Development of an online calculator for the prediction of seizure freedom following pediatric hemispherectomy using the Hemispherectomy Outcome Prediction Scale (HOPS).

OBJECTIVES: Although hemispheric surgeries are among the most effective procedures for drug-resistant epilepsy (DRE) in the pediatric population, there is a large variability in seizure outcomes at the group level. A recently developed HOPS score provides individualized estimation of likelihood of seizure freedom to complement clinical judgement. The objective of this study was to develop a freely accessible online calculator that accurately predicts the probability of seizure freedom for any patient at 1-, 2-, and 5-years post-hemispherectomy. METHODS: Retrospective data of all pediatric patients with DRE and seizure outcome data from the original Hemispherectomy Outcome Prediction Scale (HOPS) study were included. The primary outcome of interest was time-to-seizure recurrence. A multivariate Cox proportional-hazards regression model was developed to predict the likelihood of post-hemispheric surgery seizure freedom at three time points (1-, 2- and 5- years) based on a combination of variables identified by clinical judgment and inferential statistics predictive of the primary outcome. The final model from this study was encoded in a publicly accessible online calculator on the International Network for Epilepsy Surgery and Treatment (iNEST) website (https://hops-calculator.com/). RESULTS: The selected variables for inclusion in the final model included the five original HOPS variables (age at seizure onset, etiologic substrate, seizure semiology, prior non-hemispheric resective surgery, and contralateral fluorodeoxyglucose-positron emission tomography [FDG-PET] hypometabolism) and three additional variables (age at surgery, history of infantile spasms, and magnetic resonance imaging [MRI] lesion). Predictors of shorter time-to-seizure recurrence included younger age at seizure onset, prior resective surgery, generalized seizure semiology, FDG-PET hypometabolism contralateral to the side of surgery, contralateral MRI lesion, non-lesional MRI, non-stroke etiologies, and a history of infantile spasms. The area under the curve (AUC) of the final model was 73.0%. SIGNIFICANCE: Online calculators are useful, cost-free tools that can assist physicians in risk estimation and inform joint decision-making processes with patients and families, potentially leading to greater satisfaction. Although the HOPS data was validated in the original analysis, the authors encourage external validation of this new calculator.

Relationship between vestibular schwannoma and endolymphatic hydrops.

BACKGROUND: Both vestibular schwannoma (VS) and Meniere's disease (MD) patients underwent hydrops MRI to clarify the relationship between VS and endolymphatic hydrops (EH). METHODS: Eighty patients with VS or MD underwent an inner ear test battery followed by hydrops MRI, and were then divided into 3 groups. Group A comprised 58 MD patients (62 ears) with positive EH but negative VS. Group B included 18 VS patients (18 ears) with negative EH, while Group C consisted of 4 patients (4 ears) who had VS concomitant with EH. Another 14 MD patients who tested negative for EH on hydrops MRI were initially excluded from this cohort, but were later included for comparison. RESULTS: The decreasing prevalence of EH at the cochlea, saccule and utricle in Group A was identified in 59 (95%), 42 (68%) and 40 (65%) ears, respectively, mimicking a declining sequence of abnormality rates running from audiometry (86%), cervical vestibular-evoked myogenic potential (cVEMP) test (55%) to the ocular (oVEMP) test (53%). However, such decreasing trend was not identified in Groups B and C. In Groups C and A combined, 4 (6%) of 62 EH patients had concomitant VS. Conversely, 4 (18%) of 22 VS patients in Groups C and B combined had concurrent EH. CONCLUSION: A very low (6%) rate of VS in EH patients indicates that VS in EH patients may be coincidental. In contrast, EH was identified in 18% prevalence of VS patients, mirroring the 22% prevalence of cochlear EH demonstrated in VS donors through histopathological studies.

Limitation of updated MR images on the vestibular hydrops in Meniere's disease.

PURPOSE: This study adopted the cervical and ocular vestibular-evoked myogenic potential (cVEMP and oVEMP) tests in Meniere's disease (MD) patients to correlate them with vestibular endolymphatic hydrops (EH) on MR images. METHODS: A total of 25 patients with unilateral definite MD identified by positive cochlear hydrops on MR images were enrolled. All patients underwent audiometry, cVEMP test and oVEMP test, followed by MR imaging for confirmation. RESULTS: A significantly declining sequence of abnormality rates in MD patients was identified from the audiometry (92%), cVEMP test (52%) to the oVEMP test (40%), which was consistent with a significantly decreasing order of prevalence of EH on MR images running from the cochlea (100%), saccule (56%) to the utricle (52%). The cVEMP test for detecting the saccular hydrops revealed a sensitivity of 62%, while the oVEMP test for assessing the utricular hydrops showed a sensitivity of 70%. However, correlating VEMP results with vestibular hydrops did not show any significant relationship. In addition, mean hearing level (MHL) at four frequencies (500, 1000, 2000, and 3000 Hz) of Grade I cochlear hydrops (51 ± 19 dB) did not significantly differ from Grade II cochlear hydrops (53 ± 19 dB). CONCLUSION: Limitations of the updated MR imaging for visualizing the hydrops comprised: (1) failure to correlate vestibular hydrops with VEMP results, and (2) failure to correlate grade of cochlear hydrops with MHL. The reason is probably because updated MR imaging fails to identify distorted contour of the cochlea/utricle/saccule. Further advanced technique using ultrahigh resolution of fine structures in the inner ear compartments is essential to promote a wider use of MR imaging.

Novel lissencephaly-associated DCX variants in the C-terminal DCX domain affect microtubule binding and dynamics.

OBJECTIVE: Pathogenic variants in DCX on the X chromosome lead to lissencephaly and subcortical band heterotopia (SBH), brain malformations caused by neuronal migration defects. Its product doublecortin (DCX) binds to microtubules to modulate microtubule polymerization. How pathogenic DCX variants affect these activities remains not fully investigated. METHODS: DCX variants were identified using whole exome and Sanger sequencing from six families with lissencephaly/SBH. We examined how these variants affect DCX functions using microtubule binding, regrowth, and colocalization assays. RESULTS: We found novel DCX variants p.Val177AlafsTer31 and p.Gly188Trp, as well as reported variants p.Arg196His, p.Lys202Met, and p.Thr203Ala. Incidentally, all of the missense variants were clustered on the C-terminal DCX domain. The microtubule binding ability was significantly decreased in p.Val177AlafsTer31, p.Gly188Trp, p.Lys202Met, and previously reported p.Asp262Gly variants. Furthermore, expression of p.Val177AlafsTer31, p.Gly188Trp, p.Arg196His, p.Lys202Met, and p.Asp262Gly variants hindered microtubule growth in cells. There were also decreases in the colocalization of p.Val177AlafsTer31, p.Thr203Ala, and p.Asp262Gly variants to microtubules. SIGNIFICANCE: Our results indicate that these variants in the C-terminal DCX domain altered microtubule binding and dynamics, which may underlie neuronal migration defects during brain development.

Magnetic resonance imaging: Role on diagnosing all types of endolymphatic hydrops.

BACKGROUND/PURPOSE: This study utilized the recently advanced technique in MR imaging to establish its role on diagnosing all types of endolymphatic hydrops (EH). METHODS: Twenty-two patients (26 ears) with clinical EH were admitted due to acute hearing loss and/or vertiginous attack. Each patient underwent an inner ear test battery comprising audiometry, cervical and ocular vestibular-evoked myogenic potential tests, and caloric test, followed by MR imaging for confirmation. RESULTS: Of the 22 clinical EH patients, 12 patients were referred to primary EH (Meniere's disease), 8 patients were secondary EH (including delayed EH in 5), and 2 patients were EH of embryopathic origin. MR imaging of 26 affected ears demonstrated EH in the cochlea only for 14 ears, in the utricle and saccule only for 1 ear, and in all three endorgans for 3 ears, accounting for a sensitivity of 69% (18/26). The 8 affected ears showing negative MR images were EH patients with hearing recovery 1, just after vertiginous attack 3, and chronic low-tone hearing loss 4. In contrast, 3 out of 18 unaffected ears demonstrated asymptomatic EH in the cochlea, representing a specificity of 83% (15/18). CONCLUSION: The sensitivity and specificity of MR imaging for confirming all types of EH were 69% and 83%, respectively. Although diagnostic criteria can identify primary and delayed EH, MR imaging may provide a supplementary tool for diagnosing secondary, embryopathic, or asymptomatic EH, if patients are not with hearing recovery, chronic low-tone hearing loss, or just after vertiginous attack.

Comparison of the real-world effectiveness of vertical versus lateral functional hemispherotomy techniques for pediatric drug-resistant epilepsy: A post hoc analysis of the HOPS study.

OBJECTIVE: This study was undertaken to determine whether the vertical parasagittal approach or the lateral peri-insular/peri-Sylvian approach to hemispheric surgery is the superior technique in achieving long-term seizure freedom. METHODS: We conducted a post hoc subgroup analysis of the HOPS (Hemispheric Surgery Outcome Prediction Scale) study, an international, multicenter, retrospective cohort study that identified predictors of seizure freedom through logistic regression modeling. Only patients undergoing vertical parasagittal, lateral peri-insular/peri-Sylvian, or lateral trans-Sylvian hemispherotomy were included in this post hoc analysis. Differences in seizure freedom rates were assessed using a time-to-event method and calculated using the Kaplan-Meier survival method. RESULTS: Data for 672 participants across 23 centers were collected on the specific hemispherotomy approach. Of these, 72 (10.7%) underwent vertical parasagittal hemispherotomy and 600 (89.3%) underwent lateral peri-insular/peri-Sylvian or trans-Sylvian hemispherotomy. Seizure freedom was obtained in 62.4% (95% confidence interval [CI] = 53.5%-70.2%) of the entire cohort at 10-year follow-up. Seizure freedom was 88.8% (95% CI = 78.9%-94.3%) at 1-year follow-up and persisted at 85.5% (95% CI = 74.7%-92.0%) across 5- and 10-year follow-up in the vertical subgroup. In contrast, seizure freedom decreased from 89.2% (95% CI = 86.3%-91.5%) at 1-year to 72.1% (95% CI = 66.9%-76.7%) at 5-year to 57.2% (95% CI = 46.6%-66.4%) at 10-year follow-up for the lateral subgroup. Log-rank test found that vertical hemispherotomy was associated with durable seizure-free progression compared to the lateral approach (p = .01). Patients undergoing the lateral hemispherotomy technique had a shorter time-to-seizure recurrence (hazard ratio = 2.56, 95% CI = 1.08-6.04, p = .03) and increased seizure recurrence odds (odds ratio = 3.67, 95% CI = 1.05-12.86, p = .04) compared to those undergoing the vertical hemispherotomy technique. SIGNIFICANCE: This pilot study demonstrated more durable seizure freedom of the vertical technique compared to lateral hemispherotomy techniques. Further studies, such as prospective expertise-based observational studies or a randomized clinical trial, are required to determine whether a vertical approach to hemispheric surgery provides superior long-term seizure outcomes.

Hemispherectomy Outcome Prediction Scale: Development and validation of a seizure freedom prediction tool.

OBJECTIVE: To develop and validate a model to predict seizure freedom in children undergoing cerebral hemispheric surgery for the treatment of drug-resistant epilepsy. METHODS: We analyzed 1267 hemispheric surgeries performed in pediatric participants across 32 centers and 12 countries to identify predictors of seizure freedom at 3 months after surgery. A multivariate logistic regression model was developed based on 70% of the dataset (training set) and validated on 30% of the dataset (validation set). Missing data were handled using multiple imputation techniques. RESULTS: Overall, 817 of 1237 (66%) hemispheric surgeries led to seizure freedom (median follow-up = 24 months), and 1050 of 1237 (85%) were seizure-free at 12 months after surgery. A simple regression model containing age at seizure onset, presence of generalized seizure semiology, presence of contralateral 18-fluoro-2-deoxyglucose-positron emission tomography hypometabolism, etiologic substrate, and previous nonhemispheric resective surgery is predictive of seizure freedom (area under the curve = .72). A Hemispheric Surgery Outcome Prediction Scale (HOPS) score was devised that can be used to predict seizure freedom. SIGNIFICANCE: Children most likely to benefit from hemispheric surgery can be selected and counseled through the implementation of a scale derived from a multiple regression model. Importantly, children who are unlikely to experience seizure control can be spared from the complications and deficits associated with this surgery. The HOPS score is likely to help physicians in clinical decision-making.

Targeting MAPK/NF-κB Pathways in Anti-Inflammatory Potential of Rutaecarpine: Impact on Src/FAK-Mediated Macrophage Migration.

Studies have discovered that different extracts of Evodia rutaecarpa and its phytochemicals show a variety of biological activities associated with inflammation. Although rutaecarpine, an alkaloid isolated from the unripe fruit of E. rutaecarpa, has been exposed to have anti-inflammatory properties, the mechanism of action has not been well studied. Thus, this study investigated the molecular mechanisms of rutaecarpine (RUT) in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. RUT reserved the production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor (TNF-α), and interleukin (IL)-1ß in the LPS-induced macrophages. RUT showed an inhibitory effect on the mitogen-activated protein kinases (MAPKs), and it also inhibited nuclear transcription factor kappa-B (NF-κB) by hindering IκBα and NF-κB p65 phosphorylation and p65 nuclear translocation. The phospho-PI3K and Akt was concentration-dependently suppressed by RUT. However, RUT not only suggestively reduced the migratory ability of macrophages and their numbers induced by LPS but also inhibited the phospho-Src, and FAK. Taken together, these results indicate that RUT participates a vital role in the inhibition of LPS-induced inflammatory processes in RAW 264.7 macrophages and that the mechanisms involve PI3K/Akt and MAPK-mediated downregulation of NF-κB signaling pathways. Notably, reducing the migration and number of cells induced by LPS via inhibiting of Src/FAK pathway was also included to the anti-inflammatory mechanism of RUT. Therefore, RUT may have potential benefits as a therapeutic agent against chronic inflammatory diseases.

The Antithrombotic Agent Pterostilbene Interferes with Integrin αIIbß3-Mediated Inside-Out and Outside-In Signals in Human Platelets.

Platelets play a crucial role in the physiology of primary hemostasis and pathological processes such as arterial thrombosis; thus, developing a therapeutic target that prevents platelet activation can reduce arterial thrombosis. Pterostilbene (PTE) has remarkable pharmacological activities, including anticancer and neuroprotection. Few studies have reported the effects of pterostilbene on platelet activation. Thus, we examined the inhibitory mechanisms of pterostilbene in human platelets and its role in vascular thrombosis prevention in mice. At low concentrations (2-8 µM), pterostilbene strongly inhibited collagen-induced platelet aggregation. Furthermore, pterostilbene markedly diminished Lyn, Fyn, and Syk phosphorylation and hydroxyl radical formation stimulated by collagen. Moreover, PTE directly hindered integrin αIIbß3 activation through interfering with PAC-1 binding stimulated by collagen. In addition, pterostilbene affected integrin αIIbß3-mediated outside-in signaling, such as integrin ß3, Src, and FAK phosphorylation, and reduced the number of adherent platelets and the single platelet spreading area on immobilized fibrinogen as well as thrombin-stimulated fibrin clot retraction. Furthermore, pterostilbene substantially prolonged the occlusion time of thrombotic platelet plug formation in mice. This study demonstrated that pterostilbene exhibits a strong activity against platelet activation through the inhibition of integrin αIIbß3-mediated inside-out and outside-in signaling, suggesting that pterostilbene can serve as a therapeutic agent for thromboembolic disorders.

Iron-Catalyzed Cleavage Reaction of Keto Acids with Aliphatic Aldehydes for the Synthesis of Ketones and Ketone Esters.

The radical-radical coupling reaction is an important synthetic strategy. In this study, the iron-catalyzed radical-radical cross-coupling reaction based on the decarboxylation of keto acids and decarbonylation of aliphatic aldehydes to obtain valuable aryl ketones is reported for the first time. Remarkably, when tertiary aldehydes were used as carbonyl sources, ketone esters were selectively obtained instead of ketones. The gram-scale preparation of aryl ketone through this strategy was easily achieved by using only 3 mol % of the iron catalyst. As a proof-of-concept, the bioactive molecule flurprimidol was synthesized in two steps by using this strategy.

Exposure Duration and History of Hypertension Predicted Neurological Sequelae in Patients with Carbon Monoxide Poisoning.

BACKGROUND: Carbon monoxide poisoning (COP) accounts for a large number of emergency department visits worldwide and is fatal in many cases. In surviving patients, neurological sequelae (NS) attributable to cerebral hypoxia are the most devastating outcome, but reliable predictors are limited. Therefore, we conducted a study to identify predictors of NS in patients with COP and evaluate their effects. METHODS: In this retrospective case-control study, we identified patients with COP in a medical center in Southern Taiwan between January 2005 and December 2014. Cases were patients with NS, and controls were patients without NS. We obtained information on potential predictors of NS from medical records and evaluated their association with NS, including demographic characteristics, exposure source, suicide attempts, duration of exposure (by tertile), histories, symptoms, signs, laboratory data, treatment, and the length of hospital stay. RESULTS: We included 371 patients with COP. Of them, 93 developed NS, and their mean ages (41.4 ± 14.7 years vs. 39.7 ± 14.2 years) and proportions of males (59.1% vs. 58.6%) were similar to those in the 298 controls. Multivariate logistic regression showed that a history of hypertension (adjusted odds ratio = 2.1; 95% confidence interval = 1.0, 4.5) and a longer duration of carbon monoxide exposure (adjusted odds ratio = 1.7; 95% confidence interval = 1.1, 2.8; the longest tertile [>5 hours] vs. the other two tertiles [≤5 hours]) were independent predictors for NS, but not the level of carboxyhemoglobin. CONCLUSIONS: This study identified two independent predictors for NS that may be useful for public healthcare workers and physicians in predicting outcomes and deciding on treatment strategies for COP patients.

Exercise Preconditioning Attenuates Neurological Injury by Preserving Old and Newly Formed HSP72-Containing Neurons in Focal Brain Ischemia Rats.

Background: Exercise preconditioning (EP+) is a useful and important procedure for the prevention of stroke. We aimed to ascertain whether EP+ protects against ischemic brain injury by preserving heat shock protein (HSP) 72-containing neurons in ischemic brain tissues. Methods: Adult male Sprague-Dawley rats (n=240) were used to assess the contribution of HSP72-containing neurons to the neuroprotective effects of EP+ on ischemic brain injury caused by transient middle cerebral artery occlusion. Results: Significant (P<0.05) increases in the percentages of both old HSP72-containing neurons (NeuN+HSP72 double positive cells) (18~20% vs. 40~50%) and newly formed HSP72-containing neurons (BrdU+NeuN+HSP72 triple positive cells); (2~3% vs. 16~20%) after 3 weeks of exercise coincided with significant (P<0.05) reductions in brain ischemia volume (250 mm3 vs. 100 mm3), brain edema (78% vs. 74% brain water content), blood-brain barrier disruption (1.5 µg/g vs. 0.7 µg/g tissue Evans Blue dye extravasation) and neurological motor deficits (neurological severity scores of 12 vs. 6 and maximal angles of 60° vs. 20°) in brain ischemia rats. Reductions in the percentages of both old (from 40~50% to 10~12%) and newly formed (from 18~20% to 5~7%) HSP72-containing neurons by gene silencing with an intracerebral injection of pSUPER small interfering RNA showed a significant (P<0.05) reversal in the neuroprotective outcomes. Our data provide an inverse correlation between the EP+-mediated increases in both old and newly formed HSP72-containing neurons and the extent of cerebral ischemic injury. Conclusions: The percentages of both old and newly formed HSP72-containing neurons are inversely correlated with the outcomes of ischemic brain injury. Additionally, preischemic treadmill exercise improves the outcomes of ischemic brain injury by preserving both the old and newly formed HSP72-containing neurons in rats.

A de novo SCN8A heterozygous mutation in a child with epileptic encephalopathy: a case report.

BACKGROUND: Epilepsy is a complex disorder caused by various factors, including genetic aberrance. Recent studies have identified an essential role of the sodium channel Nav1.6, encoded by the gene SCN8A, in epileptic encephalopathy. CASE PRESENTATION: Using parent-offspring trio targeted-exome sequencing, we identified a de novo heterozygous missense mutation c.3953A > G (p.N1318S) in SCN8A in a 3-year-and-9-month Chinese female patient with early infantile epileptic encephalopathy and a normal magnetic resonance imaging of the brain. CONCLUSIONS: This de novo mutation was only detected in the patient but not in her parents. Bioinformatic analysis indicates the pathogenicity of this mutation. Administration of the sodium channel blocker well controlled seizures in the patient. Therefore, we recommend trio targeted-exome sequencing as a routine method for pathogenic variant screening in patients with intractable epilepsy and a normal MRI.

Application of a multidimensional computerized adaptive test for a Clinical Dementia Rating Scale through computer-aided techniques.

BACKGROUND: With the increasingly rapid growth of the elderly population, individuals aged 65 years and above now compose 14% of Taiwanese citizens, thereby making Taiwanese society an aged society. A leading factor that affects the elderly population is dementia. A method of precisely and efficiently examining patients with dementia through multidimensional computer adaptive testing (MCAT) to accurately determine the patients' stage of dementia needs to be developed. This study aimed to develop online MCAT that family members can use on their own computers, tablets, or smartphones to predict the extent of dementia for patients responding to the Clinical Dementia Rating (CDR) instrument. METHODS: The CDR was applied to 366 outpatients in a hospital in Taiwan. MCAT was employed with parameters for items across eight dimensions, and responses were simulated to compare the efficiency and precision between MCAT and non-adaptive testing (NAT). The number of items saved and the estimated person measures was compared between the results of MCAT and NAT, respectively. RESULTS: MCAT yielded substantially more precise measurements and was considerably more efficient than NAT. MCAT achieved 20.19% (= [53 - 42.3]/53) saving in item length when the measurement differences were less than 5%. Pearson correlation coefficients were highly consistent among the eight domains. The cut-off points for the overall measures were - 1.4, - 0.4, 0.4, and 1.4 logits, which was equivalent to 20% for each portion in percentile scores. Substantially fewer items were answered through MCAT than through NAT without compromising the precision of MCAT. CONCLUSIONS: Developing a website that family members can use on their own computers, tablets, and smartphones to help them perform online screening and prediction of dementia in older adults is useful and manageable.

Modulation of parietal cytokine and chemokine gene profiles by mesenchymal stem cell as a basis for neurotrauma recovery.

BACKGROUND & PURPOSE: Following traumatic brain injury (TBI), primary mechanical injury to the brain may cause blood-brain-barrier damage followed by secondary injury, ultimately culminating in cell death. We aimed to test whether one injection of mesenchymal stem cells (MSC) derived from the human umbilical cord can modulate brain cytokine and chemokine gene profiles and attenuate neurological injury in rats with TBI. METHODS: One-day post-TBI, the injured rats were treated with one injection of MSC (4 × 106/rat, i.v.). Three days later, immediately after assessment of neurobehavioral function, animals were sacrificed for analysis of neurological injury (evidenced by both brain contusion volume and neurological deficits) and parietal genes encoding 84 cytokines and chemokines in the injured brain by qPCR methods. RESULTS: Three days post-TBI, rats displayed both neurological injury and upgrade of 11 parietal genes in the ipsilateral brain. One set of 8 parietal genes (e.g., chemokine [C-X-C motif] ligand 12, platelet factor 4, interleukin-7, chemokine [C-C motif] ligand (CCL)19, CCL 22, secreted phosphoprotein 1, pro-platelet basic protein 1, and CCL 2) differentially upgraded by TBI was related to pro-inflammatory and/or neurodegenerative processes. Another set of 3 parietal genes up-graded by TBI (e.g., glucose-6-phosphate isomerase, bone morphogenetic protein (BMP) 2, and BMP 4) was related to anti-inflammatory/neuroregenerative events. Administration of MSC attenuated neurological injury, down-regulated these 8 parietal pro-inflammatory genes, and up-regulated these 3 parietal anti-inflammatory genes in the rats with TBI. CONCLUSION: Our data suggest that modulation of parietal cytokines and chemokines gene profiles by MSC as a basis for neurotrauma recovery.

Esculetin, a Coumarin Derivative, Prevents Thrombosis: Inhibitory Signaling on PLCγ2-PKC-AKT Activation in Human Platelets.

Esculetin, a bioactive 6,7-dihydroxy derivative of coumarin, possesses pharmacological activities against obesity, diabetes, renal failure, and cardiovascular disorders (CVDs). Platelet activation plays a major role in CVDs. Thus, disrupting platelet activation represents an attractive therapeutic target. We examined the effect of esculetin in human platelet activation and experimental mouse models. At 10-80 µM, esculetin inhibited collagen- and arachidonic acid-induced platelet aggregation in washed human platelets. However, it had no effects on other agonists such as thrombin and U46619. Esculetin inhibited adenosine triphosphate release, P-selectin expression, hydroxyl radical (OH·) formation, Akt activation, and phospholipase C (PLC)γ2/protein kinase C (PKC) phosphorylation, but did not diminish mitogen-activated protein kinase phosphorylation in collagen-activated human platelets. Platelet function analysis indicated that esculetin substantially prolonged the closure time of whole blood. In experimental mice, esculetin significantly increased the occlusion time in thrombotic platelet plug formation and reduced mortality associated with acute pulmonary thromboembolism. However, it did not prolong the bleeding time. This study demonstrates that esculetin inhibits human platelet activation via hindering the PLCγ2-PKC cascade, hydroxyl radical formation, Akt activation, and ultimately suppressing platelet activation. Therefore, esculetin may act as an essential therapeutic agent for preventing thromboembolic diseases.

The Novel Effect of Immunomodulator-Glatiramer Acetate on Epileptogenesis and Epileptic Seizures.

BACKGROUND/AIMS: Immunological mechanisms can be triggered as a response to central nervous system insults and can lead to seizures. In this study an investigation was made to determine if glatiramer acetate (GA), an immunomodulator currently used in the treatment of multiple sclerosis, could protect rats from pilocarpine-induced seizures and chronic epilepsy. METHODS: Two groups of adult male Sprague-Dawley rats, experimental (GA) and control, were used in the study. The systemic IL-1α and IL-1ß levels at baseline were checked as well as status epilepticus (SE), and the spontaneous recurrent seizure (SRS) stage by enzyme-linked immunosorbent assay. The GA group was given GA (150 µg/kg, ip) and the control group was given a saline injection prior to pilocarpine-induced seizures. Seizure susceptibility, severity and mortality were evaluated, using Racine seizure classification and hippocampal damage was evaluated by Nissl staining. The GA group received GA (150 µg/kg/day, ip) daily after SE, and the chronic spontaneous seizures were evaluated by long-term video recording, and mossy fiber sprouting was evaluated by Timm staining. The IL-1α and IL-1ß levels were correlated with seizure activities. The TNF-α level in the hippocampus was determined at the SRS stage by immunohistochemistry. The effect of GA on ionic currents and action potentials (APs) in NG108-15 differentiated neurons was investigated using patch-clamp technology. RESULTS: It was found that latency to severe seizures was significantly longer in the GA (p < 0.01) group, which also had SE of shorter duration and less frequent SRS (p < 0.01). GA attenuated acute hippocampal neuron loss and chronic mossy fiber sprouting in the CA3 and the SRS-reduction correlated with the reduction of IL-1α, but not with IL-1ß or TNF-α levels. Mechanistically, GA reduced the peak amplitude of voltage-gated Na+ current (INa), with a negative shift in the inactivation curve of INa and reduced the amplitude of APs along with decreased firing of APs. CONCLUSION: GA might serve as a neuroexcitability modulator which attenuates pilocarpine-induced acute and chronic excitotoxicity. Sodium channel attenuation was partially independent of the immunomodulatory effect.

Multiple Actions of Rotenone, an Inhibitor of Mitochondrial Respiratory Chain, on Ionic Currents and Miniature End-Plate Potential in Mouse Hippocampal (mHippoE-14) Neurons.

BACKGROUND/AIMS: Rotenone (Rot) is known to suppress the activity of complex I in the mitochondrial chain reaction; however, whether this compound has effects on ion currents in neurons remains largely unexplored. METHODS: With the aid of patch-clamp technology and simulation modeling, the effects of Rot on membrane ion currents present in mHippoE-14 cells were investigated. RESULTS: Addition of Rot produced an inhibitory action on the peak amplitude of INa with an IC50 value of 39.3 µM; however, neither activation nor inactivation kinetics of INa was changed during cell exposure to this compound. Addition of Rot produced little or no modifications in the steady-state inactivation curve of INa. Rot increased the amplitude of Ca2+-activated Cl- current in response to membrane depolarization with an EC50 value of 35.4 µM; further addition of niflumic acid reversed Rot-mediated stimulation of this current. Moreover, when these cells were exposed to 10 µM Rot, a specific population of ATP-sensitive K+ channels with a single-channel conductance of 18.1 pS was measured, despite its inability to alter single-channel conductance. Under current clamp condition, the frequency of miniature end-plate potentials in mHippoE-14 cells was significantly raised in the presence of Rot (10 µM) with no changes in their amplitude and time course of rise and decay. In simulated model of hippocampal neurons incorporated with chemical autaptic connection, increased autaptic strength to mimic the action of Rot was noted to change the bursting pattern with emergence of subthreshold potentials. CONCLUSIONS: The Rot effects presented herein might exert a significant action on functional activities of hippocampal neurons occurring in vivo.

Structure-Antiplatelet Activity Relationships of Novel Ruthenium (II) Complexes: Investigation of Its Molecular Targets.

The regulation of platelet function by pharmacological agents that modulate platelet signaling has proven to be a positive approach to the prevention of thrombosis. Ruthenium complexes are fascinating for the development of new drugs, as they possess numerous chemical and biological properties. The present study aims to evaluate the structure-activity relationship (SAR) of newly synthesized ruthenium (II) complexes, TQ-1, TQ-2 and TQ-3 in agonists-induced washed human platelets. Silica gel column chromatography, aggregometry, immunoblotting, NMR, and X-ray analyses were performed in this study. Of the three tested compounds, TQ-3 showed a concentration (1-5 µM) dependent inhibitory effect on platelet aggregation induced by collagen (1 µg/mL) and thrombin (0.01 U/mL) in washed human platelets; however, TQ-1 and TQ-2 had no response even at 250 µM of collagen and thrombin-induced aggregation. TQ-3 was effective with inhibiting collagen-induced ATP release, calcium mobilization ([Ca2+]i) and P-selectin expression without cytotoxicity. Moreover, TQ-3 significantly abolished collagen-induced Lyn-Fyn-Syk, Akt-JNK and p38 mitogen-activated protein kinases (p38 MAPKs) phosphorylation. The compound TQ-3 containing an electron donating amino group with two phenyl groups of the quinoline core could be accounted for by its hydrophobicity and this nature might be the reason for the noted antiplatelet effects of TQ-3. The present results provide a molecular basis for the inhibition by TQ-3 in collagen-induced platelet aggregation, through the suppression of multiple machineries of the signaling pathway. These results may suggest that TQ-3 can be considered a potential agent for the treatment of vascular diseases.