Abstract
Purpose: :
Last year we showed that one month after intravitreal injection of an AAV expressing an allotopic version of the human LHON R340H mutant ND4 induced optic neuropathy in mice. We have now describe the mechanisms of the injury and enumerate the histopathology of the retina and optic nerve at 6 months.
Methods: :
Twenty DBA/1J mice received intravitreal injections of 2 ul of an allotopic version of the LHON mutant ND4, encoding for the histidine substitution at amino acid 340, into the right eyes and AAV–GFP into the left eyes. One and 6 months later the optic nerves and retina were excised and examined by light and transmission electron microscopy. RGCs, myelin fiber area and optic nerve head area were enumerated by computerized morphometric analysis. The mechanisms by which the allotopic mutant ND4 induced RGC injury was tested by measurements of ATP synthesis using complex I substrates malate and pyruvate, apoptosis by TUNEL assay, reactive oxygen species by the fluorescent dyes DHE (superoxide) and DCDFA (hydrogen peroxide) and mitochondrial membrane potential by MitoTracker Red.
Results: :
The allotopic version of LHON mutant ND4 resulted in mitochondrial and cell toxicity, caused optic nerve head edema, retinal ganglion cells loss, as well as optic nerve degeneration and demyelination. Relative to control eyes inoculated with GFP, RGC counts were reduced by 9% (p>0.05) 1 month after injection with mutant R340H ND4. Loss of RGCs was progressive and increased to 24% (p<0.001) at 6 months. In the optic nerve myelin fiber area was reduced by 23% (p<0.001) at one month and increased to 35% (p<0.001) 6 months after injection of the mutant ND4. Indicative of optic disk edema, optic nerve head areas increased by 69% (p<0.001) at one month, but diminished to 28% (p>0.05)at 6 months. The rate of ATP synthesis was not reduced by infection of RGCs with AAV expressing the mutant ND4. However, generation of hydrogen peroxide was increased by 24% (p>0.05), and superoxide by 33% (p<0.05). Mitochondrial membrane potential was reduced by 55% (p<0.00) and TUNEL positive cells increased 40% (p<0.01) .
Conclusions: :
Mutant human LHON ND4 induces a progressive degeneration of RGCs in the mouse eye mediated predominantly by ROS injury inducing apoptosis rather than by loss of oxidative phosphorylation.
Keywords: neuro-ophthalmology: optic nerve • mitochondria • ganglion cells