• Energy Research

Ethanol intoxication is a common comorbidity in traumatic brain injury. To date, the effect of ethanol on TBI pathogenic cascades and resulting outcomes remains debated. A closed blunt weight-drop murine TBI model has been implemented to investigate behavioral (by sensorimotor and neurological tests), and neuro-immunological (by tissue cytokine arrays and immuno-histology) effects of ethanol intoxication on TBI. The effect of the occurrence of traumatic intracerebral hemorrhage was also studied. The results indicate that ethanol pretreatment results in a faster and better recovery after TBI with reduced infiltration of leukocytes and reduced microglia activation. These outcomes correspond to reduced parenchymal levels of GM-CSF, IL-6 and IL-3 and to the transient upregulation of IL-13 and VEGF, indicating an early shift in the cytokine profile towards reduced inflammation. A significant difference in the cytokine profile was still observed 24h post injury in the ethanol pretreated mice, as shown by the delayed peak in IL-6 and by the suppression of GM-CSF, IFN-γ, and IL-3. Seven days post-injury, ethanol-pretreated mice displayed a significant decrease both in CD45+ cells infiltration and in microglial activation. On the other hand, in the case of traumatic intracerebral hemorrhage, the cytokine profile was dominated by KC, CCL5, M-CSF and several interleukins and ethanol pretreatment did not produce any modification. We can thus conclude that ethanol intoxication suppresses the acute neuro-inflammatory response to TBI, an effect which is correlated with a faster and complete neurological recovery, whereas, the presence of traumatic intracerebral hemorrhage overrides the effects of ethanol.

Ethanol intoxication (EI) is a frequent comorbidity of traumatic brain injury (TBI), but the impact of EI on TBI pathogenic cascades and prognosis is unclear. Although clinical evidence suggests that EI may have neuroprotective effects, experimental support is, to date, inconclusive. We aimed at elucidating the impact of EI on TBI-associated neurological deficits, signaling pathways, and pathogenic cascades in order to identify new modifiers of TBI pathophysiology. We have shown that ethanol administration (5 g/kg) before trauma enhances behavioral recovery in a weight-drop TBI model. Neuronal survival in the injured somatosensory cortex was also enhanced by EI. We have used phospho-receptor tyrosine kinase (RTK) arrays to screen the impact of ethanol on TBI-induced activation of RTK in somatosensory cortex, identifying ErbB2/ErbB3 among the RTKs activated by TBI and suppressed by ethanol. Phosphorylation of ErbB2/3/4 RTKs were upregulated in vGlut2+ excitatory synapses in the injured cortex, including excitatory synapses located on parvalbumin (PV)-positive interneurons. Administration of selective ErbB inhibitors was able to recapitulate, to a significant extent, the neuroprotective effects of ethanol both in sensorimotor performance and structural integrity. Further, suppression of PV interneurons in somatosensory cortex before TBI, by engineered receptors with orthogonal pharmacology, could mimic the beneficial effects of ErbB inhibitors. Thus, we have shown that EI interferes with TBI-induced pathogenic cascades at multiple levels, with one prominent pathway, involving ErbB-dependent modulation of PV interneurons.

AbstractSystemic inflammatory consequences remain a significant burden after traumatic brain injury (TBI), with almost all organs affected. The spleen is connected with the brain by autonomic innervation and by soluble mediators, and the cross-talk between brain and spleen may be important to establish the systemic inflammatory response to TBI. Ethanol intoxication, the most common comorbidity of TBI, is posited to influence the peripheral inflammatory response either directly or through the brain-spleen cross-talk. Here we show that TBI causes a substantial change in transcription of genes associated with dendritic cells activation in the spleen, in particular a FLT3/FLT3L induction 3h after TBI, which was enhanced by EI. The FLT3L induction was associated with the phosphorylation of FLT3 receptor in CD11c+ dendritic cells, which enhanced the protein synthesis of a subset of mRNAs, as shown by the increase in pS6, peIF2A levels in dendritic cells. This corresponded to the upregulation of proteins associated with maturation process and immunostimulatory properties such MHC-II, LAMP1 and CD68, and of pro-inflammatory cytokines such as TNFα. Notably, EI enhanced the maturation of dendritic cells. However, whereas TBI decreases expression of the adrenergic 2b receptors on dendritic cells, EI increased it, thus augmenting the chances of cross-talk regulation of immune function by the autonomic system. In conclusion, this data indicates that TBI induces a fast maturation of the immunomodulatory functions of dendritic cells which is associated by FLT3/FLT3L signaling and which is enhanced by EI prior to TBI.

Traumatic brain injury (TBI) may cause damage to distant organs. Acute ethanol intoxication (EI) induces complex local and systemic anti-inflammatory effects and influences the early outcomes of traumatized patients. Here, we evaluated its effects on the BI-induced expression of local inflammatory mediators in the trauma-remote organs the lungs and liver. Male mice were exposed to ethanol as a single oral dose (5g·kg–1, 32%) before inducing a moderate blunt TBI. Sham groups underwent the same procedures without TBI. Ether 3 or 6h after the TBI, the lung and liver were collected. The gene expression of HMGB1, IL-6, MMP9, IL-1β, and TNF as well as the homogenate protein levels of receptor for advanced glycation end products (RAGE), IL-6, IL-1β, and IL-10 were analyzed. Liver samples were immunohistologically stained for HMGB1. EI decreased the gene expressions of the proinflammatory markers HMGB1, IL-6, and MMP9 in the liver upon TBI. In line with the reduced gene expression, the TBI-induced protein expression of IL-6 in liver tissue homogenates was significantly reduced by EI at 3h after TBI. While the histological HMGB1 expression was enhanced by TBI, the RAGE protein expression in the liver tissue homogenates was diminished after TBI. EI reduced the histological HMGB1 expression and enhanced the hepatic RAGE protein expression at 6h post TBI. With regard to the lungs, EI significantly reduced the gene expressions of HMGB1, IL-6, IL-1β, and TNF upon TBI, without significantly affecting the protein expression levels of inflammatory markers (RAGE, IL-6, IL-1β, and IL-10). At the early stage of TBI-induced inflammation, the gene expression of inflammatory mediators in both the lungs and liver is susceptible to ethanol-induced remote effects. Taken together, EI may alleviate the TBI-induced pro-inflammatory response in the trauma-distant organs, the lungs and liver, via the HMGB1-RAGE axis.

Abstract Background Blood-based biomarkers have proven to be a reliable measure of the severity and outcome of traumatic brain injury (TBI) in both murine models and patients. In particular, neuron-specific enolase (NSE), neurofilament light (NFL) and S100 beta (S100B) have been investigated in the clinical setting post-injury. Ethanol intoxication (EI) remains a significant comorbidity in TBI, with 30–40% of patients having a positive blood alcohol concentration post-TBI. The effect of ethanol on blood-based biomarkers for the prognosis and diagnosis of TBI remains unclear. In this study, we investigated the effect of EI on NSE, NFL and S100B and their correlation with blood–brain barrier integrity in a murine model of TBI. Methods We used ultra-sensitive single-molecule array technology and enzyme-linked immunosorbent assay methods to measure NFL, NSE, S100B and claudin-5 concentrations in plasma 3 hours post-TBI. Results We showed that NFL, NSE and S100B were increased at 3 hours post-TBI. Interestingly, ethanol blood concentrations showed an inverse correlation with NSE but not with NFL or S100B. Claudin-5 levels were increased post-injury but no difference was detected compared to ethanol pretreatment. The increase in claudin-5 post-TBI was correlated with NFL but not with NSE or S100B. Conclusions Ethanol induces an effect on biomarker release in the bloodstream that is different from TBI not influenced by alcohol. This could be the basis of investigations into humans.

Ethanol intoxication is a risk factor for traumatic brain injury (TBI) but clinical evidence suggests that it may actually improve the prognosis of intoxicated TBI patients. We have employed a closed, weight-drop TBI model of different severity (2cm or 3cm falling height), preceded (-30min) or followed (+20min) by ethanol administration (5g/Kg). This protocol allows us to study the interaction of binge ethanol intoxication in TBI, monitoring behavioral changes, histological responses and the transcriptional regulation of a series of activity-regulated genes (immediate early genes, IEGs). We demonstrate that ethanol pretreatment before moderate TBI (2cm) significantly reduces neurological impairment and accelerates recovery. In addition, better preservation of neuronal numbers and cFos+cells was observed 7days after TBI. At transcriptional level, ethanol reduced the upregulation of a subset of IEGs encoding for transcription factors such as Atf3, c-Fos, FosB, Egr1, Egr3 and Npas4 but did not affect the upregulation of others (e.g. Gadd45b and Gadd45c). While a subset of IEGs encoding for effector proteins (such as Bdnf, InhbA and Dusp5) were downregulated by ethanol, others (such as Il-6) were unaffected. Notably, the majority of genes were sensitive to ethanol only when administered before TBI and not afterwards (the exceptions being c-Fos, Egr1 and Dusp5). Furthermore, while severe TBI (3cm) induced a qualitatively similar (but quantitatively larger) transcriptional response to moderate TBI, it was no longer sensitive to ethanol pretreatment. Thus, we have shown that a subset of the TBI-induced transcriptional responses were sensitive to ethanol intoxication at the instance of trauma (ultimately resulting in beneficial outcomes) and that the effect of ethanol was restricted to a certain time window (pre TBI treatment) and to TBI severity (moderate). This information could be critical for the translational value of ethanol in TBI and for the design of clinical studies aimed at disentangling the role of ethanol intoxication in TBI.

Traumatic brain injury (TBI) and ethanol intoxication (EI) frequently coincide, particularly in young subjects. However, the mechanisms of their interaction remain poorly understood. Among other pathogenic pathways, TBI induces glial activation and neuroinflammation in the hippocampus, resulting in acute and chronic hippocampal dysfunction. In this regard, we investigated the role of EI affecting these responses unfolding after TBI. We used a blunt, weight-drop approach to model TBI in mice. Male mice were pre-administered with ethanol or vehicle to simulate EI. The neuroinflammatory response in the hippocampus was assessed by monitoring the expression levels of >20 cytokines, the phosphorylation status of transcription factors and the phenotype of microglia and astrocytes. We used AS1517499, a brain-permeable STAT6 inhibitor, to elucidate the role of this pathway in the EI/TBI interaction. We showed that TBI causes the elevation of IL-33, IL-1β, IL-38, TNF-α, IFN-α, IL-19 in the hippocampus at 3 h time point and concomitant EI results in the dose-dependent downregulation of IL-33, IL-1β, IL-38, TNF-α and IL-19 (but not of IFN-α) and in the selective upregulation of IL-13 and IL-12. EI is associated with the phosphorylation of STAT6 and the transcription of STAT6-controlled genes. Moreover, ethanol-induced STAT6 phosphorylation and transcriptional activation can be recapitulated in vitro by concomitant exposure of neurons to ethanol, depolarization and inflammatory stimuli (simulating the acute trauma). Acute STAT6 inhibition prevents the effects of EI on IL-33 and TNF-α, but not on IL-13 and negates acute EI beneficial effects on TBI-associated neurological impairment. Additionally, EI is associated with reduced microglial activation and astrogliosis as well as preserved synaptic density and baseline neuronal activity 7 days after TBI and all these effects are prevented by acute administration of the STAT6 inhibitor concomitant to EI. EI concomitant to TBI exerts significant immunomodulatory effects on cytokine induction and microglial activation, largely through the activation of STAT6 pathway, ultimately with beneficial outcomes.