abstract

Pyridostigmine bromide (PB), a cholinesterase inhibitor used as a prophylactic against nerve agents, has been reported to produce neuronal apoptosis in rats. The goal of this study was to determine if PB produced similar levels of apoptosis in a C57BL/6J mouse model. Since the ability of PB to cross the blood-brain barrier is controversial, we used physostigmine (PHY), a cholinesterase inhibitor that readily crosses the blood-brain barrier, as a positive control.  Following subcutaneous exposure to either PB or PHY, we examined coronal sections of the brain for evidence of apoptosis at time points ranging from 8 hours to one week. Results obtained using the standard terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) assay revealed high levels of non-specific staining in the dentate gyrus, striatum, and cerebral cortex. When the assay was modified by the addition of a lipid extraction step, reductions in incubation times, and modifications to a wash buffer to eliminate nonspecific binding, no apoptosis was detected in any brain area following treatment with PB, PHY, or saline. To verify that the modified protocol was capable of detecting low levels of apoptosis, we used 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) treatment to identify TUNEL positive cells in the substantia nigra of mice with this assay.  

 

 

INTRODUCTION

During the 1991 Gulf War soldiers were exposed to the physical and emotional stresses associated with combat and to a variety of chemicals and drugs. One of these drugs, pyridostigmine bromide (PB), is a reversible cholinesterase inhibitor that was used as a prophylactic agent against nerve gas attack.  Months to years later some veterans began to exhibit a variety of neurological and neuromuscular symptoms subsequently termed Gulf War Syndrome [Haley et al., 1997, Haley et al., 2002].  PB contains a quaternary amine group that prevents the compound from crossing the blood brain barrier under normal physiological conditions and is used clinically in the treatment of myasthenia gravis [Taylor, 1990]. Despite clinical evidence that PB does not produce neurological side effects [Taylor, 1990] the compound has been proposed to be a causative agent of Gulf War Syndrome. This hypothesis is based upon data showing increased blood brain barrier permeability to PB in rats following stress in the form of a forced swim test [Friedman et al., 1996].  However, this finding is also controversial as several groups have reported seeing no effect of PB on brain cholinesterase activity following stress [Lallement et al., 1998; Grauer et al., 2000; Sinton et al., 2000; Song et al., 2002; Tian et al., 2002].  
            Exposure to organophosphate (OP) cholinesterase inhibitors is known to produce neurotoxicity in rats and to induce apoptosis in neurons.  During development, exposure to OPs, such as the commonly used pesticides chlorpyrifos and parathion, has been shown to induce neurotoxicity.  For example, parathion exposure causes necrosis in the dentate gyrus and CA4 regions of the hippocampus in rat pups [Veronesi and Pope, 1990], while chlorpyrifos exposure in neonatal rats (post-natal days 1—4) has been shown to decrease cell numbers in the brainstem during exposure and in the forebrain shortly after the exposure had ended (15—20 days of age) [Campbell et al., 1997].  Chlorpyrifos also produced a significant increase in the number of apoptotic cells in embryonic neuroepithelial cultures [Roy et al., 1998] and induced apoptosis in cultured rat cortical neurons [Caughlan et al., 2004]. Neonatal exposure to low levels of chlorpyrifos also produces neurobehavioral deficits [Dam et al., 2000].  We were unable to find comparable studies using mice in the literature.

In contrast, exposure to carbamate cholinesterase inhibitors has not been associated with neuronal toxicity.  While there are several reports of behavioral changes following exposure to PB [Servatius et al., 1998, van Haaren et al., 1999, Wolthuis and Vanwersch, 1984, Blick et al., 1994], only one study has reported that PB causes apoptosis at a dose that did not produce mortality in rats [Li et al., 2000].  Yet the rates of apoptosis reported in the cortex, hippocampus, and caudate in this study were as high or higher as those seen in a study that showed apoptosis in brain following a high dosage (1.8 X LD₅₀ of the organophosphate DFP, with steps taken to minimize mortality [Kim et al., 1999]. 

Therefore, the present study was undertaken to determine if treatment with 10 mg/Kg/D of PB produced neuronal apoptosis in C57BL/6J mice.  The terminal deoxynucleotidyl transferase-mediated dUTP biotin nick-end labeling (TUNEL) reaction is one method of detecting cell death. It does not, however, differentiate between apoptosis and necrosis, which would require the use of one of several other techniques to differentiate between these modes of cell death. The plan in this study was to utilize such a technique once cell death was confirmed. However, since no cell death was detected utilizing the TUNEL assay a second technique was not employed in this study.

Two independent controls were employed to provide data that were critical for interpreting our results.  Physostigmine (PHY), another carbamate cholinesterase inhibitor that readily crosses the blood brain barrier, was used to determine whether access to the brain contributed to results obtained with PB. Since 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been shown to cause apoptosis in the substantia nigra of C57BL/6J mice 72 hours after a subcutaneous injection [Spooren et al., 1998], it was used as a positive control to demonstrate that the TUNEL reaction was working.

 

MATERIALS AND METHODS

Experimental animals

Male C57BL/6J mice weighing approximately 25 grams (Harlan Sprague Dawley, Indianapolis, IN), were housed separately under a 12:12 light-dark cycle with light onset at 6:00 AM. Water and laboratory rodent chow were provided ad libitum.

Time course and treatment groups

Mice were treated with 10 mg/kg/D PB, 2.88 mg/kg/D PHY, or saline. Drug delivery for all time points other than 8-hours was by subcutaneously implanted Alzet mini-osmotic pumps (Model 1007D, 0.5 µl/hr; Durect Corp., Cupertino, CA). For the 8-hour time point, drug was administered by two subcutaneous injections of 1.667 mg/kg PB, 0.480 mg/kg PHY or saline four hours apart following a mock surgery to mimic treatment for other time points. Anesthesia for the surgeries was a ketamine/xylazine (6:1) mixture injected intramuscularly.

Mice were sacrificed after 8, 24, 48 hours or 7 days of treatment with PB or PHY.  Each treatment group for the 8, 24 and 48-hour time points contained four animals. Treatment groups for the day 7 time points each contained 10 animals. 

In the event that our treatment groups did not show any evidence of TUNEL staining, we felt that it was essential to use a positive control to demonstrate that this outcome was not due to the inability of the methodology used to detect apoptosis.  Therefore, we used as our positive control a published protocol, which had been shown to produce apoptosis in the substantia nigra of C57Bl/6J mice following a single bolus of MPTP [Spooren et al., 1998].  This was done in a separate group of mice (n=4) injected subcutaneously with 40 mg/kg 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP).

Tissue collection and handling

Saline, PB, and PHY treated animals were sacrificed by decapitation at time points described above. MPTP treated animals were sacrificed by decapitation 72 hours post injection. Trunk blood was collected and analyzed for cholinesterase activity. Brains were removed, quickly frozen in isopentane, and stored at -80°C until sectioned. 10 µm sections were prepared, air dried, and stored at -20°C until analyzed.

Blood cholinesterase activity

Cholinesterase activity was determined by a modified version of the colorimetric assay of Ellman (Ellman et al., 1961)using a Packard Fusion™ Microplate Analyzer.   Fresh whole blood was diluted 1:100 immediately before initiating measurement with 0.1 M NaPO₄ pH 7.4 buffer containing 0.5% Tween 20.  Blood cholinesterase activity was determined at 25°C using acetylthiocholine as the substrate. 

Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling analysis

Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) reactions were initially done as described by the manufacturer utilizing the Trevigen NeuroTACS2 kit (Gaithersburg, MD). Briefly, the 3’ OH of fragmented DNA was labeled with biotinylated dNTP by terminal deoxynucleotidyl transferase. Cells were then exposed to streptavidin-HRP conjugate and diaminobenzoic acid. Nuclei were stained with the Blue Counterstain reagent in the NeuroTACS kit. Labeled cells were then visualized under a light microscope.  Two to five fields of view were captured with an Optronics Magnifier camera mounted on a Leica DMR fluorescence microscope. Apoptotic and non-apoptotic cells in a given field were manually counted. Cells which stained brown by the TUNEL assay (TUNEL positive) were judged apoptotic; non-apoptotic cells remained blue. Adobe Illustrator software was used to mark each cell as it was counted and thus ensure all cells were counted only once.

Modifications to the NeuroTACS2 protocol

Initial experiments employing the standard NeuroTACS2 protocol resulted in TUNEL positive staining of the negative controls. Following discussions with the technical support staff at Trevigen several changes were made to the standard protocol to reduce non-specific binding known to occur between the avidin-biotin label used and plasma membranes (Wilchek and Bayer, 1988), . These changes were implemented one at a time until the negative controls showed no TUNEL positive staining. A chloroform/methanol lipid extraction step was added to the protocol. Each sample was dipped briefly (3—5 seconds) in chloroform and then in methanol (10 minutes). Samples were fixed in 4% formaldehyde for 10 minutes and permeabilized in 1:200 Proteinase K solution for 7 minutes. The buffer for the wash step following the Streptavidin-HRP incubation step contained 1% bovine serum albumin and 0.05% Tween-20. The incubation time in the diaminobenzoic acid solution was limited to 1 minute. Cells were counterstained in Blue Counterstain solution for 5 seconds. The remaining steps of the manufacturer’s protocol were unchanged.

Statistical Analysis

Statistical significance was determined by an Analysis of Variance (ANOVA). A Least Significance Difference (LSD) analysis was then used to separate differences in means.

 

RESULTS

The doses of PB and PHY that were used in this study were adequate to inhibit blood cholinesterase by 40—60% and 20% respectively (Figure 1).  The goal was to produce a 30-40% inhibition of blood cholinesterase to match the levels of inhibition seen in the soldiers that received PB as a prophylactic treatment. Because PHY crosses the blood-brain barrier easily, we chose to use a dose of PHY that produced less inhibition of blood cholinesterase than the dose of PB used to avoid mortality. 

Utilizing the protocol supplied by the kit manufacturer produced high levels of TUNEL positive cells in the dentate gyrus region of the hippocampus, cortex, and striatum at day 7 of PB exposure. Typical results obtained using these protocols before modification are labeled “Before” in figure 2 (Figure 2A, C, E; Table 1). While the levels of cell death observed are comparable to published values [Li et al., 2000], it seems unlikely that such high levels of widespread neuronal death lasting for up to a month do not produce any animal mortality. Since the avidin-biotin labeling used in this protocol is known to produce high levels of nonspecific binding to the plasma membrane [Wilchek and Bayer, 1988], a chloroform/methanol lipid extraction step was added prior to fixation and 1% bovine serum albumin and 0.05% Tween-20 were added to the wash buffer following the streptavidin-HRP incubation step.  Lipid extraction and treatment with bovine serum albumin has been shown to significantly reduce nonspecific binding [Bayer et al., 1979; Kulomaa et al., 1979; Asou et al., 1983].  When we repeated the study using these two modifications to the manufacturer’s published protocol combined with the reduced incubation times described, no TUNEL positive cells were observed in the dentate gyrus, cortex, and striatum at day 7 of PB exposure. Typical results from the protocol after modification are labeled “After” in figure 2 (Figure 2B, D, F; Table 1). 

To demonstrate that the modified TUNEL protocol was still capable of detecting cell death, mice were treated with MPTP, a compound known to cause apoptosis in the substantia nigra of these mice 72 hours following a single bolus [Spooren et al., 1998]. As expected, treatment with MPTP induced cell death in the substantia nigra (Figure 3B). An average of 17 cells per brain section was TUNEL positive, consistent with published values [Spooren et al., 1998]. The MPTP induced cell death was confined to the substantia nigra with no cell death detected in the dentate gyrus, striatum or cortex. Cell death was detected in the substantia nigra only after treatment with MPTP and not following treatment with saline or PB (Figure 2A and 2C, respectively).   These results confirm that the modified protocol detected apoptotic cells.

Due to the rapid time course of apoptotic cell death, it can be difficult to detect in vivo unless one knows the appropriate time frame in which to look. Samples were collected at day 7 as this is the time point at which apoptosis had been reported in rats following PB exposures [Li et al., 2000].  However, particular subsets of cells may be especially susceptible to treatment by cholinesterase inhibitors and undergo cell death in a matter of hours following exposure. To study this possibility we examined neural tissue 8, 24 and 48 hours after exposure to PB and PHY.  No evidence of apoptosis was detected following exposure to either agent, however, at these time points (Table 2).

 

DISCUSSION AND CONCLUSIONS

Our results are consistent with the fundamental differences in the reported effects of the two different classes of cholinesterase inhibitors on neuronal cells.  Organophosphate pesticides induce nuclear condensation, caspase-3 activation, DNA fragmentation and laddering typical of apoptotic cell death in SH-SY5Y human neuroblastoma cells [Carlson et al., 2000]. Brain tissue from rats treated orally with organophosphate pesticides showed increased production of reactive oxygen species, lipid peroxidation, and DNA single strand breaks indicative of increased oxidative stress and cellular damage [Bagchi et al., 1995].

In contrast, carbamate-type inhibitors of cholinesterase, due to the reversible nature of binding to the cholinesterase, have been developed as neuroprotective agents.   PB is used clinically in the treatment of myasthenia [Taylor, 1990] and as a prophylactic against organophosphate nerve gas attack [Sapolsky, 1998].  Tacrine has been shown to attenuate hydrogen peroxide-induced apoptosis in a rat pheochromocytoma line PC12 [Wang et al., 2002].  The neuroprotective effect of carbamate cholinesterase inhibitors has also been demonstrated in vivo reducing ischemia-induced death of pyramidal cells in gerbils [Tanaka et al., 1995] and rats [Park et al., 2000].

In conclusion, our initial findings of apoptosis in several areas of the mouse brain following treatment with PB were apparently due to nonspecific binding of avidin-biotin, producing false-positive results. Although these results are consistent with a study done in rats [Li et al., 2000], the lack of reported mortality or observable behavioral changes in these animals led us to question these findings. We also noticed that our negative controls were positive for apoptosis providing further evidence that these results were suspect. Subsequent changes to the protocol reduced the nonspecific binding to undetectable levels except in the MPTP-treated positive controls.  It is possible that the species difference could account for the difference in findings, however, this seems unlikely since exposure to the organophosphate, sarin, at doses sufficient to reduce AChE levels in several brain areas in rats failed to produce apoptosis after 10 days the brain regions studied [Henderson et al., 2002]. Elimination of nonspecific binding during the TUNEL reaction appears to be critical to obtaining reproducible results when examining cellular death in the brain.

 

Acknowledgments

This work was supported by the Biomedical Sciences Ph.D. Program at Wright State University and US Department of Defense Contract DAMD17-00-C-0020.  The authors wish to thank Mary Key for her help establishing the tissue sectioning protocol.

 

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