MILAB brings off microdialysis experiments, elaborating on design, stereotaxic surgery, and optimization of microdialysis procedure in accordance to particular needs.
Microdialysis is one of the most powerful neurochemistry techniques, which allows the monitoring of changes in the extracellular content of endogenous and exogenous substances in the brain of living animals. The strength as well as wide applicability of this experimental approach are based on the bulk theory of brain neurotransmission (Anderzhanova and Wotjak, 2013).
When designing microdialysis experiments, some specific issues should be considered:
- What is (are) the targeting region(s) and substance(s) of interest?
- What kind of behavioral or pharmacological challenges will be applied?
- Are acute or sub-chronic (up to 4 days, depending on brain structure) experiments planned?
- Are freely moving or anaesthetized animals suitable?
- Will conventional microdialysis or no-net-flux microdialysis be employed?
- Is local pharmacological treatment (reverse dialysis) desirable?
- What are the features of the MD probes (dimensions, membrane material, MWCO, supplier)?
The microdialysis procedure can be combined with a behavioral test, e.g., open field test, fear conditioning-fear extinction procedure, FST and, respectively, bring behavioral data.
MiLAB equipment allows to run up to 4-6 animals at the same time. Each setup includes all essential components of perfusion/ fraction collection and can be adjusted according to the needs of an actual experiment:
- Multisyringe pumps (“TSE”, “Harvard apparatus”)
- Refrigerated fraction collectors (“Univentor”)
- Platforms (20 cm x 20 cm, adjustable level)
- Perfusion line: stand (custom-made), swivel (“Eicom”) and counterbalance (“Intech”)
- Perfusion liquid switches (“Univentor")
Stereotaxic surgery is used to implant microdialysis probe guide cannula (or microdialysis probe in case of acute experiments in anesthetized animals). This is a routine technique slightly modified because of a guide cannula and microdialysis probe dimensions and requirements for continuous perfusion during an experiment. Anesthesia/analgesia with Isoflurane and Metacam® is used that allows fast animal recovery within 3-4 days.
HPLC/ECD is a most sensitive method to measure the content of monoamines and their metabolites in microdialysates (dopamine, norepinephrine, serotonin, DOPAC, HVA, 5-HIAA, and MHPG). Both HPLC/ECD and HPLC/FLD are employed to measure the content of neuroactive amino acids (glutamate, aspartate, GABA, glycine, taurine, proline). Acetylcholine can be detected with HPLC/ ECD when the perfusion liquid is supplied with acetylcholine esterase inhibitors. Microdialysis gives a unique opportunity to monitor the levels of free corticosterone, which can be detected in microdialysates (brain and peripheral tissues) with RIA or ELISA.