Complex alterations in inflammatory pain and analgesic sensitivity in young and ageing female rats: involvement of ASIC3 and Nav1.8 in primary sensory neurons.

OBJECTIVE AND DESIGN: Our aim was to determine an age-dependent role of Nav1.8 and ASIC3 in dorsal root ganglion (DRG) neurons in a rat pre-clinical model of long-term inflammatory pain. METHODS: We compared 6 and 24 months-old female Wistar rats after cutaneous inflammation. We used behavioral pain assessments over time, qPCR, quantitative immunohistochemistry, selective pharmacological manipulation, ELISA and in vitro treatment with cytokines. RESULTS: Older rats exhibited delayed recovery from mechanical allodynia and earlier onset of spontaneous pain than younger rats after inflammation. Moreover, the expression patterns of Nav1.8 and ASIC3 were time and age-dependent and ASIC3 levels remained elevated only in aged rats. In vivo, selective blockade of Nav1.8 with A803467 or of ASIC3 with APETx2 alleviated mechanical and cold allodynia and also spontaneous pain in both age groups with slightly different potency. Furthermore, in vitro IL-1ß up-regulated Nav1.8 expression in DRG neurons cultured from young but not old rats. We also found that while TNF-α up-regulated ASIC3 expression in both age groups, IL-6 and IL-1ß had this effect only on young and aged neurons, respectively. CONCLUSION: Inflammation-associated mechanical allodynia and spontaneous pain in the elderly can be more effectively treated by inhibiting ASIC3 than Nav1.8.

Planificación anticipada de los cuidados y del uso de recursos hospitalarios en pacientes con COVID-19 en un programa de atención integrada para adultos mayores frágiles en residencias / Advance care planning and use of health resources among COVID-19 nursing home patients in an integrated health care program for frail elders

Objetivos: Evaluar la magnitud y la trascendencia de un proceso de estratificación y planificación anticipada de los cuidados y el uso de recursos hospitalarios en pacientes con COVID-19 en un programa de atención integrada para pacientes adultos mayores frágiles en residencias. Material y métodos: Estudio de cohorte prospectiva de pacientes >64 años con COVID-19 en un programa en residencias, desde marzo a septiembre de 2020. Se evaluó la identificación de pacientes con necesidad de abordaje paliativo (NECPAL positivo), la realización de la planificación anticipada del cuidado y su registro visible en la historia clínica electrónica. Se evaluó la utilización de cuidados intensivos. Resultados: Trescientos setenta y cuatro pacientes tuvieron COVID-19: el 88% eran mujeres, mediana de edad 88 años. El 79% tenían necesidades de abordaje paliativo, de los cuales el 68% tenía registrada la planificación anticipada del cuidado (p<0,001) en su historia clínica electrónica. Solamente el 1% de los pacientes con necesidad de abordaje paliativo con criterios de severidad utilizó cuidados intensivos. La mortalidad global fue del 25%. De los fallecidos, el 74% tenían criterios de severidad (p<0,001) y el 90% tenían necesidad de abordaje paliativo (p<0,001). Conclusiones: Realizar un proceso de atención basado en la estratificación e identificación de pacientes con necesidad de abordaje paliativo y la planificación anticipada del cuidado, con registro en un lugar central y visible de la historia clínica electrónica, podría mejorar la calidad y la seguridad de la atención, adecuando los recursos a las necesidades del paciente en todo momento y especialmente en situaciones de emergencia sanitaria. (AU)

Objectives: To estimate the magnitude and importance of a process of stratification and advance care planning and the use of health resources, among patients in an integrated health care program for frail elderls in nursing homes,that were referred to the hospital with COVID-19. Material and methods: Prospective cohort study of patients >64 years old with COVID-19 infection, in a health care program in nursing homes (from 3/15/2020 to 9/15/2020). The identification of patients with palliative needs, the performing and visible registration in electronic health records of the advance care planning and the use of intensive care were assessed. Results: We included 374 COVID-19 patients. 88% were women, the median age was 88 years old. The 79% were patients with palliative needs, of which 68% had the advance care planning (P<.001) registered in the electronic health record. Only 1% of patients with palliative needs and severity criteria were admitted to the intensive care unit. Overall mortality was 25%. Of those who died, 74% had severity criteria (P<.001) and 90% had palliative needs (P<.001). Conclusions: Carrying out a care process based on identification of patients with palliative needs and advance care planning and a central and visible registration of advance care planning in health records, could improve the quality and safety of care and optimize the use of intensive care health resources at all times and especially in public health emergencies. (AU)

[Advance care planning and use of health resources among COVID-19 nursing home patients in an integrated health care program for frail elders]. / Planificación anticipada de los cuidados y del uso de recursos hospitalarios en pacientes con COVID-19 en un programa de atención integrada para adultos mayores frágiles en residencias.

OBJECTIVES: To estimate the magnitude and importance of a process of stratification and advance care planning and the use of health resources, among patients in an integrated health care program for frail elderls in nursing homes,that were referred to the hospital with COVID-19. MATERIAL AND METHODS: Prospective cohort study of patients >64 years old with COVID-19 infection, in a health care program in nursing homes (from 3/15/2020 to 9/15/2020). The identification of patients with palliative needs, the performing and visible registration in electronic health records of the advance care planning and the use of intensive care were assessed. RESULTS: We included 374 COVID-19 patients. 88% were women, the median age was 88 years old. The 79% were patients with palliative needs, of which 68% had the advance care planning (P<.001) registered in the electronic health record. Only 1% of patients with palliative needs and severity criteria were admitted to the intensive care unit. Overall mortality was 25%. Of those who died, 74% had severity criteria (P<.001) and 90% had palliative needs (P<.001). CONCLUSIONS: Carrying out a care process based on identification of patients with palliative needs and advance care planning and a central and visible registration of advance care planning in health records, could improve the quality and safety of care and optimize the use of intensive care health resources at all times and especially in public health emergencies.

Age-dependent and modality-specific changes in the phenotypic markers Nav1.8, ASIC3, P2X3 and TRPM8 in male rat primary sensory neurons during healthy aging.

The effects during healthy aging of the tetrodotoxin-resistant voltage-gated sodium channel 1.8 (Nav1.8), the acid-sensing ion channel-3 (ASIC3), the purinergic-receptor 2X3 (P2X3) and transient receptor potential of melastatin-8 (TRPM8) on responses to non-noxious stimuli are poorly understood. These effects will influence the transferability to geriatric subjects of findings obtained using young animals. To evaluate the involvement of these functional markers in mechanical and cold sensitivity to non-noxious stimuli and their underlying mechanisms, we used a combination of immunohistochemistry and quantitation of immunostaining in sub-populations of neurons of the dorsal root ganglia (DRG), behavioral tests, pharmacological interventions and Western-blot in healthy male Wistar rats from 3 to 24 months of age. We found significantly decreased sensitivity to mechanical and cold stimuli in geriatric rats. These behavioural alterations occurred simultaneously with differing changes in the expression of Nav1.8, ASIC3, P2X3 and TRPM8 in the DRG at different ages. Using pharmacological blockade in vivo we demonstrated the involvement of ASIC3 and P2X3 in normal mechanosensation and of Nav1.8 and ASIC3 in cold sensitivity. Geriatric rats also exhibited reductions in the number of A-like large neurons and in the proportion of peptidergic to non-peptidergic neurons. The changes in normal sensory physiology in geriatric rats we report here strongly support the inclusion of aged rodents as an important group in the design of pre-clinical studies evaluating pain treatments.

Glial-derived neurotrophic factor regulates the expression of TREK2 in rat primary sensory neurons leading to attenuation of axotomy-induced neuropathic pain.

TREK2 is a member of the 2-pore domain family of K+ channels (K2P) preferentially expressed by unmyelinated, slow-conducting and non-peptidergic isolectin B4-binding (IB4+) primary sensory neurons of the dorsal root ganglia (DRG). IB4+ neurons depend on the glial-derived neurotrophic factor (GDNF) family of ligands (GFL's) to maintain their phenotype. In our previous work, we demonstrated that 7 days after spinal nerve axotomy (SNA) of the L5 DRG, TREK2 moves away from the cell membrane resulting in a more depolarised resting membrane potential (Em). Given that axotomy deprives DRG neurons from peripherally-derived GFL's, we hypothesized that they might control the expression of TREK2. Using a combination of immunohistochemistry, immunocytochemistry, western blotting, in vivo pharmacological manipulation and behavioral tests we examined the ability of the GFL's (GDNF, neurturin and artemin) and their selective receptors (GFRα1, GFRα2 and GFRα3) to regulate the expression and function of TREK2 in the DRG. We found that TREK2 correlated strongly with the three receptors normally and ipsilaterally for all GFR's after SNA. GDNF, but not NGF, neurturin or artemin up-regulated the expression of TREK2 in cultured DRG neurons. In vivo continuous, subcutaneous administration of GDNF restored the subcellular distribution of TREK2 ipsilaterally and reversed mechanical and cold allodynia 7 days after SNA. This is the first demonstration that GDNF controls the expression of a K2P channel in nociceptors. As TREK2 controls the Em of C-nociceptors affecting their excitability, our finding has therapeutic potential in the treatment of chronic pain.

Human adipose-derived mesenchymal stem cell-conditioned medium ameliorates polyneuropathy and foot ulceration in diabetic BKS db/db mice.

BACKGROUND: Diabetic polyneuropathy (DPN) is the most common and early developing complication of diabetes mellitus, and the key contributor for foot ulcers development, with no specific therapies available. Different studies have shown that mesenchymal stem cell (MSC) administration is able to ameliorate DPN; however, limited cell survival and safety reasons hinder its transfer from bench to bedside. MSCs secrete a broad range of antioxidant, neuroprotective, angiogenic, and immunomodulatory factors (known as conditioned medium), which are all decreased in the peripheral nerves of diabetic patients. Furthermore, the abundance of these factors can be boosted in vitro by incubating MSCs with a preconditioning stimulus, enhancing their therapeutic efficacy. We hypothesize that systemic administration of conditioned medium derived from preconditioned MSCs could reverse DPN and prevent foot ulcer formation in a mouse model of type II diabetes mellitus. METHODS: Diabetic BKS db/db mice were treated with systemic administration of conditioned medium derived from preconditioned human MSCs; conditioned medium derived from non-preconditioned MSCs or vehicle after behavioral signs of DPN was already present. Conditioned medium or vehicle administration was repeated every 2 weeks for a total of four administrations, and several functional and structural parameters characteristic of DPN were evaluated. Finally, a wound was made in the dorsal surface of both feet, and the kinetics of wound closure, re-epithelialization, angiogenesis, and cell proliferation were evaluated. RESULTS: Our molecular, electrophysiological, and histological analysis demonstrated that the administration of conditioned medium derived from non-preconditioned MSCs or from preconditioned MSCs to diabetic BKS db/db mice strongly reverts the established DPN, improving thermal and mechanical sensitivity, restoring intraepidermal nerve fiber density, reducing neuron and Schwann cell apoptosis, improving angiogenesis, and reducing chronic inflammation of peripheral nerves. Furthermore, DPN reversion induced by conditioned medium administration enhances the wound healing process by accelerating wound closure, improving the re-epithelialization of the injured skin and increasing blood vessels in the wound bed in a skin injury model that mimics a foot ulcer. CONCLUSIONS: Studies conducted indicate that MSC-conditioned medium administration could be a novel cell-free therapeutic approach to reverse the initial stages of DPN, avoiding the risk of lower limb amputation triggered by foot ulcer formation and accelerating the wound healing process in case it occurs.

Cutaneous inflammation differentially regulates the expression and function of Angiotensin-II types 1 and 2 receptors in rat primary sensory neurons.

Neuropathic and inflammatory pain results from cellular and molecular changes in dorsal root ganglion (DRG) neurons. The type-2 receptor for Angiotensin-II (AT2R) has been involved in this type of pain. However, the underlying mechanisms are poorly understood, including the role of the type-1 receptor for Angiotensin-II (AT1R). Here, we used a combination of immunohistochemistry and immunocytochemistry, RT-PCR and in vitro and in vivo pharmacological manipulation to examine how cutaneous inflammation affected the expression of AT1R and AT2R in subpopulations of rat DRG neurons and studied their impact on inflammation-induced neuritogenesis. We demonstrated that AT2R-neurons express C- or A-neuron markers, primarily IB4, trkA, and substance-P. AT1R expression was highest in small neurons and co-localized significantly with AT2R. In vitro, an inflammatory soup caused significant elevation of AT2R mRNA, whereas AT1R mRNA levels remained unchanged. In vivo, we found a unique pattern of change in the expression of AT1R and AT2R after cutaneous inflammation. AT2R increased in small neurons at 1 day and in medium size neurons at 4 days. Interestingly, cutaneous inflammation increased AT1R levels only in large neurons at 4 days. We found that in vitro and in vivo AT1R and AT2R acted co-operatively to regulate DRG neurite outgrowth. In vivo, AT2R inhibition impacted more on non-peptidergic C-neurons neuritogenesis, whereas AT1R blockade affected primarily peptidergic nerve terminals. Thus, cutaneous-induced inflammation regulated AT1R and AT2R expression and function in different DRG neuronal subpopulations at different times. These findings must be considered when targeting AT1R and AT2R to treat chronic inflammatory pain. Cover Image for this issue: doi: 10.1111/jnc.14737.

72-hour hospital readmission of older people after hospital discharge with home care services.

In this retrospective cohort study in Argentina, risk factors for hospital readmission of older adults, within 72 hours after hospital discharge with home care services, were analyzed. Fifty-three percent of unplanned emergency room visits within 72 hours after hospital discharge resulted in hospital readmissions, 65% of which were potentially avoidable. By multivariate logistic regression, low functionality, pressure ulcers, and age over 83 years predicted hospital readmission among emergency room attendees. It is important to identify and analyze barriers in current home care services and the high-risk population of hospital readmission to improve the strategies to avoid adverse outcomes.

Changes in the expression of the potassium channels TASK1, TASK3 and TRESK in a rat model of oral squamous cell carcinoma and their relation to malignancy.

OBJECTIVES: Potassium channels have been proposed to promote cancer cell proliferation and metastases. Thus, we investigated the expression pattern of three 2-pore domain potassium channels (K2Ps) TASK1, TASK3 and TRESK in advanced oral squamous cell carcinoma (OSCC), the commonest oral malignancy. DESIGN: We used 4-nitroquinoline-1-oxide (4-NQO) to induce high grade OSCC in male adult rats. We then used immunohistochemistry and Western blotting to study the distribution and expression pattern of TASK1, TASK3 and TRESK in normal versus cancerous tissue. We also examined the expression of ß-tubulin III (ß-tub3), a marker associated with resistance to taxane-based chemotherapy and poor patient prognosis, and its correlation with the K2Ps. Finally, we studied the expression of TASK1, TASK3 and TRESK in human samples of SCC of oral origin. RESULTS: We found that TASK3 was significantly up-regulated whereas TASK1 and TRESK were both significantly down-regulated in advanced, poorly differentiated OSCC. Both, rat and human SCC showed a significant increase in the expression of ß-tub3. Interestingly, the expression of the latter correlated positively and significantly with TASK3 and TRESK but not TASK1 in rat OSCC. Our initial results showed a similar pattern of up and down regulation and correlation with ß-tub3 for these three K2Ps in human SCC. CONCLUSIONS: The changes in expression and the co-localization with a marker of resistance to taxanes like ß-tub3 turn TASK1, TASK3 and TRESK into potentially new prognostic tools and possibly new therapeutic targets for OSCC.

MipLAAO, a new L-amino acid oxidase from the redtail coral snake Micrurus mipartitus.

L-amino acid oxidases (LAAOs) are ubiquitous enzymes in nature. Bioactivities described for these enzymes include apoptosis induction, edema formation, induction or inhibition of platelet aggregation, as well as antiviral, antiparasite, and antibacterial actions. With over 80 species, Micrurus snakes are the representatives of the Elapidae family in the New World. Although LAAOs in Micrurus venoms have been predicted by venom gland transcriptomic studies and detected in proteomic studies, no enzymes of this kind have been previously purified from their venoms. Earlier proteomic studies revealed that the venom of M. mipartitus from Colombia contains ∼4% of LAAO. This enzyme, here named MipLAAO, was isolated and biochemically and functionally characterized. The enzyme is found in monomeric form, with an isotope-averaged molecular mass of 59,100.6 Da, as determined by MALDI-TOF. Its oxidase activity shows substrate preference for hydrophobic amino acids, being optimal at pH 8.0. By nucleotide sequencing of venom gland cDNA of mRNA transcripts obtained from a single snake, six isoforms of MipLAAO with minor variations among them were retrieved. The deduced sequences present a mature chain of 483 amino acids, with a predicted pI of 8.9, and theoretical masses between 55,010.9 and 55,121.0 Da. The difference with experimentally observed mass is likely due to glycosylation, in agreement with the finding of three putative N-glycosylation sites in its amino acid sequence. A phylogenetic analysis of MmipLAAO placed this new enzyme within the clade of homologous proteins from elapid snakes, characterized by the conserved Serine at position 223, in contrast to LAAOs from viperids. MmipLAAO showed a potent bactericidal effect on S. aureus (MIC: 2 µg/mL), but not on E. coli. The former activity could be of interest to future studies assessing its potential as antimicrobial agent.

Isolation and Functional Characterization of an Acidic Myotoxic Phospholipase A2 from Colombian Bothrops asper Venom.

Myotoxic phospholipases A2 (PLA2) are responsible for many clinical manifestations in envenomation by Bothrops snakes. A new myotoxic acidic Asp49 PLA2 (BaCol PLA2) was isolated from Colombian Bothrops asper venom using reverse-phase high performance liquid chromatography (RP-HPLC). BaCol PLA2 had a molecular mass of 14,180.69 Da (by mass spectrometry) and an isoelectric point of 4.4. The complete amino acid sequence was obtained by cDNA cloning (GenBank accession No. MF319968) and revealed a mature product of 124 amino acids with Asp at position 49. BaCol PLA2 showed structural homology with other acidic PLA2 isolated from Bothrops venoms, including a non-myotoxic PLA2 from Costa Rican B. asper. In vitro studies showed cell membrane damage without exposure of phosphatidylserine, an early apoptosis hallmark. BaCol PLA2 had high indirect hemolytic activity and moderate anticoagulant action. In mice, BaCol PLA2 caused marked edema and myotoxicity, the latter seen as an increase in plasma creatine kinase and histological damage to gastrocnemius muscle fibers that included vacuolization and hyalinization necrosis of the sarcoplasm.

Cutaneous inflammation regulates THIK1 expression in small C-like nociceptor dorsal root ganglion neurons.

Tandem pore-domain Halothane Inhibited K+ channel (THIK1) is a two-pore-domain potassium channel (K2P) present in dorsal root ganglia (DRG). We previously demonstrated that THIK1 mRNA levels in the DRG dropped ipsilaterally 1day after CFA-induced cutaneous inflammation (CFA1). In this study we aimed to identify the currently unknown DRG subpopulations expressing THIK1, and to investigate the relationship between the channel and both inflammatory and spontaneous pain in normal rats. Using a combination of immunohistochemistry, western blotting and behavioural tests, we found that all small neurons and large groups of medium and large DRG neurons express THIK1. Myelinated and unmyelinated fibers, nerve endings in the skin and lamina I and II of the spinal cord also express the channel. THIK1 staining co-localizes with IB4-binding and trkA suggesting that the channel is expressed by nociceptors. At CFA1, both cytoplasmic and edge (membrane-associated) THIK1 staining were significantly reduced only in small neurons ipsilaterally compared to normal. At 4days after inflammation (CFA4), edge THIK1 staining levels in small neurons decreased bilaterally compared to normal. Medium and large size DRG neurons showed no change in THIK1 expression either at CFA1 or CFA4. Ipsilateral (but not contralateral) mean %intensities of THIK1 in small neurons at CFA1 correlated strongly negatively with spontaneous foot lifting (SFL) duration (a marker of spontaneous pain). Thus, nociceptors express THIK1 that can be regulated by cutaneous inflammation. Finally, in vivo siRNA knockdown of THIK1 resulted in longer SFL duration than siRNA scramble-treated rats. Taken together our evidence suggests a potential involvement for THIK1 in pain processing following inflammation.

Preconditioning of adipose tissue-derived mesenchymal stem cells with deferoxamine increases the production of pro-angiogenic, neuroprotective and anti-inflammatory factors: Potential application in the treatment of diabetic neuropathy.

Diabetic neuropathy (DN) is one of the most frequent and troublesome complications of diabetes mellitus. Evidence from diabetic animal models and diabetic patients suggests that reduced availability of neuroprotective and pro-angiogenic factors in the nerves in combination with a chronic pro-inflammatory microenvironment and high level of oxidative stress, contribute to the pathogenesis of DN. Mesenchymal stem cells (MSCs) are of great interest as therapeutic agents for regenerative purposes, since they can secrete a broad range of cytoprotective and anti-inflammatory factors. Therefore, the use of the MSC secretome may represent a promising approach for DN treatment. Recent data indicate that the paracrine potential of MSCs could be boosted by preconditioning these cells with an environmental or pharmacological stimulus, enhancing their therapeutic efficacy. In the present study, we observed that the preconditioning of human adipose tissue-derived MSCs (AD-MSCs) with 150µM or 400µM of the iron chelator deferoxamine (DFX) for 48 hours, increased the abundance of the hypoxia inducible factor 1 alpha (HIF-1α) in a concentration dependent manner, without affecting MSC morphology and survival. Activation of HIF-1α led to the up-regulation of the mRNA levels of pro-angiogenic factors like vascular endothelial growth factor alpha and angiopoietin 1. Furthermore this preconditioning increased the expression of potent neuroprotective factors, including nerve growth factor, glial cell-derived neurotrophic factor and neurotrophin-3, and cytokines with anti-inflammatory activity like IL4 and IL5. Additionally, we observed that these molecules, which could also be used as therapeutics, were also increased in the secretome of MSCs preconditioned with DFX compared to the secretome obtained from non-preconditioned cells. Moreover, DFX preconditioning significantly increased the total antioxidant capacity of the MSC secretome and they showed neuroprotective effects when evaluated in an in vitro model of DN. Altogether, our findings suggest that DFX preconditioning of AD-MSCs improves their therapeutic potential and should be considered as a potential strategy for the generation of new alternatives for DN treatment.

Nociceptor-like rat dorsal root ganglion neurons express the angiotensin-II AT2 receptor throughout development.

AT2 receptor (AT2R) plays a functional role in foetal development. Its expression declines in most tissues soon after birth but stays high in sensory areas of the adult nervous system. In the dorsal root ganglia (DRG) the expression pattern of AT2R during development and the identity of the subpopulation expressing it remain unknown. Using a combination of semi-quantitative PCR, western blotting and immunohistochemistry we examined the expression of AT2R at mRNA and protein levels in rat DRGs from embryonic day 15 (E15) until postnatal day 30 (PN30). We found that both AT2R mRNA and protein levels exhibited only minor (statistically non-significant) fluctuations from E15 to PN30. Detailed quantitative analysis of ABC/DAB AT2R staining showed a) that the receptor was present in most neurons at E15 and E18 and b) that postnatally it was predominantly expressed by small DRG neurons. Given that small neurons are putative C-nociceptors and the proposed role of AT2R in neuropathic pain, we next examined whether these AT2R-positive neurons co-localized with Ret and trkA embryonically and with IB4-binding postnatally. Most AT2R-positive neurons expressed trkA embryonically and bound IB4 postnatally. We found strong positive statistically highly significant correlations between AT2R cytoplasmic%intensities and trkA at E15/E18 and with Ret only at E18. Cytoplasmic AT2R also strongly and positively correlated with IB4-binding at PN3, 15 and 30. Our demonstration that a subpopulation of C-nociceptor-like neurons expresses AT2R during development supports a role for this receptor in neuropathic pain.

TREK2 expressed selectively in IB4-binding C-fiber nociceptors hyperpolarizes their membrane potentials and limits spontaneous pain.

Ongoing/spontaneous pain behavior is associated with ongoing/spontaneous firing (SF) in adult DRG C-fiber nociceptors (Djouhri et al., 2006). Causes of this SF are not understood. We show here that conducting (sometimes called uninjured) C-nociceptors in neuropathic pain models with more hyperpolarized resting membrane potentials (Ems) have lower SF rates. Understanding the control of their Ems may therefore be important for limiting pathological pain. We report that TREK2, a leak K(+) channel, is selectively expressed in IB4 binding rat C-nociceptors. These IB4(+) C-neurons are ∼10 mV more hyperpolarized than IB4(-) C-neurons in vivo (Fang et al., 2006). TREK2 knockdown by siRNA in these neurons in culture depolarized them by ∼10 mV, suggesting that TREK2 is responsible for this ∼10 mV difference. In vivo, more hyperpolarized C-nociceptor Ems were associated with higher cytoplasmic edge-TREK2 expression (edge-TREK2). Edge-TREK2 decreased in C-neurons 7 d after axotomy, and their Ems depolarized by ∼10 mV. This again supports a contribution of TREK2 to their Ems. These relationships between (1) Em and TREK2, (2) SF rate and Em, and (3) spontaneous pain behavior and C-nociceptor SF rate suggested that TREK2 knockdown might increase spontaneous pain. After CFA-induced inflammation, spontaneous foot lifting (a measure of spontaneous pain) was (1) greater in rats with naturally lower TREK2 in ipsilateral small DRG neurons and (2) increased by siRNA-induced TREK2 knockdown in vivo. We conclude that TREK2 hyperpolarizes IB4 binding C-nociceptors and limits pathological spontaneous pain. Similar TREK2 distributions in small DRG neurons of several species suggest that these role(s) of TREK2 may be widespread.

HCN1 and HCN2 in Rat DRG neurons: levels in nociceptors and non-nociceptors, NT3-dependence and influence of CFA-induced skin inflammation on HCN2 and NT3 expression.

I(h), which influences neuronal excitability, has recently been measured in vivo in sensory neuron subtypes in dorsal root ganglia (DRGs). However, expression levels of HCN (hyperpolarization-activated cyclic nucleotide-gated) channel proteins that underlie I(h) were unknown. We therefore examined immunostaining of the most abundant isoforms in DRGs, HCN1 and HCN2 in these neuron subtypes. This immunostaining was cytoplasmic and membrane-associated (ring). Ring-staining for both isoforms was in neurofilament-rich A-fiber neurons, but not in small neurofilament-poor C-fiber neurons, although some C-neurons showed cytoplasmic HCN2 staining. We recorded intracellularly from DRG neurons in vivo, determined their sensory properties (nociceptive or low-threshold-mechanoreceptive, LTM) and conduction velocities (CVs). We then injected fluorescent dye enabling subsequent immunostaining. For each dye-injected neuron, ring- and cytoplasmic-immunointensities were determined relative to maximum ring-immunointensity. Both HCN1- and HCN2-ring-immunointensities were positively correlated with CV in both nociceptors and LTMs; they were high in Aß-nociceptors and Aα/ß-LTMs. High HCN1 and HCN2 levels in Aα/ß-neurons may, via I(h), influence normal non-painful (e.g. touch and proprioceptive) sensations as well as nociception and pain. HCN2-, not HCN1-, ring-intensities were higher in muscle spindle afferents (MSAs) than in all other neurons. The previously reported very high I(h) in MSAs may relate to their very high HCN2. In normal C-nociceptors, low HCN1 and HCN2 were consistent with their low/undetectable I(h.) In some C-LTMs HCN2-intensities were higher than in C-nociceptors. Together, HCN1 and HCN2 expressions reflect previously reported I(h) magnitudes and properties in neuronal subgroups, suggesting these isoforms underlie I(h) in DRG neurons. Expression of both isoforms was NT3-dependent in cultured DRG neurons. HCN2-immunostaining in small neurons increased 1 day after cutaneous inflammation (CFA-induced) and recovered by 4 days. This could contribute to acute inflammatory pain. HCN2-immunostaining in large neurons decreased 4 days after CFA, when NT3 was decreased in the DRG. Thus HCN2-expression control differs between large and small neurons.

Leak K⁺ channel mRNAs in dorsal root ganglia: relation to inflammation and spontaneous pain behaviour.

Two pore domain potassium (K2P) channels (KCNKx.x) cause K⁺ leak currents and are major contributors to resting membrane potential. Their roles in dorsal root ganglion (DRG) neurons normally, and in pathological pain models, are poorly understood. Therefore, we examined mRNA levels for 10 K2P channels in L4 and L5 rat DRGs normally, and 1 day and 4 days after unilateral cutaneous inflammation, induced by intradermal complete Freund's adjuvant (CFA) injections. Spontaneous foot lifting (SFL) duration (spontaneous pain behaviour) was measured in 1 day and 4 day rats <1h before DRG harvest. mRNA levels for KCNK channels and Kv1.4 relative to GAPDH (n=4-6 rats/group) were determined with real-time RT-PCR. This study is the first to demonstrate expression of THIK1, THIK2 and TWIK2 mRNA in DRGs. Abundance in normal DRGs was, in descending order: Kv1.4>TRESK(KCNK18)>TRAAK(KCNK4)>TREK2(KCNK10)=TWIK2(KCNK6)>TREK1 (KCNK2)=THIK2(KCNK12)>TASK1(KCNK3)>TASK2(KCNK5)>THIK1(KCNK13)=TASK3(KCNK9). During inflammation, the main differences from normal in DRG mRNA levels were bilateral, suggesting systemic regulation, although some channels showed evidence of ipsilateral modulation. By 1 day, bilateral K2P mRNA levels had decreased (THIK1) or increased (TASK1, THIK2) but by 4 days they were consistently decreased (TASK2, TASK3) or tended to decrease (excluding TRAAK). The decreased TASK2 mRNA was mirrored by decreased protein (TASK2-immunoreactivity) at 4 days. Ipsilateral mRNA levels at 4days compared with 1 day were lower (TRESK, TASK1, TASK3, TASK2 and THIK2) or higher (THIK1). Ipsilateral SFL duration during inflammation was positively correlated with ipsilateral TASK1 and TASK3 mRNAs, and contralateral TASK1, TRESK and TASK2 mRNAs. Thus changes in K2P mRNA levels occurred during inflammation and for 4 K2P channels were associated with spontaneous pain behaviour (SFL). K2P channels and their altered expression are therefore associated with inflammation-induced pain.

La decisión de entrar a un tratamiento de adicciones: motivación propia e influencia de terceros / The decision to enter an addiction treatment: self motivation and others' influence

En este artículo se presentan los resultados de un estudio acerca de la importancia que tienen los terceros significativos en la decisión de entrar a un tratamiento de adicciones tomada por un sujeto que ha desarrollado una adicción. Interesa especialmente entender cómo esta participación de terceros significativos influye en la percepción de libertad de elegir del sujeto con el problema. A partir de esto se discute la noción de motivación a tratarse.

This article presents the results of a research about the importance of significant others over the decision to enter an addiction treatment, taken by an individual who has developed an addiction. Especially, it becomes interesting to understand how the participation of these significant others influence the perception of frees choice of the individual who carries the addiction. From this point, the notion of motivation is discussed.

Bacterial lipopolysaccharide regulates nociceptin expression in sensory neurons.

Nociceptin/orphanin FQ (N/OFQ) is an opioid-related peptide that is markedly up-regulated in sensory neurons in vivo following peripheral inflammation and plays a key role in pain physiology. To identify substances that up-regulate N/OFQ expression in sensory neurons, we carried out an in vitro screen using purified adult mouse dorsal root ganglion (DRG) neurons and identified the potent proinflammatory agent bacterial lipopolysaccharide (LPS) as a very effective inducer of N/OFQ. The robust response of these neurons to LPS enabled us to identify the components of a putative neuronal LPS receptor complex. In contrast to the immune system, where the functional LPS receptor complex is composed of CD-14 together with either MD-2 and TLR4 on myeloid cells or the homologous receptors MD-1 and RP105 on mature B cells, DRG neurons express the unusual combination of CD-14, TLR4, and MD-1. Blocking antibodies against TLR4 and MD-1 prevented induction of N/OFQ by LPS, and, in immunoprecipitation experiments, MD-1 coprecipitated with TLR4. Our findings suggest that LPS regulates N/OFN expression in sensory neurons via a novel combination of LPS receptor components and demonstrate for the first time a direct action of a key initiator of innate immune responses on neurons.