Relationship between Local Cerebral Blood Flow and Metabolism during Mild and Moderate Hypothermia in Rats

After obtaining approval from the institutional animal care committee (Regierungspräsidium Karlsruhe, Germany), the experiments were performed on 48 male Sprague-Dawley rats weighing 326 ± 29 g (Charles River Deutschland, Sulzfeld, Germany). Animals were kept under temperature-controlled environmental conditions on a 14:10 light:dark cycle, were fed a standard diet (Altromin C 1000; Altromin, Lage, Germany), and were allowed free access to food and water ad libitum  until the experiments were started.

Rats were placed in a small box and anesthetized by inhalation of a gas mixture of isoflurane (1 minimum alveolar concentration [MAC]), oxygen (40%), and air (remainder). Afterward, 1 MAC of isoflurane (Forene; Abbott, Wiesbaden, Germany) was administered using a precalibrated vaporizer (Fortec; Cyprane Keighley, United Kingdom) by inhalation via  a nose cone. MAC values were corrected for reduced actual body temperatures as determined by Vitez et al.  10Isoflurane, 1 MAC, corresponds to 1.2% at 37.5°C, 1.03% at 35°C, and 0.84% at 32°C using a fresh gas flow of 2 l/min. Tracheostomy and cannulation of the right femoral artery and vein were performed using polyethylene catheters (PE-160 and PE-50; Labokron, Sinsheim, Germany). Mean arterial blood pressure and heart rate were registered continuously by a quartz pressure transducer (Hewlett-Packard, Palo Alto, CA). Animals were mechanically ventilated (Small Animal Ventilator, KTR4; Hugo Sachs Electronic, March, Germany). Artificial ventilation was performed using capnometric control of end-tidal carbon dioxide partial pressure (Capnometer, Heyer, Bad Ems, Germany) and continuous pulse oximetry (Onin 8600V Pulse Oximeter; Onin Medical Inc., Plymouth, MN). Arterial blood gases were checked in a pH/blood gas analyzer (AVL Gas Check 939; AVL, Graz, Austria). Pericranial temperature was measured using ultrafast microthermocouple probes (IT-23, diameter 0.3 mm; Almemo 2290-3S Thermometer, Hugo Sachs Elektronic), introduced through the masseter muscle to the outside of the base of the rat skull. Rectal temperature was measured simultaneously, and body temperature was kept constant at 37–37.5°C with a temperature-controlled heating pad during the surgical preparation (Harvard Ltd., Kent, United Kingdom). Physiologic parameters were assessed and recorded just before the determination of CBF or CGU.

After completion of the surgical preparation, animals were randomly assigned to one of the following four groups:

• 1. Normothermic, conscious control animals (n = 12). These animals were allowed to recover from anesthesia after instrumentation of the femoral vessels. Thereafter, the animals were placed in a rat restrainer and were studied approximately 60 min after recovery from anesthesia. The temperature-controlled heating pad was only used during the surgical preparation, and physiologic temperature regulation was not influenced after recovery from anesthesia.

• 2. Normothermic, anesthetized animals (n = 12). In these rats, anesthesia was maintained at 1.2% isoflurane (1 MAC), and body temperature was kept constant at 37.5°C using a hollow plastic spiral surrounding the entire animal body, which was continuously perfused with water of target temperature. Animals were mechanically ventilated to maintain an arterial carbon dioxide partial pressure (Pa^CO2) of 40 mmHg, and experiments were started after a 60-min stabilization period.

• 3. Mildly hypothermic, anesthetized, animals (n = 12). Animals assigned to this group were first allowed to cool down passively and then were actively cooled until the target temperature was reached (approximately 30 min). Active cooling was also performed using the hollow plastic spiral, continuously perfused with water of target temperature. After achieving the target temperature of 35°C, pericranial temperature was maintained constant for 60 min until the radioactive tracer was administered. Blood gases were corrected for actual body temperature, and ventilatory support was adjusted to maintain a Pa^CO2of 40 mmHg (after correction for individual core temperature, i.e. , p  H-stat management).

• 4. Moderately hypothermic, anesthetized animals (n = 12). Animals assigned to this group were treated as described for the mildly hypothermic group, with the exception that body temperature was decreased to 32°C.

In each group, the 12 rats were randomized to be used either for the autoradiographic determination of LCBF (n = 6) or for the measurement of LCGU (n = 6) according to the methods described by Sokoloff et al.  11and Sakurada et al.  12Previous studies have validated these autoradiographic methods for a wide range of body and brain temperatures down to a body temperature of 9°C. 13–15 

For the measurement of LCGU, 125 μCi/kg body weight of 2-[1-¹⁴C]deoxy-D-glucose (specific activity, 50–56 mCi/mmol; New England Nuclear, Dreieich, Germany) was injected as a pulse via  the femoral venous catheter over 20 s, and timed arterial blood samples of 80 μl were collected through the arterial catheter at 15, 30, and 45 s and at 1, 2, 3, 5, 7.5, 10, 15, 25, 35, and 45 min. The blood samples were immediately centrifuged and stored on ice until assays for plasma 2-[1-¹⁴C]deoxy-D-glucose and glucose concentrations were performed. Immediately after the final arterial blood sample was collected, the animal was decapitated and the brain was rapidly removed and frozen in isopentane chilled to −60°C.

For the measurement of LCBF, 100 μCi/kg body weight of 4-iodo[N -methyl-¹⁴C]antipyrine (specific activity, 54 mCi/mmol; Amersham-Buchler, Braunschweig, Germany) dissolved in 1 ml saline was infused continuously at a progressively increasing infusion rate for 1 min via  the femoral venous catheter. The progressively increasing infusion rate, a modification of the method described previously, 12was chosen to minimize equilibration of rapidly perfused tissues with arterial blood during the period of measurement. During the 1-min infusion period, 14–20 timed blood samples were collected in drops from the free-flowing arterial catheter directly onto filter paper disks (1.3 cm in diameter) that had been prepared in small plastic beakers and weighed. The samples were weighed, and radioactivity was estimated with a liquid scintillation counter (TriCarb 4000 series; Canberra Packard, Frankfurt, Germany) after extraction of the radioactive compound with ethanol. After the 1-min infusion and sampling period, the animal was decapitated and the brain was removed as quickly as possible and frozen in isopentane chilled to −60°C. In both the 2-[1-¹⁴C]deoxy-D-glucose and 4-iodo[N -methyl-¹⁴C]antipyrine experiments, the frozen brains were coated with chilled embedding medium (Lipshaw, Detroit, MI), stored at −80°C in plastic bags, divided in 20-μm sections at −20°C in a cryostat, and autoradiographed along with precalibrated [¹⁴C]methyl methacrylate standards.

Local tissue concentrations of [¹⁴C] were determined from the autoradiographs by densitometric analysis. LCGU and LCBF were calculated from the local concentrations of [¹⁴C] and the time courses of the plasma [¹⁴C]deoxyglucose and iodo[¹⁴C]antipyrine concentrations, including corrections for the lag and washout in the arterial catheter. 11The washout correction rate constant was 100/min, and the brain–blood partition coefficient for iodo[¹⁴C]antipyrine was 0.9 in our rats. 16 

Autoradiographic images were converted to digitized optical density images by an image processing system (MCID; Imaging Research, St. Catharines, Canada). For measurements of separate brain structures, an ellipsoid cursor was used and adjusted to the size of the individual region. For measurement of mean global CBF or mean global CGU (referred to hereafter as mean CBF and mean CGU), coronal sections were analyzed as a whole at distances of 200 μm, and the values were summarized to obtain the area-weighted means of all measured sections. 17 

Differences between the experimental groups were evaluated by analysis of variance, and Bonferroni correction was used when multiple comparisons were performed. Data are presented as mean ± SD, and a P  value < 0.05 was considered statistically significant. The overall relationship between LCGU and LCBF in the examined structures of the brain (fig. 1) was assessed by the least-squares fit of the data to y = ax + b, where x is the mean LCGU in a given region and y is the mean LCBF in that same area. Contrasts of slopes of the LCBF/LCGU regression lines were tested by common t  test statistics with Bonferroni correction for multiple comparisons. Because of the limitations of this kind of analysis, an additional, more rigorous statistical approach using log-transformed data were applied, examining the relationship of LCBF and LCGU by a repeated measure of the analysis of variance according to McCulloch et al.  18and Ford et al.  19For this analysis, a computer software package (BMDP2v; BMDP Statistical Software Inc., Los Angeles, CA) considering interanimal variability and enabling the detection of heterogeneities in the relationship between LCGU and LCBF was used.

All animals survived the surgical procedure. Physiologic variables of conscious, anesthetized, and hypothermic rats are given in table 1. No statistically significant differences in baseline values were observed between all four groups. Compared with normothermic conscious rats, mildly hypothermic animals showed slightly, although significantly reduced, heart rate and pH, whereas arterial oxygen tension and plasma glucose concentrations increased. In the moderately hypothermic group, mean arterial pressure reached baseline values of conscious controls, and hematocrit increased slightly compared with the normothermic conscious rats (table 1).

Cerebral blood flow was measured for the entire brain, as well as regionally for 37 different brain structures. The results are shown in table 2. Compared with normothermic conscious animals, mean CBF was not significantly altered during isoflurane anesthesia (1 MAC). Mean CBF was significantly increased during mild hypothermia (P < 0.05) but did not differ from normothermic conscious controls in the moderately hypothermic group. On a local level, no significant reduction in LCBF was observed during mild or moderate hypothermia in any structure compared with normothermic anesthetized animals. LCBF significantly increased in 9 of 37 structures during mild hypothermia and in 1 structure during moderate hypothermia compared with normothermic anesthetized animals (P < 0.05).

In addition to blood flow, mean CGU and LCGU were measured in the four different groups of rats. The results are shown in table 3. During isoflurane anesthesia (1 MAC), mean CGU was reduced from 56 ± 3 to 31 ± 5 μmol · 100 g⁻¹· min⁻¹compared with the conscious group (45%;P < 0.05). No further reduction in mean CGU was achieved by cooling to 35°C (32 ± 3 μmol · 100 g⁻¹· min⁻¹;P = nonsignificant). Moderate hypothermia resulted in an additional significant reduction of mean CGU to 16 ± 3 μmol · 100 g⁻¹· min⁻¹(48%vs.  isoflurane and 71%vs.  conscious group;P < 0.05). The local changes observed in the 37 brain structures are also shown in table 3. Compared with the normothermic anesthetized group, mild hypothermia did not induce significant changes in LCGU within any individual structure investigated. This result is congruent with the lack of change in mean CGU in this group. Moderate hypothermia induced a significant reduction in LCGU compared with the normothermic anesthetized group in 32 of 37 brain regions examined (P < 0.05). LCGU was reduced by > 50% in 15 of these 32 brain structures and by 20–50% in 17 structures.

The relationship between LCBF and LCGU is plotted in figure 1. A strong linear relationship was found between LCBF and LCGU in the conscious and anesthetized normothermic groups, as well as in both hypothermic groups. This is indicated by the correlation coefficients between LCBF and LCGU for normothermic conscious (r = 0.94), normothermic anesthetized (r = 0.94), and hypothermic rats (35°C: r = 0.92; 32°C: r = 0.95). These correlation coefficients were significantly different from zero (P < 0.05).

The ratio between LCBF and LCGU is reflected by the slope of the individual LCBF–LCGU regression line (fig. 1). Isoflurane anesthesia caused a significant increase in the slope of the regression line from 1.4 in the normothermic conscious animals to 2.4 ml/μmol after anesthesia induction (P < 0.05). Mild and moderate hypothermia further reset the ratio of LCBF to LCGU, and the slope of the regression line increased significantly to 3.6 ml/μmol (P < 0.05). No significant difference was found between the slopes of the regression lines of mildly and moderately hypothermic rats.

The main finding of the present study was that a strong linear relationship between LCBF and metabolism is maintained during normothermic isoflurane anesthesia (1 MAC), as well as during mild and moderate hypothermic isoflurane anesthesia. Anesthesia resulted in a significant resetting of the slope of the LCBF-to-LCGU regression line to a higher level. During mild hypothermia, a further significant increase in the LCBF-to-LCGU slope was observed when compared with normothermic anesthesia. No additional increase in the slope of the LCBF-to-LCGU regression line was found during moderate hypothermia compared with mild hypothermia. Therefore, a marked reduction in mean CGU at unchanged or even increased mean CBF does not necessarily indicate flow/metabolism uncoupling, because a strong linear relationship is maintained on a local level, suggesting intact coupling.

In this context, the following issues need consideration: (1) relationship between CBF and metabolism as reported in previous studies; (2) influence of acid-base management during hypothermia on CBF; (3) applicability of autoradiographic methods during hypothermia; (4) role of the flow–metabolism relationship in the neuroprotection induced by hypothermia; and (5) limitations of the study.

In the present study, using p  H-stat management, mean CBF was unchanged during anesthesia (26%) and was significantly increased (42%;P < 0.05) or remained stable during mild or moderate hypothermia, respectively, compared with conscious controls. No reductions in mean CBF or LCBF were observed during mild or moderate hypothermia compared with conscious controls. The response of mean CBF to hypothermia in the normal brain is still a matter of a controversial discussion. It seems to depend on the acid-base management strategy used and the presence or absence of anesthesia. 9Although several investigators have reported hypothermia-induced reductions in CBF, 20–22Verhaegen et al.  23demonstrated in anesthetized rats that hypothermia reduces CBF mainly during α-stat management, whereas CBF remains similar to normothermic controls during p  H-stat management. This is in accordance with the clinical observation of a higher CBF in patients undergoing hypothermic cardiopulmonary bypass using p  H-stat compared with α-stat management. 7,24Therefore, the major reason for the increased CBF in the present study seems to be the use of p  H-stat management in anesthetized rats.

In the present study, mean CGU was reduced to approximately one half during normothermic isoflurane anesthesia. No further reduction was observed during mild hypothermia, whereas moderate hypothermia resulted in a further significant depression of CGU to approximately one half of the normothermic anesthetized values. These findings are in accordance with the literature. Previous studies in rats reported that isoflurane (1 MAC) induces CGU reductions of approximately 40%, 9,17whereas moderate hypothermia alone reduces LCGU by 13–42% in conscious rats. 14Furthermore, the magnitude of LCGU suppression by hypothermia has been reported to be relatively independent of the acid-base management strategy used. 25 

By combining LCBF and LCGU data, regression lines were constructed, demonstrating a maintained strong relationship between LCBF and LCGU during normothermic anesthesia and mild or moderate hypothermic anesthesia. During normothermic isoflurane anesthesia, the LCBF-to-LCGU ratio increased. This relative hyperperfusion was further augmented by hypothermia. No differences were observed in the LCBF-to-LCGU ratio between mild and moderate hypothermia. Despite this relative hyperperfusion, the physiologic flow/metabolism relationship was maintained on a local level during normothermic and hypothermic isoflurane anesthesia. In two clinical studies, an uncoupling of CBF and metabolism has been reported during hypothermia and p  H-stat management. 7,8Both investigations 7,8were performed in patients during hypothermic cardiopulmonary bypass and showed a marked increase in CBF (argon washin technique or ¹³³Xenon clearance) together with a marked decrease in CMR of oxygen and cerebral glucose uptake, 7which has been interpreted as uncoupling. This conclusion was based on the analysis of global values of CBF and CMR of oxygen. However, the present study shows that a marked decrease of mean CGU together with an unchanged or even increased mean CBF does not necessarily indicate uncoupling, because a strong linear relationship between CBF and CGU is observed on a local level.

Due to the interdependence of pH, Pa^CO2, and CBF, the method chosen for acid-base management during hypothermia is of crucial importance for the interpretation of CBF measurements. At a reduced blood temperature, the solubility of carbon dioxide is markedly increased. If the total carbon dioxide content of the blood remains constant, the Pa^CO2is reduced during hypothermia. Two different strategies for the acid-base management during hypothermia have been proposed. In α-stat management, Pa^CO2is maintained at 40 mmHg when measured at 37°C. The dissociation fraction of the imidazole moiety of histidine is kept constant, and the pH is changed in parallel to the changes in the neutral pH of water. In p  H-stat management, Pa^CO2is maintained at 40 mmHg when corrected to the patient’s actual body temperature. Because temperature correction results in reduced Pa^CO2(because of increased gas solubility at the lower temperature), the p  H-stat strategy requires controlled hypoventilation or addition of carbon dioxide to the inspired gas. As a consequence, the net effect of p  H-stat management is an increase in CBF. 26Therefore, p  H-stat management used in the present study may have caused the significantly higher CBF values of the anesthetized mildly hypothermic rats compared with the anesthetized normothermic rats. There is considerable debate concerning the choice of α-stat or p  H-stat management for the prevention and amelioration of ischemia, e.g. , during hypothermic cardiopulmonary bypass. p  H-stat management is associated with higher CBF, resulting in an increased intensity of brain cooling to the desired level of cerebral hypothermia. 27Furthermore, during p  H-stat management, cortical deoxygenation 27and oxyhemoglobin affinity 28are decreased compared with α-stat management. Disadvantages of p  H-stat management are an actually decreased p  H and increased intracranial blood volume. p  H-stat management was chosen for the present study to achieve a more rapid and homogeneous temperature profile throughout the entire rat brain. By the increased values of CBF, p  H-stat management might reduce the risk of temperature gradients within the brain, which could result in gradients of LCBF and LCGU.

In the present study, autoradiographic methods were used for the quantification of CBF 12and CGU. 11These techniques are based on kinetic models that may need modification during hypothermia. For the autoradiographic determination of LCGU, a model is used in which the major part of radiolabeled 2-deoxyglucose has to be phosphorylated after the experimental period of 45 min. The enzymatic process of phosphorylation may be slowed down by hypothermia, which could result in an overestimation of CGU. However, Nakashima et al.  15have demonstrated during hypothermia (25°C) that all of the labeled 2-deoxyglucose in brain is phosphorylated after the 45-min waiting period. The investigators concluded that no adaptations in the method of Sokoloff et al.  11are necessary to autoradiographically assess LCGU in hypothermic rats. The [¹⁴C]iodoantipyrine was used for the measurement of LCBF in the present study. The distribution of [¹⁴C]iodoantipyrine is mainly diffusion-dependent. With the relatively small changes in body temperature, negligible changes in [¹⁴C]iodoantipyrine diffusion can be expected. Consequently, the [¹⁴C]-iodoantipyrine method has been used for determination of CBF even in hibernating squirrels at a core temperature of 9°C. 13Autoradiographic techniques have previously been used in several studies of hypothermia. 14,15,29 

The aim of clinical hypothermia is the protection of neurons compromised by ischemia. 3–5Several mechanisms are under discussion that could mediate such a protective effect. One potential mechanism is the reduction of cerebral metabolism. In the present study, mean CGU was reduced by approximately 50% during moderate hypothermia (32°C) when compared with normothermic isoflurane anesthesia. Such a strong reduction of CGU points to reduced cerebral metabolism as an important mechanism of brain protection by moderate hypothermia. On the contrary, mean CGU during mild hypothermia (35°C) remained unchanged when compared with normothermic anesthetized animals. Under these conditions, a reduction of cerebral metabolism cannot be considered as the primary cause of hypothermic neuroprotection. Because several studies have demonstrated that mild hypothermia also reduces cell death or even improves neurologic outcome, 30,31other mechanisms, such as a reduced release of excitatory neurotransmitters or oxygen free radical or an inhibition of protein kinase C, must be considered. 32–35Furthermore, the resetting of the ratio of CBF to CGU to higher values observed in the present study might exert beneficial effects by improving oxygen availability within the brain. Therefore, CMR reduction seems to contribute to neuroprotection during moderate hypothermia, but additional mechanisms, such as a reduced release of excitotoxins, seem to be related to the beneficial effects of mild hypothermia.

One limitation of the present study is that, because of animal welfare, the effects of mild hypothermia on LCGU and LCBF were studied during anesthesia. However, the use of conscious animals would result in shivering and stress during cooling, which could induce additional changes in CBF and CGU. Furthermore, anesthesia is also used in the clinical setting when hypothermia is induced for the prevention of brain damage.

A second limitation of the present study is that brain temperature was not measured directly. 36Direct measurement of brain temperature by implanted probes was avoided because of a potential tissue damage that could result in altered values of CBF and CGU. Tissue damage was avoided in the present study by the use of ultrafast microthermocouple probes that were advanced through the masseter muscle to the outside of the base of the rat skull. 1By this procedure, the temperature was recorded as close to the brain as possible without potential traumatic damage.

A third possible limitation of the study might be the statistical approach chosen for data analysis. Assessing the relationship between LCBF and LCGU by linear regression analysis and the derived correlation coefficients is open to criticism. A fundamental assumption of this analytical approach is that observations on different regions of the brain are statistically independent. Because observations are repeated on multiple regions of all animals and only the average values for each region are used, the real uncertainty about the relationship between CGU and CBF might be greater than the regression analysis suggests. 18To exclude erroneous data interpretation by inappropriate statistical analysis, a second approach proposed by McCulloch et al.  18and Ford et al.  19was used in this study. This approach examines the LCBF/LCGU relationship by a repeated-measure analysis of variance using log-transformed data and yielded the same results.

In conclusion, the decrease of mean CGU at unchanged or increased mean CBF observed during normothermic and hypothermic isoflurane anesthesia using p  H-stat management does not necessarily indicate uncoupling of blood flow and metabolism in the brain. Local analysis shows a strong linear relationship between LCBF and LCGU, suggesting an intact coupling that is reset to a higher blood flow/metabolism ratio. Therefore, mild and moderate hypothermia during p  H-stat management did not interfere with the local relationship between CBF and metabolism.