Periischemic Cerebral Blood Flow (CBF) Does Not Explain Beneficial Effects of Isoflurane on Outcome from Near-complete Forebrain Ischemia in Rats

The following study was approved by the Duke University Animal Care and Use Committee. Male Sprague-Dawley rats (8–10 weeks old; Harlan Sprague-Dawley, Indianapolis, IN) underwent fasting for 12–16 h but had free access to water. The animals were then anesthetized with 5% isoflurane in oxygen. After orotracheal intubation, the lungs were mechanically ventilated (30% O²–balance N²). The inspired isoflurane concentration was reduced to 2.0–2.5%. Surgery was performed using the aseptic technique, and all surgical fields were infiltrated with 0.1–0.2 ml lidocaine, 1%. Cannulation was performed on the tail artery and used for blood pressure monitoring and arterial blood sampling. In addition, catheters were placed via  surgical incision in the right femoral vein and artery for isotope infusion and collection of timed arterial blood samples, respectively. Via  a ventral neck incision, cannulation was performed on the right jugular vein for drug infusion and blood withdrawal. The common carotid arteries were encircled with suture. The vagus nerves and cervical sympathetic plexus were left intact. Muscle paralysis was provided by a 1 mg intravenous bolus of succinylcholine, repeated as necessary to allow control of ventilation during ischemia. Pilot studies had been performed to ensure that rats would not exhibit an escape response in the absence of succinylcholine, given the respective anesthetic regimens. Bilateral cortical electroencephalographic (EEG) responses were continuously monitored during the experiment from active subdermal electrodes positioned over the parietal cortex bilaterally, a reference electrode placed on the nasion, and a ground lead positioned in the tail.

A 22-gauge needle thermistor (Model 524; YSI Co., Yellow Springs, OH) was percutaneously placed adjacent to the skull beneath the temporalis muscle, and pericranial temperature was servoregulated at 37.5 ± 0.1°C by surface heating or cooling. Heparin, 50 IU, was administered intravenously. After surgical preparation, a 20-min interval was allowed for physiologic stabilization.

For determination of preischemic CBF (just before ischemia), rats were randomly assigned to one of two groups:

• fentanyl–N²O or isoflurane (n = 9): Isoflurane was discontinued. An intravenous infusion of fentanyl was begun (10 μg/kg bolus followed by 25μg · kg⁻¹· h⁻¹). The inspiratory gas mixture was changed to 30% O²–70% N²O.

• Isoflurane (n = 11). Isoflurane, 1.4%, was inspired in 30% O²–balance N².

For determination of intraischemic CBF (at 8 min after onset of ischemia), rats were randomly assigned to one of three groups:

• Fentanyl–N²O (n = 5). Isoflurane was discontinued. An intravenous infusion of fentanyl was begun (10μg/kg bolus followed by 25μg · kg⁻¹· h⁻¹). The inspiratory gas mixture was changed to 30% O²–70% N²O.

• Isoflurane (n = 5). Isoflurane, 1.4%, was inspired in 30% O²–balance N².

• Isoflurane + trimethaphan (n = 5). Isoflurane, 1.4%, was inspired in 30% O²–balance N². Trimethaphan (2.5 mg intravenously) was administered at the onset of ischemia.

For determination of postischemic CBF (30 min after onset of reperfusion), rats were randomly assigned to one of three groups:

• Fentanyl–N²O (n = 9). Isoflurane was discontinued. An intravenous infusion of fentanyl was begun (10μg/kg bolus followed by 25μg · kg⁻¹· h⁻¹). The inspiratory gas mixture was changed to 30% O²–70% N²O.

• Isoflurane (n = 8). Isoflurane, 1.4%, was inspired in 30% O²–balance N²;

• Isoflurane + trimethaphan (n = 8). Isoflurane, 1.4%, was inspired in 30% O²–balance N². Trimethaphan (2.5 mg intravenously) was administered at the onset of ischemia.

A 30-min interval was allowed to establish the respective anesthetic states. Minute ventilation was adjusted to maintain partial pressure of arterial carbon dioxide (Paco²) within 36–42 mmHg.

Ischemia was induced as follows. Mean arterial pressure (MAP) was reduced by venous blood withdrawal to 30 mmHg, followed by bilateral carotid occlusion using temporary aneurysm clips. 6,11In animals that were used for measurements of postischemic CBF, ischemia persisted for 10 min. To discontinue ischemia, shed blood was reinfused, and the aneurysm clips were removed from the carotid arteries. NaHCO³(0.3 mEq intravenously) was administered to counteract systemic acidosis.

At the respective CBF measurement intervals, 100 μCi/kg of 4-iodo-n-methyl-[¹⁴C]antipyrine (¹⁴C-IAP, specific activity 60 mCi/mmol, American Radiolabeled Chemicals, Inc., St. Louis, MO) was infused intravenously over 60 s in a 60-step ramp (10 μl/min–610 μl/min) to produce an increasing ¹⁴C-IAP arterial concentration. Twelve timed, 20-μl arterial blood samples (one sample every 5 s) were collected during ¹⁴C-IAP infusion for determination of arterial ¹⁴C activity. Immediately after the last blood sample and at completion of isotope infusion, all animals were decapitated and brains were rapidly frozen in 2-methylbutane (−40°C). Brain sections and blood samples were prepared for autoradiographic analysis according to previously described methods. 12–14Autoradiographic images of six anatomically standardized brain sections were analyzed by an observer blind to experimental group. Regional CBF values were determined from at least three sections, and values for each of the six different regions for each animal were averaged. In addition, autoradiographic density histograms were compiled for each region of interest to determine the percentage cross-sectional area within each region where flow was less than 15 ml · 100 gm⁻¹· min⁻¹.

In separate analysis for each measurement interval (i.e. , before, during, and after ischemia) physiologic and regional CBF values were compared among the treatment groups by one-way analysis of variance. Post hoc  test for between group difference was performed using the Fisher protected least-significant difference test when indicated by a significant F ratio. Statistical significance was assumed when P < 0.05. Values are reported as mean ± SD. Statistical evaluations were performed using StatView 5.0 (SAS Institute Inc., Cary, NC).

Physiologic values are presented in table 1. Pre- and postischemic MAP was less in rats anesthetized with isoflurane than in those administered fentanyl–N²O (P ≤ 0.04). During the ischemic interval, EEG isoelectricity was present in all animals that were included in later analysis. Five animals (three in the isoflurane group, two in the fentanyl–N²O group) were excluded because the criterion of intraischemic EEG isoelectricity was not reached. Pericranial temperature was controlled in all animals at 37.5 ± 0.2°C.

Regional CBF values for six selected regions are presented in figure 1. Preischemic CBF did not differ between isoflurane and fentanyl–N²O anesthetized animals in any region.

During ischemia, no difference in blood flow was detected among groups in any region. The ischemic insult caused uniform blood flow reduction in the caudoputamen, cortex, and hippocampus to 5% or less of preischemic values. Thalamic blood flow was reduced to 10% or less of preischemic values. Blood flow in the cerebellum and pontine nucleus was attenuated but preserved well above critical flow levels. In these caudal regions, less than 5% of tissue had blood flow values less than 15 ml · 100 gm⁻¹· min⁻¹.

At 30 min after onset of reperfusion, there were no differences among groups for mean blood flow values in the caudoputamen, cortex, hippocampus, or thalamus. Flow remained reduced by 50–65% relative to preischemic values in all three groups. In all groups, less than 15% of cross-sectional area in the caudoputamen, cortex, hippocampus, and thalamus had flow less than 15 ml · 100 gm⁻¹· min⁻¹. However, differences between groups for percent cross-sectional area with flow less than 15 ml · 100 gm⁻¹· min⁻¹were observed in cortex (fentanyl–N²O = 15%vs.  isoflurane + trimethaphan = 3%;P = 0.01; and isoflurane = 11%vs.  isoflurane + trimethaphan = 3%;P = 0.03) and hippocampus (fentanyl–N²O = 11%vs.  isoflurane + trimethaphan = 6%;P = 0.02). Blood flow in the brain stem and cerebellum returned to greater than 80% of preischemic values without differences among groups.

Isoflurane is a frequently used volatile anesthetic with cerebrovasodilatory effects and has repeatedly been shown to improve outcome in laboratory models of cerebral ischemia. 1–4In a recent study, isoflurane improved histologic outcome after severe forebrain ischemia when compared with fentanyl–N²O anesthesia, an effect that was completely reversed by sympathetic ganglionic blockade with trimethaphan. 5The purpose of this investigation was to evaluate whether beneficial effects of isoflurane after near-complete ischemia in rats could be attributed to its effects on CBF and whether the presence of trimethaphan would alter CBF during or after ischemia. The results of this study show no CBF differences between anesthetic groups in any structure evaluated before or during ischemia. Trimethaphan administered to animals anesthetized with isoflurane did not affect intraischemic CBF. Although pronounced delayed postischemic hypoperfusion was observed in all groups, neither anesthetics nor trimethaphan where found to significantly alter this event.

Anesthetic conditions and doses chosen for this study were based on two recent studies that showed a neuroprotective effect of 1.4% isoflurane when compared with fentanyl–N²O during near-complete forebrain ischemia. 4,5Known physiologic determinants of CBF, such as Paco², partial pressure of arterial oxygen (Pao²), and temperature, were kept within physiologic ranges in all animals. Rats anesthetized with isoflurane showed a consistent pattern of lower preischemic and postischemic MAP when compared with rats anesthetized with fentanyl–N²O. However, substantially greater differences in MAP are necessary to alter histologic outcome in this model. 15During ischemia, MAP was held at 30 mmHg in all groups. This, in combination with bilateral carotid occlusion, resulted in consistent EEG isoelectricity in all animals included in the study. Miura et al.  4have also shown that the near-complete ischemic injury, as used in the current experiment, is uniformly severe among subjects in different anesthetic groups. Cortical and hippocampal EEG recordings in that study were homogeneous with respect to presence of isoelectricity, and all animals underwent ischemic depolarization. However, the CBF thresholds for EEG isoelectricity might be significantly higher than those necessary to elicit depletion of high-energy brain phosphates, disturbances of cellular ion homeostasis, and neuronal cell death. 11,16,17 

The preischemic CBF values in rats anesthetized with 1 minimum alveolar concentration (MAC) isoflurane are in agreement with previous reports. 18–21Preischemic CBF in rats anesthetized with isoflurane did not differ significantly from those administered fentanyl–N²O. As expected from previous studies of near-complete forebrain ischemia in rats, 6,7intraischemic CBF values in the hippocampus, cortex, and caudoputamen were reduced to 5% or less of baseline values in all three groups. Choice of anesthetic or presence of trimethaphan in rats anesthetized with isoflurane did not have a significant impact on this parameter. Accordingly, we can refute the hypothesis that isoflurane has an impact on intraischemic CBF that alters the severity of the ischemic insult.

Although severe postischemic hypoperfusion was observed at 30 min after the end of ischemia, there was also no effect of either anesthetics or trimethaphan on the absolute severity of this condition. The only differences detected were in the amount of tissue with postischemic CBF less than 15 ml · 100 gm⁻¹· min⁻¹between fentanyl–N²O and isoflurane-plus-trimethaphan–treated animals (cortex and hippocampus) and isoflurane- and isoflurane-plus-trimethaphan–treated animals (hippocampus), but not between isoflurane- and fentanyl–N²O-treated rats. However, because isoflurane-anesthetized animals previously had better histologic outcomes than fentanyl–N²O-treated and isoflurane-plus-trimethaphan –treated animals (with no difference between the latter two groups), 5we conclude that these minor flow differences do not impact histologic outcome significantly.

The syndrome of delayed postischemic hypoperfusion characterizes a phase of increased vascular resistance after an initial phase of postischemic reactive hyperemia. 22Postischemic hypoperfusion has been described in rat models of focal, 23incomplete, 24and complete global 25ischemia, but the underlying mechanisms are understood incompletely. Possible explanations include an imbalance of preserved autoregulation and diminished carbon dioxide reactivity, resulting in arteriolar vasoconstriction 22; expression of endothelial adhesion molecules, with consequent aggregation of polymorphonuclear leukocytes 26and formation of endothelial microvilli 27; and increased blood viscosity or microvascular compression by extracellular edema and glial swelling. 28The magnitude of hypoperfusion observed in the current study is comparable with previous investigations. Neither vasodilative effects of isoflurane nor sympatholytic properties of trimethaphan were able to attenuate this postischemic disturbance in CBF. The latter observation is in agreement with Blomquist et al. , 25who reported that lesioning of the locus coeruleus does not influence postischemic hypoperfusion in rats after complete global ischemia.

In summary, this study compared regional CBF before, during, and after near-complete forebrain ischemia in rats anesthetized with either isoflurane (with and without trimethaphan) or fentanyl–N²O. The results of this study present convincing evidence that previously reported neuroprotective effects of isoflurane and reversal of this protection by trimethaphan cannot be explained by differential vasodilative effects of the anesthetics or trimethaphan during severe forebrain ischemia or postischemic delayed hypoperfusion. Mechanisms other than effects on CBF are responsible for beneficial effects of isoflurane in this model.

The authors thank Ann D. Brinkhous, Technician, Department of Anesthesiology, Duke University Medical Center, Durham, North Carolina, for expert technical assistance.