Novel microdialysis method to assess neuropeptides and large molecules in free-moving mouse. Neuroscience

Department of Clinical Gene Therapy, Osaka University, Yamadaoka, Osaka 565-0871, Japan.
Neuroscience (Impact Factor: 3.36). 07/2011; 186:110-9. DOI: 10.1016/j.neuroscience.2011.04.035
Source: PubMed


Microdialysis is a powerful in vivo technique for the continuous sampling of small molecules within the extracellular fluid space. However, efforts to collect larger molecules have met with little success. To identify biologically active larger molecules in free-moving animals would be of great benefit. For this purpose, we have developed a novel microdialysis method that allows consistent recovery of large molecules from the brain interstitial space in the awake, free-moving mouse. Using a new "vent" probe with a push-pull perfusion system, the present study successfully demonstrated in vivo sampling of pathophysiologically important macromolecules in free-moving mouse brain. This sampling system allowed monitoring of the dynamic changes in their concentrations. Overall, this novel microdialysis system would provide the opportunity to identify the expression patterns of pathophysiologically important proteins in a variety of physiological and pathological processes for a better understanding of various diseases.

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    • "To measure the levels of soluble Aβ1–42 in the hippocampus ISF of awake, freely moving 5XFAD mice, microdialysis was performed as previously described (Takeda et al., 2011) with some modifications. Probe implantation was performed as previously described (Cirrito et al., 2003). "
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    ABSTRACT: The purpose of this study was to evaluate the therapeutic potential of human induced pluripotent stem (iPS) cell-derived macrophage-like cells for Alzheimer’s disease (AD). In previous studies, we established the technology to generate macrophage-like myeloid lineage cells with proliferating capacity from human iPS cells, and we designated the cells iPS-ML. iPS-ML reduced the level of Aβ added into the culture medium, and the culture supernatant of iPS-ML alleviated the neurotoxicity of Aβ. We generated iPS-ML expressing the Fc-receptor-fused form of a single chain antibody specific to Aβ. In addition, we made iPS-ML expressing Neprilysin-2 (NEP2), which is a protease with Aβ-degrading activity. In vitro, expression of NEP2 but not anti-Aβ scFv enhanced the effect to reduce the level of soluble Aβ oligomer in the culture medium and to alleviate the neurotoxicity of Aβ. To analyze the effect of iPS-ML expressing NEP2 (iPS-ML/NEP2) in vivo, we intracerebrally administered the iPS-ML/NEP2 to 5XFAD mice, which is a mouse model of AD. We observed significant reduction in the level of Aβ in the brain interstitial fluid following administration of iPS-ML/NEP2. These results suggested that iPS-ML/NEP2 may be a potential therapeutic agent in the treatment of AD.
    Full-text · Article · Oct 2014 · Stem Cell Research
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    • "However, the comparatively large size of lipidated apoE particles (~300 kDa) has impeded the utility of microdialysis to similarly assess apoE metabolism within the brain. A recent report demonstrated that a high molecular weight cut-off (MWCO) microdialysis probe was capable of collecting small (<30 kDa) proteins from the brain ISF[15]. We hypothesized that such a probe might be useful for collecting lipidated lipoproteins. "
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    ABSTRACT: Background The APOE4 allele variant is the strongest known genetic risk factor for developing late-onset Alzheimer’s disease. The link between apolipoprotein E (apoE) and Alzheimer’s disease is likely due in large part to the impact of apoE on the metabolism of amyloid β (Aβ) within the brain. Manipulation of apoE levels and lipidation within the brain has been proposed as a therapeutic target for the treatment of Alzheimer’s disease. However, we know little about the dynamic regulation of apoE levels and lipidation within the central nervous system. We have developed an assay to measure apoE levels in the brain interstitial fluid of awake and freely moving mice using large molecular weight cut-off microdialysis probes. Results We were able to recover apoE using microdialysis from human cerebrospinal fluid (CSF) in vitro and mouse brain parenchyma in vivo. Microdialysis probes were inserted into the hippocampus of wild-type mice and interstitial fluid was collected for 36 hours. Levels of apoE within the microdialysis samples were determined by ELISA. The levels of apoE were found to be relatively stable over 36 hours. No apoE was detected in microdialysis samples from apoE KO mice. Administration of the RXR agonist bexarotene increased ISF apoE levels while ISF Aβ levels were decreased. Extrapolation to zero-flow analysis allowed us to determine the absolute recoverable concentration of apoE3 in the brain ISF of apoE3 KI mice. Furthermore, analysis of microdialysis samples by non-denaturing gel electrophoresis determined lipidated apoE particles in microdialysis samples were consistent in size with apoE particles from CSF. Finally, we found that the concentration of apoE in the brain ISF was dependent upon apoE isoform in human apoE KI mice, following the pattern apoE2>apoE3>apoE4. Conclusions We are able to collect lipidated apoE from the brain of awake and freely moving mice and monitor apoE levels over the course of several hours from a single mouse. Our technique enables assessment of brain apoE dynamics under physiological and pathophysiological conditions and in response to therapeutic interventions designed to affect apoE levels and lipidation within the brain.
    Full-text · Article · Apr 2013 · Molecular Neurodegeneration
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    • "In the majority of hitherto performed studies one or a few small biochemical substances have been exclusively in focus. There are a few attempts to analyze larger molecules, e.g., proteins, with various results [7]–[10]. Proteins can act as signal substances, activate the formation of algesics and control nociceptive processes. "
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    ABSTRACT: Microdialysis (MD) of the trapezius muscle has been an attractive technique to investigating small molecules and metabolites in chronic musculoskeletal pain in human. Large biomolecules such as proteins also cross the dialysis membrane of the catheters. In this study we have applied in vivo MD in combination with two dimensional gel electrophoresis (2-DE) and mass spectrometry to identify proteins in the extracellular fluid of the trapezius muscle. Dialysate from women with chronic trapezius myalgia (TM; n = 37), women with chronic wide spread pain (CWP; n = 18) and healthy controls (CON; n = 22) was collected from the trapezius muscle using a catheter with a cut-off point of 100 kDa. Proteins were separated by two-dimensional gel electrophoresis and visualized by silver staining. Detected proteins were identified by nano liquid chromatography in combination with tandem mass spectrometry. Ninety-seven protein spots were identified from the interstitial fluid of the trapezius muscle; 48 proteins in TM and 30 proteins in CWP had concentrations at least two-fold higher or lower than in CON. The identified proteins pertain to several functional classes, e.g., proteins involved in inflammatory responses. Several of the identified proteins are known to be involved in processes of pain such as: creatine kinase, nerve growth factor, carbonic anhydrase, myoglobin, fatty acid binding protein and actin aortic smooth muscle. In this study, by using in vivo microdialysis in combination with proteomics a large number of proteins in muscle interstitium have been identified. Several of the identified proteins were at least two-fold higher or lower in chronic pain patients. The applied techniques open up for the possibility of investigating protein changes associated with nociceptive processes of chronic myalgia.
    Full-text · Article · Dec 2012 · PLoS ONE
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