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Effect of botulinum toxin A on the intraocular pressure and retinal ganglion cells in animal model

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Investigative Ophthalmology & Visual Science Draft

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Moschos, Marilita; University of Athens, Ophthalmology Gatzioufas, Zisis; Institute of Experimental Ophthalmology, University of Muenster Stupp, Tobias; Institute of Experimental Ophthalmology, University of Muenster Margetis, Ioannis; 2 Department of Ophthalmology, University of Athens Charalambous, Peter; Institute of Experimental Ophthalmology, University of Muenster Thanos, Solon; Institute of Experimental Ophthalmology, University of Muenster

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Abstract:

Purpose. The purpose of our study was to investigate the effect of an inadvertent intravitreal injection of botulinum toxin A (BTA) on the intraocular pressure (IOP) and the retinal ganglion cells (RGCs) in an animal model. Methods Intravitreal injection of 10L in two doses, 5 and 10 IU, were given into the left eye of two groups of Sprague-Dawleys rats. The same volume of balanced salt solution (BSS) was injected in a third group of rats. Each group consisted of six rats, with normal IOP. IOP was measured preoperatively as well as one week, two weeks and four weeks postoperatively. Retinas were stained in vivo using a retrograde labelled technique. Afterwards, retinas were microsurgically removed and the density of RGCs was determined under fluorescent microscopy. Subsequently, immunohistochemistry was performed using antibodies against rhodopsin as well as against the retinal glial fibrillary acidic protein (GFAP) which is an indirect marker of ganglion cell death. Results. Over an observation of four weeks, we documented an IOP decrease in both BTA groups and an IOP increase in BSS group. The differences were not statisticall significant (Kruskal-Wallis test p>0.05). All retinas displayed the same immunostaining pattern for rhodopsin. Minimal signals for GFAP were detected in all retinas Conclusions. Our findings indicate that diffusion of BTA or even inadvertent intravitreal injection of BTA in neuroophthalmological patients is probably without a severe impact on the IOP or a toxic effect on the RGCs.

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Effect of botulinum toxin A on the intraocular pressure and retinal ganglion cells in animal model Running title: Effect of botulinum toxin on IOP and RGCs

Zisis Gatzioufas MD, PhD1, Marilita M. Moschos MD, PhD2, Tobias Stupp MD1, Ioannis Margetis MD2, Peter Charalambous MD1, Solon Thanos MD, PhD 1

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Institute of Experimental Ophthalmology, University of Muenster, Muenster, Germany

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Department of Ophthalmology, University of Athens, Athens, Greece

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Corresponding author: Marilita M Moschos MD, PhD 6, Ikarias Str. Ekali Athens 14578 Tel. ++30 2104122139 FAX ++30 210 4113563 Email [email protected] Word count: 2184

Conflict of interest: The authors declare no conflict of interest. Financial support: None.

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ABSTRACT Purpose. The purpose of our study was to investigate the effect of an inadvertent intravitreal injection of botulinum toxin A (BTA) on the intraocular pressure (IOP) and the retinal ganglion cells (RGCs) in an animal model. Methods Intravitreal injection of 10L in two doses, 5 and 10 IU, were given into the left eye of two groups of Sprague-Dawleys rats. The same volume of balanced salt solution (BSS) was injected in a third group of rats. Each group consisted of six rats, with normal IOP. IOP was measured preoperatively as well as one week, two weeks and four weeks postoperatively. Retinas were stained in vivo using a retrograde labelled technique. Afterwards, retinas were microsurgically removed and the density of RGCs was determined under fluorescent microscopy. Subsequently, immunohistochemistry was performed using antibodies against rhodopsin as well as against the retinal glial fibrillary acidic protein (GFAP) which is an indirect marker of

ganglion cell death.

Results. Over an observation of four weeks, we documented an IOP decrease in both BTA groups and an IOP increase in BSS group. The differences were not statisticall significant (Kruskal-Wallis test p>0.05). All retinas displayed the same immunostaining pattern for rhodopsin. Minimal signals for GFAP were detected in all

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Conclusions. Our findings indicate that diffusion of BTA or even inadvertent intravitreal injection of BTA in neuroophthalmological patients is probably without a severe impact on the IOP or a toxic effect on the RGCs.

Key words: botulinum toxin A, rat, intraocular pressure, retinal ganglion cells

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Introduction Botulinum toxin A (BTA) is a potent neurotoxin used to treat a variety of neurological and neuro-ophthalmological conditions such as paralytic strabismus, hemifacial spasm and blepharospasm, as well as thyroid-associated orbitopathy.1,2 The risk of inadvertent intraocular BTA injection, though minimal, is not neglectable, in particular for the unexperienced neurologist. Scleral perforation and vitreous haemorrhage have been reported after BTA injections of extraocular muscles for strabismus.3,4 Moreover, the potential ocular perforation through tissue infiltration after repeated BTA injections of extraocular muscles in patients who undergo a systematic therapy increases the interest for the influence of BTA on ocular physiology. In the present study we investigated the effect of BTA on intraocular pressure and retinal ganglion cells using an animal model.

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Methods

Eight-week-old female Sprague Dawleys rats with an average body weight of 200 g and normal IOP were anesthetized by a peritoneal injection of a mixture of ketamine (40 mg/kg) and xylazine (10 mg/kg). The pupils were dilated with Mydriaticum 0,1%. Under an operation microscope, with the aid of a glass micropipette, 10L of two doses of BTA (5 and 10 IU) were injected in the vitreous of the left rat eye in two groups with six rats each. The same amount of balanced salt solution (BSS-sham condition) was injected intravitreally in the left eye of six additional rats serving as control group. Before the BTA and saline injection, an equal quantity of aqueous

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humor was aspired from the anterior chamber. Postoperatively, gentamycin ointment was applied once. The intraocular pressure (IOP) was measured at 8 pm with the aid of a Tono-Pen XL preoperatively as well as one week, two weeks, and four weeks postoperatively. Ten IOP readings were recorded each time and the average value was estimated. All procedures were performed in accordance with the ARVO guidelines on the use of animals in research. After the fourth postoperative week all rats were euthanized and the eyes were removed surgically. After retinas were microsurgically isolated, they were frozen en block in liquid nitrogen, transferred to the cryostat (Leika Jung CM 1500; Leica Microsystems, Wetzlar, Germany) and cut into midsagittal frozen sections (12m thick). Sections were collected on clean gelatine-coated glass slides and dried for 2 hours at room temperature, fixed in cold methanol 99% for 10 in -20 washed C, three times for 5 min each in phosphate-buffered saline (PBS) and blocked with 10% fetal calf serum (FCS) for 30 min. Immunofluorescence staining was performed using a primary antibody against rhodopsin (polyclonal rabbit anti-rhodopsin antibody; Novus Biologicals, Littleton, CO, USA) and glial fibrillary acidic protein (GFAP) (polyclonal rabbit anti-GFAP, Novus Biologicals, Littleton, CO, USA). GFAP is a marker of glial formation and therefore its expression indicates indirectly the presence of retinal ganglion cell death. Primary antibodies were diluted in 10% FCS (1:100) and the sections incubated overnight at 4 After rinsing the slides three C. times for 5 min each in PBS, the sections were incubated for 30 with the secondary antibodies (goat anti-rabbit Cy2-conjugated antibody; Abcam Inc., Cambridge, MA, USA). Thereafter, it was once again washed three times for 5 each in PBS and finally mounted in anti-fading solution for fluorescence microscopy (Mowiol; Merck, Darmstadt, Germany) containing bisbenzimide H 33258 (DAPI; Sigma, St. Louis,
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MO, USA) and observed with the aid of a fluorescence microscope (Axiophot; Carl Zeiss Meditec, Oberkochen, Germany) using appropriate filters (395-440 nm for Dapi and 450-490nm for Cy2). Photography was achieved digitally with a camera attached to the microscope (AxioCam; Carl Zeiss Meditec, Oberkochen, Germany). Retinal ganglion cells (RGCs) were retrogradely labelled with the fluorescent dye Fluoro-Gold (Abcam, MA, USA) by injection into the superior colliculus contralateral to the experimental eye, as previously described.5 This technique enabled the exclusive labelling of RGCs in a uniform manner across the entire retina, thus facilitating their quantification on retinal flatmounts. Six rats of each group were used in every single experiment. Animals were euthanized by CO2 inhalation 7 days after the retrograde labelling, which is the time required for the fluorescent dye to be transported from the axonal terminals of RGCs to their somas in the retina. Subsequently the eyes were enucleated and retinas were microsurgically isolated. Afterwards, flatmounts retinal were fixed in 4% paraformaldehyde overnight at 4 and assayed for RGC density. Cells were visualized under fluorescence microscopy (Axiophot; Carl Zeiss Meditec, Oberkochen, Germany). Five areas per retinal quadrant, each corresponding to an area of 0.098 mm2, at five different eccentricities of the retinal radius were examined and micro-photographs were obtained. The optic disc served as the point of reference. Micro-photographs were analysed using a computer-based analysis program (AlphaEaseFC by Alpha Innotech Corporation, CA, USA) and RGCs counts were calculated as mean value SD for every quadrant. For each retina, the total number of ganglion cells counted was divided by the area analyzed in order to determine the ganglion cell density (RGCs per square millimeter) for this retina. The average density of RGCs was compared between all groups.

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Statistical analysis was performed with Kruskal-Wallis test and results were considered significant for p<0.05. All data for RGCs quantification are expressed as mean valueSD.

Results The mean IOP values in all groups are shown in Table 1. The baseline IOP was not significantly different between the three groups (p>0.05). The IOP decrease in both study groups and the IOP increase in the control group were not significant (all p>0.05). The IOP decrease was not dosage-dependent (all p>0.05). All three groups displayed a strong signal for rhodopsin located in the photoreceptor layer (Fig. 1B-C, 1E-F). Immunostaining for GFAP was minimal in all groups, restricted in the ganglion cell layer as expected (Fig. 1H-I, 1K-L). A total of 192446 mm-2 RGCs were labeled in the sham group compared to 189415 mm-2 in the 5IU BTA-group and 187052 mm-2 in the 10IU BTA-group (Fig. 2). The differences were not statistically significant (all p>0.05).

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Discussion

The main finding of this study was that IOP is not significantly changed by intravitreal injection of BTA in the normotensive eye. This suggests that an inadvertent injection of BTA is probably not harmful with respect to IOP. Furthermore, the intravitreal injection of BTA did not induce marked retinal ganglion cell death, and thereby it did not produce significant glial formation. Cholinergic receptors (muscarinic receptors as well as nicotinic receptors) have been detected in the human cornea, iris-ciliary body complex, lens, and retina, determining a variety of ocular functions such as eye growth, tears fluid production, lens cell signalling, and phototransduction.6,7 The presence of muscarinic receptors in
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the trabecular meshwork tissue has been also demonstrated; muscarinic agonists increase outflow facility in human eye by a direct stimulation of the outflow tissues even in the absence of an intact ciliary body.8 Recently, we documented that the trabecular expression of cholinergic receptors is significantly reduced in a genetic model of congenital glaucoma.9 Moreover, Ellis et al. stated that muscarinic agonists reduce IOP not only by ciliary muscle contraction and increased outflow but also by decreasing aqueous humor production through a Na, K-ATPase inhibition in the ciliary body, modulated by acetylcholine.10 Also, BTA inhibits presynaptic cholinergic transmission when injected intramuscularly causing a temporary flaccid paralysis lasting 6 to 8 weeks.11

In our experiment, we injected BTA intravitreally in normotensive rat eyes, simulating an inadvertent ocular perforation after therapeutic use of BTA injection. In the BTA-injected rats, the IOP was slightly reduced; the IOP difference within and between the study groups was not statistically significant. In the sham condition group, a slight IOP increase occurred; we attributed this statistically insignificant increase to surgical manipulations. Our results are in agreement with Wienkers et al. who did not, however, examine different doses of the toxin and their dose of 0.5 ng was relatively small.12 It seems that the cholinergic regulation of the ciliary trabecular meshwork may have differential levels of control maintaining a remarkable IOP balance under modified cholinergic activation. This finding suggests that a potential inadvertent ocular perforation when performing a therapeutic BTA periorbital injection as well as the BTA diffusion after uneventful injection do not probably have a relevant impact on the IOP. Finally, our immunostaining experiments revealed a normal pattern for rhodopsin in all rat retinas treated with BTA, wheareas the GFAP signal was comparable to that of control rat retinas, indicating that BTA injections in standard
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dosage do not induce glial formation in the photoreceptor layer or in the retinal ganglion cell layer. Previous animal studies had already shown that an intravitreal injection of BTA does not affect visual evoked potentials and electroretinography, evidence which support of our results.13,13,15 Retrograde RGCs labelling confirmed that the density of RGCs in retinas treated with BTA was comparable to that of normal controls. In conclusion we suggest that the therapeutic periorbital and periocular use of BTA in movement disorders or similar neuroophthalmological dysfunctions is safe with regard to integrity and vitality of retinal ganglion cells. Nevertheless, further basic research studies are required in order to evaluate the effect of BTA on the retinal structure and function.

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References

1. Boulos PR, Hardy I. Thyroid-associated orbitopathy: a clinicopathologic and therapeutic review. Curr Opin Ophthalmol 2004;15:389-400. 2. ODay J. Use of botulinum toxin in neuro-ophthalmology. Curr Opin Ophthalmol 2001;12:419-422

3. Murray T. Strabismus: Challenges and trends. Eye 1993;7:332-340. 4. Liu M, Lee HC, Hertle RW, Ho AC. Retinal detachment from inadvertent intraocular injection of botulinum toxin A. Am J Ophthalmol 2004;137:201-202. 5. Naskar R, Wissing M, Thanos S. Detection of early neuron degeneration and accompanying microglial responses in the retina of a rat model of glaucoma. Invest Ophthalmol Vis Sci 2000;43:2962-2968

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6. Sastry BV. Cholinergic systems and multiple cholinergic receptors in ocular tissues. J Ocul Pharmacol 1985;1:201-226.

7. Dunkan G, Collison DJ. Role of the non-neuronal cholinergic system in the eye: a review. Life Sci 2003;72:2013-2019. 8. Erickson KA, Schroeder A. Direct effects of muscarinic agents on the outflow pathways in human eyes. Invest Ophthalmol Vis Sci 2000;41:1743-1748. 9. Gatzioufas Z, Charalambous P, Seitz B, et al. Cholinergic inhibition by botulinum toxin in a rat model of congenital glaucoma raises intraocular pressure. Br J Ophthalmol 2008;92:826-31.

10. Ellis DZ, Nathanson JA, Rabe J, Sweadner KJ. Carbachol and nitric oxide inhibition of Na, K-ATPase activity in bovine ciliary processes. Invest Ophthalmol Vis Sci 2001;42:2625-2631.

11. Meunier F. A., Schiavo G. and Molgo J. Botulinum neurotoxins: from paralysis to recovery of functional neuromuscular transmission. J Physiol Paris 2002;96:105 113.

12. Wienkers K, Helveston EM, Ellis FD, Cadera W. Botulinum toxin injection into rabbit vitreous. Ophthalmic Surg 1984;15:310-314.

13. Hoffman RO, Archer SM, Zirkelbach SL, Helveson EM. The effect of intravitreal botulinum toxin on rabbit visual evoked potential. Ophthalmic Surg 1987;18:118119. 14. Kutluk S, Akar S, Topcu M, Kural G. Effect of botulinum toxin injections into rabbit eyes. Strabismus 1999;7:221-226. 15. Li S, Mizota A, Adachi-Usami E. Effects of intravitreal injection of botulinum toxin on the electroretinogram of rats. Ophthalmic Res 1999;31:392-398.

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Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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Legends

Figure 1. Immunohistochemical detection of rhodamin and glial fibrillary acidic protein (GFAP) in rat retina after intravitreal injection of botulinum toxin A (BTA) and balanced salt solution (BSS), respectively. B-rh: rhodamin signal in rat retina after injection with BTA 10IU, S-rh: rhodamin signal in rat retina after injection with BSS (sham condition), B-GFAP: GFAP signal in rat retina after injection with BTA 10IU, SGFAP: GFAP signal in rat retina after injection with saline, GCL: ganglion cell layer, IPL: inner plexiform layer, INL: inner nuclear layer. OPL: outer plexiform layer, ONL: outer nuclear layer, PRL: photoreceptor layer. Cell nuclei are stained blue (Dapi). Positive immunolabeling appears green (Cy2). Rat retinas treated with BTA demonstrated a strong signal for rhodamin in the photoreceptor layer (Fig. 1B-C), as well as rat retinas treated with BSS (Fig. 1E-F). GFAP immunostaining was limited in the ganglion cell layer in both groups, as expected (Fig. 1H-I and 1K-L). Control retinas were not treated with the primary antibodies and therefore showed no signal

(Fig 1N-O). Scale bar: 100m.

Figure 2. Retrogradely labelled retinal ganglion cells (RGCs) by fluorescent dye in rats after intravitreal injection of balanced salt solution (BSS-control) as well as in rats after intravitreal injection of botulinum toxin A (BTA) 5IU and 10IU respectively. C: control group, BTA: botulinum toxin A, 5IU: BTA dosage of 5 IU, 10IU: BTA dosage of 10 IU, RGCs density: number of retinal ganglion cells per mm2. The density of RGCs was 192446 mm-2 in control group (Fig. 2A and 2D), 189415 mm-2 in the 5IU BTA-group (Fig. 2B and 2E) and 187052 mm-2 in the 10IU BTA-group (Fig. 2C and 2F). The differences were not statistically significant (Kruskal-Wallis test, all
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p>0.05). RGCs density is expressed in number of cells mm-2 (meanSD). A retinal flatmount is demonstrated in Fig. 2G. Magnification x 20 in Fig. 2A, 2B and 2C. Magnification x 40 in Fig. 2D, 2E and 2F.

Table 1. Mean value of the IOP (mmHg) in three groups of rats before and after intravitreal injection of balanced salt solution (sham condition) or botulinum toxin A (BTA) in two different dosages (5 IU and 10 IU respectively). The baseline IOP was not significantly different between the three groups (Kruskal-Wallis test, all p>0.05).

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Table 1. Mean value of the IOP (mmHg) in three groups of rats before and after intravitreal injection of balanced salt solution (sham condition) or botulinum toxin A (BTA) in two different dosages (5 IU and 10 IU respectively). The baseline IOP was not significantly different between the three groups (Kruskal-Wallis test, all p>0.05).

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