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Analysis of preseizure electrocorticographic changes in rats during hyperbaric oxygenation
EXPERIMENTAL
NEUROLOGY
Analysis
NOEMI
46, 9-19 (1975)
of Preseizure in Rats During
RADAY,
NISSIM
Department of Neurology University Hospital,
Electrocorticographic Changes Hyperbaric Oxygenation DAN HAREL,
CONFORTI, and
of Experimental University-Hadassah Jerusalem, Israel
Laboratory
and Hebrew Received
August
AND
SYLVAN
LAVY
1
Neurology, Hadassnh Medical School,
1.5,1974
The most defined and reproducible electroencephalographic manifestation of oxygen poisoning in animals culminates in a generalized synchronous electrical discharge and very often in clinical convulsions. The amplitude and frequency of the electrocortical activity (EEG) in the preseizure phase was studied in unanesthetized rats during exposure to hyperbaric oxygen. The preseizure EEG (electrocorticogram) of rats breathing 6 ATA (atmospheres absolute) oxygen showed reduction in the mean frequency starting 7 min before the appearance of the first electrical discharge and a decrease of the mean amplitude starting about 3 min before the onset of the first electrical discharge. Our findings demonstrated that there is a progressive deterioration of the cortical electrical activity in rats exposed to high oxygen pressure. This suggests that the toxic effect of oxygen on the brain is cumulative and interferes with its normal metabolic and electrochemical processes.
INTRODUCTION Deterknation of the threshold of toxicity in the central nervous system is of great importance in the use of hyperbaric oxygen. Several criteria have been suggestedby different investigators as signs of oxygen poisoning, including clinical convulsive seizures (1, 3, 4) ; postexposure mortality (4, 7, Q ; and residual neurological damage (2). However, clinical convulsive seizures are not consistent and difficulties arise in interpreting the exact time of their onset. Also, partial seizures often occur and someanimals never show a fully developed epileptic seizure. Therefore the electrical activity of the brain under high oxygen pressure has been repeatedly investigated (5, 14, 15). 1 Present address of Dr. Hare1 is: Department Abba Khoushy Medical School. 9 Copyright All rif$ts
s
1975 by Academic Press, Inc. reproduction in any form reserved.
of Neurology,
Rothschild
Hospitd:,
10
RADAY
FIG. 1. Typical electrocorticographic a-right side; b-left side. Upper two pressure ; lower two pairs : appearance six ATA oxygen. Calibrations : 2 set ;
ET AL.
recording between temporal and occipital areas. pairs of records ; control EEG in atmospheric of the first electrical discharge in exposure to 100 +v.
The most clearly defined and reproducible electroencephalographic manifestation of oxygen poisoning in animals exposed to high oxygen pressure is progressive deterioration of the electrical activity culminating in generalized synchronous electrical discharges, recorded both from the cerebral cortex and from various subcortical structures (9, 14). These electrical changes comprise an early, accurate, and sensitive criterion for oxygen toxicity (10). Careful analysis of the EEG in animals exposed to high oxygen pressure have revealed that electroencephalographic changes occurred even before the appearance of the first electrical discharge (5, 15). In the present study, we have analyzed these preseizure EEG changes with the aid of a computer, in an effort to characterize them and to determine the exact time of their onset. In order to determine whether the early EEG changes in rats exposed to high oxygen pressure are specific signs of oxygen toxicity, a second group of animals in which convulsions were induced by injection of picrotoxin or Metrazol were studied for comparison. MATERIALS
AND METHODS
Experiments were performed on 19 male rats (Wistar strain) weighing 250-300 g. Animals were fed on commercial laboratory animal food ad lib. Using a stereotaxic instrument, four stainless steel insulated electrodes were chronically implanted on the posterior cortex of each animal for electroencephalographic recordings. Recording was bipolar between temporal and occipital areas (right and left). Implantation was carried out under Nembutal anesthesia (30 mg/kg, ip) . The electrodes were connected to a microminiature connector that was fixed by dental cement to the skull.
HYPERBARIC
11
OXYGEN
Experiments commenced after a postoperative recovery period of 1 wk. In no instance was there postoperative neuro&jcal damage. During the experiment the rat was awake and could freely walk in the pressure chamber. The EEG was recorded on an eight-channel III D
l
-14
l
-13
-12
-11
-10
-9
-8
-7
-6
-5
-4
-3
-2
-1
0
LO 30 l0 IO 0 IO 20
30 LO SC i0 70 BO 90 -16
-15
-14
-13
-12
-11
-10
-9
-8
-7
-6
-5
-4
-3
-2
-1
2. Percentage difference between the number of waves (l-25 cycles/set) and the mean number of waves during spontaneous activity. Upper : control in atmospheric pressure ; lower: during exposure to 6 ATA oxygen. Vertical axes: percentage dif. . ference from control. Horizontal axes: minutes. Arrow: first electrrcal dtscharge (t = 0). FIG.
RADAY
’
-12
-11
-10
-9
-8
-7
-6
ET AL.
-5
-4
-3
-2
-1
I 0
min
PICROTOXIN(5.4,q/kg)
FIG. 3. Percentage difference between the number of waves (l-25 cycles/set) and the mean number of waves during spontaneous activity. Upper : control ; lower : after injection of picrotoxin. The first electrical discharge at t = o.
console model Grass electroencephalograph and was simultaneously recorded on magnetic tape FM (Hewlett Packard) through an a-c preamplifier (Grass P511) ; frequency range l-100 Hz. A SO-Hz filter was used.
HYPERBARIC
13
OXYGEN
The pressure chamber (TCAHO Hyperbaric Experimental ChamberGaleazzi Roberto, Italy, 160 liter capacity), was first flushed three times with pure oxygen by raising the pressure to 2 ATA (atmospheres absolute)
‘
-6
:
min
-4
-3
-2
-1
METRAZOL( 14mg’kg)
-s’ ,fn 4
0
A
1 -i -2 -1
0
FIG. 4. Percentage difference between the number of waves (l-25 cycles/set) the mean number of waves during spontaneous activity. Upper : control ; lower injection of Metrazol. The first electrical discharge at t = o.
and : after
14
RADAY
0 1 -
ET AL.
Significant Metrazol
First electrical discharge Control
- - - Treatment
ULicr \ ‘b-- )A
\\
\
‘w ’
ot oxin
0’
-’ 7
4
:
:
:
:
:
:
:
16
Oxygen
-12 -1’1-10 -9 -8 -7 -6 -5 -4 -3 -2 -1 min FIG. 5. Mean frequency of EEG in rats exposed to six ATA oxygen as compared with the mean frequency obtained after administration of picrotoxin and Metrazol.
and releasing to 1 ATA ; then the pressure was raised to 6 ATA oxygen in 5 min (rate of compression and decompressionwas 1 ATA/min). It was not necessary to add soda lime or to constantly flow oxygen through the chamber becausethe CO2 production of a rat is negligible relative to the enormous volume of the chamber. Experiments were performed at a room temperature of 20-25C. Seven of the rats were exposed to’ six ATA until the appearance of the first electrical discharge, at which time decompressionwas carried out. The time of exposure was measured from the moment when six ATA was
HYPERBARIC
15
OXYGEN
reached. Experiments never resulted in mortality or in residual neurological damage. The EEG activity was continuously recorded for a control period of 15 min of air breathing, and then at six ATA oxygen until the appearance of the first electrical discharge. Only those rats whose EEG showed signs of seizure activity within 30 min of exposure to 6 ATA oxygen were included in the study. In a second group of rats convulsions were produced by injecting either picrotoxin (5.4 mg/kg, ip) in six animals or Metrazol (14.0 mg/kg, ip) in six other rats. The rats of these two comparison groups, and the record in the opened hyperbaric chamber (atmospheric pressure) were kept during the injection in order to expose them to the same sensory milieu (temperature, light, noise) as the rats subjected to high oxygen pressure. Data from the two groups were subjected to the same analysis. The EEG activity was transferred from magnetic tape to an analog-digital converter (PDP-15) through a high pass filter (cut off 0.5 Hz) in order to avoid slow base line shifts. Rate of sampling was lOO/sec. Amplitude and frequency were analyzed using “period-amplitude analysis” based on a zero crossing method. The basic measurement procedure used in this experiment was a modification of a technique described by Legewie and Probst (13). The data were divided into five frequency bands-delta (l-3 cycle/set) ;
I -7
-6
-5
-4
-3
-2
-1 min
0
-7
-6
-5
-4
-3
-2
-1
‘0
mln
FIG. 6. The frequencyof appearance of eachfrequencybandof the EEG in atrnosphericpressureand in exposureto six ATA 02.
16
RADAY
Q Significant
ET AL.
PcO.05
4 First electrical discharge
Metrazol
,700
-Control --- Treatment
,
:
:
:
:
:
J&Erotoxin
min FIG. 7. Mean amplitudeof EEG in rats exposedto six ATA oxygen as compared with the EEG obtainedafter administrationof Metrazol and pictroxin.
theta (4-7 cycles/set), alpha (8-13 cycles/set) , beta (14-25 cycles/set) and total (l-25 cycle/set) . The mean amplitude was calculated separately for each frequency band. Analyzed data were available for oscilloscopic display, numerical printout, and magnetic tape storage for subsequent statistical calculations. The reference point in time (t = o) was the first electrical discharge. Since our analysis refers to the preconvulsive stage, our time scale was negative. RESULTS The mean latency from the time of exposure to 6 ATA oxygen until the appearance of the first electrical discharge was 12.5 -L 1.9 min. Figure 1 is a typical record which shows the control EEG at atmospheric pressure
and the appearance of the first electrical discharge in six ATA oxygen. Exposure to six ATA caused a consistent change in the pattern of distribution of frequencies in the EEG before the onset of seizure activity. This is shown in Fig. 2, which plots for seven rats the percentage difference between the number of waves within a bandwidth of l-25 Hz (10 set bins), and the mean number of waves during spontaneous activity as recorded during air breathing at atmospheric pressure (zero line). The control graph shows that during air breathing, fluctuation about the mean was t20%. However, during exposure to 6 ATA, the total number of waves in the EEG of each rat was such that the entire distribution was diverted downward so as to fluctuate between +lOY, and -90% relative to control. This pattern was not a generalized feature of development of seizures. Thus, Figs. 3 and 4 show that injection of picrotoxin or metrazol did not result in a significant change in the distribution, but rather, that the dispersion of points was random (*3OoJo relative to control) before and after drug administration. The consistent patterns of these curves permit us to use mean values for the frequency analysis which is shown in Figs. 5 and 6. Figure 5 again shows that during the 7 min at 6 ATA oxygen which preceded the first electrical discharge, there was a significant (p < 0.05) decrease in the mean frequency of the EEG. The t-test analysis revealed no significant differences in mean frequency between the control period and the period leading up to the onset of metrazol or picrotoxin induced discharges. Figure 6, which shows the analysis for each frequency band, reveals that during the preconvulsive period in 6 ATA oxygen, there was a decrease in the number of theta, beta, and alpha waves and a slight increase in the number of delta waves. The preseizure EEG of animals breathing 6 ATA 02 showed reduction in the mean amplitude which became statistically significant 3 min before onset of first electrical discharge (Fig. 7). However, the amplitude began to increase just before the onset of the first electrical discharge. Our results also show that no statistically significant change in mean amplitude was observed during the preconvulsive stage of animals injected with Metrazol and picrotoxin (Fig. 7). DISCUSSION Our findings demonstrate that during exposure to hyperbaric oxygen EEG abnormalities precede the first electrical discharge by up to 7 min. The significant reduction in the mean frequency and amplitude of the electrical activity of the brain permits us to assume that the toxic effect of oxygen on the brain is cumulative and interferes progressively with the normal activity of the brain. Our results also revealed a significant difference between the preconvulsive electrical activity in Metrazol and picro-
18
RADAY
ET AL.
toxin induced seizures and those of high oxygen pressure. Both Metrazol and picrotoxin showed no significant change in mean amplitude and mean frequency during the preconvulsive period. It appears that under these experimental conditions there is an almost sudden change in the normal electrical equilibrium of the brain and the first electrical discharge, consisting of high amplitude and sharp activity, represents the first sign of brain dysfunction. The physiological mechanism underlying the convulsive consequences of exposure to high oxygen pressure remain unknown. While our results do not directly provide such mechanisms, they do suggest that the cerebral abnormality associated with oxygen toxicity evolves gradually over several minutes culminating in seizures. This sequence is most consistent with those biochemical theories which attribute the toxicity of oxygen at high pressure to a disturbance in the cellular metabolism and normal functioning of the CNS. Many workers pointed out that most enzymes containing sulfhydryl groups necessary for their activity were inactivated by high oxygen pressure (6, 11). Among the enzyme systems the most interesting are glutamic acid decarboxylase (16) and acetylcholinesterase (17) which play a role in the level of ceurotransmitters, and Na-K-Mg ATPase (12) whose inactivation will disturb active transport. Therefore, it seems more likely to accept any theory which explains the development of the preconvulsive and convulsive changes by interference with the normal metabolic and electrochemical processes of the whole brain. REFERENCES 1. BEAN, J. W. 194.5. Effects of oxygen at increased pressure. Physiol. Rev. 25: 1-169. 2. BEAN, J. W., and E. C. SIEGFRIELL 1945. Transient and permanent after effects of exposure to oxygen at high pressure. Anzcr. J. Physiol. 143: 656665. 3. BERT, P. 1943. “Barometric Pressure : Research in Experimental Physiology.” M. A. Hitchcock and F. A. Hitchcock [trans.]. College Book Co., Columbus, Ohio. 4. BURNS, J. D. 1972. Conqentration-dependent attenuation of hyperbaric oxygen toxicity. Aerosp. Med. 43 : 989-992. 5. COHN, R., and I. GERSH. 1945. Changes in brain potentials during conclusions induced by oxygen under pressure. J. Newoplzysiol. 8: 155-160. 6. DAVIES, H. C., and R. E. DAVIES. 1965. Biochemical aspects of oxygen poisoning, pp. 1047-1058. In “Handbook of Physiology.” Sect. 3 :, “Respiration.” W. 0. Fenn and H. Rahn [Eds.]. Amer. Physiol. Sot., Washington. 7. GERSCHMAN, R., D. L. GILBERT, S. W. NYE, P. W. NADIG, and W. D. FENN. 1954. Role of adrenalectomy and adrenal cortical hormones in oxygen poisoning. Amer. J. Physiol. 178: 346-351. 8. GERSCHMAN, R., D. L. GILBERT, and D. CACCAMISE. 1958. Effect of various substances on survival times of mice exposed to different high oxygen pressure. Amer. J. Physiot. 192 : 563-571.
HYPERBARIC
OXYGEN
19
9. HAREL, D., D. KEREM, and S. LAVY. 1969. The influence of high oxygen pressures on the electrical activity of the brain. Electroenceph. Clin. Neurophysiol. 26: 310-317. 10. HAREL, D., and S. LAVY. 1971. Changes in electrical activity of the brain as a criterion of hyperbaric oxygen toxicity in the central nervous system. J. Life sci. 1: 111-114. 11. HAUGAARD, N. 1968. Cellular mechanisms of oxygen toxicity. Physiol. Rev. 48 : 311373. 12. KOEHLER, G., and S. F. GOTTLIEB. 1972. Effects of increased tensions of 02, N: and He on the activity of a Na-K-Mg ATPase of rat intestine. Aerosfi. Med. 43 : 269-273. 13. LEGEWIE, H., and W. PROBST. 1969. On-line analysis of EEG with a small computer (period-amplitude analysis). Electrocnceph. Clin. Neurophysiol. 27 : 533-535. 14. Ruccr, F. S., M. L. GIRETTI, and M. LA ROCCA. 1967. Changes in electrical activity of the cerebral cortex and of some subcortical centers in hyperbaric oxygen. Electroemcph. C/in. ATcurophysiol. 22 : 231-238. 15. SONNENSCHEIN, R. R., and S. N. STEIN. 1953. Electrical activity of the brain in acute oxygen poisoning. Electrorncejh. Clin. Ncztrophysiol. 5 : 521-524. 16. TUNNICLIFF, G., M. URTON, and J. D. WOOD. 1973. Susceptibility of chick brain L-glutamic acid decarboxylase and other neurotransmitter enzymes to hyperbaric oxygen in vitro. Biochem. Pharwtacol. 22 : 501-505. 17. ZIRKLE, L. G., C. E. MENGEL, B. D. HORTON, and E. J. DUFFY. 1965. Studies of oxygen toxicity in the central nervous system. Acrosf. Med. 36: 1027-1032.
NEUROLOGY
Analysis
NOEMI
46, 9-19 (1975)
of Preseizure in Rats During
RADAY,
NISSIM
Department of Neurology University Hospital,
Electrocorticographic Changes Hyperbaric Oxygenation DAN HAREL,
CONFORTI, and
of Experimental University-Hadassah Jerusalem, Israel
Laboratory
and Hebrew Received
August
AND
SYLVAN
LAVY
1
Neurology, Hadassnh Medical School,
1.5,1974
The most defined and reproducible electroencephalographic manifestation of oxygen poisoning in animals culminates in a generalized synchronous electrical discharge and very often in clinical convulsions. The amplitude and frequency of the electrocortical activity (EEG) in the preseizure phase was studied in unanesthetized rats during exposure to hyperbaric oxygen. The preseizure EEG (electrocorticogram) of rats breathing 6 ATA (atmospheres absolute) oxygen showed reduction in the mean frequency starting 7 min before the appearance of the first electrical discharge and a decrease of the mean amplitude starting about 3 min before the onset of the first electrical discharge. Our findings demonstrated that there is a progressive deterioration of the cortical electrical activity in rats exposed to high oxygen pressure. This suggests that the toxic effect of oxygen on the brain is cumulative and interferes with its normal metabolic and electrochemical processes.
INTRODUCTION Deterknation of the threshold of toxicity in the central nervous system is of great importance in the use of hyperbaric oxygen. Several criteria have been suggestedby different investigators as signs of oxygen poisoning, including clinical convulsive seizures (1, 3, 4) ; postexposure mortality (4, 7, Q ; and residual neurological damage (2). However, clinical convulsive seizures are not consistent and difficulties arise in interpreting the exact time of their onset. Also, partial seizures often occur and someanimals never show a fully developed epileptic seizure. Therefore the electrical activity of the brain under high oxygen pressure has been repeatedly investigated (5, 14, 15). 1 Present address of Dr. Hare1 is: Department Abba Khoushy Medical School. 9 Copyright All rif$ts
s
1975 by Academic Press, Inc. reproduction in any form reserved.
of Neurology,
Rothschild
Hospitd:,
10
RADAY
FIG. 1. Typical electrocorticographic a-right side; b-left side. Upper two pressure ; lower two pairs : appearance six ATA oxygen. Calibrations : 2 set ;
ET AL.
recording between temporal and occipital areas. pairs of records ; control EEG in atmospheric of the first electrical discharge in exposure to 100 +v.
The most clearly defined and reproducible electroencephalographic manifestation of oxygen poisoning in animals exposed to high oxygen pressure is progressive deterioration of the electrical activity culminating in generalized synchronous electrical discharges, recorded both from the cerebral cortex and from various subcortical structures (9, 14). These electrical changes comprise an early, accurate, and sensitive criterion for oxygen toxicity (10). Careful analysis of the EEG in animals exposed to high oxygen pressure have revealed that electroencephalographic changes occurred even before the appearance of the first electrical discharge (5, 15). In the present study, we have analyzed these preseizure EEG changes with the aid of a computer, in an effort to characterize them and to determine the exact time of their onset. In order to determine whether the early EEG changes in rats exposed to high oxygen pressure are specific signs of oxygen toxicity, a second group of animals in which convulsions were induced by injection of picrotoxin or Metrazol were studied for comparison. MATERIALS
AND METHODS
Experiments were performed on 19 male rats (Wistar strain) weighing 250-300 g. Animals were fed on commercial laboratory animal food ad lib. Using a stereotaxic instrument, four stainless steel insulated electrodes were chronically implanted on the posterior cortex of each animal for electroencephalographic recordings. Recording was bipolar between temporal and occipital areas (right and left). Implantation was carried out under Nembutal anesthesia (30 mg/kg, ip) . The electrodes were connected to a microminiature connector that was fixed by dental cement to the skull.
HYPERBARIC
11
OXYGEN
Experiments commenced after a postoperative recovery period of 1 wk. In no instance was there postoperative neuro&jcal damage. During the experiment the rat was awake and could freely walk in the pressure chamber. The EEG was recorded on an eight-channel III D
l
-14
l
-13
-12
-11
-10
-9
-8
-7
-6
-5
-4
-3
-2
-1
0
LO 30 l0 IO 0 IO 20
30 LO SC i0 70 BO 90 -16
-15
-14
-13
-12
-11
-10
-9
-8
-7
-6
-5
-4
-3
-2
-1
2. Percentage difference between the number of waves (l-25 cycles/set) and the mean number of waves during spontaneous activity. Upper : control in atmospheric pressure ; lower: during exposure to 6 ATA oxygen. Vertical axes: percentage dif. . ference from control. Horizontal axes: minutes. Arrow: first electrrcal dtscharge (t = 0). FIG.
RADAY
’
-12
-11
-10
-9
-8
-7
-6
ET AL.
-5
-4
-3
-2
-1
I 0
min
PICROTOXIN(5.4,q/kg)
FIG. 3. Percentage difference between the number of waves (l-25 cycles/set) and the mean number of waves during spontaneous activity. Upper : control ; lower : after injection of picrotoxin. The first electrical discharge at t = o.
console model Grass electroencephalograph and was simultaneously recorded on magnetic tape FM (Hewlett Packard) through an a-c preamplifier (Grass P511) ; frequency range l-100 Hz. A SO-Hz filter was used.
HYPERBARIC
13
OXYGEN
The pressure chamber (TCAHO Hyperbaric Experimental ChamberGaleazzi Roberto, Italy, 160 liter capacity), was first flushed three times with pure oxygen by raising the pressure to 2 ATA (atmospheres absolute)
‘
-6
:
min
-4
-3
-2
-1
METRAZOL( 14mg’kg)
-s’ ,fn 4
0
A
1 -i -2 -1
0
FIG. 4. Percentage difference between the number of waves (l-25 cycles/set) the mean number of waves during spontaneous activity. Upper : control ; lower injection of Metrazol. The first electrical discharge at t = o.
and : after
14
RADAY
0 1 -
ET AL.
Significant Metrazol
First electrical discharge Control
- - - Treatment
ULicr \ ‘b-- )A
\\
\
‘w ’
ot oxin
0’
-’ 7
4
:
:
:
:
:
:
:
16
Oxygen
-12 -1’1-10 -9 -8 -7 -6 -5 -4 -3 -2 -1 min FIG. 5. Mean frequency of EEG in rats exposed to six ATA oxygen as compared with the mean frequency obtained after administration of picrotoxin and Metrazol.
and releasing to 1 ATA ; then the pressure was raised to 6 ATA oxygen in 5 min (rate of compression and decompressionwas 1 ATA/min). It was not necessary to add soda lime or to constantly flow oxygen through the chamber becausethe CO2 production of a rat is negligible relative to the enormous volume of the chamber. Experiments were performed at a room temperature of 20-25C. Seven of the rats were exposed to’ six ATA until the appearance of the first electrical discharge, at which time decompressionwas carried out. The time of exposure was measured from the moment when six ATA was
HYPERBARIC
15
OXYGEN
reached. Experiments never resulted in mortality or in residual neurological damage. The EEG activity was continuously recorded for a control period of 15 min of air breathing, and then at six ATA oxygen until the appearance of the first electrical discharge. Only those rats whose EEG showed signs of seizure activity within 30 min of exposure to 6 ATA oxygen were included in the study. In a second group of rats convulsions were produced by injecting either picrotoxin (5.4 mg/kg, ip) in six animals or Metrazol (14.0 mg/kg, ip) in six other rats. The rats of these two comparison groups, and the record in the opened hyperbaric chamber (atmospheric pressure) were kept during the injection in order to expose them to the same sensory milieu (temperature, light, noise) as the rats subjected to high oxygen pressure. Data from the two groups were subjected to the same analysis. The EEG activity was transferred from magnetic tape to an analog-digital converter (PDP-15) through a high pass filter (cut off 0.5 Hz) in order to avoid slow base line shifts. Rate of sampling was lOO/sec. Amplitude and frequency were analyzed using “period-amplitude analysis” based on a zero crossing method. The basic measurement procedure used in this experiment was a modification of a technique described by Legewie and Probst (13). The data were divided into five frequency bands-delta (l-3 cycle/set) ;
I -7
-6
-5
-4
-3
-2
-1 min
0
-7
-6
-5
-4
-3
-2
-1
‘0
mln
FIG. 6. The frequencyof appearance of eachfrequencybandof the EEG in atrnosphericpressureand in exposureto six ATA 02.
16
RADAY
Q Significant
ET AL.
PcO.05
4 First electrical discharge
Metrazol
,700
-Control --- Treatment
,
:
:
:
:
:
J&Erotoxin
min FIG. 7. Mean amplitudeof EEG in rats exposedto six ATA oxygen as compared with the EEG obtainedafter administrationof Metrazol and pictroxin.
theta (4-7 cycles/set), alpha (8-13 cycles/set) , beta (14-25 cycles/set) and total (l-25 cycle/set) . The mean amplitude was calculated separately for each frequency band. Analyzed data were available for oscilloscopic display, numerical printout, and magnetic tape storage for subsequent statistical calculations. The reference point in time (t = o) was the first electrical discharge. Since our analysis refers to the preconvulsive stage, our time scale was negative. RESULTS The mean latency from the time of exposure to 6 ATA oxygen until the appearance of the first electrical discharge was 12.5 -L 1.9 min. Figure 1 is a typical record which shows the control EEG at atmospheric pressure
and the appearance of the first electrical discharge in six ATA oxygen. Exposure to six ATA caused a consistent change in the pattern of distribution of frequencies in the EEG before the onset of seizure activity. This is shown in Fig. 2, which plots for seven rats the percentage difference between the number of waves within a bandwidth of l-25 Hz (10 set bins), and the mean number of waves during spontaneous activity as recorded during air breathing at atmospheric pressure (zero line). The control graph shows that during air breathing, fluctuation about the mean was t20%. However, during exposure to 6 ATA, the total number of waves in the EEG of each rat was such that the entire distribution was diverted downward so as to fluctuate between +lOY, and -90% relative to control. This pattern was not a generalized feature of development of seizures. Thus, Figs. 3 and 4 show that injection of picrotoxin or metrazol did not result in a significant change in the distribution, but rather, that the dispersion of points was random (*3OoJo relative to control) before and after drug administration. The consistent patterns of these curves permit us to use mean values for the frequency analysis which is shown in Figs. 5 and 6. Figure 5 again shows that during the 7 min at 6 ATA oxygen which preceded the first electrical discharge, there was a significant (p < 0.05) decrease in the mean frequency of the EEG. The t-test analysis revealed no significant differences in mean frequency between the control period and the period leading up to the onset of metrazol or picrotoxin induced discharges. Figure 6, which shows the analysis for each frequency band, reveals that during the preconvulsive period in 6 ATA oxygen, there was a decrease in the number of theta, beta, and alpha waves and a slight increase in the number of delta waves. The preseizure EEG of animals breathing 6 ATA 02 showed reduction in the mean amplitude which became statistically significant 3 min before onset of first electrical discharge (Fig. 7). However, the amplitude began to increase just before the onset of the first electrical discharge. Our results also show that no statistically significant change in mean amplitude was observed during the preconvulsive stage of animals injected with Metrazol and picrotoxin (Fig. 7). DISCUSSION Our findings demonstrate that during exposure to hyperbaric oxygen EEG abnormalities precede the first electrical discharge by up to 7 min. The significant reduction in the mean frequency and amplitude of the electrical activity of the brain permits us to assume that the toxic effect of oxygen on the brain is cumulative and interferes progressively with the normal activity of the brain. Our results also revealed a significant difference between the preconvulsive electrical activity in Metrazol and picro-
18
RADAY
ET AL.
toxin induced seizures and those of high oxygen pressure. Both Metrazol and picrotoxin showed no significant change in mean amplitude and mean frequency during the preconvulsive period. It appears that under these experimental conditions there is an almost sudden change in the normal electrical equilibrium of the brain and the first electrical discharge, consisting of high amplitude and sharp activity, represents the first sign of brain dysfunction. The physiological mechanism underlying the convulsive consequences of exposure to high oxygen pressure remain unknown. While our results do not directly provide such mechanisms, they do suggest that the cerebral abnormality associated with oxygen toxicity evolves gradually over several minutes culminating in seizures. This sequence is most consistent with those biochemical theories which attribute the toxicity of oxygen at high pressure to a disturbance in the cellular metabolism and normal functioning of the CNS. Many workers pointed out that most enzymes containing sulfhydryl groups necessary for their activity were inactivated by high oxygen pressure (6, 11). Among the enzyme systems the most interesting are glutamic acid decarboxylase (16) and acetylcholinesterase (17) which play a role in the level of ceurotransmitters, and Na-K-Mg ATPase (12) whose inactivation will disturb active transport. Therefore, it seems more likely to accept any theory which explains the development of the preconvulsive and convulsive changes by interference with the normal metabolic and electrochemical processes of the whole brain. REFERENCES 1. BEAN, J. W. 194.5. Effects of oxygen at increased pressure. Physiol. Rev. 25: 1-169. 2. BEAN, J. W., and E. C. SIEGFRIELL 1945. Transient and permanent after effects of exposure to oxygen at high pressure. Anzcr. J. Physiol. 143: 656665. 3. BERT, P. 1943. “Barometric Pressure : Research in Experimental Physiology.” M. A. Hitchcock and F. A. Hitchcock [trans.]. College Book Co., Columbus, Ohio. 4. BURNS, J. D. 1972. Conqentration-dependent attenuation of hyperbaric oxygen toxicity. Aerosp. Med. 43 : 989-992. 5. COHN, R., and I. GERSH. 1945. Changes in brain potentials during conclusions induced by oxygen under pressure. J. Newoplzysiol. 8: 155-160. 6. DAVIES, H. C., and R. E. DAVIES. 1965. Biochemical aspects of oxygen poisoning, pp. 1047-1058. In “Handbook of Physiology.” Sect. 3 :, “Respiration.” W. 0. Fenn and H. Rahn [Eds.]. Amer. Physiol. Sot., Washington. 7. GERSCHMAN, R., D. L. GILBERT, S. W. NYE, P. W. NADIG, and W. D. FENN. 1954. Role of adrenalectomy and adrenal cortical hormones in oxygen poisoning. Amer. J. Physiol. 178: 346-351. 8. GERSCHMAN, R., D. L. GILBERT, and D. CACCAMISE. 1958. Effect of various substances on survival times of mice exposed to different high oxygen pressure. Amer. J. Physiot. 192 : 563-571.
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9. HAREL, D., D. KEREM, and S. LAVY. 1969. The influence of high oxygen pressures on the electrical activity of the brain. Electroenceph. Clin. Neurophysiol. 26: 310-317. 10. HAREL, D., and S. LAVY. 1971. Changes in electrical activity of the brain as a criterion of hyperbaric oxygen toxicity in the central nervous system. J. Life sci. 1: 111-114. 11. HAUGAARD, N. 1968. Cellular mechanisms of oxygen toxicity. Physiol. Rev. 48 : 311373. 12. KOEHLER, G., and S. F. GOTTLIEB. 1972. Effects of increased tensions of 02, N: and He on the activity of a Na-K-Mg ATPase of rat intestine. Aerosfi. Med. 43 : 269-273. 13. LEGEWIE, H., and W. PROBST. 1969. On-line analysis of EEG with a small computer (period-amplitude analysis). Electrocnceph. Clin. Neurophysiol. 27 : 533-535. 14. Ruccr, F. S., M. L. GIRETTI, and M. LA ROCCA. 1967. Changes in electrical activity of the cerebral cortex and of some subcortical centers in hyperbaric oxygen. Electroemcph. C/in. ATcurophysiol. 22 : 231-238. 15. SONNENSCHEIN, R. R., and S. N. STEIN. 1953. Electrical activity of the brain in acute oxygen poisoning. Electrorncejh. Clin. Ncztrophysiol. 5 : 521-524. 16. TUNNICLIFF, G., M. URTON, and J. D. WOOD. 1973. Susceptibility of chick brain L-glutamic acid decarboxylase and other neurotransmitter enzymes to hyperbaric oxygen in vitro. Biochem. Pharwtacol. 22 : 501-505. 17. ZIRKLE, L. G., C. E. MENGEL, B. D. HORTON, and E. J. DUFFY. 1965. Studies of oxygen toxicity in the central nervous system. Acrosf. Med. 36: 1027-1032.