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 clean the following instruments for the laminectomy: large scissors, scalpel and a bone cutter. Perform laminectomy with a custom-made bone cutter designed to reduce the risk of harming the spinal cord during the laminectomy (Figure 1).</li>
	<li>Set-up the 3D-frame used for positioning and stabilization of the animal. The custom-made frame is built with the elements of an External Fixator Hoffman 3 in association with forceps, which have been curved in order to fit the lumbar spine of the animal.</li>
	<li>Prepare the weight-drop device (impactor) used for spinal cord biomechanical injury. 
	NOTE: The custom-made impaction device was designed with a 3D software and printed in 3D.</li>
	<li>Turn on the ultrasound machine.</li>
	<li>Prepare the kit for reconstitution of the contrast agent. 
	NOTE: The kit includes 1 vial containing 25 mg of lyophilised powder, 1 pre-filled syringe containing 5 ml sodium chloride and one mini-spike transfer system (Figure 2). The steps for reconstitution of the contrast agent are detailed below (in section 5).</li>
</ol>
2. Jugular Vein Catheterization (Figure 3)
<ol>
	<li>Anesthetize the animal with 4% isoflurane. Place the animal in supine position. Confirm proper anesthetization by ensuring that the animal is unresponsive when the paws are pinched with a forceps. Apply vet ointment on eyes to prevent dryness while under anesthesia.</li>
	<li>Shave the neck and clean the skin. Make an incision on the midline of the neck. Retract the sternocleidomastoidian muscle in order to find the internal jugular vein. Tighten a ligature at the rostral part of the vein.</li>
	<li>Apply a microvascular clamp on the vein, 1 cm below the ligature. Pass another thread around the vein, just below the clamp with the knot ready to be tighten when the clamp is released.</li>
	<li>Open the wall of vein (venotomy) between the clamp and the rostral ligature. Introduce a 14 G catheter in the lumen of the vein and push it toward the heart.</li>
	<li>When it comes up against the clamp, release the latter and push the catheter further. Secure the catheter in the vein, by firmly tightening the knot on the vein with the catheter inside.</li>
	<li>Assess patency of the catheter by withdrawing a small amount of venous blood in the catheter and subsequently then flushing it with heparinized saline. This prevents obstruction of the catheter by a potential blood clot.</li>
	<li>Connect flexible tubing to the catheter for further injection of contrast agent (microbubbles). Keep it closed (sealed) until ready for use.</li>
</ol>
3. Accessing the Spine, Laminectomy and Rat Positioning (in the 3D-frame)
<ol>
	<li>Place the animal in a flat prone horizontal position. Shave and clean the back (thoracic region) of the animal.</li>
	<li>Identify the last rib (the XIIIth in the rat) by palpation (Figure 4). This allows one to estimate the location of the XIIIth thoracic vertebra (ThXIII).</li>
	<li>Make a 4 cm skin incision on the midline, centered on ThXIII. Open the skin incision as well as the underlying bursa. Observe the aponeurosis of back muscles as well as the tips of the vertebral spine processes.</li>
	<li>Carefully localize the spine process of ThXIII by palpating the XIIIth ribs. 
	NOTE: The XIIIth rib is connected to ThXIII and therefore represents an easy to locate anatomic landmark for the identification of ThXIII. This step enables the localization of the ThXII to ThIX spinous process as well as L1 and L2 (first and second lumbar vertebras).</li>
	<li>Cut the muscular aponeurosis and detach the muscles on either side to expose the spinous processes, the laminas and the facet joints from ThIX to L2. Expose the lateral aspects of L1 and L2 by detaching muscles from the transverse processes.</li>
	<li>Hook the animal&#8217;s incisor teeth on the 3D-frame to secure the position (Figure 5). Clamp the L1 and L2 vertebras with the modified forceps. Connect the modified forceps to the 3D-frame in order to stabilize the animal.</li>
	<li>Gently pull caudally the forceps holding the lumbar spine in order to tighten the whole spine and to elevate the thorax from the bench. 
	NOTE: With the described arrangement the animal should be able to breath. Furthermore, despite respiratory movements of the rib cage, the spine and the spinal cord should also remain immobile.</li>
	<li>Remove the spinous processess from ThIX to ThXII. Gently insert the inferior blade of the bone cutter beneath the left lamina of ThXII and then close the bone cutter in order to cut the lamina (Figure 6).</li>
	<li>Repeat the same manoeuvre for the right lamina and successively remove the posterior arch. Repeat the previous steps for the vertebras ThXI to ThIX in order to achieve a four-level laminectomy. Remove both facet joints for each vertebra. 
	NOTE: Throughout the procedure, clean the operative field from local bleeding. For that, use cotton swabs&#160;and irrigation with tepid saline. Hemostasis systematically occurs within minutes.</li>
</ol>
4. CEU Probe Positioning
<ol>
	<li>Cover the dura mater with ultrasound gel. This allows effective transmission of the ultrasound waves between the probe and the spinal cord (Figure 7).</li>
	<li>Stabilize the ultrasound probe with a clamp that can be subsequently connected to the 3D-frame by a jointed arm. Manually position the probe. Ensure that the probe is oriented to obtain an oblique longitudinal sagittal slice. In a correct position, the spinal cord is strictly horizontal on the image and the central canal of the spinal cord is visible along the full segment of the spinal cord. 
	NOTE: Positioning should be guided by the real-time B-mode image displayed on the screen of the ultrasound machine. The focal distance of the ultrasound probe should be aligned with the central canal of the spinal cord. At this time, the posterior aspect of the spinal cord is accessible which will ultimately allow for positioning of the impactor.</li>
	<li>When optimal, lock the jointed arm to stabilize the position.</li>
</ol>
5. Preparation of Contrast Agent - Microbubble Reconstitution
<ol>
	<li>Using the contents of a commercial reconstitution kit and connect the plunger rod by fastening it tightly into the syringe (clockwise). Open the transfer system blister and remove the syringe tip cap. Open the transfer system cap and connect the syringe to the transfer system (fasten tightly).</li>
	<li>Remove the protective disk from the vial. Slide the vial into the transparent sleeve of the</li>
	<li>transfer system and press firmly to lock the vial in place.</li>
	<li>Empty the contents of the syringe into the vial by pushing on the plunger rod. Shake vigorously for 20 sec&#160;to mix all the contents in the vial to obtain a white milky homogeneous liquid.</li>
	<li>Invert the system and carefully withdraw the contrast agent into the syringe. Unscrew the syringe from the transfer system. After reconstitution (as directed), 1 ml of the resulting dispersion contains 8 &#181;l sulphur hexafluoride in the microbubbles. Draw the suspension of microbubbles into a 100 ml syringe. Insert the 100 ml syringe into the electric pump. Close the lid.</li>
	<li>Start constant agitation of the reconstituted microbubbles. Obtained constant agitation by slow rotation of the syringe, which maintains the microbubble suspension. Connect the pump to the jugular catheter through the flexible tubing. Set the ultrasound machine to &#8220;Harmonic Mode&#8221;. 
	NOTE: The latter corresponds to the mode in which the microbubbles can be specifically detected and visualized. This mode has a low mechanical index, which doesn&#8217;t destroy the microbubbles as opposed to the B-mode.</li>
	<li>Purge the catheter by infusing a first dose (400 &#181;l) of contrast agent. During this first infusion, check that the microbubbles do appear on the ultrasound screen. This confirms that the whole circuit (from the syringe to the rat&#8217;s bloodstream) is intact and open.</li>
	<li>Set the ultrasound machine to &#8220;B-mode&#8221; to visualize the spinal cord parenchyma and the destruction of the few microbubbles remaining in the bloodstream. The high frequency of the &#8220;B-Mode&#8221; transmits high energy to the microbubbles, which enables them to breakdown.</li>
	<li>Let the animal lay still for approximately 30 min. This period allows for the stabilization of the haemodynamic parameters.</li>
</ol>
6. Assessment of SCBF in the Intact Spinal Cord
<ol>
	<li>Set the ultrasound machine to the &#8220;Harmonic Mode&#8221;. Start simultaneously (1) infusion of contrast agent (400 &#181;l) and (2) the chronometer. 
	NOTE: During the infusion, the concentration of microbubbles in the bloodstream should increase, enabling the contrast imagining of the spinal cord (Figure 8). Since the microbubbles are quickly destroyed, the blood concentration of microbubbles starts decreasing once the injection is completed which generates a progressive decrease in contrast visualization of the spinal cord.</li>
	<li>After 1 min, select (press) the &#8220;Clip Store&#8221; button on the ultrasound machine. This will enable one to save 1 min of raw ultrasound data and the imaging video recording (that was previously displayed on the ultrasound screen).</li>
	<li>Set the ultrasound machine to &#8220;B-Mode&#8221;. This will eliminate the remaining microbubbles.</li>
</ol>
7. Experimental SCI
<ol>
	<li>Using the micromanipulator connected to the 3D-frame, position the weight-drop impaction device so that the tip of the impactor comes in contact with the dura mater (on the spinal cord midline), at the junction between ThX and ThXI (Figure 9). 
	NOTE: This level should correspond to the middle of the segment of spinal cord observed with the ultrasound device. The striker and the body of the impactor are 8 mm in diameter. The tip of the impactor, which will generate the injury, is 3 mm in diameter.</li>
	<li>Place the striker of the impaction device at a 10 cm-high position. Induce the experimental SCI by releasing the striker of the impaction device. The striker falls and releases the impactor, injuring the spinal cord. The custom made impaction delivers an impact equivalent to a 10 g weight dropped from a height of 10 cm.</li>
</ol>
8. Assessment of SCBF 5 min Post-SCI
<ol>
	<li>Repeat the steps described in section 6 (Assessment of SCBF). The microbubbles will be unable to pass through the damaged microvasculature and the injury epicenter will remain dark (Figure 10).</li>
</ol>
9. Animal Sacrifice
<ol>
	<li>Euthanize the animal with intra-peritoneal lethal injection of pentobarbital (100 mg).</li>
</ol>
10. Quantification of SCBF by Offline Analysis
<ol>
	<li>Start the Ultra-Extend Software used for quantification (on ultrasound machine). Select &#8220;file&#8221; and then select previously saved raw data and open the associated files. Activate the &#8220;quantification mode&#8221; by pressing (selecting) the &#8220;Chi Q&#8221; button. Select &#8220;Set ROI&#8221; (button) and choose the circular shape.</li>
	<li>Select &#8220;Draw ROI&#8221; (button) and draw seven adjacent circular regions of interest (ROI) on the spinal cord (Figure 11). Open the menu &#8220;Fitting&#8221; and choose the function &#8220;Curve value&#8221;. Observe the software displaying several curves, each corresponding to the changes of microbubbles concentration inside a ROI. 
	NOTE: Each curve has a &#8220;perfusion-deperfusion&#8221; profile. The first phase of the curve is flat and corresponds to the period before the arrival of microbubbles. In the second phase, the concentration of microbubbles quickly increases as a result of the infusion. In the third phase, which begins when the infusion is complete, the concentration of microbubbles progressively decreases as they disintegratse in the bloodstream.</li>
	<li>Place the first vertical line at the beginning of the second phase of the curve and select &#8220;SET&#8221;. This informs the software where to begin analysis.</li>
	<li>Place the second vertical line at the end of the recording and once again select &#8220;SET&#8221;. This informs the software where to stop analysis.</li>
	<li>Look at the &#8220;Cv&#8221; menu and record the &#8220;AUC&#8221; value, which corresponds to the &#8220;Area Under the Curve&#8221; analyzed. This value is proportional to the SCBF inside the corresponding ROI.</li>
</ol>

<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 ...

Watch this Scientific Journal Video about Contrast Enhanced Ultrasound Imaging for Assessment of Spinal Cord Blood Flow in Experimental Spinal Cord Injury at JoVE.com

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 ...

contrast-enhanced-ultrasound-imaging-for-assessment-spinal-cord-blood

Research

JoVE Journal

Medicine

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.

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

Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia3. Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI4-5. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically5. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery4-5. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.

We describe two tactile sensory testing methods for acute or chronic periods of spinal cord injury in rats. These validated procedures can detect the development and maintenance of allodynia-like sensations.

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory ...

A Contusion Model of Severe Spinal Cord Injury in Rats

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is described to obtain a consistent model of severe SCI. Use of a stereotactic frame and computer controlled impactor allows for creation of reproducible injury. Hypothermia and urinary tract infection pose significant challenges in the post-operative period. Careful monitoring of animals with daily weight recording and bladder expression allows for early detection of post-operative complications. The functional results of this contusion model are equivalent to transection models. The contusion model can be utilized to evaluate the efficacy of both neuroprotective and neuroregenerative approaches.

A contusion model of severe spinal cord injury is described. Detailed pre-operative, operative and post-operative steps are described to obtain a consistent model.

The translational potential of novel treatments should be investigated in severe spinal cord injury (SCI) contusion models. A detailed methodology is ...

Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment (RMCA) in Individuals with Chronic Spinal Cord Injury

During breathing, activation of respiratory muscles is coordinated by integrated input from the brain, brainstem, and spinal cord. When this coordination is disrupted by spinal cord injury (SCI), control of respiratory muscles innervated below the injury level is compromised1,2 leading to respiratory muscle dysfunction and pulmonary complications. These conditions are among the leading causes of death in patients with SCI3. Standard pulmonary function tests that assess respiratory motor function include spirometrical and maximum airway pressure outcomes: Forced Vital Capacity (FVC), Forced Expiratory Volume in one second (FEV1), Maximal Inspiratory Pressure (PImax) and Maximal Expiratory Pressure (PEmax)4,5. These values provide indirect measurements of respiratory muscle performance6. In clinical practice and research, a surface electromyography (sEMG) recorded from respiratory muscles can be used to assess respiratory motor function and help to diagnose neuromuscular pathology. However, variability in the sEMG amplitude inhibits efforts to develop objective and direct measures of respiratory motor function6. Based on a multi-muscle sEMG approach to characterize motor control of limb muscles7, known as the voluntary response index (VRI)8, we developed an analytical tool to characterize respiratory motor control directly from sEMG data recorded from multiple respiratory muscles during the voluntary respiratory tasks. We have termed this the Respiratory Motor Control Assessment (RMCA)9. This vector analysis method quantifies the amount and distribution of activity across muscles and presents it in the form of an index that relates the degree to which sEMG output within a test-subject resembles that from a group of healthy (non-injured) controls. The resulting index value has been shown to have high face validity, sensitivity and specificity9-11. We showed previously9 that the RMCA outcomes significantly correlate with levels of SCI and pulmonary function measures. We are presenting here the method to quantitatively compare post-spinal cord injury respiratory multi-muscle activation patterns to those of healthy individuals.

Evaluation of Respiratory Muscle Activation Using Respiratory Motor Control Assessment RMCA in Individuals with Chronic Spinal Cord Injury

The purpose of this publication is to present our original work on a multi-muscle surface electromyographic approach to quantitatively characterize respiratory muscle activation patterns in individuals with chronic spinal cord injury using vector-based analysis.

During breathing, activation of respiratory muscles is coordinated by integrated input from the brain, brainstem, and spinal cord. When this coordination ...

Implanting Glass Spinal Cord Windows in Adult Mice with Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as multiple sclerosis. Here we present a low-cost and reproducible glass window implantation protocol that is suitable for intravital microscopy and studying the dynamics of spinal cord cytoarchitecture with subcellular resolution in live adult mice with EAE. Briefly, we surgically expose the vertebrae T12-L2 and construct a chamber around the exposed vertebrae using a combination of cyanoacrylate and dental cement. A laminectomy is performed from T13 to L1, and a thin layer of transparent silicone elastomer is applied to the dorsal surface of the exposed spinal cord. A modified glass cover slip is implanted over the exposed spinal cord taking care that the glass does not directly contact the spinal cord. To reduce the infiltration of inflammatory cells between the window and spinal cord, anti-inflammatory treatment is administered every 2 days (as recommended by ethics committee) for the first 10 days after implantation. EAE is induced only 2-3 weeks after the cessation of anti-inflammatory treatment.
Using this approach we successfully induced EAE in 87% of animals with implanted windows and, using Thy1-CFP-23 mice (blue axons in dorsal spinal cord), quantified axonal loss throughout EAE progression. Taken together, this protocol may be useful for studying the recruitment of various cell populations as well as their interaction dynamics, with subcellular resolution and for extended periods of time. This intravital imaging modality represents a valuable tool for developing therapeutic strategies to treat autoimmune demyelinating diseases such as EAE.

We describe a method for implanting and maintaining glass windows over the exposed spinal cords of adult mice for studying experimental autoimmune encephalomyelitis. These windows provide chronic optical access to the spinal cord for monitoring cell dynamics at the subcellular level in live animals using two-photon microscopy.

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as ...

Controlled Cortical Impact Model for Traumatic Brain Injury

Every year over a million Americans suffer a traumatic brain injury (TBI). Combined with the incidence of TBIs worldwide, the physical, emotional, social, and economical effects are staggering. Therefore, further research into the effects of TBI and effective treatments is necessary. The controlled cortical impact (CCI) model induces traumatic brain injuries ranging from mild to severe. This method uses a rigid impactor to deliver mechanical energy to an intact dura exposed following a craniectomy. Impact is made under precise parameters at a set velocity to achieve a pre-determined deformation depth. Although other TBI models, such as weight drop and fluid percussion, exist, CCI is more accurate, easier to control, and most importantly, produces traumatic brain injuries similar to those seen in humans. However, no TBI model is currently able to reproduce pathological changes identical to those seen in human patients. The CCI model allows investigation into the short-term and long-term effects of TBI, such as neuronal death, memory deficits, and cerebral edema, as well as potential therapeutic treatments for TBI.

Traumatic brain injuries (TBIs) remain a serious health problem. Using the controlled cortical impact surgery model, research on the effects of TBI and possible treatment methods may be performed.

Every year over a million Americans suffer a traumatic brain injury (TBI).  Combined with the incidence of TBIs worldwide, the physical, emotional, ...

A Radio-telemetric System to Monitor Cardiovascular Function in Rats with Spinal Cord Transection and Embryonic Neural Stem Cell Grafts

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter connected to a radio transmitter into the femoral artery of rats that underwent a T4 spinal cord transection with or without grafting of embryonic brainstem-derived neural stem cells expressing green fluorescent protein. Compared to other methods such as cannula insertion or tail-cuff, telemetry is advantageous to continuously monitor blood pressure and heart rate in freely moving animals. It is also capable of long term multiple data acquisitions. In spinal cord injured rats, basal cardiovascular data under unrestrained condition and autonomic dysreflexia in response to colorectal distension were successfully recorded. In addition, cardiovascular parameters before and after SCI can be compared in the same rat if a transmitter is implanted before a spinal cord transection. One limitation of the described telemetry procedure is that implantation in the femoral artery may influence the blood supply to the ipsilateral hindlimb.

We present a protocol for using a radio-telemetric system to record cardiovascular parameters in T4 spinal cord transected rats eight weeks after embryonic brainstem neural stem cell grafting into the lesion site. Telemetry is an advanced technique to accurately evaluate cardiovascular function in conscious freely moving spinal cord injured rats.

High thoracic or cervical spinal cord injury (SCI) can lead to cardiovascular dysfunction. To monitor cardiovascular parameters, we implanted a catheter ...

Synergetic Use of Neural Precursor Cells and Self-assembling Peptides in Experimental Cervical Spinal Cord Injury

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. Clinically, more than 50% of SCI affect the cervical spine1. As a consequence of the primary injury, a cascade of secondary mechanisms including inflammation, apoptosis, and demyelination occur finally leading to tissue scarring and development of intramedullary cavities2,3. Both represent physical and chemical barriers to cell transplantation, integration, and regeneration. Therefore, shaping the inhibitory environment and bridging cavities to create a supportive milieu for cell transplantation and regeneration is a promising therapeutic target4. Here, a contusion/compression model of cervical SCI using an aneurysm clip is described. This model is more clinically relevant than other experimental models, since complete transection or ruptures of the cord are rare. Also in comparison to the weight drop model, which in particular damage the dorsum columns, circumferential compression of the spinal cord appears advantageous. Clip closing force and duration can be adjusted to achieve different injury severity. A ring spring facilitates precise calibration and constancy of clip force. Under physiological conditions, synthetic self-assembling peptides (SAP) self-assemble into nanofibers and thus, are appealing for application in SCI5. They can be injected directly into the lesion minimizing damage to the cord. SAPs are biocompatible structures erecting scaffolds to bridge intramedullary cavities and thus, equip the damaged cord for regenerative treatments. K2(QL)6K2 (QL6) is a novel SAP introduced by Dong et al.6 In comparison to other peptides, QL6 self-assembles into &#946;-sheets at neutral pH6.14 days after SCI, after the acute stage, SAPs are injected into the center of the lesion and neural precursor cells (NPC) are injected into adjacent dorsal columns. In order to support cell survival, transplantation is combined with continuous subdural administration of growth factors by osmotic micro pumps for 7 days.

Treating cervical spinal cord injury with both self-assembling peptides (SAP) and neural precursor cells (NPC), together with growth factors, is a promising approach to promote regeneration and recovery. A contusion/compression aneurysm clip rat model of cervical SCI and combined treatment involving SAP injection and NPC transplantation is established.

Spinal cord injuries (SCI) cause serious neurological impairment and psychological, economic, and social consequences for patients and their families. ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

Calibrated Forceps Model of Spinal Cord Compression Injury

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The calibrated forceps model of compression injury is a convenient, low cost, and very reproducible animal model for SCI. We used a pair of modified forceps in accordance with the method published by Plemel et al. (2008) to laterally compress the spinal cord to a distance of 0.35 mm. In this video, we will demonstrate a dorsal laminectomy to expose the spinal cord, followed by compression of the spinal cord with the modified forceps. In the video, we will also address issues related to the care of paraplegic laboratory animals. This injury model produces mice that exhibit impairment in sensation, as well as impaired hindlimb locomotor function. Furthermore, this method of injury produces consistent aberrations in the pathology of the SCI, as determined by immunohistochemical methods. After watching this video, viewers should be able to determine the necessary supplies and methods for producing SCI of various severities in the mouse for studies on SCI and/or treatments designed to mitigate impairment after injury.

Spinal cord injury models should be highly reproducible. We demonstrate that the calibrated forceps compression model of spinal cord injury is an easy to use surgical method for generating reproducible injuries to the murine spinal cord.

Compression injuries of the murine spinal cord are valuable animal models for the study of spinal cord injury (SCI) and spinal regenerative therapy. The ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...