Caspase-3 Activity in the Rat Amygdala Measured by Spectrofluorometry After Myocardial Infarction

Myocardial infarction (MI) is not only followed by impaired cardiac function but also by apoptosis in the amygdala, a brain region involved in the behavioral consequences of MI. This protocol describes how to induce MI, collect amygdala tissue and measure caspase-3 activity, a marker of apoptosis, therein.

The overall goal of this procedure is to measure caspase-3 activity in the rat amygdala after a myocardial infarction using spectrofluorometry. This method can answer key questions in the field of behavioral neural science, such as the consequences of a myocardial infarction on cognitive performance, mental health, the aging process, and sleep. The main advantage of this technique is that it is simple, rapid, and reliable.

Generally, individuals new to this method would struggle because it requires the dexterity for surgery and tissue sampling. Demonstrating the procedure will be Kim Gilbert, a research assistant in our laboratory. After inducing anesthesia according to approved institutional protocols, confirm proper anesthetization through the absence of the paw pinch reflex.

Intubate the rat with endotracheal tubing and place the animal in the ventral decubitus position on a heating pad to maintain body temperature at 37 degrees Celsius. Connect the endotracheal tubing to an anesthesia machine dispensing 2%isofluorane. Next, prepare the surgical site with chlorhexidine gluconate and isopropyl alcohol.

And apply ophthalmic ointment to both eyes to prevent dryness. Place a sterile drape over the animal to create a sterile field around the surgical site. And place the required sterile surgical instruments on another sterile field next to the animal.

Next, incise the skin with a number 10 scalpel blade or scissors. Use scissors or a hemostatic clamp to peel off the muscle tissue. Then use scissors or a hemostatic clamp to open the thoracic wall and position the chest retractor, and open the pericardium with the hemostatic clamp.

To induce coronary occlusion, first loop a 360 millimeter long four zero silk suture around the descending coronary artery and through the contiguous myocardial tissue. Insert both ends of the silk suture into a 14-gauge 1.25 centimeter long plastic tube. Pull both ends of the silk suture and push the tube down against the artery to occlude it.

Secure the occlusion by clamping the plastic tube with a hemostatic clamp and maintain the occlusion for 40 minutes. After 40 minutes, release the occlusion by removing the hemostatic clamp and then the flexible tubing and silk suture. Close the thorax with two zero suture and place a flexible catheter through the thoracic cavity and draw air out of the thorax with a 10 milliliter syringe to prevent pneumothorax.

Stitch the muscle with four zero silk suture and the skin with three zero silk suture. Before closing the last skin suture, again draw air out of the thorax using the 10 milliliter syringe and catheter. Finish closing the skin and then stop the isofluorane.

Place the rat in a clean cage and monitor during recovery from anesthesia. Administer analgesia with repeat dosing every eight hours. After humanely decapitating the animal according to approved procedures, place the head of the rat on a dish placed on crushed ice.

Use scissors to open the skull. Then use rongeurs to detach the bone sheaves without mutilating the underlying tissue by pulling upwards. Next, place the flat blade of a spatula between the bottom of the skull and the posterior ventral surface of the brain and detach the brain from the skull by gently pushing the blade forward, lifting the brain out of the skull.

Place the brain on its dorsal surface. Identify the hypothalamus in front of the cerebellum and cut the brain coronally in front of the posterior end of the hypothalamus and also behind the anterior end. Identify the bilateral amygdala as two small spheres underneath the temporal lobes just next to the hypothalamus.

Then flip the brain flat on its frontal end with the dorsal surface away from the experimenter. Next, separate the hemispheres and remove the cortex from the continuous amygdala. When the amygdala is revealed, cut away the amygdala with a scalpel.

Cut along the dark suture that runs across the amygdala to separate the basolateral and central medial parts and then place the two parts in separate labeled tubes on ice. This step has to be done as fast as possible to prevent deterioration of enzymes. After repeating the dissection procedure with the other hemisphere, immerse all four vials in liquid nitrogen for one minute and then store in the negative 80 degree Celsius freezer.

Begin by adding 150 microliters of lysis buffer to each five to 10 milligrams of sample on ice. Sonicate each sample on ice at maximal intensity for five seconds. Then incubate on ice for 30 minutes.

During the incubation, vortex the sample for five seconds every five minutes. Then, perform three freeze-thaw cycles by placing samples alternately in liquid nitrogen and on a thermostatically controlled heating plate set at 37 decrees Celsius. After the final freeze thaw cycle, centrifuge the tissue samples at 1300 Gs at four degrees Celsius for 10 minutes.

Next, carefully aspirate the supernatant and transfer to a fresh tube on ice. After quantitating protein according to the instructions in the written protocol, add 25 micrograms of protein to a reaction tube containing 0.8 microliter of 10 millimolar Ac-DEVD-AMC and reaction buffer for a final volume of 200 microliters. For negative reaction samples, combine 25 micrograms of protein with one microliter of 800 micromolar Ac-DEVD-CHO and 0.8 microliters of 10 millimolar Ac-DEVD-AMC.

Incubate all samples and controls in the dark for three hours at 37 degrees Celsius. After the incubation time has elapsed, stop the reaction with 600 microliters of 0.4 molar glycine and 0.4 molar sodium hydroxide at pH 10 in each sample and control. Add two milliliters of distilled water to each reaction in a glass cuvette.

Quantify fluorescence by spectrofluorometry. Read controls and samples for 10 seconds with one reading every second. Finally, quantify specific activity in each sample according to the formula now shown on screen.

This histogram shows caspase-3 activity in the amygdala of rats that have undergone myocardial infarction. Data is expressed as the percentage of mean activity in sham controls set to 100%Each group consisted of eight rats. The asterisk indicates a significant between group difference with a p value of less than 0.05.

Once mastered, this technique can be done in seven hours excluding the interval between myocardial infarction surgery and tissue sampling. After watching this video, you should have a good understanding of how to induce a myocardial infarction in a rat and measure caspase-3 activity in the rat amygdala using spectrofluorometry.