An in vivo rat model of isolated intestinal
ischemia-perfusion was developed. This is used to compare the effects of crosslinked
hemoglobin (PolyHb) versus crosslinked hemolobin-
superoxide dismutase-
catalase (
PolyHb-SOD-CAT) on
free radical generation in
ischemia-reperfusion. Fasted, anesthetized male Sprague Dawley rats underwent midline
laparotomy with cannulation of the abdominal aorta and inferior vena cava.
Ligation was carried out at the renal pedicles bilaterally and the aorta and vena cava proximally at the diaphragm and distally above the femoral bifurcation. The system was flushed of blood with 20 ml of
lactated Ringer's solution. The portal vein was then cannulated with distal clamping at the porta hepatis so that isolated intestinal perfusion could be achieved with the aorta as the inlet and the portal vein as the outlet. Following a 90 minute ischemic time, perfusates containing modified
hemoglobin (5 g/dl) and
4-hydroxybenzoate (5 mM) were infused at 0.8 ml/min for 10 min. Portal vein effluent samples were collected at 2.5 minute intervals.
Hydroxyl radical generation was assessed by an aromatic hydroxylation technique with
4-hydroxybenzoate (4HB). Reaction of
hydroxyl radical with 4HB produces 3,4 dihydroxybenzoate (3,4 DHBA). In the PolyHb group, the levels of 3,4-DHBA increased 10.75-13.58 x-fold above pre-perfusion values compared to 2.25-3.75 x-fold in
PolyHb-SOD-CAT group. This indicates that
PolyHb-SOD-CAT is effective in reducing in vivo
hydroxyl radical generation following reperfusion. Since
free radicals may play a major role in the pathogenesis of
ischemia-reperfusion injury, this suggests a role for
PolyHb-SOD-CAT as a possible protective perfusate in intestinal
reperfusion injury.