Here we investigated whether the loss of ceruloplasmin ferroxidase activity in the CSF of PD patients was accompanied by NGR-motifs deamidation and gain of function.Ceruloplasmin, a ferroxidase present in cerebrospinal fluid (CSF), plays a role in iron homeostasis protecting tissues from oxidative damage. Its reduced enzymatic activity was reported in Parkinson’s disease (PD) contributing to the pathological iron accumulation. We previously showed that ceruloplasmin is modified by oxidation 962NGR-motif which is usually hidden within the ceruloplasmin structure, and the gain of integrin-binding function. These effects occur owing to the presence of abnormal levels of hydrogen peroxide we detected in the CSF of PD patients. Interestingly, the pathological CSF's environment of PD patients promoted the same modifications in the exogenously added ceruloplasmin, which in turn resulted in loss of ferroxidase-activity and acquisition of integrin-binding properties.We have found that endogenous ceruloplasmin in the CSF of PD patients showed structural changes, deamidation of the We show that in pathological oxidative environment of PD-CSF the endogenous ceruloplasmin, in addition to loss-of-ferroxidase function, is modified as to gain integrin-binding function. These findings, beside the known role of ceruloplasmin in iron homeostasis, might have important pathogenic implications due to the potential triggering of signals mediated by the unusual integrin binding in cells of central nervous system. Furthermore, there are pharmacological implications because, based on data obtained in murine models, the administration of ceruloplasmin has been proposed as potential therapeutic treatment of PD, however, the observed CSF's pro-oxidant properties raise the possibility that in human the ceruloplasmin-based therapeutic approach might not be efficacious.The online version of this article (doi:10.1186/s13024-015-0055-2) contains supplementary material, which is available to authorized users. Ceruloplasmin (Cp) is a ferroxidase enzyme expressed on the surface of astrocytes and secreted as a soluble form in the cerebrospinal fluid (CSF) by choroid plexus epithelial cells . CerulopisoAsp, isoD) formation [568N and 962N), that are part of Asparagine-Glycine-Arginine-(NGR)-sequences, can deamidate with a fast kinetics [isoDGR, a motif capable of recognizing the Arginine-Glycin-AsparticAcid-(RGD)-binding site of integrins [in vitro NGR-deamidation in ceruloplasmin is associated with gain of integrin-binding and intracellular signalling functions, that through FAK1, ERK1/2, Akt and MAPK involvement may regulate gene activation, cell cycle, proliferation, and actin cytoskeleton rearrangement [568N is exposed on the protein surface and can rapidly deamidate in vitro, deamidation of 962N requires protein oxidation and structural changes, being this residue buried within the protein tertiary structure [962NGR-motif and gain of integrin binding function, owing to the presence of abnormal levels of hydrogen peroxide in the CSF. Interestingly, the pathological environment in the CSF of PD patients promotes the same modifications in the exogenously added ceruloplasmin, with the consequent loss of ferroxidase activity and gain of integrin binding properties, which in turn rise warnings about a potential therapeutic use of ceruloplasmin in patients.The loss of ceruloplasmin-ferroxidase activity observed in PD patients might be the consequence of the protein oxidation we observed  likely oormation . In partkinetics  leading ntegrins , 16. Accangement . While 5tructure . These n962NGR- to isoDGR-motif transition, as reported for ceruloplasmin in vitro [962NGR-motifs may take place in vivo in the patients. Quantitative mass spectrometry (MS) analysis performed by parallel reaction monitoring (PRM) on the CSF of healthy subject  and PD patients  confirmed that the deamidation of the endogenous ceruloplasmin 962NGR-motif was significantly higher (p = 0.0094) in PD patients compared to healthy subjects . No correlations have been found between the rate of Cp-deamidation and the demographic/clinical parameters evaluated in patients , nor correlation has been found with the samples' time of storage at −80 °C (not shown). Considering the physiological ceruloplasmin concentration of about 1.5 μg/ml in the CSF [962DGR and 962isoDGR forms, and that only the latter is responsible for the integrin-binding, we can hypothesize, according to the reported 50-75 % conversion of NGR toward isoDGR [in vitro oxidized/deamidated ceruloplasmin to this integrin [isoDGR) available determined the capture and detection of this protein in very small amounts. Moreover, competition with other integrin-binding molecules present in CSF  was a further constraint on adhesive-ceruloplasmin capture. Interestingly, if the CSFs were aged in vitro for 9 days, a significant αvβ6-integrin binding activity  was induced exclusively in the CSF from PD patients    or patients with peripheral neuropathies (PN) or PD, and analysed it before and after incubation for different times. Ceruloplasmin aged for 9 days in PD-CSF, but not in H-CSF or PN-CSF, showed a higher sensitivity to limited trypsin proteolysis and a significant reduction of ferroxidase activity (11) Fig.  and b. T64) Fig. . These fp = 0.0091 and p = 0.0083 at 9 and 12 days, respectively) , but not by a control ARA-peptide  Fig. . The binide Fig. , indicatTo assess the contribute of oxidative stress in structural and functional changes observed after ceruloplasmin aging in PD-CSF, we analysed ceruloplasmin carbonylation before and after 9 days of incubation, as read-out of protein oxidation promoted by reactive oxygen species. Western blot (WB) analysis showed a weak carbonylation at time 0 in PN-CSF and PD-CSF, but not in H-CSF Fig. . Remarkap = 0.009)  and added to H-CSF  compared to H-CSF or PN-CSF (p < 0.0001)  Fig. . This ob09) Fig. . Of noteCSF Fig. , indicat962NGR-deamidation and gain of integrin-binding properties.These findings support the hypothesis that hydrogen peroxide was crucially involved in ceruloplasmin oxidation and in the induction of structural changes necessary for loss of ferroxidase activity, In vivo NGR-deamidation, on the contrary to what was observed in vitro [962NGR-site and not, or markedly less, on the surface-exposed 568NGR. However, this finding is conceivable because asparagine deamidation depends on neighboring residues as well as on secondary and tertiary structure of the protein, and is also affected by environmental factors such as ionic strength and pH [962NGR-deamidation requires a conformational change to occur [In vivo, asparagine deamidation has been reported in pathological conditions associated with oxidative stress as for example for Tau protein and β-amyloid peptides in Alzheimer's disease [In this study we demonstrated that, in addition to the oxidative modifications and the loss of ferroxidase activity already reported –7, the cin vitro , occurs h and pH , 20. Theh and pH , 20. As to occur , this imto occur , 21. In  disease –25. Thus disease –7. This in vitro deamidation of ceruloplasmin's NGR-motifs promotes the gain of integrin-binding function, and that the interaction of deamidate ceruloplasmin with integrins resulted in an intracellular signal transduction that affects cell physiology [isoDGR isoform), and the sensitivity of the method used for binding detection. In addition, we also hypothesized that, in vivo, as soon as the ceruloplasmin is modified, and thus becomes able to bind the integrins, it is sequestered by the surrounding, integrin-expressing tissues. In fact, in vitro in the absence of sequestering integrin-expressing tissue, the CSF from PD patients aged for 9 days showed the acquisition of the αvβ6-integrin binding activity. Interestingly the gain of function was induced exclusively in the CSF from PD patients and not in the control CSFs from healthy subjects and PN patients. This rule out the induction of methodological-artificial oxidative conditions and suggested that the PD pathological environment was able to promote a gain-of-integrin binding activity. Whether the ceruloplasmin gain of integrin-binding function observed in PD-CSF has favourable or detrimental effects on disease progression is difficult to predict at present and need further investigations. However, since we reported that integrin binding by deamidated ceruloplasmin is able to activate in vitro an intracellular signaling cascades, which in turn could affect cell proliferation and cytoskeletal re-organization [We have previously shown that ysiology , 14. Thenization –28. Seveex vivo in PD-CSF at higher rates than in normal-CSF and PN-CSF used as control neurological disease. As for the endogenous ceruloplasmin, the deamidation of spiked ceruloplasmin occurs primarily at the hidden 962NGR-site, which implies the requirement of conformational changes as supported by the different sensitivity to limited proteolysis observed. These evidences suggest as a logical consequence that PD-CSF contains compounds capable of fostering these changes. Of note, we found that the PD-CSF contains high levels of hydrogen peroxide, a highly reactive oxygen species, and that catalase, an enzyme that converts hydrogen peroxide into water, prevented both the inhibition of ferroxidase activity and the acquisition of ceruloplasmin integrin-binding properties. Therefore, hydrogen peroxide known to be overproduced in neurodegeneration [The second major finding of this work is that the CSF of PD patients contains elements capable of promoting structural and functional modifications in ceruloplasmin, including, oxidation/carbonylation, loss of ferroxidase activity, asparagine deamidation at the NGR-site and acquisition of integrin-binding properties. Indeed, NGR-deamidation of spiked ceruloplasmin was fostered neration , seems tneration , 21. Theneration . Interesneration . Thus, pneration –34. Simineration –25. The neration . Furtherneration  (revieweneration ). Anotheneration , 39, migex vivo aging in PD-CSF compared to both H-CSF and PN-CSF supports the hypothesis of a generally accelerated protein aging in neurodegenerative diseases promoted by the pathological environment [in vivo [The faster kinetic of ceruloplasmin’s NGR-motifs deamidation and loss of ferroxidase activity observed during ironment , 13. The[in vivo , made co568NGR- and 962NGR-motifs across different species is not maintained in mouse and rat; in these species the sequences are 568DGR- and 962NGK- respectively, the first not prone to isomerize to isoDGR- and the second not described to be able to generate an integrin binding-motif when deamidated , peripheral neuropathies patients  and healthy subjects . All patients and controls were at first diagnosis and drug-free. Patients affected by probable idiopathic PD according to the current diagnostic criteria [CSF samples were obtained from the Institute of Experimental Neurology-Biobank (San Raffaele Scientific Institute) after approval from the hospital’s ethical committee and informed consent from patients. The analyzed CFSs were from Parkinson’s disease patients , and for patients age (H vs. PD and PD vs. PN groups comparison), whereas H vs. PN showed a p = 0.0317 (Mann–Whitney test). Demographic and clinical features of PD patients and control groups are summarized in Table Healthy CSFs were from subjects who underwent lumbar puncture on account of a suspected neurological disease and who proved to be free from pathological alterations after complete CSF analysis and clinic-neuroimaging assessment. All controls reported negative history of neurologic disorders and were normal on neurological examination. Exclusion criteria for all groups were: HIV/HCV-seropositivity; previous cerebral ischemic events; severe metabolic disorders. No statistical differences among all groups were found for sex distribution (Fischer’s exact test), for total protein concentration  to eliminate cells; then samples, were either immediately used or stored at −80 °C in a N2-supplemented atmosphere to avoid the exposure to oxidative-environment. No differences in results were observed in the different assays using fresh or stored CSF. For hydrogen peroxide quantification, the samples have been used within 6 months of storage and CSF aliquots were used only once to avoid repeated thawing/freezing cycles. CSF samples’ blood contamination was rule-out in all samples as no hemoglobin was detected by both WB and spectrophotometric analysis at 415 nm . Due to the limited amount of CSF samples available, and depending on the sample’s volume necessary for the different assays, not all the patients and controls were used in each experiment; numbers of subjects used in the different assays are indicated in the figure legends.CSF samples (2–3 ml) were collected for bio-banking purposes by lumbar puncture and the first drops were discarded to avoid the artificial blood contamination. One ml of each CSF sample was made available for this study . We set up a 40–50 min procedure for collection, processing and storage at −80 °C all CSF samples. Within this time frame samples remained at room temperature for 15–20 min for pneumatic tube transport, and at 4 °C for further 20–35 min during which the samples were centrifuged (800 x In vitro oxidation and deamidation of ceruloplasmin (Cp-ox/AmBic) was achieved by incubation (16 h at 37 °C) in 100 mM ammonium bicarbonate buffer pH 8.5, containing 10 mM hydrogen peroxide [Purified human ceruloplasmin  was added to CSFs  and incubated at 37 °C to the indicate time points under nitrogen-conditioned atmosphere, to avoid the exposure to atmospheric oxidative environment; in selected experiments, incubation was performed in presence of catalase . After incubation in CSFs, spiked ceruloplasmin was immunoprecipitated using protein-G agarose beads (Invitrogen) coated with polyclonal sheep anti-ceruloplasmin antibody (Ab)  cross-linked with 20 mM dimethyl-pimelimidate (Sigma-Aldrich). This polyclonal Ab anti-ceruloplasmin was selected for its ability to equally immunoprecipitate both resting- and oxidized-ceruloplasmin  was added to 96-well plates and incubated 16 h at 4 °C; subsequent steps were carried out at 20 °C. After blocking with 3 % BSA-PBS, the plates were filled  with CSF samples either from healthy or pathological subjects, or in selected experiments with CSF samples previously supplemented with ceruloplasmin or with Cp-ox/AmBic and incubated for 2 h. Binding was detected using the sheep polyclonal anti-human ceruloplasmin Ab  followed by a secondary HRP-conjugate Ab (Abcam) and by o-phenylendiamine chromogenic substrate. Competitive binding assays were performed by mixing 30 μg/ml of either the acetyl-CisoDGRCGVRSSSRTPSDKY peptide (isoDGR-peptide) or the control peptide CARACGVRSSSRTPSDKY (ARA-peptide) [Recombinant αvβ6 integrin   with CSFdigestion buffer  and incubated with trypsin  for 2 h at 20 °C. Samples were desalted  and injected in a capillary chromatographic system . Peptide separations occurred on a 25 cm reverse phase silica capillary column, packed with 3-μm ReproSil-Pur 120 C18-AQ. A gradient of acetonitrile eluents was used to achieve separation (0.15 μL/min flow rate). MS analysis was performed by nanoLC-MS/MS using an LTQ-Orbitrap (Thermo Scientific) equipped with a nano-electrospray ion source (Proxeon Biosystems). Full scan spectra were acquired with the lock-mass option, resolution set to 60,000, and mass range from m/z 300 to 1750 Da. The ten most intense doubly and triply charged ions were selected and fragmented in the ion trap. All MS/MS samples were analyzed using Mascot  search engine to search the UniProt_Human Complete Proteome_cp_hum_2013_12. Searches were performed with 3-missed cleavages allowed, N-terminus-acetylation, methionine oxidation and deamidation of asparagine/glutamine as variable modifications. Mass tolerance was set to 5 ppm and 0.6 Da for precursor and fragment ions, respectively. To quantify deamidation the raw data were loaded into the MaxQuant software v1.3.0.5. Label-free protein quantification was based on the intensities of precursors. Peptides and proteins were accepted with a FDR of 0.01, two minimum peptides per protein with one unique peptide.For MS analysis, we used a protocol set up as to avoid experimental protein oxidation and/or deamidation . In briedigestion buffer and incubated 12 h at 20 °C in the presence of trypsin 1:10 (w/w). The tryptic mixture was desalted and injected in a capillary chromatographic system  for peptide separations on a 10 cm reverse phase silica capillary column, packed with 1.9-μm ReproSil-Pur 120 C18-AQ. A 60 min-gradient of acetonitrile was used to achieve separation (0.30 μL/min flow rate). Parallel reaction monitoring (PRM) analyses were performed using a Q-Exactive mass spectrometer (Thermo Scientific). The acquisition method combined two scan events corresponding to a full scan MS  and a PRM event  which targeted the precursor ions of the peptides at their relevant charge states in 1 min window. Using trypsin-digested purified Cp and Cp-ox/AmBic, a local spectral library was created to generate reference MS/MS spectra (b- and y-fragment ions) and to determine the elution time for each peptide. Data processing was performed with Skyline software, freely available [962NGR- and 962DGR-containing peptides. Five technical replicates for each sample were run.CSF samples (70 μl each) were diluted (1:2) with vailable . The quavailable . The useCeruloplasmin immunoprecipitated from CSF after incubation was resolved by 10 %-acrylamide SDS-PAGE, and analyzed by WB as described in  with the4 and analysed after 1 h with a solution of 1 mM Btp in acetate buffer, pH 6.2. Decrease in Btp-Fe2+ complex absorbance at 535 nm derives from ferrous iron oxidation into ferric form (Fe3+). Ferroxidase activity was reported as percentages of the activity recorded for 1.25 μg of untreated ceruloplasmin.Ferroxidase activity of spiked ceruloplasmin after aging in CSF was evaluated by bathophenanthroline (Btp) assay as described in  on the idigestion buffer followed by incubation for 2 h at 20 °C in the presence of agarose-immobilized trypsin (0.5 U) (Sigma-Aldrich). Trypsin was removed by centrifugation, and samples were analysed by SDS-PAGE and WB.Spiked ceruloplasmin after aging in CSF was immunoprecipitated from CSF and 100 ng were resuspended in 25 μl of The carbonylation level of ceruloplasmin immunopreciptated from CSF after aging, was analyzed as in  with the2O2 and a non-fluorescent probe (AmplexRed®) that produces a fluorescent compound. The analysis was performed on either freshly collected CSF or samples stored at −80 °C for less than 6 months and that were not previously thawed. Samples were analyzed in triplicate diluting CSF 1:100 in the assay buffer according to the manufacturer protocol, and H2O2 concentration was evaluated as fluorescence at 590 nm.Hydrogen peroxide concentration in the CSFs was evaluated using the Hydrogen Peroxide Assay Kit (BioVision) that is based on the reaction between the Ht-test, if the data passed the normality test for Gaussian distribution as assessed by the Kolmogorov-Smirnov test, or were evaluated by Mann Whitney test; two-tailed p value was used for the comparison of two means and standard error. In all analyses, p < 0.05 was considered to be statistically significant. The analysis was performed with GraphPad Prism V5 software.Sex distribution was analyzed by using Fisher’s exact test and two-tailed p value. Continuous data were evaluated by unpaired student’s


Rupture of an atherosclerotic plaque is the primary cause of acute cardiovascular and cerebrovascular syndromes. Early and non-invasive detection of vulnerable atherosclerotic plaques (VP) would be significant in preventing some aspects of these syndromes. As a new contrast agent, dimercaptosuccinic acid (DMSA) modified ultra-small super paramagnetic iron oxide (USPIO) was synthesized and used to identify VP and rupture plaque by magnetic resonance imaging (MRI).Atherosclerosis was induced in male New Zealand White rabbits by feeding a high cholesterol diet (n = 30). Group A with atherosclerosis plaque (n = 10) were controls. VP was established in groups B (n = 10) and C (n = 10) using balloon-induced endothelial injury of the abdominal aorta. Adenovirus-carrying p53 genes were injected into the aortic segments rich in plaques after 8 weeks. Group C was treated with atorvastatin for 8 weeks. Sixteen weeks later, all rabbits underwent pharmacological triggering, and imaging were taken daily for 5 d after DMSA-USPIO infusion. At the first day and before being killed, serum MMP-9, sCD40L, and other lipid indicators were measured.DMSA-USPIO particles accumulated in VP and rupture plaques. Rupture plaques appeared as areas of hyper-intensity on DMSA-USPIO enhanced MRI, especially T2*-weighted sequences, with a signal strength peaking at 96 h. The group given atorvastatin showed few DMSA-USPIO particles and had lower levels of serum indicators. MMP-9 and sCD40L levels in group B were significantly higher than in the other 2 groups (P <0.05).After successfully establishing a VP model in rabbits, DMSA-USPIO was used to enhance MRI for clear identification of plaque inflammation and rupture. Rupture plaques were detectable in this way probably due to an activating inflammatory process. Atorvastatin reduced the inflammatory response and stabilizing VP possibly by decreasing MMP-9 and sCD40L levels. Atherosclerosis (AS) is a pathological progression of chronic vascular inflammatory with a long time-course based on vascular lesions; it can cause severe complication such as myocardial infarction and stroke . TherefoSeveral imaging approaches have been developed to detect VP, but early identification of plaques at high risk of plaque rupturing and triggering thrombosis within a short space of time remains a challenge. Angiography is the most common technique used to assess cardiovascular risk by imaging arterial stenosis, but with the limitation that it cannot assess those lesions most prone to early rupture ; it alsoMRI, with no ionizing radiation, but with repeatability, can provide 3-dimension more stereoscopic information, and is quicker than traditional histological detection. Macrophages are important in the rupture of VP . With thConsidering the widespread use of dextran-coated particles in biological applications, it is surprising that the nature of the interaction between this coating and iron oxide surface has not been more extensively investigated . The preHyperlipidemia is a major risk factor for AS. It can make the blood flow rate slow and increase viscosity, thus making lipid more easily deposited in the vessel wall, gradually forming part of atherosclerotic plaques.A VP model widely reported can be the successful establishment of lesions through balloon-induced vascular endothelium injury, and transfecting the abdominal aorta with the p53 gene in rabbits given a high fat diet , 16. ChaInflammation plays an integral part in the development and progression of VP, leading eventually to plaque instability. How can we evaluate inflammation in the living body? Certainly many inflammatory indicators are known, including C-reactive protein, interleukin 6, soluble CD40 ligand (sCD40L), matrix metalloproteinase-9 (MMP-9) and others. The key treatment for stabilizing VP is the inhibition of inflammation . HoweverAtorvastatin, a hydroxymethylglutaryl-CoA redutase inhibitor, is one of the most widely used statins in the primary and secondary prevention for coronary heart disease. New clinical guides point out that atorvastatin has the effect of being anti-inflammatory, lowering lipid, stabling plaque and even reversing it. Atorvastatin can also reduce plaque destabilization by downregulating matrix metalloproteinase (MMP)  and proiWe established an atherosclerotic rabbit model of VP by fed high cholesterol diet combined with balloon injury in the abdominal aortic and locally injection with recombinant p53 adenovirus in the atherosclerotic plaques. We used Chinese Russell’s viper venom and histamine to trigger plaques as a result of acute plaque rupture and thrombosis model formation which can not be operated on human body. We used dimercaptosuccinic acid (DMSA) instead of dextran and its derivatives to identify plaque inflammation by the marked accumulation of DMSA-USPIO within macrophages in VP and rupture plaques. Whether atorvastatin reduces the vulnerability of rabbits to abdominal aorta injury by decreasing the levels of MMP-9 and sCD40L is unknown.Thirty male New Zealand white rabbits  were included in the study. The Principle of Laboratory Animal Care published by the National Institute of Health in 1996 was followed. Approval for the experimental protocol was granted by the Animal Care Committee of Xuzhou Medical College. The rabbits were given 1 week to adapt to their new environment and were raised separately in an air-conditioned room.They were randomly divided into 3 groups each of 10 animals: group A, control; group B, VP model; group C, VP and atorvastatin. The rabbits were allowed water ad libitum. Group A were fed on a hyper-cholesterolemic diet  of 150 g per kg per day for 8 weeks. Groups B and C had balloon-induced endothelial injury of the abdominal aorta, followed by right femoral artery ligation. In brief, the rabbits were anesthetized through the injection of 30mg/kg sodium pentobarbital via rabbit ear vein. Then the puncture of the right femoral artery was performed and a balloon catheter of 3.5mm diameter and 15mm length was introduced into the aorta about 20cm along the 0.014inch guide wire. The balloon was inflated by injecting distilled water up to 8 atmospheres and then was pulled back to the common iliac artery. The balloon was repeatedly pulled back for three times in order to injure abdominal aortic endothelium. The femoral artery was ligated and intramuscular injection of penicillin was given to prevent infection. They were also fed a hypercholesterolemic diet. After 8 weeks, group C were given atorvastatin  was injected at 0.1 mmol per kg in an ear vein before the rabbits were MRI scanned, which was repeated at 24, 48, 72, 96 and 120 h to follow changes in the signal-to-noise ratio (SNR) in the lesions.MRI scanning was done with a 1.5-tesla clinical system  before pharmacological triggering to measure the signal strength of each sequence in the plaques. The sequence parameter of MRI settings is shown in SNR was determined as follows:SNR=SI/the scan . The datthe scan :EI=, triglyceride (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were measured from mainline from ear edge vein at 0 and 16 weeks. Serum at 0 and 16 weeks was collected by centrifugation (10 min at 3000 rpm), and stored at -80°C until analysis MMP-9 and sCD40L using the appropriate ELISAs .At necropsy, abdominal tissues were removed and washed in saline. The aorta and iliofemoral arteries were opened for inspection. Arterial tissue sections (4 μm) were obtained from the aortic segments of 25 rabbits, yielding a total of 350 segments. The abdominal aortas were excised and cut into 3 segments. One segment was placed in cold phosphate-buffered saline, cleaned of adherent connective tissue, and fixed in 4% formaldehyde for 24 h for histopathological examination. After fixation, the second segment was embedded in paraffin and cut into 5 μm sections stained with hematoxylin and eosin (H&E), Masson and Prussian blue.The distances from the abdominal aorta and the iliac bifurcation were used as internal reference points to compare the MRI slices with histology. The aortas were marked with suture ligatures on the right artery of the iliac bifurcation over the total length imaged by MRI. The abdominal aorta was cut into 4 mm segments, and compared with the MRI slices.The third segment was fixed in 3% glutaraldehyde with 1% osmium tetroxide post-fixation. They were rinsed, dehydrated and embedded in Spurr epoxy resin. After trimming, ultrathin sections (70 nm) were cut, and double stained with uranylacetate and lead citrate. The ultrastructure was examined in a transmission electron microscope  and rupture plaques photographed.Statistical analyses used SPSS 16.0 software . Quantitative variables are expressed as mean ± SD. Statistical comparisons were made by T-Tests and Two-way Analysis of Variance. P<0.05 was considered a statistically significant difference.Animals model was successfully generated in 25  of the 30 animals (83.3%). One animal were died of diarrhoea in group A, 2 died of plaque rupture in group B, and 2 died of diarrhoea and serious liver damage in group C.Different plaque components at MR contrasted distinctively with each other. The image of plaques in T1WI and PDWI showed a slightly higher signal and an equal signal in T2WI. A decrease of the signal strength was not obvious on spectral presaturation inversion recovery in T1WI . The imaThe image of plaques in T1WI and PDW shows a slightly higher signal and an equal signal in T2WI . There wA high-cholesterol diet led as anticipated to lipid disorders. Compared with the baseline levels, the blood lipid level significantly increased in all 3 groups by 16 weeks. The level in group B were clearly higher than in group A at 16 weeks, but had decreased in group C that was given atorvastatin . MMP-9 aHistologyically, only 1 rabbit plaque was confirmed as a VP in the 9 rabbits of group A. The rest of the abdominal aortas formed thick fibrous caps and smaller lipid nuclear atherosclerotic plaques. Group B had larger lipid-core plaques but with thin fibrous caps. Post drug treatment, 6 of the 8 rabbit (75%) in group B had formed a total of 37 VPs. All in group C had stable plaques. The data indicate that the VP model had successfully induced plaque rupture by pharmacological triggering in the rabbits. Histopathological analysis can differentiate stable from vulnerable plaques.International consensus on the integrity database will currently be defined as VPs that have a tendency to burst, are prone to the risk of thrombosis and/or progress quickly to critical lesions. Naghavi et al.  establisA comparison of foam cells and collagen fibers among the 3 groups based on the different stains is shown in In group B (b), more lysosomes and macrophages were present than in groups A (a) and C (c). Increased fat and iron particles were apparent in the macrophages of group B (b) .mean cutoff values predicting rupture plaque events in group B, we used the SNRmean values in the arterial segments before drug triggering to plot ROC curves  were synthesized by a novel oxidation co-precipitation method in H2O/DMSO mixing system, where DMSO acts simultaneously as an oxidant. The second step is when OA was replaced by DMSA to obtain water-soluble nanoparticles. The mean diameter of the Fe3O4 core in DMSA-USPIO is 5.56 nm. Moreover, their ultra-small hydrodynamic size helps the particles avoid the reticulo-endothelial system and have a long half-life in circulation. As our result represented, we find much DMSA-USPIO particle accumulation in similar regions of the plaque and even rupture plaques within macrophages. DMSA-USPIO-enhanced MRI can be applied to detect macrophages in atherosclerotic plaques, and to examine the vulnerability of plaques and characteristics of plaques rupture.DMSA-USPIO is a new MRI contrast agent that was synthesized through a 2-step process . In the Morishige et al  has repo18F-FDG uptake by the arterial wall is a non-invasive technique that determines vascular inflammation and the risk of atherosclerotic plaque rupture [18F-FDG uptake over time as long as 12 months. His data distinguishes 18F-FDG uptake in calcified and non-calcified segments of the arteries. PET may provide invaluable information regarding the cellular, metabolic and molecular composition of these plaques, and thus distinguish vulnerable from stable plaques. Quanming Zhao [mean cutoff values for predicting thrombosis events; the sensitivity and specificity were 75.4% and 88.5%, respectively, compared with our SNRmeans of 75.6% and 89.3%, respectively. Because of the high cost and ionizing radiation detrimental to human health, PET may be less suitable than DMSA-USPIO enhanced MRI.PET is increasingly being used in following atheroma inflammation- a factor for high risk plaque. Measurement of  rupture , 33. It ing Zhao  used PETMany researchers agree that the expression of MMP-9 increases in plaque shoulders, regions of foam cell accumulation and microvasculature. Macrophages can also secrete and synthesize proteases, such as catheptic enzyme and MMPs. Deguchiet  showed b’s effect. Atorvastatin significantly reduces sCD40L and MMP-9 concentrations and activity by inhibiting VP progression, and enhances the stability of atherosclerotic plaques as seen from the histopathology of the abdominal aorta.Atorvastatin has the effect of lowering lipid levels. It is one of the most widely used statins in the primary and secondary prevention of coronary artery heart disease. Atorvastatin may exert anti-inflammatory effects on phospholipases that contribute to its therapeutic benefit after acute coronary syndrome. Combined with the levels of serum inflammatory indicators, we may infer that this is the most likely mechanism of atorvastatinSeveral limitations need to be addressed before the current imaging approach can be used in patients with atherosclerosis to follow plaque inflammation with time and the efficacy of treatments aimed at plaque stabilization. First, we need to determine whether there is an influence of USPIO long-term persistence (of organisms) in the circulation. The exact metabolic and excretion processes need to be more fully determined. Its toxicity and sensitization testing also need attention. From a technical standpoint, image resolution needs to be improved due to factors such as MRI gauge field strength and coil affecting it.We have successfully established the VP model in rabbits with the application of balloon injury to the vascular endothelium, feeding a high-fat diet, and locally transfection with exogenous recombinant wild-type P53 gene. MDSA-USPIO particles can be ingested by most macrophages. MDSA-USPIO enhanced MRI scanning provides a new method for the early detection of VP and even rupture plaques, and has high specificity and sensitivity. Atorvastatin can transform unstable into stable plaques possibly by not only decreasing the blood lipid levels, but by decreasing MMP-9 and sCD40L expression.S1 TableAt least one of the major criterias that can indicate the high vulnerability of plaque.(DOC)Click here for additional data file.


In microwave and terahertz frequency band, a textured metal surface can support spoof surface plasmon polaritons (SSPPs). In this paper, we explore a SSPPs waveguide composed of a metal block with pyramidal grooves. Under the deep subwavelength condition, theoretical formulas for calculation of dispersion relations are derived based on the modal expansion method (MEM). Using the obtained formulas, a general analysis is given about the properties of the SSPPs in the waveguides with upright and downward pyramidal grooves. It is demonstrated that the SSPPs waveguides with upright pyramidal grooves give better field-confinement. Numerical simulations are used to check the theoretical analysis and show good agreement with the analytical results. In addition, the group velocity of the SSPPs propagating along the waveguide is explored and two structures are designed to show how to trap the SSPPs on the metal surface. The calculation methodology provided in this paper can also be used to deal with the SSPPs waveguides with irregular grooves. In optical frequency band, a smooth dielectric-metal interface can sustain surface plasmon polaritons (SPPs), which can propagate along the surface with high field-confinement. In microwave and terahertz frequency range, the metal behaves like a perfect electrical conductor (PEC) and SPPs cannot be confined on a smooth metal surface. However, a textured dielectric-metal interface can bound another kind of surface electromagnetic waves, which are called spoof SPPs (SSPPs)123456735678101112A metal block drilled with one-dimensional periodic grooves on the surface is one representative among various SSPPs waveguides3514151661819y direction. d. Each pyramidal groove can be divided into a group of slim rectangular grooves which are stacked sequentially. The number of the rectangular grooves is denoted by N , respectively. In this paper, we regard the pyramidal groove to be upright or downward if its width increases or decreases gradually from top to bottom. If they are equal to each other, the pyramidal groove becomes a rectangular groove. These three types of pyramidal grooves are displayed in 3521λ0 ≫ d > a , the dispersion relation of the SSPPs is expressed as N = 4 in . Correspa1 < a2 and a1 > a2, respectively. It is convenient to divide the space into four regions . The electromagnetic field of the SSPPs propagating along x direction satisfies Hy in regions I, II, III and IV can be written asFirst, we consider the case that the pyramidal groove is composed of two rectangular grooves, as depicted in βn = 2πn/d (|β| ≤ π/d) are the propagation constants and z direction. n are integers and denote the diffraction orders. k = 2, 3) correspond to the eigen-functions along x-direction within the rectangular grooves in regions II and III, given bywhere γm are constants associated with the mode number m. γm are equal to 1 if m = 0 and 1/2 if m ≠ 0. The wave vectors where The electric field component in the four regions can be obtained straight forwardly through Hy and Ex must satisfy the boundary condition of electromagnetic fields. Noting that Hy are not continuous at the bottoms of the grooves, we obtain an equation set composed of five equations by applying the continuity condition at the interface z = 0, −h1 and −(h1 + h2). The condition that makes the equations set solvable can yield the dispersion relation for the waveguide. The dispersion formulas we obtain are of very complicated forms and they are different for the waveguides with a1 < a2 and a1 > a2. Like the waveguide with rectangular grooves322λ0 ≫ d > ak−1 to simplify the formulas, under which the high-order modes of electromagnetic expansions in the three regions can be neglected. In this case, the dispersion relations have the same form for the cases of a1 < a2 and a1 > a2, which is written asBoth a1 = a2, Obviously, when a2 = 0, it reduces further toand when h1 + h2 or h1, respectively.β of the SSPPs sustained by the waveguide with pyramidal grooves are decided by the groove widths  and depths . When the groove depth h (h = h1 + h2) is small, only a fundamental SSPPs mode can exist. If the groove is deep enough, high-order SSPPs modes will appear. Each SSPPs mode corresponds to a frequency band that does not overlap with the others. For the fundamental mode, we can deduce from β increases with the increasing of a2 and decreasing of a1. As a consequence, the waveguide with upright pyramidal grooves has a larger propagation constant β than that of the downward pyramidal grooves with reversed widths. The values of β for the case of rectangular grooves (a1 = a2) will fall in between the other two types. From β means higher field-confinement in z direction. Namely, waveguides with upright pyramidal grooves has better field-confinement than those with downward pyramidal grooves. As the case becomes different and complicated when considering high-order modes, we will focus our discussions on the fundamental mode in this paper.From d is set to be 1 mm. The depth of the pyramidal groove is fixed at h = h1 + h2 = 10d to guarantee the deep subwavelength condition23h1 = h2. Five sets of , , , ,  and , are taken for groove widths. The simulated dispersion relations are calculated using finite integration method. To make it more practical, we set the waveguide thickness to be 20d instead of infinity in the simulation. It has been demonstrated that the dispersion relations hardly change when the thickness is larger than 20da1 < a2 are always lower than those for a1 > a2, which is consistent with our theoretical analysis.To confirm the theoretical analysis above, we give some examples and compare with the simulated results. In all designs, the period Ex distributions of SSPPs on three different waveguides whose  are ,  and , respectively. The three Ex distributions are evaluated at 6.38 GHz, 6.84 GHz and 6.90 GHz. The corresponding propagation constants are π/d, 0.176π/d and 0.118π/d, respectively. The propagation constant remarkably increases when the mouth of the groove shrinks. The field distributions in For the SSPPs waveguides drilled with pyramidal grooves that have more than two layers, the dispersion relation of the SSPPs can also be obtained by using the MEM. The direct derivation could be very complicated. In this paper, we utilize an analogy method. Comparing a1 and its depth h1,2 satisfiesIt indicates that the two-layer groove is equivalent to a single-layer groove whose width is N = 2 can be extended to those with N > 2. First, we use an equivalent rectangular groove to replace the Nth and (N−1)th layers. The width and depth of the equivalent groove are Based on this thought, the theoretical formulas for the waveguides with N − 2)th layer and the equivalent layer could be replaced by a new equivalent groove whose width and depth are denoted as Then the . For the waveguide with upright pyramidal grooves, the width of the top rectangular groove is set as 0.1 mm. The widths of the rectangular grooves below increase evenly with a step of 0.1 mm. The waveguide with downward pyramidal grooves is just the opposite. N = 3 and N = 8. It is easy to identify that the analytical results match the simulated results well, which validates our analogy. Still, the field-confinement of the waveguide with upright pyramidal grooves is better than that with downward pyramidal grooves. Additionally, as the number of the layers increases, the field-confinement of the waveguide with upright pyramidal grooves gets better, while the field-confinement of its counterpart becomes weaker. The field-confinement properties for the different types of grooves can be easily understood if we equate the multi-layered groove to a single straight groove. The equivalent depth for the upright pyramidal groove is obviously larger than its counterpart. The deeper rectangular grooves correspond to higher field-confinement. Therefore, the waveguide with upright pyramidal grooves is of the highest field-confinement.Likewise, we use the simulated results to verify such analogy. The period is still fixed at d, 5d, 3d, 1.5d and 0.8d. It can be seen that the analytical results move away from the simulated results gradually as the groove depths decrease. Fortunately, the differences between the analytical and simulated results are always within an acceptable range even when the depth is smaller than the period. It suggests that our theoretical formulas can be used to describe the characteristics of the waveguide with a proper precision.All the waveguides discussed above have the same groove depth that decuples the period. In this case, the analytical and simulated results for the fundamental modes are in complete agreement. However, as groove depths of the waveguides decrease, the eigen-frequency will increase and the error brought by the deep subwavelength approximation will grow. In a1,3,5 = 0.2 mm and a2,4,6 = 0.4 mm. The other has the widths a1,3,5 = 0.4 mm and a2,4,6 = 0.2 mm. The period cells of the two waveguides are shown in the insets. We can see that the analytical results are consistent with the simulated results. It indicates that our theoretical formulas are applicable to waveguides with multi-layer rectangular grooves whose widths do not change evenly. To verify the application of our formulas to high-order SSPPs modes, we also illustrate the dispersion curves for the 1st and 2nd modes in For the waveguide where the width of the grooves changes continuously from top to bottom, it is hard to get a single formula for the dispersion relation. An alternate method is to divide the groove into stacked slim rectangular layers and then calculate the dispersion relation using the analogy methods provided above. Two examples are given to check the validity of the proposed method.at = 0.2 mm and bt = 0.8 mm, respectively. The second one is textured with slanted groovesas = 0.2 mm. The orientation angle is denoted with θ, which takes 10° here. Both waveguides have a period constant d = 1 mm and the groove depths are ht = 10d and hs = 5d, respectively. First, we use numerical simulation to get the dispersion relations of the SSPPs modes. The simulated results are denoted by symbols in N slim rectangular layers, as shown in the insets of ht/N. The width of the top rectangular groove is at. The widths below increase evenly with a step of (bt−at)/N. For the slanted groove, the width and depth of the N th rectangular groove are 0.5as/cosθ and 0.5assinθ, respectively. The rectangular layers above have the same width and depth, which are obtained throughThe first one is a SSPPs waveguide with upright trapezoidal grooves18andN takes 4, 8 and 16. It can be seen that the analytical results approach to the simulation results gradually as the layer number increases. Specially, when N = 16, the two results are nearly the same. It indicates that the dispersion relations for the waveguides with continuously changed grooves can be described by stacked layered grooves and can be calculated using the analogy method. This also explains why the dispersion curves of the waveguides with rectangular grooves lie between those with upright and downward trapezoidal grooves18respectively. The dispersion relations for the equivalent waveguides are calculated using the theoretical formulas. The curves in vg, given by vg = dω/dβ (ω is the angular frequency), is less than the speed of light. As illustrated in When SSPPs propagate along the waveguide drilled with grooves, their group velocity The group velocity derived from the whered = 1 mm, h = 10d and h1 = h2. We consider two cases here. In the first case, a1 is fixed at 0.4 mm. We calculate the group velocity vg as a2 increases from 0.1 mm using a2 approaches to the asymptotic width, vg tends to zero. The higher frequency corresponds to a smaller asymptotic width. In the second case, we fix a2 at 0.4 mm and calculate vg as a1 decreases from 0.9 mm using We still set d. The right waveguide has fixed a1 = 0.4 mm, while its a2 increases evenly from 0.1 mm to 0.9 mm. The left one has constant a2 = 0.4 mm, while its a1 decreases evenly from 0.9 mm to 0.1 mm. We use the finite integration method to simulate the propagation of SSPPs. A plane wave propagating in x direction serves as the wave source. E|) at 6 GHz and 7.5 GHz, respectively. The insets depict the one-dimensional |E| distributions along x direction 0.01 mm above the waveguide surface. It is easy to identify that the light is trapped at the groove where the group velocity approaches zero, which is consistent with the results in Utilizing the changing of the group velocity with groove widths, we design two waveguides to trap SSPPs, as shown in We have studied the waveguides corrugated with pyramidal grooves and provided a general method to calculate the dispersion relations for the waveguides with various types of grooves. According to the theoretical formulas we obtained, the width and depth of the rectangular groove in each layer can determine the dispersion relations. This explains why the groove shapes can influence the field-confinement of the SSPPs. Even though our theoretical formulas are derived for the waveguide with pyramidal grooves, it can be applied to waveguides with irregular grooves since a groove in any shape can be divided into slim layers. In addition, the formulas are also applied to high-order SSPPs modes.λ0 ≫ d, the dispersion relations obtained by our theoretical formulas have some deviation with the exact results. Fortunately, the deviation is within an acceptable range. The approximate dispersion relation formulas can always be used to explore the characteristics of the waveguide.It is worth to note that these conclusions above are based on the deep subwavelength approximation. For waveguides that do not satisfy the condition Another issue is that the metal is assumed as a PEC in the analysis for simplicity. Practically, we have to take into account the finite conductivity of the waveguide. In this case, the propagation constant of SSPPs becomes a complex value. Its real and imaginary parts represent propagation and loss, respectively. It has been demonstrated that the real part is close to that under the PEC approximation15For the waveguide drilled with rectangular grooves, the dispersion relations, especially the asymptotic frequencies, are not sensitive to the groove width. Therefore, the rainbow-trapping effect in waveguides is always realized by changing the groove depths in previous literatures262728The MEM is a numerical method to solve the Maxwell’s equations. It is widely used to deal with the transmission and scattering problems of the electromagnetic waves in the waveguides and photonic crystals. The main idea of the MEM is as follows. First, we use the Maxwell’s equations to find a set of complete functions in each region of the scattering structure. Then we use the superposition of these functions to meet the corresponding boundary conditions of scattering field and establish the coupling equations. Finally, we solve the coupled equations to get the expansion coefficient of each region, which represent the information related to scattering. In this paper, we use the MEM for modes analysis and do not solve the expansion coefficient of each region. The dispersion formulas of the propagation modes can be obtained through the condition that makes the continuity condition at work.Analogy is a process of transferring information or meaning from a particular subject to another. In a narrower sense, analogy is an inference from one particular to another particular, which is opposed to deduction, induction and abduction. In this paper, we use the analogy method to get the dispersion formulas for the waveguide with pyramidal grooves that consist of multi-layer rectangular grooves. This method avoids complicated formulas derivation.The simulated dispersion relations and field distributions of the SSPPs are obtained with the finite integration method. In the simulation, for dispersion relations, the metal was treated as a perfect electric conductor (PEC) and only a period cell is used. In the periodic direction, the “periodic” boundary condition is used. While in the other directions, we use the “electric” boundary conditions. Eigen-mode Solver is used for solving. In the simulation for field distributions, we use the boundary condition of “open (add space)” and the Time Domain Solver.How to cite this article: Tian, L. et al. Investigation of mechanism: spoof SPPs on periodically textured metal surface with pyramidal grooves. Sci. Rep.6, 32008; doi: 10.1038/srep32008 (2016).


Slowed gait in patients with Parkinson’s disease (PD) can be improved when patients synchronize footsteps to isochronous metronome cues, but limited retention of such improvements suggest that permanent cueing regimes are needed for long-term improvements. If so, music might make permanent cueing regimes more pleasant, improving adherence; however, music cueing requires patients to synchronize movements to the “beat,” which might be difficult for patients with PD who tend to show weak beat perception. One solution may be to use high-groove music, which has high beat salience that may facilitate synchronization, and affective properties, which may improve motivation to move. As a first step to understanding how beat perception affects gait in complex neurological disorders, we examined how beat perception ability affected gait in neurotypical adults. Synchronization performance and gait parameters were assessed as healthy young adults with strong or weak beat perception synchronized to low-groove music, high-groove music, and metronome cues. High-groove music was predicted to elicit better synchronization than low-groove music, due to its higher beat salience. Two musical tempi, or rates, were used: (1) preferred tempo: beat rate matched to preferred step rate and (2) faster tempo: beat rate adjusted to 22.5% faster than preferred step rate. For both strong and weak beat-perceivers, synchronization performance was best with metronome cues, followed by high-groove music, and worst with low-groove music. In addition, high-groove music elicited longer and faster steps than low-groove music, both at preferred tempo and at faster tempo. Low-groove music was particularly detrimental to gait in weak beat-perceivers, who showed slower and shorter steps compared to uncued walking. The findings show that individual differences in beat perception affect gait when synchronizing footsteps to music, and have implications for using music in gait rehabilitation. AlthougApart from greater beat salience, high levels of groove might also improve gait by modulating an individual’s affective state. In particular, high-groove music elicits higher arousal as well as a positive affective state  with low-groove music than with high-groove music, due to the low beat salience and low arousal properties of low-groove music. To evaluate whether gait changes in the high-groove condition were primarily due to high beat salience or physiological/arousal factors, we compared the high groove and metronome conditions. Metronomes have the highest beat salience, as only the beat is present. However, metronomes have not been shown to modulate physiological arousal or affective state. Thus, if high-groove music elicits similar gait changes as metronome cues, the changes are likely due to high beat salience. If high-groove music alters gait more than metronome cues, then the affective properties of high-groove music likely also contribute to gait changes.Beat perception ability was assessed with a perceptual beat alignment test (BAT), which measures beat perception in music with no motor requirement, and is sensitive to individual differences in beat perception ability in the general population Iversen, . We hypoForty-three healthy undergraduate psychology students from the University of Western Ontario with self-reported normal hearing  participated in this study for course credit. One participant was excluded due to incomplete data. The study was approved by the Human Research Ethics Committee at the University of Western Ontario. All participants provided written informed consent.We used the BAT from the Goldsmiths Music Sophistication Index v1.0  Groove: how much did the music make you want to move? 1 = did not want to move, 10 = very much wanted to move. (2) Familiarity: how familiar are you with the music clip? 1 = not at all familiar to me, 10 = very familiar to me. (3) How enjoyable is this piece of music? 1 = not at all enjoyable, 10 = very enjoyable. Based on individual ratings, the three music clips rated as lowest on groove and the three music clips rated as highest on groove were selected for each participant. To reduce the likelihood that familiarity with the music clip would confound the results, only low familiarity clips (ratings < 4 on familiarity) were used for the step synchronization task. Ratings data for the stimuli are listed in the Supplementary Material.First, each participant’s preferred gait step rate (number of steps per minute) during uncued walking was determined by having the participant walk eight lengths of a 16 foot Zeno pressure sensor walkway  in silence. Previous studies using similar walkways have shown that six walks results in a sufficient number of steps for reliable estimation of gait parameters  and period-matching performance (the extent to which the tempo of the steps matches the tempo of the beats). However, we were only able to evaluate period matching performance, not phase-matching performance for the following reasons. First, it is unknown whether subjects aim to synchronize the first contact time of their step , the last contact time of their step , or some time point between the heel-strike and the toe-offset, to the beat. Synchronizing the heel-strike or the toe-offset can result in significant differences in synchronization accuracy  was assessed using the interbeat interval deviation (IBI deviation), which quantified how well step tempo was matched to the beat tempo on each trial  by Cue  by Tempo  mixed-measures ANOVAs were conducted for each gait parameter of interest. The Greenhouse-Geisser correction was applied when Mauchly’s test of sphericity was significant. Pairwise comparisons using Dunn-Sidak corrections for multiple comparisons identified significant differences between conditions.M = 0.69, SD = 0.15, consistent with previous findings  and weak beat-perceivers . The use of the 0.65 split was justified by similar BAT median scores in other versions of the BAT  than weak beat-perceivers , although this difference was not statistically significant .Beat alignment test scores were calculated as the proportion correct from all 17 trials. Scores ranged from 0.47 to 1, Iversen, . A splitIversen, . Strong F = 5.92, p = 0.02, η2p = 0.13]. A significant main effect of Cue  indicates that period-matching accuracy differed between cue types. Period matching was best for metronome cues (86.53 ± 12.06), followed by high-groove music (121.44 ± 12.43), and was worst for low-groove music (151.79 ± 14.04). All conditions differed significantly from each other. Period matching was significantly less accurate for low-groove music compared to high-groove music (p = 0.001), and for low-groove music compared to metronome cues (p < 0.001). Period matching was also significantly less accurate for high-groove music compared to metronome cues (p = 0.003). The Cue × Beat Perception interaction was not significant: , indicating that period matching in strong and weak beat-perceivers was similarly affected by the different cue types.Figure F = 5.79, p = 0.02, η2p = 0.13]. A significant main effect of Cue  indicated that variability differed between cue types. Period matching was least variable for metronome cues (64.73 ± 6.71), followed by high-groove music (96.68 ± 5.87), and was most variable for low-groove music (125.50 ± 11.33). All conditions differed significantly from each other. Period matching was more variable for low-groove music compared to high-groove music (p = 0.036), and more variable for low-groove music compared to metronome cues (p < 0.001). Period matching was more variable for high-groove music than for metronome cues (p < 0.001). The Cue × Beat Perception interaction was not significant .The variability of period matching was indicated by the standard deviation of the IBI deviation  = 5.15, p = 0.029, η2p = 0.11] and near significant for step length . The main effects of Beat Perception were not qualified by any significant interactions with Cue or Tempo.Figure F = 11.027, p < 0.001, η2p = 0.22] and step length . This interaction was because high-groove music elicited similar stride velocity and step length to metronome cues at preferred tempo , but elicited slower and shorter steps than metronome cues at the faster tempo . Low-groove music elicited significantly slower and shorter steps than high-groove music and metronome  = 4.62, p < 0.001, step length t(41) = 2.43, p = 0.019] and at faster tempo .There were significant Cue × Tempo interactions for stride velocity . This three-way interaction was because, at preferred tempo, weak beat-perceivers slowed step times more than strong beat-perceivers with low-groove music  but not with high-groove music  and metronome cues . At the faster tempo, weak beat-perceivers sped up step times more than strong beat-perceivers with metronome cues  but not with low-groove music  or high-groove music . With faster tempo metronome cues, the markedly briefer step times in weak beat-perceivers were accompanied by reduced step length, and thus, the increase in stride velocity to fast metronome cues  = 4.41, p = 0.04, η2p = 0.099] and Cue  were qualified by a significant three-way Beat Perception × Cue × Tempo interaction . At preferred tempo, weak beat-perceivers increased stride width more than strong beat-perceivers with low-groove music , but not with high-groove music , or with metronome cues . At the faster tempo, weak beat-perceivers increased stride width more than strong beat-perceivers with high-groove music , but not with low-groove music , or with metronome cues .Wider strides, longer double support time, and greater stride length variability indicate greater attentional demands and greater cautiousness during gait  = 2.05, p = 0.16, η2p = 0.05], and there were no significant interactions between Beat Perception and other factors. There was a significant main effect of Cue , which was not qualified by any interactions, as both strong and weak beat-perceivers decreased double support time more with metronome cues than with low-groove music (p = 0.001) or high-groove music (p = 0.049).For double support time, the main effect of Beat Perception was not significant  was qualified by a significant Cue x Beat Perception interaction , as weak beat-perceivers showed greater increases in stride length variability than strong beat-perceivers with low-groove music , and metronome cues , but not with high-groove music .For stride length variability, the marginally significant main effect of Beat Perception [In summary, weak beat-perceivers showed larger increases in stride width and stride length variability than strong beat-perceivers, and this pattern of results was particularly evident with low-groove music.The primary finding of this study is that individual differences in beat perception ability determined whether gait was maintained or impaired by synchronizing to music and metronome cues. Weak beat-perceivers showed slower gait than strong beat-perceivers, perhaps because weak beat-perceivers had more difficulty in synchronizing footsteps to the beat, as shown by poorer performance in period matching their step tempo to the cue tempo. Conversely, strong beat-perceivers showed faster gait than weak beat-perceivers, perhaps because strong beat-perceivers found it easier to synchronize footsteps to the beat, as shown by better period-matching performance in strong beat-perceivers than weak beat-perceivers. Collectively, these findings suggest that beat perception ability may affect outcomes in music-based gait rehabilitation, particularly in patients with PD, who show beat perception deficits  were evident even with metronome cues, when there was no need to extract the beat structure. Such findings appear at first glance to contradict previous studies that report that weak beat-perceivers show intact synchronization performance when tapping to metronome cues , and thus foot contact times can be longer than the interbeat interval. This contrasts with assessments of synchronization performance with finger tapping, in which the finger contact time is brief and discrete. In bipedal gait, participants could synchronize any timepoint between the first contact time  and the last contact time of the step (the toe-off). Synchronizing using the heel-strike or the toe-off can result in significant differences in synchronization accuracy (Chen et al., The primary finding of this study is that beat perception ability affects whether gait performance is impaired or maintained when synchronizing footsteps to the beat in music. When synchronizing to auditory cues, strong beat-perceivers either maintained or improved gait performance, whereas weak beat-perceivers showed slower, more cautious gait, particularly with low-groove music, in which beat locations are less salient. High-groove music and metronome cues generally resulted in better gait performance than low-groove music in both weak and strong beat-perceivers. Taken together, these findings suggest that tailoring auditory cues according to patient beat perception ability in gait rehabilitation might elicit better outcomes.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.http://www.frontiersin.org/Journal/10.3389/fnhum.2014.00811/abstractThe Supplementary Material for this article can be found online at Click here for additional data file.


The ideal tracer for studying CSF dynamics is CSF itself. In time spatial labeling inversion pulse (Time-SLIP), MR radiofrequency pulses convert specific volumes of CSF into an endogenous tracer. CSF dynamics can then be observed under physiological and pathophysiological conditions. A gate-free and fast image acquisition technique like Time-SLIP is necessary to visualize natural CSF motion, whose behavior varies with cardiac pulsation and respiration.To study physiological variability in CSF motion using the MRI Time-SILP method.A real-time Time-SLIP balanced steady state free precession (bSSFP) sequence was used on 1.5T and 3T MRI scanners. Acquisition time for each image was approximately 130msec. Serial images were obtained one to five seconds after the labeling pulse. Pulsatile CSF motions over four to five cardiac strokes were analyzed.Considerable pulsatile CSF motion variability was observed in normal physiological brains as well as pathophysiological  brains.Real-time MR imaging is necessary to investigate natural pulsatile CSF motion. Averaging over multiple pulsatile CSF motions potentially wipes out natural physiological variability in CSF motion.


Cerebrospinal fluid (CSF) leakage of the temporal bone region is defined as abnormal communications between the subarachnoidal space and the air-containing spaces of the temporal bone. CSF leak remains one of the most frequent complications after VS surgery. Radiotherapy is considered a predisposing factor for development of temporal bone CSF leak because it may impair dural repair mechanisms, thus causing inadequate dural sealing. The authors describe the case of a 47-year-old man with a massive effusion of CSF which extended from the posterior and lateral skull base to the first cervical vertebrae; this complication appeared after a partial enucleation of a vestibular schwannoma (VS) with subsequent radiation treatment and second operation with total VS resection.  Cerebrospinal fluid (CSF) leakage in the temporal bone region is defined as an abnormal communication between the subarachnoidal space and the air-containing spaces of the temporal bone , 2. It mSome authors have suggested that radiotherapy (RT) may be considered a predisposing factor for development of temporal bone CSF leakage , 5–7. InThe authors present a rare case of a massive temporal bone CSF leak which occurred after surgery to partially enucleate a vestibular schwannoma (VS), subsequent radiotherapy, and further surgical treatment consisting of total VS resection.A 47-year-old Italian man came to our Department in January 2012 complaining of vertigo and headache. He showed a left facial nerve paralysis evaluated as grade V according to the House-Brackmann scale. He was surgically treated in 2009 by means of a partial intracapsular enucleation technique via a retrosigmoid approach for a left VS in another hospital. Postoperative adjuvant fractionated stereotactic radiotherapy (SRT) with a total dose of 40–50 Gy, administered in 20–25 fractions over a 5- to 6-week period, was performed.Due to VS tumor regrowth, causing cerebellar compression and perilesional brainstem edema, the patient underwent a second neurosurgical operation by retrosigmoid approach in October 2011 with total VS removal. Preoperative ventriculoperitoneal shunt was placed in order to prevent a brain herniation. No apparent postoperative complications occurred; however, cystic areas of CSF leakage consequent to a defect of dural repair were visible on a subsequent control MRI .After 3 months of follow-up, the patient started to complain of worsening headache and severe dizziness. Moreover, CSF rhinorrhea was reported. Brain MRI with contrast medium performed in our Department showed a massive CSF leak which measured approximately 5.6 cm and 8.4 cm along its the transversal  and longTo treat the massive CSF leak, equine pericardium grafts were used to repair the posterior fossa dural defect and a subtotal petrosectomy –13 with After surgery, there was a marked improvement of vertiginous symptoms and headache. No new episodes of CSF rhinorrhea occurred.At the most recent postoperative follow-up, 4-years after surgery, MRI showed no CSF leak recurrence and no parenchymal lesions .Cerebrospinal fluid leakage in the temporal bone region is an uncommon condition with multiple potential etiologies . It mostMost dural defects can be easily managed during the initial operation; however, in some cases they may be up to 2 cm in diameter, cannot be sealed off spontaneously, or remain unrecognized until later –26.This condition usually presents with nonspecific symptoms such as aural fullness, tinnitus, headache, and vertigo. Rhinorrhea, otorrhea, or middle ear effusion could, in certain cases, camouflage CSF leakage , 13, 27.We describe the case of a 47-year-old man with a massive effusion of CSF extending from the posterior and lateral skull base to the first cervical vertebrae, which appeared after partial enucleation of a VS with subsequent radiation treatment and a second operation consisting of a total VS resection.Imaging studies are essential for determining the site of the CSF leak in the temporal bone. High-resolution noncontrast CT (HRCT) is considered the best initial diagnostic study. HRCT usually identifies any bony defects of the temporal bone but might not demonstrate the site of the dural tear , 18, 27.MRI is a sensitive and accurate technique for detection of CSF leaks which usually appear hyperintense on T2-weighted images and hypointense on T1-weighted sequences: it also offers excellent anatomical detail without risk of radiation .In the differential diagnosis of temporal bone CSF collections, subdural hygroma, meningocele, and arachnoid cyst should all be considered. Magnetic resonance imaging is able to distinguish isolated temporal bone CSF leakage from middle ear/mastoid encephalocele because the CSF signal is usually bright on T-2 weighted images, and the encephalocele appears contiguous with the brain , 18, 26.Important risk factors for a postoperative CSF leak after VS surgery do exist , 21. In In addition to the above some authors have claimed that an important risk factor related to CSF leak formation seems to be adjuvant RT , 7, 27.Radiotherapy is considered a predisposing factor to temporal bone CSF leak development because it can impair the dural repair mechanisms, causing an insufficient dural sealing , 7. AfteRecognizing CSF risk factors can allow surgeons to better counsel patients regarding the risks of VS surgery and potentially modify perioperative management in order to reduce the risk of developing a postoperative CSF leak , 19.Subtotal petrosectomy with obliteration of the middle ear cleft seems to be an efficient solution for stubborn CSF otorrhea , 29, 30.In patients who must be submitted to subtotal petrosectomy, a preoperative high-resolution CT scan is mandatory , 13, 18.We believe that in patients submitted to radiotherapy, dural repair after new surgical treatment may be less efficient with the possibility of subsequent massive CSF leakage. In the light of the increasingly important roles played today by RT in the definitive or adjuvant management of vestibular schwannomas, this aspect should not be underestimated. Therefore, surgical reconstruction of the skull base dura should be done more carefully in patients who previously underwent RT. In the event of massive CSF leakage, it is mandatory not to delay surgical obliteration of all accessible pneumatized spaces in the petrous bone, in order to close all possible fistulas and to prevent meningitis. Our therapeutic strategy consisting of subtotal petrosectomy, demonstrated good therapeutic efficacy with improvement of clinical symptoms and a low risk of CFS leak recurrence even after a long follow-up.


This laboratory-based investigation compares the topographic outcomes of conventional penetrating keratoplasty with that of a novel procedure in which donor corneas are cross-linked prior to keratoplasty.Penetrating keratoplasty procedures with continuous running sutures were carried out in a porcine whole globe model. Sixty eyes were randomly paired as ‘donor’ and ‘host’ tissue before being assigned to one of two groups. In the cross-linked group, donor corneas underwent riboflavin/UVA cross-linking prior to being trephined and sutured to untreated hosts. In the conventional keratoplasty group, both host and donor corneas remained untreated prior to keratoplasty. Topographic and corneal wavefront measurements were performed following surgery, and technical aspects of the procedure evaluated.p < 0.005). Mean wavefront astigmatism was also significantly reduced in the cross-linked donor group 4.71 D (SD 2.1) vs. 8.29D (SD 3.6) in the conventional keratoplasty group (p < 0.005). Mean RMS higher order aberration was significantly lower in the cross-linked donor group at 1.79 um (SD 0.98), vs. 3.05 um (SD 1.9) in the conventional keratoplasty group (P = 0.02). Qualitative analysis revealed less tissue distortion at the graft-host junction in the cross-linked group.Mean keratometric astigmatism was significantly lower in the cross-linked donor group at 3.67D (SD 1.8 D), vs. 8.43 D (SD 2.4 D) in the conventional keratoplasty group (Cross-linking of donor corneas prior to keratoplasty reduces intraoperative induced astigmatism and aberrations in an animal model. Further studies are indicated to evaluate the implications of this potential modification of keratoplasty surgery. Corneal transplant or keratoplasty procedures are well recognised in the management of axial opacity of the cornea to restore vision . Both peCorneal cross-linking using UVA-activated riboflavin is a relatively recent but established procedure for the prevention of progression of corneal ectasia due to keratoconus . The treThe induction of regular and irregular astigmatism in the visual axis of transplanted donor corneas is a function of a number of biomechanical considerations, particularly related to the alignment and tension along the point of attachment to the donor, i.e., the graft-host interface. Any mismatch between the shape of donor and host trephination will result in induction of irregularity , 11, 12.Given these considerations, we hypothesized that it might be expected that the induction of irregular and regular astigmatism within the donor cornea may be inversely related to the modulus of elasticity, in such a way that a more easily deformable donor cornea would be expected to exhibit higher levels of induced regular and irregular astigmatism than a stiffer donor cornea. If this were the case, we further hypothesized that increasing the stiffness of the donor cornea, by cross-linking it prior to keratoplasty, may result in a reduction of induced central regular and irregular astigmatism. If so, this novel procedure might provide a simple means to mitigate some of the visually limiting irregularity in corneal transplantation procedures.The animal experiments in this paper comply with the Principles of Laboratory Animal Care , the OPRR Public Health Service Policy on the Humane Care and Use of Laboratory Animals (revised 1986), and the U.S. Animal Welfare Act, as amended, as well as specific national laws.Sixty fresh porcine eyes were collected within 3 h of death from animals slaughtered for non-research purposes in an EC licensed abattoir . Thirty eyes were designated as ‘donor’ tissue and the remaining 30 eyes as ‘host’ tissue. The donor and host eyes were randomly paired and allocated into two groups. In the cross-linked group, the donor cornea was de-epithelialized and immersed in commercial riboflavin 0.1 % with dextran 20 % solution , for between 30 min and 12 h. The donor eyes in the conventional keratoplasty group were also de-epithelialized and simultaneously immersed in 20 % dextran T500 solution . Host eyes were prepared identically to the conventional donor eyes.2 UVA light for 30 min using a commercially available cross-linking lamp . During the cross-linking procedure, riboflavin eye drops were applied every 5 min. During the corresponding period, the donor corneas in the conventional keratoplasty group and the host corneas were maintained at ambient temperature and not exposed to any light.All eyes were maintained in a light-excluded environment during this phase to prevent inadvertent cross-linking of the riboflavin-treated corneas. In keeping with the conventional protocol for in vivo cross-linking of keratoconus corneas , the croA keratoplasty procedure was subsequently performed for both groups (15 cross-linked donor keratoplasties and 15 standard untreated donor keratoplasties). All keratoplasty procedures in the study were carried out by a single surgeon (AM). Both donor and host globes were trephined using a 7.5 mm freehand corneal trephine and completed with corneal scissors. Sodium hyaluronate 1 % viscoelastic  was applied to maintain the anterior chamber and donors from both groups that were sutured using a standardised continuous single running 10/0 nylon suture with 16 bites under microscopic visualisation. No suture adjustment or keratoscopic visualisation was performed to avoid operator bias. In order to avoid distortion of topography induced by viscoelastic, this was aspirated via a small paracentesis and replaced with balanced salt solution . The globe was then inflated through this paracentesis to approximately physiological pressure determined digitally. The use of Tono-Pen tonometry  was avoided due to concern that measurement itself might affect the corneal topography, while Goldmann applanation tonometry was not technically feasible. A small amount of artificial tear solution (hypromellose 0.3 %) was applied to the surface of each cornea to prevent drying and allow the reliable acquisition of topographic data. The typical appearance of eyes following cross-linked donor keratoplasty and conventional keratoplasty is shown in Fig. Topography of the transplanted corneas was performed using a vertically mounted, intraoperative, high resolution, small cone placido topographer . To prevent distortion of the eyes during measurement, the globes were carefully mounted in a corneal viewing chamber (with the cornea facing upwards). The eyes were then rotationally aligned using the position of insertion of the extraocular muscles, and manually aligned with the red reflex to maintain axial orientation prior to measurement. Measurements were repeated six times for each specimen. The automated software was used to centre the horizontal alignment prior to capture. Following capture, the three readings with the least traceable ring images were discarded, and the remainder analysed. Automated analysis of topographic parameters and map were carried out, as well as corneal wavefront analysis of higher order aberrations including peak to valley (PV) and root mean square (RMS) values.P = 0.209 t-test).Simulated keratometry values derived from the topography were analysed. Mean Keratometric value was 30.9 D  in the cross-linked donor group and 34.6 D  in the conventional keratoplasty group. There was no significant difference between groups (p < 0.005 t-test). Astigmatic measurement was also derived from the wavefront analysis, since this is a composite value derived across the assessment area, and may provide a more accurate assessment in highly irregular or aberrated corneas. Mean wavefront astigmatism was 4.71 D  in the cross-linked donor group, and 8.29D  in the conventional keratoplasty group. The mean wavefront astigmatism was also significantly lower in the cross-linked donor group (p < 0.005 t-test)  in the cross-linked donor group, and 8.43 D  in the conventional keratoplasty group. Keratometric mean astigmatism was significantly lower in the cross-linked donor group . Mean P-V aberration was 11.4 um (SD 6.7 range 4.1 to 29.9 um) in the cross-linked donor group and 23.4 um (SD 17.8 um range 5.3 to 57 um) in the conventional keratoplasty group, significantly lower in the cross-linked donor group (P = 0.029 t-test). Both coma and spherical aberration were independently analysed due to the visual relevance post keratoplasty. Mean coma aberration in the cross-linked and conventional keratoplasty groups was 1.01 um  vs. 0.75 um (SD 0.67 range 0.1 to 2.3 um), while mean spherical aberration was −0.26 um (SD 0.5 range −1.4 to 0.3 um) vs. −0.09 um (SD 0.57 range −1.6 to 0.6 um), respectively. Neither coma nor spherical aberration differed significantly between groups  , while it was 3.05 um (SD 1.9 range 0.8 to 6.5 um) in the conventional keratoplasty group. RMS aberration was significantly lower in the cross-linked donor group (Topographic maps were additionally analysed qualitatively. In particular, the pattern of ring images at the graft-host junction was evaluated since the reflex in this area was beyond the range of automated assessment by the topographer. There was a distinct dissimilarity noted between cross-linked and non-cross-linked donors. At the graft-host junction, the local distortion of placido reflex due to compression by each suture bite was a subjectively larger in terms of area and magnitude in the conventional keratoplasty donors, compared to the cross-linked donors. There was also a localised ring of steepening in a ‘doughnut’ shape, in the sutured area of conventional keratoplasty donors, which was largely absent in the cross-linked cases. A typical representative sample of ring images from conventional and cross-linked donors is presented in Fig. Due to the altered appearance of the cross-linked donor corneas, the surgical operators could not be blinded to the donor group. A number of observations were made in relationship to the surgical aspects of keratoplasty in cross-linked versus conventional donors. Alignment and approximation of the sutured edges was much easier in the cross-linked group, since the tissue was more resistant to deformation and maintained shape during the passage of the needle. This was particularly noticeable during passage of the cardinal suture bites, where alignment can be challenging in keratoplasty procedures as the donor tissue is not yet fixed in place. There was a slightly increased resistance to the passage of the needle and suture in the cross-linked group, but this did not affect the ease of surgery. Achieving a watertight wound was also easier in the cross-linked group, since there was less distortion on application of suture tension and the wound was less affected by suture positioning and alignment. One negative aspect with the cross-linked donor keratoplasty procedure was the tendency to override the host tissue if vertically misaligned or over tightened, since, unlike in conventional keratoplasty donors, the compressed edge did not broaden under tension, while the host edge was deformed.The primary objective of this study was to evaluate the possibility of reducing regular and irregular astigmatism and aberrations by cross-linking donor corneas prior to keratoplasty. To our knowledge this is the first report to evaluate corneal cross-linking of donor corneas for keratoplasty. The findings of this study support the hypothesis that altering the biomechanical rigidity of a keratoplasty donor may reduce the induced regular and irregular astigmatism of the axial refracting surface by lessening the effect of forces at the graft host junction.We thus found significantly reduced values for all parameters evaluating regular astigmatism: simulated keratometry, as well as more composite geometric measures including Maloney best fit cylinder, and the reconstructed corneal wavefront astigmatism. All of these suggest that cross-linking the donor cornea may indeed reduce the regular astigmatism induced during a keratoplasty procedure. The magnitude of this effect was somewhat unexpected prior to commencing the study, since our expectation was that cross-linking would significantly reduce local changes in shape, by evenly distributing forces, and, thus, have an effect mainly on irregular astigmatism and high order aberrations, rather than reducing regular astigmatism. Nonetheless, the donor cross-linking procedure had a marked effect on regular astigmatism, with a reduction of less than half in all measured parameters. It is also noteworthy that no suture adjustment or intraoperative keratometry was performed, as would be usual in such procedures, thus, the level of astigmatism overall might be expected to be lower still if applied to clinical procedures.Our primary motivation for developing the procedure however, was more to address irregular astigmatism and high order aberrations. These are a significant issue affecting visual outcomes of keratoplasty procedures, since they are not easily corrected and relate to the limited visual outcomes in a significant proportion of cases. RMS and P-V measures of HOA were significantly reduced to approximately one half and one third respectively of the values in the conventional keratoplasty group. The Maloney topographic irregularity index was also significantly lower in the cross-linked donors. These changes would be expected to lead to considerably better visual outcomes if mirrored in human keratoplasty procedures.A secondary objective was to assess whether cross-linking would require any alteration of the surgical technique for keratoplasty. However the procedure required no significant changes to technique and was, if anything, easier to perform using cross-linked donors as alignment and placement of suture passes in more rigid tissue was simpler. The potential override of donor was easily avoided by attention to suture depth. In this regard, matched trephination techniques such as femtosecond laser trephination might be of particular benefit.The main limitation of this study is the applicability of findings to a proposed human surgical procedure due to the biomechanical differences between human donor and porcine corneas . Human dAlthough the effects of donor cross-linking on keratoplasty have not been evaluated previously, cross-linking has previously been applied in the setting of existing keratoplasty without concern. Spadea and Paroli investigated combined photorefractive keratectomy and cross-linking after lamellar keratoplasty in 14 eyes, with no noted adverse effect at a mean of 15 months . Labiris