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

Podsumowanie

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.

Streszczenie

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 µl) 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 “ischemic penumbra zone”. This tool is of major interest for assessing the effects of therapies aimed at limiting ischemia and the resulting tissue necrosis subsequent to SCI.

Wprowadzenie

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 deficit^2,3.

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 “ischemic penumbra zone”. 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 microspheres^7,8 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, photoplethysmography^14,15 and in-vivo light microscopy¹⁶ have a very limited depth/area of measurement^11-13.

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 μm and 90% having a diameter less than 6 μm) 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.

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Protokół

NOTE: The methods described in this manuscript were approved by the bioethics committee of the Lariboisière School of Medicine, Paris, France (CEEALV/2011–08-01).

1. Instrument Preparation

1. 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).
2. 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).
3. 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.
4. 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.
5. Turn on the ultrasound machine.
6. 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).

2. Jugular Vein Catheterization (Figure 3)

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.
7. Connect flexible tubing to the catheter for further injection of contrast agent (microbubbles). Keep it closed (sealed) until ready for use.

3. Accessing the Spine, Laminectomy and Rat Positioning (in the 3D-frame)

1. Place the animal in a flat prone horizontal position. Shave and clean the back (thoracic region) of the animal.
2. 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).
3. 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.
4. 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).
5. 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.
6. Hook the animal’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.
7. 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.
8. 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).
9. 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 and irrigation with tepid saline. Hemostasis systematically occurs within minutes.

4. CEU Probe Positioning

1. Cover the dura mater with ultrasound gel. This allows effective transmission of the ultrasound waves between the probe and the spinal cord (Figure 7).
2. 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.
3. When optimal, lock the jointed arm to stabilize the position.

5. Preparation of Contrast Agent - Microbubble Reconstitution

1. 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).
2. Remove the protective disk from the vial. Slide the vial into the transparent sleeve of the
3. transfer system and press firmly to lock the vial in place.
4. Empty the contents of the syringe into the vial by pushing on the plunger rod. Shake vigorously for 20 sec to mix all the contents in the vial to obtain a white milky homogeneous liquid.
5. 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 µl 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.
6. 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 “Harmonic Mode”.
   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’t destroy the microbubbles as opposed to the B-mode.
7. Purge the catheter by infusing a first dose (400 µl) 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’s bloodstream) is intact and open.
8. Set the ultrasound machine to “B-mode” to visualize the spinal cord parenchyma and the destruction of the few microbubbles remaining in the bloodstream. The high frequency of the “B-Mode” transmits high energy to the microbubbles, which enables them to breakdown.
9. Let the animal lay still for approximately 30 min. This period allows for the stabilization of the haemodynamic parameters.

6. Assessment of SCBF in the Intact Spinal Cord

1. Set the ultrasound machine to the “Harmonic Mode”. Start simultaneously (1) infusion of contrast agent (400 µl) 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.
2. After 1 min, select (press) the “Clip Store” 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).
3. Set the ultrasound machine to “B-Mode”. This will eliminate the remaining microbubbles.

7. Experimental SCI

1. 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.
2. 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.

8. Assessment of SCBF 5 min Post-SCI

1. 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).

9. Animal Sacrifice

1. Euthanize the animal with intra-peritoneal lethal injection of pentobarbital (100 mg).

10. Quantification of SCBF by Offline Analysis

1. Start the Ultra-Extend Software used for quantification (on ultrasound machine). Select “file” and then select previously saved raw data and open the associated files. Activate the “quantification mode” by pressing (selecting) the “Chi Q” button. Select “Set ROI” (button) and choose the circular shape.
2. Select “Draw ROI” (button) and draw seven adjacent circular regions of interest (ROI) on the spinal cord (Figure 11). Open the menu “Fitting” and choose the function “Curve value”. Observe the software displaying several curves, each corresponding to the changes of microbubbles concentration inside a ROI.
   NOTE: Each curve has a “perfusion-deperfusion” 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.
3. Place the first vertical line at the beginning of the second phase of the curve and select “SET”. This informs the software where to begin analysis.
4. Place the second vertical line at the end of the recording and once again select “SET”. This informs the software where to stop analysis.
5. Look at the “Cv” menu and record the “AUC” value, which corresponds to the “Area Under the Curve” analyzed. This value is proportional to the SCBF inside the corresponding ROI.

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Wyniki

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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Dyskusje

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...

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Materiały

Name	Company	Catalog Number	Comments
External Fixator Hoffman 3	Stryker, Kalamazoo, USA		Modular system used to build the custom made 3D frame and the jointed arm holding the ultrasound probe
Toshiba Applio	Toshiba, Tokyo, Japan		Ultrasound machine
Sonovue	Bracco, Milan, Italy		Contrast agent : microbubbles
Vueject pump	Bracco, Milan, Italy		Electric pump for infusion of microbubbles bolus
Aquasonic Ultrasound Gel	Parker Laboratories, Fairfield, NJ, USA		Ultrasound gel used to transmit the ultrasound waves
Isovet	Piramal Healthcare, Mumbai, India		Isoflurane used for anesthesia
Ultra Extend	Toshiba, Tokyo, Japan		Software used for quantification of spinal cord blood flow
Mastercraft Five-piece Mini-pliers Set, Product #58-4788-6	Canadian Tire, Toronto, Canada		Set of pliers for Do-it-yourself job