The vasculitis diagnosed specifically in the post-partum period are less well known. We report here such a case followed by a descriptive review of the literature.A 25 year-old French nurse reported abrupt-onset musculoskeletal pain 15 days after delivery of her first infant. Her first pregnancy was uneventful. The physical examination yielded only bilateral conjunctivitis and purpuric eruption of lower limbs, and complementary investigations evidenced pulmonary renal syndrome in connection with the diagnosis of Granulomatosis with Polyangiitis.We screened previous articles in Medline database using keywords (i) “post-partum” or “puerperium” (ii)”peripartum” (iii) “pregnancy” associated with “vasculitis”. Full texts were obtained from case reports or cases series whose title or abstract included keywords of interest (or synonyms). These references were secondarily excluded if the diagnosis of vasculitis was not confirmed or made before or more than 6 months after delivery and if vasculitis occurred only in the new born or affected only the cerebral vasculature or the retina.n = 10), Eosinophilic granulomatosis with polyangiitis (n = 9), Granulomatosis with Polyangiitis (n = 7), Periarteritis Nodosa (n = 5) and Behcet’s disease (n = 4). The first symptom occurred before delivery in 26 (59.1%) and maternal death occurred in 4 (9.1%) out of the 44 primary vasculitis cases.Fifty-six cases of vasculitis diagnosed in the post-partum period were included, 50 (89.3%) were secondary to an immunological process and 44 corresponded to primary vasculitis, 4 were secondary to Systemic Lupus Erythematosus, 1 to cryoglobulinaemia and 1 to cryoglobulinaemia associated with inflammatory bowel disease. The main primary vasculitis diagnosed were Takayasu Arteritis (The vasculitis diagnosed in the post-partum period were mainly primary vasculitis among patients in whom the diagnosis had not been made during pregnancy. In less than half of cases and as described in ours, there were no reported symptoms before delivery. Therefore, the physicians should pay attention to symptoms and keep a high degree of suspicion for vasculitis before as well as after delivery. Systemic vasculitis represent a wide range of diseases that can be either primary as well as secondary to other processes such as drug exposure, connective tissue disease or infection . DespiteWhile previously discouraged, pregnancy is now described among women suffering from non-infectious systemic vasculitis . The outHowever, there are less data concerning systemic vasculitis diagnosed specifically in the post-partum period. We describe here a case of primary systemic vasculitis diagnosed in the post-partum period, without any symptoms during pregnancy, before we make a descriptive review of the literature focusing on vasculitis diagnosed specifically in the post-partum period.2 weekly during four weeks followed by maintenance therapy with 500 mg every six months) were prescribed to the patient, who gave her written consent to report her case.A 25 year-old French nurse reported abrupt-onset musculoskeletal pain 15 days after delivery of her first infant. She had no past medical history and did not report any tobacco smoking, intravenous drug use or trip abroad. Her first pregnancy was uneventful except gestational diabetes. She gave birth to a healthy female neonate after an uncomplicated vaginal delivery. Fifteen days later, she reported diffuse joint pain without swelling. The pain was maximum in the morning and the joints affected were knees, ankles, wrists and shoulders. Bilateral conjunctivitis then purpuric eruption of lower limbs occurred one month after delivery. Despite a 14-day course of amoxicilline plus clavulanate (without any effect on symptoms), she was admitted to hospital because of hypochromic microcytic anaemia  associated with thrombocytosis  and elevated Serum C reactive protein  and ferritin  values. Physical examination evidenced only aphthae and small petechiae on the inner part of the leg, but no joint effusion or fever. Blood Creatinine level and proteinuria were 96 µmol/L (45 < N < 80 µmol/L) and 3000 mg/day (N < 150 mg/day) respectively. Chest X-Ray and Ct chest Scan yielded bilateral cavitary lung lesions  to define which kinds of vasculitis were diagnosed specifically in the post-partum period (ii) to estimate how many cases of vasculitis did not have any symptoms before delivery (iii) to describe the outcome of vasculitis diagnosed in the post-partum period.We conducted a review of cases of vasculitis whose diagnosis was made in the post-partum period. We first screened previously published articles in Medline database using keywords “post-partum” or “puerperium” associated with “vasculitis” or “vasculitides” without time limits. Articles whose title or abstract included keywords of interest “post-partum” and/or “vasculitis”  were selected (YNG). Full text versions were obtained and only case reports or cases series with available clinical data were included; review without case reports, epidemiological works without clinical data and animal data were not included. These references were secondarily excluded (i) in case of duplicate references (ii) if the diagnosis of vasculitis was not confirmed (iii) if the diagnosis of vasculitis was made before or more than 6 months after successful delivery of a living new-born (excluding miscarriage) (iv) if vasculitis occurred only in the new born but not in the mother (v) if vasculitis related to an immunological process only affected the cerebral vasculature or the retina.A second search in Medline using keywords “peripartum” associated with “vasculitis” or “vasculitides” without time limits was then conducted. A third search in Medline using keywords “pregnancy” associated with “vasculitis” or “vasculitides” without time limits but restricted to case reports was also conducted. A last search was conducted by checking references of previously selected references. In all these cases, the same methodology stated above to include and exclude references was applied (YNG).The flow chart of selected references is depicted in Fig. Fifty-six cases out of 56 references were included Fig. . Among tn = 10) n = 10) [Among the 44 primary vasculitis diagnosed in the post-partum period, the first symptom occurred before delivery in 26 cases (59.1%) –58, 60. In this review, the systemic vasculitis diagnosed in the post-partum period (i.e. during the first 6 months following delivery) were mainly due to an immunological process and corresponded to primary vasculitis in more than 75% of cases. All kinds of vasculitis have been described  but the five most common diagnoses were Takayasu Arteritis, Eosinophilic granulomatosis with polyangiitis, Granulomatosis with Polyangiitis, Periarteritis Nodosa and Behcet’s Disease. In less than half of cases, there were no symptoms reported before delivery, but in the other half, reported symptoms did not lead to a diagnosis before delivery and to the exclusion from this review (see exclusion criteria above). The maternal mortality of the primary vasculitis diagnosed in the post-partum period was less than 10%, but the maternal mortality of the vasculitis secondary to Systemic Lupus Erythematosus and cryoglobulinaemia appeared higher, within limitations of a low number of cases.Among primary vasculitis (which accounted for the majority of vasculitis diagnosed in the post-partum period), the distribution of the diagnosis was close to that observed during pregnancy , suggestThe most frequently described vasculitis in our review and in that of Gatto and colleagues focusing on pregnancy  was Takand and 3rd most common vasculitis in our review versus 4th and 3rd in the review of Gatto and colleagues [th more common diagnosis versus 2nd in the review of Gatto and colleagues [Periarteritis nodosa was the main vasculitis involving medium arteries in our review and in that of Gatto . The mailleagues ) was undWithin limitations of a low number of cases, the vasculitis secondary to Systemic Lupus Erythematosus and cryoglobulinaemia seemed to have a higher occurrence of symptoms after delivery and a higher mortality than primary vasculitis. In those latter, the diagnosis was not made before delivery even if the first symptoms occurred before delivery in 59.1% of cases. The occurrence of neonatal vasculitis was only observed in two infants born to mothers with vasculitis symptoms before delivery , 38. AllThe main limitation of this review remains its non-exhaustive nature. Even if we try to build a systematic reproducible review, some relevant references were found out of the scope of the search we made . ConversIn conclusion, the data from this review suggested that the vasculitis diagnosed in the post-partum period were mainly primary vasculitis among patients in whom the diagnosis has not been made during pregnancy. In less than half of cases and as described in ours, there were no reported symptoms before delivery. Therefore, the physicians should pay attention to symptoms and keep a high degree of suspicion for vasculitis before as well as after delivery.


Dietary biomarkers can potentially overcome the limitations of self-reported dietary data. While in ecology and archaeology, stable isotope ratios of carbon and nitrogen are widely used as biomarkers, this is not the case in nutrition research. Since the abundance of the 13C and the 15N isotope differ in food sources from plant and animal origin, stable isotope ratios of carbon and nitrogen (δ13C and δ15N) may differ in human biological material. Here, we investigated the stable isotope ratios of nitrogen and carbon in serum and urine from vegans and omnivores.Measurement of δ15N and δ13C in serum and 24 h urine was performed by Elemental Analyzer–Isotope Ratio Mass Spectrometer in the cross-sectional study “Risks and Benefits of a Vegan Diet”. The study included 36 vegans and 36 omnivores with a median age of 37.5 years (matched for age and sex), who adhered to their diet for at least 1 year.Both δ15N and δ13C were significantly lower in both the serum and 24 h urine of vegans compared to omnivores. δ15N either in serum or urine had 100% specificity and sensitivity to discriminate between vegans and omnivores. Specificity of δ13C was also > 90%, while sensitivity was 93% in serum and 77% in urine.δ15N both in serum and urine was able to accurately identify vegans and thus appears to be a promising marker for dietary habits.The online version contains supplementary material available at 10.1007/s00394-022-02992-y. Dietary intake is widely recognized as one of the most important lifestyle factors that influence both human health and planetary health. Meat intake in particular has been linked to non-communicable diseases, and its production has implications for land and water use, as well as greenhouse gas production . Diets tEven though the importance of diet in relation to health outcomes has been identified, challenges concerning the validity and reliability of dietary intake data continue to undermine research in this field. Methods for assessing dietary intake typically involve self-report and rely on memory recall and objectivity. Dietary intake methods based on self-reported data are prone to be influenced by factors such as the social desirability of foods, lack of memory or lack of consciousness that a food item(s) have been consumed, and lack of ability to estimate portion sizes or amounts of foods consumed . Thus, tStable isotope ratios are among the biomarkers that have been investigated as indicators of meat and fish , and addIn biology, stable isotopes have been also used to characterize trophic positions in the food web. Due to greater retention of the heavier 15N isotope than the lighter 14 N isotope in the production of nitrogenous waste, the nitrogen ratio of 15N to 14N (δ15N) shows a stepwise enrichment from food producers to food consumers and is therefore indicative of relative trophic position , 11, 12.12C rather than 13C in the photosynthesis when trapping/converting C from CO2 reflected by a lower δ13C value. C4 plants, among them sugar cane, corn and sorghum, have δ13C values approximately 12–13‰ higher than C3 plants. This difference can be used to measure the consumption of added sugar made from either sugar cane or corn while added sugar produced from sugar beet (C3 plant) would not show any difference in the δ13C [While the δ15N ratio in a food web reflects the trophic position, differences in the δ13C are more dependent on the type of plants consumed. C3 plants  to a greater degree than C4 plants utilize the the δ13C , 13, 14.Stable isotope ratios as dietary biomarkers can be measured in different tissues or body fluids, including skin , urine , 17, finHere, we investigate in an exploratory manner whether the stable isotope ratios of δ13C and δ15N in serum and 24 h urine can distinguish between healthy vegans and omnivores in a cross-sectional study. In addition, we investigate the discriminative power of δ13C and δ15N in comparison to two other dietary biomarkers for dairy and meat intake, plasma pentadecanoic acid (15:0) and plasma 1-methylhistidine [This is a cross-sectional study investigating the nutritional status of vegans, compared to omnivores. The recruitment process for healthy volunteers, aged 30–60 years, is described elsewhere . BrieflyAs an inclusion criterion, vegans should follow the vegan diet for at least a year, and omnivores should consume at least three times meat or two times meat and two times processed meat per week. Dietary intakes were recorded using three-day weighed food protocols. With the help of the German Nutrient Database  Version 3.02, the mean daily intake of food items, macronutrients and micronutrients was calculated. Information about age, educational attainment, and lifestyle factors were collected using tablet-based questionnaires. Height and body weight, waist circumference, and blood pressure were measured using standardized methods.From all participants in the study, 60 mL of blood was obtained. Blood lipids and creatinine were measured in a certified routine laboratory  by standard methods on the same day of blood collection. 24 h urine was collected by the participants, and urine creatinine concentrations were determined also determined on the day of the visit to the study center. All other biochemical analyses were performed on samples stored at a temperature of − 80 °C. Urine was collected on the days prior to the visit to the study center and were done from Sunday to Thursday.n = 40) − 20.58 ± 0.07 ‰; calibrated value: δ15N − 3.16 ‰, measured value (n = 31) − 3.23 ± 0.06 ‰)(Fisher Scientific) was incorporated into every run. δ13C of both reference materials and quality control sample were calibrated to the VPDB scale using LSVEC  and NBS-19  . The δ15N values were calibrated to the AIR scale using USGS40  and USGS41  . Analytical precision was based on repeated analyses of quality assurance material JALA (Fisher Scientific).Stable isotope ratios δ13C and δ15N were measured at the Stable Isotope Laboratory at the University of Oslo (UIO:CLIPT), using a method described by Kraft . Brieflyhttp://www.bevital.no). 1-methylhistidine was quantified using an isotope-labeled internal standard to an existing assay utilizing liquid chromatography combined with tandem mass spectrometry, as previously described [The fatty acid pentadecanoic acid (15:0) was measured as % of all fatty acids in plasma phospholipids at the German Institute of Human Nutrition Potsdam-Rehbrücke (Germany). The method for 15:0 measurement was described previously by Weitkunat . In addiescribed .The study was powered by a primary research question about differences in bone health between vegans and omnivores. Data were analysed exploratory to answer the research questions.T-test  or Kruskal–Wallis test  for continuous variables. Normal distribution of variables was prooven using the Shapiro–Wilk test, which indicated the non-normal distribution of δ13C, δ15N, 15:0 and 1-methylhistidine. Spearman correlations were calculated to investigate potential correlations between isotopes and variables of interest. To study the discrimination performance of biomarkers  regarding the dietary group (vegan vs. omnivorous diet), receiver operating characteristic curves (ROC) were plotted using the R package ROCit with a parametric binormal approach. The ROC curve represents a plot of sensitivity versus false-positive rate (1-specificity) of logistic regression prediction models (Diet ~ Exposure). The area under the ROC curve (AUC) represents the probability that the prediction model assigns a true vegan as vegan compared to an omnivore. The AUC may range from 0.5 indicating no discrimination to 1.0 indicating perfect discrimination. Scatter plots were used to derive cut-offs for the discrimination analysis (sensitivity and specificity).Variables were reported using mean and standard deviation (SD) for normally distributed variables, median and interquartile range (IQR) for non-normally distributed variables, and relative percentages for categorical variables. Differences between vegans and omnivores were tested using a Chi-Square test for categorical variables and a Student’s For statistical analyses of data, SAS software  and R software (version 3.6.3) was used. Even though the analyses in this report are exploratory, a p value of 0.05 was regarded as significant .In total, 72 healthy volunteers, 36 vegans and 36 omnivores (each 50% men) were included. Median age was 37.5 years (min–max: 30 –57), and median duration of vegan diet was almost 5 years. Main characteristics of the study sample are presented in Table Table The biomarkers 15:0  and 1-methylhistidine in plasma were also highly significantly different between vegans and omnivores, with higher values in omnivores compared to vegans for both biomarkers.r = 0.78), which was much weaker in vegans (r = 0.36). Correlation of δ15N in serum with δ15N in urine was, however, similar in both omnivores (r = 0.59) and vegans (r = 0.58). The strength of the correlations of both δ15N and δ13C with other factors such as age, BMI, or lipids, differed in the omnivorous group compared with the vegan group  .Thus, lower δ15N in vegans may reflect the consumption of plant-based protein sources instead of animal protein with higher δ15N. We observed lower urinary δ15N than plasma δ15N (both in vegans and omnivores) which may reflect to a stronger extent the preferred excretion of 14 N . While 1In comparison to the δ15N ratio, specificity, and sensitivity of the δ13C ratio to distinguish between vegans and omnivores were lower, but comparable to two other biomarkers of animal food intake. The fatty acid, 15:0, has been suggested as a biomarker of dairy intake and has shown a good correlation with reported dairy intakes in epidemiologic studies , althougAlthough the cross-sectional nature and sample size of our study precludes more advanced statistical analyses, the findings are nonetheless promising and should inform future work investigating reliable biomarkers of dietary intake and patterns. The results warrant therefore confirmation in studies with a more advanced study design. Of note, we relied on self-reported dietary habits when grouping the participants into a vegan or omnivorous diet. However, the risk of misclassification in this study seems to be low as participants filled in 3 days of dietary records and the call for participation was specifically addressing vegan diet and omnivorous diet, including the duration of a vegan diet and the requirement of 3 or more meat consumption occasions per week.In conclusion, the RBVD study included strict definitions of a vegan and omnivorous diet, implemented different nutritional status measurements, and provided the opportunity for stable isotope ratios and dietary biomarkers such as 15:0 percentage or 1-methylhistidine concentration to be investigated together for the first time. Using these biomarkers in combination may be promising and help to master the challenge to distinguish between for instance vegetarian and flexitarian diets, even if the absolute differences in stable isotopes between vegans and omnivores were small. Further research should focus on the added value of these combinations of biomarkers to monitor dietary changes, and whether stable isotope ratios alone or in combination with other biomarkers provide greater sensitivity, specificity, and ultimately reliability and reproducibility when distinguishing between omnivores, vegetarians, and vegans.Supplementary file1 (JPG 95 KB)Below is the link to the electronic supplementary material.


Without aggressive treatment, pulmonary arterial hypertension (PAH) has a 5-year mortality of approximately 40%. A patient’s response to vasodilators at diagnosis impacts the therapeutic options and prognosis. We hypothesized that analyzing perfusion images acquired before and during vasodilation could identify characteristic differences between PAH and control subjects.13NN PET imaging of pulmonary perfusion and ventilation. The total spatial heterogeneity of perfusion  and its components in the vertical (CV2Qvgrad) and cranio-caudal (CV2Qzgrad) directions, and the residual heterogeneity (CV2Qr), were assessed at baseline and while breathing oxygen and nitric oxide (O2 + iNO). The length scale spectrum of CV2Qr was determined from 10 to 110 mm, and the response of regional perfusion to O2 + iNO was calculated as the mean of absolute differences. Vertical gradients in perfusion (Qvgrad) were derived from perfusion images, and ventilation-perfusion distributions from images of 13NN washout kinetics.We studied 5 controls and 4 subjects with PAH using HRCT and 2 + iNO significantly enhanced perfusion distribution differences between PAH and controls, allowing differentiation of PAH subjects from controls. During O2 + iNO, CV2Qvgrad was significantly higher in controls than in PAH (0.08 (0.055–0.10) vs. 6.7 × 10–3 (2 × 10–4–0.02), p < 0.001) with a considerable gap between groups. Qvgrad and CV2Qtotal showed smaller differences: − 7.3 vs. − 2.5, p = 0.002, and 0.12 vs. 0.06, p = 0.01. CV2Qvgrad had the largest effect size among the primary parameters during O2 + iNO. CV2Qr, and its length scale spectrum were similar in PAH and controls. Ventilation-perfusion distributions showed a trend towards a difference between PAH and controls at baseline, but it was not statistically significant.OPerfusion imaging during O2 + iNO showed a significant difference in the heterogeneity associated with the vertical gradient in perfusion, distinguishing in this small cohort study PAH subjects from controls. Pulmonary arterial hypertension (PAH) is a progressive disease with a 5-year mortality of approximately 40% . Wide vaIn contrast to global parameters such as mPAP or pulmonary vascular resistance (PVR), imaging of pulmonary perfusion provides insights into regional changes associated with the disease process and its heterogeneity affecting the perfusion distribution inside the lungs. However, it is unclear if pulmonary perfusion imaging can detect substantial differences between PAH and healthy controls or assess regional changes in response to acute vasodilators such as inhaled nitric oxide and oxygen. In healthy lungs, there is a vertical gradient in perfusion , 6 inclu2 + iNO) to differentiate active effects of vasoconstriction [Here, we used positron-emission tomography and high-resolution computer tomography (PET-CT) imaging characterize the spatial distributions of pulmonary perfusion and ventilation in patients with PAH. We compared the patterns of perfusion while breathing air and during inhalation of both oxygen and inhaled nitric oxide . Due to the known limitations of impedance cardiography, a trend rather than an absolute change in cardiac output, was monitored. For assessment of within-subject changes in perfusion and perfusion heterogeneity compared to baseline and for the comparison of regional perfusion between PAH and controls, we used values of mean-normalized regional perfusion obtained directly from imaging data. Pulse oximetry and heart rate were continuously monitored from a fingertip pulse oximeter.PAH subjects receiving vasodilators as part of their treatment were maintained on those therapies during the day of study.13NN-saline PET scan was acquired starting with an equivalent breath-hold at MLV. Simultaneously with the beginning of the breath-hold we injected a bolus of 13NN dissolved in saline (25 mL) and started the acquisition of the dynamic PET scan. After 30 s of breath-hold, we asked the subject to resume breathing while the PET scan acquisition continued. Total acquisition time of the PET scan was 6 min and 40 s. During this first PET-CT imaging sequence the subject was breathing air. After it was completed, subjects were administered a gas mixture  of balance gas oxygen with 30 ppm inhaled nitric oxide (O2 + iNO) through a one-way valve with a mouthpiece and nose clip. After at least 5 min of breathing O2 + iNO, we repeated the PET-CT imaging sequence while the subject continued to breath the O2 + iNO gas mixture. Given the sample size and the desire to prevent carry over effects from the inhaled nitric oxide, all subjects underwent baseline imaging followed by imaging with iNO without randomization of order of imaging.A catheter was placed in an antecubital vein for radioisotope injection. Subjects were positioned supine and with arms abducted on the table of the PET-CT scanner . Relative lung volume was continuously measured using impedance plethysmography . After calibration of the plethysmograph, mean lung volume (MLV) was determined during 30 s of steady tidal breathing prior to imaging. Image acquisition started a ‘topogram’ CT scan to determine the field of view for PET and HRCT scans. In preparation for an HRCT scan, the subject was asked to inhale to total lung capacity (TLC), followed by slow exhalation, and to hold the breath when the impedance plethysmography signal showed that MLV was reached. During the breath-hold the HRCT scan was acquired. Following this, a dynamic 3). The PET field of view included on average 75% of the CT volume of the thorax. Dynamic PET scans were reconstructed with filtered back-projection and attenuation corrections using the HRCT scan. The dynamic PET scans yielded a stack of 19 images , each image a 3D matrix of 128 × 128 × 81 voxels (voxel size 5.3456 × 5.3456 × 2.0250 mm3). The PET scans have a full-width at half maximum (FWHM) resolution of 6 mm  so that the effective resolution with a contrast recovery coefficient of 80% at two times FWHM is approximately 12 mm. PET images were filtered before image analysis using a moving average filter of five slices (~ 10 mm) in the axial direction and a filter with 10 mm diameter within slices, which is comparable to the effective resolution of the scans.HRCT scans  were reconstructed with filtered back-projection  images using the equation:tis) images were calculated using Ftis = 1 − Fgas.Image segmentation software Apollo  was used to identify the lung region of interest (ROI) in the HRCT scans. Using custom software tools written in Matlab , the lung ROIs were converted to ROI masks for PET scan analysis. Additionally, HRCT scans were converted into gas fraction   to selecartments , 25. Theectively , 26. Not2 = (standard deviation/mean)2) using a previously developed and validated method for assessment of noiseless CV2 of PET scans based on theoretical properties of measurement errors and empirical assessment of imaging noise [2Qimage) is the sum of the variance of the true signal (perfusion) and the variance of the imaging noise CV2Qimage = CV2Qtotal + CV2noise [2noise using multiple PET images during the tracer plateau at the end of the breath-hold period and calculated the total spatial heterogeneity of 13NN images of relative perfusion, excluding the imaging noise, as CV2Qtotal = CV2Qimage − CV2noise. In the supine position, gravitational forces typically cause a gradient in perfusion in the dorsoventral direction with higher perfusion in the dependent  regions than in the non-dependent  regions, which can be a substantial component of CV2Qtotal. We separated the total spatial heterogeneity of perfusion into components caused by systematic gradients in the vertical direction (CV2Qvgrad), the axial  direction (CV2Qzgrad), and the remaining, or residual heterogeneity (CV2Qr). Thus, CV2Qtotal = CV2Qvgrad + CV2zgrad + CV2Qr. The value of CV2Qr was further split into its components due to spatial variations in perfusion across the length scale spectrum [Heterogeneity of perfusion was determined as the square of the coefficient of variation  were determined. Then the gradients were subtracted voxel-by-voxel from the 3D perfusion scan, and each of the resulting transverse slices of the scan were low-pass filtered at 10, 30, 50, 70, 90 and 110 mm with correction for edge effects [2 was calculated for each filter size, and the contribution of each length scale ranges were calculated as differences between successive filter sizes, e.g. CV2Q10-30 = CV2(10 mm) −  CV2(30 mm). Overall, small length scales of a spectrum starting at 10 mm correspond to variation among volumes of approximately 1 mL, which corresponds to about 5 acinar units; whereas large length scales of the spatial spectrum correspond to heterogeneity due to variation in structural, functional and disease processes among larger regions such as segments or subsegments.For each condition, the length scale spectrum of perfusion was quantified as follows: First, the vertical and the axial gradients (z direction) in perfusion  to correct for potential tissue displacement between the two perfusion images. First, we identified the transformation field that mapped the HRCT scan taken at MLV while breathing O2 + iNO to the corresponding HRCT scan taken at MLV while breathing air. The same method has been successfully used before to determine the regional volumetric deformation and regional lung strain during mechanical ventilation [2 + iNO PET scan to map its voxels to the corresponding voxels of the air PET scan correcting differences in tissue location. This correction is essential for an assessment of changes between PET scans at the voxel level. Images representing the relative changes in regional perfusion between baseline and O2 + iNO were calculated as voxel-by-voxel differences between the mean-normalized perfusion at air and the mean-normalized perfusion at O2 + iNO.For the assessment of changes in perfusion at the voxel level, we performed elastic image registration using the Advanced Normalization Tools  histograms of the number of voxels in relation to relative perfusion and relative height from the most dorsal point of the lung mask and the most ventral point. Difference maps of the perfusion-height distributions between air and O2 + iNO were calculated at the voxel level as mean absolute deviations. Contributions of imaging noise [2 + iNO includes the sum of the variances of imaging noise in the two original images. The variance of the imaging PET noise, which has a normal distribution, was determined, using a previously described method for noise assessment using multiple time frames of a PET scan [Mean absolute changes in regional perfusion between air and Ong noise  to the mPET scan , and sub2Qtotal) and its components, including the heterogeneity generated by the vertical gradient in perfusion (CV2Qvgrad) and the residual perfusion heterogeneity (CV2Qr) and its length scale components. In secondary analyses, we assessed various regional perfusion and ventilation metrics. We performed two-sided T-tests to evaluate whether there was a difference in means of each metric between study groups and for baseline vs. O2 + iNO within study groups. Additionally, we obtained Cohen’s d effect size estimates for the primary parameters. All analyses were performed using R Statistical Software (v4.1.3) [Our primary outcomes of interest were the noise corrected values of total spatial heterogeneity of perfusion (CV(v4.1.3) . Effect (v4.1.3) . Statist2 + iNO for both groups. However, there were no significant differences in cardiac output trends between groups at rest or while breathing O2 + iNO. All PAH subjects were on combination therapy with a PDE5 inhibitor and endothelin receptor antagonist, and two PAH subjects were also on an inhaled prostacyclin or a prostacyclin receptor agonist.Nine subjects were studied: 5 controls, and 4 subjects with PAH Table . Subject2Qtotal) for the PAH subjects was similar to that of the controls (p = 0.51). However, while breathing O2 + iNO, the PAH subjects had a lower CV2Qtotal  compared to control counterparts  and the heterogeneity generated by that gradient (CV2Qvgrad) were significantly greater in the control group compared with the PAH group   , –3.25  and 2.23 , respectively  relative to the non-dependent  regions Fig. , Table 22 + iNO administration   gradient in perfusion to total heterogeneity was small, and there was no significant difference between the groups regardless of O2Qr) when compared to controls (p = 0.41) at baseline. These similarities in CV2Qr remained consistent while breathing O2 + iNO. To further analyze the residual spatial heterogeneity in perfusion, we assessed the variations in perfusion across the length scale spectrum. At baseline and while breathing O2 + iNO, PAH subjects had similar residual heterogeneity across the spectrum of length scales of 10–110 mm , when compared to control subjects . There was no significant difference observed in the overall mean specific ventilation (stis) was similar between control and PAH subjects.Alveolar ventilation  CV2Qvgrad during O2 + iNO had the highest effect size for PAH vs. controls. (3) All subjects in our study responded to O2 + iNO with changes in regional perfusion, including the two PAH subjects who were non-responders according to vasoreactivity testing. (4) PAH subjects had significantly lower vertical gradients in perfusion than controls and such gradients in 3 of the 4 subjects were unaffected by O2 + iNO. (5) PAH subjects had residual perfusion heterogeneity across the length-scale spectrum that was similar to that of controls.We report a detailed analysis of regional perfusion and ventilation imaging data in PAH and control subjects obtained with PET-CT, revealing unique perfusion characteristics in PAH subjects that may not be reflected in routine PA pressure measurements. We found that: (1) Breathing O2 + iNO leading to perfusion redistributions. The significant difference in CV2Qvgrad between PAH subjects and controls suggests that perfusion imaging during O2 + iNO has the potential to be as a biomarker for PAH, that could lead to earlier detection and thus improve patient survival [2 + iNO and air allows identifying responders among PAH patients. The only confirmed responder in our study showed a greater response in CV2Qvgrad compared to the other PAH subjects, but it is a single case.An unexpected finding was the magnitude of the vasodilation in healthy controls during Osurvival . It rema2 + iNO in those with PAH, indicating that despite measured changes in regional perfusion with vasodilation, PAH subjects did not show normal vertical perfusion patterns. In fact, the vertical gradient in perfusion was in some subjects nearly absent in the dependent part of the lungs, as demonstrated with perfusion-height mapping  as well as abnormalities of the vessels along the pathways. In control subjects, O2-iNO had no effect on the residual heterogeneity of perfusion CV2Qr  . Additiourements . Howevertography . In contod gases . In PAH,ve found –52, but ve found , 50 and 13NN imaging technique has been discussed in detail [13NN tracer kinetics during the breath hold may affect the accuracy of noise-free CV2 parameters of the perfusion heterogeneity, and errors in image registration causing small misalignments could affect the assessment of voxel-by-voxel changes in perfusion. However, such errors only diminish differences among groups and would bias our results toward the null hypothesis of statistical tests. Additionally, an analysis of the reproducibility of CV2Qtotal, excluding imaging noise, in our previous validation showed a standard deviation of ± 8.4% in repeated imaging [Despite our study’s strengths, some important limitations should be considered. Limitations of the n detail , 22. Brin detail . The eff imaging . Third,  imaging  and diff imaging  are asso2-iNO inhalation showed in this small cohort study a large effect size for the heterogeneity associated with the vertical gradient in perfusion, CV2Qvgrad, for PAH followed in magnitude by the vertical gradient in perfusion. PAH subjects could be discriminated from healthy controls showing the potential of CV2Qvgrad measured during O2-iNO inhalation to be a biomarker for PAH. Further studies are needed to investigate NO inhalation in clinically available imaging modalities such as dual-energy CT to translate our findings into clinics.Perfusion imaging during O


Increased low frequency cortical oscillations are observed in people with neuropathic pain, but the cause of such elevated cortical oscillations and their impact on pain development remain unclear. By imaging neuronal activity in a spared nerve injury (SNI) mouse model of neuropathic pain, we show that neurons in dorsal root ganglia (DRG) and somatosensory cortex (S1) exhibit synchronized activity after peripheral nerve injury. Notably, synchronized activity of DRG neurons occurs within hours after injury and 1-2 days before increased cortical oscillations. This DRG synchrony is initiated by axotomized neurons and mediated by local purinergic signaling at the site of nerve injury. We further show that synchronized DRG activity after SNI is responsible for increasing low frequency cortical oscillations and synaptic remodeling in S1, as well as for inducing animals’ pain-like behaviors. In naive mice, enhancing the synchrony, not the level, of DRG neuronal activity causes synaptic changes in S1 and pain-like behaviors similar to SNI mice. Taken together, these results reveal the critical role of synchronized DRG neuronal activity in increasing cortical plasticity and oscillations in a neuropathic pain model. These findings also suggest the potential importance of detection and suppression of elevated cortical oscillations in neuropathic pain states. 2+ imaging of DRG sensory neurons in a mouse model of neuropathic pain, and show that synchronized activity of DRG neurons occurs within hours after injury and 1-2 days before increased cortical oscillations are observed.Increased low frequency cortical oscillations are observed in neuropathic pain. Here the authors perform Ca Low-frequency cortical oscillations are often enhanced in neuropathic pain patients and animal models10. Activity synchrony of spinal dorsal horn neurons is also augmented under pain condition and after noxious stimuli in rodents14. In addition, synchronized neuronal activity is elevated between pain-related spinal15 and supraspinal regions18. In the primary somatosensory cortex (S1), layer 2/3 (L2/3) pyramidal neurons undergo a prolonged period of increased basal activity during sustained pain states20 that is associated with an increase in synchronization22. Although the increase of neuronal synchronization is widespread during the development of neuropathic pain, the mechanisms and functional impact of such elevated activity synchrony remain unknown.Neuropathic pain arises as a debilitating consequence of peripheral nerve injury or insult. The mechanisms underlying neuropathic pain remain unclear, and existing treatments are largely ineffective23. DRG neurons exhibit spontaneous hyperactivity upon nerve injury24 or insult25, likely due to aberrant signaling at the injured site26, as well as maladaptive changes of receptors, enzymes and ion channels in the ganglia27. The enhanced DRG neuronal activity could further trigger plastic changes in the upstream pain pathway, including spinal cord, subcortical and cortical regions31, leading to the development of chronic pain33. Nevertheless, whether and how DRG neurons contribute to increased cortical oscillations and plasticity under chronic pain conditions remain unclear.The primary sensory neurons in dorsal root ganglia (DRG) detect external stimuli and relay the signal to the central nervous system (CNS)In this study, we took advantage of a newly-developed method to monitor activity of DRG neurons in awake mice during neuropathic pain development. We show synchronized activation of DRG neurons occurs rapidly in a spared nerve injury (SNI) mouse model of pain. Importantly, the synchrony, not the level, of DRG neuronal activity after SNI is critical for increased cortical plasticity, enhanced cortical oscillations, and pain hypersensitivity. Our findings reveal the important role of DRG activity synchrony in promoting cortical plasticity and oscillations in neuropathic pain development. They also suggest that early detection and suppression of increased cortical oscillations could serve as an important means for the diagnosis and treatment of chronic pain.35, in which axotomy of tibial and common peritoneal branches of sciatic nerve resulted in mechanical allodynia  pyramidal neurons in S1 of head-fixed mice with in vivo two-photon CaSNI Fig. . Using PSNI Fig. . The levSNI Fig. , but notSNI Fig. . In addiSNI Fig. . Given t41. DRG neurons are the first-order sensory neurons for detecting external stimuli and for conveying signals to CNS43. Suppressing DRG neuronal activity has been shown to ameliorate chronic pain development46. To investigate the potential contribution of DRG neuronal activity to enhanced cortical activity synchrony, we performed Ca2+ imaging of lumbar 4 (L4) DRG sensory neurons in awake mice using a recently developed imaging technique25. Imaging was performed after exposing the left L4 DRG and fixing the spine between L3–L5 with a custom-made metal sheet (see methods). We found that the integrated Ca2+ activity of DRG neurons was significantly higher within 3 hours after SNI when compared to pre-SNI  was locally applied to the injured sciatic nerve 3 days after SNI, the level and synchrony of DRG neuronal activity reduced significantly Fig. . ConcomiThy1-GCaMP6 mice to retrogradely label their somas in lumbar 4 DRG  into the tibial branch of sciatic nerve in DRG Fig. . The actDRG Fig.  or spareDRG Fig. . FollowiDRG Fig. . In contDRG Fig. , both thDRG Fig. . Thus, aDRG Fig. .Fig. 3Ax26. To explore whether ATP at the site of nerve injury may contribute to the elevated level and synchrony of DRG neuronal activity after SNI, we measured the ATP level in living mice by monitoring luminescence resulting from ATP-triggered luciferase breakdown of luciferin48. Real-time imaging showed that the ATP level after surgery was substantially higher at the injury site in SNI mice as compared to that in sham mice  to the injured sciatic nerve. Three to five minutes after ATP application in SNI mice, we found a marked increase in the level and synchrony of DRG neuronal activity  for 3 days . Notably, we found that local application of BzATP increased the level but not synchrony of DRG activity  to L3–L5 DRGs or ATP  to the sciatic nerve three times a day over 3 days in naive mice. To apply BzATP to L3-5 DRGs, three silicone catheters with 0.5 mm in inner diameter were placed to the posterior edge of the surface of three DRGs, respectively. The catheters were fixed to the vertebrae column to avoid rotation and loosening (see methods). We then performed Ca S1 Fig. . As expevel Fig.  and syncvel Fig.  in L2/3 vel Fig.  when comvel Fig. . BecauseMoreover, repeated imaging of dendritic spines of L5 pyramidal neurons showed that spine formation was significantly increased 3 days after repeated ATP treatment in the sciatic nerve as compared to the controls with saline treatment Fig. . No signNotably, repeated peripheral ATP treatment also decreased the paw withdrawal threshold substantially in naive mice, whereas BzATP application had no effects on the animals’ paw withdrawal threshold Fig. . In addi58. The mechanisms and functional impacts of enhanced cortical oscillations remain unknown. By monitoring neuronal population activity in DRG and S1 in awake, behaving mice, we show that synchronized activity of DRG neurons occurs within hours after injury and 1–2 days prior to the increase in activity synchrony of L2/3/5 pyramidal neurons and low-frequency cortical oscillations in S1. Neuronal activity synchrony in DRG is initiated by axotomized neurons and facilitated by local purinergic signaling at the site of nerve injury. Importantly, the synchrony, not level, of DRG neuronal activity is critical for cortical plasticity, mechanical hypersensitivity, and spontaneous pain-like behavior. These findings reveal the important role of the synchronized activity of DRG neurons in enhanced cortical oscillations and neuronal plasticity in S1 in the development of neuropathic pain.Increased cortical synchrony is common in patients as well as animal models of neuropathic pain60. However, due to technical difficulties, the DRG neuronal activity has not been investigated in awake animals under pain conditions. Our discovery of DRG neuronal synchrony after SNI benefits from the recent technological advancement of DRG imaging in awake-behaving mice25, as DRG neuronal activity is largely blocked in animals under anesthesia61. In addition to SNI mice, we found that the level and synchrony of DRG neuronal activity were also significantly higher in mice after plantar Complete Freund’s Adjuvant (CFA) or formalin injection as compared to pre-injection  at molecular and cellular levels. While our study identifies DRG synchronized activity as the driving force for cortical changes and increased S1 oscillations, the mechanistic links between DRG synchrony and S1 oscillations remain to be established in the future. In the present study, we employed a drug delivery approach for DRG synchrony and asynchrony induction. Since the expression of opsins in DRG neurons combined with transdermal light illumination could successfully modulate DRG neuronal activity and pain intensity77, such an optogenetic approach may be more precise for DRG synchrony induction and mechanistic studies in the future.Although our results revealed the causal relationship between DRG synchrony, pyramidal neuron synchrony and 4–8 Hz oscillations in S1, how synchronized DRG activity enhance S1 oscillations over extended periods of time is complicated and likely involves progressive changes of multiple cell types  or 10 µL formalin (2%) solution was injected under the plantar surface of the left hindpaw with a 30-gauge needle.Inflammatory pain was induced by subcutaneous CFA25. In brief, mice were placed in a 2.9-cm-diameter transparent plastic cylinder to attenuate motion artifacts. The cylinder was then mounted onto a heavy metal base. Before imaging, mice were habituated for at least 30 min. Thy1-GCaMP6s mice were used for Ca2+ imaging of afferent sensory somas in the DRG. Imaging was performed using a Bruker two-photon system equipped with a DeepSee Ti:sapphire laser (Spectra Physics) tuned to 920 nm. The average laser power on the sample was ~20–30 mW. Images were collected at frame rates of 1.3–1.7 Hz at a resolution of 512 × 512 pixels using a ×25 objective  immersed in artificial cerebrospinal fluid (ACSF) and with a 1× digital zoom. Image acquisition was performed using Bruker PrairieView software.In vivo imaging of L4 DRG in awake mice was performed according to method described recently2+ imaging in L2/3 of S1 hindlimb (HL) was performed in awake mice through a thinned skull window53. In brief, mice were anesthetized with an intraperitoneal injection of 100 mg/kg ketamine and 15 mg/kg xylazine (KX), and the head was shaved and the skull surface was exposed with a midline scalp incision. After removing the periosteum tissue, a head holder was attached to the skull surface with cyanoacrylate glue (Loctite 495) and dental acrylic cement. After identifying the hindlimb region of S1 in the contralateral hemisphere on the basis of stereotaxic coordinates , a high-speed micro-drill was used to thin a circular area (~0.2 mm in diameter) to a thickness of ~20 μm. The exposed skull was covered with a thin layer of Kwik-Sil® silicone elastomer. Imaging was performed 24 hours after surgery in well-habituated mice. Before imaging, the skull was immersed in ACSF and the head-restrained animal was then placed on the stage of a two-photon laser scanning microscopy. In some experiments  immersed in ACSF. A 3× digital zoom was used to yield high-magnification images  suitable for quantification of dendritic spines. For multiple imaging, the above procedure was repeated and the localization of the same region was facilitated by low-magnification image stack at 1× digital zoom  and with reference to vascular landmarks under the thinned skull area. The animal was head-restrained during image acquisition, which lasted ~30 minutes. The scalp incision was immediately sutured after each imaging session.The surgical procedure for chronic transcranial two-photon imaging has been described previouslyd-Luciferase/luciferin solution  was prepared with 1× DPBS  solution. A volume of 100 μL was administered systemically via retro-orbital injection when the mice were maintained under isoflurane anesthesia. Bioluminescence images were taken using an IVIS Spectrum with 60 second exposure time, medium binning (8), and f/stop 1. Light outputs were quantified using Living Image 4.5.5 .ATP released from the sciatic nerve and surrounding tissue was imaged by an IVIS Spectrum . 25. In brief, mice were deeply anesthetized with KX and placed under a stereomicroscope . A 1-cm-long incision was made in the dorsal skin at the L3–L5 level of the spine, and the muscles along the lateral aspects of L3–L5 were detached and then retracted with a custom-made metal sheet. To expose the left L4 DRG, we aligned the long bracket of the imaging device with the left side of L4–L5 vertebrae and short bracket with the right side, and then registered the two brackets with a locking screw. After trimming articular processes around the DRG, a thin layer of Kwik-Sil® silicone elastomer and a round coverslip was placed on top. For applying drugs to the DRG, a semicircular coverslip was used to cover 2/3 of DRG with 1/3 of DRG and 1-mm-long spinal nerve exposed. The exposed nerve tissue was protected with 1% agarose. The coverslip was secured to the vertebrae mount by cyanoacrylate and dental acrylic. Throughout the surgical procedure and recovery, the animal’s body temperature was maintained at ~37 oC.Surgical procedure for DRG window implantation was performed as described recently2+ imaging of L2/3 pyramidal neurons in S1, two ECoG electrodes were placed in the area close to S1 . Two copper wires were placed in the nuchal muscles for electromyographic (EMG) recordings. Recording sessions began 3-5 days after electrode implants. Connectors from ECoG electrode were tethered to preamplifier headstage leading to a multichannel amplifier. Simultaneously recorded ECoG and EMG signals were amplified 500 times  and ECoG data were analyzed using custom code in MATLAB . The code, a demo, and instructions for analysis are provided in Supplementary Software Under KX anesthesia, mice were head-fixed with a stereotaxic apparatus. A small skin incision was used to expose the skull and a small area of skull (~1 mm in diameter) over S1 contralateral to the left hind paw  was thinned with a high-speed drill and carefully removed with forceps. Two electrodes for ECoG recording were made by soldering one end of an epoxy-coated silver wire  to a female miniature multichannel connector. The electrodes were inserted above the dura mater of the cortex with the tips separated from each other by ~500 μm. The electrodes were fixed by cyanoacrylate-based glue and dental cement. When simultaneously performing Ca2+ activity of L2/3 pyramidal neurons in S1, 11.46 sec = 10 imaging frames) or 10 sec time intervals during awake resting state (no noticeable movement and sleep) in each animal. Relative power values were generated at a sampling frequency of 5 kHz between 0.5 and 40 Hz. Mean power spectra density was calculated between 0.5–4 (delta band), 4–8 (theta band), and 8–12 Hz .Fast Fourier transformation was performed to convert ECoG waveform from the time domain to the frequency domain, yielding power spectra density using 11.46 sec  was injected  into the trunk of tibial nerve using a glass microelectrode over a period of 8–10 min.Mice were anesthetized with isoflurane and a 0.8-cm-long incision was made on the dorsal skin of the left thigh. The muscles attached to the femur bone were detached and distal 2/3 of the femur was exposed. A high-speed drill was used to create two 0.5-mm-diameter holes on the distal 1/3 and 1/4 of the femur with. With the aid of these two holes, a plastic bottle  without lid and bottom was tied to the femur using the 6–0 suture , A-317491 , or apyrase  were locally applied with 100 μL microinjector to the sciatic nerve at the incision site. Entry point was located medial to linea aspera (third trochanter) of the fumer that was easily identified. ATP (5–30 mM), A-317491 (200 μM) and apyrase (400 U/mL) were dissolved directly in 0.9% saline solution to final concentrations. ATP and A-317491 were applied once (30 μL), and apyrase was applied four times within 30 minutes (50 μL every 10 min). To examine the effect of BzATP on DRG neuronal activity by in vivo DRG imaging, distal 1/3 of DRG were exposed for drug delivery. BzATP  was locally applied to distal 1/3 of DRG during in vivo imaging . To examine the behavioral effects of DRG asynchrony induction, BzATP  was delivered via intrathecally implanted catheter, with three tips placed adjacent to the left lumbar 3–5 DRGs respectively.85. In short, a series of von Frey fibers  were presented in a consecutive ascending order. In the absence of paw withdrawal response, the next stronger stimulus was presented; in the event of paw withdrawal, the next weaker stimulus was chosen. After the response threshold was first crossed, 6 data points were counted, at which time the 2 responses straddling the threshold were retrospectively designated as the first 2 responses of the series of 6. 50% response threshold was calculated as: 50% g threshold = (10 Xf[ + κδ])/10,000 where Xf = value (in log units) of the final von Frey hair used; κ = tabular value for the pattern of positive/negative responses86; and δ = mean difference (in log units) between stimuli .For mechanical allodynia test, mice were individually placed into transparent plastic boxes (10 cm × 7 cm × 7 cm) over a mesh table and habituated for at least 30 min prior to testing procedures. Dixon’s up and down method was employed to measure the animals’ paw withdrawal threshold87. Briefly, the test apparatus consisted of a shuttle-box that was divided into two compartments of the same size  by a guillotine door (4 cm wide × 6 cm high). Each compartment had different visual and textured cues . Mice were habituated to the apparatus for 10 min for 3 consecutive days prior to conditioning. In the pre-conditioning phase (day 0), animals were placed in the apparatus and the door was removed to allow animals free access to both chambers for 10 min. The time spent in each compartment was recorded. The animals showing strong unconditioned preference (>400 s) were discarded. On day 1, mice were randomly assigned for SNI or sham surgery . Membranes were blocked with 3% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 (pH = 7.40) for 1 hour at room temperature. Membranes were first incubated with rabbit anti-P2X3  on an orbital shaker in 4 °C cold room overnight, then further incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody  for 2 hours at room temperature and visualized by western peroxide reagent and luminol/enhancer reagent . Images were generated with the ChemiDoc XRS System with Image Lab software (Bio-Rad). Intensities of protein bands were quantified using the Image Lab software. Values of P2X3 proteins were normalized to those of β-actin.A length of 0.5 mm of the injured nerve trunk proximal to DRG was collected for Western blot assay. Protein samples were prepared and loaded onto a 4–15% stacking/7.5% separating SDS-polyacrylamide gel  as reported previously2+ activity, regions of interest (ROIs) corresponding to visually identifiable somas of DRG neurons were selected for quantification at the depth of 30–200 μm below the surface of DRG. ROIs for L2/3 somas in S1 were selected at the depth of 200–250 µm below the pial surface. The fluorescence in each soma was measured with NIH ImageJ by averaging all pixels within the ROI covering the soma. The ΔF/F0 was calculated as ΔF/F0 = (F−F0)/F0, where F0 is the baseline fluorescence signal averaged over a 2-s period corresponding to the lowest fluorescence signal over the 2.5-min recording period. Integrated Ca2+ activity is the sum of ΔF/F0 over 2.5 min. Active somas were defined with the criteria that Ca2+ transient was beyond the threshold of three times the standard deviation of the baseline.To analyze Ca2+ transients were included in the analysis. We calculated the cross-correlation over the time course of 150 seconds. To determine the significance of the correlation at zero time lag we used bootstrap shuffling. Specifically, we shuffled the activity of one cell by a random amount relative to the activity of the paired cell and then re-calculated the cross-correlation for the shuffled data. We repeated this process 500 times and calculated the number of times the value of the cross-correlation at zero time lag for the shuffled data was higher than the original data. If the value of the shuffled data was higher than the original data in <5% of the shuffles, we considered the correlation between the cell pair to be significant. Data analysis was performed using toolboxes and custom code in MATLAB.We calculated the cross-correlation for all cell pairs that were imaged simultaneously. Only cells displaying active Ca53. Briefly, image stacks were analyzed using NIH Image J software. For each dendritic segment analyzed, filopodia were identified as long, thin protrusions with a ratio of head diameter to neck diameter <1.2:1 and a ratio of length to neck diameter >3:1. The remaining protrusions were classified as spines. Spines were considered the same between views if their positions remained the same distance from relative adjacent landmarks. Spines were considered different if they were >0.7 μm away from their expected positions based on the first view. More than 150 spines were analyzed from each animal. The minimum length of dendritic branches included in the analysis was over 30 µm. The formation or elimination rates of spines were measured as the number of spines formed or eliminated divided by the number of spines existing in the first view.The procedure for quantifying spine dynamics has been described in our previous studiest test. One-way or two-way analysis of variance followed by Dunnett’s or Tukey’s test was also used to compare the significance between various groups. Significant levels were set at P ≤ 0.05. All statistical analyses were performed using GraphPad Prism.Summary data are presented as means ± sem. Sample sizes were chosen to ensure adequate power with the statistical tests while minimizing the number of animals used in compliance with ethical guidelines. No samples or animals that were successfully imaged or measured were excluded from the analysis. The variance was similar between the groups that were statistically compared. Tests for differences between two populations were performed using unpaired or paired Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Software 1Reporting Summary


Coupled with triboelectric nanogenerator, it could form a self-charged conformable triboiontronic device. Furthermore, the 3D aerogel scales up areal power density up to 1.3 mW cm−2 purely from ion gradient based on nanoconfined enhancement from graphene oxide that can operate under −40 °C and overcome humidity limitations, enabling to power the future implantable electronics in human-machine interface.Iontronics focuses on the interactions between electrons and ions, playing essential roles in most processes across physics, chemistry and life science. Osmotic power source as an example of iontronics, could transform ion gradient into electrical energy, however, it generates low power, sensitive to humidity and can’t operate under freezing point. Herein, based on 2D nanofluidic graphene oxide material, we demonstrate an ultrathin (∼10 µm) osmotic power source with voltage of 1.5 V, volumetric specific energy density of 6 mWh cm Osmotic power source based on 2D nanofluidic graphene oxide could overcome humidity and temperature limitations due to high areal power density purely from ion gradient. Here, authors couple it with triboelectric nanogenerator, and demonstrate a self-chargeable conformable tribo-iontronic device. Iontronics couple the electron/ion charge transfers and exchange signals at the interface of electronic/ionic conductors, differentiating them from most electronics using just electrons and/or holes as the dominating charge carriers7. Bioinspired nanofluidic iontronics could have compatible signals with neurons to enable implantable iontronic devices or even neuronal-computer interfaces3. Enhanced sensitivity of tactile sensor8 and pressure sensor9 could also be obtained by iontronic films. Unusual behavior of ion transport kinetics in channels narrower than the Debye length of electrolyte has been observed, the surface charges on the inner walls of nanofluidic channels repel ions of the same charge and attract counter ions, making them the dominating charge carriers10. Such unipolar ion transport can enhance ionic conductivity up to several orders of magnitude, which breaks the conventional continuum-based theory11. Recently, a variety of unusual ionic phenomena such as highly selective ion sieving12, ultrafast ion transport13, and anomalous increase of capacitance in nanopores15 were all observed due to the enhanced diffusion related to the strong ion-ion correlations under severe nanoconfinement16.In biological cellular membranes, ion-specific pores permit certain types of ions flow across the membrane driven by osmotic energy from ion gradient, responsible for nervous impulses, muscle contractions and physiological sensing. The osmotic energy could be generated based on either pressure-retarded osmosis (PRO) or reverse electrodialysis (RED)17 and controllable ion transport properties18. It had also been reported to provide nanoconfined charging dynamic medium to be potentially used in many applications such as molecular sieving with ultrafast speed13 and voltage gating devices19 etc. The unipolar ion transport within 2D nanofluidic material and asymmetric charge distribution could be used to generate osmotic energy21. The asymmetric charge distribution could be introduced by wettability gradient22 as observed in the power generation from GO and reduced GO junctions under moisture23, as well as by induction of oppositely charged bilayer polyelectrolyte film that could generate a peak of 1.38 V at relatively humidity (RH) of 85%24. Osmotic energy originated from such charge gradient and regulated ion transport25 in nanoconfined structures has been observed by examples from nanostructured carbon materials26, graphene single microelectrode27, MoS2 nanopores28, boron nitride nanotube25, nanostructured silicon29, protein nanowires30 to membranes based on MoS231, cellulose32, silk33 and MXene/Kevlar nanofiber composites34 etc. Among above examples, nanofluidic channels with tailored ion transport dynamics could enable high-performance RED. However, their power densities are generally small, ranging from 10 µW cm−3 to 4 mW cm−3 in recent reports35. In addition, fabrication of osmotic power source based on nanofluidic materials typically relies on expensive deposition/lithography techniques or nanoporous templates with sophisticated growth and processing steps. Such fabrication methods usually have better defined geometries, but limited applications due to the cost and sophistication. Jiang et al.36 reviewed progress on enhanced ion regulation in nanofluidic devices for osmotic energy conversion. The essential challenges on their real world applications lie in the small energy and power densities, operation limitations on humidity and temperature, and feasibility of large-scale production.Graphene oxide (GO) as 2D nanofluidic material with negative surface charge from carboxyl and hydroxyl groups etc. has shown special affinity to waterHere, we show a flexible, ultrathin and printable GO-based triboiontronics and osmotic energy power source based on the ion gradient and the fine-tuned interfacial electrochemical reactions. To maximize the ionic power, a modular design osmotic energy power source with current enhancement purely from ion gradient is made from GO aerogels and self-healing ionogels based on room temperature ionic liquids (RTILs). The GO-based osmotic energy device and triboiontronics developed in this paper could operate under harsh environment regardless of low humidity and subzero temperature.15. Graphene nanopores37 were found to preferentially transporting K+ over its counter anions such as Cl- with selectivity ratios over 100 and hydrated K+ diffuses orders magnitude more quickly than most hydrated ions37 within the 2D nanofluidic channels. It was reported that potassium hydroxide (KOH) could partially remove the oxygen-containing groups of GO sheets through a series deoxygenation reaction leaving cations between graphene layers39. FTIR (Fourier-Transform Infrared Spectrometer) characterization and the related empirical structural formula of GO and rGO were shown in Supplementary Fig. + was observed to migrate from rGO to GO when solid-state GO/rGO junction was formed at ambient humidity environment in our previous work40. The electrical conductivity of rGO was measured to be about 0.06 S m−1, falling in the conductivity range of rGO by KOH treatment (0.02–1.55 S m−1)39. In this paper, rGO containing large amount of K+ was chosen as the cation reservoir in the iontronic device. The schematic of the planar osmotic power source and its mechanism is shown in Fig. 41 was used as charge collectors and K+ will transport from rGO through 2D nanofluidic channels of GO to the cathode side of Au electrode under humidity. The scanning electron microscopy (SEM) image  characteristics of the osmotic power sources based on 2D nanofluidic material exhibited strikingly nonlinear effects, which may be due to the asymmetric transport of K+ through the GO and rGO junction (GO/rGO). The open circuit voltages (VOC) and short circuit currents (ISC) could be obtained by reading the intercepts on the current and voltage axes by applying a sweeping voltage from 2 V to −2 V. VOC of the Au/GO/rGO/Au power source was about 1.2 V at room temperature under RH around 70% as shown in Supplementary Fig. 40. RTILs are molten salts with a melting point close to or below room temperature43. Unlike organic electrolytes, RTIL will not evaporate away in the encapsulated power source and it can also significantly accelerate charging dynamics in nanopores44. Different RTILs, 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide (BMIMTFSI), triethylsulfonium bis(trifluoromethylsulfonyl)imide (TESTFSI) and 1-ethyl-3-methylimidazolium tetrafluoroborate (EMIMBF4) were tested, and they have increasing ionic conductivity at room temperature. TESTFSI was finally chosen for the development of anti-freezing osmotic power source since it has a relatively high ionic conductivity of 7.1 mS cm−1 with a low melting point of −35.5 °C45. Addition of RTIL enhanced the current but the VOC of Au/GO/RTIL/rGO/Au kept the same around 1.2 V  aqueous solution was sprayed on top of Au at the cathode side before deposition of GO. The multimeter measurement showed voltage reached around 1.5 V  and without AgNO3 (Au/GO/RTIL/rGO/Au). It was shown that there is no Faradaic current coming from electrolysis of water in a sweep voltage range between −2 V to 2 V  generated from ion gradient and the redox potential (Eredox) from electrochemical reactions. Eredox may come from the reduction of Ag+ on the cathode side between GO and Au interface, which is about 0.4 V as calculated in the Ediff comes from harvesting the Gibbs free energy existing in the ion gradient and it can be estimated by the Nernst equation. Owing to the large salt intake and unimpeded water permeation, highly concentrated solutions that were close to saturation were formed within rGO, maintaining a large concentration gradient and enabling ultrafast ion permeation of hydrated K+ from rGO to GO driven by the gradient through 2D nanofluidic channels. The planar osmotic power source is in a solid-state form and it is impractical to calculation the concentration, however, the VOC of 1.2 V from Au/GO/rGO/Au power source came mainly from Ediff since there were no redox reactions in it. Thus, the theoretical VOC of the Au/AgNO3/GO/rGO/Au power source is calculated to be 1.6 V that matches the measurement of 1.5 V. It also provides a paradigm where the Voc of the osmotic power source could be tuned by tailoring interfacial electrochemical redox reactions. Electrochemical impedance spectrum (EIS) is capable of high precision and is frequently used for the evaluation of heterogeneous charge-transfer parameters. The Nyquist plot , strong ionic rectification-like curve appeared below 0.2 V. The largest ion current from the Au/AgNO3/GO/RTIL/rGO/Au may be due to the combination of ion gradient current from Ediff and the Faradaic current from Eredox. Inset of Supplementary Fig. +/Ag redox potential. It seemed that 2D nanofludic channels enabled the confined electrochemical reactions at the interface between GO and Au charge collector. The image in Supplementary Fig. 3 coating underneath GO on the cathode side, from crystal salts of AgNO3 before discharge reduced to metallic silver particles after multi-cycles of discharge.The current-voltage  in the glovebox . It had been kept in the glovebox for half year, but when it was taken out, the Voc is still around 1.4 V  as the freshly made osmotic power source  tape was chosen as the triboelectric material, which also served as the encapsulation layer. Rectangle copper foils with dimensions of 2 × 2 cm by 50 μm thickness was attached on the PI tape as electrode. The total thickness of the TENG was <200 µm and it could be connected to the osmotic power source to form an ultrathin device that is conformal to human body  to form a self-charging triboiontronic device. Iontronics are effective in modulating electrical properties through the electric double layer (EDL) assisted with ion migration. GO with abundant Kody Fig. . In ordeody Fig. . The vol49 as shown in Fig. + were confirmed by the SEM images and energy dispersive X-Ray spectroscopy (EDS) results . GO aerogels with different GO:LAA ratio was fabricated to investigate the influence of the LAA concentration. As shown in Fig. −1 is the disorder-induced band (D peak) and the peak close to 1580 cm−1 is the graphite band (G peak)39. The area ratio of two peaks (ID/IG) decreases from 2.15 to 1.55 with increasing LAA concentration, meaning the structure disorder of GO aerogels decreases with increasing reduction degree. This is consistent with the morphology characterized by SEM  model50 was used to simulate the osmotic potential from the ion gradient of the 2D nanofluidic material and it also showed that the appropriate pore size for membrane-scale osmotic energy conversion should be around 2 nm, matching our experimental results.To extend the nanoconfinement enhancement in 3D, we then further developed a nanoporous GO aerogel-based osmotic power source with self-healing ionogel electrolytes. Two GO aerogels with different ion concentrations were sandwiched between charge collectors and separated by an ionogel consisting of RTIL and polymer matrix. GO aerogel was fabricated through a self-assembled chemical reduction and freeze-drying processlts Fig. . The eleVOC of 0.679 V and ISC of 402 μA  and 8 times higher than that of the ink-jet printed moisture-enabled power source (2.5 mW cm−3) reported before40. The improved electrochemical performance might come from the large surface area of the 3D aerogel architecture and the superior ionic conductivity of ionogel electrolyte. The optimized LAA concentration and aerogel thickness were used in the following study. To study the effects of the K+ concentration, GO aerogels were soaked in KOH solutions with different concentrations during the preparing process. As shown in Supplementary Fig. VOC of the power sources , which is consistent with our previous results and other reports in power sources utilizing the ion gradient51. This also means that by selecting aerogels with different cation concentrations as building blocks, the voltage output could be customized based on the energy requirement. The directional ion migration needs to be converted to electron transportation to power external circuits. This electrochemical process is usually based on the chemical redox reactions or the physical adsorption of ions on the electrode surface. CV of such devices with and without RTIL in Supplementary Fig. I–V characteristics show that the ISC increases with increasing ionic conductivity while the VOC remains almost the same  test is −72 °C, significantly lower than −19.7 °C for the pure PVDF-HFP polymer  with poly(vinylidene fluoride-co-hexafluoropropylene) (PVDF-HFP) was used here. It is reported that the cations in the ionic liquid and the PVDF-HFP polymer chain in the fluoro-elastomers could interact via ion-dipole interactions, giving rise to self-healing capability−1. Considering the restoration of both stress and strain, the mechanical self-healing efficiency was defined as the proportion of toughness restored relative to the original toughness55. As shown in Supplementary Fig. 53. Compared with the pristine ionogels before being cut, the healed ionogels maintained similar Young’s modulus and tensile stress curves . Such effective self-healing capability might be attributed to the highly reversible ion-dipole interactions of the ionogel53.Such ionogel could be cut and then placed together, exhibiting fast self-healing capability at ambient environment shown in Supplementary Fig. VOC of 0.6 V and the ISC of 440 μA with the maximum volumetric specific power density of 12 mW cm−3. The modular design could enable the construction of custom-built power sources with pre-fabricated components and meet the energy requirements of various integrated electronic systems. Proof of concept of the plug-unplug modular design in the osmotic power source was demonstrated by comparing the output power for the self-healing ionogel before and after being cut. As shown in Supplementary Fig. VOC of 0.62 V and the ISC of 410 μA with its power density still around 11.8 mW cm−3 vs. 12.0 mW cm−3  of various osmotic power sources with different materials were compared in Supplementary Table A modular design concept could be demonstrated Fig. , in whicm−3 Fig.  from theame Fig. . In contame Fig. , while tame Fig. . A repeaces Fig. , providi−3) of the planar osmotic power source is comparable to thin-film lithium batteries and the power density (28 mW cm−3) is as high as supercapacitors. Such printable planar power source could be conveniently integrated into other electric circuit and/or devices. Self-charging triboiontronic device with total thickness less than 200 µm was shown by coupling energy harvesting TENG with ultrathin GO osmotic power source, which was conformal to various irregular surface. Generally speaking, the voltage from the GO osmotic power source mainly comes from K+ concentration gradient (Ediff) between rGO and GO. The output current can be enhanced either by confined electrochemical redox reactions between GO and Au charge collector in the planar power source, or by amplifying ion current through incorporating nanoporous GO aerogels and self-healing ionogels with programmable conductivities in 3D. The osmotic power source based on GO aerogels with nanoconfined structure (pore size ∼2 nm) can convert the chemical potential energy of the ion gradient to electric energy through ion transportation driven purely by the ion gradient. RTIL accelerates the kinetics of ion diffusion in nanoconfined channels and it also imparts the osmotic power source to overcome the operation limitation on humidity and temperature. Benefiting from the antifreeze RTIL ionogels, the power source could operate even at the temperature of −40 °C, bringing it further step towards practical applications. The output performance of such power source could be tailored based on demands of the electronic systems by selecting pre-fabricated building blocks like Lego. An Origami inspired foldable power source was constructed demonstrating the handy assembling process and abundant potential designs for powering electronic devices by ionic power. Modular design power source offers areal power density of 1.3 mW cm−2 at ambient environment making salinity gradient power economically viable. This work advances the fundamental understanding of function of 2D nanofluidic material in iontronics and the iontronics based on GO has distinct advantages such as low cost, safe, ease of scaling up to support high ionic currents and flexibility. By combining 2D nanofluidic material in such ion gradient system, it also opens up abundant potential designs for development of ionic diode/transistor, ionic circuits and photo-induced iontronics etc. The iontronic devices possess the ability to regulate ion flow in direction and magnitude, and they especially could find applications in the development of wearable or implantable iontronic devices or even neuronal-computer interfaces in the future.GO is an ideal candidate to fabricate iontronics due to the surface charge and the 2D nanofluidic channels could be readily formed from GO when restacked. Such nanoscale confinement is very similar to the transport of ions passing through nanometer-sized biological channels accounting for a wide array of physiological processes. The asymmetric charge distribution of GO/rGO and ion transport inside GO lead to the osmotic energy. Remarkably, the osmotic power source reported in this paper yielded ionic currents in the microampere regime, which are orders of magnitude higher than those reported previously. The volumetric specific energy density , H3PO4 (2 mL) and H2SO4 (21 mL) were stirred together for 2 h in an ice bath, and KMnO4 (3 g) was added slowly. The solution was then transferred to a 35 °C water bath and stirred for ca. 0.5 h to form a thick paste. Deionized water (200 ml) and H2O2 (10 mL) were added successively at 95 °C, and the color of solution turned to bright yellow from dark brown. After several centrifugations to wash away soluble salts and incompletely reacted graphite. The brown-yellow clarified liquid collected is the graphene oxide dispersion and the upper liquid was freeze-dried to form GO powder. All related chemicals of H2SO4, KMnO4, H2O2, H3PO4 etc. were purchased from Sigma-Aldrich and were used as received. The GO solution (5 mg mL−1) was prepared by dissolving the GO with deionized water. The rGO solution was prepared by adding 0.1 mol L−1 KOH into the GO solution (5 mg mL−1) with a 1:2 (vol/vol) ratio. KOH, acetone, LAA, BMIMTFSI, TESTFSI and EMIMBF4 etc. were purchased from Sigma-Aldrich. PVDF-HFP was purchased from 3 M Company. Deionized (DI) water, having a resistivity of above 18 MΩ cm−1 was collected from a Mili-Q Biocel system. The target material Au (99.9999%) was purchased from ZhongNuo Advanced Material (Beijing) Technology Co., Ltd.GO was prepared from graphite powders  using a modified Hummers method2 laser cutter system was used to cut the PET film into predesigned patterns as the shadow mask, and then it was pasted on the no patterned PET film with the Kapon both side tape. Au was then deposited on the exposed PET substrate with the aid of the mask via the magnetron sputtering film deposition system (Denton Discovery 635). GO and then rGO solutions were deposited onto PET substrate with sputtered gold strips as charge collectors.Commercial polyethylene terephthalate (PET) film was thoroughly cleaned by abundant acetone, alcohol and deionized water with a bath sonicator. A computer-controlled commercial CO49. GO aerogel was then obtained through a freeze-drying process to remove the excessive water while maintaining the ordered interconnected network with limited sheet restacking or structure collapsing. For the GO + KOH aerogel, GO hydrogels were immersed into different concentrations of KOH aqueous solution  at room temperature for overnight before freeze-drying. To form the ionogel film, PVDF-HFP was dissolved by acetone and mixed uniformly with TESTFSI  at room temperature. After obtained through a casting and drying process at 75 °C for 48 h in a vacuum oven, the ionogel film was prepared.GO aerogel was fabricated through a self-assembled chemical reduction and freeze-drying process. The GO sheets with abundant functional groups could be partially reduced forming a GO hydrogel in a glass vial under a mild condition in the presence of leukocyte ascorbic acid (LAA)The prepared GO aerogels and GO + KOH aerogels were pressed with 10 MPa for 10 min and cut into circular electrodes with the diameter of 16 mm. For mechanism study, GO aerogel electrodes and GO + KOH aerogel electrodes were clamped between two conductive charge collectors on polyethylene glycol terephthalate substrates and separated by the cellulose separator soaked with RTIL electrolyte to avoid direct contact. For foldable power source, firstly the electrodes were put onto the substrate with screen-printed silver patterns. Then the RTIL and polymer mixture were drop-casted on top of the electrodes and subsequently dried in the vacuum oven at 55 °C for 2 days to remove acetone solvent.2 physisorption at 77 K. Raman spectra were collected with the LabRAM HR Evolution (Horiba) with a 532 nm laser. Fourier-transform infrared (FTIR) spectra were collected by Bruker VERTEX80v.The resistivity and the thickness of the aerogels were tested by the four-point probe method and the digimatic indicator , respectively. The DSC measurement were performed using the DSC Q2000 from −90 °C to 30 °C with the speed of 5 °C min–1. For the TENG characterization, A step motor (LinMot E1100) was used to provide the input of mechanical motions. The voltage and current output were recorded by a Keithley electrometer 6514. The tensile experiments were performed by an Instron E3000 instrument. Zeta potential were obtained by using a Beckman coulter instrument (Delsamax Pro).Morphologies of devices were observed by the SEM  with the 5.0 kV accelerating voltage, 10 μA emission current and EDS was used for the element analysis. AFM was performed at room temperature with an Asylum Research MFP-3D in tapping mode with a scanning rate of 0.2 Hz. Dektak XT™ stylus profiler (Brucker) was used to profile the morphology of the planar osmotic power sources and provides information on volume values for the calculation of specific energy and power density. All electrochemical measurement were carried out with the electrochemical workstation (Multi Autolab M204). Electrochemical impedance spectrum (EIS) was measured on an extended range from 1 MHz to 0.01 Hz. Temperature and relative humidity were controlled by an environmental simulation test chamber (Vötsch Technik). The BET measurement was carried out on a MicrotracBEL BELSORP-max instrument by NFurther information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Reporting Summary