This protocol provides real-time analysis of two major bioenergetic pathways:mitochondrial respiration and glycolysis in freshly dissected ex vivo mouse retinal tissues. Traditional seahorse analysis was focused on cultured monolayered cells, while this protocol allows researchers to study mitochondrial activity in freshly dissected tissues, specifically the retina. This technique is used to study mitochondria's activities and glycolysis flags in retinas of inherited retinal degeneration mouse models.
It is a useful tool to retinal research and also has potential to be applied to other type of tissue to develop potential therapies. The most difficult part of this protocol is to obtain quality punch discs from freshly dissected mouse retina, so it's recommended that the individual who will perform this technique has good retinal dissecting skills. The day before the experiment, open the sensor cartridge cassette and add one milliliter of calibration medium to each well of the utility plate.
Incubate overnight in a carbon dioxide-free incubator at 37 degrees Celsius to activate the fluorophores. On the day of the experiment, prepare assay medium in dilute compound drugs from stock. Warm up the assay medium in the 37 degrees Celsius water bath.
Set up the assay program on the machine as described in the text manuscript. Open the lid of the cassette containing mesh inserts. Pipette eight microliters of the coating mix to each mesh insert, then use a pipette tip to gently spread the droplet around to distribute the coating mix equally throughout the mesh insert.
Close the cassette and allow the mesh inserts to incubate at room temperature for at least 25 minutes for absorption. After incubation, wash the mesh inserts by pipetting four milliliters of the assay medium directly onto the inserts. Gently shake the cassette to ensure all mesh inserts are washed with the assay medium.
To prepare the retinal punch, enucleate the eyes from an adult euthanized mouse and place them into ice cold PBS in a Petri dish, then place the Petri dish under a dissection microscope. Using microscissors, cut off the optic nerve, then carefully remove the extrarectus muscles attached outside the eyeball. Using a 30 gauge needle, punch a hole at the edge of the cornea.
Then use fine dissection microscissors to make a circular cut along the edge of the cornea. Using sharp dissection forceps, remove the cornea, lens, and the vitreous humor from the eye cup. Make several small cuts on the scleral layer at the rim of the eye cup using fine dissection microscissors.
Use two sharp dissection forceps to hold onto the scleral tissue at each side and pull on the scleral layer very carefully to remove it from the neural retina. Repeat this around the eye cup until all sclera is removed and an intact retinal cup is obtained. Make radial cuts on the retinal cup to flatten it and generate several distinct sections using dissection microscissors.
Next, using a one millimeter diameter biopsy puncher, cut one retinal disc from each section of the flattened retinal cup. Punch at an equal distance from the optic nerve head. Transfer the pre-coated mesh inserts into the dissection Petri dish using forceps.
With the help of two super fine eyelash brushes, place the retinal punch disc at the center of the mesh insert with the ganglion cell layer side down touching the mesh and the photoreceptor layer facing up. To load the sensor cartridge, take the hydrated sensor cartridge plate cassette out of the 37 degree Celsius incubator. After removing the hydro booster cover, place the sensor cartridge back on the utility plate.
Load the desired volume of injection compound solutions into appropriate ports by holding the pipette tip at a 45 degree angle, inserting the pipette tip halfway into an injection port with the bevel of the tip against the opposite wall of the injection port. Gently load the compound into each port. Load the sensor cartridge into the machine for calibration by clicking start run button to open the machine tray.
Place the sensor cartridge into the machine and click I am ready button to start calibration. Load desired volume of the assay medium into each well of the islet capture plate. Using forceps, grab the rim of the mesh insert containing retinal punch discs and take it out from the Petri dish.
Lightly tap the bottom of the mesh insert on an absorbing wipe tissue to remove extra liquid and put it into the well of the islet capture plate. Repeat this step until all mesh inserts with retinal punches are placed into the islet capture plate filling the background correction wells and blank wells with empty mesh inserts. Using two Graefe forceps, press the rim of each mesh insert carefully and gently, making sure that these are securely inserted at the bottom of the islet capture plate.
Place the loaded islet capture plate into a 37 degree Celsius incubator for five minutes to warm up, then eject the utility plate after the calibration is complete. Replace it with the islet capture plate containing retinal punches and resume the assay run by clicking the load cell plate button. After the run is complete, eject the sensor cartridge and islet capture plate containing retinal punches by clicking eject button, then click the done button so that the data is automatically saved as a ASYR file and screen change automatically to result display.
The mitochondrial stress assay showing an OCR trace and glycolytic rate assay showing both OCR and ECAR trace were performed using freshly dissected one millimeter retinal punch discs. In the mitochondrial stress assay, Bam15 was injected to uncouple proton gradient from mitochondrial ATP production, resulting in the increase of OCR to maximal level. Rotenone and antimycin A were injected to inhibit mitochondria respiration at complex one and complex three respectively, resulting in a drop in the OCR to the minimal level.
The difference between the maximal level of OCR and the last measurement of the basal OCR level reflects mitochondrial reserve capacity. In the glycolytic rate assay, injection of rotenone and antimycin A shuts down mitochondrial respiration and drives glycolysis higher. Glycolysis is seized by injecting 2-deoxy-d-glucose, which competes with glucose for hexokinase binding, causing the ECAR to drop to the minimal level.
The difference between the maximal level of glyco ECAR in the last measure of glyco ECAR basal level reflects the glycolysis reserve capacity. To produce reliable data, it's critical to obtain good quality of retinal punch discs and to limit the total dissecting time within 1.5 hour. Following the experiment, one can quantify the DNA of protein content of a retinal disc in a well and use that to normalize the seahorse data.
So this protocol was utilized to study development, aging, and disease in the retina. Tissue preference or reliance for different fuel substrates such as glucose or fatty acids was also analyzed.
