Neuroprotective Effect of Sulfhydryl Reduction in a Rat
Optic Nerve Crush Model
Kyle I. Swanson, Christopher R. Schlieve, Christopher J. Lieven, and Leonard A. Levin
PURPOSE. The signaling of retinal ganglion cell (RGC) death
after axotomy is partly dependent on the generation of reactive
oxygen species. Shifting the RGC redox state toward reduction
is protective in a dissociated mixed retinal culture model of
axotomy. The hypothesis for the current study was that tris(2-
carboxyethyl)phosphine (TCEP), a sulfhydryl reductant, would
protect RGCs in a rat optic nerve crush model of axotomy.
METHODS. RGCs of postnatal day 4 to 5 Long-Evans rats were
retrogradely labeled with the fluorescent tracer DiI. At approx-
imately 8 weeks of age, the left optic nerve of each rat was
crushed with forceps and, immediately after, 4 ?L of TCEP (or
vehicle alone) was injected into the vitreous at the pars plana
to a final concentration of 6 or 60 ?M. The right eye served as
the control. Eight or 14 days after the crush, the animals were
killed, retinal wholemounts prepared, and DiI-labeled RGCs
counted. Bandeiraea simplicifolia lectin (BSL-1) was used to
RESULTS. The mean number of surviving RGCs at 8 days in eyes
treated with 60 ?M TCEP was significantly greater than in the
vehicle group (1250 ? 156 vs. 669 ? 109 cells/mm2; P ?
0.0082). Similar results were recorded at 14 days. Labeling was
not a result of microglia phagocytosing dying RGCs. No toxic
effect on RGC survival was observed with TCEP injection
CONCLUSIONS. The sulfhydryl-reducing agent TCEP is neuropro-
tective of RGCs in an optic nerve crush model. Sulfhydryl
oxidative modification may be a final common pathway for the
signaling of RGC death by reactive oxygen species after
axotomy. (Invest Ophthalmol Vis Sci. 2005;46:3737–3741)
diverse as glaucoma, optic neuritis, and traumatic optic neu-
ropathy have as a common feature the eventual death of retinal
ganglion cells (RGCs) in the eye.1Death commonly occurs
through apoptosis, an orderly cell suicide process, and, in most
cases, optic neuropathies are initiated by damage to RGC
axons.2–4RGC axotomy results in apoptosis.2–5Results from
our laboratory suggest that reactive oxygen species (ROS) are
part of the signaling pathway in cell death after axonal injury
iseases of the optic nerve (optic neuropathies) are a cause
of blindness in millions of people. Optic neuropathies as
(Lieven CJ, et al. IOVS 2003;44:ARVO E-Abstract 835).6–9We
previously demonstrated that RGC survival after axotomy de-
pends critically on the redox state of the cell and that shifting
the redox state toward mild reduction is protective in a disso-
ciated mixed retinal culture model. In particular, tris(2-car-
boxyethyl)phosphine (TCEP), a sulfhydryl reductant that does
not contain oxidizable sulfhydryls, maintained long-term sur-
vival of RGCs as potently as neurotrophic factors in mixed
retinal culture.8Because of these findings, we hypothesized
that the oxidative modification of sulfhydryl-containing pro-
teins by ROS is a mechanism for signaling apoptosis. However,
it is also possible that sulfhydryl reduction neuroprotection
arises from the mechanical effects of enzymatic dissociation in
the preparation of mixed retinal cultures, not from axotomy.
To distinguish these possibilities, we tested whether the po-
tent reducing agent TCEP would protect RGCs in a rat optic
nerve crush model of axotomy. We found that not only was
TCEP neuroprotective in an in vivo model of RGC axonal
damage, but it also prevented sulfhydryl oxidation in a cell-free
All experiments were performed in accordance with the U.S. Public
Health Service Policy on Humane Care and Use of Laboratory Animals,
the National Institutes of Health Guide for the Care and Use of Labo-
ratory Animals, the ARVO Statement for the Use of Animals in Oph-
thalmic and Vision Research, and institutional, federal, and state guide-
lines regarding animal research.
The fluorescent tracer 1,1?-dioctadecyl-3,3,3?,3?-tetramethylindocarbo-
cyanine perchlorate (DiI) was obtained from Molecular Probes (Eu-
gene, OR). Balanced saline solution and triple antibiotic ophthalmic
ointment were obtained from Wilson Ophthalmic (Mustang, OK).
Fluorescein-conjugated Bandeiraea simplicifolia lectin I (BSL-I) was
obtained from Vector Laboratories (Burlingame, CA). Paraformalde-
hyde and Triton X-100 were obtained from Fisher Scientific (Pitts-
burgh, PA). TCEP, along with all other reagents unless otherwise
noted, was obtained from Sigma-Aldrich (St. Louis, MO).
The ability of TCEP to act as a sulfhydryl-reducing agent was tested by
measuring its capacity to reduce disulfides to sulfhydryls in a DTNB
assay10modified from that described by Ellman.11Absorbance was
measured at 405 nm instead of the usual 412 nm (Fig. 1).
RGCs were retrogradely labeled by stereotactic injection of the fluo-
rescent tracer DiI, dissolved in dimethylformamide, into the superior
colliculi of anesthetized postnatal day 4 to 5 Long-Evans rats. DiI is
taken up by the synaptic terminals of RGCs in the superior colliculi and
then transported back through the optic nerve to the RGC somas in the
From the Department of Ophthalmology and Visual Science, Uni-
versity of Wisconsin Medical School, Madison, Wisconsin.
Supported by National Eye Institute Grant R01EY12492, the Glau-
coma Foundation, the Retina Research Foundation, and an unrestricted
departmental grant from Research to Prevent Blindness, Inc. KIS was
supported by a Hilldale Research Grant. LAL is a Research to Prevent
Blindness Dolly Green Scholar.
Submitted for publication February 5, 2005; revised May 17, 2005;
accepted July 21, 2005.
Disclosure: K.I. Swanson, None; C.R. Schlieve, None; C.J.
Lieven, None; L.A. Levin (P)
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked “advertise-
ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Corresponding author: Leonard A. Levin, University of Wisconsin
Medical School, 600 Highland Avenue, Madison, WI 53792-4673.
Investigative Ophthalmology & Visual Science, October 2005, Vol. 46, No. 10
Copyright © Association for Research in Vision and Ophthalmology
Optic Nerve Crush Surgery and
Surgeries were conducted on adult rats aged 8 and 12 weeks that had
previously received DiI injections. All surgeries were performed asep-
tically and on the left eye only. The right eye was not manipulated.
Animals were anesthetized with ketamine (80 mg/kg) and xylazine (8
mg/kg) intraperitoneally. A limited lateral canthotomy was performed.
The conjunctiva was then incised at the limbus and the sub-Tenon
space was bluntly dissected posteriorly. Intravitreal injections were
performed just posterior to the pars plana with a 5 ?L syringe (Ham-
ilton, Reno, NV) and a 33-gauge needle. The following were slowly
injected in a volume of 4 ?L: (1) sterile balanced saline solution (n ?
12); (2) 85 ?M TCEP dissolved in balanced saline (n ? 5); or (3) 850
?M TCEP dissolved in balanced saline (n ? 12). Assuming the vitreous
volume of an adult rat eye to be approximately 56 ?L,12the final
intravitreal concentration of TCEP for groups 2 and 3 was approxi-
mately 6 and 60 ?M, respectively. Optic nerve crushes were per-
formed according to our published methods.13The muscle cone was
entered and the optic nerve was exposed. The axons of the optic nerve
were then crushed with fine forceps for 5 seconds, 2 mm posterior to
the globe, under direct visualization. Interruption of the RGC axons
was judged to be a separation of the proximal and distal optic nerve
ends within an intact meningeal sheath. This procedure spares the
meningeal vessels that carry the arterial circulation to the retina,
interruption of which would result in retinal infarction. The skin was
then closed with sutures, and ophthalmic neomycin, polymyxin B
sulfates, and bacitracin zinc antibiotic ointment were applied to the
wound. The rats were given an intraperitoneal injection of buprenor-
phine (0.02 mg/kg) for analgesia and returned to the cage. Rats with
any kind of postoperative complication (e.g., cataract) were excluded
from analysis. Typically, six animals were operated on in each session.
To detect any toxic effects of TCEP alone, some animals were injected
with 60 ?M (final concentration) TCEP (n ? 2) or balanced saline
solution (n ? 2) without subsequent optic nerve crush. All animals
were observed to eat and drink normally after recovering from anes-
BSL-I Staining and Retinal Wholemounts
Eight or 14 days after optic nerve transection and/or intravitreal injec-
tion, the rats were euthanatized with controlled flow CO2. The eyes
were rapidly enucleated, rinsed, punctured with a needle through the
pupil, and then fixed for 1 hour in freshly prepared 4% paraformalde-
hyde (PFA). The retinas were dissected, washed with phosphate-buff-
ered saline (PBS), and permeabilized in 0.2% Triton X-100 for 15
minutes. After another PBS wash, the retinas were stained with fluo-
rescein-conjugated BSL-I (1:200) for 2 hours to label microglia, which
can phagocytose DiI-containing apoptotic RGCs and thereby be con-
fused with RGCs.14The retinas were washed again and postfixed with
4% PFA for 15 minutes. After a final wash, four cuts were made with
fine iris scissors from the edge to the center of the retinas, to flatten
them. They were mounted with the RGCs facing up on glass slides in
glycerol, and the coverslip was sealed with clear nail polish. The slides
were stored in the dark at 4°C until analysis.
Determination of RGC Density
Retinas were imaged with a digital camera (Axiocam HRc) attached to
a fluorescence microscope (Axiophot; Carl Zeiss Meditec, Inc., Dublin,
CA). Images were acquired (Axiovision 3.1 software; Carl Zeiss Med-
itec, Inc.) at 100? with a resolution of 1.0638-?m/pixel, without
binning. RGCs were identified by the presence of retrogradely trans-
ported cytoplasmic DiI, which appeared reddish orange when viewed
with rhodamine filters under epifluorescence. Fluorescein-conjugated
BSL-I-labeled cells appeared green when viewed with fluorescein fil-
ters. The density of RGCs/mm2was determined by counting labeled
DiI cells in three areas per retinal quadrant at three different eccen-
tricities of the retinal radius for a total of 12 regions per retina.
Automated counting was done with ImageJ, using the Analyze Particles
function (available by ftp at zippy.nimh.nih.gov/ or at http://rsb.info.
nih.gov/nih-image; developed by Wayne Rasband, National Institutes
of Health, Bethesda, MD). Cells positive for both DiI and BSL-I, deter-
mined by bright yellow labeling when DiI (red) and BSL-I (green)
images were merged (Fig. 2B, right), were subtracted from the total DiI
count. On average, approximately 1300 RGCs were counted per con-
trol eye. An observer masked to treatment or the presence of optic
nerve crush performed all cell counts.
Mean results were compared by using Student’s unpaired t-test. P ?
0.05 was considered significant.
Effect of Intravitreal Injection of TCEP on the
Survival of RGCs after Optic Nerve Crush
Compared with Vehicle Alone
Preservation of the retinas was excellent, with no signs at the
macroscopic or microscopic level of retinal infarction. The
mean number of surviving RGCs in eyes treated with 60 ?M
TCEP and subjected to optic nerve crush was 1250 ? 156
cells/mm2at 8 days and 1286 ? 141 cells/mm2at 14 days.
These counts were significantly greater than the mean RGC
counts in eyes treated with balanced saline solution and sub-
jected to nerve crush, which was 669 ? 109 cells/mm2at 8
days (P ? 0.0082; Fig. 2C) and 470 ? 202 cells/mm2at 14 days
(P ? 0.035; Fig. 2D). However, a 10 times lower dose of TCEP
(6 ?M) had no significant effect on RGC viability at 8 days
(457 ? 124 cells/mm2vs. 669 ? 109 cells/mm2; P ? 0.22). In
the nonsurgical control eyes, the mean number of RGCs was
1705 ? 104 cells/mm2, which is comparable to the average
number of RGCs (1710 ? 73 cells/mm2) reported by Klo ¨cker
et al.,15who used DiI to label RGCs. As would be expected, the
number of RGCs in eyes with crushed optic nerves treated
with balanced saline solution was significantly lower than the
number in uncrushed optic nerves at 8 (P ? 0.0000004) and 14
(P ? 0.01) days.
The increased survival of axotomized RGCs treated with
TCEP is not explained by erroneous identification of phago-
cytic microglia as RGCs, as those were specifically identified.
sulfhydryls in aqueous solutions. (B) TCEP reduces the disulfide con-
taining Ellman’s reagent (DTNB) in a linear fashion, forming a product
that can be quantified by measuring its absorbance at 405 nm.
(A) TCEP is a sulfhydryl reductant that irreversibly reduces
3738 Swanson et al.
IOVS, October 2005, Vol. 46, No. 10
Fluorescein-labeled BSL-I, a lectin that binds to a carbohydrate
on microglia, was used to label the latter. The percentage of
dual-labeled cells was 6.4% in the retinas with optic nerve
crush treated with balanced saline solution, 2.7% in the retinas
with optic nerve crush treated with TCEP, and 1.1% in the
control retinas. The increased survival was also not due to the
neuroprotective effects of anesthesia16(ketamine is an antag-
onist of the N-methyl-D-aspartate receptor, and xylazine is an
?2-adrenergic receptor agonist), since identically anesthetized
animals in the balanced saline solution group had significantly
lower RGC counts than did animals in the TCEP group.
Affect of TCEP Injection Alone on the Number of
In trials involving intravitreal injections without optic nerve
crush surgery, the mean number of RGCs in the TCEP group
was 1781 ? 82 cells/mm2. The balanced saline solution group
had an average of 1689 ? 176 cells/mm2. There was no
significant difference in the survival of RGCs in eyes receiving
balanced saline solution or TCEP injections without the optic
nerve crush injury (P ? 0.70). Moreover, neither of the groups
receiving injections differed significantly from the control eyes
in these trials, which had a mean of 1764 ? 278 cells/mm2(Fig.
In the present study, the sulfhydryl-reducing agent TCEP was
neuroprotective for at least 14 days in an optic nerve crush
model of RGC axotomy. Optic nerve crush experiments pro-
vide a more realistic model of acute optic neuropathies than
does acute dissociation followed by tissue culture, because the
insults caused by enzymatic disruption and the artificial culture
environment (primarily media and substrate) are eliminated.
This study thus provides evidence that the neuroprotection
due to the sulfhydryl-reducing agent TCEP is not an artifact of
cell culture. There was no RGC loss caused by injection of
TCEP into the eye alone, making it unlikely that TCEP itself has
toxic effect on RGCs.
TCEP, which irreversibly reduces disulfides to sulfhydryls in
aqueous solutions (Fig. 1), has several advantages over other
possible sulfhydryl reductants, such as 2-mercaptoethanol and
dithiothreitol (DTT). TCEP is soluble in water and is cell per-
meable. At higher temperatures, TCEP is relatively stable com-
pared with DTT. Furthermore, unlike 2-mercaptoethanol and
DTT, TCEP itself has no sulfhydryl groups that can be oxidized
and is therefore less likely to be oxidized by ROS.17Accord-
ingly, we have previously shown that RGC survival in mixed
culture is higher with TCEP treatment than with DTT treat-
optic nerve crush was significantly
increased when the crush was pre-
ceded by an intravitreal injection of
TCEP in a balanced saline solution.
(A) Schematic drawing of the exper-
iment. (B) Representative photomi-
crographs of retinas that were retro-
gradely labeled with DiI by injection
in the superior colliculus, to identify
RGCs, and the cells stained with
BSL-I to identify possible phagocytic
microglia. Control eyes had no injec-
tion. TCEP eyes had the optic nerve
crushed, immediately followed by in-
travitreal injection of 4 ?L TCEP (85
or 850 ?M) in balanced saline solu-
tion, resulting in a calculated final
concentration of 6 or 60 ?M TCEP.
Balanced saline–treated eyes com-
bined optic nerve crush with an in-
travitreal injection of balanced saline
solution alone. Retinas were har-
vested 8 or 14 days after surgery. The
density of RGCs per square millime-
ter was determined by counting la-
beled DiI cells in three areas per ret-
eccentricities of the retinal radius for
a total of 12 regions per retina. Cells
positive for both DiI and BSL-I, deter-
mined by bright yellow labeling
when DiI (red) and BSL-I (green) im-
ages were merged, were subtracted
from the total DiI count (arrowhead
in the merged balanced saline solu-
tion image). (C) Survival of RGCs after optic nerve crush was significantly increased by intravitreal injection of TCEP compared with vehicle alone.
Results are expressed as mean cells per square millimeter ? SEM. *Significance of comparison with control (uncrushed) optic nerves in the first
column. RGC survival at days after optic nerve crush with intravitreal TCEP (60 ?M) was significantly greater than with intravitreal balanced saline
solution or TCEP (6 ?M) at 8 days. No protective effect was seen with 6 ?M TCEP, compared with balanced saline solution. *P ? 0.05; **P ? 0.01;
***P ? 0.001. (D) The protective effects of intravitreal injection of TCEP persist to 14 days. RGC survival with intravitreal TCEP (60 ?M) after optic
nerve crush was significantly greater than with intravitreal balanced saline solution, at both 8 and 14 days. Data in columns 1 to 3 are the same
as in columns 1,2, and 4 of (C). *P ? 0.05; **P ? 0.01; ***P ? 0.001. (E) TCEP (60 ?M) injection alone did not adversely affect the number of
surviving RGCs in eyes that did not undergo optic nerve crush. Counts were obtained after 8 days. Results are expressed as mean cells per square
millimeter ? SEM. There is no significant difference between any of the conditions.
RGC survival 8 days after
IOVS, October 2005, Vol. 46, No. 10
Ganglion Cell Neuroprotection with Sulfhydryl Reduction 3739
Caution should be used in translating these results for po-
tential use in clinical practice. First, although TCEP was neu-
roprotective in this study, the concentration used was rela-
tively high (60 ?m), and a 10-fold lower concentration (6 ?M)
was not neuroprotective. We used an estimate of 56 ?L for the
vitreous volume of an adult rat eye to calculate a 60-?M final
concentration of TCEP in the eye, based on the anatomic
volume.12However, Dureau et al.18reported that the effective
vitreous volume (i.e., taking into account the volume of distri-
bution within the vitreous gel) is closer to 13 ?L. In that case,
the RGCs would be exposed to a TCEP concentration of ap-
proximately 260 ?M. Previous experiments in our laboratory,
in which mixed retinal cultures were used to test TCEP neu-
roprotection, set 100 ?M as the final concentration.8Achieving
these relatively high concentrations would probably be clini-
cally unfeasible with topical or systemic application. Instead,
we developed sulfhydryl-reducing agents that are neuroprotec-
tive at picomolar concentrations, to extend this therapeutic
mechanism to the clinical arena (Schlieve CR, et al. IOVS 2005;
46;ARVO E-Abstract 188; Wisconsin Alumni Research Founda-
tion, patent pending).
Second, the neuroprotective effect of TCEP was investi-
gated in the optic nerve crush model, which results in an acute
and complete transection of all RGC axons. Although the
experimental paradigm in the present study is most similar to
severe traumatic optic neuropathy,19most human optic neu-
ropathies are partial and proceed over days (e.g., optic neuri-
tis) to years (e.g., open-angle glaucoma). Also, given that mam-
malian central nervous system axons do not ordinarily
regenerate, even maintaining RGC survival by inhibiting sulf-
hydryl oxidative modification would not translate into mainte-
nance of visual function. Nonetheless, it possible that the
ability to pharmacologically maintain RGC viability in the face
of an overwhelming acute axonal injury would translate to an
increased resistance to a lower level chronic injury. Testing
this hypothesis would require studies in animal models (e.g.,
rodent experimental autoimmune encephalomyelitis or ocular
There are several possible mechanisms by which a sulfhy-
dryl-reducing agent like TCEP could protect RGCs from axonal
injury. Axotomy induces apoptosis in RGCs,2–5,20partly medi-
ated by blocked retrograde transport of neurotrophic factors21
or decreased levels of endogenous ocular neurotrophins.22,23
Intraocular administration of neurotrophins (e.g., brain-derived
neurotrophic factor; BDNF) delays RGC death after axotomy in
adult rats,24,25and gene delivery of BDNF to the retina or the
RGC increases survival in experimental glaucoma.26,27
Yet RGC axotomy induces changes in responsiveness to
neurotrophins independent of neurotrophin deprivation,14in-
dicating that axotomy can signal changes at the cell body
independent of neurotrophin deprivation. Apoptosis may arise
from a signal generated directly by the injury,28or some other
yet to be defined mechanism. One possibility is that ROS serve
as a signaling molecule to transduce the effect of axonal injury.
ROS are intracellular signaling molecules in several cell
types.29–32One ROS, superoxide anion, is generated by sym-
pathetic neurons deprived of nerve growth factor33,34and can
be identified a few hours after neurotrophin deprivation. Thus
ROS appear to contribute to cell death, not only by directly
participating in the destruction of the cell via oxidative modi-
fication of structural macromolecules, but also by activating
the apoptotic pathway.35
Our previous findings that RGC survival in rats is dependent
on the redox state, with a mildly reduced cellular environment
being most conducive for cellular survival, is supported by
similar findings by Castagne and Clarke36in chick retina. Ox-
idative stress due to ROS can result in the oxidation of sulfhy-
dryl groups in protein cysteines. This effect could result in the
formation of intramolecular and intermolecular disulfide cross-
links that would affect the conformation of critical signaling
proteins37and lead to apoptosis. We do not know the specific
targets for TCEP that are involved in its ability to prevent RGC
death after axotomy, but assume that they are sulfhydryl-con-
taining proteins that are oxidized in RGC apoptosis signaling.
Candidate molecules include components of the mitochondrial
permeability transition pore38and protein tyrosine phospha-
tases.39We are currently involved in a proteomic approach to
identifying proteins specifically involved in RGC death after
optic nerve crush. It is also possible that TCEP rescues RGCs by
inducing less specific survival mechanisms. However, TCEP
does not protect against other common modes of cell death,
such as death induced by the protein kinase inhibitor stauro-
sporine or by PK11195, which binds to the peripheral benzo-
diazepine receptor and opens the mitochondrial permeability
TCEP is neuroprotective for RGCs in the rat optic nerve crush
model, without significant toxicity. Achieving a better under-
standing of the mechanism by which TCEP increases survival
of RGCs requires determining the target of TCEP’s action,
which is likely to be one or more sulfhydryl-containing pro-
teins that TCEP protects from oxidative modification.
1. Levin LA, Clark JA, Johns LK. Effect of lipid peroxidation inhibition
on retinal ganglion cell death. Invest Ophthalmol Vis Sci. 1996;
2. Garcia-Valenzuela E, Gorczyca W, Darzynkiewicz Z, Sharma SC.
Apoptosis in adult retinal ganglion cells after axotomy. J Neuro-
3. Berkelaar M, Clarke DB, Wang YC, Bray GM, Aguayo AJ. Axotomy
results in delayed death and apoptosis of retinal ganglion cells in
adult rats. J Neurosci. 1994;14:4368–4374.
4. Levin LA, Louhab A. Apoptosis of retinal ganglion cells in anterior
ischemic optic neuropathy. Arch Ophthalmol. 1996;114:488–
5. Quigley HA, Nickells RW, Kerrigan LA, Pease ME, Thibault DJ,
Zack DJ. Retinal ganglion cell death in experimental glaucoma and
after axotomy occurs by apoptosis. Invest Ophthalmol Vis Sci.
6. Lieven CJ, Vrabec JP, Levin LA. The effects of oxidative stress on
mitochondrial transmembrane potential in retinal ganglion cells.
Antioxid Redox Signal. 2003;5:641–646.
7. Nguyen SM, Alexejun CN, Levin LA. Amplification of a reactive
oxygen species signal in axotomized retinal ganglion cells. Anti-
oxid Redox Signal. 2003;5:629–634.
8. Geiger LK, Kortuem KR, Alexejun C, Levin LA. Reduced redox
state allows prolonged survival of axotomized neonatal retinal
ganglion cells. Neuroscience. 2002;109:635–642.
9. Kortuem K, Geiger LK, Levin LA. Differential susceptibility of
retinal ganglion cells to reactive oxygen species. Invest Ophthal-
mol Vis Sci. 2000;41:3176–3182.
10. Sedlak J, Lindsay RH. Estimation of total, protein-bound, and non-
protein sulfhydryl groups in tissue with Ellman’s reagent. Anal
11. Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys.
12. Berkowitz BA, Lukaszew RA, Mullins CM, Penn JS. Impaired hya-
loidal circulation function and uncoordinated ocular growth pat-
terns in experimental retinopathy of prematurity. Invest Ophthal-
mol Vis Sci. 1998;39:391–396.
13. Levin LA, Schlamp CL, Spieldoch RL, Geszvain KM, Nickells RW.
Identification of bcl-2 family genes in the rat retina. Invest Oph-
thalmol Vis Sci. 1997;38;2545–2553
3740Swanson et al.
IOVS, October 2005, Vol. 46, No. 10
14. Shen S, Wiemelt AP, McMorris FA, Barres BA. Retinal ganglion cells Download full-text
lose trophic responsiveness after axotomy. Neuron. 1999;23:285–
15. Klo ¨cker N, Zerfowski M, Gellrich NC, Bahr M. Morphological and
functional analysis of an incomplete CNS fiber tract lesion: Graded
crush of the rat optic nerve. J Neurosci Methods. 2001;110:147–
16. Ozden S, Isenmann S. Neuroprotective properties of different
anesthetics on axotomized rat retinal ganglion cells in vivo. J Neu-
17. Getz EB, Xiao M, Chakrabarty T, Cooke R, Selvin PR. A comparison
between the sulfhydryl reductants tris(2-carboxyethyl)phosphine
and dithiothreitol for use in protein biochemistry. Anal Biochem.
18. Dureau P, Bonnel S, Menasche M, Dufier JL, Abitbol M. Quantita-
tive analysis of intravitreal injections in the rat. Curr Eye Res.
19. Levin LA, Beck RW, Joseph MP, Seiff S, Kraker R. The treatment of
traumatic optic neuropathy: the International Optic Nerve Trauma
Study. Ophthalmology. 1999;106:1268–1277.
20. Rehen SK, Linden R. Apoptosis in the developing retina: paradox-
ical effects of protein synthesis inhibition. Braz J Med Biol Res.
21. Aguayo AJ, Clarke DB, Jelsma TN, Kittlerova P, Friedman HC, Bray
GM. Effects of neurotrophins on the survival and regrowth of
injured retinal neurons. Ciba Foundation Symp. 1996;196:135–
22. Moretto G, Xu RY, Walker DG, Kim SU. Co-expression of mRNA
for neurotrophic factors in human neurons and glial cells in cul-
ture. J Neuropathol Exp Neurol. 1994;53:78–85.
23. Lambert W, Agarwal R, Howe W, Clark AF, Wordinger RJ. Neuro-
trophin and neurotrophin receptor expression by cells of the
human lamina cribrosa. Invest Ophthalmol Vis Sci. 2001;42:2315–
24. Mansour-Robaey S, Clarke DB, Wang YC, Bray GM, Aguayo AJ.
Effects of ocular injury and administration of brain-derived neuro-
trophic factor on survival and regrowth of axotomized retinal
ganglion cells. Proc Natl Acad Sci USA. 1994;91:1632–1636.
25. Peinado-Ramon P, Salvador M, Villegas-Perez MP, Vidal-Sanz M.
Effects of axotomy and intraocular administration of NT-4, NT-3,
and brain-derived neurotrophic factor on the survival of adult rat
retinal ganglion cells: a quantitative in vivo study. Invest Ophthal-
mol Vis Sci. 1996;37:489–500.
26. Martin KR, Quigley HA, Zack DJ, et al. Gene therapy with brain-
derived neurotrophic factor as a protection: retinal ganglion cells
in a rat glaucoma model. Invest Ophthalmol Vis Sci. 2003;44:
27. Wang N, Zeng M, Ruan Y, et al. Protection of retinal ganglion cells
against glaucomatous neuropathy by neurotrophin-producing, ge-
netically modified neural progenitor cells in a rat model. Chin Med
J (Engl). 2002;115:1394–1400.
28. Rajan P, Stewart CL, Fink JS. LIF-mediated activation of STAT
proteins after neuronal injury in vivo. Neuroreport. 1995;6:2240–
29. Lander HM. An essential role for free radicals and derived species
in signal transduction. FASEB J. 1997;11:118–124.
30. Finkel T. Signal transduction by reactive oxygen species in non-
phagocytic cells. J Leukoc Biol. 1999;65:337–340.
31. Cooper CE, Patel RP, Brookes PS, Darley-Usmar VM. Nanotrans-
ducers in cellular redox signaling: modification of thiols by reac-
tive oxygen and nitrogen species. Trends Biochem Sci. 2002;27:
32. Levonen AL, Patel RP, Brookes P, et al. Mechanisms of cell signal-
ing by nitric oxide and peroxynitrite: from mitochondria to MAP
kinases. Antioxid Redox Signal. 2001;3:215–229.
33. Greenlund LJ, Deckwerth TL, Johnson EM. Superoxide dismutase
delays neuronal apoptosis: a role for reactive oxygen species in
programmed neuronal death. Neuron. 1995;14:303–315.
34. Dugan LL, Creedon DJ, Johnson EM Jr, Holtzman DM. Rapid sup-
pression of free radical formation by nerve growth factor involves
the mitogen-activated protein kinase pathway. Proc Natl Acad Sci
35. Kane DJ, Sarafian TA, Anton R, et al. Bcl-2 inhibition of neural
death: decreased generation of reactive oxygen species. Science.
36 Castagne V, Clarke PG. Axotomy-induced retinal ganglion cell
death in development: its time- course and its diminution by anti-
oxidants. Proc R Soc Lond B Biol Sci. 1996;263:1193–1197.
37. Park C, Raines RT. Adjacent cysteine residues as a redox switch.
Protein Eng. 2001;14:939–942.
38. McStay GP, Clarke SJ, Halestrap AP. Role of critical thiol groups on
the matrix surface of the adenine nucleotide translocase in the
mechanism of the mitochondrial permeability transition pore. Bio-
chem J. 2002;367:541–548.
39. Xu D, Rovira II, Finkel T. Oxidants painting the cysteine chapel:
redox regulation of PTPs. Dev Cell. 2002;2:251–252.
40. Vrabec JP, Lieven CJ, Levin LA. Cell type-specific opening of the
retinal ganglion cell mitochondrial permeability transition pore.
Invest Ophthalmol Vis Sci. 2003;44:2774–2782.
IOVS, October 2005, Vol. 46, No. 10
Ganglion Cell Neuroprotection with Sulfhydryl Reduction 3741