Anatomic Pathway of Fluid Leakage in Fluid-OverloadPulmonary Edema in Mice

Kokichi Yoneda, MD

Mice were given an intravenous injection of isotonic saline containing horseradish per-oxidase (HRP) as an ultrastructural marker in an attempt to determine the site of fluidleakage from the vascular space to the air space in the lung. The localization of HRP wasstudied by ultrastructural histochemistry. When injected in a small volume of saline (0.1ml), HRP was confined in the vascular space. When the volume of saline was increased to1.0 ml, the reaction product of HRP was found first in the intercellular junctions of thearterial endothelium and then through the arterial wall. The reaction product was tracedfrom the arterial wall to the peribronchiolar tissue, bronchiolar wall, and the intercellularspace of the bronchiolar epithelium. HRP was seen in direct contact with the air space inthe bronchiole. It is suggested that in fluid-overload pulmonary edema, fluid leaksthrough the arterial wall to the peribronchiolar tissue and then into the intercellularspace of the bronchiolar epithelium. Alveolar edema is probably a result of the backflowof fluid from the bronchiole. (Am J Pathol 1980, 101:7-16)

IN SPITE OF extensive studies, the route of fluid movement inthe lung, especially in pulmonary edema, remains unsettled. Physiologicstudies indicate that the site of fluid leakage appears to be in vessels ex-posed to alveolar pressure.' These vessels include most of the alveolar cap-illary network and also some small arteries and veins.2 Morphologic stud-ies have not succeeded in identifying the exact site of fluid leakage.Schneeberger-Keeley and Karnovsky suggested the intercellular junctionsof the alveolar capillary endothelium as the site of leakage.3 This concepthas been challenged by others.4

Also unsettled is the site of leakage at the respiratory surface. Thereseems to be a general agreement concerning the impermeability of the al-veolar epithelium.56 Staub and colleagues proposed that the leakage oc-curs at the respiratory surface proximal to the alveolus.7 Fishman andPietra, however, reported that the bronchiolar epithelium is imper-meable.8

All the morphologic studies so far have been based on examination ofrandomly sampled specimens. Simionescu and colleagues have shown inthe mouse diaphragm that the venular endothelium is the most permeableand that the pathway consists of transendothelial channels of pinocytotic

From the Veterans Administration Medical Center and the Department of Pathology, Universityof Kentucky College of Medicine, Lexington, Kentucky.

Supported by the Medical Research Service of the Veterans Administration.Accepted for publication March 31, 1980.Address reprint requests to Dr. K. Yoneda, Pathology Service (1 13CD), Veterans Administration

Medical Center, Lexington, KY 40507.

0002-9440/80/1008-0007 7

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vesicles.9 Their studies indicate the significance of the systematic examina-tion of microvasculature. With this in mind, we tried to evaluate the mor-phologic pathway of fluid movement in mice with high intravascular pres-sure induced by an overloading of saline containing HRP as anultrastructural marker.

Materials and MethodsAdult male Swiss-Webster mice weighing 20-25 g were used for the experiment. Mice

were chosen because of the lack of histamine release on intravenous injection of horse-radish peroxidase (HRP) in the dosage used in this experiment.'0 The mice were anesthe-sized with an intraperitoneal injection of sodium pentobarbital (10 mg/100 g bodyweight). The left femoral vein was exposed through an incision in the left inguinal region,and HRP (Sigma Chemical Co., St. Louis, Mo, Type VI) dissolved in isotonic saline wasinjected through this vein with a 30-gauge needle attached to a tuberculin syringe. Thetrachea was exposed by an incision in the cervical region, and the fixative (3% glutaralde-hyde in 0.1 M cacodylate buffer, pH 7.4) was instilled into the lung through the tracheawith a canula. The amounts of saline and HRP injected and the time intervals from injec-tion to fixation were as follows: Two mice each were given injections of 1 mg HRP in 0.1ml saline and killed after 0, 15, and 30 seconds, 1, 2, and 5 minutes, and 24 hours. Twomice each injected with 10 mg HRP in 1.0 ml saline were killed after 15 and 30 secondsand after 1, 2, and 5 minutes. Two mice given injections of 10 mg HRP in 1.0 ml salinewere observed until their death, which occurred 7 and 8 minutes after injection. As con-trols, mice were given injections of saline alone (0.1 or 1.0 ml) and killed after the sametime intervals as the experimental groups. Two mice that received 1.0 ml saline alonewere kept for observation until they expired after 8 and 9 minutes. After 1.5 ml of the fixa-tive was instilled, the chest cavity was opened through a midline incision. Pulmonary ar-teries and veins were clamped. The chest cavity was flooded with the fixative, and thelungs were kept in situ for 10 minutes. Thereafter the lungs and trachea were excised enbloc and immersed in the fixative for 2 hours at 4 C. After fixation, the lung was sliced by aSorvall TC2 Tissue Sectioner (DuPont Instruments Co., Newton, Conn) at a setting of 50,s. The tissue slices thus obtained were washed several times in 0.05 M Tris buffer, pH 7.6,and were preincubated in 0.1% 3,3'-diaminobenzidine tetrahydrochloride in 0.05 M Trisbuffer for 1 hour at 22 C in a Dubinoff metabolic shaker. After 1 hour, 0.1% hydrogen per-oxide was added to the medium and the incubation was continued for 2 hours more." Af-ter incubation, the tissue slices were rinsed 3 times in 0.1 M cacodylate buffer and placedin 1% osmium tetroxide in 0.1 M cacodylate buffer, pH 7.4, for 1 hour at room temper-ature. They were then dehydrated through graded ethanols and embedded in epoxy resin.Tissue sections, 1.0 ,u thick, were slightly stained with alkaline toluidine blue. Thin sec-tions were cut with an LKB ultratome III and examined with a Philips 300 electron micro-scope with or without lead citrate staining.The identification of the segments of pulmonary microvasculature was according to the

method of Rhodin. 2 When we combined light and electron microscopy, small arteries andveins could be easily identified. Our examination was limited essentially to small arteries,capillaries, and veins. Although they may be of great importance, arterioles and venuleswere not examined as such except when they were encountered by chance because of dif-ficulty in positive identification in a given section.

Results

Among the control animals, the mice that received 0.1 ml saline failedto show any light- or electron-microscopic changes in the lung. The mice

Vol. 101, No. 1 FLUID-OVERLOAD PULMONARY EDEMA IN MICE 9October 1 980

that received 1.0 ml saline showed progressive widening of the peri-bronchiolar tissue after 2 minutes. At 5 minutes after injection, some ofthe alveoli were filled with fluid. On electron microscopic examination,the first change noticed was a prominence of pinocytotic vesicles in thearterial endothelium. This was followed by separation of smooth musclecells and collagen fibers in the arterial wall and peribronchiolar con-nective tissue.Among the experimental animals, the mice that received 1 mg HRLP in

0.1 ml saline showed the reaction product demonstrated as an electron-dense granular precipitate in the vascular lumen as early as 30 seconds af-ter injection. The precipitates of HRP were also seen in the pinocytoticvesicles of the endothelial cells throughout the microvasculature (Figures1 and 2). Throughout the experiment, HRP, when administered in thesmaller dosage, could not be seen outside the vascular lumen. In the micethat received 10 mg HRP in 1.0 ml saline, the small arteries showed thereaction product outside the endothelium at 2 minutes after injection. Inthe endothelium of the small artery, two kinds of transendothelial chan-nels were recognized labeled with HRP: one was the intercellular junctionof the endothelial cells stained with HRP without interruption (Figure 3);the other was a chain of pinocytotic vesicles filled with the reaction prod-uct and traversing the endothelium (Figure 4). Although no quantitativeevaluation was attempted concerning the relative density of these twostructures, connections by intercellular junctions appeared to be more nu-merous in a given artery. There was a pool of HRP in the subendothelialspace where either of these two channels connected to the internal elasticlamina (Figures 3 and 4). The reaction product was recognized as a linedelineating the internal elastic lamina, but the lamina itself did not showany HRP staining. The intima and media were connected by the reactionproduct at the occasional gaps of the elastic lamina where the cytoplasmof the smooth muscle cell protruded into the intima and connected di-rectly with the endothelial cell (myoendothelial junction 12). In the mediaand adventitia of small arteries, the precipitate was seen between thesmooth muscle cells and collagen fibers. The smooth muscle cells showednumerous pinocytotic vesicles filled with HRP on the cell surface. Capil-lary and venous endothelium showed the reaction product in luminalpinocytotic vesicles and in the luminal portion of the intercellular junc-tions (Figure 5). At 2 minutes after injection, the epiluminal portion of theintercellular junction was free of HRP, as were the alveolar basementmembranes and media of small veins.At 5 minutes after injection, the reaction product was seen in the peri-

bronchiolar tissue surrounding small arteries and bronchioles (Figure 6).

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Nearby alveolar basement membrane also showed the precipitate. In thebronchiolar wall, the reaction product formed a continous line, separatingeach epithelial cell. This line of HRP reached the luminal surface of thebronchiole without interruption (Figure 7). At the alveolar level the reac-tion product was seen in the pinocytotic vesicles and intercellular junc-tions of the endothelial cells and on the basement membrane, which nonecould be seen in the intercellular junction of the alveolar epithelial cell. Insmall veins HRP was seen in the adventitia and media separating smoothmuscle cells and collagen fibers. In intima pinocytotic vesicles showed thereaction product transendothelially. Occasionally, the intercellular junc-tions showed uninterrupted staining.

Discussion

In the present study HRP did not penetrate through the endotheliumwhen given in a small volume of saline (0.1 ml). When given in a large vol-ume (1.0 ml), however, HRP rapidly penetrated through the endothelium.HRP was traced through the arterial endothelium at the intercellularjunctions, through the elastic lamina at the myoendothelial junctions, andthrough the media at the intercellular space of smooth muscle cells. Onthe respiratory surface, HRP tracing was seen in the intercellular space ofthe bronchiolar epithelium.Two kinds of transendothelial channels were recognized: intercellular

junctions and the chains of pinocytotic vesicles. Although no quantitativeevaluation was performed, chains of pinocytotic vesicles appeared less nu-merous than intercellular junctions. When given in a small volume of sa-line, HRP stained only the luminal portion of the intercellular junctions.When given in a large volume, the transient expansion of the intra-vascular fluid volume itself, or a presumed high intravascular pressure"stretched" the intercellular junctions. This phenomenon has been de-scribed not only in the lung3"13 but also in the cerebral circulation."4 Thepoint of interest in this study is the microvascular segment, where thisstretching of the intercellular junction occurs, namely, small arteries. Pre-vious studies reported this phenomenon in the alveolar capillary endothe-lium.3"13 The difference may be due to the volume of saline employed orthe animal species. In the present study, the intercellular junctions of thearterial endothelium were stained first with HRP, and those of the capil-lary endothelium followed later. Another explanation for this discrepancymay be a possible backflow of HRP along the basement membrane, as sug-gested by Wissig and Williams.4 If the stretching of the intercellular junc-tion is due to the contraction of the endothelial cells, the arterial inter-cellular junctions appear to be more likely to stretch than those of the

Vol. 101, No. 1 FLUID-OVERLOAD PULMONARY EDEMA IN MICE 11October 1 980

capillary endothelium, because of the abundance of the myofibrils in thearterial endothelial cells."5 Further studies are required to clarify thispoint.

After passing through the endothelium, HRP appeared to pool at thepoint where the endothelial channels joined the internal elastic lamina.This pool may correspond to the subendothelial blebs described by othersin various forms of pulmonary edema."6-9 This probably indicates the rel-atively less permeable nature of elastic lamina. We found traces of HRPonly at the gaps of elastic lamina and none within the lamina itself. Themain pathway through the elastic lamina appears at the myoendothelialjunctions. Once through the elastic lamina, HRP staining was traced inthe intercellular space of the smooth muscle cells.

Concerning the fluid pathway on the air surface, it has previously beenshown that the alveolar epithelium is impermeable.56 Our findings areconsistent with these studies. We have shown, however, the presence ofHRP in the intercellular space of the bronchiolar epithelium in directcontact with the air surface. No HRP precipitate could be seen in thebronchiolar lumen. This is probably due to washing out during fixation be-cause of the tracheobronchial instillation of the fixative. Therefore, we in-terpret our finding as indicative of fluid leakage at the bronchiolar sur-face. Fishman and Pietra, on the other hand, reported that in dogs, thebronchiolar epithelium is impermeable to fluid.8 This difference may bedue to the difference of animal species or experimental conditions.

Morphologic studies of pulmonary vascular permeability have been fo-cused on the alveolar capillary, while other areas of the microvasculaturehave been neglected. In systemic circulation, Simionescu and colleagueshave shown that the venule is the most permeable in the micro-circulation.9 Giacomelli and colleagues showed that small arteries are thesite of leakage in the cerebral circulation in hypertensive animals.'4 In thelung, Pietra and colleagues reported a leaky condition of bronchial veinsin endotoxin shock.20 All of these studies employed different animals, dif-ferent organs, and different experimental conditions and present an ap-parent confusion concerning the site of fluid leakage in the micro-circulation. Close evaluation of these studies, however, demonstrate acertain pattern. The study by Pietra and colleagues deals with a vascularreaction mediated by a humoral factor of inflammation 20 and consists inpart of the inflammatory reaction of the vasculature.2" The study of Sim-ionescu and colleagues deals with the macromolecule transport in a nor-motensive steady state.9 The present study, as well as the one by Giaco-melli and colleagues, deals with a high intravascular pressure state.Although the arterial wall may appear as an unlikely site of fluid leakage,

12 YONEDA American Journalof Pathology

the transmural leakage of fluid through small pulmonary arteries was sug-gested by Severinghaus in a high-altitude pulmonary edema.22 It is tempt-ing to speculate that the site of fluid leakage is in small arteries in hemo-dynamic pulmonary edema and that it is in small veins and venules inhigh-permeability pulmonary edema. The difference in macromoleculecontents in these two conditions suggests different pathways of fluid leak-age. Detailed studies of the intercellular junctions in various segments ofthe pulmonary microvasculature will clarify these problems.','

Based on the findings in this study, we suggest that in fluid-overloadpulmonary edema in mice, the arterial endothelium is the site of fluidleakage from the vascular space and that the bronchiolar epithelium is thesite of leakage at the air surface. The alveolar flooding is probably a resultof the fluid backflow from the bronchiole into the alveoli, as suggested byStaub and colleagues.7

References

1. Woolverton WC, Brigham KL, Staub NC: Effect of positive pressure breathing onlung lymph flow and water content in sheep. Circ Res 1978, 42:550-557

2. Iliff LD: Extra-alveolar vessels and edema development in excised dog lungs. CircRes 1971, 28:524-532

3. Schneeberger-Keeley EE, Karnovsky MJ: The ultrastructural basis of alveolar-cap-illary membrane permeability to peroxidase used as a tracer. J Cell Biol 1968,37:781-793

4. Wissig SL, Williams MC: Permeability of muscle capillaries to microperoxidase. JCell Biol 1978, 76:341-359

5. Taylor AE, Gaar KA Jr: Estimation of equivalent pore radii of pulmonary capillaryand alveolar membranes. Am J Physiol 1970, 218:1133-1140

6. Schneeberger EE: Barrier function of intercellular junctions in adult and fetallungs, Pulmonary Edema. Edited by AP Fishman, AM Renkin. Bethesda, AmericanPhysiological Society, 1979, pp 21-37

7. Staub NC, Gee M, Vreim C: Mechanism of alveolar flooding in acute pulmonaryedema, Lung Liquids. Edited by R Porter, M O'Conner. Amsterdam, Elsevier Pub-lishing Co., 1976, pp 255-272

8. Fishman AP, Pietra GG: Hemodynamic pulmonary edema,6 pp 79-969. Simionescu N, Simionescu M, Palade GE: Structural basis of permeability in se-

quential segments of the microvasculature of the diaphragm: II. Pathways followedby microperoxidase across the endothelium. Microvasc Res 1978, 15:17-36

10. Cotran RS, Karnovsky MJ: Vascular leakage induced by horseradish peroxidase inthe rat. Pro Soc Exp Biol Med 1967, 126:557-561

11. Graham RC Jr, Karnovsky MJ: The early stages of absorption of injected horse-radish peroxidase in the proximal tubules of mouse kidney: Ultrastructural cyto-chemistry by a new technique. J Histochem Cytochem 1966, 14:291-302

12. Rhodin JA: Microscopic anatomy of the pulmonary vascular bed in the cat lung.Microvasc Res 1978, 15:169-193

13. Pietra GG, Szidon JP, Leventhal MM, Fishman AP: Hemoglobin as a tracer in he-modynamic pulmonary edema. Science 1969, 166:1643-1646

14. Giacomelli F, Wiener J, Spiro D: The cellular pathology of experimental hyper-

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tension: V. Increased permeability of cerebral arterial vessels. Am J Pathol 1970,59:133-159

15. Kuhn C III: The cells of the lung and their organelles, Biochemical Basis of Pulmo-nary Function. Edited by RG Crystal. New York, Dekker, 1976, pp 3-48

16. Cunningham AL, Hurley JV: Alpha-naphthyl-thiourea-induced pulmonary oedemain the rat: A trophical and electron-microscope study. J Pathol 1972, 106:25-36

17. Hatakeyama S, Shigei T: Comparative study of the fine structural changes of alveo-lar wall in adrenaline and ANTU-pulmonary edema of the rat. Jpn J Pharmacol1968, 18:279-280

18. Harrison LH, Beller JJ, Hinshaw LB, Coalson JJ, Greenfield LJ: Effects of endo-toxin on pulmonary capillary permeability, ultrastructure and surfactant. Surg Gyne-col Obstet 1969, 129:723-733

19. Finegold MJ: Interstitial pulmonary edema: An electron microscopic study of thepathology of staphylococcal enterotoxemia in rhesus monkeys. Lab Invest 1967,16:912-924

20. Pietra GG, Szidon JP, Carpenter HA, Fishman AP: Bronchial venular leakage dur-ing endotoxin shock. Am J Pathol 1974, 77:387-406

21. Ryan GB, Majno G: Acute inflammation: A review. Am J Pathol 1977,86:183-27622. Severinghaus JW: Transarterial leakage: A possible mechanism of high altitude

pulmonary oedema, High Altitude Physiology: Cardiac and Respiratory Aspects. Ed-ited by R Porter, J Knight. Edinburgh, Churchill Livingston, 1971, pp 61-77

23. Schneeberger EE, Karnovsky MJ: Substructure of intercellular junctions in freeze-fractured alveolar-capillary membranes of mouse lung. Circ Res 1976, 38:404-411

24. Inoue S, Michel RP, Hogg JC: Zonulae occludentes in alveolar epithelium and cap-illary endothelium of dog lungs studied with the freeze-fracture technique. J Ultra-struct Res 1976, 56:215-225

AcknowledgmentThe author thanks Ms. Mary G. Birk for excellent technical assistance.

Figure 1-Alveolar capillary in a mouse given an injection of 1.0 mg HRP. HRP is confined in the vascu-lar space. (x7000) Figure 2-Small artery in the same mouse as in Figure 1. HRP is confined inthe luminal portion of the intercellular junction (arrow). (x1 2,000)

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Figure 3-Arterial endothelium in a mouse given an injection of 10 mg HRP at 2 minutes afterinjection. HRP completely stains the intercellular junction (arrow). (x30,000) Figure 4-Same as Figure 4, showing a chain of pinocytotic vesicles stained with HRP (arrow).(x 32,000)

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Figure 5-Venular endothelium in a mouse given an injection of 10 mg HRP at 2 minutes afterinjection. HRP is confined in the lumen. (x 10,000) Figure 6-Peribronchiolar tissue in amouse given an injection of 10 mg HRP at 5 minutes after injection. Notice HRP staining ofarterial and bronchiolar walls. A = artery; BL = bronchiolar lumen. (x750) Figure 7-Ahigher magnification of Figure 6. HRP directly reaches the bronchiolar lumen (arrow).(x8000)