Tsai Yi-Hua, González Eduardo A, Grodzki Ana C G, Bruun Donald A, Saito Naomi H, Harvey Danielle J, Lein Pamela J
Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, Davis, CA, United States.
Department of Public Health Sciences, School of Medicine, University of California, Davis, Davis, CA, United States.
Front Toxicol. 2024 Apr 30;6:1360359. doi: 10.3389/ftox.2024.1360359. eCollection 2024.
Acute intoxication with high levels of organophosphate (OP) cholinesterase inhibitors can cause cholinergic crisis, which is associated with acute, life-threatening parasympathomimetic symptoms, respiratory depression and seizures that can rapidly progress to status epilepticus (SE). Clinical and experimental data demonstrate that individuals who survive these acute neurotoxic effects often develop significant chronic morbidity, including behavioral deficits. The pathogenic mechanism(s) that link acute OP intoxication to chronic neurological deficits remain speculative. Cellular senescence has been linked to behavioral deficits associated with aging and neurodegenerative disease, but whether acute OP intoxication triggers cellular senescence in the brain has not been investigated. Here, we test this hypothesis in a rat model of acute intoxication with the OP diisopropylfluorophosphate (DFP). Adult male Sprague-Dawley rats were administered DFP (4 mg/kg, s.c.). Control animals were administered an equal volume (300 µL) of sterile phosphate-buffered saline (s.c.). Both groups were subsequently injected with atropine sulfate (2 mg/kg, i.m.) and 2-pralidoxime (25 mg/kg, i.m.). DFP triggered seizure activity within minutes that rapidly progressed to SE, as determined using behavioral seizure criteria. Brains were collected from animals at 1, 3, and 6 months post-exposure for immunohistochemical analyses of p16, a biomarker of cellular senescence. While there was no immunohistochemical evidence of cellular senescence at 1-month post-exposure, at 3- and 6-months post-exposure, p16 immunoreactivity was significantly increased in the CA3 and dentate gyrus of the hippocampus, amygdala, piriform cortex and thalamus, but not the CA1 region of the hippocampus or the somatosensory cortex. Co-localization of p16 immunoreactivity with cell-specific biomarkers, specifically, NeuN, GFAP, S100β, IBA1 and CD31, revealed that p16 expression in the brain of DFP animals is neuron-specific. The spatial distribution of p16-immunopositive cells overlapped with expression of senescence associated β-galactosidase and with degenerating neurons identified by FluoroJade-C (FJC) staining. The co-occurrence of p16 and FJC was positively correlated. This study implicates cellular senescence as a novel pathogenic mechanism underlying the chronic neurological deficits observed in individuals who survive OP-induced cholinergic crisis.
高剂量有机磷酸酯（OP）胆碱酯酶抑制剂急性中毒可导致胆碱能危象，这与急性、危及生命的拟副交感神经症状、呼吸抑制和癫痫发作有关，癫痫发作可迅速进展为癫痫持续状态（SE）。临床和实验数据表明，从这些急性神经毒性作用中存活下来的个体往往会出现严重的慢性疾病，包括行为缺陷。将急性OP中毒与慢性神经功能缺损联系起来的致病机制仍具有推测性。细胞衰老与衰老和神经退行性疾病相关的行为缺陷有关，但急性OP中毒是否会触发大脑中的细胞衰老尚未得到研究。在此，我们在急性二异丙基氟磷酸酯（DFP）中毒大鼠模型中验证这一假设。成年雄性Sprague-Dawley大鼠给予DFP（4mg/kg，皮下注射）。对照动物给予等体积（300μL）的无菌磷酸盐缓冲盐水（皮下注射）。随后两组动物均注射硫酸阿托品（2mg/kg，肌肉注射）和2-解磷定（25mg/kg，肌肉注射）。根据行为癫痫标准确定，DFP在数分钟内引发癫痫活动，并迅速进展为SE。在暴露后1、3和6个月从动物收集大脑，用于对细胞衰老生物标志物p16进行免疫组织化学分析。虽然在暴露后1个月没有细胞衰老的免疫组织化学证据，但在暴露后3个月和6个月，海马体、杏仁核、梨状皮质和丘脑的CA3区和齿状回中p16免疫反应性显著增加，但海马体CA1区或躯体感觉皮质未增加。p16免疫反应性与细胞特异性生物标志物（特别是NeuN、GFAP、S100β、IBA1和CD31）的共定位显示，DFP动物大脑中p16的表达具有神经元特异性。p16免疫阳性细胞的空间分布与衰老相关β-半乳糖苷酶的表达以及通过FluoroJade-C（FJC）染色鉴定的退化神经元重叠。p16和FJC的共现呈正相关。本研究表明细胞衰老作为一种新的致病机制，是OP诱导的胆碱能危象存活者中观察到的慢性神经功能缺损的基础。
