Wallerian-like axonal degeneration in the optic nerve after excitotoxic retinal insult: an ultrastructural study

Abstract

Background: Excitotoxicity is involved in the pathogenesis of a number neurodegenerative diseases, and axonopathy is an early feature in several of these disorders. In models of excitotoxicity-associated neurological disease, an excitotoxin delivered to the central nervous system (CNS), could trigger neuronal death not only in the somatodendritic region, but also in the axonal region, via oligodendrocyte N-methyl-D-aspartate (NMDA) receptors. The retina and optic nerve, as approachable regions of the brain, provide a unique anatomical substrate to investigate the "downstream" effect of isolated excitotoxic perikaryal injury on central nervous system (CNS) axons, potentially providing information about the pathogenesis of the axonopathy in clinical neurological disorders.Herein, we provide ultrastructural information about the retinal ganglion cell (RGC) somata and their axons, both unmyelinated and myelinated, after NMDA-induced retinal injury. Male Sprague-Dawley rats were killed at 0 h, 24 h, 72 h and 7 days after injecting 20 nM NMDA into the vitreous chamber of the left eye (n = 8 in each group). Saline-injected right eyes served as controls. After perfusion fixation, dissection, resin-embedding and staining, ultrathin sections of eyes and proximal (intraorbital) and distal (intracranial) optic nerve segments were evaluated by transmission electron tomography (TEM).

Results: TEM demonstrated features of necrosis in RGCs: mitochondrial and endoplasmic reticulum swelling, disintegration of polyribosomes, rupture of membranous organelle and formation of myelin bodies. Ultrastructural damage in the optic nerve mimicked the changes of Wallerian degeneration; early nodal/paranodal disturbances were followed by the appearance of three major morphological variants: dark degeneration, watery degeneration and demyelination.

Conclusion: NMDA-induced excitotoxic retinal injury causes mainly necrotic RGC somal death with Wallerian-like degeneration of the optic nerve. Since axonal degeneration associated with perikaryal excitotoxic injury is an active, regulated process, it may be amenable to therapeutic intervention.

Figure 1

LM appearance of resin-embedded semi-thin section of saline control normal inner retina stained with Toluidine blue stain. INFL: inner nerve fibre layer, GCL: ganglion cell layer, IPL: inner plexiform layer. Ganglion cell layer shows prominent nuclei of RGCs (white stars) and amacrine cell (red stars). Bar = 10 μm.

Figure 2

Ultrastructural appearances of normal RGCs from saline injected eye. Typical RGCs are seen (A, Bar = 5 μm) with normal nuclei (B, Bar = 2 μm), endoplasmic reticulum (C, Bar = 1.7 μm) and mitochondrion (D, Bar = 20 nm).

Figure 3

Ultrastructural appearance of RGCs 24 hrs after NMDA injection shows (A, Bar = 5 μm) early dense appearance of the cell (rgc) cytoplasm with dendritic swelling (d) and normal euchromatic nucleus with a prominent nucleolus (B, Bar = 2 μm). Notice the dilatation of mitochondrion (m) and endoplasmic reticulum (ER) as well as some scattered ribosomes (r) in the cytoplasm (C, Bar = 2 μm).

Figure 4

Ultrastructure appearance of RGCs 72 hrs after NMDA injection. Neurons are found at different stages of degeneration and the neuronal death is morphologically 'necrotic'. Figure A shows the hyperconvoluted nucleus and cytoplasm filled with multiple vacuoles, ribosomes and swollen organelle. Figure B shows severe necrotic cell death where the cell has lost its architecture and converted into debris. There is no demarcation between cytoplasm and electron-dense bodies. The cytoplasm is studded with the multi-laminated myelin figures and damaged organelles (Bars = 2 μm); Nu = nucleus, ER = endoplasmic reticulum, ga = golgi apparatus, m = mitochondria, v = vesicles, r = ribosomes.

Figure 5

NMDA-induced ultrastructural changes in RGCs at 7 days where necrotic cell in the form of highly electron-dense neuronal debris (Red star, A & B, Bar = 2 μm) is seen lying adjacent to numerous membrane-bound microtubule-rich neuritic processes (C, red triangle, Bar = 2 μm) identified as dendrites under high power (D, red triangle, Bar = 500 nm); Figure E and F show reactive microglia surrounding the dendritic sprouts. (Bars = 2 μm).

Figure 6

TEM of the parallel running longitudinal sections of the intraorbital axons in saline injected control retina immediately after the injection. A few fibres cut to a substantial length showed varicosities (red star) and intervaricosity regions simultaneously (Bar = 1 μm).

Figure 7

EM of the retro-orbital distal segment of rat optic nerve of the saline injected control animal immediately after the injection. Axoplasm of the myelinated axons contain numerous neurofilaments, microtubules, mitochondria and various other organelles. The transverse sections (A, Bar = 5 μm and B, Bar = 2 μm) show compact arrangement of the myelin lamellae around the axons in the internodal regions. The longitudinal sections show parallel running myelinated axons (C, Bar = 5 μm). Axon-myelin relationship in the nodal-paranodal region is better appreciated at very high magnification (D, Bar = 1 μm) Here, myelin terminal loops are seen attached to the paranodal axolemma on either side of the node.

Figure 8

Ultrastructural appearances of axonal swellings in the transverse sections of distal segment of rat optic nerve after 72 hrs of NMDA injection. The major change observed is the appearance of swollen axons (A, Bar = 10 μm). The axoplasm of these axonal swellings show abnormal collection of altered tubulovesicular structures (B-D, Bars = 2 μm), cytoskeletal disintegration (C-F, Bars = 2 μm), and multilayered whorled masses (C & F, Bars = 2 μm), which are seen to be arising from the inner layers of the myelin (F, Bar = 2 μm).

Figure 9

Ultrastructural appearance of hyperdense axons in distal optic nerves seen 72 hrs after NMDA injection (Bars = 2 μm). Note that despite the extent of the changes, some adjacent axons still appear normal.

Figure 10

Ultrastructure appearances of distal segment of rat optic nerve after 72 hrs of NMDA injection. Transverse sections show separation and vacuolation of myelin sheath (A & B). Longitudinal sections display nodal blebs (C, arrows), abnormal accumulation of altered organelles (D, arrow), myelin whorls (E, arrow) arising from the inner myelin and forming mesaxon (F, arrow). The cytoskeleton surrounding the myelin whorls appears normal at this stage (E & F). Bars = 2 μm.

Figure 11

TEM of distal segment of rat optic nerve after 7 days of NMDA injection. Transverse (A, Bar = 20 μm & B, Bar = 5 μm) and longitudinal sections (C, Bar = 20 μm & D = Bar = 5 μm) show the fibres undergoing dark degeneration with most axons showing hyperdense axoplasm (red arrows). Longitudinal profile show the focal axonal swelling and hyperdense axoplasm in the same axon (D, arrows). Axon towards the end stage of degeneration (E, Bar = 1 μm) show nearly collapsed axon structure and the myelin debris phagocytosed by the astrocytes (F, Bar = 5 μm). Last series of photographs (G1, G2, G3, Bar = 2 μm) represent axoplasm in various stages of dissolution.