Caspase-3-independent photoreceptor degeneration by N-methyl-N-nitrosourea (MNU) induces morphological and functional changes in the mouse retina.
BACKGROUND
Retinal degeneration is followed by significant changes in the structure and function of photoreceptors in humans and several genetic animal models. However, it is not clear whether similar changes occur when the degeneration is induced pharmacologically. Therefore, our aim was to investigate the influence of retinotoxic N-methyl-N-nitrosourea (MNU) on the function, morphology and underlying molecular pathways of programmed cell death.


METHODS
C57/BL6 mice were injected with different doses of MNU, and function was determined by analysing optokinetic reflex measurements and cued water maze results at several time points post-injection. Morphometric measurements were also taken from H&E-stained paraffin eye sections. TUNEL-positive cells and caspase-3 and -6 were detected by immunohistochemistry. To assess the molecular changes leading to cell death, qRT-PCR from neurosensory retina mRNA was performed.


RESULTS
The application of MNU led to an instant decrease in function and a delayed decrease in the thickness of the retinal outer nuclear layer. These responses were observed in the absence of any structural changes in the retinal pigment epithelium. The degeneration of the photoreceptor cell layer was highest with 60 mg/kg MNU. The assessment of TUNEL-positive cells visualised cell death after treatment, but no detectable caspase-3 activity was observed concomitant with these changes. qRT-PCR revealed the possible involvement of the inflammatory mediator caspase-1 and endoplasmic reticulum stress-mediated apoptosis by caspase-12.


CONCLUSIONS
MNU leads to the dose-dependent degeneration of photoreceptor cells in mice by caspase-3-independent pathways and is, therefore, a suitable model to study retinal degeneration in an animal model.
Department of Ophthalmology, Inselspital, University of Bern, Freiburgstrasse 14, 3010, Bern, Switzerland.
Rahel
R
Zulliger
Stéphanie
S
Lecaudé
Sylvie
S
Eigeldinger-Berthou
Ute E K
UE
Wolf-Schnurrbusch
Volker
V
Enzmann
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3 / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D008297:Male; D008770:Methylnitrosourea / Q000633:toxicity; D051379:Mice; D008810:Mice, Inbred C57BL; D020419:Photoreceptor Cells, Vertebrate / Q000201:enzymology / Q000473:pathology*; D011597:Psychomotor Performance / Q000502:physiology; D012162:Retinal Degeneration / Q000139:chemically induced / Q000201:enzymology / Q000503:physiopathology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014792:Visual Acuity / Q000502:physiology
D008770:Methylnitrosourea; C505409:Casp3 protein, mouse; D053148:Caspase 3
Comp Biochem Physiol B Biochem Mol Biol. 2008 Sep;151(1):10-27
18602321
Prog Neurobiol. 1999 Jun;58(3):279-95
10341364
Invest Ophthalmol Vis Sci. 2004 Dec;45(12):4611-6
15557474
Vision Res. 2002 Feb;42(4):517-25
11853768
Cell Mol Life Sci. 2005 Jan;62(2):227-38
15666094
Exp Eye Res. 2007 Feb;84(2):285-92
17137578
Lancet. 2006 Nov 18;368(9549):1795-809
17113430
Pathol Int. 1997 Jun;47(6):377-83
9211525
Ann N Y Acad Sci. 2003 Dec;1010:186-94
15033718
J Toxicol Sci. 2008 Oct;33(4):493-501
18827450
J Biol Chem. 2002 Sep 13;277(37):34287-94
12097332
Br J Cancer. 1970 Sep;24(3):588-98
4319944
Biochem Pharmacol. 2008 Dec 1;76(11):1374-82
18762176
Exp Neurol. 2007 Mar;204(1):39-48
17069801
Acta Biochim Biophys Sin (Shanghai). 2005 Nov;37(11):719-27
16270150
Exp Cell Res. 1986 Jun;164(2):273-81
2940104
J Neurosci Methods. 1984 May;11(1):47-60
6471907
Cancer Res. 2008 Jun 15;68(12):4491-3
18559490
Toxicol Pathol. 2008 Dec;36(7):926-31
18827072
Brain Res Brain Res Rev. 2001 Aug;36(1):60-90
11516773
Exp Eye Res. 2006 Mar;82(3):441-8
16171805
Scand J Immunol Suppl. 1992;11:192-4
1514040
Cell Mol Life Sci. 2005 Sep;62(17):1913-24
15990959
Lab Invest. 1999 Nov;79(11):1359-67
10576206
Mol Neurobiol. 2008 Dec;38(3):253-69
18982459
Biochem Pharmacol. 2008 Dec 1;76(11):1365-73
18761323
Cell Host Microbe. 2008 Sep 11;4(3):198-208
18779046
Ophthalmic Res. 2005 Mar-Apr;37(2):72-82
15746562
In Vivo. 1996 Sep-Oct;10(5):483-8
8899426
Cancer Biol Ther. 2006 Jul;5(7):763-5
16921264
Immunol Rev. 2009 Jan;227(1):95-105
19120479