Thymus quinquecostatus Celak. (thyme oils) obtained from Loess Plateau in China had been studied. 130 constituents of thyme oils were determined using gas chromatography-mass spectrometry (GC–MS) and carvacrol ethyl ether was firstly reported as a new natural product, which has been used as a synthetic flavoring substance with no safety concern. The thyme oils showed the anti-oxidant activity using 2,2 diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS), ferric reducing antioxidant power (FRAP) and thiobarbituric acid reactive substances (TBARS) and conferred protection against oxidative stress in zebrafish. In addition, a class of carvacrol analogues was found to develop as potential natural antioxidant products of thyme oils from Loess Plateau by the correlation analysis. YL-thyme oil performed the best antioxidant activity in this research, which could be recommended as preferred sources of thyme oils. Furthermore, YL-thyme oil exhibited a potent antioxidant capacity by reactive oxygen species (ROS) scavenging, enhancing the endogenous antioxidant system, inhibiting lipid peroxidation and activation of Keap1/Nrf2 pathway in zebrafish.Chemical profile and antioxidant potency of essential oils (EOs) of  Most of ROS such as superoxide anion (O2−), hydroxyl radical (OH·), hydrogen peroxide (H2O2) and organic peroxides, are generated in the cells by mitochondrial respiratory chain2. Excessive ROS can damage cell functionality as they can harm cellular lipids, proteins and DNA3. The organism contains a complex and carefully balanced cascade of antioxidant enzymes, of which the superoxide dismutase (SOD) and catalase (CAT) are regarded as the first line of the antioxidative enzyme system against ROS such as superoxide and H2O2 generated during oxidative stress4. The kelch-like ECH-associated protein 1/nuclear factor E2-related factor 2 (Keap1/Nrf2) signaling pathway plays a central role in protecting the body from oxidative damage. The Nrf2 transcription factor is tightly regulated by the repressor protein, Keap1, in the cytoplasm. Under oxidative stress, Nrf2 is released from the Nrf2/Keap1 complex and is translocated to the nucleus. Subsequently, it binds to antioxidant-response elements (AREs) and upregulates the expression of downstream genes, such as Sod1, Cat and Hmox1, which in turn, activates cellular antioxidant defensive capacity5.Oxidative stress is a phenomenon associated with pathogenetic mechanisms of several diseases including atherosclerosis, neurodegenerative diseases, cancer, diabetes mellitus, inflammatory diseases, as well as psychological diseases or aging processes. It is defined as an imbalance between production of free radicals and reactive metabolites, so-called oxidants or ROS6. The genus Thymus belongs to the Lamiaceae family, and contains more than 300 species of aromatic perennial herbaceous plants with valuable medicinal properties7. T. quinquecostatus, commonly called thyme (‘thyme’ in this study refers to T. quinquecostatus), was distributed in the northwest of China, mainly in the Loess Plateau. It is locally used as a seasoning and also has natural preservative effects which makes it widely used as a green harmless flavor additive. Thyme was widely used to treat stroke, cold, dyspepsia, toothache, acute gastroenteritis, hypertension, chronic eczema and other diseases as folk medicines and was used alone or in combination with other herbal medicines for cancer treatment. In our previous researches, the antioxidant activities of different polar fractions of T. quinquecostatus had been evaluated. Meanwhile, chemical compositions, like flavonoids and phenolic acids, were also identified and full-scale qualitative and quantitative methods were successfully applied to the quality evaluation of T. quinquecostatus9.The beneficial effects of antioxidants on the maintenance of health in human have become an important subject that has engaged many scientists across the world over the last decade. The antioxidants from plants have been reported to inhibit the propagation of free radical reactions and protect the human body from various diseases10. To our best knowledge, remarkable chemo diversity of the same plant species has been reported due to the differences in chemotype, plant genotype, harvesting time, cultivation and climatic peculiarities and other factors; therefore, investigation of the same plant species from different geographic zones may considerably expand the existing knowledge on essential oils (EOs).Thyme oil has been widely used in food industry, pharmaceutical and cosmetic due to its various biological activities, including antioxidant, anti-inflammatory, antitumor and antimicrobial effectsAnd previous reports mainly focused on thyme oils from one certain district, while the diversity of chemical compositions in thyme oils collected from different producing areas has not yet been studied and the relationship between thyme oils compositions and biological activities is rarely mentioned. Moreover, most of the antioxidant researches on thyme oils focused on in vitro studies and the underlying mechanism of antioxidant activity of thyme oils is still unclear.Thymus genus plants as well as the variety of factors, which may influence EO compositions and their biological activities, T. quinquecostatus cultivated in the Loess Plateau of four main producing regions, namely YL (Shaanxi province), JB (Shaanxi province), QY (Gansu province) and LD (Ningxia Hui Autonomous Region) had been selected for the present study. Combined with statistical analysis, the compositions of thyme oils from the four producing areas were comparatively analyzed and the correlations between these thyme oil components and anti-oxidative activities were also discussed. Besides, their antioxidant potentials were evaluated by the in vitro assays  as well as in the in vivo zebrafish model. Moreover, the effects of thyme oils on expression of genes related to oxidative stress were investigated. This study provides a better understanding of antioxidant capacities of thyme oils and the underlying molecular mechanisms. Figures Considering polymorphism of T. quinquecostatus were collected from YL  and JB  in Shaanxi province, QY  in Gansu province and LD  in Ningxia Hui Autonomous Region in the northwest of China in the month of August. They were carefully identified by Dr. Shengjun Ma (Beijing University of Chinese Medicine). Those samples were separately dried in shade and deposited at the Lab of Engineering Research Center of GAP for Chinese Crude Drugs, Ministry of Education, Beijing University of Chinese Medicine.The aerial parts of n-hexane. Subsequently, the extracts were exsiccated by anhydrous sodium sulphate and stored in a stopper vials at 4 °C in a refrigerator until the time of analysis.Dry samples (300 g) of thyme from the four producing areas were severally soaked in deionized water at room temperature about 25 °C to stand overnight, and then were subjected to hydrodistillation in a modified clevenger-type apparatus for 10 h. The obtained EOs were extracted with 6–C24) was also analyzed using the above conditions to determine the retention index (RI).EOs were analyzed by an HP 5890 gas chromatograph (GC) using an HP 5973 mass selective detector  with the electron ionization mode (70 eV). The capillary column was a HP-5MS . The temperature of the inlet and detector, injection volume and split ratio were set to 260 °C, 260 °C, 1 µL (TBME solution), 40 : 1, respectively. The oven temperature started at 50 °C, ramped to 100 °C at a rate of 5 °C/min, ramped to 150 °C at a rate of 3 °C/min, and ramped to 250 °C at a rate of 5 °C/min, then held at 250 °C for 30 min. Helium (99.999%) carrier gas was kept with a constant flow of 1.2 mL/min. A standard n-alkane mixture (C6–C24) at the same chromatographic conditions, referring to the Van Den Dool method11. The data were analyzed by the Xcalibur 1.1 software, compared with NIST/EPA/NIH database  and several references13. The book “Identification of Essential Oils Components by Gas Chromatogrtaphy/Mass Spectrometry, 4th edition”  equally played a vital role in identifying the individual compounds.The thyme oils were identified by the retention indices (RI), relative to the series of alkanes (C14. 0.1 mL thyme oils at various concentrations of (0.02–5.67 mg/mL) were added in 0.1 mL ethanol solution of DPPH (0.001 mg/mL) acquired from Sigma-Aldrich . The absorbance of each solution was measured at 517 nm, with ethanol being a blank. Ascorbic acid was used as a positive control. Simple regression analysis was used to derive the IC50 value.The DPPH test was carried out as described before15. An ABTS stock solution was prepared by dissolving ABTS (Sigma-Aldrich) (7 mM) in water. An ABTS radical cation (ABTS·+) solution was then prepared by mixing the ABTS stock solution with potassium persulfate solution (2 mM) in equal quantities and in the dark at room temperature. The ABTS·+ solution was diluted with PBS  to an absorbance of 0.70 ± 0.02 at 734 nm and stabilized at 30 °C. Next, 0.1 mL thyme oils at different concentrations (0.02–2.32 mg/mL) were mixed with 0.1 mL dilution ABTS•+ solution with ethanol; after 10 min, the absorbance of the mixture was measured at 734 nm in the dark at room temperature. As a positive control, ascorbic acid was chosen to be employed. Simple regression analysis was used to derive the IC50 value.The antioxidant activity of the samples was determined by using the method described previously16. 5 mL of freshly prepared FRAP reagent and 20 uL thyme oils (2.32–16.87 mg/mL) were mixed and the resulting solution was vortexed and then incubated at room temperature for 70 min. After the incubation, the absorbance was measured at 594 nm. Ferrous sulfate solutions (FeSO4) (1–33 mmol·L−1) were used to obtain the standard curve and trolox (0.975 mg/mL) was used as the positive control. The results were expressed as mmol of ferrous sulfate equivalents per gram per liter of thyme oils. The ferric reducing activity of all samples was expressed as FeSO4 equivalent (mmol·L−1·g−1DW).FRAP activity was estimated by the assay described by Benzie and Strain (1996)17. Briefly, thiobarbituric acid reacts with malondialdehyde (MDA) to form a diadduct, a pink chromogen, which can be detected spectrophotometrically at 535 nm. 0.2 mL thyme oils at different concentrations (0.18–1.82 mg/mL) were mixed with 10% liver homogenate (each 100 mL homogenate solution contains 10.0 g mouse liver), then 0.1 mL of FeSO4 · 7H2O (5 mM) and 0.1 mL H2O2 (100 mM) were added to initiate lipid peroxidation. After incubation for 60 min at 37 °C, the reaction was stopped by addition of 0.3 mL 0.68% thiobarbituric acid in TCA-HCl (16.8% w/v trichloroacetic acid in 0.125 N HCl). The reaction mixtures were heated for 60 min at 95 °C. The samples were cooled and centrifuged, and the absorbance of the supernatants was measured at 535 nm. Simple regression analysis was used to derive the IC50 value. The results were compared to that of ascorbic acid employed as the reference.The TBARS assay is widely used to measure lipid oxidation and antioxidant activity in food and physiological systems. Modified TBARS were used to measure the lipid peroxide using mouse liver homogenates as lipid-rich mediacy17 transgenic zebrafish lines were obtained from Drug Screening Platform of Shandong Academy of Sciences . The adult zebrafish used in this study were reared at 28 ± 0.5 °C with a 14 : 10 h light–dark cycle in an automatic zebrafish housing system  with fish water . Embryos were collected and maintained in the zebrafish spawning box after natural spawning induced by light in the morning and then maintained in a light incubator at 28 °C until 24 h post fertilization (hpf). At the end of the experiments, all zebrafish were euthanized with an overdose of tricaine (200 mg/L).The adult zebrafish AB strain and Tg (krt4:NTR-hKikGR)18, normal developing embryos were selected under a stereomicroscope  at 24 hpf and were randomly tranferred to 24-well plates at a density of 15 per well. Thyme oils were diluted with fish water to different concentrations  in each well to a final volume of 2 mL. Mortality rates were investigated till 48 hpf. Also, morphological changes of craniofacial features, brain, kidney, yolk sac, swim bladder, pericardium and body shape were monitored under the stereomicroscope. And the value of 10% lethal concentration (LC10) and 1% lethal concentration (LC1) were calculated. In subsequent experiments, thyme oils were used at concentrations of 1/3 LC1, LC1, LC10.As depicted in previous researchcy17 zebrafish (15 embryos/well) were added to 24-well plates, and incubated with or without metronidazole (MTZ) (5 mM) (Sigma-Aldrich). Thyme oils were diluted with fish water to different concentrations  to a final volume of 2 mL. After 24 h of incubation, zebrafish larvae were anesthetized using 0.16% tricaine, and then fluorescence was detected by FSX100 Bio Imaging Navigator Equipment. N-acetyl-l-cysteine (L-NAC) (0.2 mM) was used as a positive control. L-NAC is a classical antioxidant that can reduce the damage to cells caused by oxidative stress and has been widely used as an antioxidant positive control in vivo20. The relative antioxidant capacity of the thyme oils can be determined by counting the number of fluorescence spots using Image—Pro Plus software. A fixed square region (18.00 cm × 6.00 cm) of the images on zebrafish body was selected and calculated the number of fluorescence.24-hpf Tg (krt4: NTR-hKikGR)21. All treatment solutions were diluted to different concentrations using fish water. 24-hpf AB zebrafish (15 embryos/well) were added to 24-well plates, and incubated with or without thyme oils  to a final volume of 2 mL for 1 h. Then, 2,2-Azobis (2-amidinopropane) dihydrochloride (AAPH) (12 mM) was added to incubate for 24 h. L-NAC (0.2 mM) was used as a positive control. A ROS kit  was used for analyzing generation of ROS in larvae at 48 hpf. The treatment groups were treated with 30 μM DCFH-DA and incubated in the dark at 28 ± 0.5 °C for 40 min. The zebrafish larvae were then washed with fish water three times and were observed using a fluorescence microscope . Fluorescence intensity of each zebrafish larva was determined using the Image—Pro Plus software.The dichloro-dihydro-fluorescein diacetate (DCFH-DA) method was used to quantify intracellular ROS levels18. Briefly, 24-hpf AB zebrafish were transferred into six-well plates (100 larvae/3 well/group), and incubated with or without AAPH (12 mM) and YL-thyme oils  to a final volume of 5 mL. L-NAC (0.2 mM) was used as a positive control. All treatment solutions were diluted to final different concentrations using fish water. Larvae in each group were pooled together and homogenized on ice with 500 μL ice-cold physiological saline at 48 hpf. The supernatants were collected for analysis of antioxidative enzyme and lipid peroxidation activities after centrifugation at 3,500 rpm for 15 min at 4 °C. SOD, CAT activity and MDA levels were assessed using commercial kits (Nanjing Jiancheng Bioengineering Institute) in accordance with the manufacturer’s protocols. SOD and CAT activity were expressed as unit per milligram protein and MDA levels were showed as nmol per milligram protein. And the protein content was determined using the bicinchoninic acid method22.The test was carried out as described before260/OD280 ratio. Then the synthesis of cDNA was carried out by using the HiScript II Q RT SuperMix , and RT-PCR was done by using a RT-PCR system  using the SYBR Green mix . Thermal cycling was set at 95 °C for 5 s and 60 °C for 30 s with 40 cycles. Transcription of target genes was calculated using the 2−ΔΔCt method. β-actin was chosen as the house-keeping gene. The primer sequences of the genes that were detected are shown in Table 24-hpf AB zebrafish were transferred into six-well plates (30 embryos/well), and incubated with or without AAPH 12 mM) and YL-thyme oils  to a final volume of 5 mL. L-NAC (0.2 mM) was used as a positive control. All treatment solutions were diluted to final different concentrations using fish water. Total zebrafish RNA was extracted from 30 larvae (48 hpf) using the TRIzol reagent  following the manufacturer’s instructions. RNA concentrations and quality were evaluated on the basis of OD2 mM and p was less than 0.05 or 0.01. Removal of the compounds with concentrations less than 0.1% had no effect on the overall analysis. Therefore, the thyme volatile constituents with the content more than 0.1% were evaluated using Principal Component Analysis (PCA) and Hierarchical Clustering Analysis (HCA), which could highlight the similarity and dissimilarity of the EO constituents and expound the active ingredients of thyme oils. Correlation analyses were performed by using a two-tailed Pearson’s correlation test. All statistical analyses were carried out by using OriginPro 2017 and GraphPad Prism (version 6.01).All the assays were conducted in triplicate and results were expressed as mean values ± standard deviation (SD). Analysis of variance (ANOVA) test was performed to check significant differences. Comparison between the groups was performed using Student–Newman–Keuls (SNK). Differences from controls were considered significant when T. quinquecostatus were hydrodistilled and produced a yellow EOs with characteristic odor, with the yield of 4.67 mL/kg (YL), 2.33 mL/kg (JB), 5.00 mL/kg (QY) and 2.67 mL/kg (LD). Total ion chromatograms (TICs) given by GC–MS of the thyme oils was shown in Fig. The aerial parts of Among the EO components with the content greater than 0.1%, 1,8-cineole, linalool, terpinen-4-ol, γ-terpinene, borneol, β-bisabolene and α-pinene were the common ingredients in the thyme oils from the four regions.T. vulgaris L. with a percentage of 9.8%23. Furthermore, 1,8-cineole has been widely used in perfume industry and pharmaceutical industry24. Compared to the essential oil of thyme (T. vulgaris L. and T. zygis L.) in International Standard , the percentage of linalool (1.22%–12.80%) and terpinen-4-ol (1.03%–10.56%) in this study were far in excess of this standard. Meanwhile, the content of γ-terpinene (0.15%–11.11%) and α-pinene (0.07%–1.04%) were in accordance with International Standard .The content of 1,8-cineole (1.84%–10.16%), which is the highest in the QY-thyme oil (10.16%), was similar to that from 25. Additionally, β-bisabolene is a popular resource for the synthesis of many natural products and it has been approved as a food additive in Europe26. The content of β-bisabolene was the highest in QY-thyme oil (2.84%), followed by that of LD-thyme oil (2.36%), JB-thyme oil (1.03%) and YL-thyme oil (1.12%).The borneol was observed in amounts of 6.50% in YL-thyme oil, similar to that of JB-thyme oil (5.61%). It showed the content of 1.52% in the QY-thyme oil, similar to that of LD-thyme oil (1.38%). Nowadays, it is a famous compound in prescription for cardiovascular and cerebrovascular diseases27. Additionally, JB-thyme oil displayed the highest content of terpinen-4-ol (10.56%) while 4.96% in YL-thyme oil. Thymol, the isomeric of carvacrol, was observed as a dominant compound (16.32%) in QY-thyme oil. It exhibited significantly antifungal, antibacterial and anti-inflammatory properties, together with anticonvulsant and antiepileptogenic activities as adjuvant to AEDs28. Finally, there were two components that had not been identified. One compound  composed 54% of the total volatile ingredients in LD-thyme oil. And another one  accounted for 10.16% of the volatile composition in YL-thyme oil.Notably, the carvacrol ethyl ether was firstly discovered as a new natural product and its amount is significant in YL-thyme oil (31.80%) and JB-thyme oil (23.32%). It is the pale yellow clear oily liquid and has spicy odor. Carvacrol ethyl ether has been used as a synthetic flavoring substance with no safety concern at current levels of intake evaluated by the Joint FAO/WHO Expert Committee on Food Additives (JECFA). The interesting component ρ-vinyl guaiacol was indicated with the highest content in QY-thyme oil (23.55%), followed by that of YL-thyme oil (3.97%). Vinyl guaiacol appeared as colorless or canary yellow liquid that mostly found in the volatile from corn alcohol fermentation and it is a high value-added product widely used in cosmetic, pharmaceutical and chemical industriesThe content of compounds greater than 0.1% of the thyme oils had been used for chemometric analysis. Both the chemical composition and their content of the total EOs from the four regions were obviously different. It was interesting that the thyme oils from JB and YL were in great similarity. It might result from the close distance between JB and YL, as well as the similar growing conditions such as climate, strength of illumination, humidity of soil and air etc. Thus, growing condition might be the main influencing factor to the chemo-variation in the thyme oils produced from the four regions.29. In present study, score plots were created in order to assess clustering tendency of samples, and three significant principal components (PCs) explained 40.56% (PC1), 33.63% (PC2) and 25.80% (PC3) of total variance . By plotting the PCA scores, it is possible to visually assess similarities between samples and determine whether samples can be grouped or notThe loading vectors responsible for the separations were distributed as Fig. 30. Using Dlinkage = 22.9 as a criterion for the selection of the number of groups, three clusters were obtained: Cluster I (QY-thyme oil), Cluster II (LD-thyme oil) and Cluster III (JB-thyme oil and YL-thyme oil)  Fig. d and the31. In this study, the in vitro antioxidant activity of thyme oils was evaluated using the DPPH, ABTS, FRAP and TBARS methods.Phytochemicals are known to have a complex nature hence no single assay will accurately reflect all of the radical sources or all antioxidants in a mixed or complex system50 = 0.026 mg/mL), the YL-thyme oil possessed the highest antioxidant activity (IC50 = 0.512 mg/mL), followed by 0.530 mg/mL in JB-thyme oil, 0.905 mg/mL in LD-thyme oil and 0.931 mg/mL in QY-thyme oil , which indicated that chemical compositions of thyme oils greatly affected the antioxidant activity.Characterized by being simple, rapid, accurate and highly sensitive3+-TPTZ] complex and produces a colored ferrous tripyridyltriazine [Fe2+-TPTZ], so FRAP is a reasonable screen for the ability to maintain redox status in cells or tissues31. Calibration curves (Y = 0.0379X + 0.0103), in the range of 1 mmol·L−1–33 mmol·L−1, showed good linearity (R2 ≥ 0.9992). The reducing power capacities results follow the same pattern as in case of radical scavenging assay, in which the YL-thyme oil was the most active one (2.917 mmol·L−1·g−1DW), followed by that from JB (2.549 mmol·L−1·g−1DW), LD (2.413 mmol·L−1·g−1DW) and QY (1.956 mmol·L−1·g−1DW). Comparing these results with the standard antioxidant trolox (7.28 mmol·L−1·g−1DW), it was revealed that the thyme oils had significantly lower reducing potential than the standard  among the tested thyme oils were found.Lipid peroxidation is an oxidative alteration of polyunsaturated fatty acids in the cell membranes that generates a number of degradation products. It can be initiated when fatty acids or fatty acyl side chains are attacked by free radicals. This oxidation process could affect the structural and functional damage of biomolecule50 values of YL-thyme oils with that of the authentic antioxidant ascorbic acid, it was found that the antioxidant activity of (IC50 = 0.080 mg/mL) was quite comparable with that of ascorbic acid (IC50 = 0.058 mg/mL). At a concentration of 0.563 mg/mL, the inhibition effect of YL-thyme oil was 88.90%. The others were ranked in the following order: JB-thyme oil (IC50 = 0.198 mg/mL), QY-thyme oil (IC50 = 0.241 mg/mL) and LD-thyme oil (IC50 = 0.376 mg/mL) , FRAP as well as TBARS. In contrast to this finding, β-bisabolene was inversely and strongly related to the DPPH . This inverse relationship would indicate that the higher content of the β-bisabolene did not exert protection, but actually promoted oxidation. Interestingly, carvacrol ethyl ether presented statistically significant positive correlations with DPPH  and the Code of Federal Regulations lists it as a synthetic flavoring substance that could be safely used in foods (21CFR172.515). Carvacrol methyl ether  and borneol  both showed statistically significant positive correlation with DPPH. All the compounds mentioned above are on the list of flavoring substances provided by Regulation (EC) No 2232/96 of the European Parliament and of the Council, which was introduced in Annex I to Regulation (EC) No 1334/2008 of the European Parliament and of the Council.Furthermore, it is important to correlate and understand which compounds contribute to the different antioxidant assays, showing specific antioxidant potential for the different radicals depending on their chemical structure. The correlations between thyme oil components and antioxidant activities were reported in Table p < 0.05), carvacrol acetate  and dauca-5,8-diene  was obvious. This tendency is explained by the high scavenging power of these components. Similarly, the latter two were considerably correlated with TBARS  and these observations are consistent with published data36. However, for FRAP, all of them were not statistically significant. These compounds mentioned above were found in the highest amounts in the YL and JB thyme-oil. This indicated that the higher level of these components is likely to be closely related to the relatively high antioxidant activity of YL and JB-thyme oil. These differences observed between assays are related to the individual molecular structure of each compound which indicated that stereoisomerism, functional groups distribution and any other structural parameters such as the oxidation state of the C ring, the hydroxylation and methylation pattern also are expected to affect the final value. In particular, there were good correlations of carvacrol ethyl ether both with IC50 value of DPPH and ABTS and carvacrol acetate both with IC50 value of ABTS and TBARS, which could be recommended as an indicative component for the antioxidant capacity. It is acknowledged that carvacrol is used to defined the quality of the Thymus species and widely used in the food industry as antimicrobial and antioxidant agent. Thus, in the present work, carvacrol methyl ether, carvacrol ethyl ether and carvacrol acetate were all found to be significantly associated with antioxidant activity, implying the carvacrol analogues as potential natural antioxidants of thyme oils from Loess Plateau in China.The correlation between ABTS and compounds, including isobornyl acetate cy17 is established which overexpresses the NTR-hKikGR fusion protein under the control of the skin-specific krt4 promoter. It provides as a unique quantitative and fast tool to study the signaling molecules which modulate skin apoptosis in living animals37. The fluorescence spots of zebrafish are shown in Fig. The in vivo capability of thyme oils on inhibiting oxidative stress was further evaluated using MTZ-induced oxidative insult in Tg (krt4: NTR-hKikGR)The antioxidative effect of thyme oil on ROS production in zebrafish larvae was examined by detection of DCFDA. Fluorescent photographs of larvae showed that the untreated zebrafish had low fluorescence intensity, whereas AAPH-induced group generated a marked increase in the observed fluorescence intensity, indicating that ROS was produced in the presence of AAPH in the zebrafish larvae.The scavenging efficacy of the thyme oil on the ROS production in AAPH-induced zebrafish larvae was measured. At 48 hpf, the larvae in the YL-thyme oil-treatment and JB-thyme oil-treatment groups  were examined and shown in Fig. 5. Moreover, Nrf2/Hmox1 pathway is widely studied in vertebrates and play a pivotal role in neurodegenerative disorders. Hmox1, as Nrf2-dependent gene, provides cytoprotective effect and play a crucial role in the development of oxidative and age-related disorders40. Also, Hmox1 is known to play an important role in cellular protection against oxidative insult in cardiovascular disease, including diabetes, and in the alleviation of vascular diseases and various chemical reagents were known to facilitate antioxidant response via activating Hmox1 expression41. In the present study, YL-thyme oil-treated zebrafish larvae exhibited significant downregulation of Keap1 expression and upregulation of Nrf2 expression; furthermore, the expression level of Sod1, Cat and Hmox1 were significantly increased in the treatment of YL-thyme oil groups. AAPH-induction decreased the expression of Sod1, Cat, Nrf2 and Hmox1 in zebrafish by 1.39, 1.41, 2.03 and 2.02 folds, respectively and increased Keap1 levels by 3.47 folds when compared to control group. In the YL-thyme oil-treatment group and L-NAC-treatment group, the expression levels of genes encoding the Sod1 (Fig. In order to investigate the mechanism of YL-thyme oil-treated antioxidant activity, the mRNA expression levels of genes related to the antioxidant activities were measured Fig. . Nrf2 anod1 Fig. a and Nrfod1 Fig. d were siod1 Fig. b and Hmood1 Fig. e were siod1 Fig. c. Taken T. quinquecostatus. One new natural compound, carvacrol ethyl ether, was firstly discovered in the thyme oils. It has been used as a synthetic flavoring substance with no safety concern. The thyme oils from the four regions were classified into three groups by chemometric analysis, out of which the borneol, isobornyl acetate, carvacrol methyl ether, ρ-vinyl guaiacol, thymol, carvacrol ethyl ether and linalool contributed the most to the classification, indicating that these compounds may be used for the geographical markers of the thyme oils. The YL-thyme oil was found to produce superior EO yields and possess the strongest anti-oxidant activities in vitro and in vivo, suggesting that domestication of this thyme oil is economical for the food industry. It was also proved with correlation analysis that borneol, carvacrol methyl ether, carvacrol ethyl ether, carvacrol acetate, isobornyl acetate and dauca-5,8-diene could be the characteristic constituents responsible for the antioxidant effect of the thyme oils. This indicated that the carvacrol analogues might be developed as potential natural antioxidants of thyme oils from Loess Plateau in China. Furthermore, Keap1/Nrf2 pathway contributed to the anti-oxidant processes of YL-thyme oil. Taken together, our results suggest that thyme oil could be useful as the natural antioxidant and have the potentiality of enhancing the protective effect against oxidative stress. The findings of the present study can provide new insights for enhancing the valuable components and antioxidant capacity of this plant in their EOs. Therefore, this study could lay foundation for the applications of thyme oils in food, pharmaceutical and cosmetic industry, and also provide promising sources for natural antioxidants and a new possible breakthrough point for further bioactivity studies of thyme. Admittedly, there are limitations to the current study. CC-MS has been used to quantified the thyme oils components and we didn’t examine the data further by GC-FID which might be more precise than GC–MS in quantifying complex mixtures. In addition, we cannot completely determine the major contribution of components for the antioxidant activities of thyme oils. The potential antioxidants, especially the carvacrol analogues from the correlation analysis should be assessed in future studies. Furthermore, further studies can be performed to delineate other candidate genes and signaling pathways, especially those closely correlated with anti-oxidation, in thyme oils-mediated effects.This is the first study on the chemical constituents, antioxidant activities and biological mechanism of EOs of All experimental procedures were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and approved by the Experimental Animal Ethics Committee of the Academic Committee of Beijing University of Chinese Medicine.


This study analyzed microvesicles and exosomes, called as extracellular vesicles (EVs) excreted in serum and cerebrospinal fluid (CSF) from patients with cerebral or gestational toxoplasmosis.Clinical samples from 83 individuals were divided into four groups. Group I, 20 sera from healthy individuals and pregnant women (seronegative for toxoplasmosis); group II, 21 sera from seropositive patients for toxoplasmosis ; group III, 26 CSF samples from patients with cerebral toxoplasmosis/HIV co-infection (CT/HIV) (seropositive for toxoplasmosis); and group IV, 16 CSF samples from seronegative patients for toxoplasmosis, but with HIV infection and other opportunistic infections (OI/HIV). Serum and CSF samples were ultracentrifuged to recover EVs. Next, vesicle size and concentration were characterized by Nanoparticle Tracking Analysis (NTA).10 EVs/mL) were statically higher than of non-infected individuals (mean: 5.9 x 109 EVs/mL). Concentrations of CSF-derived EVs were almost similar in both groups. CT/HIV (mean: 2.9 x 109 EVs/mL) and OI/HIV (mean: 4.8 x 109 EVs/mL). Analyses by NTA confirmed that CSF-derived EVs and serum-derived EVs had size and shape similar to microvesicles and exosomes. The mean size of EVs was similar in serum and CSF. Thus, the concentration, and not size was able distinguish patients with toxoplasmosis than healthy individuals. Presence of exosomes was also confirmed by transmission electron microscopy and evidence of tetraspanins CD63 and CD9 in immunoblotting. Relative expressions of miR-146a-5p, miR-155-5p, miR-21-5p, miR-29c-3p and miR-125b-5p were estimated in exosomal miRNA extracted of EVs.Concentrations of serum-derived EVs from toxoplasmosis patients  up-expressed miR-125b-5p and miR-146a-5p. CSF-derived EVs from CT/HIV patients) up-expressed miR-155-5p and miR-21-5p and were unable to express miR-29c-3p.These data suggest the participation of EVs and exosomal miRNAs in unbalance of immune response as elevation of TNF-α, IL-6; and downregulation of IFN-γ in cerebral and gestational forms of toxoplasmosis. Toxoplasma gondii is an infection asymptomatic in immunocompetent hosts. Chronic phase is characterized by persistence of encysted parasites in brain and muscle. In asymptomatic or subclinical form, mild symptoms occur during first few weeks [Toxoplasmosis, caused by ew weeks –3. Neverew weeks ,5. Infecew weeks –8. Oculaew weeks ,10. Reacew weeks –14.T. gondii induces changes in immune response of infected hosts in both, innate and adaptive ones. Thus, infected hosts develop life-long protective immunity against re-infection against. Consequently, antigen secretion is essential in boosting the immune system since they stimulate the T and B-cell responses [esponses ,15. Thesesponses –18.Extracellular vesicles (EVs) are a group of small structures that are released by cells . EVs areEVs are purified by ultracentrifugation or chromatography. Among methods for examination include the electron microscopy and Nanoparticle Tracking Analysis (NTA) . EVs comT. gondii and hosts is not yet established. Regarding on T. gondii, proteomic profile of exosomes containing a wide range of proteins [T. gondii derived-EVs (exosomes and microvesicles) contain miRNA and they were immunologically recognized by host immune response inducing humoral and cellular responses as IL-10, TNF-α, and iNOS [T. gondii infection alters cell proliferation mechanisms of infected cells and exosomes secreted by T. gondii-infected cells can mediate such changes to neighboring cells [T. gondii infection and EVs derived of humans is unknown. Thus, this study was aimed to characterize these nanoparticles, including microvesicles and exosomes excreted in serum and cerebrospinal fluid (CSF) from patients with cerebral or gestational toxoplasmosis.The participation of EVs in interaction between proteins . T. gondand iNOS ,31. Exosand iNOS ,33. T. gng cells –36. HoweThe Ethic Committees of Instituto Adolfo Lutz (CONEP-IAL/SES number: 2922263), Instituto Emilio Ribas (CONEP-IIER number: 1133380) and Faculdade de Medicina de São José do Rio Preto (CEP-FAMERP number: 712142) by Plataforma Brasil (from Brazilian Ministry) approved this study, with waive of the patient consent, which was performed according recommendations of Plataforma Brasil.This study analyzed human clinical samples from 83 individuals (41 serum samples and 42 CSF) divided into four groups.Group I was formed of 20 serum samples from healthy individuals and pregnant women. All of them were seronegative for toxoplasmosis in ELISA. In period of blood collection, none of them was in treatment for any infection or disease.T. gondii antibodies were underwent to amniocentesis and peripheral blood for PCR. Before collection pregnant women were informed of the procedure and signing informed-consent forms.Group II was formed of 21 serum samples from seropositive patients for toxoplasmosis . Clinical diagnosis was established by clinical manifestations, and laboratorial, by ELISA, and qPCR. HIV-infected patients were admitted and treated at Instituto de Infectologia Emilio Ribas, in São Paulo, Brazil. Clinical diagnosis of cerebral toxoplasmosis in HIV-infected patients was based on: 1) progressive neurological deficits; 2) contrast-enhancing mass lesion(s) on computed tomography and/or magnetic resonance imaging; and 3) successful clinical and radiological response to antiparasitic treatment within 10–14 days ,14. PregGroup III was composed of 26 CSF samples from patients with cerebral toxoplasmosis/HIV co-infection (CT/HIV). The diagnosis was determined by clinical manifestations, ELISA, and qPCR, as described in Group II item.Group IV was formed of 16 CSF samples from seronegative patients for toxoplasmosis, but with HIV infection and other opportunistic infections (OI/HIV). These HIV-infected patients, also, were admitted and treated at Instituto de Infectologia Emilio Ribas, in São Paulo, Brazil. Negative diagnosis for toxoplasmosis was evaluated by ELISA, while the other opportunistic infections, by clinical, laboratorial and radiological features.All clinical samples  were sent to laboratory within 48 hours after collection and immediately processed.T. gondii genome [The laboratorial diagnosis for toxoplasmosis in the 83 clinical samples was evaluated by ELISA using a tachyzoite lysate antigen (TLA) as described before ,40. In si genome .CSF (600 μL) and serum (300 μL) samples were centrifuged at 13,500 x g for 15 minutes, to remove pellets, containing dead cells and debris. The supernatants containing 250 μL and 500 μL of serum and CSF, respectively, were transferred into Ultra-Clear Centrifuge tubes (6 mL tube for SW-55 rotor),  and the volume completed until 6 mL with filtered phosphate-buffered saline (PBS), pH 7.2. The samples were ultracentrifuged at 100,000 x g for 60 minutes at 25° C in a Beckman® Coulter L8-80M ultracentrifuge. The pellets, containing EVs were resuspended in 100 μL of filtered PBS and stored at—20 °C until analysis.™, NTA 3.0). NanoSITE-NTA computes size and number of particles based on their measured Brownian motion. Capturing and analyzing settings were done according to the equipment protocol. The analyses of concentration and size were performed with coefficient and hydrodynamic radius that were determined using the Stokes–Einstein equation, produced in NanoSight NS300 instrument. Results displayed as a particle size distribution. Data were presented as the average and standard deviation of three video recordings of 30–60 seconds per sample. Since NTA is accurate between particle concentrations in the range of 2 x 107 to 2 x 109/mL, EV samples were diluted before analysis in filtered PBS and the relative concentration calculated according to the dilution factor.After ultracentrifugation, concentration (particles/mL) and particle size (nm) CSF- derived EVs and serum-derived EVs were evaluated by nanoparticle tracking analysis (NTA) using the NanoSight NS300 instrument  for 1 hour. One drop of suspension was put on EM grid and performed by negative staining technique with 2% potassium phosphotungstate at pH 6,8, as previously described . Grids wSerum-derived EVs and CSF-derived EVs were solubilized in lysis buffer , boiled and run in 10% polyacrylamide-SDS gels. The investigation of EV proteins was done after silver staining. With the intention to determine exosomes in purified EVs by immunoblotting, EV proteins were transferred to nitrocellulose membranes. After blocked (for 60 minutes with 5% skim milk-PBS), membranes were incubated with anti-CD63 and anti-CD9 antibodies (Invitrogen®) diluted 1: 500 at 4° C for 18 hours. These antibodies recognize specifically the endosome-specific tetraspanins CD9 and CD63 . After washes, the membranes were incubated for 1 hour at room temperature with a goat horseradish peroxidase-conjugated anti-mouse IgG diluted (1:500) (Sigma®) in 5% skim milk-PBS. Bound antibodies were visualized using the quimioluminescence western blotting substrate  and registered in Blot Scanner (C-DiGit®) according to the manufacturer’s instructions.Caenorhabditis elegans miRNA  were spiked to each sample . RNA pellets were dissolved in DEPEC (RNase-free) water (30 μL). Next, 2 μL RNA including miRNA were reverse-transcripted (RT) using the Taqman®Advanced miRNA cDNA Synthesis kit (Life Technologies) according to manufacturing instructions. The reactions were performed in a Veriti® 96-Well Thermal Cycler (Applied Biosystems®) following four steps under the following thermal conditions: 45 minutes at 37° C, 10 minutes at 65º for poly (A) tailing reaction; 60 minutes at 16° C for ligation reaction; 15 minutes at 42° C, 5 minutes at 85º for reverse transcription reaction; and 5 minutes at 95° C, followed by 14 cycles of 95° C for 3 seconds, 60º C for 30 seconds; and a stop reaction at 99° C for 10 minutes for miR-Amp reaction. cDNA samples were stored at -70° C until use in qPCR assays.Total RNA including miRNA were extracted from serum-derived EVs (100μL). and CSF-derived EVs (100μL). Previously, to minimize RNA degradation by RNases during the experimental process, all materials and working surfaces were cleaned using RNaseZap® RNase Decontamination Solution (AmbionTM) prior to handling the samples. Purifications were performed using the miRNeasy Mini Kit (Qiagen), according to manufacturing instructions. EVs were mixed with a denaturing buffer in volumes described in the manufacture’s protocols. The homogenate was incubated at room temperature for 5 minutes. As internal control for extractions, 25 fmol of synthetic ™ Real-Time PCR Systems (Applied Biosystems®) using the following thermal profile in Fast mode: 95° C for 20 seconds, followed by 40 cycles at 95° C for 1 second and 60° C for 20 seconds. In each reaction a negative control was added (mix only). Negative results (without PCR amplification) were repeated until three times.Assays were performed using 5 target genes . Gene expression of each sample was performed in duplicate by qPCR in a custom assay, produced by Applied Biosystems®. qPCR amplification mixtures contained 5 μL of 2X TaqMan® Fast Advanced Master Mix and 0.5 μL TaqMan® Advanced miRNA Assays (both Applied Biosystems®) for expression of each target and reference gene (Cel-miR-39). The characteristics of genes used as molecular markers, including Assay IDs are shown in T) values for each sample. The mean of CT value was calculated after qPCR results (in duplicate) for each sample. CT values were transformed into RQ by comparative CT method (2-ΔΔCT) as described by Livak and Schimittgen [T method, the expression values of calibrators are considerate as 1.0.miRNA levels (five genes) were expressed as “Relative Quantification” (RQ). qPCR amplification plots reflect the fluorescent signal in each cycle and were determined as threshold cycle . Statistical analyses were performed using Graph Pad Prism software version 6.0 .Comparisons of EV concentration and EV size between groups were determined by Mann–Whitney test, one-tailed The clinical and laboratorial data of the 83 clinical samples used in this study are described in 10 EVs/mL) were statistically higher than seronegative individuals (mean: 5.9 x 109 EVs/mL).After purification by ultracentrifugation, CSF derived EVs and serum-derived EVs were analyzed in NanoSight (size and concentration). 10 EVs/mL) than CT/HIV patients (mean: 2.0 x 1010 EVs/mL), but without statistical difference.The data produced by NTA were, also, used to compare the concentrations of serum-derived EVs of patients according clinical form of toxoplasmosis. 9 EVs/mL) than the 16 OI/HIV patients (mean: 4.8 x 109 EVs/mL), but without statistical difference.Analyzes in CSF were distinct from sera ones, since is unusual to obtain CSF samples from health individuals. Thus, as criterion, CSF samples collected from patients with other opportunistic diseases and AIDS were used as control to compare with those from CT/HIV group. In the next step was the particle size was investigated by NTA. Values represent the mean of three reads in NanoSight (concentration and size). Regarding to size, NTA results were confirmed after analysis by TEM and immunoblotting. Microvesicles and exosomes have different transmembrane proteins. Exosomes have tetraspanins, including CD9 and CD63, which were detected by immunoblotting. Serum-derived EVs were separated by 10% SDS-PAGE and stained by silver . After tNext, the relative expressions of miR-146a-5p, miR-155-5p, miR-21-5p, miR-29c-3p and miR-125b-5p were estimated in miRNA purified from serum-derived EVs and CSF-derived EVs by qPCR. CSF-derived EVs from CT/HIV patients up-expressed miR-155-5p and miR-21-5p, 6.4 and 6.2 times more than calibrators  and miR-146a-5p (1.3 times) were similarly expressed in both groups of EVs. CSF-derived EVs were unable to express miR-29c-3p .T. gondii infection, parasite EVs stimulate the host immune system to produce host EVs (including microvesicles and exosomes). Infected host cells secret EVs with pathogen molecules able to induce modifications in uninfected cells or may serve as antigen presenters for the immune system [T. gondii EVs contain specific proteins as surface antigens, which are release by tachyzoites and compose the excreted-secreted antigens. These proteins participate of tachyzoite invasion, replication within host cells and are recognized by host immune system [Among EV functions produced in humans include the transference of different macromolecules and biomarkers interacting with specific molecules of immune system ,21–23. Ie system . At the e system ,31,43,44The evidence of exosomes/microvesicles in clinical samples includes: purification of EVs by ultracentrifugation; demonstration of exosomal proteins as tetraspanins; and presence of characteristic structures (in size and shape) by NTA and electron microscopy. For analyses of microvesicles and exosomes, purifications were performed in CSF and serum samples by ultracentrifugation. In this protocol, both types of EVs were recovered. Serum was chosen to minimize residual platelets that occur with standard plasma protocols ,46.As this article was the first that evaluated EVs purified from infected human with parasitic infection, this discussion was based in studies that described functions of human EVs produced in other diseases.Concentrations of serum-derived EVs of seropositive patients were higher than those of non-infected individuals. Among those infected, pregnant women developing acute infection produced more EVs than CT/HIV patients, but without differences statistically significant. As the mean size of EVs was similar in both groups, these data suggested that only the concentration is able to distinguish patients with toxoplasmosis than healthy individuals. Similar results were shown in plasmas of patient with papillary thyroid carcinoma .Analyzes of CSF-derived EVs were distinct from those of serum. As is unusual to obtain CSF from health individuals, CSF collected of patients with other opportunistic diseases and AIDS were used as negative control. Concentrations of CSF-derived EVs were almost similar in both groups, but lower than those of sera. Similar results were shown before ,49.EVs are produced also by neurons, microglia, and dendritic cells in the CNS . NeverthThe investigation of particle size by NTA confirmed that EVs derived, serum and CSF had size and shape characteristic of microvesicles and exosomes. The average size of EVs was similar in serum and CSF groups. The majority of particles were microvesicles in sera (180–290 nm) and in CSF (162–300 nm). Presence of exosomes was also confirmed by size and shape in TEM and the evidence of the tetraspanins CD63 and CD9 confirmed in immunoblotting after SDS-PAGE.Small RNAs participate in transcriptional gene regulation in cell development and other biological processes such as cell differentiation, proliferation and apoptosis . Among sHIV/AIDS patients with low number of CD4 normally develop opportunistic infections. At the same time, EVs can cause inflammations in HIV/AIDS patients via host miRNAs and viral miRNAs ,55. HoweSerum-derived EVs from CT/HIV patients and pregnant women, with acute toxoplasmosis up-expressed miR-125b-5p and miR-146a-5p, when compared with serum-derived EVs of healthy individuals. Elevation of TNF-α, IL-6, and downregulation of IFN-γ, as already reported in AIDS patients, marked upregulation of miRNA 125b-5p ,57. In tNevertheless, CSF-derived EVs of both groups of patients, CT/HIV and OI/HIV expressed equality miR-125b-5p, and miR-146a-5p. CSF-derived EVs of CT/HIV patients compared with those of OI/HIV patients up-expressed miR-155-5p and miR-21-5p. These data can suggest the induction of pro-inflammatory cytokine expression caused by miR-155 in CT/HIV co-infection that was more intense than those from other co-infections analyzed here. At the same time, the up-expression of miR-21-5p suggested the impact on the balance between Th1 and Th2 ,59.miR-21-5p expression by serum-derived EVs was almost similar in both patient groups (with or without toxoplasmosis). Similar results were observed by Akers et al.  when stuSerum-derived EVs in patients with toxoplasmosis down-expressed miR-29c-3p. CSF-derived EVs were unable to express miR-29c-3p. IFN-γ has a critical role in the immune response in intracellular infections and miR29c-3p participates as a regulator of Th1 and IFN-γ in innate and adaptive immunity. According other authors ,61,62, dFinally, all these data together demonstrate the presence of microvesicles and exosomes expressing exosomal miRNAs in patients with cerebral or gestational toxoplasmosis Exosomes containing miRNA can participate in modulation of cellular response. Results demonstrated the presence of a heterogeneous population of EVs in serum and CSF samples.S1 Data(ZIP)Click here for additional data file.S2 Data(ZIP)Click here for additional data file.S3 Data(ZIP)Click here for additional data file.S4 Data(ZIP)Click here for additional data file.S5 Data(ZIP)Click here for additional data file.S6 Data(ZIP)Click here for additional data file.S7 Data(ZIP)Click here for additional data file.S8 Data(ZIP)Click here for additional data file.S1 File(PDF)Click here for additional data file.S1 Fig(PPTM)Click here for additional data file.S2 Fig(PPTM)Click here for additional data file.S1 Results(XLSX)Click here for additional data file.


After surgery, 5 (1.4%) patients were treated for middle ear effusion, and another 5 (1.4%) patients experienced transient CSF rhinorrhea, which was spontaneously resolved within 1 to 7 days. No patients required additional treatments for CSF leakage. Conclusion: A simple technique using pulling stay sutures to stop bleeding from the dural edges and placing a wet cottonoid on the exposed dura can make primary dural closure easier.Background: No dural substitute has proven to be complication-free in a large clinical trial, even suggesting some benefit during watertight closure. However, primary dural closure is not always possible due to dural shrinkage from electrocautery for dural bleeding. Objective: This study is performed to analyze the clinical outcomes related to cerebrospinal fluid (CSF) leakage after microvascular decompression (MVD) using a simple surgical technique. Methods: Three hundred and sixty consecutive cases were treated with MVD and followed up for more than one month after surgery. Bleeding from the cut veins during dural incision was controlled by pulling stay sutures instead of electrocautery to avoid dural shrinkage. Additionally, a wet cottonoid was placed on the cerebellar side dural flap to prevent dural dehydration. During dural closure, primary dural closure was always attempted. If not possible, a “plugging muscle” method was used for watertight dural closure. Results: The mean age was 54.1 ± 10.8 years , and 238 (66.1%) were female. Primary MVD was performed in 345 (95.8%) patients. The mean operation time (from skin incision to skin closure) was 96.7 ± 33.0 min . Primary dural closure was possible in 344 (95.6%) patients. The “plugging muscle method” was performed more frequently in patients older than 60 years  than in younger cases  ( Cerebrospinal fluid (CSF) leakage is one of the most common complications after microvascular decompression (MVD) for neurovascular cross compression syndrome, including hemifacial spasm (HFS), trigeminal neuralgia, and glossopharyngeal neuralgia. Current treatments comprise reducing CSF pressure by continuous lumbar drainage or repeated spinal taps and administering antibiotics to prevent infections . FailureTo prevent CSF leakage after MVD, it has been highly emphasized by Peter J. Jannetta to ensure watertight dural closure . AdditioAlthough many dural replacements have been introduced and used to ensure watertight dural closure, no substitute has proven to be complication-free in a large clinical trial, even suggesting some benefit ,3. ThereIn this study, clinical outcomes related to CSF leakage after retrosigmoid craniectomy with MVD were analyzed after using a simple technique to maintain the integrity of the dural flap, allowing for primary dural closure.Between 2010 and January 2019, 360 consecutive cases were treated with retrosigmoid craniectomy, with MVD for HFS in 309 (85.8%) patients, trigeminal neuralgia in 50 (13.9%) patients, and glossopharyngeal neuralgia in one (0.3%) patient, and they were followed up more than one month after surgery. A retrospective review of medical records was performed to identify patients who experienced CSF leakage, including CSF rhinorrhea, otorrhea, pseudomeningocele, and/or incisional leak, during the initial hospital stay or by the first postoperative clinical follow-up usually at one month after surgery. The primary outcome was the primary dural closure rate using the surgical technique described below. Additionally, the author defined the secondary outcome as persistent CSF leakage that needs management with additional neurosurgical intervention such as continuous lumbar drainage, repeated spinal taps, and/or neurosurgical revision operation. Moreover, all patients with symptomatic CSF leakage were examed and assessed by otolaryngologists.We collected all of the patient data based on information contained in hospital electronic medical records and followed the case record form, which was approved by the institutional review board. As a retrospective study, there was no risk to the subjects (minimum risk study), and the IRB committee approved (B-1903-528-105) the exemption of consent from the subjects.All MVDs were performed by one neurosurgeon (JHH) and are described elsewhere . With thA dural incision along the posterior margin of the sigmoid sinus was made approximately 2–3 mm away from the sinus margin for later dural repair. Stay sutures were performed along with the incised sinus-side dura mater as close possible as to the sinus margin and were pulled tightly to maximize visualization of an intracranial surgical corridor while minimizing cerebellar retraction. During the dural incision, the dural veins were often cut, and they bled into the intracranial surgical corridor. At that time, instead of electrocautery, one or two stay sutures were performed just around the cut vein. Additionally, pulling these stay sutures tightly, as usual, could quickly cease bleeding from the cut veins B–E.After completing the stay sutures F, a wet The offending vessel(s) were decompressed from the cranial nerves after the exploration of the whole intracranial portion of each involved cranial nerve, including the root exit/entry zone and cisternal segment.After that main procedure, the operator washed out the surgical field by copious normal saline irrigation. It facilitates the elimination of almost all of the blood clots within the surgical corridor and inside the field. Through this process, the authors tried to prevent the increase in ICP that could be caused by secondary CSF dynamics change with residual blood components after surgery.During the closure following the completion of the intradural procedures, primary dural closure was always attempted using interrupted sutures H. If pri®; Nycomed, Linz, Austria), actually prepared to control bleeding from the dural sinuses during craniectomy, were applied and overlapped in two or three layers on the dural incision site for additional sealing with the yellow-coated part facing the dura  bone cement. Finally, the deep and superficial muscles, fascia, and skin were approximated layer by layer.Provided the postoperative course was uneventful, the patients were discharged between 48 and 72 h after surgery.p-value of 0.05 was regarded as statistically significant. Continuous variables were analyzed by Student’s t-test, and categorical variables were compared using the chi-squared test. Data analysis was performed using statistical software .A 2-sided Among 360 cases treated with MVD, 238 (66.1%) were female. The mean age was 54.1 ± 10.8 years . MVD for the right side was performed in 179 (49.7%) patients. Primary MVD was performed in 345 (95.8%) patients. Revision MVD was performed for 15 (4.2%) patients (2 referred from other institutes after primary MVD). The mean time for the entire decompression procedure (from skin incision to skin closure) was 96.7 ± 33.0 min . The median and mode of the length of hospital stay after surgery was 3 days and 2 days, respectively.p = 0.039, chi-squared test). Female patients showed a lower primary dural closure rate  than male patients ; however, the rate did not reach statistical significance (p = 0.191). Revision MVD and left or right directionality did not affect the primary dural closure rate.Primary dural closure was possible in 344 (95.6%) cases. The “plugging muscle” method was required in 16 (4.4%) cases due to dural tearing or injury in the dural outer layer and/or the whole-layer defect when removing the inner table of the skull using a Kerrison punch or high-speed diamond drill, especially around the sigmoid sinus. The plugging muscle method was performed more frequently in patients older than 60 years  than in younger cases   was confirmed in 5 (1.4%) patients with severe postoperative ear fullness. One of them who complaining of discomfort experienced myringotomy for the patient’s request and took oral antibiotics for two days. The remaining patients did not require any additional intervention for their MEE, which was resolved during observation.Additionally, the other 5 (1.4%) patients exhibited transient watery rhinorrhea after surgery. One patient visited the emergency room with watery rhinorrhea at postoperative day (POD) 3, the day after discharge. Upon arrival, the watery rhinorrhea had already stopped and never recurred. Two patients described watery rhinorrhea at first clinical follow up after surgery that started at about POD 3 or 4 and lasted for approximately one week. They did not visit the hospital for evaluation of their rhinorrhea, and their watery rhinorrhea stopped spontaneously with a sense of pressure drop in their ears. One patient showed high fever and watery rhinorrhea at POD 2. Clinical and laboratory examinations, including CSF study, were done; however, there was no evidence of meningitis except atelectasis on chest X-ray. Watery rhinorrhea continued intermittently for four days and then stopped without any additional intervention. The last patient exhibited a small amount of watery rhinorrhea once at POD 3. The watery rhinorrhea never developed again. However, two days after discharge or POD 5, the patient visited the emergency room with a high fever and chills. Empirical intravenous antibiotics were used for one week to treat aseptic meningitis, which was revealed on CSF examination and culture study. Therefore, no patient displayed the secondary outcome, which is defined in this study as persistent CSF leakage such as CSF rhinorrhea, otorrhea, pseudomeningocele, and/or incisional leak managed with additional intervention.®; Nycomed, Linz, Austria) is applied to the dural incision site after use for hemostasis, and to the opening of the mastoid air cell, which enhances the watertight primary dural closure. Then, cranioplasty using artificial bone cement, which eliminates dead space on the craniectomy site, is performed. Finally, the soft tissues, from muscles to the skin, are closed watertight. With each step, the author could significantly reduce the occurrence of persistent CSF leakage that requires lumbar drainage or surgical intervention.In this article, the author describes a straightforward and rapid technique to prevent CSF leakage that can occur after MVD through retrosigmoid craniectomy. To prevent CSF leakage, the surgeon should try various methods at each stage of surgery, but primary closure is fundamental. Besides, the author describes the surgical technique that enables primary dural closure in detail. After that, the extra fibrinogen/thrombin-based collagen fleece (TachoCombIt is crucial to achieving tight and reliable dural closure while performing retromastoid craniectomy with MVD. The CSF leakage leads to increased morbidity, prolongation of hospital stay, and enhanced costs, as well as the need for revision surgery . The preSince the early days of neurosurgery, watertight dural closure has been advocated to avoid CSF leakage . PrimaryFirst, the meningeal veins arise from the plexiform vessels in the dura mater. Additionally, they run the space between the inner and outer layers of the dura mater and drain into the dural sinuses. Thus, a dural incision along the margin of the sigmoid sinus often causes bleeding from the incised dural edges due to cutting off the meningeal veins draining to the sigmoid sinus. The electrocautery of the bleeding point of the dural edges results in shrinkage of the dura mater. Instead, pulling stay sutures tightly, just near the cut veins, typically to maximally visualize the surgical corridor, can compress the inner and outer layers of the dura mater. Compressing these two layers of the dura mater collapses the edge of the cut veins, easily stopping bleeding without the help of electrocautery. One report has suggested that it is necessary to shift the dural incision much closer to the venous sinuses to ensure primary dural closure . HoweverA hemoclip can also be used instead of electrocautery for bleeding from the dural edges; however, removal of the used hemoclip after the intracranial procedure can result in another type of dural tearing, especially in cases of old age. In the elderly, the dura can be particularly fragile and adherent to the bone . In the Second, the exposed dura, especially the cerebellar side, can dry up, which also makes primary dural closure difficult. A wet cottonoid placed on the exposed cerebellar side dura prevents the dura from being dehydrated by air and the light of the microscope . AdditioThird, approximately 1 × 2-cm-sized pieces of fibrinogen/thrombin-based collagen fleece were applied on the dural incision site and bony edges in two or three layers in this study. In fact, the fleece was prepared for the control of bleeding from the dural sinuses during craniectomy. After surgery, the remaining pieces of the fleece were used for additional sealing of the dural closure site and bone edges. Recently, similar techniques have been introduced ,13. EvenAdditionally, in one of the above studies, the fleece was used on the brain-side dura and skull side dura together . HoweverFinally, even with this surgical technique, middle ear effusion and CSF rhinorrhea developed, although the CSF leakage defined in this case series did not occur. Although the events resolved spontaneously without additional intervention such as continuous lumbar drainage, repeated tapping, or surgical intervention, patients could develop these events that might be caused by irrigation fluid or CSF entering through the mastoid air cells during the procedure rather than the postoperative CSF leakage, considering that all the events stopped spontaneously. Thus, the opened mastoid air cells should be thoroughly sealed with bone wax as Dr. Jannetta emphasized the sealing of the opened mastoid air cells, or “wax the bone edges on the way in and on the way out” .However, an issue that should be resolved concerns the timing of CSF leakage after MVD and additional intervention for CSF leakage. The case of CSF leakage that occurs after several years after MVD has been reported, but is very rare . In geneThe cranioplasty for bone defects is also crucial for preventing CSF leaks. As mentioned above, the CSF leakage was significantly lower in the group that filled bone defects using bone cement than titanium mesh ,16,17,18Besides the above, various factors in each step during MVD though retrosigmoid craniectomy, such as the sealing of opened mastoid air cells, deep muscle closure, and surgeons’ experiences and skills, are thought to contribute to preventing postoperative CSF leakage. Therefore, surgeons should perform all steps cautiously during surgical closure to prevent CSF leakage completely. Nonetheless, primary dural closure might be crucial as a first step in preventing CSF leakage.A simple technique using pulling stay sutures to stop bleeding from the dural edges and placing a wet cottonoid on the exposed dura can make primary dural closure easier. The application of fibrinogen/thrombin-based collagen fleece on the dural closure site and bone edges, and elimination of dead space by bone cement cranioplasty might enhance positive effects on primary dural closure in terms of the prevention of CSF leakage after MVD.


Accumulating reports have suggested that α-synuclein is involved in the pathogenesis of Alzheimer’s disease (AD). As the cerebrospinal fluid (CSF) α-synuclein has been suggested as a potential biomarker of AD, this study was set out to test whether CSF α-synuclein is associated with other AD biomarkers and could predict neurodegeneration and clinical progression in non-demented elders.The associations between CSF α-synuclein and other AD biomarkers were investigated at baseline in non-demented Chinese elders. The predictive values of CSF α-synuclein for longitudinal neuroimaging change and the conversion risk of non-demented elders were assessed using linear mixed effects models and multivariate Cox proportional hazard models, respectively, in the Alzheimer’s disease Neuroimaging Initiative (ADNI) database.The CSF α-synuclein levels correlated with AD-specific biomarkers, CSF total tau and phosphorylated tau levels, in 651 Chinese Han participants (training set). These positive correlations were replicated in the ADNI database . Using a longitudinal cohort from ADNI, the CSF α-synuclein concentrations were found to increase with disease severity. The CSF α-synuclein had high diagnostic accuracy for AD based on the “ATN”  system (A + T+ versus A − T − control) . Moreover, CSF α-synuclein predicted longitudinal hippocampus atrophy and conversion from MCI to AD dementia.CSF α-synuclein is associated with CSF tau levels and could predict neurodegeneration and clinical progression in non-demented elders. This finding indicates that CSF α-synuclein is a potentially useful early biomarker for AD.The online version contains supplementary material available at 10.1186/s40035-020-00222-1. Alzheimer’s disease (AD) is the leading cause of dementia in the elderly and is clinically characterized by a gradual decline in memory and other cognitive functions. However, less than half of the patients with dementia have received a formal diagnosis in Europe and the USA . The patα-Synuclein is best known for its roles in Parkinson’s disease (PD) and dementia with Lewy bodies (DLB), and has also been reported to be implicated in AD pathogenesis . PatientIn this study, we explored the associations between CSF α-synuclein and other AD biomarkers in the non-demented Chinese elderly. We also tested whether CSF α-synuclein was altered in patients with AD and with different pathophysiological profiles of AD based on the “ATN” classifications, and its associations with other AD biomarkers, cognitive decline and imaging evidence of neurodegeneration in the Alzheimer’s Disease Neuroimaging Initiative (ADNI) database. The value of CSF α-synuclein as a predictor of disease progression and neurodegeneration at the presymptomatic stage of AD was also investigated.Six hundred and fifty-one non-demented participants were from the Chinese Alzheimer’s Biomarker and Lifestyle (CABLE) study. The CABLE is a large-cohort study mainly focusing on Alzheimer’s risk factors and biomarkers in Chinese elderly adults. The participants in the CABLE study were recruited at Qingdao Municipal Hospital, consisting of cognitively normal (CN) and mild cognitive impairment (MCI) individuals. All participants were Han Chinese in origin and aged 50–90 years. The controls had Mini-Mental State Examination (MMSE) scores of 24 or higher, with lower scores indicating more impairment and higher scores less impairment , and a Clinical Dementia Rating (CDR) score of 0, where lower scores indicate less impairment and higher scores more impairment . The patients with MCI had MMSE scores of 24 or higher, an objective memory loss tested by delayed recall of the Wechsler Memory Scale (WMS) logical memory II , a CDR score of 0.5, preserved activities of daily living, and absence of dementia. The exclusion criteria were: (1) central nervous system infection, head trauma, epilepsy, multiple sclerosis or other major neurological disorders; (2) major psychological disorders ; (3) severe systemic diseases  that may affect CSF or blood levels of AD biomarkers including Aβ and tau; and (4) family history of genetic disease. All participants underwent clinical and neuropsychological assessments, biochemical testing, as well as blood and CSF sample collection. Demographic information, AD risk factor profile and medical history were also collected by a comprehensive questionnaire and an electronic medical record system.adni.loni.usc.edu), an independent replication cohort. The ADNI was launched in 2003 as a public-private partnership under the leadership by Michael W. Weiner, MD, with a primary goal to test whether magnetic resonance imaging (MRI), positron emission tomography (PET), biological markers, as well as clinical and neuropsychological assessment can be combined to measure the progression of MCI and early AD. For up-to-date information, see www.adni-info.org.Data were obtained from the ADNI database  with no progression to AD dementia during at least 2-year follow-up, and progressive MCI (pMCI) with progression to AD dementia during at least 2-year follow-up. As a result, totally 4 groups were included: CN control, sMCI group, pMCI group and AD group. As to the “ATN” binary  categories, amyloid positive (A+) and negative (A-) were separated by a cutoff value of 192 pg/ml for CSF Aβ level; Tau pathology positive (T+) and negative (T-) were separated by a cutoff value of 23 pg/ml for CSF phosphorylated tau (p-tau) level.Our ADNI cohort included all the CN controls, MCI patients and AD patients with available baseline samples for CSF α-synuclein. The inclusion/exclusion criteria are described at The CABLE study was approved by the Institutional Ethics Committee of Qingdao Municipal Hospital. Written informed consent was obtained from all study participants directly or from their guardians. The ADNI study was approved by the Institutional Review Board at each of the participating centers, and all participants provided written informed consent.CSF was collected by lumbar puncture through the L3/L4 interspace and gently mixed to avoid gradient effects. The samples were then centrifuged at 2000 g for 10 min to remove cells and other insoluble materials, stored in 1-ml aliquots at − 80°C until use for Aβ and tau analysis. CSF was sampled between 08:00 and 09:00 in the morning taking into account the possible circadian rhythm effect.In the CABLE study, the concentrations of CSF Aβ42, total tau (t-tau), p-tau and CSF total α-synuclein were measured separately using an enzyme-linked immunosorbent assay (ELISA) kit , according to the manufacturer’s instructions. The samples and standards were measured in duplicate to generate an average value for the statistical analyses.42, t-tau and p-tau were measured at the ADNI biomarker core (University of Pennsylvania) using the multiplex xMAP Luminex platform  with the INNOBIA AlzBio3 kit . The CSF neurofilament light chain (NFL) concentrations were measured using a commercial ELISA kit (Uman Diagnostics). The plasma NFL concentrations were measured using an NFL kit , transferred onto the ultrasensitive single-molecule array platform using a home brew kit . The levels of CSF total α-synuclein concentrations in the ADNI cohort were measured by the Luminex MicroPlex Microspheres , using the biotinylated goat anti-human α-syn antibody  as the detection antibody. The α-synuclein Luminex assay demonstrated a low day-to-day and plate-to-plate signal variety. The accuracy of the assay was further determined by the recovery of spiked α-synuclein protein, which was close to 93%.In the ADNI database, CSF AβStructural MRI was performed only in the ADNI participants using a Siemens Trio 3.0 T scanner or Vision 1.5 T scanner . The regional volume estimates for the 1.5 and 3.0 T MRI images were processed with the Free-surfer software package version 4.3 and 5.1 image processing frameworks, respectively. The hippocampus and ventricles were selected as the regions of interest.APOE ε4 genotype (with CSF α-synuclein as a predictor). In the ADNI database, associations between CSF α-synuclein concentrations and the diagnostic groups were tested in an analysis of covariance model adjusted for age, gender, educational level and APOE ε4 genotype. The effect of different CSF analytes on the risk of conversion to AD was assessed with the logistic regression analysis. The receiver-operator curves and the area under the curves were derived from the predictive probabilities of the logistic regression models. We tested the associations of CSF α-synuclein concentrations with longitudinal cognition and brain structure using the linear mixed-effects models. These models had random intercepts and slopes for time and an unstructured covariance matrix for the random effects and included the interaction between (continuous) time and CSF α-synuclein as predictor with adjustment for confounders. All tests were 2-sided. Statistical significance was set at P < 0.05. All regression analyses were corrected for age, gender, educational level, diagnosis, and APOE ε4 genotype. The following variables were natural log-transformed to ensure normality: CSF α-synuclein, p-tau, t-tau, and Aβ levels, and hippocampus volume. All statistical analyses were performed using R version 3.4.0 (R Foundation).The associations between CSF α-synuclein and demographic factors were analyzed with the Mann-Whitney test and the Spearman rank correlation test. The associations of CSF α-synuclein with CSF Aβ42, t-tau, and p-tau levels were analyzed with the linear regression after adjustment for age, gender, educational level, diagnosis and We included 651 non-demented elders from the CABLE study, consisting of 457 CN controls  and 194 MCI patients  (Table P < 0.001) and p-tau  among the non-demented participants. However, there was no association between CSF α-synuclein and CSF Aβ level at baseline. In addition, the same associations were found in the CN group and the MCI group  and tested their relationships Table . We founup Table .Table 2APOE ε4 allele (69.23%) and the CN group had the lowest frequency (23.85%). There was no significant difference in the educational level (P = 0.16) or age (P = 0.53) among these four groups. Furthermore, AD patients had lower MMSE scores compared with the MCI patients and CN controls (P < 0.01).Three hundred and eighty-two subjects from the ADNI database were included Table . This coP < 0.001) and p-tau  in the whole cohort. However, there was no association between CSF α-synuclein and CSF Aβ level at baseline. The same results were obtained in the MCI group  and CN controls . In addition, the CSF α-synuclein concentration was associated with CSF NFL concentration in non-demented elders . However, there was no association between CSF α-synuclein and plasma NFL  and the sMCI group  , A+ controls (P < 0.001), and A- MCI group (P < 0.001) , A+ controls (P < 0.01), and the A- MCI group (P = 0.02). The CSF α-synuclein level was also significantly different between the A + T+ group and the A-T- group (P < 0.0001)  of the baseline model containing CSF α-synuclein, age at baseline, gender, educational level and APOE ε4 genotype was 0.76 in predicting the onset of AD among the CN controls, and the AUC was further increased by the inclusion of CSF tau/Aβ ratio (AUC = 0.88)  Fig. S. As expe8) Fig. S. In the 8) Fig. S.APOE ε4 genotype. The baseline CSF α-synuclein concentration was found to be significantly associated with the hippocampal volume    Table .In this study, we found that the CSF α-synuclein concentration (1) was associated with CSF t-tau and p-tau levels among the non-demented elderly adults, (2) was elevated in the AD dementia group and the Aβ/tau-positive group compared with the control group, and (3) could predict hippocampal atrophy and the conversion from MCI to AD dementia. Taken together, these findings suggest that CSF α-synuclein is a very early and potentially presymptomatic biomarker for AD. This biomarker may be helpful for AD diagnosis and prediction of disease progression and staging of AD even in the preclinical stage.“Pure” AD is characterized by the presence of both diffused neuritic plaques and intracellular neurofibrillary tangles, with a lack of abnormal α-synuclein inclusions or neuritis. However, more than 50% of AD patients exhibit excessive brain accumulation of α-synuclein-positive Lewy bodies, particularly in the amygdala , 20. TheAPOE ε4 genotype had good performance in predicting progression from CN to pMCI or AD. Recently, the NIA-AA committee has recommended a different definition of AD by pathophysiology, independent of the clinical symptoms. They proposed that as long as biomarker evidence of Aβ and tau pathology was present simultaneously, the term “Alzheimer’s disease” would be applied. Here the CSF α-synuclein model had high diagnostic accuracy for patients with the diagnosis of AD based on the “ATN” system (A + T+) vs controls (A-T-) .Consistent with most studies, our study showed that CSF α-synuclein was higher in the AD group compared with the CN controls and MCI group. A possible hypothesis is that the higher level of α-synuclein could induce a decrease in some proteins in synaptic vesicle and alterations of the protein composition of synaptic vesicles, thus causing neuronal damage in AD, which in turn increases the release of α-synuclein from damaged cells into the CSF , 25. As 40 and Aβ42 peptides [1–42 is sufficient to induce the intracellular accumulation of α-synuclein, whereas interaction with Aβ1–40 is not [Many lines of evidence have suggested that the pathological α-synuclein, Aβ and tau have synergistic adverse effects to promote the aggregation of each other, thereby amplifying the neuronal damage , 26–31. peptides . This ma0 is not . In our Moreover, α-synuclein has also being observed in progressive supranuclear palsy  and fronImportantly, we found that the CSF α-synuclein levels might correlate with AD severity and progression, which was consistent with a recent study indicating that increased α-synuclein displayed a stronger association with cognitive impairment than soluble Aβ and tau levels . It has CSF α-synuclein was associated with CSF t-tau and p-tau levels among the non-demented elderly adults. In the ADNI database, CSF α-synuclein concentrations were increased with the severity of the disease. CSF α-synuclein predicted longitudinal hippocampus atrophy and conversion from MCI to AD dementia. The current findings suggest CSF α-synuclein as a very early and potentially presymptomatic biomarker for AD, a prognostic marker in the clinic, and an outcome measure in clinical trials.Additional file 1: Supplementary Figure S1. Association between CSF α-synuclein and CSF/plasma NFL concentration. Linear regression trend lines are shown in blue. These regression lines were unadjusted, while the corresponding analysis in the text was adjusted for age, sex, educational level, and APOE ε4 genotype. CSF/plasma NFL and CSF a-synuclein concentrations on the y- and x-axes were logarithmic.Additional file 2: Supplementary Figure S2. Receiver operating curves to assess the diagnostic accuracy of CSF α-synuclein and other biomarkers for AD dementia. Receiver operating curves of logistic regression model were controlled for age at baseline, gender, educational level and APOE ε4 genotype.Additional file 3: Supplementary Figure S3. Conversion from CN to pMCI as predicted by baseline CSF biomarkers. Receiver operating curves of the logistic regression model controlling for age at baseline, gender, educational level and APOE ε4 genotype for predicting conversion to pMCI among people with normal cognitive function.Additional file 4: Supplementary Figure S4. Conversion from Aβ-negative status to Aβ-positive group as predicted by the baseline CSF biomarkers. Receiver operating curves of the logistic regression model were controlled for age at baseline, gender, educational level and APOE ε4 genotype for predicting conversion to Aβ-positive status among the Aβ-negative group.Additional file 5: Supplementary Figure S5. Receiver operating curves for predicting conversion from AD pathophysiology (tau and amyloid-β) negative to positive. The baseline model included age at baseline, gender, educational level and APOE ε4 genotype.