Our protocol provides a reproducible method for real-time assessment of spinal cord microcirculation. It offers the ability to evaluate therapeutic approaches on spinal cord microcirculation and the ischemia reperfusion conditions. Our technique is easy to learn, minimally invasive, and offer us the opportunity to assess spinal cord microperfusion in parallel with cerebrospinal fluid pressure and macrohemodynamics in a large animal model.
Demonstrating the procedure will be Dr.Christoph Behem and Dr.Till Friedheim, specialists for anesthesiology and scientific members of our laboratory. Additional demonstrators will be Michael Graessler, resident in anesthesiology. After confirming an appropriate level of sedation in a three-month-old 40 kilogram pig, place the animal in the right lateral position and flex the animal's back to widen the space between the vertebrae.
Surgically expose the paravertebral area for the preparation of spinous processes and vertebral arches and place a vascular 14 gauge peripheral vein catheter paramedian into the spinal cord at the level of thoracic vertebra 13 to 14 or lumbar vertebra one to two between two vertebral arches. Replace the needle with the laser Doppler needle probe and connect the probe to the designated hard and software to test the signal quality. A stable signal with a moderate pulsatility should be observed.
Carefully fix the probe with sutures and use panning to prevent the probe from dislocating or kinking. For percutaneous placement of cerebrospinal fluid drainage for measuring and controlling cerebrospinal pressure, identify the level of lumbar four to five or L5 to 6. Puncture the skin and the subcutaneous space with the introducer needle and remove the inlay needle.
Place a saline-filled syringe onto the introducer needle and use constant pressure to carefully introduce the needle into the epidural space. Once a loss of resistance is felt, indicating a correct epidural placement, insert the inlay needle and advance the needle two to three millimeters into the intrathecal space. Introduce the drainage up to a 20 centimeter depth.
Attach a Luer lock adapter and verify the position by careful aspiration of the liquor. Then carefully fix the drainage with sutures and connect the drainage to the cerebrospinal fluid drainage system. Next, expose the skull behind the left ear and use a six millimeter drill attachment to carefully perform a drill hole trepanation of the skull.
Introduce a second laser Doppler probe directly into the brain and use sutures to carefully fix the probe in place. Connect the probe to the hard and software to test the signal quality. A stable signal with moderate pulsatility should be observed.
Disconnect all of the probes and with the help of four to five assistants, carefully place the animal in a supine position using padding, taking care not to disturb the probes. Reconnect the probes and check signal quality. Then start continuous cerebrospinal fluid drainage with a target pressure of 10 millimeters of mercury and a drainage volume of 20 milliliters per hour.
After performing a mini laparotomy, increase the fraction of inspired oxygen to one and administer a 0.1 milligrams per kilogram of pancuronium intravenously. Gently dissect the sternum from the surrounding tissue, then perform retrosternal compress placement to prevent injuries. After stopping ventilation, divide the bone with an oscillating saw and restart ventilation with a reduced fraction of inspired oxygen to 0.3.
Use electrocautery to reduce bleeding and seal the sternum with bone wax. Carefully mobilize the base of the left lung and divide the left lateral part of the diaphragm to facilitate surgical exposure. Gently retract the left lung to expose the descending aorta proximal to the celiac trunk and divide the surrounding tissue.
Place an Overholt around the descending aorta to ensure proper exposure and attach a flow probe around the descending thoracic aorta, then confirm the presence of a good quality signal and attach a vessel loop around the descending aorta distal to the flow probe to mark the area of aortic cross-clamping. After acquisition of a baseline measurement, administer consecutive volume loading steps of seven milliliters per kilogram of hydroxyethyl starch colloid over a period of five minutes per step. Five minutes of equilibration between each step until the increase in cardiac output is less than 15%After completion of the hemodynamic optimization, repeat the measurements.
Next, place an aortic clamp at the previously marked area on the supraceliac aorta to induce ischemia reperfusion in one, two, five, 10, and 30-minute intervals for a total of 48 minutes. Continue aortic cross-clamping after each interval. After a maximum of five minutes or after normalization of femoral artery flow, perform manual inflow occlusion of the inferior vena cava to prevent blood pressure increases of greater than 100 milliliters of mercury mean arterial pressure and repeat the measurements at the end of the 30-minute clamping interval prior to reperfusion.
When the measurements have been obtained, gradually open the clamp to ensure hemodynamic stability and administer seven milliliters per kilogram of hydroxyethyl starch colloids and additional bolus injections of 10 to 20 micrograms of norepinephrine and/or epinephrine for stabilization. Adjust the respiratory rate to ensure normocapnia as necessary. After one hour, repeat the measurements.
After obtaining the post-reperfusion measurements, repeat the volume loading as demonstrated and obtain another set of measurements. 4-1/2 hours after the induction of ischemia reperfusion, obtain the final set of measurements. Here, examples of real-time spinal cord microcirculatory recordings in combination with cerebral microcirculatory and macrohemodynamic recordings during aortic cross-clamping for ischemia induction, as well as during unclamping and reperfusion can be observed.
The disruption of the descending aortic flow was followed by a marked decrease in spinal cord flux while pressure in the ascending aortic increased. Reperfusion led to opposite effects. Statistical analysis of the macro and microcirculatory parameters indicate a marked reduction of spinal cord flux during ischemia.
In contrast, cerebral flux markedly increased during ischemia as indicated by the estimated marginal means and their confidence intervals. This was accompanied by an increase in heart rate, arterial pressure and systemic vascular resistance, whereas cardiac output and stroke volume decreased. Fluorescent microsphere analysis revealed a marked decrease in spinal cord microcirculatory blood flow in the lower spinal cord, while no significant change was observed in the upper spinal cord.
Reperfusion led to opposite effects. Although there was a further decrease in cardiac output, stroke volume and arterial pressure at the end of the protocol, the spinal cord flux and spinal cord microcirculatory blood flow were stable. The insertion of the spinal cord needle probe needs profound knowledge of the anatomy.
Additionally, you need very high technical skills in the handling of the probe. Multiple attempts must be avoided to prevent injuries to the spinal cord, which could affect the methodology.
