Adeno-associated viral-mediated catalase expression suppresses optic neuritis in experimental allergic encephalomyelitis

Abstract

Suppression of oxidative injury by viral-mediated transfer of the human catalase gene was tested in the optic nerves of animals with experimental allergic encephalomyelitis (EAE). EAE is an inflammatory autoimmune disorder of primary central nervous system demyelination that has been frequently used as an animal model for the human disease multiple sclerosis (MS). The optic nerve is a frequent site of involvement common to both EAE and MS. Recombinant adeno-associated virus containing the human gene for catalase was injected over the right optic nerve heads of SJL/J mice that were simultaneously sensitized for EAE. After 1 month, cell-specific catalase activity, evaluated by quantitation of catalase immunogold, was increased approximately 2-fold each in endothelia, oligodendroglia, astrocytes, and axons of the optic nerve. Effects of catalase on the histologic lesions of EAE were measured by computerized analysis of the myelin sheath area (for demyelination), optic disc area (for optic nerve head swelling), extent of the cellular infiltrate, extravasated serum albumin labeled by immunogold (for blood-brain barrier disruption), and in vivo H2O2 reaction product. Relative to control, contralateral optic nerves injected with the recombinant virus without a therapeutic gene, catalase gene inoculation reduced demyelination by 38%, optic nerve head swelling by 29%, cellular infiltration by 34%, disruption of the blood-brain barrier by 64%, and in vivo levels of H2O2 by 61%. Because the efficacy of potential treatments for MS are usually initially tested in the EAE animal model, this study suggests that catalase gene delivery by using viral vectors may be a therapeutic strategy for suppression of MS.

Figure 1

Restriction map of the rAAV-Cat construct. TR, terminal repeat; Pcmv, CMV immediate early promoter/enhancer; SD/SA, SV40 late viral promoter gene 16S/19S splice donor and acceptor signals; catalase, human gene for catalase; pA2, bovine growth hormone polyadenylation signal from pRc/CMV (Invitrogen).

Figure 2

Bargraphs showing ≈2-fold increases in the mean number of catalase immunogold particles within astrocytes (astro), oligodendrocytes (oligo), microdendroglia (micro), axons, and endothelia (endo) with inoculations of rAAV-Cat (A). Bargraphs showing catalase inoculations had the following effects on EAE: reduced demyelination (increased myelin areas) (B), reduced optic nerve head edema (smaller areas) (C), decreased optic nerve cell count (D), reduced extravasated immunogold-labeled serum albumin (suppressed disruption of the BBB) (E), and reduced in vivo levels of H₂O₂ in the optic nerve head (ONH), retrobulbar optic nerve (RON) and the optic nerve sheath (ONS) (F). ∗, statistically significant.

Figure 3

Transmission electron micrographs of the optic nerve showing increased levels of catalase immunogold (arrows) are evident within the cytoplasm and nuclei of glial cells and within axons after rAAV-Cat inoculation (×2,500) (A), relative to that seen with rAAV-gfp inoculation (×2,500) (B). Less demyelinated axons were evident with rAAV-Cat inoculation (×2,500) (C), whereas naked axons (arrows) and those with thin sheaths of myelin were prominent in the control nerves that received rAAV-gfp (×2,500) (D). The rAAV-Cat inoculation reduced extravasation of immunogold-labeled serum albumin (arrows) that was predominantly confined to the intravascular compartment (×3,500) (E). Comparison with control nerves (inoculated with rAAV-gfp) showing marked extravasation of immunogold-labeled serum albumin (arrows) into the perivascular space (×4,000) (F). AAV catalase gene inoculation reduced in vivo levels of H₂O₂ (arrows) (×2,500) (G), whereas more H₂O₂ reaction product particles (arrows) were evident in the control nerves (×2,500) (H). A, axon; E, endothelial cell; G, glial cell; L, lumen; IC, inflammatory cell.