Name: scleral cross-linking using riboflavin and ultraviolet-a radiation for prevention of axial myopia in a rabbit model
Uploaded: 2016-04-03T14:00:00
Description: Watch this Scientific Journal Video about Scleral Cross-linking Using Riboflavin and Ultraviolet-A Radiation for Prevention of Axial Myopia in a Rabbit Model at JoVE.com

The authors are thankful to Ms. Dalia Sela and Mr. Emi Sharon for their professional and excellent technical work in the laboratory.

Animals were treated in accordance with the ARVO resolution on the use of animals in research. The study protocol was approved by the institutional Committee for Laboratory Animal Research (approval no. 022-4598-2; 021211).
1. Preparation for Surgery #1
<ol>
	<li>Weigh and anesthetize a 13 day-old New Zealand white rabbit with an intramuscular injection of ketamine hydrochloride 100 mg/ml (100 mg/kg) and xylazine hydrochloride 25 mg/ml (12.5 mg/kg). Those doses are used due to the length of the surgical procedur...

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Certain+biomechanical+properties+and+cross+linking+of+the+scleral+shell+of+the+eye+in+progressive+myopia.">Certain biomechanical properties and cross linking of the scleral shell of the eye in progressive myopia.</a> Proceedings of XIVth Congress on Biomechanics. Paris: International Society of Biomechanics. , 616-617 (1993).</li><li>Wollensak, G., Sp&#246;rl, E., Seiler, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Riboflavin+/+ultraviolet-A-induced+collagen+crosslinking+for+the+treatment+of+keratoconus.">Riboflavin / ultraviolet-A-induced collagen crosslinking for the treatment of keratoconus.</a> Am. J. Ophthalmol. 135, 620-627 (2003).</li><li>Wollensak, G., Sp&#246;rl, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagencrosslinking+of+human+and+porcine+sclera.">Collagencrosslinking of human and porcine sclera.</a> J. Cataract Refract. Surg. 30, 689-695 (2004).</li><li>Wollensak, G., Iomdina, E., Dittert, D. -. D., Salamatina, O., Stoltenburg, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-linking+of+scleral+collagen+in+the+rabbit+using+riboflavin+and+UVA.">Cross-linking of scleral collagen in the rabbit using riboflavin and UVA.</a> Acta Ophthalmol. 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We present the first in vivo study of the prevention of axial myopia in a rabbit model using cross-linking technology with riboflavin and UVA irradiation. Although different laboratory animals can be used in this type of study, we chose rabbits mostly due to the size of the eyes and the need to perform crosslinking on the scleral surface.
We found that exposing the rabbit sclera and suturing the upper and lower eyelids to be challenging procedures. We recommend trimming the lid margin...

Myopia is the most common of the refractive disorders. The prevalence of myopia in the USA and Europe is reported to be around 30%, and in Asian countries it affects up to 60% of the general population1,2. Myopic progression occurs in up to 50% of myopes, usually at a rate of around -0.5 dioptres over a two-year interval3. The health costs imposed by myopia are considerable, including expenses for spectacles, contact lenses and refractive surgery and costs related to the increased health risks of glaucoma, cataract, retinal detachment and visual impairment4-6.
In animal studies of myopia, sight reduction is induced by eyelid suturing7-10, placement of an occluder at a short distance from the eye and corneal tattooing11. However, for artificial myopia to occur in these studies, the occlusion process has to be performed on very young animals, as no sight deprivation experiments carried out on adult specimens have proved successful.
One of the important features of severe myopia is a pathological change of the sclera with progressive thinning of the sclera, probably due to a disturbed feedback mechanism of emmetropization after visual deprivation12 or due to some metabolic disorder of the sclera, such as in Ehlers-Danlos syndrome13. Ultimately, both mechanisms lead to stretching and thinning of the sclera, retina and choroid due to structural abnormalities of the myopic sclera such as a decreased collagen fiber diameter14,15 and disturbances in fibrillogenesis16.
Several studies have shown that impaired collagen crosslinking is an important factor in the weakening process of the myopic sclera17-18. Wollensak et al.19-21 induced collagen crosslinking by applying the photosensitizer riboflavin and ultraviolet-A (UVA) irradiation (370 nm) and noted a significant, 157% increase in the rigidity of porcine and human sclera in vitro19 and a 465% increase in rabbit scleral rigidity in vivo (Young&#39;s modulus)20. Crosslinking also had a long-term effect on rabbit sclera in vivo: rigidity increased by 320.4% after 3 days, 277.6% after 4 months, and 502% after 8 months (Young&#39;s modulus)22.
Therapeutic attempts to arrest myopic progression have been published23-26 but the success of these methods is controversial. No efficient means of preventing progressive myopia has been found to date.
The etiology of myopia is still controversial, and its treatment poses a challenge. On the basis of these findings, it is hypothesized that scleral crosslinking can serve as a means for sclera-based treatment of myopic progression. The purpose of this study is to examine the scleral collagen crosslinking effect on the development of axial myopia induced by visual axis occlusion.

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			<td height="21" style="height:21px;width:244px;">2-0 braided silk non-needled sutures&#160;</td>
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			<td height="42" style="height:42px;width:244px;">0.1% dextran-free riboflavin-5-phosphate 1mg:1ml</td>
			<td style="width:137px;">Concept for Pharmacy Ltd</td>
			<td style="width:119px;">D2-5025</td>
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			<td height="21" style="height:21px;">UV A (370nm) light source&#160;</td>
			<td>O/E LAND Inc</td>
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			<td height="21" style="height:21px;">Beveled down custom made fiber optic&#160;</td>
			<td>Prizmatix Ltd</td>
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			<td height="49" style="height:49px;width:244px;">26G lacrimal cannula&#160;</td>
			<td style="width:137px;">Beaver-visitec International Ltd.&#160;</td>
			<td style="width:119px;">REF581276</td>
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			<td height="21" style="height:21px;width:244px;">13 days old rabbits</td>
			<td style="width:137px;">Harlan&#160;</td>
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			<td height="23" style="height:23px;width:244px;">Ultrasonic biometer</td>
			<td>Allergan-Humphrey</td>
			<td style="width:119px;">820-519</td>
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			<td height="21" style="height:21px;width:244px;">Skin marker</td>
			<td style="width:137px;">Devon</td>
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scleral cross-linking using riboflavin and ultraviolet-a radiation for prevention of axial myopia in a rabbit model

Myopic individuals, especially those with severe myopia, are at higher-than-normal risk of cataract, glaucoma, retinal detachment and chorioretinal abnormalities. In addition, pathological myopia is a common irreversible cause of visual impairment and blindness1-3. Our study demonstrates the effect of scleral crosslinking using riboflavin and ultraviolet-A radiation on the development of axial myopia in a rabbit model. The axial length of the eyeball was measured by A-scan ultrasound in New Zealand white rabbits aged 13 days (male and female). The eye then underwent 360&#176; conjunctival peritomy with scleral crosslinking, followed by tarsorrhaphy. Axial elongation was induced in 13 day-old New Zealand rabbits by suturing their right eye eyelids (tarsorrhaphy). The eyes were divided into quadrants, and every quadrant had two scleral irradiation zones, each with an area of 0.2 cm&#178; and a radius of 4 mm. Crosslinking was performed by dropping 0.1% dextran-free riboflavin-5-phosphate onto the irradiation zones 20 sec before ultraviolet-A irradiation and every 20 sec during the 200 sec irradiation time. UVA radiation (370 nm) was applied perpendicular to the sclera at 57 mW/cm&#178; (total UVA light dose, 57 J/cm&#178;). Tarsorrhaphies were removed on day 55, followed by repeated axial length measurements. This study demonstrates that scleral crosslinking with riboflavin and ultraviolet-A radiation effectively prevents occlusion-induced axial elongation in a rabbit model.

Figures 1 and 2 graphically demonstrate the axial length measurements of two groups. Group 1 rabbits underwent scleral crosslinking and tarsorrhaphy on the right eye while the left eye was not operated on (Figure 1). Group 2 rabbits underwent only peritomy and tarsorrhaphy on the right eye while the left eye was not operated on (Figure 2).
In group 1, which unde...

Watch this Scientific Journal Video about Scleral Cross-linking Using Riboflavin and Ultraviolet-A Radiation for Prevention of Axial Myopia in a Rabbit Model at JoVE.com

Scleral Cross-linking Using Riboflavin and Ultraviolet-A Radiation for Prevention of Axial Myopia in a Rabbit Model

We demonstrate the effect of scleral crosslinking with riboflavin and UVA on an axial elongation rabbit eye. Axial elongation was induced in 13 day-old New Zealand rabbits (male and female) by suturing their right eye eyelids (tarsorrhaphy).

Myopic individuals, especially those with severe myopia, are at higher-than-normal risk of cataract, glaucoma, retinal detachment and chorioretinal ...

The overall goal of this procedure is to observe the effects of scleral cross-linking using riboflavin and ultraviolet-A radiation to induce axial myopia in a rabbit model. This method can help answer key questions in the scleral cross-linking field, such as, how can UVA radiation be directed to specific scleral areas. The main advantage of this technique is that it uses UVA radiation, fiber optics, and surgery to reach sceral areas of interest.Assisting the surgical procedure will be Orly Gal-Or, a resident in Rabin Medical Center Ophthalmology Department. Before beginning the procedure, confirm a lack of response to pain reflex and use a sterile cotton swab to apply a small amount of ophthalmic ointment to the non-operated eye. Then, apply topical anesthesia to each eye.And placing the probe perpendicular to the central cornea, use an Ultrasound A scanner to perform three axial length measurements of the eyes. Next, under an ophthalmic operating microscope, use surgical ophthalmic forceps and scissors to make a 360-degree conjunctival peritomy. Use a muscle hook to locate the four extraocular rectus muscles.Then, isolate the muscles with two zero braided silk non-needle sutures to create four quadrants. Using a skin marker, designate one irradiation zone at the equatorial sclera, and one irradiation zone at the posterior sclera in each quadrant. Then, load a three-to five-milliliter syringe with 0.1 percent dextran-free riboflavin 5 phosphate photo-sensitizer solution and attach a 26 gauge lacrimal cannula.Now, turn on an irradiation device with a 370 nanometer UVA light source connected to a beveled down custom made fiber optic, and set the device to 57 milliwatts per square centimeter. Twenty seconds before beginning the irradiation, move the eyeball by pulling the silk sutures in the opposite direction of the quadrant to be treated, and apply photo-sensitizer solution onto the irradiation zone. Then, use the custom made fiber optic to irradiate the selected zone for 200 seconds, applying photo-sensitizer solution to the zone every 20 seconds.After all four quadrants have been irradiated, trim the lid margins with scissors. Then, use four zero ivory colored braided silk to gently suture the upper and lower eyelids. Apply a small amount of ophthalmic ointment onto the lid margin, and place the rabbit under a heat lamp.When the animal is fully recovered, place it into a clean cage with new bedding. Fifty-five days after the cross-linking, use surgical ophthalmic scissors to remove the tarsorrhaphy. Then, perform three axial length measurements on each eye as just demonstrated.In animals that underwent scleral cross-linking and tarsorrhaphy, the mean axial length decreased after eyelid suture, compared to the unoperated eye. In animals that underwent only peritomy and tarsorrhaphy, however, the mean axial length increased in the experimental eye. Indeed, eyes that underwent scleral cross-linking exhibited a lower axial length increase overall, compared to eyes that were peritomized only.While the between-group difference in the mean axial length of the eyes that did not undergo surgery was not statistically significant. While attempting this procedure, it's important to remember to prepare all the necessary equipment in advance. After its development, this technique paved the way for researchers in the field of myopia, who explored the effects of riboflavin and UVA radiation on the retina, choroid, and sclera.After watching this video, you should have a good understanding of how to visualize specific scleral areas of interest, and to apply focused UVA radiation treatments. Don't forget that performing surgeries on such small eyes with delicate rectus muscles can be extremely challenging and that the learning curve for perfecting the technique should be accepted. The implications of this technique extend towards the prevention of pathological myopic complications.Though this method can provide insight into the prevention of axial myopia, it can also be applied to other conditions such as thin scleral areas.

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