Inhibition of calcium-stimulated ATPase in the hen brain P2 synaptosomal fraction by organophosphorus esters: relevance to delayed neuropathy

Organophosphorus (OP) compounds have been reported to inhibit Ca/Mg-ATPase, but the relevance of this inhibition to organophosphate-induced delayed neuropathy (OPIDN) has not been explored. To determine if inhibition of this enzyme was related to the development of OPIDN, neuropathic and nonneuropathic OP compounds were sted for their ability to inhibit Ca-stimulated ATPase activity in the P2 synaptosomal fraction from hen brain. Following in vitro exposure to 10(-3) to 10(-5) M OP compounds, Ca-stimulated ATPase activity was inhibited by chlorpyrifos, chlorpyrifos-oxon, phenyl saligenin phosphate (PSP), and tri-o-tolyl phosphate (TOTP), but not by parathion, paraoxon, or diisopropyl fluorophosphate (DFP). Further investigation of inhibition induced by chlorpyrifos determined that inhibition was noncompetitive with respect to calcium and ATP. OP compound hydrophobicity was well correlated with in vitro inhibition of Ca-stimulated ATPase, suggesting that OP compounds interact with membrane lipids, and this interaction may contribute to the noncompetitive inhibition of Ca-stimulated ATPase that was observed. Subsequent to in vivo exposure, DFP, but not PSP, produced inhibition of Ca-stimulated ATPase activity in the hen brain P2 synaptosomal fraction. These data indicate that inhibition of Ca-stimulated ATPase activity is not correlated to neuropathic potential and demonstrate that inhibition of Ca/Mg-ATPase is not responsible for OPIDN.     

Potentiation of Organophosphorus-Induced Delayed Neurotoxicity Following Phenyl Saligenin Phosphate Exposures in 2-, 5-, and 8-Week-Old Chickens

Phenylmethylsulfonyl fluoride (PMSF), a nonneuropathic inhibitorof neurotoxk esterase (NTE), is a known potentiator of organophosphorus-induceddelayed neurotoxicity (OPIDN)- The ability of PMSF posttreatment(90 mg/kg, sc, 4 hr after the last PSP injection) to modifydevelopment of delayed neurotoxicity was examined in 2-, 5-,and 8-week-old White Leghorn chickens treated either one, two,or three times (doses separated by 24 hr) with the neuropathicOP compound phenyl saligenin phosphate (PSP, 5 mg/kg, sc). NTEactivity was measured in the cervical spinal cord 4 hr afterthe last PSP treatment. Development of delayed neurotoxicitywas measured over a 16-day postexposure period. All PSP-treatedgroups exhibited >97% NTE inhibition regardless of age ornumber of OP treatments. Two-week-old birds did not developclinical signs of neurotoxicity in response to either singleor repeated OP treatment regimens nor following subsequent treatmentwith PMSF. Five-week-old birds were resistant to the clinicaleffects of a single PSP exposure and were minimally affectedby repeated doses. PMSF posttreatment, however, significantlyamplified the clinical effects of one, two, or three doses ofPSP. A single exposure to PSP induced slight to moderate signsof delayed neurotoxicity in 8-week-old birds with more extensiveneurotoxicity being noted following repeated dosing. As with5-week-old birds, PMSF exacerbated the clinical signs of neurotoxicitywhen given after one, two, or three doses of PSP in 8-week-oldbirds. Axonal degeneration studies supported the clinical findings:PMSF posttreatment did not influence the degree of degenerationin 2-week-old chickens but resulted in more severe degeneration(relative to PSP only exposure) in cervical cords from both5- and 8-week-old birds. The results indicate that PMSF doesnot alter the progression of delayed neurotoxicity in very young(2 weeks of age) chickens but potentiates PSP-induced delayedneurotoxicity in the presence of 0–3% residual NTE activityin older animals. We conclude that posttreatment with neuropathicor nonneuropathic NTE inhibitors, following virtually completeNTE inhibition by either single or repeated doses of a neuropathicagent in sensitive age groups, can modify both the clinicaland morphological indices of delayed neurotoxicity. This studyfurther supports the hypothesis that potentiation of OPIDN occursthrough a mechanism unrelated to NTE.     

Potentiation of organophosphorus-induced delayed neurotoxicity by phenylmethylsulfonyl fluoride

It is well known that pretreatment with the serine esterase inhibitor phenylmethylsulfonyl fluoride (PMSF) can protect experimental animals from organophosphorus-induced delayed neurotoxicity (OPIDN), presumably by blocking the active site of neurotoxic esterase (NTE) such that binding and "aging" of the neuropathic OP is thwarted. We report here that while PMSF (60 mg/kg, sc) given 4 h before the neuropathic organophosphate (OP) mipafox (50 mg/kg, im) completely prevented the clinical expression of OPIDN in hens, the identical PMSF treatment markedly amplified the delayed neurotoxicity (relative to hens treated with OP only) if administered 4 h after mipafox (5 or 50 mg/kg, im). Moreover, in a separate experiment using diisopropylphosphorofluoridate (DFP) as the neurotoxicant in place of mipafox, posttreatment with PMSF 4 h after DFP (0.5 mg/kg) also accentuated the severity of ataxia. These data indicate that PMSF only protects against OPIDN if given prior to exposure to the neurotoxicant; treatment with PMSF after OP exposure critically exacerbates the delayed neurotoxicity from exposure to organophosphorus compounds.     

Further studies toward a mouse model for biochemical assessment of neuropathic potential of organophosphorus compounds

Inhibition and aging of neuropathy target esterase (NTE) by neuropathic organophosphorus (OP) compounds triggers OP compound‐induced delayed neuropathy (OPIDN), whereas inhibition of acetylcholinesterase (AChE) produces cholinergic toxicity. The neuropathic potential of an OP compound is defined by its relative inhibitory potency toward NTE vs. AChE assessed by enzyme assays following dosing in vivo or after incubations of direct‐acting compounds or active metabolites with enzymes in vitro. The standard animal model of OPIDN is the adult hen, but its large size and high husbandry costs make this species a burdensome model for assessing neuropathic potential. Although the mouse does not readily exhibit clinical signs of OPIDN, it displays axonal lesions and expresses brain AChE and NTE. Therefore, the present research was performed as a further test of the hypothesis that inhibition of mouse brain AChE and NTE could be used to assess neuropathic potential using mouse brain preparations in vitro or employing mouse brain assays following dosing of OP compounds in vivo. Excellent correlations were obtained for inhibition kinetics in vitro of mouse brain enzymes vs. hen brain and human recombinant enzymes. Furthermore, inhibition of mouse brain AChE and NTE after dosing with OP compounds afforded ED₅₀ ratios that agreed with relative inhibitory potencies assessed in vitro. Taken together, results with mouse brain enzymes demonstrated consistent correspondence between in vitro and in vivo predictors of neuropathic potential, thus adding to previous studies supporting the validity of a mouse model for biochemical assessment of the ability of OP compounds to produce OPIDN. Copyright © 2014 John Wiley & Sons, Ltd.     

Phenylmethylsulfonyl fluoride protects rats from Mipafox-induced delayed neuropathy

Initiation of organophosphorus-induced delayed neuropathy (OPIDN) is thought to consist of two molecular events involving the phosphorylation of the target enzyme, neurotoxic esterase, or neuropathy target enzyme (NTE), and a subsequent “aging” reaction which transforms the inhibited NTE into a charged moiety critical to the neuropathic process. Compounds that inhibit NTE but cannot age because of their chemical structure abort this two-stage initiation process, and when administered before a neurotoxic organophosphorus compound (OP), protect against the neuropathy by blocking NTE's active site (Johnson, 1970). In support of this, we report that prior exposure to a nonaging NTE inhibitor, phenylmethylsulfonyl fluoride (PMSF), protects rats from neurological damage after subsequent exposure to a neurotoxic OP, Mipafox. Adult, male, Long Evans rats were exposed to either PMSF (250 mg/kg, sc) or to Mipafox (15 mg/kg, ip) and a time course of brain NTE inhibition and recovery was defined. A separate group of PMSF-treated rats was exposed to Mipafox when brain NTE inhibition was 87.7 ± 2.3%. Conversely, another group of rats, pretreated with Mipafox, was dosed with PMSF when NTE inhibition was 90.2 ± 0.8%. A third group of animals, treated with PMSF, was exposed to Mipafox 14 days later, when NTE activity had recovered to within 10 ± 4.2% of control amounts. Histopathological survey (14 to 21 days post-exposure) indicated severe cervical cord damage (damage score ≥3) in the follwing frequencies: PMSF, 0%; Mipafox, 85%; PMSF-4 hr-Mipafox, 0%; Mipafox-4 hr-PMSF, 100%; PMSF-14 days-Mipafox, 75%; controls, 0%. These data indicate that PMSF pretreatment protects rats against Mipafox-induced neurological damage and that the timing of administration and order of presentation are critical to this protection. These results support the hypothesis that the initiation of OPIDN is a multistage event involving inhibition and aging, and that these stages are experimentally separable.     

Toxicity of repeated doses of organophosphorus esters in the chicken

Hens were repeatedly exposed to paraoxon (PO, phosphonothioic acid, diethyl paranitrophenyl ester), the chemical warfare agent VX/phosphorofluoridic acid, methyl-S-(2-[bis(1-methylethyl)amino/ethyl)O-ethyl ester], or the neuropathic DFP [phosphorofluoridic acid, bis(1-methylethyl)ester] as evidence was sought for nerve or other tissue damage following long-term treatments at high dose levels. Thirty-day and 90-d trials were performed in which each bird was injected 3 or 5 times per week with atropine as protection, weighed, their eggs collected, and their blood enzymes (cholinesterases creatine kinase, and lactic dehydrogenase) and locomotion periodically examined. Muscle and brain enzymes were assayed at the end of the experiments. Doses of PO and VX were at or above LD50 levels. DFP doses were lowered with each run to estimate a no-observable-effect level for organophosphate-induced delayed-neuropathy (OPIDN). No abnormalities attributable to repeated exposures to either PO or VX were found, even though acute, short-term symptoms of toxicity appeared after each injection. No evidence for OPIDN was obtained with repeated exposures to PO and VX under conditions where OPIDN was caused by DFP. Histological signs of OPIDN appeared in the spinal cord without gross symptoms of ataxia following repeated treatments of 25 mg/kg of DFP. The results of one experiment suggested that exposure to protective injections of atropine delays the appearance of the locomotor symptoms of the DFP-induced neuropathy.     

Characterization of binding sites for diisopropyl phosphorofluoridate in spinal cord cytosol

Gel filtration chromatography was performed on cytosol preparation of hen spinal cord to find molecular target(s) for organophosphorus-induced delayed neurotoxicity (OPIDN). Three binding peaks of [(3)H]diisopropyl phosphorofluoridate (DFP), an organophosphate that induces OPIDN, were separated from the cytosol preparation. The activities of acetylcholinesterase (AChE) and neuropathy target esterase (NTE) that has been proposed as a screening method for OPIDN eluted in the fractions within these two DFP binding peaks. However, the other peak had none of the activities of AChE and NTE. Therefore, this DFP binding proteins in cytosol may be peculiar to the pathogenesis of OPIDN.     

Effects of fenthion, fenitrothion and desbromoleptophos on gait, acetylcholinesterase, and neurotoxic esterase in young chicks after in ovo exposure

Previous studies have demonstrated that gait is affected in chicks exposed to organophosphorus esters (OPs) that induce delayed neurotoxicity (OPIDN) in adult hens. To investigate the developmental relationship between such functional deficits and OPIDN, chicks were exposed to 3 OPs with different OPIDN potential. Desbromoleptophos (DBL) induces OPIDN in adult hens; fenthion (FEN) has uncertain OPIDN potential; fenitrothion (FTR) does not induce OPIDN. Chicks were treated by injection into the egg on day 15 of incubation, after the presumed period of OP-induced structural teratogenesis. AChE and neurotoxic esterase (NTE) were assayed during incubation and in parallel with post-hatching evaluations of gait. DBL, 125 mg/kg in ovo, caused paralysis in 70% of chicks after hatching. The gait of surviving chicks was affected for at least 6 weeks and marked by toes curling under. NTE was inhibited until 10 days post-hatching and AChE until hatching. FEN did not inhibit NTE significantly, but AChE was significantly inhibited until hatching. Chicks exposed as embryos to FEN were hyperactive and aggressive. Gait was still affected 6 weeks after treatment with 3 mg/kg FEN. FTR at 125 mg/kg inhibited AChE until day 10 post-hatching, but neither inhibited NTE nor affected gait. The growth of OP-exposed chicks was not significantly decreased, so the decreased length and increased width of the stride could not be ascribed to stunted growth. We conclude that OPs cause irreversible effects on gait that are not related to their defined neurotoxic effects, since altered gait (1) occurs below the age of sensitivity to OPIDN, (2) is seen in the absence of NTE inhibition and (3) does not invariably accompany AChE inhibition.     

Age sensitivity to organophosphate-induced delayed polyneuropathy. Biochemical and toxicological studies in developing chicks

Young animals are resistant to organophosphate-induced delayed polyneuropathy (OPIDP). The putative target protein in the nervous system for initiation of OPIDP in the adult hen is an enzyme called Neuropathy Target Esterase (NTE), which is dissected by selective inhibitors among nervous tissue esterases hydrolysing phenyl valerate (PV). We report here that the pool of PV-esterases sensitive to paraoxon was different in peripheral nerves of chicks as compared to that of hens while that of brain and spinal cord was not. NTE activity decreased with age in brain, spinal cord and peripheral nerve, but its sensitivity to several inhibitors remained unchanged. In the adult hen more than 70% inhibition of peripheral nerve NTE by neuropathic OPs is followed by deficit of retrograde axonal transport, axonal degeneration and paralysis. Similar NTE inhibition in 40-day-old or younger chicks however is not followed by changes in retrograde axonal transport nor by OPIDP. Chicks aged 60 to 80 days are only marginally sensitive to a single dose of DFP otherwise clearly neuropathic to hens. In vitro and in vivo phosphorylation by DFP and subsequent aging of brain NTE is similar both in chicks and in hens. The recovery of NTE activity monitored in vivo after inhibition by DFP is faster (half-life of about 3 days) in chick peripheral nerves as compared to chick brain, hen brain and hen peripheral nerve (half-life of about 5 days). It is concluded that the reduced sensitivity to OPIDP in chicks is not due to differences in OP-NTE interactions. The resistance might be explained by a more efficient repair mechanism, as suggested by the faster recovery of peripheral nerve NTE activity.     

2,5-Hexanedione-induced changes in the monomeric neurofilament protein content of rat spinal cord fractions

Quantitative morphometric analyses have demonstrated that axon atrophy is the primary neuropathic feature in the CNS and PNS of rats intoxicated with 2,5-hexanedione (HD). Axon caliber is maintained by the exchange of mobile neurofilament (NF) subunits with the stationary polymer and, therefore, HD might produce atrophy by disrupting cytoskeletal turnover. To evaluate this possibility, groups of rats were exposed to HD at dosing schedules (175 mg/kg x 101 days or 400 mg/kg x 26 days) that produced moderate levels of neurological deficits and prevalent axon atrophy in spinal cord white matter tracts. Lumbar spinal cord regions from HD-intoxicated rats and their age-matched controls were Triton-extracted and separated by differential fractionation into a low-speed, insoluble pellet (P1) of NF polymer and a high-speed supernatant fraction (S2), which presumably contained mobile monomer. Cytoskeletal protein contents (NF-L, -M, -H, and beta-tubulin) in each fraction were determined by immunoblot analysis. Results show that regardless of HD dose-rate, the NF polymer in P1 remained unaffected, although soluble monomer in the S2 fraction was depleted significantly (60-80% reduction). Fractional beta-tubulin contents were inconsistently affected and abnormal higher-molecular-weight NF proteins were detected in the P1 fraction only. Studies with antibodies directed against phosphorylated (RT97) and nonphosphorylated (SMI32) epitopes on NF-H and measurements of corresponding isoelectric range suggested that alterations in phosphorylation were not involved. The selective depletion of Triton-soluble protein suggested that HD adduction of NFs interfered with the dynamic interactions of the polymeric and mobile monomeric pools.     

Protein levels of neurofilament subunits in the hen central nervous system following prevention and potentiation of diisopropyl phosphorofluoridate (DFP)-induced delayed neurotoxicity(1).

Diisopropyl phosphorofluoridate (DFP) is an organophosphorus ester, which produces delayed neurotoxicity (OPIDN) in hens in 7-14 days. OPIDN is characterized by mild ataxia in its initial stages and severe ataxia or paralysis in about 3 weeks. It is marked by distal swollen axons, and exhibits aggregations of neurofilaments (NFs), microtubules, proliferated smooth endoplasmic reticulum, and multivesicular bodies. These aggregations subsequently undergo disintegration, leaving empty varicosities. Previous studies in this laboratory have shown an increased level of medium-molecular weight NF (NF-M) and decreased levels of high- and low-molecular weight NF (NF-H, NF-L) proteins in the spinal cord of DFP-treated hens. The main objective of this investigation was to study the effect of DFP administration on NF subunit levels when OPIDN is prevented or potentiated by pretreatment or post-treatment with phenylmethylsulfonyl fluoride (PMSF), respectively. Hens pretreated or post-treated with PMSF were killed 1, 5, 10, and 20 days after the last treatment. The alteration in NF subunit protein levels observed in DFP-treated hen spinal cords was not observed in protected hens. Estimation of NFs in the potentiation experiments, however, showed a different pattern of alteration in NF subunit levels. The results showed that an alteration in NF subunit levels in DFP-treated hens might be related to the development of OPIDN, since these changes were suppressed in PMSF-protected hens. However, results from PMSF post-treated hen spinal cords suggested that potentiation of OPIDN by PMSF was mediated by a mechanism different from that followed by DFP alone to produce OPIDN.     

DFP initiated early alterations of PKA/p-CREB pathway and differential persistence of β-tubulin subtypes in the CNS of hens contributes to OPIDN

Organophosphorus ester-induced delayed neurotoxicity (OPIDN) is a neurodegenerative disorder characterized by ataxia progressing to paralysis with a concomitant central and peripheral distal axonapathy. Diisopropylphosphorofluoridate (DFP) produces OPIDN in the chicken, which results in mild ataxia in 7-14 days and severe paralysis as the disease progresses with a single dose. White leghorn layer hens were treated with DFP (1.7 mg/kg, sc) after prophylactic treatment with atropine (1 mg/kg, sc) in normal saline and eserine (1 mg/kg, sc) in dimethyl sulfoxide. Control groups were treated with vehicle propylene glycol (0.1 mL/kg, sc), atropine in normal saline and eserine in dimethyl sulfoxide. The hens were sacrificed at different time points such as 2, 4, and 8 h, as well as 1, 2, 5, 10 and 20 days, and the tissues from cerebrum, midbrain, cerebellum brainstem and spinal cord were quickly dissected and frozen for protein (western) and mRNA (northern) studies. Subcellular fractionation, SDS-PAGE and immunoblotting of the nuclear and supernatant fractions using standard protocols from spinal cord and cerebrum showed differential expression of protein levels of PKA, CREB and phosphorylated CREB (p-CREB). There was an increase in PKA level in spinal cord nuclear fraction after 4 h (130 ± 5%) and 8 h (133 ± 6 %), while cerebrum nuclear fraction showed decrease (77 ± 5%) at 4 h and remained at the same level at 8 h. No change was seen in either spinal cord or cerebrum soluble fraction at any time points. There was an increase in CREB level in the spinal cord supernatant (133 ± 3%) after 5 days, while nuclear and supernatant fraction of the cerebrum did not show any alterations at any time point. p-CREB was induced in the spinal cord nuclear fraction at 1 day (150 ± 3%) and 5 days (173±±7%) of treatment, in contrast to the decreased levels p-CREB (72 ± 4%) at 10 days in cerebrum nuclear fraction. Supernatant fraction of spinal cord and cerebrum did not show any changes in pCREB at time points studied. Similarly another set of animals were treated with DFP and perfused using standard protocols and immunohistochemistry for p-CREB in the brain and spinal cord confirmed the overall protein expression pattern identified by western analysis. Expression of β-tubulin subtypes (1, 2, 3, and 4), studied by Northern blotting showed complex and differential pattern, while immunohistochemistry of the anti-β-tubulin for the entire period of OPIDN developmental stages showed early induction and persistence even in the disintegrating axonal and non-neuronal structures of the CNS. These data thus strongly suggest that early cytoskeletal damage at molecular level mediated by PKA/p-CREB pathways leads to the culmination of gross (microscopically observable) level cytoskeletal changes in various components of central nervous system (CNS), consistent with our earlier findings. Thus, the differential protein expression of PKA, CREB, p-CREB and β-tubulin subtypes appear to contribute to the initiation, progression and development of OPIDN, probably by recruiting other molecular pathways specific to various components of nervous system.     

Neuropathy target esterase in mouse whole blood as a biomarker of exposure to neuropathic organophosphorus compounds

The adult hen is the standard animal model for testing organophosphorus (OP) compounds for organophosphorus compound‐induced delayed neurotoxicity (OPIDN). Recently, we developed a mouse model for biochemical assessment of the neuropathic potential of OP compounds based on brain neuropathy target esterase (NTE) and acetylcholinesterase (AChE) inhibition. We carried out the present work to further develop the mouse model by testing the hypothesis that whole blood NTE inhibition could be used as a biochemical marker for exposure to neuropathic OP compounds. Because brain NTE and AChE inhibition are biomarkers of OPIDN and acute cholinergic toxicity, respectively, we compared NTE and AChE 20‐min IC₅₀ values as well as ED₅₀ values 1 h after single intraperitoneal (i.p.) injections of increasing doses of two neuropathic OP compounds that differed in acute toxicity potency. We found good agreement between the brain and blood for in vitro sensitivity of each enzyme as well for the ratios IC₅₀(AChE)/IC₅₀(NTE). Both OP compounds inhibited AChE and NTE in the mouse brain and blood dose‐dependently, and brain and blood inhibitions in vivo were well correlated for each enzyme. For both OP compounds, the ratio ED₅₀(AChE)/ED₅₀(NTE) in blood corresponded to that in the brain despite the somewhat higher sensitivity of blood enzymes. Thus, our results indicate that mouse blood NTE could serve as a biomarker of exposure to neuropathic OP compounds. Moreover, the data suggest that relative inhibition of blood NTE and AChE provide a way to assess the likelihood that OP compound exposure in a susceptible species would produce cholinergic and/or delayed neuropathic effects. Copyright © 2016 John Wiley & Sons, Ltd.     

三邻甲苯磷酸酯暴露鸡脊髓组织中Cofilin-1b的差异表达

目的从三邻甲苯磷酸酯(TOCP)暴露鸡的脊髓组织中筛选可能与调控微丝解聚作用相关的差异表达蛋白,为探讨有机磷化合物诱发的迟发性神经毒性(OPIDN)作用机制提供靶蛋白依据。方法 42只罗曼鹤母鸡随机分成1000 mg/kg TOCP组、预先给予40 mg/kg苯甲基磺酰氟(PMSF)后再投1000 mg/kg TOCP的干预组和生理盐水对照组,每组14只。染毒第5和20天,每组分别处死4只鸡,低温环境下分离脊髓,提取总蛋白。利用双向电泳结合质谱分析技术,筛选和鉴定可能与调控微丝解聚作用相关的差异表达蛋白。结果 TOCP组鸡于染毒第7日前后出现进行性共济失调和肌无力等OPIDN典型症状,起病从下肢远端部分开始且病变程度随时间逐渐加重直至全瘫,而其他组鸡在实验观察期间未见OPIDN症状。TOCP组鸡于暴露第5天,分别与对照组和PMSF前干预组比较,其脊髓组织肌动蛋白解聚因子Cofilin-1b分别下调3.4倍和2.8倍,且有统计学意义(差异表达<0.5或差异表达>2),而PMSF前干预组与对照组比较,鸡脊髓组织Cofilin-1b的表达差异无统计学意义。在TOCP暴露第20天,TOCP组鸡脊髓组织Cofilin-1b表达与其他两组比较尽管有下降趋势,但无显著性变化。结论 TOCP暴露能导致鸡脊髓神经组织Cofilin-1b表达在早期显著下调,且该蛋白表达下调可能与微丝骨架结构紊乱及其OPIDN诱发机制有关。     

Early differential cell death and survival mechanisms initiate and contribute to the development of OPIDN: a study of molecular, cellular, and anatomical parameters

Organophosphorus-ester induced delayed neurotoxicity (OPIDN) is a neurodegenerative disorder characterized by ataxia progressing to paralysis with a concomitant central and peripheral, distal axonapathy. Diisopropylphosphorofluoridate (DFP) produces OPIDN in the chicken that results in mild ataxia in 7-14 days and severe paralysis as the disease progresses with a single dose. White leghorn layer hens were treated with DFP (1.7 mg/kg, sc) after prophylactic treatment with atropine (1 mg/kg, sc) in normal saline and eserine (1 mg/kg, sc) in dimethyl sulfoxide. Control groups were treated with vehicle propylene glycol (0.1 ml/kg, sc), atropine in normal saline and eserine in dimethyl sulfoxide. The hens were euthanized at different time points such as 1, 2, 5, 10 and 20 days, and the tissues from cerebrum, midbrain, cerebellum, brainstem and spinal cord were quickly dissected and frozen for mRNA (northern) studies. Northern blots were probed with BCL2, GADD45, beta actin, and 28S RNA to investigate their expression pattern. Another set of hens was treated for a series of time points and perfused with phosphate buffered saline and fixative for histological studies. Various staining protocols such as Hematoxylin and Eosin (H&E); Sevier-Munger; Cresyl echt Violet for Nissl substance; and Gallocynin stain for Nissl granules were used to assess various patterns of cell death and degenerative changes. Complex cell death mechanisms may be involved in the neuronal and axonal degeneration. These data indicate altered and differential mRNA expressions of BCL2 (anti apoptotic gene) and GADD45 (DNA damage inducible gene) in various tissues. Increased cell death and other degenerative changes noted in the susceptible regions (spinal cord and cerebellum) than the resistant region (cerebrum), may indicate complex molecular pathways via altered BCL2 and GADD45 gene expression, causing the homeostatic imbalance between cell survival and cell death mechanisms. Semi quantitative analysis revealed that the order of severity of damage declines from the spino-cerebellar, ventral, and dorsal tract respectively, suggesting neuroanatomical specificity. Thus, early activation of cell death and cell survival processes may play significant role in the clinical progression and syndromic clinical feature presentation of OPIDN.     

Neuregulin-1 is neuroprotective in a rat model of organophosphate-induced delayed neuronal injury

Current medical countermeasures against organophosphate (OP) nerve agents are effective in reducing mortality, but do not sufficiently protect the CNS from delayed brain damage and persistent neurological symptoms. In this study, we examined the efficacy of neuregulin-1 (NRG-1) in protecting against delayed neuronal cell death following acute intoxication with the OP diisopropylflurophosphate (DFP). Adult male Sprague-Dawley rats were pretreated with pyridostigmine (0.1 mg/kg BW, i.m.) and atropine methylnitrate (20 mg/kg BW, i.m.) prior to DFP (9 mg/kg BW, i.p.) intoxication to increase survival and reduce peripheral signs of cholinergic toxicity but not prevent DFP-induced seizures or delayed neuronal injury. Pretreatment with NRG-1 did not protect against seizures in rats exposed to DFP. However, neuronal injury was significantly reduced in most brain regions by pretreatment with NRG-1 isoforms NRG-EGF (3.2 μg/kg BW, i.a) or NRG-GGF2 (48 μg/kg BW, i.a.) as determined by FluroJade-B labeling in multiple brain regions at 24 h post-DFP injection. NRG-1 also blocked apoptosis and oxidative stress-mediated protein damage in the brains of DFP-intoxicated rats. Administration of NRG-1 at 1 h after DFP injection similarly provided significant neuroprotection against delayed neuronal injury. These findings identify NRG-1 as a promising adjuvant therapy to current medical countermeasures for enhancing neuroprotection against acute OP intoxication.     

The relationship between neurological damage and neurotoxic esterase inhibition in rats acutely exposed to tri-ortho-cresyl phosphate

A rodent model of organophosphorus-induced delayed neuropathy (OPIDN) has been developed using Long-Evans adult male rats exposed to tri-ortho-cresyl phosphate (TOCP). In the present study an attempt was made to relate neurochemical with neuropathological changes in rats exposed to single dosages of TOCP ranging from 145 to 3480 mg/kg. The degree of neurotoxic esterase (NTE) inhibition, measured at 20 and 44 hr and at 14 days postexposure was correlated with the appearance of spinal cord pathology 14 days postexposure in a separate group of similarly dosed rats. Those dosages that inhibited mean NTE activity in spinal cord greater than or equal to 72% and brain greater than or equal to 66% of control values within 44 hr postexposure produced marked spinal cord pathology 14 days postexposure in greater than or equal to 90% of similarly dosed animals. In contrast, dosages of TOCP which inhibited mean NTE activity in the spinal cord less than or equal to 65% and in the brain less than or equal to 57% produced spinal cord pathology in less than or equal to 15% of the animals. These data indicate that NTE inhibition may be used as a biochemical predictor for TOCP-induced neurological damage in rats.     

Neurotoxicity of acute and repeated treatments of tabun, paraoxon, diisopropyl fluorophosphate and isofenphos to the hen.

The neuropathic potential of acute and repeated exposures of the phosphoramidates tabun (GA) and isofenphos (IFP), of diisopropyl fluorophosphate (DFP) and paraoxon (PO) were examined in the hen with treatments for up to 90 days via intramuscular injections of the highest tolerated doses with atropine protection. Plasma acetylcholinesterase (AChE), non-specific butyrylcholinesterase (BChE) and creatine kinase (CK) activities were measured in order to monitor whether the compounds were present at biologically active concentrations. Locomotor behavior was observed and tissues from the peripheral and central nervous systems were examined for signs of organophosphate-induced delayed neuropathy (OPIDN). No behavioral or histological evidence of OPIDN was observed after treatments with GA, IFP, PO, saline or atropine sulfate. DFP-treated birds displayed locomotor and neuropathological signs of OPIDN with a no effect level (NOEL) between 25 and 50 micrograms/kg.     

磁共振成像在分析脊髓栓系综合征神经损伤因素中的价值

目的研究腰骶椎磁共振成像(MRI)在评估脊髓栓系综合征(TCS)神经功能损伤中的价值,分析影响神经系统损害的因素。方法搜集TCS患者58例,按年龄分为小儿及成人两组,行MRI腰骶椎脊髓及终丝成像。神经损伤的轻重程度依临床表现分为4级。在MRI图像中观察脊髓圆锥的位置、形态及脊椎的全貌,按圆锥在椎管内的位置分为高低两级。结果58例TCS患者脊髓圆锥位置均低于L2椎体下缘,小儿组合并脊柱侧弯或腰椎生理曲度加大或硬膜囊扩张的脊柱畸形患者神经损伤程度较未合并脊柱畸形患者轻,差异有统计学意义(P=0.0035)。小儿组脊髓圆锥的位置较成人组低、神经损伤的程度较成人组严重,差异均有统计学意义。结论MRI可全面显示脊髓圆锥、终丝及周围组织形态,能为TCS患者脊髓张力的判断及神经损伤的评估提供重要的影像学依据。     

Acute and delayed effects of fenthion in young chicks

The effects of desbromoleptophos, fenitrothion, and fenthion on brain acetylcholinesterase (AChE), brain neurotoxic esterase (NTE), and walking were investigated in immature chicks, below the age of organophosphorus ester-induced delayed neurotoxicity (OPIDN). Seventy-five milligrams per kilogram of the delayed neurotoxicant desbromoleptophos (DBL) and 100 mg/kg of the nonneurotoxicant fenithrothion (FTR) were given orally to 8-d-old chicks. Five milligrams per kilogram of the suspected neurotoxicant fenthion (FEN) was administered topically for 7 d, in 4 different age groups. Behavioral testing was performed for treated and control chicks on various days after treatment. Brain NTE and AChE assays were carried out for treated and control chicks on each day of behavioral testing. NTE and AChE inhibition were around 80 and 50%, respectively, 24 h after treatment, for the chicks treated with DBL. NTE returned to normal levels by 20 d and AChE by 6 d after treatment. FTR caused 56% AChE inhibition but not NTE inhibition 24 h after treatment. NTE inhibition for the FEN-treated chicks never exceeded 25% during the whole period of the experiment, whereas 65 and 54% inhibition of AChE was seen in two age groups. DBL and FEN significantly altered the gait of treated chicks, but the non-OPIDN-inducing FTR did not. FEN-treated chicks developed an atypical ataxia at the normal age for onset of sensitivity to OPIDN. Minimal NTE inhibition, long latency for the development of ataxia, and immaturity of the chicks at treatment distinguish FEN-induced functional deficits from classical OPIDN.