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Clinical and Experimental Ophthalmology 2015; 43: 742–748 doi: 10.1111/ceo.12547

Original Article Chemical and material communication between the optic nerves in rats Shuo Yang PhD,1,2 Heng He PhD,1 Ying Zhu PhD,1 Xing Wan PhD,1 Long-Fang Zhou PhD,1 Juan Wang PhD,2 Wen-Feng Wang PhD,1 Lei Liu MD2 and Bin Li MD1 Departments of 1Ophthalmology and 2Optometry and Ophthalmology Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, China

Key words:

axoplasmic transport, binocular, material communication, optic nerve.

ABSTRACT Background: To examine interactions between optic nerves. Methods: A total of 24 Sprague–Dawley rats received unilateral intravitreal injections. The rats were equally divided into four groups: group A was administered an adeno-associated virus (AAV) carrying an exogenous gene (ND4; rAAV-ND4); group B, AAV carrying a green fluorescent protein (GFP; rAAV-GFP); group C, fluorogold (FG) nerve tracer dye; and group D, phosphate-buffered saline (PBS) as a control. Two weeks later, GFP expression was evaluated in both retinas and optic nerves of group B rats after frozen sectioning. The presence of FG was also evaluated in group C optic nerves by fluorescent microscopy after frozen sectioning. Four weeks after injection, ND4 expression was evaluated in both eyes of groups A and D using western blotting and immunofluorescence. Results: FG was observed in the optic chiasm posterior segment along the optic nerve of injected eyes. Some FG reached the anterior optic nerve of the non-injected eye. GFP fluorescence was observed only in the retina of the injected eye but not in the contralateral retina or either optic nerve. ND4 expression was significantly different between

injected and non-injected eyes but not between the non-injected eyes in groups A and D. Conclusion: Unilaterally injected material can reach the contralateral optic nerve through axoplasmic transport. It is possible that this the only mechanism by which the optic nerves directly communicate.

INTRODUCTION In our previous gene therapy studies for Leber’s hereditary optic neuropathy (LHON), we were surprised to find that visual function improved in both eyes following unilateral intravitreal injection of an adeno-associated virus (AAV) carrying the exogenous ND4 gene (AAV2-ND4). This result led us to further investigate the mechanism by which visual function in the non-injected eye improved. In the visual pathway anatomy, the eyes are anatomically connected through the optic nerves.1,2 However, it remains unknown whether material communication also occurs between the optic nerves. Influences of the contralateral eye are observed in nearly all LHON patients in the clinical setting. When one eye suffers a visual function loss, the vision in the other eye soon deteriorates. We used to believe that this successive decrease in visual function2 resulted from an asymmetrical impact of the LHON-associated mitochondrial gene mutation in the optic nerves. However, it is possible that close material and/or functional connections between

■ Correspondence: Professor Bin Li, Department of Ophthalmology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jie-fang Road, Wuhan, Hubei Province, China. E-mail: [email protected] Received 14 November 2014; accepted 30 April 2015. Conflict of interest: None. Funding sources: The work was supported by the National Nature Science Foundation of China (Grants No. 81271015). © 2015 Royal Australian and New Zealand College of Ophthalmologists

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optic nerves exist. If this is the case, damage to one optic nerve could lead to subsequent damage of the other optic nerve. We hypothesize that optic nerves interact with each other and, just as optic nerves may negatively affect each other, they may also positively affect each other. When the function of one optic nerve improves, the function of the contralateral optic nerve may also improve. Here, we tested this hypothesis by performing unilateral intravitreal injection of fluorogold (FG), a neural tracer that can be taken up by retinal ganglion cells (RGCs), in Sprague–Dawley (SD) rats. Two weeks after injection, direct substance exchange between optic nerves was evaluated by following the FG tract. We also examined whether or not an AAV carrying an exogenous gene could affect the non-injected eye. Expression of ND4 and green fluorescent protein (GFP) was examined 4 weeks after unilateral intravitreal injection of rAAV-ND4 and rAAV-GFP, respectively.

kg). A topical anaesthetic (proparacaine HCl) was administered to the cornea after pupil dilation with bistropamide. Under strictly sterile conditions, a 33-gauge needle attached to a syringe (Hamilton Company, Bonaduz, Switzerland) was inserted through the pars plana at the corneoscleral limbus into the vitreous cavity. The right eye was always the operative eye and was covered with chlortetracycline HCl ocular ointment following injection. A volume of 5 μL of fluid was injected. Groups A and B received 5 × 109 mol/L of rAAV-ND4 and rAAV-GFP, respectively. Group C received a 2% FG solution (Fluoro-Gold in 0.9% saline containing 1% dimethyl sulfoxide and 1% Triton X-100; Fluorochrome, LLC, Denver, CO, USA). Group D received phosphatebuffered saline (PBS) and served as a control group for ND4 evaluations.

METHODS

Rats in group C were sacrificed 2 weeks after FG injection. The eyes were fixed by cardiac perfusion of 4% paraformaldehyde (pH = 7.4), as previously described.4 The integrated optic nerves were carefully removed and placed in a 4% paraformaldehyde fixing solution for 6 h at 4°C. The optic nerves were then placed in 30% sucrose at 4°C for 8 h for dehydration. Finally, longitudinal sections (thickness = 10 μm) of the optic nerves were cut using a freezing microtome (Leica Biosystems, Wetzlar, Germany). Fluorescence microscopy (BX51, Olympus, Tokyo, Japan) was used to image tissue FG.

Animals In total, 18 8-week-old male SD rats (obtained from the Animal Experimental Center, Huazhong University of Science and Technology, Wuhan, China) were used in this study. The rats weighed approximately 200 g at the time of receipt. The Hospital Ethics Committee of Huazhong University of Science and Technology approved this study. The animals were cared for in accordance with the Guidelines for the Care and Use of Laboratory Animals and all possible measures were taken to minimize suffering and the number of rats used. All animal procedures were performed in accordance with the Association of Assessment and Accreditation of Laboratory Animal Care guidelines and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The rats were killed with an overdose of sodium pentobarbital (100 mg/kg body weight).

Viral vectors A genetically engineered AAV2/2 virus encoding the human ND4 gene fused to the cyclooxygenase 10 (COX10) mitochondrial targeting sequence and COX10 3' untranslated region was constructed. Viral functionality was verified by examining the capacity to induce ND4 mRNA transcription and ND4 protein expression in transduced human embryonic kidney 293 cells.3

Intravitreal injection Rats were anaesthetized with an intramuscular injection of 2% sodium pentobarbital sodium (45 mg/

Distribution of FG in retina and optic nerve tissue

GFP expression The rats in group B were sacrificed 2 weeks after intravitreal injection of rAAV-GFP. Both eyes and optic nerves were removed and placed in 4% paraformaldehyde for 12 h. The eyes were then placed in a 30% sucrose solution at 4°C for 8 h for dehydration. The anterior segment was removed to obtain an eyecup preparation. Frozen sections of retinas and optic nerves were then cut (thickness = 10 μm) with a freezing microtome and GFP was imaged with fluorescence microscopy.

ND4 expression Animals receiving intravitreal injections of rAAVND4 were sacrificed 4 weeks after intravitreal injection. The anaesthesia, euthanasia, perfusion and tissue preparation techniques were the same as those described for FG and GFP analyses. Briefly, rats were killed with an intraperitoneal injection of 2% sodium pentobarbital (45 mg/kg) followed by

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cardiac perfusion with warm saline and 4% paraformaldehyde. Both eyes were enucleated and placed in 4% paraformaldehyde for 12 h at 4°C. The tissue was then dehydrated in 30% sucrose at 4°C for 8 h. Anterior segment tissues (i.e. cornea and lens) and the vitreous body were carefully removed. The remaining posterior eyecup containing the retina was then immersed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH = 7.4) for 30 min at 4°C. The eyecups were directly embedded in Optimal Cutting Temperature medium (Tissue-Tek OCT Compound, Sakura-Finetek Japan, Tokyo, Japan). The immunofluorescence assay for ND4 was performed on freshly cut, frozen retinal sections that had been serially sectioned into 10-μm-thick slices. Retinal sections were incubated for 30 min with 0.03% Triton (9.97 mL of PBS: 0.03 mL of Triton) at room temperature. After three 5-min washes with PBS, sections were blocked and permeabilized in 3% goat serum (Biodesign International, Saco, ME, USA) and 0.2% Triton X-100 for 1 h. Following three more PBS washes, the sections were incubated with a mouse monoclonal antibody to ND4 (1:100, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight. The following steps were performed in a dark room to prevent retinal light exposure. Sections were rinsed in PBS and incubated with a fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin G (1:100, Jackson Immunoresearch Lab. Inc., West Grove, PA, USA) for 1 h. The stained sections were again rinsed and mounted in a mounting medium containing 4',6-diamidino-2phenylindole. Antifading mounting medium (BIOS Biological Technology, Wuhan, China) was used to reduce fluorochrome quenching during fluorescence microscopy analyses. Slides were visualized and photographed at 400× magnification using confocal fluorescence microscopy (Olympus, Hamburg, Germany).

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Western blot analyses of ND4 The method of western blot was performed as previously described.5,6 Total protein was extracted from retinal tissue in 300 μL of RIPA Lysis Buffer (Beyotime Institute of Biotechnology, Nantong, China). After the sample was centrifuged at 1000 g for 3 min, protein extracts were diluted at a 1:1 ratio with sample buffer (126 mM Tris HCl, pH = 6.8, containing 20% glycerol, 4% sodium dodecyl sulfate [SDS], 0.005% bromophenol blue and 5% 2-mercaptoethanol) and boiled for 3 min. Samples were fractionated according to size on a 12.5% SDSpolyacrylamide gel, transferred to a nitrocellulose membrane (Millipore, Billerica, MA, USA) and probed with polyclonal anti-ND4 (1:200, Santa Cruz Biotechnology). A secondary antibody (goat

Figure 1. Fluorogold (FG) distribution (yellow arrows) in the optic nerve and optic chiasm after injection (n = 6 rats). Panoramic views of frozen sections obtained from the optic chiasm and along the nerve of the injected eye (right side) and the contralateral optic nerve (a–d). The FG moved along the ipsilateral optic nerve, through the optic chiasm, and into the posterior segment of contralateral optic nerve. Only a small amount of FG is present on the same side of the optic nerve, with the majority of FG in the contralateral optic nerve. Trace amounts of FG are also present in front of the optic chiasm. Scale bar = 100 μm.

© 2015 Royal Australian and New Zealand College of Ophthalmologists

Communication between the optic nerves anti-rabbit IgG [Boster Biological Technology, Wuhan, China] diluted to 1:50 000) was applied and the chemiluminescent signal was measured. The same membrane was reused to detect glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal control by incubation with a mouse anti-human GAPDH antibody (Zhixian, Hangzhou, China). Bands observed on photographic films were automatically analysed using image analysis software. The integrated optical density of each protein band was normalized to that of the GAPDH band from the same sample.

Statistical analyses All values are presented as the mean ± standard deviation. Statistical analyses were performed using one-way analysis of variance with the Bonferroni multiple comparison test when comparing three or more groups and with Student’s t-test when comparing two groups. Statistical significance was defined as P < 0.05.

745 sectioning. The FG tracer was found in longitudinal sections of the optic nerve (Fig. 1). The FG moved along the optic nerve in the injected eye and was shunted at the optic chiasm so that a small amount entered the ipsilateral optic nerve, but most of it entered the contralateral optic nerve. A small amount of tracer also entered the anterior portion of the contralateral optic nerve (Fig. 1a–d).

GFP in the retina Retinal fluorescence in animals injected with rAAVGFP was examined after frozen sectioning. Fluorescence was observed in retinal sections of the injected eye (Fig. 2a), but not in sections of the non-injected eye (Fig. 2b). Additionally, GFP fluorescence was not detectable in frozen optic nerve sections or in the optic chiasm (Fig. 3). These results show that rAAVGFP transduced the retina of the injected eye, but not the non-injected eye.

RESULTS

ND4 expression in the retina

Distribution of FG in the optic nerve

Four weeks after intravitreal injection of rAAV-ND4 or PBS, retinal expression of ND4 was evaluated by immunofluorescence. In rats receiving rAAV-ND4, ND4 expression was greatly increased in the injected eye (Fig. 4a–c), but not in the non-injected eye (Fig. 4d–f). In rats receiving PBS, ND4 expression did not increase in either eye (Fig. 4g–l). Western blots were also used to examine changes in ND4 expression (Fig. 5). The ND4 expression was significantly higher in eyes receiving rAAV-ND4 than in non-injected eyes (P < 0.01). Additionally, ND4 expression was not significantly different between non-injected eyes and those administered PBS (P > 0.05). This finding indicates that rAAV-ND4 only transduced the injected eye.

Two weeks after FG injection, the optic nerve was observed by fluorescence microscopy after frozen

Figure 2. Confocal microscope image of sectioned Sprague– Dawley rat retinas with adeno-associated virus (AAV)-mediated expression of exogenous green fluorescent protein (eGFP) (n = 6 rats). GFP is present in the retina of the AAV-eGFP-injected eye (a), but not in the retina of the contralateral eye (b). Scale bar = 100 μm.

DISCUSSION We found that material intravitreally injected into one eye can reach the contralateral optic nerve via axoplasmic transport (Fig. 6). In glaucoma and optic Figure 3. Representative fluorescence microscope images of a frozen section of an optic nerve and chiasm of a Sprague–Dawley rat that underwent intravitreal injection of adeno-associated virus expressing exogenous green fluorescent protein (GFP, n = 6 rats). No GFP is present in the optic nerve (a, 50× magnification), or in the optic chiasm (b, 100× magnification). Scale bar = 100 μm.

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Figure 4. ND4 immunofluorescence detected in rats injected with adeno-associated virus (AAV) expressing ND4 in one eye. Sections of the injected eye (a–c), contralateral eye (d–f), control eye (phosphate-buffered saline injection, g–i) and contralateral control eye (j–l) are shown. The ND4 protein is shown in green in the figure (marked by FITC), and nuclei appear in blue (marked by 4',6-diamidino-2-phenylindole). There were three rats in each study group. Scale bar = 100 μm.

nerve research, FG is used to observe RGC innervation and to count RGCs because it is taken up by RGCs. In our study, FG was transported along the optic nerve to the optic chiasm. Interestingly, most of the FG was transported to the contralateral optic

Figure 5. Expression of ND4 in the retinas of Sprague–Dawley rats following intravitreal injection of adeno-associated virus carrying the ND4 gene (AAV – ND4) and phosphate-buffered saline (PBS). (a) Western blot in the AAV – ND4 injection and PBS injection groups. (b) Summary of ND4/GAPDH detection in both the injected and contralateral eyes. ND4 protein expression was significantly higher in the injected eye than in the contralateral, noninjected eye in the AAV-ND4 group (P = 0.0025), but there was no significant difference between the non-injected eye in the PBS group and the non-injected eye in the AAV-ND4 group (P = 0.2248). There were three rats in each study group. **P < 0.01.

nerve. This finding confirms that material exchange does occur between the optic nerve axons. This material exchange could help explain the pathological link between eyes in LHON patients. Pernet and Schwab7 examined optic nerve injury and repair and showed that nerve fibers from one eye cross the optic chiasm into the contralateral optic nerve and project in the direction of the contralateral retina. This finding provides anatomical support for our hypothesis, that is that the two optic nerves can exchange material through axoplasmic transport and interact with each other. Our results may provide a reasonable explanation for why, in clinical practice, the

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Communication between the optic nerves

Figure 6. Schematic diagram of suggested material communication between the optic nerves. After intravitreal injection of fluorogold (FG), the FG moved along the optic nerve to the optic chiasm, where the majority of it remained in the nerve of the injected eye. However, a fair amount of FG crossed into the nerve from the contralateral eye or traveled back up the nerve of the contralateral eye.

contralateral eye can be affected by monocular treatment or illness. When LHON patients develop optic nerve lesions, axoplasmic transport may be affected.8–12 Similarly, when LHON patients have concurrent eye disease, injury to one optic nerve may affect the other optic nerve, resulting in contralateral eye disease. However, in most clinical cases, when blindness or vision loss occurs in one eye, the other eye is not affected. It is important to understand the connection between the two eyes because the communication between the optic nerves may influence the process of some eye diseases. A better understanding of the material transfer between the optic nerves may thus lead to new therapies and preventative measures against ocular diseases. Many studies in the field of gene therapy have documented that rAAV can carry exogenous genes to treat ophthalmologic diseases, including congenital amaurosis, LHON and retinitis pigmentosa. It is known that rAAV can enter every retinal layer, where it can stably express its transgene payload. In our study, we aimed to explore whether or not rAAV could travel from the injected eye to the contralateral eye. When AAV-GFP and AAV-ND4 were adminis-

747 tered unilaterally via intravitreal injection, neither GFP nor ND4 expression increased in the noninjected eye. It is possible that the genetic material was not transported along the nerve to the RGC cell body, although we did not explore this aspect in the present study. It is also possible that the communication between the optic nerves could have a selective mechanism, where only certain materials are exchanged between the nerves. These possibilities will be evaluated in further studies. Regardless, our present findings indicate that other than axonal material transport through the optic nerve, there are no other direct pathways for contact between the eyes. Therefore, when ocular disease is present, visual function loss in the fellow eye must occur via axonal material exchange in the optic nerve. In addition to the potential pathological aspects, optic nerve communication may also be beneficial to the fellow eye. Testa et al.13 administered monocular gene therapy to five LHON patients in a 3-year clinical trial. As expected, vision improved in the treated eye; however, it also surprisingly improved in the untreated eye. Eye movements have also been examined in a canine model of LHON.14 Following treatment, the visual and eye movement function improved and nystagmus decreased. These changes would indirectly affect the contralateral eye, but not all LHON patients have nystagmus. Ashtari et al.15 argued that visual function improvements in the contralateral eye result from improvements in central pivot; however, such a mechanism would not contradict the results of our study. One surprising finding of the current study was that more FG reached the contralateral optic tract than the ipsilateral optic tract posterior to the optic chiasm. This phenomenon may have resulted from unequal optic nerve fiber distribution at the optic chiasm. In SD rats, 95% of unilateral optic nerve fibers cross to the contralateral side, with only 5% of fibers projecting to the same side.16,17 This nerve distribution pattern markedly differs from that in humans, where approximately 50% of optic nerve fibers cross to the opposite side at the optic chiasm. Given that the anatomical and physiological differences between rodents and humans are substantial,18–22 the transport mechanisms between the optic nerves require further study.

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13. Testa F, Maguire AM, Rossi S et al. Three-year follow-up after unilateral subretinal delivery of adenoassociated virus in patients with Leber congenital amaurosis type 2. Ophthalmology 2013; 120: 1283–91. 14. Jacobs JB, Dell’Osso LF, Hertle RW et al. Eye movement recordings as an effectiveness indicator of gene therapy in RPE65-deficient canines: implications for the ocular motor system. Invest Ophthalmol Vis Sci 2006; 47: 2865–75. 15. Ashtari M, Cyckowski LL, Monroe JF et al. The human visual cortex responds to gene therapy–mediated recovery of retinal function. J Clin Invest 2011; 121: 2160–8. 16. Lund RD. Uncrossed visual pathways of hooded and albino rats. Science 1965; 149: 1506–7. 17. Petros TJ, Rebsam A, Mason CA. Retinal axon growth at the optic chiasm: to cross or not to cross. Annu Rev Neurosci 2008; 31: 295–315. 18. Jeffery G, Harman AM. Distinctive pattern of organisation in the retinofugal pathway of a marsupial: II. Optic chiasm. J Comp Neurol 1992; 325: 57–67. 19. Jeffery G. Distribution and trajectory of uncrossed axons in the optic nerves of pigmented and albino rats. J Comp Neurol 1989; 289: 462–6. 20. Neveu MM, Jeffery G. Chiasm formation in man is fundamentally different from that in the mouse. Eye 2007; 21: 1264–70. 21. Reese BE. Development of the retina and optic pathway. Vision Res 2011; 51: 613–32. 22. Prasad S, Galetta SL. Anatomy and physiology of the afferent visual system. Handb Clin Neurol 2011; 102: 3–19.

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Chemical and material communication between the optic nerves in rats.

To examine interactions between optic nerves...
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