Abstract
Purpose: :
Celecoxib® is a 382 Dalton (Da) selective cyclooxygenase 2 (COX-2) inhibitor with potentially neuroprotective properties. Using the optic nerve crush model, we investigate retinal ganglion cell (RGC) survival after topical administration. Thereby, we prove a new mass spectrometry (MS) based approach to analyze the bioavailability of Celecoxib in aqueous humor and retinal tissue.
Methods: :
Unilateral Optic nerve crush was performed in 20 Lewis rats. In ten animals, 0.1% Celecoxib was applied topically (30 µl, n=10) in the crushed eye twice a day. The other 10 animals served as control without treatment. Fundus photographies were taken through a surgical microscope regularly. After ten days, neuronal survival was analyzed through cresyl stained retinal flatmounts (cells/mm2), axon counts after toluidine blue staining or via Brn-3a immunostaining in retinal cross-sections. Aqueous humor was collected post mortem and biopsied retinal tissues were prepared in a raw and rapid extraction method with chloroform for ESI- MS analysis via LTQ Orbitrap. Pure Celecoxib was measured before the complex samples to assure Celecoxib's peak signature and the exact weight.
Results: :
No major differences were visible in the fundi throughout the study. A significant reduction of neuronal loss (p=0.005) and axon loss (p<0.001) was detected in the topically treated group (2272±636 cells/mm2; 548±126 axons) compared to the control (1985±470 cells/mm2; 265±128 axons). Thereby, the analysis of Brn-3a labeled cells in cross-sections approved a reduction of RGC around 10%. ESI-MS measurement of pure Celecoxib revealed a peak at 382.08 Da. In retinal tissues of topically treated eyes, a relatively low intensive peak of exactly 382.08 Da was also available. The agent was absent in aqueous humor.
Conclusions: :
Topically applied selective COX-2 inhibitor promotes RGC survival after optic nerve crush. Moreover, the availability of Celecoxib in retinal tissues could be proven by MS. The reason for the relatively low intensity of the Celecoxib peak is still unclear and might be due to the extraction method. Over all, this new MS approach seems suitable for testing bioavailability of therapeutic agents in ocular tissues, especially with further improvement of the extraction method and later quantification.
Keywords: ganglion cells • neuroprotection