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

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Dierlijke protocollen waarbij proefdieren gevolgd door de National Institutes of Health (NIH) vastgestelde richtsnoeren en door de Animal Care en gebruik Comité van kapitaal medische universiteit zijn goedgekeurd.
De procedures van de invoering van SCI en meten van BF van het ruggenmerg met behulp van laser Doppler apparatuur hieronder beschreven werden gebruikt in een gepubliceerde studie1.
1. voorbereiding voor de operatie
<ol><li>Bereid...

<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. 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=Melatonin+prevents+blood+vessel+loss+and+neurological+impairment+induced+by+spinal+cord+injury+in+rats.">Melatonin prevents blood vessel loss and neurological impairment induced by spinal cord injury in rats.</a> J Spinal Cord Med. , 1-8 (2016).</li><li>Han, S., 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=Rescuing+vasculature+with+intravenous+angiopoietin-1+and+alpha+v+beta+3+integrin+peptide+is+protective+after+spinal+cord+injury.">Rescuing vasculature with intravenous angiopoietin-1 and alpha v beta 3 integrin peptide is protective after spinal cord injury.</a> Brain. 133 (Pt 4), 1026-1042 (2010).</li><li>Gerzanich, V., 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=De+novo+expression+of+Trpm4+initiates+secondary+hemorrhage+in+spinal+cord+injury.">De novo expression of Trpm4 initiates secondary hemorrhage in spinal cord injury.</a> Nat Med. 15 (2), 185-191 (2009).</li><li>Phillips, J. P., Cibert-Goton, V., Langford, R. M., Shortland, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion+assessment+in+rat+spinal+cord+tissue+using+photoplethysmography+and+laser+Doppler+flux+measurements.">Perfusion assessment in rat spinal cord tissue using photoplethysmography and laser Doppler flux measurements.</a> Journal of Biomedical Optics. 18 (3), 037005 (2013).</li><li>Garcia-Lopez, P., Martinez-Cruz, A., Guizar-Sahagun, G., Castaneda-Hernandez, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+spinal+cord+injury+changes+the+disposition+of+some,+but+not+all+drugs+given+intravenously.">Acute spinal cord injury changes the disposition of some, but not all drugs given intravenously.</a> Spinal Cord. 45 (9), 603-608 (2007).</li><li>Brown, A., Nabel, A., Oh, W., Etlinger, J. D., Zeman, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion+imaging+of+spinal+cord+contusion:+injury-induced+blockade+and+partial+reversal+by+&#946;2-agonist+treatment+in+rats.">Perfusion imaging of spinal cord contusion: injury-induced blockade and partial reversal by &#946;2-agonist treatment in rats.</a> Journal of Neurosurgery-Spine. 20 (2), 164-171 (2014).</li><li>Olive, J. L., McCully, K. K., Dudley, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+flow+response+in+individuals+with+incomplete+spinal+cord+injuries.">Blood flow response in individuals with incomplete spinal cord injuries.</a> Spinal Cord. 40 (12), 639-645 (2002).</li><li>Yamada, T., 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=Spinal+cord+blood+flow+and+pathophysiological+changes+after+transient+spinal+cord+ischemia+in+cats.">Spinal cord blood flow and pathophysiological changes after transient spinal cord ischemia in cats.</a> Neurosurgery. 42 (3), 626-634 (1998).</li><li>Gordeeva, A. 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Y., 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=Pre-treatment+with+LCZ696,+an+orally+active+angiotensin+receptor+neprilysin+inhibitor,+prevents+ischemic+brain+damage.">Pre-treatment with LCZ696, an orally active angiotensin receptor neprilysin inhibitor, prevents ischemic brain damage.</a> Eur J Pharmacol. 762, 293-298 (2015).</li><li>Vertiz-Hernandez, 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=L-arginine+reverses+alterations+in+drug+disposition+induced+by+spinal+cord+injury+by+increasing+hepatic+blood+flow.">L-arginine reverses alterations in drug disposition induced by spinal cord injury by increasing hepatic blood flow.</a> J Neurotrauma. 24 (12), 1855-1862 (2007).</li><li>Garcia-Lopez, P., Martinez-Cruz, A., Guizar-Sahagun, G., Castaneda-Hernandez, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+spinal+cord+injury+changes+the+disposition+of+some,+but+not+all+drugs+given+intravenously.">Acute spinal cord injury changes the disposition of some, but not all drugs given intravenously.</a> Spinal Cord. 45 (9), 603-608 (2007).</li><li>Li, Z., 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=Post+pressure+response+of+skin+blood+flowmotions+in+anesthetized+rats+with+spinal+cord+injury.">Post pressure response of skin blood flowmotions in anesthetized rats with spinal cord injury.</a> Microvasc Res. 78 (1), 20-24 (2009).</li><li>Boyle, N. 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. J., 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=Combination+of+cilostazol+and+clopidogrel+attenuates+rat+critical+limb+ischemia.">Combination of cilostazol and clopidogrel attenuates rat critical limb ischemia.</a> J Transl Med. 10, 164 (2012).</li><li>Basso, D. M., Beattie, M. S., Bresnahan, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graded+histological+and+locomotor+outcomes+after+spinal+cord+contusion+using+the+NYU+weight-drop+device+versus+transection.">Graded histological and locomotor outcomes after spinal cord contusion using the NYU weight-drop device versus transection.</a> Experimental Neurology. 139 (2), 244-256 (1996).</li><li>Basso, D. M., Beattie, M. S., Bresnahan, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Sensitive+and+Reliable+Locomotor+Rating-Scale+for+Open-Field+Testing+in+Rats.">A Sensitive and Reliable Locomotor Rating-Scale for Open-Field Testing in Rats.</a> Journal of Neurotrauma. 12 (1), 1-21 (1995).</li><li>Oberg, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+motion--a+disturbance+in+the+laser-Doppler+blood+flow+signal?.">Tissue motion--a disturbance in the laser-Doppler blood flow signal?.</a> Technol Health Care. 7 (2-3), 185-192 (1999).</li><li>Tenland, T., Salerud, E. G., Nilsson, G. E., Oberg, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+temporal+variations+in+human+skin+blood+flow.">Spatial and temporal variations in human skin blood flow.</a> Int J Microcirc Clin Exp. 2 (2), 81-90 (1983).</li><li>Kernick, D. P., Tooke, J. E., Shore, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biological+zero+signal+in+laser+Doppler+fluximetry+-+origins+and+practical+implications.">The biological zero signal in laser Doppler fluximetry - origins and practical implications.</a> Pflugers Arch. 437 (4), 624-631 (1999).</li><li>Rudolph, A. M., Heymann, M. 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. Z., 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=Dynamics+of+Microvascular+Blood+Flow+and+Oxygenation+Measured+Simultaneously+in+Human+Skin.">Dynamics of Microvascular Blood Flow and Oxygenation Measured Simultaneously in Human Skin.</a> Microcirculation. 21 (6), 562-573 (2014).</li><li>Li, Z. Y., 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=Post+pressure+response+of+skin+blood+flowmotions+in+anesthetized+rats+with+spinal+cord+injury.">Post pressure response of skin blood flowmotions in anesthetized rats with spinal cord injury.</a> Microvascular Research. 78 (1), 20-24 (2009).</li><li>Muck-Weymann, M. E., 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=Respiratory-dependent+laser-Doppler+flux+motion+in+different+skin+areas+and+its+meaning+to+autonomic+nervous+control+of+the+vessels+of+the+skin.">Respiratory-dependent laser-Doppler flux motion in different skin areas and its meaning to autonomic nervous control of the vessels of the skin.</a> Microvasc Res. 52 (1), 69-78 (1996).</li><li>Stefanovska, A., Bracic, M., Kvernmo, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wavelet+analysis+of+oscillations+in+the+peripheral+blood+circulation+measured+by+laser+Doppler+technique.">Wavelet analysis of oscillations in the peripheral blood circulation measured by laser Doppler technique.</a> Ieee Transactions on Biomedical Engineering. 46 (10), 1230-1239 (1999).</li></ol>

Een paar details moeten opgemerkt worden bij het uitvoeren van dit protocol. Ten eerste, het proces van anesthesie en chirurgie moet worden uitgevoerd zo snel en elegant mogelijk om te minimaliseren van de geïntroduceerde stress bij het dier. Verklein de verstoring van de resultaten en het dier in een relatief rustige en stabiele staat te houden. Ten tweede moet meer aandacht uitgaan naar het bloeden tijdens de meting met behulp van laser Doppler apparatuur omdat bloed potentieel met de lezing interfereren kon. Tot slot...

Het weefsel van het ruggenmerg is zeer gevacuoliseerd en zeer gevoelig aan hypoxie geïnduceerd door dwarslaesie (SCI). Onze eerdere studies is gebleken dat de doorbloeding van het ruggenmerg aanzienlijk werd verlaagd na hersenschudding schade1,2, die kunnen worden gerelateerd aan het tekort aan motor functie. Recente studies hebben aangetoond dat de integriteit van bloedvaten na SCI goed gecorreleerd zijn met de verbetering van de sensorische motorische functie3 is. Er werd gemeld dat verbeterde vasculariteit witte stof, niet indirect leidt tot verbeterde functie4zou kunnen redden. Daarom leek het onderhoud van het ruggenmerg na letsel perfusie van primair belang voor het behoud van de haalbaarheid en functionaliteit.
De effecten van verschillende behandelingen op de perfusie na SCI zijn bestudeerd door talrijke onderzoekers met behulp van een verscheidenheid van technieken in experimentele modellen van SCI5,6,7. Laser Doppler, als een gevestigde techniek, was ongetwijfeld een handige methode voor het kwantificeren van de perfusie van verschillende dierlijke en menselijke studies8,9,10,11. De techniek is gebaseerd op de meting van de Doppler shift12 geïnduceerd door rode bloedcellen te verplaatsen naar het verhelderend licht. Sinds de commercialisering van de techniek in de vroege jaren 1980, grote vooruitgang geboekt in lasertechnologie, glasvezel en signaalverwerking voor het meten van de perfusie door laser Doppler instrumenten13, waardoor LDF in een betrouwbare technologie.
In de huidige studie, werden beide methoden van laser Doppler meting toegepast om te evalueren van de bloedstroom (BF) in de spinale koorden van concussive ratten. Vanwege de noninvasive aard van de technologie en de eenvoudige installatie biedt ons protocol een gevoelige, snelle en betrouwbare methode voor BF metingen van het ruggenmerg. Met deze methode kunt en wat nog belangrijker is, longitudinaal onderzoek naar de post concussive SCI BF zonder dierlijke offer op elk tijdstip.
Vanwege de mogelijkheid om te beoordelen van de BF van het weefsel en snelle veranderingen in de perfusie tijdens de stimulatie, is het mogelijk om de toepassing van dit protocol om te evalueren van cerebrale BF14,15 , alsmede het meten van andere weefsels zoals lever16, 17, huid18,19en20van de darm. In het model van een rat van voorbijgaande occlusie van de middelste cerebrale slagader, werden de laser Doppler lezingen gebruikt om de juiste verlaging van het tarief van de BF tot niveaus die worden verwacht in de ischemische penumbra-14. Bij ratten die kritische ledemaat ischemie (CLI) inductie hebben ondergaan, werd laser Doppler scannen hind-limb BF vóór en na de ingreep CLI om en te observeren tijdens verschillende perioden na behandeling21toegepast. Bovendien, afhankelijk de biologische beschikbaarheid en metabole Goedkeuringvande sommige drugs van hepatische BF, die werd ontdekt door LDF16. Daarom kon LDF veel worden gebruikt in experimentele model, farmacokinetische en farmacodynamische evaluatie.

<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 flowmetrie (LDF) is een noninvasive methode voor het meten van bloed stroom (BF), waardoor het beter voor het meten van microcirculatory veranderingen van het ruggenmerg. In dit artikel was ons doel om het gebruik van zowel Laser Doppler Beeldvorming en toezicht voor het analyseren van de verandering van BF na dwarslaesie. Zowel de laser Doppler-imagescanner en de sonde/monitor wordt voor elke uitlezing werkten. De gegevens van LDPI verstrekt een lokale distributie van BF, die gaf een overzicht van perfusie rond de site van de schade en het toegankelijk voor vergelijkende analyse van BF onder verschillende locaties gemaakt. Door intens de indringende oppervlakte te meten over een periode van tijd, werd een gecombineerde sonde gebruikt om tegelijk meten de BF en zuurstof saturatie van het ruggenmerg, tonen algemene ruggenmerg perfusie en zuurstoftoevoer. LDF zelf heeft een aantal beperkingen, zoals de relatieve flux, gevoeligheid voor beweging en biologische nul signaal. Echter, de technologie is toegepast in de klinische en experimentele studie vanwege de eenvoudige installatie en snelle meting van BF.

LDPI werd gebruikt voor het meten van BF in het ruggenmerg, die langs de rostraal-caudal as van het ruggenmerg werd gekwantificeerd door de winning van lineaire profielen (Figuur 4). Figuur 5A en 5B van de figuur vertegenwoordigen de flux beeldvorming van het ruggenmerg van de sham-Fractie en SCI groep, respectievelijk. Figuur 5C en figuur 5D</strong...

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

Hier presenteren we een combinatie van laser Doppler perfusie imaging (LDPI) en lokale bloed stromen en zuurstof verzadiging (SO2), evenals een gestandaardiseerde procedure voor de invoering van ruggenmerg laser Doppler perfusie toezicht (LDPM) voor het meten van spinal cord trauma's op rat.

Met behulp van Laser Doppler Imaging en toezicht voor het analyseren van ruggenmerg microcirculatie in 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

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