Here, we use a mouse model (DBA/2J) to readdress the location of insult(s) to retinal ganglion cells (RGCs) in glaucoma. We localize an early sign of axon damage to an astrocyte-rich region of the optic nerve just posterior to the retina, analogous to the lamina cribrosa. In this region, a network of astrocytes associates intimately with RGC axons. Using BAX-deficient DBA/2J mice, which retain all of their RGCs, we provide experimental evidence for an insult within or very close to the lamina in the optic nerve. We show that proximal axon segments attached to their cell bodies survive to the proximity of the lamina. In contrast, axon segments in the lamina and behind the eye degenerate. Finally, the Wld^s allele, which is known to protect against insults to axons, strongly protects against DBA/2J glaucoma and preserves RGC activity as measured by pattern electroretinography. These experiments provide strong evidence for a local insult to axons in the optic nerve.

Using a variety of double and triple labeling techniques, we have reevaluated the death of retinal neurons in a mouse model of hereditary glaucoma. Cell-specific markers and total neuron counts revealed no cell loss in any retinal neurons other than the ganglion cells. Within the limits of our ability to define cell types, no group of ganglion cells was especially vulnerable or resistant to degeneration. Retrograde labeling and neurofilament staining showed that axonal atrophy, dendritic remodeling, and somal shrinkage (at least of the largest cell types) precedes ganglion cell death in this glaucoma model. Regions of cell death or survival radiated from the optic nerve head in fan-shaped sectors. Collectively, the data suggest axon damage at the optic nerve head as an early lesion, and damage to axon bundles would cause this pattern of degeneration. However, the architecture of the mouse eye seems to preclude a commonly postulated source of mechanical damage within the nerve head.

Astrocytes respond to all forms of CNS insult and disease by becoming reactive, a nonspecific but highly characteristic response that involves various morphological and molecular changes. Probably the most recognized aspect of reactive astrocytes is the formation of a glial scar that impedes axon regeneration. Although the reactive phenotype was first suggested more than 100 years ago based on morphological changes, the remodeling process is not well understood. We know little about the actual structure of a reactive astrocyte, how an astrocyte remodels during the progression of an insult, and how populations of these cells reorganize to form the glial scar. New methods of labeling astrocytes, along with transgenic mice, allow the complete morphology of reactive astrocytes to be visualized. Recent studies show that reactivity can induce a remarkable change in the shape of a single astrocyte, that not all astrocytes react in the same way, and that there is plasticity in the reactive response.

Reactive gliosis is a complex process that involves changes in gene expression and morphological remodeling. The mouse optic nerve, where astrocytes, microglia and oligodendrocytes interact with retinal ganglion cell axons and each other, is a particularly suitable model for studying the molecular mechanisms of reactive gliosis. We triggered gliosis at the mouse optic nerve head by retro orbital nerve crush. We followed the expression profiles of 14,000 genes from 1 day to 3 months, as the optic nerve formed a glial scar. The transcriptome showed profound changes. These were greatest shortly after injury; the numbers of differentially regulated genes then dropped, returning nearly to resting levels by 3 months. Different genes were modulated with very different time courses, and functionally distinct groups of genes responded in partially overlapping waves. These correspond roughly to two quick waves of inflammation and cell proliferation, a slow wave of tissue remodeling and debris removal, and a final stationary phase that primarily reflects permanent structural changes in the axons. Responses from astrocytes, microglia and oligodendrocytes were distinctively different, both molecularly and morphologically. Comparisons to other models of brain injury and to glaucoma indicated that the glial responses depended on both the tissue and the injury. Attempts to modulate glial function after axonal injuries should consider different mechanistic targets at different times following the insult.

Purpose. To establish the morphologic changes of astrocytes in the glial lamina of glaucomatous mice. Methods. A strain of mice that expresses GFP in individual astrocytes (hGFAPpr-GFP) was crossed into the DBA/2J strain that develops glaucoma. In the resulting strain (D2.hGFAPpr-GFP) we assessed the severity of glaucoma by staining the retina for neurofilaments and counting the neurons of the retinal ganglion cell layer. We observed the morphology of astrocytes in the glial lamina of the optic nerves. Results. D2.hGFAPpr-GFP mice developed glaucoma in an age-dependent manner. Astrocytes in the glial lamina showed morphologic changes that correlated with the severity of glaucoma. The cells showed thickening of processes from 1.3 ± 0.28 μm in nondiseased animals to 1.71 ± 0.46 μm in eyes with moderate glaucoma and 2.1 ± 0.42 μm in those with severe glaucoma. Their spatial coverage, as determined by their convex polygon area, was reduced in eyes with severe glaucoma. The astrocytes in severely glaucomatous optic nerves also showed simplification of their processes. In 6-month-old mice with no obvious signs of degeneration in the retina, we found astrocytes with appendages growing out of primary astrocyte processes into the axon bundles. This localized hypertrophy of processes was never observed in the hGFAPpr-GFP strain. Conclusions. Confirming results after optic nerve crush, astrocytes in glaucomatous optic nerves had thickened and simplified processes, and reduced spatial coverage. We also found evidence of localized sprouting of new processes in early stages of the disease, before detectable changes in ganglion cell number.