and right b r a c h i a l a r t e r i e s t i e d o f f externally with s u r g i c a l tubing around each forelimb. A mid-line ventral i n c i s i o n exposed the abdominal ca v i t y , the abdominal aorta was clamped at the l e v e l of the adrenals and the l i v e r was minced to allow free drainage of the perfusate. Clearance of the pulmonary vasculature was then accomplished by perfusion at 130 mmHg of 30 ml of heparinized saline (5,000 U -USP/litre) (Organon) containing 20 ug/kg nitroprusside (Roche), the l a t t e r included to compensate for the vasoconstriction which occurs when aldehyde f i x a t i v e s are perfused (48). At a flow rate of approximately 15 ml/minute at 130 mmHg pressure, 30 ml of saline i s delivered i n about 2 minutes. After clearance of the vasculature, 150 ml of the appropriate f i x a t i v e was perfused at the same pressure. In a l l cases, care was taken to ensure approximately neutral pH (7-3) - 21 -and s l i g h t hypertonicity (360 mOsm) of the f i x a t i v e , as recommended by G i l and Weibel (50). The addition of sucrose (48) to increase perfusate osmolarity was thus necessary i n some cases. Perfusion f i x a t i o n of the entire respiratory tree was con-tinued f o r 15 minutes, a f t e r which the chest was opened and the trachea and lungs removed. Airways and parenchyma were s l i c e d and immersed i n ic e - c o l d f i x a t i v e with the most anterior part of the trachea discarded to avoid catheter-induced mucosal trauma. For l i g h t microscopy, most tissue was trimmed into small blocks and post-fixed i n i c e - c o l d 100$ ethanol f o r 24 hours (93), although some was also post-fixed f o r 2 hours i n i c e - c o l d 2$ g l u t a r -aldehyde i n 0.1 M Na-cacodylate buffer (pH 7.4) and dehydrated i n a graded series of ethanols. Tracheal rings and lung blocks were then transferred d i r e c t l y into 100$ glycolmethacrylate (Polysciences JB-4 embedding k i t ) (62) and embedded i n the p l a s t i c . For routine l i g h t microscopic examination, 3 u sections were cut on a S o r v a l l JB-4 microtome, stained with e i t h e r Toluidine Blue 0 or a l c o h o l i c PAS (93) f o r dextran demonstration (see next s e c t i o n ) , and viewed on a Zeiss Universal l i g h t microscope. For fluorescence microscopy, 10 u sections were cut and examined on the same microscope using an HB0,_- UV l i g h t source and a blue interference f i l t e r set at 455-490A. D i f f e r e n t i a l interference contrast (DIC) microscopy was also done on the same sections using Zeiss/Nomarski d i f f e r e n t i a l interference equipment f o r transmitted l i g h t (2). - 22 -For u l t r a s t r u c t u r a l observation, tissue was diced into small blocks ( l x l mm) and post-fixed i n the appropriate i c e - c o l d f i x a t i v e (see subsequent sections) f o r 2-3 hours. In order to minimize post-mortem tracer d i f f u s i o n , the usually routine buffer and d i s t i l l e d water washes were omitted, and the tissue immediately dehydrated r a p i d l y (34) and embedded i n Spurr r e s i n . En bloc s t a i n i n g with uranyl acetate was also avoided due to the dextran d i f f u s i o n problems and the loss i n o v e r a l l contrast seen i n sections so treated (25). Sections of 60-90 nm thickness ( s i l v e r - g r e y interference colors) were cut using glass knives f i t t e d to a Reichert 0M-4 ultramicrotome, and mounted on either naked 200-mesh copper grids or formvar-coated 75-mesh grids (J.B. EM Se r v i c e s ) . Thin sections were then stained with lead c i t r a t e (133) and examined on a P h i l i p s EM-400 electron microscope. Thick sections (1 u) were stained with Toluidine Blue 0 (88) and examined with the l i g h t microscope. 5. Light Microscopic Demonstration of Dextran -The Mowry-Millican Method According to the o r i g i n a l technique of dextran demonstration (93), p a r a f f i n sections are treated f o r 2 hours with 1% p e r i o d i c acid i n 90% ethanol, a f t e r which the r e s u l t i n g polyaldehydes of dextran are insoluble and may be stained a magenta color with aqueous S c h i f f ' s reagent. However, the PAS reaction i s by no means s p e c i f i c f o r dextran, so f o r p o s i t i v e tracer i d e n t i f i c a t i o n , sections - 23 -stained f o r dextran are always compared to sections exposed to water before periodic acid oxidation, a treatment which s e l e c t i v e l y removes the dextran. Other PAS-positive polysaccharides such as glycogen are retained a f t e r such treatment. In addition, sections of tissue from animals not given dextran are also stained with the Mowry-Millican technique to determine i f there are any n a t u r a l l y occurring dextran- l i k e substances i n the tissue under study. Two modifications to t h i s method were required, the f i r s t due to the nature of the embedding material used —JB-4 p l a s t i c . The highly cross-linked nature of t h i s material (as compared to p a r a f f i n ) necessitated longer washout times to s e l e c t i v e l y remove the dextran. Rather than the few minutes allowed for p a r a f f i n , JB-4 sections of tissue exposed to dextran were routinely floated on water fo r 24 hours before periodic acid oxidation. The second modification was due to the i n i t i a l glutaraldehyde perfusion f i x a t i o n of the a i r -ways and lung parenchyma; non-specific PAS-staining could be encoun-tered due to free aldehyde groups involved i n unipointal f i x a t i o n (35). The technique chosen to block t h i s f a l s e PAS-positivity was to treat the sections with a saturated solution of 2,4-dinitrophenyl-hydrazine (2,4-DNPH) i n 15% acetic acid just p r i o r to periodic acid oxidation (39), which did r e s u l t i n a marked decrease i n the non-s p e c i f i c pink color i n sections i n preliminary studies. - 24 -B. S p e c i f i c Experiments 1. Fixation Technique Adequate preservation of the u l t r a s t r u c t u r a l d e t a i l of guinea pig respiratory mucosa and mucous layers depends, as stated, on a combination of f i x a t i v e nebulization and perfusion. Yet the f i x a t i v e " c o c k t a i l " c o n s i s t i n g of 1.5% formaldehyde, 2.5% glutaraldehyde, 0.66% OsO^ and 2-3 mg% lead c i t r a t e i n 0.1 M Na-cacodylate buffer developed by Palade and associates (123,124) to s t a b i l i z e and s t a i n dextran cannot be perfused for two reasons. F i r s t l y , since f i x i n g by vascular perfusion requires a much larger volume of f i x a t i v e than conventional immersion techniques, the cost per animal f o r the osmium tetroxide alone would be excessive (approximately $16.00). Moreover, i n order to minimize tissue edema or shrinkage caused by hypo- or hypertonic solutions r e s p e c t i v e l y , the osmolarity of the perfusate should be s l i g h t l y higher than that of blood plasma (approximately 330 mOsm) (48). Secondly, therefore, the osmolarity of Palade's c o c k t a i l , 1500 mOsm as measured by the freezing-point depression method (129), i s too high f o r perfusion purposes. With these complications i n mind, a series of experiments was designed to examine the best method of f i x a t i o n of guinea p i g airways and lung parenchyma i n terms of s i m p l i c i t y , low cost, dextran s t a b i l i z a t i o n and preservation of u l t r a s t r u c t u r a l d e t a i l . Eleven animals were divided randomly into two groups: one fixed by immer-sion combined with nebulization, and the other with perfusion-- 25 -nebulization (see Table I ) . Both groups received a 0.3 ml 20% unlabelled dextran T40 i n s t i l l a t e , and neither were exposed to ci g a r e t t e smoke. Immersion f i x a t i o n afforded the use of i c e - c o l d Palade's c o c k t a i l ( i n Na-cacodylate buffer, pH 7.4, 3 hours) (124) as the primary f i x a t i v e , with either 70% ethanol (for dextran p r e c i -p i t a t i o n ) or a modified Karnovsky's (73) f i x a t i v e {1% paraformal-dehyde, 2% glutaraldehyde i n Na-cacodylate buffer, pH 7.3) as the nebulant. Perfusion t r i a l s u t i l i z e d v a r i a t i o n s on Palade's c o c k t a i l as the nebulant and perfusate, with and without formaldehyde (which contributes most to the t o t a l t o n i c i t y of the mixture) and ruthenium red. The l a t t e r has been shown by Luft (81,82) to s t a b i l i z e a v a r i e t y of carbohydrates and i s p a r t i c u l a r l y e f f e c t i v e i n v i t r o i n p r e c i p i t a t i n g dextran and nasal mucous (81). Following primary f i x a t i o n , a l l perfusion-fixed tissues were post-fixed for 3 hours i n two changes of i c e - c o l d Palade's c o c k t a i l (124) and, together with immersion-fixed tissues, processed f o r l i g h t and electron microscopy as previously described. Electron microscopic evaluation of dextran deposition was then performed on at least two separate grids from each block, with at least two blocks examined from each animal. 2. Dextran Tracer Studies Twenty-six guinea pigs were divided randomly into four groups as indicated i n Table I I . Following exposure to 200 puffs (10 c i g a r -ettes) of smoke (or a i r i n sham-smoked groups), the animals were - 26 -anaesthetized, tracheostomized, cannulated and exposed to either 0.3 ml of sonicated 20$ dextran (la b e l l e d or unlabelled as indicated) i n 0.9% saline or simply to normal saline v i a the tracheostomy tube. One animal i n the sham-smoked, non-dextran exposed group received an i n s t i l l a t e of 0.3 ml 10$ polyethylene-glycol (PEG) i n 0.9$ saline to determine the e f f e c t s of a viscous PAS-negative s o l u t i o n on the res p i r a t o r y mucosa. The airways and lung parenchyma were then f i x e d with a combination of f i x a t i v e nebulization and vascular perfusion (treatment 6 i n Table I ) , post-fixed i n Palade's c o c k t a i l , and processed for l i g h t and electron microscopy. Once again, at l e a s t two separate grids from each block and at least two blocks from each animal were examined q u a l i t a t i v e l y with the electron microscope for evidence of dextran penetration into the re s p i r a t o r y epithelium. - 27 -RESULTS A. F i x a t i o n Technique In general, immersion f i x a t i o n (Table I, treatments 1-3) of guinea p i g trachea and lungs proved unacceptable f o r preservation of fine structure: mitochondrial swelling, c r i s t a e disruption, a r t i -f a c t u a l membrane d i l a t a t i o n and nuclear pyknosis of tracheal e p i t h e l i a l c e l l s and Type I and II pneumocytes were evident i n a l l treatments regardless of nebulant composition. Tissues f i x e d by treatment 1 i n which no f i x a t i v e was nebulized showed the most marked c h a r a c t e r i s t i c s of c e l l death and lack of an airway mucous layer, the l a t t e r of which i s important for tracer l o c a l i z a t i o n i n sham-smoked animals. Only one animal was given a nebulant of 70% ethanol with the hope of p r e c i p i t a t i n g the dextran i n vivo, as t h i s treatment proved f a t a l . Treatment 3 resulted i n considerably less u l t r a -s t r u c t u r a l evidence of c e l l death and a remarkably i n t a c t mucous layer, however severe tracheal e p i t h e l i a l c e l l shrinkage i n both animals was noted even at l i g h t microscopic l e v e l . In view of the nebulant's high osmolarity (approximately 1000 mOsm), t h i s r e s u l t i s not unexpected. U l t r a s t r u c t u r a l evaluation of dextran d i s t r i b u t i o n on the airway surface was d i f f i c u l t i n a l l three treatments, due to a r t i f a c t u a l extraction of c e l l u l a r materials and extensive c e l l death. - 28 -Perfusion-fixed airways and lung parenchyma exhibited much more acceptable u l t r a s t r u c t u r e , notably lacking i n ind i c a t i o n s of c e l l i n j u r y . A l l treatments (4-6) preserved the airway mucous layer to a s a t i s f a c t o r y (although inconsistent) degree, thereby allowing the assessment of dextran s t a b i l i z a t i o n . More s p e c i f i c a l l y , tissues fixed by treatment 4 showed considerable tracheal e p i t h e l i a l c e l l shrinkage, once again l i k e l y due to the high osmolarity of the nebulant (1500 mOsm). In addition, t h i n sections of tissue from t h i s treatment demonstrated a general lack of contrast which could not be improved by longer s t a i n i n g times. Dextran was observed to be present i n the airway mucous layer and alveolar spaces of the lung, however neither i t s electron density nor degree of deposition was judged to be s a t i s f a c t o r y as compared to previous studies (25,58, 124). Tissue contrast was considerably improved by the addition of osmium to the perfusate i n treatment 5, and e p i t h e l i a l c e l l shrinkage was minimized by the removal of formaldehyde and lead c i t r a t e from the nebulant. These nebulant modifications permitted an increase i n the glutaraldehyde concentration i n the nebulizer (from 2.5% to 5%) without an appreciable r i s e i n osmolarity, an a l t e r a t i o n which may contribute to dextran s t a b i l i z a t i o n i n the mucous layer (G.E. Palade, pers. comm.). The further addition of ruthenium red to both the nebulant and perfusate (treatment 6) resulted i n even more dextran retention i n the airway mucous layer and lung, and did not a l t e r t r a c e r s t a i n i n g c h a r a c t e r i s t i c s . The unlabelled dextran T40 appeared - 29 -as highly electron-dense i r r e g u l a r aggregates of p a r t i c l e s 10-30 nm i n diameter (see F i g . 11), s i m i l a r i n appearance to that reported by other workers (58,123,124). Despite these improvements i n f i x a t i o n q u a l i t y , i t i s s t i l l f e l t that the u l t r a s t r u c t u r a l contrast of e s p e c i a l l y the tracheo-bronchial epithelium i s somewhat poorer than i n routine f i x a t i o n procedures; the omission of en bloc s t a i n i n g with 2% OsO^ and uranyl acetate i s undoubtedly responsible. Moreover, the carbo-hydrate retention procedures employed both i n f i x a t i o n and processing s t i l l resulted i n considerable v a r i a t i o n i n dextran retention, even between tissue blocks of the same animal. Light microscopic analysis of these same tissues i n ethanol-fixed specimens did not show t h i s degree of v a r i a t i o n . B. Dextran Tracer Studies 1. Unlabelled Dextrans a) Light microscopy The normal l i g h t microscopic architecture of guinea pig trachea and lung as manifested by Toluidine Blue 0 and Mowry-M i l l i c a n s t a i n i n g techniques i s shown i n Figs. 1-3, micrographs of sections from sham-smoked animals receiving the saline i n s t i l l a t e , and i n Fig. 7. The l a t t e r i s a c t u a l l y a Mowry-Millican washout section from a dextran-exposed animal, however a l c o h o l i c PAS-treated tis s u e from non-exposed animals i s morphologically i d e n t i c a l . The - 30 -pseudostratified nature of the c i l i a t e d , columnar tracheal epithelium (Fig. 1) and the PAS-positive mucous layer l i n i n g the a p i c a l surface ( F i g . 2) are c l e a r l y v i s i b l e . The glycoproteins of the goblet c e l l s also exhibit an intense p o s i t i v e PAS reaction (Fig. 2). The sub-mucosa underlying the prominent basement membrane contains the r i c h vascular network that supplies nutrients to the epithelium. More d i s t a l l y , the lung a l v e o l i show the c h a r a c t e r i s t i c l i n i n g Type I and II pneumocytes c l o s e l y appositioned to the a l v e o l a r c a p i l l a r i e s ( F i g . 3 ) . The lung parenchyma i n general shows a very weak PAS r e a c t i v i t y ( F ig. 7). Animals exposed to c i g a r e t t e smoke alone exhibited a very much thickened airway mucous layer (20-50 u i n the trachea, as opposed to the normal value of approximately 10 u) (79), as well as some of the more subtle inflammatory changes c i t e d by Hulbert et a l (63). Animals exposed to the tracers (T10 and T40) showed large, often spherical droplets of highly PAS-positive dextran along airway and a l v e o l a r surfaces i n tissue sections stained by the Mowry-M i l l i c a n method (Figs. 4 and 6 ) . In a l l cases, these deposits were absent i n washout sections (Figs. 5 and 7), thus confirming t h e i r i d e n t i t y as dextran. The heavy tracer deposition seen i n ethanol-fixed specimens contrasted sharply with the almost t o t a l absence of dextran i n tissues f i x e d with glutaraldehyde. No difference was seen i n tracer deposition at the l i g h t microscopic l e v e l between smoked and sham-smoked guinea pigs, and the animal given the PAS-- 31 -negative PEG showed no morphological deviations at any airway l e v e l from s a l i n e - i n s t i l l e d animals, other than a somewhat more d i s c o n t i n -uous mucous layer. Varying degrees of tracheal mucous disruption were also noted i n a l l animals exposed to dextran. Perhaps s u r p r i s i n g , however, was the presence of dextran i n the submucosal blood vessels yet not i n the tracheal mucosa i t s e l f ( F i g . 4 - arrow). These pink p a r t i c u l a t e s , not seen i n washout sections ( Fig. 5) or i n animals r e c e i v i n g the s a l i n e i n s t i l l a t e ( F i g . 2), seemed to p r e f e r e n t i a l l y l o c a l i z e along the abluminal side of these blood vessels a l l around the tracheal r i n g . In an e f f o r t to determine i f t h i s tracer l o c a l i z a t i o n was due to t r a n s e p i t h e l i a l transport of the probe molecule, one animal was given dextran T2000 (molecular weight 2,000,000). Light microscopic examination of the trachea from t h i s animal also showed dextran i n the submucosal blood vessels, q u a l i t a t i v e l y to the same extent as with TlO and T40. Dextran aggregates were also observed i n the a d v e n t i t i a l adipose tissue surrounding the tracheal r i n g i n sections from a l l dextran-treated animals. In the lung, the tracer was seen mainly confined to the alveolar space ( F i g . 6), although occasionally i t s presence i n the vascular space could be detected. b) Electron microscopy Negatively-stained preparations of sonicated or heated dextran showed the d i s s o l u t i o n of the polymer to be incomplete i n - 32 -some preparations. Figures 8 and 9 show two representative electron micrographs, both of the same magnification, of d i l u t e d 20% dextran T40 solutions negatively-stained with uranyl acetate a f t e r i d e n t i c a l sonication conditions. The p a r t i c l e s i n F i g . 8 average approximately 10 nm i n diameter and thus probably represent i n d i v i d u a l dextran molecules, while those i n F i g . 9 are four times t h i s s i z e (approxi-mately 40 nm). Other grids of the same preparations demonstrate s i m i l a r p a r t i c l e s i z e discrepancies as do TlO and l a b e l l e d dextran solutions. Although these estimates were made only semi-quanti-t a t i v e l y without the aid of a carbon grating r e p l i c a to c a l i b r a t e the electron microscope, the differences are pronounced. Dispersion and si z e d i s t r i b u t i o n of dextran p a r t i c l e s seen i n p o s i t i v e l y - s t a i n e d sections of tracheal epithelium were also studied ( F i g . 11). The aggregates of densely-stained dextran T40 ranged from approximately 10-30 nm i n diameter, based on comparison with the glycogen p a r t i c l e s (with a diameter of 30 nm) (108) i n the a p i c a l cytoplasm of tracheal e p i t h e l i a l c e l l s ( F ig. 11 - i n s e t ) . I n c i d e n t a l l y , the abundance of these glycogen deposits i n comparison to those of r o u t i n e l y - f i x e d tissues serves witness to the success of the carbohydrate retention procedures employed i n t h i s study. The s t r i k i n g decrease i n cytoplasmic glycogen i n airway e p i t h e l i a l c e l l s of smoking dogs reported by Frasca and co-workers (43) was not seen i n the smoke-exposed guinea pigs i n t h i s study. As expected, dextran TlO aggregates (seen i n F i g . 12 along the bronchial epithelium) had - 33 -a more f i n e l y granular homogenous appearance than T40 and f o r the most part were well below 10 nm i n diameter. The detailed u l t r a s t r u c t u r e of the guinea p i g tracheal e p i -thelium has been previously reviewed (66,67) and therefore requires l i t t l e discussion. Figure 10 shows a t y p i c a l a p i c a l j u n c t i o n a l complex between c i l i a t e d tracheal e p i t h e l i a l c e l l s from an animal which received no smoke exposure and a saline i n s t i l l a t e . Note the region i n which the two plasma membranes fuse to form the b a r r i e r -l i k e t i g ht junction and immediately below, the intermediate junction and desmosome, both of which p a r t i c i p a t e i n c e l l adhesion. No u l t r a -s t r u c t u r a l differences i n junctional morphology were noted between sham-smoked and smoked animals. Lining the airway epithelium i s the mucous layer (not v i s i b l e i n F i g . 10), generally recognized as co n s i s t i n g of an outer g e l - l i k e epiphase and an inner aqueous hypo-phase ( 7 8 ) . The l a t t e r , characterized by a loose network of osmo-p h i l i c material (80), i s seen i n F i g . 14. In thin sections of tissue from sham-smoked dextran T10 and T40 treated animals, the tracer was seen to be l o c a l i z e d along the airway epithelium within the remnants of the epiphase (Figs. 11 and 12). Some dextran aggregates were also present amongst the c i l i a and adjacent to the m i c r o v i l l i , however no tracer was ever seen i n the i n t e r c e l l u l a r space at any airway l e v e l i n sham-smoked animals. Vesicular uptake of the tracer by the tracheal epithelium was only very r a r e l y seen i n sham-smoked and smoked animals. - 34 -Dextran was also seen along the abluminal walls of the tracheal submucosal blood vessels at the electron microscopic l e v e l . No evidence of tracer uptake, either by phagocytes (eg, PMN's) or by endothelial plasmalemmal v e s i c l e s was observed. In the lung parenchyma of sham-smoked animals, dextran T10 and T40 (Fig. 13) deposits lined the a l v e o l a r space and could occasionally be seen within the alveolar c a p i l l a r i e s . Smoke-exposed animals showed no q u a l i t a t i v e evidence of increased dextran depos-i t i o n i n the microvasculature. No i n d i c a t i o n of tracer transport, e i t h e r p a r a c e l l u l a r l y through junctions between Type I and II pneumo-cytes, or i n t r a c e l l u l a r l y by micropinocytosis could be detected i n smoked or sham-smoked animals. Some phagocytosis by a l v e o l a r macro-phages was seen with a l l dextrans, however no tracer uptake by Type I or II pneumocytes was observed. Of the s i x guinea pigs which were given the unlabelled dextran i n s t i l l a t e 1/2-hour a f t e r smoking, two of four r e c e i v i n g the T40 and one of two receiving the T10 showed extensive penetration of the tracer into the i n t e r c e l l u l a r space of the tracheal epithelium. Figure 14 i l l u s t r a t e s the p a r t i c u l a t e nature of the T40 molecules i n the a p i c a l i n t e r c e l l u l a r space just below the l e v e l of the t i g h t junction. Heavy dextran deposition was also noted between the numerous i n t e r d i g i t a t i o n s surrounding the basal c e l l s of the epithelium (Fig. 15). As reported by other workers (26,124), the one-step f i x a t i v e of Palade (Palade's c o c k t a i l ) (123) imparts enough - 35 -electron density to the tracer so that i t i s well v i s i b l e i n unstained sections, as i s seen i n F i g . 16. No quantitation of probe molecule penetration was done i n t h i s i n v e s t i g a t i o n , however, as with HRP studies (W.C. Hulbert, pers. comm.), varying degrees of tracer permeation were observed i n a given length of tracheal e p i -thelium. The f i n e l y granular appearance of TlO was also i l l u s t r a t e d i n areas where t h i s tracer entered the tracheal e p i t h e l i a l i n t e r -c e l l u l a r space, both a p i c a l l y ( F i g . 17) and basally ( F i g . 18). No q u a l i t a t i v e differences i n tracer penetration between TlO and T40 were noted. In neither case did the intrapulmonary airway or alveolar epitheliums of smoke-exposed animals ex h i b i t , electron microscopically, increased permeabilities to dextran. In addition, although no morphometric measurements were made, there was no obvious u l t r a s t r u c t u r a l evidence of increased numbers of pinocytic v e s i c l e s or increased f r a c t i o n s of these v e s i c l e s opening into the i n t e r -c e l l u l a r space i n smoke-exposed versus sham-smoked guinea pigs. 2. FITC-labelled Dextran Tissue from sham-smoked animals which received the s a l i n e i n s t i l l a t e exhibited very low lev e l s of auto-fluorescence when viewed by fluorescence microscopy. Sections from smoke-exposed animals (Figs. 19 and 20) showed l o c a l i z e d areas of greater fluorescence, p a r t i c u l a r l y i n the airway mucous layers and a p i c a l cytoplasms of tracheal e p i t h e l i a l c e l l s ( F i g . 19 - curved arrow). - 36 -Animals given FITC-dextran T40 presented s p h e r i c a l , highly fluorescent deposits i n the mucous layer of airways and along the alveolar surfaces of the parenchyma (Figs. 21 and 25). Figure 21 i s a fluorescence micrograph of a section of trachea from a smoke-exposed animal and c l e a r l y shows the fluorescent p a r t i c u l a t e s i n the much-thickened mucous layer. Erythrocytes (presumably a r t i f a c t u a l l y introduced by trauma during surgery) and inflammatory c e l l s , best i d e n t i f i e d i n the DIC micrograph of the same s t r i p of epithelium ( F i g . 22, arrowheads), are also contained i n the mucous. The p a r t i c u l a t e nature of the dextran tracer i s also enhanced by t h i s mode of microscopy, as seen i n the same f i g u r e . No p a r t i c l e s (or fluorescent deposits) were observed i n the submucosal blood vessels of the immersion-fixed trachea (Figs. 21 and 22), however some were seen surrounding the tracheal a d v e n t i t i a . In order to most e f f e c t i v e l y evaluate penetration of the fluorescent probe, the negatives of the fluorescence and DIC micro-graphs of a d i f f e r e n t region of trachea from the same animal as i n Figs. 21 and 22 were printed together, y i e l d i n g a composite (Fig. 24). In t h i s and a l l other f i e l d s of airway epithelium of t h i s animal f i x e d f o r l i g h t microscopy, no i n t e r c e l l u l a r fluorescence was detected. F i n a l l y , i n order to unequivocally confirm the i d e n t i t y of the pink p a r t i c u l a t e s presumed to be dextran by routine l i g h t micros-copy, the same 10 u sections seen i n Fi g s . 21 and 22 were stained by the Mowry-Millican method (Fig. 23). By ca r e f u l comparison i t can - 37 -be concluded that the droplets s t a i n i n g pink by the Mowry-Millican technique are i n fact the same droplets that fluoresce under fluorescence microscopy. Sections of lung parenchyma from guinea pigs r e c e i v i n g the FITC-dextran T40 i n s t i l l a t e (both sham-smoked and smoke-exposed animals) demonstrated extensive deposits of often large (up to 10 u i n diameter) fluorescent aggregates along the alveolar surface ( F i g . 25). Figure 28 i s a DIC-fluorescence microscopy composite of a tissue section from the smoke-exposed animal, and shows the trac e r to be r e s t r i c t e d mainly to the alveolar space, accounting f o r the extreme thickness of the section. A possible explanation f o r the large s i z e of the dextran droplets i s that the tracer may be combined with some other substance such as surfactant, the release of which from Type II pneumocytes i s known to be stimulated by dextran (E.Y. Chi, pers. comm.). Cross-sections of FITC-dextran-coated a l v e o l a r macrophages also appear to be present i n F i g . 25 (arrow). No difference i n FITC-dextran deposition between sham-smoked and smoke-exposed animals was observed. Figure 27 i s a l i g h t micrograph of the same lung section stained by the Mowry-Millican technique, again corroborating the i d e n t i t y of the pink p a r t i c u l a t e s as dextran. At the electron microscope l e v e l , as reported by other in v e s t i g a t o r s (56,64), FITC-dextrans d i f f e r n e g l i g i b l y from t h e i r unstained counterparts i n terms of molecular diameter and s t a i n i n g c h a r a c t e r i s t i c s . The single animal given the fluorescent tracer - 38 -that was perfusion-fixed f o r electron microscopy showed no pene-t r a t i o n of the dextran into the i n t e r c e l l u l a r space of the airway epithelium. 3. Iron-Dextran Other than the occasional brown pigmentation seen i n heavy deposits, the l i g h t microscopy of iron-dextran was unremarkable. The Fe^Oji l a D e l a i <^ n°t i n t e r f e r e with Mowry-Millican s t a i n i n g of the tra c e r . Electron microscopic analysis of unstained preparations of iron-dextran-TIO on formvar-coated grids ( F i g . 29, inset) showed the ir o n core of t h i s complex to be 30-50 nm i n diameter. As noted by Marshall and Rutherford (84), there was a tendency f o r these mole-cules to aggregate into large masses when v i s u a l i z e d by negative-s t a i n i n g . In p o s i t i v e l y - s t a i n e d t h i n sections (Fig. 29), Fe^O^-dextran-TlO appeared as regular, s p h e r i c a l , highly electron-dense p a r t i c l e s 40-60 nm i n diameter along the airway epithelium (compare to glycogen i n a p i c a l cytoplasm) and within the alveolar space. Unstained t h i n sections generally showed a larger, more e l e c t r o n -dense tra c e r than with unlabelled dextrans. No evidence of i n t e r -c e l l u l a r penetration of the probe at any airway l e v e l was seen i n smoke-exposed animals. - 39 -TABLE I Experiments f o r Determining the Most E f f e c t i v e F i x a t i o n Method f o r Dextran S t a b i l i z a t i o n Treatment No. Animals Fixation - E.M. Immersion Perfusion Nebulant F i x a t i v e Nebulant Perfusate 1 2 2 1 3 2 H 2 5 2 6 2** Palade's 70$ EtOH Palade's 1$ form + Palade's 2$ glut Palade's 3$ glut 5$ g l u t + 3% g l u t .8$ 0s04 .1$ 0s0*» 5$ gl u t + 3% glut .8$ 0s04 + .1$ 0s04 0.5$ RR*** 0.5$ RR* * A l l tissues subsequently post-fixed Palade's c o c k t a i l . ** These animals also served as sham-smoked dextran-treated controls i n dextran tr a c e r experiments. A l l animals received 0.3 ml 20$ dextran-T40 i n s t i l l a t e . None received c i g a r e t t e smoke. *** RR = Ruthenium Red - 40 -TABLE II Number of Animals Used i n Each Treatment  f o r Dextran (dx) Tracer Studies Sham-smoked Smoked** No dx PEG saline Dx* 1 - Dx-T2000 3 - Dx-T40 2 - Dx-TIO 2 - FITC-DX-T40 4 - Dx-T40 10 2 - Dx-TIO 2 - FITC-DxT40-l LM 1 EM 1 - F e ^ - D x - T l O 2 - Fe O^-Dx-TlO * 0.3 ml 20% dextran i n s t i l l e d . ** Each animal exposed to 200 puffs of cig a r e t t e smoke. - 41 -PLATE I Figure 1: Light micrograph of normal guinea pig tracheal e p i -thelium from a sham-smoked non-tracer-exposed animal. Along the luminal surface of the c i l i a t e d pseudostrat-i f i e d epithelium (E) i s the 10 u thick mucous layer (M). A submucosal mucous gland (MG) i s seen i n trans-verse section opening into the tracheal lumen (L). TBO s t a i n . (LP = lamina propria; S = submucosa). 1 cm. bar = 25 u; magnification = 400X. Figure 2: Light micrograph of a section of normal guinea pig tracheal epithelium stained by the Mowry-Millican technique. The highly PAS-positive glycoproteins of the goblet c e l l s (GC) are e s p e c i a l l y obvious, as i s the _ mucous layer (M) which forms a continuous "blanket" along the tracheal e p i t h e l i a l surface. (BM = basement membrane). 1 cm. bar = 20 u; magnification = 500X. Figure 3: Light micrograph of normal guinea pig lung parenchyma showing a l v e o l a r (AS) and vascular (VS) spaces. Sur-factant-producting type II pneumocytes (arrowhead) can be seen i n the niches of the a l v e o l a r walls. TBO s t a i n . 1 cm. bar = 25 u; magnification = 400X. - 43 -PLATE II Figure 4: Light micrograph of a Mowry-Millican treated section of tracheal epithelium from a dextran-exposed animal which received no smoke. Pink-purple dextran droplets can be seen amongst the c i l i a (arrowhead) along the tracheal e p i t h e l i a l surface, however no tracer penetration into the i n t e r c e l l u l a r space i s evident. Submucosal blood vessels show dextran deposition along t h e i r abluminal walls (arrow). 1 cm. bar = 20 u; magnification = 500X. Figure 5: Light micrograph of a Mowry-Millican treated washout section of tracheal epithelium from the same animal as i n F i g . 4. The absence of pink p a r t i c u l a t e s along the e p i t h e l i a l surface and within the submucosal blood vessels confirms the i d e n t i t y of the p a r t i c u l a t e s i n F i g . 4 as dextran. 1 cm. bar = 13 u; magnification = 790X. Figure 6: Light micrograph of a Mowry-Millican treated section of lung parenchyma from a dextran-exposed animal which received no smoke. PAS-positive dextran droplets l i n e the alveolar space (arrowhead). 1 cm. bar = 20 u; magnification = 500X. Figure 7: Light micrograph of a Mowry-Millican treated washout section of lung parenchyma from the same animal as i n F i g . 6. No dextran p a r t i c l e s can be seen. 1 cm. bar = 20 u; magnification = 500X. - 45 -PLATE III Figure 8: Electron micrograph of a negatively-stained preparation of sonicated unlabelled dextran T40. Average p a r t i c l e diameter i s 10 nm, suggesting that these p a r t i c l e s represent i n d i v i d u a l molecules. 2 cm. bar = 0.22 u; magnification = 92,000X. Figure 9: Electron micrograph of negatively-stained sonicated unlabelled dextran T40 prepared i n an i d e n t i c a l manner to that i n F i g . 8. Average p a r t i c l e diameter i s 40 nm. Incomplete d i s s o l u t i o n of the polymer i s the probable reason f o r these aggregates. 2 cm. bar = 0.22 u; magnification = 92.000X. Figure 10: High power electron micrograph of a j u n c t i o n a l complex between two c i l i a t e d tracheal e p i t h e l i a l c e l l s of a sham-smoked animal which received no dextran. Note the t i g h t junction (TJ), intermediate junction ( I J ) , and desmosome (D) which comprise the a p i c a l complex. (BB = basal body; C = c i l i u m ; L = tracheal lumen; M = mitochondria; MV = m i c r o v i l l i ; R = c i l i a r y r o o t l e t s ) . 2 cm. bar = 0.35 u; magnification = 56,800X. _ 47 -PLATE IV Figure 11: Low power electron micrograph of unlabelled dextran T40 along the tracheal e p i t h e l i a l surface of a sham-smoked animal which received the tracer i n s t i l l a t e . Note the dextran p a r t i c l e s (Dx) i n the remnants of the epiphase and amongst the c i l i a (C), yet not i n the i n t e r c e l l u l a r space (arrowheads). The inset shows glycogen (G) at the same magnification i n the a p i c a l cytoplasm of a tracheal e p i t h e l i a l c e l l . (CC = c i l i a t e d c e l l ; MV = m i c r o v i l l i ) . 1.5 cm. bar = 0.90 u; magnification = 16,500X. Inset: 1 cm. bar = 0.6 u; magnification = 16.500X. Figure 12: Low power electron micrograph of unlabelled dextran T40 (Dx) along the bronchial e p i t h e l i a l surface of a sham-smoked animal which received the tracer i n s t i l l a t e . Note the absence of dextran i n the i n t e r c e l l u l a r space (arrowheads). (C = c i l i a ; M = mitochondria). 1.5 cm. bar = 0.65 u; magnification = 22,900X. Figure 13: Low power electron micrograph of unlabelled dextran T40 along the alveolar epithelium of a sham-smoked animal which received the tracer i n s t i l l a t e . The tracer p a r t i c l e s (arrowheads) l i n e the alveolar space (AS) but are excluded from the vascular space (VS). (Ly = lymphocyte; T2 = type II pneumocyte). 2 cm. bar = 5.12 u; magnification = 3.900X. - 49 -PLATE V Figure 14: High power electron micrograph of tracheal epithelium from a smoke-exposed animal which received the dextran i n s t i l l a t e . The unlabelled dextran T40 has penetrated into the i n t e r c e l l u l a r space (arrowheads) and i s seen at higher magnification below the l e v e l of the t i g h t junction (TJ) i n the i n s e t . The loose network of electron-dense material along the a p i c a l e p i t h e l i a l surface i s the hypophase of the airway mucous layer. (CC = c i l i a t e d c e l l ; L = tracheal lumen; M = mito-chondria; NCC = n o n - c i l i a t e d c e l l ) . 2 cm. bar = 0.68 u; magnification = 29.400X. Inset: 1.5 cm. bar = 0.17 u; magnification = 86,800X. 50 - 51 -PLATE VI Figure 15: Low power electron micrograph of the basal region of tracheal epithelium from a smoke-exposed animal that received the tracer i n s t i l l a t e . The unlabelled dextran T40 (Dx) has penetrated into the basal i n t e r c e l l u l a r space and beyond the basement membrane (BM) into the lamina propria (LP). (E = eosinophil; N = nucleus of basal c e l l ) . 2 cm. bar = 1.7 u; magnification = 12.000X. Figure 16: Low power electron micrograph of an unstained section of tracheal epithelium s i m i l a r to that i n F i g . 15. (BM = basement membrane; Dx = dextran; E = eosinophil; LP = lamina propria; N = n u c l e i of basal c e l l s ) . 2 cm. bar = 1.7 u; magnification = 12,000X. - 53 -PLATE VII Figure 17: High power electron micrograph showing penetration of unlabelled dextran TlO (Dx) into the a p i c a l i n t e r -c e l l u l a r space of tracheal epithelium from a smoke-exposed animal which received the tracer i n s t i l l a t e . (BB = basal bodies; L = tracheal lumen; MV = micro-v i l l i ) . 2 cm: bar = 0.35 u; magnification = 53.200X. Figure 18: Low power electron micrograph of the basal region of tracheal epithelium from a smoke-exposed animal which received the tracer i n s t i l l a t e . Note the penetration of the unlabelled dextran T40 (Dx) into the i n t e r -c e l l u l a r space between the basal c e l l s (BC). (RER = rough endoplasmic reticulum). 2 cm. bar = 1.0 u; magnification = 20,000X. - 55 -PLATE VIII Figure 19: Fluorescence micrograph of tracheal epithelium from a smoke-exposed animal which received no dextran. The epithelium (E) and submucosa (S) exhibit low l e v e l s of auto-fluorescence, however the a p i c a l cytoplasms of tracheal e p i t h e l i a l c e l l s show l o c a l i z e d areas of greater fluorescence (curved arrow), presumably from the absorption of cigarette smoke products. (TL = tracheal lumen). 1.5 cm. bar = 30 u; magnification = • ' 500X. Figure 20: Fluorescence micrograph of lung parenchyma from a smoke-exposed animal which received no dextran. Low l e v e l s of tissue auto-fluorescence are evident. (AS = alveolar space; VS = vascular space). 1.5 cm. bar = 30 u; magnification = 500X. 56 - 57 -PLATE IX Figure 21: Fluorescence micrograph of tracheal epithelium from a smoke-exposed animal which received FITC-dextran. Note the thickened mucous layer (M) containing the highly fluorescent FITC-dextran (Dx), and the absence of tracer penetration into the underlying epithelium (E). (S = submucosa). 1 cm. bar = 20 u; magnification = 500X. Figure 22: DIC micrograph of the same region of the section i n F i g . 21. The p a r t i c u l a t e nature of the FITC-dextran tracer (Dx) i s evident i n the mucous layer (M), as are a r t i f a c t u a l l y - i n t r o d u c e d erythrocytes (arrowheads). (E = epithelium; S = submucosa). 1 cm. bar = 20 u; magnification = 500X. Figure 23: Light micrograph of the same region of the section i n Fig s . 20 and 21 stained with the Mowry-Millican tech-nique. The p a r t i c l e s which fluoresce i n F i g . 21 are the same ones which s t a i n dark pink i n t h i s f i g u r e . (Dx = FITC-dextran; E = epithelium; M = mucous layer; S = submucosa). 1 cm. bar = 20 u; magnification = 500X. - 59 -PLATE X Figure 24: Composite of DIC and fluorescence micrographs of tracheal epithelium from a smoke-exposed animal which received FITC-dextran. Although the fluorescent tracer is seen extensively within the mucous layer, no inter-cellular penetration can be detected. 2 cm. bar = 29 u; magnification = 690X. 60 - 61 -PLATE XI Figure 25: Fluorescence micrograph of lung parenchyma from a smoke-exposed animal which received FITC-dextran. The highly fluorescent tracer droplets (arrowheads) l i n e the alveolar space (AS), but are excluded from the vascular space (VS). Note the FITC-dextran coated macrophage (arrow) i n the alveolar space. 1 cm. bar = 20 u; magnification = 500X. Figure 26: DIC micrograph of the same region of the section i n F i g . 25. The FITC-dextran droplets (arrowheads) appear i n r e l i e f l i n i n g the alveolar space (AS) and coating macrophages (arrow). (VS = vascular space). 1 cm. bar = 20 u; magnification = 500X. Figure 27: Light micrograph of the same region of the section i n Fi g s . 25 and 26 stained with the Mowry-Millican tech-nique. Once again, the fluorescent p a r t i c l e s i n F i g . 25 s t a i n dark pink (arrowheads) when the section i s treated by the Mowry-Millican method as i n t h i s f i g u r e . (AS = alveolar space; VS = vascular space; arrow = FITC-dextran coated macrophage). 1 cm. bar = 20 u; magnification = 500X. - 63 -PLATE XII Figure 28: Composite of DIC and fluorescence micrographs of lung parenchyma from a smoke-exposed animal which received FITC-dextran. The tracer i s seen to be confined to the alveolar space, accounting f o r the extreme thickness (10 u) of the section. 2 cm. bar = 29 u; magnification = 690X. 64 - 65 -PLATE XIII Figure 29: High power electron micrograph of the tracheal epithe-l i a l surface of a smoke-exposed animal which received an Fe^O^-dextran-TlO i n s t i l l a t e . The s i z e of the iron-dextran p a r t i c l e s (FeDx) amongst the c i l i a (C) can be estimated by comparison with the glycogen (G) i n the a p i c a l cytoplasm. Note the absence of the tracer i n the i n t e r c e l l u l a r space (arrowheads). The inset shows unstained iron-dextran p a r t i c l e s from the sonicated i n s t i l l a t e . Average p a r t i c l e diameter i s 40 nm. (TJ = t i g h t j u n ction). 2 cm. bar = 0.18 u; magnifi-cation = 112,400X. Inset: 1.5'cm. bar = 0.14 u; magnification = 134,000X. 66 - 67 -DISCUSSION A. Fi x a t i o n The unique f i x a t i o n requirements of guinea pig airway and lung parenchyma and tissues containing dextran necessitated a detailed study of the appropriate method to preserve re s p i r a t o r y e p i t h e l i a l u l t r a s t r u c t u r e before commencing with the tracer i n v e s t i -gations. The immersion f i x a t i o n procedures, so less expensive and complex than f i x a t i v e perfusion that they were attempted despite previous reports of poor r e s u l t s (62,63), proved inadequate for preservation of the f i n e structure of guinea p i g trachea and lungs even when combined with nebulization of the f i x a t i v e . The vast improvement i n the q u a l i t y of f i x a t i o n by perfusion (treatments 4-6, Table I) was not unexpected, as such techniques allow a rapid and uniform f i x a t i v e penetration p r i o r to anoxic i n j u r i e s to the tissues and prevent d i f f u s i o n and t r a n s l o c a t i o n of c e l l u l a r materials (48). Moreover, the lungs are f i x e d i n f l a t e d by perfusion, and f l u i d s and c e l l u l a r elements present on airway and a l v e o l a r surfaces are often preserved (49,50,80). The presence of a granular, osmophilic mucous layer along the tracheal luminal surface, p a r t i c u l a r l y i n animals i n which t h i s layer was not disturbed by tracer i n s t i l l a t i o n , i s thus not s u r p r i s i n g . Mucous layer thickness, corresponding c l o s e l y to measurements of Luchtel (79) i n rabbit pulmonary airways, ranged from 10-12 u i n the trachea to approximately 2 u i n the bronchioles. - 68 -The addition of ruthenium red to the f i x a t i v e s (treatment 6, Table I) proved e f f e c t i v e i n s t a b i l i z i n g the dextran tracer i n the carbohydrate-rich mucous layer. The precise mechanism of action of t h i s inorganic c a t i o n i c dye i s not known, however i t s c a p a b i l i t y of binding by e l e c t r o s t a t i c forces with anionic compounds (such as acid mucosubstances) seems s i g n i f i c a n t . Although ruthenium red has been used extensively i n " f i x i n g " and s t a i n i n g e x t r a c e l l u l a r carbohydrates (14,81,82,120), only Luchtel (80) has used i t to v i s u a l i z e u l t r a -s t r u c t u r a l l y the glycoproteins of re s p i r a t o r y airway mucous. The present study d i f f e r s i n that p o s t - f i x a t i o n i n ruthenium red-OsO^ was not employed as a carbohydrate s t a i n , as i t was thought that t h i s might i n t e r f e r e with the s e l e c t i v e s t a i n i n g of dextran with Palade's c o c k t a i l . S i g n i f i c a n t l y , almost a l l studies using dextran as a macro-molecular probe were concerned with s t a b i l i z i n g the tracer i n an e a s i l y cross-linked, high protein environment (eg, plasma (1,25,56, 64,123,124), lymph (75), or i n t r a c e l l u l a r ^ (26,29,58)). Indeed, the one inquiry i n which an attempt was made to l o c a l i z e dextran i n a protein-poor environment (aqueous humour of the eye) (131) proved inconclusive due to post-mortem tracer d i f f u s i o n , thus a t t e s t i n g to the high water s o l u b i l i t y of the carbohydrate. Even when u t i l i z i n g the rigorous carbohydrate retention techniques of t h i s study, then, some dextran d i f f u s i o n could be expected while t r y i n g to s t a b i l i z e the tracer e x t r a c e l l u l a r l y with aqueous E.M. f i x a t i v e s i n the protein-poor tracheal mucous layer. Yet i t must also be remembered that - 69 -w i t h mass t r a c e r s such as HRP, d i f f u s i o n o f the t r a c e r (41) and t h e r e a c t i o n p r o d u c t (99) a r e w e l l r e c o g n i z e d d i s a d v a n t a g e s . The v a s c u l a r p e r f u s i o n f i x a t i o n t e c h n i q u e d o e s , however , e x h i b i t drawbacks as b o t h i n c o n s i s t e n t f i x a t i o n (130) and p o o r u l t r a -s t r u c t u r a l t i s s u e c o n t r a s t (49) have been r e p o r t e d . The l a t t e r was p a r t i c u l a r l y common i n t h i s s t u d y , and a l t h o u g h p a r t l y a t t r i b u t e d to the l a c k o f en b l o c t i s s u e s t a i n i n g , was a l s o u n d o u b t e d l y due t o t h e r e l a t i v e l y d i s t a n t l o c a t i o n o f the t r a c h e o b r o n c h i a l e p i t h e l i u m from the v a s c u l a t u r e ( 4 9 ) . O c c a s i o n a l l y , some f r a g m e n t a t i o n o f t h e a l v e o l a r w a l l s was a l s o n o t e d , however , g e n e r a l l y t h e l u n g showed o n l y c l e a r a n c e o f b l o o d e l ements from the m i c r o v a s c u l a t u r e , and t h e r e l a t i v e l y s m a l l a l v e o l a r s p a c e s wh ich t y p i f y l u n g s p e r f u s i o n f i x e d a t a c o n s t a n t p r e s s u r e ( 4 8 ) . The p r e s e r v a t i o n o f the e x t r a c e l l u l a r d u p l e x l i n i n g l a y e r o f a l v e o l a r e p i t h e l i u m was n e v e r c o n s i s t e n t , i n c o n t r a s t w i t h the e l e g a n t r e s u l t s o f W e i b e l and G i l u s i n g a s l i g h t l y d i f f e r e n t p e r f u s i o n t e c h n i q u e ( 5 0 , 1 4 0 ) . B . U n l a b e l l e d D e x t r a n T r a c e r S t u d i e s 1. L i g h t M i c r o s c o p y R e s p i r a t o r y e p i t h e l i u m from g u i n e a p i g s p e r f u s i o n - f i x e d w i t h g l u t a r a l d e h y d e - O s O j j - r u t h e n i u m red c o c k t a i l s and t h e n p o s t - f i x e d i n 100$ e t h a n o l w i t h o u t g r a d e d d e h y d r a t i o n showed the u s u a l m o r p h o -l o g i c a l f e a t u r e s o f t h i s s y s t e m , s u r p r i s i n g l y , w i t h o u t e v i d e n c e o f a r t i f a c t u a l t i s s u e s h r i n k a g e . H i s t o l o g i c s e c t i o n s o f t h e t r a c h e o -b r o n c h i a l mucosa r e v e a l e d the mucous l a y e r t o be c o n t i n u o u s , i n - 70 -accordance with the findings of Luchtel (80) and Sturgess (128). The glycoproteins of goblet c e l l s stained highly PAS-positive as described by Reid and Jones (106), and the lung parenchyma exhibited the anticipated weak PAS-positivity. Animals exposed to c i g a r e t t e smoke showed the thickened tracheal mucous layer caused by secretory c e l l discharge (106) and the resultant apparent decrease i n goblet c e l l number (63,105), although neither was quantified i n t h i s study. The large, s p h e r i c a l , highly PAS-positive deposits seen along the re s p i r a t o r y epithelium of tissue sections from tracer-exposed animals were i d e n t i f i e d as dextran by t h e i r magenta colour (93) and by t h e i r absence both i n sections from non-exposed animals and i n washout sections from exposed animals. Chondrocytic glycogen, how-ever, was not removed from sections exposed to water, thus i l l u -s t r a t i n g the s p e c i f i c i t y of the treatment (93). As i t was suggested that the viscous tr a c e r f l u i d may be causing disruption and beading of the mucous layer and hence the PAS-positive droplets, a PAS-negative, viscous l i q u i d , PEG, was i n s t i l l e d into the airways, and no pink droplets were produced. Comparison of the appearance of dextran i n t h i s study with previous reports i s d i f f i c u l t , as most inve s t i g a t o r s have chosen to r e s t r i c t t h e i r t r a c e r demonstration to electron microscopy. Yet i n a study of the h i s t o l o g i c a l l o c a l i z a t i o n of dextran i n tissues of Korean battle c a s u a l t i e s , Vickery (13*0 did observe the same large magenta-coloured droplets as seen i n lungs of guinea pigs exposed to - 71 -the t r a c e r . That the probe molecule was seen to be deposited i n the d i s t a l lung i n t h i s study i s i n t e r e s t i n g , as analogous studies with HRP show t h i s tracer to be r e s t r i c t e d to the upper airways (20,111) a f t e r the i n s t i l l a t i o n of an equivalent volume. Presumably the d i f f e r e n t chemical composition and physical properties of dextran are responsible. Although some lobules of the lungs were shown to be dextran-free, t h i s non-uniformity of deposition i s to be expected with i n t r a t r a c h e a l i n s t i l l a t i o n techniques (22). Despite i t s apparent i n s e n s i t i v i t y i n detecting small amounts of dextran, both generally i n aldehyde-fixed tissues and i n t e r c e l l u -l a r l y i n smoke-exposed animals, the Mowry-Millican technique i s s t i l l u seful as an i n d i c a t o r of r e l a t i v e dextran deposition between experi-mental animals. It must also be remembered that i n the HRP studies, i n t e r c e l l u l a r penetration of the tracer also could not be v i s u a l i z e d at the l i g h t microscopic l e v e l (J.C. Hogg, pers. comm.). The presence of i n t r a t r a c h e a l l y - i n s t i l l e d dextran i n the submucosal blood vessels of the res p i r a t o r y airways was a serious threat to i t s use as a tracer f o r tracheobronchial mucosal perme-a b i l i t y studies, e s p e c i a l l y i f t h i s deposition occurred following tr a c e r transport through the epithelium, e i t h e r p a r a c e l l u l a r l y or i n t r a c e l l u l a r l y . The observation that a molecule of 2,000,000 molecular weight (dextran T2000) was d i s t r i b u t e d i n these blood vessels i n a q u a l i t a t i v e l y s i m i l a r manner to a molecule of 40,000 molecular weight (dextran T40) seemed to obviate t h i s p o s s i b i l i t y , - 72 -however a more detailed electron microscopic examination was necessary. In no case was there any u l t r a s t r u c t u r a l evidence of either v e s i c u l a r or p a r a c e l l u l a r transport of dextran across the tracheal epithelium of animals showing dextran i n submucosal blood vessels. Furthermore, no tracer was seen i n micropinocytic v e s i c l e s of submucosal blood vessel endothelial c e l l s or as membrane-bound cytoplasmic in c l u s i o n s i n phagocytes. Given that dextrans of molec-u l a r weight 40,000 have been shown to stimulate phagocytosis (24) and to be taken up by endothelial plasmalemmal v e s i c l e s within three minutes a f t e r intravenous i n f u s i o n (124), i t i s therefore u n l i k e l y that the tracer observed i n the submucosal blood vessels a r r i v e d there by physiological means. Thus, the p o s s i b i l i t y that the tracer reached the vasculature by increased transport through s p e c i a l i z e d areas such as bronchus-associated lymphoid tissue (BALT) or through l o c a l i z e d areas of sloughing or increased c e l l turnover (20) seems less l i k e l y . Instead, observations of dextran i n other a r t i f a c t u a l areas i n the present study such as surrounding the tracheal a d v e n t i t i a , would tend to favour post-mortem tracer d i f f u s i o n as the mechanism f o r the dextran deposition along the abluminal walls of the sub-mucosal blood vessels. In support of t h i s concept, various other authors (10,26,64,131) have also reported a r t i f a c t u a l dextran d i f f u s i o n from e x t r a c e l l u l a r tissue compartments. Cryostat sections were cut from frozen tissue from animals given a dextran i n s t i l l a t e - 73 -i n t h i s i n v e s t i g a t i o n i n an a t t empt t o l o c a l i z e the t r a c e r b e f o r e d i f f u s i o n c o u l d o c c u r , however t h e M o w r y - M i l l i c a n t e c h n i q u e c o u l d not be used under such c o n d i t i o n s . N e v e r t h e l e s s , as l o n g as t h e t r a c e r movement i s p o s t - m o r t e m and i s c o n f i n e d to the t i s s u e s p a c e s ( e g , t r a c h e a l l u m e n , v a s c u l a t u r e and a r o u n d the c i r c u m f e r e n c e o f t h e t r a c h e a l r i n g ) , q u a n t i t a t i v e and q u a l i t a t i v e d e t e r m i n a t i o n o f t r a c h e o b r o n c h i a l m u c o s a l p e r m e a b i l i t y would r e m a i n u n a f f e c t e d . 2 . E l e c t r o n M i c r o s c o p y The f u n d a m e n t a l p h y s i c a l p r o p e r t y w h i c h d i c t a t e s the p o t e n t i a l u s e f u l n e s s o f a m a c r o m o l e c u l a r t r a c e r i s m o l e c u l a r d i a m e t e r . In t h i s s t u d y , i f the i n s t i l l e d p o l y m e r s a r e above t h e t h e o r e t i c a l p o r e d i a -m e t e r (15 nm) (17) o f t h e t r a c h e a l e p i t h e l i u m t h r o u g h w h i c h p a r a -c e l l u l a r HRP t r a n s p o r t o c c u r s i n normal a n i m a l s , i t i s l e s s l i k e l y t h a t the d e x t r a n w i l l be a b l e t o p e n e t r a t e c i g a r e t t e smoke-exposed e p i t h e l i u m . Yet n e g a t i v e l y - s t a i n e d p r e p a r a t i o n s o f s o n i c a t e d o r h e a t e d d e x t r a n i n s t i l l a t e s show a wide v a r i a b i l i t y i n the d i s s o c i -a t i o n o f the t r a c e r . F i g u r e 9 shows a g g r e g a t e s o f T40 m o l e c u l e s whose d i a m e t e r s c l e a r l y exceed 15 nm; such i n c o n s i s t e n t d i s s o l u t i o n o f t h e t r a c e r c o u l d w e l l a c c o u n t f o r t h e i n c o n s i s t e n t e l e c t r o n m i c r o -s c o p i c d e m o n s t r a t i o n o f i n c r e a s e d t r a c h e a l m u c o s a l p e r m e a b i l i t y f o l l o w i n g smoke e x p o s u r e . F o r example , t h e s o l u t i o n s n e g a t i v e l y -s t a i n e d i n F i g . 8 ( i n d i v i d u a l m o l e c u l e s ) and F i g . 9 ( l a r g e a g g r e g a t e s ) were i n s t i l l e d i n t o smoke-exposed a n i m a l s t h a t e x h i b i t e d t r a c e r p e n e -t r a t i o n and no t r a c e r p e n e t r a t i o n r e s p e c t i v e l y . The p a r t i c l e s i n - 74 -F i g . 8 a v e r a g e a p p r o x i m a t e l y 10 nm i n d i a m e t e r , and a r e t h u s i n c l o s e agreement w i t h t h e t h e o r e t i c a l m o l e c u l a r d i a m e t e r o f T40 (7-8 nm) based on m o l e c u l a r w e i g h t (52) and d i f f u s i o n c o - e f f i c i e n t s ( 5 3 ) . P r e v i o u s w o r k e r s u s i n g t h e more e f f i c i e n t p r o b e - t y p e s o n i c a t o r s ( 1 0 , 2 5 , 5 8 ) o r a d j u s t a b l e b a t h - t y p e s o n i c a t o r s ( 1 2 3 , 1 2 4 ) e x p e r i e n c e d o n l y s l i g h t v a r i a b i l i t y i n the s i z e o f t r a c e r p a r t i c l e s seen i n n e g a t i v e s t a i n i n g . A more d e t a i l e d s t u d y o f t h e r e l a t i o n s h i p between d i s s o l u t i o n c o n d i t i o n s and the s i z e o f t r a c e r p a r t i c l e s seen i n n e g a t i v e s t a i n i n g needs t o be u n d e r t a k e n t o c l a r i f y t h i s p o i n t . (See a l s o Addendum) P o s i t i v e l y - s t a i n e d d e x t r a n T l O and T40 a g g r e g a t e s i n t r a c h e a l t h i n s e c t i o n s a l s o c o n s i d e r a b l y exceeded t h e i r p r e d i c t e d s i z e based on m o l e c u l a r w e i g h t ( 5 2 ) , y e t c l o s e l y matched p a r t i c l e s i z e s i n o t h e r s t u d i e s ( 2 6 , 5 8 , 1 2 3 ) . T h i s a g g r e g a t i o n a p p a r e n t l y o c c u r s d u r i n g f i x a t i o n ( 2 5 , 1 2 4 ) . F u r t h e r m o r e , t h e i r r e g u l a r o u t l i n e o f t h e d e x t r a n p a r t i c l e s (as compared t o t h e i r r e l a t i v e l y s p h e r i c a l shape i n n e g a t i v e l y - s t a i n e d p r e p a r a t i o n s ) has been a t t r i b u t e d to t h e i r e x t e n d e d s t r u c t u r e ( l o n g c h a i n s w i t h f e w « ? ^ - l : 3 b r a n c h p o i n t s ) , w h i c h r e n d e r s them s e n s i t i v e t o s t r e s s e s i n t h e i r e n v i r o n m e n t d u r i n g t h e f i x a t i o n o f s u r r o u n d i n g p r o t e i n s ( 1 2 3 ) . The e x c l u s i o n o f d e x t r a n f rom the t r a c h e o b r o n c h i a l e p i t h e l i u m o f sham-smoked g u i n e a p i g s c o n f i r m s the f i n d i n g s o f p r e v i o u s i n v e s t i -g a t o r s u s i n g HRP as a t r a c e r ( 1 9 , 6 3 , 1 2 2 ) . D e s p i t e the somet imes p o o r p r e s e r v a t i o n o f the t r a c h e a l mucous l a y e r , d e x t r a n was a lways seen - 75 -l i n i n g the airway luminal surface, whereas i n the absence of mucous, HRP was d i f f i c u l t to demonstrate (W.C. Hulbert, unpublished data). Since dextran i s v i s u a l i z e d d i r e c t l y ( i . e . non-enzymatically) as disc r e t e p a r t i c l e s , i t may prove easier to p o s i t i v e l y i d e n t i f y than the d i f f u s e electron-dense HRP reaction product. Uptake of exogenous p a r t i c l e s by the tracheobronchial e p i -thelium has been a l t e r n a t e l y confirmed and denied by previous i n v e s t i g a t o r s . Richardson (111) demonstrated that HRP could cross guinea pig tracheobronchial epithelium v i a goblet c e l l endocytosis, yet i n a l a t e r study (109) claimed i t was a slow (30-60 minutes) uptake by brush c e l l s which accounted f o r the transport. Ranga et a l (103,104) also showed evidence of goblet c e l l uptake of HRP, however Watson and Brain (139) demonstrated conclusively that i n mouse airway epithelium, i t i s a l l e p i t h e l i a l c e l l s except goblet c e l l s which pinocytose iron oxide p a r t i c l e s . Of the HRP studies, only one (109) included control tissue from guinea pigs unexposed to the tracer, hence the peroxidase a c t i v i t y observed i n tissues from the other investigations could i n fact be endogenous (28,143). Goblet c e l l s have been shown to be p a r t i c u l a r l y peroxidase-positive (28). The experimental evidence from our laboratory (19,20,63,122), however, indicates that only very minimal tracer uptake by the tracheobronchial epithelium occurs, and i n t h i s sense, dextran behaves l i k e HRP. Diffuse tracer uptake by senescent c e l l s has been - 76 -described both i n r e s p i r a t o r y (104, W.C. Hulbert, unpublished data) and i n t e s t i n a l (32,40) e p i t h e l i a l permeability studies using HRP, and was also observed i n the present study with dextran. Whereas i n previous HRP studies of acute cigarette smoke-induced tracheobronchial mucosal permeability changes (19,63), a l l smoke-exposed animals exhibited some degree of e p i t h e l i a l tracer penetration, only three of s i x exposed animals i n the present inves-t i g a t i o n showed unlabelled dextran to be present i n the tracheal i n t e r c e l l u l a r space. As mentioned, incomplete d i s s o l u t i o n of the tra c e r i s a possible cause of t h i s discrepancy, however one cannot rule out that the period of maximum e p i t h e l i a l permeability following smoke exposure i s d i f f e r e n t f o r dextran than that for HRP (30 minutes) (63). Moreover, the 15 minutes allowed i n t h i s study fo r dextran movement through the mucous layer and into the i n t e r -c e l l u l a r space a f t e r i t s i n s t i l l a t i o n may not be optimal, given the d i f f e r e n t chemical composition and physical c h a r a c t e r i s t i c s of dextran as opposed to HRP. Conversely, F i g s . 15 and 16 show dextran extending beyond the basement membrane into the lamina propria (rarely seen with HRP) (20), however whether such examples are those of unusually dramatic permeability increases remains to be seen. Inci d e n t a l l y , as reported by other i n v e s t i g a t o r s (25,124), dextran molecules were never observed crossing the basement membrane i t s e l f . The s i m i l a r carbohydrate nature of tracer and basal lamina i s thought to render s e l e c t i v e s t a i n i n g of dextran d i f f i c u l t i n t h i s l o c a t i o n (25). - 77 -S t a t i s t i c a l considerations also made the v i s u a l i z a t i o n of i n t e r c e l l u l a r dextran penetration more d i f f i c u l t . Screening of E.M. tissue blocks f o r the presence of dextran was problematic, since 1 u sections from these blocks could not be stained by the Mowry-Millican technique. Epoxy resins such as Spurr's are notoriously impermeable to s t a i n i n g solutions, and the one method described i n the l i t e r a t u r e f o r PAS-staining of araldite-embedded thick tissue sections (38) could not be made to work with Spurr sections. With respect to problems such as these, one author has commented that "... the main d i f f i c u l t y i n loc a t i n g ( i n t r a t r a c h e a l l y ) administered p a r t i c l e s i n electron micrographs i s the sheer improbability of f i n d i n g any i n sections taken at random" (127). Of i n t e r e s t are the recent i n t e s t i n a l e p i t h e l i a l permeability studies (40,107) that show HRP to be present i n the hyperpermeable epithelium along the m i c r o v i l l a r brush border and deep within the i n t e r c e l l u l a r space, but not i n the tight j unctional zones themselves. Although not reported i n t h e i r texts, a s i m i l a r picture of HRP pene-t r a t i o n can be seen i n electron micrographs from hyperpermeable guinea pig respiratory epithelium investigations (19,20,122), and F i g . 14 shows the same to be true f o r dextran. The absence of tracer p a r t i c u l a t e s within the tight j unctional zone could be explained by f o c a l a l t e r a t i o n s i n the i n t e g r i t y of t i g h t junctions, allowing the tracer to enter the i n t e r c e l l u l a r space and appear i n sections below unaltered t i g h t junctions (107,122). I t would appear that the T10 - 78 -i n F i g . 17 , however , e x t e n d s up i n t o the most a p i c a l r e g i o n o f t h e i n t e r c e l l u l a r s p a c e . As m e n t i o n e d , no r e l e a s e o f e n d o c y t o t i c v e s i c l e s i n t o the l a t e r a l i n t e r c e l l u l a r space (15) was seen i n s e c t i o n s from smoked o r sham-smoked a n i m a l s . Perhaps most s u r p r i s i n g was the l a c k o f t r a c e r p e n e t r a t i o n i n the i n t r a p u l m o n a r y a i r w a y s o f smoke-exposed a n i m a l s . A p a r t f rom an e a r l y s t u d y by S i m a n i , Inoue and Hogg (122) w h i c h showed c h r o n i c smoke e x p o s u r e (50-300 c i g a r e t t e s ) o f g u i n e a p i g s t o have a more pronounced p e r m e a b i l i t y - a l t e r i n g e f f e c t a t the b r o n c h i o l a r r a t h e r t h a n l a r g e a i r w a y l e v e l , a l l r e s p i r a t o r y e p i t h e l i a l p e r m e a b i l i t y i n v e s t i g a t i o n s have been l i m i t e d t o t h e t r a c h e a . Y e t t h e n a t u r e o f s m a l l a i r w a y s d i s e a s e s u g g e s t s t h a t the d i s t a l a i r w a y s may be somehow more s u s c e p t i b l e t o i r r i t a n t s , and thus may e x p r e s s p o t e n -t i a l l y r e v e r s i b l e i n f l a m m a t o r y changes b e f o r e l a r g e r b r o n c h i o l e s , b r o n c h i , e t c . (33,98). F o r example , t h e somewhat l e s s u n i f o r m a i r w a y f l u i d l i n i n g may be much more permeable to i n h a l e d p a r t i c l e s t h a n the t h i c k e r , c o n t i n u o u s mucous l a y e r o f the more p r o x i m a l a i r w a y s ( 7 7 ) . In a d d i t i o n , S c h n e e b e r g e r (116) has p r e s e n t e d p r e l i m i n a r y e v i d e n c e , based on q u a n t i t a t i v e f r e e z e - f r a c t u r e t e c h n i q u e s , t h a t the i n t r a -pu lmonary a i r w a y e p i t h e l i u m o f the r a t may i n f a c t be more permeable t h a n t h e e x t r a p u l m o n a r y a i r w a y e p i t h e l i u m . Q u a l i t a t i v e l y , d e x t r a n d e p o s i t i o n a p p e a r e d no l e s s e x t e n s i v e i n the s m a l l a i r w a y s t h a n i n the l a r g e i n t h i s s t u d y . D a t a f rom p r e v i o u s i n v e s t i g a t i o n s i n d i c a t e t h a t p a r t i c l e r e t e n t i o n t i m e s i n - 79 -the d i s t a l airways are greater than those i n the upper regions of the tracheobronchial tree (77), however, i n general, clearance mechanisms are much less e f f i c i e n t i n i n t r a t r a c h e a l i n s t i l l a t i o n studies (23) and a f t e r acute cigarette smoke exposure (138). Hence, any permeability changes at the bronchiolar l e v e l could p o t e n t i a l l y have been detected. A smaller "pore s i z e " f o r respiratory epithelium at t h i s l e v e l could be postulated f o r the lack of dextran penetration, however the most l i k e l y p o s s i b i l i t y i s that of less smoke reaching the d i s t a l lung under acute exposure conditions. The extensive f i l t e r i n g systems i n the nasal c a v i t i e s of guinea pigs are well documented (90) and could severely reduce the e f f e c t i v e smoke dose to the smaller airways a f t e r only ten c i g a r e t t e s . Even under chronic smoke exposure conditions i n r a t s , J e f f e r y and Reid (69) observed l i t t l e change i n d i s t a l bronchiolar morphology despite well-characterized e p i t h e l i a l hypertrophy and hyperplasia i n extra-pulmonary bronchi. Oral delivery devices, such as that developed f o r aerosol exposure f o r rabbits (55), bypass the nasal f i l t e r and would allow smoke to be delivered to the lower res p i r a t o r y t r a c t i n large doses. The heavy deposition of dextran i n the lungs of smoke and sham-smoked animals can be l a r g e l y a t t r i b u t e d to the e f f e c t s of gravity (23) and the small amounts of tracer seen i n the a l v e o l a r microvasculature of both groups to post-mortem tracer d i f f u s i o n . The l a t t e r statement i s corroborated by the lack of evidence f o r - 80 -i n t e r c e l l u l a r o r p a r a c e l l u l a r t r a n s p o r t o f d e x t r a n a c r o s s t h e a l v e o l a r e p i t h e l i u m . T h i s i s not s u r p r i s i n g s i n c e , by c o n v e n t i o n a l p o r e a n a l y s i s , t h e p e n e t r a t i o n o f h y d r o p h i l i c m o l e c u l e s o c c u r s t h r o u g h aqueous c h a n n e l s w i t h a d i a m e t e r o f o n l y 1 nm i n n o r m a l a l v e o l a r e p i t h e l i u m ( 4 6 ) . The p h a g o c y t o s i s o f d e x t r a n p a r t i c l e s by a l v e o l a r macrophages was a l s o a n t i c i p a t e d , as t h i s i s the main a l v e o l a r c l e a r a n c e mechan i sm. C o n s i d e r a b l y more p h a g o c y t i c d e x t r a n u p t a k e might have o c c u r r e d had l a r g e r p a r t i c l e s been used i f s u c h u p t a k e by macrophages i s optimum f o r 2 u p a r t i c l e s as some a u t h o r s s u g g e s t ( 7 7 ) . The u p t a k e o f t r a c e r p a r t i c l e s by Type I and I I pneumocytes r e m a i n s c o n t r o v e r s i a l ( 1 2 ) , but was not seen i n t h i s s t u d y . I n c r e a s e s i n a l v e o l a r e p i t h e l i a l p e r m e a b i l i t y i n r e s p o n s e t o c i g a r e t t e smoke have been r e p o r t e d by Jones and c o - w o r k e r s (70) i n 99m humans u s i n g the t r a c e r T c DPTA ( d i e t h y l e n e t r i a m i n e p e n t a -a c e t a t e ) , a ^ - e m i t t i n g r a d i o - i s o t o p e o f v e r y s m a l l m o l e c u l a r we ight (492 d a l t o n s ) . S i m i l a r l y , N i c k e r s o n e t a l (97) p r e s e n t e d morpho-l o g i c a l e v i d e n c e o f i n c r e a s e d v e s i c u l a r t r a n s p o r t o f cy tochrome C ( m o l e c u l a r we ight 1 2 , 5 2 3 , d i a m e t e r 3 nm) a c r o s s Type I pneumocytes o f r a b b i t l u n g s f o l l o w i n g 48 h o u r s o f e x p o s u r e t o 100% oxygen a t one a t m o s p h e r e . I t would seem, t h e n , t h a t i f any a l v e o l a r e p i t h e l i a l p e r m e a b i l i t y changes were t o be d e t e c t e d i n g u i n e a p i g s exposed t o c i g a r e t t e smoke, d e x t r a n m o l e c u l e s o f s m a l l d i a m e t e r ( e g , T l O , m o l e -c u l a r d i a m e t e r a p p r o x i m a t e l y 4 nm) and more c h r o n i c smoke e x p o s u r e c o n d i t i o n s would have t o be u s e d . - 81 -C. FITC-Dextran Tracer Studies As c i g a r e t t e smoke products are known to fluoresce even at very high d i l u t i o n s ( 8 6 , 8 7 ) , a detailed examination of tissues from smoke-exposed, s a l i n e - i n s t i l l e d animals was necessary to e s t a b l i s h baseline fluorescence before analyzing sections from animals given FITC-dextran. As reported by Mellors ( 8 6 ) , fluorescent smoke products were observed i n the mucous layer and a p i c a l cytoplasm of tracheal e p i t h e l i a l c e l l s ( F i g . 1 9 ) , however p o s i t i v e i d e n t i f i -cation of alveolar macrophages said to contain the fluorescent p o l y c y c l i c hydrocarbons of tobacco smoke as seen i n human smokers ( 1 3 2 ) was d i f f i c u l t . Low e f f e c t i v e smoke dose to the d i s t a l lung may once again be responsible. No difference was apparent i n FITC-dextran retention between sham-smoked and smoke-exposed animals, despite the previously mentioned c i l i o s t a t i c e f f e c t of cigarette smoke. Incomplete disso-l u t i o n of the probe molecules may be the reason no tracheal e p i t h e l i a l tracer penetration was seen, ei t h e r at the l i g h t or electron microscopic l e v e l . Many aggregates were seen i n ngatively-stained preparations of FITC-dextran. The great s e n s i t i v i t y of fluorescence microscopy might, however, allow l i g h t microscopic detection of i n t e r c e l l u l a r FITC-dextran i n future studies i f disso-l u t i o n d i f f i c u l t i e s can be overcome. The i n t e n s i t y of fluorescence of the tracer varies with the degree of s u b s t i t u t i o n (number of molecules of FITC per glucose residue) of each l o t ( 3 7 ) . - 82 -The absence of FITC-dextran from the tracheal submucosal blood vessels of the tissues immersion-fixed for fluorescence micro-scopy would be predicted i f such deposition occurs by post-mortem t r a c e r d i f f u s i o n . With immersion techniques, blood i s fixed i n the vessels and therefore would not allow movement of dextran into the vascular space. It would seem, then, that the demonstrated ease of detection of FITC-dextrans, both by fluorescence-DIC and electron microscopy, t h e i r low cost and ease of synthesis, and t h e i r propensity f o r quan-t i t a t i v e determination i n blood and tissue, make these tracers extremely useful f o r tracheobronchial mucosal permeability studies, provided d i s s o l u t i o n inconsistencies can be minimized. The f l u o r e s -cent properties of c i g a r e t t e smoke products also allow crude assess-ment of the extent of respiratory t r a c t smoke exposure. D. Iron-Dextran Tracer Studies Since pharmaceutical preparations of iron-dextran ('Imferon 1 and 'Imposil 1, Fisons Pharmaceuticals Ltd.) are used c l i n i c a l l y f o r the treatment of iron-deficiency anemia (112), much experimental work has centered on the physical c h a r a c t e r i s t i c s and metabolism of these complexes. Electron microscopically, Imferon p a r t i c l e s (mole-cular weight 180,000) (112) consist of small (3.0 nm) (84,94), s p h e r i c a l , electron-dense i r o n cores surrounded by electron-trans-parent sheaths. The l a t t e r , presumed to be dextran, increase the - 83 -o v e r a l l molecular dimensions to approximately 11 x 7 nm (84). Due to i t s r e l a t i v e l y low cost and i n e r t nature, Imferon has been used almost exclu s i v e l y i n iron-dextran tracer studies (9,74,94). Imposil, with i t s larger, r o d - l i k e complexes (84), has been u t i l i z e d only r a r e l y (76). No published i n v e s t i g a t i o n s to date have examined the transmission E.M. c h a r a c t e r i s t i c s of Fe^O^-dextran-TlO. Direct electron microscopy of our c o l l o i d a l iron-dextran complexes revealed that the ir o n core alone of the iron-dextran (30-50 nm i n diameter) greatly exceeds the siz e of HRP and the calculated pore s i z e of tracheal epithelium (15 nm diameter) (17). As shadow casting would probably ind i c a t e the actual molecular s i z e to be somewhat l a r g e r (84), i t can s a f e l y be concluded that i r o n -dextran complexes of t h i s nature are unsuitable to demonstrate permeability changes i n cigarette smoke-exposed guinea pig tracheo-bronchial epithelium. P o s i t i v e l y - s t a i n e d thin sections ( F i g . 29) confirm the probe molecule's large size i n comparison to the t i g h t junction and i n t e r c e l l u l a r space regions. Yet i n terms of ease of detection (both q u a l i t a t i v e l y and qu a n t i t a t i v e l y ) iron-dextrans do o f f e r some advantages over t h e i r unlabelled counterparts, although at the l i g h t microscopic l e v e l FITC-dextrans remain the tracer of choice. With t h i s i n mind, further experimentation with the smaller tracer molecules of Imferon could be b e n e f i c i a l . - 84 -CONCLUSION Horseradish peroxidase has proven i t s e l f extremely useful as an electron-dense tracer i n tracheobronchial mucosal permeability studies. Its high cost, however, l i m i t s i t s use to small volumes and unphysiological exposure methods. Dextrans present an i n e r t , cheap a l t e r n a t i v e tracer a v a i l a b l e i n a wide range of molecular s i z e s , suitable f or nebulization (and therefore p h y s i o l o g i c a l l y r e a l i s t i c d i s t a l airway deposition) and use i n human experimentation. U t i l i z i n g s p e c i a l l y designed perfusion f i x a t i o n and carbo-hydrate retention techniques, unlabelled dextran TlO and T40 can be v i s u a l i z e d at the l i g h t microscopic l e v e l through s t a i n i n g with a modified a l c o h o l i c PAS-reaction, the Mowry-Millican technique. The high water s o l u b i l i t y of dextran was exemplif ied by a moderate degree of post-mortem tracer d i f f u s i o n , however such a r t i f a c t s are also well-recognized with mass tracers such as HRP. For the most part, though, dextran TlO and T40 were l o c a l i z e d along the respiratory epithelium of the airways and lung parenchyma. That no diffe r e n c e i n dextran e p i t h e l i a l penetration was seen at the l i g h t microscopic l e v e l between sham-smoked and smoke-exposed animals serves witness to the i n s e n s i t i v i t y of the Mowry-Millican technique, and necessi-tates u l t r a s t r u c t u r a l examination of the t i s s u e s . S i m i l a r l y , assessment of HRP penetration at the l i g h t microscopic l e v e l i s dubious at best. - 85 -Whereas HRP can only be v i s u a l i z e d electron m i c r o s c o p i c a l l y as the end-product of a cytochemical reaction, dextran molecules can be stained i n d i v i d u a l l y and d i r e c t l y with Palade's f i x a t i v e c o c k t a i l . P o s t - f i x a t i o n of guinea pig trachea and lungs i n t h i s aldehyde-OsO^-lead c i t r a t e mixture presented the unlabelled dextrans as i r r e g u l a r , highly electron-dense aggregates of p a r t i c l e s within the i n c o n s i s t e n t l y preserved airway mucous layer and a l v e o l a r spaces of lung parenchyma. The exclusion of the t r a c e r from the r e s p i r a t o r y epithelium confirms r e s u l t s from HRP studies, however, contrary to the l a t t e r , only one-half of animals exposed to the smoke from ten ciga r e t t e s showed dextran i n t e r c e l l u l a r penetration. Incomplete polymer d i s s o l u t i o n i n some i n s t i l l a t e s , as shown by negative s t a i n i n g i s thought to be the major reason for t h i s discrepancy and requires further improvements. No endocytotic uptake of dextran by the respiratory epithelium was noted, however t h i s process i s s t i l l i n dispute amongst other investigators using HRP. Furthermore, no intrapulmonary airway or a l v e o l a r t r a n s e p i t h e l i a l tracer transport was observed, although low smoke dosage at these l e v e l s could be responsible. Investigation of small airways disease mechanisms w i l l thus require either bypassing the h i g h l y - e f f i c i e n t nasal f i l t e r of guinea pigs, or more chronic smoke exposure. With respect to quantitative analysis of tracheobronchial mucosal permeability changes, only l a b e l l e d dextrans may be measured i n plasma by means as s e n s i t i v e as the immunochemical methods f o r - 86 -HRP. Iron-dextran TlO consists of molecules too large f o r p r a c t i c a l use with smoke-exposed epithelium, although the smaller molecular weight commercial preparations deserve further experimentation. 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V i c k e r y A L : The f a t e o f d e x t r a n i n t i s s u e s o f the a c u t e l y wounded. A s t u d y o f the h i s t o l o g i c l o c a l i z a t i o n o f d e x t r a n i n t i s s u e s o f K o r e a n b a t t l e c a s u a l t i e s . Am J P a t h o l 32: 161-184, 1956. 135. Wade J B , K a r n o v s k y M J : F r a c t u r e f a c e s o f o s m o t i c a l l y - d i s r u p t e d z o n u l a e o c c l u d e n t e s . J C e l l B i o l 62: 344-353, 1974. 136. W a l l e n i u s G : D e x t r a n as a t r a c e r i n g l o m e r u l a r p e r m e a b i l i t y . A c t a C h i r S c a n d S u p p l 211: I-83, 1953. 137. W a l l e n i u s G: R e n a l c l e a r a n c e o f d e x t r a n as a measure o f g l o m e r u l a r p e r m e a b i l i t y . A c t a Soc Med U p p s a l S u p p l 4: 1-61, 1954. 138. Wanner A: C l i n i c a l a s p e c t s o f m u c o c i l i a r y t r a n s p o r t . Am Rev Resp D i s 116: 73-125, 1977. 139. Watson A Y , B r a i n J D : Uptake o f i r o n o x i d e a e r o s o l s by mouse a i r w a y e p i t h e l i u m . Lab I n v e s t 40: 450-456, 1979. - 98 -140 . W e i b e l E R , G i l J : E l e c t r o n m i c r o s c o p i c d e m o n s t r a t i o n o f an e x t r a c e l l u l a r d u p l e x l i n i n g l a y e r o f a l v e o l i . Resp P h y s i o l 4: 42 -57 , 1968. 141 . Widdicombe J G : R e f l e x c o n t r o l o f t r a c h e o b r o n c h i a l smooth musc le i n e x p e r i m e n t a l and human a s t h m a , f rom L i c h t e n s t e i n L M , A u s t e n KF ( e d s ) : Asthma: P h y s i o l o g y , Immunopharmacology and T r e a t m e n t , p p . 225-231 . Academic P r e s s , New Y o r k , 1977 . 142 . W i l e s , F J , F a v r e MH: C h r o n i c o b s t r u c t i v e l u n g d i s e a s e i n g o l d m i n e r s from W a l t o n WH: I n h a l e d P a r t i c l e s I V . Permagon P r e s s , O x f o r d , 1975. 143• W i l l i a m s MC, Benson B J : Immunocytochemica l l o c a l i z a t i o n and i d e n t i f i c a t i o n o f the major s u r f a c t a n t p r o t e i n i n a d u l t r a t l u n g . J H i s t o c h e m Cytochem 29: 291-305 , 1981. 1 4 4 . Yamaguchi H , U s u i H , T u r i k a t a C , T a j i m a T : S t u d i e s o n p r o l i -f e r a t i o n o f b r o n c h i o l a r e p i t h e l i a f o l l o w i n g a d m i n i s t r a t i o n o f h i g h doses o f N a 2 - E D T A . Exp P a t h 18: 477-486, 1980. - 99 -ADDENDUM During review of t h i s thesis by committee members, i t was, pointed out that since dextran molecules are normally completely i n so l u t i o n i n s a l i n e , the Stokes' r a d i i (or ESR) of the polymers i s the accepted method of expressing molecular s i z e . Molecular exclu-sion chromatography i s required to y i e l d ESR values, however a crude assessment of molecular si z e can be obtained by the negative s t a i n i n g method described i n the text. Granted, a d d i t i o n a l forces may come int o play during dehydration of the dextran solution on the coated g r i d , yet these can be minimized by using an extremely d i l u t e tracer solution, as i n t h i s study (approx. 0.5$). Furthermore, based on the data of Simionesu and Palade (123), there e x i s t s a high degree of c o - r e l a t i o n (r = 0.99) between ESR values and molecular diameters calculated from negatively stained specimens of dextrans of varying molecular weight (see Table i A ) . Secondly, a point concerning the d i s s o l u t i o n of dextrans requires c l a r i f i c a t i o n . Although low molecular weight dextrans (T10 to T70) are highly water-soluble, microscopic "pockets" of undissolved dextrans may s t i l l form i f extreme care i s not taken to bring the polymer slowly into s o l u t i o n . Such aggregates may then presumably be dispersed by b o i l i n g or sonicating the s o l u t i o n . Unfortunately, a low e f f i c i e n c y bath-type sonicator was used i n t h i s - 100 -study to enhance d i s s o l u t i o n , r e s u l t i n g i n the persistence of undis-solved dextran aggregates such as those seen i n Figure 9. Previous inve s t i g a t o r s u t i l i z e d the more e f f i c i e n t sonicators of the probe (10,25,58) or adjustable bath (123,124) type, and hence experienced l i t t l e v a r i a b i l i t y i n polymer dimensions as seen with negative s t a i n i n g . I t should be pointed out, however, that once dextran molecules are completely i n so l u t i o n , no aggregation occurs. - 101 -Table IA: Co-relation between Einstein-Stokes' Radius (ESR) values and molecular diameters calculated from negatively stained  specimens of dextrans of varying molecular weight Mol. Wt. of Theoretical Molecular P a r t i c l e Diameter © o Dextran Diameter (A) * from neg. s t a i n i n g (A)+ 250,000 215 197 + 37 75,000 116 124 + 29 40,000 85 82 + 14 20,000 60 48 + 11 r = 0.99 * estimated as double of ESR + mean measurements + standard deviation Data from Simionescu and Palade (123). 

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