This approach provides a reproducible and minimally invasive way to elevate IOP for extended periods of time. This will help us better understand glaucoma. An advantage of this technique, is that the IOP elevation is easily reversible, allowing researchers to investigate the abilities of the eyes to recover from chronic pressure elevation.
A visual demonstration of this method is critical as suturing and tying a tension knot are difficult to learn from reading a manuscript alone. To fine assist, we recommend that one new to this technique, practice suturing under microscope. Begin by swaddling the awake rat in a piece of soft cloth to calm the animal.
Gently hold the torso in one hand, with the animals back resting against the investigators chest, and expose the head and neck. Use the other hand to bring the rebound tonometer near the rats eye, so that the tip of the interocular probe is approximately two to three millimeters away, and perpendicular to the corneal apex. Wait a few seconds for the rat to calm, then press the measurement button once.
Observe the top of the IOP probe gently hit the corneal apex once, and hear the rebound tonometer beep. After one or two seconds, repeat the measurement. Make a total of 10 measurements to derive an average IOP value for the time point.
Reset the tonometer after the fifth reading. Once your are monitoring, measure IOP in the same time of day. And under consistence lighting conditions, to minimize variation due to a IOP cycle.
Cover the anesthetized rodent with a sterile fenestrated surgical drape, exposing the eye to be sutured. Next, after ensuring a surgical plane of anesthesia, use 7-10 nylon suture to perform a purse-string suture on the bulbar conjunctiva around the globe, by weaving the 7-10 suture parallel and 2 milliliters posterior to the limbus. Take care not to penetrate his sclera.
A sudden pupillary dilation during the surgical procedure indicates the sclera has likely been penetrated. Anchor the suture on the rats conjunctiva using five to six anchor points. Avoid direct compression on the major episcleral veins by threading the suture underneath the conjunctiva at the crossing of these veins.
Fasten the purse-strings suture by tying a slip knot. Then tie a simple knot and keep it loose. To avoid an excessively high post-surgical IOP spike, have an assistant measure the IOP.
If the IOP is found to be too high, adjust the slipknot by partially releasing the tension on one end of the suture. After the desired IOP of 30 to 60 millimeters of mercury is achieved, tie off the second knot while maintaining a continuous pulling force on the end of that suture. After the second knot has been tightened, trim the ends of the suture to minimize any foreign body sensation.
Monitor the animal during recovery from general anesthesia. When working with mice, place a 10-0 nylon suture at one millimeter posterior to the limbus. Use four to five anchor points to anchor the suture on the mouse's conjunctiva.
Then fasten the purse-string suture by tying a slipknot. After tying a simple knot and keeping it loose, measure the IOP with the help of an assistant. To sustain long term IOP and vision, it's important to ensure that the knot is tight enough when fastening the purse-string suture.
Here we demonstrate how to tie a significant knot on a model eye, in order to generate enough tension. After the desired IOP is reached, tie off the second knot, while maintaining a continuous pulling force on that end of the suture. In this graph individual ocular hypertensive eyes are represented by red symbols, and control eyes by gray symbols.
The circumlimbal suture raised intraocular pressure for 8 weeks in rats. IOP remained unchanged in contralateral control eyes. The positive scotopic threshold response, reflects retinal ganglion cell health, and was found to be reduced in the ocular hypertensive eyes indicated by the red line;relative to the control eyes indicated by the black line.
There was also a small reduction of the ERG a and b-wave, which is likely to reflect a mild disfunction of the photo receptors and bipolar cells, respectively. The largest deficit however, was found in the positive scotopic threshold response. Confirming preferential ganglion cell disfunction following the mild, chronic IOP elevation.
While attempting this procedure, it is important to remember to tighten the slipknot with adequate inward compression. The most common reason that IOP fails to rise in the longterm, is that the knot becomes loosened over time. Following this procedure, all modification like suture removal, to normalize intraocular pressure, can be performed to answer additional questions like whether regular ganglion cells can recover from sustain IOP elevation.
And what are the mechanisms that involve this recovery?
