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The effect of hyperbaric oxygen on regional brain and spinal cord levels of nitric oxide metabolites in rat
Brain Research Bulletin 75 (2008) 668–673
Research report
The effect of hyperbaric oxygen on regional brain and spinal cord levels of nitric oxide metabolites in rat Yusuke Ohgami a , Eunhee Chung a , Donald Y. Shirachi c , Raymond M. Quock a,b,∗ a
Department of Pharmaceutical Sciences, College of Pharmacy, Washington State University, Pullman, WA 99164-6534, USA b Center for Integrated Biotechnology, Washington State University, Pullman, WA 99164-6534, USA c Chico Hyperbaric Center, Chico, CA 95926-3509, USA Received 18 April 2007; received in revised form 5 November 2007; accepted 6 November 2007 Available online 3 December 2007
Abstract Hyperbaric oxygen (HBO2 ) therapy is reported to be beneficial in transient brain ischemia. The present study was conducted to determine the influence of HBO2 on metabolites of nitric oxide (NO) in brain and spinal cord of rats. Rats were exposed to room air (RA), normobaric air (NBA), normobaric oxygen (NBO2 ), hyperbaric air (HBA) or HBO2 , the last two conditions at 2.5 ATA (atmosphere absolute) for 60 min. The results demonstrate that, compared to the NBA control, oxygen alone generally reduced tissue levels of NOx − (nitrite plus nitrate). On the other hand, 2.5 ATA alone tended to have a slight, if any, effect on tissue levels of NOx − . The combination of oxygen and pressure (i.e., HBO2 ) generally led to an increase in tissue levels of NOx − . Based on these findings, it is concluded that HBO2 appears to markedly increase NO function most notably in the corpus striatum, brainstem, cerebellum and spinal cord. © 2007 Elsevier Inc. All rights reserved. Keywords: Hyperbaric oxygen; Nitric oxide; Rats
1. Introduction Hyperbaric oxygen (HBO2 ) therapy is a medical treatment that administers 100% oxygen at a controlled pressure (greater than sea level) for a prescribed amount of time (usually 60–90 min). Increasing the atmospheric pressure of the oxygen that is inhaled dramatically increases the tissue concentration of oxygen [40]. This increase in tissue oxygen is presumed to be the reason for the clinical improvement observed when patients are exposed to HBO2 therapy, such as in decompression sickness; carbon monoxide poisoning and smoke inhalation; gas gangrene; acute traumatic ischemia; wound-healing; necrotizing soft tissue infections; radiation tissue damage; compromised skin grafts; and thermal burns [14]. Among reported beneficial effects of HBO2 is improvement in neurological and cognitive function following cerebral ischemia [20,24,27,34,35]. The mechanism(s) by which these clinical improvements occur is still not clear. It has been sug-
gested that superoxide and/or hydrogen peroxide might be responsible for some of the beneficial effects of HBO2 therapy [19]. Another strong candidate that is implicated in the effects of HBO2 is nitric oxide (NO). Recently, it was shown by in vivo rat brain microdialysis that HBO2 (3 ATA for 2 h) increased NO metabolites six- and fourfold in the hippocampus and striatum, respectively [13]. In more recent studies utilizing NO-sensitive electrodes placed in the cerebral cortex, there was a dose–response elevation in levels of NO at 2.0 ATA and 2.8 ATA in the presence of 100% oxygen [38,39]. This would suggest NO might play a role in the clinical improvements. The purpose of this study was to determine whether exposure to HBO2 increases NOx − levels (as an index of NO production) in different rat brain regions and spinal cord and ascertain whether this is a generalized phenomenon or whether it is sitespecific. 2. Methods
∗ Corresponding author. Current address: Department of Pharmaceutical Sciences, College of Pharmacy, Washington State University, P.O. Box 646534, Pullman, WA 99164-6534, USA. Tel.: +1 509 335 5545; fax: +1 509 335 5902. E-mail address: [email protected] (R.M. Quock).
0361-9230/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2007.11.002
2.1. Animals Male Sprague Dawley rats, 350–400 g body weight, were obtained from Harlan Laboratories (Indianapolis, IN). Rats were housed in groups of two in
Y. Ohgami et al. / Brain Research Bulletin 75 (2008) 668–673 polycarbonate bins (38 cm L × 20 cm W × 24 cm H) in the Wegner Hall Vivarium with access to food (Purina Chow) and water ad libitum. The facility was maintained on a 12-h light:dark cycle (lights on 0700–1900 h) under standard conditions (22 ± 1 ◦ C room temperature, 33% humidity). Rats were transported to the laboratory 2–3 h prior to testing. Experiments were conducted routinely between 1300 and 1600 h. The experimental protocol followed in this research was approved by an institutional animal care and use committee and is in compliance with The Guide for the Care and Use of Laboratory Animals (U.S. National Research Council, 1996). All care was taken to minimize pain and discomfort in the experimental animals.
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2.4. Statistical analysis of data The levels of NO2 − , NO3 − and NOx − in the cerebral cortex, corpus striatum, hippocampus, brainstem, cerebellum and spinal cord of room air control and NBA groups were analyzed by Student’s t-test. The levels of NOx − in different brain regions and spinal cord of NBA, NBO2 , HBA and HBO2 groups were analyzed using a two-way analysis of variance (ANOVA). In Table 1, the levels of NO2 − , NO3 − and NOx − in different brain regions and spinal cord of different treatment groups were analyzed using a one-way ANOVA followed by a post hoc Tukey’s HSD test for parametric multiple comparisons.
3. Results 2.2. Exposure to hyperbaric oxygen Rats were randomly assigned to five different test groups of 8–17 rats each: RA (room air) for 60 min; NBA (normobaric air), compressed (room) air at 1 absolute atmospheres (ATA) for 60 min; NBO2 (normobaric oxygen), 100% oxygen at 1 ATA for 60 min; HBA (hyperbaric air), compressed air at 2.5 ATA for 60 min; and HBO2 (hyperbaric oxygen), 100% oxygen at 2.5 ATA for 60 min. Rats were placed in a B-11 research hyperbaric chamber (Reimers Systems, Inc., Lorton, VA). The pressure inside was increased to 2.5 ATA at a rate of 0.5 ATA/min with 100% oxygen or compressed air (21% oxygen) and was maintained for 60 min. The chamber was ventilated with 100% oxygen or compressed air at a flow rate of 20 l/min to minimize carbon dioxide accumulation. Rats were allowed to breathe spontaneously. After completion of the HBO2 exposure, the chamber was then decompressed at a rate of 1 ATA/min. The HBA group was exposed to compressed air at 2.5 ATA for 60 min. The NBO2 group was exposed to 100% oxygen at 1.0 ATA for 60 min. The NBA group was exposed to compressed air at 1.0 ATA for 60 min. The RA group was placed in the unsealed hyperbaric chamber open to room air for 60 min. Immediately following the 60-min exposure period, all rats were killed by decapitation under isoflurane anesthesia. The brains were quickly removed and dissected into five regions: cerebral cortex, corpus striatum, hippocampus, brainstem and cerebellum. The remaining brain tissue was discarded. Spinal cords of decapitated rats were pushed out of the cranial end of the spinal column by forced injection of saline into the caudal end. These tissue samples were weighed and homogenized in 10 volumes of saline. After centrifugation (15,000 × g for 20 min at 4 ◦ C), the supernatant was separated for determination of NOx − levels and protein assay. The samples were immediately stored on dry ice and later frozen at −80 ◦ C until analyzed. The protein content of the supernatant was determined using the bicinchoninic acid (BCA) method and a commercially available assay kit (Pierce Chemical Co., Rockford, IL) with bovine albumin as a standard. The levels of NO metabolites were expressed in terms of pmol/mg protein.
2.3. Determination of NOx − levels The previously frozen tissue samples were thawed and diluted 50% with 100% methanol. After centrifugation (3000 × g for 5 min at 4 ◦ C), 20 l of the supernatant was injected into the HPLC system. Tissue levels of NOx − were determined by measuring the formation of both stable oxidation products of nitrite (NO2 − ) and nitrate (NO3 − ), using an ENO-20 NOx analyzer (Eicom, Kyoto, Japan). NO2 − and NO3 − in the samples were separated by a reverse-phase separation column packed with polystyrene polymer (NO-PAK, 4.6 mm × 50 mm, Eicom), and NO3 − was reduced to NO2 − in a reduction column packed with copper-plated cadmium filings (NO-RED, Eicom). NO2 − was mixed with a Griess reagent to form a purple azo dye in a reaction coil. The separation and reaction columns and the reaction coil were placed in a column oven that was set at 35 ◦ C. The absorbance of the color of the product dye at 540 nm was measured using a flow-through spectrophotometer. The mobile phase consisted of 10% methanol containing 0.15 M NaCl–NH4 Cl and 0.5 g/l of 4Na–EDTA. The Griess reagent, consisting of 1.25% HCl containing 5 g/l sulfanilamide with 0.25 g/l N-naphthylethylenediamine, was delivered at a rate of 0.1 ml/min. The contamination of nitrite and nitrate in artificial CSF and the reliability of the reduction column were examined in each experiment.
The effects of room air, compressed air, oxygen, pressure and HBO2 on NO2 − , NO3 − and NOx − levels for each brain region or spinal cord are shown in Table 1. There was no significant difference between RA and NBA groups in their levels of NO2 − , NO3 − and NOx − in any of the different brain regions or spinal cord (as analyzed by Student’s t-test). Therefore, the effects of NBA, NBO2 , HBA and HBO2 on levels of NO2 − , NO3 − and NOx − in different brain regions and spinal cord were analyzed by a two-way ANOVA. Each tissue exhibited a unique pattern of significant main and interaction effects of oxygen and pressure. For example, in the cerebral cortex, there was no independent effect of oxygen and atmospheric pressure on levels of NO2 − nor was there any interaction between the two [Foxygen = 3.738, P = 0.0591; Fpressure = 1.221, P = 0.2748; Foxygen×pressure = 0.8693, P = 0.3558]. In contrast, there was a significant effect of both oxygen and hyperbaric pressure on NO3 − levels as well as a significant interaction between the two [Foxygen = 50.64, P < 0.0001; Fpressure = 24.1, P < 0.0001; Foxygen×pressure = 24.61, P < 0.0001]. On the other hand, in the hippocampus, there was a significant effect of both oxygen and hyperbaric pressure on NO2 − (but not NO3 − ) as well as a significant interaction between the two [NO2 − : Foxygen = 48.28, P < 0.0001; Fpressure = 24.38, P < 0.0001; Foxygen×pressure = 89.9, P < 0.0001; Foxygen = 0.04226, P = 0.838; Fpressure = 4.406, P = 0.0411; Foxygen×pressure = 2.136, P = 0.1504]. The relative proportions of NO2 − and NO3 − within tissues were variable; therefore, the best index of total NO production is the sum of both products (NO2 − + NO3 − = NOx − ). In four of the brain regions – cerebral cortex, corpus striatum, brainstem and cerebellum – there was a significant effect of oxygen and hyperbaric pressure on NOx − levels as well as a significant interaction between the two: cerebral cortex [Foxygen = 14.23, P = 0.0004; Fpressure = 30.3, P < 0.0001; Foxygen×pressure = 15.01, P = 0.0003]; corpus striatum [Foxygen = 31.71, P < 0.0001; Fpressure = 91.19, P < 0.0001; Foxygen×pressure = 191.1, P < 0.0001]; brainstem [Foxygen = 36.19, P < 0.0001; Fpressure = 53.75, P < 0.0001; Foxygen×pressure = 98.52, P < 0.0001]; and cerebellum [Foxygen = 13.03, P = 0.0007; Fpressure = 17.32, P = 0.0001; Foxygen×pressure = 30.75, P < 0.0001]. But, in case of the hippocampus, there was a significant effect of only hyperbaric pressure [Foxygen = 0.005649, P > 0.05; Fpressure = 4.786, P < 0.05; Foxygen×pressure = 2.677, P > 0.05]. On the other hand, in spinal cord there was a significant effect of hyperbaric
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Table 1 Effect of room air, compressed air, oxygen, 2.5 atmosphere absolute (ATA) and combined oxygen and 2.5 ATA on the mean levels of nitrite (NO2 − ), nitrate (NO3 − ) and NOx − (NO2 − plus NO3 − ) in the regional brain and spinal cord in rat Control (RA) Mean ± S.E.M. (N = 8)
Effect of compressed air (NBA)
Effect of 2.5 ATA alone (HBA)
Effect of O2 alone (NBO2 )
Effect of O2 plus 2.5 ATA (HBO2 )
%
Mean ± S.E.M. (N = 17)
%
Mean ± S.E.M. (N = 9)
%
Mean ± S.E.M. (N = 9)
%
Mean ± S.E.M. (N = 9–17)
%
Cerebral cortex NO2 − 32.7 ± 1.1 NO3 − 1617.2 ± 15.7 NOx − 1649.9 ± 16.4
100 100 100
33.2 ± 2.7 1624.4 ± 36.5 1657.6 ± 35
101 100 100
36.1 ± 5.6 604.3 ± 92.7*,§ 640.4 ± 97.1*,§
110 37 39
27.2 ± 1.4 1847.1 ± 138.1§ 1874.4 ± 139.3§
83 114 114
35.6 ± 1.3 1852.5 ± 166.8† 1888 ± 167†
109 115 114
Corpus striatum NO2 − 66.9 ± 4.9 NO3 − 1652.2 ± 38.6 NOx − 1719 ± 37
100 100 100
69.2 ± 1.8 1641.5 ± 20.9 1710.7 ± 21
103 99 100
110 52 55
82.6 ± 2.8† 1225.2 ± 62.9*,§ 1307.9 ± 64.4§
124 74 76
105.6 ± 7.6*,§ 3028.2 ± 123*,§,†,# 3133.9 ± 124.5*,§,†,#
158 183 182
Hippocampus NO2 − 50.3 ± 3.4 NO3 − 2322.2 ± 75.2 2372.5 ± 77.8 NOx −
100 100 100
50.6 ± 1.1 2376.5 ± 179.2 2427.1 ± 178.8
101 102 102
41.7 ± 3.7 2064.2 ± 115.7 2105.9 ± 117.6
83 89 89
34.7 ± 0.7§,† 2495.9 ± 80.5 2530.6 ± 80.5
69 107 107
92.4 ± 4.5*,§,# 2731.2 ± 188.4† 2823.6 ± 189.2
184 118 119
Brainstem NO2 − NO3 − NOx −
100 100 100
18.4 ± 1.1 901.6 ± 5.1 920.1 ± 4.7
94 102 102
32.1 ± 2.6*,§ 525.7 ± 22§ 557.9 ± 24.5§
163 60 62
28.5 ± 0.5*,§ 651.3 ± 16.5 679.7 ± 16.7
144 74 75
35.5 ± 2*,§,# 2121 ± 131.8*,§,†,# 2156.5 ± 132.6*,§,†,#
180 241 239
Cerebellum NO2 − 36.5 ± 1.9 NO3 − 1021.8 ± 147.7 NOx − 1058.3 ± 148.3
100 100 100
37.2 ± 1.8 1036.5 ± 11 1073.7 ± 10.9
102 101 101
36.7 ± 2 793.8 ± 66.4 830.5 ± 67.9
100 78 78
33.4 ± 1.8 866.4 ± 42 899.8 ± 40.9
92 85 85
48.3 ± 2.2*,§,†,# 2002 ± 176.2*,§,†,# 2050.3 ± 176.1*,§,†,#
132 196 194
Spinal cord NO2 − 42.1 ± 1.1 NO3 − 1399.4 ± 33 NOx − 1441.5 ± 33.2
100 100 100
41.6 ± 2.6 1410.9 ± 34.5 1452.4 ± 33.4
99 101 101
40.9 ± 3 700.9 ± 39*,§ 741.8 ± 41.9*,§
97 50 51
45 ± 2 1202.8 ± 92.7† 1247.9 ± 91.4†
107 86 87
32.7 ± 2# 1754.6 ± 103.4*,§,†,# 1787.3 ± 104.1*,§,†,#
78 125 124
19.7 ± 0.7 881.7 ± 27.2 901.5 ± 27.7
73.3 ± 10.4 867.3 ± 58*,§ 940.6 ± 68.1*,§
Abbreviations: RA, room air; NBA, normobaric air; NBO2 , normobaric oxygen; HBA, hyperbaric air; HBO2 , hyperbaric oxygen, unit of measurement: pmol/mg protein, significance of difference: *P < 0.05 vs. RA, § P < 0.05 vs. NBA, † P < 0.05 vs. NBO2 , # P < 0.05 vs. HBA (Tukey’s HSD test).
pressure and interaction between oxygen and hyperbaric pressure [Foxygen = 1.652, P > 0.05; Fpressure = 39.81, P < 0.0001; Foxygen×pressure = 87.98, P < 0.0001]. It is clear that 2.5 ATA hyperbaric pressure alone (HBA) did not appreciably alter levels of NOx − , compared to NBA levels. In fact, NOx − levels were significantly decreased in some brain regions, notably the corpus striatum, brainstem, cerebellum and spinal cord. When animals were exposed to 100% oxygen at 2.5 ATA (HBO2 ), the NOx − levels were increased in all brain regions and the spinal cord. If HBO2 effects were compared with NBA data, the NOx − levels were significantly increased in the corpus striatum, brainstem, cerebellum and spinal cord but not in cerebral cortex or hippocampus. If the HBO2 effects were compared with the NBO2 data (100% oxygen at 1 ATA), the NOx − levels were significantly elevated in all brain regions and the spinal cord. 4. Discussion NO is a free radical molecule that is involved in a variety of signal transduction processes in the body. In the periphery, NO is primarily the endothelially derived mediator of vasodilation and a regulator and effector of the immune response. In the CNS, NO has even more important functions as an intercellular signaling molecule that has been implicated in synaptic
plasticity, pain modulation, behavioral regulation and neuroprotection/neurotoxicity [7,17]. NO in the brain is the product of the enzymatic action of all three isozymes of nitric oxide synthase (NOS): neuronal, endothelial and inducible. NOx − levels in the brain largely reflects the metabolites of neuronally, endothelially and inducibly derived NO [42]; however, it has been demonstrated brain NOx − may be primarily an index of neuronal NO function [32]. It is apparent that serum NOx − is formed from a variety of sources; however, the literature shows that the brain levels of NOx − are unrelated to serum levels [26,29]. It appears that the brain is highly compartmentalized by its blood–brain barrier and that NOx − in the brain does reflect products of NO metabolism centrally [16]. A seminal work by [32] demonstrated variation in absolute levels and the regional distribution of nitric oxide synthase enzyme activity in rat brain. Enzyme activities ranged from approximately 5–10 nmol/(min g) tissue in the thalamus, cerebral cortex, and pons-medulla to nearly 50 nmol/(min g) tissue. The regional distribution of NOS enzyme activity was generally consistent with the reported distribution in rat brain of NADPH diaphorase [37], neuronal NOS antibody staining [30] and in situ hydridization to neuronal NOS mRNA [6]. It is posited that the uneven distribution of NOx − in the brain regions and spinal cord reflects regional brain NO func-
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tion. A histochemical/immunohistochemical study reported comparable distribution patterns of NOS enzyme and NOstimulated cyclic GMP accumulation [37], the enzyme guanylyl cyclase being the most likely target of NO [41]. The findings support the hypothesis that NO generated in the brain functions primarily as a mediatory of intercellular signaling [15,28]. Our studies show a heterogeneous distribution of NO2 − , NO3 − and NOx − in five brain regions and the spinal cord. The range of NO2 − levels were 20–70 pmol/mg protein (0.07–0.19 pmol/mg wet tissue), the range of NO3 − levels was 880–2300 pmol/mg protein (1.78–7.99 pmol/mg wet tissue), and the range of NOx − levels was 900–2400 pmol/mg protein (1.85–8.15 pmol/mg wet tissue). Comparable levels of NOx − have been reported in rat cerebral cortex [2,3], corpus striatum [4,23], hippocampus [22] and cerebellum [36]. The literature also shows that HBO2 exposures of durations comparable to this study causes a decrease in cerebral blood flow (CBF) which would be correlated to reduction in endothelial NO activity and, hence, reduction in NOx − levels. We hypothesize that increases in regional brain and spinal cord levels of NOx − of HBO2 -exposed animals is indicative of an increase in neuronal NO function. Experiments are planned to determine the effects of selective neuronal, endothelial and inducible NOS-inhibitors on the regional brain and spinal cord HBO2 -induced increases in NOx − levels. HBO2 increases oxygen tension PO2 in blood but reduces blood flow by means of O2 -induced vasoconstriction [10]. The effect of HBO2 on cerebral blood flow is pressure-dependent. HBO2 therapy generally employs no higher than 2.5 ATA; atmospheres greater than 2.5 ATA enter the realm of O2 toxicity. O2 toxicity is attributed to overproduction of NO through increased neuronal and/or endothelial NOS activity. One possible mechanism is that increased NO causes overactivation of the glutamate–NMDA system, which is known to produce neurotoxicity [43]. Another mechanism involves NO-mediated increases in the cerebral blood flow, which causes hyperoxygenation of tissues with increased generation of highly reactive and toxic oxygen radicals [8]. There appears to be a key role of nitric oxide synthase (NOS) in the effects of HBO2 [40]. Exposure to HBO2 increases steady state levels of NO in the cerebral cortex (as measured by NO-sensitive electrodes in anesthetized rats); this increase in NO levels was inhibited by the neuronal NOS (nNOS) inhibitor 7-nitroindazole [38,39]. By contrast, in the present study, exposure to HBO2 in conscious rats significantly increased NOx − in selected brain regions. Exposure to HBO2 caused greater increase in periaortic levels of NO in wild-type and endothelial NOS (eNOS) knockout mice than in nNOS knockout mice, further underscoring the predominant role of nNOS activity in contributing to the total NO increase [38]. Brain in vivo microdialysis experiments in rats have also shown that HBO2 exposure markedly increased levels of NO2 − and NO3 − in dialysates from rat parietal cortex, hippocampus and corpus striatum; these increases were completely blocked by NOS-inhibitors [13,18,33]. Increased
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pressure alone failed to appreciably alter dialysate levels of NOx − [13]. NO has been implicated in adult neurogenesis in the mouse [25] and guinea pig hippocampus [21]. In addition a cGMP signaling system appears to be involved in neurite outgrowth and survival in hippocampal and dopaminergic neurons in vitro [11]. These studies suggest a role of NO in brain plasticity after injury. These processes might be involved where long-term HBO2 therapy appeared to reverse memory loss in chronic stroke and traumatic brain-injured patients [20,35]. In addition it should be noted that the experimental conditions of this study differ quite significantly from those in which oxygen toxicity relative to NO has been demonstrated at 5–6 ATA for 60–75 min but not at 4 ATA [9]. This is commensurate with our studies (2.5 ATA) in which we did not observe any signs which might have indicated any seizure effects during the 60-min HBO2 treatment. The finding that exposure to 100% oxygen alone (NBO2 ) for 60 min significantly lowered the NOx − levels in all brain regions and the spinal cord except in the hippocampus was contrary to what was anticipated. Why this effect occurred is not immediately clear. However, there is a superoxide (O2 − )/NO biological balance wherein changes in levels of one can influence levels and function of the other [5,12]. Both hyperoxia and O2 − have been shown to inactivate NO [31]. The abolition of the vasodilating effect of NO donors by HBO2 exposure suggests that an interaction between NO and O2 − might be responsible for the HBO2 -induced reduction in cerebral blood flow [8]. The data from this study supports the concept that the possible effects of HBO2 treatment in animal and clinical studies occurs via a complex set of biochemical reactions involving molecular oxygen and nNOS, resulting in the formation of nitric oxide [1]. The experimental HBO2 conditions of this study are within those used in earlier studies which demonstrated the synthesis of NO in vivo [13,39]. The results of the present study show that HBO2 exposure has a major effect on regional brain and spinal cord levels of NOx − . Compared to normobaric air, oxygen under normobaric conditions (NBO2 ) generally caused a reduction in tissue NOx − levels in all nervous tissues, more drastic in some tissues (cerebral cortex, spinal cord) than others. Exposure to compressed air at 2.5 ATA pressure (HBA) tended to reduce NOx − levels in all regions except the cerebral cortex and hippocampus. Exposure to 100% oxygen and 2.5 ATA pressure (HBO2 ) markedly increased NOx − in all areas; the increase was more acute in some tissues (corpus striatum, brainstem, cerebellum and spinal cord) than others (cerebral cortex and hippocampus). The findings of this research show that NO function in different nervous tissues may be differentially affected by exposure to HBO2 . Conflict of interest None.
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[18] S. Hagioka, Y. Takeda, S. Zhang, T. Sato, K. Morita, Effects of 7nitroindazole and N-nitro-l-arginine methyl ester on changes in cerebral blood flow and nitric oxide production preceding development of hyperbaric oxygen-induced seizures in rats, Neurosci. Lett. 382 (2005) 206–210. [19] J. Hink, E. Jansen, Are superoxide and/or hydrogen peroxide responsible for some of the beneficial effects of hyperbaric oxygen therapy? Med. Hypoth. 57 (2001) 764–769. [20] M.L. Hoggard, D.Y. Shirachi, K.E. Johnson, S. Hannigan-Downs, The effect of hyperbaric oxygen therapy on improvement of speech, language, and cognitive deficits observed in a traumatic brain injury patient, Abst. Undersea Hyperbar. Med. Assoc. 32 (2005) 276. [21] A.T. Islam, A. Kuraoka, M. Kawabuchi, Morphological basis of nitric oxide production and its correlation with the polysialylated precursor cells in the dentate gyrus of the adult guinea pig hippocampus, Anat. Sci. Int. 78 (2003) 98–103. [22] S.O. Jacobsson, G.E. Cassel, S.A. Persson, Increased levels of nitrogen oxides and lipid peroxidation in the rat brain after soman-induced seizures, Arch. Toxicol. 73 (1999) 269–273. [23] A. Jelenkovic, B. Janac, V. Pesic, D.M. Jovanovic, I. Vasiljevic, Z. Prolic, Effects of extremely low-frequency magnetic field in the brain of rats., Brain Res. Bull. 68 (2006) 355–360. [24] J.P. Kapp, Neurological response to hyperbaric oxygen—a criterion for cerebral revascularization, Surg. Neurol. 15 (1981) 43–46. [25] B. Moreno-Lopez, J.A. Noval, L.G. Gonzalez-Bonet, C. Estrada, Morphological bases for a role of nitric oxide in adult neurogenesis, Brain Res. 869 (2000) 244–250. [26] I. Nagatomo, W. Hashiguchi, M. Tominaga, M. Uchida, Y. Akasaki, M. Takigawa, Long-term maternal ethanol administration decreases nitric oxide metabolites in brains of postnatal mice, Neurosci. Res. Commun. 28 (1991) 165–172. [27] R.A. Neubauer, E. End, Hyperbaric oxygenation as an adjunct therapy in strokes due to thrombosis, Stroke 11 (1980) 297–300. [28] H. Prast, A. Philippu, Nitric oxide as modulator of neuronal function, Prog. Neurobiol. 64 (2001) 51–68. [29] K. Rejdak, A. Petzold, M.A. Sharpe, M. Smith, G. Keir, Z. Stelmasiak, E.J. Thompson, G. Giovannoni, Serum and urine nitrate and nitrite are not reliable indicators of intrathecal nitric oxide production in acute brain injury, J. Neurol. Sci. 208 (2003) 1–7. [30] J. Rodrigo, D.R. Springall, O. Uttenthal, M.L. Bentura, F. Abadia-Molina, V. Riveros-Moreno, R. Mart´ınez-Murillo, J.M. Polak, S. Moncada, Localization of nitric oxide synthase in the adult rat brain, Phil. Trans. R. Soc. London B 345 (1994) 175–221. [31] G.M. Rubanyi, P.M. Vanhoutte, Superoxide anions and hyperoxia inactivate endothelium-derived relaxing factor, Am. J. Physiol. 250 (1986) H822–H827. [32] M. Salter, C. Duffy, J. Garthwaite, P.J.L.M. Strijbos, Ex vivo measurement of brain tissue nitrite and nitrate accurately reflects nitric oxide synthase activity in vivo, J. Neurochem. 66 (1996) 1683–1690. [33] T. Sato, Y. Takeda, S. Hagioka, S. Zhang, M. Hirakawa, Changes in nitric oxide production and cerebral blood flow before development of hyperbaric oxygen-induced seizures in rats, Brain Res. 918 (2001) 131–140. [34] D.Y. Shirachi, M.L. Hoggard, K.E. Johnson, The effect of hyperbaric oxygen therapy on a 3 year post-stroke aphasic patient, in: Proc. XIVth Int. Congr. Hyperbaric Med., vol. 14, 2003, pp. 153–156. [35] D.Y. Shirachi, M.L. Hoggard, K.E. Johnson, The improvement of speech and language deficits in 3 aphasic stroke patients by HBO therapy—a preliminary study, in: Proc. XVth Int. Congr. Hyperbaric Med., vol. 15, 2005, p. 214. [36] E. Siles, E. Martinez-Lara, A. Canuelo, M. Sanchez, R. Hernandez, J.C. Lopez-Ramos, M.L. Del Moral, F.J. Esteban, S. Blanco, J.A. Pedrosa, J. Rodrigo, M.A. Peinado, Age-related changes of the nitric oxide system in the rat brain, Brain Res. 29 (2002) 385–392. [37] E. Southam, J. Garthwaite, The nitric oxide-cyclic GMP signaling pathway in rat brain, Neuropharmacol. 32 (1993) 1267–1277. [38] S.R. Thom, V.M. Bhopale, D. Fisher, Y. Manevich, P.L. Huang, D.G. Buerk, Stimulation of nitric oxide synthase in cerebral cortex due to elevated partial pressures of oxygen: an oxidative stress response, J. Neurobiol. 51 (2002) 85–100.
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Research report
The effect of hyperbaric oxygen on regional brain and spinal cord levels of nitric oxide metabolites in rat Yusuke Ohgami a , Eunhee Chung a , Donald Y. Shirachi c , Raymond M. Quock a,b,∗ a
Department of Pharmaceutical Sciences, College of Pharmacy, Washington State University, Pullman, WA 99164-6534, USA b Center for Integrated Biotechnology, Washington State University, Pullman, WA 99164-6534, USA c Chico Hyperbaric Center, Chico, CA 95926-3509, USA Received 18 April 2007; received in revised form 5 November 2007; accepted 6 November 2007 Available online 3 December 2007
Abstract Hyperbaric oxygen (HBO2 ) therapy is reported to be beneficial in transient brain ischemia. The present study was conducted to determine the influence of HBO2 on metabolites of nitric oxide (NO) in brain and spinal cord of rats. Rats were exposed to room air (RA), normobaric air (NBA), normobaric oxygen (NBO2 ), hyperbaric air (HBA) or HBO2 , the last two conditions at 2.5 ATA (atmosphere absolute) for 60 min. The results demonstrate that, compared to the NBA control, oxygen alone generally reduced tissue levels of NOx − (nitrite plus nitrate). On the other hand, 2.5 ATA alone tended to have a slight, if any, effect on tissue levels of NOx − . The combination of oxygen and pressure (i.e., HBO2 ) generally led to an increase in tissue levels of NOx − . Based on these findings, it is concluded that HBO2 appears to markedly increase NO function most notably in the corpus striatum, brainstem, cerebellum and spinal cord. © 2007 Elsevier Inc. All rights reserved. Keywords: Hyperbaric oxygen; Nitric oxide; Rats
1. Introduction Hyperbaric oxygen (HBO2 ) therapy is a medical treatment that administers 100% oxygen at a controlled pressure (greater than sea level) for a prescribed amount of time (usually 60–90 min). Increasing the atmospheric pressure of the oxygen that is inhaled dramatically increases the tissue concentration of oxygen [40]. This increase in tissue oxygen is presumed to be the reason for the clinical improvement observed when patients are exposed to HBO2 therapy, such as in decompression sickness; carbon monoxide poisoning and smoke inhalation; gas gangrene; acute traumatic ischemia; wound-healing; necrotizing soft tissue infections; radiation tissue damage; compromised skin grafts; and thermal burns [14]. Among reported beneficial effects of HBO2 is improvement in neurological and cognitive function following cerebral ischemia [20,24,27,34,35]. The mechanism(s) by which these clinical improvements occur is still not clear. It has been sug-
gested that superoxide and/or hydrogen peroxide might be responsible for some of the beneficial effects of HBO2 therapy [19]. Another strong candidate that is implicated in the effects of HBO2 is nitric oxide (NO). Recently, it was shown by in vivo rat brain microdialysis that HBO2 (3 ATA for 2 h) increased NO metabolites six- and fourfold in the hippocampus and striatum, respectively [13]. In more recent studies utilizing NO-sensitive electrodes placed in the cerebral cortex, there was a dose–response elevation in levels of NO at 2.0 ATA and 2.8 ATA in the presence of 100% oxygen [38,39]. This would suggest NO might play a role in the clinical improvements. The purpose of this study was to determine whether exposure to HBO2 increases NOx − levels (as an index of NO production) in different rat brain regions and spinal cord and ascertain whether this is a generalized phenomenon or whether it is sitespecific. 2. Methods
∗ Corresponding author. Current address: Department of Pharmaceutical Sciences, College of Pharmacy, Washington State University, P.O. Box 646534, Pullman, WA 99164-6534, USA. Tel.: +1 509 335 5545; fax: +1 509 335 5902. E-mail address: [email protected] (R.M. Quock).
0361-9230/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2007.11.002
2.1. Animals Male Sprague Dawley rats, 350–400 g body weight, were obtained from Harlan Laboratories (Indianapolis, IN). Rats were housed in groups of two in
Y. Ohgami et al. / Brain Research Bulletin 75 (2008) 668–673 polycarbonate bins (38 cm L × 20 cm W × 24 cm H) in the Wegner Hall Vivarium with access to food (Purina Chow) and water ad libitum. The facility was maintained on a 12-h light:dark cycle (lights on 0700–1900 h) under standard conditions (22 ± 1 ◦ C room temperature, 33% humidity). Rats were transported to the laboratory 2–3 h prior to testing. Experiments were conducted routinely between 1300 and 1600 h. The experimental protocol followed in this research was approved by an institutional animal care and use committee and is in compliance with The Guide for the Care and Use of Laboratory Animals (U.S. National Research Council, 1996). All care was taken to minimize pain and discomfort in the experimental animals.
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2.4. Statistical analysis of data The levels of NO2 − , NO3 − and NOx − in the cerebral cortex, corpus striatum, hippocampus, brainstem, cerebellum and spinal cord of room air control and NBA groups were analyzed by Student’s t-test. The levels of NOx − in different brain regions and spinal cord of NBA, NBO2 , HBA and HBO2 groups were analyzed using a two-way analysis of variance (ANOVA). In Table 1, the levels of NO2 − , NO3 − and NOx − in different brain regions and spinal cord of different treatment groups were analyzed using a one-way ANOVA followed by a post hoc Tukey’s HSD test for parametric multiple comparisons.
3. Results 2.2. Exposure to hyperbaric oxygen Rats were randomly assigned to five different test groups of 8–17 rats each: RA (room air) for 60 min; NBA (normobaric air), compressed (room) air at 1 absolute atmospheres (ATA) for 60 min; NBO2 (normobaric oxygen), 100% oxygen at 1 ATA for 60 min; HBA (hyperbaric air), compressed air at 2.5 ATA for 60 min; and HBO2 (hyperbaric oxygen), 100% oxygen at 2.5 ATA for 60 min. Rats were placed in a B-11 research hyperbaric chamber (Reimers Systems, Inc., Lorton, VA). The pressure inside was increased to 2.5 ATA at a rate of 0.5 ATA/min with 100% oxygen or compressed air (21% oxygen) and was maintained for 60 min. The chamber was ventilated with 100% oxygen or compressed air at a flow rate of 20 l/min to minimize carbon dioxide accumulation. Rats were allowed to breathe spontaneously. After completion of the HBO2 exposure, the chamber was then decompressed at a rate of 1 ATA/min. The HBA group was exposed to compressed air at 2.5 ATA for 60 min. The NBO2 group was exposed to 100% oxygen at 1.0 ATA for 60 min. The NBA group was exposed to compressed air at 1.0 ATA for 60 min. The RA group was placed in the unsealed hyperbaric chamber open to room air for 60 min. Immediately following the 60-min exposure period, all rats were killed by decapitation under isoflurane anesthesia. The brains were quickly removed and dissected into five regions: cerebral cortex, corpus striatum, hippocampus, brainstem and cerebellum. The remaining brain tissue was discarded. Spinal cords of decapitated rats were pushed out of the cranial end of the spinal column by forced injection of saline into the caudal end. These tissue samples were weighed and homogenized in 10 volumes of saline. After centrifugation (15,000 × g for 20 min at 4 ◦ C), the supernatant was separated for determination of NOx − levels and protein assay. The samples were immediately stored on dry ice and later frozen at −80 ◦ C until analyzed. The protein content of the supernatant was determined using the bicinchoninic acid (BCA) method and a commercially available assay kit (Pierce Chemical Co., Rockford, IL) with bovine albumin as a standard. The levels of NO metabolites were expressed in terms of pmol/mg protein.
2.3. Determination of NOx − levels The previously frozen tissue samples were thawed and diluted 50% with 100% methanol. After centrifugation (3000 × g for 5 min at 4 ◦ C), 20 l of the supernatant was injected into the HPLC system. Tissue levels of NOx − were determined by measuring the formation of both stable oxidation products of nitrite (NO2 − ) and nitrate (NO3 − ), using an ENO-20 NOx analyzer (Eicom, Kyoto, Japan). NO2 − and NO3 − in the samples were separated by a reverse-phase separation column packed with polystyrene polymer (NO-PAK, 4.6 mm × 50 mm, Eicom), and NO3 − was reduced to NO2 − in a reduction column packed with copper-plated cadmium filings (NO-RED, Eicom). NO2 − was mixed with a Griess reagent to form a purple azo dye in a reaction coil. The separation and reaction columns and the reaction coil were placed in a column oven that was set at 35 ◦ C. The absorbance of the color of the product dye at 540 nm was measured using a flow-through spectrophotometer. The mobile phase consisted of 10% methanol containing 0.15 M NaCl–NH4 Cl and 0.5 g/l of 4Na–EDTA. The Griess reagent, consisting of 1.25% HCl containing 5 g/l sulfanilamide with 0.25 g/l N-naphthylethylenediamine, was delivered at a rate of 0.1 ml/min. The contamination of nitrite and nitrate in artificial CSF and the reliability of the reduction column were examined in each experiment.
The effects of room air, compressed air, oxygen, pressure and HBO2 on NO2 − , NO3 − and NOx − levels for each brain region or spinal cord are shown in Table 1. There was no significant difference between RA and NBA groups in their levels of NO2 − , NO3 − and NOx − in any of the different brain regions or spinal cord (as analyzed by Student’s t-test). Therefore, the effects of NBA, NBO2 , HBA and HBO2 on levels of NO2 − , NO3 − and NOx − in different brain regions and spinal cord were analyzed by a two-way ANOVA. Each tissue exhibited a unique pattern of significant main and interaction effects of oxygen and pressure. For example, in the cerebral cortex, there was no independent effect of oxygen and atmospheric pressure on levels of NO2 − nor was there any interaction between the two [Foxygen = 3.738, P = 0.0591; Fpressure = 1.221, P = 0.2748; Foxygen×pressure = 0.8693, P = 0.3558]. In contrast, there was a significant effect of both oxygen and hyperbaric pressure on NO3 − levels as well as a significant interaction between the two [Foxygen = 50.64, P < 0.0001; Fpressure = 24.1, P < 0.0001; Foxygen×pressure = 24.61, P < 0.0001]. On the other hand, in the hippocampus, there was a significant effect of both oxygen and hyperbaric pressure on NO2 − (but not NO3 − ) as well as a significant interaction between the two [NO2 − : Foxygen = 48.28, P < 0.0001; Fpressure = 24.38, P < 0.0001; Foxygen×pressure = 89.9, P < 0.0001; Foxygen = 0.04226, P = 0.838; Fpressure = 4.406, P = 0.0411; Foxygen×pressure = 2.136, P = 0.1504]. The relative proportions of NO2 − and NO3 − within tissues were variable; therefore, the best index of total NO production is the sum of both products (NO2 − + NO3 − = NOx − ). In four of the brain regions – cerebral cortex, corpus striatum, brainstem and cerebellum – there was a significant effect of oxygen and hyperbaric pressure on NOx − levels as well as a significant interaction between the two: cerebral cortex [Foxygen = 14.23, P = 0.0004; Fpressure = 30.3, P < 0.0001; Foxygen×pressure = 15.01, P = 0.0003]; corpus striatum [Foxygen = 31.71, P < 0.0001; Fpressure = 91.19, P < 0.0001; Foxygen×pressure = 191.1, P < 0.0001]; brainstem [Foxygen = 36.19, P < 0.0001; Fpressure = 53.75, P < 0.0001; Foxygen×pressure = 98.52, P < 0.0001]; and cerebellum [Foxygen = 13.03, P = 0.0007; Fpressure = 17.32, P = 0.0001; Foxygen×pressure = 30.75, P < 0.0001]. But, in case of the hippocampus, there was a significant effect of only hyperbaric pressure [Foxygen = 0.005649, P > 0.05; Fpressure = 4.786, P < 0.05; Foxygen×pressure = 2.677, P > 0.05]. On the other hand, in spinal cord there was a significant effect of hyperbaric
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Table 1 Effect of room air, compressed air, oxygen, 2.5 atmosphere absolute (ATA) and combined oxygen and 2.5 ATA on the mean levels of nitrite (NO2 − ), nitrate (NO3 − ) and NOx − (NO2 − plus NO3 − ) in the regional brain and spinal cord in rat Control (RA) Mean ± S.E.M. (N = 8)
Effect of compressed air (NBA)
Effect of 2.5 ATA alone (HBA)
Effect of O2 alone (NBO2 )
Effect of O2 plus 2.5 ATA (HBO2 )
%
Mean ± S.E.M. (N = 17)
%
Mean ± S.E.M. (N = 9)
%
Mean ± S.E.M. (N = 9)
%
Mean ± S.E.M. (N = 9–17)
%
Cerebral cortex NO2 − 32.7 ± 1.1 NO3 − 1617.2 ± 15.7 NOx − 1649.9 ± 16.4
100 100 100
33.2 ± 2.7 1624.4 ± 36.5 1657.6 ± 35
101 100 100
36.1 ± 5.6 604.3 ± 92.7*,§ 640.4 ± 97.1*,§
110 37 39
27.2 ± 1.4 1847.1 ± 138.1§ 1874.4 ± 139.3§
83 114 114
35.6 ± 1.3 1852.5 ± 166.8† 1888 ± 167†
109 115 114
Corpus striatum NO2 − 66.9 ± 4.9 NO3 − 1652.2 ± 38.6 NOx − 1719 ± 37
100 100 100
69.2 ± 1.8 1641.5 ± 20.9 1710.7 ± 21
103 99 100
110 52 55
82.6 ± 2.8† 1225.2 ± 62.9*,§ 1307.9 ± 64.4§
124 74 76
105.6 ± 7.6*,§ 3028.2 ± 123*,§,†,# 3133.9 ± 124.5*,§,†,#
158 183 182
Hippocampus NO2 − 50.3 ± 3.4 NO3 − 2322.2 ± 75.2 2372.5 ± 77.8 NOx −
100 100 100
50.6 ± 1.1 2376.5 ± 179.2 2427.1 ± 178.8
101 102 102
41.7 ± 3.7 2064.2 ± 115.7 2105.9 ± 117.6
83 89 89
34.7 ± 0.7§,† 2495.9 ± 80.5 2530.6 ± 80.5
69 107 107
92.4 ± 4.5*,§,# 2731.2 ± 188.4† 2823.6 ± 189.2
184 118 119
Brainstem NO2 − NO3 − NOx −
100 100 100
18.4 ± 1.1 901.6 ± 5.1 920.1 ± 4.7
94 102 102
32.1 ± 2.6*,§ 525.7 ± 22§ 557.9 ± 24.5§
163 60 62
28.5 ± 0.5*,§ 651.3 ± 16.5 679.7 ± 16.7
144 74 75
35.5 ± 2*,§,# 2121 ± 131.8*,§,†,# 2156.5 ± 132.6*,§,†,#
180 241 239
Cerebellum NO2 − 36.5 ± 1.9 NO3 − 1021.8 ± 147.7 NOx − 1058.3 ± 148.3
100 100 100
37.2 ± 1.8 1036.5 ± 11 1073.7 ± 10.9
102 101 101
36.7 ± 2 793.8 ± 66.4 830.5 ± 67.9
100 78 78
33.4 ± 1.8 866.4 ± 42 899.8 ± 40.9
92 85 85
48.3 ± 2.2*,§,†,# 2002 ± 176.2*,§,†,# 2050.3 ± 176.1*,§,†,#
132 196 194
Spinal cord NO2 − 42.1 ± 1.1 NO3 − 1399.4 ± 33 NOx − 1441.5 ± 33.2
100 100 100
41.6 ± 2.6 1410.9 ± 34.5 1452.4 ± 33.4
99 101 101
40.9 ± 3 700.9 ± 39*,§ 741.8 ± 41.9*,§
97 50 51
45 ± 2 1202.8 ± 92.7† 1247.9 ± 91.4†
107 86 87
32.7 ± 2# 1754.6 ± 103.4*,§,†,# 1787.3 ± 104.1*,§,†,#
78 125 124
19.7 ± 0.7 881.7 ± 27.2 901.5 ± 27.7
73.3 ± 10.4 867.3 ± 58*,§ 940.6 ± 68.1*,§
Abbreviations: RA, room air; NBA, normobaric air; NBO2 , normobaric oxygen; HBA, hyperbaric air; HBO2 , hyperbaric oxygen, unit of measurement: pmol/mg protein, significance of difference: *P < 0.05 vs. RA, § P < 0.05 vs. NBA, † P < 0.05 vs. NBO2 , # P < 0.05 vs. HBA (Tukey’s HSD test).
pressure and interaction between oxygen and hyperbaric pressure [Foxygen = 1.652, P > 0.05; Fpressure = 39.81, P < 0.0001; Foxygen×pressure = 87.98, P < 0.0001]. It is clear that 2.5 ATA hyperbaric pressure alone (HBA) did not appreciably alter levels of NOx − , compared to NBA levels. In fact, NOx − levels were significantly decreased in some brain regions, notably the corpus striatum, brainstem, cerebellum and spinal cord. When animals were exposed to 100% oxygen at 2.5 ATA (HBO2 ), the NOx − levels were increased in all brain regions and the spinal cord. If HBO2 effects were compared with NBA data, the NOx − levels were significantly increased in the corpus striatum, brainstem, cerebellum and spinal cord but not in cerebral cortex or hippocampus. If the HBO2 effects were compared with the NBO2 data (100% oxygen at 1 ATA), the NOx − levels were significantly elevated in all brain regions and the spinal cord. 4. Discussion NO is a free radical molecule that is involved in a variety of signal transduction processes in the body. In the periphery, NO is primarily the endothelially derived mediator of vasodilation and a regulator and effector of the immune response. In the CNS, NO has even more important functions as an intercellular signaling molecule that has been implicated in synaptic
plasticity, pain modulation, behavioral regulation and neuroprotection/neurotoxicity [7,17]. NO in the brain is the product of the enzymatic action of all three isozymes of nitric oxide synthase (NOS): neuronal, endothelial and inducible. NOx − levels in the brain largely reflects the metabolites of neuronally, endothelially and inducibly derived NO [42]; however, it has been demonstrated brain NOx − may be primarily an index of neuronal NO function [32]. It is apparent that serum NOx − is formed from a variety of sources; however, the literature shows that the brain levels of NOx − are unrelated to serum levels [26,29]. It appears that the brain is highly compartmentalized by its blood–brain barrier and that NOx − in the brain does reflect products of NO metabolism centrally [16]. A seminal work by [32] demonstrated variation in absolute levels and the regional distribution of nitric oxide synthase enzyme activity in rat brain. Enzyme activities ranged from approximately 5–10 nmol/(min g) tissue in the thalamus, cerebral cortex, and pons-medulla to nearly 50 nmol/(min g) tissue. The regional distribution of NOS enzyme activity was generally consistent with the reported distribution in rat brain of NADPH diaphorase [37], neuronal NOS antibody staining [30] and in situ hydridization to neuronal NOS mRNA [6]. It is posited that the uneven distribution of NOx − in the brain regions and spinal cord reflects regional brain NO func-
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tion. A histochemical/immunohistochemical study reported comparable distribution patterns of NOS enzyme and NOstimulated cyclic GMP accumulation [37], the enzyme guanylyl cyclase being the most likely target of NO [41]. The findings support the hypothesis that NO generated in the brain functions primarily as a mediatory of intercellular signaling [15,28]. Our studies show a heterogeneous distribution of NO2 − , NO3 − and NOx − in five brain regions and the spinal cord. The range of NO2 − levels were 20–70 pmol/mg protein (0.07–0.19 pmol/mg wet tissue), the range of NO3 − levels was 880–2300 pmol/mg protein (1.78–7.99 pmol/mg wet tissue), and the range of NOx − levels was 900–2400 pmol/mg protein (1.85–8.15 pmol/mg wet tissue). Comparable levels of NOx − have been reported in rat cerebral cortex [2,3], corpus striatum [4,23], hippocampus [22] and cerebellum [36]. The literature also shows that HBO2 exposures of durations comparable to this study causes a decrease in cerebral blood flow (CBF) which would be correlated to reduction in endothelial NO activity and, hence, reduction in NOx − levels. We hypothesize that increases in regional brain and spinal cord levels of NOx − of HBO2 -exposed animals is indicative of an increase in neuronal NO function. Experiments are planned to determine the effects of selective neuronal, endothelial and inducible NOS-inhibitors on the regional brain and spinal cord HBO2 -induced increases in NOx − levels. HBO2 increases oxygen tension PO2 in blood but reduces blood flow by means of O2 -induced vasoconstriction [10]. The effect of HBO2 on cerebral blood flow is pressure-dependent. HBO2 therapy generally employs no higher than 2.5 ATA; atmospheres greater than 2.5 ATA enter the realm of O2 toxicity. O2 toxicity is attributed to overproduction of NO through increased neuronal and/or endothelial NOS activity. One possible mechanism is that increased NO causes overactivation of the glutamate–NMDA system, which is known to produce neurotoxicity [43]. Another mechanism involves NO-mediated increases in the cerebral blood flow, which causes hyperoxygenation of tissues with increased generation of highly reactive and toxic oxygen radicals [8]. There appears to be a key role of nitric oxide synthase (NOS) in the effects of HBO2 [40]. Exposure to HBO2 increases steady state levels of NO in the cerebral cortex (as measured by NO-sensitive electrodes in anesthetized rats); this increase in NO levels was inhibited by the neuronal NOS (nNOS) inhibitor 7-nitroindazole [38,39]. By contrast, in the present study, exposure to HBO2 in conscious rats significantly increased NOx − in selected brain regions. Exposure to HBO2 caused greater increase in periaortic levels of NO in wild-type and endothelial NOS (eNOS) knockout mice than in nNOS knockout mice, further underscoring the predominant role of nNOS activity in contributing to the total NO increase [38]. Brain in vivo microdialysis experiments in rats have also shown that HBO2 exposure markedly increased levels of NO2 − and NO3 − in dialysates from rat parietal cortex, hippocampus and corpus striatum; these increases were completely blocked by NOS-inhibitors [13,18,33]. Increased
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pressure alone failed to appreciably alter dialysate levels of NOx − [13]. NO has been implicated in adult neurogenesis in the mouse [25] and guinea pig hippocampus [21]. In addition a cGMP signaling system appears to be involved in neurite outgrowth and survival in hippocampal and dopaminergic neurons in vitro [11]. These studies suggest a role of NO in brain plasticity after injury. These processes might be involved where long-term HBO2 therapy appeared to reverse memory loss in chronic stroke and traumatic brain-injured patients [20,35]. In addition it should be noted that the experimental conditions of this study differ quite significantly from those in which oxygen toxicity relative to NO has been demonstrated at 5–6 ATA for 60–75 min but not at 4 ATA [9]. This is commensurate with our studies (2.5 ATA) in which we did not observe any signs which might have indicated any seizure effects during the 60-min HBO2 treatment. The finding that exposure to 100% oxygen alone (NBO2 ) for 60 min significantly lowered the NOx − levels in all brain regions and the spinal cord except in the hippocampus was contrary to what was anticipated. Why this effect occurred is not immediately clear. However, there is a superoxide (O2 − )/NO biological balance wherein changes in levels of one can influence levels and function of the other [5,12]. Both hyperoxia and O2 − have been shown to inactivate NO [31]. The abolition of the vasodilating effect of NO donors by HBO2 exposure suggests that an interaction between NO and O2 − might be responsible for the HBO2 -induced reduction in cerebral blood flow [8]. The data from this study supports the concept that the possible effects of HBO2 treatment in animal and clinical studies occurs via a complex set of biochemical reactions involving molecular oxygen and nNOS, resulting in the formation of nitric oxide [1]. The experimental HBO2 conditions of this study are within those used in earlier studies which demonstrated the synthesis of NO in vivo [13,39]. The results of the present study show that HBO2 exposure has a major effect on regional brain and spinal cord levels of NOx − . Compared to normobaric air, oxygen under normobaric conditions (NBO2 ) generally caused a reduction in tissue NOx − levels in all nervous tissues, more drastic in some tissues (cerebral cortex, spinal cord) than others. Exposure to compressed air at 2.5 ATA pressure (HBA) tended to reduce NOx − levels in all regions except the cerebral cortex and hippocampus. Exposure to 100% oxygen and 2.5 ATA pressure (HBO2 ) markedly increased NOx − in all areas; the increase was more acute in some tissues (corpus striatum, brainstem, cerebellum and spinal cord) than others (cerebral cortex and hippocampus). The findings of this research show that NO function in different nervous tissues may be differentially affected by exposure to HBO2 . Conflict of interest None.
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