Has Free Radical Release Across the Brain After Carotid Endarterectomy Traditionally Been Underestimated?
Significance of Reperfusion Hemodynamics
Background and Purpose— Ischemia-reperfusion is an established paradigm for the induction of neuro-oxidative stress. The present report highlights the limitations associated with the measurement of free radical exchange across the human brain after carotid endarterectomy if reperfusion hemodynamics are not taken into account.
Summary of Report— Only 2 human studies have reported local changes in the arterio-jugular bulb venous concentration difference (a-vdiff) of free radicals during carotid endarterectomy. The authors reported either no change or only a very minor trans-cerebral release during the course of reperfusion, which was unexpected. However, consistent with other surgical models of ischemia-reperfusion, reperfusion would have been expected to increase plasma volume consistent with reflow-hemodilution. This would artifactually dilute the local concentration of free radicals, attenuate the a-vdiff and thus underestimate the “true” magnitude of cerebral free radical release.
Conclusion— After correction for reflow-hemodilution, the cerebral generation of free radicals after carotid endarterectomy is likely to be significantly more pronounced than previously documented in humans.
Carotid endarterectomy remains the most frequently performed noncardiac vascular procedure clinically proven to reduce the incidence of ipsilateral strokes in patients with carotid stenoses ≥70%.1 However, surgery is not without its complications with cerebral hyperperfusion syndrome, cerebral embolism, cognitive impairment, intracerebral hemorrhage, seizures, and recurrent stenosis among some of the neurovascular problems encountered postoperatively.1
Because surgery involves obligatory ischemia-reperfusion (I-R), an established paradigm for the induction of neuro-oxidative stress, a pathophysiological role for free radicals has been suggested. Modest antioxidant defenses, high mitochondrial density, abundance of redox-active transition metal ions, reactive microglia, auto-oxidizable neurotransmitters, and neuronal membrane lipids rich in polyunsaturated fatty acid side chains exposed to a high-mass-specific oxygen flux, render the reperfused brain exquisitely sensitive to oxidative damage.2
Blood Flow and Free Radical Exchange: Some Critical Considerations
Unlike the recent measurements across the exercising muscle bed3,4 and surgical repair site after abdominal aortic aneurysm and infra-inguinal bypass repair,5 there are no published human studies to date that have documented free radical exchange kinetics across the cerebral circulation during surgery. This measurement requires the simultaneous sampling of arterial (inflow) and jugular bulb venous (outflow) blood most proximal to the brain and regional assessment of flow to calculate the “rate” of generation (identified as a negative arterio-jugular bulb venous concentration difference [a-vdiff] implying net release assuming no change in the arterial concentration) of oxidants or consumption (identified as a positive a-vdiff, thus implying net uptake) of antioxidants downstream of the surgical repair site. This direct approach in, albeit different, human models of I-R has allowed us to demonstrate how free radical and antioxidant exchange kinetics can be manipulated by changes in the activity of mitochondrial respiration4 and after antioxidant prophylaxis.5
Research has traditionally relied on the systemic “accumulation” of indirectly measured, often unreliable biological “footprints” of lipid peroxidation confined to the mixed venous circulation distal to the target organ of interest thus complicating previous interpretation of the source and mechanisms associated with free radical generation during cerebral I-R. Only 2 human studies have documented regional changes in the a-vdiff of redox-reactive biomarkers during carotid endarterectomy, although blood flow measurements were not documented thus preventing calculation of regional exchange kinetics.6,7
Bacon et al6 applied electron paramagnetic resonance (EPR) spectroscopy, the only molecular technique sine qua non that exists for the direct detection and characterization of free radical species. However, using an ex vivo spin-trapping technique that involved the molecular detection of α-phenyl-tert-butylnitrone (PBN) adducts, they could not detect any significant release (indicated by an unchanged a-vdiff) of free radicals during reperfusion despite a subtle indication of an increased jugular venous concentration, the significance of which may have been masked. In contrast, they observed an increase in the a-vdiff of plasma antioxidants suggesting a net uptake or consumption, which was interpreted as evidence for oxidative stress. This was later confirmed by Weigand et al,7 who also demonstrated a trans-cerebral release of malondialdehyde and cell adhesion molecules implying endothelial activation.
Reperfusion Hemodynamics: Significance of Reflow-Hemodilution
These important human studies documenting changes in the a-vdiff of reactive biomarkers6,7 are of particular significance because they imply that regional oxidative stress is indeed a metabolic feature of cerebral I-R, which has important implications for the future design of therapeutic antioxidant strategies aimed at improving patient recovery. However, because these studies did not take into account hemodynamic changes during reperfusion, specifically reflow-hemodilution, it is possible that the magnitude of free radical release was underestimated.
Surgical repair of the carotid stenosis results in a transient increase in cerebral blood flow even in patients without cerebral hyperperfusion syndrome confirmed by transcranial Doppler ultrasonography and single photon emission computed tomography.8 As a consequence of the rapid restoration of normal perfusion pressure, the jugular bulb venous effluent and to a lesser extent, arterial inflow is subject to a transient shift of fluids into the intravascular space causing a net hemodilution. This reflow-hemodilution artifact needs to be taken into account when measuring the concentration of plasma or serum-borne solutes. The decrease in the arterial concentration of hemoglobin 15 minutes after reperfusion (from 10.6 to 10.1 g/dL, the statistical significance of which was not assessed) observed by Weigand et al7 provides some indirect evidence for this hemodilution effect that in other clinical models of surgical I-R can be considerably more marked and as a consequence, lead to an underestimation of oxidant release, or, alternatively, overestimation of antioxidant consumption.5
Correcting for Blood Volume-Shifts
Simply by measuring the a-v concentration of hemoglobin and hematocrit, relative changes in plasma volume can be calculated according to established methods9 and solute concentrations normalized for any hemodynamic changes encountered during the course of reperfusion. As proof-of-concept, albeit in a different surgical model of I-R (abdominal aortic aneurysm repair), the findings illustrated in the Figure highlight the interpretive problems encountered when solute concentrations are not corrected for volume shifts.5
This specific example presents typical EPR spectra of PBN adducts obtained from the common iliac artery and iliac vein in one subject after abdominal aortic aneurysm repair (Figure, A). Blood samples were obtained immediately on clamp release and subsequent restoration of blood flow. The ex vivo PBN “spin-trapping” technique that permits direct molecular detection of predominantly lipid-derived free radicals using X-band EPR spectroscopy has been described in detail elsewhere3,4 and was similar to the methods described by Bacon et al.6
Compared with baseline control measurements (before vascular clamp application), reperfusion increased plasma volume, as indicated by changes in hemoglobin and hematocrit concentrations, by 41% and 44% in the arterial and venous circulation, respectively. Note the much larger EPR signal amplitude (proportional to spin density or concentration) in the venous site indicating a net release of free radicals across the surgical repair site. When samples were corrected for this reflow-hemodilution (Figure, B), the normalized concentration of free radicals and, hence, a-vdiff was markedly greater. Based on a measured cardiac output of 5 L/min, the net release of free radicals increased from 7285 arbitrary units/min (uncorrected) to 10 740 arbitrary units/min (corrected).
Thus, failure to normalize for volume shifts translated into a 47% underestimation of the rate of free radicals released across the surgical repair site. Alternatively, any increase observed in the a-vdiff of nonenzymic antioxidants may simply prove an artifact of reflow-hemodilution and not simply reflect net uptake or consumption (interpreted as oxidative stress) as has traditionally been reported in the literature. Although abdominal aortic aneurysm repair represents a very different model of surgical I-R characterized by greater bulk blood flow, the principle of reflow-hemodilution remains the same and as such represents a useful “proof-of-concept” example because there are no existing data available that permit direct calculation of plasma volume expansion after carotid endarterectomy surgery.
Consistent with other surgical models of I-R, the restoration of blood flow during the course of carotid endarterectomy would be expected to increase plasma volume resulting in a reflow-hemodilution. This would artifactually dilute the blood-borne concentration of free radicals, particularly those confined to the local venous circulation, which has not traditionally been considered. The subsequent shift in the a-vdiff to more positive values may be misinterpreted as evidence for either reduced free radical release or, alternatively, increased antioxidant consumption across the cerebral circulation. Thus, in light of these observations, we suspect that after adequate correction for reflow-hemodilution and simultaneous measurement of cerebral blood flow, the trans-cerebral release of free radicals reported in the literature has probably been underestimated. Antioxidant prophylaxis therefore may be considered an eminently justifiable means of improving patient functional recovery after carotid endarterectomy.
- Received October 11, 2006.
- Revision received January 11, 2007.
- Accepted January 12, 2007.
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