The following protocols are accepted under the University of Maryland&#39;s Institutional Animal Care and Use Committee as well as the Institutional Review Board. The protocols follow federal, state and local standards, and the guidelines set out by the University of Maryland Policy on Biosafety.
1. Dissection of Eye Sample
<ol>
	<li>Obtain an eye sample from local slaughterhouse or tissue bank. If an entire eye globe is obtained, immediately extract the lens, attached zonules, and v...

<ol>
	<li>Von Helmholtz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uber+die+akkommodation+des+auges.">Uber die akkommodation des auges.</a> Arch Ophthal. 1, 1-74 (1855).</li><li>Schachar, R. A., Black, T. D., Kash, R. L., Cudmore, D. P., Schanzlin, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+accommodation+and+presbyopia+in+the+primate.">The mechanism of accommodation and presbyopia in the primate.</a> Ann Ophthalmol. 27, 58-67 (1995).</li><li>Glasser, A., Campbell, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presbyopia+and+the+optical+changes+in+the+human+crystalline+lens+with+age.">Presbyopia and the optical changes in the human crystalline lens with age.</a> Vision Res. 38 (2), 209-229 (1998).</li><li>Reilly, M. A., Hamilton, P. D., Perry, G., Ravi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+behavior+and+natural+and+refilled+porcine+lenses+in+a+robotic+lens+stretcher.">Comparison of the behavior and natural and refilled porcine lenses in a robotic lens stretcher.</a> Exp Eye Res. 88, 483-494 (2009).</li><li>Langenbucher, A., Huber, S., Nguyen, N. X., Seitz, B., Gusek-Schneider, G. C., K&#252;chle, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+accommodation+after+implantation+of+an+accommodating+posterior+chamber+intraocular+lens.">Measurement of accommodation after implantation of an accommodating posterior chamber intraocular lens.</a> J Cataract Refract Surg. 29 (4), 677-685 (2003).</li><li>Ehrmann, K., Ho, A., Parel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+analysis+of+the+accommodative+apparatus+in+primates.">Biomechanical analysis of the accommodative apparatus in primates.</a> Clin Exp Optom. 91 (4), 411 (2008).</li><li>Pinilla Cort&#233;s, 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=Experimental+Protocols+for+Ex+Vivo+Lens+Stretching+Tests+to+Investigate+the+Biomechanics+of+the+Human+Accommodation+Apparatus.">Experimental Protocols for Ex Vivo Lens Stretching Tests to Investigate the Biomechanics of the Human Accommodation Apparatus.</a> Invest Ophthalmol Vis Sci. 56 (5), 2926 (2015).</li><li>Fisher, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+elastic+constants+of+the+human+lens.">The elastic constants of the human lens.</a> J Physiol. 212 (1), 147-180 (1971).</li><li>Eppig, T., 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=Biomechanical+eye+model+and+measurement+setup+for+investigating+accommodating+intraocular+lenses.">Biomechanical eye model and measurement setup for investigating accommodating intraocular lenses.</a> Z Med Ohys. 23 (2), 144-152 (2013).</li><li>Manns, F., Parel, , 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=Response+of+Human+and+Monkey+Lenses+in+a+Lens+Stretcher.">Response of Human and Monkey Lenses in a Lens Stretcher.</a> Invest Ophthalmol Vis Sci. 48 (7), 3260 (2007).</li><li>Scarcelli, G., Kim, P., Yun, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+measurement+of+age-related+stiffening+in+the+crystalline+lens+by+Brillouin+optical+microscopy.">In vivo measurement of age-related stiffening in the crystalline lens by Brillouin optical microscopy.</a> Biophys J. 101 (6), 1539-1545 (2011).</li><li>Besner, S., Scarcelli, G., Pineda, R., Yun, S. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Brillouin+Analysis+of+the+Aging+Crystalline+Lens.">In Vivo Brillouin Analysis of the Aging Crystalline Lens.</a> Invest Ophthalmol Vis Sci. 57 (13), 5093 (2016).</li><li>Cortes, 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=Experimental+Protocols+for+Ex+Vivo+Lens+Stretching+Tests+to+Investigate+the+Biomechanics+of+the+Human+Accommodation+Apparatus.">Experimental Protocols for Ex Vivo Lens Stretching Tests to Investigate the Biomechanics of the Human Accommodation Apparatus.</a> Invest Ophthalmol Vis Sci. 56 (5), 2926-2932 (2015).</li><li>Bernal, A., Parel, J. -. M., Manns, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+Posterior+Zonular+Fiber+Attachment+on+the+Anterior+Hyaloid+Membrane.">Evidence for Posterior Zonular Fiber Attachment on the Anterior Hyaloid Membrane.</a> Invest Ophthalmol Vis Sci. 47 (11), 4708 (2006).</li><li>Kammel, R., Ackermann, R., Mai, T., Damm, C., Nolte, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pig+Lenses+in+a+Lens+Stretcher.">Pig Lenses in a Lens Stretcher.</a> Optom Vis Sci. 89 (6), 908-915 (2012).</li><li>Hahn, J., 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=Measurement+of+Ex+Vivo+Porcine+Lens+Shape+During+Simulated+Accommodation,+Before+and+After+fs-Laser+Treatment.">Measurement of Ex Vivo Porcine Lens Shape During Simulated Accommodation, Before and After fs-Laser Treatment.</a> Invest Ophthalmol Vis Sci. 56 (9), 5332-5343 (2015).</li><li>D'Antin, J. C., Cortes, L. P., Montenegro, G. A., Barraquer, R. I., Michael, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+portable+manual+stretching+device+to+simulate+accommodation.">Evaluation of a portable manual stretching device to simulate accommodation.</a> Acta Ophthalmol. 93 (255), (2015).</li><li>Pierscionek, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+response+of+human+lenses+to+stretching+forces.">Age-related response of human lenses to stretching forces.</a> Exp Eye Res. 60 (3), 325-332 (1995).</li><li>Marussich, 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=Measurement+of+Crystalline+Lens+Volume+During+Accommodation+in+a+Lens+Stretcher.">Measurement of Crystalline Lens Volume During Accommodation in a Lens Stretcher.</a> Invest Ophthalmol Vis Sci. 56 (8), 4239 (2015).</li><li>Martinez-Enriquez, E., P&#233;rez-Merino, P., Velasco-Ocana, M., Marcos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OCT-based+full+crystalline+lens+shape+change+during+accommodation+in+vivo.">OCT-based full crystalline lens shape change during accommodation in vivo.</a> Biomed Opt Exp. 8 (2), 918-933 (2017).</li></ol>

We have devised a novel method to provide an accurate and efficient way of studying the accommodation ability of the lens by utilizing a dual-piece clamping mechanism to couple the stretcher to the sample. During accommodation, the lens relaxes, and the diameter decreases in response to relaxation of the zonular fibers1,2,4,19. The method focuses on this phenomenon by clamping and controlling t...

Accommodation is the process by which the human eye is able to dynamically adjust the shape of its crystalline lens to see objects at far or close distances in sharp focus. Accommodation is an intrinsically biomechanical process. Upon neural stimulus, the ciliary muscles produce a force onto the ciliary body and to the zonule fibers that attach to the circumference of the lens capsule1,2. While there are different theories behind the biomechanics of accommodation, the most widely accepted is the Helmholtz hypothesis. According to the hypothesis, the lens is in a natural stretched state, corresponding to the thinnest shape of the lens which is optimal for the focus of distant objects. To change focus to closer objects, the ciliary muscles contract and the zonular fibers are relaxed. In turn, the lens thickens, increasing the anterior and posterior surface curvatures. This corresponds to an increase in dioptric power which is necessary for near vision, therefore, a shorter focal length1.
The ability to accommodate is compromised over time via a condition named presbyopia. Affecting everyone by age 50, presbyopia makes the eye unable to dynamically change focus from far to close distances3. To combat presbyopia, current methods are passive including corrective lenses and bifocals. While increasing one&#39;s ability to focus on close objects at few planes, such passive treatments cannot restore the dynamic focus ability of the lens4,5. In order to treat presbyopia efficiently, or possibly prevent it, there is an ongoing need to better understand accommodation.
To study lens accommodation, a number of devices have been developed to simulate the phenomenon ex vivo4,6,7,8,9. Spinning disks were first introduced to monitor the stretching of the lens via centrifugal forces8. To more faithfully replicate the phenomenon, lens stretching devices were gradually introduced and innovated. Using a lens stretcher, Manns et al. characterized the force required to accommodate the lens while correlating such to lens power and equatorial diameter9. Current understanding is that the lens stiffens with age, resulting in a reduced change in lens shape in response to an equal force from the ciliary body3,10,11,12.
Current lens stretchers often involve a complex setup, implementing electronics and programmable stretching rates, and requires the entire cadaver eyeball6,7,10,13. This requirement increases the cost per experiment to over $500.00 per eye and decreases sample availability. Here we present a method to replicate lens accommodation at low cost as the eye posterior totals around $200.00. While less sophisticated than many devices used today, the technique is much more cost effective and adoptable without compromising results. This method is centered around a manual lens stretcher (MLS) depicted in Figure 1, and uses a unique clamping system on the zonular fibers and a radial twisting method to expand the diameter of the lens. The physiological accuracy of the protocol is validated by the findings of Bernal et al., who studied the pathway by which the anterior and posterior zonular fibers are connected to the lens capsule14. Using the design of custom shoes which only require the lens, zonule, and ciliary body, we aimed to study lens biomechanics by replicating physiological accommodation.

<table border="0" cellpadding="0" cellspacing="0" style="width:856px;" width="856">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:191px;">Manual Lens Stretcher</td>
			<td style="width:233px;">Bioniko</td>
			<td style="width:132px;">MLS</td>
			<td style="width:300px;">Different animal species will require different shoe sizes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Porcine Eye Samples</td>
			<td>George G. Ruppersberger; slaughterhouse</td>
			<td>N/A</td>
			<td>Whole eyeballs were obtained</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human Eye Samples</td>
			<td>The National Disease Research Interchange</td>
			<td>N/A</td>
			<td>Posterior poles without corneas were ordered</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dissecting Scissors (5 1/2&#39;&#39; Straight)</td>
			<td>Electron Microsopy Sciences</td>
			<td>72960</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tissue Forceps (4 1/2&#39;&#39;)</td>
			<td>Electron Microsopy Sciences</td>
			<td>72960</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">iPhone 6s</td>
			<td>Apple</td>
			<td>N/A</td>
			<td>Any imaging system with ~0.1 mm resolution will work</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Hypochorite</td>
			<td>Clorox</td>
			<td>Clorox Regular-Bleach</td>
			<td>Any disinfectant will work</td>
		</tr>
	</tbody>
</table>

simulating the mechanics of lens accommodation via a manual lens stretcher

The goal of this protocol is to mimic the biomechanics of physiological accommodation in a cost-efficient, practical manner. Accommodation is achieved through the contraction of the ciliary body and relaxation of zonule fibers, which results in the thickening of the lens necessary for near vision. Here, we present a novel, simple method in which accommodation is replicated by tensing the zonules connected to the lens capsule via a manual lens stretcher (MLS). This method monitors the radial stretching achieved by a lens when subjected to a consistent force and allows for a comparison of accommodating lenses, which can be stretched, to non-accommodating lenses, which cannot be stretched. Importantly, the stretcher couples to the zonules directly, and not to the sclera of the eye, thus only requiring the lens, zonules, and ciliary body rather than the entire globe sample. This difference can significantly decrease the cost of acquiring donor cadaver lenses by about 62% compared to acquiring an entire globe.

Porcine eyes, a common sample for studying presbyopia via lens stretching4,15, were obtained, (n = 10) from a local slaughterhouse and this protocol was used to observe the accommodation ability of the lenses. Figure 5A shows the comparison of the porcine lens before and after stretching via the MLS. There was an average 0.19 &#177; 0.07 mm increase in lens radius when stretched (...

Watch this Scientific Journal Video about Simulating the Mechanics of Lens Accommodation via a Manual Lens Stretcher at JoVE.com

Simulating the Mechanics of Lens Accommodation via a Manual Lens Stretcher

We present an efficient method of studying lens accommodation by using a manual lens stretcher. The protocol mimics physiological accommodation by pulling the zonules connected around the lens capsule, thereby, stretching the lens.

The goal of this protocol is to mimic the biomechanics of physiological accommodation in a cost-efficient, practical manner. Accommodation is achieved ...

The overall goal of this technique is to provide a more feasible and inexpensive method for accurately studying lens biomechanics. This method can help answer key questions in the ocular biomechanics field about how the lens changes with the loss of accommodation and the onset of presbyopia. The main advantage of this technique is that it accurately monitors the ability of the lens to accommodate at a low material cost and a high procedural robustness.After obtaining the eye sample, firmly grasp the side of the eye and use a razor blade to make a small incision along the side three millimeters away from the cornea deep enough to reach the vitreous. Using sterilized scissors, carefully continue the incision around the circumstance of the eye using the forceps to remove the posterior eye tissue once it has been excised. Isolate the lens, zonules, and the attached vitreous using forceps as necessary to disconnect the zonules and the ciliary body from the ciliary muscles while maintaining connection with the lens.Then remove the excess vitreous so the lens can lay flat on the manual lens stretcher. Take care to keep the lens, zonules, and a minimal amount of vitreous intact while removing the excess vitreous. To assemble the stretcher, insert 10 millimeter shoe bottoms and their corresponding shoe tops into the bottom plate of the stretcher such that a five milliliter gap remains between the back wall of the shoe indent and the shoe itself.Using curved forceps, place the extracted lens face up in the middle of the bottom plate so that the shoes are supporting the lens over the central hole. Snap the corresponding top of the shoes into place clipping only the zonules and the vitreous. Then insert the plates into the plate case and the stopper screw into the hole located on the side of the bottom plate and insert the plate case into the base.To measure the lens, place an imaging system directly above the apparatus at the appropriate distance for obtaining images and place a ruler in the field of view for the accurate sizing and scaling of the images in post processing. Next, firmly yet smoothly rotate the wrench in a clockwise direction to stretch the lens and obtain images of the lens. To ensure sample-to-sample consistency, it is essential to rotate the wrench smoothly while stretching the lens.When all of the images have been acquired, rotate the wrench in the counterclockwise direction to restore the sample to its resting state and obtain the images of the final resting state of the lens. To analyze the data, upload the images to the appropriate image analysis software and use the point to select at least 40 points around the circumference of the lens. Under the analyze menu, select measure to yield the location of each selected point and fit the location points with MathLab to yield a radius and chi square of the fit.Then use the photographed ruler to convert the pixel radius and error into metrics and perform paired two-tailed t-test analysis to compare an individual lens before and after stretching. In this representative analysis of 10 porcine eye samples, the lenses demonstrated an over 4%increase from their original radii validating the lens stretching protocol with both literature consistency and accuracy. When human lenses were tested, a dramatic decrease in the elasticity of the lens was observed in correlation with the age of the donor as expected from previous lens elasticity studies.Once mastered, this lens stretching technique can be completed in less than 10 minutes if it is performed properly. While stretching the lens, it's important to remember to provide a continuous wrenching force until the stopper screw has been reached to minimize the variability between samples. Using this manual lens stretching technique, we can monitor the lens stiffness of patients with a variety of disease pathologies including patients with diabetes, cataracts, or presbyopia.After its development, this technique paved the way for researchers in the field of ocular biomechanics to explore the properties of the lens during the loss of accommodation and/or onset phases of presbyopia. After watching this video, you should have a good understanding of how to use a manual lens stretcher to obtain valuable biomechanical insight about the lens. Don't forget that working with biological tissue can be extremely hazardous and that precautions such as wearing proper laboratory attire and sterilizing all of the instruments prior to use should always be taken while performing this procedure.

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Research

JoVE Journal

Bioengineering

6.7K 次观看.  University of Maryland. The overall goal of this technique is to provide a more feasible and inexpensive method for accurately studying lens biomechanics. This method can help answer key questions in the ocular biomechanics field about how the lens changes with the loss of accommodation and the onset of presbyopia. The main advantage of this technique is that it accurately monitors the ability of the lens to accommodate at a low material cost and a high procedural robustness.After obtaining the eye sample, fi...