| Schizophrenia is a mental disease whose etiology has not been fullyelucidated. Before, it was generally believed that schizophrenia was afunctional disease and patients with schizophrenia had no structural change.However, with the development of science and technology, more and moreevidences showed that schizophrenia was associated with the alterations inbrain structure. Some researchers reported that there were regional thinningof cerebral cortex, elevation in neuron density in cerebral cortex ofschizophrenia while the number of neurons in cerebral cortex did not changesignificantly. Neuroimaging study showed that there was alterations in fiberbundles in white matter and damage in corticocortical structural integrity inschizophrenia. Moreover, pathological results showed that there wereswelling and loss of oligodendrocytes, impairment of myelin in cerebralcortex of schizophrenia. Gene microarray analysis showed that myelinassociated gene was down-regulated. These studies have observedsignificant demyelination in the white matter and cerebral cortex of schizophrenia, and it is generally believed that the connectivity dysfunctioncaused by demyelination plays an important role in the pathogenesis ofschizophrenia. However, up to now, existing researches at home and abroadhad no report investigating the myelinated fiber and myelin sheaths changesin cerebral cortex of schizophrenia with three-dimensional quantitativemethods. In this study, therefore, we used recognized cuprizone-inducedschizophrenia-like animal model to investigate the change of the cortexvolume, the myelinated fibers and myelin sheaths in cerebral cortex withtransmission electron microscope (TEM) and stereological methods.PARTⅠ1. Materials and methods1.1Experimental animal6weeks old male C57BL/6mice (provided by experimental animalcenter of Chongqing Medical University) were divided into CPZ group andcontrol group.9mice in CPZ group were administrated with0.2%CPZmixed into standerd rodent chow,10mice in control group were fed standerdlab chow for6weeks. Animal weight was weighed every2or3days.1.2Behavior testThe behavior tests for the mice in CPZ group and control group wereperformed in6weeks to detect the behavior changes after CPZadministration for6weeks. Behavior tests include open-field test, elevatedplus maze test, Morris water maze, hole-board test, rota-rod test. 1.3After the behavior tests, the mice were anesthetized for perfusionfixation. Then, the brain was removed and the immunohistochemicalmethod was used to observe the demyelination changes in cerebral cortex.1.4One of the cerebral hemisphere was randomly selected and1mmcontinuous brain slices were made. Then, the total volume of cerebralcortex was estimated.5mice were randomly selected in each group. Then,5tissue blocks were randomly sampled from the cerebral cortex in eachanimal. One ultra-thin section was cut from each block and10fields wererandomly selected to photograph under TEM at a magnification of6,000×.50photos were shot in each animal.1.5The cortex volume, the total volume, the total length and the meandiameter of the myelinated fibers in the cortex were measured withstereological methods.2. Results2.1Changes in the animal body weight: After the start of the experiment,the body weight of mice in CPZ group was significantly reduced in the firsttwo weeks, then slowly restored. The body weight of mice in control groupwas significantly higher than the body weight of mice in CPZ group.2.2The results of of behavior tests: Compared with the mice in controlgroup, the activities in the central area of open-field test and the activitiesin the open arm of elevated plus maze test were significantly increased inthe mice of CPZ group. There were no significant differences in Morris water maze test, hole-board test and rota-rod test between the two groups.2.3Immunohistochemical staining: Compared with mice in control group,obvious demyelination in cerebral cortex was observed in CPZ group.2.4Stereological results: There was no significant difference in the totalvolume of cerebral cortex between the two groups (P>0.05). Comparedwith control group, the total volume of the myelinated fibers in the cerebralcortex of CPZ group was significantly decreased by69.8%(P<0.01). Thetotal length of the myelinated fibers in the cerebral cortex of CPZ groupwas significantly decreased by68.9%when compared with control group(P<0.01). Compared with control group, the total length of the myelinatedfibers with a diameter of0.2to0.4μm，0.4to0.6μm，0.6to0.8μm in thecerebral cortex of CPZ group were significantly decreased by4.317km,3.313km and0.940km, respectively (P<0.01). The mean diameter of themyelinated fibers in the cerebral cortex of CPZ group was significantlyincreased by17.8%when compared with the control group (P<0.01).PART Ⅱ1Materials and methods1.1The animals were the same as in Part Ⅰ.1.2Electron microscope samples were the same as in Part Ⅰ.15～20fields of view were randomly photographed under TEM at a magnificationof175000and90photos were shot in each animal. The ultrastructures ofthe myelinated fibers in cortex were observed in electron microscope photos.1.3The total volume, thickness, inner perimeter and outer perimeter of themyelin sheaths in cerebral cortex were measured with stereologicalmethods. Thickness, inner perimeter, outer perimeter of the myelin sheathsin cerebral cortex, the difference and the ratio between inner perimeter andouter perimeter in different diameter range were calculated.2. Results2.1The observation of electron microscope photos: Compared with controlgroup, obvious demyelination was observed in CPZ group, which includedobvious lamellar separation phenomenon, myelin and axonal separationphenomenon and vacuolar changes.2.2Stereological results：Compared with control group, the total volume ofthe myelin sheaths in the cerebral cortex of CPZ group was significantlydecreased by44%(P<0.05), and the mean thickness of myelin sheaths wassignificantly increased by31.5%(P<0.01). There was no significantdifference in mean outer perimeter of myelin sheaths, but there wassignificant difference trend in inner perimeter of myelin sheaths betweenthe two groups (P=0.054). The difference between inner perimeter andouter perimeter of myelin sheaths in the cerebral cortex of CPZ group wassignificantly increased by0.249μm when compared with the control group(P<0.01). The ratio between inner perimeter and outer perimeter of myelinsheath in the cerebral cortex of CPZ group was significantly decreased by 11.6%when compared with the control group (P<0.01).Compared with control group, the thickness of myelin sheaths in thecerebral cortex of CPZ group with the diameter of less than0.8μm (exceptfor0.2～＜0.3μm) was significantly increased. The outer perimeter ofmyelin sheaths in the cerebral cortex of CPZ group with the diameter of0.2～＜0.3μm and0.8～＜0.9μm was significantly decreased. The innerperimeter of myelin sheaths in the cerebral cortex of CPZ group with thediameter of0～＜0.3μm and0.8～＜1.0μm was significantly decreased.The difference between inner perimeter and outer perimeter in the cerebralcortex of CPZ group with the diameter of0～＜0.2μm,0.4～＜0.5μm and0.6～＜0.7μm was significantly increased, while the difference with thediameter of≥1.0μm was significantly decreased. The ratio between innerperimeter and outer perimeter of the myelinated fibers in the cerebral cortexof CPZ group with the diameter of less than0.7μm (except for0.3～＜0.4μm) was significantly decreased, while the ratio with the diameter of≥1.0μm was significantly increased (P<0.05).General Conclusions1. The present study showed that mouse fed with0.2%CPZ can appearschizophrenia-like behavior changes.2. The present study provided the basic data of the cortex volume, the totallength, the total volume and the mean diameter of the myelinated fibers, the total volume, the thickness, inner perimeter, outer perimeter of myelinsheaths in the cerebral cortex of0.2%CPZ-treated mice. These data willprovide important baseline values for future studies.3. The present study indicated that the total volume and the total length ofmyelinated fibers in the cerebral cortex of CPZ group were significantlydecreased when compared with control group. The loss of myelinatedfibers with small diameter contributed to the decrease of the total length ofmyelinated fibers in cerebral cortex.4. The present study found the loss of the myelin sheaths in the cerebralcortex of CPZ group, which provided direct, quantitative basis fordemyelination hypothesis of schizophrenia. |
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