的放射自显影测量[<sup> 14</sup> J] -Iodoantipyrine大鼠脑继中央脑卒中后疼痛
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
中风出血已被证明是谁的神经性疼痛患者遭受超过8％的发生，4称为中央中风后疼痛（CPSP）1-3 CPSP可以从体感功能障碍，从而诱发过敏和异常性造成的。但是，体感功能障碍的CPSP病理生理机制仍然不明朗。例如，躯体感觉的损失从出血脑区的神经元传入神经阻滞造成的。痛觉过敏可由中央伤害感受神经元或中央去抑制，5，6，但所涉及的CPSP症状神经基板保持未知的过度兴奋引起的。一些研究表明，背外侧前额叶皮层（DPFC），喙前扣带皮层（ACC），杏仁核，海马，脑导水管周围灰质（PAG），喙腹内侧髓质，以及它们相互连接的调解处理痛觉7附加被证明LY，内侧前额叶皮质（内侧前额叶皮质）-amygdala电路参与疼痛相关的看法。在CPSP的病理生理机制8数据是多种多样的，神经基板的CPSP的激活需要进一步推敲。
[14 C] -Iodoantipyrine（IAP）的摄取来间接观察区域脑血流量（rCBF），假设大脑活动和CBF之间的关系。尽管[14 C] -IAP不能实时评估脑活动，如与功能性磁共振成像（fMRI），它具有若干优点。例如，[14 C] -IAP适合测量自发过程的病理状态中发生脑活动。9此外，[14 C] -IAP摄取没有麻醉测量。它还成本低于其它成像方法，包括磁共振成像和正电子发射断层扫描（PET）。的[14 C] -IAP方法已经建议适合measurin克自发性疼痛（ 例如 ，CPSP）由丘脑的腹侧基底核（VB）的病变引起的。9
本协议描述了如何执行[14 C] -IAP的方法来评估由丘脑VB的病变动物模型引起CPSP的神经基础的参与。该技术提供了确定所依据的行为和神经元水平CPSP症状的病理生理机制的一种方式。
Protocol
Representative Results
Discussion
在行为测试中，CPSP集团展出在冯弗雷测试热痛试验和机械力在基线和缩足阈值的降低周1 – 5，发现与先前的研究相一致14
的[14 C] -IAP方法依赖于大脑图像的不同的脑切片的定量分析的像素强度。为了评估在大脑的图像数据，所述像素信号强度被定义。在本研究中，对环境的背景信号定义为25.811 – 46.979 CPM。在[14 C] -IAP 0.001微居里滤纸的信号被定义为42 CP…
Disclosures
The authors have nothing to disclose.
Acknowledgements
本研究是由国家科学委员会授予柏庄徐中雄博士（NSC 99-2320-B-001-016-MY3，NSC 100-2311-B-001-003-MY3和NSC 102-2320-支持B-001-026-MY3）。这项工作是在生物医学科学，并获得资助中央研究院研究所进行。
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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