* Department of Neurobiology, The Weizmann Institute of Science, 76100 Rehovot, Israel; Department of Internal Medicine E, Rabin Medical Center, Beilinson Campus, Petah-Tikva 49100, Israel; and
Israel Defense Force, Medical Corps, Chaim Sheba Medical Center, Tel Hashomer 52621 Israel
1 To whom correspondence should be addressed at Department of Neurobiology, The Weizmann Institute of Science, 76100 Rehovot, Israel. Fax: 972 8 9346018. E-mail: michal.schwartz{at}weizmann.ac.il.
Received March 24, 2005; accepted June 7, 2005
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ABSTRACT |
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Key Words: organophosphate; neuroprotection; neurodegeneration; protective autoimmunity.
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INTRODUCTION |
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Physiological compounds (such as glutamate and nitric oxide), normally pivotal for proper functioning of the brain, are cytotoxic when present in abnormally high concentrations (Barger and Basile, 2001; Choi, 1988
; Fonnum, 1984
) and are likely to participate in secondary degenerative processes. There is evidence, for example, that the glutamatergic system participates in the induction of seizures by nerve agents and contributes to the ensuing neuropathology (McDonough and Shih, 1993
; Olney et al., 1986
). Because the excessive presence of glutamate and of some other physiological compounds is known to cause neuronal death, these observations prompted a search for a therapeutic approach to ward off intoxication by OPs in the CNS, with the aim of preventing or minimizing the toxicity-induced spread of damage (McDonough and Shih, 1993
; Sparenborg et al., 1992
).
Recent data from our laboratory, obtained from rat and mouse models, suggest that the peripheral adaptive immune system, in the form of T cells specifically directed against autoantigens residing in sites of CNS damage, plays a key role in helping the CNS to withstand the degenerative consequences of insults in general and glutamate toxicity in particular (Kipnis et al., 2001; Mizrahi et al., 2002
; Schori et al., 2001b
). Moreover, boosting of this T cell-mediated autoimmune response by immunization with glatiramer acetate (Copolymer 1; Cop-1), a synthetic copolymer that cross-reacts weakly with a wide range of CNS self-antigens, helps to reduce neuronal loss without incurring the risk of autoimmune disease induction (Angelov et al., 2003
; Benner et al., 2004
; Kipnis et al., 2000
; Schori et al., 2001a
).
The aim of this study was to determine whether vaccination with Cop-1 can protect mice against neuronal death after their exposure to OPs. In general, exposure to OPs intoxicates both the central and the peripheral nervous systems. However, because we were interested in examining immune system participation in protecting the organism against the effects of OPs on the CNS, and in order to simplify the experimental conditions, the model chosen for the study was one of direct CNS exposure to OPs by injection of diisopropyl fluorophosphate (DFP) into the mouse eye (Yoles and Schwartz, 1998). We found that the DFP injection resulted in dose-dependent lethality of retinal ganglion cells (RGCs), and that significantly more RGCs died in mice deprived of T cells (nu/nu) than in the wild type. Moreover, when mice were directly exposed to intoxication by glutamate, treatment with DFP delayed RGC death relative to that observed in untreated mice, suggesting that at least part of the loss of CNS neurons caused by OP intoxication is the result of secondary degenerative processes known to be amenable to neuroprotective therapy (Kornhuber et al., 1994
). Moreover, because of the multiple factors involved in degeneration, an immune-based approach is likely to provide the most comprehensive protection possible.
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MATERIALS AND METHODS |
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DFP injection and treatment with Cop-1 or MK-801.
With the aid of a binocular microscope, the right eye of the anesthetized mouse was punctured in the upper part of the sclera with a 27-gauge needle, and a 10-µl Hamilton syringe with a 30-gauge needle was inserted as far as the vitreal body. Mice were injected with a total volume of 1 µl of DFP (Sigma, St. Louis, MO) dissolved at different concentrations in saline. Seven days before, immediately after, or 48 h after DFP injection, the mice were immunized subcutaneously in the flank with 75 µg of Cop-1 (Teva Pharmaceuticals, Petah Tikva, Israel) solubilized in phosphate-buffered saline (PBS). On the day of DFP injection, some of the mice were injected ip with 1 mg/kg of MK-801 (Sigma). Control mice were injected with PBS.
Labeling of retinal ganglion cells.
RGCs were labeled, 72 h before tissue excision, with a fluorescent dye injected stereotactically into the superior colliculus. For this purpose, mice were anesthetized and placed in a stereotactic device. The skull was exposed and kept dry and clean, and the bregma was identified and marked. The designated point of injection was 2.92 mm posterior to the bregma, 0.5 mm lateral to the midline, and at a depth of 2 mm from the brain surface. A window was drilled in the scalp above the designated coordinates in the right and left hemispheres. The neurotracer dye FluoroGold (5% solution in saline; Fluorochrome, Denver, CO) was applied (1 µl, at a rate of 0.5 µl/min in each hemisphere) using a Hamilton syringe, and the skin over the wound was sutured.
Assessment of retinal ganglion cell survival.
At the end of the experimental period the mice were given a lethal dose of pentobarbitone (170 mg/kg). Their eyes were enucleated, and the retinas were detached and prepared as flattened whole mounts in 4% paraformaldehyde in PBS. Labeled cells from four to six fields of identical size (0.076 mm2) were counted. The counted fields were located at approximately the same distance from the optic disk (0.3 mm) to allow for variations in RGC density as a function of distance from the optic disk. Fields were counted under fluorescence microscope (magnification x800) by observers blinded to the treatment received by the mice. The average number of RGCs per field was calculated for each retina. The number of RGCs in the contralateral (uninjured) eye was also counted and served as an internal control.
Histological analysis.
Two weeks after injection of DFP or saline and immunization with Cop-1 or PBS, the mice were killed as described above, and their eyes were removed and fixed in formaldehyde (4% in PBS) for 48 h at 4°C. Sections (10 µm thick) were embedded in paraffin and stained with hematoxylin and eosin (H&E).
Statistical analysis.
One-way ANOVA was used for overall comparison of means. Dunnett test was preformed to compare each treatment to the control. Tukey-Kramer test was used for simultaneous comparison of several means. Comparison in cases where only two treatments were involved was preformed by t-test.
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RESULTS |
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DISCUSSION |
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Recent studies have suggested that OPs invoke changes in brain neurotransmitters and peptides, leading to death of neurons in the CNS (McDonough and Shih, 1993). Attempts to reduce the damage caused by OP intoxication have focused on immediate emergency treatment with atropine, oxime, and an anticonvulsant. No acceptable treatment is currently available for counteracting the toxic effects of glutamate, a major causative factor in the spread of damage and neuronal loss observed in a number of acute and chronic brain disorders, irrespective of the primary etiology. Moreover, as a consequence of exposure of the CNS to OPs, as with any CNS insult, numerous additional mediators of toxicity contribute to the self-perpetuating degenerative process. Studies in our laboratory suggest that well-controlled levels of activated autoimmune T cells, upon encountering microglia, confer on them a protective phenotype that is capable in part, via production of insulin-like growth factor I, of counteracting the cytotoxic environment and supporting neuronal survival (Shaked et al., 2005
). It therefore seems feasible that a post-intoxication vaccination, administered in the subacute and not in the hyperacute phase (Okano et al., 2003
), might offer a new therapeutic strategy for reducing nerve damage that inevitably follows the initial insult.
Studies by our group have shown that the peripheral adaptive immune system plays a key role in an individual's ability to withstand the consequences of a CNS insult (including direct exposure to glutamate) (Moalem et al., 1999; Schori et al., 2001a
). They also showed that the number of neurons that survive axotomy or direct glutamate intoxication is strain dependent, and that strains which are genetically endowed with the ability to withstand the injury lose their protective advantage if they lack mature T cells (Kipnis et al., 2001
; Schori et al., 2001b
). The T cells that participate in the ability to withstand glutamate toxicity or other CNS insult were found to be directed against self-antigens residing in the site of damage. Further studies by our group suggested that T cells will be beneficial for neural tissue, provided that the timing and intensity of their activities are well-controlled (Fisher et al., 2001
; Hauben et al., 2001
; Shaked et al., 2005
); if poorly controlled, the same T cells will lead to development of an autoimmune disease. One way to ensure that autoimmunity will be kept under control is by using weak agonists of relevant self-antigens as therapeutic vaccines to boost the T cell-mediated protective effect (Mizrahi et al., 2002
; Schori et al., 2001a
). An example of such an agonist is Cop-1, which cross-reacts weakly with a wide range of self-reactive antigens (Hafler, 2002
; Kipnis and Schwartz, 2002
), thereby circumventing the tissue specificity barrier.
In the present work we showed that the direct result of local administration of OPs to the mouse eye was a loss of RGCs, demonstrating that OPs, known to cause damage to the brain, can also damage the visual system. The DFP-induced damage appears to trigger a glutamatergic-dependent toxic pathway, since it was partially prevented here by i.p. injection of the NMDA-receptor antagonist MK-801. The choice of this antagonist rather than alternative or additional glutaminergic antagonists was based on our previous finding that MK-801 blocks glutamate toxicity in the same strain of mice as that used in this study, (Schori et al., 2002).
It was encouraging to find here that boosting of the immune system by vaccination with Cop-1 increases the animal's ability to withstand intoxication if administered before (and not only after) exposure to the intoxicating agents (Moalem et al., 1999; Schori et al., 2001a
). This finding is in agreement with those obtained in other studies. (Angelov et al., 2003
; Bakalash et al., 2003
; Benner et al., 2004
; Kipnis et al., 2000
; Schori et al., 2001a
). This finding implies that vaccination, unlike pharmacological treatments, can be administered not only for therapeutic purposes but also prophylactically. However, a late vaccination was ineffective, suggesting that the therapeutic effect is limited to the subacute phase that follows intoxication. Moreover, harnessing of the immune system for therapeutic purposes provides a cell-mediated therapy, resulting in protection that is more likely to comprehensively counteract a battery of toxicity mediators than pharmaceutical monotherapies. Vaccination with Cop-1, for example, results in increased homing of relevant T cells to their specific self-antigens residing at the lesion site in the CNS (Bakalash et al., 2003
; Kipnis et al., 2000
; Moalem et al., 2000
; Schori et al., 2001b
). Once locally activated there (by their encounter with the relevant self-antigens presented to them on antigen-presenting cells), the T cells produce cytokines and growth factors that boost the ability of the resident glial cells to buffer the potentially harmful environmental conditions induced by the injury (Butovsky et al., 2001
; Shaked et al., 2005
).
Cop-1 has been approved by the United States Food and Drug Administration for human use. It is therefore worth exploring the possibility of developing it (once the correct dosage protocols are established) as a protective injection after OP intoxication, in addition to the current basket of therapies used for this purpose. Other powerful vaccinations with similar compounds are currently under investigation.
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ACKNOWLEDGMENTS |
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