The Studies Of The Magnetic Contrast Agent For MRI Diagnosis, Medicine And Cell Therapy Of Alzheimer’s Disease

BackgroundsAlzheimer’s disease (AD) is a neurodegenerative disease with the feature of chronicprogressive dementia and is one of the most common causes for senile dementia. Themorbidity increases with age and great financial load is brought to society and families.However, the diagnosis and therapy of AD remain limited. This study is to find the possiblemethod and direction through discussing the intense research in AD. In terms of diagnosis,magnetic resonance imaging (MRI) is a promising technique in detection of Aβ deposits inAD. It has been proved that using the MRI contrast agent can shorten the scanning time andprovide a specific image to identify various structures. In terms of therapy, the protection ofvascular function may be the new target in the early stage of AD and the celltransplantation may be the development direction to repair the neuron loss in the late stageof AD in the future.Objectives1. To develop an ultrasmall superparamagnetic iron oxide (USPIO) nanoparticlescoupled with functional protein as a novel MRI contrast agent to detect the Aβ deposits.2. To find whether s-(-)-3-n-butylphthalide, which can protect the vascule and is usedfor ischemia clinically, could be applied for AD through the observation of the effects onthe Aβ-damaged cerebral microvascular endothelial cells (CMECs). 3. Neural stem cells (NSCs) are the applied form in cell transplantation for diseases ofcentral nervous system. The increase of neuronal differentiation rate of NSCs remains ablock, particular in a damage environment. The study is to see the effects of CMECs on theneurogenesis of NSCs with or without Oxygen-glucose deprivation (OGD).Methods1. We developed a novel MRI contrast agent USPIO nanoparticles coupledAβ(16-20)(KLVFF) as a probe for connecting to Aβ deposits and HIV-1trans-activatingtranscriptor-protein transduction domain (Tat-PTD) for the penetration of blood-brainbarrier (BBB). The penetration, toxicity and binding affinity of the contrast agent in vitrohave been studied. The contrast agents were injected into the transgenic mice for livingbrain MRI imaging and histological studies.2. Establish the method for primary culture of CMECs. After detecting the specificityof CMECs, they were pre-treated with s-(-)-3-n-butylphthalide on different concentrationsand then were damaged by Aβ. The cell viability and apoptosis rates were observed and theconcentrations of NO, inducible nitric oxide synthase (iNOS) and endothelial nitric oxidesynthase (eNOS) were detected.3. Survival and proliferation of NSCs were studied after CMECs and NSCs wereco-cultured. Removing the CMECs, NSCs were maintained in differentiation culture withor without treatment of OGD to observe the differentiation rates. Mouse embryo fibroblast(MEF) cells co-cultured with NSCs were used as the control groups.Results1. The coupling efficiency of protein can reach97.75%for Tat-PTD-USPIO-Aβ(16-20).The developed MRI contrast agents can pass through biological membranes, bind to the Aβdeposits and decrease the MRI signal of the tissue in vitro and in vivo. Compare toAβ(16-20) or Aβ(1-40) alone, no significant toxicity of Tat-PTD and Aβ(16-20) was foundwhen they coupled with USPIO nanoparticles.2. Successfully establish the method of primary culture of CMECs from newborn mice and the positive rate was95.5%. In the intervention research of s-(-)-3-n-butylphthalide, thecell viability increased and the apoptosis rates decreased. The secretion of NO and iNOSwere increased. There were no significantly changes of the secretion of eNOS in all groups.3. NSCs co-cultured with CMECs have an increase in size and number compared tothose co-cultured with MEF cells. They also have an increase in neuronal differentiationrates with or without OGD.Conclusions1. The novel developed MRI contrast agent by USPIO nanoparticles coupled withfunctional protein can pass through BBB and detect Aβ deposits in AD animal models byMRI in vivo. This novel MRI contrast agent may help in the evaluation and development ofpotential novel therapeutic interventions to reduce amyloid deposits in AD experimentalanimals.2. The s-(-)-3-n-butylphthalide could protect CMECs from the damage of Aβ throughincreasing the expression and secretion of iNOS, in turn the NO. The effect was morestabilized under the large concentration. Hence, the s-(-)-3-n-butylphthalide may be acandidate for the therapy of AD through the effect on the protection of vascule.3. This study provides the evidence that OGD cannot alter the effects of CMECs inpromoting the neuronal differentiation potential of NSCs. These findings may haveimportant implications for the development of new cell therapies for the diseases of centralnervous system.Innovation points1. A novel MRI nano-contrast agent was developed and may contribute to the diagnosisof AD and also help in the evaluation and development of potential novel therapeuticinterventions.2. Established an economical, simple and repetitive primary culture with high positiverate for CMECs.3. According to the recent opinion of AD, the drug used for ischemia clinically was discussed for the application of AD and that may shorten the development time for new ADdrugs.3. In the cell transplantation, the study of the neuronal differentiation rate of NSCs in adamage environment is more significant to the transplantation therapy.