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

Summary

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.

Abstract

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.

Introduction

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 cel....

Protocol

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

1. Animals

1. 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 °C and a 14/10 hr light/dark cycle ¹⁵.
2. Follow the animal care guidelines of the involved institutions for the animal experiments after approval by the Cantonal Veterinary Office.

2. MNU Treatment

1. Prepare water contain....

Results

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 – 10 minutes for each zebrafish, which tolerated the procedure well. Visual acuity before MNU exposure was 0.577 ± 0.014 cycles/degree (c/d). Figure 1 shows the visual acuity course after t.......

Discussion

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 s.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acetic acid	Sigma-Aldrich, Buchs, Switzerland	A6283
Ammonia	Sigma-Aldrich, Buchs, Switzerland	294993	0.80%
Bovine serum albumine (BSA)	Sigma-Aldrich, Buchs, Switzerland	05470
Dako Pen	Dako, Glostrup, Danmark	S2002
DAPI mounting medium	Vector Labs, Burlingame, CA, USA	H-1200
Eosin	Sigma-Aldrich, Buchs, Switzerland	45260
Ethanol	Sigma-Aldrich, Buchs, Switzerland	2860	100%, 96%, 70%
Ethylenediaminetetraacetic acid (EDTA)	Sigma-Aldrich, Buchs, Switzerland	ED
Ethyl 3-aminobenzoate methanesulfonate salt	Sigma-Aldrich, Buchs, Switzerland	E10521	Tricaine
Eukitt	Sigma-Aldrich, Buchs, Switzerland	3989
Goat anti-rabbit Alexa 594	Life Technologies, Zug, Switzerland	A11012
Goat normal serum	Dako, Glostrup, Danmark	X0907
Hydrochloric acid	Sigma-Aldrich, Buchs, Switzerland	320331	0.20%
In situ Cell Death Detection Kit	Roche Applied Sciences, Rotkreuz, Switzerland	11684795910	TUNEL Kit
Mayer's hemalum solution	Merck, Darmstadt, Germany	109249
Methylnitrosourea (MNU)	Sigma-Aldrich, Buchs, Switzerland	N4766	Toxic
OptoMotry	CerebralMechanics, Lethbridge, AB, Canada	n.a.
Paraformaldehyde (PFA)	Sigma-Aldrich, Buchs, Switzerland	P6148
Phosphate buffered saline (PBS)	Sigma-Aldrich, Buchs, Switzerland	P5368
Proteinase K	Dako, Glostrup, Danmark	S3004
Rabbit anti-PCNA	Santa Cruz Biotechnology, Santa Cruz, USA	sc-33756
Superfrost Plus glass slides	Gehard Menzel GmbH, Braunschweig, Germany	10149870
Tris buffered saline (TBS)	Sigma-Aldrich, Buchs, Switzerland	P5912
Trizma base	Sigma-Aldrich, Buchs, Switzerland	T1503
Tween 20	Sigma-Aldrich, Buchs, Switzerland	P1379
Xylene	Sigma-Aldrich, Buchs, Switzerland	534056
Zebrafish (Danio rerio) AB (Oregon) strain	University of Fribourg, Dept. of Biology	n.a.	Own fish facility