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Method Article

Müller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

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DOI:

10.3791/56249

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October 27th, 2017

 ,  ,  ,  , 
1Department of Ophthalmology, University Hospital of Bern, University of Bern, 2Department of Clinical Research, University of Bern, 3Graduate School for Cellular and Biomedical Sciences, University of Bern

In This Article

Summary

The zebrafish is a popular animal model to study mechanisms of retinal degeneration/regeneration in vertebrates. This protocol describes a method to induce localized injury disrupting the outer retina with minimal damage to the inner retina. Subsequently, we monitor in vivo the retinal morphology and the Müller glia response throughout retinal regeneration.

Abstract

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after severe damage. Investigating the regeneration pathways in zebrafish might bring new insights to develop innovative strategies for the treatment of retinal degenerative diseases in mammals. Herein, we focused on the induction of a focal lesion to the outer retina in adult zebrafish by means of a 532 nm diode laser. A localized injury allows investigating biological processes that take place during retinal degeneration and regeneration directly at the area of damage. Using non-invasive optical coherence tomography (OCT), we were able to define the location of the damaged area and monitor subsequent regeneration in vivo. Indeed, OCT imaging produces high-resolution, cross-sectional images of the zebrafish retina, providing information which was previously only available with histological analyses. In order to confirm the data from real-time OCT, histological sections were performed and regenerative response after the induction of the retinal injury was investigated by immunohistochemistry.

Introduction

Vision is probably the most essential sense of the human being and its impairment has a high socio-economic impact. In the industrialized world, retinal degenerative diseases account for the majority of vision loss and blindness among the adult population1. Retinitis pigmentosa (RP) is the most common inherited cause of blindness in people between the ages of 20 and 60, affecting approximately 1.5 million people worldwide2,3. It is a heterogeneous family of inherited retinal disorders characterized by progressive loss of the photoreceptors (PRs) followed by degeneration of retinal pigment epithelium and, subsequently, gliosis and remodeling of inner neurons4. The course of the disease can be explained by the incremental loss of the two PR cell types, usually starting with rods, which are responsible for achromatic vision in dim light, and cones, which are essential for color vision and visual acuity5. A single gene defect is sufficient to cause RP. So far more than 130 mutations in over 45 genes have been associated with the disease6. This leads to varying disease phenotypes and is one reason that gene therapy is non-generalizable and thus an intricate therapeutic approach. Therefore, there is an urgent need to develop new general therapeutic approaches to treat retinal degenerations in blinding diseases.

Retinal degeneration often involves PR loss; therefore, PR cell death is a hallmark of the degenerative processes in the retina7. It has already been demonstrated that PR cell death stimulates Müller glia cell (MC) activation and proliferation8. MCs, the major glial cell type in the vertebrate retina, were once considered to be nothing more than a "glue" between retinal neurons. In recent years, many studies have shown that MCs act as more than mere structural support9. Among the different functions, MCs participate also in neurogenesis and repair10. Indeed, in response to diffusible factors from the degenerating retina, MCs significantly increase glial fibrillary acidic protein (GFAP) expression. Therefore, GFAP labelling can be used as a marker for MC activation as a secondary response to retinal injury and degeneration11.

Recently, we developed a novel adaptation of focal injury using a laser to induce retinal degeneration in zebrafish (Danio rerio). Focal injury is advantageous for studying certain biological processes such as the migration of cells into the injured site and the precise timing of events that take place during retinal regeneration12. Furthermore, the zebrafish has become important in visual research because of the similarities between its visual system and that of other vertebrates. Gross morphological and histological features of human and teleost retinae display few differences. Accordingly, human and zebrafish retinae contain the same major cell classes organized in the same layered pattern, where light-sensing photoreceptors occupy the outermost layer, while the retinal projection neurons, the ganglion cells, reside in the innermost neuronal layer, proximal to the lens. The retinal interneurons, the amacrine, bipolar, and horizontal cells, localize in between the photoreceptor and ganglion cell layers13. Furthermore, the zebrafish retina is cone-dominated and therefore closer to the human retina than, for example, the intensively studied rodent retina. A fascinating difference between teleost and mammals is the persistent neurogenesis in fish retina and retinal regeneration after damage. In zebrafish, MCs can dedifferentiate and mediate regeneration in injured retina14,15. In chicken, MCs have some capacity also to re-enter the cell cycle and to dedifferentiate. Following retinal injury in adult fish, MCs adopt certain characteristics of progenitor and stem cells, migrate to the damaged retinal tissue and produce new neurons16. Gene expression profiling of mammalian MCs revealed unexpected similarities to retinal progenitors, and evidence for intrinsic neurogenic potential of MCs in the chicken, rodent, and even human retina is growing17. Nevertheless, why the regenerative response in birds and mammals is lower compared with the robust response in fish is not yet understood. Therefore, understanding the endogenous repair mechanisms in zebrafish may suggest strategies for stimulating retinal regeneration in mammals and humans. Employing the endogenous repair mechanism of MCs as a therapeutic tool for the treatment of patients with retinal degeneration would have an outstanding impact for our society.

Herein, we provide the steps necessary to employ the degeneration/regeneration model in ophthalmic research. We focused first on inducing focal damage in the neurosensory retina, then on the imaging of events developing at the injury site, and finally visualizing involvement of the adjacent MCs. The general protocol is relatively easy to perform and opens a wide variety of possibilities for evaluating the retina afterwards.

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Protocol

All experiments adhered to the Statement for the Use of Animals in Ophthalmic and Vision Research of the Association for Research in Vision and Ophthalmology (ARVO) and respect the related regulations of the governmental authorities.

1. Animals

  1. Maintain TgBAC (gfap:gfap-GFP) zebrafish 167 (AB) strain aged between 6 - 9 months under standard conditions in water with a temperature of 26.5 °C and a 14/10 h light/dark cycle18.
  2. Follow the animal care guidelines of the involved institutions for the animal experiments after approval by the governmental authorities.

2. Reversible Systemic Anesthesia

  1. Prepare the stock solution of ethyl 3-aminobenzoate methanesulfonate salt (tricaine) by dissolving 400 mg of tricaine powder in 97.9 mL of tank water and 2.1 mL of 1 M of Tris buffered saline (TBS). Adjust to pH 7.0 with 1 M Tris (pH 9) and store at 4 °C in the dark up to one month.
    NOTE: Tricaine should be prepared in water like the natural living conditions of the animal, preferably original tank water should be used.
  2. Dilute the stock solution 1:25 in tank water and use immediately.
  3. Place the zebrafish into a 10 cm Petri dish containing 50 mL of anesthesia solution until they become immobile and do not respond to external stimuli (approximately 2 - 5 min, depending on weight and age).
  4. Transfer each fish by hand to a custom-made silicone pin holder for laser treatment (Figure 1A).
    CAUTION! The fish can remain anesthetized outside of the tank for up to 10 min only.
  5. To reverse anesthesia after the treatment and/or imaging, place the zebrafish in container containing tank water.
  6. To support recovery, create a flow of fresh tank water over the gills by moving the zebrafish back and forth in the water.

3. Laser Focal Injury on Retina

NOTE: A 532 nm diode laser is used to create focal light damage on the retina of the zebrafish. The experimental set-up of the laser enables the establishment of a reproducible focal retinal injury in adult zebrafish.

  1. Set up the output power of the laser: 70 mW; aerial diameter: 50 µm; pulse duration: 100 ms.
    CAUTION! The use of laser light requires appropriate personal protection and labelling of the area.
  2. Apply 1 - 2 drops of 2% hydroxypropylmethylcellulose topically in the eye before treatment and use a 2.0 mm fundus laser lens to focus the laser-aiming beam on the retina.
    CAUTION! hydroxypropylmethylcellulose drops are viscous and may cause problems in breathing if it goes on the gills.
  3. Place four laser spots around the optic nerve on the left eye and use the right, untreated eye as internal control.

4. In vivo Imaging of the Retinal Morphology

  1. On day 0, visualize the zebrafish retina directly after the laser induction without reviving them from anesthesia. At all other time points, employ general anesthesia (see Section 2: Reversible systemic anesthesia). Place the immobilized zebrafish on a custom-made silicone pin holder (Figure 1B, B.1).
  2. To obtain optimal images, cut a commercially available hydrogel contact lens to fit the zebrafish eye (Ø = 5.2 mm, r = 2.70 mm, center thickness = 0.4 mm) by means of a hole punch. Fill the concave surface of the lens with methylcellulose and place it over the cornea.
  3. Equip the OCT system with a 78D non-contact slit lamp lens.
  4. Focus the infrared (IR) image in the IR + OCT mode (Figure 2A) to visualize the fundus of the eye and take the IR pictures by clicking the "Acquire" button (Figure 2B) to localize the laser spots on the retina using the system's software.
  5. Visualize a three-dimensional section of the retinal layers in the IR + OCT mode and take the pictures by clicking the "Acquire" button (Figure 2B). Observe the severity of injury in the outer nuclear layer (ONL) (see Section 3: Laser focal injury on retina) in these images.
  6. To reverse anesthesia after the treatment and/or imaging place the zebrafish in a container containing tank water.
  7. To support recovery, create a flow of fresh tank water over the gills by moving the zebrafish back and forth in the water.
  8. Perform similar in vivo imaging of retinal morphology on day 1, 3, 7, 14 and week 6.

5. Hematoxylin & Eosin (H&E) Staining

  1. Euthanize zebrafish by submersion into cold (4 °C) anesthesia solution on ice for at least 10 min and enucleate the eyes immediately by means of small curved forceps.
  2. Fix the whole eyes in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) at 4 °C for 20 h and then dehydrate the samples in a graded alcohol series (xylene 100% for 5 min twice, ethanol 100% for 5 min twice, ethanol 96% for 3 min twice and ethanol 70% 3 min once).
    CAUTION! PFA can be irritating to the eyes, nose, and upper respiratory track. PFA is a known human carcinogen and a suspected reproductive hazard.
  3. Embed the samples in paraffin, cut 5 µm sections at the level of the optic nerve head and mount them on glass slides.
  4. Stain the deparaffinized sections with 0.1% acid hematoxylin solution for 5 min and dip the slides two times in distilled water after dipping the slides in hydrochloric acid mix (2 mL 25% HCl in 250 mL distilled water) and ammonia mix (2 mL 25% ammonia in 250 mL distilled water). Stain the sections with eosin G aqueous solution 0.5% for 3 min after development of the hematoxylin staining by tap water for at least 10 min.
  5. Mount the dehydrated slides in acrylic resin mounting medium and observe the slides under the light microscope.

6. Immunohistochemistry for the MC Activation

  1. Heat the deparaffinized sections in antigen retrieval buffer (Tris-EDTA + 0.05% non-ionic detergent, pH 9.0) for 3 min in an appropriate steamer or a microwave for 10 - 15 min and wash three times with TBS for 5 min each.
  2. Circle the sections with a silicone pen and add 100 µL blocking solution (TBS + 10% goat normal serum + 1% bovine serum albumin, pH 7.6) at room temperature for 1 h.
  3. Stain with anti-glial fibrillary acidic protein (GFAP) rabbit polyclonal antibody and with anti-glutamine synthetase (GS) mouse monoclonal antibody, both in a 1:200 dilution (40 µL per sample). Incubate the slide in a humidified chamber at 4 °C overnight. Wash three times with TBS + 0.1 % Tween 20 for 5 min each.
  4. Finish visualization with the appropriate secondary antibodies. This protocol used a goat anti-rabbit IgG H&L green secondary antibody for GFAP and a goat anti-mouse IgG H&L bright red for GS, both in a 1:500 dilution at room temperature for 1 h.
  5. Mount the slides with mounting medium containing DAPI and observe the slides under the fluorescence microscope.

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Results

Real-time OCT: In order to analyze the role of MCs in retinal repair, we used a laser injury model inducing a well-delineated zone of damage in the zebrafish retina. The site of damage was imaged by means of in vivo OCT for the first time (Day 0) within 60 minutes following the injury (Figure 3). To compensate for the optics of the fish eye, a custom-made contact lens was placed on the cornea. Immediately after laser treatment, a dif...

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Discussion

Retinal regeneration/degeneration in the zebrafish has been investigated by different approaches such as cytotoxin-mediated cell death22, mechanical injury23, and thermal injury24. We employed a 532 nm diode laser to damage the zebrafish retina. Thereby, our model offers several advantages. For instance, we rapidly created a well-defined area of injury localized in the outer retina, specifically in the PRs layer. Furthermore, this experimental set-up...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

We thank Martin Zinkernagel, MD, PhD and Miriam Reisenhofer, PhD for her scientific input at establishing the model and Federica Bisignani for her excellent technical assistance.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Acid hematoxylin solutionSigma-Aldrich, Buchs, Switzerland2852
AlbuminSigma-Aldrich, Buchs, SwitzerlandA07030
Bovine serum albumin (BSA)Sigma-Aldrich, Buchs, Switzerland5470
Dako PenDako, Glostrup, DanmarkS2002
DAPI mounting mediumVector Labs, Burlingame, CA, USAH-1200
Eosin G aqueous solution 0.5%Carl Roth, Arlesheim, SwitzerlandX883.2
EthanolSigma-Aldrich, Buchs, Switzerland2860
Ethylene diamine tetraacetic acid (EDTA)Sigma-Aldrich, Buchs, SwitzerlandED
EukittSigma-Aldrich, Buchs, Switzerland3989
Goat anti-rabbit IgG H&L Alexa Fluor® 488Life Technologies, Zug, SwitzerlandA11008
Goat anti-mouse IgG H&L Alexa Fluor® 594Life Technologies, Zug, SwitzerlandA11020
Goat normal serumDako, Glostrup, DanmarkX0907
Hydrogel contact lensJohnson & Johnson AG, Zug, Switzerlandn.a.1-Day Acuvue Moist
Hydroxypropylmethylcellulose 2%OmniVision, Neuhausen, Switzerlandn.a.Methocel 2%
Ethyl 3-aminobenzoate methanesulfonateSigma-Aldrich, Buchs, SwitzerlandA5040Tricaine, MS-222
Visulas 532sCarl Zeiss Meditec AG, Oberkochen, Germanyn.a.532 nm laser
Mouse anti-GS monoclonal antibodyMillipore, Billerica, MA, USAMAB302
HRA + OCT Imaging SystemHeidelberg Engineering, Heidelberg, Germanyn.a.Spectralis
Heidelberg Eye ExplorerHeidelberg Engineering, Heidelberg, Germanyn.a.Version 1.9.10.0
Paraformaldehyde (PFA)Sigma-Aldrich, Buchs, SwitzerlandP5368
Phosphate buffered saline (PBS)Sigma-Aldrich, Buchs, SwitzerlandP5368
Rabbit anti-GFAP polyclonal antibodyInvitrogen, Waltham, MA, USA180063
Silicone pin holderHuco Vision AG Switzerlandn.a.Cut by hand from silicone pin mat of the sterilization tray accordingly.
Slit lamp BM900Haag-Streit AG, Koeniz, Switzerlandn.a.
Slit lamp adapterIridex Corp., Mountain View, CA, USAn.a.
Superfrost Plus glass slidesGehard Menzel GmbH, Braunschweig, Germany10149870
TgBAC (gfap:gfap-GFP) zf167 (AB) strainKIT, Karlsruhe, Germany15204http://zfin.org/ZDB-ALT-100308-3
Tris buffered saline (TBS)Sigma-Aldrich, Buchs, SwitzerlandP5912
Tween 20Sigma-Aldrich, Buchs, SwitzerlandP1379
78D non-contact slit lamp lensVolk Optical, Mentor, OH, USAV78C
XyleneSigma-Aldrich, Buchs, Switzerland534056
Ocular fundus laser lensOcular Instruments, Bellevue, WA, USAOFA2-0
2100 RetrieverAptum Biologics Ltd., Southampton, United KingdomR2100-EUSteamer

References

  1. Haddad, S., Chen, C. A., Santangelo, S. L., Seddon, J. M. The genetics of age-related macular degeneration: a review of progress to date. Surv. Ophthalmol. 51 (4), 316-363 (2006).
  2. Stefano Ferrari, S., Di Iorio, E., Barbaro, V., Ponzin, D., Sorrentino, F. S., Parmeggiani, F. Retinitis Pigmentosa: Genes and Disease Mechanisms. Curr Genomics. 12 (4), 238-249 (2011).
  3. Berson, E. L. Retinitis pigmentosa. The Friedenwald Lecture. Invest Ophthalmol Vis Sci. 34 (5), 1659-1676 (1993).
  4. Strettoi, E. A Survey of Retinal Remodeling. Front Cell Neurosci. 9, 494(2015).
  5. Hartong, D. T., Berson, E. L., Dryja, T. P. Retinitis pigmentosa. Lancet. 368, 1795-1809 (2006).
  6. Wang, D. Y., Chan, W. M., Tam, P. O., Baum, L., Lam, D. S., Chong, K. K., Fan, B. J., Pang, C. P. Gene mutations in retinitis pigmentosa and their clinical implications. Clin Chim Acta. 351 (1-2), 5-16 (2005).
  7. Pierce, E. A. Pathways to photoreceptor cell death in inherited retinal degenerations. BioEssays. 23, 605-618 (2001).
  8. Tackenberg, M. A., Tucker, B. A., Swift, J. S., Jiang, C., Redenti, S., Greenberg, K. P., Flannery, J. G., Reichenbach, A., Young, M. J. Muller cell activation, proliferation and migration following laser injury. Mol. Vis. , 1886-1896 (2009).
  9. Newman, E., Reichenbach, A. The Müller cell: a functional element of the retina. Trends Neurosci. 19 (8), 307-312 (1996).
  10. Kubota, R., Hokoc, J. N., Moshiri, A., McGuire, C., Reh, T. A. A comparative study of neurogenesis in the retinal ciliary marginal zone of homeothermic vertebrates. Brain Res Dev Brain Res. 134, 31-41 (2002).
  11. Zhao, T. T., Tian, C. Y., Yin, Z. Q. Activation of Müller cells occurs during retinal degeneration in RCS rats. Adv Exp Med Biol. 664, 575-583 (2010).
  12. DiCicco, R. M., Bell, B. A., Kaul, C., Hollyfield, J. G., Anand-Apte, B., Perkins, B. D., Tao, Y. K., Yuan, A. Retinal Regeneration Following OCT-Guided Laser Injury in Zebrafish. Invest Ophthalmol Vis Sci. 55 (10), 6281-6288 (2014).
  13. Bilotta, J., Saszik, S. The zebrafish as a model visual system. Int. J. Dev. Neurosci. , 621-629 (2001).
  14. Fausett, B. V., Goldman, D. A role for alpha1 tubulin-expressing Müller glia in regeneration of the injured zebrafish retina. J Neurosci. 26 (23), 6303-6313 (2006).
  15. Yurco, P., Cameron, D. A. Responses of Müller glia to retinal injury in adult zebrafish. Vision Res. 45, 991-1002 (2005).
  16. Ashutosh, P. J., Roesch, K., Cepko, C. L. Development and neurogenic potential of Müller gial cells in the vertebrate retina. Prog Retin Eye Res. 28 (4), 249-262 (2009).
  17. Xia, X., Ahmad, I. Unlocking the Neurogenic Potential of Mammalian Müller Glia. Int J Stem Cells. 9 (2), 169-175 (2016).
  18. Brand, M., Granato, M., Nüsslein-Volhard, C. Keeping and raising zebrafish. Zebrafish: A Practical Approach. Nüsslein-Volhard, C., Dahm, R. , IRL Press. 7-38 (2002).
  19. Riepe, R. E., Norenburg, M. D. Müller cell localisation of glutamine synthetase in rat retina. Nature. 268 (5621), 654-655 (1977).
  20. Derouiche, A., Rauen, T. Coincidence of L-glutamate/L-aspartate transporter (GLAST) and glutamine synthetase (GS) immunoreactions in retinal glia: evidence for coupling of GLAST and GS in transmitter clearance. J Neurosci Res. 42 (1), 131-143 (1995).
  21. Bignami, A., Dahl, D. The radial glia of Müller in the rat retina and their response to injury. An immunofluorescence study with antibodies to the glial fibrillary acidic (GFA) protein. Exp Eye Res. 28 (1), 63-69 (1979).
  22. Sherpa, T., Fimbel, S. M., Mallory, D. E., Maaswinkel, H., Spritzer, S. D., Sand, J. A., Li, L., Hyde, D. R., Stenkamp, D. L. Ganglion cell regeneration following whole-retina destruction in zebrafish. Dev Neurobiol. 68 (2), 166-181 (2008).
  23. Cameron, D. A., Carney, L. H. Cell mosaic patterns in the native and regenerated inner retina of zebrafish: implications for retinal assembly. J Comp Neurol. 416 (3), 356-367 (2000).
  24. Raymond, P. A., Barthel, L. K., Bernardos, R. L., Perkowski, J. J. Molecular characterization of retinal stem cells and their niches in adult zebrafish. BMC Dev Biol. 6, 36(2006).
  25. Bailey, T. J., Davis, D. H., Vance, J. E., Hyde, D. R. Spectral-domain optical coherence tomography as a noninvasive method to assess damaged and regenerating adult zebrafish retinas. Invest Ophthalmol Vis Sci. 53 (6), 3126-3138 (2012).
  26. Koinzer, S., Saeger, M., Hesse, C., Portz, L., Kleemann, S., Schlott, K., Brinkmann, R., Roider, J. Correlation with OCT and histology of photocoagulation lesions in patients and rabbits. Acta Ophthalmol. 91 (8), e603-e611 (2013).
  27. Wan, J., Zheng, H., Chen, Z. L., Xiao, H. L., Shen, Z. J., Zhou, G. M. Preferential regeneration of photoreceptor from Müller glia after retinal degeneration in adult rat. Vision Res. (2), 223-234 (2008).
  28. Thomas, J. L., Thummel, R. A novel light damage paradigm for use in retinal regeneration studies in adult zebrafish. J Vis Exp. (80), e51017(2013).

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Zebrafish RetinaLaser InjuryOptical Coherence TomographyRetinal RegenerationImmunohistochemistryH E StainingTricaine AnesthesiaFundus Laser LensGlial Fibrillary Acidic Protein
Müller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish
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