No funding was received for this manuscript.

All procedures performed in the following protocol involving human participants were in accordance with the ethical standards of the institutional research committee of the University of Cologne and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
1. Prosthetic eye customization
<ol>
	<li>Select one of the &#34;half-done&#34; cryolite glass eyes based on the best matching iris color to the healthy fellow eye of the patient (Figure 3).</li>
	<li>Examine the fitting of the current prosthetic eye. To do so, let the patient look straight ahead. Pay special attention to the retention of the prosthesis, the viewing direction, the eye lid contour (ptosis, entropion, and ectropion), as well as to the size and volume (exophthalmos and enophthalmos) of the current prosthesis.</li>
	<li>Remove the current prosthetic eye with help of a contact lens suction cup for hard contact lenses.</li>
	<li>Examine the anophthalmic eye socket without the prosthesis and pay attention to a potential inflammation of conjunctiva, the volume filling of the orbital implant, if the orbital implant is visible through the conjunctiva, and if the fornices and sulci are deep enough for a good fitting prosthesis. If there are any major concerns regarding one of these points, an examination by an ophthalmic surgeon should be performed before manufacturing a new prosthesis.</li>
	<li>Take the selected &#34;half-done&#34; cryolite glass eye with the ocularist forceps and in the other hand take a hollow skewer that will be used later as a mouthpiece for blowing the glass prosthesis. Heat both slowly to 600 &#176;C with a Bunsen burner while continuously rotating it, and melt the skewer at the open end of the &#34;half-done&#34; cryolite glass eye. Open the forceps and lay it down.</li>
	<li>Heat the &#34;half-done&#34; cryolite glass eye continuously (Figure 4). Using the healthy fellow eye as a model for the color, shape, and quantity of the conjunctival vessels, draw the vessels on the white sclera with heated glass stems in different colors (mostly red, brown, or yellow) (Figure 5).</li>
	<li>Heat the whole &#34;half-done&#34; cryolite glass eye while continuously rotating it so that the drawn vessels merge with the white cryolite glass and to produce a very smooth surface.</li>
	<li>Modify the shape and the volume of the of the cryolite glass prosthetic eye by suction and blowing in the mouthpiece. Keep rotating the glass eye in the flame of the Bunsen burner from time to time. Use the old prosthesis as a template for this step, but if necessary, modify the shape and the volume of the new prosthesis based on the findings of the previous examinations.</li>
	<li>Heat a transparent glass stem and melt it at the pupil of the cryolite glass prosthetic eye while continuously rotating the glass eye (Figure 6).</li>
	<li>While continuously rotating the &#34;half-done&#34; glass prosthetic eye, melt the glass at the rear of the prosthesis (Figure 6 and Figure 7) and reduce the volume of the rear by suction with help of the mouthpiece so that the back side shape is nearly equal to the sample prosthesis or the desired shape.</li>
	<li>Melt the glass stem at the front side away and heat the front side of the prosthesis again to produce a very smooth surface (Figure 8).</li>
	<li>Take the front side of the prosthesis with the forceps again, form the final shape of the back side with help of the skewer (Figure 9), and then melt the skewer away (Figure 10).</li>
	<li>Heat the whole prosthesis for fire polishing again, especially at the back side and rotate the prosthesis until the surface is very smooth all over.</li>
	<li>Put the prosthesis in a preheated metal container and let it slowly cool down (Figure 11).</li>
	<li>Insert the prosthesis and check the fitting as described in step 1.2 (Figure 12).</li>
	<li>If necessary, modify the shape of the prosthesis again (repeat steps 1.8&#8211;1.15).</li>
</ol>

<ol>
	<li>Hintschich, C., Baldeschi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rehabilitation+of+anophthalmic+patients.+Results+of+a+survey.">Rehabilitation of anophthalmic patients. Results of a survey.</a> Ophthalmologe. 98 (1), 74-80 (2001).</li><li>Rokohl, A. C., Mor, J. M., Trester, M., Koch, K. R., Heindl, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rehabilitation+of+Anophthalmic+Patients+with+Prosthetic+Eyes+in+Germany+Today+-+Supply+Possibilities,+Daily+Use,+Complications+and+Psychological+Aspects.">Rehabilitation of Anophthalmic Patients with Prosthetic Eyes in Germany Today - Supply Possibilities, Daily Use, Complications and Psychological Aspects.</a> Klinische Monatsbl&#228;tter Augenheilkunde. 236 (1), 54-62 (2019).</li><li>Rokohl, A. C., Koch, K. R., Trester, M., Heindl, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryolite+glass+ocular+prostheses+and+coralline+hydroxyapatite+implants+for+eye+replacement+following+enucleation.">Cryolite glass ocular prostheses and coralline hydroxyapatite implants for eye replacement following enucleation.</a> Ophthalmologe. 115 (9), 793-794 (2018).</li><li>Rokohl, A. C., 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=Concerns+of+anophthalmic+patients-a+comparison+between+cryolite+glass+and+polymethyl+methacrylate+prosthetic+eye+wearers.">Concerns of anophthalmic patients-a comparison between cryolite glass and polymethyl methacrylate prosthetic eye wearers.</a> Graefe's Archive for Clinical and Experimental Ophthalmology. 256 (6), 1203-1208 (2018).</li><li>Rokohl, A. C., Trester, M., Pine, K. R., Heindl, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+of+breakage+of+cryolite+glass+prosthetic+eyes.">Risk of breakage of cryolite glass prosthetic eyes.</a> Graefe's Archive for Clinical and Experimental Ophthalmology. 257 (2), 437-438 (2019).</li><li>den Tonkelaar, I., Henkes, H. E., van Leersum, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herman+Snellen+(1834-1908)+and+Muller's+'reform-auge'.+A+short+history+of+the+artificial+eye.">Herman Snellen (1834-1908) and Muller's 'reform-auge'. A short history of the artificial eye.</a> Documenta Ophthalmologica. 77 (4), 349-354 (1991).</li><li>Koch, K. 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=Ocular+prosthetics.+Fitting,+daily+use+and+complications.">Ocular prosthetics. Fitting, daily use and complications.</a> Ophthalmologe. 113 (2), 133-142 (2016).</li><li>Pine, K. R., Sloan, B. H., Jacobs, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Clinical ocular prosthetics. 1st ed. , (2015).</li><li>Buckel, M., Bovet, 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+eye+as+an+art+form:+the+ocular+prosthesis.">The eye as an art form: the ocular prosthesis.</a> Klinische Monatsbl&#228;tter Augenheilkunde. 200 (5), 594-595 (1992).</li><li>Rokohl, A. C., 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=Concerns+of+Anophthalmic+Patients+Wearing+Cryolite+Glass+Prosthetic+Eyes.">Concerns of Anophthalmic Patients Wearing Cryolite Glass Prosthetic Eyes.</a> Ophthalmic Plastic and Reconstructive Surgery. 34 (4), 369-374 (2018).</li><li>Rokohl, A. C., 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=Cryolite+glass+prosthetic+eyes-the+response+of+the+anophthalmic+socket.">Cryolite glass prosthetic eyes-the response of the anophthalmic socket.</a> Graefe's Archive for Clinical and Experimental Ophthalmology. , (2019).</li><li>Thiesmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motility+and+lid+changes+with+coralline+hydroxyapatite+orbital+implants+and+cryolite+glass+ocular+prostheses.">Motility and lid changes with coralline hydroxyapatite orbital implants and cryolite glass ocular prostheses.</a> Ophthalmologe. 115 (9), 794-796 (2018).</li><li>Thiesmann, R., Anagnostopoulos, A., Stemplewitz, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+results+of+the+compatibility+of+a+coralline+hydroxyapatite+implant+as+eye+replacement.">Long-term results of the compatibility of a coralline hydroxyapatite implant as eye replacement.</a> Ophthalmologe. 115 (2), 131-136 (2018).</li><li>Chin, K., Margolin, C. B., Finger, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+ocular+prosthesis+insertion+improves+quality+of+life+after+enucleation.">Early ocular prosthesis insertion improves quality of life after enucleation.</a> Optometry. 77 (2), 71-75 (2006).</li><li>Pine, K., Sloan, B., Stewart, J., Jacobs, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concerns+of+anophthalmic+patients+wearing+artificial+eyes.">Concerns of anophthalmic patients wearing artificial eyes.</a> Clinical and Experimental Ophthalmology. 39 (1), 47-52 (2011).</li><li>Pine, K. R., Sloan, B., Jacobs, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosocial+profile+of+New+Zealand+prosthetic+eye+wearers.">Biosocial profile of New Zealand prosthetic eye wearers.</a> New Zealand Medical Journal. 125 (1363), 29-38 (2012).</li><li>Pine, K. R., Sloan, B., Stewart, J., Jacobs, R. 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+response+of+the+anophthalmic+socket+to+prosthetic+eye+wear.">The response of the anophthalmic socket to prosthetic eye wear.</a> Clinical and Experimental Optometry. 96 (4), 388-393 (2013).</li><li>Pine, K. R., Sloan, B. H., Jacobs, R. 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Alexander C. Rokohl, Joel M. Mor, Niklas Loreck, Konrad R. Koch, and Ludwig M. Heindl have no financial or proprietary interest in any material or method mentioned in the article. The participant in this study was recruited from the Trester-Institute for Ocular Prosthetics and Artificial Eyes in Cologne that is owned and operated by Marc Trester.&#160;

Following enucleation with an orbital implant, a conformer has to be inserted for two weeks (Figure 1) in order to prevent scarring of the conjunctival fornices and subsequent inserting of a prosthesis2,3,4,7,12,13. Because an early ocular prosthesis insertion improves quality of life after enucl...

The purpose of the present manuscript is to comprehensively demonstrate the technique of manufacturing a customized cryolite glass prosthetic that is long forgotten outside the German-speaking countries (Figure 1). This manuscript also focuses on major advantages of this technique. These include a very smooth surface of the prosthesis due to fire polishing, the light weight of the prosthesis due to the hollow design, high levels of patient satisfaction, and the need of only one appointment for manufacturing of the customized prosthesis1,2,3,4,5. This article also gives ophthalmologists better insights into ocularistic care in order to improve essential interprofessional collaboration1,2,3,4,5.
In 1832, the glassblower Ludwig Uri M&#252;ller from Thuringia, Germany, developed the cryolite glass prosthetic eye based on the class-leading models made in France4. Benefits of cryolite glass included a better look, better tolerability, easier processing, and longer durability than previous glass eyes4,6,7,8. Herman Snellen, a Dutch eye surgeon, used this cryolite glass to produce a lightweight hollow prosthetic eye in 18804,6,7,8. This lightweight prosthetic eye, the Snellen &#8216;reform eye&#8217;, increased the volume of prosthetic eyes, resulting in better fitting into larger eye sockets following the introduction of enucleation procedures made possible by the development of anesthesia and asepsis4,8. Twenty years later, cryolite glass had become the most commonly used material for prosthetic eyes. Germany developed into the manufacturing center of prosthetic eyes globally2,4,5,7,8. At the start of the second world war, German cryolite glass eyes became unavailable outside of the German-speaking area. Therefore, (poly)methyl methacrylate (PMMA) became a substitute material for prosthetic eyes4,7,8, and today PMMA is the most commonly used material for prosthetic eyes globally4,5,8. Notwithstanding, in German speaking countries, over 90% of ocularists still manufacture customized prostheses using the cryolite glass from Thuringia2,3,4,5,7,8,9,10,11,12,13. Each customized cryolite glass prosthetic eye is produced in two major steps: the first step is to produce a &#34;half-done&#34; cryolite glass eye that conforms to a white sphere with an iris and a pupil (Figure 2). The second and decisive step is to customize the &#34;half-done&#34; cryolite glass prosthetic eye for the respective patient. To that end, a &#34;half-done&#34; cryolite glass eye is selected from thousands (Figure 3) based on the best matching iris color to the patient&#39;s healthy fellow eye.
The following protocol presents customizing a selected &#34;half-done&#34; cryolite glass eye for a specific patient. This step lasts about 25&#8211;35 min.

<table border="0" cellpadding="0" cellspacing="0" style="width:1129px;" width="1130">
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			<td height="21" style="height:21px;width:697px;">Bunsen burner with gas and air flow over a fire-resistant worktop made from anodised stainless steel</td>
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			<td height="21" style="height:21px;">Hollow skewer</td>
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			<td height="21" style="height:21px;">Ocularist forceps</td>
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			<td height="21" style="height:21px;">Preheated metal container to 500 degree celsius</td>
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			<td height="21" style="height:21px;">Pre-produced &#34;half-done&#34; cryolite glass eye</td>
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			<td height="21" style="height:21px;">Transparent glass stem</td>
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		</tr>
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			<td height="21" style="height:21px;">Various preproduced glass stems in different colors</td>
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customizing a cryolite glass prosthetic eye

In Germany, Austria, and Switzerland, over 90% of ocularists still manufacture customized prostheses using cryolite glass from Thuringia. The present manuscript demonstrates this long-forgotten technique in detail. This manuscript shows some major advantages of manufacturing prosthetic eyes using cryolite glass in comparison to poly(methyl methacrylate) (PMMA). These advantages include a lighter weight of the prosthesis, higher levels of patient satisfaction, and only one appointment necessary for the customized manufacturing. Potential risk of breakage seems not to be a critical disadvantage for glass prosthetic eye wearers. However, in some patients, manufacturing a well-fitting prosthetic eye is not possible or reasonable due to anophthalmic socket complications such as post nucleation socket syndrome, scarred fornices, or an orbital implant exposure. This article gives ophthalmologists a better insight into ocularistic care in order to improve the essential interprofessional collaboration between ocularists and ophthalmologists.

Optimal results include a new prosthetic cryolite glass eye that fits very well, is comfortable, has a good motility, and the appearance with the prosthetic eye, including the eye lid contour, is nearly symmetrical to the healthy fellow eye (Figure 12).
Suboptimal results can result if the new prosthetic cryolite glass eye fits and is comfortable, but there are concerns regarding the cosmetic results. If a prosthesis does not fit perfectly, the appearance, includi...

Watch this Scientific Journal Video about Customizing a Cryolite Glass Prosthetic Eye at JoVE.com

Customizing a Cryolite Glass Prosthetic Eye

This manuscript shows each step of customizing a cryolite glass prosthetic eye including some major advantages of the use of cryolite glass for manufacturing an eye prosthesis compared to poly(methyl methacrylate). In addition, this manuscript gives ophthalmologists better insight into the ocularistic care that could improve interprofessional collaboration.

In Germany, Austria, and Switzerland, over 90% of ocularists still manufacture customized prostheses using cryolite glass from Thuringia. The present ...

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10.5K Views.  Faculty of Medicine and University Hospital Cologne. This manuscript shows each step of customizing a cryolite glass prosthetic eye including some major advantages of the use of cryolite glass for manufacturing an eye prosthesis compared to poly(methyl methacrylate). In addition, this manuscript gives ophthalmologists better insight into the ocularistic care that could improve interprofessional collaboration.

Video: Customizing a Cryolite Glass Prosthetic Eye

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are administered 1. This uniqueness has made the cornea pocket assay one of the most used models for angiogenesis studies. The cornea composes multiple layers of cells. It is therefore possible to embed a pellet containing the angiogenic factor of interest in the cornea to investigate its angiogenic effect 2,3. Here, we provide a step by step demonstration of how to (I) produce the angiogenic factor-containing pellet (II) embed the pellet into the cornea (III) analyze the angiogenesis induced by the angiogenic factor of interest. Since the basic fibroblast growth factor (bFGF) is known as one of the most potent angiogenic factors 4, it is used here to induce angiogenesis in the cornea.

The cornea is unique in that it lacks vascular tissues. However, robust blood vessel growth and survival can be induced in the cornea by potent angiogenic factors. Therefore, the cornea can provide with us a valuable tool for angiogenic studies. This protocol demonstrates how to perform the mouse model of cornea pocket assay and how to assess the angiogenesis induced by angiogenic factors using this model.

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are ...

Multifocal Electroretinograms

A limitation of traditional full-field electroretinograms (ERG) for the diagnosis of retinopathy is lack of sensitivity. Generally, ERG results are normal unless more than approximately 20% of the retina is affected. In practical terms, a patient might be legally blind as a result of macular degeneration or other scotomas and still appear normal, according to traditional full field ERG. An important development in ERGs is the multifocal ERG (mfERG). Erich Sutter adapted the mathematical sequences called binary m-sequences enabling the isolation from a single electrical signal an electroretinogram representing less than each square millimeter of retina in response to a visual stimulus1.
Results that are generated by mfERG appear similar to those generated by flash ERG. In contrast to flash ERG, which best generates data appropriate for whole-eye disorders. The basic mfERG result is based on the calculated mathematical average of an approximation of the positive deflection component of traditional ERG response, known as the b-wave1. Multifocal ERG programs measure electrical activity from more than a hundred retinal areas per eye, in a few minutes. The enhanced spatial resolution enables scotomas and retinal dysfunction to be mapped and quantified.
In the protocol below, we describe the recording of mfERGs using a bipolar speculum contact lens.
Components of mfERG systems vary between manufacturers. For the presentation of visible stimulus, some suitable CRT monitors are available but most systems have adopted the use of flat-panel liquid crystal displays (LCD). The visual stimuli depicted here, were produced by a LCD microdisplay subtending 35 - 40 degrees horizontally and 30 - 35 degrees vertically of visual field, and calibrated to produce multifocal flash intensities of 2.7 cd s m-2. Amplification was 50K. Lower and upper bandpass limits were 10 and 300 Hz. The software packages used were VERIS versions 5 and 6.

The development of the multifocal electroretinogram (mfERG) is an important advance in the diagnosis and characterization of retinopathy. Multifocal electroretinograms are a mathematical average of an approximation of a b-wave. Software programs can derive ERGs from more than a hundred retinal areas in a few minutes per eye. Scotomas and retinal dysfunction can be mapped and quantified.

A limitation of traditional full-field electroretinograms (ERG) for the diagnosis of retinopathy is lack of sensitivity.  Generally, ERG results are ...

Corneal Donor Tissue Preparation for Endothelial Keratoplasty

Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes1,2. Since its inception, traditional full thickness corneal transplantation has been the treatment to restore sight in those limited by corneal disease. Some disadvantages to this approach include a high degree of post-operative astigmatism, lack of predictable refractive outcome, and disturbance to the ocular surface. The development of Descemet's stripping endothelial keratoplasty (DSEK), transplanting only the posterior corneal stroma, Descemet's membrane, and endothelium, has dramatically changed treatment of corneal endothelial disease. DSEK is performed through a smaller incision; this technique avoids 'open sky' surgery with its risk of hemorrhage or expulsion, decreases the incidence of postoperative wound dehiscence, reduces unpredictable refractive outcomes, and may decrease the rate of transplant rejection3-6.
Initially, cornea donor posterior lamellar dissection for DSEK was performed manually1 resulting in variable graft thickness and damage to the delicate corneal endothelial tissue during tissue processing. Automated lamellar dissection (Descemet's stripping automated endothelial keratoplasty, DSAEK) was developed to address these issues. Automated dissection utilizes the same technology as LASIK corneal flap creation with a mechanical microkeratome blade that helps to create uniform and thin tissue grafts for DSAEK surgery with minimal corneal endothelial cell loss in tissue processing.
Eye banks have been providing full thickness corneas for surgical transplantation for many years. In 2006, eye banks began to develop methodologies for supplying precut corneal tissue for endothelial keratoplasty. With the input of corneal surgeons, eye banks have developed thorough protocols to safely and effectively prepare posterior lamellar tissue for DSAEK surgery. This can be performed preoperatively at the eye bank. Research shows no significant difference in terms of the quality of the tissue7 or patient outcomes8,9 using eye bank precut tissue versus surgeon-prepared tissue for DSAEK surgery. For most corneal surgeons, the availability of precut DSAEK corneal tissue saves time and money10, and reduces the stress of performing the donor corneal dissection in the operating room. In part because of the ability of the eye banks to provide high quality posterior lamellar corneal in a timely manner, DSAEK has become the standard of care for surgical management of corneal endothelial disease.
The procedure that we are describing is the preparation of the posterior lamellar cornea at the eye bank for transplantation in DSAEK surgery (Figure 1).

Endothelial corneal transplantation is a surgical technique for treatment of posterior corneal diseases. Mechanical microkeratome dissection to prepare tissue results in thinner, more symmetric grafts with less endothelial cell loss and improved outcomes. Dissections can be performed at the eye bank prior to corneal transplantation surgery.

Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes1,2. Since its inception, traditional full ...

Implanting Glass Spinal Cord Windows in Adult Mice with Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as multiple sclerosis. Here we present a low-cost and reproducible glass window implantation protocol that is suitable for intravital microscopy and studying the dynamics of spinal cord cytoarchitecture with subcellular resolution in live adult mice with EAE. Briefly, we surgically expose the vertebrae T12-L2 and construct a chamber around the exposed vertebrae using a combination of cyanoacrylate and dental cement. A laminectomy is performed from T13 to L1, and a thin layer of transparent silicone elastomer is applied to the dorsal surface of the exposed spinal cord. A modified glass cover slip is implanted over the exposed spinal cord taking care that the glass does not directly contact the spinal cord. To reduce the infiltration of inflammatory cells between the window and spinal cord, anti-inflammatory treatment is administered every 2 days (as recommended by ethics committee) for the first 10 days after implantation. EAE is induced only 2-3 weeks after the cessation of anti-inflammatory treatment.
Using this approach we successfully induced EAE in 87% of animals with implanted windows and, using Thy1-CFP-23 mice (blue axons in dorsal spinal cord), quantified axonal loss throughout EAE progression. Taken together, this protocol may be useful for studying the recruitment of various cell populations as well as their interaction dynamics, with subcellular resolution and for extended periods of time. This intravital imaging modality represents a valuable tool for developing therapeutic strategies to treat autoimmune demyelinating diseases such as EAE.

We describe a method for implanting and maintaining glass windows over the exposed spinal cords of adult mice for studying experimental autoimmune encephalomyelitis. These windows provide chronic optical access to the spinal cord for monitoring cell dynamics at the subcellular level in live animals using two-photon microscopy.

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as ...

A Standardized Obstacle Course for Assessment of Visual Function in Ultra Low Vision and Artificial Vision

We describe an indoor, portable, standardized course that can be used to evaluate obstacle avoidance in persons who have ultralow vision. Six sighted controls and 36 completely blind but otherwise healthy adult male (n=29) and female (n=13) subjects (age range 19-85 years), were enrolled in one of three studies involving testing of the BrainPort sensory substitution device. Subjects were asked to navigate the course prior to, and after, BrainPort training. They completed a total of 837 course runs in two different locations. Means and standard deviations were calculated across control types, courses, lights, and visits. We used a linear mixed effects model to compare different categories in the PPWS (percent preferred walking speed) and error percent data to show that the course iterations were properly designed. The course is relatively inexpensive, simple to administer, and has been shown to be a feasible way to test mobility function. Data analysis demonstrates that for the outcome of percent error as well as for percentage preferred walking speed, that each of the three courses is different, and that within each level, each of the three iterations are equal. This allows for randomization of the courses during administration.
Abbreviations: 
preferred walking speed (PWS) 
course speed (CS) 
percentage preferred walking speed (PPWS)

We describe an indoor, portable, standardized course that can be used to evaluate obstacle avoidance in persons who have ultralow vision. The course is relatively inexpensive, simple to administer, and has been shown to be reliable and reproducible.

We describe an indoor, portable, standardized course that can be used to evaluate obstacle avoidance in persons who have ultralow vision. Six sighted ...

Corneal Donor Tissue Preparation for Descemet's Membrane Endothelial Keratoplasty

Descemet&#8217;s Membrane Endothelial Keratoplasty (DMEK) is a form of corneal transplantation in which only a single cell layer, the corneal endothelium, along with its basement membrane (Descemet&#39;s membrane) is introduced onto the recipient&#39;s posterior stroma3. Unlike Descemet&#8217;s Stripping Automated Endothelial Keratoplasty (DSAEK), where additional donor stroma is introduced, no unnatural stroma-to-stroma interface is created. As a result, the natural anatomy of the cornea is preserved as much as possible allowing for improved recovery time and visual acuity4. Endothelial Keratoplasty (EK) is the procedure of choice for treatment of endothelial dysfunction. The advantages of EK include rapid recovery of vision, preservation of ocular integrity and minimal refractive change due to use of a small, peripheral incision1. DSAEK utilizes donor tissue prepared with partial thickness stroma and endothelium. The rapid success and utilization of this procedure can be attributed to availability of eye-bank prepared precut tissue. The benefits of eye-bank preparation of donor tissue include elimination of need for specialized equipment in the operating room and availability of back up donor tissue in case of tissue perforation during preparation. In addition, high volume preparation of donor tissue by eye-bank technicians may provide improved quality of donor tissue. DSAEK may have limited best corrected visual acuity due to creation of a stromal interface between the donor and recipient cornea. Elimination of this interface with transplantation of only donor Descemet&#39;s membrane and endothelium in DMEK may improve visual outcomes and reduce complications after EK5. Similar to DSAEK, long term success and acceptance of DMEK is dependent on ease of availability of precut, eye-bank prepared donor tissue. Here we present a stepwise approach to donor tissue preparation which may reduce some barriers eye-banks face in providing DMEK grafts.

Corneal Donor Tissue Preparation for Descemet&#039;s Membrane Endothelial Keratoplasty

Endothelial keratoplasty is a surgical technique continuously evolving as advancements result in transplantation of thinner grafts with each succession1. Here we present a technique for controlled manual tissue dissection to allow safe and repeatable separation of endothelium and Descemet&#39;s membranes from donor corneoscleral buttons for transplantation2.

Descemet&#8217;s Membrane Endothelial Keratoplasty (DMEK) is a form of corneal transplantation in which only a single cell layer, the corneal endothelium, ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, diabetic retinopathy, age-related macular degeneration (AMD) and other ocular inflammatory conditions. It may also help to monitor the therapeutic responses in the eye. The proper labeling of the blood cells, coupled with live-cell imaging of the labeled cells, allows for the investigation of the flow dynamics in the retinal and choroidal circulation. Here, we describe the standardized protocols of 1.5% indocyanine green (ICG) and 1% sodium fluorescein labeling of mice erythrocytes and leukocytes, respectively. Scanning laser ophthalmoscopy (SLO) was applied to visualize the labeled cells in the retinal circulation of C57BL/6J mice (wild type). Both methods demonstrated distinct fluorescently labeled cells in the mouse retinal circulation. These labeling methods can have wider applications in various ocular disease models.

Live-cell imaging of the labeled blood cells in ocular circulation can provide information about inflammation and ischemia in diabetic retinopathy and age-related macular degeneration. A protocol to label blood cells and image the labeled cells in the retinal circulation is described.

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, ...

Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant for targeting neovascular glaucoma, a sight-threatening complication of diabetic retinopathy. This method is based on the induction of iris vascular response by a series of self-sealing uveal punctures on BALB/c mice and takes advantage of the postpartum maturation of mouse ocular vasculature. Mouse pups undergo uveal punctures from postnatal day 12.5, when the pups naturally open their eyes, until postnatal day 24.5. Due to the transparency of the cornea, iris vasculature can be analyzed easily through time by noninvasive in vivo methods. Furthermore, the semitransparent iris of BALB/c mice can be flatmounted for detailed immunohistologic analysis with minimal non-specific background staining. In this model, angiogenesis is mainly driven by the inflammatory and plasminogen activating systems. The puncture-induced model is the first to induce iris neovascularization in small rodents, and has the advantage of allowing direct noninvasive in vivo analysis of the angiogenic process. Moreover, the model can be combined with angiogenic modulating substances, which highlights its potential in the study of angiogenesis with an in vivo perspective.

Iris neovascularization, a common complication of ischemic retinal disease, may lead to sight-threatening neovascular glaucoma. Here, we describe a murine protocol for inducing experimental iris neovascularization that may be used for noninvasive evaluation of angiogenesis-modulating substances.

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant ...

Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic step. Despite advances in our understanding of DED, significant knowledge gaps remain. Advances are limited in part due to the lack of informative animal models. The authors recently reported on a method of DED induced by injecting all orbital lacrimal gland (LG) tissues with the lectin concanavalin A. Here, we report a novel model of aqueous-deficient DED based on the surgical resection of all orbital LG (dacryoadenectomy) tissues. Both methods use rabbits because of their similarity to human eyes in terms of the size and structure of the ocular surface. One week after removal of the nictitating membrane, the orbital superior LG was surgically removed under anesthesia, followed by removal of the palpebral superior LG, and finally removal of the inferior LG. Dacryoadenectomy induced severe DED, evidenced by a marked reduction in the tear break up time test and the Schirmer&#39;s tear test, and significantly increased tear osmolarity and rose bengal staining. Dacryoadenectomy-induced DED lasted at least eight weeks. There were no complications and animals tolerated the procedure well. The technique can be mastered relatively easily by those with adequate surgical experience and appreciation of the relevant rabbit anatomy. Since this model recapitulates the features of human aqueous-deficient DED, it is suitable for studies of ocular surface homeostasis, DED, and candidate therapeutics.

A novel approach is presented to induce chronic dry eye disease in rabbits by surgically removing all orbital lacrimal glands. This method, distinct from those previously reported, produces a stable, reproducible model of aqueous deficient dry eye well suited to study tear physiology and pathophysiology and ocular therapeutics.

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic ...