Biology Reference
In-Depth Information
4.4.2. Determination
of Relative Recovery
of the Probe
1. Position the CMA probe (as during priming) in a microcentrifuge
tube containing standard solution.
2. Run the micropump at the fl ow rate of 2
l/min.
3. Place the probe outlet tubing in a vial placed on ice and collect
sample every 10 min for 30 min (a fraction collector can be
used for this purpose; CMA 142 Microfraction Collector).
4. Place all the three dialysate tubes at 4°C.
μ
1. Place the rat on the stereotaxic table. Make a small burr hole at
the right sensorimotor cortex; from bregma; posterior, 0.9 mm;
lateral, 4.0 mm; depth, 2.0 mm.
2. Using a cannula guide insert the microdialysis probe in the
burr hole and fi x it in this position via stereotaxic adaptor and
connecting rod.
3. Place the probe outlet into sample vial and start the
micropump.
4.4.3. Probe Implantation
1. Insert the microdialysis probe in animal's brain and collect
dialysis samples every 10 min at the fl ow rate of 2
4.4.4. Determination
of Time Required
to Recover from Probe
Insertion Trauma
μ
l/min) for
180 min. Store at 4°C.
2. Analyze all samples for the difference of metabolites level (see
below).
3. The time after insertion where the analytes fi rst reach the basal
levels reported in literature (see above) is the recovery time.
4. Repeat this determination in at least fi ve animals to confi rm the
recovery time in your experiments.
1. Induce SAH and start collecting dialysate keeping the fl ow rate
2
4.4.5. Microdialysis After
SAH
l/min and fractionation interval 10 min.
Note: Animals are anesthetized and body temperature is main-
tained via a homeothermic blanket (Harvard Apparatus) dur-
ing microdialysis. For extended sampling (beyond 6 h) CMA
120 System for freely moving animals should be considered.
2. Store samples in 4°C till analyzed.
Note: samples should be stored at 4°C only if their analysis is
to be performed the same day; otherwise, they should be stored
at −70°C.
μ
1. Components of dialysate and their relative concentrations are
detected via CMA analyzer. This machine can detect up to four
analytes simultaneously. The order of analysis should be such
that relative recovery of the probe is read fi rst and then the
samples.
2. Alternatively, dialysate can be analyzed via HPLC analysis with
suitable detector.
4.4.6. Dialysate Analysis
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