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Behavioral effects of 7-nitroindazole on hyperbaric oxygen toxicity
Physiology & Behavior 76 (2002) 611 – 616
Behavioral effects of 7-nitroindazole on hyperbaric oxygen toxicity Finn Konow Jellestada,*, Hilde Gundersenb a
Division of Physiological Psychology, Department of Biological and Medical Psychology, University of Bergen, Aarstadvn. 21, N-5009 Bergen, Norway b Division of Cognitive Neuroscience, Department of Biological and Medical Psychology, University of Bergen, Aarstadvn. 21, N-5009 Bergen, Norway Received 13 June 2001; received in revised form 25 March 2002; accepted 22 April 2002
Abstract The aim of this study was to evaluate the role of nitric oxide (NO) upon hyperbaric oxygen (HBO) toxicity in male Sprague – Dawley rats during exposure to 0.5 MPa > 99% O2. In the first experiment, the selective neuronal NO synthase inhibitor 7-nitroindazole (7-NI) was injected intraperitoneally (ip) in 15 rats. Another 15 rats received vehicle injections of peanut oil intraperitoneally. Latency to observable tonic-clonic convulsions and motor activity during the HBO exposure were scored and compared between the control group and the 7-NI group. The results showed that injection of 7-NI (30 mg/kg) significantly prolonged the latency to observable tonic-clonic convulsions. The 7-NI group also showed a significant decrease in motor activity compared with the control group. A second experiment was performed to measure the effect of 7-NI injections upon open-field activity during normobaric conditions. Twenty-four male Sprague – Dawley rats were randomly divided into three groups, each consisting of eight rats receiving 30 mg/kg 7-NI injections, 10 mg/kg 7-NI injections or vehicle injections of peanut oil intraperitoneally, respectively. The results showed that injection of 7-NI led to a significant dose-dependent reduction in horizontal and vertical activities. This study shows that 7-NI prolongs the latency to hyperoxia-induced seizures. However, it also demonstrates that 7-NI in doses ranging from 30 to 10 mg/kg has a secondary effect upon motor behavior in general. It can therefore not be ruled out that the protective effect of 7-NI upon HBO intoxication is partly due to reduced motor activity. D 2002 Elsevier Science Inc. All rights reserved. Keywords: 7-Nitroindazole; Hyperbaric oxygen; Convulsions; Open-field activity; Nitric oxide
1. Introduction Hyperbaric oxygen (HBO) is used in medical therapy as well in professional and military diving [15,23]. The use of HBO, however, has several adverse consequences that limit its usefulness. Actually, HBO exposure of sufficient pressure and duration can result in CNS toxicity ranging from mild neurological symptoms to serve tonic-clonic convulsions [1,8,14]. Physiologically, CNS intoxication is reflected in pathological EEG changes (increase in slow wave activity and decrease in a activity), changes in cerebral blood flow (CBF) and metabolic rate [7,30,31]. HBO exposure leads to an initial cerebral vasoconstriction that is time and pressure limited [3,30], probably reflecting a compensatory mechanism for the increasing oxygen pressure. Secondary vasodilatation is correlated with changes in EEG and hyperoxia-induced seizures [2]. Bert first described the toxic effects of HBO in 1878. However, the mechanism responsible for the hyperoxia-
* Corresponding author. Tel.: +47-55-58-62-28; fax: +47-55-58-98-72. E-mail address: [email protected] (F.K. Jellestad).
induced hemodynamic changes and the neurological manifestations are still not fully understood, although several hypotheses have been suggested. Recent studies have suggested that nitric oxide (NO) may be involved in the onset of HBO toxicity [4,18]. There are several reports indicating an increase in NO generation under HBO conditions [9,14,18]. Zhang et al. [35] have published data that demonstrate a significant increase in latency to the development of behaviorally observable tonic-clonic convulsions during HBO exposure by injecting a NO synthase inhibitor (NOSi). Three isoforms of NOS have been identified, one neurospecific (nNOS), one endothelial specific (eNOS) and one immunospecific (iNOS). Zhang’s study and other previous HBO studies have mainly used unspecific NOS inhibitors such as L-NAME [4,19,20] and L-NNA [33,35], which inhibit eNOS and nNOS [26]. Prolonged latency to HBO toxicity may therefore be a result of eNOS inhibition as well as of nNOS inhibition. To evaluate the role of NO in the pathophysiology of hyperoxia-induced seizures, we used the selective nNOS inhibitor 7-nitroindazole (7-NI). 7-NI is believed to be specific for nNOS and is therefore specifically adapted for CNS research [26]. Bitterman and Bitterman [4] have earlier
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used 7-NI as a NOSi under hyperbaric conditions and demonstrated prolonged latency to changes in EEG. Other factors than elevated oxygen partial pressure (pO2) may contribute to the development of oxygen toxicity. Reduced motor activity under pressure increases the latency to hyperoxia-induced seizures [22]. Previous studies report that injection of 7-NI decreases motor activity in rats [25,33] and in mice [13], while others report no effect on motor performance in chicks [16] and in rats [17]. The aim of this study was to evaluate the protective effect of the selective nNOS inhibitor 7-NI upon oxygen intoxication during HBO exposure. In addition, we wanted to evaluate whether 7-NI has any effect upon motor activity either during HBO exposure or under normobaric conditions as reflected in open-field behavior.
2. Materials and methods 2.1. Experiment 1 2.1.1. Animals The experiment was carried out on male Sprague – Dawley rats weighing 380 –440 g, kept in separate cages on a 12-h day– night cycle. The rats had ad libitum access to commercial food and tap water. 2.1.2. Drugs 7-NI (C7H5N3O2) (Sigma) was used as NO synthase inhibitor. 7-NI (30 mg/kg) was dissolved in peanut oil and sonicated. Both 7-NI and vehicle were injected intraperitoneally 30 min before the hyperbaric exposure. 2.1.3. Apparatus A 30-l steel chamber (length: 78 cm, diameter: 25 cm) certified for dives down to 50 MPa was used in the experiment. The chamber contained sensors that register oxygen level, humidity, temperature and pressure. This information was continually monitored and stored on a computer. Each rat was monitored and videotaped during the HBO exposure. 2.1.4. Experimental procedures Thirty rats were randomly assigned to a control (n = 15) and a 7-NI group (n = 15) before the HBO exposure. All rats were handled on 4 consecutive days before testing. During the HBO exposure, the rats were kept in a 2-l Plexiglas cage (length: 21 cm, width: 10 cm) inside the pressure chamber, which allowed free movement. The chamber was sealed and flushed with pure oxygen of medical quality until the >99% oxygen level was obtained. This was followed by compression to 0.5 MPa with a compression rate of 0.1 MPa/min. Maximum time at 0.5 MPa >99% O2 was 45 min. Decompression rate was linear with 0.025 MPa/min and was started immediately after the first behaviorally observable tonicclonic convulsion sign of epileptic seizure activity, eventually at maximal time at 0.5 MPa.
The duration of the latent period was measured from the time the rats reached the pressure of 0.5 MPa and until the appearance of observable tonic-clonic convulsions. Seizure latency for rats that did not convulse was sat to 45 min. Motor activity was scored during the compression and at stable pressure until the appearance of observable tonicclonic convulsions. For rats that did not convulse, motor activity was scored until maximal time at stable pressure (45 min). Motor activity was defined as horizontal and vertical behaviors in the chamber and calculated in percent of total time spent in the chamber. 2.2. Experiment 2 2.2.1. Animals Twenty-four male Sprague – Dawley rats weighing 350– 400 g were used in this experiment. Housing conditions were the same as in Experiment 1. 2.2.2. Drugs 7-NI from the same batch as in Experiment 1 was used and prepared in the same way. Two different doses of 7-NI were applied in this experiment (10 and 30 mg/kg) to detect possible dose-dependent effects upon open-field motor activity. Both 7-NI and vehicle (equivalent to the volume of the 30 mg/kg 7-NI dose) were injected intraperitoneally 30 min before the open-field test. 2.2.3. Apparatus Open-field testing was performed in a 1 1-m plywood box with 40-cm high walls. The floor was painted flat black and divided into 25 squares (20 20 cm) by white lines. The room was darkened and light was provided by two 20-W red light bulbs. A white noise generator masked eventual auditory disturbances. The behavior of the rats was recorded by a video camera attached to the ceiling. The camera was connected to a video recorder and a monitor in an adjoining room. 2.2.4. Experimental procedures The rats were randomly assigned to three groups: a control group (n = 8), a 10 mg/kg 7-NI group (n = 8) and a 30 mg/kg 7-NI group (n = 8). All rats were handled on 4 consecutive days before testing, as in Experiment 1. An open-field test session lasted for 15 min. Open-field behavior is defined as horizontal activity, vertical activity and horizontal speed. Horizontal activity was scored as crossing of one square with all four legs. Total number of crossed squares during the test was counted. Vertical activity was recorded as the number of times the rats reared on the hind legs. Horizontal speed was calculated as the number of squares crossed per second. All rats were tested once. 2.2.5. Statistical methods The statistical significance of differences between groups was determined using one-way ANOVA and planned com-
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Fig. 1. Mean latency to observable tonic-clonic convulsions in the control and 7-NI groups. Maximum time at 0.5 MPa >99% O2 was 45 min. Vertical lines mark standard deviations. ** P < .001 (ANOVA).
parisons. All behavioral data were tested with Levene’s test of homogeneity of variance. An a level of .05 was used for all statistical tests. Data were expressed as the mean value ± S.D. Correlation analyses were performed with Pearson Product – Moment Correlation.
3. Results 3.1. Experiment 1 3.1.1. Ambient parameters There were no significant differences between the 7-NI and the control groups with regard to relative humidity (95.9 ± 5.1% vs. 98.2 ± 3.4%) or temperature (25.2 ± 0.6 vs. 25.6 ± 0.6 C) during the HBO exposure. 3.1.2. Latency to observable tonic-clonic convulsions Injection of 7-NI led to a significantly longer latency to the onset of observable tonic-clonic convulsions compared with the control group [ F(1,26) = 94.165, P < .0001] (see Fig. 1). Only 10 out of 15 rats in the 7-NI group showed tonic-clonic seizures compared with 13 out of 13 in the control group. Two rats in the control group were excluded due to technical reasons during the compression.
Fig. 2. Motor activity in the control and 7-NI groups during the HBO exposure before hyperoxia-induced seizures or until maximal time at 0.5 MPa. Vertical lines mark standard deviations. ** P < .001 (ANOVA).
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Fig. 3. Total number of crossed squares during the open-field test in the control group and after injection of 7-NI. Vertical lines marks standard deviations. ** P < .001 (planned comparisons).
3.1.3. Motor activity Injection of 7-NI led to a decrease in motor activity during the HBO exposure compared with the control group (see Fig. 2). One-way ANOVA showed significant difference between the control group and the 7-NI group [ F(1,25) = 123.2, P < .0001]. Correlation analysis showed also that high motor activity was related to short latency to observable behavioral epileptic symptoms (n = 25, r = .73, P < .05). 3.2. Experiment 2 3.2.1. Open field Injection of 7-NI led to a significant dose-dependent reduction of horizontal activity in the open field (see Fig. 3). One-way ANOVA showed significant differences between the three groups [ F(2,21) = 65.667, P < .0001]. Planned comparisons showed differences between control and 10 mg/kg 7-NI [ F(1,21) = 36.496, P < .0001], between control and 30 mg/kg 7-NI [ F(1,21) = 131.202, P < .0001] and between the two 7-NI groups [ F(1,21) = 29.301, P < .0001]. Injection of 7-NI also led to a significant dose-dependent reduction of vertical activity in the open field (see Fig. 4). One-way ANOVA showed significant differences between the three groups [ F(2,21) = 29.857, P < .0001]. Planned comparisons showed differences between control and 10 mg/kg 7-NI [ F(1,21) = 17.038, P < .0001], between control and
Fig. 4. Total of number of rearings (vertical activity) during the open-field test in the control group and after injection of 7-NI. Vertical lines marks standard deviations. ** P < .001 (planned comparisons).
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Fig. 5. Horizontal speed in the control group and after injection of 7-NI. Vertical lines marks standard deviations. ** P < .001 (planned comparisons).
30 mg/kg 7-NI [ F(1,21) = 59.619, P < .0001] and between the two 7-NI groups [ F(1,21) = 12.914, P < .0001]. One-way ANOVA also revealed significant differences in horizontal speed between the three groups [ F(2,21) = 23.01, P < .0001] (see Fig. 5). Planned comparisons showed differences between control and 30 mg/kg 7-NI [ F(1,21) = 42.726, P < .0001] and between the two 7-NI groups [ F(1,21) = 23.448, P < .0001]. There was no difference between the control group and the 10 mg/kg 7-NI group.
4. Discussion Injection of the selective nNOS inhibitor 7-NI significantly prolonged the latency to observable tonic-clonic seizures. The present results confirm recent findings by Bitterman and Bitterman [4] using 7-NI and other previous studies using unspecific NOSi [4,19,20,33,35]. Injection of 7-NI led in addition to a reduction in motor activity, and there was also a significant correlation between high motor activity and short latency to observable tonic-clonic convulsions. This latter finding is similar to that of Kafka et al. [21] who investigated the effect of treadmill exercise upon oxygen intoxication in rats. In the second experiment of our study, injection of 7-NI led to a dose-dependent reduction in horizontal and vertical open-field activities under normobaric conditions. Based on several studies, it seems reasonable to conclude that NO generated by nNOS is involved in the mechanism of HBO toxicity. Demchenko et al. [9] showed that increases in regional CBF (rCBF) during HBO were associated with large increases in NO production. They suggest that increased NO overproduction initiates CNS O2 toxicity by increasing rCBF. Similarly, Sato et al. [28] showed, by using microdialysis technique, that the level of NO metabolites are closely related to an increase in CBF before the appearance of EEG discharges. Chavko et al. [6] also showed that nNOS protein, determined by Western blot, was higher 1 and 2 days after HBO induced seizures, while eNOS and iNOS were unchanged. They also reported that 7-NI had a clear protective effect against HBO seizures. Accordingly, 7-NI may have other properties that also can delay onset of hyperoxia-induced tonic-clonic seizures.
Previous studies show that injection of 7-NI leads to inhibition of monoamine oxidase (MAO) [10] and that MAO inhibitors prolong the onset to hyperoxia-induced seizures [5]. Other studies show that the unspecific NOS inhibitor L-NAME has antioxidant properties that not are related no nNOS [29] and that injection of L-NAME reduces the corticosteroid level [20]. We cannot exclude that 7-NI have such properties, which would account for the protective effect of 7-NI. Although several studies show that 7-NI has a clear protective effect with regard to oxygen intoxication, most probably by prolonging vasoconstriction, 7-NI has also several other CNS and behavioral effects. In our study, injection of 7-NI had a clear depressive effect upon motor activity during HBO exposure. To our knowledge, motor activity during hyperbaric exposure after injection of NOS inhibitors has not been thoroughly investigated by others. Whether the reduced motor activity is due to a general CNS depression or to a more specific peripheral effect is difficult to conclude at the present stage. There are findings supporting both these notions. A previous study showed that injection of 7-NI induced a loss of the righting reflex in rats. The loss of the righting reflex may indicate that 7-NI exerts a prominent depression of the CNS and has therefore been suggested as an index of a narcotic effect [12]. Another study suggested that injection of 7-NI leads to sedation [32]. Sedation and reduced motor activity have been reported after injection of iNOS under normobaric conditions [27,32]. In addition to motor activity, hyperoxia-induced seizures can be potentiated by increased anxiety and stress, indicated by increased activity in the CNS [22]. A previous study in our laboratory showed that injection of L-NAME leads to reduced locomotor activity in the open field and to a decrease in plasma corticosterone levels, reflecting a lowered activation level in the CNS [20]. It is possible that injection of 7-NI also leads to a decrease in corticosterone level, which may indicate reduced anxiety and thereby contribute to the prolonged latency to HBO intoxication. Other studies have shown that injection of 7-NI has anxiolytic-like properties in several rodent models of anxiety, and there are several reports indicating that NO may be involved in the mechanism of anxiety [32,34]. The assumption that inhibition of NO synthesis can have an anxiolyticlike effect is feasible when taking into account the close interaction between NMDA and NOS and the well-established anxiolytic-like profile of distinct NMDA antagonists and the anxiogenic-like action of NMDA [11]. Therefore, we cannot exclude that an anxiolytic-like property of 7-NI may contribute to the prolonged latency of hyperoxiainduced seizures. Our open-field data showed that injection of 7-NI under normobaric conditions leads to a significant dose-dependent decrease in horizontal and vertical activities. In addition, injection of 30 mg/kg 7-NI leads to a reduction in horizontal speed (number of squares crossed per second) in the open
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field test. Our results are in agreement with some previous studies [25,32] and could indicate that 7-NI also has some peripheral effects. Reduced horizontal activity, vertical activity and horizontal speed in the open-field test may indicate that 7-NI reduces skeletal muscle tonus. This suggestion is supported by a previous in vitro study, which showed that injection of 7-NI leads to a reduction of maximal contraction velocity and to a reduction of isometric force in the extensor digitorum longus muscle [24]. An unpublished study in our laboratory showed in addition that injection of 7-NI led to a reduction in the acoustic startle response. This further supports the notion that 7-NI induces reduced muscle tonus and that this may contribute to the reduced motor activity both under hyperbaric and normobaric conditions. In conclusion, the protective effect of 7-NI suggests that NO generated by nNOS is involved in HBO toxicity and that reduced motor activity during HBO exposure may contribute to the protective effect of 7-NI. At any rate, our behavioral data suggest that 7-NI has effects that may be secondary to those solely related to nNOS regulation. Whether reduced motor activity is induced by sedation, reduced anxiety or by reduced muscle tone is beyond the scope of this paper. Because the decrease in horizontal and vertical activities under normobaric conditions was dose dependent, it could be interesting for further research to investigate the latency to hyperoxia-induced seizures after injection of 10 mg/kg 7-NI. References [1] Ballentine JD. Pathology of oxygen toxicity. New York: Academic Press, 1982. [2] Bean JW, Ligell J, Burgess DW. Cerebral O2, CO2, regional cerebral vascular control, and hyperbaric oxygenation. J Appl Physiol 1972; 32: 650 – 7. [3] Bergo¨ GW, Tyssebotn I. The effect of 5bar oxygen on systemic hemodynamic variables and local cerebral blood flow in conscious rats. Undersea Biomed Res 1992;19:339 – 54. [4] Bitterman N, Bitterman H. L-Arginine – NO pathway and CNS oxygen toxicity. J Appl Physiol 1998;84:1633 – 8. [5] Blenkarn DG, Schanberg SM, Saltzman HA. Cerebral amines and acute hyperbaric oxygen toxicity. J Pharmacol Exp Ther 1968;166: 346 – 53. [6] Chavko M, Xing G-Q, Keyser DO. Increased sensitivity to seizures in repeated exposure to hyperbaric oxygen: role of NOS activation. Brain Res 2001;900:227 – 33. [7] Clark JM. Oxygen toxicity. In: Bennet PB, Elliot DH, editors. The physiology and medicine of diving. 4th ed. London: Saunders, 1993. p. 121 – 69. [8] De Martino G, Luchetti M, De Rosa RC. Toxic effects of oxygen. In: Oriani G, Marroni A, Wattel F, editors. Handbook on hyperbaric medicine. Berlin: Springer-Verlag, 1996. p. 59 – 74. [9] Demchenko IT, Boso AE, O’Neill TJ. Nitric oxide and cerebral blood flow responses to hyperbaric oxygen. J Appl Physiol 2000;88:1381 – 9. [10] Desvignes C, Bert L, Vinet L, Renaud B, Lamba´s-Sen´as L. Evidence that the neuronal nitric oxide synthase inhibitor 7-nitroindazole inhibits monoamine oxidase in the rat: in vivo effects on extracellular striatal dopamine and 3,4 dihydroxyphenylacetic acid. Neurosci Lett 1999;264:5 – 8.
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Behavioral effects of 7-nitroindazole on hyperbaric oxygen toxicity Finn Konow Jellestada,*, Hilde Gundersenb a
Division of Physiological Psychology, Department of Biological and Medical Psychology, University of Bergen, Aarstadvn. 21, N-5009 Bergen, Norway b Division of Cognitive Neuroscience, Department of Biological and Medical Psychology, University of Bergen, Aarstadvn. 21, N-5009 Bergen, Norway Received 13 June 2001; received in revised form 25 March 2002; accepted 22 April 2002
Abstract The aim of this study was to evaluate the role of nitric oxide (NO) upon hyperbaric oxygen (HBO) toxicity in male Sprague – Dawley rats during exposure to 0.5 MPa > 99% O2. In the first experiment, the selective neuronal NO synthase inhibitor 7-nitroindazole (7-NI) was injected intraperitoneally (ip) in 15 rats. Another 15 rats received vehicle injections of peanut oil intraperitoneally. Latency to observable tonic-clonic convulsions and motor activity during the HBO exposure were scored and compared between the control group and the 7-NI group. The results showed that injection of 7-NI (30 mg/kg) significantly prolonged the latency to observable tonic-clonic convulsions. The 7-NI group also showed a significant decrease in motor activity compared with the control group. A second experiment was performed to measure the effect of 7-NI injections upon open-field activity during normobaric conditions. Twenty-four male Sprague – Dawley rats were randomly divided into three groups, each consisting of eight rats receiving 30 mg/kg 7-NI injections, 10 mg/kg 7-NI injections or vehicle injections of peanut oil intraperitoneally, respectively. The results showed that injection of 7-NI led to a significant dose-dependent reduction in horizontal and vertical activities. This study shows that 7-NI prolongs the latency to hyperoxia-induced seizures. However, it also demonstrates that 7-NI in doses ranging from 30 to 10 mg/kg has a secondary effect upon motor behavior in general. It can therefore not be ruled out that the protective effect of 7-NI upon HBO intoxication is partly due to reduced motor activity. D 2002 Elsevier Science Inc. All rights reserved. Keywords: 7-Nitroindazole; Hyperbaric oxygen; Convulsions; Open-field activity; Nitric oxide
1. Introduction Hyperbaric oxygen (HBO) is used in medical therapy as well in professional and military diving [15,23]. The use of HBO, however, has several adverse consequences that limit its usefulness. Actually, HBO exposure of sufficient pressure and duration can result in CNS toxicity ranging from mild neurological symptoms to serve tonic-clonic convulsions [1,8,14]. Physiologically, CNS intoxication is reflected in pathological EEG changes (increase in slow wave activity and decrease in a activity), changes in cerebral blood flow (CBF) and metabolic rate [7,30,31]. HBO exposure leads to an initial cerebral vasoconstriction that is time and pressure limited [3,30], probably reflecting a compensatory mechanism for the increasing oxygen pressure. Secondary vasodilatation is correlated with changes in EEG and hyperoxia-induced seizures [2]. Bert first described the toxic effects of HBO in 1878. However, the mechanism responsible for the hyperoxia-
* Corresponding author. Tel.: +47-55-58-62-28; fax: +47-55-58-98-72. E-mail address: [email protected] (F.K. Jellestad).
induced hemodynamic changes and the neurological manifestations are still not fully understood, although several hypotheses have been suggested. Recent studies have suggested that nitric oxide (NO) may be involved in the onset of HBO toxicity [4,18]. There are several reports indicating an increase in NO generation under HBO conditions [9,14,18]. Zhang et al. [35] have published data that demonstrate a significant increase in latency to the development of behaviorally observable tonic-clonic convulsions during HBO exposure by injecting a NO synthase inhibitor (NOSi). Three isoforms of NOS have been identified, one neurospecific (nNOS), one endothelial specific (eNOS) and one immunospecific (iNOS). Zhang’s study and other previous HBO studies have mainly used unspecific NOS inhibitors such as L-NAME [4,19,20] and L-NNA [33,35], which inhibit eNOS and nNOS [26]. Prolonged latency to HBO toxicity may therefore be a result of eNOS inhibition as well as of nNOS inhibition. To evaluate the role of NO in the pathophysiology of hyperoxia-induced seizures, we used the selective nNOS inhibitor 7-nitroindazole (7-NI). 7-NI is believed to be specific for nNOS and is therefore specifically adapted for CNS research [26]. Bitterman and Bitterman [4] have earlier
0031-9384/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 3 1 - 9 3 8 4 ( 0 2 ) 0 0 7 6 5 - 5
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used 7-NI as a NOSi under hyperbaric conditions and demonstrated prolonged latency to changes in EEG. Other factors than elevated oxygen partial pressure (pO2) may contribute to the development of oxygen toxicity. Reduced motor activity under pressure increases the latency to hyperoxia-induced seizures [22]. Previous studies report that injection of 7-NI decreases motor activity in rats [25,33] and in mice [13], while others report no effect on motor performance in chicks [16] and in rats [17]. The aim of this study was to evaluate the protective effect of the selective nNOS inhibitor 7-NI upon oxygen intoxication during HBO exposure. In addition, we wanted to evaluate whether 7-NI has any effect upon motor activity either during HBO exposure or under normobaric conditions as reflected in open-field behavior.
2. Materials and methods 2.1. Experiment 1 2.1.1. Animals The experiment was carried out on male Sprague – Dawley rats weighing 380 –440 g, kept in separate cages on a 12-h day– night cycle. The rats had ad libitum access to commercial food and tap water. 2.1.2. Drugs 7-NI (C7H5N3O2) (Sigma) was used as NO synthase inhibitor. 7-NI (30 mg/kg) was dissolved in peanut oil and sonicated. Both 7-NI and vehicle were injected intraperitoneally 30 min before the hyperbaric exposure. 2.1.3. Apparatus A 30-l steel chamber (length: 78 cm, diameter: 25 cm) certified for dives down to 50 MPa was used in the experiment. The chamber contained sensors that register oxygen level, humidity, temperature and pressure. This information was continually monitored and stored on a computer. Each rat was monitored and videotaped during the HBO exposure. 2.1.4. Experimental procedures Thirty rats were randomly assigned to a control (n = 15) and a 7-NI group (n = 15) before the HBO exposure. All rats were handled on 4 consecutive days before testing. During the HBO exposure, the rats were kept in a 2-l Plexiglas cage (length: 21 cm, width: 10 cm) inside the pressure chamber, which allowed free movement. The chamber was sealed and flushed with pure oxygen of medical quality until the >99% oxygen level was obtained. This was followed by compression to 0.5 MPa with a compression rate of 0.1 MPa/min. Maximum time at 0.5 MPa >99% O2 was 45 min. Decompression rate was linear with 0.025 MPa/min and was started immediately after the first behaviorally observable tonicclonic convulsion sign of epileptic seizure activity, eventually at maximal time at 0.5 MPa.
The duration of the latent period was measured from the time the rats reached the pressure of 0.5 MPa and until the appearance of observable tonic-clonic convulsions. Seizure latency for rats that did not convulse was sat to 45 min. Motor activity was scored during the compression and at stable pressure until the appearance of observable tonicclonic convulsions. For rats that did not convulse, motor activity was scored until maximal time at stable pressure (45 min). Motor activity was defined as horizontal and vertical behaviors in the chamber and calculated in percent of total time spent in the chamber. 2.2. Experiment 2 2.2.1. Animals Twenty-four male Sprague – Dawley rats weighing 350– 400 g were used in this experiment. Housing conditions were the same as in Experiment 1. 2.2.2. Drugs 7-NI from the same batch as in Experiment 1 was used and prepared in the same way. Two different doses of 7-NI were applied in this experiment (10 and 30 mg/kg) to detect possible dose-dependent effects upon open-field motor activity. Both 7-NI and vehicle (equivalent to the volume of the 30 mg/kg 7-NI dose) were injected intraperitoneally 30 min before the open-field test. 2.2.3. Apparatus Open-field testing was performed in a 1 1-m plywood box with 40-cm high walls. The floor was painted flat black and divided into 25 squares (20 20 cm) by white lines. The room was darkened and light was provided by two 20-W red light bulbs. A white noise generator masked eventual auditory disturbances. The behavior of the rats was recorded by a video camera attached to the ceiling. The camera was connected to a video recorder and a monitor in an adjoining room. 2.2.4. Experimental procedures The rats were randomly assigned to three groups: a control group (n = 8), a 10 mg/kg 7-NI group (n = 8) and a 30 mg/kg 7-NI group (n = 8). All rats were handled on 4 consecutive days before testing, as in Experiment 1. An open-field test session lasted for 15 min. Open-field behavior is defined as horizontal activity, vertical activity and horizontal speed. Horizontal activity was scored as crossing of one square with all four legs. Total number of crossed squares during the test was counted. Vertical activity was recorded as the number of times the rats reared on the hind legs. Horizontal speed was calculated as the number of squares crossed per second. All rats were tested once. 2.2.5. Statistical methods The statistical significance of differences between groups was determined using one-way ANOVA and planned com-
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Fig. 1. Mean latency to observable tonic-clonic convulsions in the control and 7-NI groups. Maximum time at 0.5 MPa >99% O2 was 45 min. Vertical lines mark standard deviations. ** P < .001 (ANOVA).
parisons. All behavioral data were tested with Levene’s test of homogeneity of variance. An a level of .05 was used for all statistical tests. Data were expressed as the mean value ± S.D. Correlation analyses were performed with Pearson Product – Moment Correlation.
3. Results 3.1. Experiment 1 3.1.1. Ambient parameters There were no significant differences between the 7-NI and the control groups with regard to relative humidity (95.9 ± 5.1% vs. 98.2 ± 3.4%) or temperature (25.2 ± 0.6 vs. 25.6 ± 0.6 C) during the HBO exposure. 3.1.2. Latency to observable tonic-clonic convulsions Injection of 7-NI led to a significantly longer latency to the onset of observable tonic-clonic convulsions compared with the control group [ F(1,26) = 94.165, P < .0001] (see Fig. 1). Only 10 out of 15 rats in the 7-NI group showed tonic-clonic seizures compared with 13 out of 13 in the control group. Two rats in the control group were excluded due to technical reasons during the compression.
Fig. 2. Motor activity in the control and 7-NI groups during the HBO exposure before hyperoxia-induced seizures or until maximal time at 0.5 MPa. Vertical lines mark standard deviations. ** P < .001 (ANOVA).
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Fig. 3. Total number of crossed squares during the open-field test in the control group and after injection of 7-NI. Vertical lines marks standard deviations. ** P < .001 (planned comparisons).
3.1.3. Motor activity Injection of 7-NI led to a decrease in motor activity during the HBO exposure compared with the control group (see Fig. 2). One-way ANOVA showed significant difference between the control group and the 7-NI group [ F(1,25) = 123.2, P < .0001]. Correlation analysis showed also that high motor activity was related to short latency to observable behavioral epileptic symptoms (n = 25, r = .73, P < .05). 3.2. Experiment 2 3.2.1. Open field Injection of 7-NI led to a significant dose-dependent reduction of horizontal activity in the open field (see Fig. 3). One-way ANOVA showed significant differences between the three groups [ F(2,21) = 65.667, P < .0001]. Planned comparisons showed differences between control and 10 mg/kg 7-NI [ F(1,21) = 36.496, P < .0001], between control and 30 mg/kg 7-NI [ F(1,21) = 131.202, P < .0001] and between the two 7-NI groups [ F(1,21) = 29.301, P < .0001]. Injection of 7-NI also led to a significant dose-dependent reduction of vertical activity in the open field (see Fig. 4). One-way ANOVA showed significant differences between the three groups [ F(2,21) = 29.857, P < .0001]. Planned comparisons showed differences between control and 10 mg/kg 7-NI [ F(1,21) = 17.038, P < .0001], between control and
Fig. 4. Total of number of rearings (vertical activity) during the open-field test in the control group and after injection of 7-NI. Vertical lines marks standard deviations. ** P < .001 (planned comparisons).
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Fig. 5. Horizontal speed in the control group and after injection of 7-NI. Vertical lines marks standard deviations. ** P < .001 (planned comparisons).
30 mg/kg 7-NI [ F(1,21) = 59.619, P < .0001] and between the two 7-NI groups [ F(1,21) = 12.914, P < .0001]. One-way ANOVA also revealed significant differences in horizontal speed between the three groups [ F(2,21) = 23.01, P < .0001] (see Fig. 5). Planned comparisons showed differences between control and 30 mg/kg 7-NI [ F(1,21) = 42.726, P < .0001] and between the two 7-NI groups [ F(1,21) = 23.448, P < .0001]. There was no difference between the control group and the 10 mg/kg 7-NI group.
4. Discussion Injection of the selective nNOS inhibitor 7-NI significantly prolonged the latency to observable tonic-clonic seizures. The present results confirm recent findings by Bitterman and Bitterman [4] using 7-NI and other previous studies using unspecific NOSi [4,19,20,33,35]. Injection of 7-NI led in addition to a reduction in motor activity, and there was also a significant correlation between high motor activity and short latency to observable tonic-clonic convulsions. This latter finding is similar to that of Kafka et al. [21] who investigated the effect of treadmill exercise upon oxygen intoxication in rats. In the second experiment of our study, injection of 7-NI led to a dose-dependent reduction in horizontal and vertical open-field activities under normobaric conditions. Based on several studies, it seems reasonable to conclude that NO generated by nNOS is involved in the mechanism of HBO toxicity. Demchenko et al. [9] showed that increases in regional CBF (rCBF) during HBO were associated with large increases in NO production. They suggest that increased NO overproduction initiates CNS O2 toxicity by increasing rCBF. Similarly, Sato et al. [28] showed, by using microdialysis technique, that the level of NO metabolites are closely related to an increase in CBF before the appearance of EEG discharges. Chavko et al. [6] also showed that nNOS protein, determined by Western blot, was higher 1 and 2 days after HBO induced seizures, while eNOS and iNOS were unchanged. They also reported that 7-NI had a clear protective effect against HBO seizures. Accordingly, 7-NI may have other properties that also can delay onset of hyperoxia-induced tonic-clonic seizures.
Previous studies show that injection of 7-NI leads to inhibition of monoamine oxidase (MAO) [10] and that MAO inhibitors prolong the onset to hyperoxia-induced seizures [5]. Other studies show that the unspecific NOS inhibitor L-NAME has antioxidant properties that not are related no nNOS [29] and that injection of L-NAME reduces the corticosteroid level [20]. We cannot exclude that 7-NI have such properties, which would account for the protective effect of 7-NI. Although several studies show that 7-NI has a clear protective effect with regard to oxygen intoxication, most probably by prolonging vasoconstriction, 7-NI has also several other CNS and behavioral effects. In our study, injection of 7-NI had a clear depressive effect upon motor activity during HBO exposure. To our knowledge, motor activity during hyperbaric exposure after injection of NOS inhibitors has not been thoroughly investigated by others. Whether the reduced motor activity is due to a general CNS depression or to a more specific peripheral effect is difficult to conclude at the present stage. There are findings supporting both these notions. A previous study showed that injection of 7-NI induced a loss of the righting reflex in rats. The loss of the righting reflex may indicate that 7-NI exerts a prominent depression of the CNS and has therefore been suggested as an index of a narcotic effect [12]. Another study suggested that injection of 7-NI leads to sedation [32]. Sedation and reduced motor activity have been reported after injection of iNOS under normobaric conditions [27,32]. In addition to motor activity, hyperoxia-induced seizures can be potentiated by increased anxiety and stress, indicated by increased activity in the CNS [22]. A previous study in our laboratory showed that injection of L-NAME leads to reduced locomotor activity in the open field and to a decrease in plasma corticosterone levels, reflecting a lowered activation level in the CNS [20]. It is possible that injection of 7-NI also leads to a decrease in corticosterone level, which may indicate reduced anxiety and thereby contribute to the prolonged latency to HBO intoxication. Other studies have shown that injection of 7-NI has anxiolytic-like properties in several rodent models of anxiety, and there are several reports indicating that NO may be involved in the mechanism of anxiety [32,34]. The assumption that inhibition of NO synthesis can have an anxiolyticlike effect is feasible when taking into account the close interaction between NMDA and NOS and the well-established anxiolytic-like profile of distinct NMDA antagonists and the anxiogenic-like action of NMDA [11]. Therefore, we cannot exclude that an anxiolytic-like property of 7-NI may contribute to the prolonged latency of hyperoxiainduced seizures. Our open-field data showed that injection of 7-NI under normobaric conditions leads to a significant dose-dependent decrease in horizontal and vertical activities. In addition, injection of 30 mg/kg 7-NI leads to a reduction in horizontal speed (number of squares crossed per second) in the open
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field test. Our results are in agreement with some previous studies [25,32] and could indicate that 7-NI also has some peripheral effects. Reduced horizontal activity, vertical activity and horizontal speed in the open-field test may indicate that 7-NI reduces skeletal muscle tonus. This suggestion is supported by a previous in vitro study, which showed that injection of 7-NI leads to a reduction of maximal contraction velocity and to a reduction of isometric force in the extensor digitorum longus muscle [24]. An unpublished study in our laboratory showed in addition that injection of 7-NI led to a reduction in the acoustic startle response. This further supports the notion that 7-NI induces reduced muscle tonus and that this may contribute to the reduced motor activity both under hyperbaric and normobaric conditions. In conclusion, the protective effect of 7-NI suggests that NO generated by nNOS is involved in HBO toxicity and that reduced motor activity during HBO exposure may contribute to the protective effect of 7-NI. At any rate, our behavioral data suggest that 7-NI has effects that may be secondary to those solely related to nNOS regulation. Whether reduced motor activity is induced by sedation, reduced anxiety or by reduced muscle tone is beyond the scope of this paper. Because the decrease in horizontal and vertical activities under normobaric conditions was dose dependent, it could be interesting for further research to investigate the latency to hyperoxia-induced seizures after injection of 10 mg/kg 7-NI. References [1] Ballentine JD. Pathology of oxygen toxicity. New York: Academic Press, 1982. [2] Bean JW, Ligell J, Burgess DW. Cerebral O2, CO2, regional cerebral vascular control, and hyperbaric oxygenation. J Appl Physiol 1972; 32: 650 – 7. [3] Bergo¨ GW, Tyssebotn I. The effect of 5bar oxygen on systemic hemodynamic variables and local cerebral blood flow in conscious rats. Undersea Biomed Res 1992;19:339 – 54. [4] Bitterman N, Bitterman H. L-Arginine – NO pathway and CNS oxygen toxicity. J Appl Physiol 1998;84:1633 – 8. [5] Blenkarn DG, Schanberg SM, Saltzman HA. Cerebral amines and acute hyperbaric oxygen toxicity. J Pharmacol Exp Ther 1968;166: 346 – 53. [6] Chavko M, Xing G-Q, Keyser DO. Increased sensitivity to seizures in repeated exposure to hyperbaric oxygen: role of NOS activation. Brain Res 2001;900:227 – 33. [7] Clark JM. Oxygen toxicity. In: Bennet PB, Elliot DH, editors. The physiology and medicine of diving. 4th ed. London: Saunders, 1993. p. 121 – 69. [8] De Martino G, Luchetti M, De Rosa RC. Toxic effects of oxygen. In: Oriani G, Marroni A, Wattel F, editors. Handbook on hyperbaric medicine. Berlin: Springer-Verlag, 1996. p. 59 – 74. [9] Demchenko IT, Boso AE, O’Neill TJ. Nitric oxide and cerebral blood flow responses to hyperbaric oxygen. J Appl Physiol 2000;88:1381 – 9. [10] Desvignes C, Bert L, Vinet L, Renaud B, Lamba´s-Sen´as L. Evidence that the neuronal nitric oxide synthase inhibitor 7-nitroindazole inhibits monoamine oxidase in the rat: in vivo effects on extracellular striatal dopamine and 3,4 dihydroxyphenylacetic acid. Neurosci Lett 1999;264:5 – 8.
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