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 care guidelines of the involved institutions for the animal experiments after approval by the governmental authorities.</li>
</ol>
2. Reversible Systemic Anesthesia
<ol>
	<li>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 &#176;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.</li>
	<li>Dilute the stock solution 1:25 in tank water and use immediately.</li>
	<li>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).</li>
	<li>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.</li>
	<li>To reverse anesthesia after the treatment and/or imaging, place the zebrafish in container containing tank water.</li>
	<li>To support recovery, create a flow of fresh tank water over the gills by moving the zebrafish back and forth in the water.</li>
</ol>
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.
<ol>
	<li>Set up the output power of the laser: 70 mW; aerial diameter: 50 &#181;m; pulse duration: 100 ms. 
	CAUTION! The use of laser light requires appropriate personal protection and labelling of the area.</li>
	<li>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.</li>
	<li>Place four laser spots around the optic nerve on the left eye and use the right, untreated eye as internal control.</li>
</ol>
4. In vivo Imaging of the Retinal Morphology
<ol>
	<li>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).</li>
	<li>To obtain optimal images, cut a commercially available hydrogel contact lens to fit the zebrafish eye (&#216; = 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.</li>
	<li>Equip the OCT system with a 78D non-contact slit lamp lens.</li>
	<li>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 &#34;Acquire&#34; button (Figure 2B) to localize the laser spots on the retina using the system&#39;s software.</li>
	<li>Visualize a three-dimensional section of the retinal layers in the IR + OCT mode and take the pictures by clicking the &#34;Acquire&#34; 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.</li>
	<li>To reverse anesthesia after the treatment and/or imaging place the zebrafish in a container containing tank water.</li>
	<li>To support recovery, create a flow of fresh tank water over the gills by moving the zebrafish back and forth in the water.</li>
	<li>Perform similar in vivo imaging of retinal morphology on day 1, 3, 7, 14 and week 6.</li>
</ol>
5. Hematoxylin &#38; Eosin (H&#38;E) Staining
<ol>
	<li>Euthanize zebrafish by submersion into cold (4 &#176;C) anesthesia solution on ice for at least 10 min and enucleate the eyes immediately by means of small curved forceps.</li>
	<li>Fix the whole eyes in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) at 4 &#176;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.</li>
	<li>Embed the samples in paraffin, cut 5 &#181;m sections at the level of the optic nerve head and mount them on glass slides.</li>
	<li>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.</li>
	<li>Mount the dehydrated slides in acrylic resin mounting medium and observe the slides under the light microscope.</li>
</ol>
6. Immunohistochemistry for the MC Activation
<ol>
	<li>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.</li>
	<li>Circle the sections with a silicone pen and add 100 &#181;L blocking solution (TBS + 10% goat normal serum + 1% bovine serum albumin, pH 7.6) at room temperature for 1 h.</li>
	<li>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 &#181;L per sample). Incubate the slide in a humidified chamber at 4 &#176;C overnight. Wash three times with TBS + 0.1 % Tween 20 for 5 min each.</li>
	<li>Finish visualization with the appropriate secondary antibodies. This protocol used a goat anti-rabbit IgG H&#38;L green secondary antibody for GFAP and a goat anti-mouse IgG H&#38;L bright red for GS, both in a 1:500 dilution at room temperature for 1 h.</li>
	<li>Mount the slides with mounting medium containing DAPI and observe the slides under the fluorescence microscope.</li>
</ol>

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The authors have nothing to disclose.

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&amp;amp;L Alexa Fluor&amp;reg; 488</td><td>Life Technologies, Zug, Switzerland</td><td>A11008</td><td></td></tr><tr><td>Goat anti-mouse IgG H&amp;amp;L Alexa Fluor&amp;reg; 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 &amp;amp; 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 ...

mller-glia-cell-activation-laser-induced-retinal-degeneration

Research

JoVE Journal

Neuroscience

9.7K Views.  University of Bern. De zebravis is een populaire diermodel te bestuderen van de mechanismen van Retina degeneratie/regeneratie in gewervelden. Dit protocol beschrijft een methode om gelokaliseerde letsel verstoren het buitenste netvlies met minimale schade aan het innerlijke netvlies veroorzaken. Vervolgens controleren we in vivo de retinale morfologie en de reactie hierop van Müller, glia hele netvlies regeneratie.

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

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

Developmental Biology

Motor Nerve Transection and Time-lapse Imaging of Glial Cell Behaviors in Live Zebrafish

The nervous system is often described as a hard-wired component of the body even though it is a considerably fluid organ system that reacts to external stimuli in a consistent, stereotyped manner, while maintaining incredible flexibility and plasticity. Unlike the central nervous system (CNS), the peripheral nervous system (PNS) is capable of significant repair, but we have only just begun to understand the cellular and molecular mechanisms that govern this phenomenon. Using zebrafish as a model system, we have the unprecedented opportunity to couple regenerative studies with in vivo imaging and genetic manipulation. Peripheral nerves are composed of axons surrounded by layers of glia and connective tissue. Axons are ensheathed by myelinating or non-myelinating Schwann cells, which are in turn wrapped into a fascicle by a cellular sheath called the perineurium. Following an injury, adult peripheral nerves have the remarkable capacity to remove damaged axonal debris and re-innervate targets. To investigate the roles of all peripheral glia in PNS regeneration, we describe here an axon transection assay that uses a commercially available nitrogen-pumped dye laser to axotomize motor nerves in live transgenic zebrafish. We further describe the methods to couple these experiments to time-lapse imaging of injured and control nerves. This experimental paradigm can be used to not only assess the role that glia play in nerve regeneration, but can also be the platform for elucidating the molecular mechanisms that govern nervous system repair.

Although the peripheral nervous system (PNS) is capable of significant repair after injury, little is known about the cellular and molecular mechanisms that govern this phenomenon. Using live, transgenic zebrafish and a reproducible nerve transection assay, we can study dynamic glial cell behaviors during nerve degeneration and regeneration.

The  nervous system is often described as a hard-wired component of the body even  though it is a considerably fluid organ system that reacts to external ...

A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, photoreceptor degeneration triggers M&#252;ller glial cells to re-enter the cell cycle and produce transient-amplifying progenitors. These progenitors continue to proliferate as they migrate to the damaged area, where they ultimately give rise to new photoreceptors. Currently, there are two widely-used LIRD paradigms, each of which results in varying degrees of photoreceptor loss and corresponding differences in the regeneration response. As more genetic and pharmacological tools are available to test the role of individual genes of interest during regeneration, there is a need to develop a robust LIRD paradigm. Here we describe a LIRD protocol that results in widespread and consistent loss of both rod and cone photoreceptors in which we have combined the use of two previously established LIRD techniques. Furthermore, this protocol can be extended for use in pigmented animals, which eliminates the need to maintain transgenic lines of interest on the albino background for LIRD studies.

Multiple light damage protocols have been described to damage photoreceptors and consequently induce a retinal regeneration response in adult zebrafish. This protocol describes an improved method that can be used in pigmented animals and that damages the vast majority of rod and cone photoreceptors across the entire retina.

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, ...

Confocal Microscope-Based Laser Ablation and Regeneration Assay in Zebrafish Interneuromast Cells

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally replenished in non-mammalian vertebrates such as zebrafish. The zebrafish lateral line system is a useful model for characterizing sensory hair cell regeneration. The lateral line is comprised of hair cell-containing organs called neuromasts, which are linked together by a string of interneuromast cells (INMCs). INMCs act as progenitor cells that give rise to new neuromasts during development. INMCs can repair gaps in the lateral line system created by&#160;cell death. A method is described here for selective INMC ablation using a conventional laser-scanning confocal microscope and transgenic fish that express green fluorescent protein in INMCs. Time-lapse microscopy is then used to monitor INMC regeneration and determine the rate of gap closure. This represents an accessible protocol for cell ablation that does not require specialized equipment, such as a high-powered pulsed ultraviolet laser. The ablation protocol may serve broader interests, as it could be useful for the ablation of additional cell types, employing a tool set that is already available to many users. This technique will further enable the characterization of INMC regeneration under different conditions and from different genetic backgrounds, which will advance the understanding of sensory progenitor cell regeneration.

Laser ablation is a widely applicable technology for studying regeneration in biological systems. The presented protocol describes use of a standard laser-scanning confocal microscope for laser ablation and subsequent time-lapse imaging of regenerating interneuromast cells in the zebrafish lateral line.

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally ...

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., M&#971;ller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.

In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

Biology

Stab Wound Injury Model of the Adult Optic Tectum Using Zebrafish and Medaka for the Comparative Analysis of Regenerative Capacity

While zebrafish have a superior capacity to regenerate their central nervous system (CNS), medaka has a lower CNS regenerative capacity. A brain injury model was developed in the adult optic tectum of zebrafish and medaka and comparative histological and molecular analyses were performed to elucidate the molecular mechanisms regulating the high regenerative capacity of this tissue across these fish species. Here a stab wound injury model is presented for the adult optic tectum using a needle and histological analyses for proliferation and differentiation of the neural stem cells (NSCs). A needle was manually inserted into the central region of the optic tectum, and then the fish were intracardially perfused, and their brains were dissected. These tissues were then cryosectioned and evaluated using immunostaining against the appropriate NSC proliferation and differentiation markers. This tectum injury model provides robust and reproducible results in both zebrafish and medaka, allowing for comparing NSC responses after injury. This method is available for small teleosts, including zebrafish, medaka, and African killifish, and enables us to compare their regenerative capacity and investigate unique molecular mechanisms.

A mechanical brain injury model in the adult zebrafish is described to investigate the molecular mechanisms regulating their high regenerative capacity. The method explains to create a stab wound injury in the optic tectum of multiple species of small fish to evaluate the regenerative responses using fluorescent immunostaining.

While zebrafish have a superior capacity to regenerate their central nervous system (CNS), medaka has a lower CNS regenerative capacity. A brain injury ...

Triggering Cell Stress and Death Using Conventional UV Laser Confocal Microscopy

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and cell-cell interactions in the CNS in real-time. Previously, studying these cell-specific responses after injury often required complicated setups or the transfer of cells or animals into different, non-physiological environments, confounding immediate and short-term analysis. For example, drug-mediated ablation approaches often lack the specificity that is required to study single-cell responses and immediate cell-cell interactions. Similarly, while high-power pulsed laser ablation approaches provide very good control and tissue penetration, they require specialized equipment that can complicate real-time visualization of cellular responses. The refined UV laser ablation approach described here allows researchers to stress or kill an individual cell in a dose- and time-dependent manner using a conventional confocal microscope equipped with a 405-nm laser. The method was applied to selectively ablate a single neuron within a dense network of surrounding cells in the zebrafish spinal cord. This approach revealed a dose-dependent response of the ablated neurons, causing the fragmentation of cellular bodies and anterograde degeneration along the axon within minutes to hours. This method allows researchers to study the fate of an individual dying cell and, importantly, the instant response of cells-such as microglia and astrocytes-surrounding the ablation site.

Targeted manipulations to cause directed stress or death in individual cells have been relatively difficult to accomplish. Here, a single-cell-resolution ablation approach to selectively stress and kill individual cells in cell culture and living animals is described based on a standard confocal UV laser.

Using a standard confocal setup, a UV ablation method can be utilized to selectively induce cellular injury and to visualize single-cell responses and ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

Methylnitrosourea (MNU)-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics

Retinal degenerative diseases, e.g. retinitis pigmentosa, with resulting photoreceptor damage account for the majority of vision loss in the industrial world. Animal models are of pivotal importance to study such diseases. In this regard the photoreceptor-specific toxin N-methyl-N-nitrosourea (MNU) has been widely used in rodents to pharmacologically induce retinal degeneration. Previously, we have established a MNU-induced retinal degeneration model in the zebrafish, another popular model system in visual research.
A fascinating difference to mammals is the persistent neurogenesis in the adult zebrafish retina and its regeneration after damage. To quantify this observation we have employed visual acuity measurements in the adult zebrafish. Thereby, the optokinetic reflex was used to follow functional changes in non-anesthetized fish. This was supplemented with histology as well as immunohistochemical staining for apoptosis (TUNEL) and proliferation (PCNA) to correlate the developing morphological changes.
In summary, apoptosis of photoreceptors occurs three days after MNU treatment, which is followed by a marked reduction of cells in the outer nuclear layer (ONL). Thereafter, proliferation of cells in the inner nuclear layer (INL) and ONL is observed. Herein, we reveal that not only a complete histological but also a functional regeneration occurs over a time course of 30 days. Now we illustrate the methods to quantify and follow up zebrafish retinal de- and regeneration using MNU in a video-format.

Methylnitrosourea MNU-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics

Herein we demonstrate quantification of retinal de- and regeneration and its impact on visual function using N-methyl-N-nitrosourea in the adult zebrafish. Loss of visual acuity and decreased photoreceptor numbers were followed by proliferation in the inner nuclear layer. Complete morphological and functional regeneration occurred 30 days after the initial treatment.

Retinal degenerative diseases, e.g. retinitis pigmentosa, with resulting photoreceptor damage account for the majority of vision loss in the industrial ...

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform (RpEGEN) for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent retinal and vascular tissues. At present, the limited reparative capacity of mammalian RPE, which is restricted to small injuries, has hindered progress to understanding in vivo RPE regenerative processes. Here, a detailed methodology is provided to facilitate the study of in vivo RPE repair utilizing the zebrafish, a&#160;vertebrate model capable of robust tissue regeneration. This protocol describes a transgenic nitroreductase/metronidazole (NTR/MTZ)-mediated injury paradigm (rpe65a:nfsB-eGFP), which results in ablation of the central two-thirds of the RPE after 24 h treatment with MTZ, with subsequent tissue recovery. Focus is placed on RPE ablations in larval zebrafish and methods for testing the effects of pharmacological compounds on RPE regeneration are also outlined. Generation and validation of RpEGEN, a MATLAB script created to automate quantification of RPE regeneration based on pigmentation, is also discussed. Beyond active RPE repair mechanisms, this protocol can be expanded to studies of RPE degeneration and injury responses as well as the effects of RPE damage on adjacent retinal and vascular tissues, among other cellular and molecular processes. This zebrafish system holds significant promise in identifying genes, networks, and processes that drive RPE regeneration and RPE disease-related mechanisms, with the long-term goal of applying this knowledge to mammalian systems and, ultimately, toward therapeutic development.

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform RpEGEN for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

This protocol describes the methodology to genetically ablate the retinal pigment epithelium (RPE) using a transgenic zebrafish model. Adapting the protocol to incorporate signaling pathway modulation using pharmacological compounds is extensively detailed. A MATLAB platform for quantifying RPE regeneration based on pigmentation was developed and is presented and discussed.

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent ...

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

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