Experimental Study On Graded Optic Nerve Injury And Regeneration In Rats

ObjectiveTo found a calibrated animal model of graded optic nerve injury (ONI) in rats focusing on quantification of injury intensity, injury severity and the correlation between them; also to investigate the secondary injury of retinal ganglion cell (RGC) and axon, nerve regeneration ability and gene expression profiling following partial ONI.Methods1. A pair of cross-action forceps and a pair of artery clips with constant pressures are used to clip rat optic nerves with variable durations to found graded ONI animal models. The injury severities are measured by the count of RGCs retrogradely labeled by FluoroGold and axons by transmission electron microscopy (TEM). Ocular blood supply is evaluated by transcardiacly perfused Luxol Fast Blue post-injury.2. The secondary injury following graded ONIs is reflected by time course, survival ratio and loss ratio of RGCs and axons. The regeneration is observed by TEM and explored by immunohistochemical detection of growth-associated protein-43 (GAP-43) by TEM and modified Glees silver impregnation on which the regeneration index (RI) is based to evaluate the regenerating ability following graded ONI.3. Gene expression patterns in axotomized optic nerves and retinae are investigated with high-density DNA microarrays.Results1. The graded ONI animal models are successfully founded in rats without retinal ischeia post-trauma. The injury intensities can be well-defined by impulse or averaged impulse, while injury severity by the count of RGCs retrogradely labeled by FluoroGold and axons by TEM. RGCs count serves better than axon count. The severer the injury intensity, the less the number of RGCs and axons. The correlation between injury impulse and RGCs is fit for power function.2. Secondary injury following graded ONI perpetuates further loss of RGCs and axons. The declining trend is fit for exponential pattern with an acute loss phase within two weeks after injury and an ensued slowly loss phase lasting months, while this process is self-limiting after mild optic nerve injury.3. There are many clustered, regional distributed bundles of putative unmyelinated regenerating axons in post-injury region. GAP-43 is expressed in both paranodal region of nerve fibers proximal to the lesion site and fine unmyelinated fibers distal to the lesion site.4. Regeneration index (RI) can well-reflect regenerating ability following graded ONI. Different graded injury is followed different regenerating ability, mild injury with better ability.5. A large-scale gene expression profiling is investigated following partial ONI, including those genes whose functions are related with cell survival, cytoskeleton, extracellular matrix and cell adhension, free radicals and oxidative damage, energy and metabolism, inflammation, neurotransmission and ion transport, signal transduction, structural protein, transcription and translation.Conclusions1. A calibrated graded ONI animal model is successfully founded byclipping optic nerves with a pair of cross-action forceps. The model can be evaluated with impulse, averaged impulse and RGCs count standing for injury intensity and injury severity respectively.2. Different graded ONI ensues different secondary reaction and regenerating ability, mild injury with a self-limiting secondary injury and a better regenerating ability.3. A global view on the gene expression pattern following ONI is crucial to discover the mechanism of post-injury reaction and regeneration.