The Time Course of Gene Expression during Reactive Gliosis in the Optic Nerve

Abstract

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.

Figure 1. The anatomy of the ONH.

A. The optic nerve of the left eye was exposed superiorly by blunt dissection of the conjunctiva, and crushed at about 1 mm behind the globe. The white arrow points at the optic nerve. B. The isolated eye, optic nerve and ONH. The black arrowhead points at the crush site. The bracket and the black arrow indicate the focus of this study – the ONH and the myelination transition zone. A magnified view of this region is shown in the lower right insert. Note the transition from translucent ONH to opaque myelination transition zone. Also note that some of the sclera was left intentionally on this ONH for demonstration purpose, but it was further removed from all the ONHs used in RNA extraction. The upper left insert shows the schematic view of the optic nerve crush. The region circled with the red line is displayed in C–F. C–F. The distribution of the glial cells in the normal ONH. Immunohistochemistry against cell markers for astrocyte (GFAP), microglia (IBA1) and oligodendrocyte (MBP) was performed in longitudinal sections. The retina is on the left side in each image. The white circle in F outlines the tissue used in the gene expression assays. Scale bar, 100 microns.

Figure 2. The timeline of retinal ganglion cell axon degeneration after optic nerve crush.

The localization of the crush site and the glial lamina, where the microscopic images were taken, are indicated in the cartoon. Ganglion cell axon degeneration at the ONH was evaluated by immunohistochemistry against neurofilament SMI32. The retina is to the left of the image, and the crush site is to the right of the image. The axon swelling (arrowheads) was already visible at the distal end (right edge of the image) of the ONH 1 day after the crush, and spread to the proximal end (left edge of the image) at 3 days. The axon degeneration became severe at 1 week with most of remaining axons appearing swollen and fragmented. By 3 weeks, almost all the axons were lost. Scale bar, 50 microns.

Figure 3. The loss of ganglion cells in the retina.

A. Ganglion cell soma degeneration was estimated by counting the neurons in the mid-peripheral retina with the nuclear dye To-Pro1. B. The crushed eyes had a clear reduction 1 week after crush and reached a plateau (about 40% neuron loss in ganglion cell layer) by 3 weeks. The contralateral eyes were not different from the naïve controls at any time point. (* Two-tailed paired t-test p<0.05).

Figure 4. Microarray data of the expression levels of glial marker genes after crush.

Overall, there was significant down-regulation in astrocyte markers and up-regulation in microglia markers, but both returned to baseline levels at 3 weeks. The down-regulation in oligodendrocyte markers did not recover. The smooth lines represent the naïve and contralateral control samples and the lines with dot markers represent crushed samples. They were both normalized to the mean of all the naïve controls and the contralateral controls of the same gene.

Figure 5. The morphology of astrocytes and microglia after crush.

The localization of the crush sites is indicated with white bars. Astrocyte at the crush site degenerated and formed a GFAP-IR free zone within a few days. Astrocytes from both the proximal and distal sides of the optic nerve then migrated into this zone and rebuilt the architecture. The GFAP-IR at the ONH intensified after crush, indicating reactive astrogliosis. There were high density of IBA1-IR positive microglia cells/macrophages at the crush site. They were strongly activated and had retracted processes and enlarged cell bodies. This reactive morphology resolved with time. The microglia located further away from the crush site, both distally and proximally, including the ONH, retained stratified morphology. Scale bar, 200 microns.

Figure 6. Clustering analysis of the samples and the genes based on the 2056 probe sets that were differentially expressed after crush.

Each column is one array sample. Each row is one probe set that had been normalized to have mean 0 and standard deviation 1. The crushed samples (filled squares above the heatmap) from each time point formed unique subgroups before merging into one big cluster, which was distinctly different from the cluster of the naïve and the contralateral controls (open squares). The genes changed expression levels in a time dependent manner. They were divided into 8 major groups (A–H) based on their expression patterns. The pathways that were enriched in each group were listed in Table 2.

Figure 7. Microarray data of the expression levels of selected genes in the enriched pathways.

Panels A–H corresponded to Groups A–H in Figure 5 and Table 2. The x-axes were time points after crush −1 day (1D), 3 days (3D), 1 week (1W), 3 weeks (3W), and the y-axes were mean fold changes between the crushed samples and the contralateral controls.

Figure 8. Microglia/macrophages proliferated after crush injury.

Immunohistochemistry against BrdU, IBA1 and GFAP were performed in longitudinally sectioned optic nerves. A1–4. Contralateral control, no BrdU-IR was detected. B1–4. 1 week after crush, BrdU-IR colocalized with IBA1-IR. Arrowheads point at the crush site. C1–3. Magnified views of the three outlined regions in B4. C1 is the ONH, C2 is the crush site, and C3 is distal optic nerve. D1–3. Transvers sections of the ONH at the three locations indicated in C1. D1 is pre-lamina, D2 is the glial lamina, and D3 is post-lamina. Arrowheads in C1–3 and D1–3 point at colocalized BrdU-IR and IBA1-IR. The insert in D2 shows one microglia/macrophage with elongated BrdU-IR positive nuclei, indicating active cell division. Scale bars are 200 microns in A and B, 50 microns in C and D, and 20 microns in D2 insert.

Figure 9. The up-regulation of tissue remodeling genes.

A. Microarray data of the mRNA levels in crushed eyes normalized to the contralateral control eyes. B. qRT-PCR confirmation of the results in A. C. TNC protein levels increased in ONH at 3 days after crush. Images show the longitudinal sections at low magnification (first row, scale bar 200 microns) and at high magnification (second row, scale bar 50 microns), and the transvers sections through the unmyelinated glial lamina (bottom row, scale bar 50 mcirons). D. THBS1 protein levels peaked at 3 days after crush (scale bar 200 microns).

Figure 10. Ganglion cell survival after optic nerve crush in thrombospondin-1 knock out mice.

Cells in the ganglion cell layer were counted in mid-periphery with the nuclear dye To-Pro1. Differences in cell survival between C57bl/6 and the thrombospondin-1 knock-out mice were not significant.

Figure 11. The heterogeneity of the glia cells at the ONH and the investigation of cell specific responses.

A. Acutely dissociated ONH astrocytes from the crushed and the contralateral control eyes were collected for single-cell PCR. Astrocytes were identified based on the morphology and the expression of tomato marker (bottom row). B. Agarose gel showing results of single-cell RT-PCR. Presence of astrocyte marker (Gfap) and absence of microglia marker (CD45) and oligodendrocyte marker (Mbp) confirmed the identity of the cells as astrocytes. Tnc was not detectable in control astrocytes, but was highly expressed in most of the astrocytes after crush. C. Astrocyte marker AQP4 was expressed in the myelinated optic nerve, in the distal region of the ONH and in the retina, but not in the astrocytes in the proximal section of the ONH. Scale bar, 100 microns.

Figure 12. Comparison of gene expression levels between ONHs in D2.hGFAPpr-EGFP mice (A, B and C) and ONHs after crush (A’, B’ and C’).

Gene expression levels in the D2 mice were determined by qRT-PCR. Each independent sample contained 3–4 ONHs with the same retina and optic nerve grades. There were total 2 no glaucoma (Nog), 3 moderate glaucoma (Mod) and 3 severe glaucoma (Sev) samples. The expression levels of each gene were normalized to the mean of the no glaucoma samples. Gene expression levels after crush were from the microarray data and the expression levels of the crushed eyes were normalized to the contralateral controls. Results were presented as mean with 90% lower and upper confidence bounds. Many genes showed similar responses after these two injuries. The genes that had different responses were highlighted with black frames.