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.

MRI assessment of cerebral blood flow after experimental traumatic brain injury combined with hemorrhagic shock in mice.

Secondary insults such as hypotension or hemorrhagic shock (HS) can greatly worsen outcome after traumatic brain injury (TBI). We recently developed a mouse combined injury model of TBI and HS using a controlled cortical impact (CCI) model and showed that 90 minutes of HS can exacerbate neuronal death in hippocampus beneath the contusion. This combined injury model has three clinically relevant phases, a shock, pre hospital, and definitive care phases. Mice were randomly assigned to four groups, shams as well as a CCI only, an HS only, and a CCI+HS groups. The CCI and HS reduced cerebral blood flow (CBF) in multiple regions of interest (ROIs) in the hemisphere ipsilateral and contralateral to injury. Hemorrhagic shock to a level of ∼30 mm Hg exacerbated the CCI-induced CBF reductions in multiple ROIs ipsilateral to injury (hemisphere and thalamus) and in the hemisphere contralateral to injury (hemisphere, thalamus, hippocampus, and cortex, all P<0.05 versus CCI only, HS only or both). An important effect of HS duration was also seen after CCI with maximal CBF reduction seen at 90 minutes (P<0.0001 group-time effect in ipsilateral hippocampus). Given that neuronal death in hippocampus is exacerbated by 90 minutes of HS in this model, our data suggest an important role for exacerbation of posttraumatic ischemia in mediating the secondary injury in CCI plus HS. In conclusion, the serial, non invasive assessment of CBF using ASL-MRI (magnetic resonance imaging with arterial spin labeling) is feasible in mice even in the complex setting of combined CCI+HS. The impact of resuscitation therapies and various mutant mouse strains on CBF and other outcomes merits investigation in this model.

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.

Blast exposure in rats with body shielding is characterized primarily by diffuse axonal injury.

Blast-induced traumatic brain injury (TBI) is the signature insult in combat casualty care. Survival with neurological damage from otherwise lethal blast exposures has become possible with body armor use. We characterized the neuropathologic alterations produced by a single blast exposure in rats using a helium-driven shock tube to generate a nominal exposure of 35 pounds per square inch (PSI) (positive phase duration ∼ 4 msec). Using an IACUC-approved protocol, isoflurane-anesthetized rats were placed in a steel wedge (to shield the body) 7 feet inside the end of the tube. The left side faced the blast wave (with head-only exposure); the wedge apex focused a Mach stem onto the rat's head. The insult produced ∼ 25% mortality (due to impact apnea). Surviving and sham rats were perfusion-fixed at 24 h, 72 h, or 2 weeks post-blast. Neuropathologic evaluations were performed utilizing hematoxylin and eosin, amino cupric silver, and a variety of immunohistochemical stains for amyloid precursor protein (APP), glial fibrillary acidic protein (GFAP), ionized calcium-binding adapter molecule 1 (Iba1), ED1, and rat IgG. Multifocal axonal degeneration, as evidenced by staining with amino cupric silver, was present in all blast-exposed rats at all time points. Deep cerebellar and brainstem white matter tracts were most heavily stained with amino cupric silver, with the morphologic staining patterns suggesting a process of diffuse axonal injury. Silver-stained sections revealed mild multifocal neuronal death at 24 h and 72 h. GFAP, ED1, and Iba1 staining were not prominently increased, although small numbers of reactive microglia were seen within areas of neuronal death. Increased blood-brain barrier permeability (as measured by IgG staining) was seen at 24 h and primarily affected the contralateral cortex. Axonal injury was the most prominent feature during the initial 2 weeks following blast exposure, although degeneration of other neuronal processes was also present. Strikingly, silver staining revealed otherwise undetected abnormalities, and therefore represents a recommended outcome measure in future studies of blast TBI.

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.

Regional calcineurin subunit B isoform expression in rat hippocampus following a traumatic brain injury.

Calcineurin subunit isoforms are implicated in long term potentiation, long term depression, and structural plasticity. Calcineurin inhibitors benefit axonal damage, cellular dysfunction, and cognitive outcomes in animal models of traumatic brain injury (TBI). Distribution of the catalytic calcineurin A subunit is altered and calcineurin activity increased following fluid percussion injury. Alterations in calcineurin subunit A isoform distribution within the hippocampus also occur post controlled cortical impact (CCI) demonstrating a reduction in catalytic subunit distribution in CA1-2 dendritic fields. Furthermore the effect of TBI on the regulatory subunit, calcineurin B, is unknown. Understanding the role of both subunits is necessary to effectively target alterations in calcineurin signaling as current calcineurin inhibitors, such as cyclosporin A and FK-506, rely upon binding sites on both subunits for complete inhibition. The effect of moderate CCI on the expression and distribution of calcineurin B isoforms within the hippocampus was examined at 2h and 2weeks post injury. Calcineurin B isoforms showed increased expression throughout the CA1 and CA2 while there was a decrease in expression within the ipsilateral dentate gyrus. Alterations in CnB isoform expression within the CA1, CA1-2, and dentate gyrus have significant implications for persistent hippocampal dysfunction following TBI. Regional changes in regulatory subunit expression may alter the effect of calcineurin inhibitors regionally following a traumatic brain injury.

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.

Expression of protein phosphatase 2B (calcineurin) subunit A isoforms in rat hippocampus after traumatic brain injury.

Calcineurin (CaN) is a calcium/calmodulin-dependent phosphatase directly activated by calcium as a result of neuronal activation that is important for neuronal function. CaN subunit isoforms are implicated in long-term potentiation (LTP), long-term depression (LTD), and structural plasticity. CaN inhibitors are also beneficial to cognitive outcomes in animal models of traumatic brain injury (TBI). There are known changes in the CaN A (CnA) subunit following fluid percussion injury (FPI). The CnA subunit has two isoforms: CnAalpha and CnAbeta. The effect of moderate controlled cortical impact (CCI) on distribution of CnA isoforms was examined at 2 h and 2 weeks post-injury. CnA distribution was assayed by immunohistochemistry and graded for non-parametric analysis. Acutely CnA isoforms showed reduced immunoreactivity in stratum radiatum processes of the ipsilateral CA1 and CA1-2. There was also a significant alteration in the immunoreactivity of both CnA isoforms in the ipsilateral dentate gyrus, predominantly within the hidden blade. Alterations in CnA isoform regional distribution within the CA1, CA1-2, and dentate gyrus may have significant implications for persistent hippocampal dysfunction following TBI, including dysfunction in hippocampal plasticity. Understanding alterations in CnA isoform distribution may help improve the targeting of current therapeutic interventions and/or the development of new treatments for 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.

Starving neurons show sex difference in autophagy.

Sex-dependent differences in adaptation to famine have long been appreciated, thought to hinge on female versus male preferences for fat versus protein sources, respectively. However, whether these differences can be reduced to neurons, independent of typical nutrient depots, such as adipose tissue, skeletal muscle, and liver, was heretofore unknown. A vital adaptation to starvation is autophagy, a mechanism for recycling amino acids from organelles and proteins. Here we show that segregated neurons from males in culture are more vulnerable to starvation than neurons from females. Nutrient deprivation decreased mitochondrial respiration, increased autophagosome formation, and produced cell death more profoundly in neurons from males versus females. Starvation-induced neuronal death was attenuated by 3-methyladenine, an inhibitor of autophagy; Atg7 knockdown using small interfering RNA; or L-carnitine, essential for transport of fatty acids into mitochondria, all more effective in neurons from males versus females. Relative tolerance to nutrient deprivation in neurons from females was associated with a marked increase in triglyceride and free fatty acid content and a cytosolic phospholipase A2-dependent increase in formation of lipid droplets. Similar sex differences in sensitivity to nutrient deprivation were seen in fibroblasts. However, although inhibition of autophagy using Atg7 small interfering RNA inhibited cell death during starvation in neurons, it increased cell death in fibroblasts, implying that the role of autophagy during starvation is both sex- and tissue-dependent. Thus, during starvation, neurons from males more readily undergo autophagy and die, whereas neurons from females mobilize fatty acids, accumulate triglycerides, form lipid droplets, and survive longer.

Hemorrhagic shock after experimental traumatic brain injury in mice: effect on neuronal death.

Traumatic brain injury (TBI) from blast injury is often complicated by hemorrhagic shock (HS) in victims of terrorist attacks. Most studies of HS after experimental TBI have focused on intracranial pressure; few have explored the effect of HS on neuronal death after TBI, and none have been done in mice. We hypothesized that neuronal death in CA1 hippocampus would be exacerbated by HS after experimental TBI. C57BL6J male mice were anesthetized with isoflurane, mean arterial blood pressure (MAP) was monitored, and controlled cortical impact (CCI) delivered to the left parietal cortex followed by continued anesthesia (CCI-only), or either 60 or 90 min of volume-controlled HS. Parallel 60- or 90-min HS-only groups were also studied. After HS (+/-CCI), 6% hetastarch was used targeting MAP of > or =50 mm Hg during a 30-min Pre-Hospital resuscitation phase. Then, shed blood was re-infused, and hetastarch was given targeting MAP of > or =60 mm Hg during a 30-min Definitive Care phase. Neurological injury was evaluated at 24 h (fluorojade C) or 7 days (CA1 and CA3 hippocampal neuron counts). HS reduced MAP to 30-40 mm Hg in all groups, p < 0.05 versus CCI-only. Ipsilateral CA1 neuron counts in the 90-min CCI+HS group were reduced at 16.5 +/- 14.1 versus 30.8 +/- 6.8, 32.3 +/- 7.6, 30.6 +/- 2.2, 28.1 +/- 2.2 neurons/100 mum in CCI-only, 60-min HS-only, 90-min HS-only, and 60-min CCI+HS, respectively, all p < 0.05. CA3 neuron counts did not differ between groups. Fluorojade C staining confirmed neurodegeneration in CA1 in the 90-min CCI+HS group. Our data suggest a critical time window for exacerbation of neuronal death by HS after CCI and may have implications for blast injury victims in austere environments where definitive management is delayed.

Biomarkers of primary and evolving damage in traumatic and ischemic brain injury: diagnosis, prognosis, probing mechanisms, and therapeutic decision making.

PURPOSE OF REVIEW: Emerging data suggest that biomarkers of brain injury have potential utility as diagnostic, prognostic, and therapeutic adjuncts in the setting of traumatic and ischemic brain injury. Two approaches are being used, namely, assessing markers of structural damage and quantifying mediators of the cellular, biochemical, or molecular cascades in secondary injury or repair. Novel proteomic, multiplex, and lipidomic methods are also being applied. RECENT FINDINGS: Biochemical markers of neuronal, glial, and axonal damage such as neuron-specific enolase, S100B, and myelin basic protein, respectively, are readily detectable in biological samples such as serum or cerebrospinal fluid and are being studied in patients with ischemic and traumatic brain injury. In addition, a number of studies have demonstrated that novel tools to assess simultaneously multiple biomarkers can provide unique insight such as details on specific molecular participants in cell death cascades, inflammation, or oxidative stress. SUMMARY: Multifaceted cellular, biochemical, and molecular monitoring of proteins and lipids is logical as an adjunct to guiding therapies and improving outcomes in traumatic and ischemic brain injury and we appear to be on the verge of a breakthrough with the use of these markers as diagnostic, prognostic, and monitoring adjuncts, in neurointensive care.

Identification of poly-ADP-ribosylated mitochondrial proteins after traumatic brain injury.

Poly-ADP-ribosylation is a post-translational modification performed by poly(ADP-ribose) polymerases (PARP), involved in many diverse cellular functions including DNA repair, transcription, and long-term potentiation. Paradoxically, PARP over-activation under pathologic conditions including traumatic brain injury (TBI) results in cell death. We previously demonstrated that intra-mitochondrial poly-ADP-ribosylation occurs following excitotoxic and oxidative injury in vitro. Here we sought to identify mitochondrial proteins modified by poly-ADP-ribosylation after TBI in vivo. Poly-ADP-ribosylation within mitochondria from injured brain after experimental TBI in rats was first verified using western blot and immuno-electron microscopy. Poly-ADP-ribosylated mitochondrial proteins identified using a targeted proteomic approach included voltage-dependent anion channel-1, mitofilin, mitochondrial stress proteins, and the electron transport chain components F1F0 ATPase, cytochrome c oxidase, and cytochrome c reductase. To examine the functional consequences of mitochondrial poly-ADP-ribosylation, isolated rat brain mitochondria were exposed to conditions of nitrosative stress known to activate PARP. PARP activation-induced reductions in State 3 respiration were prevented by the PARP-1 inhibitor 5-iodo-6-amino-1,2-benzopyrone or exogenous poly(ADP-ribose) glycohydrolase. As the effects of PARP activation on mitochondrial respiration appear regulated by poly(ADP-ribose) glycohydrolase, a direct effect of poly-ADP-ribosylation on electron transport chain function is suggested. These findings may be of relevance to TBI and other diseases where mitochondrial dysfunction occurs.

Autophagy is increased after traumatic brain injury in mice and is partially inhibited by the antioxidant gamma-glutamylcysteinyl ethyl ester.

Autophagy is a homeostatic process for recycling of proteins and organelles, induced by nutrient deprivation and regulated by oxygen radicals. Whether autophagy is induced after traumatic brain injury (TBI) is not established. We show that TBI in mice results in increased ultrastructural and biochemical evidence of autophagy. Specifically, autophagosomal vacuoles and secondary lysosomes were frequently observed in cell processes and axons in ipsilateral brain regions by electron microscopy, and lipidated microtubule-associated protein light chain 3, a biochemical footprint of autophagy referred to as LC3 II, was increased at 2 and 24 h after TBI versus controls. Since oxygen radicals are believed to be important in the pathogenesis of TBI and are essential for the process of starvation-induced autophagy in vitro, we also sought to determine if treatment with the antioxidant gamma-glutamylcysteinyl ethyl ester (GCEE) reduced autophagy and influenced neurologic outcome after TBI in mice. Treatment with GCEE reduced oxidative stress and partially reduced LC3 II formation in injured brain at 24 h after TBI versus vehicle. Treatment with GCEE also led to partial improvement in behavioral and histologic outcome versus vehicle. Taken together, these data show that autophagy occurs after experimental TBI, and that oxidative stress contributes to overall neuropathology, in part by initiating or influencing autophagy.

Local administration of the poly(ADP-ribose) polymerase inhibitor INO-1001 prevents NAD+ depletion and improves water maze performance after traumatic brain injury in mice.

Poly(ADP-ribose) polymerase-1 (PARP-1) is an enzyme best known for its role in DNA repair and as a mediator of NAD+ depletion and energy failure-induced cell death. We tested the effect of the potent and selective ideno-isoquinolone PARP-1 inhibitor INO-1001 after controlled cortical impact (CCI) in mice. Anesthetized adult male mice were subjected to moderate CCI (velocity 6 m/sec, depth 1.2 mm) or sham-injury. Immediately after CCI or sham-injury mice received either INO-1001 (1.6 mg/kg) or vehicle via intracerebral injection (5 microl over 5 min) in a randomized fashion. At 2 h, contused brain tissue was dissected and NAD+ levels were measured. Separate mice underwent neuropathological outcome tests that included spatial memory acquisition (Morris water maze days 14-20), and assessment of contusion volume and hippocampal cell death at day 21. Local treatment with INO-1001 preserved brain NAD+ levels 2 h after CCI (vehicle = 67 +/- 7.6, INO-1001 = 95.8 +/- 4.4 % uninjured hemisphere; n = 6/group, p = 0.03). In the Morris water maze, treatment with INO-1001 reduced the latency to find the hidden platform and increased the time spent in the target quadrant versus vehicle after CCI (n = 11/group, p < or = 0.05). Histological damage did not differ between vehicle and INO-1001-treated mice after CCI. Treatment with INO-1001 prevented NAD+ depletion and improved outcome, although modestly, identifying PARP-mediated energy failure as a contributor to the pathological sequelae of TBI. Further study testing the effects of PARP inhibitors is warranted, specifically in models of brain injury where energy failure is seen.

A gel-based proteomic comparison of human cerebrospinal fluid between inflicted and non-inflicted pediatric traumatic brain injury.

Traumatic brain injury (TBI) is the most common cause of traumatic death in infancy, and inflicted TBI (iTBI) is the predominant cause. Like other central nervous system pathologies, TBI changes the composition of cerebrospinal fluid (CSF), which may represent a unique clinical window on brain pathophysiology. Proteomic analysis, including two-dimensional (2-D) difference in gel electrophoresis (DIGE) combined with mass spectrometry (MS), was used to compare the CSF protein profile of two pooled samples from pediatric iTBI (n = 13) and non-inflicted TBI (nTBI; n = 13) patients with severe injury. CSF proteins from iTBI and nTBI were fluorescently labeled in triplicate using different fluorescent Cy dyes and separated by 2-D gel electrophoresis. Approximately 250 protein spots were found in CSF, with 90% between-gel reproducibility of the 2-D gel. Following in-gel digestion, the tryptic peptides were analyzed by MS for protein identification. The acute phase reactant, haptoglobin (HP) isoforms, showed an approximate fourfold increase in nTBI versus iTBI. In contrast, the levels of prostaglandin D(2) synthase (PGDS) and cystatin C (CC) were 12-fold and sevenfold higher in iTBI versus nTBI, respectively. The changes of HP, PGDS, and CC were confirmed by Western blot. These initial results with conventional gel-based proteomics show new protein changes that may ultimately help to understand pathophysiological differences between iTBI and nTBI.

Gel-based hippocampal proteomic analysis 2 weeks following traumatic brain injury to immature rats using controlled cortical impact.

Traumatic brain injury (TBI) to postnatal day 17 rats has been shown to produce acute changes in hippocampal global protein levels and spatial learning and memory deficits. The purpose of the present study was to analyze global hippocampal protein changes 2 weeks after a moderate ipsilateral controlled cortical impact in postnatal day 17 rats using 2-dimensional difference gel electrophoresis and mass spectrometry. Paired sham and ipsilateral injured hippocampal lysates were independently labeled with different fluorescent cyanine dyes and coseparated within the same immobilized pH gradient strips and slab gel based on isoelectric point and molecular mass. Significant changes in key proteins involved in glial and neuronal stress, oxidative metabolism, calcium uptake and neurotransmitter function were found 2 weeks after injury, and their potential roles in hippocampal plasticity and cognitive dysfunction were discussed.

Immunohistochemical analysis of histone H3 acetylation and methylation--evidence for altered epigenetic signaling following traumatic brain injury in immature rats.

Posttranslational modifications (PTMs) of histone proteins may result in altered epigenetic signaling after pediatric traumatic brain injury (TBI). Hippocampal histone H3 acetylation and methylation in immature rats after moderate TBI were measured and decreased only in CA3 at 6 h and 24 h with persistent methylation decreases up to 72 h after injury. Decreased histone H3 acetylation and methylation suggest altered hippocampal CA3 epigenetic signaling during the first hours to days after TBI.

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.