| BackgroundCPZ is a copper ion chelator.,in 1966 Carlton observed that after administration of CPZ,mice exhibited a decrease in serum copper and demyelination of brain white matter.Thereafter,CPZ was widely used in the following three decades,for study demyelinating disease pathology,chemical molecular mechanism and treatment.CPZ-induced white matter demyelination is a reversible injury.After cessation of feeding with CPZ feed,spontaneous whitening of myelin may occur in the white matter of mice,which allows the model to simulate the characteristics of acute onset and relapse of MS.And it has obvious pathological similarity with MS type 3,so it is widely used in the research of MS pathogenesis and treatment.To study the demyelinating lesions induced by CPZ,the lesions and prognosis of different parts of white matter can be produced in different animal germlines,ages,different modes of administration and doses,but there is no uniform model standard in China.The use of standard animal models is more conducive to the unity and judgment of experimental results.C57BL/6J mice are the most common animal species in the CPZ model,but it is still controversial whether the demyelination and spontaneous remyelination in the CPZ model have gender differences.Is it a good model to study MS gender differences?It needs further research.As a chelating agent for copper ions,CPZ is still unknown in the physical and chemical behavior and toxicity mechanism in vivo.The physical and chemical behaviors and toxic mechanisms of CPZ in vivo can be unveiled,and the model can be better utilized,and give clues to the etiology of demyelinating diseases.Objectives:To establish a standard CPZ-induced demyelination model of degeneration to explore whether there is a male-female anomaly in this model in this strain of C57BL/6J mice and to provide a reference for selecting appropriate animal models for MS gender differential studies.To further explore whether the copper,iron and zinc ion concentrations in mouse brain tissue under standard modeling are changed.It is inferred whether the cause of CPZ is dependent on the physicochemical properties of the copper chelator,which provides a reference for the further exploration of the physical and chemical behavior of CPZ in vivo.Methods:1.Established degeneration model induced by CPZ Sixteen male C57BL/6J mice were randomly selected as model group and eight as control group.The control group was fed with conventional feed,and the model group was fed with 0.2%CPZ feed for 6 weeks.At the end of the 6th week,the model group and the control group were tested for motor coordination and then sacrificed for brain tissue.The brain tissues were stained withLFB,ratio of apoptosis of brain cells and MBP immunohistochemical staining,and the cumulative optical density was calculated.2.Sex difference of demyelination CPZ-induced in C57BL/6J mice 48 C57BL/6J mice,male and female,were randomly divided into two groups:32 were randomly selected as model group,16 were control group(group A),and the control group was fed regularly.The model group was fed with food containing CPZ for 6 weeks.When the model was successfully established,They were randomly divided into demyelination group(B group)and remyelination group(C group).Group A and B were to take brain tissue after 6 weeks.All mice take exercise coordination ability test before sacrifice.group C was continued to feed with routine feeding for 6 weeks,then was sacrificed after the coordination ability test.All brain tissues were examined for LFB staining,MBP immunohistochemical staining,and apoptosis ratio.3.The changes of copper,iron and zinc ion content in brain tissue of C57BL/6J mice in demyelination model induced by CPZ 48 C57BL/6J mice were randomly divided into experimental group and control group.The control group was fed with conventional diet.The experimental group was fed with diet containing with 0.2%CPZ.The mice were sacrificed for brain tissues on the 2nd,4th,6th and 12th week respectively.Brain tissue sections were stained with LFB and immunohistochemical staining with MBP.The contents of total copper,total iron and total zinc in brain stem,cerebellum,basal ganglia and cerebral cortex were measured by flame atomic absorption spectrometry.Statistical analysisSPSS 21.0 statistical software was used for data analysis.Data are presented as mean±standard deviation(x±s),that between groups using one-way analysis of variance,two independent samples t-test group,P<0.05 for the difference was significant.Results:1.Compared with the control group,the movement time of the model group on the rotating rod was shortened,and the difference was significant(P<0.05).Compared with the control group,the number of falling of the model group on the rotating rod increased,and the difference was significant(P<0.05).The results of LFB staining were observed under light microscope.Compared with the control group,the nerve fiber structure of the model group was disordered,and the myelin staining was different.The myelin sheath was swollen and fractured.Visible vacuole formation was observed.Compared with the control group,the ratio of neuronal apoptosis in the cerebral corpus callosum of model mice increased,the difference was significant(P<0.05);the optical density of MBP accumulated in the experimental group decreased significantly in the control group,the difference was significant(P<0.05).2.Compared with group A,the exercise coordination ability and MBP content in group B were significantly decreased(P<0.05),and the ratio of brain cell apoptosis and the degree of demyelination lesions were significantly increased(P<0.05).The motor coordination ability and MBP content in group C were significantly higher than those in group B(P<0.05).The ratio of brain cell apoptosis and the score of demyelination lesions were lower than those in group B(P<0.05).There was no significant difference in the coordinating ability of the males and females,the degree of demyelination lesions,the ratio of brain cell apoptosis and the content of MBP in group B(P>0.05).Compared with group C,the female group had better exercise coordination ability,lower score of demyelination lesions and higher MBP content than the male group,and there was no significant difference in brain cell apoptosis ratio.3.In the experimental group,demyelination did not occur in the corpus callosum at 2th week.Compared with the control group,there was no significant difference in total copper content in the brain stem,cerebellum,and prefrontal cortex of the experimental group(P<0.05),but this time the total copper content in the basal ganglia was significantly higher than that in the control group(P<0.05).At the 4th week,the corpus callosum showed incomplete demyelination in the experimental group.At this time,the brain stem and basal ganglia of the experimental group,the total copper content was increased compared with the control group,the difference was significant(P<0.05),but there was no significant difference in total copper content of the cerebellum and prefrontal cortex between the experimental group and control group.At the sixth week,the corpus callosum was completely demyelinated in the experimental group.Compared with the control group,the total copper content in the brainstem and basal ganglia of the experimental group increased,the difference was significant(P<0.05),but the total copper content in the cerebellum and prefrontal cortex had no significant difference(P<0.05);at the 12th week,in the experimental group,the content of corpus callosum brain cells was significantly reduced and the interstitium was increased.At this time,the content of copper in the brain stem,cerebellum,and basal ganglia was higher than that in the control group.The difference was statistically significant(P<0.05).There was no significant difference in total copper content of the cortex between experimental group and the control group(P>0.05).There was no significant difference in the total iron of the brain stem,cerebellum,basal ganglia and prefrontal cortex between the experimental group and the control group,the 2th week,the 4th week and the 6th week(P>0.05).At the 12th week,the total iron in the brain stem,cerebellum,and basal ganglia in the experimental group was significantly higher than that in the control group(P<0.05),but there was no significant difference in the prefrontal cortex(P>0.05).At all times,there was no significant difference in total zinc content of brain stem,cerebellum,basal ganglia and prefrontal cortex between the experimental group and the control group(P>0.05).conclusion1.Feeding 8 weeks old C57BL/J mice with 0.2%CPZ diet for 4-6 weeks can induce extensive demyelination of the white matter.The model has a simple modeling process,good repeatability,no complicated operation,and low price.The short experimental period and the easy detection of evaluation indicators are good animal models for the study of demyelinating diseases.2.CPZ-induced C57BL/6J mouse white matter demyelination disease was no difference between male and female,but in the myelin regeneration period,female mice have better myelination ability.3.CPZ as a copper ion chelator does not reduce the copper content in brain tissue.Instead,it causes a rise in the total copper content in brain tissue.This may be due to the fact that CPZ interferes with the normal transport of copper ions inside and outside the cell,resulting in copper in the brain tissue,abnormal deposition.Since the demyelination of white matter CPZ-induced is not caused by copper deficiency,More experiments are needed to further explore the physicochemical behavior of CPZ in vivo in order to unveil the mechanism of demyelination.The deposition of iron ions is most likely caused by the release of iron-rich oligodendrocytes following apoptosis. |
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