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Changes in Hippocampal Monoamine Concentration Following Halothane Anesthesia and Concussion Gerald Eschun, B.Sc. Med., Dwight Parkinson, M.D., and Jerry Vriend, Ph.D. Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada
Eschun G, Parkinson D, Vriend J. Changes in hippocampal monoamine concentration following halothane anesthesia and concussion. Surg Neurol 1992;37:101-5. T h e concentration of norepinephrine in the hippocampus
of rats anesthetized with halothane (Wyeth-Ayerst, Philadelphia, Pa) is found to be markedly increased, presumably due to the stress of handling and administering t h e anesthetic. This increased norepinephrine concentration persists for about 50 minutes but is obliterated when the anesthetized rat is concussed. This 50-minute period corresponds to the time it takes for a rat (or human), comatose for 1-2 seconds following concussion, to regain normal memory. No changes in 3,4-dihydroxybenzeneacetic acid (DOPAC), 3-(3,4-dihydroxyphenyl) alanine (L-DOPA), and 3,4-dihydroxybenzylamine (DHBA) were noted. 5Hydroxy indole acetic acid (HIAA) showed a depression at 5 minutes and again at 30 minutes, changes that were consistent but not considered statistically significant. KEY WORDS: Concussion; Halothane; Monoamines; Hippocampus; Memory
T h e amnesic period following cerebral concussion has been well verified but little studied [8,13-18, 23,31, 32,36]. Like other aspects of the p h e n o m e n o n known as concussion, the pathophysiology whereby billions of neurons suddenly lose then rapidly regain their functions is unknown. This subsequent transient disturbance of m e m o r y is equally poorly understood. T h e r e is no universally accepted definition of coma [16], nor of concussion, nor of m e m o r y [42]. In this article coma is somatic flaccidity, with regular, unassisted respirations and no response to pain. Concussion means a brief ( 1 - 2 seconds) period of coma resulting from a sufficient amount of cerebral acceleration (in this case 50 G), presumably a transient disturbance of function without structural damage. Memory refers to central
Address reprint requests to." Dwight Parkinson, M.D., 128 Basic Sciences Building, 730 William Avenue, Winnipeg, Manitoba R3E OW3, Canada. Received February 26, 1991; accepted July 19, 1991.
¢~ 1992 by Elsevier Science Publishing Co., Inc.
nervous system functions of the rat necessary to learn and r e m e m b e r a multiple pathway maze devoid of any olfactory clues. The purpose of this project is to define the changes in concentrations of the monoamines following concussion in a region of brain strongly associated with m e m ory, the hippocampus [49], over the time required for m e m o r y to return to normal. Materials and Methods
Stage 1 The first step was to examine the effects of anesthesia alone on m e m o r y and on the metabolites. Because of its ease of administration, short duration of action, and rapid reversibility, halothane (the only volatile anesthetic known to undergo reductive metabolism) [6] was selected. Ten white, male Sprague-Dawley rats, weighing 1 5 0 - 1 8 0 g, were used in this phase of the experiment. Each animal was trained in a series of multiple-choice mazes, and his behavior and performance were recorded as to learning times, best performance times, and m e m ory spans. Water deprivation for 22 hours was used, with water as the maze reward. The maze was thoroughly cleaned and dried between trials. When each animal was reliably able to perform two completely different mazes at an optimal or baseline performance, each was randomly assigned to an experimental or control group. The maze details, the training methods, the performance and m e m o r y recording, and evaluation were the same as reported previously [32]. Animals in the experimental group (n = 5) were anesthetized by spraying halothane onto a gauze mask. The anesthetic was terminated when the animal was comatose for 2 seconds, sufficient time to deliver the concussion in the second stage of the experiment. Control animals (n = 5) were handled in an identical manner, but water was sprayed on the gauze instead of halothane. U p o n cessation of the anesthesia (or sham anesthesia), the rats were placed back in the starting box and confronted with a maze they had performed at baseline minutes before anesthesia. They were also challenged with an unfamiliar maze pattern. 0090-3019/92/$5.00
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Results An experienced handler can pick up and hold a rat, with no evidence o f fear or struggling. Even when the nose cone is placed on the rat, there is no struggling, but when halothane is sprayed on the gauze, there is violent struggling for 3 - 4 seconds before the animal becomes limp. The sham-anesthetized animals remain calm as water is sprayed on the gauze.
Stage 1 Figure I. Frozen coronal section photographed and enlarged ( x 10). A dime is placed over the site of the l-mm biopsy punch. The original dorsal ventral diameter is I cm.
Stage 2 The second stage consisted of an identical procedure except that the test group (n = 5) was concussed as soon as comatose from the anesthesia, and the control group (n = 5) was sham concussed (50-G acceleration applied to shoulder). All were placed immediately back in the starting box.
Stage 3 The third stage consisted of killing groups (n = 5) of anesthetized, nonconcussed rats immediately, then subsequent groups (n = 5) at intervals of 5 minutes to a total of 50 minutes after the anesthetic.
Back in the starting box immediately after being rendered comatose for 1 - 2 seconds by halothane anesthesia, the rat requires about 10 seconds to regain normal posture, gait, and speed of locomotion, at which point he runs the maze at the same speed and accuracy as before. But, most dramatically, during that 10-second recovery time he will begin crawling and staggering slowly but accurately along the maze. It is most evident that his m e m o r y for that maze has returned before complete m o t o r function returns. H e will begin learning an unfamiliar maze immediately upon somatic recovery (10 seconds) in a manner that does not vary from his previous performance. In no aspect of the maze performances did we observe any difference between the preanesthesia and postanesthesia performance, nor between the anesthetized animals and the nonanesthetized controls. Thus, the brief halothane anesthesia did not affect the m e m o r y needed to execute successfully a maze pattern or to learn a new maze.
Stage 2 Stage 4 T h e fourth stage consisted of killing groups (n = 5) of anesthetized, concussed rats immediately, then subsequent groups (n = 5) at intervals of 5 minutes to a total of 50 minutes after the concussion. Previous work had established that cerebral acceleration of 50 G would consistently result in I - 2 seconds of coma, with no detectable residual other than 5 0 - 6 0 minutes of m e m o r y disturbance [31,33,36]. All the rats were killed by guillotine; the heads were immediately placed in liquid nitrogen, then dissected on dry ice. Care was taken to sample consistently the same hippocampal regions (CA-1 and CA-4), including part of the dentate blade, using the dissection microscope and a 1-mm punch (Figure 1). Samples were stored at - 8 0 ° C until analysis was performed using standard methodology of high-performance liquid chromatography (HPLC) with electrochemical detection.
The series (n = 5) of anesthetized and concussed rats regained full locomotor function in about 10 seconds, as did the nonconcussed, anesthetized series (n = 5) and the anesthetized, sham-concussed controls (n = 5). However, the concussed test group behaved in the maze as though they had never seen it before [36], and it was 5 0 - 6 0 minutes before they could run the maze previously run in their o p t i m u m time and before they could begin to learn a new maze as well as they could before concussion. Thus, the significant difference between the two groups was the 5 0 - 6 0 - m i n u t e period of deficient m e m o r y and learning in the concussed animals.
Stages 3 and 4 From HPLC analysis, significant changes in hippocampal norepinephrine (NE) in the anesthetized, nonconcussed and anesthetized, concussed animals were demonstrated (Figure 2). Five minutes postanesthesia, NE levels peak
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Figure 2. Norepinephrine concentration in picograms per milligram (pg/ mg), vertical axis; time in minutes, horizontal axis. Closed circles, anesthetized and not concussed; open circles, anesthetized and concussed," - 5, time of anesthesia or sham anesthesia: O, time of concussion or sham concussion. The slight difference in starting points is explained by the change in HPLC columns and~or buffer from one day to the next. A N O V A : F ~ 7~71. significance with 11 degrees of freedom, p ~ 0.001. Student's t-test." first peak significant, p ~ 0.05; second peak borderline,"from second peak to end point significant,-p ~ 0.05, with 7 degrees of freedom.
at approximately 1050 mg/pg and fall to control values at 20 minutes. This peak is absent in the anesthetized,
concussed specimens. A smaller peak is present at 25 minutes in both the concussed and nonconcussed groups o f animals before levels become stable at about 50 minutes. It is noted that the NE levels in the hippocampus are substantially lower throughout the 50-minute period in the concussed animals than in the nonconcussed control groups (Figure 2). Changes in the levels of 5-HIAA, a breakdown product of serotonin, initially fall from control values with halothane anesthesia and rise to near control values at 15 to 20 minutes. Levels fall again to an ebb at 30 minutes before beginning to rise once more. The changes are consistent in all animals but are not considered statistically significant. There were no significant changes in the amounts of L-DOPA, D O P A C , D H B A , or homo vanylmandelic acid noted. Because of time constraints, serotonin was not recorded (over 45 minutes per observation coming off the column).
Discussion
The reduction of NE and return to normal within 50 minutes following concussion correspond with a previously noted, 50-minute metabolite disturbance following concussion [34,35]. Since discovery of the anesthetic properties of halothane in 1957 [48], there has been an enormous amount of excellent investigation into its usage and actions [50,51]. This information, plus the recognition o f NE as an important transmitter in the
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processing and storage o f memory [2-5,19,45], is all relevant to our present study. There is very little in the literature specifically addressing the effects of a n e s t h e s i a on memory in humans or rats. T h e r e are some studies that have investigated the psychological effects o f halothane [11], and studies reporting 30-minute retrograde and antegrade memory disturbance following midazolam for induction [39]. Some limited observations on the aged indicate it would be very difficult to separate the anesthetic effects from the effects of the displacement to a hospital environment, along with the worry, anxiety, pain, and medication associated with a surgical procedure [7,20,40]. Townes et al [52] reported that following halothane anesthesia for minor surgery, memory was disturbed in young adults for 24 hours and advised that significant judgments should not be made within 8 days. Bruce et al [9] reported that 4-hour exposure to trace amounts of halothane impaired short-term memory in humans. Others have found that prolonged exposure to subanesthetic concentrations of halothane resulted in decreased dopamine concentration of up to 41% in the ventral tegmentum in rats but had no effect on NE [18]. There is no evidence that halothane affects, one way or the other, the synthesis of NE [30], nor does halothane increase the plasma NE, whereas cyclopropane does [38]. The turnover rate of NE is approximately 0.25 ~g/g/h in the rat brain [10,28,29]. Minimal anesthetic requirements are increased by anything that increases the concentration of NE, and are decreased by anything that decreases the NE concentration [22,25,27,47 ]. Unless pretreated with opioids [ 12 ], restraint (hand holding) and halothane anesthesia are forms of stress for the rat, increasing plasma NE up to 2000% and increasing the NE in the nucleus accumbens, central gray, and locus ceruleus, but not in 17 other brain areas tested (the hippocampus was not listed) [41]. Thus, the very act of anesthetizing increases the concentration of the catechols, which are recognized as important in memory functions. It is not known whether this excess is balanced by the stress requirements or whether an excess remains that might facilitate memory and learning. That we found no measurable maze memory disturbance following halothane anesthesia in rats is contrary to the findings o f T o w n e s et al [52] and Bruce et al [9] in humans following halothane exposure, but supports the opinion of Riss et al [40]. It is true that maze memory, learning, and performance by the rat [21] does not approximate the complete spectrum of human memory function. Pure, isolated memory loss in the otherwise completely normal individual occurs, if ever, in very few situations, possibly in early Alzheimer's disease, in Korsakoffs psychosis, in temporal lobe lesions, and tran-
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siently following the coma of concussion and electric shock therapy (ECT). T h e short-term coma produced by ECT [1,24,45,46] and concussion [8,14-16,18,23, 31,32,36] have some similarities: a virtual instantaneous onset and comparatively rapid recovery of all measurable functions other than memory. T h e patient transiently resembles the early Alzeheimer's and Korsakoff's patient. Memory in humans is never uniformly lost nor retained. Events that make a dramatic impact on the senses or that have been reinforced or repeated many times may surface from a period when other events are submerged. Similar events impact differently on different individuals. Thus, in some ways the laboratory animal with the controls available is a better model than the human. The material for measurement is uniform, in this case a maze performance, and the postevent times are easily measured. It is obvious that any memory-related function in the animal is not speech related, whereas most m e m o r y related functions in humans are. While extensive and fascinating work has been done to separate the features of learning and m e m o r y [21], the first is not possible without the second. After concussion, the human, within a few seconds, recovers speech, locomotion, primary skills, grooming, etc., which are "solidly" learned functions dependent on m e m o r y , while recollection of happenings and lesser experiences, particularly those occurring within the past hour, requires a disproportionately long while to recover. (Even walking must, to some extent, depend on r e m e m b e r i n g how to walk). Events immediately before the loss of consciousness are never retrieved. The boxer never r e m e m b e r s the blow that knocked him out, but r e m e m b e r s his boxing skills often well enough to go on and win. The dramatic discrepancy in recovery times of the neuronal circuits responsible for "solid" memory-related functions and other m e m o r y functions is possibly explained on continuing plasticity of the m e m o r y circuits. During development, genetically determined, new dendritic attachments occur rapidly, enhanced by action potentials. The development of new connections is stopped by genetic determination after which the circuits are "hard wired" [2]. The circuits for learning and m e m o r y must remain plastic or new learning could not occur. Long-term m e m o r y must be transmitted from protein to protein, possibly by means of R N A or genome, as neuron proteins are constantly being replaced. Shortterm m e m o r y probably depends on transient chemical perturbation at the transmitter site [1,26], and NE is probably involved. The study by Pearce et al [37] is most significant in that they found halothane had no effect on evoked excitatory responses or on long-term potentia-
Eschun et al
tion recording from the CA-1 region with stimulation of the contralateral CA-3 region, whereas paired pulse depression was significantly prolonged.
Summary Following administration of halothane anesthesia there is a double-peaked surge of N E concentration lasting for about 50 minutes, during which there is no demonstrable disturbance of memory. With the addition of concussion, the concentration of N E is decreased for the same period, during which m e m o r y is grossly disturbed. The plasticity of the neuronal circuits for m e m o r y related functions is not as disproportionately disturbed following halothane anesthesia in rats as it is following equivalent depths and times of coma following concussion in rats and humans, and following ECT in humans [43,44]. In most instances, m e m o r y is the last function to return following loss of consciousness. H o w e v e r , following halothane anesthesia, it appears to be the first function to recover. This work is part of Gerald Eschun's University of Manitoba B.Sc. Med. thesis, supported by White Cross Guild and in part by MHSC Research Foundation. References i. Abrams R. Electro convulsive therapy. In: Abrams R, ed. Electro convulsive therapy. Oxford: Oxford University Press, !988:135. 2. AgranoffBW. Learning and memory. In: Seigel G, AgranoffBW, Albers RW, Molinoff P, eds. Basic neurochemistry. New York: Raven, 1988:915-27. 3. Axelrod J. Biochemistry of the catecholamines. Ann Rev Biochem 1971 ;40:465. 4. Axelrod J. The fate of noradrenaline in the sympathetic neurons. Harvey Lecture 1973;67:175. 5. Axelrod J. O-Methylation of catecholamines in vivo and in vitro. Science 1957:1:126-40. 6. Barash PG, Cullen BE, Stoelting RK, eds. Clinical anesthesia. Philadelphia: JB Lippincott Company, 1989:326. 7. Bedfi~rd D. Adverse effects of anesthesia on old people. Lancet 1955;2:259. 8. Binder LM. Neurobehavioral recovery after mild head injury. J Neurosurg 1987;67:785-6. 9. Bruce DL, Bach MJ, ArbitJ. Trace anesthetic effects on perceptual, cognitive and motor skills. Anesthesiology 1974;40:453-8. 111. Carlsson A, Bedard P, Davis JN, Kehr W, Lindquist M, Magnusson T. Physiological control of 5-HT synthesis and turnover in the brain. Proc lnternat Cong Pharmacol 1973;4:286-98. 11. Davison LA, SteinhelberJC, Eger El, Stevens WC. Psychological effects of halothane and isoflurane anesthesia. Anesthesiology 1975;43:313-24. 12. DeLange S, Stanley TGH, Boscoe JM, deBruijn N, Berman L, Robertson D. Catecholamine and cortisol responses to sufentanilO~ and alfentanil-O, anesthesia during coronary artery surgery. Can Anaesth SocJ 1983;30:248. 15. Dixon K. Thc amnesia of concussion. Lancet 1962;2:1359-60.
C h a n g e s in M o n o a m i n e C o n c e n t r a t i o n
14. Enzenauer RW, Montrey JS, Enzenauer RJ, Mauldin M. Boxing related injuries in the US Army. JAMA 1989;261:1463-6. 15. Fisher CM. Concussion amnesia. Neurol Minn 1966; 16:826-30. 16. Frowein RA. Classification of coma. Acta Neurochir (Wien/ 1976;34:5-10. 17. Gerbich SG, PriestJD, BoenJR, Straub CP, Maxwell RE. Concussion incidences and severity in secondary school varsity football players. Am J Pub Health 1983;73:1370-5. 18. Gottesfeld Z, Patsalos PN, Rigor BM, Wiggins RC. Chronic subanaesthetic halothane exposure causes selective alteration in neurotransmitters system in discrete brain regions. Exp Neurol 1982;76:168-80. 19. HaycockJW, van Buskirk R, RyanJR, McCaughJL. Enhancement of retention with centrally administered catechotamines. Esp Neurol 1977;54:199-208. 20. Hughes D, Valentin N, Danielsen U, Dryberg V, Changes in memory following general or spinal anaesthesia for hip arthroplasty. Anaesthesia 1988;43:114-227. 21. Huppert FA, Piercy M. Dissociation between learning and re membering in organic amnesia. Nature (London) 1978;275:317. 22. Johnson RW, Way WL, Miller RD. Alteration of anesthetic requirement by amphetamine. Anesthesiology 1972;36:357-153. 23. Levin HS, High WM, Eawing-Cobbs L, Fletcher JM, Eisenberg HM, Homer EG. Memory functioning during the first year after closed head injury in children and adolescents. Neurosurgery 1988;22:1043-52. 24. Merskey H. In: Merskey H, ed. Psychiatric illness. London: Bailliere Tindall, 1988:289. 25. Miller RD, Way WL, Eger El. The effects of alpha-methyldopa, reserpine, guanethedine, and iproniazid on minimum alveolar anesthetic requirement (MAC). Anesthesiology 1968;29:1153-8. 26. Mizumori SJ, Channon V, Rosenweig MR, Bennett EL. Short anti long term working memory in the rat. Behav Neurosci 1987: 101:782-87. 27. Mueller RA, Smith RD, Spruill WA, Breese GR. Central monoaminergic neuronal effects on minimum alveolar concentration (MAC) of halothane and cyclopropane in rats. Anesthesiol 1975;42:143-52. 28. Nagatsu T, Levitt M, Udenfriend S. Tyrosine hydroxylase--the initial step in NE biosynthesis. J Biol Chem 1964;239:2911). 29. Neff H, Ngai SH, Wang CT, Costa E. Calculation of the rate of catecholamine synthesis from the rate of conversion of 14Ctyrosine to catecholamines: effect of adrenal demedullation on the synthesis rate. Mol Pharmacol 1969;5:90. 30. Ngai SH. Enzymes in anesthesiology. In: Foldes FF, ed. Enzymes in anesthesiology. New York: Springer, 1989:238. 31. Parkinson D. The biomechanics of concussion. In: Little JF, ed. Clinical neurosurgery, Volume 29. Baltimore: Williams and Wil kins, 1981:131-45. 32. Parkinson D. Concussion. Proc Mayo Clin 1977;52:492-6. 33. Parkinson D, Jell RM. Concussion. Acceleration limits causing concussion. Surg Neurol 1988;30:102-7. 34. Parkinson D, Vriend J. Changes in neurotransmitters following concussion (abstract). J Am Assoc Anat (Cincinnati) 1988 73A-4A.
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35. Parkinson D, West M, Havlicek V, Labella FS. Cerebral concussion in rats rapidly induces hypothalamic specific effects on opiate and cholinergic receptors. Brain Res 1981;225:271-7. 3(5. Parkinson D, West M, Pathiraja T. Concussion: comparison of humans and rats. Neurosurgery 1978;3:176-80. 37. Pearce RA, StringerJL, Lothman EW. Effect of volatile anesthesia on synaptic transmission in the rat hippocampus. Anesthesiology 1989;71:591-8. 38. Price HL, Linde HW,Jones RE, Black GW, Price ML. Sympathoadrenal responses to general anesthesia in man and their relation to hemodynamics. Anesthesiology 1959;20:563. 39. ReitanJA, Porter W, Braunstein M. Comparison of psychomotor skills and amnesia after induction of anesthesia with midazolam and thiopental. Anesthesia Analog 1986;65:933-7. 40. Riss J, Lomholt B, Haxholot O, Hehlet H. Immediate and long term mental recovery from general versus epidural anesthesia in elderly patients. Acta Anaesthesiol Stand 198.3;27:44-9. 41. Roizen MF, Shnider SM, Frazer B. Effect of anesthetics on the sympathetic nervous system: is it relevant to the practice of anesthesia. In: Fink RB, ed. Molecular mechanisms of anesthesia. Progress in anesthesiology, volume 2. New York: Raven, 1988: 447-82. 42. Shimamura AP, Squire LR. Korsakoffs syndrome: the relation between anterograde amnesia and remote memory impairment. Behav Neurosci 1986b;1110:165-7I). 43. Squire LR Comparisons between forms of amnesia: some deficits are unique to Korsakoff's syndrome. J Exp Psychol 1982;8: 560-71. .44. Squire LR. The neurophysiology of memory dysfunction and its assessment. In: Grant I, Adams K, eds. Neuropsychological assessment of neuropsychiatric disorders. New York: Oxford University Press, 1986:268-99. 45. Squire LR, Slater P. Anterograde and retrograde memory impairment in chronic amnesia. Neuropsychologia 1978;16: 313 22.
46. Squire LR, Slater P, Miller PL. Retrograde amnesia and bilateral electro convulsive therapy. Long term follow-up. Arch Gen Psychiatry 1981:38:89-95. 47. Stoelting RK, Creasser CW, Martz RZ. Effect of cocaine administration on halothane MAC in dogs. Anesthesia Analog (Cleveland) 1975;54:~22 4. ,18. Suckling CW. Some chemical and physical factors in developing of fluothane. B r J Anaes 1957;28:466. 49. Tamura R, Onon T, Fukuda M, Nakamura K. Recognition of egocentric and allocentric visual and auditory space by neurons in the hippocampus of monkeys. Neuroscience Lett 1990;109: 293-8. 50. Tarka M. Eftects of mechanical ventilation and halothane on pulmonary serotonin removal in dogs. Acta Anaesthesiol Scand 1985;29: ~1)0. 51. Theye RA, Michenfelder J D The effect of halothane on canine cerebral metabolism. Anesthesiology 1968;26:1113. 52. Townes BD, Dikemann SS, Bledsoe SW, Hornbein TF, Martin DC, Janesheski JA. Neuropsychological changes in a young healthy population after controlled hypotensive anesthesia. Anesthesia Analog 1986;65:955-9.
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Changes in Hippocampal Monoamine Concentration Following Halothane Anesthesia and Concussion Gerald Eschun, B.Sc. Med., Dwight Parkinson, M.D., and Jerry Vriend, Ph.D. Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada
Eschun G, Parkinson D, Vriend J. Changes in hippocampal monoamine concentration following halothane anesthesia and concussion. Surg Neurol 1992;37:101-5. T h e concentration of norepinephrine in the hippocampus
of rats anesthetized with halothane (Wyeth-Ayerst, Philadelphia, Pa) is found to be markedly increased, presumably due to the stress of handling and administering t h e anesthetic. This increased norepinephrine concentration persists for about 50 minutes but is obliterated when the anesthetized rat is concussed. This 50-minute period corresponds to the time it takes for a rat (or human), comatose for 1-2 seconds following concussion, to regain normal memory. No changes in 3,4-dihydroxybenzeneacetic acid (DOPAC), 3-(3,4-dihydroxyphenyl) alanine (L-DOPA), and 3,4-dihydroxybenzylamine (DHBA) were noted. 5Hydroxy indole acetic acid (HIAA) showed a depression at 5 minutes and again at 30 minutes, changes that were consistent but not considered statistically significant. KEY WORDS: Concussion; Halothane; Monoamines; Hippocampus; Memory
T h e amnesic period following cerebral concussion has been well verified but little studied [8,13-18, 23,31, 32,36]. Like other aspects of the p h e n o m e n o n known as concussion, the pathophysiology whereby billions of neurons suddenly lose then rapidly regain their functions is unknown. This subsequent transient disturbance of m e m o r y is equally poorly understood. T h e r e is no universally accepted definition of coma [16], nor of concussion, nor of m e m o r y [42]. In this article coma is somatic flaccidity, with regular, unassisted respirations and no response to pain. Concussion means a brief ( 1 - 2 seconds) period of coma resulting from a sufficient amount of cerebral acceleration (in this case 50 G), presumably a transient disturbance of function without structural damage. Memory refers to central
Address reprint requests to." Dwight Parkinson, M.D., 128 Basic Sciences Building, 730 William Avenue, Winnipeg, Manitoba R3E OW3, Canada. Received February 26, 1991; accepted July 19, 1991.
¢~ 1992 by Elsevier Science Publishing Co., Inc.
nervous system functions of the rat necessary to learn and r e m e m b e r a multiple pathway maze devoid of any olfactory clues. The purpose of this project is to define the changes in concentrations of the monoamines following concussion in a region of brain strongly associated with m e m ory, the hippocampus [49], over the time required for m e m o r y to return to normal. Materials and Methods
Stage 1 The first step was to examine the effects of anesthesia alone on m e m o r y and on the metabolites. Because of its ease of administration, short duration of action, and rapid reversibility, halothane (the only volatile anesthetic known to undergo reductive metabolism) [6] was selected. Ten white, male Sprague-Dawley rats, weighing 1 5 0 - 1 8 0 g, were used in this phase of the experiment. Each animal was trained in a series of multiple-choice mazes, and his behavior and performance were recorded as to learning times, best performance times, and m e m ory spans. Water deprivation for 22 hours was used, with water as the maze reward. The maze was thoroughly cleaned and dried between trials. When each animal was reliably able to perform two completely different mazes at an optimal or baseline performance, each was randomly assigned to an experimental or control group. The maze details, the training methods, the performance and m e m o r y recording, and evaluation were the same as reported previously [32]. Animals in the experimental group (n = 5) were anesthetized by spraying halothane onto a gauze mask. The anesthetic was terminated when the animal was comatose for 2 seconds, sufficient time to deliver the concussion in the second stage of the experiment. Control animals (n = 5) were handled in an identical manner, but water was sprayed on the gauze instead of halothane. U p o n cessation of the anesthesia (or sham anesthesia), the rats were placed back in the starting box and confronted with a maze they had performed at baseline minutes before anesthesia. They were also challenged with an unfamiliar maze pattern. 0090-3019/92/$5.00
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Results An experienced handler can pick up and hold a rat, with no evidence o f fear or struggling. Even when the nose cone is placed on the rat, there is no struggling, but when halothane is sprayed on the gauze, there is violent struggling for 3 - 4 seconds before the animal becomes limp. The sham-anesthetized animals remain calm as water is sprayed on the gauze.
Stage 1 Figure I. Frozen coronal section photographed and enlarged ( x 10). A dime is placed over the site of the l-mm biopsy punch. The original dorsal ventral diameter is I cm.
Stage 2 The second stage consisted of an identical procedure except that the test group (n = 5) was concussed as soon as comatose from the anesthesia, and the control group (n = 5) was sham concussed (50-G acceleration applied to shoulder). All were placed immediately back in the starting box.
Stage 3 The third stage consisted of killing groups (n = 5) of anesthetized, nonconcussed rats immediately, then subsequent groups (n = 5) at intervals of 5 minutes to a total of 50 minutes after the anesthetic.
Back in the starting box immediately after being rendered comatose for 1 - 2 seconds by halothane anesthesia, the rat requires about 10 seconds to regain normal posture, gait, and speed of locomotion, at which point he runs the maze at the same speed and accuracy as before. But, most dramatically, during that 10-second recovery time he will begin crawling and staggering slowly but accurately along the maze. It is most evident that his m e m o r y for that maze has returned before complete m o t o r function returns. H e will begin learning an unfamiliar maze immediately upon somatic recovery (10 seconds) in a manner that does not vary from his previous performance. In no aspect of the maze performances did we observe any difference between the preanesthesia and postanesthesia performance, nor between the anesthetized animals and the nonanesthetized controls. Thus, the brief halothane anesthesia did not affect the m e m o r y needed to execute successfully a maze pattern or to learn a new maze.
Stage 2 Stage 4 T h e fourth stage consisted of killing groups (n = 5) of anesthetized, concussed rats immediately, then subsequent groups (n = 5) at intervals of 5 minutes to a total of 50 minutes after the concussion. Previous work had established that cerebral acceleration of 50 G would consistently result in I - 2 seconds of coma, with no detectable residual other than 5 0 - 6 0 minutes of m e m o r y disturbance [31,33,36]. All the rats were killed by guillotine; the heads were immediately placed in liquid nitrogen, then dissected on dry ice. Care was taken to sample consistently the same hippocampal regions (CA-1 and CA-4), including part of the dentate blade, using the dissection microscope and a 1-mm punch (Figure 1). Samples were stored at - 8 0 ° C until analysis was performed using standard methodology of high-performance liquid chromatography (HPLC) with electrochemical detection.
The series (n = 5) of anesthetized and concussed rats regained full locomotor function in about 10 seconds, as did the nonconcussed, anesthetized series (n = 5) and the anesthetized, sham-concussed controls (n = 5). However, the concussed test group behaved in the maze as though they had never seen it before [36], and it was 5 0 - 6 0 minutes before they could run the maze previously run in their o p t i m u m time and before they could begin to learn a new maze as well as they could before concussion. Thus, the significant difference between the two groups was the 5 0 - 6 0 - m i n u t e period of deficient m e m o r y and learning in the concussed animals.
Stages 3 and 4 From HPLC analysis, significant changes in hippocampal norepinephrine (NE) in the anesthetized, nonconcussed and anesthetized, concussed animals were demonstrated (Figure 2). Five minutes postanesthesia, NE levels peak
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i
i
,
i
5 10 15 20 25 30 35 40 45 50 55 60 time (mln)
Figure 2. Norepinephrine concentration in picograms per milligram (pg/ mg), vertical axis; time in minutes, horizontal axis. Closed circles, anesthetized and not concussed; open circles, anesthetized and concussed," - 5, time of anesthesia or sham anesthesia: O, time of concussion or sham concussion. The slight difference in starting points is explained by the change in HPLC columns and~or buffer from one day to the next. A N O V A : F ~ 7~71. significance with 11 degrees of freedom, p ~ 0.001. Student's t-test." first peak significant, p ~ 0.05; second peak borderline,"from second peak to end point significant,-p ~ 0.05, with 7 degrees of freedom.
at approximately 1050 mg/pg and fall to control values at 20 minutes. This peak is absent in the anesthetized,
concussed specimens. A smaller peak is present at 25 minutes in both the concussed and nonconcussed groups o f animals before levels become stable at about 50 minutes. It is noted that the NE levels in the hippocampus are substantially lower throughout the 50-minute period in the concussed animals than in the nonconcussed control groups (Figure 2). Changes in the levels of 5-HIAA, a breakdown product of serotonin, initially fall from control values with halothane anesthesia and rise to near control values at 15 to 20 minutes. Levels fall again to an ebb at 30 minutes before beginning to rise once more. The changes are consistent in all animals but are not considered statistically significant. There were no significant changes in the amounts of L-DOPA, D O P A C , D H B A , or homo vanylmandelic acid noted. Because of time constraints, serotonin was not recorded (over 45 minutes per observation coming off the column).
Discussion
The reduction of NE and return to normal within 50 minutes following concussion correspond with a previously noted, 50-minute metabolite disturbance following concussion [34,35]. Since discovery of the anesthetic properties of halothane in 1957 [48], there has been an enormous amount of excellent investigation into its usage and actions [50,51]. This information, plus the recognition o f NE as an important transmitter in the
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processing and storage o f memory [2-5,19,45], is all relevant to our present study. There is very little in the literature specifically addressing the effects of a n e s t h e s i a on memory in humans or rats. T h e r e are some studies that have investigated the psychological effects o f halothane [11], and studies reporting 30-minute retrograde and antegrade memory disturbance following midazolam for induction [39]. Some limited observations on the aged indicate it would be very difficult to separate the anesthetic effects from the effects of the displacement to a hospital environment, along with the worry, anxiety, pain, and medication associated with a surgical procedure [7,20,40]. Townes et al [52] reported that following halothane anesthesia for minor surgery, memory was disturbed in young adults for 24 hours and advised that significant judgments should not be made within 8 days. Bruce et al [9] reported that 4-hour exposure to trace amounts of halothane impaired short-term memory in humans. Others have found that prolonged exposure to subanesthetic concentrations of halothane resulted in decreased dopamine concentration of up to 41% in the ventral tegmentum in rats but had no effect on NE [18]. There is no evidence that halothane affects, one way or the other, the synthesis of NE [30], nor does halothane increase the plasma NE, whereas cyclopropane does [38]. The turnover rate of NE is approximately 0.25 ~g/g/h in the rat brain [10,28,29]. Minimal anesthetic requirements are increased by anything that increases the concentration of NE, and are decreased by anything that decreases the NE concentration [22,25,27,47 ]. Unless pretreated with opioids [ 12 ], restraint (hand holding) and halothane anesthesia are forms of stress for the rat, increasing plasma NE up to 2000% and increasing the NE in the nucleus accumbens, central gray, and locus ceruleus, but not in 17 other brain areas tested (the hippocampus was not listed) [41]. Thus, the very act of anesthetizing increases the concentration of the catechols, which are recognized as important in memory functions. It is not known whether this excess is balanced by the stress requirements or whether an excess remains that might facilitate memory and learning. That we found no measurable maze memory disturbance following halothane anesthesia in rats is contrary to the findings o f T o w n e s et al [52] and Bruce et al [9] in humans following halothane exposure, but supports the opinion of Riss et al [40]. It is true that maze memory, learning, and performance by the rat [21] does not approximate the complete spectrum of human memory function. Pure, isolated memory loss in the otherwise completely normal individual occurs, if ever, in very few situations, possibly in early Alzheimer's disease, in Korsakoffs psychosis, in temporal lobe lesions, and tran-
104
Surg Neurol 1992;37:101-5
siently following the coma of concussion and electric shock therapy (ECT). T h e short-term coma produced by ECT [1,24,45,46] and concussion [8,14-16,18,23, 31,32,36] have some similarities: a virtual instantaneous onset and comparatively rapid recovery of all measurable functions other than memory. T h e patient transiently resembles the early Alzeheimer's and Korsakoff's patient. Memory in humans is never uniformly lost nor retained. Events that make a dramatic impact on the senses or that have been reinforced or repeated many times may surface from a period when other events are submerged. Similar events impact differently on different individuals. Thus, in some ways the laboratory animal with the controls available is a better model than the human. The material for measurement is uniform, in this case a maze performance, and the postevent times are easily measured. It is obvious that any memory-related function in the animal is not speech related, whereas most m e m o r y related functions in humans are. While extensive and fascinating work has been done to separate the features of learning and m e m o r y [21], the first is not possible without the second. After concussion, the human, within a few seconds, recovers speech, locomotion, primary skills, grooming, etc., which are "solidly" learned functions dependent on m e m o r y , while recollection of happenings and lesser experiences, particularly those occurring within the past hour, requires a disproportionately long while to recover. (Even walking must, to some extent, depend on r e m e m b e r i n g how to walk). Events immediately before the loss of consciousness are never retrieved. The boxer never r e m e m b e r s the blow that knocked him out, but r e m e m b e r s his boxing skills often well enough to go on and win. The dramatic discrepancy in recovery times of the neuronal circuits responsible for "solid" memory-related functions and other m e m o r y functions is possibly explained on continuing plasticity of the m e m o r y circuits. During development, genetically determined, new dendritic attachments occur rapidly, enhanced by action potentials. The development of new connections is stopped by genetic determination after which the circuits are "hard wired" [2]. The circuits for learning and m e m o r y must remain plastic or new learning could not occur. Long-term m e m o r y must be transmitted from protein to protein, possibly by means of R N A or genome, as neuron proteins are constantly being replaced. Shortterm m e m o r y probably depends on transient chemical perturbation at the transmitter site [1,26], and NE is probably involved. The study by Pearce et al [37] is most significant in that they found halothane had no effect on evoked excitatory responses or on long-term potentia-
Eschun et al
tion recording from the CA-1 region with stimulation of the contralateral CA-3 region, whereas paired pulse depression was significantly prolonged.
Summary Following administration of halothane anesthesia there is a double-peaked surge of N E concentration lasting for about 50 minutes, during which there is no demonstrable disturbance of memory. With the addition of concussion, the concentration of N E is decreased for the same period, during which m e m o r y is grossly disturbed. The plasticity of the neuronal circuits for m e m o r y related functions is not as disproportionately disturbed following halothane anesthesia in rats as it is following equivalent depths and times of coma following concussion in rats and humans, and following ECT in humans [43,44]. In most instances, m e m o r y is the last function to return following loss of consciousness. H o w e v e r , following halothane anesthesia, it appears to be the first function to recover. This work is part of Gerald Eschun's University of Manitoba B.Sc. Med. thesis, supported by White Cross Guild and in part by MHSC Research Foundation. References i. Abrams R. Electro convulsive therapy. In: Abrams R, ed. Electro convulsive therapy. Oxford: Oxford University Press, !988:135. 2. AgranoffBW. Learning and memory. In: Seigel G, AgranoffBW, Albers RW, Molinoff P, eds. Basic neurochemistry. New York: Raven, 1988:915-27. 3. Axelrod J. Biochemistry of the catecholamines. Ann Rev Biochem 1971 ;40:465. 4. Axelrod J. The fate of noradrenaline in the sympathetic neurons. Harvey Lecture 1973;67:175. 5. Axelrod J. O-Methylation of catecholamines in vivo and in vitro. Science 1957:1:126-40. 6. Barash PG, Cullen BE, Stoelting RK, eds. Clinical anesthesia. Philadelphia: JB Lippincott Company, 1989:326. 7. Bedfi~rd D. Adverse effects of anesthesia on old people. Lancet 1955;2:259. 8. Binder LM. Neurobehavioral recovery after mild head injury. J Neurosurg 1987;67:785-6. 9. Bruce DL, Bach MJ, ArbitJ. Trace anesthetic effects on perceptual, cognitive and motor skills. Anesthesiology 1974;40:453-8. 111. Carlsson A, Bedard P, Davis JN, Kehr W, Lindquist M, Magnusson T. Physiological control of 5-HT synthesis and turnover in the brain. Proc lnternat Cong Pharmacol 1973;4:286-98. 11. Davison LA, SteinhelberJC, Eger El, Stevens WC. Psychological effects of halothane and isoflurane anesthesia. Anesthesiology 1975;43:313-24. 12. DeLange S, Stanley TGH, Boscoe JM, deBruijn N, Berman L, Robertson D. Catecholamine and cortisol responses to sufentanilO~ and alfentanil-O, anesthesia during coronary artery surgery. Can Anaesth SocJ 1983;30:248. 15. Dixon K. Thc amnesia of concussion. Lancet 1962;2:1359-60.
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