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

The authors have no acknowledgements.

Animal protocols involving experimental animals followed guidelines established by the National Institutes of Health (NIH) and were approved by the Animal Care and Use Committee of Capital Medical University.
The procedures of introducing SCI and measuring BF of spinal cord using laser Doppler equipment described below were used in a published study1.
1. Preparation for the Surgery
<ol>
	<li>Prepare pentobarbital sodium solution 3% (w/v) in...

<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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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scanning+laser+Doppler+is+a+useful+technique+to+assess+foot+cutaneous+perfusion+during+femoral+artery+cannulation.">Scanning laser Doppler is a useful technique to assess foot cutaneous perfusion during femoral artery cannulation.</a> Critical Care. 3 (4), 95-100 (1999).</li><li>Emmanuel, A. V., Chung, E. A. L., Kamm, M. A., Middleton, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+gut-specific+autonomic+testing+and+bowel+dysfunction+in+spinal+cord+injury+patients.">Relationship between gut-specific autonomic testing and bowel dysfunction in spinal cord injury patients.</a> Spinal Cord. 47 (8), 623-627 (2009).</li><li>Sheu, J. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+circulation+of+the+fetus+in+utero.+Methods+for+studying+distribution+of+blood+flow,+cardiac+output+and+organ+blood+flow.">The circulation of the fetus in utero. Methods for studying distribution of blood flow, cardiac output and organ blood flow.</a> Circ Res. 21 (2), 163-184 (1967).</li><li>Dubory, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contrast+Enhanced+Ultrasound+Imaging+for+Assessment+of+Spinal+Cord+Blood+Flow+in+Experimental+Spinal+Cord+Injury.">Contrast Enhanced Ultrasound Imaging for Assessment of Spinal Cord Blood Flow in Experimental Spinal Cord Injury.</a> Jove-Journal of Visualized Experiments. (99), e52536 (2015).</li><li>Kuliga, K. 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A few details should be noticed when performing this protocol. Firstly, the process of anesthesia and surgery should be carried out as quickly and elegantly as possible to minimize the introduced stress to the animal. To reduce disturbance to the results, keep the animal in a relatively peaceful and stable state. Secondly, more attention should be paid to bleeding during the measurement using laser Doppler equipment, since blood could potentially interfere with the reading. Finally, during the data recording, animals sho...

The tissue of the spinal cord is highly vascularized and extremely sensitive to hypoxia induced by spinal cord injury (SCI). Our previous studies showed that blood flow of the spinal cord was significantly decreased after concussion injury1,2, which might be related to the deficit of motor function. Recent studies have shown that the integrity of blood vessels following SCI is well-correlated with the improvement of sensory motor function3. It has been reported that improved vascularity might rescue white matter, indirectly leading to improved function4. Therefore, the maintenance of post-injury spinal cord perfusion appeared to be of primary importance for preserving viability and functionality.
The effects of various treatments on perfusion after SCI have been examined by numerous investigators using a variety of techniques in experimental models of SCI5,6,7. Laser Doppler, as a well-established technique, was undoubtedly a useful method for quantifying perfusion in several animal and human studies8,9,10,11. The technique is based on measuring the Doppler shift12 induced by moving red blood cells to the illuminating light. Since the commercialization of the technique in the early 1980s, great progress has been made in laser technology, fiber optics and signal processing for measuring perfusion by laser Doppler instruments13, which made LDF into a reliable technology.
In the current study, both methods of laser Doppler measurement&#160;were applied to evaluate blood flow (BF) in the spinal cords of concussive rats. Due to the noninvasive nature of the technology and its simple setup, our protocol provides a sensitive, rapid and reliable method for BF measurements of the spinal cord. More importantly, this method allows longitudinal study of BF post concussive SCI without animal sacrifice at each time point.
Due to the ability to assess the BF of the tissue and fast changes of perfusion during stimulation, it is possible to apply this protocol to evaluate cerebral BF14,15 as well as measure other tissues such as liver16,17, skin18,19, and bowel20. In a rat model of transient occlusion of the middle cerebral artery, the laser Doppler readings were used to ensure proper reduction of the BF rate to levels that are expected in the ischemic penumbra14. In rats which have undergone critical limb ischemia (CLI) induction, laser Doppler scanning was applied to observe hind limb BF&#160;before and after the CLI procedure and during different periods after treatment21. Additionally, the bioavailability and metabolic clearance of some drugs depended on hepatic BF, which was detected by LDF16. Therefore, LDF could be widely used in experimental model, pharmacodynamic, and pharmacokinetic evaluation.

<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

Laser Doppler flowmetry (LDF) is a noninvasive method for blood flow (BF) measurement, which makes it preferable for measuring microcirculatory alterations of the spinal cord. In this article, our goal was to use both Laser Doppler imaging and monitoring to analyze the change of BF after spinal cord injury. Both the laser Doppler image scanner and the probe/monitor were being employed to obtain each readout. The data of LDPI provided a local distribution of BF, which gave an overview of perfusion around the injury site and made it accessible for comparative analysis of BF among different locations. By intensely measuring the probing area over a period of time, a combined probe was used to simultaneously measure the BF and oxygen saturation of the spinal cord, showing overall spinal cord perfusion and oxygen supply. LDF itself has a few limitations, such as relative flux, sensitivity to movement, and biological zero signal. However, the technology has been applied in clinical and experimental study due to its simple setup and rapid measurement of BF.

LDPI was used to measure BF in the spinal cord, which was quantified along the rostral-caudal axis of the spinal cord by extracting linear profiles (Figure 4). Figure 5A and Figure 5B represent the flux imaging of the spinal cord of the sham group and SCI group, respectively. Figure 5C and Figure 5D represent the altering BF along the rostra...

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

Here we present a combination of laser Doppler perfusion imaging (LDPI) and laser Doppler perfusion monitoring (LDPM) to measure spinal cord local blood flows and oxygen saturation (SO2), as well as a standardized procedure for introducing spinal cord trauma on rat.

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.

Research

JoVE Journal

Behavior

How to Detect Amygdala Activity with Magnetoencephalography using Source Imaging

In trace fear conditioning a conditional  stimulus (CS) predicts the occurrence of the unconditional stimulus (UCS),  which is presented after a brief ...

Measurement of Fronto-limbic Activity Using an Emotional Oddball Task in Children with Familial High Risk for Schizophrenia

Adolescence is a critical developmental period where the early symptoms of schizophrenia frequently emerge. First-degree relatives of people with ...

Uncovering Beat Deafness: Detecting Rhythm Disorders with Synchronized Finger Tapping and Perceptual Timing Tasks

A set of behavioral tasks for assessing perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) is presented here ...

Behavioral and Locomotor Measurements Using an Open Field Activity Monitoring System for Skeletal Muscle Diseases

The open field activity monitoring system comprehensively assesses locomotor and behavioral activity levels of mice. It is a useful tool for assessing ...

Using Fiberless, Wearable fNIRS to Monitor Brain Activity in Real-world Cognitive Tasks

Functional Near Infrared Spectroscopy (fNIRS) is a neuroimaging technique that uses near-infrared light to monitor brain activity. Based on neurovascular ...

Treating Low Back Pain in Failed Back Surgery Patients with Multicolumn-lead Spinal Cord Stimulation

Failed back surgery syndrome (FBSS) refers to persistent, chronic pain following spinal surgery. Spinal cord stimulation with dorsal epidural leads can be ...

Paradigms of Lower Extremity Electrical Stimulation Training After Spinal Cord Injury

Skeletal muscle atrophy, increased adiposity and reduced physical activity are key changes observed after spinal cord injury (SCI) and are associated with ...

Simultaneous Recordings of Cortical Local Field Potentials and Electrocorticograms in Response to Nociceptive Laser Stimuli from Freely Moving Rats

Electrocortical responses, elicited by laser heat pulses that selectively activate nociceptive free nerve endings, are widely used in many animal and ...

Using Facial Electromyography to Assess Facial Muscle Reactions to Experienced and Observed Affective Touch in Humans

"Affective" touch is believed to be processed in a manner distinct from discriminatory touch and to involve activation of C-tactile (CT) afferent fibers. ...

Using Practice Testing, Public Speaking, and Source Monitoring to Examine the Influences of Learning Strategies and Stress on Episodic Memory

Prior research demonstrated that learning information via retrieval practice, which entails studying and taking practice tests, resulted in less memory ...