The overall goal is to induce a transient ischemic event in the brain to simulate the cerebral insult caused by cardiac arrest and resuscitation. First systemic hypotension is induced by the withdrawal of blood from the femoral artery. Next, the carotid arteries are occluded to restrict blood flow to the brain.
The brain is maintained in an ischemic state for eight minutes by tight control of systemic blood pressure. Finally, the clips are removed and the withdrawn blood is reintroduced. Ultimately, neuronal damage can be quantified at different time points following ischemia.
The main advantage of this technique over existing methods like cardiac arrest and resuscitation, is that the cerebral ischemic event can be simulated without the mortality and comorbidities associated with systemic ischemia. Visual demonstration of this surgery allows us to emphasize the importance of particular procedural nuances to ensure reproducibility Begin by making a vascular catheter. To do this, cut an eight inch length of polyethylene 50 tubing and insert a blunted 23 gauge needle into one end.
Secure this needle into one port of a three-way stop cock and place. Another three-way stop cock on the opposite port. Heparinized the stop cocks and catheter by running heparin saline solution throughout.
Next, connect a 10 milliliter saline syringe onto the stop cock, perpendicular and distal to the catheter. Connect the stop cock distal to the catheter to the pressure transducer tube. Fill the system with saline and remove any air bubbles.
Intubate an anesthetized rat to allow for the continuous administration of anesthesia. After administering an IP injection of ketamine, reduce the isof fluorine to 1.5%and monitor the level of anesthesia throughout the procedure. Once anesthetized shave the neck region and the right pelvis where the thigh meets the abdomen, place the rat in a supine position on a heated blanket.
After applying lubricant to the eyes and inserting a rectal thermometer, scrub the incision areas with repeated applications of Betadine and 70%ethanol. Place a surgical field over the rat and cut holes to expose the incision areas. After confirming the proper depth of anesthesia, a scalpel to make a midline incision along the neck blunt dissect through the tissue to reach the sterno hyoid and the sterno mastoid muscles.
Carefully separate the muscle by blunt dissection until the common carotid artery is visualized. Isolate the carotid arteries on both sides by passing a silk suture underneath. Clear away any fascia from the vessels at the indentation where the hind limb muscles meet the abdomen.
Make a second incision. Dissect beneath the abdominal muscle along the thigh muscles until you reach the inguinal ligament. Exposing the femoral neurovascular bundle.
Carefully isolate the femoral artery by passing a silk suture underneath and cla away any fascia leaving five to seven millimeters of exposed vessel. Once exposed, tie a permanent knot at the distal end of the artery. Place another silk suture around the proximal end of the artery and tie a loose knot.
Next, apply traction on the suture at the proximal end to occlude blood flow and make a small incision across the top of the vessel with ophthalmic scissors. Use a vascular introducer to insert the catheter tubing into the vessel. Once the catheter is in place, tie a loose knot around the vessel and catheter to secure.
Insert the catheter tubing into the vessel, seven to nine millimeters past the vessel incision and toward the midline. Remove the traction from the vessel and allow it to line naturally. After administering 0.3 milliliters of heparin, flush away any blood from the catheter with a small amount of saline to prevent clotting.
Finally, turn on the pressure monitor and calibrate the equipment. To ensure consistency between procedures, it's important to keep detailed records of the core temperature, blood pressure, and other physiological variables during surgery. When ready to begin, connect a heparinized syringe to the stop cock perpendicular and proximal to the catheter.
Set a timer for one minute and open the stop cock so that blood can be withdrawn. Withdrawal the blood slowly. Within one minute, seven to nine millimeters of blood should be removed.
The MAP should then drop to around 30 millimeters of mercury, the target blood pressure to achieve ischemia. Next, apply hemostatic clips to the carotid arteries and start a timer that is set for eight minutes. Immediately check the blood pressure if the MAP is not at 30 millimeters of mercury, infuse or withdraw blood slowly until the correct level is reached.
Make adjustments throughout ischemia to maintain this level. Keep the blood at 37 degrees Celsius to avoid cooling before it can be re-infused at the end of the eight minute ischemic period. Retrieve the warm blood, withdrawn earlier for reperfusion, remove the hemostatic eclipse and re-infuse the blood slowly at about two milliliters per minute when the blood has been reintroduced, remove the cannula from the femoral artery and secure two separate knots proximal to the incision on the artery.
Suture the incisions with a discontinuous pattern using an inverse cutting needle with 5.0 Vicryl suture. When the wound is closed, turn off the iso fluorine to wean the rat off the ventilator and administer an analgesic. When voluntary respiration returns.
Extubate the animal and place in a heated recovery cage. Monitor the animal for signs of distress until fully recovered at the desired time point. After ischemia, fix the brain in 4%Paraform aldehyde.
After trans cardio perfusion, the brain sample is later sliced into sections and stained with crestal violet image. J.A free program offered by the National Institutes of Health is used to quantify the viable neurons which represent the extent of brain damage. First, open the image with the cell counter plugin.
Next, select a marking color and count the neurons within the boundaries of the scale. Bar neurons are counted based on simple inclusion criteria for neuronal morphology. That is a large parametal shape or exclusion criteria for microglial, astrocyte morphology, or small round, or long tubular shapes.
After recording the neuron counts save the cell counter window. The mean of all images from each animal will provide a single mean neuron count, which is the quantitative measure of CA one hippocampal damage. The example shows the injury produced by eight minutes of global brain ischemia 14 days after reperfusion.
Here the hippocampus from a sham operated rat exhibits normal morphology, including an intact ca one. In contrast damages localized to the ca one region of the hippocampus. In rats with ischemia and reperfusion shown here are representative counting windows in a sham operated control animal and an animal exposed to global brain ischemia.
The injured CA one displays a staining that includes microglia and astrocytes, which appear small and tubular or teardrop shaped. These can be distinguished from intact hippocampal neurons, which are large with a circular or parametal shape. Finally, this data demonstrates that the neuronal counts are much lower in the CA one region of ischemic animals compared to control.
After watching this video, you should have an understanding of how to create transient global brain ischemia in the rat and how to quantify the associated injury. Success requires delicate surgical skills and a strict maintenance of anesthesia, hemodynamics, and physiological parameters After its development. This technique paved the way for researchers to explore the cerebral events that occur following global brain ischemia in patients suffering.
Cardiac arrest followed by resuscitation.
