title: Photoreceptor degeneration by intravitreal injection of N-methyl-N-nitrosourea (MNU) in rabbits: a pilot study.
abstract: BACKGROUND
  Pilot study on the attempt to induce selective photoreceptor degeneration in the rabbit eye by intravitreal injection of MNU, facing the difficulties of the evaluation of retinal degeneration by different in-vivo and in-vitro methods in such a large eye animal model.
  
  
  METHODS
  Eight pigmented Chinchilla Bastard rabbits were injected intravitreally with MNU (1 × 1mg/kg body weight (BW), 1 × 2mg/kg BW, 3 × 3mg/kg BW, 1 × 4mg/kg BW, 1 × 6mg/kg BW, and 1 × DMSO + PBS as control). One, 2, and 3 weeks after injection, the effects on the rabbit retina were examined in vivo using clinical observation (macroscopic images, funduscopy, weighing of the animals), measurement of intraocular pressure (IOP), full-field Electroretinography (ffERG), and spectral-domain Optical Coherence Tomography (sd-OCT). After 3 weeks follow-up, blood samples were taken to evaluate the general health status of the animals, and immunohistochemistry (IH) was performed on sections obtained from six different regions throughout the whole retina to evaluate MNU effects in more detail.
  
  
  RESULTS
  It was difficult to observe the effects of MNU on retinal structure by OCT in vivo. Only the temporal quadrant of the retina could be visualized. Therefore, it was indispensible to evaluate the effects of MNU on the retina in vitro by examining six areas of the retina using immunohistochemistry. Furthermore, immunohistochemistry plays a decisive role to evaluate the effects on retinal cells other than photoreceptors while in H&E staining, namely the cell count of the ONL can be observed. The results obtained in vivo and in vitro in this study mainly follow the results of a previous study in mice. The low doses of MNU (1, 2 mg/kg BW) had no effects on retinal function and morphology, while high doses (4, 6 mg/kg BW) led to retinal changes in combination with significant side-effects (e.g., cataractous changes). Injection of 3 mg/kg BW MNU induced selective photoreceptor degeneration. However, the degree of degeneration varied between different parts of the same retina and between retinae of different animals. In two of three animals, a complete loss of ERG potentials was observed. Negative effects on the contralateral eye or on general welfare of the animal were never observed.
  
  
  CONCLUSIONS
  In rabbits, the intravitreal injection of 3 mg/kg BW MNU leads to selective but inhomogeneous photoreceptor degeneration.
authors.affiliation: Department of Ophthalmology, RWTH Aachen University, Pauwelsstr. 30, 52074, Aachen, Germany.|Clinical Department of Small Animals, University of Leipzig, Leipzig, Germany.
authors.forename: Sarah
authors.initials: S
authors.lastname: Rösch
authors.affiliation: Department of Ophthalmology, RWTH Aachen University, Pauwelsstr. 30, 52074, Aachen, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Werner
authors.affiliation: Institute of Complex Systems, Cellular Biophysics, ICS-4, Forschungszentrum Jülich GmbH, Jülich, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Müller
authors.affiliation: Department of Ophthalmology, RWTH Aachen University, Pauwelsstr. 30, 52074, Aachen, Germany. pwalter@ukaachen.de.
authors.forename: Peter
authors.identifier: http://orcid.org/0000-0001-8745-6593
authors.initials: P
authors.lastname: Walter
mesh_terms: D000818:Animals; D017209:Apoptosis; D004195:Disease Models, Animal; D004596:Electroretinography; D005260:Female; D007150:Immunohistochemistry; D058449:Intravitreal Injections; D008770:Methylnitrosourea / Q000008:administration & dosage / Q000633:toxicity*; D020419:Photoreceptor Cells, Vertebrate / Q000187:drug effects / Q000473:pathology*; D010865:Pilot Projects; D011817:Rabbits; D012162:Retinal Degeneration / Q000139:chemically induced / Q000175:diagnosis* / Q000503:physiopathology; D041623:Tomography, Optical Coherence
chemical_list: D008770:Methylnitrosourea
keywords: Intravitreal injection; N-methyl-N-nitrosourea (MNU); Rabbit; Retinal degeneration