Name: m&#252;ller glia cell activation in a laser-induced retinal degeneration and regeneration model in zebrafish
Uploaded: 2017-10-27T14:00:00
Description: Watch this Scientific Journal Video about MÃ¼ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish at JoVE.com

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.

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
<ol>
	<li>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 &#176;C and a 14/10 h light/dark cycle18.</li>
	<li>Follow the animal ca...

<ol>
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An immunofluorescence study with antibodies to the glial fibrillary acidic (GFA) protein.</a> Exp Eye Res. 28 (1), 63-69 (1979).</li><li>Sherpa, T., Fimbel, S. M., Mallory, D. E., Maaswinkel, H., Spritzer, S. D., Sand, J. A., Li, L., Hyde, D. R., Stenkamp, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ganglion+cell+regeneration+following+whole-retina+destruction+in+zebrafish.">Ganglion cell regeneration following whole-retina destruction in zebrafish.</a> Dev Neurobiol. 68 (2), 166-181 (2008).</li><li>Cameron, D. A., Carney, L. 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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...

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 M&#252;ller glia cell&#160;(MC) activation and proliferation8. MCs, the major glial cell type in the vertebrate retina, were once considered to be nothing more than a &#34;glue&#34; 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.

<table>
	<tbody>
		<tr>
			<td>Acid hematoxylin solution</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>2852</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>A07030</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>5470</td>
			<td></td>
		</tr>
		<tr>
			<td>Dako Pen</td>
			<td>Dako, Glostrup, Danmark</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI mounting medium</td>
			<td>Vector Labs, Burlingame, CA, USA</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin G aqueous solution 0.5%</td>
			<td>Carl Roth, Arlesheim, Switzerland</td>
			<td>X883.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>2860</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene diamine tetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>ED</td>
			<td></td>
		</tr>
		<tr>
			<td>Eukitt</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>3989</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-rabbit IgG H&#38;L Alexa Fluor&#174; 488</td>
			<td>Life Technologies, Zug, Switzerland</td>
			<td>A11008</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG H&#38;L Alexa Fluor&#174; 594</td>
			<td>Life Technologies, Zug, Switzerland</td>
			<td>A11020</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat normal serum</td>
			<td>Dako, Glostrup, Danmark</td>
			<td>X0907</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogel contact lens</td>
			<td>Johnson &#38; Johnson AG, Zug, Switzerland</td>
			<td>n.a.</td>
			<td>1-Day Acuvue Moist</td>
		</tr>
		<tr>
			<td>Hydroxypropylmethylcellulose 2%</td>
			<td>OmniVision, Neuhausen, Switzerland</td>
			<td>n.a.</td>
			<td>Methocel 2%</td>
		</tr>
		<tr>
			<td>Ethyl 3-aminobenzoate methanesulfonate</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>A5040</td>
			<td>Tricaine, MS-222</td>
		</tr>
		<tr>
			<td>Visulas 532s</td>
			<td>Carl Zeiss Meditec AG, Oberkochen, Germany</td>
			<td>n.a.</td>
			<td>532 nm laser</td>
		</tr>
		<tr>
			<td>Mouse anti-GS monoclonal antibody</td>
			<td>Millipore, Billerica, MA, USA</td>
			<td>MAB302</td>
			<td></td>
		</tr>
		<tr>
			<td>HRA + OCT Imaging System</td>
			<td>Heidelberg Engineering, Heidelberg, Germany</td>
			<td>n.a.</td>
			<td>Spectralis</td>
		</tr>
		<tr>
			<td>Heidelberg Eye Explorer</td>
			<td>Heidelberg Engineering, Heidelberg, Germany</td>
			<td>n.a.</td>
			<td>Version 1.9.10.0</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>P5368</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>P5368</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-GFAP polyclonal antibody</td>
			<td>Invitrogen, Waltham, MA, USA</td>
			<td>180063</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone pin holder</td>
			<td>Huco Vision AG Switzerland</td>
			<td>n.a.</td>
			<td>Cut by hand from silicone pin mat of the sterilization tray accordingly.</td>
		</tr>
		<tr>
			<td>Slit lamp BM900</td>
			<td>Haag-Streit AG, Koeniz, Switzerland</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Slit lamp adapter</td>
			<td>Iridex Corp., Mountain View, CA, USA</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus glass slides</td>
			<td>Gehard Menzel GmbH, Braunschweig, Germany</td>
			<td>10149870</td>
			<td></td>
		</tr>
		<tr>
			<td>TgBAC (gfap:gfap-GFP) zf167 (AB) strain</td>
			<td>KIT, Karlsruhe, Germany</td>
			<td>15204</td>
			<td>http://zfin.org/ZDB-ALT-100308-3</td>
		</tr>
		<tr>
			<td>Tris buffered saline (TBS)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>P5912</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>78D non-contact slit lamp lens</td>
			<td>Volk Optical, Mentor, OH, USA</td>
			<td>V78C</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>534056</td>
			<td></td>
		</tr>
		<tr>
			<td>Ocular fundus laser lens</td>
			<td>Ocular Instruments, Bellevue, WA, USA</td>
			<td>OFA2-0</td>
			<td></td>
		</tr>
		<tr>
			<td>2100 Retriever</td>
			<td>Aptum Biologics Ltd., Southampton, United Kingdom</td>
			<td>R2100-EU</td>
			<td>Steamer</td>
		</tr>
	</tbody>
</table>

m&#252;ller glia cell activation in a laser-induced retinal degeneration and regeneration model in zebrafish

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.

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...

Watch this Scientific Journal Video about MÃ¼ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish at JoVE.com

M&#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

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 M&#252;ller glia response throughout retinal regeneration.

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after ...

The overall goal of this video is to show how to monitor cellular changes in vivo following a focal, laser-induced retinal damage in Zebrafish. This model can help you answer key questions of retinal regeneration, such as morphological changes, kinetics, as well as cell types involved. The main advantage of this technique is that by inducing a focal injury, biological processes can be investigated directly at the site of injury.Though this method can provide insight into regeneration of Zebrafish retina, it can also be applied to study the repair mechanism in different animal models. Prepare a stock solution of anesthetic by dissolving 400 milligrams of tricaine powder in 97.9 millimeters of tank water, and 2.1 millimeters of one molar TBS. Adjust to pH 7.0 with one molar tris at pH 9.To make the working solution, dilute the tricaine stock solution 1 to 25 in tank water and transfer 50 milliliters to a Petri dish. Then, place the Zebrafish into the anesthetic solution for two to five minutes until they become immobile and do not respond to external stimuli. Transfer each fish by hand to a custom made silicone pin holder for laser treatment.Appropriate anesthesia is critical for the well-being of the animal and the success of procedure. Therefore, freshly prepared tricaine solution is pivotal, and the time out of the water should not far exceed 10 minutes. Set up the output power of the 532 nanometer dialed laser to 70 milliwatts, the pulse duration to 100 milliseconds, and the aerial diameter to 50 microns.Then, apply one to two drops of 2%hydroxypropyl methylcellulose to the eye. When apply methylcell to the eye, ensure that the viscous solution doesn't go in the gills. Next, use a 2 millimeter fundus laser lens to focus the laser aiming beam on the retina.Place four laser spots around the optic nerve on the left eye, and use the right, untreated eye as internal control. Immediately after laser induction, place the still anesthetized Zebrafish on a custom made silicone pin holder in the imaging area. To obtain optimal images, cut a commercially available hydrogel contact lens to fit the Zebrafish eye by means of a hole punch.Fill the concave surface of the lens with methylcellulose, then place it over the cornea. Equip the optical coherence tomography system with a 78D non-contact slit lamp lens. Focus the infrared image in IR plus OCT mode to visualize the fundus of the eye.Then, take the IR pictures by clicking the acquire button to localize the laser spots on the retina. Then, visualize a three dimensional section of the retinal layers in IR plus OCT mode, and take the pictures by clicking the acquire button. Observe the severity of injury in the outer nuclear layer in these images.To reverse anesthesia after treatment and imaging, place the zebrafish in a container containing tank water. To support recovery, create a flow of fresh tank water over the gills by moving the zebrafish back and forth in the water. Immediately after laser treatment, a diffuse hyper-reflective signal was localized to the outer retina.It extended from the retinal pigmented epithelium to the outer plexiform layer. The diffuse hyper-reflective signal is absent in the retina from the control un-lesioned eye. A similar diffuse hyper-reflective signal detected on day one after injury.After day three, this diffuse signal became more organized and dense. It was consistently seen in the outer nuclear layer, extending into the photoreceptor layer. Following the first week, there was a significant decrease in average lesion size, and only a small hyper-reflective signal was detected.Starting from day 14 until the latest time point investigated, the laser spots were no longer visible in IR and OCT images, and the morphology of the retina is comparable to the control side, seen here. H&E staining was employed to investigate the extent and kinetics of retinal degeneration and regeneration. This image shows the control section.This image shows H&E staining three days after injury, when maximum photoreceptor loss is apparent. Immunohistochemistry to visualize the glial cell markers, glutamine synthetase, in red, and glial fibrillary acidic protein, in green, was performed. This image shows a control retina where there is very little green fluorescence, which is indicative of GFAP.The GFAP signal was up-regulated at day three post injury, whereas the red glutamine synthetase signal remained unchanged. Following this procedure, other methods like two photon microscopy as well as cellular and molecular analysis can be performed in order to study the involvement of molecular cells in endogenous repair mechanisms. After watching this video, you should have a good understanding how to induce focal damage to the zebrafish retina and to monitor in vivo the following degenerative and regenerative processes.

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