Randall A. Ruppel

Assessment of Antioxidant Reserves and Oxidative Stress in Cerebrospinal Fluid after Severe Traumatic Brain Injury in Infants and Children

Studies in experimental traumatic brain injury (TBI) support a key role for oxidative stress. The degree of oxidative injury in clinical TBI, however, remains to be defined. We assessed antioxidant defenses and oxidative stress in pediatric TBI by applying a comprehensive battery of assays to cerebrospinal fluid samples. Using a protocol approved by our institutional review board, 87 cerebrospinal fluid samples from 11 infants and children with severe TBI (Glasgow Coma Scale score ≤8) and 8 controls were studied. Cerebrospinal fluid was drained as standard care after TBI. CSF was assessed on d 1, 2, and 5–7 after ventricular drain placement. Biochemical markers of oxidative stress included F2-isoprostane and protein sulfhydryl (detected by ELISA and fluorescence assay, respectively). Antioxidant defenses were measured by determination of total antioxidant reserve (via chemiluminescence assay), and ascorbate (via HPLC) and glutathione (via fluorescence assay) concentrations. Free radical production (ascorbate radical) was assessed by electron paramagnetic resonance spectroscopy. F2-isoprostane was markedly increased versus control, maximal on d 1 (93.8 ± 30.8 pg/mL versus 7.6 ± 5.1 pg/mL, p < 0.05). Total antioxidant reserve was reduced versus control. Reduction was maximal on d 5–7 (81.8 ± 3.7 μM versus 178.9 ± 2.2 μM, p < 0.05). Ascorbate was remarkably reduced (53.8 ± 8 μM versus 163.8 ± 21 μM on d 1, p < 0.05). Ascorbate depletion was likely associated with its free radical oxidation, as evidenced by electron paramagnetic resonance spectroscopy. Glutathione levels increased on d 1, then decreased versus control (0.19 ± 0.05 μM versus 1.2 ± 0.16 μM, p < 0.05). This is the first comprehensive study of antioxidant reserve and oxidative injury in clinical TBI. Progressive compromise of antioxidant defenses and evidence of free radical–mediated lipid peroxidation are noted. These markers could be used to monitor antioxidant strategies in clinical trials.

Biochemical, cellular, and molecular mechanisms in the evolution of secondary damage after severe traumatic brain injury in infants and children: Lessons learned from the bedside.

Objective To present a state-of-the-art review of mechanisms of secondary injury in the evolution of damage after severe traumatic brain injury in infants and children. Data Sources We reviewed 152 peer-reviewed publications, 15 abstracts and proceedings, and other material relevant to the study of biochemical, cellular, and molecular mechanisms of damage in traumatic brain injury. Clinical studies of severe traumatic brain injury in infants and children were the focus, but reports in experimental models in immature animals were also considered. Results from both clinical studies in adults and models of traumatic brain injury in adult animals were presented for comparison. Data Synthesis Categories of mechanisms defined were those associated with ischemia, excitotoxicity, energy failure, and resultant cell death cascades; secondary cerebral swelling; axonal injury; and inflammation and regeneration. Conclusions A constellation of mediators of secondary damage, endogenous neuroprotection, repair, and regeneration are set into motion in the brain after severe traumatic injury. The quantitative contribution of each mediator to outcome, the interplay between these mediators, and the integration of these mechanistic findings with novel imaging methods, bedside physiology, outcome assessment, and therapeutic intervention remain an important target for future research.

Increased adenosine in cerebrospinal fluid after severe traumatic brain injury in infants and children: association with severity of injury and excitotoxicity.

Objectives To measure adenosine concentration in the cerebrospinal fluid of infants and children after severe traumatic brain injury and to evaluate the contribution of patient age, Glasgow Coma Scale score, mechanism of injury, Glasgow Outcome Score, and time after injury to cerebrospinal fluid adenosine concentrations. To evaluate the relationship between cerebrospinal fluid adenosine and glutamate concentrations in this population. Design Prospective survey. Setting Pediatric intensive care unit in a university-based children’s hospital. Patients Twenty-seven critically ill infants and children who had severe traumatic brain injury (Glasgow Coma Scale <8), who required placement of an intraventricular catheter and drainage of cerebrospinal fluid as part of their neurointensive care. Interventions None. Measurements and Main Results Patients ranged in age from 2 months to 14 yrs. Cerebrospinal fluid samples (n = 304) were collected from 27 patients during the first 7 days after traumatic brain injury. Control cerebrospinal fluid samples were obtained from lumbar puncture on 21 infants and children without traumatic brain injury or meningitis. Adenosine concentration was measured by using high-pressure liquid chromatography. Adenosine concentration was increased markedly in cerebrospinal fluid of children after traumatic brain injury vs. controls (p < .001). The increase in cerebrospinal fluid adenosine was independently associated with Glasgow Coma Scale ≤4 vs. >4 and time after injury (both p < .005). Cerebrospinal fluid adenosine concentration was not independently associated with either age (≤4 vs. >4 yrs), mechanism of injury (abuse vs. other), or Glasgow Outcome Score (good/moderately disabled vs. severely disabled, vegetative, or dead). Of the 27 patients studied, 18 had cerebrospinal fluid glutamate concentration previously quantified by high-pressure liquid chromatography. There was a strong association between increases in cerebrospinal fluid adenosine and glutamate concentrations (p < .005) after injury. Conclusions Cerebrospinal fluid adenosine concentration is increased in a time- and severity-dependent manner in infants and children after severe head injury. The association between cerebrospinal fluid adenosine and glutamate concentrations may reflect an endogenous attempt at neuroprotection against excitotoxicity after severe traumatic brain injury.

Endothelin-1 Is Increased in Cerebrospinal Fluid and Associated with Unfavorable Outcomes in Children after Severe Traumatic Brain Injury

Severe pediatric traumatic brain injury (TBI) is associated with unfavorable outcomes secondary to injury from activation of the inflammatory cascade, the release of excitotoxic neurotransmitters, and changes in the reactivity of cerebral vessels, causing ischemia. Hypoperfusion of injured brain tissues after TBI is also associated with unfavorable outcomes. Therapeutic hypothermia is an investigational treatment strategy for use in patients with severe TBI that has shown differential effects on various cerebrospinal fluid (CSF) mediators in pediatric patients. Endothelin-1 (ET-1) is a powerful vasoconstrictor that exerts its effects on the cerebrovascular endothelium for sustained periods after TBI. The purpose of this study was to determine if CSF concentrations of ET-1 are increased after severe TBI in children, and if they are associated with demographics and outcomes that are affected by therapeutic hypothermia. This was an ancillary study to a prospective, randomized-controlled trial of early hypothermia in a tertiary care pediatric intensive care unit. Children (n = 34, age 3 months-15 years) suffering from severe TBI were randomized to hypothermia (n = 19) and normothermia (n = 15) as part of the efficacy study. Children undergoing diagnostic lumbar puncture (n = 11) to rule out infection were used as controls. Patients received either mild to moderate hypothermia (32-33°C) or normothermia as part of their treatment protocol. CSF was serially collected during the first 5 days after TBI. ET-1 concentrations were quantitated in patient and control CSF samples by a validated ELISA in duplicate with a limit of quantification of 0.195 pg/mL. CSF ET-1 concentrations were increased by two- to threefold in children after TBI compared to controls, and the increase was sustained for up to 5 days post-TBI. This relationship was not affected by hypothermia, and there were no differences in ET-1 response between children with inflicted and accidental TBI. Group-based trajectory analysis revealed two distinct groups with similar ET-1 levels over time. Univariate analysis showed a significant association between ET-1 levels and Glasgow Outcome Scale (GOS) scores, for which higher ET-1 levels over time were associated with unfavorable outcomes. ET-1 is increased in children with severe TBI and is associated with unfavorable outcomes. This increase in ET-1 may mediate the hypoperfusion or cerebrovascular dysfunction accompanying severe TBI in children. Importantly, hypothermia does not affect the brains ET-1 response as measured in the CSF.

Induction of the stress response after inflicted and non-inflicted traumatic brain injury in infants and children.

Rapid induction of 72-kD heat shock protein (Hsp70) is a key component of the stress response and is seen after a variety of insults to the brain including experimental hyperthermia, ischemia, seizures, and traumatic brain injury (TBI). Little is known about the endogenous stress response in pediatric patients after brain injury. Accordingly, the concentration of Hsp70 was determined in 61 cerebrospinal fluid (CSF) samples from 20 infants and children after TBI. Peak Hsp70 level were increased in TBI patients vs. controls (4.60 [1.49-78.99] vs. 2.18 [1.38-4.25] ng/mL, respectively, median (range), p = 0.01) and occurred most often on day 1 after injury. Strikingly, CSF levels of Hsp70 were positively and independently associated with inflicted vs. non-inflicted TBI (7.03 [2.30-27.22] vs. 2.06 [1.06-78.99] ng/mL, respectively, p = 0.05). Endogenous Hsp70 expression was confirmed by Western blot and immunocytochemistry using brain tissue samples removed from patients who underwent decompressive craniotomy for refractory intracranial hypertension or at autopsy. These data suggest that the endogenous stress response, as measured and quantified by the Hsp70 concentration in CSF, occurs in infants and children after TBI. The endogenous stress response is more robust in victims of child abuse, compared with patients with accidental TBI, supporting age-dependence or a difference in either injury frequency, duration, severity, or mechanism in this subgroup of TBI patients. Further studies are needed to determine the role of Hsp70 in both non-inflicted and inflicted TBI in infants and children.

CEREBRAL RESUSCITATION AFTER TRAUMATIC BRAIN INJURY AND CARDIOPULMONARY ARREST IN INFANTS AND CHILDREN IN THE NEW MILLENNIUM

As outlined in Figure 1, it is likely that a series of interventions beginning in the field and continuing through the emergency department, ICU, rehabilitation center, and possibly beyond, will be needed to optimize clinical outcome after severe TBI or asphyxial CA in infants and children. Despite the many differences between these two important pediatric insults, it is likely that many of the therapies targeting neuronal death, in either condition, will need to be administered early after the insult, possibly at the injury scene. Even cerebral swelling, a pathophysiologic derangement routinely treated in the PICU, almost certainly is better prevented rather than treated. Finally, this review includes, for one of the first times, a brief discussion of additional horizons in the management of patients with severe brain injury, namely, manipulation of the injured circuitry and stimulation of regeneration. Further research is needed to define better the pathobiology of these two important conditions at the bedside, to understand the optimal application of contemporary therapies, and to develop and apply novel therapies. The tools necessary to carry out these studies are materializing, although the obstacles are great. This difficult but important challenge awaits further investigation by clinician-scientists in pediatric neurointensive care.

Critical mechanisms of secondary damage after inflicted head injury in infants and children

A number of critical mechanisms are involved in the pathophysiology of inflicted head injury. Excitotoxicity, oxidative stress, inflammation, programmed cell death, and mediators of blood flow and metabolism all contribute to secondary injury after abusive head trauma. These mechanisms are reviewed and the implications for clinical practice discussed.

Increased adrenomedullin in cerebrospinal fluid after traumatic brain injury in infants and children.

Adrenomedullin is a recently discovered 52-amino acid peptide that is a potent vasodilator and is produced in the brain in experimental models of cerebral ischemia. Infusion of adrenomedullin increases regional cerebral blood flow and reduces infarct volume after vascular occlusion in rats, and thus may represent an endogenous neuroprotectant. Disturbances in cerebral blood flow (CBF), including hypoperfusion and hyperemia, frequently occur after severe traumatic brain injury (TBI) in infants and children. We hypothesized that cerebrospinal fluid (CSF) adrenomedullin concentration would be increased after severe TBI in infants and children, and that increases in adrenomedullin would be associated with alterations in CBF. We also investigated whether posttraumatic CSF adrenomedullin concentration was associated with relevant clinical variables (CBF, age, Glasgow Coma Scale [GCS] score, mechanism of injury, and outcome). Total adrenomedullin concentration was measured using a radioimmunometric assay. Sixty-six samples of ventricular CSF from 21 pediatric patients were collected during the first 10 days after severe TBI (GCS score < 8). Control CSF was obtained from children (n = 10) undergoing lumbar puncture without TBI or meningitis. Patients received standard neurointensive care, including CSF drainage. CBF was measured using Xenon computed tomography (CT) in 11 of 21 patients. Adrenomedullin concentration was markedly increased in CSF of infants and children after severe TBI vs control (median 4.5 versus 1.0 fmol/mL, p < 0.05). Sixty-two of 66 CSF samples (93.9%) from head-injured infants and children had a total adrenomedullin concentration that was greater than the median value for controls. Increases in CSF adrenomedullin were most commonly observed early after TBI. CBF was positively correlated with CSF adrenomedullin concentration (p < 0.001), but this relationship was not significant when controlling for the effect of time. CSF adrenomedullin was not significantly associated with other selected clinical variables. We conclude adrenomedullin is markedly increased in the CSF of infants and children early after severe TBI. We speculate that adrenomedullin participates in the regulation of CBF after severe TBI.

Cerebrospinal fluid procalcitonin and severe traumatic brain injury in children.

Objective To determine the relationship between cerebrospinal fluid procalcitonin concentration and severe traumatic brain injury in children. Design Prospective, observational clinical study. Setting A multidisciplinary, tertiary-care pediatric intensive care unit. Patients Twenty-eight patients who required external ventricular drainage for management of severe traumatic brain injury (Glasgow Coma Scale score of <8) and 22 control patients for whom lumbar cerebrospinal fluid evaluation excluded possible meningitis. Interventions Standard intracranial pressure-directed neurointensive care, including intraventricular catheter placement and continuous cerebrospinal fluid drainage, was used to manage patients with severe traumatic brain injury. Measurements and Main Results Demographic data including age, mechanism of injury, time of injury, initial Glasgow Coma Scale score, and outcome were collected. Cerebrospinal fluid procalcitonin concentration was determined by immunoluminometric assay. Initial cerebrospinal fluid procalcitonin concentration (median [range]) in patients with severe traumatic brain injury was increased greater than three-fold vs. controls (0.41 ng/mL [0.15–2.14] vs. 0.12 ng/mL [0.00–0.24], p < .001). Initial cerebrospinal fluid procalcitonin concentration among patients with abusive head trauma (0.31 ng/mL [0.29–0.50]) also was increased vs. controls (p < .05), although this increase was less robust than patients with accidental trauma (0.41 ng/mL [0.15–2.14], p < .001 vs. controls). Additional examination of key demographic and outcome variables with a generalized linear regression model was performed for patients with severe traumatic brain injury. Univariate analysis revealed that both time after injury (p < .01) and abusive head trauma as a mechanism of injury (p < .001) were associated with attenuation of the increased cerebrospinal fluid procalcitonin response after traumatic brain injury. Conclusion Cerebrospinal fluid procalcitonin concentration is increased in children after traumatic brain injury. The attenuated increase in cerebrospinal fluid procalcitonin among victims of abusive head trauma warrants further study because it may reflect impairment of endogenous neuroprotective mechanisms or delay in seeking medical attention. The significance of these observations remains to be determined as future studies elucidate the physiologic and mechanistic properties of procalcitonin.

Increased Adrenomedullin in Cerebrospinal Fluid After Traumatic Brain Injury in Children: A Preliminary Report

Adrenomedullin is a recently discovered 52-amino-acid peptide that is a potent vasodilator. Infusion of adrenomedullin increases regional cerebral blood flow and reduces infarct volume after vascular occlusion in rats. Adrenomedullin may represent an endogenous neuroprotectant since it is increased after focal brain ischemia. Cerebral hypoperfusion is present after traumatic brain injury (TBI) in children. We hypothesized that adrenomedullin levels would be increased in children with severe TBI. Total adrenomedullin concentrations were measured using a radioimmunometric assay. Thirty-six samples of ventricular cerebrospinal fluid (CSF) from 10 pediatric patients were collected during the first 10 days after severe TBI (GCS < 8). Control CSF was obtained from 5 children undergoing lumbar puncture, who had normal CSF parameters and no evidence of central nervous system infection. Patients underwent standard neuro-intensive care, including cerebrospinal fluid drainage. Data were analyzed using a univariate regression model. Adrenomedullin concentration was markedly elevated in CSF of children following TBI versus control (mean level 10.65 vs 1.51 fmol/ml, p = 0.006). All 36 case samples had an adrenomedullin concentration above the median value for the controls (1.52 fmol/ml). We conclude adrenomedullin is elevated in the CSF of children following severe TBI. We speculate that it participates in the endogenous response to cerebral hypoperfusion after TBI.