Microelectrodes in the Microcirculation

1
Microelectrodes in the Microcirculation
The Microcirculatory Society, Inc. 1954-2004
Half a Century of Discovery and Innovation
Donald G. Buerk
Department of Physiology, University of
Pennsylvania School of Medicine, Philadelphia, PA
19104
Recessed Oxygen Microelectrodes
Abstract
Since this was a micro- review, many other papers
of historical relevance by the numerous members
of the Society who have used oxygen
microelectrodes could not be cited in this
poster. Interested readers can find a more
complete survey on oxygen microelectrode
techniques (Buerk, 2004). On a personal note, it
was a sincere honor and privilege to have started
my career in Bill Whalens laboratory in 1969. He
was a true physiologist with great integrity and
abundant enthusiasm, and we all mourn his passing
last August, 2003. Also, I would like to
acknow-ledge my graduate mentor, Thomas K.
Goldstick, an expert on oxygen electrodes and
oxygen transport theory, who is another man with
high scientific integrity. Both were members of
the Microcirculatory Society during my formative
years, and it has been a highly rewarding
experience to follow in their footsteps.
It should be no surprise that one of the earliest
uses of micro-electrodes in the microcirculation
was by the esteemed physiologist, Eugene M.
Landis. He used fluid filled glass micropipettes
with tips as small as 5 microns to penetrate
small blood vessels in the frog mesentery
(Landis, 1926). The micropipettes were
pressurized by a fluid reservoir with an
adjustable height. By observing whether fluid
flowed into the vessel, or whether red blood
cells flowed into the pipette, and skillfully
balancing out the pressure difference, he was the
first to characterize the distribution of blood
pressure along the microvasculature. He also
studied transcapillary fluid exchange by
occluding the distal end of capillaries using a
fine glass rod with a small ball at the end, and
observing the movement and change in spacing of
individual red blood cells in the occluded
capillary. Dyes were microinjected into the
microcirculation in some of his experiments. He
also conducted a study on the effects of hypoxia
on capillary permeability (Landis, 1928). These
elegant microelectrode methods were extended to
mammalian studies, and were used for measurements
in humans including patients with Raynauds
disease. In a series of papers, summarized in his
classic review paper (Landis, 1934), he provided
convincing experimental evidence proving Ernest
Starlings hypothesis for fluid movement due to
osmotic pressure differences postulated in the
1890s. One of Landis methods was miniaturized
by Zweifach and Intaglietta (1968), who used a
glass microneedle with a double bend near the tip
to study capillary fluid exchange by making a
more localized occlusion. The accuracy of the
micropipette pressure measurement technique was
significantly improved by a servo-nulling method
(Weiderhielm et al., 1964), and the theory behind
the measurement was developed (Fox and
Weiderhielm, 1973) to understand possible errors
due to ionic gradient effects with very small
(less than 0.1 micron) tips. Improved
instrumentation using the servo-null method with
a negative capacitance input amplifier allowed
Intaglietta and Tompkins (1971) to use
microelectrodes with 1 micron tip diameters to
measure intravascular pressures. Fronek and
Zweifach (1974) used 3 to 5 micron diameter tips
to quantify upstream and downstream resistances
in the cat mesentery. Micropipette techniques
were further modified over the years to perform
even more elaborate investigations, particularly
in the laboratory of Brian R. Duling at the
University of Virginia. These techniques included
cannulation and perfusion of isolated
microvessels (Duling et al., 1981), and direct
sampling of the blood in microvessels (Dejardins
and Duling, 1987) to confirm that the discharge
hematocrit is in fact equal to the systemic
hematocrit, and that the apparent reduction in
hematocrit seen under the microscope does not
violate the law of mass conservation.


If you accept the time worn axiom (as I do) that
you need the right tool to do the job right, and
you really cant see what you are working on
without a microscope, then you might agree that
microelectrodes are the right tools for studying
the micro-circulation. This has certainly proven
to be true for a number of distinguished members
of the Microcirculatory Society, who perfected a
variety of different microelectrode techniques.
Practical uses for these tiny tools range from
microneedles to mechanically compress individual
capillaries as a way to study fluid exchange, to
fluid filled open tip micropipettes to measure
blood pressure in arterioles and venules, or to
alter the local chemical environment or deliver
drugs to individual blood vessels by
microperfusion. Electrochemical sensors were also
miniaturized, and early oxygen microelectrode
measurements in the microcirculation were
discussed as part of a methods workshop sponsored
by the Microcirculatory Society at the University
of Arizona in 1971. The desire to measure other
physiologically relevant chemical species was
also expressed in this workshop, and subsequently
pursued over the years in many microcirculation
laboratories. A history of these efforts will be
reviewed, especially with regard to the recessed
oxygen microelectrode that many members of the
Microcirculatory Society have used at one point
or another during their careers.

Fig. 1. Sketch of the Whalen-Nair type recessed
oxygen microelectrode (Whalen et al., 1967). In
1965, the Microcirculatory Society held a
symposium on blood flow and fluid exchange at the
Federation Proceedings. At that meeting, William
J. Whalen published an abstract on an innovative
recessed oxygen microelectrode design (Fig. 1).
Whalen et al. (1967) reported that oxygen
reduction currents for the recessed cathode were
nearly two orders of magnitude lower than a 1
micron tip needle-type oxygen microelectrode
(Fatt, 1964). Since the recessed oxygen
microelectrode could be made with tip diameters
less than 2 microns, it was possible to make
intracellular PO2 measurements with excellent
temporal and spatial resolution. Extremely low
usage of oxygen due to the electrochemical
reaction meant that measurement errors caused by
depletion of oxygen around the tip would be
negligible. Later, a paper modeling the recessed
cathode by Schneiderman and Goldstick (1978)
explained how to optimize the membrane and depth
of the recess to minimize measurement artifacts.
It took a few years to recognize that the
recessed cathode design is superior to membrane
covered needle type oxygen microelectrodes. By
1971, tissue PO2 was among the topics at a
workshop held at the University of Arizona that
was organized by Paul C. Johnson and Harold
Wayland, with a final report published in
MIcrovascular Research (Zweifach, 1972). Whalen
described how to fabricate the fragile glass
microelectrode. Some of his earliest tissue PO2
measurements were in skeletal muscle (Whalen et
al., 1973), and in the beating heart with a
spring mounted microelectrode (Whalen, 1971). He
observed that mean tissue PO2 was lower than the
venous blood PO2, which seemed controversial at
the time, but is consistent with oxygen transport
models. Duling described microcirculatory
prepara-tions in which he had measured PO2, and
pointed out that there was a very heterogeneous
distribution of PO2 values in the
microcirculation, so that placement of the
microelectrode was critical. For example, Duling
and Berne (1971) mapped in vivo perivascular PO2
values along arterioles and venules in the
hamster cheek pouch, finding significant losses
along the microvascular network. These
longitudinal oxygen gradients are predicted from
mathematical models and their existence has been
confirmed by optical measurements.
References
Buerk DG. Measuring tissue PO2 with
microelectrodes. Meth. Enzymol. 381665-689,
2004. Desjardins C, Duling BR. Microvessel
hematocrit measurement and implications for
capillary oxygen transport. Am. J. Physiol.
252H494-503, 1987. Duling BR, Berne RM.
Longitudinal gradients in periarteriolar oxygen
tension. A possible mechanism for the
participation of oxygen in local regulation of
blood flow. Circ. Res. 27669-678, 1970. Duling
BR, Gore RW, Dacey RG Jr, Damon DN. Methods for
isolation, cannulation, and in vitro study of
single microvessels. Am. J. Physiol. 241H10-116,
1981. Fatt I. An ultramicro oxygen electrode. J.
Appl. Physiol. 19326-329, 1964. Fox JR,
Weiderhielm CA. Characteristics of the
servo-controlled micropipette pressure system.
Microvas. Res. 5324-325, 1973. Fronek K,
Zweifach BW. Pre- and postcapillary resistances
in cat mesentery. Microvas. Res. 7351-361,
1974. Intaglietta M, Tompkins WR. Micropressure
measurement with smaller cannulae. Microvas. Res.
3211-214, 1971. Landis EM. The capillary
pressure in frog mesentery as determined by
micro-injection methods. Am. J. Physiol.
75548-570, 1926. Landis EM. Micro-injection
studies of capillary permeability. III. The
effects of lack of oxygen on the permeability of
the capillary wall to fluid and to plasma
proteins. Am. J. Physiol. 83528-542, 1928.
Landis EM. Capillary pressure and capillary
permeability. Physiol. Rev. 14404-481,
1934. Schneiderman G, Goldstick TK. Oxygen
electrode design criteria and performance
characteristics recessed cathode. J. Appl.
Physiol. 45145-154, 1978. Weiderhielm CA,
Woodbury JW, Kirk S, Rushmer RF. Pulsatile
pressure in the microcirculation of the frogs
mesentery. Am. J. Physiol. 207173-176,
1964. Whalen WJ, Riley J, Nair P. A
microelectrode for measuring intracellular PO2.
J. Appl. Physiol. 23798-801, 1967. Whalen WJ.
Intracellular PO2 in heart and skeletal muscle.
Physiologist 1469-82, 1971. Whalen WJ, Nair P,
Ganfield RA. Measurements of oxygen tension in
tissues with a micro oxygen electrode. Microvas.
Res. 5254-262, 1973. Zweifach BW. Report on
Workshop on the Microcirculatory approach to
peripheral vascular function. Microvas. Res.
4306-318, 1972. Zweifach BW, Intaglietta M.
Mechanics of fluid movement across single
capillaries in the rabbit. Microvas. Res.
183-101, 1968.
A Micro- Review
As we celebrate the formation of the
Microcirculatory Society 50 years ago, you might
be astounded to discover that a keyword search
for microcirculation on MEDLINE for the years
1950-59 returns exactly zero publications. A
search on the International Sciences Institute
(ISI) database is a bit more representative with
15 returns for the decade. The first half of the
1960s (1960-1964) lists only 11 papers on
MEDLINE and 83 citations on ISI. However, our
societys rich history of innovation and
discovery can not be explored by performing
simple literature searches. One must be aware of
the individuals who have worked in this field.
For example, if you search ISI for citations to
papers by Benjamin W. Zweifach, one of our most
prolific founding members, you would find 57
entries for the period 1950-59. Similarly, the
keyword microelectrodes on MEDLINE turns up
zero for the period from 1950-1964, and only 11
in 1965-1969. The number of citations in ISI for
the 1950s is 51, with 100 from the 1960s. Most
of these papers are related to electrophysio-logic
al measurements. If you combine keywords
microcircu-lation and microelectrodes you
will find zero publications until you reach 1990.
This cant be right, you might deduce, especially
if you have been working in this field, as I
have, for over 30 years.