Medidas autoradiográficas de [<sup> 14</sup> C] -Iodoantipyrine no cérebro de um rato Seguindo Central pós-AVC Dor
Summary
We present a protocol to measure [14C]-iodoantipyrine (IAP) uptake and assess the activation of neural substrates that are involved in central post-stroke pain (CPSP) in a rodent model.
Abstract
Approximately 8% of stroke patients present symptoms of central post-stroke pain (CPSP). CPSP is associated with allodynia and hypersensitivity to nociceptive stimuli. Although some studies have shown that neuropathic pain may involve the dorsolateral prefrontal cortex, rostral anterior cingulate cortex, amygdala, hippocampus, periaqueductal gray, rostral ventromedial medulla, and medial thalamus, the neural substrates and their connections that mediate CPSP remain unclear. [14C]-Iodoantipyrine (IAP) uptake can be measured to evaluate spontaneously active pain. It can be used to assess the activation of neural substrates that may be involved in CPSP in an animal model. The [14C]-IAP method in rats is less expensive to perform compared with other brain mapping techniques. The present [14C]-IAP protocol is used to measure the activation of neural substrates that are involved in CPSP that is induced by lesions of the ventral basal nucleus (VB) of the thalamus in a rodent model.
Introduction
Hemorragia acidente vascular cerebral foi demonstrado que ocorrem em mais do que 8% de pacientes que sofrem de dor neuropática, a que se refere dor pós-derrame como central (CPSP). 1-3 CPSP pode resultar de disfunção somatossensorial, induzindo, assim, a hipersensibilidade e a alodinia. 4 Contudo , os mecanismos fisiopatológicos da disfunção somatossensorial em CPSP permanecem incertas. Por exemplo, a perda de sensações somáticas resulta de desaferentação neuronal na área do cérebro hemorrágico. A hiperalgesia pode ser causada pela hiper-excitabilidade de neurónios nociceptivos centrais ou desinibição Central, 5, 6, mas os substratos neurais que estão envolvidos nos sintomas de CPSP permanecem desconhecidos. Alguns estudos sugeriram que o córtex pré-frontal dorsolateral (CPFD), rostral do córtex cingulado anterior (ACC), a amígdala, o hipocampo, substância cinzenta periaquedutal (PAG), medula ventromedial rostral, e suas conexões com o outro mediar processamento nociceptivo. 7 adicionaiscircuitos -amygdala ly, medial córtex pré-frontal (mPFC) foram mostrado para ser envolvido na percepção relacionados com a dor. 8 Os dados sobre os mecanismos fisiopatológicos da CPSP são diversas, ea ativação de substratos neurais no CPSP precisa de uma análise mais aprofundada.
[14 C] -Iodoantipyrine captação (IAP) é usado para observar indirectamente o fluxo sanguíneo cerebral regional (FSCr), assumindo uma relação entre a atividade cerebral e CBF. Apesar de [14 C] -IAP não permite avaliar a actividade cerebral em tempo real, tal como acontece com a ressonância magnética (IRMf), tem várias vantagens. Por exemplo, [14 C] -IAP é adequado para a medição que ocorre espontaneamente eventos cerebrais durante estados patológicos. 9 Além disso, a [14 C] -IAP absorção é medida sem anestesia. Ele também custa menos do que outros métodos de imagem, incluindo fMRI e tomografia por emissão de positrões (PET). O [C 14] -IAP método tem sido sugerido para ser apropriado para measuring dor espontânea (p.ex., CPSP) que é induzido pelas lesões do núcleo basal ventral (VB) do tálamo. 9
O presente protocolo descreve como realizar a [14 C] -IAP método para avaliar o envolvimento de substratos neurais de CPSP que é induzido pelas lesões da VB do tálamo em um modelo animal. A técnica oferece uma maneira de determinar os mecanismos fisiopatológicos subjacentes a sintomas CPSP nos níveis comportamentais e neuronais.
Protocol
Representative Results
Discussion
Nos testes comportamentais, o grupo CPSP exibiram reduções do limiar de retirada da pata no teste de dor térmica e força mecânica no teste de von Frey no início do estudo e semanas 1 – 5. Os resultados foram consistentes com um estudo anterior 14.
O [C 14] -IAP método baseia-se na intensidade de pixel de imagens do cérebro para a análise quantitativa de diferentes fatias de cérebro. Para avaliar os dados das imagens do cérebro, a intensidade do sinal de pixel…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
O presente estudo foi apoiado por bolsas do Conselho Nacional de Ciência para Dr. Bai-Chuang Shyu (NSC 99-2320-B-001-016-MY3, NSC 100-2311-B-001-003-MY3 e NSC 102-2320- B-001-026-MY3). Este trabalho foi realizado no Instituto de Ciências Biomédicas, que receberam financiamento da Academia Sinica.
Materials
| Anesthetic: | |||
| Isoflurane | Halocarbon Products Corporation | NDC 12164-002-25 | 4% |
| Surgery | |||
| homeothermic blanket system | Harvard Apparatus | Model 50–7079 | body temperature were maintained at 36.5-37.5°C. |
| 10µl micro syringe | Hamilton | 80008, Model 1701SN | injected with collagenase |
| polyethylene-50 tubing | Becton, Dickinson and Company | 427411 | catheterized into external jugular vein |
| 1 c.c syringe | Terumo Medical Products | SS-01T | injected with 14C-IAP and saline. |
| saline (Sodium Chloride 0.9gm) | Taiwan Biotech Co., LTD. | 100-130-0201 | To flush the tube |
| Drugs: | |||
| type 4 collagenase | Sigma | C5138-500MG | 0.125 U |
| Gentamicin | Sigma | G1264-250MG | 6 mg/kg |
| Heparin | Sigma | H9399 | 20 U/ml; 0.1 ml/day |
| 14C-iodoantipyrine (IAP) | PerkinElmer | NEC712 | 125 mCi/kg in 300 ml of 0.9% saline |
| Potassium chloride | Merck | 1.04936.1000 | 3 M |
| Behavior system: | |||
| von Frey esthesiometer | Fabrication Enterprises, Inc. | Baseline Tactile Monofilaments 12-1666 | mechanical hyperalgesia was assessed by measuring the withdrawal response to a mechanical stimulus |
| plantar test apparatus | IITC Life Science | IITC 390G Plantar Test | Thermal hyperalgesia was assessed by measuring the hind paw withdrawal latency in response to radiant heat. |
| Brain slice: | |||
| Optimal Cutting Temperature compound | Sakura Fintek Inc | 4583 | embedded the brain |
| dry ice | frozen in dry ice/methylbutane (approximately −55°C) | ||
| methylbutane | Sigma | M32631-1L | frozen in dry ice/methylbutane (approximately −55°C) |
| Cryostat | Leica Biosystems Nussloch GmbH, Germany | Leica CM1850 | Coronal brain slice were sectioned on this machine. |
| Data analyze | |||
| exposure cassettes with a phosphor screen | Amersham Biosciences | 20 cm x 25 cm | The slices were dried on glass slides and placed alongside five standard filter papers with graded radioactivity. All of the slides were exposed to the cassettes at −20°C. |
| γ-counter | Beckman Coulter | Beckman LS 6500 Liquid Scintillation Counter | To measure the radioactivity count of the filter papers. |
| Typhoon 9410 Variable Mode Imager | GMI, Inc. | WS-S9410 | To read phosphor screen which was exposed by brain slice |
| Statistical Parametric Mapping (SPM) | Wellcome Centre for Neuroimaging | version 8 | all of the brains were averaged to create the final brain template. To determine significant differences between the images in these two groups, the images were derived by subtracting the sham group from the CPSP group. |
| ImageJ | http://imagej.nih.gov/ij | version 1.46 | Adjacent sections were aligned both manually and using Stack- Reg, an automated pixel-based registration algorithm in ImageJ software. All of the original three-dimensionally reconstructed brains were smoothed and normalized to the reference rat brain model. |
| Matlab | MathWorks | version 2009b | used Pearson correlation coefficients to examine the relationships between the CPSP and sham groups. An inter-regional correlation matrix was calculated across animals from each group. |
| Pajek | http://Pajek.imfm.si/ | version 3.06 | Graphical theoretical analysis was performed on networks defined by the above correlation matrices using Pajek software. |
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