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 astrocytes are a pathological hallmark of many CNS injuries and neurodegenerations. They are characterized by hypertrophy of the soma and processes and an increase in the expression of glial fibrillary acidic protein. Because the cells obscure each other in immunostaining, little is known about the behavior of a single reactive astrocyte, nor how single astrocytes combine to form the glial scar. We have investigated the reaction of fibrous astrocytes to axonal degeneration using a transgenic mouse strain expressing enhanced green fluorescent protein in small subsets of astrocytes. Fibrous astrocytes in the optic nerve and corpus callosum initially react to injury by hypertrophy of the soma and processes. They retract their primary processes, simplifying their shape and dramatically reducing their spatial coverage. At 3 d after crush, quantitative analysis revealed nearly a twofold increase in the thickness of the primary processes, a halving of the number of primary processes leaving the soma and an eightfold reduction in the spatial coverage. In the subsequent week, they partially reextend long processes, returning to a near-normal morphology and an extensive spatial overlap. The resulting glial scar consists of an irregular array of astrocyte processes, contrasting with their original orderly arrangement. These changes are in distinct contrast to those reported for reactive protoplasmic astrocytes of the gray matter, in which the number of processes and branchings increase, but the cells continue to maintain nonoverlapping individual territories throughout their response to injury.

Reactive remodeling of optic nerve head astrocytes is consistently observed in glaucoma and other optic nerve injuries. However, it is unknown whether this reactivity is beneficial or harmful for visual function. In this study, we used the Cre recombinase (Cre)–loxP system under regulation of the mouse glial fibrillary acidic protein promoter to knock out the transcription factor signal transducer and activator of transcription 3 (STAT3) from astrocytes and test the effect this has on reactive remodeling, ganglion cell survival, and visual function after experimental glaucoma and nerve crush. After injury, STAT3 knockout mice displayed attenuated astrocyte hypertrophy and reactive remodeling; astrocytes largely maintained their honeycomb organization and glial tubes. These changes were associated with increased loss of ganglion cells and visual function over a 30-day period. Thus, reactive astrocytes play a protective role, preserving visual function. STAT3 signaling is an important mediator of various aspects of the reactive phenotype within optic nerve astrocytes.

Much of what we know about astrocyte form and function is derived from the study of gray matter protoplasmic astrocytes, whereas white matter fibrous astrocytes remain relatively unexplored. Here, we used the ribotag approach to isolate ribosome-associated mRNA and investigated the transcriptome of uninjured fibrous astrocytes from three regions: unmyelinated optic nerve head, myelinated optic nerve proper, and corpus callosum. Astrocytes from each region were transcriptionally distinct and we identified region-specific astrocyte genes and pathways. Energy metabolism, particularly oxidative phosphorylation and mitochondrial protein translation emerged as key differentiators of astrocyte populations. Optic nerve astrocytes expressed higher levels of neuroinflammatory pathways than corpus callosum astrocytes and we further identified CARTPT as a new marker of optic nerve head astrocytes. These previously uncharacterized transcriptional profiles of white matter astrocyte types reveal their functional diversity and a greater heterogeneity than previously appreciated.

The optic nerve head is thought to be the site of initial injury to retinal ganglion cell injury in glaucoma. In the initial segment of the optic nerve directly behind the globe, the ganglion cell axons are unmyelinated and come into direct contact to astrocytes, suggesting that astrocytes may play a role in the pathology of glaucoma. As in other parts of the CNS, optic nerve head astrocytes respond to injury by characteristic changes in cell morphology and gene expression profile. Using RNA-sequencing of glaucomatous optic nerve heads, single-cell PCR, and an in-vivo assay, we demonstrate that an up-regulation of astrocytic phagocytosis is an early event after the onset of increased intraocular pressure. We also show that astrocytes in the glial lamina of the optic nerve are apparently functionally heterogeneous. At any time, even in naïve nerves, some of the cells show signs of reactivity—process hypertrophy, high phagocytic activity, and expression of genetic markers of reactivity whereas neighboring cells apparently are inactive. A period of increased intraocular pressure moves more astrocytes towards the reactive phenotype; however, some cells remain unreactive even in glaucomatous nerves.

Background: The optic nerve is an important tissue in glaucoma and the unmyelinated nerve head region remains an important site of many early neurodegenerative changes. In both humans and mice, astrocytes constitute the major glial cell type in the region, and in glaucoma they become reactive, influencing the optic nerve head (ONH) microenvironment and disease outcome. Despite recognizing their importance in the progression of the disease, the reactive response of optic nerve head astrocytes remains poorly understood. Methods: To determine the global reactive response of ONH astrocytes in glaucoma we studied their transcriptional response to an elevation in IOP induced by the microbead occlusion model. To specifically isolate astrocyte mRNA in vivo from complex tissues, we used the ribotag method to genetically tag ribosomes in astrocytes, restricting analysis to astrocytes and enabling purification of astrocyte-associated mRNA throughout the entire cell, including the fine processes, for bulk RNA-sequencing. We also assessed the response of astrocytes in the more distal myelinated optic nerve proper (ONP) as glaucomatous changes manifest differently between the two regions. Results: Astrocytes of the optic nerve exhibited a region-specific and temporally distinct response. Surprisingly, ONH astrocytes showed very few early transcriptional changes and ONP astrocytes demonstrated substantially larger changes over the course of the experimental period. Energy metabolism, particularly oxidative phosphorylation and mitochondrial protein translation emerged as highly upregulated processes in both ONH and ONP astrocytes, with the former showing additional upregulation in antioxidative capacity and proteolysis. Interestingly, optic nerve astrocytes demonstrated a limited neuroinflammatory response, even when challenged with a more severe elevation in IOP. Lastly, there were a greater number of downregulated processes in both astrocyte populations compared to upregulated processes. Conclusion: Our findings demonstrate an essential role for energy metabolism in the response of optic nerve astrocytes to elevated IOP, and contrary to expectations, neuroinflammation had a limited overall role. The transcriptional response profile is supportive of the notion that optic nerve astrocytes have a beneficial role in glaucoma. These previously uncharacterized transcriptional response of optic nerve astrocytes to injury reveal their functional diversity and a greater heterogeneity than previously appreciated.