Multiple High-resolution Microscopic Imaging Applied On Alzheimer's Disease Mice Treated With Vanadium Compounds

With the development of biomedical photonics,high-resolution omnidirectional imaging and real-time in-vivo detection technology have gradually been applied to biological samples to three-dimensionally view their fine structure changes and dynamically track the pathological development of mice in vivo or under the treatment of drugs.With the combination of tissue optical transparency technology and microscopic optical imaging technology,highly transparent biological tissues were obtained that were available for getting deep-level,multiple-angle,high-resolution and three-dimensional images of intact samples or thier fine structures.Two-photon microscopic imaging technology can dynamically track the altered images of neurons in the brain of living animals.The 18F-FDG-PET dynamic scanning can monitor glucose uptake and metabolism in organs of living animals.Alzheimer's disease(AD)is a neurodegenerative disease commonly occurred in the elderly.The pathological features of AD are mainly characterized by the massive deposition of ?-amyloid(A?)in the brain to form senile plaque(SP),and the hyperphosphorylation of tau protein to result in neurofibrillary tangles(NFTs),the death of neurons and the loss of synapses.The main clinical symptoms of AD patients are memory loss,cognitive dysfunction,weak judgment and comprehension,and low self-care ability.With the increasing number of aging populations in the world,the number of AD patients is also increasing.However,there has been a lack of safe and effective therapeutic drugs for the prevention of AD and the treatment of clinical drugs.Therefore,the prevention of AD and the treatment of drug research is a hot topic in this field.Vanadium,as a necessary trace element for the human body,mainly exists in brain,liver,bone,myocardium and other organs,which plays an extremely important role in maintaining the normal function of the immunesystem.Bis(ethylmaltolato)oxidovanadium(IV)(BEOV)has been reported to have an insulin-like hypoglycemic effect in rodent models.However,whether it has any effect on AD has not been reported yet.Methods: In this study,triple transgenic AD model mice(3×Tg-AD)were mated with fluorescent mouse YFP to obtain AD-YFP mice with both the AD pathogenic gene and the YFP fluorescent protein gene and raised to 6 months of age.After treatment with 0.2 mol/L and 1.0 mol/L of BEOV for 32 weeks,the brains of AD-YFP mice were transparentized using tissue optical transparency technology and further using optical projection tomography and laser confocal microscopy to get the 3D stereo imaging of mouse brain neurons,A? plaques and astrocytes.Meanwhile,2.5-month-old AD-YFP mice were selected,and the cranial window technique was used in combination with two-photon microscopic live imaging to dynamically observe the dynamic changes of dendritic spines in brain neurons of living mice.In this study,6-month-old APP/PS1 and 3×Tg-AD mice were treated with BEOV at concentrations of 0.2mol/L and 1.0 mol/L for 13 weeks,respectively.Transmission electron microscope was used to observe the morphological structure of synapses.18F-FDG-PET scanning also was used to detect the glucose uptake and metabolism in mouse brain.The behavioral test(Morris water maze,open field test,step-down avoidance test)to study the effects of BEOV on learning cognitive and memory abilities in mice.Western blot,immunofluorescence staining,and other biochemical analysis method were used to study the effects of BEOV on neuroinflamation,A? cascade,Tauopathy pathway,endoplasmic reticulum stress,and insulin signalling pathways.Results: Our study combinates tissue optical transparency technology and optical imaging technology to detect the effect of BEOV on AD with high resolution and all-round detection.,and combinates open skull technology and two-photon in vivo microscopic imaging technology to monitor the effect of BEOV on AD in real time.On this basis,the molecular mechanism of the effectof BEOV on AD was further investigated.1)Establishing a variety of new optical imaging technologies and find that BEOV has a significant therapeutic effect on AD.Tissue optical transparency technology was used to prepare the transparent mouse brain,and optical projection tomography and laser confocal microscopy were used to perform three-dimensional imaging of YFP-AD fluorescent mouse brain slices.The imaging results showed that A? plaques in the hippocampus and cortex of AD-YFP mice were significantly increased,the number of neurons was reduced,and neuroinflammation-related astrocytes were increased when compared with YFP mice.Transparent brain imaging in YFP-AD fluorescence mice showed that A? plaques in the brain of mice were significantly reduced,neuron loss were also inhibited and inflammation levels were reduced after BEOV treatment.Furthermore,the changes of dendritic spines in the brain of 2.5 month old AD-YFP mice treated or untreated with BEOV were dynamically observed by using craniotomy under a two-photon microscope.After BEOV treatment,the loss of dendritic spines were inhibited in the mice brains.GCa MP6 virus was injected into the cortex of 3×Tg-AD mice to monitor calcium signal activity,with stereotaxic coordinates 0-2mm anterior and 0-2mm lateral to bregma.It was found that BEOV can effectively regulate calcium signal in neurons of AD mice.Meanwhile,transmission electron microscopy was used to detect the number of synapses in the brain of AD mice,and it was found that BEOV also significantly prevented the damage and reduction of synapse.2)Confirming the molecular mechanism of BEOV in prevention and treatment of AD.After 13 weeks of BEOV treatment,the behavioral test experiments of APP/PS1 and 3×Tg-AD AD model mice showed that BEOV significantly improved the learning and memory ability and exploration ability of mice.18F-FDG-PET scan showed that BEOV significantly increased the glucose uptake and metabolic level in the brain of AD mice.BEOV can significantly reduce A? plaques in the hippocampus and cortex of AD mice,A?levels in primary neurons,and the expression levels of key proteins including APP,s APP?,BACE1 in A? pathway.Meanwhile it can increase the expression levels of proteins related to A? metabolism,such as PPAR? gamma and IDE.BEOV can also significantly inhibit the activity of PTP1 B and the phosphorylation level of tau,increase the activity of IR and AKT,and reduce the activity of GSK3?.Molecular mechanism research shows that BEOV has multiple targets for AD.1)By activating PPAR?,it can inhibit BACE1 expression,promote IDE expression,and reduce A? aggregation.2)By inhibiting the expression of PTP1 B,it can inhibit JAK2/STAT3/SOCS1 pathway,activate insulin signaling,regulate AKT/GSK3? signaling pathway,and reduce the excessive phosphorylation of tau.3)By activating PPAR?,it inhibits the endoplasmic reticulum stress caused by A? in the hippocampus and cortex of AD mice and reduces neuronal apoptosis.4)BEOV can also significantly inhibit A? cascade reaction and plaque formation in the olfactory bulb of AD mice,inhibit Tau hyperphosphorylation pathway and nerve fiber tangles,and improve the function of olfactory bulb in mice.Conclusions: This study successfully established a variety of AD mouse brain in vitro and in vivo optical imaging technology,and applied it to study the efficacy of BEOV.For the first time,multi-angle omni-directional high resolution imaging recorded the inhibition of A? plaque and the protection of neurons by BEOV in the pathological process of AD.For the first time,it was observed that BEOV protect the dendritic spines of neurons in the cerebral cortex of AD mice and regulate the calcium signal in vivo.The process of improving brain glucose metabolism by BEOV was also recorded in real time.On this basis,the present study further revealed the multi-target molecular mechanism of BEOV in the prevention and treatment of AD.For the first time,it proved that BEOV can reduce endoplasmic reticulum stress caused by A?through activating PPAR?,reduce the number of apoptotic neurons,and slow down the pathological process of AD.It was revealed that by inhibitingJAK2/STAT3/SOCS1 signaling pathways and improving insulin resistance in the brain,BEOV can inhibit PTP1 B expression,increase the sensitivity of insulin receptor,regulate the AKT/GSK3? signaling pathways,reduce the excessive phosphorylated tau protein,thus inhibiting various pathological changes in the hippocampal cortex and olfactory bulb of AD mice,and improving the function of learning and memory ability and the olfactory bulb in AD mice.The above results provide a solid theoretical foundation and experimental basis for BEOV to become a multi-target effective drug for the prevention and treatment of AD.