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Femoral Artery Ligation and Hind Limb Muscle pO2 in Different Mouse Strains: an EPR Oximetry and Laser Doppler Investigation

 Armin Helisch1, Nadeem Khan2, Huagang Hou2, and Harold M. Swartz2
1Department of Medicine, Dartmouth-Hitchcock Medical Center, Lebanon, NH 03756, USA
2EPR Center for the Study of Viable Systems, Dartmouth Medical School, Hanover, NH 03755, USA;

 

INTRODUCTION

For hemodynamic reasons the development of true collateral arteries, or arteriogenesis, is clearly the most effective compensatory
vascular growth process after occlusion of a major artery. However, there is still some controversy about how collateral arteries develop.
It often has been assumed that collaterals grow de novo, even though this really has never been proven to be the mechanism.
As recently reviewed, in some animal models of coronary and hind limb ischemia it has been shown that collateral arteries can develop
by positive remodeling of preexisting vessels.1 There also is evidence that preexistent collateral vessels exist in humans. While hypoxia is
an important factor for angiogenesis, whether hypoxia plays a major role for arteriogenesis still is controversial. It has been suggested
that collaterals often grow in non-ischemic tissue and that their growth is primarily related to hemodynamic forces of increased blood flow.
In a rabbit femoral artery ligation model of collateral growth, no evidence for a role of ischemia was detected using an assay
for mRNA levels of hypoxia inducible genes.2

Mice have become a very attractive model for studying vascular growth, as transgene technology for mice is widely available
and therefore should help to define the roles of genes involved in collateral growth. Evidence for differences in blood flow recovery
as measured by Laser Doppler imaging and hemoglobin oxygen saturation as assessed by non-invasive tissue oximetry in the distal hind limbs
after femoral artery occlusion in different inbred strains of mice has been reported, with C57BL/6 mice recovering faster and more completely
than BALB/c mice.3,4 However, there are unexplained discrepancies between strain-dependent perfusion differences observed immediately after
femoral artery ligation and the lack of any differences in average diameters of preexisting collaterals on the surface of the adductor muscles.3
More recently we found with postmortem angiography that collateral dependent filling of the distal hind limbs immediately after femoral
artery ligation is lower in BALB/c mice than in C57BL/6; thus C57BL/6 mice seem to have better developed preexistent collaterals than BALB/c mice.4
Furthermore, we have observed tissue necrosis in the areas of collateral growth in the adductor muscle group of BALB/c mice,
suggesting significant local ischemia in the area of collateral growth at least in this mouse strain.4

The goal of this study was to test the following questions pertaining to the mouse femoral artery ligation model: (1) Is there any local hypoxia
in the areas of collateral growth in the proximal hind limbs of BALB/c and C57BL/6 mice? (2) Do BALB/c mice have more hypoxia compared to C57BL/6 mice,
even though they have no evidence for increased collateral growth? (3) How severe is the hypoxia in the distal hind limbs,
where predominantly angiogenesis occurs, compared to the proximal hind limbs, where the collateral arteries grow?

 

MATERIALS AND METHODS

Animals and Surgery

Six male BALB/c and six C57BL/6 mice (Charles River Laboratories, Wilmington, MA) were used in this study. The experimental techniques
and protocol were approved by the Dartmouth College Animal Care and Use Program. Three mice of each strain were used for measurements
in a particular muscle group: gastrocnemius or adductor muscles. The mice were anesthetized using 1.5-2.0 % isoflurane, and approximately 100 µg of EMS char
was injected in the left and right hind limbs. After 14 days of implantation, the baseline pO2 was measured. Femoral artery ligations were performed
(6/0 silk, Ethicon) just distal to the deep femoral artery and proximal to the a. genus descendens in the right hind limb, while only sham ligations
were performed in the left hind limb (day 0).  The tissue pO2 was measured after ligation at days 1, 2, 3, 5, 7, 14 and 21.
The mice were maintained with 1.25-1.5% of isoflurane with 26% oxygen through a sealed nose cone at a rate of 2 liters/minute
throughout the experiment. The animal temperature was maintained by using a heated water blanket and warm air forced through the gap of the magnet.  

               

EPR Measurements

In vivo EPR measurements were carried out on a 1.2 GHz (L - band) EPR spectrometer.  The mice were placed in the magnet, and the extended
loop resonator was placed directly above the site of EMS implantation. Typical settings for the spectrometer were: incident microwave power, 80 mW;
magnetic field center, 400 gauss; scan range, 20 gauss; and scan time 12-15 seconds. Modulation amplitude was set at less than one-third
of the EPR line width. Usually 6-9 spectra were averaged to achieve a better signal to noise ratio.

 

RESULTS

The changes in pO2 observed in gastrocnemius muscle of BALB/c and C57BL/6 mice are shown in Figures 1a and 1b respectively
and the changes observed in adductor muscles are shown in Figures 2a and 2b respectively.

 

 

 

 

 

 


      Fig. 1a


 

 

 

 

 


Fig. 1b
 Figure 1: (a) Gastrocnemius muscle pO2 before and after femoral artery ligation in Balb/c (n=2) and in (b) C57BL/6 mice (n=3), Mean ± SE.

 

 

 

 


      Fig. 2a

 

 

 

 


     Fig. 2b
     Figure 2: (a) Adductor muscle pO2 before and after femoral artery ligation in Balb/c (n = 3) and in (b) C57BL/6 mice (n = 3), Mean ± SE

 

DISCUSSION

The baseline pO2 values in all the four sets of mice were 20 – 30 mm Hg and were similar in both hind limbs, which is as expected
and indicates the validity of the methodology of EPR oximetry for this type of experiment. A significant transient decrease in baseline pO2
 was observed in the proximal and distal muscles of the hind limbs of both mouse strains on the ligated side, without any significant changes
on the sham ligated side (control). In C57BL/6 mice the decrease in pO2 seemed to be more delayed. In the distal gastrocnemius C57BL/6 mice
appeared to have a faster recovery of their pO2 values. These are encouraging results and indicate that we can follow changes in pO2
in these muscle groups before and during compensatory vascular growth after ligation. We therefore plan further experiments to obtain
data for both pO2 and relative blood perfusion rates (as assessed by laser Doppler imaging) with a larger number of animals, and then to study
the molecular biology of the process.

 

1.Helisch A, Schaper W. Arteriogenesis: The development and growth of collateral arteries. Microcirculation, 2003; 10: 83-97.

2.Deindl E, Buschmann I, Hoefer IE, Podzuweit T, Boengler K, Vogel S, van Royen N, Fernandez B, Schaper W.
Role of ischemia and of hypoxia-inducible genes in arteriogenesis after femoral artery occlusion in the rabbit. Circ Res. 2001; 89: 779-86.

3.Scholz D, Ziegelhoeffer T, Helisch A, Wagner S, Friedrich C, Podzuweit T, Schaper W. Contribution of arteriogenesis and angiogenesis
to postocclusive hindlimb perfusion in mice. J.  Mol.Cell Cardiol. 2002;34: 775-87.

4.Helisch A, Wagner S, Brandt U, Heil M, Ziegelhoeffer T, Bachmann G, Schaper W. Genetic background is a major determinant of arteriogenesis
after femoral artery ligation in inbred mice. J Am Coll Cardiol. 2003; 41: 279A.

 

ACKNOWLEDGMENTS

This work was supported by a NHLBI grant, RO1 HL53793, “ Angiongenesis in Myocardial Ischemia”, by a NIH (NIBIB) grant PO1 EB002180,
“Measurement of pO2 in Tissues In Vivo and In Vitro,” and used the facilities of the EPR Center for the Study of Viable Systems
supported by NIH (NIBIB) grant P41 EB002032.

 

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