This protocol describes the use of a modified T-maze to evaluate functional learning/memory in asphyxia cardiac arrest-induced cerebral ischemia.
The overall goal of this modified T-maze is to evaluate functional learning and memory after cardiac arrest induced cerebral ischemia. These modified team mates can help answer hippocampus function in disease that cause short-term memory deficits, such as cardiac arrest. The major advantage of the modified T-maze is it's relatively simple design.
That is powerful enough to measure functional learning memory after ischemia. Additionally the data analysis is simple and straight forward. Since asphyxial cardiac arrest mainly results in short-term memory deficits, the modified T-maze can also be applied to other central nervous system diseases, that cause short-term memory deficits.
Such as Alzheimers disease, Parkinson disease and transient ischemic attack. After femoral and arterial and venous catheterization, adjust physiological parameters as needed by modulating stroke volume, oxygen, or nitrous oxide levels. Then use a one milliliter syringe connected with a 23 gauge lure stub adaptor, to administer vecuronium bromide via femoral vein and wait for two minutes.
Ensure that the blood pressure is at or around 100 millimeters of mercury. Induce apnea for six minutes by disconnecting the endotracheal tube from the ventilator. Further block the endotracheal tube by a one milliliter syringe to ensure complete apnea.
During the last minute of apnea, adjust the respiratory rate of the ventilator to 80 breaths per minute and increase oxygen to two liters per minute with zero percent nitrous oxide to blow out any remaining isoflurane or nitrous oxide remaining in the ventilator. After the six minutes of apnea, remove the one milliliter syringe from the endotracheal tube. Reconnect the endotracheal tube to the ventilator.
Next, use a one milliliter syringe connected with a 23 gauge lure stub adaptor to administer epinephrine via femoral vein. Administer manual chest compressions by the thumb, index and middle finger on the animals chest, in a light circular motion on the X and Z axis, until the return of spontaneous circulation. Then use another one milliliter syringe connected with a 23 gauge lure stub adapter to administer sodium bicarbonate via femoral vein, immediately after return to spontaneous circulation, to alleviate respiratory acidosis.
10 minutes after resuscitation, measure blood gases again to determine the acid base status. Use a hemostat to clamp the femoral artery and vein. Slowly and gently remove arterial and venous catheters using blunt tip forceps.
Then legate femoral artery and vein with a 5 O silk suture to prevent bleeding. Close the skin overlying the surgical site with a 3 O silk suture using the interrupted suturing technique, to minimize the chance of wound reopening. After the rat starts breathing on its own, 30 to 60 minutes after resuscitation, disconnect from the ventilator and gently remove the endotracheal tube.
Place the rat in the baby incubator overnight. Finally transfer the rat to the individual cage and return the rat to the animal facility with regular chow and water. On the day before surgery, handle each rat for five minutes and never elevate the rat from the cage when handling.
Then gently pick it up by it's tail with one hand, while the other hand supports it's legs, and let it jump from the hand to the cage five times. Separate each rat into individual cages so they will not dominate for food and or fight. Then three days after sham or ACA surgery, transfer the rat with the cage into a quiet and dark room before the start of the first run.
Only turn on a low power desk lamp, and place it in the corner of the testing room to maintain minimum illumination. Allow the rat to adapt to darkness for 10 minutes. Next spread a thin layer of bedding to cover the entire floor of the maze and place the rat at the start arm.
Allow each rat three minutes to explore the right or left goal arm. After the rat commits to one of the goal arms, when all four paws of the rat have entered the goal arm, lock the T junction between the start arm and the opposing goal arm to prevent the rat from entering the opposing goal arm. Leave the rat in the maze for 30 seconds.
Then pick up the rat and place it back in it's cage for approximately 30 seconds. Then remove the T junction block from the T-maze. Place the rat at the start arm again and repeat the test to determine spontaneous alternation.
Have rats perform four runs per day with a 10 minute break after the first two runs. Finally, change the bedding during the 10 minute break and between animals to eliminate scent bias. Clean the T-maze with 75%ethanol followed by distilled water at the end of each experimental day.
Results from the spontaneous alternation test suggest that the alternation rate from three consecutive days in the ACA group was significantly lower compared to the control group. This is due to the fact that rats subjected to ACA developed an increase in side preference compared to controls. After watching this video, you should have a good understanding of how to utilize this modified T-maze to evaluate short-term memory deficit after cerebral ischemia in a rodent.
While attempting this modified T-maze, it is important to gently block the opposite arm after the rat enters one of the goal arms. Additionally, T-maze should be performed in dark room with minimal illumination, which reduces rat anxiety.
