\set{final}

\def\Author{Qi}
\def\author{qi}
\def\vol{13}
\def\year{2007}
\def\anum{1}
\def\pages{1-11}
\def\txt_title{Dual gene therapy with extracellular superoxide dismutase and catalase attenuates experimental optic neuritis}
\def\txt_authors{Xiaoping Qi, William W. Hauswirth, John Guy}

\def\rcvd{24 April 2006}
\def\accept{4 January 2007}
\def\publ{5 January 2007}
\def\pdfsize{}
\def\PMID{}


\include{mvstyle.hsm}

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\article{

\title{Dual gene therapy with extracellular superoxide dismutase and
catalase attenuates experimental optic neuritis}

\authors{Xiaoping Qi,\sup{1} William W. Hauswirth,\sup{1,2}
\mailto{johnguy@eye.ufl.edu}{John Guy}\sup{1,3}}

\institutions{\sup{1}Departments of Ophthalmology, Molecular Genetics
and \sup{2}Microbiology, \sup{3}Neurology, University of Florida,
College of Medicine, Gainesville, FL}

\correspondence{Dr. J Guy, Department of Ophthalmology, University of
Florida, College of Medicine, Gainesville, FL, 32610; Phone: (352)
392-3451; FAX: (352) 392-7839; email: johnguy@eye.ufl.edu}

\abstract

\abs_purpose{To ameliorate experimental optic neuritis by combining
scavenging of superoxide by germ line increases in the extracellular
superoxide dismutase (ECSOD) and hydrogen peroxide by viral-mediated
gene transfer of the human catalase gene.}

\abs_methods{The human catalase gene inserted into recombinant
adeno-associated virus (rAAV) was injected into the right eyes of
transgenic mice overexpressing human ECSOD and wild-type littermates.
Animals were simultaneously sensitized for experimental autoimmune
encephalomyelitis (EAE) and then sacrificed one month later. The effects
of antioxidant genes (ECSOD and catalase) on the histologic lesions of
EAE were measured by computerized analysis of myelin area, optic disc
area, extent of the cellular infiltrate, cerium derived H\sub{2}O\sub{2}
reaction product and extravasation of serum albumin detected by
immunogold.}

\abs_results{Combined scavenging of H\sub{2}O\sub{2} and superoxide with
ECSOD and catalase suppressed demyelination by 72%, 54% due to catalase,
and 19% due to ECSOD. Disruption of the blood-brain barrier was reduced
63% by the combined effects of catalase and ECSOD, 35% due to catalase
and 29% due to ECSOD.}

\abs_conclusions{Transgene modulation of antioxidant enzyme defenses
against both superoxide and its metabolite H\sub{2}O\sub{2} provide a
substantial suppressive effect against EAE in the optic nerve that may
be a new therapeutic strategy for suppression of optic neuritis and
multiple sclerosis.}

\introduction

\p{Experimental autoimmune encephalomyelitis (EAE) is an autoimmune
inflammatory disorder leading to primary central nervous system
demyelination. EAE has been frequently used as an animal model for
testing treatments against multiple sclerosis (MS) [1-16]. The optic
nerve is a frequent site of involvement in both EAE and MS [17-23].
Reactive oxygen species (ROS) such as superoxide, hydrogen peroxide,
nitric oxide and peroxynitrite are mediators of demyelination and
disruption of the blood-brain barrier (BBB) in EAE [24-31]. The role
ROS play in altering BBB permeability and demyelination has been
inferred from the beneficial effect of monotherapy with free radical
scavengers or antioxidants on EAE [27-31]. ROS scavengers include
catalase and superoxide dismutase (SOD). SOD dismutes superoxide to
hydrogen peroxide (H\sub{2}O\sub{2}) and catalase detoxifies the
H\sub{2}O\sub{2} to H\sub{2}O and O\sub{2}.}

\p{In a prior study, we targeted a single ROS, hydrogen peroxide, for
detoxification by catalase gene inoculationn [23]. It reduced
demyelination of the optic nerve by 38%. An approximately one-third
suppressive effect on disease activity is achieved by currently
available treatments for MS by utilizing a single drug [32]. Some
studies have suggested that combination therapy may have a better
suppressive effect on MS than monotherapy [33,34] although this is not
always the case [35]. Here, we attempt to further ameliorate EAE by
assessing the additional protective effects on experimental optic
neuritis of combining in vivo scavenging of superoxide by germ line
increases in the extracellular superoxide dismutase (ECSOD) and
scavenging of hydrogen peroxide by viral mediated gene transfer of the
human catalase gene.}

\methods

\subsection{Recombinant adeno-associated virus}

\p{The adeno-associated virus (AAV) vector pTR-UF was used to accept the
human catalase cDNA at the Not1 and Sal1 sites. The resulting pTR-CAT
plasmid were amplified, then purified and packaged into serotype 2 rAAV.
Briefly, recombinant AAV was purified through iodixanol step gradients
and heparin-agarose affinity columns and assayed as previously
described [36]. Each virus preparation contained 10\sup{11} to
10\sup{12} particles per milliliter and 10\sup{9} to 10\sup{10}
infectious center units per milliliter.}

\subsection{Induction of EAE and intraocular injections}

\p{Two \mu l of recombinant adeno-associated virus (rAAV) catalase were
injected into the vitreous cavity of 20 transgenic ECSOD mice,
overexpressing human extracellular superoxide dismutase (ECSOD; a
generous gift of Dr. James Crapo) and 20 wild-type littermates were also
injected with AAV-catalase as controls. Briefly, ECSOD transgenic mice
were constructed by injection of DNA containing the entire human ECSOD
cDNA driven by a human \beta-actin promoter that was injected into
pronuclei of fertilized eggs that were isolated from mice
[(C57BL/6xC3H)F1x(C57BL/6xC3H)F1]. Surviving eggs were implanted into
pseudopregnant foster mothers to generate offspring containing the ECSOD
transgene. Mice expressing human ECSOD were identified using Southern
blot analysis of DNA extracted from the tail and probed with the entire
human ECSOD cDNA [37,38]. EAE was induced in the mice by sensitization
with 0.2 cc of ultrasonically homogenized spinal cord emulsion in
complete Freunds adjuvant (Difco, Detroit, MI) that was injected
subdermally into the nuchal area [30]. Mice were maintained in
veterinarian-supervised animal care facilities that are fully accredited
by the American Association of Laboratory Animal Science and they were
humanely cared for in full compliance with ARVO guidelines.}

\subsection{Immunobloting and immunohistochemistry}

\p{Retinal ganglion cells (RGC-5) were grown in Dulbecco's Modified
Eagle Medium (DMEM; Fisher Scientific) supplemented with 10%
heat-inactivated fetal bovine serum and 1% penicillin streptomycin
(Sigma) at 37 \deg C with 5% CO\sub{2}. Cells were grown in 15 cm dishes
that were infected with AAV containing the human catalase cDNA at
multiplicities of infection (MOI) of 5,000 particles per cell. Controls
were infected with AAV-GFP. Two days after AAV infections, cells were
harvested. Briefly this involved washing the trypsinized cells in cold
PBS, then manually homogenizing them. For immunodetection, 15 mg of
homogenated protein was separated on a 10% SDS polyacrylamide gel and
electro-transferred to a polyvinylidene fluoride membrane (BioRad,
Hercules, CA). We immunostained the membrane with monoclonal
anti-catalase antibodies (Sigma-Aldrich, St. Louis, MO, C0979, mol wt,
60 kDa) and then goat anti-mouse IgG horseradish peroxidase (HRP)
conjugated secondary antibodies (Sigma-Aldrich). We detected complexes
using the enhanced chemiluminesence (ECL) system (Amersham Pharmacia
Biotech, Piscataway, NJ). Anti-mouse \beta-actin antibody was used as an
internal control for protein loading.}

\p{One month after AAV and EAE inoculations, mice were overdosed with
sodium pentobarbital (0.3 mg/g body weight). They were then perfused by
cardiac puncture with fixative consisting of 4% paraformaldehyde in 0.1
M PBS buffer (pH 7.4). The eyes with attached optic nerves were
dissected out of ten ECSOD mice and ten littermates. The specimens were
further processed by immersion fixation in 2.5% gluteraldehyde,
postfixed in 1% osmium tetroxide, 0.1 M sodium cacodylate-HCl buffer (pH
7.4), 7% sucrose in the cold, and then dehydrated through an ethanol
series to propylene oxide, infiltrated, and embedded in epoxy resin that
was polymerized at 60 \deg C overnight. For immunocytochemistry, tissue
specimens from the other ten ECSOD mice and ten littermates were
postfixed in 5.0% acrolein, 0.1 M sodium cacodylate-HCl buffer (pH 7.4)
and 7% sucrose and then dehydrated through an ethanol series and
embedded in LR-White (Ted Pella, Redding, PA) that was polymerized at
50\deg C overnight. Semi-thin longitudinal sections (0.5 \mu m) of the
optic nerve head and retrobulbar nerve were stained with toluidine blue
for light microscopic examination. Ultrathin sections (90 nm) were
placed on nickel grids for immunocytochemistry. Nonspecific binding of
antibodies was blocked by 5% normal goat serum in 0.01 M Tris-buffered
saline, (pH 7.2) or 2% teleost gelatin and 2% nonfat dry milk in 0.01 M
TBS (pH 7.2) with TBST for 30 min for albumin immunostaining. They were
then reacted with a rabbit anti-albumin antibody or an ECSOD antibody (a
generous gift of Dr. Stephan Marklund) that recognizes the human ECSOD
[39], but not the murine ECSOD (personal communications with Dr.
Marklund) in the same buffer for 2 h at room temperature.}

\p{After washes in 0.1 M PBS, the specimens were reacted with the
secondary goat anti-rabbit IgG antibodies conjugated to 10 nm gold or
Cy3 for immunofluorescence microscopy. After washes in buffer, grids
were rinsed in deionized water. For examination at low magnification
transmission electron microscopy, the immunogold particles were enlarged
by silver enhancement using a kit (Ted Pella, Redding, PA) according to
the manufacturer's specifications. To check for nonspecific binding of
the secondary antibody, control specimens were incubated in the buffer,
followed by the gold-labeled or Cy3 labeled antibody. Immunolabeled and
control specimens were photographed by transmission electron microscopy
without poststaining.}

\subsection{Morphometric analysis}

\p{Morphometric analysis was performed in masked fashion as previously
described [23]. Briefly, images of toluidine blue stained sections of
the optic nerve were captured with a video camera mounted on a light
microscope and then the digital image was entered into computer memory.
After initial calibration with a stage micrometer, the optic nerve head
areas were manually traced using NIH IMAGE software and a MacIntosh
Computer (Apple, Cupertino, CA). The number of glial cells and
inflammatory cells in the retrobulbar optic nerve were also quantitated
by thresholding of the darker staining cell nuclei. Using electron
microscopy, identification of glial cells in the optic nerve was based
on morphologic criteria. Fibrous astrocytes were identified by their
round or elliptical nuclei with few clumps of chromatin in a relatively
light karyoplasm that was surrounded by a voluminous pale cytoplasm with
long processes and glial filaments. Inflammatory cells were identified
by the prominent clumping of nuclear chromatin, ribosome rich cytoplasm
that was clearly more electron dense than that of astrocytes and the
presence of phagosomes with engulfed myelin debris.}

\p{Optic nerve specimens were examined without poststaining using a
Hitachi H-7000 transmission electron microscope (Tokyo, Japan) operating
at 75 kV. Photographs were made at a magnification of 2,500X. For
quantitative analysis, micrographs of each optic nerve were digitized
into computer memory by using a UMAX scanner (UMAX Data Systems,
Fremont, CA). Extravasated serum albumin immunogold or H\sub{2}O\sub{2
}derived cerium perhydroxide were quantitated by thresholding the
respective elements. Mean particle counts for each nerve were obtained
by taking the mean value of the 10 micrographs. Each mean value was
expressed as the number of elements per unit area. The extent of
demyelination was quantitated by threshold measurements of the electron
dense myelin sheaths that were derived from the axonal transmission
electron micrographs for each optic nerve. Again using NIH IMAGE
software, we utilized the threshold feature to outline the myelin
sheaths for each micrograph. Glial or inflammatory cells that were also
highlighted in the thresholded micrographs were removed manually by
using the eraser function. To calculate the outlined myelin area we used
the software "analyze" feature. Increases in myelin sheath area (less
demyelination) thereby indicated a beneficial treatment effect. Grouped
student t-tests were used to assess differences in the myelin areas,
optic nerve head areas, optic nerve cell counts, hydrogen peroxide
reaction product and extravasated albumin immunogold between the
transgenic ECSOD mice and the wild-type littermates.}

\results

\subsection{ECSOD and catalase expression}

\p{Expression of the human ECSOD was evident in the optic nerves of
transgenic ECSOD mice (\figref{1}{A}), but not in the wild-type
littermates (\figref{1}{B}). As implied by its name, ECSOD immunogold
localized to the perivascular space and endothelial cells in the optic
nerve (\figref{1}{C}) and peripapillary choroid (\figref{1}{E}). It was
also represented in the meninges comprising the optic nerve sheath of
ECSOD mice (\figref{1}{F}). Human ECSOD immunogold was absent in the
perivascular space of the littermates (\figref{1}{D}). This distribution
of ECSOD mirrored the presence of H\sub{2}O\sub{2} in the EAE optic
nerve. Immunobloting showed that retinal ganglion cells infected with
rAAV containing the gene for human catalase had increased catalase
expression relative to controls infected with AAV-GFP (\figref{1}{G}).}

\subsection{Demyelination}

\p{Light microscopy of the EAE optic nerves revealed foci of
demyelination, the hallmark of the histopathology of EAE and MS, was
evidenced by loss of toluidine blue staining. This finding was seen to
some degree in all animals sensitized for EAE. Transmission electron
microscopy clearly demonstrated the benefits of anti-ROS gene therapy.
Illustrative of the benefits of double protection, the right eyes of
ECSOD mice that received the AAV-catalase gene inoculation had much less
myelin fiber injury than the unprotected left eyes of wild-type mice. A
representative micrograph of the right eyes of ECSOD mice further
protected by catalase shows a near normal complement of optic nerve
fibers with relatively preserved myelin lamellae (\figref{2}{A}). These
findings sharply contrasted with the unprotected left eyes of wild-type
littermates where myelin and fiber loss was severe (\figref{2}{B}). Here
remaining axons were degenerating, enveloped by thin sheaths of myelin,
or naked. Optic nerve fibers were replaced by a proliferation of
astroglial processes.}

\p{Measurements of myelin area in wild-type mice not sensitized for EAE
revealed a mean of 439,683 \mu m\sup{2}. This value was comparable to the
normal unsensitized ECSOD transgenic mouse with a value of 424,878
\mu m\sup{2}. Relative to unsensitized wild-type mice, the myelin area of
unprotected wild-type mice induced with EAE (Wt OS) was reduced by 55%
(p\lt 0.03). Quantitative analysis confirmed that in vivo scavenging of
hydrogen peroxide by viral mediated catalase gene transfer and
superoxide scavenging by ECSOD resulted in a mean myelin area of 340,236
\mu m\sup{2} (ECSOD OD), thus reducing demyelination by 72% relative to a
value of 197,517 \mu m\sup{2} (Wt OS) for untreated wild-type mice with EAE
(p\lt 0.01; \figref{2}{C-D}). This combined effect was greater than that
of superoxide scavenging alone as ECSOD mice had 19% more myelin (less
demyelination) with a mean myelin area of 235,123 \mu m\sup{2} (ECSOD OS)
than the mean of 197,517 \mu m\sup{2} (Wt OS) for unprotected wild-type
mice (p\lt 0.05). It was also greater than the solo effect of catalase
mediated scavenging of hydrogen peroxide with a mean myelin area of
304,190 \mu m\sup{2} (Wt OD) versus 197,517 \mu m\sup{2} (Wt OS) that reduced
demyelination by 54% (p\lt 0.01). Relative to unsensitized wild-type
mice, myelin area was reduced by 23% even with combined scavenging
(ECSOD OD). However, this difference was not statistically significant.
Clearly, double protection against superoxide and H\sub{2}O\sub{2} by
ECSOD and catalase offered the best protection against the most
desirable parameter sought after for treatment, amelioration of myelin
fiber injury in the EAE optic nerve. The solo effect of catalase was
better than that of ECSOD.}

\subsection{Optic disc edema}

\p{Optic disc edema, seen ophthalmoscopically in approximately one-third
of patients with acute optic neuritis or multiple sclerosis, was evident
in EAE animals in which lateral displacement of the peripapillary retina
and filling of the optic cup indicated optic nerve head swelling at the
light microscopic level. Relative to ECSOD and catalase-protected nerves
(ECSOD OD; \figref{3}{A}), optic nerve head swelling was most severe in
the unprotected nerves of wild-type mice (Wt OS; \figref{3}{B}). The
combined effects of in vivo scavenging of hydrogen peroxide by viral
mediated catalase gene transfer and superoxide scavenging by germ line
increases of ECSOD reduced optic nerve head edema by 34% with a mean
optic nerve head area of 29,821 \mu m\sup{2} (ECSOD OD) relative to
wild-type mice with a mean value of 45,354 \mu m\sup{2} (Wt OS; p\lt
0.01; \figref{3}{C-D}). This combined effect was greater than that of
superoxide scavenging alone. ECSOD mice had 16% less optic disc edema
with a mean optic nerve head area of 38,092 \mu m\sup{2} (ECSOD OS)
relative to a mean of 45,354 \mu m\sup{2} (Wt OS) for wild-type mice
(p\lt 0.05). Clearly, the additional benefit of ECSOD was not much
greater than the solo effect of catalase that reduced optic disc edema
by 32% with a mean optic nerve head area of 30,754 \mu m\sup{2} (Wt OD)
relative to 45,354 \mu m\sup{2} for untreated eyes (Wt OS; p\lt 0.01).}

\subsection{Optic nerve cell count}

\p{Mononuclear inflammatory cells and reactive astroglial cells
predominantly involved the retrobulbar optic nerve of mice inoculated
for EAE. ECSOD eyes inoculated with catalase had the greatest decrease
in optic nerve cellularity. A representative light micrograph shows no
inflammatory cells and a relatively normal complement of astroglial
nuclei. (\figref{4}{A}). In contrast, the unprotected nerves revealed
many mononuclear inflammatory cells in addition to the astroglia
(\figref{4}{B}). The combined effects of in vivo scavenging of hydrogen
peroxide by viral mediated catalase gene transfer and superoxide
scavenging suppressed the optic nerve cell count by 27% with mean of 243
cells per 10\sup{5} \mu m\sup{2} (ECSOD OD) relative to 335 cells per
10\sup{5} \mu m\sup{2} (Wt OS) for untreated wild-type mice (p\lt 0.01)
(\figref{4}{C-D}). This combined effect was greater than that of
superoxide scavenging alone as ECSOD mice (ECSOD OS) had 17% less
inflammation with a mean cell count of 276 cells per 10\sup{5} \mu m\sup{2}
versus a mean of 335 cells per 10\sup{5} \mu m\sup{2} for wild-type mice
(Wt OS; p\lt 0.05). It was slightly greater than the solo effect of
catalase mediated scavenging of hydrogen peroxide with a mean cell count
of 258 cells per 10\sup{5} \mu m\sup{2} (Wt OD) relative to 335 cells per
10\sup{5} \mu m\sup{2} (Wt OS) that reduced inflammation by 23% (p\lt
0.01).}

\subsection{H\sub{2}O\sub{2}}

\p{Electron dense cerium derived H\sub{2}O\sub{2} reaction product in
the EAE optic nerve was greatest in the meninges of the optic nerve
sheath, but was also evident in the optic nerve head and retrobulbar
nerve, thus it was similar to the distribution of the ECSOD.
Transmission electron micrographs show cerium perhydroxide reaction
product in the lumen and perivascular space of ECSOD mice
(\figref{5}{A}) is increased relative to wild-type mice (\figref{5}{B}).
In the retrobulbar optic nerve of AAV-catalase gene inoculated eyes of
ECSOD mice (ECSOD OD) a mean of 62 cerium perhydroxide particles per
2.6x10\sup{6} \mu m\sup{2} represented a 5% increase of H\sub{2}O\sub{2}
counts relative to 59 particles per 2.6x10\sup{6} \mu m\sup{2} for
unprotected nerves of wild-type mice (Wt OD; \figref{5}{C-D}). This
slight difference was not statistically significant. Catalase gene
inoculation into the eyes of wild-type mice (Wt OD) with a value of 38
particles per 2.6x10\sup{6} \mu m\sup{2} decreased H\sub{2}O\sub{2}
counts by 35% relative to the unprotected eyes of wild-type mice (Wt
OS), but this difference was not statistically significant. However,
cerium perhydroxide reaction product particles increased by 72% in the
left eyes of ECSOD mice (ECSOD OS) with a mean of 106 particles per
2.6x10\sup{6} \mu m\sup{2} relative to unprotected left eyes of
wild-type mice (Wt OS; p\lt 0.01).}

\subsection{Blood-brain barrier}

\p{Disruption of the blood-brain barrier (BBB), a hallmark of optic
neuritis and MS, was seen in all animals sensitized for EAE. A standard
marker of BBB disruption is the extravasation of serum albumin that was
detected by immunolabeling. Transmission electron microscopy of the
optic nerves revealed albumin immunogold labeling in all animals with
EAE. Extravasated albumin immunogold in the perivascular compartment
located the foci of BBB disruption in EAE. Albumin immunogold confined
to the intravascular compartment indicated normal integrity of the BBB.
\figref{6} shows representative transmission electron micrographs of the
optic nerve of ECSOD mice inoculated with rAAV-catalase exhibiting less
extravasated serum albumin (\figref{6}{A}) than the control left eyes of
wild-type littermates in which accumulation of extravasated albumin
immunogold in the perivascular space is evident (\figref{6}{B}).
AAV-delivered catalase to the right eyes of transgenic ECSOD mice (ECSOD
OD) reduced disruption of the BBB by 63%, with a mean value of 62
extravasated immunogold particles per 2.6x10\sup{6} \mu m\sup{2}
relative to the left eyes of wild-type mice (Wt OS) with a mean value of
167 extravasated immunogold particles per 2.6x10\sup{6} \mu m\sup{2}
(p\lt 0.01; \figref{6}{C-D}). This combined effect was greater than a
value of 108 extravasated immunogold particles per 2.6x10\sup{6} \mu
m\sup{2} for catalase gene transfer to the eyes of wild-type mice (Wt
OD) that suppressed BBB disruption by 35% relative to unprotected left
eyes (Wt OS; p\lt 0.05). It was also greater than 119 extravasated
immunogold particles per 2.6x10\sup{6} \mu m\sup{2} for the solo effect
of germ line increases in ECSOD (ECSOD OS) that suppressed disruption of
the BBB by 29% relative to untreated left eyes of wild-type littermates
(Wt OS; p\lt 0.05). Thus, combined scavenging of superoxide by ECSOD and
hydrogen peroxide by catalase gene transfer restored integrity to the
BBB.}

\discussion

\p{Our results demonstrate that most parameters of experimental optic
neuritis were substantially ameliorated by genetically induced expansion
of two key antioxidant enzymes, catalase and ECSOD. Previously, we had
demonstrated the beneficial effects of detoxification of a single ROS
(H\sub{2}O\sub{2}) by catalase gene monotherapy. However, catalase only
suppressed demyelination by 38% [23,30]. Since multiple ROS are likely
involved in the pathogenesis of EAE and MS, it was reasonable to expect
that detoxifying several detrimental ROS would have the best suppressive
effect on disease activity. Here by combining ROS scavenging with ECSOD
and catalase, we achieved a 72% reduction in the most important
parameter of EAE studied, myelin injury to the optic nerve. This result
was substantially better than the solo effect of catalase or ECSOD. Dai
and coworkers attempted this approach in an animal model of
antigen-induced arthritis in rodents [40]. In their study combined
scavenging with ECSOD and catalase did not have an additive protective
effect on arthritis though each antioxidant gene had a protective effect
as in our study, in which each also suppressed demyelination and
disruption of the blood-brain barrier. Still, we found that the ECSOD
did not exert a suppressive effect on optic nerve head swelling. Since
optic nerve head edema is predominantly due to swelling of axons rather
than accumulation of extracellular fluid, lack of intra-axonal ECSOD
likely contributed to the relative lack of protection at the nerve
head.}

\p{The linearity of the combined protective effect of ECSOD and catalase
that suppressed blood-brain barrier disruption and demyelination in our
study suggests that superoxide and hydrogen peroxide each has a direct
detrimental effect on blood vessels and myelin in the EAE optic nerve.
Still, superoxide and hydrogen peroxide can combine to generate a highly
toxic metabolite, the hydroxyl radical. While scavenging of either
reactant may then suppress hydroxyl radical formation, combined
scavenging of either reactant may have no additional benefit. Clearly,
this was not the case here. By detoxifying H\sub{2}O\sub{2} to
relatively nontoxic byproducts the solo effect of catalase was better
than that of ECSOD that had the least protective effect. Several factors
may have contributed to this result. Dismutation of superoxide by ECSOD
increased extracellular H\sub{2}O\sub{2} levels. Our prior publications
have demonstrated the adverse impact of hydrogen peroxide on
experimental optic neuritis [31,41]. Thus, accumulation of
H\sub{2}O\sub{2} in the extracellular compartment may have partially
dampened the suppressive effect that dismutation of superoxide by ECSOD
had on experimental optic neuritis. Still despite the increase in
H\sub{2}O\sub{2}, ECSOD had a modest suppressive effect. In addition,
the suppressive effect of ECSOD on experimental optic neuritis may have
occurred perhaps by reducing generation of other pathogenic ROS. With
less superoxide available to react with nitric oxide, levels of highly
toxic peroxynitrite may have been suppressed and levels of nitric oxide
increased [42]. Nitric oxide and peroxynitrite, formed by the reaction
of nitric oxide and superoxide, also play a role in the pathogenesis of
EAE and MS [43-45].}

\p{The antioxidant enzymes catalase and SOD can be used to target ROS
for destruction, thus suppress tissue injury. However, there are
limitations to the use of the proteins themselves as treatment agents in
EAE and MS. First, the antioxidant enzyme (SOD) must be administered
daily, even with conjugation of polyethylene glycol, to prolong the
half-life of the enzyme [31,46]. Second, exogenous SOD or catalase are
effective only during the periods of active BBB disruption when these
high molecular weight proteins are able to penetrate the central nervous
system (CNS) [47]. Finally, optic neuritis recurs in part due to the
inability of the protein to cross the BBB after integrity is
restored.\sup{47} Genetic augmentation of cellular defenses against
superoxide and hydrogen peroxide helps surmount these limitations. Since
transgene expression following delivery with the AAV vector is
relatively long-lived, a single treatment may be sufficient. Still,
treatment by intraocular injection incurs some risk, even with an AAV
vector that is relatively nonpathogenic.}

\p{Our findings in acute EAE suggest that genetic amplification of
cellular defenses against ROS may have a role in attenuating CNS injury
associated with optic neuritis and perhaps MS. Determining whether
comparable levels of protection are also maintained against the repeated
demyelinating inflammation of chronic relapsing EAE may be key, if
anti-ROS gene transfers are to be applied in a clinical setting. While
the 72% suppressive effect on demyelination achieved here is promising,
our next steps are to tackle the pitfalls inherent in dual AAV infection
of the retina and optic nerve and to demonstrate a protective effect by
AAV mediated transfer of both genes (catalase and SOD), or perhaps even
a chimeric SOD [48] on chronic EAE.}

\acknowledgements

\p{We wish to thank Mabel Wilson for editing of the manuscript.}

\p{The work was supported by NIH EY 07982 (Dr. Guy), EY 11123, NS 36302
(Dr. Hauswirth) and Research to Prevent Blindness.}

\p{W.W.H. and the University of Florida could be entitled to patent
royalties for inventions related to this work and W.W.H and the
University of Florida both own equity in a company that may
commercialize some of the technology described herein.}

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\endreferences

}

\beginfigures

\figfile{1}{
\figtitle{1}{Expression of extracellular superoxide dismutase and
catalase}

\p{Immunofluorescence micrographs show expression of the human ECSOD
(arrows) in the optic nerve of a representative transgenic ECSOD mouse
(\panel{A}), but it is absent in the optic nerve of a wild-type
littermate (\panel{B}). Transmission electron micrograph of the
retrobulbar optic nerve of a transgenic ECSOD mouse reveals ECSOD
immunogold (arrows) in the perivascular space and endothelia of the
optic nerve (\panel{C}), peripapillary choroid (\panel{E}) and optic
nerve sheath (\panel{F}). Human ECSOD is absent in wild-type littermates
(\panel{D}). Immunobloting shows increased catalase expression in
cultured retinal ganglion cells infected with rAAV containing the gene
for human catalase, relative to control RGC-5 cells infected with
AAV-GFP (\panel{G}). E represents endothelial cell. RBC represents red
blood cell. L represents lumen, Cat represents catalase, gfp represents
green fluorescent protein.}

\ctr{\jpgimage{1}{800}{1215}{158}}

}

\figfile{2}{
\figtitle{2}{Suppression of demyelination}

\p{Representative transmission electron micrographs of the retrobulbar
optic nerve show many normal fibers and substantially less demyelinated
and thinly myelinated axons following rAAV-catalase inoculation of the
right eyes of transgenic ECSOD mice (\panel{A}), relative to the
unprotected left eyes of wild-type littermates in whom marked fiber
loss, naked axons and those with thin sheaths of myelin were prominent
ultrastructural findings (\panel{B}). The barplot shows mean myelin
areas of the retrobulbar optic nerve protected by both ECSOD and
catalase (ECSOD OD), catalase (Wt OD), ECSOD (ECSOD OS) and unprotected
EAE (Wt OS; \panel{C}). Barplot (\panel{D}) illustrates the preservation
of myelin induced by ECSOD and catalase, catalase or ECSOD relative to
unprotected nerves. Asterisk (*) represents p\lt 0.05, double asterisks
(**) represents p\lt 0.01, Ax represents axon, As represents astrocyte
process.}

\ctr{\jpgimage{2a}{500}{701}{128}}

\ctr{\gifimage{2b}{500}{578}{23}}

}

\figfile{3}{
\figtitle{3}{Suppression of optic disc swelling}

\p{Representative light micrographs showing catalase and SOD suppressed
optic nerve head edema (\panel{A}) relative to the unprotected optic
nerve (arrows) exhibiting marked swelling of the optic nerve head
(\panel{B}). The barplot of mean optic nerve head (ONH) areas shows that
optic nerve head swelling (smaller ONH area) was reduced by combined
ECSOD and catalase (ECSOD OD) treatment, catalase treatment (Wt OD),
ECSOD treatment (ECSOD OS), but it was greatest with no treatment (Wt
OS; \panel{C}). Barplot (\panel{D}) illustrates the reduction in ONH
swelling induced by ECSOD and catalase, catalase or ECSOD relative to
unprotected nerves. Asterisk (*) represents p\lt 0.05, double asterisks
(**) represents p\lt 0.01.}

\ctr{\jpgimage{3a}{500}{335}{46}}

\ctr{\gifimage{3b}{500}{588}{26}}

}

\figfile{4}{
\figtitle{4}{Suppression of cellular infiltration}

\p{Representative light micrographs show that cellular infiltration in
the retrobulbar optic nerve is reduced by double protection with ECSOD
and catalase (\panel{A}) relative to the unprotected optic nerve
(\panel{B}). The barplot shows the mean optic nerve cell count of the
retrobulbar optic nerve protected by both ECSOD and catalase (ECSOD OD),
catalase (Wt OD), ECSOD (ECSOD OS) and unprotected EAE (Wt OS;
\panel{C}). Barplot (\panel{D}) illustrates the reduction in the optic
nerve cell count induced by both ECSOD and catalase, catalase or ECSOD
relative to unprotected nerves. Asterisk (*) represents p\lt 0.05 double
asterisks (**) represents p\lt 0.01, A represents astrocyte, M
represents mononuclear inflammatory cell.}

\ctr{\jpgimage{4a}{500}{812}{113}}

\ctr{\gifimage{4b}{500}{542}{23}}

}

\figfile{5}{
\figtitle{5}{Reactive oxygen species in the optic nerve}

\p{Transmission electron micrographs show electron dense cerium
perhydroxide (arrows) formed by the reaction of cerium chloride and
endogenous hydrogen peroxide is more prominent in the optic nerves of
animals protected by ECSOD (ECSOD OS; \panel{A}) relative to unprotected
nerves (Wt OS; \panel{B}). The barplot shows mean cerium perhydroxide
particle counts in the retrobulbar optic nerves protected by ECSOD and
catalase (ECSOD OD), catalase (Wt OD), ECSOD (ECSOD OS) or unprotected
EAE (Wt OS; \panel{C}). Barplot (\panel{D}) illustrates the relative
changes in H\sub{2}O\sub{2} reaction product counts in nerves treated
with ECSOD and catalase, catalase or ECSOD relative to the unprotected
nerves. Asterisk (*) represents p\lt 0.05, L represents lumen.}

\ctr{\jpgimage{5a}{500}{735}{126}}

\ctr{\gifimage{5b}{500}{568}{25}}

}

\figfile{6}{
\figtitle{6}{Restoration of blood-brain barrier integrity}

\p{Transmission electron micrographs show that the combined effects of
catalase and ECSOD markedly decreased extravasation of serum albumin
labeled by immunogold (arrows) from the vessel lumen into the
perivascular space (\panel{A}), relative to perivascular accumulation of
labeled serum albumin in the unprotected optic nerve (\panel{B}). The
barplot shows mean extravasated albumin immunogold counts in the
retrobulbar optic nerves protected by ECSOD and catalase (ECSOD OD),
catalase (Wt OD), ECSOD (ECSOD OS) or unprotected EAE (Wt OS;
\panel{C}). Barplot (\panel{D}) illustrates the decrease in extravasated
albumin immunogold in nerves treated with ECSOD and catalase, catalase
or ECSOD relative to the unprotected nerves. Asterisk (*) represents
p\lt 0.05, double asterisks (**) represents p\lt 0.01, L represents
lumen.}

\ctr{\jpgimage{6a}{500}{659}{96}}

\ctr{\gifimage{6b}{500}{525}{21}}

}