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The so-called neutral red vacuome and the golgi apparatus.

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I N TROD UCT 1ON
Sirice Rensley (’10) aiinoiinced his vacuolar theory of Ihc
nature of the canaliciilar apparatus in aiiirnal cells, the theory
has been appropriatcd in toto by Quillierniond ( ’Ti),wlio uses
in place of the term ‘vaenole’ the term ‘vacuome,’ applied by
Dangeard to the ciisemble of vacuolar structures in a vegetable
oell. Guilliermorid and his pupils iouiid tliwt this vacuolar
system in the plant cell frequently segregated neutral red
vjlieii applied as a vital dye t o the living cell, arid this method
has been of great service to him and his associates in estendiiig the factual background of the vacuolar hypothesis which
he terms the ‘vacuome’ hypothesis. This hypopthesis has
been accepted in rather motlified form by Parat ( ’24, ’26) and
his pupils, 77’110 havc studied extensively tlie system o i bodies
appearing in ariiiiiul cells after a vital staining with rieatral
red. Parat’s views, in a concentrated form are: that the
neutral red-staiiied bodies found under these circumstarices
in animal cells are the vacnome, arid that this vacuome is tlie
Golgi apparatus, although in his recent articles lie bas enlarged this hypothesis t o include extravacuolar deposits associated with functional activity.
The vacuome hypothesis known t o tlie majority of workers
with animal cells is not based oil tlie vaciiome of Gnilliermond,
but on the neutral red bodies of Parat. Accordingly, we find
these neutral red bodies termed vacuomc and their interpre95
T H E A X l T O Y I C ~ L RECORU. VOL. 6 2 , KO.
1
96
HIU C. CHANG
tation taken for granted. It should he noted lliat the validity
of this position of P a r a t depends upon the validity of his assumption that neutral red per se is ail indicator of primary
vacuolar spaces in the cytoplasm I f this be not true, Parat
is not eiilitled to call his bodies stained with neutral red by the
term vacuome, which has a different significance iii the plant
kingdom, and it is on the validity of' this assumption that
much of the iiivestigatioii coriceriiiiig canalicular apparatus
and Golgi apparatus in animals cells binges.
It should bc pointed out that the adequacy of the vacuolar
thcory of the nature of the Chlgi apparatus does not in any
sense depend upon the correctness of this assumption.
Xeutral red bodies are either pre-existent vacuolar spaces,
or they are not. I n the latter case they are, ips0 facto, not
vacuoles in the sciise of Berisley axid Ouilliermond. The validity of Pnrat's primary assumption has been vigorously
attacked by various workers, in particular by Ludford ('30),
by I3cams ( '30 a mid b ) , and by Weatherford ( '32). Reams
lias attacked this point of 17im-in a series of interesting papers
iii which lie has shown that the ~ieutralred bodies once dcmoiistrsted can be blacliencd with osmic acid like the Golgi apparatus, but ofteii the two images do not coincide with o i i ~
another. Ile suggests that the neutral red bodies d o not represent pre-existent elements, but are due to the reaction between
the dye and the cell.
Michaelis ( '00) has shocvn aiid Weatherford ( '3%)has recently confirmed the fact that the cells of the liver contain in
the living conditioii granules which readily stain with neutral
red. They coiiicide also in position mith the Golgi apparatus,
as demonstrated by the I<olatchev method. Ludford ('30)
studied the pancreas of the mouse after vital staining with
iieatral red. IIe says :
Following the injection of neutral red iiito liviiig mice, dye
droplets appear ill the acinar cells. I have not seen them appear when the pancreas is teased out j n saline contaiiiing
neutral red and examined uiider the microscope at room
temperature, although by this method secretion granules arc
ultimately stained. Such experiences suggest that the formation of the dye droplets is brought about by the vital activity
of the cells and is not due to a passive staining of prcformetl
droplets.
After osmic impregnation, he noted that the osrnicated reticul a r structure (the Golgi apparatus) was broken up and among
the strands occurred large osmicated bodies. The appearance
presented suggested that tlie dye di-oplcts had caused the
reticular structure of Oolgi to become dilated in places and
lielice destroyed its continuity. H e thus confirmed, in general,
the findings of Beams, but differed from his view that tlic
iieutral red bodies were never fused with the Golgi apparatus.
Ludf ord concedes in this paper that the canalicular apparatus
seen in mitochondria1 preparations is the same as the Golgi
apparatus seen iii silver and osmic preparations, but insists
that neutral red bodies belono. t o a differelit category.
?
The qaestion of the pre-existence of the neutral red bodies
seen in the preparations made according to the method of
P a r a t is of paramount importance t o the vacuome hypothesis
as enunciated by him. If the neutral red bodies are newformed elements contingent on the action of the dyc on the
cell, then the vaciionie hypothesis of P a r a t immediately goes
to ground. This demonstration, however, does not affect the
validity of the vacuolar hypothesis as first enunciated by
Bensley.
O’Learg- ( ’SO), studying iinder high powers of the microscope the pancreas of the white mouse in the living animal b~
the method introdiiced by Cove11 (’28), fouiid that in the beta
cells of the islands of Langerhaiis licutral red granules developed after injection of neutral red and could 11e seen independently of the canalicular spaces pi*eviously observed by
Berisley in these cells, indicating an indepclldeiiee of the
neutral red graniiles and tlie caiialiciilar system.
The whole confusion lias risen from the application of the
term vaciiome to the structures stainable by neutral red mithout first dcmonstratiiig the homology of these structures with
the vacuolai. system of the plant cell. b‘or the solutioii of this
98
HITJ C. CFIAR’G
problem it is necessary to consider the well-known fact that
neutral red stains a vast category of iiitracellular materials.
T o these belong tlie phagocytosed material in tlie protoplasm
of the histiocFte, the rosette of the monocytc, the neutral red
granules of Michaelis in the liver cell, arid so forth. Cells of
this sort which alreadJ- coritaiii granules staiiiable with neutral
red are not satisfactory material f o r tlie elucidation of tlie
problem. 1 have re-stnclied this question, using tlie paiicreas
of the mouse a s the experimental material.
IIATERTMA A N D JfETHODS
F o r tlie demonstration 01 neutral red bodies, neutral red
in 1t o 5 per ceiit solution in Ringer’s fluid was injected subcataiieoizsly. The paiicreas was observed i n the living aninials
during the process of tlie experiment, and for osmication t o
demolistrate the Golgi apparatus, the pancreas was put into
Rlann’s osmic liichloride solution and kept in tlie dark for
various periods, up t o 2 weeks.
OBSERVATIONS
I n the pancreas of the living mouse neutral red bodies begin t o appear about 15 minutes after iiijectioii. At first only
ARBREVIATTONS FOR A L L FIGURES
c.t., connectivc tissue
m., cutirlr
4, Golgi appaiatus
Iy”’., lym~rlloc!,t c
n.g., negative Golgi
n.r.g., neutral red granules
r.b.c., red blood cell
e, zvniogcn granules
[i’ig. 1 Aciiins frotii the pancwas of a niouse staiiied intravital!y with iieutial
red and then fixed for 5 hours i n Ilurin’4 solution. Nvutral rcd bodies blackened
particiilarly on the surfare.
E’ig.2 Same preparation as figure 1, except that the pancreas was fixrd in
Mann’s solution for 3 days. l T e u t r a l red bodies more intcnselg stained black, hut
with still peripheral Rtaining. Slight irregularity of outline. Golgi apparatus heginning t o blncken in t h e form of strings among the zymogen granules.
Fig. 3 Hame material as figures 1 and 2, but kept in Mann’s solution f o r 15
days. Both Golgi apparatus and neutral red bodies are blackcned.
Fig. 4 ].+ithelium of epididymis t u h l r of inousc. Vitally staiiied in neutral
red, fixed in Marin’s solutioii f o r 24 hours. Many neutral red bodies and f a t
droplets blarkcncd. Golgi apparatus appears in the form of canals unstained
~ v i t hoymic.
NEUTRAL RED ‘YACUOME’ AND GOLGI APPARATUS
99
100
I-IIU C. C l i A N G
two or three a r e risible in the zone between the ii~icle~is
aiid
the zymogen granules. The size of the granules aiid their
number may be increased by extending the time or b37 incareasing the concentration of tlie dye. Two or three of tlie
granules may fuse together to make a larger one. I n the
larger neutral red granules smaller arid more deeply stained
red particles maj7 be seen. Two or t h e e liours after the
neutral red lias been injected into the aiiimal iieutral red
granules a s large a s the uucleus may be seen. After prolonged
observation racuoles appear in the cytoplasm, hnt they do not
stain with neutral red.
Figures 1, 2 and 3 show thcl progress of hlackeiiiiig of
neutral red bodies and the Golgi apparatus, respectively, in
preparations of the pancreas of a mouse which had been injected with a solution of neutral red subcntaneoiisl~1 4 hours
before killing. Thc preparations, after removal from the animal, were placed in Alanii's osmic biclilorjde solution, and then
transferred to chrom-sublimate for further fixation. I n the
first of these preparations the neutral red bodies oiily are
blackened, the hlackeniiig being more intense on the surface
of tlie granule. 111 the seeoiid, tlic ricutral red granules are
much darker, although the staining still remains peripheral,
and the Golgi apparatus is beginning to appear a s isolated
threads, paley stained. In the third preparation the lieu tral
red granules a r e intensely siaiiiecl and the Golgi apparatus j s
present a s headed strings tvliich tend t o form a network.
Thc neutral red granules and the Golgi apparatiis in these
prcparatioris occupy diff creiit positions in the cell, the Golgi
apparatus being iiear the frce border arid the zymogen granules nearer the nuclens.
Figares 4 and 5 relxesent a similar progress in the staining of the epithelial cells of the epididymis of tlie mousc. The
animal iii this case has been injected with iieiitral red solution
and then transferred to Jla~in'ssolution for 24 hours, and 6
days, respectively. In the first of these (fig. 4) the neutral red
bodies which occupy both the area ahore the micleus and the
base of the cell a r e already hlaclrened after 24 hours. I n
NEUTRAL XED k . 4 C T T O M E ’ A N D GOLGI BPPARATVS
101
Fig. 5 Epitheliuni of cpididyinis from same animal as iigurc 4. Impregnation
i n hlann’s solution for G daps. Gtrlgi apparatus more strongly staincd t h a n neutral
red bodies, xiid occupy a different loc~ationin the cell.
E’ig. 6 Intestinal epithelium of the mouse. Preparation the same a q figure 6.
In this ease the neutral rcd bodies, iiitcnsely staincd, show composite structure and
occupy a p s i t i o n iii the eel1 iieaier the frec border tliaii the Golgi apparatus,
which is partially iinpregiiated as discoiitinuous threads aiid giaiinles.
102
H l U C. C H A X G
these prcparatiom tlie Golgi apparatns is also apparcnt as in
the form of canals w-hich a r e negativc to osmic. I n the 6-day
preparation both tlie Golgi al)paratns and thc neutral red
granules are blackened. It is seen that these two elcmcnts do
not occupy the same portion of thc cell, the neutral red pranules being nearer the nucleus and the Golgi apparatus nearer
the surface of the cell, After this prolonged immersion in the
osmic acid solution, the Golgi apparatus which in the 24-day
preparation showed in ihc negative can~licnlarform appears
a s threads which are milch blacker than the neutral red bodies
on the same cell.
Similar results were obtained by the same technique with
the intesiinal epithelium, but in this case the neutral red
bodies occupy for the most part the superficial segment of the
cell, the Golgi apparatus occupying the iiit erveriing space bctween tlie neutral red area and the nucleus.
In all these preparations it is to be noted that the neutral
..
red bodies are composite structures containing granules in
their interior which a r e more deeply staining willi iieutral red
than the main mass of tlic granule. Tn these preparations
also, some of the blackened material is fat, and, in addition,
i n tlie cells of tlie epididpmis tnhule are vacnole-like spaces
containing particles \yhich blacken with osmic acid.
STJXhIARP
In the fresh pancreas cells of Chc mouse, in thc! intestinal
epithelium and in the cells of the spiiial ganglia, neutral red
granules do not exist a s independent pre-exihtent bodies.
After the intravjtal injection of neutral red? granules stained
with this dye appear in the cytoplasm in these several sites.
The place of clioicc f o r the formation of the neutral red
the Golgi apparatus, or
granules is in tlie region occupied
ncar it. The inference drawn by others that the Golgi apparatus is necessary t o the formation of the neuiral red bodies, I
cannot accept. There is a specialized arca of thc cytoplasm
which is most fa\-orable f o r the formation of neutral red
bodies which may coincide with tlie position of tlie Qolgi ap-
NEUTRAL RED ‘VACUOXE’ AND COLGI APPARATUS
103
paratus, as claimed by Ludford, or overlap it, as demonstrated
by Beams, or be entirely outside of it as some of these preparations show.
The neutral red bodies are entirely new-formed structures
and depend upon a protective reaction of the cytoplasm
agahst the foreign material.
ACKNOWLEDGMENT
The author wishes to record his deepest appreciation of the
unusual opportunity afforded him by the Rockefeller Foundation. And he is under great obligation to Professor Bensley,
who has spent valuable hours f o r suggestions and painstaking
reading and rearranging of the proof.
LITERATURE CITED
BEAMS,H. W. 1 9 3 0 a Studies on thte vacuome and the Golgi apparatus in the
acinar cells of the pancreas of the rat. Anat. Rec., vol. 45, p. 137.
1930 b Golgi apparatus, caiialicular apparatus, vacuome, and mitochondria in the islets of Langerhans of the albino rat. Anat. Bee.,
vol. 46, p. 305.
BENSLEY,
R. R. 1910 On the nature of the canalicular apparatus of animal cells.
Biol. Bull., vol. 19, p. 179.
COYELL,W. P., AND G. H. Scorn 1928 Experimental study of relation between
granules stainable with neutral red and Golgi apparatus in nerve cells.
Anat. Rec., vol. 38, p. 377.
GUILLIERMOND,
A. 1927 Recherches SUI l’appareil de Golgi dam les cellules
vbgbetales et sur ses relations avec le vacuome. Arch. d’Anat. Micr.,
vol. 23, p. 1.
LUDFORD,
R. J. 1930 The vital staining of normal and malignant cells. 111. Vital
staining of acinar cells of the pancreas, and its bearing on the theories
of vital staining with basic dyes. Proc. Roy. Soe., 13, vol. 107, p. 101.
MICHAELIS, L. 1900 Die vitale Farbung, eine Darstellungsmethode der Zellgranula. Arch. mikrosk. Anat. u. Entwickelungsg., Bd. 55, S. 558.
O’LIF~RY,
J. L. 1930 An experimental study on the islet cells of the pancreas in
vivo. Anat. Rec., vol. 45, p. 27.
PARAT,
hl. 1926 Sur la constitution de l’appareil de Golgi e t de l’idiosome;
vrais et faux dictyosomes. G. R. Acad. de Sei., T. 92.
PAEAT,M., AND J. PAINLEV*1924 Apparcil reticulaire interne de Golgi,
trophosponge de Holmgren, et vacuome. C . R. Acad. d. Sci., T. 179,
p. 844.
WEATHERFORD,
H. L. 1932 The Golgi apparatus and vital staining of the amphibian and reptilian liver. Zeitschr. f . Zellforsch. u. mikrosk. Anat.,
Ed. 15, S. 343.
THE ANATOYIOAL RECORD, VOL. 62, NO. 1
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