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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 worldwide^2,3. 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 retina^14,15. 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.

Protokół

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.

Wyniki

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...

Dyskusje

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...

Materiały

Name	Company	Catalog Number	Comments
Acid hematoxylin solution	Sigma-Aldrich, Buchs, Switzerland	2852
Albumin	Sigma-Aldrich, Buchs, Switzerland	A07030
Bovine serum albumin (BSA)	Sigma-Aldrich, Buchs, Switzerland	5470
Dako Pen	Dako, Glostrup, Danmark	S2002
DAPI mounting medium	Vector Labs, Burlingame, CA, USA	H-1200
Eosin G aqueous solution 0.5%	Carl Roth, Arlesheim, Switzerland	X883.2
Ethanol	Sigma-Aldrich, Buchs, Switzerland	2860
Ethylene diamine tetraacetic acid (EDTA)	Sigma-Aldrich, Buchs, Switzerland	ED
Eukitt	Sigma-Aldrich, Buchs, Switzerland	3989
Goat anti-rabbit IgG H&L Alexa Fluor® 488	Life Technologies, Zug, Switzerland	A11008
Goat anti-mouse IgG H&L Alexa Fluor® 594	Life Technologies, Zug, Switzerland	A11020
Goat normal serum	Dako, Glostrup, Danmark	X0907
Hydrogel contact lens	Johnson & Johnson AG, Zug, Switzerland	n.a.	1-Day Acuvue Moist
Hydroxypropylmethylcellulose 2%	OmniVision, Neuhausen, Switzerland	n.a.	Methocel 2%
Ethyl 3-aminobenzoate methanesulfonate	Sigma-Aldrich, Buchs, Switzerland	A5040	Tricaine, MS-222
Visulas 532s	Carl Zeiss Meditec AG, Oberkochen, Germany	n.a.	532 nm laser
Mouse anti-GS monoclonal antibody	Millipore, Billerica, MA, USA	MAB302
HRA + OCT Imaging System	Heidelberg Engineering, Heidelberg, Germany	n.a.	Spectralis
Heidelberg Eye Explorer	Heidelberg Engineering, Heidelberg, Germany	n.a.	Version 1.9.10.0
Paraformaldehyde (PFA)	Sigma-Aldrich, Buchs, Switzerland	P5368
Phosphate buffered saline (PBS)	Sigma-Aldrich, Buchs, Switzerland	P5368
Rabbit anti-GFAP polyclonal antibody	Invitrogen, Waltham, MA, USA	180063
Silicone pin holder	Huco Vision AG Switzerland	n.a.	Cut by hand from silicone pin mat of the sterilization tray accordingly.
Slit lamp BM900	Haag-Streit AG, Koeniz, Switzerland	n.a.
Slit lamp adapter	Iridex Corp., Mountain View, CA, USA	n.a.
Superfrost Plus glass slides	Gehard Menzel GmbH, Braunschweig, Germany	10149870
TgBAC (gfap:gfap-GFP) zf167 (AB) strain	KIT, Karlsruhe, Germany	15204	http://zfin.org/ZDB-ALT-100308-3
Tris buffered saline (TBS)	Sigma-Aldrich, Buchs, Switzerland	P5912
Tween 20	Sigma-Aldrich, Buchs, Switzerland	P1379
78D non-contact slit lamp lens	Volk Optical, Mentor, OH, USA	V78C
Xylene	Sigma-Aldrich, Buchs, Switzerland	534056
Ocular fundus laser lens	Ocular Instruments, Bellevue, WA, USA	OFA2-0
2100 Retriever	Aptum Biologics Ltd., Southampton, United Kingdom	R2100-EU	Steamer