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Originally published In Press as doi:10.1167/iovs.08-2404 on September 4, 2008
 (Investigative Ophthalmology and Visual Science. 2009;50:424-431.)
© 2009 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.08-2404
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Immediate Upregulation of Proteins Belonging to Different Branches of the Apoptotic Cascade in the Retina after Optic Nerve Transection and Optic Nerve Crush

Marta Agudo,1,2 Maria C. Pérez-Marín,1 Paloma Sobrado-Calvo,1 Ulrika Lönngren,3 Manuel Salinas-Navarro,1 Isabel Cánovas,1 Francisco M. Nadal-Nicolás,1,2 Jaime Miralles-Imperial,1 Finn Hallböök,3 and Manuel Vidal-Sanz1

1From the Departamento de Oftalmología, Facultad de Medicina, Universidad de Murcia, Murcia, Spain; 2Servicio Murciano de Salud, Hospital Universitario Virgen de la Arrixaca, Murcia, Spain; and the 3Department of Neuroscience, Unit for Developmental Neuroscience, Uppsala University, Uppsala, Sweden.

PURPOSE. To further investigate the molecular signals underlying optic nerve (ON) injury, the authors analyzed in adult control, ON-transected, and ON-crushed retinas the expression pattern and time-course regulation of the following proteins, all of which are linked to apoptosis through different pathways: Stat 1, caspase 11 (inflammation and death), cathepsins C and B (lysosomal death pathway), calpain 1 (endoplasmic reticulum stress), calreticulin (apoptosis marker), Jun (early response), and aryl hydrocarbon receptor (cell cycle arrest).

METHODS. Adult female rats were subjected to intraorbital optic nerve transection (IONT) or intraorbital optic nerve crush (IONC). Protein from naive and ON-injured adult rat retinas was extracted at different times postlesion, and Western blotting experiments were performed. For immunohistofluorescence analyses, retinal ganglion cells (RGCs) were retrogradely identified with fluorogold applied to the superior colliculi 1 week before injury.

RESULTS. Western blotting analyses revealed upregulation of all the analyzed proteins as early as 12 hours postlesion (hpl), peaking at 48 hpl, in agreement with our previous RNA study findings. Furthermore, immunohistofluorescence to radial sections showed that all but Stat 1 were expressed by the primarily injured neurons, the RGCs, as seen by colocalization with fluorogold.

CONCLUSIONS. All analyzed proteins were upregulated in the retina after IONT or IONC as early as 12 hpl, indicating that ON injury regulates several branches of the apoptotic cascade and suggesting that commitment to death might be an earlier event than previously anticipated.






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