Enriched populations of rat pituitary thyrotrophs in monolayer culture.

Rat anterior pituitary cells were dissociated and subjected to 4 h of unit gravity sedimentation. Eighty-five 8-ml fractions were collected, and nine pooled fractions were placed in monolayer culture for 1 week. The attached cells were immunocytochemically stained for TSH using an antiserum previously shown to be specific for the beta subunit of TSH and all culture media saved for subsequent assay of TSH. Thyrotrophs were localized both immunocytochemically and by radioimmunoassay to 1-2 fractions from the top of the sedimentation chamber. Typically, it was found that 60-80% of the cells in these fractions were immunocytochemically identified as thyrotrophs. Electron microscopic observations indicated that such cells displayed the classical morphological features associated with thyrotrophs. The enriched thyrotroph cultures responded to 1.0 mM dibutyryl cyclic AMP with an increased release of TSH indicating that the intracellular secretory machinery was not altered by the procedures employed. In all cases, the basal TSH secretion decreased with time in culture. In addition, the fractions containing the most TSH in the culture media were usually one fraction lighter (lower sedimentation rate) than the fractions which were found immunocytochemically to contain the highest percentage of thyrotrophs. The results suggest the possibility of two functionally distinct thyrotroph cell types as has been suggested for the pituitary somatotrophs.

Plasma beta-endorphin in neonates: effect of prematurity, gender, and respiratory status.

A consecutive cohort of 87 infants (46 infants less than 37 weeks gestational age and 41 term infants greater than or equal to 37 weeks gestation) admitted to the Neonatal Intensive Care Unit (NICU) and a convenience cohort of 27 term well babies at the University of Nebraska Medical Center (Omaha, NE) were evaluated for plasma beta-endorphin (beta E) levels during the first 4 h after birth. Demographic data, maternal history, and respiratory status at the time of sampling as well as development of documented apneic episodes during the initial hospitalization were analyzed for all infants. All NICU infants had higher plasma beta E levels than the control infants. Premature infants had significantly higher neonatal plasma beta E levels than term infants in either the control or NICU groups, but the response was gender specific; premature males had higher plasma beta E than premature females (P = 0.008). Perinatal stress, including respiratory problems, was associated with the increase in plasma beta E, but prematurity and being male were significantly predictors of an elevated plasma beta E level. Immaturity in respiratory control, as evaluated by the development of documented apneic episodes during the infant's initial hospitalization, did not correlate with an elevated perinatal plasma beta E level.

Rhabdomyolysis and myoglobinemia in neonates.

Serial myoglobin determinations were made in 20 neonates during the first week of life to determine whether birth asphyxia results in ischemic damage to muscle with the subsequent pathologic release of myoglobin. Serum myoglobin values were significantly elevated in asphyxiated infants compared with control infants. High myoglobin values correlated with a longer duration of oliguria in the neonatal intensive care unit population. The value of urine dipstick testing for myoglobinuria screening was also evaluated. Infants with elevated myoglobin values were more likely to have a strongly positive urine dipstick for occult blood in the first 48 hours of life. These data suggest that ischemic damage to muscle with pathologic release of myoglobin occurs in the neonatal period and that urine dipstick testing provides a reasonable screening examination for myoglobinuria.

Plasma fentanyl levels in infants undergoing extracorporeal membrane oxygenation.

Plasma levels of fentanyl were analyzed in 12 infants undergoing extracorporeal membrane oxygenation who received a fentanyl bolus (5 to 10 micrograms/kg) followed by infusion at 1 to 6.3 micrograms/kg/hr. Fentanyl levels, averaging 11 samples/infant, were measured by radioimmunoassay (mean 19.7 +/- 35.7 ng/ml; n = 140). Eight of the infants, all with a primary diagnosis other than congenital diaphragmatic hernia, survived with relatively short (< 7 days) courses on extracorporeal membrane oxygenation; this group of infants did not develop tolerance to fentanyl and could be maintained on infusion rates of < 5 micrograms/kg/hr throughout. The four infants with congenital diaphragmatic hernia had longer extracorporeal membrane oxygenation runs and three did not survive; their plasma fentanyl levels were consistently higher and while the infusion rates were higher early on extracorporeal membrane oxygenation, they did not exceed 7 micrograms/kg/hr and actually decreased after 5 days on extracorporeal membrane oxygenation. Five infants (42%) received lorazepam in addition to fentanyl for at least one sampling time. The fentanyl infusion dose and plasma level were higher in the congenital diaphragmatic hernia nonsurvivors who did not receive lorazepam (p < 0.001). A decrease in fentanyl clearance correlated with renal dysfunction (p < 0.01). A bolus of fentanyl followed by infusion of relatively low doses (1 to 5 micrograms/kg/hr) provides adequate analgesia for infants on extracorporeal membrane oxygenation, particularly when it is supplemented with intravenous lorazepam whenever needed to control infant movement.

Prostaglandin F1 alpha levels during and after neonatal extracorporeal membrane oxygenation.

Infants receiving extracorporeal membrane oxygenation therapy undergo long-term cardiopulmonary bypass, are systemically heparinized, and frequently receive platelet transfusions. Prostacyclin is a powerful inhibitor of platelet aggregation as well as a potent vasodilator. The levels of its stable metabolite prostaglandin F1 alpha increase significantly in children undergoing cardiopulmonary bypass during heart operations but decrease to preoperative levels after bypass. To determine the effect of long-term bypass on prostacyclin levels, multiple plasma samples were analyzed in 10 human neonates both during extracorporeal membrane oxygenation therapy and within 24 hours after extracorporeal membrane oxygenation. Prostaglandin F1 alpha, the stable metabolite of prostacyclin, was quantitated by radioimmunoassay in picograms per milliliter. Prostaglandin F1 alpha levels were elevated while the patients received extracorporeal membrane oxygenation therapy but decreased with duration of extracorporeal membrane oxygenation. In most infants, prostaglandin F1 alpha levels rose again during weaning from extracorporeal membrane oxygenation and remained elevated for 24 hours after extracorporeal membrane oxygenation. Extracorporeal membrane oxygenation course influenced circulating prostaglandin F1 alpha levels. Fluctuating prostaglandin F1 alpha levels are of clinical significance in the management of vasomotor tone and platelet function, common problems in the care and the prevention of hemorrhage in these critically ill infants.

Serum concentrations of prostacyclin and thromboxane in children before, during, and after cardiopulmonary bypass.

Twenty-six consecutive pediatric patients undergoing reparative procedures necessitating cardiopulmonary bypass were prospectively studied to determine changes in serum levels of 6-keto-prostaglandin F1 alpha and thromboxane B2. Cardiac lesions included acyanotic lesions (five patients), obstructive lesions (10 patients), and right-to-left shunts (11 patients). There was a significant (p less than 0.05) increase in 6-keto-prostaglandin F1 alpha from preoperative levels measured at the time of arterial and venous cannula insertion. This concentration was maintained throughout cardiopulmonary bypass and remained significantly elevated (p less than 0.001) in the recovery room, but returned to preoperative levels by the morning after the operation. Preoperative levels of thromboxane B2 varied widely and were not significantly different from intraoperative levels. The postoperative levels of thromboxane B2, however, were significantly different (p less than 0.05) from the intraoperative levels. In the pediatric age group undergoing cardiopulmonary bypass, 6-keto-prostaglandin F1 alpha and thromboxane B2 change during bypass but do not significantly differ when preoperative levels are compared to postoperative values.

The antioxidant properties of zinc and metallothionein.

Support for the hypothesis that metallothionein isoforms participate in intracellular defense against reactive oxygen and nitrogen species is derived from observations that substances causing oxidative stress, such as ethanol and iron, and agents involved in inflammatory processes, such as interleukin-1 and tumour necrosis factor alpha, induce the synthesis of metallothionein. Moreover, animals deficient in metallothionein isoforms exhibit greater susceptibility to oxidative stress; metallothionein genes are transcriptionally activated in cells and tissues during oxidative stress; and over expression of metallothionein reduces the sensitivity of cells and tissues to free radical-induced injury. In this study, we have shown that the i.c.v. administration of ZnSO4 increases the synthesis of metallothionein I mRNA and metallothionein II mRNA. In addition, the i.c.v. administration of ZnSO4 enhances the concentration of zinc and in direct proportion the synthesis of metallothionein mRNAs. Agents known to generate free radicals and to cause oxidative stress such as 6-hydroxydopamine, iron, hydrogen peroxide, and various alcohols lead to induction of metallothionein in the hippocampal neurons in primary culture and in Chang liver cells in culture. In view of the fact that zinc and 6-hydroxydopamine induce the level of brain metallothionein and its mRNAs and zinc and metallothionein concentrations vary in different regions of the brain, it is postulated that metallothionein may play a major role in nullifying the iron-mediated generation of free radicals and in protecting against oxidative stress in the brain.

Intravenous meperidine during labor: a randomized comparison between nursing- and patient-controlled administration.

Preliminary reports about patient-controlled analgesia during labor have been promising. The purpose of this investigation was to compare our experience with meperidine given intravenously by the patient versus by a nurse. Sixty-four healthy women beginning active labor (cervical dilation 3 cm) at term were randomly assigned to either self-administer a 10-mg dose as often as every 20 minutes or have a nurse administer 25-50 mg every 3 hours as requested. The total meperidine dose and consumption rates were greater when administered by the patient than by a nurse. Maternal side effects occurred with similar frequency in both groups, and pain relief was judged to be equivalent. Maternal and umbilical serum concentrations of meperidine at delivery increased in the patient-controlled group if active labor lasted longer than 2 hours. Neonatal naloxone therapy was used more often when meperidine was administered by the patient than by a nurse (five of 31, 16%, versus three of 33, 10%, respectively). Self-administration of intravenous meperidine by the laboring patient was not found to be advantageous over nursing administration, and may pose an increased threat to the infant.

Synaptic density in the arcuate nucleus of female rats approaching middle age.

Synaptic number and synapses/neuronal cell membrane were evaluated from ultrastructural micrographs of the arcuate nucleus from 90-, 120-, 180-, and 240-day female rats grouped into ovary-intact or ovariectomized animals treated with peanut oil vehicle or estradiol benzoate (10 microg/100 g body weight) for 3 days. In ovary-intact rats, synaptic density was significantly less in middle aged 240 day animals than in 90-, 120-, or 180-day animals with greatest decrease occurring between 180- and 240-day animals. Ovary-intact and ovariectomized animals treated with estradiol benzoate had significantly higher sera estradiol levels, but the estradiol was ineffective in increasing synaptic density in the middle aged animals. Logistical regression confirmed a correlation between a decrease in synapses and increasing age but not estradiol treatment.

Evaluation of platelet function in pregnancy. Comparative studies in non-smokers and smokers.

We have evaluated the possible role of platelet functional abnormalities as a contributory cause of thrombosis during pregnancy and to the increased fetal mortality and morbidity among women who smoke. Fifty-three pregnant women were enrolled and evaluated on two separate prenatal visits held between the 20th and 36th week of pregnancy and, when possible, post partum. Smoking status was evaluated by personal statement and alveolar carbon monoxide levels. Women in the smoking group deliberately avoided cigarettes for at least 20 minutes before sampling. Plasma levels of beta-thromboglobulin, thromboxane B2, and 6-Keto PGF1 alpha were evaluated. A significant increase in 6-Keto PGF1 alpha was noted among smoking women as pregnancy advanced. 6-Keto PGF1 alpha levels decreased among non-smoking women while beta-thromboglobulin increased significantly between the 20th and 33rd week of pregnancy in non-smokers. Platelet aggregation, both in platelet rich plasma and in whole blood (by impedance aggregometry), was evaluated by five different parameters and four different aggregating agents. Significant differences between the non-smoking and smoking pregnant women were noted for selected age cohorts and aggregating agents. An increase in platelet reactivity among smokers was observed in whole blood by impedance aggregometry with adenosine diphosphate and in two age cohorts using platelet rich plasma. In two groups in which aggregation was significantly accelerated among non-smokers, epinephrine was used as the aggregating agent.

Development of secondary sex characteristics in male rats after fetal and perinatal cimetidine exposure.

Cimetidine has been reported to cause antiandrogenic effects in male pups of female rats receiving cimetidine during gestation. Because of conflicting reports of cimetidine causing permanent antiandrogenic effects in male rats, we studied the sexual development of male rats born to females receiving cimetidine. Water or water and cimetidine (194 mg/kg of body weight per day) were administered to pregnant rats from day 12 of gestation through weaning. A total of 130 male pups were studied. Testicular prostate gland/seminal vesicle weights, anogenital distance, serum testosterone and dihydrotestosterone levels, and seminiferous tubule areas were compared between the two groups. Transfer of cimetidine across the placenta and though breast milk was confirmed by HPLC analysis of serum from female littermates at 0, 10, and 20 days of age. With the exception of a smaller anogenital distance (p < 0.03) and a lower anogenital index (p < 0.05) in the cimetidine-exposed newborn rats, no statistically significant differences were observed in the measured parameters between the cimetidine-exposed and control groups. Cimetidine exposure during the fetal and perinatal periods did not alter the development of secondary sex characteristics in male rat pups.

Effects of asphyxia on telencephalic microvessels of premature beagle pups.

The authors identified alterations in the brain microvasculature of the premature Beagle pup that could be attributed to asphyxia and potentially associated with the etiology of intraventricular hemorrhage (IVH). Beagle pups were delivered by cesarean section six days before their predicted whelping date and exposed to an alternating protocol of asphyxial episodes known to produce IVH. Data from the telencephalic microvasculature at both the germinal matrix (an area susceptible to IVH) and the borderzone cerebral cortex (an area rarely involved in hemorrhage) were analyzed. The germinal matrix microvessels responded differently to asphyxia than the cortical vessels. The germinal matrix microvessels maintained junctional complexes and intraluminal microvilli, and increased in cross-sectional luminal area following asphyxia; however, total vessel cross-sectional area did not change. After the asphyxial insult, cortical microvessels no longer exhibited intraluminal microvilli and increased in both total cross-sectional area and luminal area. The number of junctional complexes in cross-sections of cortical vessels was significantly reduced due at least in part to the narrowing of the endothelial cell lateral borders. The data provide indirect evidence to substantiate vasodilation speculated to be due to an increased cerebral blood flow to telencephalic vessels (both the germinal matrix and cerebral cortex) following asphyxia. The data also suggest differential rates of maturation between germinal matrix and cortical microvessels.

Predicting outcome of care in the neonatal intensive care unit.

The accuracy of a neonatal intensive care unit (NICU) staff in predicting the outcome and length of stay of infants admitted to the NICU, and the factors they felt were important in making these predictions, were evaluated. This prospective study used a questionnaire to survey 44 nurses, residents, and attending neonatologists working in the NICU about the predicted outcomes of 52 infants admitted to the NICU at the University of Nebraska Medical Center over a 1-month period. Factors previously identified by the staff as important indicators of infant outcome were assigned points of importance by the respondents, and specific values for these factors were recorded for each infant. The NICU staff indicated that gestational age was the most important indicator of infant outcome. The attending neonatologists placed more value on gestational age (analysis of variance [ANOVA] P less than .0001) than did the nurses or residents. Among the staff groups, there were significant differences in the weight or points assigned to a given factor for pulmonary function, renal function, number of resuscitations, and the "other" category (ANOVA P less than .05). The nurses ranked pulmonary function criteria lower than the residents did (P less than .04), but considered renal function more important than either the residents (P = .005) or the attending neonatologists considered it to be (P = .01). The number of resuscitations was ranked higher by the nursing staff than by other groups, whereas the attending neonatologists assigned less importance to the "other" category.(ABSTRACT TRUNCATED AT 250 WORDS)

Metallothionein induction in rat hippocampal neurons in primary culture.

Primary cultures of hippocampal neurons are important for the study of trace elements in epileptogenesis. We developed a model system for culturing hippocampal neurons on poly-L-lysine in Iscove's modification of Dulbecco's MEM (IMDM) supplemented with K+, D-glucose, glutamine, insulin, p-amino benzoic acid, transferrin, BSA, beta-estradiol, gentamycin, and fungizone. Neurons were identified by histochemical staining for cholinesterase. Zinc at concentrations of 10(-9) to 10(-6) M induced metallothionein in hippocampal neuronal cultures. Maximum metallothionein induction occurred after 48 hrs incubation with zinc.

Telencephalic microvessels of premature beagle pups.

The ultrastructural morphologic and morphometric characteristics of the telencephalic microvasculature of both germinal matrix and border-zone cerebral cortex are analyzed from premature beagle puppies delivered by caesarean section in order to determine whether differences in the matrical vessels could account for their susceptibility to intraventricular hemorrhage. The germinal matrix microvessels are immature, since they contain intraluminal microvilli and junctional complexes (primarily tight junctions) that are not continuous between the lateral endothelial cell walls. The number of junctional complexes per vessel cross-section is less than either cortical vessels from the same premature animals or tissue from either site in full-term animals. Morphometric data on the cross-sectional area of vessels from the two sites indicate that the germinal matrix vessels have a significantly larger cross-sectional area than the cortical vessels. The increase is due almost exclusively to a larger endothelial cell area per cross section. This pattern is an altogether different one from that seen in the term animals. There the germinal matrix vessels exhibit a steadily decreasing luminal area from birth through 72 hr, whereas endothelial cell cross-sectional area is static. Here the luminal cross-sectional area is smaller, but both endothelial cell and total vessel cross-sectional areas are significantly larger. Structural components of the blood-brain barrier undergo active modifications during the late prenatal period that are distinct from the modifications during the immediate postnatal period.

Role of calcium in acute stimulated release of prolactin from neoplastic GH3 cells.

Using a calcium-sensitive electrode to monitor calcium movements, we found that neoplastic GH3 cells experienced a net accumulation of calcium when exposed to elevated (50 mM) K+. Acute prolactin (PRL) release was also stimulated under these conditions. Both calcium uptake and PRL release could be blocked by the calcium antagonist methoxyverapamil (D-600). Thyrotropin-releasing hormone (TRH) also stimulated PRL release but had no effect on cellular calcium accumulation. Likewise, D-600 had no effect on TRH-induced PRL release. Such results indicate that enhanced secretory activity does not require an increase in intracellular calcium content. The observation that secretagogues do not stimulate PRL release in the absence of extracellular calcium was investigated. When GH3 cells were placed in a Ca-free medium, they underwent a prompt and sustained loss of cellular calcium. The loss of such intracellular calcium could be blocked with D-600. We conclude that the inability of TRH to stimulate the release of PRL in Ca-free medium is due to the loss of intracellular calcium and not to the absence of external calcium per se.

Vasoconstriction in telencephalic microvessels: a response to one model for intraventricular hemorrhage in beagle pups.

Telencephalic microvessels were morphologically and morphometrically evaluated in beagle pups at 24, 48, and 72 hours of age following an experimental insult consisting of exposure to acute hypovolemic hypotension followed by rapid corrective blood reinfusion. Animals were anesthetized throughout the procedure and maintained for 75 minutes prior to sacrifice. Microvessels from the germinal matrix, where intraventricular hemorrhage (IVH) most commonly occurs, were compared to those of the adjacent cerebral cortex and choroid plexus. Perfusion, fixation and processing of the tissue followed our previously published protocol (Leuschen et al.: Anatomical Record 208:435-443, 1984). Both germinal matrix and cortical microvessels had collapsed lumens and normal endothelial cell morphology; mitochondria contained distinct cristae, junctional complexes were present and a basement lamina completely surrounded most vessels. Morphometric data on matrical and cortical microvessels supported vasoconstriction. Microvessels from choroid plexus were not vasoconstricted; tight junctions were infrequently seen and microvesicular elements were present. An increased cerebral blood flow to the germinal matrix and the adjacent cerebral cortex has been reported during and immediately following hypovolemic hypotensive insult. By 75 minutes following insult, our ultrastructural studies support a rebound phenomena including vasoconstriction of microvessels. The resulting vasoconstriction and associated cerebral ischemia may play an important role in subsequent tissue damage to the matrical area and ultimately to the etiology of IVH.

PhDs in pediatric departments.

A survey on PhDs in academic pediatric departments was sent to members of the Association of Medical School Pediatric Department Chairmen (70% responded). Significant numbers (14.5%) of pediatric faculty members hold PhDs. Another 3.6% hold PhDs/MDs. Research is their major responsibility in every subspecialty except psychology. The majority of PhDs (51%) are assistant professors (8% tenured) in contrast to 37% of MD faculty (14% tenured). The majority of PhDs at full professor (56%) are tenured compared with only 44% of MDs. Separate criteria exist for promotion for PhDs in 42% of departments. For promotion to associate professor, the mean number of refereed publications is 18 (median, 15); for full professor, the number ranges from ten to 50 (median, 40). Extramural research funding carries the highest weight in evaluating research efforts. PhDs could impact significantly on long-range planning in academic pediatric departments.