Assessment of Global Ischemia - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 363

4.3. Materials
and Instruments

1. Perfusate: Krebs-Ringer bicarbonate buffer consisting of [in
mM]: 122 NaCl, 25 NaHCO 3 , 3 KCl, 1.3 CaCl 2 , 1.2 MgSO 4 ,
and 0.4 KH 2 PO 3 . Warmed at 37°C.
2. Standard solution: In Krebs-Ringer bicarbonate buffer: lactate
0.1 mM/L, pyruvate 10

4.3.1. Solutions

μ M/L, glutamate: 10 μ M/L, and

glucose: 10 mM/L.
3. 70% Ethanol.

1. Micro pump (Harvard Apparatus, South Nantick, MA, USA).
2. Homeothermic blanket (Harvard Apparatus).
3. CMA microdialysis analyzer (CMA microdialysis; Solon).

4.3.2. Instruments

1. CMA 12 probe (membrane length 2 mm, outer diameter
0.5 mm and cut-off weight 20,000 Da; CMA microdialysis;
Solon).
2. Guide Cannula (CMA microdialysis; Solon).
3. FEP tubing (CMA microdialysis; Solon).
4. Plastic or glass sample collection vials (Fisher scientifi c, USA).
5. 1.5-ml microcentrifuge tubes (Fisher scientifi c, USA).
6. Small animal stereotaxic frame, adaptor and a connecting
Rod.

4.3.3. Probe Placement
and Sampling Aids

This step prepares the microdialysis probe for use. New probe
needs to be fl ushed with 100% ethanol and from thereon it is kept
immersed in 100% ethanol when not in use. Priming procedure
clears out ethanol and fi lls the dead space in the tubing with Krebs-
Ringer solution. Steps are as follows:

4.4. Procedures
4.4.1. Probe Priming

1. Connect the inlet and outlet of the microdialysis probe with
FEP tubing. The size of inlet and outlet tubing depends upon
the distance of probe to the micropump and to the sample col-
lection vial, respectively.
2. Take a syringe fi ll with Krebs-Ringer and load it into the
micropump.
3. Connect the inlet tubing of the probe to the syringe.
4. Take 1.5-ml microcentrifuge tube, fi ll it with Krebs-Ringer
solution, and place it in a stand (in vitro stand from CMA/
microdialysis AB can be used).
5. Position the probe such that its tip and the shaft are dipped in
the microcentrifuge tube (probe clip may be used; CMA/
microdialysis).
6. Attach the inlet channel of the probe to the syringe and fl ush
at the rate of 10-15

l/min till Krebs-Ringer solution starts
coming out of the tube. Check that there are no air bubbles in
the line. The probe in now primed.

μ