Voluntary Exercise Promotes Glymphatic Clearance of Amyloid Beta and Reduces the Activation of Astrocytes and Microglia in Aged Mice methods

[
  {
    "@context": "https://schema.org",
    "@type": "HowTo",
    "name": "Voluntary Exercise Promotes Glymphatic Clearance of Amyloid Beta and Reduces the Activation of Astrocytes and Microglia in Aged Mice methods",
    "description": "Evidence-backed execution summary for Voluntary Exercise Promotes Glymphatic Clearance of Amyloid Beta and Reduces the Activation of Astrocytes and Microglia in Aged Mice methods from Voluntary Exercise Promotes Glymphatic Clearance of Amyloid Beta and Reduces the Activation of Astrocytes and Microglia in Aged Mice.",
    "totalTime": "PT14535M",
    "step": [
      {
        "@type": "HowToStep",
        "position": 1,
        "name": "Materials and Methods",
        "text": "This study was approved by the Animal Research Committee of the First Affiliated Hospital of Sun Yat-sen University (Guangzhou, China; committee's reference number: [2013]97). All efforts were made to minimize the number and suffering of animals used in this study. Twenty-four male C57BL/6J mice obtained from the Animal Center of Sun Yat-sen University and 12 Thy1-GFP transgenic mice obtained from the Model Animal Research Center of Nanjing University (stock number: 003782) were used. All the animals were used at 14-16 months of age, and were housed under a 12:12 h light-dark cycle (light from 07:00 to 19:00), with controlled temperature and humidity, and given food and water ad libitum."
      },
      {
        "@type": "HowToStep",
        "position": 2,
        "name": "Voluntary Wheel Training",
        "text": "The animals were randomly divided into two groups, the control group and the physical training (running) group, and each group included 12 C57BL/6J and six Thy1 - GFP male mice. In the control group, the mice were housed in polypropylene cages (36 cm L × 20 cm W × 14 cm H). The mice in the training group were housed in polypropylene cages of the same size, with a 16 cm diameter running wheel, which rotated when a mouse climbed onto the wheel voluntarily. The mice were housed under these conditions for 6 weeks (Littlefield et al., )."
      },
      {
        "@type": "HowToStep",
        "position": 3,
        "name": "Morris Water Maze",
        "text": "The 12 C57BL/6J mice in each group performed the water-maze task, according to the protocol of van Praag et al. ( ) and Akers et al. ( ). The maze consisted of a circular tub (120 cm in diameter, 50 cm in height) with a white circular platform (10 cm). The tub was surrounded by a curtain located about 1 m from the tub wall, which was painted with distinct geometric cues. The water (24 ± 1°C) was made opaque with white tempera paint to conceal the platform. Over five consecutive days, the platform was submerged 1 cm beneath the surface of the water in the center of one quadrant of the pool. Twenty-four mice underwent four trials (up to 60 s each) per day, starting from each of four different locations in the pool. The animals that failed to locate the platform within the allotted 60 s were gently guided to the platform. All the mice remained on the platform for 10 s at the en..."
      },
      {
        "@type": "HowToStep",
        "position": 4,
        "name": "In Vivo Two-Photon Imaging of Glymphatic Pathway Clearance",
        "text": "For two-photon imaging, the mice were anesthetized with chloral hydrate (4.2%, 0.01 ml/g), and placed securely in a stereotaxic device, with the skull level between the bregma and lambda. The stereotaxic coordinates of the right parietal cortex were 2 mm lateral to the midline and 1.7 mm ahead of the lambdoid (Harvey et al., ). A thin cranial window (3 mm in diameter; Golshani and Portera-Cailliau, ) was constructed for in vivo two-photon imaging."
      },
      {
        "@type": "HowToStep",
        "position": 5,
        "name": "In Vivo Two-Photon Imaging of Glymphatic Pathway Clearance",
        "text": "To evaluate the PVS-ISF exchange, fluorescein isothiocyanate (FITC; 70 kDa; Sigma-Aldrich, Darmstadt, Germany) was dissolved in artificial CSF (ACSF) at a concentration of 1%. A microsyringe (BASi, West Lafayette, IN, USA) was inserted into the cisterna magna, and 10 µl of FITC-dextran was injected within a period of 10 min. To visualize the vasculature, 0.2 ml of rhodamine B (RD; 1% in saline; Sigma) was injected intravenously immediately before imaging."
      },
      {
        "@type": "HowToStep",
        "position": 6,
        "name": "In Vivo Two-Photon Imaging of Glymphatic Pathway Clearance",
        "text": "Two-photon imaging was performed with a custom-built two-photon laser scanning microscope (Leica) at a wavelength of 800 nm and a laser scanning system (Coherent, Santa Clara, CA, USA) equipped with a water immersion objective (25 × ). To monitor the clearance of ovalbumin injected into the brain parenchyma, lateral images of the xyz stacks (512 × 512 pixels, 2 µm resolution) were taken up to 300 µm below the cortical surface. The stacks were collected at 5, 15, 30, 45 and 60 min after the injection of the dye."
      },
      {
        "@type": "HowToStep",
        "position": 7,
        "name": "Analysis of BBB Permeability",
        "text": "As described previously, RD (1% in saline) was injected intravenously to visualize the brain vasculature while evaluating the PVS-ISF exchange. Fluorescent intravascular dyes were used to detect leakage from the vasculature (Burgess et al.,; He et al., ). Therefore, we selected the red fluorescence channel for detection and analyzed the total fluorescence intensity in the extravascular compartment (Nhan et al., ). Images were collected at 0, 5, 15, 30 and 60 min after the injection of fluorescent dextran, and were shown as both three-dimensional (3D) stacks and xyz stacks."
      },
      {
        "@type": "HowToStep",
        "position": 8,
        "name": "Histology",
        "text": "For the histological analysis, the dendrites and dendritic spines of 12 Thy1-GFP mice ( n = 6 per group) were detected, and 12 C57BL/6J mice ( n = 6 per group) were used for other histological analyses ( n = 6 per group), including enzyme-linked immunosorbent assays (ELISAs) that detected Aβ1-40 and Aβ1-42. The mice were perfused with 50 ml of ice-cold saline, followed by 50 ml of 4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS; pH 7.4). Their brains were removed and incubated overnight in 4% paraformaldehyde, and then dehydrated in 20%-30% sucrose in PBS. Coronal brain slices (40 µm or 10 µm thick) from the right parietal cortex were sectioned with a frozen microtome (Leica) at intervals of 100 µm to produce consecutive frozen sections. For immunofluorescent staining, the sections were boiled in citric acid buffer (pH 6.0)..."
      }
    ],
    "tool": [
      {
        "@type": "HowToTool",
        "name": "In Vivo Two-Photon Imaging of Glymphatic Pathway Clearance"
      },
      {
        "@type": "HowToTool",
        "name": "Morris Water Maze"
      },
      {
        "@type": "HowToTool",
        "name": "In Vivo Two-Photon Imaging of Glymphatic Pathway Clearance"
      },
      {
        "@type": "HowToTool",
        "name": "In Vivo Two-Photon Imaging of Glymphatic Pathway Clearance"
      },
      {
        "@type": "HowToTool",
        "name": "In Vivo Two-Photon Imaging of Glymphatic Pathway Clearance"
      },
      {
        "@type": "HowToTool",
        "name": "In Vivo Two-Photon Imaging of Glymphatic Pathway Clearance"
      },
      {
        "@type": "HowToTool",
        "name": "Histology"
      },
      {
        "@type": "HowToTool",
        "name": "Histology"
      }
    ],
    "supply": [
      {
        "@type": "HowToSupply",
        "name": "Histology"
      },
      {
        "@type": "HowToSupply",
        "name": "ELISAs"
      },
      {
        "@type": "HowToSupply",
        "name": "Amyloid Plaque Deposition"
      }
    ],
    "isBasedOn": {
      "@type": "ScholarlyArticle",
      "headline": "Voluntary Exercise Promotes Glymphatic Clearance of Amyloid Beta and Reduces the Activation of Astrocytes and Microglia in Aged Mice",
      "datePublished": "2017",
      "author": [
        {
          "@type": "Person",
          "name": "Xiao-fei He"
        },
        {
          "@type": "Person",
          "name": "Dong-xu Liu"
        },
        {
          "@type": "Person",
          "name": "Qun Zhang"
        },
        {
          "@type": "Person",
          "name": "Feng-ying Liang"
        },
        {
          "@type": "Person",
          "name": "Guang-yan Dai"
        },
        {
          "@type": "Person",
          "name": "Jin-sheng Zeng"
        },
        {
          "@type": "Person",
          "name": "Zhong Pei"
        },
        {
          "@type": "Person",
          "name": "Guang-qing Xu"
        },
        {
          "@type": "Person",
          "name": "Yue Lan"
        }
      ],
      "identifier": "10.3389/fnmol.2017.00144"
    }
  },
  {
    "@context": "https://schema.org",
    "@type": "BreadcrumbList",
    "itemListElement": [
      {
        "@type": "ListItem",
        "position": 1,
        "name": "Experiments",
        "item": "https://replicatescience.com/experiments"
      },
      {
        "@type": "ListItem",
        "position": 2,
        "name": "Voluntary Exercise Promotes Glymphatic Clearance of Amyloid Beta and Reduces the Activation of Astrocytes and Microglia in Aged Mice methods",
        "item": "https://replicatescience.com/experiments/voluntary-exercise-promotes-glymphatic-clearance-of-amyloid-beta-and-reduces-the-activation-of-astrocytes-and-microglia-in-aged-mice-methods-xiao-fei-he-pmc5437122/voluntary-exercise-promotes-glymphatic-clearance-of-amyloid-beta-and-reduces-the-activation-of-astro-mlph8ayn"
      }
    ]
  }
]