The overall goal of the following experiment is to examine the systemic and regional hemodynamic changes that occur during the asphyxiation and reoxygenation processes and the respective effects of applicable interventions. This is achieved by first surgically preparing the animal with vascular, tracheal, carotid mesenteric, renal, and pulmonary catheters for induction and monitoring of the hypoxia. Next alveolar hypoxia is induced, which causes severe hypoxemia producing clinical asphyxia in the animal.
Finally, the hypoxic process is abruptly terminated after two hours by reoxygenation the animal with 100%oxygen for 30 minutes, and then 21%oxygen for 3.5 hours, simulating resuscitation of an asphyxiated neonate. This method can be used to monitor the progressive development of cardiogenic shock, hypotension, and severe metabolic acidosis that occur in response to two hours of severe hypoxia. The implications of this technique extend towards the therapy of neonatal asphyxia because of how closely the method simulates the asphyxia in a clinical setting, giving significant translational value to the collected data.
So generally, individuals new to this method will struggle because the technical challenges of the surgical procedure and the medical knowledge required to perform the hypoxia re oxygenation maneuvers. In the video, Dr.David Bacon will be demonstrating the procedure along with Dr.Rich Dip Gill, a graduate student from the Neural science Laboratory. The the protocol shown here is a non survival procedure.
Monitor the oxygen saturation and vital signs of the anesthetized newborn piglet by pulse, oximetry, and cardiopulmonary monitor respectively. Maintain the rectal temperature at 38 to 40 degrees Celsius with a heating blanket and radiant warmer. To prepare the animal for placement of the vascular catheters, make a two to three centimeter long incision.
In the right groin, dissect one centimeter of the right femoral artery and one centimeter of the right femoral vein. Put 2 3 0 strings around each vessel for right femoral venous catheterization. Legate the distal end of the vein.
Insert an argyle catheter to 15 centimeters. Placing the catheter at the right atrium. Tie both strings to secure the catheter for right femoral arterial catheterization.
Ligate the distal end of the artery. Lift up the proximal string to stop the blood flow. Insert an argyle catheter to five centimeters placing the arterial catheter at the infrarenal aorta for continuous mean arterial pressure measurement and blood sampling.
Then tie both strings to secure the catheter and close the skin. To prepare the animal for mechanical ventilation, make a long two to three centimeter horizontal incision in the neck. After dissecting and exposing one centimeter of the trachea, put 2 1 0 strings around the trachea.
Next, insert a three zero endotracheal tube at one centimeter into the trachea. Connect the tube to a ventilator and commence mechanical ventilation. Tie the one zero strings to secure the endotracheal tube.
After dissecting and exposing the common carotid artery, encircle the vessel with a two RB transit time ultrasound flow Probe to continuously measure the blood flow. After dosing the animal with extra anesthesia, lay the animal at the right lateral position and make a long subcostal flank incision and carefully dissect the muscle layers. Then expose the abdominal aorta, minimizing vascular handling and lymphatic injury.
Now dissect 0.5 to one centimeter of the superior mesenteric artery, and put a three SB transonic flow probe around it. Next, dissect 0.5 to one centimeter of the left renal artery and put a two SB transonic flow probe around it. Then close the skin and secure the flow probe using adhesive tape as necessary.
After dosing the piglet with extra anesthesia and performing a thoracotomy at the left fourth intercostal space, use a dental swab to press down the left lung and increase the oxygen as necessary. Although placing the pulmonary arterial catheter and flow probe are the most difficult parts of the experimental procedure, the lation of the patent ductus arterials is, is important for the use of pulmonary arterial flow as a surrogate of cardiac output. Thus, special care with the following steps will result in a more accurate simulation of clinical hypoxia.
Then open the pericardium. Identify the ductus arteriosis, which runs from the aorta to the pulmonary artery. The ductus arteriosis may be ligated placing a thick three zero silk tie at its origin.
Next, free the main pole mony artery and then pass a vascular sling under the artery using a thick zero tie. Then use a five zero proline suture at the base of the artery to create a per string for the placement of the pulmonary artery catheter. Now take a 20 gauge angio with three side holes at less than one centimeter from the tip of the catheter and insert it through the per string to a maximum of one centimeter.
Then check the catheter for free flow of venous blood. Connect the catheter to the pressure transducer and check for pulmonary artery pressure and waveform. Next, tighten the purse string to secure the pulmonary catheter.
Then place a six SB transonic flow probe around the main pulmonary artery. Finally, place ultrasonic gel between the flow probe and the artery to allow for optimal signal transduction and cover the wound with moist saline gauze oes to induce hypoxia. Decrease the inspired oxygen concentration to 12%by increasing the concentration of inhaled nitrogen gas.
Then adjust the inspired oxygen concentration between 10 and 15%to obtain an arterial partial pressure of oxygen of 20 to 40 millimeters of mercury and arterial oxygen saturation of 30 to 40%for two hours. Next, perform an arterial blood analysis to assess arterial partial pressure of carbon dioxide and adjust the ventilator rate accordingly. Continue to monitor for changes in blood flow at the common carotid, superior mesenteric, and left renal arteries.
During the second hour of hypoxia, the hypoxic stress is increased to steadily lower cardiac output to 30 to 40%of baseline mean arterial pressure to 30 to 35 millimeters of mercury and arterial pH to 6.95 to 7.05. Increase the inspired oxygen concentration abruptly to 100%by discontinuing nitrogen gas while continuing pure oxygen. Note the dramatic recovery of the cardiac output, mean arterial pressure and other hemodynamic parameters.
Continue resuscitation with 100%oxygen for half an hour following this time period. Quickly reduce the inspired oxygen concentration to 21%The induction of hypoxemia in the newborn piglet over the first hour of hypoxia results in an increase in the cardiac output or pulmonary arterial flow to 130%to 140%of baseline. Typically, the cardiac output reaches its peak compensation between the first 30 and 60 minutes of hypoxia.
An increase in heart rate during hypoxia is also observed. Further blood flow becomes centralized, resulting in a decreased mesenteric and renal perfusion. At the same time, a preserved or increased common carotid arterial flow is observed during the second hour of hypoxia.
There is a steady decrease of cardiac output, a development of hypotension and a slowing of heart rate with or without arrhythmia. Hypoxia also induces pulmonary hypotension with increased pulmonary artery pressure as seen here in this final figure. After watching this video, you should have a good understanding of how to continuously monitor and measure the systemic and regional hemodynamic state in a newborn piglet.
Once mastered, this technique can be done in 75 minutes if it is performed properly Following the procedure. Other methods, like the placement of free radical sensors at the cerebral cortex can be performed to answer additional questions like, what is the role of free radical generation in hypoxic ischemic encephalopathy?
