Techniques
Microdialysis
Dr. Cirrito pioneered the microdialysis technology to longitudinally measure protein within the brain extracellular fluid, or interstitial fluid (ISF) in awake and freely-moving mice (Cirrito et al. 2003). For example, in vivo microdialysis measures brain ISF Aβ every 30 minutes for up to 3 days in a single mouse. This enables us to manipulate a multitude of factors in a mouse, such as synaptic activity, signaling pathways or behaviors, to determine how each alters the amount of Aβ, but also the kinetics/rates of those changes over time. Only nine other groups in the world perform Aβ microdialysis; seven of which visited Washington University to learn the technique directly from our group. We have used microdialysis to assess the effect of various genes, proteins, drugs, and behaviors on Aβ metabolism (Cirrito et al., 2003; DeMattos et al. 2004; Cirrito et al., 2005; Kang et al. 2009)
We recently developed a new “large-pore microdialysis technique” that measures large proteins within the brain extracellular space. Microdialysis requires that a molecule diffuse through a semi-permeable membrane on the probe in order to be collected and measured; this typically limits recovery to only small molecules and peptides. Our standard probes contain a 38 kiloDalton (kDa) molecular weight cut-off (MWCO) membrane whereas our newer probes utilize a 1,000 kDa MWCO membrane (or a MegaDalton MWCO, hence why we call the probes “MegaProbes”). The new probes recovery large proteins from the ISF such as tau, α-synuclein, soluble APP fragments, antibodies, and apolipoprotein E among others.Using an approach called “reverse microdialysis,” we can also add a drug to the microdialysis probe perfusion buffer in order to locally deliver the compound directly to the brain at the same time as we measure Aβ, thereby bypassing the blood-brain barrier.
In some case we attached platinum-iridium wires to the microdialysis probe to record electrical activity (local field potentials or depth EEG) in the brain at the same time and place as we measure proteins.
Micro-immunoelectrode
We recently developed a novel micro-immunoelectrode (MIE) technology to measure ISF Aβ levels every 60 seconds over time (described in detail below in Fig) (Prabhulkar et al., 2012; Yuede et al., 2016). MIEs utilize amperometry, an electrochemical method that measures the oxidation of molecules. The general approach has been used for decade s to measure the concentration and dynamics of small molecules, such as dopamine, in animal models using a small carbon fiber microelectrode implanted into the brain. Drs. Cirrito and Yuede collaborated with Dr. Chenzhong Li at Florida International University in Miami to adapt the approach to measure proteins by attaching antibodies to the electrode surface to concentrate specific targets at the tip. Several national security biohazard detectors, such as for anthrax, utilize amperometry in combination with antibodies to make chip-based sensors specific for a protein. Those sensors are typically biased for ultra-high sensitivity instead of detecting a range of concentrations (for national security it generally does not matter how much anthrax is present; even a little anthrax is bad!). The MIEs use a similar amperometric, antibody-based approach to measure Aβ, however our sensors can 1) quantify a wide range of concentrations, 2) take longitudinal measurements, and3) are only 10µm diameter and 40µm long so they are implantable directly into brain. A drawback to MIEs is that they only last for 3 hours, which means they complement the microdialysis technology, but cannot replace it based on longevity.
(A) Anti-Aβ antibodies are covalently-attached to the carbon fiber electrode surface, and then the unbound space is blocked with BSA. MIEs are then inserted into living acute brain slices or implanted into the hippocampus of APP/PS1 mice. An electrode held at 0.65V induces oxidation of tyrosine amino acids. Oxidation releases an electron that the MIE detects as current. The inclusion of an anti-Aβ antibody is a critical factor that determines specificity of the electrode. (B) Scanning electron microscopy (SEM) image of carbon fiber pulled within a glass capillary tube.