Novel Brain-Penetrating Oxime Acetylcholinesterase Reactivators Attenuate Organophosphate-Induced Neuropathology in the Rat Hippocampus

Abstract

Organophosphate (OP) anticholinesterases cause excess acetylcholine leading to seizures which, if prolonged, result in neuronal damage in the rodent brain. Novel substituted phenoxyalkyl pyridinium oximes have previously shown evidence of penetrating the rat blood-brain barrier (BBB) in in vivo tests with a sarin surrogate (nitrophenyl isopropyl methylphosphonate, NIMP) or the active metabolite of the insecticide parathion, paraoxon (PXN), by reducing the time to cessation of seizure-like behaviors and accumulation of glial fibrillary acidic protein, whereas 2-PAM did not. The neuroprotective ability of our lead oximes (15, 20, and 55) was tested using NeuN, Nissl, and Fluoro-Jade B staining in the rat hippocampus. Following lethal-level subcutaneous challenge with NIMP or PXN, rats were intramuscularly administered a novel oxime or 2-PAM plus atropine and euthanized at 4 days. There were statistically significant increases in the median damage scores of the NeuN-stained NIMP, NIMP/2-PAM, and NIMP/Oxime 15 groups compared with the control whereas the scores of the NIMP/Oxime 20 and NIMP/Oxime 55 were not significantly different from the control. The same pattern of statistical significance was observed with PXN. Nissl staining provided a similar pattern, but without statistical differences. Fluoro-Jade B indicated neuroprotection from PXN with novel oximes but not with 2-PAM. The longer blood residence times of Oximes 20 and 55 compared with Oxime 15 might have contributed to their greater efficacy. These results suggest that novel oximes 20 and 55 were able to penetrate the BBB and attenuate neuronal damage after NIMP and PXN exposure, indicating potential broad-spectrum usefulness.

Keywords: acetylcholinesterase inhibitors; neuropathology; neuroprotection; organophosphate; oxime reactivators.

Figure 1.

Structure of novel substituted phenoxyalkyl pyridinium oximes where n is the number of C’s in the alkyl chain and R is the substitution on the phenoxy moiety.

Figure 2.

Relative neuropathology observed in the CA1 region of rats challenged subcutaneously with organophosphates (OP) (either nitrophenyl isopropyl methyl phosphonate [NIMP] at 0.6 mg/kg or paraoxon [PXN] at 0.8 mg/kg) with some later receiving pralidoxime (2-PAM), Oxime 15, Oxime 20, or Oxime 55. Control animals received Multisol. Sequential CA1 hippocampal sections from the same rat were stained with NeuN or Nissl. For NeuN, n was from 7 to 14. For Nissl, n was from 6 to 11. The symbol ▼ indicates a statistically significant difference between the median of this group and that of the control group; bars reflect SD. (For Nissl PXN-Oxime 20 group, all observations were 0, so no SD was calculated.)

Figure 3.

Representative sections of the CA1 hippocampal regions from rats challenged subcutaneously with either nitrophenyl isopropyl methyl phosphonate (NIMP) at 0.6 mg/kg or paraoxon (PXN) at 0.8 mg/kg with some receiving Oxime 20 (OX20) or pralidoxime (2-PAM). Control animals received Multisol. Rats were perfused at the 4-day time point and brains were stained with NeuN. Photomicrographs are at 20× magnification.

Figure 4.

Male Sprague Dawley rats received a single dose of Oxime 15, 20, or 55 by IM (50 mg/kg) injection. Each data point represents the mean ± SD of n = 3 rats, except for the IM dose of Oxime 15 in which one rat was considered an outlier and data from this animal were not included in additional calculations. Samples collected prior to dose administration were <lower limit of quantitation.