Increased intraocular pressure (IOP) leads, by an unknown mechanism, to apoptotic retinal ganglion cell (RGC) death in glaucoma. We now report cleavage of the autoinhibitory domain of the protein phosphatase calcineurin (CaN) in two rodent models of increased IOP. Cleaved CaN was not detected in rat or mouse eyes with normal IOP. In in vitro systems, this constitutively active cleaved form of CaN has been reported to lead to apoptosis via dephosphorylation of the proapoptotic Bcl-2 family member, Bad. In a rat model of glaucoma, we similarly detect increased Bad dephosphorylation, increased cytoplasmic cytochrome c (cyt c), and RGC death. Oral treatment of rats with increased IOP with the CaN inhibitor FK506 led to a reduction in Bad dephosphorylation and cyt c release. In accord with these biochemical results, we observed a marked increase in both RGC survival and optic nerve preservation. These data are consistent with a CaN-mediated mechanism of increased IOP toxicity. CaN cleavage was not observed at any time after optic nerve crush, suggesting that axon damage alone is insufficient to trigger cleavage. These findings implicate this mechanism of CaN activation in a chronic neurodegenerative disease. These data demonstrate that increased IOP leads to the initiation of a CaN-mediated mitochondrial apoptotic pathway in glaucoma and support neuroprotective strategies for this blinding disease.retina ͉ optic nerve ͉ apoptosis

Background-Extracellular deposition of low-density lipoprotein (LDL) in the arterial wall is an essential early step in atherosclerosis. This process preferentially occurs at arterial branch points, reflecting a regional variation in lipoprotein-arterial wall interactions. In this study, we characterized the submicron microstructure of arterial wall collagen and elastin to evaluate its potential role in regional LDL deposition. Methods and Results-With 2-photon microscopy, we used the intrinsic optical properties of collagen and elastin to determine the arterial wall macromolecular microstructure in fresh porcine and murine arteries. This optical approach generated unique nondestructive en face 3-dimensional views of the wall. The collagen/elastin microstructure was found to vary with the topology of the arterial bed. A nearly confluent elastin surface layer was present throughout but was missing at atherosclerosis-susceptible branch points, exposing dense collagen-proteoglycan complexes. In LDL binding studies, this luminal elastin layer limited LDL penetration, whereas its absence at the branches resulted in extensive LDL binding. Furthermore, LDL colocalized with proteoglycans with a sigmoidal dose dependence (inflection point, Ϸ130 mg LDL/dL). Ionic strength and competing anions studies were consistent with the initial interaction of LDL with proteoglycans to be electrostatic in nature. Conclusions-This optical sectioning approach provided a robust 3-dimensional collagen/elastin microstructure of the arterial wall in fresh samples. At atherosclerosis-susceptible vascular branch points, the absence of a luminal elastin barrier and the presence of a dense collagen/proteoglycan matrix contribute to increased retention of LDL. (Circulation.

Retinal ganglion cells (RGCs) are the only output neurons of the retina, and their degeneration after damage to the optic nerve or in glaucoma is a well established system for studying apoptosis in the central nervous system. Frequently used procedures for assessing RGC number in retinal flat mounts suffer from two problems: RGC densities are not uniform across retinal flat mounts, and density measures may therefore not reflect total number, and flat mounts do not allow efficient use of tissue.To overcome these problems we developed a stereological method for efficiently assessing RGC number in cryostat sections of the retina. We empirically demonstrate that only ~1:20 sections need be assessed to accurately estimate the total number of RGCs in the rat retina, providing ample tissue for additional studies in the same retina and saving considerably on more exhaustive sampling strategies. Using this method, we estimate that there are 86,282 ± 4,759 RGCs in the normal BrownNorway rat retina. These counts match well with estimates of axon counts in optic nerve. In a pilot study of experimental glaucoma, we determined a reduction of RGCs to 53,862 ± 4272 (p<0.05). The current technique should prove advantageous to assess neuroprotective strategies in these experimental models.