In Vivo Amyloid-beta Clearance Assay

In vivo Aβ₄₀ clearance was investigated using the
BCI method as described previously.^{36 (link)} Animals
were anesthetized followed by the insertion of a stainless steel guide
cannula into the right caudate nucleus of mice brains. A tracer fluid
(0.5 μL) containing ¹²⁵I-Aβ₄₀ (30
nM, PerkinElmer, MA) and ¹⁴C-inulin (0.02 mCi, American
Radiolabeled Chemicals, MO) prepared in extracellular fluid buffer
(ECF) was microinjected. Thirty minutes later, brains were rapidly
collected. One hemisphere of the brain was used for ¹²⁵I-Aβ₄₀ analysis and the second hemisphere was used
for microvessels isolation as described below. Calculations of ¹²⁵I-Aβ₄₀ clearance were performed as described
previously.^{36 (link)} Using trichloroacetic acid
(TCA) precipitation intact (precipitate) and degraded (supernatant) ¹²⁵I-Aβ₄₀ were determined in brain tissue
using a Wallac 1470 Wizard Gamma Counter (PerkinElmer, MA). ¹⁴C-Inulin in the precipitate and supernatant were also determined
using a Wallac 1414 WinSpectral Counter (PerkinElmer). The ¹²⁵I-Aβ₄₀ brain clearance index (BCI_total(%)) and clearance of ¹²⁵I-Aβ₄₀ across
BBB (BCI_BBB(%)) were determined as described previously.^{36 (link)}