Atherosclerosis is a major cause of mortality and morbidity, which is mainly driven by complications such as myocardial infarction and stroke. These complications are caused by thrombotic arterial occlusion localized at the site of high-risk atherosclerotic plaques, of which early detection and therapeutic stabilization are urgently needed. Here we show that near-infrared autofluorescence is associated with the presence of intraplaque hemorrhage and heme degradation products, particularly bilirubin by using our recently created mouse model, which uniquely reflects plaque instability as seen in humans, and human carotid endarterectomy samples. Fluorescence emission computed tomography detecting near-infrared autofluorescence allows in vivo monitoring of intraplaque hemorrhage, establishing a preclinical technology to assess and monitor plaque instability and thereby test potential plaque-stabilizing drugs. We suggest that near-infrared autofluorescence imaging is a novel technology that allows identification of atherosclerotic plaques with intraplaque hemorrhage and ultimately holds promise for detection of high-risk plaques in patients. Atherosclerosis diagnosis relies primarily on imaging and early detection of high-risk atherosclerotic plaques is important for risk stratification of patients and stabilization therapies. Here Htun et al. demonstrate that vulnerable atherosclerotic plaques generate near-infrared autofluorescence that can be detected via emission computed tomography. Some plaques remain quiescent and stable for years and others become unstable and vulnerable, ultimately leading to plaque rupture. Plaque rupture is the main cause of thrombosis and occlusion of arteries leading to potentially catastrophic outcomes such as stroke and myocardial infarction (MI), the latter being the most frequent single reason of death7. The development of a method to reliably identify high-risk atherosclerotic plaques is seen as one of the major quests of contemporary cardiovascular medicine. It would provide the opportunity to apply prophylactic treatment, either pharmacological or interventional, and thus to prevent e.g. MI and its associated substantial morbidity and mortality.Although atherosclerosis is usually described as a single disease entity with significant morbidity and mortality worldwide, not all atherosclerotic lesions or plaques are the same with vast differences in their composition and natural history13. However, this technology was limited by poor specificity, as low wavelength fluorescence is not exclusively emitted by lipid core associated materials, as well as poor signal-to-noise ratio and weak tissue penetration caused by hindering tissue autofluorescence and strong photon absorption15. In contrast, our study demonstrates that autofluorescence in the NIR range uniquely characterizes atherosclerotic plaques with intraplaque hemorrhage and may allow classification of atherosclerotic plaques into lesions of low or high risk for future cardiovascular events.Significant efforts have been made to characterize and risk stratify carotid and coronary atherosclerotic plaques using various imaging technologies. We here propose the use of near-infrared autofluorescence (NIRAF) for the identification of high-risk atherosclerotic plaques. Investigators previously used low-wavelength (300–400 nm) fluorescence technology to assess plaque composition, detecting fluorescence signals of collagen, elastin, and oxidized low density lipoprotein, typically as components of the lipid core16. This mouse model was instrumental for the description of NIRAF as a potential indicator of an atherosclerotic plaque’s risk in causing future cardiovascular events. In addition to human CEA samples, this model allowed us to identify intraplaque hemorrhage and heme degradation products as sources of the detected NIRAF signal. Besides establishing fluorescence emission tomography (FLECT) as a preclinical tool for the detection of plaques with intraplaque hemorrhage and its potential use in testing and monitoring of plaque-stabilizing drugs, our report has the potential to support technical developments for imaging technologies that will ultimately allow to risk stratify atherosclerotic plaques in patients and, in particular, to identify plaques that are prone to cause cardiovascular events.We have previously developed a mouse model that exhibits unstable plaques as seen in humansBoth human and murine atherosclerotic samples were used in this study. With informed consent, human CEA specimens were collected from patients who presented to the Alfred Hospital, Melbourne, Australia with clinical indications for CEA.p < 0.0001, unpaired Student’s t-test).To investigate human atherosclerotic plaques, 50 CEA samples freshly taken from patients in the operating theater were immediately scanned using the Odyssey Imaging System before being snap-frozen and stored at −80 °C prior to further analysis. Of which, 26 samples were from patients who presented with symptoms such as stroke or transient ischemic attacks, while 24 samples were from asymptomatic patients. Patients with symptoms can be assumed to harbor more complicated plaques, whereas asymptomatic patients underwent endarterectomy because they had high-grade, severely stenotic lesions . Hence the CEA samples obtained will exhibit a mixture of advanced atherosclerosis including histological characteristics of unstable as well as stable plaque pathology. Thirty-one out of 50 CEA samples collected (62%) showed significant intrinsic autofluorescence (mean fluorescence intensity (MFI)>20) in the NIR range  utilized an excitation laser source of 785 nm to detect autofluorescence emission signals. This was achieved by integrating the area under the curve between 4000 and 150 cmion Fig. . To furtion Fig. . Seg-a sion Fig. . Overall16. This murine model is unique in its reflection of histological plaque instability as seen in humans. Briefly, a surgical ligation procedure . Seven weeks postoperatively, disruption of fibrous caps, intraplaque hemorrhage, intraluminal thrombosis, neovascularization, and other characteristics of plaque instability/rupture as described in humans were seen in these mice16  of atherosclerosis in mice as described by Chen et al.16, and scanned freshly with the Odyssey Imaging System  in contrast to histologically stable plaques, which showed only minimal fluorescence   Fig. . The hea15) Fig. . Serial 15) Fig. . These r17, recorded at the same excitation wavelength. The spectra obtained from the fluorescent plaques in mice and humans reveal a mixture of heme products as evidenced by the position of ν4, which appears between 1360 and 1371 cm−1 and is assigned to a symmetric pyrrole half-ring stretching vibration17. This band is known as the oxidation state marker band because it is sensitive to the oxidation state of iron17. In oxygenated hemoglobin, which is in the ferric low spin state, this band normally appears between 1376 and 1370 cm−1. In deoxygenated hemoglobin, where the Fe ion is in the ferrous high spin state, this band normally appears between 1360 and 1350 cm−1. Figure −1 and a shoulder at 1372 cm−1, indicating this contains a mixture of ferrous and ferric iron. The appearance of ν4 between 1360 and 1371 cm−1 in the fluorescent plaques indicates that the heme-based compounds in these plaques are also likely to be a mixture of oxidation states as this lies between the wave number values expected for ferric and ferrous heme. The likely origin of these mixed redox states of heme in atheromatous plaques is the oxidation and reduction of hemoglobin at the sites of intraplaque hemorrhage. A number of other strong heme bands are also observed in the spectra obtained from the plaques. These include bands at 1620 cm−1, 1582 cm−1, 1544 cm−1 assigned to C=C and C–C bonds of the porphyrin backbone and other bands at 753 and 666 cm−1 assigned to pyrrole breathing and deformation modes, respectively17. Due to the inherent nature of atherosclerosis that contains a mixture of different biological tissues, the Raman signals of heme degradation products from the plaques are likely to be shifted to some extent by co-existing compounds within the plaque rather than exhibiting classical Raman spectra of pure chemicals.Once we had established NIRAF to be associated with high-risk atherosclerotic plaques, we searched for the source of NIRAF using chemical fingerprinting; comparing Raman signals obtained from fluorescent atheromatous plaques to those from non-fluorescent plaques. To record resonance Raman spectra, we had to choose an excitation wavelength well apart from the NIR fluorescence and hence we applied 413 nm. Resonance Raman spectra were recorded using a Spectra Physics Stabilite 2017 argon ion laser system coupled to a Renishaw Raman 2000 spectrometer and interfaced to a Leica Raman microscope. Resonance Raman spectra from the fluorescent atheromatous plaques showed distinct peaks Fig.  that cloIn the event of intraplaque hemorrhage, inflammatory cells e.g. macrophages phagocytose extravasated red blood cells (RBCs), leading to induction of heme oxygenase-1 (HO-1). Heme derived from the ingested RBC hemoglobin is then metabolized to biliverdin via the enzyme HO-1. Biliverdin is subsequently reduced to bilirubin via biliverdin reductase. In order to investigate this further, we obtained pure standards of hemoglobin, protoporphyrin IX, biliverdin and bilirubin to cover the whole spectrum of heme metabolism) and determined their Raman spectra. The spectra of biliverdin and bilirubin differed markedly from those of hemoglobin and protoporphyrin IX because the porphyrin structure is no longer present in biliverdin and bilirubin. Overall, Raman spectra of atherosclerotic plaques with NIRAF are clearly dominated by hemoglobin signals Fig. .Based on the resonance Raman spectra obtained from atheromatous plaques, intraplaque hemorrhage is considered to be the likely source of the autofluorescence signals. Hence fluorescence characteristics at the NIR range of blood and its related products  were investigated using the Odyssey Imaging System.Samples of fresh and old blood obtained from mice were also scanned using the Odyssey System. No fluorescence signals were detected in the NIR range, indicating blood by itself, either fresh or old, is not responsible for the NIRAF found in atherosclerotic plaques  was then explored. While hemoglobin, ferrous-stabilized hemoglobin and biliverdin did not exhibit any fluorescence on the Odyssey System, the heme precursor protoporphyrin IX and the final heme degradation product bilirubin revealed intrinsic fluorescence in the NIR range Fig. .Fig. 4Bi19 and bilirubin concentration (represented as absorbance at 476 nm) in the extracted fluid was measured with a Cary 60 UV–vis Spectrophotometer (Agilent Technologies). As demonstrated in Fig. Seven human CEA plaque samples with NIRAF and seven samples of human carotid arterial segments without NIRAF were excised and weighed (120 mg each). A standard chloroform-based extraction was used to extract bilirubin from these samples16, collected at 1, 4, and 7 weeks post TS surgery, was determined using liquid chromatography–tandem spectrometry (LC–MS/MS) on an Agilent 1290 binary pump connected to a 6490 triple quadrupole mass spectrometer . The content of heme, biliverdin and bilirubin in stable plaques remained low and unchanged at all time points  in small amounts of tissues, such as plaques of the TS mouse model. The heme, biliverdin and bilirubin content in histologically defined unstable (segment I) and stable plaques (segment V)nts Fig. . In contnts Fig. . Heme annts Fig. . At 7 weUsing the IVIS Lumina Series II Imaging System  we compared the NIRAF characteristics of hemoglobin and bilirubin over a wide range of excitation wavelength. Whereas hemoglobin shows again no NIRAF, bilirubin shows NIRAF at a broad range of different excitation wavelengths  with detection of NIR signals above 810 nm  after staining CEA samples with Alexa 647 dye-labeled anti-bilirubin antibody  was performed to test the presence of iron/hemosiderin in human CEA specimens with or without NIRAF . Staining was strongly positive in the CEA samples with NIRAF and only minimal in the CEA samples without NIRAF, indicating that areas of fluorescence match the areas of intraplaque hemorrhage  impeded atherosclerotic lesion progression into vulnerable plaques in ApoE−/− mice, resulting in reduced size of the necrotic cores, less intraplaque lipid accumulation and an increased ratio of fibrous cap thickness/intimal vascular smooth muscle cells, whereas inhibition of HO-1 by zinc protoporphyrin IX (ZnPPIX) augmented plaque instability. We hypothesized that the manipulation of HO-1 activity by ZnPPIX and CoPPIX will be an ideal test to evaluate whether the newly detected NIRAF characteristic would be suitable to detect and monitor changes in histological plaque instability.Heme oxygenase 1 expression has been demonstrated in atherosclerotic lesions in both animal and human studies and it has been implicated to be atheroprotective−/− mice (6 weeks on western diet at 12 weeks of age). CoPPIX (HO-1 inducer) and ZnPPIX (HO-1 inhibitor) were used for HO-1 enzyme modulation. Two weeks after the TS surgery, intraperitoneal injections of heme oxygenase modulators  at pH 7.4) were performed as described by Cheng et al.22 every second day for 5 weeks. The mice were sacrificed after 5 weeks of intraperitoneal injections. The timeline of this experiment is shown in Fig. 2+/Mg2+) at pH 7.4).Tandem stenosis surgery was performed on ApoEThe unstable plaques (segment I of the TS model) from all three groups of mice were freshly harvested and imaged using the Odyssey Imaging System. The ZnPPIX group showed significantly higher fluorescence intensity (MFI ± SEM of 3.05 ± 0.38) compared to the other two groups (MFI ± SEM of 1.90 ± 0.19 in the CoPPIX group and MFI ± SEM of 2.08 ± 0.23 in the vehicle group) Fig. . The carThese findings indicate that changes in histologically defined plaque instability can be detected and monitored by NIRAF measurements.16). The subsequent CT images confirmed NIRAF signals are localized to segment I of the carotid artery  was used to obtain three-dimensional NIRAF images of mice with the TS model, exhibiting high-risk atherosclerotic plaques. NIRAF was detected in TS mice exhibiting intraplaque hemorrhage Fig. , but notery Fig. . To furtery Fig. . Mice weery Fig. . Represeery Fig.  either wery Fig. .Fig. 8Fl13, fluorescence measurements of atherosclerotic plaques in the NIR range reported here has substantial technical advantages compared to lower wavelength imaging. NIR fluorescence imaging (650–900 nm) encounters markedly less photon absorption, has better tissue penetration and less background autofluorescence of tissues15. Various extrinsic fluorophores for a variety of molecular targets  have been tested in animal models of atherosclerotic plaque development in mice27 and New Zealand rabbits26 with a focus on intravascular probes. Our study describes and characterizes NIR fluorescence signals in atheromatous plaques without the requirement of external fluorophores. The areas of NIRAF coincide with the areas of intraplaque hemorrhage with higher concentrations of bilirubin, the major breakdown product of heme. Our study indicates the source of NIRAF likely to be the heme degradation product bilirubin.Although some of the components of plaques such as collagen, elastin, some extracellular lipids and ceroids/lipofuscin have been shown to exhibit intrinsic fluorescence when excited in the violet and blue range (325–475 nm)33. Some of the NIR dyes were synthesized using porphyrin-based compounds29. Bilirubin was also known to exhibit intrinsic fluorescence in the 400–500 nm range34 and was recently also reported to have an emission peak at 660 nm while excited at 615 nm, which is consistent with our findings35. Naturally occurring protoporphyrin IX, a precursor of heme, showed autofluorescence in the NIR spectrum36. Fluorescence signals of these compounds are not constant in intensity and can change markedly with protein binding. Bilirubin’s fluorescence is enhanced with albumin binding39 or binding with metals . Even interaction with human erythrocyte ghosts seems to enhance the fluorescence of bilirubin42. In our experiments, bilirubin displayed NIRAF on the Odyssey Infrared Imaging System. Bilirubin is sufficient for NIRAF generation, although other atheroma-based molecules, including the heme precursor protoporphyrin IX, could be additional sources of NIRAF. Hence it is likely that heme degradation products accumulate in the areas of intraplaque hemorrhage and mixed with various proteins, lipids and necrotic debris then become a source of NIRAF.Porphyrin derivatives have long been described as fluorophores with emission spectra reaching red and infrared rangesInterestingly, Raman spectra collected from plaques with NIRAF showed predominantly hemoglobin signals, rather than bilirubin signals. Most likely the Raman spectra are overloaded by hemoglobin. Mass spectrometry-based measurements of heme and bilirubin in unstable carotid plaques from segment I of TS mice Fig.  showed m45 and it is believed to be caused either by rupture of thin fibrous caps or by leaky vessels as a result of extensive neovascularisation of unstable atheromatous plaques46. Intraplaque hemorrhage has been shown not only to be a common feature of larger arteries but also of high-risk coronary plaques. Kolodgie et al.47 found that 77% of the high-risk, thin-cap fibroatheromas exhibit intraplaque hemorrhage. In a study of 57 patients who died of coronary artery disease, Virmani et al.48 found extravasated erythrocytes in coronary plaques of 84% of patients. Recent studies indicate that intraplaque hemorrhage could also be a trigger, rather than just a marker, of plaque instability, by playing a major role in plaque progression with resultant accumulation of erythrocyte membranes, cholesterol crystal generation and retention, enlargement of necrotic cores, promoting oxidant and proteolytic activities, leukocyte infiltration, and inflammation in plaques51.Intraplaque hemorrhage is one of the classical histological features of plaque instability52. Despite extensive advances in the field of cardiovascular medicine and imaging technologies, the ability for early detection of the high-risk plaques remains elusive. Several invasive plaque-imaging techniques have been developed with the aim to detect the various characteristics of histologically defined unstable plaques (such as TCFA). However, most of these currently used methods are able to detect only some, but not all, histological features of unstable plaques.There are several histological features that characterise an unstable and high-risk atherosclerotic plaque: Thin fibrous cap of <65 µm (giving rise to the term thin-cap fibroatheroma (TCFA)); large necrotic core; increased plaque inflammation; positive vascular remodeling; increased neovascularization by vasa vasorum; and intraplaque hemorrhage53 showed the major adverse cardiovascular event rate for non-culprit lesions, which were classified as TCFA, exhibiting a plaque burden of >70% and minimal luminal area of  ≤ 4 mm2, was 18.2% at 3.4 years follow-up. While this clearly demonstrates that the use of IVUS-VH has some value in predicting future adverse cardiovascular events, it is still far from ideal with a relatively poor predictive value, highlighting its limitations in detecting all of the histological criteria of unstable plaques.Using intravascular ultrasound with virtual histology (IVUS-VH) Stone et al.57. Fujii et al.58 found that the mean value for minimum fibrous-cap thickness within TCFAs was 57.4 ± 5.4 µm in 35 MI patients, and 55.9 ± 7.3 µm in 20 stable angina patients (p = 0.4), indicating that by itself TCFA, detected by OCT, is probably not sufficient to differentiate low-risk from high-risk atherosclerotic plaques.Optical coherence tomography (OCT) with its high resolution (10–15 µm) is well-suited for detection of thin fibrous caps, macrophages and necrotic core but is not well designed for reliable detection of positive remodeling or plaque burden61. Madder et al.62 recently published a study using NIRS to identify the composition of target lesions in patients with acute coronary syndrome (28 patients) vs. stable angina (32 patients). Nearly 50% of patients with stable angina showed lipid core plaques in this study, casting doubts on its ability to identify the plaques with high risk for cardiovascular events.Near-infrared spectroscopy (NIRS) has been shown to be able to detect lipid core-containing plaques in both, ex vivo and in vivo studies63.Other emerging methods including angioscopy  and thermography (detects metabolic activity of the plaque) are reporting individual characteristics of atherosclerotic plaques but have not been proven to provide predictive value yet. Intravascular application of Raman spectroscopy is also technically challenging due to its low signal-to-noise ratio and poor tissue penetration66. But MRI of coronary arteries has been challenging due to their small diameter, deep location in the chest and their motions with cardiac cycle and diaphragmatic movements67. Intravascular MRI to image vulnerable plaques, especially in the coronary arteries, is still at an early development stage although holding promise to ultimately being able to detect intraplaque hemorrhage.Non-invasive MRI has been used to identify intraplaque hemorrhage and vasa vasorum in the carotid arteries with a prognostic significance26. However, it should be noted that these rabbit models and other animal models of atherosclerosis may not represent unstable atherosclerotic plaques as seen in our TS mouse model or in humans. Hence plaques in rabbit and other larger animal models may not harbor significant areas of intraplaque hemorrhage, which we see as the central prerequisite for NIRAF . While the higher emission wavelength (750–800 nm range) may be well suited for targeted NIR fluorescence agents, it might be less suited for detection of NIRAF, such as successfully reported at a range between 633 and 700 nm69. Further in vivo intracoronary imaging studies based on these findings will help to determine the clinically most suitable NIRAF detection wavelengths for the identification of high-risk atherosclerotic plaques.Very recently, a pioneering dual-modality OCT-NIRAF (excitation 633 nm and detection between 675 and 950 nm) optical catheter system has been developed for the investigation of human coronary arteries71. However, in general implanting stents is not without risks, including death, MI, emergency bypass surgery, stroke and bleeding as well as stent thrombosis (acute or late), with a very high mortality rate. Hence for prophylactic stenting  of silent, non-obstructive lesions to become a feasible treatment option, a reliable imaging technology is required that has a high predictive value for the risk of cardiovascular complications.While clinicians and scientists alike are pursuing to improve intravascular imaging technologies to identify high-risk plaques, there are some studies already investigating the feasibility, safety and efficacy of pre-emptive, prophylactic stenting in potentially high-risk, but non-obstructive coronary lesions72, none of these technologies is generally accepted to reliably detect high-risk plaques. We here describe NIRAF, which characterizes high-risk plaques with intraplaque hemorrhage both in mice and humans, as such potentially highly promising technology. We also identify a potential source of NIRAF, bilirubin; being generated by heme degradation. Our study demonstrating NIRAF in the areas of intraplaque hemorrhage, which is clearly a critical element of plaque instability, provides a much-needed new foundation in the field of intravascular imaging of high-risk plaques. Fluorescence imaging in preclinical models can now be used to assess and monitor potential plaque stabilizing drugs, a technology long sought-after by the pharmaceutical industry. In humans, the feasibility of NIRAF measurements to risk stratify atherosclerotic plaques and thus to provide a basis for decision-making on preventative treatment will have to be tested in large randomized and controlled clinical trials. Such trials will then have to determine whether NIRAF quantification holds up to the promise of ultimately making a difference in cardiovascular mortality and morbidity.Although some of the currently available intravascular imaging modalities or their combination have their own merits and are able to identify some aspects of high-risk plaques In our study, a cut-off in MFI was used to categorize whether or not human CEA samples had significant NIRAF. Although this cut-off was chosen according to a standard dye intensity and based on comparisons with underlying plaque histology, it is to some extent arbitrary. Hence, the percentage of samples defined to be positive for NIRAF is dependent on this cut-off definition. However, a more meaningful cut-off point can only be defined via follow-up studies determining the occurrence of adverse cardiovascular events correlated to various levels of NIRAF.The current study describes NIRAF as a feature of atherosclerotic plaques that contain intraplaque hemorrhage. Our histological characterization indicates that these plaques are unstable and prone to rupture. However, this does not exclude that some plaques without intraplaque hemorrhage and thus without NIRAF can also cause cardiovascular complications. Ultimately, a definitive association of NIRAF with cardiovascular risk can only be made with certainty, if natural history studies with clinical follow-up are obtained. Such studies are warranted to determine the clinical relevance of NIRAF for the management of atherosclerotic disease and its potential impact on cardiovascular mortality and morbidity.Human CEA specimens were collected immediately after being taken out at the operating rooms from patients who presented to the Alfred Hospital, Melbourne, Australia with clinical indications for CEA. The project was approved by The Alfred Hospital Ethics Committee  and all the participants signed the informed consent form. All animal experiments were approved by the Alfred Medical Research Education Precinct (AMREP) Animal Ethics Committee .−1. The dye IRDye800CW (LI-COR Biotechnology) was used as a standard for comparison. All figures and fluorescence intensity measurements using the Odyssey Infrared Imaging System were taken using the 800 nm channel unless specified otherwise.The Odyssey Infrared Imaging System is a closed chamber analyzer that was originally designed for the characterization and quantification of protein bands in gels. It is a bottom up scanning process made possible by two solid state diode lasers with an excitation at 685 nm in the 700 Channel or an excitation at 785 nm in the 800 channel. The detector uses a silicon avalanche photodiode with a resolution of 21–337 µm and adjustable focusing range at 0–4 mm, the scanning speed is between 5 and 40 cm sThe fluorescence signals from human and murine samples were analyzed using Odyssey software version 3.0.30. For NIRAF analysis, samples were typically prepared fresh. However, paraffin-embedded samples also demonstrated NIRAF. Settings were adjusted to eliminate background fluorescence for image presentation. Absolute MFI values were used for statistical assessment. For human CEA samples, although determined in comparison to IRDye800CWl as a standard dye and to histological characteristics of plaque instability, a relatively arbitrary cut-off fluorescence unit of 20 was used to define the presence ( ≥ 20) or absence (<20) of relevant NIRAF of the samples.−1. The integration time for a single spectrum was 0.3 s with a spectral resolution of 3 cm−1. To generate autofluorescence images the integrated area between 4000 and 150 cm−1 was calculated, which corresponds to integrating the region between 794 and 1144 nm, when taking into account the Stokes shift from the 785 nm excitation line. The grating spectrograph was calibrated using the Raman scattering line produced by a silicon wafer (520.7 cm−1). Following excitation at 785 nm, emission fluorescence of the sample between 794 and 1144 nm was detected and presented as shown. Because of the strong fluorescence in this range, no Raman bands were observed and the resulting images represent autofluorescence from the sample.Raman autofluorescence imaging of tissue sections was performed on a WITec confocal CRM alpha 300 Raman microscope. The spectrometer was equipped with a Toptica diode laser operating at 785 nm and a back illuminated CCD detector, cooled to −60 °C. The laser was coupled to a microscope via a single mode optical fiber with a diameter of 100 μm and the typical power at the sample was 5 mW using a ×50 objective. The back scattered light was focused onto a multi-mode fiber (100 μm diameter) coupling to a 300 line grating spectrograph. The system was equipped with a long Pass Raman Filter E-Grade and a Razor Edge Dichroic Beamsplitter with high transmission starting at 65 cmHuman CEA samples (with or without autofluorescence) and carotid segments of mice that had undergone TS surgery (generating segments with or without autofluorescence), embedded in “optimal cutting temperature” compound  were sectioned at 5 µm (for human samples) and 40 µm (for murine samples), respectively using the Leica CM1950 cryostat . The samples were then mounted on microscope glass slides . Raman signals were obtained at 14 mW with a 413 nm excitation using the Renishaw InVia confocal micro-Raman system  with the Innova Ar/Kr ion laser 514/413 nm as a laser source. The data were analyzed by OPUS 6.0 software (Bruker Optik GmbH).2+ and Mg2+ in Eppendorf tubes. One milliliter of chloroform (Sigma-Aldrich) was added into each tube and vortexed vigorously for 30 s. The tubes were then centrifuged at 1000 × g for 10 min. The organic phase was carefully collected and transferred to new tubes for analysis. The tubes were protected from light with aluminum foil.The whole extraction procedure was performed with minimal exposure to light since bilirubin is light sensitive. The tissue segments were homogenized manually in 1 ml of PBS without Ca73.The Cary 60 UV–vis Spectrophotometer  was used to measure bilirubin concentration in the extracted fluid. The baseline measurements were performed using chloroform first and the absorbance at 476 nm (representing bilirubin concentration) was measured for each sample. A protocol used by a routine laboratory for the quantification of bilirubin in CSF (xanthochromia) was followedg for 10 min at 4 °C and 5 μl of the resulting supernate subjected to LC–MS/MS analysis using an Agilent 1290 binary pump HPLC couple to an Agilent 6490 triple quadrupole mass spectrometer.Mouse unstable  and stable plaques  collected at 1, 4 and 7 weeks post-TS surgery were homogenized in 100 or 200 μl ice-cold PBS , respectively. Homogenization was performed on ice for 3 min using a 0.2 ml Potter-Elvehjem micro tissue grinder (Wheaton). Internal standards  were then added to 50 μl of homogenates followed by 2.25 μl of 0.2 M EDTA. The mixture was mixed for 5 s and then 391 μl of ice-cold methanol containing 0.2 M EDTA added, the mixture vigorously mixed for 30 s and left on ice for 5 min. The extract was then centrifuged at 13,000 × −1. The gradient was as follows: 0–100% B over 8 min, 100% B for the subsequent 8 min, returning to 0% B over 1 min and then equilibrating at 0% B for 3 min. Following separation, flow was directed into the triple quadrupole mass spectrometer with parameters set as follows: gas temperature = 200 °C; gas flow = 15 l min−1; nebulizer pressure = 35 psi; sheath gas heater = 395 °C; sheath gas flow = 11 l min−1; capillary voltage = 3500 V and nozzle voltage = 1500 V. Pigments and internal standards were detected by multiple reaction monitoring (MRM) in positive ion mode using the above general mass spectrometry parameters with fragmentor voltage set at 380 V and cell accelerator voltage at 7 V. In each case, the fragment ions generated by collision-induced dissociation of the [M + H]+ ions were used for quantification. MRM transitions for the target analytes were: heme (m/z 616.2→557.2) with collision energy (CE) = 41 V; biliverdin (m/z 583.3→297.2) with CE = 33, bilirubin (m/z 585.3→299.2) with CE = 17 V; n-methyl-protoporphyrin IX (m/z 577.7→503.3) with CE = 45; meso-biliverdin (m/z 587.7→300.3) with CE = 41 and meso-bilirubin (m/z 589.7→301.1) with CE = 17. All analytes were quantified against authentic commercial standards obtained from Frontier Scientific and data was normalized to tissue wet weight.The pigments and internal standards were separated on a C-18 column  using a gradient of mobile phase A (59.5% water/40% acetonitrile/0.5% acetic acid) and mobile phase B (7.5% water/92% acetonitrile/0.5% acetic acid) at a flow rate of 0.2 ml min−/− mice were placed on a western diet for 13 weeks (6 weeks before and 7 weeks after TS surgery), as described previously. On the day of imaging, mice were anesthetized, shaved and imaged in the FLECT scanner  to obtain 3-dimensional acquisition using a 730 nm excitation laser, 803 nm filter with 29 slices at 1 mm spacing and 29 source angles per slice. The reconstruction was performed using 1 mm3 grid with attenuation correction at 1000 iterations to generate the reconstructed three-dimensional image of each scan. Following the FLECT scan, the Computed Tomography (CT) scan was performed for each animal using settings of 45 kV, 1500 ms & 360 projections to generate the anatomical image, which was overlayed onto its respective FLECT image.ApoETetrapyrole-containing compounds used in this manuscript were all purchased from Sigma-Aldrich, USA): Bilirubin (cat. no.: B4126), Hemoglobin (cat. no.: H7379), Ferrous stabilized hemoglobin (cat. no.: H0267), Biliverdin hydrochloride (cat. no.: 30891), Protoporphyrin IX (cat. no.: P8293), Protoporphyrin IX cobalt chloride (cat. no.: C1900), and Protoporphyrin IX zinc(II) (cat. no.: 282820).Fluorescence images were recorded on a DeltaVision Widefield microscope (Applied Precision) using a ×60 1.42 NA oil immersion objective, a Cy5 filter set  for Alexa 647 fluorescence and a Cy7 filter set  for NIR autofluorescence. The system is equipped with a Photometrics HQ camera. Images were recorded with 4 × 4 pixel binning and critical illumination to be able to detect the NIR autofluorescence signal. Bright field images were recorded along with fluorescence images. Image processing was performed using the open-source image analysis software Fiji.−1). For old blood with and without anticoagulant, blood remained 45 min at room temperature. Syringes with blood were scanned using the Odyssey Infrared Imaging System at 21 μm resolution.Fresh whole blood was obtained from mice via cardiac puncture using 1 ml Terumo syringes with 25 G needles. Heparin was used as anticoagulant at a total volume of 100 μl . After the blocking steps, the sections were treated with primary antibodies for 1 h. Anti-bilirubin , anti-hemoglobin subunit alpha  and anti-glycophorin A  were used as primary antibodies to detect intraplaque hemorrhage. Sections were subsequently incubated with corresponding biotinylated secondary antibodies for 30 min, followed by incubation with the avidin-biotin-peroxidase system . The reaction products were stained with a DAB substrate kit for peroxidase (Vector Laboratories) to generate a brown reaction product. Then sections were counterstained with hematoxylin. Negative controls were done identical to the above protocol, except for incubation with PBS instead of primary antibodies.Iron stain kit from Sigma-Aldrich, Australia was used for the procedure. The 5 μm paraffin sections of human CEA samples were deparaffinized and then hydrated with deionised water. The samples were then treated with Working Iron Stain Solution prepared by mixing equal volumes of potassium ferrocyanide solution and hydrochloric acid solution. The slides were then rinsed with deionized water and stained with Working Pararosaniline solution (prepared by 1 ml of Pararosaniline solution to 50 ml of water) for 5 min. The slides were rinsed again with deionized water and hydrated with alcohol and xylene before examining for Prussian blue reaction (bright blue staining of iron).Hemoglobin and bilirubin (50 μg each) were discretely embedded in an agarose phantom (10% w/v) before imaging in the IVIS Lumina Series II Imaging System . Fluorescence images were obtained by a charge-coupled device (CCD) camera using the XFO-12 fluorescence equipment with appropriate filter combinations . Reflected light photographic pictures were taken during illumination with white light-emitting diode. Both fluorescence and photographic images were overlayed, processed and analyzed using Living Image 4.4 software.−1) and xylazine (20 mg kg−1) mixture through intraperitoneal injection. An incision was made in the neck and the right common carotid artery was dissected from circumferential connective tissues. A TS with 150 μm outer diameters was introduced with the distal point 1 mm from the carotid artery bifurcation and the proximal point 3 mm from the distal stenosis. The stenosis diameter was obtained by placing a 6-0 blue braided polyester fiber suture (TI.CRON 0.7 Metric) around the carotid artery together with a 150 μm needle  that was tied to it and later removed. Animals were euthanized at 7 weeks after surgery.At 12 weeks of age, 6 weeks after commencement of HFD, ApoE−/− mice (C57BL/6J background) were anaesthetized by ketamine  was used for all statistical analyses. Unpaired Student’s The data that support the findings of this study are available from the corresponding author upon reasonable request.Supplementary InformationSupplementary Movie 1Supplementary Movie 2


A 45-year-old woman with type 1 diabetes and chronic renal failure on dialysis underwent simultaneous pancreas-kidney transplantation from a brain dead donor. On postoperative day 15, acute generalized peritonitis was diagnosed and emergency laparotomy was performed. Perforation of the donor duodenum was found, which had apparently resulted from duodenal compression by the tip of the intestinal fistula tube placed for decompression. The perforation was sutured and the intestinal fistula tube was exchanged. Following this, perforation repeatedly recurred at the same site and open repair at laparotomy was required a total of four times. The fourth operation involved both suturing the perforation and covering it with ileum, after which there was no further recurrence. The patient was discharged on posttransplantation day 219, with the pancreas and kidney grafts both functioning well. This report presents a rare complication of simultaneous pancreas-kidney transplantation. In Japan, the Organ Transplant Act was revised in July 2010, leading to an increase of patients undergoing simultaneous pancreas and kidney transplantation from brain dead donors, which has been established as surgical treatment for diabetes . Here weA 45-year-old woman was diagnosed with type I diabetes at the age of 16 years and hyperthyroidism at age 20, as well as acute pancreatitis at 22 years, caesarean section at 30 years, and gastric ulcer at the age of 38.She received insulin therapy for diabetes. At the age of 33 years, she started hemodialysis due to chronic renal failure caused by diabetic nephropathy. Hypoglycemic episodes were frequent. She was admitted to our department for simultaneous pancreas and kidney transplantation from a brain dead donor .2. Her total insulin dose was 27 units/day . Hemoglobin A1c (HbA1c) was 8.2% , glycoalbumin was 34.1%, and C-peptide was <0.0099 nmol/L. In addition, anti-insulin antibodies were <0.4 kU/L, antiglutamic acid decarboxylase antibodies were 6.5 kU/L, and anti-islet antigen-2 antibodies were <0.4 kU/L.On admission, she was 156 cm tall, weighed 54.0 kg, and had a body mass index of 22.2 kg/mSurgery. The pancreas was transplanted into the right iliac fossa and the kidney was transplanted into the left iliac fossa. To drain exocrine secretions, a Y-limb was created at the terminal ileum by the Roux-en-Y method and was anastomosed side-to-side to the donor duodenum following vascular anastomosis. The donor portal vein was anastomosed to the recipient's right external iliac vein, and the celiac artery and a Carrel patch extending from the superior mesenteric artery were each anastomosed to the recipient's right external iliac artery. The pancreatic graft was placed inside the abdominal cavity, as it was large relative to the recipient. Since the donor duodenum was distended, an intestinal fistula tube was placed for decompression of the intestinal tract. A 16 Fr Salem sump tube was inserted from the distal part of the ileal Y-limb, and the tip was advanced beyond the anastomosis and fixed inside the donor duodenum.The total ischemic time was 9 hours and 24 minutes for the pancreas and 10 hours and 54 minutes for the kidney. Blood loss was 370 g. Postoperative Course. The postoperative course is outlined in On postoperative day 14, fever (temperature ≥ 38°C) and abdominal pain developed. On postoperative day 15, there were signs of peritoneal irritation. Abdominal CT revealed ascites and free intraperitoneal gas. Generalized peritonitis due to gastrointestinal perforation near the pancreatic graft was diagnosed and emergency laparotomy was performed. The previous surgical wound in the right lower abdomen was reopened. The abdominal cavity contained intestinal fluid, and perforation of the donor duodenum was confirmed . PerforaThe initial postoperative course was favorable and oral intake was started on day 17 after reoperation, but bowel obstruction developed five days later. It seemed to be caused by adhesions and resolved after decompression with a gastric tube. Oral intake was started again on day 25 following removal of the gastric tube, but fever occurred after six days. Peritonitis was diagnosed two days later and emergency laparotomy was performed via the previous surgical wound (second reoperation). Since there was reperforation of the donor duodenum at the previous perforation site, a 3.5 mm silicon tube (Lilia drain) was inserted through the perforation into the duodenum as a second intestinal tube and was anchored with a purse-string suture.On day 7 after the second reoperation, peritonitis recurred and another emergency laparotomy was performed via the same surgical wound in the right lower abdomen (third reoperation). Leakage of pancreatic juice was observed from the site where the silicon tube had been inserted into the donor duodenum. This tube was removed and omental patch repair was performed. The intestinal tube was replaced by a new 9 Fr jejunostomy catheter (3 mm outer diameter).On day 5 after the third reoperation, peritonitis was diagnosed again and yet another emergency laparotomy was performed (fourth reoperation). This time, a midline abdominal incision was made, in addition to reopening the wound in the right lower abdomen. It was found that pancreatic juice was leaking from the edge of the omental patch. The patch was completely removed, and, after extensively peeling off the adhesions around the donor duodenal perforation, it was closed with a simple two-layer suture. Then adhesions were peeled off the terminal ileum and fixed over the sutured duodenal perforation as a small intestinal patch. Damage to the ascending colon, which had probably occurred when adhesions were being removed, was also managed by two-layer suture closure.2, urinary protein was negative, C-peptide was 0.53 nmol/L, fasting blood glucose was 116 mg/dL, and HbA1c was 5.0%. The total insulin dose at discharge was 11 units/day (7 units of Novolin R short-acting insulin and 4 units of Apidra ultra-rapid-acting insulin). Two months after discharge, she was readmitted and the intestinal tube was removed uneventfully.GFO was started on day 45 after the fourth reoperation. Around this time, neutropenia and thrombocytopenia developed but resolved after discontinuation of valganciclovir and proton pump inhibitor therapy. On day 69 after the fourth reoperation, a nonfat digested liquid diet was started. Food intake was commenced on day 78, followed by slow transition from a liquid to a solid diet, and central venous nutrition was stopped on day 89. She was discharged on day 121 after the last operation (posttransplantation day 219). At discharge, serum creatinine was 0.61 mg/dL the estimated glomerular filtration rate was 82.1 mL/min/1.73 mAs of July 2016, our hospital has performed a total of 60 pancreatic transplantations, including 11 from nonbeating heart donors and 49 from brain dead donors. However, this is our first experience of a patient with perforation of the donor duodenum. Our literature search revealed only 8 reported cases worldwide of donor duodenum perforation after pancreatic transplantation  [4–11].In these 9 patients, including our case, perforation occurred from 14 days to 27 months postoperatively. The cause of perforation was rejection in three patients, CMV infection in two patients, and donor duodenal ulcer, intestinal obstruction by an internal hernia, ischemic necrosis at the anastomosis, and compression necrosis due to the tip of an intestinal decompression tube in one patient each. Although we did not perform histopathological examination, rejection was considered unlikely in the present case because function of the transplanted kidney and pancreas was satisfactory. Moreover, CMV prophylaxis was initiated at an early stage, and the CMV antigenemia test and quantitative polymerase chain reaction for CMV-DNA were both negative, so CMV infection was excluded. Ischemic necrosis was also excluded from the macroscopic appearance of the donor duodenum. Accordingly, we concluded that perforation was due to sustained compression of the duodenal wall by the tip of the intestinal tube, which was also suggested by the intraoperative findings. The tube was initially placed away from the duodenal wall, but the donor duodenum may have shortened as edema resolved, allowing the tip of the catheter to compress the duodenal wall and cause perforation. The efficacy of the intestinal tube in pancreas transplant is not established. We opted for the tube insertion because of distention of duodenum but these series of perforation might be avoided without the insertion.Among the previously reported cases, surgical treatment included simple closure of the perforation in three patients, removal of the donor duodenum in two patients, and total graft removal in three patients. Thus, pancreatic graft preservation was possible in six patients, including our patient. It was reported that 70% of patients who developed intra-abdominal infection after pancreatic transplantation required graft removal . In JapaIn the present patient, preservation of the grafted pancreas was achieved by performing laparotomy four times to repair perforation of the donor duodenum causing acute generalized peritonitis, which resulted from the tip of an intestinal tube compressing the duodenal wall after pancreas transplantation. We reported this case because it represents a rare and interesting complication of pancreatic transplantation.


This paper proposes a nonlinear correlation-based wavelet scale selection technology to select the effective wavelet scales for the estimation of handgrip force from surface electromyograms (SEMG). The SEMG signal corresponding to gripping force was collected from extensor and flexor forearm muscles during the force-varying analysis task. We performed a computational sensitivity analysis on the initial nonlinear SEMG-handgrip force model. To explore the nonlinear correlation between ten wavelet scales and handgrip force, a large-scale iteration based on the Monte Carlo simulation was conducted. To choose a suitable combination of scales, we proposed a rule to combine wavelet scales based on the sensitivity of each scale and selected the appropriate combination of wavelet scales based on sequence combination analysis (SCA). The results of SCA indicated that the scale combination VI is suitable for estimating force from the extensors and the combination V is suitable for the flexors. The proposed method was compared to two former methods through prolonged static and force-varying contraction tasks. The experiment results showed that the root mean square errors derived by the proposed method for both static and force-varying contraction tasks were less than 20%. The accuracy and robustness of the handgrip force derived by the proposed method is better than that obtained by the former methods. Various dynamic handgrip tasks are required in a number of applications, including manual fabrication and machining, handling tools, monitoring for surgery, human–robot interactions and so on ,2,3. UsuIn previous studies, a number of correlations between SEMG of a target muscle and force output of a target task have been discussed. In muscle contraction, the activation of motor units within a target muscle follows a mechanism (motor unit rotation and substitution) to maintain a given force. However, muscle fatigue resulting from prolonged muscle contractions can reduce the force generation performance of the target muscles. To overcome the decline in performance, more fresh motor units are recruited to replace the burden of fatigued motor units and compensate for a lack of performance ,9,11,12.Many researchers used Fast Fourier Transform (FFT) to observe that the power spectrum of SEMG shifts to a lower frequency range when complex noises  develops ,19,20,21Many CWT-based methods, such as frequency band analysis, have been utilized to obtain a suitable wavelet scale for estimating accurate force from SEMG. These methods have used a single scale or particular WSC to analyze and filter the original signal ,22. An oThe purpose of this paper is to find an appropriate WSC for accurate evaluation of handgrip force from SEMG during force-varying muscle contractions of long duration. The suitable WSC was obtained by using the sequence combination analysis (SCA) method based on Monte Carlo sensitivity analysis. As a simple and reliable sensitivity analysis method, Monte Carlo simulation can calculate the degree of nonlinearity between variables and the actual target output, regardless of how many factors the variable contains ,27. We fTwenty healthy participants (16 men and 4 women), whose ages ranged from 22 to 31 years, participated in the experiment. At this stage, we did not consider the differences between males and females. Ten subjects (group A: 8 men and 2 women) were used for determining the suitable WSC for estimating handgrip force in the force-varying contractions as a training group. The other ten subjects (group B: 8 men and 2 women) were used as a validation group, to evaluate the performance of the suitable WSC. Before the experiment, all participants were informed of the experiment process and the risk of muscle fatigue, and agreed to perform the experiment. 2 for each electrode and a center-to-center recording distance of 20 mm. Electrode locations were determined in accordance with a previous study [In the gripping tasks, SEMG data was recorded from the flexor digitorum superficialis (FDS) muscle and extensor carpi radialis (ECR) muscle of the forearm, as shown in us study ,29. EMG us study . The forAt the beginning of the experiment, each participant was asked to perform a maximal voluntary contraction task (MVC) three times, and from this, the average value of maximum grip strength (kg) and maximum amplitude (mV) of the SEMG were calculated. In the experiment, the participants in Group A first performed the force-varying analysis task to obtain the suitable WSC using the proposed method. Then, all participants in Groups A and B performed the static and force-varying validation tasks to evaluate the performance of the proposed method. The force-varying analysis task, static validation task, and force-varying validation task are defined as follows.Participants in Group A were required to perform a force-varying fluctuating contraction. In the force-varying analysis task, a series of varying handgrip forces, occurring within ten minutes, was increased or decreased respectively by squeezing and releasing the dynamometer. Participants were asked to increase their handgrip force gradually to provide an adequate correlation between handgrip force and SEMG for each minute, as shown in Static tasks were performed at four force levels, which included 20%, 30%, 50%, and 70% of the MVC in a random order during the isometric contraction ,29. The In the force-varying validation task, a force level that varied between 0% and 80% of the MVC was performed over a 10-min period. Before the force-varying validation task, the participant rested for 60 min. A sample of the recorded handgrip force and SEMG for both muscles is illustrated in To accurately estimate the handgrip force from the SEMG signals during the long duration force-varying grip motion, this paper utilized the Monte Carlo sensitivity analysis to determine the nonlinear degree of the relationship between each wavelet scale and handgrip force from the initial nonlinear SEMG-handgrip force relationship, and used a nonlinear correlation-based scale-selection method (SCA) to obtain the suitable WSC of the scale.e(1), …, e(P), the response of handgrip force F(estimation) is predicted by:P is the total number of wavelet scales, and the vectors of coefficients p, which are collected from the original SEMG n,x with data size, N, through CWT and root mean square (RMS)*) indicates the complex conjugate [MThe initial nonlinear SEMG-handgrip force relationship was built using the ten wavelet (db2) scales  from the SEMG signals and handgrip force collected from the force-varying analysis tasks of group A. These wavelet scales are most relevant to handgrip force . This moonjugate . Then, t(200 ms)  was used(1)CWT was used to convert the original SEMG signals to wavelet coefficients of the ten scales in the force-varying analysis task.(2)Wavelet coefficients of the ten scales normalized by MVC data were used to build the initial nonlinear SEMG-handgrip force relationship.(3)A specific number of random variables were used to conduct a simulation of the handgrip force in the initial nonlinear relationship.(4)Mean square deviation was used to calculate the simulated sensitivity value corresponding to each correlation between ten scales and handgrip force.(5)The sensitivity value of each correlation was normalized by the sum of the sensitivity values of the ten scales.(6)Steps 1–5 were repeated for every minute in the force-varying analysis task.For a nonlinear index, the sensitivity analysis is important . In contr, that was between 100 to 140,000, with an interval of 100. By using this range, r, the sensitivity of each wavelet scale was obtained from the Monte Carlo simulation at each given variable quantity and these sensitivities were calculated by first order differential calculation.p = 1, 2,…, 10, is the ordinal number of the wavelet scale, r = 100, 200..., 140,000, and iy(p) is the sensitivity value of the wavelet scale of p with a specific random variable number (r = i × 100). Then, the sum, id(integration), of the sensitivity differences under a variable number for Group A can be calculated as follows:In this paper, the random variable was distributed evenly in a specific range (0–100) as a basic step to realize the Monte Carlo simulation. In each iteration of the simulation, each model parameter used a given number of random variables to obtain the expected sensitivity value. The given number of random variables was obtained by a finite difference-based convergence experiment for all participants in Group A . This exBased on the Monte Carlo simulation, we obtained the nonlinear degree of each scale-handgrip force correlation through the force-varying analysis tasks. To choose the suitable WSC, we designed a set of scale permutations and combination rules based on the mean value of sensitivity of each scale. The mean sensitivity value was calculated from all participants in Group A. The distribution and mean sensitivity value of each scale is shown in In this paper, two former methods were utilized to be compared with the proposed method. Formerly, force estimation was realized by calculating the amplitude of the SEMG signal, for example, using RMS and the pre-defined SEMG-force relationship. Former method A builds a linear SEMG-force relationship by using the normalized SEMG signal, which is bandpass-filtered between 15 and 450 Hz .F is the sample data of the observed griping force from the grip dynamometer and F(estimation) is the predicted force from SEMG. The sampling number of handgrip force is To obtain the estimated error, a comparison between the estimated and actual handgrip force was conducted using the RMSE:From the results shown in The performance evaluation consisted of a varying RMSE process of the ten WSCs during the force-varying validation task. In the force varying validation task, the experimental results  show thaThe proposed method has a significant performance improvement for fatigued muscle contractions with long durations see B,D. The Compared to the first minute, the estimated accuracy of former method B in the last minute decreased by an average of 11% in the force range of 10–40% of MVC. From the perspective of sensitivity of each scale to handgrip force, this is because the wavelet scales 2 and 3 have lower sensitivities to gripping force prediction than the other scales and thus, cannot characterize flexibles change during force-varying contractions for low forces. The signal noise induced by the intramuscular electrophysiological effect and motion artifacts has consequences for the correlation between the two scales and handgrip force and further reduces the estimated performance of former method B. Previous studies have shown that low strength muscle contractions are mainly controlled by slow-twitch muscle fibers (approximately 143 Hz of SEMG), and high strength muscle contractions are mainly controlled by fast-twitch muscle fibers (approximately 397 Hz) ,35. In fIn contrast to the former methods, the proposed method can effectively inhibit the influence of complex electrophysiological noises on the predicted accuracy of the handgrip force from SEMG during long duration fatiguing muscle contractions. However, the prediction errors for low-level forces are greater than those for high level forces. The reason for this is that the intramuscular electrophysiological effect and motion artifacts can reduce the degree of correlation between different levels of force and SEMG to varying degrees. During the long duration muscle contractions, all participants overcame fatigue to produce a specified handgrip force with extra shaking of the forearm. Motion artifact noise may be caused by the shaking of these forearm muscles. When the fatigue reduces the force generated from the muscle, the rotation and substitution between fatigued motor units and fresh motor units is performed to generate and maintain the requisite force level. In this situation, the complex electrophysiology within muscles affects the changes in the SEMG signal. Thus, the intramuscular electrophysiological effect and motion artifacts ultimately change the amplitude and frequency of the SEMG signal. In this paper, we proposed a nonlinear correlation-based method to select appropriate wavelet scales to accurately estimate handgrip force from SEMG during a force-varying muscle contraction of long duration. The sensitivity analysis of the Monte Carlo simulation was used to explore the nonlinear correlation between ten wavelet scales and handgrip force. A nonlinear, correlation-based SCA method was proposed to determine the suitable WSC. The results derived via SCA indicated that the suitable scales for the extensor muscles is WSC VI, consisting of scales 1 to 5, and WSC V including scales 1 to 6 is suitable for flexor muscles. The appropriate WSC, obtained by the proposed method, was compared to two former methods through a set of static and force-varying contraction tasks. The experimental results showed that the proposed method has a better estimation performance than the former methods, especially for low level contractions .The proposed method enhances the performance of grip strength estimation from SEMG. However, the actual working muscles not only provide force output but also drive limb posture changes. Further research using the proposed nonlinear correlation-based scale selection method is needed to explore the correlation between wavelet scales of SEMG and muscle loads or limb posture in various work tasks, such as human–robot interactions, and gait analysis based on SEMG of lower limb muscles. An accurate force estimation based only on electromyography data can be used for gait analysis instead of using inverse dynamics or similar methods ,37. In a


There was no difference in plasma/CSF C1q or C3 between older adults and youth. In 18–24-year-old youth, a nearly significant (p = 0.052) elevation of CSF C1q expression was observed in cognitively impaired subjects compared to cognitively normal subjects. Further investigation into the role of the CNS complement system in the neuropathogenesis of HIV is warranted and should be considered in a developmentally specific context.The complement system (C1q/C3) is a key mediator of synaptic pruning during normal development. HIV inappropriately induces C1q and C3 production in the brain, and reduces neuronal complement inhibition. HIV may thus alter neural connectivity in the developing brain by excessively targeting synapses for elimination. The resultant pattern of neuronal injury may fundamentally alter neurodevelopmental and cognitive processes differentially across ages. This study aimed to (1) measure the association between the cerebrospinal fluid (CSF) complement factors (C1q/C3) and a marker of neuronal injury (NFL) in HIV+ subjects; (2) quantify the differences in CSF C1q/C3 between HIV+ youth and older adults; and (3) define the relationship between CSF C1q/C3 and cognitive impairment in each age group. We performed a retrospective cross-sectional study of 20 HIV+ 18–24-year-old youth and 20 HIV+ 40–46-year-old adults with varying levels of cognitive impairment enrolled in the CNS Antiretroviral Therapy Effects Research study. We quantified C3, C1q, and NFL by ELISA in paired CSF/plasma specimens. We found that CSF C1q correlates with NFL in all subjects not receiving antiretroviral therapy ( The complement system is a key mediator of synaptic pruning during normal development  is a structural protein specific to neurons that is released into the CSF and blood following axonal disruption Julien . In HIV In order to begin to examine the role of the CNS complement system as one mechanistic player in HIV neuropathogenesis, we sought to elucidate the relationship between complement factors and neuronal damage in HIV infection across the developmental age span. Specifically, we measured the correlations among CSF and plasma complement factors (C1q and C3) and NFL as a marker of neuronal injury. We then examined the differences in complement protein concentrations in youth and older adults, and finally explored the relationship between these markers and cognitive function in each age group.We performed a retrospective cross-sectional study using biological samples  and data from 40 HIV+ subjects enrolled in the CNS HIV Antiretroviral Therapy Effects Research (CHARTER) cohort of the NIMH/NINDS/NIH. Characteristics of the CHARTER cohort are described elsewhere  certified, or CLIA equivalent, medical center laboratory. Plasma HIV RNA measurements  were quantified by a RT-PCR ultrasensitive assay  in a central lab . Paired CSF/plasma samples were assayed for C3  and C1q . CSF samples were assayed for NFL . Previous work by our group did not demonstrate measurable quantities of NFL in plasma  and an instrumental activities of daily living (IADL) questionnaire . Non-parametric analysis methods were used given small sample sizes to improve accuracy of statistical estimates and reduce sensitivity of analyses to any statistical outliers. Continuous variables were described using median and intraquartile range (IQR), and intergroup differences were evaluated using the Wilcoxan rank-sum tests and Kruskal-Wallis tests. Categorical variables were described using counts and percents, and intergroup differences were compared using the Chi-square test. Spearman’s correlation coefficients were used for correlations between biomarkers. Statistical significance was determined a priori as a two-tailed p = 0.005), and a subsequent lower nadir CD4+ T lymphocyte count . Other markers of systemic infection  were similar between the different age groups. Older adults trended towards more frequent current ART use , likely related to duration of time linked into appropriate care and counseling. Younger adults had higher Wide Range Achievement Test (WRAT) scores , suggesting a higher level of premorbid cognitive functioning despite comparable total years of education. It is unclear if this difference is simply reflective of the population currently being infected with HIV, or if it is age-related. However, all neurocognitive data are adjusted for age and demographic factors, making the latter possibility less likely. HAND diagnoses were comparable between the two age groups. Finally, no subjects were using illicit drugs at the time of this study.Cohort demographics, clinical, and laboratory characteristics of everyone and of each specific age group are summarized in Table n = 17) did approach statistical significance . We subsequently performed an exploratory analysis eliminating outlier NFL values  that demonstrated a significant relationship between CSF C1q and NFL    with the entire cohort, and 2) restricted to subjects not receiving ART in order to avoid any potential confounding effects of ART on expression of biomarkers of neuroimmune dysregulation or neuronal injury. There were no significant correlations between NFL and either CSF complement marker in the overall cohort. However, the correlation between CSF C1q and NFL in subjects off ART (p = 0.052) elevation of CSF C1q expression was observed in cognitively impaired subjects compared with cognitively normal subjects  . Of note, four of the 40 total subjects were not classified for HAND so were therefore excluded from this analysis. In 18–24 year-old youth, a nearly significant (p = 0.001) , demonstrating a consistent relationship between systemic immune status and risk of CNS neuronal injury previously demonstrated in studies of older-aged HIV cohorts  between older adults and youth, on or off ART (data not shown). NFL was significantly lower in youth compared to older adults across the cohort  Fig. . FurtherThis is the first study to examine CSF complement protein expression as a novel immunologic mediator of HIV-associated neuronal damage in human subjects. It is also the first study to address the possibility of an age-specific ontogenetic neuropathogenesis of acquired HIV infection. We have demonstrated a potential positive correlation in this small cohort between CSF expression of C1q and NFL in subjects of all ages not receiving ART. These data suggest that C1q in CSF may be a correlate or biomarker for neuronal injury across the age span in HIV. Furthermore, in 18–24-year-old youth we found a nearly significant elevation of CSF C1q expression in cognitively impaired subjects to cognitively normal subjects. This second observation may reflect an age-specific vulnerability to complement-mediated cognitive dysfunction. Finally, we have confirmed a positive correlation between CSF expression of C1q and C3, suggesting our data are likely demonstrating true differences in activation of the complement system, rather than anomalous assay results. Borderline statistical significance may thus reflect actual biological significance but too small of a sample size for definitive results.C1q is a 460 kDa hexameric glycoprotein that serves as a pattern recognition molecule and the first recognition subcomponent of the complement classical pathway. It binds HIV, IgG, IgM, and many other ligands and receptors largely via a gC1q domain  and CD4+ T lymphocyte count. This observation supports the idea that at least part of HIV neuropathophysiology occurs independently of peripheral disease and is clinically consistent with the fact that mild forms of HAND persist in the antiretroviral era in subjects with good systemic viral control.There have been several recent compelling studies examining the presence of systemic inflammatory markers in youth with different developmental synaptopathies, including perinatally acquired HIV. One such study demonstrated an inverse correlation between serum markers of three combined pro-inflammatory markers  and processing speed, suggesting that residual inflammation may contribute to neurocognitive deficits despite well-controlled HIV disease in the periphery , because there are fewer specimens available from younger adults in the CHARTER cohort. Thus, overall analyses and sub-analyses with even smaller numbers  may be underpowered, and truly significant associations may therefore be underestimated. Second, while 19/36 subjects did have cognitive impairment (four were not classified), most of these (17/19) were mild (ANI). Therefore, linear correlations of complement markers with degree of impairment are limited. This is why we chose to use cognitive impairment as a binary variable in the majority of our analyses. Third, because of these small numbers and the cross-sectional nature of this correlation study, factors confounding the relationship between complement proteins and NFL are not accounted for in statistical associations. However, univariate analysis did not suggest relationships between complement proteins and systemic markers of disease, or peripheral complement levels, so not including these variables in subsequent models likely had little effect on the associations examined. In addition, causality cannot be inferred in a cross-sectional study. Finally, in our statistical analysis, we had several outlier values for NFL that we were not able to biologically explain why they otherwise differed from the remainder of the cohort. While the non-parametric analyses we used with our data should minimize the impact of outliers on statistical associations, a larger cohort would allow us in the future to determine if these subjects have a different specific biological reason for elevations in NFL, or if they were indeed spurious.Our study had several limitations. First, because we wanted to specifically include a comparable number of 18–22-year-old youth compared to older adults, the overall cohort size was relatively small (We chose to investigate the role of the complement system in HIV neuropathogenesis based on several recent articles by Stevens et al.  and Step


It was found that acacetin (0.3–3 μM) significantly decreased the apoptosis and reactive oxygen species production induced by hypoxia/reoxygenation injury in cardiomyocytes and H9C2 cardiomyoblasts via reducing the pro-apoptotic proteins Bax and cleaved-caspase-3 and increasing the anti-apoptotic protein Bcl-2. In addition, acacetin not only suppressed the release of pro-inflammatory cytokines TLR-4 and IL-6 induced by hypoxia/reoxygenation injury, but also increased the secretion of anti-inflammatory cytokine IL-10. Moreover, acacetin increased Nrf2 and HO-1 in a concentration-dependent manner, and rescued SOD1 and SOD2 reduction induced by hypoxia/reoxygenation insult. These beneficial effects of acacetin disappeared in cells with silenced Nrf2, suggesting that Nrf2 activation participates in the cardioprotective effect of acacetin against hypoxia/reoxygenation insult. However, acacetin-induced Nrf2 activation was not observed in cells with silenced AMPK and in ventricular tissues of rat hearts treated with the AMPK inhibitor Compound C and subjected to ischemia/reperfusion injury. Our results demonstrate for the first time that AMPK-mediated Nrf2 activation is involved in the cardiomyocytes protection of acacetin against hypoxia/reoxygenation injury by activating a series of intracellular signals involved in anti-oxidation, anti-inflammation, and anti-apoptosis.The present study investigates the potential signal pathway of acacetin in cardioprotection against ischemia/reperfusion injury using an  Ischemic cardiomyopathy is the leading cause of mortality and morbidity around the world . It is gex vivo and in vivo rat models by increasing the anti-oxidants superoxide dismutase-2 (SOD2) and thioredoxin, and decreasing inflammation and apoptosis  was synthesized in the laboratory as described previously in the United States patent . DulbeccThe anti-SOD2 (sc-133134), anti-Bcl-2 (sc-7382), anti-Bax (sc-493), anti-TLR-4 (sc-293072), anti-IL-10 (sc-365858), anti-AMPK (sc-25792), anti-pAMPK (sc-33524), anti-JNK (sc-571), anti-pJNK (sc-12882), and anti-β-actin (sc-130300) antibodies were purchased from Santa Cruz Biotechnology. The anti-Nrf2 (PB0327), anti-HO-1 (PB0212), anti-SOD1 (BA1401) and anti-IL-6 (PB0061) antibodies were from Wuhan Boster Biological Technology, Ltd. , while the anti-cleaved caspase-3 antibody (9661S), anti-P38 (8690S), anti-pP38 (4511S), anti-ERK1/2 (4695S), anti-pERK1/2 (9101S), anti-Akt (9272S), anti-pAkt (4060S) antibodies were obtained from Cell Signaling Technology .3 tissue chunks, and then digested with PBS containing collagenase II (0.1%) for 15–20 min at 37°C. The supernatants were transferred to a fresh sterile tube, the DMEM (containing 10% FBS) was added to stop digestion. The tissue chunks were then digested for 10 min with fresh Accutase (eBioscience) for two to three times  rats  were from Beijing Vital River Laboratory Animal Technology, Co.  and mated naturally to produce offspring in Laboratory Animal Center of Xiamen University. The animals were cared for following the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health . The hearts of 1- to 3-day-old neonatal rats were surgically removed under sterile conditions, washed three times with cold PBS, minced into ∼1 mmee times . After e2 in DMEM supplemented with 10% FBS, 100 μg/ml streptomycin, and 100 units/ml penicillin. When cells grew to 70–80% confluence, they were pretreated with acacetin  or vehicle (DMSO) for 4 h (a time point with better efficacy in cardiomyocytes protection). The cells were then exposed to an anaerobic medium (serum and glucose free) in a hypoxia incubator chamber  with an anoxic mixture gas (95% N2 and 5% CO2) for 18 h at 37°C followed by reoxygenation for 6 h with fresh culture medium (95% air and 5% CO2) to simulate ischemia/reperfusion injury in isolated hearts and/or in anesthetized rats  were cultured at 37°C with 95% air and 5% COzed rats .The flow cytometry analysis  was employed to determine cell viability, apoptosis, and intracellular ROS level as described previously . For celFor ROS production determination, neonatal rat cardiomyocytes, and H9C2 cardiomyoblasts were cultured in the absence and presence of acacetin. After hypoxia/reoxygenation exposure, the cells were incubated with DCFH-DA (10 μM) at 37°C for 30 min and intracellular ROS level was determined by a flow cytometer .TM reagents , and the signals were captured with chemiluminescence detection system . The density of the band was analyzed with image analysis software.The Western blots were employed using the procedure described previously  to deterThe experiments on silencing Nrf2 or AMPK were performed in H9C2 cardiomyoblasts as described previously . BrieflyThe myocardial ischemia/reperfusion model in anesthetized rats was established as described previously  to confiP < 0.05 was considered as statistically significant.Data analysis was performed with GraphPad Prism 5.0 . Results are presented as mean ± SEM. One-way ANOVA followed by Bonferroni-test was used for comparisons of multiple groups. A value of Figure 1A shows the flow cytometry graphs of neonatal rat cardiomyocytes without or with hypoxia/reoxygenation exposure in the absence or presence of 3 μM acacetin. Cell viability was 83% with 11.7% early apoptosis under control conditions. Hypoxia/reoxygenation insult decreased the viability to 69.1% and increased early apoptosis to 18.7%. Acacetin at 3 μM partially reversed the reduced viability to 75.6% and the increased early apoptosis to 15.3%. Figure 1B illustrates the mean percent values of cell viability and apoptosis. Acacetin at 0.3–3 μM increased cell viability and decreased early apoptosis. A statistically significant effect was observed at 3 μM (P < 0.05 vs. hypoxia/reoxygenation).Acacetin had no effect on cell viability in H9C2 cardiomyoblasts with 24 h incubation . Statistical analysis  shows that cell viability was decreased from 96.5 ± 0.6% of control to 68.5 ± 1.8% , and the late apoptotic cells were increased from 1.3 ± 0.2% of control to 21.2 ± 1.3% (P < 0.01 vs. control), and the dead cells from 0.9 ± 0.4% of control to 7.1 ± 1.6% (P < 0.01 vs. control) in cells with hypoxia/reoxygenation exposure. Acacetin at 0.3, 1, and 3 μM antagonized the reduction of cell viability and the increased apoptotic cells and dead cells. Significant effect was observed at 1 and 3 μM for cell viability and late apoptotic cells  and at 3 μM for dead cells .In H9C2 cardiomyoblasts, hypoxia/reoxygenation insult decreased cell viability and increased late apoptosis and dead cells, which were partially antagonized by pre-incubation of 3 μM acacetin , hypoxia/reoxygenation insult, as in ischemia/reperfusion insult , in which hypoxia/reoxygenation decreased Bcl-2 and increased Bax and cleaved caspase-3. Acacetin treatment antagonized the alterations by hypoxia/reoxygenation insult. These results confirmed that acacetin may antagonize the apoptosis induced by hypoxia/reoxygenation insult, as previously observed in rat hearts subjected to ischemia/reperfusion insult . In primary neonatal rat cardiomyocytes , hypoxia/reoxygenation caused a significant increase of IL-6 and TLR-4 and decrease of IL-10, and these effects were reversed by acacetin. Similar results were seen in H9C2 cardiomyoblasts , in which the increased IL-6 and TLR-4 and the decreased IL-10 were significantly reversed by acacetin. These results suggest that acacetin-induced anti-inflammatory effect may participate in the anti-apoptosis in both primary neonatal rat cardiomyocytes and H9C2 cardiomyoblasts subjected to hypoxia/reoxygenation insult.To investigate whether anti-inflammation is involved in the anti-apoptotic effect of acacetin in cells subjected to hypoxia/reoxygenation insult, the expression levels of the pro-inflammatory factors IL-6 and TLR-4 and the anti-inflammatory factor IL-10 were determined in primary neonatal rat cardiomyocytes and H9C2 cardiomyoblasts without or with hypoxia/reoxygenation exposure in the absence and presence of 0.3, 1, and 3 μM acacetin , which was decreased by 3 μM acacetin to 156.4 ± 4.5% of control . Similar results were observed in H9C2 cardiomyoblasts , in which acacetin (3 μM) reduced the ROS level from 676.3 ± 90.8% of control in cells with hypoxia/reoxygenation insult to 313.1 ± 38.9%  . These results indicate that acacetin significantly inhibits ROS production in both primary neonatal rat cardiomyocytes and H9C2 cardiomyoblasts subjected to hypoxia/reoxygenation insult.It is generally believed that ROS play a crucial role in mediating ischemia/reperfusion or hypoxia/reoxygenation injury by initiating inflammation and apoptosis. To study whether the protective effect of acacetin is related to inhibiting intracellular ROS production, the cells were loaded with dichlorofluorescin  and ROS It is well-recognized that intracellular ROS level is dependent on Nrf2 to regulate endogenous antioxidants, e.g., HO-1, SOD1, and SOD2, etc. . We therFigure 5). Hypoxia/reoxygenation insult stimulated an increase in expression of Nrf2 and HO-1 in both types of cells. Acacetin at 0.3–3 μM induced an additional increase in a concentration-dependent manner. However, hypoxia/reoxygenation insult slightly decreased SOD1 and SOD2, and the reduction was reversed in cells treated with 3 μM acacetin. These results suggest that upregulation of Nrf2, HO-1, SOD1, and SOD2 may be involved in acacetin protection of cardiomyocytes against hypoxia/reoxygenation in primary neonatal rat cardiomyocytes and H9C2 cardiomyoblasts.The effect of acacetin on Nrf2/HO-1 antioxidative pathway was then determined in primary neonatal rat cardiomyocytes and H9C2 cardiomyoblasts subjected to hypoxia/reoxygenation insult . In addition, intracellular ROS production induced by hypoxia/reoxygenation insult was reduced by acacetin in cells transfected with control siRNA ; however, in cells transfected with Nrf2 siRNA, hypoxia/reoxygenation-induced a greater increase of ROS level , which cannot be decreased by acacetin. These results suggest that cardiomyocytes protection of acacetin against hypoxia/reoxygenation is related to Nrf2 activation.Hypoxia/reoxygenation insult resulted in the reduced cell viability and increased apoptotic cell population, acacetin (3 μM) antagonized these effects in cells transfected with control siRNA, but not in cells transfected with Nrf2 siRNA . Nrf2 was significantly increased by 3 μM acacetin in cells transfected with control siRNA; silencing Nrf2 with Nrf2 siRNA removes acacetin effect and suggests that the target molecule of acacetin may be Nrf2 . Interestingly, HO-1 expression was significantly decreased in cells transfected with Nrf2 siRNA. In cells transfected with control siRNA, HO-1 was enhanced by acacetin . This further indicates that acacetin-induced HO-1 increase is dependent on Nrf2 activation.Nrf2 regulates the expression of downstream proteins . Therefore, these proteins related to apoptosis and inflammation were determined in H9C2 cardiomyoblasts transfected with control siRNA or Nrf2 siRNA molecules and subjected to hypoxia/reoxygenation insult , suggesting they are also regulated by Nrf2. SOD1 and SOD2 were increased by acacetin in cells transfected with control siRNA, but not in cells transfected with Nrf2 siRNA, which supports the notion that SOD1 and SOD2 are also regulated by acacetin via activating Nrf2.On the other hand, the expression of SOD1 and SOD2 was slightly reduced in cells transfected with Nrf2 siRNA , indicating that anti-apoptosis and anti-inflammation effects of acacetin are secondary to its activation of Nrf2 and subsequent anti-oxidation and reduction of intracellular ROS level.In addition, the beneficial effects of acacetin for reducing Bax, cleaved caspase-3, TLR-4 and IL-6, and upregulating Bcl-2 and IL-10 in cells subjected to hypoxia/reoxygenation insult disappeared in cells with silenced Nrf2 , suggesting that AMPK may be an activator of Nrf2 signal pathway. To confirm whether acacetin mediates Nrf2 activation through AMPK, the effect of acacetin on Nrf2 expression was determined in cells transfected with siRNA targeting AMPKα. Acacetin (3 μM) increased Nrf2 expression in cells transfected with scramble control siRNA. Interestingly, Nrf2 expression was significantly reduced in cells transfected with siAMPKα and acacetin no longer increased Nrf2 in these cells . The results indicate that acacetin mediates the activation of Nrf2 through AMPK.Interestingly, acacetin increased pAMPKα in a concentration dependent manner , acacetin induced an additional increase of Nrf2 level (P < 0.01 vs. sham); however, the increase of Nrf2 by acacetin was not observed in rats treated with the AMPK inhibitor Compound C. The results from the in vivo animal support the notion that cardioprotection of acacetin against ischemia/reperfusion injury is mediated by AMPK/Nrf2 signal pathway.The AMPK involvement of acacetin in cardiomyocytes protection against hypoxia/reoxygenation insult through Nrf2 was further confirmed in anesthetized rats subjected to ischemia/reperfusion injury, in which the animal was pretreated with the AMPK inhibitor Compound C (1 mg/kg). Figure 10).In the present study, we have demonstrated that the natural flavone acacetin significantly antagonizes hypoxia/reoxygenation-induced reduction of cell viability and increase of apoptosis in primary neonatal rat cardiomyocytes and H9C2 cardiomyoblasts. The cardioprotective effect of acacetin against hypoxia/reoxygenation insult is related to inhibiting ROS production. Acacetin activates pAMPKα which increases the master anti-oxidant regulator Nrf-2, thereby exerting anti-inflammatory and anti-apoptotic effects via increasing the anti-inflammatory cytokine IL-10 and the anti-apoptotic kinase Bcl-2 and decreasing the pro-inflammatory cytokines TLR-4 and IL-6 and the pro-apoptotic kinases Bax and cleaved caspase-3 (Kur (ultra-rapidly activating delayed rectifier potassium current) or Kv1.5, IK.ACh (acetylcholine-activated potassium current), Ito (transient outward potassium current) or Kv4.3   broadly distributed in plant pigments, and is responsible for many of the colors in nature . In addior Kv4.3 , 2013 ancurrent) , acaceticurrent) , inflammcurrent) , and acucurrent)  in mousecurrent)  demonstrpoptosis . Howeverex vivo hearts with ischemia/reperfusion injury (It has been well-documented that ROS and redox signaling are involved in myocardial ischemia-reperfusion injury and cardioprotection . ROS aren injury , acacetiOur results showed that the inhibition of ROS production by acacetin is clearly related to the activation of Nrf2. It is well-recognized that Nrf2 is a key transcription factor that exists in all types of cells and regulates the expression of antioxidant pathway proteins that protect against oxidative damage triggered by tissue injury . Nrf2 isNrf2 has been considered to be a drug target in the treatment of cardiomyocytes injury, cardiac dysfunction and other disorders . Nrf2 acSeveral natural compounds, e.g., quercetin , danshenThe observation that pAMPK is increased by acacetin is supported by a recent report in 3T3-L1 preadipocytes , in whicIn this study, we did not explore how acacetin crosses the cell membrane to induce activation of the signaling pathways, how it interacts with any protein or receptor on the cell membrane, how much extracellular flavone enters into the cell, and how it is metabolized during ischemia exposure. However, these limitations would not affect the conclusion that acacetin protects cardiomyocytes against hypoxia/reoxygenation insult by activating a series of intracellular beneficial molecules.Collectively, the present study provides the novel information that the natural flavone acacetin confers significant cardiomyocytes protection against hypoxia/reoxygenation insult and also ischemia/reperfusion injury via AMPK-mediated activation of Nrf2 signaling pathway. It may be a promising drug candidate that can be used for managing ischemic cardiac disorders in addition to atrial fibrillation.W-YW, GL, YWu, YWang, and G-RL conceived and designed the experiments. W-YW, Y-DL, Y-KC, CW, and Y-XH performed the experiments. W-YW, L-JJ, and G-RL analyzed the data. W-YW and G-RL wrote the paper. All authors approved the submission.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.


The closely related herpes simplex viruses 1 and 2 can cause inflammations of the central nervous system (CNS), where type 1 most often manifest as encephalitis (HSE), and type 2 as meningitis (HSM). HSE is associated with severe neurological complications, while HSM is benign in adults. We proposed that studying the chemokine and cytokine production in cerebrospinal fluid (CSF) and serum could indicate why two closely related viruses exhibit different severity of their accompanied CNS inflammation.n = 14) and HSM (n = 20) in the initial stage of disease were analyzed and compared to control subjects without viral central nervous system infections and to levels in serum.Secretion patterns of 30 chemokines and 10 cytokines in CSF of adult patients with acute HSE  contains supplementary material, which is available to authorized users. Herpes simplex viruses (HSV) may cause inflammations in the central nervous system (CNS), manifesting either as encephalitis or meningitis. Encephalitis is most often caused by herpes simplex virus type 1 (HSV-1), whereas meningitis primarily is caused by HSV type 2 (HSV-2) . Women aHSV-1 and HSV-2 are double-stranded DNA viruses with approximately 50% DNA homology. Both viruses initially infect mucosal tissue and later establish latent infections in the CNS . HSV-1 mLeukocyte entry into the CNS is restricted by the blood-brain barrier (BBB) and/or the blood-cerebrospinal fluid barrier (BCSFB); hence, the CNS is considered immune privileged. However, immune surveillance continuously takes place in the CNS even in non-inflammatory states, suggesting controlled mechanisms of leukocyte trafficking over the barriers and into the cerebrospinal fluid (CSF) –22. InflTo identify parameters associated with the recruitment of immune cells to the CNS in HSE and HSM, we conducted analysis of 30 chemokines and 10 cytokines in the CSF of adult patients with HSE or HSM and compared to control subjects without viral CNS infections. To analyze potential in- and/or efflux of chemokines/cytokines between CSF and serum, we compared paired CSF and serum samples from these patients. We found that most measured chemokines and cytokines increased in the CSF of both HSE and HSM patients. However, immune cells move toward a chemokine gradient, which imply that levels in CSF need to exceed those in serum for potent cell migration into the CNS. This criterion was filled for CXCL8, CXCL9, and CXCL10 in both HSE and HSM. Two chemokines distinguished HSE from HSM; CCL8 and in particular CXCL11 was only expressed above serum levels in the CSF of HSM but not HSE patients. We thus conclude that HSM, despite being a more benign disease, is characterized by a stronger and a more diverse inflammatory CSF chemokine response.n = 35). None of the control patients were diagnosed with any neurologic condition including infection or autoimmunity during a 1-year follow-up period. As none of the patients had pleocytosis, we find it unlikely that they had an undiagnosed CNS infection. The cerebrospinal fluid samples were centrifuged 1500 rpm for 5 min to remove cells. Eight patients with HSE and 8 patients with HSM had paired CSF and serum samples (±2 days). In the control group, 3 persons had paired CSF and serum, while CSF was obtained from 30 persons, and additional sera from 2 persons. All HSE and HSM samples were obtained during acute infection within 20 days after symptom onset and stored in −70 °C. The medical records were obtained for registration of clinical data including laboratory results on CSF albumin levels, pleocytosis, and treatment.Patients with suspected HSE or HSM were admitted to the Department of Infectious Diseases, Sahlgrenska University Hospital, Gothenburg, Sweden. Patients with HSE had clinical signs of encephalitis with fever, disorientation, altered consciousness, paresis, seizures and/or dysphasia, and MR or CT-changes compatible with herpes encephalitis. Patients with HSM had clinical signs of meningitis with headache, nausea and/or vomiting, and pleocytosis in lumbar puncture. HSE and HSM, respectively, were confirmed on admission to the hospital by detection of HSV-1 or HSV-2 DNA by quantitative in-house TaqMan PCR methods . Cerebro6 cells/L and for polynuclear cells <3 × 106 cells/L [Patients with HSE  had the median age of 64.5 years (range 27–89 years). Patients were treated with acyclovir 10–15 mg/kg 3 times daily intravenously for 2–3 weeks. Patients with HSM  had the median age of 42.5 years (range 31–64 years). Treatment was given after lumbar puncture and serum sampling with oral valaciclovir 1 g 3 times daily for 1 week, initially replaced by intravenously acyclovir 5 mg/kg 3 times daily in case of difficulties with oral administration. Control subjects  had the median age of 53 years (range 21–91 years). Routine clinical analysis data of albumin levels and number of mono- and polynuclear cells in CSF were retrieved from medical records of HSE and HSM patients. Reference values for healthy individuals are for albumin <320 mg/L (15–45 years) and <420 mg/L (>45 years), for mononuclear cells <5 × 10 cells/L .Thirty chemokines and 10 cytokines  were detected and quantified in duplicate samples of serum or CSF using Bio-Plex Pro™ Assay (Bio-Rad Laboratories) according to the manufacturer’s instructions. Briefly, samples and standards were mixed with capture beads, were incubated in the dark in room temperature (RT) for 1 h on a shaker at 850 rpm, and were subsequently washed. Biotin-labeled antibodies were added to the capture beads and incubated for 30 min as described above. Following the washing step capture bead/biotin antibody complexes were incubated with Streptavidin-PE for 10 min. Complexes were washed and measured by a Bio-Plex 200 System (Bio-Rad Laboratories) with 5-parameter logistics standard curves which were used for interpolation of chemokine and cytokine levels.U test. For multiple comparisons of the three groups (HSE and HSM patients and control subjects) Kruskal-Wallis’ non-parametric test with Dunn’s post-test was used (n = 8–33/group). Statistical differences between serum and CSF in patient groups were calculated using Wilcoxon matched-pairs signed rank test (n = 8/group). Non-parametric Spearman’s correlation coefficient was used to calculate correlation between serum and CSF (n = 8/group). All statistical analysis was performed using GraphPad Prism version 6 (GraphPad Software).Normal distribution was tested using Shapiro-Wilk normality test. Albumin levels and mono- and polynuclear cell numbers were compared using Mann-Whitney The levels of albumin and the number of mono- and polynuclear cells in CSF were measured during routine clinical analysis and are summarized in Table n = 5–33). In the CC chemokine group five chemokines  were constitutively expressed in CSF . 28 out of 30 chemokines increased significantly in CSF of HSE and/or HSM patients compared to the levels in control patients without CNS infections  as well as HSE    Fig. , Table 2ly) Fig. . There w01) Fig.  with a C01) Fig. .Fig. 2Lep = <0.0001)  Fig. , Table 201) Fig. , with a 01) Fig. , Table 2Of the remaining measured chemokines most increased in CSF of both HSM and HSE, excluding only CCL2, CCL27, and CXCL12, but the levels were overall modest Table . In serup = 0.02, Additional file Most cytokines analyzed were induced in CSF of both HSM and HSE patients Table . IFN-γ, n = 8/group) were analyzed. Overall, the constitutive levels of chemokines were higher in serum compared to CSF  and CXCL13 (r = 0.76) in HSE and for CCL7 (r = 0.83) and CCL24 (r = 0.79) in HSM. There was also inverse correlations for CXCL6 (r = −0.81), CXCL16 (r = −0.86), IL-10 (r = −0.74), and IL-16 (r = −0.74) in HSE Additional file 2: Figure S1.n = 6-7) and HSM (n = 7-17) patients compared to healthy controls (n = 7-15). Data are presented as individual values with medians indicated by horizontal bars. CSF comparisons were performed using Kruskal-Wallis’ non-parametric test with Dunn’s post-test. Statistical analysis was performed using GraphPad Prism version 6 (GraphPad Software). Abbreviations: CSF, cerebrospinal fluid; HSE, herpes simplex encephalitis; HSM, herpes simplex meningitis. (PPTX 1582 kb)Levels of (A) CXCL10, (B) CXCL11 and (C) CCL8 in CSF of female HSE (