Name: using laser doppler imaging and monitoring to analyze spinal cord microcirculation in rat
Uploaded: 2018-05-30T13:00:00
Description: Watch this Scientific Journal Video about Using Laser Doppler Imaging and Monitoring to Analyze Spinal Cord Microcirculation in Rat at JoVE.com

著者の謝辞があります。

実験動物を含む動物のプロトコルは健康国立研究所 (NIH) によって確立されたガイドラインに従い、動物のケアと使用首都医科大学委員会によって承認されました。
科学を導入し、レーザー以下ドプラ装置による脊髄の BF を測定の手順は、発行された研究1で使用されました。
1. 手術の準備
<ol><li>生理食塩水でペントバルビ タール...

<ol>
	<li>Jing, Y. L., Bai, F., Chen, H., Dong, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meliorating+microcirculatory+with+melatonin+in+rat+model+of+spinal+cord+injury+using+laser+Doppler+flowmetry.">Meliorating microcirculatory with melatonin in rat model of spinal cord injury using laser Doppler flowmetry.</a> Neuroreport. 27 (17), 1248-1255 (2016).</li><li>Jing, Y. 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いくつかの詳細は、このプロトコルを実行するときに気づいたする必要があります。まず、麻酔や手術のプロセス実施されなければならない限り迅速かつエレガントな動物に導入されたストレスを最小限に抑えるため可能な限り。結果への影響を減らすためには、比較的平和で安定した状態で動物を飼います。第二に、血が読み取りを妨げている可能性があるのでレーザー ドップラー装置を...

脊髄の組織は、高度に血管や脊髄損傷 (SCI) による低酸素に非常に敏感です。私たちの以前の研究は、脊髄の血流は脳震盪傷害1,2, 運動機能の欠損に関連する可能性があります後低下したことを示した。最近の研究は、SCI に続く血管の整合性が感覚運動機能3の改善と相関関係にあることを示しています。それは、改良された血管が直接ない機能の改善4につながる白質を救助可能性があります報告されています。したがって、受傷後の脊髄灌流の維持の重要性と機能を維持するために登場。
SCI5,6、7の実験モデルでさまざまな技術を使用して多くの研究者によって様々 な処理 SCI 後血流に及ぼす影響を調べました。レーザーのドップラー、十分に確立された技術として、いくつかの動物と人間の研究8,9,10,11の灌流を定量化するための有用な方法は間違いなくだった。技術は、照明の光に赤色の血液細胞の移動によるドップラー シフト12の測定に基づいています。1980 年代初頭の技術の実用化、以来、レーザー ドップラー計測器13LDF に信頼性の高い技術にしたレーザー技術、光ファイバーと血流の測定用信号処理で大きな進歩しました。
現在の研究では、レーザー ドップラー計測の両方のメソッドは震とう性のラットの脊髄で血流 (BF) を評価するために適用されました。技術で、簡単にセットアップの非侵襲的性質では、我々 のプロトコルは、脊髄の BF 測定高感度、迅速かつ信頼性の高い方法を提供します。もっと重要なは、このメソッドでは、各時点での動物の犠牲のない BF ポスト震とう性科学に関する縦断的研究ができます。
能力が組織の BF と刺激の間に血流の急激な変化を評価するため、測定肝16,などの他の組織と同様、脳 BF14,15を評価するこのプロトコルを適用することが可能です。17、肌18,19、および腸20。ラット一過性中大脳動脈閉塞モデル、レーザーのドップラー測定値が虚血性ペナンブラ14に期待されているレベルに BF 率の適切な削減を確保するため使用されました。肢虚血 (CLI) 誘導を受けたラット、レーザーのドップラー スキャン前に CLI 処理後と治療21後さまざまな期間中に後肢 BF を観察する適用しました。また、バイオアベイラビリティといくつかの薬物の代謝クリアランスは、LDF16によって検出された肝 BF に依存。したがって、LDF は実験モデル、薬力学及び薬物動態学的評価で広く使用できます。

<table>
	<tbody>
		<tr>
			<td>Laser Doppler Line Scanner</td>
			<td>Moor Instruments</td>
			<td>moorLDLS2</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Doppler Monitor</td>
			<td>Moor Instruments</td>
			<td>moorVMS-LDF</td>
			<td></td>
		</tr>
		<tr>
			<td>Probe for Monitor</td>
			<td>Moor Instruments</td>
			<td>VP3</td>
			<td>Blunt needle end delivery probe</td>
		</tr>
		<tr>
			<td>Impactor</td>
			<td>Precision Systems and Instrumentation</td>
			<td>IH-0400</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenobarbital sodium</td>
			<td>Sigma-Aldrich</td>
			<td>P3761</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Sigma-Aldrich</td>
			<td>B-908</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Becton Dickinson Medica (s) Pte.Ltd</td>
			<td>300841</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical suture needles with thread</td>
			<td>Shanghai Pudong Jinhuan Medical Products Co., Ltd</td>
			<td></td>
			<td>18T0329 (batch number) /4-0</td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Operation instrument factory of Shanghai Medical Instrument Co., Ltd.</td>
			<td>J11030 4#</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blade</td>
			<td>Operation instrument factory of Shanghai Medical Instrument Co., Ltd.</td>
			<td>J12130 20#</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophthalmic forceps</td>
			<td>Operation instrument factory of Shanghai Medical Instrument Co., Ltd.</td>
			<td>JD1040</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemostatic forceps</td>
			<td>Operation instrument factory of Shanghai Medical Instrument Co., Ltd.</td>
			<td>J31050</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzyl penicillin sodium</td>
			<td>North China Pharmaceutical Co., Ltd</td>
			<td></td>
			<td>F6072116 (batch number)</td>
		</tr>
		<tr>
			<td>75% alcohol</td>
			<td>Dezhou Anjie Gaoke disinfection products Co., Ltd</td>
			<td></td>
			<td>150421R (batch number)</td>
		</tr>
		<tr>
			<td>Iodine</td>
			<td>Shandong Lierkang Medical Technology Co., Ltd</td>
			<td></td>
			<td>20170102 (batch number)</td>
		</tr>
		<tr>
			<td>Rat</td>
			<td>Laboratory Animal Center, The Academy of Millitery Medical Sciences</td>
			<td></td>
			<td>Sprague-Dawly (rat strain)</td>
		</tr>
	</tbody>
</table>

using laser doppler imaging and monitoring to analyze spinal cord microcirculation in rat

レーザードップラー (LDF) は、脊髄の微小循環の変化を測定することをお勧めになる血流量 (BF) 測定の非侵襲的方法です。この記事では、私たちの目標は、レーザー ドップラー イメージングと監視の両方を使用して、脊髄損傷後の BF の変化を分析するでした。各読み出しを取得するレーザー ドップラー画像スキャナーとプローブ/モニターの両方を採用されていた。LDPI のデータは、BF、損傷部位の周囲の血流の概要を与えた、アクセシブルに BF の異なる場所の間での比較分析のためのローカルの配布を提供します。期間にわたって測定プローブ領域ない激しくすることにより複合プローブは全体的な脊髄の血流と酸素供給を示す脊髄の BF と酸素飽和度を同時に測定していました。自体 LDF の動きに対する感度と生物ゼロ信号相対磁束などのいくつかの制限があります。しかし、技術は、単純なセットアップと BF の迅速測定法による臨床的および実験的研究で適用されています。

LDPI は、直線プロファイル (図 4) を抽出することによって脊髄の尾側吻側軸に沿って定量化された脊髄の BF を測定に使用されました。図 5 aと図 5 bは、偽のグループおよび SCI グループの脊髄の磁束イメージングをそれぞれ表します。図 5と図 5は、偽のグ?...

Watch this Scientific Journal Video about Using Laser Doppler Imaging and Monitoring to Analyze Spinal Cord Microcirculation in Rat at JoVE.com

Using Laser Doppler Imaging and Monitoring to Analyze Spinal Cord Microcirculation in Rat

ここでレーザー血流イメージング (LDPI) の組み合わせを提案して、脊髄を導入するための標準化された手順だけでなく、局所血流と酸素飽和度 (その2) をコード レーザー血流モニタリング脊髄を測定する (LDPM)ラットの外傷。

レーザーのドップラー イメージングおよび監視を使用したラット脊髄の微小循環の分析

Laser Doppler flowmetry (LDF) is a noninvasive method for blood flow (BF) measurement, which makes it preferable for measuring microcirculatory ...

The overall goal of this procedure is to present a combination of laser doppler imaging and monitoring to measure spinal cord local blood flows and oxygen saturation, as well as a standardized procedure for introducing spinal cord trauma on a rat. This method can help answer key questions in the field of micro circulation such as how blood flow redistributed over time and its impact on the surrounding area after spinal cord injury. The main advantage of this technique is by using both laser doppler imaging and monitoring device we could rapidly derive data of the local distribution of the blood flow as well as the blood flow variation over a period of time, which allows more sophisticated analysis of the blood flow in the problem area.Begin by checking the anesthetized rat for a surgical plane of anesthesia by the absence of a reaction to a toe pinch. Shave the dorsal area of the rat from the lower back to the neck. Then place the rat dorsal side up on a 40-degrees Celsius heating pad to maintain a constant body temperature.Sterilize the shaved area by wiping with cotton balls soaked in iodine followed by 75%alcohol. After making a skin incision over the laminectomy site covering thoracic vertebrae T7 to T11, cut the attached muscles on both sides from T8 to T10 to expose the spinous processes, the laminae, and the facet joints. Use the scalpel to make incisions that disconnect the junction between T10 and T11.Further expose the junction by carefully dissecting the muscle layer away to expose the bone. Use the scissors to further clear muscle away from the lamina and around the pedicle with small snips. This will open a small space between the vertebrae at T10 and T11.Slowly and delicately insert hemostatic forceps into this gap and break the pedicle. Repeat on the other side. Expose the spinal cord and carefully lift and break off the lamina without leaving free or jagged bone fragments behind.Repeat the process to further remove T9 and T8 laminae. An impactor may now be used to induce concussion injury in experimental groups. To scan the exposed spinal cord, place the rat dorsal side up on a black, non-reflective background, then set up the scanning parameters in the software by clicking Measure to enter the measurement graphical user interface and then the Scanner Setup button to open the Scanner Setup interface.To scan small areas such as in this experiment, click Scan Size and Display options and select High Resolution for a fine scanning mode with higher resolution. Click on the Image Scan option to check the scanning parameters. Check the live video image by clicking on the Video and Distance option.Position the scanner 10 to 13 centimeters above the surgical window and move the background with the animal to center the exposed spinal cord on the scanning window. Select the Auto Distance function to fine adjust the scanning height, which should be kept consistent across all measurements in the experiment. Use a non-reflective cover with a window to expose only the surgical area to further minimize background and mark the animal's direction.Click on Repeat Scan to set the number of scans, then click Okay to open the Repeat Scan interface. Click the Start button to start scanning. The whole process will take approximately three to four minutes.A scanner monitor with a VP3 blunt needle and delivery probe is used here to monitor blood flow and oxygen saturation over time. Begin by attaching the probe perpendicular to a stereotaxic instrument to set up the monitoring equipment. Put the rat on the stereotaxic apparatus, dorsal side up, and underlay the animal with a small piece of Styrofoam to level the exposed spinal cord.Examine the incision and remove any excessive liquid or blood using a cotton pad. Use the apparatus's x-and y-axes to locate the probe to two millimeters rostral to the center point of the exposed spinal cord or lesion point and devoid the central vein. Use the z-axis to slowly lower the probe to the level just touching the surface of the spinal cord.The probe should just touch the surface of the spinal cord but not be so loose as to allow bright light to escape from the side of the contact point. Proper positioning is critical for adapting and implementing the monitoring protocol. The probe should be perpendicular to the measured surface and excessive pressure should be avoided.To record data, click on the New Experiment button in the software to open the setup interface. Under the General option, check the system configuration, then click Next. In the Display setup, select the channel for blood flow and oxygen saturation and click Next.Input file information and click Next to enter the data recording interface. Click on the green triangle button to start recording data from the probe. Once the signal is stable, record data for eight consecutive minutes, then lift the probe and remove the animal from the stereotaxic apparatus.After suturing the muscle and the skin, place the rat on its side in a clean cage on a heating pad, avoiding contact between the surgery site and the cage bottom. Monitor the animal until it wakes up from anesthesia to ensure no post-surgery bleeding and that the sutures remain closed. These images represent the flux imaging of the spinal cord of the sham group on the left and the spinal cord injury group on the right.Flux imaging demonstrated that spinal cord injury induced reduction of blood flow and blood flow of the epicenter was lower than rostral cord and caudal cord. These images show the alternating blood flow along the rostral-caudal axis of the spinal cord of the sham group on the left and the SCI group on the right. As shown here, spinal cord blood flow of the spinal cord injury group significantly decreased compared to the sham group.Simultaneously, the oxygen saturation of the spinal cord was remarkably lower after spinal cord concussion, which was consistent with the change in blood flow after injury. After watching this video, you should have a good understanding of how to measure blood flow of the spinal cord using both laser doppler perfusion imaging and the laser doppler perfusion monitoring. Once mastered, the surgery of introducing spinal cord injury can be done in 20 minutes and the measurements, including use both laser doppler instruments, will take roughly 20 minutes if it is performed properly.While attempting this procedure, it's important to remember to use a non-reflective cover with a window to expose only the surgical area to further minimize background and mark the animal's direction. This technique paved the way for researchers in the field of microcirculation to explore the changes of blood flow for its pharamacodynamics and pharmacokinetics evaluation.

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レーザーのドップラー イメージングおよび監視を使用したラット脊髄の微小循環の分析

レーザーのドップラーイメージングおよび監視を使用したラット脊髄の微小循環の分析