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]-1a, IL-6, IL-10, and macrophage inflammatory protein 1 alpha ) were increased; by 24 h, only MIP-1a 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.

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.

Objective Resuscitation of hemorrhagic hypotension after traumatic brain injury (TBI) is challenging. A hemoglobin (Hb)-based oxygen carrier (HBOC) may offer advantages. The novel therapeutic HBOC, polynitroxylated pegylated Hb (PNPH) may represent a neuroprotective HBOC for TBI resuscitation. Hypotheses 1) PNPH is a unique non-neurotoxic HBOC 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 (MAP) than lactated Ringer’s (LR) or Hextend (HEX) and confer neuroprotection in a mouse model of TBI plus hemorrhagic hypotension. Design Prospective randomized controlled experimental study. Setting University center. Measurements and Main Results In rat primary cortical neuron cultures, control bovine Hb was neurotoxic (LDH release; MTT assay) at concentrations from 12.5 to 0.625µM, while polyethylene-glycol (Peg)-conjugated Hb showed intermediate toxicity. PNPH was not neurotoxic (p<0.05 vs bovine Hb and Peg-Hb; all concentrations). PNPH conferred neuroprotection in in vitro neuronal injury (glutamate/glycine exposure and neuronal stretch), as assessed via LDH, and MTT all p<0.05 vs control. C57BL6 mice received controlled cortical impact followed by hemorrhagic hypotension (2mL/100g, MAP ~35–40 mmHg) for 90 min. Mice were resuscitated (MAP >50 mmHg for 30 min) with LR, HEX, or PNPH, then shed blood was re-infused. MAPs, resuscitation volumes, blood gasses, glucose and lactate were recorded. Brain sections at 7d were examined via H&E and Fluoro-Jade C (FJC, identifying dying neurons) staining in CA1 and CA3 hippocampus. Resuscitation with PNPH or HEX required less volume than LR (both p<0.05). PNPH but not HEX improved MAP vs. LR (p<0.05). Mice resuscitated with PNPH had fewer FJC+ neurons in CA1 vs. HEX and LR, and CA3 vs. HEX (p<0.05). Conclusion PNPH is a novel neuroprotective HBOC in vitro and in vivo that may offer unique advantages for TBI resuscitation.

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.

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 ¼ 30) underwent controlled cortical impact (CCI) and 90 min of volumecontrolled 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.

Objective The magnitude and role of the cellular immune response following pediatric traumatic brain injury (TBI) remains unknown. We tested the hypothesis that macrophage/microglia and T-cell activation occurs following pediatric TBI by measuring cerebrospinal fluid (CSF) levels of sCD163 and ferritin, and sIL-2Rα, respectively, and determined whether these biomarkers were associated with relevant clinical variables and outcome. Design Retrospective analysis of samples from an established, single-center CSF bank. Setting Pediatric Intensive Care Unit (PICU) in a tertiary Children’s Hospital Patients Sixty-six pediatric patients after severe TBI (Glasgow coma scale score [GCS]<8) age 1 mo-16 y and 17 control patients age 1 mo-14 y. Measurements and Main Results CSF levels of sCD163, ferritin, and sIL-2Rα were determined by ELISA at 2 time points (t1=17±10, t2=72±15 h) for each TBI patient. CSF sCD163, ferritin, and sIL2Rα levels after TBI were compared with controls and analyzed for associations with age, patient sex, initial GCS, diagnosis of abusive head trauma (AHT), the presence of hemorrhage on computerized tomography scan, and Glasgow outcome scale score (GOS). CSF sCD163 was increased in TBI patients at t2 vs. t1 and controls (95.4[21.8–134.0] vs. 31.0[5.7–77.7] and 27.8[19.1–43.1] ng/ml, respectively; median[IQ]; P<0.05). CSF ferritin was increased in TBI patients at t2 and t1 vs. controls (8.3[7.5–19.8] and 8.9[7.5–26.7] vs. [7.5[0.0–0.0] ng/ml, respectively; P<0.05). CSF sIL-2Rα in TBI patients at t2 and t1 were not different vs. controls. Multivariate regression revealed associations between high ferritin and age ≤ 4 y, lower GCS, AHT, and unfavorable GOS. Conclusions Children with TBI demonstrate evidence for macrophage activation after TBI, and in terms of CSF ferritin, this appears more prominent with young age, initial injury severity, AHT, 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 TBI.

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.

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.