Abstract
Purpose: :
Increased intraocular pressure (IOP) leads to increased levels of extracellular ATP in the eye. As stimulation of the P2X7 receptor for ATP can elevate intracellular calcium and death of retinal ganglion cells, this increased ATP may be detrimental. The enzyme ecto-nucleoside triphosphate diphosphohydrolase 1 (NTPDase1) acts as a marker for extracellular ATP. NTPDase1 is increased in the retinas from primates and rats with experimental glaucoma. As the optic nerve plays a central role in the ganglion cell pathology in glaucoma, this study asked whether expression of the NTPDase1 was also increased in the optic nerve of rats with experimental glaucoma.
Methods: :
Brown Norway rats received unilateral episcleral vein injections of hypertonic saline. IOP was monitored with TonoLab tonometer 4-6 x weekly and animals were sacrificed 14 days after IOP elevation. For quantitative immunoblots, eyes were fast frozen and protein extracted from 3 mm of optic nerve proximal to the sclera was probed with a polyclonal antibody to rat NTPDase1. Staining was quantified in 3 independent immunoblots from 6 optic nerve pairs. Immunohistochemical analysis was performed on perfused tissue to localize the antigen in the optic nerve.
Results: :
The IOP was elevated in the treated eye in each of 6 rats; the mean pressure over 14 days was 28.03 ± 3.08 mm Hg in experimental and 18.57 ± 0.26 mm Hg in controls. Immunoblots detected only one band at 78 kDa, as predicted for the functional glycosylated form of NTPDase1 protein. Bands were significantly more intense in material from experimental eyes as compared to control (1.58 ± 0.16, Mean ± SE, P<0.05). Intensity increased in proportion to the magnitude of pressure rise. NTPDase1 staining in the optic nerve was brightest in endothelial cells, with preliminary examination suggesting an increase in diffused staining throughout the nerve in glaucomatous eyes.
Conclusions: :
Expression of NTPDase1 is increased in the optic nerve from rats with experimental glaucoma, consistent with a chronic increase in extracellular levels of ATP. Whether this increase contributes to axonal injury in glaucoma, or whether it reflects damage due to other causes, remains to be determined.
Keywords: ganglion cells • intraocular pressure • adenosine