[Analysis of Diagnosis and Treatment of 6 Cases of Cerebral Amyloid Angiopathy-related Inflammation].

Cerebral amyloid angiopathy associated with inflammation (CAA-ri) is characterized by an inflammatory response to the vascular deposits of ß-amyloid within the brain that is a very rare subtype of cerebral amyloid angiopathy.The most common clinical manifestation of CAA-ri was headache, epilepsy, and cognitive dysfunction. Magnetic resonance imaging (MRI) showed focal or multiple white matter lesions, lobar intracerebral hemorrhage, extensive cortical or subcortical microbleeds. We reported 6 cases of probable CAA-ri diagnosed and treated in our hospital from January 2017 to September 2019 according to the revised diagnostic criteria in 2016.We found that 5 patients also had microbleeds and cortical superficial siderosison T2 and fluid-attenuated inversion recovery (FLAIR), suggesting that if the patients had a long course of disease, older age and heavy microbleeds load, the lesions could be found in the routine MRI, which is a clue for the diagnosis of CAA-ri. Clinicians should attach great importance to this phenomenon, and can further verify by susceptibility weighted imaging (SWI).

[Establishment of STO Cell Lines Expressing Green Fluorescent Protein and Mouse Leukemia Inhibitory Factor].

OBJECTIVE: To establish the STO cell lines expressing green fluorescent protein (GFP) and mouse leukemia inhibitory factor (LIF) , and try to culture the mouse embryonic stem cells (mESCs) by using the established STO-GFP-mLIF cells as the feeder layer. METHODS: The lentiviral particles containing GFP and mLIF and puromycin-resistance gene were constructed and transduced into STO cell lines. The cell lines stably expressing GFP and mLIF genes were screened out. The expression level of the inserted exogenous LIF gene was tested by Western blot and ELISA. The STO-GFP-mLIF cells were treated with different concentrations of mitomycin C (5, 10, 15, 20 µg/ml) for different time (1.5, 2.5, 3, 3.5 hours) to prepare feeder layers and the cell proliferation level on feeder layer was observed. Mouse embryonic stem cells were cultured on mitomycin C-treated feeder layer and the growth of cell colonies was observed. RESULTS: The expression level of LIF protein in STO-GFP-mLIF cells was up-regulated, as compared with STO cells (P＜0.05). It was confirmed that the optimal concentration and time for inhibiting the proliferetion of STO-GFP-mLIF cells by mitomycin C were 10 µg/ml and 3 hours respectively. The observation also found that the embryonic stem cells could develop into typic "birdnest" shaped stem cell colony on mitomycin C-treated feeder layer. CONCLUSION: The stable STO cell lines effectively expressing green fluorescent protein and mouse leukemia inhibitory factor have been established successfully, which can maintain the undifferentiated state of mouse embryonic stem cells.

[Comparison of Different Leaching Methods for Heavy Metals in Sludge Fly Ash and Comprehensive Toxicity Evaluation].

Fly ash from sludge incineration was separated into five different sizes (<1 µm, 1-2.5 µm, 2.5-10 µm, 10-50 µm, and > 50 µm) by high-precision air classification equipment. The leaching of heavy metals was contrastively studied using the HJT 299-2007-sulfuric acid/nitric acid method, HJ 557-2009-Horizontal Oscillation Method, toxicity characteristic leaching procedure (TCLP), and European standard protocol (EN 12457-3) for the different size fractions of the fly ash. Based on the leaching results, an evaluation method for the comprehensive toxicity of heavy metal leaching was established. The results show that the content of heavy metals and the amount of leaching from the fly ash decrease with the increase in fly ash particle size. The leaching of the heavy metals Zn and Cu in the < 1 µm particle size range of TCLP leaching method was the highest, at 107.34 mg·kg-1 and 318 mg·kg-1, respectively. The TCLP and sulfuric acid/nitric acid methods of heavy metal leaching were more effective than the EU (EN 12457-3) and horizontal oscillation methods. According to the value of OPTI, the OPTI value of < 10 µm fly ash was much larger than that of fly ash that was > 10 µm. This indicated that the fly ash of particle size < 10 µm was more toxic and more harmful.

[Effect of Endomorphin-1 on Maturation and Expression of TLR4 in Peripheral Blood Dendritic Cells Induced by High Glucose].

OBJECTIVE: To investigate the effects of endomorphin-1 (EM-1) on the maturation phenotype, cytokine secretion, T cell proliferation and TLR4 expression in human peripheral blood dendritic cells (PBDCs) stimulated and induced by high glucose, and to explore the regulatory mechanism of EM-1 on DC immune function. METHODS: Peripheral blood mononuclear cells (PBMNCs) were induced into immature dendritic cells (imDCs). The high glucose was used as the stimulating factor, and the EM-1 was used as the interventional factor. Then, the experiments were divided into normal glucose group (NG group), high glucose group (HG group), high glucose plus EM-1 group (EM group) and high glucose plus EM-1 and naloxone group (Nal group), respectively. The PBDC's phenotype changes were detected by flow cytometry; ELISA was used to detect the changes of cytokines secreted by PBDCs co-cultured with autologous lymphocytes; CFSE was used to detect the proliferation of T lymphocytes. TLR4 expression on PBDC surface was detected by RT-PCR. RESULTS: Compared with HG group, the expression of PBDC surface molecules CD86, CCR7 and CD36 was up-regulated in EM group (P<0.01), while the change of CD83 expression was not statistically significant. However, IL-12 and IL-10 secreted by PBDCs and the proliferation index of T-lymphocytes stimulated by PBDCs were both decreased in EM group. Compared with EM group, the expression of CD86, CCR7 and CD36 was decreased in Nal group (P<0.01), while the expression of CD83 was almost unchanged (P>0.05). T-lymphocyte proliferation index was increased very significantly in Nal group (P<0.01). The gray ratio of TLR4 in HG group was higher than that in NG group, while the gray ratio in EM group's was very significantly lower than that in HG group's (P<0.01). These results indicate that the high glucose can promote the expression of PBDC TLR4, while the EM-1 inhibits the expression of TLR4. CONCLUSION: EM-1 up-regulates the expression of PBDC surface molecules CD86, CCR7 and CD36 stimulated and induced by high glucose, but inhibites the induction of PBDC to maturity by high glucose. And the secreted inflammatory cytokines IL-12 and IL-10 inhibites the proliferation of T lymphocytes derived from PBDCs, while naloxone inhibites the effect of EM-1. EM-1 inhibites the expression of TLR4 on PBDC surface induced by high glucose.

[Role of mitochondrial permeability transition pore in mediating the effect of endomorphin-1 postconditioning against myocardial ischemia-reperfusion injury in rats].

OBJECTIVE: To investigate the role of mitochondrial permeability transition pore (MPTP) opening in mediating the effect of endomorphine-1 postconditioning to alleviate myocardial ischemia-reperfusion (IR) injury in rats. METHODS: Forty-five male SD rats were randomized equally for sham operation, myocardial IR injury, endomorphin-1 postconditioning, atractyloside (a MPTP opener) postconditioning, or endomorphin-1 + atractyloside postconditioning. The hemodynamic param-eters of the rats were monitored in real time via carotid artery cannulation to the left ventricle. After reperfusion, plasma samples were collected for biochemical analyses. The size of myocardial infarct area was detected using Evans blue and TTC double staining, and the myocardial expressions of apoptosis-related proteins Bax, Bcl-2 and cleaved caspase-3 were analyzed using Western blotting. RESULTS: Myocardial IR injury resulted in significantly decreased heart rate and blood pressure in the rats (P<0.05). Compared with those in IR group, the rats with endomorphin-1 postconditioning showed significantly increased heart rate and blood pressure (P<0.05), lowered contents or activities of LDH, CK-MB, cTnI, IL-6, TNF-α, Cyt-C and MDA in the plasma (P<0.05), increased plasma SOD activity (P<0.05), reduced size of myocardial infarction, decreased myocardial expression of Bax and cleaved caspase-3 protein (P<0.05), and increased myocardial expression of Bcl-2 protein (P<0.05). All these changes induced by endomorphin-1 were obviously reversed by atractyloside postconditioning (P<0.05). CONCLUSION: Endomorphin-1 postconditioning protects against myocardial IR injury in rats probably by inhibiting the opening of MPTP and reducing cardiac myocyte apoptosis via down-regulating cleaved caspase-3 expression.

Pretreatment with intravenous levetiracetam in the rhesus monkey Coriaria lactone-induced status epilepticus model.

PURPOSE: To investigate the antiepileptic and protective effects of intravenous levetiracetam (iv LEV) in the rhesus monkey model of acute status epilepticus (SE). METHODS: Thirty minutes before intraperitoneal induction of SE by Coriaria lactone (CL), rhesus monkeys were treated with LEV (15 or 150 mg/kg) delivered intravenously as a single bolus. CL dose and epileptic behavior were recorded. Electroencephalography (EEG) was performed before and during the experiment. All rhesus monkeys were killed after 1-month video monitoring and processed for pathological investigation of neuronal injury, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining, and glial fibrillary acidic protein (GFAP) staining. RESULTS: No animal exhibited spontaneous seizures during 1-month video monitoring. Development of acute SE was significantly inhibited in the group given 150 mg/kg LEV, compared with controls and the 15 mg/kg LEV group. Delayed latency, reduction of SE duration, decreased cumulative time of tonic convulsions, slight severity of SE, and a high CL induction dose were observed in the high LEV dose group (p<0.05). The EEG showed less frequent epileptic discharges in the group administered with 150 mg/kg LEV. Hematoxylin and eosin (H&E) staining, ultrastructural examination, TUNEL and GFAP staining revealed serious damage, including neuron loss, swollen mitochondrion, and strong positivity for TUNEL in the hippocampus and thalamus of controls, whereas moderate damage in the group administered with 15 mg/kg LEV, and very mild damage in the 150 mg/kg LEV group. Gliosis was found in the hippocampus of controls, not in the LEV groups and normal rhesus monkey. CONCLUSION: The study supports the antiepileptic and protective effect of pretreatment with intravenous LEV in rhesus monkey model with SE.

A novel kindling model of temporal lobe epilepsy in rhesus monkeys induced by Coriaria lactone.

One of the major challenges in developing novel therapeutics for human epileptic disorders derives from the limitation of knowledge of the processes by which epilepsy is generated (epileptogenesis). Furthermore, the inability to obtain human samples at the early stage of epilepsy hinders studies designed to further understand epileptogenesis. Thus, an effective animal model is critical for studies investigating this process. The purpose of this study was to establish a new primate kindling model of temporal lobe epilepsy (TLE) as an animal model of epileptogenesis. Here, repeated injections of Coriaria lactone (CL) at a subthreshold dose elicited partial seizures that culminated in secondarily generalized tonic-clonic seizures. The sequence of events and features of the behaviors observed in this model simulated those observed in human TLE. Electroencephalogram monitoring revealed the temporal lobe origins of the epileptiform potentials, which were consistent with the behavioral changes observed. A total of 7 rhesus monkeys (78%) were kindled with a median of 48 (41 to 60) CL injections. Both the seizure-induction and mortality rates were dose-dependent. A CL injection at 1.50mg/kg showed the lowest animal mortality rate (0%) and the highest seizure-induction rate (100%). Extensive kindling by CL injections with a median of 97 injections (overkindling) subsequently resulted in the recurrence of spontaneous seizures in rhesus monkeys with frequency patterns that were similar to those observed in human TLE. In addition, rhesus monkeys subjected to large numbers of kindling stimuli displayed mitochondrial damage and astrocyte activation in a pattern that was similar to the neuropathological changes characteristic of human TLE. Thus, a kindling TLE model in rhesus monkeys representing a primate animal model of epileptogenesis was established for the first time using repeated intramuscular injections of Coriaria lactone. This model was easily and efficiently performed and resulted in behavioral, electrographical, and anatomical characteristics of human TLE. Thus, this model might be used in future investigations of the mechanisms involved in the epileptogenesis of TLE and in the development of new antiepileptic drugs.

Aberrant glutamate receptor 5 expression in temporal lobe epilepsy lesions.

Glutamate receptor 5 (GluR5) plays a role as an excitatory regulator of synaptic transmission and plasticity; however, its exact role in the pathological mechanism underlying epilepsy is not fully known. We investigated GluR5 expression in resected brain tissues from humans with temporal lobe epilepsy (TLE) and from a macaque model of Coriaria lactone-induced TLE. GluR5 was upregulated in the hippocampus, but not in the temporal neocortex, of patients with TLE compared to the control group. In contrast, GluR5 expression in the hippocampus of macaques treated with Coriaria lactone was not upregulated compared to the control. In addition, mossy fiber sprouting in the hippocampus of TLE patients was correlated with GluR5 upregulation, whereas mossy fiber sprouting was not observed in the macaque model lacking GluR5 upregulation, suggesting that GluR5 function is mainly associated with mossy fiber sprouting.

Suppression of the multidrug transporter P-glycoprotein using RNA interference in cultured rat astrocytes induced by coriaria lactone.

The overexpression of the multidrug resistance gene (MDR-1) and its translational product p-glycoprotein (P-gp) may play an important role in pharmacoresistant epilepsy. We established the rat astrocyte model overexpressing P-gp induced by coriaria lactone and successfully nucleofected it with the siRNA-hairpin expression vector pSIREN-shuttle designed to target MDR-1B mRNA. The mRNA expression of MDR-1B gene was mostly knock down by 67.70% (p<0.01). The expression of P-gp in experimental group was significantly lower than that in negative control (p<0.05), and the rhodamine efflux ratio of experimental group (23.08%) was remarkably lower than that of negative control (78.35%, p<0.01). We first employed RNA interfering to the drug resistance reversal of refractory epilepsy and this may provide a new way for refractory epilepsy remedy.

Economic burden of epilepsy in a developing country: a retrospective cost analysis in China.

PURPOSE: To study the cost of epilepsy in China, and, therefore, provide essential information on the burden of the disease to individuals and society. METHODS: A cost-of-illness study was performed on a retrospective cohort of medically treated patients. Data on clinical characteristics, utilization of sources, and costs were collected from 289 patients in a standardized format. RESULTS: Direct medical care costs was Chinese yuan, renminbi (RMB) 2,529 (USD 372) per year per patient, of which antiepileptic drugs (RMB 1,651 or USD 243) accounted for the major cost component. Nonmedical direct costs were much less than direct health care costs, averaging approximately RMB 756 (USD 111). Costs due to loss of productivity averaged approximately RMB 1,968 (USD 289) per patient per year. Taken together, the overall mean annual cost for epilepsy per patient in our series was approximately RMB 5,253 (USD 773), and these costs accounted for more than half of the mean annual income. Total cost was significantly associated with disease severity and different responses to drug treatment. In addition, new antiepileptic drugs and the number of drugs taken were closely related with the drug cost. CONCLUSION: The results indicate that the economic burden of epilepsy to both Chinese patients and the nation is heavy, and the composition proportions of the costs in China have many similar features and some noteworthy differences with that of other countries.

[Construction of recombinant adenovirus vector for RNA interference with multidrug resistance MDR1 gene].

OBJECTIVE: To construct the recombinant adenoviral RNA interference (RNAi) vector in order to inhibit the expression of multidrug resistance MDR1 gene, and probe whether gene therapy for multidrug resistance of epilepsy is feasibility. METHODS: Three target sequences for short hairpin RNA (shRNA) expression were selected and designed according to MDR1 gene sequence of rat. Annealed oligos were inserted into the downstream of treated pSIREN-shuttle U6 promoter to construct RNAi plasmid pSIREN-shuttle-MDR1. Next, MDR1 shRNA sequence was cloned to pAdeno-X, a transfer vector of adenovirus, to produce the pAdeno-MDR1, which was then packed and amplified in HEK293 cells. Further the recombinant adenovirus was purified by CsCl and used to infect the rat astrocytes with P170-glucoprotein (P-gp) over-expression which have been induced by coriaria lactone (CL). RESULTS: It was confirmed by restriction digestion, PCR and sequencing that MDR1 shRNA expression structure was correctly cloned to pSIREN-shuttle and pAdeno-X vector respectively. Virus titer was 6 x 10(9) pfu/mL. The interference efficiency of pAdeno-MDR1 to the expression drop of multidrug resistance gene in astrocyte model neared to 100%. CONCLUSION: RNAi adenovirus vector of rat MDR1 gene has been constructed and found its high interference efficiency. It is the essential building required for the remedy of refractory epilepsy and the research on mechanism of multidrug resistance.

[Migration characteristics of heavy metals during sintering of fly ash from municipal solid waste incinerator].

The sintering process is used to stabilize the heavy metal in fly ash from municipal solid waste incinerator (MSWI). Migration characteristics of 6 targeted heavy metals (Cd, Pb, Cu, Ni, Cr and Zn) in the sintering process of MSWI fly ash were investigated by experiments. Effect of several factors including sintering temperature, residence time, molding pressure and particle diameter on the migration of heavy metals were discussed in details. The results show that cadmium and lead are volatile metals, while nickel, copper, chromium and zinc belong to involatilizable metals. The effects of sintering temperature, residence time, molding pressure and particle diameter on the stabilizing efficiency differ from each other. The study shows that most of heavy metals are stabilized during the sintering process. The stabilizing efficiency of heavy metals was different for different elements.