It is now accepted that white matter abnormalities play an important role in demyelinating diseases and a wide range of psychiatric disorders. Experimental demyelination (especially induced by cuprizone) has been investigated extensively. However, details regarding demyelination and ultrastructural changes of myelinated fibers have not been previously reported. Therefore, we determined the extent of demyelination using quantitative stereology. Mice exposed to cuprizone in the current study showed abnormal anxiety‐like behavior without impaired spatial learning or memory. The myelinated fibers in whole corpus callosum of mice exposed to cuprizone showed extensive myelin deficiencies and occasional axonal injuries. The total length of the myelinated fibers in whole corpus callosum of mice exposed to cuprizone was significantly decreased by 45% compared with control mice. The loss of myelinated fibers was mainly due to the marked loss of the fibers with a diameter of 0.4 to 0.8 μm. The g‐ratio of the myelinated fibers in the corpus callosum of mice exposed to cuprizone (0.69 ± 0.02) was significantly decreased compared with control mice (0.76 ± 0.02). These results might help us to further understand the role of white matter abnormalities in demyelinating diseases or a wide range of psychiatric disorders. © 2016 Wiley Periodicals, Inc.

目的:研究双环己酮草酰二腙(cuprizone,CPZ)诱导C57BL/6小鼠建立的精神分裂症样模型小鼠海马内有髓神经纤维体积改变及其与行为学改变之间的关系.方法:19只雄性6周龄小鼠,随机分为实验组(9只)和对照组(10只),实验组用含0.2％(质量分数百分比,w/w)CPZ的混合鼠饲料饲喂6周,建立精神分裂症样小鼠模型;对照组用标准鼠饲料饲喂6周.运用旷场实验、高架十字迷宫实验、Morris水迷宫实验、转棒实验和探孔实验测试行为学改变;髓鞘碱性蛋白(myelin basic protein,MBP)免疫组化染色及透射电镜定性观察髓鞘结构变化;体视学方法测量并计算海马总体积及海马内有髓神经纤维体积;统计学方法分析行为学改变与海马内有髓神经纤维体积改变之间的关系.结果:与对照组相比,实验组小鼠体质量下降,差异具有统计学意义(P＜0.05);行为学实验结果表明:实验组小鼠存在异常的焦虑行为(P=0.018)及空间认知能力障碍(P=0.037),但运动能力(P=0.443)、探索习性(P=-0.306)及学习记忆能力未见受损(P=0.462);MBP免疫组化染色发现海马区域免疫阳性染色变浅;透射电镜定性观察发现海马内有髓神经纤维存在脱髓鞘改变;体视学定量测定发现,实验组小鼠海马总体积未发生显著性改变(P=0.955),而海马内有髓神经纤维体积减小(P=0.009);统计学相关分析发现,闭臂路程百分比和海马内有髓神经纤维体积呈正相关(rs=0.83,P=0.003),开臂路程百分比和海马内有髓神经纤维体积呈负相关(rs=-0.65,P=0.043).结论:进一步证实CPZ模型小鼠可以出现类似精神分裂症样症状的行为学改变及海马内有髓神经纤维存在脱髓鞘改变,并且发现海马内有髓神经纤维体积和行为学表现间具有一定的相关性.

Objective To explore an appropriate dose of cuprizone(CPZ) for the CPZ-fed animal model as a novel schizophrenia animal model,and to investigate the possible relationship among CPZ dose,demyelination of nerve fibers in hippocampus and behavior alternations in CPZ-fed mice.Methods C57BL /6 mice were fed with 0.2%,0.3% and 0.4%(w / w) of CPZ for 6 weeks,respectively,and the mice without any treatment were used as control.The body weight,survival time and behavior changes were compared between the CPZ-fed mice and the control mice.The myelin basic protein(MBP)-positive nerve fibers in CA1 and DG regions of hippocampus were stained with immunohistochemical methods,and the mean optical density(OD) of MBP-positive nerve fibers in the hippocampal CA1 and DG regions of each group were calculated and compared.The changes of the myelinated nerve fibers in CA1 and DG regions of hippocampus between the control group and 0.2% CPZ group were investigated with a stereological method.Results The survive time of the 0.4% CPZ group was significantly less than that of control group(P < 0.01).The body weight of the CPZ-fed groups was significantly lower than that of the control group(P < 0.05),and the weight loss increased along with the increase of CPZ doses.In open field test,the mice fed with 0.2% of CPZ spent significantly more time in the central area of box compared with the control group(P < 0.05).The ODs of MBP-positive fibers in CA1 region of hippocampus in the 0.2% CPZ group and 0.3% CPZ group were significantly lower than that in the control group(P < 0.01).The OD of MBP-positive fibers in DG region of hippocampus in the 0.2% CPZ group was significantly lower than that in the control group and 0.3% CPZ group(P < 0.01).The volumes of CA1 and DG regions of the hippocampus in the 0.2% CPZ group were not significantly different from those in the control group(P > 0.05).The length density and total length of the myelinated nerve fibers in CA1 and DG regions of hippocampus in the 0.2% CPZ group were significantly less than those in the control group(P < 0.05).Conclusion 0.2% CPZ(w / w) was the appropriate dose for CPZ-fed animal model to mimic schizophrenia.The myelinated fibers in CA1 and DG regions of hippocampus undergo demyelination when mice are exposed to 0.2% CPZ for 6 weeks.

Objective To investigate the changes of cerebral cortex volume and myelinated fibers in the cerebral cortices of cuprizone(CPZ)-induced schizophrenia-like mice.Methods Six-week old male C57BL/6 mice were divided into a CPZ group and a control group.The mice in the CPZ group were fed with mixed standard rodent chow containing 0.2% CPZ,while those in the control group were fed with standard lab chow.After six weeks,behavioral tests were performed to confirm the success of schizophrenia-like animal model.Then the cerebral cortex volume and myelinated fibers in the cerebral cortices were determined with transmission electron microscopy and stereological methods.Results Mice in the CPZ group showed schizophrenia-like performance.Stereological results showed that there was no significant change in the volume of the cerebral cortex between CPZ group and control group(P>0.05).Compared with the control group,the length density and total length of myelinated fibers in the cerebral cortices of the CPZ group significantly decreased by 64.3% and 68.9%,respectively(P<0.01).Compared with the control group,the mean diameter of myelinated fibers in the CPZ group significantly increased by 17.8%(P<0.01),and the total length of the myelinated fibers with diameters of 0.2 to less than 0.4 μm,0.4 to less than 0.6 μm and 0.6 to less than 0.8 μm significantly decreased by 4.317,3.313 and 0.940 km,respectively(P<0.01).There was no significant difference in the total length of other myelinated fibers between the two groups.Conclusion In CPZ group,the total length of myelinated fibers in cerebral cortex decreases but the mean diameter of the myelinated fibers increases,which is due to the loss of small-diameter myelinated fibers.

Objective To investigate the changes of the myelinated fibers in the corpus callosum of the cuprizone(CPZ)-induced mouse model of schizophrenia,and the relationship between the demyelination changes and the behavioral abnormalities in this animal model.Methods Nineteen male C57BL/6 mice were randomly divided into 2 groups,control group(n=10) and CPZ-treated group(n=9).The mice in the later group were fed with a diet of 0.2%(w/w) CPZ mixed into standard rodent chow for 6 weeks.The mice in control group were fed standard lab chow and lab water.The behavioral changes of the animals were investigated with a series of behavioral tests.The changes of the corpus callosum and the myelinated fibers in the corpus callosum were studied with immunohistochemical assay,transmission electron microscopy and stereological methods.Results The CPZ-treated mice displayed more activities in the central area of open field(P<0.05)and in the open arms of elevated plus maze(P<0.05),which suggested that the anxiety behavior in the CPZ-treated mice was reduced when compared to the control.Morris water maze demonstrated that there was no working memory deficit in the CPZ-treated mice compared to the control(P>0.05).There was no abnormality in the motor function and basic exploration ability in the CPZ-treated mice when compared to the control.There were demyelination changes in the corpus callosum of the CPZ-treated mice.There was no significant difference in the total volume of the corpus callosum between the CPZ-treated mice and the control(12.66±1.07 vs 13.53±2.79 mm3).The length density and total length of the myelinated fibers in the corpus callosum were significantly decreased in CPZ-treated mice than control mice(0.70±0.17 vs 1.47±0.17 km/mm3,9.06±2.56 vs 19.75±3.70 km,P<0.05).Conclusion CPZ-treated mice display schizophrenia-like behaviors and demyelination change in the corpus callosum.