In vivo microdialysis has enabled collection of molecules present in brain interstitial fluid (ISF) from awake, freely-behaving animals. In order to analyze relatively large molecules in ISF, the current article specifically focuses on the microdialysis protocol using probes with high molecular weight cut off membranes.
In vivo microdialysis is a powerful technique to collect ISF from awake, freely-behaving animals based on a dialysis principle. While microdialysis is an established method that measures relatively small molecules including amino acids or neurotransmitters, it has been recently used to also assess dynamics of larger molecules in ISF using probes with high molecular weight cut off membranes. Upon using such probes, microdialysis has to be run in a push-pull mode to avoid pressure accumulated inside of the probes. This article provides step-by-step protocols including stereotaxic surgery and how to set up microdialysis lines to collect proteins from ISF. During microdialysis, drugs can be administered either systemically or by direct infusion into ISF. Reverse microdialysis is a technique to directly infuse compounds into ISF. Inclusion of drugs in the microdialysis perfusion buffer allows them to diffuse into ISF through the probes while simultaneously collecting ISF. By measuring tau protein as an example, the author shows how its levels are altered upon stimulating neuronal activity by reverse microdialysis of picrotoxin. Advantages and limitations of microdialysis are described along with the extended application by combining other in vivo methods.
ISF comprises 15-20% of total brain volume and offers a microenvironment critical for signal transduction, substrate transport and waste clearance1. Therefore, the ability of collecting ISF from living animals will provide greater implications for various biological processes as well as disease mechanism. In vivo microdialysis is one of the few methods that sample and quantify extracellular molecules from ISF from awake, freely moving animals and thereby serves as a useful tool in neuroscience research field2,3. In this method, microdialysis probes with semipermeable membranes are inserted in the brain and perfused with perfusion buffer at the relatively slow flow rate (0.1-5 µL/min). During this perfusion, extracellular molecules in ISF passively diffuse into the probe according to the concentration gradient and collect as a dialysate. Although this article focuses on the method to sample ISF in the brain, both the principle and the method can be applied to other organs by appropriate modification if necessary.
Microdialysis was first employed in the early 1960s, and since then it has been extensively used to collect small molecules including amino acids or neurotransmitters in brain. However, recent commercial availability of microdialysis probes with high-molecular weight cut off membranes (100 kDa-3 MDa) has extended its application to relatively larger proteins in ISF as well4,5,6,7. The studies using these probes has led to the finding that proteins such as tau or α-synuclein that were long thought to be exclusive cytoplasmic are also physiologically present in ISF4,5,8.
One of the difficulties using microdialysis probes with large cut off membranes (typically over 1,000 kDa) is that they are more susceptible to ultrafiltration fluid loss due to the inner pressure accumulated in the probes. Microdialysis probes used here have a unique structure to avoid this issue. The pressure will not be built up due to this structure, thus microdialysis with these probes should be operated in a "push-pull" mode using a syringe pump to perfuse the probes (=push) and a roller/peristaltic pump to collect the dialysate coming from the probe outlet (=pull)9 (Although it needs both push and pull pumps, due to pressure cancelling vent holes present in the probes, the system is technically only driven by the pull pump). This article starts with the stereotaxic surgery of a guide cannula implantation and describes how to set up microdialysis lines in order to collect ISF through microdialysis probes with 1,000 kDa cut-off membranes.
All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of the Graduate School of Medicine at the University of Tokyo.
1. Pre-surgical Procedure
2. Stereotaxic Surgery for Guide Cannula Implantation
3. Microdialysis Setup
To stimulate or inhibit neuronal activity in reverse microdialysis11,12,13, picrotoxin, GABAA receptor antagonist or tetrodotoxin, Na+ channel blocker have been used. It has been shown that tau release is stimulated by increase of neuronal activity13,14. Consistent with these previous observations, when 50 µM picrotoxin (PTX) was administered via reverse microdialysis (see Discussion for more details) in awake C57B6/J mice increased ISF endogenous tau levels measured by ELISA compared to vehicle control (DMSO) (Figure 2).
Figure 1: Surgery setup and the construction of microdialysis circuits. (A) Set up a stereotaxic assembly for a guide implantation using a cap nut, a stereotaxic adaptor and a guide cannula. (B) Stereotaxic surgery to implant a guide cannula. The guide cannula is inserted in the brain at 12 degrees with respect to vertical. (C) Construction of an inlet tubing and an outlet tubing. Inlet line consists of 70 cm FEP tubing (JF-70) and the outlet line consists of JF-70 and the roller tube and 1/2 of FEP tubing. The connection needles are used for each connection. (D) Once the entire tubing is filled with perfusion buffer, stop the pump and replace the connection needle between inlet line and outlet line with an activated microdialysis probe. Make sure to connect the end of inlet tubing to the inlet port of a probe (longer port) and connect the end of outlet tubing with outlet port of a probe (shorter port). Please click here to view a larger version of this figure.
Figure 2: Representative data showing ISF tau changes upon picrotoxin reverse microdialysis. Picrotoxin (PTX, 50 µM) or DMSO were delivered into the hippocampus of C57B6/J mice via reverse microdialysis. Picrotoxin rapidly increased ISF endogenous tau levels from its basal levels (i.e., the average tau concentrations during 3 h before PTX administration) compared to DMSO treatment. Data is shown as mean±SEM, n = 3 for DMSO, n = 4 for PTX. Please click here to view a larger version of this figure.
Microdialysis with high molecular weight cut off membranes has to be operated by a push-pull mode, thus it is critical that the flow rate is accurate and constant. The inaccuracy in the flow rates can be the cause of air bubble generation and inconsistency in the sample concentration. If the flow is inconsistent, check all connections for leakage. If the problem still persists, it may be necessary to re-start with new probes and tubings.
Microdilaysis probes are continuously perfused by the perfusion buffer. Therefore, there is not sufficient time for extracellular molecules to reach a full equilibrium in the perfusion buffer. As a consequence, the concentration in the dialysate is much lower than its actual concentration in ISF. In order to estimate the true concentration of molecules in ISF, a zero flow method is often used3,5,15. This method measures the concentration by altering the flow rate. There is an inverse relationship between the concentration and the flow rate. Therefore, the concentration of target molecules can be determined by extrapolating the exponential curve back to the theoretical zero flow, which represents the perfect recovery of the molecules. However, the recovery can be influenced by various factors such as interaction with membranes or hydrophobicity of molecules or interaction with other proteins, and one should keep in mind that the concentration at the theoretical zero may not be necessarily equivalent to its true concentration in ISF.
A probe insertion causes acute local injury in brain. From this reason, the first several fractions are typically excluded from further analysis. In fact, the levels of ISF tau protein in hippocampus are not stable in the first 9-15 hour likely because the acute injury causes non-specific release of tau into ISF (the author's unpublished observation). Recovery after probe insertion injury varies from target to target. Pilot studies should be performed to determine when each target reaches a steady-state level to determine when samples should be analyzed. This determines the "start point" for microdialysis sampling. An endpoint for sampling is when the target is no longer at the steady state (typically 3-5 days after microdialysis probe implantation; not guide implantation). Start and end for each target should be determined empirically by each lab for each target.
Microdialysis is especially useful when combined with drug treatment. Drugs can be administered systemically or directly infused into ISF. Reverse microdialysis is one method that directly infuses small compounds via probes. Dialysis occurs bi-directionally. Therefore, inclusion of drugs in the perfusion buffer allows its diffusion though microdialysis probes to the surrounding tissue. This method enabled the local administration of small compound and simultaneous collection of ISF. Reverse microdialysis is less invasive compared to a pressure injection through cannula and can maintain more constant concentration of drugs in target area.
Before starting reverse microdialysis, the internal volume of entire tubing should be taken into account to calculate the timing of treatment and sample collection. For example, the internal volume of entire tubing used in this article is 91.5 µL (Refer to the Table of Materials for internal volume of each tubing). Therefore, when the flow rate is 1.0 µL/min, it takes 91.5 min for the perfusion buffer containing drugs to reach the fraction collector. On the other hand, when drugs are delivered systemically, the inner volume of the outlet tubing (56.5 µL in this case) should be considered.
In vivo microdialysis method offers several advantages over other techniques. For example, while brain homogenates likely consist of both extracellular and intracellular proteins, microdialysis specifically collects extracellular proteins from ISF. Second, a single microdialysis probe can collect ISF at the different time points from the same animal. Target concentration in each sample can be normalized to a % baseline. This normalizes absolute concentration between animals so relative differences can be compared. This increases power of a study and typically reduces animal number needed for an experiment when comparing relative differences in protein levels following drug administration/manipulation. On the other hand, the major limitation of microdialysis is the restriction of the size of the target molecule.
Microdialysis can be combined with other in vivo techniques. For example, performing microdialysis in regulatable transgenic mice can estimate in vivo turnover of target protein16. The combination with EEG, measurement was used to measure electric activity during reverse microdialysis11. And optogenetics enabled manipulation of neuronal activity in brain while simultaneously collecting ISF17.
The authors have nothing to disclose.
This work was supported by ''Grant-in-Aid for Scientific Research on Innovative Areas (Brain Protein Aging and Dementia Control)(15H01552) from MEXT and Grant-in-Aid for Young Scientists (B) (16K20969). The author thanks Dr. David M. Holtzman and Dr. John R. Cirrito for the technical advices during the development of this method.
The Univentor 820 Microsampler | Univentor | 8303002 | Refrigerated fraction collector |
Syringe pump | KD scientific | KDS-101 | |
Roller pump | Eicom microdialysis | ERP-10 | |
Raturn Stand-Alone System | BASi | MD-1409 | Free-moving system |
Dual species cage kit | BASi | CX-1600 | |
AtmosLM Microdialysis probe (shaft length 8 mm, membrane length 2 mm) | Eicom microdialysis | PEP-8-02 | Shaft length for a probe, a guide, a dumy probe and a stereotaxic adaptor should be identical. |
Microdialysis guide (shaft length 8 mm) | Eicom microdialysis | PEG-8 | |
Microdialysis dummy probe (shaft length 8 mm) | Eicom microdialysis | PED-8 | |
Bone screw | BASi | MD-1310 | |
Super bond C&B set | Sunmedical | Dental cement | |
Small animal Stereotaxic Instrument with digital display console | Kopf | Model 940 | Stereotaxic apparatus |
Mouse and neonatal rat adaptor | Stoelting | 51625 | |
Standard Ear Bars and Rubber Tips for Mouse Stereotaxic | Stoelting | 51648 | |
Albumin solution from bovine serum | Sigma | A7284-50ML | 30% BSA solution |
FEP tubing (70 cm) | Eicom microdialysis | JF-10-70 | Internal volume = 0.5 µL/cm |
Teflon tubing (50 cm) | Eicom microdialysis | JT-10-50 | Internal volume = 0.08 µL/cm |
Byton tube | Eicom microdialysis | JB-30 | |
Intramedic luer stab adaptor 23G | BD | 427565 | Blunt end needle |
Roller tube | Eicom microdialysis | RT-5S | Internal volume = 4 µL |
Cap nut | Eicom microdialysis | AC-5 | |
0.25 mL microcentrifuge tube with cap | QSP | 503-Q | Tubes for fraction collector |
Sterotaxic adaptor (shaft length 8 mm) | Eicom microdialysis | PESG-8 | |
Connection needle | Eicom microdialysis | RTJ | |
Mouse animal collar | BASi | MD-1365 | |
High Speed Rotary Micromotor kit | FOREDOM | K.1070 | Drill |
Picrotoxin | Sigma | P1675 | |
Screw driver for bone screws | |||
Scalpel | |||
Cotton swab | |||
Surgical clipper |