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 procedure.&#160;&#160;Assure appropriate level of anesthesia by the lack of a pain reflex.</li>
	<li>Using a sterile cotton swab, apply a small amount of ophthalmic ointment to the non-operated eye to prevent the cornea from drying out. Apply 0.9% normal saline drops onto the operated eye cornea during the operation to prevent the corneas from drying out.</li>
	<li>Perform three axial length measurements in each eye after topical anesthesia (oxybuprocaine hydrochloride 0.4%) using an ultrasound A-scanner and then average the measurements. Apply the probe perpendicularly to the central cornea.</li>
</ol>
2. Surgery #1 - Pre Cross Linking Step
<ol>
	<li>Using surgical ophthalmic forceps and scissors under an ophthalmic operating microscope, make a 360&#176; conjunctival peritomy with surgical ophthalmic forceps and scissors.</li>
	<li>Using an angled forceps or a muscle hook identify the four extraocular rectus muscles and isolate them with a 2-0 braided silk non-needled sutures. 
	NOTE: The silk sutures will help with moving the globe in the desired direction. The eyeball is divided into quadrants between the four rectus muscles (four quadrants).
	<ol>
		<li>Mark with a skin marker two zones in each quadrant, one at the equatorial sclera and one at the posterior sclera. These are the irradiation zones.</li>
	</ol>
	</li>
	<li>Prepare a 3 ml to 5ml syringe containing 0.1% dextran-free riboflavin-5-phosphate. Connect the syringe to a 26 G lacrimal cannula or 25 G tapered hydrodelineator.</li>
	<li>Prepare the irradiation device which includes a UV A (370 nm) light source connected to a beveled down custom-made fiber optic. Following the measurement of energy power and calibration (see NOTE below), set the device to 57 mW/cm&#178;. 
	NOTE: Calibration was carried out commercially by the manufacturer. This method provides a total UVA light dose of 57 J/cm&#178;. (57 mW/cm2 on 0.2 cm2 is 11.4 mW/0.2 cm2 using 11.4 mW/0.2 cm2 for 200 sec is a cumulative load of 2.2 J per 0.2 cm2 spot)</li>
</ol>
3. Surgery #1 &#8211; Cross Linking
<ol>
	<li>Move the eyeball by pulling the silk sutures in the opposite direction of the chosen quadrant to be treated (e.g.,&#160;Pull up and nasal when treating the lower temporal quadrant). Perform the eyeball movement using a 2-0 braided silk non-needled suture which isolates each rectus muscle.</li>
	<li>Apply photosensitizer solution containing 0.1% dextran-free riboflavin-5-phosphate onto the irradiation zone 20 sec before irradiation starts.</li>
	<li>Irradiate the irradiation zone using a beveled down custom made fiber optic for an irradiation period of 200 sec. Measure the area of each zone as 0.2 cm&#178; with a radius of 4 mm.</li>
	<li>Apply photosensitizer solution containing 0.1% dextran-free riboflavin-5-phosphate onto the irradiation zone every 20 sec during the 200 sec irradiation period. 
	NOTE: Perform the irradiation and the dropping of riboflavin simultaneously by two surgeons.</li>
	<li>Repeat steps 3.1-3.4 for each of the four eyeball quadrants.</li>
</ol>
4. Surgery # 1 - Post Cross Linking Step
<ol>
	<li>Shave the fur around the eye.&#160;Trim the lid margins using surgical ophthalmic scissors and then gently suture the upper and the lower eyelids using 4-0 braided silk ivory color - 3/8 circle reverse cutting- 13 mm length C-3 needle (tarsorrhaphy). Apply a small amount of ophthalmic ointment on the lid margin at the end of the procedure (chloramphenicol 5%).</li>
</ol>
5. Surgery #1 - Post Operative Care
<ol>
	<li>Keep the animals under a heating lamp during recovery. Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency.</li>
	<li>Place the animal in a clean cage with new bedding until it is fully recovered. After recovery, the animal will be returned to the animal room.&#160;Return the animal to his mother only when awake.&#160;</li>
	<li>During the first 48 hr, examine the animals concerning the general condition and signs of illness or infections. Administer analgesia as per section 1.&#160;Administer analgesia as needed if a change in the rabbit behavior is observed. (Metamizole drops 50mg/kg every 6 hr).</li>
</ol>
6. Surgery #2
<ol>
	<li>Fifty-five days after surgery #1, weigh and anesthetize the rabbit with an intramuscular injection of ketamine hydrochloride 100 mg/ml (100 mg/kg) and xylazine hydrochloride 25 mg/ml (12.5 mg/kg).</li>
	<li>Using surgical ophthalmic scissors under an ophthalmic operating microscope, remove the tarsorrhaphy. Perform three axial length measurements on each eye after topical anesthesia (oxybuprocaine hydrochloride 0.4%) using an ultrasound A-scanner and then average the measurements. Apply the probe perpendicularly to the central cornea.</li>
	<li>Euthanize the rabbits with intraperitoneal phenobarbital sodium (200 mg/1.5 kg body weight).</li>
</ol>

<ol>
	<li>McBrien, N. A., Gentle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+sclera+in+the+development+and+pathological+complications+of+myopia.">Role of the sclera in the development and pathological complications of myopia.</a> Prog. Retin. Eye Res. 22, 307-338 (2003).</li><li>Saw, S. M., Gazzard, G., Au Eong, K. -. G., Tan, D. T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myopia:+attempts+to+arrest+progression.">Myopia: attempts to arrest progression.</a> Br. J. Ophthalmol. 86, 1306-1311 (2002).</li><li>Bullimore, M. A., Jones, L. A., Moeschberger, M. L., Zadnik, K., Payor, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+retrospective+study+of+myopia+progression+in+adult+contact+lens+wearers.">A retrospective study of myopia progression in adult contact lens wearers.</a> Invest Ophthalmol Vis Sci. 43, 2110-2113 (2002).</li><li>Rose, K., Smith, W. E., Morgan, I., Mitchell, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+increasing+prevalence+of+myopia:+implications+for+Australia.">The increasing prevalence of myopia: implications for Australia.</a> Clin. Exp. Ophthalmol. 29, 116-120 (2001).</li><li>Saw, S. M., Gazzard, G., Shih-Yen, E. C., Chua, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myopia+and+associated+pathological+complications.">Myopia and associated pathological complications.</a> Ophthal. Physiol. Opt. 25, 381-391 (2005).</li><li>Tano, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathologic+myopia+-+where+are+we+now.">Pathologic myopia - where are we now.</a> Am. J. Ophthalmol. 134, 645-660 (2002).</li><li>Greene, P. R., Guyton, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+of+rhesus+lid-suture+myopia.">Time course of rhesus lid-suture myopia.</a> Exp. Eye Res. 42, 529-534 (1986).</li><li>McBrien, N. A., Norton, T. 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+development+of+experimental+myopia+and+ocular+component+dimensions+in+monocularly+lid-sutured+tree+shrews+(Tupaia+belangeri).">The development of experimental myopia and ocular component dimensions in monocularly lid-sutured tree shrews (Tupaia belangeri).</a> Vision Res. 32, 843-852 (1992).</li><li>McKanna, J. A., Casagrande, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+lens+development+in+lid-suture+myopia.">Reduced lens development in lid-suture myopia.</a> Exp. Eye Res. 26, 715-723 (1978).</li><li>Shapiro, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+visual+deprivation+and+myopia.">Experimental visual deprivation and myopia.</a> Doc. Ophthalmol. Proc. Ser. 28, 193-195 (1981).</li><li>Wiesel, T. N., Raviola, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increase+in+axial+length+of+the+macaque+monkey+eye+after+corneal+opacification.">Increase in axial length of the macaque monkey eye after corneal opacification.</a> Invest. Ophthalmol. Vis. Sci. 18, 1232-1236 (1979).</li><li>Weiss, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unilateral+high+myopia:+optical+components,+associated+factors+and+visual+outcomes.">Unilateral high myopia: optical components, associated factors and visual outcomes.</a> Br J Ophthalmol. 87, 1025-1031 (2003).</li><li>Mechanic, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crosslinking+of+collagen+in+a+heritable+disorder+of+connective+tissue:+Ehlers-Danlos+syndrome.">Crosslinking of collagen in a heritable disorder of connective tissue: Ehlers-Danlos syndrome.</a> Biochem. Biophys. Res. Com. 47, 267-272 (1972).</li><li>Curtin, B. J., Iwamoto, T., Renaldo, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+and+staphylomatous+sclera+of+high+myopia.+An+electron+microscopic+study.">Normal and staphylomatous sclera of high myopia. An electron microscopic study.</a> Arch. Ophthalmol. 97, 912-915 (1979).</li><li>Liu, K. R., Chen, M. S., Ko, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscopic+studies+of+the+scleral+collagen+fibre+in+excessively+high+myopia.">Electron microscopic studies of the scleral collagen fibre in excessively high myopia.</a> J. Formosan Med. Assoc. 85, 1032-1038 (1986).</li><li>Funata, M., Tokoro, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scleral+change+in+experimentally+myopic+monkeys.">Scleral change in experimentally myopic monkeys.</a> Graefes Arch. Clin. Exp. Ophthalmol. 228, 174-179 (1990).</li><li>McBrien, N. A., Norton, T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+collagen+cross-linking+increases+form+deprivation+myopia+in+tree+shrew.">Prevention of collagen cross-linking increases form deprivation myopia in tree shrew.</a> Exp. Eye Res. 59, 475-486 (1994).</li><li>Iomdina, E. N., Daragan, V. A., Ilyina, E. 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. Scand. 83, 477-482 (2005).</li><li>Wollensak, G., Iomdina, E. <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+biomechanical+properties+of+rabbit+sclera+after+collagen+crosslinking+using+riboflavin+and+ultraviolet+(UVA).">Long-term biomechanical properties of rabbit sclera after collagen crosslinking using riboflavin and ultraviolet (UVA).</a> Acta Ophthalmol. 87, 193-198 (2009).</li><li>Wildsoet, C. F., Norton, T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+controlling+myopia+progression.">Toward controlling myopia progression.</a> Optom. Vis. Sci. 76, 341-342 (1999).</li><li>Avetisov, E. S., Tarutta, E. P., Iomdina, E. N., Vinetskaya, M. I., Andreyeva, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonsurgical+and+surgical+methods+of+sclera+reinforcement+in+progressive+myopia.">Nonsurgical and surgical methods of sclera reinforcement in progressive myopia.</a> Acta Ophthalmol. Scand. 75, 618-623 (1997).</li><li>Thompson, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+sclera+reinforcement+technique.">A simplified sclera reinforcement technique.</a> Am. J. Ophthalmol. 86, 782-790 (1978).</li><li>Whitmore, W. G., Curtin, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scleral+reinforcement:+two+case+reports.">Scleral reinforcement: two case reports.</a> Ophthalmic Surg. 18, 503-505 (1987).</li><li>Dotan, A., Kremer, I., Livnat, T., Zigler, A., Weinberger, D., Bourla, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scleral+cross-linking+using+riboflavin+and+ultraviolet-A+radiation+for+prevention+of+progressive+myopia+in+a+rabbit+model.">Scleral cross-linking using riboflavin and ultraviolet-A radiation for prevention of progressive myopia in a rabbit model.</a> Exp Eye Res. 127, 190-195 (2014).</li></ol>

The authors declare that they have no competing financial interests.

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 margins and using the sutures mentioned in our protocol to ensure that the eyelids are firmly closed for the given period without need for corrections.
One important thing to consider is the need to calibrate the irradiation device and the fiber optic for the required power energy.
H&#38;E histological slides revealed no toxic changes to the retina on eyes that underwent the crosslinking procedure. Further investigations should be performed to address the potential toxicity of riboflavin and UVA irradiation on the retina, choroid and sclera, with a focus on position, amount of energy and exposure time.
Several limitations of this study warrant consideration. Only preoperative and postoperative axial lengths were measured. The axial length of the eyeball was measured by A-scan ultrasound. We did not evaluate biometric properties, refractive analysis or biomechanical properties of the sclera.
Taken together, we think that the effect of scleral crosslinking with riboflavin and UVA on the axial elongation of the rabbit eye will have a positive impact on myopia research.

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.

<table><tbody><tr><td>2-0 braided silk non-needled sutures&amp;nbsp;</td><td>ETHICON</td><td>W193</td><td></td></tr><tr><td>4-0 braided silk ivory color&amp;nbsp;</td><td>ETHICON</td><td>W816</td><td></td></tr><tr><td>0.1% dextran-free riboflavin-5-phosphate 1 mg:1 ml</td><td>Concept for Pharmacy Ltd</td><td>D2-5025</td><td></td></tr><tr><td>UV A (370 nm) light source&amp;nbsp;</td><td>O/E LAND Inc</td><td>NCSU033B</td><td></td></tr><tr><td>Beveled down custom made fiber optic&amp;nbsp;</td><td>Prizmatix Ltd</td><td></td><td></td></tr><tr><td>26 G lacrimal cannula&amp;nbsp;</td><td>Beaver-visitec International Ltd.&amp;nbsp;</td><td>REF581276</td><td></td></tr><tr><td>25 G tapered hydrodelineator [Blumenthal]&amp;nbsp;</td><td>Beaver-visitec International Ltd.&amp;nbsp;</td><td>REF585107</td><td></td></tr><tr><td>13 days old rabbits</td><td>Harlan&amp;nbsp;</td><td>1NZWR40</td><td></td></tr><tr><td>Ultrasonic biometer</td><td>Allergan-Humphrey</td><td>820-519</td><td></td></tr><tr><td>Skin marker</td><td>Devon</td><td>4237101664X</td><td></td></tr></tbody></table>

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 underwent scleral crosslinking and tarsorrhaphy, the mean axial length in the right eye measured 10.68 &#177; 0.74 mm before eyelid suture and 14.29 &#177; 0.3 mm 55 days later, for a mean difference of 3.61 &#177; 0.76 mm. Corresponding values in the unoperated/unsutured left eye were 10.70 &#177; 0.79 mm and 15.14 &#177; 0.32 mm, for a mean difference of 4.44 &#177; 0.81 mm (Figure 1). Comparison of the axial lengths of the sutured and unsutured eyes at the end of the occlusion phase revealed a lesser net increase in the sutured eyes.
In group 2, which underwent only peritomy and tarsorrhaphies, mean axial length in the right eye measured 10.50 &#177; 0.67 mm before eyelid suture and 15.69 &#177; 0.39 mm 55 days later, for a mean difference of 5.19 &#177; 0.85 mm. Corresponding values in the unoperated/unsutured left eye were 10.54 &#177; 0.71 mm and 14.74 &#177; 0.38 mm, for a mean difference of 4.20 &#177; 0.67 mm (Figure 2). Comparison of the axial lengths of the sutured and unsutured eyes at the end of the occlusion phase revealed a greater net increase in the sutured eyes.
Comparison of the mean change in axial length of the right eyes between group 2 (5.19 &#177; 0.85 mm) and group 1 (3.61 &#177; 0.76 mm) yielded a significantly lower value at the end of the occlusion phase (55 days) in the eyes that underwent the crosslinking procedure (p &#60;0.001, nonparametric Mann-Whitney test). The between-group difference in mean axial length in the left eyes (4.20 &#177; 0.67 mm vs. 4.44 &#177; 0.81 mm) was not statistically significant (p = 0.39, Mann-Whitney non-parametric test).
<img alt="Figure 1" src="/files/ftp_upload/53201/53201fig1.jpg" /> 
Figure 1. Right eye axial measurements before and after scleral crosslinking and tarsorrhaphy vs. left eye axial measurements. The mean axial length of the right eye before scleral crosslinking and tarsorrhaphy (RE start) and after removal of the tarsorrhaphy 55 days later (RE end). The mean axial length of the left eye at baseline (LE start) and 55 days later (LE end). The left eye was not operated on and was left open. The error bars indicates standard error of the mean. (Re-printed with permission from reference27). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53201/53201fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53201/53201fig2.jpg" /> 
Figure 2. Right eye axial measurements before and after tarsorrhaphy vs. left eye axial measurements. The mean axial length of the right eye before tarsorrhaphy (RE start) and after removal of the tarsorrhaphy 55 days later (RE end). The mean axial length of the left eye at baseline (LE start) and 55 days later (LE end).The left eye was not operated on and was left open. The error bars indicates standard error of the mean. (Re-printed with permission from reference27). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53201/53201fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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 ...

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JoVE Journal

Medicine

8.8K Views.  Rabin Medical Center, Beilinson Campus. 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).

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

Novel Photoacoustic Microscopy and Optical Coherence Tomography Dual-modality Chorioretinal Imaging in Living Rabbit Eyes

Photoacoustic ocular imaging is an emerging ophthalmic imaging technology that can noninvasively visualize ocular tissue by converting light energy into sound waves and is currently under intensive investigation. However, most reported work to date is focused on the imaging of the posterior segment of the eyes of small animals, such as rats and mice, which poses challenges for clinical human translation due to small eyeball sizes. This manuscript describes a novel photoacoustic microscopy (PAM) and optical coherence tomography (OCT) dual-modality system for posterior segment imaging of the eyes of larger animals, such as rabbits. The system configuration, system alignment, animal preparation, and dual-modality experimental protocols for in vivo, noninvasive, label-free chorioretinal imaging in rabbits are detailed. The effectiveness of the method is demonstrated through representative experimental results, including retinal and choroidal vasculature obtained by the PAM and OCT. This manuscript provides a practical guide to reproducing the imaging results in rabbits and advancing photoacoustic ocular imaging in larger animals.

This manuscript describes the novel setup and operating procedure of a photoacoustic microscopy and optical coherence tomography dual-modality system for noninvasive, label-free chorioretinal imaging of larger animals, such as rabbits.

Photoacoustic ocular imaging is an emerging ophthalmic imaging technology that can noninvasively visualize ocular tissue by converting light energy into ...

Engineering

Three Different Protocols of Corneal Collagen Crosslinking in Keratoconus: Conventional, Accelerated and Iontophoresis

Keratoconus is a bilateral and progressive corneal ectasia. In order to slow down its progression, corneal collagen cross-linking (CXL) has recently been introduced as an efficient treatment option. In biological and chemical sciences, crosslinking refers to new chemical bonds formed between reactive molecules. Hence, the aim of corneal collagen CXL is to synthetically increase the formation of crosslinks between collagen fibrils in the corneal stroma. Despite the fact that the efficiency of the conventional CXL (C-CXL) protocol has already been shown in several clinical studies, it might benefit from improvements in duration of the procedure and removal of corneal epithelium. Hence, in order to provide a coherent evaluation of two new and optimized CXL protocols, we studied keratoconus patients who had undergone one of the three CXL treatments: iontophoresis (I-CXL), accelerated CXL (A-CXL), and conventional CXL (C-CXL). A-CXL is a 6 time faster CXL procedure using a ten time higher UVA irradiance but still including an epithelium removal. Iontophoresis is a transepithelial non-invasive technique in which a small electric current is applied to improve riboflavin penetration throughout the cornea. Using anterior segment optical coherence tomography (AS OCT) and in vivo confocal microscopy (IVCM), we conclude that regarding the depth of treatment penetration, conventional CXL protocol remains the standard for treating progressive keratoconus. Accelerated CXL seems to be a quick, effective and safe alternative to treat thin corneas. The use of iontophoresis is still being investigated and should be considered with greater caution.

Corneal collagen cross-linking (CXL) is the only conservative treatment currently available to halt keratoconus progression by improving the biomechanical rigidity of the corneal stroma. The aim of this manuscript is to highlight the methods of three different protocols of CXL: conventional CXL (C-CXL), accelerated CXL (A-CXL), and iontophoresis CXL (I-CXL).

Keratoconus is a bilateral and progressive corneal ectasia. In order to slow down its progression, corneal collagen cross-linking (CXL) has recently been ...

Use of Rabbit Eyes in Pharmacokinetic Studies of Intraocular Drugs

The intraocular route of drug administration enables the delivery of high concentrations of therapeutic drugs, while minimizing their systemic absorption. Several drugs are administered into the anterior chamber or vitreous, and the intraocular injection has been effective in curing various intraocular diseases. Rabbit eyes have been widely used for ophthalmic research, as the animal is easy to handle and economical compared to other mammals, and the size of a rabbit eye is similar to that of a human eye. Using a 30 G needle, drugs can be injected into the intracameral and intravitreal spaces of rabbit eyes. The eyeballs are then frozen until analysis, and can be divided into the aqueous humor, vitreous, and retina/choroid. The vitreous and retina/choroid samples can be homogenized and solubilized before analysis. Then, immunoassays can be performed to measure the concentrations of intraocular drugs in each compartment. Appropriate pharmacokinetic models can be used to calculate several parameters, such as the half-life and maximum concentration of the drug. Rabbit eyes can be a good model for pharmacokinetic studies of intraocular drugs.

Rabbits are widely used to study the pharmacokinetics of intraocular drugs. We describe a method for conducting pharmacokinetic studies of intraocular drugs using rabbit eyes.

The intraocular route of drug administration enables the delivery of high concentrations of therapeutic drugs, while minimizing their systemic absorption. ...

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 ...

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy include photochemical cross-linking and tissue cross-linking (TXL) methods. Such methods for inducing mechanical tissue property changes are being employed to the cornea in corneal thinning (mechanically weakened) disorders such as keratoconus as well as the sclera in progressive myopia, where thinning and weakening of the posterior sclera occurs and likely contributes to axial elongation. The primary target proteins for such tissue strengthening are fibrillar collagens which constitute the great majority of dry weight proteins in the cornea and sclera. Fortuitously, fibrillar collagens are the main source of second harmonic generation signals in the tissue extracellular space. Therefore, modifications of the collagen proteins, such as those induced through cross-linking therapies, could potentially be detected and quantitated through the use of second harmonic generation microscopy (SHGM). Monitoring SHGM signals through the use of a laser scanning microscopy system coupled with an infrared excitation light source is an exciting modern imaging method that is enjoying widespread usage in the biomedical sciences. Thus, the present study was undertaken in order to evaluate the use of SHGM microscopy as a means to measure induced cross-linking effects in ex vivo rabbit sclera, following an injection of a chemical cross-linking agent into the sub-Tenon&#39;s space (sT), an injection approach that is standard practice for causing ocular anesthesia during ophthalmologic clinical procedures. The chemical cross-linking agent, sodium hydroxymethylglycinate (SMG), is from a class of cosmetic preservatives known as formaldehyde releasing agents (FARs). Scleral changes following reaction with SMG resulted in increases in SHG signals and correlated with shifts in thermal denaturation temperature, a standard method for evaluating induced tissue cross-linking effects.

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking TXL for Myopia

This protocol describes techniques for evaluating chemical cross-linking of the rabbit sclera using&#160;second harmonic generation imaging and differential scanning calorimetry.

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy ...

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 ...

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, and result in a large, persistent corneal epithelial defect. Epithelial defect with the following corneal opacity and peripheral neovascularization result in irreversible visual impairment and hinder future management, especially keratoplasty. Since the animal model can be used as an effective drug development platform, models of corneal injury to the mouse and alkali burn to rabbit corneal epithelium are developed here. New Zealand white rabbit is used in the alkali burn model. Different concentrations of sodium hydroxide can be applied onto the central circular area of the cornea for 30 s under intramuscular and topical anesthesia. After copious isotonic normal saline irrigation, residual loose corneal epithelium was removed with corneal burr deep down to the Bowman's layer within this circular area. Wound healing was documented by fluorescein staining under Cobalt blue light. C57BL/6 mice were used in the traumatic model of murine corneal epithelium. The murine central cornea was marked using a skin punch, 2 mm in diameter, and then debrided by a corneal rust ring remover with a 0.5 mm burr under a stereomicroscope. These models can be prospectively used to validate the therapeutic effect of eye drops or mixed agents such as stem cells, which potentially facilitate corneal epithelial regeneration. By observing corneal opacity, peripheral neovascularization, and conjunctival congestion with stereomicroscope and imaging software, therapeutic effects in these animal models can be monitored.

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Here, animal models based on mouse and rabbit are developed for mechanical and chemical injury of corneal epithelium to screen new therapeutics and the underlying mechanism.

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, ...

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, which leads to subsequent degeneration of vital retinal structures (e.g., photoreceptors) causing severe vision impairment. Similarly, RPE-loss and decrease in visual acuity is seen in long-term follow up of patients with advanced wet age-related macular degeneration (AMD) receiving intravitreal anti-vascular endothelial growth factor (VEGF) treatment. Therefore, on the one hand, it is fundamental to efficiently derive RPE cells from an unlimited source that could serve as replacement therapy. On the other hand, it is important to assess the behavior and integration of the derived cells in a model of the disease entailing surgical and imaging methods as close as possible to those applied in humans. Here, we provide a detailed protocol based on our previous publications that describes the generation of a preclinical model of GA using the albino rabbit eye, for evaluation of the human embryonic stem cell derived retinal pigment epithelial cells (hESC-RPE) in a clinically relevant setting. Differentiated hESC-RPE are transplanted into naive eyes or eyes with NaIO3-induced GA-like retinal degeneration using a 25 G transvitreal pars plana technique. Evaluation of degenerated and transplanted areas is performed by multimodal high-resolution non-invasive real-time imaging.

Retinal pigment epithelial cells could serve as a cell-replacement therapy for the advanced form of dry age-related macular degeneration. This protocol describes the generation of a large-eyed model of geographic atrophy and the subretinal transplantation of human embryonic stem cell-derived retinal pigment epithelial cells into this model of disease.

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, ...

Biology

Excimer Laser Multifocal Double Aspheric Ablation for Presbyopia Correction

Correction of Presbyopia by Monocular Bi-Aspheric Ablation Profile

The study aims to evaluate visual acuity and objective visual quality before and after the monocular bi-aspheric ablation profile for correction of presbyopia surgery. This prospective self-control study included 20 cases and 38 eyes of patients who underwent monocular bi-aspheric ablation profile correction of myopia with presbyopia at the Eye Hospital of Shandong University of Traditional Chinese Medicine from January 2023 to January 2024. These patients were selected for observation, and each patient's preoperative and postoperative uncorrected distance visual acuity (UDVA), uncorrected near visual acuity (UNVA), corrected distance visual acuity (CDVA), spherical aberration (SA) (within 6 mm), horizontal and vertical coma (within 6 mm), and corneal aspheric index (Q-value) (within 6 mm) were evaluated. Statistical data analysis was performed at different time points before and after the operation. There were statistically significant differences in UDVA between dominant and non-dominant eyes before and after surgery (Z = -3.784, p &lt; 0.001; Z = -3.817, p &lt; 0.001). Post-operatively, 90% of the non-dominant eyes achieved UNVA of J1 and above, and 95% of the bilateral eyes achieved UNVA of J1 and above. Significant differences were found in the SA of the dominant eyes, which showed a positive increase (Z= -3.784, p &lt; 0.001); however, compared with the dominant eye, the SA of the non-dominant eye was negatively increased, but the difference was not statistically significant (p = 0.08). There was a significant difference in the vertical coma of the dominant eye before and after the operation, but there was no significant difference in non-dominant eyes. There was no significant difference in the change of binocular horizontal coma before and after the operation. There were significant changes in the Q value of both eyes before and after the operation (Z = -3.923, p &lt; 0.001; Z = -3.51, p &lt; 0.001). After the monocular bi-aspheric ablation profile, the cornea of the non-dominant eye showed a prolate shape, negative SA increased, and the UDVA and UNVA improved after the operation.

Author Spotlight: Advancements in Refractive Surgical Correction for Presbyopia and Exploring Postoperative Visual Acuity

This article provides a step-by-step guide to correcting presbyopia with a monocular bi-aspheric ablation profile.

The study aims to evaluate visual acuity and objective visual quality before and after the monocular bi-aspheric ablation profile for correction of ...

Rabbit Eye Dissection and Cryosectioning for Immunohistochemistry Studies

Obtaining High-Quality Cryosections of Whole Rabbit Eye

This protocol describes how to obtain high-quality retinal cryosections in larger animals, such as rabbits. After enucleation, the eye is briefly immersed in the fixative. Then, the cornea and iris are removed and the eye is left overnight for additional fixation at 4 &#176;C. Following fixation, the lens is removed. The eye is then placed in a cryomold and filled with an embedding medium. By removing the lens, the embedding medium has better access to the vitreous and leads to better retinal stability. Importantly, the eye should be incubated in embedding medium overnight to allow complete infiltration throughout the vitreous. Following overnight incubation, the eye is frozen on dry ice and sectioned. Whole retinal sections may be obtained for use in immunohistochemistry. Standard staining protocols may be utilized to study the localization of antigens within the retinal tissue. Adherence to this protocol results in high-quality retinal cryosections that may be used in any experiment utilizing immunohistochemistry.

Author Spotlight: Optimizing Retinal Tissue Processing for Immunohistochemistry in Rabbit Models

This protocol describes a reliable method for obtaining high-quality cryosections of whole rabbit eyes. It details rabbit eye dissection, fixation, embedding, and sectioning procedures, which may be easily adapted for use in any study utilizing immunohistochemistry in larger eyes.

This protocol describes how to obtain high-quality retinal cryosections in larger animals, such as rabbits. After enucleation, the eye is briefly immersed ...

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

Summary

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Chapters in this video

Novel Photoacoustic Microscopy and Optical Coherence Tomography Dual-modality Chorioretinal Imaging in Living Rabbit Eyes

Three Different Protocols of Corneal Collagen Crosslinking in Keratoconus: Conventional, Accelerated and Iontophoresis

Use of Rabbit Eyes in Pharmacokinetic Studies of Intraocular Drugs

A Step by Step Protocol for Subretinal Surgery in Rabbits

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia

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

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

Correction of Presbyopia by Monocular Bi-Aspheric Ablation Profile

Obtaining High-Quality Cryosections of Whole Rabbit Eye