Following traumatic brain injury (TBI), individuals over 65 years of age show increased mortality and worse functional outcomes compared to younger persons. As neuroinflammation is a key pathobiological mechanism of secondary injury after TBI, we examined how aging affects posttraumatic microglial responses and functional outcomes. Young (12-week-old) and aged (18month-old) male C57Bl/6 mice were subjected to moderate-level controlled cortical impact (CCI) or sham surgery and neurological function was evaluated. At 72 hours post-injury, brain, blood, and spleen leukocyte counts were assessed ex vivo using flow cytometry. Aged mice demonstrated more severe deficits in forelimb grip strength, balance and motor coordination, spontaneous locomotor activity, and anxiety-like behavior. These animals also exhibited more robust microglial proliferation and significantly higher numbers of brain-infiltrating leukocytes. Microglia in aged mice showed impairments in phagocytic activity and higher production of interleukin-1β (IL-1β). Infiltrating myeloid cells in aged TBI mice also had deficits in phagocytosis, but showed diminished pro-inflammatory cytokine production and greater reactive oxygen species production. Expression of several senescence markers (Bcl-2, p16 ink4a , p21 cip1a , lipofuscin, and H2AX (pS139)) was increased with age and/or TBI in both microglia and injured cortex. Although there was no difference in the number of circulating blood neutrophils as a function of age, young mice exhibited more pronounced TBI-induced splenomegaly and splenic myeloid cell expansion. Thus,

The inflammatory response to moderate-severe controlled cortical impact (CCI) in adult male mice has been shown to exhibit greater glial activation compared with age-matched female mice. However, the relative contributions of resident microglia and infiltrating peripheral myeloid cells to this sexually dimorphic neuroinflammatory responses remains unclear. Here, 12-week-old male and female C57Bl/6 mice were subjected to sham or CCI, and brain samples were collected at 1, 3, or 7 days post-injury for flow cytometry analysis of cytokines, reactive oxygen species (ROS), and phagocytosis in resident microglia (CD45 int CD11b+) versus infiltrating myeloid cells (CD45 hi CD11b+). Motor (rotarod, cylinder test), affect (open field), and cognitive (Y-maze) function tests also were performed. We demonstrate that male microglia had increased phagocytic activity and higher ROS levels in the non-injured brain, whereas female microglia had increased production of tumor necrosis factor (TNF) a and interleukin (IL)-1b. Following CCI, males showed a significant influx of peripheral myeloid cells by 1 day post-injury followed by proliferation of resident microglia at 3 days. In contrast, myeloid infiltration and microglial activation responses in female CCI mice were significantly reduced. No sex differences were observed for TNFa, IL-1b, transforming growth factor b, NOX2, ROS production, or phagocytic activity in resident microglia or infiltrating cells at any time. However, across these functions, infiltrating myeloid cells were significantly more reactive than resident microglia. Female CCI mice also had improved motor function at 1 day postinjury compared with male mice. Thus, we conclude that sexually dimorphic responses to moderate-severe CCI result from the rapid activation and infiltration of pro-inflammatory myeloid cells to brain in male, but not female, mice.

Background Excessive iron contributes to oxidative stress after central nervous system injury. NADPH oxidase (NOX) enzymes are upregulated in microglia after pro-inflammatory activation and contribute to oxidative stress. The relationship between iron, microglia, NOX, and oxidative stress is currently unclear. Methods We evaluated the effects of iron on lipopolysaccharide (LPS)-activated microglia and its secondary effect within neuronal co-cultures. Further, NOX2 and four specific inhibitors were tested to evaluate the relationship with the reactive oxygen species (ROS)-producing enzymes. Results An iron dose-dependent increase in ROS production among microglia treated with LPS was identified. Interestingly, despite this increase in ROS, inflammatory polarization alterations were not detected among the microglia after exposure to iron and LPS. Co-culture experimentation between primary neurons and exposed microglia (iron and LPS) significantly reduced neuronal cell number at 24 h, suggesting a profound neurotoxic effect despite the lack of a change in polarization phenotype. NOX2 and NOX4 inhibition significantly reduced ROS production among microglia exposed to iron and LPS and reduced neuronal damage and death in response to microglial co-culture. Conclusions In conclusion, iron significantly increased ROS production and neurotoxicity without exacerbating LP-activated microglia phenotype in vitro, suggesting that iron contributes to microglia-related oxidative stress, and this may be a viable therapeutic target for injury or neurodegeneration. Further, this study highlights both NOX2 and NOX4 as potential therapeutic targets in the treatment of iron-induced microglia-related inflammation and neurotoxicity. Electronic supplementary material The online version of this article (10.1186/s12974-019-1430-7) contains supplementary material, which is available to authorized users.

Background Sex and age are emerging as influential variables that affect spinal cord injury (SCI) recovery. Despite a changing demographic towards older age at the time of SCI, the effects of sex or age on inflammation remain to be elucidated. This study determined the sex- and age-dependency of the innate immune response acutely after SCI. Methods Male and female mice of ages 4- and 14-month-old received T9 contusion SCI and the proportion of microglia, monocyte-derived macrophages (MDM), and neutrophils surrounding the lesion were determined at 3- and 7-day post-injury (DPI) using flow cytometry. Cell counts of microglia and MDMs were obtained using immunohistochemistry to verify flow cytometry results at 3-DPI. Microglia and MDMs were separately isolated using fluorescence-activated cell sorting (FACS) at 3-day post-injury (DPI) to assess RNA expression of 27 genes associated with activation, redox, and debris metabolism/clearance. Results Flow cytometry revealed that being female and older at the time of injury significantly increased MDMs relative to other phagocytes, specifically increasing the ratio of MDMs to microglia at 3-DPI. Cell counts using immunohistochemistry revealed that male mice have more total microglia within SCI lesions that can account for a lower MDM/microglia ratio. With NanoString analyses of 27 genes, only 1 was differentially expressed between sexes in MDMs; specifically, complement protein C1qa was increased in males. No genes were affected by age in MDMs. Only 2 genes were differentially regulated in microglia between sexes after controlling for false discovery rate, specifically CYBB (NOX2) as a reactive oxygen species (ROS)-associated marker as well as MRC1 (CD206), a gene associated with reparative phenotypes. Both genes were increased in female microglia. No microglial genes were differentially regulated between ages. Differences between microglia and MDMs were found in 26 of 27 genes analyzed, all expressed higher in MDMs with three exceptions. Specifically, C1qa, cPLA2, and CD86 were expressed higher in microglia. Conclusions These findings indicate that inflammatory responses to SCI are sex-dependent at both the level of cellular recruitment and gene expression.

Angiopoietin-1 (Ang-1) is a well-known endothelial growth factor, but its effects on neurons have yet to be elucidated. We show that Ang-1 is rapidly downregulated in the injured brain after controlled cortical impact (CCI), a mouse experimental traumatic brain injury (TBI) model and in etoposide-induced neuronal apoptosis in vitro. Ang-1 treatment inhibits etoposide-induced upregulation of proapoptotic B-cell lymphoma 2 (Bcl-2) family members Noxa, p53 upregulated modulator of apoptosis (Puma), Bcl-2 interacting mediator of cell death (Bim), and Bcl-2-associated X protein (Bax); reduces markers of caspase-dependent (cytochrome c release/caspase activation) and caspase-independent (apoptosis-inducing factor release) pathways; and limits neuronal cell death. Ang-1 treatment phosphorylates receptors Tunica interna endothelial cell kinase 2 (Tie2), and β1-integrin and limits the etoposide-induced decrease in protein kinase B (Akt) activity. Blocking Tie2 and β1-integrin signaling reduces Ang-1 neuroprotective effects. After both TBI and etoposide treatment microRNA (miR)-711 are upregulated, consistent with its putative role as a negative regulator of Ang-1. We show that miR-711 directly targets the Ang-1 messenger RNA (mRNA), decreasing Ang-1 expression. Increased levels of miR-711 and Ang-1 mRNA are found in the RNA-induced silencing complex complex site of miR-mediated degradation of target mRNAs after etoposide treatment and the miR-711mimic downregulates Ang-1. Administration of miR-711 inhibitor elevates Ang-1 after TBI whereas Ang-1 administration increases Akt activation; reduces Puma, Noxa, Bim, and Bax levels; and attenuates caspase-dependent and -independent neuronal apoptosis 24 h after TBI. Ang-1 also attenuates neuronal degeneration, increases gene expression of molecules that maintain blood-brain barrier integrity, and reduces post-traumatic lesion volume/edema 24 h after TBI. Although we only observed short-term neuroprotective effects after Ang-1 administration, miR-711-dependent downregulation of Ang-1, followed by Akt pathway inhibition, may play a role in neuronal cell death after neuronal injury in vitro and after experimental TBI.

Structure-switching sensors utilize recognition elements that undergo a conformation change upon target binding that is converted into a quantitative signal. Electrochemical, aptamer-based sensors achieve detection of analytes through a conformation change in an electrode-bound, oligonucleotide aptamer by measuring changes in electron transfer efficiencies. The analytical performance of these sensors is related to the magnitude of the conformation change of the aptamer. The goal of the present work is to develop a general method to predictably tune the analytical performance (sensitivity and linear range) of electrochemical, aptamer-based sensors by utilizing a mixture of rationally-designed aptamer sequences that are specific for the same target but with different affinities on the same electrode surface. To demonstrate control over sensor performance, we developed heterogeneous sensors for two representative small molecule targets (adenosine triphosphate; tobramycin). We demonstrate that mixtures of modified sequences can be used to tune the affinity, dynamic range, and sensitivity of the resulting sensors predicted by a bi-Langmuir-type isotherm.

Advanced age at the time of spinal cord injury (SCI) exacerbates damage from reactive oxygen species (ROS). Mechanisms underlying this age-dependent response are not well understood and may arise from decreased antioxidant defense. We investigated how spinal cord levels of the antioxidant glutathione (GSH), and its regulation, change with age and SCI. GSH is used by GSH peroxidase to sequester ROS and is recycled by GSH reductase. Male and female, 4- and 14-month-old (MO) mice received a 60 kDyn contusion SCI, and the levels of GSH and its regulatory enzymes were evaluated at one and three days post-injury (dpi). The mice with SCI were treated with N-acetylcysteine-amide (NACA; 150 mg/kg), a cysteine supplement that increases GSH, to determine effects on functional and histological outcomes. GSH was decreased with older age in sham mice, and an SCI-dependent depletion was observed in 4-MO mice by three dpi. Neither age nor injury affected the abundance of proteins regulating GSH synthesis or recycling. GSH peroxidase activity, however, increased after SCI only in 4-MO mice. In contrast, GSH peroxidase activity was increased in 14-MO sham mice, indicating that spinal cords of older mice have an elevated oxidative state. Indeed, 14-MO sham mice had more oxidized protein (3-nitrotyrosine [3-NT]) within their spinal cords compared with 4-MO sham mice. Only 4-MO mice had significant injury-induced increases in 3-NT at three dpi. NACA treatment restored GSH and improved the redox environment in injured 4- and 14-MO mice at one dpi; however, three days of NACA delivery did not improve motor, sensory, or anatomical deficits at 28 dpi in 4-MO mice and trended toward toxicity in all outcomes in 14-MO mice. Our observation suggests that GSH levels at acute stages of SCI play a minimal role in age-dependent outcomes reported after SCI in mice. Collective results implicate elements of injury occurring after three dpi, such as inflammation, as key regulators of age-dependent effects.

Traumatic brain injury (TBI) activates multiple neuronal cell death mechanisms, leading to post-traumatic neuronal loss and neurological deficits. TBI-induced cell cycle activation (CCA) in post-mitotic neurons causes regulated cell death involving cyclin-dependent kinase (CDK) activation and initiation of an E2F transcription factor-mediated pro-apoptotic program. Here we examine the mechanisms of CCA-dependent neuronal apoptosis in primary neurons in vitro and in mice exposed to controlled cortical impact (CCI). In contrast to our prior work demonstrating robust neuroprotective effects by CDK inhibitors after TBI, examination of neuronal apoptotic mechanisms in E2F1−/−/E2F2−/− or E2F2−/− transgenic mice following CCI suggests that E2F1 and/or E2F2 likely play only a modest role in neuronal cell loss after brain trauma. To elucidate more critical CCA molecular pathways involved in post-traumatic neuronal cell death, we investigated the neuroprotective effects and mechanisms of the potent CDK inhibitor CR8 in a DNA damage model of cell death in primary cortical neurons. CR8 treatment significantly reduced caspase activation and cleavage of caspase substrates, attenuating neuronal cell death. CR8 neuroprotective effects appeared to reflect inhibition of multiple pathways converging on the mitochondrion, including injury-induced elevation of pro-apoptotic Bcl-2 homology region 3 (BH3)-only proteins Puma and Noxa, thereby attenuating mitochondrial permeabilization and release of cytochrome c and AIF, with reduction of both caspase-dependent and -independent apoptosis. CR8 administration also limited injury-induced deficits in mitochondrial respiration. These neuroprotective effects may be explained by CR8-mediated inhibition of key upstream injury responses, including attenuation of c-Jun phosphorylation/activation as well as inhibition of p53 transactivation of BH3-only targets.