Alteration in Extracellular Armno Acids After Traumatic Spinal Cord Injury S. Scott Panter, PhD, Sabrina W. Yum, MD, and Alan I. Faden, M D It has recently been demonstrated that N-methyl-Daspartate antagonists limit tissue damage after spinal cord trauma, implicating excitatory amino acids in the secondary injury response. To determine whether spinal cord trauma alters the concentrations of extracellular amino acids, microdialysis was conducted in spinal cord during and after administration of impact trauma Extracellular concentrations of excitatory, inhibitory, and nontransmitter amino acids were elevated after trauma, with the degree of increase related to severity of injury. Moderate trauma resulted in an immediate but transient increase (200-40096)in the extracellular levels of all amino acids measured. Severe trauma produced a more prolonged and significant increase (400-630’96)in the concentrations of extracellular amino acids, including aspartate and glutamate. These results are consistent with the hypothesis that excitatory amino acids may contribute to delayed tissue injury after central nervous system trauma. Panter SS, Yum SW, Faden AI. Alteration in extracellular amino acids after traumatic spinal cord injury. Ann Neurol 1990;27:96-99
White New Zealand male rabbits (weighing approximately 3.5-4.0 kg) were anesthetized with isoflurane (3%), and anesthesia was maintained (1.0% isoflurane)throughout all experiments. Animals were subsequently intubated, paralyzed
with gallamine triethiodide ( 3 mg/kg, IV), and maintained on a respirator. After placement of an arterial (ear artery) line used for blood gas and blood pressure monitoring, each animal was immobilized in a spinal cord stereotaxicapparatus (David Kopf Instruments, Tujunga, CA), and an incision was made over the lumbar section of the spinal cord. A 2segment laminectomy was performed at L3-L4. Arterial blood gases were maintained in the normal range through respirator adjustments and use of sodium bicarbonate. A microdialysis probe (4-mm membrane length; BioAnalytical Systems, West Lafayette, IN) was fastened to a micromanipulator and positioned at a 60-degree angle, 1 mm lateral to midline, a distance of 5 mm into the spinal cord (Fig 1). Throughout the entire procedure, the flow of perfusate (distilled water) through the microdialysis probe was maintained at 2 pYmin using a precision syringe pump (BioAnalytical Systems). The first 10 pl (corresponding to the void volume of the probe) was discarded, and subsequent samples were collected for a minimum period of 5 minutes. All samples were analyzed for amino acids (data presented here) and monovalent and divalent cations (analysis in progress). In order to measure cation changes, it was necessary to use distilled water as the dialysis perfusate. Pilot studies have shown that there are no differences between basal amino acid levels from microdialysis experiments using a distilled water perfusate and those obtained using physiological saline (n = 4). After insertion of the probe, tissue was allowed to normalize for a minimum of 20 minutes. Impact trauma to the spinal cord was produced using a weight-drop technique, as previously described in detail C5J.In brief, a 20-gm weight is
From the Center for Neural Injury, Department of Neurology, University of California at San Francisco, and Veterans Administration Medical Center, San Francisco, CA.
Address correspondence to Dr Faden, Neurology Service (127), Veterans Administration Medical Center, 4150 Clement St, San Francisco, CA 94 12 1.
Impact injuries to the spinal cord involve both primary
and secondary injury processes El}. Many factors have been proposed to contribute to the pathophysiology of the delayed injury response, including endogenous opioids { 2 } , ion fluxes [S-51, and changes in membrane fatty acid composition [b}. Recently, excitatory amino acids have also been implicated as mediators of secondary tissue damage after brain and spinal cord trauma {7-91. Excitatory amino acids have been shown to produce delayed toxicity in tissue culture systems {lo}, and the resulting cell death appears to be caused, in part, by calcium-related mechanisms. Tissue damage after central nervous system (CNS) injury and cell death after extracellular excitatory amino acid exposure are both attenuated by treatment with N-methylDaspartate (NMDA) receptor antagonists 17, 8, 11141. Increased extracellular levels of excitatory amino acids are found following experimental brain trauma {S, 91. In the present series of experiments, we used microdialysis techniques to address the hypothesis that extracellular levels of aspartate and glutamate are elevated after spinal cord trauma, with the degree of elevation being related to severity of injury.
Methods
Received for publication Apr 19, 1989, and in revised form Jun 13. Accepted for publication Jun 19, 1989.
96
analysis of variance, and post hoc comparisons of group means were performed using Fisher's protected least squares difference [l5} at a significance level of p < 0.05.
Fig I. A schematic representation of the position of the microdialysis probe in relation to the spinal cord and the area of injury. Stippling indicates areas within the spinal cord. dropped a distance of 2.0 cm or 7.5 crn through a fiberglass guide tube onto a Teflon impact plate that rests on the exposed dura. This drop produces moderate (40 gm-cm, n = 5) or severe (150 gm-cm, n = 6) degrees of tissue injury, respectively, as determined by behavioral and histological outcome. Control animals (n = 5) were subjected to laminectomy but were not injured. Amino acids were separated by high-performance liquid chromatography, detected electrochemically,and quantitated via peak areas as previously described 18). Amino acid data, expressed as concentration in dialysate, were studied using
8o
1
Results The insertion of the dialysis probe caused a small increase in the extracellular concentrations of all amino acids. Within 20 minutes of the insertion of the microdialysis probe, extracellular levels of amino acids had decreased to concentrations that were maintained during the experimental time period (Fig 2). After impact trauma, the extracellular concentrations of all amino acids immediately increased, but the degree as well as the pattern of increase was related to severity of injury (see Fig 2, Table). After a 40 gmcm trauma, extracellular amino acids peaked within 10 minutes. Extracellular concentrations of all amino acids, except glutamine and threonine, were significantly ( p < 0.05) elevated over control levels (Table). Fig 2. Eflect of two different levels of impact injuv to spinal cord on extracellular concentrations of aspartate, glutamate, glycine, and gamma aminobutyric acid (GABA).Baseline amino acid concentrations were designated as those detected at timepoint 0, which corresponds to the time at which trauma was administered. a = d;fferentfrom controlsubject, p < 0.05;b = d$ifwent from 40-gm-cm injuty, p < 0.05.
AS PARTATE
T
*O0 150
1
GLYCINE T
CONTROL 40 GM-CM
150 GM-CM
&--.
50
I
0
20 40 TIME (MINUTES)
60
40
GLUTAMATE
I
I
0
20
40 TIME (MINUTES)
I
60
1
0
I
i
60
GABA
1
I
1
I
20 40 TIME (MINUTES)
0
I
20 40 TIME (MINUTES)
I
60
.
Panter et al: Amino Acids and Injury 97
Mean Maximal Increases in Extracellular Amino Acids After Spinal Cord Trauma
Injury Severity (% increase) Amino Acid
40 gm-cm
Aspartate Glutamate Glutamine Serine Threonine Glycine AIanine Taurine Gamma aminobutyric acid
26 1 357 409 275 280 330 375 425 458
150 gm-cm
542 410 ,631 616 575 454 630 597 472
"The concentrations of 9 different amino acids were determined in samples of spinal cord extracellular fluid obtained by microdialysis before, during, and after trauma. The data from moderate (40 gmcm) injury (n = 5 ) represent extracellular amino acids observed 10 minutes after trauma. Following a severe (150 gm-cm) injury (n = 6), however, extracellular amino acid concentrations did not peak until 20 minutes after trauma.
With the exception of alanine, amino acid levels recovered to pretrauma levels by 20 minutes after trauma After a 150 gm-cm injury, levels of extracellular amino acids increased but did not peak until 20 minutes after trauma (see Fig 2, Table). At the first postinjury timepoint (10 minutes), significant ( p < 0.05) increases were observed for aspartate, glutamate, serine, glycine, taurine, and alanine. By 20 minutes, all amino acids except gamma aminobutyric acid (GABA) were significantly different from control levels, and all except taurine and GABA were significantly higher than extracellular levels attained after a 40 gm-cm trauma. Extracellular levels of most amino acids remained significantly elevated for at least 30 minutes after trauma.
Discussion The microdialysis studies of the current report were conducted to determine the changes in extracellular amino acids after spinal cord trauma. Trauma caused a rapid and significant increase in the extracellular levels of 9 different amino acids, with the degree of increase related to the degree of injury. Although no previous reports have evaluated posttraumatic alterations of extracellular amino acids in spinal cord, a number of studies have reported either tissue andor extracellular concentrations of amino acids after ischemia or hypoglycemia [lb). Seven of the 9 amino acids examined in the current report have been examined previously: aspartate, glutamate, GABA, taurine, alanine, glycine, and threonine [1720). Regardless of the type of injury administered, extracellular amino acid concentrations rapidly increased for the duration of injury, and when the tissue
98 Annals of Neurology Vol 27 No 1 January 1990
condition was normalized (during reperfusion or after reinstitution of normoglycemia), levels of extracellular amino acids declined rapidly. In the one case in which ischemia was irreversible (caused by cardiac arrest), the concentration of amino acids in the dialysate continued to rise throughout the experimental course, which was 20 to 25 minutes 1171. The data from these earlier reports agree with the finding of the current experimental series and suggest that primary tissue damage causes extracellular amino acid changes that may be independent of the specific insult. Spinal cord trauma did not affect amino acids selectively; rather, there was an increase in the extracellular concentrations of all amino acids measured (see Table). A trauma-related disruption of the blood-spinal cord barrier could result in a significant movement of free amino acids from plasma into the extracellular space of the spinal cord, yielding a generalized increase in the extracellular concentration of amino acids. Except for 3 amino acids (alanine, threonine, and serine), the peak concentrations of extracellular amino acids detected in the current study (adjusted to compensate for incomplete recovery) were well above those reported in rat plasma 121, 22). Consequently, free amino acids from plasma may leak into the extracellular space of spinal cord after trauma, but the high extracellular amino acid concentrations observed in the present study suggest that a major proportion of elevated extracellular amino acids arise from the cells of the spinal cord itself. Of the 9 amino acids examined in the present study, increases in the extracellular levels of GABA, taurine, serine, threonine, alanine, and glutamine have not been reported to be toxic. An additional amino acid, glycine, is generally considered to be inhibitory, but a recent study indicates that glycine may potentiate excitotoxicity by significantly increasing receptor affinity for NMDA agonists E23). However, because the glycine-induced enhancement of agonist binding in vitro saturates at low micromolar concentrations, the relevance of this observation to in vivo systems remains to be determined. Aspartate and glutamate are excitatory amino acids that are thought to have toxic effects in the central nervous system [24). Experiments conducted on cells in culture have shown that excitatory amino acids themselves or their analogues exacerbate 2 different processes of cellular injury [lo, 121. First, excitatory amino acids are thought to mediate significant ion shifts that cause cellular edema. Secondly, excitotoxins promote intracellular calcium increases, which may damage membranes, activate proteolysis, cause irreversible protein cross-linking, and result in cellular death. Many of these changes may be attenuated by NMDA-receptor antagonists [12, 241. Treatments with NMDA antagonists in vivo have
been shown to reduce CNS injury after ischemia 1141, hypoglycemia {13f, traumatic brain injury [8, 251, and spinal cord trauma 17, 111. The latter studies have shown that both competitive and noncompetitive NMDA antagonists improve outcome after brain or spinal cord trauma in rats. In addition, administration of NMDA itself adjacent to the injured area of spinal cord significantly worsens neurological outcome, but its stereoisomer N-methyl-L-aspartate, which is less active at NMDA receptors, did not affect outcome 171. These animal studies provide strong evidence that excitatory amino acids, through actions at NMDA receptors, may contribute to secondary tissue injury. The present experiments show that extracellular levels of excitatory amino acids increase after spinal cord trauma in proportion to severity of injury and are consistent with the hypothesis that excitatory amino acids mediate, in part, secondary injury after spinal cord trauma. We thank Dr J. B. Justice, Jr, for his advice on microdialysis, and Vicky Cardenas, Michael Daly, and Peter Halt for technical assistance. We adhered to the principles enumerated in the Guidefor the Care and Use of Laboratory Aninals, prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Resources, National Research Council (DHEW publication no. [NIH] 85-23, 1985).
This research was supported in part by National Institutes of Health Program Center grant NS14543-09, Centers for Disease Control grant R49lCCR902269, and by a Veterans Administration Merit Review Grant to AIF.
References 1. Faden AI. Recent pharmacological advances in experimental
2.
3.
4.
5.
6.
7.
spinal injury: theoretical and methodological considerations. Trends Neurosci 1983;6:3 7 5 -3 7 7 Faden AI, Molineaux CJ, Rosenberger JG, et al. Endogenous opioid immunoreactivity in rat spinal cord following traumatic injury. Ann Neurol 1985;17:386-390 Young W. Blood flow, metabolism and neurophysiologic mechanisms of spinal cord injury. In: Becker DP, PovlishockJT, eds. CNS trauma status report. Washington, DC: NIH-NINCDS, 1985~463-473 Vink R, McIntosh TK, Demediuk P, et al. Decline in intracellular free MgfZ is associated with irreversible tissue injury after brain .trauma. J Biol Chem 1988;263:757-761 Vink R, Yum SW, Lemke M, et al. Traumatic spinal cord injury in rabbits decreases invarellular free magnesium concentration as measured by 31PMRS. Brain Res 1989;490144-147 Demediuk P, Saunders RD, Anderson DK, et al. Membrane lipid changes in laminectomized and traumatized cat spinal cord. Proc Natl Acad Sci USA 1985;82:7071-7075 Faden AI, Simon RP. A potential role for excitotoxins in the pathophysiology of spinal cord injury. Ann Neurol 1988;23: 623-626
8. Faden AI, Demediuk P, Panter SS, Vink P. The role of excitatory amino acids and NMDA receptors in traumatic brain injury. Science 1989;244:798-800 9. Katayama Y, Cheung MK, Gorman L, et al. Increase in extracellular glutamate and associated massive ionic fluxes following concussive brain injury. SOCNeurosci Abstr 1988;14:1154 10. Choi DW. Ionic dependence of glutamate neurotoxicity. J Neurosci 1987;7:369-379 11. Faden AI, Lemke M, Simon RP, Noble LJ. N-methyl-Daspartate antagonist MK-801 improves outcome following traumatic spinal cord injury in rats: behavioral, anatomical, and neurochemical studies. J Neurotrauma 1988;5:27-37 12. Choi DW. Calcium-mediated neurotoxicity: relationship to specific channel types and role in ischemic damage. Trends Neurosci 1988;11:465-469 13. Wieloch T. Hypoglycemia-induced neuronal damage prevented by an N-methyl-Daspartate antagonist. Science 1985;230:681683 14. Simon RP, Swan JH, Griffiths T, Meldrum BS. Blockade of Nmethyl-D-aspartate receptors may protect against ischemic damage in the brain. Science 1984;226:850-852 15. Snedecor GW, Cochran WG. Statistical methods. Ames: Iowa State University Press, 1980:333-337 16. Auer RN,Siesjo BK. Biological differences between ischemia, hypoglycemia, and epilepsy. Ann Neurol 1988;24:699-707 17. Korf J, Klein HC, Venema K, Postema F. Increases in striatal and hippocampal impedance and extracellular levels of amino acids by cardiac arrest in freely moving rats. J Neurochem 1988;50:1087-1096 8. Hagberg H, Lehmann A, Sandberg M, et al. Ischemia-induced shift of inhibitory and excitatory amino acids from intra- to extracellular compartmests. J Cereb Blood Flow Metab 1985; 5:413-419 9. Globus MY-T, Busto R, Dietrich WD, et al. Effect of ischemia on the in vivo release of striatal dopamine, glutamate, and gamma-aminobutyric acid studied by intracerebralmicrodialysis. J Neurochem 1988;51:1455-1464 20. Butcher SP, Sandberg M, Hagberg H, Hamberger A. Cellular origins of endogenous amino acids released into the extracellular fluid of the rat striatum during severe insulin-induced hypoglycemia J Neurochem 1987;48:722-728 21. Lunn PG, Whitehead RG, Baker BA. The relative effects of a low-protein-high-carbohydrate diet on the free amino acid composition of liver and muscle. Br J Nutr 1976;36:219230 22. Clarke DD, Lajtha AL, Maker HS. Intermediary metabolism. In: Siege1 GJ, Agranoff BW, Albers RW, et al, eds. Basic neurochemistry, 4th ed. New York: Raven, 1989341-564 23. Ransom RW, Stec NL. Cooperative modulation of C3H]MK801 binding to the N-methyl-Baspartate receptor-ion channel complex by L-glutamate, glycine, and polyamines. J Neurochem 1988;512330-836 24. Rothman SM, Olney JW. Excitotoxicity and the NMDA receptor. Trends Neurosci 1987;10:299-302 25. Hayes RL, Jenkins LW, Lyeth BG, et al. Pretreatment with phencyclidine, an N-methyl-Gaspartate antagonist, attenuates long-term behavioral deficits in the rat produced by traumatic brain injury. J Neurotrauma 1988;5:259-274
Panter et al: Amino Acids and Injury 99
White New Zealand male rabbits (weighing approximately 3.5-4.0 kg) were anesthetized with isoflurane (3%), and anesthesia was maintained (1.0% isoflurane)throughout all experiments. Animals were subsequently intubated, paralyzed
with gallamine triethiodide ( 3 mg/kg, IV), and maintained on a respirator. After placement of an arterial (ear artery) line used for blood gas and blood pressure monitoring, each animal was immobilized in a spinal cord stereotaxicapparatus (David Kopf Instruments, Tujunga, CA), and an incision was made over the lumbar section of the spinal cord. A 2segment laminectomy was performed at L3-L4. Arterial blood gases were maintained in the normal range through respirator adjustments and use of sodium bicarbonate. A microdialysis probe (4-mm membrane length; BioAnalytical Systems, West Lafayette, IN) was fastened to a micromanipulator and positioned at a 60-degree angle, 1 mm lateral to midline, a distance of 5 mm into the spinal cord (Fig 1). Throughout the entire procedure, the flow of perfusate (distilled water) through the microdialysis probe was maintained at 2 pYmin using a precision syringe pump (BioAnalytical Systems). The first 10 pl (corresponding to the void volume of the probe) was discarded, and subsequent samples were collected for a minimum period of 5 minutes. All samples were analyzed for amino acids (data presented here) and monovalent and divalent cations (analysis in progress). In order to measure cation changes, it was necessary to use distilled water as the dialysis perfusate. Pilot studies have shown that there are no differences between basal amino acid levels from microdialysis experiments using a distilled water perfusate and those obtained using physiological saline (n = 4). After insertion of the probe, tissue was allowed to normalize for a minimum of 20 minutes. Impact trauma to the spinal cord was produced using a weight-drop technique, as previously described in detail C5J.In brief, a 20-gm weight is
From the Center for Neural Injury, Department of Neurology, University of California at San Francisco, and Veterans Administration Medical Center, San Francisco, CA.
Address correspondence to Dr Faden, Neurology Service (127), Veterans Administration Medical Center, 4150 Clement St, San Francisco, CA 94 12 1.
Impact injuries to the spinal cord involve both primary
and secondary injury processes El}. Many factors have been proposed to contribute to the pathophysiology of the delayed injury response, including endogenous opioids { 2 } , ion fluxes [S-51, and changes in membrane fatty acid composition [b}. Recently, excitatory amino acids have also been implicated as mediators of secondary tissue damage after brain and spinal cord trauma {7-91. Excitatory amino acids have been shown to produce delayed toxicity in tissue culture systems {lo}, and the resulting cell death appears to be caused, in part, by calcium-related mechanisms. Tissue damage after central nervous system (CNS) injury and cell death after extracellular excitatory amino acid exposure are both attenuated by treatment with N-methylDaspartate (NMDA) receptor antagonists 17, 8, 11141. Increased extracellular levels of excitatory amino acids are found following experimental brain trauma {S, 91. In the present series of experiments, we used microdialysis techniques to address the hypothesis that extracellular levels of aspartate and glutamate are elevated after spinal cord trauma, with the degree of elevation being related to severity of injury.
Methods
Received for publication Apr 19, 1989, and in revised form Jun 13. Accepted for publication Jun 19, 1989.
96
analysis of variance, and post hoc comparisons of group means were performed using Fisher's protected least squares difference [l5} at a significance level of p < 0.05.
Fig I. A schematic representation of the position of the microdialysis probe in relation to the spinal cord and the area of injury. Stippling indicates areas within the spinal cord. dropped a distance of 2.0 cm or 7.5 crn through a fiberglass guide tube onto a Teflon impact plate that rests on the exposed dura. This drop produces moderate (40 gm-cm, n = 5) or severe (150 gm-cm, n = 6) degrees of tissue injury, respectively, as determined by behavioral and histological outcome. Control animals (n = 5) were subjected to laminectomy but were not injured. Amino acids were separated by high-performance liquid chromatography, detected electrochemically,and quantitated via peak areas as previously described 18). Amino acid data, expressed as concentration in dialysate, were studied using
8o
1
Results The insertion of the dialysis probe caused a small increase in the extracellular concentrations of all amino acids. Within 20 minutes of the insertion of the microdialysis probe, extracellular levels of amino acids had decreased to concentrations that were maintained during the experimental time period (Fig 2). After impact trauma, the extracellular concentrations of all amino acids immediately increased, but the degree as well as the pattern of increase was related to severity of injury (see Fig 2, Table). After a 40 gmcm trauma, extracellular amino acids peaked within 10 minutes. Extracellular concentrations of all amino acids, except glutamine and threonine, were significantly ( p < 0.05) elevated over control levels (Table). Fig 2. Eflect of two different levels of impact injuv to spinal cord on extracellular concentrations of aspartate, glutamate, glycine, and gamma aminobutyric acid (GABA).Baseline amino acid concentrations were designated as those detected at timepoint 0, which corresponds to the time at which trauma was administered. a = d;fferentfrom controlsubject, p < 0.05;b = d$ifwent from 40-gm-cm injuty, p < 0.05.
AS PARTATE
T
*O0 150
1
GLYCINE T
CONTROL 40 GM-CM
150 GM-CM
&--.
50
I
0
20 40 TIME (MINUTES)
60
40
GLUTAMATE
I
I
0
20
40 TIME (MINUTES)
I
60
1
0
I
i
60
GABA
1
I
1
I
20 40 TIME (MINUTES)
0
I
20 40 TIME (MINUTES)
I
60
.
Panter et al: Amino Acids and Injury 97
Mean Maximal Increases in Extracellular Amino Acids After Spinal Cord Trauma
Injury Severity (% increase) Amino Acid
40 gm-cm
Aspartate Glutamate Glutamine Serine Threonine Glycine AIanine Taurine Gamma aminobutyric acid
26 1 357 409 275 280 330 375 425 458
150 gm-cm
542 410 ,631 616 575 454 630 597 472
"The concentrations of 9 different amino acids were determined in samples of spinal cord extracellular fluid obtained by microdialysis before, during, and after trauma. The data from moderate (40 gmcm) injury (n = 5 ) represent extracellular amino acids observed 10 minutes after trauma. Following a severe (150 gm-cm) injury (n = 6), however, extracellular amino acid concentrations did not peak until 20 minutes after trauma.
With the exception of alanine, amino acid levels recovered to pretrauma levels by 20 minutes after trauma After a 150 gm-cm injury, levels of extracellular amino acids increased but did not peak until 20 minutes after trauma (see Fig 2, Table). At the first postinjury timepoint (10 minutes), significant ( p < 0.05) increases were observed for aspartate, glutamate, serine, glycine, taurine, and alanine. By 20 minutes, all amino acids except gamma aminobutyric acid (GABA) were significantly different from control levels, and all except taurine and GABA were significantly higher than extracellular levels attained after a 40 gm-cm trauma. Extracellular levels of most amino acids remained significantly elevated for at least 30 minutes after trauma.
Discussion The microdialysis studies of the current report were conducted to determine the changes in extracellular amino acids after spinal cord trauma. Trauma caused a rapid and significant increase in the extracellular levels of 9 different amino acids, with the degree of increase related to the degree of injury. Although no previous reports have evaluated posttraumatic alterations of extracellular amino acids in spinal cord, a number of studies have reported either tissue andor extracellular concentrations of amino acids after ischemia or hypoglycemia [lb). Seven of the 9 amino acids examined in the current report have been examined previously: aspartate, glutamate, GABA, taurine, alanine, glycine, and threonine [1720). Regardless of the type of injury administered, extracellular amino acid concentrations rapidly increased for the duration of injury, and when the tissue
98 Annals of Neurology Vol 27 No 1 January 1990
condition was normalized (during reperfusion or after reinstitution of normoglycemia), levels of extracellular amino acids declined rapidly. In the one case in which ischemia was irreversible (caused by cardiac arrest), the concentration of amino acids in the dialysate continued to rise throughout the experimental course, which was 20 to 25 minutes 1171. The data from these earlier reports agree with the finding of the current experimental series and suggest that primary tissue damage causes extracellular amino acid changes that may be independent of the specific insult. Spinal cord trauma did not affect amino acids selectively; rather, there was an increase in the extracellular concentrations of all amino acids measured (see Table). A trauma-related disruption of the blood-spinal cord barrier could result in a significant movement of free amino acids from plasma into the extracellular space of the spinal cord, yielding a generalized increase in the extracellular concentration of amino acids. Except for 3 amino acids (alanine, threonine, and serine), the peak concentrations of extracellular amino acids detected in the current study (adjusted to compensate for incomplete recovery) were well above those reported in rat plasma 121, 22). Consequently, free amino acids from plasma may leak into the extracellular space of spinal cord after trauma, but the high extracellular amino acid concentrations observed in the present study suggest that a major proportion of elevated extracellular amino acids arise from the cells of the spinal cord itself. Of the 9 amino acids examined in the present study, increases in the extracellular levels of GABA, taurine, serine, threonine, alanine, and glutamine have not been reported to be toxic. An additional amino acid, glycine, is generally considered to be inhibitory, but a recent study indicates that glycine may potentiate excitotoxicity by significantly increasing receptor affinity for NMDA agonists E23). However, because the glycine-induced enhancement of agonist binding in vitro saturates at low micromolar concentrations, the relevance of this observation to in vivo systems remains to be determined. Aspartate and glutamate are excitatory amino acids that are thought to have toxic effects in the central nervous system [24). Experiments conducted on cells in culture have shown that excitatory amino acids themselves or their analogues exacerbate 2 different processes of cellular injury [lo, 121. First, excitatory amino acids are thought to mediate significant ion shifts that cause cellular edema. Secondly, excitotoxins promote intracellular calcium increases, which may damage membranes, activate proteolysis, cause irreversible protein cross-linking, and result in cellular death. Many of these changes may be attenuated by NMDA-receptor antagonists [12, 241. Treatments with NMDA antagonists in vivo have
been shown to reduce CNS injury after ischemia 1141, hypoglycemia {13f, traumatic brain injury [8, 251, and spinal cord trauma 17, 111. The latter studies have shown that both competitive and noncompetitive NMDA antagonists improve outcome after brain or spinal cord trauma in rats. In addition, administration of NMDA itself adjacent to the injured area of spinal cord significantly worsens neurological outcome, but its stereoisomer N-methyl-L-aspartate, which is less active at NMDA receptors, did not affect outcome 171. These animal studies provide strong evidence that excitatory amino acids, through actions at NMDA receptors, may contribute to secondary tissue injury. The present experiments show that extracellular levels of excitatory amino acids increase after spinal cord trauma in proportion to severity of injury and are consistent with the hypothesis that excitatory amino acids mediate, in part, secondary injury after spinal cord trauma. We thank Dr J. B. Justice, Jr, for his advice on microdialysis, and Vicky Cardenas, Michael Daly, and Peter Halt for technical assistance. We adhered to the principles enumerated in the Guidefor the Care and Use of Laboratory Aninals, prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Resources, National Research Council (DHEW publication no. [NIH] 85-23, 1985).
This research was supported in part by National Institutes of Health Program Center grant NS14543-09, Centers for Disease Control grant R49lCCR902269, and by a Veterans Administration Merit Review Grant to AIF.
References 1. Faden AI. Recent pharmacological advances in experimental
2.
3.
4.
5.
6.
7.
spinal injury: theoretical and methodological considerations. Trends Neurosci 1983;6:3 7 5 -3 7 7 Faden AI, Molineaux CJ, Rosenberger JG, et al. Endogenous opioid immunoreactivity in rat spinal cord following traumatic injury. Ann Neurol 1985;17:386-390 Young W. Blood flow, metabolism and neurophysiologic mechanisms of spinal cord injury. In: Becker DP, PovlishockJT, eds. CNS trauma status report. Washington, DC: NIH-NINCDS, 1985~463-473 Vink R, McIntosh TK, Demediuk P, et al. Decline in intracellular free MgfZ is associated with irreversible tissue injury after brain .trauma. J Biol Chem 1988;263:757-761 Vink R, Yum SW, Lemke M, et al. Traumatic spinal cord injury in rabbits decreases invarellular free magnesium concentration as measured by 31PMRS. Brain Res 1989;490144-147 Demediuk P, Saunders RD, Anderson DK, et al. Membrane lipid changes in laminectomized and traumatized cat spinal cord. Proc Natl Acad Sci USA 1985;82:7071-7075 Faden AI, Simon RP. A potential role for excitotoxins in the pathophysiology of spinal cord injury. Ann Neurol 1988;23: 623-626
8. Faden AI, Demediuk P, Panter SS, Vink P. The role of excitatory amino acids and NMDA receptors in traumatic brain injury. Science 1989;244:798-800 9. Katayama Y, Cheung MK, Gorman L, et al. Increase in extracellular glutamate and associated massive ionic fluxes following concussive brain injury. SOCNeurosci Abstr 1988;14:1154 10. Choi DW. Ionic dependence of glutamate neurotoxicity. J Neurosci 1987;7:369-379 11. Faden AI, Lemke M, Simon RP, Noble LJ. N-methyl-Daspartate antagonist MK-801 improves outcome following traumatic spinal cord injury in rats: behavioral, anatomical, and neurochemical studies. J Neurotrauma 1988;5:27-37 12. Choi DW. Calcium-mediated neurotoxicity: relationship to specific channel types and role in ischemic damage. Trends Neurosci 1988;11:465-469 13. Wieloch T. Hypoglycemia-induced neuronal damage prevented by an N-methyl-Daspartate antagonist. Science 1985;230:681683 14. Simon RP, Swan JH, Griffiths T, Meldrum BS. Blockade of Nmethyl-D-aspartate receptors may protect against ischemic damage in the brain. Science 1984;226:850-852 15. Snedecor GW, Cochran WG. Statistical methods. Ames: Iowa State University Press, 1980:333-337 16. Auer RN,Siesjo BK. Biological differences between ischemia, hypoglycemia, and epilepsy. Ann Neurol 1988;24:699-707 17. Korf J, Klein HC, Venema K, Postema F. Increases in striatal and hippocampal impedance and extracellular levels of amino acids by cardiac arrest in freely moving rats. J Neurochem 1988;50:1087-1096 8. Hagberg H, Lehmann A, Sandberg M, et al. Ischemia-induced shift of inhibitory and excitatory amino acids from intra- to extracellular compartmests. J Cereb Blood Flow Metab 1985; 5:413-419 9. Globus MY-T, Busto R, Dietrich WD, et al. Effect of ischemia on the in vivo release of striatal dopamine, glutamate, and gamma-aminobutyric acid studied by intracerebralmicrodialysis. J Neurochem 1988;51:1455-1464 20. Butcher SP, Sandberg M, Hagberg H, Hamberger A. Cellular origins of endogenous amino acids released into the extracellular fluid of the rat striatum during severe insulin-induced hypoglycemia J Neurochem 1987;48:722-728 21. Lunn PG, Whitehead RG, Baker BA. The relative effects of a low-protein-high-carbohydrate diet on the free amino acid composition of liver and muscle. Br J Nutr 1976;36:219230 22. Clarke DD, Lajtha AL, Maker HS. Intermediary metabolism. In: Siege1 GJ, Agranoff BW, Albers RW, et al, eds. Basic neurochemistry, 4th ed. New York: Raven, 1989341-564 23. Ransom RW, Stec NL. Cooperative modulation of C3H]MK801 binding to the N-methyl-Baspartate receptor-ion channel complex by L-glutamate, glycine, and polyamines. J Neurochem 1988;512330-836 24. Rothman SM, Olney JW. Excitotoxicity and the NMDA receptor. Trends Neurosci 1987;10:299-302 25. Hayes RL, Jenkins LW, Lyeth BG, et al. Pretreatment with phencyclidine, an N-methyl-Gaspartate antagonist, attenuates long-term behavioral deficits in the rat produced by traumatic brain injury. J Neurotrauma 1988;5:259-274
Panter et al: Amino Acids and Injury 99
