18 kDa Translocator protein (TSPO) is upregulated in rat brain after peripheral nerve injury and downregulated by diroximel fumarate.

Neuroimmune signaling is a key process underlying neuropathic pain. Clinical studies have demonstrated that 18 kDa translocator protein (TSPO), a putative marker of neuroinflammation, is upregulated in discrete brain regions of patients with chronic pain. However, no preclinical studies have investigated TSPO dynamics in the brain in the context of neuropathic pain and in response to analgesic treatments. We used positron emission tomography-computed tomography (PET-CT) and [18F]-PBR06 radioligand to measure TSPO levels in the brain across time after chronic constriction injury (CCI) of the sciatic nerve in both male and female rats. Up to 10 weeks post-CCI, TSPO expression was increased in discrete brain regions, including medial prefrontal cortex, somatosensory cortex, insular cortex, anterior cingulate cortex, motor cortex, ventral tegmental area, amygdala, midbrain, pons, medulla, and nucleus accumbens. TSPO was broadly upregulated across these regions at 4 weeks post CCI in males, and 10 weeks in females, though there were regional differences between the sexes. Using immunohistochemistry, we confirmed TSPO expression in these regions. We further demonstrated that TSPO was upregulated principally in microglia in the nucleus accumbens core, and astrocytes and endothelial cells in the nucleus accumbens shell. Finally, we tested whether TSPO upregulation was sensitive to diroximel fumarate, a drug that induces endogenous antioxidants via nuclear factor E2-related factor 2 (Nrf2). Diroximel fumarate alleviated neuropathic pain and reduced TSPO upregulation. Our findings indicate that TSPO is upregulated over the course of neuropathic pain development and is resolved by an antinociceptive intervention in animals with peripheral nerve injury.

Interleukin-1beta and inflammasome expression in spinal cord following chronic constriction injury in male and female rats.

Females represent a majority of chronic pain patients and show greater inflammatory immune responses in human chronic pain patient populations as well as in animal models of neuropathic pain. Recent discoveries in chronic pain research have revealed sex differences in inflammatory signaling, a key component of sensory pathology in chronic neuropathic pain, inviting more research into the nuances of these sex differences. Here we use the chronic constriction injury (CCI) model to explore similarities and differences in expression and production of Inflammatory cytokine IL-1beta in the lumbar spinal cord, as well as its role in chronic pain. We have discovered that intrathecal IL-1 receptor antagonist reverses established pain in both sexes, and increased gene expression of inflammasome NLRP3 is specific to microglia and astrocytes rather than neurons, while IL-1beta is specific to microglia in both sexes. We report several sex differences in the expression level of the genes coding for IL-1beta, as well as the four inflammasomes responsible for IL-1beta release: NLRP3, AIM2, NLRP1, and NLRC4 in the spinal cord. Total mRNA, but not protein expression of IL-1beta is greater in females than males after CCI. Also, while CCI increases all four inflammasomes in both sexes, there are sex differences in relative levels of inflammasome expression. NLRP3 and AIM2 are more highly expressed in females, whereas NLRP1 expression is greater in males.

Voluntary wheel running prevents formation of membrane attack complexes and myelin degradation after peripheral nerve injury.

Regular aerobic activity is associated with a reduced risk of chronic pain in humans and rodents. Our previous studies in rodents have shown that prior voluntary wheel running can normalize redox signaling at the site of peripheral nerve injury, attenuating subsequent neuropathic pain. However, the full extent of neuroprotection offered by voluntary wheel running after peripheral nerve injury is unknown. Here, we show that six weeks of voluntary wheel running prior to chronic constriction injury (CCI) reduced the terminal complement membrane attack complex (MAC) at the sciatic nerve injury site. This was associated with increased expression of the MAC inhibitor CD59. The levels of upstream complement components (C3) and their inhibitors (CD55, CR1 and CFH) were altered by CCI, but not increased by voluntary wheel running. Since MAC can degrade myelin, which in turn contributes to neuropathic pain, we evaluated myelin integrity at the sciatic nerve injury site. We found that the loss of myelinated fibers and decreased myelin protein which occurs in sedentary rats following CCI was not observed in rats with prior running. Substitution of prior voluntary wheel running with exogenous CD59 also attenuated mechanical allodynia and reduced MAC deposition at the nerve injury site, pointing to CD59 as a critical effector of the neuroprotective and antinociceptive actions of prior voluntary wheel running. This study links attenuation of neuropathic pain by prior voluntary wheel running with inhibition of MAC and preservation of myelin integrity at the sciatic nerve injury site.

HDAC6 Inhibition Reverses Cisplatin-Induced Mechanical Hypersensitivity via Tonic Delta Opioid Receptor Signaling.

Peripheral neuropathic pain induced by the chemotherapeutic cisplatin can persist for months to years after treatment. Histone deacetylase 6 (HDAC6) inhibitors have therapeutic potential for cisplatin-induced neuropathic pain since they persistently reverse mechanical hypersensitivity and spontaneous pain in rodent models. Here, we investigated the mechanisms underlying reversal of mechanical hypersensitivity in male and female mice by a 2 week treatment with an HDAC6 inhibitor, administered 3 d after the last dose of cisplatin. Mechanical hypersensitivity in animals of both sexes treated with the HDAC6 inhibitor was temporarily reinstated by a single injection of the neutral opioid receptor antagonist 6ß-naltrexol or the peripherally restricted opioid receptor antagonist naloxone methiodide. These results suggest that tonic peripheral opioid ligand-receptor signaling mediates reversal of cisplatin-induced mechanical hypersensitivity after treatment with an HDAC6 inhibitor. Pointing to a specific role for δ opioid receptors (DORs), Oprd1 expression was decreased in DRG neurons following cisplatin administration, but normalized after treatment with an HDAC6 inhibitor. Mechanical hypersensitivity was temporarily reinstated in both sexes by a single injection of the DOR antagonist naltrindole. Consistently, HDAC6 inhibition failed to reverse cisplatin-induced hypersensitivity when DORs were genetically deleted from advillin+ neurons. Mechanical hypersensitivity was also temporarily reinstated in both sexes by a single injection of a neutralizing antibody against the DOR ligand met-enkephalin. In conclusion, we reveal that treatment with an HDAC6 inhibitor induces tonic enkephalin-DOR signaling in peripheral sensory neurons to suppress mechanical hypersensitivity.SIGNIFICANCE STATEMENT Over one-fourth of cancer survivors suffer from intractable painful chemotherapy-induced peripheral neuropathy (CIPN), which can last for months to years after treatment ends. HDAC6 inhibition is a novel strategy to reverse CIPN without negatively interfering with tumor growth, but the mechanisms responsible for persistent reversal are not well understood. We built on evidence that the endogenous opioid system contributes to the spontaneous, apparent resolution of pain caused by nerve damage or inflammation, referred to as latent sensitization. We show that blocking the δ opioid receptor or its ligand enkephalin unmasks CIPN in mice treated with an HDAC6 inhibitor (latent sensitization). Our work provides insight into the mechanisms by which treatment with an HDAC6 inhibitor apparently reverses CIPN.

Grazing management for soil carbon in Australia: A review.

The livestock industry accounts for a considerable proportion of agricultural greenhouse gas emissions, and in response, the Australian red meat industry has committed to an aspirational target of net-zero emissions by 2030. Increasing soil carbon storage in grazing lands has been identified as one method to help achieve this, while also potentially improving production and provision of other ecosystem services. This review examined the effects of grazing management on soil carbon and factors that drive soil carbon sequestration in Australia. A systematic literature search and meta-analysis was used to compare effects of stocking intensity (stocking rate or utilisation) and stocking method (i.e, continuous, rotational or seasonal grazing systems) on soil organic carbon, pasture herbage mass, plant growth and ground cover. Impacts on below ground biomass, soil nitrogen and soil structure are also discussed. Overall, no significant impact of stocking intensity or method on soil carbon sequestration in Australia was found, although lower stocking intensity and incorporating periods of rest into grazing systems (rotational grazing) had positive effects on herbage mass and ground cover compared with higher stocking intensity or continuous grazing. Minimal impact of grazing management on pasture growth rate and below-ground biomass has been reported in Australia. However, these factors improved with grazing intensity or rotational grazing in some circumstances. While there is a lack of evidence in Australia that grazing management directly increases soil carbon, this meta-analysis indicated that grazing management practices have potential to benefit the drivers of soil carbon sequestration by increasing above and below-ground plant production, maintaining a higher residual biomass, and promoting productive perennial pasture species. Specific recommendations for future research and management are provided in the paper.

The Neuroimmunology of Chronic Pain: From Rodents to Humans.

Chronic pain, encompassing conditions, such as low back pain, arthritis, persistent post-surgical pain, fibromyalgia, and neuropathic pain disorders, is highly prevalent but remains poorly treated. The vast majority of therapeutics are directed solely at neurons, despite the fact that signaling between immune cells, glia, and neurons is now recognized as indispensable for the initiation and maintenance of chronic pain. This review highlights recent advances in understanding fundamental neuroimmune signaling mechanisms and novel therapeutic targets in rodent models of chronic pain. We further discuss new technological developments to study, diagnose, and quantify neuroimmune contributions to chronic pain in patient populations.

Suppression of active phase voluntary wheel running in male rats by unilateral chronic constriction injury: Enduring therapeutic effects of a brief treatment of morphine combined with TLR4 or P2X7 antagonists.

The present series of studies examine the impact of systemically administered therapeutics on peripheral nerve injury (males; unilateral sciatic chronic constriction injury [CCI])-induced suppression of voluntary wheel running, across weeks after dosing cessation. Following CCI, active phase running distance and speed are suppressed throughout the 7-week observation period. A brief course of morphine, however, increased active phase running distance and speed throughout this same period, an effect apparent only in sham rats. For CCI rats, systemic co-administration of morphine with antagonists of either P2X7 (A438079) or TLR4 ((+)-naloxone) (receptors critical to the activation of NLRP3 inflammasomes and consequent inflammatory cascades) returned running behavior of CCI rats to that of shams through 5+ weeks after dosing ceased. This is a striking difference in effect compared to our prior CCI allodynia results using systemic morphine plus intrathecal delivery of these same antagonists, wherein a sustained albeit partial suppression of neuropathic pain was observed. This may point to actions of the systemic drugs at multiple sites along the neuraxis, modulating injury-induced, inflammasome-mediated effects at the injured sciatic nerve and/or dorsal root ganglia, spinal cord, and potentially higher levels. Given that our data to date point to morphine amplifying neuroinflammatory processes put into motion by nerve injury, it is intriguing to speculate that co-administration of TLR4 and/or P2X7 antagonists can intervene in these inflammatory processes in a beneficial way. That is, that systemic administration of such compounds may suppress inflammatory damage at multiple sites, rapidly and persistently returning neuropathic animals to sham levels of response.

How Modelers Model: the Overlooked Social and Human Dimensions in Model Intercomparison Studies.

There is a growing realization that the complexity of model ensemble studies depends not only on the models used but also on the experience and approach used by modelers to calibrate and validate results, which remain a source of uncertainty. Here, we applied a multi-criteria decision-making method to investigate the rationale applied by modelers in a model ensemble study where 12 process-based different biogeochemical model types were compared across five successive calibration stages. The modelers shared a common level of agreement about the importance of the variables used to initialize their models for calibration. However, we found inconsistency among modelers when judging the importance of input variables across different calibration stages. The level of subjective weighting attributed by modelers to calibration data decreased sequentially as the extent and number of variables provided increased. In this context, the perceived importance attributed to variables such as the fertilization rate, irrigation regime, soil texture, pH, and initial levels of soil organic carbon and nitrogen stocks was statistically different when classified according to model types. The importance attributed to input variables such as experimental duration, gross primary production, and net ecosystem exchange varied significantly according to the length of the modeler's experience. We argue that the gradual access to input data across the five calibration stages negatively influenced the consistency of the interpretations made by the modelers, with cognitive bias in "trial-and-error" calibration routines. Our study highlights that overlooking human and social attributes is critical in the outcomes of modeling and model intercomparison studies. While complexity of the processes captured in the model algorithms and parameterization is important, we contend that (1) the modeler's assumptions on the extent to which parameters should be altered and (2) modeler perceptions of the importance of model parameters are just as critical in obtaining a quality model calibration as numerical or analytical details.

Important constraints on soil organic carbon formation efficiency in subtropical and tropical grasslands.

More than 10% of Australia's 49 M ha of grassland is considered degraded, prompting widespread interest in the management of these ecosystems to increase soil carbon (C) sequestration-with an emphasis on long-lived C storage. We know that management practices that increase plant biomass also increase C inputs to the soil, but we lack a quantitative understanding of the fate of soil C inputs into different soil organic carbon (SOC) fractions that have fundamentally different formation pathways and persistence in the soil. Our understanding of the factors that constrain SOC formation in these fractions is also limited, particularly within tropical climates. We used isotopically labelled residue (13 C) to determine the fate of residue C inputs into short-lived particulate organic matter (POM) and more persistent mineral-associated organic matter (MAOM) across a broad climatic gradient (ΔMAT 10°C) with varying soil properties. Climate was the primary driver of aboveground residue mass loss which corresponded to higher residue-derived POM formation. In contrast, MAOM formation efficiency was constrained by soil properties. The differential controls on POM and MAOM formation highlight that a targeted approach to grassland restoration is required; we must identify priority regions for improved grazing management in soils that have a relatively high silt+clay content and cation exchange capacity, with a low C saturation in the silt+clay fraction to deliver long-term SOC sequestration.

A role for neuroimmune signaling in a rat model of Gulf War Illness-related pain.

More than a quarter of veterans of the 1990-1991 Persian Gulf War suffer from Gulf War Illness (GWI), a chronic, multi-symptom illness that commonly includes musculoskeletal pain. Exposure to a range of toxic chemicals, including sarin nerve agent, are a suspected root cause of GWI. Moreover, such chemical exposures induce a neuroinflammatory response in rodents, which has been linked to several GWI symptoms in rodents and veterans with GWI. To date, a neuroinflammatory basis for pain associated with GWI has not been investigated. Here, we evaluated development of nociceptive hypersensitivity in a model of GWI. Male Sprague Dawley rats were treated with corticosterone in the drinking water for 7 days, to mimic high physiological stress, followed by a single injection of the sarin nerve agent surrogate, diisopropyl fluorophosphate. These exposures alone were insufficient to induce allodynia. However, an additional sub-threshold challenge (a single intramuscular injection of pH 4 saline) induced long-lasting, bilateral allodynia. Such allodynia was associated with elevation of markers for activated microglia/macrophages (CD11b) and astrocytes/satellite glia (GFAP) in the lumbar dorsal spinal cord and dorsal root ganglia (DRG). Additionally, Toll-like receptor 4 (TLR4) mRNA was elevated in the lumbar dorsal spinal cord, while IL-1ß and IL-6 were elevated in the lumbar dorsal spinal cord, DRG, and gastrocnemius muscle. Demonstrating a casual role for such neuroinflammatory signaling, allodynia was reversed by treatment with either minocycline, the TLR4 inhibitor (+)-naltrexone, or IL-10 plasmid DNA. Together, these results point to a role for neuroinflammation in male rats in the model of musculoskeletal pain related to GWI. Therapies that alleviate persistent immune dysregulation may be a strategy to treat pain and other symptoms of GWI.

Drainage increases CO2 and N2 O emissions from tropical peat soils.

Tropical peatlands are vital ecosystems that play an important role in global carbon storage and cycles. Current estimates of greenhouse gases from these peatlands are uncertain as emissions vary with environmental conditions. This study provides the first comprehensive analysis of managed and natural tropical peatland GHG fluxes: heterotrophic (i.e. soil respiration without roots), total CO2 respiration rates, CH4 and N2 O fluxes. The study documents studies that measure GHG fluxes from the soil (n = 372) from various land uses, groundwater levels and environmental conditions. We found that total soil respiration was larger in managed peat ecosystems (median = 52.3 Mg CO2  ha-1  year-1 ) than in natural forest (median = 35.9 Mg CO2  ha-1  year-1 ). Groundwater level had a stronger effect on soil CO2 emission than land use. Every 100 mm drop of groundwater level caused an increase of 5.1 and 3.7 Mg CO2  ha-1  year-1 for plantation and cropping land use, respectively. Where groundwater is deep (≥0.5 m), heterotrophic respiration constituted 84% of the total emissions. N2 O emissions were significantly larger at deeper groundwater levels, where every drop in 100 mm of groundwater level resulted in an exponential emission increase (exp(0.7) kg N ha-1  year-1 ). Deeper groundwater levels induced high N2 O emissions, which constitute about 15% of total GHG emissions. CH4 emissions were large where groundwater is shallow; however, they were substantially smaller than other GHG emissions. When compared to temperate and boreal peatland soils, tropical peatlands had, on average, double the CO2 emissions. Surprisingly, the CO2 emission rates in tropical peatlands were in the same magnitude as tropical mineral soils. This comprehensive analysis provides a great understanding of the GHG dynamics within tropical peat soils that can be used as a guide for policymakers to create suitable programmes to manage the sustainability of peatlands effectively.

Stereochemistry and innate immune recognition: (+)-norbinaltorphimine targets myeloid differentiation protein 2 and inhibits toll-like receptor 4 signaling.

Deregulation of innate immune TLR4 signaling contributes to various diseases including neuropathic pain and drug addiction. Naltrexone is one of the rare TLR4 antagonists with good blood-brain barrier permeability and showing no stereoselectivity for TLR4. By linking 2 naltrexone units through a rigid pyrrole spacer, the bivalent ligand norbinaltorphimine was formed. Interestingly, (+)-norbinaltorphimine [(+)-1] showed ∼25 times better TLR4 antagonist activity than naltrexone in microglial BV-2 cell line, whereas (-)-norbinaltorphimine [(-)-1] lost TLR4 activity. The enantioselectivity of norbinaltorphimine was further confirmed in primary microglia, astrocytes, and macrophages. The activities of meso isomer of norbinaltorphimine and the molecular dynamic simulation results demonstrate that the stereochemistry of (+)-1 is derived from the (+)-naltrexone pharmacophore. Moreover, (+)-1 significantly increased and prolonged morphine analgesia in vivo. The efficacy of (+)-1 is long lasting. This is the first report showing enantioselective modulation of the innate immune TLR signaling.-Zhang, X., Peng, Y., Grace, P. M., Metcalf, M. D., Kwilasz, A. J., Wang, Y., Zhang, T., Wu, S., Selfridge, B. R., Portoghese, P. S., Rice, K. C., Watkins, L. R., Hutchinson, M. R., Wang, X. Stereochemistry and innate immune recognition: (+)-norbinaltorphimine targets myeloid differentiation protein 2 and inhibits toll-like receptor 4 signaling.

Oral Dimethyl Fumarate Reduces Peripheral Neuropathic Pain in Rodents via NFE2L2 Antioxidant Signaling.

BACKGROUND: Available treatments for neuropathic pain have modest efficacy and significant adverse effects, including abuse potential. Because oxidative stress is a key mechanistic node for neuropathic pain, the authors focused on the master regulator of the antioxidant response-nuclear factor erythroid 2-related factor 2 (NFE2L2; Nrf2)-as an alternative target for neuropathic pain. The authors tested whether dimethyl fumarate (U.S. Food and Drug Administration-approved treatment for multiple sclerosis) would activate NFE2L2 and promote antioxidant activity to reverse neuropathic pain behaviors and oxidative stress-dependent mechanisms. METHODS: Male Sprague Dawley rats, and male and female wild type and Nfe2l2 mice were treated with oral dimethyl fumarate/vehicle for 5 days (300 mg/kg; daily) after spared nerve injury/sham surgery (n = 5 to 8 per group). Allodynia was measured in von Frey reflex tests and hyperalgesia in operant conflict-avoidance tests. Ipsilateral L4/5 dorsal root ganglia were assayed for antioxidant and cytokine/chemokine levels, and mitochondrial bioenergetic capacity. RESULTS: Dimethyl fumarate treatment reversed mechanical allodynia (injury-vehicle, 0.45 ± 0.06 g [mean ± SD]; injury-dimethyl fumarate, 8.2 ± 0.16 g; P < 0.001) and hyperalgesia induced by nerve injury (injury-vehicle, 2 of 6 crossed noxious probes; injury-dimethyl fumarate, 6 of 6 crossed; P = 0.013). The antiallodynic effect of dimethyl fumarate was lost in nerve-injured Nfe2l2 mice, but retained in nerve-injured male and female wild type mice (wild type, 0.94 ± 0.25 g; Nfe2l2, 0.02 ± 0.01 g; P < 0.001). Superoxide dismutase activity was increased by dimethyl fumarate after nerve injury (injury-vehicle, 3.96 ± 1.28 mU/mg; injury-dimethyl fumarate, 7.97 ± 0.47 mU/mg; P < 0.001). Treatment reduced the injury-dependent increases in cytokines and chemokines, including interleukin-1ß (injury-vehicle, 13.30 ± 2.95 pg/mg; injury-dimethyl fumarate, 6.33 ± 1.97 pg/mg; P = 0.022). Injury-impaired mitochondrial bioenergetics, including basal respiratory capacity, were restored by dimethyl fumarate treatment (P = 0.025). CONCLUSIONS: Dimethyl fumarate, a nonopioid and orally-bioavailable drug, alleviated nociceptive hypersensitivity induced by peripheral nerve injury via activation of NFE2L2 antioxidant signaling. Dimethyl fumarate also resolved neuroinflammation and mitochondrial dysfunction-oxidative stress-dependent mechanisms that drive nociceptive hypersensitivity after nerve injury.

Lovastatin inhibits Toll-like receptor 4 signaling in microglia by targeting its co-receptor myeloid differentiation protein 2 and attenuates neuropathic pain.

There is growing interest in drug repositioning to find new therapeutic indications for drugs already approved for use in people. Lovastatin is an FDA approved drug that has been used clinically for over a decade as a lipid-lowering medication. While lovastatin is classically considered to act as a hydroxymethylglutaryl (HMG)-CoA reductase inhibitor, the present series of studies reveal a novel lovastatin effect, that being as a Toll-like receptor 4 (TLR4) antagonist. Lovastatin selectively inhibits lipopolysaccharide (LPS)-induced TLR4-NF-κB activation without affecting signaling by other homologous TLRs. In vitro biophysical binding and cellular thermal shift assay (CETSA) show that lovastatin is recognized by TLR4's coreceptor myeloid differentiation protein 2 (MD-2). This finding is supported by molecular dynamics simulations that lovastatin targets the LPS binding pocket of MD-2 and lovastatin binding stabilizes the MD-2 conformation. In vitro studies of BV-2 microglial cells revealed that lovastatin inhibits multiple effects of LPS, including activation of NFkB; mRNA expression of tumor necrosis factor-a, interleukin-6 and cyclo-oxygenase 2; production of nitric oxide and reactive oxygen species; as well as phagocytic activity. Furthermore, intrathecal delivery of lovastatin over lumbosacral spinal cord of rats attenuated both neuropathic pain from sciatic nerve injury and expression of the microglial activation marker CD11 in lumbar spinal cord dorsal horn. Given the well-established role of microglia and proinflammatory signaling in neuropathic pain, these data are supportive that lovastatin, as a TLR4 antagonist, may be productively repurposed for treating chronic pain.

Repeated Morphine Prolongs Postoperative Pain in Male Rats.

BACKGROUND: Opioids are effective postoperative analgesics. Disturbingly, we have previously reported that opioids such as morphine can worsen inflammatory pain and peripheral and central neuropathic pain. These deleterious effects are mediated by immune mediators that promote neuronal hyperexcitability in the spinal dorsal horn. Herein, we tested whether perioperative morphine could similarly prolong postoperative pain in male rats. METHODS: Rats were treated with morphine for 7 days, beginning immediately after laparotomy, while the morphine was tapered in a second group. Expression of genes for inflammatory mediators was quantified in the spinal dorsal horn. In the final experiment, morphine was administered before laparotomy for 7 days. RESULTS: We found that morphine treatment after laparotomy extended postoperative pain by more than 3 weeks (time × treatment: P < .001; time: P < .001; treatment: P < .05). Extension of postoperative pain was not related to morphine withdrawal, as it was not prevented by dose tapering (time × treatment: P = .8; time: P < .001; treatment: P = .9). Prolonged postsurgical pain was associated with increased expression of inflammatory genes, including those encoding Toll-like receptor 4, NOD like receptor protein 3 (NLRP3), nuclear factor kappa B (NFκB), caspase-1, interleukin-1ß, and tumor necrosis factor (P < .05). Finally, we showed that of preoperative morphine, concluding immediately before laparotomy, similarly prolonged postoperative pain (time × treatment: P < .001; time: P < .001; treatment: P < .001). There is a critical window for morphine potentiation of pain, as a 7-day course of morphine that concluded 1 week before laparotomy did not prolong postsurgical pain. CONCLUSIONS: These studies indicate the morphine can have a deleterious effect on postoperative pain. These studies further suggest that longitudinal studies could be performed to test whether opioids similarly prolong postoperative pain in the clinic.

Morphine paradoxically prolongs neuropathic pain in rats by amplifying spinal NLRP3 inflammasome activation.

Opioid use for pain management has dramatically increased, with little assessment of potential pathophysiological consequences for the primary pain condition. Here, a short course of morphine, starting 10 d after injury in male rats, paradoxically and remarkably doubled the duration of chronic constriction injury (CCI)-allodynia, months after morphine ceased. No such effect of opioids on neuropathic pain has previously been reported. Using pharmacologic and genetic approaches, we discovered that the initiation and maintenance of this multimonth prolongation of neuropathic pain was mediated by a previously unidentified mechanism for spinal cord and pain-namely, morphine-induced spinal NOD-like receptor protein 3 (NLRP3) inflammasomes and associated release of interleukin-1ß (IL-1ß). As spinal dorsal horn microglia expressed this signaling platform, these cells were selectively inhibited in vivo after transfection with a novel Designer Receptor Exclusively Activated by Designer Drugs (DREADD). Multiday treatment with the DREADD-specific ligand clozapine-N-oxide prevented and enduringly reversed morphine-induced persistent sensitization for weeks to months after cessation of clozapine-N-oxide. These data demonstrate both the critical importance of microglia and that maintenance of chronic pain created by early exposure to opioids can be disrupted, resetting pain to normal. These data also provide strong support for the recent "two-hit hypothesis" of microglial priming, leading to exaggerated reactivity after the second challenge, documented here in the context of nerve injury followed by morphine. This study predicts that prolonged pain is an unrealized and clinically concerning consequence of the abundant use of opioids in chronic pain.

Effect of urbanization on soil methane and nitrous oxide fluxes in subtropical Australia.

Increasing population densities and urban sprawl are causing rapid land use change from natural and agricultural ecosystems into smaller, urban residential properties. However, there is still great uncertainty about the effect that urbanization will have on biogeochemical C and N cycles and associated greenhouse gas (GHG) budgets. We aimed to evaluate how typical urbanization related land use change in subtropical Australia affects soil GHG exchange (N2 O and CH4 ) and the associated global warming potential (GWP). Fluxes were measured from three land uses: native forest, a long-term pasture, and a turf grass lawn continuously over two years using a high-resolution automated chamber system. The fertilized turf grass had the highest N2 O emissions, dominated by high fluxes >100 g N2 O-N day-1 immediately following establishment though decreased to just 0.6 kg N2 O-N ha-1 in the second year. Only minor fluxes occurred in the forest and pasture, with the high aeration of the sandy topsoil limiting N2 O emissions while promoting substantial CH4 uptake. Native forest was consistently the strongest CH4 sink (-2.9 kg CH4 -C ha-1  year-1 ), while the pasture became a short-term CH4 source after heavy rainfall when the soil reached saturation. On a two-year average, land use change from native forest to turf grass increased the non-CO2 GWP from a net annual GHG sink of -83 CO2 -e ha-1  year-1 to a source of 245 kg CO2 -e ha-1  year-1 . This study highlights that urbanization can substantially alter soil GHG exchange by altering plant soil water use and by increasing bulk density and inorganic N availability. However, on well-drained subtropical soils, the impact of urbanization on inter-annual non-CO2 GWP of turf grass was low compared to urbanized ecosystems in temperate climates.

Assessing uncertainties in crop and pasture ensemble model simulations of productivity and N2 O emissions.

Simulation models are extensively used to predict agricultural productivity and greenhouse gas emissions. However, the uncertainties of (reduced) model ensemble simulations have not been assessed systematically for variables affecting food security and climate change mitigation, within multi-species agricultural contexts. We report an international model comparison and benchmarking exercise, showing the potential of multi-model ensembles to predict productivity and nitrous oxide (N2 O) emissions for wheat, maize, rice and temperate grasslands. Using a multi-stage modelling protocol, from blind simulations (stage 1) to partial (stages 2-4) and full calibration (stage 5), 24 process-based biogeochemical models were assessed individually or as an ensemble against long-term experimental data from four temperate grassland and five arable crop rotation sites spanning four continents. Comparisons were performed by reference to the experimental uncertainties of observed yields and N2 O emissions. Results showed that across sites and crop/grassland types, 23%-40% of the uncalibrated individual models were within two standard deviations (SD) of observed yields, while 42 (rice) to 96% (grasslands) of the models were within 1 SD of observed N2 O emissions. At stage 1, ensembles formed by the three lowest prediction model errors predicted both yields and N2 O emissions within experimental uncertainties for 44% and 33% of the crop and grassland growth cycles, respectively. Partial model calibration (stages 2-4) markedly reduced prediction errors of the full model ensemble E-median for crop grain yields (from 36% at stage 1 down to 4% on average) and grassland productivity (from 44% to 27%) and to a lesser and more variable extent for N2 O emissions. Yield-scaled N2 O emissions (N2 O emissions divided by crop yields) were ranked accurately by three-model ensembles across crop species and field sites. The potential of using process-based model ensembles to predict jointly productivity and N2 O emissions at field scale is discussed.

Protraction of neuropathic pain by morphine is mediated by spinal damage associated molecular patterns (DAMPs) in male rats.

We have recently reported that a short course of morphine, starting 10days after sciatic chronic constriction injury (CCI), prolonged the duration of mechanical allodynia for months after morphine ceased. Maintenance of this morphine-induced persistent sensitization was dependent on spinal NOD-like receptor protein 3 (NLRP3) inflammasomes-protein complexes that proteolytically activate interleukin-1ß (IL-1ß) via caspase-1. However, it is still unclear how NLRP3 inflammasome signaling is maintained long after morphine is cleared. Here, we demonstrate that spinal levels of the damage associated molecular patterns (DAMPs) high mobility group box 1 (HMGB1) and biglycan are elevated during morphine-induced persistent sensitization in male rats; that is, 5weeks after cessation of morphine dosing. We also show that HMGB1 and biglycan levels are at least partly dependent on the initial activation of caspase-1, as well as Toll like receptor 4 (TLR4) and the purinergic receptor P2X7R-receptors responsible for priming and activation of NLRP3 inflammasomes. Finally, pharmacological attenuation of the DAMPs HMGB1, biglycan, heat shock protein 90 and fibronectin persistently reversed morphine-prolonged allodynia. We conclude that after peripheral nerve injury, morphine treatment results in persistent DAMP release via TLR4, P2X7R and caspase-1, which are involved in formation/activation of NLRP3 inflammasomes. These DAMPs are responsible for maintaining persistent allodynia, which may be due to engagement of a positive feedback loop, in which NLRP3 inflammasomes are persistently activated by DAMPs signaling at TLR4 and P2X7R.