Michael J. Bell

Determining the Fetal Inflammatory Response in an Experimental Model of Intrauterine Inflammation in Rats

Intrauterine infection is a risk factor for developmental brain injuries in childhood. A variety of cytokines known to be toxic to developing brain cells have been isolated from mothers or children at risk for developmental disabilities, and these cytokines have been proposed as mediators of these injuries. We have developed a model of intrauterine inflammation that damages the developing white matter and we now hypothesize that selected cytokines are increased after our experimental inflammatory stimulus. Timed-pregnant Fischer 344 and Lewis rats were injected with 0.1 mg/kg of lipopolysaccharide (LPS) into the cervix at E15. Tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), IL-6, and IL-10 were measured in homogenates of fetal brain and placenta at serial time periods within the first 24 h after the inflammatory stimulus. TNF-α was increased 20-fold in the placenta and more than 5-fold in the fetal brain after the stimulus. IFN-γ was only increased within the fetal brain (20-fold) and IL-6 was only increased in the placenta (10-fold). IL-10 was mildly increased in the placenta and was decreased slightly in the fetal brain. Our observations show that an intrauterine inflammatory stimulus can cause large increases in Th1 cytokines within the fetal brain. The placenta can produce selected cytokines but fails to produce IFN-γ, suggesting that the fetal immune system produces this cytokine in response to our stimulus. By studying placental and brain cytokine responses in models such as ours, the mechanisms responsible for the damage to developing white matter can be determined.

A tertiary care center's experience with therapeutic hypothermia after pediatric cardiac arrest*

Objective: To describe the use and feasibility of therapeutic hypothermia after pediatric cardiac arrest. Design: Retrospective cohort study. Setting: Pediatric tertiary care university hospital. Patients: Infants and children (age 1 wk to 21 yrs) without complex congenital heart disease with return of spontaneous circulation after in-hospital or out-of-hospital cardiac arrest from 2000 to 2006. Intervention: None. Measurements and Main Results: We studied 181 patients after cardiac arrest, of which 91% were asphyxial in etiology (vs. cardiac) and 52% occurred in-hospital. Overall survival to hospital discharge was 45%. Forty patients received therapeutic hypothermia; all were admitted during or after 2002. Sixty percent of patients in the therapeutic hypothermia group had an initial temperature <35°C. The median therapeutic hypothermia target temperature was 34.0°C (33.5–34.8°C), was reached by 7 hrs (5–8 hrs) after admission in patients who were not hypothermic on admission, and was maintained for 24 hrs (16–48 hrs). Re-warming lasted 6 hrs (5–8 hrs). In the therapeutic hypothermia group, temperature <32°C occurred in 15% of patients and was associated with higher hospital mortality (29% vs. 11%; p = .02). Patients treated with therapeutic hypothermia differed from those treated with standard therapy, with more un-witnessed cardiac arrest (p = .04), more doses of epinephrine to achieve return of spontaneous circulation (p = .03), and a trend toward more out-of-hospital cardiac arrests (p = .11). After arrest, therapeutic hypothermia patients received more frequent electrolyte supplementation (p < .05). Standard therapy patients were twice as likely as therapeutic hypothermia patients to have a fever (>38°C) after arrest (37% vs. 18%; p = .02) and trended toward a higher rate of re-arrest (26% vs. 13%; p = .09). Rates of red blood cell transfusions, infection, and arrhythmias were similar between groups. There was no difference in hospital mortality (55.0% therapeutic hypothermia vs. 55.3% standard therapy; p = 1.0), and 78% of the therapeutic hypothermia survivors were discharged home (vs. 68% of the standard therapy survivors; p = .46). In multivariate analysis, mortality was independently associated with initial hypoglycemia or hyperglycemia, number of doses of epinephrine during resuscitation, asphyxial etiology, and longer duration of cardiopulmonary resuscitation, but not treatment group (odds ratio for mortality in the therapeutic hypothermia group, 0.47; p = .2). Conclusions: This is the largest study reported on the use of therapeutic mild hypothermia in pediatric cardiac arrest to date. We found that therapeutic hypothermia was feasible, with target temperature achieved in <3 hrs overall. Temperature below target range was associated with increased mortality. Prospective study is urgently needed to determine the efficacy of therapeutic hypothermia in pediatric patients after cardiac arrest.

Interleukin-6, interleukin-8, and a rapid and sensitive assay for calcitonin precursors for the determination of bacterial sepsis in febrile neutropenic children.

Objective: Children with cancer often develop febrile illnesses after cytotoxic chemotherapy. Determining which children have serious bacterial infections in this vulnerable period would be valuable. We evaluated the ability of a rapid and sensitive assay for the concentration of calcitonin precursors (CTpr) as a sensitive diagnostic marker for bacterial sepsis in febrile, neutropenic children and determined the utility of measuring cytokines to improve the predictive value of this approach. Design: Prospective cohort study. Setting: Academic children’s hospital. Patients: Fifty-six children (aged 5 months to 17 yrs) with a known malignancy who presented with fever and neutropenia. Interventions: Serial blood samples were obtained (admission, 24 hrs, and 48 hrs), and concentrations of CTpr, interleukin-6, and interleukin-8 were determined. Demographic and laboratory data from the patients were collected from the medical record. Measurements and Main Results: Sixteen (29%) of the children met the criteria for bacterial sepsis. Plasma levels of CTpr and interleukin-8, but not interleukin-6, were increased at all time points in children with sepsis compared with those without sepsis. CTpr at 24 and 48 hrs after admission were reliable markers for sepsis (area under the curve = 0.92 and 0.908, respectively). Logistic regression using CTpr at 24 hrs in addition to interleukin-8 at 48 hrs produced the best-fit models associated with sepsis. Using cutoff values of CTpr >500 pg/mL and interleukin-8 >20 pg/mL produced a screening test for sepsis with 94% sensitivity and 90% specificity. Conclusions: Our data show the utility of a rapid and sensitive assay for CTpr combined with interleukin-8 as a highly sensitive and specific diagnostic marker of bacterial sepsis in febrile, neutropenic children. The use of these markers as a clinical tool may allow for better prognostication for clinicians and may eventually lead to more targeted therapies for this heterogeneous population.

Interstitial brain adenosine and xanthine increase during jugular venous oxygen desaturations in humans after traumatic brain injury.

ObjectiveAdenosine decreases the cerebral metabolic rate for oxygen and increases cerebral blood flow, and it may play an important role in cerebrometabolic and cerebrovascular responses to hypoperfusion after traumatic brain injury. Jugular venous oxygen saturation is monitored after traumatic brain injury to assess brain oxygen extraction, and desaturations may reflect secondary brain insults. We hypothesized that brain interstitial adenosine and related purine metabolites would be increased during jugular venous oxygen saturation desaturations (<50%) and determined associations between the purines, lactate, and glucose to assess the role of adenosine during secondary insults in humans. DesignStudy of critically ill adults with severe traumatic brain injury. SettingAdult neurointensive care unit. PatientsWe prospectively defined periods of normal saturation and desaturation in six patients after severe traumatic brain injury. InterventionsDuring these periods, cerebral microdialysis samples of brain interstitial fluid were collected, and adenosine and purine metabolites were measured by high-pressure liquid chromatography. Measurements and Main Results Adenosine increased 3.1-fold and xanthine increased 2.5-fold during desaturation periods (both p < .05 vs. normal saturation period, signed rank). Adenosine, xanthine, hypoxanthine, and cyclic-adenosine monophosphate correlated with lactate over both study periods (r2 = .32, .14, .31, .07, and .26, respectively, all p < .05, Pearson product moment correlation). ConclusionThe marked increases in interstitial brain adenosine that occur during jugular venous oxygen desaturations suggest that adenosine may play an important role during periods of secondary insults after traumatic brain injury. The correlation of these metabolites with lactate further suggests that adenosine is increased during periods of enhanced glycolytic metabolism.

Increases in serum levels of troponin I are associated with cardiac dysfunction and disease severity in pediatric patients with septic shock.

Objectives: Myocardial cell injury may contribute to cardiac dysfunction in septic shock. Troponin I is a biochemical marker of myocardial cell injury and death. We hypothesized that troponin I is increased in pediatric patients with septic shock and correlates with cardiac dysfunction and disease severity. Design: Prospective, observational study. Setting: Children’s medical center. Patients: Twenty-three patients with septic shock and cardiovascular failure were enrolled. Measurements and Main Results: Serum troponin I was measured at admission and serially over 72 hrs. Within 24 hrs of study enrollment, echocardiograms were performed to determine left ventricular ejection fraction, systolic fractional shortening, heart rate corrected mean velocity of circumferential fiber shortening, and end-systolic wall stress. Requirement for inotropic support (stratified as low, moderate, or high), number of organ system failures, and other demographic data (including Pediatric Risk of Mortality III) were collected. Troponin I was increased on admission in 13 of 23 patients (57%) and at 12 hrs in ten of 22 patients (46%). In all cases, troponin I was maximal within 12 hrs of admission. Admission troponin I was inversely correlated to ejection fraction and fractional shortening and directly correlated to wall stress. Patients who had increased admission troponin I had lower heart rate corrected mean velocity of circumferential fiber shortening (preload and heart rate independent measure of left ventricular systolic function) and higher wall stress (measure of afterload) compared with patients with normal troponin I. Admission troponin I correlated with Pediatric Risk of Mortality III and organ system failure but did not correlate with requirement for inotropic support. Conclusions: Troponin I was increased in >50% of septic children early in their illness. Increased admission troponin I was associated with decreased measures of systolic cardiac function, as measured by echocardiography, and correlated with severity of illness. Early myocardial cell injury may contribute to the development of subsequent organ failure in septic shock, and measuring troponin I on admission may be helpful in assessing severity of sepsis.

Quinolinic acid in the cerebrospinal fluid of children after traumatic brain injury.

OBJECTIVE To measure quinolinic acid, a macrophage-derived neurotoxin, in the cerebrospinal fluid (CSF) of children after traumatic brain injury (TBI) and to correlate CSF quinolinic acid concentrations to clinically important variables. DESIGN A prospective, observational study. SETTING The pediatric intensive care unit in Childrens Hospital of Pittsburgh, a tertiary care, university-based childrens hospital. PATIENTS Seventeen critically ill children following severe TBI (Glasgow Coma Scale score <8) whose care required the placement of an intraventricular catheter for continuous drainage of CSF. INTERVENTIONS None. MEASUREMENTS AND MAIN RESULTS Patients ranged in age from 2 mos to 16 yrs (mean 6.0 yrs). CSF was collected immediately on placement of the ventricular catheter and daily thereafter. Quinolinic acid concentration was measured by gas chromatography/mass spectroscopy in 69 samples (4.0 +/- 0.4 [SEM] samples per patient). CSF quinolinic acid concentration progressively increased after injury (p = .034, multivariate analysis) and was increased in nonsurvivors vs. survivors (p = .002, multivariate analysis). CSF quinolinic acid concentration was not associated with age. Although overall CSF quinolinic acid concentration was not associated with shaken injury (p = .16, multivariate analysis), infants suffering with shaken infant syndrome had increased admission CSF quinolinic acid concentrations compared with children with accidental mechanisms of injury (p = .027, Mann-Whitney Rank Sum test). CONCLUSIONS A large and progressive increase in the macrophage-derived neurotoxin quinolinic acid is seen following severe TBI in children. The increase is strongly associated with increased mortality. Increased CSF quinolinic acid concentration on admission in children with shaken infant syndrome could reflect a delay in presentation to medical attention or age-related differences in quinolinic acid production. These findings raise the possibility that quinolinic acid may play a role in secondary injury after TBI in children and suggest an interaction between inflammatory and excitotoxic mechanisms of injury following TBI.

Neurological injury markers in children with septic shock.

Objective: To determine whether known serum markers of neurologic injury are increased in children with septic shock. Design: Prospective, observational study. Setting: Tertiary-care, pediatric intensive care unit. Patients: Two cohorts of children (n = 24) with septic shock were prospectively enrolled within 24 hrs of their diagnosis. In cohort 1, serum markers (S100&bgr;, neuron-specific enolase [NSE], and glial fibrillary acidic protein [GFAP]) were determined (n = 18). In cohort 2, in addition to serum markers, urine S100&bgr; and GFAP were determined, and continuous electroencephalography (cEEG) was performed. Children who presented to the emergency room with a fever served as controls (n = 32). Children with known neurologic conditions were excluded. Interventions: None. Measurements and Main Results: Serum and urine were collected daily for up to 7 days or until pediatric intensive care unit discharge. Biomarker concentrations were determined by commercially available enzyme-linked immunosorbent assays. cEEG was performed on days 1, 2, 4, and 7 in a 16-channel montage for at least 6 hrs. Physical examinations did not reveal focal neurologic deficits. Children with septic shock demonstrated increased serum S100&bgr; and NSE compared with controls (mean ± sem: 10.5 &mgr;g/L ± 2.4 vs. .9 &mgr;g/L ± .1, p < .001; 96.6 &mgr;g/L ± 8.9 vs. 4.0 &mgr;g/L ± 1.3, p < .001, respectively). Serum GFAP was detectable in five septic children and none of the controls. In cohort 2, urine of four patients demonstrated measurable S100&bgr; levels, and GFAP was detected in one child (nonsurvivor). cEEG demonstrated moderate to severe encephalopathy in all children studied. Conclusions: Markers of neurologic injuries are increased in children with septic shock. This may indicate subclinical injuries that are either transient or permanent. Studies that correlate the long-term neurologic outcome of children with these markers are needed to identify children at risk for neurologic injuries from septic shock.

Cerebrospinal fluid mitochondrial DNA: a novel DAMP in pediatric traumatic brain injury.

ABSTRACT Danger-associated molecular patterns (DAMPs) are nuclear or cytoplasmic proteins that are released from the injured tissues and activate the innate immune system. Mitochondrial DNA (mtDNA) is a novel DAMP that is released into the extracellular milieu subsequent to cell death and injury. We hypothesized that cell death within the central nervous system in children with traumatic brain injury (TBI) would lead to the release of mtDNA into the cerebrospinal fluid (CSF) and has the potential to predict the outcome after trauma. Cerebrospinal fluid was collected from children with severe TBI who required intracranial pressure monitoring with Glasgow Coma Scale (GCS) scores of 8 or less via an externalized ventricular drain. Control CSF was obtained in children without TBI or meningoencephalitis who demonstrated no leukocytes in the diagnostic lumbar puncture. The median age for patients with TBI was 6.3 years, and 62% were male. The common mechanisms of injury included motor vehicle collision (35.8%), followed by falls (21.5%) and inflicted TBI (19%); six children (14.2%) died during their intensive care unit course. The mean CSF mtDNA concentration was 1.10E+05 ± 2.07E+05 and 1.63E+03 ± 1.80E+03 copies/&mgr;L in the pediatric TBI and control populations, respectively. Furthermore, the mean CSF mtDNA concentration in pediatric patients who later died or had severe disability was significantly higher than that of the survivors (1.63E+05 ± 2.77E+05 vs. 5.05E+04 ± 6.21E+04 copies/&mgr;L) (P < 0.0001). We found a significant correlation between CSF mtDNA and high mobility group box 1, another prototypical DAMP, concentrations (&rgr; = 0.574, P < 0.05), supporting the notion that both DAMPs are increased in the CSF after TBI. Our data suggest that CSF mtDNA is a novel DAMP in TBI and appears to be a useful biomarker that correlates with neurological outcome after TBI. Further inquiry into the components of mtDNA that modulate the innate immune response will be helpful in understanding the mechanism of local and systemic inflammation after TBI.

The Th1 versus Th2 cytokine profile in cerebrospinal fluid after severe traumatic brain injury in infants and children

Objective To further characterize the Th1 (proinflammatory) vs. the Th2 (antiinflammatory) cytokine profile after severe traumatic brain injury (TBI) by quantifying the ventricular cerebrospinal fluid concentrations of Th1 cytokines (interleukin [IL]-2 and IL-12) and Th2 cytokines (IL-6 and IL-12) in infants and children. Design Retrospective study. Setting University childrens hospital. Patients Twenty-four children hospitalized with severe TBI (admission Glasgow Coma Scale score, <13) and 12 controls with negative diagnostic lumbar punctures. Interventions All TBI patients received standard neurointensive care, including the placement of an intraventricular catheter for continuous drainage of cerebrospinal fluid. Measurements and Main Results Ventricular cerebrospinal fluid samples (n = 105) were collected for as long as the catheters were in place (between 4 hrs and 222 hrs after TBI). Cerebrospinal fluid samples were analyzed for IL-2, IL-4, IL-6, and IL-12 concentrations by enzyme-linked immunoassay. Peak and mean IL-6 (335.7 ± 41.4 pg/mL and 259.5 ± 37.6 pg/mL, respectively) and IL-12 (11.4 ± 2.2 pg/mL and 4.3 ± 0.8 pg/mL, respectively) concentrations were increased (p < .05) in children after TBI vs. controls (2.3 ± 0.7 pg/mL and 1.0 ± 0.5 pg/mL) for IL-6 and IL-12, respectively. In contrast, peak and mean IL-2 and IL-4 concentrations were not increased in TBI children vs. controls. Increases in the cerebrospinal fluid concentration of IL-6 were significantly associated with admission Glasgow Coma Scale score of ≤4 and age of ≤4 yrs. Increases in cerebrospinal fluid IL-4 and IL-12 were associated with child abuse as an injury mechanism (both p ≤ .05 vs. accidental TBI). Conclusions This study confirms that IL-6 levels are increased in cerebrospinal fluid after TBI in infants and children. It is the first report of increased IL-12 levels in cerebrospinal fluid after TBI in infants and children. Further, it is the first to report on IL-2 and IL-4 levels in pediatric or adult TBI. These data suggest that selected members of both the Th1 and Th2 cytokine families are increased as part of the endogenous inflammatory response to TBI. Finally, in that both IL-6 and IL-12 (but neither IL-2 nor IL-4) can be produced by astrocytes and/or neurons, a parenchymal source for cytokines in the brain after TBI may be critical to their production in the acute phase after TBI.

Brain injuries and neurological system failure are the most common proximate causes of death in children admitted to a pediatric intensive care unit

Objective: Mortality rates from critical illness in children have declined over the past several decades, now averaging between 2% and 5% in most pediatric intensive care units. Although these rates, and mortality rates from specific disorders, are widely understood, the impact of acute neurologic injuries in such children who die and the role of these injuries in the cause of death are not well understood. We hypothesized that neurologic injuries are an important cause of death in children. Design: Retrospective review. Setting: Pediatric intensive care unit at Childrens Hospital of Pittsburgh, an academic tertiary care center. Patients: Seventy-eight children who died within the pediatric intensive care unit from April 2006 to February 2008. Interventions: None. Measurements and Main Results: Data regarding admission diagnosis, presence of chronic illness, diagnosis of brain injury, and cause of death were collected. Mortality was attributed to brain injury in 65.4% (51 of 78) of deaths. Ninety-six percent (28 of 29) of previously healthy children died with brain injuries compared with 46.9% (23 of 49) of chronically ill children (p < .05). The diagnosed brain injury was the proximate cause of death in 89.3% of previously healthy children and 91.3% with chronic illnesses. Pediatric intensive care unit and hospital length of stay was longer in those with chronic illnesses (38.8 ± 7.0 days vs. 8.9 ± 3.7 days and 49.2 ± 8.3 days vs. 9.0 ± 3.8 days, p < .05 and p < .001, respectively). Conclusion: Brain injury was exceedingly common in children who died in our pediatric intensive care unit and was the proximate cause of death in a large majority of cases. Neuroprotective measures for a wide variety of admission diagnoses and initiatives directed to prevention or treatment of brain injury are likely to attain further improvements in mortality in previously healthy children in the modern pediatric intensive care unit.