ToxSci Advance Access originally published online on February 19, 2004
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Toxicological Sciences 78, 241-247 (2004)
Toxicological Sciences vol. 78 no. 2 © Society of Toxicology; all rights reserved.
Interactive Effects of Paraoxon and Pyridostigmine on Blood-Brain Barrier Integrity and Cholinergic Toxicity
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* Neurotoxicology Laboratory, Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University, Stillwater, Oklahoma 74078; Kennedy Krieger Institute and Department of Environmental Health Sciences, School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205
Received October 8, 2003; accepted January 10, 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The effect of the organophosphorous insecticide paraoxon on the integrity of the blood-brain barrier (BBB) and permeability of pyridostigmine (PYR), a peripheral inhibitor of cholinesterase activity, was examined in Long Evans rats. The integrity of the BBB was examined by measuring the number of capillaries leaking horseradish peroxidase, which was injected into the heart. Treatment with paraoxon at 100 µg/kg, intramuscularly, resulted in a 3- to 4-fold increase in the number of leaky capillaries in young rats (25 to 30 days old) but not in older rats (90 days old). Interestingly, young rats treated with PYR (30 mg/kg, po) 50 min before treatment with paraoxon showed an inhibited effect of paraoxon on the BBB. Furthermore, no increase in the degree of inhibition of acetylcholinesterase activity was observed in young rats treated with PYR before paraoxon compared with young rats treated with paraoxon alone. Cholinergic toxicity, as assessed by changes in behavior, was not observed in young rats treated with paraoxon alone; but, slight signs of cholinergic toxicity were observed in rats treated with PYR. Young rats treated with both PYR and paraoxon did not exhibit more extensive signs of toxicity than rats treated with paraoxon alone or PYR alone. The results indicate that treatment with paraoxon can compromise BBB permeability at dosages that do not induce cholinergic toxicity, but only in young rats. Also, PYR pre-exposure appears to protect the BBB from the paraoxon-induced alterations.
Key Words: paraoxon; pyridostigmine bromide; blood-brain barrier; cholinesterase; Gulf War illness.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The blood-brain barrier (BBB) protects the central nervous system from changes occurring in the composition of the blood that might influence neural activity. An acute increase in permeability of the BBB, as in certain traumas and diseases, may cause death from swelling or hemorrhaging. A subtle increase in permeability may result in cells, blood-derived proteins, and small ions crossing into the parenchyma (Farkas and Luiten, 2001
). Inflammation, stroke, and seizures have been shown to increase permeability of the BBB (Belayev et al., 1996; de Vries et al., 1997; Huber et al., 2002; Sahin et al., 2003). Indeed, the etiology of multiple sclerosis might be due to an immune reaction caused by a compromised BBB (Smith and McDonald, 1999). Several neurodegenerative diseases have been suggested to involve the BBB. As recently noted (Farkas and Luiten, 2001), increases in permeability of the BBB might not be the primary event, rather a secondary event that is required to promote neurodegenerative diseases such as Alzheimer's disease. Possibly, very small increases in permeability would produce a loss of nutrients, such as glucose, that would result in the death of neighboring cells.
Environmental factors have been shown to affect the integrity of the BBB. For example, exposure to high levels of lead produces a breakdown in the BBB in children, resulting in edema that could be fatal (Goldstein, 1984). Interestingly, the effects of lead on the BBB are observed only in children and, experimentally, only in preweanling rats (Pentschew and Garro, 1966; Toews et al., 1978). Also, organophosphorous toxicants used in chemical warfare, such as soman and sarin, have been repeatedly shown to cause a breakdown in the BBB in adult rats (Abdel-Rahman et al., 2002; Carpentier et al., 1990; Gupta et al., 1999), but studies on organophosphorous insecticides are less convincing. Quinalphos was shown to increase the permeability of the BBB in 10-day-old rats (Gupta et al., 1999), but exposure to metrifonate did not affect the BBB permeability in rats weighing 200 g (Rakonczay and Papp, 2001). Paraoxon was found to increase permeability in 200 g rats but at dosages that caused seizures (Ashani and Catravas, 1981). The factors influencing the effect of organophosphorous insecticides on the BBB are mostly unknown. For example, no study, that we are aware of, has examined developmental sensitivity of the BBB. Organophosphorous toxicants appear to affect the BBB by inducing seizures (Ashani and Catravas, 1981; Carpentier et al., 1990; Grange-Messent et al., 1999).
The increase in permeability of the BBB would be expected to result in entry into the brain of chemicals that are normally excluded. Indeed, a hypothesis for explaining some Gulf War illnesses states that a compromise in the BBB that is induced by physical stress and/or exposure to chemical agents resulted in increased uptake of pyridostigmine (PYR; Friedman et al., 1996; Hanin, 1996), which was taken by soldiers in the Persian Gulf War to prevent cholinergic toxicity from possible exposure to organophosphorus nerve agents. Numerous studies do not support, however, the finding that physical stress affects the entry of PYR into the brain (Grauer et al., 2000; Lallement et al., 1998; Ovadia et al., 2001; Shaikh et al., 2003; Sinton et al., 2000; Song et al., 2002; Tian et al., 2002).
In light of the inconsistencies that have been reported regarding the effects of organophosphorous insecticides on the BBB, we examined the BBB in young and older rats exposed to paraoxon. Also, we indirectly tested the hypothesis put forth for explaining Gulf War illnesses, which is that exposure to organophosphorous toxicants resulted in increased uptake of PYR into the brain because of a breakdown in the BBB integrity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals.
Horseradish peroxidase (HRP, type II), pyridostigmine bromide (PYR, 3-dimethylaminocarbonyloxy-N-methylpyridinium bromide), glucose oxidase, and 3,3'-diaminobenzidine (DAB) were purchased from Sigma Chemical Co. (St. Louis, MO). Paraoxon (O,O-diethyl-O-(p-nitrophenyl) phosphate) was purchased from Chem Service (West Chester, PA). Antibody against rat albumin was obtained from Accurate Scientific (Westbury, NY). [3H]Acetylcholine iodide (specific activity = 82.0 mCi/mmol) was obtained from New England Nuclear Co. (Boston, MA). All chemicals were reagent grade. PYR and paraoxon were prepared in 0.9% saline (w/v) fresh on the day of experiment. DAB was prepared immediately before the start of the reaction for peroxidase histochemistry.
Maintenance and treatment of rats.
Male, Long Evans rats were used throughout the experiment. Different groups of rats were used for each assay. Rats were maintained and handled according to the NIH/NRC Guide for the Care and Use of Laboratory Animals and reviewed by the Institutional Animal Care and Use Committees at Oklahoma State University and Johns Hopkins University. Rats were given free access to food and water and isolated from environmental stress. PYR (30 mg/kg, po) in saline or vehicle was administered to rats by gavage 50 min prior to paraoxon treatment. Paraoxon (100 µg/kg, im) in saline or vehicle alone was injected into the thigh muscle.
Assay for leaky capillaries.
The number of leaky capillaries was measured as an indicator of compromised BBB integrity. Rats were anesthetized with 200 µl xylaket/100 g body weight and given diethyl ether by inhalation at 10 min after treatment with paraoxon. Then, 200 µl HRP (40 mg/ml) in 2% Evans blue solution was injected into the left ventricle over a 10-s period. The eyes, skin, feet, and tail of the rats turned blue as a result of Evans blue circulating throughout the whole body. Rats were decapitated at 1 min after HRP injection.
The histochemical stain for visualizing HRP in sections of brain was performed as previously described (Stewart et al., 1992). Brains were removed (surface blood was cleaned with tissue) and then placed overnight in glutaraldehyde (2.5% in 0.1 M phosphate buffer, pH 7.4) followed by paraformaldehyde (4%) for at least 8 h. Sections (70 µm) from each treatment group were collected, placed on ice, and sequentially washed with phosphate-buffered saline (PBS), 4% paraformaldehyde in PBS, PBS, and washed twice with 0.1 M Tris buffer (pH 7.4). The sections were then treated with 0.5% cobalt chloride in 0.1 M Tris buffer for 10 min at room temperature. Sections were washed three times with 0.1 M Tris buffer and twice with 0.1 M phosphate buffer (pH 7.4). Substrate (50 mg DAB, 40 mg ammonium chloride, 200 mg ß-D-glucose, and 3 mg glucose oxidase in 100 ml of 0.1 M phosphate buffer) was added to the slices that were then incubated in the dark at 37°C for 1 to 2 h until a dark brown/black color was noted. Slices were mounted on glass slides and a coverslip was positioned with 90% glycerol. Slides were dried for 10 min prior to observation under a light microscope.
Two investigators, who were unaware of the experimental conditions, counted the number of capillaries leaking HRP in four sections of the hippocampus and striatum (x10 magnification) from each rat. Leaks were recognized by the presence of HRP immediately outside of the capillary wall (Fig. 1). A mean of the percentage increase ± SEM was computed by dividing the number of leaks in treated rats by the number in controls, multiplied by 100.
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Immunocytochemistry for albumin extravasation.
Rats were treated with paraoxon, anesthetized with xylaket, and perfused after 10 min with fixative (4% paraformaldehyde in 0.15 M phosphate buffer, pH 7.2) through the heart. Brains were excised and placed in fixative for 72 h and incubated 2 days at 4°C in 30% sucrose in PBS. Sections were cut to 25 µm with a cryostat and dried. Next, sections were incubated with 10 mM sodium citrate buffer, pH 6.0, and subjected to microwave radiation for 90 s, cooled, and treated for 16 h with 0.2% papain in 0.01 N HCl at room temperature. After washing three times in PBS for 5 min, sections were treated with 0.5% Triton X-100 in PBS for 60 min. After three washings with PBS, sections were blocked for 60 min in 5% normal goat serum with 0.5% Triton X-100. The sections were washed and incubated with rabbit antialbumin diluted 1:1000 in PBS/Triton X-100 overnight at 4°C. The sections were given three incubations, 20 min each, in PBS. The antibody-antigen complex was visualized with the ABC technique with a biotinylated goat antirabbit IgG and avidin-labeled horseradish peroxidase. After washing, the sections were incubated with diaminobenzidine at 0.5 mg/ml in 50 mM Tris, pH 7.7, for 10 min at room temperature.
Cholinergic signs of toxicity.
Rats were observed and evaluated by two investigators for the signs of cholinergic toxicity including involuntary movements and salivation, lacrimation, urination, and defecation (SLUD), according to previously described methods (Liu and Pope, 1996; Moser et al., 1998). Involuntary movements were quantified and scored as follows: 2 = normal quivering of vibrissae, head, and limbs; 3 = mild, fine tremor seen in the forelimbs and head; 4 = whole body tremor; 5 = myoclonic jerks; and 6 = clonic convulsions. Overt secretion from autonomic dysfunction was scored as follows: 1 = normal, no excessive secretion; 2 = slight, 1 SLUD sign, or very mild multiple signs; 3 = moderate, multiple, overt SLUD signs; and 4 = severe, multiple, extensive SLUD signs.
Assay for cholinesterase (ChE) activity.
For ChE evaluation, rats were treated identically as described for assaying leaky capillaries except that rats were not injected with HRP. Frontal and temporal cortex brain tissues were separated, collected on ice, and washed with 0.9% saline to remove surface blood. Brain tissues were stored at –70°C and thawed on the day of the assay. Tissues were homogenized in 50 mM potassium phosphate buffer by Polytron PT-3000 homogenizer (Brinkman Instruments, Westbury, NY) at 28,000 rpm for 20 s. ChE activity was radiometrically evaluated (Johnson and Russell, 1975) using 1 mM [3H]-acetylcholine as the substrate, as described previously (Tian et al., 2002), and expressed as nmol/min/mg protein and percentage of control activity. Protein was determined with the Folin phenol reagent (Lowry et al., 1951).
Statistical analysis.
Functional and behavioral signs of toxicity were expressed as median ± interquartile range (IQR) and analyzed for significance by the Pearson Chi-square test. ChE activity was analyzed by two-way analysis of variance (ANOVA) followed by linear contrasts using JMP statistical software (SAS Institute, Inc., Cary, NC). Leaky capillaries were also analyzed by two-way ANOVA followed by Tukey's posthoc test to compare different treatments. A p value > 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Paraoxon Increased the Number of Leaky Capillaries
One objective of our study was to determine whether an organophosphate anticholinesterase could increase leaks without inducing seizures and other signs of cholinergic toxicity. To accomplish this objective, male rats (25–30 days of age) were injected with a moderately low dosage (100 µg/kg, im) of paraoxon, and BBB permeability of horseradish peroxidase (HRP) was evaluated. Figure 1 shows that, at 10 min after injection, leaks were observed in several brain regions including hippocampus, striatum, and cortex. Leaks differed in size and density even in the same brain region. A few leaks were observed in sections from the control rats, which were treated with saline (not shown).
Extravasation of Albumin in Rats Treated with Paraoxon
To verify the effects of paraoxon on the BBB, extravasation of albumin was examined because serum proteins leak into the brain when BBB integrity is compromised. Figure 2 shows albumin leaking from capillaries in the parenchyma of the cortex and striatum in rats treated with paraoxon. No staining in the parenchyma was observed in controls. Staining was basically restricted to the vessel lumen, most likely because of some albumin binding to the vessel wall that was not washed by perfusion (Fig. 2A). Massive diffusion was observed in some areas, as an intense reaction product (Fig. 2B), or very light staining (Fig. 2C), possibly due to several leaky capillaries, while the leaks were more localized in other areas (Fig. 2D).
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Evaluation of Leaky Capillaries in Young and Older Rats after Treatment with Paraoxon
Previous studies have shown a maturational decrease in sensitivity to cholinergic toxicity of many organophosphorus insecticides (Benke and Murphy, 1974
; Brodeur and Dubois, 1963; Pope et al., 1991). To determine whether BBB modulation by paraoxon also displays a maturational difference in sensitivity, the effectiveness of paraoxon (100 µg/kg, im) to induce leaky capillaries in young (25 to 30 days of age) and older (90 days of age) rats was compared in the hippocampus and striatum. Figure 3 shows that a significant increase in leaky capillaries was observed in the young rats compared with the older rats.
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The Interactive Effects of PYR and Paraoxon on ChE Activity
The increase in albumin leaking into the brain suggests that other impermeable chemicals would also leak in the young rats. PYR, for example, is impermeable but might enter the brain after exposure to paraoxon. The permeability of PYR was examined indirectly by measuring ChE activity in rats treated with a high dosage of PYR (30 mg/kg, po) 50 min before paraoxon treatment. Treatment with PYR and paraoxon produced significant inhibition of ChE activity in peripheral tissues (Fig. 4). ChE activity was inhibited by more than 80% in blood and diaphragm of rats treated with PYR, whereas a 40–65% inhibition was observed in these same tissues of rats treated with paraoxon. In the brain, PYR did not inhibit ChE activity. Paraoxon, however, caused about 54–58% ChE inhibition in frontal and temporal cortex tissues. When PYR was given to rats before paraoxon treatment, the degree of ChE inhibition in blood and diaphragm was similar to that noted in rats given PYR alone. Interestingly, ChE activity was significantly higher in the temporal cortex in rats given PYR before paraoxon compared to rats given paraoxon alone, and a similar difference between the same treatment groups approached significance (p = 0.07) in the frontal cortex.
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The Interactive Effects of PYR and Paraoxon on Leaky Capillaries
Because the combination of PYR and paraoxon did not produce greater inhibition of ChE activity than paraoxon alone, we examined whether PYR modulated the effects of paraoxon on leaky capillaries. Interestingly, fewer leaks were noted in the hippocampus and striatum of rats treated with PYR prior to paraoxon than in rats treated with paraoxon alone (Fig. 5).
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Functional and Behavioral Measurements in Rats Treated with ChE Inhibitors
As described above, the increase in leaky capillaries in rats treated with organophosphorous nerve agents has been previously associated with severe cholinergic toxicity, i.e., seizures and convulsions (Carpentier et al., 1990
). Rats injected with paraoxon at 100 µg/kg did not, however, display any overt signs of cholinergic toxicity such as involuntary movements and SLUD signs (Fig. 6). Rats given PYR showed slight but significant signs of cholinergic toxicity (SLUD signs median score = 2, involuntary movement median score = 3). Very similar signs of toxicity were noted in rats given both paraoxon and PYR (SLUD signs median score = 2, involuntary movement median score = 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As previously stated, one of the goals of our study was to examine the integrity of the BBB in young and older rats exposed to the organophosphate paraoxon. Previous investigators have shown an association between exposure to high dosages of organophosphorus toxicants and increased number of leaky capillaries in the brain (Abdel-Rahman et al., 2002
; Ashani and Catravas, 1981; Carpentier et al., 1990), which would be expected to allow the entry of drugs that normally do not enter the brain. Importantly, a greater uptake of PYR, a drug given prophylactically to soldiers to attenuate the effects of nerve agent exposure, could potentially have contributed to some Gulf War illnesses (Friedman et al., 1996).
There were two novel findings of our study. Firstly, we found that dosages of paraoxon that increased the number of leaky capillaries did not induce behavioral changes that are characteristic of cholinergic toxicity. Two different assays, HRP leaking from capillaries and the extravasation of albumin, verified the increase in leaky capillaries. Several studies have demonstrated increases in leaky capillaries in rats treated with organophosphorous toxicants such as sarin, soman, and paraoxon. For example, rats exposed to soman or paraoxon combined with the peripherally acting ChE inhibitor phospholine iodide displayed significantly greater inhibition of brain ChE compared to rats exposed to soman or paraoxon alone (Ashani and Catravas, 1981). The authors suggested that the greater inhibition of ChE was due to increased entry of phospholine iodide into the brain. Exposure to high concentrations of soman (Carpentier et al., 1990) produced extravasation of exogenous tracer Evans blue and endogenous immunoprotein IgG into the brains of rats. In rats exposed to 0.9 x LD50 of soman, leaky brain capillaries were observed by measuring the uptake of Evans blue, HRP, and quaternary 3H-hexamethonium leaks into the brain (Petrali et al., 1991). Interestingly, these studies suggested that increased BBB permeability was related to the induction of seizures; but, the dosage of paraoxon used in our study did not elicit behavioral changes such as seizures that are characteristic of cholinergic toxicity. Furthermore, in the studies of rats exposed to soman (Carpentier et al., 1990), leaky capillaries were not observed in the hippocampus, another result that is in contrast with our observations.
A possible explanation for these differences is the respective ages of the rats in the different studies. Whereas studies found an association between seizures and breakdown in the BBB in adult rats, we found leaky capillaries after exposure to paraoxon in rats 25 to 30 days of age but not 90 days of age. It is possible the BBB of the young rats displays a greater sensitivity to the effects of paraoxon than that of the older rats, so that lower dosages increase leaky capillaries but do not produce clinical signs of cholinergic toxicity. Although by postnatal day 25 the BBB is impermeable to proteins (Saunders et al., 2000) and the level of expression of nutrient transporters, such as the glucose transporter, has reached a plateau (Vannucci and Simpson, 2003), studies have shown that other components of the BBB continue to mature. For example, the multidrug resistance glycoprotein reaches a plateau by postnatal day 40 (Matsuoka et al., 1999), and the growth of hydroxyproline content and thickening of capillary basement membranes continue until postnatal day 45 (Betz and Goldstein, 1981). The components of the BBB that are affected and responsible for the leaks are unknown, but they might be more susceptible to the signals evoked by paraoxon in young rats compared to older ones.
The second novel finding of our study was that the increase in leaky capillaries in young rats was not apparently associated with greater inhibition of ChE when rats were pretreated with PYR. Increases in leaky capillaries would be expected to result in greater uptake of PYR into the brain and greater inhibition of ChE. Some studies have suggested that Gulf War illnesses are due to elevated inhibition of brain ChE, which resulted from a breakdown in the BBB integrity due to stress or exposure to organophosphorus toxicants and treatment with PYR (Abou-Donia et al., 1996; Friedman et al., 1996); but, other studies disagree (Grauer et al., 2000; Lallement et al., 1998; Song et al., 2002; Tian et al., 2002). Like the latter group of studies, we were unable to find evidence of greater ChE inhibition in rats treated with paraoxon following pretreatment with PYR compared to rats treated with paraoxon alone.
It appears, however, paradoxical that exposure to paraoxon increases the entry of horseradish peroxidase but not PYR. One explanation is that PYR is also affecting the integrity of the BBB by increasing the secretion of glucocorticoids, which are often given to patients with brain injury or infection to reduce edema (Rhodes, 2003). Glucocorticoids have been shown to have protective effects on the BBB, possibly by increasing the levels of proteins that comprise tight junctions (Antonetti et al., 2002; Heiss et al., 1996; Ikeda et al., 1999; Romero et al., 2003). Peripheral cholinergic stimulation, for example, by the administration of peripherally active neostigmine in rats (Kolta and Soliman, 1981) and PYR in humans (Murialdo et al., 1993; Risch et al., 1986), increases levels of glucocorticoids. Possibly, the inhibition of paraoxon-mediated increase in leaky capillaries in rats pretreated for 50 min with PYR was due to increases in corticosterone. Paraoxon alone might also increase the release of glucocorticoids and attenuate its effects on the BBB. In our studies, the length of time between treatment with paraoxon and assaying leaky capillaries was only 10 min, though, which might not be sufficient time to increase glucocorticoids and affect the BBB.
In summary, our study shows that the integrity of the BBB is compromised in young rats injected with paraoxon at a dosage that does not induce clinical signs of cholinergic toxicity. The sensitivity of BBB modulation by paraoxon appears developmentally regulated. Also, pretreatment with PYR before paraoxon exposure did not enhance cholinergic toxicity, rather such pretreatment appeared to attenuate the effects of paraoxon on the BBB. These findings may have implications for the continued use of PYR as a prophylactic agent for nerve agent exposures.
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ACKNOWLEDGMENTS |
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This research was supported by the U.S. Army (DAMD 17-00-1-0070), Oklahoma State University Board of Reagent, NIEHS Center Grant 03819, and NIH R01NS32148-09A1S1.
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NOTES |
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1 To whom correspondence should be addressed at Kennedy Krieger Institute, Johns Hopkins University, Baltimore, MD 21205. Fax: (443) 923-2695. E-mail: bressler{at}kennedykrieger.org
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