Caffeine Inhibits Activation of the NLRP3 Inflammasome via Autophagy to Attenuate Microglia-Mediated Neuroinflammation in Experimental Autoimmune Encephalomyelitis.

The activation of microglia is an important cause of central nervous system (CNS) inflammatory cell infiltration and inflammatory demyelination in experimental autoimmune encephalomyelitis (EAE). Furthermore, the proinflammatory response induced by the NLR family pyrin domain containing 3 (NLRP3) inflammasome can be amplified in microglia after NLRP3 inflammasome activation. Autophagy is closely related to the inflammatory response. Caffeine exerts anti-inflammatory and autophagy-stimulating effects, but the specific mechanism remains unclear. This study examined the mechanism underlying the anti-inflammatory effect of caffeine on EAE. In this study, C57BL/6 mice were immunized to induce EAE and treated with caffeine to observe its effect on prognosis. The effects of caffeine on autophagy and inflammation were also analysed in mouse primary microglia (PM) and the BV2 cell line. The data demonstrated that caffeine reduced the clinical score, the infiltration of inflammatory cells, the demyelination level, and the activation of microglia in EAE mice. Furthermore, caffeine increased the LC3-II/LC3-I levels and decreased the NLRP3 and P62 levels in EAE mice, whereas the autophagy inhibitor 3-methylamine (3-MA) blocked these effects. In vitro, caffeine promoted autophagy by suppressing the mechanistic target of rapamycin (mTOR) pathway and inhibited activation of the NLRP3 inflammasome. However, autophagy-related gene 5 (ATG5)-specific siRNA abolished the anti-inflammatory effect of caffeine treatment in PM and BV2 cells. Taken together, these data suggest that caffeine exerts a newly discovered effect on EAE by reducing NLRP3 inflammasome activation via the induction of autophagy in microglia.

MBNL1 reverses the proliferation defect of skeletal muscle satellite cells in myotonic dystrophy type 1 by inhibiting autophagy via the mTOR pathway.

Skeletal muscle atrophy is one of the clinical symptoms of myotonic dystrophy type 1 (DM1). A decline in skeletal muscle regeneration is an important contributor to muscle atrophy. Skeletal muscle satellite cells (SSCs) drive skeletal muscle regeneration. Increased autophagy can reduce the proliferative capacity of SSCs, which plays an important role in the early regeneration of damaged skeletal muscle in DM1. Discovering new ways to restore SSC proliferation may aid in the identification of new therapeutic targets for the treatment of skeletal muscle atrophy in DM1. In the pathogenesis of DM1, muscleblind-like 1 (MBNL1) protein is generally considered to form nuclear RNA foci and disturb the RNA-splicing function. However, the role of MBNL1 in SSC proliferation in DM1 has not been reported. In this study, we obtained SSCs differentiated from normal DM1-04-induced pluripotent stem cells (iPSCs), DM1-03 iPSCs, and DM1-13-3 iPSCs edited by transcription activator-like (TAL) effector nucleases (TALENs) targeting CTG repeats, and primary SSCs to study the pathogenesis of DM1. DM1 SSC lines and primary SSCs showed decreased MBNL1 expression and elevated autophagy levels. However, DM1 SSCs edited by TALENs showed increased cytoplasmic distribution of MBNL1, reduced levels of autophagy, increased levels of phosphorylated mammalian target of rapamycin (mTOR), and improved proliferation rates. In addition, we confirmed that after MBNL1 overexpression, the proliferative capability of DM1 SSCs and the level of phosphorylated mTOR were enhanced, while the autophagy levels were decreased. Our data also demonstrated that the proliferative capability of DM1 SSCs was enhanced after autophagy was inhibited by overexpressing mTOR. Finally, treatment with rapamycin (an mTOR inhibitor) was shown to abolish the increased proliferation capability of DM1 SSCs due to MBNL1 overexpression. Taken together, these data suggest that MBNL1 reverses the proliferation defect of SSCs in DM1 by inhibiting autophagy via the mTOR pathway.

Clinical Characteristics and Functional Outcomes of Wake-Up Stroke in the Chinese Population.

BACKGROUND: Wake-up stroke (WUS) accounts for up to 25% of all new ischemic strokes, but debate exists regarding whether WUS differs from non-WUS in previous studies. Our study aimed to investigate the proportion of WUS cases and to examine differences in clinical characteristics and outcomes in these two groups. METHODS: Data from acute ischemic stroke patients who presented to the First Affiliated Hospital of Chongqing Medical University between April 2017 and September 2017 were prospectively collected. Admission demographic information, clinical and radiological characteristics, and 3-month functional outcomes were assessed and compared between patients with WUS and those with non-WUS. Poor functional outcome was defined as modified Rankin Scale ≥ 3 at the 90-day follow-up. Risks of poor outcomes for WUS were estimated with logistic regression analysis. RESULTS: A total of 473 eligible patients were included, of which 132 had been diagnosed with WUS (27.9%). Forty WUS patients had poor functional outcomes and 92 WUS patients had good functional outcomes. WUS and non-WUS patients were similar in regard to stroke risk factors, severity, etiology, and prognosis at 90 days (p > 0.05), but WUS patients were more likely to have had previous stroke (p < 0.001) and a tendency of higher albumin levels (p = 0.051). WUS patients show significant differences in terms of age, gender, prior stroke, atrial fibrillation, impaired consciousness at admission, levels of albumin and triglycerides, stroke severity, and stroke etiology between the good outcome group and the poor outcome group (p < 0.05). Multivariate logistic regression analysis showed that age (odds ratio [OR] 1.079, 95% confidence interval [CI] 1.021-1.141; p < 0.05), previous stroke (OR 4.017, 95% CI 1.197-13.484; p < 0.05), and admission National Institutes of Health Stroke Scale (NIHSS) score ≥5 (OR 5.453, 95% CI 1.510-19.696; p < 0.05) were independently associated with an unfavorable outcome of WUS. CONCLUSIONS: WUS accounts for 27.9% of 473 ischemic strokes in the Chinese population. WUS and non-WUS patients were similar in terms of stroke risk factors, severity, etiology, and early outcomes. Age, previous stroke, and a high admission NIHSS score were independent risk factors for unfavorable outcomes in patients with WUS.

A Novel Scale for Assessment of Stroke Severity at Symptom Onset: Correlation With Neurological Deterioration and Outcome.

Objective: To propose a novel scale for the assessment of stroke severity at symptom onset and to investigate whether it is associated with ultra-early neurological deterioration (UND) and functional outcomes. Methods: The Chongqing Stroke Scale (CQSS) was constructed based on key aspects of history, emphasizing language, motor function, and level of consciousness to yield a total 0-11 scale. The diagnostic performance of the CQSS was assessed in 215 ischemic stroke patients between June 2017 and October 2017 in a tertiary hospital. Patients were included if they presented within 24 h after onset of symptoms and they or their witness can recall the scenario at symptom onset. UND was defined as an increase ≥2 points on the CQSS between symptom onset and admission. Functional outcomes were assessed using the 3-month modified Rankin scale. The correlation between the CQSS score and baseline National Institutes of Health Stroke Scale (NIHSS) score was assessed. The sensitivity, specificity, and positive and negative predictive values of CQSS for the outcomes were calculated. Logistic regression was used to test the association between the CQSS score and functional outcomes. Results: A total of 215 patients with available CQSS scores were included. Baseline CQSS scores at symptom onset were correlated with the admission NIHSS score (r = 0.56, p < 0.001) and functional outcome at 3 months (r = 0.47, p < 0.001). Baseline CQSS ≥ 6 was an independent predictor of functional outcome at 3 months (odds ratio, 12.61; 95% confidence interval 5.68-27.97, p < 0.001). UND was observed in 20 (9.30%) patients. The 90-day mortality was significantly higher in patients with UND than those without UND (25.0 vs. 8.2%, p < 0.001). After adjusting for age, admission systolic blood pressure, hypertension, and diabetes, UND independently predicted poor functional outcome in the multivariate logistic regression model (odds ratio, 9.69; 95% confidence interval 3.19-29.45, p < 0.001). Conclusions: The newly developed CQSS is a simple and easy-to-perform scale that allows a quantitative evaluation of the stroke severity at symptom onset and an assessment of UND before hospital admission. It is associated with NIHSS and predicts functional outcome in patients with acute ischemic stroke.

Total Small Vessel Disease Burden Predicts Functional Outcome in Patients With Acute Ischemic Stroke.

Background: Cerebral small vessel disease (SVD) is generally considered as a cause of stroke, disability, gait disturbances, vascular cognitive impairment, and dementia. The aim of this study was to investigate whether the total SVD burden can be used to predict functional outcome in patients with acute ischemic stroke. Methods: From April 2017 to January 2018, consecutive patients with acute ischemic stroke who underwent baseline MRI scan were evaluated. The functional outcome was assessed using the modified Rankin Scale (mRS) at 90 days and defined as i) excellent outcome (mRS ≤ 1) and ii) good outcome (mRS ≤ 2). Brain MRI was performed and assessed for lacunes, white matter hyperintensities (WMH), and enlarged perivascular spaces (EPVS). The total SVD burden was calculated based on lacunes, WMH, and EPVS and then summed up to generate an ordinal "total SVD burden" (range 0-3). Bivariate logistic regression models were used to identify the association between SVD and functional outcome. Results: A total of 416 patients were included in the final analysis; 44.0, 33.4, 19.2, and 3.4% of the patients had 0, 1, 2, and 3 features of SVD, respectively. In regard to individual SVD feature, lacunes (OR: 0.48, 95% CI: 0.32-0.71; OR: 0.49, 95% CI: 0.31-0.77) and WMH (OR: 0.53, 95% CI: 0.34-0.82; OR: 0.53, 95% CI: 0.33-0.85) were negatively associated with excellent outcome and good outcome. As to the total burden of SVD, three SVD features had strongest negative associations with functional outcomes (excellent outcome, OR: 0.13, 95% CI: 0.03-0.48; good outcome, OR: 0.18, 95% CI: 0.06-0.54). After adjustment for potential confounders, a high SVD burden (3 features, OR: 0.07, 95% CI: 0.01-0.41) and the score of total SVD burden (OR: 0.64, 95% CI: 0.44-0.93) remained negatively associated with excellent outcome. Conclusion: Total SVD burden negatively associated with functional outcome at 3 months in patients with acute ischemic stroke and is superior to individual SVD feature in prediction of functional outcome. MRI-based assessment of total SVD burden is highly valuable in clinical management of stroke victims and could help guide the allocation of resources to improve outcome.

Comparison of Satellite Sign and Island Sign in Predicting Hematoma Growth and Poor Outcome in Patients with Primary Intracerebral Hemorrhage.

OBJECTIVE: Satellite sign (SS) and island sign (IS) are novel noncontrast computed tomography (CT) predictors of hematoma growth. The aim of this study was to compare diagnostic performance of IS and SS in predicting hematoma growth and functional outcome in patients with intracerebral hemorrhage (ICH). METHODS: The study included patients with ICH who underwent baseline CT scan within 6 hours of symptom onset and follow-up CT scan within 36 hours after initial CT between July 2012 and April 2017. Sensitivity, specificity, positive predictive value, and negative predictive value of IS and SS in predicting hematoma growth and functional outcome were assessed. Accuracy of the 2 signs in predicting hematoma growth and functional outcome was analyzed using receiver operating characteristic analysis. Association between the presence of IS and SS and ICH growth was assessed using multivariate logistic regression. RESULTS: Of 307 patients with ICH, IS was observed in 46 patients (15.0%), and SS was observed in 151 patients (49.2%). Rates of hematoma growth were 40.4% in SS+ patients, 91.3% in IS+ patients, 18.4% in SS-IS- patients, 21.1% in SS+IS- patients, 100% in SS-IS+ patients, and 90.5% in SS+IS+ patients. After adjusting for potential confounders, IS remained an independent predictor for hematoma growth and poor functional outcome. The area under the curve of IS was significantly larger than the area under the curve of SS in predicting hematoma growth (P = 0.001). CONCLUSIONS: IS seems to be an optimal shape irregularity imaging marker for predicting hematoma growth and functional outcome in patients with ICH.

Black Hole Sign Predicts Poor Outcome in Patients with Intracerebral Hemorrhage.

BACKGROUND: In spontaneous intracerebral hemorrhage (ICH), black hole sign has been proposed as a promising imaging marker that predicts hematoma expansion in patients with ICH. The aim of our study was to investigate whether admission CT black hole sign predicts hematoma growth in patients with ICH. METHODS: From July 2011 till February 2016, patients with spontaneous ICH who underwent baseline CT scan within 6 h of symptoms onset and follow-up CT scan were recruited into the study. The presence of black hole sign on admission non-enhanced CT was independently assessed by 2 readers. The functional outcome was assessed using the modified Rankin Scale (mRS) at 90 days. Univariate and multivariable logistic regression analyses were performed to assess the association between the presence of the black hole sign and functional outcome. RESULTS: A total of 225 patients (67.6% male, mean age 60.3 years) were included in our study. Black hole sign was identified in 32 of 225 (14.2%) patients on admission CT scan. The multivariate logistic regression analysis demonstrated that age, intraventricular hemorrhage, baseline ICH volume, admission Glasgow Coma Scale score, and presence of black hole sign on baseline CT independently predict poor functional outcome at 90 days. There are significantly more patients with a poor functional outcome (defined as mRS ≥4) among patients with black hole sign than those without (84.4 vs. 32.1%, p < 0.001; OR 8.19, p = 0.001). CONCLUSIONS: The CT black hole sign independently predicts poor outcome in patients with ICH. Early identification of black hole sign is useful in prognostic stratification and may serve as a potential therapeutic target for anti-expansion clinical trials.

Blend sign predicts poor outcome in patients with intracerebral hemorrhage.

INTRODUCTION: Blend sign has been recently described as a novel imaging marker that predicts hematoma expansion. The purpose of our study was to investigate the prognostic value of CT blend sign in patients with ICH. OBJECTIVES AND METHODS: Patients with intracerebral hemorrhage who underwent baseline CT scan within 6 hours were included. The presence of blend sign on admission nonenhanced CT was independently assessed by two readers. The functional outcome was assessed by using the modified Rankin Scale (mRS) at 90 days. RESULTS: Blend sign was identified in 40 of 238 (16.8%) patients on admission CT scan. The proportion of patients with a poor functional outcome was significantly higher in patients with blend sign than those without blend sign (75.0% versus 47.5%, P = 0.001). The multivariate logistic regression analysis demonstrated that age, intraventricular hemorrhage, admission GCS score, baseline hematoma volume and presence of blend sign on baseline CT independently predict poor functional outcome at 90 days. The CT blend sign independently predicts poor outcome in patients with ICH (odds ratio 3.61, 95% confidence interval [1.47-8.89];p = 0.005). CONCLUSIONS: Early identification of blend sign is useful in prognostic stratification and may serve as a potential therapeutic target for prospective interventional studies.

Minocycline Promotes Neurite Outgrowth of PC12 Cells Exposed to Oxygen-Glucose Deprivation and Reoxygenation Through Regulation of MLCP/MLC Signaling Pathways.

Minocycline, a semi-synthetic second-generation derivative of tetracycline, has been reported to exert neuroprotective effects both in animal models and in clinic trials of neurological diseases. In the present study, we first investigated the protective effects of minocycline on oxygen-glucose deprivation and reoxygenation-induced impairment of neurite outgrowth and its potential mechanism in the neuronal cell line, PC12 cells. We found that minocycline significantly increased cell viability, promoted neurite outgrowth and enhanced the expression of growth-associated protein-43 (GAP-43) in PC12 cells exposed to oxygen-glucose deprivation/reoxygenation injury. In addition, immunoblots revealed that minocycline reversed the overexpression of phosphorylated myosin light chain (MLC) and the suppression of activated extracellular signal-regulated kinase 1/2 (ERK1/2) caused by oxygen-glucose deprivation/reoxygenation injury. Moreover, the minocycline-induced neurite outgrowth was significantly blocked by Calyculin A (1 nM), an inhibitor of myosin light chain phosphatase (MLCP), but not by an ERK1/2 inhibitor (U0126; 10 µM). These findings suggested that minocycline activated the MLCP/MLC signaling pathway in PC12 cells after oxygen-glucose deprivation/reoxygenation injury, which resulted in the promotion of neurite outgrowth.

Blend Sign on Computed Tomography: Novel and Reliable Predictor for Early Hematoma Growth in Patients With Intracerebral Hemorrhage.

BACKGROUND AND PURPOSE: Early hematoma growth is not uncommon in patients with intracerebral hemorrhage and is an independent predictor of poor functional outcome. The purpose of our study was to report and validate the use of our newly identified computed tomographic (CT) blend sign in predicting early hematoma growth. METHODS: Patients with intracerebral hemorrhage who underwent baseline CT scan within 6 hours after onset of symptoms were included. The follow-up CT scan was performed within 24 hours after the baseline CT scan. Significant hematoma growth was defined as an increase in hematoma volume of >33% or an absolute increase of hematoma volume of >12.5 mL. The blend sign on admission nonenhanced CT was defined as blending of hypoattenuating area and hyperattenuating region with a well-defined margin. Univariate and multivariable logistic regression analyses were performed to assess the relationship between the presence of the blend sign on nonenhanced admission CT and early hematoma growth. RESULTS: A total of 172 patients were included in our study. Blend sign was observed in 29 of 172 (16.9%) patients with intracerebral hemorrhage on baseline nonenhanced CT scan. Of the 61 patients with hematoma growth, 24 (39.3%) had blend sign on admission CT scan. Interobserver agreement for identifying blend sign was excellent between the 2 readers (κ=0.957). The multivariate logistic regression analysis demonstrated that the time to baseline CT scan, initial hematoma volume, and presence of blend sign on baseline CT scan to be independent predictors of early hematoma growth. The sensitivity, specificity, positive and negative predictive values of blend sign for predicting hematoma growth were 39.3%, 95.5%, 82.7%, and 74.1%, respectively. CONCLUSIONS: The CT blend sign could be easily identified on regular nonenhanced CT and is highly specific for predicting hematoma growth.

Intraventricular Hemorrhage and Early Hematoma Expansion in Patients with Intracerebral Hemorrhage.

Intraventricular hemorrhage is associated with poor functional outcomes in patients with intracerebral hemorrhage (ICH). We aimed to investigate the association between intraventricular hemorrhage and early hematoma expansion in patients with ICH. Patients with ICH who underwent a baseline CT scan within six hours after onset of symptoms were included. The follow-up CT scan was performed within 24 hours after the baseline CT scan. Univariate and multivariable logistic regression were used to assess the relationship between presence of intraventricular hemorrhage and early hematoma expansion. A total of 160 patients were included in the study. Significant hematoma growth was observed in 52 (32.5%) patients presenting within six hours after onset of symptoms. Intraventricular hemorrhage was observed in 66 (41.25%) patients with ICH. Multivariate analyses demonstrated that a short time from onset to baseline CT scan, the initial hematoma volume, and the presence of intraventricular hemorrhage on follow-up CT scan were independently associated with hematoma enlargement. The presence of intraventricular hemorrhage on follow-up CT scan can be associated with hematoma expansion in patients with ICH.

[ERKl/2 signaling pathway mediates heme oxygenase-1 up-regulation by minocycline in PC12 cells exposed to oxygen glucose deprivation].

OBJECTIVE: To investigate the effects of minocycline in promoting the survival of pheochromocytoma (PCI2) cells exposed to oxygen glucose deprivation (OGD) and explore the underlying mechanisms. METHODS: An in vitro cell model of cerebral ischemia was established by OGD for 6 h in PCI2 cells with pretreatment with minocycline or an ERK1/2 inhibitor. At 24 h after OGD injury, the cells were evaluated for cell viability by MTT assay and expressions of heme oxygenase-I (HO-I) and phospholylated extracellular signal-regulated protein kinase 1/2 (ERK1/2) by Western blotting. RESULTS: The cell viability decreased dramatically following OGD. Pretreatment with minocycline (O.I-IO JJ.mol/L) induced a significant increase in the cell viability after OGD and caused up-regulation of HO-I protein and enhanced ERK1/2 phospholylation, and the effects were especially obvious with 1 JJ.mol/L minocycline and were abolished by inhibition of ERK1/2 activity with UOI26 (IO JJ.mol/L). CONCLUSION: Minocycline can protect PCI2 cells against OGD-induced toxicity by up-regulating HO-I protein expression through ERKl/2 signaling pathways.

Neuroelectrophysiological characteristics of Hirayama disease: report of 14 cases.

BACKGROUND: Hirayama disease is a juvenile muscular atrophy of the distal upper extremities and affects mainly young males. The present study aimed to investigate the neuroelectrophysiological characteristics of Hirayama disease. METHODS: We retrospectively analyzed the neural conduction velocity (NCV) parameters and needle-electrode electromyograms (EMG) of 14 patients with Hirayama disease. According to the clinical features of the patients, NCV was performed on affected upper-limb including median nerves and ulnar nerves, while EMG was selectively performed on upper and lower extremities, sternocleidomast and thoracic paraspinal muscles. RESULTS: The median nerves of all affected upper limbs of patients with Hirayama disease had normal conduction velocities and compound motor action potentials (CMAPs). The ulnar nerves of all affected upper limbs also had normal conduction velocities. Of the 16 measured ulnar nerves of the affected upper limbs, eight had normal CMAPS, while the other eight showed CMAPs below the normal value by < 20%. All patients had neurogenic injury on the affected side in muscles innervated by anterior horn cells at the lower cervical region (C7-8, T1). Four patients had unilateral upper-limb muscle neurogenic injury on the affected side. Seven patients had bilateral upper-limb muscle neurogenic injury, while only two patients experienced bilateral upper-limb muscle atrophy/weakness. The other three patients showed extensive neurogenic injury (unilateral upper-limb muscle atrophy/weakness in one patient, bilateral symptoms in the other two patients). CONCLUSIONS: Electromyographic examination showed that the majority of Hirayama disease patients exhibited characteristic segmental injury in the anterior horn of the lower cervical region, while a few patients exhibited extensive neurogenic injury. These data suggest that the actual influence of Hirayama disease may be more extensive than indicated by the clinical presentations.

Prognosis study of 324 cases with spontaneous intracerebral hemorrhage in Chongqing, China.

We aimed to investigate the clinical characteristics of intracerebral hemorrhage (ICH) in Chongqing City, China, and evaluate some factors predicting the prognosis of ICH. We collected 324 cases with spontaneous intracerebral hemorrhage in our hospital from January 2008 to November 2009. Univariate variance analyses were used for comparison of characteristics. Odds ratios (ORs) were calculated by logistic regression. Potential confounders were adjusted, including gender, age, smoking, and drinking status. Hypertension was the major cause of the spontaneous intracerebral hemorrhage, accounting for 75% of all patients in this study. Hemorrhages were located in lobes (22.5%), basal ganglia (65.3%), cerebral ventricles (2.6%), cerebellum (4.2%), and brain stem (7.4%). Serum glucose and conscious status were independently associated with in-hospital mortality after ICH. Comparing subjects who died in the hospital to those who survived, the adjusted ORs of serum glucose were 1.248 (95% CI 1.013-1.537, p=0.037), and the adjusted ORs of consciousness status were 1.995 (95% CI 1.519-2.621, p<0.001). In China, spontaneous intracerebral hemorrhage is mostly caused by hypertension and is usually located in the basal ganglia. Serum glucose and consciousness status independently predict the prognosis of ICH.

Small hairpin RNA interference of the Nogo receptor inhibits oxygen-glucose deprivation-induced damage in rat hippocampal slice cultures.

In adult mammals, CNS damage does not repair well spontaneously. The Nogo receptor (NgR) signaling pathway prevents axonal regrowth and promotes neuronal apoptosis. This pathway, and pathways like it, may be part of the reason why nerves do not regrow. A number of preclinical experiments inhibiting portions of the NgR pathway have yielded limited induction of nerve repair. Here, we developed a small hairpin RNA (shRNA) to knock down NgR expression. With the use of rat hippocampal slices in tissue culture, we induced neuronal damage similar to that of ischemia-reperfusion injury by exposing the cultured tissues to oxygen-glucose deprivation. We then assayed the effect of NgR knockdown in this model system. Adenovirally delivered NgR shRNA decreased NgR mRNA and protein expression. Thirty minutes of oxygen-glucose deprivation resulted in widespread tissue damage, including apoptosis and loss of neurite extension, 72 h after termination of oxygen-glucose deprivation. The NgR shRNA knockdown reduced, but did not eliminate, the effects of oxygen-glucose deprivation. Thus, NgR shRNA shows promise as a potential tool for the treatment of nerve damage.

Effects of carbon disulfide on the expression and activity of nitric oxide synthase in rat hippocampus.

BACKGROUND: Carbon disulfide (CS(2)) is a commonly used organic solvent. Many epidemiological investigations and animal experiments have indicated that learning and memory ability can be affected to different degrees after long-term exposure to CS(2), but the mechanisms are still unclear. The aim of this study was to explore the possible mechanisms of CS(2)-related impairment of the learning and memory ability of rats, by investigating the effects of CS(2) on nitric oxide synthase (NOS) activity and NOS mRNA expression in rat hippocampus. METHODS: Rat models of toxicity were generated by inhalation of various doses of CS(2). After two months of inhaling intoxication, the activities of constitutive NOS (cNOS) and induced NOS (iNOS) in the hippocampus were measured. The levels of neuronal NOS (nNOS) mRNA and iNOS mRNA were measured by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). RESULTS: cNOS activity was significantly decreased compared with controls, while iNOS activity was changed only slightly. CS(2) treatment significantly decreased nNOS mRNA levels. iNOS mRNA levels were significantly increased only at higher doses of CS(2). CONCLUSION: The effect of CS2 on learning and memory ability in rats is related to the activity of NOS and the expression of nNOS in the hippocampus.