Publications by authors named "Linda R Watkins"

258 Publications

Toll-like receptor 2 and 4 antagonism for the treatment of experimental autoimmune encephalomyelitis (EAE)-related pain.

Brain Behav Immun 2021 Mar 7;93:80-95. Epub 2021 Jan 7.

Department of Psychology and Neuroscience, University of Colorado, Boulder, CO, United States; The Center for Neuroscience, University of Colorado, Boulder, CO, United States.

Neuropathic pain is a major symptom of multiple sclerosis (MS) with up to 92% of patients reporting bodily pain, and 85% reporting pain severe enough to cause functional disability. None of the available therapeutics target MS pain. Toll-like receptors 2 and 4 (TLR2/TLR4) have emerged as targets for treating a wide array of autoimmune disorders, including MS, as well as having demonstrated success at suppressing pain in diverse animal models. The current series of studies tested systemic TLR2/TLR4 antagonists in males and females in a low-dose Myelin oligodendrocyte glycoprotein (MOG) experimental autoimmune encephalomyelitis (EAE) model, with reduced motor dysfunction to allow unconfounded testing of allodynia through 50+ days post-MOG. The data demonstrated that blocking TLR2/TLR4 suppressed EAE-related pain, equally in males and females; upregulation of dorsal spinal cord proinflammatory gene expression for TLR2, TLR4, NLRP3, interleukin-1β, IkBα, TNF-α and interleukin-17; and upregulation of dorsal spinal cord expression of glial immunoreactivity markers. In support of these results, intrathecal interleukin-1 receptor antagonist reversed EAE-induced allodynia, both early and late after EAE induction. In contrast, blocking TLR2/TLR4 did not suppress EAE-induced motor disturbances induced by a higher MOG dose. These data suggest that blocking TLR2/TLR4 prevents the production of proinflammatory factors involved in low dose EAE pathology. Moreover, in this EAE model, TLR2/TLR4 antagonists were highly effective in reducing pain, whereas motor impairment, as seen in high dose MOG EAE, is not affected.
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http://dx.doi.org/10.1016/j.bbi.2020.12.016DOI Listing
March 2021

Postoperative cognitive dysfunction is made persistent with morphine treatment in aged rats.

Neurobiol Aging 2021 02 24;98:214-224. Epub 2020 Nov 24.

Institute for Behavioral Medicine Research, The Ohio State University, Columbus, OH, USA; Department of Psychology and Neuroscience, Center for Neuroscience, University of Colorado Boulder, Boulder, CO, USA; Department of Psychiatry and Behavioral Health, The Ohio State University, Columbus, OH, USA; Department of Neuroscience, The Ohio State University, Columbus, OH, USA; Chronic Brain Injury Program, Discovery Themes Initiative, The Ohio State University, Columbus, OH, USA. Electronic address:

Postoperative cognitive dysfunction (POCD) is the collection of cognitive impairments, lasting days to months, experienced by individuals following surgery. Persistent POCD is most commonly experienced by older individuals and is associated with a greater vulnerability to developing Alzheimer's disease, but the underlying mechanisms are not known. It is known that laparotomy (exploratory abdominal surgery) in aged rats produces memory impairments for 4 days. Here we report that postsurgical treatment with morphine extends this deficit to at least 2 months while having no effects in the absence of surgery. Indeed, hippocampal-dependent long-term memory was impaired 2, 4, and 8 weeks postsurgery only in aged, morphine-treated rats. Short-term memory remained intact. Morphine is known to have analgesic effects via μ-opioid receptor activation and neuroinflammatory effects through Toll-like receptor 4 activation. Here we demonstrate that persistent memory deficits were mediated independently of the μ-opioid receptor, suggesting that they were evoked through a neuroinflammatory mechanism and unrelated to pain modulation. In support of this, aged, laparotomized, and morphine-treated rats exhibited increased gene expression of various proinflammatory markers (IL-1β, IL-6, TNFα, NLRP3, HMGB1, TLR2, and TLR4) in the hippocampus at the 2-week time point. Furthermore, central blockade of IL-1β signaling with the specific IL-1 receptor antagonist (IL-1RA), at the time of surgery, completely prevented the memory impairment. Finally, synaptophysin and PSD95 gene expression were significantly dysregulated in the hippocampus of aged, laparotomized, morphine-treated rats, suggesting that impaired synaptic structure and/or function may play a key role in this persistent deficit. This instance of long-term memory impairment following surgery closely mirrors the timeline of persistent POCD in humans and may be useful for future treatment discoveries.
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http://dx.doi.org/10.1016/j.neurobiolaging.2020.11.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7870544PMC
February 2021

Experimental autoimmune encephalopathy (EAE)-induced hippocampal neuroinflammation and memory deficits are prevented with the non-opioid TLR2/TLR4 antagonist (+)-naltrexone.

Behav Brain Res 2021 01 6;396:112896. Epub 2020 Sep 6.

Department of Psychology and Neuroscience, University of Colorado, Boulder, CO, USA; The Center for Neuroscience, University of Colorado, Boulder, CO, USA; Institute for Behavioral Medicine Research, The Ohio State University, Columbus, OH, USA; Department of Psychiatry and Behavioral Health, The Ohio State University, Columbus, OH, USA; Chronic Brain Injury Program, Discovery Themes Initiative, The Ohio State University, Columbus, OH, USA; Department of Neuroscience, The Ohio State University, Columbus, OH, USA. Electronic address:

Multiple sclerosis (MS) is associated with burdensome memory impairments and preclinical literature suggests that these impairments are linked to neuroinflammation. Previously, we have shown that toll-like receptor 4 (TLR4) antagonists, such as (+)-naltrexone [(+)-NTX], block neuropathic pain and associated spinal inflammation in rats. Here we extend these findings to first demonstrate that (+)-NTX blocks TLR2 in addition to TLR4. Additionally, we examined in two rat strains whether (+)-NTX could attenuate learning and memory disturbances and associated neuroinflammation using a low-dose experimental autoimmune encephalomyelitis (EAE) model of MS. EAE is the most commonly used experimental model for the human inflammatory demyelinating disease, MS. This low-dose model avoided motor impairments that would confound learning and memory measurements. Fourteen days later, daily subcutaneous (+)-NTX or saline injections began and continued throughout the study. Contextual and auditory-fear conditioning were conducted at day 21 to assess hippocampal and amygdalar function. With this low-dose model, EAE impaired long-term, but not short-term, contextual fear memory; both long-term and short-term auditory-cued fear memory were spared. This was associated with increased mRNA for hippocampal interleukin-1β (IL-1β), TLR2, TLR4, NLRP3, and IL-17 and elevated expression of the microglial marker Iba1 in CA1 and DG regions of the hippocampus, confirming the neuroinflammation observed in higher-dose EAE models. Importantly, (+)-NTX completely prevented the EAE-induced memory impairments and robustly attenuated the associated proinflammatory effects. These findings suggest that (+)-NTX may exert therapeutic effects on memory function by dampening the neuroinflammatory response in the hippocampus through blockade of TLR2/TLR4. This study suggests that TLR2 and TLR4 antagonists may be effective at treating MS-related memory deficits.
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http://dx.doi.org/10.1016/j.bbr.2020.112896DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7572683PMC
January 2021

Targeted interleukin-10 plasmid DNA therapy in the treatment of osteoarthritis: Toxicology and pain efficacy assessments.

Brain Behav Immun 2020 11 13;90:155-166. Epub 2020 Aug 13.

Xalud Therapeutics, Inc., Berkeley, CA, USA.

Osteoarthritis results in chronic pain and loss of function. Proinflammatory cytokines create both osteoarthritis pathology and pain. Current treatments are poorly effective, have significant side effects, and have not targeted the cytokines central to osteoarthritis development and maintenance. Interleukin-10 is an anti-inflammatory cytokine that potently and broadly suppresses proinflammatory cytokine activity. However, interleukin-10 protein has a short half-life in vivo and poor joint permeability. For sustained IL-10 activity, we developed a plasmid DNA-based therapy that expresses a long-acting human interleukin-10 variant (hIL-10var). Here, we describe the 6-month GLP toxicology study of this therapy. Intra-articular injections of hIL-10var pDNA into canine stifle joints up to 1.5 mg bilaterally were well-tolerated and without pathologic findings. This represents the first long-term toxicologic assessment of intra-articular pDNA therapy. We also report results of a small double-blind, placebo-controlled study of the effect of intra-articular hIL-10var pDNA on pain measures in companion (pet) dogs with naturally occurring osteoarthritis. This human IL-10-based targeted therapy reduced pain measures in the dogs, based on veterinary and owner ratings, without any adverse findings. These results with hIL-10var pDNA therapy, well-tolerated and without toxicologic effects, establish the basis for clinical trials of a new class of safe and effective therapies for OA.
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http://dx.doi.org/10.1016/j.bbi.2020.08.005DOI Listing
November 2020

Acute stress induces the rapid and transient induction of caspase-1, gasdermin D and release of constitutive IL-1β protein in dorsal hippocampus.

Brain Behav Immun 2020 11 1;90:70-80. Epub 2020 Aug 1.

Department of Psychology and Neuroscience, Center for Neuroscience, University of Colorado Boulder, Boulder, CO, United States.

The proinflammatory cytokine interleukin (IL)-1β plays a pivotal role in the behavioral manifestations (i.e., sickness) of the stress response. Indeed, exposure to acute and chronic stressors induces the expression of IL-1β in stress-sensitive brain regions. Thus, it is typically presumed that exposure to stressors induces the extra-cellular release of IL-1β in the brain parenchyma. However, this stress-evoked neuroimmune phenomenon has not been directly demonstrated nor has the cellular process of IL-1β release into the extracellular milieu been characterized in brain. This cellular process involves a form of inflammatory cell death, termed pyroptosis, which involves: 1) activation of caspase-1, 2) caspase-1 maturation of IL-1β, 3) caspase-1 cleavage of gasdermin D (GSDMD), and 4) GSDMD-induced permeability of the cell membrane through which IL-1β is released into the extracellular space. Thus, the present study examined whether stress induces the extra-cellular release of IL-1β and engages the above cellular process in mediating IL-1β release in the brain. Male Sprague-Dawley rats were exposed to inescapable tailshock (IS). IL-1β extra-cellular release, caspase-1 activity and cleavage of GSDMD were measured in dorsal hippocampus. We found that exposure to IS induced a transient increase in the release of IL-1β into the extracellular space immediately after termination of the stressor. IS also induced a transient increase in caspase-1 activity prior to IL-1β release, while activation of GSDMD was observed immediately after termination of the stressor. IS also increased mRNA and protein expression of the ESCRTIII protein CHMP4B, which is involved in cellular repair. The present results suggest that exposure to an acute stressor induces the hallmarks of pyroptosis in brain, which might serve as a key cellular process involved in the release of IL-1β into the extracellular milieu of the brain parenchyma.
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http://dx.doi.org/10.1016/j.bbi.2020.07.042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7544655PMC
November 2020

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.

J Neurosci Res 2020 Jun 13. Epub 2020 Jun 13.

Laboratories of Neuroimmunology, Department of Symptom Research, University of Texas MD Anderson Cancer Center, Houston, TX, USA.

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.
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http://dx.doi.org/10.1002/jnr.24645DOI Listing
June 2020

Acute stress induces chronic neuroinflammatory, microglial and behavioral priming: A role for potentiated NLRP3 inflammasome activation.

Brain Behav Immun 2020 10 30;89:32-42. Epub 2020 May 30.

Department of Psychology and Neuroscience, Center for Neuroscience, University of Colorado Boulder, Boulder, CO 80301, USA.

Prior exposure to acute and chronic stressors potentiates the neuroinflammatory and microglial pro-inflammatory response to subsequent immune challenges suggesting that stressors sensitize or prime microglia. Stress-induced priming of the NLRP3 inflammasome has been implicated in this priming phenomenon, however the duration/persistence of these effects has not been investigated. In the present study, we examined whether exposure to a single acute stressor (inescapable tailshock) induced a protracted priming of the NLRP3 inflammasome as well as the neuroinflammatory, behavioral and microglial proinflammatory response to a subsequent immune challenge in hippocampus. In male Sprague-Dawley rats, acute stress potentiated the neuroinflammatory response (IL-1β, IL-6, and NFκBIα) to an immune challenge (lipopolysaccharide; LPS) administered 8 days after stressor exposure. Acute stress also potentiated the proinflammatory cytokine response (IL-1β, IL-6, TNF and NFκBIα) to LPS ex vivo. This stress-induced priming of microglia also was observed 28 days post-stress. Furthermore, challenge with LPS reduced juvenile social exploration, but not sucrose preference, in animals exposed to stress 8 days prior to immune challenge. Exposure to acute stress also increased basal mRNA levels of NLRP3 and potentiated LPS-induction of caspase-1 mRNA and protein activity 8 days after stress. The present findings suggest that acute stress produces a protracted vulnerability to the neuroinflammatory effects of subsequent immune challenges, thereby increasing risk for stress-related psychiatric disorders with an etiological inflammatory component. Further, these findings suggest the unique possibility that acute stress might induce innate immune memory in microglia.
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http://dx.doi.org/10.1016/j.bbi.2020.05.063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7572608PMC
October 2020

Activation of sphingosine-1-phosphate receptor subtype 1 in the central nervous system contributes to morphine-induced hyperalgesia and antinociceptive tolerance in rodents.

Pain 2020 09;161(9):2107-2118

Department of Pharmacology and Physiology, Saint Louis University School of Medicine, St. Louis, MO, United States.

Abstract: Morphine-induced alterations in sphingolipid metabolism in the spinal cord and increased formation of the bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P) have been implicated in the development of morphine-induced hyperalgesia (OIH; increased pain sensitivity) and antinociceptive tolerance. These adverse effects hamper opioid use for treating chronic pain and contribute to dependence and abuse. S1P produces distinct effects through 5 G-protein-coupled receptors (S1PR1-5) and several intracellular targets. How S1P exerts its effects in response to morphine remains unknown. Here, we report that S1P contributes to the development of morphine-induced hyperalgesia and tolerance through S1P receptor subtype 1 (S1PR1) signaling in uninjured male and female rodents, which can be blocked by targeting S1PR1 with S1PR1 antagonists or RNA silencing. In mouse neuropathic pain models, S1PR1 antagonists blocked the development of tolerance to the antiallodynic effects of morphine without altering morphine pharmacokinetics and prevented prolonged morphine-induced neuropathic pain. Targeting S1PR1 reduced morphine-induced neuroinflammatory events in the dorsal horn of the spinal cord: increased glial marker expression, mitogen-activated protein kinase p38 and nuclear factor κB activation, and increased inflammatory cytokine expression, such as interleukin-1β, a cytokine central in the modulation of opioid-induced neural plasticity. Our results identify S1PR1 as a critical path for S1P signaling in response to sustained morphine and reveal downstream neuroinflammatory pathways impacted by S1PR1 activation. Our data support investigating S1PR1 antagonists as a clinical approach to mitigate opioid-induced adverse effects and repurposing the functional S1PR1 antagonist FTY720, which is FDA-approved for multiple sclerosis, as an opioid adjunct.
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http://dx.doi.org/10.1097/j.pain.0000000000001888DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7554181PMC
September 2020

The behavioral and neurochemical effects of an inescapable stressor are time of day dependent.

Stress 2020 07 20;23(4):405-416. Epub 2020 Jan 20.

Department of Psychology and Neuroscience, Center for Neuroscience, University of Colorado Boulder, Boulder, CO, USA.

Circadian rhythms are ∼24 h fluctuations in physiology and behavior that are synchronized with the light-dark cycle. The circadian system ensures homeostatic balance by regulating multiple systems that respond to environmental stimuli including stress systems. In rats, acute exposure to a series of uncontrollable tailshocks (inescapable stress, IS) produces an anxiety and depression-like phenotype. Anxiety- and fear-related behavioral changes produced by IS are driven by sensitization of serotonergic (5-hydroxytryptamine, 5-HT) neurons in the dorsal raphe nucleus (DRN). Because the circadian and serotonergic systems are closely linked, here we tested whether the DRN-dependent behavioral and neurochemical effects of IS are time of day dependent. Exposure to IS during the light (inactive) phase elicited the expected changes in mood related behaviors. In contrast, rats that underwent IS during the dark (active) phase were buffered against stress-induced changes in juvenile social exploration and shock-elicited freezing, both DRN-dependent outcomes. Interestingly, behavioral anhedonia, which is not a DRN-dependent behavior, was comparably reduced by stress at both times of day. Neurochemical changes complimented the behavioral results: IS-induced activation of DRN 5-HT neurons was greater during the light phase compared to the dark phase. Additionally, 5-HT1AR and 5-HTT, two genes that regulate 5-HT activity were up-regulated during the middle of the light cycle. These data suggest that DRN-dependent behavioral outcomes of IS are time of day dependent and may be mediated by circadian gating of the DRN response to stress.Lay summaryHere we show that the time of day at which a stressor occurs impacts the behavioral and neurochemical outcomes of the stressor. In particular, animals appear more vulnerable to a stressor that occurs during their rest phase. This work may have important implications for shift-workers and other populations that are more likely to encounter stressors during their rest phase.
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http://dx.doi.org/10.1080/10253890.2019.1707180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7335331PMC
July 2020

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

Brain Behav Immun 2019 11 19;82:432-444. Epub 2019 Sep 19.

Key Laboratory of Molecular Pharmacology and Drug Evaluation, Ministry of Education, Yantai University, Yantai 264005, China; Laboratory of Chemical Biology, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China; Department of Applied Chemistry and Engineering, University of Science and Technology of China, Hefei 230026, China. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbi.2019.09.013DOI Listing
November 2019

Oxycodone, fentanyl, and morphine amplify established neuropathic pain in male rats.

Pain 2019 11;160(11):2634-2640

Department of Symptom Research, University of Texas MD Anderson Cancer Center, Houston, TX, United States.

Opioids are widely prescribed for chronic pain, including neuropathic pain, despite growing evidence of long-term harm. Previous preclinical studies have documented exacerbation of nociceptive hypersensitivity, including that induced by peripheral nerve injury, by morphine. The present series of behavioral studies sought to replicate and extend our prior research, which demonstrated a multimonth exacerbation of nociceptive hypersensitivity by a 5-day course of morphine initiated 10 days after nerve injury. The current studies demonstrate that enduring exacerbation of nociceptive hypersensitivity is not restricted to morphine, but rather is also created by the clinically relevant opioids fentanyl and oxycodone when these are likewise administered for 5 days beginning 10 days after nerve injury. Furthermore, enduring exacerbation of nociceptive hypersensitivity is also observed when the same dosing regimen for either morphine, fentanyl, or oxycodone begins 1 month after nerve injury. Finally, a striking result from these studies is that no such exacerbation of nociceptive hypersensitivity occurs when either morphine, fentanyl, or oxycodone dosing begins at the time of nerve injury. These results extend our previous findings that morphine exacerbates nociceptive hypersensitivity to the clinically relevant opioids fentanyl and oxycodone when administered after the development of nociceptive hypersensitivity, while also providing possible clinically relevant insight into when these opioids can be safely administered and not exacerbate neuropathic pain.
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http://dx.doi.org/10.1097/j.pain.0000000000001652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7053537PMC
November 2019

Methamphetamine Activates Toll-Like Receptor 4 to Induce Central Immune Signaling within the Ventral Tegmental Area and Contributes to Extracellular Dopamine Increase in the Nucleus Accumbens Shell.

ACS Chem Neurosci 2019 08 17;10(8):3622-3634. Epub 2019 Jul 17.

Department of Psychology and Neuroscience and the Center for Neuroscience , University of Colorado at Boulder , Boulder , Colorado 80309 , United States.

Methamphetamine (METH) is a globally abused, highly addictive stimulant. While investigations of the rewarding and motivational effects of METH have focused on neuronal actions, increasing evidence suggests that METH can also target microglia, the innate immune cells of the central nervous system, causing release of proinflammatory mediators and therefore amplifying the reward changes in the neuronal activity induced by METH. However, how METH induces neuroinflammatory responses within the central nervous system (CNS) is unknown. Herein, we provide direct evidence that METH creates neuroinflammation, at least in part, via the activation of the innate immune Toll-like receptor 4 (TLR4). Biophysical studies revealed that METH bound to MD-2, the key coreceptor of TLR4. Molecular dynamics simulations showed METH binding stabilized the active heterotetramer (TLR4/MD-2) conformation. Classic TLR4 antagonists LPS-RS and TAK-242 attenuated METH induced NF-κB activation of microglia, whereas added MD-2 protein boosted METH-induced NF-κB activation. Systemically administered METH (1 mg/kg) was found to specifically up-regulate expression of both CD11b (microglial activation marker) and the proinflammatory cytokine interleukin 6 (IL-6) mRNAs in the ventral tegmental area (VTA), but not in either the nucleus accumbens shell (NAc) or prefrontal cortex (PFC). Systemic administration of a nonopioid, blood-brain barrier permeable TLR4 antagonist (+)-naloxone inhibited METH-induced activation of microglia and IL-6 mRNA overexpression in VTA. METH was found to increase conditioned place preference (CPP) as well as extracellular dopamine concentrations in the NAc, with both effects suppressed by the nonopioid TLR4 antagonist (+)-naloxone. Furthermore, intra-VTA injection of LPS-RS or IL-6 neutralizing antibody suppressed METH-induced elevation of extracellular NAc dopamine. Taken together, this series of studies demonstrate that METH-induced neuroinflammation is, at least in part, mediated by TLR4-IL6 signaling within the VTA, which has the downstream effect of elevating dopamine in the NAc shell. These results provide a novel understanding of the neurobiological mechanisms underlying acute METH reward that includes a critical role for central immune signaling and offers a new target for medication development for treating drug abuse.
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http://dx.doi.org/10.1021/acschemneuro.9b00225DOI Listing
August 2019

TDP-43 knockdown causes innate immune activation via protein kinase R in astrocytes.

Neurobiol Dis 2019 12 21;132:104514. Epub 2019 Jun 21.

Departement of Integrative Physiology, University of Colorado Boulder, Boulder, CO 80309, USA.

TAR-DNA binding protein 43 (TDP-43) is a multifunctional RNA binding protein directly implicated in the etiology of amyotrophic lateral sclerosis (ALS). Previous studies have demonstrated that loss of TDP-43 function leads to intracellular accumulation of non-coding repetitive element transcripts and double-stranded RNA (dsRNA). These events could cause immune activation and contribute to the neuroinflammation observed in ALS, but this possibility has not been investigated. Here, we knock down TDP-43 in primary rat astrocytes via siRNA, and we use RNA-seq, immunofluorescence, and immunoblotting to show that this results in: 1) accumulation of repetitive element transcripts and dsRNA; and 2) pro-inflammatory gene and protein expression consistent with innate immune signaling and astrocyte activation. We also show that both chemical inhibition and siRNA knockdown of protein kinase R (PKR), a dsRNA-activated kinase implicated in the innate immune response, block the expression of all activation markers assayed. Based on these findings, we suggest that intracellular accumulation of endogenous dsRNA may be a novel and important mechanism underlying the pathogenesis of ALS (and perhaps other neurodegenerative diseases), and that PKR inhibitors may have the potential to prevent reactive astrocytosis in ALS.
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http://dx.doi.org/10.1016/j.nbd.2019.104514DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6834892PMC
December 2019

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

FASEB J 2019 08 4;33(8):9577-9587. Epub 2019 Jun 4.

Laboratory of Chemical Biology, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, China.

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 . 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.
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http://dx.doi.org/10.1096/fj.201900173RRRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6988860PMC
August 2019

Glucocorticoids mediate stress induction of the alarmin HMGB1 and reduction of the microglia checkpoint receptor CD200R1 in limbic brain structures.

Brain Behav Immun 2019 08 9;80:678-687. Epub 2019 May 9.

Department of Psychology and Neuroscience, University of Colorado Boulder, Boulder, CO 80301, USA; Center for Neuroscience, University of Colorado Boulder, Boulder, CO 80301, USA.

Exposure to stressors primes neuroinflammatory responses to subsequent immune challenges and stress-induced glucocorticoids (GCs) play a mediating role in this phenomenon of neuroinflammatory priming. Recent evidence also suggests that the alarmin high-mobility group box-1 (HMGB1) and the microglial checkpoint receptor CD200R1 serve as proximal mechanisms of stress-induced neuroinflammatory priming. However, it is unclear whether stress-induced GCs play a causal role in these proximal mechanisms of neuroinflammatory priming; this forms the focus of the present investigation. Here, we found that exposure to a severe acute stressor (inescapable tailshock) induced HMGB1 and reduced CD200R1 expression in limbic brain regions and pharmacological blockade of GC signaling (RU486) mitigated these effects of stress. To confirm these effects of RU486, adrenalectomy (ADX) with basal corticosterone (CORT) replacement was used to block the stress-induced increase in GCs as well as effects on HMGB1 and CD200R1. As with RU486, ADX mitigated the effects of stress on HMGB1 and CD200R1. Subsequently, exogenous CORT was administered to determine whether GCs are sufficient to recapitulate the effects of stress. Indeed, exogenous CORT induced expression of HMGB1 and reduced expression of CD200R1. In addition, exposure of primary microglia to CORT also recapitulated the effects of stress on CD200R1 suggesting that CORT acts directly on microglia to reduce expression of CD200R1. Taken together, these findings suggest that GCs mediate the effects of stress on these proximal mechanisms of neuroinflammatory priming.
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http://dx.doi.org/10.1016/j.bbi.2019.05.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6662571PMC
August 2019

Microglia: Neuroimmune-sensors of stress.

Semin Cell Dev Biol 2019 10 9;94:176-185. Epub 2019 Jan 9.

Department of Psychology and Neuroscience, University of Colorado Boulder, Boulder, CO, 80309, USA; Center for Neuroscience, University of Colorado Boulder, Boulder, CO, 80309, USA.

Exposure to stressors disrupts homeostasis and results in the release of stress hormones including glucocorticoids, epinepherine and norepinepherine. Interestingly, stress also has profound affects on microglia, which are tissue-resident macrophages in the brain parenchyma. Microglia express a diverse array of receptors, which also allows them to respond to stress hormones derived from peripheral as well as central sources. Here, we review studies of how exposure to acute and chronic stressors alters the immunophenotype and function of microglia. Further, we examine a causal for stress hormones in these effects of stress on microglia. We propose that microglia serve as immunosensors of the stress response, which puts them in the unique position to sense and respond rapidly to alterations in homeostasis and integrate the neural response to threats.
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http://dx.doi.org/10.1016/j.semcdb.2019.01.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6614020PMC
October 2019

Spinal Cord Injury in Rats Disrupts the Circadian System.

eNeuro 2018 Nov-Dec;5(6). Epub 2018 Dec 21.

Department of Psychology and Neuroscience, University of Colorado Boulder, Boulder, Colorado 80301.

Spinal cord injury (SCI) perturbs many physiological systems. The circadian system helps maintain homeostasis throughout the body by synchronizing physiological and behavioral functions to predictable daily events. Whether disruption of these coordinated daily rhythms contributes to SCI-associated pathology remains understudied. Here, we hypothesized that SCI in rats would dysregulate several prominent circadian outputs including glucocorticoids, core temperature, activity, neuroinflammation, and circadian gene networks. Female and male Sprague Dawley rats were subjected to clinically relevant thoracic 9 moderate contusion SCI (or laminectomy sham surgery). Diurnal measures-including rhythms of plasma corticosterone (CORT), body temperature, and activity (using small implanted transmitters), and intraspinal circadian and inflammatory gene expression-were studied prior to and after surgery. SCI caused overall increases and disrupted rhythms of the major rodent glucocorticoid, CORT. Presurgery and sham rats displayed expected rhythms in body temperature and activity, whereas rats with SCI had blunted daily rhythms in body temperature and activity. In parallel, SCI disrupted intraspinal rhythms of circadian clock gene expression. Circadian clock genes can act as transcriptional regulators of inflammatory pathways. Indeed, SCI rats also showed dysregulated rhythms in inflammatory gene expression in both the epicenter and distal spinal cord. Our data show that moderate SCI in rats causes wide-ranging diurnal rhythm dysfunction, which is severe at acute time points and gradually recovers over time. Normalizing post-SCI diurnal rhythms could enhance the recovery of homeostasis and quality of life.
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http://dx.doi.org/10.1523/ENEURO.0328-18.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6325559PMC
March 2019

Spinal Cord Injury in Rats Dysregulates Diurnal Rhythms of Fecal Output and Liver Metabolic Indicators.

J Neurotrauma 2019 06 9;36(12):1923-1934. Epub 2019 Jan 9.

1 Center for Neuroscience, University of Colorado Boulder, Boulder, Colorado.

Spinal cord injury (SCI) dysregulates metabolic homeostasis. Metabolic homeostasis is optimized across the day by the circadian system. Despite the prevalence of metabolic pathologies after SCI, post-SCI circadian regulation of metabolism remains understudied. Here, we hypothesized that SCI in rats would disrupt circadian regulation of key metabolic organs, leading to metabolic dysregulation. Female and male Sprague-Dawley rats received moderate thoracic (T)-9 contusion SCI (or sham surgery). First, SCI disrupted diurnal rhythms in two metabolic behaviors: fecal production and food intake rhythms were ablated acutely. SCI also expedited whole-gut transit time. In parallel, acute SCI increased plasma glucose. Diurnal glucose storage-release cycles regulated by the liver were disrupted by SCI, which also increased liver glucose metabolism messenger RNAs (mRNAs). Further, SCI disrupted liver clock gene expression and suppressed inflammatory gene rhythms. Together, our novel data suggest that SCI disrupts typical metabolic and circadian function. Improving post-SCI metabolic function could enhance recovery of homeostasis.
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http://dx.doi.org/10.1089/neu.2018.6101DOI Listing
June 2019

A single peri-sciatic nerve administration of the adenosine 2A receptor agonist ATL313 produces long-lasting anti-allodynia and anti-inflammatory effects in male rats.

Brain Behav Immun 2019 02 16;76:116-125. Epub 2018 Nov 16.

Department of Psychology and Neuroscience, University of Colorado, Boulder, CO, USA; The Center for Neuroscience, University of Colorado, Boulder, CO, USA.

Neuropathic pain is a widespread problem which remains poorly managed by currently available therapeutics. Peripheral nerve injury and inflammation leads to changes at the nerve injury site, including activation of resident and recruited peripheral immune cells, that lead to neuronal central sensitization and pain amplification. The present series of studies tested the effects of peri-sciatic nerve delivery of single doses of adenosine 2A receptor (AR) agonists on pain and neuroinflammation. The data provide converging lines of evidence supportive that AR agonism at the site of peripheral nerve injury and inflammation is effective in suppressing ongoing neuropathic pain. After AR agonism resolved neuropathic pain, a return of pain enhancement (allodynia) was observed in response to peri-sciatic injection of H-89, which can inhibit protein kinase A, and by peri-sciatic injection of neutralizing antibody against the potent anti-inflammatory cytokine interleukin-10. AR agonist actions at the nerve injury site suppress neuroinflammation, as reflected by decreased release of interleukin-1β and nitric oxide, as well as decreased sciatic expression of markers of monocytes/macrophages and inducible nitric oxide synthase. Taken together, the data are supportive that AR agonists, acting at the level of peripheral nerve injury, may be of therapeutic value in treating chronic pain of neuroinflammatory origin.
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http://dx.doi.org/10.1016/j.bbi.2018.11.011DOI Listing
February 2019

Circadian misalignment has differential effects on affective behavior following exposure to controllable or uncontrollable stress.

Behav Brain Res 2019 02 9;359:440-445. Epub 2018 Oct 9.

Department of Psychology and Neuroscience, Center for Neuroscience, Muenzinger Building, UCB 345, University of Colorado Boulder, Boulder, CO, 80309, USA.

In modern 24 h society, circadian disruption is pervasive, arising from night shift work, air travel across multiple time zones, irregular sleep schedules, and exposure to artificial light at night. Disruption of the circadian system is associated with many adverse health consequences, including mood disorders. Here we investigate whether inducing circadian misalignment using a phase advance protocol interferes with the ability to cope with a stressor, thereby increasing susceptibility to the negative consequences of stress. Male rats were maintained on a standard 12:12 light: dark (LD) cycle or subjected to a chronic phase advance (CPA) protocol involving 4 weekly 6 h phase shifts (earlier light onset) of the LD cycle. Rats were then exposed to escapable stress (ES), inescapable stress (IS), or no stress (home cage control; HC) and performance on juvenile social exploration and active escape learning in the two-way shuttlebox test was assessed 24 h and 48 h following stress, respectively. CPA alone had no effect on pre-stress juvenile social exploration, and it also did not interfere with the protective effect of ES on the stress-induced reduction in juvenile social exploration. In contrast, CPA impaired escape learning in the two-way shuttlebox to the same extent as IS in all subjects, regardless of stress history. Additionally, CPA produced somatic alterations that included increased body mass, increased epididymal adiposity, and decreased adrenal mass. These data indicate that CPA differentially modulated the stress-protective effects of behavioral control depending on the type of affective behavior examined.
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http://dx.doi.org/10.1016/j.bbr.2018.10.013DOI Listing
February 2019

Mycobacterium vaccae immunization protects aged rats from surgery-elicited neuroinflammation and cognitive dysfunction.

Neurobiol Aging 2018 11 24;71:105-114. Epub 2018 Jul 24.

Department of Psychology and Neuroscience, University of Colorado Boulder, Boulder, CO, USA.

Aging is a major risk factor for developing postoperative cognitive dysfunction. Neuroinflammatory processes, which can play a causal role in the etiology of postoperative cognitive dysfunction, are potentiated or primed as a function of aging. Here we explored whether exposure to a microorganism with immunoregulatory and anti-inflammatory properties, Mycobacterium vaccae NCTC 11659 (M. vaccae), could ameliorate age-associated neuroinflammatory priming. Aged (24 months) and adult (3 months) male F344XBN rats were immunized with heat-killed M. vaccae (3 injections, once per week) before undergoing a laparotomy or anesthesia control procedure. Aged, but not young rats, showed postoperative learning/memory deficits in a fear-conditioning paradigm. Importantly, M. vaccae immunization protected aged rats from these surgery-induced cognitive impairments. M. vaccae immunization also shifted the aged proinflammatory hippocampal microenvironment toward an anti-inflammatory phenotype. Furthermore, M. vaccae immunization reduced age-related hyperinflammatory responses in isolated hippocampal microglia. Overall, our novel data suggest that M. vaccae can induce an anti-inflammatory milieu in the aged brain and thus mitigate the neuroinflammatory and cognitive impairments induced by surgery.
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http://dx.doi.org/10.1016/j.neurobiolaging.2018.07.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6162105PMC
November 2018

Could Probiotics Be Used to Mitigate Neuroinflammation?

ACS Chem Neurosci 2019 01 15;10(1):13-15. Epub 2018 Aug 15.

Center for Neuroscience , University of Colorado Boulder , Boulder , Colorado 80309 , United States.

Neuroinflammatory processes promote a constellation of neurochemical and hormonal changes resulting in profound effects on motivational states (anhedonia), mood (depression and anxiety disorders), and cognitive function (decrements in learning and memory). The enhancement of immunoregulatory processes, such as increasing anti-inflammatory cytokines in the CNS, is a highly effective strategy for curtailing neuroinflammation. Here, we explore recent evidence that probiotic treatment approaches might be an effective strategy to induce long-lasting immunoregulation in the CNS and thus mitigate the neural and behavioral effects of neuroinflammation.
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http://dx.doi.org/10.1021/acschemneuro.8b00386DOI Listing
January 2019

Immunization with Mycobacterium vaccae induces an anti-inflammatory milieu in the CNS: Attenuation of stress-induced microglial priming, alarmins and anxiety-like behavior.

Brain Behav Immun 2018 10 26;73:352-363. Epub 2018 May 26.

Center for Neuroscience, University of Colorado Boulder, Boulder, CO 80309, USA; Department of Integrative Physiology, University of Colorado Boulder, Boulder, CO 80309, USA; Department of Physical Medicine & Rehabilitation, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA; Center for Neuroscience, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA; Rocky Mountain Mental Illness Research Education and Clinical Center (MIRECC), Denver Veterans Affairs Medical Center (VAMC), Denver, CO 80220, USA; Military and Veteran Microbiome Consortium for Research and Education (MVM-CoRE), Denver, CO 80220, USA.

Exposure to stressors induces anxiety- and depressive-like behaviors, which are mediated, in part, by neuroinflammatory processes. Recent findings demonstrate that treatment with the immunoregulatory and anti-inflammatory bacterium, Mycobacterium vaccae (M. vaccae), attenuates stress-induced exaggeration of peripheral inflammation and stress-induced anxiety-like behavioral responses. However, the effects of M. vaccae on neuroimmune processes have largely been unexplored. In the present study, we examined the effect of M. vaccae NCTC11659 on neuroimmune regulation, stress-induced neuroinflammatory processes and anxiety-like behavior. Adult male rats were immunized 3× with a heat-killed preparation of M. vaccae (0.1 mg, s.c.) or vehicle. M. vaccae induced an anti-inflammatory immunophenotype in hippocampus (increased interleukin (Il)4, Cd200r1, and Mrc1 mRNA expression) and increased IL4 protein 8 d after the last immunization. Central administration of recombinant IL4 recapitulated the effects of M. vaccae on Cd200r1 and Mrc1 mRNA expression. M. vaccae reduced basal levels of genes (Nlrp3 and Nfkbia) involved in microglial priming; thus, we explored the effects of M. vaccae on stress-induced hippocampal microglial priming and HMGB1, which mediates priming. We found that M. vaccae blocked stress-induced decreases in Cd200r1, increases in the alarmin HMGB1, and priming of the microglial response to immune challenge. Furthermore, M. vaccae prevented stress-induced increases in anxiety-like behavior. The present findings suggest that M. vaccae enhances immunomodulation in the CNS and mitigates the neuroinflammatory and behavioral effects of stress, which may underpin its capacity to impart a stress resilient phenotype.
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http://dx.doi.org/10.1016/j.bbi.2018.05.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6129419PMC
October 2018

Post-stroke Intranasal (+)-Naloxone Delivery Reduces Microglial Activation and Improves Behavioral Recovery from Ischemic Injury.

eNeuro 2018 Mar-Apr;5(2). Epub 2018 Apr 18.

Institute of Biotechnology, HiLIFE Unit, University of Helsinki, 00014, Finland.

Ischemic stroke is the leading cause of disability, and effective therapeutic strategies are needed to promote complete recovery. Neuroinflammation plays a significant role in stroke pathophysiology, and there is limited understanding of how it affects recovery. The aim of this study was to characterize the spatiotemporal expression profile of microglial activation and whether dampening microglial/macrophage activation post-stroke facilitates the recovery. For dampening microglial/macrophage activation, we chose intranasal administration of naloxone, a drug that is already in clinical use for opioid overdose and is known to decrease microglia/macrophage activation. We characterized the temporal progression of microglia/macrophage activation following cortical ischemic injury in rat and found the peak activation in cortex 7 d post-stroke. Unexpectedly, there was a chronic expression of phagocytic cells in the thalamus associated with neuronal loss. (+)-Naloxone, an enantiomer that reduces microglial activation without antagonizing opioid receptors, was administered intranasally starting 1 d post-stroke and continuing for 7 d. (+)-Naloxone treatment decreased microglia/macrophage activation in the striatum and thalamus, promoted behavioral recovery during the 14-d monitoring period, and reduced neuronal death in the lesioned cortex and ipsilateral thalamus. Our results are the first to show that post-stroke intranasal (+)-naloxone administration promotes short-term functional recovery and reduces microglia/macrophage activation. Therefore, (+)-naloxone is a promising drug for the treatment of ischemic stroke, and further studies should be conducted.
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http://dx.doi.org/10.1523/ENEURO.0395-17.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5952324PMC
January 2019

MicroRNA-124 and microRNA-146a both attenuate persistent neuropathic pain induced by morphine in male rats.

Brain Res 2018 08 1;1692:9-11. Epub 2018 May 1.

Department of Psychology and Neuroscience, and The Center for Neuroscience, University of Colorado, Boulder, CO, USA.

We have recently reported that a short course of morphine, starting 10 days 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 microglial reactivity and Toll-like receptor 4 signaling. Given that microRNAs (miRNAs) such as miR-124 and miR-146a possess the ability to modulate such signaling, we directly compared their function in this model. We found that both miRNAs reversed established allodynia in our model of morphine-induced persistent sensitization. The efficacy of miR-124 and miR-146a were comparable, and in both cases allodynia returned within hours to days of miRNA dosing conclusion. Our findings demonstrate that miRNAs targeting Toll-like receptor signaling are effective in reversing neuropathic pain, which underscores the clinical potential of these non-coding RNAs.
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http://dx.doi.org/10.1016/j.brainres.2018.04.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5976546PMC
August 2018

A novel platform for in vivo detection of cytokine release within discrete brain regions.

Brain Behav Immun 2018 07 17;71:18-22. Epub 2018 Apr 17.

ARC Centre of Excellence in Nanoscale Biophotonics (CNBP), Macquarie University, Sydney, Australia; Graduate School of Biomedical Engineering, The University of New South Wales, Sydney, Australia. Electronic address:

Mounting evidence indicates that cytokines secreted by innate immune cells in the brain play a central role in regulating neural circuits that subserve mood, cognition, and sickness responses. A major impediment to the study of neuroimmune signaling in healthy and disease states is the absence of tools for in vivo detection of cytokine release in the brain. Here we describe the design and application of a cytokine detection device capable of serial monitoring of local cytokine release in discrete brain regions. The immunocapture device consisted of a modified optical fiber labeled with a capture antibody specific for the pro-inflammatory cytokine interleukin-1 beta (IL-1β). Using a sandwich immunoassay method, in vitro data demonstrate that the sensing interface of the modified optical fiber has a linear detection range of 3.9 pg mL-500 pg mL and spatial resolution on the order of 200-450 μm. Finally, we show that the immunocapture device can be introduced into a perforated guide cannula for repeated analyte measurements in vivo. An increase in fluorescence detection of spatially localized intrahippocampal IL-1β release was observed following a peripheral lipopolysaccharide challenge in Sprague-Dawley rats. This novel immunosensing technology represents an opportunity for unlocking the function of neuroimmune signaling.
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http://dx.doi.org/10.1016/j.bbi.2018.04.011DOI Listing
July 2018

Repeated Morphine Prolongs Postoperative Pain in Male Rats.

Anesth Analg 2019 01;128(1):161-167

From the Department of Psychology and Neuroscience, and The Center for Neuroscience, University of Colorado, Boulder, Colorado.

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-1β, 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.
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http://dx.doi.org/10.1213/ANE.0000000000003345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7054903PMC
January 2019

DREADDed microglia in pain: Implications for spinal inflammatory signaling in male rats.

Exp Neurol 2018 06 9;304:125-131. Epub 2018 Mar 9.

Department of Psychology and Neuroscience, The Center for Neuroscience, University of Colorado, Boulder, CO, USA.

The absence of selective pharmacological tools is a major barrier to the in vivo study of microglia. To address this issue, we developed a G- and G-coupled Designer Receptor Exclusively Activated by a Designer Drug (DREADD) to enable selective stimulation or inhibition of microglia, respectively. DREADDs under a CD68 (microglia/macrophage) promoter were intrathecally transfected via an AAV9 vector. Naïve male rats intrathecally transfected with G (stimulatory) DREADDs exhibited significant allodynia following intrathecal administration of the DREADD-selective ligand clozapine-N-oxide (CNO), which was abolished by intrathecal interleukin-1 receptor antagonist. Chronic constriction injury-induced allodynia was attenuated by intrathecal CNO in male rats intrathecally transfected with G (inhibitory) DREADDs. To explore mechanisms, BV2 cells were stably transfected with G or G DREADDs in vitro. CNO treatment induced pro-inflammatory mediator production per se from cells expressing G-DREADDs, and inhibited lipopolysaccharide- and CCL2-induced inflammatory signaling from cells expressing G-DREADDs. These studies are the first to manipulate microglia function using DREADDs, which allow the role of glia in pain to be conclusively demonstrated, unconfounded by neuronal off-target effects that exist for all other drugs that also inhibit glia. Hence, these studies are the first to conclusively demonstrate that in vivo stimulation of resident spinal microglia in intact spinal cord is a) sufficient for allodynia, and b) necessary for allodynia induced by peripheral nerve injury. DREADDs are a unique tool to selectively explore the physiological and pathological role of microglia in vivo.
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http://dx.doi.org/10.1016/j.expneurol.2018.03.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5916033PMC
June 2018

Neuroinflammatory priming to stress is differentially regulated in male and female rats.

Brain Behav Immun 2018 05 7;70:257-267. Epub 2018 Mar 7.

Department of Psychology and Neuroscience, University of Colorado, Boulder, CO 80309, USA.

Exposure to stressors can enhance neuroinflammatory responses, and both stress and neuroinflammation are predisposing factors in the development of psychiatric disorders. Females suffer disproportionately more from several psychiatric disorders, yet stress-induced changes in neuroinflammation have primarily been studied in males. Here we tested whether exposure to inescapable tail shock sensitizes or 'primes' neuroinflammatory responses in male and female rats. At 24 h post-stress, male and female rats exposed to a peripheral immune challenge enhanced neuroinflammatory responses and exacerbated anxiety- and depressive-like behaviors. These changes are likely glucocorticoid dependent, as administering exogenous CORT, caused a similar primed inflammatory response in the hippocampus of male and female rats. Further, stress disinhibited anti-inflammatory signaling mechanisms (such as CD200R) in the hippocampus of male and female rats. In males, microglia are considered the likely cellular source mediating neuroinflammatory priming; stress increased cytokine expression in ex vivo male microglia. Conversely, microglia isolated from stressed or CORT treated females did not exhibit elevated cytokine responses. Microglia isolated from both stressed male and female rats reduced phagocytic activity; however, suggesting that microglia from both sexes experience stress-induced functional impairments. Finally, an immune challenge following either stress or CORT in females, but not males, increased peripheral inflammation (serum IL-1β). These novel data suggest that although males and females both enhance stress-induced neuroinflammatory and behavioral responses to an immune challenge, this priming may occur through distinct, sex-specific mechanisms.
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http://dx.doi.org/10.1016/j.bbi.2018.03.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5953809PMC
May 2018

Dissecting the Innate Immune Recognition of Opioid Inactive Isomer (+)-Naltrexone Derived Toll-like Receptor 4 (TLR4) Antagonists.

J Chem Inf Model 2018 04 11;58(4):816-825. Epub 2018 Apr 11.

Laboratory of Chemical Biology, Changchun Institute of Applied Chemistry , Chinese Academy of Sciences , Changchun , Jilin , 130022 , China.

The opioid inactive isomer (+)-naltrexone is one of the rare Toll-like receptor 4 (TLR4) antagonists with good blood-brain barrier (BBB) permeability, which is a lead with promising potential for treating neuropathic pain and drug addiction. (+)-Naltrexone targets the lipopolysaccharides (LPS) binding pocket of myeloid differentiation protein 2 (MD-2) and blocks innate immune TLR4 signaling. However, the details of the molecular interactions of (+)-naltrexone and its derivatives with MD-2 are not fully understood, which hinders the ligand-based drug discovery. Herein, in silico and in vitro assays were performed to elucidate the innate immune recognition of the opioid inactive (+)-isomers. The results showed that the conserved LPS binding pocket of MD-2 accommodated these opioid inactive (+)-isomers. The calculated binding free energies of (+)-naltrexone and its derivatives in complex with MD-2 correlated well with their experimental binding affinities and TLR4 antagonistic activities. Hydrophobic residues in the MD-2 cavity interacted directly with these (+)-naltrexone based TLR4 antagonists and principally participated in ligand binding. Increasing the hydrophobicity of substituted group at N-17 improved its TLR4 antagonistic activity, while charged groups disfavored the binding with MD-2. Molecular dynamics (MD) simulations showed the binding of (+)-naltrexone or its derivatives to MD-2 stabilized the "collapsed" conformation of MD-2, consequently blocking the binding and signaling of TLR4. Thermodynamics and dynamic analysis showed the topology of substituted group at N-17 of (+)-naltrexone affected the binding with MD-2 and TLR4 antagonistic activity. This study provides a molecular insight into the innate immune recognition of opioid inactive (+)-isomers, which would be of great help for the development of next-generation of (+)-opioid based TLR4 antagonists.
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http://dx.doi.org/10.1021/acs.jcim.7b00717DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6700752PMC
April 2018