APPENDIX I-Z: Song, et al, Interactive Effects of Paraoxon and Pyridostigmine on Blood‑Brain Barrier Integrity and Cholinergic Toxicity, Toxicological Sciences 78, 241‑247 (2004)

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Toxicological Sciences 78, 241‑247 (2004)

Toxicological Sciences vol. 78 no. 2 8 Society of Toxicology; all rights reserved.

Interactive Effects of Paraoxon and Pyridostigmine on Blood‑Brain Barrier Integrity and Cholinergic Toxicity

Xun Song*, Carey Pope*, Ramesh Murthy{dagger}, Jamaluddin Shaikh*, Bachchu Lal{dagger} and Joseph P. Bressler{dagger},{ddagger},1

* Neurotoxicology Laboratory, Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University, Stillwater, Oklahoma 74078; {dagger} Kennedy Krieger Institute and {ddagger} Department of Environmental Health Sciences, School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205

Received October 8, 2003; accepted January 10, 2004

ABSTRACT

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

REFERENCES

ABSTRACT

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.

INTRODUCTION

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, 2001Go). Inflammation, stroke, and seizures have been shown to increase permeability of the BBB (Belayev et al., 1996Go; de Vries et al., 1997Go; Huber et al., 2002Go; Sahin et al., 2003Go). Indeed, the etiology of multiple sclerosis might be due to an immune reaction caused by a compromised BBB (Smith and McDonald, 1999Go). Several neurodegenerative diseases have been suggested to involve the BBB. As recently noted (Farkas and Luiten, 2001Go), 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, 1984Go). Interestingly, the effects of lead on the BBB are observed only in children and, experimentally, only in preweanling rats (Pentschew and Garro, 1966Go; Toews et al., 1978Go). 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., 2002Go; Carpentier et al., 1990Go; Gupta et al., 1999Go), 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., 1999Go), but exposure to metrifonate did not affect the BBB permeability in rats weighing 200 g (Rakonczay and Papp, 2001Go). Paraoxon was found to increase permeability in 200 g rats but at dosages that caused seizures (Ashani and Catravas, 1981Go). 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, 1981Go; Carpentier et al., 1990Go; Grange‑Messent et al., 1999Go).

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., 1996Go; Hanin, 1996Go), 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., 2000Go; Lallement et al., 1998Go; Ovadia et al., 2001Go; Shaikh et al., 2003Go; Sinton et al., 2000Go; Song et al., 2002Go; Tian et al., 2002Go).

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.

MATERIALS AND METHODS

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., 1992Go). 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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FIG. 1. Horseradish peroxidase leaking from brain capillaries. Rats, 25 to 30 days of age, were injected with HRP into the left ventricle at 10 min after exposure to 100 μg/kg paraoxon injected im. Then, 70 μm sections were prepared from hippocampus, cortex, and striatum and incubated with a histochemical stain for HRP, as described in Materials and Methods. (A and D) Cortex; (B and E) hippocampus; (C and F) striatum; (A‑C) sections taken from control rats; (D‑F) sections from rats treated with paraoxon.

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, 1996Go; Moser et al., 1998Go). 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 B70°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, 1975Go) using 1 mM [3H]‑acetylcholine as the substrate, as described previously (Tian et al., 2002Go), and expressed as nmol/min/mg protein and percentage of control activity. Protein was determined with the Folin phenol reagent (Lowry et al., 1951Go).

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.

RESULTS

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 (25B30 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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FIG. 2. Extravasation of albumin from brain capillaries. Rats were treated as described in Figure 1 and perfused with 0.1 M phosphate buffer, pH 7.4, with 4% paraformaldehyde. Brains were removed and cryoprotected with sucrose. Next, 25 μm sections from cortex and striatum were incubated with a rabbit antibody against rat albumin, which was followed by the ABC technique and peroxidase staining. Staining was restricted to the lumen in the (A) control, whereas staining in the parenchyma was observed in (B‑D) 25‑ to 30‑day‑old rats treated with 100 μg paraoxon.

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, 1974Go; Brodeur and Dubois, 1963Go; Pope et al., 1991Go). 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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FIG. 3. Leaky capillaries in young and older rats. Rats, 25 to 30 days and 90 days of age, were given an injection of HRP into the left ventricle at 10 min after an im injection of 100 μg/kg paraoxon or vehicle (control). The number of leaky capillaries was counted in four sections of 70 μm thickness cut from striatum and hippocampus after staining for HRP. The data are expressed as the mean of the percentage increase in leaks in paraoxon‑treated rats compared to controls " SEM; n = 6. *p > 0.05 when comparing paraoxon‑treated 25‑/30‑day‑old to 90‑day‑old rats with ANOVA analysis and Tukey's posthoc test.

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 40B65% 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 54B58% 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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FIG. 4. ChE activity in peripheral tissue and brain. Paraoxon (PO) or saline (as controls) was injected into 25‑/30‑day‑old rats (n = 7) at 50 min after administering either saline or PYR (30 mg/kg) by gavage. ChE activity was measured at 10 min after injecting PO. The data are expressed as a mean " SEM percentage of inhibition in paraoxon‑treated rats compared to controls. ChE activity (nmol/min/mg protein) in control rats was as follows: frontal cortex, 26.7 " 2.82; temporal cortex, 15.5 " 1.50; diaphragm, 3.29 " 0.38; and blood, 0.17 " 0.03. *p > 0.05, PO alone compared to PO + PYR.

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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FIG. 5. Leaky capillaries in rats pretreated with PYR. Rats were treated as described in Figure 4. The number of leaky capillaries was counted in four, 70 μm sections cut from striatum and hippocampus after staining for HRP. The data are expressed as the mean of the percentage increase in leaks in paraoxon‑treated rats compared to controls or leaks in rats treated with PYR + PO compared to PYR alone " SEM; n = 6. *p > 0.05, PO alone compared to PO + PYR in the hippocampus or striatum with ANOVA analysis and Tukey's posthoc test.

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., 1990Go). 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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FIG. 6. Clinical signs of cholinergic toxicity. Rats (n = 14/group) were treated with PYR or saline for 50 min followed by paraoxon or saline, as described in Figure 4. Rats were observed for SLUD signs and involuntary movement (IM) 10 min after treatment with PO. Scores were expressed as median " interquartile range. *p > 0.05 compared to control rats.

DISCUSSION

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., 2002Go; Ashani and Catravas, 1981Go; Carpentier et al., 1990Go), 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., 1996Go).

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, 1981Go). 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., 1990Go) 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., 1991Go). 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., 1990Go), 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., 2000Go) and the level of expression of nutrient transporters, such as the glucose transporter, has reached a plateau (Vannucci and Simpson, 2003Go), 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., 1999Go), and the growth of hydroxyproline content and thickening of capillary basement membranes continue until postnatal day 45 (Betz and Goldstein, 1981Go). 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., 1996Go; Friedman et al., 1996Go); but, other studies disagree (Grauer et al., 2000Go; Lallement et al., 1998Go; Song et al., 2002Go; Tian et al., 2002Go). 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, 2003Go). 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., 2002Go; Heiss et al., 1996Go; Ikeda et al., 1999Go; Romero et al., 2003Go). Peripheral cholinergic stimulation, for example, by the administration of peripherally active neostigmine in rats (Kolta and Soliman, 1981Go) and PYR in humans (Murialdo et al., 1993Go; Risch et al., 1986Go), 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.

ACKNOWLEDGMENTS

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.

NOTES

1 To whom correspondence should be addressed at Kennedy Krieger Institute, Johns Hopkins University, Baltimore, MD 21205. Fax: (443) 923‑2695. E‑mail: bressler@kennedykrieger.org

REFERENCES

Abdel‑Rahman, A., Shetty, A. K., and Abou‑Donia, M. B. (2002). Acute exposure to sarin increases blood brain barrier permeability and induces neuropathological changes in the rat brain: Dose‑response relationships. Neuroscience 113, 721B741.[CrossRef][ISI][Medline]

Abou‑Donia, M. B., Wilmarth, K. R., Abdel‑Rahman, A. A., Jensen, K. F., Oehme, F. W., and Kurt, T. L. (1996). Increased neurotoxicity following concurrent exposure to pyridostigmine bromide, DEET, and chlorpyrifos. Fundam. Appl. Toxicol. 34, 201B222.[CrossRef][ISI][Medline]

Antonetti, D. A., Wolpert, E. B., DeMaio, L., Harhaj, N. S., and Scaduto, R. C., Jr. (2002). Hydrocortisone decreases retinal endothelial cell water and solute flux coincident with increased content and decreased phosphorylation of occludin. J. Neurochem. 80, 667B677.[CrossRef][ISI][Medline]

Ashani, Y., and Catravas, G. N. (1981). Seizure‑induced changes in the permeability of the blood‑brain barrier following administration of anticholinesterase drugs to rats. Biochem. Pharmacol. 30, 2593B2601.[CrossRef][ISI][Medline]

Belayev, L., Busto, R., Zhao, W., and Ginsberg, M. D. (1996). Quantitative evaluation of blood‑brain barrier permeability following middle cerebral artery occlusion in rats. Brain Res. 739, 88B96.[CrossRef][ISI][Medline]

Benke, G. M., and Murphy, S. D. (1974). Anticholinesterase action of methyl parathion, parathion, and azinphosmethyl in mice and fish: Onset and recovery of inhibition. Bull. Environ. Contam. Toxicol. 12, 117B122.[CrossRef][ISI][Medline]

Betz, A. L., and Goldstein, G. W. (1981). Developmental changes in metabolism and transport properties of capillaries isolated from rat brain. J. Physiol. 312, 365B376.[Abstract/Free Full Text]

Brodeur, J., and Dubois, K. P. (1963). Comparison of acute toxicity of anticholinesterase insecticides to weanling and adult male rats. Proc. Soc. Exp. Biol. Med. 114, 509B511.[Medline]

Carpentier, P., Delamanche, I. S., Le Bert, M., Blanchet, G., and Bouchaud, C. (1990). Seizure‑related opening of the blood‑brain barrier induced by soman: Possible correlation with the acute neuropathology observed in poisoned rats. Neurotoxicology 11, 493B508.[ISI][Medline]

de Vries, H. E., Kuiper, J., de Boer, A. G., Van Berkel, T. J., and Breimer, D. D. (1997). The blood‑brain barrier in neuroinflammatory diseases. Pharmacol. Rev. 49, 143B155.[Abstract/Free Full Text]

Farkas, E., and Luiten, P. G. (2001). Cerebral microvascular pathology in aging and Alzheimer's disease. Prog. Neurobiol. 64, 575B611.[CrossRef][ISI][Medline]

Friedman, A., Kaufer, D., Shemer, J., Hendler, I., Soreq, H., and Tur‑Kaspa, I. (1996). Pyridostigmine brain penetration under stress enhances neuronal excitability and induces early immediate transcriptional response. Nat. Med. 2, 1382B1385.[CrossRef][ISI][Medline]

Goldstein, G. W. (1984). Brain capillaries: A target for inorganic lead poisoning. Neurotoxicology 5, 167B176.[ISI][Medline]

Grange‑Messent, V., Bouchaud, C., Jamme, M., Lallement, G., Foquin, A., and Carpentier, P. (1999). Seizure‑related opening of the blood‑brain barrier produced by the anticholinesterase compound, soman: New ultrastructural observations. Cell. Mol. Biol. (Noisy‑le‑grand) 45, 1B14.

Grauer, E., Alkalai, D., Kapon, J., Cohen, G., and Raveh, L. (2000). Stress does not enable pyridostigmine to inhibit brain cholinesterase after parenteral administration. Toxicol. Appl. Pharmacol. 164, 301B304.[CrossRef][ISI][Medline]

Gupta, A., Agarwal, R., and Shukla, G. S. (1999). Functional impairment of blood‑brain barrier following pesticide exposure during early development in rats. Hum. Exp. Toxicol. 18, 174B179.[Abstract/Free Full Text]

Hanin, I. (1996). The Gulf War, stress, and a leaky blood‑brain barrier. Nat. Med. 2, 1307B1308.[CrossRef][ISI][Medline]

Heiss, J. D., Papavassiliou, E., Merrill, M. J., Nieman, L., Knightly, J. J., Walbridge, S., Edwards, N. A., and Oldfield, E. H. (1996). Mechanism of dexamethasone suppression of brain tumorBassociated vascular permeability in rats. Involvement of the glucocorticoid receptor and vascular permeability factor. J. Clin. Invest. 98, 1400B1408.[Abstract/Free Full Text]

Huber, J. D., Hau, V. S., Borg, L., Campos, C. R., Egleton, R. D., and Davis, T. P. (2002). Blood‑brain barrier tight junctions are altered during a 72‑h exposure to {gamma}‑carrageenanBinduced inflammatory pain. Am. J. Physiol. Heart Circ. Physiol. 283, H1531BH1537.[Abstract/Free Full Text]

Ikeda, T., Xia, X. Y., Xia, Y. X., and Ikenoue, T. (1999). Hyperthermic preconditioning prevents blood‑brain barrier disruption produced by hypoxia‑ischemia in newborn rat. Brain Res. Dev. Brain Res. 117, 53B58.[Medline]

Johnson, C. D., and Russell, R. L. (1975). A rapid, simple radiometric assay for cholinesterase, suitable for multiple determinations. Anal. Biochem. 64, 229B238.[CrossRef][ISI][Medline]

Kolta, M. G., and Soliman, K. F. (1981). Effect of peripheral cholinergic activation on the adrenal cortex function. Endocr. Res. Commun. 8, 239B246.[ISI][Medline]

Lallement, G., Foquin, A., Baubichon, D., Burckhart, M. F., Carpentier, P., and Canini, F. (1998). Heat stress, even extreme, does not induce penetration of pyridostigmine into the brain of guinea pigs. Neurotoxicology 19, 759B766.[ISI][Medline]

Liu, J., and Pope, C. N. (1996). Effects of chlorpyrifos on high‑affinity choline uptake and [3H]hemicholinium‑3 binding in rat brain. Fundam. Appl. Toxicol. 34, 84B90.[CrossRef][ISI][Medline]

Lowry, O. H., Rosenbrough, N. J., Farr, A. L., and Randall, J. R. (1951). Protein measurements with Folin phenol reagent. J. Biol. Chem. 193, 265B275.[Free Full Text]

Matsuoka, Y., Okazaki, M., Kitamura, Y., and Taniguchi, T. (1999). Developmental expression of P‑glycoprotein (multidrug resistance gene product) in the rat brain. J. Neurobiol. 39, 383B392.[CrossRef][ISI][Medline]

Moser, V. C., Chanda, S. M., Mortensen, S. R., and Padilla, S. (1998). Age‑ and gender‑related differences in sensitivity to chlorpyrifos in the rat reflect developmental profiles of esterase activities. Toxicol. Sci. 46, 211B222.[Abstract/Free Full Text]

Murialdo, G., Fonzi, S., Torre, F., Costelli, P., Solinas, G., Tosca, P., Di Paolo, E., Porro, S., Zerbi, F., and Polleri, A. (1993). Effects of pyridostigmine, corticotropin‑releasing hormone and growth hormoneBreleasing hormone on the pituitary‑adrenal axis and on growth hormone secretion in dementia. Neuropsychobiology 28, 177B183.[CrossRef][ISI][Medline]

Ovadia, H., Abramsky, O., Feldman, S., and Weidenfeld, J. (2001). Evaluation of the effect of stress on the bloodBbrain barrier: Critical role of the brain perfusion time. Brain Res. 905, 21B25.[CrossRef][ISI][Medline]

Pentschew, A., and Garro, F. (1966). Lead encephalomyelopathy of the suckling rat and its implications on the porphyrinopathic nervous diseases: With special reference to the permeability disorders of the nervous system capillaries. Acta Neuropathol. 6, 266B278.[CrossRef][Medline]

Petrali, J. P., Maxwell, D. M., Lenz, D. E., and Mills, K. R. (1991). Effect of an anticholinesterase compound on the ultrastructure and function of the rat blood‑brain barrier: A review and experiment. J. Submicrosc. Cytol. Pathol. 23, 331B338.[ISI][Medline]

Pope, C. N., Chakraborti, T. K., Chapman, M. L., Farrar, J. D., and Arthun, D. (1991). Comparison of in vivo cholinesterase inhibition in neonatal and adult rats by three organophosphorothioate insecticides. Toxicology 68, 51B61.[CrossRef][ISI][Medline]

Rakonczay, Z., and Papp, H. (2001). Effects of chronic metrifonate treatment on cholinergic enzymes and the blood‑brain barrier. Neurochem. Int. 39, 19B24.[CrossRef][ISI][Medline]

Rhodes, J. K. (2003). Actions of glucocorticoids and related molecules after traumatic brain injury. Curr. Opin. Crit. Care 9, 86B91.[CrossRef][Medline]

Risch, S. C., Janowsky, D. S., Mott, M. A., Gillin, J. C., Kalir, H. H., Huey, L. Y., Ziegler, M., Kennedy, B., and Turken, A. (1986). Central and peripheral cholinesterase inhibition: Effects on anterior pituitary and sympathomimetic function. Psychoneuroendocrinology 11, 221B230.[CrossRef][ISI][Medline]

Romero, I. A., Radewicz, K., Jubin, E., Michel, C. C., Greenwood, J., Couraud, P. O., and Adamson, P. (2003). Changes in cytoskeletal and tight junctional proteins correlate with decreased permeability induced by dexamethasone in cultured rat brain endothelial cells. Neurosci. Lett. 344, 112B116.[CrossRef][ISI][Medline]

Sahin, D., Ilbay, G., and Ates, N. (2003). Changes in the blood‑brain barrier permeability and in the brain tissue trace element concentrations after single and repeated pentylenetetrazole‑induced seizures in rats. Pharmacol. Res. 48, 69B73.[CrossRef][ISI][Medline]

Saunders, N. R., Knott, G. W., and Dziegielewska, K. M. (2000). Barriers in the immature brain. Cell Mol. Neurobiol. 20, 29B40.[CrossRef][ISI][Medline]

Shaikh, J., Karanth, S., Chakraborty, D., Pruett, S., and Pope, C. N. (2003). Effects of daily stress or repeated paraoxon exposures on subacute pyridostigmine toxicity in rats. Arch. Toxicol. 77, 576B583.[CrossRef][ISI][Medline]

Sinton, C. M., Fitch, T. E., Petty, F., and Haley, R. W. (2000). Stressful manipulations that elevate corticosterone reduce blood‑brain barrier permeability to pyridostigmine in the rat. Toxicol. Appl. Pharmacol. 165, 99B105.[CrossRef][ISI][Medline]

Smith, K. J., and McDonald, W. I. (1999). The pathophysiology of multiple sclerosis: The mechanisms underlying the production of symptoms and the natural history of the disease. Philos. Trans. R Soc. Lond. B Biol. Sci. 354, 1649B1673.[CrossRef][ISI][Medline]

Song, X., Tian, H., Bressler, J., Pruett, S., and Pope, C. (2002). Acute and repeated restraint stress have little effect on pyridostigmine toxicity or brain regional cholinesterase inhibition in rats. Toxicol. Sci. 69, 157B164.[Abstract/Free Full Text]

Stewart, P. A., Farrell, C. R., Farrell, C. L., and Hayakawa, E. (1992). Horseradish peroxidase retention and washout in blood‑brain barrier lesions. J. Neurosci. Methods 41, 75B84.[CrossRef][ISI][Medline]

Tian, H., Song, X., Bressler, J., Pruett, S., and Pope, C. N. (2002). Neither forced running nor forced swimming affect acute pyridostigmine toxicity or brain‑regional cholinesterase inhibition in rats. Toxicology 176, 39B50.[CrossRef][ISI][Medline]

Toews, A. D., Kolber, A., Hayward, J., Krigman, M. R., and Morell, P. (1978). Experimental lead encephalopathy in the suckling rat: Concentration of lead in cellular fractions enriched in brain capillaries. Brain Res. 147, 131B138.[CrossRef][ISI][Medline]

Vannucci, S. J., and Simpson, I. A. (2003). Developmental switch in brain nutrient transporter expression in the rat. Am. J. Physiol. Endocrinol. Metab. 285, E1127BE1134.[Abstract/Free Full Text]