Abstract
Purpose: :
Following optic nerve stroke (AION), retinal ganglion cells (RGCs) die. We wanted to determine if stroke–affected RGCs undergo visible stress prior to death. We used a cfos/lacZ X Thy–1/CFP double transgenic mouse model to directly visualize RGCs and to compare early cell stress and later cell death after rodent anterior ischemic optic neuropathy (rAION).
Methods: :
Mouse rAION was induced as previously described (Goldenberg–Cohen et al, IOVS, Vol.46, 2005). Doubly transgenic mice were generated by cross breeding the cfos promotor/lacz expressor and Thy–1 promotor/Cyan Fluorescent Protein (CFP) expressors on a C57B6/6J background. Whole mouse retina was examined at 3 days and 21days post rAION induction. CFP expression was viewed directly with a confocal microscope at 405nm emission. LacZ expression was examined with whole mount immunohistochemistry using a mouse anti–ß–galactosidase antibody and a secondary antibody conjugated with Texas Red, using a confocal microscope with at emission 543nm. Direct lacZ activity was also evaluated using the BlueII reagent. To compare optic nerve damage and RGC loss, we used both toluidine blue staining and transmission electron–microscopy (TEM) to examine optic nerve cross sections of rAION induced mice.
Results: :
Immunohistochemistry showed regional ß–galactosidase expression in RGCs 3 days post rAION induction. Strong lacZ expression was seen centrally and in a regional radial pattern, and gradually faded out at the periphery of the affected regions. Direct lacZ activity (ß–galactosidase activity) was a much less robust marker of total cell stress than immunohistochemical methods. CFP expression 21 days post rAION again showed a radial loss of cells. Toluidine blue stained optic nerve cross sections showed both central and peripheral axonal loss. TEM examination also revealed regional axonal degeneration.
Conclusions: :
Our results show that the pattern of ß–galactosidase–positive cells overlap well with CFP positive cells, indicating ganglion cell specific staining. Thus, cfos activation indicates early RGC stress. The distribution and pattern of ß–galactosidase positive cells is similar to that seen with ultimate loss of CFP positive cells. This correlation suggests that many of the cells which are under stress immediately after axonal stroke may later die. The radial pattern of loss suggests that there is regional stereotopic representation and distribution from the mouse optic nerve to the retina.
Keywords: ganglion cells • microscopy: confocal/tunneling • immunohistochemistry