Supported in part by National Institute of Health contracts R01-EY13178, and P30-EY08098 (Bethesda, MD), the Eye and Ear Foundation (Pittsburgh, PA), and unrestricted grants from Research to Prevent Blindness (New York, NY).

Ethics Statement: Approval was obtained from the Institutional Review Board of the University of Pittsburgh School of Medicine before subject recruitment began. All subjects provided written informed consent before participation in the study.
1. Data Acquisition
<ol>
	<li>Pressure Elevation
	<ol>
		<li>Take baseline measurements (IOP and OCT measurements) by instilling one drop of 0.5% proparacaine into the eye. Wait 3 min for efficacy.</li>
		<li>Gently apply pressure to the temporal sclera with the ophthalmodynamometer, 30 g in cohort 1, and 5 and then 10 g in cohort 2. Then take the desired (IOP and OCT) measurements as follows
		<ol>
			<li>IOP Measurements
			<ol>
				<li>Measure baseline IOP. Elevate pressure as described in section 1.1.</li>
				<li>In cohort 1 instill one drop of 0.5% proparacaine, allow 3 min for efficacy, and apply 30 g of pressure to the sclera. While pressure is being applied, measure IOP using the tonometer following manufacturer&#8217;s instructions.</li>
				<li>In cohort 2, instill one drop of 0.5% proparacaine, allow 3 min for efficacy, and apply 5 g of scleral pressure using a ophthalmodynamometer. Measure IOP during pressure elevation using the tonometer following manufacturer&#8217;s instructions.
				<ol>
					<li>Wait 5 min after the 5 g measurement.</li>
					<li>Apply 10 g of scleral pressure by ophthalmodynamometer, and measure IOP during pressure elevation using the tonometer following manufacturer&#8217;s instructions. Record the IOP and condition (i.e., baseline or 10 g) in the study record.</li>
				</ol>
				</li>
			</ol>
			</li>
			<li>OCT Scanning
			<ol>
				<li>Seat the subject at the OCT scanner. Enter patient demographic data for new subjects, or recall demographic data from the OCT database for previously scanned subjects.</li>
				<li>Select the anterior segment 512 x 128 scan protocol. Center the eye in the video image window. Decrease the distance between the scanner and eye until the cornea cross-sectional image appears in the scan window</li>
				<li>Using verbal commands position the temporal limbus to the center of the scan window by directing patient gaze in the nasal direction</li>
				<li>Acquire the baseline scan, and review the scan for quality. IF acceptable, save, and if not acceptable, repeat this step.
				<ol>
					<li>Accept scans if there are no blinks, and the angle is visualized throughout the volume without drifting off of the edge of the image or flipping at the top.</li>
				</ol>
				</li>
				<li>Instill one drop of 0.5% proparacaine, allow 3 min for efficacy, and repeat steps 1.3.2 through 1.3.5.</li>
				<li>For cohort 1, apply 30 g&#160;of scleral pressure by ophthalmodynamometer, and acquire the scan while pressure is being applied. Remove the pressure and review the scan for quality. IF acceptable, save, and if not acceptable, repeat this step.</li>
				<li>For cohort 2, apply 5 g of scleral pressure by ophthalmodynamometer, and acquire the scan while pressure is being applied. Remove the pressure and review the scan for quality. IF acceptable, save, and if not acceptable, repeat this step.</li>
				<li>Wait 5 min allowing the eye to recover from the 5 g pressure perturbation.</li>
				<li>For cohort 2, apply 10 g of scleral pressure by ophthalmodynamometer, and acquire the scan while pressure is being applied. Remove the pressure and review the scan for quality. IF acceptable, save, and if not acceptable, repeat this step.</li>
				<li>Record the scan time, condition (i.e., baseline or 10 g) and location in the study record.</li>
			</ol>
			</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Data Processing
<ol>
	<li>Plug a high-capacity USB storage device into the OCT. Select &#8220;Export&#8221; from the Records menu on the OCT. Designate a file location for exported files on the USB drive. De-select the &#8220;.zip&#8221; option. Enter the patient name for scans to be exported, and select the scans to be exported. Initiate the export.</li>
	<li>When the export completes, unmount the USB drive and remove from the OCT. Plug the high-capacity USB drive containing the exported images into the image processing workstation.</li>
	<li>Launch the image processing program in this case ImageJ.</li>
	<li>Import the raw image data; select &#8220;File -&#62; Import -&#62; Raw&#8221; from the file menu. Select the file with ending in &#8220;_cube_raw.img&#8221; to be processed from the USB drive.</li>
	<li>Save the imported file using a new name, so that the original image data is saved unaltered (http://www.ori.dhhs.gov/education/products/RIandImages).</li>
	<li>Enter the import parameters as follows, Image type: 8 bit, Width: 512, Height: 1,024, Offset: 0; Number of images: 128.</li>
	<li>Select &#8220;Plugins -&#62; Registration -&#62; StackReg&#8221; from the plugins menu. And then select the &#8220;Rigid Body&#8221; option. Then select &#8220;File -&#62; Save As -&#62; TIFF&#8221; to save the aligned stack.</li>
	<li>Select &#8220;Process -&#62; Filters -&#62; Mean 3D&#8221; from the process menu. Enter the parameters X = 1, Y = 1, and Z = 1 as the filter options. Repeat this step twice.</li>
	<li>Select &#8220;File -&#62; Save As -&#62; TIFF&#8221; to save the averaged stack. Spin the mouse wheel to move to frame 1 of the active stack.</li>
	<li>Select &#8220;Process -&#62; Enhance Local Contrast (CLAHE)&#8221; from the Processing menu. Use the parameters block size: 31, histogram bins: 256, Maximum Slope: 5, Mask: none, and check the &#8220;fast&#8221; option. Use the right arrow key to advance to the next frame.</li>
	<li>Move to frame 2 of the active stack and repeat the Process-&#62; Enhance Local Contrast (CLAHE). Repeat until all frames have had contrast enhanced.</li>
	<li>Select &#8220;File -&#62; Save As -&#62; TIFF&#8221; to save the contrast-enhanced stack. Then select &#8220;Image -&#62; Adjust -&#62; Size&#8221; from the Image menu. Deselect the &#8220;Constrain Aspect Ratio&#8221; option, then enter Width: 2,048 and height: 1,024 values.</li>
	<li>Select &#8220;Image -&#62; Transform -&#62; Flip Vertically&#8221; from the Image menu. Then select &#8220;Analyze -&#62; Set Scale&#8221; from the Analyze menu. Enter Distance in pixels: 2,048, Known distance: 4,000, and Pixel Aspect Ratio: 1, and click OK. Select &#8220;File -&#62; Save As -&#62; TIFF&#8221; to save the calibrated 1:1 aspect ratio stack.</li>
	<li>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.</li>
	<li>Press the left arrow key 15 times to go to the first measurement frame. Select the &#8220;Freehand Selections&#8221; tool from the toolbar.</li>
	<li>Place the mouse in the center of SC and press the up arrow. Repeat this step until SC fills the screen.</li>
	<li>Manually segment SC by circling the boundary with the mouse. Hold the control (Ctrl) key and press D for the current image frame. Hold the Ctrl key, and press M. Transcribe the measurement of SC cross-sectional area and measurement frame number to the analysis spreadsheet. De-select the outlined area. Press the right arrow key 3 times. Repeat this step until SC has been measured in 10 frames.</li>
	<li>Press the left arrow key 30 times to return to the first measurement frame.</li>
	<li>Select the straight segment tool from the toolbar.</li>
	<li>Draw a straight line from the anterior-most to posterior-most locations on SC. Hold the control key and press D for the current frame only. Hold the control key and press M. Transcribe SC length and frame number to the analysis spreadsheet. De-select the outlined area. Press the right arrow key 3 times. Repeat this step until SC length has been measured in the same 10 frames as the SC cross-sectional area.</li>
	<li>Insert the equation SC-IOWD = SC-CSA / axial length into the analysis spreadsheet to calculate average SC inner-wall to outer-wall distance (SC-IOWD) by dividing the area measurements by the length measurements.</li>
	<li>Press the left arrow key 30 times to return to the first measurement frame.</li>
	<li>Select the straight segment tool from the toolbar.</li>
	<li>Draw a straight line from the anterior-most on SC to the border of the trabecular meshwork and anterior chamber. Ensure that the line is perpendicular to the border. Hold Ctrl and press D, then M.</li>
	<li>Draw a line from the posterior-most location of SC and the border of the TM and the anterior chamber. Ensure that the line perpendicular to the border. Hold Control and press D, then M.</li>
	<li>Draw a line from the center of SC and the border of the TM and the anterior chamber. Ensure that the line perpendicular to the border. Hold the control key and press D for the current frame only.</li>
	<li>Transcribe the three TM thickness measurements and frame number to the analysis spreadsheet. To do this press the right arrow key 3 times. Repeat this step until TM thickness has been measured in the same 10 frames as the SC cross-sectional area.</li>
</ol>

<ol>
	<li>Quigley, H. A., Broman, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+number+of+people+with+glaucoma+worldwide+2010+and+2020.">The number of people with glaucoma worldwide 2010 and 2020.</a> The British journal of ophthalmolog. 90, 262-267 (2006).</li><li>Sommer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+intraocular+pressure+and+primary+open+angle+glaucoma+among+white+and+black+Americans.+The+Baltimore+Eye+Survey.">Relationship between intraocular pressure and primary open angle glaucoma among white and black Americans. The Baltimore Eye Survey.</a> Archives of ophthalmolog. 109, 1090-1095 (1991).</li><li>Sommer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Racial+differences+in+the+cause-specific+prevalence+of+blindness+in+east+Baltimore.">Racial differences in the cause-specific prevalence of blindness in east Baltimore.</a> The New England journal of medicin. 325, 1412-1417 (1991).</li><li>Leske, M. C., Connell, A. M., Wu, S. Y., Hyman, L., Schachat, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+intraocular+pressure.">Distribution of intraocular pressure.</a> The Barbados Eye Study. Archives of ophthalmolog. 115, 1051-1057 (1997).</li><li>Leske, M. C., Wu, S. Y., Hennis, A., Honkanen, R., Nemesure, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+factors+for+incident+open-angle+glaucoma:+the+Barbados+Eye+Studies.">Risk factors for incident open-angle glaucoma: the Barbados Eye Studies.</a> Ophthalmolog. 115, 85-93 (2008).</li><li>Mitchell, P., Lee, A. J., Rochtchina, E., Wang, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open-angle+glaucoma+and+systemic+hypertension:+the+blue+mountains+eye+study.">Open-angle glaucoma and systemic hypertension: the blue mountains eye study.</a> Journal of glaucom. 13, 319-326 (2004).</li><li>Mitchell, P., Smith, W., Attebo, K., Healey, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+of+open-angle+glaucoma+in+Australia.+The+Blue+Mountains+Eye+Study.">Prevalence of open-angle glaucoma in Australia. The Blue Mountains Eye Study.</a> Ophthalmolog. 103, 1661-1669 (1996).</li><li>Gabelt, B., Kaufman, P., Kaufman, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Adler's Physiology of the Ey. , 237-289 (2003).</li><li>Grant, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+aqueous+perfusion+in+enucleated+human+eyes).">Experimental aqueous perfusion in enucleated human eyes).</a> Archives of ophthalmolog. 69, 783-801 (1963).</li><li>Jocson, V. L., Sears, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+aqueous+perfusion+in+enucleated+human+eyes.+Results+after+obstruction+of+Schlemm's+canal.">Experimental aqueous perfusion in enucleated human eyes. Results after obstruction of Schlemm's canal.</a> Archives of ophthalmolog. 86, 65-71 (1971).</li><li>Maepea, O., Bill, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressures+in+the+juxtacanalicular+tissue+and+Schlemm's+canal+in+monkeys.">Pressures in the juxtacanalicular tissue and Schlemm's canal in monkeys.</a> Experimental eye researc. 54, 879-883 (1992).</li><li>Johnstone, M. A., Grant, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure-dependent+changes+in+structures+of+the+aqueous+outflow+system+of+human+and+monkey+eyes.">Pressure-dependent changes in structures of the aqueous outflow system of human and monkey eyes.</a> American journal of ophthalmolog. 75, 365-383 (1973).</li><li>Overby, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+mechanobiology+of+Schlemm's+canal+endothelial+cells+in+glaucoma.">Altered mechanobiology of Schlemm's canal endothelial cells in glaucoma.</a> Proceedings of the National Academy of Sciences of the United States of Americ. , (2014).</li><li>Allingham, R. R., de Kater, A. W., Ethier, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schlemm's+canal+and+primary+open+angle+glaucoma:+correlation+between+Schlemm's+canal+dimensions+and+outflow+facility.">Schlemm's canal and primary open angle glaucoma: correlation between Schlemm's canal dimensions and outflow facility.</a> Experimental eye researc. 62, 101-109 (1996).</li><li>Camras, L. J., Stamer, W. D., Epstein, D., Gonzalez, P., Yuan, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+trabecular+meshwork+stiffness+on+outflow+facility+in+normal+human+and+porcine+eyes.">Differential effects of trabecular meshwork stiffness on outflow facility in normal human and porcine eyes.</a> Investigative ophthalmolog., &amp; visual scienc. 53, 5242-5250 (2012).</li><li>Last, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastic+modulus+determination+of+normal+and+glaucomatous+human+trabecular+meshwork.">Elastic modulus determination of normal and glaucomatous human trabecular meshwork.</a> Investigative ophthalmolog., &amp; visual. 52, 2147-2152 (2011).</li><li>Lutjen-Drecoll, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+morphology+of+the+trabecular+meshwork+in+primate+eyes.">Functional morphology of the trabecular meshwork in primate eyes.</a> Progress in retinal and eye researc. 18, 91-119 (1999).</li><li>Li, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pilocarpine-induced+dilation+of+Schlemm's+canal+and+prevention+of+lumen+collapse+at+elevated+intraocular+pressures+in+living+mice+visualized+by+OCT.">Pilocarpine-induced dilation of Schlemm's canal and prevention of lumen collapse at elevated intraocular pressures in living mice visualized by OCT.</a> Investigative ophthalmolog., &amp; visual scienc. 55, 3737-3746 (2014).</li><li>Francis, A. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphometric+analysis+of+aqueous+humor+outflow+structures+with+spectral-domain+optical+coherence+tomography.">Morphometric analysis of aqueous humor outflow structures with spectral-domain optical coherence tomography.</a> Investigative ophthalmolog., &amp; visual. 53, 5198-5207 (2012).</li><li>Kagemann, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+visualization+of+aqueous+humor+outflow+structures+in-situ+in+humans.">3D visualization of aqueous humor outflow structures in-situ in humans.</a> Experimental eye researc. 93, 308-315 (2011).</li><li>Kagemann, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+assessment+of+Schlemm's+canal+by+spectral-domain+optical+coherence+tomography.">Identification and assessment of Schlemm's canal by spectral-domain optical coherence tomography.</a> Investigative ophthalmolog., &amp; visual. 51, 4054-4059 (2010).</li><li>Kagemann, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IOP+Elevation+Reduces+Schlemm's+Canal+Cross-sectional+Area.">IOP Elevation Reduces Schlemm's Canal Cross-sectional Area.</a> Investigative ophthalmolog &amp; visual scienc. , (2014).</li></ol>

Dr. Schuman received royalties for intellectual property licensed by Massachusetts Institute of Technology and Massachusetts Eye and Ear Infirmary to Zeiss, Inc.

The present technique leverages non-invasive observation of the mechanical response of soft tissue to quantify SC collapse. Future work using human cadaver eyes is needed to calibrate tissue deflections actual tissue stiffness after dissection. But, such studies will suffer the same limitations of previous outflow models; specifically, that the contributions of living muscle to tissue tension will not be present. Further calibration in a living mammalian eye model may allow calibration of imaging&#160;and direct measurements of stiffness of the TM.
There are several limitations to the technique. It has yet to be demonstrated on other OCT platforms. The literature suggests that the same structures may be visualized on other OCT devices, however sensitivity to changes associated with acute IOP elevation on those devices has yet to be demonstrated in human eyes. The present device was used out of convenience, as no additional optics are required for anterior segment scanning. The greatest challenge to this work is identification of SC within the scans. It is impossible to definitely identify SC within a single slice. Interrogation of the volume is required to first locate the area of tissue containing SC. Its identity is then confirmed by observation of collector channel ostia, and interconnection of the various segments of SC that appear slice to slice. In our experience, SC will present as between 0 to 4 openings within the limbus that can merge into single large openings near a collector channel ostium, or collapse to a pinched section of complete closure.
The greatest significance of this break-through technique is that there is no other option for the assessment of TM stiffness in situ. The morphology and stiffness of the TM correlate with outflow facility14,15,&#160;emphasizing the need to measure the biomechanical characteristics of outflow pathway. In the future, such measurements may provide insights currently unavailable in the management of glaucoma.

Glaucoma is the world&#8217;s second leading cause of irreversible blindness1. Elevated intraocular pressure (IOP) is a major causal risk factor for the presence and progression of glaucoma2-7.&#160;IOP is regulated by balance between the formation and outflow of aqueous humor8.&#160;The locations of greatest outflow resistance are the juxtacanicular tissue and the inner wall of Schlemm canal (SC), the interface between SC and the trabecular meshwork (TM) 9-11.&#160;While TM stiffness may contribute to the prevention of SC collapse in the face of IOP elevation,&#160;Overby et al. 12&#160;recently demonstrated that gene expression in glaucoma is altered, resulting in increased SC endothelial stiffening, impeding formation of pores, leading to IOP elevation in glaucomatous eyes13.&#160;TM morphology and stiffness correlate with outflow facility14,15,&#160;emphasizing the need to measure its biomechanical characteristics.
Atomic force microscopy measurements of the TM show elevated stiffness in eyes donated by glaucoma patients (81 kPa) compared with eyes from donors without glaucoma (4.0 kPa) 16,&#160;but these measurements were made in dissected ex vivo tissues. The posterior TM is anchored into the ciliary muscle via anterior tendons of the longitudinal muscle cells which insert into the outer lamellated and cribiform TM17.&#160;Ciliary muscle (CM) activity may increase TM tautness, mimicking elevated TM stiffness17.&#160;The ability to observe alterations in resistance to SC collapse induced by perturbations of smooth muscle has been shown in an animal model18.&#160;We have demonstrated the ability to non-invasively image the primary aqueous humor outflow system in living human eyes distal to and including SC using spectral domain optical coherence tomography (OCT) 19-21.&#160;Using this technique, we have demonstrated the ability to quantify the morphometric response of the TM and SC to acute IOP elevation22.
The overall goal of the method described herein was to quantify the morphometric response of the living outflow tract to acute IOP elevation in living tissues in situ. This technique has the advantage of examining the TM under physiological conditions, which includes contributions of both contractile fiber activity within the TM and CM to TM stiffness, as compared to published measurements made in dissected tissues. The rationale behind applying this technique to observation of the mechanical TM response is that it provides us with otherwise unavailable insights into the mechanical behavior of the TM, which we now know to be linked directly to outflow resistance and IOP regulation13.&#160;To discern the contribution of contractile tissues to overall stiffness, a small cohort of subjects was examined without and with suppression of smooth muscle activity by administration of tropicamide.

<table><tbody><tr><td>Spectral Domain OCT</td><td>Zeiss</td><td>Cirrus</td><td></td></tr><tr><td>Imaging Workstation</td><td>Apple</td><td>iMac</td><td></td></tr><tr><td>Ophthalmodynamometer</td><td>Baillairt Matalene Ophthalmodynamometer, Surgical instruments CO., Inc. New York, NY</td><td></td><td></td></tr><tr><td>Image Processing Program</td><td>rsb.info.nih.gov/ij</td><td>ImageJ, FIJI</td><td></td></tr></tbody></table>

trabecular meshwork response to pressure elevation in the living human eye

The mechanical characteristics of the trabecular meshwork (TM) are linked to outflow resistance and intraocular pressure (IOP) regulation. The rationale behind this technique is the direct observation of the mechanical response of the TM to acute IOP elevation. Prior to scanning, IOP is measured at baseline and during IOP elevation. The limbus is scanned by spectral-domain optical coherence tomography at baseline and during IOP elevation (ophthalmodynamometer (ODM) applied at 30 g force). Scans are processed to enhance visualization of the aqueous humor outflow pathway using ImageJ. Vascular landmarks are used to identify corresponding locations in baseline and IOP elevation scan volumes. Schlemm canal (SC) cross-sectional area (SC-CSA) and SC length from anterior to posterior along its long axis are measured manually at 10 locations within a 1 mm segment of SC. Mean inner to outer wall distance (short axis length) is calculated as the area of SC divided by its long axis length. To examine the contribution of adjacent tissues to the effect IOP elevations, measurements are repeated without and with smooth muscle relaxation with instillation of tropicamide. TM migration into SC is resisted by TM stiffness, but is enhanced by the support of its attachment to adjacent smooth muscle within the ciliary body. This technique is the first to measure the living human TM response to pressure elevation in situ under physiological conditions within the human eye.

Using these data acquisition and image analysis techniques, the effects of small and large changes in IOP on outflow tract morphological parameters such as SC cross-sectional area are obtained (Figure 1). We can see that high levels of IOP increase produce an observable collapse of SC, as represented by a large reduction in cross-sectional area. The eye appears to be able to accommodate small increases in IOP, as evidenced by the lack of change in SC-CSA (Figure 1). These results show that the technique is capable of quantifying the morphometric response of the outflow tract to an acute IOP challenge. No other family of technologies or techniques provides both visual and quantitative information about outflow tract biomechanics.
Throughout the study, no significant change in TM thickness was observed. In response to a 23 mmHg IOP increase, SC inner to outer wall distance was reduced by 5.03 &#181;m. Without and with suppression of smooth muscle activity, a 6 mmHg increase in IOP caused SC inner to outer wall distance to decrease by 0.18 &#181;m and 2.34 &#181;m respectively. In addition, baseline SC-CSA dropped from 4,597 &#177; 2,503 &#181;m2 to 3,588 &#177; 1,198 &#181;m2 (mean &#177; standard deviation) with smooth muscle activity suppression. Together, with the insertion of anterior tendons from the ciliary muscle which insert into the outer lamellated and cribiform TM17, this implies a control system to maintain SC patency involving smooth muscle. Further study is merited.
<img alt="Figure 1" src="/files/ftp_upload/52611/52611fig1.jpg" /> 
Figure 1. Schlemm&#8217;s canal area versus intraocular pressure in living eyes.&#160;Schlemm&#8217;s canal (SC) cross-sectional areas from the two cohorts of subjects are provided. Error bars present 1 standard error in intraocular pressure (IOP) on the X-axis, and SC area on the Y-axis.

Watch this Scientific Journal Video about Trabecular Meshwork Response to Pressure Elevation in the Living Human Eye at JoVE.com

Trabecular Meshwork Response to Pressure Elevation in the Living Human Eye

Trabecular meshwork (TM) migration into Schlemm&#8217;s canal space can be induced by acute pressure elevation by ophthalmodynamometer, and observed by spectral domain optical coherence tomography. The goal of this method is to quantify the morphometric response of the living outflow tract to acute pressure elevation in living tissues in situ.

The mechanical characteristics of the trabecular meshwork (TM) are linked to outflow resistance and intraocular pressure (IOP) regulation. The rationale ...

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9.9K Views.  University of Pittsburgh School of Medicine. Trabecular meshwork (TM) migration into Schlemm’s canal space can be induced by acute pressure elevation by ophthalmodynamometer, and observed by spectral domain optical coherence tomography. The goal of this method is to quantify the morphometric response of the living outflow tract to acute pressure elevation in living tissues in situ.

Video: Trabecular Meshwork Response to Pressure Elevation in the Living Human Eye

Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present here a protocol for elevating intraocular pressure (IOP) in the rat, by injecting magnetic microspheres into the anterior chamber of the eye. The use of magnetic particles allows the user to manipulate the beads into the iridocorneal angle, thus providing a very effective blockade of fluid outflow from the trabecular meshwork. This leads to long-lasting IOP rises, and eventually neuronal death in the ganglion cell layer (GCL) as well as optic nerve pathology, as seen in patients with the disease. This method is simple to perform, as it does not require machinery, specialist surgical skills, or many hours of practice to perfect. Furthermore, the pressure elevations are very robust, and reinjection of the magnetic microspheres is not usually required unlike in some other models using plastic beads. Additionally, we believe this method is suitable for adaptation for the mouse eye.

We present a method for inducing elevated intraocular pressure (IOP), by injecting magnetic microspheres into the rat eye, to model glaucoma. This leads to strong pressure rises, and extensive neuronal death. This protocol is easy to perform, does not require repeat injections, and produces stable long-lasting IOP rises.

Progress in understanding the pathophysiology, and providing novel treatments for glaucoma is dependent on good animal models of the disease. We present ...

A Magnetic Microbead Occlusion Model to Induce Ocular Hypertension-Dependent Glaucoma in Mice

The use of rodent models of glaucoma has been essential to understand the molecular mechanisms that underlie the pathophysiology of this multifactorial neurodegenerative disease. With the advent of numerous transgenic mouse lines, there is increasing interest in inducible murine models of ocular hypertension. Here, we present an occlusion model of glaucoma based on the injection of magnetic microbeads into the anterior chamber of the eye using a modified microneedle with a facetted bevel. The magnetic microbeads are attracted to the iridocorneal angle using a handheld magnet to block the drainage of aqueous humour from the anterior chamber. This disruption in aqueous dynamics results in a steady elevation of intraocular pressure, which subsequently leads to the loss of retinal ganglion cells, as observed in human glaucoma patients. The microbead occlusion model presented in this manuscript is simple compared to other inducible models of glaucoma and also highly effective and reproducible. Importantly, the modifications presented here minimize common issues that often arise in occlusion models. First, the use of a bevelled glass microneedle prevents backflow of microbeads and ensures that minimal damage occurs to the cornea during the injection, thus reducing injury-related effects. Second, the use of magnetic microbeads ensures the ability to attract most beads to the iridocorneal angle, effectively reducing the number of beads floating in the anterior chamber avoiding contact with other structures (e.g., iris, lens). Lastly, the use of a handheld magnet allows flexibility when handling the small mouse eye to efficiently direct the magnetic microbeads and ensure that there is little reflux of the microbeads from the eye when the microneedle is withdrawn. In summary, the microbead occlusion mouse model presented here is a powerful investigative tool to study neurodegenerative changes that occur during the onset and progression of glaucoma.

Here, we present a protocol to induce ocular hypertension in the murine eye that results in the loss of retinal ganglion cells as observed in glaucoma. Magnetic microbeads are injected into the anterior chamber and attracted to the iridocorneal angle using a magnet to block the outflow of aqueous humour.

The use of rodent models of glaucoma has been essential to understand the molecular mechanisms that underlie the pathophysiology of this multifactorial ...

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the brain for visual perception. Damage to RGCs and their axons leads to vision loss and/or blindness. Although the specific cause of glaucoma is unknown, the primary risk factor for the disease is an elevated intraocular pressure. Glaucoma-inducing procedures in animal models are a valuable tool to researchers studying the mechanism of RGC death. Such information can lead to the development of effective neuroprotective treatments that could aid in the prevention of vision loss. The protocol in this paper describes a method of inducing glaucoma - like conditions in an in vivo rat model where 50 &#181;l of 2 M hypertonic saline is injected into the episcleral venous plexus. Blanching of the vessels indicates successful injection. This procedure causes loss of RGCs to simulate glaucoma. One month following injection, animals are sacrificed and eyes are removed. Next, the cornea, lens, and vitreous are removed to make an eyecup. The retina is then peeled from the back of the eye and pinned onto sylgard dishes using cactus needles. At this point, neurons in the retina can be stained for analysis. Results from this lab show that approximately 25% of RGCs are lost within one month of the procedure when compared to internal controls. This procedure allows for quantitative analysis of retinal ganglion cell death in an in vivo rat glaucoma model.

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma is characterized by damage to retinal ganglion cells. Inducing glaucoma in animal models can provide insight into the study of this disease. Here, we outline a procedure that induces loss of RGCs in an in vivo rat model and demonstrates the preparation of whole-mount retinas for analysis.

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the ...

A Model of Glaucoma Induced by Circumlimbal Suture in Rats and Mice

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known risk factor for glaucoma. This protocol demonstrates a step-by-step guide on this technique in Long Evans rats and C57BL/6 mice. Under general anesthesia, a "purse-string" suture is applied on the conjunctiva, around the equator and behind the limbus of the eye. The fellow eye serves as an untreated control. Over the duration of our study, which was a period of 8 weeks for rats and 12 weeks for mice, IOP remained elevated, as measured regularly by rebound tonometry in conscious animals without topical anesthesia. In both species, the sutured eyes showed electroretinogram features consistent with preferential inner retinal dysfunction. Optical coherence tomography showed selective thinning of the retinal nerve fiber layer. Histology of the rat retina in cross-section found reduced cell density in the ganglion cell layer, but no change in other cellular layers. Staining of flat-mounted mouse retinae with a ganglion cell specific marker (RBPMS) confirmed ganglion cell loss. The circumlimbal suture is a simple, minimally invasive and cost-effective way to induce ocular hypertension that leads to ganglion cell injury in both rats and mice.

Chronic ocular hypertension is induced by applying a circumlimbal suture in rats and mice, leading to functional and structural deterioration of the retinal ganglion cells consistent with glaucoma.

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known ...

Translaminar Autonomous System Model for the Modulation of Intraocular and Intracranial Pressure in Human Donor Posterior Segments

There is a current unmet need for a new preclinical human model that can target disease etiology ex vivo using intracranial pressure (ICP) and intraocular pressure (IOP) which&#160;can identify various pathogenic paradigms related to the glaucoma pathogenesis. Ex vivo human anterior segment perfusion organ culture models have previously been successfully utilized and applied as effective technologies for the discovery of glaucoma pathogenesis and testing of therapeutics. Preclinical drug screening and research performed on ex vivo human organ systems can be more translatable to clinical research. This article describes in detail the generation and operation of a novel ex vivo human translaminar pressure model called the translaminar autonomous system (TAS). The TAS model can independently regulate ICP and IOP using human donor posterior segments. The model allows for studying pathogenesis in a preclinical manner. It can reduce the use of living animals in ophthalmic research. In contrast to in vitro experimental models, optic nerve head (ONH) tissue structure, complexity, and integrity can also be maintained within the ex vivo TAS model.

We describe and detail the use of the translaminar autonomous system. This system utilizes the human posterior segment to independently regulate the pressure inside the segment (intraocular) and surrounding the optic nerve (intracranial) to generate a translaminar pressure gradient that mimics features of glaucomatous optic neuropathy.

There is a current unmet need for a new preclinical human model that can target disease etiology ex vivo using intracranial pressure (ICP) and intraocular ...

Neuroscience

Biomechanical Characterization of Porcine Ocular Lens with Automated Compression

Automated Compression Testing of the Ocular Lens

The biomechanical properties of the ocular lens are essential to its function as a variable power optical element. These properties change dramatically with age in the human lens, resulting in a loss of near vision called presbyopia. However, the mechanisms of these changes remain unknown. Lens compression offers a relatively simple method for assessing the lens' biomechanical stiffness in a qualitative sense and, when coupled with appropriate analytical techniques, can help quantify biomechanical properties. A variety of lens compression tests have been performed to date, including both manual and automated, but these methods inconsistently apply key aspects of biomechanical testing such as preconditioning, loading rates, and time between measurements. This paper describes a fully automated lens compression test wherein a motorized stage is synchronized with a camera to capture the force, displacement, and shape of the lens throughout a preprogrammed loading protocol. A characteristic elastic modulus may then be calculated from these data. While demonstrated here using porcine lenses, the approach is appropriate for the compression of lenses of any species.

Author Spotlight: Advancing Understanding of Age-Related Lens Stiffness Changes 

We present an automated method for characterizing the effective elastic modulus of an ocular lens using a compression test.

The biomechanical properties of the ocular lens are essential to its function as a variable power optical element. These properties change dramatically ...

Bioengineering

Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic nerve and retinal ganglion cell (RGC) degeneration, resulting in visual dysfunction by interrupting the transmission of visual information from the eye to the brain. Elevated intraocular pressure is the most important risk factor; thus, several models of ocular hypertension have been developed in rodents by either genetic or experimental approaches to investigate the causes and effects of the disease. Among those, some limitations have been reported such as surgical invasiveness, inadequate functional assessment, requirement of extensive training, and highly variable extension of retinal damage. The present work characterizes a simple, low-cost, and efficient method to induce ocular hypertension in rodents, based on low-temperature, full-circle cauterization of the limbal vascular plexus, a major component of aqueous humor drainage. The new model provides a technically easy, noninvasive, and reproducible subacute ocular hypertension, associated with progressive RGC and optic nerve degeneration, and a unique post-operative clinical recovery rate that allows in vivo functional studies by both electrophysiological and behavioral methods.

The goal of this protocol is to characterize a novel model of glaucomatous neurodegeneration based on 360&#176; thermic cauterization of limbal vascular plexus, inducing subacute ocular hypertension.

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic ...

Laser Capture Microdissection of Highly Pure Trabecular Meshwork from Mouse Eyes for Gene Expression Analysis

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great tool for the study of the molecular mechanisms underlying cell differentiation and the development and progression of various diseases, including glaucoma. Glaucoma, which comprises a family of progressive optic neuropathies, is the most common cause of irreversible blindness worldwide. Structural changes and damage within the trabecular meshwork (TM) can result in increased intraocular pressure (IOP), which is a major risk factor for developing glaucoma. However, the precise molecular mechanisms involved are still poorly understood. The ability to perform gene expression analysis will be crucial in obtaining further insights into the function of these cells and its role in the regulation of IOP and glaucoma development. To achieve this, a reproducible method for isolating highly enriched TM from frozen sections of mouse eyes and a method for downstream gene expression analysis, such as RT-qPCR and RNA-Seq is needed. The method described herein is developed to isolate highly pure TM from mouse eyes for downstream digital PCR and microarray analysis. In addition, this technique can be easily adapted for the isolation of other highly enriched ocular cells and cell compartments that have been difficult to isolate from mouse eyes. The combination of LCM and RNA analysis can contribute to a more comprehensive understanding of the cellular events underlying glaucoma.

Here, we describe a protocol for a reproducible laser capture microdissection (LCM) for isolating trabecular meshwork (TM) for downstream RNA analysis. The ability to analyze changes in gene expression in the TM will help in understanding the underlying molecular mechanisms of TM-related ocular diseases.

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great ...

Biology

 Induction of Elevated Intraocular Pressure in a Rat Model

<a href='https://app-jove-com.remotexs.ntu.edu.sg/v/63442/full-circle-cauterization-limbal-vascular-plexus-for-surgically'>Source: Lani-Louzada, R., et al., Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents. J. Vis. Exp. (2022)</a>This video demonstrates the induction and monitoring of elevated intraocular pressure (IOP) in a rat model. The process involves cauterizing the limbal blood vessels, resulting in vessel occlusion and elevated IOP. The rat is then allowed to recover, and IOP is regularly monitored in both the experimental and control eyes for comparison.

Source: Lani-Louzada, R., et al., Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents. J. Vis. Exp. (2022)This ...

JoVE Encyclopedia of Experiments

Neuropathology

Cerebrovascular Diseases

 Developing a Translaminar Pressure Model Using a Translaminar Autonomous System

<a href='https://app-jove-com.remotexs.ntu.edu.sg/v/61006/translaminar-autonomous-system-model-for-modulation-intraocular'>Source: Sharma, T. P., et al.,&#160;Translaminar Autonomous System Model for the Modulation of Intraocular and Intracranial Pressure in Human Donor Posterior Segments. J. Vis. Exp.&#160;(2020).</a>This video demonstrates the procedure of preparing a human eye to study the effects of translaminar pressure on retinal ganglion cell signaling by simulating intraocular and intracranial pressures in a translaminar autonomous system.

Source: Sharma, T. P., et al.,&#160;Translaminar Autonomous System Model for the Modulation of Intraocular and Intracranial Pressure in Human Donor ...

Trabecular Meshwork Response to Pressure Elevation in the Living Human Eye

Summary

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Chapters in this video

Experimental Glaucoma Induced by Ocular Injection of Magnetic Microspheres

A Magnetic Microbead Occlusion Model to Induce Ocular Hypertension-Dependent Glaucoma in Mice

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

A Model of Glaucoma Induced by Circumlimbal Suture in Rats and Mice

Translaminar Autonomous System Model for the Modulation of Intraocular and Intracranial Pressure in Human Donor Posterior Segments

Automated Compression Testing of the Ocular Lens

Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents

Laser Capture Microdissection of Highly Pure Trabecular Meshwork from Mouse Eyes for Gene Expression Analysis

Induction of Elevated Intraocular Pressure in a Rat Model

Developing a Translaminar Pressure Model Using a Translaminar Autonomous System