Ion current and action potential alterations in peripheral neurons subject to uniaxial strain.

Peripheral nerves, subject to continuous elongation and compression during everyday movement, contain neuron fibers vital for movement and sensation. At supraphysiological strains resulting from trauma, chronic conditions, aberrant limb positioning, or surgery, conduction blocks occur which may result in chronic or temporary loss of function. Previous in vitro stretch models, mainly focused on traumatic brain injury modelling, have demonstrated altered electrophysiological behavior during localized deformation applied by pipette suction. Our aim was to evaluate the changes in voltage-activated ion channel function during uniaxial straining of neurons applied by whole-cell deformation, more physiologically relevant model of peripheral nerve trauma. Here, we quantified experimentally the changes in inwards and outwards ion currents and action potential (AP) firing in dorsal root ganglion-derived neurons subject to uniaxial strains, using a custom-built device allowing simultaneous cell deformation and patch clamp recording. Peak inwards sodium currents and rectifying potassium current magnitudes were found to decrease in cells under stretch, channel reversal potentials were found to be left-shifted, and half-maximum activation potentials right-shifted. The threshold for AP firing was increased in stretched cells, although neurons retained the ability to fire induced APs. Overall, these results point to ion channels being damaged directly and immediately by uniaxial strain, affecting cell electrophysiological activity, and can help develop prevention and treatment strategies for peripheral neuropathies caused by mechanical trauma.

Aligned electrospun fibers for neural patterning.

OBJECTIVES: To test a 3D approach for neural network formation, alignment, and patterning that is reproducible and sufficiently stable to allow for easy manipulation. RESULTS: A novel cell culture system was designed by engineering a method for the directional growth of neurons. This uses NG108-15 neuroblastoma x glioma hybrid cells cultured on suspended and aligned electrospun fibers. These fiber networks improved cellular directionality, with alignment angle standard deviations significantly lower on fibers than on regular culture surfaces. Morphological studies found nuclear aspect ratios and cell projection lengths to be unchanged, indicating that cells maintained neural morphology while growing on fibers and forming a 3D network. Furthermore, fibronectin-coated fibers enhanced neurite extensions for all investigated time points. Differentiated neurons exhibited significant increases in average neurite lengths 96 h post plating, and formed neurite extensions parallel to suspended fibers, as visualized through scanning electron microscopy. CONCLUSIONS: The developed model has the potential to serve as the basis for advanced 3D studies, providing an original approach to neural network patterning and setting the groundwork for further investigations into functionality.

Engineering a uniaxial substrate-stretching device for simultaneous electrophysiological measurements and imaging of strained peripheral neurons.

Peripheral nerves are continuously subjected to mechanical strain during everyday movements, but excessive stretch can lead to damage and neuronal cell functionality can also be impaired. To better understand cellular processes triggered by stretch, it is necessary to develop in vitro experimental methods that allow multiple concurrent measurements and replicate in vivo mechanical conditions. Current commercially available cell stretching devices do not allow flexible experimental design, restricting the range of possible multi-physics measurements. Here, we describe and characterise a custom-built uniaxial substrate-straining device, with which neurons cultured on aligned patterned surfaces (50 µm wide grooves) can be strained up to 70% and simultaneously imaged with widefield and confocal imaging (up to 100x magnification). Furthermore, direct and indirect electrophysiological measurements by patch clamping and calcium imaging can be made during strain application. We characterise the strain applied to cells cultured in deformable wells by using finite element method simulations and experimental data, showing local surface strains of up to 60% with applied strains of up to 25%. We also show how patterned substrates do not alter the mechanical properties of the system compared to unpatterned surfaces whilst still inducing a homogeneous cell response to strain. The characterisation of this device will be useful for research into investigating the effect of whole-cell mechanical stretch on neurons at both single cell and network scales, with applications found in peripheral neuropathy modelling and in platforms for preventive and regenerative studies.

Membrane Mechanical Properties Regulate the Effect of Strain on Spontaneous Electrophysiology in Human iPSC-Derived Neurons.

Peripheral nerves contain neuron fibers vital for movement and sensation and are subject to continuous elongation and compression during everyday movement. At supraphysiological strains conduction blocks occur, resulting in permanent or temporary loss of function. The mechanisms underpinning these alterations in electrophysiological activity remain unclear; however, there is evidence that both ion channels and network synapses may be affected through cell membrane transmitted strain. The aim of this work was to quantify the changes in spontaneous activity resulting from application of uniaxial strain in a human iPS-derived motor neuron culture model, and to investigate the role of cell membrane mechanical properties during cell straining. Increasing strain in a custom-built cell-stretching device caused a linear decrease in spontaneous activity, and no immediate recovery of activity was observed after strain release. Imaging neuronal membranes with c-Laurdan showed changes to the lipid order in neural membranes during deformation with a decrease in lipid packing. Neural cell membrane stiffness can be modulated by increasing cholesterol content, resulting in reduced stretch-induced decrease of membrane lipid packing and in a reduced decrease in spontaneous activity caused by mechanical strain. Together these results indicate that the mechanism whereby cell injury causes impaired transmission of neural impulses may be governed by the mechanical state of the cell membrane, and contribute to establishing a direct relationship between neural uniaxial straining and loss of spontaneous neural activity.

Strain partitioning between nerves and axons: Estimating axonal strain using sodium channel staining in intact peripheral nerves.

BACKGROUND: Peripheral nerves carry afferent and efferent signals between the central nervous system and the periphery of the body. When nerves are strained above physiological levels, conduction blocks occur, resulting in debilitating loss of motor and sensory function. Understanding the effects of strain on nerve function requires knowledge of the multi-scale mechanical behaviour of the tissue, and how this is transferred to the cellular environment. NEW METHOD: The aim of this work was to establish a technique to measure the partitioning of strain between tissue and axons in axially loaded peripheral nerves. This was achieved by staining extracellular domains of sodium channels clustered at nodes of Ranvier, without altering tissue mechanical properties by fixation or permeabilisation. RESULTS: Stained nerves were imaged by multi-photon microscopy during in situ tensile straining, and digital image correlation was used to measure axonal strain with increasing tissue strain. Strain was partitioned between tissue and axon scales by an average factor of 0.55. COMPARISONS WITH EXISTING METHODS: This technique allows non-invasive probing of cell-level strain within the physiological tissue environment. CONCLUSIONS: This technique can help understand the mechanisms behind the onset of conduction blocks in injured peripheral nerves, as well as to evaluate changes in multi-scale mechanical properties in diseased nerves.

Evidences of the Effect of GO and rGO in PCL Membranes on the Differentiation and Maturation of Human Neural Progenitor Cells.

The effect of doping graphene oxide (GO) and reduced graphene oxide (rGO) into poly(ε-caprolactone) (PCL) membranes prepared by solvent induced phase separation is evaluated in terms of nanomaterial distribution and compatibility with neural stem cell growth and functional differentiation. Raman spectra analyses demonstrate the homogeneous distribution of GO on the membrane surface while rGO concentration increases gradually toward the center of the membrane thickness. This behavior is associated with electrostatic repulsion that PCL exerted toward the polar GO and its affinity for the non-polar rGO. In vitro cell studies using human induced pluripotent cell derived neural progenitor cells (NPCs) show that rGO increases marker expression of NPCs differentiation with respect to GO (significantly to tissue culture plate (TCP)). Moreover, the distinctive nanomaterials distribution defines the cell-to-nanomaterial interaction on the PCL membranes: GO nanomaterials on the membrane surface favor higher number of active matured neurons, while PCL/rGO membranes present cells with significantly higher magnitude of neural activity compared to TCP and PCL/GO despite there being no direct contact of rGO with the cells on the membrane surface. Overall, this work evidences the important role of rGO electrical properties on the stimulation of neural cell electro-activity on PCL membrane scaffolds.

Probing multi-scale mechanics of peripheral nerve collagen and myelin by X-ray diffraction.

Peripheral nerves are continuously subjected to mechanical forces, both during everyday movement and as a result of traumatic events. Current mechanical models focus on explaining the macroscopic behaviour of the tissue, but do not investigate how tissue strain translates to deformations at the microstructural level. Predicting the effect of macro-scale loading can help explain changes in nerve function and suggest new strategies for prevention and therapy. The aim of this study was to determine the relationship between macroscopic tensile loading and micro scale deformation in structures thought to be mechanically active in peripheral nerves: the myelin sheath enveloping axons, and axially aligned epineurial collagen fibrils. The microstructure was probed using X-ray diffraction during in situ tensile loading, measuring the micro-scale deformation in collagen and myelin, combined with high definition macroscopic video extensiometry. At a tissue level, tensile loading elongates nerves axially, whilst simultaneously compressing circumferentially. The non-linear behaviour observed in both directions is evidence, circumferentially, that the nerve core components have the ability to rearrange before bearing load and axially, of a recruitment process in epineurial collagen. At the molecular level, axially aligned epineurial collagen fibrils are strained, whilst the myelin sheath enveloping axons is compressed circumferentially. During induced compression, the myelin sheath shows high circumferential stiffness, indicating a possible role in mechanical protection of axons. The myelin sheath is deformed from low loads, despite the non-linearity of whole tissue compression, indicating more than one mechanism contributing to myelin compression. Epineurial collagen shows similar load-bearing characteristics to those of other collagenous connective tissues. This new microstructural knowledge is key to understand peripheral nerve mechanical behaviour, and will support new regenerative strategies for traumatic and repetitive injury.

Rapid and efficient differentiation of functional motor neurons from human iPSC for neural injury modelling.

Primary rodent neurons and immortalised cell lines have overwhelmingly been used for in vitro studies of traumatic injury to peripheral and central neurons, but have some limitations of physiological accuracy. Motor neurons (MN) derived from human induced pluripotent stem cells (iPSCs) enable the generation of cell models with features relevant to human physiology. To facilitate this, it is desirable that MN protocols both rapidly and efficiently differentiate human iPSCs into electrophysiologically active MNs. In this study, we present a simple, rapid protocol for differentiation of human iPSCs into functional spinal (lower) MNs, involving only adherent culture and use of small molecules for directed differentiation, with the ultimate aim of rapid production of electrophysiologically functional cells for short-term neural injury experiments. We show successful differentiation in two unrelated iPSC lines, by quantifying neural-specific marker expression, and by evaluating cell functionality at different maturation stages by calcium imaging and patch clamping. Differentiated neurons were shown to be electrophysiologically altered by uniaxial mechanical deformation. Spontaneous network activity decreased with applied stretch, indicating aberrant network connectivity. These results demonstrate the feasibility of this rapid, simple protocol for differentiating iPSC-derived MNs, suitable for in vitro neural injury studies focussing on electrophysiological alterations caused by mechanical deformation or trauma.

Coincidence Detection of Membrane Stretch and Extracellular pH by the Proton-Sensing Receptor OGR1 (GPR68).

The physical environment critically affects cell shape, proliferation, differentiation, and survival by exerting mechanical forces on cells. These forces are sensed and transduced into intracellular signals and responses by cells. A number of different membrane and cytoplasmic proteins have been implicated in sensing mechanical forces, but the picture is far from complete, and the exact transduction pathways remain largely elusive. Furthermore, mechanosensation takes place alongside chemosensation, and cells need to integrate physical and chemical signals to respond appropriately and ensure normal tissue and organ development and function. Here, we report that ovarian cancer G protein coupled receptor 1 (OGR1) (aka GPR68) acts as coincidence detector of membrane stretch and its physiological ligand, extracellular H+. Using fluorescence imaging, substrates of different stiffness, microcontact printing methods, and cell-stretching techniques, we show that OGR1 only responds to extracellular acidification under conditions of membrane stretch and vice versa. The level of OGR1 activity mirrors the extent of membrane stretch and degree of extracellular acidification. Furthermore, actin polymerization in response to membrane stretch is critical for OGR1 activity, and its depolymerization limits how long OGR1 remains responsive following a stretch event, thus providing a "memory" for past stretch. Cells experience changes in membrane stretch and extracellular pH throughout their lifetime. Because OGR1 is a widely expressed receptor, it represents a unique yet widespread mechanism that enables cells to respond dynamically to mechanical and pH changes in their microenvironment by integrating these chemical and physical stimuli at the receptor level.

Engineered method for directional growth of muscle sheets on electrospun fibers.

Research on the neuromuscular junction (NMJ) and its function and development spans over a century. However, researchers are limited in their ability to conduct experimentation on this highly specialized synapse between motor neurons and muscle fibers, as NMJs are not easily accessible outside the body. The aim of this work is to provide a reliable and reproducible muscle sheet model for in vitro NMJ study. A novel culture system was designed by engineering a method for the directional growth of myofiber sheets, using muscle progenitor cells cultured on electrospun fiber networks. Myoblastic C2C12 cells cultured on suspended aligned fibers were found to maintain directionality, with alignment angle standard deviations approximately two-thirds lower on fibers than on regular culture surfaces. Morphological studies found nuclei and cytoskeleton aspect ratios to be elongated by 20 and 150%, respectively. Furthermore, neurons were shown to form innervation patterns parallel to suspended fibers when co-cultured on developed muscle sheets, with alignment angle standard deviations three times lower compared with those on typical surfaces. The effect of agrin on samples was quantified through the slow release of agrin medium, encapsulated in alginate pellets and imbedded within culture chambers. Samples exposed to agrin showed significantly increased percentage of AChR-covered area. The developed model has potential to serve as the basis for synaptogenesis and NMJ studies, providing a novel approach to bio-artificial muscle alignment and setting the groundwork for further investigations in innervation. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1165-1176, 2018.

Probing multi-scale mechanical damage in connective tissues using X-ray diffraction.

The accumulation of microstructural collagen damage following repetitive loading is linked to painful and debilitating tendon injuries. As a hierarchical, semi-crystalline material, collagen mechanics can be studied using X-ray diffraction. The aim of the study was to describe multi-structural changes in tendon collagen following controlled plastic damage (5% permanent strain). We used small angle X-ray scattering (SAXS) to interrogate the spacing of collagen molecules within a fibril, and wide angle X-ray scattering (WAXS) to measure molecular strains under macroscopic loading. Simultaneous recordings of SAXS and WAXS patterns, together with whole-tissue strain in physiologically hydrated rat-tail tendons were made during increments of in situ tensile loading. Results showed that while tissue level modulus was unchanged, fibril modulus decreased significantly, and molecular modulus significantly increased. Further, analysis of higher order SAXS peaks suggested structural changes in the gap and overlap regions, possibly localising the damage to molecular cross-links. Our results provide new insight into the fundamental damage processes at work in collagenous tissues and point to new directions for their mitigation and repair. STATEMENT OF SIGNIFICANCE: This article reports the first in situ loading synchrotron studies on mechanical damage in collagenous tissues. We provide new insight into the nano- and micro-structural mechanisms of damage processes. Pre-damaged tendons showed differential alteration of moduli at macro, micro and nano-scales as measured using X-ray scattering techniques. Detailed analysis of higher order diffraction peaks suggested damage is localised to molecular cross-links. The results are consistent with previous X-ray scattering studies of tendons and also with recent thermal stability studies on damaged material. Detailed understanding of damage mechanisms is essential in the development of new therapies promoting tissue repair.

Atrial supersensitivity to noradrenaline in stressed female rats.

Stress can change the responses to catecholamines in many tissues. The aim of this study was to investigate the influence of the estrous cycle on the sensitivity of right atria to noradrenaline in female rats subjected to acute swimming stress. Female Wistar rats in proestrus, estrus, metestrus or diestrus were submitted to a 50 min-swimming session. Immediately after the exercise, the rats were killed and their right atria were mounted for isometric recording of the spontaneous beating rate. Concentration-effect curves to noradrenaline were obtained before and after the inhibition of neuronal uptake with phenoxybenzamine (10 microM) and of extraneuronal uptake with estradiol (5 microM). Acute swimming stress did not change the right atrial sensitivity to noradrenaline in rats in estrus, metestrus and diestrus. However, swimming stress produced supersensitivity to noradrenaline in proestrus (pD(2) control: 7.14 +/- 0.03 vs. pD(2) swimming: 7.55 +/- 0.04; p<0.05). This supersensitivity was still observed after uptake inhibition. When catecholamine uptake was inhibited, the concentration-effect curve to noradrenaline was shifted to the left 2.5-fold in the proestrus control group and 1.7-fold in the proestrus stress group (p<0.05). In conclusion, the estrous cycle influenced the acute stress-induced atrial supersensitivity to noradrenaline.

Levantamento epidemiológico de cárie dentária em escolares de 5 e 12 anos de idade do município de Cascavel, PR / Epidemiological survey of dental caries in children with 5 and 12 years of age of Cascavel, PR

Por solicitação da divisão de saúde bucal da Secretaria Municipal de Saúde de Cascavel-PR, o curso de odontologia da Universidade Estadual do Oeste do Paraná (UNIOESTE) executou o primeiro levantamento epidemiológico na área de Saúde Bucal. A pesquisa foi realizada em crianças nas idades de 5 e 12 anos e foi utilizado os índices epidemiológicos ceo e CPO-D das escolas estaduais e municipais escolhidas através de sorteio contemplando todas as regiões do município. Os resultados obtidos através do levantamento produziram informações sobre as condições de saúde bucal da população de Cascavel. O ceo teve média de 2,42 e o CPO-D foi de 1,91, sendo maior na região Central. Embora os resultados apresentem valores menores que os do SB BRASIL 2000, o trabalho foi de grande valia para a criação e manutenção de uma base de dados contribuindo na perspectiva da estruturação de um sistema de vigilância epidemiológica em saúde bucal.

At the request of the Division of Oral Health of the Municipal Health Department of Cascavel-PR, the Dentistry course at the Universidade Estadual do Oeste do Paraná (UNIOESTE) performed the first epidemiological survey in the area of Oral Health. The survey was conducted in children aged 5 and 12 years and was used epidemiological indices dtmf and DMFT indexes of state and municipal schools chosen by lottery covering all regions of the city. The results obtained through the survey yielded information on the oral health status of the population of Cascavel. The dtmf had a mean of 2.42 and DMFT was 1.91, being higher in the Central region. Although the results showed lower values than those of SB BRASIL 2000, the work was valuable for the creation and maintenance of a database contributing the perspective of designing a surveillance system in oral health.

Estudo morfométrico do plexo mientérico do duodeno de ratos submetidos ao alcoolismo / Morphometric study of duodenum myenteric plexus in rats submitted to alcoholism

O etanol é uma molécula que se difunde facilmente pelas membranas celulares e no citosol leva a produção de compostos tóxicos que podem alterar as proteínas celulares e o DNA. Assim, o objetivo deste estudo foi avaliar os efeitos do etanol sobre os parâmetros corporais e a morfometria dos neurônios mientéricos do duodeno de ratos submetidos ao alcoolismo crônico. Foram utilizados 14 Rattus norvegicus, mantidos durante 16 semanas, divididos em dois grupos: Grupo controle (n=7), que recebeu ração padronizada e água ad libitum, e Grupo experimental (n=7), que recebeu a mesma ração e aguardente de cana em concentrações crescentes ad libitum. Ao final do período, os ratos foram anestesiados para a realização da laparatomia e retirada do duodeno, que foi submetido à realização de preparados de membrana e corados com Giemsa. Os dados foram submetidos ao teste estatístico t (Student) com nível de significância de 5%. Os animais alcoolizados apresentaram médias inferiores na ingesta líquida, sólida, crescimento e peso corporal, sendo que a diferença entre as médias foi significativa (p<0,005). Nos animais do GE a área do pericário foi 40,6% menor que o GC, do núcleo 70,5% menor no GE e do citosol 26% menor no GE, sendo a diferença estatisticamente significante para as três regiões celulares observadas (p<0,05). Logo, o consumo crônico de etanol por 16 semanas interferiu na ingestão e ganho de peso dos animais e provocou redução na área do pericário, núcleo e citosol dos neurônios mientéricos do duodeno de ratos.

Ethanol is a molecule that diffuses easily through cellular membranes, and in cytosol leads to the production of toxic compounds that can alter cellular proteins and DNA. Thus, the aim of this study was to evaluate the effects of ethanol on body parameters and morphometry of duodenum myenteric neurons of rats submitted to chronic alcoholism. We used 14 Rattus norvegicus, which were kept for 16 weeks, divided into two groups: control group (n = 7), which received standard feed and water ad libitum, and experimental group (n = 7), which received the same feed and sugar cane brandy in increasing concentrations ad libitum. At the end of the period, the rats were anesthetized for the realization of laparotomy and duodenum removal, which was submitted to preparations of membrane and stained with Giemsa. The data were submitted to statistical test t (Student) with a significance level of 5%. The intoxicated animals showed lower average in liquid and solid intake, growth and body weight, and the difference between both groups average was significant (p <0.005). In animals of the EG, the area of pericardium was 40.6% lower than the CG, the nucleus was 70.5% lower in the EG, and cytosol was 26% lower in the EG. The difference is statistically significant for all the three cellular regions observed (p <0.05). Therefore, the chronic consumption of ethanol for 16 weeks interfered with intake and weight gain of animals, and caused a reduction in the area of perikarion, nucleus and cytosol of duodenum myenteric neurons in rats.

A case of renal cancer with TFE3 gene fusion in an elderly man. Clinical, radiological and surgical findings.

Sporadic cases of a particular group of renal cancers associated with a translocation involving Xp11.2, known as the TFE3 transcription factor gene, have been reported in the last 20 years. The group was also classified in 2004 WHO kidney carcinoma classifications. A 79-year-old male patient was investigated at the outpatient department for gross intermittent hematuria. Sonography showed a spherical left kidney with increased total size, without evidence of the corticomedullary differentiation due to parenchymal dyshomogeneity with a neoplasm aspect. CT confirmed the sonographic left kidney findings and showed gross node involvement. Angiography did not show any pathological arterial circulation, but massive thrombotic involvement of the renal vein was evident. Radical nephrectomy with thrombectomy and staging lymphectomy were performed. At pathological examination the kidney parenchyma was completely substituted by white firm tissue. Microscopically the tumor was composed of papillary structures lined by epithelial cells with a clear cytoplasm. Multiple node metastases were found. Immunohistochemical examination showed negativity for epithelial markers (cytokeratin and epithelial membrane antigen) and reactivity for CD10 and TFE3. The genetic and histological aspects of this rare tumor are reported. In addition, we describe clinical, radiological and surgical findings.

Análise morfométrica dos neurônios da curvatura gástrica maior glandular de ratos induzidos ao alcoolismo / Morphometric analysis of gastric neurons of the greater curvature glandular of rats induced to alcoholism

Objetivo: analisar os efeitos do álcool sobre a morfometria dos neurônios do plexo mioentérico da curvatura gástricamaior glandular de ratos submetidos à ingestão crônica de álcool. Método: utilizados 14 ratos Wistar, com 90 dias,distribuídos em: Controle (GC, n=7) e Experimental (GE, n=7). O GC recebeu durante 120 dias ração para roedorese água ad libitum, enquanto o GE recebeu ração e aguardente de cana ad libitum, diluída em água em concentraçõescrescentes de 5% até 30%. A curvatura maior do estômago glandular foi submetida à realização de preparados demembrana de acordo com o método de Giemsa. Foram mensurados 40 campos microscópicos de cada animal. Os cálculosestatísticos realizados utilizando-se o software Prisma 2.0, por meio do teste T de Student com nível de significânciade 5%. Resultados: verificou-se que média da área dos neurônios no GC foi de 43,56±2,69 ?m2 e no GE de 56,03±2,16 ?m2. Quando comparados os pesos finais dos dois grupos observamos que os animais experimentais tiveram umaredução de peso corporal de 18,94% em relação aos controles, com p=0.0002. Conclusão: a ingesta crônica de álcoolem ratos aumentou a área neuronal da curvatura maior do estômago glandular

Objective: to analyze the alcohol effects on the neurons? morphology in the myenteric plexus of the greater gastriccurvature of rats? glandular stomach subjected to chronic alcohol ingestion. Method: 14 Wistar rats with 90 days wereused, distributed into two groups: Control Group (CG n=7) and Experimental Group (EG n = 7). CG animals receivedfood and water ad libitum during 120 days, while EG animals received food and sugar cane brandy ad libitum dilutedin water at increasing concentrations from 5% to 30%. The greater gastric curvature of rats, glandular stomach wassubjected to implementation of membrane preparations according to the Giemsa?s method. 40 microscopic s weremeasured for each animal. Statistical calculations were performed using Prism 2.0 software and t-Student test withsignificance level of 5%. Results: it was identified that neurons? average area in CG was 43,56 ± 2,69 ?m2 and 56,03 ±2,16 ?m2 in EG. When comparing both groups? final weights it was observed that experimental animals had a reductionof body weight of 18.94% when compared to the control group, p = 0.0002. Conclusion: chronic alcohol ingestion inrats increased greater gastric curvature neurons of glandular stomach

Influence of orofacial pain, in superficial and deep tissue, on the anxiety levels in rats / Influência da dor orofacial, em tecidos superficiais e profundos, no nível de ansiedade em ratos

Purpose: The present study aimed to determine the influence of orofacial pain in superficial and deep tissue on anxiety levels in male Wistar rats. Methods: Orofacial pain was induced by injecting 50 µL of 1% formalin in the lip (superficial tissue) and in the temporomandibular joint (TMJ) (deep tissue) of male Wistar rats. The anxiety level was measured by the elevated plus maze (EPM) test. The standard measures (number of entries into open and enclosed arms, and time spent in opened ones), were recorded. The three measures of plus-maze behavior calculated were the percentage of entries into the openarms (% EOA) (100 X open/total), the percentage of time spent in the open arms (% TSOA) (100 X open/total) and the number of entries into the closed arms (NECA). Data were analyzed with ANOVA and Tukey's tests. Results: The formalin injection in the lip and in the TMJ region significantly increased the anxiety level as measured by the percentage of time spent inside and the number of entries in the open arms of the EPM test, but did not increase the general activity measured by the number of entrances in the closed and opened arms. Anxiety response was significantly reduced by Diazepam® administration (1 mg/kg) before the formalin injection to lip/TMJ. Conclusion: The results suggest that the orofacial nociceptive response induced by the injection of 1% formalin in the lip or TMJ region increases the anxiety level in rats and that Diazepam® can reduce it.

Objetivo: Este estudo objetivou determinar a influência da dor orofacial, em tecidos superficiais e profundos, sobre o nível de ansiedade em ratos machos Wistar. Metodologia: A dor orofacial foi induzida pela injeção de 50 µL formalina a 1%, na região de lábio (tecido superficial) e da articulação temporomandibular (ATM) (tecido profundo) de ratos machos Wistar. A ansiedade foi medida utilizando o teste do labirinto em cruz elevado (LCE). Resultados: Os resultados demonstraram que as injeções de formalina no lábio e na ATM aumentaram significativamente o nível de ansiedade, medido pela porcentagem de tempo gasto nos braços abertos e pela porcentagem de entrada nos braços abertos do LCE, masnão aumentou a atividade geral medida pelo número de entradas nos braços abertos e fechados do LCE. A ansiedade foi significantemente reduzida pela administração de Diazepam® (1 mg/kg), previamente à injeção de formalina no lábio e na ATM. Conclusão: Os resultados sugerem que a dor orofacial induzida pela injeção de formalina 1% na região do lábio e da ATM é capaz de aumentar o nível de ansiedade em ratos e que a administração de Diazepam® pode reduzir esta resposta.

Análise quantitativa dos neurônios mioentéricos da curvatura gástrica menor do estômago glandular de ratos induzidos à carência proteica / Quantitative analysis of the myenteric neurons of the smaller gastric curvature of the glandular stomach in rats submitted to protein deficiency

A deficiência nutricional pode causar nas células a perda de sua capacidade de absorção, de gerar energia, bem como de mediar o transporte de pequenas moléculas e de sintetizar macromoléculas. Objetivou-se quantificar os neurônios mioentéricos da curvatura gástrica menor do estômago glandular de ratos controle e de ratos submetidos a carência protéica. Foram utilizados 10 ratos machos, com 90 dias de idade, divididos em grupos controle (GC) e experimental (GE). O GC recebeu, durante 120 dias, ração com 22% de proteina e água, enquanto GE recebeu ração com teor proteico de 4% e água. Após os experimentos os animais foram submetidos à eutanásia, laparotomia vertical e retirada do estômago. Realizou-se a microdissecação sob estereomicroscópico dos segmentos, com a retirada da túnica mucosa e da tela submucosa. Foi utilizada a técnica de coloração de Giemsa para evidenciar os neurônios mientéricos. Os neurônios foram contados com auxílio de um microscópio de luz com aumento de 400 x, com 40 campos de cada animal. Os cálculos estatísticos foram realizados por meio do teste ?t? de Student, na forma média ± erro padrão da média, com nível de significância de 5%. No grupo controle foram encontrados 23,51±2,7 neurônios e no grupo experimental 21,38±1,7. Verificou-se que as diferenças não são estatisticamente significantes. Podemos verificar que a dieta imposta ocasionou um quadro de desnutrição que não provocou alterações quantitativas nos neurônios mioentéricos da curvatura gástrica menor do estômago glandular.

Nutritional deficiency creates conditions in which the cells lose their capability of absorption and energy generation, as well as their capability to mediate the transport of small molecules and digest macromolecules. The objective of this study was to quantify the myenteric neurons of the smaller gastric curvature of the glandular stomach of control rats and rats submitted to protein deficiency. Ten 90-day-old male rat were divided into control group (CG) and experimental group (EG). The CG received ration with 22% of protein and water during 120 days, while the EG received ration with the protein percentage at 4% and water. After the experiment, the animals were submitted to euthanasia, vertical laparotomy and each animal had its stomach removed. Micro-dissection was performed under stereomicroscopic of the segments with the removal of the mucosa and the tela submucosa. The Giemsa staining technique was used to evidence the myenteric neurons. The neurons were counted, with the aid of a 400X optical microscope ? 40 s from each animal. The statistical calculations were performed through the T-test, mean ± standard deviation, with a 5% significance level. In the GC, 23.51 ± 2.7 neurons were found, and 21.38 ± 1.7 in the EG. Differences were not verified to be statistically significant. We can verify that the imposed diet resulted in malnutrition which did not induce quantitative changes in the myenteric neurons of the smaller gastric curvature of the glandular stomach.

Influência do ciclo estral sobre a sensibilidade da resposta cronotrópica à norepinefrina em ratas submetidas a estresse oxidativo / Influence of estrous cycle on the sensitivity of cronotropic answer to norepinephrine in rats submitted to acute stress

O estresse pode alterar a sensibilidade da resposta cronotrópica às catecolaminas em vários tecidos. O objetivo deste estudo foi avaliar a sensibilidade à norepinefrina (NE) em átrios direitos de ratas submetidas ao estresse agudo por nataçäo nas fases de estro e proestro. Ratas Wistar em estro ou proestro foram submetidas a um sessäo de 50min de nataçäo, após a qual foram anestesiadas e sacrificadas. Os átrios direitos destes animais e de ratas controle foram isolados para a obtençäo de curvas concentraçäo-efeito à NE antes e após o bloqueio dos sistemas de metabolizaçäo das catecolaminas (fenoxibenzamina + estradiol). Os dados foram analisados por ANOVA ou teste t de Student. Näo houve diferenças...

Análise do significado do tempo de imobilidade em modelos experimentais de natacão / Analysis of the meaning of the immobility time in swimming experimental models

O objetivo do trabalho foi testar a hipótese de que a interpretação do tempo de imobilidade (desamparo aprendido ou adaptação) pode variar conforme o modelo utilizado (teste da natação forçada ou estresse por natação). Foram analisados o tempo de imobilidade (TI) e a mobilização de glicogênio de ratos submetidos à natação em dois protocolos: estresse por natação (EN) e teste da natação forçada (TNF). Também comparamos os efeitos da desipramina e diazepam. Os experimentos foram filmados para análise do TI. Os ratos, após a sessão de natação, foram sacrificados e amostras do fígado e músculos foram preparadas para quantificação do glicogênio. O TI foi menor no EN comparado ao TNF (pÍ0.001)...