On optic nerve injury : Experimental studies on axonal regeneration in the adult mammalian CNS

Abstract: Optic nerve injury may occur after a trauma to the head and in many cases it causes partial or total blindness. Today, these patients cannot be offered any specific treatment to restore visual function. The aim of this thesis was to analyze the neurobiological events after such an injury, and study the role of current and suggested methods of treatment. The optic nerve, a part of the central nervous system, has a morphology similar to that of white matter elsewhere in the CNS. In this project, we developed a standardized and easily reproducible crush lesion model of the optic nerve. The optic nerve of the adult Sprague-Dawley rat was exposed in the orbita, using a microsurgical technique, and a crush injury was made with a fine pair of custom-designed forceps, set to provide a fixed pressure of 0.6N for 10 seconds. The temporal neuropathological pattern, focusing on axonal degeneration, regeneration and glial reactions, was documented by various methods, including morphology, immunohistochemistry and in situ hybridization. Electrophysiological measurements of visual evoked potentials from implanted electrodes were made to assess optic nerve function. After the lesion, we found that the microglial and astroglial cells proliferated and hypertrophied, and later formed a dense glial scar at the site of injury. The number of viable retinal neural cells declined, paralleled by Wallerian degeneration of most of the optic nerve axons. A slight regenerative response was seen proximal to the lesion, but it never extended distal to the site of injury, probably because of the inhibitory effects of the glial scar and myelin debris. The complement system, a part of the innate immune system, was activated in the optic nerve following injury. Microglial cells seemed to play a significant role in complement activation, at least by synthesis of complement factor C3. The lytic end-product of the complement cascade, the membrane attack complex, MAC, was found to target axons and structures at the site of the lesion. This suggests that the activated complement system is involved in secondary insults and affects the outcome. The complement regulatory compound, Clusterin, which inhibits MAC formation, was upregulated in astrocytes in the injured optic nerve, which would suggest a protective role in counteracting activation of endogenous complement. The balance between activation and regulation of the complement cascade may affect the degenerative events that follow optic nerve injuries, and indicate that the administration of complement inhibitors may reduce secondary damage. One treatment suggested for optic nerve lesions is the administration of high doses of methylprednisolone, a treatment that has membrane-stabilizing effects and suppresses lipid peroxidation. The administration of methylprednisolone had no effects on axonal degeneration, regeneration, macrophage recruitment, glial reactions or function, as monitored by visual evoked potentials, although some minor effects might escape detection. On the basis of these findings, methylprednisolone can hardly be regarded as the sole treatment of a severe optic nerve injury. Macrophages seem to play a decisive role in helping axonal regrowth after injury, by the removal of inhibitory debris and the synthesis of various growth factors. The injured CNS does not normally permit the recruitment of many macrophages after an injury, which makes it different from the peripheral nervous system, a system in which regeneration is more likely to succeed. By stimulating the endogenous macrophage response with a group B streptococcus exotoxin, the macrophages increased in size and numbers throughout the lesioned optic nerve. Animals receiving this bacterial exotoxin showed sprouts in and distal to the lesion, with regrowth of axons crossing the glial scar. This finding seemed to be due, at least partly, to stimulation of the macrophages, although other unknown effects cannot be ruled out. A crush injury of the optic nerve can be due to displaced fragments of fractured bone after a head trauma. It is still disputed whether decompressive surgery should be performed early, because of insufficient scientific data. To simulate this situation, we placed a ligature around the optic nerve to compress it partly for various times, up to 24 h. A partial injury for 24 h caused a compact glial scar at the site of the lesion that prevented axonal regrowth, while a similar one for up to 6 h led to less marked gliosis, permeable to sprouts and axonal regrowth. These results suggest that early (within 6 h) decompressive surgery has advantages and can be recommended for these lesions. In summary, this thesis focuses on methods that minimize axonal damage, and new ones that favor axonal regeneration after optic nerve injury. It describes at least three ways to induce regrowth of axons in the injured optic nerve. First, by controlling the complement system, beneficial effects can be preserved while reducing secondary axonal insults caused by the formation of MAC. Second, by stimulating macrophageal phagocytosis, inhibitory tissue debris is more efficiently removed and the weak spontaneous regenerative response can be increased. Third, early decompressive surgery seems to reduce the number of injured axons, and results in a less compact glial scar that permits sprouting beyond the lesion.