The overall goal of this procedure is to assess spinal cord blood flow or SCBF. This is accomplished by first inserting a catheter into the jugular vein of a rat. Next, the animal is stabilized in an experimental frame, a laminectomy is performed and the ultrasound probe is positioned.
A contrast agent is then prepared and blood flow is assessed on the intact spinal cord by injecting microbubbles, then an experimental spinal cord injury is performed, followed by a second injection of microbubbles to assess blood flow in the injured spinal cord. Ultimately, ultrasound is used to show changes in blood flow as a result of spinal cord injury. The main advantage of this technique over existing methods like laser dipple, is that it allows in vivo temporal spatial quantification of spinal cord blo flow while providing morphological assessment of the spinal cord parenchyma.
After preparing the instruments, reagents 3D frame and weight drop device and anesthetizing the animal according to the text protocol. Confirm proper anesthesia by using forceps to pinch the paws. Apply vet ointment to the eyes to prevent dryness.
Shave the neck and use a Betadine solution and ethanol to clean the skin. Make a left-sided incision on the neck. Then retract the soft tissues to find the jugular vein.
Next, insert a ligature at the rostral part of the vein and tighten it. Pass another thread around the vein just below the first ligature With the knot ready to be tightened, open the wall of the vein below the rostral ligature. Then introduce a 14 gauge catheter into the lumen of the vein and push it towards the heart.
Push the catheter further. Secure the catheter in the vein by firmly tightening the knot on the vein with the catheter inside. Now, assess the patency of the catheter by pressing the plunger rod to check that there is no resistance.
Using heparinized saline to flush it. This prevents obstruction of the catheter from a potential blood clot. Connect flexible tubing to the catheter for further injection of contrast agent, keep it closed until ready to use to carry out a laminectomy.
Place the animal in a flat prone horizontal position. Shave and clean the thoracic region on the back of the animal palpate to identify the last rib. To estimate the location of the 13th thoracic vertebrae, make a four centimeter skin incision on the midline centered on TH 13, and open the skin and the underlying bursa.
Carefully palpate the 13th ribs to localize the spine process of TH 13. Next, cut the muscular a neurosis and detach the muscles on either side to expose the spinous processes, the laminas and the facet joints from TH nine to L two. Expose the lateral aspects of L one and L two by detaching muscles from the transverse processes.
Then hook the animals incisor teeth on the 3D frame. To secure the position, use the modified forceps to clamp the L one and L two vertebrae and connect the forceps to the 3D frame. In order to stabilize the animal while holding the lumbar spine, gently pull the forceps coddly in order to tighten the whole spine and to elevate the thorax from the bench.
Now remove the spinous processes from TH nine to TH 12, and then gently insert the inferior blade of the bone cutter beneath the left lamina of TH 12. Then close the bone cutter in order to cut the lamina. Repeat the same maneuver for the right lamina and remove the posterior arch.
Then repeat for the TH 11 to TH nine vertebrae to achieve a four level laminectomy. Finally, remove both facet joints for each vertebrae. To position the CEU probe, begin by using ultrasound gel to cover the dura mater.
Connect a clamp to the 3D frame to stabilize the ultrasound probe. Then manually position the probe. When the position is optimal.
Lock the jointed arm to stabilize it from the contents of a microbubble reconstitution kit. Connect the plunger rod by fastening it tightly into the syringe. Open the transfer system blister and remove the syringe cap.
Open the transfer system cap and connect the syringe to the transfer system. Remove the protective disc from the vial. Then slide the vial into the transparent sleeve of the transfer system, and press firmly to lock the vial in place.
Empty the contents of the syringe into the vial by pushing on the plunger broad shake vigorously for 20 seconds to mix all the contents in the vial. To obtain a white milky homogeneous liquid. Invert the system and carefully withdraw the contrast agent into the syringe.
Then unscrew the syringe from the transfer system. After reconstituting the microbubbles as directed, draw the suspension of microbubbles into a 20 milliliter syringe. Insert the 20 milliliter syringe into the electric pump and close the lid.
Next, start constant agitation of the reconstituted microbubbles by slowly rotating the syringe. Connect the pump to the jugular catheter through the flexible tubing. Set the ultrasound machine to harmonic mode.
Purge the catheter by infusing a first dose of contrast agent. Check that the microbubbles appear on the ultrasound screen. Set the ultrasound machine to B mode to visualize the spinal cord parenchyma and the destruction of the few microbubbles remaining in the bloodstream.
Let the animal lay still for approximately 30 minutes. For stabilization of hemodynamic parameters, set the ultrasound machine to harmonic mode simultaneously start infusion of the contrast agent and the chronometer. After one minute, press the clip store button on the ultrasound machine.
Then set the ultrasound machine to be mode using the micro manipulator 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 at the junction between TH 10 and TH 11. Now, position the striker of the impaction device 10 centimeters high, induce the experimental spinal cord injury or SCI by releasing the Stryker of the impaction device, enabling the Stryker to fall and release the impactor, injuring the spinal cord.
Repeat the infusion of contrast agent as just demonstrated. To quantify SCBF offline, start the ultra extend software used for quantification on the ultrasound machine. Activate quantification mode by pressing the Kai Q button.
Select draw ROI and draw five adjacent circular regions of interest on the spinal cord. Look at the CV menu and record the a UC value, which corresponds to the area under the curve analyzed. Using the protocol demonstrated in this video, it is possible to map the SEBF along a longitudinal spinal cord sagittal segment.
As shown in this figure in the intact spinal cord, there appear to be SCBF irregularities within the parenchyma due to the variable distribution of radi medullary arteries from one animal to another that reach the anterior spinal artery and therefore provide a blood supply to the spinal cord parenchyma. After SCI, real-time, CEU imaging shows a deficiency in circulation at the injury epicenter. The epicenter remains dark as there is no active blood flow.
Analysis of the blood flow using several ROIs shows three unique blood flow territories. At the level of the epicenter, the blood flow is the lowest with a mean decrease of about minus 90%in the territories adjacent to the epicenter, corresponding to the ischemic penumbra zone where neuroprotective therapies should be targeted. SCBF was also significantly decreased in the most remote areas corresponding to intact tissue.
SCBF is preserved After its development. This technique paved the way for researchers in the field of spinal cord injury to explore secondary injury and ischemia in rats.
