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(Investigative Ophthalmology and Visual Science. 2005;46:3737-3741.)
© 2005 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.05-0155
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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

From the Department of Ophthalmology and Visual Science, University of Wisconsin Medical School, Madison, Wisconsin.


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 Conclusion
 References
 
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 approximately 8 weeks of age, the left optic nerve of each rat was crushed with forceps and, immediately after, 4 µL of TCEP (or vehicle alone) was injected into the vitreous at the pars plana to a final concentration of 6 or 60 µM. 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 identify microglia.

RESULTS. The mean number of surviving RGCs at 8 days in eyes treated with 60 µM 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 alone.

CONCLUSIONS. The sulfhydryl-reducing agent TCEP is neuroprotective 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.

Diseases of the optic nerve (optic neuropathies) are a cause of blindness in millions of people. Optic neuropathies as diverse as glaucoma, optic neuritis, and traumatic optic neuropathy have as a common feature the eventual death of retinal ganglion cells (RGCs) in the eye.1 Death commonly occurs through apoptosis, an orderly cell suicide process, and, in most cases, optic neuropathies are initiated by damage to RGC axons.2 3 4 RGC axotomy results in apoptosis.2 3 4 5 Results from our laboratory suggest that reactive oxygen species (ROS) are part of the signaling pathway in cell death after axonal injury (Lieven CJ, et al. IOVS 2003;44:ARVO E-Abstract 835).6 7 8 9 We previously demonstrated that RGC survival after axotomy depends critically on the redox state of the cell and that shifting the redox state toward mild reduction is protective in a dissociated mixed retinal culture model. In particular, tris(2-carboxyethyl)phosphine (TCEP), a sulfhydryl reductant that does not contain oxidizable sulfhydryls, maintained long-term survival of RGCs as potently as neurotrophic factors in mixed retinal culture.8 Because of these findings, we hypothesized that the oxidative modification of sulfhydryl-containing proteins 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 potent 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 assay.


   Methods
Top
 Abstract
 Methods
Results
 Discussion
 Conclusion
 References
 
Animals
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 Laboratory Animals, the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and institutional, federal, and state guidelines regarding animal research.

Reagents
The fluorescent tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) was obtained from Molecular Probes (Eugene, 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). Paraformaldehyde and Triton X-100 were obtained from Fisher Scientific (Pittsburgh, PA). TCEP, along with all other reagents unless otherwise noted, was obtained from Sigma-Aldrich (St. Louis, MO).

DTNB Assay
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 assay10 modified from that described by Ellman.11 Absorbance was measured at 405 nm instead of the usual 412 nm (Fig. 1) .



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  		FIGURE 1. (A) TCEP is a sulfhydryl reductant that irreversibly reduces sulfhydryls in aqueous solutions. (B) TCEP reduces the disulfide containing Ellman’s reagent (DTNB) in a linear fashion, forming a product that can be quantified by measuring its absorbance at 405 nm.

 
RGC Labeling
RGCs were retrogradely labeled by stereotactic injection of the fluorescent 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 eye.

Optic Nerve Crush Surgery and Intravitreal Injections
Surgeries were conducted on adult rats aged 8 and 12 weeks that had previously received DiI injections. All surgeries were performed aseptically 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 µL syringe (Hamilton, Reno, NV) and a 33-gauge needle. The following were slowly injected in a volume of 4 µL: (1) sterile balanced saline solution (n = 12); (2) 85 µM TCEP dissolved in balanced saline (n = 5); or (3) 850 µM TCEP dissolved in balanced saline (n = 12). Assuming the vitreous volume of an adult rat eye to be approximately 56 µL,12 the final intravitreal concentration of TCEP for groups 2 and 3 was approximately 6 and 60 µM, respectively. Optic nerve crushes were performed according to our published methods.13 The 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 buprenorphine (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 µM (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 anesthesia.

BSL-I Staining and Retinal Wholemounts
Eight or 14 days after optic nerve transection and/or intravitreal injection, 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% paraformaldehyde (PFA). The retinas were dissected, washed with phosphate-buffered saline (PBS), and permeabilized in 0.2% Triton X-100 for 15 minutes. After another PBS wash, the retinas were stained with fluorescein-conjugated BSL-I (1:200) for 2 hours to label microglia, which can phagocytose DiI-containing apoptotic RGCs and thereby be confused with RGCs.14 The 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 Meditec, Inc.) at 100x with a resolution of 1.0638-µm/pixel, without binning. RGCs were identified by the presence of retrogradely transported 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 filters. The density of RGCs/mm2 was determined by counting labeled DiI cells in three areas per retinal quadrant at three different eccentricities 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, determined 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 control eye. An observer masked to treatment or the presence of optic nerve crush performed all cell counts.



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  		FIGURE 2. RGC survival 8 days after optic nerve crush was significantly increased when the crush was preceded by an intravitreal injection of TCEP in a balanced saline solution. (A) Schematic drawing of the experiment. (B) Representative photomicrographs of retinas that were retrogradely 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 injection. TCEP eyes had the optic nerve crushed, immediately followed by intravitreal injection of 4 µL TCEP (85 or 850 µM) in balanced saline solution, resulting in a calculated final concentration of 6 or 60 µM TCEP. Balanced saline–treated eyes combined optic nerve crush with an intravitreal injection of balanced saline solution alone. Retinas were harvested 8 or 14 days after surgery. The density of RGCs per square millimeter was determined by counting labeled DiI cells in three areas per retinal quadrant at three different eccentricities of the retinal radius for a total of 12 regions per retina. Cells positive for both DiI and BSL-I, determined by bright yellow labeling when DiI (red) and BSL-I (green) images were merged, were subtracted from the total DiI count (arrowhead in the merged balanced saline solution 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 µM) was significantly greater than with intravitreal balanced saline solution or TCEP (6 µM) at 8 days. No protective effect was seen with 6 µM 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 µM) 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 µM) 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.

 
Statistical Analysis
Mean results were compared by using Student’s unpaired t-test. P < 0.05 was considered significant.


   Results
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 Abstract
 Methods
 Results
Discussion
 Conclusion
 References
 
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 µM TCEP and subjected to optic nerve crush was 1250 ± 156 cells/mm2 at 8 days and 1286 ± 141 cells/mm2 at 14 days. These counts were significantly greater than the mean RGC counts in eyes treated with balanced saline solution and subjected to nerve crush, which was 669 ± 109 cells/mm2 at 8 days (P = 0.0082; Fig. 2C ) and 470 ± 202 cells/mm2 at 14 days (P = 0.035; Fig. 2D ). However, a 10 times lower dose of TCEP (6 µM) had no significant effect on RGC viability at 8 days (457 ± 124 cells/mm2 vs. 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 Klöcker et al.,15 who 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 phagocytic microglia as RGCs, as those were specifically identified. 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 antagonist of the N-methyl-D-aspartate receptor, and xylazine is an {alpha}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 Surviving RGCs
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. 2E) .


   Discussion
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 Abstract
 Methods
 Results
 Discussion
Conclusion
 References
 
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 provide 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 permeable. At higher temperatures, TCEP is relatively stable compared 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.17 Accordingly, we have previously shown that RGC survival in mixed culture is higher with TCEP treatment than with DTT treatment.8

Caution should be used in translating these results for potential use in clinical practice. First, although TCEP was neuroprotective in this study, the concentration used was relatively high (60 µm), and a 10-fold lower concentration (6 µM) was not neuroprotective. We used an estimate of 56 µL for the vitreous volume of an adult rat eye to calculate a 60-µM final concentration of TCEP in the eye, based on the anatomic volume.12 However, Dureau et al.18 reported that the effective vitreous volume (i.e., taking into account the volume of distribution within the vitreous gel) is closer to 13 µL. In that case, the RGCs would be exposed to a TCEP concentration of approximately 260 µM. Previous experiments in our laboratory, in which mixed retinal cultures were used to test TCEP neuroprotection, set 100 µM as the final concentration.8 Achieving these relatively high concentrations would probably be clinically unfeasible with topical or systemic application. Instead, we developed sulfhydryl-reducing agents that are neuroprotective 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 Foundation, patent pending).

Second, the neuroprotective effect of TCEP was investigated 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,19 most human optic neuropathies are partial and proceed over days (e.g., optic neuritis) to years (e.g., open-angle glaucoma). Also, given that mammalian central nervous system axons do not ordinarily regenerate, even maintaining RGC survival by inhibiting sulfhydryl oxidative modification would not translate into maintenance 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 hypertension).

There are several possible mechanisms by which a sulfhydryl-reducing agent like TCEP could protect RGCs from axonal injury. Axotomy induces apoptosis in RGCs,2 3 4 5 20 partly mediated 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 25 and 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,14 indicating that axotomy can signal changes at the cell body independent of neurotrophin deprivation. Apoptosis may arise from a signal generated directly by the injury,28 or 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 30 31 32 One ROS, superoxide anion, is generated by sympathetic neurons deprived of nerve growth factor33 34 and 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 modification 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 Clarke36 in chick retina. Oxidative stress due to ROS can result in the oxidation of sulfhydryl 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 proteins37 and 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-containing proteins that are oxidized in RGC apoptosis signaling. Candidate molecules include components of the mitochondrial permeability transition pore38 and protein tyrosine phosphatases.39 We 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 staurosporine or by PK11195, which binds to the peripheral benzodiazepine receptor and opens the mitochondrial permeability transition pore.40


   Conclusion
Top
 Abstract
 Methods
 Results
 Discussion
 Conclusion
References
 
TCEP is neuroprotective for RGCs in the rat optic nerve crush model, without significant toxicity. Achieving a better understanding 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 proteins that TCEP protects from oxidative modification.


   Footnotes
 
Supported by National Eye Institute Grant R01EY12492, the Glaucoma 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 "advertisement" 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.


   References
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 Abstract
 Methods
 Results
 Discussion
 Conclusion
 References
 

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