Hyperoncotic enhancement of pulmonary growth after fetal tracheal occlusion: a comparison between dextran and albumin

Hyperoncotic Enhancement of Pulmonary Growth After Fetal Tracheal Occlusion: A Comparison Between Dextran and Albumin By Robert Chang, Makoto Komura, Steven Andreoli, Markus Klingenberg, Russell Jennings, Jay Wilson, and Dario Fauza Boston, Massachusetts

Background/Purpose: This study was aimed at comparing albumin and dextran as intrapulmonary hyperoncotic enhancers of fetal lung growth after tracheal occlusion. Methods: Fetal lambs (n ⫽ 27) were divided proportionally into 5 groups: group I consisted of sham-operated controls; group II underwent tracheal occlusion (TO); groups III, IV, and V underwent TO and intratracheal infusion of 60 mL of either saline, 6% dextran-70, or 25% albumin, respectively. Multiple fetal lung analyses were performed near term. Statistical analysis was by 1-way analysis of variance (ANOVA) and post-hoc analyses by the Bonferroni correction for multiple comparisons (P ⬍ .05). Results: The lung volume-to-body weight ratio was significantly higher in groups IV and V than in all other groups with no differences between groups II and III, nor between groups IV and V. Airspace fraction was not significantly different

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LINICAL EXPERIENCE with fetal tracheal occlusion has shown that it does not always lead to accelerated lung growth. Contributing factors to this finding include the short window commonly present between fetal intervention and the all-pervading postoperative premature labor, the physiologic decrease in lung liquid secretion observed in the latter stages of gestation and, possibly, fetal stress.1-3 In a previous effort to maximize the reliability of fetal tracheal occlusion, we have shown that intrapulmonary dextran delivery enhances lung growth acceleration after this procedure, likely because it leads to an active expansion of lung liquid volume through positive oncotic pressure.4 Dextran administration, however, is not without potentially relevant side effects, such as significant functional

among the groups, nor was there any evidence of alveolar cellular damage. Type-II pneumocyte density was higher in group I than in groups II, IV, and V, with no differences among the latter 3 groups. Lung liquid biochemical profile was normal in all groups. Conclusions: Albumin is as effective as dextran as an intrapulmonary hyperoncotic booster of lung growth acceleration after fetal tracheal occlusion, with no lasting effects on its fetal lung liquid levels. As a naturally occurring oncotic agent, albumin may be a safer option in the clinical application of this therapeutic concept. J Pediatr Surg 39:324-328. © 2004 Elsevier Inc. All rights reserved. INDEX WORDS: Congenital diaphragmatic hernia, pulmonary hypoplasia, tracheal occlusion, fetal surgery, lung development, fetus, albumin, dextran.

impairment of the reticuloendothelial system (RES), inflammation, unwarranted plasma expansion, and others.5-9 In addition, dextran’s half life is quite variable, and it is likely to be eliminated at a much slower rate from the alveolar lumen than after intravenous administration.6,10,11 This study was aimed at determining whether an oncotic agent naturally occurring in the fetal lung liquid, namely albumin, could also be used to maximize fetal pulmonary growth in this setting. MATERIALS AND METHODS

From the Departments of Surgery, Children’s Hospital and Harvard Medical School; the Advanced Fetal Care Center; and the Harvard Center for Minimally Invasive Surgery, Boston, MA. Presented at the 34th Annual Meeting of the American Pediatric Surgical Association, Fort Lauderdale, Florida, May 25-28, 2003. This research was supported by grants from the United States Surgical Corporation and The Children’s Hospital Surgical Foundation. Address reprint requests to Dario O. Fauza, MD, Children’s Hospital, 300 Longwood Ave, Fegan 3, Boston, MA 02115. © 2004 Elsevier Inc. All rights reserved. 0022-3468/04/3903-0014$30.00/0 doi:10.1016/j.jpedsurg.2003.11.007

The Harvard Medical School (HMS) animal management program is sanctioned by the American Association for the accreditation of Laboratory Animal Care (AAALAC, file #000009) and meets National Institutes of Health Standards as set forth in the Guide for the Care and Use of Laboratory Animals (National Research Council Publication, revised 1996). The current study was approved by the HMS Standing Committee on Animals, under protocol # 03355. Time-dated pregnant ewes at 120 to 129 days gestation (mean, 124 ⫾ 2 days) were anesthetized with 2% to 4% halothane (Halocarbon Laboratories, River Edge, NJ) and received 1 g of cefazolin (BMH, Philadelphia, PA) intravenously before surgical manipulation. The bicornuate uterus was exposed through a midline laparotomy. Fetal lambs (n ⫽ 27) then were divided into 5 groups: group I (n ⫽ 5) consisted of sham-operated controls; group II (n ⫽ 5) underwent simple tracheal occlusion (TO) as previously described12,13, group III (n ⫽ 5) received TO and 60 mL of saline injected into the trachea; group IV (n ⫽ 6) underwent TO and intratracheal infusion of 60 mL of pH-adjusted, isosmotic 6% dextran 70 in 0.9% NaCl (B. Braun Medical, Irvine, CA); and group V (n ⫽ 6) underwent TO and intratracheal infusion of 60 mL of 25% human albumin (Buminate, Baxter Health-

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care, Deerfield, IL). The amniotic fluid, which had been previously removed and kept at 37°C, was reinfused into the amniotic cavity, together with 500 mg of Cefazolin. The gestational membranes and uterine wall were closed in 1 layer with a TA 90-mm Titanium surgical stapler (United States Surgical Corp [USSC], Norwalk, CT). The mother’s abdomen was closed in layers. On the first postoperative day, all ewes received 1.2 million units of benzathine penicillin intramuscularly (Wyeth Laboratories, Philadelphia, PA). All fetuses were delivered by cesarean section near full term, 14 to 18 days postoperatively (mean 15.7 ⫾ 1 days), immediately after maternal death with a lethal dose of Somlethal (J. A. Webster, Sterling, MA). There was no difference in the duration of the postoperative period among groups by repeated measures analysis of variance (ANOVA).

Lung Preparation and Analysis Each lamb was weighed and had its chest opened through a median sternotomy. Lung liquid was aspirated through the trachea and analyzed on a Stat Profile Ultra blood gas and electrolyte analyzer (Nova Biomedical Corp, Waltham, MA). Lung liquid albumin levels were determined using established techniques.14 The trachea and both lungs then were removed en bloc and inflated with saline at 20 cm H2O pressure. Lung volumes were determined by water displacement of the inflated lung. Samples of lung tissue then were taken from standard positions on the periphery of the right and left apical and diaphragmatic lobes and fixed in 10% neutral-buffered formalin (Sigma, St Louis, MO). Morphometric analysis within the intra-acinar region of the lung was performed using a Zeiss laboratory microscope (Carl Zeiss, Jena, Germany) with a projection head engraved with a 42-point coherent test lattice15 at a magnification of 200x. Airspace fraction analysis consisted of counting test points falling on airspace and alveolar wall tissue. Lung tissue was fixed and embedded for electron microscopy analysis as previously described.13 Silver sections were cut with a LKB ultramicrotome (LKB, Sweden), collected on 200-mesh copper grids, and stained with uranyl acetate and lead citrate. All samples were viewed and micrographs obtained with a Zeiss EM10 transmission electron microscope (Carl Zeiss, Jena, Germany).

Surfactant Protein-B Immunohistochemistry Lung specimens were analyzed for the presence of surfactant protein-B (SP-B), a selective marker expressed by type II pneumocytes. Lung samples were formalin fixed, paraffin embedded, and then cut into 5-␮m sections. The slides were heat fixed for 1 hour in an 110°C oven. Next, samples were rinsed with a “blocking solution” consisting of 2% donkey serum (Sigma) and 2% bovine serum albumin (Sigma) in Dulbecco’s phosphate-buffered saline (PBS; Sigma) for 30 minutes, washed again in PBS, and incubated for 4 hours with a 1:150 dilution of polyclonal anti–SP-B antibody (Chemicon International, Temecula, CA). After further PBS washings, an antirabbit secondary antibody was added (Vector Laboratories, Burlingame, CA) for 1 hour. The slides were then rinsed 3 times in blocking solution, mounted in DAPI fluorescence medium (Vector Laboratories) and viewed with a Zeiss fluorescence microscope (Carl Zeiss). Standard positive and negative control slides were used in the preparation.

Quantitation of Type II Pneumocytes Five random sections of lung from each specimen were analyzed. Five slides from each section were studied, with a total of 10 fields per slide. Quantitative analysis was performed with a Nikon Eclipse E 800 microscope (Nikon Corp, Tokyo, Japan) coupled with a computerized digital-enabled image capture program (Spot 2E, Diagnostic Instruments, Sterling Heights, MI). Computer-assisted methods then were used to quantitate positively stained cells, which were identified by

Fig 1. The lung volume-to-body weight ratio was significantly higher in the groups that underwent tracheal occlusion plus intrapulmonary delivery of dextran 70 (TO/Dx) or albumin (TO/Alb) than in all other groups (*) and significantly lower in the sham operated group (C) than in all other groups (**). There was no difference between the group that underwent simple tracheal occlusion (TO) and the group that had tracheal occlusion plus intrapulmonary delivery of saline (TO/S), nor between TO/Dx and TO/Alb.

their brown hue. A ratio was derived with the positive cells in the numerator over the total amount of cells per field. Permanent photomicroscope images were taken, and the identified cells were confirmed via manual inspection by 3 independent, blinded examiners.

Statistical Analysis Statistical analysis was performed by 1-way ANOVA, with post-hoc analyses by the Bonferroni correction for multiple comparisons as a means to compare individual groups. Significance was set at P values of less than .05.

RESULTS

The lung volume to body weight ratio (LV:BW) was significantly different among the groups (P ⬍ .001; Fig 1). Pairwise comparisons of LV:BW showed that it was significantly higher in groups IV and V than in all other groups and lower in group I than in all other groups (Fig 1). However, there was no difference between groups II and III, nor between groups IV and V (Fig 1). Airspace fraction analysis showed no significant difference among all groups (Fig 2). Histologic appearance was normal and indistinguishable in all lung samples studied. This observation, combined with the airspace fraction data, shows preserved maturation pattern and absence of emphysematous changes in all groups. Density of type-II pneumocytes was significantly different among groups. Pairwise comparisons showed that it was higher in group I (0.10 ⫾ 0.02) than in all other groups studied, namely II (0.04 ⫾ 0.01), IV (0.05 ⫾ 0.02), and V (0.05 ⫾ 0.01). There was no significant difference between groups II, IV, and V (group III was not studied for pneumocyte type-II). Electron microscopy results showed no signs of cellular edema, no ultrastructural damage of the alveolar-

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Fig 2. Airspace fraction determinations did not differ significantly among all groups. C, sham-operated controls; TO, tracheal occlusion; TO/S, tracheal occlusion plus intrapulmonary delivery of saline; TO/ Dx, tracheal occlusion plus intrapulmonary delivery of dextran 70; TO/Alb, tracheal occlusion plus intrapulmonary delivery of 25% albumin.

capillary membrane, nor any evidence of type-II cell apoptosis in lung samples (Fig 3). There were no differences in lung liquid osmolarity, pH level, and electrolyte (Na⫹, K⫹, and Cl⫺) concentrations among all groups. Albumin levels were measured in groups I (3.24 g/dL ⫾ 0.72) and V (3.46 g/dL ⫾ 1.20), which were not significantly different. DISCUSSION

The acceleration of pulmonary growth observed after fetal tracheal occlusion depends on sustained intrapulmonary distension by retained lung liquid, which is secreted actively by the alveolar-capillary membrane.12,13,16,17 Erratic postoperative lung liquid production, however, has been considered one of the reasons why clinical application of fetal tracheal occlusion has

met with limited success thus far.18 In the current study, we have shown that albumin can be as effective as dextran in maximizing control over lung liquid volume expansion through positive intrapulmonary oncotic pressure, also without any evidence of cell damage. As to type II pneumocytes, given that they compose the pulmonary component of the RES and that significant functional impairment of the RES has been described after extensive intravenous dextran therapy,11 one could speculate that the response of these cells to dextran delivery into the fetal lung would be unique. However, our data showed that the known depression on the density of type-II pneumocytes caused by tracheal occlusion alone19,20 was not distinctly affected by intrapulmonary delivery of either dextran or albumin, at least in the short term. Albumin is responsible for up to 80% of plasma’s colloid osmotic pressure.21 It has been extensively used as a plasma expander in a variety of clinical entities for many decades.22,23 Yet, 60% to 70% of all albumin is located in the extravascular space, such as the interstitial fluid and, in the fetus, the lung liquid.17,21,24 The 25% albumin used in this study has an oncotic pressure that is roughly 5 times greater than an equivalent volume of plasma, while maintaining isosmolarity.25 When injected intravenously, this formulation will draw approximately 3.5 times its volume of additional fluid into the circulation.25 Thus, perhaps our finding that the effect of 25% albumin was comparable to that dextran 70 should not be surprising. The half-life of albumin after intravenous administration has been shown to be 15 to 20 days.22 Although there are little data on intrapulmonary fetal metabolism of albumin, previous work has shown that it is primarily metabolized by the alveolar epithelium via a

Fig 3. Transmission electron micrographs of the lung. (A) Tracheal occlusion plus dextran and (B) tracheal occlusion plus albumin. There are no signs of cellular edema nor ultrastructural damage of the alveolar-capillary membrane (original magnification x2,200).

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receptor-mediated transcellular endocytotic process.26 This could explain why the animals that received albumin had lung liquid levels of this protein similar to those of the sham-operated controls. Finally, unlike dextran and other volume expanders substitutes, albumin does not lead to inflammation.27 Current survival rates of postnatal care for congenital diaphragmatic hernia often reach close to 90% at referral centers.28,29 Yet, because of its potential to reduce the morbidity of select newborns with severe pulmonary hypoplasia associated with this disease, refinements in

fetal tracheal occlusion therapy continue to be pursued. As part of this effort, this study suggests that, as a naturally occurring oncotic agent in the fetal lung liquid, albumin may be a safer option for hyperoncotic enhancement of lung growth acceleration after temporary fetal tracheal occlusion. ACKNOWLEDGMENT The authors thank Jeffrey Pettit for his excellence in laboratory assistance.

REFERENCES 1. Mescher EJ, Platzker AC, Ballard PL, et al: Ontogeny of tracheal fluid, pulmonary surfactant, and plasma corticoids in the fetal lamb. J Appl Physiol 39:1017-1021, 1975 2. Kitterman JA, Ballard PL, Clements JA, et al: Tracheal fluid in fetal lambs: Spontaneous decrease prior to birth. J Appl Physiol 47:985-989, 1979 3. Brown MJ, Olver RE, Ramsden CA, et al: Effects of adrenaline and of spontaneous labour on the secretion and absorption of lung liquid in the fetal lamb. J Physiol 344:137-152, 1983 4. Dzakovic A, Kaviani A, Jennings RW, et al: Positive intrapulmonary oncotic pressure enhances short-term lung growth acceleration after fetal tracheal occlusion. J Pediatr Surg 37:1007-1010, 2002 5. Walkley JW, Tillman J, Bonnar J: The persistence of dextran 70 in blood plasma following its infusion, during surgery, for prophylaxis against thromboembolism. J Pharm Pharmacol 28:29-31, 1976 6. Klotz U, Kroemer H: Clinical pharmacokinetic considerations in the use of plasma expanders. Clin Pharmacokinet 12:123-135, 1987 7. Griffel MI, Kaufman BS: Pharmacology of colloids and crystalloids. Crit Care Clin 8:235-253, 1992 8. Wagner BK, D’Amelio LF: Pharmacologic and clinical considerations in selecting crystalloid, colloidal, and oxygen-carrying resuscitation fluids, Part 1. Clin Pharm 12:335-346, 1993 9. Rainey T, Read C: Pharmacology of colloids and crystaloids, in Chernow B (ed): The Pharmacologic Approach to the Critically Ill Patient. ed 3. Baltimore, MD, Williams & Wilkins, 1994, pp 272-290 10. Terg R, Miguez CD, Castro L, et al: Pharmacokinetics of Dextran-70 in patients with cirrhosis and ascites undergoing therapeutic paracentesis. J Hepatol 25:329-333, 1996 11. Ginz HF, Gottschall V, Schwarzkopf G, et al: [Excessive tissue storage of colloids in the reticuloendothelial system]. Anaesthesist 47:330-334, 1998 12. Wilson JM, DiFiore JW, Peters CA: Experimental fetal tracheal ligation prevents the pulmonary hypoplasia associated with fetal nephrectomy: Possible application for congenital diaphragmatic hernia. J Pediatr Surg 28:1433-1439; discussion 1439-1440, 1993 13. DiFiore JW, Fauza DO, Slavin R, et al: Experimental fetal tracheal ligation reverses the structural and physiological effects of pulmonary hypoplasia in congenital diaphragmatic hernia. J Pediatr Surg 29:248-256; discussion 256-257, 1994 14. Assessment of fetal lung maturity. Vol 230, American College of Obstetrics and Gynecology Educational Bulletin, 1996 15. Weibel ER: Stereologic methods, in Weibel ER (ed): Practical

Methods for Biological Morphometry. Vol. I. San Diego, CA, Academic Press, 1989, pp 63-236 16. DiFiore JW, Fauza DO, Slavin R, et al: Experimental fetal tracheal ligation and congenital diaphragmatic hernia: A pulmonary vascular morphometric analysis [see comments]. J Pediatr Surg 30:917923; discussion 923-924, 1995 17. Olver RE, Strang LB: Ion fluxes across the pulmonary epithelium and the secretion of lung liquid in the foetal lamb. J Physiol 241:327-357, 1974 18. Harrison MR, Mychaliska GB, Albanese CT, et al: Correction of congenital diaphragmatic hernia in utero IX: Fetuses with poor prognosis (liver herniation and low lung-to-head ratio) can be saved by fetoscopic temporary tracheal occlusion. J Pediatr Surg 33:1017-1022; discussion 1022-1023, 1998 19. O’Toole SJ, Sharma A, Karamanoukian HL, et al: Tracheal ligation does not correct the surfactant deficiency associated with congenital diaphragmatic hernia. J Pediatr Surg 31:546-550, 1996 20. O’Toole SJ, Karamanoukian HL, Irish MS, et al: Tracheal ligation: The dark side of in utero congenital diaphragmatic hernia treatment. J Pediatr Surg 32:407-410, 1997 21. Tullis JL: Albumin. I. Background and use. JAMA 237:355360, 1977 22. Peters T: Serum albumin, in Putnam F (ed): The Plasma Proteins. Vol 1 (ed 2). New York, NY, Academic Press, 1975, pp 133-181 23. Finlayson J: Albumin products. Semin Thromb Hemost 6:85120, 1980 24. Peters T: Serum albumin, in Putnam F (ed): The Plasma Proteins. New York, NY, Academic Press, 1975, pp 133-181 25. Finlayson J: Albumin products. Semin Thromb Homeost 6:85120, 1980 26. Kim KJ, Matsukawa Y, Yamahara H, et al: Absorption of intact albumin across rat alveolar epithelial cell monolayers. Am J Physiol: Lung Cell Mol Physiol 284:L458-L465, 2003 27. Rhee P, Wang D, Ruff P, et al: Human neutrophil activation and increased adhesion by various resuscitation fluids. Crit Care Med 28:74-78, 2000 28. Muratore CS, Wilson JM: Congenital diaphragmatic hernia: Where are we and where do we go from here? Semin Perinatol 24:418-428, 2000 29. Boloker J, Bateman DA, Wung JT, et al: Congenital diaphragmatic hernia in 120 infants treated consecutively with permissive hypercapnea/spontaneous respiration/elective repair. J Pediatr Surg 37:357-366, 2002

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Discussion A. Flake (Philadelphia, PA): That was a very nice study. In our clinical experience with tracheal occlusion, one of the big problems was not lack of lung growth, it was too rapid lung growth and that induced hydrops and ultimately preterm delivery in fetuses that had the best lung growth. I would ask first, is this really something we want to do? And secondly, can you explain why you see type 2 pneumocytes in those numbers in your study as opposed to most of the tracheal occlusion work that suggests that you lose type 2 pneumocytes? R. Chang (response): Thank you for your questions. In regard to your second part first, we saw a decrease in type 2 pneumocyte populations as a lot of other groups have suggested suggesting an altered maturation pattern. We are simply saying that there is no difference when we added the hyperoncotic agents, dextran, or albumin, compared with an occlusion group. With regard to your first question, while that may be the case, we do feel that there is a segment of the CDH population that would benefit from some lung growth and obviously the other parts of that clinical problem need to be investigated.

J. M. Laberge (Montreal, Quebec): I just want to know how you determine the amount that you would put. The 60 cc, what was this based on? R. Chang (response): The 60 cc was our best estimate of about 20% of lung volume at term in these animals. It was a conservative amount that we used. We did not want to be too aggressive and perhaps add half the lung volume, and we did not want to do too little to perhaps not show a change, so it was just an estimate. C. Stolar (New York: NY): When you talk about conventional lung morphometrics, what do you mean by that? In other words, are you talking about square meters of valvular surface area per gram of lung or something else? And, when you talk about the growth of the lung, what happens to the blood supply? What happens to the capillary growth because it should grow concomitantly with the air space? R. Chang (response): Thank you for your questions, Dr Stolar. For the purpose of this study we attempted just to prove the principle of hyperoncotic enhancement and so we elected to use lung volume to body weight as well as air space fraction analysis using a 200X slide.