Cerebrospinal Fluid Markers of Macrophage and Lymphocyte Activation After Traumatic Brain Injury in Children.

OBJECTIVES: The magnitude and role of the cellular immune response following pediatric traumatic brain injury remains unknown. We tested the hypothesis that macrophage/microglia and T-cell activation occurs following pediatric traumatic brain injury by measuring cerebrospinal fluid levels of soluble cluster of differentiation 163 and ferritin and soluble interleukin-2 receptor α, respectively, and determined whether these biomarkers were associated with relevant clinical variables and outcome. DESIGN: Retrospective analysis of samples from an established, single-center cerebrospinal fluid bank. SETTING: PICU in a tertiary children's hospital. PATIENTS: Sixty-six pediatric patients after severe traumatic brain injury (Glasgow Coma Scale score < 8) who were 1 month to 16 years old and 17 control patients who were 1 month to 14 years old. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Cerebrospinal fluid levels of soluble cluster of differentiation 163, ferritin, and soluble interleukin-2 receptor α were determined by enzyme-linked immunosorbent assay at two time points (t1 = 17 ± 10 hr; t2 = 72 ± 15 hr) for each traumatic brain injury patient. Cerebrospinal fluid levels of soluble cluster of differentiation 163, ferritin, and soluble interleukin-2 receptor α after traumatic brain injury were compared with controls and analyzed for associations with age, patient sex, initial Glasgow Coma Scale score, diagnosis of abusive head trauma, the presence of hemorrhage on CT scan, and Glasgow Outcome Scale score. Cerebrospinal fluid level of soluble cluster of differentiation 163 was increased in traumatic brain injury patients at t2 versus t1 and controls (median, 95.4 ng/mL [interquartile range, 21.8-134.0 ng/mL] vs 31.0 ng/mL [5.7-77.7 ng/mL] and 27.8 ng/mL [19.1-43.1 ng/mL], respectively; p < 0.05). Cerebrospinal fluid level of ferritin was increased in traumatic brain injury patients at t2 and t1 versus controls (8.3 ng/mL [<7.5-19.8 ng/mL] and 8.9 ng/mL [<7.5-26.7 ng/mL] vs <7.5 ng/mL below lower limit of detection, respectively; p < 0.05). Cerebrospinal fluid levels of soluble interleukin-2 receptor α in traumatic brain injury patients at t2 and t1 were not different versus controls. Multivariate regression revealed associations between high ferritin and age 4 years or younger, lower Glasgow Coma Scale score, abusive head trauma, and unfavorable Glasgow Outcome Scale score. CONCLUSIONS: Children with traumatic brain injury demonstrate evidence for macrophage activation after traumatic brain injury, and in terms of cerebrospinal fluid ferritin, this appears more prominent with young age, initial injury severity, abusive head trauma, and unfavorable outcome. Further study is needed to determine whether biomarkers of macrophage activation may be used to discriminate between aberrant and adaptive immune responses and whether inflammation represents a therapeutic target after traumatic brain injury.

Complications associated with prolonged hypertonic saline therapy in children with elevated intracranial pressure.

OBJECTIVES: Safe upper limits for therapeutic hypernatremia in the treatment of intracranial hypertension have not been well established. We investigated complications associated with hypernatremia in children who were treated with prolonged infusions of hypertonic saline. DESIGN: Retrospective chart analysis. SETTING: PICU in university-affiliated children's hospital. PATIENTS: All children from 2004 to 2009 requiring intracranial pressure monitoring (external ventricular drain or fiberoptic intraparenchymal monitor) for at least 4 days who were treated with hypertonic saline infusion for elevated intracranial pressure and did not meet exclusion criteria. INTERVENTION: Continuous hypertonic saline infusion on a sliding scale was used to achieve target sodium levels that would keep intracranial pressure less than 20 mm Hg once the conventional therapies failed. MEASUREMENTS AND MAIN RESULTS: Eighty-eight children met inclusion criteria. Etiologies of elevated intracranial pressure included trauma (n = 48), ischemic or hemorrhagic stroke (n = 20), infection (n = 8), acute disseminated encephalomyelitis (n = 5), neoplasm (n = 2), and others (n = 5). The mean peak serum sodium was 171.3 mEq/L (range, 150-202). The mean Glasgow Outcome Score was 2.8 (± 1.1) at time of discharge from the hospital. Overall mortality was 15.9%. Children with sustained (> 72 hr) serum sodium levels above 170 mEq/L had a significantly higher occurrence of thrombocytopenia (p < 0.001), renal failure (p < 0.001), neutropenia (p = 0.006), and acute respiratory distress syndrome (p = 0.029) after controlling for variables of age, gender, Pediatric Risk of Mortality score, duration of barbiturate-induced coma, duration of intracranial pressure monitoring, vasopressor requirements, and underlying pathology. Children with sustained serum sodium levels greater than 165 mEq/L had a significantly higher prevalence of anemia (p < 0.001). CONCLUSIONS: Children treated by continuous hypertonic saline infusion for intracranial hypertension whose serum sodium levels exceeded certain thresholds experienced significantly more events of acute renal failure, thrombocytopenia, neutropenia, anemia, and acute respiratory distress syndrome than those whose sodium level was maintained below these thresholds.

Polynitroxylated pegylated hemoglobin: a novel neuroprotective hemoglobin for acute volume-limited fluid resuscitation after combined traumatic brain injury and hemorrhagic hypotension in mice.

OBJECTIVE: Resuscitation of hemorrhagic hypotension after traumatic brain injury is challenging. A hemoglobin-based oxygen carrier may offer advantages. The novel therapeutic hemoglobin-based oxygen carrier, polynitroxylated pegylated hemoglobin (PNPH), may represent a neuroprotective hemoglobin-based oxygen carrier for traumatic brain injury resuscitation. HYPOTHESES: 1) PNPH is a unique non-neurotoxic hemoglobin-based oxygen carrier in neuronal culture and is neuroprotective in in vitro neuronal injury models. 2) Resuscitation with PNPH would require less volume to restore mean arterial blood pressure than lactated Ringer's or Hextend and confer neuroprotection in a mouse model of traumatic brain injury plus hemorrhagic hypotension. DESIGN: Prospective randomized, controlled experimental study. SETTING: University center. MEASUREMENTS AND MAIN RESULTS: In rat primary cortical neuron cultures, control bovine hemoglobin was neurotoxic (lactate dehydrogenase release; 3-[4,5-dimethylthiazol-2-yl-]-2,5-diphenyltetrazolium bromide assay) at concentrations from 12.5 to 0.625 µM, whereas polyethylene glycol-conjugated hemoglobin showed intermediate toxicity. PNPH was not neurotoxic (p<.05 vs. bovine hemoglobin and polyethylene glycol hemoglobin; all concentrations). PNPH conferred neuroprotection in in vitro neuronal injury (glutamate/glycine exposure and neuronal stretch), as assessed via lactate dehydrogenase and 3-[4,5-dimethylthiazol-2-yl-]-2,5-diphenyltetrazolium bromide (all p<.05 vs. control). C57BL6 mice received controlled cortical impact followed by hemorrhagic hypotension (2 mL/100 g, mean arterial blood pressure ∼35-40 mm Hg) for 90 min. Mice were resuscitated (mean arterial blood pressure>50 mm Hg for 30 min) with lactated Ringer's, Hextend, or PNPH, and then shed blood was reinfused. Mean arterial blood pressures, resuscitation volumes, blood gasses, glucose, and lactate were recorded. Brain sections at 7 days were examined via hematoxylin and eosin and Fluoro-Jade C (identifying dying neurons) staining in CA1 and CA3 hippocampus. Resuscitation with PNPH or Hextend required less volume than lactated Ringer's (both p<.05). PNPH but not Hextend improved mean arterial blood pressure vs. lactated Ringer's (p<.05). Mice resuscitated with PNPH had fewer Fluoro-Jade C positive neurons in CA1 vs. Hextend and lactated Ringer's, and CA3 vs. Hextend (p<.05). CONCLUSIONS: PNPH is a novel neuroprotective hemoglobin-based oxygen carrier in vitro and in vivo that may offer unique advantages for traumatic brain injury resuscitation.

Hemorrhagic shock shifts the serum cytokine profile from pro- to anti-inflammatory after experimental traumatic brain injury in mice.

Secondary insults, such as hemorrhagic shock (HS), worsen outcome from traumatic brain injury (TBI). Both TBI and HS modulate levels of inflammatory mediators. We evaluated the addition of HS on the inflammatory response to TBI. Adult male C57BL6J mice were randomized into five groups (n=4 [naïve] or 8/group): naïve; sham; TBI (through mild-to-moderate controlled cortical impact [CCI] at 5 m/sec, 1-mm depth), HS; and CCI+HS. All non-naïve mice underwent identical monitoring and anesthesia. HS and CCI+HS underwent a 35-min period of pressure-controlled hemorrhage (target mean arterial pressure, 25-27 mm Hg) and a 90-min resuscitation with lactated Ringer's injection and autologous blood transfusion. Mice were sacrificed at 2 or 24 h after injury. Levels of 13 cytokines, six chemokines, and three growth factors were measured in serum and in five brain tissue regions. Serum levels of several proinflammatory mediators (eotaxin, interferon-inducible protein 10 [IP-10], keratinocyte chemoattractant [KC], monocyte chemoattractant protein 1 [MCP-1], macrophage inflammatory protein 1alpha [MIP-1α], interleukin [IL]-5, IL-6, tumor necrosis factor alpha, and granulocyte colony-stimulating factor [G-CSF]) were increased after CCI alone. Serum levels of fewer proinflammatory mediators (IL-5, IL-6, regulated upon activation, normal T-cell expressed, and secreted, and G-CSF) were increased after CCI+HS. Serum level of anti-inflammatory IL-10 was significantly increased after CCI+HS versus CCI alone. Brain tissue levels of eotaxin, IP-10, KC, MCP-1, MIP-1α, IL-6, and G-CSF were increased after both CCI and CCI+HS. There were no significant differences between levels after CCI alone and CCI+HS in any mediator. Addition of HS to experimental TBI led to a shift toward an anti-inflammatory serum profile--specifically, a marked increase in IL-10 levels. The brain cytokine and chemokine profile after TBI was minimally affected by the addition of HS.

Screening of biochemical and molecular mechanisms of secondary injury and repair in the brain after experimental blast-induced traumatic brain injury in rats.

Abstract Explosive blast-induced traumatic brain injury (TBI) is the signature insult in modern combat casualty care and has been linked to post-traumatic stress disorder, memory loss, and chronic traumatic encephalopathy. In this article we report on blast-induced mild TBI (mTBI) characterized by fiber-tract degeneration and axonal injury revealed by cupric silver staining in adult male rats after head-only exposure to 35 psi in a helium-driven shock tube with head restraint. We now explore pathways of secondary injury and repair using biochemical/molecular strategies. Injury produced ∼25% mortality from apnea. Shams received identical anesthesia exposure. Rats were sacrificed at 2 or 24 h, and brain was sampled in the hippocampus and prefrontal cortex. Hippocampal samples were used to assess gene array (RatRef-12 Expression BeadChip; Illumina, Inc., San Diego, CA) and oxidative stress (OS; ascorbate, glutathione, low-molecular-weight thiols [LMWT], protein thiols, and 4-hydroxynonenal [HNE]). Cortical samples were used to assess neuroinflammation (cytokines, chemokines, and growth factors; Luminex Corporation, Austin, TX) and purines (adenosine triphosphate [ATP], adenosine diphosphate, adenosine, inosine, 2'-AMP [adenosine monophosphate], and 5'-AMP). Gene array revealed marked increases in astrocyte and neuroinflammatory markers at 24 h (glial fibrillary acidic protein, vimentin, and complement component 1) with expression patterns bioinformatically consistent with those noted in Alzheimer's disease and long-term potentiation. Ascorbate, LMWT, and protein thiols were reduced at 2 and 24 h; by 24 h, HNE was increased. At 2 h, multiple cytokines and chemokines (interleukin [IL]-1α, IL-6, IL-10, and macrophage inflammatory protein 1 alpha [MIP-1α]) were increased; by 24 h, only MIP-1α remained elevated. ATP was not depleted, and adenosine correlated with 2'-cyclic AMP (cAMP), and not 5'-cAMP. Our data reveal (1) gene-array alterations similar to disorders of memory processing and a marked astrocyte response, (2) OS, (3) neuroinflammation with a sustained chemokine response, and (4) adenosine production despite lack of energy failure-possibly resulting from metabolism of 2'-3'-cAMP. A robust biochemical/molecular response occurs after blast-induced mTBI, with the body protected from blast and the head constrained to limit motion.

Severe brief pressure-controlled hemorrhagic shock after traumatic brain injury exacerbates functional deficits and long-term neuropathological damage in mice.

Hypotension after traumatic brain injury (TBI) worsens outcome. We published the first report of TBI plus hemorrhagic shock (HS) in mice using a volume-controlled approach and noted increased neuronal death. To rigorously control blood pressure during HS, a pressure-controlled HS model is required. Our hypothesis was that a brief, severe period of pressure-controlled HS after TBI in mice will exacerbate functional deficits and neuropathology versus TBI or HS alone. C57BL6 male mice were randomized into four groups (n=10/group): sham, HS, controlled cortical impact (CCI), and CCI+HS. We used a pressure-controlled shock phase (mean arterial pressure [MAP]=25-27 mm Hg for 35 min) and its treatment after mild to moderate CCI including, a 90 min pre-hospital phase, during which lactated Ringer's solution was given to maintain MAP >70 mm Hg, and a hospital phase, when the shed blood was re-infused. On days 14-20, the mice were evaluated in the Morris water maze (MWM, hidden platform paradigm). On day 21, the lesion and hemispheric volumes were quantified. Neuropathology and hippocampal neuron counts (hematoxylin and eosin [H&E], Fluoro-Jade B, and NeuN) were evaluated in the mice (n=60) at 24 h, 7 days, or 21 days (n=5/group/time point). HS reduced MAP during the shock phase in the HS and CCI+HS groups (p<0.05). Fluid requirements during the pre-hospital phase were greatest in the CCI+HS group (p<0.05), and were increased in HS versus sham and CCI animals (p<0.05). MWM latency was increased on days 14 and 15 after CCI+HS (p<0.05). Swim speed and visible platform latency were impaired in the CCI+HS group (p<0.05). CCI+HS animals had increased contusion volume versus the CCI group (p<0.05). Hemispheric volume loss was increased 33.3% in the CCI+HS versus CCI group (p<0.05). CA1 cell loss was seen in CCI+HS and CCI animals at 24 h and 7 days (p<0.05). CA3 cell loss was seen after CCI+HS (p<0.05 at 24 h and 7 days). CA1 cell loss at 21 days was seen only in CCI+HS animals (p<0.05). Brief, severe, pressure-controlled HS after CCI produces robust functional deficits and exacerbates neuropathology versus CCI or HS alone.

Adenosine A1 receptor activation as a brake on the microglial response after experimental traumatic brain injury in mice.

We reported that adenosine A(1) receptor (A(1)AR) knockout (KO) mice develop lethal status epilepticus after experimental traumatic brain injury (TBI), which is not seen in wild-type (WT) mice. Studies in epilepsy, multiple sclerosis, and neuro-oncology suggest enhanced neuro-inflammation and/or neuronal death in A(1)AR KO. We hypothesized that A(1)AR deficiency exacerbates the microglial response and neuronal damage after TBI. A(1)AR KO and WT littermates were subjected to mild controlled cortical impact (3 m/sec; 0.5 mm depth) to left parietal cortex, an injury level below the acute seizure threshold in the KO. At 24 h or 7 days, mice were sacrificed and serial sections prepared. Iba-1 immunostaining was used to quantify microglia at 7 days. To assess neuronal injury, sections were stained with Fluoro-Jade C (FJC) at 24 h to evaluate neuronal death in the hippocampus and cresyl violet staining at 7 days to analyze cortical lesion volumes. We also studied the effects of adenosine receptor agonists and antagonists on (3)H-thymidine uptake (proliferation index) by BV-2 cells (immortalized mouse microglial). There was no neuronal death in CA1 or CA3 quantified by FJC. A(1)AR KO mice exhibited enhanced microglial response; specifically, Iba-1 + microglia were increased 20-50% more in A(1)AR KO versus WT in ipsilateral cortex, CA3, and thalamus, and contralateral cortex, CA1, and thalamus (p < 0.05). However, contusion and cortical volumes did not differ between KO and WT. Pharmacological studies in cultured BV-2 cells indicated that A(1)AR activation inhibits microglial proliferation. A(1)AR activation is an endogenous inhibitor of the microglial response to TBI, likely via inhibition of proliferation, and this may represent a therapeutic avenue to modulate microglia after TBI.

Resuscitation of traumatic brain injury and hemorrhagic shock with polynitroxylated albumin, hextend, hypertonic saline, and lactated Ringer's: Effects on acute hemodynamics, survival, and neuronal death in mice.

Outcome after traumatic brain injury (TBI) is worsened by hemorrhagic shock (HS), but the optimal resuscitation approach is unclear. In particular, treatment of TBI patients with colloids remains controversial. We hypothesized that resuscitation with the colloids polynitroxylated albumin (PNA) or Hextend (HEX) is equal or superior to resuscitation with the crystalloids hypertonic (3%) saline (HTS) or lactated Ringer's solution (LR) after TBI plus HS in mice. C57/BL6 mice (n = 30) underwent controlled cortical impact (CCI) and 90 min of volume-controlled HS (2 mL/100 g). The mice were randomized to resuscitation with LR, HEX, HTS, or PNA, followed by 30 min of test fluid administration targeting a mean arterial pressure (MAP) of >50 mm Hg. Shed blood was re-infused to target a MAP >70 mm Hg. At 7 days post-insult, hippocampal neuron counts were assessed in hematoxylin and eosin-stained sections to quantify neuronal damage. Prehospital MAP was higher, and prehospital and total fluid requirements were lower in the PNA and HEX groups (p < 0.05 versus HTS or LR). Also, 7-day survival was highest in the PNA group, but was not significantly different than the other groups. Ipsilateral hippocampal CA1 and CA3 neuron loss did not differ between groups. We conclude that the colloids PNA and HEX exhibited more favorable effects on acute resuscitation parameters than HTS or LR, and did not increase hippocampal neuronal death in this model.