Keri L. Janesko

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.

Marked gender effect on lipid peroxidation after severe traumatic brain injury in adult patients.

Striking gender differences have been reported in the pathophysiology and outcome of acute neurological injury. Greater neuroprotection in females versus males may be due, in part, to direct and indirect sex hormone-mediated antioxidant mechanisms. Progesterone administration decreases brain levels of F(2)-isoprostane, a marker of lipid peroxidation, after experimental traumatic brain injury (TBI) in male rats, and estrogen is neuroprotective in experimental neurological injury. In this study, we evaluated the effect of gender on lipid peroxidation, as assessed by cerebrospinal fluid (CSF) levels of F(2)-isoprostane, after severe TBI in humans. Lipid peroxidation was assessed in CSF from 68 adults enrolled in two randomized controlled trials evaluating the effect of therapeutic hypothermia after severe TBI (Glasgow coma scale [GCS] score </= 8). Patients treated with hypothermia (n = 41, 12 females, 29 males) were cooled to 32-33 degrees C (within approximately 6 h) for either 24 or 48 h and then re-warmed. F(2)-isoprostane levels were assessed by ELISA in ventricular CSF samples (n = 199) on day 1, 2, and 3. The association between age, GCS score, time, gender, treatment, duration of treatment, core temperature at the time of CSF sampling, secondary hypoxemia, and CSF F(2)-isoprostane level was assessed by multivariate and dichotomous analyses. F(2)-isoprostane was approximately 2-fold higher in males than females (145.8 +/- 39.6 versus 75.4 +/- 16.6 pg/mL, day 1 p = 0.018). An effect of time after injury (p = 0.007) was reflected by a marked early peak in F(2)-isoprostane (day 1). CSF F(2)-isoprostane was also associated with hypoxemia (p = 0.04). Hypothermia tended to decrease F(2)-isoprostane levels only in males on d1 after TBI. To our knowledge, this is the first study showing gender differences in lipid peroxidation after clinical TBI. Lipid peroxidation occurs early after severe TBI in adults and is more prominent in males vs females. These results established that gender is an important consideration in clinical trial design, particularly in the case of antioxidant strategies.

Adenosine A1 receptor knockout mice develop lethal status epilepticus after experimental traumatic brain injury.

Adenosine, acting at A1 receptors, exhibits anticonvulsant effects in experimental epilepsy—and inhibits progression to status epilepticus (SE). Seizures after traumatic brain injury (TBI) may contribute to pathophysiology. Thus, we hypothesized that endogenous adenosine, acting via A1 receptors, mediates antiepileptic benefit after experimental TBI. We subjected A1-receptor knockout (ko) mice, heterozygotes, and wild-type (wt) littermates (n = 115) to controlled cortical impact (CCI). We used four outcome protocols in male mice: (1) observation for seizures, SE, and mortality in the initial 2 h, (2) assessment of seizure score (electroencephalogram (EEG)) in the initial 2 h, (3) assessment of mortality at 24 h across injury levels, and (4) serial assessment of arterial blood pressure, heart rate, blood gases, and hematocrit. Lastly, to assess the influence of gender on this observation, we observed female mice for seizures, SE, and mortality in the initial 2 h. Seizure activity was noted in 83% of male ko mice in the initial 2 h, but was seen in no heterozygotes and only 33% of wt (P < 0.05). Seizures in wt were brief (1 to 2 secs). In contrast, SE involving lethal sustained (>1 h) tonic clonic activity was uniquely seen in ko mice after CCI (50% incidence in males), (P < 0.05). Seizure score was twofold higher in ko mice after CCI versus either heterozygote or wt (P < 0.05). An injury-intensity dose–response for 24 h mortality was seen in ko mice (P < 0.05). Physiologic parameters were similar between genotypes. Seizures were seen in 100% of female ko mice after CCI versus 14% of heterozygotes and 25% wt (P < 0.05) and SE was restricted to the ko mice (83% incidence). Our data suggest a critical endogenous anticonvulsant action of adenosine at A1 receptors early after experimental TBI.

Enhanced Oxidative Stress in iNOS-Deficient Mice after Traumatic Brain Injury: Support for a Neuroprotective Role of iNOS

Studies in experimental traumatic brain injury (TBI) suggest both deleterious and protective effects of inducible nitric oxide synthase (iNOS). Early after injury, iNOS may be detrimental via formation of peroxynitrite and iNOS inhibitors are protective. In contrast, we reported impaired long-term functional outcome after TBI in iNOS knockout (ko) versus wild-type (wt) mice. To elucidate potential neuroprotective and neurotoxic mechanisms for iNOS, we studied nitric oxide formation by electron paramagnetic resonance (EPR) spectroscopy using diethyldithiocarbamate—iron (DETC—Fe) as a spin trap and markers of nitrosative (S-nitrosothiol (RSNO, Fluorescent assay); nitrotyrosine (3NT, ELISA)) and oxidative stress (ascorbate, HPLC) at 72 h after controlled cortical impact (CCI) in iNOS ko and wt and in uninjured iNOS ko and wt mice. 3NT immunostaining with macrophage and myeloperoxidase (MPO) dual labeling was also assessed in brain sections. Brain DETC—Fe—NO low-temperature EPR signal intensity was ∼2-fold greater in wt versus iNOS ko at 72 h after CCI. Ascorbate levels decreased in injured hemisphere in wt and iNOS ko versus uninjured —this decrease was more pronounced in iNOS ko. In wt mice, RSNO and 3NT levels were increased after CCI versus uninjured (50% and 400%, respectively, P<0.05). RSNO levels were not increased in iNOS ko after CCI. Nitrotyrosine levels increased after CCI in wt and ko versus respective uninjured —this increase was more pronounced in wt (2.34±0.95 versus 1.27±0.49 pmol/mg protein, P<0.05). Increased 3NT immunoreactivity was detected in wt versus iNOS ko at 72 h after CCI, and colocalized with macrophage marker and MPO. Our data support a role for iNOS-derived NO as an endogenous antioxidant after CCI. iNOS also contributes protein nitrosylation and nitration. Colocalization of 3NT with macrophages and MPO suggests generation of nitrating agents by macrophages and/or phagocytosis of nitrated proteins.

F2-isoprostane and neuron-specific enolase in cerebrospinal fluid after severe traumatic brain injury in infants and children.

It has been hypothesized that oxidative stress plays an important role in mediating secondary damage after traumatic brain injury (TBI). To study the relationship between lipid peroxidation, clinical variables, and neuronal damage in pediatric TBI, we measured levels of F2-isoprostane, a marker of lipid peroxidation, and neuron-specific enolase (NSE), a marker of neuronal damage, in serial cerebrospinal fluid (CSF) samples from 23 infants and children with severe TBI (Glasgow Coma Scale score <8). These were compared to CSF samples from 10 uninjured pediatric controls. On d1 after injury, F2-isoprostane was increased 6-fold vs. control (36.59+/-8.96 pg/ml vs. 5.64+/-8.08 pg/ml, p=0.0035) and NSE was increased 10-fold (100.62+/-17.34 ng/ml vs. 8.63+/-2.76 ng/ml, p=0.0002). Multivariate analysis of F2-isoprostane levels and selected clinical variables showed a trend toward an inverse association with time after injury (p=0.0708). Multivariate analysis of NSE levels and selected variables showed a positive association between d1 NSE and F2-isoprostane (p=0.0426). CSF F2-isoprostane increases early after TBI in infants and children and is correlated with NSE, supporting a role for oxidative stress in the evolution of secondary damage early after severe TBI in infants and children.

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.

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.

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.

Hyperglycolysis is exacerbated after traumatic brain injury with fentanyl vs. isoflurane anesthesia in rats

Despite common use of narcotics in the clinical management of severe traumatic brain injury (TBI), in experimental models rats treated with fentanyl have exhibited worse functional outcome and more CA1 hippocampal death than rats treated with standard isoflurane anesthesia. We hypothesized that greater post-traumatic excitotoxicity, reflected by cerebral glucose utilization (CMRglu), may account for detrimental effects of fentanyl vs. isoflurane. Rats were anesthetized with either isoflurane (1% by inhalation) or fentanyl (10 mcg/kg iv bolus then 50 mcg/kg/h infusion). 14C-deoxyglucose autoradiography was performed 45 min after controlled cortical impact (CCI) to left parietal cortex (n=4 per anesthetic group) or in uninjured rats after 45 min of anesthesia (n=3 per anesthetic group). Uninjured rats treated with fentanyl vs. isoflurane showed 35-45% higher CMRglu in all brain structures (p<0.05) except CA3. After TBI in rats treated with isoflurane, CMRglu increased significantly only in ipsilateral CA1 and ipsilateral parietal cortex (p<0.05 vs. isoflurane uninjured). Conversely, after TBI in rats treated with fentanyl, CMRglu increased markedly and bilaterally in CA1 and CA3 (p<0.05 vs. fentanyl uninjured), but not ipsilateral parietal cortex. In contralateral CA1, CMRglu was nearly two times greater after TBI in fentanyl vs. isoflurane treated rats (p<0.05). Hyperglycolysis was exacerbated in CA1 and CA3 hippocampus after TBI in rats treated with fentanyl vs. isoflurane anesthesia. This post-traumatic hyperglycolysis suggests greater excitotoxicity and concurs with reports of worse functional outcome and more CA1 hippocampal death after TBI with fentanyl vs. isoflurane anesthesia.

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.