David Shellington

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 Ringers 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.

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 Ringers 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 &mgr;M, 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 Ringers, 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 Ringers (both p < .05). PNPH but not Hextend improved mean arterial blood pressure vs. lactated Ringers (p < .05). Mice resuscitated with PNPH had fewer Fluoro-Jade C positive neurons in CA1 vs. Hextend and lactated Ringers, 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.

Differences in medical therapy goals for children with severe traumatic brain injury-an international study.

Objectives: To describe the differences in goals for their usual practice for various medical therapies from a number of international centers for children with severe traumatic brain injury. Design: A survey of the goals from representatives of the international centers. Setting: Thirty-two pediatric traumatic brain injury centers in the United States, United Kingdom, France, and Spain. Patients: None. Interventions: None. Measurements and Main Results: A survey instrument was developed that required free-form responses from the centers regarding their usual practice goals for topics of intracranial hypertension therapies, hypoxia/ischemia prevention and detection, and metabolic support. Cerebrospinal fluid diversion strategies varied both across centers and within centers, with roughly equal proportion of centers adopting a strategy of continuous cerebrospinal fluid diversion and a strategy of no cerebrospinal fluid diversion. Use of mannitol and hypertonic saline for hyperosmolar therapies was widespread among centers (90.1% and 96.9%, respectively). Of centers using hypertonic saline, 3% saline preparations were the most common but many other concentrations were in common use. Routine hyperventilation was not reported as a standard goal and 31.3% of centers currently use PbO2 monitoring for cerebral hypoxia. The time to start nutritional support and glucose administration varied widely, with nutritional support beginning before 96 hours and glucose administration being started earlier in most centers. Conclusions: There were marked differences in medical goals for children with severe traumatic brain injury across our international consortium, and these differences seemed to be greatest in areas with the weakest evidence in the literature. Future studies that determine the superiority of the various medical therapies outlined within our survey would be a significant advance for the pediatric neurotrauma field and may lead to new standards of care and improved study designs for clinical trials.

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 Ringers 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.

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 Ringers 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.

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 &agr;, 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 &agr; 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 &agr; 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 &agr; 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.

An analysis of cervical spine magnetic resonance imaging findings after normal computed tomographic imaging findings in pediatric trauma patients: ten-year experience of a level I pediatric trauma center.

BACKGROUND A variety of radiologic screening protocols exist for evaluation of pediatric trauma patients with potential cervical spine (c-spine) injuries. The purpose of this study was to describe findings on c-spine magnetic resonance imaging (MRI) after previously normal c-spine computed tomographic (CT) scan findings at a Level 1 trauma center. METHODS A retrospective chart review of trauma patients evaluated at Rady Children’s Hospital, San Diego, between January 2000 and February 2010 was conducted. Trauma patients who were younger than 18 years, placed in c-spine precautions, had a normal c-spine CT scan, who subsequently had a c-spine MRI were included. The sample was subdivided into patients who underwent CT scans between January 1, 2000 to July 31, 2005 (early group), and August 1, 2005 to February 28, 2010 (late group), to compare results between different CT scan resolutions. RESULTS A total of 173 patients met inclusion criteria. With 100% of patients demonstrating normal c-spine CT scan findings, 83% of c-spine MRI findings were also negative (p < 0.001). Thirty patients (17%) demonstrated significant abnormalities on MRI. Of the 30, 5 (2.9%) required operative c-spine stabilization. Eighty-five patients underwent CT scan in the early group, and 88 in the late group. All 5 patients with unstable injuries not discovered on CT scan were from the early group, compared with none in the late group (p = 0.027). CONCLUSION Our results suggest that high-resolution CT scan with sagittal and coronal reconstructions may be comparable with MRI for the detection of unstable c-spine injuries in pediatric trauma patients. Although minimizing CT scan radiation exposure remains essential, high-resolution c-spine CT scan may allow for earlier c-spine clearance with reduction of associated hard collar comorbidities in centers where MRI is not available or in situations where the patient’s clinical stability precludes obtaining MRI. LEVEL OF EVIDENCE Diagnostic study, level III.

Heat Illness and Hypothermia

Children have impaired ability for effective thermoregulation. Environmental exposures and impaired intrinsic thermoregulation can lead to the catastrophic consequences from heat and cold injuries. The most severe form of heat related injury, heat stroke, involves extreme hyperthermia, neurologic impairment and progression to systemic inflammation, disseminated intravascular coagulation and multiple organ dysfunction syndrome (MODS). The mechanism of injury involves the inflammatory cascade, heat shock proteins, gut endotoxin translocation, and the systemic effects of direct thermal injury. Immediate rapid cooling and support of organ function are the mainstays of treatment. Accidental cold injuries commonly present with associated trauma. Regional frostbite and systemic deterioration are progressive with prolonged exposures. Predictable clinical sequelae including neurologic depression and eventual cardiopulmonary arrest occur if uninterrupted. Aggressive rewarming techniques including extracorporeal methods have the potential for excellent neurologic outcomes even in severe cases.

1312: TRANSIENT CENTRAL DIABETES INSIPIDUS IN AN 8 YEAR-OLD WITH A CONCUSSION – A CASE REPORT

Introduction: Central diabetes insipidus (DI) has been identified as a result of traumatic brain injury. One retrospective study conducted over a 4-year period found a 2.9% incidence of central DI in traumatic brain injury patients. Another study found that 26% of its population developed central DI