Retinal Ganglion Cell Survival and Axon Regeneration after Optic Nerve Transection is Driven by Cellular Intravitreal Sciatic Nerve Grafts

Abstract

Neurotrophic factors (NTF) secreted by Schwann cells in a sciatic nerve (SN) graft promote retinal ganglion cell (RGC) axon regeneration after either transplantation into the vitreous body of the eye or anastomosis to the distal stump of a transected optic nerve. In this study, we investigated the neuroprotective and growth stimulatory properties of SN grafts in which Schwann cells had been killed (acellular SN grafts, ASN) or remained intact (cellular SN grafts, CSN). We report that both intravitreal (ivit) implanted and optic nerve anastomosed CSN promote RGC survival and when simultaneously placed in both sites, they exert additive RGC neuroprotection. CSN and ASN were rich in myelin-associated glycoprotein (MAG) and axon growth-inhibitory ligand common to both the central nervous system (CNS) and peripheral nervous system (PNS) myelin. The penetration of the few RGC axons regenerating into an ASN at an optic nerve transection (ONT) site is limited into the proximal perilesion area, but is increased >2-fold after ivit CSN implantation and increased 5-fold into a CSN optic nerve graft after ivit CSN implantation, potentiated by growth disinhibition through the regulated intramembranous proteolysis (RIP) of p75^NTR (the signalling trans-membrane moiety of the nogo-66 trimeric receptor that binds MAG and associated suppression of RhoGTP). Mϋller cells/astrocytes become reactive after all treatments and maximally after simultaneous ivit and optic nerve CSN/ASN grafting. We conclude that simultaneous ivit CSN plus optic nerve CSN support promotes significant RGC survival and axon regeneration into CSN optic nerve grafts, despite being rich in axon growth inhibitory molecules. RGC axon regeneration is probably facilitated through RIP of p75^NTR, which blinds axons to myelin-derived axon growth-inhibitory ligands present in optic nerve grafts.

Keywords: CNS; axon regeneration; neuroprotection; neurotrophic factors; optic nerve; optic nerve transection; peripheral nerve grafts; retinal ganglion cells.

Figure 1

Control and experimental groupings (see text). (i) Control (CON)/intravitreal (ivit) Sterispon (_S)/optic nerve transection (ONT), (ii) ivit acellular sciatic nerve grafts (_ASN)/ONT; (iii) ivit_S/ONT_ASN; (iv) ivit_ASN/ONT_ASN; (v) ivit cellular sciatic nerve grafts (_CSN)/ONT; (vi) ivit_CSN/ONT_ASN; (vii) ivit_ASN/ONT_CSN; (viii) ivit_CSNONT_CSN.

Figure 2

Fluorogold (FG)⁺ retinal ganglion cells (RGC) at 21 days after grafting in retinal wholemounts from: (A) CON/intact, CON/ivit_S/ONT, ivit_ASN/ONT, ivit_S/ONT_ASN, ivit_S/ONT_CSN, ivit_CSN/ONT, ivit_CSN/ONT_ASN, ivit_ASN/ONT_CSN, and ivit_CSN/ONT_CSN groups. Note that the ivit_CSN/ONT_CSN group show the greatest RGC neuroprotection (scale bars = 20 μm). (B) Quantification of numbers of FG⁺ RGC in retinal wholemounts confirms that RGC survival is increased in all ivit_CSN paradigms with the greatest survival observed in the ivit_CSN/ONT_CSN group. n = 16 retinae/group; ** p < 0.001; *** p < 0.0001, ANOVA.

Figure 3

Müller cell/astrocyte activation in the retina at 21 days after grafting. Representative images of glial fibrillary acidic protein (GFAP)⁺ astrocytes (arrowheads) and activated Müller cell processes (arrows) in: (A) CON/intact, (B) CON/ivit_S/ONT, (C) ivit_ASN/ONT, (D) ivit_S/ONT_ASN, (E) ivit_S/ONT_CSN, (F) ivit_CSN/ONT, (G) ivit_CSN/ONT_ASN, (H) ivit_ASN/ONT_CSN, and (I) ivit_CSN/ONT_CSN groups. Note the increased glial activation after ivit_CSN grafting (scale bars = 50 μm). (J) Quantification of the number of Müller cell processes confirms that ivit_CSN promotes glial activation in the retina. n = 16 retinae/group; *** p < 0.0001, ANOVA.

Figure 4

Macrophages accumulate in the retina in eyes containing ivit_CSN implants at 21 days after grafting. ED1-HRP⁺ macrophages in the vitreous body and retina in: (A) CON/intact, (B) CON/ivit_S/ONT, (C) ivit_ASN/ONT, (D) ivit_S/ONT_ASN, (E) ivit_S/ONT_CSN, (F) ivit_CSN/ONT, (G) ivit_CSN/ONT_ASN, (H) ivit_ASN/ONT_CSN, and (I) ivit_CSN/ONT_CSN groups. ED1⁺ macrophages were present in both (J) ivit_CSN, and (K) ivit_ASN implants; ED1⁺ macrophages (red) at the anastomosis site of (L) ONT_CSN; (M) ivit_CSN/ONT_ASN; (N) ivit_CSN/ONT_CSN groups (scale bars in A–K = 50 μm; in L–N = 100 μm). n = 16 retinae/group.

Figure 5

Growth inhibitory ligands in the optic nerve, cellular sciatic nerve (CSN), and acellular sciatic nerve (ASN) grafts at 21 days after grafting. (A) Nogo-A, MAG, and CSPG were all present in optic nerve, CSN, and ASN whilst Nogo-C was not present in optic nerve. (B) Densitometry to confirm the levels of Nogo-A, Nogo-C, MAG, and CSPG in optic nerve, ASN, and CSN lysates (n = 6/per group, 3 independent repeats). ** p < 0.001, ANOVA. β-actin was used as a loading control.

Figure 6

Regeneration of rhodamine B (RhB)⁺ RGC axons (red) into cellular sciatic nerve (CSN) or acellular sciatic nerve (ASN) grafts at 21 days after optic nerve transection (ONT). Representative images of longitudinal sections of optic nerve and graft in (A), ivit_CSN/ONT_ASN and (B), ivit_CSN/ONT_CSN groups—note the presence throughout the length of the ONT_CSN graft of RhB⁺ (red) regenerating RGC axons (open arrows) and their limited penetration into the ONT_ASN graft (A,B insets), optic nerve GFAP⁺ astrocyte processes (green) do not enter ONT_ASN grafts, but do enter the proximal region of the ONT_CSN graft (closed arrow) (scale bars in A, B, and D = 200 µm; broken line indicates anastomosis site). (C) Quantification of the number of axons extending 400, 800, and 1200 μm past the anastomosis site into the ONT_CSN graft (*** p < 0.0001). A few axons penetrate the junctional zone of ONT_ASN grafts compared to the numbers extending for long distances into ONT_CSN grafts. (D), anastomosis site in an ivit_CSN/ONT_CSN rat showing the laminin⁺ basal lamina of the optic nerve vasculature (arrow heads) and Schwann cell basal lamina tubes aligned in parallel arrays in the ONT_CSN graft (arrow), with which (E) regenerating RGC axons are associated with the extent of GFAP⁺ astrocyte process (green) invasion and the growth of RhB⁺ RGC axons (red) (scale bars = 150 µm; the broken line indicates the anastomosis site).

Figure 7

Axon regeneration promoted by ivit_CSN is correlated with reduced levels of RhoGTP and regulated intramembranous proteolysis (RIP) of p75^NTR in the optic nerve and retina. In both the retina (A,C) and optic nerve (B,D) p75 intracellular domain (p75_ICD) accumulates only in ivit_CSN/ONT_CSN group. RhoGTP is activated in the retina and optic nerve and remains high in ivit_ASN/ONT_CSN group and significantly suppressed in ivit_CSN/ONT_CSN group (E). β-actin was used as a loading control. *** p < 0.0001, n = 8 retinae/ON pooled/treatment, three independent repeats).