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OBJECTIVE - To identify clinical factors associated with pulmonary hypertension (PH) and mortality in patients with congenital diaphragmatic hernia (CDH).
STUDY DESIGN - A prospective cohort of neonates with a diaphragm defect identified at 1 of 7 collaborating medical centers was studied. Echocardiograms were performed at 1 month and 3 months of age and analyzed at a central core by 2 cardiologists independently. Degree of PH and survival were tested for association with clinical variables using Fischer exact test, χ(2), and regression analysis.
RESULTS - Two hundred twenty patients met inclusion criteria. Worse PH measured at 1 month of life was associated with higher mortality. Other factors associated with mortality were need for extracorporeal membrane oxygenation, patients inborn at the treating center, and patients with a prenatal diagnosis of CDH. Interestingly, patients with right sided CDH did not have worse outcomes.
CONCLUSIONS - Severity of PH is associated with mortality in CDH. Other factors associated with mortality were birth weight, gestational age at birth, inborn status, and need for extracorporeal membrane oxygenation.
Copyright © 2013 Mosby, Inc. All rights reserved.
Skeletal muscle adaptation to chronic hypoxia includes loss of oxidative capacity and decrease in fiber size. However, the diaphragm may adapt differently since its activity increases in response to hypoxia. Thus, we hypothesized that chronic hypoxia would not affect endurance, mitochondrial function, or fiber size in the mouse diaphragm. Adult male mice were kept in normoxia (control) or hypoxia (hypoxia, FIO(2) = 10%) for 4 weeks. After that time, muscles were collected for histological, biochemical, and functional analyses. Hypoxia soleus muscles fatigued faster (fatigue index higher in control, 21.5 ± 2.6% vs. 13.4 ± 2.4%, p < 0.05), but there was no difference between control and hypoxia diaphragm bundles. Mean fiber cross-sectional area was unchanged in hypoxia limb muscles, but it was 25% smaller in diaphragm (p < 0.001). Ratio of capillary length contact to fiber perimeter was significantly higher in hypoxia diaphragm (28.6 ± 1.2 vs. 49.3 ± 1.4, control and hypoxia, p < 0.001). Mitochondrial respiration rates in hypoxia limb muscles were lower: state 2 decreased 19%, state 3 31%, and state 4 18% vs. control, p < 0.05 for all comparisons. There were similar changes in hypoxia diaphragm: state 3 decreased 29% and state 4 17%, p < 0.05. After 4 weeks of hypoxia, limb muscle mitochondria had lower content of complex IV (cytochrome c oxidase), while diaphragm mitochondria had higher content of complexes IV and V (F (1)/F (0) ATP synthase) and less uncoupling protein 3 (UCP-3). These data demonstrate that diaphragm retains its endurance during chronic hypoxia, apparently due to a combination of morphometric changes and optimization of mitochondrial energy production.
OBJECTIVE - To summarize the state of research in maternal-fetal surgery regarding the surgical repair of abnormalities in fetuses in the womb.
DATA SOURCES - We searched MEDLINE from 1980 to 2010 for studies of maternal-fetal surgery for the following conditions: twin-twin transfusion syndrome, obstructive uropathy, congenital diaphragmatic hernia, myelomeningocele, thoracic lesions, cardiac malformations, and sacrococcygeal teratoma.
METHODS OF STUDY SELECTION - We used pilot-tested data collection forms to screen publications for inclusion and to extract data. We compiled information about how fetal diagnoses were defined, maternal inclusion criteria, type of surgery, study design, country, setting, comparators used, length of follow-up, outcomes measured, and adverse events.
TABULATION, INTEGRATION, AND RESULTS - Two reviewers independently extracted data and discordance was resolved by a third party. Of 1,341 articles located, we retained 258 (comprising 166 unique study populations). Three studies were randomized controlled trials; the majority of the evidence was observational (116 case series [70%], 36 retrospective [22%], and 11 prospective [7%] cohorts). Twin-twin transfusion is the most studied condition, with 84 studies including 2,532 pregnancies. Fewer than 500 pregnancies are represented in the literature for each of the other conditions except congenital diaphragmatic hernia (n=503). Inclusion criteria were poorly specified. Outcomes typically measured were survival to birth, preterm birth, and neonatal death. Longer-term outcomes were sparse but included pulmonary, renal, and neurologic status and developmental milestones. Maternal outcome data were rare.
CONCLUSION - Although developing rapidly, maternal-fetal surgery research has yet to achieve the typical quality of studies and aggregate strength of evidence needed to optimally inform care.
The mitochondrial content of skeletal muscles is proportional to activity level, with the assumption that intrinsic mitochondrial function is the same in all muscles. This may not hold true for all muscles. For example, the diaphragm is a constantly active muscle; it is possible that its mitochondria are intrinsically different compared with other muscles. This study tested the hypothesis that mitochondrial respiration rates are greater in the diaphragm compared with triceps surae (TS, a limb muscle). We isolated mitochondria from diaphragm and TS of adult male Sprague Dawley rats. Mitochondrial respiration was measured by polarography. The contents of respiratory complexes, uncoupling proteins 1, 2, and 3 (UCP1, UCP2, and UCP3), and voltage-dependent anion channel 1 (VDAC1) were determined by immunoblotting. Complex IV activity was measured by spectrophotometry. Mitochondrial respiration states 3 (substrate and ADP driven) and 5 (uncoupled) were 27 ± 8% and 24 ± 10%, respectively, lower in diaphragm than in TS (P < 0.05 for both comparisons). However, the contents of respiratory complexes III, IV, and V, UCP1, and VDAC1 were higher in diaphragm mitochondria (23 ± 6, 30 ± 8, 25 ± 8, 36 ± 15, and 18 ± 8% respectively, P ≤ 0.04 for all comparisons). Complex IV activity was 64 ± 16% higher in diaphragm mitochondria (P ≤ 0.01). Mitochondrial UCP2 and UCP3 content and complex I activity were not different between TS and diaphragm. These data indicate that diaphragm mitochondria respire at lower rates, despite a higher content of respiratory complexes. The results invalidate our initial hypothesis and indicate that mitochondrial content is not the only determinant of aerobic capacity in the diaphragm. We propose that UCP1 and VDAC1 play a role in regulating diaphragm aerobic capacity.
AIM - The liver kinase B1 (LKB1)/AMP-activated protein kinase (AMPK) signalling pathway is a major regulator of skeletal muscle metabolic processes. During exercise, LKB1-mediated phosphorylation of AMPK leads to its activation, promoting mitochondrial biogenesis and glucose transport, among other effects. The roles of LKB1 and AMPK have not been fully characterized in the diaphragm.
METHODS - Two methods of AMPK activation were used to characterize LKB1/AMPK signalling in diaphragms from muscle-specific LKB1 knockout (KO) and littermate control mice: (1) acute injection of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and (2) 5-min direct electrical stimulation of the diaphragm. Diaphragms were excised 60 min post-AICAR injection and immediately after electrical stimulation.
RESULTS - AMPK phosphorylation increased with AICAR and electrical stimulation in control but not KO mice. Acetyl CoA carboxylase phosphorylation increased with AICAR in control but not KO mice, but increased in both genotypes with electrical stimulation. While the majority of mitochondrial protein levels were lower in KO diaphragms, uncoupling protein 3, complex I and cytochrome oxidase IV protein levels were not different between genotypes. KO diaphragms have a lower percentage of IIx fibres and an elevated percentage of IIb fibres when compared with control diaphragms. While in vitro peak force generation was similar between genotypes, KO diaphragms fatigued more quickly and had an impaired ability to recover.
CONCLUSION - LKB1 regulates AMPK phosphorylation, mitochondrial protein expression, fibre type distribution, as well as recovery of the diaphragm from fatigue.
Chronic hypoxia reduces aerobic capacity (mitochondrial content) in limb skeletal muscles, and one of the causes seems to be decreased physical activity. Diaphragm and other respiratory muscles, however, may have a different pattern of adaptation as hypoxia increases the work of breathing. Thus, we hypothesized that chronic hypoxia would not reduce mitochondrial content in mouse diaphragm. Adult male C57BL/6J mice were kept in normoxia (Fi(O(2)) = 21%, control) or normobaric hypoxia (Fi(O(2)) = 10%, hypoxia) for 1, 2, and 4 wk. Mice were then killed, and the diaphragm and gastrocnemius muscles collected for analysis. In the diaphragm, cytochrome c oxidase histochemistry showed less intense staining in the hypoxia group. The total content of subunits from the electron transport chain, pyruvate dehydrogenase kinase 1 (PDK1), and voltage-dependent anion channel 1 (VDAC1) was evaluated by Western blot. These proteins decreased by 25-30% after 4 wk of hypoxia (P < 0.05 vs. control for all comparisons), matching a comparable decrease in diaphragmatic mitochondrial volume density (control 33.6 +/- 5.5% vs. hypoxia 26.8 +/- 6.7%, P = 0.013). Mitochondrial volume density or protein content did not change in gastrocnemius after hypoxia. Hypoxia decreased the content of peroxisome proliferator-activated receptor gamma (PPARgamma) and PPARgamma cofactor 1-alpha (PGC-1alpha) in diaphragm but not in gastrocnemius. PGC-1alpha mRNA levels in diaphragm were also reduced with hypoxia. BCL2/adenovirus E1B interacting protein 3 (BNIP-3) mRNA levels were upregulated after 1 and 2 wk of hypoxia in diaphragm and gastrocnemius, respectively; BNIP-3 protein content increased only in the diaphragm after 4 wk of hypoxia. Contrary to our hypothesis, these results show that chronic hypoxia decreases mitochondrial content in mouse diaphragm, despite the increase in workload. A combination of reduced mitochondrial biogenesis and increased mitophagy seems to be responsible for the decrease in mitochondrial content in the mouse diaphragm after hypoxia.
Nicotinic receptors (nAchRs) are responsible for fast excitatory signaling by the neurotransmitter acetylcholine (Ach). They are present on the postsynaptic membrane at neuromuscular junctions (NMJs) and also at brain synapses. Alpha-bungarotoxin (alpha-BTX), a high-affinity nAchR antagonist, inhibits Ach binding and neurotransmission. Here we demonstrate biotinylated alpha-BTX, bound to native mouse diaphragm nAchRs, can be quantified and visualized ex vivo using streptavidin-conjugated quantum dots. This approach provides a novel methodology for the direct assessment of the presence and mobility of neurotransmitter receptors in native tissue.
Congenital diaphragmatic hernia (CDH) is a vexing anomaly that manifests with variable pulmonary compromise in neonates. More than one-third of neonates with CDH require extracorporeal membrane oxygenation (ECMO) for refractory pulmonary hypertension (PHN). To assess the outcome of neonates having CDH repair on ECMO, we reviewed our experience for babies treated between 1992 and 2003. Of 97 neonates with CDH, 40 required ECMO, and 30 were repaired on bypass. Eighteen were supported by veno-venous bypass (VV) and 12 by veno-arterial bypass (VA). While on ECMO, transfusion requirements increased twofold postoperatively (15 to 33 cc x kg(-1) day(-1), P = 0.03) and then significantly decreased after decannulation (1.5 cc x kg(-1) x day(-1), P < 0.01). Non-intracranial hemorrhage occurred in 7 (23%) infants and intracranial hemorrhage in 3 (10%). Twelve (40%) infants died; one (3%) on ECMO secondary to refractory PHN. The mean length of stay for the 18 (60%) survivors was 48 days. Comparisons between survivors and nonsurvivors showed a significantly increased mortality for infants placed on VA bypass (P < 0.01). However, no other variable was predictive of survival. We conclude that CDH repair on ECMO is technically feasible, shows similar survival to the Extracorporeal Life Support Organization (ELSO) registry, and is associated with few bleeding complications.