Fructose-1,6-bisphosphate protects astrocytes from hypoxic damage.

To determine the effects of glucose and fructose-1,6-bisphosphate (FDP) on hypoxic cell damage, primary cultures of astrocytes were incubated for 18 h in an air-tight chamber that had been flushed with 95% N2/5% CO2 for 15 min before it was sealed. Cultures containing 7.5 mM glucose without FDP or FDP without glucose showed evidence of significant cell injury after 18 h of hypoxia (increased lactate dehydrogenase content in the culture medium; cell edema and disruption by phase-contrast microscopy). Cultures exposed to glucose + FDP had normal lactate dehydrogenase concentrations and appeared normal microscopically. Maximal protection of hypoxic cells occurred at 6.0 mM FDP. Lactate concentrations of the culture medium of hypoxic cells increased 2.5 times above normoxic control values when glucose was present, but neither FDP alone nor glucose + FDP caused the lactate concentrations to increase further. This implies that anaerobic glycolysis was not increased by adding FDP to the medium. Cell volumes (water space) measured with [14C]-3-0-methyl-D-glucose were normal with glucose + FDP in the culture medium of hypoxic cells but were significantly larger than normal when glucose alone was present. Increases in cell volume paralleled changes in lactate dehydrogenase in the culture medium. Uptake of [14C]FDP occurred rapidly in normoxic cells and was maximal after 5 min of incubation. The data indicate that the presence of glucose + FDP in the culture medium protects primary cultures of hypoxic astrocytes from cell damage.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypoxia-induced dysfunctions and injury of astrocytes in primary cell cultures.

The effects of severe hypoxia were studied in a primary culture of astrocytes prepared from newborn rat cerebral cortex. Hypoxia was created by placing cultures in an airtight chamber that was flushed with 95% N2/5% CO2 for 15 min before being sealed. The hypoxic environment was maintained constant for up to 24 h. During the first 12 h of hypoxia, astrocytes showed no morphological changes by phase-contrast microscopy. After 18 h of hypoxia, some astrocytes in culture became swollen and started to detach from the culture dish. All cells in the culture were destroyed after 24 h of hypoxia. The lactate dehydrogenase level in the culture medium increased more than tenfold between 12 and 24 h of hypoxia. Glutamate uptake was inhibited 80% by similar hypoxic conditions. The cell volume of astrocytes, as measured by 3-O-methyl-[14C]-D-glucose uptake, was increased. These observations suggested cell membrane dysfunction. The malondialdehyde level of hypoxic cultures increased two-fold after 24 h of hypoxia. Verapamil (0.5 mM), furosemide (1 mM), indomethacin (1 mM), MgCl2 (10 mM), and mannitol (10 mM) reduced but never completely abolished the release of lactate dehydrogenase from hypoxic astrocytes. These data suggest multifactorial causes for severe injury in hypoxic astrocytes.

Effects of hypoxic hypoxia on cerebral phosphate metabolites and pH in the anesthetized infant rabbit.

The effects of hypoxic hypoxia on high-energy phosphate metabolites and intracellular pH (pHi) in the brain of the anesthetized infant rabbit were studied in vivo using 31P nuclear magnetic resonance spectroscopy. Five 10- to 16-day-old rabbits were anesthetized with 1.5% halothane. Ventilation was controlled to maintain normocarbia. Inspired O2 fraction was adjusted to produce three states of arterial oxygenation: hyperoxia (PaO2 greater than 250 mm Hg), normoxia (PaO2 approximately 100 mm Hg), and hypoxia (PaO2 25-30 mm Hg). During hypoxia, blood pressure was kept within 20% of control values with a venous infusion of epinephrine. During hyperoxia, the phosphocreatine-to-ATP ratio was 0.86, a value that is 2-2.5 times less than that reported for adults. During normoxia, ATP decreased by 20% and Pi increased by 90% from hyperoxia values. During 60 min of hypoxia, the concentrations of high-energy phosphate metabolites did not change, but intracellular and arterial blood pH (pHa) decreased significantly. When hyperoxia was reestablished, pHi returned to normal and pHa remained low. These results suggest that during periods of hypoxemia, the normotensive infant rabbit maintains intracellular concentrations of cerebral high-energy phosphates better than has been reported for adult animals.

Comparison of noninvasive and direct measurements of intracranial pressure.

After creating "fontanelles" in infant rhesus monkeys, we measured intracranial pressure of these monkeys directly with an indwelling cannula and indirectly with a Ladd 1700 sensor. Values obtained with the Ladd sensor placed over the shaven fontanelle closely approximate directly measured pressure in acute experiments in these animals and in human cadavers. Application of the device is simple and application pressure need not always be the same for multiple determinations. The response time is 1.6 cm H2O/sec, which is adequate to record changes in intracranial pressure caused by changes in the partial pressure of CO2 in arterial blood.

Pneumothorax in the respiratory distress syndrome: incidence and effect on vital signs, blood gases, and pH.

We determined the incidence of pneumothorax in 295 infants (mean birthweight, 1,917 gm) with the respiratory distress syndrome (RDS) treated according to the same protocol. Fifty-five infants (mean birthweight, 1,594 gm) developed pneumothorax (incidence, 19%); incidence varied with severity of RDS and intensity of respiratory assistance. Pneumothorax occurred in 3.5% (2 of 58) of infants who received no assisted ventilation and in 11% (14 of 124) of infants who received continuous positive airway pressure (CPAP) as the only form of assisted ventilation; the difference between these two groups is not significant. Forty-nine infants initially treated with CPAP later required mechanical ventilation with positive end-expiratory pressure (PEEP). Pneumothorax occurred in 12 of the 49 (24%) and in 21 of 64 (33%) of those infants initially treated with PEEP; the incidence of pneumothorax for both these groups was significantly higher than for those treated with no assisted ventilation or CPAP only. To assess the value of frequent measurement of vital signs, blood gas tensions, and pH in the recognition of pneumothorax, we analyzed these variables by the cumulative sum statistical technique. We noted the following significant changes associated with pneumothorax: arterial blood pressure, heart rate, and respiratory rate decreased in 77% of cases; pulse pressure narrowed in 51% of cases; Po2 decreased in 17 of 20 cases in which ventilatory settings were constant for at least three hours prior to pneumothorax. However, pH and Pco2 showed consistent changes. Frequent measurements of vital signs and Po2 aid in the early diagnosis of pneumothorax.

Cardiorespiratory status of erythroblastotic newborn infants: III. Intravascular pressures during the first hours of life.

We measured aortic and central venous pressures beginning soon after birth in 40 prematurely born infants with moderate or severe erythroblastosis fetalis, including 13 with severe and 10 with mild hydrops fetalis. All but four were asphyxiated at birth and this affected intravascular pressures. Before resuscitation, aortic or central venous pressure or both were elevated in more than one third. All but two of the remaining infants had normal initial pressures. Following resuscitation which relieved acidosis, hypoxia, and anemia, but did not reduce blood acidosis, hypoxia, and anemia, but did not reduce blood volume, the high pressures usually fell to normal and occasionally to subnormal levels, normal pressures fell to subnormal in almost one half, and those with initial subnormal pressures remained hypotensive. In all, 40% were hypotensive after resuscitation; treatment with blood volume expanders consistently returned these pressures to normal. Only two of the 13 severely hydropic infants and none of the mildly hydropic had findings indicative of hypervolemia and myocardial failure which persisted after treatment of asphyxia.

Mean airway pressure and mean alveolar pressure during high-frequency jet ventilation in rabbits.

Mean airway pressure underestimates mean alveolar pressure during high-frequency oscillatory ventilation. We hypothesized that high inspiratory flows characteristic of high-frequency jet ventilation may generate greater inspiratory than expiratory pressure losses in the airways, thereby causing mean airway pressure to overestimate, rather than underestimate, mean alveolar pressure. To test this hypothesis, we ventilated anesthetized paralyzed rabbits with a jet ventilator at frequencies of 5, 10, and 15 Hz, constant inspiratory-to-expiratory time ratio of 0.5 and mean airway pressures of 5 and 10 cmH2O. We measured mean total airway pressure in the trachea with a modified Pitot probe, and we estimated mean alveolar pressure as the mean pressure corresponding in the static pressure-volume relationship to the mean volume of the respiratory system measured with a jacket plethysmograph. We found that mean airway pressure was similar to mean alveolar pressure at frequencies of 5 and 10 Hz but overestimated it by 1.1 and 1.4 cmH2O at mean airway pressures of 5 and 10 cmH2O, respectively, when frequency was increased to 15 Hz. We attribute this finding primarily to the combined effect of nonlinear pressure frictional losses in the airways and higher inspiratory than expiratory flows. Despite the nonlinearity of the pressure-flow relationship, inspiratory and expiratory net pressure losses decreased with respect to mean inspiratory and expiratory flows at the higher rates, suggesting rate dependence of flow distribution. Redistribution of tidal volume to a shunt airway compliance is thought to occur at high frequencies.(ABSTRACT TRUNCATED AT 250 WORDS)

Pulmonary mechanics during rapid mechanical ventilation in rabbits with saline-lavaged lungs.

Infants with respiratory failure are frequently mechanically ventilated at rates exceeding 60 breaths/min. We analyzed the effect of ventilatory rates of 30, 60, and 90 breaths/min (inspiratory times of 0.6, 0.3, and 0.2 s, respectively) on the pressure-flow relationships of the lungs of anesthetized paralyzed rabbits after saline lavage. Tidal volume and functional residual capacity were maintained constant. We computed effective inspiratory and expiratory resistance and compliance of the lungs by dividing changes in transpulmonary pressure into resistive and elastic components with a multiple linear regression. We found that mean pulmonary resistance was lower at higher ventilatory rates, while pulmonary compliance was independent of ventilatory rate. The transpulmonary pressure developed by the ventilator during inspiration approximated a linear ramp. Gas flow became constant and the pressure-volume relationship linear during the last portion of inspiration. Even at a ventilatory rate of 90 breaths/min, 28-56% of the tidal volume was delivered with a constant inspiratory flow. Our findings are consistent with the model of Bates et al. (J. Appl. Physiol. 58: 1840-1848, 1985), wherein the distribution of gas flow within the lungs depends predominantly on resistive factors while inspiratory flow is increasing, and on elastic factors while inspiratory flow is constant. This dynamic behavior of the surfactant-depleted lungs suggests that, even with very short inspiratory times, distribution of gas flow within the lungs is in large part determined by elastic factors. Unless the inspiratory time is further shortened, gas flow may be directed to areas of increased resistance, resulting in hyperinflation and barotrauma.

Activation of the neuroprotective ERK signaling pathway by fructose-1,6-bisphosphate during hypoxia involves intracellular Ca2+ and phospholipase C.

The mechanism of the neuroprotective action of the glycolytic pathway intermediate fructose-1,6-bisphosphate (FBP) may involve activation of a phospholipase-C (PLC) dependent MAP kinase signaling pathway. In this study, we determined whether FBP's capacity to decrease delayed cell death in hippocampal slice cultures is dependent on PLC signaling or activation of the intracellular Ca(2+)-MEK/ERK neuroprotective signaling cascade. FBP (3.5 mM) reduced delayed death from oxygen/glucose deprivation in CA1, CA3 and dentate neurons in slice cultures. The phospholipase-C inhibitor U73122 and the MEK1/2 inhibitor U0126 prevented this protection. In hippocampal and cortical neurons, FBP increased phospho-ERK1/2 (p42/44) immunostaining during hypoxic, but not normoxic conditions. Increased phospho-ERK immunostaining was dependent on PLC and also on MEK 1/2, an upstream regulator of ERK. Further, we found that FBP enhancement of phospho-ERK immunostaining depended on [Ca(2+)](i): PLC inhibition and the IP(3) receptor blocker xestospongin C prevented FBP from increasing [Ca(2+)](i) and increasing phospho-ERK levels. However, while FBP-induced increases in [Ca(2+)](i) were blocked by xestospongin and a PLC inhibitor, [Ca(2+)](i) increases induced by the neuroprotective growth factor BDNF were not prevented. We conclude that during hypoxia FBP initiates a series of neuroprotective signals which include PLC activation, small increases in [Ca(2+)](i), and increased activity of the MEK/ERK signaling pathway.

Neuroprotection and intracellular Ca2+ modulation with fructose-1,6-bisphosphate during in vitro hypoxia-ischemia involves phospholipase C-dependent signaling.

The neuroprotectant fructose-1,6-bisphosphate (FBP) preserves cellular [ATP] and prevents catastrophic increases in [Ca2+]i during hypoxia. Because FBP does not enter neurons or glia, the mechanism of protection is not clear. In this study, we show that FBP's capacity to protect neurons and stabilize [Ca2+]i during hypoxia derives from signaling by a phospholipase-C-intracellular Ca2+-protein kinases pathway, rather than Ca2+ chelation or glutamate receptor inhibition. FBP reduced [Ca2+]i changes in hypoxic hippocampal neurons, regardless of [Ca2+]e, and preserved cellular integrity as measured by trypan blue or propidium iodide exclusion and [ATP]. FBP also prevented hypoxia-induced increases in [Ca2+]i when glucose was absent and when [Ca2+]e was increased to negate Ca2+ chelation by FBP. These protective effects were observed equally in postnatal day 2 (P2) and P16 neurons. Inhibiting glycolysis with iodoacetate eliminated the protective effects of FBP in P16 neurons. FBP did not alter Ca2+ influx stimulated by brief applications of NMDA or glutamate during normoxia or hypoxia, but did reduce the increase in [Ca2+]i produced by 10 min of glutamate exposure during hypoxia. Because FBP increases basal [Ca2+]i and stimulates membrane lipid hydrolysis, we tested whether FBP's protective action was dependent on phospholipase C signaling. The phospholipase C inhibitor U73122 prevented FBP-induced increases in [Ca2+]i and eliminated FBP's ability to stabilize [Ca2+]i and increase survival during anoxia. Similarly, FBP's protection was eliminated in the presence of the mitogen/extracellular signal protein kinase (MEK) inhibitor U0126. We conclude that FBP may produce neuroprotection via activation of neuroprotective signaling pathways that modulate Ca2+ homeostasis.

Fructose-1,6-bisphosphate reduces ATP loss from hypoxic astrocytes.

Hypoxia caused injury and metabolic dysfunction of astrocytes, as indicated by a time-dependent loss of lactate dehydrogenase (LDH) activity and ATP content. The combination of 3.5 mM fructose-1,6-bisphosphate (FBP) and 7.5 mM glucose (GLC) reduced the decrease of ATP and prevented the loss of LDH. These data indicate that the combination of GLC + FBP protects astrocytes from hypoxia. The results also suggest that the maintainance of ATP concentration is the mechanism by which FBP prevents hypoxic injury.

Protection of astrocytes by fructose 1,6-bisphosphate and citrate ameliorates neuronal injury under hypoxic conditions.

Fructose 1,6-bisphosphate (FBP) protects astrocytes from hypoxic injury in vitro. To determine whether FBP and citrate (inhibitors of phosphofructokinase) ameliorate hypoxia-induced injury to neurons and, if they do, whether the protective effects are a direct result of their actions on neurons or a consequence of their actions on astrocytes, we added FBP or citrate to the media of normoxic and hypoxic 'pure', mixed and co-culture systems. FBP (3.5 mM) and citrate (10 microM-2 mM) decreased release of LDH from astrocytes following 24 h of hypoxia. Eight hours of hypoxia killed pure neuronal cultures and neither FBP nor citrate prevented this death. However, in mixed and co-culture systems, FBP and citrate increased neuronal viability (as determined by the ratio of live-to-total cells), even after 47 h of hypoxia. In co-culture, following 24 h of hypoxia, both FBP and citrate reduced neuronal release of LDH and neuronal death. Fluorocitrate, a suicidal-inhibitor of aconitase, also protected astrocytes, but not neurons, from hypoxia in 'pure' culture, presumably by increasing intracellular citrate concentrations through inhibition of the catalysis of citrate to isocitrate We conclude that FBP and citrate attenuate hypoxic neuronal injury through their effects on astrocytes.

Fructose-1,6-bisphosphate after hypoxic ischemic injury is protective to the neonatal rat brain.

Fructose-1,6-bisphosphate (FBP) has been shown to attenuate central nervous system injury in adult animals. We evaluated whether FBP given after an ischemic-hypoxic insult is protective to the developing brain in a neonatal rat model of hypoxia-ischemia. Postnatal day 7 rat pups were subjected to focal ischemia followed by global hypoxia and then administered either FBP or saline intraperitoneally. A dose of 500 mg/kg or greater of FBP significantly reduced the amount of injury such that 55% of FBP- vs. 17% of saline-treated rats had no injury; 6% of FBP- and 47% of saline-treated rats had severe damage (P = 0.004). There was less infarcted brain in FBP-treated rats (12 +/- 11% vs. 37 +/- 32%; P = 0.005); and fewer FBP-treated rats had > 30% ipsilateral cortical injury (12% of FBP- vs. 50% of saline-treated rats; P = 0.002). FBP lowered serum calcium levels during the first 24 h after the insult without significant changes in ionized calcium or osmolarity. These results indicate that FBP treatment administered systemically after hypoxia-ischemia reduces CNS injury in neonatal rats.

Effects of neuroprotective dose of fructose-1,6-bisphosphate on hypoxia-induced expression of c-fos and hsp70 mRNA in neonatal rat cerebrocortical slices.

In situ hybridization (ISH) measurements of c-fos and hsp70 expression were made in brain slice studies of hypoxia, with or without fructose-1,6-bisphosphate (FBP) pretreatment. Each experiment used eighty 350 microns thick cerebrocortical slices, obtained from twenty 7-day old rats. Thirty minute periods of hypoxia were followed by 8 h of hyperoxic perfusion. Slices were removed at eight predetermined times, and processed for ISH and immunohistochemistry. In three of six hypoxia experiments, slices were pretreated for 60 min with 2 mM FBP, a condition known to maintain ATP level in brain slices during hypoxia. In three other hypoxia experiments slices received no pretreatment. In two control experiments slices were perfused for 11.5 h without hypoxia. In control experiments, hsp70 mRNA was barely detectable in slices at all times, although moderate c-fos mRNA expression occurred at 1 h after decapitation. Hypoxia produced a modest but statistically significant increase in c-fos mRNA and hsp70 mRNA induction 4 h following reoxygenation. At all times after hypoxia, FBP pretreatment reduced expression of c-fos and hsp70 mRNA. The absence of hsp70 mRNA in control slices suggests that intracellular protein denaturation was minimal in this preparation. In slices made hypoxic, the decrease in c-fos and hsp70 mRNA caused by FBP pretreatment suggests ameliorated progression towards injury. Immunohistochemistry showed no HSP70 protein at any time following hypoxia, with or without FBP pretreatment, presumably due to delayed HSP70 protein synthesis, or to a block in translation, as observed in vivo in other studies.

Response of cerebral endothelial cells to hypoxia: modification by fructose-1,6-bisphosphate but not glutamate receptor antagonists.

Damage to the cerebral endothelium from ischemia could exacerbate brain injury by altering vascular integrity, but little is known concerning the response of cerebral endothelial cells to hypoxia. To address this issue, cerebral capillary endothelial cells were isolated from 14-day-old rats, grown to confluence, and placed in hypoxic chambers for up to 62 h. Cells were undamaged by 24 hours of hypoxia as assessed by lactate dehydrogenase release and ethidium bromide staining, but 48 h resulted in marked damage. Hypoxia was probably exacerbated by hypoglycemia because glucose levels fell to < 1 mM by 24 h, at which point ATP levels began to fall in hypoxic cultures (3.25 +/- 1.48 nmol/mg protein; mean +/- S.D.) relative to normoxic cultures (9.52 +/- 1.41 nmol/mg protein). Cells treated with 4 mM fructose-1,6-bisphosphate (FBP) had significantly less damage at 48 h of hypoxia than controls. FBP had little effect on rate of glucose depletion from the media, but ATP depletion due to hypoxia was significantly less. Thus, the protective effect of FBP may be mediated by the ability of treated cells to maintain higher ATP levels. Unlike FBP, glutamate receptor antagonists including MK-801, NBQX, DNQX, and kynurenic acid were ineffective in ameliorating hypoxia-induced endothelial cell injury.

Plasma norepinephrine levels, vagal tone index, and flexor reflex threshold in premature neonates receiving intravenous morphine during the postoperative period: a pilot study.

OBJECTIVE: The goal of this study was to evaluate the effects of a single dose of intravenous morphine on postoperative pain in extremely premature neonates after thoracotomy. DESIGN: Descriptive correlational study. PATIENTS: Twenty-four critically ill mechanically ventilated premature neonates with a mean gestational age of 26.1 +/- 2.1 (SD) weeks and a postnatal age of 13.8 +/- 8.1 (SD) days. OUTCOME MEASURES: Plasma norepinephrine (NE) levels, vagal tone index (VTI), and flexor reflex threshold were measured preoperatively, immediately before, and 20 and 60 minutes after the administration of the first postoperative dose of morphine (0.1 mg/kg). RESULTS: One-way repeated-measures ANOVA revealed no significant change in plasma NE levels from baseline levels (df[2,32] = 2.40, p = 0.11). Pre- and postmorphine VTI values were significantly lower than preoperative values (df[3,60] = 6.04, p = 0.0012), but no significant differences were found between pre- and postmorphine VTI values. Neonates (n = 10) who had a flexor reflex response during the postoperative period demonstrated no significant differences in the force required to elicit a flexor reflex across the four measurements (df[3,27] = 0.76, p = 0.53); however, the flexor reflex responses were significantly less vigorous during the postoperative period than at baseline. CONCLUSIONS: Findings from this pilot study suggest that the dose of morphine commonly used to treat postoperative pain in premature neonates does not affect NE, VTI, and flexor reflex threshold values within 1 hour of administration.

Resection of distal tracheal stenosis in a baby with agenesis of the lung.

A newborn infant with agenesis of the left lung and critical distal tracheal stenosis required tracheal resection and reanastomosis. This case illustrates the combined use of flow-volume curves, radiology and endoscopy in the diagnosis and management of airway obstruction in infants. It also establishes the feasibility of complete tracheal reconstruction in small babies.

The demographics of inpatient pediatric anesthesia: implications for credentialing policy.

STUDY OBJECTIVE: To examine the demographics of inpatient anesthesia care for infants and children in a specific region to determine if there were sufficient numbers of procedures to permit credentialing to take place, as a first step in understanding the consequences of implementing credentialing policies based on caseload. DESIGN: Retrospective computerized review of discharge abstracts. SETTING: All hospitals in northern California. MEASUREMENTS AND MAIN RESULTS: Surgical procedures and date of surgery were linked to create "procedure-days." Each procedure-day counted as one anesthesia case. Annual hospital caseloads (procedure-days) were tabulated for three separate age subgroups under six years of age. The proximity of hospitals with smaller surgical volumes to those with larger volumes was determined. Of the 205 hospitals in the region, 162 had at least one procedure-day for children less than 6 years of age for a total of 14,435 procedure-days (anesthesia cases). For each of three age groups studied--0 to 6 months, 7 to 24 months, and 25 to 72 months--85%, 90%, and 81%, respectively, of hospitals had caseloads of 1 to 50 per year. When procedure days from all three age groups were totalled, 59% of hospitals had less than 20 cases per year and 72% of hospitals had less than 50 cases per year; 86% of hospitals had less than 100 cases per year. Of hospitals with less than 100 cases per year, 75% were within 50 miles of a hospital with more than 100 cases. CONCLUSIONS: Performance based credentialing for pediatric anesthesia based on caseload may be problematic for many hospitals due to the distribution of cases: a majority of hospitals care for a few children, and most children are cared for in a few hospitals.