Ischemia in Rabbit Limbs
Oleg Y. Grinberg1, Huagang Hou1,
Stalina A. Grinberg1, Karen L. Moodie2,
Bruce J. Friedman2,
Mark J Post3, Harold M. Swartz1
1EPR Center for the Study of Viable Systems, Dartmouth
Medical School, Hanover, NH USA;
2Dartmouth-Hitchcock Medical Center, Lebanon, NH;
3Department
of Physiology and of Biomedical Technology, Maastricht University,
Universiteitssingel 50,
6229 ER Maastricht, The Netherlands
INTRODUCTION
Atherosclerotic obstruction
of a large conduit artery is an important cause of acute ischemia in limbs. In
the absence of collaterals
and with insufficient angiogenesis, ischemia may
result in continuous pain, inadequate wound healing, and potential necrosis of
the extremity.
The lack of reliable methods that directly measure regional limb
oxygen tension (pO2) is an obstacle to the evaluation of recently
developed
molecular strategies to reverse severe limb ischemia. EPR oximetry
has been proven successful in making repeated measurements of the actual
oxygen
tension (pO2) in different tissues. Once the oxygen sensitive
material is implanted, this technique can provide repeated non-invasive
measurements of the pO2 at specific sites within tissue in vivo. In
this study the effects of
femoral ligature on pO2 in New
Zealand white male
rabbits (n=7) was monitored by EPR oximetry with EMS char for 2 weeks. We also examined whether a correlation
exists between the pO2
in muscles as measured by EPR oximetry and
blood flow as measured using microspheres.
METHODS
Animal Model
For the initial surgery the
animals were anesthetized, intubated, and placed on a respirator. Two incisions
in each thigh were made
to access the femoral artery and its side branches. For
initial flow measurements, non-radioactive microspheres were injected into the
left
ventricle to provide adequate mixing of the spheres, while a reference
blood sample was drawn from the femoral artery. A 4 Fr JR4 catheter
was
advanced into the descending aorta through the repositioned arterial sheath and
a selective iliac angiogram was made at the side of ligation.
Then the contralateral femoral artery was dissected and ligated at two positions spaced
1.5 cm apart and distal from the internal femoral artery
and proximal from the
trifurcation. The angiogram was repeated, and the microsphere injection
repeated with a different isotope. For euthanasia,
animals were anesthetized
and heparinized. The contralateral femoral artery was cannulated for withdrawal
of blood samples and the saphenous
artery on the ligated femoral artery side
was cannulated for monitoring peripheral pressure. The flow in each
iliac artery was measured
with a T601 flowmeter (Transonic Systems,
Inc). Systemic pressures, peripheral capillary pressure, and flow were analyzed
on-line by a Micro-Med system.
Implantation of EMS Char
EMS
wood char was sterilized by autoclaving. The material was pre-calibrated using
a standard procedure. Three weeks before surgery
the rabbits were anesthetized
by mouth cone with an inhalation gas mixture (isofluorane 1.1%-1.2%,
oxygen/nitrogen 26%/74%), and EMS
wood char was implanted in 4 muscles of two
legs (2-3 mg each implant): #1 peroneus, #2 gastrocnemius, #3 gluteus, #4
adductor. The material
was deposited by pushing a plunger through the barrel of
an 18-gauge needle. The depth of implantation was 2-3 mm. A baseline pO2
reading
was taken just before surgery. Then pO2 measurements were
made after surgery and two weeks later just before euthanasia.
Measurement of pO2 in Muscles Using EPR Oximetry
When measurements of the pO2
were desired the animal was anesthetized as before, placed between the poles of
the magnet of |
the EPR spectrometer, and a detector in the form of an extended
loop was placed on the surface of the leg over each region of muscle where
the
material was located. Body temperature was maintained within normal range on a
heated pad and a rectal probe monitored the core temperature
of the rabbit.
Typically four EPR spectra were collected every five minutes. The EPR line
widths were converted to pO2 using a calibration curve
determined
for EMS char.
Calculation of Flow
For the calculation of sample
flows, we used the mean sample activity (Am) per gram of
muscle weight and related this to total flow (Ft)
per
gram of muscle weight, which allowed the calculation of sample flow
(Fs) using the following equation: Fs=Ft/AmxAs.
This correlates well
with the calculation of Fs from
sample activity (As), reference sample activity (Ar),
weight of the reference sample (Wr), and time of
reference
sample withdrawal (t), according to the following
equation: Fs=As/ArxWr/t.
RESULTS AND DISCUSSION
Using EPR oximetry and
radioactive microspheres, pO2 and regional blood flow were measured
in the same rabbit muscles in non-occluded
and occluded legs before surgery,
post surgery, and two weeks after surgery. We were able to follow the effects
of ligation on the pO2 in four
different muscle groups and found
that these differed significantly. A
correlation between and blood flow in the ligated legs for each type of muscle
was observed. Decreases in pO2 in the non-occluded leg
were observed as well, indicating that the catheterization of the artery for blood withdraw
also
results in lower oxygen supply into the muscles.
CONCLUSIONS
The results with EPR oximetry
were consistent with the information obtained with measurements of blood flow.
The data with EPR oximetry,
however, provided additional information and were
able to be carried out repeatedly in the same animals in four different muscle
groups. This study
demonstrates, for the first time, the applicability of EPR
oximetry in animals larger than rodents.