Systemic inflammation following traumatic injury and its impact on neuroinflammatory gene expression in the rodent brain.

BACKGROUND: Trauma can result in systemic inflammation that leads to organ dysfunction, but the impact on the brain, particularly following extracranial insults, has been largely overlooked. METHODS: Building upon our prior findings, we aimed to understand the impact of systemic inflammation on neuroinflammatory gene transcripts in eight brain regions in rats exposed to (1) blast overpressure exposure [BOP], (2) cutaneous thermal injury [BU], (3) complex extremity injury, 3 hours (h) of tourniquet-induced ischemia, and hind limb amputation [CEI+tI+HLA], (4) BOP+BU or (5) BOP+CEI and delayed HLA [BOP+CEI+dHLA] at 6, 24, and 168 h post-injury (hpi). RESULTS: Globally, the number and magnitude of differentially expressed genes (DEGs) correlated with injury severity, systemic inflammation markers, and end-organ damage, driven by several chemokines/cytokines (Csf3, Cxcr2, Il16, and Tgfb2), neurosteroids/prostaglandins (Cyp19a1, Ptger2, and Ptger3), and markers of neurodegeneration (Gfap, Grin2b, and Homer1). Regional neuroinflammatory activity was least impacted following BOP. Non-blast trauma (in the BU and CEI+tI+HLA groups) contributed to an earlier, robust and diverse neuroinflammatory response across brain regions (up to 2-50-fold greater than that in the BOP group), while combined trauma (in the BOP+CEI+dHLA group) significantly advanced neuroinflammation in all regions except for the cerebellum. In contrast, BOP+BU resulted in differential activity of several critical neuroinflammatory-neurodegenerative markers compared to BU. t-SNE plots of DEGs demonstrated that the onset, extent, and duration of the inflammatory response are brain region dependent. Regardless of injury type, the thalamus and hypothalamus, which are critical for maintaining homeostasis, had the most DEGs. Our results indicate that neuroinflammation in all groups progressively increased or remained at peak levels over the study duration, while markers of end-organ dysfunction decreased or otherwise resolved. CONCLUSIONS: Collectively, these findings emphasize the brain's sensitivity to mediators of systemic inflammation and provide an example of immune-brain crosstalk. Follow-on molecular and behavioral investigations are warranted to understand the short- to long-term pathophysiological consequences on the brain, particularly the mechanism of blood-brain barrier breakdown, immune cell penetration-activation, and microglial activation.

Systemic inflammation induced from remote extremity trauma is a critical driver of secondary brain injury.

Blast exposure, commonly experienced by military personnel, can cause devastating life-threatening polysystem trauma. Despite considerable research efforts, the impact of the systemic inflammatory response after major trauma on secondary brain injury-inflammation is largely unknown. The aim of this study was to identify markers underlying the susceptibility and early onset of neuroinflammation in three rat trauma models: (1) blast overpressure exposure (BOP), (2) complex extremity trauma (CET) involving femur fracture, crush injury, tourniquet-induced ischemia, and transfemoral amputation through the fracture site, and (3) BOP+CET. Six hours post-injury, intact brains were harvested and dissected to obtain biopsies from the prefrontal cortex, striatum, neocortex, hippocampus, amygdala, thalamus, hypothalamus, and cerebellum. Custom low-density microarray datasets were used to identify, interpret and visualize genes significant (p < 0.05 for differential expression [DEGs]; 86 neuroinflammation-associated) using a custom python-based computer program, principal component analysis, heatmaps and volcano plots. Gene set and pathway enrichment analyses of the DEGs was performed using R and STRING for protein-protein interaction (PPI) to identify and explore key genes and signaling networks. Transcript profiles were similar across all regions in naïve brains with similar expression levels involving neurotransmission and transcription functions and undetectable to low-levels of inflammation-related mediators. Trauma-induced neuroinflammation across all anatomical brain regions correlated with injury severity (BOP+CET > CET > BOP). The most pronounced differences in neuroinflammatory-neurodegenerative gene regulation were between blast-associated trauma (BOP, BOP+CET) and CET. Following BOP, there were few DEGs detected amongst all 8 brain regions, most were related to cytokines/chemokines and chemokine receptors, where PPI analysis revealed Il1b as a potential central hub gene. In contrast, CET led to a more excessive and diverse pro-neuroinflammatory reaction in which Il6 was identified as the central hub gene. Analysis of the of the BOP+CET dataset, revealed a more global heightened response (Cxcr2, Il1b, and Il6) as well as the expression of additional functional regulatory networks/hub genes (Ccl2, Ccl3, and Ccl4) which are known to play a critical role in the rapid recruitment and activation of immune cells via chemokine/cytokine signaling. These findings provide a foundation for discerning pathophysiological consequences of acute extremity injury and systemic inflammation following various forms of trauma in the brain.

MicroRNA-7 attenuates secondary brain injury following experimental intracerebral hemorrhage via inhibition of NLRP3.

BACKGROUND AND PURPOSE: The pathophysiological mechanisms underlying brain injury resulting from intracerebral hemorrhage (ICH) remain incompletely elucidated, and efficacious therapeutic interventions to enhance the prognosis of ICH patients are currently lacking. Previous research indicates that MicroRNA-7 (miR-7) can suppress the expression of Nod-like receptor protein 3 (NLRP3), thereby modulating neuroinflammation in Parkinson's disease pathogenesis. However, the potential regulatory effects miR-7 on NLRP3 inflammasome after ICH are yet to be established. This study aims to ascertain whether miR-7 mitigates secondary brain injury following experimental ICH by inhibiting NLRP3 and to investigate the underlying mechanisms. METHODS: An ICH model was established by stereotaxically injecting 100 µL of autologous blood into the right basal ganglia of Sprague-Dawley (SD) rats. Subsequently, these rats were allocated into three groups: sham, ICH + Vehicle, and ICH + miR-7, each comprising 18 animals. Twelve hours post-modeling, rats received intraventricular injections of 10 µL physiological saline, 10 µL phosphate, and 10 µL phosphate-buffered saline solution containing 0.5 nmol of miR-7 mimics, respectively. Neurological function was assessed on day three post-modeling, followed by euthanasia for brain tissue collection. Brain water content was determined using the dry-wet weight method. The expression of inflammatory cytokines in cerebral tissues surrounding the hematoma was analyzed through immunohistochemistry and Western blot assays. These cytokines were re-evaluated using Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Moreover, bioinformatics tools were employed to predict miR-7's binding to NLRP3. A wild-type luciferase reporter gene vector and a corresponding mutant vector were constructed, followed by transfection of miR-7 mimics into HEK293T cells to assess luciferase activity. RESULTS: Our study demonstrates that the administration of miR-7 mimics markedly reduced neurological function scores and attenuated brain edema in rats following ICH. A significant upregulation of NLRP3 expression in microglia/macrophage adjacent to the hematoma was observed, substantially reduced after the treatment with miR-7 mimics. Furthermore, this intervention ameliorated neurodegenerative changes and effectively decreased the protein and mRNA levels of pro-inflammatory cytokines, namely TNF-α, IL-1ß, IL-6, and Caspase1, in the cerebral tissues proximate to the hematomas. In addition, miR-7 mimics distinctly inhibited the luciferase activity associated with the wild-type reporter gene, an effect not mirrored in its mutant variant. CONCLUSIONS: The miR-7 suppressed NLRP3 expression in microglia/macrophage to reduce the production of inflammatory cytokines, leading to conducting certain neuroprotection post-ICH in rats.

Association between the C-reactive protein to albumin ratio and poor clinical outcome in patients with spontaneous intracerebral hemorrhage.

BACKGROUND: The C-reactive protein-to-albumin ratio (CAR) is a novel prognostic biomarker of systemic inflammation and nutritional status. The association between CAR and the long-term outcome of spontaneous intracerebral hemorrhage (ICH) remains unclear. METHODS: From January 2014 to September 2016, 497 patients with spontaneous ICH were enrolled in our study from 13 hospitals in Beijing. According to the CAR quartiles, patients were classified into four groups (Q1-Q4). Logistic regression was applied to analyze the relationship between different CAR levels and main outcome (90-day and 1-year mRS 4-6). Restricted cubic splines and receiver operating characteristic (ROC) curves of CAR for poor clinical outcomes were assessed. RESULTS: In the multivariate logistic regression model, compared with the lowest quartile of CAR, the adjusted odds ratios of the Q2, Q3, and Q4 group for 90-day mRS score of 4-6 were 3.64 (1.61-8.23), 3.83 (1.67-8.77), and 8.91 (3.85-20.64). In terms of 1-year mRS score of 4-6, compared with the lowest quartile of CAR, the adjusted odds ratios of the Q3 and Q4 group were 3.31 (1.33-8.22) and 6.87 (2.81-16.78). CONCLUSIONS: A high CAR level was associated with a high risk of long-term adverse prognosis in patients with ICH, and the risk of ICH poor outcome increased steadily with CAR rising in a certain range, and maintained in a high level thereafter.

Early Brain Injury After Subarachnoid Hemorrhage: Incidence and Mechanisms.

Aneurysmal subarachnoid hemorrhage is a devastating condition causing significant morbidity and mortality. While outcomes from subarachnoid hemorrhage have improved in recent years, there continues to be significant interest in identifying therapeutic targets for this disease. In particular, there has been a shift in emphasis toward secondary brain injury that develops in the first 72 hours after subarachnoid hemorrhage. This time period of interest is referred to as the early brain injury period and comprises processes including microcirculatory dysfunction, blood-brain-barrier breakdown, neuroinflammation, cerebral edema, oxidative cascades, and neuronal death. Advances in our understanding of the mechanisms defining the early brain injury period have been accompanied by improved imaging and nonimaging biomarkers for identifying early brain injury, leading to the recognition of an elevated clinical incidence of early brain injury compared with prior estimates. With the frequency, impact, and mechanisms of early brain injury better defined, there is a need to review the literature in this area to guide preclinical and clinical study.

Receptor-interacting protein 3-phosphorylated Ca2+ /calmodulin-dependent protein kinase II and mixed lineage kinase domain-like protein mediate intracerebral hemorrhage-induced neuronal necroptosis.

Necroptosis-mediated cell death is an important mechanism in intracerebral hemorrhage (ICH)-induced secondary brain injury (SBI). Our previous study has demonstrated that receptor-interacting protein 1 (RIP1) mediated necroptosis in SBI after ICH. However, further mechanisms, such as the roles of receptor-interacting protein 3 (RIP3), mixed lineage kinase domain-like protein (MLKL), and Ca2+ /calmodulin-dependent protein kinase II (CaMK II), remain unclear. We hypothesized that RIP3, MLKL, and CaMK II might participate in necroptosis after ICH, including their phosphorylation. The ICH model was induced by autologous blood injection. First, we found the activation of necroptosis after ICH in brain tissues surrounding the hematoma (propidium iodide staining). Meanwhile, the phosphorylation and expression of RIP3, MLKL, and CaMK II were differently up-regulated (western blotting and immunofluorescent staining). The specific inhibitors could suppress RIP3, MLKL, and CaMK II (GSK'872 for RIP3, necrosulfonamide for MLKL, and KN-93 for CaMK II). We found the necroptosis surrounding the hematoma and the concrete interactions in RIP3-MLKL/RIP3-CaMK II also both decreased after the specific intervention (co-immunoprecipitation). Then we conducted the short-/long-term neurobehavioral tests, and the rats with specific inhibition mostly had better performance. We also found less blood-brain barrier (BBB) injury, and less neuron loss (Nissl staining) in intervention groups, which supported the neurobehavioral tests. Besides, oxidative stress and inflammation were also alleviated with intervention, which had significant less reactive oxygen species (ROS), tumor necrosis factor (TNF)-α, lactate dehydrogenase (LDH), Iba1, and GFAP surrounding the hematoma. These results confirmed that RIP3-phosphorylated MLKL and CaMK II participate in ICH-induced necroptosis and could provide potential targets for the treatment of ICH patients.

Coagulopathy in Isolated Traumatic Brain Injury: Myth or Reality.

INTRODUCTION: Traumatic Brain Injury (TBI) has been shown to be associated with altered hemostasis and coagulopathy, that correlates with worsening secondary injury and clinical outcomes. Isolated Traumatic Brain Injury (iTBI), that is TBI without significant extracranial injuries, has also been shown to be associated with systemic coagulopathy and derangements in hemostasis. METHODS: Literature Review. RESULTS: Present your results in logical sequence in the text, tables, and figures, giving the main or most important findings first. Do not repeat all the data in the tables or figures in the text; emphasize or summarize only the most important observations. Provide data on all primary and secondary outcomes identified in the Methods section. Give numeric results not only as derivatives (e.g. percentages) but also as the absolute numbers from which the derivatives were calculated, and specify the statistical significance attached to them, if any. DISCUSSION: In this review, we provide an overview of the pathophysiology of the hemostatic disturbances caused by iTBI, review key clinical findings and discrepancies in the way this question has been approached, describe the use and role of global viscoelastic assays such as the thromboelastrogram, and detail principles for reversal of pre-injury blood thinners. CONCLUSIONS: iTBI is clearly associated with the development of coagulopathy, but the extent to which it occurs is confounded by the fact that many of the studies have included patients with moderate extracranial trauma into the iTBI category. The coagulopathy itself has been better studied in preclinical models, and the mechanisms driving it suggest a pattern consistent with disseminated intravascular coagulation with hyperfibrinolysis. We provide pragmatic clinical takeaways and suggestions for future research.

Dietary (Poly)phenols in Traumatic Brain Injury.

Traumatic brain injury (TBI) remains one of the leading causes of death and disability in young adults worldwide. Despite growing evidence and advances in our knowledge regarding the multifaceted pathophysiology of TBI, the underlying mechanisms, though, are still to be fully elucidated. Whereas initial brain insult involves acute and irreversible primary damage to the brain, the processes of subsequent secondary brain injury progress gradually over months to years, providing a window of opportunity for therapeutic interventions. To date, extensive research has been focused on the identification of druggable targets involved in these processes. Despite several decades of successful pre-clinical studies and very promising results, when transferred to clinics, these drugs showed, at best, modest beneficial effects, but more often, an absence of effects or even very harsh side effects in TBI patients. This reality has highlighted the need for novel approaches that will be able to respond to the complexity of the TBI and tackle TBI pathological processes on multiple levels. Recent evidence strongly indicates that nutritional interventions may provide a unique opportunity to enhance the repair processes after TBI. Dietary (poly)phenols, a big class of compounds abundantly found in fruits and vegetables, have emerged in the past few years as promising agents to be used in TBI settings due to their proven pleiotropic effects. Here, we give an overview of the pathophysiology of TBI and the underlying molecular mechanisms, followed by a state-of-the-art summary of the studies that have evaluated the efficacy of (poly)phenols administration to decrease TBI-associated damage in various animal TBI models and in a limited number of clinical trials. The current limitations on our knowledge concerning (poly)phenol effects in TBI in the pre-clinical studies are also discussed.

Clinical potential of automated convolutional neural network-based hematoma volumetry after aneurysmal subarachnoid hemorrhage.

OBJECTIVES: Cerebrospinal fluid hemoglobin has been positioned as a potential biomarker and drug target for aneurysmal subarachnoid hemorrhage-related secondary brain injury (SAH-SBI). The maximum amount of hemoglobin, which may be released into the cerebrospinal fluid, is defined by the initial subarachnoid hematoma volume (ISHV). In patients without external ventricular or lumbar drain, there remains an unmet clinical need to predict the risk for SAH-SBI. The aim of this study was to explore automated segmentation of ISHV as a potential surrogate for cerebrospinal fluid hemoglobin to predict SAH-SBI. METHODS: This study is based on a retrospective analysis of imaging and clinical data from 220 consecutive patients with aneurysmal subarachnoid hemorrhage collected over a five-year period. 127 annotated initial non-contrast CT scans were used to train and test a convolutional neural network to automatically segment the ISHV in the remaining cohort. Performance was reported in terms of Dice score and intraclass correlation. We characterized the associations between ISHV and baseline cohort characteristics, SAH-SBI, ventriculoperitoneal shunt dependence, functional outcome, and survival. Established clinical (World Federation of Neurosurgical Societies, Hunt & Hess) and radiological (modified Fisher, Barrow Neurological Institute) scores served as references. RESULTS: A strong volume agreement (0.73 Dice, range 0.43 - 0.93) and intraclass correlation (0.89, 95% CI, 0.81-0.94) were shown. While ISHV was not associated with the use of antithrombotics or cardiovascular risk factors, there was strong evidence for an association with a lower Glasgow Coma Scale at hospital admission. Aneurysm size and location were not associated with ISHV, but the presence of intracerebral or intraventricular hemorrhage were independently associated with higher ISHV. Despite strong evidence for a positive association between ISHV and SAH-SBI, the discriminatory ability of ISHV for SAH-SBI was insufficient. The discriminatory ability of ISHV was, however, higher regarding ventriculoperitoneal shunt dependence and functional outcome at three-months follow-up. Multivariate survival analysis provided strong evidence for an independent negative association between survival probability and both ISHV and intraventricular hemorrhage. CONCLUSIONS: The proposed algorithm demonstrates strong performance in volumetric segmentation of the ISHV on the admission CT. While the discriminatory ability of ISHV for SAH-SBI was similar to established clinical and radiological scores, it showed a high discriminatory ability for ventriculoperitoneal shunt dependence and functional outcome at three-months follow-up.

A mechanistic model of calcium homeostasis leading to occurrence and propagation of secondary brain injury.

Secondary brain injury (SBI) refers to new or worsening brain insult after primary brain injury (PBI). Neurophysiological experiments show that calcium (Ca2+) is one of the major culprits that contribute to neuronal damage and death following PBI. However, mechanistic details about how alterations of Ca2+ levels contribute to SBI are not well characterized. In this paper, we first build a biophysical model for SBI related to calcium homeostasis (SBI-CH) to study the mechanistic details of PBI-induced disruption of CH, and how these disruptions affect the occurrence of SBI. Then, we construct a coupled SBI-CH model by formulating synaptic interactions to investigate how disruption of CH affects synaptic function and further promotes the propagation of SBI between neurons. Our model shows how the opening of voltage-gated calcium channels (VGCCs), decreasing of plasma membrane calcium pump (PMCA), and reversal of the Na+/Ca2+ exchanger (NCX) during and following PBI, could induce disruption of CH and further promote SBI. We also show that disruption of CH causes synaptic dysfunction, which further induces loss of excitatory-inhibitory balance in the system, and this might promote the propagation of SBI and cause neighboring tissue to be injured. Our findings offer a more comprehensive understanding of the complex interrelationship between CH and SBI.NEW & NOTEWORTHY We build a mechanistic model SBI-CH for calcium homeostasis (CH) to study how alterations of Ca2+ levels following PBI affect the occurrence and propagation of SBI. Specifically, we investigate how the opening of VGCCs, decreasing of PMCA, and reversal of NCX disrupt CH, and further induce the occurrence of SBI. We also present a coupled SBI-CH model to show how disrupted CH causes synaptic dysfunction, and further promotes the propagation of SBI between neurons.

Role of mass effect and trehalose on early erythrolysis after experimental intracerebral hemorrhage.

The mechanisms of brain injury after intracerebral hemorrhage (ICH) involve mass effect-induced primary injury and secondary injury caused by a pathologic response to the hematoma. Considerable attentions have recently been paid to the mechanisms and therapeutic strategy for secondary brain injury due to no overall benefit from early surgery compared with initial conservative treatment. However, it is unclear whether there is a causal relationship between mass effect and secondary brain injury. Here, the role of mass effect on early erythrolysis after experimental ICH was investigated based on the poly(N-isopropylacrylamide) (PNIPAM) ICH model. Autologous blood and PNIPAM hydrogel were co-injected into the right basal ganglia of rats to induce different degrees of mass effect, but with a constant hematoma. The influences of different mass effect and time courses on erythrolysis and brain damages after ICH were investigated. Furthermore, the protective effect of trehalose against erythrolysis after ICH was evaluated. The results showed that mass effect caused erythrocyte morphological change at 24 hr after ICH. The released hemoglobin was quantitatively evaluated by a polynomial concerning with the mass effect, the volume of hematoma, and the time of ICH. An obvious increase in heme oxygenase-1 (HO-1) and ionized calcium binding adaptor molecule-1 (Iba-1) expression, iron deposition, cell death, and neurological deficits was observed with increasing mass effect. Moreover, trehalose alleviated brain injury by inhibiting erythrolysis after ICH. These data demonstrated that mass effect accelerated the erythrolysis and brain damages after ICH, which could be relieved through trehalose therapy.

MyD88-TLR4-dependent choroid plexus activation precedes perilesional inflammation and secondary brain edema in a mouse model of intracerebral hemorrhage.

BACKGROUND: The functional neurological outcome of patients with intracerebral hemorrhage (ICH) strongly relates to the degree of secondary brain injury (ICH-SBI) evolving within days after the initial bleeding. Different mechanisms including the incitement of inflammatory pathways, dysfunction of the blood-brain barrier (BBB), activation of resident microglia, and an influx of blood-borne immune cells, have been hypothesized to contribute to ICH-SBI. Yet, the spatiotemporal interplay of specific inflammatory processes within different brain compartments has not been sufficiently characterized, limiting potential therapeutic interventions to prevent and treat ICH-SBI. METHODS: We used a whole-blood injection model in mice, to systematically characterized the spatial and temporal dynamics of inflammatory processes after ICH using 7-Tesla magnetic resonance imaging (MRI), spatial RNA sequencing (spRNAseq), functional BBB assessment, and immunofluorescence average-intensity-mapping. RESULTS: We identified a pronounced early response of the choroid plexus (CP) peaking at 12-24 h that was characterized by inflammatory cytokine expression, epithelial and endothelial expression of leukocyte adhesion molecules, and the accumulation of leukocytes. In contrast, we observed a delayed secondary reaction pattern at the injection site (striatum) peaking at 96 h, defined by gene expression corresponding to perilesional leukocyte infiltration and correlating to the delayed signal alteration seen on MRI. Pathway analysis revealed a dependence of the early inflammatory reaction in the CP on toll-like receptor 4 (TLR4) signaling via myeloid differentiation factor 88 (MyD88). TLR4 and MyD88 knockout mice corroborated this observation, lacking the early upregulation of adhesion molecules and leukocyte infiltration within the CP 24 h after whole-blood injection. CONCLUSIONS: We report a biphasic brain reaction pattern after ICH with a MyD88-TLR4-dependent early inflammatory response of the CP, preceding inflammation, edema and leukocyte infiltration at the lesion site. Pharmacological targeting of the early CP activation might harbor the potential to modulate the development of ICH-SBI.

A model of metabolic supply-demand mismatch leading to secondary brain injury.

Secondary brain injury (SBI) is defined as new or worsening injury to the brain after an initial neurologic insult, such as hemorrhage, trauma, ischemic stroke, or infection. It is a common and potentially preventable complication following many types of primary brain injury (PBI). However, mechanistic details about how PBI leads to additional brain injury and evolves into SBI are poorly characterized. In this work, we propose a mechanistic model for the metabolic supply demand mismatch hypothesis (MSDMH) of SBI. Our model, based on the Hodgkin-Huxley model, supplemented with additional dynamics for extracellular potassium, oxygen concentration, and excitotoxity, provides a high-level unified explanation for why patients with acute brain injury frequently develop SBI. We investigate how decreased oxygen, increased extracellular potassium, excitotoxicity, and seizures can induce SBI and suggest three underlying paths for how events following PBI may lead to SBI. The proposed model also helps explain several important empirical observations, including the common association of acute brain injury with seizures, the association of seizures with tissue hypoxia and so on. In contrast to current practices which assume that ischemia plays the predominant role in SBI, our model suggests that metabolic crisis involved in SBI can also be nonischemic. Our findings offer a more comprehensive understanding of the complex interrelationship among potassium, oxygen, excitotoxicity, seizures, and SBI.NEW & NOTEWORTHY We present a novel mechanistic model for the metabolic supply demand mismatch hypothesis (MSDMH), which attempts to explain why patients with acute brain injury frequently develop seizure activity and secondary brain injury (SBI). Specifically, we investigate how decreased oxygen, increased extracellular potassium, excitotoxicity, seizures, all common sequalae of primary brain injury (PBI), can induce SBI and suggest three underlying paths for how events following PBI may lead to SBI.

Microglia as target for anti-inflammatory approaches to prevent secondary brain injury after subarachnoid hemorrhage (SAH).

BACKGROUND: Microglia-driven cerebral spreading inflammation is a key contributor to secondary brain injury after SAH. Genetic depletion or deactivation of microglia has been shown to ameliorate neuronal cell death. Therefore, clinically feasible anti-inflammatory approaches counteracting microglia accumulation or activation are interesting targets for SAH treatment. Here, we tested two different methods of interference with microglia-driven cerebral inflammation in a murine SAH model: (i) inflammatory preconditioning and (ii) pharmacological deactivation. METHODS: 7T-MRI-controlled SAH was induced by endovascular perforation in four groups of C57Bl/6 mice: (i) Sham-operation, (ii) SAH naïve, (iii) SAH followed by inflammatory preconditioning (LPS intraperitoneally), and (iv) SAH followed by pharmacological microglia deactivation (colony-stimulating factor-1 receptor-antagonist PLX3397 intraperitoneally). Microglia accumulation and neuronal cell death (immuno-fluorescence), as well as activation status (RT-PCR for inflammation-associated molecules from isolated microglia) were recorded at day 4 and 14. Toll-like receptor4 (TLR4) status was analyzed using FACS. RESULTS: Following SAH, significant cerebral spreading inflammation occurred. Microglia accumulation and pro-inflammatory gene expression were accompanied by neuronal cell death with a maximum on day 14 after SAH. Inflammatory preconditioning as well as PLX3397-treatment resulted in significantly reduced microglia accumulation and activation as well as neuronal cell death. TLR4 surface expression in preconditioned animals was diminished as a sign for receptor activation and internalization. CONCLUSIONS: Microglia-driven cerebral spreading inflammation following SAH contributes to secondary brain injury. Two microglia-focused treatment strategies, (i) inflammatory preconditioning with LPS and (ii) pharmacological deactivation with PLX3397, led to significant reduction of neuronal cell death. Increased internalization of inflammation-driving TLR4 after preconditioning leaves less receptor molecules on the cell surface, providing a probable explanation for significantly reduced microglia activation. Our findings support microglia-focused treatment strategies to overcome secondary brain injury after SAH. Delayed inflammation onset provides a valuable clinical window of opportunity.

SRC-3 Deficiency Exacerbates Neurological Deficits in a Mouse Model of Intracerebral Hemorrhage: Role of Oxidative Stress.

Intracerebral hemorrhage (ICH) causes long term neurological abnormality or death. Oxidative stress is closely involved in ICH mediated brain damage. Steroid receptor cofactor 3 (SRC-3), a p160 family member, is widely expressed in the brain and regulates transactivation of Nrf2, a key component of antioxidant response. Our study aims to test if SRC-3 is implicated in ICH mediated brain injury. We first examined levels of SRC-3 and oxidative stress in the brain of mice following ICH and analyzed their correlation. Then ICH was induced in wild type (WT) and SRC-3 knock out mice and how SRC-3 deletion affected ICH induced brain damage, oxidative stress and behavioral outcome was assessed. We found that SRC-3 mRNA and protein expression levels were reduced gradually after ICH induction in WT mice along with an increase in oxidative stress levels. Correlation analysis revealed that SRC-3 mRNA levels negatively correlated with oxidative stress. Deletion of SRC-3 further increased ICH induced brain edema, neurological deficit score and oxidative stress and exacerbated ICH induced behavioral abnormality including motor dysfunction and cognitive impairment. Our findings suggest that SRC-3 is involved in ICH induced brain injury, probably through modulation of oxidative stress.

Role of zinc dyshomeostasis in inflammasome formation in cultured cortical cells following lipopolysaccharide or oxygen-glucose deprivation/reperfusion exposure.

Exposure of mouse mixed cortical cell cultures to lipopolysaccharide (LPS) resulted in inflammasome formation in neurons and astrocytes, as indicated by increases in the levels of NLRP3, ASC, caspase-1, and IL-1ß. LPS exposure concurrently increased the level of free zinc in the cytosol of both cell types. Addition of the membrane-permeant zinc chelator TPEN blocked the increases in the levels of NLRP3 and caspase-1 as well as the release of inflammatory cytokines, indicating a role for increased zinc in LPS-induced inflammasome formation. Oxygen-glucose deprivation (OGD), a cellular model of hypoxia, also induced inflammasome formation and zinc dyshomeostasis in cortical cells, effects that were abolished upon zinc chelation with TPEN. A similar mechanism appeared to be at work in vivo. Whereas intraperitoneal injection of LPS in mice resulted in inflammasome formation and microglial activation in the brain, it caused little induction of inflammasome formation in ZnT3-null mice, which lack synaptic zinc, suggesting a specific role for synaptic zinc in LPS-induced formation of inflammasomes in the mouse brain.

Orexin A alleviates neuroinflammation via OXR2/CaMKKß/AMPK signaling pathway after ICH in mice.

BACKGROUND: Orexins are two neuropeptides (orexin A, OXA; orexin B, OXB) secreted mainly from the lateral hypothalamus, which exert a wide range of physiological effects by activating two types of receptors (orexin receptor 1, OXR1; orexin receptor 2, OXR2). OXA has equal affinity for OXR1 and OXR2, whereas OXB binds preferentially to OXR2. OXA rapidly crosses the blood-brain barrier by simple diffusion. Many studies have reported OXA's protective effect on neurological diseases via regulating inflammatory response which is also a fundamental pathological process in intracerebral hemorrhage (ICH). However, neuroprotective mechanisms of OXA have not been explored in ICH. METHODS: ICH models were established using stereotactic injection of autologous arterial blood into the right basal ganglia of male CD-1 mice. Exogenous OXA was administered intranasally; CaMKKß inhibitor (STO-609), OXR1 antagonist (SB-334867), and OXR2 antagonist (JNJ-10397049) were administered intraperitoneally. Neurobehavioral tests, hematoma volume, and brain water content were evaluated after ICH. Western blot and ELISA were utilized to evaluate downstream mechanisms. RESULTS: OXA, OXR1, and OXR2 were expressed moderately in microglia and astrocytes and abundantly in neurons. Expression of OXA decreased whereas OXR1 and OXR2 increased after ICH. OXA treatment significantly improved not only short-term but also long-term neurofunctional outcomes and reduced brain edema in ipsilateral hemisphere. OXA administration upregulated p-CaMKKß, p-AMPK, and anti-inflammatory cytokines while downregulated p-NFκB and pro-inflammatory cytokines after ICH; this effect was reversed by STO-609 or JNJ-10397049 but not SB-334867. CONCLUSIONS: OXA improved neurofunctional outcomes and mitigated brain edema after ICH, possibly through alleviating neuroinflammation via OXR2/CaMKKß/AMPK pathway.

MitoQ attenuates brain damage by polarizing microglia towards the M2 phenotype through inhibition of the NLRP3 inflammasome after ICH.

Microglial phenotype plays an important role in secondary injury after intracerebral haemorrhage (ICH), with M1 microglia promoting inflammatory injury and M2 microglia inhibiting neuroinflammation and promoting haematoma absorption. However, there is no effective intervention for regulating the phenotypic transformation of microglia after ICH. This study aimed to elucidate the protective effect of MitoQ, a selective mitochondrial ROS antioxidant, against microglial M1 state polarization and secondary brain injury. The in vivo data showed that MitoQ attenuated neurological deficits and decreased inflammation, oedema and haematoma volume after ICH. In addition, MitoQ decreased the expression of M1 markers and increased the expression of M2 markers both in vivo and in vitro after ICH. Mechanistically, MitoQ blocked overproduction of mitochondrial ROS and activation of the NLRP3 inflammasome in FeCl2-treated microglia. Moreover, NLRP3 siRNA shifted FeCl2-treated microglia from the M1 to the M2 cells, revealing that MitoQ-induce polarization states may be mediated by the mitochondrial ROS/NLRP-3 pathway. In summary, MitoQ alleviates secondary brain injury and accelerates haematoma resolution by shifting microglia towards the M2 phenotype after ICH.

Thromboelastometry as a Comprehensive Assessment of Hypercoagulation After Aneurysmal Subarachnoid Hemorrhage: A Case Report and Literature Review.

Subarachnoid hemorrhage after cerebral aneurysm rupture (aSAH) leads to delayed cerebral ischemia (DCI) in 25-35% of surviving patients. It is believed that DCI has a multifactorial etiology, including vasospasm. Furthermore, aSAH is associated with the development of hypercoagulation and microthrombosis; thus, its pharmacological correction may help to prevent DCI. We encountered a case where hypercoagulation was detected using rotational thromboelastometry (ROTEM), although the standard coagulation test results were within the normal ranges. Based on reviews of viscoelastic tests in cases of aSAH, ROTEM could be more sensitive to hypercoagulation after aSAH, compared to standard coagulation testing.

The Importance of Probe Location for the Interpretation of Cerebral Microdialysis Data in Subarachnoid Hemorrhage Patients.

BACKGROUND: There is no uniform definition for cerebral microdialysis (CMD) probe location with respect to focal brain lesions, and the impact of CMD-probe location on measured molecule concentrations is unclear. METHODS: We retrospectively analyzed data of 51 consecutive subarachnoid hemorrhage patients with CMD-monitoring between 2010 and 2016 included in a prospective observational cohort study. Microdialysis probe location was assessed on all brain computed tomography (CT) scans performed during CMD-monitoring and defined as perilesional in the presence of a focal hypodense or hyperdense lesion within a 1-cm radius of the gold tip of the CMD-probe, or otherwise as normal-appearing brain tissue. RESULTS: Probe location was detected in normal-appearing brain tissue on 53/143 (37%) and in perilesional location on 90/143 (63%) CT scans. In the perilesional area, CMD-glucose levels were lower (p = 0.003), whereas CMD-lactate (p = 0.002), CMD-lactate-to-pyruvate-ratio (LPR; p < 0.001), CMD-glutamate (p = 0.002), and CMD-glycerol levels (p < 0.001) were higher. Neuroglucopenia (CMD-glucose < 0.7 mmol/l, p = 0.002), metabolic distress (p = 0.002), and mitochondrial dysfunction (p = 0.005) were more common in perilesional compared to normal-appearing brain tissue. Development of new lesions in the proximity of the CMD-probe (n = 13) was associated with a decrease in CMD-glucose levels, evidence of neuroglucopenia, metabolic distress, as well as increasing CMD-glutamate and CMD-glycerol levels. Neuroglucopenia was associated with poor outcome independent of probe location, whereas elevated CMD-lactate, CMD-LPR, CMD-glutamate, and CMD-glycerol levels were only predictive of poor outcome in normal-appearing brain tissue. CONCLUSIONS: Focal brain lesions significantly impact on concentrations of brain metabolites assessed by CMD. With the exception of CMD-glucose, the prognostic value of CMD-derived parameters seems to be higher when assessed in normal-appearing brain tissue. CMD was sensitive to detect the development of new focal lesions in vicinity to the neuromonitoring probe. Probe location should be described in the research reporting brain metabolic changes measured by CMD and integrated in statistical models.