Synergistic effect of glucose and prolactin on GLUT2 expression in cultured neonatal rat islets

We studied the synergistic effect of glucose and prolactin (PRL) on insulin secretion and GLUT2 expression in cultured neonatal rat islets. After 7 days in culture, basal insulin secretion (2.8 mM glucose) was similar in control and PRL-treated islets (1.84 ± 0.06% and 2.08 ± 0.07% of the islet insulin content, respectively). At 5.6 and 22 mM glucose, insulin secretion was significantly higher in PRL-treated than in control islets, achieving 1.38 ± 0.15% and 3.09 ± 0.21% of the islet insulin content in control and 2.43 ± 0.16% and 4.31 ± 0.24% of the islet insulin content in PRL-treated islets, respectively. The expression of the glucose transporter GLUT2 in B-cell membranes was dose-dependently increased by exposure of the islet to increasing glucose concentrations. This effect was potentiated in islets cultured for 7 days in the presence of 2 µg/ml PRL. At 5.6 and 10 mM glucose, the increase in GLUT2 expression in PRL-treated islets was 75% and 150% higher than that registered in the respective control. The data presented here indicate that insulin secretion, induced by different concentrations of glucose, correlates well with the expression of the B-cell-specific glucose transporter GLUT2 in pancreatic islets

Aversive stimulation during the stress-hyporesponsive period does not affect the number of corticotroph cells in neonatal male rats

Immunohistochemistry was used to evaluate the effects of neonatal handling and aversive stimulation during the first 10 days of life on the number of corticotrophs in the anterior lobe of the pituitary of 11-day-old male Wistar rats. Since adult rats handled during infancy respond with reduced corticosterone secretion in response to stressors and with less behavior inhibition in novel environments, we assumed that neonatal stimulation could affect pituitary morphology during this critical period of cell differentiation. Three groups of animals were studied: intact (no manipulation, N = 5), handled (N = 5) and stimulated (submitted to 3 different aversive stimuli, N = 5). The percentage of ACTH-immunoreactive cells in the anterior lobe of the pituitary (number of ACTH-stained cells divided by total number of cells) was determined by examining three slices per pituitary in which a minimum of 200 cells were counted by two independent researchers. Although animals during the neonatal period are less reactive to stress-like stimulation in terms of ACTH and corticosterone secretion, results showed that the relative number of ACTH-stained cells of neonatal handled (0.25 ± 0.01) and aversive stimulated (0.29 ± 0.03) rats was not significantly different from intact (0.30 ± 0.03) animals. Neonatal stimulation may have a differential effect on the various subpopulations of corticotroph cells in the anterior pituitary

Development of the insulin secretion mechanism in fetal and neonatal rat pancreatic B-cells: response to glucose, K+, theophylline, and carbamylcholine

We studied the development of the insulin secretion mechanism in the pancreas of fetal (19- and 21-day-old), neonatal (3-day-old), and adult (90-day-old) rats in response to stimulation with 8.3 or 16.7 mM glucose, 30 mM K+, 5 mM theophylline (Theo) and 200 µM carbamylcholine (Cch). No effect of glucose or high K+ was observed on the pancreas from 19-day-old fetuses, whereas Theo and Cch significantly increased insulin secretion at this age (82 and 127% above basal levels, respectively). High K+ also failed to alter the insulin secretion in the pancreas from 21-day-old fetuses, whereas 8.3 mM and 16.7 mM glucose significantly stimulated insulin release by 41 and 54% above basal levels, respectively. Similar results were obtained with Theo and Cch. A more marked effect of glucose on insulin secretion was observed in the pancreas of 3-day-old rats, reaching 84 and 179% above basal levels with 8.3 mM and 16.7 mM glucose, respectively. At this age, both Theo and Cch increased insulin secretion to close to two-times basal levels. In islets from adult rats, 8.3 mM and 16.7 mM glucose, Theo, and Cch increased the insulin release by 104, 193, 318 and 396% above basal levels, respectively. These data indicate that pancreatic B-cells from 19-day-old fetuses were already sensitive to stimuli that use either cAMP or IP3 and DAG as second messengers, but insensitive to stimuli such as glucose and high K+ that induce membrane depolarization. The greater effect of glucose on insulin secretion during the neonatal period indicates that this period is crucial for the maturation of the glucose-sensing mechanism in B-cells.

Histopathological diagnosis of intra- and extrahepatic neonatal cholestasis

The histopathology of the liver is fundamental for the differential diagnosis between intra- and extrahepatic causes of neonatal cholestasis. However, histopathological findings may overlap and there is disagreement among authors concerning those which could discriminate between intra- and extrahepatic cholestasis. Forty-six liver biopsies (35 wedge biopsies and 11 percutaneous biopsies) and one specimen from a postmortem examination, all from patients hospitalized for neonatal cholestasis in the Pediatrics Service of Hospital de Clínicas de Porto Alegre, were prospectively studied using a specially designed histopathological protocol. At least 4 of 5 different stains were used, and 46 hepatic histopathological variables related to the differential diagnosis of neonatal cholestasis were studied. The findings were scored for severity on a scale from 0 to 4. Sections which showed less than 3 portal spaces were excluded from the study. Sections were examined by a pathologist who was unaware of the final diagnosis of each case. Bile tract permeability was defined by scintigraphy of the bile ducts and operative cholangiography. The F test and discriminant analysis were used as statistical methods for the study of the hepatic histopathological variables. The chi-square method with Yates correction was used to relate the age of the patients on the date of the histopathological study to the discriminatory variables between intra- and extrahepatic cholestasis selected by the discriminant function test. The most valuable hepatic histopathological variables for the discrimination between intra- and extrahepatic cholestasis, in decreasing order of importance, were periportal ductal proliferation, portal ductal proliferation, portal expansion, cholestasis in neoductules, foci of myeloid metaplasia, and portal-portal bridges. The only variable which pointed to the diagnosis of intrahepatic cholestasis was myeloid metaplasia. Due to the small number of patients who were younger than 60 days on the date of the histopathological study (N = 6), no variable discriminated between intra- and extrahepatic cholestasis before the age of 2 months and all of them, except for the portal expansion, were discriminatory after this age. In infants with cholestasis, foci of myeloid metaplasia, whenever present in the liver biopsy, suggested intrahepatic cholestasis. Periportal ductal proliferation, portal ductal proliferation, portal expansion, cholestasis in neoductules, portal cholestasis and portal-portal bridges suggested extrahepatic obstructive cholestasis.

Effects of sciatic-conditioned medium on neonatal rat retinal cells in vitro

Schwann cells produce and release trophic factors that induce the regeneration and survival of neurons following lesions in the peripheral nerves. In the present study we examined the in vitro ability of developing rat retinal cells to respond to factors released from fragments of sciatic nerve. Treatment of neonatal rat retinal cells with sciatic-conditioned medium (SCM) for 48 h induced an increase of 92.5 ± 8.8% (N = 7 for each group) in the amount of total protein. SCM increased cell adhesion, neuronal survival and glial cell proliferation as evaluated by morphological criteria. This effect was completely blocked by 2.5 µM chelerythrine chloride, an inhibitor of protein kinase C (PKC). These data indicate that PKC activation is involved in the effect of SCM on retinal cells and demonstrate that fragments of sciatic nerve release trophic factors having a remarkable effect on neonatal rat retinal cells in culture.

Neonatal handling induces anovulatory estrous cycles in rats

Since previous work has shown that stimulation early in life decreases sexual receptiveness as measured by the female lordosis quotient, we suggested that neonatal handling could affect the function of the hypothalamus-pituitary-gonadal axis. The effects of neonatal handling on the estrous cycle and ovulation were analyzed in adult rats. Two groups of animals were studied: intact (no manipulation, N = 10) and handled (N = 11). Pups were either handled daily for 1 min during the first 10 days of life or left undisturbed. At the age of 90 days, a vaginal smear was collected daily at 9:00 a.m. and analyzed for 29 days; at 9:00 a.m. on the day of estrus, animals were anesthetized with thiopental (40 mg/kg, ip), the ovaries were removed and the oviduct was dissected and squashed between 2 glass slides. The number of oocytes of both oviductal ampullae was counted under the microscope. The average numbers for each phase of the cycle (diestrus I, diestrus II, proestrus and estrus) during the period analyzed were compared between the two groups. There were no significant differences between intact and handled females during any of the phases. However, the number of handled females that showed anovulatory cycles (8 out of 11) was significantly higher than in the intact group (none out of 10). Neonatal stimulation may affect not only the hypothalamus-pituitary-adrenal axis, as previously demonstrated, but also the hypothalamus-pituitary-gonadal axis in female rats.

Stimulatory effect of dexamethasone on angiotensin-converting enzyme in neonatal rat cardiac myocytes

Angiotensin-converting enzyme (ACE) plays a central role in cardiac remodeling associated with pathological conditions such as myocardial infarction. The existence of different cell types in the heart expressing components of the renin-angiotensin system makes it difficult to evaluate their relative role under physiological and pathological conditions. Since myocytes are the predominant cellular constituent of the heart by mass, in the present study we studied the effects of glucocorticoids on ACE activity using well-defined cultures of neonatal rat cardiac myocytes. Under steady-state conditions, ACE activity was present at very low levels, but after dexamethasone treatment ACE activity increased significantly (100 nmol/l after 24 h) in a time-dependent fashion. These results demonstrate the influence of dexamethasone on ACE activity in rat cardiac myocytes. This is consistent with the idea that ACE activation occurs under stress conditions, such as myocardial infarction, in which glucocorticoid levels may increase approximately 50-fold.

Reduction of intraspecific aggression in adult rats by neonatal treatment with a selective serotonin reuptake inhibitor

Most studies suggest that serotonin exerts an inhibitory control on the aggression process. According to experimental evidence, this amine also influences growth and development of the nervous tissue including serotoninergic neurons. Thus, the possibility exists that increased serotonin availability in young animals facilitates a long-lasting effect on aggressive responses. The present study aimed to investigate the aggressive behavior of adult rats (90-120 days) treated from the 1st to the 19th postnatal day with citalopram (CIT), a selective serotonin reuptake inhibitor (20 mg/kg, sc, every 3 days). Aggressive behavior was induced by placing a pair of rats (matched by weight) in a box (20 x 20 x 20 cm), and submitting them to a 20-min session of electric footshocks (five 1.6-mA - 2-s current pulses, separated by a 4-min intershock interval). When compared to the control group (rats treated for the same period with equivalent volumes of saline solution), the CIT group presented a 41.4% reduction in the duration of aggressive response. The results indicate that the repeated administration of CIT early in life reduces the aggressive behavior in adulthood and suggest that the increased brain serotoninergic activity could play a role in this effect.

Neonatal handling and the expression of immunoreactivity to tyrosine hydroxylase in the hypothalamus of adult male rats

Neonatal handling has long-lasting effects on behavior and stress reactivity. The purpose of the present study was to investigate the effect of neonatal handling on the number of dopaminergic neurons in the hypothalamic nuclei of adult male rats as part of a series of studies that could explain the long-lasting effects of neonatal stimulation. Two groups of Wistar rats were studied: nonhandled (pups were left undisturbed, control) and handled (pups were handled for 1 min once a day during the first 10 days of life). At 75-80 days, the males were anesthetized and the brains were processed for immunohistochemistry. An anti-tyrosine hydroxylase antibody and the avidin-biotin-peroxidase method were used. Tyrosine hydroxylase-immunoreactive (TH-IR) neurons were counted bilaterally in the arcuate, paraventricular and periventricular nuclei of the hypothalamus in 30-µm sections at 120-µm intervals. Neonatal handling did not change the number of TH-IR neurons in the arcuate (1021 ± 206, N = 6; 1020 ± 150, N = 6; nonhandled and handled, respectively), paraventricular (584 ± 85, N = 8; 682 ± 62, N = 9) or periventricular (743 ± 118, N = 7; 990 ± 158, N = 7) nuclei of the hypothalamus. The absence of an effect on the number of dopaminergic cells in the hypothalamus indicates that the reduction in the amount of neurons induced by neonatal handling, as shown by other studies, is not a general phenomenon in the brain.

Fuzzy expert system in the prediction of neonatal resuscitation

In view of the importance of anticipating the occurrence of critical situations in medicine, we propose the use of a fuzzy expert system to predict the need for advanced neonatal resuscitation efforts in the delivery room. This system relates the maternal medical, obstetric and neonatal characteristics to the clinical conditions of the newborn, providing a risk measurement of need of advanced neonatal resuscitation measures. It is structured as a fuzzy composition developed on the basis of the subjective perception of danger of nine neonatologists facing 61 antenatal and intrapartum clinical situations which provide a degree of association with the risk of occurrence of perinatal asphyxia. The resulting relational matrix describes the association between clinical factors and risk of perinatal asphyxia. Analyzing the inputs of the presence or absence of all 61 clinical factors, the system returns the rate of risk of perinatal asphyxia as output. A prospectively collected series of 304 cases of perinatal care was analyzed to ascertain system performance. The fuzzy expert system presented a sensitivity of 76.5% and specificity of 94.8% in the identification of the need for advanced neonatal resuscitation measures, considering a cut-off value of 5 on a scale ranging from 0 to 10. The area under the receiver operating characteristic curve was 0.93. The identification of risk situations plays an important role in the planning of health care. These preliminary results encourage us to develop further studies and to refine this model, which is intended to implement an auxiliary system able to help health care staff to make decisions in perinatal care.

Air pollution and neonatal deaths in São Paulo, Brazil

Air pollution has been associated with health effects on different age groups. The present study was designed to assess the impact of daily changes in air pollutants (NO2, SO2, CO, O3, and particle matter (PM10)) on total number of daily neonatal deaths (those that occur between the first and the 28th days of life) in São Paulo, from January 1998 to December 2000, since adverse outcomes such as neonatal deaths associated with air pollution in Brazil have not been evaluated before. Generalized additive Poisson regression models were used and nonparametric smooth functions (loess) were adopted to control long-term trend, temperature, humidity, and short-term trends. A linear term was used for holidays. The association between air pollutants and neonatal deaths showed a short time lag. Interquartile range increases in PM10 (23.3 µg/m³) and SO2 (9.2 µg/m³) were associated with increases of 4% (95% CI, 2-6) and 6% (95% CI, 4-8), respectively. Instead of adopting a two-pollutant model we created an index to represent PM10 and SO2 effects. For an interquartile range increase in the index an increase of 6.3% (95% CI, 6.1-6.5) in neonatal deaths was observed. These results agree with previous studies performed by our group showing the deleterious effects of air pollutants during the perinatal period. The method reported here represents an alternative approach to analyze the relationship between highly correlated pollutants and public health problems, reinforcing the idea of the synergic effects of air pollutants in public health.

Neonatal administration of citalopram delays somatic maturation in rats

We investigated the somatic maturation of neonate rats treated during the suckling period with citalopram, a selective serotonin reuptake inhibitor. Groups with 6 male neonates were randomly assigned to different treatments 24 h after birth. Each litter was suckled by one of the dams until the 21st postnatal day. Body weight, head axis and tail length were measured daily from the 1st to the 21st postnatal day. Time of ear unfolding, auditory conduit opening, incisor eruption, and eye opening was determined. Pups received 5 mg (Cit5), 10 mg (Cit10) or 20 mg/kg (Cit20) citalopram sc, or saline (0.9% NaCl, w/v, sc). Compared to saline, body weight was lower (24.04%, P < 0.01) for Cit10 from the 10th to the 21st day and for Cit20 from the 6th to the 21st day (38.19%, P < 0.01). Tail length was reduced in the Cit20 group (15.48%, P < 0.001) from the 8th to the 21st day. A reduction in mediolateral head axis (10.53%, P < 0.05) was observed from the 11th to the 21st day in Cit10 and from the 6th to the 21st day in Cit20 (13.16%, P < 0.001). A reduction in anteroposterior head axis was also observed in the Cit20 group (5.28%, P < 0.05) from the 13th to the 21stday. Conversely, this axis showed accelerated growth from the 12th to the 21stday in the Cit5 group (13.05%, P < 0.05). Auditory conduit opening was delayed in the Cit5 and Cit20 groups and incisor eruption was delayed in all citalopram groups. These findings show that citalopram injected during suckling to rats induces body alterations and suggest that the activity of the serotoninergic system participates in growth mechanisms.

Umbilical cord blood and neonatal endothelin-1 levels in preterm newborns with and without respiratory distress syndrome

Increased pulmonary vascular resistance in preterm newborn infants with respiratory distress syndrome is suggested, and endothelin-1 plays an important role in pulmonary vascular reactivity in newborns. We determined umbilical cord blood and neonatal (second sample) levels of endothelin-1 in 18 preterm newborns with respiratory distress syndrome who had no clinical or echocardiographic diagnosis of pulmonary hypertension and 22 without respiratory distress syndrome (gestational ages: 31.4 ± 1.6 and 29.3 ± 2.3 weeks, respectively). Umbilical cord blood and a second blood sample taken 18 to 40 h after birth were used for endothelin-1 determination by enzyme immunoassay. Median umbilical cord blood endothelin-1 levels were similar in both groups (control: 10.9 and respiratory distress syndrome: 11.4 pg/mL) and were significantly higher than in the second sample (control: 1.7 pg/mL and respiratory distress syndrome: 3.5 pg/mL, P < 0.001 for both groups). Median endothelin-1 levels in the second sample were significantly higher in children with respiratory distress syndrome than in control infants (P < 0.001). There were significant positive correlations between second sample endothelin-1 and Score for Neonatal Acute Physiology and Perinatal Extension II (r = 0.36, P = 0.02), and duration of mechanical ventilation (r = 0.64, P = 0.02). A slower decline of endothelin-1 from birth to 40 h of life was observed in newborns with respiratory distress syndrome when compared to controls. A significant correlation between neonatal endothelin-1 levels and some illness-severity signs suggests that endothelin-1 plays a role in the natural course of respiratory distress syndrome in preterm newborns.

Distribution of microglial cells in the cerebral hemispheres of embryonic and neonatal chicks

The distribution, morphology and morphometry of microglial cells in the chick cerebral hemispheres from embryonic day 4 (E4) to the first neonatal day (P1) were studied by histochemical labeling with a tomato (Lycopersicon esculentum) lectin. The histochemical analysis revealed lectin-reactive cells in the nervous parenchyma on day E4. Between E4 (5.7 ± 1.35 mm length) and E17 (8.25 ± 1.2 mm length), the lectin-reactive cells were identified as ameboid microglia and observed starting from the subventricular layer, distributed throughout the mantle layer and in the proximity of the blood vessels. After day E13, the lectin-reactive cells exhibited elongated forms with small branched processes, and were considered primitive ramified microglia. Later, between E18 (5.85 ± 1.5 mm cell body length) and P1 (3.25 ± 0.6 mm cell body length), cells with more elongated branched processes were observed, constituting the ramified microglia. Our findings provide additional information on the migration and differentiation of microglial cells, whose ramified form is observed at the end of embryonic development. The present paper focused on the arrangement of microglial cells in developing cerebral hemispheres of embryonic and neonatal chicks, which are little studied in the literature. Details of morphology, morphometry and spatial distribution of microglial cells contributed to the understanding of bird and mammal central nervous system ontogeny. Furthermore, the identification and localization of microglial cells during the normal development could be used as a morphological guide for embryonic brain injury researches.

Effects of systemic administration of ciliary neurotrophic factor on Bax and Bcl-2 proteins in the lumbar spinal cord of neonatal rats after sciatic nerve transection

Ciliary neurotrophic factor (CNTF) is a cytokine that plays a neuroprotective role in relation to axotomized motoneurons. We determined the effect of daily subcutaneous doses of CNTF (1.2 µg/g for 5 days; N = 13) or PBS (N = 13) on the levels of mRNA for Bcl-2 and Bax, as well as the expression and inter-association of Bcl-2 and Bax proteins, and the survival of motoneurons in the spinal cord lumbar enlargement of 2-day-old Wistar rats after sciatic nerve transection. Five days after transection, the effects were evaluated on histological and molecular levels using Nissl staining, immunoprecipitation, Western blot analysis, and reverse transcriptase-polymerase chain reaction. The motoneuron survival ratio, defined as the ratio between the number of motoneurons counted on the lesioned side vs those on the unlesioned side, was calculated. This ratio was 0.77 ± 0.02 for CNTF-treated rats vs 0.53 ± 0.02 for the PBS-treated controls (P < 0.001). Treatment with CNTF modified the level of mRNA, with the expression of Bax RNA decreasing 18% (with a consequent decrease in the level of Bax protein), while the expression of Bcl-2 RNA was increased 87%, although the level of Bcl-2 protein was unchanged. The amount of Bcl-2/Bax heterodimer increased 91% over that found in the PBS-treated controls. These data show, for the first time, that the neuroprotective effect of CNTF on neonatal rat axotomized motoneurons is associated with a reduction in free Bax, due to the inhibition of Bax expression, as well as increased Bcl-2/Bax heterodimerization. Thus, the neuroprotective action of the CNTF on axotomized motoneurons can be related to the inhibition of this apoptotic pathway.