RESUMO
Second-order spinal cord excitatory neurons play a key role in spinal processing and transmission of pain signals to the brain. Exogenously induced change in developmentally imprinted excitatory neurotransmitter phenotypes of these neurons to inhibitory has not yet been achieved. Here, we use a subpial dorsal horn-targeted delivery of AAV (adeno-associated virus) vector(s) encoding GABA (gamma-aminobutyric acid) synthesizing-releasing inhibitory machinery in mice with neuropathic pain. Treated animals showed a progressive and complete reversal of neuropathic pain (tactile and brush-evoked pain behavior) that persisted for a minimum of 2.5 months post-treatment. The mechanism of this treatment effect results from the switch of excitatory to preferential inhibitory neurotransmitter phenotype in dorsal horn nociceptive neurons and a resulting increase in inhibitory activity in regional spinal circuitry after peripheral nociceptive stimulation. No detectable side effects (e.g., sedation, motor weakness, loss of normal sensation) were seen between 2 and 13 months post-treatment in naive adult mice, pigs, and non-human primates. The use of this treatment approach may represent a potent and safe treatment modality in patients suffering from spinal cord or peripheral nerve injury-induced neuropathic pain.
Assuntos
Neuralgia , Nociceptores , Animais , Técnicas de Transferência de Genes , Camundongos , Neuralgia/etiologia , Neuralgia/terapia , Células do Corno Posterior , Medula Espinal , Corno Dorsal da Medula Espinal , SuínosRESUMO
In the rat, tail nerves are the longest peripheral nerves in their body. We suggest that ventral caudal nerve (VCN) may serve as a model for studying nerve injury and long distance regeneration. For this purpose, we have studied the anatomy and morphometry of the VCN in control animals. 10â¯cm long segment of the VCN was removed, and transversal sections were collected at 10â¯mm distances. The myelinated axons were counted, and the series of data were used to characterize the craniocaudal tapering of the nerve. In a separate group of animals, retrograde tracing with Fluorogold was used to localize and quantitate the spinal neurons projecting their axons into the VCN. After complete nerve transection, the time course of histopathological changes in the distal segment was studied. The primary goal was to define the time needed for axonal disintegration. In later periods, axonal debris removal and rearrangement of tissue elements was documented. After compression injury (axonotmesis), Wallerian degeneration was followed by spontaneous regeneration of axons. We show that the growing axons will span the 10â¯cm distance within 4-8 weeks. After different survival periods, the numbers of regenerating axons were counted at 10â¯mm distances. These data were used to characterize the dynamics of axonal regeneration during 4 months' survival period. In the present study we show that axonal regeneration across 10â¯cm distance can be studied and quantitatively analyzed in a small laboratory animal.
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Measuring viscosity in volumes smaller than a microliter is a challenging endeavor. A new type of microscopic viscometers is presented to assess the viscosity of Newtonian liquids. Micron-sized flexible polymer cantilevers are created by two-photon polymerization direct laser writing. Because of the low stiffness and high elasticity of the polymer material the microcantilevers exhibit pronounced Brownian motion when submerged in a liquid medium. By imaging the cantilever's spherically shaped end, these fluctuations can be tracked with high accuracy. The hydrodynamic resistance of the microviscometer is determined by fitting the power spectral density of the measured fluctuations with a theoretical frequency dependence. Validation measurements in water-glycerol mixtures with known viscosities reveal excellent linearity of the hydrodynamic resistance to viscosity, allowing for a simple linear calibration. The stand-alone viscometer structures have a characteristic size of a few tens of microns and only require a very basic external instrumentation in the form of microscopic imaging at moderate framerates (~ 100 fps). Thus, our results point to a practical and simple to use ultra-low volume viscometer that can be integrated into lab-on-a-chip devices.
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Precisely controlled manipulation of nonadherent single cells is often a pre-requisite for their detailed investigation. Optical trapping provides a versatile means for positioning cells with submicrometer precision or measuring forces with femto-Newton resolution. A variant of the technique, called indirect optical trapping, enables single-cell manipulation with no photodamage and superior spatial control and stability by relying on optically trapped microtools biochemically bound to the cell. High-resolution 3D lithography enables to prepare such cell manipulators with any predefined shape, greatly extending the number of achievable manipulation tasks. Here, it is presented for the first time a novel family of cell manipulators that are deformable by optical tweezers and rely on their elasticity to hold cells. This provides a more straightforward approach to indirect optical trapping by avoiding biochemical functionalization for cell attachment, and consequently by enabling the manipulated cells to be released at any time. Using the photoresist Ormocomp, the deformations achievable with optical forces in the tens of pN range and present three modes of single-cell manipulation as examples to showcase the possible applications such soft microrobotic tools can offer are characterized. The applications describe here include cell collection, 3D cell imaging, and spatially and temporally controlled cell-cell interaction.
Assuntos
Pinças Ópticas , Análise de Célula Única , Humanos , Robótica/instrumentação , Elasticidade , AnimaisRESUMO
Spinal cord injury (SCI) resulting from trauma decreases the quality of human life. Numerous clues indicate that the limited endogenous regenerative potential is a result of the interplay between the inhibitory nature of mature nervous tissue and the inflammatory actions of immune and glial cells. Knowledge gained from comparing regeneration in adult and juvenile animals could draw attention to factors that should be removed or added for effective therapy in adults. Therefore, we generated a minimal SCI (mSCI) model with a comparable impact on the spinal cord of Wistar rats during adulthood, preadolescence, and the neonatal period. The mechanism of injury is based on unilateral incision with a 20 ga needle tip according to stereotaxic coordinates into the dorsal horn of the L4 lumbar spinal segment. The incision should harm a similar amount of gray matter on a coronal section in each group of experimental animals. According to our results, the impact causes mild injury with minimal adverse effects on the neurological functions of animals but still has a remarkable effect on nervous tissue and its cellular and humoral components. Testing the mSCI model in adults, preadolescents, and neonates revealed a rather anti-inflammatory response of immune cells and astrocytes at the lesion site, as well as increased proliferation in the central canal lining in neonates compared with adult animals. Our results indicate that developing nervous tissue could possess superior reparative potential and confirm the importance of comparative studies to advance in the field of neuroregeneration.
Assuntos
Animais Recém-Nascidos , Proliferação de Células , Modelos Animais de Doenças , Ratos Wistar , Traumatismos da Medula Espinal , Animais , Traumatismos da Medula Espinal/imunologia , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologia , Proliferação de Células/fisiologia , Ratos , Medula Espinal/patologia , Medula Espinal/imunologia , Astrócitos/patologia , FemininoRESUMO
The basic features of bronchial asthma are dyspnea with wheezing and objectively confirmed obstructive respiratory disorder reversible after inhalation of bronchodilators. In stable intermittent bronchial asthma, these features are not present; therefore confirmation of asthma consists of the presence of bronchial hyperresponsiveness (BHR). In the present study, there were 902 bronchoprovocation tests performed for the verification of BHR. A significant criterium for BHR is a decrease of FEV(1) of 20% from the baseline level. Every test either positive or negative was finished with inhalation of four doses of salbutamol through a spacer. We obtained 675 bronchoprovocation tests negative and 227 positive. Among the 675 subjects with a negative test there were 49 subjects who after inhalation of salbutamol had an increase in FEV(1) of ≥20% above baseline. The bronchodilatatory response of these 49 subjects, makes one think about the so-called latent bronchospasm present already at baseline, limiting further constriction during bronchoprovocation tests. The detection of such latent bronchospasm in BHR increases the number of patients with an objectively confirmed bronchial asthma from 25.0% to 30.5%. Our results suggest that bronchodilation test be performed in all patients with suspected bronchial asthma to allow detecting latent bronchospasm as an initial stage of the disease.
Assuntos
Asma/fisiopatologia , Hiper-Reatividade Brônquica/etiologia , Adolescente , Adulto , Idoso , Testes de Provocação Brônquica , Feminino , Volume Expiratório Forçado , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
This study aims to examine experimental conditions in which active particles are forced by their surroundings to move forward and backward in a continuous oscillatory manner. The experimental design is based on using a vibrating self-propelled toyrobot called hexbug, which is placed inside a narrow channel closed on one end by a rigid moving wall. Using the end-wall velocity as a controlling factor, the main forward mode of the hexbug movement can be turned to mostly rearward mode. We investigate the bouncing hexbug motion on both experimental and theoretical grounds. The Brownian model of active particles with inertia is employed in the theoretical framework. The model itself uses a pulsed Langevin equation in order to simulate abrupt changes in velocity that mimic hexbug propulsion in the moments when its legs make contact with the base plate. Significant directional asymmetry is caused by the legs bending backward. We demonstrate that the simulation successfully reproduces the experimental characteristics of hexbug motion after regressing the spatial and temporal statistical characteristics, especially when directional asymmetry is under consideration.
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We present a method that allows preparing histological sections from large blocks of nervous tissue embedded in epoxy resin. Resin-embedding provides excellent resolution especially for the myelin-rich white matter and is often being used for visualizing the myelinated axons in peripheral nerves. However, because of the limited penetration of the reagents, only very small tissue specimens can be processed in this way. Here, we describe a method that enables to embed large specimens and their sectioning on a standard sliding microtome. To process the large specimens, modifications in several steps of the processing technique had to be made. In this paper we demonstrate, that with this technique 1-3 µm thick transversal sections can be prepared from the resin-embedded specimens as large as rat brain hemisphere. Such a large section allows simultaneously: 1.) overviewing and delineating the gross anatomical structures, and 2.) observing the subcellular details at the highest possible optical magnifications. Such a large section with excellent resolution allows application of unbiased stereological methods and reliable quantification of very small objects within the area of interest.
Assuntos
Axônios/metabolismo , Resinas Epóxi , Bainha de Mielina/metabolismo , Inclusão do Tecido/métodos , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Limite de Detecção , Microscopia/métodos , Microscopia/normas , Nervos Periféricos/citologia , Nervos Periféricos/metabolismo , Ratos , Inclusão do Tecido/normasRESUMO
Photopolymer nanowires prepared by two-photon polymerization direct laser writing (TPP-DLW) are the building blocks of many microstructure systems. These nanowires possess viscoelastic characteristics that define their deformations under applied forces when operated in a dynamic regime. A simple mechanical model was previously used to describe the bending recovery motion of deflected nanowire cantilevers in Newtonian liquids. The inverse problem is targeted in this work; the experimental observations are used to determine the nanowire physical characteristics. Most importantly, based on the linear three-parameter solid model, we derive explicit formulas to calculate the viscoelastic material parameters. It is shown that the effective elastic modulus of the studied nanowires is two orders of magnitude lower than measured for the bulk material. Additionally, we report on a notable effect of the surrounding aqueous glucose solution on the elasticity and the intrinsic viscosity of the studied nanowires made of Ormocomp.
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BACKGROUND: In cases of acute surgery or trauma, the most effective method of increasing the level of estrogen in postmenopausal women is its administration immediately pre- or postsurgery. However, in our previous study (J Surg Res 2008; 147:117-122) we showed that postsurgical administration of nonspecific estrogen receptor (ER) agonist decreases wound tensile strength. Therefore, the aim of this study was to evaluate whether this effect is mediated via the alpha or beta ER. MATERIALS AND METHODS: Three months prior to the wound healing experiment, 18 rats were anesthetized and underwent ovariectomy (OVX), while another six rats were sham operated. Two parallel full thickness skin incisions were performed on the back of each rat. Doses of 1mg/kg of either PPT (ER-alpha agonist) or DPN (ER-beta agonist) were administered to 12 OVX rats for 6 d postoperatively, whereas all other animals received vehicle. After 6 d, all animals were sacrificed and samples removed for wound tensile strength measurement and histologic evaluation. RESULTS: The mean wound tensile strength of PPT-treated rats (6.8+/-1.9 g/mm2) was significantly lower compared with all other groups (P<0.05). No significant differences were observed between DPN-treated (8.9+/-2.2 g/mm2), non-OVX vehicle-treated (8.7+/-2.0 g/mm2), and OVX vehicle-treated (9.1+/-1.7 g/mm2) rats. Nevertheless, no remarkable differences were found between groups during histologic evaluation. CONCLUSION: Our results indicate that the wound tensile strength decrease is mediated through the alpha rather than beta ER.
Assuntos
Receptor alfa de Estrogênio/agonistas , Receptor beta de Estrogênio/agonistas , Terapia de Reposição de Estrogênios , Resistência à Tração/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Animais , Feminino , Nitrilas/farmacologia , Ovariectomia , Fenóis/farmacologia , Pirazóis/farmacologia , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Pele/lesões , Pele/patologiaRESUMO
Nocturnal cardiac arrhythmias (NCA) were analyzed in patients with sleep apnea/hypopnea syndrome (SAHS) and controls. Occurrence and severity of NCA were compared in 33 SAHS patients and 16 control subjects, matched for cardiovascular risk factors. Continuous overnight polysomnography provided ECG, respiratory and sleep parameters for a comparative analysis. Various types and severity of NCA were detected already in moderate SAHS (apnea/hypopnea index = 26 ±15.6/h), reflecting the respiratory and atherosclerotic changes. Moderately severe arrhythmias, represented with benign and 2 complex types were caused by hypoxemia characterized by AHI, minimal SaO2, and lower values after desaturation. Three-time higher prevalence of complex arrhythmias in SAHS patients was not significantly different by usual statistical comparison, likely due to a low number of controls and a joint occurrence of various types and complex severity of arrhythmias in some patients. Therefore, a complex assessment of different types and varying severity of arrhythmias would require a scale specifically constructed for their evaluation.
Assuntos
Arritmias Cardíacas/etiologia , Hipóxia/complicações , Síndromes da Apneia do Sono/complicações , Adulto , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Gene silencing with virally delivered shRNA represents a promising approach for treatment of inherited neurodegenerative disorders. In the present study we develop a subpial technique, which we show in adult animals successfully delivers adeno-associated virus (AAV) throughout the cervical, thoracic and lumbar spinal cord, as well as brain motor centers. One-time injection at cervical and lumbar levels just before disease onset in mice expressing a familial amyotrophic lateral sclerosis (ALS)-causing mutant SOD1 produces long-term suppression of motoneuron disease, including near-complete preservation of spinal α-motoneurons and muscle innervation. Treatment after disease onset potently blocks progression of disease and further α-motoneuron degeneration. A single subpial AAV9 injection in adult pigs or non-human primates using a newly designed device produces homogeneous delivery throughout the cervical spinal cord white and gray matter and brain motor centers. Thus, spinal subpial delivery in adult animals is highly effective for AAV-mediated gene delivery throughout the spinal cord and supraspinal motor centers.
Assuntos
Esclerose Lateral Amiotrófica/terapia , Dependovirus/metabolismo , Inativação Gênica , Técnicas de Transferência de Genes , Neurônios Motores/patologia , Degeneração Neural/terapia , Pia-Máter/patologia , Medula Espinal/patologia , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/fisiopatologia , Animais , Atrofia , Progressão da Doença , Potencial Evocado Motor , Feminino , Regulação da Expressão Gênica , Humanos , Inflamação/patologia , Interneurônios/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Desenvolvimento Muscular , Degeneração Neural/genética , Degeneração Neural/fisiopatologia , Pia-Máter/fisiopatologia , Primatas , Dobramento de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/administração & dosagem , Medula Espinal/diagnóstico por imagem , Medula Espinal/fisiopatologia , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , SuínosRESUMO
Ependymal cells (EC) in the spinal cord central canal (CC) are believed to be responsible for the postnatal neurogenesis following pathological or stimulatory conditions. In this study, we have analyzed the proliferation of the CC ependymal progenitors in adult rats processed to compression SCI or enhanced physical activity. To label dividing cells, a single daily injection of Bromo-deoxyuridine (BrdU) was administered over a 14-day-survival period. Systematic quantification of BrdU-positive ependymal progenitors was performed by using stereological principles of systematic, random sampling, and optical Dissector software. The number of proliferating BrdU-labeled EC increased gradually with the time of survival after both paradigms, spinal cord injury, or increased physical activity. In the spinal cord injury group, we have found 4.9-fold (4 days), 7.1-fold (7 days), 4.9-fold (10 days), and 5.6-fold (14 days) increase of proliferating EC in the rostro-caudal regions, 4 mm away from the epicenter. In the second group subjected to enhanced physical activity by running wheel, we have observed 2.1-2.6 fold increase of dividing EC in the thoracic spinal cord segments at 4 and 7 days, but no significant progression at 10-14 days. Nestin was rapidly induced in the ependymal cells of the CC by 2-4 days and expression decreased by 7-14 days post-injury. Double immunohistochemistry showed that dividing cells adjacent to CC expressed astrocytic (GFAP, S100beta) or nestin markers at 14 days. These data demonstrate that SCI or enhanced physical activity in adult rats induces an endogenous ependymal cell response leading to increased proliferation and differentiation primarily into macroglia or cells with nestin phenotype.
Assuntos
Células-Tronco Adultas/fisiologia , Epêndima/fisiologia , Epêndima/fisiopatologia , Compressão da Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/fisiopatologia , Animais , Bromodesoxiuridina , Contagem de Células , Proliferação de Células , Imuno-Histoquímica , Masculino , Atividade Motora , Ratos , Ratos Wistar , Canal Medular/fisiologia , Canal Medular/fisiopatologia , Vértebras TorácicasRESUMO
Using a rat model of ischemic paraplegia, we examined the expression of spinal AMPA receptors and their role in mediating spasticity and rigidity. Spinal ischemia was induced by transient occlusion of the descending aorta combined with systemic hypotension. Spasticity/rigidity were identified by simultaneous measurements of peripheral muscle resistance (PMR) and electromyography (EMG) before and during ankle flexion. In addition, Hoffman reflex (H-reflex) and motor evoked potentials (MEPs) were recorded from the gastrocnemius muscle. Animals were implanted with intrathecal catheters for drug delivery and injected with the AMPA receptor antagonist NGX424 (tezampanel), glutamate receptor 1 (GluR1) antisense, or vehicle. Where intrathecal vehicle had no effect, intrathecal NGX424 produced a dose-dependent suppression of PMR [ED50 of 0.44 microg (0.33-0.58)], as well as tonic and ankle flexion-evoked EMG activity. Similar suppression of MEP and H-reflex were also seen. Western blot analyses of lumbar spinal cord tissue from spastic animals showed a significant increase in GluR1 but decreased GluR2 and GluR4 proteins. Confocal and electron microscopic analyses of spinal cord sections from spastic animals revealed increased GluR1 immunoreactivity in reactive astrocytes. Selective GluR1 knockdown by intrathecal antisense treatment resulted in a potent reduction of spasticiy and rigidity and concurrent downregulation of neuronal/astrocytic GluR1 in the lumbar spinal cord. Treatment of rat astrocyte cultures with AMPA led to dose-dependent glutamate release, an effect blocked by NGX424. These data suggest that an AMPA/kainate receptor antagonist can represent a novel therapy in modulating spasticity/rigidity of spinal origin and that astrocytes may be a potential target for such treatment.
Assuntos
Astrócitos/metabolismo , Rigidez Muscular/metabolismo , Espasticidade Muscular/metabolismo , Receptores de AMPA/biossíntese , Receptores de AMPA/genética , Isquemia do Cordão Espinal/metabolismo , Animais , Astrócitos/citologia , Células Cultivadas , Regulação da Expressão Gênica/fisiologia , Masculino , Rigidez Muscular/etiologia , Rigidez Muscular/genética , Espasticidade Muscular/etiologia , Espasticidade Muscular/genética , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/fisiologia , Medula Espinal/citologia , Medula Espinal/metabolismo , Isquemia do Cordão Espinal/complicações , Isquemia do Cordão Espinal/genéticaRESUMO
The c-fos gene expression method was used to localize brainstem neurons functionally related to the tracheal-bronchial cough on 13 spontaneously breathing, pentobarbitone anesthetized cats. The level of Fos-like immunoreactivity (FLI) in 6 animals with repetitive coughs (170+/-12) induced by mechanical stimulation of the tracheobronchial mucosa was compared to FLI in 7 control non-stimulated cats. Thirty-four nuclei were compared for the number of labeled cells. Enhanced cough FLI was found bilaterally at following brainstem structures, as compared to controls: In the medulla, FLI was increased in the medial, interstitial and ventrolateral subnuclei of the solitary tract (p < 0.02), in the retroambigual nucleus of the caudal medulla (p < 0.05), in the ambigual, paraambigual and retrofacial nuclei of the rostral medulla along with the lateral reticular nuclei, the ventrolateral reticular tegmental field (p < 0.05), and the raphe nuclei (p < 0.05). In pons, increased FLI was detected in the lateral parabrachial and Kölliker-Fuse nuclei (p < 0.01), in the posteroventral cochlear nuclei (p < 0.01), and the raphe midline (p < 0.05). Within the mesencephalon cough-related FLI was enhanced at the rostral midline area (p < 0.05), but a decrease was found at its caudal part in the periaqueductal gray (p < 0.02). Results of this study suggest a large medullary - pontine - mesencephalic neuronal circuit involved in the control of the tracheal-bronchial cough in cats.
Assuntos
Anestesia , Tronco Encefálico/metabolismo , Brônquios/fisiopatologia , Tosse/patologia , Tosse/fisiopatologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Animais , Pressão Sanguínea/fisiologia , Mapeamento Encefálico , Tronco Encefálico/patologia , Gatos , Tosse/etiologia , Feminino , Regulação da Expressão Gênica , Masculino , Neurônios/patologia , Estimulação Física/efeitos adversos , Proteínas Proto-Oncogênicas c-fos/genéticaRESUMO
A novel rotary viscometer--developed for the determination of rheologic properties of liquid/air interface layers--is presented. The instrument can be used to measure the shear viscosity and the shear elasticity of liquid surfaces. It contains a rotor floating on the liquid surface which is rotated by means of an electromagnetic torque. A torsion filament is used to calibrate the applied torque. The viscosity data are obtained on the basis of the Navier-Stokes equation solved for the rotation of a cylinder touching the surface of water and submerged into the water. The time behavior of the surface viscosity of films gradually formed from solutions of some proteins as well as their activation energy is presented.
RESUMO
According to previous opinion, the derivation of neurons and glia from the central canal (CC) lining of the spinal cord in rodents should occur in the embryonic period. Reports of the mitotic activity observed in the lining during postnatal development have often been contradictory, and proliferation was ascribed to the generation of ependymocytes, which are necessary for the elongation of CC walls. Our study quantifies the intensity of proliferation and determines the cellularity of the CC lining in reference to lumbar spinal segment L4 during the postnatal development of rats. The presence of dividing cells peaks in the CC lining on postnatal day 8 (P8), with division occurring in 19.2% ± 3.2% of cells. In adult rats, 3.6% ± 0.9% of cells still proliferate, whereas, in mice, 10.3% ± 2.3% of cells at P8 and only 0.6% ± 0.2% of cells in the CC lining in adulthood are proliferating. In the rat, the length of the cell cycle increases from 100.3 ± 35.7 hours at P1 to 401.4 ± 80.6 hours at P43, with a sudden extension between P15 and P22. Despite the intensive proliferation, the total cellularity of the CC lining at the L4 spinal segment significantly descended in from P8 to P15. According to our calculations, the estimated cellularity was significantly higher compared with the measured cellularity of the CC lining at P15. Our results indicate that CC lining serves as a source of cells beyond ependymal cells during the first postnatal weeks of the rat. J. Comp. Neurol. 525:693-707, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Proliferação de Células , Medula Espinal/citologia , Medula Espinal/crescimento & desenvolvimento , Animais , Animais Recém-Nascidos , Bromodesoxiuridina , Ciclo Celular , Epêndima/citologia , Epêndima/crescimento & desenvolvimento , Imunofluorescência , Antígeno Ki-67/metabolismo , Vértebras Lombares , Camundongos Endogâmicos BALB C , Microscopia Confocal , Neuroglia/citologia , Neurônios/citologia , Ratos Wistar , Especificidade da Espécie , Fatores de TempoRESUMO
In experimental and clinical studies, an objective assessment of peripheral muscle resistance represents one of the key elements in determining the efficacy of therapeutic manipulations (e.g. pharmacological, surgical) aimed to ameliorate clinical signs of spasticity and/or rigidity. In the present study, we characterize a newly developed limb flexion resistance meter which permits a semi-automated, computer-controlled measurement of peripheral muscle resistance (PMR) in the lower extremities during a forced flexion of the ankle in the awake rat. Ischemic paraplegia was induced in Sprague-Dawley rats by transient aortic occlusion (10 min) in combination with systemic hypotension (40 mm Hg). After ischemia the presence of spasticity component was determined by the presence of an exaggerated EMG activity recorded from gastrocnemius muscle after nociceptive or proprioceptive afferent activation and by velocity-dependent increase in muscle resistance. Rigidity was induced by high dose (30 mg/kg, i.p.) of morphine. Animals with defined ischemic spasticity or morphine-induced rigidity were then placed into a plastic restrainer and a hind paw attached by a tape to a metal plate driven by a computer-controlled stepping motor equipped with a resistance transducer. The resistance of the ankle to rotation was measured under several testing paradigms: (i) variable degree of ankle flexion (40 degrees, 50 degrees, and 60 degrees), (ii) variable speed/rate of ankle flexion (2, 3, and 4 sec), (iii) the effect of inhalation anesthesia, (iv) the effect of intrathecal baclofen, (v) the effect of dorsal L2-L5 rhizotomy, or (vi) systemic naloxone treatment. In animals with ischemic paraplegia an increased EMG response after peripheral nociceptive or proprioceptive activation was measured. In control animals average muscle resistance was 78 mN and was significantly increased in animals with ischemic spasticity (981-7900 mN). In ischemic-spastic animals a significant increase in measured muscle resistance was seen after increased velocity (4 > 3 > 2 sec) and the angle (40 degrees > 50 degrees > 60 degrees) of the ankle rotation. In spastic animals, deep halothane anesthesia, intrathecal baclofen or dorsal rhizotomy decreased muscle resistance to 39-80% of pretreatment values. Systemic treatment with morphine induced muscle rigidity and corresponding increase in muscle resistance. Morphine-induced increase in muscle resistance was independent on the velocity of the ankle rotation and was reversed by naloxone. These data show that by using this system it is possible to objectively measure the degree of peripheral muscle resistance. The use of this system may represent a simple and effective experimental tool in screening new pharmacological compounds and/or surgical manipulations targeted to modulate spasticity and/or rigidity after a variety of neurological disorders such as spinal cord traumatic or ischemic injury, multiple sclerosis, cerebral palsy, or Parkinson's disease.
Assuntos
Eletromiografia/instrumentação , Eletromiografia/métodos , Isquemia/diagnóstico , Rigidez Muscular/diagnóstico , Paraplegia/diagnóstico , Processamento de Sinais Assistido por Computador , Anestésicos Inalatórios/farmacologia , Animais , Baclofeno/farmacologia , Doença Crônica , Halotano/farmacologia , Injeções Espinhais , Isquemia/complicações , Isquemia/fisiopatologia , Morfina/farmacologia , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Relaxantes Musculares Centrais/farmacologia , Rigidez Muscular/induzido quimicamente , Rigidez Muscular/tratamento farmacológico , Músculo Esquelético/fisiopatologia , Entorpecentes/farmacologia , Paraplegia/etiologia , Paraplegia/fisiopatologia , Ratos , Rizotomia , Medula Espinal/efeitos dos fármacos , Medula Espinal/patologia , Medula Espinal/fisiopatologia , Raízes Nervosas Espinhais/cirurgiaRESUMO
Computer-based visualization of large tissue volumes with high resolution based on composing series of high-resolution confocal images is presented. GlueMRC and LinkMRC programs are introduced, implementing composition of overlapping series of optical sections captured by a confocal microscope, registration and subsequent composition of successive confocal stacks. Both programs are using an interactive approach in combination with automatic algorithms for image registration. Further, the method for obtaining surface renderings of microscopical structure under study is described. For this purpose, structure contours visible in the sections are interactively digitized using a Colon plug-in module running in Ellipse environment. Then the coordinates of the contours are processed by special modules in the graphic programming environment IRIS Explorer and the structure surface is rendered. The method is shown on the 3-D reconstruction of the capillary bed of human placental villi and chick embryonic gut and its vascular bed.
Assuntos
Capilares/ultraestrutura , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Software , Animais , Embrião de Galinha , Vilosidades Coriônicas/irrigação sanguínea , Vilosidades Coriônicas/ultraestrutura , Sistema Digestório/irrigação sanguínea , Sistema Digestório/embriologia , Sistema Digestório/ultraestrutura , Humanos , Microscopia Confocal/métodosRESUMO
Systematic study in animals indicated, that in addition to cough there are 2 distinct airway reflexes. The aspiration reflex (AspR) characterized by rapid and strong gasp-like inspiration provoked by stimulation of nasopharynx, nasal phyltrum or auricle of ear. The expiration reflex (ExpR) manifests by prompt expiration, induced by laryngeal stimulation. Both reflexes strongly activate the brainstem inspiratory or expiratory generators, respectively, and inhibit the opposite respiratory and various functional disorders. This paper indicates several functional disorders occurring during manipulation with airways in anaesthesiological practice, which can be influenced positively or negatively by application of these special reflexes (asphyxia, breath-holding, laryngospasm, bronchospasm, sleep apnoea episodes, arrhythmia, collapse, etc.). The AspR, ExpR and CR (cough reflex) have important clinical relevance in anaesthesia and emergency medicine applicable also in domestic therapy and in hardly accessible places particularly by application of ICT (Information & Communication Technologies) using a mobile connection of the patient with the remote hospital centre.