Role of spinal microglia in myositis-induced central sensitisation: An immunohistochemical and behavioural study in rats

There is increasing evidence that spinal glial cells play an important role in chronic pain states. However, so far no data on the role of microglia in muscle pain are available. The aim of the present study was to investigate the involvement of spinal microglial cells in chronic muscle pain. In a rat model of chronic muscle inflammation (injection of complete Freunds adjuvant into the gastrocnemius-soleus muscle) alterations of microglia were visualized with quantitative OX-42 immunohistochemistry in the dorsal horn of the segments L4 and L5 12 days after induction of inflammation. In behavioural experiments the influence of chronic intrathecally applied minocycline - a specific microglia inhibitor - or an antibody against tumour necrosis factor-alpha (TNF-alpha: a cytokine released from microglia) on pain-related behaviour was investigated after 1, 3, 6, and 12 days. The immunhistochemical data show that in the deep laminae of the spinal dorsal horn microglial cells reacted with morphological changes to the muscle inflammation. Following inflammation, the mean boundary length surrounding the OX-42 immunostained area was significantly shorter. This indicates that microglial cells were activated by the myositis and withdrew their processes. Chronic intrathecal administration of minocycline or anti TNF-alpha with an osmotic mini-pump largely normalised the inflammation-induced changes in spontaneous exploratory behaviour and attenuated the hypersensitivity to mechanical stimulation. Both the immunohistochemical and behavioural data show that spinal microglial cells are involved in nociceptive processes in the cause of a chronic muscle inflammation. (C) 2008 European Federation of International Association for the Study of Pain Chapters. Published by Elsevier Ltd. All rights reserved.

Putative antinociceptive action of nitric oxide in the caudal part of the spinal trigeminal nucleus during chronic carrageenan-induced arthritis in the rat temporomandibular joint

In order to investigate a putative role for nitric oxide (NO) in the central nociceptive processing following carrageenan-induced arthritis in the rat temporomandibular joint (TMJ), we analyzed the immunoreactivity, gene expression and activity of nitric oxide synthases (NOS) in the caudal part of the spinal trigeminal nucleus (Sp5C) during the acute (24 h), chronic (15 days) and chronic-active (14 days-24 h) arthritis. In addition, evaluation of head-withdrawal threshold was carried out in all phases of arthritis under chronic inhibition of nNOS with the selective inhibitor 7-nitroindazole (7-NI). Neurons with nNOS-like immunoreactivity (nNOS-LI) were concentrated mainly in the lamina II of the Sp5C, showing no significant statistical difference during arthritis. Only a discrete percentage of nNOS-LI neurons expressed Fos immunoreactivity. The mRNA expression for both nNOS and endothelial nitric oxide synthases (eNOS) presented no noticeable differences among the groups. No expression of inducible nitric oxide synthase (iNOS) was detected in the Sp5C by either immunohistochemistry or reverse-transcription polymerase chain reaction (RTPCR). Ca(2+)-dependent NOS activity in the ipsilateral Sp5C was significantly higher (108.3 +/- 49.2%; P<0.01) in animals during the chronic arthritis. Interestingly, this increased activity was completely abolished 24 h later, in the chronic-active arthritis. Finally, head-withdrawal threshold decreased significantly in the chronic arthritis in animals under 7-NI chronic inhibition. In conclusion, nNOS immunoreactivity and mRNA expression are stable in the Sp5C during TMJ arthritis evolution, but its activity significantly increases in the chronic-phases supporting an antinociceptive role of the nNOS as evidenced by pain threshold experiment. (C) 2009 Elsevier B.V. All rights reserved.

Neurodegeneration and Increased Production of Nitrotyrosine, Nitric Oxide Synthase, IFN-gamma and S100 beta Protein in the Spinal Cord of IL-12p40-Deficient Mice Infected with Trypanosoma cruzi

Background/Aim: Chagas` disease is caused by Trypanosoma cruzi and occurs in most Latin American countries. The protozoan may colonize the central nervous system (CNS) of immune-compromised human hosts, thus causing neuronal disorders. Systemic control of the intracellular forms of the parasite greatly depends on the establishment of a TH1 response and subsequent nitric oxide (NO) release. At the CNS, it is known that low concentrations of NO promote neuronal survival and growth, while high concentrations exert toxic effects and neuron death. Accounting for NO production by astrocytes is the glia-derived factor S100 beta, which is overproduced in some neurodegenerative diseases. In the current work, we studied the expression of NO, interferon (IFN)-gamma and S100 beta in the spinal cord tissue of IL-12p40KO mice infected with T. cruzi, a model of neurodegenerative process. Methods: IL-12p40KO and wild-type (WT) female mice infected with T. cruzi Sylvio X10/4 (10(5) trypomastigotes, intraperitoneally) were euthanized when IL-12p40KO individuals presented limb paralysis. Spinal cord sections were submitted to immunohistochemical procedures for localization of neurofilament, laminin, nitrotyrosine, NO synthases (NOS), IFN-gamma and S100 beta. The total number of neurons was estimated by stereological analysis and the area and intensity of immunoreactivities were assessed by microdensitometric/morphometric image analysis. Results: No lesion was found in the spinal cord sections of WT mice, while morphological disarrangements, many inflammatory foci, enlarged vessels, amastigote nests and dying neurons were seen at various levels of IL-12p40KO spinal cord. Compared to WT mice, IL-12p40KO mice presented a decrement on total number of neurons (46.4%, p<0.05) and showed increased values of immunoreactive area for nitrotyrosine (239%, p<0.01) and NOS (544%, p<0.001). Moreover, the intensity of nitrotyrosine (16%, p<0.01), NOS (38%, p<0.05) and S100 beta (21%, p<0.001) immunoreactivities were also augmented. No IFN-gamma labeled cells were seen in WT spinal cord tissue, contrary to IL-12p40KO tissue that displayed inflammatory infiltrating cells and also some parenchymal cells positively labeled.Conclusion: We suggest that overproduction of NO may account for neuronal death at the spinal cord of T. cruzi-infected IL-12p40KO mice and that IFN-gamma and S100 beta may contribute to NOS activation in the absence of IL-12. Copyright (C) 2009 S. Karger AG, Basel

Flexibility and inhibitor binding in Cdc25 phosphatases

Cdc25 phosphatases involved in cell cycle checkpoints are now active targets for the development of anti-cancer therapies. Rational drug design would certainly benefit from detailed structural information for Cdc25s. However, only apo- or sulfate-bound crystal structures of the Cdc25 catalytic domain have been described so far. Together with previously available crystalographic data, results from molecular dynamics simulations, bioinformatic analysis, and computer-generated conformational ensembles shown here indicate that the last 30-40 residues in the C-terminus of Cdc25B are partially unfolded or disordered in solution. The effect of C-terminal flexibility upon binding of two potent small molecule inhibitors to Cdc25B is then analyzed by using three structural models with variable levels of flexibility, including an equilibrium distributed ensemble of Cdc25B backbone conformations. The three Cdc25B structural models are used in combination with flexible docking, clustering, and calculation of binding free energies by the linear interaction energy approximation to construct and validate Cdc25B-inhibitor complexes. Two binding sites are identified on top and beside the Cdc25B active site. The diversity of interaction modes found increases with receptor flexibility. Backbone flexibility allows the formation of transient cavities or compact hydrophobic units on the surface of the stable, folded protein core that are unexposed or unavailable for ligand binding in rigid and densely packed crystal structures. The present results may help to speculate on the mechanisms of small molecule complexation to partially unfolded or locally disordered proteins.

Canine spinal cord neuron and axon myelin sheath morphometry

This inedited morphometric study has been developed from healthy canine spinal cord neuron cytoplasm and nucleus, and white matter axonal myelin sheath, from cervical, thoracic and lumbar regions. For the morphometric study, the parameters were area, perimeter, maximum and minimum diameters and roundness for neurons and myelin thickness for axon. For each parameter, 300 neurons were analysed. The results revealed that lumbar neurons had the highest mean values for the analysed parameters, indicating the presence of large neurons in this region, with large axons as a result of myelin thickness, which is proportional to axon calibre. We conclude that these morphometric results can contribute for the establishment of normal patterns, for canine spinal cord cervical, thoracic and lumbar segments.

Effect of intravenous alizapride on spinal morphine-induced pruritus

Background. This double-blind study was undertaken to determine whether alizapride inhibits spinal morphine-induced pruritus.Methods. Eighty-four patients undergoing Caesarean section under spinal anaesthesia (100 mg of hyperbaric lidocaine 5% plus morphine 0.2 mg) were randomly allocated to one of two groups. just after birth, alizapride-50 mg (alizapride group) or metoclopramide 10 mg (metoclopramide group) were injected i.v. Patients were assessed after surgery for pruritus (absent, mild, moderate or severe) or other untoward symptoms.Results. In the metoclopramide group, pruritus was absent in 5 (12%) patients, mild in 23 (55%), moderate in 11 (26%), and severe in 3 (7%), while in the alizapride group, these incidences were, respectively, 5 (12%), 33 (79%), 4 (10%), and 0 (P=0.045, chi(2)-test). There was no difference in the incidence of side-effects, which were all minor.Conclusions. Alizapride reduced the severity of morphine-induced pruritus.

The effects of intrathecal administration of betamethasone over the dogs' spinal cord and meninges

OBJETIVO: Determinar possíveis alterações clínicas e histológicas determinadas pela administração da betametasona no espaço subaracnóideo de cães. MÉTODOS: Vinte e um cães foram incluídos no estudo de forma aleatória e encoberta. Depois de anestesiados, os cães foram submetidos a punção subaracóidea com injeção de 1 ml da solução sorteada. Os animais receberam solução salina 0,9% em G1, betametasona na dose de 1,75 mg em G2 e betametasona na dose de 3,5 mg em G3. Todos os animais foram mantidos em observação clínica por 21 dias, sendo posteriormente sacrificados. Porções da medula espinhal e sacral foram removidas para análise histológica por microscopia óptica. RESULTADOS: Não foram detectadas alterações clínicas em quaisquer dos animais incluídos no estudo. da mesma forma, nenhum animal do G1 apresentou alterações histológicas. Infiltração inflamatória foi observada em dois cães, um do G2 e outro e G3. No cão do G2 onde a infiltração inflamatória foi observada ocorreu, conjuntamente, hemorragia e necrose. em dois cães, um de G2 e outro de G3, observou-se discreta fibrose e espessamento da aracnóide, sendo focal em um e difusa no outro. CONCLUSÃO: A administração subaracnóidea de betametasona determinou alterações histológicas em medula e meninges de alguns dos cães envolvidos no estudo.

Preoperative warming combined with intraoperative skin-surface warming does not avoid hypothermia caused by spinal anesthesia in patients with midazolam premedication

CONTEXTO E OBJETIVO: Hipotermia inadvertida no perioperatório é freqüente durante anestesia subaracnóidea e após a administração de midazolam. O objetivo foi avaliar os efeitos do aquecimento da pele no intra-operatório, associado ou não ao aquecimento da pele durante o período de 45 minutos no pré-operatório, na prevenção de hipotermia intra- e pós-operatória determinada pela anestesia subaracnóidea em pacientes com medicação pré-anestésica com midazolam. TIPO DE ETUDO E LOCAL: Estudo prospectivo e aleatório, realizado no Hospital das Clínicas, Universidade Estadual Paulista (Unesp), Botucatu, SP. MÉTODOS: O estudo foi realizado em 30 pacientes com estado físico ASA (da Sociedade Norte-americana de Anestesiologistas) I e II submetidos à cirurgia eletiva do abdômen. Como medicação pré-anestésica, utilizou-se o midazolam, 7,5 mg via intramuscular (IM) e anestesia subaracnóidea padrão. em 10 pacientes (Gcontrole) utilizou-se isolamento térmico passivo; 10 pacientes (Gpré+intra) foram submetidos a aquecimento ativo no pré- e intra-operatório; e 10 pacientes (Gintra) foram aquecidos ativamente somente no intra-operatório. RESULTADOS: Após 45 minutos de aquecimento no pré-operatório, os pacientes do Gpré+intra apresentaram temperatura central mais elevada em relação aos dos grupos não aquecidos antes da anestesia (p < 0,05) mas não no início da cirurgia (p > 0,05). Os pacientes que receberam aquecimento no intra-operatório apresentaram temperatura central mais elevada no final da cirurgia em relação aos de Gcontrole (p < 0,05). Todos os pacientes estavam hipotérmicos na admissão da sala de recuperação pós-anestésica (temperatura central < 36º C). CONCLUSÕES: 45 minutos de aquecimento no pré-operatório combinado com aquecimento no intra- operatório não evita, mas minimiza a ocorrência de hipotermia determinada pela anestesia subaracnóidea em pacientes que receberam midazolam como medicação pré-anestésica.

The Effects of Subarachnoid Administration of Preservative-Free S(+)-Ketamine on Spinal Cord and Meninges in Dogs

BACKGROUND: The N-methyl-D-aspartate receptor antagonist ketamine and its active enantiomer, S(+)-ketamine, have been injected in the epidural and subarachnoid spaces to treat acute postoperative pain and relieve neuropathic pain syndrome. In this study we evaluated the effects of a single dose of preservative-free S(+)-ketamine, in doses usually used in clinical practice, in the spinal cord and meninges of dogs.METHODS: Under anesthesia (IV etomidate (2 mg/kg) and fentanyl (0.005 mg/kg), 16 dogs (6 to 15 kg) were randomized to receive a lumbar intrathecal injection (L5/6) of saline solution of 0.9% (control group) or S(+)-ketamine 1 mg/kg(-1) (ketamine group). All doses were administered in a volume of 1 mL over a 10-second interval. Accordingly, injection solution ranged from 0.6% to 1.5%. After 21 days of clinical observation, the animals were killed; spinal cord, cauda equine root, and meninges were removed for histological examination with light microscopy. Tissues were examined for demyelination (Masson trichrome), neuronal death (hematoxylin and eosin) and astrocyte activation (glial fibrillary acidic protein).RESULTS: No clinical or histological alterations of spinal tissue or meninges were found in animals from either control or ketamine groups.CONCLUSION: A single intrathecal injection of preservative-free S(+)-ketamine, at 1 mg/kg-1 dosage, over a concentration range of 6 to 15 mg/mL injected in the subarachnoid space in a single puncture, did not produce histological alterations in this experimental model. (Anesth Analg 2012;114:450-55)

Effects on mother and fetus of epidural and combined spinal-epidural techniques for labor analgesia

OBJETIVO: A peridural (AP) e a técnica de duplo bloqueio (DB) são utilizadas em analgesia para o trabalho de parto. Este estudo comparou os efeitos na mãe e no feto de ambas as técnicas em analgesia e anestesia para o parto. MÉTODOS: Quarenta parturientes ASA I e II receberam por via peridural 15 ml de ropivacaína a 0,125% (grupo AP) e 5 µg de sufentanil com 2,5 mg bupivacaína por via subaracnóidea (grupo DB). Foram avaliados: intensidade de dor, altura do bloqueio sensitivo, tempo de latência, bloqueio motor, duração da analgesia de parto, tempo para a resolução do parto, hipotensão materna e presença de prurido. Os recém-nascidos foram avaliados pelo índice de Apgar e escore da capacidade adaptativa e neurológica (ECAN), método de Amiel-Tison. RESULTADOS: Não houve diferenças significativas entre os grupos na intensidade da dor, no tempo de latência, no nível do bloqueio sensitivo e no índice de Apgar. O bloqueio motor, a duração da analgesia e o tempo para resolução do parto foram maiores no grupo DB, do qual sete parturientes apresentaram prurido leve. ECAN foi maior no grupo AP após meia hora, duas horas e 24 horas. Noventa e cinco por cento dos recém-nascidos do grupo AP e 60% do grupo DB foram considerados neurologicamente vigorosos ao exame de 24 horas. CONCLUSÃO: As duas técnicas mostraram-se eficazes para analgesia do trabalho de parto. As parturientes do grupo DB apresentaram prurido e trabalho de parto mais prolongado. Recém-nascidos de mães que receberam analgesia de parto via peridural apresentaram melhor ECAN.

Postoperative renal function evaluation, through RIFLE criteria, of elderly patients who underwent femur fracture surgery under spinal anesthesia

Introduction. The postoperative acute renal failure (ARF) incidence in different kinds of surgery has rarely been studied. Age, cardiac dysfunction, previous renal dysfunction, intraoperative hypoperfusion, and use of nephrotoxic medications are mentioned as risk factors for ARF at the postoperative period. The postoperative ARF definition was based on the creatinine increase by the RIFLE classification (R = risk, I = injury, F = failure, L = loss, E = end stage), which corresponds to a 1.5 creatinine increase, two to three times, respectively, above the basal value. This study aimed to evaluate the postoperative ARF incidence in elderly patients who underwent femur fracture surgery under subarachnoid anesthesia and stratify it by the RIFLE criteria. Methods. Ninety patients older than 65 years under spinal anesthesia with fixed dosage of 15 mg of 0.5% isobaric bupivacaine associated with morphine 50 g were studied. Immediate postoperative creatinine was considered basal and compared with maximal creatinine evaluated at 24, 48, and 72 postoperative hours. Results. The mean age of the patients was 80.27 years. ARF incidence was 24.44% and stratified this way: R = 21.11% and I = 3.33%. Conclusions. In conclusion, the postoperative ARF incidence after femur fracture surgery in patients over 65 years was 24.44%. By analyzing the stratification based on the RIFLE classification, the incidence was categorized as Risk (R) = 21.11% and Injury (I) = 3.33%.

Putative antinociceptive action of nitric oxide in the caudal part of the spinal trigeminal nucleus during chronic carrageenan-induced arthritis in the rat temporomandibular joint

In order to investigate a putative role for nitric oxide (NO) in the central nociceptive processing following carrageenan-induced arthritis in the rat temporomandibular joint (TMJ), we analyzed the immunoreactivity, gene expression and activity of nitric oxide synthases (NOS) in the caudal part of the spinal trigeminal nucleus (Sp5C) during the acute (24 h), chronic (15 days) and chronic-active (14 days-24 h) arthritis. In addition, evaluation of head-withdrawal threshold was carried out in all phases of arthritis under chronic inhibition of nNOS with the selective inhibitor 7-nitroindazole (7-NI). Neurons with nNOS-like immunoreactivity (nNOS-LI) were concentrated mainly in the lamina II of the Sp5C, showing no significant statistical difference during arthritis. Only a discrete percentage of nNOS-LI neurons expressed Fos immunoreactivity. The mRNA expression for both nNOS and endothelial nitric oxide synthases (eNOS) presented no noticeable differences among the groups. No expression of inducible nitric oxide synthase (iNOS) was detected in the Sp5C by either immunohistochemistry or reverse-transcription polymerase chain reaction (RTPCR). Ca(2+)-dependent NOS activity in the ipsilateral Sp5C was significantly higher (108.3 +/- 49.2%; P<0.01) in animals during the chronic arthritis. Interestingly, this increased activity was completely abolished 24 h later, in the chronic-active arthritis. Finally, head-withdrawal threshold decreased significantly in the chronic arthritis in animals under 7-NI chronic inhibition. In conclusion, nNOS immunoreactivity and mRNA expression are stable in the Sp5C during TMJ arthritis evolution, but its activity significantly increases in the chronic-phases supporting an antinociceptive role of the nNOS as evidenced by pain threshold experiment. (C) 2009 Elsevier B.V. All rights reserved.

Flexibility and Torsional Strength of ProTaper and ProTaper Universal Rotary Instruments Assessed by Mechanical Tests

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)