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ABSTRACT: Prenatal tracheal occlusion (TO) promotes lung growth and is applied clinically in fetuses with severe congenital diaphragmatic hernia. Limited data are available regarding the effect of duration of TO on lung development. Our objective was to evaluate the effects of long (2 and 2.5 days) versus short (1 day) TO on lung development in rats with nitrofen-induced diaphragmatic hernia.
Nitrofen was administered on embryonic day (ED) 9 and fetal TO performed either on ED18.5, 19 or 20 (term = 22 days). Sham-operated and untouched littermates served as controls. On ED21, lungs were harvested and only fetuses with a left-sided diaphragmatic defect were included in further analyses.
Lung-body-weight ratio incrementally increased with the duration of TO. Increased proliferation following long TO was confirmed by immunohistochemistry and qRT-PCR for the proliferation marker Ki-67. Irrespective of duration, TO induced more complex airway architecture. Medial wall thickness of pulmonary arteries was thinner after long rather than short TO.
In the nitrofen rat model of congenital diaphragmatic hernia, a longer period of TO leads to enhanced lung growth and less muscularized pulmonary arteries.
Prenatal Diagnosis 11/2011; 32(1):39-44. · 2.11 Impact Factor
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Human gene therapy 04/2011; 22(4):383-5. · 4.20 Impact Factor
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ABSTRACT: The fetal response to small tendon injury results in regenerative or scarless healing and is characterized by a markedly diminished cellular inflammatory response, lack of fibroplasia, and restoration of normal tissue architecture. We hypothesized that an increasing fetal tendon wound size would lead to increased wound inflammation and a change from regenerative to reparative healing and scar formation. We created small or large tendon wounds in early gestation fetal sheep and used histology to assess tissue architecture, immunohistochemistry to assess the cellular inflammatory response, ovine-specific gene microarrays, and real-time reverse transcription-polymerase chain reaction to measure the gene expression in response to injury. Small tendon wounds showed a regenerative healing phenotype with orderly deposition of collagen fibers while large tendon wounds showed disorderly collagen deposition consistent with scar formation. Small tendon wounds had few inflammatory cells at 7 and 28 days after injury, whereas the large wounds showed a significant inflammatory cell infiltrate at 7 days that resolved by 28 days. At 3 days, the differential expression of genes involved in the response to injury and inflammation were seen between large and small tendon wounds. By real-time polymerase chain reaction at 7 days, large tendon wounds also had significantly increased expression of interleukin-6, interleukin-8, transforming growth factor-β1, and transforming growth factor-β3, compared with the small wounds. Increasing the fetal tendon wound size results in increased proinflammatory gene expression, inflammatory cell infiltration, and a change from regenerative to reparative healing. This model allows the process of regenerative healing to be examined without the confounding variable of gestational age.
Wound Repair and Regeneration 08/2010; 18(5):543 - 549. · 2.91 Impact Factor
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ABSTRACT: Objectives: The adult response to myocardial infarction results in inflammation, scar formation, left ventricular dilatation, and loss of regional and global function. Regenerative scarless healing has been demonstrated in foetal dermis and tendon and is associated with diminished inflammation. We hypothesised that following foetal myocardial infarction, there would be minimal inflammation, regenerative healing, and preservation of function. Methods: Anteroapical myocardial infarction encompassing 20% of the left ventricle was created in adult or early gestation foetal sheep. Myocardial function was serially assessed using quantitative echocardiography. Infarct architecture was examined histologically for evidence of scar formation. Cellular inflammation, cellular proliferation, and apoptosis were assessed using immunohistochemistry. Results: In the adult sheep 4 weeks following myocardial infarction, there was a significant decline in ejection fraction (EF) (41±7.4% to 26±7.4%, p<0.05), and the akinetic myocardial segment increased in size (6.9±0.8 cm to 7.9±1.1 cm, p<0.05). By contrast, there was no decline in the foetal EF (53±8.1% to 55±8.8%) and no akinetic foetal myocardial segment 4 weeks post-infarction. The foetal infarcts lacked an inflammatory cell infiltrate and healed with minimal fibrosis, compared with the adults. Foetal infarcts also demonstrated 5-bromo-2'-deoxyuridine (BrdU)+ proliferating cells, including cardiomyocytes, within the infarct. Conclusions: These data demonstrate that the foetal response to myocardial infarction is dramatically different from the adult and is characterised by minimal inflammation, lack of fibrosis, myocardial proliferation and restoration of cardiac function. Diminished inflammation is associated with foetal regenerative cardiac healing following injury. Understanding the mechanisms involved in foetal myocardial regeneration may lead to applications to alter the adult response following myocardial infarction.
European journal of cardio-thoracic surgery: official journal of the European Association for Cardio-thoracic Surgery 05/2010; 38(6):691-8. · 2.40 Impact Factor
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ABSTRACT: The aim of this study is to examine structure of lung parenchyma (Pp) and muscularization of pulmonary arterioles (PAs) in human fetuses that underwent tracheal occlusion (TO) therapy for severe congenital diaphragmatic hernia (CDH).
Fifteen fetuses underwent TO, with 5 survivors (Am J Obstet Gynecol. 2000:183;1059-1066). Paraffin-embedded lung specimens from 7 of 10 nonsurvivors (CDH-TO) and 6 age-matched fetuses (CDH) were available for morphometric analysis, which included measurements of point fraction of lung Pp and surface density. The PAs were categorized according to external diameter (<70 microm and 70 <or= 140 microm). Percent medial wall thickness (%MWT) was calculated by dividing raw measurements of MWT by external diameter.
Gestational age at TO was 27.6 +/- 0.9 weeks with a mean duration of TO of 32.6 +/- 6.8 days. Gestational age at delivery (CDH-TO 31.9 +/- 0.9 vs CDH 35.4 +/- 1.8 weeks; P = .18) and postnatal survival time (CDH-TO 20.5 +/- 6.0 vs CDH 18.6 +/- 7.8 days; P = .85) were not significantly different between groups. Tracheal occlusion significantly increased the lung-to-body weight ratio (CDH-TO 13.0 +/- 2.2 vs CDH 6.6 +/- 0.9; P = .02). Tracheal occlusion tended to decrease right-lung Pp (CDH-TO 54.6% +/- 2.6% vs CDH 65.7% +/- 5.9%; P = .05), whereas left-lung Pp was not different between groups (CDH-TO 63.0% +/- 3.5% vs CDH 66.7% +/- 4.1%; P = .51). Surface density of airspaces was not different between groups in either left (CDH-TO 171.3 +/- 16.1 cm(-1) vs CDH 151.1 +/- 8.1 cm(-1); P = .34) or right (CDH-TO 172.0 +/- 10.6 cm(-1) vs CDH 160.8 +/- 3.6 cm(-1); P = .33) lungs. The %MWT in small and large PA was similar between groups.
Open prenatal TO in human fetuses increased lung growth, as evidenced by an increase in lung weight, but did not improve parenchymal structure or muscularization of PAs.
Journal of Pediatric Surgery 10/2008; 43(10):1767-75. · 1.45 Impact Factor
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ABSTRACT: The present study investigates a new surgical approach in the treatment of left diaphragmatic hernia in fetal sheep. We postulated that unlike tracheal occlusion where both lungs are occluded, selective left main bronchus occlusion (LMBO) would accelerate growth of only the left lung, reduce left visceral herniation, and recover space in the both thoraces necessary for lung development.
Left-sided congenital diaphragmatic hernia (CDH) was surgically created in 8 fetal lambs at approximately 65 days of gestation; in 4 of these animals, LMBO was performed at approximately 118 days. Four sham-operated animals served as controls. Lambs were delivered by hysterotomy at 137 days. We measured lung-to-body weight ratios, alveolar surface area, septal wall thickness, and AE2 cell density in the left and right lungs.
Left main bronchus occlusion increased (P < .05) left lung growth causing severe right mediastinal shift but failed to reduce herniated abdominal viscera in 3 of 4 lambs. Wet lung-to-body weight ratio in LMBO group was similar to that of the control group; however, this was achieved by overgrowth of left lung, whereas the right wet lung-to-body weight ratio remained low. In the LMBO group, right lung AE2 cell density was higher than that of control group and not different to that of the CDH group.
Left main bronchus occlusion failed to restore normal pulmonary development in CDH.
Journal of Pediatric Surgery 05/2008; 43(4):620-6. · 1.45 Impact Factor
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ABSTRACT: In fetal sheep with surgically created diaphragmatic hernia (DH), tracheal occlusion (TO) can restore lung growth but does not ameliorate the increase in inter-alveolar wall thickness (T(W)). We determined whether prenatal exposure to glucocorticoids (GC) could reduce T(w) in fetuses with DH undergoing TO. At 65 days of gestation, DH was created in 12 fetal sheep, and TO subsequently performed at 110 days (DH/TO). Six of these fetuses were exposed to betamethasone (DH/TO + GC; 0.5 mg/kg; maternal, IM) 48 hr before delivery; Sham operated fetuses (n = 7) served as controls. At 139 days, we measured alveolar surface density (S(V)), parenchymal tissue fraction, T(W), alveolar type 2 (AE2) cell density and lung surfactant protein (SP) mRNA expression. Prenatal GC decreased T(W) and S(V) by 33% and 27% respectively, and increased fixed lung volume (by 55%), AE2 cell density and partially restored SPmRNA expression. Our data indicate that prenatal exposure to GC can reverse some of the negative effects of prolonged fetal TO. We hypothesize that a GC-induced reduction in lung liquid volume during TO contributes, in part, to the observed increase in AE2 cell density and SPmRNA expression.
Pediatric Pulmonology 01/2007; 41(12):1188-96. · 2.53 Impact Factor
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ABSTRACT: Fetal tracheal occlusion (TO) accelerates lung growth and can reverse severe lung hypoplasia associated with diaphragmatic hernia (DH), however, lung compliance (Cl) and respiratory gas exchange remain abnormal. We determined the individual and combined effects of prenatal glucocorticoids (GC) and exogenous surfactant therapy (S) on postnatal pulmonary function in lambs with DH that underwent prolonged TO. DH was created in 22 fetal sheep at 65 d of gestation and TO performed at 110 d. Eleven DH/TO animals received prenatal GC (betamethasone, 0.5 mg/kg) 48 h before delivery; six GC-treated and five non-GC lambs were administered surfactant (Infasurf, 3 mg/kg) at birth. Six sham-operated lambs served as controls. Lambs were delivered at 139 d gestation and ventilated for 2 h. GC or surfactant therapy alone significantly improved respiratory gas exchange, Cl, and ventilatory efficiency index. Total lung capacity was normalized only in DH/TO lambs that received both GC and S.
Pediatric Research 09/2006; 60(2):131-5. · 2.70 Impact Factor
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ABSTRACT: Mesenchymal nuclear transcription factors (MNTF) are involved in lung development and maturation and regulate surfactant protein (SP) expression. Prolonged (>2 weeks) fetal tracheal occlusion (TO) has been shown to accelerate lung growth and inhibit pulmonary surfactant synthesis. The effects of TO on SP expression and MNTF, however, have not been formally assessed. The objectives of this study were to evaluate the effects of short-term (3 days) TO on normal lung growth and protein expression of pulmonary MNTF involved in SP synthesis.
At E19 (term, 22 days), 2 fetuses per time-dated Sprague-Dawley rats underwent either TO (n = 23) or a sham (n = 22) operation. Lungs were harvested 72 hours post surgery. Pulmonary SP-A; SP-B; SP-C messenger RNA (mRNA) expression; and SP-A and SP-B, Hoxb5, thyroid transcription factor 1, and retinoic X receptor-alpha protein expression were analyzed.
Lung weight was significantly increased by TO (TO 0.32 +/- 0.02g vs SHAM 0.14 +/- 0.01 g; P < .001), resulting in 123% increase of the lung-to-body-weight ratio. No difference of SP-A-mRNA (177 +/- 4.3 TO vs 169 +/- 4.4 SHAM; P = .25), SP-B-mRNA (87.7 +/- 0.2 TO vs 87.4 +/- 0.02 SHAM; P = .33), and SP-C-mRNA (186.5 +/- 3.2 TO vs 183.2 +/- 2.7 SHAM; P = .45) expression was found. Surfactant protein A (175.6 +/- 25.3 TO vs 192.5 +/- 19.8 SHAM; P = .59) and SP-B (163.4 +/- 5.2 TO vs 166.8 +/- 9.3 SHAM; P = .75) protein expression were similar in both groups; however, Hoxb5 (70.3 +/- 18.9 TO vs 130.6 +/- 5.1 SHAM; P = .02) and thyroid transcription factor 1 (102.6 +/- 19 TO vs 181.1 +/- 6.3 SHAM; P = .007) expression were significantly decreased. Retinoic X receptor-alpha expression tended to be increased by TO (171.9 +/- 6.0 TO vs 155.4 +/- 6.7 SHAM; P = .06).
Short-term TO late in gestation induces rapid lung growth. Surfactant protein-mRNA and protein expression are not significantly altered. Thyroid transcription factor 1 and Hoxb5 are down-regulated by TO, suggesting that duration and timing of occlusion are important in balancing the effects of TO on lung growth vs lung maturation.
Journal of Pediatric Surgery 04/2006; 41(4):774-80. · 1.45 Impact Factor
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ABSTRACT: Congenital diaphragmatic hernia (CDH) impairs fetal lung growth and increases the density of alveolar epithelial type 2 (AE2) cells. There is controversy whether surfactant protein (SP) expression is altered in CDH. The primary aim of this study was to assess SP expression (mRNA and protein) in the left and right lungs of fetal sheep with and without a diaphragmatic hernia (DH). Left-sided DH was created in four fetal sheep at 65 days of gestational age (g.a.). Sham-operated animals were used as controls. At 138 days g.a., lungs were harvested and the following parameters were measured: SP-A, -B, and -C mRNA expression (Northern blot), SP-A and -B expression (Western blot), and AE2 cell density (immunohistochemistry). The lung weight-to-body weight ratio was reduced by 42% in DH animals. The left-to-right lung weight ratio was lower in DH animals (0.47 +/- 0.03 vs. 0.69 +/- 0.03), indicative of asymmetric lung growth. SP-A, -B, and -C mRNA expression were increased by 61.7%, 32.9%, and 75.5%, respectively, in the left lungs of DH animals. SP-A and SP-B were also increased in DH. In the right lung, SP expression (mRNA and protein) was not different between groups. AE2 cell density was higher (by 67%) in the left but not right lungs of DH animals. Although DH in fetal sheep results in significant lung hypoplasia, SP expression is not reduced. On the contrary, SP expression was increased in the ipsilateral lung of fetuses with left-sided DH. Furthermore, AE2 cell density is increased in DH, suggesting that the increase in SP mRNA and protein levels is due to increases AE2 cell number. Our data further support the premise that fetal lung hypoplasia favors an AE2 phenotype.
Pediatric Pulmonology 05/2005; 39(4):359-67. · 2.53 Impact Factor
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ABSTRACT: In utero repair of myelomeningocele (MMC) in humans spares distal neurologic function, reverses the hindbrain herniation component of the Arnold-Chiari II malformation (ACM), and reduces the rate of postnatal shunt placement. The authors hypothesized that extravasation of cerebrospinal fluid (CSF) from the lumbar spinal cord results in herniation. This hypothesis was tested by assessing the impact of a spinal cord myelotomy on hindbrain anatomy in fetal sheep.
A MMC lesion was created surgically in 34 fetal sheep at 75 days' gestation by excision of the L1-L5 lamina, the exposed dura, and surrounding tissues. A lumbar level myelotomy was performed in 28 of the 34 fetuses to open the central canal of the spinal cord to enhance egress of CSF through the MMC defect and potentially induce hindbrain herniation. At 102 days' gestation, a repair of the MMC lesion was performed in 14 fetuses with a myelotomy. Fetuses underwent autopsy at 102, 114, 120, or 140 days' gestation. Control animals underwent 2 unrelated fetal surgical procedures at approximately 70 and 110 days' gestation. The incidence of hindbrain herniation, ventricular size, biparietal diameter, brain weight, and brain anatomy were compared between the different animal groups.
After MMC creation, significant cerebellar tonsillar herniation was observed in 85% of fetuses that underwent creation of a myelotomy; none of the lambs without a myelotomy (n = 6) had hindbrain herniation. At autopsy, cerebellar tonsillar herniation was present at the time of MMC repair (102 days' gestation), 2 weeks after MMC repair, but was reversed 3 weeks post-MMC repair. At birth, tonsillar herniation was absent, and hindbrain anatomy was restored in 88% of the fetuses with a myelotomy that underwent fetal MMC repair. No significant differences in brain weight and ventricular size was observed between animals with and without MMC repair.
Adding a myelotomy to the sheep model of MMC leads to hindbrain herniation that is similar to that observed in the human ACM. These experiments support the hypothesis that leakage of CSF through the exposed central canal alters the normal CSF hydrodynamics, resulting in cerebellar tonsillar herniation. Fetal MMC repair reverses hindbrain herniation and restores gross anatomy of the vermis.
Journal of Pediatric Surgery 04/2003; 38(3):451-8; discussion 451-8. · 1.45 Impact Factor
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ABSTRACT: Prolonged fetal tracheal occlusion (TO) accelerates lung growth but leads to loss of alveolar epithelial type II (AE2) cells. In contrast, temporary TO leads to recovery of AE2 cells and their ability to produce surfactant. The aim of this study was to determine the effects of temporary TO in fetal sheep with lung hypoplasia on postnatal lung function, structure, and surfactant protein mRNA expression. Diaphragmatic hernia (DH) was created in 22 fetal sheep at 65 days of gestation. TO was performed between 110 days of gestation and full term (DH/TO, n = 7) and between 110 and 130 days of gestation (DH/TO+R, n = 6). Sham-operated fetuses (n = 11) served as controls. Lambs were delivered at approximately 139 days of gestation, and blood gas tensions were monitored over a 2-h resuscitation period. Temporary TO increased growth of the hypoplastic lung and restored surfactant protein mRNA expression and AE2 cell density but did not improve respiratory function above that of animals that underwent prolonged TO; DH/TO and DH/TO+R lambs were hypoxic and hypercapnic compared with Sham animals. Lung compliance remained low in DH/TO+R lambs, most likely as a consequence of the persistent increase in alveolar wall thickness in these animals.
Journal of Applied Physiology 04/2003; 94(3):1054-62. · 3.75 Impact Factor
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ABSTRACT: The fetal response to small tendon injury results in regenerative or scarless healing and is characterized by a markedly diminished cellular inflammatory response, lack of fibroplasia, and restoration of normal tissue architecture. We hypothesized that an increasing fetal tendon wound size would lead to increased wound inflammation and a change from regenerative to reparative healing and scar formation. We created small or large tendon wounds in early gestation fetal sheep and used histology to assess tissue architecture, immunohistochemistry to assess the cellular inflammatory response, ovine-specific gene microarrays, and real-time reverse transcription-polymerase chain reaction to measure the gene expression in response to injury. Small tendon wounds showed a regenerative healing phenotype with orderly deposition of collagen fibers while large tendon wounds showed disorderly collagen deposition consistent with scar formation. Small tendon wounds had few inflammatory cells at 7 and 28 days after injury, whereas the large wounds showed a significant inflammatory cell infiltrate at 7 days that resolved by 28 days. At 3 days, the differential expression of genes involved in the response to injury and inflammation were seen between large and small tendon wounds. By real-time polymerase chain reaction at 7 days, large tendon wounds also had significantly increased expression of interleukin-6, interleukin-8, transforming growth factor-β1, and transforming growth factor-β3, compared with the small wounds. Increasing the fetal tendon wound size results in increased proinflammatory gene expression, inflammatory cell infiltration, and a change from regenerative to reparative healing. This model allows the process of regenerative healing to be examined without the confounding variable of gestational age.
Wound Repair and Regeneration 18(5):543-9. · 2.91 Impact Factor
