Direct optic nerve sheath (DONS) application of Schwann cells prolongs retinal ganglion cell survival in vivo

Cell-based therapies are increasingly recognized as a potential strategy to treat retinal neurodegenerative disease. Their administration, however, is normally indirect and complex, often with an inability to assess in real time their effects on cell death and their migration/integration into the host retina. In the present study, using a partial optic nerve transection (pONT) rat model, we describe a new method of Schwann cell (SC) delivery (direct application to injured optic nerve sheath, SC/DONS), which was compared with intravitreal SC delivery (SC/IVT). Both SC/DONS and SC/IVT were able to be assessed in vivo using imaging to visualize retinal ganglion cell (RGC) apoptosis and SC retinal integration. RGC death in the pONT model was best fitted to the one-phase exponential decay model. Although both SC/DONS and SC/IVT altered the temporal course of RGC degeneration in pONT, SC/DONS resulted in delayed but long-lasting effects on RGC protection, compared with SC/IVT treatment. In addition, their effects on primary and secondary degeneration, and axonal regeneration, were also investigated, by histology, whole retinal counting, and modelling of RGC loss. SC/DONS was found to significantly reduce RGC apoptosis in vivo and significantly increase RGC survival by targeting secondary rather than primary degeneration. Both SC/DONS and SC/IVT were found to promote RGC axonal regrowth after optic nerve injury, with evidence of GAP-43 expression in RGC somas and axons. SC/DONS may have the potential in the treatment of optic neuropathies, such as glaucoma. We show that SC transplantation can be monitored in real time and that the protective effects of SCs are associated with targeting secondary degeneration, with implications for translating cell-based therapies to the clinic.In the central (CNS) and peripheral (PNS) nervous systems, injury from initial lesions can lead to widespread damage to neurons beyond the primary injury site; a phenomenon known as ‘secondary degeneration''. Studies in spinal cord injury have revealed secondary rather than primary degeneration to be the major contributor to neuronal death and functional impairment, and it is increasingly recognized as a therapeutic target.^1,2 Secondary degeneration also occurs in optic neuropathies, including glaucoma, ischaemic optic neuropathy, and Leber''s hereditary optic neuropathy.^{3, 4, 5} Retinal neuronal loss in these conditions is reported to occur long after the initial insult,⁶ implying that secondary mechanisms may have an important role in optic neuropathic damage and that targeting of secondary neuronal loss may represent a novel therapeutic strategy.Partial optic nerve transection (pONT) represents a reliable and reproducible model for studying secondary degeneration, in which a primary lesion is only made to dorsal axons and leaves those in ventral optic nerve (ON) intact but vulnerable to secondary degeneration.^4,7 Secondary degeneration is thought to be initiated by a cascade of reactive metabolic events, including glutamate excitotoxicity, Ca²⁺ overload, excess free radical formation, oxidative stress, mitochondrial dysfunction, and increased proteoglycan expression, leading to cell death.^{7, 8, 9, 10, 11, 12, 13, 14} Activated astrocytes are reported to be a major contributor to spreading and acceleration of secondary degeneration.^8,9As in most CNS pathways, the mature ON possesses only a limited ability to repair itself after injury, resulting in permanent vision loss due to the death of retinal ganglion cells (RGCs), the retinal output neurons that transmit visual information to the brain.¹⁵ Compared with the CNS, the PNS has a remarkable ability to regrow after injury, a process in which Schwann cells (SCs) are thought to have a key role.^16,17SCs are the principal glia of the PNS and support normal neuronal function.^18,19 Upon axonal injury, SCs are reported to shed their myelin sheaths and de-differentiate into progenitor stem cells, which are capable of replacing damaged tissue and providing a permissive environment for neuronal survival and axonal regrowth.^18,19 SCs are believed to achieve this through releasing neurotrophic factors and producing cell adhesion molecules and extracellular matrix components.²⁰ The neuroprotective and regenerative mechanisms between SCs and neurons are thought to operate on a local basis via adhesion molecules, allowing contact-mediated signalling between cells,^16,17,20,21 and extracellular free ligands, facilitating specific binding to the receptors in the target neurons.^16,17,20 However, a novel regulatory mechanism has emerged, representing a more efficient and advanced communication machinery, that is, vesicular transfer between SCs and axons.¹⁶ We have recently demonstrated that the highly efficient response of SCs to PN injury is triggered by Ephrin-B/EphB2 signalling in fibroblasts, which guide SC sorting and migration during nerve repair.²¹Due to the regenerative ability of SCs in PNS repair, transplantation of SCs to the injured ON has been previously attempted.^{22, 23, 24, 25, 26, 27, 28} To date, however, the protective effects of SCs on retinal neurons have been only assessed after either intravitreal administration or suturing artificial SC grafts onto transected ON, using postmortem histological observations, with incomplete delineation of the mechanisms involved.^{22, 23, 24, 25, 26, 27, 28}Here we use a pONT model to investigate a new method of SC delivery (direct application to injured ON sheath, SC/DONS), using in vivo imaging and histological techniques, and compare its effects on RGC apoptosis and loss to intravitreal SC delivery (SC/IVT). Furthermore, we analyse whether these actions target primary or secondary degeneration, to determine their potential in the treatment of optic neuropathy.