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I i~, Fa 1,. i` b•, THE COUNCIL FOR TOBACCO RE$EARCH-U.S.A., INC. ~ Dec. 10. 1984 Memorandum 7.b: Dr. S.C. Srnmers and, Staff Flroen: D. H. Fbrd Ne: Site visit with Drs. N.C. Staub and R. Conhaim, University of California, Cardiovascular Research Institute, Nov. 14, 1984. Grant No. 1595 R1 entitled "Alveolar-airway barrier permiability to liquid and macrcanrolecules in dog and sheep lung.' Goal: zb understand how liquid accum:lates in the interstitial space around -ainrays and blood vessels leading to or frcm alveoli to eventually flood the alveolar spaces and produce puLmonary edena. These investigators are attempti to' find the site where fluid leaks fran the interstitium into the airways. The; are enploying dog and sheep lungs and 'backfilling' the airways with flufd con- taining evans Blue tagged a7Jnunen and observing Where it leaks back into the interstial spaces...a sort of reverse flooding process. 7hey hope that this model reflects what actually. occurs in the genesis of pulironary edema in life. Ubether or not such a'reversefilling' process will truely define the sites where edema fluid leaks from the interstitiom into the airways *+e+si*+Q t o be detenained. Questions beinq considered- 1. kftt is the storage trolwre of fluid which niay aoc:mulate in the inter- stitial space before it 'floods' over into the airways? How can one measure it4 2. AIlmt causes pulimnary edema to occur following head injury? Are there neural pathways involved. 3. What is the site of leakage froan the interstitium into the airways (at the alveol i or soere<saheres along the bronchiolar tubes )? 4. How permiable is the leakage site and how large a molecule can pass through the pores where leakage occurs? Soeae answers to the above questions are beginning to aocimotlate as indicat by the following data: Data a..^cunulated-presented by Dr. Conhaim, since Dr. Staub aas not able to present a't the s_ ite visit: 7heir major progress, as `cated in e recent pr report has been to develop their model of perivascular-peribronchiolar fluid cu formation. The model provides the basis for the projects on liquid pressure an volume cs`as++-ene*+t, pore size and site of fluid leakage. Initially it appears that the volume of fluid aacuan:lated in the dog 1unc,r alLmst twice that occuring in sheep. Fluid volurie is determined by point countia on sections wherein the presence of the fluid in the interstitial space is indii

2 by the presence of Evans Blue tagged alblanen 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 taater/alburen/dye mixtur4 Further, the longer the tine of exposure, the greater was the volume of fluid in the cuff spaa (Fig.2). Also note that in dogs irore fluid accwmilat.ed around the srtall than the large vessels (Table 1). With sheep, the smaller vessels also tended to have larger c,-uffs, but there were more large vessels with cuffs (100%) than smaller vessels (22%) (Table 2). Studies on pore size for leakage suggest that there are sare holes with a radius as large as 15 me whicF4 will permit liposames to leak through from the airways into the interstititnn as well as pores with radii as small as 3.5 nm, %hich still permit albumin to leak through. Water, with a molecular radius of 2 ran will of course pass through both. Ihe.irr calculations have lead theln to assume that there may be some pores with radi as large as 75 nm. lfiey hope to resolve the probl®n of determining the sizes of the various pores through which leakage can occur by using latex particles of various size. These particles will be eoate( with the albumin tagged with Evans Blue. Z'he site of fluid leakage does not appear to be in the alveolar sacs since ferritin part~cles (5.5 run diamete.r) were shown by FM studies to not be able to pass from the alveolar space into the interstitium. However, since such ferritin particles were observed to penetrate the interstitiwn surrowxding the bronchiolar tubes, it would appear that at least some leakage oocurs along this seynent of the airway. Further, some ferritin particles were observed within the intracelluL clefts between the epithelial cells, suggesting that the site of leakage is bstwex cells. Coemnent: Drs. Staub and Oonhaim appear to have made smre progress on a projec in which Dr. Oonhaim appears to be the primary investigator. However, they have a long way to go before they begin to obtain the A*+swe*s they seeY.. FUrther, I am not sure that their mxlel wherein the interstitial spaces are filled with fluiK a*+A*+nP+^ -Aich is the reverse of the way it would normally occur will provide ansr relevant to what occurs in vivo. The study is possibly worth continued support during the R2 year. Hawever,~if considerable progress has nat occurred by the end of the third year, I would doubt if continued support would permit conclusive results to be obtained. DF/ff D. FUB

~ j FU~,U I ~ MEASUREMENT OF INTERSTITIAL LIOUID VOLUME BY POINT COUNTING Interstitial Cuff Interstitial Cuff Volume Cuff Volume !i lung Vol ume 4 . . . 0 Airway ( +) Counting Point 0 vessel 34 2f 1t 04 1 2 3 4 5 6 7 8 910111213141516 No. of Transparencies Counted 1 17 I FL

4 3 CUFF AREA 2 VESS 1 SFUJ'~1~r, Staub, Norman C. CTR Grant # 1595 EFFECT OF INFLATION T1ME ON PERIVASCULAR CUFF SIZE T T h ` 1.015 0.5 - 1.0 >l.0 VESSEL DIAMETERS. MU" 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

~'.U TA-6 L Q PERIURSCULRR FLUID CUFF PDPULRTION IN LI QUI D I NFLRTED OQG LUNG LOBES , RATIO # CUFFS AVERAGE FRACTION OF CUFF-TO-VESSEL VESSELS WITH 7- Vessel Di em ' , (mm) PA'st PV's" PA's PV's ~ 1 min Inflation (1)" 1 (0.5 0.94 (2)§ 0.9110. 1 (3) 0.30 0.08 0.5-1.0 0.36 t 0.1 M 0.56 t 0.4 (14) 0.75 0.75 0 1 44 0 0 2 (5) 0 1 (13) 33 0 0 33 0 89 C > . . 3 . . 3 . . . 3-6 min Inflation (2) <0.5 3.8 ± 3.2 (22) 2.6 s 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 t0.9(18) 1.3 :0.6(15) 0.94 1.0 15-20 min Inflation (2) <0.5 4.0 ± 2.1 (18) 3.2 12.2 (44) 0.63 0.45 0.5-1'.0 3.7 t2.5(17) 2.2 10.5 (10) 0.84 0.79" > 1.0 2.6 t 1.4 (9) 2.2 10.6 (15) 0.70 0.75 45-300 min Inflation (4) <0.5 3.7 t 1.5 (38) 3.5 t 1.3 (78) 0.91 0.38 0.5-1.0 4.0 t 1.7 (34) 3.2 3 1.4 (64) 1.0 0.97 > 1.0 3.5 12.4 (15) 3.0 3 1.1 (37) 1.0 1.0 ~: meen 3 s.d.;': pulmonary arterys;-": pulmonary veins; -: no. of lobes; 6: no. of cutfs. pA: P..1,~..~.,.Y j,4#.oa,:r p v= pM1w.,r.wy vP.4Fs

TAb%.& X SF PERIUASCULRR FLUID CUFF POPULRTION IN LiqUID INFLATED SHEEP HEEP LUNGS AVERAGE CUFF-TO-VESSEL RATIO ~ FRACTION OF VESSELS WITH CUFFS Vessel Diem, (mm) PA'st PV's* PA's PV's 3 min Inflation (I)" < 0.5 0.5-1.0 No fluid cuffs were present after 3 min of inflation. > 1.0 C 15-20 min Infletion (1) <0.5 2.0 3 1.2 (17) 3.3 t 2.3 (7) 0.28 0.33 0.5-1.0 2.7 3 2.5 (20) 1.1 s 1.0 (1) 0.47 0.33 > 1.0 1.4 3 1.3 (36) 1.1 10.5 (2) 0.90 0.67 Y 30 min Inflation (1) <0.5 4.4 t 4.4 (27) 2.1 3 1.6 (3) 0.66 0.1 0.5-1.0 3.9 13.5 (20) 2.9 14.1 (5) 1.0 1.0 >1.0 2.9 1 1.6 (15) 0.6 30.3(3) 1.0 1.0 f 45-180 min Infletion (3) <0.5 4.1 ± 2.1 (56) 2.1 ± 1.2 (15) 0.5 0.26 0.5-1.0 3.7 :t2.1 (35) 0.8 t0.101) 1.0 1.0 >1.0 1.8 ± 1.1 (51) 1.0 t 1.5 (20) 1.0, 1.0 a : mean *- s.d.;': pulmonary arterys; *: pulmonary veins; • no. of lungs; g: no. of cuffs. do1F + JP Vjw..% a+, , AwIUA1es QV + ()..lY..w." vuawv. 6