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

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 ofboth 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

100 ©

90 +

80 +

70 +

Percentof 60 +

Fetuses

Delivered 50 +

Stillborn

40 +

30 4

20 +

10 +

0 I I

Salazar, INTRAUTERINE REPAIR, CLEFT LIP 39

14 15 16 17 18 19 20

Day Laparotomy Performed

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.

HoparEn GM. (1981). Antenatal diagnosis and treatment offetal skeletal malformations. JAMA 246: 1079-1083.

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.