Acute inflammatory profiles differ with sex and age after spinal cord injury.

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.

Mitochondria exert age-divergent effects on recovery from spinal cord injury.

The extent that age-dependent mitochondrial dysfunction drives neurodegeneration is not well understood. This study tested the hypothesis that mitochondria contribute to spinal cord injury (SCI)-induced neurodegeneration in an age-dependent manner by using 2,4-dinitrophenol (DNP) to uncouple electron transport, thereby increasing cellular respiration and reducing reactive oxygen species (ROS) production. We directly compared the effects of graded DNP doses in 4- and 14-month-old (MO) SCI-mice and found DNP to have increased efficacy in mitochondria isolated from 14-MO animals. In vivo, all DNP doses significantly exacerbated 4-MO SCI neurodegeneration coincident with worsened recovery. In contrast, low DNP doses (1.0-mg/kg/day) improved tissue sparing, reduced ROS-associated 3-nitrotyrosine (3-NT) accumulation, and improved anatomical and functional recovery in 14-MO SCI-mice. By directly comparing the effects of DNP between ages we demonstrate that mitochondrial contributions to neurodegeneration diverge with age after SCI. Collectively, our data indicate an essential role of mitochondria in age-associated neurodegeneration.

Delayed Azithromycin Treatment Improves Recovery After Mouse Spinal Cord Injury.

After spinal cord injury (SCI), macrophages infiltrate into the lesion and can adopt a wide spectrum of activation states. However, the pro-inflammatory, pathological macrophage activation state predominates and contributes to progressive neurodegeneration. Azithromycin (AZM), an FDA approved macrolide antibiotic, has been demonstrated to have immunomodulatory properties in a variety of inflammatory conditions. Indeed, we previously observed that post-SCI AZM treatment reduces pro-inflammatory macrophage activation. Further, a combined pre- and post-injury treatment paradigm improved functional recovery from SCI. Therefore, for the current study, we hypothesize that post-injury AZM treatment will improve recovery from SCI. To test this hypothesis, we examined the therapeutic potential of delayed AZM treatment on locomotor, sensory, and anatomical recovery. We administered AZM beginning 30-min, 3-h, or 24-h following contusion SCI in female mice, and then daily for 7 days. AZM administration beginning 30-min and 3-h post-injury improved locomotor recovery with increased stepping function relative to vehicle controls. Further, delaying treatment for 30-min after SCI significantly reduced lesion pathology. Initiating AZM treatment 24-h post-injury was not therapeutically effective. Regardless of the timing of the initial treatment, AZM did not statistically reduce the development of neuropathic pain (mechanical allodynia) nor increase neuron survival. Collectively, these results add to a growing body of evidence supporting AZM's translational potential as a therapeutic agent for SCI and other neuroinflammatory conditions in which patients currently have very few options.

Docosahexaenoic acid decreased neuroinflammation in rat pups after controlled cortical impact.

Traumatic brain injury (TBI) is the leading cause of acquired neurologic disability in children, yet specific therapies to treat TBI are lacking. Therapies that decrease the inflammatory response and enhance a reparative immune action may decrease oxidative damage and improve outcomes after TBI. Docosahexaenoic acid (DHA) modulates the immune response to injury in many organs. DHA given in the diet before injury decreased rat pup cognitive impairment, oxidative stress and white matter injury in our developmental TBI model using controlled cortical impact (CCI). Little is known about DHA effects on neuroinflammation in the developing brain. Further, it is not known if DHA given after developmental TBI exerts neuroprotective effects. We hypothesized that acute DHA treatment would decrease oxidative stress and improve cognitive outcome, associated with decreased pro-inflammatory activation of microglia, the brain's resident macrophages. METHODS: 17-day-old rat pups received intraperitoneal DHA or vehicle after CCI or SHAM surgery followed by DHA diet or continuation of REG diet to create DHACCI, REGCCI, SHAMDHA and SHAMREG groups. We measured brain nitrates/nitrites (NOx) at post injury day (PID) 1 to assess oxidative stress. We tested memory using Novel Object Recognition (NOR) at PID14. At PID 3 and 7, we measured reactivity of microglial activation markers Iba1, CD68 and CD206 and astrocyte marker GFAP in the injured cortex. At PID3, 7 and 30 we measured mRNA levels of inflammation-related genes and transcription factors in flow-sorted brain cells. RESULTS: DHA decreased oxidative stress at PID1 and pro-inflammatory microglial activation at PID3. CCI increased mRNA levels of two interferon regulatory family transcription factors, blunted by DHA, particularly in microglia-enriched cell populations at PID7. CCI increased mRNA levels of genes associated with "pro- " and "anti-" inflammatory activity at PID3, 7 and 30. Most notably within the microglia-enriched population, DHA blunted increased mRNA levels of pro-inflammatory genes at PID 3 and 7 and of anti-inflammatory genes at PID 30. Particularly in microglia, we observed parallel activation of pro-inflammatory and anti-inflammatory genes. DHA improved performance on NOR at PID14 after CCI. CONCLUSIONS: DHA decreased oxidative stress and histologic and mRNA markers of microglial pro-inflammatory activation in rat pup brain acutely after CCI associated with improved short term cognitive function. DHA administration after CCI has neuroprotective effects, which may result in part from modulation of microglial activation toward a less inflammatory profile in the first week after CCI. Future and ongoing studies will focus on phagocytic function and reactive oxygen species production in microglia and macrophages to test functional effects of DHA on neuroinflammation in our model. Given its favorable safety profile in children, DHA is a promising candidate therapy for pediatric TBI.