Trabecular Meshwork Response to Pressure Elevation in the Living Human Eye

The overall goal of this procedure is to quantify the morphometric response of the living outflow tract to acute IOP elevation in living human eyes in situ by examination of the trabecular meshwork under physiological conditions. This is accomplished by first measuring the pressure within the eye at baseline and imaging the limbus, the location of the aqueous humor outflow pathway by optical coherence tomography. The second step is to repeat pressure measurements and limbus scans while IOP is being elevated with the application of five and 10 grams of pressure to the lateral sclera by ophthalm dynamometer.

Next after relaxing smooth muscle with one drop of tropic aide. Pressure measurements and limbus scans are repeated without. And during IOP elevation image data is then exported and processed, and outflow pathway structure morphology measured comparison of morphological change in response to IOP elevation with and without smooth muscle relaxation provides insights into the mechanical properties of the trabecular meshwork.

The main advantage this technique has over other techniques, such as atomic force microscopy, is that we're measuring the trabecular meshwork in the living eye and examining the mechanical response to IOP perturbation. That response includes the influence of the surrounding smooth muscle that supports it. Generally, individuals that are new to this method will initially struggle because there's no fixation, target, and fixation nor eye position in this method is primarily adjusted by verbal command.

Furthermore, when we apply the abdominal dynamometer, what happens is that it causes a slight shift in eye position. Therefore, the scan must be realigned before image acquisition. Begin with pressure elevation.

A baseline pressure is first taken using Goldman application Chronometry put a drop of 0.5%propane onto the eye and wait three minutes. Divide the test group into two cohorts. Cohort one gets 30 grams of pressure and cohort two gets five grams of pressure.

Gently apply pressure to the temporal sclera with the ophthalm dynamometer. Then wait five minutes and apply 10 grams of pressure to the second cohort and take another measurement being the elevated pressure measurement. Now perform optical coherence tomography scanning of the subject's eye seat, the subject at the scanner, and enter their demographic data.

Next, use verbal commands to center the scan on the temporal limbus by directing the patient's gaze in the nasal direction. Once the temporal limbus is centered, take a baseline scan. Now put a drop of 0.5%propane on the eye.

Wait three minutes and take another scan while applying pressure using an almo dynamometer cohort, one gets 30 grams of pressure and cohort two gets five grams of pressure. For the second cohort, wait five minutes from the initial pressure application and apply 10 grams of pressure while taking a third scan of the eye. As before, only a good scan is acceptable.

After exporting the data and importing into Image J, convert the image into a TIFF image stack and use the enhanced local contrast process with the following settings. Parameter block size of 31, Instagram bins at 256, maximum slope at five masks set to none, and set the fast option as enabled. Do this on each frame of the image stack and save the changes.

Then adjust the size of the aspect ratio to 2048 By 10 24, flip the images vertically. Set the scale distance to 2048. Set the known distance to 4, 000 and set the pixel aspect ratio to one.

The scans can then be analyzed for vessel crossings. Slowly spin the mouse wheel to visually examine the scans to identify a distinctive vessel crossing. To serve as a point of reference within scans, record the image number and reference frame number in the analysis spreadsheet.

Scrolling through the frames in the scan volume serves two purposes. First, we're looking for distinctive landmarks in the aqueous vessels. These will be used to align scan volumes taken at different times.

Secondly, as we scroll through the volume, we're integrating in our mind the morphology of the aqueous vessels and using that morphology to distinguish aqueous vessels from Schlemm's canal. Using the freehand selection tool, focus on the Schlemm canal First, take measurements of its cross-sectional area. Manually segment the canal by circling the boundary with the mouse.

Hold down CTRL D for the current image frame to document the location of the segmentation line. Then hold down CTRL M to automatically measure the area of the Schlemm canal. When magnified, we see that there's no distinctive boundary of Schlemm's canal.

For that reason, a full width half max approach is used a priori as a guideline to determine the boundary for segmentation. Now copy the Schlemm canal cross-sectional area and measurement frame number to the analysis spreadsheet. Next, deselect the outlined area and press the right arrow.

Key three times to advance three image frames. Repeat this process until the Schlemm canal cross-sectional area has been measured in 10 frames. Now press the left arrow key to return to the first measurement frame and start to measure the Schlemm canal's length.

Select the straight segment tool from the toolbar. Draw a straight line from the anterior most to posterior most locations on the Schlemm canal on the current frame. Hold down control D to document the location of the segmentation line.

Then hold down control M to measure the schlemm canal's length. Then copy the Schlemm canal length and frame number to the analysis spreadsheet, deselect the length and in every third image where the volume was measured, take the canal length measurement. Lastly, measure the trabecular meshwork thickness in three samples.

Return to the first measurement frame and draw a straight line from the anterior most position on the canal to the border of the trabecular meshwork and interior chamber. Ensure that the line is perpendicular to the border. Now hold down control and press D and then press M to get the measurement of the line.

Always copy these measurements to the database along with which frame they're taken from. Second, draw a line from the center of the canal to the border of the meshwork and the anterior chamber. The line must be perpendicular to the border.

Then hold the control key and press D for the current frame only. Thirdly, draw a line from the posterior most location of the canal to the border of the meshwork with the anterior chamber. Also, make certain that this line is perpendicular to the border.

Then hold down control and press D and then press M.Repeat taking these three thickness measurements at each of the other previously analyzed nine frames. Then proceed with analyzing the data using the described protocols, the effects of small and large changes in interocular pressure on outflow, tracted, morphological parameters such as Schlemm canal, cross-sectional area were obtained. High levels of pressure increase produce an observable collapse of the canal while small increases of interocular pressure were accommodated Once mastered, this technique can be performed within 10 to 15 minutes if performed properly.