Intrauterine Repair of a Lesion Resembling Cleft Lip and its Effect on Maternal Behavior in Rats ALBERTO SALAZAR, M.D. Two studies were done to determine the ideal day to perform an in- trauterine procedure and to explore maternal behavior toward repaired cleft lip offspring rats. The first study showed that the best time to cre- ate a surgical cleft lesion was on the 19th day of pregnancy. Thus, for the second study rat fetuses were operated in utero on the 19th day of gestation, and the pups were delivered by cesarean section. One group was obtained with a lesion resembling cleft lip and a second group with repair of such lesion. Nonoperated litter mates were used as a con- trol group. Two animals of each type were placed inside a cage, and the order in which the mother retrieved them and carried them to the nest was noted. Statistical analysis showed that retrieving them was not at random. All controls and 14 out of 18 treated animals were retrieved, but only 4 out of 18 of the clefted group were retrieved. KEY WORDS: cleft lip, intrauterine repair, rat behavior Experimental intrauterine surgery has been used for fetal physiological studies (Kraner and Parshall, 1968), but recently prenatal surgery has been proposed as future treatment for problems such as congenital diaphragmatic hernia (Harri- son, et al 198la; Harrison et al 1981b), hydrocephalus, and spina bifida (Hodgen, 1981). Robinson and Goss (1981) proposed intrauter- ine repair of cleft lip as a possible treatment be- cause of facility of healing of surgical wounds in fetal rats. Hallock (1985) corrected a cleft lip in utero in mice and confirmed the absence of scarring as Robinson and Goss had mentioned. The surgical results presented by those authors were considered only from a histological point of view; however, the maternal response toward the treated and clefted pups is also of interest. Two studies are reported here. The first one was to determine the ideal day to perform the Dr. Salazar is Professor of Surgery at the Facultad de Medi- cina Universidad Nacional Autonoma de Mexico and works at the Instituto Nacional de Enfermedades Respiratorias, Secretaria de Salud. This work was also supported by the Instituto Nacional de Ciencias y Tecnologia, Sistema Nacional para el Desar- rollo Integral de la Familia. This paper was presented at the Vth International Congress on Cleft Palate and Related Craniofacial anomalies. Septem- ber 2-7, 1985, Montecarlo, Monaco. 38 intrauterine procedure, according to the lowest death rate. A section in the upper lip of rat fe- tuses was removed from the 14th through the 20th gestational day. The second study was designed to explore maternal behavior toward three categories of offspring: normal, surgically induced cleft lip, and repaired surgically induced cleft lip. MATERIALS AND METHODS The first study involved 140 female, virgin, Wistar rats. They weighed 230 to 250 g and were 3 months old. These rats were mated by the har- em system of five females with two males. For the first study, seven groups of 20 mem- bers each were formed. Group one underwent laparotomy 14 days after impregnation. Group two after 15 days and so on (Fig. 1). The laparotomy was done under ether anesthesia. Two fetuses were operated in each rat; those most proximal to each ovary were chosen be- cause of easy manipulation. A purse suture 1 cm in diameter with 7-0 silk was applied to the free uterine wall, followed by the section of both the uterine muscle and the fetal membranes in the area surrounded by the silk suture. Then the fe- tus' oral region was exposed, and a square-like piece of tissue (3 X 3 mm) was excised in the midline of the upper lip. Afterward the fetus was
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Intrauterine Repair of a Lesion Resembling Cleft Lip and its
Effect on Maternal Behavior in Rats
ALBERTO SALAZAR, M.D.
Two studies were done to determine the ideal day to perform an in-
trauterine procedure and to explore maternal behavior toward repaired
cleft lip offspring rats. The first study showed that the best time to cre-ate a surgical cleft lesion was on the 19th day of pregnancy. Thus, forthe second study rat fetuses were operated in utero on the 19th day
of gestation, and the pups were delivered by cesarean section. Onegroup was obtained with a lesion resembling cleft lip and a second groupwith repair of such lesion. Nonoperated litter mates were used as a con-
trol group. Two animals of each type were placed inside a cage, and
the order in which the mother retrieved them and carried them to the
nest was noted. Statistical analysis showed that retrieving them wasnot at random. All controls and 14 out of 18 treated animals wereretrieved, but only 4 out of 18 of the clefted group were retrieved.
KEY WORDS: cleft lip, intrauterine repair, rat behavior
Experimental intrauterine surgery has been
used for fetal physiological studies (Kraner and
Parshall, 1968), but recently prenatal surgery has
been proposed as future treatment for problems
such as congenital diaphragmatic hernia (Harri-
son, et al 198la; Harrison et al 1981b),
hydrocephalus, and spina bifida (Hodgen, 1981).
Robinson and Goss (1981) proposed intrauter-
ine repair of cleft lip as a possible treatment be-
cause of facility of healing of surgical wounds
in fetal rats. Hallock (1985) corrected a cleft lip
in utero in mice and confirmed the absence of
scarring as Robinson and Goss had mentioned.
The surgical results presented by those authors
were considered only from a histological point
of view; however, the maternal response toward
the treated and clefted pups is also of interest.
Two studies are reported here. The first one
was to determine the ideal day to perform the
Dr. Salazar is Professor of Surgery at the Facultad de Medi-cina Universidad Nacional Autonoma de Mexico and worksat the Instituto Nacional de Enfermedades Respiratorias,Secretaria de Salud.This work was also supported by the Instituto Nacional
de Ciencias y Tecnologia, Sistema Nacional para el Desar-rollo Integral de la Familia.
This paper was presented at the Vth International Congresson Cleft Palate and Related Craniofacial anomalies. Septem-ber 2-7, 1985, Montecarlo, Monaco.
38
intrauterine procedure, according to the lowest
death rate. A section in the upper lip of rat fe-
tuses was removed from the 14th through the
20th gestational day. The second study was
designed to explore maternal behavior toward
three categories of offspring: normal, surgically
induced cleft lip, and repaired surgically induced
FIGURE 1 Percent of operated fetuses that were stillborn when delivered on the 21st day of pregnancy.
replaced in the original position inside the uter-
us and the purse suture was closed (Fig. 2).
For the second study, the fetuses were oper-
ated on the 19th day of gestation; those clefted
(N= 18) were obtained in the same way as those
in the first study. The pups considered as treat-
ed (N= 18) were obtained by replacing the sec-
tioned lip piece and suturing it back with two
simple points of 10-0 nylon monofilament.
Nonoperated litter mates (N= 18) were used as
controls. On the 21st day the fetuses were deli-
vered by cesarean section (Fig. 3). Two pups
of each type were placed away from the nest
(made of wood shaving) in the edges of stained
polycarbonate cages 42 x 21 ® 20 cm. The
order in which the pups were placed was ran-
domly determined in four cases and by rotating
the pups in the other five cases (Fig. 4). Foster
mothers were used to avoid the effect on their
behavior of cesarean section. These foster
mothers made their nest and delivered normally
their own pups 12 hours before exposure to the
experimental offspring.
Retrieving to the nest was noted. The obser-
vation was done by the same person in a dark
room under a red light for a 2-hour period.
RESULTS
The results showed a 100 percent mortality
when fetuses were operated on the 14th to 15th
day of pregnancy; those operated on the 16th and
FIGURE 2 Rat fetus operated on 19th day of gestation
by resecting and replacing one-half of the upper lip (sutur-
ing with two points of 10-0 nylon monofilament).
40 Cleft Palate Journal, January 1988, Vol. 25 No. 1
FIGURE 3 Examples of rat pups classified as "treat-ed" (top and right) and as "cleft" (bottom left).
17th days had death rates of 97 and 81 percent
respectively. The lowest death rates of 30, 7, and
15 percent were obtained on the 18th, 19th, and
20th days of pregnancy (see Fig. 1).
a) Random distribution
A statistical difference in death rate at proba-
bility level of 0.001 was seen among groups
operated on the 14th through the 20th day. This
difference is remarkable from the 18th day
through the 20th day when compared with days
14 through 17 as shown in Figure 1.
Figure 5 shows the percentage of nonoperat-
ed fetuses that were stillborn; these were litter-
mates of the operated animals.
The lowest death rate was obtained when the
procedure was done on the 18th or 19th day of
pregnancy (1 and 2 percent respectively) with a
significance level of p< 0.05 when groups
operated on the 18th, 19th, and 20th days were
compared with those operated on the 14th, 15th,
16th, and 17th days (Fig. 5).
In the second study, the behavior of the foster
mothers was initially indistinctive toward the ex-
perimental pups (two clefted, two treated, and
two controls), touching, smelling, and licking
them with no apparent order. However, as is
shown in Table 1, when retrieving the pups to
the nest, only two mothers retrieved the whole
litter. Three carried all controls and treated
animals but none with clefts. Four retrieved the
control pups and one of two treated pups but none
of the clefted pups.
To summarize, all controls were carried to the
nest; 14 of 18 treated animals were also carried,
and only 4 of 18 of the clefted group were
retrieved. A Chi-square test showed a statistical
difference at p < 0.001 level between the treated
group and the clefted group.
Nest C Nest C Nest C Nest C
T T T T
C
Co T Co Co T Co Co T Co Co T Co
1 2 3 4
b) Predetermined sequential distribution
Nest C Nest Co Nest T Nest C Nest Co
C Co C Co
T C Co T C
Co Co T T T C C C Co Co Co T T T C
5 6 8 9
FIGURE 4 Distribution of clefts (C), treated (T) and control (Co) pups in cages.
Salazar, INTRAUTERINE REPAIR, CLEFT Lp 41
TABLE 1 Order for Retrieving Clefted (C), Treated (T), and Control (Co) Pups
Foster Order of RetrievalMotherNumber 1st 2nd 3rd 4th 5th 6th
1 T Co Co C T C
2 Co T Co
3 Co T Co T
4 Co T Co
5 _ Co Co T
6 T Co Co T
7 T T Co C Co C
8 T T Co Co
9 Co Co T
10 4
9 L
3 L
7 L.Percent
of. 6 +
Stillborn
Non- 5 L
Operated
Litter a L
Mates
3 L
o J
1 4
014 15 16 17 18 19 20
Day Laparotomy Performed
FIGURE 5 Percent of nonoperated fetuses that were stillborn when delivered on the 21st day of pregnancy.
42 Cleft Palate Journal, January 1988, Vol. 25 No. 1
-DIsCcUssION
In a previous unpublished experiment, mother
rat behavior was evaluated by the method of
Galler and Propert (1982). The behaviors meas-
ured were active nursing, retrieving and licking,
number of pups in contact with the mother, and
whether or not the mother was in the nest. In that
study the authors placed the pups (clefted, treat-
ed, and controls) together in the nest, instead of
placing them at the edges of the cage. Maternal
behavior was good except toward clefted pups,
which could not be nursed. They were found
dead 24 hours later. Therefore, in the present
experiment the evaluation of retrieving was em-
ployed.
The fetuses were recovered at 21st day of
gestation, 48 hours after the operation. In this
time, the epithelial healing is almost complete
as reported in other studies (Robinson and Goss
1981; Hallock, 1985). However, it could be
necessary to study the histological course of heal-
ing 48 hours after operation (in postnatal life).
There was a gradient of acceptance-rejection
in which the first three places in retrieving were
occupied by control and some treated animals.
We found some treated and clefted pups carried
in fourth to sixth places, and finally a rejection
level in which clefted pups were not retrieved.
According to these findings we suggest that the
majority of mother rats reject pups with lesions
resembling cleft lip and that the repair of this le-
sion seems to be a useful method to avoid mater-
nal rejection in this model.
REFERENCES
GALLER JR, PROPERT K. (1982). Early maternal behaviorspredictive of the survival of suckling rats with intergener-ational malnutrition. J Nutr 112: 332-337.
Harrock GG. (1985). In utero cleft lip repair in A/J mice.Plast Rec Surg 75: 785-790.
Harrison MR, Ross NA, DE LorIimIER AA. (1981a). Cor-rection of congenital diaphragmatic hernia in utero. III. De-velopment of a successful surgical technique usingabdominoplasty to avoid compromise of umbilical bloodflow. J Pediatr Surg 16: 934-941.
HaArRIsON MR, GorBuUs MS, FILLY RA. (1981b). Manage-ment of the fetus with a correctable congenital defect.JAMA 246: 774-777.
KraANER KL, ParsHALL CJ. (1968). Experimental proceduresand surgical techniques performed on intrauterine fetalanimals. In: Gay WI, ed. Methods of animal experimenta-tion. New York and London: Academic Press, 211-239. Edit-ed by: William I. Gay.
Rosinson BW, Goss AN. (1981). Intrauterine healing offetal rat cheek wounds. Cleft Palate J 18: 251-255.