Standardization of pulmonary ventilation technique using volume-controlled ventilators in rats with congenital diaphragmatic hernia

OBJECTIVE: To standardize a technique for ventilating rat fetuses with Congenital Diaphragmatic Hernia (CDH) using a volume-controlled ventilator.METHODS: Pregnant rats were divided into the following groups: a) control (C); b) exposed to nitrofen with CDH (CDH); and c) exposed to nitrofen without CDH (N-). Fetuses of the three groups were randomly divided into the subgroups ventilated (V) and non-ventilated (N-V). Fetuses were collected on day 21.5 of gestation, weighed and ventilated for 30 minutes using a volume-controlled ventilator. Then the lungs were collected for histological study. We evaluated: body weight (BW), total lung weight (TLW), left lung weight (LLW), ratios TLW / BW and LLW / BW, morphological histology of the airways and causes of failures of ventilation.RESULTS: BW, TLW, LLW, TLW / BW and LLW / BW were higher in C compared with N- (p <0.05) and CDH (p <0.05), but no differences were found between the subgroups V and N-V (p> 0.05). The morphology of the pulmonary airways showed hypoplasia in groups N- and CDH, with no difference between V and N-V (p <0.05). The C and N- groups could be successfully ventilated using a tidal volume of 75 ìl, but the failure of ventilation in the CDH group decreased only when ventilated with 50 ìl.CONCLUSION: Volume ventilation is possible in rats with CDH for a short period and does not alter fetal or lung morphology.

A simple model for the estimation of congenital malformation frequency in racially mixed populations

A simple model is proposed, using the method of maximum likelihood to estimate malformation frequencies in racial groups based on data obtained from hospital services. This model uses the proportions of racial admixture, and the observed malformation frequency. It was applied to two defects: postaxial polydactyly and cleft lip, the frequencies of which are recognizedly heterogeneous among racial groups. The frequencies estimated in each racial group were those expected for these malformations, which proves the applicability of the method.

Ultrastructural study of the neovagina following the utilization of human amniotic membrane for treatment of congenital absence of the vagina

We present an ultrastructural study of the utilization of human amniotic membrane in the treatment of congenital absence of the vagina in 10 patients. All patients were surgically treated with application of an amniotic membrane graft using the modified McIndoe and Bannister technique. Sixty days after surgery, samples of the vaginal neo-epithelium were collected for transmission electron microscopy analysis. The ultrastructural findings consisted of a lining of mature squamous epithelium indicating the occurrence of metaplasia of the amniotic epithelium into the vaginal epithelium. The cells were arranged in layers as in the normal vaginal epithelium, i.e., superficial, intermediate and deep layers. There were desmosomes and cytoplasmic intermediate cytokeratin filaments, as well as some remnant features of the previous amniotic epithelium. These findings suggest that human amniotic membrane is able to complete metaplasia into squamous cells but the mechanism of this cellular transformation is unknown

Phenotype and genotype correlation of the microconversion from the CYP21A1P to the CYP21A2 gene in congenital adrenal hyperplasia

Deficiency of 21-hydroxylase is the most common form of congenital adrenal hyperplasia (CAH-21OH). We determined by allele-specific PCR the frequency of microconversion in the CYP21A2 gene in 50 Brazilian patients with the classical (salt wasting: SW and simple virilizing: SV) forms and nonclassical (NC) form of CAH-21OH and correlated genotype with phenotype. Genotypes were classified into three mutation groups (A, B, and C) based on the amount of enzymatic activity in in vitro studies using adrenal cells. In 94 unrelated alleles, we diagnosed 76% of the affected alleles after screening for 7 microconversions. The most frequent point mutations observed in this series were I172N (19%), V281L (18%), and IVS2,A/C>G,-12 (15%). In the SW form, the most frequent mutation was IVS2,A/C>G,-12 (38%), in the SV form it was I172N (53%), and in the NC form it was V281L (57.7%). We observed a good correlation between genotype and phenotype. Discordance between genotype and phenotype was found in one SV patient with a mild mutation in one of the alleles (R356W/V281L). However, we cannot rule out the presence of an additional mutation in these alleles. We also observed a good correlation of genotype with 17alpha-hydroxyprogesterone, testosterone, and androstenedione levels. The severity of external genitalia virilization correlated with the severity of mutation. In conclusion, the frequencies described in the present study did not differ from worldwide studies, including the Brazilian population. The few differences observed may reflect individual sample variations. This new Brazilian cohort study suggests the presence of new mutations in Brazilian patients with different forms of CAH-21OH.

Impact of congenital calcitonin deficiency due to dysgenetic hypothyroidism on bone mineral density

The objective of the present study was to determine the effect of chronic calcitonin deficiency on bone mass development. The results of 11 patients with thyroid dysgenesis (TD) were compared to those of 17 normal individuals (C) and of 9 patients with other forms of hypothyroidism (OH): 4 with hypothyroidism due to inborn errors of thyroid hormone synthesis and 5 with Hashimoto's thyroiditis. The subjects received an intravenous calcium stimulus and blood was collected for the determination of ionized calcium (Ca2+), calcitonin, and intact parathyroid hormone. Bone mineral density (BMD) was determined by dual-energy X-ray absorptiometry. After calcium administration the levels of Ca2+ in the two groups of hypothyroidism were significantly higher than in the normal control group (10 min after starting calcium infusion: C = 1.29 ± 0.08 vs TD = 1.34 ± 0.03 vs OH = 1.34 ± 0.02 mmol/l; P < 0.05), and only the TD group showed no calcitonin response (5 min after starting calcium infusion: C = 27.9 ± 5.8 vs TD = 6.6 ± 0.3 vs OH = 43.0 ± 13.4 ng/l). BMD values did not differ significantly between groups (L2-L4: C = 1.116 ± 0.02 vs TD = 1.109 ± 0.03 vs OH = 1.050 ± 0.04 g/cm²). These results indicate that early deficiency of calcitonin secretion has no detrimental effect on bone mass development. Furthermore, the increased calcitonin secretion observed in patients with inborn errors of thyroid hormone biosynthesis does not confer any advantage in terms of BMD.

Congenital ventricular septal defects and prenatal exposure to cyclooxygenase inhibitors

Ventricular septal defects (VSDs) are common congenital abnormalities which have been reported to be associated with maternal fever and various environmental factors. The aim of the present study was to evaluate the effect of prenatal exposure to cyclooxygenase (COX) inhibitors on heart defects. A retrospective statistical analysis was performed using data collected in our laboratory during various teratological studies carried out on albino CRL:(WI)WUBR Wistar strain rats from 1997 to 2004. The observations were compared with concurrent and historic control data, as well as findings from other developmental toxicological studies with selective and nonselective COX-2 inhibitors. Despite the lack of significant differences in the frequency of VSDs between drug-exposed and control groups, statistical analysis by the two-sided Mantel-Haenszel test and historical control data showed a higher incidence of heart defects in offspring exposed to nonselective COX inhibitors (30.06/10,000). Unlike other specific inhibitors, aspirin (46.26/10,000) and ibuprofen (106.95/10,000) significantly increased the incidence of the VSD when compared with various control groups (5.38-19.72/10,000). No significant differences in length or weight were detected between fetuses exposed to COX inhibitors and born with VSD and non-malformed offsprings. However, a statistically significant increase of fetal body length and decrease of body mass index were found in fetuses exposed to COX inhibitors when compared with untreated control. We conclude that prenatal exposure to COX inhibitors, especially aspirin and ibuprofen, increased the incidence of VSDs in rat offspring but was not related to fetal growth retardation.

Reduced folate carrier 1 (RFC1) is associated with cleft of the lip only

In this report, we have reanalyzed genotyping data in a collection of families from South America based on maternal origin. Genotyping analysis was performed at the Craniofacial Anomalies Research Center at the University of Iowa. These genotypes were derived from genomic DNA samples obtained from blood spots from children born with isolated orofacial clefts in 45 hospitals located in eight countries (Argentina, Bolivia, Brazil, Chile, Ecuador, Paraguay, Uruguay, and Venezuela) collaborating with ECLAMC (Latin American Collaborative Studies of Congenital Malformations) between January 1998 and December 1999. Dried blood samples were sent by regular mail to the Laboratory of Congenital Malformations, Federal University of Rio de Janeiro. Previous findings suggested that mitochondrial haplotype D is more commonly found among cleft cases born in South America. We hypothesized that association of certain genes may depend upon the ethnic origin, as defined by population-specific markers. Therefore, we tested if markers in MTHFR (5,10-methylenetetrahydrofolate reductase) and RFC1 (reduced folate carrier 1) were associated with oral clefts, depending on the maternal origin defined by the mitochondrial haplotype. Transmission distortion of alleles in MTHFR C677T and RFC1 G80A polymorphic variants was tested in 200 mother/affected child pairs taking into consideration maternal origin. RFC1 variation was over-transmitted to children born with cleft lip only (P = 0.017) carrying mitochondrial DNA haplotypes other than haplotype D. Our results provide a new indication that variation in RFC1 may contribute to cleft lip only. Future studies should investigate the association between oral clefts and RFC1 based on more discrete phenotypes.

Plasma von Willebrand factor as a predictor of survival in pulmonary arterial hypertension associated with congenital heart disease

Biomarkers have been identified for pulmonary arterial hypertension, but are less well defined for specific etiologies such as congenital heart disease-associated pulmonary arterial hypertension (CHDPAH). We measured plasma levels of eight microvascular dysfunction markers in CHDPAH, and tested for associations with survival. A cohort of 46 inoperable CHDPAH patients (age 15.0 to 60.2 years, median 33.5 years, female:male 29:17) was prospectively followed for 0.7 to 4.0 years (median 3.6 years). Plasma levels of von Willebrand factor antigen (VWF:Ag), tissue plasminogen activator (t-PA) and its inhibitor (PAI-1), P-selectin, reactive C-protein, tumor necrosis factor alpha, and interleukin-6 and -10 were measured at baseline, and at 30, 90, and 180 days in all subjects. Levels of six of the eight proteins were significantly increased in patients versus controls (13 to 106% increase, P < 0.003). Interleukin-10 level was 2.06 times normal (P = 0.0003; Th2 cytokine response). Increased levels of four proteins (t-PA, PAI-1, P-selectin, and interleukin-6) correlated with disease severity indices (P < 0.05). Seven patients died during follow-up. An average VWF:Ag (mean of four determinations) above the level corresponding to the 95th percentile of controls (139 U/dL) was independently associated with a high risk of death (hazard ratio = 6.56, 95%CI = 1.46 to 29.4, P = 0.014). Thus, in CHDPAH, microvascular dysfunction appears to involve Th2 inflammatory response. Of the biomarkers studied, plasma vWF:Ag was independently associated with survival.

Plasmatic ADAMTS-13 metalloprotease and von Willebrand factor in children with cyanotic congenital heart disease

Changes in plasma von Willebrand factor concentration (VWF:Ag) and ADAMTS-13 activity (the metalloprotease that cleaves VWF physiologically) have been reported in several cardiovascular disorders with prognostic implications. We therefore determined the level of these proteins in the plasma of children with cyanotic congenital heart disease (CCHD) undergoing surgical treatment. Forty-eight children were enrolled (age 0.83 to 7.58 years). Measurements were performed at baseline and 48 h after surgery. ELISA, collagen-binding assays and Western blotting were used to estimate antigenic and biological activities, and proteolysis of VWF multimers. Preoperatively, VWF:Ag and ADAMTS-13 activity were decreased (65 and 71% of normal levels considered as 113 (105-129) U/dL and 91 ± 24% respectively, P < 0.003) and correlated (r = 0.39, P = 0.0064). High molecular weight VWF multimers were not related, suggesting an interaction of VWF with cell membranes, followed by proteolytic cleavage. A low preoperative ADAMTS-13 activity, a longer activated partial thromboplastin time and the need for cardiopulmonary bypass correlated with postoperative bleeding (P < 0.05). Postoperatively, ADAMTS-13 activity increased but less extensively than VWF:Ag (respectively, 2.23 and 2.83 times baseline, P < 0.0001), resulting in an increased VWF:Ag/ADAMTS-13 activity ratio (1.20 to 1.54, respectively, pre- and postoperative median values, P = 0.0029). ADAMTS-13 consumption was further confirmed by decreased ADAMTS-13 antigenic concentration (0.91 ± 0.30 to 0.70 ± 0.25 µg/mL, P < 0.0001) and persistent proteolysis of VWF multimers. We conclude that, in pediatric CCHD, changes in circulating ADAMTS-13 suggest enzyme consumption, associated with abnormal structure and function of VWF.

VEGF receptor expression decreases during lung development in congenital diaphragmatic hernia induced by nitrofen

Changes in vascular endothelial growth factor (VEGF) in pulmonary vessels have been described in congenital diaphragmatic hernia (CDH) and may contribute to the development of pulmonary hypoplasia and hypertension; however, how the expression of VEGF receptors changes during fetal lung development in CDH is not understood. The aim of this study was to compare morphological evolution with expression of VEGF receptors, VEGFR1 (Flt-1) and VEGFR2 (Flk-1), in pseudoglandular, canalicular, and saccular stages of lung development in normal rat fetuses and in fetuses with CDH. Pregnant rats were divided into four groups (n=20 fetuses each) of four different gestational days (GD) 18.5, 19.5, 20.5, 21.5: external control (EC), exposed to olive oil (OO), exposed to 100 mg nitrofen, by gavage, without CDH (N-), and exposed to nitrofen with CDH (CDH) on GD 9.5 (term=22 days). The morphological variables studied were: body weight (BW), total lung weight (TLW), left lung weight, TLW/BW ratio, total lung volume, and left lung volume. The histometric variables studied were: left lung parenchymal area density and left lung parenchymal volume. VEGFR1 and VEGFR2 expression were determined by Western blotting. The data were analyzed using analysis of variance with the Tukey-Kramer post hoc test. CDH frequency was 37% (80/216). All the morphological and histometric variables were reduced in the N- and CDH groups compared with the controls, and reductions were more pronounced in the CDH group (P<0.05) and more evident on GD 20.5 and GD 21.5. Similar results were observed for VEGFR1 and VEGFR2 expression. We conclude that N- and CDH fetuses showed primary pulmonary hypoplasia, with a decrease in VEGFR1 and VEGFR2 expression.

Early neonatal echocardiographic findings in an experimental rabbit model of congenital diaphragmatic hernia

This study aimed to demonstrate that congenital diaphragmatic hernia (CDH) results in vascular abnormalities that are directly associated with the severity of pulmonary hypoplasia and hypertension. These events increase right ventricle (RV) afterload and may adversely affect disease management and patient survival. Our objective was to investigate cardiac function, specifically right ventricular changes, immediately after birth and relate them to myocardial histological findings in a CDH model. Pregnant New Zealand rabbits underwent the surgical procedure at 25 days of gestation (n=14). CDH was created in one fetus per horn (n=16), and the other fetuses were used as controls (n=20). At term (30 days), fetuses were removed, immediately dried and weighed before undergoing four-parameter echocardiography. The lungs and the heart were removed, weighed, and histologically analyzed. CDH animals had smaller total lung weight (P<0.005), left lung weight (P<0.005), and lung-to-body ratio (P<0.005). Echocardiography revealed a smaller left-to-right ventricle ratio (LV/RV, P<0.005) and larger diastolic right ventricle size (DRVS, P<0.007). Histologic analysis revealed a larger number of myocytes undergoing mitotic division (186 vs 132, P<0.05) in CDH hearts. Immediate RV dilation of CDH hearts is related to myocyte mitosis increase. This information may aid the design of future strategies to address pulmonary hypertension in CDH.

Histidine-tryptophan-ketoglutarate solution decreases mortality and morbidity in high-risk patients with severe pulmonary arterial hypertension associated with complex congenital heart disease: an 11-year experience from a single institution

Cardioplegic reperfusion during a long term ischemic period interrupts cardiac surgery and also increases cellular edema due to repeated solution administration. We reviewed the clinical experiences on myocardial protection of a single perfusion with histidine-tryptophan-ketoglutarate (HTK) for high-risk patients with severe pulmonary arterial hypertension associated with complex congenital heart disease. This retrospective study included 101 high-risk patients undergoing arterial switch operation between March 2001 and July 2012. We divided the cohort into two groups: HTK group, myocardial protection was carried out with one single perfusion with HTK solution; and St group, myocardial protection with conventional St. Thomas' crystalloid cardioplegic solution. The duration of cardiopulmonary bypass did not differ between the two groups. The mortality, morbidity, ICU stay, post-operative hospitalization time, and number of transfusions in HTK group were lower than those in St group (P<0.05). Univariate and multivariate analysis showed that HTK is a statistically significant independent predictor of decreased early mortality and morbidity (P<0.05). In conclusion, HTK solution seems to be an effective and safe alternative to St. Thomas' solution for cardioplegic reperfusion in high-risk patients with complex congenital heart disease.