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Histological aspects of the function of the malpighian body in the living frog's kidney based on studies with the fluorescence microscope.

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HISTOLOGICAL ASPECTS OF THE FUNCTION OF
THE MALPIGHIAN BODY I N T H E LIVING FROG’S
KIDNEY, BASED ON STUDIES WITH THE
FLUORESCENCE MICROSCOPE
EDWARD SINGER
WITH THE TECHNICAL ASSISTANCE O F ELIZABETH B. CUZZORT
Biological Laboratory, Cold Spring Harbor and Research Laboratory, St. Clare’s
Hospitat, New Pork
ELEVEN FIGURES
The results of the experiments of many investigators of
recent years show that there are so many divergent factors
that influence the different tissues crowded into the microscopical malpighian body, that one cannot suspect its function
from its morphological aspect in fixed sections ; or vice versa,
one cannot attempt to deduce its histological appearance from
its function. Fluorescence microscopy makes it possible t o
observe the cytological details and study simultaneously the
function of the malpighian body, as has been reported in
previous papers. This method also permits at the same time
an analysis of the chemical and physical changes that perpetually occur in the living cells but does not interfere with
the function of the cells or disturb their structure. Thus by
showing differences in cells that appear homogeneous in white
light, the use of fluorescent light permits a closer correlation
of structural changes and function than could be made by
previous direct examination of living glomeruli in white light.
‘This investigation has been made with the assistance of a grant from the
Diamond Jubilee Fund, New York.
The author wishes to acknowledge the courtesy of Dr. Hugo Fricke, director of
the biophysics laboratory at Cold Spring Harbor, f o r permitting the use of the
facilities of his laboratory. We are also indebted t o Prof. Max Haitinger of
Vienna who supplied us with many of the dyes used in the experiments,
Acknowledgment is due also to Zeiss Company, New York, who constructed
parts of the instrument according to my plans.
343
T H E ANATOMICAL RECORD, VOL. 66, NO. 3
344
EDWARD SINGER AND ELIZABETH B. CTJZZORT
MATERIAL USED
The frogs used in our experiments were of three species :
Rana catesbiana, R. pipiens, and R. clamitans. These frogs,
weighing from 25 to 250 gm., were caught on the grounds of
the biological laboratory in the months of July, August, and
September, and were kept and fed in captivity for a few days,
as we noticed that the excitement due to capture caused an
interference with the glomerular circulation. The malpighian
bodies of these three species did not show structural differences but varied in size, those of Rana catesbiana being
largest and best suited for examination; therefore our experiments were chiefly made on this species of frog, and all
the illustrations of the malpighian bodies were made from
their kidneys.
It was necessary to discard a number of these frogs, as
they were found to have a general parasitic infection, a fact
that was called attention t o by Oliver and Smith ('30). We
found, however, upon the examination of three frogs that the
parasitic infection, if not very extensive, did not interfere
with the normal function of the malpighian body.
In forty-six of the experiments we were able to conduct observations from 6 to 8 hours without any interference that
would have disturbed the working plan of the experiment.
METHOD
In preparing the animals for observation, the same method
was employed that we used in our previous experiments in
which the kidney of the animal was exposed by a ventral incision of the abdominal wall after carefully ligating all blood
vessels in order to prohibit bleeding. To expose the ventral
side of the kidney the intestines were pushed aside with cotton
moistened with Ringer's solution. Both sexes were used for
the experiments, and though the ovaries of the females and
the enlarged testicles of the male during the spawning period
made it difficult to focus on the kidney, these organs were not
excised as we noticed that their incision had a reflex effect
upon the glomerular capillaries thereby interfering with the
circulation.
HISTOLOGY OF THE LIVING MALPIGHIAN BODY
345
Most of the observations were conducted on the malpighian
bodies situated between the lateral margin of the kidney and
the suprarenal gland, as in this part of the kidney the cytological details were best observed, since here the malpighian
bodies lie closest to the surface.
Observations on the malpighian bodies were made in reflected white light and in their fluorescent light, which was
excited by ultraviolet illumination.
OBSERVATIONS
1. The rnalpigh/im bodies ifi white light
The malpighian bodies of healthy frogs uninfluenced by any
substance will be described first as they appeared in a reflected white light, as their appearance in this light will form
a basis for comparison for further observations.
I n this light the capillary loops and their continuous layer
of endothelial cells were visible with their nuclei slightly protruding into the lumen of the capillaries. The outside covering of the capillaries, the visceral layer of the glomerular
epithelium, appeared as a homogeneous layer of uniform
thickness over the capillaries. No cell boundaries or structures of any kind could be seen. It could be observed that
the capillary tufts form lobes that hang into the capsular
space from a common body. This common body contains the
afferent and efferent vessels. The afferent vessel lies closer
to the ventral side of the kidney and therefore is more superficial in this position than the efferent ;hence it may be traced
for a greater distance. Bauman’s capsule appeared as a thin
membrane of squamous cells with their nuclei bulging into the
capsular space in bead-like protuberances. The capsular
space seen between the lobes of the glomerulus and the capsule
was darker and duller than its boundaries.
The only function that could be observed with any great
accuracy in white light was the circulation that is continuous
under normal conditions. The speed of the circulation may
vary, but all glomeruli have the same speed at the same time.
346
EDWARD SINGER AND ELIZABETH B. CUZZORT
The circulation, however, may be stopped easily by interferences that do not have any effect on the other blood vessels
of the kidney.
2.
The rnalpighian bodies in autofluorescent light
The autofluorescence of the malpighian bodies is very
slight ; therefore all the experiments were made by inducing
fluorescence by means of different substances. Our choice of
fluorsecent substances was guided by our past experiences
with them, by Dh6r6's work ('33) and by Haitinger's studies
on fluorescent dyes ('34). These substances have to be
classified, as to their ability to show fluorescence in neutral,
in acid, and in alkaline solutions, as to their chemical properties, and as to their behavior in the electrical field of the blood
plasma. All of these points must be taken into consideration
in any attempt to correlate the appearance of fluorescence
staining with function.
3. T h e rnalpighiam bodies in induced fllzcorescerct light
Descriptions of the malpighian body as it appeared in induced fluorescent light will be given best by grouping our
findings according to the substances employed to induce
fluorescence. The optimum amount of each substance that
could be injected was determined and the strength of the
solution was made accordingly. Thus the relation between
the weight of the animal and the water incorporated with the
injection of various substances remained constant. The
majority of the fluorescent colors of the various substances
show only in a very definite part of the malpighian body in
ultraviolet light, and this appearance of selective fluorescence
staining depends upon the functional state of the glomerulus
and also upon the property of the substance.
HISTOLOGY OF THE LIVING MUPIGHIAN BODY
347
TABLE 1
ACID
suBSTbWms
ELEC'IILICAL
OIiARQI IN
BUXD
PLASMA
Aesculine
~Uranine
FLIJOlLESCENCIC COWIE IN ULl'BAVIOIaET LIQHT
COLOB IN
WHITl
LIQHT
....
Positive
Positive
Greenish
Acriflavine
Negative
Yellow
~Thioflavine
Negative
Neutral Alcaline
solution solution
Acid
solution
Adsorbate
Solid
Blue
Yellow
Blue
DulI
yellow
Greenish
yellow
Blueish
yellow
Purplish
blue
Blueish
yellow
Strong
orange
Strong
orange
Strong
yellow
Strong
yellow
green
Red
~
Blue
Green
Blue
Yellow
Blue
Green
AWALINE
SUBSTANCES
__
8.
Thiazolyellow
Primulin
yellow
Negative
-
Phosphine-
Negative
Negative
...
Yellow
Strong
green
Yellowish
Faint
green
Faint
0.
Berberine
Negative
_sulphate
_
Geranine 0. Negative
....
Green
Strong
green
__
Yellow
Strong Strong
blue
blue ___
Yellow Purple Faint Faint red
red
___
Yellowish Strong Strong Blueish
blue
white
purple
Reddish
....
Faint
Strong
yellow
green
...
Yellow
Strong
yellow
Faint Faint red Faint red
blue
Yellow
Red
1. Aescuhe. One cubic centimeter saturated solution of
Aesculine for each 100 gm. of the weight of the animal was
injected into a lymph sac.
The mode of appearance of this substance was described
previously in another paper (Singer, '33) where we showed
that the glomerular capillaries are clearly visible until the
filtration of Aesculine reached the same concentration in the
capsular space as it had in the capillaries. This time using
lenses with higher numerical aperture we could also distinguish that the entire capillary wall became luminous, showing the glomerular epithelium as a homogeneous covering
but without any cell differentiations. The cells of the
capsule, however, were visible. One minute after this took
place all details of the glomerulus disappeared and the
348
EDWARD SINGER AND ELIZABETH B. CUZZORT
glomerulus looked like a luminous blue bulb. The uniformity
of the color was disturbed only by the circulating blood corpuscles, erythrocytes looking dark, the leucocytes and lymphocytes appearing brilliantly sparkling. As the extra-glomerular vessels stay clearly visible all the time, the disappearance of the glomerular capillaries indicates that the
capsular space has the same concentration of Aesculine as
the blood. This picture of the glomerulus showed only
slight variations, as the exposure to light affected it in two
ways. After illumination for 15 to 20 minutes the cell
boundaries and other cytological details became clearer and
after that slightly hazy. The blood circulation or urine Htration, however, was never observed to suffer any changes.
The same phenomenon is also characteristic of the tissues
that are exposed to illumination without any fluorescent
substances. This phenomenon was noted by Wels and
Jokisch ('29, '31) in their studies on sperm and ova, and they
also mention that the autofluorescence of the cells increases
during irradiation by ultraviolet light without any damaging
influence on the vitality of the cell. Aesculine is, therefore,
especially adapted for the study of elimination in the
glomerulus, but its ability to bring out cytological differences,
is not as good as that of other substances.
2. Urawine. One cubic centimteer of 1:1000 solution for
each 100 gm.of the weight of the animal was injected into the
lymph sac.
The behavior of Uranine is similar to that of Aesculine but
causes a better cell definition. However, this substance i8
more toxic than Aesculine.
3. ThioflaviHe 8. One cubic centimeter of a 4:1000 solution for each 100 gm. of the weight of the animal was injected
into the lymph sac.
The glomerular capillaries appeared almost instantaneously in a light greenish-yellow color that was much brighter
than the surrounding blue-violet tissues. The capillary tufts
were clearly defined, as the entire capillary wall and the
glomerular epithelium was stained. The blood which circulates in the capillaries had a more yellowish color than the
HISTOLOGY O F THE LIVING MALPIGHIAN BODY
349
wall itself. The fluid in the capsular space was dark purple
(fig. 2). In this case, the fluorescent color of both, tissue and
dye, being blue we do not have color changes resulting from
the mixing of different fluorescent colors. The color changes
in the fluorescence of Thioflavine S. must be explained by a
phenomenon that we would call according to Haitinger and
Hamper1 fluorescence metachromasy, that is, a color change
in the fluorescene of the dye according to the pH and electric
properties of the cell, and also to whether the dye is adsorbed
or absorbed into the tissue elements.
Thioflavine S. is a dye that is especially adapted to show
the blood vessels and blood serum. The pictures obtained by
it are clearer than any histological slide could offer. This
clear-cut picture, however, lasted only 3 to 5 minutes, as the
dye is photosensitive, and exposure to light changed the
greenish color to yellow in the capillary wall, and also made
it considerably duller. The fluorescence fast became diffuse
and the cytological details *disappeared. At the same time
the circulation in the glomerulus stopped in a characteristic
way, the efferent vessel contracting and the glomerulus and
afferent vessel filling with blood. The glomerulus that was
filling the capsule, especially when curare was injected previously, shrank and its wall appeared considerably thinner as
the staining of the epithelium disappeared. When this occurred, the internal margin of Bauman’s capsule became very
distinctly yellow (fig. 5). If the exposure to light continues,
the glomerulus may lose its fluorescence in ultraviolet light
entirely, and the malpighian body may appear as a dark disc,
in which the shrunken coils of capillaries were visible only in
white light. This phenomenon showed that this dye besides
being photosensitive has a photodynamic action and makes
the glomerular capillaries photosensitive also. It had a
similar effect on the liver, but it did not seem to affect the
color or appearance of the tubuli or their contents. Thus
it seems that blood serum in vivo stimulates photosensitization; while according to Tappeiner (’23) serum in vitro
retards the photodynamic effects. Non-illuminated parts re-
350
EDWARD SINGER AND ELIZABETH B. CUZZORT
mained intact and were used for further study 2 to 4 hours after
the injection.
4. Thiuxol yellow. One cubic centimeter of 1:1000 solution for each 100 gm.of the weight of the animal was injected
into the lymph sac.
It stained the glomeruli with a blue-violet color, but its
fluorescence was not strong enough and soon became diffuse ;
thus it was discarded after a few trials f o r use in the glomerular experiments.
5. Primzclin yellow. One cubic centimeter of 1:1000 solution f o r eaeh 100 p.of the weight of the animal was injected into the lymph sac.
This like the previous dye did not stain the malpighian
bodies well, although in other structures the cells contained
well-stained nuclei.
6. Phosphime 0. One cubic centimeter of 1:1000 solution
for each 100 gm.of the weight of the animal was injected into
the lymph sac.
The endothelial cells in the glomerular capillaries and the
capsule were clearly visible, as they became a greenish yellow
when stained by Phosphine 0. Leucocytes were brilliantly
white, but the fluorescent color of this dye did not show in the
blood serum or in the tubular fluid.
7. Acriflaviae. One cubic centimeter of 1:1000 solution
for each 100 gm.of the weight of the animal was injected into
the lymph sac.
Acriflavine stained the nuclei in the glomerulus if it was
injected alone, but the picture appeared very dark. If, however, Acriflavine was injected following an injection of
Aesculine, a very good picture of the glomerular epithelial
Fig. 1 Malpighian body of a liviiig frog which was injected with Aesculine
and Acriflavine showing the cell boundaries of the individual glomerular epithelial
cells. The dark space between the tubules is the distended capsular space.
Fig.2 Capsular space and glomerular tufts of a living frog as they appear
in the fluorescent light of Thioflavine S. in the first 3 minutes of the ultraviolet
irradiation. The walls of the capillaries are thick as the glomerular epithelium
and the glomerular endothelium are stained. The numerous capillary tufts are
crowded.
HISTOLOGY OF THE L I V I S G ;ZIATAPIGHIAW BODY
T H E ANATOMICAI, RECORD, !'OL 6 6 , NO. 3
351
352
RIIWARD SINGER A N D ELIZABETH B. CTTZZORT
cells could 1)c seen (fig. 1). This indicates that Acriflaviiic
was contained in the cytoplasm, but it needed the illuniinatiori of another substance to make it visible, a s it did not show
fluorcscencc by itself in the cytoplasm.
8. H'erherin,e sulplaate. Onc cubic centimeter of 1: 100
solution for each 100 gin. of the weight of the animal was injected into thc lymph sac.
rl'liis is a photodynamic dye which before being subjected
to long irradiation stained thc nuclei. After irradiation of
I0 to 35 minutes the nuclei became dark and the cytoplasm
was stained. When the cytoplasm begin to show staining, the
glomcrular cndothelial cells became visible.
9. Gwan,ine G. One cubic centimeter of 1: 1000 solutioii
for each 100 gm. of the weight of the animal was injected into
tlic lyiripli sac.
The fluorescence of this dye mas hardly distinguishable.
Only R slight staiiiing of the nuclei was visible.
H,>h,avior o f the malpighialz bodies ulzder the iiiflueiace of
swake ~ t n o mcaffeine
,
awd cziraril, and thP ef'ect of these
ngrrrts on t h e flzcorcscewe stal'niitg
Of the subsiances we have already mentioned, only Tliioflavinc S. i s deleterious to the vitality of the tissue. I t s toxic
efiects were definitely connected with the illumination, for
whcre there was no illumination or only a short exposure to
irradiation, 110 damaging effects were detected. The above
suhstances, which were also used by other investigators,
prodiicc injuries to the malpighiun bodies or interfcre with
thcir function independcnt of the illumination.
1 . I'enom of ('rotalirs adawaanteus (Texas rattlesnake).
To obswve the effects of this venom two methods of injection
were used :
a. For each 100 gm. of the weight of the animal 0.20 to
0.30 mg. of the d r i d vciiioin was dissolvecl in water which was
half the amourit of the required volume of solution. The
other half of the required volume was obtained by adding
glycerine. The dose was divided into thrce parts and given in
4.
HISTOLOGY OF THE LIVING MALPIGHIAN
BODY
353
Fig. 3 Malpighinn body of a liring frog, whieli was injcetcd with the vciiom
of Crotalus atlamanteus tlircc time8 within 3 hours, during which time i t was
under continuous microscopic observation in thc fluoresecnce staiuiug of Thioflavine S. It shows the slirunkeii glonicrular r:ipillaries with brightly fluorescent
endothelial cells. A t t h e lower p a r t of t h e capsular space a luminous M B ~ S is
visible. A t the right upper part of thc capsular spare is the affrrcnt and a t the
left tho efferent vrssel.
tlii*cc scparatc injcctioiis within 3 liours’ tinie, during whicli
thc frog was uiider continuous iriicroscopical c~xaniiiiatioil.
By using this method we wcre able to observe the immediate
iw~ctioiiof the malpigliian body to the d e c k of the vciioi~~.
Td’ollowing the injection of tlw vciiorn the walls of the capillary tufts aiid other hlood vcssels became very distinct aiid
w r c ~ r c sliarp
~
aiitf hrillimtly lumiiious in white light. Two or
3 houi*s after tlie iitjcctioii tlic glomcrular tufts becanie ver:s1vollc11. Their w~allswcrc thickened but their lumiiia did iiot
sccwi to be iir any way influcnced and permitted a very fast
c*ircnlation of blood. I n some of the malpighian bodies tlie
glomcrulus filled the ciitire capsule, lcaviirg hardly any capsular sriacc; whilc in others a larger spacc ii-as seen (fig. 3). Tn
the lattcr the swollen loops of the gloincruli hung loosely into
tlic capsular space like fingers of‘ a heavy glove, liming large
spaces between thc loops. This swelling, as shown in figure 4,
may mist in sonic glomeruli for scvcral days. Iii other
glomeruli, it has been observed that these swolleii loops slowly
tlraw together until finally all space disappeared bctweeii
t h i n . At this stage the glomerulus looked like ail irregularly
shaped mass. IIowever, the loops occasioiially parted agaiii
with a sutldeii cxpaiisioii, wlieii the capsular wall bii rst.
Burstiiig was obsei-vcd 4 to 5 hours after the first ~ciioiriinjection.
microscopic section of a ruptured capsule is shown
iii fiyure 8. A t the siirne time it conld IN? observed also that
the tufts of other glomeruli with intact capsule expanded b?
jerk)- movements. Each pulsc wave separated tlieiii further.
aiicl further as though there would havc becii some rcsistaiice
tit tlw iicck of thc g1omc:rulus which liad to he o v ~ ~ i ~ o by
rnc
the 1iigi.horpressure of the pulse wave. This is a pheiiomcnon,
Fig. 4 Malpigliiaii hody of a liviiig frog, which was iiijcctrd aitli tlw vrnoiii
of (”I 0t:llus il(l:llll:llltPIlN Oil tl1roc su< sit (lays :iii(l o1)servcd i i i tlici fluoi
light trf Arsculiiir, oti the iiiittli (lay :iftrr the last injrctioii. It slioms swollen
l o o p (if tlie rapillary wall 1i:inging looscl\ into the capsular s p c e :ind a very
bright capsular nirni1)r:iw.
Fig. 5 ,Malpighiaii body of the same ftwg as in figure 4 after iii,jertion of
Thiofi:iviii(> K. ant1 exposure l o ultraviolet irratliation, sliowiiig tlw fatlctl fluorcsceiiw of l’liioflarinr 8. a i d stasis in tlie gloinrrular capillaries.
(3
355
356
E:I)WABD SINGER ANJ) ELIZABETH B. CUZZORT
however, which is sornetimes observed in glomeruli that were
iiot exposed to any poison (fig.6).
The glomerular circulation was affected i n the following
way: After the injection of the venom the circulation in the
glomeruli of some kidneys became jerky and intermittent.
The blood corpuscles crowded and pushed each other in an
cff‘ort to enter the capillaries, and only a few passed at a time.
An hour after the injection the blood that circulatcs in the
glonwrular capillaries contained an increased number of
white bloocl corpuscles. The white blood corpuscles circulated
on the periplierp of the blood stmam in contrast to the normal
c*irculation. (lradually a number of the white blood corpuscles
stuck t o the walls of the capillaries arid in 3 or 4 hours they
10:15
70:00
Fig. 6
72:15
m50
Cliaiigc!s ill the frcv capsu1:ir
SJ):IW.
lined the walls. 111 othcr kidneys after the injection, the circulation became more and more sluggish with the glomerular
loops d w a y s in a loonc~lyhanging pod tion.
Tiijectetl substances filtered through tlie capillaries into the
~lomeriilarcapsulc in thc first few hours, but it W R S difficult
l o decide wlicther there was any filtration after the fifth hour,
>is most of the animals arc moribund at ihis time.
h. F o r each 100 gm. of aniinal 0.06 mg. to 0.12 mg. of venom
WCIT prepared in the same way. This amount w a s injected
o n w H day for 3 consec*utivedays making a total arriouiit of
Fig. 7 .Microscopic. scction (if :I rnalpigliian body of the sniue frog as in
figi1rc.s 4 and 5 stained with H. and 15. slio\~inga granular exudate and ruptured
rapsnlo. The c,xtrcrnely dil:itcd neck is also notewort1i.v.
Fig. 8 Microscopic scction stained with H. and E. showing tlic shrunken
glouierular capsule of a frog which rcct4ved three injections of the wnorn of
(‘rotalus :rdamantcus in 3 succcssivc days :inti W R B killed t h e fourth day :ifttJr
the kist injwtion.
HISTOLOGY O F T H E LIVING MALPIGHIAR BODY
357
0.18 to 0.36 for each 100 gm. of tlie frog. Thc frogs were used
for cxpcriments or killed for dissection 3 or 4 days after thc
last injection, dcpeiidiiig upon how many days it took for
t hc? frogs to rcwh a moribund stage.
I n malpigliian bodies of kidneys that were in,jcctecl BCcording to this method, wc saw hypcraemia in the glomerular
capi1li-lric.s and a sweIIii~gof thc gloincrular loops. Injected
fluorescent substances appeared as iii tlic normal malpigliian
hoclies. Most of the glomeruli showctl good circulation, but
soriie were ~)lugg.cclwith 1)lood and staycd that way, unaffoctctl by adrciialinc or saline injections. The cntirc capillary
\w11 was illnminatecl h;v fluorescence if Thioflaviiw 8. was injcctcd arid tlic. capsiilc~w i ~ ssurrounded by ;1 luminous border.
Ilctwwn this luminous liiic and tlic sui~oundiiigv(wc1s was
i i tlctiiiite chrk violet spacc (fig. 5 ) .
111 tlic glornc~i.ul;i~~
<~ii1)sUlc
WC! occasioii;illy o b ~ e r ~ t~i l~ i i
tlia t ap1)c~nrctlw i * ylumiiious in the flnoresccnt I igh t of ‘I’liioflaviiic S. and opaque in white light. ‘l’his could have bcen
ti gloiiiernlar (?xiidate,which shows on Ihc slides made of tlicsc
kidneys or it might h a r e bccii tlint tlic swollen cilii of the ncclr
of t he nialpigliiaii body appeared in this manner for this
substance al~--ayslies opposite to tlie lower pole of the
glomerulus, whei-e cilii a r e found. Thcsc swollen rilii also
tiplictir on the slides (fig. 7 ) .
2. Oafeime. (’affeinc is an alcoloicl having a slight bluish
])urplc fluoresceiwc a i d was uscd in a solution of 4:1000
of which 1 cc. was injected for cadi 100 gm. of the wcight of
thc u i m a l into the lymph sac.
After tlic injectioii tlic circulation in thc glomerular ciq)illtirics hecnrnc much faster, even rapid. Thc corpus(~lcs,howcvcr, did not proccccl snioothly at an even specd but riiovctl
with a jerky motion. This would iiidicatc that tlicrc TWS ti
high pressure iii the ;ifferelit arterial system for which the
efierciit vessels c~ouldnot take care; or that the cffcrciit r c w c l
wzs soiiicwliat coiitractcct a t some place i i i i d this coiit radio11
liad t o I ) c b overcome'. Glomcr*ulurtufts that did not show a n y
c>irculatioiib ~ f o i ~ 110\v
h , begail to havo 21 good eir*cul*
<It‘1011.
HISTOLOGY O F THE LIVING MALl’IGRIAX BODY
359
white light we could see very clearly the glomerular
tufts with a very lumiiious iiiside margin. The inner border
of the capsulc was also very luminous. This luminous line
was surrounded by a darkcr, thicker line which, again, had a
luminous outer lining. The luminosity of these lines which
also contailled sparkling dots tirid streaks, became more and
I11
Fig. Y Malpigliian body of a liviiig frog in reflected white light after injection
of Caffeine showing thin dark outside margin and a broader luminous inside
margin on the capillary walls of the glomerulus. The capsular epithelium is
lunrinous. Its nuclei protrude into the capsular space. Around the rpithelium is
:I dark linc outside of which is aiiothcr luminous limb.
THE AXATOYICAL BECOBD, VOL. 6 6 , NO. 3
360
EDWARD SINGER AND ELIZABETH B. CUZZOItT
more sparkling during observation (fig. 9). These streaks
were also visible on the rriicroscopic section made from these
kidncys (fig. 11).
I n ultraviolet light the luminous lines in the capillaries had
a slight greenish-yellow fluoresceuce ; those on the capsule
did not show any fluorescence.
If Aesculine was in,jcctecl its fluorescence appeared almost
immediately in the cvipsular spti(~e. Tlie outline of the capsule
was violet and distiiict. The gloniernlar capillaries, liowever,
were not clistinguishable at all, evtw at the begiiiiiing, in
contrast to the glomeruli iii B kidney that did not r.cwivc any
caffeine. The capsule appeared, a s a homogeneous, lnrninoiis
bulb, in which seemiugly free blood corpuscles circulated.
The liiniinosity was higlicr than in capsulcs without caffeine,
antl it reached nearly the luminosity of the distal convoluted
tubuli (fig. 10).
Whcn Thioflavine S. was injected and its fluorescence appearcd, the picture was that of a glomerulus without Caffeine.
.?. ("umriZ. One cubic centimeter of 1: 10 of the standard
Curaril solution was irijcctcd for each 100 gm. of the weight
of the animal into thc lymph sac.
This substance did not seem to have any other effect on the
appcaraiicc or function of the glomerulus, but it should he
mentioned a s it was used to paralyze the frogs in some of the
experiments and affected the size of thc capsular space.
Capsular spacca wcre narrower in frogs that were injected
with curaril.
Fig. 10 Malpighian body of R living frog, wliicli received Caffeinr in the
fluorescent light of Aesculiw. The liaziness of the glomerular structures anti the
slmrpness of the dark capsule are noteworthy. Tlic luminosity of tlir glomrrulus
antl that of an adjoining distal eonvolutrd tubule is about the sainr.
Fig. 11 Microscwpic sertioii of the ni:~lpighian body of the sanie frog as in
figurr 10 stailid with H. :iud E. showing numerous luminous strcwks in tbc
capillar,V wall.
361
1>1S(’1788ION
7 ’ 1 1 ~/I’M capsdar. spacc awd its variatioirs
( )i11* oliscrwitioiis
of the experimciit s with fluorescent s u b
poisoiis give evidence that the size of tlic capsular
space that is unoccupicd by glomerular tufts tlepeiids on the
functional state of the entire oi-gaii of the kidney tint1 not
only oil the function of the gloiiicrulus. ‘I’hc size of t l i c
free capsular spaces i n normal malpigliian bodies is .\-PI->similar, and slight increase o r decrease in ilic size of tlic
(*apsulztrspaces owurs siinnltaneously in the glomcruli a s 1 0 1 1 ~
;is tlic ltidiicy is kept uiidcr circumstances that closclp coiiforiii
to its physiological state. Iiicreasc or decrease in thc free
capular spztcc is due to compressioii or expansion of the 10112s
of the glomerulus. This variation of the free space does iiot
a i d y iiiflneiice thc circulation. LLKU and TVhite ( ’22)
callcd attention to tlie ahove fact and we have also reported
it in a previous paper. Variations in the size of the free
catipsulttr space in the same kidney occur under pathological
o r exl)aiixioii of the xlomerulus.
cwiiclitions by the sli~*i~~Iiillg
[’athologicol factors a r e also rcsponsihlc for the tlistciition
a i d shrinkage of tlic capsule.
A diminished free capsular space occurs with the 1)eginiiing
of t l i ~tlarnagiiig cffecbt 011 thc g1oiriorulw~-tufts, or glomciw
loiiephritis caused by Crotalus adamanteus venom, for the
swolleii lobes of the glomerulus occupy more space. The free
capsular space becomes larger a few hours after the begiiining
of t h e iiiflmmiation. Y’liis iiicrease ill f r e c ciipsnlai- S ] ) H C C ~i i
t l ~ wto the compression of the glomei*ular tufts, by iiicreasccl
1)wssure of the glomerular fluid, for it c ~ i he
i seen that the
cii*cunifcwxce of the capsule does not change 1)ut tlie size
of tlw g1omcAriilus decreases. rncrcasetl l)ressm*c! ot’ the
glouic~rularfluid indicatcs that its drainage into tlie tubuli
is impaiiwl by sl g r m u l a r exudate foimed in the capsule ~ n t l
pliiggiiig thc 11cck Tuercascd iiifiltmf ion may be only
sccoiic1;irily rt1s1mi sihl o for the 11i g911ci. gl omci.ulai* pi’essiire
as when 1)i’opei.(Irwiiiage is restoiwl the glomerulus expands.
st tiiiees a i d
HISTOLOGY O F THE LIVING MALPIGHIAN BODY
363
A variety of different sizes of capsular spaces can be observed when the capsule ruptures. I n our previous experiments we have observed, as have Lucas and White ( '32), the
distention of the capsule itself. Never, however, did we observe the rupture of the capsule, even in capsules that were
more distended than they become when the frogs are poisoned
with snake venom. Rupturing of the capsule, therefore, would
indicate that snake venom has a selective deleterious effect,
not only on the glomerular capillaries as already has been
described by Pearce ('09) but also on the capsule, which
causes it to become brittle.
The size of the free capsular space also diminishes by the
use of curare. The diminishing is uniform and appears to
be due to the shrinkage of the capsule, as the lobes of the
glomerulus are close together without any interspaces. This
shrinkage is so slight, however, that we were unable to make
exact measurements.
Enlarging of the free space occurs when the glomerular
capillaries are shrunken through photosensitization. This
phenomenon we shall describe more fully in the discussion of
the glomerular tufts.
Circurnstarzces g o v e r k g the appearalzce of fluorescelzce irc
the capsular space
Whether fluorescence appears or not in the fluid in the
glomerular space after the administration of certain fluorescent substances depends upon the electric charge of the
injected substance. The hue of the color of the fluorescence
is in accordance with the di-electric constant2 of the capsular
fluid. Thus fluorescence being an optical phenomenon, is a
truer reflection of the electrical properties of the cell than
vital stains that do not have fluorescence properties.
According to the measurements made by Peterfi of the
electrical charge of the glomerular fluid and the experiments
Di-electric constant is a magnitude that regulates the electrostatic attraction or repulsion of electrically charged particles. The brightness of the shade
of fluorescence is in reverse proportion to the di-electric constant.
TEE ANATOMICAL RECORD, VOL. 66, NO.
3
364
EDWARD SINQER AND ELIZABETH B. CUZZORT
of Keller ('30) made with vital stains, the glomerular fluid
is positively charged. The results of our experiments agree
with the findings of these two investigators, for Aesculine and
Uranine, two dyes that are positively charged could be detected
immediately in the capsular space after injection, but other
dyes which were negatively charged took a long time to appear and never showed with a great degree of luminosity.
This fact is also in agreement with the findings of Peterfi
and Keller, who pointed out that the selectiveness of dyes is
diminished with their raising concentration.
The variation in the pH which plays an important role in
the conditions controlling fluorescence will be of secondary
importance, however, to the electric properties of the living
organs, as it has an effect only on the intensity or color of
the fluorescence and will not cause the fluorescence to appear
or disappear, as the range of variation in pH, of the living
tissue is very slight. The shade of the fluorescence is
governed by the di-electric constant. The color of the
fluorescence by having a darker shade in the capsular space
than in the surrounding tissues indicates a high di-electric
constant. This finding is also in harmony with the findings of
Keller and Peterfi.
The glomerular capillaries
The various tissues of the malpighian body exercise an influence on the fluoresence of the different substances. This
influence varies according to the chemical properties of the
dyes and according to the functional state of the tissues;
therefore the capillary wall will appear in different shapes
and thicknesses at different times. The width of the loops
of the glomerular capillaries can be exactly determined in
white light. The different components, however, are not
distinguishable, as the wall looks homogeneous. This wall
also appears homogeneous in ultraviolet light for a few
minutes after the injection of aesculine until the fluorescence
becomes so diffuse that the margins of the loops are indefinite.
HISTOLOGY O F THE L I V I N G M A L P I G H I A N BODY
365
This same homogeneous appearance of the entire capillary
wall is present after the injection of Thioflavine S. but later
when the tissue becomes photosensitized, only the endothelial
cells are stained and the epithelial covering is unstained.
Diminishing and disappearing of fluorescence in the epithelial
covering indicates a damage that occurs in the epithelial cells
of the capillaries. This disappearance is a complex phenomenon, and at present we are not prepared to give an explanation
for it. Effects of damage on the glomerular capillaries can
be manifested in the sharpening of their contour or swelling
of their loops. A peculiar effect of damage is that of photosensitization, which causes a vascular spasm and is accompanied by a change of color.
The nuclei of the endothelial cells are stained by all dyes
grouped in our table as having a negative electric charge
with the exception of Thioflavine S.
The cytoplasm of glomerular epithelium appears fluorescent
in ultraviolet light if Aesculine is injected previously to the
injection of Acriflavine.
The glomerular circulatiort
An even and continuous circulation is found in glomerular
capillaries of kidneys that are kept under conditions closely
approximating the physiological state of the organism as was
shown by the experiments of Tamura, Miyamura, Nishina
and Nagasawa ( '27), and as we reported in our previous paper
and for which our present experiments supply additional
evidence. I n accordance with these investigators, we also
observed the particular susceptibility of the glomerular circulation t o influences that have hardly any effect on the circulation in the blood vessels, a fact to which Richards and
Schmidt ( '24) first called attention. This susceptibility
causes a variety of interferences that can either diminish or
accelerate the speed of the circulation.
Diminished and jerky circulation is the first reaction following the injection of poisons. This jerky circulation gives
way to a rapid circulation in the later stages, regardless of
366
EDWARD SINGER AND ELIZABETH B. CUZZORT
whether vascular poisons were administered, as we observed
in these experiments or tubular poisons given, as we reported
in a previous paper.
Intermittent circulation is caused by desiccation that may
result in a stasis in the capillaries as recognized by Okkels
( '33). Stasis occurs always when hy-pertonic solutions are applied to the surface of the kidney as Ebbecke and Jaeger ( '33)
showed. The strength of the solution which causes stasis is
parallel with the water content of the animal.
Our observation showed that stasis can also be the result
of the photodynamic action of dyes. Stasis caused in this
manner is the most constant, and the manner in which it
occurs shows that it is the result of a spasm in the efferent
vessel. This would indicate that the efferent vessel alone is
capable of influencing the entire glomerular circulation, an
occurrence that Bensley ( '29) postulated after demonstrating
special cell elements around the efferent vessel which are
capable of contraction. The extra capillary pressure seems
to play a secondary and subordinate role on the circulation,
as when the capsule is extremely distended, we as well as
Ebbecke and Jaeger ( '33), observed very rapid circiilation.
SUMMARY
1. The different cellular and humoral elements of the
malpighian body were delineated by selective fluorescence
staining.
2. As the appearance of the fluorescence staining and its
color in the various elements of the malpghian body does not
depend upon the presence of the dye only but also upon the
physical and chemical properties of the structures, from the
variations of the fluorescence conclusions could be drawn regarding the qualities of the different structures.
3. The pH, the electric charge, and the di-electric constant
of the components of the malpighian body could be demonstrated.
4. Positively charged substances have fluorescence in the
capsular fluid and in the cytoplasm, negatively charged have
fluorescence in the nucleus.
HISTOLOGY O F THE L I V I N G M A L P I G H I A N BODY
367
5. The variations in the electric and chemical properties
manifested themselves in changes of the fluorescence staining.
This caused different cell structures to be visible at different
times and caused color changes in the cells and fluids.
6. In malpighian bodies of frogs poisoned with the venom
of Crotalus adamanteus or in photosensitized malpighian
bodies the glornerular and capsular epithelium showed more
extensive changes in fluorescence than other constituents of
the malpighian body.
7. The rate of the glomerular circulation is even and its
flow continuous as long as the animal is kept under circumstances closely approximating the physiological state of the
organism.
8. The glomerular circulation, as shown by Richards and
Schmidt ( '24)is affected by influences that do not change the
circulation in other blood vessels.
9. In accordance with the results of the experiments of
Oliver and Smith ( '30), Mosonyi and Voith ( '35) and Reemelin
and Isaacs ( '16) disturbances in the circulation and changes
in the constitution of the blood plasma cause greater variations in the filtration of the fluorescent substances than
changes in the histological structure of the malpighian body
which are demonstrable by microscopic sections.
LITERATURE CITED
BENSLEY,R. D. 1929 The efferent vessels of the renal glomeruli of mammals
as a mechanism for the control of glomerular activity and pressure.
Am. J. Anat., vol. 44, pp. 141-169.
BENSLEY,R. R. AND R. D. EENSLEY1930 The structure of the renal corpuscle.
Anat. Rec., vol. 47, pp. 147-175.
DE~W, CH. 1933 Naehweis der biologisch wichtigen Korper durch Fluoreseenz
and Fluorescenz Spektren. Handb. biol. Arb. -4bt. 11. Phys. Meth.,
Teil 3, Heft 4. '
EBBECKE,
U. AND A. J m m 1933 Lokale Einfliisse auf den Blutfluss im
Glomerulus der Froschniere. Pfluger 's Archiv ges. Phys., Ed. 232,
8. 36-41.
GIKLHORN,J. AND R. KELLER 1924 Uber elektive Vitalfarbungen. Vorldufige
Mitteilung. Biochem, Zeitschr., Bd. 150, S. 2-13.
HAITINQER,
MAX 1930 Methoden der Fluorewnz Analyse. Mikrochemie, Bd.
XI, Nf. Bd. V, S. 429-464.
1934 Die Methoden der Fluorescenz Mikroskopie. Handb. biol.
Arbeitsmeth. Abt. 11. Teil 3, S. 3307-3337.
368
EDWARD SINGER AND ELIZABETH B. CUZZORT
ISAACS,
R. 1917 The reaction of the kidney colloids and its bearing on renal
function. Am. J. Physiol., vol. 45, pp. 71-80.
KELLER,R. 1930 Der electrische Faktor des Wassertransports im Lichte der
Vitalfirbung. Erg. Physiol., Bd. 30, S. 294-407.
LUCAS,A. AND H. L. WHI’PE 1932 Factors responsible for the abnormal distention of the glomerular capsule in the Nccturus kidney. Anat. Rec.,
VOI.53, pp. 371-385.
MOSONYI,
J. AND L. VoITri 1935 Zur Frage der Ultrafiltration i n den
Glomerulis. Arch. exp. Path. Pharm., Bd. 177, S. 177-182.
OKKELS,H. 1933 Lokale Einfliisse auf den Blutfluss im Glomerulus der Froschniere. Pfliiger’s Arch. ges. Physiol., Ed. 232, S. 741-742.
OLIVER,J. AND P. SMITH 1930 Experimental nephritis in the frog. J. Exp.
Med., vol. 52, pp. 181-193.
PEARCE,
R. 1909 An experimental glomerular lesion caused by venom. J. Exp.
Med., vol. 11, p. 532.
REEMELIN,E. B. AND R. ISAACS
1916 A study of the conditions involved in the
accumulation of dissolved substances in the blood. Am. J. Physiol.,
VOI.42, pp. 163-174.
RICHARDS,
A. N. AND C. F. S C H X ~1924 A description of the glomerular
circulation in the frog’s kidney and observations concerning the
action of adrenalin and various other substances upon it. Am. J.
Physiol., vol. 71, pp. 17%208.
SINGER,
E. 1932 A microscope for observation of fluorescence in living tissues.
Science, vol. 75, pp. 289-291.
1933a A study of the morphological physiology ,of the frog’s
kidney with the fluorescence microscope. Anat. Rec., vol. 53, p. 37.
1933 b Observations on the frog’s kidney with the fluorescence
microscope. Am. J. Anat., vol. 53, pp. 469-495.
TAYURA,K.,. I(. MIYAM~UIW,
T. NISHINA AND H. NAGASAWA1927 The glomerular circulation i n the living frog’s kidney. Jap. J. Med. Science.
IT.Pharm., vol. 1, pp. 211-228.
TAPPEINER,
H. 1923 Methoden beim h b e i t e n mit sensibilisierenden fluoreseierenden Farbstoffen. Handb. biol. Arbeitsmeth., Abt. IV, Teil 7, S.
1070-1082.
WELS,P. AND M. JOKISCH 1939 Der Einfluss der Quartlampenbestrahlung auf
die Fluorescenz von Geweben und Zellen. Pfliiger’s Arch. ges. Physiol.,
Bd. 223, S. 369-377.
1931 Weitere Untersuchungen fiber die Fluorescene bestrahlter
Zellen. Ibid., Bd. 228, S. 671-682.
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