Aged mice are less susceptible to motion sickness and show decreased efferent vestibular activity compared to young adults.

INTRODUCTION: The efferent vestibular system (EVS) is a feedback circuit thought to modulate vestibular afferent activity by inhibiting type II hair cells and exciting calyx-bearing afferents in the peripheral vestibular organs. In a previous study, we suggested EVS activity may contribute to the effects of motion sickness. To determine an association between motion sickness and EVS activity, we examined the effects of provocative motion (PM) on c-Fos expression in brainstem efferent vestibular nucleus (EVN) neurons that are the source of efferent innervation in the peripheral vestibular organs. METHODS: c-Fos is an immediate early gene product expressed in stimulated neurons and is a well-established marker of neuronal activation. To study the effects of PM, young adult C57/BL6 wild-type (WT), aged WT, and young adult transgenic Chat-gCaMP6f mice were exposed to PM, and tail temperature (Ttail ) was monitored using infrared imaging. After PM, we used immunohistochemistry to label EVN neurons to determine any changes in c-Fos expression. All tissue was imaged using laser scanning confocal microscopy. RESULTS: Infrared recording of Ttail during PM indicated that young adult WT and transgenic mice displayed a typical motion sickness response (tail warming), but not in aged WT mice. Similarly, brainstem EVN neurons showed increased expression of c-Fos protein after PM in young adult WT and transgenic mice but not in aged cohorts. CONCLUSION: We present evidence that motion sickness symptoms and increased activation of EVN neurons occur in young adult WT and transgenic mice in response to PM. In contrast, aged WT mice showed no signs of motion sickness and no change in c-Fos expression when exposed to the same provocative stimulus.

Molecular and Functional Changes to Postsynaptic Cholinergic Signaling in the Vestibular Sensory Organs of Aging C57BL/6 Mice.

Cholinergic circuits in the central nervous system are vulnerable to age-related functional decline, but it is not known if aging impacts cholinergic signaling in the vestibular sensory organs, which are critically important to balance maintenance and visual gaze stability. We have previously shown cholinergic neurotransmission between vestibular efferent terminals and type II mechanosensory hair cells requires the alpha9 (Chrna9) nicotinic receptor subunit. Homozygous knockout of the alpha9 subunit causes vestibulo-ocular reflex adaptation deficits that mirror those observed in aged mice. This prompted examination of cholinergic signaling in the vestibular sensory organs of aged mice. We confirmed older (>24 months) mice had impaired performance in a balance beam task compared to young (3-4 months) adult mice. While there was no qualitative loss of cholinergic axon varicosities in the crista ampullaris of old mice, qPCR analysis revealed reduced expression of nicotinic receptor subunit genes Chrna1, Chrna9, and Chrna10 in the cristae of old relative to young mice. Functionally, single-cell patch clamp recordings taken from type II vestibular hair cells exposed to acetylcholine show reduced conductance through alpha9/10 subunit-containing nicotinic receptors in older mice, despite preserved passive membrane properties and voltage-activated conductances. These findings suggest that cholinergic signaling in the peripheral vestibular sensory organs is vulnerable to aging processes, manifesting in dynamic molecular and functional age-related changes. Given the importance of these organs to our everyday activities, and the dramatic increase in fall incidence in the older, further investigation into the mechanisms of altered peripheral vestibular function in older humans is warranted.

Age-related gene expression changes in lumbar spinal cord: Implications for neuropathic pain.

Clinically, pain has an uneven incidence throughout lifespan and impacts more on the elderly. In contrast, preclinical models of pathological pain have typically used juvenile or young adult animals to highlight the involvement of glial populations, proinflammatory cytokines, and chemokines in the onset and maintenance of pathological signalling in the spinal dorsal horn. The potential impact of this mismatch is also complicated by the growing appreciation that the aged central nervous system exists in a state of chronic inflammation because of enhanced proinflammatory cytokine/chemokine signalling and glial activation. To address this issue, we investigated the impact of aging on the expression of genes that have been associated with neuropathic pain, glial signalling, neurotransmission and neuroinflammation. We used qRT-PCR to quantify gene expression and focussed on the dorsal horn of the spinal cord as this is an important perturbation site in neuropathic pain. To control for global vs region-specific age-related changes in gene expression, the ventral half of the spinal cord was examined. Our results show that expression of proinflammatory chemokines, pattern recognition receptors, and neurotransmitter system components was significantly altered in aged (24-32 months) versus young mice (2-4 months). Notably, the magnitude and direction of these changes were spinal-cord region dependent. For example, expression of the chemokine, Cxcl13, increased 119-fold in dorsal spinal cord, but only 2-fold in the ventral spinal cord of old versus young mice. Therefore, we propose the dorsal spinal cord of old animals is subject to region-specific alterations that prime circuits for the development of pathological pain, potentially in the absence of the peripheral triggers normally associated with these conditions.

Reduced cortical somatostatin gene expression in a rat model of maternal immune activation.

Alterations in GABAergic interneurons and glutamic acid decarboxylase (GAD) are observed in the brains of people with schizophrenia. Studies also show increased density of interstitial white matter neurons (IWMN), including those containing GAD and somatostatin (SST) in the brain in schizophrenia. Maternal immune activation can be modelled in rodents to investigate the relationship between prenatal exposure to infections and increased risk of developing schizophrenia. We reported that maternal immune activation induced an increase in density of somatostatin-positive IWMN in the adult rat offspring. Here we hypothesised that maternal immune activation induced in pregnant rats by polyinosinic:polycytidylic acid would alter SST and GAD gene expression as well as increase the density of GAD-positive IWMNs in the adult offspring. SST gene expression was significantly reduced in the cingulate cortex of adult offspring exposed to late gestation maternal immune activation. There was no change in cortical GAD gene expression nor GAD-positive IWMN density in adults rats exposed to maternal immune activation at either early or late gestation. This suggests that our model of maternal immune activation induced by prenatal exposure of rats to polyinosinic:polycytidylic acid during late gestation is able to recapitulate changes in SST but not other GABAergic neuropathologies observed in schizophrenia.

Advantages and Limitations of Current Techniques for Analyzing the Biodistribution of Nanoparticles.

Nanomedicines are typically submicrometer-sized carrier materials (nanoparticles) encapsulating therapeutic and/or imaging compounds that are used for the prevention, diagnosis and treatment of diseases. They are increasingly being used to overcome biological barriers in the body to improve the way we deliver compounds to specific tissues and organs. Nanomedicine technology aims to improve the balance between the efficacy and the toxicity of therapeutic compounds. Nanoparticles, one of the key technologies of nanomedicine, can exhibit a combination of physical, chemical and biological characteristics that determine their in vivo behavior. A key component in the translational assessment of nanomedicines is determining the biodistribution of the nanoparticles following in vivo administration in animals and humans. There are a range of techniques available for evaluating nanoparticle biodistribution, including histology, electron microscopy, liquid scintillation counting (LSC), indirectly measuring drug concentrations, in vivo optical imaging, computed tomography (CT), magnetic resonance imaging (MRI), and nuclear medicine imaging. Each technique has its own advantages and limitations, as well as capabilities for assessing real-time, whole-organ and cellular accumulation. This review will address the principles and methodology of each technique and their advantages and limitations for evaluating in vivo biodistribution of nanoparticles.

Reduced deoxyribonuclease enzyme activity in response to high postinjury mitochondrial DNA concentration provides a therapeutic target for Systemic Inflammatory Response Syndrome.

BACKGROUND: Cell-free mitochondrial DNA (mtDNA) is proinflammatory and has been detected in high concentrations in trauma patients' plasma. Deoxyribonuclease (DNAse) is the free plasma enzyme responsible for the digestion of extracellular DNA. The relationship between mtDNA and DNAse after major trauma is unknown. We hypothesized that DNAse activity would be elevated after injury and trauma surgery and would be associated with high concentrations of extracellular DNA. METHODS: Two-year prospective study was performed on 103 consecutive trauma patients (male, 81%; age, 38 years [interquartile range, 30-59 years]; injury severity score, 18 [interquartile range, 12-26 years]) who underwent standardized major orthopedic trauma surgical interventions. Blood was collected at five perioperative time points (preoperative, postoperative, 7 hours, 24 hours, and 3 days postoperatively). Healthy control subjects (n = 20) were also sampled. Cell-free mtDNA and nuclear DNA (nDNA) were measured using quantitative polymerase chain reaction. Deoxyribonuclease was also assayed in the same plasma samples. RESULTS: Increased levels of mtDNA (from preoperative 163 ± 86 ng/mL to 3 days 282 ± 201 ng/mL, p < 0.0001) and nDNA (from preoperative 28 ± 20 ng/mL to 3 days 37 ± 27 ng/mL, p < 0.05) were present in trauma patients at all perioperative time points compared with healthy controls (mtDNA: 4 ± 2 ng/mL; nDNA: 10 ± 5 ng/mL). Deoxyribonuclease activity was lower in the trauma cohort (from preoperative 0.06 ± 0.04U/mL to 3 days 0.08 ± 0.04U/mL, p < 0.0001) compared with healthy controls (DNAse: 0.17 ± 0.03U/mL). There was no correlation between DNAse and perioperative DNA concentrations. Elevated mtDNA (but not nDNA) correlated with the development of systemic inflammatory response syndrome (SIRS) (p = 0.026) but not multiple organ failure. CONCLUSIONS: The significant perioperative elevation in plasma-free mtDNA concentration is associated with the development of SIRS. The fact that increased cell-free DNA concentrations present with significantly lower than healthy control DNAse activity suggests a potential therapeutic opportunity with DNAse administration to modulate postinjury severe SIRS. LEVEL OF EVIDENCE: Prognostic/Epidemiological, level II.

Late gestation immune activation increases IBA1-positive immunoreactivity levels in the corpus callosum of adult rat offspring.

Animal models of maternal immune activation study the effects of infection, an environmental risk factor for schizophrenia, on brain development. Microglia activation and cytokine upregulation may have key roles in schizophrenia neuropathology. We hypothesised that maternal immune activation induces changes in microglia and cytokines in the brains of the adult offspring. Maternal immune activation was induced by injecting polyriboinosinic:polyribocytidylic acid into pregnant rats on gestational day (GD) 10 or GD19, with brain tissue collected from the offspring at adulthood. We observed no change in Iba1, Gfap, IL1-ß and TNF-α mRNA levels in the cingulate cortex (CC) in adult offspring exposed to maternal immune activation. Prenatal exposure to immune activation had a significant main effect on microglial IBA1-positive immunoreactive material (IBA1+IRM) in the corpus callosum; post-hoc analyses identified a significant increase in GD19 offspring, but not GD10. No change in was observed in the CC. In contrast, maternal immune activation had a significant main effect on GFAP+IRM in the CC at GD19 (not GD10); post-hoc analyses only identified a strong trend towards increased GFAP+IRM in the GD19 offspring, with no white matter changes. This suggests late gestation maternal immune activation causes subtle alterations to microglia and astrocytes in the adult offspring.

Advantages and Limitations of Current Imaging Techniques for Characterizing Liposome Morphology.

There are currently a number of imaging techniques available for evaluating the morphology of liposomes and other nanoparticles, with each having its own advantages and disadvantages that should be considered when interpreting data. Controlling and validating the morphology of nanoparticles is of key importance for the effective clinical translation of liposomal formulations. There are a number of physical characteristics of liposomes that determine their in vivo behavior, including size, surface characteristics, lamellarity, and homogeneity. Despite the great importance of the morphology of nanoparticles, it is generally not well-characterized and is difficult to control. Appropriate imaging techniques provide important details regarding the morphological characteristics of nanoparticles, and should be used in conjunction with other methods to assess physicochemical parameters. In this review, we will discuss the advantages and limitations of available imaging techniques used to evaluate liposomal formulations.

Effects of Ageing on the Mitochondrial Genome in Rat Vestibular Organs.

BACKGROUND: Deterioration in vestibular function occurs with ageing and is linked to age-related falls. Sensory hair cells located in the inner ear vestibular labyrinth are critical to vestibular function. Vestibular hair cells rely predominantly on oxidative phosphorylation (OXPHOS) for energy production and contain numerous mitochondria. Mitochondrial DNA (mtDNA) mutations and perturbed energy production are associated with the ageing process. OBJECTIVE: We investigated the effects of ageing on mtDNA in vestibular hair and support cells, and vestibular organ gene expression, to better understand mechanisms of age-related vestibular deficits. METHODS: Vestibular hair and supporting cell layers were microdissected from young and old rats, and mtDNA was quantified by qPCR. Additionally, vestibular organ gene expression was analysed by microarray and gene set enrichment analyses. RESULTS: In contrast to most other studies, we found no evidence of age-related mtDNA deletion mutations. However, we found an increase in abundance of major arc genes near the mtDNA control region. There was also a marked age-related reduction in mtDNA copy number in both cell types. Vestibular organ gene expression, gene set enrichment analysis showed the OXPHOS pathway was down regulated in old animals. CONCLUSION: Given the importance of mtDNA to mitochondrial OXPHOS and hair cell function, our findings suggest the vestibular organs are potentially on the brink of an energy crisis in old animals.

Temporally specific miRNA expression patterns in the dorsal and ventral striatum of addiction-prone rats.

MicroRNAs (miRNAs) within the ventral and dorsal striatum have been shown to regulate addiction-relevant behaviours. However, it is unclear how cocaine experience alone can alter the expression of addiction-relevant miRNAs within striatal subregions. Further, it is not known whether differential expression of miRNAs in the striatum contributes to individual differences in addiction vulnerability. We first examined the effect of cocaine self-administration on the expression of miR-101b, miR-137, miR-212 and miR-132 in nucleus accumbens core and nucleus accumbens shell (NAcSh), as well as dorsomedial striatum and dorsolateral striatum (DLS). We then examined the expression of these same miRNAs in striatal subregions of animals identified as being 'addiction-prone', either immediately following self-administration training or following extinction and relapse testing. Cocaine self-administration was associated with changes in miRNA expression in a regionally discrete manner within the striatum, with the most marked changes occurring in the nucleus accumbens core. When we examined the miRNA profile of addiction-prone rats following self-administration, we observed increased levels of miR-212 in the dorsomedial striatum. After extinction and relapse testing, addiction-prone rats showed significant increases in the expression of miR-101b, miR-137, miR-212 and miR-132 in NAcSh, and miR-137 in the DLS. This study identifies temporally specific changes in miRNA expression consistent with the engagement of distinct striatal subregions across the course of the addiction cycle. Increased dysregulation of miRNA expression in NAcSh and DLS at late stages of the addiction cycle may underlie habitual drug seeking, and may therefore aid in the identification of targets designed to treat addiction.

Alpha-9 nicotinic acetylcholine receptors mediate hypothermic responses elicited by provocative motion in mice.

Hypothermic responses accompany motion sickness in humans and can be elicited by provocative motion in rats. We aimed to determine the potential role in these responses of the efferent cholinergic vestibular innervation. To this end, we used knockout (KO) mice lacking α9 cholinoreceptor subunit predominantly expressed in the vestibular hair cells and CBA strain as a wild-type (WT) control. In WT mice, circular horizontal motion (1Hz, 4cm radius, 20min) caused rapid and dramatic falls in core body temperature and surface head temperature associated with a transient rise in the tail temperature; these responses were substantially attenuated in KO mice; changes were (WT vs. KO): for the core body temperature-5.2±0.3 vs. -2.9±0.3°C; for the head skin temperature-3.3±0.2 vs. -1.7±0.2°C; for the tail skin temperature+3.9±1.1 vs+1.1±1.2°C. There was a close correlation in the time course of cooling the body and the surface of the head. KO mice also required 25% more time to complete a balance test. We conclude: i) that the integrity of cholinergic efferent vestibular system is essential for the full expression of motion-induced hypothermia in mice, and that the role of this system is likely facilitatory; ii) that the system is involvement in control of balance, but the involvement is not major; iii) that in mice, motion-induced body cooling is mediated via increased heat flow through vasodilated tail vasculature and (likely) via reduced thermogenesis. Our results support the idea that hypothermia is a biological correlate of a nausea-like state in animals.

Aging reduces the high-frequency and short-term adaptation of the vestibulo-ocular reflex in mice.

Prevailing evidence indicates a relatively late life decline in human vestibulo-ocular reflex (VOR) function. Although mice are commonly used in mechanistic studies of vestibular function, it remains unclear whether aging produces a corresponding decline in VOR function in mice. We sought to determine how the baseline VOR and its short-term adaptation were affected by aging. We tested 8 young (3-month old) and 8 aged (30-month old-equivalent to a ∼80-year old human) C57BL/6 mice. We measured their VOR response to whole-body static tilts and during 0.1-10 Hz whole-body sinusoidal and transient rotations before and after VOR adaptation training. Our data revealed minimal differences in static counter-tilt response between young and aged mice, but a significant deficit in baseline VOR gain in aged mice during transient rotations. Moreover, aged mice had a significant decrease in short-term VOR adaptation, particularly for training that sought to decrease the VOR response.

Perturbed cholesterol homeostasis in aging spinal cord.

The spinal cord is vital for the processing of sensorimotor information and for its propagation to and from both the brain and the periphery. Spinal cord function is affected by aging, however, the mechanisms involved are not well-understood. To characterize molecular mechanisms of spinal cord aging, microarray analyses of gene expression were performed on cervical spinal cords of aging rats. Of the metabolic and signaling pathways affected, cholesterol-associated pathways were the most comprehensively altered, including significant downregulation of cholesterol synthesis-related genes and upregulation of cholesterol transport and metabolism genes. Paradoxically, a significant increase in total cholesterol content was observed-likely associated with cholesterol ester accumulation. To investigate potential mechanisms for the perturbed cholesterol homeostasis, we quantified the expression of myelin and neuroinflammation-associated genes and proteins. Although there was minimal change in myelin-related expression, there was an increase in phagocytic microglial and astrogliosis markers, particularly in the white matter. Together, these results suggest that perturbed cholesterol homeostasis, possibly as a result of increased inflammatory activation in spinal cord white matter, may contribute to impaired spinal cord function with aging.

Rapamycin reduces motivated responding for cocaine and alters GluA1 expression in the ventral but not dorsal striatum.

The mechanistic target of rapamycin complex 1 (mTORC1) regulates synaptic protein synthesis and therefore synaptic function and plasticity. A role for mTORC1 has recently been demonstrated for addiction-related behaviors. For example, central or intra-accumbal injections of the mTORC1 inhibitor rapamycin attenuates several indices of cocaine-seeking including progressive ratio (PR) responding and reinstatement. These behavioral effects are associated with decreased mTORC1 activity and synaptic protein translation in the nucleus accumbens (NAC) and point to a possible therapeutic role for rapamycin in the treatment of addiction. Currently, it is unclear whether similar behavioral and biochemical effects can be achieved by administering rapamycin systemically, which represents a more clinically-appropriate route of administration. Here, we assessed the effects of repeated, systemic administration of rapamycin (10mg/kg, i.p.) on PR responding for cocaine. We also assessed whether systemic rapamycin was associated with changes in measures of mTORC1 activity and GluA1 expression in the ventral and dorsal striatum. We report that systemic rapamycin treatment reduced PR breakpoints to levels comparable to intra-NAC rapamycin. Systemic rapamycin treatment also reduced phosphorylated p70S6K and GluA1 AMPARs within the NAC but not dorsal striatum. Thus, systemic administration of rapamycin is as effective at reducing drug seeking behavior and measures of mTORC1 activity compared to direct accumbal application and may therefore represent a possible therapeutic option in the treatment of addiction. Possible caveats of this treatment approach are discussed.

Age-related gene expression changes in substantia nigra dopamine neurons of the rat.

Ageing affects most, if not all, functional systems in the body. For example, the somatic motor nervous system, responsible for initiating and regulating motor output to skeletal musculature, is vulnerable to ageing. The nigrostriatal dopamine pathway is one component of this system, with deficits in dopamine signalling contributing to major motor dysfunction, as exemplified in Parkinson's disease (PD). However, while the dopamine deficit in PD is due to degeneration of substantia nigra (SN) dopamine (DA) neurons, it is unclear whether there is sufficient loss of SN DA neurons with ageing to explain observed motor impairments. Instead, evidence suggests that age-related loss of DA neuron function may be more important than frank cell loss. To further elucidate the mechanisms of functional decline, we have investigated age-related changes in gene expression specifically in laser microdissected SN DA neurons. There were significant age-related changes in the expression of genes associated with neurotrophic factor signalling and the regulation of tyrosine hydroxylase activity. Furthermore, reduced expression of the DA neuron-associated transcription factor, Nurr1, may contribute to these changes. Together, these results suggest that altered neurotrophic signalling and tyrosine hydroxylase activity may contribute to altered DA neuron signalling and motor nervous system regulation in ageing.

Cell necrosis-independent sustained mitochondrial and nuclear DNA release following trauma surgery.

BACKGROUND: Mitochondrial DNA (mtDNA), a potent proinflammatory damage-associated molecular pattern, is released in large titers following trauma. The effect of trauma surgery on mtDNA concentration is unknown. We hypothesized that mtDNA and nuclear DNA (nDNA) levels would increase proportionately with the magnitude of surgery and both would then decrease rapidly. METHODS: In this prospective pilot, plasma was sampled from 35 trauma patients requiring orthopedic surgical intervention at six perioperative time points. Healthy control subjects (n = 20) were sampled. DNA was extracted, and the mtDNA and nDNA were assessed using quantitative polymerase chain reaction. Markers of cell necrosis were also assayed (creatine kinase, lactate dehydrogenase, and aspartate aminotransferase). RESULTS: The free plasma mtDNA and nDNA levels (ng/mL) were increased in trauma patients compared with healthy controls at all time points (mtDNA: preoperative period, 108 [46-284]; postoperative period, 96 [29-200]; 7 hours postoperatively, 88 [43-178]; 24 hours, 79 [36-172]; 3 days, 136 [65-263]; 5 days, 166 [101-434] [healthy controls, 11 (5-19)]) (nDNA: preoperative period, 52 [25-130]; postoperative period, 100 [35-208]; 7 hours postoperatively, 75 [36-139]; 24 hours postoperatively, 85 [47-133]; 3 days, 79 [48-117]; 5 days, 99 [41-154] [healthy controls, 29 (16-54)]). Elevated DNA levels did not correlate with markers of cellular necrosis. mtDNA was significantly elevated compared with nDNA at preoperative period (p = 0.003), 3 days (p = 0.003), and 5 days (p = 0.0014). Preoperative mtDNA levels were greater with shorter time from injury to surgery (p = 0.0085). Postoperative mtDNA level negatively correlated with intraoperative crystalloid infusion (p = 0.0017). Major pelvic surgery (vs. minor) was associated with greater mtDNA release 5 days postoperatively (p < 0.05). CONCLUSION: This pilot of heterogeneous orthopedic trauma patients showed that the release of mtDNA and nDNA is sustained for 5 days following orthopedic trauma surgery. Postoperative, circulating DNA is not associated with markers of tissue necrosis but is associated with surgical invasiveness and is inversely related to intraoperative fluid administration. Sustained elevation of mtDNA levels could be of inflammatory origin and may contribute to postinjury dysfunctional inflammation. LEVEL OF EVIDENCE: Prospective study, level III.

Nucleotide excision repair deficiency in melanoma in response to UVA.

BACKGROUND: The causative link between UV exposure and melanoma development is well known, however the mechanistic relationship remains incompletely characterised. UVA and UVB components of sunlight are implicated in melanomagenesis; however the majority of studies have focused on the effects of UVB and UVC light. Interestingly, melanoma tumour sequencing has revealed an overrepresentation of mutations signature of unrepaired UV-induced DNA damage. Repair of UVA-induced DNA damage is thought to occur primarily through the Nucleotide Excision Repair (NER) pathway, which recognises and repairs damage either coupled to transcription (Transcription Coupled Repair; TCR), or through global genome scanning (Global Genome Repair; GGR). Current literature suggests NER is deficient in melanoma, however the cause of this remains unknown; and whether reduced NER activity in response to UVA may be involved in melanoma development remains uncharacterised. In this study we aimed to determine if melanoma cells exhibit reduced levels of NER activity in response to UVA. METHODS: Melanocyte and melanoma cell lines were UVA-irradiated, and DNA damage levels assessed by immunodetection of Cyclobutane Pyrimidine Dimer (CPD) and (6-4) Photoproduct [(6-4)PP] lesions. Expression of NER pathway components and p53 following UVA treatment was quantified by qPCR and western blot. RESULTS: UVA did not induce detectable induction of (6-4)PP lesions, consistent with previous studies. Repair of CPDs induced by UVA was initiated at 4 h and complete within 48 h in normal melanocytes, whereas repair initiation was delayed to 24 h and >40 % of lesions remained in melanoma cell lines at 48 h. This was coupled with a delayed and reduced induction of GGR component XPC in melanoma cells, independent of p53. CONCLUSION: These findings support that NER activity is reduced in melanoma cells due to deficient GGR. Further investigation into the role of NER in UVA-induced melanomagenesis is warranted and may have implications for melanoma treatment.

Mitochondrial DNA neutrophil extracellular traps are formed after trauma and subsequent surgery.

INTRODUCTION: Neutrophil extracellular traps (NETs) have not been demonstrated after trauma and subsequent surgery. Neutrophil extracellular traps are formed from pure mitochondrial DNA (mtDNA) under certain conditions, which is potently proinflammatory. We hypothesized that injury and orthopedic trauma surgery would induce NET production with mtDNA as a structural component. METHODS: Neutrophils were isolated 8 trauma patients requiring orthopedic surgery postinjury and up to 5 days postoperatively. Four healthy volunteers provided positive and negative controls. Total hip replacement patients acted as an uninjured surgical control group. Neutrophil extracellular traps were visualized with DNA (Hoechst 33342TM/Sytox Green/MitoSox/MitoTracker) stains using live cell fluorescence microscopy with downstream quantitative polymerase chain reaction analysis of DNA composition. RESULTS: Neutrophil extracellular traps were present after injury in all 8 trauma patients. They persisted for 5 days postoperatively. Delayed surgery resulted in NET resolution, but they reformed postoperatively. Total hip replacement patients developed NETs postoperatively, which resolved by day 5. Quantitative polymerase chain reaction analysis of NET-DNA composition revealed that NETs formed after injury and surgery were made of mtDNA with no detectable nuclear DNA component. CONCLUSIONS: Neutrophil extracellular traps formed after major trauma and subsequent surgery contain mtDNA and represent a novel marker of heightened innate immune activation. They could be considered when timing surgery after trauma to prevent systemic NET-induced inflammatory complications.

Exercise reverses the effects of early life stress on orexin cell reactivity in male but not female rats.

Early life stress (ELS) is a known antecedent for the development of mood disorders such as depression. Orexin neurons drive arousal and motivated behaviors in response to stress. We tested the hypothesis that ELS alters orexin system function and leads to an altered stress-induced behavioral phenotype in adulthood. We also investigated if voluntary exercise during adolescent development could reverse the ELS-induced changes. Male and female Wistar rats were subjected to maternal separation stress on postnatal days (PND) 2-14. A subset of animals was given access to running wheels in late adolescence (1hr/day, PND40-70). In adulthood, rats were exposed to restraint stress and then tested on the open field (OF) and elevated plus maze (EPM). Brains were processed for Fos-protein and orexin or tyrosine hydroxylase immunohistochemistry. Restraint stress stimulated Fos-protein expression in perifornical area orexin cells, the paraventricular hypothalamic nucleus, and paraventricular thalamic nuclei, but this neuronal response was dampened in male and female rats exposed to ELS. ELS also reduced exploration in the OF, without affecting EPM behavior. These neural and behavioral changes are consistent with a depressive-like phenotype. Adolescent exercise reversed the orexin and behavioral deficits in ELS males. Exercise was not protective in females, although this may be due to sex differences in running behavior. Our findings highlight the inherent plasticity of the orexin system-a trait that may lead to a state of pathological rewiring but could also be treated using non-pharmacological approaches. We also highlight a need to better understand the sex-specific changes in orexin circuits and stress-related pathology.