Resting-state functional MRI (rs-fMRI) in rodent models have the potential to bridge invasive experiments and observational human studies, increasing our understanding of functional alterations in the brains of patients with depression. A major limitation in current rodent rs-fMRI studies is that there has been no consensus on healthy baseline resting-state networks (RSNs) that are reproducible in rodents. Therefore, the present study aimed to construct reproducible RSNs in a large dataset of healthy rats and then evaluate functional connectivity changes within and between these RSNs following a chronic restraint stress (CRS) model within the same animals.A combined MRI dataset of 109 Sprague Dawley rats at baseline and after two weeks of CRS, collected during four separate experiments conducted by our lab in 2019 and 2020, was re-analysed. The mICA and gRAICAR toolbox were first applied to detect optimal and reproducible ICA components and then a hierarchical clustering algorithm (FSLNets) was applied to construct reproducible RSNs. Ridge-regularized partial correlation (FSLNets) was used to evaluate the changes in the direct connection between and within identified networks in the same animals following CRS.Four large-scale networks in anesthetised rats were identified: the DMN-like, spatial attention-limbic, corpus striatum, and autonomic network, which are homologous across species. CRS decreased the anticorrelation between DMN-like and autonomic network. CRS decreased the correlation between amygdala and a functional complex  in the right hemisphere within the corpus striatum network. However, a high individual variability in the functional connectivity before and after CRS within RSNs was observed.The functional connectivity changes detected in rodents following CRS differ from reported functional connectivity alterations in patients with depression. A simple interpretation of this difference is that the rodent response to CRS does not reflect the complexity of depression as it is experienced by humans. Nonetheless, the high inter-subject variability of functional connectivity within networks suggests that rats demonstrate different neural phenotypes, like humans. Therefore, future efforts in classifying neural phenotypes in rodents might improve the sensitivity and translational impact of models used to address aetiology and treatment of psychiatric conditions including depression. Accumulating studies have suggested that these core symptoms of depression are associated with disrupted affective and cognitive brain networks, revealed by magnetic resonance imaging (MRI) and computational approaches that can map the brain connectome non-invasively . RestingRSNs are not unique to humans, some homologous RSNs have also been observed in healthy rodents using rs-fMRI . For exaTo date, limited rs-fMRI studies in animal models of depression have investigated functional alterations within and between RSNs in response to interventions that induce stress and anxiety . MajoritA major limitation in current rodent rs-fMRI studies is that there has been no consensus on healthy baseline RSNs that are reproducible in rodents  regardle2.2.1.All experimental procedures adhered to the ethics guideline of the University of Western Australia Animal Ethics Committee (RA/3/100/1640) and the National Health and Medical Research Council’s Australian code for the care and use of animals for scientific purposes. All investigators had obtained the Permission to Work with Animals and were trained by the UWA Program in Animal Welfare, Ethics, and Science.2.2.2.2.1.N = 12 from N = 56 from N = 41 unpublished) were sourced from the Animal Resources Centre . All rats were housed in pairs under a standard 12-h light–dark cycle with ad libitum food and water, in a temperature-controlled and spacious laboratory room located at UWA’s Animal Care Unit, M block building . All animals were habituated to the new environment for one week after their arrival and prior to experiments.The MRI data analysed in the present study was a combined dataset of rodent cohorts from previous experiments conducted by our lab in 2019 and 2020. Briefly, 109 male Sprague—Dawley rats , served as controls: they did not undergo CRS but received MRI imaging twice at the same interval applied to the intervention group.96 rats underwent CRS for 2.5 h per day for 13 consecutive days. These animals’ MRI scans were conducted at baseline (one or two days before the first CRS procedure) and post-CRS . The remaining animals  was identified and the average time elapsed was 32 min (SD = 4). After the imaging session, animals were administered a 0.15 mg/kg injection of atipamezole to antagonize medetomidine. Periodical monitoring for adverse events after MRI scan was performed in the following 24 h.Animals were transferred to the National Imaging Facility located in the Harry Perkins Institute of Medical Research  for MRI scan on the assigned day. The rat was weighed and then pre-anaesthetised in an induction box with 4% isoflurane in medical air (2 L/min). Once fully anaesthetised, the animal was transferred to a heated imaging bed and anaesthesia was initially administered through a nose cone with 4% isoflurane in medical air (1 L/min). The animal’s vital status was monitored using a PC-SAM Small Animal Monitor . Once the respiratory rate dropped to 55–60 breaths/min, isoflurane concentration was adjusted to 2% in medical air (1 L/min). After the animal was stabilized on 2% isoflurane for at least 2 min, medetomidine was delivered subcutaneously, with an initial 0.05–0.1 mg/kg bolus injection and continuous 0.15 mg/kg/h infusion. Meanwhile, the isoflurane concentration was gradually reduced to 0.5–0.75% based on the animal’s respiratory rate . The com2.2.4.Rats were scanned using a 9.4 T Bruker Biospec 94/30 pre-clinical MRI scanner  with a BGA-12SHP imaging gradient system, a 72 mm or 86 mm volume resonator transmit coil (depending on hardware availability), a rat brain surface quadrature receive coil, and Avance III console. ParaVision 6.0.1 software of the Bruker controlled scanning sequences and operation for structural MRI and rs-fMRI acquisition . High-re2.3.The workflow was remotely operated on the MASSIVE’s super-computing desktop  and most2.3.1.The pre-processing for each data package was batch processed as follows: (1). Extract DICOM  of both 2.3.2.3) was used to better match the voxel size of functional data and all subsequent processing was performed in this atlas space. Following registration, pre-statistics processing was applied with motion correction . To elaborate, upscaled functional brain images were first registered to their corresponding upscaled anatomical brain images and normalized to a Sprague–Dawley rat brain atlas, which was down sampled by a factor of eight from the Waxholm Space atlas  comprising cerebral cortex, hippocampus, amygdala, thalamus, basil ganglia, claustrum, and hypothalamus and colliculi. Most of these structures are considered components of RSNs in humans and animals, while the colliculi and piriform cortex are typically found in rodents as additional components .2.3.3.1.The ROI atlas mask was generated as follows: (1). Extract high-resolution masks for substructures of the ROI based on their label ID from the Waxholm Space atlas  and the ROI atlas mask were imported to the toolbox. The rationale for excluding rs-MRI data acquired at post-CRS is to avoid CRS-related resting-state alteration in identifying ICA template and to increase the sensitivity of further data processing in detecting group differences following CRS (N = 54 samples/group) and a cross-correlation matrix between components’ spatial maps was calculated using Pearson’s correlation. Hungarian sorting algorithm (N = 100) at the resultant dimensionality and Gaussian kernel were ready for the following processing.An optimal group-ICA dimensionality was estimated using the mICA toolbox, which estimates correlation values using random split-half sampling or test–retest analyses for a range of dimensionalities . Brieflywing CRS . Random lgorithm  was appllgorithm . The optlgorithm . Resulta2.3.3.3.Ranking and averaging independent component analysis by reproducibility (RAICAR) ranks and selects components based on the reproducibility over repeated ICA realizations, in which a cross-realization correlation matrix is constructed to align components . Each alSpatial maps of all resultant nodes were then merged using FSL/fslmerge command and parcellated using mixture modelling approach based on the thresholded z-transformed results of each node . A numer2.3.4.N = 109; de-noised and normalized functional images, spatially smoothed at resultant Gaussian kernel in section 2.3.3.2) to derive subject-specific time series for all nodes using FSL/dual regression (Z-score of brain structure covered by each node within each network were extracted and summarized in The group-ICA template for the ROI was mapped onto baseline data (gression . Images gression . These mgression . Additio2.3.5.N = 13) and CRS (N = 96) groups were compared to each other directly at the second time point. But the very different sample size  resulted in low effect sizes and thus direct comparison approach was not deemed suitable (data not shown).To compare the differences in network connectivity for the CRS group between baseline and post-CRS, as well as for the control group between baseline and the second scan session, statistical analysis was conducted in parallel. Due to the difference in the sample size , the CRS group was not compared directly with the control group. However, by analysing control and CRS group in parallel, this longitudinal (repeated measures) approach increases the statistical power as each animal was compared to itself, meaning participant/animal variables are controlled at both time points. It also adds biological relevance because it allows investigation of individual susceptibility and response, information which is relevant to future precision medicine approaches (see discussion). In an exploratory statistical analysis, the control  to derive subject-specific time series for all networks using FSL/dual regression (p < 0.05) were extracted from MATLAB and imported to RStudio (version: 2021.09.2 + 382) to estimate the effect size using ‘dabestr’ . Results showing significant differences (p < 0.05) were reported with network images, estimation plots, mean ± SD, permutation p values, and Cohen’s d with 95% CI. Raw values of significant edges between networks can be found in For the intervention group, the classified networks were merged using FSL/fslmerge and then mapped onto all data acquired at baseline and post-CRS (gression . These tgression  and ridggression . The edgdabestr’ . For the2.3.5.2.p < 0.05) within each network were reported with node images, estimation plots, mean ± SD, permutation p values, Cohen’s d with 95% CI for the intervention group, and Hedges’ g with 95% CI for the control group. Raw values of significant edges within each network can be found in For the intervention and control group, the same methods described in section 2.3.5.1 were applied to each resultant network template from section 2.3.4 to detect if there were differences in the direct connection between nodes within each network at baseline and post-CRS, respectively. Results showing significant differences  of abovementioned significant edges between and within networks were grouped into three categories at baseline and post-CRS: positive (above 0.2), none (between −0.2 and 0.2), negative (below −0.2) partial correlation . Changes3.3.1.There was no failure of convergence in the group-ICA across all dimensionality levels under four different Gaussian kernels. As shown in 3.2.The resultant 50 components  were aligned and ranked over 100 group-ICA spatial outputs with gRAICAR. Node 1 had the highest similarity and node 50 had the lowest similarity . The gRA3.3.Z-score are summarized in Network modelling based on hierarchical clustering detected four common patterns of functional connectivity in the rat brain. The data-driven approach merged the 50 nodes into four major clusters . Major s3.3.1.The network was predominantly cortical. Most of the DMN components  demonstrated bilateral symmetry in the resting-state activity. The spatial extent of resting-state activity in the cornu ammonis/CA, parietal association and visual cortex was larger in the left hemisphere, whereas the resting-state activity of temporal association, auditory, and perirhinal cortex was completely absent in the right hemisphere. Moreover, the resting-state activity of sensorimotor structures were mostly symmetrical in the network.3.3.2.The homologous brain regions presenting symmetrical resting-state activity were identified as follows: inferior colliculi, superior colliculi, posterior retrosplenial cortex, dorsal hippocampus, and thalamus. The spatial extent of resting-state activity of the visual, auditory, temporal association, postrhinal, and medial entorhinal cortex was larger in the right than left hemisphere, whilst parietal association, perirhinal, and primary somatosensory (trunk representation) cortex only had unilateral resting-state activity in the right hemisphere.3.3.3.Brain substructures demonstrating bilateral symmetry in the resting-state activity included corpus striatum , claustrum, insular, orbital, motor, secondary somatosensory cortex. The spatial extent of resting-state activity of the endopiriform nucleus, perirhinal, piriform cortex was larger in the right than left hemisphere, whereas the resting-state activity of amygdala, CA, lateral entorhinal cortex was totally absent in the left hemisphere.3.3.4.The network was predominantly subcortical. Bilateral symmetry in the resting-state activity was observed in the following subcortical structures: hypothalamus, ventral hippocampus, and substantia nigra. The spatial extent of resting-state activity of amygdala and adjacent cortical regions  was larger in the left than right hemisphere, but the endopiriform nucleus and piriform cortex only demonstrated unilateral resting-state activity in the left hemisphere.3.4.p value: 0.044; Cohen’s d: −0.32; 95% CI: −0.54 ~ −0.07). In contrast, the connection strength between the DMN-like network (network 1) and autonomic network (network 4) increased significantly with a medium effect size following CRS . In other words, the anticorrelation between DMN-like network and autonomic network decreased significantly following CRS.As shown in 3.5.p value: 0.040; Cohen’s d: −0.49; 95% CI: −0.79 ~ −0.20). Node 37 mainly consisted of bilateral cingulate cortex (posterior part) and node 46 mainly comprised frontal association, orbital, and secondary motor cortex in the left hemisphere. Moreover, the edge strength between node 4 and 6 . Node 4 was a bilateral functional complex comprising infralimbic, prelimbic, and orbital cortex. Node 6 was a unilateral functional complex in the left hemisphere, mainly including auditory, temporal association, and perirhinal cortex.Within the DMN-like network (network 1), a pair of nodes  showed a 4 and 6  increasep value: 0.0028; Cohen’s d: -0.62; 95% CI: −0.92 ~ −0.31). Node 11 was a functional complex in the left hemisphere, mainly including postrhinal, visual, auditory, temporal association, and medial entorhinal cortex. Node 42 consisted of thalamus with adjacent dentate gyrus and CA3 in the right hemisphere.Within the spatial attention-limbic network (network 2), another pair of nodes  demonstrp value: 0.0028; Cohen’s d: −0.62; 95% CI: −0.93 ~ −0.32). Node 21 mainly consisted of right amygdala with adjacent piriform cortex. Node 26 mostly comprised nucleus accumbens and ventral pallidum, with their adjacent piriform cortex in the right hemisphere. In other words, the correlation between amygdala and a functional complex  in the right hemisphere decreased significantly following CRS.Similarly, within the corpus striatum network (network 3), another pair of nodes  presenteN = 13), no changes were detected between baseline and the second scan session , except that a pair of nodes (node 5 and 26) presented a significant increase in the direct connection strength at the second scan session  with strong effect size . Node 5 mainly consisted of right insular cortex with adjacent perirhinal and secondary somatosensory cortex.For the control group (3.6.There was a high inter-subject variability in the changes of edge strength within the DMN-like network (network 1) and spatial attention-limbic network (network 2). As shown in 4.The main aims of the present study were to use a large dataset to first identify reproducible RSNs in healthy Sprague Dawley rats and then evaluate CRS effects on the functional connectivity in these RSNs within the same animals. The key findings revealed four large-scale networks in the anesthetised rats, including a DMN-like network, a spatial attention-limbic network, a corpus striatum network, and an autonomic network. At a group level, CRS decreased the anticorrelation between the DMN-like network and the autonomic network. In addition, CRS decreased the correlation between the amygdala and a functional complex  in the right hemisphere within the corpus striatum network. However, high individual variability in the functional connectivity before and after CRS within RSNs were observed.4.1.r > 0.70) at a dimensionality of 10 under four Gaussian kernels may indicate that the cross-correlation analysis by mICA toolbox  identified here based on the hierarchical analysis are consistent with previously published rodent studies using seed-based approaches or group ICA analysis with a low dimensionality from 10 to 30 . Hippoca4.2.2.The components of this network are similar to what has been observed in awake rodents from other studies . The col4.2.3.The network is one of the basal ganglia networks observed in previous rodent and human studies . The str4.2.4.The networks are commonly observed in awake and anesthetized rodents as an ICA component . The key4.3.The decreased anticorrelation between the whole DMN-like network and subcortical autonomic network in the present study has not previously been reported. Although the mechanism underlying the anticorrelation between the DMN-like and autonomic network is unknown, a meta-analysis study has suggested that these two networks are both associated with autonomic regulation in humans . MoreoveThis is also the first time that a decreased correlation between the amygdala and a functional complex  in the right hemisphere within the corpus striatum network is reported in an animal model. These structures are parts of the reward system, the nucleus accumbens receives glutamatergic inputs from the basolateral amygdala and project GABAergic signals to the ventral pallidum . The nucIn contrast with the present results, some human neuroimaging studies have reported that depression is commonly associated with hyperconnectivity within the DMN, as well as aberrant cross-network interaction among DMN, central executive and salience networks . A simpl4.4.Additional limitations are worth mentioning. First, only male late adolescent/young adult Sprague–Dawley rats were used in the analysis due to data availability. This means that the brains of these animals were still developing during the CRS intervention and may have confounded interpretation of the network changes . HoweverMoreover, the level of anaesthesia is a key consideration in MRI studies and the present study applied combined use of isoflurane and medetomidine at doses, which have been found to provide stable anaesthesia with the least impact on brain activity . At the Z-scores are low. Finally, only hierarchical clustering analysis was applied to classify the networks. Future efforts in systematically comparing the differences on the RSN classification with other algorithms, such as the fuzzy-c-means clustering algorithm  to address the uncertainty resulting from regions where multiple nodes overlap and lgorithm  and deeplgorithm , are nec5.The present study is the first to construct reproducible RSNs in anaesthetized rats through identifying optimal and reproducible functional components, and in turn evaluate functional connectivity changes in the RSNs following a CRS model within the same animals. The key findings revealed four large-scale networks that are homologous across species, decreased anticorrelation between DMN-like and autonomic network, decreased correlation between amygdala and a functional complex  in the right hemisphere within the corpus striatum network. Moreover, high inter-subject variability of functional connectivity is observed within networks, indicating rats may demonstrate different neural phenotypes as humans. Therefore, future efforts in classifying neural phenotypes in rodents might improve the sensitivity and translational impact of models used to address aetiology and treatment of psychiatric conditions including depression.The original contributions presented in the study are included in the article/The animal study was reviewed and approved by University of Western Australia Animal Ethics Committee (RA/3/100/1640).TD and JR conceived and designed the study. LH and SB collected a subset of data that is not previously published. TD carried out the analysis with guidance from BS and TR, and wrote the first draft of the manuscript. All authors contributed to the article and approved the submitted version.JR is supported by a Senior Fellowship from MSWA and the Perron Institute for Neurological and Translational Science. TD is supported by the Australian Government International Research Training Program scholarship, and Byron Kakulas Prestige scholarship.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


A Chinese herbal formula, Tiaopi Xiezhuo decoction (TXD), is developed from a classical Chinese prescription Sanhuang Xiexin decoction.To investigate the regulatory effect of TXD on gut dysbiosis, as a treatment of constipation in patients with peritoneal dialysis (PD).The chemical content of TXD was assessed by high-performance liquid chromatography. A total of 29 PD patients were enrolled and treated with TXD orally (3 g crude drug/each/twice/day) for 3 months. Blood and faecal samples were collected at the beginning and end, to determine the changes in biochemical characteristics and gut microbial composition. The stool conditions were asked to be scored. Additional 30 healthy individuals were recruited as a control for the analysis of gut microbiota.p < 0.01), increased 2.6-fold sloppy stools (p < 0.05) and eliminated hard stool completely (p < 0.01). The analysis of gut microbiota showed that, compared to the healthy group, the microbial richness was reduced in PD patients. After a 3-month TXD treatment, this reduced richness was raised, and Paraprevotella clara, Lachnospiraceae bacterium 2-146FA, Phascolarctobaterium succinatutens, Lachnospiraceae bacterium 2-1-58FAA, Fusobacterium mortiferum, and Prevotella copri were accumulated in the intestinal flora. Furthermore, the bacterial species enriched by TXD correlated with the improvement of constipation.Although having no significant effects on serum biochemical characteristics, 3-month TXD intervention improved constipation in PD patients: decreased 80% abdominal distention (TXD treatment may improve constipation by modulating gut dysbiosis in PD patients. These findings provide data to support the further application of TXD in the adjuvant treatment of PD. Peritoneal dialysis (PD) is one of the three main alternative therapies for end-stage kidney disease (ESKD). PD uses the patient’s peritoneal tissue to exchange water, creatinine, urea, electrolytes, glucose, and other small and medium-sized toxins between the capillaries on either side of the peritoneum and dialysate , Coptis chinensis Franch (Ranunculaceae), and Zingiber officinale Roscoe (Zingiberaceae) at a ratio of 1:1:1. Among them, R. palmatum is a traditional herbal medicine for the treatment of constipation. Our clinical data showed that TXD significantly improved gastrointestinal symptoms in PD patients , a Chinese herbal formula, is developed from the historical Sanhuang Xiexin decoction which was first described in the R. palmatum (1 g/bag), C. chinensis (0.5 g/bag), and Z. officinale (0.5 g/bag) at one bag of each for a time, which were produced by Jiangyin Tianjiang Pharmaceutical Co., Ltd. According to the manufacturer’s protocols, the roots of each herb have been decocted, filtered, concentrated and dried, and made into granules, respectively; each bag of granules is equivalent to 3 g of a crude drug; qualities of granules, such as appearance, determination of content, size of granule, solubility, hygroscopicity, heavy metals, toxic elements, pesticide residues, and microbial limit, were controlled rigorously according to the 2015 Chinese pharmacopoeia.Tiaopi Xiezhuo decoction (TXD) is made by commercial granules of The chemical content of TXD solution was assessed by high-performance liquid chromatography (HPLC) using a COSMOSIL 5C18-MS-II column  on an Agilent 1290 Infinity II LC system . The granules of TXD were dissolved in 50 mL of deionized water. After being filtered through a 0.45 µm membrane, 5 μL of TXD solution was carefully transferred to sample vials for HPLC analysis. The mobile phase consisted of acetonitrile  as solvent A and 0.2% formic acid  in water as solvent B. Then the elution was performed in mobile phase gradient (A:B) of 2:98, 15:85, 30:70 and 98:2 at a rate of 1 mL/min with UV detection at 280 nm. Standard epiberberine or berberine  were prepared in methanol at a concentration of 1 mg/mL and eluted in the mobile phase.We recruited 29 PD patients and 30 healthy individuals for this study. Among them, 29 randomized PD patients aged 18–80 years old, who had received stable PD for longer than 2 months, were enrolled on the peritoneal dialysis clinic of Guangdong Provincial Hospital of Traditional Chinese Medicine between December 2017 to December 2018; 30 healthy individuals were recruited from the families of PD patients, 18–80 years old, who have no chronic kidney diseases, cardiovascular diseases or cancers. We excluded subjects who showed infection within 1 month, had diarrhoea within 2 weeks and used hormones, antibiotics, or intestinal flora-regulatory drugs in the previous 3 months. We also excluded those with inflammatory bowel disease, intestinal malignancies, and tuberculosis. All the participants completed the ‘Information Collection Form’ upon enrollment in the study, stated that they were willing to cooperate with this research, and signed an informed consent form. The protocol was approved by the ethics committee of the Guangdong Provincial Hospital of Traditional Chinese Medicine (B2017-052.2-02), and the project has been registered at the China Clinical Trial Registration Center (ChiCTR-INR-17013624). The trial was performed in accordance with Good Clinical Practice guidelines. The clinical trial flow diagram is shown in As shown in At the enrollment and follow-up visits (M0-M3), all 29 PD patients were asked to fill out forms ranking their stool experience, characterized as abdominal distention, sloppy stool, and hard stool, and provide a 0–4 score indicating light to heavy . The operational taxonomic units and α- or β-diversity indices were calculated based on the OTUs data using QIIME (version 1.8.0). Principal coordinates analysis (PCoA) was used to compare the community composition of gut bacteria between groups. Linear discriminant analysis Effect Size (LEfSe) was used to identify the specific species with the most significant differences. The HUMAnN2 method was used to directly compare metagenomics to the metabolic pathways of response genes. Metabolic pathways were mainly interpreted by referring to the MetaCYC database.p < 0.05.Statistical analyses were performed using SPSS 18.0 statistical software . Data are presented as mean value ± standard error for normally distributed variables, or as medians for non-normally distributed variables. Spearman rank correlation was determined, using GraphPad Prism software , to analyze the relationship between the enriched species and clinical data. Statistical significance was set at R. palmatum , increased 2.6-fold sloppy stools (p < 0.05) and eliminated hard stool completely  and diminished beneficial bacteria (Bacteroides and Alistipes); the analysis of the biochemical metabolic functions of intestinal flora showed that their metabolic pathways lead to the disordered metabolism of creatinine and arginine, as well as excessive toxin synthesis (E. coli is closely related to peritonitis in patients. A study reports that E. coli is the main cause of Gram-negative peritonitis (59.2%), which is often associated with a high probability of mortality and technique failure , which are involved in gut barrier integrity maintenance, glucose and lipid metabolism, and immunity regulations , enriches beneficial bacteria including Lactobacilli and Bifidobacteria, and elevates SCFA production in the colon (Habtemariam TXD is developed from the historical Sanhuang Xiexin Decoction, and consists of analysis , there aPrevotella is a common spore-free Gram-negative anaerobic bacterium. It is widely found in the oral cavity, intestinal tract, female genital tract, and other parts of the healthy human body, and constitutes the normal flora in these areas (Ley Prevotella is more common in populations that consume a plant-rich diet, suggesting that it is a beneficial microbe (Wu GD et al. Prevotella can use xylan, xylose, and carboxymethylcellulose to produce high levels of short-chain fatty acids (Flint et al. P. copri was found to improve glucose tolerance in a mouse model (Kovatcheva-Datchary et al. Prevotella has also been reported to be associated with immune diseases, especially rheumatoid arthritis, female genital tract inflammation, and oral infection (Kim and Kim P. copri was enriched by TXD and positively correlated with the improvement of constipation in PD patients, but we also found it was positively correlated with the serum levels of phosphate (Prevotella in CKD.Among the species enriched by TXD, reas Ley . As membhosphate , which aE. coli and enriched multiple beneficial species in patients with PD. It is important to note that intestinal dysbiosis, especially E. coli, is associated with an increased risk of peritonitis (Kosmadakis et al. Our data demonstrated that the 3-month intervention of TXD decreased the abundance of Clostridium and Bifidobacterium species (Zoppi et al. Bifidobacterium, Lactobacillus, Bacteroides, and Clostridium species, and increased levels of Enterobacteriaceae were observed in adult constipated patients (Khalif et al. Prevotella, and an increased level of several species of Firmicutes, including Lactobacillus (Zhu et al. P. copri, F. mortiferum, Lachnospiraceae, V. parvula, and P. clara. Most of these bacteria are reported to produce SCFA. Whether the beneficial effect of TXD on constipation is associated with SCFA levels in the colon requires further exploration in patients with PD.Clinical data have shown that constipation is one of the most common gastrointestinal disorders among patients with CKD (Kosmadakis et al. This study is an exploratory work to investigate the beneficial effects of TXD on patients with PD. Our data suggest that TXD treatment may improve constipation by modulating gut microbiota. These findings provide data to support the further application of TXD in the adjuvant treatment of PD.


The role of inflammation and the correlation between inflammatory markers and type 2 diabetes mellitus (T2DM) and chronic kidney disease (CKD) have been studied. In clinical work, a large number of T2DM patients complicated with CKD, but the cause of CKD was not clear. Our study aimed to evaluate the relationship between monocyte-to-lymphocyte ratio (MLR) and mortality in T2DM patients with CKD. The data from Medical Information Mart for Intensive Care III was analyzed. The primary outcome was 90-day all-cause mortality; the secondary outcomes were the length of ICU stay, hospital mortality and 30-day all-cause mortality. Cox regression was used to evaluate the association between MLR and 90-day mortality. We performed subgroup analyses to determine the consistency of this association, and used Kaplan–Meier survival curve to analysis the survival of different levels of MLR. A total of 1830 patients were included in study retrospectively. The length of ICU stay, 30-day all-cause mortality, and 90-day all-cause mortality in the MLR > 0.71 group were significantly higher than those in the MLR < 0.28 and 0.28 ≤ MLR ≤ 0.71 group. In Cox regression analysis, high MLR level was significantly associated with increased greater risk of 90-day all-cause mortality. The adjusted HR (95%CIs) for the model 1, model 2, and model 3 were 2.429 (1.905–3.098), 2.070 (1.619–2.647), and 1.898 (1.478–2.437), respectively. Subgroup analyses also showed the consistency of association between MLR and 90-day all-cause mortality. The Kaplan–Meier survival curve analysis revealed that MLR > 0.71 had worst prognosis. In T2DM patients with CKD in the intensive care unit, high MLR was significantly associated with increased risk 90-day all-cause mortality. Diabetes mellitus is the main cause of CKD and the main risk factor for progression from CKD to end stage renal disease2. A study showed that type 2 diabetes patients with CKD reduced life expectancy by 16 years than that of patients without CKD, and resulted in higher medical cost burden and mortality4. In patients with type 2 diabetes mellitus (T2DM) and CKD, the body is in an inflammatory state, and monocyte infiltration is stimulated by intermediate products such as advanced glycation end-products and immune complexes to aggravate cell damage, thus accelerating disease deterioration6.A study predicts that the global population of diabetes will increase to 783 million by 2045. The study found that the prevalence of chronic kidney disease (CKD) among diabetic patients in the United States during 2015–2020 was still high (64–81.6/1000), and with the significant increase of the prevalence of diabetes, the prevalence of CKD will be further increased8. Monocyte to lymphocyte ratio (MLR) can reflect the degree of inflammation better than only high monocytes and low lymphocytes. High MLR was found to be significantly associated with increased risk of adverse cardiovascular outcomes in patients with atherosclerosis and could be used as a prognostic indicator of the degree of coronary artery stenosis9. Further, another study demonstrated that MLR was significantly associated with increased risk of the incidence of acute kidney injury in intensive care unit (ICU) patients and may become an effective biomarker10. Besides, a study showed that high MLR was an independent risk factor for stroke associated pneumonia and had predictive value for the severity of pneumonia in stroke associated pneumonia patients11.Inflammation usually causes monocytes increased and lymphocytes decreased. Studies found that high monocyte and low lymphocyte values were positively associated with increased cardiovascular disease and mortalityIn clinical work, a large number of T2DM patients complicated with CKD, but the cause of CKD was not clear. Patients were complicated with more underlying diseases in ICU, such as T2DM, CKD, hypertension and others, but we often ignored these problems in treatment, because there was lack of clinical prognostic research on T2DM patients complicated with CKD. The body of patients with T2DM and CKD was in a state of micro inflammation. As a new inflammatory indicator, MLR had also been confirmed to be related to the prognosis of ICU patients. Therefore, in our study, we explored the relationship between MLR and 90-day all-cause mortality of T2DM patients with CKD in ICU. We hope to provide doctors with reliable data related to the prognosis of T2DM patients with CKD.12. First, we selected patients according to the following inclusion and exclusion criteria. Second, we extracted demographic information, clinical laboratory data and related scoring information from the database. According to the interquartile ranges (IQRs) of MLR value, we divided into three groups: MLR < 0.28, 0.28 ≤ MLR ≤ 0.71 and MLR > 0.71. Then, we used Cox regression to develop a prediction model for the 90-day mortality. And subgroup and Kaplan–Meier analysis were used to evaluate the differences in different MLR levels in 90 day mortality of patients.We obtained data from the Medical Information Mart for Intensive Care III (MIMIC-III) that contained information about 50,000 patients, respectively2 for 3 months or more13. (4) Missing value of lymphocyte and monocyte; (5) less than 48 h in ICU; (6) missing data for more than 5% of patients. Finally, 1830 patients were included in this study  patients admitted to the ICU for the first time; (2) ICD code is T2DM. Exclusion criteria: (1) less than 18 years of age; (2) non-type 2 diabetes mellitus; (3) without CKD, CKD was diagnosed that glomerular filtration rate (GFR) below 60 mL/min/1.73 m patientsWe extracted the variables from the database with structured query language in PostgreSQL, including age, gender, weight, coronary artery disease (CAD), chronic heart failure (CHF), hypertension, CKD stage, sequential organ failure assessment (SOFA), hemoglobin (HGB), platelet, monocytes, lymphocytes, potassium, sodium, phosphate, creatinine, blood urea nitrogen (Bun), albumin (ALB), alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate transaminase (AST), glucose, Prothrombin time (PT), partial thromboplastin time (PTT) and estimate-GFR (eGFR). The formula of GFR estimation wasThe informed consent was waived by the Institutional Review Boards of Beth Israel Deaconess Medical Center  and the Massachusetts Institute of Technology . The patient’s information has been standardized and the project did not affect clinical care, so requirement for individual patient consent was waived.Continuous variables were expressed as mean ± standard deviations or IQRs, and classification variables were expressed as totals and percentage (%). The chi-square test was utilized to compare classified variables between groups. The Wilcoxon rank-sum test was used for non-normally distributed continuous variables. The student t-test and analysis of variance were used for normally distributed continuous variables. Simple and multivariable Cox regressions were used to identify predictors of 90-day all-cause mortality, which expressed as the hazard ratio (HR) and 95% confidence interval (CI). In order to reduce the impact of confounding factors, we constructed three Cox regression models by including covariates with p values < 0.05 in the simple Cox analyses or for importance of clinical concern. We brought the MLR into the cox model in the form of every 0.1 increase in MLR. Kaplan–Meier survival analysis was used to determine the difference MLR level in 90-day all-cause mortality between the three groups. We performed stratification analyses to assess the association of MLR with 90-day all-cause mortality across various subgroups classified by age, gender, hypertension, CHF, CAD, SOFA score, weight, CKD stage, phosphate, Bun, ALB, ALT, ALP and PTT. p < 0.05 was considered statistically significant. The statistical analyses were conducted by using the Stata software version 16.0 .According to the MLR, 1830 patients were enrolled and were divided into the three groups: 454 patients were in the low-MLR group (MLR < 0.28), 919 patients were in the mid-MLR group (0.28 ≤ MLR ≤ 0.71), and 457 patients were in the high-MLR group (MLR > 0.71). Patients with higher MLR were trend to be male, higher weight and CKD 4 stage; Moreover, these patients had higher SOFA score, potassium, phosphate, creatinine, Bun, ALP and PT; They also had lower sodium and eGFR    Table . Adjust We performed subgroup analyses to determine the consistency of association between MLR and risk of 90-day all-cause mortality Table . We founThe patients were divided into three groups based on MLR level. The Kaplan–Meier survival curve analysis revealed that MLR > 0.71 had worst prognosis. Patients in higher MLR group had significantly higher 90-day all-cause mortality than low and mid-MLR groups  .MLR had been used as a biomarker of inflammation in various studies due to its ease of obtaining from blood parameters29. And a study also showed that higher MLR is associated with increased risks of both CVD events and infectious disease hospitalization in dialysis patients30. Similarly, in our study, we also found that the higher MLR group had higher length of stay in the ICU, hospital mortality, 30-day and 90-day all-cause mortality. Furthermore, the Kaplan–Meier survival curve analysis revealed that MLR > 0.71 had worst prognosis. After adjusting for confounding variables, higher MLR was also found to be a predictor of increased atherosclerosis in patients with diabetes31. Similarly, we controlled for confounding variables by multivariable Cox regression analysis and also found that high MLR was an independent risk factor for 90-day mortality. In addition, subgroup analysis also showed that high MLR was an effective predictor of 90-day mortality in T2DM patients with CKD under various specific conditions. MLR is superior to other conventional markers because it is cheap, effective, and easy to obtain. Meanwhile, MLR is more stable and conducive to clinical application compared with a single indicator.A multicenter retrospective cohort study showed that MLR > 0.45 was significantly associated with HR for CVD mortality of 1.45 at the commencement of peritoneal dialysisIn our study, several limitations were observed as follows: First, A single-centric retrospective study had the possibility of selection bias. Second, some unrecorded clinical information, such as the condition before entered to the ICU, may affect the outcome. Third, there were uncontrollable confounding factors affecting monocyte and lymphocyte counts, such as the use of drug and unspecified comorbidities. Finally, the underlying mechanism between MLR and prognosis could not be determined. Therefore, a large multicenter prospective study should be designed to confirm the above results and further study the mechanism.In this study, we demonstrated that higher MLR was significantly associated with an increased risk of 90-day all-cause mortality in T2DM patients with CKD in ICU. MLR could be served as a predictive and effective marker due to its inexpensive and reliability.


The aim of this meta-analysis is to evaluate the impact of light at night (LAN) exposure on the risk of breast cancer across varying factors.We conducted a systematic search of literature up to July 15, 2023, including PubMed, Cochrane Library, and Embase databases, using keywords related to breast cancer and LAN exposure. Cohort study and case–control study literature on night light exposure and breast cancer risk were included. Statistical analyses were performed using Stata software version 17.0. To address heterogeneity among different studies, we employed a random-effects model for analysis and assessed publication bias using funnel plots and Egger’s test.2 = 31.3%, p < 0.001). In the subgroup analysis, the results of the analysis for study types  and the results for light exposure types  are presented. In the analysis conducted for continents, the highest breast cancer risk was observed in the Asian population  and in the analysis of estrogen receptor status . We also conducted an analysis on menopausal status and various lifestyles but did not find any statistically significant findings.We included 13 case–control and 8 cohort studies with 734,372 participants worldwide. In the Newcastle-Ottawa Scale (NOS) assessments, the average score was 7.43 (ranging from 5 to 9). The overall meta-analysis demonstrated a significant association between exposure to LAN and risk of breast cancer (RR = 1.12; 95% CI: 1.06–1.17; IOur study demonstrates that LAN exposure is associated with an increased risk of breast cancer, particularly in the Asian population. Among the existing hypotheses, the idea that LAN exposure leads to a decrease in melatonin is widely accepted. However, until the mechanism of this effect is clearly elucidated, it is not recommended to take melatonin supplements for breast cancer prevention without medical advice. We hope to conduct more high-quality research, especially concerning the investigation of other environmental confounding factors, to further advance this field. The incidence and mortality of breast cancer have increased among women worldwide , 2. In 2Breast cancer, as a highly heterogeneous and multifactorial disease , has beeLight pollution is an emerging environmental issue that has intensified with urbanization and industrialization. Over the past few decades, light pollution has posed an increasing threat to human health . BetweenExisting hypotheses suggest that exposure to LAN may inhibit the natural surge of melatonin during the night , 54, theA series of epidemiological studies have examined the impact of LAN exposure on breast cancer risk, considering factors such as classification of this exposure, hormone receptor status, race, menopausal status, and others. In various studies examining different types of LAN exposure, previous research has reported a positive association between outdoor LAN exposure and breast cancer risk , 47. HowIn the past 2 years, a recent meta-analysis included an analysis of BMI and reanalyzed the impact of breast cancer risk in different estrogen receptor status populations . Additio22.1The present meta-analysis was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses PRISMA;  guidelin2.2We searched the PubMed, Cochrane Library, and Embase databases up to July 15, 2023. We employed Medical Subject Headings (MeSH) and relevant keywords. We drew upon the previous methodologies of meta-analysis. , 59–64. 2.3The study selection process was carried out by two reviewers (LZN and LZL) in accordance with the predetermined inclusion and exclusion criteria. Both reviewers independently screened the literature and removed duplicate and irrelevant articles based on titles and abstracts. Subsequently, the full texts of potentially eligible articles were obtained and thoroughly reviewed to ensure the inclusion of all relevant studies. Any disagreements between the two reviewers were resolved through discussion until a consensus was reached.2.4The inclusion criteria were as follows: (a) inclusion of an independent exposure variable pertaining to LAN exposure; (b) consideration of an outcome variable related to breast cancer; (c) incorporation of case–control or cohort study designs. The exclusion criteria were as follows: (a) reviews, conference proceedings, or commentaries; (b) duplicate literature (retaining the variant with the most comprehensive information).2.5The data extraction process was conducted independently by the first reviewer (LZN), and upon its completion, cross-verification and consultation was performed with the second reviewer (LZL). A predesigned data extraction form was utilized, and the following data were extracted: study type (case–control or cohort), first author, publication year, source of cases, source of controls, number of cases, number of controls, exposure definition, breast cancer definition, classification of exposure, information collection period and scope, baseline age, and values of outcome variables (odds ratio or hazard ratio) determined based on various confounding factors. Any potential discrepancies were addressed through discussions with LZL to achieve a consensus.2.6The quality of our studies was evaluated using the Newcastle-Ottawa Scale (NOS) , which e2.72 values (The adjusted relative ratios (RRs) and 95% confidence intervals (CIs) for each observational study were used to assess the association between breast cancer incidence and LAN exposure. Heterogeneity was evaluated using I2 values  and Coch2 values . Further2 values . Based o2 values , we used2 values . The pri33.1A total of 2,964 observational studies were initially identified after excluding duplicates. After conducting title and abstract screening, 2,871 articles were subsequently excluded. An in-depth review was conducted on 93 articles, and after the further exclusion of 72 articles, 21 studies were eligible for the meta-analysis . The pri3.2The 21 studies included in this study were published between 2001 and 2023 and involved a total of 734,372 participants. Of these studies, 11 originated from North America , 52, 3 fn = 9) (n = 10) (n = 14) (n = 14)  , 50, 52,(n = 10) , 51. Add(n = 14) , 50, 51,(n = 14) , 50, 52.n = 4) of studies examined the habit of watching TV while sleeping (n = 5) looked at the use of bedroom shutters (n = 3) explored the habit of waking up and turning on lights  assessments conducted on the various studies, the average score for the included research was 7.43 (ranging from 5 to 9). A score of 7 is considered as the cut-off point for high-quality research. Studies with a score of 7 or above are regarded as of higher quality. The scores of the included studies are illustrated in 3.42 = 31.3%, p < 0.001; A comprehensive analysis of 21 studies  revealed3.52 = 0%) was observed for continents, white race, and postmenopausal individuals, which was considered the main factor contributing to the slight heterogeneity observed in the overall analysis.We conducted subgroup analyses on the 21 included studies to explore the impact of various factors on the incidence of breast cancer . Based o2 = 0.0%, p < 0.001) was significantly higher than that in North America . However, the breast cancer incidence rate in Europe , despite being analyzed, did not exhibit any statistically significant difference. When considering racial differences, the incidence of breast cancer was higher in the white population , with no statistically significant difference observed in the Black population . Additionally, subgroup analysis for menopausal status did not yield statistically significant differences between the premenopausal  and postmenopausal groups .Regarding confounding factors, in our subgroup analyses that focused on different continents, we observed a clear disparity in breast cancer incidence rates associated with exposure to the LAN. Specifically, the breast cancer incidence rate in Asia  had a slightly higher promoting effect on breast cancer incidence than indoor LAN exposure . In the analysis of estrogen receptor status, LAN exposure exhibited a slightly stronger impact on breast cancer risk for cases with estrogen receptor positivity  compared to cases with estrogen receptor negativity . Subgroup analyses for different study types revealed that LAN exposure had a higher promoting effect on individual breast cancer incidence in case–control studies  compared to cohort studies .We also conducted analyses on various lifestyle factors summarized in the literature. When considering TV on while sleeping as an effect-modifying factor, the impact of LAN exposure on breast cancer did not show statistical significance . This observation holds true for “Turn on the light when you wake up”  and “Bedroom shutters (open)” . The complete dataset can be found in In terms of effect-modifying factors, similar to the meta-analysis conducted in 2021 , we cond3.6p = 0.746). Similar findings of bias were observed across various subgroups . Notably, our findings demonstrated a more substantial risk association among the case–control study population, contradicting the results reported by Urbano et al.  (n = 2)  , and sub (n = 2) , 47. Whi (n = 2) , we coul (n = 2)  was used (n = 2) , 50, 51, (n = 2)  categori (n = 2)  used onl (n = 2) , we have5Our meta-analysis provides evidence to support the hypothesis that exposure to LAN is associated with an elevated risk of breast cancer incidence, thereby adding valuable insights to the existing body of research. Although we did not observe a statistically significant correlation between lifestyle factors and the association between LAN and breast cancer incidence, our findings indicate a stronger relationship in certain subgroups, such as study populations located in Asia or investigations focusing on outdoor LAN exposure. These results contribute to the identification of important avenues for future research and offer meaningful recommendations.ZIL: Conceptualization, Data curation, Formal analysis, Software, Supervision, Validation, Writing – original draft, Writing – review & editing. ZHL: Conceptualization, Funding–acquisition, Writing – review & editing. HC: Data curation, Investigation, Supervision, Writing – review & editing. YL: Writing – review & editing-Methodology/Investigation. NT: Investigation, Supervision, Writing – review & editing. HL: Investigation, Supervision, Writing – review & editing.


The Alberta Stroke Program Early Computed Tomography Score (ASPECTS) was designed for semi-quantitative assessment of early ischemic changes on non-contrast computed tomography (NCCT) for acute ischemic stroke (AIS). We evaluated two automated ASPECTS software in comparison with reference standard.NCCT of 276 AIS patients were retrospectively reviewed (March 2018–June 2020). A three-radiologist consensus for ASPECTS was used as reference standard. Imaging data from both baseline and follow-up were evaluated for reference standard. Automated ASPECTS were calculated from baseline NCCT with 1-mm and 5-mm slice thickness, respectively. Agreement between automated ASPECTS and reference standard was assessed using intra-class correlation coefficient (ICC). Correlation of automated ASPECTS with baseline stroke severity (NIHSS) and follow-up ASPECTS were evaluated using Spearman correlation analysis.p < 0.05). Automated ASPECTS derived from 5-mm slice thickness images by software B showed higher sensitivity (0.56 vs. 0.51), higher specificity (0.87 vs. 0.81), higher accuracy (0.80 vs. 0.73), and higher AUC (0.71 vs. 0.66) than those using 1-mm slice thickness images (p < 0.05). Automated ASPECTS were significantly associated with baseline NIHSS and follow-up ASPECTS.In score-based analysis, automated ASPECTS calculated from 5-mm slice thickness images agreed well with reference standard . Bland–Altman analysis revealed that the mean differences between automated ASPECTS and reference standard were ≤ 0.6. In region-based analysis, automated ASPECTS derived from 5-mm slice thickness images by software A showed higher sensitivity (0.60 vs. 0.54), lower specificity (0.91 vs. 0.94), and higher AUC (0.76 vs. 0.74) than those using 1-mm slice thickness images (Automated ASPECTS showed good reliability and 5 mm was the optimal slice thickness. The Alberta Stroke Program Early Computed Tomography Score (ASPECTS) was introduced by PA Barber in 2000 and was designed for semi-quantitative assessment of early ischemic changes (EIC) on non-contrast computed tomography (NCCT) images of patients with acute ischemic stroke (AIS) . Since 2Studies investigating the use of artificial intelligence technology for automated determination of ASPECTS have yielded robust results . SeveralValidation studies of automated ASPECTS software also apply different reference standards for ASPECTS . DiffereThe aim of this study was to assess the impact of different slice thicknesses of NCCT images on the performance of RAPID and NBC software, and to investigate the differences in the clinical utility of automated ASPECTS. Images with different slice thicknesses were reconstructed from the raw data of the same patient. A three-radiologist consensus for ASPECTS was used as the reference standard. Imaging data from both baseline and follow-up were evaluated for reference standard.The study protocol was approved by the Medical Ethics Committee of our hospital (Number 2020-380). The requirement for informed consent was waived off by the institutional review board owing to the retrospective nature of the analysis.The present study was designed to evaluate the reliability and accuracy of automated ASPECTS based on images with slice thicknesses of 5 mm and 1 mm. Baseline ASPECTS were manually calculated from the baseline NCCT by two radiologists and automatically by the two software packages. Follow-up ASPECTS were generated by a single radiologist using follow-up CT/MRI. Reference standard of ASPECTS was defined by three radiologists. Imaging data from both baseline multimodal CT and follow-up CT/MRI were evaluated for the reference standard. Baseline ASPECTS were compared with the reference standard by means of score-based and region-based analysis. The correlation of baseline ASPECTS with baseline stroke severity (NIHSS) and follow-up ASPECTS were evaluated using Spearman correlation analysis. A schematic illustration of the study design is shown in We retrospectively enrolled patients with AIS who underwent multimodal CT scanning and received intravenous thrombolysis (IVT) or mechanical thrombectomy (MT) at the emergency department of our hospital between March 2018 and June 2020. Patients who qualified the following criteria were included: (1) age ≥ 18 years (2) availability of baseline multimodal CT images; (3) baseline NCCT images with 5 mm and 1 mm slice thickness reconstructed from raw data. The exclusion criteria were: (1) patients with hemorrhage, trauma, brain tumor, or encephalitis; (2) patients with bilateral AIS; (3) unqualified image quality for multimodal CT; (4) no follow-up CT/MRI obtained 24 h after baseline NCCT. The detailed flowchart of the patient selection process is shown in Baseline multimodal CT imaging included brain NCCT, head and neck CT angiography (CTA), and brain CT perfusion (CTP). All baseline multimodal CT images were acquired using a 256-channel scanner . Follow-up CT/MRI was obtained 24 h after the baseline NCCT. The scanning parameters for NCCT were: 120 kV, 200 mAs. The scanning parameters for CTP were 80 kV, 80 mAs, and 64 mm × 1.25 mm detector collimation and scan duration of 60 s. The scanning parameters for CTA were 100 kV, 266 mAs.To define the reference standard, two radiologists  independently reviewed the baseline multimodal CT and follow-up CT/MRI data. In the event of disagreement, a consensus score was provided by a third radiologist . Meeting criteria 1 + 3 + 4 or 2 + 3 + 4 was defined as EIC on baseline NCCT: (1) presence of hypodensity and/or loss of gray-white differentiation on baseline NCCT , (2) relThree months later, the baseline NCCT images with slice thickness of 5 mm were retrospectively assessed by two radiologists  without access to other imaging modality or clinical information, except for the lesion side. The ASPECTS calculated by the two radiologists were designated as RAD_9Y and RAD_5Y, respectively  and NBC (NeuBrainCARE) software supplied by the scanner’s manufacturers  were used to automatically calculate ASPECTS. Both RAPID and NBC can analyze images with different slice thickness. Datasets with slice thicknesses of 2–3 mm and 5 mm are recommended for RAPID and NBC, respectively.RAPID applies non-rigid registration with a standardized atlas to outline the 10 ASPECTS regions on each hemisphere. It measures the Hounsfield units (HU) and other parameters for the regions, then classifies each region as either normal or abnormal using a machine learning–based algorithm (random forest classifier) , 8. FinaNBC applies a non-rigid algorithm based on a hybrid supervised convolutional neural network (CNN) with a standardized atlas to outline the 10 ASPECTS regions on each hemisphere . It extrThe ASPECTS automatically calculated using RAPID and NBC was designated as RAPID_5mm, RAPID_1mm, NBC_5mm, and NBC_1mm, according to the slice thickness .Continuous variables are presented as mean ± standard deviation (SD) or as median (interquartile range), while categorical data are presented as frequencies (percentage). In score-based analysis, Bland–Altman plots were used to illustrate differences between automated ASPECTS, manual ASPECTS, and the reference standard. The intra-class correlation coefficients (ICCs) were used to examine the agreement between automatic ASPECTS, manual ASPECTS, and the reference standard. Accuracy, sensitivity, specificity, and area under the curve (AUC) of the receiver-operating characteristics (ROC) curve were used to assess the performance of automated ASPECTS and manual ASPECTS methods compared to the reference standard at the region level. Correlation of the baseline and follow-up ASPECTS with the baseline neurological severity (NIHSS score) was estimated using Spearman correlation. Two-tailed A total of 276 eligible patients were retrospectively identified . Baseline NCCT was obtained within a median time of 239 min from last known well (IQR 146–404 min); the median NIHSS score was 13 (IQR 9–16) and median consensus ASPECTS was 8.5 (IQR 6–10). The clinical and imaging characteristics are summarized in Bland–Altman analysis of the ASPECTS revealed that the mean differences between RAPID_5mm, RAPID_1mm, NBC_5mm, NBC_1mm, RAD_5Y, RAD_9Y, and reference standard were 0.3, 0.6, 0.1, −0.3, 0.9, and 0.6 points, respectively .Automated ASPECTS derived from 5-mm slice thickness images showed higher agreement with reference standard than the scores derived from 1-mm slice thickness images (as shown in p < 0.05), while RAPID_5mm showed better performance with higher sensitivity, lower specificity, and higher AUC than RAPID_1mm . The detailed results for correlation coefficients (rho) are shown in There was a significant correlation between baseline ASPECTS and follow-up ASPECTS as well as baseline stroke severity measured by NIHSS score . Our study demonstrated that slice thickness had an influence on the automatic ASPECTS results. The ASPECTS automatically calculated from images with 5-mm slice thickness performed similar at score level and superior at region level to those calculated with 1-mm slice thickness.Head NCCT is the most common examination for patients with AIS  and CT sThere is no gold standard for ASPECTS assessment  and the p > 0.05) and region level (p < 0.05). The reason for different conclusions may be the scanning protocol  . Our stu’s study : 120 kV,’s study : hybrid ’s study . There iASPECTS is highly correlated with baseline neurological severity in AIS. e-ASPECTS derived from 1-mm slice thickness images showed the highest correlation with the baseline NIHSS score . Of noteAccurate segmentation of ASPECTS region is the basis for region specific accuracy of automated ASPECTS. At region level, automated ASPECTS show greater differences on sensitivity than specificity and accuracy. NBC showed higher specificity and lower sensitivity, while RAPID showed lower specificity and higher sensitivity for detection of EIC in deep regions. NBC showed lower specificity and higher sensitivity, while RAPID showed higher specificity and lower sensitivity for detection of EIC in cortical regions. NBC and RAPID did not perform better than experienced radiologists for detection of EIC in cortical regions. Variability in region level results may be due to inconsistent definition of the region boundaries, especially for IC and M4-6 . The volSome limitations of our study should be considered. Firstly, we used imaging data from a single center. NCCT image acquisition and quality tend to vary in different centers. However, the sample size of this study was relatively large. Secondly, this study did not analyze the correlation between automated ASPECTS and prognosis. Several factors are known to affect the functional outcomes of AIS, such as onset time, NIHSS score, age, occlusion site, and treatment protocol. We included patients who received IVT or MT; hence we did not assess the prognostic value of ASPECTS. However, we analyzed the correlation between baseline NIHSS score, follow-up ASPECTS, and baseline ASPECTS.Automated ASPECTS produced robust scores with a stringent scanning protocol . Our results indicate that slice thickness impacts the identification of EIC by automated ASPECTS, and that the optimal slice thickness is 5 mm. Different software offer different advantages for assessment of deep regions and cortical regions.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Medical Ethics Committee of the First Hospital of Jilin University Clinical trial and research approval. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.DT and ZC: full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. ZS, FL, and LL: data collection. DT and ZC: study concept and design. ZC: drafting of the manuscript. DT: critical revision of the manuscript for important intellectual content. ML and SW: statistical analysis. ZC: study supervision. DT, ZC, and WW: CT scanning protocol. WW, YLi, and YLuo: supplying software. All authors contributed to the article and approved the submitted version.This study has received funding by the Jilin Province Special Scientific Research Project of Health (2018SCZWSZX-001), with additional funding support from the Jilin Province Science and Technology Development Plan Project (20200201303JC).YLi was an employee of Neusoft Medical Systems Co., Ltd. WW was an employee of Philips Healthcare.The remaining 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Hypercalcemia of malignancy (HCM) is a common complication seen in patients with cancer and is associated with high morbidity and mortality. Current long-term medical therapy for HCM focuses on inhibiting bone resorption with bisphosphonates or denosumab, which have the rare complication of osteonecrosis of the jaw. This case illustrates cinacalcet as an effective therapy for severe HCM resulting from PTH-related peptide in the setting of osteonecrosis of the jaw. Although the mechanism of action remains unclear, cinacalcet has been successful in other HCM cases even if not associated with elevated PTH-related peptide. Endocrinologists are frequently consulted for evaluation and management for hypercalcemia of malignancy (HCM). The mechanisms by which HCM occur are elevated PTH-related protein (PTHrP), cytokine-induced local osteolysis, tumoral production of 1,25-dihydroxyvitamin D, or ectopic PTH. Initial workup for HCM involves checking a PTH level to rule out primary hyperparathyroidism or ectopic PTH production. Subsequent evaluation with PTHrP, 25-hydroxyvitamin D, and 1-25-dihydroxyvitamin D levels aid in determining HCM from non-PTH etiologies.Ideal management of HCM is treatment of the underlying malignancy until remission is achieved. Because the treatment course may be prolonged or remission is not achieved, medications can be required to control calcium. Immediate hypercalcemia treatment is focused on promoting renal calcium excretion with IV fluids and the possible addition of loop diuretics or calcitonin . ContinuA 57-year-old woman with heart failure with a reduced ejection fraction (HFrEF) of 30% to 35% treated with furosemide and recurrent left-sided breast cancer, estrogen receptor-positive and human epidermal growth factor receptor 2 negative, stage 4, presented to the hospital from oncology infusion clinic because of hypercalcemia.Her breast cancer was initially treated with bilateral mastectomy, chemotherapy , and radiation. She was then on tamoxifen, which was later changed to anastrazole, for continued management. She became uninsured after 3 years and was unable to continue follow up or medications. Eight years later, in 2017, she presented to the hospital with abdominal pain and distension resulting from tumor burden from breast cancer recurrence. Bone biopsy of a hip lesion confirmed metastatic disease. She was diagnosed with stage 4 breast cancer with bone metastasis and started on chemotherapy  and denosumab 120 mg monthly . Of noteDuring her denosumab holiday, she developed symptomatic hypercalcemia, with corrected calcium, 14.8 mg/dL (3.69 mmol/L) ; PTH, 10 pg/mL (1.1 pmol/L) ; PTHrP, 74.4 pg/mL (7.9 pmol/L) ; and 25-hydroxyvitamin D 40 ng/mL (99.8 nmol/L) . Monthly denosumab was restarted in July 2020 for hypercalcemia. The patient had recurrent episodes of hypercalcemia that required additional management with 2 infusions of zoledronic acid the following year. This combination antiresorptive therapy improved her hypercalcemia.In early 2022, she had jaw pain and swelling that prompted concerns for ONJ leading to a referral to oral maxillofacial surgery (OMFS) and cessation of denosumab therapy. She received IV fluids intermittently at the infusion center when she was due for chemotherapy. By May 2022, when she presented for her infusion, she reported nausea, vomiting, mild confusion, palpitations, and altered vision described as “silver dots.” Her calcium levels were found to be greater than 13 mg/dL (3.24 mmol/L) and she was transferred to the hospital.On presentation to the hospital, her corrected calcium was 15.5 mg/dL (3.87 mmol/L), PTH was 22.8 pg/mL (2.4 pmol/L), and 25-hydroxyvitamin D 22.0 ng/mL (54.9 nmol/L). On physical examination, she was edentulous with bone exposure measuring 2 mm with expressible pus and no sign of volume overload or other abnormality. Repeat computed tomography maxillofacial scan with IV contrast showed extensive lytic and sclerotic changes involving the mandibular body bilaterally that was compatible with osteonecrosis and possible superimposed chronic osteomyelitis. Additionally, a pathologic fracture at the junction of the right mandibular body and angle was seen. She had a significant progression of her dental disease compared with the imaging 3 months prior. PTHrP and 1,25-dihydroxyvitamin D laboratory tests were undertaken but test results returned after the patient’s discharge.OMFS, along with the internal medicine team, initiated treatment for osteomyelitis. OMFS recommended no further antiresorptive therapy in the setting of severe ONJ. Calcitonin and continuous IV fluids decreased calcium levels to 13.0 mg/dL (3.24 mmol/L). Endocrinology was consulted to determine further treatment options. Extensive discussion occurred with the patient and her adult daughter regarding her treatment history, current situation, and inability to use first-line therapy for her hypercalcemia. Considering the severity of the hypercalcemia, off-label use of cinacalcet was discussed. The primary medicine team, patient, and adult daughter agreed with trial of cinacalcet 30 mg twice a day. Calcitonin was stopped after 3 days, and IV fluids were continued at decreasing rates while she was encouraged to increase her oral water intake . ThroughHer corrected calcium decreased to 11.4 mg/dL (2.84 mmol/L) with IV fluids and cinacalcet. Intravenous fluid rate was not increased because of risk of volume overload HFrEF; furosemide was continued at the same dose. As her calcium levels decreased, she felt mentally clearer, nausea and vomiting subsided, and she was able to eat and drink. Three days after initiation of cinacalcet, IV fluids were stopped, and her corrected calcium level remained stable at 11.4 mg/dL (2.84 mmol/L) . She wasHer insurance did not cover cinacalcet for this off-label use, but the county health care pharmacy allowed her to pay for her prescription on a payment plan and subsequently found that a medication coupon would make it more affordable. Five months after initiation of cinacalcet 30 mg twice daily, her corrected calcium decreased further to 9.4 mg/dL (2.35 mmol/L). Oncology continued to monitor her cancer with imaging and, on its assessment, there was no progression or remission of metastatic disease. More than a year after being followed by the endocrinology clinic, her cinacalcet was decreased to 30 mg daily. But she began to miss appointments with endocrinology. Her calcium levels were reviewed from the laboratory tests she got monthly before her chemotherapy, and it remained in the normal range. She was called when she missed appointments and confirmed she continued to take the cinacalcet 30 mg daily. Although repeat PTHrP and PTH were ordered by endocrinology, these were not drawn. OMFS continued treatment of her ONJ with further debridement procedures, and she completed IV antibiotics for osteomyelitis.Zoledronic acid and denosumab are frequently used in metastatic bone disease, HCM, and osteoporosis. Because of the high and frequent dosages required of denosumab and bisphosphonates, ONJ is more common in those being treated for HCM compared with osteoporosis. The risk of ONJ is mitigated with dental hygiene and oral health monitoring in those with HCM before initiation of denosumab or bisphosphonate therapy. Yet, as shown with this case, even with planned monitoring and evaluation, adverse reactions can occur and limit the available options for management of HCM.The Journal of Clinical Endocrinology & Metabolism HCM guideline, cinacalcet is primarily recommended for HCM from parathyroid carcinoma with a comment that there are case reports that its use results in decreased calcium levels in those with nonparathyroid carcinomas [Cinacalcet is most used in secondary hyperparathyroidism for patients on dialysis, HCM secondary to parathyroid carcinoma, and in primary hyperparathyroidism for those who are not surgical candidates . Althougrcinomas . Callaghrcinomas . Cases orcinomas .Cinacalcet lowers calcium levels through G protein-coupled transmembrane cell-surface calcium-sensing receptor, primarily located on chief cells of the parathyroid gland, activation of which inhibits PTH secretion and enhances renal urinary calcium excretion . AlthougAlthough there is not a clear mechanism of action widely identified to explain why cinacalcet has been an effective way to normalize calcium in those with nonparathyroid carcinoma HCM, multiple cases of refractory HCM have been successfully treatment with cinacalcet , 7‐9. ThImportance of early dental clinical and imaging evaluation when antiresorptive medications are possibly needed for hypercalcemia of malignancy (HCM) or bone metastasis treatment.Cinacalcet has been an effective, off-label, therapy for HCM resulting from elevated PTH-related peptide or 1,25 dihydroxyvitamin D.The mechanism of action for cinacalcet in treatment of HCM is not well understood but possible reasons for its effect include increased endogenous calcitonin levels, decreased calcium absorption, and increased calcium urinary excretion.All authors made individual contributions to authorship. L.V. and T.C. were involved in the diagnosis and management of this patient and in manuscript submission.