Abstract
Convulsions and encephalopathy are frequent complications of childhood shigellosis. We studied the role of nitric oxide (NO) in Shigella-related seizures in an animal model. Pretreatment of mice with Shigella dysenteriae 60R sonicate elevated serum NO levels and enhanced the convulsive response to pentylenetetrazole (PTZ), as indicated by a higher mean convulsion score and a higher number of mice responding with seizures. Treatment of the mice with S-methylisothiourea sulfate (SMT), a potent inhibitor of inducible NO synthase (NOS), prevented the elevation of serum NO levels and concomitantly reduced the enhanced response to PTZ. The mean convulsion scores were 0.7, 0.7, 1.3, and 0.8 for mice treated with saline, saline and SMT, S. dysenteriae 60R sonicate, and S. dysenteriae 60R sonicate with SMT, respectively (P = 0.001 for 60R sonicate versus saline and P = 0.013 for 60R sonicate versus 60R sonicate with SMT). The corresponding seizure rates were 40, 44, 75, and 47% for saline, saline with SMT, S. dysenteriae 60R sonicate, and S. dysenteriae 60R sonicate with SMT, respectively (P = 0.0004 for 60R sonicate versus saline and P = 0.005 for 60R sonicate versus 60R sonicate with SMT). In contrast, injection of N-nitro-l-arginine, a selective inhibitor of constitutive NOS, neither abolished the elevation of serum NO nor attenuated the enhancement of seizures. These findings indicate that NO, induced by S. dysenteriae 60R sonicate, is involved in enhancing the susceptibility to seizures caused by S. dysenteriae.
Shigellosis, the acute gastroenteritis caused by Shigella species, is often accompanied by neurologic complications (1, 2, 5). The most frequent complications are convulsions and encephalopathy, which can be fulminant, leading rapidly to unconsciousness and death (11). Typically, the neurologic disorders appear very early in the course of the disease, often before the onset of diarrhea (2, 11). Neurologic complications have also been reported in infections caused by certain enterohemorrhagic Escherichia coli strains (14, 17).
The pathogenesis of Shigella- and E. coli-associated neurologic disturbances is unclear. Shiga toxin (ST), the main toxic product of Shigella dysenteriae, and two very similar toxins, Shiga-like toxins (SLTs) 1 and 2, produced by certain E. coli strains (for review, see reference 24), have been implicated because of their neurotoxicity in laboratory animals (3, 4, 7, 8, 15, 27).
Recent data indicate that lipopolysaccharide (LPS) acts in concert with ST and SLTs in pathological processes. Barrett et al. showed that the toxicity of SLT in mice was macrophage dependent (3) and that LPS either increased or decreased SLT toxicity in mice and rabbits depending on the time of its application (4). In a comparison of LPS-responding and LPS-nonresponding mice infected with either SLT-producer or SLT-nonproducer E. coli strains, Karpman et al. observed the most severe systemic manifestations in the LPS-responding mice inoculated with SLT-producing E. coli (17).
Our team, using a model of pentylenetetrazole (PTZ)-induced seizures, had previously shown that preinjection of mice with crude preparations of S. dysenteriae 60R (a producer of ST) or with E. coli H-30 (a producer of SLT) reduced the threshold to PTZ-induced seizures (34). The increased sensitivity to PTZ could be mimicked by pretreating the mice with ST together with LPS, but not with either of them alone (34). Employing this model, we have further demonstrated that tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) play an important role in the enhanced seizure response of mice to PTZ after administration of S. dysenteriae (33).
LPS, TNF-α, and IL-1β themselves (12, 30), as well as ST, as we had shown previously (32), induce another host mediator—nitric oxide (NO). NO is well recognized as an important messenger in the peripheral and central nervous systems (6, 10). In the brain, NO plays an essential role in the control of blood flow. As an excitatory neurotransmitter involved in synaptic plasticity, it influences complex neural functions, such as brain development, memory formation, and behavior. Overproduction of NO, however, has been linked to neurotoxicity during ischemia, some forms of neurodegenerative brain diseases, and induction of seizures (10).
NO is produced in many cell types and organs by NO synthases (NOSs), which convert l-arginine to l-citrulline and NO. There are two types of NOSs: a constitutive NOS (cNOS), which is regulated by changes in intracellular calcium; and an inducible NOS (iNOS), which is stimulated during infection and inflammatory processes (21). Both types are present in the brain: in endothelial cells and certain neurons, NO is catalyzed by constitutive endothelial or brain NOSs, and in microglia and astrocytes, it is catalyzed by iNOS in response to LPS, IL-1β, and gamma interferon (9, 19).
Results of studies on the role of NO in convulsions have been contradictory, indicating either anticonvulsive or proconvulsive activity, depending on the model employed (18, 29). These studies examined the role of NO produced by cNOS, but not under conditions in which increased NO levels are achieved by stimulation of iNOS.
In the present study, we employed the PTZ-induced seizure model to investigate the role of NO induced by S. dysenteriae in the enhanced susceptibility to seizures after S. dysenteriae administration shown in our previous study (34).
(This work was presented in part at the 39th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, Calif., September 1999.)
MATERIALS AND METHODS
Mice.
ICR outbred male mice 25 to 28 days old (20 to 25 g) were maintained under standard conditions.
Materials.
PTZ, S-methylisothiourea sulfate (SMT), N-nitro-l-arginine (NNA), and flavin adenine dinucleotide disodium salt (FAD) were purchased from Sigma Chemical Co. (St. Louis, Mo.). Aspergillus nitrate reductase, NADPH, lactate dehydrogenase (LDH [bovine muscle]), and pyruvic acid sodium salt were purchased from Boehringer (Mannheim, Germany).
Preparation of bacterial sonicate.
Strain 60R of S. dysenteriae serotype 1 was grown in syncase broth for 48 h with shaking, lysed by sonication, and filter sterilized as described previously (26). The bacterial sonicate was analyzed for protein content, cytotoxic activity, and lethality in mice (34).
PTZ-induced convulsion.
Induction of seizures with PTZ was performed as described previously (34). Groups of six to eight mice were inoculated intraperitoneally (i.p.) with PTZ (50 mg/kg of body weight) and observed for their reaction for 10 min. The reaction included several phases: unresponsiveness, mild contractions, clonic seizures, and tonic seizures (forelegs and then hind legs rigidly extended to the rear), occasionally followed by death. For statistical analysis, each phase was given a numeric score (23): unresponsiveness = 0, mild contractions = 1, clonic seizures = 2, tonic seizures = 3, and death = 4. The response of each mouse was scored according to the highest phase reached, and a mean seizure severity score was calculated for each group.
Enhancement of PTZ-induced seizures.
Groups of six to eight mice were inoculated with 1,000 50% cytotoxic doses (∼4 50% lethal doses [LD50]) of S. dysenteriae 60R sonicate. Saline-treated mice were used as controls. At 7 h after injection, the mice were inoculated with PTZ (50 mg/kg) and scored for their response compared with that of controls (33, 34).
NO measurement.
An indirect determination of NO was performed by measuring the combined amounts of the stable products of NO, nitrite (NO2), and nitrate (NO3), in the mouse serum. Nitrate was first reduced to nitrite by using Aspergillus nitrate reductase, according to the procedure previously described (13). Nitrite levels were then determined by a colorimetric method whereby equal volumes of sample serum and Griess reagent (1% sulfanilamide and 0.1% naphthyl-ethylenediamine in 2% H3PO4) were mixed. The A540 was measured, and nitrite concentrations were calculated from a standard curve of NaNO2.
Treatment with NOS inhibitors.
SMT was injected i.p. at a dose of 2.0 mg/kg of body weight. Groups of six to eight mice were injected with S. dysenteriae 60R sonicate or saline and subsequently treated with SMT or saline 2 h later. At 5 h after injection of SMT (or saline), the mice were treated with PTZ and scored for their response, as described above. NNA was injected i.p. at a dose of 2.5 mg/kg of body weight. Groups of six to eight mice preinjected with S. dysenteriae 60R sonicate or saline were treated with NNA or saline 3, 5, and 6.5 h later. At 30 min after injection of the final NNA (or saline) dose, the mice were treated with PTZ, as described above.
Statistical analysis.
The difference in the incidence of seizures among the various groups in each experiment was analyzed by chi-square test for multiple comparisons or by Fisher's exact test, as appropriate. Convulsion scores were compared by two-tailed unpaired t test or one-way analysis of variance, as appropriate.
RESULTS
Induction of NO production by S. dysenteriae 60R sonicate.
An i.p. injection of S. dysenteriae 60R sonicate rapidly induced NO in mouse circulation. An increase in the concentration of the two stable products of NO, NO−2 and NO−3, was detectable 3 h after injection, peaking at 5 h and returning to baseline levels at 24 h (Fig. 1). The mean ± standard error (SE) serum NO levels 1, 3, 5, 8, and 24 h after 60R sonicate administration were 88 ± 10, 141 ± 46, 650 ± 218, 167 ± 29, and 63 ± 47 μM, respectively.
FIG. 1.
Serum NO production in response to S. dysenteriae 60R sonicate (4 LD50 i.p.). Measurement of the stable products of NO, NO−2, and NO−3, was performed by using nitrate reductase and the colorimetric assay with Griess reagent, as described in Materials and Methods. Each point represents the average of three animals.
Inhibition of NO production by inhibitors of NOSs.
Production of NO in response to S. dysenteriae 60R sonicate was inhibited by injection of the potent iNOS inhibitor SMT (31) (2 mg/kg) 2 h after bacterial administration. The mean ± SE serum NO levels 7 h after 60R sonicate injection were 150 ± 25, 90 ± 13, 535 ± 15, and 90 ± 15 μM for mice treated with saline alone, saline with SMT, 60R sonicate with saline, or 60R sonicate followed by SMT, respectively (P = 0.001 for 60R sonicate versus saline, and P = 0.0001 for 60R sonicate versus 60R sonicate with SMT) (Fig. 2A). There was no similar significant reduction of NO production following treatment with the highly selective brain and endothelial cNOS inhibitor NNA (22) (2.5 mg/kg) (Fig. 2B).
FIG. 2.
Effect of treatment with NOS inhibitors on serum NO levels 7 h after S. dysenteriae administration. Mice were injected (i.p.) with saline alone, saline with NOS inhibitor, S. dysenteriae 60R sonicate with saline, or S. dysenteriae 60R sonicate with NOS inhibitor. Each point represents the mean of five determinations. (A) Mice treated with SMT. P = 0.001 for S. dysenteriae sonicate versus saline and P = 0.0001 for S. dysenteriae sonicate versus S. dysenteriae sonicate with SMT. SMT (2 mg/kg) was administered 2 h after S. dysenteriae sonicate. (B) Mice treated with NNA. P = 0.0001 for S. dysenteriae sonicate versus saline and is nonsignificant for S. dysenteriae sonicate versus S. dysenteriae sonicate with NNA-treated mice. NNA (2.5 mg/kg) was administered 3, 5, and 6 1/2 h after S. dysenteriae 60R sonicate.
Effect of NOS inhibitors on enhancement of PTZ-induced seizures by S. dysenteriae.
Pretreatment of mice with S. dysenteriae 60R sonicate enhanced the seizure response to PTZ as early as 7 h after administration. This was indicated by a higher mean convulsion score (Fig. 3) as an indication of the severity of seizures and a higher number of mice responding with seizures compared with control (saline-pretreated) mice (Table 1).
FIG. 3.
Effect of treatment with NOS inhibitors on the enhanced severity of PTZ-induced seizures by S. dysenteriae 60R sonicate. Mice were injected (i.p.) with saline alone, saline with NOS inhibitor, S. dysenteriae 60R sonicate with saline, or S. dysenteriae 60R sonicate with NOS inhibitor; PTZ was injected (i.p.) at 7 h. (A) Mice treated with SMT. P = 0.001 for S. dysenteriae sonicate (n = 53) versus saline (n = 57), and P = 0.013 for S. dysenteriae sonicate versus S. dysenteriae sonicate with SMT (n = 51). SMT (2 mg/kg) was administered 2 h after S. dysenteriae 60R sonicate. (B) Mice treated with NNA. NNA (2.5 mg/kg) was administered 3, 5, and 6 1/2 h after S. dysenteriae 60R sonicate.
TABLE 1.
Effect of treatment with SMT and NNA on S. dysenteriae 60R sonicate-enhanced rate of PTZ-induced seizures
| Treatmenta
|
No. of mice with seizures/ no. tested (%) at +7 hb | P | ||||
|---|---|---|---|---|---|---|
| 0 h | +2 h | +3 h | +5 h | +6 1/2 h | ||
| Saline | Saline | 23/57 (40) | ||||
| Saline | SMT | 26/59 (44) | ||||
| S. dysenteriae 60R sonicate | Saline | 40/53 (75) | 0.0004c | |||
| S. dysenteriae 60R sonicate | SMT | 24/51 (47) | 0.005d | |||
| Saline | Saline | Saline | Saline | 7/16 (44) | ||
| Saline | NNA | NNA | NNA | 6/16 (38) | ||
| S. dysenteriae 60R sonicate | Saline | Saline | Saline | 13/16 (81) | 0.03c | |
| S. dysenteriae 60R sonicate | NNA | NNA | NNA | 15/16 (94) | 0.3e | |
SMT (2.0 mg/kg) and NNA (2.5 mg/kg) were injected i.p. after administration of S. dysenteriae sonicate at the time points indicated. PTZ (50 mg/kg) was injected i.p. 7 h after S. dysenteriae sonicate.
Data are presented as the number of mice responding with seizures (score ≥1)/number of mice tested.
S. dysenteriae 60R sonicate versus saline-treated mice.
S. dysenteriae 60R sonicate versus S. dysenteriae 60R sonicate- and SMT-treated mice.
S. dysenteriae 60R sonicate- versus S. dysenteriae 60R sonicate- and NNA-treated mice.
To determine whether NO, which is induced after S. dysenteriae sonicate administration, contributes to the enhanced response to PTZ, mice were treated with selective inhibitors of NOSs. Injection of SMT, the iNOS inhibitor (2.0 mg/kg), 2 h after injection of bacterial sonicate significantly reduced the enhanced response to PTZ (Fig. 3A). The mean ± SE convulsion scores were 0.7 ± 0.12, 0.7 ± 0.10, 1.3 ± 0.13, and 0.8 ± 0.12 for mice treated with saline, saline with SMT, 60R sonicate, and 60R sonicate followed by SMT, respectively (P = 0.001 for 60R sonicate versus saline and P = 0.013 for 60R sonicate versus 60R sonicate with SMT). The corresponding seizure rates for the four groups (Table 1) were 40% (23 of 57 saline-treated mice), 44% (26 of 59 saline- and SMT-treated mice), 75% (40 of 53 60R sonicate-treated mice) and 47% (24 of 51 of mice treated with 60R sonicate and SMT; P = 0.0004 for 60R sonicate versus saline and P = 0.005 for 60R sonicate versus 60R sonicate with SMT).
In contrast to the results obtained with SMT, treatment of the mice with NNA, the cNOS inhibitor (2.5 mg/kg) at 3, 5, and 6.5 h after S. dysenteriae 60R sonicate administration did not attenuate the increased response to PTZ (Fig. 3B). The mean ± SE convulsion scores for mice treated with saline, saline with NNA, 60R sonicate, and 60R sonicate followed by NNA were 0.8 ± 0.25, 0.6 ± 0.17, 1.5 ± 0.20, and 1.6 ± 0.15, respectively. The higher number of mice responding with seizures was also not affected by treatment with NNA (Table 1). The corresponding seizure rates were 44% (7 of 16 saline-treated mice), 38% (6 of 16 saline- and SMT-treated mice), 81% (13 of 16 60R sonicate-treated mice), and 94% (15 of 16 mice treated with 60R sonicate and NNA).
DISCUSSION
Our data show that treatment of mice with the iNOS inhibitor SMT completely prevents the elevation of NO levels in serum after S. dysenteriae sonicate administration and concomitantly reduces the enhanced response to PTZ. In contrast, NNA, an inhibitor of brain and endothelial cNOS, neither abolished the induction of NO nor attenuated the PTZ enhancement of the seizures. These findings strongly imply that NO, which is induced by exposure to the S. dysenteriae 60R sonicate, plays an important role in the sensitization of the central nervous system to convulsive activity.
NO has been linked to epileptic activity through the formation of cyclic GMP (cGMP). Stimulation of the brain N-methyl-d-aspartate (NMDA) receptors with glutamate or excitatory amino acids increases calcium influx, which results in activation of cNOS and formation of NO. NO in turn activates guanylate cyclase to synthesize cGMP, which is assumed to initiate seizures (10).
NO modulates experimentally induced seizures in a complex manner. In addition to stimulation of cGMP, NO has several other actions: it blocks NMDA receptors in a negative feedback manner, thereby attenuating excitable activity (20); promotes and suppresses glutamate release (25); and reduces the receptor activity of the inhibitory γ-aminobutyric acid (GABA) neurotransmitter (28). PTZ induction of seizures may be related to the antagonistic activity of the compound at the GABA-A receptor and to its activation of the NMDA receptor (16). Thus, the induced NO may augment the capability of PTZ to induce seizures at both pathways.
The various investigations of the role of NO in epileptic activity employed mainly proconvulsive drugs and inhibitors of NOS. The results were contradictory, indicating both an anticonvulsive role and a proconvulsive role of NO, depending on the model used (18, 29). Some of the authors postulated that multiple factors, such as the specific proconvulsive drug, the type of NOS inhibitor, their concentrations, mode of application, and specific strain or species used, affect the results. These studies, however, evaluated the involvement of NO produced by cNOS during epileptic activity. To the best of our knowledge, ours is the first study to demonstrate a role for NO produced by iNOS in the induction of seizures.
We found that NO induced by S. dysenteriae sonicate is proconvulsive. However, the relevance of S. dysenteriae-induced NO elevation to other infectious diseases associated with seizures is as yet unclear. In our previous study, we found that the enhancement of seizures is a result of mutual actions of ST and LPS. Injection of LPS alone, which also induced high levels of NO, was not sufficient for a significant increase in convulsion rates (34). Thus, it is possible that like in earlier studies of cNOS-produced NO in seizure modulation, which had different results according to the model used, the exact role of NO produced by iNOS is determined by the nature of the infection. Multiple factors, such as the specific pathological processes, the interaction of NO with other induced factors, and the amount or site of NO release, may all determine whether NO is proconvulsive or anticonvulsive.
The exact mode of interaction between ST and LPS that renders the central nervous system vulnerable to convulsive activity is unclear. Our results showed that their activity is at least partly mediated by NO induction. ST is transported very rapidly to the central nervous system, where it binds to endothelial and neuron cells and exerts cytotoxic activity (7, 8, 27). We speculate that NO, which is itself neurotoxic, not only modulates seizures in various mechanisms, but may also augment cell damage.
In conclusion, using the PTZ-induced seizure mouse model, we have shown that NO, which is induced by S. dysenteriae, plays a proconvulsive role in the process that leads to the enhancement of PTZ-induced seizures caused by administration of S. dysenteriae. Similar mechanisms may be involved in the neurologic manifestations of human shigellosis, enterohemorrhagic E. coli infection, and possibly other infectious diseases.
ACKNOWLEDGMENTS
We thank Charlotte Sachs and Gloria Ginzach of the Editorial Board, Rabin Medical Center, Beilinson Campus, for assistance.
This study was supported by Tel Aviv University Research Foundation grant 572/96.
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