What IS Microdialysis Sampling?

In vivo Microdialysis in Rat
Subcutaneous Space and Brain
The Good, the Bad, and the Ugly about
Microdialysis Sampling
Julie A. Stenken (jstenken@uark.edu)
Department of Chemistry and Biochemistry
University of Arkansas

Gaining Access to In Vivo Chemistry
• Blood Sampling – allows temporal resolution
– Problem: Drug or analyte sample is far away from
the targeted site.
• Urine Collection – allows accumulation
information
– Problem: Removed from target site.
• Biopsy of Organs – allows information at
target site and can give whole body chemical
information
– Problem: Loss of temporal resolution and requires
many experimental animals or subjects.
– Not easy to perform in humans.

Microdialysis Probe – Transport
~ 1 ml/min
DP
D
D
DP
P
D
P
D
DP
DP
DP
D
DP
D P D
DP
P P P
PERFUSATE DIALYSATE
D
P
D
Length 1 –30 mm
D
D
D
DP
D
P
DP
o.d. 200-500
microns

What IS Microdialysis Sampling?
• Microdialysis sampling is a minimally
invasive collection technique
• Microdialysis allows sampling of ONLY the
ECF space
– Total tissue concentrations, e.g., concentrations
inside cells is not possible with this method
• Uses small hollow fiber membranes
• Fluid is passed through at low µL/min flow
rates
• The applicability of microdialysis sampling to
any particular bioanalytical application
requires several pre-requisites

Microdialysis Popularity?
• Dialysates can typically be considered analytically clean,
i.e., no further sample clean-up necessary. Fast
equilibration
• Allows animal to serve as its own control.
• Has been implanted in numerous tissue spaces in rats
including: brain, blood, liver, muscle, kidney, skin, lung,
intestine, tumors, etc.
• Probes have been modified for use with genetically
engineering mice: principally brain
• Has been used in humans: brain, blood, lung, skin, and
tumors.
• Local substrate infusion followed by either metabolite or
pharmacodynamic effect (e.g., release of endogenous
substances) is possible.

www.microdialysis.se
Probes for Research
Rat Brain Mouse Brain Peripheral Tissues

Probe Information
J.A. Stenken (2006) Microdialysis Sampling, in Encyclopedia of Medical Devices,
J.Webster, Ed

Probe Information
• Probes are commercially available
• Many individual investigators make in-house
probes
– Brain: D. Jolly and P.Vezina (1996) J.Neurosci. Methods 68(2),
259-267
– Brain: S. Steffes and M. Sandstrom (1998) Journal of
Undergraduate Neuroscience Education (JUNE), Fall 2008,
7(1):A33-A47
– Peripheral Tissue: Several papers by Malonne I. Davies
• M.I. Davies and C.E. Lunte, Drug Metab. Disp., 23 (1995)
1072-1079.

Important Microdialysis Sampling
Considerations
• Microdialysis sampling only provides access to the
extracellular fluid space (ECF) environment
– Examples:
• Neurotransmitters: Dopamine, Serotonin, Glutamate
• Pharmaceutical compounds
• Peptides and proteins that are released in signaling.
• Molecular targets that reside inside the cell (e.g., NF-kB)
cannot be quantified with this approach.
• Membrane molecular weight cutoff is not an absolute
number.
– Good recovery is typically achieved with low molecular weight
solutes (i.e., analyte molecular weight

Molecular Weight Cutoff and Collection
J.A. Stenken (2006) Microdialysis Sampling, in Encyclopedia of Medical Devices,
J.Webster, Ed
K. Snyder, et al., Analyst, 2001, 126, 1261-1268

Fluids/Flow Rate Issues

Calculated Relative Recovery %
Flow Rate and Extraction Efficiency
Extraction. Efficiency
100
80
60
40
20
0
C C
outlet inlet
C C
sample inlet
1.0 E-5 cm 2 /s (e.g., glucose)
1.0 E-6 cm 2 /s (e.g., neuropeptide)
2.7 E-7 cm 2 /s (e.g., TNF-)
0 1 2 3 4 5
Perfusion Flow Rate (µL/min)
J.A. Stenken. (2006) Microdialysis Sampling, Encyclopedia of Medical Devices and Instrumentation, 2nd
Edition, Volume 4. John G. Webster (Editor). John Wiley & Sons, Inc. Hoboken, NJ. pp. 400-420.

Fluid Issues with 100 kDa MWCO or
Larger Dialysis Membranes
• Research for collection of peptides and proteins
requires larger MWCO membranes (100 kDa or
greater)
• ULTRAFILTRATION is a problem with these
membranes
– Fluid will be lost
– Membrane appears to “sweat”
• Becomes a greater issue in awake animal work
• Counter this with osmotic agents
– Dextran-60 (or -70) ~ 5 to 6 wt %
– Albumin
– Must verify experimentally

In vivo Microdialysis
Probe Implant

RECOVERY
Hydrophobic Analytes
Log P = -0.5
DELIVERY

Docetaxel – More Hydrophobic –
BUT – Microdialysis Works
Log P = 2.6

Docetaxel
NOTE(!): Standards were prepared from a stock solution containing 100% methanol.
Standards ranged from 0.5% to 7% methanol based on dilutions reported.

Some Analytical
Chemistry Issues

• LC-MS
Small Molecules
– Mass spectrometers don’t like a lot of salt
– Usually dialysate samples are treated like plasma
samples and undergo extraction into an organic
solvent

Large Molecules
• Very important to test molecular weight cutoff
(MWCO) of membrane
– Just recently, we have not been able to collect CCL2
(MCP-1) a 14 kDa protein through a 55 kDa MWCO
membrane
• For proteins size and shape affect recovery
– IL-6 and IL-10 have similar MW, but different
geometry.
• IL-6 is recovered through 100 kDa probes
• IL-10 is hard to recover through 100 kDa probes

Cytokine Detection

KC/GRO (pg/mL)
IL-1 (pg/mL)
12000 Right Probe
Left Probe
10000
8000
6000
4000
2000
0
35
30
25
20
15
10
5
KC/GRO
Multiplexed Cytokine Analysis
0 20 40 60 80 100 120 140 160 180 200
IL-1
Right Probe
Left Probe
Time (min)
0
0 20 40 60 80 100 120 140 160 180 200
Time (min)
IL-6 (pg/mL)
IL-10 (pg/mL)
50 RightProbe
LeftProbe
45
40
35
30
25
20
15
10
1400
1300
1200
1100
1000
900
800
700
600
500
400
300
200
100
IL-10
5
0 20 40 60 80 100 120 140 160 180 200
IL-6
Right Probe
Left Probe
Time (min)
0
0 20 40 60 80 100 120 140 160 180 200
Time (min)

Enhancement of Cytokine Detection
WITHOUT TRAPPING
IL-2
IL-4
IL-5
IFN-
TNF-
PBS
Perfusate Dialysate
~ 1 ml/min
Flow rate
WITH TRAPPING
Length 1 –30 mm
o.d.
200-500 mM
Perfusate Dialysate

Summary
• Microdialysis has been widely used in animal
studies
– Over 12,000 citations in Medline
• Hydrophobic analytes cause some problems
– Must test prior to going to animals
• Proteins can be difficult to collect with
microdialysis
– Luminex assays allow multiplexed detection

People
• Dan Loegering, Ph.D.,
AMC
• Michelle Lennartz, Ph.D.,
AMC
• Tim Sellati, Ph.D., AMC
• Xiaoping Ao, Ph.D. (Post
Doc U Michigan)
• Amy Steuerwald, M.S.
(NYS D.O.H.)
• Xiangdan Wang, Ph.D.
(Genentech)
• X. Susan Mou, Ph.D.
(Rhodes Technologies)
• Jia Duo
• Heidi Fletcher
• Ying Wang
• Yuexi Wang
Acknowledgements
People
• Randy Espinal Cabrera
• Tony Herbaugh
• Miles Ritter
• Brandon Suttles
• Mayen Udoetuk
• Summer 2008 REU
• Will Newhart
• Chris Ladner
$$$
• NIH EB-001441
• NIH DA-020577
• U of A Start Up
• Arkansas Biosciences Institute