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Notes on the application of experimental methods upon mammalian embryos.

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SOTES ON THE APPLICATION OF E X P E R I h l ~ N T R L
METHODS UPON ilIAAI,llAI, IAN ft:1IBRYOS
J. 8. NICHOLAS
Anatomical Laboratorg, Scahool of M e d i r i : ~ p , 77niwrsity of Pittsburgh, and
Biologzcal Laboratory, C o l d Sprtng Harboi, Loirg Iala7it/, S c w Yorl;
While the experimental method has been applied to many
forms among the vertebrates in which the developmental
mechanics of the embryo have been studied, by f a r the
greater amount of work has been carried out upon the ampliibia which lend tlicmselves most readily to this form of
experimentation. The mammalian embryo lias been praetically untouched because of the many natural difficulties which
arc present.
Recent studies upon the transplantation of eyes in adult
mammals ?IF Roppgnyi ('23) have rendered it imper at'1 V 8
that more should be known about the eal.1:- mechaiiics of development ill the mammal. The possihili ties of regeneration
which may be present at some early stage of mammalian
development must be determined. The time of permanent
differentiation of parts which cannot be changed by esperimental procedure should be ascertained.
It was with these factors in mint1 that the studies nhicli
are liere reported were begun two years ago a t the University
of Pittsburgh School of ;Iledicine,l the present report constituting only the method of approach to the problem and
pointing out some of the future possibilities to which this
method is capablc of npplication.
Tlie studies so far conductctl upon the mammalian fetus
are discussed in a paper by Swensen ( '25). He cites Bichat,
The experiments were pcrformed in the An:ttomical Lahoratorp, School of
Yedicine, Uniwrsity of Pittsburgh.
38.5
I’rcyer, ant1 Zuntz as having made observations upon maml y
His own eoritriliution deals with tlie
mals a t ail ~ ~ r stage.
possihility of direct observations upon rat embryos after ereatiiig cerc~bralaiiaemia by tlie ligation of the carotids. He
was able to stimulate the embryos both mechanically and
clcctrically by manipulating the embryos in a water-bath. A11
tlie experiments cited -:01 him are those in which tlic fetus
has Ixxn removed from the uterus, the placental site being
nntlisturberl, and placed in a fluid medium f o r study. Uiider
these conditions, the time of ohserration is necessarily limitc.tl. The ref~rericesare iiot a t present a t hand aiid I refer
t o them onlj- as cited from Swenseii’s article.
U y own studies are concerned with the reaction of the
fetiie after operation. The prcliminary study inrolres tlie
followiiig questions :
1.Can a mammalian embryo be subjected to operative procedure in utero and remain viable through the completion
of tlic gestatioii period?
2. What are tlie possibilities of wound healing: a ) by the
em1)ryo ; 71) in tlie embryonic coverings ; c ) in tlie uterine wall ?
3. What is the best method of operative approach which
will give tlie best results with the minimum of manipulation
a n d the grcatest completeness of surgical repair?
The present paper is an aiiswror to tlie first and third questioils, leaving tlie secoiid f o r it mom complete cliscussioii in
aiio tli cr cominniiication.
MhTEllIAL
8x1) YETHODS
The rat is a 1-ery suitalde animal for experimentation of
this kind. Tlie animal is exceedingly resistant to ordiiiary
iiif e(+fioiis,is capable of rather long periods of aiiaesthetizatioii 1%-ithontIml effects, mcl recovcrs readilj- from the
operatioii.
Tlic aiiimals are prepared for operation by preliminary
niiacsthesia, at which time tlie area of operation is prepared
b y sliaviiig. Tlie skin area is tlicii wasliecl and ptiiiitetl with
iotliiic. An incision is made throngli tlic skin and superficial
FOETAL OPEBATIOKS
387
fascia extending from just below the margin of the ribs to
a point just anterior to the posterior limb. This incision is
slightly lateral t o the long muscles of the back. A second
incision is made somewhat lateral to the first incision through
the underlying muscle layer, and this layer is dissected even
farther laterally before making the final incision into the
abdominal cavity.2
With ordinary care there is little bleeding to occlude visibility in the field of operation. The incision exposes the lower
portion of the kidney and slight retraction of the wound edges
brings the horn of the uterus of the operated side into view.
The ovary also is in plain sight. The uterine horn is then
grasped with a pair of fine forceps and brought to the level
of the incision for inspection. If embryos are found in the
tube, one is chosen for the operation and is brought to the
previously prepared skin surface, upon which it is laid for
subsequent manipulation.
The embryo is now in plain view, is clearly visible through
the uterine wall, and is in position f o r the operation. The
placental site is carefully noted and carefully avoided during
the operation. A small slit is made in the uterine wall and
the amniotic sac is punctured to permit the drainage of the
amniotic fluid. This relieves the tension upon the uterine
wall. The incision for removing the amniotic fluid should
be made as nearly as possible over the area of the embryo
upon which the subsequent operation is to be made. The
embryo is fairly movable inside the uterine wall after the
drainage of the amniotic fluid, and it is possible to move
the part to be operated to a more favorable position even if
the initial uterine incision should not be in such location.
The incision in the uterine wall must be as small as possible
with the smaller embryos. An incision made with a fine needle
* The posterior method of approach as used in mammalian operations first
appears in the literature in Biedl’s work. I t has since then been quite widely
applied to different types of procedure. The recent publications using this method
a r e those of MacDowcll, Anat. ~ I c c . , vol. 27, and Davenport, Jour. Esp. Zool.,
vol. 42.
388
,J. S. KIC’ITOLM
i+ wfficiciit t o drtiiii tlic amniotic fluid from c1rnl)ryos of
t\\-elve to forirteen (lays ~ r i t lalso to permit the operation
upon the embryo. If the incision is too large, thcre is a
grcbat possibility that tlie embryo may be extruded through
thc opcning hy contraction of the uterine musculature. Once
estrided, it is practically impossible to replace the embrj-o
w i t h tlie uterus and also to repair the tear in the wall.
~ s t i ~ u s i oofn tlw embryo generally dislodges the placental
site, and ilic animal is lost.
Jndivid~ialswhich have been extruded from the uterus have
remained viahle for several hours, provided the placental
attaclimcnt has not been disturbed. Experiments to be reported later will deal with the conditions occurring in animals
which liare heen removed from the uterus and permitted to
witlcrgo development within the abdominal cavity.
In the experiments lierc reported, t>lie operations were
c>;iri.irtl on within tlic utcirinc wall, and the orgaii, a limb,
tail, 01’ eye 1*cmovec1tliroiigli tlic prirnary incision in the uteriiic. \I a l l .
I4;xcision of parts was accomplished by initial ligation with
i t sul),sccjiientcut i i i tlie c a w of the limb arid the tail, lmt canttitig \\.it11 ;I fine pair of iridtvtomy scissors iit the case of tlic
(’ye. The first two opcratioiis critail no loss of foetal i)lood,
t h e h c c w n d occasions a small aniourit of hemorrhage, wlii(~li,
1 1 0 ~ w v ~is~ rcaxily
,
chec*kctlby the iiitroduction of a small cotto11
sy)ollgSe.
-1ftei. tlw clxcaisioii of a part, tlic fetus is mowcl abont i n
the iitcrus so tliat thc operated area is not in proximity with
thc opcwiiig in tlie uterine wall. The incisiori is then snrgi-
rq)aired.
Stcys in surgicd repair arc 21s follows: No attempt is
rnacle to repair the amniotic sac. Whether this is healed and
t l i e amniotic fluid rcplacetl in the processes of healing cannot
at present be stated. Attempted repair causes more serious
damage than permitting the amnion to go unrepaired. If
the emln-yos are in the early stages of development, it is better
not to attempt repair of the uterine wall. I n all animals,
cid I
FOETAL OPEHATIORS
389
however, operated after the sixteenth day of development
it is necessary to suture the uterine wall due to the increase
in size of the uterine incision during manipulation of the
embryo. I n this case the uterine wall may be closed by suture, using fine silk thread and the smallest obtainable
needles; the uterine horn and its contents are then replaced
within the abdomiiial cavity and the repair of the body wall is
undertaken.
In the rat care must be taken in the repair of the body wall.
The superficial stitches are often removed by the animal
and, unless the underlying repair has been carefully performed, there is danger of herniation. It was f o r this specific
ieason that the initial incision was made a8 described above.
‘i’he repair of this incision entails a double overlapping of
the tissues concerned. The lower muscle layer is joined with
a continuous suture, a second series of interrupted sutures
joins the edges of the second muscle layer. A final series of
interrupted sutures joins the edges of the skin and superficial fascia. After completing the final skin suture, the -\vound
area is cleaned and tlie animal returned to its cage.
The first group of experiments was planned to sho\v the
possibilities of this type of operation and to give some indication of the future value of such procedure. The operations
upon rats have formed the bulk of the preliminary work and
is the group considered in this communication. Operations
of a similar nature have been conducted upon cats with promising results.
Since the feasibility of the operation is the first big cluestion, the actual operation served only to demark a certain
animal from the rest of the litter. This was accomplished by
the excision of a leg o r of the tail. The experiments fall
naturally into groups according to the age of the embryo.
The results tabulated in table 1 show that the operation is
feasible and that a fair percentage of animals operated in
utero may be recovered a t the end of the normal gestation
period. If, as has been clone in subsequent experiments. it is
desired t o study the emhryonic reactions during a short inter-
390
J.
S. NIClIOLAS
\-a1 of developmcnt after operation, the operated fctus can
he recovercd in every case by reopcrating tlie mother and
removing the fetus at the end of several days’ development
suhseyncnt t o the first opcratioii.
The age of embryos, unfortunately, has not been accurately
timed. The age given in table 1 is an approximation based
011 tlie general size and development of the embryo.
This
has heen checked against the Wistar table for weight, which
S(’FWH vcry well f o r the immediate problem. If a control
animal is desired as a check on the age of the embrvo at thc
time of operation, an embryo may be removed from the tube
in which thc operated animal is contained. Preferably, this
is the animal attached next to the operated animal.
The ativisahlc method of approach would seem, at first
sight, to be tlirougli the anterior abdominal wall. This would
give a widc field of operation and would expose both horns
of the uterus with their contained embryos. All of the initial
espwiments wcre performed in this way. It was found, however, that the posterolateral approach is f a r better for the
following reasons :
1. The amount of shock incitloiit to handling of the viscera
is aroidcd. Such liaiidling of visccra is necessary in all anterior operations in order to inspect the uterine contents.
2. There is less displacement of parts and less necessary
maiiipidatioii of the uterus itself, for the irterine horns arc
f o n n d practically in contact with the body wall.
3 . T l d i i i g of the hocly wall mid its repair is more complete.
The \s-ouiid can be kept clcaner and is less apt to become
infected through contact with the cage flooring.
4. -1 better and stronger surgical repair can he secnrccl
through an overlapping of the muscular layers.
5. While the field of operation is necessarily more restricted, it is not advisable to operate too many embryos, f o r
the increased shock is generally productive of abortion.
I have purposely omitted from table 1the list of ~~nsuccessful cxperiments perf ormcd before the development of the
present technique. It is sufficient to state that a minimum
391
FOETAL OPEI'lATIONG
of manipulation is neccssay to give posit.ive resu1t.s. Multiple operat,ions, while t,hey are not impossible, increase in
direct ratio the chalices of losing the whole experiment.
The removal of all embryos from oiie horn of t.lie uterus,
with the exception of the one animal opera.ted in order t.o
secure for t,he one operated greater nutribional possibilit.ies,
is not pra.ctically sound because of the inc,rea.sein amount of
operative shock.
TABLE 1
...
ANIIAIS
OPKHATED
~~~~
~
1
.
~~~
A G E OF
EMBRYO
I
I
~
ur1u.S
. ..
~
~~~
I)AW BEFOKE
.
BIRTH
I
~~~
1d'ITP.K BORN
..
.
~
i
. ..
.
. .. .
. . ..
REMARKS
I
~
1 4 lost
I 2 recorered
with litter
j 5 lost
( 4 recovered, :dl viable
3 born dead, 2 lost
6 recovered, all viable
i born dead, G lost
14 recovered, all riable
9lostl
Sornetimee premature 5( 16
recorered, all viable
\
8
lost
(+enerallypreinature 1 1 4 recovered, all viable
-
1
29
i
2
Embryos are recorded as lost when absent from the litter upon inspection as
soon as possible after birth. Since, unfortunately, it is impossible to have these
animals under constant observations, many embryos were lost which either were
born dead or else were so slightlp viable that they failed t o survive and were
c:rtm before the next o h e r r a t i o n was m:ide.
The first series of experiments in which the rat was used
were those in which timed embryos were secured by surgical
procedure. A pregnant female was operated through the
anterior abdominal wall, one horn of the uterus was ligated
near its junction with the body of the uterus and removed
completely. Its contents were removed and preserved. The
embryos in the remaining horn were permitted to continue
development and be born a t the close of the normal gestation
period. The difference in time between the date of operation
%Four animals of this series were observed after birth, hut were lost in an
attempt t o hale them reared with the litter.
the (late of birth of the litter wlien subtracted from the
total gestat,ioii period gi;vcs tlic actual age of tlie embryos
rcmo\-etl 1))- operation. This age value has beeu cliccketl
against selreral embryos in which tlic copulation time w a s
obst*r\-edand also by comparison with the Wistar series (The
Rat, 11. 23, also p. 65, tables 4.6 and 47, 1915 cd.). The differeiices in time between these groups fall well within the normal limits of error. This method is peculiarly applicablc to
scriiriiig graded series of rat embryos f o r class work.
1;lsrcSssivc maiiipalation of tlic abdominal viscera or an
0lWl.a tioii iuvolring a numher of incisions through the
atc.riiic \ v d l caii al\\-a.;s cause a1)ortiou. Roth of these coilclitioiis ciiusc an excessive amoniit of shock. So long as tlic
opt-rativc field is restricted and thc placental site is uiitlisturl)cbtl, there is little danger of abortion except in tlic last
two ( l a p hefore tlie close of gestation. At this time mi operat i o n of any sort npot~the mother is apt to bring : h u t the
pi-c.in;tti1rc 1)irtli of tho lit tcr. Tlic? young, while prcmatnr.el,v
h i - n , a i ’ c g~iicrallyvia Me and c a n he rearctl.
T h t h of tlie fctus in ixtcro is geiicrally the rcsult of t h c
tlisloclgmciit of the placenta Pro~iithc? utcriiie w d l (luring
mniii1)nlation of the uterus. Sovci~alohservations have l w n
s;ccni*tvl iii n-liicli the fetus has midergone partial resorptiou
\ritIiiii t l i ~iitcrus. Thc fetus may become so adlierent to the
i l t oriiie -\vallI)ccausci of healing after operat ion that i t caiiiiot
l)c tlisloclgctl n~ tlic closc of gesf ation. This contlitioii has
Iwtw found in tlic opcrcxtions upoii the earlier stagcs. l h twyos operated later in development generally are horn with
the rest of the litter, even though they have not snrvived
the cwiitlitions occurring subsec~ucii
t to operation. The u s e
of suture material in the uterine wall tcncls to incrcase the
possihility of adhesion due to decreased distcnsibilitj- of
tlie nteriiic wall.
Because of the resistance which the rat seems to have to
orciinary pyogeiiic infection, it has been found that strict
astqitic precautions are not necessary. Experiments in which
simply oi*diiicirycleanliness of materials has been practiced
aiitl
FOETAL OPEFlATIONS
393
produce as great a percentage of positive results as those
in which strict asepsis has been attempted. From this standpoint, the rat is an ideal animal for this type of experiment a ti on.
Tlic operation upon the embryo and tlie cutting of the
uterine wall have no apparent effect upon the future litterbeariiig possibilities of the mother. Sevcral of my animals
which have been under observation in regard t o this point
have given birth to normal-sized litters since the time of
operation. Three of these have been reoperated and have
given birth to tlic opcrated litter and also to subsequent
normal litters.
The conditions tending to reduce the number of operated
young which can be recovered at birth are due to several
factors. The young which have been operated in utero are
perceptibly weaker than their unopcmtcd litter mates. They
are, therefore, less capable of being carried over the earl:critical period. There is more neglect of the litter than is
usual, cluc to tlie normal seqiielae of operation. Frequently
function of the mammary gland is retarded and nutrition is
not available during the first days after birth. This is probably the main reason for the lack of maternal interest in the
litter. Cannibalism on the part of the mother is not infrequent. Many animals which probably were viable at birth
succumbed sliortly after and were disposed of by the mother
before observations could be made. Tlic absence of a limb
further lcsseris the chances of existence of tlic operated
animal.
Since tlie cxperiments here reported deal only with the
possibilities of completing the normal gestation period with
viability at birth after an intrauterine operation, it is not
necessary to detail other experiments now under way which
deal with thc results of extirpation and the possibilities of
transplantation in this form. Experiments upon the limb and
upon the nervous system have been performed arid the prcliminary results indicate that there is a large field in this
form for the study of the reactions occurring during mammalian development.
394
J. S. XICHOIAS
SUMMARY
;. It is possible t o operate upon tlie fetus of the rat arid to
sewre thcse animals, operated in utero, alirc! at the end of
tlic riormal gestation period.
2. The rat is exceedingly applicable to this type of expcrimcritatioii f o r the follomiiig reasom :
a. Extraordinary resistance to ordinary iiifectioris occiirring after operation.
1). The size of the embryo is suc~lithat it can readily be
liandled.
(*. Tlie period of gestation is slioi*t and results are fairly
(1 uicklp oh t ained.
(7. The pregnant animal posscsscs coiisiderable ability to
withstand the operative shock without aborting.
3. The experiments so far conducted S ~ O Rthat a limb may
bc extirpated, the tail may be excised, and the eye may be
c*stirpated and that the reactions of tlie embryo to these types
of operation maF he studied throughout subsequent developmcii t .
LITERATCRE: CITED
BIUIAT,S . 1803 A llgemeiiir Anntomic niigewniidt auf die Z’li~siologia untl
Arziieiwissensc11:tft. e l ~ e i .v. Pfitfi., Leipxig.
~ ) O N I I . D S O N , 11. 11. 1916 The rat, 1st rd. 1’hil:idelphia.
1ioPPAss-1, THsonoR 1923 T)ie 1~cpl:i11tatio1i
voii Augeii. Arch. f . m i e l . Anat.
11. f . Entw.niecli., Hd. 99.
I’I~E\
E.K. i V . 1885 Spwielle l’lipsiologie d r s Embr? o. L e i p i g .
SUTSSXK,E. A . 1925 The i i ~ eof rc.rel)r:d m n e m i a in exprriiiiei~tnl eml>r.gcl
loyic~~
stuclies
l
upon mnmmals. AII:L~.Rrc., to]. d0.
Z u s ~ z 1877 ltespiration tles Skugetllier-Yctos.
l’fliip,r ‘F Arch. f. {I. grs.
I’lrysiol., Bd. 14.
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