Open globe injuries can lead to permanent loss of vision if left untreated. This anterior segment organ culture model allows assessment of these injuries within the need for animal testing. This protocol allows tracking open globe injuries, and therapeutics, to stabilize the injury for three or more days with an organ culture platform.
Demonstrating the procedure will be Trent Pearson, a research technician from Dr.Boice laboratory. To begin, lay the surgical drape and the instruments required for the gross dissection. Set up the bio safety cabinet for visualizing the eyes on the dissecting microscope.
Set up the bio safety cabinet for dish assembly. Set up the open globe injury induction workspace inside the bio safety cabinet. Position the cross tracking vice on the lab jack, in front of the puncture platform.
After removing the extra orbital tissues and washing the eyes with antibiotic antimycotic PBS, to hemisect the eye place the eye on the antibiotic antimycotic PBS soaked gauze. Use a sterile razorblade or scalpel to create an incision near the equator of the eye. Then hemisect the eye using curved surgical scissors to isolate the anterior eye.
Use micro scissors as a shovel to scoop the vitreous humor from the anterior segment and remove the lens from the anterior segment with micro scissors. Gradually cut the iris to the iris root radially until the trabecular meshwork is visible. Extend the cut 360 degrees around the iris, to expose the entire trabecular meshwork region and clean up any remaining residual iris covering the trabecular meshwork if necessary.
Trim the ciliary body remnants posterior to the trabecular meshwork region leaving only a one millimeter thin band of the tissue. After placing the anterior segment in the Petri dish. Wet a cotton swab in the media and gently dab in the center of the cornea to remove any pigment.
Hold the eyes with forceps and remove extra pigment around the sclera by wiping. Place the inverted anterior segment over the elevated region of the bottom dish, ensuring that the cornea remains in the center of the elevated region. Position the clamping ring on top of the newly placed anterior segment, and gently tighten the screws with the L key.
Attach both the fluidic connectors with O-rings to threaded ports on the bottom of the dish. To the first fluidic connector attach 18 gauge 90 degree bent needle hub, a length of tubing, an 18 gauge needle hub, a nylon syringe filter, a three-way valve and a 20 millimeter syringe filled with media. To the second fluidic connector attach 18 gauge 90 degree bent needle hub, a length of tubing, an 18 gauge needle hub, a three-way valve as demonstrated for the first fluid connector and then attach the barrel portion of a sterile 10 milliliter syringe, which will act as a reservoir to catch the liquid and bubbles.
Keeping the three-way valves open appropriately to the syringes, gently push the media through the system, using the first fluidics connector port. To inflate the anterior segment, fill the tubing with media and eventually fill the reservoir. Remove the bubbles by gently pushing the media into the dish and invert the dish to push out the bubbles.
Place the anterior segment organ culture dish into the cell culture incubator. Direct the tubing lines out through the bottom of the incubator door to prevent interference with the door opening and closing. Position the tubing lines with the reservoir at the pressure transducer instrument.
Connect the side three way valve to the pressure transducer set up while PBS flows through the line to avoid the air bubbles from entering the tubing lines. Inside the prepared bio safety cabinet for the open injury induction set the pressure regulator on the puncture platform to 50 pound force per square inch for generating adequate puncture force on the objects of up to 4.5 millimeters in diameter. Position the cross tracking vice on the lab jack in front of the puncture platform.
Extend the piston arm to its maximal distance and position the corneal apex within the one millimeter region of the puncturing object, retract the piston arm and bring the anterior segment one centimeter closer to the puncturing object. To fire the puncture device, turn on the device and open the solenoid valve with the second switch on the device. To retract the device from the eye, press the second switch, verify proper injury induction by visual inspection and media leaking from the injury site.
After the surgery, the corneal epithelium integrity was assessed via imaging using optical coherence tomography. The injured anterior segment tissue was imaged immediately after injury and 72 hours post-injury. The controlled eyes show no noticeable disruption in the cornea while clear injuries were located through the cornea after the injury.
From the top-down maximum intensity projection, it was evident that injuries were irregular in shape and size, but the injury size was decreased over 72 hours. Interocular pressure was observed for the first three days before the surgery to determine pressure stability in the acceptable range. In the representative results three out of five eyes fall within the acceptable interocular pressure range.
The interocular pressure changes due to the open globe injury and therapeutic intervention. After the open globe injury induction, the pressure significantly decreased and remained low until the experiment was complete. Treatment of the injured tissues with dermabond adhesive resulted in an increased interocular pressure compared to untreated tissue, which indicates that this method can be used to measure the efficacy of the therapeutics for restoring the intraocular pressure.
The initial setup of the segment in organ culture is critical for the long-term stability of the tissue. Over tightening the ring can cause stress on the tissue and reduce compliance.
