Post-Intoxication Vaccination for Protection of ...

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Nerve agents are highly toxic organophosphates (OPs) that can cause severe damage to the central and peripheral nervous systems. The central nervous ...
TOXICOLOGICAL SCIENCES 87(1), 163–168 (2005) doi:10.1093/toxsci/kfi237 Advance Access publication June 23, 2005

Post-Intoxication Vaccination for Protection of Neurons against the Toxicity of Nerve Agents Hadas Schori,* Eyal Robenshtok,† Michal Schwartz,*,1 and Ariel Hourvitz‡ *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 Received March 24, 2005; accepted June 7, 2005

The nerve agents sarin, soman, tabun, and VX are highly toxic organophosphates (OPs) that inhibit the enzyme cholinesterase. Exposure to these compounds causes a progression of toxic signs, including hypersecretions, fasciculations, tremors, convulsions, coma, and death. Each of these manifestations was thought to result solely from hyperactivity of the cholinergic system due to cholinesterase inhibition (McDonough and Shih, 1997). Recent studies have suggested, however, that OPs can also cause secondary events in the central nervous system (CNS) leading to seizures and consequently to nerve pathology (Lemercier et al., 1983). 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. 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: [email protected].

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

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Nerve agents are highly toxic organophosphates (OPs) that can cause severe damage to the central and peripheral nervous systems. The central nervous system insult results in seizures and neuronal death. The glutamatergic system apparently contributes to the neuropathology. Using a model of OP intoxication causing death of retinal ganglion cells in the mouse eye, we show here that intoxication is exacerbated if the mice are devoid of mature T cells. The retinal neurons could be protected from these effects by vaccination, 7 days before or immediately after intoxication, with the copolymer glatiramer acetate (Cop-1), recently found to limit the usual consequences of an acute glutamate insult to the eye. These findings underlie a new therapeutic approach to protection against OP intoxication, based on the rationale that boosting of the adaptive immunity recruited at the site of intoxication helps the local cellular machinery such as resident microglia to withstand the neurotoxic effects. Key Words: organophosphate; neuroprotection; neurodegeneration; protective autoimmunity.

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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. MATERIALS AND METHODS

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-ll Hamilton syringe with a 30-gauge needle was inserted as far as the vitreal body. Mice were injected with a total volume of 1 ll 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 lg 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.

RESULTS

Injection of DFP into the mouse eye, used here as a simple in vivo model that allows direct quantification of neuronal damage and protection, caused a dose-dependent loss of RGCs. Each group of DFP-injected mice was compared to a group of saline-injected mice. In agreement with a previous report (Schori et al., 2002), the mean number of surviving RGCs in the saline-injected eyes did not differ from that in the noninjected eyes (Fig. 1A). RGC death occurred mainly during the second week after intoxication, suggesting that DFP intoxication triggered a delayed process of degeneration (Fig. 1B). The toxic effect of the DFP injection was partially blocked by i.p. injection of the NMDA-receptor antagonist MK-801 ( p < 0.05; Student’s t-test) (Fig. 2), suggesting that at least some of the

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 ll, at a rate of 0.5 ll/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 3800) 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 lm 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.

FIG. 1. Dose- and time-dependent death of retinal ganglion cells induced by intravitreal DFP injection in C57Bl/6J mice. (A) Mice received a single injection of DFP (5.4 or 0.54 nmol), and surviving RGCs were counted 21 days later. Bars represent the number of surviving RGCs per mm2 (mean ± SEM) (n ¼ 8–9 in each group). (B) Mice received a single injection of DFP (5.4 nmol), and surviving RGCs were counted at different times after the injection. Bars represent the number of surviving RGCs per mm2 (mean ± SEM; n ¼ 6–7 in each group). To stabilize variances, log transformation was applied (*indicates a significant difference in level of 5% relative to control saline-injected group; Dunnett test).

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Animals. Animals were handled according to the regulations formulated by the Institutional Animal Care and Use Committee. Wild-type C57Bl/6J mice and wild-type or nude Balb/c mice, aged 8–13 weeks, were supplied by the Animal Breeding Center of The Weizmann Institute of Science and housed in light- and temperature-controlled rooms. Prior to all experiments the mice were anesthetized by intraperitoneal (ip) administration of ketamine 80 mg/kg and xylazine 16 mg/kg. Previous studies in our laboratory have shown that ketamine does not have a neuroprotective effect in the NMDA toxicity model (Schori et al., 2002).

Comparison in cases where only two treatments were involved was preformed by t-test.

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induced RGC death was induced by a self-perpetuating process secondary to DFP intoxication and was mediated, at least in part, by glutamate. To determine whether the ability of the neurons to withstand DFP toxicity is dependent only on local physiological mechanisms (cellular, biochemical, or molecular) or, as in the case of optic nerve injury or glutamate toxicity (Fisher et al., 2001; Mizrahi et al., 2002; Schori et al., 2002), is aided by support from the systemic immune system, we compared RGC survival in two strains previously shown to differ in their T celldependent ability to withstand the degenerative effects of optic nerve crush and glutamate toxicity. As in those earlier studies (Kipnis et al., 2001), we found here that intravitreal injection of DFP caused significant RGC death in the wild-types of both C57Bl/6J and Balb/c strains relative to that in strain-matched controls with undamaged eyes (Fig. 3). However, Balb/c mice were significantly (Tukey-Kramer test) better able than C57Bl/6J mice to withstand the toxicity. To determine whether this strain-related difference in susceptibility to the toxin is systemic in origin, which would imply that resistance to the toxic effect is immune dependent, we examined whether the observed advantage of the Balb/c mice is eliminated if these mice are deprived of T cells (nu/nu). The number of surviving RGCs was significantly (Tukey-Kramer test) smaller in nu/nu Balb/c than in matched wild-type controls and did not differ from the number in the C57Bl/6J wild type (Fig. 3). Cop-1 has been shown to protect RGCs against the consequences of glutamate toxicity and axonal injury (Kipnis et al., 2000; Schori et al., 2001a). To determine whether Cop-1 is also protective against DFP intoxication, we examined the toxic effect of DFP in mice that were immunized with this copolymer 7 days before, immediately after, or 48 h after DFP

intoxication. Cop-1 reduced the toxicity by 20% when given prior to or immediately after DFP intoxication, but had no effect if given after 48 h. Each group of Cop-immunized mice was compared to a group that had received the DFP injection only (Dunnett test; Fig. 4). To assess whether the overall morphology of the retinas and not only the RGCs was affected by the DFP-intoxication we carried out histological analysis. Comparison of undamaged eye (Fig. 5A) to DFP-intoxicated eyes with (Fig. 5B) and without (Fig. 5C) revealed that DFP did not affect the overall morphology of the retina and that loss of RGCs was indeed attenuated by the Cop-1 treatment.

DISCUSSION

A correlation between OP poisoning and retinal damage was reported more than 10 years ago, but until recently there were hardly any further studies of the retinal effect (Boyes et al., 1994; Imai et al., 1983). Here we focused on OP-induced damage to the retina, and not only to the brain as in most of the previous studies on OP intoxication (Damodaran et al., 2002). Moreover, since RGC loss was found here to be significantly greater in the absence of T cells, the number of neurons that survived OP intoxication is evidently dependent, at least in part, on the integrity of the adaptive immune system and, in particular, on the spontaneous ability of the animal to harness a systemic adaptive immune response. In wild-type mice, neuronal survival was significantly increased after a single injection of Cop-1 given either before or immediately after exposure to DFP. Recent studies have suggested that OPs invoke changes in brain neurotransmitters and peptides, leading to death of

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FIG. 2. DFP-induced death of neurons is mediated by glutamate. C57Bl/6J mice (n ¼ 14 in each group) were injected i.p. with the glutamate antagonist MK-801 (1 mg/kg) immediately after intravitreal injection of DFP (5.4 nmol). Surviving RGCs were counted 21 days later. The number of surviving RGCs per mm2 (mean ± SEM) was significantly higher in the MK-801-injected mice than in control mice without MK-801 (*p < 0.05, two-tailed t-test).

FIG. 3. DFP toxicity is T-cell dependent and varies between strains. DFP (5.4 nmol) was injected intravitreally into wild-type C57Bl/6J and Balb/c mice and nu/nu Balb/c mice. Surviving RGCs were counted 21 days later. Significantly more neurons survived in the wild-type than in the nu/nu Balb/c mice, and in the wild-type Balb/c mice than in the C57Bl/6J mice. Bars represent the number of surviving RGCs per mm2 (mean ± SEM; n ¼ 5–7 in each group). To stabilize variances, log transformation was applied. Significant differences relative to the saline-injected control group at the 5% level (Dunnett’s test) are indicated by (*) above each bar. A significance difference between Balb/c wild-type and Balb/c nu/nu mice at the 5% level (TukeyKramer’s test) is indicated by (**).

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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,

FIG. 5. Morphological manifestation of DFP intoxication and Cop-1 protection. C57Bl/6J mice were immunized with 75 lg of Cop-1 in PBS or were injected with PBS alone (control) and, immediately afterward, received an intravitreal injection of DFP. After 21 days the mice were killed, and the treated eyes were removed and processed for histology. H&E-stained retinal sections (10 lm thick) of DFP-injected and control mice are shown. Bar ¼ 50 lm. (A) Normal eye. (B) Eye of a mouse injected with DFP and immunized with Cop-1. (C) Eye of a mouse injected with DFP and not immunized. Arrows point to the RGC layer.

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FIG. 4. Active immunization with Cop-1 induces neuroprotection against the effects of intravitreally injected DFP in C57Bl/6J mice. (A) C57Bl/6J mice were immunized with 75 lg of Cop-1 in PBS 7 days before, immediately after, or 48 h after intravitreal injection of 5.4 nmol DFP (n ¼ 7–8 mice in each group). Surviving RGCs were counted 21 days after DFP injection. RGC survival was calculated as the number of neurons that survived, expressed as a percentage of the number amenable to protection [(XY) / (ZY) 3 100, where X, Y, and Z are the numbers of RGCs in immunized mice, in nonimmunized mice, and in uninjured mice, respectively]. In mice immunized with Cop-1 on the day of the insult or 7 days earlier, the number of surviving RGCs per mm2 (mean ± SEM) was significantly higher than that in the mice that received DFP only (n ¼ 6–8 mice in each group). No protective effect was seen in mice immunized 48 h after the insult. To stabilize variances, log transformation was applied. A significant difference at the 5% level relative to the nonvaccinated DFP-injected control group (Dunnett’s test) is indicated by (*).

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 postintoxication 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

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grants awarded to M. Schwartz by Teva Pharmaceutical Industries, Proneuron Biotechnologies Inc. and the Ministry of Defense, IDF Medical Corps Scientific Committee Award. Conflict of interest: none declared.

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ACKNOWLEDGMENTS

Kipnis, J., Yoles, E., Schori, H., Hauben, E., Shaked, I., and Schwartz, M. (2001). Neuronal survival after CNS insult is determined by a genetically encoded autoimmune response. J. Neurosci. 21, 4564–4571.

We thank Smith for editing the manuscript and A. Shapira for animal maintenance. M. Schwartz holds the Maurice and Ilse supported in part by

Kornhuber, J., Weller, M., Schoppmeyer, K., and Riederer, P. (1994). Amantadine and memantine are NMDA receptor antagonists with neuroprotective properties. J. Neural Transm. Suppl. 43, 91–104.

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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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Schori, H., Kipnis, J., Yoles, E., WoldeMussie, E., Ruiz, G., Wheeler, L. A., and Schwartz, M. (2001a). Vaccination for protection of retinal ganglion cells against death from glutamate cytotoxicity and ocular hypertension: Implications for glaucoma. Proc. Natl. Acad. Sci. U.S.A. 98, 3398–3403.