(Stroke. 1995;26:1908-1915.)
© 1995 American Heart Association, Inc.
Articles |
AMPA Antagonist LY293558 Does Not Affect the Severity of Hypoxic-Ischemic Injury in Newborn Pigs
From the Departments of Pediatrics (M.H.L., M.H., D.M.P.), Medicine (X.Q.L.), and Pathology (E.E.S.), University of Mississippi School of Medicine, Jackson.
Correspondence to Michael H. LeBlanc, MD, Department of Pediatrics, University of Mississippi Medical Center, 2500 No State St, Jackson, MS 39216-4505. E-mail leblanc@fiora.umsmed.edu.
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Background and Purpose LY293558 is a systemically active
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)
excitatory amino acid antagonist. AMPA
antagonists have shown promise in several adult
hypoxic-ischemic brain injury models, and we wanted to see
if this work could be extended to a newborn animal. Methods Seventy-six (ß error <.10) 0- to 3-day-old piglets under 1.5% isoflurane anesthesia underwent placement of carotid snares and arterial and venous catheters. While paralyzed with succinylcholine under 0.5% isoflurane, 50% nitrous oxide, piglets were randomly assigned to receive either 5 mg/kg or 15 mg/kg of LY293558 or saline at time -10 minutes and again 10 hours later. At time 0, both carotid arteries were clamped, and blood was withdrawn to reduce the blood pressure to two thirds of normal. At time 15 minutes, inspired oxygen was reduced to 6%. At time 30 minutes, the carotid snares were released, the withdrawn blood was reinfused, and the oxygen was switched to 100%. On the third day after the hypoxic-ischemic injury, the animals were killed by perfusion of the brain with 10% formalin. Brain pathology was scored by a blinded observer.
Results There were no significant differences between the drug-treated and control groups.
Conclusions The systemically active AMPA antagonist LY293558, when given at a dose of 5 mg/kg or 15 mg/kg before injury and 10 hours later, does not affect the severity of hypoxic-ischemic brain injury in newborn piglets. Neither AMPA receptor activity nor NMDA receptor activity are important in brain injury in this model.
Key Words: hypoxia • pigs • excitatory amino acids • newborn
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Introduction |
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Excitatory amino acids are thought to be important in the pathophysiology of hypoxic-ischemic brain injury. The current working hypothesis is that when neuron energy stores become depleted, and active transport of ions to maintain an electrochemical gradient is impossible, the neurons depolarize. Excitatory amino acids are released in response to neuronal depolarization. Reuptake of glutamate by both neurons and glia is also an energy-dependent process that is inhibited by cellular energy depletion. Thus, excitatory amino acids accumulate. The excitatory amino acids glutamate and aspartate open receptor-mediated calcium channels and allow flooding of extracellular calcium into the intercellular milieu.1 Although at low levels intercellular calcium is an important second messenger, at high levels it can activate proteolytic and lipolytic enzymes that are responsible for cell apoptosis2 3 4 5 and cause release of free radicals from mitochondria6 and other sources within the cell.7 These processes can lead to cell death.5 8 The three major excitatory amino acid receptors that are linked to ion channels are the N-methyl-D-aspartate (NMDA) receptors, the
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors,
and the kainate receptors.9 The AMPA receptors open
both sodium channels and sodium-calcium channels. The NMDA
receptors regulate sodium-calcium–permeable
channels.10 NMDA antagonists have been useful
in focal cerebral ischemia but in general must be administered
before the ischemic insult to be effective.11 AMPA
receptor antagonists have shown promise in both focal and
global ischemia models and in treatment both before and for
extended periods after injury.11 12 We have previously studied MK801, an NMDA receptor antagonist, in a piglet hypoxic ischemia model.13 In that study, although MK801 caused profound sedation in the piglets that lasted for the entire 3 days of reperfusion, the agent offered no neuroprotection. Because our model resulted in damaged neurons throughout the brain, it is perhaps more akin to a global ischemia model, and thus perhaps an AMPA receptor antagonist would be more useful. At the time this study was begun, the most commonly used systemically active AMPA antagonist, NBQX,14 was no longer available through Nova Nordix and had not yet become available commercially from other companies. However, another intriguing AMPA receptor antagonist, (3SR,4aRS,6RS,8aRS)6-[2-(1H-tetrazol-5-yl)ethyl] decahydroisoquinoline-3-carboxylic acid (LY293558), was described by Paul Ornstein et al15 of Eli Lilly and shown to be a specific AMPA inhibitor with little or no action on the NMDA receptor in mice. After beginning this study, we were excited to learn that Bullock et al16 had shown LY293558 to be effective in focal ischemia in a cat model. Although we have been unable to develop a system for measuring serum levels of the compound, its sedative effects can be monitored clinically. In pilot experiments in piglets, one dose of 5 mg/kg of the compound caused mild but definite sedation and incoordination, apparent within 5 minutes, that lasted for approximately 10 hours. Thus, initial experiments were performed using a dose of 5 mg/kg given 10 minutes before initiation of hypoxic ischemia. The second dose was given 10 hours later. When this proved unsuccessful, additional studies were performed at a higher dose on the basis of the study by Bullock et al.
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This protocol was approved by our institutional committee on animal use and performed in accordance with institutional guidelines.
Following our previously described protocol,13 17 38 0- to
3-day-old piglets were removed from their mothers on the day of the
experiment and were randomly assigned to either a LY293558 or vehicle
group (Fig 1
). Sample size was chosen to provide a 90%
chance of detecting a difference in pathological scores one half as
large as those seen in our previous study.17 Piglets were
anesthetized with 1.5% isoflurane and 50% nitrous oxide,
intubated, and ventilated with a Harvard Rodent Ventilator adjusted to
obtain an initial PCO2 of approximately 5.3
kPa (40 mm Hg). Catheters were placed in the superficial artery of the
right rear leg and in the right external jugular vein with a sterile
technique. A snare was placed around both carotid arteries. After
surgery was completed, the isoflurane was reduced to 0.5%, and the
animal was paralyzed with an infusion of 0.5 mg/kg per minute of
succinylcholine. Both isoflurane and nitrous oxide have effects on
cerebral blood flow, metabolism, and the response of the
brain to ischemia,18 19 as do most anesthetics.
The animal's rectal temperature was maintained at 38.0°C with a
servo-controlled infrared lamp.
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After the initial surgery was performed, at experimental time of -10 minutes, those piglets assigned to the LY293558 group intravenously received 5 mg/kg of LY293558 dissolved in saline (low dose). Those piglets assigned to the control group received an equivalent value of saline. At time -5 minutes, baseline measurements were taken. Baseline measurements included arterial blood gases, arterial blood pressure, rectal temperature, oral temperature, heart rate, and levels of hematocrit and serum glucose. This set of measurements was repeated at 0, 15, 30, 35, 45, 60, and 90 minutes. At time 0, 700 U/kg heparin was injected, and the carotid arteries were ligated by pulling the snares snugly around them. Blood was withdrawn from the arterial catheter into syringes to reduce the arterial pressure to approximately two thirds of control levels and maintain it at that level. Isoflurane was discontinued at 10 minutes, by which time the animal had been rendered unconscious by the ischemia (pilot studies). Fifteen minutes after the carotid ligation and the reduction of blood pressure to two thirds of normal, the animal was switched from ventilation with 50% nitrous oxide and 50% O2 to a gas mixture containing 70% nitrous oxide, 22% N2, 2% CO2, and 6% O2. This reduced arterial PO2 to approximately 3.3 kPa (25 mm Hg) within 1 to 2 minutes. Succinylcholine was discontinued at 20 minutes (it is required until this time to prevent gasping). At an experimental time of 30 minutes, after 15 minutes of hypoxia, the animal was reoxygenated by switching the inspired gas from 6% O2 to 100% O2, releasing the carotid ligatures, and reinfusing the blood that had been previously withdrawn. The effect of reoxygenation with 100% O2 on neurological outcome of hypoxic ischemia is controversial.20 21 We confirmed at autopsy that patency of the carotids was reestablished. At 10 hours after reoxygenation, an additional dose of 5 mg/kg LY293558 or vehicle was given. A second set of 38 piglets was randomized to receive either 15 mg/kg of LY293558 (high dose) at -10 minutes and 10 hours or an equal value of vehicle (high-dose control).
Piglets were nursed in cages. Warmth was provided by heat lamp. From 2
to 17 hours after reoxygenation, they received 5%
dextrose intravenously at 8 cc/h. They were then fed 60 cc
artificial piglet formula by gavage every 6 hours. Rectal temperature
was measured at 2, 8, and 20 hours after
reoxygenation. Neurological examination was made by
the staff performing the experiment at 2 hours after
reoxygenation. Neurological examinations were
performed by a blinded observer at 1, 2, and 3 days after
reoxygenation. The results were recorded and
scored from 5 to 20, with 20 being normal and 5 being brain dead
according to a standard scoring system (Table 1).
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Three days after the experiment, with piglets under isoflurane and nitrous oxide anesthesia, the chest was opened, the carotid artery cannulated, and the brain perfused with 10% formalin after flushing with 30 cc of saline. Formalin was continued until the effluent from the right atrium was clear, thus preserving the brain and killing the animal. Good preservation of both sides of the brain in piglets is achieved using this technique. The brain was then removed and preserved in formalin for later pathological examination. If a piglet died before completion of the 3 days, its carcass was stored in the refrigerator until morning, after which a gross autopsy was performed and its brain preserved in formalin.
After preservation in formalin, the brains of all piglets were cut,
fixed, and stained. A coronal section was taken at the level of the
optic chiasm, and another was taken approximately 3 mm back to
demonstrate the cerebral cortex, the hippocampus, and the basal
ganglia. Paraffin sections were stained with hematoxylin and eosin and
examined with light microscopy. Each section was graded on a scale of 1
to 10 (with 10 being normal) by a pathologist blinded to the
experimental group of piglets. The scoring system is shown in Table 2.
Cellular changes were classified as hypoxic (considered by the
pathologist to be potentially reversible, scores 5 to 9 depending on
size of involved area) or necrotic (thought to be clearly irreversible,
scores 1 to 4 depending on size of involved area). The final score was
the sum of the scores of each of the three tissues. Hypoxic changes
were largely shrunken hyperchromatic neurons but also included enlarged
perivascular spaces and eosinophilic-staining neurons with
pyknotic nuclei, while necrotic changes included loss of neurons with
glial and vascular proliferation and increased macrophage
activity in the tissue.
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In the development of this protocol, the period of ischemia was gradually lengthened from 10 minutes to the 30 minutes used in this experiment, with observation of neurological and pathological changes until definite and reproducible changes were seen. Representative histological sections have been published.17 A subgroup of pigs from previous experiments performed using this protocol13 was randomized to a sham procedure and subjected to blinded neurological and pathological examination. Their data are included for comparison.
Arterial serum glucose was measured by the glucose oxidase
technique.22 Blood gases and pressures were measured using
standard techniques.13 Neurological and pathological
examination comparisons were made between the two doses of LY293558 and
the control groups using a Kruskal-Wallis nonparametric
ANOVA,23 with P<.05 considered statistically
significant. Since the two control groups were not significantly
different, they were added together. Results from the two scores, since
they are ordinal variables, are presented as median (25th
percentile, 75th percentile). Physiological
variables were analyzed by multiple-measures ANOVA for
significant group or group-by-time differences
(P<.05), and if these were present, individual time
points were compared with ANOVA using Dunnett's test with Bonferroni
correction for multiple time points (P<.05). Results for
physiological variables are presented
as mean±SE. Death rates were compared using the
2 test.
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Results |
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The piglets receiving 15 mg/kg LY293558 (n=19) weighed 1423±73 g and were 1.5±0.2 days old, and those receiving 5 mg/kg LY293558 (n=19) weighed 1538±74 g and were 1.8±0.2 days old. Those in the control group (n=38) weighed 1471±53 g and were 1.6±0.15 days old. Results for arterial pH, PCO2, PO2, and blood pressure are shown in Fig 2
. Arterial pH was higher in the
15-mg/kg group than in either the control or the low-dose group
(P<.05). Group-by-time differences were also
statistically significantly different (P<.000001). The only
individual time point that showed statistically significant differences
was at 35 minutes, when the high-dose group value was significantly
greater than that of the control group. There were no significant group
differences for PO2,
PCO2, or blood pressure.
Group-by-time differences were statistically significant for
PCO2 (P<.00001) and blood
pressure (P=.006) but not PO2. The
only significant point difference was at 17 minutes for
PCO2, when both drug group values were
significantly higher than those of the control group. Changes in all
four variables over time are highly statistically significant
(P<.000001). Fig 3 shows changes in rectal
and oral temperatures, heart rate, and glucose over time. There were no
significant group differences for any of these variables. Although
the group-by-time differences for rectal temperature and
glucose were not statistically significant, those for oral temperature
(P<.05) and heart rate (P<.000001) were
statistically significant. Statistically significant point differences
were seen only for heart rate, where at 15 minutes both drug group
values were significantly lower than those of the control group and at
45 minutes values of the high-dose experimental group were
significantly higher than those of the control group. Changes in all
four variables over time were highly statistically significant
(P<.000001).
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and dark lines indicate 15-mg/kg LY293558 group;
and light lines, 5-mg/kg LY293558 group; and 
and dashed
lines, control group. *Statistically significant difference from the
control groups (P<.05).
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The blood volume withdrawn to induce a decrease in blood pressure to two thirds of normal at 30 minutes was 34.5±2.4 cc/kg in the control group (n=38), 26.4±2.8 cc/kg in the 5-mg/kg group (n=19), and 19.6±3.3 cc/kg in the 15-mg/kg group (n=19). The blood volume withdrawn was significantly smaller in the high-dose group than in the control group (P<.01). There were no significant differences among the groups or between groups by time for hematocrit value. Hematocrit level at 30 minutes was 27±1 in the high-dose group (n=19), 26±0.9 in the low-dose group (n=19), and 25±0.6 in the control group (n=38) (P=NS).
Rectal temperature was also measured at 2, 8, and 20 hours after reoxygenation. In the high-dose group, rectal temperatures were 38.1±0.1°C at 2 hours (n=19), 35.8±0.7°C at 8 hours (n=12), and 36.6±0.8°C at 20 hours (n=15). In the low-dose experimental group, rectal temperature was 38.4±0.2°C at 2 hours (n=19), 37.2±0.3°C at 8 hours (n=16), and 37.2±0.3°C at 20 hours (n=15), while in the control group the temperatures were 38.1±0.13°C (n=38), 37.6±0.3°C (n=29), and 37.4±0.2°C (n=28) at 2, 8, and 20 hours, respectively. There were no statistically significant differences among the three groups for rectal temperature at 2, 8, or 20 hours.
Results of the neurological examination are shown in Table 3. In the low-dose group, there was no significant
change in neurological function caused by the drug. In the
high-dose group, neurological examination scores in the
drug-treated group were lower both at 2 and 24 hours
(P<.01). Neurological scores from sham-operated piglets
not subjected to hypoxia or ischemia (n=10) were 20
(20, 20) as previously reported.13
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At the high dose, the drug has a profound sedative effect. The time to return of blink reflex after reoxygenation in a subset of the high-dose protocol increased from 59±4 minutes in the control group (n=10) to 82±7 in the drug group (n=14) (P<.05), and the time to return to even minimal respiration increased from 55±4 minutes in the control group (n=19) to 74±6 minutes (P<.05) in the high-dose experimental group (n=18). In the high-dose experiment, it was necessary to ventilate the drug-treated piglets for 2 to 6 hours longer than our usual 2 hours to keep them from dying of apnea. Given the long duration of action of the drug seen in our pilot studies, it is not surprising that the effect is still present at the 1-day examination. By days 2 and 3, although the neurological examination showed a trend toward lower scores in the high-dose drug-treated group, this difference was no longer statistically significant.
Pathological examination scores are shown in Table 4. In
the upper half of Table 4, the value for all pigs is shown; in the
lower half of the table, the values are shown for only those pigs
surviving the full 72 hours and receiving brain preservation at death.
Our scores for the cortex, hippocampus, and basal ganglia are given as
well as values for the sum of the scores for the three tissues. There
were no statistically significant differences among the experimental
and control groups in brain pathological damage. In the low-dose
experiment, 14 of the 19 experimental-group piglets and 13 of the
19 controls died before the third day and brain preservation at death
(P=NS). In the high-dose experiment, 9 of the 19
drug-treated piglets and 10 of the 19 control animals died before
completing the 3 days (P=NS). Thus, the death rate was 9 of
19 (47%) in the high-dose group, 14 of 19 (73%) in the
low-dose group, and 23 of 38 (60%) in the controls
(P=NS). Pathological examination results for sham piglets
not subjected to hypoxic-ischemic injury were cortex, 9 (9,
9); basal ganglion, 10 (9, 10); hippocampus, 10 (9.5, 10); and sum, 29
(27.5, 29) as previously reported (n=11 for all).13
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LY293558 is clearly a centrally active agent in piglets. As has been described by other investigators,24 anesthesia was prolonged by the use of this agent. In addition, neurological examination scores of the piglets showed evidence of sedation even 1 day after dosing. Thus, the activity of the agent on the central nervous system of the piglet has been clearly documented in this study. Because sedation was seen at the same doses used for AMPA antagonism in studies showing neuroprotection,16 we would presume LY293558 is active against AMPA receptors in piglet brain. We did see some effects on heart rate and blood volumes withdrawn to induce hypotension, as would be expected from the described effect of AMPA receptor antagonists on the sympathetic nervous system.25 26 Less blood volume had to be withdrawn to produce hypotension in the high-dose–treated group because of decreased sympathetic tone.27 Abolition of the baroreceptor heart response28 by LY293558 reduced the tachycardia induced by hypovolemia and the bradycardia caused by volume expansion. Because blood pressure was controlled in this experiment during the hypoxic-ischemic phase, the disruption of sympathetic nerve activity was unlikely to have affected the pathological results. The brain is relatively insensitive to sympathetic vasoconstriction.
Why then is the newborn piglet unaffected by AMPA antagonists while other models show such promising effects? There seems to be agreement that 30 to 90 mg/kg NBQX, an AMPA antagonist, is protective if given before or after 10 minutes of four-vessel occlusion brain ischemia in adult rats.12 29 30 31 32 33 34 Similar results are seen in the adult gerbil two-vessel occlusion experiment.14 35 36 Protection by AMPA antagonists from focal ischemia caused by middle cerebral artery occlusion has been shown in cats16 and rats37 38 39 by most but not all investigators.40 Using 10 minutes of bilateral carotid artery occlusion with hypotension to produce injury in adult rats, Nellgard and Wieloch11 found hippocampal CA1 protection when 30 mg/kg NBQX was given at reperfusion and for 6 hours afterward at 4.5 mg/kg per hour. This is the adult animal model most closely resembling our protocol. Aoki et al41 demonstrated lack of efficacy of AMPA antagonists in older piglets with injury induced by hypothermic circulatory arrest using acute metabolic recovery as the outcome variable. Aoki et al used a 25-mg/kg dose of NBQX given before injury. This is in direct contrast to the results of Redmond et al,42 who showed a protective effect in dogs of 3 mg/kg NBQX given after injury by hypothermic cardiac arrest. In the only neonatal study to date, Hagberg et al43 produced brain injury in 7-day-old rat pups by unilateral carotid ligation followed by 1.5 hours of 8% oxygen. They reported a weak protective effect in response to 40 mg/kg NBQX given after injury. This dose produced death in 26% of the treated rat pups. Arguably, their exclusion of the dead pups from analysis may be the only reason even a weak effect was seen. It is reasonable to assume that the dying pups had the most severe brain damage. We have not studied a piglet model of unilateral carotid ligation and several hours of hypoxia. Would this have altered our results? Seven-day-old rats are at their period of maximum susceptibility to direct AMPA excitotoxicity44 because of developmental changes in their AMPA receptors.45 Susceptibility to direct AMPA excitotoxicity has not been measured in piglets. Human newborns, newborn pigs, and 7-day-old rats are all at their maximal period of developmental brain growth.46 They may, however, be at very different developmental stages with respect to AMPA receptor development. Puka-Sundvall et al47 showed that even in newborn rats, where excitatory NMDA amino acid inhibitors are effective in preventing hypoxic brain injury,48 49 NMDA antagonists do not prevent the decreased extracellular calcium that is hypothesized to be critical for their action. Cherici et al50 have shown in newborn rat pups that hypoxia does not cause increased glutamate release. Similarly, Pastuszko51 found glutamate is elevated in the brain in only 40% of piglets during hypoxia. In these studies a more severe hypoxic stress might have been needed to produce glutamate release52 or calcium influx. Elevated excitatory amino acid levels are seen in the cerebrospinal fluid of human infants after asphyxic brain injury.53 54
Neither NMDA receptor antagonists13 nor AMPA receptor antagonists affect the outcome of hypoxic-ischemic brain injury in the newborn piglet model used in this study. Why excitatory amino acids are not involved in the pathophysiology of hypoxic-ischemic brain injury in this model is unclear. Both NMDA receptors55 and AMPA receptors56 have been isolated from pig brain and are similar to the receptors seen in rats. NMDA receptors have been measured in newborn pig brain,57 and the number of receptors is similar to that in adult pigs,58 although less than half that of receptors seen in adult rats.58 Measurement of AMPA receptors in newborn pig brain still needs to be made. It is possible that differences in receptor number or activity in the newborn pig explain the negative results seen in this study. The clear sedative effect of the AMPA antagonist at doses used in other studies would seem to imply AMPA activity in the brain of newborn pigs. It is possible, however, that LY293558 acts on the pig brain by other mechanisms. Certainly, measurement of AMPA receptors activity in newborn pig brain is now of great importance.
The only agent that is known to affect the outcome of hypoxic ischemia in the newborn piglet model presented in this article is glucose. Glucose enhances brain damage and insulin prevents it.17 The hypothesized mode of action of glucose is by increasing intracellular acidosis during the ischemic period. NMDA receptors are known to be inhibited by acidosis.59 The AMPA receptor is also inhibited by acidosis, although at a much lower pH.60 However, in the adult rat four-vessel occlusion model in which AMPA antagonists are effective, hyperglycemia exacerbates injury and hypoglycemia prevents it.61 It is very likely that some aspect of the animal model is critical to the differences described, either the age or species or the specifics of how the injury was induced. This leads us to counsel caution in extrapolating results from one model of hypoxic-ischemic brain injury to other models and species.
Received April 28, 1995; revision received June 26, 1995; accepted July 12, 1995.
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