We thank Monika Kilchenmann, Federica Bisignani and Agathe Duda for their 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).
1. Animals
<ol>
	<li>Maintain the wild-type zebrafish (Danio rerio) of the AB (Oregon) strain aged between 6-12 months under standard conditions in water with a temperature of 26.5 &#176;C and a 14/10 hr light/dark cycle 15.</li>
	<li>Follow the animal care guidelines of the involved institutions for the animal experiments after approval by the Cantonal Veterinary Office.</li>
</ol>
2. MNU Treatment
<ol>
	<li>Prepare water containing 150 mg/L dry substance of N-methyl-N-nitrosourea (MNU). CAUTION! MNU is toxic; may cause cancer, heritable genetic damage or harm to the unborn child.</li>
	<li>Incubate zebrafish in water containing the MNU for 60 min at room temperature.</li>
	<li>Rinse zebrafish quickly with fresh water and put the fish to a new fish tank without MNU. Maintain fish under standard conditions as long as desired for the experiments.</li>
</ol>
3. Visual Acuity Measurement
<ol>
	<li>Start the optomotor system, choose &#39;testing&#39; from the menu and set the options to &#8220;Simple Staircase&#8221;, &#8220;Acuity (Freq)&#8221; and &#8220;Randomized/Separate&#8221; 16.</li>
	<li>Install an infusion bottle filled with 500 ml water about 1 m above the optomotor system.</li>
	<li>Place one zebrafish in the examination chamber and connect it to the infusion bottle. Place the examination chamber in the optomotor system.</li>
	<li>Start the measurement and observe the eye movement in real time on the computer screen. Thereby, a positive response (&#8220;yes&#8221;) is defined as consecutive saccades in the correct direction, whereas a negative response (&#8220;no&#8221;) represents random eye movements similar to the ones observed with stationary gratings. If the eyes of the zebrafish exhibit three or more subsequent optokinetic responses (OKR), press &#39;yes&#39;; if not, press &#39;no&#39;.</li>
	<li>Extract the visual acuity, which has been calculated by the software by determining the threshold of the spatial frequency of the optokinetic stimulus, under &#8220;Results&#8221; in the menu.</li>
</ol>
4. Histology
<ol>
	<li>Euthanize the zebrafish by an overdose of ethyl 3-aminobenzoate methanesulfonate (200-300 mg/L) and enucleate the eyes.</li>
	<li>Fix the whole eyes in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) at 4 &#176;C for 12 hr and then dehydrate the samples in a graded alcohol series.</li>
	<li>Embed the samples in paraffin, cut 5 &#181;m sections through the optic nerve head (ONH) and mount them on a glass slides.</li>
	<li>Stain the deparaffized paraffin sections with hemalum for 4 min and dip the slides two times in distilled water followed by 0.2% hydrochloric acid and 0.8% ammonia. Stain the sections with eosin for 3 min after development of the hemalum staining in tap water for at least 10 min (H&#38;E).</li>
	<li>Embed the dehydrated slides in mounting medium and observe the slides under the light microscope.</li>
</ol>
5. Immunohistochemistry
<ol>
	<li>Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)
	<ol>
		<li>Incubate the deparaffinized sections with 20 &#181;g/ml proteinase K at room temperature for 15 min. Wash the sections three times with Tris buffered saline (TBS) for 5 min each.</li>
		<li>Incubate the sections with 50 &#181;l TUNEL reaction mixture (10% labeling solution and 90% enzyme solution) in a humidified chamber at 37 &#176;C for 60 min. Wash three times with TBS for 5 min each.</li>
		<li>Mount the slides with mounting medium containing DAPI and observe the slides under the fluorescence microscope.</li>
	</ol>
	</li>
	<li>Proliferating Cell Nuclear Antigen (PCNA) staining
	<ol>
		<li>Boil the deparaffinized sections in antigen retrieval buffer (Tris-EDTA + 0.05% Tween 20, pH 9.0) for 3 min and wash three times with TBS for 5 min each. Block with 100 &#181;l blocking solution (TBS + 10% goat normal serum + 1% bovine serum albumin, pH7.6) at room temperature for 1 hr.</li>
		<li>Stain with anti-PCNA primary antibodies in a 1:200 dilution in a humidified chamber at 4 &#176;C overnight. Wash three times with TBS + Tween 20 for 5 min each.</li>
		<li>Finish the detection with the appropriate secondary antibodies in a 1:500 dilution at room temperature for 1 hr.</li>
		<li>Mount the slides with mounting medium containing DAPI and observe the slides under the fluorescence microscope.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Haddad, S., Chen, C. A., Santangelo, S. L., Seddon, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+age-related+macular+degeneration:+a+review+of+progress+to+date.">The genetics of age-related macular degeneration: a review of progress to date.</a> Surv. Ophthalmol. 51 (4), 316-363 (2006).</li><li>Bhatti, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinitis+pigmentosa,+pigmentary+retinopathies,+and+neurologic+diseases.">Retinitis pigmentosa, pigmentary retinopathies, and neurologic diseases.</a> Curr. Neurol. Neurosci. Rep. 6 (5), 403-413 (2006).</li><li>Hartong, D. T., Berson, E. L., Dryja, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinitis+pigmentosa.">Retinitis pigmentosa.</a> Lancet. 368, 1795-1809 (2006).</li><li>Tsubura, A., Yoshizawa, K., Kuwata, M., Uehara, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+retinitis+pigmentosa+induced+by+MNU;+disease+progression,+mechanisms+and+therapeutic+trials.">Animal models for retinitis pigmentosa induced by MNU; disease progression, mechanisms and therapeutic trials.</a> Histol. Histopathol. 25, 233-248 (2010).</li><li>Machida, K., Urano, K., Yoshimura, M., Tsutsumi, H., Nomura, T., Usui, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carcinogenic+comparative+study+on+rasH2+mice+produced+by+two+breeding+facilities.">Carcinogenic comparative study on rasH2 mice produced by two breeding facilities.</a> J. Toxicol. Sci. 33, 493-501 (2008).</li><li>Morton, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-Methyl-N-Nitrosourea+(MNU):+A+positive+control+chemical+for+p53+/-+mouse+carcinogenicity+studies.">N-Methyl-N-Nitrosourea (MNU): A positive control chemical for p53+/- mouse carcinogenicity studies.</a> Toxicol. Pathol. 36, 926-931 (2008).</li><li>Terracini, B., Testa, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carcinogenicity+of+a+single+administration+of+N-nitrosomethylurea:+a+comparison+between+newborn+and+5-week-old+mice+and+rats.">Carcinogenicity of a single administration of N-nitrosomethylurea: a comparison between newborn and 5-week-old mice and rats.</a> 24, 588-598 (1970).</li><li>Zulliger, R., Lecaud&#233;, S., Eigeldinger-Berthou, S., Wolf-Schnurrbusch, U. E. K., Enzmann, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase-3-independent+photoreceptor+degeneration+by+N-methyl-N-nitrosourea+(MNU)+induces+morphological+and+functional+changes+in+the+mouse+retina.">Caspase-3-independent photoreceptor degeneration by N-methyl-N-nitrosourea (MNU) induces morphological and functional changes in the mouse retina.</a> Graefes Arch. Clin. Exp. Ophthalmol. 249, 859-869 (2011).</li><li>Tappeiner, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+rod+regeneration+in+a+novel+zebrafish+retinal+degeneration+model+using+N-methyl-N-nitrosourea+(MNU).">Characteristics of rod regeneration in a novel zebrafish retinal degeneration model using N-methyl-N-nitrosourea (MNU).</a> PLOS One. 12, (2013).</li><li>Bilotta, J., Saszik, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+as+a+model+visual+system.">The zebrafish as a model visual system.</a> Int. J. Dev. Neurosci. 19, 621-629 (2001).</li><li>Fleisch, C., Neuhauss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+Behavior+in+Zebrafish.">Visual Behavior in Zebrafish.</a> Zebrafish. 3, 191-201 (2006).</li><li>Hodel, C., Neuhauss, S. C. F., Biehmaier, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+and+development+of+light+adaptation+processes+in+the+outer+zebrafish+retina.">Time course and development of light adaptation processes in the outer zebrafish retina.</a> InterScience. 288, 653-662 (2006).</li><li>Gemberling, M., Bailey, T. J., Hyde, D. R., Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+as+a+model+for+complex+tissue+regeneration.">The zebrafish as a model for complex tissue regeneration.</a> Trends Genet. 29 (11), 611-620 (2013).</li><li>Brockerhoff, S. E., Fadool, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+photoreceptor+degeneration+and+regeneration+in+zebrafish.">Genetics of photoreceptor degeneration and regeneration in zebrafish.</a> Cell Mol. Life Sci. 68, 651-659 (2011).</li><li>Brand, M., Granato, M., N&#252;sslein-Volhard, C., N&#252;sslein-Volhard, C., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keeping+and+raising+zebrafish.">Keeping and raising zebrafish.</a> Zebrafish: A Practical Approach. , 7-38 (2002).</li><li>Tappeiner, C., Gerber, S., Enzmann, V., Balmer, J., Jazwinska, A., Tschopp, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+acuity+and+contrast+sensitivity+of+adult+zebrafish.">Visual acuity and contrast sensitivity of adult zebrafish.</a> Front. Zool. 9 (1), 10 (2012).</li><li>Bailey, T. J., Fossum, S. L., Fimbel, S. M., Montgomery, J. E., Hyde, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inhibitor+of+phagocytosis,+O-phospho-L-serine,+suppresses+M&#252;ller+glia+proliferation+and+cone+cell+regeneration+in+the+light-damaged+zebrafish+retina.">The inhibitor of phagocytosis, O-phospho-L-serine, suppresses M&#252;ller glia proliferation and cone cell regeneration in the light-damaged zebrafish retina.</a> Exp. Eye Res. 91, 601-612 (2010).</li><li>Nelson, C. M., Hyde, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M&#252;ller+glia+as+a+source+of+neuronal+progenitor+cells+to+regenerate+the+damaged+zebrafish+retina.">M&#252;ller glia as a source of neuronal progenitor cells to regenerate the damaged zebrafish retina.</a> Adv. Exp. Med. Biol. 723, 425-430 (2012).</li><li>Prusky, G. T., Alam, N. M., Beekman, S., Douglas, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+quantification+of+adult+and+developing+mouse+spatial+vision+using+a+virtual+optomotor+system.">Rapid quantification of adult and developing mouse spatial vision using a virtual optomotor system.</a> Invest. Ophthalmol Vis. Sci. 45 (12), 4611-4616 (2004).</li><li>Beck, J. C., Gilland, E., Tank, D. W., Baker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+the+ontogeny+of+optokinetic+and+vestibulo-ocular+behaviors+in+zebrafish,+medaka,+and+goldfish.">Quantifying the ontogeny of optokinetic and vestibulo-ocular behaviors in zebrafish, medaka, and goldfish.</a> J. Neurophysiol. 92 (6), 3546-3561 (2004).</li><li>Fimbel, S. M., Montgomery, J. E., Burket, C. T., Hyde, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+Inner+Retinal+Neurons+after+Intravitreal+Injection+of+Ouabain+in.">Regeneration of Inner Retinal Neurons after Intravitreal Injection of Ouabain in.</a> Zebrafish. J. Neurosci. 27, 1712-1724 (2007).</li><li>Sherpa, T., et al. <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, 166-181 (2008).</li><li>Yurco, P., Cameron, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Responses+of+M&#252;ller+glia+to+retinal+injury+in+adult+zebrafish.">Responses of M&#252;ller glia to retinal injury in adult zebrafish.</a> Vision Res. 45, 991-1002 (2005).</li></ol>

The authors have nothing to disclose.

Previously, our group has transferred the MNU model of photoreceptor degeneration from rodents into the zebrafish system 9. The ensuing events were described and followed for up to 30 days. In this time period complete retinal morphological degeneration and regeneration occurred after the initial treatment. First, histology reveals a reduced rod cell count from day 3 on with a minimum at day 8. Correspondingly, TUNEL staining identifies apoptosis of rod photoreceptors 3 days after MNU treatment. Regeneration starts in the INL at day 3, with a maximum at day 5. In the ONL, proliferating cells can be observed until the end of the observation period with a maximum at day 5. This is in contrast to the INL where no increased proliferation was observed after day 15. The ciliary marginal zone is the only location of the retina where some PCNA positive cells were found in the untreated retina. This indicates an area with constantly retinal neurogenesis throughout lifetime 14. The identification of cell types involved in the regeneration of pharmacologically damaged photoreceptors in zebrafish was not in the focus of this manuscript. However, in previous publications the process has been traced to regenerating cells in the INL that have been suggested to be M&#252;ller cells 9,17,18.
In the present study, we have also evaluated functional changes in the MNU-induced zebrafish model. To quantify visual function in adult fish, assessment of the OKR has been employed, one of several techniques to reliably measure the visual acuity (VA) of small animals 19. Thereby, the OKR, based on an innate behavior, does not need prior time-consuming training of the animals 20. Furthermore, as the OKR can be repeatedly elicited independently of the animals&#8217; cooperation, and the large eyes can easily be observed, assessment of the OKR is an ideal method to follow visual acuity during degeneration and regeneration in zebrafish. OKR measurements in the present study revealed a decreasing visual acuity until day 3, followed by a complete restoration of the visual function on day 30. This is in accordance with the histological de- and regenerative changes after MNU application that shows a maximum of apoptosis at day 3.
A benefit of the model is its simplicity as MNU is fast to prepare and can be directly dissolved in the water tank of the zebrafish. Treatment of zebrafish with MNU is performed in one hour. The number of fish that can be treated en masse is only limited by the size of the fish tank. Overall, this allows simple induction of retina degeneration in a great number of zebrafish without the need of manipulation of individual fish. Therefore, this model is less invasive than other models of retina degeneration that use intravitreal or surgical induced techniques 21-23 and it may thereby exhibit a better reproducibility.
In general, this zebrafish MNU model is a versatile tool to induce photoreceptor-specific degeneration and to visualize the following regeneration not only morphologically but also functionally. Therefore, we are confident that the model helps to better understand the degeneration and regeneration process in the sensory retina in general and opens the possibility to compare its outcome with the mammalian system.

Vision is the most essential sense for the human being and its impairment has a high socio-economic impact. In the developed world, retinal degenerative diseases are the leading cause of vision loss and blindness among older adults 1. The cause of most degenerative retinal diseases is only partly understood and therapeutical solutions to regain lost vision are very limited. Retinitis pigmentosa is a typical example of a retinal degenerative disease with primary photoreceptor loss 2-3. N-methyl-N-nitrosourea (MNU) induces retinal degeneration and is therefore widely used in rodents to model diseases with primary photoreceptor cell death 4. It is an alkylating agent and leads to benign and malignant tumors, which usually appear several months after exposure 5-7. In addition, it causes specific photoreceptor cell death within a short term observation period. Thereby, the loss of the retinal layer structure and significant retinal thinning were observed in a concentration-dependent manner. Retinal glia cells were activated, but no changes in the retinal pigment epithelium (RPE) were found. Endoplasmic reticulum (ER) stress-related apoptosis appears to be the main pathway of MNU action in the retina 8.
We have recently introduced MNU as a chemical model to induce photoreceptor degeneration in zebrafish 9. Amongst other reasons, the zebrafish (Danio rerio) has become important in visual research because of the similarities of its visual system to that of other vertebrates 10. The outer retina contains the photoreceptors, which can be grouped into four different cone types with peak sensitivities in the ultraviolet, short, middle, and long wavelength of the visible spectra and one rod photoreceptor type. In the inner nuclear layer (INL), the cell bodies of bipolar, horizontal, and amacrine interneurons are found, as well as the cell soma of Muller glia cells. In the outer plexiform layer (OPL) the synaptic contacts between photoreceptors and the inner retina are formed, whereas the cell layer closest to the lens is the ganglion cell layer (GC), which components form long axons comprising the optic nerve and the optic tract. Synaptic contacts between ganglion cells and the cells in the inner nuclear layer are formed in the inner plexiform layer (IPL) 11. The RPE lies outside the neurosensory retina and surrounds the photoreceptor outer segments with long apical microvilli 12. Furthermore, the zebrafish is highly regenerative and able to regrow lesioned brain, retina, spinal cord, heart, and other tissues 13. When retinal damage occurs, cells in the INL, which are thought to be M&#252;ller cells, are activated and have the potential to differentiate into various retinal cell types. In addition, they also generate rod progenitors, which are located in the ONL. Another source that supplies the retina of adult zebrafish with new cells is the ciliary marginal zone. This source is needed to achieve a constant density of rod photoreceptors in the continuously growing zebrafish eye 14.
The MNU model can be used as a simple and reproducible degeneration/regeneration approach for retinal tissue. Due to certain similarities of biological processes in zebrafish and in humans this could open the doors to identify involved cell death pathways and to screen potential neuroprotective drugs. Based on a previous study from our group, we now illustrate the methods of this MNU-induced zebrafish model of retinal de- and consequent regeneration including functional changes with according laboratory videos 9.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Acetic acid</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>A6283</td>
			<td></td>
		</tr>
		<tr >
			<td >Ammonia</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>294993</td>
			<td>0.80%</td>
		</tr>
		<tr >
			<td >Bovine serum albumine (BSA)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>05470</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</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>45260</td>
			<td></td>
		</tr>
		<tr >
			<td >Ethanol</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>2860</td>
			<td>100%, 96%, 70%</td>
		</tr>
		<tr >
			<td >Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>ED</td>
			<td></td>
		</tr>
		<tr >
			<td >Ethyl 3-aminobenzoate methanesulfonate salt</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>E10521</td>
			<td>Tricaine</td>
		</tr>
		<tr >
			<td >Eukitt</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>3989</td>
			<td></td>
		</tr>
		<tr >
			<td >Goat anti-rabbit Alexa 594</td>
			<td>Life Technologies, Zug, Switzerland</td>
			<td>A11012</td>
			<td></td>
		</tr>
		<tr >
			<td >Goat normal serum</td>
			<td>Dako, Glostrup, Danmark</td>
			<td>X0907</td>
			<td></td>
		</tr>
		<tr >
			<td >Hydrochloric acid</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>320331</td>
			<td>0.20%</td>
		</tr>
		<tr >
			<td >In situ Cell Death Detection Kit</td>
			<td>Roche Applied Sciences, Rotkreuz, Switzerland</td>
			<td>11684795910</td>
			<td>TUNEL Kit</td>
		</tr>
		<tr >
			<td >Mayer&#39;s hemalum solution</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>109249</td>
			<td></td>
		</tr>
		<tr >
			<td >Methylnitrosourea (MNU)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>N4766</td>
			<td>Toxic</td>
		</tr>
		<tr >
			<td >OptoMotry</td>
			<td>CerebralMechanics, Lethbridge, AB, Canada</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr >
			<td >Paraformaldehyde (PFA)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>P6148</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 >Proteinase K</td>
			<td>Dako, Glostrup, Danmark</td>
			<td>S3004</td>
			<td></td>
		</tr>
		<tr >
			<td >Rabbit anti-PCNA</td>
			<td>Santa Cruz Biotechnology, Santa Cruz, USA</td>
			<td>sc-33756</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 >Tris buffered saline (TBS)</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>P5912</td>
			<td></td>
		</tr>
		<tr >
			<td >Trizma&#210; base</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr >
			<td >Tween 20</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr >
			<td >Xylene</td>
			<td>Sigma-Aldrich, Buchs, Switzerland</td>
			<td>534056</td>
			<td></td>
		</tr>
		<tr >
			<td >Zebrafish (Danio rerio) AB (Oregon) strain</td>
			<td>University of Fribourg, Dept. of Biology</td>
			<td>n.a.</td>
			<td>Own fish facility</td>
		</tr>
	</tbody>
</table>

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.

Visual acuity:
The experimental set-up [spatial frequency: 0.042 circles/degree (c/d); contrast: 100%; drift speed: 20 degrees/second (d/src); back light luminance: 152 cd/m2] of this study enabled OKR assessment of adult zebrafish. The mean duration of VA measurement was about 5 &#8211; 10 minutes for each zebrafish, which tolerated the procedure well. Visual acuity before MNU exposure was 0.577 &#177; 0.014 cycles/degree (c/d). Figure 1 shows the visual acuity course after the application of 150 mg/L MNU. Starting from day 1, the measurements revealed a marked decrease in visual acuity, reaching minimum values at day 3. Beginning from day 8, an increase of visual acuity occurs, showing a full recovery of visual acuity 30 days after MNU exposure. For statistical analysis a one-way ANOVA followed by Bonferroni&#8216;s multiple comparison test has been applied. Thereby, the differences in visual acuity between baseline and measurements after treatment was significant for days 1, 3, 5, and 8 (p &#60;0.001, each), but not for days 15 and 30.
Histological assessment of the retinal degeneration:
H&#38;E staining was used to quantify morphological changes in one eye of the zebrafish per time point (n = 3) at baseline as well as 3, 8, 15, and 30 days after treatment (Figure 2). Histology was performed as described by Tappeiner et al.9. Thereby, retinal degeneration including disruption of the INL and cavity formation of the ONL was observed starting at day 3. The number of cells in the ganglion cell layer (GC), the inner nuclear layer (INL) and the number of rod (RN) and cone (CN) photoreceptors were determined manually 250 &#181;m from the center of the ONH on both sides of the eye section (size of the counted area refers to a retinal section of 100 &#181;m length). The degeneration was followed for 30 days after treatment with the maximum of rod ONL loss found on day 8. Thereby, the number of rod photoreceptors (RN) decreased to 82% on day 3, 71% on day 8 and 77% on days 15 and 30 of the original number. Furthermore, accumulation of cell clusters was found, mainly in the INL.
Quantification of apoptosis:
TUNEL staining was performed according to the manufacturer (In situ Cell Death Detection Kit; Roche Applied Sciences, Rotkreuz, Switzerland). The observed retinal degeneration after MNU treatment was caused by apoptosis. This was revealed by positive TUNEL staining in different cell layers in the sensory retina. For quantification, the number of TUNEL-positive cells was determined manually at two retinal areas of 450 &#956;m length each, starting at the periphery of the retina. Thereby, the majority of TUNEL-positive cells was found in the ONL at day 3. However, dying cells were also seen in the INL at that time point (Figure 3). No relevant TUNEL-positive cells were detectable before treatment.
Quantification of cell proliferation:
To assess proliferation after cell death, retinas were stained for proliferating cell nuclear antigen (PCNA) as described by Tappeiner et al. 9. For quantification, the number of PCNA-positive cells was determined manually as described above. Cell death was followed by induced proliferation seen as PCNA-positive cells (Figure 4). Thereby, the maximum of proliferating cells was measured in the INL on day 5. Additionally, cells in the ONL (mainly rods) showed positive staining for PCNA until the end of the study (day 30). Few PCNA-positive cells in the ciliary marginal zone were detectable before and at all time-points after treatment.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51909/51909fig1highres.jpg" width="500" /> 
Figure 1: Visual acuity. Measurement of the visual acuity in adult zebrafish after the application of 150 mg/L MNU. Significant decrease of visual acuity with a minimum at day 3 post treatment was followed by full recovery, beginning on day 5, until day 30 (mean &#177; SD; n = 3).
<img alt="Figure 2" fo:content-width="7in" src="/files/ftp_upload/51909/51909fig2highres.jpg" width="700" /> 
Figure 2: Histology. Examples of H&#38;E stained histological sections of the zebrafish retina at baseline (A) and 3 (B), 8 (C), 15 (D), and 30 (E) days after MNU treatment. CN, cone nuclei; RN, rod nuclei; INL, inner nuclear layer; GC, ganglion cell layer. The arrows mark cell clusters in the INL. Scale bar equals 50 &#181;m.
<img alt="Figure 3" fo:content-width="3in" src="/files/ftp_upload/51909/51909fig3highres.jpg" width="300" /> 
Figure 3: TUNEL-positive cells. TUNEL-positive cells (red) in the zebrafish retina at day 3 after MNU treatment. The cells were mainly located in the ONL but were also found in the INL. Panel A depicts the control, whereas panel B shows the images after MNU treatment. Scale bar indicates 50 &#181;m.
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51909/51909fig4highres.jpg" width="500" /> 
Figure 4: PCNA-positive cells. PCNA-positive cells (red) in the zebrafish retina at day 5 after MNU treatment compared to the control sample. Panel A depicts the control, whereas panel B shows the images after MNU treatment. Scale bar indicates 100 &#181;m.

Watch this Scientific Journal Video about Methylnitrosourea MNU-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics at JoVE.com

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.

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 ...

methylnitrosourea-mnu-induced-retinal-degeneration-regeneration

Nanyang Technological University

Research

JoVE Journal

Biology

11.4K Views.  University of Bern. 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.

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

In Utero Intraventricular Injection and Electroporation of E16 Rat Embryos

In-utero in-vivo injection and electroporation of the embryonic rat neocortex provides a powerful tool for the manipulation of individual progenitors lining the walls of the lateral ventricle. This technique is now widely used to study the processes involved in corticogenesis by over-expressing or knocking down genes and observing the effects on cellular proliferation, migration, and differentiation. In comparison to traditional knockout strategies, in-utero electroporation provides a rapid means to manipulate a population of cells during a specific temporal window. In this video protocol, we outline the experimental methodology for preparing rats for surgery, exposing the uterine horns through laporatomy, injecting DNA into the lateral ventricles of the developing embryo, electroporating DNA into the progenitors lining the lateral wall, and caring for animals post-surgery. Our laboratory uses this protocol for surgeries on E15-E21 rats, however it is most commonly performed at E16 as shown in this video.

In-utero in-vivo injection and electroporation of the embryonic rat neocortex provides a powerful tool for the manipulation of individual progenitors ...

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a 'trunk' fragment is cut from an intact worm, a new 'head' will regenerate anteriorly and a 'tail' will regenerate posteriorly restoring the original 'head-to-tail' polarity of body structures prior to amputation (Figure 1A).
Regeneration is driven by planarian stem cells, known as 'neoblasts' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity2-9.
The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters10, represent an important system for studying evolution of the central nervous system11, 12 and have behavioral screening potential13, 14. 
Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs8, 9, 15. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

An attractive model for studying stem cell differentiation within a live animal is the planarian flatworm. Regeneration is studied by simple amputation experiments that are easily performed in a basic laboratory and are amenable to pharmacological and genetic (in vivo RNAi) manipulation as detailed by protocols in this article.

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

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 ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.17&#160;that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Immunolabelling Myofiber Degeneration in Muscle Biopsies

The necrosis of muscle fibres (myonecrosis) plays a central role in the pathogenesis of several muscle conditions, including muscular dystrophies. Therapeutic options addressing the causes of muscular dystrophy pathogenesis are expected to alleviate muscle degeneration. Therefore, a method to assay and quantify the extent of cell death in muscle biopsies is needed. Conventional methods to observe myofiber degeneration in situ are either poorly quantitative or rely on the injection of vital dyes. In this article, an immunofluorescence protocol is described that stains necrotic myofibers by targeting immunoglobulin G (IgG) uptake by myofibers. The IgG uptake method is based on cell features characterizing the necrotic demise, including 1) the loss of plasma membrane integrity with the release of damage-associated molecular patterns and 2) the uptake of plasmatic proteins. In murine cross-sections, the co-immunolabelling of myofibers, extracellular matrix proteins, and mouse IgG allows clean and straightforward identification of myofibers with necrotic fate. This simple method is suitable for quantitative analysis and applicable to all species, including human samples, and does not require the injection of vital dye. The staining of necrotic myofibers by IgG uptake can also be paired with other co-immunolabelling.

Described here is a protocol for direct immunolabelling of necrotic myofibers in muscle cryosections. Necrotic cells are permeable to serum proteins, including immunoglobulin G (IgG). Revealing the uptake of IgG by myofibers allows the identification and quantification of myofibers that undergo necrosis regardless of muscle condition.