Application Of Magnetic Resonance Tracking Exogenous Labeled Cells To Cell Therapy Of Alzheimer's Disease

ObjectiveAlzheimer' s disease ( AD) is a kind of neural degenerating disease which is common among the old people. In clinic, the early symptom is memory deficit, and then the progressing decline of aptitude, loss of language and spatial recognition.The most obvious pathological and histological characters of AD are extra -cellular senile plaques ( SP) and intra - neuronal neurofibrillary tangles (NET). These pathologic characters are seen in most parenchyma, but serious in neocortex, hippocampus and hypothalamus, etc. The biochemical basis of these pathological changes is the extra - cellular deposition of beta - amyloid ( AS ) and intra - cellular excess phosphorylation of Tau. At present, the pathogenic factors and pathogenesis of Alzheimer' s disease is still unclear, but most scholars think it' s relationship with many factors.The core component of SP is Aβ peptide consisting of 39 -43 amino acids. The deposition of AS may be the common route of all factors leading to AD. Studies have indicted that AS has neurotoxicity which can induce the necrosis, apoptosis and amyloid of neurons. The neurotoxicity of AS has been commonly accepted as the pivotal factor in the generation and progress of AD.According to the amyloid cascade hypothesis, aggregated Aβ in senile plaques are an early and necessary event in AD. Therefore, the inhibition of its deposition in the brain and the removing AS from the brain are important for preventing and treating AD. Recent studies have suggested that Aβ is removed mainly by non - cellular and cellular ( glial) removal pathways in vivo. The non- cellular pathways include: ( 1) extracellularly secreted peptidases, such as neprilysin and insulin - degrading enzyme (IDE) , which degrade extracellular Ap peptides;and (2) the efflux of A(3 through transport and drainage from the brain parenchyma into the circulation. Among cellular removal pathways, microglia may be useful because they have a strong ability to remove Ap deposits, although microglia display both benefit and unbenefit aspects.In brain of AD, extracellular Ap deposits are markedly accumulated and associated with microglia. This involvement of microglia has led to the suggestion that they function to phagocytose A(3 peptide and/or participate in inflammatory activation. As demonstrated by a previous report using brain unfixed sections from both AD cases and APP transgenic mice demonstrated that exogenous-ly added microglia were capable to gathered senile plaques and phagocytosed Ap peptides with addition of Ap antibody. Recently, the importance of Ap clearance has been brought to the forefront by the finding that the lightening of AD pathology and reducing of senile plaques, induced by Ap - vaccine therapy, are due to microglial phagocytosis through Fc receptors, and/or sequester plasma Ap peptides. Thus, microglia are thought to uptake Ap using Fc receptor on its membrane. It suggested that microglia can phagocytosed Ap specially. Recently, Akiyama and McGeer showed that reduction of senile plaques occurred in a cortical area affected by incomplete ischemia in a typical AD. In those areas, Ap fragments were observed in activated microglia. Thus, they suggested that microglia activated by other stimuli, such as inflammation or brain damage, had also ability to phagocytose Ap non - specifically.. These findings suggested that microglia can phagocytose and clean Ap by specific and/or nonspecific routes to have effect on AD therapy. But for cell therapy, blood - brain barrier has an obstructive action for exogenous cells to enter into the brain. Sawada et al. have recently reported that some type of microglia can pass through intact BBB. If such type of microglia can be selected, the possibility is raised that mi-croglia/macrophages might be applied for the treatment of AD.The therapeutic use of microglia has recently received some attention for the treatment of brain diseases, but for therapeutic application of microglia, it is of important to observe the location and distribution of transplanted cells non - in-vasively. In present, few non - invasive techniques exist for monitoring the cells after administration. Recent advance in magnetic resonance imaging ( MRI) give a method to it.In the present study, we present a technique using magnetic resonance imaging (MRI) to track microglia after they are injected into the animal. We labeled microglia expressing enhanced green fluorescent protein ( EGFP) with su-perparamagnetic iron oxide (SPIO) using the hemagglutinating virus of Japan envelope ( HVJ - E) vector. Several transfection agents have previously been employed to incorporate paramagnetic Fe3+ particles into cells. These have included carboxyterminated dendrimer, FuGene, Superfect, Polyfect, PLUS/lipo-fectamine, Effectene, and poly - L - lysine. Since most such agents have the same cationic charge as Fe + , their efficiency is reduced to some extent. Because the HVJ - E vector has no charge and uses membrane fusion activity to transfer SPIO into cells. The efficiency of transfection is high. The labeled microglia were injected into the left ventricle of the heart of normal mice and/or the carotid artery of the rats which had been micro - injected A(3 42 and saline into the left and right hippocampus of rats, respectively. We present to track the ex-ogenously administered microglia in the brain of animals to validate the usefulness of MR tracking for non - invasive monitoring of exogenous cells in living animals and evaluate the promising future for microglia/macrophages as therapeutic tools for brain disease.MethodsMouse microglia cell line (EOC 13. 31 ,ATCC,USA) was used in this stud-y. We transfected a construct consisting of chicken - actin promoter (CAP) and the reporter gene, enhanced green fluorescent protein ( EGFP) , into cultured microglia by using the transfection reagent, hemagglutinating virus of Japan envelope ( HVJ - E, GenomONE, Japan). After the transfection, we screened for microglia consistently expressing EGFP by adding Genetecih (500|xg/ml) into the medium.For MRI, microglia consistently expressing EGFP were further labeled withSPIO using the HVJ - E vector. Then microglia which were double labeled with EGFP and SPIO were injected into the left ventricle of normal mouse or the internal carotid artery of rat whose left hippocampus was pre - injected with A|3 42One day or two days after microglial injection, magnetic resonance images were acquired with a 7T Unity Inova magnetic resonance (MR) scanner (Vari-an, Palo Alto, USA ). During the MRI sessions, the spontaneously breathing animals were anesthetized with 1.5% isoflurane in 50% O2 and 50% N2.After obtaining MR images, sections of animal brains were stained by histo-chemistry method or observed under confocal microscopy. The results were compared with MR images.Three hippocampal sections (20|xm) for each rat were visualized using a fluorescent microscope ( Olympus DC50/IX70, Japan ). The hippocampus was divided into the square area (113,400jxm ). Fluorescent cells in the whole hippocampal region were counted in the both sides, and the data were converted into the number of cells per square mm. All data are shown as mean ± SEM. Statistical significance was assessed using Students -1 test. Significance was set at P<0.05.Some sections were incubated overnight at 4 C with mouse monoclonal antibody against A(3 (4G8, Signet Laboratory Inc. ,USA,1:1000) , followed by incubating with Cy5 - labeled secondary antibody ( Chemicon, USA, 1:100) for 3 hours at room temperature. Photographs were taken under a laser confocal microscope (LSM510 META,Germany). For controls, sections were stained with the omission of the primary antibody or using the A antibody pre - absorbed with 20|xg /ml of Ap peptide.Some sections were reacted with peroxidase labeled anti mouse IgG complex (Histofine, Nichirei, Japan, 1:20 ) after incubating overnight with 4G8 ( 1: 1000). A brown color was developed with 0.02% 3, 3' - diaminobenzidine in 50mM Tris - HC1 buffer (pH 7.6). The sections were then incubated for 30 minutes with 1% potassium ferrocyanide in 1% HC1 at room temperature, to stain iron particles a blue color (Prussian blue stain).For double staining of iron particles and endothelium of mouse brain ves-sels, sections were stained with Prussian blue and then were incubated overnight at 4 X. with a rat monoclonal anti - CD34 antibody (ljxg/mL,BO Biosciences Pharmingen, USA). After washing with 0. 1M phosphate buffered saline containing 0. 3% Triton - X100 ( PBST,pH 7. 4) , sections were incubated for 4 h with Alexa594 anti - rat IgG ( 1:500, Molecular Probe, USA ). After washing with PBST, sections were observed under fluorescent microscopy (Olympus, Japan).Results1. MRI detected exogenously administered microglia migrating into the brain of normal mice after SPIO - containing microglia were injected intra - cardially. MRI revealed some dark points in mouse brain.2. In mouse brain, histochemistry staining of SPIO -containing microgia located at the same positions as which were showed by MRI.3. Most of SPIO - containing microglia were in parenchyma of mouse brain. No embolism of SPIO - containing microglia was seen in vessels.4. MRI revealed clear signal changes attributable to SPIO - containing microglia aggregation in AS - injected areas of living rat' s brain.5. Histochemical examination demonstrated that intra - arterially injected microglia positive for EGFP and SPIO had accumulated at sites of AS deposition in the rat hippocampus. Few exogenous mocroglia wvt, seen at sites of saline injection.6. Counted under fluoresent microscopy, the number of EGFP - positive cells at sites of AS injection was much higher than that of saline injection.7. Some exogenous microglia phagocytosed distributed AS deposits. AS signals were detected in these microglia.Conclusion1. Intra - cardially injected exogenous microglia can pass through the intact blood - brain barrier and enter into the parenchyma of normal mouse brain.? 10-2. Intra - arterially injected exogenous microglia accumulate around A(3 deposits in the hippocampus of rat and take some distributed A{3.3. MRI can be used as a non - invasive means of detecting transplanted microglia, with the potential for future clinical application in cell therapy of AD.