Caudal DMN neurons innervate the spleen and release CART peptide to regulate neuroimmune function.

BACKGROUND: Inflammation is a fundamental biological response to injury and infection, which if unregulated can contribute to the pathophysiology of many diseases. The vagus nerve, which primarily originates from the dorsal motor nucleus (DMN), plays an important role in rapidly dampening inflammation by regulating splenic function. However, direct vagal innervation of the spleen, which houses the majority of immune and inflammatory cells, has not been established. As an alternative to direct innervation, an anti-inflammatory reflex pathway has been proposed which involves the vagus nerve, the sympathetic celiac ganglion, and the neurotransmitter norepinephrine. Although sympathetic regulation of inflammation has been shown, the interaction of the vagus nerve and the celiac ganglia requires a unique interaction of parasympathetic and sympathetic inputs, making this putative mechanism of brain-spleen interaction controversial. BODY: As neuropeptides can be expressed at relatively high levels in neurons, we reasoned that DMN neuropeptide immunoreactivity could be used to determine their target innervation. Employing immunohistochemistry, subdiaphragmatic vagotomy, viral tract tracing, CRISPR-mediated knock-down, and functional assays, we show that cocaine and amphetamine-regulated transcript (CART) peptide-expressing projection neurons in the caudal DMN directly innervate the spleen. In response to lipopolysaccharide (LPS) stimulation, CART acts to reduce inflammation, an effect that can be augmented by intrasplenic administration of a synthetic CART peptide. These in vivo effects could be recapitulated in cultured splenocytes, suggesting that these cells express the as yet unidentified CART receptor(s). CONCLUSION: Our results provide evidence for direct connections between the caudal DMN and spleen. In addition to acetylcholine, these neurons express the neuropeptide CART that, once released, acts to suppress inflammation by acting directly upon splenocytes.

Endoplasmic Reticulum Stress Contributes to the Loss of Newborn Hippocampal Neurons after Traumatic Brain Injury.

Adult hippocampal neurogenesis has been shown to be required for certain types of cognitive function. For example, studies have shown that these neurons are critical for pattern separation, the ability to store similar experiences as distinct memories. Although traumatic brain injury (TBI) has been shown to cause the loss of newborn hippocampal neurons, the signaling pathway(s) that triggers their death is unknown. Endoplasmic reticulum (ER) stress activates the PERK-eIF2α pathway that acts to restore ER function and improve cell survival. However, unresolved/intense ER stress activates C/EBP homologous protein (CHOP), leading to cell death. We show that TBI causes the death of hippocampal newborn neurons via CHOP. Using CHOP KO mice, we show that loss of CHOP markedly reduces newborn neuron loss after TBI. Injured CHOP mice performed significantly better in a context fear discrimination task compared with injured wild-type mice. In contrast, the PERK inhibitor GSK2606414 exacerbated doublecortin cell loss and worsened contextual discrimination. Administration of guanabenz (which reduces ER stress) to injured male rats reduced the loss of newborn neurons and improved one-trial contextual fear memory. Interestingly, we also found that the surviving newborn neurons in brain-injured animals had dendritic loss, which was not observed in injured CHOP KO mice or in animals treated with guanabenz. These results indicate that ER stress plays a key role in the death of newborn neurons after TBI. Further, these findings indicate that ER stress can alter dendritic arbors, suggesting a role for ER stress in neuroplasticity and dendritic pathologies.SIGNIFICANCE STATEMENT The hippocampus, a structure in the temporal lobe, is critical for learning and memory. The hippocampus is one of only two areas in which neurons are generated in the adult brain. These newborn neurons are required for certain types of memory, and are particularly vulnerable to traumatic brain injury (TBI). However, the mechanism(s) that causes the loss of these cells after TBI is poorly understood. We show that endoplasmic reticulum (ER) stress pathways are activated in newborn neurons after TBI, and that manipulation of the CHOP cascade improves newborn neuron survival and cognitive outcome. These results suggest that treatments that prevent/resolve ER stress may be beneficial in treating TBI-triggered memory dysfunction.

A role for autophagy in long-term spatial memory formation in male rodents.

A hallmark of long-term memory formation is the requirement for protein synthesis. Administration of protein synthesis inhibitors impairs long-term memory formation without influencing short-term memory. Rapamycin is a specific inhibitor of target of rapamycin complex 1 (TORC1) that has been shown to block protein synthesis and impair long-term memory. In addition to regulating protein synthesis, TORC1 also phosphorylates Unc-51-like autophagy activating kinase-1 (Ulk-1) to suppress autophagy. As autophagy can be activated by rapamycin (and rapamycin inhibits long-term memory), our aim was to test the hypothesis that autophagy inhibitors would enhance long-term memory. To examine if learning alters autophagosome number, we used male reporter mice carrying the GFP-LC3 transgene. Using these mice, we observed that training in the Morris water maze task increases the number of autophagosomes, a finding contrary to our expectations. For learning and memory studies, male Long Evans rats were used due to their relatively larger size (compared to mice), making it easier to perform intrahippocampal infusions in awake, moving animals. When the autophagy inhibitors 3-methyladenine (3-MA) or Spautin-1 were administered bilaterally into the hippocampii prior to training in the Morris water maze task, the drugs did not alter learning. In contrast, when memory was tested 24 hours later by a probe trial, significant impairments were observed. In addition, intrahippocampal infusion of an autophagy activator peptide (TAT-Beclin-1) improved long-term memory. These results indicate that autophagy is not necessary for learning, but is required for long-term memory formation.

Activation of Alpha 7 Cholinergic Nicotinic Receptors Reduce Blood-Brain Barrier Permeability following Experimental Traumatic Brain Injury.

Traumatic brain injury (TBI) is a major human health concern that has the greatest impact on young men and women. The breakdown of the blood-brain barrier (BBB) is an important pathological consequence of TBI that initiates secondary processes, including infiltration of inflammatory cells, which can exacerbate brain inflammation and contribute to poor outcome. While the role of inflammation within the injured brain has been examined in some detail, the contribution of peripheral/systemic inflammation to TBI pathophysiology is largely unknown. Recent studies have implicated vagus nerve regulation of splenic cholinergic nicotinic acetylcholine receptor α7 (nAChRa7) signaling in the regulation of systemic inflammation. However, it is not known whether this mechanism plays a role in TBI-triggered inflammation and BBB breakdown. Following TBI, we observed that plasma TNF-α and IL-1ß levels, as well as BBB permeability, were significantly increased in nAChRa7 null mice (Chrna7(-/-)) relative to wild-type mice. The administration of exogenous IL-1ß and TNF-α to brain-injured animals worsened Evans Blue dye extravasation, suggesting that systemic inflammation contributes to TBI-triggered BBB permeability. Systemic administration of the nAChRa7 agonist PNU-282987 or the positive allosteric modulator PNU-120596 significantly attenuated TBI-triggered BBB compromise. Supporting a role for splenic nAChRa7 receptors, we demonstrate that splenic injection of the nicotinic receptor blocker α-bungarotoxin increased BBB permeability in brain-injured rats, while PNU-282987 injection decreased such permeability. These effects were not seen when α-bungarotoxin or PNU-282987 were administered to splenectomized, brain-injured rats. Together, these findings support the short-term use of nAChRa7-activating agents as a strategy to reduce TBI-triggered BBB permeability. SIGNIFICANCE STATEMENT: Breakdown of the blood-brain barrier (BBB) in response to traumatic brain injury (TBI) allows for the accumulation of circulating fluids and proinflammatory cells in the injured brain. These processes can exacerbate TBI pathology and outcome. While the role of inflammation in the injured tissue has been examined in some detail, the contribution of peripheral inflammation in BBB breakdown and ensuing pathology has not been well defined. We present experimental evidence to indicate that the stimulation of nicotinic acetylcholine α7 receptors (nAChRa7s) can reduce peripheral inflammation and BBB breakdown after TBI. These results suggest that activators of nAChRa7 may have therapeutic utility for the treatment of TBI.

Wnt3a, a Protein Secreted by Mesenchymal Stem Cells Is Neuroprotective and Promotes Neurocognitive Recovery Following Traumatic Brain Injury.

Intravenous administration of bone marrow derived mesenchymal stem cells (MSCs) has been shown to reduce blood brain barrier compromise and improve neurocognition following traumatic brain injury (TBI). These effects occur in the absence of engraftment and differentiation of these cells in the injured brain. Recent studies have shown that soluble factors produced by MSCs mediate a number of the therapeutic effects. In this study, we sought to determine if intravenous administration of MSCs (IV-MSCs) could enhance hippocampal neurogenesis following TBI. Our results demonstrate that IV-MSC treatment attenuates loss of neural stem cells and promotes hippocampal neurogenesis in TBI injured mice. As Wnt signaling has been implicated in neurogenesis, we measured circulating Wnt3a levels in serum following IV-MSC administration and found a significant increase in Wnt3a. Concurrent with this increase, we detected increased activation of the Wnt/ß-catenin signaling pathway in hippocampal neurons. Furthermore, IV recombinant Wnt3a treatment provided neuroprotection, promoted neurogenesis, and improved neurocognitive function in TBI injured mice. Taken together, our results demonstrate a role for Wnt3a in the therapeutic potential of MSCs and identify Wnt3a as a potential stand-alone therapy or as part of a combination therapeutic strategy for the treatment of TBI. Stem Cells 2016;34:1263-1272.

Traumatic brain injury decreases AMP-activated protein kinase activity and pharmacological enhancement of its activity improves cognitive outcome.

Prolonged metabolic suppression in the brain is a well-characterized secondary pathology of both experimental and clinical traumatic brain injury (TBI). AMP-activated kinase (AMPK) acts as a cellular energy sensor that, when activated, regulates various metabolic and catabolic pathways to decrease ATP consumption and increase ATP synthesis. As energy availability after TBI is suppressed, we questioned if increasing AMPK activity after TBI would improve cognitive outcome. TBI was delivered using the electromagnetic controlled cortical impact model on male Sprague-Dawley rats (275-300 g) and C57BL/6 mice (20-25 g). AMPK activity within the injured parietal cortex and ipsilateral hippocampus was inferred by western blots using phospho-specific antibodies. The consequences of acute manipulation of AMPK signaling on cognitive function were assessed using the Morris water maze task. We found that AMPK activity is decreased as a result of injury, as indicated by reduced AMPK phosphorylation and corresponding changes in the phosphorylation of its downstream targets: ribosomal protein S6 and Akt Substrate of 160 kDa (AS160). Increasing AMPK activity after injury using the drugs 5-amino-1-ß-d-ribofuranosyl-imidazole-4-carboxamide or metformin did not affect spatial learning, but significantly improved spatial memory. Taken together, our results suggest that decreased AMPK activity after TBI may contribute to the cellular energy crisis in the injured brain, and that AMPK activators may have therapeutic utility. Increased phosphorylation of Thr172 activates AMP-activated protein kinase (AMPK) under conditions of low cellular energy availability. This leads to inhibition of energy consuming, while activating energy generating, processes. Hill et al., present data to indicate that TBI decreases Thr172 phosphorylation and that its stimulation by pharmacological agents offers neuroprotection and improves memory. These results suggest that decreased AMPK phosphorylation after TBI incorrectly signals the injured brain that excess energy is available, thereby contributing to the cellular energy crisis and memory impairments.

TIMP3 Attenuates the Loss of Neural Stem Cells, Mature Neurons and Neurocognitive Dysfunction in Traumatic Brain Injury.

Mesenchymal stem cells (MSCs) have been shown to have potent therapeutic effects in a number of disorders including traumatic brain injury (TBI). However, the molecular mechanism(s) underlying these protective effects are largely unknown. Herein we demonstrate that tissue inhibitor of matrix metalloproteinase-3 (TIMP3), a soluble protein released by MSCs, is neuroprotective and enhances neuronal survival and neurite outgrowth in vitro. In vivo in a murine model of TBI, intravenous recombinant TIMP3 enhances dendritic outgrowth and abrogates loss of hippocampal neural stem cells and mature neurons. Mechanistically we demonstrate in vitro and in vivo that TIMP3-mediated neuroprotection is critically dependent on activation of the Akt-mTORC1 pathway. In support of the neuroprotective effect of TIMP3, we find that intravenous delivery of recombinant TIMP3 attenuates deficits in hippocampal-dependent neurocognition. Taken together, our data strongly suggest that TIMP3 has direct neuroprotective effects that can mitigate the deleterious effects associated with TBI, an area with few if any therapeutic options.

Intrahippocampal glutamine administration inhibits mTORC1 signaling and impairs long-term memory.

The mechanistic Target of Rapamycin Complex 1 (mTORC1), a key regulator of protein synthesis and cellular growth, is also required for long-term memory formation. Stimulation of mTORC1 signaling is known to be dependent on the availability of energy and growth factors, as well as the presence of amino acids. In vitro studies using serum- and amino acid-starved cells have reported that glutamine addition can either stimulate or repress mTORC1 activity, depending on the particular experimental system that was used. However, these experiments do not directly address the effect of glutamine on mTORC1 activity under physiological conditions in nondeprived cells in vivo. We present experimental results indicating that intrahippocampal administration of glutamine to rats reduces mTORC1 activity. Moreover, post-training administration of glutamine impairs long-term spatial memory formation, while coadministration of glutamine with leucine had no influence on memory. Intracellular recordings in hippocampal slices showed that glutamine did not alter either excitatory or inhibitory synaptic activity, suggesting that the observed memory impairments may not result from conversion of glutamine to either glutamate or GABA. Taken together, these findings indicate that glutamine can decrease mTORC1 activity in the brain and may have implications for treatments of neurological diseases associated with high mTORC1 signaling.

Prolonging the survival of Tsc2 conditional knockout mice by glutamine supplementation.

The genetic disease tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by loss of function mutations in either TSC1 (hamartin) or TSC2 (tuberin), which serve as negative regulators of mechanistic target of rapamycin complex 1 (mTORC1) activity. TSC patients exhibit developmental brain abnormalities and tuber formations that are associated with neuropsychological and neurocognitive impairments, seizures and premature death. Mechanistically, TSC1 and TSC2 loss of function mutations result in abnormally high mTORC1 activity. Thus, the development of a strategy to inhibit abnormally high mTORC1 activity may have therapeutic value in the treatment of TSC. mTORC1 is a master regulator of growth processes, and its activity can be reduced by withdrawal of growth factors, decreased energy availability, and by the immunosuppressant rapamycin. Recently, glutamine has been shown to alter mTORC1 activity in a TSC1-TSC2 independent manner in cells cultured under amino acid- and serum-deprived conditions. Since starvation culture conditions are not physiologically relevant, we examined if glutamine can regulate mTORC1 in non-deprived cells and in a murine model of TSC. Our results show that glutamine can reduce phosphorylation of S6 and S6 kinase, surrogate indicators of mTORC1 activity, in both deprived and non-deprived cells, although higher concentrations were required for non-deprived cultures. When administered orally to TSC2 knockout mice, glutamine reduced S6 phosphorylation in the brain and significantly prolonged their lifespan. Taken together, these results suggest that glutamine supplementation can be used as a potential treatment for TSC.

Disruption of the perineuronal net in the hippocampus or medial prefrontal cortex impairs fear conditioning.

The perineuronal net (PNN) surrounds neurons in the central nervous system and is thought to regulate developmental plasticity. A few studies have shown an involvement of the PNN in hippocampal plasticity and memory storage in adult animals. In addition to the hippocampus, plasticity in the medial prefrontal cortex (mPFC) has been demonstrated to be critical for the storage of long-term memory, particularly memories for temporally separated events. In the present study, we examined the role of PNN in the acquisition and retention of memories for trace (in which the conditioned and unconditioned stimuli are temporally separated) and delayed (in which the conditioned and unconditioned stimuli overlap) fear conditioning in both the hippocampus and the mPFC. Consistent with a role for the hippocampus in memory storage in both delayed and trace fear conditioning, removal of hippocampal PNN disrupted contextual and trace fear memory. Disruption of the PNN in the mPFC impaired long-term trace and conditioned stimulus (CS)-elicited fear memory in the trace fear conditioning task. Interestingly, CS-elicited fear memory was also impaired when a delayed fear conditioning paradigm was used. These findings further support a role for the PNN in neural plasticity and implicate PNN-regulated plasticity in neocortical memory storage.

Lysophospholipids Are Associated With Outcomes in Hospitalized Patients With Mild Traumatic Brain Injury.

Mild traumatic brain injury (mTBI) accounts for 70-90% of all TBI cases. Lipid metabolites have important roles in plasma membrane biogenesis, function, and cell signaling. As TBI can compromise plasma membrane integrity and alter brain cell function, we sought to identify circulating phospholipid alterations after mTBI, and determine if these changes were associated with clinical outcomes. Patients with mTBI (Glasgow Coma Score [GCS] ≥13 and loss of consciousness <30 min) were recruited. A total of 84 mTBI subjects were enrolled after admission to a level I trauma center, with the majority having evidence of traumatic intracranial hemorrhage on brain computed tomography (CT). Plasma samples were collected within 24 h of injury with 32 mTBI subjects returning at 3 months after injury for a second plasma sample to be collected. Thirty-five healthy volunteers were enrolled as controls and had a one-time blood draw. Lipid metabolomics was performed on plasma samples from each subject. Fold change of selected lipid metabolites was determined. Multivariable regression models were created to test associations between lipid metabolites and discharge and 6-month Glasgow Outcomes Scale-Extended (GOSE) outcomes (dichotomized between "good" [GOSE ≥7] and "bad" [GOSE ≤6] functional outcomes). Plasma levels of 31 lipid metabolites were significantly associated with discharge GOSE using univariate models; three of these metabolites were significantly increased, while 14 were significantly decreased in subjects with good outcomes compared with subjects with poor outcomes. In multivariable logistic regression models, higher circulating levels of the lysophospholipids (LPL) 1-linoleoyl-glycerophosphocholine (GPC) (18:2), 1-linoleoyl-GPE (18:2), and 1-linolenoyl-GPC (18:3) were associated with both good discharge GOSE (odds ratio [OR] 12.2 [95% CI 3.35, 58.3], p = 5.23 × 10-4; OR 9.43 [95% CI 2.87, 39.6], p = 7.26 × 10-4; and OR 5.26 [95% CI 1.99, 16.7], p = 2.04 × 10-3, respectively) and 6-month (OR 4.67 [95% CI 1.49, 17.7], p = 0.013; OR 2.93 [95% CI 1.11, 8.87], p = 0.039; and OR 2.57 [95% CI 1.08, 7.11], p = 0.046, respectively). Compared with healthy volunteers, circulating levels of these three LPLs were decreased early after injury and had normalized by 3 months after injury. Logistic regression models to predict functional outcomes were created by adding each of the described three LPLs to a baseline model that included age and sex. Including 1-linoleoyl-GPC (18:2) (8.20% improvement, p = 0.009), 1-linoleoyl-GPE (18:2) (8.85% improvement, p = 0.021), or 1-linolenoyl-GPC (18:3) (7.68% improvement, p = 0.012), significantly improved the area under the curve (AUC) for predicting discharge outcomes compared with the baseline model. Models including 1-linoleoyl-GPC (18:2) significantly improved AUC for predicting 6-month outcomes (9.35% improvement, p = 0.034). Models including principal components derived from 25 LPLs significantly improved AUC for prediction of 6-month outcomes (16.0% improvement, p = 0.020). Our results demonstrate that higher plasma levels of LPLs (1-linoleoyl-GPC, 1-linoleoyl-GPE, and 1-linolenoyl-GPC) after mTBI are associated with better functional outcomes at discharge and 6 months after injury. This class of phospholipids may represent a potential therapeutic target.

A Combination of Low Doses of Lithium and Valproate Improves Cognitive Outcomes after Mild Traumatic Brain Injury.

The prevalence of mild traumatic brain injury (mTBI) is high compared with moderate and severe TBI, comprising almost 80% of all brain injuries. mTBI activates a complex cascade of biochemical, molecular, structural, and pathological changes that can result in neurological and cognitive impairments. These impairments can manifest even in the absence of overt brain damage. Given the complexity of changes triggered by mTBI, a combination of drugs that target multiple TBI-activated cascades may be required to improve mTBI outcomes. It has been previously demonstrated that cotreatment with the U.S. Food and Drug Administration (FDA)-approved drugs lithium plus valproate (Li + VPA) for 3 weeks after a moderate-to-severe controlled cortical impact injury reduced cortical tissue loss and improved motor function. Since both lithium and valproate can exhibit toxicity at high doses, it would be beneficial to determine if this combination treatment is effective when administered at low doses and for a shorter duration, and if it can improve cognitive function, after a mild diffuse TBI. In the present study, we tested if the combination of low doses of lithium (1 mEq/kg or 0.5 mEq/kg) plus valproate (20 mg/kg) administered for 3 days after a mild fluid percussion injury can improve hippocampal-dependent learning and memory. Our data show that the combination of low-dose Li + VPA improved spatial learning and memory, effects not seen when either drug was administered alone. In addition, postinjury Li + VPA treatment improved recognition memory and sociability and reduced fear generalization. Postinjury Li + VPA also reduced the number of anti-ionized calcium binding adaptor molecule 1 (Iba1)-positive microglia counted using a convolutional neural network, indicating a reduction in neuroinflammation. These findings indicate that low-dose Li + VPA administered acutely after mTBI may have translational utility to reduce pathology and improve cognitive function.

The evolving pathophysiology of TBI and the advantages of temporally-guided combination therapies.

Several clinical and experimental studies have demonstrated that traumatic brain injury (TBI) activates cascades of biochemical, molecular, structural, and pathological changes in the brain. These changes combine to contribute to the various outcomes observed after TBI. Given the breadth and complexity of changes, combination treatments may be an effective approach for targeting multiple detrimental pathways to yield meaningful improvements. In order to identify targets for therapy development, the temporally evolving pathophysiology of TBI needs to be elucidated in detail at both the cellular and molecular levels, as it has been shown that the mechanisms contributing to cognitive dysfunction change over time. Thus, a combination of individual mechanism-based therapies is likely to be effective when maintained based on the time courses of the cellular and molecular changes being targeted. In this review, we will discuss the temporal changes of some of the key clinical pathologies of human TBI, the underlying cellular and molecular mechanisms, and the results from preclinical and clinical studies aimed at mitigating their consequences. As most of the pathological events that occur after TBI are likely to have subsided in the chronic stage of the disease, combination treatments aimed at attenuating chronic conditions such as cognitive dysfunction may not require the initiation of individual treatments at a specific time. We propose that a combination of acute, subacute, and chronic interventions may be necessary to maximally improve health-related quality of life (HRQoL) for persons who have sustained a TBI.

Randomized trial to evaluate nutritional status and absorption of enteral feeding after brain death.

CONTEXT: Catecholamines and inflammatory mediators, with elevated levels after brain death, are associated with reduced function and survival of transplanted organs. Enteral nutrition reduces tissue damage and may benefit organs. OBJECTIVE: To evaluate the effects of immunomodulating enteral nutrition in organ donors. DESIGN: Prospective, randomized, open-label study. SETTING: Intensive care unit. PATIENTS: Thirty-six brain-dead organ donors. INTERVENTIONS: Donors were randomized to receive enteral nutrition containing omega-3 polyunsaturated fatty acid, antioxidants, and glutamine or standard care (fasting). Donors received hormonal replacement therapy of corticosteroid, levothyroxine, dextrose, and insulin. MAIN OUTCOME MEASURES: Gastrointestinal assimilation (measured by 13 carbon-labeled uracil breath analysis), quantity of organs recovered, resting energy expenditure, urine level of urea nitrogen, and serum levels of albumin, prealbumin, interleukin 6, tumor necrosis factor-α, and C-reactive protein were evaluated. RESULTS: Thirteen patients (36%) assimilated 13C-labeled uracil. Resting energy expenditure was significantly higher than predicted between 10 and 14 hours after baseline in 33 donors (P= .007). Other measures were not conclusively different between fed and fasting groups. No adverse events occurred that were related to the enteral feeding. CONCLUSIONS: About 30% of donors metabolized 13C-labeled uracil, although no difference in oxidation rate was found between fasting and fed donors. Corticosteroid administration lowers plasma levels of interleukin 6 and most likely contributes to greater than predicted resting energy expenditure. Thus energy needs may not be met during fasting if hormones are given. Consequences of this possible energy deficit warrant further study.

Enhanced presynaptic mitochondrial energy production is required for memory formation.

Some of the prominent features of long-term memory formation include protein synthesis, gene expression, enhanced neurotransmitter release, increased excitability, and formation of new synapses. As these processes are critically dependent on mitochondrial function, we hypothesized that increased mitochondrial respiration and dynamics would play a prominent role in memory formation. To address this possibility, we measured mitochondrial oxygen consumption (OCR) in hippocampal tissue punches from trained and untrained animals. Our results show that context fear training significantly increased basal, ATP synthesis-linked, and maximal OCR in the Shaffer collateral-CA1 synaptic region, but not in the CA1 cell body layer. These changes were recapitulated in synaptosomes isolated from the hippocampi of fear-trained animals. As dynamin-related protein 1 (Drp1) plays an important role in mitochondrial fission, we examined its role in the increased mitochondrial respiration observed after fear training. Drp1 inhibitors decreased the training-associated enhancement of OCR and impaired contextual fear memory, but did not alter the number of synaptosomes containing mitochondria. Taken together, our results show context fear training increases presynaptic mitochondria respiration, and that Drp-1 mediated enhanced energy production in CA1 pre-synaptic terminals is necessary for context fear memory that does not result from an increase in the number of synaptosomes containing mitochondria or an increase in mitochondrial mass within the synaptic layer.

Epigenetic Modifications and Their Potential Contribution to Traumatic Brain Injury Pathobiology and Outcome.

Epigenetic information is not permanently encoded in the DNA sequence, but rather consists of reversible, heritable modifications that regulate the gene expression profile of a cell. Epigenetic modifications can result in cellular changes that can be long lasting and include DNA methylation, histone methylation, histone acetylation, and RNA methylation. As epigenetic modifications are reversible, the enzymes that add (epigenetic writers), the proteins that decode (epigenetic readers), and the enzymes that remove (epigenetic erasers) these modifications can be targeted to alter cellular function and disease biology. While epigenetic modifications and their contributions are intense topics of current research in the context of a number of diseases, including cancer, inflammatory diseases, and Alzheimer disease, the study of epigenetics in the context of traumatic brain injury (TBI) is in its infancy. In this review, we will summarize the experimental and clinical findings demonstrating that TBI triggers epigenetic modifications, with a focus on changes in DNA methylation, histone methylation, and the translational utility of the universal methyl donor S-adenosylmethionine (SAM). Finally, we will review the evidence for using methyl donors as possible treatments for TBI-associated pathology and outcome.

Metformin Reduces Repeat Mild Concussive Injury Pathophysiology.

Mild traumatic brain injury (mTBI) can initiate complex pathophysiological changes in the brain. Numerous cellular and molecular mechanisms underlying these pathologic changes are altered after injury, including those involved in energy utilization, excitotoxicity, ionic disturbances, and inflammation. It is thought that targeting multiple mechanisms may be necessary to produce meaningful reductions in brain pathology and to improve outcome. Previous studies have reported that the anti-diabetic drug metformin can also affect inflammatory, cell survival, and metabolic outcomes, possibly by acting on multiple targets and/or pathways. We therefore questioned whether metformin treatment can reduce pathology after repeat mild closed head injury (rmCHI) in male C57Bl/6 mice. We found that metformin, administered acutely after each head impact, resulted in markedly reduced white matter damage, astrogliosis, loss of hippocampal parvalbumin neurons, and improved mitochondrial function. In addition, both motor and cognitive functions were significantly improved when tested after discontinuation of the treatment. These studies suggest that metformin may be beneficial as a treatment for repeat concussion.

A novel strategy to activate cytoprotective genes in the injured brain.

The transcription factor nuclear factor E2-related factor 2 (Nrf2) regulates the expression of multiple cytoprotective genes that have been shown to offer protection in response to a number of insults. The present study describes a novel strategy to increase expression of Nrf2-responsive genes in brain injured mice. Under normal conditions, the adapter protein Kelch-like ECH-associated protein 1 (Keap1) binds to Nrf2 and promotes its proteosomal degradation in the cytoplasm. The amino acid sequence DEETGE, located at amino acid 77-82 of Nrf2, is critical for Nrf2-Keap1 interaction, and synthetic peptides containing this sequence can be used to disrupt the complex in vitro. We observed that intracerebroventricular (i.c.v.) infusion of a peptide containing the DEETGE sequence along with the cell transduction domain of the HIV-TAT protein (TAT-DEETGE) into brain-injured mice did not increase the mRNA levels for Nrf2-driven genes. However, when a calpain cleavage sequence was introduced between the TAT sequence and the DEETGE sequence, the new peptide (TAT-CAL-DEETGE) increased the mRNA levels of these genes. Increased gene expression was not observed when the TAT-CAL-DEETGE peptide was injected into uninjured animals. Furthermore, injection of TAT-CAL-DEETGE peptides before or after brain injury reduced blood-brain barrier compromise, a prominent secondary pathology that negatively influences outcome. The present strategy to increase Nrf2-responsive gene expression can be adapted to treat other insults or diseases based on their underlying mechanism(s) of cellular damage.

Insulin-Like Growth Factor-2 (IGF-2) Does Not Improve Memory in the Chronic Stage of Traumatic Brain Injury in Rodents.

Persistent cognitive impairment(s) can be a significant consequence of traumatic brain injury (TBI) and can markedly compromise quality of life. Unfortunately, identifying effective treatments to alleviate memory impairments in the chronic stage of TBI has proven elusive. Several studies have demonstrated that insulin-like growth factor-2 (IGF-2) can enhance memory in both normal animals and in experimental models of disease. In this study, we questioned whether IGF-2, when administered before learning, could enhance memory performance in the chronic stage of TBI. Male C57BL/6 mice (n = 7 per group) were injured using an electronic cortical impact injury device. Four months later, mice were tested for their cognitive performance in the fear memory extinction, novel object recognition (NOR), and Morris water maze tasks. Twenty minutes before each day of training, mice received a subcutaneous injection of either 30 µg/kg of IGF-2 or an equal volume of vehicle. Memory testing was carried out 24 h after training in the absence of the drug. Uninjured sham animals treated with IGF-2 (or vehicle) were trained and tested in the fear memory extinction task as a positive control. Our data show that although IGF-2 (30 µg/kg) improved memory extinction in uninjured mice, it was ineffective at improving fear memory extinction in the chronic stage of TBI. Similarly, IGF-2 administration to chronically injured animals did not improve TBI-related deficits in either NOR or spatial memory. Our results indicate that IGF-2, administered in the chronic stage of injury, is ineffective at enhancing memory performance and therefore may not be a beneficial treatment option for lingering cognitive impairments after a TBI.

Serum IL-6: a candidate biomarker for intracranial pressure elevation following isolated traumatic brain injury.

BACKGROUND: Increased intracranial pressure (ICP) is a serious, life-threatening, secondary event following traumatic brain injury (TBI). In many cases, ICP rises in a delayed fashion, reaching a maximal level 48-96 hours after the initial insult. While pressure catheters can be implanted to monitor ICP, there is no clinically proven method for determining a patient's risk for developing this pathology. METHODS: In the present study, we employed antibody array and Luminex-based screening methods to interrogate the levels of inflammatory cytokines in the serum of healthy volunteers and in severe TBI patients (GCSor= 25 mm Hg had significantly higher IL-6 levels within the first 17 hours of injury as compared to the patients whose ICP remained 128 pg/ml correctly identified 85% of isolated TBI patients who subsequently developed elevated ICP, and values between these cut-off values correctly identified 75% of all patients whose ICP remained