Data-driven approach to integrating genomic and behavioral preclinical traumatic brain injury research.

Understanding recovery from TBI is complex, involving multiple systems and modalities. The current study applied modern data science tools to manage this complexity and harmonize large-scale data to understand relationships between gene expression and behavioral outcomes in a preclinical model of chronic TBI (cTBI). Data collected by the Moody Project for Translational TBI Research included rats with no injury (naïve animals with similar amounts of anesthetic exposure to TBI and sham-injured animals), sham injury, or lateral fluid percussion TBI, followed by recovery periods up to 12 months. Behavioral measures included locomotor coordination (beam balance neuroscore) and memory and cognition assessments (Morris water maze: MWM) at multiple timepoints. Gene arrays were performed using hippocampal and cortical samples to probe 45,610 genes. To reduce the high dimensionality of molecular and behavioral domains and uncover gene-behavior associations, we performed non-linear principal components analyses (NL-PCA), which de-noised the data. Genomic NL-PCA unveiled three interpretable eigengene components (PC2, PC3, and PC4). Ingenuity pathway analysis (IPA) identified the PCs as an integrated stress response (PC2; EIF2-mTOR, corticotropin signaling, etc.), inflammatory factor translation (PC3; PI3K-p70S6K signaling), and neurite growth inhibition (PC4; Rho pathways). Behavioral PCA revealed three principal components reflecting the contribution of MWM overall speed and distance, neuroscore/beam walk, and MWM platform measures. Integrating the genomic and behavioral domains, we then performed a 'meta-PCA' on individual PC scores for each rat from genomic and behavioral PCAs. This meta-PCA uncovered three unique multimodal PCs, characterized by robust associations between inflammatory/stress response and neuroscore/beam walk performance (meta-PC1), stress response and MWM performance (meta-PC2), and stress response and neuroscore/beam walk performance (meta-PC3). Multivariate analysis of variance (MANOVA) on genomic-behavioral meta-PC scores tested separately on cortex and hippocampal samples revealed the main effects of TBI and recovery time. These findings are a proof of concept for the integration of disparate data domains for translational knowledge discovery, harnessing the full syndromic space of TBI.

Traumatic brain injury induces region-specific glutamate metabolism changes as measured by multiple mass spectrometry methods.

The release of excess glutamate following traumatic brain injury (TBI) results in glutamate excitotoxicity and metabolic energy failure. Endogenous mechanisms for reducing glutamate concentration in the brain parenchyma following TBI are poorly understood. Using multiple mass spectrometry approaches, we examined TBI-induced changes to glutamate metabolism. We present evidence that glutamate concentration can be reduced by glutamate oxidation via a "truncated" tricarboxylic acid cycle coupled to the urea cycle. This process reduces glutamate levels, generates carbon for energy metabolism, leads to citrulline accumulation, and produces nitric oxide. Several key metabolites are identified by metabolomics in support of this mechanism and the locations of these metabolites in the injured hemisphere are demonstrated by MALDI-MS imaging. The results of this study establish the advantages of multiple mass spectrometry approaches and provide insights into glutamate metabolism following TBI that could lead to improved treatment approaches.

MicroRNA sequencing of rat hippocampus and human biofluids identifies acute, chronic, focal and diffuse traumatic brain injuries.

High-throughput sequencing technologies could improve diagnosis and classification of TBI subgroups. Because recent studies showed that circulating microRNAs (miRNAs) may serve as noninvasive markers of TBI, we performed miRNA-seq to study TBI-induced changes in rat hippocampal miRNAs up to one year post-injury. We used miRNA PCR arrays to interrogate differences in serum miRNAs using two rat models of TBI (controlled cortical impact [CCI] and fluid percussion injury [FPI]). The translational potential of our results was evaluated by miRNA-seq analysis of human control and TBI (acute and chronic) serum samples. Bioinformatic analyses were performed using Ingenuity Pathway Analysis, miRDB, and Qlucore Omics Explorer. Rat miRNA profiles identified TBI across all acute and chronic intervals. Rat CCI and FPI displayed distinct serum miRNA profiles. Human miRNA profiles identified TBI across all acute and chronic time points and, at 24 hours, discriminated between focal and diffuse injuries. In both species, predicted gene targets of differentially expressed miRNAs are involved in neuroplasticity, immune function and neurorestoration. Chronically dysregulated miRNAs (miR-451a, miR-30d-5p, miR-145-5p, miR-204-5p) are linked to psychiatric and neurodegenerative disorders. These data suggest that circulating miRNAs in biofluids can be used as "molecular fingerprints" to identify acute, chronic, focal or diffuse TBI and potentially, presence of neurodegenerative sequelae.

Peroxynitrite decomposition catalyst reduces vasopressin requirement in ovine MRSA sepsis.

BACKGROUND: Sepsis is one of the most frequent causes of death in the intensive care unit. Host vascular hypo-responsiveness to vasopressors during septic shock is one of the challenging problems. This study tested the hypothesis that adjunct therapy with peroxynitrite decomposition catalyst (WW-85) would reduce arginine vasopressin (AVP) requirements during sepsis resuscitation, using ovine sepsis model. METHODS: Thirteen adult female Merino sheep, previously instrumented with multiple vascular catheters, were subjected to "two-hit" (cotton smoke inhalation and intrapulmonary instillation of live methicillin-resistant Staphylococcus aureus; 3.5 × 1011 colony-forming units) injury. Post injury, animals were awakened and randomly allocated to the following groups: (1) AVP: injured, fluid resuscitated, and titrated with AVP, n = 6 or (2) WW-85 + AVP: injured, fluid resuscitated, treated with WW-85, and titrated with AVP, n = 7. One-hour post injury, a bolus intravenous injection of WW-85 (0.1 mg/kg) was followed by a 23-h continuous infusion (0.02 mg/kg/h). Titration of AVP started at a dose of 0.01 unit/min, when mean arterial pressure (MAP) decreased by 10 mmHg from baseline, despite aggressive fluid resuscitation, and the rate was further adjusted to maintain MAP. After the injury, all animals were placed on a mechanical ventilator and monitored in the conscious state for 24 h. RESULTS: The injury induced severe hypotension refractory to aggressive fluid resuscitation. High doses of AVP were required to partially attenuate the sepsis-induced hypotension. However, the cumulative AVP requirement was significantly reduced by adjunct treatment with WW-85 at 17-24 h after the injury (p < 0.05). Total AVP dose and the highest AVP rate were significantly lower in the WW-85 + AVP group compared to the AVP group (p = 0.02 and 0.04, respectively). Treatment with WW-85 had no adverse effects. In addition, the in vitro effects of AVP on isolated artery diameter changes were abolished with peroxynitrite co-incubation. CONCLUSIONS: The modulation of reactive nitrogen species, such as peroxynitrite, may be considered as a novel adjunct treatment option for septic shock associated with vascular hypo-responsiveness to vasopressors.

MicroRNA profiling identifies a novel compound with antidepressant properties.

Patients with traumatic brain injury (TBI) are frequently diagnosed with depression. Together, these two leading causes of death and disability significantly contribute to the global burden of healthcare costs. However, there are no drug treatments for TBI and antidepressants are considered off-label for depression in patients with TBI. In molecular profiling studies of rat hippocampus after experimental TBI, we found that TBI altered the expression of a subset of small, non-coding, microRNAs (miRNAs). One known neuroprotective compound (17ß-estradiol, E2), and two experimental neuroprotective compounds (JM6 and PMI-006), reversed the effects of TBI on miRNAs. Subsequent in silico analyses revealed that the injury-altered miRNAs were predicted to regulate genes involved in depression. Thus, we hypothesized that drug-induced miRNA profiles can be used to identify compounds with antidepressant properties. To confirm this hypothesis, we examined miRNA expression in hippocampi of injured rats treated with one of three known antidepressants (imipramine, fluoxetine and sertraline). Bioinformatic analyses revealed that TBI, potentially via its effects on multiple regulatory miRNAs, dysregulated transcriptional networks involved in neuroplasticity, neurogenesis, and circadian rhythms- networks known to adversely affect mood, cognition and memory. As did E2, JM6, and PMI-006, all three antidepressants reversed the effects of TBI on multiple injury-altered miRNAs. Furthermore, JM6 reduced TBI-induced inflammation in the hippocampus and depression-like behavior in the forced swim test; these are both properties of classic antidepressant drugs. Our results support the hypothesis that miRNA expression signatures can identify neuroprotective and antidepressant properties of novel compounds and that there is substantial overlap between neuroprotection and antidepressant properties.

Traumatic brain injury induces long-lasting changes in immune and regenerative signaling.

There are no existing treatments for the long-term degenerative effects of traumatic brain injury (TBI). This is due, in part, to our limited understanding of chronic TBI and uncertainty about which proposed mechanisms for long-term neurodegeneration are amenable to treatment with existing or novel drugs. Here, we used microarray and pathway analyses to interrogate TBI-induced gene expression in the rat hippocampus and cortex at several acute, subchronic and chronic intervals (24 hours, 2 weeks, 1, 2, 3, 6 and 12 months) after parasagittal fluid percussion injury. We used Ingenuity pathway analysis (IPA) and Gene Ontology enrichment analysis to identify significantly expressed genes and prominent cell signaling pathways that are dysregulated weeks to months after TBI and potentially amenable to therapeutic modulation. We noted long-term, coordinated changes in expression of genes belonging to canonical pathways associated with the innate immune response (i.e., NF-κB signaling, NFAT signaling, Complement System, Acute Phase Response, Toll-like receptor signaling, and Neuroinflammatory signaling). Bioinformatic analysis suggested that dysregulation of these immune mediators-many are key hub genes-would compromise multiple cell signaling pathways essential for homeostatic brain function, particularly those involved in cell survival and neuroplasticity. Importantly, the temporal profile of beneficial and maladaptive immunoregulatory genes in the weeks to months after the initial TBI suggests wider therapeutic windows than previously indicated.

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries.

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast's cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p < 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p < 0.05) higher than in the Sham bTBI groups. MCA's collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA's collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Modulation of Peroxynitrite Reduces Norepinephrine Requirements in Ovine MRSA Septic Shock.

Vascular hypo-responsiveness to vasopressors during septic shock is a challenging problem. This study is to test the hypothesis that reactive nitrogen species (RNS), such as peroxynitrite, are major contributing factors to vascular hypo-responsiveness in septic shock. We hypothesized that adjunct therapy with peroxynitrite decomposition catalyst (PDC) would reduce norepinephrine requirements in sepsis resuscitation. Fourteen female Merino sheep were subjected to a "two-hit" injury (smoke inhalation and endobronchial instillation of live methicillin-resistant Staphylococcus aureus [1.6-2.5 × 10 CFUs]). The animals were randomly allocated to control: injured, fluid resuscitated, and titrated norepinephrine, n = 7; or PDC: injured, fluid resuscitated, titrated norepinephrine, and treated with PDC, n = 7. One-hour postinjury, an intravenous injection of PDC (0.1 mg/kg) was followed by a continuous infusion (0.04 mg/kg/h). Titration of norepinephrine started at 0.05 mcg/kg/min based on their mean arterial pressure. All animals were mechanically ventilated and monitored in the conscious state for 24 h. The mean arterial pressure was well maintained in the PDC with significantly less norepinephrine requirement from 7 to 23 h after injury compared with control. Total norepinephrine dose, the highest norepinephrine rate, and time on norepinephrine support were also significantly lower in PDC. Modified sheep organ failure assessment scores at 6 to 18 h postinjury were significantly lower in PDC compared with control. PDC improved survival rate at 24 h (71.4% vs. 28.6%). PDC treatment had no adverse effects. In conclusion, the modulation of RNS may be considered an effective adjunct therapy for septic shock, in the case of hypo-responsiveness to norepinephrine.

Publisher Correction: Evidence linking microRNA suppression of essential prosurvival genes with hippocampal cell death after traumatic brain injury.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Proteomic changes in traumatic brain injury: experimental approaches.

PURPOSE OF REVIEW: The underlying mechanisms responsible for chronic and progressive neurological damage after traumatic brain injury (TBI) are poorly understood, and therefore, current treatment options are limited. Proteomics is an emerging methodology to study changes to the TBI proteome in both patients and experimental models. RECENT FINDINGS: Although experimentally complex, mass spectrometry-based proteomics approaches are converging on a set of common methods. However, these methods are being applied to an increasingly diverse range of experimental models and types of injury. SUMMARY: In this review, our aim is to briefly describe experimental TBI models and the underlying methods common to most proteomic approaches. We will then review a series of articles that have recently appeared in which these approaches have been applied to important TBI questions. We will summarize several recent experimental studies, and suggest how the results of these emerging studies might impact future research as well as patient treatment.

Pre-Clinical Testing of Therapies for Traumatic Brain Injury.

Despite the large number of promising neuroprotective agents identified in experimental traumatic brain injury (TBI) studies, none has yet shown meaningful improvements in long-term outcome in clinical trials. To develop recommendations and guidelines for pre-clinical testing of pharmacological or biological therapies for TBI, the Moody Project for Translational Traumatic Brain Injury Research hosted a symposium attended by investigators with extensive experience in pre-clinical TBI testing. The symposium participants discussed issues related to pre-clinical TBI testing including experimental models, therapy and outcome selection, study design, data analysis, and dissemination. Consensus recommendations included the creation of a manual of standard operating procedures with sufficiently detailed descriptions of modeling and outcome measurement procedures to permit replication. The importance of the selection of clinically relevant outcome variables, especially related to behavior testing, was noted. Considering the heterogeneous nature of human TBI, evidence of therapeutic efficacy in multiple, diverse (e.g., diffuse vs. focused) rodent models and a species with a gyrencephalic brain prior to clinical testing was encouraged. Basing drug doses, times, and routes of administration on pharmacokinetic and pharmacodynamic data in the test species was recommended. Symposium participants agreed that the publication of negative results would reduce costly and unnecessary duplication of unsuccessful experiments. Although some of the recommendations are more relevant to multi-center, multi-investigator collaborations, most are applicable to pre-clinical therapy testing in general. The goal of these consensus guidelines is to increase the likelihood that therapies that improve outcomes in pre-clinical studies will also improve outcomes in TBI patients.

Nano-Pulsed Laser Therapy Is Neuroprotective in a Rat Model of Blast-Induced Neurotrauma.

We have developed a novel, non-invasive nano-pulsed laser therapy (NPLT) system that combines the benefits of near-infrared laser light (808 nm) and ultrasound (optoacoustic) waves, which are generated with each short laser pulse within the tissue. We tested NPLT in a rat model of blast-induced neurotrauma (BINT) to determine whether transcranial application of NPLT provides neuroprotective effects. The laser pulses were applied on the intact rat head 1 h after injury using a specially developed fiber-optic system. Vestibulomotor function was assessed on post-injury days (PIDs) 1-3 on the beam balance and beam walking tasks. Cognitive function was assessed on PIDs 6-10 using a working memory Morris water maze (MWM) test. BDNF and caspase-3 messenger RNA (mRNA) expression was measured by quantitative real-time PCR (qRT-PCR) in laser-captured cortical neurons. Microglia activation and neuronal injury were assessed in brain sections by immunofluorescence using specific antibodies against CD68 and active caspase-3, respectively. In the vestibulomotor and cognitive (MWM) tests, NPLT-treated animals performed significantly better than the untreated blast group and similarly to sham animals. NPLT upregulated mRNA encoding BDNF and downregulated the pro-apoptotic protein caspase-3 in cortical neurons. Immunofluorescence demonstrated that NPLT inhibited microglia activation and reduced the number of cortical neurons expressing activated caspase-3. NPLT also increased expression of BDNF in the hippocampus and the number of proliferating progenitor cells in the dentate gyrus. Our data demonstrate a neuroprotective effect of NPLT and prompt further studies aimed to develop NPLT as a therapeutic intervention after traumatic brain injury (TBI).

Impact & Blast Traumatic Brain Injury: Implications for Therapy.

Traumatic brain injury (TBI) is one of the most frequent causes of combat casualties in Operations Iraqi Freedom (OIF), Enduring Freedom (OEF), and New Dawn (OND). Although less common than combat-related blast exposure, there have been significant numbers of blast injuries in civilian populations in the United States. Current United States Department of Defense (DoD) ICD-9 derived diagnoses of TBI in the DoD Health Care System show that, for 2016, severe and moderate TBIs accounted for just 0.7% and 12.9%, respectively, of the total of 13,634 brain injuries, while mild TBIs (mTBIs) accounted for 86% of the total. Although there is a report that there are differences in the frequency of long-term complications in mTBI between blast and non-blast TBIs, clinical presentation is classified by severity score rather than mechanism because severity scoring is associated with prognosis in clinical practice. Blast TBI (bTBI) is unique in its pathology and mechanism, but there is no treatment specific for bTBIs-these patients are treated similarly to TBIs in general and therapy is tailored on an individual basis. Currently there is no neuroprotective drug recommended by the clinical guidelines based on evidence.

Effects of Mild Blast Traumatic Brain Injury on Cerebral Vascular, Histopathological, and Behavioral Outcomes in Rats.

To determine the effects of mild blast-induced traumatic brain injury (bTBI), several groups of rats were subjected to blast injury or sham injury in a compressed air-driven shock tube. The effects of bTBI on relative cerebral perfusion (laser Doppler flowmetry [LDF]), and mean arterial blood pressure (MAP) cerebral vascular resistance were measured for 2 h post-bTBI. Dilator responses to reduced intravascular pressure were measured in isolated middle cerebral arterial (MCA) segments, ex vivo, 30 and 60 min post-bTBI. Neuronal injury was assessed (Fluoro-Jade C [FJC]) 24 and 48 h post-bTBI. Neurological outcomes (beam balance and walking tests) and working memory (Morris water maze [MWM]) were assessed 2 weeks post-bTBI. Because impact TBI (i.e., non-blast TBI) is often associated with reduced cerebral perfusion and impaired cerebrovascular function in part because of the generation of reactive oxygen and nitrogen species such as peroxynitrite (ONOO-), the effects of the administration of the ONOO- scavenger, penicillamine methyl ester (PenME), on cerebral perfusion and cerebral vascular resistance were measured for 2 h post-bTBI. Mild bTBI resulted in reduced relative cerebral perfusion and MCA dilator responses to reduced intravascular pressure, increases in cerebral vascular resistance and in the numbers of FJC-positive cells in the brain, and significantly impaired working memory. PenME administration resulted in significant reductions in cerebral vascular resistance and a trend toward increased cerebral perfusion, suggesting that ONOO- may contribute to blast-induced cerebral vascular dysfunction.

Effects of AAV-mediated knockdown of nNOS and GPx-1 gene expression in rat hippocampus after traumatic brain injury.

Virally mediated RNA interference (RNAi) to knock down injury-induced genes could improve functional outcome after traumatic brain injury (TBI); however, little is known about the consequences of gene knockdown on downstream cell signaling pathways and how RNAi influences neurodegeneration and behavior. Here, we assessed the effects of adeno-associated virus (AAV) siRNA vectors that target two genes with opposing roles in TBI pathogenesis: the allegedly detrimental neuronal nitric oxide synthase (nNOS) and the potentially protective glutathione peroxidase 1 (GPx-1). In rat hippocampal progenitor cells, three siRNAs that target different regions of each gene (nNOS, GPx-1) effectively knocked down gene expression. However, in vivo, in our rat model of fluid percussion brain injury, the consequences of AAV-siRNA were variable. One nNOS siRNA vector significantly reduced the number of degenerating hippocampal neurons and showed a tendency to improve working memory. GPx-1 siRNA treatment did not alter TBI-induced neurodegeneration or working memory deficits. Nevertheless, microarray analysis of laser captured, virus-infected neurons showed that knockdown of nNOS or GPx-1 was specific and had broad effects on downstream genes. Since nNOS knockdown only modestly ameliorated TBI-induced working memory deficits, despite widespread genomic changes, manipulating expression levels of single genes may not be sufficient to alter functional outcome after TBI.

Evidence linking microRNA suppression of essential prosurvival genes with hippocampal cell death after traumatic brain injury.

The underlying molecular mechanisms of how dysregulated microRNAs (miRNAs) cause neurodegeneration after traumatic brain injury (TBI) remain elusive. Here we analyzed the biological roles of approximately 600 genes - we previously found these dysregulated in dying and surviving rat hippocampal neurons - that are targeted by ten TBI-altered miRNAs. Bioinformatic analysis suggests that neurodegeneration results from a global miRNA-mediated suppression of genes essential for maintaining proteostasis; many are hub genes - involved in RNA processing, cytoskeletal metabolism, intracellular trafficking, cell cycle progression, repair/maintenance, bioenergetics and cell-cell signaling - whose disrupted expression is linked to human disease. Notably, dysregulation of these essential genes would significantly impair synaptic function and functional brain connectivity. In surviving neurons, upregulated miRNA target genes are co-regulated members of prosurvival pathways associated with cellular regeneration, neural plasticity, and development. This study captures the diversity of miRNA-regulated genes that may be essential for cell repair and survival responses after TBI.

Persistent Behavioral Deficits in Rats after Parasagittal Fluid Percussion Injury.

Although traumatic brain injury (TBI) is now considered a chronic disease, few studies have investigated the long-term behavioral deficits elicited by a well-established rodent model of injury. Here we evaluate behavioral measures, commonly used in TBI research, to determine which tests are useful for studying long-term effects of brain injury in rats. Male Sprague-Dawley rats were handled and pre-trained to neurological, balance, and motor coordination tests prior to receiving parasagittal fluid-percussion injury (FPI), sham injury, or maintenance as naïve cohorts. Rats underwent neuroscore, beam-balance, and beam-walk tests for 3 days after injury. Subsequently, in separate groups at 3, 6, or 12 months, they were re-tested on the same tasks followed by a working memory version of the Morris water maze. On post-injury days (PIDs) 1-3, significant effects of injury on neuroscore, beam-balance, and beam-walk were observed. Differences in the beam-walk task were not detectable at any of the later time-points. However, deficits persisted in beam-balance out to 3 months and neuroscore out to 6 months. Working memory deficits persisted out to 12 months, at which time a reference memory deficit was also evident. These data suggest that balance and motor coordination recovered more quickly than neurological deficits. Furthermore, while deficits in working memory remained stable over the 12-month period, the late onset of the reference memory deficit points to the progressive nature of the injury, or an age/TBI interaction. In conclusion, standard behavioral tests are useful measures of persistent behavioral deficits after parasagittal FPI and provide evidence that TBI is a chronic condition that can change over time and worsen with age.

Human Neural Stem Cell Transplantation-Mediated Alteration of Microglial/Macrophage Phenotypes after Traumatic Brain Injury.

Neural stem cells (NSCs) promote recovery from brain trauma, but neuronal replacement is unlikely the sole underlying mechanism. We hypothesize that grafted NSCs enhance neural repair at least partially through modulating the host immune response after traumatic brain injury (TBI). C57BL/6 mice were intracerebrally injected with primed human NSCs (hNSCs) or vehicle 24 h after a severe controlled cortical impact injury. Six days after transplantation, brain tissues were collected for Western blot and immunohistochemical analyses. Observations included indicators of microglia/macrophage activation, M1 and M2 phenotypes, axonal injury detected by amyloid precursor protein (APP), lesion size, and the fate of grafted hNSCs. Animals receiving hNSC transplantation did not show significant decreases of brain lesion volumes compared to transplantation procedures with vehicle alone, but did show significantly reduced injury-dependent accumulation of APP. Furthermore, intracerebral transplantation of hNSCs reduced microglial activation as shown by a diminished intensity of Iba1 immunostaining and a transition of microglia/macrophages toward the M2 anti-inflammatory phenotype. The latter was represented by an increase in the brain M2/M1 ratio and increases of M2 microglial proteins. These phenotypic switches were accompanied by the increased expression of anti-inflammatory interleukin-4 receptor α and decreased proinflammatory interferon-γ receptor ß. Finally, grafted hNSCs mainly differentiated into neurons and were phagocytized by either M1 or M2 microglia/macrophages. Thus, intracerebral transplantation of primed hNSCs efficiently leads host microglia/macrophages toward an anti-inflammatory phenotype that presumably contributes to stem cell-mediated neuroprotective effects after severe TBI in mice.