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Photoreceptor degeneration by intravitreal injection of N-methyl-N-nitrosourea (MNU) in rabbits: a pilot study

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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.

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References

  1. Berson EL (1993) Retinitis pigmentosa. The Friedenwald Lecture. Invest Ophthalmol Vis Sci 34:1659–1676

    CAS  PubMed  Google Scholar 

  2. Gekeler F, Kobuch K, Schwahn HN, Stett A, Shinoda K, Zrenner E (2004) Subretinal electrical stimulation of the rabbit retina with acutely implanted electrode arrays. Graefes Arch Clin Exp Ophthalmol 242:587–596

    Article  PubMed  Google Scholar 

  3. Jones BW, Marc RE (2005) Retinal remodeling during retinal degeneration. Exp Eye Res 81:123–137

    Article  CAS  PubMed  Google Scholar 

  4. Menzel-Severing J, Laube T, Brockmann C, Bornfeld N, Mokwa W, Mazinani B, Walter P, Roessler G (2012) Implantation and explantation of an active epiretinal visual prosthesis: 2-year follow-up data from the EPIRET3 prospective clinical trial. Eye 26:501–509

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Feucht M, Laube T, Bornfeld N, Walter P, Velikay-Parel M, Hornig R, Richard G (2005) Entwicklung einer epiretinalen Prothese zur stimulation der humanen Netzhaut. Ophthalmologe 102:688–691

    Article  CAS  PubMed  Google Scholar 

  6. Roessler G, Laube T, Brockmann C, Kirschkamp T, Mazinani B, Goertz M, Koch C, Krisch I, Sellhaus B, Trieu HK, Weis J, Bornfeld N, Röthgen H, Messner A, Mokwa W, Walter P (2009) Implantation and explantation of a wireless epiretinal retina implant device: observations during the EPIRET3 prospective clinical trial. Invest Ophthalmol Vis Sci 50:3003–3008

    Article  PubMed  Google Scholar 

  7. Wang W, Fernandez de Castro J, Vukmanic E, Zhou L, Emery D, Demarco PJ, Kaplan HJ, Dean DC (2011) Selective rod degeneration and partial cone inactivation characterize an iodoacetic acid model of swine retinal degeneration. Invest Ophthalmol Vis Sci 52:7917–7923

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Rösch S, Johnen S, Mazinani B, Müller F, Pfarrer C, Walter P (2015) The effects of iodoacetic acid on the mouse retina. Graefes Arch Clin Exp Ophthalmol 253(1):25–35

    Article  PubMed  Google Scholar 

  9. Tsubura A, Yoshizawa K, Kiuchi K, Moriguchi K (2003) N-Methyl-N-nitrosourea-induced retinal degeneration in animals. Acta Histochem Cytochem 36:263–270

    Article  CAS  Google Scholar 

  10. Rösch S, Johnen S, Mataruga A, Müller F, Pfarrer C, Walter P (2014) Selective photoreceptor degeneration by intravitreal injection of N-methyl-N-nitrosourea. Invest Ophthalmol Vis Sci 55(3):1711–1723

    Article  PubMed  Google Scholar 

  11. Schreiber D, Jänisch W, Warzok R, Tausch H (1969) Induction of brain and spinal cord tumors in rabbits with N-methyl-N-nitrosourea. Z Gesamte Exp Med 150:76–86

    Article  CAS  PubMed  Google Scholar 

  12. Durairaj C, Shah J, Senapati S, Kompella UB (2009) Prediction of vitreal half-life based on drug physicochemical properties: quantitative structure–pharmacokinetic relationships (QSPKR). Pharm Res 26(5):1236–1260

  13. Ye YF, Gao YF, Xie HT, Wang HJ (2014) Pharmacokinetics and retinal toxicity of various doses of intravitreal triamcinolone acetonide in rabbits. Mol Vis 20:629–636

    PubMed  PubMed Central  Google Scholar 

  14. Wood D, Wood J (1975) Pharmacologic and biochemical considerations of dimethyl sulfoxide. Ann N Y Acad Sci 243:7–19

    Article  CAS  PubMed  Google Scholar 

  15. Marmor MF, Fulton AB, Holder GE, Miyake Y, Brigell M, Bach M (2009) ISCEV Standard for full-field clinical electroretinography (2008 update). Doc Ophthalmol 118:69–77

    Article  CAS  PubMed  Google Scholar 

  16. Hirota R, Kondo M, Ueno S, Sakai T, Koyasu T, Terasaki H (2012) Photoreceptor and post-photoreceptoral contributions to photopic ERG a-wave in rhodopsin P347L transgenic rabbits. Invest Ophthalmol Vis Sci 53(3):1467–1472

    Article  CAS  PubMed  Google Scholar 

  17. Cang B, Hawes NL, Hurd RE, Davisson MT, Nusinowitz S, Heckenlively JR (2002) Retinal degeneration mutants in the mouse. Vision Res 42:517–525

    Article  Google Scholar 

  18. Chang B, Hawes NL, Pardue MT et al (2007) Two mouse retinal degenerations caused by missense mutations in the beta-subunit of rod cGMP phosphodiesterase gene. Vision Res 47:624–633

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Wang S, Lu B, Lund RD (2005) Morphological changes in the Royal College of Surgeons rat retina during photoreceptor degeneration and after cell-based therapy. J Comp Neurol 491:400–417

    Article  PubMed  Google Scholar 

  20. Cuenca N, Pinilla I, Sauvé Y, Lund R (2005) Early changes in synaptic connectivity following progressive photoreceptor degeneration in RCS rats. Eur J Neurosci 22:1057–1072

    Article  PubMed  Google Scholar 

  21. Rösch S, Aretzweiler C, Müller F, Walter P (2016) Evaluation of retinal function and morphology of the pink-eyed Royal College of Surgeons (RCS) rat: a comparative study of in vivo and in vitro methods. Curr Eye Res. doi:10.1080/02713683.2016.1179333

    PubMed  Google Scholar 

  22. Kinoshita J, Iwata N, Maejima T, Imaoka M, Kimotsuki T, Yasuda M (2015) N-Methyl-N-nitrosourea-induced acute alteration of retinal function and morphology in monkeys. Invest Ophathlmol Vis Sci 56(12):7146–7158

    Article  CAS  Google Scholar 

  23. Taylor L, Arnér K, Ghosh F (2016) N-methyl-N-nitrosourea-induced neuronal cell death in a large animal model of retinal degeneration in vitro. Exp Eye Res 148:55–64

    Article  CAS  PubMed  Google Scholar 

  24. Chen YY, Liu SL, Hu DP, Xing YQ, Shen Y (2014) N-methyl-N-nitrosourea-induced retinal degeneration in mice. Exp Eye Res 121:102–113

    Article  CAS  PubMed  Google Scholar 

  25. Reisenhofer M, Balmer J, Zulliger R, Enzmann V (2015) Multiple programmed cell death pathways are involved in N-methyl-N-nitrosourea-induced photoreceptor degeneration. Graefes Arch Clin Exp Ophthalmol 253(5):721–731

    Article  CAS  PubMed  Google Scholar 

  26. Aplin FP, Luu CD, Vessey KA, Guymer RH, Shepherd RK, Fletcher EL (2014) ATP-induced photoreceptor death in a feline model of retinal degeneration. Invest Ophthalmol Vis Sci 55(12):8319–8329

    Article  CAS  PubMed  Google Scholar 

  27. Hikichi T, Ueno N, Chakrabarti B, Trempe CL (1996) Vitreous changes during ocular inflammation induced by interleukin 1 beta. Jpn J Ophthalmol 40(3):297–302

    CAS  PubMed  Google Scholar 

  28. Williams DL (2007) Rabbit and rodent ophthalmology. EJCAP 17(3):242–252

    Google Scholar 

  29. Reichenbach A, Schnitzer J, Friedrich A, Ziegert W, Brückner G, Schober W (1991) Development of the rabbit retina. Anat Embryol 183:287–297

    Article  CAS  PubMed  Google Scholar 

  30. Eisner G, Bachmann E (1974) Vergleichende morphologische Spaltlampenuntersuchung des Glaskörpers von Schaf, Schwein, Hund, Affen und Kaninchen. Graefes Arch Clin Exp Ophthalmol 192:9–17

    Article  CAS  Google Scholar 

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Acknowledgments

The authors thank Prof. Dr. R. Tolba and the Institute for Laboratory Animal Science of the University Hospital RWTH Aachen for the great opportunity to access one of their laboratories and to perform our experiments under special safety precautions. We further thank Jakob Becker and Detlef Emonts-Holley (University Hospital RWTH Aachen) for their support in preparing images for OCT and funduscopy. And we thank Christoph Aretzweiler (Institute of Complex Systems, Cellular Biophysics, ICS-4, Forschungszentrum Jülich GmbH) for the excellent support in the preparation of the eyes and in immunohistochemical stainings.

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Authors and Affiliations

  1. Department of Ophthalmology, RWTH Aachen University, Pauwelsstr. 30, 52074, Aachen, Germany

    Sarah Rösch, Claudia Werner & Peter Walter

  2. Institute of Complex Systems, Cellular Biophysics, ICS-4, Forschungszentrum Jülich GmbH, Jülich, Germany

    Frank Müller

  3. Clinical Department of Small Animals, University of Leipzig, Leipzig, Germany

    Sarah Rösch

Authors
  1. Sarah Rösch
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  2. Claudia Werner
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  4. Peter Walter
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Corresponding author

Correspondence to Peter Walter.

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Funding

The Deutsche Forschungsgemeinschaft (DFG) provided financial support in the form of research grants to PW (WA 1472/6-1) and FM (MU 3036/3-1 and 3036/2-1). The sponsor had no role in the design or conduct of this research.

Conflict of interest

All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge, or beliefs) in the subject matter or materials discussed in this manuscript.

Animal experiments

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

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Rösch, S., Werner, C., Müller, F. et al. Photoreceptor degeneration by intravitreal injection of N-methyl-N-nitrosourea (MNU) in rabbits: a pilot study. Graefes Arch Clin Exp Ophthalmol 255, 317–331 (2017). https://doi.org/10.1007/s00417-016-3531-7

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  • DOI: https://doi.org/10.1007/s00417-016-3531-7

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