Neuroscienee Letters, 136 (1992) 165-168
165
© 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00
NSL 08426
Pretreatment with NMDA antagonists limits release of excitatory amino acids following traumatic brain injury S. Scott Panter" and Alan I. Faden b aBlood Research Division, Letterman Army Institute of Research, San Francisco, CA 94129 (USA) and bDepartment of Neurology and Pharmacology, Georgetown University School of Medicine, Washington, DC 20007 (USA) (Received 7 August 1991; Revised version received 25 October 1991; Accepted 27 November 1991)
Key words: CGS-19755; Dextrorphan; Extracellular excitatory amino acid; Hippocampus; Microdialysis; N-Methyl-D-aspartate antagonist; Traumatic brain injury After central nervous system (CNS) trauma, there are marked elevations in the extracellular levels of excitatory amino acids (EAA), which are believed to contribute to delayed tissue damage. Administration of N-methyl-D-aspartate (NMDA) receptor antagonists reduces injury severity after brain or spinal cord trauma, presumably by blocking the postsynaptic NMDA receptor. In the present studies, levels of extracellular amino acids were monitored by microdialysis during, and after, a moderately severe fluid-percussion brain injury to rats. Pretreatment (15 min prior to injury) with the non-competitive NMDA antagonist dextrorphan or the competitive NMDA antagonist CGS 19755 significantly attenuated the post-traumatic increase in extracellular glutamate. Pretreatment with dextrorphan attenuated the post-traumatic increase in extracellular levels of aspartate; although these differences did not reach significance when examined as absolute values, they were significant when analyzed as percent increase over pre-trauma baseline levels. These results are consistent with recent experiments and suggest that NMDA antagonists may limit the release of glutamate and aspartate after trauma through a presynaptic mechanism.
Excitatory amino acids (EAA) appear to contribute to the pathophysiology of central nervous system (CNS) injury [1, 5, 6]. Elevated extracellular concentrations of aspartate and glutamate have been detected after neurotrauma [6, 19, 21] or brain ischemia [2, 7, 9]. Administration of competitive or non-competitive NMethyl-D-aspartate (NMDA) antagonists improves outcome following traumatic brain injury (TBI) [6, 11, 15, 17], spinal cord injury (SCI) [4, 5, 8] or cerebral ischemia [1, 10, 22]. The beneficial effect of the NMDA antagonists is presumed to be mediated through postsynaptic actions. However, recent evidence from cell culture studies suggests that certain effects of NMDA antagonists may be mediated through presynaptic mechanisms [18]. In the current study, the non-competitive NMDA antagonist dextrorphan [6] and the competitive NMDA antagonist CGS 19755 [12] were administered 15 min prior to fluid-percussion induced brain injury in order to determine whether such treatment modifies EAA release. Sprague-Dawley rats (males, weighing 425-430 g) were anesthetized with pentobarbital (70 mg/kg, i.p.) and Correspondence: A.I. Faden, Georgetown University Medical Center, 3900 Reservoir Rd., Med./Dent. Bldg, NW-101, Washington, DC 20007, USA.
intubated. After bilateral femoral cutdown, an arterial line (for monitoring blood pressure) and a venous line (for drug administration) were inserted. Following surgery, anesthesia was continuously maintained throughout the procedure by continuous infusion ofpentobarbital (15 mg/kg/h, i.v.). Although brain temperature was not monitored, a heating pad was used to maintain body temperature. A microdialysis probe was stereotaxically placed through a burr hole in the temporal region to position the tip of the probe in hippocampal regions CA2 and CA3, just below the trauma site, as previously described [6]. Fluid-percussion induced TBI was induced through a second craniotomy site centered over the left parietal cortex, as detailed elsewhere [6, 16]. Although the presence of the probe may have contributed to post-traumatic tissue injury in the present experiments, the probe was not removed during trauma because of the potential problem of reinsertion artifact and because we were interested in measuring early post-traumatic changes. Microdialysis studies were performed using probes (4 mm membrane length, 0.6 mm o.d.) and a precision syringe pump manufactured by Carnegie Medicin, AB (Sweden) and purchased from Bioanalytical Systems (Lafayette, IN). The flow rate of dialysate (distilled
166 water) t h r o u g h the probe was 2/al/min. Distilled water was used as the dialysate, consistent with prior studies in this model [6]; use o f distilled water, rather than an isotonic solution, does not affect the baseline levels or posttraumatic increase o f glutamate or aspartate in this model [20]. Immediately following insertion o f the probe, dialysate flow was started and continued t h r o u g h o u t the duration o f the experiment. After a 1 h equilibration period, sample collection was initiated. Fifteen minutes prior to the administration o f trauma, either dext r o r p h a n (10 mg/kg, i.v., n=4), C G S 19755 (30 mg/kg, i.v., n--5), or saline (1 ml, i.v., n=4) was administered. Following trauma, samples were collected into microfuge tubes at 10 min intervals for 1 h, after which time samples were collected at 30 min intervals for an additional 3 h. The concentrations of amino acids in each sample were measured by high performance liquid c h r o m a t o g r a phy, using the m e t h o d previously described [6]. All data are expressed as the concentration o f amino acid present in the dialysate. Statistical analyses were performed using analysis o f variance. Post hoc comparisons o f g r o u p means were performed using Fisher's protected least squares difference [23] at a significance level o f P
165
© 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00
NSL 08426
Pretreatment with NMDA antagonists limits release of excitatory amino acids following traumatic brain injury S. Scott Panter" and Alan I. Faden b aBlood Research Division, Letterman Army Institute of Research, San Francisco, CA 94129 (USA) and bDepartment of Neurology and Pharmacology, Georgetown University School of Medicine, Washington, DC 20007 (USA) (Received 7 August 1991; Revised version received 25 October 1991; Accepted 27 November 1991)
Key words: CGS-19755; Dextrorphan; Extracellular excitatory amino acid; Hippocampus; Microdialysis; N-Methyl-D-aspartate antagonist; Traumatic brain injury After central nervous system (CNS) trauma, there are marked elevations in the extracellular levels of excitatory amino acids (EAA), which are believed to contribute to delayed tissue damage. Administration of N-methyl-D-aspartate (NMDA) receptor antagonists reduces injury severity after brain or spinal cord trauma, presumably by blocking the postsynaptic NMDA receptor. In the present studies, levels of extracellular amino acids were monitored by microdialysis during, and after, a moderately severe fluid-percussion brain injury to rats. Pretreatment (15 min prior to injury) with the non-competitive NMDA antagonist dextrorphan or the competitive NMDA antagonist CGS 19755 significantly attenuated the post-traumatic increase in extracellular glutamate. Pretreatment with dextrorphan attenuated the post-traumatic increase in extracellular levels of aspartate; although these differences did not reach significance when examined as absolute values, they were significant when analyzed as percent increase over pre-trauma baseline levels. These results are consistent with recent experiments and suggest that NMDA antagonists may limit the release of glutamate and aspartate after trauma through a presynaptic mechanism.
Excitatory amino acids (EAA) appear to contribute to the pathophysiology of central nervous system (CNS) injury [1, 5, 6]. Elevated extracellular concentrations of aspartate and glutamate have been detected after neurotrauma [6, 19, 21] or brain ischemia [2, 7, 9]. Administration of competitive or non-competitive NMethyl-D-aspartate (NMDA) antagonists improves outcome following traumatic brain injury (TBI) [6, 11, 15, 17], spinal cord injury (SCI) [4, 5, 8] or cerebral ischemia [1, 10, 22]. The beneficial effect of the NMDA antagonists is presumed to be mediated through postsynaptic actions. However, recent evidence from cell culture studies suggests that certain effects of NMDA antagonists may be mediated through presynaptic mechanisms [18]. In the current study, the non-competitive NMDA antagonist dextrorphan [6] and the competitive NMDA antagonist CGS 19755 [12] were administered 15 min prior to fluid-percussion induced brain injury in order to determine whether such treatment modifies EAA release. Sprague-Dawley rats (males, weighing 425-430 g) were anesthetized with pentobarbital (70 mg/kg, i.p.) and Correspondence: A.I. Faden, Georgetown University Medical Center, 3900 Reservoir Rd., Med./Dent. Bldg, NW-101, Washington, DC 20007, USA.
intubated. After bilateral femoral cutdown, an arterial line (for monitoring blood pressure) and a venous line (for drug administration) were inserted. Following surgery, anesthesia was continuously maintained throughout the procedure by continuous infusion ofpentobarbital (15 mg/kg/h, i.v.). Although brain temperature was not monitored, a heating pad was used to maintain body temperature. A microdialysis probe was stereotaxically placed through a burr hole in the temporal region to position the tip of the probe in hippocampal regions CA2 and CA3, just below the trauma site, as previously described [6]. Fluid-percussion induced TBI was induced through a second craniotomy site centered over the left parietal cortex, as detailed elsewhere [6, 16]. Although the presence of the probe may have contributed to post-traumatic tissue injury in the present experiments, the probe was not removed during trauma because of the potential problem of reinsertion artifact and because we were interested in measuring early post-traumatic changes. Microdialysis studies were performed using probes (4 mm membrane length, 0.6 mm o.d.) and a precision syringe pump manufactured by Carnegie Medicin, AB (Sweden) and purchased from Bioanalytical Systems (Lafayette, IN). The flow rate of dialysate (distilled
166 water) t h r o u g h the probe was 2/al/min. Distilled water was used as the dialysate, consistent with prior studies in this model [6]; use o f distilled water, rather than an isotonic solution, does not affect the baseline levels or posttraumatic increase o f glutamate or aspartate in this model [20]. Immediately following insertion o f the probe, dialysate flow was started and continued t h r o u g h o u t the duration o f the experiment. After a 1 h equilibration period, sample collection was initiated. Fifteen minutes prior to the administration o f trauma, either dext r o r p h a n (10 mg/kg, i.v., n=4), C G S 19755 (30 mg/kg, i.v., n--5), or saline (1 ml, i.v., n=4) was administered. Following trauma, samples were collected into microfuge tubes at 10 min intervals for 1 h, after which time samples were collected at 30 min intervals for an additional 3 h. The concentrations of amino acids in each sample were measured by high performance liquid c h r o m a t o g r a phy, using the m e t h o d previously described [6]. All data are expressed as the concentration o f amino acid present in the dialysate. Statistical analyses were performed using analysis o f variance. Post hoc comparisons o f g r o u p means were performed using Fisher's protected least squares difference [23] at a significance level o f P
