Measurements of Dynamics
of Oxygen Concentrations in Wounds by EPR Oximetry
H. Hou1, O.Y. Grinberg1, S. Grinberg1, N. Rosen2, H.W. Hopf2, H.M. Swartz1
1EPR Center for the Study of Biological System,
Dartmouth Medical School, Hanover, NH, USA;
2 Wound Healing
Laboratory, Department of Anesthesia and Surgery,
University of California
School of Medicine, San Francisco, CA, USA
INTRODUCTION
The healing of surgical wounds requires
an adequate supply of oxygen and other nutrients necessary for collagen
synthesis and wound repair. Since it has been shown that an adequate oxygen
supply is particularly important to healing,
it would be of value to measure
the dynamics of oxygen in clinical wounds.
Wound oxygen tensions have previously been measured by methods such as
aspiration of fluid from the wound
dead space or microelectrodes inserted into
the dead space of wounds. But these techniques do not permit repeated
measurements and have some technical limitations in regard to accuracy.
One
of the advantages of EPR oximetry is that it can be used to make non-invasive,
accurate, and repeated measurements
of the actual oxygen tension at specific
sites within live tissue. We used an EPR method in which the oxygen sensitive
material
was placed in an oxygen permeable Teflon tube that was positioned
within the silastic mesh cylinder that established the wound,
using the method
developed by Hunt et al. This enabled us to measure oxygen levels in the wound
dead space, at several
definable sites and time points. This also should make
it feasible to calculate the gradients from the center to the edge of the wound.
We also tested the response of the wound to changes in oxygen tension by
varying the breathing gas between room air and pure oxygen.
METHODS
Preparation of Wound Cylinder
Wound cylinders were manufactured with
0.25 Silastic wire mesh and capped with silicone disks on each end.
Cylinders
were 35 mm long and 10 mm in diameter. Two
1- 2 mm long aggregates of LiPc
crystals (total about 200µg) were placed into
thin oxygen permeable Teflon
tubing and fixed by sutures. The distance between the two aggregates of LiPc
was 10-12 mm.
This tubing then was placed inside of the Silastic
mesh cylinder
and one end was fixed at the center of the cylinder.
Wound Cylinder Implantation
Two male Sprague-Dawley rats were
anesthetized with 1.25-1.5% isoflurane in 26% oxygen. Two longitudinal dorsal
midline
incisions were made. Blunt dissection created a space for cylinder
implantation. Each incision was used to place 2 cylinders,
one on each side, a
total of 4 cylinders per animal. Wounds were closed with 4-0 nylon sutures.
EPR Measurements
After
5 days of implantation the animal was anesthetized with 1.5% isoflurane in 21%
oxygen and restrained and placed
between the poles of the magnet of the EPR
spectrometer; the external loop was placed on the surface of the back over the
region
of the skin where the cylinder was located. In this study we made
measurements for 4-6 minutes in each point in two rats
each breathing two
different concentrations of oxygen. After base line measurement (FiO2 = 0.21), the animals were allowed
to stabilize
for 90 min, during which time FiO2 was maintained at 1.0, and then
measurements were carried out for 4-6 minutes
in each point at 1.0 FiO2.
The measurements were repeated at days 5, 8, and 10 after implantation in rat #
1 and at 5, 8, 10, 25, 59,
and 76 days in rat # 2. During the
EPR measurements the rectal temperature was maintained at around 37.0 - 38.0
°C
by using a heating pad.
Sampling of Wound Fluid
On
day 10 after EPR oximetry
measurement, the fluids in the cylinders in
rat # 1 were
withdrawn with a 20 gauge
needle
and a syringe for biological. Cylinders yielded 0.6 - 0.8 ml of fluid
depending on the day sampled. Each cylinder was sampled only once.
RESULTS
The
graph shows the mean oxygen values measured on days 5 – 76. The data also showed a significant
difference in wound pO2
when the breathing gas was changed from 21%
oxygen to 100% oxygen on days 5 to days 25 postimplantation, while there
was no
significant difference on days 59 to days 76 postimplantation. Gross
examination of Silastic tubes and the surrounding tissues
10 days and 76 days
after implantation in two rats revealed a connective tissue layer around the
tube, with much more at day 76.
We speculate that the decrease in the response
of pO2 of older wounds (day 59 to day 76) before and after 100%
oxygen breathing
is probably due to the presence of the dense layer of
connective tissue.
CONCLUSIONS
The results indicate that this methodology can be used to obtain good data on pO2 in wounds with EPR. These results also show that there is a significant effect of time on oxygen tension in this wound model. The methods that were used would be quite feasible for use in human subjects, and plans for such studies are being developed in collaboration with two established clinical groups who study wound healing.
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Figure. Mean oxygen values
measured within wound cylinders (all points) on days after
implantation. All values
given for wound pO2 as mm Hg. Mean ±SD. *Values significantly
elevated
compared with FiO2 = 0.21.