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*Department of Intensive Care, Gunma University, School of Medicine, Gunma, Japan; and
Department of Anesthesiology, Gunma University, Graduate School of Medicine, Gunma, Japan
Address correspondence and reprint requests to Yuji Kadoi, MD, Department of Intensive Care, Gunma University, School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan. Address e-mail to kadoi{at}med.gunma-u.ac.jp
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Abstract |
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IMPLICATIONS: We examined whether selective inducible nitric oxide synthase inhibition can prevent the neurological changes induced by sepsis and found that it could not improve neurological dysfunction.
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Introduction |
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An increase in the amount of nitric oxide (NO) produced by the inducible form of NO synthase (iNOS) might play a central role in the pathophysiology of sepsis (4–6). An excess of NO contributes to severe hypotension and to vascular hyporeactivity to vasoconstrictor drugs, which finally causes MOF (4–6). In addition, NO plays a key role in the regulation of CBF and autoregulation, blood flow metabolism coupling, neurotransmission, memory formation, modulation of neuroendocrine functions, and behavioral activity (4–8). Szabo (7) noted in 1996 that peripheral systemic inflammatory conditions such as endotoxemia do not appear to cause major alterations in the expression of NOS isoforms in neural or glial elements (9), probably because of resistance of the blood-brain barrier to circulating cytokines. However, as in the blood vessels of the periphery, lipopolysaccharide (LPS) induces iNOS in the vascular smooth muscle cells of cerebrovascular blood vessels. In contrast, Wong et al. (10) demonstrated that iNOS could be induced in the vascular and neuronal structures of the rat brain by endotoxin. Harada et al. (11) have also reported that a large dose of LPS activates iNOS gene expression and NOS activity in the paraventricular nucleus. These reports suggest that an excess of NO mediated by iNOS plays a pivotal role in the pathogenesis of CNS dysfunction. We surmised that NO plays a role in CNS dysfunction during sepsis. The effects of NO might be exerted through reacting with oxidants to produce the reactive nitrogen intermediate peroxynitrite (OONO–) (4,12), which is a key cause of injury associated with increased NO production in shock or sepsis (4,12).
The cecal ligation and puncture (CLP) sepsis model described by Wichterman et al. (13) involves an endogenous septic focus, and shock develops over a prolonged time course. Our previous studies showed that the CLP model resembled the type of septic shock that can develop in humans (2,3). We evaluated the time course of changes in iNOS activity and examined whether NO is involved in the development of neurological dysfunction by using the CLP rat model of septic shock.
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Methods |
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Sepsis was induced by CLP as described by Wichterman et al. (13). Male Wistar rats were randomly divided into Group 1, control; Group 2, sham; Group 3, CLP; and Group 4, CLP + L-NIL [L-N6-(1-iminoethyl)-lysine, a selective iNOS inhibitor].
Rats (n = 10 per group) were anesthetized with pentobarbital (10 mg/kg intraperitoneally). A 2-cm midline abdominal incision was cut at the level of the cecum, which was then extracted and ligated with 5-0 silk just below the ileocecal valve. The cecum was punctured twice with a 22-gauge needle, and a small amount of cecal content was expressed. The perforated cecum was returned to the abdominal cavity, and the abdomen was sutured closed in 2 layers with 3-0 silk. This experimental CLP model resulted in 10%–20% lethality at 24 h (2,3).
CLP was omitted from the sham-operated model. We intraperitoneally injected L-NIL (4 mg/kg) in 1 mL of saline immediately after CLP to produce the CLP+ L-NIL group. Strunk et al. (14) reported that intraperitoneal injection of L-NIL (4 mg/kg) prevented hypotension and metabolic derangements in septic rats and completely blocked the immunohistochemical staining for nitrotyrosine in rat aorta. Moore et al. (15) reported that L-NIL has a 50% inhibitory concentration of 3.3 µM for mouse iNOS, compared with a 50% inhibitory concentration of 92 µM for rat brain constitutive NOS (cNOS), indicating that L-NIL is 28-fold more selective for iNOS. After the surgical procedure, all animals were housed in cages with access to food and water ad libitum.
Control, sham, and CLP rats were neurologically assessed at 1, 6, 12, and 24 h after the surgery and before the brain was removed. The Pinna reflex was assessed by lightly touching the auditory meatus of the ear to elicit a vigorous head shake (simple nonpostural somatomotor function). The corneal reflex was evaluated by lightly touching the cornea with a cotton swab to elicit a head shake (simple nonpostural somatomotor function). The paw or tail flexion reflex was assessed by briefly pinching (0.2 kg/mm2) the hindpaw or tail to elicit a withdrawal response (simple postural somatomotor function). The righting reflex was tested by placing the animal on its back and measuring the time taken to return to a spontaneous upright position (complex postural somatomotor function). The escape response was assessed by briefly pinching (0.2 kg/mm2) the tail of the animal to elicit locomotive activity away from the noxious stimulus (complex postural somatomotor function).
To exclude the influence of blood sampling on hemodynamic variables and neurological assessment, we also measured hemodynamic or biochemical variables in another series of animals (n = 10 per group). These animals were grouped and treated to represent the control, sham, and CLP animals described above. To extrapolate data from these to the experimental animals, the rats were exposed to identical experimental conditions for systemic measurements. Hemodynamics were measured by inserting a 2F high-fidelity micromanometer catheter into the right carotid artery and then advancing it into the left ventricle (LV), where it was secured. The position was confirmed by a characteristic decrease in diastolic blood pressure that occurred when the catheter was passed across the aortic valve into the LV cavity. The free ends of the catheter were tunneled subcutaneously into the dorsal neck. Catheters in the carotid artery were connected to a hemodynamic monitoring system (AP-601 G; Nihon Koden, Tokyo, Japan). The catheter was flushed with heparinized saline before use. We measured heart rate (HR), mean arterial blood pressure (MAP), LV end-diastolic pressure (LVEDP), and maximal rate of change in LV pressure (±dp/dt max) at 1, 6, 12, and 24 h after surgery.
We measured the plasma concentrations of lactate, glutamine oxaloacetate transaminase, glutamine pyruvate transaminase, ammonia, glucose, creatinine, pancreatic lipase, epinephrine (E), norepinephrine (NE), tumor necrosis factor (TNF)-
, and interleukin (IL)-1ß and the partial pressure of arterial blood gases at 1, 6, 12, and 24 h after the surgery in blood (total, 2.0 mL) collected from the carotid artery. The partial pressure of the arterial blood gases was analyzed by using an acid-base laboratory machine (ABL3; Radiometer, Copenhagen, Denmark). The plasma E and NE concentrations were measured by high-performance liquid chromatography. Plasma TNF- activity was quantified by measuring cytotoxicity against L929 cells in rabbit serum. Serum IL-1ß was quantified by using a competitive radioligand binding assay.
Plasma levels of nitrate and nitrite, which indicate the biosynthesis of NO, were measured at 1, 6, 12, and 24 h after surgery, as described previously (16). Samples collected in tubes containing EDTA were centrifuged at 4000g for 20 min. After plasma nitrate was reduced to nitrite by using nitrate reductase (670 mU/mL) and reduced nicotinamide adenine dinucleotide phosphate (160 µM) at room temperature for 16–17 h, the supernatant was decanted. Triplicate samples (100 µL per tube) were diluted to 1 mL with double-distilled water, and then 100 µL of fresh Greiss reagent (1% sulfanilamide in 5% concentrated H3PO4 acid and 0.1% naphthylethylenediamine dihydrochloride in H2O) was added. The nitrite concentration was measured at 550 nm against a standard curve of sodium nitrite.
The calcium-independent conversion of L-arginine to L-citrulline in cortex homogenates indicates iNOS activity (14). Brain cortex sections (n = 10 per group; n = 2 at 1 h; n = 2 at 6 h; n = 3 at 12 h; n = 3 at 24 h) at 1, 6, 12, and 24 h after surgery were scraped into 50 mM Tris-HCl containing 0.1 mM EDTA and 1 mM phenylmethylsulfonyl fluoride (pH 7.4) and homogenized in the same buffer on ice. Homogenates (50 µL) were added to 10-mL tubes (warmed to 37°C) that contained 100 µL of reaction buffer (10 µL of [3H]L-arginine; 150–200 cpm/pM), reduced nicotinamide adenine dinucleotide phosphate (1 mM), calmodulin (30 µM), tetrahydrobiopterin (5 µM), and EGTA (2 µM). The samples were incubated for 30 min at 37°C. The reaction was terminated by adding cold (4°C) stop buffer (pH 5.5), 100 mM HEPES, and 12 mM EDTA. Reaction mixtures were applied to columns containing Dowex 50 W (8% cross-linked; Na+ form), and eluted [3H]L-citrulline activity was measured with a liquid scintillation counter (Aloka 650; Aloka Co., Tokyo, Japan). Enzyme activity is expressed as femtomoles of L-citrulline produced per milligram of total protein per minute. Protein was measured by the Bradford method by using bovine serum albumin as the standard (Bio-Rad Inc., Richmond, CA).
The nitrotyrosine content in the cortex was assayed before and at 1, 6, 12, and 24 h after surgery by using standard sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and immunoblotting techniques as described previously (17). An anti-nitrotyrosine polyclonal antibody was purchased from Upstate Biotechnology (Lake Placid, NY).
The cortex was homogenized in ice-cold buffer (25 mM Tris-HCl [pH 7.5], 5 mM EDTA, 5 mM EGTA, 20 mg/mL leupeptin, 20 mg/mL benzamidine, and 1 mM phenylmethylsulfonyl fluoride) for 30 s with a Polytron and centrifuged for 30 min at 50,000g. The supernatant was centrifuged again at 137,000g. The pellet was resuspended in lysis buffer with 250 mM NaCl, homogenized, and centrifuged once again at 137,000g. Sodium chloride was added to a final concentration of 50 mM, and all samples were equilibrated with 0.5 mL of 50% (vol/vol) DEAE-Sephacel (pH 7.0) for 15 min on ice. The slurry was poured into small columns and eluted with 0.5 mL of lysis buffer. The eluate volume was reduced by filtration through a microconcentrator (Centricon 30; Amicon) to 100 µL, and then the protein concentration was measured by the Bradford method by using bovine serum albumin as the standard (Bio-Rad Inc., Richmond, CA) (2,3). For protein blotting, 20 µg of protein was resolved by SDS/polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose membranes in blotting buffer containing 25 mM Tris hydroxymethylaminomethane, 192 mM glycine, 0.02% SDS, and 20% methanol (pH 8.3) by using standard blotting wells (NA-1512; Nippon-Endo Inc., Tokyo, Japan). Nonspecific binding was blocked with 5% nonfat milk for 1 h at room temperature, and then blots were incubated with an anti-nitrotyrosine polyclonal antibody in Tris-buffered saline containing 0.05% Tween 20 diluted 1:500. The level of immunoreactivity was detected after sequential incubations with biotinylated goat anti-rabbit immunoglobulin G, streptavidin-biotinylated alkaline phosphatase complex, and alkaline phosphatase color development buffer. Incu- bation and washes proceeded according to the manufacturer’s recommendations (Bio-Rad Inc., Hercules, CA). The relative densities of the blots were determined by using an image analyzer (Personal Densitometer; Molecular Dynamics Inc., Sunnyvale, CA).
All data are presented as arithmetic means ± SD. After equal variance among groups was confirmed with the Bartlett test, analysis of variance with multiple comparisons was performed. Scheffé’s method compared means. Statistical significance was established at P < 0.05. All statistical analyses were performed with the software StatView 5.0 (Abacus Concepts, Berkeley, CA).
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Results |
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and IL-1ß did not appear to differ in the control and sham groups throughout the study. TNF- was increased at 6 h in the CLP and CLP + L-NIL groups and peaked at 12 h after the surgery. IL-1ß was increased at 12 h and peaked at 24 h after the surgery. Table 3 shows the neurological assessment of the four groups at 24 h. There were no changes in consciousness reflex in the four groups at 1, 6, or 12 h (data not shown). The consciousness reflex significantly decreased in the CLP and CLP + L-NIL groups at 24 h after the surgery.
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Discussion |
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We found that the hemodynamic changes in the CLP rat consisted of an early hyperdynamic phase, followed by a hypodynamic phase during late sepsis. Tang and Liu (18) reported that the hyperdynamic phase was characterized by increases in body temperature, HR, cardiac output, and LV +dp/dt max, whereas the hypodynamic phase was characterized by decreases in body temperature, HR, MAP, cardiac output, LV +dp/dt max, and –dp/dt max. Although we did not measure cardiac output in this study, the hemodynamic changes that we observed were consistent with those reported by Tang and Liu (18). In addition, the changes observed in this study closely resembled the biphasic septic syndrome seen in clinical sepsis. Therefore, the CLP model is suitable for evaluating the development of septic shock in humans.
The increased levels of circulating nitrite/nitrate during sepsis indicate that NO is involved in the cardiovascular alterations associated with septic shock (4–7). Strunk et al. (14) reported that plasma nitrite/nitrate concentration in rats increased up to fourfold at six hours after an intraperitoneal injection of an endotoxin. Schwartz et al. (19) showed that NO levels were increased in rats exposed to LPS. In contrast, Vromen et al. (20) reported that CLP caused increased plasma nitrite/nitrate levels (up to 59 µM at 24 hours) and that the degree of NO production was approximately 30% of that observed several hours after LPS administration. The NO concentration identified in the present study peaked 12 hours after surgery at levels that were lower than those in other reports. This might be attributable to the differences in species and in the septic shock model. Javeshghani and Magder (16) found no changes in the NO concentration after LPS injection in pigs and concluded that caution should be used when the septic response in rodents is extrapolated to other species.
Mice lacking iNOS are resistant to endotoxin-induced mortality and vascular hypocontractility (21,22), supporting the notion that iNOS plays a key role in endotoxin shock. Ruetten et al. (23) reported that endotoxemia for six hours was associated with a significant increase in iNOS activity in the lung and liver, which could be reduced by an iNOS inhibitor. Strunk et al. (14) reported that selective iNOS prevented hypotension and metabolic derangements in septic rats without affecting endothelium-dependent vasodilation. The activity of iNOS in the brain is controversial. Van Dam et al. (9) reported that the brain cells of rats treated with endotoxin were not iNOS positive. In contrast, Wong et al. (10) demonstrated that endotoxin induced iNOS in neuronal structures of the rat brain. Satta et al. (24) demonstrated a significant increase in iNOS in the hypothalamus and pituitary after various doses of endotoxin. Moreover, Harada et al. (11) reported that a large dose of LPS activated iNOS gene expression and NOS activity in the paraventricular nucleus. Our findings were consistent with these reports. In addition, the selective iNOS inhibitor (L-NIL) prevented iNOS activity from increasing in the brain and restored the hemodynamics induced by sepsis at 12 and 24 hours after CLP. Our data reinforced the notion that a large amount of NO in the brain is induced by an increase in iNOS activity.
OONO–, which is formed from the reaction between NO and superoxide, can cause diffuse cellular damage by nitrating important cellular proteins and by breaking DNA strands, which in turn activates poly(adenosine diphosphate ribose) synthetase (4,12). OONO– is difficult to measure, but because it interacts with tyrosine residues to form nitrotyrosine, this molecule represents evidence of OONO– formation. Strunk et al. (14) immunohistochemically identified a substantial increase in nitrotyrosine levels in the vicinity of large blood vessels at six hours after LPS administration. The present study is the first to evaluate OONO– production in the brain during sepsis. Substantial evidence indicates that the effects of prolonged NO overproduction are partially mediated by the highly cytotoxic agent OONO–. Szabo (4) demonstrated that OONO– causes DNA strand breakage with the subsequent suicide activation of poly(adenosine triphosphate ribosyltransferase) and showed that, during endotoxemia, poly(adenosine triphosphate ribosyltransferase) inhibition improves vascular reactivity and prolongs survival. We found that a selective iNOS inhibitor prevented the OONO– level from increasing in the brain, which was consistent with the findings of Szabo (4) and Strunk et al. (14). However, the selective iNOS inhibitor did not improve the neurological dysfunction induced by sepsis. These findings indicate that iNOS and nitrotyrosine accumulation do not play key roles in sepsis-induced CNS dysfunction. How CNS dysfunction is induced by sepsis remains unknown (2,3). Papadopoulos et al. (25) noted that the most immediate and serious complication of septic encephalopathy is impaired consciousness, for which the patient may require ventilation.
The etiology of septic encephalopathy involves reduced CBF and oxygen extraction by the brain, cerebral edema, and disruption of the blood-brain barrier. These may arise from the action of inflammatory mediators on the cerebrovascular endothelium, abnormal neurotransmitter composition of the reticular activating system, impaired astrocyte function, and neuronal degeneration. Voigt et al. (26) reported that short-term oxidative stress might be an important factor in the development of septic encephalopathy, possibly through dysregulation of the blood-brain barrier. Further study is required to clarify the mechanisms involved in NO formation and neurological dysfunction during sepsis.
Vincent et al. (6) reviewed the role of three major NOS isoforms—iNOS, cNOS, and endothelial NOS—in septic shock and suggested that NO produced by iNOS or cNOS can have both beneficial and detrimental effects on many organ systems in sepsis. Attempts to nonselectively block all its actions may therefore not yield positive results on outcome. In this study, we examined the iNOS activity and the effects of a selective iNOS inhibitor on neurological dysfunction induced by sepsis.
Some investigators see neurologic dysfunction as an early sign of sepsis. In this study, the neurologic signs appeared much later (24 hours) than the hemodynamic changes. It remains controversial whether hemodynamic changes, especially alterations in CBF, could potentially contribute to septic encephalopathy (1,2,25). Papadopoulos et al. (25) stated that although CBF is reduced to 
62% of normal in septic patients, this decrease in CBF does not appear to be enough to threaten neuronal viability or to cause electroencephalographic changes.
Strunk et al. (14) found that OONO– formed at six hours after LPS administration in parallel with an increase in the plasma NO concentration. Why the time courses of peak NO concentration and nitrotyrosine formation differ remains unknown. However, Liu et al. (27) reported that OONO– is formed at least six hours after spinal cord injury, whereas the NO concentration increases immediately after spinal cord injury.
In conclusion, selective iNOS inhibition restores the hemodynamic changes induced by sepsis but does not improve neurological dysfunction.
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Acknowledgments |
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The authors thank Forte Inc. (Tokyo, Japan) for assistance with the manuscript.
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References |
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-aminobutyric acid receptor system in experimentally induced septic shock in rats. Crit Care Med 1996; 24: 298–305.[ISI][Medline]
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