Measuring pain and nociception: Through the glasses of a computational scientist. Transdisciplinary overview of methods

In a healthy state, pain plays an important role in natural biofeedback loops and helps to detect and prevent potentially harmful stimuli and situations. However, pain can become chronic and as such a pathological condition, losing its informative and adaptive function. Efficient pain treatment remains a largely unmet clinical need. One promising route to improve the characterization of pain, and with that the potential for more effective pain therapies, is the integration of different data modalities through cutting edge computational methods. Using these methods, multiscale, complex, and network models of pain signaling can be created and utilized for the benefit of patients. Such models require collaborative work of experts from different research domains such as medicine, biology, physiology, psychology as well as mathematics and data science. Efficient work of collaborative teams requires developing of a common language and common level of understanding as a prerequisite. One of ways to meet this need is to provide easy to comprehend overviews of certain topics within the pain research domain. Here, we propose such an overview on the topic of pain assessment in humans for computational researchers. Quantifications related to pain are necessary for building computational models. However, as defined by the International Association of the Study of Pain (IASP), pain is a sensory and emotional experience and thus, it cannot be measured and quantified objectively. This results in a need for clear distinctions between nociception, pain and correlates of pain. Therefore, here we review methods to assess pain as a percept and nociception as a biological basis for this percept in humans, with the goal of creating a roadmap of modelling options

Small fibre neuropathy : expanding the clinical pain universe

Small fibre neuropathy (SFN) is a disorder of thinly myelinated A\u3b4 and unmyelinated C fibres. SFN is clinically dominated by neuropathic pain and autonomic complaints, leading to a significant reduction in quality of life. According to international criteria, the diagnosis is established by the assessment of intra-epidermal nerve fibre density and/or quantitative sensory testing. SFN is mainly associated with autoimmune diseases, sodium channel gene variants, diabetes mellitus, and vitamin B12 deficiencies, although in more than one-half of patients no etiology can be identified. Recently, gain-of-function variants in the genes encoding for the Nav 1.7, Nav 1.8 and Nav 1.9 sodium channel subunits have been discovered in SFN patients, enlarging the spectrum of underlying conditions. Sodium channel gene variants associated with SFN can lead to a diversity of phenotypes, including different pain distributions and presence or absence of autonomic symptoms. This suggests that SFN is part of a clinical continuum. New assessments might contribute to a better understanding of the cellular and molecular substrates of SFN and might provide improved diagnostic methods and trial designs in the future. Identification of the underlying mechanisms may inform the development of drugs that more effectively address neuropathic pain and autonomic symptoms of SFN

Painful and Painless Diabetic Neuropathies: What Is the Difference?

Purpose of Review: The prevalence of diabetes mellitus and its chronic complications are increasing to epidemic proportions. This will unfortunately result in massive increases in diabetic distal symmetrical polyneuropathy (DPN) and its troublesome sequelae, including disabling neuropathic pain (painful-DPN), which affects around 25% of patients with diabetes. Why these patients develop neuropathic pain, while others with a similar degree of neuropathy do not, is not clearly understood. This review will look at recent advances that may shed some light on the differences between painful and painless-DPN. RECENT FINDINGS: Gender, clinical pain phenotyping, serum biomarkers, brain imaging, genetics, and skin biopsy findings have been reported to differentiate painful- from painless-DPN. Painful-DPN seems to be associated with female gender and small fiber dysfunction. Moreover, recent brain imaging studies have found neuropathic pain signatures within the central nervous system; however, whether this is the cause or effect of the pain is yet to be determined. Further research is urgently required to develop our understanding of the pathogenesis of pain in DPN in order to develop new and effective mechanistic treatments for painful-DPN

Cellular and molecular mechanisms of inflammatory arthritis and fibromyalgia

In Study I, we examined the impact of the hR100E-NGF mutation on inflammatory pain and bone erosion in both female and male mice. Our findings indicate that the hR100E-NGF mutation did not affect the development of the peripheral sensory nervous system at the lumbar DRG, sciatic nerve, ankle joint, or glabrous skin. Moreover, hR100E-NGF mice displayed sensory thresholds similar to those of the hWT-NGF mice in response to mechanical, heat, or cold stimulation under normal conditions. The hR100E-NGF and hWT-NGF mice developed comparable mechanical and heat sensitivity impairments after the intra-articular injection of complete Freund’s adjuvant. Notably, the hR100E-NGF mice were insensitive to nociceptive stimulation in the deeper tissues assessed by weight bearing and gait analysis. Furthermore, mRNA analysis from the inflamed joint showed a differential sex-dependent gene expression profile between hR100E-NGF female and male mice. Finally, the hR100E-NGF female but not the male mice were protected against the CFA-bone erosion. These data collectively demonstrate that the R100E NGF mutation effectively protects against joint pain-like behaviors in both male and female mice while providing bone protection exclusively to female mice in a monoarthritis model. We propose that manipulating the signaling of NGF and its receptors in a manner similar to the R100E mutation could be a promising approach to treating chronic pain and maintaining bone health, particularly in women. Study II investigated the effects of injecting purified IgG from fibromyalgia (FM) patients and healthy controls (HC) in mice. We found that the injection of FM IgG but not IgG from healthy controls (HC) induces pressure, mechanical, and cold hypersensitivity in mice that were coupled to enhanced nociceptor responsiveness to mechanical and cold stimulation. The FM IgG-injected mice also developed impaired muscular strength and decreased locomotor activity. Moreover, FM IgG bound and stimulated satellite glial cells (SGCs) in vivo and in vitro. No FM or HC IgG accumulation was found in the brain or spinal cord of the injected mice. Our study also demonstrated that FM IgG can bind to satellite glial cells and neurons in the human DRG. In addition, we observed a significant reduction in the intraepidermal nerve fiber density in the mice 14 days after the FM IgG injection. Our results suggest that transferring FM IgG into mice can replicate some peripheral FM symptoms. This study can provide a valuable animal model for studying the peripheral physiology of FM. Our discovery could significantly advance the understanding and treatment of fibromyalgia and other related conditions. However, more research is needed to understand the cellular and molecular mechanisms involved in FM-IgG-mediated changes in mice. Study III aimed to investigate the frequency of anti-satellite glial cell (SGC) antibodies and the antibody association with the disease severity in FM patients. We used serum (Karolinska Institutet, Sweden; n=30/group) and plasma (McGill University, Canada; n=35/group) samples collected from FM patients and HCs. Our results showed a higher binding intensity of the FM IgG to SGC in vitro. Furthermore, the frequency of SGC bound to FM IgG was significantly higher than HC IgG-treated cells. These findings correlated with pain intensity and fibromyalgia impact questionnaire scores (FIQ, questionnaire was only assessed in the Karolinska cohort). Further cluster analysis separated the FM group into severe and mild groups. Additionally, we found that serum from FM patients contains IgG that binds in greater proportion to SGC in the human DRG, measured by higher signal intensity. There were no differences in the binding intensity to neuronal cell bodies or axons between FM and HC serum samples. Finally, the previous results were confirmed using an FM serum sample with high levels of anti-SGC antibodies in 5 more human DRGs. To summarize, our report indicates that levels of anti-human SGC and anti-mouse SGC antibodies are elevated in patients with FM, which are linked to a more severe form of the disease. Patient stratification based on their profile of anti-SGC antibodies might benefit from therapies aiming to decrease circulating IgG or prevent IgG binding. Our results point to the possible involvement of anti-SGC antibodies and SGCs in the severity of FM; however, more in-depth studies are necessary to elucidate the antigen or antigens expressed in the SGC that bind to the circulating anti-SGC antibodies. In Study IV, we aimed to explore the neuroimmune signature of the FM skin. We processed 16 FM and 16 HC sex-matched skin biopsies by immunohistochemistry. Using a pan-neuronal marker, we found lower intraepidermal nerve fiber density (IENFD) in the FM compared with HC skin. Moreover, the length and volume of dermal NF200+ nerve profiles were significantly elevated, but we found no changes in the length of dermal or epidermal Gap43+ nerve profiles in the FM group. Similarly, we found no changes in the total volume of CD31+ blood vessels between FM and HC skin. Our results showed that the density of non-nerve associated S100b+, CD68+, and CD163+ cells was significantly lower in the FM skin. Furthermore, the dermal CD117+FcERI+ mast cells in the dermis of FM patients were significantly increased compared with the HCs. Additionally, we found similar densities of CD207+, CD3+, or Neutrophil elastase+ cells between FM and HC skin biopsies. mRNA analysis of FM skin showed no changes in Cd68, Cd163, Cx3cr1, or FceR1 mRNA levels between FM and HC skin. In summary, this study reveals crucial dermal and epidermal changes in FM skin, particularly regarding nerve fibers and certain immune cell populations. These findings are highly relevant as they provide deeper insights into the complex interactions between the nervous and immune systems in FM. Understanding these changes could be key to developing more effective treatments for FM, focusing on both the neuropathic and immune components of the disease

A role for the hearing gene Cib2 in somatic sensation

Hearing and touch are two of our senses that rely on mechanotransduction to convert mechanical stimulus into electrochemical activity. Several studies have demonstrated that touch and hearing share common genetic determinants; mutations in genes causing deafness are also associated with reduced tactile sensitivity. Furthermore, transcriptome analysis from several studies has revealed that the same genes are expressed in both systems. However, the role of these genes has yet to be determined. In this work, I demonstrated that the calcium and integrin binding protein 2 (CIB2), a key component of mechanotransduction in the auditory system, is also implicated in somatosensory mechanosensation. CIB2 is an auxiliary subunit of the mechanotransduction channel in the auditory system that modulates channel function and determines correct channel positioning. Mutations in Cib2 cause hearing loss in humans and mice, as well as the cessation of mechanotransduction in the auditory system. By applying immunohistochemistry, I demonstrated the expression of CIB2 in a subset of DRG neurons, mainly nociceptors and, to a lesser extent, mechanoreceptors. CIB2 was also present in the terminal endings of sensory neurons that innervate skin end organs. In particular, CIB2 was found in terminal afferents innervating Meissner’s corpuscles and hair follicles, both associated with rapidly adapting mechanoreceptors. The impact of CIB2 on the physiological properties of different types of mechanoreceptors and nociceptors was investigated using an ex-vivo skin nerve preparation from the saphenous and tibial nerve. I showed that genetic ablation of Cib2 had a profound impact on rapidly adapting mechanoreceptors (RAMs). In a velocity and vibration experiment, RAMs lacking Cib2 exhibited dramatically higher frequency sensitivity. Thus, CIB2 influences RAM adaption rate and functions as a brake on their velocity sensitivity. CIB2 exhibits structural similarities with KChIP proteins, which are like CIB2, calcium sensors. Additionally, KChIP proteins have been shown to modulate Kv4 channel surface expression, assembly, and kinetics, leading us to the hypothesis that CIB2 influences RAM adaptation by interacting with and regulating a potassium channel. KCNQ4 inhibition or genetic deletion in RAMs had previously been shown to enhance velocity and vibration responses, similarly to observations in Cib2 knockout mice. I found that inhibiting KCNQ4 did not affect RAM responses in Wnt1-Cre; Cib2fl/ fl mice, showing that in mice lacking Cib2, KCNQ4 does not function effectively in RAMs. We were able to study the influence of CIB2 on KCNQ4 kinetics by employing a whole-cell patch clamp and transiently overexpressing CIB2 and KCNQ4 in the CHO cell line. We discovered that the deactivation time for KCNQ4 was slower after CIB2 overexpression. Furthermore, KCNQ4 mRNA was found in 60% of Cib2-positive neurons. Thus, we conclude that CIB2 interacts with and modulates KCNQ4. The absence of CIB2 in C-fibers, clearly altered their physiological properties. C-fires lacking Cib2 showed signs of sensitization as they displayed a prominent increase in after-charge responses. However, genetic deletion of Cib2 did not affect another type of nociceptor, the Aδ nociceptors. Finally, we assessed the behavioral impact of peripheral Cib2 ablation on light touch and mechanical pain. By using multiple behavioral assays, I found that mice lacking Cib2 exhibit a profound loss of touch sensation. Therefore, I performed an electrical search experiment using an ex-vivo skin nerve preparation to search for mechanically insensitive fibers and found that half of the Aβ-fibers were mechanically silent. These findings suggest that CIB2 is interacting with a mechanosensitive channel, the nature of which is yet to be investigated. This study allowed us to conclude that CIB2 interacts with and modulates ion channels (potassium and mechanosensitive channels) in the somatosensory system and therefore regulates sensory mechanotransduction. We hypothesize that CIB2, as in the auditory system, is an auxiliary subunit of channels in the somatosensory system.Hören und Tasten sind zwei unserer Sinne, die auf Mechanotransduktion beruhen, um mechanische Reize in elektrochemische Aktivität umzuwandeln. Mehrere Studien haben gezeigt, dass Tastsinn und Gehör gemeinsame genetische Determinanten haben; Mutationen in Genen, die Taubheit verursachen, werden auch mit einer verringerten Tastsensibilität in Verbindung gebracht. Darüber hinaus haben Transkriptomanalysen in mehreren Studien ergeben, dass in beiden Systemen die gleichen Gene exprimiert werden. Die Rolle dieser Gene ist jedoch noch nicht geklärt. In dieser Arbeit habe ich gezeigt, dass das Calcium- und Integrin-bindende Protein 2 (CIB2), eine Schlüsselkomponente der Mechanotransduktion im auditorischen System, auch an der somatosensorischen Mechanosensation beteiligt ist. CIB2 ist eine Hilfsuntereinheit des Mechanotransduktionskanals im auditorischen System, die die Kanalfunktion moduliert und die korrekte Kanalpositionierung bestimmt. Mutationen in Cib2 führen bei Menschen und Mäusen zu Hörverlust und zur Unterbrechung der Mechanotransduktion im auditorischen System. Mit Hilfe der Immunhistochemie konnte ich die Expression von CIB2 in einer Untergruppe von DRG-Neuronen nachweisen, hauptsächlich in Nozizeptoren und in geringerem Maße in Mechanorezeptoren. CIB2 war auch in den Endigungen der sensorischen Neuronen vorhanden, die die Endorgane der Haut innervieren. Insbesondere wurde CIB2 in terminalen Afferenzen gefunden, die Meissner-Körperchen und Haarfollikel innervieren, die beide mit schnell adaptierenden Mechanorezeptoren assoziiert sind. Die Auswirkungen von CIB2 auf die physiologischen Eigenschaften verschiedener Arten von Mechanorezeptoren und Nozizeptoren wurden anhand eines Ex-vivo-Hautnervenpräparats aus dem Nervus saphenus und dem Nervus tibialis untersucht. Ich konnte zeigen, dass die genetische Ablation von Cib2 eine tiefgreifende Auswirkung auf die sich schnell anpassenden Mechanorezeptoren (RAMs) hat. In einem Geschwindigkeits- und Vibrationsexperiment zeigten RAMs, denen Cib2 fehlte, eine dramatisch höhere Frequenzempfindlichkeit. Somit beeinflusst CIB2 die Anpassungsgeschwindigkeit der RAMs und wirkt als Bremse für ihre Geschwindigkeitsempfindlichkeit. CIB2 weist strukturelle Ähnlichkeiten mit KChIP-Proteinen auf, die wie CIB2 Calcium-Sensoren sind. Darüber hinaus wurde gezeigt, dass KChIP-Proteine die Oberflächenexpression, den Zusammenbau und die Kinetik von Kv4-Kanälen modulieren, was uns zu der Hypothese führt, dass CIB2 die RAM-Anpassung durch Interaktion mit einem Kaliumkanal und dessen Regulierung beeinflusst. Die Hemmung oder genetische Deletion von KCNQ4 in RAMs hatte zuvor gezeigt, dass sich die Reaktionen auf Geschwindigkeit und Vibration verstärken, ähnlich wie bei Cib2-Knockout-Mäusen beobachtet wurde. Ich fand heraus, dass die Hemmung von KCNQ4 die RAM-Reaktionen in Wnt1-Cre; Cib2fl/ fl-Mäusen nicht beeinflusste, was zeigt, dass KCNQ4 in Mäusen, denen Cib2 fehlt, nicht effektiv in RAMs funktioniert. Wir konnten den Einfluss von CIB2 auf die Kinetik von KCNQ4 untersuchen, indem wir eine Ganzzell-Patch-Clamp-Anlage einsetzten und CIB2 und KCNQ4 in CHO-Zellen vorübergehend überexprimierten. Wir entdeckten, dass die Deaktivierungszeit für KCNQ4 bei CIB2-Überexpression langsamer war. Außerdem wurde KCNQ4 mRNA in 60% der Cib2-positiven Neuronen gefunden. Daraus schließen wir, dass CIB2 mit KCNQ4 interagiert und es moduliert. Das Fehlen von CIB2 in C-Fasern veränderte eindeutig deren physiologische Eigenschaften. C-Fasern, denen Cib2 fehlte, zeigten Anzeichen einer Sensibilisierung, da sie eine deutliche Zunahme der Nachladungsreaktionen zeigten. Die genetische Deletion von Cib2 hatte jedoch keine Auswirkungen auf einen anderen Nozizeptortyp, die Aδ-Nozizeptoren. Schließlich untersuchten wir die verhaltensbezogenen Auswirkungen der peripheren Cib2-Ablation auf leichte Berührungen und mechanische Schmerzen. Mit Hilfe mehrerer Verhaltenstests stellte ich fest, dass Mäuse, denen Cib2 fehlt, einen tiefgreifenden Verlust des Tastsinns aufweisen. Daher führte ich ein elektrisches Suchexperiment mit einem ex-vivo Hautnervenpräparat durch, um nach mechanisch unempfindlichen Fasern zu suchen, und stellte fest, dass die Hälfte der Aβ-Fasern mechanisch stumm waren. Diese Ergebnisse deuten darauf hin, dass CIB2 mit einem mechanosensitiven Kanal interagiert, dessen Natur noch untersucht werden muss. Diese Studie erlaubt uns die Schlussfolgerung, dass CIB2 mit Ionenkanälen (Kalium- und mechanosensitiven Kanälen) im somatosensorischen System interagiert und diese moduliert und somit die sensorische Mechanotransduktion reguliert. Wir stellen die Hypothese auf, dass CIB2, wie im auditorischen System, eine Hilfsuntereinheit von Kanälen im somatosensorischen System ist

Avaliação do efeito da administração aguda pregabalina na ativação do córtex somatosensitivo e motor esquerdo de fibromialgicas por meio da espectroscopia infravermelha funcional (fNIRS)

A Fibromialgia (FM) é uma síndrome que se caracteriza por dor crônica difusa, fadiga, transtornos do sono e alterações de humor. Embora sua fisiopatologia não esteja totalmente elucidada, o processo neurobiológico parece envolver alterações do córtex sensitivo e motor e de suas conexões com estruturas subcorticais que constituem a neuromatriz da dor, assim como alterações neuropáticas periféricas. Sabe-se que o aumento do cálcio intracelular acima de certo limiar, pode ser parte do processo dependente de atividade que leva à sensibilização central, por aumento do influxo de cálcio por meio de canais NMDA, AMPA, canais dependentes de voltagem, e por liberação de reservas intracelular microssomais. A sensibilização central pode ser também interpretada como um processo de plasticidade mal-adaptativa que sustenta circuitos da dor e de seus correlatos. Por tanto, o córtex sensitivo e motor tem sido alvo diagnóstico e terapêutico para o estudo e tratamento da dor crônica. Dentre as múltiplas estratégias farmacológicas tem sido preconizado o uso da pregabalina, aprovado pela Food and Drug Administration (FDA) dos EUA para uso no tratamento de fibromialgia em 2007. A pregabalina age inibindo os canais de cálcio pré sinápticos dependentes de voltagem por se ligar à proteína auxiliar alfa-2-delta. In vitro, este fármaco reduz a liberação de neurotransmissores cálcio-dependentes incluindo glutamato, norepinefrina, calcitonina e substância P, estes neurotransmissores têm sido associados à sensibilização do sistema nervoso. Portanto, considerando o potencial neuromodulador da pregabalina baseado no seu mecanismo de ação, é um fármaco atrativo para avaliar o papel da modulação do córtex sensitivo e motor na fisiopatolgia da FM. No entanto, para que se avance no conhecimento do efeito dos fármacos na função encefálica, necessitamos utilizar recursos de neuroimagem que sejam exequíveis em contextos diversos, e que permitam mensurar o efeito dos fármacos dinamicamente. Dentre os recursos de imagem existe a Functional Near Infrared Spectroscopy (fNIRS) que permite avaliar ativação cortical por meio da mudança no consumo local de oxigênio que acompanha o disparo neuronal regional, mensurado pelas mudanças na concetração da oxi- e desoxi-hemoglobina. Com estas considerações, hipotetizamos que a modulação farmacológica induzida pela pregabalina poderia ser mensurada, clinicamente por meio de testes psicofísicos da dor (que avaliam vias nociceptivas associadas a termoreceptores e barorreceptores) e à nível de neuroimagem por meio do fNIRS. Por se tratar de um mesmo sistema com potencial de ser avaliado de forma virtualmente simultânea, hipotetizamos também que existirá uma associação entre as modulações clínicas (testes psicofísicos) e neurológicas (fNIRS do córtex sensitivo e motor primários). Desta forma, neste estudo avaliou-se o efeito de pregabalina (150 mg) em dose única em fibromiálgicas e controles saudáveis. Em ambos os grupos a pregabalina foi comparada ao placebo, num desenho de estudo randomizado, duplo-cego, cruzado. Avaliou-se o efeito das intervenções, intra e inter-grupos, na ativação cortical de maneira indireta, pela concetração da oxi-hemoglobina durante testes psicofísicos da dor por meio meio do Quantitative Sensory Testing (QST) e algometria de pressão, que foram comparados com a ativação cortical durante uma tarefa motora de percussão dos dedos da mão (left hand finger tapping). Foram estudadas mulheres com idade entre 18 e 65 anos, 17 fibromiálgicas e 10 controles saudáveis. Os parâmetros do QST foram avaliados uma hora após dose única de 150 mg de pregabalina. Resultados: Na linha de base, as fibromiálgicas apresentaram alterações no QST sugestivas de lesão de fibras finas: o limiar de detecção de calor (HDT, do inglês Heat Detection Threshold) foi maior que nas controles (35,53 ºC ± 3,22 vs. 33,33 ºC ± 0,85; p<0,05), enquanto o limiar de dor por pressão (PPT, do inglês Pain Pressure Threshold) foi menor (2,44 kg/cm2 ± 1,08 vs. 4,32 kg/cm2 ± 1,45; P<0,01). Não foram observadas diferenças nos outros componentes do QST, nem mudanças com a pregabalina. Quando comparados com as saudáveis, nas fibromiálgicas o HDT, limiar de dor por calor (HPT) e a tolerância ao calor (HT) evocaram activação nos giros frontal médio, precentral e póscentral, porém, de menor amplitude do que as controles. Depois da administração da pregabalina, aumentou a ativação em responsta ao HDT, mas não teve correlação com o valor do limiar. Já o HPT mostrou se correlacionar de forma inversa com a ativação nos giros frontal superior (rs=-0,552, p=0,033) e precentral (rs=-0,545, p=0,036) na linha de base e após pregabalina (rs=-0,52, p=0,047). A HT também apresentou uma correlação inversa com os giros frontal superior (rs=-0,645, p=0,032) e precentral (rs=-0,655, p=0,029), mas neste caso, esta correlação desapareceu após ter recebido pregabalina. A ativação cortical pelo PPT não detectou diferenças entre fibromiálgicas e controles. Conclusões: O perfil nos testes psicofísicos nas pacientes apresenta correlação com sua ativação cortical. As alterações nos testes sugerem alterações de fibras finas nociceptivas, o que é explicado por um componente de neuropatia periférica, que na fibromialgia é acompanhado por diminuição da ativação em áreas sensitivas e motoras, e aumento da ativação em áreas associadas com processamento cognitivo da dor, cuja atividade foi elevada com a pregabalina. Quando comparadas às controles, nas fibromiálgicas a HT recrutou mais áreas associada ao processamento cognitivo da dor, o que fortalece a hipótese a favor da existência do componente de sensibilização central na fibromialgia. Desta forma, estes achados reforçam a provável coexistência de alterações periféricas e centrais na fisiopatologia da fibromialgia.Fibromyalgia is a syndrome characterized by presenting chronic diffuse pain, fatigue, mood and sleep disturbances. Although its pathophysiology has not been totally elucidated yet, the neurobiological processes seems to involve funciontal alterations of the sensorimotor cortex and its conections with subcortical structures (related to the pain neuronal matrix), and also, with quantitative and qualitative alterations in fine sensitive fibers from the peripheral nervous system. It is known that increased intracellular calcium above certain threshold might be part of a process activity-dependant that leads to central sensitization, due to elevated calcium influx through NMDA and AMPA channels, as well as voltage-dependent channels, and also due to relase of intracellular microsomal reserves. Central sensitization can also be interpreted as a maladaptive plasticity that sustains pain circuits and its correlated. Thus, the sensorimotor cortex has been a diagnostic and therapeutic target for the study and treatment of chronic pain. Among the multiple pharmacological strategies, the use of pregabalin has been recommended and approved by the Food and Drug Administration (FDA) of the United States of America for treatment of patients with fibromyalgia since 2007. Pregabalin acts by inhibiting voltage-dependant pre-synaptic calcium channels by binding to the auxiliary protein alfa-2-delta. In vitro, this drug reduces the liberation of neurottransmissors that depend on calcium, and that include glutamate, norepinephrine, calcitonin and P-substance. All the latter mentioned neurotransmitters are associated with the central nervou system sensitization. Thus, considering its potential as neuronal modulation, taking into accout tis mechanisms of action, the pregabalin is an appealing drug to study the role of the modulation fo the sensorimotor cortex in the pathophysiology of fibromyalgia. Nevertheless, to incrase the knowledge about the effect of drugs on the cortical function, we need to use feasible neuroimaging resources able to be applied in diverse contexts, and that allow to measure the effect of the drugs in real time. Among the neuroimaging resources, there is the Functional Near Infrared Spectroscopy (fNIRS), which allows to assess cortical activation estimating the uptake of regional oxygen, that accompanies local neuronal firing. The fNIRS measures changes in the concentration of oxy and desoxy hemoglobin. Given these considerations, we hypothezise that the pharmacological modulation induced by pregabalin could be measured, clinically through psychophysical pain testing, and at the neuroimaging level using fNIRS. Given that it is about the same system with the potential to be assessed in complementary and virtually simultaneous ways, we also hypothesize that there still could exist an association between the clinical modulation (psychophysical tests) and cortical sensorimotor activation (assessed by fNIRS). In this way, this study appraised the effect of a single dose of pregabalin (150 mg) in the cortical activation and psicophysical pain testing in fibromyalgic and in healthy subjects. In both groups, pregabalin was compared to placebo, in a randomized, double-blinded, cross-over trial design. We assessed the effect of pregabalin, within and between-groups, on the cortical activity in an indirect way via the changes in oxy-hemoglobin upon heat and pressure stimuliation inside a protocol of QST, and also compared the psychophysical pain tests results with the performance during a Left Hand Fingertapping Task. We studied women aging 18 to 65, 17 of them with fibromyalgia and 10 healthy controls. QST parameters were assessed one hour after a single dose of 150 mg of pregabalin. Results: At baseline, patients with fibromyalgia presented QST alterations suggestive of fine nerve fibers lesion: baseline HDT was higher in fibromyalgia (35.53±3.22 vs. 33.33±0.85, P<0.05), while PPT was lower (2.44±1.08 vs. 4.32±1.45, P<0.01) than healthy volunteers, but did not change with pregabalin. When compared to healthy subjects, HDT, HPT, and HT evoked smaller activation in the middle frontal, pre- and post-central gyri in fibromyalgia, that increased after pregabalin (only for HDT-induced activation), but that was not correlated to the HDT. HPT was inversely correlated to the activation in the superior frontal (rs=-0.552, p=0.033) and precentral gyri (rs=-0.545, p=0.036), remaining unchanged after pregabalin (rs=-0.52, p=0.047). HT was inversely correlated to the middle frontal (rs=-0.645, p=0.032) and precentral gyri activation (rs=-0.655, p=0.029), but was no longer correlated after pregabalin. PPT cortical activation did not differ between fibromyalgia and healthy volunteers. Conclusions: The psychophysical pain testing profile in fibromyalgia has a cortical correlate. Alterations in tests for small fibers support its probable peripheral neuropathic component, and was accompanied by decreased activation in sensorimotor areas but increased in pain-related cognitive processing cortexes, and whose activity is increased by pregabalin. Also, upon HT fibromyalgia patients recruited more areas related to pain cognitive processing, which could favor the hypothesis of a component of central sensitization in fibromyalgia, and which was poorly modulated by pregabalin. Taken together, these findings support the co-existence of both, peripheral and central alterations in fibromyalgia

Peripheral mechanisms of cancer-induced bone pain

With the rise in cancer rates and better treatments which prolong life expectancy for patients, the prevalence of cancer associated pain is also increasing. Many cancer patients suffer from painful metastases to the bone, which have a significant impact on quality of life and constitute an economic burden on society. Opioids, the most widely used treatment for metastatic cancer pain, especially in advanced stages of the disease, are associated with severe side effects. While preclinical work suggests anti-NGF therapy may be a useful strategy for malignant bone pain relief, it’s efficacy in the clinic has been disappointing. Like for other forms of chronic pain, the establishment and maintenance of metastatic bone pain depend on peripheral inputs. Elucidating novel peripheral mechanisms and targets for therapy remains a key challenge in the field. This thesis investigates peripheral mechanisms involved in cancer-induced bone pain, by using molecular, transgenic and in vivo imaging approaches, providing new evidence for peripheral changes in cancer-induced bone pain and potential targets for further therapeutic investigation. Firstly, we explored the role of the voltage gated sodium channel Nav1.9, which together with Nav1.7 and Nav1.8 is preferentially expressed in the peripheral nervous system, in metastatic cancer pain. Nav1.9 does not play a role in bone cancer pain, as identified in global knockout mice. Through microarray analysis we identified novel genes and pathways which are dysregulated in the peripheral nervous system in cancer-induced bone pain. A large proportion of differentially expressed genes were microRNAs, suggesting large changes at the posttranscriptional level. Five identified protein coding genes had been previously associated with pain (Adamts5, Adcyap1, Calca, Gal, Nts), but only neurotensin and galanin have been described in the context of cancer-induced bone pain. The three other genes may constitute novel targets for analgesia. Secondly, we investigated the molecular profile of mouse bone marrow afferent neurons by using transgenic mouse reporter lines. Contrary to previous reports in the literature based on immunohistochemistry, we found more than three quarters of bone marrow afferents express the nociceptive neuron’s marker Nav1.8. Additionally, bone afferents never expressed the marker parvalbumin, indicating they are not involved in proprioception. Thirdly, using in vivo calcium imaging we found no increase in the excitability of bone marrow afferents in cancer-bearing animals. However, a larger proportion of cutaneous afferents responded to mechanical stimulation in animals with metastatic bone pain, reflecting behavioural mechanical hypersensitivity. Cutaneous afferents showed increased calcium transients to both thermal and mechanical stimuli in animals with cancer-induced bone pain, suggesting hyperexcitability in the peripheral nervous system contributes to secondary hyperalgesia. Fourthly, based on clinical and pre-clinical evidence of pain relief by osteoclast targeting agents, we wondered if increased activity of these cells alone is sufficient to induce bone pain. While a localized increase in osteoclast activity could not be achieved, a widespread model of osteoclast activation through multiple nuclear factor κB ligand (RANKL) injections, resulted in decreased bone mineral density, without producing any symptoms of pain. Pain may be induced only after reaching a certain threshold of osteoclast activity, or additional changes in the bone microenvironment are needed for a phenotypic switch from physiologic to inflammatory osteoclast

Intensity-dependent modulation of cortical somatosensory processing during external, low-frequency peripheral nerve stimulation in humans.

External low-frequency peripheral nerve stimulation (LFS) has been proposed as a novel method for neuropathic pain relief. Previous studies have reported that LFS elicits long-term depression-like effects on human pain perception when delivered at noxious intensities, while lower intensities are ineffective. To shed light on cortical regions mediating the effects of LFS, we investigated changes in somatosensory-evoked potentials (SEPs) during four LFS intensities. LFS was applied to the radial nerve (600 pulses, 1 Hz) of twenty-four healthy participants at perception (1×), low (5×), medium (10×) and high intensities (15× detection threshold). SEPs were recorded during LFS, and averaged SEPs in 10 consecutive one-minute epochs of LFS were analysed using source dipole modelling. Changes in resting electroencephalography (EEG) were investigated after each LFS block. Source activity in the midcingulate cortex (MCC) decreased linearly during LFS, with greater attenuation at stronger LFS intensities, and in the ipsilateral operculo-insular cortex during the two lowest LFS stimulus intensities. Increased LFS intensities resulted in greater augmentation of contralateral primary sensorimotor cortex (SI/MI) activity. Stronger LFS intensities were followed by increased alpha (9-11 Hz) band power in SI/MI and decreased theta (3-5 Hz) band power in MCC. Intensity-dependent attenuation of MCC activity with LFS is consistent with a state of long-term depression. Sustained increases in contralateral SI/MI activity suggests that effects of LFS on somatosensory processing may also be dependent on satiation of SI/MI. Further research could clarify if the activation of SI/MI during LFS competes with nociceptive processing in neuropathic pain