The distributions of mitochondria and sodium channels reflect the specific energy requirements and conduction properties of the human optic nerve head

Abstract

Aim: To study the normal distributions of mitochondria and voltage gated Na+ channels in the human optic nerve head in order to gain insight into the potential mechanisms of optic nerve dysfunction seen in the inherited optic neuropathies.

Methods: Five fresh frozen human optic nerves were studied. Longitudinally orientated, serial cryosections of optic nerve head were cut for mitochondrial enzyme histochemistry and immunolabelling for cytochrome c oxidase (COX) subunits and voltage gated Na+ channel subtypes (Na(v) 1.1, 1.2, 1.3, and 1.6).

Results: A high density of voltage gated Na+ channels (subtypes Na(v) 1.1, 1.3, and 1.6) in the unmyelinated, prelaminar, and laminar optic nerve was found. This distribution co-localised both with areas of high COX activity and strong immunolabelling for COX subunits I and IV.

Conclusions: Increased numbers of mitochondria in the prelaminar optic nerve have previously been interpreted as indicating a mechanical hold up of axoplasmic flow at the lamina cribrosa. These results suggest that this increased mitochondrial density serves the higher energy requirements for electrical conduction in unmyelinated axons in the prelaminar and laminar optic nerve and is not a reflection of any mechanical restriction. This could explain why optic neuropathies typically occur in primary inherited mitochondrial diseases such as Leber's hereditary optic neuropathy, myoclonic epilepsy with ragged red fibres (MERRF), and Leigh's syndrome. Secondary mitochondrial dysfunction has also been reported in dominant optic atrophy, Friedreich's ataxia, tobacco alcohol amblyopia, Cuban epidemic optic neuropathy, and chloramphenicol optic neuropathy. These diseases are rare but these findings challenge the traditional theories of optic nerve structure and function and may suggest an alternative approach to the study of commoner optic neuropathies such as glaucoma.

Figure 1

Histology, enzyme histochemistry and immunohistochemistry on 10 µm, serial, cryostat sections of optic nerve from a 45 year old woman. (A) Haematoxylin and eosin preparation showing preservation of morphology. (B) Van Gieson preparation to demonstrate connective tissue fibres (red). The position of the lamina cribrosa is indicated by the arrow. (C) Weigert’s iron haematoxylin preparation to demonstrate myelin (dark blue). Note that myelination commences abruptly in the retrolaminar position. (D) COX enzyme histochemistry showing strong reaction in the unmyelinated portion of the optic nerve extending into the lamina cribrosa but halting abruptly in the retrolaminar position. (E) IHC for COX subunit IV showing that the distribution of the protein co-localises with enzyme activity. (F) IHC for Na_v 1.1 showing that the distribution of this voltage gated Na⁺ channel parallels that of the mitochondrial markers. (G) IHC for Na_v 1.2 showing a very pale, diffuse labelling pattern with no predilection for either prelaminar or retrolaminar positions. (H) IHC for Na_v 1.6 showing strong immunolabelling in unmyelinated optic nerve. (I) Representative negative control for IHC where the primary antibody was omitted.

Figure 2

Control experiment to show immunoreagents have access to the myelinated portion of the optic nerve. (A) IHC for neurofilament protein (200 kDa) showing a strong, fibrous immunolabelling pattern in myelinated optic nerve. (B) IHC for Na_v 1.6. (C) COX subunit IV, showing pale immunolabelling.