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Studies of the lining of the pulmonary alveolus of normal lungs of adult animals.

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Dcpnrttnent of Anatomy, The Universily of Clircccgo
In the c1evelo~)mentof a iicw techiiique for demonstrating
the ground substance in the fine reticular framework of various organs, some light has been thrown on the structure of the
wall of the pulmoiiary alveolus. The technique is as follows :
A mouse is injected intravenously (through the tail rein)
with 100 mg. of gold sodium thiosnlphatc in 1 cc. of distilled
water. If it is not possible t o inject it all into the tail rein,
ihe rest is given intraperitoiieally. The mousc dies in 20
minutes from asphyxia. The blood seems to thicken aiid circulation becomes sluggish althougli the heart contiiiues to
beat. Respiration fails and the mouse goes into slight convulsions. Whcu the chest wall is opened, the lungs appear less
collapsed than usual.
Pieces of tissue are fixed in 10 per cent neutral formalin,
dehydrated mitliout washing and embedded in paraffin and
sectioned, After the para611 has been removed from tlie
section, it may be stained with toliiidiiie blue accordiiig to the
technique described in an earlier paper (S. H, BensleT, ’34).
The ground substance is stained metacliromatically pink while
the cells axid their nuclei are blue. Such a preparation of the
mouse’s luiig shows a greater amount of meiachromaticallp
stainiiig ground substance than usual. It appears to have
heen swollen by the intravenous tliiosulphate and fixed in that
TFThen tlie alveolar wall in a freshly stained preparation
mounted in water is analyzed under the oil immersion lens, it
is seeii to be composed of a central layer of ground substance.
Some times the central layer is in relalion t o capillaries on
either side of it, and sometimes it lies adjacent to the air space
on one side with a capillarly interrening betweeii it and the
alveolar space on the other. Often the capillaries are completely surrounded by the pink ground substance. But in
almost every case, a thin blue membrane intervenes between
the ground substance and the alveolar spaces. Where no
ground substance is seen snperficial to a capillary, this membrane is distinguishable from the thin capillary eiidothelium
only as a doublc line, save where it bulges to include a
The chief advantage of this technique seems to lie in tlie
swelling of the connective tissue ground sribstaiice, thereby
enlarging structures ~7hichotherwise collapse or shrink at
death or oil fixation. It, has been particularly of advantage
in the study of the alveolar malls of the lung and the siiiusoids
of the liver.
T o determine whether this membrane is indeed a cellular
one or not, the method used by almost all investigators of the
lung, namely the attempt to outline cells with silver and
photograpliic developrneiit, was tried with some modificat'lolls
and improvements. Fortunately the guinea pig was chosen as
the experimental animal. Instead of pouring the silver solution down the trachea, the roots of the lung mere ligated and
the silver solutioiis introduced by stick injectioii with a hppodermic needle into the lmig substance until the lung was
moderately distended. Pieces were then fixed in 10 per cent
formalin, run np, embedded in celloidin or paraffin, sectioned,
developed with photographic developer and staiiied OF examined unstained. Frozen sections were made, developed and
examined at once.
Varions silver soliitioiis were used : Silver citrate, made according to the technique described in an earlier paper
(Bensley, 'as),silver nitrate in concentrations from 5 per
cent to 10 per cent, and Kalitype solution made as follows:
Ferrir oxalate
Oxalic acid
Silver iiitrate
Distilled water
15.6 gm.
6.25 gm.
The ferric oxalate is dissolved with the oxalic acid in warm
water, then filtered and the silver nitrate added.
I n thcse experiments the outlining of cells is most complete
and cleaii following the silver citrate iiijections. Though even
here the areas of complete outlining are patchy. In favorable
areas from snch a section developed with fine grain photographic developer, the endothelial cells of the capillaries are
outlined. If the outlining is complete, as is the case in the
larger vessels, the cells are similar in size and distribution to
those in ordinary capillaries in other parts of ihe body.
Where tlie capillaries are stretched and thin, the eiidothelial
outlines appear a s two thick black lilies, one at each periphery
of the capillary, extending. parallel to the long axis of the
capillary along its whole extent.
The apparent complexity of the capillary network is consideral)ly reduced by stretching the alvcoli, which is accomplished by stick injection. I n the collapsed lung the capillaries, distended, convoluted and superimposed, oiic o n the
other, present the picture of a very rich vascular bed, obscuring other structures. When tlie capillaries are stretched,
straightened and collapsed, the aiiastornoses are spread apart
and the intercapillary spaces are increased.
But there are more thaii endothelial cell outlilies in sixch a
section. I n tliiii sections where the alveolar walls are cut
laiigentially o r obliquely, sheets of cells whose outliiies are in
one plane of focus under oil immersion are seen, overlying or
iinderlying the capillaries (fig. 1). The area over which they
extend excludes the possibility of their being eiidothelial cells
of the collapsed capillaries viewed from the surface. Uoreover, they do not correspond to the pattern of eiidothelial cells
seen in sections where 0111;~the capillaries arc completely
That tliese outlines a r e not reticular fibers is evident from
a section of the same material which had been toned in gold
chloride to protect the original silver outlines, and subsequently the reticular fibers impregnated by tlie Bielschowslq
method. Here the ontliiies of the epithelial cells a r e quite indcpeiident of the reticular fibers definitely overly them, and
tlie patterns of the two a r e in no way similar (fig. 2 ) .
TTnfortanately, the silver citrate solution inhibits the adsorption of dyes in these areas and the cells and their nuclei
a r e oiily faintly stained, if at all. However, with the use of
the Kalitype solution, though the cells are less clearly outlined,
they stain more readily, and in a section from such material
sheets of these large cells faintly outlined with silver yet contiguous, sometimes containing a resicular nucleus, a r e seen
lining the alveolar space (fig. 3).
In espcriments on the rabbit, rat mid mouse, this definite
outlining of the epithelial cells has not been a s clearly or completely obtained. Here the picture is complicated by tlie
presence of small, heavily stained and outlined cells, incompletely outlined plates and all intermediate forms (fig. 4).
This may be clue to some fault in technique, namely, insufficieiit
injection of silver solution or to a slight structural difference
in the epithelial cells. Some e\-ideiice in support of the latter
view is afforded by findings in some subsequent experiments.
Some further evidence of the existelice of a n epithelial membrane between the capillaries and the lumen of the alveolus
has been afforded by a stadr of the distribution of chlorides
in interalveolar septa.
Fig. 1 Canieia lucida drawing of an alveolar wall, cut tangentially, in a
section of guinea pig lung which was stick injcctcd with silver citrate solutinn.
fixed i n formalin, embedded, sectioned a t 5 p and de\clopcd with photographic
developcr, X 1440.
Fig. 2 Camera lucida draiTing of an alveolar wall, cut tangcutially, in a section
of guinez pig lung whic.11 was stielc injcrted with silver eitrate solntioa, fixed in
formalin, embedded, sectioned, rlereloped and iriiprcgnated by the Xielschowsky
nirthod for reticular fibers. x 1140.
Fig. 3 Caincra lueida drawing of a n alveolar wall, Lut tangcntially, in a
section of guinea pig lung which was stick injected with Kalitype solution, fixed
in formalin, emlwdded, sectioned, dcvelopcd, and stained with Ehrlich’s acid
hematoxglin. X 1440.
A new technique for the demonstration of chlorides in cells
and tissues was snggested by a stuclcrit in chemistry, MF.
David M. Ritter. T!i titrating sodium chloride with silver
iiitrate a few drops of a neutral solution of dichlorfluorescciii
are used as the indicator. When the end-point is reached (in
Fig. 4 Camera lucida drawing of an alveolar mall, vieivcd from its surface,
i n a 50 p paraffin section of a young rabbit’s lung which was stick injected with
silver citrate solution, fixed in fornialin, embedded, scctioned and developed with
pliotographic developer. Notc the small dark heavily outlincd cells, and the large
X 1440.
the presence of a slight excess of silver ions) the flocculent
precipitate of silver chloride assumes a pinkish color due to
the adsorption of the dichlorfluorescein to the positively
charged silver chloride molecule to form a complex molecule
which gives a distinctive color (Kolthoff, Lauer and Sunde,
Tllis reactioll as applied to the lung of the rabbit iii the
f ollowiiig way. A 1 per cent, solntion of clichlorfluorescein
was injected intravenously into the animal until it mas quite
yellow. The animal was then killed aiid a 10 per cent solution
of silver nitrate OF silver citrate solution was introduced
either intratracheally or by stick iiijectioii into the lung suhstalice until the lung w7as moderately disteiicicd. h period of
about 20 minutes mas required for the color reaction to reach
its maximum. Pieces of lung werc then fixed in 10 per ccnt
neutral formalin aiid frozen sections were made. Tlie sections
were deliydrateci and cleared in absolute alcohol and iso-safrol
a i d mounted in balsam. Some pieces were fixed in formalin,
dehydrated aiicl embedded in paraffin rapidly.
This color reaction is not pcrmaiient hut is masked aiid
finally lost with the browning and then blackening of the
silver. This change is somewhat hastened by embedding in
paraffin. Therefore, frcsll or frozen sections examined immediately arc best to indicate the presence of chlorides.
This reactioii i n tissues is not a microchemical stain in the
sbi-ict sense of the word. It does detect the presence of
chlorides. Bat as in the case of a gelatin film, the chlorides
are mobilized h;v the silver and tend t o migrate toward the
periphery of the cell.
\.\'it11 this technique mesothelial cells and cndothelial cells
are brilliantly and completely outlined in pink. The bronchiolar epithelium is also outlined in pink at the free borders,
much like the terminal bars in the intestinal cells. The deeper
cell outlines are fainter and less complete. The epithelial cells
of the alwoliis, however, are outlined only by a pink stippling.
The cytoplasm of these cells also shom a piiik stippliiig; the
nuclei are large, oval, clear and give a rather rich stippled
chloride reaction.
If the reaction is allowed t o continue in the excess of silver
solution, the mcsothelial and eiidothelial outlines become black,
those of the broiichiolar epithelium dark brown to black and
the ground substance shows a heavy brown precipitnte. The
periphery of the small cells in the lining of the alveoli become
darkened to a deep brown or black, while the larger epitlielial
cells wliich complete the lining of the alveolus maintain their
pinkish stippling for a iiiuch longer time ; this, however,
eventually turns black aiid these cells may become outlined
in black. (With the silver stick injections these small cells
were heavily stained and outlined wliile the larger cells appeared as pale plates outlined in black.)
On closer iiispection of such material, the capillaries can
be identified by the black outlines of their endothelial cells.
Overlying these and ensheathing them stretch the pink
stippled alveolar epithelial cells, whose outlines are difficult
to determine except by a slightly richer stippling along the
borders of the cell. The smaller, heavily outlined cells seem
to be cells udiich have not shared in the stretching effect of the
larger plates and therefore map protrude into the lumen of
tlic alveolus and appear in shapc and outliiiiag much like tlie
free surfaces of the broncliiolar epithelium, when viewed from
the surface.
From these observations it seems that two processes are
taking place. The first is the formatioii of silver chloride dic4ilorfluoresccin7 which subsequently becomes blackened. The
second is the ‘impregiiation’ of the tissues by reduced silver.
It appears that while the cenient substances of niesothelial
cells, endothelial cells, and bronchial-epithelial cells, and tlie
ground substaiice are rich in chlorides, the cement substance
of the alveolar epithelium, if present, is poor in chlorides.
The cytoplasm of these epithelial cells, however, contains a
considerable amount of chlorides. They are outlined by
silver, by the migration of the chlorides to the periphery of
the cells aiid perhaps by tlie adsorption of reduced colloidal
silver provided by photographic development. This may account f o r the variable pictures which a r e obtaiiied with the
silver solutions.
In summarizing, we may saj- that the pulmonary alveoli of
adult mammals a r e lined by a continuous cellular membrane.
This membrane is best visnalized if the alveoli a r e distended
before fixation. The ground substance of the alveolar septa
may be made to swell with intravenous gold sodium thiosulphate which prevents the lung from collapsing as much as
usual. Subsequently the membrane may be differentiated
from the capillary endothelium by staining the intervening
ground substance metachromatically.
These cells may be outlined in the guinea pig lung fairly
easily and completely by stick injection of the lung with silver
citrate solution and subsequent photographic development,
and less completely in the rabbit, rat and mouse. Stick injection seems preferable to intratracheal injection because it replaces the air and fluid from below and thereby the solution
comes in direct contact with the cells over a more 'extensive
area in greater concentration. Moreover, the stick injection
tends to produce a better distention of the alveoli.
The epithelial cells of the pulmonary alveoli may be further
identified by their colored chloride reaction, when dichlorfluorescein is injected intravenously into the living animal and
a stick injection of the lung with excem of silver solution is
made. The reddish stippled cells are seen overlying and ensheathing the capillaries whose endothelial cells are outlined
in black.
ROBERTDANIEL 1929 The efferent vesseIs of the renal glomeruli of
mammah as a mechanism for the control of glomerular activity and
pressure. Am. J. Anat., vol. 44, p, 146.
HOLTON1934 On the presence, properties and distribution
of the intercellular ground substance of loose connective tissue. Anat.
Ree., vol. 60, p. 96.
I. M., w. hf. LAWRAND c. J. SUNDE 1929 The use of dichlorofluorescein as an adsorption indicator for thc argento-metric titration
of chlorides. J. Am. Chem. Soe., vol. 51, pp. 3273-3277.
64, NO. 1, A N D SUPPLFAfENP NO. 1
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adults, pulmonaria, animals, lung, lining, norman, studies, alveolus
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