Tobacco Documents Online

THE COUNCIL FOR TOBACCO RESEARCH-U.S.A., INC. Dec. 10, 1984 Me.crorandum Zb: Dr. S.C. Scamiers and Staff Frcun: D.H. Ford Re: Site visit with Drs. N.C. Staub and R. Conhaim, University of California, Cardiovascular Research Institute, Nov. 14,,1984. Grant No. 150,5 R1 entitled "Alveolar-airway barrier permiability to liquid and macromnlecules in dog and sheep lung." Goal: To understand how liquid accumulates in the interstitial space around airways and blood vessels leading to or from alveoli to eventually flood the alveolar spaces and produce pulmonary edema. These investigators are attempting to find the site where fluid leaks from the interstititun into the airways. They are errploying dog and sheep lungs and 'backfilling' the airways with fluid con- taining evans Blue tagged albumen and observing where it leaks back into the interstial spaces...a sort of reverse flooding process. They hope that this model reflects what actually occurs in the genesis of pulmonary edema in life. 4hether or not such a'reversefilling' process will truely define the sites where edema fluid leaks from the interstitium into the airways remains to be determined. Questions being considered: 1. M-iat is the storage volume of fluid which may accumalate in the inter- stitial space before it 'floods' over into the airways? How can one measure it? 2. Mat causes pulmonary edema to occur following head injury? Are there neural pathways involved. 3. Miat is the site of leakage from the interstitium into the airways (at the alveoli or somewheres along the bronchiolar tubes)? 4. How permiable is the leakage site and how large a molecule can pass through the pores where leakage occurs? Some answers to the above questions are beginning to accumulate as indicated by the following data: Data accumulated-presented by Dr. Conhaim, since Dr. Staub was not able to present at the site visit: Their major progress, as indicated in the recent progress report has been to develop their mcdel of perivascular-peribronchiolar fluid cuff formation. The mxiel provides the basis for the projects on liquid pressure and volum° measurement, pore size and site of fluid leakage. Initially it appears that the volume of fluid accumulated in the dog lung is al.nnst twice that occuring in sheep. Fluid volume is determined by point counting on sections wherein the presence of the fluid in the interstitial space is indicated

2 by the presence of Evans Blue tagged albumen which leaked into these spaces with the water (Fig. 1). (Note that these 'cuff' interstitial spaces were filled in reverse following airway filling with the water/albucten/dye mixture). Further, the longer the time of exposure, the greater was the volume of fluid in the cuff space (Fig.2). Also note that in dogs more fluid accumulated around the small than the large vessels (Table 1). With sheep, the smaller vessels also tended to have larger cuffs, but there were irore large vessels with cuffs (100%) than smaller vessels (22$~ (Table 2). Studies on pore size for leakage suggest that there are some holes with a radius as large as 15 nm which will permit liposomes to leak through from the airways into the interstitium as well as pores with radii as small as 3.5 nm, which still permit albumin to leak through. Water, with a Mlecular radius of 2 nm will of course pass through both. Their calculations have lead them to assume that there may be some pores with radi as large as 75 nm. They hope to resolve the problem of determining the sizes of the various pores through which leakage can occur by using latex particles of various size. These particles will be coated with the albumin tagged with Evans Blue. The site of fluid leakage does not appear to be in the alveolar sacs since ferritin particles (5.5 nm diameter) were shown by IIM studies to not be able to pass from the alveolar space into the interstitium. However, since such ferritin particles were observed to penetrate the interstitium surrounding the bronchiolar tubes, it would appear that at least some leakage occurs along this segment of the airway. Further, some ferritin particles were observed within the intracellula2 clefts between the epithelial cells, suggesting that the site of leakage is between cells. Comme.nt: Drs. Staub and Conhaim appear to have made some progress on a project in which Dr. Conhaim appears to be the primary investigator. However, they have a long way to go before they begin to obtain the answers they seek. Further, I am not sure that their m)del wherein the interstitial spaces are filled with fluid in a manner which is the reverse of the way it would normally occur will provide answers relevant to what occurs in vivo. The study is possibly worth continued support during the R2 year. However, if considerable progress has not occurred by the end of the third year, I would doubt if continued support would permit conclusive results to be obtained. DF/ff D. FORD

MEASUREMENT OF INTERSTITIAL LIQUID C VOLUME BY POINT COUNTING 0 0 Airway Vessel Interstitial Cuff ( + ) Counting Point Interstitial Cuff Volume 4 ~1 Lung Volume 3 2 1+ p 1 0 1 0 r - rT+ r - i i i i + ~ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 No. of Transparencies Counted Ft~ I.

C C CUFF AREA VESSEL AREA Staub, Norman C. CTR Grant # 1595 EFFECT OF INFLATION T1ME ON PERIVASCULAR CUFF SIZE 4 T T 3 2 I 1 1 <0.5 0.5 - 1.0 0 VESSEL DIAMETERS, MM Effect of time on size of perivascular fluid cuffs in liquid inflated dog lungs. Size is expressed as cuff-to-vessel ratio, which is the size of the fluid cuff divided by the size of the vessel it surrounds. Data are shown for vessels of three size ranges, corresponding to the range of diameters used in modeling cuff formation. Inflation times ranged from 1 to 300 min in 8 lungs. One hundred vessels were measured in each lung. Bars: + 1 s.d. i.

PER i URSCULflR f LUt D CUFF PDPULflT10N I N LIQUID INFLflTED DUG LUNG LDBES Vessel Diam, (mm) AVERAGE FRACTION OF CUFF-TO-VESSEL VESSELS WITH RATIO * CUFFS PA'st PV's* PA"s PV's 1 min Inflation (1)'" < 0.5 0.94 (2)9 0.91 ± 0.1 (3) 0.30 0.08 0.5-1.0 0.36 ± 0.1 (3) 0.56 ± 0.4 (14) 0.75 0.75 > 1.0 0.44 ± 0.2 (5) 0.33 ± 0.1 (13) 0.33 0.89 3-6 min Inflation (2) - <0.5 3.8 ± 3.2 (22) 2.6 ± 1.4 (62) 0.95 0.59 0.5-1.0 1.8 ± 1.3 (10) 2.2 ± 1.3 (27) 1.0 1.0 >1.0 1.4 ±0.9(18) 1.3 ±0.6(15) 0.94 1.0 15-20 min Inflation (2) <0.5 4.0 ± 2.1 (18) 3.2 ± 2.2 (44) 0.63 0.45 0.5-1.0 3.7 ± 2.5 (17) 2.2 ± 0.5 (18) 0.84 0.79 > 1.0 2.6 ± 1.4 (9) 2.2 ± 0.6 (15) 0.70 0.75 45-300 min Inflation (4) <0.5 3.7 ± 1.5 (38) 3.5 ± 1.3 (78) 0.91 0.38 0.5-1.0 4.0 ± 1.7 (34) 3.2 ± 1.4 (64) 1.0 0.97 > 1.0 3.5 ± 2.4 (15) 3.0 ± 1.1 (37) 1.0 1.0 x: meon ± s.d.;': pulmonbry orterys; *: pulmonary veins; -: no. of lobes; s: no. of cuffs. P/~= P..1W..r...,.Y f6,a-t.u4tes p v = vei ps

7 a~3>-c 4 PER I UHSCULAR FLU I D CUFF POPULHTI DN IN L IQU I D INFLATED SHEEP LUNGS AVERAGE FRACTION OF CUFF-TO-VESSEL VESSELS WITH RATIO # CUFFS Vessel Diam, (mm) PA'st PV's* PA's PV's 3 min Inflation (1)" < 0.5 0.5-1.0 No fluid cuffs were present after 3 min of inflation. > 1.0 15-20 min Inflation (1) <0.5 2.0 ± 1.2 (17) 3.3 ± 2.3 (7) 0.28 0.33 0.5-1.0 2.7 ± 2.5 (20) 1.1 ± 1.0 (1) 0.47 0.33 > 1.0 1.4 ± 1.3 (36) 1.1 ± 0.5 (2) 0.90 0.67 30 min Inflation (1) <0.5 4.4 ± 4.4 (27) 2.1 ± 1.6 (3) 0.66 0.1 0.5-1.0 3.9 ± 3.5 (20) 2.9 ± 4.1 (5) 1.0 1.0 > 1.0 2.9 ± 1.6 (15) 0.6 ± 0.3 (3) 1.0 1.0 45-180 min Inflation (3) <0.5 4.1 ± 2.1 (56) 2.1 ± 1.2 (15) 0.5 0.26 0.5-1.0 3.7 ± 2.1 (35) 0.8 ± 0.1 0 1) 1.0 1.0 > 1.0 1.8 ± 1.1 (51) 1.0 ± 1.5 (20) 1.0 1.0 mean ± s.d.;': pulmonary arterys; *: pulmonary veins; -: no. of lungs; s: no. of cuffs. 04 , d) wl vV-n -I A Yi'~Gdl~ S A V= v.a. 1..,5 . •