Neonatal Day Research Articles

Perinatal development of the rat kidney: apoptosis and epidermal growth factor.

Localization of apoptotic cells in the kidney of perinatal rats was examined by the terminal deoxynucleotidyl transferase-mediated d-UTP-biotin nick end labeling (TUNEL) method and electron microscopy. Perinatal changes in the percentage of kidney cells with DNA fragmentation were determined by flow cytometric analysis. Through observation of two successive sections, the relationship between the localization of the epidermal growth factor receptor (EGFR) positive cells and TUNEL positive cells in the kidney was determined. From fetal day 18 to neonatal day 5, TUNEL positive cells were noted in immature glomeruli, collecting ducts and interstitium. Electron microscopically, chromatin condensed nuclei and apoptotic bodies were seen in the same tissue component as the TUNEL positive cells. The percentage of DNA fragmented cells significantly increased from fetal days 18 to 20 and significantly decreased from fetal days 20 to 22, while they still remained low in the neonatal period. The TUNEL positive cells in immature glomeruli and collecting ducts were not reactive to the EGFR antibody. The TUNEL positive cells were not observed in the proximal tubular cells, which were positive to EGFR antibody. These results indicate that apoptotic cells are present in the kidney throughout the perinatal period in the rat and that EGF plays an important role in perinatal development of the rat kidney.

Adopt a wait-and-see attitude for patent processus vaginalis in neonates

Purpose: The purpose of this paper is to describe the ultrasonographic findings of the patent processus vaginalis (PPV) in neonates. Methods: The patency of the processus vaginalis was examined by ultrasonography in 117 neonates. The ultrasonographic findings, with increment and decrement of the intraabdominal pressure, were categorized into 6 types as follows: type I, the intraabdominal organ is observed; type II, cystic PPV; type III, the PPV is widened with abdominal pressure increment, the length is ≥20 mm; type IV, the PPV contains moving fluid without PPV widening; type V, the PPV is widened with abdominal pressure increment, the length is less than 20 mm; type VI, others. The authors we regarded types I to IV as PPV with inguinal hernia. Results: Twenty-two of 40 neonates with a birth weight under 2,500 g had PPV, including 8 with type I. Twenty of 37 premature neonates 22 to 37 gestational weeks had PPV, including 8 with type I. Eighty-one percent (13 of 16) of PPV in low-birth-weight neonates and 91% (10 of 11) in premature neonates closed spontaneously. The median ages at the time of spontaneous regression of PPV were 242 days in low birth weight neonates and 262 days in premature neonates. Conclusions: Most premature or low-birth-weight neonates with PPV regress spontaneously. The inguinal hernia in neonates (especially in premature or low-birth-weight neonates) should be observed until at least 9 months of age without attempting hernia repair.

Immunological basis of septal fibrosis of the liver in Capillaria hepatica-infected rats.

Rats infected with the helminth Capillaria hepatica regularly develop septal fibrosis of the liver similar to that induced by repeated ip injections of pig serum. Fibrosis starts when the focal parasitic lesions begin to show signs of resorption, thus suggesting an immunologically mediated pathogenesis of this fibrosis. To explore this possibility, the development of C. hepatica-related hepatic fibrosis was observed in rats exposed to worm antigens from the first neonatal day onward. Wistar rats (150 g) were either injected ip with an extract of C. hepatica eggs (protein concentration: 1 mg/ml) or received immature eggs by gavage from the first neonatal day until adult life and were then infected with 500 embryonated eggs. Changes were monitored on the basis of serum levels of anti-worm antibodies and hepatic histopathology. Rats submitted to immunological oral tolerance markedly suppressed C. hepatica-related serum antibodies and septal fibrosis of the liver when infected with the helminth later on. Tolerance trials with ip injections of worm antigens gave essentially negative results. The partial suppression of septal fibrosis of the liver after the induction of immunological tolerance to C. hepatica antigens in rats indicates an immunological basis for the fibrosis and emphasizes the importance of immunological factors in the pathogenesis of hepatic fibrosis.

Necrotizing enterocolitis in full-term infants

Objectives Although necrotizing enterocolitis (NEC) is primarily a disease of prematurity, full-term infants account for approximately 10% of cases. Previous studies have reported conflicting results regarding NEC in full-term (FT) versus preterm (PT) infants. A review of all infants diagnosed with NEC at our institution over the past 3 decades was performed to identify factors associated with this disease in full-term neonates. Methods The charts of all infants with definitive NEC from January 1, 1972 through January 1, 2001 were reviewed. Two hundred seventy-seven patients made up the study group: 251 PT and 26 FT infants. Data regarding demographics, clinical presentation, management, outcome, and other variables were collected. FT and PT infants were compared. Results Mean gestational age and birth weight in the FT group were 39.3 weeks and 3,132 g versus 30.2 weeks and 1,396 g for PT infants. Apgar scores were similar. Mean age at diagnosis was 5 days in FT versus 13 days in PT neonates ( P < .001). Enteral nutrition was initiated earlier in FT infants (1.6 days v 3.1 days; P < .001), and FT infants were discharged an average of 14 days earlier than PT infants ( P value not significant). Factors predisposing to NEC were found in 62% (16 of 26) of patients—heart disease in 6 infants and other conditions in 10 patients. Cardiac disease was found significantly more often (23% v 10%; P = .027) in FT infants. Survival rate was 65% (17 of 26) in the FT group versus 69% (173 of 251) in the PT infants ( P value not significant). Conclusions FT infants with NEC differ from their PT counterparts in several distinct ways. FT neonates had NEC at a significantly earlier age, perhaps owing to earlier initiation of feeding. There was a correlation between age at which feeding was begun and age of onset of NEC. Additionally, an association between cardiac disease and development of NEC in term infants was shown. Predisposing factors were present in a majority of FT infants. In contrast to other reports, the outcome of NEC in full-term infants was no better than for PT infants.

Maternal Autoantibody Triggers De Novo T Cell-Mediated Neonatal Autoimmune Disease

Although human maternal autoantibodies may transfer transient manifestation of autoimmune disease to their progeny, some neonatal autoimmune diseases can progress, leading to the loss of tissue structure and function. In this study we document that murine maternal autoantibody transmitted to progeny can trigger de novo neonatal pathogenic autoreactive T cell response and T cell-mediated organ-specific autoimmune disease. Autoantibody to a zona pellucida 3 (ZP3) epitope was found to induce autoimmune ovarian disease (AOD) and premature ovarian failure in neonatal, but not adult, mice. Neonatal AOD did not occur in T cell-deficient pups, and the ovarian pathology was transferable by CD4(+) T cells from diseased donors. Interestingly, neonatal AOD occurred only in pups exposed to ZP3 autoantibody from neonatal days 1-5, but not from day 7 or day 9. The disease susceptibility neonatal time window was not related to a propensity of neonatal ovaries to autoimmune inflammation, and it was not affected by infusion of functional adult CD4(+)CD25(+) T cells. However, resistance to neonatal AOD in 9-day-old mice was abrogated by CD4(+)CD25(+) T cell depletion. Finally, neonatal AOD was blocked by Ab to IgG-FcR, and interestingly, the disease was not elicited by autoantibody to a second, independent native ZP3 B cell epitope. Therefore, a new mechanism of neonatal autoimmunity is presented in which epitope-specific autoantibody stimulates de novo autoimmune pathogenic CD4(+) T cell response.

Dexrazoxane does not protect against doxorubicin-induced damage in young rats.

Doxorubicin (DOX), an anticancer drug, causes a dose-dependent cardiotoxicity. Some evidence suggests that female children have an increased risk for DOX-mediated cardiac damage. To determine whether the iron chelator dexrazoxane (DXR) could reduce DOX-induced cardiotoxicity in the young, we injected day 10 neonate female and male rat pups with a single dose of saline or DOX, DXR, or DXR + DOX (20:1). We followed body weight gain with growth, measured cardiac hypertrophy after a 2-wk swim exercise program, markers of apoptosis (Bcl-2, BAX, BNIP1, caspase 3 activation), oxidative stress (heme oxygenase 1, protein carbonyl levels), the chaperone protein clusterin, and the transcriptional activator early growth response gene-1 (Egr-1) in hearts of nonexercised and exercised rats on neonate day 38. All DOX-alone and DXR + DOX-treated rats showed decreased weight gain, with female rats affected earlier than male rats. DXR-alone, DOX-alone, and DXR + DOX-treated rats had an increased heart weight-to-body weight (heart wt/body wt) ratio after the exercise program with female rats showing the largest increase in heart wt/body wt. Drug-treated females also showed increased cardiac apoptosis, as measured by the increased expression of the proapoptotic proteins BAX and BNIP1 and the appearance of caspase 3 activation products, and oxidative stress, as measured by increased heme oxygenase 1 expression, and reduced Egr-1 and clusterin expression when compared with the similarly treated male rats. We conclude that DXR preinjection did not reduce DOX-induced noncardiac and cardiac damage and that young female rats were more susceptible to DXR and DOX toxicities than age-matched male rats.

Metabotropic glutamate receptor 5-regulated Elk-1 phosphorylation and immediate early gene expression in striatal neurons.

The Galphaq protein-coupled metabotropic glutamate receptor subtype-5 (mGluR5) is densely expressed in medium spiny projection neurons of striatum. Emerging evidence suggests a significant role of mGluR5 in the addictive plasticity of striatal neurons that is likely derived from inducible cellular gene expression related to stimulation of mGluR5 and associative signaling proteins. In this study, we found that activation of mGluR5 with a selective agonist (RS)-2-chloro-5-hydroxy-phenylglycine (CHPG) induced a rapid and transient phosphorylation of a transcription regulator Elk-1 in cultured striatal neurons from rat E19 embryos or neonatal day-1 pups. The Elk-1 phosphorylation was dose-dependent and occurred in neurochemically identified GABAergic neurons, but not glia. A series of experiments further demonstrated that the CHPG-stimulated Elk-1 phosphorylation was mediated through selective activation of mGluR5-regulated phospholipase C and associative second messenger system, i.e. 1,4,5,-triphosphate-sensitive Ca2+ release. Moreover, the Elk-1 phosphorylation was partially dependent on mGluR5-mediated co-activation of NMDA, but not kainate/AMPA receptors and L-type voltage-operated Ca2+ channels. Using an immediate early gene c-fos as a report of inducible gene expression, we found that CHPG induced marked c-fos mRNA expression. The c-fos induction kinetically corresponded to the Elk-1 phosphorylation and was attenuated by antisense oligonucleotides that selectively knocked down Elk-1 proteins. These results indicate that glutamatergic tone on mGluR5 is positively coupled to Elk-1 phosphorylation in striatal neurons via multiple signaling mechanisms involving Ca2+ release and NMDA activation, and the mGluR5-mediated Elk-1 phosphorylation facilitates gene transcription.

Neonatal ontogeny of murine arylamine N-acetyltransferases: implications for arylamine genotoxicity.

Age-related changes in the expression of xenobiotic biotransformation enzymes can result in differences in the rates of chemical activation and detoxification, affecting responses to the therapeutic and/or toxic effects of chemicals. Despite recognition that children and adults may exhibit differences in susceptibility to chemicals, information about when in development specific biotransformation enzymes are expressed is incomplete. N-acetyltransferases (NATs) are phase II enzymes that catalyze the acetylation of arylamine and hydrazine carcinogens and therapeutic drugs. The postnatal expression of NAT1 and NAT2 was investigated in C57Bl/6 mice. Hepatic NAT1 and NAT2 messenger RNAs (mRNAs) increased with age from neonatal day (ND) 4 to adult in a nonlinear fashion. The presence of functional proteins was confirmed by measuring NAT activities with the isoform selective substrates p-aminobenzoic acid and isoniazid, as well as the carcinogens 2-aminofluorene and 4-aminobiphenyl (4ABP). Neonatal liver was able to acetylate all of the substrates, with activities increasing with age. Protein expression of CYP1A2, another enzyme involved in the biotransformation of arylamines, showed a similar pattern. The genotoxicity of 4ABP was assessed by determining hepatic 4ABP-DNA adducts. There was an age-dependent increase in 4ABP-DNA adducts during the neonatal period. Thus, developmental increases in expression of NAT1 and NAT2 genes in neonates are associated with less 4ABP genotoxicity. The age-related pattern of expression of biotransformation enzymes in mice is consistent with human data for NATs and suggests that this may play a role in developmental differences in arylamine toxicity.

The growth factor midkine is modulated by both glucocorticoid and retinoid in fetal lung development.

The glucocorticoids (GC) and retinoids (RA) modulate branching morphogenesis and cytodifferentiation in the developing lung. We investigated downstream target genes that link glucocorticoid stimulation to the achievement of a mature lung in glucocorticoid receptor (GR) knockout mice. All GR(null) mice and approximately 80% of mice homozygous for a hypomorphic allele (GR(hypo)) die shortly after birth of respiratory failure. cDNA microarray analysis showed organ-specific upregulation of the retinoic acid responsive gene midkine (MK) and its chondroitin-sulfate binding partner PG-M/versican at fetal day 18 and at neonatal day 1 in lungs of GR(hypo) mice, and at neonatal day 1 in lungs of GR(null) mice. By contrast, lung MK and PG-M/versican were downregulated in these mice at fetal day 16.5. In situ hybridization studies showed a dramatic decrease in MK and PG-M/versican RNA between days 16.5 and 17.5 in GR(WT) but not in GR(null) mice. Continued diffuse and robust expression of MK protein was observed in GR(null) mice at neonatal day 1. These findings suggest that MK may contribute to the dysmature lung phenotype in GR-deficient mice. Exposure of cultured day 21 fetal rat lung cells to GC downregulated MK, whereas RA enhanced MK expression. Our findings demonstrate the coincident modulation of expression of MK at the same developmental time point by both GC and RA, providing a potential mechanism for the integration of GC and RA effects on fetal lung development.

Perinatal outcomes in monoamniotic gestations

Objective: A comprehensive review of monoamniotic twin gestations reported between 1990 and 2002 was performed to estimate current perinatal mortality and morbidity rates, as well as the predictivevalue of an antenatal diagnosis of cord entanglement for poor obstetric outcomes.Method: A Medline literature review using the search term 'monoamniotic' and limited to articles published inthe English language between 1990 and 2002 was performed.Results: A total of 133 continuing, non-conjoined twin monoamniotic pregnancies with delivery information were identified. Perinatalloss per 2-week interval was relatively constant at 2-4% from 15 to 32 weeks. However, of the 131 fetuses reaching 33 weeks, the percentage loss significantly increased to 11.0% at 33-35 weeks and 21.9%at 36-38 weeks compared to that at 30-32 weeks. Overall perinatal mortality was 23.3%. Of all losses, 61.2% involved both twins and 38.8% involved only one fetus. Cord entanglements were documented antenatallyin 22.6% of reports. There was a statistically significant decrease in the average number of neonatal intensive care unit days for non-anomalous neonates (10.6 ± 7.7 vs. 32.6 ± 32.0), averagegestational age at the time of delivery (30.4 ± 7.6 vs. 32.6 ± 4.1), as well as a decrease in the prevalence of total (8.3% vs. 27.7%) and non-anomalous (7.0% vs. 21.6%) perinatal mortalityin pregnancies with an antenatal diagnosis of cord entanglement compared to those without the antenatal diagnosis of cord entanglement. The presence of fetal anomalies was associated with a 42.9% perinatalmortality rate.Conclusions: Contrary to previous reports, there is a significant increase in the incidence of perinatal loss beyond 32 weeks among monoamniotic twins, suggesting that deliveryafter corticosteroid therapy should be strongly considered at this gestational age.

Phosphorylation of cAMP response element-binding protein in cultured striatal neurons by metabotropic glutamate receptor subtype 5.

The metabotropic glutamate receptor subtype 5 (mGluR5) is densely expressed in striatal projection neurons. As a G protein-coupled receptor, mGluR5 may initiate an intracellular cascade that conveys extracellular signals to gene expression. This study investigated the possible role of mGluR5 in the inducible phosphorylation of a nuclear transcription factor, cAMP response element-binding protein (CREB), in primary cultures of striatal neurons from rat E19 embryos or neonatal day-1 pups. We found that selective activation of mGluR5 with a selective agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) induced a rapid and transient increase in phosphorylated CREB immunoreactivity in striatal neurons as analyzed by both immunocytochemistry and western blot. The increase in CREB phosphorylation was concentration-dependent, and seen in neurochemically identified GABAergic neurons. Pre-treatment with the mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP) blocked the CHPG phosphorylation of CREB. In contrast, the mGluR1 antagonist, 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOet) did not alter CHPG-stimulated CREB phosphorylation. The mGluR5 antisense oligonucleotides, but not their controls, selectively reduced basal mGluR5 levels as well as CREB phosphorylation in response to CHPG addition. Lastly, using an immediate early gene c-fos as a reporter of inducible gene expression downstream to phosphorylated CREB, we found that CHPG induced a rapid and transient increase in c-fos mRNA levels in cultured neurons as revealed by quantitative in situ hybridization. The increase in c-fos was kinetically correlated well with the CREB phosphorylation and blocked by MPEP and the CREB antisense oligonucleotides. These results demonstrate a positive linkage from surface mGluR5 to CREB phosphorylation, which is able to facilitate immediate gene expression in striatal neurons.

Repeated neonatal pain influences maternal behavior, but not stress responsiveness in rat offspring

Early preterm neonates in the Neonatal Intensive Care Unit (NICU) are subjected to repeated painful procedures which could sensitize their responses to pain and potentiate neuroendocrine and behavioral responses to subsequent stressors in the long-term. In this study, we used the model of the neonatal rat to test the effects of repeated pain during the first 2 weeks of life on neuroendocrine responses (CRF, ACTH and corticosterone) to stressors varying in intensity and on maternal behavior in the postnatal period. To closely mimic the type of repeated painful stimulus experienced by preterm neonates (i.e., heelstick), neonatal rats aged day 2–14 were submitted daily to having their rear heels warmed to 34 °C and pricked (handled and pain, HP) or not (handled, H) with a needle. For the procedure, all pups were separated from their mothers for a total period of 15 min and reunited afterwards. Unhandled (UH) pups not subjected to daily maternal separation were used as controls. On days 6 and 12, litters from the HP and H groups were videotaped for 90 min upon return with the mother and maternal behavior was analyzed. Frequency of ultrasonic vocalizations (USV) were recorded during the procedure and upon return of pups with the mother. On day 15 and 20, rat pups from all groups were exposed to a 3-min ether vapor stressor or to an openfield for 10 min. Plasma ACTH and corticosterone concentrations were determined at 0, 5, 30, 60 and 120 min after stress onset. Our results show that repeated pain did not modify body weight of the pups, however, on day 6 of life, maternal pup grooming was increased significantly ( P<0.05) in the HP group compared to the H group. Frequency of USV was not changed between H and HP rats either during the separation or after reunion with the mother. Plasma ACTH and corticosterone levels under basal or stimulated conditions were not different between UH, H and HP groups. However, the UH pups showed a tendency towards higher ACTH secretion after stress compared to H and HP groups. These results suggest that repeated pain during the first 2 weeks of life in the rat does not lead to significant changes in stress responsiveness in 2-week-old pups, but we suggest that changes in mother–pup interaction (increased grooming) might act as a buffer on the cumulative effect of pain on stress responsiveness.

Prospective delivery of reliably dated term infants of diabetic mothers without determination of fetal lung maturity: comparison to historical control

Objective: To determine whether omitting fetal lung maturity (FLM) testing prior to delivery in term pregnancies complicated by gestational (GDM) and pregestational diabetes mellitus would increase the risk of neonatal respiratory distress syndrome (RDS). Methods: In a 2-year study (1990-91), 1457 pregnant women with accurately dated pregnancies were enrolled after 37 completed weeks and prospectively followed through delivery without FLM testing (study group). The prevalence of RDS and other neonatal outcomes was compared with a historical control group (n = 713, 1988-89) who had undergone determination of lecithin/sphingomyelin ratio prior to delivery at term. Logistic regression analysis was performed to determine independent predictors of RDS. Results: The study group compared to the control group had less severe diabetes: diet-controlled GDM, 35% vs. 18%, respectively; insulin-requiring GDM, 42% vs. 42%, respectively; undiagnosed type-2 diabetes, 14% vs. 31%, respectively; and pre-existing diabetes, 9.6% vs. 8.8%, respectively, p < 0.001. RDS rates in the study group (0.8%) and control group (1.0%) were not significantly different, nor were rates of resuscitation at delivery, neonatal intensive care admission or hospitalization days. Logistic regression analysis found only Cesarean delivery to be independently predictive (adjusted OR 2.21, 95% CI 2.04-2.27) of RDS. Non-predictive variables included FLM testing, diabetic classification, insulin use, poor third-trimester glycemic control, chronic hypertension, pre-eclampsia, labor, neonatal gender, gestational age or large-for-gestational-age fetuses. Conclusions: Routine FLM testing did not change the RDS prevalence in reliably dated, term infants of diabetic mothers and should be abandoned. Delivery by Cesarean section was associated with increased RDS.

Characterization of uterine leiomyomas in CD-1 mice following developmental exposure to diethylstilbestrol (DES).

Experimental animal and clinical studies have well established the association of prenatal exposure to diethylstilbestrol (DES) and the subsequent development of reproductive tract abnormalities, including poor reproductive outcome and neoplasia. Overwhelmingly, the focus has been on DES-induced epithelial lesions, particularly vaginal adenosis and adenocarcinoma; however, uterine smooth muscle cells are also recognized as cellular targets of DES. This descriptive report characterizes uterine leiomyomas that occur in outbred CD-1 mice following exposure to DES prenatally on days 9 to 16 of gestation or on neonatal days 1 to 5. These DES-induced uterine leiomyomas have typical histomorphologic, and some immunohistochemical characteristics of spontaneously occurring uterine smooth muscle tumors of B6C3F1 mice previously described in our laboratory, and they are also similar to uterine leiomyomas (fibroids) commonly observed in premenopausal women.

Clinical Factors Influencing Blood Pressure in the Neonate

After completing this article, readers should be able to: 1. Describe the association between gestational age, birthweight, and postnatal age and blood pressure in the neonate. 2. Delineate the primary clinical consideration in an infant who has hypotension. 3. Describe the controversy surrounding the relationship between antenatal steroid therapy and neonatal blood pressure. 4. Delineate the aspects of management of respiratory illnesses in the neonate that may be associated with altered blood pressure. 5. List factors that may cause altered blood pressure in the neonate. Few aspects of neonatal care have generated as much controversy as the assessment of blood pressure (BP) and need for treatment of perceived abnormalities of this physiologic variable. Familiarity with clinical situations in which BP may be low should allow anticipation of this common clinical problem and timely intervention when such treatment is necessary (Tables 1 and 2). In this article, we review factors that have been shown to have the greatest effect on BP. View this table: Table 1. Some Risk Factors for Shock in the Newborn View this table: Table 2. Some Causes of Cardiogenic Shock in the Newborn A direct reading from an indwelling arterial catheter represents the “gold standard” for measuring BP in the neonate, and this method should be used whenever possible. Obviously, arterial access may not always be available, and the ability to monitor BP noninvasively using an oscillometric technique represents a major advancement in neonatal care. The correlation between direct and indirect methods generally has been good. Disparities in results have been related to various factors, including inappropriate cuff width-to-arm ratio or problems with the arterial catheter-transducer system, such as air bubbles or clots. In general, indirect determinations are higher than those obtained directly, often by 3 to 5 mm Hg. The individual contributions of birthweight and gestational age to BP are difficult to delineate. Several groups of investigators have shown that BP …

Toxicogenomic effects of neonatal exposure to diethylstilbestrol on mouse testicular gene expression in the long term: a study using cDNA microarray analysis.

We examined the effect of neonatal exposure to diethylstilbestrol (DES) on mouse testicular gene expression, using in-house mouse fetus (day 14.5) cDNA microarrays. Newborn male ICR mice were exposed to DES (50 microg/mouse/day) from neonatal day 1 to 5. Differential expression was detected in 14 genes in 4-week-old (day 28) mouse testes by cDNA microarray analysis; 11 genes (AI035263, AU080565, AU080361, AU080678, AI131681, AU080631, AA986882, AI037066, AA986537, AI156816, and AI596237) were up-regulated and three genes (AI131656, AI118968, and AI117606) were down-regulated in DES-treated mouse testes. Higher expression levels of the former eight genes, out of the up-regulated genes picked-up by the microarray, were also confirmed by reverse transcription and real-time polymerase chain reaction (real-time RT-PCR) analysis. However, the differential expression of other genes could not be confirmed. Real-time RT-PCR analysis also revealed that expression levels of the eight genes were still higher in DES-treated testes at 8 and 12 weeks of age. Our results suggest that cDNA microarray analysis is a useful method by which a large number of gene expressions are simultaneously detected and changes in gene expression are screened. In addition, our results suggest that these genes, whose expressions are changed in the testes of adult mice by fetal or neonatal exposure to exogenous chemicals, might be candidates for predictive biological markers.

Neonatal development of projections to the basolateral amygdala from prefrontal and thalamic structures in rat.

Recently an animal model for neurodevelopmental disorders has been developed. In this model the effects of an early neonatal [postnatal day 7 (Pd7)] basolateral amygdala lesion are compared with the effects of a lesion later in life (Pd21). Early amygdala damage results in enduring behavioral disturbances that become more manifest after puberty. These disturbances were not present in animals lesioned at Pd21. Accordingly it was postulated that the early damage may affect the neuroanatomical and neurochemical organization and functioning of other brain structures. To obtain information on the innervation of the amygdala during normal development, we used the retrograde tracer fluoro-gold. From neonatal day 7 onward (studied until Pd19), retrogradely labeled cells were present in the caudal and rostral thalamus, the substantia innominata, and the prefrontal but not the caudal cortex. Development of the topography of the projecting cells differed substantially for the thalamic regions and substantia innominata vs. the cortical regions. In thalamic regions and substantia innominata, no changes were observed during the studied period (Pd7-Pd9). In the prefrontal cortex, the number of labeled cells increased (from Pd7 to Pd13), the topography of the location of the cells changed from unilateral to bilaminar (from Pd9 to Pd13), and the number of subareas in which the cells were present increased (from Pd7 to Pd13). In the caudal cortex, relatively few cells were present up to Pd15. From Pd17 onward, a bilaminar topography of the location of the cells was observed. These data provide information on the circuitry that may be involved in the aberrant neurodevelopment of neonatally amygdala-lesioned rats, which has been proposed as an animal model for neurodevelopmental psychopathological disorders.

OGG1 mRNA expression and incision activity in rats are higher in foetal tissue than in adult liver tissue while 8-oxo-2′-deoxyguanosine levels are unchanged

This study was set up to investigate the relationships between the formation and removal of DNA damage in form of 8-oxodeoxyguanosine (8-oxodG) in neonatal (day 16 of gestation) as compared to adult rats. The hypothesis addressed was whether the rapidly dividing foetal tissue has an enhanced requirement of DNA repair providing protection against potentially mutagenic DNA damages such as 8-oxodG. The activity of the primary 8-oxodG-repair protein OGG1 was measured by a DNA incision assay and the expression of OGG1 mRNA was measured by Real-Time PCR normalised to 18S rRNA. The tissue level of 8-oxodG was measured by HPLC-ECD. We found a 2–3-fold increased incision activity in the foetal control tissue, together with a 3–15-fold increase in mRNA of OGG1 as compared to liver tissue from adult rats. The levels of 8-oxodG in the foetal tissue were unaltered as compared to the adult groups. To increase the levels of 8-oxodG, the rats received an injection (i.p.) of the hepatotoxin 2-nitropropane. The compound induced significant levels of 8-oxodG in male rat livers 5 h after the injection and in the foetuses 24 h after the injection, while the female rats showed no increase in 8-oxodG. The incision activity was slightly depressed in both male and female liver tissue and in the foetal tissue 5 h after the injection, but significantly increased from 5 to 24 h after the injection. However, it did not reach levels significantly above the control levels. In conclusion, this study confirms that foetal tissue has increased levels of OGG1 mRNA and correspondingly an enhanced incision activity on an 8-oxodG substrate in a crude tissue extract.

Overexpression of c-Myc in β-Cells of Transgenic Mice Causes Proliferation and Apoptosis, Downregulation of Insulin Gene Expression, and Diabetes

To test the hypothesis that c-Myc plays an important role in beta-cell growth and differentiation, we generated transgenic mice overexpressing c-Myc in beta-cells under control of the rat insulin II promoter. F(1) transgenic mice from two founders developed neonatal diabetes (associated with reduced plasma insulin levels) and died of hyperglycemia 3 days after birth. In pancreata of transgenic mice, marked hyperplasia of cells with an altered phenotype and amorphous islet organization was displayed: islet volume was increased threefold versus wild-type littermates. Apoptotic nuclei were increased fourfold in transgenic versus wild-type mice, suggesting an increased turnover of beta-cells. Very few cells immunostained for insulin; pancreatic insulin mRNA and content were markedly reduced. GLUT2 mRNA was decreased, but other beta-cell-associated genes (IAPP [islet amyloid pancreatic polypeptide], PDX-1 [pancreatic and duodenal homeobox-1], and BETA2/NeuroD) were expressed at near-normal levels. Immunostaining for both GLUT2 and Nkx6.1 was mainly cytoplasmic. The defect in beta-cell phenotype in transgenic embryos (embryonic days 17-18) and neonates (days 1-2) was similar and, therefore, was not secondary to overt hyperglycemia. When pancreata were transplanted under the kidney capsules of athymic mice to analyze the long-term effects of c-Myc activation, beta-cell depletion was found, suggesting that, ultimately, apoptosis predominates over proliferation. In conclusion, these studies demonstrate that activation of c-Myc in beta-cells leads to 1) increased proliferation and apoptosis, 2) initial hyperplasia with amorphous islet organization, and 3) selective downregulation of insulin gene expression and the development of overt diabetes.

Prospective delivery of reliably dated term infants of diabetic mothers without determination of fetal lung maturity: comparison to historical control

Objective: To determine whether omitting fetal lung maturity (FLM) testing prior to delivery in term pregnancies complicated by gestational (GDM) and pregestational diabetes mellitus would increasethe risk of neonatal respiratory distress syndrome (RDS).Methods: In a 2-year study (1990-91), 1457 pregnant women with accurately dated pregnancies were enrolled after 37 completed weeks andprospectively followed through delivery without FLM testing (study group). The prevalence of RDS and other neonatal outcomes was compared with a historical control group (n = 713, 1988-89) who hadundergone determination of lecithin/sphingomyelin ratio prior to delivery at term. Logistic regression analysis was performed to determine independent predictors of RDS.Results: The study groupcompared to the control group had less severe diabetes: diet-controlled GDM, 35% vs. 18%, respectively; insulin-requiring GDM, 42% vs. 42%, respectively; undiagnosed type-2 diabetes, 14% vs. 31%, respectively;and pre-existing diabetes, 9.6% vs. 8.8%, respectively, p < 0.001. RDS rates in the study group (0.8%) and control group (1.0%) were not significantly different, nor were rates of resuscitationat delivery, neonatal intensive care admission or hospitalization days. Logistic regression analysis found only Cesarean delivery to be independently predictive (adjusted OR 2.21, 95% CI 2.04-2.27) of RDS.Non-predictive variables included FLM testing, diabetic classification, insulin use, poor third-trimester glycemic control, chronic hypertension, pre-eclampsia, labor, neonatal gender, gestational age orlarge-for-gestational-age fetuses.Conclusions: Routine FLM testing did not change the RDS prevalence in reliably dated, term infants of diabetic mothers and should be abandoned. Delivery byCesarean section was associated with increased RDS.