磁気マイクロビーズ閉塞モデルマウスにおける緑内障高眼圧症依存誘導すること

The overall goal of this surgical procedure is to induce ocular hypertension-dependent glaucoma by injecting magnetic micro-beads into the anterior chamber of the mouse eye. This method can help answer key questions in the glaucoma field, such as what early pathological changes occur in the eye prior to overt retinal ganglion cell death. The main advantage of this technique is that inter-ocular pressure can be reliably elevated to where causing minimal damage to the eye during the procedure.

People new to this method will struggle with puncturing the cornea during the injection, and damaging nearby structures, such as the lens, due to an improperly prepared micro-needle, or an incorrect positioning of the micro-needle. To begin, use a capillary puller to pull a micro-needle from a Bohr silicate glass capillary. Under a microscope, use a sharp blade to carefully create an opening at the tip of the micro-needle, so that the tip has an elliptical shape with a major and a minor axis diameter of approximately 190 micrometers and 70 micrometers, respectively.

In the micro-pipette beveling system, place the micro-needle at a 20 degree angle relative to the beveling plate, so that the micro-needle opening is touching the plate. Bevel for approximately 10 minutes using drops of water until the edges are flat and smooth. Next, rotate the needle to bevel the two edges surrounding the opening until the micro-needle tip is sharp.

Then, with an aerosol duster, clean all debris and water from the micro-needle tip opening. Carefully examine the finished micro-needle under a microscope. Discard micro-needles with fractures.

The following procedure requires two people to complete. When a particular action is to be performed by a specific person, the person to perform the action will be indicated as Person 1"or Person 2"Prepare the magnetic micro-bead solution according to the text protocol. To measure baseline inter-ocular pressure, or IOP in an awake mouse, gently restrain the mouse by holding the skin between the ears.

Place the mouse on the bench top so that the animal is comfortable and the eyes are accessible. Apply one drop of proparacaine hydrochloride on the cornea, then hold the tonometer perpendicular to the corneal surface, and take at least three sets of 10 consecutive readings per eye to obtain an IOP average. Next, after anesthetizing the mouse according to the text protocol, administer 0.05 milligrams per kilogram of body weight of buprenorphine subcutaneously.

Then treat the eye with a tropicamide eye drop to induce pupil dilation to aid with the visualization of the positioning of the micro-needle during injection. Apply a topical ointment on the contra-lateral eye to prevent drying of the cornea during the procedure. Now, attach a sterile micro-needle to the injection assembly of the micro-syringe pump.

Then transfer the anesthetized mouse to the operating platform. Under the microscope, have Person 1 ensure that the pupil is fully dilated and that the ocular muscles are relaxed so that there is no eye movement to ensure stability during the procedure. Use absorbent swabs to gently wipe the tropicamide eye drop from the eye.

Have Person 2 mix the magnetic microbead solution by pipetting up and down. Then, using the micro-syringe pump, load the micro-needle with 1.5 microliters of the homogenized magnetic micro-bead solution. Ensure that there are no air bubbles at the tip of the micro-needle.

As quickly as possible, position the needle at a 45 degree angle anteriorly, relative to the limbus. Have Person 1 use the plastic forceps to support the eye, keeping the angle between the micro-needle and the plastic forceps at approximately 90 degrees. Using the micro-syringe pump, have Person 2 gently puncture the cornea with the loaded micro-needle, so that the tip of the micro-needle enters the anterior chamber while avoiding the lens, iris and posterior chamber.

Without moving the mousehead, have Person 1 place the magnet beside the eye opposite the needle tip. To attract the magnetic microbeads into the anterior chamber, and minimize the contact of the beads with the inner surface of the cornea. With Person 1 continuing to hold the magnet, have Person 2 use the micro-syringe pump to inject 1.5 microliters of the magnetic bead solution into the anterior chamber over a period of 15 to 30 seconds.

Once the full volume of beads has been injected, slowly withdraw the micro-needle from the eye. To avoid reflux of the micro-beads, have Person 1 continue to hold the magnet next to the eye for an additional 30 to 60 seconds to attract the beads into the anterior chamber. Then, use the magnet to attract the beads to the iridocorneal angle while avoiding attracting the beads to the inner surface of the cornea, where they may stick.

Ensure that the beads form an evenly distributed ring around the circumference of the anterior chamber. Afterwards, treat the operated eye with an antibiotic eye drop to minimize the risk of infection. Allow the mouse to recover on a heat pad until fully awake with its operated eye facing upwards to prevent infection, and the accumulation of beads to the inner surface of the cornea.

After at least two days of recovery, measure IOP as demonstrated earlier in the video, and monitor at least once per week or more as needed at the same time of day. Analyze the results according to the text protocol. The injection of magnetic micro-beads into the anterior chamber of adult mice described in this protocol, resulted in a robust and reproduce-able elevation of IOP.

As seen here, one week after the procedure, IOP increased from an average baseline of 10, plus or minus 0.6 millimeters of mercury, to 19, plus or minus 0.5 millimeters of mercury in hypertensive eyes. This table shows that IOP stabilized thereafter, and remained elevated at an average of 20 millimeters of mercury for at least six weeks, the longest time point examined in this study. The average peak IOP, in micro-bead injected eyes at two, three and six weeks after surgery, was 25 millimeters of mercury.

To assess retinal ganglion cell, or RGC loss, the number of BRN-3A positive cells was quantified on flat mounts of retinas at one, two, three and six weeks after induction of ocular hypertension. Although significant IOP elevation was detected at one week, no significant loss of RGC soma was observed within the first two weeks. However, significant cell death was observed at three and six weeks post-operation.

In this figure, toluidine blue stained optic nerve cross-sections show a substantial loss of RGC axons at three and six weeks compared to intact optic nerves, indicating that this operation results in axonal loss as observed in glaucoma. Once mastered, the injection of one mouse can be done in 15 minutes. While attempting this procedure, it's important to take the appropriate measures to ensure the same number of micro-beads are injected into the anterior chamber.

This will reduce the variation that may occur between animals. After watching the video, you should have a good understanding of how to induce ocular hypertension by injecting magnetic micro-beads into the anterior chamber of the mouse eyes.