Robert D. Bart

Differential Effects of cAMP in Neurons and Astrocytes ROLE OF B-RAF

Mitogen-activated protein kinase (MAPK) activation provides cell type-specific signals important for cellular differentiation, proliferation, and survival. Cyclic AMP (cAMP) has divergent effects on MAPK activity depending on whether signaling is through Ras/Raf-1 or Rap1/B-raf. We found that central nervous system-derived neurons, but not astrocytes, express B-raf. In neurons, cAMP activated MAPK in a Rap1/B-raf-dependent manner, while in astrocytes, cAMP decreased MAPK activity. Inhibition of MAPK in neurons decreased neuronal growth factor-mediated survival, and activation of MAPK by cAMP analogues rescued neurons from death. Furthermore, constitutive expression of B-raf in astrocytoma cells increased MAPK activation, as seen in neurons, and enhanced proliferation. These data provide the first experimental evidence that B-raf is the molecular switch which dominantly permits differential cAMP-dependent regulation of MAPK in neuronsversus astrocytes, with important implications for both survival and proliferation.

Serum creatinine as stratified in the RIFLE score for acute kidney injury is associated with mortality and length of stay for children in the pediatric intensive care unit.

Objective:To evaluate the ability of the RIFLE criteria to characterize acute kidney injury in critically ill children. Design:Retrospective analysis of prospectively collected clinical data. Setting:Multidisciplinary, tertiary care, 20-bed pediatric intensive care unit. Patients:All 3396 admissions between July 2003 and March 2007. Interventions:None. Measurements and Main Results:A RIFLE score was calculated for each patient based on percent change of serum creatinine from baseline (risk = serum creatinine ×1.5; injury = serum creatinine ×2; failure = serum creatinine ×3). Primary outcome measures were mortality and intensive care unit length of stay. Logistic and linear regressions were performed to control for potential confounders and determine the association between RIFLE score and mortality and length of stay, respectively.One hundred ninety-four (5.7%) patients had some degree of acute kidney injury at the time of admission, and 339 (10%) patients had acute kidney injury develop during the pediatric intensive care unit course. Almost half of all patients with acute kidney injury had their maximum RIFLE score within 24 hrs of intensive care unit admission, and approximately 75% achieved their maximum RIFLE score by the seventh intensive care unit day. After regression analysis, any acute kidney injury on admission and any development of or worsening of acute kidney injury during the pediatric intensive care unit stay were independently associated with increased mortality, with the odds of mortality increasing with each grade increase in RIFLE score (p < .01). Patients with acute kidney injury at the time of admission had a length of stay twice that of those with normal renal function, and those who had any acute kidney injury develop during the pediatric intensive care unit course had a four-fold increase in pediatric intensive care unit length of stay. Also, other than being admitted with RIFLE risk score, an independent relationship between any acute kidney injury at the time of pediatric intensive care unit admission, any acute kidney injury present during the pediatric intensive care unit course, or any worsening RIFLE scores during the pediatric intensive care unit course and increased pediatric intensive care unit length of stay were identified after controlling for the same high-risk covariates (p < .01). Conclusions:RIFLE criteria serves well to describe acute kidney injury in critically ill pediatric patients.

Apolipoprotein E-Deficient Mice Have Increased Susceptibility To Focal Cerebral Ischemia

Recent evidence suggests that apolipoprotein E (ApoE) plays a role in neurologic disease. This experiment compared the neurologic and histologic outcome of ApoE-deficient mutant and wild-type mice subjected to a 60- or 90-minute episode of middle cerebral artery filament occlusion and a recovery interval of 24 hours. With 60 minutes of ischemia, there was no mortality. Apolipoprotein E-deficient mice had larger infarcts (cortex: ApoE deficient = 20 mm3 ± 12, wild-type = 9 ± 7 mm3, P = 0.03; subcortex: ApoE deficient = 22 ± 7 mm3, wild-type = 16 ± 7 mm3, P = 0.07). Hemiparesis was less severe in wild-type animals (P = 0.02). After 90 minutes of ischemia, mortality in ApoE-deficient mice (n = 10) was 40% versus 0% in wild-type mice (n = 10; P = 0.09). Intraparenchymal hemorrhage was found in 3 of the 4 dead mice. No difference in cortical (ApoE deficient = 37 ± 8 mm3; wild-type = 31 ± 18 mm3; P = 0.49) or subcortical (ApoE deficient = 30 ± 11 mm3; wild-type = 32 ± 18 mm3; P = 0.78) infarct volumes was present among survivors. ApoE-deficient mice had a prolonged activated partial thromboplastin time and increased fibrinogen concentration. This data supports the hypothesis that apolipoprotein E plays a role in the pathophysiology of ischemic brain damage.

Apolipoprotein E Isoform-Specific Differences in Outcome from Focal Ischemia in Transgenic Mice:

Apolipoprotein E (apoE), a 34-kD glycosylated lipid-binding protein, is expressed as three common isoforms in humans (E2, E3, or E4). Clinical evidence suggests that the apoE genotype (APOE) may be a risk factor for poor outcome after acute central nervous system injury. This was examined further in transgenic mice constructed with the human APOE3 or APOE4 gene under the control of human promoter and tissue expression elements. Presence of human apoE3 and apoE4 proteins in brains of human APOE homozygous transgenic mice was confirmed by Western blotting. APOE3 (n = 12) and APOE4 (n = 10) mice underwent 60 minutes of middle cerebral artery occlusion. After 24-hour recovery, infarct size was measured. Infarct volumes (mean ± standard deviation) were smaller in the APOE3 group (cortex: APOE3 = 18 ± 4 mm3; APOE4 = 30 ± 11 mm3, P = 0.04; subcortex: APOE3 = 12 ± 4 mm3; APOE4 = 18 ± 4 mm3, P = 0.003). Hemiparesis was less severe in APOE3 mice (P = 0.02). These data indicate that human isoform-specific effects of apoE are relevant to acute pathomechanisms of focal ischemic brain damage when examined in the mouse. APOE transgenic mice may provide an appropriate model to examine the mechanistic basis for the differential effects of human apoE isoforms in acute central nervous system injury.

Differential Effects of Anesthetic Agents on Outcome from Near-complete but Not Incomplete Global Ischemia in the Rat

BackgroundIt has been postulated that anesthetic agents that reduce cerebral metabolic rate will protect the brain against ischemia when electroencephalographic (EEG) activity is persistent, but will provide no protection when ischemia is severe enough to cause EEG isoelectricity. No outcome studies

Mice overexpressing extracellular superoxide dismutase have increased resistance to focal cerebral ischemia.

Transgenic mice, which had been transfected with the human extracellular superoxide dismutase gene, causing an approximate five-fold increase in brain parenchymal extracellular superoxide dismutase activity, were used to investigate the role of extracellular superoxide dismutase in ischemic brain injury. Transgenic (n = 21) and wild-type (n = 19) mice underwent 90 min of intraluminal middle cerebral artery occlusion and 24 h of reperfusion. Severity of resultant hemiparesis and cerebral infarct size were measured. Wild-type mice had larger infarcts (cortex: wild type =37+/-14 mm3, transgenic = 27+/-13 mm3, P=0.03; subcortex: wild type = 33+/-14 mm3, transgenic = 23+/-10 mm3, P = 0.02). Neurological scores, however, were similar (P = 0.29). Other mice underwent autoradiographic determination of intra-ischemic cerebral blood flow. The volume of tissue at risk of infarction (defined as volume of tissue where blood flow was <25 ml/100g/min) was similar between groups (cortex: wild type = 51+/-15 mm3, transgenic = 47+/-9 mm3, P=0.65; subcortex: wild type = 39+/-16 mm3, transgenic= 37+/-17 mm3, P=0.81). These results indicate that antioxidant scavenging of free radicals by extracellular superoxide dismutase plays an important role in the histological response to a focal ischemic brain insult.

Comparison of the Pulse Oximetric Saturation/Fraction of Inspired Oxygen Ratio and the Pao2/Fraction of Inspired Oxygen Ratio in Children

BACKGROUND Although diagnostic criteria for acute lung injury (ALI) and ARDS are clear, invasive arterial sampling is required for computation of Pao(2)/fraction of inspired oxygen (Fio(2)) [PF] ratios. The pulse oximetric saturation (Spo(2))/Fio(2) (SF) ratio may be a reliable noninvasive alternative to the PF ratio for identifying children with lung injury. METHODS We electronically queried blood gas measurements from two tertiary care pediatric ICUs (PICUs). Included in the analysis were corresponding measurements of Spo(2), Pao(2), and Fio(2) charted within 15 min of each other when Spo(2) values were between 80% and 97%. Computed PF and SF ratios were compared to identify threshold values for SF ratios that correspond to PF criteria for ALI (< or = 300) and ARDS (< or = 200). Data from one PICU were used for derivation and validated with measurements from the second PICU. RESULTS From the 1,298 observations in the derivation data set, SF ratio could be predicted by the regression equation SF = 76 + 0.62 x PF (p < 0.0001, R(2) = 0.61). SF ratios of 263 and 201 corresponded to PF ratios of 300 and 200, respectively. The ALI SF cutoff of 263 had 93% sensitivity and 43% specificity, and the ARDS cutoff of 201 had 84% sensitivity and 78% specificity. Applying these values to the 1,845 observations in the validation data set yielded a sensitivity of 86% and specificity of 47% for ALI and a sensitivity of 68% and specificity of 84% for ARDS. CONCLUSION SF ratio is a reliable noninvasive marker for PF ratio to identify children with ALI or ARDS.

Lung cancer and cyclooxygenase-2

Lung cancer is by far the leading cause of cancer-related death. Overall survival is poor and has not improved substantially over the last half century. It is clear that new approaches are needed and these should include prevention, screening for early detection, and novel treatments based on our understanding of the molecular biology of this disease. Recently attention has been drawn to the role of the cyclooxygenase (COX) enzyme and its involvement in tumorigenesis. Investigations have documented two isoforms, COX-1 and COX-2, encoded by different genes. COX-1 is constitutively expressed in most tissues and appears to be responsible for the production of prostaglandins mediating normal physiologic functions, such as the maintenance of gastric mucosa and regulation of renal blood flow. In contrast, COX-2 is normally undetectable in most tissues, and is induced by cytokines, growth factors, oncogenes, and tumor promoters. A growing body of evidence indicates COX-2 plays a key role in lung cancer, and can serve as a potential marker of prognosis in this disease. Furthermore, the recent availability of COX-2 inhibitor medications offers a unique opportunity to interfere with the development of lung cancer and the progression of metastasis. Because COX-2 inhibitors have been demonstrated to interfere with tumorigenesis, the COX-2 enzyme may be an attractive target for therapeutic and chemoprotective strategies in lung cancer patients.

Interactions between Hypothermia and the Latency to Ischemic Depolarization Implications for Neuroprotection

Background The authors postulated that hypothermic neuroprotection can be attributed to a delayed onset of ischemic depolarization. Methods Halothane‐anesthetized rats were prepared for near‐complete forebrain ischemia. Direct current (DC) potential microelectrodes were placed in hippocampal CA1. The pericranial temperature was maintained at 31 [degree sign] Celsius, 33 [degree sign] Celsius, 35 [degree sign] Celsius, or 37 [degree sign]C (n = 6 per group). Bilateral carotid occlusion with systemic hypotension was initiated for 10 min. The time to onset of the DC shift was recorded. In a second experiment, rats were assigned to 37 [degree sign]C or 31 [degree sign]C for 10 min of ischemia, or to 31 [degree sign]C for 14 min of ischemia (n = 8 per group). These durations of ischemia were defined to allow 9 min of ischemic depolarization in the 37 [degree sign] Celsius‐10 min and 31 [degree sign] Celsius‐14 min groups. Neurologic and histologic outcomes were examined 7 days later. Results Hippocampal CA1 time to depolarization increased with decreasing temperature (P < 0.0001). Time to depolarization was increased by approximately 4 min in the rats maintained at 31 [degree sign]C compared with those at 37 [degree sign] Celsius. Time to repolarization during reperfusion was not affected by temperature. Increasing the duration of ischemia from 10 min to 14 min with the pericranial temperature maintained at 31 [degree sign]C resulted in a duration of depolarization that was equivalent in the 37 [degree sign] Celsius‐10 min and 31 [degree sign] Celsius‐14 min groups. However, hippocampal CA1 damage was not increased (31 [degree sign] Celsius‐10 min = 4 +/‐ 1% dead neurons; 31 [degree sign] Celsius‐14 min = 6 +/‐ 1% dead neurons, 95% CI, ‐1% to 3%; 37 [degree sign] Celsius‐10 min = 90 +/‐ 17% dead neurons). Conclusions Despite similar durations of DC depolarization, outcome in hypothermic rats was markedly improved compared with normothermic rats. This indicates that hypothermic neuroprotection can be attributed to mechanisms other than the delay in time to onset of ischemic depolarization.

Neuroprotective Effect of NMDA Receptor Glycine Recognition Site Antagonism Persists When Brain Temperature is Controlled

Several lines of inquiry have indicated that glycine plays an important role in both glutamatergic neurotransmission and pathophysiology of cerebral ischemia. However, subacute outcome trials demonstrating the efficacy of glycine antagonists as neuroprotectants have not been performed with rigorous control of brain temperature. In this study, we investigated the effect of N-methyl-D-aspartate (NMDA) receptor glycine recognition site antagonism in a temperature-controlled rodent model of transient focal ischemia. Male Wistar rats underwent 75 min of intraluminal middle cerebral artery occlusion (MCAO). During MCAO and the first 24 h of reperfusion, rats (n = 10) were administered either vehicle or the glycine antagonist 5-nitro-6,7-dichloro-2,3-quinoxalinedione (ACEA 1021) i.v. as a bolus infusion of 5 mg/kg followed by 3.5 mg/kg/h (Low-Dose) or 10 mg/kg followed by 7 mg/kg/h (High-Dose) for 24 h. Cortical temperature was controlled at 38.0 ± 0.1°C during MCAO and the first 6 h of reperfusion. A 7-day recovery interval was allowed. Mean total infarct volume was reduced by ∼ 40% in both high- and low-dose groups (p < 0.01). The preponderance of infarct reduction occurred in the cortex (p < 0.01). Neurologic function correlated with the size of cerebral infarct (p = 0.001). Neurologic grade was similarly improved by treatment with either dose (p = 0.01). These results demonstrate that neuroprotection achieved by antagonism of the glycine recognition site persists when brain temperature is controlled, indicating a potent mechanism of action other than attenuating a hyperthermic response to ischemia.