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The effect of buffered phosphate solutions upon a thin layer of living vascular tissue in moat chambers introduced into the rabbit's ear.

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THE EFFECT OF BUFFERED PHOSPHATE SOLUTIONS UPON A THIN LAYER O F LIVING, VASCULAR
TISSUE I N MOAT CHAMBERS INTRODUCED INTO
THE RABBIT'S EAR a
RICHARD G. ABELL
Awatom~alLaboratory, University of PenwyEvania Sahool of Medicine
FOUR FIGURES
That phosphate buffers produce toxic symptoms when introduced into the body in sufficient concentration is well known.
The cause of this toxicity, however, is somewhat obscure, and
has been variously attributed to the phosphate ions in combination with the pH of the solution in which they exist, to the
sodium or potassium ions contained in the buffers rather than
to the phosphate ions, and to other changes in the blood
plasma and tissue fluid.
The present technic offers the possibility of submitting a
thin layer of living vascular tissue to the action of buffered
phosphate solutions, and at the same time of observing with
the high powers of the microscope the effect of such solutions
upon the tissue. The reaction of the tissue to phosphate solutions having the same pH as the blood, and to others differing
in pH from the blood, can thus be determined by direct observation. So also can the effect of solutions of sodium and
potassium chloride containing the same concentration of
sodium and potassium ions as the buffers, and of others con'This work was aided by a grant made to the department of anatomy at the
University of Pennsylvania by the Rockefeller Foundation, under which the author
held a fellowship.
lBrief reference to some of the results obtained in this study waa made by
AbeU and Clark ('33) at the 1933 meeting of the American Association of
Anatomists.
61
52
RICHARD G. ABELL
taining even greater concentrations of these ions. I n a similar manner, the effect upon the tissue of the osmotic pressure
of the solutions can be determined.
Many experiments concerning the toxicity of phosphate
solutions are contained in the literature, but so f a r as the
author is aware, with the exception of a preliminary report
by Abell and Clark ( ’33), direct microscopic observations of
the sort to be presented in this paper have not previously
been described.
MATERIAL AND METHODS
Information concerning the construction, installation, and
care of moat chambers may be secured from the original description of the chamber by Abell and Clark (’32). Briefly,
such a chamber contains a very thin, transparent space called
a ‘bay.’ Following introduction of the chamber into a hole
cut through the ear of a rabbit, living tissue, continuous with
the subcutaneous tissue of the ear, grows into the bay through
two entrance holes at the proximal end. At its distal end the
bay communicates with a reservoir, or ‘moat,’ into which
chemical solutions may be placed and from which they may be
subsequently removed, through two permanently installed access cannulae, Chemical solutions introduced into the moat
pass by diffusion into the bay, and there interact with the
living cells and tissue normally present during the vascularization of the chamber (compare fig. 1). Since the bay is
transparent, and very thin, the effect of the solution upon the
tissue can be observed with the high powers of the microscope.
Buffered solutions of phosphate at pH 7.4 and 6.2 were used
in the experiments to be described. The one at pH 7.4 was
prepared by adding 39.50 cc. of 0.2 M NaOH to 50 cc. of 0.2 M
KH,PO,, that at p H 6.2 by the addition of 8.60 cc. of 0.2 M
NaOH to 50 cc. of 0.2 MKH,PO,, as described by Wilson
(’28). When the hydrogen ion concentration of the phosphate
solutions thus prepared differed from that desired-as shown
by standard buffers, the pH of which had been determined
electrometrically-such small corrections as necessary were
PHOSPHATE SOLUTlONS A N D LITING TISSUES
53
made, bromthymol blue and phenol red being u s e d as indicators.
The method of installation of the phosphate solutions within
the moat and of subsequently removing them from it was the
same as that described for soliitioiis of methyleiie blue by the
author ( ’34).
Before introclizctioii into the moat, the phosphate soliitions
were sterilized by boiling. 7JpOli cooling, the pH was again
measured. No corrections were necessary a t this time.
Records of growth of blood vessels into the chaiiibers were
made with a Leitz ‘clrawing eyepiece’ at 2- to M a y intervals
during the period preceding tlie iiistallation of the phosphate
solutions within the moats, a period wliich varied iii length,
in all of the chambers but one, from 23 t o 30 days. Photomicrographs of the tissues in the bays were taken almost
every day throughout the prc-introductorj- periods. Microscopic and photomicrographic records were lilicmise made €011o wing int r odu ct i on of the ph o spli at e solut iom s wit liiii t lie
moats, i n one instance such records being continued for more
than a year. The techiiic emploj-ed in the experiments to he
described does not involve the introduction of a needle into
the tissue itself. C’onseqnently the microscopic picture produced bp iiitroductioii of the phosphate solntioiis is free from
the effect upon tlie tissue of direct meclianical manipulatioii.
It is a real pleasure to express to Dr. Eliot R. Clark my deep
appreciation for encouragement and advice given throughout
the course of these experiments.
OBSEBVA TTONS
1. Tntroduction into f fie moat of bicferpd piiospkate solnt i o m at pH 7.4. Phosphate solntions at this pH were intro-
duced into the moats of three chambers, designated as
chambers 7/30/31L, 1/23/32R, and 2/17/32L. I n each case
the installation of the buffer within the moat was followed by
certain definite reactions of the tissue. These reactions may,
perhaps, best be described by referring to the individual experiments.
T l I H A N A T O M I C I L RhCORD, VOL. 6 4 , KO.
1, AND
STJPPLEMENT N O
1
54
RICHARD G . ABELL
The phosphate solution MYIS introduced into the moat of
chamber 2/17/32L 23 clays following the entrance of the first
blood capillaries into the bay. During this 23-day period
drawing ocular and photomicrographic records allow that the
vessels advanced uninterruptedly toward the moat, the most
distal capillaries being approximately 0.53 mm. from the moat
at the time of iiistallation of the phosphate solution mithin it,
(compare fig. 1,a). A drawing eypiece record showing the
peripheral capillary pattern was completd just before iiitroduction of the solution into tlie moat. lmmediately following
introduction of the buffer, the image of the peripheral Iressels
was projected upon this record. No alteration of the position
of the capillaries had occurred, and careful microscopic esamination showed no irritation of any of tlic tissue in the bay.
Similar results were also secured in the case of the other
chambers in these experiments.
Alicroscopic observatioiis made 22 hours following the installation of the phosphate solution within the moat showed
that the most distal of the capillaries in the bay had been injured for a distance of approximately 0.2 mm. Manj- erythrocytes and leukocytes were present in front of the new line of
functional peripheral capillaries, and sticking of leukocytes to
the enclothelium of the inost distal capillaries and to that of
tlie veiiulcs and veins collecting blood from the periphery was
extensive.3 &licroscopic examination showed that the diame ters of tlie most distal of tlic capillaries, arid of the venules
collecting blood from these capillaries, were greater than hefore introduction of tlie bnffer. Oiie of the two large arterioles
which snppliccl blood to the peripheral tissne was more widely
distended than at ariy previous time, aiid the rate of hlood flow
through it, and in general all of the vessels in the bay, was
more rapid than before tlie introduction of the phosphate solution into the moat. It appears probable that the increase in
Observations on the sticking of leukocytes t o the cndothelml wall h a w been recently presciited and discussed by E. R. and E. L. Clark ( ' 3 5 a and h). They
produce evidence which indicates t h a t progressive grades of leukocyte sticking
and emigration are associated with progressive changes in endothelium.
PHOSPHATE SOLUTIONS h N D LIVING TISSUES
55
diameter of the arteriole proximally was due t o the effect of
tlic phosphate solution upon its distal ciid, dilation at the
periphery decreasing the resistance to blood Bow, thus allowing more blood to enter the arteriole. The passage of a
greater amount of blood into the arteriole was evidently responsible for its proximal dilation, which was therefore
probably secondary in nature, and not due to the direct effect
of the phosphate solution, although this altcrnative caiinot be
completely ruled out.
Approximately 70 p in front of the most distal of the functional capillaries, and exteiidiiig from this point toward the
,
numbers of small
moat for a distance of about 1 6 0 ~ large
granules and irregular masses of granular material could he
observed extending from one side of tlie bay to the other
(compare fig. 1,b).
Tm7o days later these granules and irregular masses were
still present. At this time, the phosphate solution having becn
lcft continuously within the moat, with free access to the
tissue, thc most distal of the functional capillaries on tlie right
side of the bay were 180 p farther from the moat than on tlie
day following introduction of the buff er-on the opposite side
of the bay 46 1-1. I n the space thus left betweeii the masses of
granular material preseiit the day after introduction of the
phosphate solution, and the iiem position of the distal vessels
in the bay, fresh, graiiular masses of precipitate could be observed. During the succeediiig 3 days gradual precipitate
formation continued, the masses of precipitate increasing in
size by fusion. It was observed that at this time tlie precipitate was of glistening appearance.
Precipitate formation continued until the time of rcmoval
of the phosphate solution from the moat, 20 days after its installation within it (possibly to some extent even after this
time), and resultcd in the formation of three roughly distinguishable TOWS of precipitate, corresponcling to different
stages in the witltdrawal of the peripheral circiilatioii away
from the source of phosphate solution, i.e., the moat (compare
figs. 1,b and 2). This recession of thc peripheral circulation
56
XICHARD G. ABELL
was evidently due iii part t o actual iiijnry, possibly destruction, of thc most advanced capillaries bj- the phosphate solution, hut probably also in part to mecliaiiical tension exerted
upon the peripheral tissue by migration of a part of tlie proximal tissue toward the eiitraiice holes, particiilarly on the right
side of the bap (compare fig.2, c).
Xasses of precipitate formed in the bay of chamber
2/17/32L are shown in figures 1,b, 2, aiid 3. The photomicrographs shown in figures 1,b a i d 2 were taken during the
process of precipitate formation. Figure 3 is a photomicrograph taken 65 days following tlie removal of tlie phosphate
solution from the moat. Dnriiig the iiitcrrd betwcen tlic
time of removal of the phosphate solution and tlic time this
last photomicrograph ix-ils taken, tlie precipitate Iiacl undergone practically 110 change in appearance, aiid formed a
harrier which prevented forward growth of the ressels. The
precipitate formed in this cliamber persisted as a burrier
which blood capillaries did not penetrate tliroughont the entire
survival period of the chamber-more thaii 13 months following the time of precipitate f o r r n ~ t i o i i - a l t h o ~ isprout
~ ~ ~ s from
maiiy of the peripheral capillary loops grew up t o this barrier.
Tissue in contact with the precipitate garc no evidence of
being irritated by it.
Owing to ihc shallowiiess of the hay of chamber 2/17/33L
(depth approximately 40 p), the precipitate could be observed
with tlie greatest clearness. Two forms could be disFig. 1 a, A ~~hotoinic.rojira~~h,
showing blood vessels and other strw turcs in the
bay of chamber 2/17/32L, just before introduction of a phosphate buffer (pII 7.4)
into the moat. Eraekets E are placed 1 x 1 0 ~tlie edges of the two entrance holes
through which tlic new tissue inIaded the bay. The bay extends from the proxiinal
line D t o the distal line HB. The m o a t communicates with t h r bay a t t h e line
BB. The arrowq pointing u p ~ a r dare placed next a1terioles, ,4; tllovc pointing
downnard next veins, V. X 19.3. b, h photomicrograph, showing blood resscls
and other structures in the bay of chamber 2 / l i / 3 2 L , 22 houri following installation of the phosphate buffer ( p I I 7.4) within the moat. The extcnt of injury
of the tissue a t this t i m e can b e seen bp comparing the position of the peripheral
capillary loops i n b, x i t h those in a. The dark masses just beyoiid the most advanced capillaries, which form two more or less distinct rows, x and y, are composed chiefly of granulai material prwipitatcd by the phosphate huffcr, and of
emigrated Icukocytes. X 19.3.
PHOSPEIBTE SOLTJTIONS ANT) LIVING TISSUES
57
58
EICIiARU G. ISELL
tinguished. Oiie consisted of irregular, glistening, granular,
amorphous bodies, clumped together t o form larger masses.
The other mas composed of rounded structures, glassy in appearance and showing conceiitric rings, as though laid do.lr.11
in layers. Iiicomplete fusion of these rounded, spherule-like
bodies had resulted in the formation of double and multiple
structures (fig. 3).
Results similar to those described for chamber 2/17/32L
were also secured with chamber 7/30/31L. In the case of
this chamber, the phosphate solution was not iiitroducecl into
the moat until 11months and 3 weeks after the appearance of
the first vessels in the hay. The bay of this chamber was so
thin at the end toward the moat, approximately oiily 18 p deep,
Fig. 2 a, A photomicrograph, showing the distal tissue in the bay of chamber
3/17/32L and the precipitate being formed jiist in front of thc distal tissue.
This photomicrograph was taken G days following installation of a phospliate buffer
(pH 7.4) wthin the moat. C’ompare with figure 1,b, a photomicrograph taken
22 hours subsequent to introduction of t h r sanie buffer into the moat. X 23.3.
h, -4 photomicrograph, sliolving tlic distal tissue in thc bay of chamlm 2/17/32L
and the prec4ipitate being formed in front of the distal tirsue. This photornjcrograph was takcn 10 days followjng the installation of tlic phosphatc buffer mithin
the moat. X 23.3. c, A photomicrograph, showing the distal tissuc i n the bay of
chamber 0,/17/32L and the precipitate formed in front of the distal tissue b y
reaction of the buffcr (pH 7.4) with some substance or suE,stanccs in the bay.
This photomicrograph tyas taken 20 days following installation of the phosphate
buffer (pH 7.4) writhin the moat. The letters x, y and z ifidicnte roughlv distinguishable rows of precipitate. The buffered phosphate solution was removed
from t h e moat immediately after this photomicrogra~ihwas taken, and RingerLocke’s solution introduced in its place. x 23.3.
Fig. 3 A pliotoinicmgraph, showing a t high magnification t h a t part of the
precipitate and peripheral tissue contained in chamber 2/17/3211 which is within
the rectangle in figure 2 , c. One of the spherule-like rnaaws of precipitate is
designated by M. The positiori of sonre of the granular, auiorphous bodies is
shown by N. This photomicrograph was taken 65 daTs after removal of the
phosphate buffer, pH 7.4 at the time of its introduction, Prom the moat. The
precipitate reniained undissolved by the tissue. Thirteen and one-half months
after the time of formation of tlic precipitate thc chamber w a y removed froin the
ear of the rabbit, and the nixsses of precipitate analyzed. They contained
phosphorus. X 307.
Fig. 4 A niass of graiiiilar, amorphous precipitate formed withiii the hay of
chamber 1/23/32R, subsequent to introduction of a phosphate buffer (pH 6 2 )
into the moat. Living tissue has coniple+ely surrounded this mass of precipitatc,
which later dissolred. This photomicrograph mas taken 23 days following the
reninval of the buffer from the moat. X 307.
PHOSPHATE SOLUTIOXS AND L I V I N G TISSGES
59
60
JXICHAIlD G . A4EELL
that the blood capillaries were unable to approach closer to
the moat than 1.37 mm. In spite of this extreme thinness of
the bay, between the vessels and the moat, precipitate formatioii occurred.
It was observed that precipitate formation was not limited
to the region of injured tissue proximal to the original position of the peripheral capillary loops. Precipitate was also
formed in the region just in front of these capillai-y loops.
The distance beyond tlic original peripheral capillary loops to
the line at which such precipitate has been obserrecl to form
varies from approximately 75 to 230 p. This region is one
which coiltailis sprouts from the distal capilla1.27 loops, many
living white blood cells, and large numbers of fibroblasts.
Some of these structures were injured, others possiblp destroyed, by the buffers during precipitate formation. It is not
a t present l~iiowiiwhether any precipitate mas formed within
uninjnrecl tissue either distal or proximal to the peripheral
capillary loops.
2. I&roduc.tioiz i t i t n t h e woat of u liujf"ered phosphate solutio+z at pH 6.2. The phosphate solution at this pH was introduced into chamber 1/23,/32R, the bay of wliieli was approximately 100 p deep. Both drawing eyepiece and photomicrographic records show that the tissue advancccl uninterruptedly
before introdiiction of the phosphate solution into tlie moat.
At the time of introdidion of tlie phosphate solution, two
main crests of vascular tissue, h and C, extended for a considerable distance in front of the djstal tissue in other parts of the
bay. The most advanced capillary loops of the larger of the
crests, crest A, %[-ere0.33 mm. from the moat a t the time of the
introduction of tlie phosphate solution into the chamber, those
between the crests 0.73 mm., while those adjacent to the smaller
of the crests, C, were 1.22 mm. from the moat.
Microscopic observations made 2 days following the installation of the buffer vvithiii the moat showed that some of the
peripheral capillaries in the bay had been injured, others
probably destroyed, during this time, this result being similar
to that secured following introduction of the phosphate solu-
PHOSPHATE SOLUTIONS AND L M N G TISSUES
61
tion at pH 7.4 into chamber 2/17/32L and 7/30/31R, the injury
and destruction being, however, somewhat more extensive in
places in the present case. Destruction of the capillaries of
the crests, which had been closer to the moat than capillaries
in other parts of the bay, was most extensive. The vessels of
the crest that had been closest to the moat, crest A, had undergone disintegration for a distance of 1.086 mm. proximal to
the tip of the crest. Similar destruction of vessels of crest C
was observed. Between the crests, injury to the vessels was
slight, while beside the base of the smaller of them, C, none
of the vessels showed any visible effect of the phosphate solution, the peripheral capillaries beside the base of this crest
having been farther from the moat than those in any other
parts of the bay.
Arterioles which supplied blood to the tissue of the crests
were more widely distended than at any previous time, and
the more peripheral of the capillary loops and the venules collecting blood from these loops were noticeably dilated. The
circulation through the arterioles supplying the peripheral
crests, the distal capillaries, and the venules and veins collecting blood from these capillaries was much more rapid than
before the introduction of the buffer into the moat. I n contrast to the change in size of these arterioles, the arteriole
which supplied the peripheral tissue beside the base of crest
C, where no destruction of vessels had occurred, had undergone little or no change in diameter.
Microscopic examination of the vessels showed extensive
sticking of leukocytes to the endothelium of the peripheral
capillaries and to that of the venules and veins collecting blood
from these peripheral capillaries.
As in the cases of introduction of the buffered phosphate
solutions a t pH 7.4, large numbers of erythrocytes and leukocytes were present in front of the new line of functional
peripheral capillaries, and for a distance of about 0.8 mm.
proximal to this new line of functional capillaries many
freshly emigrated leukocytes could be seen in the tissue outside of the vessels.
62
RICHARD 0. ABELL
The chamber contained several lymphatic vessels, all of
which were separated from the moat by several rows of intact
blood capillaries. No injury to any of these lymphatics could
be observed. The number of leukocytes in them was no
greater than normal, and none contained any erythrocytes.
Just in front of the most peripheral of the intact vessels of
the crests, and between these crests, masses of irregular,
granular, clumped precipitate could be observed. None of
this precipitate appeared to be present in the form of spherules. Little or no precipitate could be seen in front of the
vessels at the base of crest C, and none was observed in any
of the tissue separated from the moat by circulating capillaries.
The phosphate solution was removed from the moat 50
hours after being installed within it, and Ringer-Locke’s solution introduced in its place. The pH of the phosphate solution taken from the moat was measured, and was found to have
remained unchanged while the solution was in the moat.
Twenty-four hours after removal of the phosphate solution the rate of circulation had decreased considerably, and the
sticking of leukocytes to the endothelium of the blood vessels
was not as pronounced as on the preceding day. During the
next 48 hours the rate of circulation and size of arterioles returned to the normal condition. At the end of this time
sticking of ledocytes to the endothelium was no more pronounced than before the injection of the phosphate solution
into the moat.
Five days following the removal of the phosphate solution
from the moat new sprouts could be seen growing from the
peripheral capillaries, but during the succeeding 10 days the
vessels of the crests grew forward only approximately 78 p.
This is 27 p less than the average distance that the vessels advanced per diem during the 30-day period preceding the introduction of the buffer into the moat. I n contrast to this abnormally slow rate of forward growth, the peripheral capillaries beside crest C, in front of which little or no precipitate
had been formed, and which had given little evidence of being
PHOSPHATE SOLUTIONS AND LIVING TISSUES
63
affected by the phosphate solution, advanced 1.1mm. during
the 15-day period following removal of the buffer from the
moat. At the end of this time they had grown beyond the
peripheral capillaries in the other regions of the bay distal to
which extensive precipitates had been formed.
As the tissue grew forward, it surrounded many of the
masses of precipitate. The presence in the tissue of these
masses of precipitate did not seem to irritate the vessels in any
way. Many cases were studied in which capillaries had grown
close to such precipitated masses, yet showed no abnormal
stickiness of their endothelium toward leukocytes, or other
signs of irritation. Figure 4 shows such a mass of precipitate within the living tissue.
Many of these masses of precipitate within the tissue were
gradually absorbed, so that 42 days following the removal of
the phosphate solution from the moat a considerable portion
of the precipitate had disappeared. Some of the masses of
precipitate were less soluble than others.
3. Ilztroductiom ilzto the moat of solutions of sodium and
potassium chloride. I n order to determine whether the
sodium and potassium ions present in the buffers had been responsible for the injury of the tissue and formation of the
precipitate, a solution of sodium and potassium chloride containing the same concentration of sodium and potassium ions
as the phosphate solution at pH 7.4 was installed within the
moat of chamber 1/23/32R after much of the precipitate previously formed in this chamber by the phosphate solution at
p H 6.2 had been dissolved. None of the vessels were destroyed, and no precipitate was formed. A phosphate solution
at p H 7.4, subsequently introduced into the moat, injured the
peripheral tissue for a distance of 0.77 mm. proximal to the
most advanced capillaries, and caused the formation of a precipitat e.
A solution of sodium and potassium chloride containing approximately twice the concentration of sodium and potassium
ions as did the phosphate solution at pH 7.4 was introduced
into chamber 7/30/31L, before injection of any phosphate
64
RICHARD G. ABELL
solution into the moat. No precipitate was formed and no
vessels were destroyed. As in the case of the experiment
noted above, a phosphate solution at pH 7.4,subsequently introduced into the moat, produced typical injury to the tissue
and resulted in the formation of a precipitate.
4. AfiPzalysis of the precipit,ate formed i.n-the bay subsequaat
to iw%vductiofiinto the moat of a buffered phosphate solution
at pH 7.4. In the case of one chamber, chamber 2/17/32L,
the precipitate formed subsequent to introduction of the phosphate buffer at pH 7.4 was analyzed for phosphorous. This
was done in the following way. Thirteen and one-half months
after the formation of the precipitate, the chamber was cut
from the ear of the rabbit, cocaine being used as the local
anaesthetic, and care being taken not to disturb either the precipitate or the tissue in the bay. The top of the chamber was
removed, thus exposing the precipitate and the tissue in the
bay. The bay was then flooded with 2 M nitric acid, followed
by a solution of ammonium nitrate and a 3 per cent solution
of ammonium molybdate (Wilson, '28). The masses of precipitate turned yellow. So also did the tissue, probably because of the production of ammonium phosphomolybdate.
The color of the masses of precipitate was slightly more intense than that of the tissue.
On the following day, the masses of precipitate were removed from the chamber. Care was taken not to include
material other than the masses of precipitate that had been
formed by interaction of the buffer with some substance or
substances in the bay. The masses of precipitate were then
washed with water. The yellow color paled decidedly, but did
not disappear entirely. Following the addition of 2 M nitric
acid and solutions of ammonium nitrate and ammonium moIybdate, the masses of precipitate became as yellow in color
as they had been before washing with water. It would thus
appear that the precipitate formed as the result of introduction of the buffered phosphate solation into the moat contained
phosphorous.
PHOSPHATE SOLUTIONS AND LIVING TISSUES
65
DISCUSSION
That the phosphate solutions introduced into moat chambers
in the present experiments actually passed to the region of
living cells and tissue in the bay, is indicated by the increased
stickiness of certain of the vessels toward leukocytes, the injury to a part of the peripheral tissue, and the formation of
masses of precipitate.
The work of Starkenstein ( '14), Binger ('17), Tisdall ( '22),
and others, demonstrates that solutions of sodium phosphate,
injected into animals, may produce toxic symptoms. Binger
('17) suggests that the toxicity may be due to the specific
action of the phosphate ions in combination with the reaction
of the solution in which they exist, and states that it is intimately associated with a decrease in serum calcium, but not dependent upon this alone. Greenwald ('18) asserts that the
phosphate ion is not particularly toxic, and attributes the effects of injection of sodium phosphate solutions to the changes
in reaction and osmotic pressure produced by them in the
blood plasma and in the tissue fluids, and "to the increased
concentration, both absolutely and relatively to the other
cations, of the sodium ions." Tisdall ( '22) considers the increased sodium concentration combined with the lowered concentration of calcium as the most important factor in the production of the toxic symptoms, while Underhill, Gross and
Cohen ('23) believe that the phosphate ion plays no specific
role in the production of the toxemia, except in so f a r as it
may aid in causing a diminution in calcium and thus disturb
the normal ionic equilibrium. They agree with Greenwald and
Tisdall that the symptoms are due to sodium, and in their experiments also to potassium poisoning. On the other hand,
Elias and Kornfeld ('23) report that the tetany produced in
patients by the injection of solutions of sodium phosphate is
due to the phosphate ions, and not to the hydrogen ion concentration or to the sodium ions contained in the solutions.
I n the present experiments it is clear that the sodium and
potassium ions were not responsible, at least not directly, for
the injury to the living tissue, since solutions of sodium and
THHl ANATOMICAL RECORD, VOL.
64, NO. 1, AXD SUPPLEMENT NO, 1
66
RICHARD 0. ABELL
potassium chloride containing even greater amolxnts of sodium
and potassium than did the phosphate solutions failed to produce such an effect. The injury of the tissue by the phosphate
solutions at pH 7.4 as well as at pH 6.2, indicates that the
toxicity of such solutions, when introduced into moat
chambers, does not depend upon the hydrogen ion concentration within the limits used in the present experiments, though
it is possible that the somewhat greater extent of tissue injury
in parts of chamber 1/23/32R by the phosphate solution at
p H 6.2 may have been due to the greater concentration of
hydrogen ions contained in this solution. I n a series of experiments, the author has introduced into moat chambers
Ringer-Locke 's solutions of greater and of less osmotic concentration than the phosphate solutions described in ihis paper.
Such solutions did not visibly injure any of the tissue in t.he
bay. It would thus appear that the phosphate ions in the
phosphate buffers introduced into moat chambers in the
present experiments exerted a toxic effect upon the living
tissue. Injury to the tissue concomitantly with the formation
of masses of precipitate suggests that, as in the case of the
experiments reported by Binger ('17), the toxicity may have
been associated with a diminution of the calcium ion concentration, such diminution having been caused by the excess
of phosphate ions. Whether or not the toxicity was due to an
effect of the phosphate ions exerted in some other manner than
by a disturbance of the normal ionic equilibrium is not evident.
There appears to be little doubt that the phosphate ions
contained in the phosphate solutions were responsible for the
formation of the precipitate, since masses of precipitate were
produced by introduction of phosphate solutions at pH 7.4 as
well as at pH 6.2, and since none were formed subsequent to
injection of sodium and potassium chloride solutions containing even greater concentrations of sodium and potassium ions
than did the phosphate solutions.
According to Peters and Van Slyke ( '32), the serum and
other body fluids are probably normally effectively saturated
with Cas(PO,),, so that any excess of either calcium or phosphate ions would, in time, cause precipitation, were not such
FIBERS IN TISSUE CULTURE
67
excess removed. I n the case of the plasma, such removal according to these investigators, is normally presumably effected
through excretory channels or by precipitation of the excess
Caa(P04)2in the bones. I n the present experiments the circulation passing through the most advanced vessels was evidently unable to remove the phosphate solution with sufticient
rapidity to prevent injury to the peripheral tissue, the phosphate ion concentration becoming sufficiently high to cause
precipitation even in the absence of bone. Since each of the
phosphate solutions used contained a large physiological excess of phosphate ions, it appears probable that the precipitates produced were composed partly of calcium phosphate.
Such a conclusion is borne out by analysis of the precipitates
formed in chamber 2/17/32L for phosphorous. No analysis
for calcium was made, but it is well known that excess of
phosphate ions precipitates calcium within the pH range used
in the present experiments, and vice versa.
Sendroy and Hastings (’27) found that the addition of
solid Cas(P04)2to horse serum ‘in vitro’ resulted in a rapid
precipitation of CaCO,. This suggests that a part of the precipitate formed following introduction of the phosphate solutions into moat chambers may have been calcium carbonate.
I n this connection the experiments of Wells and Mitchell ( ’10)
are of interest. These investigators observed that subsequent
to calcium phosphate or calcium carbonate implantation into
animals, in time carbonate was taken up by the phosphate and
phosphate by the carbonate.
The amorphous, granular, clumped precipitates formed in
the present experiments resemble those of calcium phosphate
described by Watt (’23), precipitated ‘in vitro’ in colloidal
solutions. Such precipitates were found by Watt to be
constantly granular and amorphous in character, the amorphous granules gathering in clumps and masses. Calcium
carbonate, precipitated in the presence of colloids, was observed by Watt to separate out and persist in the form of
spherules, some of which had slightly irregular peripheries
and appeared to be built up of concentric layers. The similarity between such spherules and those observed in the
‘PHB ANATOMICAL RECORD, VOL. 64, NO. 1, AND BUPPLEXRNT N O . 1
68
RICHARD Q. ABELL
present experiments is suggestive. It should be noted that
spherules were not observed in chamber 1/23/32R following
introduction of the phosphate solution at p H 6.2. This would
seem to be of some interest, since Watt reports that spherules
are less likely to form at higher hydrogen ion concentrations.
Areas of pathologic calcification taken from the pineal
gland, the choroid plexus, and various arteries were observed
by Watt ( '27) to contain calcium phosphate and calcium carbonate in forms which resembled those previously described
for these salts precipitated in colloidal solutions. The precipitates formed in the present experiments are strikingly
similar in appearance to those observed by Watt in these areas
of pathologic calcification.
Wells ('11) refers to the highly insoluble character of the
calcium salts of pathologic calcifications, while Watt ('25)
states that the deposits of calcified areas, once formed, tend
to persist. Shelly ('31) described calcified nodules in the
subcutaneous tissues of the arm and buttocks. The history of
the patient suggests that the deposits may have been highly
insoluble. Many of the masses of precipitate formed in moat
chambers in the present experiments were extremely insoluble,
some persisting for several months, others remaining undissolved throughout the entire survival period of the chamber
-in one instance more than 13 months. Some of the masses
of precipitate were absorbed within a few weeks, however,
particularly in the case of those formed by the phosphate
solution at pH 6.2. Wells refers to the work of von Werra
(1882) as evidence that calcification deposits are not necessarily permanent, and to that of Maximow ('06)' who observed that even if such deposits have undergone secondary
ossification they may disappear within a year. The present
experiments suggest that differences in solubility of the deposits of pathologic calcifications may be related to the pH at
which the deposits were formed.
The studies of Klotz ( '05), Wells ( '06), and others show
clearly that pathological deposition of calcium generally occurs only in tissues that have suffered either a partial or a
PHOSPHATE SOLUTIONS AND LNINQ TISSUES
69
complete loss of vitality. This may also have been the case
with the formation of masses of precipitate in the present experiments. The cells and tissues in the regions in which
masses of precipitate subsequently appeared were living and
active before introduction of the phosphate solutions into the
moat. In most instances they were evidently injured during
the process of precipitate formation.
I n the case of ‘metastatic calcification’ Wells (’11) states
that “whenever from any cause the proportion of calcium
present in the blood is so great that it requires the effect of
both the colloids and of the CO, in maximum concentration to
keep it in solution; then the calcium salts are deposited in those
points in the body where the CO, content of the fluids is least.”
The present work suggests that a similar precipitation might
occur without increase of the calcium ion concentration if the
phosphate ion concentration became sufliciently high.
By means of the present technic it is possible not only to
observe, with the high powers of the microscope, including oil
immersion lenses, the actual formation of precipitates which
resemble closely those of pathologic calcification, but also to
modify at will the chemical environment of the tissue, with the
purpose of throwing further light upon the factors which are
responsible for such calcification.
The formation of masses of precipitate for a short distance
in front of the original peripheral capillary loops, as well as
throughout the region of injured tissue, is of interest in the
light of the observation, reported by the author (’34), that
granules in certain cells in this same general region stain a
brilliant blue following introduction of solutions of methylene
blue into moat chambers. At a greater distance beyond the
peripheral capillary loops than approximately 293 p the
methylene blue was reduced within all of the living cells, indicating a decrease in oxygen concentration with increased distance from the distal capillary loops. The present experiments might indicate a similar decrease in calcium ion concentration.
70
RICHARD Gi. ABELL
That methylene blue, following introduction into moat
chambers, is prevented from diffusing proximally throughout
the tissue in the bay because of removal of the dye by blood
passing through the most advanced rows of peripheral capillaries, was shown by the author ('34). Sticking of leukocytes
to the endothelium of the venules and veins that collected blood
from the peripheral capillaries, but not in others, suggests
that the phosphate solutions used in the present experiments
also entered the peripheral capillaries. Removal of the solutions by the blood passing through these capillaries, possibly
also the formation of masses of precipitate which interfered
with the free access of the phosphate solutions to the tissue,
evidently prevented more general diffusion of the solutions
throughout the bay, and accounts for limitation of precipitate
formation and injury of vessels to a narrow strip of peripheral
tissue.
That the concentration of phosphate solution which reached
those capillary loops in the bay that were closest to the moat
was greater than that which reached other peripheral capillary
loops located in the bay at greater distances from the moat,
is shown by the experiment in which the phosphate solution
at pH 6.2 was introduced into chamber 1/23/32R, since the
extent of tissue injury and precipitate formation was greatest
in those regions of peripheral tissue that were closest to the
moat, and since terminal arterioles and peripheral capillary
loops closest to the moat became dilated, evidently because of
the effect of the phosphate solution upon them, while the more
proximally placed of the terminal arterioles and capillary
loops changed little, if any, in caliber. Similar but not such
striking results were also secured with the other chambers.
SUMMARY
1. This paper describes experiments in which buffered
phosphate solutions at pH 7.4 and 6.2 were introduced into
moat chambers. Such solutions, placed within the moat, pass
by diffusion into the bay, and there interact with the thin
layer of living cells and tissue normally present during the
PHOSPHATE SOLUTIONS A N D LIVING TISSUES
71
growth of blood vessels and other structures in the chamber.
The effect of the phosphate solutions upon the living tissues
within the chambers was observed with the high powers of the
microscope.
2. Phosphate buffers, of the pH and concentration used in
the present experiments, injure the peripheral tissue in the
bay and cause the formation of masses of precipitate in the
region of injured tissue.
3. The most advanced of the distal tissue was injured
proximally for a distance of 1.22 111111. following introduction
of the buffer at pH 6.2. Subsequent to installation of the
buffer at pH 7.4, the distal tissue was injured in no case for
more than a distance of 0.77 mm.
4. The distance beyond the original distal capillary loops
to the line at which precipitate has been observed to form
varies from 75 p to 230 p. It is not at present certain whether
precipitate formation occurs in the uninjured tissue proximal
to the most advanced of the peripheral functional capillaries,
although masses of precipitate have been frequently observed
within the proximal living tissue.
5. Injury to the peripheral tissue was accompanied by dilation of the arterioles which supplied this tissue with blood;
by increased stickiness toward leukocytes manifested by the
endothelium of the capillaries, venules and veins which collected blood from the periphery; and with emigration of
leukocytes from the distal capillaries.
6. The results indicate that the phosphate solutions entered
the most advanced capillaries and were removed by the blood
circulating through them. The solutions were not removed
with sufficient rapidity, however, to prevent the phosphate ion
concentration-possibly also in the case of the buffer at pH
6.2, the hydrogen ion concentration-from becoming injuriously high.
7. The concentration of phosphate solution that reached
those capillary loops closest to the moat was greater than that
which reached other distal capillary loops, located at greater
distances from the moat.
72
RICHARD G. B E L L
8. Injury to the living tissue was limited to a narrow strip
of peripheral tissue, evidently because of removal of the phosphate solutions by the most advanced capillaries, possibly also
because of the formation of masses of precipitate which interfered with the free access of the phosphate solutions to the
tissue.
9. Masses of precipitate formed subsequent to installation
of the buffer at pH 6.2 were gradually absorbed by the living
tissue, but those produced following introduction of the buffer
at pH 7.4were much less soluble, many remaining in contact
with living tissue practically unchanged f o r months-in the
case of one chamber, for more than a year. Once formed, the
masses of precipitate exerted no observable injurious effect
upon the tissue. Such masses of precipitate resemble closely
those of pathological calcification. Analysis of the precipitate
formed by the phosphate solution at pH 7.4 showed that it
contained phosphorous, and it appears likely that the precipitates formed by the phosphate solutions at both pH 6.2 and
7.4 were composed partly of calcium phosphate. They may
also have contained calcium carbonate.
LITERATURE CITED
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living cells and tissues in the transparent moat chamber introduced
into the rabbit’s ear. Anat. Bee., vol. 60, p. 161.
ABELL,
&CHARD G., AND ELIOT
R. CLARK 1932 A method of studying the effect
of chemicals upon living cells and tissues in the moat chamber, a transparent chamber inserted i n the rabbit’s ear. Anat. Rec., vol. 53, p. 121.
1933 Some studies on the reaction between living cells and chemical
substances in the ‘moat’ chamber introduced into the rabbit’s ear.
Anat. Rec., vol. 55, supplement, p. 1.
BINGEE,CARL 1917 Toxicity of phosphates in relation to blood calcium and
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KLOTZ,OSKAR 1905 Studies upon calcareous degeneration. I. The process of
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1925 The deposition of calcium phosphate and calcium carbonate
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1927 Deposition of calcium salts in areas of oalciflcation. Arch.
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