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.

Objective To explore the role of white matter injuries in the schizophrenia induced by the NMDA re-ceptor antagonist. Methods Adult male C57BL/6J mice (8 week old) were equally divided into four groups. One group was sub-chronically treated with saline solution, and the other three groups were intraperitoneally treated with MK-801 at dose of 0.025 mg/mL (M1), 0.050 mg/mL (M2) and 0.100 mg/mL (M3) in a volume 10 ml per kilogram body weight. All ani-mals were tested using Morris water maze at the 9th-15th day and using the Hole Board exploration as well as Rota Rod performance tests on the 16th day. The myelin basic protein (MBP) and the ultrastructure of the myelin sheaths in the cor-pus callosum were then examined using immunohistochemical methods, transmission electron microscope technique and stereological methods. Results The repeated sub-chronic MK-801 treatment did not induce impairment of spatial learning and memory in Morris water maze. The MK-801 treatment at 0.25 mg/kg and 1.00 mg/kg but not at 0.50 mg/kg resulted in less exploration to a new environment. The myelin staining with anti-MBP antibody was less intense in all three schizo-phrenic groups when compared to saline control group (P<0.01). Furthermore, MK-801 treatment caused pathological al-terations of the myelin sheaths including segmental demyelination of myelinated fibers and splitting of myelin sheath lamel- lae in schizophrenic groups. The ratio of the injured myelinated nerve fibers in the corpus callosum of MK-801 treated mice [M3 group, (22.42 ± 4.24)%] was significantly higher when compared to the control mice [(3.84 ± 1.35)%,P<0.01)]. Conclusions The present study demonstrated the white matter damages, mainly low MBP expression and segmental demye-lization in the corpus callosum in the mice sub-chronic treated with MK-801, indicating that the white matter changes might be involved in the schizophrenia induced by NMDA antagonist.

In this study, the effect of enriched environment (EE) on the spatial learning of aged rats was examined, and then the effects of EE on the aged corpus callosum (CC) were investigated by means of the modern stereological methods. We found that EE significantly improved the spatial learning of aged rats. The CC volume, the total volume of the myelinated fibers and total volume of the myelin sheaths in the CC, the total length of the myelinated fibers in the CC of enriched rats were significantly increased when compared to standard rats. The increase of the myelinated fibers in enriched rat CC might provide one of the structural bases for the enrichment‐related improvement of the spatial learning. This study provided, to the best of our knowledge, the first evidence of environmental enrichment‐induced increases of the CC and the myelinated fibers in the CC of aged rats. Anat Rec, , 2012. © 2012 Wiley Periodicals Inc.

There has been no study investigating the effects of enriched environment on the capillaries of cortex with new stereological methods. In the present study, both 14 month female and male Sprague-Dawley rats were randomly divided into enriched environment (EE) rats and standard environment (SE) rats. EE rats were reared in enriched environment and SE rats were reared in standard environment for 4 months. The effects of short-term enriched environment on the cortex volume and on the total volume, total length, total surface area and mean diameter of the capillaries in the cortex of mid-aged Sprague-Dawley rats were quantitatively investigated with immunohistochemistry technique and unbiased stereological methods. There were no significant differences in the cortex volume, the total length and total surface area of the capillaries in the cortex between EE rats and SE rats. The total volume of the capillaries in the cortex of female EE rats and male EE rats was significantly increased when compared to female SE rats and male SE rats. The mean diameter of the capillaries in the cortex of female EE rats was significantly decreased when compared to that in female SE rats, but there was no significant difference in the mean diameter of the capillaries in the cortex between male EE rats and male SE rats. The present results indicate that enriched environment had a positive effect on the capillaries in the cortex of middle-aged rats. The present study might provide an important morphological basis for searching the ethology strategy to delay the progress of brain aging in the future.

In the present study, tissue blocks were randomly sampled from the entire hippocampus of 6-week-old Long-Evans rats. Isotropic, uniform and random sections, 60 nm thick, were prepared by isector. Fifteen fields of view were randomly selected for each section and photographed using a transmission electron microscope. The mean internal and external diameters of the myelin sheaths were obtained by measuring the longest profile diameter perpendicular to its longest axis. The inner and outer perimeters of the myelin sheaths were estimated using the equidistant parallel test lines. The thickness of the myelin sheaths was estimated by direct orthogonal measurements in uniform, random locations. These stereological methods should permit an unbiased quantitative assessment of changes in the myelin sheaths of myelinated fibers in the hippocampus.

It had been reported that enriched environment was beneficial for the brain cognition, neurons and synapses in cortex and hippocampus. With diffusion tensor imaging (DTI), several studies recently found the trained-induced larger corpus callosum. However, the effect of enriched environment on the oligodendrocytes in corpus callosum has not been explored with the unbiased stereological methods. In current study, the effect of enriched environment on the total number of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) positive cells in middle-aged rat corpus callosum was investigated by means of immunohistochemical techniques and the unbiased stereological methods. We found that, when compared to standard rats, the spatial learning capacity of enriched-environment rats was significantly increased. The total number of the CNPase positive cells in the corpus callosum of enriched-environment middle-aged rats was significantly increased when compared to standard rats. The present study provided, to the best of our knowledge, the first evidence of environmental enrichment-induced increases in the total number of CNPase positive cells in the corpus callosum of middle-aged rats.