What is Microdialysis?

Microdialysis is a well-established technique for sampling extracellular space in vivo over a period of time.
It is ideal for generating time-resolved data for local concentrations of multiple neurotransmitters, neuromodulators, or proteins in behaving animals.

Amuza has been working with microdialysis for decades, both supporting Eicom products and manufacturing our own. Unique among microdialysis companies, Amuza can create your entire microdialysis system, from microdialysis probes and pumps to automated HPLC systems for analysis. It’s our goal to streamline and automate your projects so that you can quickly generate the data you need without time lost to troubleshooting.

Primary benefits of Microdialysis:

• A sampling of molecules from the extracellular space in brain tissue.
• Allows changes in analyte concentration to be monitored over time.
• Subject animals can behave normally.

Recovery

Percent recovery depends on optimizing several conditions for the analysis of specific molecules. For example, if you want to analyze neuropeptides from a section of the brain that is 3mm in diameter, then the membrane on the microdialysis probe needs to have a large pore size and the length of the membrane should be of similar diameter. Other factors, such as flow rate, perfusion liquid choice, membrane length, membrane type, tube length, back pressure, etc., also need to be optimized. Further discussed here.

Neurochemical Analysis

Microdialysis samples are referred to as microdialysate or just dialysate. The microdialysate is collected either manually or by the preferred method, a fraction collector. The sample is prepared for analysis by various detectors, including but not limited to UV, ECD, and mass spec. More details are below.

In-Depth Explanation of Microdialysis

A microdialysis probe is a small “Y” shaped catheter containing an inlet and outlet port with a fibrous, semi-permeable membrane at the bottom tip. The microdialysis probe is gently inserted through a previously surgically implanted guide cannula to allow the membrane to rest in the desired brain tissue. Perfusate mimicking the extracellular fluid (Ringer’s solution or artificial cerebrospinal fluid, aCSF), is pumped through the inlet side of the probe. Solutes in the extracellular fluid, such as neurotransmitters (serotonin, dopamine, norepinephrine, acetylcholine, GABA, histamine, glutamate, glycine, and metabolites), cytokines, proteins, and neuropeptides, passively diffuse through the membrane and are carried by perfusate through the outlet port. Samples are collected at specific time intervals for neurochemical analysis.

Eicom’s Ideal System Overview

After reading about Eicom’s Ideal system, you may have noticed some interesting traits. First, why the rotating cage? Second, why another pump for large molecules? Well, to answer these, we have to think about the basic microdialysis setup.
For each microdialysis probe, there are two thin lines of tubing necessary to transfer aCSF to and from the probe. Since the animal is freely moving, just connecting the lines directly to the animal means the lines twist when the animal rotates. This risks the lines detaching or limiting the animal’s ability to move. Liquid swivels are often used to prevent the twisting of microdialysis lines, but become cumbersome as more lines (tubing, fiber optic, or electrical) are attached to an animal. So we thought, how do we stop twisting of lines no matter how many lines are connected to the animal? We felt the solution was to combat the twisting at the source: the animal that keeps rotating. Some researchers spend time untwisting the animal by hand, we thought why not have a machine do that for us? Thus, we have a rotating cage stage. As the animal rotates, the tether twists the sensor, which triggers the stage to counter-rotate. The result is zero net rotations by the animal, therefore, no twisting to worry about. Best of all, multiple implants and tubing lines can be kept untwisted. Plus, no more cleaning swivels!

Now, why the second pump? The short answer is to remove back pressure in the outlet line. The long answer is that sometimes researchers seek to collect large molecules, but that means a large pored membrane is required. The large pores mean, if backpressure is present, more fluid will permeate across the membrane and enter the brain which can lead to increased intracranial pressure and lower recovery rates. By adding a second pump, we create a push-pull system. To adjust for even the slightest pressure difference between the pumps, we added a vent to the AtmosLM™ probes. This eliminates any pressure build-up in the probe and therefore prevents any membrane leakage into the brain.

Recovery Percentage from Microdialysis

Recovery percentage depends on several factors, such as diffusion rate, flow rate, flux, membrane length, membrane type/pore size, and perfusion liquid. The best results require optimization of each factor. For example, the lowest flow rate, longest practical membrane, and maximum sampling time will yield samples with the highest concentration of the analyte. This is because the membrane is maximizing surface area for diffusion, while the slow flow rate is giving diffusion plenty of time to equilibrate; the sampling time determines the absolute content each sample will contain. This example is a simplified understanding of the factors that must be considered.

Diffusion: The diffusion rate is inversely proportional to the molecular size. This means that as the molecular weight of the analyte increases, the diffusion rate will decrease. If the diffusion rate decreases, then the recovery percent decreases. This essentially forces a lower flow rate for large molecules. Next, hydrophobicity also plays a role in the diffusion rate and must be considered when analyzing large molecules, such as neuropeptides. Hydrophobic molecules also exhibit “sticky” characteristics which make monitoring of concentration changes (primarily decreases) difficult since these changes take significantly longer to equilibrate. The concentration of the target analyte can be depleted from the area around the probe faster than it can be supplied by the cells or diffused from surrounding extracellular fluid; This is partly because of diffusion in tissue taking longer than in aqueous solutions due to limited extracellular fluid and indirect diffusion pathways between cells. Analytes may attach to cells along the way and slow down their diffusion; Results can be misinterpreted if this principle is not considered during the study.

Flow Rate and Flux: Low flow rate creates low pressure in the probe; when using a perfusion liquid that mimics extracellular fluid besides the low pressure, there is a minimal ionic flux between the probe and the brain which positively affects diffusion. The principle of flux can estimate extracellular concentrations of the target analyte. To do this, the perfusate is spiked with a known concentration of the target analyte and then pumped through the probe. The dialysate is analyzed for differences in concentration; decreased analyte concentration in the dialysate shows that extracellular fluid has a lower concentration of the analyte, therefore, the analyte diffused from the perfusate to the extracellular fluid. If the concentration of the analyte in the dialysate was found to be higher than its initial concentration, then it can be inferred that extracellular fluid is higher in analyte concentration, therefore, the analyte diffused from the extracellular fluid into the perfusate. By adjusting the concentration of the analyte in the perfusate until there is no difference in analyte concentration between the perfusate and the dialysate, we can infer there was no net diffusion of analyte across the membrane, therefore, we have estimated the concentration of the analyte in the extracellular fluid. Keeping these factors optimized allows us to more accurately determine/calculate the absolute content of the target molecule near the probe’s surface.

Perfusion liquid: The perfusion liquid should be as close as possible in nature to the extracellular fluid. The purpose of this is to limit the ionic transfer across the membrane, which can affect diffusion rates. Many use a Ringer’s solution (148 mM NaCl, 4 mM KCl, 3 mM CaCl2) which has higher calcium and potassium levels than aCSF (148 mM NaCl, 4 mM KCl, 1.2 mM CaCl2, 0.85 mM MgCl2). The aCSF solution more closely mimics physiological salt conditions in-vivo which yields more accurate readings from the animal. For microdialysis of large molecules, an osmotic agent such as BSA (bovine serum albumin) may be added to limit fluid loss through large pore membranes. BSA also serves as a blocking agent by preventing non-specific adsorption of other peptides and proteins to the probe and tubing surfaces.

Microdialysis Probe: As mentioned above, membrane length and pore size play a major role in the recovery rate. Pore size is determined by the choice of material that is used to make the membrane, several options of membrane material can be picked depending on the application (peptide vs. monoamine). Some types of membranes are regenerated cellulose, polyacrylonitrile, polyethylene, and polyethersulfone. The membrane length should not be longer than the diameter of the target brain tissue. Most membranes will work well for low molecular weight substances, but as the molecular weight becomes larger, the pore size becomes significantly more important.

Tubing: Careful consideration of tubing length is necessary to avoid too much back pressure after the microdialysis probe. If the tubing length is too long, the backpressure from the line could cause damage to non-vented probes and result in injury to the animal. The solution is minimizing the tubing between the microdialysis probe and the collector or adding a pump that will pull the fluid away from the probe and eliminate pressure altogether. Adding a second pump is what we did for our AtmosLM™ probes. If a second pump is not an option, then limiting tubing not only minimizes back pressure, it shortens transport time from the brain to the collection well where the sample can be chilled. Unfortunately, increasing the diameter of the tube is not an option because this will increase the diffusion of analytes during transport and make precise changes in concentration difficult to determine. Large diameter tubing also increases the transport time and the risk of degradation. This brings us to the next point.

Sample collection and analysis: Once the microdialysis sample is collected via the probe, it can either be injected directly (on-line) into high-performance liquid chromatography (HPLC) or sampled into a 96-well plate via a fraction collector where it is chilled for later analysis (off-line). Depending on the analytes of interest, different analytical techniques can be used.  The most commonly used are HPLC and ELISA. For monoamines, an HPLC-ECD (electrochemical detector) or mass spectrometry are the common routes to take. Mass spectrometry is typically done when a nonspecific analysis is being performed, since it can analyze a broad array of molecules all at once. The disadvantages come with the price of the instrument or even the cost of paying to have samples analyzed. The HPLC-ECD is a much less expensive instrument and still has the sensitivity to detect neurologic concentrations of analytes. An HPLC-ECD can be set up to analyze specific molecules quickly and easily inside most labs with high sensitivity. The HPLC also does not take nearly as much experience to analyze the results compared to a mass spec, making it easy to operate with minimal training. The overall cost of running samples becomes multitudes cheaper compared to mass spectrometry. Besides this, because of the low instrument cost and maintenance cost, many researchers prefer the availability of immediate analysis of microdialysate which makes it a top contender for must-haves in a neuroscience lab. Eicom has designed an entire setup to be perhaps the most convenient system for neuroscientists to use by making our fraction collector compatible with our HPLC autosampler. Simply take the 96-well plate from the fraction collector and place it in the autosampler, create the sequence, and press start. Although an electrochemical detector is not geared for very large molecules, the HPLC can be set up to use several types of detectors, making it even more versatile for many fields of study.

Other applications of Microdialysis

Some researchers use retrodialysis to deliver a compound to the target brain tissue. This compound can be medicine, toxin, protein, etc. This reverse type of microdialysis is called retrodialysis and is done when the precise administration of a compound is necessary. The compound is placed in the perfusate and diffused across the membrane to the target tissue. For example, placing medicine in the perfusate will diffuse across the membrane of the microdialysis probe that is implanted directly in a tumor. A second microdialysis probe could be placed nearby the first probe and could collect dialysate in response to the medication administration.

Some researchers like to do bilateral microdialysis. One probe is functioning as a baseline while certain manipulations occur to the other probe.