[オートラジオグラフィー測定<sup> 14</sup> C]中央郵便ストローク痛みに続いてラット脳で-Iodoantipyrine
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
脳卒中の出血は、神経因性疼痛に苦しむ患者の8％以上で起こることが示されており、中央の脳卒中後疼痛（CPSP）と呼ばれる。1-3 CPSPそれにより過敏症および異痛症を誘発する、感覚機能不全から生じ得る。4が、CPSPにおける体性感覚機能障害の病態生理学的メカニズムは不明のまま。例えば、体性感覚の喪失は出血性脳領域の神経求心路遮断に起因します。痛覚過敏は、中央侵害受容ニューロンまたは中枢脱抑制、5、6しかしCPSP症状に関与している神経基盤は不明のままの過剰興奮によって引き起こされることがあります。いくつかの研究は、背外側前頭前野（DPFC）、吻側前帯状皮質（ACC）、扁桃体、海馬、中脳水道周囲灰白質（PAG）、吻側腹内側髄質、および互いの接続が侵害受容処理を仲介することを示唆している。追加の7LY、内側前頭前皮質（のmPFC）-amygdala回路は、疼痛関連の知覚に関与していることが示された。CPSPの病態生理学的メカニズムの8データは多様であり、かつCPSPの神経基質の活性化がさらに精査が必要です。
[14 C] -Iodoantipyrine（IAP）の取り込みは、脳活動とCBFの関係を仮定して、間接的に局所脳血流量（のrCBF）を観察するために使用されます。 [14 C] -IAPは、機能的磁気共鳴画像法（fMRI）のように、リアルタイムで脳活動を評価することはできないが、それはいくつかの利点を有します。たとえば、[14 C] -IAPは自然に測定する病理学的状態の間に、脳のイベントを発生するのに適している。9また、[14 C] -IAPの取り込みは麻酔なしで測定されます。また、fMRIの陽電子放出断層撮影（PET）を含む他の画像化方法、より少ないコスト。 [14 C] -IAP方法はmeasurinために適切であることが示唆されています視床の腹側基底核（VB）の病変によって誘発されるグラム自発痛（ 例えば 、CPSP）。9
現在のプロトコルは、動物モデルにおける視床のVBの病変によって誘発されるCPSPの神経基盤の関与を評価するための[14 C] -IAP方法を実行する方法について説明します。技術は、行動と神経細胞レベルでCPSP症状の根底にある病態生理学的メカニズムを決定する方法を提供しています。
Protocol
Representative Results
Discussion
行動試験では、CPSPグループは、ベースライン時に、von Frey試験における熱痛試験および機械的な力で足引っ込め閾値の削減と1週間展示- 。5.調査結果は先行研究と一致していたが14
[14 C] -IAP方法は、異なる脳切片の定量分析のために脳画像のピクセル強度に依存しています。脳画像のデータを評価するために、画素の信号強度を定義しました。 46.979 C…
Disclosures
The authors have nothing to disclose.
Acknowledgements
本研究は、博士白-荘Shyu（NSC 99から2320-B-001から016-MY3、NSC 100から2311-B-001-003-MY3、およびNSCに国家科学委員会の助成金によってサポートされていました102-2320- B-001-026-MY3）。この作品は、中央研究院から資金提供を受け医科学研究所、で行いました。
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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