Cuprizone   中文名称: 双环己酮草酰二腙

copper chelating agent; copper enzymes such as SOD1, cytochrome oxidase, and DβH

铜（II）与双（环己酮）草酰二腙（也称为Cuprizone/铜酮）配位，形成强烈的蓝色配合物，70多年前首次报道。铜酮反应已被用于测定痕量铜的比色试验。铜酮在C57BL/6小鼠体内的应用也会随着时间的推移导致脱髓鞘，这一结果似乎是由于铜的不平衡造成的，这使得铜酮自20世纪60年代首次使用以来，一直被用作毒性诱导脱髓鞘动物模型的主要方法。尽管人们对铜带及其结合铜的能力有广泛的兴趣，但对这种配体的铜配位性质进行结构表征的研究相对较少。在没有水介质（如纯醇）的情况下，铜和铜酸盐只形成无定形的绿色沉淀物。在水条件下，存在大量过量的铜（相对于铜），主要形成与铜-铜配合物同义的蓝色配合物。蓝色和绿色铜-铜酸盐产品的溶解度较差，对传统的结构表征方法（如x射线晶体学或核磁共振波谱学）提出了挑战。通过结合质谱、x射线吸收光谱、计算化学和其他技术，揭示了蓝色和绿色配合物的铜配位结构的自一致图像——证实蓝色配合物处于Cu（III）状态，每个金属中心含有两个水解铜酮配体，而绿色配合物代表一个扩展的低聚配合物。由间隔4.8 Å的重复Cu（II）中心组成，并由未水解的铜酸盐供体桥接。[３]

当小鼠接触Cuprizone （0.5 mg/100 mg 喂食或淡水喂养；表皮）时，小鼠的中枢神经系统会经历严重的海绵状状态，特别是在脑干和小脑白质中 [1]。

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最近的人类研究表明，少突胶质细胞的改变在精神分裂症的病理生理中起作用。我们最近的动物研究报告了暴露于Cuprizone/铜酮（Xu et al., 2009）的小鼠的一些精神分裂症样行为，Cuprizone是一种铜螯合剂，已被证明可以选择性地损伤白质。本研究旨在探讨铜酮暴露小鼠行为改变的机制，并考察抗精神病药物氟哌啶醇、氯氮平和喹硫平对小鼠行为改变的影响。给药14天的小鼠在脉冲前抑制声惊反应和前额皮质（PFC）多巴胺含量升高方面表现出缺陷，而给药加抗精神病药物的小鼠则没有这种变化。给予铜酮21天的小鼠y型迷宫自发性改变较低，而在铜酮加抗精神病药物治疗的小鼠中未见这种变化。给予铜酮28天的小鼠表现出较少的社会互动，这在给予铜酮加氯氮平/喹硫平的小鼠中没有看到，但在给予铜酮加氟哌啶醇的小鼠中看到。小鼠给予铜吡嗪42天，髓鞘丢失，PFC、尾状壳核和海马髓鞘碱性蛋白降低。铜吡酮加氯氮平/氟哌啶醇可减轻小鼠PFC白质损伤。氯氮平和喹硫平仅能减轻尾状壳核和海马白质损伤，氟哌啶醇则不能。这些结果有助于我们理解行为变化，并为抗精神病药物对铜酮暴露小鼠白质损伤的保护作用提供实验证据。[4]

Male C57BL/6 mice (6-weeks old, 20 to 22 g) were used. After an acclimatization period of 10 days, the C57BL/6 mice were given 0.2% by weight Cuprizone/CPZ in the standard powdered rodent chow for 14–42 days, during which age-matched mice received the standard chow without CPZ and were used as controls. Three independent experiments were performed for HAL, CLZ, and QUE, respectively. Each experiment consisted of four animal groups designed to examine effects of CPZ on animals’ behaviors and white matter and effects of the antipsychotics in mice without or with CPZ-exposure. For example, HAL experiment included CNT, CPZ, HAL, and CPZ+HAL groups. CNT group mice ate normal rodent chow without CPZ and received no HAL; CPZ group mice ate CPZ-containing rodent chow and received no HAL; HAL group mice ate normal rodent chow without CPZ but received HAL treatment (1 mg/kg/day, i.p.); CPZ + HAL group mice ate CPZ-containing rodent chow and received HAL treatment. The experimental period was 42 days, during which mice were subjected to behavioral tests at indicated time points. For high-performance liquid chromatography (HPLC) analysis, two additional experiments were performed, in which the same treatments (CPZ ingestion and antipsychotics administration) continued for 12 and 21 days, respectively. Each of the experiments consisted of eight animal groups (CNT, CPZ, HAL, CLZ, QUE, CPZ+HAL, CPZ+CLZ, and CPZ+QUE; 5–8 mice/group). [4]

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Prepulse inhibition test[4]
PPI refers to the inhibition of a startle reflex produced by preceding the startling stimulus, or pulse, with a weak prepulse stimulus. This test provides an operational measure of sensory gating of subjects. In our recent study, CuprizoneCPZ-exposed mice showed PPI deficits on 14th and 21st days after CPZ-exposure (Xu et al., 2009). In this study PPI test was performed on 14th day after CPZ-exposure. As described in our recent study (Xu et al., 2009), each mouse was placed into a small Plexiglas cylinder within a large sound-attenuating chamber. The cylinder was seated upon a piezoelectric transducer, which allows vibrations to be quantified and displayed on a computer. The background sound levels (74–75 dB) and calibration of the acoustic stimuli were confirmed with a digital sound level meter. After a 5-min habituation period, PPI test sessions were conducted. Each session started and ended with five startle trials (40 ms; 120 dB), respectively. Between the starting and ending startle trials, there were eight identical blocks consisting of the following five trials: a no-stimulus trial, a startle trial, and three prepulse-startle trials, each of which had a pre-pulse stimulus (3, 6, or 12 dB above the background sound levels) prior to a startle stimulus (100 ms after the prepulse). The average inter-trial interval was 15 s. Measures were taken of the startle amplitude for each trial, defined as the peak response during a 65-ms sampling window starting from the onset of a startle stimulus. Levels of PPI at each prepulse sound level were calculated as 100 × (1-averaged response amplitude in trials with a prepulse stimulus and startle stimulus/averaged response amplitude in trials with the startle stimulus alone).

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Y-maze test [4]
The Y-maze is a simple two-trial recognition test for measuring spatial recognition memory. In a previous study (Oades et al., 1985), amphetamine reduced alternation dose-dependently and HAL pretreatment inhibited this effect, suggesting that this paradigm is useful for studying spatial working memory in animal models of schizophrenia. In our recent study, Cuprizone/CPZ-exposed mice showed lower spontaneous alternation in Y-maze on 14th, 21st, 28th, 35th, and 42nd days after CPZ-exposure (Xu et al., 2009). In this study Y-maze test was performed on 21st and 42nd days after CPZ-exposure. As described in our recent study (Xu et al., 2009), each mouse was placed at the end of one arm of a symmetrical Y-maze and allowed to move freely through the maze during an 8-min test period. The total number and series of arm entries were recorded. The number of overlapping entrance sequences (e.g., ABC, BCA) defines the number of spontaneous alternations.

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Social interaction test [4]
In our recent study, CuprizoneCPZ-exposed mice showed less social interaction on 28th, 35th, and 42nd days after CuprizoneCPZ-exposure (Xu et al., 2009). In this study social interaction test was performed on 28th day after CPZ-exposure. As described in our recent study (Xu et al., 2009), pairs of unfamiliar mice (that had been housed separately) of the same experimental group were placed in the center of an open-field box (56 cm × 56 cm × 31 cm) about 10 cm apart. A video camera was placed above the open-field box to monitor the animals’ movement. During a 10 min test period, the social interaction of the animals was defined when the tested mice were closer than 5 cm (between the center point of the back of a mouse and another one) for at least 0.2 s. Each mouse was used only one time. The results were recorded and analyzed automatically by the video tracking program SMART, which counted the amount of time spent near another mouse for two mice simultaneously. Although this method only measures closeness of subjects examined, it has been used in previous studies and the closeness is believed to reflect social interactions (Shi et al., 2003; Egashira et al., 2007), which is an animal correlate of social withdrawal seen in patients with schizophrenia (Weinberger, 1987; Abi-Dargham et al., 2000). Social behavioral deficit has been reported in various animal models of schizophrenia (Shi et al., 2003; Boucher et al., 2007; Lazar et al., 2008).

[1]. Suzuki K, et al. Status spongiosus of CNS and hepatic changes induced by cuprizone (biscyclohexanone oxalyldihydrazone). Am J Pathol. 1969 Feb;54(2):307-25.
[2]. Sanadgol N, et al. Alpha-lipoic acid mitigates toxic-induced demyelination in the corpus callosum by lessening of oxidative stress and stimulation of polydendrocytes proliferation. Metab Brain Dis. 2018 Feb;33(1):27-37.
[3]. M Jake Pushie, et al. Synthesis and structural characterization of copper-cuprizone complexes. Dalton Trans. 2022 Jun 29.
[4]. Haiyun Xu, et al. Behavioral and neurobiological changes in C57BL/6 mouse exposed to cuprizone: effects of antipsychotics. Front Behav Neurosci. 2010 Mar 18;4:8.

Cuprizon is an organooxygen compound and an organonitrogen compound. It is functionally related to an alpha-amino acid.
Copper chelator that inhibits monoamine oxidase and causes liver and brain damage.

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Multiple Sclerosis (MS), is a disease that degenerates myelin in central nervous system (CNS). Reactive oxygen species (ROSs) are toxic metabolites, and accumulating data indicate that ROSs-mediated apoptosis of oligodendrocytes (OLGs) plays a major role in the pathogenesis of MS under oxidative stress conditions. In this study, we investigated the role of endogenous antioxidant alpha-lipoic acid (ALA) as ROSs scavenger in the OLGs loss and myelin degeneration during Cuprizone (cup)-induced demyelination in the experimental model of MS. Our results have shown that ALA treatment significantly increased population of mature OLGs (MOG+ cells), as well as decreased oxidative stress (ROSs, COX-2 and PGE2) and apoptosis mediators (caspase-3 and Bax/Bcl2 ratio) in corpus callosum (CC). Surprisingly, ALA significantly stimulates population of NG2 chondroitin sulfate proteoglycan positive glia (NG2+ cells or polydendrocytes), from week 4 afterward. Accordingly ALA could prevents apoptosis, delays demyelination and recruits OLGs survival and regeneration mechanisms in CC. We conclude that ALA has protective effects against toxic demyelination via reduction of redox signaling, and alleviation of polydendrocytes vulnerability to excitotoxic challenge.[2]

C14H22N4O2

278.3501

278.174

C, 60.41; H, 7.97; N, 20.13; O, 11.50

370-81-0

9723

Typically exists as White to off-white solid at room temperature

1.3±0.1 g/cm3

210-214 °C(lit.)

1.626

0.85

82.92

2

4

2

20

373

0

O=C(C(N([H])/N=C1\C([H])([H])C([H])([H])C([H])([H])C([H])([H])C\1([H])[H])=O)N([H])/N=C1\C([H])([H])C([H])([H])C([H])([H])C([H])([H])C\1([H])[H]

DSRJIHMZAQEUJV-UHFFFAOYSA-N

InChI=1S/C14H22N4O2/c19-13(17-15-11-7-3-1-4-8-11)14(20)18-16-12-9-5-2-6-10-12/h1-10H2,(H,17,19)(H,18,20)

N,N'-bis(cyclohexylideneamino)oxamide

Cuprizone; 370-81-0; Bis(cyclohexanone)oxaldihydrazone; Ethanedioic acid, bis(cyclohexylidenehydrazide); Cuprizane; Oxalic acid bis(cyclohexylidenehydrazide); Cuprizon; Biscyclohexanone oxaldihydrazone;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~6.67 mg/mL (~23.96 mM)

配方 1 中的溶解度: ≥ 0.67 mg/mL (2.41 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，将 100 μL 6.7 mg/mL 澄清 DMSO 储备液加入 900 μL 20% SBE-β-CD 生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 0.67 mg/mL (2.41 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 6.7 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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