Acta Ophthalmologica 2013
Trigeminal stereotactic electrolysis induces dry eye in mice Giulio Ferrari,1 Hiroki Ueno,2 Fabio Bignami,3 Paolo Rama3 and Reza Dana2 1
Editor, orneal nerves are known to contribute significantly to ocular surface integrity. A normal corneal innervation is required for a normal tear secretion and flow. In fact, dry eye disease is defined as a disorder of the lacrimal functional unit, an integrated system comprising the sensory and motor nerves (DEWS 2007). Corneal nerve depletion reduces tear secretion by decreasing reflex-induced lacrimal secretion and by reducing the blink rate. Moreover, corneal nerves secrete up to 17 different mediators such as nerve growth factor and others, which have profound effects on corneal surface homoeostasis (Mu¨ller et al. 2003). Damage to the sensory nerves in the ocular surface, particularly the cornea, is common in a number of conditions, including refractive surgery, keratoplasty and even normal ageing (He et al. 2010). Corneal nerve damage often occurs in concomitance with inflammation elicited by surgery or drugs; hence, it is difficult to precisely dissect the role of corneal nerves in dry eye. We recently developed a murine model of corneal denervation (TSE, Trigeminal Stereotactic Electrolysis) where we selectively ablate the ophthalmic branch of the trigeminal nerve (Ferrari et al. 2011). We used this model to test whether and to which extent selective sensory nerve ablation could induce dry eye in terms of (i) tear secretion and (ii) corneal fluorescein staining. Eight eyes of eight C57BL ⁄ 6 mice were used. Animals were examined
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doi: 10.1111/j.1755-3768.2012.02529.x
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G.B. Bietti Eye Foundation, IRCCS, Rome, Italy 2 Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts, USA 3 Eye Repair Lab, Cornea and Ocular Surface Unit, San Raffaele Scientific Institute, Milan, Italy
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Fig. 1. (Panels A, B) development of punctate keratitis following TSE-induced corneal denervation. Note the development of fluorescein positive lesions (B) that were not present before TSE treatment (A). (Panel C) the amount of corneal staining was significantly increased following TSE (p < 0.0001, paired t-test). (Panel D) tear production, measured with the phenol red test, significantly decreased following TSE (p < 0.0001, paired t-test). Vertical bars are (±) standard deviation (SD) of the mean.
before and 7 days after TSE. The effectiveness of the procedure was confirmed by the absence of blink reflex (Ferrari et al. 2011). At the end of the procedure, a complete tarsorrhaphy was performed to minimize the effect of absent blink reflex. The animals were examined using a biomicroscope, and tear production was quantified with phenol red thread test. Finally, the corneas were stained with fluorescein and scored according to the National Eye Institute Grading System. Before TSE was performed, biomicroscopy revealed a normal cornea. Corneal fluorescein staining was negative before TSE (score, 0) and phenol test was 2.59 ± 0.44 mm (mean ± SD). Corneal reflex was tested with a cotton thread and found to be present in all the eyes. Following TSE, cornea fluorescein staining score was 10.39 ± 2.20 (mean ± SD), phenol test decreased to 0.52 ± 0.36 mm (mean ± SD) and corneal reflex was absent in all the eyes (Fig. 1). After TSE, we found significant differences in both the fluorescein score (p < 0.0001) and the phenol test (p < 0.0001) compared to the tests at baseline before TSE (paired t-test).
In conclusion, sensory corneal denervation induces a form of dry eye in the TSE murine model. This occurred despite complete tarsorrhaphy, suggesting that reduction in the blink reflex was not responsible for this finding. Functional inhibition of corneal nerves with topical anaesthesia is able to reduce tear flow up to 75% (Jordan & Baum 1980). In this model, tear production was reduced by approximately five times. Our findings may be due to the reduced sensory drive from the ocular surface, which, together with a reduced trophic influence of the nerves on the ocular surface epithelium, could result in a dry eye phenotype. We suggest that the TSE model may be useful in the study of dry eye disease, in particular as a model for the dry eye patients who also demonstrate significant ‘neurotrophic’ disease, such as viral keratitis, diabetes, or after refractive surgery.
References DEWS Definition and Classification Subcommittee (2007): The definition and classification of dry eye disease: report of the
Acta Ophthalmologica 2013
Definition and Classification Subcommittee of the International Dry Eye WorkShop. Ocul Surf 5: 75–92. Ferrari G, Chauhan SK, Ueno H, Nallasamy N, Gandolfi S, Borges L & Dana R (2011): A novel mouse model for neurotrophic keratopathy: trigeminal nerve stereotactic electrolysis through the brain. Invest Ophthalmol Vis Sci 52: 2532–2539. He J, Bazan NG & Bazan HE (2010): Mapping the entire human corneal nerve architecture. Exp Eye Res 91: 513–523. Jordan A & Baum J (1980): Basic tear flow. Does it exist?. Ophthalmology 87: 920–930. Mu¨ller LJ, Marfurt CF, Kruse F & Tervo TM (2003): Corneal nerves: structure, contents and function. Exp Eye Res 76: 521– 542.
Correspondence: Giulio Ferrari, MD San Raffaele Scientific Institute Via Olgettina, 60 Milano 20132 Italy Tel: + 39 0226 436 232 Fax: + 39 022 6436 164 Email:
[email protected]
Restoration of photoreceptor structure and function in nonischaemic central retinal vein occlusion Bartosz L. Sikorski,1,2 Danuta Bukowska,2 Jakub J. Kaluzny,1 Andrzej Kowalczyk,2 and Maciej Wojtkowski2 1
Department of Ophthalmology, Nicolaus Copernicus University, Bydgoszcz, Poland 2 Institute of Physics, Nicolaus Copernicus University, Torun, Poland doi: 10.1111/j.1755-3768.2012.02516.x
Editor, he development of spectral optical coherence tomography (SOCT) has allowed in vivo ultrahigh-resolution imaging of retinal structure. In particular, the clear detection of the junction between the inner and outer photoreceptor segments (IS ⁄ OS) has become possible. Thus, in recent years, many studies
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investigating the restoration of photoreceptor ultrastructure after surgery for retinal detachment and macular holes have been conducted (Sano et al. 2009; Shimoda et al. 2010). None of them, however, was able to precisely visualize all alterations in the IS ⁄ OS throughout the entire scanned area due to the lack of appropriate three-dimensional SOCT data segmentation and flattening algorithms in commercial SOCT systems. In our study, we applied a customdesigned software, which has been previously described by our group (Szkulmowski et al. 2007; Sikorski et al. 2008, 2011), to commercially available SOCT device (Copernicus HR, Optopol, Poland) to reconstruct the distribution of the reflectivity along the IS ⁄ OS in all SOCT crosssectional images as two-dimensional reflectivity maps (RM). Thanks to this, it has become possible to correlate directly the photoreceptor reflectivity measured by SOCT with retinal sensitivity determined by microperimetry (MP-1, Nidek, Italy) within the whole scanned area of 7 · 7 mm. For the purpose of the study, the RMs of the IS ⁄ OS that also contained a small adjacent portion of the outer segments (spOS), the RMs of outer segments (OS) and retinal pigment epithelium (RPE) were created. We used them to investigate the regeneration process of photoreceptors in a patient with nonischemic central retinal vein occlusion (nCRVO) (Fig. 1C,D). A 48-year-old man had been experiencing reduced vision in the right eye for four days. His visual acuity was 20 ⁄ 40. SOCT tomograms revealed a lack of the line corresponding to the IS ⁄ OS in the central and temporal part of the macula with its partial maintenance in the fovea (Fig. 1E–G). Within this region, external limiting membrane (ELM) was shifted in the direction of the RPE. This clearly indicated that the lack of reflectivity of the IS ⁄ OS did not solely result from photoreceptor malfunction but also from their physical decomposition. When we displayed the reflectivity of the IS ⁄ OS as the RM-IS ⁄ OS-spOS, we found that the region in which SOCT did not reveal the presence of the IS ⁄ OS was surrounded by the oval area with slightly reduced reflectivity (Fig. 1H). To our surprise, the latter changes were invisible during simple assessment of SOCT
tomograms. The separate RM of the OS showed exactly the same area of decreased reflectivity as the RMIS ⁄ OS-spOS, indicating that the source of reduced reflectivity is the OS (Fig. 1I). Furthermore, the RM-RPE depicted only shadows of superficial retinal haemorrhages, in this way proving that the changes observed on the RM-OS are not a result of alterations in the IS ⁄ OS that cast shadows on deeper located layers (Fig. 1J). Simultaneously, we performed microperimetry that revealed an irregular absolute scotoma surrounded by a zone of reduced retinal sensitivity (Fig. 1A). With the course of time, however, the absolute scotoma underwent diminution and after 18 months the patient could see the light stimulus in all examined locations (Fig. 1B). The visual acuity was 20 ⁄ 20. SOCT tomograms depicted the presence of undisturbed IS ⁄ OS as well as the correct location of ELM in the entire macular area (Fig. 1K–M). The RM-IS ⁄ OS-spOS did not show any zones of the complete IS ⁄ OS reflectivity loss (Fig. 1N). The circular area of previously reduced OS reflectivity was still detectable and underwent only minor nasal limitations (Fig. 1O). Surprisingly, within this region, microperimetry did not present decreased retinal sensitivity (Fig. 1B). Our study has demonstrated that despite the restoration of photoreceptor structure and function in nCRVO, the RMs revealed persistent changes in the reflectivity of the OS. The results indicate that SOCT RMs can visualize and monitor over time subtle alterations in the OS reflectivity, which are otherwise undetectable by simple assessment of SOCT tomograms, most likely due to the low signal-to-noise ratio.
Funding This work was supported by the EuroHORCs-European Science Foundation EURYI Award (EURYI-01 ⁄ 2008-PL).
References Sano M, Shimoda Y, Hashimoto H & Kishi S (2009): Restored photoreceptor outer segment and visual recovery after macular hole closure. Am J Ophthalmol 147: 313–318. Shimoda Y, Sano M, Hashimoto H, Yokota Y & Kishi S (2010): Restoration of photo-
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