Study On Ameliorative Effects Of ScFv17 On Abnormal Gait And Related Pathological Changes Of Cerebellum In AD Transgenic Mice

Objective:Alzheimer’s disease(AD)is a neurodegenerative disease prevalent in the elderly.Its typical clinical symptoms were the impairment of cognition and memory and the gait disorder.Amyloid β(Aβ),the typical and main pathological feature of AD,is always been regarded as an important target of immunotherapy treatment of AD.Although there are many studies on the ameliorative effects of Aβ immunotherapy on the impairment of cognition and emotion,there is a little known about the effects on gait behavior.Single chain variable domain antibody fragment 17(scFv17)can significantly reduce Aβ plaques and improve the cognitive functions.In the present study,APPswe/PS1M146V/tauP301L(3xTg-AD)transgenic mice were used as AD models.A gait analyzer was used in this study as a behavioral experiment to observe the changes in the gait of 3xTg-AD mice and then to investigate the effect of scFv17 against 3xTg-AD mice gait disorder.Moreover,in order to clarify the underlying mechanisms of the effects of scFv17 on gait,we further used the various morphological methods to observe the pathological changes in cerebellum.Methods:In the present study,a selection of 6-month-old 3xTg-AD mice and C57BL/6 wild-type mice was performed.First,we observed their gait changes and found that the gait of 12-month-old 3xTg-AD mice were severely damaged.Then,the two groups of mice were randomly divided into four groups: WT+PBS,WT+scFv17,3xTg-AD+PBS and 3xTg-AD+scFv17.The gait behavior test and pathological test were performed 12 weeks after continuous administration of scFv17(1.5 mg/kg)or an equal volume of PBS(0.01 M)by nasal gavage.1.Gait AnalysisThe mice were allowed to pass through the gait analyzer’s passage freely,without pause,without interference,while high-speed video cameras were used to record the mouse foot information within a set length of 30 cm.The gait parameters were obtained after analysis made by specific analysis software,including of Stance Time Percentage,Swing Time Percentage,and Rear Track Width,to determine the balance and coordination of mice in each group.2.Pathological TestAfter perfusion of the heart,the cerebellum was embedded sagittally in the paraffin,and the following experiments were carried out after the slicing,slices stalling and slices grilling:(1)HE Staining TestThe morphological structure of the vermis of the cerebellum was observed.(2)Nissl’s Staining TestThe damages of Nissl body in Purkinje cells ofthe cerebellar vermis were analyzed to evaluate the effects of scFv17.(3)Paraffin ImmunohistochemistryAfter the baked cerebellum slice was dewaxed and washed with distilled water,they were handled by the following procedure: hot antigen repairment→endogenous peroxidase inactivation→serum blockage→primary antibody incubation→secondary antibody incubation→horseradish peroxidase labeled streptomycin avid in the third antibody treatment→coloring→coloring termination→hematoxylin counterstaining →dehydrate→transparency→seal.Later,the pathological section were scanned and photographed by the Scanscope digital pathological slide imaging system.The distribution of cortical Aβ plaques(6E10),neurofibrillary tangles(NFT)(AT8)in PCs,the expression of brain derived neurotrophic factor(BDNF)and its high-affinity receptor TrkB,and the expression of synaptophysin in the molecular layer were observed and analyzed to explore the positive roles of scFv17 on the pathological changes of cerebellum.Results:1.In the gait test,(1)we found that 12-month-old 3xTg-AD mice had wider Rear Track Width than the 6-month-old 3xTg-AD mice(P<0.001)and 12 months of age.The Rear Track Width of the 12-month-old 3xTg-AD mice was significantly wider than that of the same months old WT mice(P<0.001).Compared to 6 or 9-month-old 3xTg-AD mice,12-month-old 3xTg-AD mice had a greater percentage of stance time,a smaller percentage of swing time(P<0.001),and 12-month-old 3xTg-AD mice owned the significantly lower swing time percentage than that of mice of the same month old WT group(P<0.001),and the stance time percentage of 12-month-old 3xTg-AD mice was significantly higher than that of the same month old WT group mice(P<0.001).(2)We found that in comparison to the WT+PBS mice,the Rear Track Width of the 3xTg-AD+PBS control group mice was significantly wider(P<0.001).There was no statistical difference between the Rear Track Width of theWT+PBS mice and that of the WT+scFv17mice(P>0.05),but the Rear Track Width of the 3xTg-AD+scFv17 group was significantly smaller than that of the 3xTg-AD+PBS control group(P<0.05).(3)On the other hand,the Stance Time Percentage was increased significantly in the 3xTg-AD+PBS control group,compared to the WT+PBS mice group(P<0.001),while the Swing Time percentage was decreased significantly(P<0.001).There was no significant difference between the two kinds of time percentage parameters of the WT+PBS and WT+scFv17 mice(P>0.05),but the Stance Time Percentage of the 3xTg-AD+scFv17 group was significantly smaller than that of the 3xTg-AD+PBS control group(P<0.01),while the Swing Time Percentage of the 3xTg-AD+scFv17 group was significantly greater(P<0.001).2.In the HE staining test,the cerebellar Purkinje cells of the WT+PBS and WT+scFv17 mice groups were arranged neatly and densely.Its structure was clear and cytoplasm was red-stained.The nuclei were blue-stained and the nucleus was round or oval.Purkinje cell arrangement of 3xTg-AD+PBS group mice was sparse,fuzzy outlined.There were numerous apoptotic cells.However,compared with the 3xTg-AD+PBS group mice,the apoptosis of the 3xTg-AD+scFv17 group mice was slight and the morphology was improved.3.In the Nissl staining test,the density of Nissl-staining positive Purkinje cells was significantly lower in the 3xTg-AD+PBS control group than in the WT+PBS group(P<0.001).The density of Nissl-staining positive Purkinje cells was not significantly different between WT+PBS and WT+scFv17 groups(P>0.05),but scFv17 significantly upregulated the number of Purkinje cells in the 3xTg-AD+scFv17 group(P<0.001).4.In paraffin immunohistochemistry test,(1)the number of Aβ plaques in the cerebellar cortex of the 3xTg-AD+PBS group was significantly higher than that in WT+PBS group(P<0.001),whereas in the 3xTg-AD+scFv17 group,the number of Aβ plaques deposited in the cerebellar cortex was significantly less than that in the 3xTg-AD+PBS group(P<0.001)after chronic nasal feeding with scFv17.(2)Compared with the WT+PBS group,the 3xTg-AD+PBS group had a larger number of NFT(P<0.001)in the PCs of cerebellar vermis,while the NFT in PCs of 3xTg-AD+scFv17 group was significantly reduced when compared with 3xTg-AD+PBS group(P<0.01).(3)In the 3xTg-AD+PBS group,the BNDF expression level of the PCs layer was significantly decreased compared with that in the WT+PBS control group(P<0.001),and the PCs layer BNDF expression level in the 3xTg-AD+scFv17 group was significantly higher than that in the 3xTg-AD+PBS group(P<0.001).(4)There was no significant difference in the Trk-B expression of PCs of cerebellar vermis between the WT+PBS group and the WT+scFv17 group(P>0.05),but the Trk-B expression level of PCs was significantly decreased in the 3xTg-AD+PBS group when compared with the WT+PBS group(P<0.001),while the expression of PCs Trk-B in 3xTg-AD+scFv17 group was higher than that in 3xTg-AD+PBS group(P<0.001).(5)The relative optical density of synaptophysin-positive granules of the cerebellum vermis molecular layer in the 3xTg-AD+PBS group was significantly lower than that of the WT+PBS group(P<0.001),whereas it was significantly enhanced in the 3xTg-AD+scFv17 group(P< 0.001).Conclusion:1.The movement coordination and balance of the 12-month-old 3xTg-AD mice were severely impaired.scFv17 improved these changes obviously.2.scFv17 improved the morphological structure and reduced damages of PCs in the cerebellar vermis of 3xTg-AD mice.3.scFv17 decreased Aβ plaques and NFT in cerebellums of 3xTg-AD mice.4.scFv17 increased the expression of BDNF and its high-affinity receptor Trk-B in Purkinje Cells layer,as well as the expression of synaptophysin in the molecular layer of cerebellar vermis of 3xTg-AD mice.In summary,the ameliorative effects of scFv17 on abnormal gait might be associated with the clearance of Aβ plaques and of NFT in the cerebellar vermis cortex,which were followed by the positive effects of scFv17 against the pathological changes in cerebellum.It was suggested that scFv17 might become a new treatment strategy against gait disorder and impairment of cerebellum of AD.