Stereological Study On The Myelinated Nerve Fibers In The Hippocampal Subregions Of The CPZ-induced Schizophrenia Animal Model

Schizophrenia is a common severe psychotic disorder which affectsabout1%of the world population. Although the mechanism is now stillunknown, there is converging evidence that pathophysiology ofschizophrenia involves disturbances of brain white matter. Recent studiesshowed that the demyelination of the myelinated nerve fiber andpathological changes of oligodendrocyte existed in the hippocampus ofschizophrenia. However, there is no appropriate schizophrenic animal modelfor studying the demyelination of schizophrenic hippocampus and there areno stereological quantitative data of the myelinated nerve fibers in thehippocampal subregions, CA1and DG of schizophrenic hippocampus. Thus,it is difficult to objectively evaluate the demyelination level of hippocampusundertaken in the schizophrenia animal model. Cuprizone (CPZ) model is anovel schizophrenia model. The characteristic of this model is the centralnervous system demyelination, which can be used to study thedemyelination of hippocampus. In the first part of this study, we manipulateda series of doses of CPZ to treat the mice. By comparing the body weight, the survive time, the behavioral parameters and immunohistochemistry stanning,the best dose of CPZ was obtained, and, then the reliability of theschizophrenia animal model was evaluated by many means. In the secondpart, the stereological methods were used to investigate the quantitativechanges of the myelinated nerve fibers in the mouse hippocampal subregions,CA1and DG, of the CPZ-induced animal model.Part11Materials and methods1.1The806-week-old male C57BL/6mice were randomly divided into4groups, including control group,0.2%CPZ-treated group,0.3%CPZ-treated group and0.4%CPZ-treated group. The control mice werefed the standard chow, while0.2%CPZ-treated mice were fed the standardchow mixed with0.2%doze of cuprizone(w/w).0.3%CPZ-treated micewere fed the standard chow mixed with0.3%doze of cuprizone(w/w), and0.4%CPZ-treated mice were fed the standard chow mixed with0.4%doze ofcuprizone(w/w). The experiment lasted for6weeks.1.2The body weights of all mice were measured every3days, and thedeath date of mice were documented. At the end of the4th week of theexperiment, part of the mice were killed for further studies.1.3The Open field test, the Morris water maze test, the Turn-club testand the Hole board test were used to investigate behavior changes. 1.4The accurate positions of hippocampal subregions, CA1and DG,were identified. The Anti-MBP Immunohistochemistry staining methodswere used. The optical densities of MBP-positive myelinated nerve fibers inboth hippocampal subregions were compared.1.5The body weight, the survival time, the behavioral parameters andthe optical densities of the MBP-positive myelinated nerve fibers in mousehippocampal subregions, CA1and DG, of all groups were compared using aseries of statistical methods.2Results2.1The survive time of0.4%CPZ group was significantly less than thatof control group (P<0.01). There were no significant differences between thesurvive time of control group,0.2%CPZ group and0.3%CPZ group(P>0.05).2.2The weight of mice in the CPZ-fed groups was significant less thanthat of control group (P<0.05). The mice received higher CPZ dose hadsignificant lower weight when compared to the group received lower CPZdose.2.3In the open field test,0.2%CPZ mice spent significantly more timein the central area of box compared with control group (P<0.05).2.4ODs of MBP positive fibers in the area CA1of hippocampus of0.2%and0.3%CPZ groups were significantly less than that of control group (P<0.01), respectively. OD of MBP positive fibers in the area DG ofhippocampus in0.2%group was significantly less than that of control groupand0.3%CPZ group (P<0.01).Part21Materials and methods1.1The106-week-old male C57BL/6mice were randomly divided intocontrol group and0.2%CPZ-treated group. The control mice were fed thestandard chow, while0.2%CPZ-treated mice were fed the standard chowmixed with0.2%doze of cuprizone(w/w). The experiment lasted for6weeks1.2At the end of the6th week of the experiment, all the mice werekilled and their brains were removed. Based on the accurate identification ofthe boundary of CA1and DG, the respective volume of the mousehippocampal CA1and DG was estimated using a stereological method.1.3The tissue blocks with volume about1mm3were randomly cut fromhippocampal subregions, CA1and DG, respectively. Ultrathin sections wereobtained from these tissue blocks after microscopy processing. Everysection was viewed in a transmission electron microscope at a magnificationof6000×and20000×. Then10vision of fields were randomly photographedat a TEM magnification of6,000×. 1.4The respective stereological parameters of the myelinated nervefibers in mouse hippocampal subregions, CA1and DG, of these two groupswere estimated by means of a series of stereological methods.1.5The respective stereological parameters of the mouse hippocampalsubregions, CA1and DG, in the control mice and the0.2%CPZ mice werecompared using a series of statistical methods.2Results2.1Compared to the control group, the respective volume ofhippocampal CA1and DG of the0.2%CPZ mice did not significantlychange (P>0.05).2.2Compared to the control group, the length density and the totallength of the myelinated nerve fibers in the hippocampal CA1and DG of the0.2%CPZ mice were significantly decreased (P<0.05).2.3Compared to the control group, the volume density and the totalvolume of the myelinated nerve fibers in the hippocampal CA1and DG ofthe0.2%CPZ mice were significantly decreased (P<0.05).2.4Compared to the control group, the mean diameter of themyelinated nerve fibers in subregion CA1of the0.2%CPZ mice wasnonsignificantly changed (P>0.05), while the mean diameter of themyelinated nerve fibers in subregion DG of the0.2%CPZ mice wassignificantly decreased (P<0.05). General conclusions1. The dose of0.2%CPZ which was used to induce the schizophrenicanimal model was reliable.2. The CPZ-treated mice could have a series of schizophrenia-likealtered behaviors and the CA1and DG of CPZ-treated mouse hippocampuswere undertaking the pathological change of demyelination. TheCPZ-treated mice could be a well animal model of schizophrenia.3. The respective volume of hippocampal CA1and DG of CPZ-treatedmice did not significantly change compared to the control group.4. The length density, the total length, the volume density and the totalvolume of the myelinated nerve fibers in CPZ-treated mouse hippocampalCA1and DG were significantly decreased compared to the control group.5. There was remyelination in the hippocampus of CPZ-treated mice.6. The demyelination patterns of the hippocampal CA1and DG ofCPZ-treated mice were different. The regional differences in thedemyelination patterns are probably due to the different susceptibilities ofhippocampal subregions for CPZ and the different levels of remyelination inhippocampal areas CA1and DG.