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1 Departments of Pediatrics, 2 Anesthesiology, 3 Surgery, and 4 Neurobiology, Duke University Medical Center, Durham, North Carolina 27710; and 5 Allos Therapeutics, Denver, Colorado 80221
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Hypothermia decreases the arterial PO2 at which hemoglobin is 50% saturated (P50), increasing hemoglobin O2-binding affinity. We used RSR13, a synthetic allosteric modifier of hemoglobin that increases P50, to study the role of altered hemoglobin O2-binding affinity in mild hypothermic neuroprotection. RSR13 (150 mg/kg iv) restored P50 to normothermic values. Rats underwent 70 min of middle cerebral artery occlusion (MCAO) at 30.0, 34.0, or 37.5°C with hemoglobin saturation held at 98-100%. The 34.0°C group received RSR13 or vehicle before ischemia. After 7 days of recovery, infarct volumes were reduced in all hypothermic groups, without evidence of a detrimental effect on infarct size or neurological score as a result of P50 correction. To examine for a beneficial effect of P50 correction, ischemia duration was increased to 120 min in rats maintained at 34.0°C. Correction of P50 by RSR13 did not alter cerebral infarct sizes or neurological scores. The decrease in P50, caused by mild hypothermia, could not be associated with infarct size or neurological deficit resulting from ischemic brain hypoxia in rats.
brain; allosteric modification; hypothermia; RSR13
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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IN LABORATORY MODELS of focal and global cerebral ischemia, mild (34.0°C) and moderate (30.0°C) hypothermia have been shown to reduce brain injury (8, 14, 17, 30, 31). Although the efficacy of induced hypothermia remains unproven in humans sustaining ischemic brain injury, intense investigation continues with the hope of defining conditions where benefits may be obtained (12, 20, 34, 36, 43).
Mild hypothermia produces multiple effects on the ischemic brain. Cerebral metabolic rate is modestly reduced (29), as is the accumulation of glutamate in the extracellular space (9, 41). However, the importance of these effects has been questioned (32, 42). In vitro, mild hypothermia reduces calcium uptake by neurons (2, 6). Mild hypothermia has no effect on protein synthesis during early recirculation (4) but hastens recovery of protein synthesis several hours after reperfusion (39) and diminishes membrane-bound protein kinase C activity in selectively vulnerable regions (10). Hypothermia also reduces the cerebral formation of reactive oxygen species and nitric oxide (18, 22, 23, 26). The frequency of peri-infarct spontaneous depolarizations is also decreased in hypothermic rats (11).
Hypothermia exerts another biological effect, the significance of which has not been examined. The PO2 at which 50% of hemoglobin (Hb) binding sites are occupied by oxygen (P50) is reduced in a temperature-dependent fashion. It can be speculated that this effect on Hb O2-binding affinity may be either beneficial or detrimental to the ischemic brain. Because hypothermia increases the affinity of Hb for O2, release of O2 into tissue could be reduced. In contrast, when Hb affinity for O2 is increased, a lower tissue PO2 is required to release O2 from Hb. This may serve to preserve O2 delivery until it is released in more hypoxic tissue. Alternatively, it may be that the effect of hypothermia on P50 is negligible in the context of other major pathophysiological events initiated by an ischemic insult and/or the neuroprotective effect of hypothermia is of such magnitude that the effect of altered O2 release is negligible in comparison.
2-[4-[[(3,5-Dimethylanilino)carbonyl]methyl]phenoxy]-2-methylproprionic acid (RSR13) belongs to a unique class of synthetic compounds that allosterically modify Hb (1). RSR13 binds to a specific site in the central water cavity of deoxyhemoglobin, reducing Hb O2-binding affinity. This stabilizes deoxyhemoglobin, thereby increasing P50. Administration of RSR13 has been shown to increase tissue O2 delivery. Khandelwal et al. (24) recorded increased PO2 in murine skeletal muscle after administration of RSR13. Wei et al. (38) found decreased cerebral venous blood Hb saturation after treatment with RSR13. Finally, Watson et al. (37) found increased brain tissue PO2 in cats subjected to an ischemic insult when given RSR13.
Accordingly, RSR13 can serve as a tool to define the effect of hypothermia-induced P50 changes on brain damage resulting from a cerebral ischemic insult. We hypothesized that an RSR13-mediated correction of the hypothermia-induced decrease in P50 would alter brain injury in rats subjected to temporary middle cerebral artery occlusion (MCAO). Several experiments were performed to test this hypothesis. We first investigated the effects of RSR13 on P50 at different temperatures. Two outcome experiments were then performed to examine the effect of P50 correction with different durations of MCAO (70 and 120 min).
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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This study was approved by the Duke University Animal Care and Use Committee.
Experiment 1: Effects of Hypothermia and RSR13 on P50
Male Wistar rats (age 8-10 wk, Harlan Sprague Dawley; Indianapolis, IN) were fasted but allowed free access to water for 12-16 h before the experiment. Rats were then anesthetized with halothane (3-5%) in 100% O2. After tracheal intubation, the lungs were mechanically ventilated (50% O2-balance N2, tidal volume ~2.5 ml, respiratory rate ~60 breaths/min) to maintain normocapnia (37-42 mmHg). Inspired O2 concentration was continuously measured with a polarographic O2 monitor. The inspired halothane concentration was then reduced to 1.0-1.5%. Surgery was performed with aseptic techniques, and all surgical fields were infiltrated with 1% lidocaine. The tail artery was cannulated and used to monitor mean arterial blood pressure (MAP) and obtain samples for P50 measurement. The left jugular vein was cannulated for drug administration. During surgical preparation, rectal temperature was servoregulated at 37.5 ± 0.1°C by surface heating and cooling. Pericranial temperature was controlled at 37.5 ± 0.1°C by surface heating and cooling using a 22-gauge needle thermistor percutaneously placed adjacent to the skull. After surgical preparation, a 20-min interval was allowed for physiological stabilization.Halothane concentration was then decreased to 0.5%, and 50 µg/kg
fentanyl (Elkins-Sinn; Cherry Hill, NJ) were administered intravenously
over 10 min (4). Halothane was then discontinued, and a
continuous intravenous infusion of fentanyl was initiated at a rate of
50 µg · kg
1 · h1.
Simultaneously, either 150 mg/kg RSR13 (20 mg/ml preparation in 0.25%
NaCl, Allos Therapeutics; Denver, CO) or an equivalent volume of saline
(vehicle) was administered intravenously over 15 min (n = 4 rats/group). Five minutes after the completion of drug or vehicle
infusion, arterial blood samples were collected in triplicate in
heparinized tubes and stored on ice for subsequent measurement of
P50 by multipoint tonometry. Rats were then administered a
lethal dose of halothane.
For P50 measurements, each blood sample was divided into three portions, with each portion incubated in a separate tonometer (model IL 237, Instrumentation Laboratories; Boston, MA) with temperature controlled at either 30.0, 34.0, or 37.5°C according to established techniques (7). The blood sample in each temperature-controlled tonometer was equilibrated for 10-20 min with gas mixtures containing 2.95%, 5.85%, or 8.75% O2 in 5.8% CO2 and balance N2. These O2 concentrations achieved PO2 in whole blood of ~20, 40, and 60 mmHg, respectively. At each O2 concentration, a blood sample was removed anaerobically from the tonometer by syringe and injected into a blood gas analyzer and cooximeter to measure pH, PCO2, PO2, and oxygen saturation (SaO2), respectively.
Because P50 is the PO2 at which Hb is 50% saturated, measured arterial PO2 (PaO2) and SaO2 at each O2 concentration and nonlinear regression analysis were used to determine the P50 at each temperature. P50 values were then corrected using the appropriate temperature factors for 30.0, 34.0, or 37.5°C, supplied by Instrumentation Laboratories.
Experiment 2: Effects of Hypothermia and RSR13-Induced Increase in P50 on Injury Resulting From 70-min MCAO
Male Wistar rats (age 8-10 wk) were fasted but allowed free access to water for 12-16 h. Rats were then anesthetized with halothane (1.0-1.5%) in 100% O2. After tracheal intubation, the lungs were mechanically ventilated (tidal volume ~2.5 ml, respiratory rate ~60 breaths/min) to maintain normocapnia (37-42 mmHg). Surgery was performed with aseptic technique, and all surgical fields were infiltrated with 1% lidocaine. The tail artery was cannulated and used to monitor MAP and sample blood. The left jugular vein was cannulated for drug administration. Body temperature (both pericranial and rectal) was controlled by surface heating and cooling with a heat lamp and cooling fan controlled by a temperature regulation system (model 401 Telethermometers, Yellow Spring Instruments; Yellow Spring, OH).Animals were prepared for MCAO using modifications of techniques previously described (3, 5). After midline cervical incision, the right common carotid artery was identified. The external carotid artery was isolated, and the occipital, superior thyroid, and external maxillary arteries were ligated and divided. The internal carotid artery was dissected distally until the origin of the pterygopalatine artery was visualized. Rats were then randomly assigned to one of the following four groups.
Vehicle 37.5°C group. Pericranial and rectal temperatures were maintained at 37.5 ± 0.1°C throughout the experiment. NaCl (0.9% iv) was given before the onset of ischemia (n = 12).
Vehicle 34.0°C group. Pericranial and rectal temperatures were reduced to 34.0°C over 45 min before the onset of ischemia and maintained at 34.0 ± 0.1°C during MCAO. NaCl (0.9% iv) was given before the onset of ischemia (n = 16).
RSR13 34.0°C group. Pericranial and rectal temperatures were reduced to 34.0°C over 45 min before the onset of ischemia and maintained at 34.0 ± 0.1°C during MCAO. RSR13 (150 mg/kg iv) was given before the onset of ischemia (n = 16).
Vehicle 30.0°C group. Pericranial and rectal temperatures were reduced to 30.0°C over 45 min before the onset of ischemia and maintained at 30.0 ± 0.1°C during MCAO. NaCl (0.9%) was given before onset of ischemia (n = 16).
Before ischemia, the inspired halothane concentration was decreased to 0.5%, and 50 µg/kg fentanyl were given intravenously over 10 min. Thereafter, halothane was discontinued, and a continuous infusion of fentanyl was initiated at a rate of 50 µg · kg1 · h1 iv. Either
150 mg/kg RSR13 (Allos Therapeutics; Denver, CO; 20 mg/ml preparation
in 0.25% NaCl) or an equivalent volume of saline (3.75 ml/kg) was
given intravenously over the 15 min immediately before ischemia.
Because RSR13 is known to shift P50 to the right, pilot
studies were performed to define the fraction of inspired O2 (FIO2) at which
RSR13-treated animals would have arterial Hb SaO2
values similar to vehicle-treated normothermic rats breathing 50%
O2. That value was found to be 60% O2.
Accordingly, the lungs of rats treated with RSR13 were ventilated with
60% O2-40% N2, whereas all other
rats received 50% O2-50% N2.
FIO2 was continuously measured with a
polarographic O2 monitor.
To achieve MCAO, a 0.25-mm-diameter silicone-coated nylon filament was
inserted into the stump of the external carotid artery, passed rostral
through the internal carotid artery (23 mm from carotid bifurcation)
until a slight resistance was felt, and secured.
After 70 min of MCAO, the occlusive filament was removed.
Simultaneously, the fentanyl infusion was discontinued, and anesthesia was continued with inspired halothane at 0.7-1.0%. The animals remained anesthetized with halothane for 50 min after the conclusion of
ischemia, during which time they were rewarmed to 37.5 ± 0.1°C by surface heating as required. Fifty minutes after the onset of
reperfusion, catheters were removed, and the wounds were closed with
sutures. Halothane was discontinued, and rats were allowed to awaken.
After recovery of spontaneous ventilation, the trachea was extubated.
Each rat was placed in 100% O2 for 2 h before being returned to its home cage.
Physiological values, including SaO2 (measured by
cooximetry, OSM 3 Hemoximeter, Radiometer; Copenhagen, Denmark),
hematocrit, arterial PCO2
(PaCO2), PaO2, and arterial pH, were
measured immediately after the completion of the RSR13/vehicle infusion
(5 min before the onset of MCAO), 45 min after MCAO onset, and 50 min
after the onset of reperfusion. Arterial blood gas concentrations were not corrected for temperature. Plasma glucose was determined 45 min
after the onset of MCAO and 50 min after the onset of reperfusion. MAP
and rectal and pericranial temperatures were continuously recorded in
all groups before, during, and for 50 min after MCAO.
Seven days after ischemia, rats underwent a standardized neurological
examination designed to evaluate sensorimotor function (1). With the observer blinded to group assignment, this
test examined six different functions (spontaneous activity over 5 min,
movement symmetry, forepaw outstretching, climbing, body proprioception, and response to vibrissae touch). The individual performance in each test was rated with a three- or four-point score.
The score given to each animal at the completion of testing was the sum
of all six individual scores, 3 being the minimum (worst) and 18 being
the maximum (best) score. After neurological evaluation, animals were
weighed, anesthetized with 3% halothane, and decapitated. The brains
were removed, frozen at 
40°C in 2-methylbutane, and stored at
70°C for later analysis. Serial quadruplicate 20-µm-thick coronal
sections were taken using a cryotome at 660-µm intervals over the
rostral-caudal extent of the infarct. The sections were dried and
stained with hematoxylin and eosin.
Infarct volume was measured by digitally sampling stained sections with
a videocamera controlled by an image analyzer (M2 Turnkey System,
Imaging Research; St. Catherines, Ontario, Canada). The image of each
section was stored as a 1,280 × 960-pixel matrix. The digitized
image was then displayed on a video monitor. With the observer blinded
to experimental conditions, infarct borders in both cortex and
subcortex were individually outlined (corpus callosum and ventricles
excluded) using an operator-controlled cursor. The area of infarct (in
mm2) was determined by counting pixels contained within the
outlined regions of interest. Infarct volumes (in mm3) were
computed as running sums of infarct area multiplied by the known
interval (e.g., 660 µm) between sections over the extent of the
infarct calculated as an orthogonal projection.
Experiment 3: Effects of Hypothermia and RSR13-Induced Increase in P50 on Injury Resulting From 120-min MCAO
Pilots studies were performed to define the duration of MCAO at 34.0°C that would result in a larger size infarct in hypothermic animals than was observed in experiment 2 (see RESULTS). That interval was found to be 120 min. Additional analysis was then required to determine whether the duration of P50 correction caused by RSR13 (150 mg/kg) would persist throughout the 120-min ischemic interval. Rats (n = 6) were surgically prepared for MCAO as described above. Body temperature was reduced to 34.0°C. With the use of a protocol identical to that of experiment 2, rats received 150 mg/kg RSR13 or an equal volume of vehicle intravenously and were subjected to 120-min MCAO. Arterial blood was sampled at the end of drug infusion, 60 min after the onset of ischemia, and at the termination of ischemia. The animals were then killed, and P50 values were determined as described in experiment 1.Another set of rats was prepared for MCAO as described in experiment 2 to perform outcome analysis. After surgical preparation, the animals were randomly assigned to one of the following two groups.
Vehicle 34.0°C group. Pericranial and rectal temperatures were reduced to 34.0°C over 45 min before the onset of ischemia and maintained at 34.0 ± 0.1°C during MCAO. No RSR13 was given. Vehicle (3.75 ml/kg iv of 0.9% NaCl) was given over 15 min immediately before the onset of ischemia (n = 18).
RSR13 34.0°C group. Pericranial and rectal temperatures were reduced to 34.0°C over 45 min before the onset of ischemia and maintained at 34.0 ± 0.1°C during MCAO. RSR13 (150 mg/kg iv) was given over 15 min immediately before the onset of ischemia (n = 17).
All other aspects of physiological management, recovery, neurological evaluation, and measurement of cerebral infarct size were the same as those described for experiment 2. An additional group of rats (n = 6) was studied to provide a frame of reference that was not intended for statistical analysis. These rats were treated identical to those above except that no RSR13 was given and pericranial temperature was held at 37.5°C during ischemia and the first 50 min of reperfusion.Statistics
Physiological values and cerebral infarct volumes were compared by one-way analysis of variance (StatView 5.0, SAS; Cary, NC). Post hoc analysis was performed using Scheffé's test when indicated by a significant F-ratio. Nonparametric neurological scores were compared between groups by the Kruskal-Wallis and Mann-Whitney tests as appropriate. Physiological values and infarct volumes are reported as means ± SD. Neurological scores are reported as medians and interquartile ranges. Statistical significance was assumed when P < 0.05.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Experiment 1: Effects of Hypothermia and RSR13 on P50
In blood from animals not given RSR13, P50 values were decreased by temperature in a linear manner (40.4 ± 1.7, 27.3 ± 3.1, and 16.9 ± 3.5 mmHg at 37.5, 34.0, and 30.0°C, respectively, P < 0.001, R2 = 0.99). At each temperature, P50 was increased by RSR13 in a linear manner (54.1 ± 7.0, 36.0 ± 4.1, and 24.4 ± 3.8 mmHg at 37.5, 34.0, and 30.0°C, respectively, P < 0.001, R2 = 0.98). The difference between P50 values at 37.5°C in vehicle-treated rats (40.4 ± 1.7 mmHg) and P50 at 34.0°C in RSR13-treated rats (36.0 ± 4.1 mmHg) was not statistically significant (P = 0.10), indicating that treatment with RSR13 effectively increased hypothermic P50 values to normothermic P50 values.Experiment 2: Effects of Hypothermia and RSR13-Induced Increase in P50 on Injury Resulting From 70-min MCAO
Physiological values for the experimental groups are given in Table 1. Values were similar among groups with two exceptions. PaO2 was greater in the RSR13-treated 34.0°C group (P < 0.05). Despite the use of pilot study data and a higher FIO2, SaO2 was decreased by 1-2% in the RSR13-treated 34.0°C group versus the other three groups at all measurement intervals (P < 0.001).
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Mortality was <10% in all groups over the 7-day recovery interval.
There were differences among groups for neurological scores (vehicle
37.5°C: 13 ± 4.5; vehicle 34.0°C: 14 ± 4.5; RSR13
34.0°C: 16 ± 3; and vehicle 30.0°C: 17 ± 3, P < 0.017; Fig. 1).
Significant intergroup differences were present for the vehicle
37.5°C group versus the vehicle 30.0°C group (P = 0.003) and the vehicle 34.0°C group versus the vehicle 30.0°C group
(P = 0.032). The difference between the RSR13 34.0°C
group versus the vehicle 37.5°C group approached significance
(P = 0.064). There was no difference between the
vehicle 34.0°C and RSR13 34.0°C groups (P = 0.423).
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Cerebral infarct sizes are depicted in Fig. 1. Hypothermia was highly protective against cortical injury in all groups (vehicle 37.5°C: 61 ± 78 mm3; vehicle 34.0°C: 0 mm3; RSR13 34.0°C: 0 mm3; and vehicle 30.0°C: 0 mm3). Injury in the subcortex was also severely reduced in all hypothermic groups (vehicle 37.5°C: 58 ± 31 mm3; vehicle 34.0°C: 24 ± 11 mm3; RSR13 34.0°C: 23 ± 11 mm3; and vehicle 30.0°C:14 ± 17 mm3, P < 0.001). Significant intergroup differences were present for the vehicle 37.5°C group versus the vehicle 30.0°C group, the vehicle 37.5°C group versus the vehicle 34.0°C group, and the RSR13 34.0°C versus the vehicle 37.5°C group (P < 0.001). A difference between the RSR13 34.0°C group versus the vehicle 34.0°C group was absent (P = 0.81).
Experiment 3: Effects of Hypothermia and RSR13-Induced Increase in P50 on Injury Resulting From 120-min MCAO
There were no differences between groups for physiological values with the exception of PaO2, which was greater in the RSR13-treated rats (Table 2). In this experiment, there were no differences between groups with respect to SaO2 at any measurement interval. Data from the pilot study examining the temporal relationship between RSR13 and P50 at 34.0°C showed no effect of time, indicating persistent pharmacological correction of P50 during the 120-min hypothermic ischemic insult.
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There were no differences between groups for neurological scores
(vehicle 34.0°C: 16 ± 4; RSR13 34.0°C: 16 ± 2.25, P = 0.99; Fig. 2). There
were no differences between groups for total (vehicle 34.0°C:
103 ± 64 mm3; RSR13 34.0°C: 108 ± 65 mm3, P = 0.80), cortical (vehicle 34.0°C:
48 ± 40 mm3; RSR13 34.0°C: 56 ± 41 mm3, P = 0.77), or subcortical (vehicle
34.0°C: 55 ± 26 mm3; RSR13 34.0°C: 57 ± 29 mm3, P = 0.87) infarct volume (Fig. 2).
Total infarct volume in both 34.0°C groups was 40-45% less than
that observed in vehicle-treated rats subjected to MCAO with
temperature held at 37.5°C.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The goals of this study were twofold. First, we sought to define the mechanistic importance of a leftward shift of P50 caused by mild hypothermia in the context of transient focal cerebral ischemia. RSR13 is an effective allosteric modifier of the affinity of Hb for O2 (1). This allowed a direct test of the role of P50 in the hypothermic brain during ischemia. Second, we have previously shown that, although RSR13 does not provide independent neuroprotection from a focal ischemic insult (33), when combined with other pharmacotherapy (e.g., dizocilpine), an increase in P50 serves to reduce ischemic brain damage in normothermic animals (27). RSR13 also has been shown to augment hypothermic protection of the myocardium in a canine cardiopulmonary bypass model (25). As a result, we speculated that RSR13 might also enhance the neuroprotection provided by hypothermia. We did not, however, observe an effect of P50 correction on ischemic outcome in this series of experiments.
The 150 mg/kg dose of RSR13 was chosen for several reasons. First, this dose has been shown to favorably alter outcome in studies of both global and focal normothermic ischemia (19, 27, 33, 37). Second, this dose was found in experiment 1 to cause near-complete reversal of the P50 reduction caused by mild hypothermia. This allowed preservation of Hb saturation at values similar to those observed in animals untreated with RSR13, although a greater FIO2 was required to achieve this effect. Finally, the duration of action of RSR13 was found to be sufficient to allow P50 correction to persist throughout the entire ischemic insult.
Experiment 2 was designed to allow analysis of pharmacological correction of 34.0°C hypothermia-induced P50 reduction within the frame of reference of animals subjected to MCAO while either normothermic (37.5°C) (15, 21) or moderately hypothermic (30.0°C). Mild hypothermia (34.0°C) has been repeatedly shown to be neuroprotective against MCAO (17, 30, 31). However, we believed that the inclusion of a normothermic group would allow the magnitude of any adverse effect of hypothermic P50 correction to be held in contrast to outcome from a normothermic insult. Pilot studies were performed to define the maximal duration of MCAO at 37.5°C that would allow formation of a large infarct with >90% survival of experimental subjects given vehicle. The duration was found to be 70 min. Accordingly, all experimental groups were exposed to 70-min MCAO. Both mild (34.0°C) and moderate (30.0°C) hypothermia provided profound and similar protection against 70-min ischemic injury. With the correction of P50, there was no increase in infarct size in the 34.0°C group exposed to RSR13. From this, we can conclude that the reduction of P50 by mild hypothermia is not responsible for the beneficial effect of mild hypothermia on the ischemic brain.
Experiment 3 was designed to investigate a substantially longer interval of MCAO (i.e., 120 min), sufficient to cause a moderate size infarct at 34.0°C. In this context, there would be ample opportunity to observe either an increase or decrease in infarct size attributable to the leftward shift of the Hb-O2 dissociation curve induced by hypothermia and its correction to normothermic values by use of RSR13. Again, there was no difference between RSR13- and vehicle-treated animals for infarct size or neurological deficit, although injury in both 34.0°C groups was markedly less than that observed in a group of vehicle-treated rats concurrently studied at 37.5°C. Cumulatively, these data allow the conclusion that the effect of mild hypothermia on P50 has no importance on ischemic outcome within the conditions examined in this investigation. The effects of mild hypothermia on Hb O2-binding affinity cannot therefore be invoked as a mechanism for the neuroprotection mediated by mild hypothermia during transient ischemia. However, further study is required to determine effects of P50 correction under conditions of more severe reductions in temperature as well as effects of P50 correction on postischemic hypothermia therapeutic interventions.
Previous studies with RSR13 in our rat model of MCAO were conducted in the absence of anesthesia during normothermic ischemic insults (27, 33). Use of a radiotelemetered thermistor allowed strict control of brain temperature. In the current studies, we recognized that some form of anesthesia would be required in the hypothermic animals during MCAO. Although surgery was conducted under halothane anesthesia, this volatile anesthetic was eliminated before ischemia and substituted with fentanyl. Prior work comparing the outcome in rats subjected to MCAO while either awake or anesthetized with fentanyl found no effect of fentanyl on infarct size or neurological score despite careful temperature control during and after the ischemic insult (35). As a result, we do not believe that the anesthesia used in this study could explain the lack of effect of P50 on outcomes secondary to ischemic injury under hypothermic conditions.
Reduction of pericranial and rectal temperature from 37.5 to 34.0°C resulted in a 32% reduction in P50 in our experiment. This magnitude of effect is proportionately similar to that previously reported in humans at a variety of moderately hypothermic temperatures (3, 5, 40). The range of temperatures studied in the current experiments is of particular relevance to current clinical trials utilizing induced hypothermia in either perioperative or intensive care settings (3, 12, 20, 34, 36, 43). Greater reduction in P50 would be expected in humans subjected to cardiopulmonary bypass and deep or profound hypothermia (13). Our study did not address the potential for P50 to be relevant to outcome from ischemic insults occurring with deep or profound hypothermia. It has been argued that during profound hypothermia ~77% of metabolically available O2 is transported in the blood as dissolved O2 because of the magnitude of P50 reduction (16). It remains plausible that pharmacological correction of P50 during hypothermia more severe than that studied in the current experiments might prove to be important. Such examination could be made in a recently defined rodent recovery model of cardiopulmonary bypass in which residual neurocognitive deficits have been identified (28).
With the available data, we cannot explain the failure of correction of
P50 to alter ischemic brain injury under conditions of mild
hypothermia. Definition of effects of RSR13 on tissue PO2 would seem futile because of the absence of
effect of P50 correction on ischemic outcome. It is also
unlikely that our use of 
-stat management of arterial blood gases
(i.e., uncorrected for temperature) accounts for the absence of effect
because animals treated with either RSR13 or vehicle were managed with
the same strategy. Hematocrit and SaO2 were similar
between groups, again providing no explanation for the lack of effect
observed. A temperature reduction of 3.5°C, as occurred in our
experiment, would be expected to provide an approximate 35% reduction
in the cerebral metabolic rate for oxygen
(CMRO2) (29). It is plausible that
this magnitude of CMRO2 was sufficient to dwarf
benefits from any increase in O2 delivery obtained by
correction of P50. At the same time, demand for
O2 in the ischemic periphery (i.e., penumbra) may have
overwhelmed benefits from a shift of P50 achieved by
hypothermia. Other consequences of ischemia such as changes in pH,
bicarbonate, and CO2 in the ischemic tissue may also affect
the binding of O2 to Hb.
The results of this study are consistent with an earlier study in which normothermic rats were subjected to MCAO in the presence of sufficient RSR13 (150 mg/kg) to increase P50 by 68% above normal values. No effect of treatment was observed for infarct size or neurological score (33). At the same time, others have reported that RSR13 administered before and during normothermic permanent MCAO in cats caused a trend for increased tissue PO2 in the ischemic penumbra. Although only acute recovery (5-h survival) was assessed, RSR13 infusion reduced cerebral infarct size by 36% (37).
In conclusion, we examined interactions between P50 and the effect of altering P50 on outcomes from focal cerebral ischemia as a potential mechanism of hypothermic neuroprotection during MCAO. Although hypothermia provided substantial protection and a 32% reduction in P50, pharmacological normalization of P50 failed to alter outcomes in either an adverse or beneficial manner. We conclude that P50 changes caused by mild hypothermia do not alter brain infarct size after experimental focal ischemia and that other mechanisms must account for the benefits observed from hypothermic neuroprotection.
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ACKNOWLEDGEMENTS |
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The authors are grateful to Ann D. Brinkhous for expert technical assistance.
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FOOTNOTES |
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M. Wainwright was supported by a research fellowship award from the Department of Pediatrics at Duke University Medical Center. G. B. Mackensen was supported by a postdoctoral stipend through the German Academic Exchange Service. This study was partially supported by a grant from Allos Therapeutics, Incorporated (Denver, CO).
Address for reprint requests and other correspondence: D. S. Warner, Dept. of Anesthesiology, Box 3094, Multidisciplinary Neuroprotection Laboratories, Duke Univ. Medical Center, Durham, NC 27710 (E-mail: warne002{at}mc.duke.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpheart.00863.2001
Received 4 October 2001; accepted in final form 30 November 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Abraham, DJ,
Wireko FC,
Randad RS,
Poyart C,
Kister J,
Bohn B,
Liard J-F,
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Allosteric modifiers of hemoglobin: 2[4-[[(3,5-disubstituted anilino)carbonyl]methyl]phenoxy]-2-methylproprionic acid derivatives that lower the oxygen affinity of hemoglobin in red cell suspensions, in whole blood, and in vivo in rats.
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, Values for individual rats; horizontal
lines, group mean values for infarct sizes and median values for
neurological scores.
