Expression of Developmentally Important Axon Guidance Cues in the Adult Optic Chiasm

Abstract

Purpose: Regeneration of optic nerve axons after injury can be facilitated by several approaches, but misguidance at the optic chiasm is often observed. We characterized guidance cues in the embryonic visual system and adult optic chiasm before and after optic nerve crush (ONC) injury to better understand barriers to optic nerve regeneration in adults.

Methods: Radial glial (RC2/BLBP/Slit1), developmental (Pax2) and extracellular markers (CSPG: H2B/CS-56) were assessed in C57BL/6J mice by immunohistochemistry. RC2, BLBP, Slit1, and CSPG are known inhibitory guidance cues while Pax2 is a permissive guidance cue.

Results: At embryonic day 15.5 (E.15.5), RC2 and BLBP were identified superior to, and extending through, the optic chiasm. The optic chiasm was BLBP-ve in adult uninjured mice but BLBP+ve in adult mice 10 days after ONC injury. The reverse was true for RC2. Both BLBP and RC2 were absent in adult mice 6 weeks post-ONC. Slit1 was present in the optic chiasm midline and optic tracts in embryonic samples but was absent in uninjured adult tissue. Slit1 was observed superior to and at the midline of the optic chiasm 10 days post-ONC but absent 6 weeks after injury. Pax2 was expressed at the junction between the optic nerve and optic chiasm in embryonic brain tissue. In embryonic sections, CS-56 was observed at the junction between the optic chiasm and optic tract, and immediately superior to the optic chiasm. Both 2H6 and CS-56 staining was absent in uninjured and ONC-injured adult brains.

Conclusion: Differences in guidance cue expression during development, in adulthood and after injury may contribute to misguidance of regenerating RGC axons in the adult optic chiasm.

Figure 1

RC2 (red) and BLBP (green) staining of E15.5 mouse coronal samples. (a–aii) Images of the OC and surrounding structures. Scale bar: 500 μm. (b–bii) High-power images show the chiasm structure in detail. (ai) BLBP and (aii) RC2 positive staining was observed immediately superior to the optic chiasm and glial processes (arrows, b) can be observed interacting with the optic chiasm. Blue: DAPI. Scale bar: 100 μm.

Figure 2

RC2 (red) and BLBP (green) staining of adult uninjured and 10 days pONC mouse coronal samples. Images of the OC and surrounding structures are shown and in (a–aii) adult-uninjured and (c–cii) -injured samples. Scale bar: 500 μm. High-power images of (b–bii) adult-uninjured and (d–dii) -injured samples show the chiasm structure in detail. Blue: DAPI. Scale bar: 100 μm. RC2-positive and BLBP-negative processes were observed extending through the optic chiasm midline (arrows, bii) in uninjured adult mice; while in 10-day pONC adult mice, only BLBP-positive staining was observed (arrows, di).

Figure 3

Slit1 (green) staining of embryonic day E15.5 mouse coronal samples. Images of the embryonic OC, OT, and respective surrounding structures are shown in (a, b), respectively. Slit1 positive staining was observed in the optic chiasm midline (arrow, a) and also in more posterior sections where the optic tract is visible (arrows, b). Scale bar: 500 μm.

Figure 4

Slit1 (green) staining of adult uninjured and injured mouse in coronal samples. Images of the OC, OT, and surrounding structures in adult-uninjured samples are shown in (a, d); adult samples 10 days pONC are shown in (b, e); and adult samples 6 weeks pONC are shown in (c, f). Scale bar: 500 μm. High-power images of each dotted area are shown. Scale bar: 100 μm. Slit1-positive staining was observed at the optic chiasm midline and immediately above it 10 days pONC (b–bi), but the same was not observed in uninjured adult samples (a–ai) and 6-week pONC samples (c–ci). Interestingly, also at 10 days pONC, slit1-positive staining was present only in the right optic tract and this pattern was not observed in the other two conditions.

Figure 5

Pax2 (red) staining of E15.5, adult uninjured and adult 10-day pONC mouse coronal samples. Mouse brains were collected and sections were collected following a coronal orientation (a); images of the OC, optic nerve ON, and surrounding structures are shown in (b–f). Positive Pax2 staining is indicated by the arrows. Pax2 positive staining was observed at the junction between the optic nerves and chiasm in embryonic E15.5 samples (b); while in adult uninjured (c, d) and 10-day pONC (e, f) brain samples, positive Pax2 staining was observed evenly dispersed throughout the optic chiasm. Scale bar: 500 μm. Higher magnification images of the optic chiasm midline for both uninjured and 10-day pONC are presented in (ci–di, ei–fi), respectively. White arrows indicate positive staining. Scale bar: 100 μm.

Figure 6

CSPG (green) staining of embryonic day E15.5 mouse coronal samples. Images of the OC and surrounding structures are shown. (a) Shows 2H6 staining and (b) shows CS-56 staining. Scale bar: 500 μm. High power images in panel ai and bi show the chiasm structure in detail. Scale bar: 100 μm. Both 2H6 and CS-56 staining were detected immediately above the optic chiasm (a–ai, b–bi, respectively) and CS-56 was also detected at the connection between optic nerves and optic chiasm (stars, b).

Figure 7

CSPG (green) staining of adult uninjured and injured mouse coronal samples. Images of the OC and surrounding structures are stained with 2H6 (a, c, e), and CS-56 (b, d, f). Scale bar: 500 μm. Both guidance cue markers were not detected in adult uninjured samples (a, b), in 10-day pONC samples (c, d) and in 6-week pONC samples (e, f).

Figure 8

Quantification of DAPI-positive cells in the optic chiasm at E15.5 and in adult–uninjured and adult–injured samples. Images of the optic chiasm are shown at E15.5 in (a), with the optic chiasm outline in yellow, in adult uninjured tissue in (b), in adult injured tissue at 10 days pONC in (c) and in adult injured tissue at 6 weeks pONC in (d). The number of DAPI-positive cells in the optic chiasm were manually counted, with total numbers shown in (e) and fold-change in (f). Blue: DAPI. Scale bar: 100 μm.

Figure 9

GFAP (green) and NG2 (red) labeling in the optic chiasm at E15.5 and in adult uninjured and adult injured mouse coronal samples. Images of the optic chiasm at E15.5 are shown in (ai–aiv), in adult uninjured tissue in panel (bi–biv) and in adult injured tissue at 10 days pONC in (ci–civ) and at 6 weeks pONC in (di–div). GFAP (green) staining is shown in (aii, bii, cii, dii) and NG2 (red) staining is shown in (aiii, biii, ciii, diii). Blue: DAPI. Scale bar: 100 μm. The percentage of DAPI-positive cells colocalized with GFAP or NG2 in the optic chiasm in the adult uninjured compared to the adult injured samples was quantified and can be seen in the graph in (e).

Figure 10

Iba1 (green) and Olig2 (red) labelling in the optic chiasm at E15.5 and in adult uninjured and adult injured mouse coronal samples. Images of the optic chiasm at E15.5 are shown in (ai–aiv), in adult uninjured tissue in (bi–biv) and in adult injured tissue at 10 days pONC in (ci–civ) and at 6 weeks pONC in (di–div). Iba1 (green) staining is shown in (aii, bii, cii, dii) and Olig2 (red) staining is shown in (aiii, biii, ciii, diii). Blue: DAPI. Scale bar: 100 μm. The percentage of DAPI-positive cells colocalized with Iba1 or Olig2 in the optic chiasm in the adult uninjured compared to the adult injured samples was quantified and can be seen in the graph in panel e.