Does phenobarbital improve the effectiveness of therapeutic hypothermia in infants with hypoxic-ischemic encephalopathy?

OBJECTIVE: To determine whether phenobarbital (PB) given before therapeutic hypothermia to infants with hypoxic-ischemic encephalopathy (HIE) augments the neuroprotective efficacy of hypothermia. STUDY DESIGN: Records of 68 asphyxiated infants of 36 weeks' gestation, who received hypothermia for moderate or severe HIE were reviewed. Some of these infants received PB prophylactically or for clinical seizures. All surviving infants had later brain magnetic resonance imaging (MRI). The composite primary outcome of neonatal death related to HIE with worsening multiorgan dysfunction despite maximal treatment, and the presence of post-hypothermia brain MRI abnormalities consistent with hypoxic-ischemic brain injury, were compared between the infants who received PB before initiation of hypothermia (PB group, n=36) and the infants who did not receive PB before or during hypothermia (No PB group, n=32). Forward logistic regression analysis determined which of the pre-hypothermia clinical and laboratory variables predict the primary outcome. RESULT: The two groups were similar for severity of asphyxia as assessed by Apgar scores, initial blood pH and base deficit, early neurologic examination, and presence of an intrapartum sentinel event. The composite primary outcome was more frequent in infants from the PB group (PB 78% versus No PB 44%, P=0.006, odds ratio 4.5, 95% confidence interval 1.6 to 12.8). Multivariate analysis identified only the PB receipt before initiation of hypothermia (P=0.002, odds ratio 9.5, 95% confidence interval 2.3 to 39.5), and placental abruption to be independently associated with a worse primary outcome. CONCLUSION: PB treatment before cooling did not improve the composite outcome of neonatal death or the presence of an abnormal post-hypothermia brain MRI, but the long-term outcomes have not yet been evaluated.

Effects of therapeutic hypothermia on multiorgan dysfunction in asphyxiated newborns: whole-body cooling versus selective head cooling.

OBJECTIVE: Multiorgan dysfunction in asphyxiated newborns receiving therapeutic hypothermia achieved by either selective head cooling (SHC) or whole-body cooling (WBC) has not been well characterized. The beneficial effect of SHC in organs other than the brain may potentially be limited because unlike WBC, SHC aims to achieve effective brain cooling with less-systemic hypothermia. However, the relative effects of SHC and WBC with currently available cooling protocols on multiorgan dysfunction are unknown.The aim of this study was to compare the multiorgan dysfunction in infants receiving therapeutic hypothermia induced by either SHC or WBC. STUDY DESIGN: In 59 asphyxiated newborns who received therapeutic hypothermia by either SHC (n=31) or WBC (n=28), the severity of pulmonary, hepatic and renal dysfunction and coagulopathy and electrolyte disturbances were assessed before the start of cooling (baseline), and at specific time intervals (24, 48 and 72 h) throughout cooling. Enrollment criteria, clinical monitoring and treatment during cooling, whether SHC or WBC, were similar, as reported earlier. RESULT: The presence of clinical respiratory distress, along with the need for ventilatory support for varying duration during cooling, was similar in both the WBC and SHC groups (100 vs 94%, P=0.49, OR 1.9, 95% CI 1.5-2.5). The use of fresh frozen plasma and platelet transfusion to treat coagulopathy and thrombocytopenia was similar (WBC 48% vs SHC 58%, P=0.59, OR 0.7, 95% CI 0.2-1.9, and WBC 41% vs SHC 32%, P=0.58, OR 1.4, 95% CI 0.5-4.2, respectively), and equivalent numbers of infants from both groups were treated with vasopressors for >24 h (WBC 59% vs SHC 55%, P=0.79, OR 1.2, 95% CI 0.4-3.4). The incidence of oliguria (urine output <0.5 ml kg(-1) h(-1) for >24 h after birth) and rising serum creatinine (with maximum serum creatinine >0.9 mg dl(-1)) was also similar (WBC 18% vs SHC 39%, P=0.15, OR 0.4, 95% CI 0.1-1.3, and WBC 48% vs SHC 58%, P=0.59, OR 0.7, 95% CI 0.2-1.9, respectively). Laboratory parameters to assess the differential effect of WBC versus SHC on multiorgan dysfunction during 72 h of cooling, which include serum transaminases (serum aspartate aminotransferase and alanine aminotransferase), prothrombin time, partial thromboplastin time, INR, platelet counts, serum creatinine, serum sodium, serum potassium and serum calcium, were similar between the groups at the initiation of cooling and did not differ with the method of cooling. CONCLUSION: Multiorgan system dysfunction in asphyxiated newborns during cooling remains similar for both cooling methods. Concerns regarding a differential effect of WBC versus SHC on multiorgan dysfunction, other than of the brain, should not be a consideration in selecting a method to produce therapeutic hypothermia.

Hydrocortisone administration for the treatment of refractory hypotension in critically ill newborns.

OBJECTIVE: The purpose of this observation was to evaluate the safety and efficacy of hydrocortisone (HC) for the treatment of refractory hypotension in term and preterm infants. A secondary purpose was to determine the utility of serum cortisol concentrations in predicting the response to treatment. STUDY DESIGN: This is a retrospective observational study of 117 infants treated with a standardized HC protocol for refractory hypotension. Refractory hypotension was defined as a mean arterial pressure (MAP) less than the gestational age (GA) despite a total inotrope dose of 20 microg per kg per min. Baseline serum cortisol concentrations were determined prior to treatment with stress dose HC. RESULT: Treatment with HC increased the MAP at 2, 6, 12 and 24 h after initiation, decreased the total inotrope dose at 6, 12 and 24 h, and was associated with resolution of oliguria. There was no correlation between the pretreatment baseline cortisol concentration and GA, birth weight or the response to treatment. The incidence of grades III to IV intraventricular hemorrhage, periventricular leukomalacia, bacterial or fungal sepsis and spontaneous intestinal perforation (SIP) after HC treatment was similar to institutional historic controls prior to institution of this standardized HC protocol. CONCLUSION: HC treatment was associated with a rapid resolution of cardiovascular compromise. The incidence of significant side effects was similar to that in previously published reports, including a comparable incidence of SIP. On the basis of our results, measuring baseline serum cortisol concentration to guide the management of refractory hypotension is unwarranted.

Should amplitude-integrated electroencephalography be used to identify infants suitable for hypothermic neuroprotection?

OBJECTIVE: Amplitude-integrated electroencephalography (aEEG) has been used adjunctively to identify infants suitable for hypothermic neuroprotection following severe intrapartum asphyxia. To determine whether an early aEEG predicts short-term adverse outcome in infants with significant hypoxic-ischemic encephalopathy (HIE) evaluated for hypothermic neuroprotection. STUDY DESIGN: The aEEG recordings were obtained within 6 h of birth in infants >or=36 weeks' gestational age during evaluation for possible selective head or whole-body cooling. Recordings were subsequently re-evaluated for both background pattern and voltage abnormalities by a certified reader masked to clinical history and brain-oriented interventions. All infants with moderate or severe HIE evaluated for hypothermic neuroprotection also underwent magnetic resonance imaging (MRI) of the brain at a median postnatal age of 7 days. The predictive value using the aEEG for determining short-term dichotomous outcomes, defined as early death related to HIE, or a characteristic pattern of abnormalities consistent with hypoxic-ischemic injury on the MRI brain scans was assessed. RESULT: Fifty-four infants with moderate or severe HIE were evaluated with aEEG for hypothermic neuroprotection; 34 infants received selective head cooling, 12 infants underwent total body cooling and 8 infants were not cooled. Outcome data, available for 46 of the 54 infants, revealed a poor correlation between the early aEEG and short-term adverse outcomes, with a sensitivity of 54.8% and negative predictive value (NPV) of only 44%. CONCLUSION: Because of the poor NPV of an early aEEG for a short-term adverse outcome, its use as an 'additional selection criterion' for hypothermic neuroprotection may not be appropriate.

Subventricular zone proliferation after alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated neonatal brain injury.

The mammalian forebrain subventricular zone (SVZ) contains stem cells capable of generating new neurons and glia. Recent studies indicate that acute brain injury is a potent stimulus for SVZ stem cell proliferation. To better understand mechanisms of the SVZ response to neonatal brain injury, we used a model that focuses on a unique mechanism of vulnerability of the immature CNS, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated excitotoxicity. We previously demonstrated that intracerebroventricular injection of the glutamate analog AMPA in rats at postnatal day 7 (P7) caused bilateral periventricular gray and white matter injury. We hypothesized that excitotoxic injury would stimulate cellular proliferation in the SVZ; we used the AMPA intracerebroventricular injection model to test this hypothesis. P7 rat pups received either left or right intracerebroventricular injections of S-AMPA (2.5 nmol). Normal and PBS-injected littermates were included as controls. On P8 or P14, serial coronal sections through the SVZ were collected; an immunohistochemical assay was performed with an antibody to the cell proliferation marker Ki-67. Bilateral Ki-67+ cells/SVZ were quantitated stereologically using the optical disector method. The median number of Ki-67+ cells/SVZ was increased in the SVZ of AMPA-injected rats relative to normal controls on both P8 and P14. To evaluate neurogenesis, we assayed the expression of doublecortin, a microtubular protein expressed only by immature neurons. From P8 to P14, there was a marked increase in doublecortin immunoreactive cells in the AMPA-injected SVZ. Many Ki-67+ nuclei were immediately surrounded by doublecortin staining. This study indicates that there is a proliferative response in the immature SVZ after an excitotoxic stimulus. Our findings suggest that some of these newly generated cells differentiate as immature neurons. This model may provide information about the mechanisms that regulate SVZ responses to neonatal brain injury.

AMPA-Induced suppression of oligodendroglial gene expression in neonatal rat brain.

Oligodendroglia are susceptible to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-mediated excitotoxicity. We hypothesized that analysis of oligodendroglial gene expression would provide a sensitive indicator of acute excitotoxic oligodendroglial injury in the immature brain. AMPA (2 nmol) was injected intracerebroventricularly in 7-day-old rats. Proteolipid protein and myelin basic protein encoding mRNAs were evaluated 24-48 h later by semi-quantitative reverse transcription-polymerase chain reaction assays. AMPA markedly suppressed brain tissue content of oligodendroglial mRNAs.

Behavioral/environmental intervention improves learning after cerebral hypoxia-ischemia in rats.

BACKGROUND AND PURPOSE: In premature infants, many of whom experience ischemic brain insults, the environment of rearing influences cognitive outcome. We developed a model to evaluate the effect of rearing conditions on learning after unilateral cerebral hypoxia-ischemia (HI) in 7-day-old (P7) rats. We hypothesized that neonatal handling would benefit rats recovering from an episode of HI. METHODS: Seventeen litters of P7 Long-Evans rats underwent either HI (right carotid ligation followed by 1.5 hours in 8% O(2)) or control procedures. From P8 to P14, randomized litters were either handled (15 minutes of separation from dam per day) or nonhandled. After P55, learning was tested in the Morris water maze. To evaluate injury severity, hippocampal, cortical, and striatal volumes were measured. RESULTS: In water-maze performance, ANCOVA revealed an interaction between handling and severity of hippocampal damage. Among HI rats, handled rats learned faster when hippocampal damage was moderate (P<0.01, repeated-measures ANOVA), with no benefit when damage was mild or severe. CONCLUSIONS: These observations suggest the beneficial cognitive effect of neonatal handling was limited to animals with moderate damage. Neonatal handling in post-HI rats may be a useful model in which to study mechanisms underlying the benefits of post-HI developmental intervention.

Platelet-activating factor antagonist BN 50730 attenuates hypoxic-ischemic brain injury in neonatal rats.

Platelet-activating factor (PAF) is a lipid derived from breakdown of cell membranes that is postulated to be a mediator of cerebral ischemic injury. PAF regulates CNS gene transcription via intracellular binding sites. To test the hypothesis that PAF mediates CNS injury in part by modulating gene transcription, we evaluated the neuroprotective efficacy of the drug BN 50730, an antagonist of the intracellular (microsomal) CNS PAF binding site, in the neonatal rat model of unilateral cerebral hypoxia-ischemia. Seven-day-old rats underwent right carotid ligation followed by a 2.5-h exposure to 8% O(2), and were then treated with BN 50730 (2.5 or 25 mg/kg per dose) or vehicle, at 0 and 2 h after the end of hypoxia. Ipsilateral cortical, striatal, and hippocampal damage was quantitated either 5 d later, or at 5 wk after the insult. Treatment with BN 50730 resulted in approximately 60- 80% reduction in ipsilateral tissue loss at both times. Learning and memory were evaluated 5 wk after insult using the Morris Watermaze place navigation task. Severity of cortical and striatal damage correlated significantly with learning and memory deficits. These results support the hypothesis that PAF is a pathogenetic mediator of hypoxic-ischemic damage in the immature brain. Accumulating evidence suggests that PAF mediates its deleterious effects in the immature CNS via multiple mechanisms.

Attenuation of hypoxia-ischemia-induced monocyte chemoattractant protein-1 expression in brain of neonatal mice deficient in interleukin-1 converting enzyme.

Interleukin-1beta (IL-1beta) upregulates expression of the chemokine monocyte chemoattractant protein-1 (MCP-1) in many experimental models. In neonatal rodent brain, hypoxia-ischemia rapidly stimulates expression of this chemokine, although the role of IL-1beta in regulating this response is unknown. Interleukin-1 converting enzyme (ICE) is a cysteine protease that cleaves inactive pro-IL-1beta to generate mature IL-1beta. Neonatal mice with a homozygous deletion of ICE (ICE -/-) are resistant to moderate, but not to severe cerebral hypoxic-ischemic insults, relative to their wild-type controls. We hypothesized that their resistance to moderate hypoxic-ischemic insults is mediated by suppression of the acute inflammatory response to brain injury in the absence of IL-1beta, and that hypoxia-ischemia induced MCP-1 expression would be attenuated in ICE -/- animals. To test this hypothesis, paired litters of 9-10-day-old ICE -/- and wild-type mice underwent right carotid ligation, followed by 40, 70 or 120 min exposure to 10% O2 and ischemia-induced changes in MCP-1 mRNA and protein were compared, using a semi-quantitative reverse-transcription polymerase chain reaction assay and an ELISA, respectively. With a lesioning protocol that elicits minimal injury in wild-types (ligation+40 min 10% O2), there was an attenuation of hypoxia-ischemia-induced MCP-1 production at 8 h post-hypoxia; in contrast, in animals that underwent longer periods of hypoxia-ischemia the magnitude of injury-induced induced MCP-1 production did not differ between wild-type and ICE -/- animals. These results demonstrate both that the acute inflammatory response to hypoxia-ischemia is attenuated in ICE -/- animals, and also that hypoxic-ischemic brain injury stimulates MCP-1 expression even in the absence of IL-1beta activity.

Hypoxic-ischemic injury results in acute disruption of myelin gene expression and death of oligodendroglial precursors in neonatal mice.

Studies of ischemic brain injury in neonatal rodents have focused upon the pathophysiology of neuronal damage. Much less consideration has been given to white matter injury, even though it is a major contributor to chronic neurological dysfunction in children. In the human neonate, particularly in those born prematurely, periventricular white matter is highly susceptible to hypoxic--ischemic (H--I) injury. To understand the basis for this selective vulnerability, we examined myelin gene expression and cell death in the subventricular layer and the surrounding white matter of neonatal mice following H--I insult. Using an in situ hybridization technique that gives high resolution and is very sensitive, we examined myelin basic protein and proteolipid protein gene expression three and twenty-four hours after a H-I insult. To elicit unilateral forebrain hypoxic and ischemic injury, 9--10-day-old mice underwent right carotid artery ligation followed by timed (40--70 min) exposure to 10% oxygen. Twenty-four hours following H--I, myelin basic protein and proteolipid protein transcripts were markedly reduced in striatum, external capsule, fornix, and corpus callosum in the injured side. Three hours after lesioning (ligation+70 min hypoxic exposure) myelin basic protein gene transcripts were visibly reduced in the ipsilateral white matter tracts. Interestingly, some cells in the subventricular layer expressed proteolipid protein transcripts, and 3 h after a H--I insult they were degenerating in the injured but not contralateral side. TUNEL staining showed an increase in the number of positive cells in the injured subventricular layer and corpus callosum but the adjacent striatum did not show a corresponding change in the number of TUNEL labeled cells. Ultrastructural studies of the subventricular zone and corpus callosum 3 h after H--I revealed that many subventricular cells, glial cells in the corpus callosum, and callosal axons in the injured side had already degenerated. However, the subventricular cells, glia and axons in the contralateral corpus callosum were spared. Many cells in the injured corpus callosum exhibited a apoptotic morphology; yet more mature oligodendrocytes in this region appeared normal. Our results show that a H--I insult causes a surprisingly swift and dramatic degenerative response in the subventricular layer and adjacent white matter. Within 3 h after H--I, the programmed cell death cascade was initiated; internucleosomal DNA degradation took place in subventricular and glial cells; oligodendrocyte progenitors died and axonal degeneration in the ipsilateral corpus callosum was extensive. The swiftness of the subventricular and glial cell degeneration suggests the H--I insult directly targets glia, as well as neurons, and raises the provocative question of whether glia exert damaging effects upon neurons and axons. Since the severity of the H--I insult can be modulated by varying the duration of hypoxia, the model is ideal to study whether oligodendrocyte progenitors are more susceptible to death than mature oligodendrocytes, whether mature oligodendrocytes de-differentiate and then are induced to remyelinate surviving axons, and/or whether oligodendrocyte progenitors in the subventricular layer can be stimulated to proliferate, migrate, and remyelinate the surviving axons.

Pentoxifylline attenuates hypoxic-ischemic brain injury in immature rats.

Inflammatory mediators are implicated in the pathogenesis of ischemic injury in immature brain. The phosphodiesterase inhibitor pentoxifylline inhibits production of tumor necrosis factor-alpha and platelet-activating factor. We hypothesized that pentoxifylline treatment would attenuate hypoxic-ischemic brain injury in immature rats. Seven-day-old rats (n = 79) underwent right carotid ligation, followed by hypoxia (FiO2 = 0.08). Rats received pentoxifylline immediately before and again after hypoxia (two doses, 25-150 mg/kg/dose, n = 34), or vehicle (n = 27). In separate experiments, rats received pentoxifylline treatment (40 mg/kg/dose, n = 8), or vehicle (n = 10) immediately and again 3 h after hypoxia-ischemia. Severity of injury was assessed 5 d later by visual evaluation of ipsilateral hemisphere infarction and by measurement of bilateral hemispheric cross-sectional areas. Pentoxifylline pretreatment reduced the incidence of liquefactive cerebral infarction, from 75% in controls to 10% with pentoxifylline, 40 mg/kg/dose (p<0.001, chi2 trend test). Quantification of hemispheric areas confirmed these findings. In contrast, posthypoxic-ischemic treatment with pentoxifylline resulted in only a modest reduction in cortical damage, without an overall reduction in incidence of infarction. Phosphodiesterase inhibition may be an effective strategy to use to decrease the severity of neonatal hypoxic-ischemic brain injury. Pretreatment regimens could be clinically relevant in settings in which an increased risk of cerebral ischemia can be anticipated, such as in infants undergoing surgery to correct congenital heart disease.

Mice deficient in interleukin-1 converting enzyme are resistant to neonatal hypoxic-ischemic brain damage.

Interleukin-1 (IL-1) converting enzyme (ICE) is a cysteine protease that cleaves inactive pro-IL-1beta to active IL-1beta. The pro-inflammatory cytokine IL-1beta is implicated as a mediator of hypoxic-ischemic (HI) brain injury, both in experimental models and in humans. ICE is a member of a family of ICE-like proteases (caspases) that mediate apoptotic cell death in diverse tissues. The authors hypothesized that in neonatal mice with a homozygous deletion of ICE (ICE-KO) the severity of brain injury elicited by a focal cerebral HI insult would be reduced, relative to wild-type mice. Paired litters of 9- to 10-day-old ICE-KO and wild-type mice underwent right carotid ligation, followed by 70 or 120 minutes of exposure to 10% O2. In this neonatal model of transient focal cerebral ischemia followed by reperfusion, the duration of hypoxia exposure determines the duration of cerebral ischemia and the severity of tissue damage. Outcome was evaluated 5 or 21 days after lesioning; severity of injury was quantified by morphometric estimation of bilateral cortical, striatal, and dorsal hippocampal volumes. In animals that underwent the moderate HI insult (70-minute hypoxia), damage was attenuated in ICE-KO mice, when evaluated at 5 or 21 days post-lesioning. In contrast, in mice that underwent the more severe HI insult (120-minute hypoxia), injury severity was the same in both groups. Reductions in intra-HI CBF, measured by laser Doppler flow-metry, and intra- and post-HI temperatures did not differ between groups. These results show that ICE activity contributes to the progression of neonatal HI brain injury in this model. Whether these deleterious effects are mediated by pro-inflammatory actions of IL-1beta and/or by pro-apoptotic mechanisms is an important question for future studies.

Tat, a human immunodeficiency virus-1-derived protein, augments excitotoxic hippocampal injury in neonatal rats.

To test the hypothesis that the human immunodeficiency virus-1-derived Tat protein may cause neuronal damage in the CNS, we evaluated the neurotoxicity of recombinant human immunodeficiency virus-1-derived Tat in vivo in seven-day-old rats. The intrinsic neurotoxicity of Tat (250 ng-1 microg) and the effects of direct intra-hippocampal co-infusion of Tat with N-methyl-D-aspartate were assessed. Extent of injury in the lesioned hippocampus was evaluated five days later, based on histopathology and morphometric measurements of hippocampal volume. To confirm that any observed neurotoxic effects were attributable to Tat bioactivity, all experiments included controls that received equal amounts of heat-treated (boiled) Tat. Intra-hippocampal injection of Tat, alone, elicited minimal focal tissue damage immediately adjacent to the injection track, and no hippocampal atrophy. Co-injection of Tat (500 ng) with N-methyl-D-aspartate (5 nmol, threshold excitotoxic dose) doubled the severity of hippocampal injury, quantified by comparison of bilateral hippocampal volumes, in comparison with animals that received heat-treated Tat or saline co-injections; in animals that received injections of N-methyl-D-aspartate (5 nmol) in combination with saline, heat-treated Tat, or Tat [mean(+/-S.E.M.) % volume loss values in the lesioned hippocampus were: 11(+/-3), 11(+/-3), and 26(+/-3), respectively (P<0.002, ANOVA)]. Co-injection of 100 ng Tat with 5 nmol N-methyl-D-aspartate exacerbated the severity of excitotoxic injury to a similar extent, whereas co-injection of 20 ng Tat had no effect on N-methyl-D-aspartate-mediated injury. Treatment with the N-methyl-D-aspartate antagonist 3-((RS)-2-carboxypiperazin4-yl)-propyl-1-phosphonic acid (20 mg/kg) markedly attenuated hippocampal injury resulting from co-injection of 100 ng Tat with N-methyl-D-aspartate [mean(+/-S.E.M.) % volume loss in lesioned hippocampus: 0.1(+/-2) in 3-((RS)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid-treated vs 19(+/-3) in controls, P<0.001, ANOVA]. Co-injection of Tat had no effect on N-methyl-D-aspartate-mediated striatal damage or on alpha-amino-3-hydroxy-5-methylisoxazole-4-pro hippocampal damage. These data support the hypothesis that locally released Tat could exert neurotoxic effects, mediated by N-methyl-D-aspartate receptor activation, in vivo in the immature brain.

Tumor necrosis factor-a attenuates N-methyl-D-aspartate-mediated neurotoxicity in neonatal rat hippocampus.

Tumor necrosis factor-a TNFa. has been implicated in the pathophysiology of acute neonatal brain injury. We hypothesized that acute brain injury would induce TNFa expression and that exogenous TNFa would influence the severity of N-methyl-D-aspartate-induced tissue damage. We performed two complementary groups of experiments to evaluate the potential role s. of TNFa in a neonatal rodent model of excitotoxic injury, elicited by intracerebral injection of N-methyl-D-aspartate. We used immunohistochemistry and ELISA to evaluate N-methyl-D-aspartate-induced changes in TNFa expression, and we co-injected TNFa with N-methyl-D-aspartate, to evaluate the effect of this cytokine on the severity of tissue injury. Both intra-hippocampal and intra-striatal injection of N-methyl-D-aspartate 5 nmol. stimulated TNFa expression. Increased TNFa expression was detected 3-12 h after lesioning; TNFa was localized both in glial cells in the corpus callosum, and in cells with the morphology of interneurons in the ipsilateral hippocampus, striatum, cortex and thalamus. Intra-hippocampal or intra-striatal administration of TNFa 50 ng. alone did not elicit neuropathologic damage. In the hippocampus, when co-injected with N-methyl-D-aspartate 5 or 10 nmol., TNFa 50 ng. attenuated excitotoxic injury by 35%-57%, compared to controls co-injected with heat-treated TNFa. In contrast, in the striatum, co-injection of TNFa with N-methyl-D-aspartate had no effect on the severity of the ensuing damage. The data indicate that TNFa is rapidly produced in glial cells and neurons after an excitotoxic insult in the neonatal rat brain, and that administration of exogenous TNFa results in region-specific attenuation of excitotoxic damage. We speculate that endogenous TNFa may modulate the tissue response to excitotoxic injury in the developing brain.

Improving the timing of antibiotic administration to high-risk newborns.

OBJECTIVES: To describe the timing of initiation of administration of parenteral antibiotics to infants with suspected sepsis at birth, identify barriers to prompt administration, and assess the effectiveness of subsequent interventions designed to minimize these barriers. The goals were to administer antibiotics within 1 hour of the physician order and within 2 hours of birth with more than 80% compliance for both goals. STUDY DESIGN: Retrospective chart review and prospective interventions involved 488 infants born at the University of Michigan Medical Center with indications for antibiotic therapy at birth. After an initial audit of the charts of 56 infants and the identification of poor compliance with the goals, unit policies and educational programs were developed to facilitate timely antibiotic administration. After a second audit demonstrated improvement but failure to attain the target compliance rates, review of individual cases with the responsible physician and nurse was initiated. Time intervals between birth, writing the order for antibiotics, noting the order by the nurse, and administration of antibiotics were tracked for an additional 20 months after these interventions. RESULTS: Before the interventions, antibiotics were administered to 28% of infants within 1 hour of the physician order (mean +/- SEM 1.58 +/- 0.11 hours) and to 19% within 2 hours of birth (3.12 +/- 0.16 hr). By the conclusion of the study, antibiotics were administered to 87% (p < 0.0001) of infants within 1 hour of the physician order (0.79 +/- 0.04 hour; p < 0.001) and to 92% (p < 0.0001) within 2 hours of birth (1.26 +/- 0.06 hours; p < 0.001). CONCLUSIONS: Administration of the first dose of parenteral antibiotics to newborns with suspected sepsis at birth frequently takes more than 1 hour after the order is written and more than 2 hours after birth. Efforts to identify and minimize common barriers significantly improved the timing of antibiotic administration. Additional improvement was attained by means of continued surveillance and individual feedback to caregivers of infants when timing objectives were not fulfilled.

The non-competitive AMPA antagonist LY 300168 (GYKI 53655) attenuates AMPA-induced hippocampal injury in neonatal rodents.

In contrast with the neuroprotective efficacy of competitive and non-competitive N-methyl-D-aspartate (NMDA) antagonists versus NMDA neurotoxicity, reported neuroprotective effects of non-NMDA antagonists in limiting alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) toxicity have been less robust. We tested the effect of the non-competitive AMPA receptor antagonist LY 300168 (GYKI 53655; E. Lilly) (0.25 or 2.5 mg/kg per dose i.p. x 3 doses vs. vehicle) on AMPA-induced excitotoxic injury in postnatal day 7 (P7) rats. To assess specificity, we tested the effect of LY 300168 (2.5 mg/kg per dose x 3 doses) on NMDA-induced excitotoxic injury. P7 rats received right intrahippocampal injections of either (S)-AMPA (2.5 nmol, n = 67) or NMDA (12.5 nmol, n = 11). Injection of AMPA resulted in right hippocampal atrophy with pyramidal cell loss. LY 300168 treatment produced dose-dependent attenuation of AMPA-induced right hippocampal injury; based on comparisons with left hippocampal volumes, 2.5 nmol AMPA resulted in 42 +/- 3% (mean +/- SEM) right hippocampal volume loss in controls, but only 10 +/- 5% after LY 300168 2.5 mg/kg per dose (P < 0.001; ANOVA). LY 300168 had no effect on NMDA-induced hippocampal injury. The data support the hypothesis that drugs that allosterically regulate AMPA receptor activity can modulate the response of immature brain to AMPA-mediated injury.

TNF-alpha and IL-4 synergistically increase vascular cell adhesion molecule-1 expression in cultured vascular smooth muscle cells.

Vascular cell adhesion molecule-1 (VCAM-1) is expressed on the endothelium and vascular smooth muscle in atherosclerosis, where it is thought to recruit alpha4 integrin-positive leukocytes, which play a role in disease progression. In this study, we show an increase of VCAM-1 expression on vascular smooth muscle cells (VSMC) results in increased adhesion of alpha4 integrin-positive lymphocytes. Additionally, we examine the regulation of VCAM-1 expression by cytokines in cultured VSMC. Previously in endothelial cells, we have demonstrated that TNF-alpha increases transcription of the VCAM-1 gene, whereas IL-4 acts to increase VCAM-1 mRNA stability. The combination of a cytokine that increases transcription with a cytokine that stabilizes mRNA results in a synergistic increase in VCAM-1 expression. In this study, we show that the combination of TNF-alpha with IL-4 also resulted in a synergistic increase in VCAM-1 expression on VSMC; however, the mechanism of cytokine activation differed. In contrast to endothelial cells, IL-4 stimulated VCAM-1 gene transcription in the VSMC, but there was little effect of TNF-alpha alone. Additionally, the synergy between TNF-alpha and IL-4 appears to result, at least in part, from a cooperative transcriptional mechanism.

Increased chromosome 20 copy number detected by fluorescence in situ hybridization (FISH) in malignant melanoma.

DNA amplification is an important mechanism of tumor progression that allows cancer cells to up-regulate the expression of critical genes such as oncogenes and genes conferring drug resistance. Recent studies using comparative genomic hybridization (CGH) revealed increased DNA copies of 20q sequences in 7 melanoma cell lines and B archival metastatic melanoma lesions. To evaluate chromosome 20 abnormalities in more detail and to resolve discrepancies between karyotype and CGH findings, we performed FISH analysis of metaphase cells in 13 melanoma cell lines (including the 7 lines used for CGH) and 9 primary melanoma specimens by using a whole chromosome paint specific for chromosome 20. All 13 cell lines (100%) and 8/9 primary tumors (89%) showed extra copies of chromosome 20 relative to tumor ploidy. Additionally, 6/14 cell lines (43%) and 2/8 primary tumors (25%) showed translocated chromosome 20 material previously undetected by standard cytogenetics. Cytologic evidence for gene amplification was also found in one cell line, which contained an add(20)(p13), with additional DNA being derived from 20q sequences. These data suggest that overrepresentation of a gene or genes important for melanoma pathogenesis resides on the long arm of chromosome 20.

Cytokines and perinatal brain injury.

A rapidly expanding body of data provides support for the hypothesis that pro-inflammatory cytokines including interleukin-1 beta (IL-1 beta), and tumor necrosis factor-alpha (TNF-alpha) are expressed acutely in injured brain and contribute to progressive neuronal damage. Little is known about the pathogenetic role of these cytokines in perinatal brain injury. Recent experimental studies have incorporated two closely related in vivo perinatal rodent brain injury models to evaluate the role(s) of pro-inflammatory cytokines in the progression of neuronal injury: a perinatal stroke model, elicited by unilateral carotid artery ligation and subsequent timed exposure to 8% oxygen in 7-day-old rats, and a model of excitotoxic injury, elicited by stereotactic intra-cerebral injection of the selective excitatory amino acid agonist NMDA. Each of these lesioning methods results in reproducible, quantifiable focal forebrain injury at this developmental stage. Acute brain injury, evoked by cerebral hypoxia-ischemia or excitotoxin lesioning, results in transient marked increases in expression of IL-1 beta, and TNF-alpha mRNA in brain regions susceptible to irreversible injury, and there is evidence that pharmacological antagonism of IL-1 receptors can attenuate injury in both models. Recent studies also suggest that complementary strategies, based on pharmacological antagonism of platelet activating factor and on neutrophil depletion can also limit the extent of irreversible injury. In summary, current data suggest that pro-inflammatory cytokines contribute to the progression of perinatal brain injury, and that these mediators are important targets for neuroprotective interventions in the acute post-injury period.

gp120, a human immunodeficiency virus-1 coat protein, augments excitotoxic hippocampal injury in perinatal rats.

Recent data suggest that gp120, a human immunodeficiency virus-1 (HIV-1) coat glycoprotein that is secreted by HIV-infected cells, is neurotoxic, and that this toxicity is mediated, at least in part, by activation of N-methyl-D-aspartate-type excitatory amino acid receptors. To test this hypothesis in vivo, we examined the neurotoxicity of gp120 injected intrahippocampally, alone or co-injected with the selective excitatory amino acid agonist N-methyl-D-aspartate, in seven-day-old rats. Severity of injury in the lesioned hippocampus was assessed five days later, using three outcome measures: histopathology, hippocampal atrophy (derived from regional cross-sectional area measurements) and loss of [3H]glutamate receptor binding (based on in vitro autoradiography assays). To confirm that any observed effects were attributable to gp120 bioactivity, each group of experiments included controls that received equal amounts of heat-treated gp120. Gp120 (200 ng) elicited minimal focal pyramidal cell loss immediately adjacent to the injection track; there was no hippocampal atrophy or loss of [3H]glutamate binding. Co-injection of 50 ng gp120 with N-methyl-D-aspartate (5 nmol, threshold excitotoxic dose) increased the severity of hippocampal injury; hippocampal atrophy was greater in animals that received injections of 5 nmol N-methyl-D-aspartate in combination with 50 ng gp120 than in those that received either N-methyl-D-aspartate alone (5 nmol) or 5 nmol N-methyl-D-aspartate+50 ng heat-treated gp120 (mean+/-S.E.M. percentage reduction in injected hippocampal volume vs contralateral: N-methyl-D-aspartate, -19+/-3; N-methyl-D-aspartate+gp120, -26.8+/-2.1; N-methyl-D-aspartate+heat-treated gp120, -14.0+/-2.2; P<0.001, ANOVA). Treatment with the competitive N-methyl-D-aspartate antagonist 3-((RS)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (20mg/kg) markedly reduced the severity of injury elicited by the combination of gp120 with N-methyl-D-aspartate. These data support the hypothesis that locally secreted gp120 could exert neurotoxic effects, mediated by N-methyl-D-aspartate receptor activation, in vivo in the immature brain.