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Surgically produced congenital diaphragmatic hernia in fetal rabbits
Surgically
Produced Congenital Diaphragmatic Hernia in Fetal Rabbits
By Dario 0. Fauza, Uenis Tannuri, Ali A.R. Ayoub, Vera Luiza Capelozzi, Paulo Hilhrio N. Saldiva, and Jo80 Gilbert0 Maksoud SBo Paulo, Brazil l Animal models of congenital diaphragmatic hernia (CDH) still are indispensable for the evolution of knowledge related to this disease and to fetal surgery in general. The lamb has provided the most reliable experimental design thus far. Considering the possible advantages of using rabbits (rather than lambs) namely lower costs, no need of special veterinary facilities, smaller body size, year-round availability, higher number of fetuses per pregnancy, and shorter gestational period, a successful model of CDH was developed in fetal rabbits. Sixteen pregnant New Zealand rabbits underwent hysterotomy and fetal operation. Group 1 (6 does) underwent surgery on gestational day 20 and group 2 (10 does) on gestational day 24 or 25. The normal full gestation time is 31 to 33 days. In group 1, the CDH was created by transabdominai puncture and dilatation of the fetal diaphragm. In group 2, the CDH was created through open thoracotomy, either left or right. The fetuses were delivered by cesarean section on gestational day 30. The overall fetal survival rate was 0 for group 1 and 70% for group 2. All operated fetuses in group 2 that were born alive had CDH. The histological morphometric examinations (radial alveolar count after sustained lung expansion) of the normal and operated fetuses in group 2 showed pulmonary hypoplasia in all the lungs on the same side as the CDH (statistical analysis was performed using the Neuman-Keuls test and analysis of variance; the significance level was set at .05). There was significant arterial wall thickening in the parenchyma of the lungs ipsilateral and contralateral to the CDH (analysis by the Kruskall-Wallis test; significance level set at .05). This study demonstrates that (1) CDH can be surgically created in fetal rabbits, with high survival rates, (2) either left or right CDH is feasible in fetal rabbits, and (3) experimental CDH is associated with pulmonary hypoplasia in New Zealand rabbits operated on at 24 or 25 days’ gestation. The rabbit may provide a reliable and practical experimental model of CDH. Copyright 0 1994 by WA s8Und8rS Company INDEX WORDS: Diaphragmatic gery; lung hypoplasia.
hernia, congenital;
fetal sur-
T
HE INCIDENCE of congenital diaphragmatic hernia (CDH) is approximately 1 in 4,000 live births.*-3 Despite many relevant advances in postnatal From the Pediatric Surgery Research Laboratory, Discipline of Pediatric Surgery, and the Environmental Pollution Research Laboratory, Department of Pathology, University of Stio Paul0 Medical School, Stio Paula, Brazil. Date accepted: July 23,1993. Supported in part by grants from FAPESP and LIM-HCFMUSP. Address reprint requests to Dario 0. Fauza, MD, Department of Surgery, Fegan 433, The Children’s Hospital, 300 Longwood Ave, Boston, M-I 02115. Copyright Q 1994 by W.B. Saunders Company 0022-3468/94/2907-003.00l0 662
management and intensive care, including prolonged extracorporeal life support, the high mortality rate of newborns with this defect has not improved significantly since the first reported series of patients, more than a century ago.4-7The variable degrees of pulmonary hypoplasia and persistent fetal circulation universally seen in association with CDH are well documented, both clinically and experimentally, and are considered the main causes of death for these children. Such conditions are more severe the earlier the diaphragmatic defect arises during intrauterine life. Thus, CDH is one of the major challenges requiring either successful in utero treatment, which has already proven to be feasible,8 or postnatal lung growth stimulation, yet to be demonstrated. Since the initial clinical experience with in utero CDH repair, results have not improved significantly. To date, the procedure cannot be considered for widespread application. At the present stage of open fetal surgery, animal experimental models still play an important role in its progress. The designs using fetal lambs have proven reliable, particularly those conceming CDH.g-*3 However, because of costs and animal size, this model cannot be adopted by some research laboratories. Moreover, the availability of pregnant ewes is seasonal. A model using fetal rabbits would be less expensive, would be available year-round, would require less space and facilities, and would provide a higher number of fetuses per mother; in addition, the gestation period is shorter than that of lambs. An experimental model of left CDH in rabbits was reported in 1976.i4 However, the fetal survival rate was low, and the model failed to obtain pulmonary hypoplasia. Because pulmonary hypoplasia is essential in any experimental model of CDH, it has been considered that a rabbit model would be inappropriate for the study of this anomaly. To our knowledge, this is the first successful study of an experimental model of CDH comprising pulmonary hypoplasia in fetal rabbits. This is also the first report of surgically produced right CDH in these animals. MATERIALS AND METHODS Sixteen pregnant New Zealand rabbits were housed in separate cages, with 13-hour light/ll-hour dark cycles, and were allowed to acclimate to their new environments. They were divided in two Journaloffediefric
Surgery, Vol29, No 7 (July), 1994:
pp662-886
883
CDH MODEL IN FETAL RABBITS
groups with respect to gestational age at the time of the first operation. Six does had this operation on gestational day 20; 10 had it on gestational day 24 or 25. The normal full gestation time is 31 to 33 days. On the day of the first operation, each doe received 5 mg/kg of sodium thiobarbiturate intravenously, before the induction of general anesthesia (with 2.0% to 5.0% halothane in 02) delivered by mask. The rabbit was placed supine, with its four limbs extended. The maternal abdomen was shaved with povidoneiodine. All the procedures were performed under sterile technique. A midline laparotomy (of approximately 10 cm in length) was performed, with the umbilicus at its cranial end. The gravid bicornuate uterus was removed from the abdomen, and the fetuses in each horn were counted. The most distal fetus of the most populated horn was operated on. Only one fetus from each doe underwent surgery. If the two uterine horns had the same number of fetuses, one of them was chosen randomly. The other horn as well as the proximal portion of the one to be opened were carefully repositioned inside the maternal abdomen. The surgical field was moistened continuously with warmed sterile saline solution. One of the forelegs of the fetus (either left or right) was palpated, and a l-cm longitudinal linear hysterotomy was performed with a blade-15 knife, at the region overlying the palpated foreleg. The incision was made as far as possible from the mesometrial border of the uterus. The subjacent chorion and amnion were opened with Iris scissors (C.S.P., Sao Paula, Brazil). Hemostasis was performed by needle-tip electrocautery. Between 0.5 and 2.0 mL of the amniotic fluid were aspirated from the open uterus and not saved for later reinfusion. The previously palpated foreleg and corresponding side of the chest wall of the fetus were exposed through the hysterotomy. Depending on the gestational day of operation, either of two procedures was used. For the does whose operation was on gestational day 20, the adjacent hemiabdomen of the fetus was exposed further. A transdiaphragmatic puncture was performed with a pair of closed Addison smooth forceps entering the abdominal wall and penetrating the ipsilateral exposed hemithorax. After the penetration in the hemithorax, the route of this puncture was dilated by spreading the forceps. After removal of the forceps, the point of entry into the fetus was closed with one simple monofilament polypropylene suture. For the does that underwent surgery on gestational day 24 or 25, the fetus underwent a low lateral thoracotomy (either left or right, depending on which foreleg was exposed) performed with microsurgical scissors. Through the thoracotomy, the diaphragm was grasped with Addison or microsurgical forceps and then partially excised using microsurgical scissors. No dilatation of the opened diaphragm, nor mobilization of the abdominal viscera was performed. The thoracotomy was closed in just one layer of simple interrupted monotilament polypropylene 7-Osutures. For both groups, the final closing procedure was the same. The fetus was gently repositioned inside the uterus. Warmed sterile saline solution was infused in the amniotic cavity, until uterine repletion, while the hysterotomy was oversewn in a double-running fashion with mononylon 5-O sutures. The maternal laparotomy was closed in two layers of mononylon 4-O running sutures. Povidoneiodine was applied to the closed incision. The does were awakened and returned to their cages. On gestational day 30, the does were again submitted to the same premeditation, anesthesia, and positioning procedures. All fetuses of each doe were then delivered by cesarean section. The fetuses were killed by drowning in saline solution. Both normal and operated fetuses had both lungs, the main bronchi, and the trachea removed in just one block, after a median sternotomy and cervicotomy. For the fetuses that underwent previous surgery, the
position of herniated abdominal organs was noted before their removal. The tracheas of normal and operated fetuses were immediately cannulated and perfused with buffered 6% formaline solution at 20 cm Hz0 for 24 hours; both lungs were immersed in the same solution. After the 24 hours, lung tissue was processed according to routine histological procedures; sagittal slices (5 km in thickness) were taken from paraffin-embeded tissue and stained with H&E and Verhoeff. The tissues were grouped as follows: (a) controls: left and right lungs of normal animals (Fig 1); (b) lungs ipsilateral to the CDH (Fig 2) and (c) lungs contralateral to the CD H (Fig 3).
MorphometricIndicators Six randomly selected noncoincident regions of lung parenchyma were studied. In these areas, theoretical lines crossing the entire tung section were traced with the aid of an eyepiece having lines of 1 mm in length. The intercepts of this hne with alveoli, bronchi, and blood vessels were counted. For each region, 10 microscopic fields (100x) were examined, giving a total of 60 fields per animal. The results of each lung were averaged, so that each lung provided one data-point for alveoli, bronchi, and blood vessels. The statistical analysis was performed using the NeumanKeuls test and analysis of variance; the significance level was set a .05.
HistopathologicStudies Transversely cut intraparenchymatous arteries (ie, with less than 10% variation between the larger and smaller diameters) were divided in two groups: large arteries (external diameter 2 0.1 mm) and small arteries (external diameter
Fit 1. Microscopic field of lung parenchyma from a normal newborn rabbi (H&E, original magnification x 100).
FAUZA ET AL
884
Fig 2. Microscopic field of lung parenchyma ipsilateral to a congenital diaphragmatic hernia in a newborn rabbit (H&E, original magnification x 100).
of an integrating eyepiece of 100 points. The W:L ratio represents an estimate of arterial thickening. Because this ratio hardly could be interpreted under Gaussian approximation, it was compared among the different groups by means of the Kruskall-Wallis test, with the significance level set at .05. RESULTS
Of the six does that underwent surgery on gestational day 20, two aborted all fetuses soon after surgery, and one died on postoperative day 2. The other three carried until gestational day 30, but the operated fetuses died in utero. Consequently, there was no fetal survival in this group, as shown in Table 1. For the group that underwent surgery on gestational day 24 or 25, the overall fetal survival rate was 70% (Table 1). One doe aborted and one died, on postoperative days 2 and 1, respectively. Another doe reached gestational day 30, but its operated fetus was dead in utero. The other seven fetuses were alive at the time of cesarean section delivery, and their size was compatible with that of full-term gestation. Although no diaphragmatic dilatation nor abdominal organ mobilization had been performed, all had their only partially excised diaphragm widely opened, and all had intraabdominal organs herniated to the respective hemithorax. When the left diaphragm was opened, the organs most commonly herniated were the liver (mainIy the left lobe), stomach, and smaI1 bowel. If
Fig 3. Microscopic field of lung parenchyma contralateral to a congenital diaphragmatic hernia in a newborn rabbit (H&E, original magnification x 100).
the partial excision was on the right diaphragm, the liver (either right lobe or all lobes) was the most commonly herniated organ. The results of the morphometric procedures are depicted in Figs 4 through 6. A significant decrease (P = .0085) of alveolar intercepts was detected in all animals, in the lungs ipsilateral to the CDH (Fig 4). The controls and the lungs contralateral to the CDH were statistically similar in terms of alveolar formation (Fig 4). Although there were differences among the three groups with regard to the number of bronchial branches (Fig 5) and blood vessels (Fig 6), the number of intercepts did not differ significantly. The average W:L ratios are shown in Table 2. There was statistically significant arterial wall thickening in the lungs ipsilateral and contralateral to the CDH, mainly in the small arteries. DISCUSSION
In this study, it was clear that the lungs associated with CDH were underdeveloped. In all groups, the number of alveoli was significantly lower in the lungs Table 1. Gestational of Operation 20 24 or 25
Fetal Survival Rate and Day of Operation Fetal Survival
Day Number
of Does
Rate (%)
6
0
10
70
885
CDH MODEL IN FETAL RABBITS
200
-
175
--
150
--
125
--
100
--
75
--
50
--
25
--
ON* of alveolar intercepts NQof blood vessels intercepts
Fig 4. Average number of alveolar intercepts in each group of lungs (original magnification x100). Control, normal lungs; Contra, lungs contralateral to the CDH; Ipsi, lungs ipsilateral to the CDH. Asterisk denotes significant difference relative to the control (P = .0085).
the same side as the CDH. When the CDH was in the right hemithorax, the ipsilateral lungs were even more altered than when it was in the left hemithorax. Not surprisingly, the diminution in bronchial branches was not statistically significant; the bronchial tree develops mainly during early intrauterine life, and the surgical intervention was not successful until gestational day 24. However, it is noteworthy that such observed diminution was uniform in the lobes and involved partial loss of the smaller bronchi and bronchioli, which failed to develop. This observation is compatible with the probable arrest of growth of the bronchial tree during the “glandular” phase of lung development. The quantitative development of the vascular bed of the lungs was statistically normal, but with a certain diminution of the lobar branches, mainly in the right lungs ipsilateral to the CDH. On the other hand, the statistically significant arterial wall thickening, observed in all lungs ipsilateral and contralateral to the CDH, is compatible with increased pulmonary vascular resistance, a key component of the pathophysiology of CDH. Since de Lorimier et al first demonstrated pulmonary hypoplasia in fetal lambs with surgically proon
Fig 6. Average number of blood vessel intercepts in each group of lungs [original magnification x100). Control, normal lungs; Contra, lungs contralateral to the CDH; Ipsi, lungs ipsilateral to the CDH.
duced CDH (in 1967),9 this animal model has been essential to the development of fetal surgery in general, and particularly to the study of CDH.9-‘3 In 1976, Ohi et al first reported surgically produced left CDH in fetal rabbits,14 but they did not demonstrate pulmonary hypoplasia, and the overall fetal survival rate was 55.6%. Thus, the rabbit model has not been considered for the study of CDH. They operated on gestational days 23 to 26. We tried to operate on gestational day 20 because, as it is well known, the earlier the herniation, the more intense the pulmonary hypoplasia; but no fetuses survived. Considering the possible advantages of a rabbit model for CDH and for fetal surgery in general, we decided to try the operation on gestational day 24 or 25 once again. The results clearly showed that there was pulmonary hypoplasia in all lungs on the same side as the CDH, and the overall fetal survival rate was 70%. Why are our results different from those of Ohi et al? One possible explanation is the difference in the fixation method of the fetal lungs. Ohi et al noted that “. . . the lungs were removed and fixed in 10% neutral formalin solution. . . ,” aparently without lung expansion. We expanded all lungs for 24 hours because it is quite clear that any study of pulmonary hypoplasia becomes much more accurate if there is sustained pulmonary expansion. 12,13~15~16 If this methodological Table 2. Average Ratio of Wall Area to Luminal Area (W:L) Among the lntraparenchymatous W:Lof
W:Lof
Large Arteries
Small Arteries
Control
0.71
0.96
Contra
0.91X
i .84x
lpsi
1.39*
2.19x
Lung Group
N’ of bronchial intercepts
Fig 5. Average number of bronchial intercepts in each group of lungs (original magnification x 100). Control, normal lungs; Contra, lungs contralateral to the CDH; Ipsi, lungs ipsilateral to the CDH.
Arteries in Each Group
NOTE. Large arteries are those with an external diameter of z 0.1 mm; small arteries are those with an external diameter of ~0.1 mm. Abbreviations: Control, normal lungs; Contra, lungs contralateral to the CDH; Ipsi, lungs ipsilateral to the CDH. *P < .05 relative to control.
886
FAUZA ET AL
difference is not the reason for the distinctly different results, other possible ones are not clear. We conclude that (1) CDH can be surgically created in fetal rabbits, with high survival rates, (2) either left or right CDH is feasible in fetal rabbits, and (3) experimental CDH is associated with pulmonary hypoplasia in New Zealand rabbits operated on
at 24 or 25 days’ gestation. The fetal rabbit may provide a reliable and practical experimental model of surgically produced CDH. ACKNOWLEDGMENT The authors support.
thank B. Braun Laboratories
for the logistic
REFERENCES 1. Ravitch MM, Barton BA: The need for pediatric surgeons as determined by the volume of work and the mode of delivery of surgical care. Surgery 76754763, 1974 2. Touloukian RJ, Cole D: A state-wide survey of index pediatric surgical conditions. J Pediatr Surg 10:725-732, 1975 3. Harrison MR, Bjordal RI, Langmark F, et al: Congenital diaphragmatic hernia: The hidden mortality. J Pediatr Surg 13:227230.1979 4. Bowditch HI: A treatise on diaphragmatic hernia, I. Buffalo Med J Month Rev 9:1-39,1853 5. Bowditch HI: A treatise on diaphragmatic hernia, II. Buffalo Med J Month Rev 9:65-94,1853 6. Stolar CJH, Snedecor SM, Bartlett RH: Extracorporeal membrane oxygenation and neonatal respiratory failure. Experience from the Extracorporeal Life Support Organization. J Pediatr Surg 26:563-571,199l 7. Wilson JM, Lund DP, Lillehei CW, et al: Delayed repair and preoperative ECMO does not improve survival in high-risk congenital diaphragmatic hernia. J Pediatr Surg 27:368-375, 1992 8. Harrison MR, Langer JC, Adzick S, et al: Correction of congenital diaphragmatic hernia in utero, V. Initial clinical experience. J Pediatr Surg 25:47-57,199O
9. de Lorimier AA, Tierney OF, Parker HR: Hypoplastic lungs in fetal lambs with surgically produced congenital diaphragmatic hernia. Surgery 62:12-17,1967 10. Kent GM, Olley PM, Creighton RE, et al: Hemodynamic and pulmonary changes following creation of a diaphragmatic hernia in fetal lambs. Surgery 72:427-433,1972 11. Haller JA, Signer RD, Golladay ES, et al: Pulmonary and ductal hemodynamics in studies of simulated diaphragmatic hernia of fetal and newborn lambs. J Pediatr Surg 11:675-680, 1976 12. de Lorimier AA, Mans A, Tyler WS: Morphometry of normal and hypoplastic lungs in newborn lambs. Top Surg Res 1:431-442,1969 13. Pringle KC, Turner JW, Schofield JL, et al: Creation and repair of diaphragmatic hernia in fetal lambs: Lung development and morphology. J Pediatr Surg 19:131-140,1984 14. Ohi R, Suzuki H, Kato T, et al: Development of the lung in fetal rabbits with experimental diaphragmatic hernia. J Pediatr Surg 11:955-959,1976 15. Inselman LS, Mellins RB: Growth and development of the lung. J Pediatr 98:1-l& 1981 16. Tenbrinck R, Tibboel D, Gaillard JLJ, et al: Experimentally induced congenital diaphragmatic hernia in rats. J Pediatr Surg 25~426-429,1990
Produced Congenital Diaphragmatic Hernia in Fetal Rabbits
By Dario 0. Fauza, Uenis Tannuri, Ali A.R. Ayoub, Vera Luiza Capelozzi, Paulo Hilhrio N. Saldiva, and Jo80 Gilbert0 Maksoud SBo Paulo, Brazil l Animal models of congenital diaphragmatic hernia (CDH) still are indispensable for the evolution of knowledge related to this disease and to fetal surgery in general. The lamb has provided the most reliable experimental design thus far. Considering the possible advantages of using rabbits (rather than lambs) namely lower costs, no need of special veterinary facilities, smaller body size, year-round availability, higher number of fetuses per pregnancy, and shorter gestational period, a successful model of CDH was developed in fetal rabbits. Sixteen pregnant New Zealand rabbits underwent hysterotomy and fetal operation. Group 1 (6 does) underwent surgery on gestational day 20 and group 2 (10 does) on gestational day 24 or 25. The normal full gestation time is 31 to 33 days. In group 1, the CDH was created by transabdominai puncture and dilatation of the fetal diaphragm. In group 2, the CDH was created through open thoracotomy, either left or right. The fetuses were delivered by cesarean section on gestational day 30. The overall fetal survival rate was 0 for group 1 and 70% for group 2. All operated fetuses in group 2 that were born alive had CDH. The histological morphometric examinations (radial alveolar count after sustained lung expansion) of the normal and operated fetuses in group 2 showed pulmonary hypoplasia in all the lungs on the same side as the CDH (statistical analysis was performed using the Neuman-Keuls test and analysis of variance; the significance level was set at .05). There was significant arterial wall thickening in the parenchyma of the lungs ipsilateral and contralateral to the CDH (analysis by the Kruskall-Wallis test; significance level set at .05). This study demonstrates that (1) CDH can be surgically created in fetal rabbits, with high survival rates, (2) either left or right CDH is feasible in fetal rabbits, and (3) experimental CDH is associated with pulmonary hypoplasia in New Zealand rabbits operated on at 24 or 25 days’ gestation. The rabbit may provide a reliable and practical experimental model of CDH. Copyright 0 1994 by WA s8Und8rS Company INDEX WORDS: Diaphragmatic gery; lung hypoplasia.
hernia, congenital;
fetal sur-
T
HE INCIDENCE of congenital diaphragmatic hernia (CDH) is approximately 1 in 4,000 live births.*-3 Despite many relevant advances in postnatal From the Pediatric Surgery Research Laboratory, Discipline of Pediatric Surgery, and the Environmental Pollution Research Laboratory, Department of Pathology, University of Stio Paul0 Medical School, Stio Paula, Brazil. Date accepted: July 23,1993. Supported in part by grants from FAPESP and LIM-HCFMUSP. Address reprint requests to Dario 0. Fauza, MD, Department of Surgery, Fegan 433, The Children’s Hospital, 300 Longwood Ave, Boston, M-I 02115. Copyright Q 1994 by W.B. Saunders Company 0022-3468/94/2907-003.00l0 662
management and intensive care, including prolonged extracorporeal life support, the high mortality rate of newborns with this defect has not improved significantly since the first reported series of patients, more than a century ago.4-7The variable degrees of pulmonary hypoplasia and persistent fetal circulation universally seen in association with CDH are well documented, both clinically and experimentally, and are considered the main causes of death for these children. Such conditions are more severe the earlier the diaphragmatic defect arises during intrauterine life. Thus, CDH is one of the major challenges requiring either successful in utero treatment, which has already proven to be feasible,8 or postnatal lung growth stimulation, yet to be demonstrated. Since the initial clinical experience with in utero CDH repair, results have not improved significantly. To date, the procedure cannot be considered for widespread application. At the present stage of open fetal surgery, animal experimental models still play an important role in its progress. The designs using fetal lambs have proven reliable, particularly those conceming CDH.g-*3 However, because of costs and animal size, this model cannot be adopted by some research laboratories. Moreover, the availability of pregnant ewes is seasonal. A model using fetal rabbits would be less expensive, would be available year-round, would require less space and facilities, and would provide a higher number of fetuses per mother; in addition, the gestation period is shorter than that of lambs. An experimental model of left CDH in rabbits was reported in 1976.i4 However, the fetal survival rate was low, and the model failed to obtain pulmonary hypoplasia. Because pulmonary hypoplasia is essential in any experimental model of CDH, it has been considered that a rabbit model would be inappropriate for the study of this anomaly. To our knowledge, this is the first successful study of an experimental model of CDH comprising pulmonary hypoplasia in fetal rabbits. This is also the first report of surgically produced right CDH in these animals. MATERIALS AND METHODS Sixteen pregnant New Zealand rabbits were housed in separate cages, with 13-hour light/ll-hour dark cycles, and were allowed to acclimate to their new environments. They were divided in two Journaloffediefric
Surgery, Vol29, No 7 (July), 1994:
pp662-886
883
CDH MODEL IN FETAL RABBITS
groups with respect to gestational age at the time of the first operation. Six does had this operation on gestational day 20; 10 had it on gestational day 24 or 25. The normal full gestation time is 31 to 33 days. On the day of the first operation, each doe received 5 mg/kg of sodium thiobarbiturate intravenously, before the induction of general anesthesia (with 2.0% to 5.0% halothane in 02) delivered by mask. The rabbit was placed supine, with its four limbs extended. The maternal abdomen was shaved with povidoneiodine. All the procedures were performed under sterile technique. A midline laparotomy (of approximately 10 cm in length) was performed, with the umbilicus at its cranial end. The gravid bicornuate uterus was removed from the abdomen, and the fetuses in each horn were counted. The most distal fetus of the most populated horn was operated on. Only one fetus from each doe underwent surgery. If the two uterine horns had the same number of fetuses, one of them was chosen randomly. The other horn as well as the proximal portion of the one to be opened were carefully repositioned inside the maternal abdomen. The surgical field was moistened continuously with warmed sterile saline solution. One of the forelegs of the fetus (either left or right) was palpated, and a l-cm longitudinal linear hysterotomy was performed with a blade-15 knife, at the region overlying the palpated foreleg. The incision was made as far as possible from the mesometrial border of the uterus. The subjacent chorion and amnion were opened with Iris scissors (C.S.P., Sao Paula, Brazil). Hemostasis was performed by needle-tip electrocautery. Between 0.5 and 2.0 mL of the amniotic fluid were aspirated from the open uterus and not saved for later reinfusion. The previously palpated foreleg and corresponding side of the chest wall of the fetus were exposed through the hysterotomy. Depending on the gestational day of operation, either of two procedures was used. For the does whose operation was on gestational day 20, the adjacent hemiabdomen of the fetus was exposed further. A transdiaphragmatic puncture was performed with a pair of closed Addison smooth forceps entering the abdominal wall and penetrating the ipsilateral exposed hemithorax. After the penetration in the hemithorax, the route of this puncture was dilated by spreading the forceps. After removal of the forceps, the point of entry into the fetus was closed with one simple monofilament polypropylene suture. For the does that underwent surgery on gestational day 24 or 25, the fetus underwent a low lateral thoracotomy (either left or right, depending on which foreleg was exposed) performed with microsurgical scissors. Through the thoracotomy, the diaphragm was grasped with Addison or microsurgical forceps and then partially excised using microsurgical scissors. No dilatation of the opened diaphragm, nor mobilization of the abdominal viscera was performed. The thoracotomy was closed in just one layer of simple interrupted monotilament polypropylene 7-Osutures. For both groups, the final closing procedure was the same. The fetus was gently repositioned inside the uterus. Warmed sterile saline solution was infused in the amniotic cavity, until uterine repletion, while the hysterotomy was oversewn in a double-running fashion with mononylon 5-O sutures. The maternal laparotomy was closed in two layers of mononylon 4-O running sutures. Povidoneiodine was applied to the closed incision. The does were awakened and returned to their cages. On gestational day 30, the does were again submitted to the same premeditation, anesthesia, and positioning procedures. All fetuses of each doe were then delivered by cesarean section. The fetuses were killed by drowning in saline solution. Both normal and operated fetuses had both lungs, the main bronchi, and the trachea removed in just one block, after a median sternotomy and cervicotomy. For the fetuses that underwent previous surgery, the
position of herniated abdominal organs was noted before their removal. The tracheas of normal and operated fetuses were immediately cannulated and perfused with buffered 6% formaline solution at 20 cm Hz0 for 24 hours; both lungs were immersed in the same solution. After the 24 hours, lung tissue was processed according to routine histological procedures; sagittal slices (5 km in thickness) were taken from paraffin-embeded tissue and stained with H&E and Verhoeff. The tissues were grouped as follows: (a) controls: left and right lungs of normal animals (Fig 1); (b) lungs ipsilateral to the CDH (Fig 2) and (c) lungs contralateral to the CD H (Fig 3).
MorphometricIndicators Six randomly selected noncoincident regions of lung parenchyma were studied. In these areas, theoretical lines crossing the entire tung section were traced with the aid of an eyepiece having lines of 1 mm in length. The intercepts of this hne with alveoli, bronchi, and blood vessels were counted. For each region, 10 microscopic fields (100x) were examined, giving a total of 60 fields per animal. The results of each lung were averaged, so that each lung provided one data-point for alveoli, bronchi, and blood vessels. The statistical analysis was performed using the NeumanKeuls test and analysis of variance; the significance level was set a .05.
HistopathologicStudies Transversely cut intraparenchymatous arteries (ie, with less than 10% variation between the larger and smaller diameters) were divided in two groups: large arteries (external diameter 2 0.1 mm) and small arteries (external diameter
Fit 1. Microscopic field of lung parenchyma from a normal newborn rabbi (H&E, original magnification x 100).
FAUZA ET AL
884
Fig 2. Microscopic field of lung parenchyma ipsilateral to a congenital diaphragmatic hernia in a newborn rabbit (H&E, original magnification x 100).
of an integrating eyepiece of 100 points. The W:L ratio represents an estimate of arterial thickening. Because this ratio hardly could be interpreted under Gaussian approximation, it was compared among the different groups by means of the Kruskall-Wallis test, with the significance level set at .05. RESULTS
Of the six does that underwent surgery on gestational day 20, two aborted all fetuses soon after surgery, and one died on postoperative day 2. The other three carried until gestational day 30, but the operated fetuses died in utero. Consequently, there was no fetal survival in this group, as shown in Table 1. For the group that underwent surgery on gestational day 24 or 25, the overall fetal survival rate was 70% (Table 1). One doe aborted and one died, on postoperative days 2 and 1, respectively. Another doe reached gestational day 30, but its operated fetus was dead in utero. The other seven fetuses were alive at the time of cesarean section delivery, and their size was compatible with that of full-term gestation. Although no diaphragmatic dilatation nor abdominal organ mobilization had been performed, all had their only partially excised diaphragm widely opened, and all had intraabdominal organs herniated to the respective hemithorax. When the left diaphragm was opened, the organs most commonly herniated were the liver (mainIy the left lobe), stomach, and smaI1 bowel. If
Fig 3. Microscopic field of lung parenchyma contralateral to a congenital diaphragmatic hernia in a newborn rabbit (H&E, original magnification x 100).
the partial excision was on the right diaphragm, the liver (either right lobe or all lobes) was the most commonly herniated organ. The results of the morphometric procedures are depicted in Figs 4 through 6. A significant decrease (P = .0085) of alveolar intercepts was detected in all animals, in the lungs ipsilateral to the CDH (Fig 4). The controls and the lungs contralateral to the CDH were statistically similar in terms of alveolar formation (Fig 4). Although there were differences among the three groups with regard to the number of bronchial branches (Fig 5) and blood vessels (Fig 6), the number of intercepts did not differ significantly. The average W:L ratios are shown in Table 2. There was statistically significant arterial wall thickening in the lungs ipsilateral and contralateral to the CDH, mainly in the small arteries. DISCUSSION
In this study, it was clear that the lungs associated with CDH were underdeveloped. In all groups, the number of alveoli was significantly lower in the lungs Table 1. Gestational of Operation 20 24 or 25
Fetal Survival Rate and Day of Operation Fetal Survival
Day Number
of Does
Rate (%)
6
0
10
70
885
CDH MODEL IN FETAL RABBITS
200
-
175
--
150
--
125
--
100
--
75
--
50
--
25
--
ON* of alveolar intercepts NQof blood vessels intercepts
Fig 4. Average number of alveolar intercepts in each group of lungs (original magnification x100). Control, normal lungs; Contra, lungs contralateral to the CDH; Ipsi, lungs ipsilateral to the CDH. Asterisk denotes significant difference relative to the control (P = .0085).
the same side as the CDH. When the CDH was in the right hemithorax, the ipsilateral lungs were even more altered than when it was in the left hemithorax. Not surprisingly, the diminution in bronchial branches was not statistically significant; the bronchial tree develops mainly during early intrauterine life, and the surgical intervention was not successful until gestational day 24. However, it is noteworthy that such observed diminution was uniform in the lobes and involved partial loss of the smaller bronchi and bronchioli, which failed to develop. This observation is compatible with the probable arrest of growth of the bronchial tree during the “glandular” phase of lung development. The quantitative development of the vascular bed of the lungs was statistically normal, but with a certain diminution of the lobar branches, mainly in the right lungs ipsilateral to the CDH. On the other hand, the statistically significant arterial wall thickening, observed in all lungs ipsilateral and contralateral to the CDH, is compatible with increased pulmonary vascular resistance, a key component of the pathophysiology of CDH. Since de Lorimier et al first demonstrated pulmonary hypoplasia in fetal lambs with surgically proon
Fig 6. Average number of blood vessel intercepts in each group of lungs [original magnification x100). Control, normal lungs; Contra, lungs contralateral to the CDH; Ipsi, lungs ipsilateral to the CDH.
duced CDH (in 1967),9 this animal model has been essential to the development of fetal surgery in general, and particularly to the study of CDH.9-‘3 In 1976, Ohi et al first reported surgically produced left CDH in fetal rabbits,14 but they did not demonstrate pulmonary hypoplasia, and the overall fetal survival rate was 55.6%. Thus, the rabbit model has not been considered for the study of CDH. They operated on gestational days 23 to 26. We tried to operate on gestational day 20 because, as it is well known, the earlier the herniation, the more intense the pulmonary hypoplasia; but no fetuses survived. Considering the possible advantages of a rabbit model for CDH and for fetal surgery in general, we decided to try the operation on gestational day 24 or 25 once again. The results clearly showed that there was pulmonary hypoplasia in all lungs on the same side as the CDH, and the overall fetal survival rate was 70%. Why are our results different from those of Ohi et al? One possible explanation is the difference in the fixation method of the fetal lungs. Ohi et al noted that “. . . the lungs were removed and fixed in 10% neutral formalin solution. . . ,” aparently without lung expansion. We expanded all lungs for 24 hours because it is quite clear that any study of pulmonary hypoplasia becomes much more accurate if there is sustained pulmonary expansion. 12,13~15~16 If this methodological Table 2. Average Ratio of Wall Area to Luminal Area (W:L) Among the lntraparenchymatous W:Lof
W:Lof
Large Arteries
Small Arteries
Control
0.71
0.96
Contra
0.91X
i .84x
lpsi
1.39*
2.19x
Lung Group
N’ of bronchial intercepts
Fig 5. Average number of bronchial intercepts in each group of lungs (original magnification x 100). Control, normal lungs; Contra, lungs contralateral to the CDH; Ipsi, lungs ipsilateral to the CDH.
Arteries in Each Group
NOTE. Large arteries are those with an external diameter of z 0.1 mm; small arteries are those with an external diameter of ~0.1 mm. Abbreviations: Control, normal lungs; Contra, lungs contralateral to the CDH; Ipsi, lungs ipsilateral to the CDH. *P < .05 relative to control.
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FAUZA ET AL
difference is not the reason for the distinctly different results, other possible ones are not clear. We conclude that (1) CDH can be surgically created in fetal rabbits, with high survival rates, (2) either left or right CDH is feasible in fetal rabbits, and (3) experimental CDH is associated with pulmonary hypoplasia in New Zealand rabbits operated on
at 24 or 25 days’ gestation. The fetal rabbit may provide a reliable and practical experimental model of surgically produced CDH. ACKNOWLEDGMENT The authors support.
thank B. Braun Laboratories
for the logistic
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