Fetal tracheal occlusion in the rat model of nitrofen-induced congenital diaphragmatic hernia: tracheal occlusion reverses the arterial structural abnormality

BACKGROUND/PURPOSE: The high mortality rate of congenital diaphragmatic hernia (CDH) is ascribed generally to pulmonary hypoplasia and persistent pulmonary hypertension characterized by associated pulmonary arterial structural changes. Prenatal tracheal occlusion (TO) accelerates lung growth, but the effect of TO on pulmonary arterial structure in CDH has not been well defined. The authors hypothesized that TO could reverse the pulmonary arterial structural changes observed in CDH. To address this hypothesis, we utilized the nitrofen-induced rat model of CDH to examine the effect of TO on pulmonary arterial morphology of CDH lungs. METHODS: Left-sided CDH was induced by administering 100 mg of nitrofen to pregnant Sprague-Dawley rats on day 9 of gestation. TO was performed on day 19, and the fetuses were harvested on day 21.5 of gestation. After the ductus arteriosus was ligated, the pulmonary arteries were injected with a barium-gelatin mixture, and the lungs were inflation fixed. Coronal sections of the lungs were stained with elastin van Gieson. External diameter (ED), internal diameter (ID), and medial and adventitial wall thickness of the pulmonary arteries were measured using a computer image analyzer, and the percent medial thickness (%MT) and adventitial thickness (%AT) were calculated. The lungs from nitrofen-exposed fetuses with left-sided CDH (CDH group), trachea-occluded left-sided CDH (CDH+TO group), non-CDH (non-CDH group), and normal fetuses (normal group) were compared. RESULTS: The %MT was significantly increased in all sizes of arteries in CDH compared with non-CDH and normal groups (P < .01). Compared with the CDH group, the CDH+TO group had significantly reduced %MT in all sizes of arteries (P < .01), to values comparable or less than the non-CDH and normal groups. The %AT of the CDH group was significantly increased in larger arteries compared with non-CDH and normal control groups (P < .01). CDH+TO had significantly decreased %AT compared with CDH in both larger (P < .01), and smaller arteries (P < .05) and that was comparable with the non-CDH and normal control groups. CONCLUSIONS: TO in hypoplastic CDH lung can reverse the pulmonary arterial structural changes that are seen in the nitrofen-induced fetal rat model of CDH. These data suggest that TO may reduce pulmonary vascular reactivity, and the risk of postnatal persistent pulmonary hypertension observed in human neonates with severe CDH. J Pediatr Surg 36:839-845.     

Remodeling of pulmonary arteries in human congenital diaphragmatic hernia with or without extracorporeal membrane oxygenation

PURPOSE: The aim of this study was to describe in detail the perinatal developmental profile of the pulmonary vasculature in congenital diaphragmatic hernia (CDH) and to examine the potential beneficial effects of extracorporeal membrane oxygenation (ECMO) on the vascular morphology. Additionally the authors aimed to identify the differences in pulmonary vascular morphology among CDH cases according to the primary cause of death: either extreme lung hypoplasia (LH) or persistent pulmonary hypertension (PPH). METHODS: The authors studied autopsy sections from 30 high-risk CDH cases with respect to the pulmonary arteries in relation to gestational age (GA) and ECMO treatment. They were grouped into CDH-I: 20 cases with GA greater than 34 weeks who were not subjected to ECMO and CDH-II: 10 cases with GA greater than 34 weeks, who were subjected to ECMO for an average time of 237 hours. Five age-matched neonates who died from placental insufficiency or birth asphyxia without evidence of lung hypoplasia served as controls (CON). Medial and adventitial thicknesses of pulmonary arteries were measured in lung sections stained with Elastic van Gieson by 2 investigators blinded for the clinical data. Immunohistological staining with anti-alpha-smooth muscle actin (alpha-SMA) was performed to confirm the precise location of the arterial media before morphometry. CDH cases were subgrouped and compared according to the primary cause of death. Unpaired Student t test was used for statistics, with significant P value < or =.05. RESULTS: In CDH newborns, a significant increase in medial, adventitial, and total wall thickness was found in pulmonary arteries with an external diameter of less than 200 microm as compared with age-matched controls (P<.004, .0001, and .0009, respectively). ECMO-treated CDH newborns showed a significantly thinner arterial adventitia than CDH patients who did not receive this treatment (P<.0001), approaching normal values. However, the medial thickness remained increased. Morphometrically, no significant differences in CDH cases between patients dying of PPH or severe LH could be determined. CONCLUSIONS: (1) In CDH, there is failure of the normal arterial remodeling processes occurring in the perinatal period. (2) Pulmonary vascular morphology in CDH does not differ between the groups with lung hypoplasia or persistent pulmonary hypertension as primary cause of death. (3) Adventitial thinning of these arteries might be one of the mechanisms by which ECMO alters PPH in CDH cases.     

Correction of congenital diaphragmatic hernia in utero. IV. An early gestational fetal lamb model for pulmonary vascular morphometric analysis

Infants born with congenital diaphragmatic hernia (CDH) often have specific pathologic abnormalities of the pulmonary microcirculation that result in high pulmonary vascular resistance and extrapulmonary right-to-left shunting after birth. In an attempt to make an animal model with similar vascular changes, we created CDH in fetal lambs at 60 to 63 days gestation, repaired some at 100 to 113 days gestation, and subsequently performed morphometric analysis of the pulmonary vasculature. Creation of CDH at this early gestational age resulted in a high fetal mortality rate. In the unrepaired CDH lambs, the pulmonary vascular abnormalities were more severe in the left lung. Similar to human CDH, diaphragmatic hernia in the fetal lamb resulted in a decrease in the total size of the pulmonary vascular bed, a decrease in the number of vessels per unit area lung, and increased muscularization of the arterial tree. Fetal surgical repair of CDH restored the pulmonary arterial bed towards normal.     

Pulmonary growth and remodeling in infants with high-risk congenital diaphragmatic hernia.

Infants born with congenital diaphragmatic hernia (CDH) have pulmonary hypoplasia, but the pattern of postnatal growth in these lungs has not been documented. The lungs of 21 children dying with CDH were analyzed to determine how the pulmonary morphology changed with age. The patients were stratified into three age groups for ANOVA analysis (less than 8 days, 8 to 21 days, greater than 21 days). Morphometric techniques previously described were used. Lung volume and weight as well as pulmonary artery length and diameter increased with age (P = .04), whereas the number of airway generations was similar for each group. Radial alveolar number also increased, particularly in the contralateral lung (P = .02). The percentage of intraacinar artery muscularization decreased with age (P = .02), while larger intraacinar arteries showed a nonmuscular structure, again particularly in the contralateral lung (P = .004). It is concluded that: (1) significant lung growth does occur postnatally at the alveolar level after CDH repair; and (2) there is postnatal vascular remodelling resulting in larger and less muscular arteries. These changes should contribute to a decrease in pulmonary arterial hypertension over time. However, the time period over which these changes occur exceeds the current limitations of invasive support measures such as extracorporeal membrane oxygenation. Elucidation of the factors responsible for this growth could result in new therapeutic strategies to enhance or accelerate postnatal pulmonary development in infants with CDH.     

Use of the postductal PaO2 as a predictor of pulmonary vascular hypoplasia in infants with congenital diaphragmatic hernia

Infants with congenital diaphragmatic hernia (CDH) demonstrate a wide range of anatomic and physiologic abnormalities that result in decreased pulmonary perfusion. We have used the patients' ability to achieve at least one postductal PaO2 greater than 100 torr while on maximal ventilation with 100% oxygen during the first 24 hours of life as the clinical marker to identify the degree of pulmonary perfusion. Patients were grouped as follows: group 1 had at least one postductal PaO2 greater than 100 torr, and group 2 patients never had a postductal PaO2 above 100 torr. To see if this classification did reflect pulmonary vascular abnormalities, we compared the pulmonary arteriograms of these two groups of CDH infants for size of the main pulmonary arteries (PAs), size of the lungs, and degree of peripheral vascular obstructive disease (PVO). Infants in group 2 had significantly smaller ipsilateral and contralateral main PAs, as well as smaller ipsilateral lungs with more severe PVO. We propose the postductal PaO2 as the clinical marker for identification of the degree of pulmonary perfusion.     

Expression patterns of heat shock proteins in lungs of neonates with congenital diaphragmatic hernia.

BACKGROUND: Congenital diaphragmatic hernia (CDH) is associated in many cases with pulmonary hypertension. Currently, extracorporeal membrane oxygenation (ECMO) is one of the possible modalities of treatment of pulmonary hypertension and prevention of parenchymal lung injury in neonates with CDH. HYPOTHESIS: Molecular stress is present in the lungs of neonates with CDH. To test this hypothesis, we investigate the expression pattern of stress genes (heat shock proteins [HSPs] 27 and 70) in lungs of patients with CDH who have pulmonary hypertension, and evaluate the influence of ECMO on the expression levels of these genes to understand the underlying molecular mechanisms. DESIGN: Paraffin-embedded lung autopsy specimens from patients with CDH and lung hypoplasia who either did or did not receive ECMO treatment and age-matched controls were immunostained by means of monoclonal antihuman antibodies against HSP 70 and HSP 27, with the streptavidin-biotin complex method. SETTING: Level III academic children's hospital. MAIN OUTCOME MEASURES: Expression levels of both HSP 27 and HSP 70 were semiquantitatively evaluated in bronchial epithelium, as well as in medial smooth muscle cells (SMCs) and endothelium of large and small pulmonary arteries, by means of a score ranging from 0 to 4. Statistical analysis of the data was performed with the nonparametric Mann-Whitney test, with significant probability value at P < or = .05. RESULTS: For HSP 70, the most pronounced immunoreactivity was observed in the bronchial epithelium, followed by the medial SMCs of small arteries (of external diameter < 200 microm). The overall expression was significantly higher in patients with CDH than controls in bronchi as well as in pulmonary arteries. For HSP 27, intense expression was found in medial SMCs, followed by the bronchial epithelium in controls, with significantly increased expression in medial SMCs of large and small arteries in patients with CDH. Treatment with ECMO was associated with significantly reduced expression levels of HSP 70 in medial SMCs of both large and small arteries, whereas HSP 27 expression levels were decreased only in small arteries. In addition, the expression levels of both HSPs were significantly lower in endothelium of small arteries. CONCLUSIONS: Increased expression of HSPs in CDH points to a condition of pulmonary stress. This pulmonary stress appears to be partially ameliorated by ECMO treatment. This may point to one of the mechanisms by which ECMO alleviates pulmonary hypertension associated with CDH.     

Pulmonary vascular morphology in a fetal rabbit model for congenital diaphragmatic hernia

Background/Purpose

Although the pulmonary vascular abnormalities in congenital diaphragmatic hernia (CDH) are described from a morphologic point of view, the treatment of pulmonary hypertension (PH) remains one of the main unsolved problems in clinical daily practice. For this reason, detailed studies in well-validated animal models could still be of significance in our understanding of the pathogenesis of CDH.

Methods

In does of 23 days’ gestational age (GA), 39 fetuses underwent creation of diaphragmatic hernia (DH) and 15 fetuses a sham thoracotomy (SHAM). Thirty-nine nonoperated littermates served as internal controls (CTR). Fetuses were harvested by cesarean section on days 25, 27, 29, and 30 of gestation. Lung specimens were obtained and formalin fixed for further vascular morphometry studies.

Results

Lung vessels from DH fetuses started to show significantly smaller internal diameter (ID), external diameter (ED), larger arterial wall thickness (WT), and, in particular, a proportionally higher medial thickness (%MT) and adventitial thickness (%AT) from day 27 onward when compared with control fetuses. SHAM fetuses, which were harvested at term, showed no differences with CTR.

Conclusions

This is the first report documenting changes over time in the vascular system in a rabbit fetal model of surgically induced DH. These changes mimic pathologic findings observed in human fetuses and are also concordant with earlier observations in the surgical ovine model and the toxic nitrofen rat model.     

Enhanced expression of vascular endothelial growth factor in lungs of newborn infants with congenital diaphragmatic hernia and pulmonary hypertension

BACKGROUND: Pulmonary hypoplasia accompanied by pulmonary hypertension resistant to treatment is an important feature of congenital diaphragmatic hernia (CDH). The pathogenesis of the pulmonary vascular abnormalities in CDH remains to be elucidated at the molecular level. Vascular endothelial growth factor (VEGF), an endothelial cell specific mitogen, is known to play a role in pulmonary angiogenesis and vascular remodelling but there are no data on VEGF expression in patients with CDH. METHODS: Necroscopic lung specimens from 21 patients with CDH with lung hypoplasia and from seven age matched control newborn infants without lung hypoplasia were processed for immunohistochemical analysis using affinity purified anti-human VEGF antibodies. All the cases of CDH had pulmonary hypoplasia, indicated by a lung/body weight index of 200 microm) and small (<200 microm) pulmonary arteries, the most intense staining being in the medial smooth muscle cells of the small pulmonary arteries. Endothelial cells were positive for VEGF staining in patients with CDH but not in controls. CONCLUSIONS: This is the first study of VEGF expression in newborn infants with CDH. Increased levels of VEGF, especially in the small, pressure regulating pulmonary arteries, point to a potential role in vascular remodelling. This may reflect an unsuccessful attempt by the developing fetus to increase the pulmonary vascular bed in the hypoplastic lungs to alleviate the associated pulmonary hypertension.     

Structural remodeling of resistance arteries in uremic hypertension

BACKGROUND: Structural remodeling of the resistance vasculature is present in many forms of human and experimental hypertension. In particular, an increase in the ratio of wall thickness to lumen diameter develops, and might in itself maintain hypertension by increasing vascular resistance. Because uremia is associated with raised peripheral resistance, hypertension, and histologic changes suggestive of vascular remodeling, we sought to formally examine the structural and mechanical (elastic) properties of isolated pressurized resistance arteries in uremic hypertension. METHODS: Cremaster, cerebral and mesenteric arteries from subtotally nephrectomised Wistar-Kyoto rats, normotensive control Wistar-Kyoto rats, and spontaneously hypertensive rats were mounted on a pressure myograph and relaxed in calcium-free buffer. Wall thickness and lumen diameter were measured at increasing lumen pressures from 10 to 200 mm Hg, and from this wall:lumen ratio, wall cross-sectional area, and an index of elasticity were derived. RESULTS: In uremic hypertensive animals increased wall:lumen ratio and decreased lumen diameter was seen in cremaster and mesenteric arteries, although no significant changes were observed in cerebral arteries, compared to normotensive controls. In spontaneously hypertensive animals increased wall thickness and wall:lumen ratio was seen in cerebral and mesenteric arteries, decreased lumen diameter in cremaster and mesenteric arteries, and increased wall cross-sectional area in cerebral arteries, compared to normotensive controls. Elasticity of the arterial wall in uremic and spontaneously hypertensive animals did not differ from normotensive controls. CONCLUSION: Cremaster and mesenteric resistance arteries undergo predominantly eutrophic inward remodeling in uremic hypertension, broadly similar to that seen in spontaneous hypertension.     

Quantitative structural study of pulmonary circulation in the newborn with aortic atresia, stenosis, or coarctation.

Study of the structural features of the pulmonary circulation in various types of congenital heart disease makes it possible to correlate function and structure in the fetal and newborn lung. We applied quantitative morphometric techniques to the injected and inflated lungs of newborn infants who had died with obstruction to left ventricular outflow from aortic atresia, stenosis, or coarctation. The structure and development of the pulmonary circulation was judged by the number of arteries and veins and their size and wall structure, with particular attention to vessels within the respiratory unit. The study established for the first time that the structure of the pulmonary circulation is modified by the antenatal abnormalities in blood flow that occur through the heart and great vessels in the presence of congenital heart disease. Fetal multiplication of intra-acinar arteries in aortic atresia and stenosis is increased as also is the muscularity of both pre- and intra-acinar arteries and veins, muscle extending into smaller and more peripheral vessels than is normal at birth. When the pulmonary circulation is normal before birth but arterial pressure and flow are abnormally increased at birth, as in coarctation with patent ductus and ventricular septal defect, an increase in arterial diameter and muscularity is apparent within the first week of life.     

Bacteriology of the closed duodenal loop model of acute pancreatitis and ultrastructural changes induced in the lungs

Injection of infected human bile into a closed duodenal loop in the rat consistently produces lethal pancreatitis with severe pulmonary damage. Lung abnormalities, resembling the human adult respiratory distress syndrome (ARDS) are frequently seen in this model and are usually ascribed to the pancreatitis. Similar pulmonary problems are seen in the human form of acute pancreatitis. Recently, it has been suggested, that the lung changes in the experimental animal are more likely to be due to bacteremia than to the pancreatitis. The aim of this study was to determine whether bacteremia occurred in this model, and if so, to determine whether bacteremia alone, in the absence of pancreatitis could produce this lung damage. A group of rats underwent induction of acute pancreatitis by a closed duodenal loop method and were compared to two groups comprising a closed small bowel loop bile infusion preparation, isolated from the pancreas, and a control group of rats undergoing a "sham" gastrotomy. Both pancreatitis and closed small bowel groups of animals were found to be bacteremic when sacrificed at 6 hr. The lungs from the animals with pancreatitis were significantly heavier than those in the other groups and scanning electron micrographs of the lungs in pancreatitis showed gross abnormalities, with marked increases in the alveolar wall thickness. The lungs in the closed small bowel loop preparation were indistinguishable from a control sham gastrotomy group of non-bacteremic rats. These results indicate that although the closed duodenal loop model of pancreatitis produces bacteremia, pancreatitis is necessary for development of pulmonary abnormalities.     

The effect of gastroschisis on experimental diaphragmatic hernia in fetal rabbits

Purpose

In this study, the authors analyzed the effect of experimentally induced gastroschisis on pulmonary hypoplasia in fetal rabbits with congenital diaphragmatic hernia (CDH).

Methods

Twenty-three pregnant rabbits underwent fetal surgery on gestational day 24 through 27. A left diaphragmatic hernia was created in 1 fetus (DH group) from each rabbit, and a left diaphragmatic hernia with gastroschisis was created in another fetus (GS group). The fetuses were delivered on gestational day 27 through 33. Histologic and morphometric examination of the lungs in each group was done.

Results

In the DH group, the lungs were hypoplastic with a decrease in lung weight to body weight ratio and an increase pulmonary arterial medial wall thickness. The alveolar septae were markedly thickened with increased interstitial tissue and diminished alveolar air spaces. In the GS group, the alveolar septae were thickened but narrower than those of DH group, and air spaces were increased. The pulmonary arterial wall was markedly thickened in the DH group but only slightly thickened in the GS group.

Conclusions

Pulmonary hypoplasia seen in newborn rabbits after experimentally induced diaphragmatic hernia is less severe in those rabbits with both gastroschisis and DH.     

Short-term tracheal occlusion corrects pulmonary vascular anomalies in the fetal lamb with diaphragmatic hernia

BACKGROUND: Sustained fetal tracheal occlusion (TO) results in accelerated lung growth but causes severe type II cell depletion. Temporary TO fails to cause lung growth in a congenital diaphragmatic hernia (CDH) model but preserves type II cells and corrects pulmonary hypertension. Herein, we study the pulmonary vascular changes caused by temporary TO. METHODS: CDH was created in 12 fetal lambs (65-70 d; term, 145 days). In 6 lambs, the trachea was occluded for 2 weeks (CDH + TO; 108-122 d). Animals were killed at 136 days. The lungs were processed with elastin stains and anti-alpha-smooth muscle actin antibody. Partial or circumferential presence of inner and outer elastic lamina was used to determine muscularization of pulmonary arterioles. The percent of medial wall thickness was plotted against vessel diameter for each group. RESULTS: Lung weight/body weight was smaller in lambs with CDH (1. 35% +/- 0.56%) and CDH + TO (1.70% +/- 0.34%) than in control lambs (3.55% +/- 0.56%; P <.05, single-factor analysis of variance). The smallest muscularized vessel was 113 +/- 50 microm, and the largest nonmuscularized vessel was 138 +/- 49 microm in lambs with CDH, significantly different from control lambs (185 +/- 69 microm and 350 +/- 116 microm, respectively) and lambs with CDH + TO (185 +/- 97 microm and 245 +/- 100 microm, respectively; P <.05). In lambs with CDH, only 25% of vessels of less than 60 microm were nonmuscularized, compared with 81% in control lambs (P <.05) and 74% in lambs with CDH + TO.Conclusions. Temporary tracheal occlusion, from 108 to 122 days, corrects the abnormal muscularization of pulmonary arterioles seen in CDH. These morphometric findings parallel physiologic results at birth and further suggest that short-term occlusion, which preserves surfactant-producing type II pneumocytes without lung growth, may be sufficient to improve neonatal outcome of diaphragmatic hernia.     

Correction of congenital diaphragmatic hernia in utero. II. Simulated correction permits fetal lung growth with survival at birth

Infants with congenital diaphragmatic hernia (CDH) die because their lungs are hypoplastic. If hypoplasia is a developmental consequence of compression by herniated viscera, decompression before birth may allow pulmonary development and survival at term. A conical silicone rubber balloon progressively inflated (60 to 150 ml) in the left hemithoraxi of fetal limbs (days 100 to 145) simulated compression by growing viscera ("CDH"). Six of six neonates delivered by cesarean section died of severe respiratory insufficiency, despite maximal resuscitation. Lungs were hypoplastic. Lung weight and air capacity were significantly reduced (P less than 0.01) as compared with controls. Pressure-volume curves revealed decreased compliance and barium gelatin injections revealed decreased cross-sectional area of the pulmonary vascular bed. Deflation of the balloon at day 120 (simulated "correction") allowed sufficient lung growth and development to alleviate respiratory insufficiency and to assure survival in five of five lambs delivered by cesarean section. Simulated correction produced a significant (P less than 0.01) increase in lung weight, air capacity, compliance, and area of the pulmonary vascular bed. Efficacy of in utero correction was confirmed by three twin studies in which simulated CDH in one twin was compared with simulated correction in the other. Infants with CDH may be salvaged by in utero correction.     

Relationship between bronchial and arterial diameters in normal human lungs.

In order to find an objective method for measuring narrowing of small airways, eight lungs from four people without lung disease were inflated and fixed at 25 cm of water pressure. Eight to nine blocks were taken at random from each of six parasaggital slices from each lung, sectioned, and stained. The size of the bronchioles (airways without cartilage) in relation to their accompanying arteries was determined. The internal bronchiolar diameter was compared with three different arterial diameters (the internal, external medial, and external adventitial). The ratio of the internal bronchiolar diameter to the external adventitial arterial diameter was constant between lungs (0.62 +/- 0.02) and independent of the method of inflation or the position within the lungs. This ratio may provide a useful index of bronchiolar narrowing in disease.     

Hypoxic pulmonary vasoconstriction disappears in a rabbit model of cavopulmonary shunt

BACKGROUND: Cavopulmonary shunt is widely known as an interim staging procedure in patients with single-ventricle physiology. However, the physiologic characteristics of the pulmonary arterial system after cavopulmonary shunt are not clearly understood. In this article, we developed a rabbit cavopulmonary shunt model and studied the morphologic changes and physiologic characteristics (namely, hypoxic pulmonary vasoconstriction) of pulmonary arteries after cavopulmonary shunt. METHODS: Male Japanese white rabbits aged 12 to 16 weeks were used for the study. In 5 rabbits, the superior vena cava was anastomosed to the right pulmonary artery in an end-to-side fashion, followed by a proximal side ligation of the right pulmonary artery (cavopulmonary shunt group). In 4 rabbits, the superior vena cava and the right pulmonary artery were dissected and clamped for 10 minutes without making a cavopulmonary shunt (sham group). Two weeks after the operation, we then measured the internal diameter of the acinar (internal diameter, 164 +/- 7 microm), the lobular (305 +/- 13 microm), and the segmental (669 +/- 16 microm) pulmonary arteries in both controlled and hypoxic conditions by using a specially designed x-ray television system. Also, morphometric measurements were made in the pulmonary arteries around the terminal bronchioles. RESULTS: Two weeks after the operation, the arterial oxygen tension under room air conditions was significantly lower in the cavopulmonary shunt group than in the sham group (68.2 +/- 2.2 mm Hg vs 91.1 +/- 1.9 mm Hg; P =.01). The baseline internal diameters in the acinar and the lobular (resistance), but not the segmental (conduit), pulmonary arteries on the anastomosed side of the cavopulmonary shunt group were significantly larger than those of pulmonary arteries on the nonanastomosed side of the cavopulmonary shunt group and the sham group. Moreover, the pulmonary arteries on the anastomosed side of the cavopulmonary shunt group did not respond to hypoxia, whereas those on the nonanastomosed side of the cavopulmonary shunt and sham groups did have local internal diameter reductions in the acinar and lobular arteries (-1.1% +/- 1.0% in the anastomosed side vs -17.7% +/- 3.5% in the nonanastomosed side vs -20.9% +/- 6.1% in the sham group; P =.03). In the morphometric studies, the internal diameter of the pulmonary artery accompanying the terminal bronchiole in the anastomosed side of the cavopulmonary shunt group was significantly larger, and the ratio of medial thickness relative to the outer diameter was smaller compared with ratios in the nonanastomosed side of the cavopulmonary shunt group and the sham group. CONCLUSIONS: We developed a rabbit cavopulmonary shunt model. In the anastomosed side of the cavopulmonary shunt group, the peripheral pulmonary arteries, which contributed greatly in regulating the pulmonary vascular resistance, had a local reduction in the basal vascular tone and no hypoxic vasoconstriction 2 weeks after the operation.     

Effect of antenatal tetrandrine administration on endothelin-1 and epidermal growth factor levels in the lungs of rats with experimental diaphragmatic hernia

Purpose

The aim of this study was to evaluate the effect of the traditional Chinese medicine tetrandrine (Tet) and to determine its possible mechanism on expression of endothelin-1 (ET-1) and epidermal growth factor (EGF) in the lung of a rat model of nitrofen-induced congenital diaphragmatic hernia (CDH).

Methods

A single oral dose (115 mg/kg) of nitrofen on day 9.5 of pregnancy was maternally administered to induce CDH. Pregnant rats were divided into 4 groups on day 18.5: control (n = 5), CDH (n = 5), CDH+dexamethasone (Dex) (n = 5), and CDH+Tet (n = 5). All fetuses were delivered by cesarean delivery on day 21.5. Accordingly, there were 4 groups of fetuses: control (n = 38), CDH (n = 25), CDH+Dex (n = 21), and CDH+Tet (n = 22). Lung tissue weight (LW) and body weight (BW) of each fetus were recorded, lung histologic evaluations and ET-1 and EGF immunohistochemistry staining were performed, and image analysis was performed after lung processing.

Results

Five female rats in the control group produced 38 fetuses without CDH. CDH was observed in 68 of the 128 rat fetuses (53.1%) among the other 3 groups. The LW/BW ratio of the CDH group was significantly lower than those of the Dex and EGF groups (P < .05). The lungs of fetuses with CDH showed marked abnormal structure such as pulmonary hypoplasia and vascular remodeling, in contrast to improved pulmonary structure in lungs of fetuses in the CDH+Dex and CDH+Tet groups. Statistical differences in morphologic parameters (radial alveolar counts, percentage of alveoli, percentage of medial wall thickness, and vascular volume) were found (P < .05). The immunoreactivity of EGF and ET-1 in the CDH group was markedly stronger than that in the control, CDH+Dex, and CDH+Tet groups (P < .01). In addition, EGF and ET-1 expression in the CDH+Dex and CDH+Tet groups was stronger than that in the control group (P < .05). There was no difference in lung EGF and ET-1 immunoreactivity between CDH+Dex and CDH+Tet groups (P > .05).

Conclusion

Antenatal treatment with Tet may improve lung growth and vascular remodeling, and its mechanism seems to be involved in decreasing EGF and ET-1 expression. Tet administered maternally may be a hopeful new therapeutic option in the treatment of CDH and may be effective in helping to avoid the side effects of Dex.     

Gene transfer of hepatocyte growth factor with prostacyclin synthase in severe pulmonary hypertension of rats

Objective: Hepatocyte growth factor (HGF) is a multi-potent growth factor, which has anti-fibrotic effects for lung injuries. In this study, we investigated whether human HGF gene transfer may attenuate the medial hypertrophy of pulmonary arteries and enhance the ameliorating effect of prostacyclin in monocrotaline (MCT)-induced pulmonary hypertension in rats. Methods and results: The day before MCT injection, HVJ-liposome complex with the cDNA encoding HGF gene (H group), PGIS gene (P group), and both HGF and PGIS gene (HP group) were transfected to the liver of rats as drug delivery system for the lung. Rats transfected with control vector served as controls (C group). Twenty-eight days after MCT injection, histological examination showed marked thickening of medial wall of pulmonary arteries and right ventricular hypertrophy. Percent medial wall thickness (%WT) of peripheral pulmonary arteries, pressure ratio of the right ventricle (RV) to the left ventricle (LV), and weight ratio of the RV to the LV plus septum were significantly increased in the control. Percent medial wall thickness was significantly ameliorated in H group and HP group in comparison with C group. Pressure and weight ratio of RV to LV was significantly ameliorated in P group and HP group in comparison with C group, and was significantly ameliorated in HP group than P group. Conclusions: In vivo gene transfection with HGF gene attenuated the medial hypertrophy of pulmonary arteries and enhanced the ameliorating effect of prostacyclin for pulmonary hypertension in MCT rats. Thus, gene therapy with HGF and PGIS may be a promising strategy for severe pulmonary hypertension.     

Hypoxia and the neonatal rabbit lung: neuroendocrine cell numbers, 5-HT fluorescence intensity, and the relationship to arterial thickness.

We assessed the dynamics of neuroendocrine (NE) cell numbers, the intensity of specific 5-HT fluorescence, and the arterial medial thickness in the lungs of neonatal rabbits in normoxia and acute and chronic hypoxia. Hypoxic neonates had significantly higher NE cell numbers and medial thickness of the pulmonary arteries at 5 days of age than did normoxic controls; 1- and 3-day-old young that died in hypoxia also had significantly higher cell numbers and medial thickness than did hypoxic survivors. A decline in these cell numbers was noted between 1 and 5 days of age among normoxic young, whereas there was no significant change among hypoxic young. Medial thickness was unchanged among normoxic young but increased between 1 and 5 days of age among hypoxic survivors. A 1-day exposure to normoxia of hypoxic young four days postpartum caused a decrease in NE cell numbers and medial thickness to more normal values. Serotonin (5-HT) fluorescence intensity levels of groups of NE cells or neuroepithelial bodies (NEBs) in this group were equal to those of normal controls although these levels were decreased in early chronic hypoxia. Medial thickness and NE cell numbers were inversely correlated with serotonin levels, suggesting that serotonin may be associated with medial hypertrophy and presence of argyrophil material. Medial thickness was positively correlated with NE cell numbers. The above findings led to the following summary: pulmonary NE cells respond to changes in airway oxygen levels; hypoxia or decreased oxygen is associated with decreased cellular 5-HT content and an increase in NE cell numbers by argyrophil stain and medial thickness of pulmonary artery walls. The change to normoxia from hypoxia results in higher cellular 5-HT content and decreased NE argyrophil cell numbers along with reduced pulmonary artery wall medial thickness.