マウスの脊髄損傷のモデルとしての光血栓誘発性局所虚血
Summary
Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice.
Abstract
Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) below the site of injury. The secondary ischemia that develops following the initial mechanical insult is a serious complication of the SCI and severely impairs the function and viability of surviving neuronal and non-neuronal cells in the SC. In addition, ischemia is also responsible for the growth of lesion during chronic phase of injury and interferes with the cellular repair and healing processes. Thus there is a need to develop a spinal cord ischemia model for studying the mechanisms of ischemia-induced pathology. Focal ischemia induced by photothrombosis (PT) is a minimally invasive and very well established procedure used to investigate the pathology of ischemia-induced cell death in the brain. Here, we describe the use of PT to induce an ischemic lesion in the spinal cord of mice. Following retro-orbital sinus injection of Rose Bengal, the posterior spinal vein and other capillaries on the dorsal surface of SC were irradiated with a green light resulting in the formation of a thrombus and thus ischemia in the affected region. Results from histology and immunochemistry studies show that PT-induced ischemia caused spinal cord infarction, loss of neurons and reactive gliosis. Using this technique a highly reproducible and relatively easy model of SCI in mice can be achieved that would serve the purpose of scientific investigations into the mechanisms of ischemia induced cell death as well as the efficacy of neuroprotective drugs. This model will also allow exploration of the pathological changes that occur following SCI in live mice like axonal degeneration and regeneration, neuronal and astrocytic Ca2+ signaling using two-photon microscopy.
Introduction
外傷性脊髄損傷（SCI）は、SCの感覚と自律神経機能に影響を与える壊滅的な臨床症状です。 SCIを生き残った患者は、多くの場合、大幅に日常活動や生活1の品質に影響を与える対麻痺を衰弱が残されています。実験SCIモデルはSCIの病態生理と関連する神経修復プロセスを理解するために、科学的な調査で必要不可欠なツールとなっています。これらのモデルはまた、機能回復を目的とする種々の実験の神経保護的介入の前臨床効力を試験するために使用されています。現在、実際にSCIモデルの大部分は、機械的に破壊し、SCを傷つけるために物理的な鈍力の使用を採用しています。これらの方法は、SC 2の挫傷、圧縮、転位や離断を含みます。ことが示唆されている主要な機械的な損傷、負傷したSC内における虚血セットの形での二次損傷後 3,4。二次虚血の原因は、大規模な組織変性、実質内出血、時には組織浮腫5-7による血管の閉塞によって含まれています。 SCの整合性がさらに影響される二次的損傷の結果として、ニューロンとグリア細胞がひどく機能および生存度が損なわれ、虚血性ペナンブラ脳卒中後の成長に類似した損傷の慢性期の間に成長を梗塞につながるアポトーシスを受けます8,9。興奮毒性、フリーラジカル生成、および炎症などのいくつかのメカニズムは、SCI 10,11次の虚血性細胞死の原因であることが報告されています。また、SCの虚血は、多くの場合、患者12,13に対麻痺を引き起こす胸腹部大動脈瘤修復手術の重篤な合併症です。このような高い臨床的影響にもかかわらず、高い再現性脊髄虚血の非常にいくつかのモデルは、現在利用可能です。
NTは">光血栓（PT）は、技術が高度に再現、かなり非侵襲的である。脳14-20における局所的虚血の誘導のために一般的に使用される方法であり、脳17の露出した領域に正確な焦点虚血性病変を生成します-21。これは、ローズベンガル（RB）16-20,22又はエリスロシンBなどの光活性染料の全身投与により達成されるが、適切な光源を用いて血管の局所照射し23。色素の光活性化は、フリーラジカルの生成を引き起こします滑らかな血管内皮の完全性を破壊し、そして血小板がその後容器24によって供給される領域内の梗塞内血栓結果により血液の流れの閉塞、血栓を形成し、蓄積させる。ためには、制御を容易にします照射の強度および持続時間は、この手順は非常に均一で再現性の梗塞を生じる。また、この方法はinfarcを誘導するために使用することができます虚血の影響の空間（ 例えば 、対白質、灰白質）の理解を可能にする様々な解剖学的位置でT。現在の研究の目的は、マウスにおけるSC虚血の簡単かつ再現性の高いモデルを開発することです。我々は、マウスにおけるSC虚血のPTモデルの手順を説明しました。組織学および免疫染色の結果から、PTが効果的にSC梗塞、神経細胞脱落と反応性神経膠症を誘発することができることを実証しました。
Protocol
Representative Results
Discussion
本研究では、SCの虚血の光血栓モデルを説明しました。遺伝工学の進歩が可能SCにおける虚血性病態生理に関与する特定の遺伝子の影響を研究するために作られた市販のトランスジェニックマウスの急増がありました。研究の目的は、脊髄虚血の再現可能なマウスモデルを開発することでした。ここでは、マウスでSCIを誘導するために皮質PTモデルを適応しました。手術後のT11胸?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
この作品は、国立衛生研究所[助成金なしでサポートされていました。 R01NS069726]と援助グラントのアメリカ心臓協会グラント【グラントありません。 13GRNT17020004] SDへ。
Materials
| Rose Bengal | Sigma-Aldrich | 330000 | 20 mg/ml in sterile saline |
| C57Bl6/J | Jackson lab | 664 | 22-25g |
| Ketamine | VEDCO | NDC-50989-996-06 | 100 mg/ml |
| Xylazine | VEDCO | NDC-50989-234-11 | 100 mg/ml |
| Betadine solution | Purdue | NDC-67618-150-01 | 10% povidone iodine topical solution |
| Normal saline | Abott Laboratories | 04930-04-10 | For diluting RB, anaesthesia and for preventing tissue from drying |
| Artificial tears ointment | Rugby | NDC-0536-6550-91 | 83% white petrolatum |
| Ethanol | Decon labs.Inc | 2716 | 70% ethanol for disinfection |
| Metal halide lamp | EXFO, Canada | X-Cite 120 PC | Set power at 12% |
| Spring scissors | Fine Science Tool | 15000-10 | for minor dissection |
| Scissors (angled to side) | Fine Science Tool | 14063-011 | No. 3 handle |
| Standard scalpel | Fine Science Tool | 10003-12 | for removing muscle |
| Scalpel blade | Feather | 2976 | No. 10 |
| Forceps (curved) | Fine Science Tool | 11150-10 | for holding tissue |
| Forceps (straight) | Fine Science Tool | 11151-10 | for holding tissue |
| Needle holder | Fine Science Tool | 12002-12 | for suturing |
| Tissue adhesive glue | 3M Vetbond | 1469SB | to adhere to edges of the cut skin |
| Monofilament polypropylene | USSC Sutures | VP-521 | Size = 4-0 (for fascia) |
| Perma-hand silk | Ethicon | 683G | Size = 4-0 (for skin) |
| Micro drill | Roboz Surgical Instrument Co. Inc. | RS-6300 | with bone polishing drill bit |
| Laser doppler flowmeter | Moor Instruments | moorVMS-LDF1 | for monitoring change in blood flow |
| Heating pad | Fine Science Tool | 21052-00 | to prevent hypothermia |
| Lab-Jack | Fisher scientific | 14-673-50 | 4×4 in plate to adjust the height of the animal |
| X-Y gliding stage | Amscope | GT100 | for positioning the animal under microscope |
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