Vinyl Acetate Casts of Emphysematous Rat Lungs CARMELO COLOMBO, SALVATORE COLOMBO, and BERNARD STEINETZ x ~
Abstract Emphysema was induced in rats by chronic exposure to a papain aerosol according to established methods. The emphysematous changes were evaluated by previously described histological and biochemical parameters and by a new method employing infiltration of the lungs with vinyl acetate to make casts of the air spaces of the pulmonary tree. The weights and gross and microscopic appearance of the casts demonstrated the emphysematous changes in lungs of the papain-treated rats. Vinyl acetate casting may offer a new and useful tool in evaluating experimental induction of emphysema in rats. Keyphrases 0 Vinyl acetate-lung infiltration, casts formed, papain-induced emphysematous changes evaluated, rats Lung infiltration-vinyl acetate, casts formed, papain induced emphysematous changes evaluated, rats Emphysema, papain induced-lung changes evaluated by infiltration with vinyl acetate to form casts, rats
Pulmonary emphysema is a chronic respiratory disease characterized by a gradual destruction of alveolar walls, resulting in the formation of enlarged air spaces distal to the terminal bronchioles. Various substances have been used to induce an emphysema-like condition in laboratory animals. These substances include aerosols of papain, polymorphonuclear leukocyte hom*ogenates, and other noxious substances (1-4) and intratracheally administered papain, phytohemagglutinin, and silica dust ( 5 , 6 ) . The severity of the lung disease produced may be evaluated histologically, biochemically, or physiologically, but none of these parameters is entirely satisfactory (1,3). Latex casts were used to study acinar changes in human emphysema (7). In the present investigation, the pulmonary trees of rats were infiltrated with vinyl acetate and the resulting casts were freed from tissue by digestion in mineral acid. The technique revealed distinct differences between control and emphysematous lungs.
Table I - C o m p a r i s o n of Various Parameters in t h e Evaluation of Papain-Induced Emphysema Treatment Parameter Lung histology, alveoli/linear m m Lung p-glucuronidase, E a t 450 n m Lung weight, g/100 g body weight Vinyl acetate cast of lung Cross-sectional area, m m z Weight of cast, mg Lung capacity, vinyl acetate infused, ml 0p
Control 92 + 4
Papain Aerosol 59
f
40
0.37 f 0.02
0.73
f
0.040
0.51 0.02
0.87
f
0.06
0.29 420 2.90
0.48 320 2.03
5 f
0.020
0.02 20 t 0.14 f
?
f
30t 0.16b
< 0.01 versus control. b p < 0.05 uersus control.
and the alveoli falling on a line 0.2 mm long were counted. Ten fields per lung were randomly chosen, and 10 counts were made in each field. The average number of alveoli per linear millimeter was computed. The lungs of six rats from each group were used for 8-glucuronidase determination as previously described (3). The tracheas of the remaining six rats from each group were exposed and cannulated with 0.6-cm (0.25-in.) (i.d.) dimethicone tubing2.Vinyl acetate3 (12% stock solution) was mixed 21 (v/v) with acetone and delivered into the lungs oia the tracheal cannula from a calibrated reservoir at a height of 40 cm. The volume of vinyl acetate infused was recorded. The apparatus was washed with acetone immediately after infusion to prevent permanent clogging by the vinyl acetate. After 1hr, the lungs were carefully removed and immersed in 100 ml of concentrated hydrochloric acid in screw-capped jars. Twentyfour hours later, the remaining bits of tissue were removed by gentle washing. Then the casts were air dried and weighed prior to examination under a dissecting microscope at 40X magnification. By means of an ocular micrometer, the long and short axes of 20 alveoli were measured for each lung and the cross-sectionalareas were calculated. The alveoli to be measured were picked a t random in areas that could be clearly seen. RESULTS AND DISCUSSION
EXPERIMENTAL Male Charles River CD albino rats, -100 g, were used in two major groups of 18 rats each. Each group was divided into three subgroups of six animals each. The subgroups were used for: ( a ) lung histology, ( b ) lung p-glucuronidase activity, and (c) vinyl acetate casts of lungs. One major group of rats served as the control (water aerosol), and the other group was exposed to a 10% papain’ aerosol in a Laskin chamber as previously described (3).The animals were exposed to the aerosol twice a week for 3 weeks, after which they were sacrificed and used for the various determinations. The lungs of six rats from each group were infused with 10%neutral buffered formalin by means of a tracheal cannula attached to a reservoir; the cannula delivered the fixative at a pressure of 10 cm water. When the flow of fixative ceased, the trachea was ligated and the thoracic cavity was opened. The entire chest contents were carefully removed and placed in additional formalin. Following fixation, the lungs were routinely sectioned and stained with hematoxylin and eosin. The slides of right and left lungs of each rat were examined at lOOX magnification with an ocular micrometer, Papain-High Test, S. B. Penick and Co., New York, N.Y.
1082 /Journal of Pharmaceutical Sciences
The rats exposed to the papain aerosol developed unmistakable signs of emphysema as determined histologically and biochemically. The alveolar spaces were enlarged, leading to a 36% decrease in their number per linear millimeter (Table I). In agreement with previous findings (3),lung /3-glucuronidase activity increased by 97% in the papain-treated rats (Table I). These results correlated well with observations made on the vinyl acetate casts. Gross inspection of the casts revealed distinct differences between control and emphysematouslungs (Fig. 1).The terminal buds of lungs of exposed rats were coarser and sparser, and the bronchioles were more distended than those of the controls (Figs. 2 and 3).Fused and broken alveoli also were observed. The casts of alveolar spaces of the emphysematouslungs averaged 66%larger in cross-sectionalarea than those of controls (Table I). The casts of the emphysematouslungs also weighed less than the control casts. On the other hand, lung tissue weight increased by 71%. Previous results showed that lungs of rats exposed chronically to papain demonstrated changes resembling those seen in lungs of human patients with diagnosed emphysema (1,3,6).In the present
* Silastic tubing, Dow Corning Medical Products Division, Midland. Mich. Ward’s Natural Science Establishment, Rochester, N.Y.
Figure 1-Vinyl acetate casts of control (right) and emphysematous ( l e f t )rat lungs (approximately 2 X natural size). Note “solid” appearance of control lungs (due t o close spacing of alueoli) iv comparison with lacy appearance of emphysematous lungs.
F i gu r e 2-Vinyl acetate cast of control lung (56X magnification). Note detail of alveoli.
studies, the typical histological (dilation and depletion of alveoli) and biochemical (increased /3-glucuronidase activity) changes were observed in papain-treated rats. The vinyl acetate casts accurately reflected the emphysematous changes. The terminal buds were grossly enlarged, presumably reflecting the breakdown and coalescence of smaller alveoli. The distension of the bronchioles may have been due to a loss of elasticity together with a hyperplasia of nonfunctional parenchymal tissue, leading to obstruction of air passageways and enlargement and eventual destruction of alveoli. Emphysema has been reported to be due to loss of alveoli, resulting in enlargement of respiratory bronchioles, alveolar ducts, and lung distension ( 5 ) . In corrosion models of emphysematous human lungs, a notable abnormality was a loss of alveoli on the respiratory bronchioles which appeared as smooth and bulging structures (7), similar to what was observed in the present study. These phenomena were observable by gross inspection of the casts or by measurement of the alveolar cross-sectional area, which was increased by 66%.The decrease in functional alveolar capacity was also reflected in the 24%reduction in the weight of the lung casts and
F i g u r e 3-Vinyl acetate cast of emphysematous lung (56X magnification). Note relatiue sparseness and coarseness of alveoli i n comparison with Fig. 2.
the decreased volume of vinyl acetate required to inflate the lungs of emphysematous rats. The reduction in these parameters probably was due to nonfunctional connective tissue hyperplasia, which resulted in obstruction of the respiratory bronchioles and broken or absent alveoli. The nonfunctional tissue was, of course, later removed during hydrochloric acid digestion. Vinyl acetate casts of lungs, therefore, offer an effective, rapid, and relatively simple adjunct to histological and biochemical parameters in evaluating the severity of experimental emphysema in laboratory rats. REFERENCES
( 1 ) R. Giles, M. Finkel, and R. Leeds, Proc. SOC. E x p . Biol. Med., 134,157(1970). ( 2 ) B. Mass, T. Ikeda, D. Meranze, G. Weinbaum, and P. Kimbel, A m . R w .Resp. Dis., 103,907(1971). ( 3 ) C. Colombo and B. Steinetz, Arch. I n t . Pharmacodyn. Ther., 216,86(1975). Vol. 65, No. 7, J u l y 1976 1 1083
(4) H. Boren, Am. Reu. Resp. Dis., 92, l(1965). (5) P. Gross, E. Pfitzer, E. Tolker, M. Babyak, and M. Kaschak, Areh. Enoiron. Health, 11,50(1965). (6) H. Ito and D. Aviado, J . Pharmacol. E r p . Ther., 161, 197(1968). (7) K. K. Pump, Am. Reu. Resp. Dis., 108,610(1973).
ACKNOWLEDGMENTS AND ADDRESSES Received August 18,1975, from the Research Department, Pharrnaceutical Diuision, Ciba-Geigy Corporation, Ardsley, N Y I0502 Accepted for publication September 25,1975. To whom inquiries should be directed.
Isolation of Cannabisativine, an Alkaloid, from Cannabis sativa L. Root CARLTON E. TURNER *, MEI-FENG H. HSU Q, JOSEPH E. KNAPP *, PAUL L. SCHIFF, Jr. *, and DAVID J. SLATKIN *x
Abstract 0 An ethanol extract of the root of a Mexican variant of Cannabis satiua L. (marijuana) afforded, after partitioning and chromatography, the new spermidine alkaloid cannabisativine. Keyphrases 0 Cannabis satiua-cannabisativine isolated from ethanol extract of roots Alkaloids-cannabisativine isolated from ethanol extract of roots of Cannabis satiua Cannabisativineisolated from roots of Cannabis satiua
I The occurrence of several low molecular weight, nitrogen-containing substances in Cannabis satiua L. has 60-90') (2 liters) and methanol-water (91) (2 liters) to yield fractions been reported including choline (1-4), trigonelline (2, of 12.7 g (0.1%)and 33.4 g (0.3%),respectively. 3), muscarine (5), piperidine (6), N-(p-hydroxy-PThe aqueous methanol fraction was chromatographed on silicic phenylethy1)-p-hydroxy-trans-cinnamamide (7), acid3 (450 g, 5 X 49 cm) packed in petroleum ether (bp 60-90'). Elution with 8%methanol-chloroform afforded a residue (3.0 g), which neurine (8),L-proline (8),and L-(+)-isoleucjne betaine was dissolved in chloroform (25 ml) and partitioned with 1%hydro(9). Small amounts of four alkaloids were isolated (lo), chloric acid (3 X 25 ml). The combined acid layers (75 ml) were basibut the high-resolution and mass spectra were inconfied to pH 8 with concentrated ammonium hydroxide and partitioned clusive. with chloroform (3 X 75 ml). The combined chloroform layers were dried over anhydrous sodium sulfate and evaporated, in uacuo, at 40' Recently, the presence of a new spermidine alkaloid, cannabisativine (I) [ 13-(1,2-dihydroxyheptanyl)- to yield a white residue (48 mg). Crystallization from acetone afforded cannabisativine (29 mg), mp 1,4,5,6,7,8,9,10,11,13,16,16a-dodecahydropyrido[2,1- 167-168O; [a]::+ 55.1' (c0.53, CHCl3);U V no maximum above 210 d ] [1,5,9]triazacyclotridecin-2(3H)-one], was found in nm; IR: u,,, (KBr) 3300, 3020, 2960, 2920, 2850, 1628, 1580, 1470, the roots of this species (11). This report details the 1250,1130,1045,and 707 cm-1; NMR b 5.90 (2H, s, vinyl) and 9.6 (lH, s, broad, CONH); mass spectrum (M+):mle 381 (I%),363 (I), 352 (11, procedure used in the isolation of this substance. EXPERIMENTAL' Plant Material-Roots of a Mexican variant of C. satiua L. were used2. Extraction-Air-dried ground roots of C. satiua (11.72 kg) were extracted by percolation with methanol (150 liters). The extract was evaporated in U ~ C U Oat 40' to leave a dark-brown syrup (323 g, 2.8%). Isolation of Cannabisativine-The methanol extract (323g) was partitioned between water (4 liters) and chloroform (4 liters) to give fractions of 266.0 g (2.3%) and 47.7 g (0.4%),respectively. The chloroform fraction was then partitioned between petroleum ether (bp
Melting points were determined on a Thomas-Hoover Uni-Melt meltingpoint apparatus and are corrected. IR spectra were run in potassium bromide using a Perkin-Elmer 257 spectrophotometer. Optical rotations were determined on a Perkin-Elmer 241 polarimeter. UV spectra were run on a PerkinElmer 202 spectrophotometer. NMR spectra were obtained in deuterated chloroform on a Hitachi Perkin-Elmer R-24 spectrometer, with tetramethylsilane as the internal standard. Mass spectra were recorded on a LKB-9000 spectrometer. Lot Me-A(2)-C-69grown in 1971; voucher specimens were deposited in the herbarium of the School of Pharmacy, University of Mississippi.
1084 1Journal of Pharmaceutical Sciences
310 (2), 280 (3), 250 (64), 208 (loo),198 (6), 171 (12),129 (5), 114 (5), 112 (6), 96 (4), 94 (6), 84 (8), 80 (6), 72 (71, 70 (8), and 55 (6). The structure of this compound was recently determined to be I (11).
REFERENCES (1) E. Jahns, Arch. Pharm., 225,479(1887). (2) E. Schulze and S. Frankfurt, Ber. Dtsch. Chem. Ges., 27, 769(1894). ( 3 ) K. W. Merz and K. B. Bergner, Arch. Pharm., 278, 49(1940). (4) M. L. Mole, Jr., and C. E. Turner, Acta Pharm. Jugosl., 23, 203(1973). (5) V. Kwasniewski, Dtsch. Apothek-Ztg., 94,1177(1954). (6) Y. Obata, Y. Ishikawa. and R. Kitizawa. Bull. &r. - Chem. SOC. Jpn., 24,670(1960). (7) D. J. Slatkin. N. J. Doorenbos. L. S. Harris. A. N. Masoud. M. W. Quimby, and P. L. Schiff, Jr., J. Pharm. Sci., 60,1891(1971): (8) M. L. Mole, Jr., and C. E. Turner, ibid., 63,154(1974). (9) C. A. L. Bercht, R. J. J. C. Lousberg, F. J. E. M. Kuppers, and
:j
Mallinckrodt.
