Rozprawy doktorskie na temat „DIABETIC NEUROPATHIC PAIN”

Il dolore neuropatico è una caratteristica frequente delle neuropatie periferiche, in particolare quando sono coinvolte le piccole fibre nervose che veicolano la sensibilità termo-dolorifica. La neuropatia delle piccole fibre (SFN) tipicamente si presenta con dolore urente distale agli arti e rappresenta un buon modello per lo studio del dolore neuropatico. Mutazioni nei geni dei canali del sodio voltaggio dipendenti (VGSC) sono state descritte in sindromi algiche familiari e nell’insensibilità congenita al dolore. Più recentemente, varianti in questi geni sono state identificate in associazione a condizioni più comuni quali le neuropatie dolorose. Obiettivo di questa tesi è di indagare il rischio di dolore neuropatico in una coorte ben caratterizzata di pazienti affetti da SFN e neuropatia diabetica. Nella prima sezione sono stati indagati gli aspetti clinici mediante una dettagliata caratterizzazione fenotipica dei pazienti con sospetto di SFN e dolore neuropatico, attraverso la progettazione di un database per la raccolta sistematica dei dati, l'integrazione e la condivisione tra medici e ricercatori. Tali dati sono stati utilizzati per condurre due studi retrospettivi. Il primo ha valutato l'accuratezza diagnostica della biopsia cutanea nel tempo, confrontando i diversi valori normativi per la densità di innervazione intraepidermica adottati dal 1999 al 2019. I risultati su 439 pazienti hanno evidenziato un significativo miglioramento della specificità diagnostica della biopsia cutanea dopo l'introduzione dei valori normativi corretti per età e sesso nel 2010, con una riduzione dei falsi positivi di oltre il 50%. Il secondo studio ha indagato le variazioni circadiane dell'intensità del dolore neuropatico in una coorte di 253 pazienti con sospetta SFN dolorosa rivelando un significativo incremento dell’intensità del dolore dal mattino/pomeriggio alla sera. Lo studio ha mostrato un pattern circadiano del dolore neuropatico che potrebbe fornire una misura di outcome aggiuntiva negli studi clinici per il trattamento del dolore neuropatico nella SFN. La seconda sezione include due studi finalizzati ad identificare determinanti genetiche associate a diversi fenotipi clinici in relazione all’eziologia e alla presenza o assenza di dolore neuropatico. Una prima analisi è stata effettuata su geni candidati, ricercando varianti rare e a bassa frequenza nei geni VGSC potenzialmente esercitanti maggiore effetto sul fenotipo clinico. L'analisi condotta su 1.015 pazienti ha mostrato una maggiore frequenza di varianti rare nei geni VGSC in pazienti con dolore rispetto al fenotipo senza dolore (13,5% e 9,7%) ma nessuna differenza significativa dividendo il campione sulla base dell’eziologia diabetica o idiopatica (11,5%). Osservando la distribuzione delle varianti nei geni VGSCs, i pazienti idiopatici e quelli con dolore presentavano una frequenza significativamente più alta di varianti in SCN9A, mentre nei pazienti con diabete e in quelli senza dolore prevalevano varianti nel gene SCN10A. Tuttavia, la maggior parte di queste varianti sono state classificate come VUS (varianti di significato sconosciuto), pertanto esistono dubbi circa il loro reale significato patogenetico. Sulla base dell'ipotesi di un'architettura poligenica della neuropatia dolorosa in cui tutte le varianti, sia rare che comuni, possono contribuire al fenotipo clinico, è stato adottato un nuovo approccio per indagare il rischio di dolore neuropatico nei pazienti con diabete. E’ stato calcolato un punteggio di rischio poligenico (PRS) combinando il peso di ogni variante identificata in un pannello di 107 geni correlati al dolore, in pazienti affetti da neuropatia diabetica con e senza dolore. Il PRS è stato in grado di discriminare con una sufficiente accuratezza pazienti con dolore da quelli senza dolore. Questo studio rappresenta la prima applicazione del PRS nello studio del dolore neuropatico associato a neuropatia diabetica.
Neuropathic pain is a frequent feature in peripheral neuropathy in particular when small nerve fibers, which convey thermal and nociceptive sensations, are involved. Excruciating burning pain at feet and hand is the most common feature of small fiber neuropathy (SFN) which represents a good model for studying neuropathic pain. Voltage gated sodium channel (VGSCs) genes mutations have been found in rare familial painful disorders and more recently, variants in the same genes have been identified in idiopathic and diabetic painful neuropathies, thus widening the spectrum of genetic pain disorders. This PhD thesis aimed at investigating the risk for neuropathic pain in a well-phenotyped cohort of SFN and diabetic neuropathy patients in order to provide a clinical and genetic characterization of patients. The first section focused on the deep-phenotyping of patients with suspected SFN or neuropathic pain through the development of a database for systematic data collection, integration and sharing among clinicians and researchers. Collected data have been used to conduct two retrospective studies. The first study aimed at addressing the diagnostic accuracy of skin biopsy over time comparing the different normative values for intraepidermal nerve fiber density (IENFD) adopted from 1999 to 2019. This study, comparing skin biopsy results in 439 patients according to different cut-off values, showed a significant improvement of skin biopsy diagnostic specificity after the introduction of the age-and-sex-adjusted normative reference values in the 2010, reporting a reduction of false positive of more than 50% when compared with the cut-off values previously adopted. The second study investigated the circadian dynamics of neuropathic pain intensity scored using the numeric rating scale (PI-NRS) in a cohort of 253 patients with suspected painful SFN. This study revealed a circadian pattern of pain features, showing an increase of NRS scores towards the evening, suggesting a possible role for the intra-day PI-NRS variations as adjunctive outcome measure in clinical trials for analgesic drug in SFN-related neuropathic pain. The second section of the thesis provided a genetic characterization of SFN patients. A candidate-gene analysis has been conducted, looking for rare and low frequency genetic variants in VGSCs genes expected to have a large effect on clinical phenotype and describing their frequency in phenotypically well-defined cohorts of SFN patients. The analysis conducted on 1,015 patients grouped according to etiology and painful phenotype showed a slightly higher frequency of VGSCs variants in painful compared to painless phenotype (13.5% and 9.7%, respectively) but no significant differences between diabetes and idiopathic SFN patients (11.5%). Looking at the variants distribution in VGSCs genes, idiopathic and painful patients showed a significant higher frequency of SCN9A variants whereas diabetes and painless patients had more variants in SCN10A gene. However, concerns have been raised about the pathogenicity of single rare gain-of-function variants, since most of them were classified as VUS (variants of unknown significance). Based on these findings, we adopted a new research approach to investigate the risk of neuropathic pain in our diabetic cohort of 513 patients. The work hypothesis relied on a polygenic architecture of painful neuropathy in which all variants, whether rare or common, might contribute with a small effect size to compose the clinical phenotype. Therefore, we computed a polygenic risk score (PRS) combining the weight of each variant identified in a panel of 107 pain-related genes in diabetic neuropathy patients. The PRS was able to discriminate with sufficient accuracy painful from painless patients with an AUC of 60.3%. This study represented the first application of PRS for addressing the risk of neuropathic pain in diabetic neuropathy, pioneering the use of this tool in this clinical context.

Peripheral sensitisation and central sensitisation are implicated in the development of neuropathic pain with neuroplasticity occurring at multiple levels of the pain pathway. Hypersensitivity of the spinothalamic tract has been described in neuropathic animal models of diabetes. Spinal dorsal horn neurones of diabetic rats exhibit abnormally high spontaneous firing, suggesting an imbalance between excitatory and inhibitory signals converging within this structure. GABAergic neurones within the spinal cord and thalamus are crucial for the transmission of painful stimuli to higher centres of the brain that are involved in pain perception. GABAA receptors (GABAARs) are an important target for many clinical drugs, and certain endogenous neurosteroids act as potent allosteric modulators of these receptors. A developmental change in the rate of exponential decay of GABAergic synaptic events has been observed in other types of neurones and this may be related in part to fluctuations in endogenous neurosteroid tone. The objective of this study was to investigate changes to inhibitory neurotransmission with development in three levels of the pain pathway and to explore potential mechanisms underlying diabetic neuropathy. The whole-cell patch-clamp technique was used on slices of neural tissue. Electrophysiological recordings were obtained from wild type mice between the ages of 6 and 80 days in lamina II of the spinal cord, the nucleus reticularis (nRT) of the thalamus and the cerebral cortex. Recordings were also obtained from mice with diabetic neuropathy (ob/ob and db/db) between the ages of 60 and 80 days. Neurosteroids and their precursors were employed along with compounds that prevented their activity at the GABAAR such as ?-cyclodextrin, which is a barrel-shaped cyclic oligosaccharide with a lipophilic interior that sequesters neurosteroids. Behavioural experiments were also performed using von Frey filaments and the tail flick test to examine mechanical and thermal nociception. Recordings from the spinal cord, the thalamus and the cerebral cortex revealed that the decay time of miniature inhibitory postsynaptic currents are significantly reduced with development. The neurosteroids allopregnanolone and ganaxolone were significantly more effective in neurones from the older mice. In contrast, ?-cyclodextrin had significantly less effect in neurones from the older mice. In mature diabetic mice (ob/ob mice), the endogenous neurosteroid tone is reduced compared to control mice, but certain neurosteroid compounds have a greater effect on the GABAARs of these diabetic mice. In addition, the diabetic mice exhibit mechanical allodynia and hyperalgesia, which is responsive to exogenously applied neurosteroids. These results are consistent with the hypothesis that a dramatic reduction in endogenous neurosteroid tone occurs as development progresses and that this impacts on the exponential decay time of GABAergic mIPSCs within neurones of the pain pathway. The higher neurosteroid tone in the youngest mice may confer a degree of neural protection over the nervous system as it develops. The reduction of endogenous neurosteroid tone in diabetic mice may be associated with their hypersensitivity. It is possible that pregnane-derived neurosteroids may exert analgesic effects in pathological pain states by attempting to restore the physiological GABAergic inhibitory tone that is observed in immature animals.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-03-06T16:58:42Z No. of bitstreams: 1 Afrânio Ferreira Evangelista Avaliação...2014.pdf: 2792913 bytes, checksum: 154973247ed482dbfeac342e4b641d7c (MD5)
Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-03-06T17:00:06Z (GMT) No. of bitstreams: 1 Afrânio Ferreira Evangelista Avaliação...2014.pdf: 2792913 bytes, checksum: 154973247ed482dbfeac342e4b641d7c (MD5)
Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2015-03-06T17:01:18Z (GMT) No. of bitstreams: 1 Afrânio Ferreira Evangelista Avaliação...2014.pdf: 2792913 bytes, checksum: 154973247ed482dbfeac342e4b641d7c (MD5)
Made available in DSpace on 2015-03-06T17:01:18Z (GMT). No. of bitstreams: 1 Afrânio Ferreira Evangelista Avaliação...2014.pdf: 2792913 bytes, checksum: 154973247ed482dbfeac342e4b641d7c (MD5) Previous issue date: 2014
Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil
O diabetes é uma doença de alta prevalência que, frequentemente, induz o comprometimento do sistema nervoso periférico. Na neuropatia diabética periférica, os sintomas mais encontrados são os sensitivos, no qual a dor neuropática, condição crônica caracterizada por alodinia e hiperalgesia, é a mais debilitante. Esta, prejudica a qualidade de vida do paciente, sendo muitas vezes não responsiva aos métodos farmacológicos convencionais de tratamento. Diante desse panorama, o desenvolvimento de novas abordagens terapêuticas que possuam ação efetiva neste tipo de dor é de grande relevância. O uso da terapia celular no tratamento de lesões do sistema nervoso tem demonstrado resultados promissores e o potencial terapêutico de células-tronco na neuropatia experimental tem sido proposto. Neste estudo, avaliou-se o efeito de células-tronco mesenquimais derivadas da medula óssea (CMsMO) na neuropatia diabética periférica estabelecida em modelo experimental de diabetes induzido por estreptozotocina (ETZ). Quatro semanas após a indução do modelo por ETZ (80 mg/kg; ip; 3 dias consecutivos), os animais receberam uma administração endovenosa de CMsMO (1 x 106) ou veículo. O tratamento com gabapentina (30 mg/kg; v.o. a cada 12 horas durante seis dias consecutivos) foi usado como padrão ouro. Os limiares nociceptivos térmico e mecânico foram avaliados durante todo o período experimental (90 dias), pelos métodos de hargreaves e von Frey. A avaliação da função motora foi realizada pelo teste de rota-rod. Em diferentes tempos e para todos os grupos experimentais, foram realizadas coletas de segmentos da medula espinal (L4-L5) para dosagem de citocinas por ELISA e segmentos do nervo isquiático foram também coletados para avaliação de alterações morfológicas por microscopia óptica e eletrônica de transmissão. Os dados comportamentais demonstraram que o tratamento com CMsMO reduziu a mecanoalodinia e a hipoalgesia térmica, levando os limiares nociceptivos de animais neuropáticos a níveis similares aos de animais não neuropáticos. Do mesmo modo, a administração de CMsMO normalizou a função motora dos animais neuropáticos. Dados de microscopia mostraram que animais neuropáticos apresentaram atrofia axonal, redução do número de fibras mielínicas e aparente redução do numero de fibras amielínicas no nervo isquiático. Animais neuropáticos tratados com CMsMO tiveram menor ocorrência de atrofia axonal e não apresentaram redução do numero de fibras mielínicas ou amielínicas, em relação aos neuropáticos tratados com salina. Além disso, animais neuropáticos tratados com CMsMO apresentaram menores níveis espinais de IL-1β e TNF-α, e maiores de IL-10 e TGF-β, em relação aos animais neuropáticos não tratados. Esse conjunto de resultados indica que CMsMO produzem efeito antinociceptivo duradouro na neuropatia diabética, seguido de modificações no padrão fisiopatológico da doença, o que aponta a terapia celular como uma interessante alternativa para o controle da neuropatia diabética periférica dolorosa.
Diabetes is a highly prevalent disease which frequently compromises the peripheral nervous system. In peripheral diabetic neuropathy, the most frequent symptoms are sensitive, in which the neuropathic pain, chronic condition characterized by allodynia and hyperalgesia, is the most debilitating. Neuropathic pain affects the quality of patients’ lives, and is often not responsive to pharmacological conventional treatment methods. Against this background, the development of new therapeutic approaches that have an effective action in this type of pain is of great importance. The use of cell therapy in the treatment of lesions in the nervous system has shown promising results and the therapeutic potential of stem cells in experimental neuropathy has been proposed. In this study, we evaluated the effect of mesenchymal stem cells derived from bone marrow (CMsMO) in peripheral diabetic neuropathy established in experimental model of streptozotocin (STZ) induced diabetes in mice. Four weeks after the induction of the model by administration of STZ (80 mg/kg, ip; 3 days) the animals received an CMsMO by intravenous administration (1x106) or vehicle. The treatment with gabapentin (30 mg/kg, orally every 12 hours for six days) was used as the gold standard. The thermal and mechanical nociceptive thresholds were assessed throughout the entire experimental period (90 days), using Hargreaves and von Frey methods, respectively. Motor function evaluation of was conducted using the rotarod test. At different times, were analyzes conducted in spinal cord segments (L4-L5) to determine cytokines profile by ELISA. Sciatic nerve segments were also collected for evaluation of morphological changes by optical and electron transmission microscopy. According to the behavioral data, the CMsMO treatment reduced the mecanoalodinia and the thermal hypoalgesia, leading nociceptive thresholds of neuropathic animals to levels similar to those of non-neuropathic animals. Similarly, CMsMO administration normalized motor function of neuropathic animals. Microscopy data demonstrated that neuropathic animals had axonal atrophy and an apparent decrease of the number of myelinated fibers as well a reduction in the number of unmyelinated fibers in the sciatic nerve, but neuropathic animals treated with CMsMO had a lower incidence of axonal atrophy, showed no decrease in the number of myelinated fibers and no apparent decrease in the amount of unmyelinated fibers in relation to neuropathics treated with saline. Furthermore, neuropathic animals treated with CMsMO presented lower levels of spinal IL-1β and higher levels of TNF-α, and IL-10 and TGF-β compared to neuropathic animals that received saline. These data indicate that CMsMO produces a lasting analgesic effect in diabetic neuropathy, followed by changes in the pathophysiological disease pattern, which indicates cell therapy as an interesting alternative for the control of painful peripheral diabetic neuropathy.

The available treatments for neuropathic pain are still unsatisfactory – they cope with low efficiency and serious side effects. To our knowledge, this is a pioneering study evaluating whole-genome DNA methylation in unique populations of type 2 diabetes mellitus patients that were histopalogically diagnosed with diabetic neuropathy, with or without neuropathic pain. We provided an evidence on the significant differences in methylation patterns between painful and painless phenotypes. Epigenomes of patients from two independent cohorts were assessed in whole blood samples with Infinium Methylation EPIC BeadChip. We performed differential analysis and identified epigenetic signals that highlight the dissimilarities between painful and painless subjects. We estimated epigenetic age of the patients and evaluated eventual acceleration of biological age in painful and painless phenotypes using set of epigenetic predictors. With the differential analysis we identified 27 CpG sites that reached the level of statistical significance in both studied cohorts, presented the methylation change between painful and painless diabetic neuropathy > 1% in one of the populations and had the direction of methylation change concordant between the two cohorts. 19 of selected probes were genic and resulted in a list of 19 unique genes. Multidimensional scaling analysis confirmed the potential of generated set of CpG sites to separate painful and painless subjects and to highlight the dissimilarities between two phenotypes. Evaluation of biological age showed that there was no association between painful phenotype and acceleration of biological age expressed by any of the assessed epigenetic clocks. DNA methylation based prediction of telomere length was found to vary between painful and painless groups in both studied cohorts. Obtained results confirmed the presence of epigenetic differences between painful and painless diabetic neuropathy patients. Promising genes were identified that may be linked to neuropathic pain through DNA methylation mechanisms.

Neuropathy is a nerve disorder found in HIV disease and diabetes mellitus that indicates damage in the peripheral nervous system. Burning, tingling, stabbing, shooting, and painful sensations in the hands and feet are common symptoms of this chronic disorder, and no treatments are available that repair the nerves. The approved pain treatments are few and only available for the diabetic neuropathy population. A mixed-methods study of archival data was performed to compare patients with painful neuropathy (PN) associated with 2 diseases: HIV (HIV-PN) and diabetes mellitus (DPN). This study examined the similarities and differences of the pain narratives and common pain questionnaires from 12 HIV-PN and 11 DPN subjects. An independent t test of the Visual Analog Scale, Numeric Rating Scale, Brief Pain Intensity subscale, and the Short Form McGill Pain questionnaire failed to reject the null hypothesis that HIV-PN and DPN have equal pain levels. The qualitative analysis revealed 8 shared themes in both groups, with footwear challenges reported as the primary theme. This finding supports the many shared themes between these groups, yet education addressing these themes is minimal. One contrasting theme, privacy, was detected in the HIV-PN group, correlating statistically with the Beck Depression Inventory findings of guilt feelings. The theme of exercise was unique for the DPN group. Both groups had paralinguistic and nonverbal elements discovered in the recordings demonstrating the need for future research to explore these components. Results of education and research themes of privacy in the HIV-PN group and pain communication strategies for both groups will increase understanding of etiology, intervention, and patterns of pain for those diagnosed with neuropathy.

A neuropatia periférica diabética é caracterizada por hiperalgesia e alodínia. O receptor CB1 é o principal responsável pelo efeito dos canabinóides na via nociceptiva. A Hemopressina (Hp), é um agonista inverso do CB1, que induz antinocicepção. Neste trabalho investigamos o efeito do tratamento com Hp (2,5 mg/Kg, por 28 dias) sobre a neuropatia diabética de camundongos, induzido por estreptozotocina (200mg/kg). A Hp reverte a hipersensibilidade mecânica em camundongos com neuropatia diabética, sendo que este efeito é específico para o tratamento da nocicepção e envolve a participação de receptores CB1, astrócitos e microglia em nível espinal. A Hp também previne a desmielinização do nervo isquiático dos animais diabéticos, e auxilia na manutenção dos níveis do NGF. Ainda, a Hp participa no controle da sensibilidade ao estímulo térmico quente em animais KO MOR e participa do controle da sensibilidade mecânica de animais KO MOR diabéticos pelo aumento da dimerização de CB1-DOR na medula espinal. Revelando a Hp um candidato para fins terapêuticos.
Diabetic peripheral neuropathy is characterized by hyperalgesia and allodynia. CB1 receptors are primarily responsible for the effect of cannabinoids in nociceptive pathways. Hemopressin (Hp) is an inverse agonist of CB1, which induces antinociception. In this study we investigated the effects of treatment with Hp (2.5 mg / kg for 28 days) on mice subjected to diabetic neuropathy by streptozotocin (STZ - 200 mg/kg). Hp treatement reversed the mechanical hypersensitivity in mice with neuropathy diabetic, and this effect is specific for the treatment of nociception and involves the participation of CB1 receptors, astrocytes and microglia at the spinal level. Hp prevented demyelination of the sciatic nerve in diabetic animals, and assisted in mantaining the levels of NGF. Also, Hp participates in the control of heat sensitivity to thermal stimulus in KO MOR animals and participates in the control of mechanical sensitivity in KO MOR diabetics animals by the increase in CB1-DOR dimerization in the spinal cord. Revealing Hp as a candidate for therapeutic purposes.

Neuropathic pain is defined by the International Association for the Study of Pain (IASP) as pain that arises as a direct consequence of a lesion or disease affecting the somatosensory system. Neuropathic pain is often poorly alleviated by first-, and second-line medications recommended by IASP due to lack of efficacy and/or dose-limiting side-effects. Hence, there is an urgent need to develop novel mechanism-based therapeutic agents that are highly efficacious and well tolerated to improve relief of neuropathic pain. Palmitoylethanolamide (PEA) is the parent molecule of ALIAmides (Autacoid Local Injury Antagonism Amides), a group of endogenous fatty acid derivatives sharing anti-inflammatory and antinociceptive effects with the endocannabinoid family mainly through the down-modulation of local mast cell degranulation. Several evidences in literature show the antinociceptive effect of PEA in different animal models of pain, such as spinal cord injury, chronic constriction injury of the sciatic nerve, carrageenan-induced acute inflammation, and complete Freund’s adjuvant-induced chronic inflammation. Based on these fundings, the aim of this study is to further explore the therapeutic potentiality of PEA in resolving painful states in three very common forms of neuropathic pain in human associated to osteoarthritis, diabetes, and chemotherapy. Osteoarthritis (OA) is the most common chronic joint disease characterized by a progressive destruction of cartilage, resulting in pain, and loss of articular function. The monosodium iodoacetate (MIA) rat model of OA was used to investigate the effects of PEA. Under a chronic treatment regiment, PEA was able to completely abolish knee swelling and thermal hyperalgesia, as index of inflammation. Moreover, treatment with PEA resulted in a significant relief of mechanical allodynia, as index of neuropathic pain. Futhermore, PEA treatment completely restored locomotor functionality, and is also able to preserve cartilage from damage. Diabetes mellitus is a metabolic syndrome today affecting 382 million people. One of the most disabling long-term complications of diabetes is diabetic neuropathy. The well established streptozotocin (STZ)-induced mice model of type 1 diabetes was emploied to explore the antinociceptive effect of PEA in diabetic neurophaty. PEA relieved mechanical allodynia, counteracted nerve growth factor deficit, improved insulin level, preserved Langherans islet morphology reducing the development of insulitis in diabetic mice. Chemotherapy-induced neuropathic pain (CINP) is another common form of neurophatic pain, affecting up to 90% of patients. The effect of PEA in paclitaxel model of CINP, one of the most common used antineoplastic drugs in clinic, was investigated. Preliminary results show that PEA is able to evoke a total antiallodynic effect in CINP model, after acute administration. The results of this thesis show the pharmacological effect of PEA to relieve neuropathic pain associated to osteoarthritis, diabetes, and chemotherapy, three very common diseases in human, that lack a resolutive, and effective treatment. These findings allow us to suggest a therapeutic use of PEA in clinic.

Neuropathic pain affects up to 50% of the 29 million diabetic patients in the United States. Neuropathic pain in diabetes manifests as a disease of the peripheral and central nervous systems. The prevalence of type 2 diabetes is far greater than type 1 (90%), yet the overwhelming focus on type 1 models this has left the mechanisms of pain in type 2 diabetes largely unknown. Therefore I aimed to improve the current mechanistic understanding of pain associated with type 2 diabetes using two preclinical rodent models: Zucker Diabetic Fatty rats and db/db mice. In addition, I highlight the translational importance of simultaneous measurement of evoked/sensory and non-evoked/affective pain-related behaviors in preclinical models. This work is the first to show a measure of motivational-affective pain in a model of type 2 diabetes. I used methodological approaches including: (1) immunohistochemical and calcium imaging to assess stimulus-evoked sensitization; (2) measurement nociceptive behaviors and evoked sensory thresholds as well as pain affect using novel mechanical conflict avoidance and conditioned place preference/aversion assays; (3) pharmacological and genetic manipulation of methylglyoxal, TRPA1, AC1, and PPARγ. I hypothesized that the thiazolidinedione class of peroxisome proliferator-activated receptor gamma (PPARγ) agonists would reduce neuropathic pain-like behavior and spinal neuron sensitization in traumatic nerve injury and type 2 diabetes. As PPARγ is a nuclear receptor, and already targeted clinically to promote cellular insulin sensitization to reduce hyperglycemia, sustained changes in gene expression are widely believed to be the mechanism of pain reduction. In two separate research aims, I challenged this view and tested whether the PPARγ agonist pioglitazone would (1) rapidly alleviate neuropathic pain through a non-genomic mechanism and (2) reduce painful sensitization in nociceptive and neuropathic pain models independent from lowering blood glucose. I aimed to investigate the contribution of the glucose metabolite methylglyoxal to painful type 2 diabetes. I tested the hypothesis that methylglyoxal produces nociceptive, evoked, and affective pain that is dependent on activation of the sensory neuron cation channel TRPA1 and the secondary messenger enzyme AC1. I also tested whether pioglitazone or the novel methylglyoxal scavenging peptide GERP10 could alleviate painful type 2 diabetes.

O Diabetes mellitus (DM) é o transtorno endócrino mais comum e afeta 6% da população mundial. A neuropatia diabética é uma das complicações do DM e acomete até 60% dos doentes diabéticos. Este é um estudo descritivo que teve como objetivo avaliar as condições odontológicas e a prevalência da ardência bucal nos doentes diabéticos tipo 2 com neuropatia diabética. Foram utilizados os seguintes instrumentos de avaliação: exame sensitivo padronizado da face (algiometria), questionário RDC/TMD eixos 1 e 2, (critérios de diagnóstico em pesquisa) para o diagnóstico de disfunção temporomandibular (DTM), protocolo de avaliação de dor orofacial (EDOF-HC), questionário McGill para avaliação de dor e exame periodontal (IS índice de sangramento, PCS profundidade clínica de sondagem, PCI profundidade clínica de inserção). A prevalência de dor orofacial foi 55,2% e de ardor bucal foi 17,2%. Os valores de glicemia em jejum e HbA1c apresentaram-se acima dos valores normais. Dos doentes avaliados, 44,8% eram totalmente desdentados, 41,2% dos doentes apresentaram doença periodontal avançada, prevalência de dor miofascial mastigatória foi 31%. Traços de depressão moderada e grave e de sintomas físicos inespecíficos moderados e graves foram encontrados em 51,7% dos doentes, de incapacidade baixa e moderada em 37,9%. Observou-se comprometimento da sensibilidade dolorosa em V2 lado D (p=0,007), correlacionada com os valores de HbA1c.
Diabetes mellitus (DM) is one of the most common metabolic diseases affecting 6% of the world population. Diabetic neuropathy is one of DM complications and it affects up to 60% of diabetic patients. This descriptive study aimed to evaluate the dental characteristics and burning mouth prevalence of type 2 DM patients. The following instruments were used: pain sensory test, RDC/TMD questionnaire axis 1 and 2 (research diagnostic criteria for temporomandibular disorders), EDOF-HC protocol (for orofacial pain), Mcgills pain questionnaire and periodontal evaluation (bleeding index, clinical probing depth, clinical probing insertion). Orofacial pain was found in 55,2% of the patients, burning mouth was found in 17,2%. Fasting blood glucose and HbA1c levels were found to be higher than the recommended. 44,8% of the patients were totally edentulous, severe periodontal disease prevalence was 41,2%. Masticatory myofascial pain was found in 31% of the evaluated patients. Traces of severe and moderate depression and severe and moderate nonspecific physical symptoms were found in 51,7% of the patients; moderate and low disability in 37,9%. There was significative difference in the algometry values of the V2 right side compared to the left side and to the study group (p=0,007), with positive correlation with the increased values of the HbA1c.

>Magister Scientiae - MSc
Background: Patients who suffer from diabetic peripheral neuropathy in the leg experience a greater risk of developing gait deviations due to a decrease in strength of the lower extremities, especially the tibialis anterior and triceps surea muscle groups. Aim: The aim of the study was to determine the effect of an exercise training programme on blood pressure, fasting blood glucose, muscle strength, range of motion, balance and gait pattern deviations in patients with diabetic neuropathies. Methods: A total of fourteen participants, who had been diagnosed with diabetic peripheral neuropathy or nocturnal allodynia in either one or both extremities, were asked to participate in this study. Participants were purposively selected from two private Podiatry practices based on their signs and symptoms of diabetic neuropathy, age, gender and doctor’s clearance to participate in any form of physical activity. Dependent variables included isometric strength of the muscles surrounding the hip, knee and ankle, the range of motion of the ankle in plantarflexion and dorsiflexion using goniometry, an assessment of balance using the stork stand test, and a gait pattern analysis, using the modified Tinetti Gait pattern Assessment Scale. Study design: The study was a single-blinded, pre-test and post-test experimental study design using a quantitative approach. Intervention: The researcher (a registered biokineticist) developed a scientifically-based exercise intervention programme to specifically target the entire kinetic chain, and to reduce fall risks, improve quality of life and to assist in developing a standard protocol for patients with DPN. The intervention programme consisted of a combination of ankle, hip and knee rehabilitation, including gait pattern specific rehabilitation. The intervention took place 2-3 times a week for 45 minutes per session and was divided in four categories: Range of motion exercises, strengthening exercises, balance and proprioception and gait pattern training exercises. Results: The Mann-Whitney and Wilcoxon Sign Rank Tests were used to evaluate the differences in dependent variables from pre- to post-intervention. The level of significance was set at p<0.05. An increase in range of motion only in the left ankle dorsiflexion were observed and an increase in balance time for the left leg were observed in the intervention group after a 10-week follow up assessment. Clinical significance was observed in the intervention group, post-intervention, with a decrease in systolic (-9.09%) and diastolic blood pressure (-13.89%) and a decrease in blood glucose levels (-17.89%), however, an increase in these variables was observed in the control group post-intervention. An increase in plantarflexion, 8% (left) and 8% (right) and dorsiflexion 5.26% (left) and an 11.11% (right) increase in range of motion for both left and right ankles, and balance time for both legs, 200% (left) and 159% (right) was observed in the intervention group post-intervention. Although the muscular strength variables showed a mix of an increase and decrease in strength post-intervention in the intervention group, however a clinically significant decreased amount was observed in the control group post-intervention for the majority of muscular strength variables. Conclusions: Although not many findings of this study are statistically significant, clinical significance were observed with most of the variables of this study. The findings of this study can assist future researchers in the development of exercise interventions for patients who suffers from DPN.

Orientadores: Juliana Trindade Clemente Napimoga, Maria Cláudia Gonçalves de Oliveira Fusaro
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
Made available in DSpace on 2018-08-24T17:04:10Z (GMT). No. of bitstreams: 1 MuzilliJunior_Augusto_M.pdf: 554884 bytes, checksum: 13ac41d2adee82f2e294fb0a65c5bb5e (MD5) Previous issue date: 2014
Resumo: A doença diabetes em sua fase inicial é conhecida por induzir dor neuropática, como resultado de complicações neurovasculares mediada pela via diacilglicerol/ Proteinoquinase C (DAG/PKC). Considerando que a neuropatia induzida pelo diabetes está associada com diferentes isoformas da PKC, o objetivo deste estudo foi avaliar a expressão de diferentes isoformas da PKC nas condições dolorosas induzidas pelo diabetes tipo 1 na articulação temporomandibular (ATM) de ratos. Ratos Wistar (± 150 g, 2 meses de idade, n=4-6/grupo) foram tratados com uma injeção intraperitoneal de tampão citrato (veículo; normoglicêmicos ¿ NG) ou Estreptozotocina 75 mg/kg (diabéticos ¿ DB). As alterações dolorosas induzidas pelo diabetes tipo 1 foram avaliadas através da mensuração do comportamento nociceptivo dos animais induzido por uma injeção intra-articular de formalina 1,5% ou capsaicina 1,5% nos dias 7, 14, 21, 28, 35 ou 42 dias após a indução do diabetes ou veículo. Após a avaliação dos comportamentos nociceptivos, os animais foram mortos por anestesia e o tecido periarticular removido para análise de Western Blot. A fase inicial do diabetes tipo 1 induziu uma hiponocicepção na ATM dos ratos 7, 14, 21, 28, 35 e 42 dias depois da indução do diabetes (p<0.05: Two-way ANOVA, Bonferroni¿s test). Os animais diabéticos tratados com uma injeção intra-articular de capsaicina demonstraram hiponocicepção 7, 14, 21 e 28 dias depois da indução do diabetes (p<0.05: Two-way ANOVA, Bonferroni¿s test). A análise por Western Blot demonstrou que a expressão das proteinoquinases PKC-?1, PKC-?2, PKC-delta e PKC-epsilon no tecido periarticular foi significativamente maior nos ratos DB em comparação aos animais NG (p<0.05: Two-way ANOVA, Bonferroni¿s test), enquanto a expressão da PKC-? no tecido periarticular foi significativamente menor nos ratos DB em comparação aos animais NG (p<0,05). Os resultados sugerem que a hiponocicepção induzida pelo diabetes na ATM de ratos está associada à variação da expressão das diferentes isoformas da PKC: -?, -?1, -?2, -delta e -epsilon
Abstract: Diabetes in early phase is known to result in painful neuropathy as a result of neurovascular complications mediated by the activation of Diacylglycerol/Protein kinase C pathway (DAG/PKC). Considering that diabetes-induced neuropathy is associated with different PKC isoforms, the aim of this study was to evaluate the expression of PKC in diabetes type 1-induced pain condition in the temporomandibular joint (TMJ) of rats. Wistar rats (± 150 g, 2 month-old, n=4-6/group) were treated with an intraperitoneal injection of citrate buffer (vehicle; normoglycemic ¿ NG) or Streptozotocin 75 mg/kg (diabetic ¿ DB). Diabetes-induced pain conditions were assessed by the animals¿ nociceptive behavior induced by an intra-articular injection of 1.5% formalin or 1.5% capsaicin 7, 14, 21, 28, 35 or 42 days after the diabetic induction or vehicle. After behavioral assays, animals were terminally anesthetized and their periarticular tissue removed for Western Blot analysis. Early phase of diabetes type 1 induced hyponociception into TMJ of rats 7, 14, 21, 28, 35 and 42 days after the diabetic induction (p<0.05: Two-way ANOVA, Bonferroni¿s test). DB animals treated with an intra-articular injection of capsaicin demonstrated hyponociception 7, 14, 21 and 28 days after diabetes induction (p<0.05: Two-way ANOVA, Bonferroni¿s test). Western Blot analysis demonstrated that the expression of PKC-?1, PKC-?2, PKC-delta and PKC-epsilon into periarticular tissue was significantly higher in the DB rats than in the NG rats (p<0.05: Two-way ANOVA, Bonferroni¿s test), whereas the expression of PKC-? into periarticular tissue was significantly higher in the DB rats than in the NG rats (p<0.05). Early phase of diabetes type 1 induce hyponociception into rats TMJ that involves changes in the expression of different kinds of PKC isoforms: -?, -?1, -?2, -delta and -epsilon. OBSERVAÇÃO Tanto no resumo como no abstract, os símbolos equivalentes à "delta" e "epsilon" haviam ficado como um quadrado, sendo assim, eu escrevi por extenso o nome do símbolo, mas o ideal seria ficar como os símbolos ? e ?
Mestrado
Fisiologia Oral
Mestre em Odontologia

Diabetes mellitus is one of the most common and serious chronic disease in the world. Although the number of available agents to manage diabetes continues to rapidly expand, treatment of diabetes complications, such as neuropathy that is one of the most frequent complication of diabetes mellitus, remains a substantial challenge [Aring et al., 2005]. Pathophysiology of diabetic neuropathy is complex and not fully elucidated; it has multipathogenic mechanisms that cause a diversity of physical symptoms: allodynia, hyperalgesia, numbness and cutaneous ulceration [Vinik et al., 1995]. Persistent Neuropathic Pain (NP) interferes significantly with quality of life, impairing sleep, and emotional well-being, and is a significant causative factor for anxiety, loss of sleep, and non-compliance with treatment. Recent advances in the mechanisms involved in NP have demonstrated that pro- and anti-inflammatory cytokines produced by immune cells as well as by glia and microglia in nerve, dorsal root ganglia (DRG) and spinal cord are common denominators in neuropathic pain [Sacerdote et al., 2013; Old et al., 2015]. These start a cascade of neuroinflammation-related events that may maintain and worsen the original injury, participating in pain generation and chronicization [Valsecchi et al., 2011; Sommer and Kress, 2004; Austin and Moaelem-Taylor, 2010]. Activation of inflammatory cascade, pro- inflammatory cytokines upregulation, and neuroimmune communication pathways play a vital role in structural and functional damage of the peripheral nerves leading to the diabetic peripheral neuropathy. Unfortunately, most of the available analgesic drugs appear to be relatively ineffective in controlling diabetic neuropathic pain, both for insufficient efficacy and side effects [Galer et al., 2000; Kapur, 2003]. Thus, there is a clear need for new disease-modifying therapeutic approaches. Mesenchymal stem/stromal cells (MSCs) may offer a novel therapeutic option to treat diabetic neuropathy. MSCs modulate the nervous system injured environment and promote repair as they secrete anti-inflammatory, anti-apoptotic molecules, and trophic factors to support axonal growth, immunomodulation, angiogenesis, remyelination, and protection from apoptotic cell death [Ma et al., 2014]. Transplanted MSCs not only directly differentiate into endogenous cells on administration, but also secrete a broad range of biologically active factors, generally referred to as the MSCs secretome; in fact even if initially MSCs were proposed for cell therapy based on their differentiation potential, the lack of correlation between functional improvement and cell engraftment or differentiation at the site of injury has led to the proposal that MSCs exert their effects not through their differentiation potential but through their secreted products [Makridakis, 2016; Blaber et al., 2012]. For these reasons in the present study we analyze in a Streptozotocin mouse model of type 1 diabetes the therapeutic effect of hASC (human adipose stem/stromal cells) and their conditioned media (CM-hASC/ secretome) on allodynia and hyperalgesia, on pro- and anti- inflammatory cytokines expression in the main tissue stations involved in nociception transmission as well as in peripheral immune responses. Type 1 diabetes was induced in mice by intraperitoneal (i.p.) injection of moderate low doses of Streptozotocin (STZ, 80 mg/kg, daily for three consecutive days) while control mice were injected with vehicle (citrate buffer). In all groups, mechanical allodynia was evaluated by Von Frey test before diabetes induction and every week after STZ until the end of protocol (14 weeks after STZ). When allodynia was established (2 weeks after STZ) animals were treated with 106 hASC that have been mechanically dissociated to a single cell suspension in PBS solution with 2.5% heparin; CM-hASC from 2x106 cells was also re-suspended in PBS solution with 2.5% heparin and both hASC and CM-hASC were intravenously injected in the tail vein to mice. Animals injected with vehicle only were considered as controls. Our data demonstrated that hASC and CM-hASC treatments were able to reduce allodynia, although the effect of hASC was significantly higher than that elicited by CM-hASC. The effect of both hASC and their secretome was very fast, since a significant reduction of mechanical allodynia was evident already 3 hours after the injection. The antiallodynic effect was maximal beetwen 1 and 2 weeks after treatments and it was extremely long lasting: a significant reduction of allodynia was still present 12 weeks after a single hASC and CM-hASC treatment. Moreover, 4 weeks after the first hASC/CM-hASC treatment (6 weeks after STZ) we decided to treat again a group of diabetic animals with hASC or CM-hASC; repeated hASC treatment did not further ameliorate allodynia. On the other hand, already few hours after the second CM-hASC injection, the antiallodynic effect was significantly potentiated and it completely mimicked the effect evoked by hASC. In order to discover whether hASC and CM-hASC treatments were effective also in a more advanced stage of the disease, when a severe loss of nerve function is reported, we treated animals 6 weeks after diabetes induction. Also in this situation both treatments were efficacious in providing a fast and irreversible antiallodynic effect. Futhermore, in order to verify whether stemness is a fundamental prerequisite for obtaining pain relief a group of STZ-mice was treated with CM obtained from 2x106 human fibroblasts (CM-hF). CM-hF did not exert any effect on mechanical allodynia, demonstrating that only secretome from stem cell cultures is biologically active. It is very important also to consider preparation method of secretome, because lyophilized CM-hASC was unable to provide pain relief, suggesting that during the lyophilization process some essential bioactive factors may be lost. Moreover, since in patients sensory alterations associated to diabetic neuropathy are often diverse in order to ascertain whether the effects of hASC and CM-hASC were limited only to mechanical allodynia, we evaluated thermal hyperalgesia (hot stimuli) and thermal allodynia (cold stimuli) by plantar test and acetone test, respectively. In STZ-mice cold allodynia was present and both treatments were able to significantly reduce it. As regards to heat hyperalgesia, it was present in diabetic mice until 3 weeks from STZ administration, but subsequently we observed hypoalgesia appearance and both treatments were able to avoid hypoalgesia development; these results demostrate the ability of stem cells and their secretome to relieve and prevent the typical diabetic hypersensitivity in response to different types of stimuli. In order to evaluate the impact of treatments on pro- and anti- inflammatory cytokines, animals were sacrificed at different time points: 2 weeks after STZ, i.e. 3 hours after hASC/CM-hASC treatment; 3 weeks after STZ, i.e. 1 week from treatments and 14 weeks after STZ, i.e. 12 or 8 weeks from treatments. From each animal, sciatic nerves, dorsal root ganglia, spinal cord and spleens were collected. IL-1β, TNF-α, IL-6 and IL-10, were evaluated as protein in nervous tissues by ELISA assay. Three weeks after neuropathy induction pro-inflammatory cytokines IL-1β, TNFα and IL-6 resulted overexpressed in peripheral (sciatic nerve and DRG) and central (spinal cord) nervous system of diabetic mice, both hASC and CM-hASC were similarly able to restore pro-inflammatory cytokine levels that 1 week from treatments were back to basal levels; while in all nervous tissues IL-10 levels appeared instead significantly reduced in diabetic animals and both hASC and CM-hASC significantly increased IL-10 concentrations, reaching physiological levels in DRG and spinal cord, while it exceeded basal levels in the sciatic nerve, indicating a switch towards an anti-inflammatory environment in all these tissues. Fourteen weeks after STZ, spinal cord IL-1β, TNF-α and IL-6 levels were still significantly elevated and IL-10 levels reduced in comparison to non diabetic mice, indicating the persistence of neuroinflammation. As observed for the antiallodynic effect, also cytokine modulation induced by hASC and CM-hASC was long lasting. Twelve weeks after treatments performed 2 weeks from STZ, IL-1β, TNF-α and IL-6 levels were still significantly reduced by hASC and CM-hASC treatments, while hASC-treated mice showed a significant normalization of IL-10 levels. Similar effects were observed also in double treatments (2 and 6 weeks after STZ) and both treatments were effective in modulating cytokine levels also when they were administered in an advanced pathological state (6 weeks after STZ). Moreover, to investigate the timing of cytokines modulation exerted by both treatments IL-1 and IL-10 levels in scatic nerves, DRG and spinal cord were measured. Two weeks after diabetic induction, STZ mice were characterized by pro- inflammatory profile and only 3 hours after hASC and CM-hASC administration , both treatments were able to modulate cytokines levels. To further demonstrate the modulation of treatments on pain-related mediators we demonstrated the ability of hASC and CM-hASC to normalize calcitonin gene related peptide level (CGRP), that was elevated in DRG from diabetic animals. Moreover, we evaluated loss of nerve fibers and skin thickness 1 and 12 weeks after a single hASC/CM-hASC administration at 2 weeks after STZ. Both treatments were able to contrast loss of nerve fibers and skin thickness, although hASC treatment was more effective. Since STZ multiple low-doses protocol that we utilized is able to develop an autoimmune response against pancreatic tissue sustained by a T-helper 1 pattern of activation, we studied whether a T-helper polarization was present in splenocytes from diabetic mice and whether hASC or their secretome did exert any immunomodulatory activity. Two weeks after STZ, Con-A stimulated splenocytes released higher levels of IFN-γ, while IL-10 release was significant reduced; both hASC and CM-hASC treatments 3 hours after administration were already able to augment IL-10 levels. Th1/Th2 cytokines unbalance was more evident 3 weeks after STZ and both tretaments appeared able to restablish a correct IFNγ/IL-10 balance. When cytokine levels were measured at longer time from diabetes induction, i.e. 14 weeks after STZ, a clear shift toward a Th1 pattern, characterized by higher IFN-γ and IL-2 secretion and lower levels of IL-4 and IL-10, was present and both hASC/CM-hASC treatments were able to normalize cytokine levels. In the whole, the data indicate that both hASC and CM-hASC treatments are able to block Th1 polarization that develops in this experimental model of diabetes. Moreover, throughout the experiment, blood glucose levels and weight were monitored. In respect to non-diabetic control animals, a significant body weight loss was observed in diabetic mice, that started to be significant 3 weeks after STZ. In STZ-mice the administration of hASC or CM-hASC, 2 weeks after diabetes induction significantly prevented the loss of body weight. Neither treatments did modify blood glucose levels that were elevated in STZ-mice nor glucose tolerance test response. Moreover both hASC and CM-hASC did ameliorate nephropathy that was present in diabetic animals, indicating that the treatments may be useful for treating also other diabetes complications. Our results demonstrated that hASC can control diabetic complications such as neuropathic pain, acting on several peripheral and central mechanisms involved in development and maintenance of this condition, such as neural and immune elements. Moreover the significant new positive results observed also with hASC conditioned medium strongly suggest that their effect is likely to be mediated by their secreted products.

There is an unmet medical need for the efficient treatment of neuropathic pain, a condition that affects approximately 10% of the population worldwide. Current therapies need to be improved due to the associated side effects and lack of response in many patients. Moreover, neuropathic pain causes great suffering to patients and puts an economical burden on society. The work presented in this thesis addresses SP1-7, (Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH), a major metabolite of the pronociceptive neuropeptide Substance P (SP). SP is released in the spinal cord following a noxious stimulus and binds to the NK1 receptor. In contrast to SP, the degradation fragment SP1-7 is antinociceptive through binding to specific binding sites distinct from the NK1 receptor. The aim of this thesis was to investigate the impact of SP1-7 on neuropathic pain. To understand how SP1-7 exerts its effect, a series of N-truncated forms of the heptapeptide were biologically evaluated. A set of small high-affinity ligands was evaluated in animal models of neuropathic pain. To confirm a clinical relevance the levels of SP1-7 in human neuropathic pain were assessed incerebrospinal fluid (CSF) collected from neuropathic pain patients. The results showed that SP1-7 could alleviate thermal as well as mechanical hypersensitivity in three different animal models of neuropathic pain. C-terminal amidation was connected with increased efficacy. N-terminal truncation of SP1-7 indicated a necessity of five amino acids in order to retain biological effect. One small high-affinity ligand showed a significant anti-allodynic effect. CSF levels of SP1-7 in neuropathic pain patients were lower compared to controls. Taken together, these findings demonstrate that the formation of SP1-7 may be attenuated in neuropathic pain. C-terminal amidation and a majority of its amino acids are necessary for stability and permeability. Clearly, SP1-7 and SP1-7 mimetics with high affinity to the SP1-7 binding site ameliorate neuropathic pain-like behaviors in animal models of neuropathic pain. Overall, the findings presented in this thesis contribute to new knowledge regarding the role of SP1-7 and related analogues and fragments in neuropathic pain. In a future perspective, this could be essential for the development of efficient strategies for managing patients with neuropathic pain.

O tramadol é um analgésico de ação central eficaz na atenuação de dores agudas e crônicas, entre elas a dor neuropática em pacientes diabéticos. Encontra-se disponível na clínica como mistura de (+)-tramadol e (-)-tramadol. O tramadol é metabolizado pelo CYP2D6 em O-desmetiltramadol (M1) e pelo CYP3A4 e CYP2B6 em N-desmetiltramadol (M2). Ambos enantiômeros do tramadol e o (+)-M1 contribuem para a atividade analgésica: o (+)-tramadol e o (+)-M1 agem como agonistas do receptor -opióide; o (+)-tramadol inibe a recaptação de serotonina; e o (-)-tramadol inibe a recaptação de noradrenalina. O estudo investiga a influência do diabetes mellitus (DM) tipo 1 e tipo 2 descompensados na disposição cinética, metabolismo e farmacocinética-farmacodinâmica dos enantiômeros tramadol em pacientes com dor neuropática. Os pacientes não diabéticos (Grupo Controle, n=12), os pacientes com DM tipo 1 (n=9) e os pacientes com DM tipo 2 (n=9), todos portadores de dor neuropática e fenotipados como metabolizadores extensivos do CYP2D6, receberam dose única oral de 100 mg de tramadol racêmico. Amostras seriadas de sangue foram coletadas até 24 h após a administração do tramadol para o estudo farmacocinético e para a avaliação das concentrações de noradrenalina. A dor dos pacientes foi avaliada através da escala analógica visual de dor nos mesmos tempos de coleta de sangue. Os pacientes foram avaliados quanto à atividade in vivo do CYP3A utilizando midazolam como fármaco marcador e genotipados para o CYP2B6. As concentrações plasmáticas total e livre dos enantiômeros do tramadol, M1 e M2 foram analisadas por LC-MS/MS usando a coluna Chiralpak® AD. A disposição cinética do tramadol é enantiosseletiva nos pacientes dos Grupos Controle e DM tipo 1, com acúmulo plasmático do (+)-tramadol. O DM tipo 1, mas não o DM tipo 2, reduz a AUC do metabólito ativo (+)-M1 e simultaneamente aumenta sua fração livre. Portanto, a concentração plasmática livre do eutômero (+)-M1 permanece inalterada nos pacientes portadores de DM tipo 1 e DM tipo 2. Não foram observadas diferenças entre os Grupos Controle, DM tipo 1 e DM tipo 2 quanto às razões metabólicas plasmáticas e urinárias do metoprolol/-hidroximetoprolol e quanto ao clearance do midazolam. Correlações significativas entre as razões metabólicas de AUC (+)-tramadol/(+)-M1 ou (-)-tramadol/(-)-M1 e a atividade in vivo do CYP2D6 avaliada em plasma ou urina empregando o metoprolol como fármaco marcador sugerem a aplicação do tramadol como fármaco marcador do CYP2D6. Os dados também mostram uma tendência de aumento do clearance do (+)-tramadol e do (-)-tramadol em virtude da presença do alelo mutante T no polimorfismo 516G>T do CYP2B6. O modelo sigmóide de efeito máximo fracional foi empregado para descrever a relação farmacocinética-farmacodinâmica do tramadol em pacientes com dor neuropática, relacionando as concentrações plasmáticas livre do (+)-M1 com o efeito analgésico do tramadol. O presente estudo mostra a importância da análise da concentração livre dos enantiômeros individuais do tramadol e seus metabólitos nos estudos de farmacocinética-farmacodinâmica.
Tramadol is a centrally acting analgesic that effectively relieves acute and chronic pain, including neuropathic pain in diabetic patients. The drug is available in clinical practice as a mixture of the (+)-tramadol and (-)-tramadol enantiomers. Tramadol is metabolized by CYP2D6 to O-desmethyltramadol (M1) and by CYP3A4 and CYP2B6 to N-desmethyltramadol (M2). Both tramadol enantiomers and (+)-M1 contribute to the analgesic activity of the drug: (+)-tramadol and the (+)-M1 metabolite act as -opioid receptor agonists; (+)-tramadol inhibits serotonin reuptake; and (-)-tramadol inhibits the reuptake of norepinephrine. This study investigated the influence of uncontrolled type 1 and type 2 diabetes mellitus (DM) on the kinetic disposition, metabolism and pharmacokinetics-pharmacodynamics of tramadol enantiomers in patients with neuropathic pain. Nondiabetic patients (control group, n = 12), patients with type 1 DM (n = 9), and patients with type 2 DM (n = 9), all with neuropathic pain and phenotyped as extensive metabolizers of CYP2D6, received a single oral dose of 100 mg racemic tramadol. Serial blood samples were collected up to 24 h after administration of the drug for pharmacokinetic study and for the analysis of noradrenaline in plasma. Pain was rated on a visual analog pain scale at the same time as blood sampling. The patients were evaluated for in vivo CYP3A activity using midazolam as a probe drug and genotyped for CYP2B6. Total and unbound plasma concentrations of the tramadol, M1 and M2 enantiomers were analyzed by LC-MS/MS using a Chiralpak® AD column. The kinetic disposition of tramadol was enantioselective in the control and type 1 DM groups, with the accumulation of (+)-tramadol. Type 1, but not type 2, DM reduced the AUC of the active (+)-M1 metabolite and simultaneously increased its unbound fraction. Therefore, unbound plasma concentrations of the (+)-M1 eutomer remain unchanged in patients with type 1 and type 2 DM. No differences in the plasma and urinary metabolic ratios of metoprolol/-hydroxymetoprolol or in midazolam clearance were observed between the control, type 1 and type 2 DM groups. The significant correlations seen between (+)-tramadol/(+)-M1 or (-)-tramadol/(-)-M1 AUC metabolic ratios and in vivo CYP2D6 activity evaluated in plasma or urine using metoprolol as a probe drug suggest the application of tramadol as a marker for CYP2D6. The data also showed a trend towards increased clearance of (+)-tramadol and (-)-tramadol as a result of the presence of mutant allele T in the 516G>T polymorphism of the CYP2B6 gene. The fractional sigmoid maximum drug effect model was used to describe the pharmacokinetic-pharmacodynamic relationship of tramadol in patients with neuropathic pain, associating the unbound plasma concentrations of (+)-M1 with the analgesic effect of tramadol. The present study highlights the importance of analyzing unbound concentrations of the individual tramadol enantiomers and its metabolites in pharmacokinetic-pharmacodynamic studies.

FundaÃÃo de Amparo à Pesquisa do Estado do Amazonas
Os efeitos dos triterpenos pentaciclicos -amirina e -amirina, isolados a partir da resina de Protium heptaphyllum March. (Burseraceae), foram testados preliminarmente em modelos de nocicepÃÃo oral, sendo que -amirina apresentou significantes efeitos antinociceptivos, norteando a pesquisa com este isolado na investigaÃÃo de seus efeitos em modelos de dor orofacial induzida por capsaicina ou formalina e na dor induzida por capsaicina na cÃrnea de camundongos; na dor tÃrmica (testes de imersÃo de cauda em Ãgua quente e placa quente); e na nocicepÃÃo visceral induzida por Ãcido acÃtico 0,6%. Camundongos Swiss machos (n = 8 / grupo) foram prÃ-tratados com β-amirina (10, 30 e 100 mg / kg, v.o.), morfina (5 mg / kg, s.c.) ou controle (Ãgua destilada + 0,05% de Tween 80, v.o.), uma hora antes de capsaicina (20 L, 1,5 g) ou formalina (20L/animal) serem administradas na vibrissa direita. β-amirina tambÃm foi avaliada em teste comportamental relacionado à dor, desta vez por aplicaÃÃo tÃpica de capsaicina na conjuntiva do camundongo (âeye wiping testâ). Neste teste foi medido o tempo, em segundos, que o animal passou âlimpandoâ o olho durante um perÃodo de 10 minutos. O triterpenÃide demonstrou principalmente um efeito antinociceptivo dose-independente em todos os modelos de nocicepÃÃo testados. Na dor orofacial induzida por capsaicina, -amirina (30 e 100 mg/kg) e morfina foram mais eficazes na reduÃÃo da resposta nociceptiva. Nestas doses, as reduÃÃes foram de 81 e 90% para -amirina e 97% para morfina, respectivamente. No modelo de dor orofacial, a nocicepÃÃo produzida pela capsaicina à acompanhada por um aumento na resposta tÃrmica localizada (que foi mensurada por termometria), e reduzida significantemente pelo prÃ-tratamento dos animais com -amirina ou L-NAME, um inibidor da NOS. Em animais diabÃticos, a capsaicina injetada na vibrissa promoveu um menor grau de nocicepÃÃo orofacial comparada com os nÃo-diabÃticos. No teste da formalina, morfina e β-amirina apresentaram antinocicepÃÃo significativa reversÃvel nas duas fases por naloxona. No entanto, β-amirina (30 mg/kg) inibiu a segunda fase com maior eficiÃncia. Os valores de DE50 para β-amirina e morfina foram 16,44 mg/kg (LC 10,0-38,41) e 3 mg/kg (LC 2,5-5,0) na primeira fase e 43,37 mg/kg (LC 30,52-39,30) e 3 mg/kg (LC 2,5-5,0) na segunda fase, respectivamente. A co-administraÃÃo de β-amirina e morfina, em seus respectivos nÃveis de dose de DE50, nÃo apresentou qualquer efeito aditivo ou potencializador antinociceptivo. No entanto, as combinaÃÃes das doses DE25 e DE12,5 apresentaram uma antinocicepÃÃo comparÃvel ao efeito combinado da DE50, sugerindo que atravÃs da utilizaÃÃo de β-amirina, a dose analgÃsica de morfina poderia ser minimizada para evitar a sua alta dose e os efeitos colaterais associados. β-amirina tambÃm foi eficaz em aumentar o limiar de dor tÃrmica no teste da imersÃo da cauda (mais nÃo no teste placa quente) e, na reduÃÃo das contorÃÃes induzidas por Ãcido acÃtico. A antinocicepÃÃo produzida por β-amirina, foi significativamente bloqueada em animais prÃ-tratados com os respectivos antagonistas vermelho de rutÃnio (2 mg/kg, s.c.) e naloxona (1 mg/kg, i.p.), indicando o envolvimento de receptores da capsaicina (TRPV1) e opiÃides em seu mecanismo. No teste da formalina, de forma similar à morfina, β-amirina bloqueou significativamente a inibiÃÃo da ingestÃo alimentar associada a dor. Assim como morfina, β-amirina apresentou aÃÃo inibitÃria sobre o trÃnsito intestinal, efeito esse revertido pelo prÃ-tratamento com antagonista opiÃide nÃo seletivo, naloxona. Estes dados sugerem que β-amirina apresenta um potencial antinociceptivo comparÃvel à analgesia perifÃrica produzida pela morfina, evidencia a exploraÃÃo desta para o desenvolvimento de um analgÃsico nÃo-opiÃide Ãtil na farmacoterapia de patologias do trigÃmeo e visceral.
The effects of pentacyclic triterpene β-amiryn and β-amyrin, isolated from resin of Protium heptaphyllum March. (Burseraceae), were preliminarily showed significant tested in models of nociception oral, and antinociceptives effects, guiding the search with this isolate in the investigation of their effects in models of orofacial pain induced by capsaicin or formalin and against capsaicin-induced corneal pain; thermal pain (tail immersion test in hot water and hot-plate) and in acetic acid 0,6%-induced visceral nociception in mice. Male Swiss mice (n = 8 per group) were pre-treated with β-Amyrin (10, 30, and 100 mg/kg, p.o.), morphine (5 mg/kg, s.c.) or vehicle (distlled water + 0,05% Tween 80), one hour before the capsaicin (20 μl, 1.5 μg) or formalin (20 μl, 1.5%) injection into the right vibrissa. β-Amyrin was also assessed on pain-related behavioral test (Eye-wiping) by topical application of capsaicin (20 μl, 1.5 μg) on to the mouse conjuctiva and the time (sec) that the animal spent in eye wiping was determined during a 10 min period. The triterpenoid demonstrated mostly a dose-unrelated antinociception in all the test models of nociception. Against the orofacial pain induced by capsaicin, β-Amyrin (30 e 100 mg/kg, p.o.) and morphine showed greater potency in reducing the nociceptive response. At the doses employed, the reductions were 81 and 90% to β-Amyrin and 97% for the morphine, respectively. Capsaicin nociception in orofacial test is accompanied by a localized thermal flare (measured by thermometry), which was significantly diminished by pretreatment of animals with β-Amyrin or L-NAME, an NOS inhibitor. In four weeks diabetic mice, capsaicin injected into vibrissa pad demonstrated a lesser degree of orofacial nociception compared to non-diabetics. In formalin test, both morphine and β-Amyrin showed significant naloxone reversible antinociception in both phases. However, β-Amyrin inhibited the second phase response, more prominently, at 30 mg/kg. The caliculated ED50 values for β-Amyrin and morphine were 16,44 mg/kg (CL 10,0 - 38,41) and 3 mg/kg (CL 2,5 - 5,0) in the first phase and 43,37 mg/kg (CL 30,52 - 39,30) and 3 mg/kg (CL 2,5 - 5,0) in the second phase, respectively. Co-administration of β-Amyrin and morphine at their respective ED50 dose levels failed to demonstrate any additive or potentiating effect on anti-nociception. However, at ED25 and ED12.5 dose-combinations exhibited an antinociception that equalled their ED50 combination effect, suggesting that by the use of β-Amyrin, the analgesic dose of morphine could be minimised to avoid its high-dose-associated side-effects. Similar to morphine, β-Amyrin significantly blocked the pain-related suppression of food intake in formalin test. β-Amyrin (30 and 100 mg/kg was also effective in increasing the thermal pain threshold in hot-water tail immersion test (but not in hot-plate test), and in reducing the acetic acid-induced writhes. The antinociception produced by 30 mg/kg β-Amyrin was significantly blocked in animals pre-treated with the respective antagonists capsazepine (5 mg/kg, s.c.), and naloxone (1 mg kg/kg, i.p.), indicating the involvement of capsaicin (TRPV1) and opioid receptors in its mechanism. Like morphine, β-Amyrin showed an inhibitory effect on intestinal transit, an effect reversed by pretreatment with nonseletive opiÃide antagonist, naloxona. These data indicate that β-Amyrin has the antinociceptive potential comparable to peripheral analgesia produced by morphine that could be explored further on its suitability in developing a non-opioid analgesic useful in pharmacotherapy of trigeminal and visceral pathologies.

It is estimated that 366 million people were living with diabetes in 2011, and this is predicted to rise to 522 million by 2030. One of the most common complications of diabetes is diabetic neuropathy, where patients experience various symptoms of neuropathic pain including mechanical allodynia. Using a high fat diet (HFD) in combination with streptozotocin (STZ) produces a model of diabetes which mimics aspects of type 2 diabetes. The aim of this thesis was to characterise pain responses in the HFD/STZ model and to explore some of the peripheral and spinal mechanisms associated with the changes in somatosensory processing. The effectiveness of a variety of drugs in alleviating/preventing neuropathic pain was also investigated in this model. The effects of the HFD/STZ model on mechanical sensitivity, and changes in metabolic parameters were investigated, and it was found to cause a robust development of mechanical hypersensitivity and a large increase in plasma glucose and a contrasting decrease in plasma insulin. The impacts of the HFD/STZ model on peripheral nerve function and pathology, and spinal mechanisms of central sensitisation, were explored to help identify the mechanisms underpinning the behavioural pain phenotype in these rats. No neuronal degeneration was detected in DRGs or the spinal cord at the timepoints investigated, and a decrease in microglial activation and GFAP immunoreactivity was observed at later timepoints (day 50). Changes in neuronal responses in the dorsal horn of the spinal cord were then investigated, and there was a trend towards a decrease in mechanically evoked responses of spinal neurones in the HFD/STZ group, but no changes in the threshold for electrical activation of C-fibres, nor any significant changes to electrically evoked responses, were observed. There was no change in spontaneous firing, possibly due to the search criteria used. The effects of different types of interventions on aberrant pain responses were also investigated. As neuropathic pain often proves intractable, one of the key objectives is to develop new drugs, or to find alternative uses of current drugs, that are able to provide symptomatic relief of pain. The gold standard treatment for pain in diabetic neuropathy, pregabalin (10mgkg-1, p.o.), was effective at alleviating established mechanical hypersensitivity at day 37 in the HFD/STZ model. A novel MAGL inhibitor, MJN110 (5mgkg-1, i.p.), was found to be as effective as pregabalin in this model, highlighting a possible role for endocannabinoid modulators in providing pain relief in diabetes. The antidiabetic pioglitazone (10mgkg-1, p.o.), however, was unable to alleviate mechanical hypersensitivity when administered at day 21 for 28 days. Two other antidiabetic drugs, linagliptin (3mgkg-1, p.o.) and metformin (200mgkg-1, p.o.), did show promise in preventing the development of mechanical hypersensitivity in this model when administered from day 4, independent of glycemic control. It is worth investigating these findings further since both of these drugs are already licensed and have undergone all necessary safety testing, and so could rapidly be put to use if effective. In conclusion, this thesis has highlighted the role that the HFD/STZ model can play in investigating underlying mechanisms of diabetic peripheral neuropathic pain, and its use in exploring potential new therapeutic options for alleviating this pain.

The present study is a retrospective cohort analysis which sought to examine adherence to medications used in managing painful diabetic peripheral neuropathy (PDPN) and to determine their association with oral antidiabetic (OAD) medication adherence using the Texas Medicaid prescription claims database. The study objectives were to: 1) provide a description of PDPN and OAD medication use among the study subjects; 2) determine if PDPN medication adherence differs among individual PDPN agents (i.e., tricyclic antidepressants, gabapentin, pregabalin and duloxetine); 3) determine if pre-index OAD and post-index OAD medication adherence differs among mono, dual, and triple OAD therapies; and 4) determine if PDPN medication adherence is related to post-index OAD medication adherence while controlling for covariates. Study participants were adult (≥18 years) Medicaid beneficiaries prescribed OAD and PDPN medications. The index date was the first PDPN prescription. Data were extracted from June 1, 2003 to October 31, 2009 and prescription claims were analyzed over an 18-month study period (i.e., 6 months pre-index and 12 months post index period). Medication possession ratio (MPR) was used as a proxy measure of medication adherence. An MPR less than 80 percent was regarded as being non-adherent to prescribed medication, while an MPR greater than or equal to 80 percent was regarded as being adherent to prescribed medication. Objective 1 was addressed using descriptive statistics (i.e., mean, standard deviation, frequency). Univariate analysis (ANOVA) was employed to address Objectives 2 and 3. Multivariate analyses (i.e., multiple linear regression and logistic regression) were conducted to address Objective 4. For the logistic regression MPR was dichotomized at the cut-off value of 80 percent. A total of 4,277 patients met the study’s inclusion criteria. The overall mean MPR (±SD) for PDPN medications was 75.4 percent (±23.9). Mean MPR (±SD) was highest for duloxetine (85.6% ±18.2) and was lowest for pregabalin (69.4% ±24.9). Mean MPR differed significantly among individual PDPN medications (p<0.0001). The overall mean MPR (±SD) for OAD medications in the pre and post-index period was 73.0 percent (±24.3) and 64.5 percent (±25.6) respectively. In both pre and post-index periods, mean MPR differed significantly among mono, dual, and triple OAD therapies (p<0.0001). In the pre-index period, mean MPR (±SD) was highest for monotherapy users (75.4% ±24.7) and was lowest for triple therapy users (63.9% ±22.9). Similarly, mean MPR (±SD) was highest for monotherapy users (69.0% ±26.1) and was lowest for triple therapy users (52.9% ±21.8) in the post-index period. After controlling for the covariates, PDPN adherence (i.e., MPR) was statistically significant (p<0.0001) and positively related to post-index OAD adherence (i.e., MPR). PDPN patients who were non-adherent (i.e., MPR<80%) to their PDPN medications (or neuropathic pain medications), compared to those who were adherent (MPR≥80%), were significantly less likely to be adherent to their OAD medications [Odds Ratio (OR) = 0.626, 95% CI=0.545-0.719]. In addition, post-index OAD adherence (i.e., MPR) did not differ significantly (p>0.05) when pregabalin, duloxetine and gabapentin users were individually compared to tricyclic antidepressants users. In conclusion, PDPN patients who were adherent (i.e., MPR≥80%) to their PDPN medications, compared to those who were not adherent (i.e., MPR<80%), were more adherent to their OAD medications. Also, adherence to OAD medications was independent of the type of PDPN medication used. PDPN patients need to be educated regularly that neuropathic pain medications only relieve the pain associated with the neuropathy but achieving adequate glycemic control remains the only established approach for slowing down the progression of the neuropathy and other complications associated with the diabetes.
text

Introduction. Painful diabetic peripheral neuropathy (DPN) is a common complication of diabetes that interferes with daily living and causes severe pain. Pharmacotherapy is the accepted treatment strategy, however, this strategy is associated with high cost, minimal reductions in pain, and adverse side effects. Thus, a critical need exists to develop alternative treatment strategies. Purpose. To determine if a 12-week whole-body vibration (WBV) intervention reduces pain in adults with DPN. Methods. Twenty-one adults with physician confirmed painful DPN volunteered to take part in a 26-week time series design study. Pain was assessed with the Brief Pain Inventory Short Form [BPI-sf] and a 0-10 numeric rating scale [NRS]. The BPI-sf contains two indices that respectively measure how pain interferes with daily living and severity. The intervention began after a 12-week control period. At week 13, participants were asked to stand on a WBV machine 3 d/week for 4, 3-min bouts at 30-50 Hz with 1-min rest intervals between bouts. Pain levels were reported using the NRS before and after each bout. Results. Comparing post- to pre-intervention, BPI-sf pain interference scores decreased from 5.61±1.40 to 2.39±1.82 (p≤0.001). BPI-sf pain severity scores decreased from 5.1±0.64 to 3.1±1.87 (p≤0.01). Analyses of the NRS scores indicate that pain decreased each week following WBV and that between weeks, pain continued to decrease. Conclusion. These findings demonstrate that whole-body vibration was effective at reducing pain in a sample of adults with painful DPN.
Graduation date: 2012

碩士
國立成功大學
物理治療學系
103
Background and Purpose: Transcutaneous electrical nerve stimulation (TENS) has been used to improve heat and cold pain thresholds and decrease the pain scales in diabetic patients. However, it remains unknown whether TENS could attenuate diabetic neuropathy. Previous research has shown that hyperglycemia could change the levels of TNF-α, TNFR1, TNFR2, cleaved caspase-3, p-Akt and p-PI3k in the PN. Thus, this study investigates the effects of TENS in the pain-like behaviors of the diabetic rats and the expression of TNF-α, TNFR1, TNFR2, cleaved caspase-3, p-Akt and p-PI3k in the DRG, SCDH, SN and PN. Methods: Five experimental groups consist of control, STZ, STZ+ HF TENS, STZ+ LF TENS, and STZ+ AF TENS. TENS will be delivered to the HF, LF and AF groups for 30 minutes per day and 5 days per week for 3 weeks. The mechanical and thermal pain thresholds have been tested under the rats’ hind paws of each side. The rats were sacrificed on the 21st day since the diabetic induction. After the 30-minute post-test period of the last treatment, the L4-L6 SCDH, DRGs, SN and PN (including tibial nerve, peroneal nerve and sural nerve) are collected for protein analysis. TNF-α levels were detected by Enzyme-linked Immunosorbent Assay (ELISA) and TNFR1, TNFR2, cleaved caspase-3, p-Akt and p-PI3k expressions were analyzed by Western Blot. Results: The peaks of the mechanical withdrawal threshold and thermal withdrawal latency are shown lasting after 30 minutes until 2 hours of treatment, but they slightly decreased after 24 hours. TENS can effectively increase the mechanical withdrawal threshold for 3 weeks. However, HF TENS only increases the thermal withdrawal latency for 3 weeks. TNF-α level in the spinal cord dorsal horn and dorsal root ganglions were suppressed after the 3-week TENS treatment, but it increased in the sciatic nerve and the peripheral nerve in both high and LF groups. TNF-α level in the sciatic nerve only decreased in the AF group. Expression of TNFR1, TNFR2, cleaved caspase-3, p-Akt and p-PI3k in the spinal cord dorsal horn significantly up-regulated in the STZ and treatment groups. Expressions of TNFR1, TNFR2 and p-Akt in dorsal root ganglion up-regulated in the STZ and treatment groups as well. However, only the expressions of TNFR2 and p-PI3k in sciatic nerve increased in the STZ group. The expressions of TNFR1 and TNFR2 in sciatic nerve and TNFR1 in peripheral nerve both enhanced in the treatment groups. Conclusion: HF, LF and AF TENS can all lower mechanical allodynia in diabetes, but the decrease in TNF-α level and cell proliferation and the increase of apoptosis in LF and AF groups are more significant than HF group.