Investigating the Glial Contribution to Persistent Neuropathic Pain

Persistent neuropathic pain is the coordinated activation and sensitization of glial and neuronal elements both peripherally and centrally. Here, we have investigated the role of glial fibrillary acidic protein (GFAP)-positive astrocytes in the central nervous system (CNS) and non-myelinating Schwann cells in the peripheral nervous system (PNS) and their individual contributions to persistent neuropathic pain. We used three-dimensional reconstruction of ultrastructural data to establish the morphological relationship between astrocyte processes and incoming C and A-delta fiber synapses with second-order pain neurons. We found that not only do astrocytes contact 100% of the C and A-delta fiber glomerular synapses, but they also provide a high degree of ensheathment of each glomerulus. This encapsulation of the glomerular synapses puts astrocytes in a position to potentially modulate neuronal activity and synapse structure. Next, we used two glial-specific transgenic mouse models to interfere vesicular gliotransmitter release. However, neither blocking IP3-dependent Ca2+ release or SNARE-dependent vesicle release had any effect on nociception. We then used two glial-specific transgenic mouse models that interfere with the NFkB-COX2 inflammatory pathway and observed a robust yet temporary alleviation of pain behavior from one to five weeks post-nerve injury. This finding indicates that the NFkB-COX2 signaling pathway in GFAP-positive glia is critical to the maintenance of a specific phase of persistent neuropathic pain. GFAP-positive glia include peripheral non-myelinating Schwann cells, which ensheath unmyelinated nociceptive neurons, as well as central astrocytes, which ensheath neuronal synapses throughout the CNS. We used the tet-Off transgenic mouse expression system in a novel manner to tease apart the peripheral vs central roles of GFAP-positive cells. The administration of oxytetracycline, a blood-brain barrier impermeable analog of doxycycline, was capable of turning off transgene expression in the PNS without affecting transgene expression in the CNS. The blockade of peripheral transgene expression reversed the alleviation of pain behavior post-nerve injury in mice with suppressed NFkB activity. Thus, the suppression of NFkB in astrocytes is insufficient to relieve mechanical sensitization following nerve injury. The implicates non-myelinating Schwann cells in an important role in the maintenance of a specific phase of persistent neuropathic pain from one to five weeks post-injury.Doctor of Philosoph

Restraint-induced Corticosterone Secretion and Hypothalamic CRH mRNA Expression are Augmented During Acute Withdrawal from Chronic Cocaine Administration

Stress responses during cocaine withdrawal likely contribute to drug relapse and may be intensified as a consequence of prior cocaine use. The present study examined changes in stressor-induced activation of the hypothalamic–pituitary–adrenal (HPA) axis during acute withdrawal from chronic cocaine administration. Adult male Sprague–Dawley rats received daily administration of cocaine (30 mg/kg, i.p.) or saline for 14 days. Twenty-four hours after the last injection, rats in each group were sacrificed under stress-free conditions or following 30 min of immobilization. Plasma corticosterone (CORT) was measured in trunk-blood using radioimmunoassay, corticotropin-releasing hormone (CRH) mRNA levels in the paraventricularnucleus (PVN) of the hypothalamus were measured using in situ hybridization and glucocorticoid receptor (GR) protein expression in the pituitary gland and dissected brain regions was measured using Western blot analysis. Basal CRH mRNA in the PVN was unaltered as a result of prior cocaine administration. However, a significant increase in CRH mRNA was observed 90 min following the termination of restraint in cocaine withdrawn, but not saline-treated, rats. Basal CORT was also unaffected by prior cocaine administration, but the CORT response measured immediately after restraint was significantly augmented in cocaine-withdrawn rats. Differences in GR protein expression in number of regions implicated in negative feedback regulation of HPA function, including the hypothalamus, were not observed. These findings indicate that the HPA response to stressors is intensified during early withdrawal from cocaine administration and may be independent of changes in GR-mediated negative feedback

Nuclear Factor κB-COX2 Pathway Activation in Non-myelinating Schwann Cells Is Necessary for the Maintenance of Neuropathic Pain in vivo

Chronic neuropathic pain leads to long-term changes in the sensitivity of both peripheral and central nociceptive neurons. Glial fibrillary acidic protein (GFAP)-positive glial cells are closely associated with the nociceptive neurons including astrocytes in the central nervous system (CNS), satellite glial cells (SGCs) in the sensory ganglia, and non-myelinating Schwann cells (NMSCs) in the peripheral nerves. Central and peripheral GFAP-positive cells are involved in the maintenance of chronic pain through a host of inflammatory cytokines, many of which are under control of the transcription factor nuclear factor κB (NFκB) and the enzyme cyclooxygenase 2 (COX2). To test the hypothesis that inhibiting GFAP-positive glial signaling alleviates chronic pain, we used (1) a conditional knockout (cKO) mouse expressing Cre recombinase under the hGFAP promoter and a floxed COX2 gene to inactivate the COX2 gene specifically in GFAP-positive cells; and (2) a tet-Off tetracycline transactivator system to suppress NFκB activation in GFAP-positive cells. We found that neuropathic pain behavior following spared nerve injury (SNI) significantly decreased in COX2 cKO mice as well as in mice with decreased glial NFκB signaling. Additionally, experiments were performed to determine whether central or peripheral glial NFκB signaling contributes to the maintenance of chronic pain behavior following nerve injury. Oxytetracycline (Oxy), a blood-brain barrier impermeable analog of doxycycline was employed to restrict transgene expression to CNS glia only, leaving peripheral glial signaling intact. Signaling inactivation in central GFAP-positive glia alone failed to exhibit the same analgesic effects as previously observed in animals with both central and peripheral glial signaling inhibition. These data suggest that the NFκB-COX2 signaling pathway in NMSCs is necessary for the maintenance of neuropathic pain in vivo

Measurement of Steroid Concentrations in Brain Tissue: Methodological Considerations

It is well recognized that steroids are synthesized de novo in the brain (neurosteroids). In addition, steroids circulating in the blood enter the brain. Steroids play numerous roles in the brain, such as influencing neural development, adult neuroplasticity, behavior, neuroinflammation, and neurodegenerative diseases such as Alzheimer’s disease. In order to understand the regulation and functions of steroids in the brain, it is important to directly measure steroid concentrations in brain tissue. In this brief review, we discuss methods for the detection and quantification of steroids in the brain. We concisely present the major advantages and disadvantages of different technical approaches at various experimental stages: euthanasia, tissue collection, steroid extraction, steroid separation, and steroid measurement. We discuss, among other topics, the potential effects of anesthesia and saline perfusion prior to tissue collection; microdissection via Palkovits punch; solid phase extraction; chromatographic separation of steroids; and immunoassays and mass spectrometry for steroid quantification, particularly the use of mass spectrometry for “steroid profiling.” Finally, we discuss the interpretation of local steroid concentrations, such as comparing steroid levels in brain tissue with those in the circulation (plasma vs. whole blood samples; total vs. free steroid levels). We also present reference values for a variety of steroids in different brain regions of adult rats. This brief review highlights some of the major methodological considerations at multiple experimental stages and provides a broad framework for designing studies that examine local steroid levels in the brain as well as other steroidogenic tissues, such as thymus, breast, and prostate

What Is the Role of Astrocyte Calcium in Neurophysiology?

Astrocytes comprise approximately half of the volume of the adult mammalian brain and are the primary neuronal structural and trophic supportive elements. Astrocytes are organized into distinct nonoverlapping domains and extend elaborate and dense fine processes that interact intimately with synapses and cerebrovasculature. The recognition in the mid 1990s that astrocytes undergo elevations in intracellular calcium concentration following activation of G protein-coupled receptors by synaptically released neurotransmitters demonstrated not only that astrocytes display a form of excitability but also that astrocytes may be active participants in brain information processing. The roles that astrocytic calcium elevations play in neurophysiology and especially in modulation of neuronal activity have been intensely researched in recent years. This review will summarize the current understanding of the function of astrocytic calcium signaling in neurophysiological processes and discuss areas where the role of astrocytes remains controversial and will therefore benefit from further study

A community-based physical activity intervention to prevent mobility-related disability for retired older people (REtirement in ACTion (REACT)): Study protocol for a randomised controlled trial

© 2018 The Author(s). Background: The REtirement in ACTion (REACT) study is a multi-centre, pragmatic, two-arm, parallel-group randomised controlled trial (RCT) with an internal pilot phase. It aims to test the effectiveness and cost-effectiveness of a community, group-based physical activity intervention for reducing, or reversing, the progression of functional limitations in older people who are at high risk of mobility-related disability. Methods/design: A sample of 768 sedentary, community-dwelling, older people aged 65 years and over with functional limitations, but who are still ambulatory (scores between 4 and 9 out of 12 in the Short Physical Performance Battery test (SPPB)) will be randomised to receive either the REACT intervention, delivered over a period of 12 months by trained facilitators, or a minimal control intervention. The REACT study incorporates comprehensive process and economic evaluation and a nested sub-study which will test the hypothesis that the REACT intervention will slow the rate of brain atrophy and of decline in cognitive function assessed using magnetic resonance imaging (MRI). Outcome data will be collected at baseline, 6, 12 and 24 months for the main study, with MRI sub-study data collected at baseline, 6 and 12 months. The primary outcome analysis (SPPB score at 24 months) will be undertaken blinded to group allocation. Primary comparative analyses will be on an intention-to-treat (ITT) basis with due emphasis placed on confidence intervals. Discussion: REACT represents the first large-scale, pragmatic, community-based trial in the UK to target the non-disabled but high-risk segment of the older population with an intervention to reduce mobility-related disability. A programme that can successfully engage this population in sufficient activity to improve strength, aerobic capacity, coordination and balance would have a major impact on sustaining health and independence. REACT is also the first study of its kind to conduct a full economic and comprehensive process evaluation alongside the RCT. If effective and cost-effective, the REACT intervention has strong potential to be implemented widely in the UK and elsewhere

RR: Microglia and spinal cord synaptic plasticity in persistent pain

Microglia are regarded as macrophages in the central nervous system (CNS) and play an important role in neuroinflammation in the CNS. Microglial activation has been strongly implicated in neurodegeneration in the brain. Increasing evidence also suggests an important role of spinal cord microglia in the genesis of persistent pain, by releasing the proinflammatory cytokines tumor necrosis factor-alpha (TNF ), Interleukine-1beta (IL-1 ), and brain derived neurotrophic factor (BDNF). In this review, we discuss the recent findings illustrating the importance of microglial mediators in regulating synaptic plasticity of the excitatory and inhibitory pain circuits in the spinal cord, leading to enhanced pain states. Insights into microglial-neuronal interactions in the spinal cord dorsal horn will not only further our understanding of neural plasticity but may also lead to novel therapeutics for chronic pain management. Microglia-Synapse Interactions in Healthy CNS Microglia are derived from myeloid precursor cells in the periphery and penetrate the central nervous system (CNS) during embryogenesis Microglial processes constantly and dynamically survey their environment and interact with nearby synapses Several studies demonstrated that microglial processes can engulf synapses and participate to their phagocytic elimination in an experience-dependent manner in the mature healthy brain Nociceptive Pain and Persistent Pain Our bodies play host to a wide variety of sensory information that is detected every moment by the peripheral nervous system. Primary sensory neurons that are responsible for the detection and transduction of painful stimuli (e.g., cold, heat, mechanical, and chemical), which are somatosensory stimuli that cause potential danger to the organism, are called nociceptors Spinal Cord Microglial Activation in the Context of Persistent Pain Under pathological conditions, especially nerve injury conditions, microglia undergo "microgliosis", a complex set of changes that allow the cell to respond rapidly and perform a broad range of functions such as shielding injury sites, phagocytosing cellular debris, and releasing inflammatory signals to initiate and/or propagate the immune response. Traditionally, microgliosis has been determined by a change in morphology from ramified to amoeboid The p38 MAPK pathway can be activated by a host of molecules known to increase pain sensitivity, including the proinflammatory cytokines TNF and IL-1 , CCL2 (also known as monocyte chemoattractant protein 1 (MCP-1)), fractalkine (CX3CL1), inducible nitric oxide synthase (iNOS), and matrix metalloprotease-9 (MMP-9) as well as the ATP receptors P2X4 and P2X7 Dorsal Horn Microglial-Synapse Interactions in the Context of Persistent Pain The development of central sensitization in persistent pain is characterized by increased excitatory synaptic transmission and decreased inhibitory synaptic transmission in the dorsal horn of the spinal cord. In order to modulate pain sensitivity and participate in central sensitization, glia must interact with neural pain circuits via modulation of neurotransmission. Glial mediators can modulate synaptic transmission at very low concentrations. While neurotransmitters such as glutamate, GABA, and glycine produce synaptic effects at M concentrations, glial cytokines, chemokines, and growth factors can affect synaptic activity at nM concentrations TNF . TNF is present both in healthy brain tissue and in disease states. TNF is known to play a role in synaptic plasticity, which has been studied mainly in hippocampal slices. Glial TNF has been shown to enhance synaptic efficacy by increasing the surface expression of GluR1-possessing AMPA receptors via TNFR1-mediated PI3 K activation TNF also acts on the postsynaptic neurons in the spinal cord. In a carrageenan model of inflammation, TNF recruited Ca 2+ permeable AMPA receptors to dorsal horn neurons resulting in increased sEPSC amplitude In spinal cord slices, TNF not only enhances sEPSCs but also suppresses the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) In spinal cord slices from TNFR1 KO mice, TNF was unable to elicit increases in sEPSCs or increases in NMDA currents Long-term potentiation (LTP) in the spinal cord is implicated in pathological pain It is important to note that the effects of TNF vary among regions of the CNS particularly at high pathological concentrations. While constitutive TNF release may be permissive for plastic changes in neurotransmission, the activation of microglia can result in 10-fold higher TNF concentrations and, at these high concentrations (greater than 0.3 nM), TNF may change its mode of action IL-1 . IL-1 is induced in astrocytes, neurons, and microglia in inflammatory and neuropathic pain IL-1 also suppresses inhibitory neurotransmission in the dorsal horn. Application of IL-1 to spinal cord slices inhibits both the frequency and the amplitude of spontaneous postsynaptic currents (sIPSCs) BDNF. Brain derived neurotrophic factor (BDNF) is a secreted protein and part of the family of neurotrophins which act on neurons to promote survival, growth, and differentiation of new neurons and synapses Following nerve injury, microglia upregulate their expression of the ionotropic ATP receptor P2X4 concurrently with the development of allodynia Microglial derived BDNF contributes to pain hypersensitivity through the disinhibition of nociceptive processing in the dorsal horn. BDNF acts on lamina I pain transmission neurons; neurons that carry the output message of the dorsal horn to higher brain centers where pain is perceived. BDNF, through the activation of its receptor TrkB, decreases expression of the potassium-chloride cotransporter 2 (KCC2) resulting in a rise in intracellular chloride In naïve animals, lamina I projection neurons respond to painful but not innocuous stimuli. The suppression of inhibitory drive and thus exaggerated responses of lamina I neurons to normally noxious stimuli explains the development of hyperalgesia, an exaggerated response to normally noxious stimuli, but it does not explain the development of allodynia, the painful response to normally innocuous stimuli. Persistent pain sufferers report three cardinal features of their pain: hyperalgesia, allodynia, and spontaneous pain. In vivo recordings from lamina I projection neurons show that following nerve-injury, these neurons begin to respond to nonnoxious stimuli, increase their response to noxious stimuli, and discharge spontaneously Concluding Remarks Hyperactivity of peripheral nociceptive fibers due to inflammation or injury causes the release of factors such as CCL2, ATP, and fractalkine into the dorsal horn of the spinal cord We examined the mechanisms of three key signaling molecules released by microglial cells in the context of pain: TNF , IL-1 , and BDNF. TNF and IL-1 increase presynaptic release of excitatory neurotransmitter as well as increasing excitatory postsynaptic currents through the recruitment of 6 Neural Plasticity AMPA receptors and enhancing NMDA currents TNF , IL-1 , and BDNF also affect long-term neuronal plasticity in the dorsal horn. Activation of their respective receptors, TNFR1, IL-1, and trkB, on neurons leads to the phosphorylation of ERK which can enter the nucleus and produce phosphorylation of cAMP response element binding protein (CREB

Impact of a one-time interprofessional education event for rehabilitation after stroke for students in the health professions

© 2019 Association of Schools of Allied Health Professions, Wash., DC. AIMS: Faculty in the healthcare professions are engaging their students in a variety of interprofessional education (IPE) experiences. One such experience is a one-time IPE event, performed over 2 consecutive years by 2 different cohorts, involving 342 students in occupational therapy, physical therapy, speech-language pathology, and therapeutic recreation. The aim was to provide students with the opportunity to learn about the impact of a stroke, the rehabilitation process, the rehabilitation team and their own profession, as well as incorporating the Core Competencies for Interprofessional Collaborative Practice. METHODS: The event had 3 separate components: a small group discussion on a pre-assigned case of a patient who had a stroke, a patient panel, and a clinician panel. RESULTS: All answers to the survey questions taken by students who attended the IPE event improved significantly at posttest (p\u3c0.001). The IPE event was well received by the students (89.0% reported that the overall experience was good-excellent). CONCLUSION: This one-time event involving four health professions was successful in exposing the students to IPE

Hemophilia A and B mice, but not VWF -/-mice, display bone defects in congenital development and remodeling after injury: Bone health in FVIII FIX and VWF deficient mice

International audienceWhile joint damage is the primary co-morbidity of hemophilia, osteoporosis and osteopenia are also observed. Coagulation factor VIII deficient (FVIII-/-) mice develop an osteoporotic phenotype in the absence of induced hemarthrosis that is exacerbated two weeks after an induced joint injury. Here we have compared comprehensively the bone health of clotting factor VIII, factor IX, and Von Willebrand Factor knockout (FVIII-/-, FIX-/-, and VWF-/- respectively) mice both in the absence of joint hemorrhage and following induced joint injury. We found FVIII-/- and FIX-/- mice, but not VWF-/- mice, developmentally have an osteoporotic phenotype. Unilateral induced hemarthrosis causes further bone damage in both FVIII-/- and FIX-/- mice, but has little effect on VWF-/- bone health, indicating that the FVIII.VWF complex is not required for normal bone remodeling in vivo. To further investigate the bone healing following hemarthrosis in hemophilia we examined a two week time course using microCT, serum chemistry, and histological analysis. Elevated ratio of osteoprotegerin (OPG)/receptor activator of nuclear factor-kappa B ligand (RANKL), increased osterix+ osteoblastic cells, and decreased smoothness of the cortical bone surface were evident within several days of injury, indicative of acute heterotopic mineralization along the cortical surface. This was closely followed by increased interleukin-6 (IL-6) levels, increased osteoclast numbers, and significant trabecular bone loss. Uncoupled and disorganized bone formation and resorption continued for the duration of the study resulting in significant deterioration of the joint. Further elucidation of the shared mechanisms underlying abnormal bone homeostasis in the absence of FVIII or FIX is needed to guide evidence-based approaches to the screening and treatment of the prevalent bone defects in hemophilia A and B