Clinical background
Optic neuritis is an inflammatory disorder of autoimmune optic nerve demyelination. It is often the first clinical sign of multiple sclerosis (MS). Optic neuritis is second only to glaucoma as the most common optic neuropathy in the USA. There are approximately 25 000 cases diagnosed per year. Optic neuritis patients are young, typically more than 10 years of age but less than 50 years of age. The clinical symptomatology is characterized by sudden painful visual loss in one eye, although occasionally both eyes can be simultaneously involved. The pain is usually exacerbated by eye movement. Optic neuritis associated with transverse myelitis may be due to Devics disease.
Clinical examination of patients with acute optic neuritis usually reveals a relative afferent pupillary defect (unilateral cases or bilateral cases with asymmetric involvement), visual field deficits, loss of visual acuity, contrast sensitivity, color vision, and stereopsis. The intraocular segment of optic nerve known as the optic nerve head that is visible by clinical examination with an ophthalmoscope is normal in three-quarters of cases. When swelling of the optic nerve head is present, leakage of lipid from the vasculature of the swollen disc into the neurosensory retina sometimes results in a macular star pattern. Although this is coined neuroretinitis, retinal inflammation is typically absent.
In a landmark study of almost 500 optic neuritis patients, the Optic Neuritis Treatment Trial (ONTT), visual field defects were nonspecific in approximately half the patients. In those patients with defects considered specific for an optic neuropathy, one-third were altitudinal defects previously associated with another disorder, ischemic optic neuropathy, caused by infarction of the optic nerve head that also has swelling of the optic nerve head. Occasionally the visual field defects of optic neuritis may be a bitemporal hemianopsia or junctional scotoma mimicking a pituitary adenoma or other suprasellar mass such as an aneurysm compressing the optic chiasm. Such cases may be resolved by neuroimaging with magnetic resonance imaging (MRI) or computed tomography (CT), as done for two patients inadvertently entered into the ONTT who turned out to have a pituitary adenoma or aneurysm as the cause of their optic neuropathy. MRI is the imaging modality of choice, as it typically reveals contrast enhancement of the optic nerve characteristic of disruption of the blood–optic nerve barrier in most cases of acute optic neuritis. For visualization of contrast enhancement of the intraorbital optic nerve, a fat suppression pulse sequence is necessary. MRI also reveals asymptomatic lesions of the intracranial white matter associated with the MS and excludes compressive lesions.
Etiology
While optic neuritis may occur in isolation or in association with MS, systemic diseases such as sarcoidosis, collagen vascular disease, or the remote effects of cancer and infections including cat scratch, human immunodeficiency virus (HIV), syphilis, retinal necrosis, Whipple’s disease, or West Nile encephalitis may be masqueraders of optic neuritis. They can be elucidated by atypical features such as persistence of pain, progression of visual loss beyond 2 weeks, or lack of visual recovery after 4 weeks.
Genetics
Traditionally, optic neuritis and MS have not been considered to be genetic disorders, apart from association with certain human leukocyte antigen (HLA) haplotypes. Recently, single nucleotide polymorphisms have been detected in the mitochondrial genome of some optic neuritis patients and in the gene encoding the interleukin-7 receptor alpha chain (IL7R) in MS patients ( Box 37.1 ).
- •
Optic neuritis is an inflammatory disorder of autoimmune optic nerve demyelination that is often the first clinical sign of multiple sclerosis
- •
Optic neuritis is second only to glaucoma as the commonest optic neuropathy in the USA
- •
Traditionally, optic neuritis and multiple sclerosis have not been considered to be genetic disorders. Recently, single nucleotide polymorphisms have been detected in the mitochondrial genome of some optic neuritis patients and in the gene encoding the interleukin-7 receptor alpha chain (IL7R) in multiple sclerosis patients
Prognosis
The prognosis of visual loss in optic neuritis is typically good. Visual loss stops worsening 2 weeks after it begins and by 4 weeks most patients show signs of recovery. Recovery of vision occurs in almost all patients after their initial bout of optic neuritis. Still, 6% are left with 20/50 or worse, and 3% are devastated by 20/200 or worse, i.e., legal blindness. Even though visual acuity recovers to 20/20 in 70% of patients and even more (87%) recover to 20/25, most of these patients show signs of residual deficits in their contrast sensitivity, visual fields, and stereopsis 10 years later, thereby suggesting these troubling deficits are permanent. A single recurrence of optic neuritis is common (60% within 10 years), as are multiple recurrences. Repeated attacks can lead to more severe visual loss, and in some patients legal blindness.
Pathophysiology
The traditional view of optic neuritis and MS emphasizes demyelination as the primary event in the disease process. The targeted cells appear to be the oligodendrocytes that are responsible for producing the axon’s myelin ( Box 37.2 ). In fact, apoptosis of oligodendrocytes has been described as the earliest event in the early lesions of MS. Recently this focus has changed. Axonal and neuronal loss are increasingly recognized as the primary factors contributing to persistent deficits and disability in MS and optic neuritis, as also revealed by optical coherence tomography (OCT). Permanent disability is believed to develop when a threshold of neuronal and axonal loss is reached and compensatory responses are exhausted. There is relatively little known about the underlying molecular mechanisms involved in the neurodegeneration process and as a consequence there is no treatment for this phase of the disease. A leading hypothesis is that axons are transected by inflammatory cells (ICs). However, this does not explain the degeneration of neurons seen well before the IC infiltration or the progressive loss of function after the inflammatory phase has subsided. Mitochondria play a key role in the pathogenesis of many neurological diseases, but the role of the organelle has only recently been recognized in optic neuritis and MS.
- •
The traditional view of optic neuritis and multiple sclerosis has emphasized demyelination as the primary event in the disease process
- •
The targeted cells appear to be the oligodendrocytes that are responsible for producing the axon’s myelin. In fact, apoptosis of oligodendrocytes has been described as the earliest event in the early lesions of multiple sclerosis
- •
Recently this focus has changed. Axonal and neuronal loss are increasingly recognized as the primary factors contributing to persistent deficits and disability in multiple sclerosis and optic neuritis
Animal model
The experimental autoimmune encephalomyelitis (EAE) animal model has impacted the design and direction of both basic and clinical research to understand the pathogenesis and treatment of MS. Immunomodulatory cyclophosphamide, ciclosporin A, copolymer 1, antibodies to specific lymphocyte subsets, and immunization with T-cell receptor peptides initially evaluated in EAE have been or are being applied to MS. The EAE model has an additional important advantage over other animal models. The alterations in the permeability of the blood–brain barrier (BBB) play a major role in the pathogenesis of EAE-induced demyelination. Comparable disruption of this barrier occurs in immune-mediated disorders such as optic neuritis and MS. In fact, optic neuritis and MS are believed to be disorders of the BBB through which ICs and humoral factors producing demyelination gain access to the central nervous system (CNS). Histopathology of inactive EAE lesions shows foci of gliosis without active inflammation that are also seen in MS. Active optic nerve lesions in EAE reveal demyelination, mononuclear cell infiltration, and phagocytosis of axons and myelin by effector macrophages. These findings are also seen in MS. In EAE there is also an immunogenetically restricted recognition system that involves the major histocompatibility antigens. Helper CD4 lymphocytes first adhere to endothelial cells, and then they infiltrate the CNS. The inflammatory response is amplified by recruitment of ICs and release of mediators, such as cytokines, antibodies, and reactive oxygen species (ROS). It is presumed that similar mechanisms may contribute to the pathogenesis of MS, but this is not known because in most patients the disease is already well established at clinical presentation.
Histopathologic findings of a patient with optic neuritis exhibited inflammation and demyelination that is also seen in the EAE optic nerve. Since human pathologic material is generally unavailable, MRI has provided an important link of EAE to optic neuritis and MS. MRIs of the optic nerve showing contrast enhancement and demyelination are similar in EAE and human disease. The MRI, histopathologic, and ROS similarities of the EAE animal model to human optic neuritis suggest that EAE is the ideal model system to investigate and target the underlying mechanisms by which retinal ganglion cells and their axons are lost in optic neuritis and MS ( Box 37.3 ).
- •
The experimental autoimmune encephalomyelitis (EAE) animal model has impacted the design and direction of both basic and clinical research to understand the pathogenesis and treatment of multiple sclerosis
- •
The magnetic resonance imaging and reactive oxygen species histopathologic similarities of the EAE animal model to human optic neuritis suggest that EAE is the ideal model system to investigate and target the underlying mechanisms by which retinal ganglion cells and their axons are lost in optic neuritis and multiple sclerosis
Neurodegeneration in EAE
Axonal loss is seen in acute EAE. Loss of retinal ganglion cells (RGCs) is also common to chronic EAE as well as to relapsing/remitting EAE. The incidence of optic neuritis is very high in both model systems with one significant difference. In chronic EAE, RGC loss occurs prior to the infiltration of ICs, but not in relapsing and remitting EAE or in myelin oligodendrocyte glycoprotein (MOG)-specific T-cell receptor transgenic mice that develop isolated optic neuritis, usually without any other characteristic lesions of EAE in the brain or in the spinal cord. Mitochondrial dysfunction may play an important role in the neurodegeneration of EAE and MS and this process begins much sooner than currently believed. Mitochondria are the primary source of cellular adenosine triphosphate (ATP), energizing neurons and axons in the CNS. Current evidence implicating mitochondria is a loss of ATPase activity in MS lesions, but how? In addition, mitochondria are the primary source of cellular ROS. Increased ROS activity is linked to many neurodegenerative diseases that have as a major feature axonal and neuronal loss. Still, while ROS have been recognized among the mediators of CNS injury in EAE and MS, the contribution of mitochondria to ROS activity and cell death has received little attention. The rest of this chapter reviews the role of mitochondrial respiration, oxidative stress, and the potential effects of modulating antioxidant gene expression in the visual system of mice induced with EAE, with a focus on long-term suppression of neurodegeneration.
Mitochondrial injury in EAE starts early
ICs that cause the classical demyelination of EAE and MS are believed to be the primary source of oxidative stress. IC infiltration typically begins within 1–2 weeks of antigenic sensitization for EAE. Qi and coworkers focused on the role of the mitochondrion as a potential source of ROS and target of oxidative injury before this initial phase of disease. Mitochondria were isolated from the retina, optic nerve, brain, and spinal cord of animals 3 and 6 days after sensitization for EAE, then probed for ROS. At this early stage none of the animals exhibited any clinical signs of EAE. As an initial gauge of ROS activity they used the peroxynitrite-mediated nitration of tyrosine residues that was detected with an antibody directed against nitrotyrosine. Peroxynitrite formed by the reaction of two other ROS, superoxide and nitric oxide, has been implicated in the pathogenesis of EAE, optic neuritis, and MS. They found nitration of mitochondrial proteins in the EAE nervous system began as early as 3 days after sensitization for EAE ( Figure 37.1 ). Control specimens of unsensitized animals did exhibit some mitochondrial protein nitration indicative of the basal ROS activity that occurs under normal physiologic conditions. Peroxynitrite can inactivate proteins, but which ones?
Respiratory chain, glycolytic, and chaperone proteins are altered
Using a proteonomics approach, Qi and coworkers identified the nitrated mitochondrial proteins. In situ trypsin digests of the excised protein bands were submitted for mass spectroscopy. When the peptide fingerprints obtained were submitted for protein database sequence analysis the highest match was for mitochondrial heat shock protein 70 (mtHsp70) ( Figure 37.2A ). This chaperone is critical not only to the import of nuclear-encoded mitochondrial proteins from the cytosol, but also protein folding and assembly into the mitochondrial matrix. In vitro, loss of mtHsp70 function results in aggregation of mitochondria and profoundly alters the morphology of the organelle. Qi and coworkers showed these very same ultrastructural changes in the mitochondria of EAE axons.
Protein database sequence analysis of the other peptide fingerprints obtained ( Figure 37.2B ) included two respiratory chain complexes. They were identified as the NADPH-ubiquinone oxidoreductase B14 subunit (NDUFA6) of complex I and cytochrome c oxidase subunit IV. Loss of activity of complexes I and IV induced by ROS here potentially contributes to loss of cellular energy. NDUFA6 is critical to the assembly of the holo complex I. Protein database sequence analysis of other peptide fingerprints obtained included the calcium-transporting ATPase and the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Alterations in calcium sequestration, another important function of mitochondria, contribute to loss of mitochondrial membrane potential that can lead to cell death. In addition to its role in glycolysis, translocation of nitrosylated GAPDH to the nucleus is linked to apoptotic cell death, or necrosis with ATP depletion. Both mechanisms may induce loss of retinal ganglion cells in EAE.
ROS suppress oxidative phosphorylation
Exposure of RGCs to peroxynitrite for 24 hours suppressed the rate of ATP synthesis by 94%, relative to RGCs grown in normal culture media ( Figure 37.3 ). This degree of impairment in oxidative phosphorylation is much greater than that seen in fatal neurodegenerative diseases caused by mutated mitochondrial DNA such as maternally inherited Leigh’s syndrome and neuropathy ataxia retinitis pigmentosa or the blinding disease Leber hereditary optic neuropathy that primarily results in loss of RGCs. Thus, loss of ATP synthesis induced by ROS can potentially have a severe impact on optic nerve function and cell survival in EAE and perhaps MS.
Antioxidant gene therapy suppresses loss of OXPHOS in vitro
Cellular defenses against ROS are present to some degree in all tissues and organs. They include the antioxidant enzymes superoxide dismutase (SOD), catalase, and glutathione peroxidase. Unfortunately, there are no endogenous defenses per se against peroxynitrite. However, defense against a key reactant, superoxide, leading to peroxynitrite formation does exist in all cells. SOD dismutes superoxide to hydrogen peroxide (H 2 O 2 ) that can be further metabolized by the actions of catalase and glutathione peroxidase to water and molecular oxygen. Several isoforms of SOD exist. The manganese SOD (MnSOD), encoded by the SOD2 gene, is exclusively mitochondrial. The copper/zinc (Cu/Zn) SOD, encoded by the SOD1 gene, is primarily cytoplasmic, but it is also found in the mitochondrial and nuclear compartments.
ROS localization in EAE
In the EAE optic nerve the distribution of inflammation and peroxynitrite-mediated protein nitration is remarkably similar to findings in a human optic neuritis specimen. ROS have been linked to loss of mitochondrial membrane potential. This can initiate a cascade of events leading to death of retinal ganglion cells, whose axons comprise the optic nerve. Qi and coworkers looked at mitochondrial membrane potential ( Figure 37.4A ) in the optic nerve 6 days after antigenic sensitization for EAE. Hydrogen peroxide labeled green by dichorofluordiacetate ( Figure 37.4B ) had a heavy presence at perineural and perivascular foci with the mitochondrial membrane potential sensing dye MitoTracker red. However, at several perivascular foci where hydrogen peroxide was highly expressed, mitochondrial membrane potential was diminished or even completely lost ( Figure 37.4C ). It was also found that loss of mitochondrial membrane potential detected by MitoTracker green was associated with superoxide labeled red by dihydroethedium. Thus, in vivo mitochondrial function is altered by ROS activity.
Modulation of anti-ROS genes alters acute optic neuritis
Qi and coworkers examined the effect of antioxidant gene therapy in experimental optic neuritis. They found that, relative to the normal optic nerve ( Figure 37.5A ), a month after sensitization for EAE, filling of the optic cup and displacement of the peripapillary retina are seen in EAE ( Figure 37.5B ). This is illustrative of the histologic features of optic disc edema, also visible by ophthalmoscopy of optic neuritis patients. This finding was due predominantly to hydropic degeneration of axons. Relative to the normal retina ( Figure 37.5E ), loss of RGCs was not yet apparent ( Figure 37.5F ). Indicative of mitochondrial ROS activity, electron-dense cerium perhydroxide reaction product was found formed by the reaction of perfused cerium chloride and endogenous hydrogen peroxide within mitochondria ( Figure 37.6 ), some swollen with dissolution of cristae. These mitochondrial findings were not limited to fibers with loss of the myelin sheath, considered the hallmark of MS and EAE. They were more widespread. Myelinated axons also contained swollen mitochondria that exhibited disorganization and dissolution of cristae, some to the point that only a double membranous bag identified the organelle.
