We acknowledge Stephanie Gorgeard, Thierry Scheerlink (Toshiba France), and Christophe Lazare (Bracco France).

NOTE: The methods described in this manuscript were approved by the bioethics committee of the Lariboisi&#232;re School of Medicine, Paris, France (CEEALV/2011&#8211;08-01).
1. Instrument Preparation
<ol>
	<li>Prepare and clean the following instruments for catheter insertion: micro-forceps, micro-scissors, micro-vascular clamp, large scissors, surgical thread (Black braided silk 4-0) and a 14 G catheter. Heparinize the catheter with a heparin solution (5,000 U/ml).</li>
	<li>Prepare and cle...

<ol>
	<li>Cadotte, D. W., Fehlings, M. G. <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+injury:+a+systematic+review+of+current+treatment+options.">Spinal cord injury: a systematic review of current treatment options.</a> Clin Orthop Relat Res. 469 (3), 732-741 (2011).</li><li>Beattie, M. S., Farooqui, A. A., 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=Review+of+current+evidence+for+apoptosis+after+spinal+cord+injury.">Review of current evidence for apoptosis after spinal cord injury.</a> J Neurotrauma. 17 (10), 915-925 (2000).</li><li>MacDonald, J. W., Sadowsky, C. <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+injury.">Spinal-cord injury.</a> Lancet. 359 (9304), 417-425 (2002).</li><li>Mautes, A. E., Weinzierl, M. R., Donovan, F., Noble, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+events+after+spinal+cord+injury:+contribution+to+secondary+pathogenesis.">Vascular events after spinal cord injury: contribution to secondary pathogenesis.</a> Phys Ther. 80 (7), 673-687 (2000).</li><li>Martirosyan, N. L., 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=Blood+supply+and+vascular+reactivity+of+the+spinal+cord+under+normal+and+pathological+conditions.">Blood supply and vascular reactivity of the spinal cord under normal and pathological conditions.</a> J Neurosurg Spine. 15 (3), 238-251 (2011).</li><li>Blight, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+morphology+of+chronic+spinal+cord+injury+in+the+cat:+analysis+of+myelinated+axons+by+line-sampling.">Cellular morphology of chronic spinal cord injury in the cat: analysis of myelinated axons by line-sampling.</a> Neuroscience. 10 (2), 521-543 (1983).</li><li>Bassingthwaighte, J. B., 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=Validity+of+microsphere+depositions+for+regional+myocardial+flows.">Validity of microsphere depositions for regional myocardial flows.</a> Am J Physiol. 253 (1 Pt 2), H184-H193 (1987).</li><li>Drescher, W. R., Weigert, K. P., Bunger, M. H., Hansen, E. S., Bunger, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinal+blood+flow+in+24-hour+megadose+glucocorticoid+treatment+in+awake+pigs.">Spinal blood flow in 24-hour megadose glucocorticoid treatment in awake pigs.</a> J Neurosurg. 99 (3 Suppl), 286-290 (2003).</li><li>Golanov, E. V., Reis, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+oxygen-sensitive+neurons+of+the+rostral+ventrolateral+medulla+to+hypoxic+cerebral+vasodilatation+in+the+rat.">Contribution of oxygen-sensitive neurons of the rostral ventrolateral medulla to hypoxic cerebral vasodilatation in the rat.</a> J Physiol. 495 (Pt 1), 201-216 (1996).</li><li>Ueda, 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=Influence+on+spinal+cord+blood+flow+and+function+by+interruption+of+bilateral+segmental+arteries+at+up+to+three+levels:+experimental+study+in+dogs).">Influence on spinal cord blood flow and function by interruption of bilateral segmental arteries at up to three levels: experimental study in dogs).</a> Spine (Phila Pa 1976). 30 (20), 2239-2243 (2005).</li><li>Carlson, G. D., 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=Sustained+spinal+cord+compression:+part+II:+effect+of+methylprednisolone+on+regional+blood+flow+and+recovery+of+somatosensory+evoked+potentials).">Sustained spinal cord compression: part II: effect of methylprednisolone on regional blood flow and recovery of somatosensory evoked potentials).</a> J Bone Joint Surg Am. 85-A (1), 95-101 (2003).</li><li>Hamamoto, Y., Ogata, T., Morino, T., Hino, M., Yamamoto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+direct+measurement+of+spinal+cord+blood+flow+at+the+site+of+compression:+relationship+between+blood+flow+recovery+and+motor+deficiency+in+spinal+cord+injury.">Real-time direct measurement of spinal cord blood flow at the site of compression: relationship between blood flow recovery and motor deficiency in spinal cord injury.</a> Spine (Phila Pa 1976). 32 (18), 1955-1962 (2007).</li><li>Horn, E. M., 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=The+effects+of+intrathecal+hypotension+on+tissue+perfusion+and+pathophysiological+outcome+after+acute+spinal+cord+injury).">The effects of intrathecal hypotension on tissue perfusion and pathophysiological outcome after acute spinal cord injury).</a> Neurosurg Focus. 25 (5), E12 (2008).</li><li>Phillips, J. P., George, K. J., Kyriacou, P. A., Langford, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+photoplethysmographic+changes+using+a+static+compression+model+of+spinal+cord+injury.">Investigation of photoplethysmographic changes using a static compression model of spinal cord injury.</a> Conf Proc IEEE Eng Med Biol Soc. 2009, 1493-1496 (2009).</li><li>Phillips, J. P., George, K. J., Kyriacou, P. A., Langford, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+photoplethysmographic+changes+using+a+static+compression+model+of+spinal+cord+injury.">Investigation of photoplethysmographic changes using a static compression model of spinal cord injury.</a> Conf Proc IEEE Eng Med Biol Soc. 2009, 1493-1496 (2009).</li><li>Ishikawa, M., 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=Platelet+adhesion+and+arteriolar+dilation+in+the+photothrombosis:+observation+with+the+rat+closed+cranial+and+spinal+windows.">Platelet adhesion and arteriolar dilation in the photothrombosis: observation with the rat closed cranial and spinal windows.</a> J Neurol Sci. 194 (1), 59-69 (2002).</li><li>Soubeyrand, M., 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=Real-time+and+spatial+quantification+using+contrast-enhanced+ultrasonography+of+spinal+cord+perfusion+during+experimental+spinal+cord+injury.">Real-time and spatial quantification using contrast-enhanced ultrasonography of spinal cord perfusion during experimental spinal cord injury.</a> Spine (Phila Pa 1976). 37 (22), E1376-E1382 (1976).</li><li>Huang, L., 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=Quantitative+assessment+of+spinal+cord+perfusion+by+using+contrast-enhanced+ultrasound+in+a+porcine+model+with+acute+spinal+cord+contusion).">Quantitative assessment of spinal cord perfusion by using contrast-enhanced ultrasound in a porcine model with acute spinal cord contusion).</a> Spinal Cord. 51 (3), 196-201 (2012).</li><li>Postema, M., Gilja, O. H. <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+and+targeted+ultrasound.">Contrast-enhanced and targeted ultrasound.</a> World J Gastroenterol. 17 (1), 28-41 (2011).</li><li>Soubeyrand, M., Badner, A., Vawda, R., Chung, Y. S., Fehlings, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Very+High+Resolution+Ultrasound+Imaging+for+Real-Time+Quantitative+Visualisation+of+Vascular+Disruption+After+Spinal+Cord+Injury.">Very High Resolution Ultrasound Imaging for Real-Time Quantitative Visualisation of Vascular Disruption After Spinal Cord Injury.</a> J Neurotrauma. , (2014).</li><li>Akhtar, A. Z., Pippin, J. J., Sandusky, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+in+spinal+cord+injury:+a+review.">Animal models in spinal cord injury: a review.</a> Rev Neurosci. 19 (1), 47-60 (2008).</li></ol>

The authors declare that they have no competing financial conflict of interest. The ultrasound machine was graciously lent by the Toshiba France company. The Vueject pump was graciously lent by the Bracco France company.

Although we have described how to use CEU in a rat SCI contusion model, this protocol can be modified to fit other experimental objectives or SCI models. We have chosen to measure SCBF at only two time points (before injury and 15 min post-SCI), however the number of time points and the delay between SCBF measurements can be adapted to fulfill the needs of other studies. For example, in our previous work 17, we have measured SCBF at five successive time points throughout the first hour post-SCI. It is importan...

Traumatic spinal cord injury (SCI) is a devastating condition leading to significant impairment in motor, sensory and autonomous functions. To date, no therapy has demonstrated its efficiency in patients. For such reason, it is important to identify new techniques that will improve the assessment of potential treatments and can further elucidate injury pathiophysiology1.
SCI is divided into two sequential phases, referred to as primary and secondary injuries. The primary injury corresponds to the initial mechanical insult. Whereas the secondary injury groups a cascade of various biological events (such as inflammation, oxidative stress and hypoxia) that further contribute to the progressive expansion of the initial lesion, tissue damage and therefore neurological deficit2,3.
At the acute phase of SCI, neuroprotective therapies are aimed at reducing the secondary injury pathology and should accordingly improve neurological outcomes. Among the many secondary injury events, ischemia plays a crucial role 4,5. At the level of the SCI epicenter, the damaged parenchymal microvessels impede effective spinal cord blood flow (SCBF). Moreover, SCBF is also significantly reduced in the region surrounding the injury epicenter, an area specifically known as the &#8220;ischemic penumbra zone&#8221;. If SCBF cannot be quickly restored within these regions, ischemia can lead to supplementary parenchymal necrosis and further nervous tissue damage. As even the slightest tissue preservation can have substantial effects of function, it is of major interest to develop drugs and therapies that can reduce ischemia post-SCI. To highlight this phenomenon, previous work has shown that preservation of only 10% of myelinated axons was enough to permit walking in cats post-SCI 6.
Although several techniques have been described to assess SCBF, they all have significant limitations. For example, the use of radioactive microspheres7,8 and C14-iodopyrine autoradiography9 requires subsequent animal sacrifice and cannot be repeated at later time-points. The hydrogen clearance technique10 depends on the insertion of intraspinal electrodes, which may further damage the spinal cord. While laser Doppler imaging, photoplethysmography14,15 and in-vivo light microscopy16 have a very limited depth/area of measurement11-13.
Our team has previously shown that contrast enhanced ultrasound (CEU) imaging can be used to assess real time and in-vivo the SCBF changes in the rat spinal cord parenchyma17. It is important to note that a similar technique was applied by Huang et al. in a porcine model of SCI18. CEU applies a specific mode of ultrasound imaging which allows to associate grayscale morphological images (obtained by the conventional B-mode) with spatial distribution of blood flow 19. The SCBF imaging and quantification relies on intravascular injection of echo-contrast agents. The contrast agent is made up of sulphur hexafluoride microbubbles (mean diameter of about 2.5 &#956;m and 90% having a diameter less than 6 &#956;m) stabilized by phospholipids. The microbubbles reflect the ultrasound beam emitted by the probe thus enhancing blood echogenicity and increasing contrast of the tissues according to their blood flow. It is therefore possible to assess the blood flow in a given region of interest according to the intensity of the reflected signal. The microbubbles are also safe and they have been clinically applied in humans. The sulphur hexafluoride is quickly cleared (mean terminal half-life is 12 min) and more than 80% of the administered sulphur hexafluoride is recovered in exhaled air within 2 min after injection. This protocol provides a simple way to use CEU imaging to assess SCBF changes in rat.

<table border="0" cellpadding="0" cellspacing="0" width="1154">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:397px;">Name of Reagent/ Equipment</td>
			<td style="width:229px;">Company</td>
			<td style="width:229px;"></td>
			<td style="width:299px;">Comments/Description</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:397px;">External Fixator Hoffman 3</td>
			<td style="width:229px;">Stryker, Kalamazoo, USA</td>
			<td style="width:229px;"></td>
			<td>Modular system used to build the custom made 3D frame and the jointed arm holding the ultrasound probe</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:397px;">Toshiba Applio</td>
			<td style="width:229px;">Toshiba, Tokyo, Japan</td>
			<td style="width:229px;"></td>
			<td>Ultrasound machine</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:397px;">Sonovue</td>
			<td style="width:229px;">Bracco, Milan, Italy</td>
			<td style="width:229px;"></td>
			<td>Contrast agent : microbubbles</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:397px;">Vueject pump</td>
			<td style="width:229px;">Bracco, Milan, Italy</td>
			<td style="width:229px;"></td>
			<td>Electric pump for infusion of microbubbles bolus</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:397px;">Aquasonic Ultrasound Gel</td>
			<td style="width:229px;">Parker Laboratories, Fairfield, NJ, USA</td>
			<td style="width:229px;"></td>
			<td>Ultrasound gel used to transmit the ultrasound waves</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:397px;">Isovet</td>
			<td style="width:229px;">Piramal Healthcare, Mumbai, India</td>
			<td style="width:229px;"></td>
			<td>Isoflurane used for anesthesia</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:397px;">Ultra Extend</td>
			<td style="width:229px;">Toshiba, Tokyo, Japan</td>
			<td style="width:229px;"></td>
			<td>Software used for quantification of spinal cord blood flow</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:397px;">Mastercraft Five-piece Mini-pliers Set, Product #58-4788-6</td>
			<td style="width:229px;">Canadian Tire, Toronto, Canada</td>
			<td style="width:229px;"></td>
			<td>Set of pliers for Do-it-yourself job</td>
		</tr>
	</tbody>
</table>

contrast enhanced ultrasound imaging for assessment of spinal cord blood flow in experimental spinal cord injury

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an important target for neuroprotective therapies. Although several techniques have been described to assess SCBF, they all have significant limitations. To overcome the latter, we propose the use of real-time contrast enhanced ultrasound imaging (CEU). Here we describe the application of this technique in a rat contusion model of SCI. A jugular catheter is first implanted for the repeated injection of contrast agent, a sodium chloride solution of sulphur hexafluoride encapsulated microbubbles. The spine is then stabilized with a custom-made 3D-frame and the spinal cord dura mater is exposed by a laminectomy at ThIX-ThXII. The ultrasound probe is then positioned at the posterior aspect of the dura mater (coated with ultrasound gel). To assess baseline SCBF, a single intravenous injection (400 &#181;l) of contrast agent is applied to record its passage through the intact spinal cord microvasculature. A weight-drop device is subsequently used to generate a reproducible experimental contusion model of SCI. Contrast agent is re-injected 15 min following the injury to assess post-SCI SCBF changes. CEU allows for real time and in-vivo assessment of SCBF changes following SCI. In the uninjured animal, ultrasound imaging showed uneven blood flow along the intact spinal cord. Furthermore, 15 min post-SCI, there was critical ischemia at the level of the epicenter while SCBF remained preserved in the more remote intact areas. In the regions adjacent to the epicenter (both rostral and caudal), SCBF was significantly reduced. This corresponds to the previously described &#8220;ischemic penumbra zone&#8221;. This tool is of major interest for assessing the effects of therapies aimed at limiting ischemia and the resulting tissue necrosis subsequent to SCI.

With the protocol described above, it is possible to map the SCBF along a longitudinal spinal cord sagittal segment.
In the intact spinal cord, there appears to be SCBF irregularities within the parenchyma (Figure 12). This can be explained by the variable distribution of radiculo-medullary arteries (RMA) from one animal to another. RMA refers to segmental arteries that reach the anterior spinal artery (ASA) and therefore provide blood supply to the spinal cord parenchyma. In ...

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Contrast Enhanced Ultrasound Imaging for Assessment of Spinal Cord Blood Flow in Experimental Spinal Cord Injury

Contrast Enhanced Ultrasound imaging is a reliable in-vivo tool for quantifying spinal cord blood flow in an experimental rat spinal cord injury model. This paper contains a comprehensive protocol for application of this technique in association with a contusion model of thoracic spinal cord injury.

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an ...

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13.9K Views.  Faculté de Médecine Paris Diderot Paris VII, U942. Contrast Enhanced Ultrasound imaging is a reliable in-vivo tool for quantifying spinal cord blood flow in an experimental rat spinal cord injury model. This paper contains a comprehensive protocol for application of this technique in association with a contusion model of thoracic spinal cord injury.