Carbon monoxide reduces neuropathic pain and spinal microglial activation by inhibiting nitric oxide synthesis in mice

Background: Carbon monoxide (CO) synthesized by heme oxygenase 1 (HO-1) exerts antinociceptive effects during inflammation but its role during neuropathic pain remains unknown. Our objective is to investigate the exact contribution of CO derived from HO-1 in the modulation of neuropathic pain and the mechanisms implicated. Methodology/Principal Findings: We evaluated the antiallodynic and antihyperalgesic effects of CO following sciatic nerve injury in wild type (WT) or inducible nitric oxide synthase knockout (NOS2-KO) mice using two carbon monoxide-releasing molecules (CORM-2 and CORM-3) and an HO-1 inducer (cobalt protoporphyrin IX, CoPP) daily administered from days 10 to 20 after injury. The effects of CORM-2 and CoPP on the expression of HO-1, heme oxygenase 2 (HO-2), neuronal nitric oxide synthase (NOS1) and NOS2 as well as a microglial marker (CD11b/c) were also assessed at day 20 after surgery in WT and NOS2-KO mice. In WT mice, the main neuropathic pain symptoms induced by nerve injury were significantly reduced in a time-dependent manner by treatment with CO-RMs or CoPP. Both CORM-2 and CoPP treatments increased HO-1 expression in WT mice, but only CoPP stimulated HO-1 in NOS2-KO animals. The increased expression of HO-2 induced by nerve injury in WT, but not in NOS2-KO mice, remains unaltered by CORM-2 or CoPP treatments. In contrast, the over-expression of CD11b/c, NOS1 and NOS2 induced by nerve injury in WT, but not in NOS2-KO mice, were significantly decreased by both CORM-2 and CoPP treatments. These data indicate that CO alleviates neuropathic pain through the reduction of spinal microglial activation and NOS1/NOS2 over-expression. Conclusions/Significance: This study reports that an interaction between the CO and nitric oxide (NO) systems is taking place following sciatic nerve injury and reveals that increasing the exogenous (CO-RMs) or endogenous (CoPP) production of CO may represent a novel strategy for the treatment of neuropathic pain

The Antinociceptive Effects of JWH-015 in Chronic Inflammatory Pain Are Produced by Nitric Oxide-cGMP-PKG-KATP Pathway Activation Mediated by Opioids

Background: Cannabinoid 2 receptor (CB2R) agonists attenuate inflammatory pain but the precise mechanism implicated in these effects is not completely elucidated. We investigated if the peripheral nitric oxide-cGMP-protein kinase G (PKG)-ATP-sensitive K+ (KATP) channels signaling pathway triggered by the neuronal nitric oxide synthase (NOS1) and modulated by opioids, participates in the local antinociceptive effects produced by a CB2R agonist (JWH-015) during chronic inflammatory pain. Methodology/Principal Findings: In wild type (WT) and NOS1 knockout (NOS1-KO) mice, at 10 days after the subplantar administration of complete Freund's adjuvant (CFA), we evaluated the antiallodynic (von Frey filaments) and antihyperalgesic (plantar test) effects produced by the subplantar administration of JWH-015 and the reversion of their effects by the local co-administration with CB2R (AM630), peripheral opioid receptor (naloxone methiodide, NX-ME) or CB1R (AM251) antagonists. Expression of CB2R and NOS1 as well as the antinociceptive effects produced by a high dose of JWH-015 combined with different doses of selective L-guanylate cyclase (ODQ) or PKG (Rp-8-pCPT-cGMPs) inhibitors or a KATP channel blocker (glibenclamide), were also assessed. Results show that the local administration of JWH-015 dose-dependently inhibited the mechanical and thermal hypersensitivity induced by CFA which effects were completely reversed by the local co-administration of AM630 or NX-ME, but not AM251. Inflammatory pain increased the paw expression of CB2R and the dorsal root ganglia transcription of NOS1. Moreover, the antinociceptive effects of JWH-015 were absent in NOS1-KO mice and diminished by their co-administration with ODQ, Rp-8-pCPT-cGMPs or glibenclamide. Conclusions/Significance: These data indicate that the peripheral antinociceptive effects of JWH-015 during chronic inflammatory pain are mainly produced by the local activation of the nitric oxide-cGMP-PKG-KATP signaling pathway, triggered by NOS1 and mediated by endogenous opioids. These findings suggest that the activation of this pathway might be an interesting therapeutic target for the treatment of chronic inflammatory pain with cannabinoids

Challenges and future prospects on 3D in-vitro modeling of the neuromuscular circuit

Movement of skeletal-muscle fibers is generated by the coordinated action of several cells taking part within the locomotion circuit (motoneurons, sensory-neurons, Schwann cells, astrocytes, microglia, and muscle-cells). Failure s in any part of this circuit could impede or hinder coordinated muscle movement and cause a neu romuscular disease (NMD) or determine its severity. Studying fragments of the circuit cannot provide a comprehensive and complete view of the pathological process. We trace the historic developments of studies focused on in-vitro modeling of the spinal-locomotion circuit and how bioengineered innovative technologies show advantages for an accurate mimicking of hysiological conditions of spinal-locomotion circuit. New developments on compartmentalized microfluidic culture systems (cμFCS), the use of human induced pluripotent stem cells (hiPSCs) and 3D cell-cultures are analyzed. We finally address limitations of current study models and three main challenges on neuromuscular studies: (i) mimic the whole spinal-locomotion circuit including all cell-types involved and the evaluation of independent and interdependent roles of each one; (ii) mimic the neurodegenerative response of mature neurons in-vitro as it occurs in-vivo ; and (iii) develop, tune, implement, and combine cμFCS, hiPSC, and 3D-culture technologies to ultimately create patient-specific complete, translational, and reliable NMD in-vitro model. Overcoming these challenges would significantly facilitate understanding the events taking place in NMDs and accelerate the process of finding new therapies

Neuromuscular activity induces paracrine signaling and triggers axonal regrowth after injury in microfluidic lab‐on‐chip devices

Peripheral nerve injuries, including motor neuron axonal injury, often lead to functional impairments. Current therapies are mostly limited to surgical intervention after lesion, yet these interventions have limited success in restoring functionality. Current activity‐based therapies after axonal injuries are based on trial‐error approaches in which the details of the underlying cellular and molecular processes are largely unknown. Here we show the effects of the modulation of both neuronal and muscular activity with optogenetic approaches to assess the regenerative capacity of cultured motor neuron (MN) after lesion in a compartmentalized microfluidic‐assisted axotomy device. With increased neuronal activity, we observed an increase in the ratio of regrowing axons after injury in our peripheral‐injury model. Moreover, increasing muscular activity induces the liberation of leukemia inhibitory factor and glial cell line‐derived neurotrophic factor in a paracrine fashion that in turn triggers axonal regrowth of lesioned MN in our 3D hydrogel cultures. The relevance of our findings as well as the novel approaches used in this study could be useful not only after axotomy events but also in diseases affecting MN survival

Lack of astrocytic glycogen alters synaptic plasticity but not seizure susceptibility

Brain glycogen is mainly stored in astrocytes. However, recent studies both in vitro and in vivo indicate that glycogen also plays important roles in neurons. By conditional deletion of glycogen synthase (GYS1), we previously developed a mouse model entirely devoid of glycogen in the central nervous system (GYS1Nestin-KO). These mice displayed altered electrophysiological properties in the hippocampus and increased susceptibility to kainate-induced seizures. To understand which of these functions are related to astrocytic glycogen, in the present study, we generated a mouse model in which glycogen synthesis is eliminated specifically in astrocytes (GYS1Gfap-KO). Electrophysiological recordings of awake behaving mice revealed alterations in input/output curves and impaired long-term potentiation, similar, but to a lesser extent, to those obtained with GYS1Nestin-KO mice. Surprisingly, GYS1Gfap-KO mice displayed no change in susceptibility to kainate-induced seizures as determined by fEPSP recordings and video monitoring. These results confirm the importance of astrocytic glycogen in synaptic plasticity

Astrocytic glycogen accumulation drives the pathophysiology of neurodegeneration in Lafora disease

The hallmark of Lafora disease, a fatal neurodegenerative disorder, is the accumulation of intracellular glycogen aggregates, called Lafora bodies. Until recently, it was widely believed that brain Lafora bodies were present exclusively in neurons and thus that Lafora disease pathology derived from their accumulation in this cell population. However, recent evidence indicates that Lafora bodies are also present in astrocytes. To define the role of astrocytic Lafora bodies in Lafora disease pathology, we deleted glycogen synthase specifically from astrocytes in a mouse model of the disease (malinKO). Strikingly, blocking glycogen synthesis in astrocytes-thus impeding Lafora bodies accumulation in this cell type-prevented the increase in neurodegeneration markers, autophagy impairment, and metabolic changes characteristic of the malinKO model. Conversely, mice that overaccumulate glycogen in astrocytes showed an increase in these markers. These results unveil the deleterious consequences of the deregulation of glycogen metabolism in astrocytes and change the perspective that Lafora disease is caused solely by alterations in neuron

Involvement of Cellular Prion Protein in a-Synuclein Transport in Neurons

The cellular prion protein, encoded by the gene Prnp, has been reported to be a receptor of ß-amyloid. Their interaction is mandatory for neurotoxic effects of ß-amyloid oligomers. In this study, we aimed to explore whether the cellular prion protein participates in the spreading of a-synuclein. Results demonstrate that Prnp expression is not mandatory for a-synuclein spreading. However, although the pathological spreading of a-synuclein can take place in the absence of Prnp, a-synuclein expanded faster in PrPC-overexpressing mice. In addition, a-synuclein binds strongly on PrPC-expressing cells, suggesting a role in modulating the effect of a-synuclein fibrils

Involvement of Mechanical Cues in the Migration of Cajal-Retzius Cells in the Marginal Zone During Neocortical Development

Emerging evidence points to coordinated action of chemical and mechanical cues during brain development. At early stages of neocortical development, angiogenic factors and chemokines such as CXCL12, ephrins, and semaphorins assume crucial roles in orchestrating neuronal migration and axon elongation of postmitotic neurons. Here we explore the intrinsic mechanical properties of the developing marginal zone of the pallium in the migratory pathways and brain distribution of the pioneer Cajal-Retzius cells. These neurons are generated in several proliferative regions in the developing brain (e.g., the cortical hem and the pallial subpallial boundary) and migrate tangentially in the preplate/marginal zone covering the upper portion of the developing cortex. These cells play crucial roles in correct neocortical layer formation by secreting several molecules such as Reelin. Our results indicate that the motogenic properties of Cajal-Retzius cells and their perinatal distribution in the marginal zone are modulated by both chemical and mechanical factors, by the specific mechanical properties of Cajal-Retzius cells, and by the differential stiffness of the migratory routes. Indeed, cells originating in the cortical hem display higher migratory capacities than those generated in the pallial subpallial boundary which may be involved in the differential distribution of these cells in the dorsal-lateral axis in the developing marginal zone

iPS cell cultures from a Gerstmann-Straussler-Scheinker patient with the Y218N PRNP mutation recapitulate tau pathology

Gerstmann-Straussler-Scheinker (GSS) syndrome is a fatal autosomal dominant neurodegenerative prionopathy clinically characterized by ataxia, spastic paraparesis, extrapyramidal signs and dementia. In some GSS familiar cases carrying point mutations in the PRNP gene, patients also showed comorbid tauopathy leading to mixed pathologies. In this study we developed an induced pluripotent stem (iPS) cell model derived from fibroblasts of a GSS patient harboring the Y218N PRNP mutation, as well as an age-matched healthy control. This particular PRNP mutation is unique with very few described cases. One of the cases presented neurofibrillary degeneration with relevant Tau hyperphosphorylation. Y218N iPS-derived cultures showed relevant astrogliosis, increased phospho-Tau, altered microtubule-associated transport and cell death. However, they failed to generate proteinase K-resistant prion. In this study we set out to test, for the first time, whether iPS cell-derived neurons could be used to investigate the appearance of disease-related phenotypes (i.e, tauopathy) identified in the GSS patient

Role of gaseous neurotransmitters in the effects and expression of opioid and cannabinoid receptors during neuropathic pain

El dolor neuropàtic es causat per una lesió o malaltia del sistema nerviós somatosensiorial, i es caracteritza per la presencia d'alodínia e hiperalgèsia. Actualment el seu tractament es basa en la teràpia amb opioids, però són necessàries altes dosis per alleujar el símptomes, les quals van acompanyades de nombrosos efectes secundaris. Aleshores es necessari investigar noves dianes així com nous mecanismes per tal de millorar els tractaments amb opioids a fi d’evitar l’administració de dosis altes i els efectes no desitjats. En aquest estudi hem investigat el paper que juguen dos dels principals neurotransmissors gasosos, l'òxid nítric (NO) i el monòxid de carboni (CO) en el desenvolupament del dolor neuropàtic, així com dels seus efectes en les teràpies mediades per receptors opioids mu (MOR) i delta (DOR), i cannabinoids tipus 2 (CB2). D'aquesta manera, utilitzant la constricció crònica del nervi ciàtic com a model de dolor neuropàtic hem demostrat que: I) la via de senyalització perifèrica de l'NO-sGC-PKG, activada per NOS1 i NOS2, juga un paper essencial en el desenvolupament i l’expressió dels principals símptomes del dolor neuropàtic, II) els efectes antinociceptius perifèrics, però no sistèmics, dels agonistes MOR durant el dolor neuropàtic, es produeixen per activació de la via de senyalització de HO1/NOS1/NOS2-sGC-PKG-K+ATP, i que l'NO, sintetitzat per NOS1 i NOS2, esta implicat en la disminuïda expressió del MOR, III) els efectes antinociceptius perifèrics dels agonistes dels receptors DOR i CB2 durant el dolor neuropàtic es poden incrementar a través de l' inhibició de la via de senyalització de NOS1/NOS2-sGC-PKG. A més, l'NO sintetitzat per NOS1, es un dels responsables de la disminuïda i augmentada expressió perifèrica dels receptors DOR i CB2 respectivament durant el dolor neuropàtic, IV) la inhibició de la via de senyalització de NO-sGC-PKG-JNK evita l'aparició de tolerància als efectes antialodínics perifèrics produïts per la morfina durant el dolor neuropàtic, V) el CO, sintetitzat per HO1, inhibeix la simptomatologia del dolor neuropàtic mitjançant l'atenuació de la sobrepressió de NOS1 i NOS2 així com de l'activació microglial produïdes per la lesió nerviosa, i finalment VI) el tractament amb CO, administrat exògenament o sintetitzat endògenament per HO1, potencia els efectes antinociceptius perifèrics dels agonistes MOR, a través d'una sobrepressió perifèrica dels seus receptors i inhibint l'activació microglial. En resum, tant NO com CO tenen un paper essencial durant l’expressió del dolor neuropàtic, i tots dos son capaços de modular els efectes i l'expressió dels receptors MOR, DOR i CB2 durant el dolor neuropàtic, no obstant, mentre que els agonistes MOR produeix els seus efectes antinociceptius perifèrics a través de l'activació de la via de senyalització de HO1/NOS-sGC-PKG-K+ATP, els agonistes DOR i CB2 no utilitzen aquesta via per produir els seus efectes. Finalment, malgrat en aquest estudi es mostren diferents possibilitats d’augmentar l’acció antinociceptiva local produïda per opioids i cannabinoids i evitar el desenvolupament de tolerància durant el dolor neuropàtic, la investigació dels mecanismes d’acció d’aquests fàrmacs es indispensable per millorar la seva acció terapèutica durant el dolor neuropàtic.Neuropathic pain is caused by a lesion or disease of the somatosensory nervous system, and is characterized by the presence of allodynia and hyperalgesia. Nowadays, its treatment is based upon opioids, but high doses are necessary to alleviate symptoms and they have several undesirable side effects. Therefore, it is important to investigate new targets and mechanisms to improve the current opioid treatments in order to reduce dosage and avoid side effects. In this study, we investigated the role played by the two main gaseous neurotransmitters, nitric oxide (NO) and carbon monoxide (CO) in the development of neuropathic pain, as well as their effects in the mu (MOR) and delta (DOR) opioid, and cannabinoid 2 (CB2) receptors mediated therapies. Indeed, by using the chronic constriction of the sciatic nerve, as a mouse model of neuropathic pain, we demonstrated that: I) the peripheral NO-sGC-PKG signaling pathway, triggered by NOS1 and NOS2, plays a key role in the development and expression of the main symptoms of neuropathic pain, II) the peripheral, but not systemic, antinociceptive effects of MOR agonists during neuropathic pain are produced through the activation of the HO1/NOS1/NOS2-sGC-PKG-K+ATP signaling pathway, and the NO, synthesized by NOS1 and NOS2, is implicated in the peripheral down regulation of MOR, III) the peripheral antinociceptive effects of DOR and CB2 receptor agonists during neuropathic pain can be increased by the inactivation of the NOS1/NOS2-sGC-PKG signaling pathway. Moreover the NO, synthesized by NOS1, is implicated in the peripheral down- and up-regulation of DOR and CB2 receptor during neuropathic pain, IV) the inhibition of the NO-sGC-PKG-JNK signaling pathway avoids the development of tolerance to the local antiallodynic effects produced by morphine during neuropathic pain, V) CO, synthesized by HO1, inhibits neuropathic pain by the attenuation of NOS1/NOS2 overexpression and microglial activation induced by nerve injury, and VI) the treatment with CO, exogenously deliberated or endogenously synthesized by HO1, enhances the peripheral antinociceptive effects of MOR agonists by up-regulating the peripheral expression of MOR and inhibiting the microglial activation. In summary, both NO and CO systems have an essential role in the expression of neuropathic pain and both modulate the effects and expression of MOR, DOR and CB2 receptors after sciatic nerve injury, but while MOR elicits its peripheral antinociceptive effects through the activation of the HO1/NOS-sGC-PKG-K+ATP signaling pathway, DOR or CB2 receptor do not use this pathway to produce their effects. Finally, although this study shows different strategies to increase the local antinociceptive effects produced by opioids and cannabinoids and avoid the development of tolerance during neuropathic pain, the investigation of new mechanisms of action of these drugs is essential to improve their therapeutic actions in neuropathic pain