Fine Structural Morphometry on Biopsy Specimens of Human Lung

Fine Structural Morphometry on Biopsy Specimens of Human Lung 1. Normal Lung* Stanlexj M. Cassan, M.D., Matthew B. Divertie, M.D., F.C.C.P., Arnold L. Brown, Jr., M.D.

Stereologie techniques were applied to an electronmicroscopic study of random samples of normal lung tissue obtained from nine patients during thoracotomy. Relative fractional volumes of lung components, alveolar and capillary surface areas, surface-to-volume ratios, and arithmetic and harmonic mean thicknesses of the alveolarcapillary membranes were determined and compared with values previously obtained by others for inflated whole lung. Values for fractional volumes and absolute surface

A ccurate estimates o f m a n y dimensional

param-

eters of t h e lung w e r e n o t a v a i l a b l e until t h e advent of e l e c t r o n m i c r o s c o p i c t e c h n i q u e s and t h e

and

area were significantly influenced by changes in lung inflation imposed by the sampling technique and the use of predicted normal lung volumes for purposes of calculation. However, parameters for alveolar septal structures agreed closely with those obtained for inflated whole lung. Distribution in a volume could not be assessed, but within the limits of this study, there did not appear to be a significant difference in the width of the blood-air barrier between inflated and noninflated states.

titative m e a s u r e m e n t s of tissue structures w i t h i n alveolar septa c o m p a r e d well w i t h those

described

b y W e i b e l for inflated w h o l e lung.

application of mensural formulas previously used in fields other than m e d i c i n e . T h e D e l e s s e

1

mea-

surement of samples for m o r e than 1 0 0 years a n d has b e e n applied m o r e r e c e n t l y to lung tissue b y W e i b e l , Kistler, S c h e r l e , and F r e e r e . " 2

4

It is b a s e d on t h e

f a c t that structures dispersed in a volume are q u a n titatively represented on sections of that

volume.

Application of t h e principle to e x t r e m e l y thin sections of h o m o g e n e o u s

respiratory tissue has

per-

mitted quantitative m e a s u r e m e n t of randomly distributed alveoli in the p a r e n c h y m a of the inflated whole animal lung. T h i s m e t h o d w a s applied in t h e present study to t h e ultrastrueture of r a n d o m b i o p s y samples of normal h u m a n p u l m o n a r y alveoli in an effort to establish r e f e r e n c e values for relative f r a c tional volumes of lung c o m p o n e n t s , alveolar

and

capillary surface areas, surface-to-volume ratios, and arithmetic and h a r m o n i c m e a n thicknesses of t h e alveolar-capillary m e m b r a n e . V a l u e s o b t a i n e d

for

these p a r a m e t e r s w e r e c o m p a r e d to previously d e rived estimates for inflated w h o l e l u n g ,

2

METHODS

principle

has b e e n used b y geologists for stereologie

the frac-

tional air volumes o f w h i c h could b e used to determine indirectly the d e g r e e of lung inflation o f the

Normal lung tissue was obtained from nine patients at the time of resection of solitary circumscribed tumors (Table 1 ) . In each instance the sampled areas of pulmonary parenchyma %vere remote from the lesion itself and were obtained only from the respiratory portion of the lung where alveoli are considered randomly distributed except in the immediately subpleural zone. Tissue for electronmicroscopic study was fixed for three hours in 4 percent glutaraldehyde with 0.05 M cacodylate buffer followed by immersion for two hours in 1 percent osmium tetroxide (Dalton's solution) at pll 7.6. The tissue was then rinsed in 0.18M sucrose in 0.08M cacodylic acid for three hours, dehydrated up through absolute alcohol, passed through two changes of propylene oxide, and embedded in Epon 812 by the method of Luft. Tissue sections of approximately 600 A were made with a Porter Blum microtome using a diamond knife, and sections were mounted on copper grids. These were stained with a saturated solution of uranyl acetate followed by 0.1 percent lead citrate (modified Reynolds' stain) and examined with an RCA model EMU3E clectronmicroscope (Fig 1 ) . Tissue sections and photographic fields were randomly selected as described by Weibel and 20 photographs per patient were made by obtaining photographs of five tissue 5

H

7

8

9

2

Table 1—Clinical Data (Seven Men, Two

Women)

specimens used in this study. W h i l e distribution in Range

Mean Value

Age of patient, yr.

36-68

56

Predicted total lung eapcity, Liters

4.9-6.6

5.7

Volume of respiratory portion of lung, Liters

5.0-6.7

5.8

a volume could not b e assessed a n d relative volumes of air spaces w e r e p r e d i c t a b l y altered, q u a n °From the Mayo Clinic and Mayo Foundation, Rochester, Minn. Manuscript received August 13; accepted October 5. Reprint requests: Section of Publications, Mayo Clinic, Rochester, Minnesota 55901 CHEST, 65: 3, MARCH, 1974

FINE STRUCTURAL MORPHOMETRY OF HUMAN LUNG (1) 269

ACT

ACT ICT

•it

FIGURE 3. Diagram of alveolar tissue subdivided by lines perpendicular to alveolar surface at lateral extremes of capillaries. ACT, alveolar-capillary tissue; ICT, intercapillary tissue; Cap, capillary.

FIGURE 1. Electronmicrograph of normal human alveolar septum. A, alveolus; M, blood-air barrier ("alveolar-capillary membrane"); C, capillary; fl, red blood cell; N, intermembranous space; F, collagen; I, interstitial cell nucleus (original magnification x4,300). sections on each of four copper grids. Photographs were made at a magnification of approximately x2,200 and prints were enlarged to match a translucent plastic test pattern of 168 points and 84 lines (Fig 2 ) . Point-counting volumetry was applied to air spaces, capillaries, alveolar-capillary tissue ( A C T ) , and intercapillary tissue ( I C T ) . The latter two components were separated by lines drawn perpendicular to the alveolar epithelial surface at the lateral extremes of the alveolar capillaries (Fig 3 ) . This arbitrary subdivision of tissue components was made so that a numerical value could be obtained which would relate direct-

ly to that portion of alveolar septal tissue lying directly in the path of gas diffusion. Such a value can provide a basis for comparison with disease states in which an impairment of diffusion is known to be present. Furthermore, the relationship of alveolar-capillary to intercapillary tissue is independent of lung inflation and affords an index of the proportions of each in the septa studied. Surface areas of alveoli and capillaries were determined from the number of transections of these components by test pattern lines. Calculation of surface area, however, required knowledge of the lung volume. The use of samples of lung tissue instead of whole lung precluded estimation of the degree of lung inflation at which measurements were obtained. Consequently, a predicted value for the volume of the respiratory portion of the lung, based on the height and sex of each patient, was utilized. Predicted total lung capacity was increased by a factor of 0.12 to include tissue volume and the respiratory portion calculated as 90 percent of the combined volume. Morphometric calculation of fractional volumes and thickness of the blood-air barrier does not require a knowledge of measured total lung capacity. Except for estimation of surface area, the actual volume at which measurements are made is required only for reference purposes. The degree of inflation and fractional air volume are related but are independent of any other variable. As a result, it is possible to derive the degree of inflation based on a comparison of the fractional air volumes obtained in this study with those established by Weibel who found that at 75 percent of total lung capacity, air made up 93 percent and tissue, the remaining 7 percent of the respiratory portion of the lung. Use of a predicted lung volume for surface area determinations produces an overestimation that can be quantitated on the basis of fractional tissue volumes. Arithmetic mean thickness (AMT) of the alveolar-capillary membrane and surface-to-volume ratios were obtained by a combination of point-counting volumetry and transections of the capillary surface. Arithmetic mean thickness was determined by relating tissue volume to the enclosing air and blood surfaces without discrimination between them. This method has been used extensively by Weibel, but he has pointed out that it is more appropriate to relate tissue volume to one or the other of the bounding surfaces when problems of gas diffusion are considered. Both methods were used in this study. Harmonic mean thickness (HMT) of the alveolar-capillary membrane was calculated from harmonic mean intercept lengths determined by superimposition on photographs of a translucent plastic test pattern of 14 rows of equidistant 2

2

2

FIGURE 2. Test pattern grid for superimposition on photographs (84 lines connecting 168 equidistant points). (From Weibel ER, Kistler, GS, and Scherle W F : Practical stereological methods for morphometric cytology. / Cell Biol 30: 23, 1966, by permission of The Rockefeller University Press)

270

CASSAN, DIVERTIE, BROWN

CHEST, 65: 3, MARCH, 1974

Table 2—Formulas Used in Morphometric

Analysis

its b a s e m e n t m e m b r a n e , a n d an i n t e r m e m b r a n o u s

Key: V,=volume of specific component of lung tissue

rier is a multicellular structure and different from

s p a c e o f v a r i a b l e thickness. W h i l e t h e blood-air bar— V

= —1

t

o

1

t h e triple l a y e r e d m o l e c u l a r a r c h i t e c t u r e of a bio-

2N,V = — V = volume of respiratory portion of lung Zn

l o g i c m e m b r a n e , w e will c o n t i n u e to d e s c r i b e it as

t

x

t

_

p

j =

4N ** X

alveolar-capillary

membrane

because

of

common

u s a g e . D i m e n s i o n s of s o m e of its c o m p o n e n t s h a v e

number of test points of specific component of lung tissue

been obtained by nonmorphometric methods

and

are listed in T a b l e 7.

2Lh

V o l u m e t r i c analysis o f tissue from n o r m a l lungs in this study i n d i c a t e d an a v e r a g e f r a c t i o n a l air volume

Th = —-— P = total number of test points applied to lung tissue t

of 44.4 p e r c e n t . T h e reduction of 4 8 . 6 p e r c e n t in S,

4N„

— =

f r a c t i o n a l air s p a c e as c o m p a r e d to t h e value o f 9 3 p e r c e n t o b t a i n e d b y W e i b e l

Sx= surface area of specific component of lung tissue

t r i b u t e d in this study a m o n g

N =number of intersections of test lines with specific components x

previous 2

is dis-

the components

of

alveolar tissue, that is, A C T and I C T ( F i g 3 ) as well

Z= length of test line

as capillary s p a c e . T h e total f r a c t i o n a l v o l u m e of

n= number of test lines

these c o m p o n e n t s was previously e s t i m a t e d as

T= arithmetic mean thickness of alveolar-capillary membrane

p e r c e n t of the respiratory portion of t h e lung dist r i b u t e d equally b e t w e e n tissue ( A C T and

Th= harmonic mean thickness of alveolar-capillary membrane

2

RESULTS AND DISCUSSION

alveolar-capillary

i n d i c a t e a total f r a c t i o n a l tissue ( A C T and

ICT)

v o l u m e o f 3 7 . 1 p e r c e n t a n d f r a c t i o n a l capillary volu m e o f 18.5 p e r c e n t . T h e s e c o m p o n e n t s , therefore, h a v e i n c r e a s e d b y 3 3 . 6 p e r c e n t a n d 15 p e r c e n t , respectively, o v e r t h e previously d e s c r i b e d 3 . 5 p e r c e n t f r a c t i o n a l v o l u m e for e a c h or b y a b o u t two-thirds a n d one-third, respectively, of the d e c r e a s e in f r a c tional air volume. T h i s disproportionate distribution in t h e a b s e n c e of p a t h o l o g i c tissue c h a n g e s m a y b e a s c r i b e d to a r e d u c t i o n in capillary v o l u m e in t h e p r e p a r a t i o n o f tissue. T h e reduction in air s p a c e from 9 3 p e r c e n t to 44.4 p e r c e n t can b e shown to r e p r e s e n t approximately a 15-fold r e d u c t i o n in inflation c o m p a r e d to previous s t u d i e s . H o w e v e r , within t h e g r o u p of normal lungs 2

membrane

1 0

con-

sists of surfactant, t h e alveolar epithelium and its

t h e r e was c o n s i d e r a b l e variation in f r a c t i o n a l volu m e s o f all lung c o m p o n e n t s . T o test t h e hypothesis

b a s e m e n t m e m b r a n e , the capillary e n d o t h e l i u m a n d Table 3—Fractional

ICT)

and capillary s p a c e . V a l u e s o b t a i n e d in this study

Lh= harmonic mean intercept length *P,= number of points on ACT in this study. **N,= capillary intersections only. This formula and the formula for surface-to-volume ratio are valid only when number of test points is twice number of test lines. points. The ratio of HMT to both values of AMT was derived. Formulas used for calculation of these parameters are listed in Table 2. Mean values were obtained for all parameters. Correction factors for fixation and processing were not considered, since calculation of these factors also requires use of whole lung specimens. However, previously calculated factors for tissue similarly prepared have approximate unity. The results of these observations are contained in Tables 3 through 6. Diseased lungs prepared in identical fashion will be systematically compared at a future date.

T h e human

Table 5—Surface-to-Volume

Volumes

Ratios M e a n ± l SD

Mean +1 SD

A/ACT+1CT

0.46 + .035

Air space, %

44.4 ±3.25

A/ACT

0.80 + .054

Capillary space, %

18.5+2.00

A/ICT

1.17 ±.137

Alveolar-capillary tissue (ACT), %

21.2±1.12

C/ACT+ICT

0.37 ±.038

Intercapillary tissue (1CT), %

15.9 + 1.94

C/ACT

0.63 + .051

ACT/1CT

1.33+0.19

C/ICT

0.95 ±.138

Table 4—Surface Areas

A = alveolar surface areas; C = capillary surface areas; ACT = alveolar-capillary tissue; ICT = intercapillary tissue.

(M ) 2

Area (M ), Mean + 1 SD 2

Table 6—Mean Thickness Membrane

of

Alveolar-Capillary ( )

Alveoli, A

953 ±52.76

Capillaries, C

769 ±76.88

Arithmetic

1.665 ±0.128

Ratio: A/C

1.31+ 0.10

Harmonic

0.829 + 0.067

CHEST, 65: 3, MARCH, 1974

7

Mean ± 1 SD

FINE STRUCTURAL MORPHOMETRY OF HUMAN LUNG (1) 271

Table 7—Dimensions of Alveolar-Capillary Membrane Reference

and Its Components

(Nanometers)

Alveolar-capillary Membrane

Epithelium

Epithelial Base Membrane

Endothelium

Endothelial Base Membrane

390-2,070

249

77

238

79

40-65

110-160

20-400

110-160

60-120

46-230

25

10

Divertie & Brown

10

Schulz

11

Sehulz

12

Low

600-2,450

13

Nagaishi & associates

Karrer

75

Kisch (dogs, rabbits)

Kapanci et a l (monkeys)

Weibel

81

22

250-500

250-500

620

640

100

(mice)

Weibel & Knight (rats)

Yasuda

10

800

16

(mice)*

18

22

300-700

17

Robertson

100-500

1 4

Loosli & Baker 16

200

AMT**-1,250; HMTt-570

19

2,540

2 0

(mammals)

25-50

(mammals)

AMT**-1,500 HMTt-500 "Arithmetic mean thickness.

"Thinnest measurements.

of h o m o g e n e i t y within the group, t h e c a l c u l a t e d X value was 1,814 c o m p a r e d to a critical value f o r X of 4 3 ( w i t h 24 degrees of f r e e d o m at t h e 0 . 0 1 level of s i g n i f i c a n c e ) . T h e inhornogeneity confirmed b y this analysis is interpreted as principally due to nonuniform compression of tissue during preparation.

2

2

T h e ratio of A C T to I C T is i n d e p e n d e n t of t h e degree of lung inflation. Individual lungs, h o w e v e r , showed a significant variation in the ratio of A C T to I C T . T o test the hypothesis of h o m o g e n e i t y with t h e group, the c a l c u l a t e d X value was 3 3 3 . 9 8 c o m p a r e d to a critical value for X of 20.1 ( w i t h eight degrees of f r e e d o m at the 0.01 level of s i g n i f i c a n c e ) . T h i s represents a m u c h smaller d e g r e e of variation than was f o u n d a m o n g the fractional volumes of t h e lung. 2

2

T h e c h a n g e in total fractional tissue volume ( A C T a n d I C T ) from 3.5 p e r c e n t to 3 7 . 1 p e r c e n t of the respiratory portion of the lung b e t w e e n previously c a l c u l a t e d v a l u e s a n d the present ones represents approximately a tenfold increase due to difference in d e g r e e of inflation. S u r f a c e areas c o m p u t e d on t h e basis of p r e d i c t e d lung volumes w o u l d b e e x p e c t e d to increase b y a similar f a c t o r over previous estimates. T h i s w a s f o u n d to b e so ( T a b l e 4 ) with alveolar s u r f a c e a r e a b e i n g 9 5 3 M and capillary surface area 7 6 9 M . 2

2

2

I t has b e e n e s t i m a t e d that a b o u t two-thirds of t h e alveolar s u r f a c e represents "respiratory s u r f a c e " overlying alveolar c a p i l l a r i e s . Alveolar surface 11

272

CASSAN, DIVERTIE, BROWN

fHarmonic mean thickness. area, therefore, m i g h t b e expected to exceed capillary surface area b y 5 0 p e r c e n t ( a factor of 1 . 5 ) . T h i s p e r c e n t a g e represents an overestimate b e c a u s e t h e capillary surface is convex and thus larger than t h e surface represented b y the overlying alveolar epithelium. F u r t h e r m o r e , that portion of capillary s u r f a c e at right angles to the alveolar surface is not included w h e n considering opposing surfaces. Indeed, t h e present study shows t h e m e a n ratio of alveolar to capillary surface to b e 1.31 in normal lungs. Surface-to-volume ratios ( T a b l e 5 ) were determ i n e d by referring alveolar or capillary surface areas to total tissue volume or its components, n a m e ly, A C T and I C T . T h e results serve to emphasize t h a t in n o r m a l lungs, alveolar surface exceeds capillary surface area a n d the volume of I C T is smaller than that of A C T . T h e largest ratio, 1.17, was thus o b t a i n e d b y comparing alveolar surface to I C T . W h e n calculated with reference to both alveolar and capillary boundaries, t h e A M T of the alveolarcapillary m e m b r a n e was 2.551 fim. I f tissue volume was related only to capillary surface, a method pref e r r e d w h e n diffusion problems are being conside r e d , the A M T was x l . 6 6 5 /mi. Both values correspond in general to those established b y ourselves and others f o r t h e thickness of the blood-air barrier using b o t h m o r p h o m e t r i c and nonmorphometric t e c h n i q u e s . Values f o r animal lung have b e e n s o m e w h a t smaller w h e n m o r p h o m e t r y has been 2

CHEST, 65: 3, MARCH, 1974

used ' 1 2

but

2 2 , 2 3

Weibel

2 4

suggested

that

human

n e e d s o f different species, that of t h e pigeon b e i n g

lung would h a v e larger dimensions at t h e blood-gas

a b o u t h a l f that of t h e c h i c k e n a n d t h r e e to ten

interface. I n our study, t h e tissues of t h e alveolar-

times t h i n n e r than t h e t o a d .

capillary m e m b r a n e a p p e a r e d m o r p h o l o g i c a l l y nor-

species, h o w e v e r , m i n i m a l b a r r i e r thickness is almost

mal,

i d e n t i c a l a n d t h e ratio o f A M T to H M T is approx-

and

there

was

no

evidence

of

interstitial

edema.

2 9

Among mammalian

i m a t e l y t h e s a m e from o n e to a n o t h e r .

27

fixation

T h e results o f t h e p r e s e n t study on biopsy m a t e -

and processing h a v e b e e n f o u n d previously to a p -

rial o f n o r m a l h u m a n lung i n d i c a t e that analysis b y

proximate unity a n d should n o t a l t e r t h e s e m e a -

stereologie m e t h o d o f t h e relative volumes of tissue

surements. Variables introduced by the use of pre-

c o m p o n e n t s in alveolar septa a n d m e a s u r e m e n t of

C o r r e c t i o n factors necessitated b y tissue

2

dicted lung volumes affect s u r f a c e a r e a and tissue

t h e A M T a n d H M T o f t h e b l o o d - a i r b a r r i e r give

volume proportionately

values c o m p a r a b l e to those d e s c r i b e d b y W e i b e l

and should n o t

influence

2

determinations of thickness that d e p e n d on t h e s e

for inflated w h o l e lung. I t w o u l d seem possible to

measurements.

apply this m e t h o d o f m e a s u r e m e n t o f c o m p a r a t i v e

However,

a

disporportionate

in-

crease in alveolar s u r f a c e a r e a m i g h t o c c u r w i t h

studies of h u m a n l u n g biopsy m a t e r i a l in w h i c h

progressive

interstitial disease is present.

degrees

of

inflation,

25

resulting

in

a

lower value for A M T than would b e o b t a i n e d on uninflated l u n g .

25

T h e u s e of capillary surface a r e a

alone in the calculation o f this p a r a m e t e r has t h e advantage o f excluding t h e influence o f inflation on alveolar surface area and defining A C T in terms o f capillary size. Additionally, it permits an estimate of the thickness of that portion o f t h e alveolar s e p t u m directly r e l a t e d to gas e x c h a n g e b y excluding s e p t a l areas devoid of capillaries a n d alveolar s u r f a c e t h a t does not overlie capillary walls. T h e latter c a n a c count for up to 3 0 p e r c e n t o f total alveolar s u r f a c e area,

11

and in t h e p r e s e n t study t h e A C T / I C T r a t i o

was 1.33. V a l u e s f o r a r i t h m e t i c m e a n thickness d o not include the s u r f a c t a n t layer. W i t h m e t h o d s f o r preservation and e l e c t r o n m i c r o s c o p i c

visualization

this is e s t i m a t e d to b e 1 0 0 to 2 0 0 A thick, a u g m e n t ing t h e total thickness o f t h e blood-air b a r r i e r b y 3 percent " 2 8

2 8

H a r m o n i c m e a n thickness of t h e alveolar-capillary m e m b r a n e is d e t e r m i n e d b y a m e t h o d i n d e p e n d e n t o f point c o u n t i n g volumetry a n d s u r f a c e intersection and does not involve A C T / I C T

subdivisions.

Its

value has b e e n shown to r e l a t e m o r e directly to diffusion o f g a s e s ,

19

and in this study it was f o u n d to

b e 0.829 //.m. T h i s is slightly l a r g e r t h a n parameters mals

2 2

determined

morphometrically

similar in

ani-

and is in k e e p i n g w i t h w h a t h a d b e e n ex-

p e c t e d in h u m a n t i s s u e .

24

In t h e p r e s e n t study, t h e

ratio of A M T t o H M T varied b e t w e e n a f a c t o r of 2 and 3 depending on t h e m e t h o d used to c a l c u l a t e A M T . T h e s e values are similar to those r e p o r t e d in a number of mammalian species.

27

T h e ratio reflects

t h e f u n c t i o n a l optimization b e t w e e n structural integrity a n d gas p e r m e a b i l i t y in t h e b l o o d - a i r b a r r i e r , w h i c h appears to b e m a i n t a i n e d regardless o f t h e d e g r e e of lung inflation. W i t h t h e b u l k o f tissue in all layers c o n c e n t r a t e d at t h e thickest portion of t h e blood-air

barrier,

its

structural

integrity

can

be

maintained a t m i n i m a l e x p e n s e to its p r i m a r y f u n c tion of gas transfer. T h i c k n e s s of t h e alveolar-capillary m e m b r a n e m a y b e a d a p t e d to t h e m e t a b o l i c CHEST, 65: 3, MARCH, 1974

REFEHENCES

1 Delesse A: Prooede mechanique pour determiner la composition des roches. C R Acad Sci [D] (Paris) 25:544545, 1847 2 Weibel ER: Morphometry of the Human Lung. New York, Academic Press, Inc, 1963, p 151 3 Weibel ER, Kistler GS, Scherle W F : Practical stereological methods for morphometric cytology, J Cell Biol 30:2338, 1966 4 Freere RH, Weibel E R : Stereologie techniques in microscopy. J R Micr Soc 87:25-34, 1967 5 Sabatini DD, Bensch K, Barrnett RJ: Cytochemistry and electron microscopy: the preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. J Cell Biol 17:19-58, 1963 6 Dalton AJ: A chrome-osmium fixative for electron microscopy (abstract). Anat Rec 121:281, 1955 7 Luft J H : Improvements in epoxy resin embedding methods. J Biophys Biochem Cytol 9:409-414, 1961 8 Watson ML: Staining of tissue sections for electron microscopy with heavy metals. J Biophys Biochem Cytol 4:475-478, 1958 9 Venable JH, Coggeshall R: A simplified lead citrate stain for use in electron microscopy. J Cell Biol 25:407-408, 1965 10 Divertie MB, Brown AL J r : The fine structure of the normal human alveolocapillary membrane. JAMA 187: 938-941, 1964 11 Schulz H: Some remarks on the sub-microscopic anatomy and pathology of the blood-air pathway in the lung. In Pulmonary Structure and Function (Ciba Foundation Symposium) (de Reuck AVS, O'Connor M, eds). Boston, Little, Brown & Co, 1962, pp 205-214 12 Schulz H: Elektronenoptische Untersuchungen der normalen Lunge und der Lunge bei Mitralstenose. Virchows Arch [Pathol Anat] 328:582-604, 1956 13 Low FN: The pulmonary alveolar epithelium of laboratory mammals and man. Anat Rec 117:241-263, 1953 14 Nagaishi C, Okada Y, Ishiko S, et al: Electron microscopic observations of the pulmonary alveoli. Exp Med Surg 22:81-117, 1964 15 Loosli CA, Baker RF; The human lung: microscopic structure and diffusion. In Pulmonary Structure and Function (Ciba Foundation Symposium) (de Reuck AVS, O'Connor M, eds). Boston, Little, Brown & Co, 1962, pp 194-204 16 Karrer H E : An electronmicroscopic study of the fine structure of pulmonary capillaries and alveoli of the

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mouse: preliminary report. Johns Hopkins Med J 98:6583, 1956 Kisch B: Electron microscopy of the lungs in acute pneumonia. Exp Med Surg 18:273-296, 1960 Robertson J D : The ultrastructure of cell membranes and their derivatives. Biochem Soc Symp 16:3-43, 1959 Weibel ER, Knight BW: A morphometric study on the thickness of the pulmonary air-blood barrier. J Cell Biol 21:367-396, 1964 Kapanci Y, Weibel ER, Kaplan HP, et al: Pathogenesis and reversibility of the pulmonary lesions of oxygen toxicity in monkeys. 2. Ultrastructural and morphometric studies. Lab Invest 20:101-118, 1969 Yasuda H: Electron microscopic cytohistopathology. IV. A study of normal adult and fetus lung in mammals as revealed by electronmicroscopy. Acta Pathol Jap 8:189213, 1958 Weibel E R : Morphometric estimation of pulmonary diffusion capacity. 5. Comparative morphometry of alveolar lungs. Resp Physiol 14:26, 1972 Weibel ER: Dimensions of the tracheobronchial tree and alveoli. 2. Alveolar-capillary air-blood barrier: mammals,

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respiration and circulation. In Respiration and Circulation (Altaian PL, Smith DE, eds). Bethesda, Federation of American Societies for Experimental Biology, 1971, p 112 Weibel E R : Airways and respiratory surface. In The Lung. (Liebovv AA, Smith DE, eds). Baltimore, The Williams & Wilkins Co, 1968, pp 1-18 Untersee P, Gil J, Weibel ER: Morphometric characteristics of air-blood barrier at different degrees of inflation. Bull Physio-Pathol Resp 8:139, 1972 Gil. J, Weibel ER: Improvements in demonstration of lining layer of lung alveoli by electron microscopy. Resp Physiol 8:13-36, 1969 Weibel ER: The ultrastructure of the alveolar-capillary membrane or barrier. In The Pulmonary Circulation and Interstitial Space (Fishman AP, Hecht HH, eds). Chicago, University of Chicago Press, 1969, pp 9-27 Untersee P, Gil J, Weibel ER: Visualization of extracellular lining layer of lung alveoli by freeze-etching. Resp Physiol 13:171, 1971 Meessen H: The pathomorphology of diffusion processes in the lung. Stanf Med Bull 19:19-31, 1961

The Enigma Of Hibernomas And Pertinent Reflections One may question the merit of contemplating this benign neoplasm in view of its extremely rare occurrence in clinical practice (less than 100 reported cases). Too, skepticism may be strengthened by controversial nosologic considerations. Some regard hibernoma in humans as consisting of lipoid material which is but an immature fetal developmental phase of normal human fat tissue. It has been ascertained that in human embryos "brown fat," probably as a transition to "yellow fat," exists in the interscapular, subpleural and axillary regions. Others consider it as a vestigial structure essentially identical with the brown fat of the hibernating glands of certain animals. In the latter, the hibernating gland is of light brown color, lobulated and highly vascular. Its central mass is localized in the superior mediastinum, with extensions into the neck, axillas, the back, perirenal, retroperitoneal, inguinal and gluteal areas. Hook, W F (Proc Soc Exper Biol and Med 4 5 : 3 7 , 1 9 4 0 ) postulated that the endocrine function of this gland was the lowering of metabolism during hibernation. T h e onset of hibernation is preceded by endocrine changes, including involution of the thyroid gland, adrenal cortex, and hyperactivity of the parathyroids, and increase in the number of cells in the isles of Langerhans. Consequently, there are lowering of the body temperature, reduction in the metabolic rate and oxygen consumption, bradycardia and bradypnea with Cheyne-Stokes breathing. Induction and control of these changes originates from a putative "biologic gyrostat" in the hypothalamus, Berlin, L et al (in Larousse Encyclopedia of Animal Life, New York, McGraw-Hill, 1 9 6 7 ) mention that in 160 days a hibernating marmot loses one quarter of its original weight. During hibernation the animal lives off its own fat. These observations offered reasonable basis for the suggestion by Nelson, R A (Mayo Clin Proc 4 8 : 7 3 3 , 1 9 7 3 ) in reference to room for storage of food and water in spacecrafts making prolonged flights. "A fascinating problem would be to devise a scheme whereby these principles could be applied in prolonged space flights for humans. This regimen could be combined with a method of selecting obese astronauts and providing a quantity of calories which would be large enough to spare body

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protein but small enough to permit mobilization of adipose tissues gradually over the duration of the flight." The first case of human hibernoma was reported by Merkel, H over 60 years ago (Beitr Path Anat 3 9 : 1 5 2 , 1 9 0 6 ) . Gery, L introduced the term hibernoma a few years later (Bull Mem Soc Anat Paris 8 9 : 1 1 1 . 1914) because of the resemblance of the neoplasm to the hibernating gland of some animal species. The tumor has been observed mostly in young adults. The youngest reported case was six weeks of age, the oldest one was 60 years of age. About seven percent of human hibernomas were intrathoracic. Others were observed in the neck, interscapular area, axilla, lumbosacral and gluteal regions, popliteal space, thigh, abdominal wall, adjacent to the kidneys, and intracranially. Hibernoma is of brown, brownish-yellow to tan in color but milky-white appearance has also been noted. Thoracic hibernoma may be asymptomatic and discovered on routine x-ray examination or it may cause cough, wheezing, dyspnea and pain. It is revealed in the roentgenogram as a spherical, smooth-contoured, sharply demarcated opacity with increased radiolucency at its periphery. Resected specimens are covered by a delicate capsule, finely lobulated and of spongy consistency. Morgan et al (Thorax 2 1 : 1 8 6 , 1 9 7 2 ) reported that the phospholipid content of surgically removed thoracic hibernoma was 3 6 times greater than that in normal adipose tissue. It is well to refer to the electron microscopic findings of Levine, G D (Human Path 3 : 3 5 1 , 1 9 7 2 ) . He notes that hibernoma is "composed predominantly of multivacuolated cells with granular eosinophilic cytoplasm. All graduations are present between multivacuolated and univacuolated cells. T h e cells contain lipid vacuoles and numerous pleomorphic mitochondria; the latter account for the granular cytoplasm seen histologically and are responsible for the relatively high phospholipid content of the hibernoma." Apropos of the aforementioned article of Nelson, perhaps it is not beyond conceptual realism to anticipate that the isolation of the specific endogenous hibernation-inducing substance might lead to its appropriate use in humans. Andrew L. Banyai, M.D. CHEST, 65: 3, MARCH, 1974