Validation of mechanical, electrical and thermal nociceptive stimulation methods in horses

To validate a model for investigating the effects of analgesic drugs on mechanical, thermal and electrical stimulation testing. To investigate repeatability, sensitivity and specificity of nociceptive tests. Randomised experiment with 2 observers in 2 phases. Mechanical (M), thermal (TL) and electrical (E) stimuli were applied to the dorsal metacarpus (M-left and TL-right) and coronary band of the left thoracic limb (E) and a thoracic thermal stimulus (TT) was applied caudal to the withers in 8 horses (405 ± 43 kg). Stimuli intensities were increased until a clear avoidance response was detected without exceeding 20 N (M), 60°C (TL and TT) and 15 V (E). For each set of tests, 3 real stimuli and one sham stimulus were applied (32 per animal) using a blinded, randomised, crossover design repeated after 6 months. A distribution frequency and, for each stimulus, Chi-square and McNemar tests compared both the proportion of positive responses detected by 2 observers and the 2 study phases. The κ coefficients estimated interobserver agreement in determining endpoints. Sensitivity (384 tests) and specificity (128 tests) were evaluated for each nociceptive stimulus to assess the evaluators' accuracy in detecting real and sham stimuli. Nociceptive thresholds were 3.1 ± 2 N (M), 8.1 ± 3.8 V (E), 51.4 ± 5.5°C (TL) and 55.2 ± 5.3°C (TT). The level of agreement after all tests, M, E, TL and TT, was 90, 100, 84, 98 and 75%, respectively. Sensitivity was 89, 100, 89, 98 and 70% and specificity 92, 97, 88, 91 and 94%, respectively. The high interobserver agreement, sensitivity and specificity suggest that M, E and TL tests are valid for pain studies in horses and are suitable tools for investigating antinociceptive effects of analgesics in horses.

External signal-activated liposomal drug delivery systems

Modern drug discovery gives rise to a great number of potential new therapeutic agents, but in some cases the efficient treatment of patient may not be achieved because the delivery of active compounds to the target site is insufficient. Thus, drug delivery is one of the major challenges in current pharmaceutical research. Numerous nanoparticle-based drug carriers, e.g. liposomes, have been developed for enhanced drug delivery and targeting. Drug targeting may enhance the efficiency of the treatment and, importantly, reduce unwanted side effects by decreasing drug distribution to non-target tissues. Liposomes are biocompatible lipid-based carriers that have been studied for drug delivery during the last 40 years. They can be functionalized with targeting ligands and sensing materials for triggered activation. In this study, various external signal-assisted liposomal delivery systems were developed. Signals can be used to modulate drug permeation or release from the liposome formulation, and they provide accurate control of time, place and rate of activation. The study involved three types of signals that were used to trigger drug permeation and release: electricity, heat and light. Electrical stimulus was utilized to enhance the permeation of liposomal DNA across the skin. Liposome/DNA complex-mediated transfections were performed in tight rat epidermal cell model. Various transfection media and current intensities were tested, and transfection efficiency was evaluated non-invasively by monitoring the concentration of secreted reporter protein in cell culture medium. Liposome/DNA complexes produced gene expression, but electrical stimulus did not enhance the transfection efficiency significantly. Heat-sensitive liposomal drug delivery system was developed by coating liposomes with biodegradable and thermosensitive poly(N-(2-hydroxypropyl) methacrylamide-mono/dilactate polymer. Temperature-triggered liposome aggregation and contents release from liposomes were evaluated. The cloud point temperature (CP) of the polymer was set to 42 °C. Polymer-coated liposome aggregation and contents release were observed above CP of the polymer, while non-coated liposomes remained intact. Polymer precipitates above its CP and interacts with liposomal bilayers. It is likely that this induces permeabilization of the liposomal membrane and contents release. Light-sensitivity was introduced to liposomes by incorporation of small (< 5 nm) gold nanoparticles. Hydrophobic and hydrophilic gold nanoparticles were embedded in thermosensitive liposomes, and contents release was investigated upon UV light exposure. UV light-induced lipid phase transitions were examined with small angle X-ray scattering, and light-triggered contents release was shown also in human retinal pigment epithelial cell line. Gold nanoparticles absorb light energy and transfer it into heat, which induces phase transitions in liposomes and triggers the contents release. In conclusion, external signal-activated liposomes offer an advanced platform for numerous applications in drug delivery, particularly in the localized drug delivery. Drug release may be localized to the target site with triggering stimulus that results in better therapeutic response and less adverse effects. Triggering signal and mechanism of activation can be selected according to a specific application.

Paired associative transcranial alternating current stimulation increases the excitability of corticospinal projections in humans

Many types of non-invasive brain stimulation alter corticospinal excitability (CSE). Paired associative stimulation (PAS) has attracted particular attention as its effects ostensibly adhere to Hebbian principles of neural plasticity. In prototypical form, a single electrical stimulus is directed to a peripheral nerve in close temporal contiguity with transcranial magnetic stimulation delivered to the contralateral primary motor cortex (M1). Repeated pairing of the two discrete stimulus events (i.e. association) over an extended period either increases or decreases the excitability of corticospinal projections from M1, contingent on the interstimulus interval. We studied a novel form of associative stimulation, consisting of brief trains of peripheral afferent stimulation paired with short bursts of high frequency (≥80 Hz) transcranial alternating current stimulation (tACS) over contralateral M1. Elevations in the excitability of corticospinal projections to the forearm were observed for a range of tACS frequency (80, 140 and 250 Hz), current (1, 2 and 3 mA) and duration (500 and 1000 ms) parameters. The effects were at least as reliable as those brought about by PAS or transcranial direct current stimulation. When paired with tACS, muscle tendon vibration also induced elevations of CSE. No such changes were brought about by the tACS or peripheral afferent stimulation alone. In demonstrating that associative effects are expressed when the timing of the peripheral and cortical events is not precisely circumscribed, these findings suggest that multiple cellular pathways may contribute to a long term potentiation-type response. Their relative contributions will differ depending on the nature of the induction protocol that is used.

Modulation of human corticospinal excitability by paired associative stimulation

Paired Associative Stimulation (PAS) has come to prominence as a potential therapeutic intervention for the treatment of brain injury/disease, and as an experimental method with which to investigate Hebbian principles of neural plasticity in humans. Prototypically, a single electrical stimulus is directed to a peripheral nerve in advance of transcranial magnetic stimulation (TMS) delivered to the contralateral primary motor cortex (M1). Repeated pairing of the stimuli (i.e., association) over an extended period may increase or decrease the excitability of corticospinal projections from M1, in manner that depends on the interstimulus interval (ISI). It has been suggested that these effects represent a form of associative long-term potentiation (LTP) and depression (LTD) that bears resemblance to spike-timing dependent plasticity (STDP) as it has been elaborated in animal models. With a large body of empirical evidence having emerged since the cardinal features of PAS were first described, and in light of the variations from the original protocols that have been implemented, it is opportune to consider whether the phenomenology of PAS remains consistent with the characteristic features that were initially disclosed. This assessment necessarily has bearing upon interpretation of the effects of PAS in relation to the specific cellular pathways that are putatively engaged, including those that adhere to the rules of STDP. The balance of evidence suggests that the mechanisms that contribute to the LTP- and LTD-type responses to PAS differ depending on the precise nature of the induction protocol that is used. In addition to emphasizing the requirement for additional explanatory models, in the present analysis we highlight the key features of the PAS phenomenology that require interpretation.

Assessment of biocompatibility of selected ferroelectric ceramics

Desde há muitas décadas que é sabido que os organismos vivos, em especial os tecidos, reagem fisicamente a estímulos eléctricos, podendo esses efeitos reproduzirem-se numa libertação de químicos endógenos, ou deformar a sua estrutura física. O tecido ósseo por si só é considerado um material/tecido piezoeléctrico, deformando-se mecanicamente quando lhe é induzido um estímulo eléctrico e vice-versa, ou seja, produz um potencial eléctrico quando sofre uma tracção ou compressão mecânica. A hipótese de que um material ferroeléctrico possa vir a produzir efeitos no desempenho deste tipo de tecidos é então proposta, como por exemplo, para uma melhor, mais rápida e eficaz regeneração óssea. Estes mesmos materiais ferroeléctricos podem porventura alterar as cargas de superfície dos tecidos vivos de modo a atrair, atrasar ou até impedir o fluxo iónico de elementos químicos específicos responsáveis pelo processo de regeneração. São escolhidos então o niobato de lítio e o tantalato de lítio como cerâmicos ferroeléctricos e foi estudada pela primeira vez a sua bioactividade in vitro, esperando-se encontrar pistas relativas à sua bioactividade in vivo. Estes cerâmicos ferroeléctricos foram seleccionados devido às suas importantes propriedades piezoeléctricas e ferroeléctricas. Estas propriedades podem abrir um novo e importante leque de aplicações biomédicas caso estes cerâmicos sejam bioactivos. Este trabalho foi dividido em 3 fases: (i) sintetização dos pós de niobato de lítio e tantalato de lítio, (ii) caracterização dos pós e (iii) preparação das amostras e (iv) estudo da bioactividade destes cerâmicos ferroeléctricos. Os pós foram produzidos através de um processo simples de mistura/moagem seguido de calcinação. Foram estudadas as fases cristalinas presentes através de Difracção de raios-X (DRX) e avaliadas as características morfológicas destes pós, nomeadamente o diâmetro de partículas e área superficial específica. De modo a simular o ambiente do plasma humano, foi produzido sinteticamente um “Simulated Body Fluid” (SBF). Seguidamente as amostras foram imersas nesse ambiente líquido por 1, 3, 7, 15 e 21 dias. Após remoção dos pós foram realizadas uma série de análises de modo a estudar a sua bioactividade. De entre estes testes destacam-se a microscopia electrónica de varrimento (SEM/EDS), DRX e espectroscopia de Infravermelho por transformada de Fourier com reflectância total atenuada (FTIR-ATR). Embora não tenham sido detectadas alterações no DRX realizado aos pós, verificou-se a formação de aglomerados de fosfato de cálcio na superfície dos pós através do SEM, resultados estes, reforçados pelo EDS e FTIR-ATR. Estes precipitados de fosfato de cálcio indiciam a capacidade destes pós cerâmicos ferroeléctricos se comportarem como bioactivos em contacto com tecidos ósseos in vivo.

Compósitos CNTs/biocerâmico para a estimulação elétrica óssea in situ

The present thesis aims to develop a biocompatible and electroconductor bone graft containing carbon nanotubes (CNTs) that allows the in situ regeneration of bone cells by applying pulsed external electrical stimuli. The CNTs were produced by chemical vapor deposition (CVD) by a semi-continuous method with a yield of ~500 mg/day. The deposition parameters were optimised to obtain high pure CNTs ~99.96% with controlled morphologies, fundamental requisites for the biomedical application under study. The chemical functionalisation of CNTs was also optimised to maximise their processability and biocompatibility. The CNTs were functionalised by the Diels-Alder cycloaddition of 1,3-butadiene. The biological behaviour of the functionalised CNTs was evaluated in vitro with the osteoblastic cells line MG63 and in vivo, by subcutaneous implantation in rats. The materials did not induce an expressed inflammatory response, but the functionalised CNTs showed a superior in vitro and in vivo biocompatibility than the non-functionalised ones. Composites of ceramic matrix, of bioglass (Glass) and hydroxyapatite (HA), reinforced with carbon nanotubes (CNT/Glass/HA) were processed by a wet approach. The incorporation of just 4.4 vol% of CNTs allowed the increase of 10 orders of magnitude of the electrical conductivity of the matrix. In vitro studies with MG63 cells show that the CNT/Glass/HA composites guarantee the adhesion and proliferation of bone cells, and stimulate their phenotype expression, namely the alkaline phosphate (ALP). The interactions between the composite materials and the culture medium (α-MEM), under an applied electrical external field, were studied by scanning vibrating electrode technique. An increase of the culture medium electrical conductivity and the electrical field confinement in the presence of the conductive samples submerged in the medium was demonstrated. The in vitro electrical stimulation of MG63 cells on the conductive composites promotes the increase of the cell metabolic activity and DNA content by 130% and 60%, relatively to the non-stimulated condition, after only 3 days of daily stimulation of 15 μA for 15 min. Moreover, the osteoblastic gene expression for Runx2, osteocalcin (OC) and ALP was enhanced by 80%, 50% and 25%, after 5 days of stimulation. Instead, for dielectric materials, the stimulus delivering was less efficient, giving an equal or lower cellular response than the non-stimulated condition. The proposed electroconductive bone grafts offer exciting possibilities in bone regeneration strategies by delivering in situ electrical stimulus to cells and consequent control of the new bone tissue formation rate. It is expected that conductive smart biomaterials might turn the selective bone electrotherapy of clinical relevance by decreasing the postoperative healing times.

Effects of butorphanol on the minimum anesthetic concentration for sevoflurane in guineafowl (Numida meleagris)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Effect of intravenous propofol and remifentanil on heart rate, blood pressure and nociceptive response in acepromazine premedicated dogs

ObjectiveTo investigate the cardiorespiratory, nociceptive and endocrine effects of the combination of propofol and remifentanil, in dogs sedated with acepromazine.Study designProspective randomized, blinded, cross-over experimental trial.AnimalsTwelve healthy adult female cross-breed dogs, mean weight 18.4 +/- 2.3 kg.MethodsDogs were sedated with intravenous (IV) acepromazine (0.05 mg kg-1) followed by induction of anesthesia with IV propofol (5 mg kg-1). Anesthesia was maintained with IV propofol (0.2 mg kg-1 minute-1) and remifentanil, infused as follows: R1, 0.125 mu g kg-1 minute-1; R2, 0.25 mu g kg-1 minute-1; and R3, 0.5 mu g kg-1 minute-1. The same dogs were administered each dose of remifentanil at 1-week intervals. Heart rate (HR), mean arterial pressure (MAP), respiratory rate (f(R)), end tidal CO(2) (Pe'CO(2)), arterial hemoglobin O(2) saturation, blood gases, and rectal temperature were measured before induction, and 5, 15, 30, 45, 60, 75, 90, and 120 minutes after beginning the infusion. Nociceptive response was investigated by electrical stimulus (50 V, 5 Hz and 10 ms). Blood samples were collected for plasma cortisol measurements. Statistical analysis was performed by anova (p < 0.05).ResultsIn all treatments, HR decreased during anesthesia with increasing doses of remifentanil, and increased significantly immediately after the end of infusion. MAP remained stable during anesthesia (72-98 mmHg). Antinociception was proportional to the remifentanil infusion dose, and was considered satisfactory only with R2 and R3. Plasma cortisol concentration decreased during anesthesia in all treatments. Recovery was smooth and fast in all dogs.Conclusions and clinical relevanceInfusion of 0.25-0.5 mu g kg-1 minute-1 remifentanil combined with 0.2 mg kg-1 minute-1 propofol produced little effect on arterial blood pressure and led to a good recovery. The analgesia produced was sufficient to control the nociceptive response applied by electrical stimulation, suggesting that it may be appropriate for performing surgery.

Medidas operantes de limiar auditivo em crianças com surdez pré-lingual, usuárias de implante coclear

Limiares auditivos de crianças surdas pré-linguais usuárias de implante coclear foram avaliados com estimulação elétrica em um dos eletrodos mediais. A avaliação empregou um procedimento operante do tipo go/no go para ensinar uma discriminação simples, evidenciada por uma resposta motora, entre presença e ausência do estímulo auditivo. Estabelecida a linha de base, a manipulação na intensidade do estímulo foi implementada de acordo com o método psicofísico de escada modificado, começando por uma seqüência descendente. Os sete participantes do estudo mostraram perda da precisão no responder sob controle do estímulo quando a intensidade diminuía além de um certo valor e a precisão era recuperada quando a intensidade era novamente aumentada, o que permitiu a identificação de limiares individuais. Os resultados sugerem que o método psicofísico combinado com o procedimento operante pode ser uma alternativa viável para avaliar limiar auditivo de pessoas sem linguagem em situação clínica de regulagem do implante coclear.

Effects of remifentanil infusion regimens on cardiovascular function and responses to noxious stimulation in propofol-anesthetized cats

Objective-To evaluate the effects of 2 remifentanil infusion regimens on cardiovascular function and responses to nociceptive stimulation in propofol-anesthetized cats.Animals-8 adult cats.Procedures-On 2 occasions, cats received acepromazine followed by propofol (6 mg/kg then 0.3 mg/kg/min, IV) and a constant rate infusion (CRI) of remifentanil (0.2 or 0.3 mu g/kg/min,IV) for 90 minutes and underwent mechanical ventilation (phase I). After recording physiologic variables, an electrical stimulus (50 V; 50 Hz; 10 milliseconds) was applied to a forelimb to assess motor responses to nociceptive stimulation. After an interval (>= 10 days), the same cats were anesthetized via administration of acepromazine and a similar infusion regimen of propofol; the remifentanil infusion rate adjustments that were required to inhibit cardiovascular responses to ovariohysterectomy were recorded (phase II).Results-In phase I, heart rate and arterial pressure did not differ between remifentanil-treated groups. From 30 to 90 minutes, cats receiving 0.3 mu g of remifentanil/kg/min had no response to noxious stimulation. Purposeful movement was detected more frequently in cats receiving 0.2 mu g of remifentanil/kg/min. In phase II, the highest dosage (mean +/- SEM) of remifentanil that prevented cardiovascular responses was 0.23 +/- 0.01 mu g/kg/min. For all experiments, mean time from infusion cessation until standing ranged from 115 to 140 minutes.Conclusions and Clinical Relevance-Although the lower infusion rate of remifentanil allowed ovariohysterectomy to be performed, a CRI of 0.3 mu g/kg/min was necessary to prevent motor response to electrical stimulation in propofol-anesthetized cats. Recovery from anesthesia was prolonged with this technique.

Efeitos do citrato de sufentanil, administrado em infusão contínua, na concentração alveolar mínima (CAM) de isofluorano em felinos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Implementação de um controlador PID embarcado para o controle em malha fechada de um estimulador neuromuscular funcional

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Eletroestimulador funcional de oito canais com malha de realimentação utilizando Controlador Digital

Pós-graduação em Engenharia Elétrica - FEIS

Efeitos cardiorrespiratório e analgésico da anestesia intravenosa contínua com propofol e remifentanil em cães após medicação pré-anestésica com acepromazina

Pós-graduação em Medicina Veterinária - FMVZ

Validação de métodos nociceptivos elétrico, mecânico e térmico para avaliação de dor de equinos tratados com metadona associada à detomidinaou acepromazina

Pós-graduação em Anestesiologia - FMB