Microglial activation is not equivalent to neuroinflammation in alcohol-induced neurodegeneration: The importance of microglia phenotype.

Excessive alcohol intake, a defining characteristic of an alcohol use disorder (AUD), results in neurodegeneration in the hippocampus and entorhinal cortex that has been linked to a variety of cognitive deficits. Neuroinflammation is thought to be a factor in alcohol-induced neurodegeneration, and microglia activation is a key but not sole component of an inflammatory response. These experiments investigate the effects of ethanol exposure in a well-accepted model of an AUD on both microglial activation and blood brain barrier disruption (BBB) in order to understand their relationship to classical definitions of inflammation and alcohol-induced neurodegeneration. Following a four-day binge ethanol paradigm, rat hippocampal and entorhinal cortex tissue was examined using three distinct approaches to determine microglia phenotype and BBB disruption: immunohistochemistry, autoradiography, and ELISA. After ethanol exposure, there was an increase in [(3)H]-PK-11195 binding and OX-42 immunoreactivity indicative of microglial activation; however, microglia were not fully activated since both OX-6 and ED-1 immunoreactive microglia were absent. This data was supported by functional evidence as there was no increase in the proinflammatory cytokines IL-6 or TNF-α, but a 26% increase in the anti-inflammatory cytokine, IL-10, and a 38% increase in the growth factor, TGF-ß, seven days after exposure. Furthermore, there was no evidence of a disruption of the BBB. These data suggest that the four-day binge model of an AUD, which produces neurodegeneration in corticolimbic regions, does not elicit classical neuroinflammation but instead produces partially activated microglia. Partial activation of microglia following binge ethanol exposure suggest that microglia in this model have beneficial or homeostatic roles rather than directly contributing to neurodegeneration and are a consequence of alcohol-induced-damage instead of the source of damage.

Effects of genetic deficiency of cyclooxygenase-1 or cyclooxygenase-2 on functional and histological outcomes following traumatic brain injury in mice.

BACKGROUND: Neuroinflammation contributes to the pathophysiology of acute CNS injury, including traumatic brain injury (TBI). Although prostaglandin lipid mediators of inflammation contribute to a variety of inflammatory responses, their importance in neuroinflammation is not clear. There are conflicting reports as to the efficacy of inhibiting the enzymes required for prostaglandin formation, cyclooxygenase (COX) -1 and COX-2, for improving outcomes following TBI. The purpose of the current study was to determine the role of the COX isoforms in contributing to pathological processes resulting from TBI by utilizing mice deficient in COX-1 or COX-2. RESULTS: Following a mild controlled cortical impact injury, the amount of cortical tissue loss, the level of microglial activation, and the capacity for functional recovery was compared between COX-1-deficient mice or COX-2-deficient mice, and their matching wild-type controls. The deficiency of COX-2 resulted in a minor (6%), although statistically significant, increase in the sparing of cortical tissue following TBI. The deficiency of COX-1 resulted in no detectable effect on cortical tissue loss following TBI. As determined by 3[H]-PK11195 autoradiography, TBI produced a similar increase in microglial activation in multiple brain regions of both COX-1 wild-type and COX-1-deficient mice. In COX-2 wild-type and COX-2-deficient mice, TBI increased 3[H]-PK11195 binding in all brain regions that were analyzed. Following injury, 3[H]-PK11195 binding in the dentate gyrus and CA1 region of the hippocampus was greater in COX-2-deficient mice, as compared to COX-2 wild-type mice. Cognitive assessment was performed in the wild-type, COX-1-deficient and COX-2-deficient mice following 4 days of recovery from TBI. There was no significant cognitive effect that resulted from the deficiency of either COX-1 or COX-2, as determined by acquisition and spatial memory retention testing in a Morris water maze. CONCLUSION: These findings suggest that the deficiency of neither COX-1 nor COX-2 is sufficient to alter cognitive outcomes following TBI in mice.

Induction of miR-155 after Brain Injury Promotes Type 1 Interferon and has a Neuroprotective Effect.

Traumatic brain injury (TBI) produces profound and lasting neuroinflammation that has both beneficial and detrimental effects. Recent evidence has implicated microRNAs (miRNAs) in the regulation of inflammation both in the periphery and the CNS. We examined the expression of inflammation associated miRNAs in the context of TBI using a mouse controlled cortical impact (CCI) model and found increased levels of miR-21, miR-223 and miR-155 in the hippocampus after CCI. The expression of miR-155 was elevated 9-fold after CCI, an increase confirmed by in situ hybridization (ISH). Interestingly, expression of miR-155 was largely found in neuronal nuclei as evidenced by co-localization with DAPI in MAP2 positive neurons. In miR-155 knock out (KO) mice expression of type I interferons IFNα and IFNß, as well as IFN regulatory factor 1 and IFN-induced chemokine CXCL10 was decreased after TBI relative to wild type (WT) mice. Unexpectedly, miR-155 KO mice had increased levels of microglial marker Iba1 and increased neuronal degeneration as measured by fluoro-jade C (FJC) staining, suggesting a neuroprotective role for miR-155 in the context of TBI. This work demonstrates a role for miR-155 in regulation of the IFN response and neurodegeneration in the aftermath of TBI. While the presence of neuronal nuclear miRNAs has been described previously, their importance in disease states is relatively unknown. Here, we show evidence of dynamic regulation and pathological function of a nuclear miRNA in TBI.

The pathophysiology of traumatic brain injury in alpha7 nicotinic cholinergic receptor knockout mice.

The alpha7 nicotinic cholinergic receptor is a ligand-gated ion channel with calcium permeability similar to that of ionotrophic glutamate receptors. Previous studies from our laboratory have implicated changes in expression alpha7 nicotinic cholinergic receptors in the pathophysiology of traumatic brain injury (TBI). In rats, TBI causes a time-dependent and significant decrease in cortical and hippocampal alpha-[(125)I]-bungarotoxin (BTX) binding. We have postulated that deficits in alpha7 expression may contribute to TBI-induced cognitive impairment and that nicotinic receptor agonists can reverse alpha7 binding deficits and result in significant cognitive improvement compared to saline-treated controls. Thus, alpha7 nAChRs could be involved in a form of cholinergically mediated excitotoxicity following brain injury. In the current study, wild-type, heterozygous and null mutant mice were employed to test the hypothesis that genotypic depletion of the alpha7 receptor would render animals less sensitive to tissue loss and brain inflammation following experimental brain injury. Mice were anesthetized and subjected to a 0.5-mm cortical contusion injury of the somatosensory cortex. Brain inflammation, changes in nicotinic receptor expression and cortical tissue sparing were evaluated in wild-type, heterozygous and homozygous mice 1 week following TBI. In wild-type mice, brain injury caused a significant decrease in BTX binding in several hippocampal regions, consistent with what we have measured in rat brain following TBI. However, there were no genotypic differences in cortical tissue sparing or brain inflammation in this experiment. Although the results of this study were largely negative, it is still plausible that changes in the activity/expression of native alpha7 receptors contribute to pathophysiology following TBI. However, when null mutant mice develop in the absence of central alpha7 expression, it is possible that compensatory changes occur that confound the results obtained.

Traumatic brain injury increases levels of miR-21 in extracellular vesicles: implications for neuroinflammation.

Traumatic brain injury (TBI) is an important health concern and effective treatment strategies remain elusive. Understanding the complex multicellular response to TBI may provide new avenues for intervention. In the context of TBI, cell-cell communication is critical. One relatively unexplored form of cell-cell communication in TBI is extracellular vesicles (EVs). These membrane-bound vesicles can carry many different types of cargo between cells. Recently, miRNA in EVs have been shown to mediate neuroinflammation and neuronal injury. To explore the role of EV-associated miRNA in TBI, we isolated EVs from the brain of injured mice and controls, purified RNA from brain EVs, and performed miRNA sequencing. We found that the expression of miR-212 decreased, while miR-21, miR-146, miR-7a, and miR-7b were significantly increased with injury, with miR-21 showing the largest change between conditions. The expression of miR-21 in the brain was primarily localized to neurons near the lesion site. Interestingly, adjacent to these miR-21-expressing neurons were activated microglia. The concurrent increase in miR-21 in EVs with the elevation of miR-21 in neurons, suggests that miR-21 is secreted from neurons as potential EV cargo. Thus, this study reveals a new potential mechanism of cell-cell communication not previously described in TBI.

Granulocyte-macrophage colony stimulating factor exerts protective and immunomodulatory effects in cortical trauma.

Neurodegeneration after traumatic brain injury is facilitated by innate and adaptive immunity and can be harnessed to affect brain repair. In mice subjected to controlled cortical impact (CCI), we show that treatment with granulocyte macrophage colony stimulating factor (GM-CSF) affects regulatory T cell numbers in the cervical lymph nodes coincident with decreased lesion volumes and increased cortical tissue sparing. This paralleled increases in neurofilament and diminished reactive microglial staining. Transcriptomic analysis showed that GM-CSF induces robust immune neuroprotective responses seven days following CCI. Together, these results support the therapeutic potential of GM-CSF for TBI.

Bridge between neuroimmunity and traumatic brain injury.

The pathophysiology of degenerative, infectious, inflammatory and traumatic diseases of the central nervous system includes a significant immune component. As to the latter, damage to the cerebral vasculature and neural cell bodies, caused by traumatic brain injury (TBI) activates innate immunity with concomitant infiltration of immunocytes into the damaged nervous system. This leads to proinflammatory cytokine and prostaglandin production and lost synaptic integrity and more generalized neurotoxicity. Engagement of adaptive immune responses follows including the production of antibodies and lymphocyte proliferation. These affect the tempo of disease along with tissue repair and as such provide a number of potential targets for pharmacological treatments for TBI. However, despite a large body of research, no such treatment intervention is currently available. In this review we will discuss the immune response initiated following brain injuries, drawing on knowledge gained from a broad array of experimental and clinical studies. Our discussion seeks to address potential therapeutic targets and propose ways in which the immune system can be controlled to promote neuroprotection.

Long-term in vivo imaging of viscoelastic properties of the mouse brain after controlled cortical impact.

Traumatic brain injury (TBI) presents a variety of causes and symptoms, thus making the development of reliable diagnostic methods and therapeutic treatments challenging. Magnetic resonance elastography (MRE) is a technique that allows for a noninvasive assessment of the mechanical properties of soft biological tissue, such as tissue stiffness, storage modulus, and loss modulus. Importantly, by quantifying the changes in the stiffness of tissue that is often associated with disease, MRE is able to detect tissue pathologies at early stages. Recent improvements in instrumentation have allowed for the investigation of small samples with microscopic resolution (µMRE). We hypothesize that µMRE can sensitively detect variations in micromechanical properties in the brain caused by the compressive and shearing forces sustained during TBI. To test this hypothesis, we randomized 13 C57BL mice to receive a controlled cortical impact at a 0.5 mm or 0.75 mm depth, with both sham and naïve mice as controls. Our objective was to propagate mechanical shear waves throughout the brain for in vivo TBI µMRE imaging. The mechanical properties of the injured brain tissue were determined at days 0, 1, 7, and 28 post-injury. For both groups, we observed a significant drop in the stiffness of the impacted region immediately following the injury; the 0.75 mm animals experienced increased tissue softness that lasted longer than that for the 0.5 mm group. Although the shear stiffness, storage modulus, and loss modulus parameters all followed the same trend, the tissue stiffness yielded the most statistically significant results. Overall, this article introduces a transformative technique for mechanically mapping the brain and detecting brain diseases and injury.

Traumatic brain injury: central and peripheral role of α7 nicotinic acetylcholine receptors.

Traumatic brain injury (TBI) is a significant public health concern worldwide for which there is no cure. Once trauma has occurred, multiple biochemical pathways are set into motion that leads to a chronic, neurodegenerative condition. Two of the most widely studied pathological pathways are excitotoxicity and inflammation, processes that are influenced by α7 nicotinic acetylcholine receptors (nAChR). Previous studies have found a bilateral decrease in α7 nAChR expression in regions of the cortex and hippocampus that occurs in relation to injury severity. Subsequent studies showed that this decrease was evident in some parts of the hippocampus as early as 1 hour post-injury and remained decreased through 21 days. Other ligand-gated ion channels, such as non-α7 nAChRs and n-methyl-D-aspartate (NMDA) receptors did not show a similar widespread and consistent pattern of change following TBI, nor did the G-protein coupled muscarinic acetylcholine receptors, suggesting that the α7 nAChR could be a key mediator in the pathophysiology of traumatic brain injury. In addition to its expression in the brain, the α7 nAChR has been found outside of the central nervous system (CNS) on many different cell types, including peripheral blood leukocytes, where they have a role in the cholinergic antiinflammatory pathway, and have recently been identified on platelets where they may have a role in activation. How these receptors are regulated in response to injury has not been investigated, but could potentially serve as a marker of neurodegeneration as has been done in Alzheimer's disease and schizophrenia. In this review, we will detail the role of α7 nAChR following TBI as well as explore the evidence of this receptor subtype in regards to blood component (leukocytes and platelets) involvement and the potential influence TBI has on peripheral expression and function.

Therapeutic targets for neuroprotection and/or enhancement of functional recovery following traumatic brain injury.

Traumatic brain injury (TBI) is a significant public health concern. The number of injuries that occur each year, the cost of care, and the disabilities that can lower the victim's quality of life are all driving factors for the development of therapy. However, in spite of a wealth of promising preclinical results, clinicians are still lacking a therapy. The use of preclinical models of the primary mechanical trauma have greatly advanced our knowledge of the complex biochemical sequela that follow. This cascade of molecular, cellular, and systemwide changes involves plasticity in many different neurochemical systems, which represent putative targets for remediation or attenuation of neuronal injury. The purpose of this chapter is to highlight some of the promising molecular and cellular targets that have been identified and to provide an up-to-date summary of the development of therapeutic compounds for those targets.

Microscopic magnetic resonance elastography of traumatic brain injury model.

Traumatic brain injury (TBI) is a major cause of death and disability for which there is no cure. One of the issues inhibiting clinical trial success is the lack of targeting specific patient populations due to inconsistencies between clinical diagnostic tools and underlying pathophysiology. The development of reliable, noninvasive markers of TBI severity and injury mechanisms may better identify these populations, thereby improving clinical trial design. Magnetic resonance elastography (MRE), by assessing tissue mechanical properties, can potentially provide such marker. MRE synchronizes mechanical excitations with a phase contrast imaging pulse sequence to noninvasively register shear wave propagation, from which local values of tissue viscoelastic properties can be deduced. The working hypothesis of this study is that TBI involves a compression of brain tissue large enough to bring the material out of its elastic range, sufficiently altering mechanical properties to generate contrast on MRE measurements. To test this hypothesis, we combined microscopic MRE with brain tissue collected from adult male rats subjected to a controlled cortical impact injury. Measurements were made in different regions of interest (somatosensory cortex, hippocampus, and thalamus), and at different time points following the injury (immediate, 24 h, 7 days, 28 days). Values of stiffness in the somatosensory cortex were found to be 23-32% lower in the injured hemisphere than in the healthy one, when no significant difference was observed in the case of sham brains. A preliminary in vivo experiment is also presented, as well as alternatives to improve the faithfulness of stiffness recovery.

Melatonin and minocycline for combinatorial therapy to improve functional and histopathological deficits following traumatic brain injury.

The biochemical sequelae that follow traumatic brain injury (TBI) are numerous and affect many different brain functions at different points of time as the secondary cascades progress. The complexity of the resulting pathophysiology is such that a singular therapeutic intervention may not provide adequate benefit and a combination of drugs targeting different pathways may be needed. Two of the most widely studied injury mechanisms are oxidative stress and inflammation. Numerous studies have suggested that pharmacological agents targeting either of these pathways may produce an improvement in histological and functional outcome measures. We hypothesized that combining melatonin, a potent antioxidant, with minocycline, a bacteriostatic agent that also inhibit microglia, would provide better neuroprotection than either agent used alone. To test this hypothesis, we subjected anesthetized adult male rats to a 1.5mm controlled cortical impact and administered melatonin or vehicle in the acute post-injury period followed by daily minocycline or vehicle injections beginning the following day in a 2×2 study design. The animals were allowed to recover for 5 days before undergoing Morris water maze (MWM) testing to assess cognitive functioning following injury. There was no significant difference in MWM performance between the vehicle, melatonin, minocycline, or combination treatments. Following sacrifice and histological examination for neuroprotection, we did not observe a significant difference between the groups in the amount of cortical tissue that was spared nor was there a significant difference in [(3)H]-PK11195 binding, a marker for activated microglia. These results suggest that neither drug has therapeutic efficacy, however dosing and/or administration issues may have played a role.

Roles of neural stem cells and adult neurogenesis in adolescent alcohol use disorders.

This review discusses the contributions of a newly considered form of plasticity, the ongoing production of new neurons from neural stem cells, or adult neurogenesis, within the context of neuropathologies that occur with excessive alcohol intake in the adolescents. Neural stem cells and adult neurogenesis are now thought to contribute to the structural integrity of the hippocampus, a limbic system region involved in learning, memory, behavioral control, and mood. In adolescents with alcohol use disorders (AUDs), the hippocampus appears to be particularly vulnerable to the neurodegenerative effects of alcohol, but the role of neural stem cells and adult neurogenesis in alcoholic neuropathology has only recently been considered. This review encompasses a brief overview of neural stem cells and the processes involved in adult neurogenesis, how neural stem cells are affected by alcohol, and possible differences in the neurogenic niche between adults and adolescents. Specifically, what is known about developmental differences in adult neurogenesis between the adult and adolescent is gleaned from the literature, as well as how alcohol affects this process differently among the age groups. Finally, this review suggests differences that may exist in the neurogenic niche between adults and adolescents and how these differences may contribute to the susceptibility of the adolescent hippocampus to damage. However, many more studies are needed to discern whether these developmental differences contribute to the vulnerability of the adolescent to developing an AUD.